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Toxflush Reviews
The ToxFlush supplement encourages weight loss, and the company is claiming to help the users in losing at least up to three pounds per week. However, we have a lot to say about the ToxFlush supplement.
Click to learn more about ToxFlush Supplement.
Therefore, we are going to discuss every aspect of this supplement, and at the end of this ToxFlush reviews, we will lay a conclusion.
Are you curious to know what Toxflush is all about then you have landed at the right place? This will be the Toxflush review that you have been looking for.
However, the problem might be a little deeper than this. With years of toxins built in your body, they can prevent nutrients absorption that leads to fat build-up as well.
Hence, detoxification supplements can improve fat loss as well as remove the toxins that are hurting your body every single day. ToxFlush is your first step in detoxifying the toxins from your body.
It improves your overall health, removes toxins, and encourages fat loss for better results. Whether youre trying to go on a diet or starting a new workout routine, this supplement can help in Fixing your body.
As the supplement only takes 2 seconds to gulp, it is not even something complex. However, the biggest claim that folks at ToxFlush make is that this supplement will encourage a three-pound loss every week. Before you start jumping to conclusions, lets talk about the ingredients used in this pill.
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Toxflush Reviews– Detoxify Your Body And Helps To Lose Weight

Toxflush is a weight loss supplement that helps you shed up to 3 pounds of stubborn fat every 7 days. This Toxflush review will provide in-depth knowledge about the product, so it will be easy for you to decide whether to purchase it or not.

Product Name	Toxflush
Category	Weight loss
Main Benefits	5 second morning ritual that unlocks fat-burning blockers to burn calories faster
Ingredients	Graviola leaf, Red raspberry, Green tea, Beta Glucan, Turmeric, Pycnogenol, Essiac tea complex, Grapeseed, Mushroom complex, Quercetin Dihydrate, Pomegranate, Olive leaf
Administration Route	Oral
Dosage	2 capsules every day.
Alcohol Warning	No Restrictions
Side Effects	No Major Side Effects reported
Price	$67
Money-Back Guarantee	60 Days

What Is It?

Toxflush is a 5 second morning ritual that unlocks fat burning blockers to burn calories faster. The company behind the supplement claims that each and every ingredients used in the supplement are clinically proven to enhance metabolism and thus speeds up the fat burning process.
It also helps to support immunity and ease stress. The most important feature of Toxflush supplement is that it targets all the problems that cause weight loss resistance.
The formula includes exactly 26 ingredients that are clinically relevant and fight against insulin resistance, inflammation, and toxic build-up inside the body.
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The Ingredients

Toxflush ingredients are 100% natural and are effective in quick fat burning. They are:

Graviola Leaf

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Graviola leaves are used to make medicines. It is used to treat infections caused by bacteria and parasites. The Graviola tree leaves treat stomach ailments, fever, parasitic infections, hypertension, and rheumatism. Also, it is widely used as a sedative.

Red Raspberry

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As mentioned in Toxflush review, Raspberry helps to break down the fat within the cells more effectively, helping your body to burn fat faster. They also increase the levels of adiponectin, a hormone that helps to regulate metabolism.
Both fresh and frozen raspberries contain high amounts of fiber which could help in reducing belly fat. Eating raspberries also helps in regulating the insulin response of the body to reduce the risk of high blood sugar levels.
It also contains essential nutrients like Vitamin C, manganese, Vitamin K, etc. These nutrients help burn fat by boosting your metabolism, helps slow down your digestive process, and leaves you feeling full for longer.

Green Tea

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Green tea contains caffeine that acts as a stimulant. This caffeine has been shown to aid fat burning and improve exercise performance.
The presence of antioxidants known as catechins in green tea helps burn fat and boost metabolism and thus helps to lose weight. Green tea helps to lose abdominal fat and the effects of green tea are relatively modest.

Beta Glucan

Beta glucan aids weight loss and fat reduction in your body. It can reduce visceral fat as well as body weight, BMI, and waist circumference.
This might also help you to feel fuller for a longer period, help you eat less, which could make you lose weight. Research suggests that Beta-glucan may also reduce cholesterols and improve skin conditions.

Turmeric

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According to Toxflush review, Curcumin present in turmeric suppresses fat tissue growth and it also helps in losing weight by regulating sugar levels and further preventing insulin resistance.

Pycnogenol

This ingredient is more effective in improving blood flow and was found to have significant effect in weight loss. It may have benefits for heart and artery health too.
Pycnogenol seems to lower blood pressure and improve blood flow to the legs. It may also protect against coronary artery disease and blood clots.

Essiac Tea Complex

It is a herbal tea that can kill cancer cells, stimulate immunity and aid detoxification. Actually, Essiac tea is a blend of different herbs like burdock root, slippery elm, sheep sorrel, and Indian rhubarb.
Ancients also believed to enhance detoxification, boost immune function, and reduce inflammation. The herbs present in it are also shown to promote blood circulation, improve skin texture, and stabilize blood sugar.

Grape Seed

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Consuming grape seeds lower the amount of fat which your body absorbs from the food. It also helps to inhibit the total fat deposits in the body and the grape seed extracts can benefit you and aid your efforts to lose weight.
Supplements containing grape seed extracts in a high fat diet might normalize body weight and back weights. It may also normalize lipid concentrations and carnitine levels by controlling lipid metabolism.
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Mushroom Complex

Mushrooms provide proteins and fibers required for your body and have also been found to be beneficial for weight loss. It has unusually high levels of essential vitamins and it can also help to increase vitamin D levels.
The active ingredients in mushrooms have high molecular weight polysaccharides and this improves weight management.

Quercetin Dihydrate

It is a flavonoid that activates metabolism and may reduce weight gain by decreasing feed efficiency. Quercetin helps protect against heart disease and cancer. It can stabilize the cells that release histamine in the body and thereby have an anti-inflammatory and antihistamine effect.

Pomegranate

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They are rich in antioxidants, polyphenols and conjugated linolenic acid which help you burn fat and boost your metabolism. The juice of pomegranate helps you in suppressing appetite.

Olive Leaf

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Extract of olive leaf helps in preventing high fat, diet-induced obesity. The olive leaf extract prevents obesity by regulating the expression of genes that affect weight gain. It can also help in reducing food intake.

Arabinogalactan

It helps in boosting immune power and studies found that it increases the body’s potential to defend against common cold infection.

Cat’s Claw

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It is a herbal supplement that helps fight a range of ailments, including infections, cancer, arthritis, and Alzheimer’s disease.

