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Does mechanical shock kill animals?

Does mechanical shock kill animals?


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I heard that loss of blood kills shot animals, a lot. But a few times, I heard that mechanical shock kills animals. For example in this video (starts to play at the right spot; relevant from 14:35 to 15:03).

That loss of blood kills an animal seems pretty obvious as the brain can't function without being supplied oxygen and it can't be supplied oxygen if there is no blood to carry the oxygen.

If the mechanical shock is applied to the head, it also seems somewhat reasonable. I guess it could be that the shock destroys vessels in the brain. But other than for a headshot: Is there a biological reason why an animal couldn't continue to live after experiencing mechanical shock due to being hit by a small projectile (being hit by a truck which is able to accelerate the entire animal doesn't count)?


With a small projectile you will always get some sort of shearing forces: the tissues directly impacted will accelerate faster than adjacent tissues, and so on. This can cause tearing of many tissues, including blood vessels, but other tissues as well.

In the context of the brain, the effects of traumatic brain injury are a constant area of research, for example see here.


Heat Shock Proteins

Abstract

Heat shock proteins (HSPs) are specific proteins that are made when cells are briefly exposed to temperatures above their normal growth temperature. The synthesis of HSPs is a universal phenomenon, occurring in all plant and animal species studied, including humans. HSPs are also made by prokaryotic cells, namely, bacterial and archaean. Because HSPs can also be induced by oxidants, toxins, heavy metals, free radicals, viruses, and other stressors, they are sometimes called the ‘stress proteins’. Most HSPs are molecular chaperones, which normally promote the self-assembly of newly synthesized polypeptide chains of proteins into a native spatial structure, the assembly of their complexes, and their transport through membranes as well as their participation in signal transduction. A nonlethal increase in temperature above the physiological norm for a biological species suppresses protein synthesis in the cell, activates the heat shock factor (HSF), and enhances transcription of heat shock genes, while exposure to a lethal temperature initiates apoptosis or programmed cell death. In turn, HSPs inhibit apoptosis and provide cells with thermal stability if stress reoccurs. In so doing, chaperones prevent irreversible aggregation of unfolded proteins and assist in the restoration of their native structure and/or degradation of denatured proteins. Response to heat shock is attenuated as the cell returns to normalcy after stress has been removed, HSF transforms to its inactive form and is transported to the cytoplasm, and ‘bookmarked preinitiation complexes’ are accumulated in the promoters of the genes encoding HSPs. HSP gene polymorphisms are associated with inflammatory, autoimmune, cardiovascular, and neurodegenerative diseases and, finally, aging.


Does mechanical shock kill animals? - Biology

(This is my entry to the first “special edition” of The Giant’s Shoulders, dubbed “The Leviathan’s Shoulders”, with an emphasis on oceans and ocean life. The post is actually about a river creature, but, hey, it’s still aquatic!)

I’ve spent a lot of time talking about Michael Faraday (1791-1867) and his scientific accomplishments on this blog. His thorough investigations into the nature of electricity and magnetism paved the way for all of modern electromagnetics as well as optics, and he is rightly viewed as one of the greatest experimentalists of all time. Among his monumental works are the observation that changing magnetic fields induce electric fields (electromagnetic induction) and the observation that light polarization can be affected by an applied magnetic field (Faraday rotation).

Though it is natural to think of Faraday as a researcher of electricity alone, in his era the study of electricity connected to almost every aspect of the natural sciences. In the late 1700s Luigi Galvani had shown that an amputated frog’s leg could be made to move by electrical stimulation, demonstrating a connection between biological function and electricity. By 1800 it was known that chemical reactions can be induced by electricity, in a process known as electrolysis Faraday himself published fundamental results on electrolysis in 1834. Electricity could be connected to thermodynamics through the observation that an electrical current heats the wire it passes through (Joule heating) this process was rather mysterious because neither the origins of heat (atomic motion) nor electricity (electrons) were established in Faraday’s time.

Electricity could be generated through atmospheric, chemical, and mechanical means, and it was by no means obvious that these different sources were manifestations of the same fundamental electrical phenomenon. (In fact, Faraday himself did a significant amount of research to demonstrate that all forms of electricity are in fact the same. )

A researcher of electricity could therefore be expected to make forays into quite diverse areas of study. In 1839, Faraday published the scientific results of one of his forays, “Notice of the character and direction of the electric force of the Gymnotus,” in the Philosophical Transactions of the Royal Society (pp. 1-12).

What is the “Gymnotus”? The taxonomy of the species seems to have been changed over the years, but at this time seems to be referring to what used to be known as Gymnotus electricus, or the electric eel (image source):

In modern taxonomy, the electric eel is Electrophorus electricus, and is part of the bigger family of Gymnotidae (knifefish), which also includes all species that are now classified under the genus Gymnotus. All knifefish possess special bioelectric organs, though the electric eel (not really an “eel”) is the only one that has developed these electric organs as a weapon for hunting.

The electric eel is a freshwater fish found in the waters of South American rivers, notably the Amazon and Orinoco Rivers. They can reach up to 8 feet in length and 45 pounds in weight, and are air breathing — they come to the surface frequently for gulps of air.

The eels feed on pretty much any small creatures they can get: primarily fish, but also amphibians, birds, and even small mammals. They are able to stun their prey (and repel predators) by generating a sizable electrical shock — up to 600 volts potential difference, which could be potentially fatal for a human under the right circumstances. These shocks are produced by specialized organs in the eel that contain cells called electrocytes, each of which acts like a little battery containing about .15 volt potential difference. The eel has roughly 6000 of these electrocytes, and their electrical energy can be released at will.

The study of creatures such as the electric eel were of special interest to researchers of Faraday’s time because they provided insight into the workings of living creatures themselves scientists had long suspected that electricity played a vital role in the nervous system, though the exact nature of that role was still controversial. To quote Faraday’s introduction (all references and citations removed for clarity):

Wonderful as are the laws and phenomena of electricity when made evident to us in inorganic or dead matter, their interest can bear scarcely any comparison with that which attaches to the same force when connected with the nervous system and with life and though the obscurity which for the present surrounds the subject may for the time also veil its importance, every advance in our knowledge of this mighty power in relation to inert things, helps to dissipate that obscurity, and to set forth more prominently the surpassing interest of this very high branch of Physical Philosophy. We are indeed but upon the threshold of what we may, without presumption, believe man is permitted to know of this matter and the many eminent philosophers who have assisted in making this subject known, have, as is very evident in their writings, felt up to the latest moment that such is the case.

As well as being an amazing experimentalist, Faraday was also a wonderful writer, as the passage above shows.

Unlike a lot of his other work, however, Faraday was not exactly breaking new ground, as he himself observed:

The existence of animals able to give the same concussion to the living system as the electrical machine, the voltaic battery, and the thunder storm, being with their habits made known to us by RICHER, S’GRAVESENDE, FIRMIN, WALSH, HUMBOLDT, &c. &c., it became of growing importance to identify the living power which they possess, with that which man can call into action from inert matter, and by him named electricity. With the Torpedo this has been done to perfection, and the direction of the current of force determined by the united and successive labours of WALSH, CAVENDISH , GALVANI, GARDINI, HUMBOLDT and GAY-LUSSAC, TODD, Sir HUMPHRY DAVY, Dr. DAVY, BECQUEREL, and MATTEUCCI.

The Gymnotus has also been experimented with for the same purpose, and the investigations of WILLIAMSON, GARDEN, HUMBOLDT, FAHLBERG and GUISANI, have gone very far in showing the identity of the electric force in this animal with the electricity excited by ordinary means and the two latter philosophers have even obtained the spark.

The “torpedo” is another term for the electric ray, a genus of rays that can, like the electric eel, produce jolts for hunting or defense. Torpedos are salt water animals of a variety of species with a wide geographic distribution the largest specimens can produce a jolt of up to 220 volts, significantly less than the eel. Faraday notes, however, that the torpedo is a less hardy creature that rarely survives long in captivity,

To obtain Gymnoti has therefore been a matter of consequence and being stimulated, as much as I was honoured, by very kind communications from Baron HUMBOLDT, I in the year 1835 applied to the Colonial Office, where I was promised every assistance in procuring some of these fishes, and continually expect to receive either news of them or the animals themselves.

Luck was with Faraday, and he soon was offered a specimen to study:

A Gymnotus has lately been brought to this country by Mr. PORTER, and purchased by the proprietors of the Gallery in Adelaide Street: they immediately most liberally offered me the liberty of experimenting with the fish for scientific purposes they placed it for the time exclusively at my disposal, that (in accordance with HUMBOLDT’S directions) its powers might not be impaired: only desiring me to have a regard for its life and health. I was not slow to take advantage of their wish to forward the interests of science, and with many thanks accepted their offer. With this Gymnotus, having the kind assistance of Mr. BRADLEY of the Gallery, Mr. GASSIOT, and occasionally other gentlemen, as Professors DANIELL, OWEN and WHEATSTONE, I have obtained every proof of the identity of its power with common electricity. All of these had been obtained before with the Torpedo, and some, as the shock, circuit, and spark, with the Gymnotus but still I think a brief account of the results will be acceptable to the Royal Society, and I give them as necessary preliminary experiments to the investigations which we may hope to institute when the expected supply of animals arrive.

Faraday was a very thorough experimentalist though his observations were not necessarily groundbreaking, he wanted to provide the details so that future researchers could confirm his results and not unnecessarily duplicate them. It was in the same spirit that he would later recount his failed attempts to link gravity and electricity.

Faraday begins his account proper with a description of the animal itself and its condition when he received it:

The fish is forty inches long. It was caught about March 1838 was brought to the Gallery on the 15th of August, but did not feed from the time of its capture up to the 19th of October. From the 24th of August Mr. BRADLEY nightly put some blood into the water, which was changed for fresh water next morning, and in this way the animal perhaps obtained some nourishment. On the 19th of October it killed and eat four small fish since then the blood has been discontinued, and the animal has been improving ever since, consuming upon an average one fish daily*.

* The fish eaten were gudgeons, carp, and perch.

He was careful, according to directions he had received from others, not to exhaust the animal with frequent testing:

I first experimented with it on the 3rd of September, when it was apparently languid, but gave strong shocks when the hands were favourably disposed on the body. The experiments were made on four different days, allowing periods of rest from a month to a week between each. His health seemed to improve continually, and it was during this period, between the third and fourth days of experiment, that he began to eat.

The first time I read that paragraph, I did a double-take: Faraday would test the eel’s shocking ability by placing his bare hands on it. Remember that this is a creature that produces upwards of 600 volts per shock clearly Faraday was not working under any OSHA guidelines!

Hands would be an inadequate measuring device for a number of tests, however, so other tools were developed:

Beside the hands two kinds of collectors were used. The one sort consisted each of a copper rod fifteen inches long, having a copper disc one inch and a half in diameter brazed to one extremity, and a copper cylinder to serve as a handle, with large contact to the hand, fixed to the other, the rod firom the disc upwards being well covered with a thick caoutchouc tube to insulate that part fiomn the water. By these the states of particular parts of the fish whilst in the water could be ascertained.

The other kind of collectors were intended to meet the difficulty presented by the complete immersion of the fish in water for even when obtaining the spark itself I did not think myself justified in asking for the removal of tile animal into air. A plate of copper eight inches long by two inches and a half wide, was bent into a saddle shape, that it might pass over the fish, and inclose a certain extent of the back and sides, and a thick copper wire was brazed to it, to convey the electric force to the experimental apparatus a jacket of sheet caoutchouc was put over the saddle, the edges projecting at the bottom and the ends the ends were made to converge so as to fit in some degree the body of the fish, and the bottom edges were made to spring against any horizontal surface on which the saddles were placed. The part of the wire liable to be in the water was covered with caoutchouc.

“Caoutchouc” is another name for India rubber, and it served as an insulator. My rough sketch of this second collector is shown below:

The rubber jacket and bottom edges allowed the fish to be well insulated from the water while “clamped” in the device:

These conductors being put over the fish, collected power sufficient to produce many electric effects but when, as in obtaining the spark, every possible advantage was needful, then glass plates were placed at the bottom of the water, and the fish being over them, the conductors were put over it until the lower caoutchouc edges rested on the glass, so that the part of the animal within the caoutchouc was thus almost as well insulated as if the Gymnotus had been in the air.

Faraday’s paddles served almost as “reverse” defibrillator paddles, conveying a charge from the target instead of to the target!

Faraday’s initial tests involved demonstrating that the electricity produced by the Gymnotus was of the same nature as all other known forms of electricity. This involved showing that the Gymnotus’ electricity could perform the same set of “tricks” as other sources:

Shock. The shock of this animal was very powerful when the hands were placed in a favourable position, i. e. one on the body near the head, and the other near the tail the nearer the hands were together within certain limits the less powerful was the shock. The disc conductors conveyed the shock very well when the hands were wetted and applied in close contact with the cylindrical handles but scarcely at all if the handles were held in the dry hands in an ordinary way.

Galvanometer. Using the saddle conductors applied to the anterior and posterior parts of the Gymnotus, a galvanometer was readily affected. It was not particularly delicate for zinc and platina plates on the upper and lower surface of the tongue did not cause a permanent deflection of more than 25° yet when the fish gave a powerful discharge the deflection was as much as 30°, and in one case even 40°. The deflection was constantly in a given direction, the electric current being always from the anterior parts of the animal through the galvanometer wire to the posterior parts. The former were therefore for the time externally positive, and the latter negative.

Making a magnet. When a little helix containing twenty-two feet of silked wire wound on a quill was put into the circuit, and an annealed steel needle placed in the helix, the needle became a magnet, and the direction of its polarity in every case indicated a current from the anterior to the posterior parts of the Gymnotus through the conductors used.

Chemical decomposition. Polar decomposition of a solution of iodide of potassium was easily obtained. Three or four folds of paper moistened in the solution were placed between a platina plate and the end of a wire also of platina, these being respectively connected with the two saddle conductors. Whenever the wire was in conjunction with the conductor at the forepart of the Gymnotus, iodine appeared at its extremity but when connected with the other conductor none was evolved at the place on the paper where it before appeared. So that here again the direction of the current proved to be the same as that given by the former tests.

By this test I compared the middle part of the fish with other portions before and behind it, and found that the conductor A, which being applied to the middle was negative to the conductor B applied to the anterior parts, was, on the contrary, positive to it when B was applied to places near the tail. So that within certain limits the condition of the fish externally at the time of the shock appears to be such, that any given part is negative to other parts anterior to it, and positive to such as are behind it.

Evolution of heat. Using a HARRIS’S thermo-electrometer belonging to Mr. GASSIOT, we thought we were able in one case, namely, that when the deflection of the galvanometer was 40°, to observe a feeble elevation of temperature. I was not observing the instrument myself, and one of those who at first believed they saw the effect now doubts the result.