Panax Ginseng

It is a traditional Chinese medicine that stimulates weight loss, delays fat absorption and modifies fat formation. It also improves the blood and organ lipid profile when combined with exercise.

Lycopene

It is an antioxidant that prevents free radical damage and premature ageing of cells. It also helps in reducing the oxidative stress of the body and thus helps you lose weight.
= Click to Order ToxFlushWeight Loss Detox Supplementfrom its Official Website

What Benefits Can You Expect?

Helps to get rid of excess fat quickly
Removes toxins from your body
Increase the rate of metabolism
Boosts energy
Rejuvenates your body

Side Effects, Dosage, And How To Use It?

By analyzing Toxflush review, All the Toxflush ingredients are 100% natural and are clinically proven. So the side effects are minimal and the supplement does not contain any harmful chemicals, toxins, or additives which harms your body.
As per Toxflush official website, the supplement is manufactured in a GMP certified laboratory and it boosts the overall wellbeing. To get the desired result, you have to consume 2 capsules everyday.
= Click to Order ToxFlushWeight Loss Detox Supplementfrom its Official Website

Is It A Magic Pill?

No, Toxflush supplement is absolutely not a magic pill. Because it doesn’t deliver results within minutes or seconds. Many people with obesity related issues have felt great results with this supplement. But you have to practise simple home workouts and healthy diets along with consuming Toxflush capsules.

How Long Will It Take To See The Results?

It takes at least 2-3 months to see the results. Some of the customers have opinionated and complained that they used the pill for a long one month and it’s not working.
Believe that almost all dietary pills take a minimum of 2 months to deliver results. The ingredients present in the Toxflush supplement requires around 2 months to become effective on your body.

How Long Would The Results Stay?

The results you got by consuming the supplement for continuous 2-3 months will stay with you for a period of at least 1-2 years. If you are following strict healthy diets and routines, then the result stays longer than this.

Price And Where To Get It?

If you wish to purchase Toxflush supplements don’t head to Amazon or any other online eCommerce sites to place your order. Because it is only available through its official website page.
This is done to limit the fake unauthorized replicas of the real product. So don’t fall for scam websites that try to deceive you. Anyone can buy Biotox Gold supplement form its official website and the webpage also provides some discount packages.

Buy 1 bottle package for 30 day supply at just $67
The price of 3 bottles required for 90 days supply is $57 per bottle
You can order 6 bottles required for 180 days supply at $47 per bottle

= Click to Order ToxFlushWeight Loss Detox Supplementfrom its Official Website

Product Complaints And Customer Reviews

Most of the Toxflush customer reviews are positive. Some customers have reported a complaint about the availability of this product as the supplement is available for purchase only on its official website and it will become frequently out of stock due to the huge demand. This makes them a little uncomfortable and inconvenient.

Is The Product Scam Or Legit?

Lean Body Burn is definitely a legit product. The supplement delivers all the claims mentioned in its official website for its users. Also, the supplement includes ingredients that are scientifically backed and clinically proven.

Verdict

We would recommend buying Toxflush if you are looking for a quick fat burning supplement that provides overall wellbeing too.
= Click to Order ToxFlushWeight Loss Detox Supplementfrom its Official Website
To sum up Toxflush review, it includes natural ingredients that also help in suppressing appetite and losing weight. Coupling a good healthy eating habit and diet regimes, Toxflush can do magic on your body. so it’s definitely one we would recommend.

Two problems not commonly discussed prior to the novel Coronavirus outbreak are the emergence of infectious disease and the related increasing prevalence of antimicrobial resistance. Here, I will explain the science behind these problems and some solutions that can be driven by legislation. My background is more squarely rooted in the science, so I apologize if I lean too heavily in this area as opposed to the economics and policy focus of this subreddit. I frequent this sub and enjoy the discourse here, and in my area this is one topic that overlaps with public health policy that I am passionate about.
To understand emerging disease and antimicrobial resistance, it’s important to understand evolution
The novel coronavirus, SARS-CoV2, is an example of an emerging infectious disease. SARS-CoV2 is a disease that, prior to 2019, had not to the best of our knowledge infected a human being. The genetic makeup of the virus indicates that the virus is natural, originating likely as a bat or pangolin Coronavirus that acquired the ability to infect humans, and that it is not man-made (1). Why do new diseases come into existence? Why haven’t humans encountered all the diseases capable of infecting us? Furthermore, why do diseases that we had previously thought conquered have the newfound ability to harm us again, in spite of our advancements in antibiotic development?
The answer to these questions is partially answered by evolution. Several novel viruses, like SARS-CoV1, MERS, and SARS-CoV2, began as zoonosis: infection by a pathogen with an animal source. Viruses, though generally considered non-living, contain nucleic acid genomes (either RNA or DNA) similar to every other organism in the tree of life. This genome is subject to selective pressures, just as with every other nucleic-acid containing being, and mutates non-specifically (that is, an organism develops a mutation, then selective pressures have a positive, negative, or neutral effect on retaining or discarding the mutation). An animal coronavirus that recognizes surface molecules on animal cells that have some similarity to human cell surface molecules may only be a few small genome changes away from being capable of infecting humans. It is likely that SARS-CoV2 emerged in one of two ways: as either an animal virus that mutated within an animal that gained the ability to infect humans, or as an animal virus that jumped to humans, and within the human host was selected for the ability to infect humans (1). The advent of novel viruses is also facilitated by the horizontal transfer of genetic material between distinct viral lineages. In Influenza viruses, this can take the form of segments of genome being transferred wholesale between viruses. Influenza viruses contain a genome composed entirely of RNA in multiple segments of sequence. Segments “re-assort” when flu viruses of distinct lineage infect the same cell, and viral genomes are mixed during the process of producing new viruses. Alternatively, as would be the case in coronaviruses, recombination occurs through a mechanism not fully understood, where whole portions of genome are exchanged between viruses (2).
The problem of antimicrobial resistance is also best understood through evolution. To explain this phenomenon, I will describe mainly how resistance manifests in bacteria, but similar processes drive resistance to anti-virals, anti-fungals, and anti-parasitics. Antibiotics are largely derived from natural sources: as microbes compete for resources, there is a drive to reduce competitors numbers by killing them or inhibiting their growth. Antibiotics are typically small molecules that target essential processes for bacterial growth; commonly cell wall biosynthesis (preventing growth and division of the cell, an example being penicillin), protein synthesis (blocks the process of translation, an example being erythromycin), production of RNA (blocks the process of transcription, an example being rifampin) or production of DNA (blocks the process of replication, an example being fluoroquinolone). These antibiotics arose through selective pressures, and in response bacteria have developed systems to circumvent the deleterious effects of antibiotics. These include: rapidly excreting the antibiotic before it is capable of inhibiting growth (efflux pumps, a notable offender being Pseudomonas aeruginosa, a common pathogen in patients with cystic fibrosis), degrading the antibiotic (beta-lactamases are a class of enzyme that degrade beta-lactam family antibiotics, such as penicillin), modifying the antibiotic (the most common mechanism for aminoglycoside resistance is to chemically modify the antibiotic so it doesn’t work), or simply modifying the target (Streptococcus pneumoniae is a microbe that causes multiple diseases that is naturally resistant to beta-lactams by modification of the drug target, the aptly-named Penicilin-binding protein) (3). As humans, it has been beneficial to identify these natural compounds and use them medically to treat infection.
Bacteria have incredible genome plasticity, engaging in a process known as horizontal gene transfer (HGT; sometimes referred to as lateral gene transfer) that increases the prevalence of resistant microbes. Not all bacteria are capable of this set of processes, but importantly several medically important pathogens, such as E. coli, Salmonella, Yersinia pestis, Acinetobacter baumannii engage in processes that facilitate the transfer of genetic material between bacteria. There are several molecular mechanisms for HGT: bacteria-infecting viruses can transmit pieces of genetic material between similar bacteria (transduction), bacteria can form a bridge that transfers plasmids (conjugation; plasmids are typically circular pieces of DNA, and are typically maintained independently of the bacterial chromosome and commonly encode antibiotic resistance genes), or bacteria can simply pick up naked DNA in the environment and integrate that DNA into their chromosomes (natural transformation) (3). The effect of these processes is that, when a gene that imparts resistance to a particular antibiotic is introduced into a population, it may spread between members of the population, not just within the progeny of the cells that encode the resistance gene. This is especially true when a gene that imparts resistance is on a plasmid or is otherwise mobilizable (transposons, or jumping genes, are also common perpetrators of transmission in that they move somewhat readily and often encode drug resistance). The key point to understand here is that while genes are present in bacteria, either on a chromosome or on a mobilizable element, these genes are capable of moving to many other members of the same population.
To understand this in more practical terms, many people have undergone a course of antibiotics and experienced gastrointestinal distress or stomach pains. This can be attributed to disturbing your normal intestinal microbiome, as you kill off non-resistant bacteria. Now assume you have an infection of some sort, it could be anywhere in your body accessible to an orally administered antibiotic, and your doctor prescribes you an antibiotic. It is possible, and possibly probable, that within your gut are bacteria that harbor resistance genes. In the absence of the antibiotic, these are likely to have a neutral or possibly deleterious effect; think of this like a welder that is unable to remove his welding mask: it certainly helps when he is welding, but is cumbersome at other times of the day. Taking the antibiotic results in high selection for resistant microbes to grow and prosper. This allows the resistant bugs to soon outnumber the non-resistant bugs. Ultimately, this increases the concentration of the resistance genes in the population of microbes in your gut. Subsequent to this, you may encounter an infection of a gastrointestinal pathogen that, in infecting your gut, acquires the resistance genes that you selected for. In disseminating this pathogen, you are also disseminating this resistance gene. Additionally, and perhaps more importantly, in taking antibiotics you select for drug resistance in the opportunistic pathogens of your body, notably Clostridium dificile and Staphylococcus epidermidis. These microbes are capable of causing disease, but reside in you or on you and cause infection when conditions are optimal for their growth.
The problem of antimicrobial resistance is convergent with emerging pathogens, as many pathogens “re-emerge” as they develop resistance to antimicrobials. While TB cannot be said to be an emerging pathogen as the world has been experiencing a TB pandemic since at least the early 1800’s, TB is re-emerging in the since that increased drug resistance has led to strains of TB that are not treatable via the traditional course of antibiotics (4). Similarly, common pathogens such as E. coli, Klebsiella, and Clostridium dificile are bugs that have become increasingly resistant to the antibitoics used to treat them (5). Acinetobacter baumanii, a soil microbe with resistance to a spectrum of antibiotics, became a common Gulf and Iraq War wound infection. Many of these pathogens find a home in hospitals, where the use of antibiotics is prevalent and potential hosts are abundant. Furthermore, the recently emerged pathogen HIV, the causal agent of AIDS, is intersectional with that of antibiotic resistance, as infection with HIV increases susceptibility to bacterial infections due to reduced immune cell numbers; increased infection rates of Both issues, antibiotic resistance and emerging pathogens, pose a threat to human health the world over, and I will attempt to address both of these issues in this post.
The problem of emerging disease and antibiotic resistance is exacerbated by humans