Spark. The electric spark was obtained thus. A good magneto-electric coil, with a core of soft iron wire, had one extremity made fast to the end of one of the saddle collectors, and the other fixed to a new steel file another file was made fast to the end of the other collector. One person then rubbed the point of one of these files over the face of the other, whilst another person put the collectors over the fish, and endeavoured to excite it to action. By the friction of the files contact was made and broken very frequently and the object was to catch the moment of the current through the wire and helix, and by breaking contact during the current to make the electricity sensible as a spark. The spark was obtained four times, and nearly all who were present saw it. That it was not due to the mere attrition of the two piles was shown by its not occurring when the files were rubbed together, independently of the animal. Since then I have substituted for the lower file a revolving steel plate, cut file fashion on its face, and for the upper file wires of iron, copper and silver, with all of which the spark was obtained.

These tests established not only the identity of the Gymnotus’ shock with common electricity, but also gave insight into the location of origin of the charge and the direction of flow. In essence, the Gymnotus acts like a long voltaic pile, with the positive end at the head. When the shock is released, the current flows from the head to the tail (eel image source):

One of the big challenges of researching electricity in Faraday’s era was the lack of universally accepted standard units — such units would not be formalized until the late 1800s (and that story will appear in a later blog post). The best that researchers like Faraday could do is measure the strength of electrical signals relative to some existing and familiar electrical source:

I think a few further but brief details of experiments relating to the quantity and disposition of the electricity in and about this wonderful animal will not be out of place in this short account of its powers.

When the shock is strong, it is like that of a large Leyden battery charged to a low degree, or that of a good voltaic battery of perhaps one hundred or more pair of plates, of which the circuit is completed for a moment only. I endeavoured to form some idea of the quantity of electricity by connecting a large Leyden battery with two brass balls, above three inches in diameter, placed seven inches apart in a tub of water, so that they might represent the parts of the Gymnotus to which the collectors had been applied but to lower the intensity of the discharge, eight inches in length of six-fold thick wetted string were interposed elsewhere in the circuit, this being found necessary to prevent the easy occurrence of the spark at the ends of the collectors, when they were applied in the water near to the balls, as they had been before to the fish. Being thus arranged, when the battery was strongly charged and discharged, and the hands put into the water near the balls, a shock was felt, much resembling that from the fish and though the experiments have no pretension to accuracy, yet as the tension could be in some degree imitated by reference to the more or less ready production of a spark, and after that the shock be used to indicate whether the quantity was about the same, I think we may conclude that a single medium discharge of the fish is at least equal to the electricity of a Leyden battery of fifteen jars, containing 3500 square inches of glass coated on both sides, charged to its highest degree. This conclusion respecting the great quantity of electricity in a single Gymnotus shock, is in perfect accordance with the degree of deflection which it can produce in a galvanometer needle, and also with the amount of chemical decomposition produced in the electrolyzing experiments.

Statements like these make me appreciate even more the accomplishments of researchers like Faraday, who managed to learn so much about the physical world starting with so few foundational principles!

Faraday was also interested in how the electricity travels from the head to the tail of the fish through the water*, and devised a series of measurements to study this:

As, at the moment when the fish wills the shock, the anterior parts are positive and the posterior parts negative, it may be concluded that there is a current from the former to the latter through every part of the water which surrounds the animal, to a considerable distance from its body. The shock which is felt, therefore, when the hands are in the most favourable position, is the effect of a very small portion only of the electricity which the animal discharges at the moment, by far the largest portion passing through the surrounding water. This enormous external current must be accompanied by some effect within the fish equivalent to a current, the direction of which is from the tail towards the head, and equal to the sum of all these external forces. Whether the process of evolving or exciting the electricity within the fish includes the production of this internal current (which need not of necessity be as quick and momentary as the external one), we cannot at present say but at the time of the shock the animal does not apparently feel the electric sensation which he causes in those around him.

Faraday uses the following adorable diagram to help the reader visualize his description:

I can do no better than simply quote his explanation of the experiments:

By the help of the accompanying diagram I will state a few experimental results which illustrate the current around the fish, and show the cause of the difference in character of the shock occasioned by the various ways in which the person is connected with the animal, or his position altered with respect to it. The large circle represents the tub in which the animal is confined its diameter is forty-six inches, and the depth of water in it three inches and a half it is supported on dry wooden legs. The figures represent the places where the hands or the disc conductors were applied, and where they are close to the figure of the animal, it implies that contact with the fish was made. I will designate different persons by A, B, C, &c., A being the person who excited the fish to action.

When one hand was in the water the shock was felt in that hand only, what- ever part of the fish it was applied to it was not very strong, and was only in the part immersed in the water. When the hand and part of the arm was in, the shock was felt in all the parts immersed.

When both hands were in the water at the same part of the fish, still the shock was comparatively weak, and only in the parts immersed. If the hands were on opposite sides, as at 1, 2, or at 3, 4, or 5, 6, or if one was above and the other below at the same part, the effect was the same. When the disc collectors were used in these positions no effect was felt by the person holding them, (and this corresponds with the observation of GAY-LUSSAC on Torpedos,) whilst other persons, with both hands in at a distance from the fish, felt considerable shocks.

When both hands or the disc collectors were applied at places separated by a part of the length of the animal, as at 1, 3, or 4, 6, or 3, 6, then strong shocks extending up the arms, and even to the breast of the experimenter, occurred, though another person with a single hand in at any of these places, felt comparatively little. The shock could be obtained at parts very near the tail, as at 8, 9. I think it was strongest at about 1 and 8. As the hands were brought nearer together the effect diminished, until being in the same cross plane, it was, as before described, only sensible in the parts immersed.

B placed his hands at 10, 11, at least four inches from the fish, whilst A touched the animal with a glass rod to excite it to action B quickly received a powerful shock. In another experiment of a similar kind, as respects the non-necessity of touching the fish, several persons received shocks independently of each other thus A was at 4, 6 B at 10, 1 C at 16, 17 and D at 18, 19 all were shocked at once, A and B very strongly, C and D feebly. It is very useful, whilst experimenting with the galvanometer or other instrumental arrangements, for one person to keep his hands in the water at a moderate distance from the animal, thlat he may know and give information when a discharge has taken place.

When B had both hands at 10, 11, or at 14, 15, whilst A had but one hand at 1, or 3, or 6, the former felt a strong shock, whilst the latter had but a weak one, though in contact with the fish. Or if A had both hands in at 1, 2, or 3, 4, or 5, 6, the effect was the same.

If A had the hands at 3, 5, B at 14, 15, and C at 16, 17, A received the most powerful shock, B the next powerful, and C the feeblest.

When A excited the Gymnotus by his hands at 8, 9, whilst B was at 10, 11, the latter had a much stronger shock than the former, though the former touched and excited the animal.

A excited the fish by one hand at 3, whilst B had both hands at 10, 11 (or along), and C had the hands at 12, 13 (or across) A had the pricking shock in the immersed hand only B had a strong shock up the arms C felt but a slight effect in the immersed parts.

The experiments I have just described are of such a nature as to require many repetitions before the general results drawn from them can be considered as established nor do I pretend to say that they are anything more than indications of the direction of the force. It is not at all impossible that the fish may have the power of throwing each of its four electric organs separately into action, and so to a certain degree direct the shock, i.e. he may have the capability of causing the electric current to emanate from one side, and at the same time bring the other side of his body into such a condition, that it shall be as a non-conductor in that direction. But I think the appearances and results are such as to forbid the supposition, that he has any control over the direction of the currents after they have entered the fluid and substances around him.

These experiments are fascinating and paint an amusing picture: Faraday and up to three assistants sticking their hands in the water repeatedly, zapping themselves**. (I can’t help but visualize Faraday speaking like Christopher Guest in this classic video clip: “What did this do to you? Tell me. And remember, this is for posterity so be honest. How do you feel?”)

In addition to studying the electric powers of the Gymnotus, Faraday also indulged in some amateur behavioral observations:

This Gymnotus can stun and kill fish which are in very various positions to its own body but on one day when I saw it eat, its action seemed to me to be peculiar. A live fish about five inches in length, caught not half a minute before, was dropped into the tub. The Gymnotus instantly turned round in such a manner as to form a coil inclosing the fish, the latter representing a diameter across it a shock passed, and there in an instant was the fish struck motionless, as if by lightning, in the midst of the waters, its side floating to the light. The Gymnotus made a turn or two to look for its prey, which having found he bolted, and then went searching about for more. A second smaller fish was given him, which being hurt in the conveyance, showed but little signs of life, and this he swallowed at once, apparently without shocking it. The coiling of the Gymnotus round its prey had, in this case, every appearance of being intentional on its part, to increase the force of the shock, and the action is evidently exceedingly well suited for that purpose, being in full accordance with the well-known laws of the discharge of currents in masses of conducting matter and though the fish may not always put this artifice in practice, it is very probable he is aware of its advantage, and may resort to it in cases of need.

The Gymnotus appears to be sensible when he has shocked an animal, being made conscious of it, probably, by the mechanical impulse he receives, caused by the spasms into which it is thrown. When I touched him with my hands, he gave me shock after shock but when I touched him with glass rods, or the insulated conductors, he gave one or two shocks, felt by others having their hands in at a distance, but then ceased to exert the influence, as if made aware it had not the desired effect. Again, when he has been touched with the conductors several times, for experiments on the galvanometer or other apparatus, and appears to be languid or indifferent, and not willing to give shocks, yet being touched by the hands, they, by convulsive motion, have informed him that a sensitive thing was present, and he has quickly shown his power and his willingness to astonish the experimenter.

In concluding his article, Faraday explains that an understanding of the Gymnotus could have implications for the understanding of all living creatures:

It has been remarked by GEOFFROY ST. HILAIRE, that the electric organs of the Torpedo, Gymnotus, and similar fishes, cannot be considered as essentially connected with those which are of high and direct importance to the life of the animal, but to belong rather to the common teguments and it has also been found that such Torpedos as have been deprived of the use of their peculiar organs, have continued the functions of life quite as well as those in which they were allowed to remain. These, with other considerations, lead me to look at these parts with a hope that they may upon close investigation prove to be a species of natural apparatus, by means of which we may apply the principles of action and re-action in the investigation of the nature of the nervous influence.

The anatomical relation of the nervous system to the electric organ the evident exhaustion of the nervous energy during the production of electricity in that organ the apparently equivalent production of electricity in proportion to the quantity of nervous force consumed the constant direction of the current produced, with its relation to what we may believe to be an equally constant direction of the nervous energy thrown into action at the same time all induce me to believe, that it is not impossible but that, on passing electricity per force through the organ, a reaction back upon the nervous system belonging to it might take place, and that a restoration, to a greater or smaller degree, of that which the animal expends in the act of exciting a current, night perhaps be effected. We have the analogy in relation to heat and magnetism. SEEBECK taught us how to commute heat into electricity and PELTIER has more lately given us the strict converse of this, and shown us how to convert the electricity into heat, including both its relation of hot and cold. OERSTED showed how we were to convert electric into magnetic forces, and I had the delight of adding the other member of the full relation, by reacting back again and converting magnetic into electric forces. So perhaps in these organs, where nature has provided the apparatus by means of which the animal can exert and convert nervous into electric force, we may be able, possessing in that point of view a power far beyond that of the fish itself, to re-convert the electric into the nervous force.

This may seem to some a very wild notion, as assuming that the nervous power is in some degree analogous to such powers as heat, electricity, and magnetism. I am only assuming it, however, as a reason for making certain experiments, which, according as they give positive or negative results, will regulate further expectation. And with respect to the nature of nervous power, that exertion of it which is conveyed along the nerves to the various organs which they excite into action, is not the direct principle of life and therefore I see no natural reason why we should not be allowed in certain cases to determine as well as observe its course. Many philosophers think the power is electricity. PRIESTLEY put forth this view in 1774 in a very striking and distinct form, both as regards ordinary animals and those which are electric, like the Torpedo. Dr. WILSON PHILIP considers that the agent in certain nerves is electricity modified by vital action. MATTEUCCI thinks that the nervous fluid or energy, in the nerves belonging to the electric organ at least, is electricity. MM. PREVOST and DUMAS are of opinion that electricity moves in the nerves belonging to the muscles and M. PREVOST adduces a beautiful experiment, in which steel was magnetized, in proof of this view which, if it should be confirmed by further observation and by other philosophers, is of the utmost consequence to the progress of this high branch of knowledge. Now though I am not as yet convinced by the facts that the nervous fluid is only electricity, still I think that the agent in the nervous system may be an inorganic force and if there be reasons for supposing that magnetism is a higher relation of force than electricity, so it may well be imagined, that the nervous power may be of a still more exalted character, and yet within the reach of experiment.

There is a lot of discussion in those paragraphs, which I will attempt to summarize. Many researchers of Faraday’s era strongly suspected (correctly) that the nervous system operated by electricity or, in other words, that electricity is the “principle of life”. This suspicion was sparked by the already mentioned research of Galvani, among others. Faraday was not willing to go quite so far as to treat the human body as a purely electrical device, but thought that the “nervous fluid” could be another fundamental force, subject to laws just like electricity, magnetism, and gravity. The nervous force could then be coupled to electricity, just like magnetism is coupled to electricity.

Faraday even suggests some possibilities for future experimentation based on this principle:

The kind of experiment I am bold enough to suggest is as follows. If a Gymnotus or Torpedo has been fatigued by frequent exertion of the electric organs, would the sending of currents of similar force to those he emits, or of other degrees of force, either continuously or intermittingly in the same direction as those he sends forth, restore him his powers and strength more rapidly than if he were left to his natural repose ?

Would sending currents through in the contrary direction exhaust the animal rapidly? There is, I think, reason to believe that the Torpedo (and perhaps the Gymnotus) is not much disturbed or excited by electric currents sent only through the electric organ so that these experiments do not appear very difficult to make.

Such are some of the experiments which the conformation and relation of the electric organs of these fishes suggest, as being rational in their performance, and promising in anticipation. Others may not think of them as I do but I can only say for myself, that were the means in my power, they are the very first that I would make.

Faraday himself did not apparently indulge in many more experiments on electric fishes. We are in some sense fortunate that he did not, because one of his greatest discoveries (Faraday rotation) was still in his future. His dabbling in biology does, however, give us a fascinating snapshot of how the diverse fields of natural sciences were all closely intertwined in that era when electricity and magnetism were still relatively mysterious phenomena.

M. Faraday, “Notice of the character and direction of the electric force of the Gymnotus,” Phil. Trans. Roy. Soc. 129 (1839), 1-12

* A fish that hunted by waiting for an experimental physicist to put its hands on its head and tail would probably not survive very long.

** Faraday’s self-electrocution doesn’t quite beat the story of Jack Barnes for masochistic self-experimentation, but it comes close…


Two open doors

Although the known human coronaviruses can infect many cell types, they all mainly cause respiratory infections. The difference is that the four that cause common colds easily attack the upper respiratory tract, whereas MERS-CoV and SARS-CoV have more difficulty gaining a hold there, but are more successful at infecting cells in the lungs.

SARS-CoV-2, unfortunately, can do both very efficiently. That gives it two places to get a foothold, says Shu-Yuan Xiao, a pathologist at the University of Chicago, Illinois. A neighbour’s cough that sends ten viral particles your way might be enough to start an infection in your throat, but the hair-like cilia found there are likely to do their job and clear the invaders. If the neighbour is closer and coughs 100 particles towards you, the virus might be able get all the way down to the lungs, says Xiao.