To what extent do emerging diseases and antibiotic resistance affect humans? SARS-CoV2 has had an extensive impact on human health and living, and the response to shut down to stop the spread of the virus has had a large economic impact. It is impossible to accurately predict the threat posed by non-discovered viruses, so the next threat could be relatively benign, or truly horrific. This is not to fearmonger, there is no reason to suspect that such a virus is bound to steamroll us soon, but to say that the next plague may be brewing inside a pig in a Chinese farm or outside our homes in the bodies of ticks, and we would not know it. The US Center for Disease Control and Prevention (CDC) has published two Antibiotic Resistance Threat reports on the subject, in 2013 and 2019. In the 2013 edition, it was reported that 2 million people in the United States will acquire an antibiotic resistant infection, and that 23,000 will die as a direct result of that infection (5). While by 2019 this was realized to be an underestimation of the drug-resistant cases, new approaches had determined that the true value had lowered from 2013 to 2019, with an updated estimate of 2.8 million cases and 35,000 fatalities in 2019 (6). An excellent illustration of the problem can be found on page 28 of the 2013 report, which reports the introduction date (left) and the date at which resistance was observed on the right for crucial antibiotic groups. Commonly, within a decade of the introduction of an antibiotic, resistance emerges. This problem cannot be expected to go away on its own, and more than likely pathogens commonly thought vanquished will re-emerge with drug-resistant characteristics.
There are human processes that contribute to the emergence of disease and spread of antibiotic resistance. In China, Wet Markets bring together livestock from all over the country, creating an environment that is diverse in the microbial life that live commensally and parasitically in and on these animals. The proximity of these animals allows for the exchange of these microbes; these microbes are then capable of exchanging genetic material. As I described for Flu and Coronaviruses, viruses that come into contact within cells are capable of genetic recombination, a process that can result in viruses that are capable of infecting humans. This is not to say this is a common phenomenon, just that 1) the process is accelerated by live animal markets and 2) this practice and resulting genetic recombination of zoonotic viruses is thought to have contributed to both the original and novel SARS-CoV outbreaks.
In the United States, a textbook example of an emerging disease is Lyme Disease (7). Named for the town of Lyme, Connecticut, Lyme Disease is caused by the peculiar bacterium known as Borellia burgdorferi. Borellia is a corkscrew-shaped bacteria that is interesting for its ability to grow without iron (a key component of the immune response is the sequestration of iron away from pathogens). Lyme Disease is spread through ticks, and the number of infectious cases is exacerbated by reforestation and settlement close to wooded areas in suburban environments. As building projects move closer to forested areas, exposure to arthropod-borne illnesses will be expected to rise.
Beyond settlement and the wet market practice, the emergence of new infectious disease is complicated by global warming and healthcare practices. Global warming is hypothesized to drive heat resistance in fungi, potentially improving their capacity to grow within the human body (8). The pathogenic potential of fungi is hypothesized to be limited by the heat of the human body, and there is some speculation that global warming is a contributing factor to the emergence of the notorious fungal pathogen Candida auris (8). These claims should be taken with a grain of salt and evaluated critically, but it is possible that human-caused climate change will disturb the ecology of our planet with as of yet unforeseen consequences, among them the generation novel and resurgent diseases.
In healthcare, over-prescription of and a lack of regulation on antibiotics has caused the problem to worsen (5,6). When a patient receives an antibiotic, the drug has an effect on all microbes where the drug is bioavailable. This includes the intestines, which contain a resident population of microbes, and the skin, which contains Staphylococci resident species that prevent colonization by pathogenic strains of similar bacteria. These residents are then selected for their ability to resist the drug, causing an increase in resistance among the healthy microbiota. These resistance genes, as I have described, can then move between dissimilar bacteria in the same environment. If a harmful strain of E. coli is introduced into such an environment, for example, it has a higher likelihood of encountering and assimilating the genetic potential to resist antibiotics than in an environment that is naïve to the antibiotic. Patients are commonly prescribed antibiotics for infections that are more likely to be caused by a virus, or in instances where an infection is likely to run course without medical intervention. The increased exposure to antibiotics causes the microbiota to increase the concentration of resistance genes. Additionally, in places like India, the regulations on antibiotics are much more laxed than even the United States, where one is able to purchase over-the-counter antibiotics. This allows anyone to give themselves an incomplete course of antibiotics for any condition, even if the symptoms are not caused by an infection of any kind. Additionally, prescription antibiotics that have deteriorated with time, or are manufactured with subpar quality control resulting in lower concentrations, that remain in circulation exacerbate the problem by establishing sub-inhibitory concentrations of the antibiotic in the body and resulting in selection for resistance. Furthermore, environmental pollution of antibiotics into natural water sources and sewage results in increased environmental concentrations of resistance genes. These genes can spill into humans by exposure to microbes in these environments (9).
Agriculture provides another increase in the concentration of resistance genes (10). Livestock are fed antibiotics, which increase the weight of animals in an as-of-yet not understood mechanism. A deleterious consequence of this increase in yield with antibiotic usage is the increase in resistance in response to this widespread antibiotic usage. These resistance genes then find their way into humans, whether through ingestion of food contaminated with resistant microbes.
Science and technology can solve the problem, but face institutional and biological challenges
There are both institutional and scientific challenges to combating emerging disease and antibiotic resistance. Some of these problems are easily apparent as I have described above: countries with laxed restrictions on who can obtain antibiotics, countries where the drugs are used often over-prescribed, suburbanization, and global warming all contribute to the problem.
Scientifically, there are challenges in that novel diseases are difficult to combat. The novel Coronavirus had the precedent of other coronaviruses (i.e. SARS and MERS) that had been studied and their virology dissected, but that won’t necessarily be the case everytime a novel pathogen infects a human. A technological benefit to this problem is the use of meta-genomics, which allows for DNA/RNA sequencing without prior knowledge of the nucleic acid sequence of the genome. Within weeks of the first identification of the virus, its sequence was available to researchers. This was not the case during the outbreak of SARS-CoV1, when meta-genomics approaches such as Illumina Sequencing, NanoPore Sequencing, and Pacific Biosciences Sequencing were not available. In the event of a novel disease emergence, this information would be vital to combating the pathogen.
Despite not knowing necessarily what the next threat will be, expanding the human knowledge base on microbes is an essential component of any plan to fight emerging diseases. Any emerging disease is likely to be similar to other microbes that we have encountered, and knowledge of the physiology of these organisms helps to understand weaknesses, transmission, and potential therapeutic targets. The study of all microorganisms therefore benefits the effort to combat the next pandemic, as any one piece of information could be critical.
Surveillance is perhaps the most important tool to fight emerging infectious disease; knowing the problem exists is a crucial step to curbing spread. A recent example of successful surveillance can be seen in a recent PNAS publication regarding the presence of potential pandemic influenza in hogs, and the presence of antibodies against this particular class of flu viruses in swine workers (11). While at present it does not appear that the virus has acquired the ability to cause a pandemic, this knowledge allows for immunologists to potentially include viral antigens specific to this particular viral class in seasonal vaccines. Surveillance is critical in controlling both emerging diseases and antibiotic resistance: knowledge of what potential pathogens emerge where, and what microbes are exhibiting resistance to what drugs, can drive containment and treatment efforts.
To combat antibiotic resistance, new drugs must be developed, but there are hurdles in identification, validation, and production of new antibiotics. First, potential new antibiotics have to be either identified or designed. This often involves looking through filtered environmental samples to determine the presence of small molecules that inhibit bacterial growth, or chemically altering known drugs to circumvent drug resistance. This is not necessarily difficult, as there are microbes in the soil and water that produce potential therapeutics, but this does require both time and money, as well as the consideration that it is likely that resistance to that novel therapeutic exists in the environment from which it was pulled. New drugs must be safe, but due to the abundance of antibiotics presently in use and their historic efficacy, the standard for antibiotics to pass safety regulations is extremely high. As drug resistance becomes more common, it will become apparent that more and more side effects may have to be tolerated to prevent death due to bacterial infection. Finally, and the most important challenge to developing antibiotics is that the profit margin on antibiotics is low for drug companies in the present market, disincentivizing research and production of novel drugs.
In addition to stand-alone antibiotics, new inhibitors of resistance must be developed as well. Clavulanic acid is one such inhibitor, and is administered with the beta-lactam drug amoxicillin to improve its ability to kill bacteria. Bacteria that are resistant beta-lactams often encode enzymes called beta-lactamases. Beta-lactamases break open the active portion of the beta-lactam molecule, rendering it ineffective in attacking its target. Clavulanic acid is a beta-lactam itself, and is a target for the beta-lactamase enzyme; however, when the enzyme begins to degrade clavulanic acid, the enzyme becomes stuck at an intermediate step in the reaction, rendering the beta-lactamase enzyme useless. These drugs must also be explored and screened for in environmental samples, as well as developed. It is possible to take a rational approach to drug design, with increasing knowledge of how resistance mechanisms work. This means that scientists specifically look at, say, a beta-lactamase enzyme at the molecular level, and design a small molecule that will fit into the enzyme and block its function. Chemists then design the molecule to test its efficacy.
Ultimately, scientists either know how to solve the problem, or know how to get the tools they need to solve the problem. It is the institutional challenges that make the problem more difficult to solve.
How legislation can improve the ability of scientists to combat emerging disease and drug resistance
In discussing emerging diseases and antibiotic resistance, I try to draw parallels to the problem of global warming: a global problem with global solutions. I don’t have a novel solution to climate change to discuss here, other than to parrot this subreddit’s typical ideas, so I will omit that discussion here. That is to say, global warming is a driver for emerging infectious disease, and fighting global warming is important to combat the potential rise of fungal pathogens. I will, however, discuss some ideas for combating emerging disease and drug resistance. These ideas are mostly derived from scientists familiar with the problem,
Funding for research, basic and applied, is crucial. No bit of knowledge hurts in the fight against human disease. Learning how Alphaviruses replicate, determining the structure of E. coli outer membrane proteins, and examining the life cycle of the non-pathogen Caulobacter crescentus all contribute to the fight against the next disease. The more we know, the more powerful our vision is in understanding the inner machinations of disease. Every immune response, every molecular mechanism, and every aspect of microbial physiology is potentially a drug or vaccine target, a clue into pathogenesis, or an indication of how a bug is likely to spread. The Trump administration has not been kind to science funding (12). Science that does not appear to have benefit at first glance often does in the long run, and for this reason I will stress the importance of funding research of this sort, as well as funding applied research.
Knowing is half the battle. In combating emerging diseases, it is important to know they exist. As I have mentioned the example of recent viral surveillance with regard to the novel reassortment influenza viruses, I would like to stress the importance of funding surveillance programs in fighting emerging disease and drug resistance. There are currently US governmental surveillance programs that provide valuable information about the spread of drug resistance, such as NARMS in the United States (13).
In the United States, there is a need for greater accountability in using antibiotics. Resistance is unlikely to completely go away, even when the use of an antibiotic is discontinued, but the levels of resistant bacteria dwindle when the selective pressure is reduced. For this reason, several medical practitioners have proposed a rotating schedule of prescription antibiotics, that includes the retention of some new antibiotics from use. The reasoning for this is that, in the years following the halted use of a particular antibiotic, it is expected that the concentration of resistant bacteria will decrease. As I discussed with the example of always wearing a welding helmet, carrying resistance genes often imparts some form of growth defect on the resistant bacteria (for example, altering an essential gene targeted by an antibiotic may render the bacteria resistant, but there is a reason such a gene is essential, in that it’s required for growth; changing the gene in a substantive way may negatively impact its performance and by extension make these resistant bacteria less fit). A rotating cycle of what antibiotics are allowed to be prescribed, informed by surveillance data, would buy time for the development of new antibiotics as well. Additionally, higher standards should be required for the prescription of antibiotics, to increase accountability of physicians; these standards could involve clinically verifying the presence of susceptible bacteria prior to administering a drug in situations where the disease in not life-threatening.
There is a need to reduce the environmental pollution of drugs into sewage and natural bodies of water as well. This will require research into cost-effective methods for reducing the population of resistant bugs and drugs in these environments. In the case of natural bodies of water, a source of contamination is often factories where drugs are produced. Often, waters near these factories have high levels of antibiotics that select for resistance to develop and spread. This may require legislation to improve environmental outcomes, as well as surveillance of drug resistance gene levels and the levels of antibiotics in these waters to ensure compliance.
There is also a need to halt the use of antibiotics in treating livestock (14). Halting the use of antibiotics typically results in reductions of antibiotic resistant bug populations within a year or two (10). I don’t know of studies that estimate the economic cost of halting use of antibiotics in American meat, but in the case of Denmark, livestock production does not appear to have been significantly impacted.
I think that the most challenging problem will be for drug companies to develop new antibiotics when there is not presently a financial incentive to do so. Because antibiotics are still largely effective, and the financial benefit to adding an antibiotic to the market does not outweigh the cost to put a drug to market, there is not currently a large incentive to produce new drugs (15). To address this negative externality, it is necessary to generate financial incentives of some form for the production of new antibiotics. This may take the form of subsidizing antibiotic discovery efforts and drug safety trials; additionally, applied research with the goal of specifically finding new antibiotics should see increased funding.
To combat the problem overseas, it is obvious that obtaining an antibiotic course must occur through a doctor. This eliminates false self-diagnoses of bacterial infections. The problem of wet markets may be partially resolved by preventing animals that do not regularly contact each other from being traded and stored in the same vicinity as animals that are not typically encountered. This may involve limiting a particular wet market to the trade of animals that come from a particular geographic area, preventing geographically diverse microbes from encountering each other.
It's on all of us to stop the next pandemic:
If you made it this far, thank you reading this post and I hope that I have convinced you of the importance of this issue! There are simple steps that we can all take as consumers to reduce antimicrobial resistance: don’t take antibiotics unless prescribed by a doctor and buy meat that was produced without antibiotics. I welcome any and all criticism, and would love to hear people's ideas! Please let me know of any errors as well, or any missed concepts that I glossed over. I've been excited to give my two cents to this sub, and I don't want to mislead in any way.
Sources:
1: Andersen, KG, et al. 2020. The Proximal Origin of Sars-CoV-2. Nature Medicine 26: 450-452.
2: Su, Shou, et al. 2016. Epidemiology, Genetic Recombination, and Pathogenesis of Coronaviruses. Cell Trends in Microbiology 24(6): 490-502. https://doi.org/10.1016/j.tim.2016.03.003
3: Munita, JM; Arias, CA. 2016. Mechanisms of Antibiotic Resistance. Microbiology Spectrum VMBF-0016-2015. doi:10.1128 /microbiolspec.VMBF-0016-2015.
4: Shah, NS; et al. 2007. Worldwide Emergence of Extensively Drug-resistant Tuberculosis. Emerging Infectious Diseases 13(3): 380-387. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2725916/
5: CDC Antibiotic Threats Report, 2013. https://www.cdc.gov/drugresistance/pdf/ar-threats-2013-508.pdf
6: CDC Antibiotic Threats Report, 2019. https://www.cdc.gov/drugresistance/pdf/threats-report/2019-ar-threats-report-508.pdf
7: Barbour, AG; Fish, D. 1993. The Biological and Social Phenomenon of Lyme Disease. Science 260(5114):1610-1616. https://pubmed.ncbi.nlm.nih.gov/8503006/
8: Casadevall, A; Kontoyiannis, DP; Robert, V. 2019. On the Emergence of Candida auris: Climate Change, Azoles, Swamps, and Birds. mBio 10.1128/mBio.01397-19. https://mbio.asm.org/content/10/4/e01397-19
9: Kraemer, SA; Ramachandran, A; Perron, GG. 2019. Antibiotic Pollution in the Environment: From Microbial Ecology to Public Policy. Microorgansims 7(6): 180. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6616856/
10: Levy, S. 2014. Reduced Antibiotic Use in Livestock: How Denmark Tackled Resistance. Environmental Health Perspectives 122(6): A160-A165. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4050507/
11: Sun, H, et al. 2020. Prevalent Eurasian avian-like H1N1 swine influenza virus with 2009 pandemic viral genes facilitating human infection. Proceedings of the National Academy of Science https://doi.org/10.1073/pnas.1921186117.
12: Kaiser, J. 2020. National Institutes of Health would see 7% cut in 2021 under White House plan. Science Magazine. https://www.sciencemag.org/news/2020/02/national-institutes-health-would-see-7-cut-2021-under-white-house-plan
13: About NARMS: National Antimicrobial Resistance Monitoring System for Enteric Bacteria. https://www.cdc.gov/narms/about/index.html
14: Khachatourians, GG. 1998. Agricultural use of antibiotics and the evolution and transfer of antibiotic-resistant bacteria. CMAJ 159(9):1129-1136 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1229782/
15: Jacobs, Andrew. 2019. Crisis Looms in Antibiotics as Drug Makers Go Bankrupt. The New York Times. https://nyti.ms/366f7it