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These varying capacities might explain why people with COVID-19 have such different experiences. The virus can start in the throat or nose, producing a cough and disrupting taste and smell, and then end there. Or it might work its way down to the lungs and debilitate that organ. How it gets down there, whether it moves cell by cell or somehow gets washed down, is not known, says Stanley Perlman, an immunologist at the University of Iowa in Iowa City who studies coronaviruses.

Clemens-Martin Wendtner, an infectious-disease physician at the Munich Clinic Schwabing in Germany, says it could be a problem with the immune system that lets the virus sneak down into the lungs. Most infected people create neutralizing antibodies that are tailored by the immune system to bind with the virus and block it from entering a cell. But some people seem unable to make them, says Wendtner. That might be why some recover after a week of mild symptoms, whereas others get hit with late-onset lung disease. But the virus can also bypass the throat cells and go straight down into the lungs. Then patients might get pneumonia without the usual mild symptoms such as a cough or low-grade fever that would otherwise come first, says Wendtner. Having these two infection points means that SARS-CoV-2 can mix the transmissibility of the common cold coronaviruses with the lethality of MERS-CoV and SARS-CoV. “It is an unfortunate and dangerous combination of this coronavirus strain,” he says.

The virus’s ability to infect and actively reproduce in the upper respiratory tract was something of a surprise, given that its close genetic relative, SARS-CoV, lacks that ability. Last month, Wendtner published results 8 of experiments in which his team was able to culture virus from the throats of nine people with COVID-19, showing that the virus is actively reproducing and infectious there. That explains a crucial difference between the close relatives. SARS-CoV-2 can shed viral particles from the throat into saliva even before symptoms start, and these can then pass easily from person to person. SARS-CoV was much less effective at making that jump, passing only when symptoms were full-blown, making it easier to contain.

These differences have led to some confusion about the lethality of SARS-CoV-2. Some experts and media reports describe it as less deadly than SARS-CoV because it kills about 1% of the people it infects, whereas SARS-CoV killed at roughly ten times that rate. But Perlman says that’s the wrong way to look at it. SARS-CoV-2 is much better at infecting people, but many of the infections don’t progress to the lungs. “Once it gets down in the lungs, it’s probably just as deadly,” he says.

What it does when it gets down to the lungs is similar in some respects to what respiratory viruses do, although much remains unknown. Like SARS-CoV and influenza, it infects and destroys the alveoli, the tiny sacs in the lungs that shuttle oxygen into the bloodstream. As the cellular barrier dividing these sacs from blood vessels break down, liquid from the vessels leaks in, blocking oxygen from getting to the blood. Other cells, including white blood cells, plug up the airway further. A robust immune response will clear all this out in some patients, but overreaction of the immune system can make the tissue damage worse. If the inflammation and tissue damage are too severe, the lungs never recover and the person dies or is left with scarred lungs, says Xiao. “From a pathological point of view, we don’t see a lot of uniqueness here.”

And as with SARS-CoV, MERS-CoV and animal coronaviruses, the damage doesn’t stop with the lungs. A SARS-CoV-2 infection can trigger an excessive immune response known as a cytokine storm, which can lead to multiple organ failure and death. The virus can also infect the intestines, the heart, the blood, sperm (as can MERS-CoV), the eye and possibly the brain. Damage to the kidney, liver and spleen observed in people with COVID-19 suggests that the virus can be carried in the blood and infect various organs or tissues, says Guan Wei-jie, a pulmonologist at the Guangzhou Institute of Respiratory Health at Guangzhou Medical University, China, an institution lauded for its role in combating SARS and COVID-19. The virus might be able to infect various organs or tissues wherever the blood supply reaches, says Guan.

But although genetic material from the virus is showing up in these various tissues, it is not yet clear whether the damage there is being done by the virus or by a cytokine storm, says Wendtner. “Autopsies are under way in our centre. More data will come soon,” he says.

Whether it infects the throat or the lungs, SARS-Cov-2 breaches the protective membrane of host cells using its spike proteins (see ‘Deadly invader’). First, the protein’s receptor-binding domain latches on to a receptor called ACE2, which sits on the surface of the host cell. ACE2 is expressed throughout the body on the lining of the arteries and veins that course through all organs, but it is particularly dense on the cells lining the alveoli and small intestines.

Although the exact mechanisms remain unknown, evidence suggests that after the virus attaches itself, the host cell snips the spike protein at one of its dedicated ‘cleavage sites’, exposing fusion peptides — small chains of amino acids that help to pry open the host cell’s membrane so that the virus’s membrane can merge with it. Once the invader’s genetic material gets inside the cell, the virus commandeers the host’s molecular machinery to produce new viral particles. Then, those progeny exit the cell to go and infect others.


Defence Mechanisms and Innate Immunity

The following points highlight the top six defence mechanisms involved in innate immunity. The defence mechanisms are: 1. Physical (or Mechanical) and Chemical Barriers 2. Inflammation 3. Phagocytosis 4. The Complement System 5. Antibacterial Substances 6. Antiviral Substances.

Mechanism # 1. Physical (or Mechanical) and Chemical Barriers:

Physical (or mechanical) barriers of the host in cooperation with chemical barriers (secretions) act as the first line of defence against pathogenic microorganisms and foreign materials. These barriers include skin, mucous membranes, respiratory system, gastrointestinal tract, genitourinary tract, eye, bacteriocins, and beta-lysin and other polypeptides.

Skin, mucous membranes, respiratory system, gastrointestinal tract, genitourinary tract, and eyes are the barriers that provide both physical and chemical defence (e.g., gastric juices, lysozyme, lactoferrin, glycoproteins, urea etc.) in cooperation. In addition, bacteriocins and beta-lysin and other polypeptides are the defensive chemicals against microorganisms.

Intact skin is a very effective physical or mechanical barrier to block the entry of microbial pathogens into the body. With few exceptions the microorganisms fail to penetrate the skin because its outer layer consists of thick, closely packed cells called keratinocytes that produce keratins.

Keratins are scleroproteins comprising the main components of hair, nails, and outer skin cells. These scleroproteins are not easily degradable enzymatically by microorganisms. They resist the entry of microbe-containing water and thus function as physical barrier to microorganisms.

In addition to direct prevention of penetration, continuous shedding of the outer epithelial cells of skin removes many of those microbial pathogens that manage to adhere on the surface of the skin.

2. Mucous membranes:

Mucous membranes of various body systems such as respiratory, gastrointestinal, genitourinary, and eye prevent invasion by microorganisms with the help of their intact stratified squamous epithelium and mucous secretions, which form a protective covering that resists penetration and traps many microorganisms.

3. Respiratory system:

An average person inhales about 10,000 microorganisms per day usually at the rate of eight microorganisms per minute. These microorganisms are deposited on the moist, sticky mucosal surfaces of the respiratory tract. The mucociliary blanket of the respiratory epithelium traps the microorganism less than 10 μm in diameter and transports them by ciliary action away from the lungs.

Microorganisms larger than 10 μm normally are trapped by hairs and cilia lining the nasal cavity which beat towards the pharynx so that the mucus with its trapped microorganisms is moved towards the mouth and expelled. Coughing and sneezing also help removal of microorganisms from the respiratory tract.

They make clear the respiratory system of microorganisms by expelling air forcefully from the lungs through the mouth and nose, respectively. Salivation also washes microorganisms from the mouth and nasopharyngeal areas into the stomach.

4. Gastrointestinal system:

Microorganisms may manage to reach the stomach. Many of them are destroyed by the gastric juice of the stomach. The gastric juice is a mixture of hydrochloric acid, proteolytic enzymes, and mucus, and is very acidic with a pH 2 to 3. This juice is normally sufficient to kill most microorganisms and destroy their toxins.

Furthermore, the normal microbial population of the large intestine is extremely significant in not allowing the establishment of pathogenic microorganisms in it.

For convenience, many commensalistic microorganisms in the intestinal tract secrete metabolic products (e.g., fatty acids) that prevent “unwanted” microorganisms from becoming established in the tract. In small intestine, however, the microbial pathogens are often killed by various pancreatic enzymes, bile, and enzymes in intestinal secretions.

5. Genitourinary system:

Kidneys, ureters, and urinary bladder are sterile under normal conditions. Kidney medulla is so hypertonic that it allows only few microorganisms to survive.

Urine destroys some microorganisms due to its low pH and the presence of urea and other metabolic end-products like uric acid, hippuric acid, mucin, fatty acids, enzymes, etc. The lower urinary tract is flushed with urine eliminating potential microbial pathogens. The acidic environment (pH 3 to 5) of vagina also confers defence as it is unfavourable to most microorganisms to establish.

The conjunctiva of eye lines the interior surface of each eyelid and the exposed surface of the eyeball. It is a specialised mucus-secreting epithelial membrane and is kept moist by continuous flushing action of tears secreted by the lacrimal glands. Tears contain lysozyme and lactoferrin and thus act as physical as well as chemical barriers.

The surfaces of skin and mucous membranes are inhabited by normal microbial flora. Of this, many bacteria synthesize and release toxic proteins (e.g., colicin, staphylococcin) under the direction of their plasmids. These toxic proteins are called bacteriocins, which kill other related species thus provide an adaptive advantage against other bacteria.

8. Beta-lysin and other polypeptides:

Blood platelets release a cationic polypeptide called beta-lysin, which disrupts the plasma membrane of certain gram-positive bacteria and kills them. Leukin, cecropins, plakins, and phagocytin are some other cationic polypeptides that kill specific gram-positive bacteria. Prostatic antibacterial factor, a zinc-containing polypeptide, is an important antimicrobial chemical secreted by the prostrate glands in males.

Mechanism # 2. Inflammation (Inflammatory Response):

Inflammation (L. inflammatio = to set on fire) is an innate (nonspecific) defence response of the body to pathogenic infection or tissue injury and helps localizing the infection or injury in its local area. Many of the classic features of inflammation were described as early as 1600 BC in Egyptian papyrus writings.

In the first century AD, the Roman physician Celsus described the four cardinal signs of inflammation as redness (rubor), swelling (tumor), heat (color) and pain (dolor). In the second century AD, another physician, Galen added a fifth sign: altered function (functio laesa).

1. Major events that result in cardinal signs:

Following are the major events that result in the cardinal signs of inflammation:

(i) The redness and heat (rise in temperature) of the localized area is due to vasodilation (an increase in the diameter of blood vessels) of nearby capillaries that occurs as the vessel that carry blood away from the affected area constrict resulting in engorgement of the capillary network.

(ii) Tissue swelling occurs due to accumulation of exudates in the area of infection or injury. An increase in capillary permeability facilitates an influx of fluid and cells from engorged capillaries into the tissue. The fluid that accumulates (exudate) possesses a much higher protein content than fluid normally released from the vascular system.

(iii) Pain is due to lysis of blood cells. The lysis triggers the production of prostaglandins and bradykinin, the chemical substances that alter the threshold and intensity of the nervous system response to pain. Pain probably serves a protective role as it normally causes individual to protect the infected or injured area.

2. Mechanism of defence:

Inflammatory response is a collective term representing the complex sequence of events during inflammation. It initiates when injured tissue cells release inflammatory mediators
(chemicals). Among the inflammatory mediators are various serum proteins called acute-phase proteins the principal acute-phase proteins are histamine and kinins.

The acute-phase proteins bind to receptors on nearby capillaries and venules causing vasodilation and increased permeability which results in influx of phagocytes (e.g., neutrophils, lymphocytes monocytes and macrophages) from the blood into the tissues.

The emigration of phagocytes is a multistep process (Fig. 44.14) that includes adherence of the cells to the endothelial wall of the blood vessels (margination), followed by their emigration between endothelial cells in to the tissues (diapedesis or extravasation), and finally, their migration through the tissue to the site of the invasion (chemotaxis).

As the phagocytic cells accumulate in the site of injury and begin to phagocytose microbial pathogens, during this process they release lytic enzymes that normally damage the nearby healthy cells. Dead host cells, dead phagocytic cells, dead microbial pathogens, and the body fluid collectively form a substance called pus (the inflammatory exudate).

When the acute-phase proteins bind to receptors on nearby capillaries and venules and cause vasodilation and increased permeability, the latter enable enzymes of the blood-clotting system to enter the tissue. These enzymes activate an enzyme cascade that results in the deposition of insoluble strands of fibrin, a main constituent of a blood clot.

The fibrin strands wall off the injured area from the rest of the body and serve to prevent the spread of infection. Once the inflammatory response is subsided and the pus is removed, the infected or injured area is filled with new tissues that start normal function.

Mechanism # 3. Phagocytosis:

Phagocytosis (Gk. Phagein = to eat cyte = cell and osis = a process) is a process during which large particles and microbial cells are enclosed in a phagocytic vacuole or phagosome and ingulfed. It acts a highly efficient cellular barrier against the pathogenic microorganisms and is met out by uptake and digestion of microorganisms by a variety of cells of the body’s defence system.

Besides its contribution in defence, phagocytosis helps certain cells and even organisms (e.g., protozoa) to obtain their nutrients. However, phagocytosis was a chance discovery by E. Metchnikoff (a native of Ukraine) in 1884 who suggested that the motile cells of larvae of starfish actively sought out and engulfed foreign particles present in their environment.

The following lines are devoted in the context of the role of phagocytosis in innate (nonspecific) host defence:

1. Recognition and adherence of microorganisms:

Phagocytic cells (neutrophils, monocytes macrophages, and dendritic cells) employ two fundamental molecular mechanisms for the recognition o microbial pathogens and their adherence on phgocyte’s plasma membrane:

(i) Opsonin-dependent (opsonic) recognition (called opsonization) and

(ii) Opsonin-independent (nonopsonic) recognition.

Opsonin-dependent recognition or opsonization (Gk. opson = to prepare victim for) is a process in which the phagocytic cells readily recognize the microbial pathogens that are coated by serum components (antibodies especially lgG1 and lgG3, complement C3b, and both antibody and complement C3b) called opsonins.

The opsonins function as a bridge between the microorganism and the phagocyte by binding to he surface of microorganism at one end and to specific receptors on the phagocyte surface at the other (Fig. 44.15) and enhance phagocytosis multifold. In one study for convenience, the rate of phagocytosis of a microorganism was 4000-fold higher in the presence of opsonin than in its absence.

Opsonin-independent recognition involves the mechanism which does not involve opsonins and employs other receptors on phagocytic cells that recognise structures (adhesins) expressed on the surface of different microbial pathogens (Fig. 44.16). Important ones of such receptors are lectins, polysaccharides, glycolipids, proteolycans, lypopolysaccharides (LPS), flagellin, etc.

It is important to note that during opsonin-independent recognition a particular microbial species may display multiple adhesins, each recognised by a distinct receptor present on phagocytic cells.

2. Ingestion and digestion of microorganisms:

Adherence of microorganisms on phagocyte’s plasma membrane is followed by their ingestion and digestion. Adherence induces plasma membrane protrusions, called pseudopodia, 10 extent around the adhered microorganisms.