1) Chemistry of Life

Content

Transpiration
- Hydrogen bonds pull water up like string and leave through stoma
- Stomata: leaf pores that allow gas exchange, most are on bottom side of leaf
- Xylem: tube-shaped, nonlining, vascular system, carries water from roots to rest of plant
- Epidermis: outer layer, protects plant
- Phloem: transports food
- Parenchyma: stores food
- Transpiration: evaporation of water from leaves
- Adhesion: polar water molecules adhere to polar surfaces (sides of xylem)
- Cohesion: polar water molecules adhere to each other
- Guard cells: cells surrounding stoma, regulate transpiration through opening and closing stoma
- Turgid vs flaccid guard cells
  - Turgid swell caused by potassium ions, water potential decreases, water enters vacuoles of guard cells
  - Swelling of guard cells open stomata
- High light levels, high levels of water, low temperature, low CO2 causes opening of stomata
- Water potential: transport of water in plant governed by differences in water potential
  - Affected by solute concentration and environmental conditions
- High water potential (high free energy and more water) travels to low water potential
- Hydrophilic = attracts water, hydrophobic = repels water
Water and its Properties
- Polar molecule due to positive hydrogen and negative oxygen regions
- Negative oxygen of one molecule to positive hydrogen of another water molecule forms a hydrogen bond, which are weak individually but strong together
- Important physical properties of water:
  - Cohesion and adhesion: cohesion creates surface tension and they both allow for transpiration
  - High specific heat: enables water to absorb and lose heat slowly
  - High heat of vaporization: allows much of it to remain liquid
  - Nearly universal polar solvent: dissolves a lot of stuff
  - Flotation of ice: insulates, transportation
Biological Macromolecules
- Polymer: long molecule consisting of many similar building blocks linked by covalent bonds
- Monomer: building block of a polymer
- ATP - adenosine triphosphate, energy carrier that uses bonds between phosphates to store energy
  - Similar in structure to a ribonucleotide
- Four Types
  - Carbohydrates
  - Lipids
  - Proteins
  - Nucleic Acids

https://preview.redd.it/xp12oli61w451.png?width=1098&format=png&auto=webp&s=cc897738989258c67bcc760ba040e2cee8f7875c

Functional groups
- Hydroxyl - carbs, alcohols - OH-, O-
- Amino - proteins - NH2, NH3+
- Carboxyl - weak acids - COOH, COO-
- Sulfhydryl - proteins - SH
- Phosphatic - salts, strong acids - PO
Directionality:
- ex: glucose alpha and beta
- ex: DNA and RNA 5’ and 3’ ends
Identification of Macromolecules

https://preview.redd.it/cb3oau2j1w451.png?width=1089&format=png&auto=webp&s=409e26f32c9996a3649bad81d17ed72769955ce9

Calculations

Number of bonds
- # of molecules - 1
- i.e. 20 glucose molecules linked together would have 19 bonds
Molecular formula
- # of molecules * molecular formula - number of bonds * H20 (from hydrolysis)
- i.e. when you bond 5 glucose molecules together you have to subtract 4H2O
pH/pOH
- -log[H+] = pH
- -log[OH-] = pOH
- pH + pOH = 14
Leaf surface area
- i.e. using graph paper to find surface area
Transpiration rate
- Amount of water used / surface area / time

Labs

Transpiration Lab
- Basically you take this potometer which measures the amount of water that gets sucked up by a plant that you have and you expose the plant to different environmental conditions (light, humidity, temperature) and see how fast the water gets transpired
- Random stuff to know:
  - It’s hard to get it to work properly
  - A tight seal of vaseline keeps everything tidy and prevents water from evaporating straight from the tube, also allows for plant to suck properly
  - Water travels from high water potential to low water potential

2) Cell Structure & Function

Content

Cellular Components
- Many membrane-bound organelles evolved from once free prokaryotes via endosymbiosis, such as mitochondria (individual DNA)
- Compartmentalization allows for better SA:V ratio and helps regulate cellular processes
- Cytoplasm: thick solution in each cell containing water, salts, proteins, etc; everything - nucleus
  - Cytoplasmic streaming: moving all the organelles around to give them nutrients, speeds up reactions
- Cytosol: liquid of the cytoplasm (mostly water)
- Plasma Membrane: separates inside of cell from extracellular space, controls what passes through amphipathic area (selectively permeable)
  - Fluid-Mosaic model: phospholipid bilayer + embedded proteins
  - Aquaporin: hole in membrane that allows water through
- Cell Wall: rigid polysaccharide layer outside of plasma membrane in plants/fungi/bacteria
  - Bacteria have peptidoglycan, fungi have chitin, and plants have cellulose and lignin
  - Turgor pressure pushes the membrane against the wall
- Nucleus: contains genetic information
  - Has a double membrane called the nuclear envelope with pores
- Nucleolus: in nucleus, produces ribosomes
- Chromosomes: contain DNA
- Centrioles: tubulin thing that makes up centrosome in the middle of a chromosome
- Smooth Endoplasmic Reticulum: storage of proteins and lipids
- Rough Endoplasmic Reticulum: synthesizes and packages proteins
- Chloroplasts: photosynthetic, sunlight transferred into chemical energy and sugars
  - More on this in photosynthesis
- Vacuoles: storage, waste breakdown, hydrolysis of macromolecules, plant growth
- Plasmodesmata: channels through cell walls that connect adjacent cells
- Golgi Apparatus: extracellular transport
- Lysosome: degradation and waste management
  - Mutations in the lysosome cause the cell to swell with unwanted molecules and the cell will slow down or kill itself
- Mitochondria: powerhouse of the cell
  - Mutations in the mitochondria cause a lack of deficiency of energy in the cell leading to an inhibition of cell growth
- Vesicles: transport of intracellular materials
- Microtubules: tubulin, stiff, mitosis, cell transport, motor proteins
- Microfilaments: actin, flexible, cell movement
- Flagella: one big swim time
- Cilia: many small swim time
- Peroxisomes: bunch of enzymes in a package that degrade H202 with catalase
- Ribosomes: protein synthesis
- Microvilli: projections that increase cell surface area like tiny feetsies
  - In the intestine, for example, microvilli allow more SA to absorb nutrients
- Cytoskeleton: hold cell shape
Cellular Transport
- Passive transport: diffusion
  - Cell membranes selectively permeable (large and charged repelled)
  - Tonicity: osmotic (water) pressure gradient
- Cells are small to optimize surface area to volume ratio, improving diffusion
- Primary active transport: ATP directly utilized to transport
- Secondary active transport: something is transported using energy captured from movement of other substance flowing down the concentration gradient
- Endocytosis: large particles enter a cell by membrane engulfment
  - Phagocytosis: “cell eating”, uses pseudopodia around solids and packages it within a membrane
  - Pinocytosis: “cell drinking”, consumes droplets of extracellular fluid
  - Receptor-mediated endocytosis: type of pinocytosis for bulk quantities of specific substances
- Exocytosis: internal vesicles fuse with the plasma membrane and secrete large molecules out of the cell
- Ion channels and the sodium potassium pump
  - Ion channel: facilitated diffusion channel that allows specific molecules through
  - Sodium potassium pump: uses charged ions (sodium and potassium)
- Membrane potential: voltage across a membrane
- Electrogenic pump: transport protein that generates voltage across a membrane
- Proton pump: transports protons out of the cell (plants/fungi/bacteria)
- Cotransport: single ATP-powered pump transports a specific solute that can drive the active transport of several other solutes
- Bulk flow: one-way movement of fluids brought about by pressure
- Dialysis: diffusion of solutes across a selective membrane
Cellular Components Expanded: The Endomembrane System
- Nucleus + Rough ER + Golgi Bodies
  - Membrane and secretory proteins are synthesized in the rough endoplasmic reticulum, vesicles with the integral protein fuse with the cis face of the Golgi apparatus, modified in Golgi, exits as an integral membrane protein of the vesicles that bud from the Golgi’s trans face, protein becomes an integral portion of that cell membrane

Calculations

Surface area to volume ratio of a shape (usually a cube)
U-Shaped Tube (where is the water traveling)
- Solution in u-shaped tube separated by semi-permeable membrane
- find average of solute (that is able to move across semi permeable membrane)
- add up total molar concentration on both sides
- water travels where concentration is higher
Water Potential = Pressure Potential + Solute Potential
- Solute Potential = -iCRT
  - i = # of particles the molecule will make in water
  - C = molar concentration
  - R = pressure constant (0.0831)
  - T = temperature in kelvin

Labs

Diffusion and Osmosis
- Testing the concentration of a solution with known solutions
- Dialysis bag
  - Semipermeable bag that allows the water to pass through but not the solute
- Potato core
  - Has a bunch of solutes inside

Relevant Experiments

Lynne Margolis: endosymbiotic theory (mitochondria lady)
Chargaff: measured A/G/T/C in everything (used UV chromatography)
Franklin + Watson and Crick: discovered structure of DNA; Franklin helped with x ray chromatography