Fusion of the pseudopodia encloses the microorganisms within a membrane-bounded structure called a phagosome, which moves towards the cell interior and fuses with a lysosome to form a phagolysosome (Fig. 44.17) Lysomes contribute to the phagolysosome a variety of hydrolytic enzymes such as lysozyme, phospholipase A2, ribonuclease deoxyri- bonuclease, and proteases.

An acidic vacuolar pH favours the activity of hydrolytic enzymes. Hydrolytic enzymes digest the entrapped microorganisms. The residual contents after digestion inside the phagolysosome are then eliminated through a process called exocytosis.

Mechanism # 4. The Complement System:

The serum of the blood contains a large number (over 30) of serum proteins that circulate in an inactive state and following their initial activation by specific (adaptive) and nonspecific (innate) immunogenic mechanisms, interact in a highly regulated cascade-fashion in which the activation of one component results in the activation of next in the cascade. This cascade of scrum proteins is collectively called the complement system and the serum protein of the complement system are called complement proteins.

When the inactive forms of complement proteins are converted into active forms by various specific (adaptive) and nonspecific (innate) immunologic mechanisms, they damage the membranes of microbial pathogens either destroying them or facilitating their clearance.

Complement system may act as an effector system that is triggered by binding if antibodies to certain cell surfaces, or it may be activated by reactions between complement proteins and receptors of microbial cell walls. Reactions between complement proteins and cellular receptors trigger activation of cells of the innate or adaptive immunity.

There are three pathways of complement activation:

(i) Classical complement pathway,

(ii) Alternate complement pathway, and

(iii) Lectin complement pathway.

Although these pathways employ similar mechanisms, specific proteins are unique to the first part of each pathway. Classical pathway is involved in specific or acquire (adaptive) immunity, whereas both the alternate and lectin pathways play important role in innate (nonspecific) immunity.

Mechanism # 5. Antibacterial Substances:

Human hosts possess antibacterial substances with which they combat the continuous onslaught of bacterial pathogens. These antibacterial substances are produced either by the host itself or by certain indigenous bacteria. The important antibacterial substances are the lysozyme, bacteriocins, and beta-lysin, and other polypeptides.

Lysozyme is the enzyme that breaks the β-1, 4-glycosidic bonds between N-acetylglucosamine and N- acetylmuramic acid in peptidoglycan, the signature molecule of bacterial cell wall. This bond breakage weakens the bacterial cell wall.

Water then enters the cell, and the cell swells and eventually bursts, a process called lysis (Fig. 44.18). Lysozyme occurs in body secretions including tears, saliva, and other body fluids, and presumably functions as a major line of non-specific defence against bacterial infections.

Many of the normal bacterial flora of the host body synthesize and release plasmid-encoded toxic proteins (e.g., colicins, staphylococcin) collectively called bacteriosins that inhibit or kill closely related bacterial species or even different and may give their producers and adaptive advantage against other bacteria.

These toxic proteins are called bacteriocins to distinguish them from the antibiotics because possess a more narrow spectrum of activity than antibiotics. Bacteriocins producing genes are often present on plasmid or a transposon.

Most bacteriocins are produced by gram-negative bacteria, and are generally named after the species of the bacterial genera that produce them the bacteriocin produced by E. coli is colicin, by Bacillus subtilis is substilicin.

E. coli synthesizes colicins. Some colicins bind to specific receptors on the surface of susceptible cells and kill them by disrupting some critical cell function. For example, many colicins form channels in the plasma membrane that allows potassium ions and protons to leak out, leading to a loss of the cell’s energy forming ability. Colicin E2 (encoded by plasmid col E2) is a DNA endonuclease and cleaves DNA. Colicin E3 (encoded by plasmid Col E3) is a nuclease that cuts at a specific site in 16S rRNA and inactivates ribosomes.

Recently it has been discovered that some grain-positive bacteria produce bacteriocin-like peptides. For example, lactic acid bacteria produce Nisin A, which strongly inhibits the growth of a wide range of gram- positive bacteria.

Beta-lysin and other polypeptides:

Beta-lysin is a cationic polypeptide synthesized and released by blood platelets, and kills some gram-positive bacteria by disrupting their plasma membranes. Other cationic polypetides produced in host body include leukins, plakins, cecropins, and phagocytin. A zinc-containing polypeptide named ‘prostatic antibacterial factor’ is secreted by the prostate gland in males, and acts as an important antibacterial substance.

Mechanism # 6. Antiviral Substances:

The outcome of a virus infection is influenced by the virulence of the infecting strain and the resistance conferred by the host. Mechanisims of host resistance may be immunological or non-specific. The latter include various genetic and physiological factors such as interferons, reactive nitrogen intermediates (RNIs), defensins, and fever.

Interferons are a family of host coded proteins produced by cells on induction by viral inducers, and are considered to be the first line of defence against viral attacks. Interferon by itself has no direct effect on viruses but it acts on other cells of the same species rendering them refractory to viral infection.

On exposure to interferon, cells produce a protein (translation inhibiting protein, TIP) which selectively inhibits translation of viral mRNA without affecting cellular mRNA. Translation inhibiting protein (TIP) is actually a mixture of at least three different enzymes, namely, protein kinase, oligonucleotide synthetase, and ribonuclease (RNAse).

These enzymes together block translation of viral mRNA into viral proteins. It has also been suggested that inhibition of viral transcription may also be responsible for the antiviral activity of interferon.

Reactive nitrogen intermediates:

Macrophages (also neutrophils and mast cells) have been found recently producing reactive nitrogen intermediates (RNIs). These molecules include nitric oxide (NO) and its oxidized forms, nitrite (NO2 – ) and nitrite (NO3 – ), and are very potent cytotoxic agents.

RNIs may be either released from cells or generated within cell vacuoles. Macrophages produce RNIs from the amino acid arginine. Macrophages have been found to destruct the herpes simplex virus with the help of RNIs produced by them.

Definsins are broad-spectrum antimicrobial peptides synthesized by myeloid precursor cells during their sojourn in the bone marrow, and are then stored in the cytoplasmic granules of mature neutrophils.

Besides gram-positive and gram-negative bacteria and yeasts and moulds, defensins target some viruses. Antiviral activity of defensins involves direct neutralization of enveloped viruses non-enveloped viruses are not affected by defensins.

Fever (Elevated Body Temperature):

Fever is a physiological factor and results from disturbance in hypothalamic thermoregulatory activity leading to an increase in normal body temperature. In adult humans fever is defined as an oral temperature above 98°F (37°C) or a rectal temperature above 99.5°F (37.5°C).

In almost every instance there is a specific constituent called ‘endogenous pyrogen’ that directly triggers fever production. These pyrogens include interleukin 1 (IL-1), interleukin (IL-6), and tissue necrosis factor that are synthesized and released by host macrophages in response to pathogenic factors that include viruses, bacteria, and bacterial toxins. It has been found that fever may act as natural defence mechanism against viral infections because most viruses are inhibited by temperatures above 39°C.


4. Microfabricated Platforms for Cell Lysis

Microfluidics is one of the emerging platforms for cell lysis on a micro scale. Microfluidics is the manipulation and handling of small volumes (nano- to picoliters) of liquid in microchannels. Due to the micro scale operation regime, microfluidics is well suited for application where the sample or sample volume is small. This lowers the cost of the analysis due to low consumption of reagents [46]. Microfluidics also enables integration of different modules (or operations) into one device. For example, cells can be lysed and the intracellular products can directly be post processed (PCR or DNA isolation for diagnostics) inside the same device [47,48]. Although there have been a number of reviews on cell lysis in the past 10 years [7,8,49], some of the recent developments in the field have not been reviewed. This review will focus on the recent developments from 2014 onwards and will briefly cover the developments from before, which have been extensively surveyed. Some of the macro scale techniques have been implemented in microfabricated devices for cell lysis. Techniques such as electrical lysis methods are applicable only in the micro scale. Microfluidic lysis technology can be broadly classified into six types. They include mechanical lysis, thermal lysis, chemical lysis, optical lysis, acoustic lysis and electrical lysis.

4.1. Mechanical Lysis

Mechanical lysis in microfluidics involves physically disrupting the cell membrane using shear or frictional forces and compressive stresses. Berasaluce et al. [50] developed a miniaturized bead beating based method to lyse large cell volumes. Zirconium/silica beads were placed inside a cell lysis chamber along with a permanent magnet and actuation of an external magnetic field caused the motion of the beads inside the chamber. Figure 7 shows the various components and device assembled for cell lysis. Staphylococcus epidermidis cells were used in this study and they studied the effect of bead size, volume, flow rate and surfactant (Tween-20) on lysing efficiency. They found the optimum parameters achieved a 43% higher yield efficiency at a flow rate of 60 μL/min compared to off chip bead beating system.

Miniaturized bead beading cell lysis system: (a) various components: (1) inlet (2) outlet (3) stirring magnet (4) zirconia/silica beads (5) bead weir (6) rotating magnet and (7) electric motor coupling and (b) image of the device for lysis. Reproduced with permission from [50].

Pham et al. [51] have recently used nanotechnology to fabricate black silicon nano pillars to lyse erythrocytes in about 3 min. They fabricated these nanopillar with

12 nm tip diameter and 600 nm tall on silicon substrate using reactive ion etching technology. The authors showed that the interaction of erythrocytes cultured on nanopillar arrays causes stress induced cell deformation, rupture and lysis in about 3 min. Figure 8 shows the interaction of erythrocytes with the nanostructures.

Cell lysis using nano pillars: (a,b) top and side view of the cells interacting with the nanopillars and (c) confocal laser scanning microscopy pictures of intact, deformed and ruptured cells. Reproduced with permission from [51].

Mechanical lysis has been demonstrated by using nano-scale barb [52]. When cells are forced through small opening, high shear forces cause rupture of the cell membrane. Similar principle has been used here where “nanoknives” were fabricated in the wall of microchannels by using modified deep reactive ion etching (DRIE). Distance between these sharp edges was 0.35 μm and width of the channel was 3 μm. The lysis section of this device consisted of an array of these “nanoknives” patterned on a microchannel as shown in Figure 9 b. Human promyelocytic leukemia cells (HL-60) were used to pass through this section at sufficient velocity. The addition of this “nanoknives” pattern increased the amount of lysis. This device was used to extract protein from inside the cell. It has been estimated that as much as 99% of the cell was lysed but, only 6% protein was released.

Mechanical lysis using nanoscale barbs: (a) microfluidic device showing different inlets and outlet channels (b) schematic of the barbs (c) deep reactive ion etching (DRIE) fabricated nano-knives (d) magnified image of nano-knives patterned using DRIE technique and (e) dimensions of the nano-knives used for cell lysis. Reproduced with permission from [52].

Alternatively, mechanical impingement through collision has also been used to lyse in the microscale [53,54,55]. Cells were suspended in solution with glass beads and placed on the microfluidic compact disc (CD) device, which was then set to rotate at a very high velocity. The centrifugal force generated by the rotation, causes collision and friction between cells and beads, which results in cell lysis. Various kinds of cells including mammalian, bacteria and yeast have been lysed using this technique.

Though the efficiency of the mechanical lysis is very high, these disruption methods have some drawbacks in microscale application. Fabrication of these devices is complex as well as expensive and collecting the target materials from a complex mixture is very difficult.

4.2. Thermal Lysis

In thermal lysis, heat is supplied to the cells to denature the membrane proteins and lyse the cells. One advantage of thermal lysis is the easy integration of microfluidic devices such as polymerase chain reaction (PCR). The thermal lysis can be performed in such devices with no additional modification. The cells are generally heated above 90 ଌ and the intracellular products are cycled through different temperatures for example in a PCR device. Tsougeni et al. [56] fabricated a microfluidic device which can capture and lyse cells. They used thermal lysis at 95 ଌ for 10 min to capture and lyse bacteria. Nanostructures were fabricated in poly(methyl methacrylate) using lithography and plasma etching technique. Microfluidic PCR devices which have incorporated thermal cell lysis [57,58,59] consist of a glass chamber and a resistive heater to heat the chamber.

In general, thermal lysis is effective in a microfluidic platform, however, these devices are not suitable for sample preparation where the sample is of a large volume and cells have to be lysed from a continuous flow [29]. However, cells have to be treated with lysozyme in order to break the cell wall and make bacteria protoplast. The addition of this lysozyme is time consuming and requires complex structures. Moreover, preserving the enzyme within the device becomes problematic when the device has to be used for a long period of time. Higher lysis time and elevated power consumption are other drawbacks of this method.

4.3. Chemical Lysis

Chemical lysis methods use chemical reagents such as surfactants, lysis buffers and enzymes to solubilize lipids and proteins in the cell membrane to create pores and lyse cells. Although chemical and enzymatic methods are categorized separately in macro scale method, these two techniques are incorporated in the same group for micro scale cell lysis techniques. Buser et al. [60] lysed gram-positive bacteria (Staphylococcus aureus) and RNA virus (respiratory syncytial virus) using a dried enzyme mixture (achromopeptidase). They were able to lyse in less than a minute and then used a disposable chemical heater to deactivate the lysis enzyme. They were able to amplify (off-chip) the lysate without purification and showed the proof of principle for a point of care device for diagnostics.

Kashyap et al. [61] developed a microfluidic probe for selective local lysis of adherent cells (

300 cells) for nucleic acid analysis. Hall et al. [62] used a device for cell lysis experiment, which had two supply wells and a pressure well. Mixing of cell and lysis solution was controlled by adjusting the pressure of the wells. Three different types of solution were used—Solution A containing only SDS (detergent based reagent), Solution B containing surfactant, Triton X-100, Tween-20 with enzyme such as lysozyme, protease, proteinase K and Solution C containing an antibiotic named polymyxin B. Gram-negative and gram-positive bacteria were used for lysis. It was concluded that detergent alone was not suitable for lysis, while Solution B, a mixture of chemical surfactants and biological reagents, can disintegrate the cell membrane and lyse various kinds of bacteria. However, polymyxin B can be potentially used in microfluidic cell lysis platform only for gram-negative bacteria.

Kim et al. [63] also developed a microfluidic device with two inlets and outlets in order to develop an optimal lysis reagent for gram-negative bacteria. Heo et al. [64] demonstrated a microfluidic based bioreactor which was capable of entrapping E. coli by using hydrogel patches. Then the immobilized E. coli was lysed by using SDS as it can penetrate hydrogel. Cell lysis was accomplished within 20 min. This device was capable of cell lysis using only SDS, however, the previous one could not due to lower exposure time in chemical environment. In another study, Sethu et al. [65] also developed a microfluidic chip ( Figure 10 ) to lyse Erythrocyte in order to isolate Leukocyte. One hundred-percent recovery was possible within 40 s. The device consists of three inlet reservoirs and one outlet reservoir. One inlet was used to flow the entire blood. Second inlet was used for lysis buffer containing mainly aluminum oxide and two side channels were connected with this inlet which converged to direct the entire blood into a narrow stream. This increases the surface contact between the lysis buffer and the cells. The mixture of cells and lysis buffer was then run through a long channel with a number of “U” turns to enhance the buffer. Finally, third inlet was used to flow the phosphate buffer in order to dilute the sample for restoring the physiological concentration [66,67].

Schematic of a simple chamber and serpentine microfluidic channel for chemical lysis. Reproduced with permission from [65].

Even though chemical lysis method is widely used in many microfluidic devices, this method requires an additional time consuming step for reagents delivery. Therefore, complex microfluidics structures including injection channels and micro-mixers to homogenize the samples are needed [66,68]. After lysis, these reagents might interfere with downstream assay as it is very hard to separate the target molecules [69]. In addition, storage of these reagents is a problem which is why the device cannot be used for long time.

4.4. Optical Lysis

Optical lysis of cells involves the use of lasers and optically induced dielectrophoresis (ODEP) techniques to break open the cell membrane. In laser lysis, a shock wave created by a cavitation bubble, lysis the cell membrane. A focused laser pulse at the cell solution interface creates this cavitation bubble. In ODEP, a conductive electrode and a photoconductive layer (for example amorphous silicon) are formed on the top surface of glass slide. A non-uniform electric field is generated by shining light on the photoconductive layer which then generates a transmembrane potential across the cell membrane disrupting the cell membrane. Huang et al. [70] developed an optically induced cell lysis microfluidic chip for lysing HEK293T cells and extracting intact nucleus. They report cell lysis and nucleus separation efficiency as 78% and 80% respectively using this device.

Kremer et al. [71] lysed cells using an opto-electrical setup. They were able to lyse cells selected based on shape of the cell. They used ODEP to lyse red blood cells in a mixture of red and white blood cells. They developed a method that enabled shape-selectivity such that cells with a different geometry will lyse in a mixture of cell types. The cell with a different shape induces a non-uniform electric field which is used for lysis. Figure 11 shows the schematic of the lysis chip and lysis of differently shaped cells.

Optical cell lysis device: (a) cell lysis chip using optically induced dielectrophoresis (ODEP) (bd) cell lysis of red blood cells in a mixture of white and red blood cells and (eg) lysis of red blood cells in a mixture of red blood cells and trypanosomes. Reproduced with permission from [71].

Use of laser light to induce lysis has also been attempted in microfluidic devices. In one instance, optical lysis was induced by application of a nanosecond 532 nm laser pulse [72] which generates a microplasma locally. The plasma collapses causing cavitation, bubble expansion and its collapse as described in previous section are the main reason for a laser induced cell lysis. Various types of cell lines such as rat basophilic leukemia (RBL) [73], rat-kangaroo (Potorous tridactylis) epithelial kidney cells (PtK2) [74], and murine interleukin-3 dependent pro-B (BAF-3) [75] have been lysed by using this laser induced method. However, all these experiments had been done for single cell analysis. It has been found that when laser based lysis was incorporated with polydimethylsiloxane (PDMS) microchannel efficiency of lysis decreased [75]. It was suggested that this may be due to the deformation of PDMS walls which dissipates the mechanical energy from the bubble collapse. For that reason, high energy was required.

Ultraviolet (UV) light array combined with titanium oxide has been used to lyse the cell [76]. Titanium oxide possesses photolytic properties and excitation energy that falls within UV range. When titanium oxides are excited with UV light array, electrons in the valence band are excited to conduct ion band which results in electron–hole pairs. In aqueous environment, these electron–hole pairs react with surrounding molecules and generate free radicals such as OH, O and O2 − . These react with cell membrane and lyse the cell. E. coli cells were lysed with the above technique. A primary disadvantage of ultraviolet lysis was that the time required to lyse the cell was very high (45 min).

4.5. Acoustic Lysis

In acoustic lysis, a high energy sound wave is generated which is used for cell lysis. This surface acoustic wave (SAW) is produced on a piezoelectric substrate. An inter-digitated transducer (IDT) can be used to produce a SAW electrically with the wave propagating on the surface away from it. Taller et al. [77] have used on chip surface acoustic wave lysis for detecting exosomal RNA for pancreatic cancer study. They achieved a lysis rate of 38% using this technique. Figure 12 shows the fabricated device with the SAW transducer.

Surface acoustic wave (SAW) lysis microfluidic device: (a) assembly of device and (b,c) as fabricated device with liquid inlet and outlet for exosome lysis. Reproduced with permission from [77].

They report that the lysis of exosomes is possible due to the effects of acoustic radiation force and dielectric force acting on small particles [78,79]. The SAW device was fabricated using standard photolithography technology. Twenty pairs of titanium aluminum electrodes were patterned on top of piezoelectric lithium niobate substrate to form a single phase unidirectional SAW transducer. This transducer can generate SAW in only one direction. Raw media was exposed to SAW for 30 s at 1 W of power for lysing. The authors report that a lysis efficiency of 38% achieved using this method was sufficient for obtaining enough exosome RNA for detection.

Marentis et al. [80] lysed the eukaryotic cell as well as bacteria by using sonication. This device consists of a microfluidic channel with integrated transducer. The channel was made on glass substrate and piezoelectric transducer was made by depositing zinc-oxide and gold on quartz substrate. The transducers were driven by a sinusoidal source in the 360-MHz range. Eighty-percent lysis of HL-60 and 50% lysis of Bacillus Subtilis spores were obtained by using this device. The temperature rise due to sonication was moderated by using ice pack and cold finger. Ultrasonic horn tip and liquid region are coupled in a microfluidic chip by increasing fluidic pressure in order to increase the efficiency of lysis [81].

Reboud et al. [82] have developed a disposable microfluidic chip to detect the rodent malaria parasite Plasmodium berghei in blood. They used SAW to lyse the red blood cells and parasitic cells in a drop of blood. They report a cell lysis efficiency of more than 99.8% using their device. Xueyong et al. [83] have fabricated a SAW microfluidic device which can lyse red blood cells with high efficiency (95%).

However, sonication has limitations such as generation of heat, complex mechanism as well as expensive fabrication process. Due to this excessive heat generation denaturation of protein and excessive diffusion of the cell contents have been observed [8,84]. To reduce the operation time, cells were first treated with some weak detergent such as digitonin [8,85] before ultrasonic exposure. Digitonin weakened the cell membrane and facilitated lysis.

4.6. Electrical Lysis

In electrical method, cells are lysed by exposing them to a strong electric field. An electric field is applied across the cell membrane which creates a transmembrane potential. A potential higher than the threshold potential is required to form pores in the cell membrane. If the value of the potential is lower than the threshold potential, the pores can be resealed by the cell. On the other hand, a high enough potential can completely disintegrate the cell. At such high voltages, it is found that the electric field does not have any effect on the intracellular components [86]. Electric field is the critical parameter to lyse the cell. As higher electric field is required for cell lysis, high voltage generator is required in order to generate this high electric field in macroscale. Thus, this method is not common in macroscale. However, in microscale due to small size of the devices, higher electric field can be obtained at lower voltage. For this reason and as a method for fast and reagentless procedure of lysis, electrical lysis has achieved substantial popularity in microfluidic community.

Ameri et al. [87] used a direct current (DC) source to lyse cells in a microfluidic chip. Figure 13 shows the fabrication and working principle of their chip. Their device consists of a glass slide coated with indium tin oxide coating patterned for electrodes. The 6400-Microwell arrays are fabricated using SU-8 polymer by photolithography technique. Inlet and outlet channels are created using PDMS polymer and is sealed using a glass slide with ITO electrode for impedance measurement. Red blood cells (10 7 cells/mL) are flown through the device at 20 μL/min and dielectrophoresis (DEP) is used to immobilize the cells into the microarray. A DC voltage of 2 V for 10 s was applied to the cell for lysis. The lysis process was monitored using impedance measurement before and after lysis and a decrease in impedance suggested a complete lysis of cells. They report a lysis efficiency of 87% in their device. The authors proposed a device for cell lysis by electric fields and optical free monitoring of the lysis process on a microfluidic platform which could have potential use in the medical diagnostic field.

Electrical cell lysis device: (a) fabrication protocol of the device (b) working principle of the device and (c) microfluidic device used in the study for lysing red blood cells. Reproduced with permission from [87].

Jiang et al. [88] developed a low cost microfluidic device for cell lysis using electric fields. They applied a 10 V square pulse to lyse cells at 50% efficiency. They report a device which had the capability to lyse cells at a much lower voltage compared to a commercially available electropolator device which operated at 1000 V to lyse 200 μL of PK15 cells. They observed bubble formation in their device during cell lysis due to joule heating effect. De Lange et al. [89] have lysed cells in droplets using electric fields. They demonstrated a robust new technique for detergent free cell lysis in droplets. In their device, electric field was applied to lyse bacteria immediately before merging the cell stream with lysozyme and encapsulating the mixture in droplets. They report that with lysozyme alone the lysis efficiency is poor (less than 50%) but when combined with electric fields they were able to obtain up to 90% cell lysis efficiency. Figure 14 shows their microfluidic device for cell lysis in droplets. The authors suggest that their device could be used in applications where use of cell lysis detergents could hinder the cell analysis such as binding assays or studying the chemical activity of proteins and in mass spectroscopy studies where chemical lysis agents can hamper the results.

Electrical cell lysis microfluidic device: (A) schematic of the electrical lysis and coflow droplet generation microfluidic chip (B) actual image of the droplet generation part and (C) complete electrical lysis with electroporation channels. Reproduced with permission from [89].

Escobedo et al. [90] showed electrical lysis of cells inside a microfluidic chip using a hand held corona device. They were able to lyse baby hamster kidney cells (BHK), enhanced green fluorescent protein human-CP cells (eGFP HCP) 116 and non-adherent K562 leukemia cells completely inside a microfluidic channel. A metal electrode was embedded inside the channel which was used to discharge 10 to 30 kV to lyse the cells in less than 300 ms. Lysis was assessed by observing before and after images of cells using bright field and high speed microscope and also by cell-viability fluorescence probes. They also report no bubble formation during lysis indicating no joule heating effect thereby making this method suitable for analyzing sensitive proteins and intracellular components. Figure 15 shows the setup and results of the study.

Electrical lysis through handheld plasma device: (a) schematic of the device. Cells were lysed using a hand held corona device by applying electric field at the inlet of the device (b) bright field and fluorescent images of before and after of lysis of K562 cells. Reproduced with permission from [90].

Besant et al. [91] detected mRNA molecules of E. coli by electrochemical lysis technique. They applied a potential of 20 V, which initiated the cell lysis by producing hydroxide ions from water at cathode to break down bacterial membranes. The sensor electrodes were placed 50 μm away which was enough to detect the mRNA molecules in 10 min. They reported lysis and detection of E. coli mRNA at concentrations as low as 0.4 CFU/μL in 2 min which was relevant for clinical application in both sensitivity and time.

Gabardo et al. [92] developed a low cost and easy method to fabricate multi-scale 3D electrodes that could be used for bacterial lysis using a combination of electrical and electrochemical means. These micron-sized electrodes can be rapidly prototyped using craft cutting, polymer induced wrinkling and electro-deposition techniques. They report that these tunable electrodes performed better as compared to lithographically prepared electrodes. They were able to successfully extract nucleic acids extracted from lysed bacteria on a microfluidic platform. They reported 95% lysis efficiency at 4 V using their electrodes. Figure 16 shows the device and electrode structures.

Bacterial lysis device: (a) schematic of the lysis device (b) scanning electron micrographs of: (i) planar (ii) wrinkled and (iii) electrodeposited electrodes (c) cyclic voltammetry scan of the electrodes. Reproduced with permission from [92].

Li et al. [93] developed a double nano-electrode electrical cell lysis device to lyse single neuronal cells. Similarly, Wassermann et al. [94] showed cell specific lysis of up to 75% of the total human blood cells using SiO2 passivated electrical cell lysis electrodes at an applied voltage of 8� V. Ma et al. [95] reported a 10�-fold increase in mRNA extracted from M. smegmatis using electrical lysis in a microfluidic platform as compared to a commercial bead beading instrument. They used a 4000� V/cm field intensity to lyse the bacteria with long pulses (5 s). They report that their device can be effective for mRNA release from hard to lyse cells.

Islam et al. [96] showed the proof of concept of a simple microfluidic device for electrical lysis of larger volumes of sample. They used a nanoporous membrane sandwiched between two microfluidic channels to trap and lyse E. coli bacteria by applying 300 V. They report a lysis efficiency of 90% in less than 3 min. Figure 17 shows the schematic of the device used for lysis in their study.

Electrical cell lysis microfluidic device: (a) schematic of cell lysis device and (b) experimental setup. Reproduced with permission from [96].

Different types of voltages such as alternating current (AC) [97,98], DC pulses [99,100,101] and continuous DC voltages [102] have been used in order to lyse the cells. Along with electric field, exposure time of cells within that electric field is also an important parameter for cell lysis. It has been found that cells can be lysed by using higher electric field for short period of time as well as lower electric field for long period of time [103]. For that reason, AC and DC pulses of a higher electric field are needed as compared to a continuous DC electric field. As the electric field depends on the distance between the electrodes, microfabricated electrodes have been used during AC or DC pulses. An overview of different electrical lysis devices and the characteristics of the designed system is presented in Table 4 .

Table 4

Different electrical lysis devices used for cell lysis.

ReferenceSpeciesType of CellCell Size (μm)ElectrodeType of Voltage (AC/DC)Lysing Voltage (V)
[104]HumanHT-2910GoldAC8.5
[97]HumanA43110GoldAC20
[98]-FITC-BSA laden vesicle50ITOAC5
[99]-Leukocytes-3DDC pulse10
[100]HumanRed blood cells6𠄸Pt wireDC/AC30�
[105]BacteriaE. coli-GoldDC pulse50
[106]HamsterCHO10�Pt wireDC pulse1200
[106]BacteriaE. coli Pt wireDC930
[102]HumanRed blood cells6𠄸Pt wireDC50
[87]HumanRed blood cells6𠄸ITODC2
[96]BacteriaE. coli-Pt wireDC300

Lu et al. [104] developed a microfluidic electroporation platform in order to lyse human HT-29 cell. Microfabricated saw-tooth electrode array was used in order to intensify the electric field periodically along the channel. Seventy-four-percent efficiency was obtained for an operational voltage of 8.5 V. However, this mode of lysis is not suitable for bacteria due their sizes and shapes. Compared to mammalian cell, high electric field and longer exposure is needed to lyse bacteria. Rosa [105] developed a chip to lyse bacteria consisting of an array of circular gold electrodes. DC pulses were used and lysis with 17% efficiency was achieved by using an operational voltage 300 V. This efficiency was increased up to 80% after adding enzyme with cell solution. In 2006, Wang et al. [107] proposed application of continuous DC voltage along the channel for cell lysis. The device consists of a single channel with uniform depth and variable width. Since the electric field is inversely proportional to width of the channel, high electric field can be obtained at the narrow section of the channel. Thus, lysis occurs into a predetermined portion of the device. Exposure time of the cell to the electric field can be tuned by changing the length of this narrow section. The configuration of the device was optimized and lysis of complete E. coli bacteria was possible at 930 V. Complete disintegration of cell membrane was observed when the electric field was higher than 1500 V/cm. This device was very simple and did not need any microfabricated electrodes. Pt wires were used as electrodes. Only a power generator was needed to operate it. However, bubble generation and Joule heating issue could not be completely eliminated. Similar kind of device was used by Lee [102] where the length and width of the narrow section was modified in order to lyse mammalian cell. Bao et al. [108] also developed a device to lyse E. coli by using DC pulses. Release of intracellular materials was observed when the electric field was higher than 1000 V/cm.

In conclusion, electrical method offers a simple, fast and reagent less lysis procedure to lyse various kinds of cells. This method is also suitable for selective lysis and is compatible with other downstream assays such as amplification and separation. Although requirement of high voltage is a problem in this procedure, it can be overcome by decreasing the gap between electrodes through microfabrication. However, heat generation and formation of bubble is a major problem for electric lysis method.

4.7. Comparison of Different Microfluidic Technologies for Cell Lysis

Various microfluidic technologies for cell lysis are compared in Table 5 . The advantages and disadvantages of different methods are listed for each technique.

Table 5

Comparison of different microfluidic lysis methods. Cell lysis efficiency was determined by averaging the lysis efficiencies from the references cited. Low: 0%�% Medium: 50%�% High: 80%�%.


Cryopreservation of the Embryo: An Interplay Between Fundamental and Empirical Cryobiology

In the decade following the successful cryopreservation of mammalian sperm in 1949, a number of attempts were made to cryopreserve mammalian preimplantation zygotes and embryos. All failed. The highest reported survival based on cleavage was 1% for rabbit zygotes frozen slowly in 15% glycerol [ 63]. In 1971, Whittingham switched to polyvinylpyrrolidone (PVP) as the cryoprotectant, and reported that, using rapid cooling at 60°C/min, he was able to obtain survival of 8-cell mouse embryos cooled to −79°C and held 30 min [ 64]. However, a year later, Whittingham et al. [ 4], and subsequently others [ 65, 66], were unable to repeat these findings.

In the preceding decade, between 1963 and 1971, a degree of mechanistic understanding of cryobiological injury had emerged. In 1963, Mazur [ 67] published a mathematical model based on physical-chemical considerations showing that the probability of IIF in a cell depended on the degree to which it underwent osmotic dehydration during cooling, and that in turn depended on the water permeability of the cell, the temperature coefficient or activation energy of that water permeability, and the surface:volume ratio of the cell. Subsequent experiments with yeast and human red blood cells led to the so-called two-factor hypothesis [ 68] namely, that cells cooled too rapidly are killed by IIF and cells cooled too slowly are killed by solution-effect injury. The consequence of the interaction of these two factors is that plots of survival vs. cooling rate have the shape of an inverted “U.” An example of solution effects is the increase in salt concentration by ice formation that had been quantitatively described by Lovelock [ 69] 12 years previously. Lovelock [ 70] had also shown that protection against freezing damage by low molecular weight compounds, such as glycerol or dimethyl sulfoxide, was due to their ability to colligatively lower the salt concentration at a given subzero temperature. Then, in 19701971, experimental observations showed that plots of survival of mouse marrow stem cells [ 71] and hamster V79 cells [ 72, 73], as a function of cooling rate, also exhibited an inverted U, with highest survivals at an intermediate, optimum rate. These data provided the first direct experimental support for the applicability of the two-factor hypothesis to nucleated mammalian cells.

Whittingham's 1971 published procedure [ 64] for mouse embryos was not consistent with these concepts. Physical-chemical modeling indicated that, to avoid IIF, an assemblage of cells of the size and presumed permeability properties of mouse embryos would have to be cooled at <1°C/min, not 60°C/min [ 67]. The theory underlying protection from slow freezing solution-effect injury argued that PVP would be an ineffective CPA because of its high molecular weight and consequent impermeability. When Whittingham et al. [ 4] redesigned the protocol for mouse embryos with these fundamentals in mind, they immediately obtained high survivals.

For a number of years, the mouse embryo cryopreservation procedure published by Whittingham et al. [ 4] served as the standard for laboratories such as the Jackson Laboratory. In 1977, Willadsen [ 74] introduced a modification based on experiments with bovine and ovine morulae namely, rather than cooling the embryos slowly to −70°C, he cooled them slowly to −36° (0.3°C/min to −30°C, then 0.1°C/min to −36°C), followed by a plunge in liquid nitrogen. That modification was entirely compatible with and explicable by the underlying fundamentals. Slow cooling to −36°C was sufficient to cause most of the intracellular freezable water to flow out of the cell and freeze externally consequently, little intracellular ice formed during the subsequent plunge into liquid nitrogen. A small amount of ice or glass apparently does form during the plunge, for the Willadsen procedure generally requires that the embryos be warmed rapidly. Rapid warming minimizes devitrification and minimizes recrystallization of small, pre-existing ice crystals. As Leibo and Songsasen have reported [ 75], and as Leibo pointed out in the workshop, Willadsen's now standard procedure has been used to successfully cryopreserve embryos from some 22 mammalian species. In the case of cattle, sheep, mice, and humans, more than a million offspring have developed from embryos cryopreserved by this “simple” method.

From this success, one might conclude that the problem of embryo cryopreservation has been solved for all species but such is not the case. The standard procedure, derived from cryobiological fundamentals, was and still is unsuccessful in a number of cases, and these cases are important in germplasm preservation. They include embryos of swine (with a few exceptions), fish, and insects. In each of these species, the underlying fundamentals are not wrong, but they are overlaid by other injurious factors or blockades. Perhaps the most important of these “other factors” is chilling injury. If an embryo or oocyte is highly chill sensitive, the low cooling rate required to prevent IIF may produce such long exposure times to low temperature as to produce major chilling injury. That conflict appears to apply to embryos of the pig, zebrafish, and Drosophila, and to oocytes of many species. The proven or suggested remedy in all these cases has been to “outrun” the chilling injury by using high cooling rates to pass through the damaging temperature zone so rapidly that there is no time for injury to occur. But since high cooling rates normally induce lethal IIF, IIF has to be prevented by introducing multimolar concentrations of CPA to induce vitrification of the cell water. That, in turn, can introduce serious osmotic and toxicity problems.

In the cell types of some species, there are other overlying, sometimes multiple, injurious factors. At ovulation, oocytes of most mammalian species are arrested in metaphase II of meiosis, and cooling them to temperatures near 0°C causes disassembly of microtubules and disruption of the meiotic spindle [ 76, 77]. Furthermore, the high concentrations of CPA required for vitrification appear to harden the zona pellucida, making it difficult or impossible for the sperm to penetrate the egg [ 78]. In zebrafish embryos, one major problem is that IIF occurs at just about the same temperature as external freezing [ 9]. Put another way, the water in the zebrafish embryo can tolerate very little supercooling before it freezes intracellularly. Without supercooling, the fundamental considerations do not apply, and the classical slow freezing approach will not succeed. Finally, the zebrafish embryo is compartmentalized, and the compartments possess significant permeability barriers to water and CPA [ 79].

This leaves us with a strategic question: will research on fundamentals lead to new sets of principles to explain and overcome some or all of these injurious overlying factors, principles that are probably unrelated to the physical chemical fundamentals applied previously? Or conversely, are empirical studies more likely to lead to solutions? If the answer to this pair of questions were known, then the problems would have already been solved. It is interesting in this regard that there was a 40-yr gap between the cryopreservation of bull and mouse sperm, where research proceeded empirically, but only a 1-yr gap between the cryopreservation of mouse and bovine embryos, where success in the former was based on fundamentals.

We cite one example that could conceivably be a new set of principles applied to chilling injury. There is a correlation between the amount of intracellular lipid or yolk that embryos contain and their susceptibility to chilling injury. For example, early cleavage stages of bovine and porcine embryos contain substantial amounts of internal lipid droplets and exhibit high sensitivity to chilling injury. Mouse and human embryos contain few apparent lipid droplets, and exhibit no chilling injury. Zebrafish embryos contain large amounts of yolk and exhibit high chilling sensitivity. When lipid droplets are removed from pig embryos by differential centrifugation and micropipetting [ 80], their chilling sensitivity is markedly reduced. When yolk is removed from zebrafish embryos by similar procedures, their chilling sensitivity is also substantially reduced [ 81]. What is the causal relationship between intracellular lipid and susceptibility to chilling injury? Does the same cause and effect operate with intraembryonic yolk in zebrafish, even though there is no apparent chemical relationship between yolk and lipid droplets? A favored hypothesis is that chilling injury arises because of lipid phase changes in the plasma membrane at low temperatures. If so, why should events in intracytoplasmic lipid droplets affect the properties of the plasma membrane?


Plant Defenses Against Pathogens

Plants defend against pathogens with barriers, secondary metabolites, and antimicrobial compounds.

Learning Objectives

Identify plant defense responses to pathogens

Key Takeaways

Key Points

  • Many plants have impenetrable barriers, such as bark and waxy cuticles, or adaptations, such as thorns and spines, to protect them from pathogens.
  • If pathogens breach a plant’s barriers, the plant can respond with secondary metabolites, which are often toxic compounds, such as glycol cyanide, that may harm the pathogen.
  • Plants produce antimicrobial chemicals, antimicrobial proteins, and antimicrobial enzymes that are able to fight the pathogens.

Defense Responses Against Pathogens

Pathogens are agents of disease. These infectious microorganisms, such as fungi, bacteria, and nematodes, live off of the plant and damage its tissues. Plants have developed a variety of strategies to discourage or kill attackers.

The first line of defense in plants is an intact and impenetrable barrier composed of bark and a waxy cuticle. Both protect plants against pathogens.

A plant’s exterior protection can be compromised by mechanical damage, which may provide an entry point for pathogens. If the first line of defense is breached, the plant must resort to a different set of defense mechanisms, such as toxins and enzymes. Secondary metabolites are compounds that are not directly derived from photosynthesis and are not necessary for respiration or plant growth and development. Many metabolites are toxic and can even be lethal to animals that ingest them.

Additionally, plants have a variety of inducible defenses in the presence of pathogens. In addition to secondary metabolites, plants produce antimicrobial chemicals, antimicrobial proteins, and antimicrobial enzymes that are able to fight the pathogens. Plants can close stomata to prevent the pathogen from entering the plant. A hypersensitive response, in which the plant experiences rapid cell death to fight off the infection, can be initiated by the plant or it may use endophyte assistance: the roots release chemicals that attract other beneficial bacteria to fight the infection.

Mechanical wounding and predator attacks activate defense and protective mechanisms in the damaged tissue and elicit long-distancing signaling or activation of defense and protective mechanisms at sites farther from the injury location. Some defense reactions occur within minutes, while others may take several hours.


Physical Barriers

Physical barriers play an important role in preventing microbes from reaching tissues that are susceptible to infection. At the cellular level, barriers consist of cells that are tightly joined to prevent invaders from crossing through to deeper tissue. For example, the endothelial cells that line blood vessels have very tight cell-to-cell junctions, blocking microbes from gaining access to the bloodstream. Cell junctions are generally composed of cell membrane proteins that may connect with the extracellular matrix or with complementary proteins from neighboring cells. Tissues in various parts of the body have different types of cell junctions. These include tight junctions, desmosomes, and gap junctions, as illustrated in Figure (PageIndex<1>). Invading microorganisms may attempt to break down these substances chemically, using enzymes such as proteases that can cause structural damage to create a point of entry for pathogens.

Figure (PageIndex<1>): There are multiple types of cell junctions in human tissue, three of which are shown here. Tight junctions rivet two adjacent cells together, preventing or limiting material exchange through the spaces between them. Desmosomes have intermediate fibers that act like shoelaces, tying two cells together, allowing small materials to pass through the resulting spaces. Gap junctions are channels between two cells that permit their communication via signals. (credit: modification of work by Mariana Ruiz Villareal)

The Skin Barrier

One of the body&rsquos most important physical barriers is the skin barrier, which is composed of three layers of closely packed cells. The thin upper layer is called the epidermis. A second, thicker layer, called the dermis, contains hair follicles, sweat glands, nerves, and blood vessels. A layer of fatty tissue called the hypodermis lies beneath the dermis and contains blood and lymph vessels (Figure (PageIndex<2>)).

Figure (PageIndex<2>): Human skin has three layers, the epidermis, the dermis, and the hypodermis, which provide a thick barrier between microbes outside the body and deeper tissues. Dead skin cells on the surface of the epidermis are continually shed, taking with them microbes on the skin&rsquos surface. (credit: modification of work by National Institutes of Health)

The topmost layer of skin, the epidermis, consists of cells that are packed with keratin. These dead cells remain as a tightly connected, dense layer of protein-filled cell husks on the surface of the skin. The keratin makes the skin&rsquos surface mechanically tough and resistant to degradation by bacterial enzymes. Keratin also helps to make the outer surface of the skin relatively waterproof, this helps keep the surface of the skin dry, which reduces microbial growth. Fatty acids on the skin&rsquos surface create a dry, salty, and acidic environment that inhibits the growth of some microbes and is highly resistant to breakdown by bacterial enzymes. Sebum from the oil glands in hair follicles is an endogenous mediator, providing an additional layer of defense by helping seal off the pore of the hair follicle, preventing bacteria on the skin&rsquos surface from invading sweat glands and surrounding tissue. Certain members of the microbiome, can use lipase enzymes to degrade sebum, using it as a food source. This produces oleic acid, which creates a mildly acidic environment on the surface of the skin that is inhospitable to many pathogenic microbes. Oleic acid is an example of an exogenously produced mediator because it is produced by resident microbes and not directly by body cells. In addition, the dead cells of the epidermis are frequently shed, along with any microbes that may be clinging to them (desquamation). Shed skin cells are continually replaced with new cells from below, providing a new barrier that will soon be shed in the same way.

Perspiration (sweat) provides some moisture to the epidermis, which can increase the potential for microbial growth. For this reason, more microbes are found on the regions of the skin that produce the most sweat, such as the skin of the underarms and groin. However, in addition to water, sweat also contains substances that inhibit microbial growth, such as salts, lysozyme, and antimicrobial peptides. Sebum also serves to protect the skin and reduce water loss. Although some of the lipids and fatty acids in sebum inhibit microbial growth, sebum contains compounds that provide nutrition for certain microbes. In the ears, cerumen (earwax) exhibits antimicrobial properties due to the presence of fatty acids, which lower the pH to between 3 and 5.

Infections can occur when the skin barrier is compromised or broken. A wound can serve as a point of entry for opportunistic pathogens, which can infect the skin tissue surrounding the wound and possibly spread to deeper tissues.

Mike, a gardener from southern California, recently noticed a small red bump on his left forearm. Initially, he did not think much of it, but soon it grew larger and then ulcerated (opened up), becoming a painful lesion that extended across a large part of his forearm (Figure (PageIndex<3>)). He went to an urgent care facility, where a physician asked about his occupation. When he said he was a landscaper, the physician immediately suspected a case of sporotrichosis, a type of fungal infection known as rose gardener&rsquos disease because it often afflicts landscapers and gardening enthusiasts.

Under most conditions, fungi cannot produce skin infections in healthy individuals. Fungi grow filaments known as hyphae, which are not particularly invasive and can be easily kept at bay by the physical barriers of the skin and mucous membranes. However, small wounds in the skin, such as those caused by thorns, can provide an opening for opportunistic pathogens like Sporothrix schenkii, a soil-dwelling fungus and the causative agent of rose gardener&rsquos disease. Once it breaches the skin barrier, S. schenkii can infect the skin and underlying tissues, producing ulcerated lesions like Mike&rsquos. Compounding matters, other pathogens may enter the infected tissue, causing secondary bacterial infections.

Luckily, rose gardener&rsquos disease is treatable. Mike&rsquos physician wrote him a prescription for some antifungal drugs as well as a course of antibiotics to combat secondary bacterial infections. His lesions eventually healed, and Mike returned to work with a new appreciation for gloves and protective clothing.

Figure (PageIndex<3>): Rose gardener&rsquos disease can occur when the fungus Sporothrix schenkii breaches the skin through small cuts, such as might be inflicted by thorns. (credit left: modification of work by Elisa Self credit right: modification of work by Centers for Disease Control and Prevention)

Barriers in the Eye

Although the eye and skin have distinct anatomy, they are both in direct contact with the external environment. An important component of the eye is the nasolacrimal drainage system, which serves as a conduit for the fluid of the eye, called tears. Tears flow from the external eye to the nasal cavity by the lacrimal apparatus, which is composed of the structures involved in tear production (Figure (PageIndex<4>)). The lacrimal gland, above the eye, secretes tears to keep the eye moist. There are two small openings, one on the inside edge of the upper eyelid and one on the inside edge of the lower eyelid, near the nose. Each of these openings is called a lacrimal punctum. Together, these lacrimal puncta collect tears from the eye that are then conveyed through lacrimal ducts to a reservoir for tears called the lacrimal sac, also known as the dacrocyst or tear sac.

From the sac, tear fluid flows via a nasolacrimal duct to the inner nose. Each nasolacrimal duct is located underneath the skin and passes through the bones of the face into the nose. Chemicals in tears, such as defensins, lactoferrin, and lysozyme, help to prevent colonization by pathogens. Lysozyme cleaves the bond between NAG and NAM in peptidoglycan, a component of the cell wall in bacteria. It is more effective against gram-positive bacteria, which lack the protective outer membrane associated with gram-negative bacteria. Lactoferrin inhibits microbial growth by chemically binding and sequestering iron. This effectually starves many microbes that require iron for growth. In addition, mucins facilitate removal of microbes from the surface of the eye.

Figure (PageIndex<4>): The lacrimal apparatus includes the structures of the eye associated with tear production and drainage. (credit: modification of work by &ldquoEvidence Based Medical Educator Inc.&rdquo/YouTube)

Mucous Membranes

The mucous membranes lining the nose, mouth, lungs, and urinary and digestive tracts provide another nonspecific barrier against potential pathogens. Mucous membranes consist of a layer of epithelial cells bound by tight junctions. The epithelial cells secrete a moist, sticky substance called mucus, which covers and protects the more fragile cell layers beneath it and traps debris and particulate matter, including microbes. Mucus secretions also contain antimicrobial peptides.

In many regions of the body, mechanical actions serve to flush mucus (along with trapped or dead microbes) out of the body or away from potential sites of infection. For example, in the respiratory system, inhalation can bring microbes, dust, mold spores, and other small airborne debris into the body. The nasal cavity is lined with hairs that trap large particles, like dust and pollen, and prevent their access to deeper tissues. The nasal cavity is also lined with a mucous membrane and Bowman&rsquos glands that produce mucus to help trap particles and microorganisms for removal, a layer known as the mucociliary blanket. The viscosity and acidity of this secretion inhibits microbial attachment to the underlying cells. The upper respiratory system is under constant surveillance by mucosa-associated lymphoid tissue (MALT), including the adenoids and tonsils. Other mucosal defenses include secreted antibodies (IgA), lysozyme, surfactant, and antimicrobial peptides called defensins. The epithelial cells lining the upper parts of the respiratory tract are called ciliated epithelial cells because they have hair-like appendages known as cilia. Movement of the cilia propels debris-laden mucus out and away from the lungs. The expelled mucus is then swallowed and destroyed in the stomach, or coughed up, or sneezed out (Figure (PageIndex<5>)). This system of removal is often called the mucociliary escalator.


Figure (PageIndex<5>): This scanning electron micrograph shows ciliated and nonciliated epithelial cells from the human trachea. The mucociliary escalator pushes mucus away from the lungs, along with any debris or microorganisms that may be trapped in the sticky mucus, and the mucus moves up to the esophagus where it can be removed by swallowing.

The mucociliary escalator is such an effective barrier to microbes that the lungs, the lowermost (and most sensitive) portion of the respiratory tract, were long considered to be a sterile environment in healthy individuals. Only recently has research suggested that healthy lungs may have a small normal microbiota. Disruption of the mucociliary escalator by the damaging effects of smoking or diseases such as cystic fibrosis can lead to increased colonization of bacteria in the lower respiratory tract and frequent infections, which highlights the importance of this physical barrier to host defenses. Lastly, the outer surface of the lungs is protected with a double-layered pleural membrane, which protects the lungs and provides lubrication to permit the lungs to move easily during respiration. They also are protected by alveolar macrophages. These phagocytes efficiently kill any microbes that manage to evade the other defenses.

Like the respiratory tract, the digestive tract is a portal of entry through which microbes enter the body, and the mucous membranes lining the digestive tract provide a nonspecific physical barrier against ingested microbes. Several factors appear to work against making the mouth hospitable to certain microbes. For example, chewing allows microbes to mix better with saliva so they can be swallowed or spit out more easily. In the oral cavity, saliva contains mediators such as lactoperoxidase enzymes, and lysozyme, which can damage microbial cells. Lysozyme is part of the first line of defense in the innate immune system and cleaves linkages between N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) in bacterial peptidoglycan. Additionally, fluids containing immunoglobulins and phagocytic cells are produced in the gingival spaces. The stomach is an extremely acidic environment (pH 1.5&ndash3.5) due to the gastric juices that break down food and kill many ingested microbes this helps prevent infection from pathogens. Further down, the intestinal tract is lined with epithelial cells, interspersed with mucus-secreting goblet cells (Figure (PageIndex<6>)). This mucus mixes with material received from the stomach, trapping foodborne microbes and debris.

Figure (PageIndex<6>): Goblet cells produce and secrete mucus. The arrows in this micrograph point to the mucus-secreting goblet cells (magnification 1600⨯) in the intestinal epithelium. (credit micrograph: Micrograph provided by the Regents of University of Michigan Medical School © 2012)

Goblet cells, which are modified simple columnar epithelial cells, also line the GI tract (Figure (PageIndex<6>)). Goblet cells secrete a gel-forming mucin, which is the major component of mucus. The production of a protective layer of mucus helps reduce the risk of pathogens reaching deeper tissues. Small aggregates of underlying lymphoid tissue in the ileum, called Peyer&rsquos patches, detect pathogens in the intestines via microfold (M) cells, which transfer antigens from the lumen of the intestine to the lymphocytes on Peyer&rsquos patches to induce an immune response. The Peyer&rsquos patches then secrete IgA and other pathogen-specific antibodies into the intestinal lumen to help keep intestinal microbes at safe levels.

The mechanical action of peristalsis, a series of muscular contractions in the digestive tract, moves the sloughed mucus and other material through the intestines, rectum, and anus, excreting the material in feces. In fact, feces are composed of approximately 25% microbes, 25% sloughed epithelial cells, 25% mucus, and 25% digested or undigested food. Finally, the normal microbiota provides an additional barrier to infection via a variety of mechanisms. For example, these organisms outcompete potential pathogens for space and nutrients within the intestine. This is known as competitive exclusion. Members of the microbiota may also secrete protein toxins known as bacteriocins that are able to bind to specific receptors on the surface of susceptible bacteria.

Endothelia

The epithelial cells lining the urogenital tract, blood vessels, lymphatic vessels, and certain other tissues are known as endothelia. These tightly packed cells provide a particularly effective frontline barrier against invaders. The endothelia of the blood-brain barrier, for example, protect the central nervous system (CNS), which consists of the brain and the spinal cord. The CNS is one of the most sensitive and important areas of the body, as microbial infection of the CNS can quickly lead to serious and often fatal inflammation. The cell junctions in the blood vessels traveling through the CNS are some of the tightest and toughest in the body, preventing any transient microbes in the bloodstream from entering the CNS. This keeps the cerebrospinal fluid that surrounds and bathes the brain and spinal cord sterile under normal conditions.

In both men and women, however, the kidneys are sterile. Although urine does contain some antibacterial components, bacteria will grow in urine left out at room temperature. Therefore, it is primarily the flushing action that keeps the ureters and bladder free of microbes. The female reproductive system employs lactate, an exogenously produced chemical mediator, to inhibit microbial growth. The cells and tissue layers composing the vagina also produce glycogen, a branched and more complex polymer of glucose.

  1. Describe how the mucociliary escalator functions.
  2. What other defenses do each of the body sites have in common?
  3. Name two places you would find endothelia.

Antimicrobial Peptides

The antimicrobial peptides (AMPs) are a special class of nonspecific cell-derived mediators with broad-spectrum antimicrobial properties. Some AMPs are produced routinely by the body, whereas others are primarily produced (or produced in greater quantities) in response to the presence of an invading pathogen. Research has begun exploring how AMPs can be used in the diagnosis and treatment of disease.

AMPs may induce cell damage in microorganisms in a variety of ways, including by inflicting damage to membranes, destroying DNA and RNA, or interfering with cell-wall synthesis. Depending on the specific antimicrobial mechanism, a particular AMP may inhibit only certain groups of microbes (e.g., gram-positive or gram-negative bacteria) or it may be more broadly effective against bacteria, fungi, protozoa, and viruses. Many AMPs are found on the skin, but they can also be found in other regions of the body.

A family of AMPs called defensins can be produced by epithelial cells throughout the body as well as by cellular defenses such as macrophages and neutrophils. Defensins may be secreted or act inside host cells they combat microorganisms by damaging their plasma membranes. AMPs called bacteriocins are produced exogenously by certain members of the resident microbiota within the gastrointestinal tract. The genes coding for these types of AMPs are often carried on plasmids and can be passed between different species within the resident microbiota through lateral or horizontal gene transfer. There are numerous other AMPs throughout the body. The characteristics of a few of the more significant AMPs are summarized in Table (PageIndex<2>).

Table (PageIndex<2>): Characteristics of Selected Antimicrobial Peptides (AMPs)
AMP Secreted by Body site Pathogens inhibited Mode of action
Bacteriocins Resident microbiota Gastrointestinal tract Bacteria Disrupt membrane
Cathelicidin Epithelial cells, macrophages, and other cell types Skin Bacteria and fungi Disrupts membrane
Defensins Epithelial cells, macrophages, neutrophils Throughout the body Fungi, bacteria, and many viruses Disrupt membrane
Dermicidin Sweat glands Skin Bacteria and fungi Disrupts membrane integrity and ion channels
Histatins Salivary glands Oral cavity Fungi Disrupt intracellular function

Why are antimicrobial peptides (AMPs) considered nonspecific defenses?

Mechanical Defenses

In addition to physical barriers that keep microbes out, the body has a number of mechanical defenses that physically remove pathogens from the body, preventing them from taking up residence. We have already discussed several examples of mechanical defenses, including the shedding of skin cells, the expulsion of mucus via the mucociliary escalator, and the excretion of feces through intestinal peristalsis. Other important examples of mechanical defenses include the flushing action of urine and tears, which both serve to carry microbes away from the body. The flushing action of urine is largely responsible for the normally sterile environment of the urinary tract, which includes the kidneys, ureters, and urinary bladder. Urine passing out of the body washes out transient microorganisms, preventing them from taking up residence. The eyes also have physical barriers and mechanical mechanisms for preventing infections. The eyelashes and eyelids prevent dust and airborne microorganisms from reaching the surface of the eye. Any microbes or debris that make it past these physical barriers may be flushed out by the mechanical action of blinking, which bathes the eye in tears, washing debris away (Figure (PageIndex<7>)).

Figure (PageIndex<7>): Tears flush microbes away from the surface of the eye. Urine washes microbes out of the urinary tract as it passes through as a result, the urinary system is normally sterile.

Name two mechanical defenses that protect the eyes.

Microbiome

In various regions of the body, resident microbiota serve as an important first-line defense against invading pathogens. Through their occupation of cellular binding sites and competition for available nutrients, the resident microbiota prevent the critical early steps of pathogen attachment and proliferation required for the establishment of an infection. For example, in the vagina, members of the resident microbiota compete with opportunistic pathogens like the yeast Candida. This competition prevents infections by limiting the availability of nutrients, thus inhibiting the growth of Candida, keeping its population in check. Similar competitions occur between the microbiota and potential pathogens on the skin, in the upper respiratory tract, and in the gastrointestinal tract. The resident microbiota also contribute to the chemical defenses of the innate nonspecific host defenses.

The importance of the normal microbiota in host defenses is highlighted by the increased susceptibility to infectious diseases when the microbiota is disrupted or eliminated. Treatment with antibiotics can significantly deplete the normal microbiota of the gastrointestinal tract, providing an advantage for pathogenic bacteria to colonize and cause diarrheal infection. In the case of diarrhea caused by Clostridium difficile, the infection can be severe and potentially lethal. One strategy for treating C. difficile infections is fecal transplantation, which involves the transfer of fecal material from a donor (screened for potential pathogens) into the intestines of the recipient patient as a method of restoring the normal microbiota and combating C. difficile infections.

Table (PageIndex<3>) provides a summary of the physical defenses discussed in this section.

Table (PageIndex<3>): Physical Defenses of Nonspecific Innate Immunity


How is “Bismillah” to be said when slaughtering chickens with modern mechanical devices?

Giving the animal an electric shock before slaughtering it may kill the animal if the voltage is high, or it may cause it to lose consciousness without killing it, if the voltage is low or moderate.

If it kills it, it is not permissible to eat it because it is “dead meat” (an animal that was not slaughtered in the proper manner) according to the consensus of the fuqaha’. If it does not kill it, and it is slaughtered in the proper shar‘i manner immediately afterwards, then it is halaal and it is permissible to eat it.

Dr. Muhammad al-Ashqar (may Allah preserve him) said:

If the electric shock was fatal, then the animal is like one that has been “beaten to death” (and therefore haraam, as mentioned in al-Maa’idah 5:3). If it caused it to lose consciousness without killing it, and the animal was slaughtered in the proper shar‘i manner after that, then it is halaal. If it was not slaughtered properly but it was skinned and cut up without being slaughtered, then it is not halaal.

End quote from Majallat Majma‘ al-Fiqh al-Islami. Issue no. 10, vol. 1, p. 339

The Islamic Fiqh Council (Majma‘ al-Fiqh al-Islami) is of the view that it is not permissible to give chickens electric shocks before slaughtering them, because experience has proven that this leads to the death of a considerable number of them.

In a statement of the Islamic Fiqh Council, issued during its tenth conference in Jeddah in the Kingdom of Saudi Arabia during the period 23-28 Safar 1418 AH/28 June-3 July 1997 CE, it says the following:

Animals that are slaughtered in the proper shar‘i manner after stunning are halaal and may be eaten if technical conditions are met that ascertain that the animal was not dead before it was slaughtered. These have been defined by experts at the present time as follows:

1.The electrodes should be placed on the temples or on the forehead and back of the head

2.The voltage should be between 100 and 400 volts

3.The current should be between .75 and 1 amp for sheep, between 2 and 2.5 amps for cattle.

4.The electrical current should be applied for between 3 and 6 seconds.

(c) it is not permissible to stun an animal that is intended for slaughter by using a captive bolt pistol or bolt gun, or by gassing.

(d) It is not permissible to stun chickens by means of electric shock, because experience has proven that this leads to the death of a considerable number of them before slaughter.

(e) It is not haraam to eat animals that were slaughtered properly after being stunned by using a mixture of carbon dioxide and air or oxygen, or by using a non-penetrating bolt gun that does not lead to the death of the animal before it is properly slaughtered. End quote.

Dr. Muhammad al-Hawaari stated that stunning of chickens by means of electrocution leads to cardiac arrest in 90% of cases and to death in 10%.

See Majallat Majma‘ al-Fiqh al-Islami, issue no. 10, vol. 1, p. 411, 583

Based on that, you need to look at the electrocution asked about. If it will lead, as the Council said, to the death of a considerable number of the chickens that are not separated from the live chickens, then it is not permissible to electrocute them. But if the electrocution uses a low voltage that does not lead to that, then the slaughter is halaal.

Saying “Bismillah” is a condition of slaughter being halaal, and is not waived in the case of forgetting or ignorance, according to the more correct scholarly view. See the answer to question no. 85669.

The basic principle with regard to saying “Bismillah” is that it must be done for each individual animal with the intention of slaughtering it in the proper manner.

But in the case of mechanical devices that slaughter a large number of chickens within a short time period, the scholars have differed with regard to the way of saying “Bismillah” that is essential for the slaughter to be halaal. There are several opinions:

1.That it is sufficient for “Bismillah” to be said once by the person operating the machine, if it slaughters a number of chickens in one continuous time period. This is what has been stated in fatwas issued by the Standing Committee and in a statement issued by the Islamic Fiqh Council.

2.That it is sufficient for “Bismillah” to be said once by the person operating the machine, on condition that the specific chickens that he is going to slaughter are in front of him, such as if they are lined up on the conveyor belt and the like. This has been stated in fatwas issued by Shaykh Ibn ‘Uthaymeen (may Allah have mercy on him).

3.That saying “Bismillah” when using these machines is not possible, therefore it is not permissible to use these machine for halaal slaughter.

The more correct view is the first one, for the following reasons:

It says in Fataawa al-Lajnah ad-Daa’imah:

What is the ruling on mechanical slaughter, in which dozens of chickens are slaughtered by machines at the same time, saying “Bismillah” only once? If a person is slaughtering a large number of chickens by hand, is it acceptable for him to say “Bismillah” just once, or does he have to say it for each one individually?

Firstly: it is permissible to slaughter using modern machines on condition that (the blades) are sharp and that the oesophagus and windpipe are cut.

Secondly: if the machine slaughters a number of chickens in the same continuous length of time, it is acceptable for the person operating the machine to say “Bismillah” once when he begins to operate it with the intention of slaughtering, so long as the person operating the machine is a Muslim or kitaabi (Jewish or Christian).

Thirdly: if the person is slaughtering by hand, he must say “Bismillah” separately for each chicken he slaughters, because each chicken is a separate entity.

Fourthly: The slaughter must be done in the slaughterhouse and the windpipe and two veins, or one of them, must be cut.

Bakr Abu Zayd, Saalih al-Fawzaan, ‘Abdullah ibn Ghadyaan, ‘Abd al-‘Azeez ibn ‘Abdullah Aal ash-Shaykh

End quote from Fataawa al-Lajnah ad-Daa’imah, 22/463

It also says (22/462): is it permissible to say “Bismillah” when operating the machine which does one repeated movement. Please note that what is meant is saying “Bismillah” only once, when starting the machine for slaughter.

Answer: it is acceptable for the person who is operating the machine to say “Bismillah” once when starting it for a number of (chickens) with the intention of slaughtering them, so long as the one who is operating it is a Muslim or a Jew or a Christian.

‘Abdullah ibn Ghadyaan, ‘Abd ar-Razaaq ‘Afeefi, ‘Abd al-‘Azeez ibn ‘Abdullah ibn Baaz. End quote.

It says in the statement of the Islamic Fiqh Council quoted above:

8. The basic principle is that slaughter of poultry and other animals is to be done by hand, but there is nothing wrong with using mechanical devices to slaughter poultry so long as the conditions of shar‘i slaughter mentioned above in paragraph 2 are met. And it is acceptable to say “Bismillah” once for each batch that is to be slaughtered in a continuous session, but if there was an interruption then saying “Bismillah” must be repeated. End quote.

But the statement of the Council did not specify that saying “Bismillah” must come from the one who is operating the machine.

Dr. Muhammad Sulaymaan al-Ashqar said: Saying “Bismillah” in the case of a large number, if they are to be slaughtered by hand in the Islamic manner, may be exhausting for the slaughterman. For example, if a person has the task of slaughtering 1200 chickens per hour at a rate of one chicken every three seconds, then he would have to say “Bismillah wa Allahu akbar” 1200 times in an hour which would be exhausting and very difficult, and such burdensome difficulty is to be avoided in Islam because Allah, may He be exalted, says (interpretation of the meaning): “and has not laid upon you in religion any hardship” [al-Hajj 22:78].

Hence the Fatwa Council in Kuwait, of which I was a member at the time this fatwa was issued, stated that when slaughtering a large number of poultry it is sufficient to say “Bismillah” over them once, at the beginning, if the task is to proceed continuously without stopping. If there is a pause for some reason, then the slaughterman has to say “Bismillah” again for the remainder.

End quote from Majallat Majma‘ al-Fiqh al-Islami, issue no. 10, vol. 1, p. 346.

Shaykh Ibn ‘Uthaymeen (may Allah have mercy on him) was asked the following question:

I went to visit the National Poultry Farms and I saw how they slaughter the chickens in the beginning they suspend the chickens so that they cannot move, then they pass over the slaughterman who slaughters them without saying “Bismillah”. I asked: Why do you not say “Bismillah”? He said: Because I say “Bismillah” when I enter and I cannot say it for five hundred thousand chickens. So when I start I say, “Bismillah, Allahu akbar”, and that is sufficient. I said, Who did you ask? He said: The scholars gave me a fatwa to that effect and permitted it.

I do not know, O Shaykh, whether this action is permissible?

He replied: It is essential to say “Bismillah” over something specific, whether it is one or more. For example, if he lines up a thousand chickens then when starting the machine he says, “Bismillah”, that is sufficient. Then if he lines up another thousand chickens, for example, and starts the machine and the knives start moving, it is sufficient for him to say, “Bismillah” for this batch. And if another batch is lined up for him, he should say “Bismillah” for it.

Questioner: He says, “I say ‘Bismillah’ once and that is sufficient”?

Shaykh: Do you mean until the machine stops? No, that is not permissible, because “Bismillah” must be said over something specific.

Questioner: Another question, O Shaykh. We also went to visit Astra Farms in Tabook, where they were slaughtering quail. What do they do? They hang up these birds, then after hanging them up they pass over a machine that sprays water on the birds and stuns them somewhat, then they pass over something like a wall on which is written, “Bismillah wa Allahu akbar”, then they go to a machine that cuts off their heads. The person in charge said that this is acceptable. Is it acceptable to have the words “Bismillah wa Allah akbar” written down?

Shaykh: All of that is ignorance and now you, may Allah bless you, have to report what you and your brothers have seen in a signed statement and send it to Dar al-Ifta’, and tell them when you saw that, whether it was this year or a few years ago, so that you may discharge your duty with regard to this matter.

Questioner: O Shaykh, they say that a group of shaykhs gave them a fatwa allowing that.

Shaykh: No, some shaykhs issued a fatwa saying something other than this. Maybe he issued a fatwa saying what I have said, which is that he may collect a batch and then turn on the machine for this batch, even if he does not say “Bismillah” for each individual bird. It is similar to the case where he sees a flock of birds and shoots them and says “Bismillah”, and twenty birds fall – in that case they are halaal.

What seems most likely to be the case, and Allah knows best, is that what the shaykh (may Allah have mercy on him) mentioned about saying “Bismillah” for each specific batch of birds being slaughtered, is not essential, because saying “Bismillah” once in this case is analogous to what is done when hunting. When hunting it is not essential to say “Bismillah” for each specific target rather saying “Bismillah” is connected to the weapon. So if a person said “Bismillah” over his weapon with the intention of hunting, and he catches something other than what he aimed at, it is still halaal.

Here we will quote some useful words from Shaykh Muhammad Taqi al-‘Uthmaani (may Allah preserve him) which confirms what we have said above about the principle that “Bismillah” should be said over a specific animal (or batch) and that saying “Bismillah” just once on the part of the person operating the machine is a kind of concession that differs from the basic principle, by analogy with what is done when hunting. And he explains that there is no point in somebody standing next to the machine saying “Bismillah” when he is not actually operating it.

He said (may Allah have mercy on him):

With regard to the issue of saying “Bismillah”, it is very difficult when using this method. The first problem is identifying who is doing the slaughtering, because saying “Bismillah” is obligatory on the slaughterman to such an extent that if a man says “Bismillah” then another man does the slaughtering, that is not permissible. So the question is: Who is the slaughterer in the case of this machine? We could say that the one who starts the machine the first time is regarded as the slaughterer, because the function of the machine can only be attributed to the one who operates it, because machines are not sentient beings to which actions may be attributed. So the action is to be attributed to the one who uses them, and he becomes the doer by means of the machine. But the problem here is that the person who uses the machine at the beginning of the day, for example, only starts it once, then the machine continues running during work hours, and sometimes runs for twenty-four hours, cutting the necks of thousands of chickens. So if the person who turned it on at the beginning of the day said “Bismillah” only once, is that single utterance of “Bismillah” sufficient for thousands of chickens that are slaughtered throughout the day after turning on the machine? The apparent meaning of the Qur’aanic text (interpretation of the meaning) “Eat not (O believers) of that (meat) on which Allahs Name has not been pronounced” [al-An‘aam 6:121] indicates that each animal should have the name of Allah pronounced over it separately and be slaughtered immediately afterwards. From this the fuqaha’ derived rulings which indicate that the name of Allah should be mentioned over each animal or for each action.

I mentioned these phrases in my research and I concluded from them that the majority of imams who stipulate that the name of Allah should be pronounced at the time of slaughter stipulate that it should be said over a specific animal, and that it should be at the time of slaughter, and there should be no significant interval between saying the name of Allah and the act of slaughter. These conditions are not met in the method described in the case of machines. If the one who switches it on the first time says “Bismillah” once, that means that he did not say “Bismillah” over a particular animal, and between his saying “Bismillah” and his slaughtering of thousands of chickens there may be a lengthy interval that may last for a whole day or two days. So it appears to be the case that this single utterance of “Bismillah” is not sufficient for the slaughter of all these animals.

Then I saw some slaughterhouses in Canada, where they have a man standing beside the rotating knives, continually saying, “Bismillah Allahu akbar”. And I thought: with regard to his saying “Bismillah” carrying any weight in shar‘i terms there are the following problems:

1.The words “Bismillah” should be uttered by the slaughterer this man who is standing beside the rotating knife has nothing to do with the slaughter process he is not operating the machine or turning the knife, and the chickens come nowhere near him. Rather he is a man who is completely separated from the process of slaughter and his pronouncement of the name of Allah does not come from the slaughterer.

2.A number of chickens come to the rotating knife in a matter of seconds this man who is standing there cannot say the name of Allah over each one of these chickens without intervals.

3.This man who is standing is a human he is not an automatic machine. So he cannot do any action apart from saying “Bismillah”. He may need to do things that will distract him from saying “Bismillah”, and during that dozens of chickens may pass through the rotating knife.

There is another concern to be noted regarding the topic of saying “Bismillah” over machines, which is drawing an analogy between turning on the machine and releasing a hunting dog. It is not obligatory to say “Bismillah” when the prey animal dies rather it must be said when releasing the dog, and there may be a lengthy interval between the release of the dog and the death of the prey, and the hunting dog may kill a number of animals after being released once. So it seems that saying “Bismillah” once is sufficient for all of them to be regarded as halaal. Ibn Qudaamah (may Allah have mercy on him) said: If the hunter says “Bismillah” over one prey (when releasing his dog) but then he catches another, it is halaal, and if he says “Bismillah” over one arrow and shoots it, then he takes another and shoots it (without saying “Bismillah”), what he catches with it (the second arrow) is not halaal.

What we mentioned above has to do with necessity, and in the issue under discussion, there is no necessity. However, if we think of the need to produce a large amount within a short time, which is because of increased population and the rise in the number of consumers, and the small number of slaughtermen, and the fact that sharee‘ah waived the condition of specifying the prey in the case of hunting because it is too difficult, as Ibn Qudaamah (may Allah have mercy on him) said, and in such cases sharee‘ah allows concessions to ward off hardship, in that case we may compare the issue under discussion to the issue of necessity (as in the case of hunting) with regard to mentioning the name of Allah, so as to ward off hardship and make things easier for people. However, I am not quite certain of this conclusion, but I wanted to put it forward for discussion by the scholars to decide about it, and I have not issued any fatwa on that basis until now, especially when we have a suitable alternative to the revolving knife which will meet all of the production needs at the same time. That alternative is to remove the revolving knife from the machine and replace it with four Muslim men who could take turns in cutting the chickens’ throats whilst mentioning the name of Allah, every time the suspended chickens pass by them.

This is something that I have suggested to a large slaughterhouse on the island of Reunion, and they did that. Experience indicates that this did not reduce the rate of production at all, because these people cut the throats of the chickens within the same timeframe as the revolving knife.

End quote from Shaykh Muhammad Taqi al-‘Uthmaani, Majallat Majma‘ al-Fiqh al-Islami, issue no. 10, vol. 1, p. 541-544

1.Stunning the chickens by means of electrocution must be avoided, and it is not permissible for the organisation that is supervising slaughter to allow it unless they can be certain that it does not lead to killing any of the chickens.

2.It is sufficient for the machine operator to say “Bismillah” when switching it on, and that must be repeated after any pause.

3.There is no point in the five men beside the machine saying “Bismillah” rather this is a waste of time that should be put a stop to.

4.The organisations that supervise Islamic slaughter must pay attention to the conditions and essential guidelines on the matter, and not be careless in applying them. And they should try to arrange for the slaughter to be done by hand instead of by machines, in accordance with the suggestion made by Shaykh Muhammad Taqi al-‘Uthmaani. That is so as to do away with problems having to do with electrocution and saying “Bismillah”, and so as to avoid the possibility of the slaughter of some of the chickens being done inappropriately when passing over the rotating knife, because of differences in size among the chickens. This is a problem that some researchers have pointed out.


Watch the video: Animal electric shock (February 2023).