3) Cellular Energetics

Content

Reactions and Thermodynamics
- Baseline: used to establish standard for chemical reaction
- Catalyst: speeds up a reaction (enzymes are biological catalysts)
- Exergonic: energy is released
- Endergonic: energy is consumed
- Coupled reactions: energy lost/released from exergonic reaction is used in endergonic one
- Laws of Thermodynamics:
  - First Law: energy cannot be created nor destroyed, and the sum of energy in the universe is constant
  - Second Law: energy transfer leads to less organization (greater entropy)
  - Third Law: the disorder (entropy) approaches a constant value as the temperature approaches 0
- Cellular processes that release energy may be coupled with other cellular processes
- Loss of energy flow means death
- Energy related pathways in biological systems are sequential to allow for a more controlled/efficient transfer of energy (product of one metabolic pathway is reactant for another)
- Bioenergetics: study of how energy is transferred between living things
- Fuel + 02 = CO2 + H20
  - Combustion, Photosynthesis, Cellular Respiration (with slight differences in energy)
Enzymes
- Speed up chemical processes by lowering activation energy
- Structure determines function
- Active sites are selective
- Enzymes are typically tertiary- or quaternary-level proteins
- Catabolic: break down / proteases and are exergonic
- Anabolic: build up and are endergonic
- Enzymes do not change energy levels
- Substrate: targeted molecules in enzymatic
- Many enzymes named by ending substrate in “-ase”
- Enzymes form temporary substrate-enzyme complexes
- Enzymes remain unaffected by the reaction they catalyze
- Enzymes can’t change a reaction or make other reactions occur
- Induced fit: enzyme has to change its shape slightly to accommodate the substrate
- Cofactor: factor that help enzymes catalyze reactions (org or inorg)
  - Examples: temp, pH, relative ratio of enzyme and substrate
  - Organic cofactors are called coenzymes
- Denaturation: enzymes damaged by heat or pH
- Regulation: protein’s function at one site is affected by the binding of regulatory molecule to a separate site
- Enzymes enable cells to achieve dynamic metabolism - undergo multiple metabolic processes at once
- Cannot make an endergonic reaction exergonic
- Steps to substrates becoming products
  - Substrates enters active site, enzyme changes shape
  - Substrates held in active site by weak interactions (i.e. hydrogen bonds)
  - Substrates converted to product
  - Product released
  - Active site available for more substrate
- Rate of enzymatic reaction increases with temperature but too hot means denaturation
- Inhibitors fill the active site of enzymes
  - Some are permanent, some are temporary
  - Competitive: block substrates from their active sites
  - Non competitive (allosteric): bind to different part of enzyme, changing the shape of the active site
- Allosteric regulation: regulatory molecules interact with enzymes to stimulate or inhibit activity
- Enzyme denaturation can be reversible
Cellular Respiration
- Steps
  - Glycolysis
  - Acetyl co-A reactions
  - Krebs / citric acid cycle
  - Oxidative phosphorylation
- Brown fat: cells use less efficient energy production method to make heat
- Hemoglobin (transport, fetal oxygen affinity > maternal) and myoglobin (stores oxygen)
Photosynthesis
- 6CO2 + 6H20 + Light = C6H12O6 + 6O2
- Absorption vs action spectrum (broader, cumulative, overall rate of photosynthesis)
- Components
  - Chloroplast
  - Mesophyll: interior leaf tissue that contains chloroplasts
  - Pigment: substance that absorbs light
- Steps
  - Light-Dependent Reaction
  - Light-Independent (Dark) Reaction (Calvin Cycle)
Anaerobic Respiration (Fermentation)
- Glycolysis yields 2ATP + 2NADH + 2 Pyruvate
- 2NADH + 2 Pyruvate yields ethanol and lactate
- Regenerates NAD+

Calculations

Calculate products of photosynthesis & cellular respiration

Labs

Enzyme Lab
- Peroxidase breaks down peroxides which yields oxygen gas, quantity measured with a dye
- Changing variables (i.e. temperature) yields different amounts of oxygen
Photosynthesis Lab
- Vacuum in a syringe pulls the oxygen out of leaf disks, no oxygen causes them to sink in bicarbonate solution, bicarbonate is added to give the disks a carbon source for photosynthesis which occurs at different rates under different conditions, making the disks buoyant
Cellular Respiration Lab
- Use a respirometer to measure the consumption of oxygen (submerge it in water)
- You put cricket/animal in the box that will perform cellular respiration
- You put KOH in the box with cricket to absorb the carbon dioxide (product of cellular respiration)-- it will form a solid and not impact your results

Relevant Experiments

Engelmann
- Absorption spectra dude with aerobic bacteria

4) Cell Communication & Cell Cycle

Content

Cell Signalling
- Quorum sensing: chemical signaling between bacteria
  - See Bonnie Bassler video
- Taxis/Kinesis: movement of an organism in response to a stimulus (chemotaxis is response to chemical)
- Ligand: signalling molecule
- Receptor: ligands bind to elicit a response
- Hydrophobic: cholesterol and other such molecules can diffuse across the plasma membrane
- Hydrophilic: ligand-gated ion channels, catalytic receptors, G-protein receptor
Signal Transduction
- Process by which an extracellular signal is transmitted to inside of cell
- Pathway components
  - Signal/Ligand
  - Receptor protein
  - Relay molecules: second messengers and the phosphorylation cascade
  - DNA response
- Proteins in signal transduction can cause cancer if activated too much (tumor)
  - RAS: second messenger for growth factor-- suppressed by p53 gene (p53 is protein made by gene) if it gets too much
- Response types
  - Gene expression changes
  - Cell function
  - Alter phenotype
  - Apoptosis- programmed cell death
  - Cell growth
  - Secretion of various molecules
- Mutations in proteins can cause effects downstream
- Pathways are similar and many bacteria emit the same chemical within pathways, evolution!
Feedback
- Positive feedback amplifies responses
  - Onset of childbirth, lactation, fruit ripening
- Negative feedback regulates response
  - Blood sugar (insulin goes down when glucagon goes up), body temperature
Cell cycle
- Caused by reproduction, growth, and tissue renewal
- Checkpoint: control point that triggers/coordinates events in cell cycle
- Mitotic spindle: microtubules and associated proteins
  - Cytoskeleton partially disassembles to provide the material to make the spindle
  - Elongates with tubulin
  - Shortens by dropping subunits
  - Aster: radial array of short microtubules
  - Kinetochores on centrosome help microtubules to attach to chromosomes
- IPMAT: interphase, prophase, metaphase, anaphase, telophase
  - PMAT is mitotic cycle
- Steps
  - Interphase
  - Mitosis
  - Cytokinesis
- Checkpoints
  - 3 major ones during cell cycle:
  - cyclin-cdk-mpf: cyclin dependent kinase mitosis promoting factor
  - Anchorage dependence: attached, very important aspect to cancer
  - Density dependence: grow to a certain size, can’t hurt organs
  - Genes can suppress tumors
- G0 phase is when cells don’t grow at all (nerve, muscle, and liver cells)

Calculations

Chi-squared

Relevant Experiments

Sutherland
- Broke apart liver cells and realized the significance of the signal transduction pathway, as the membrane and the cytoplasm can’t activate glycogen phosphorylase by themselves

5) Heredity

Content

Types of reproduction
- Sexual: two parents, mitosis/meiosis, genetic variation/diversity (and thus higher likelihood of survival in a changing environment)
- Asexual: doesn’t require mate, rapid, almost genetically identitical (mutations)
  - Binary fission (bacteria)
  - Budding (yeast cells)
  - Fragmentation (plants and sponges)
  - Regeneration (starfish, newts, etc.)
Meiosis
- One diploid parent cell undergoes two rounds of cell division to produce up to four haploid genetically varied cells
- n = 23 in humans, where n is the number of unique chromosomes
- Meiosis I
  - Prophase: synapsis (two chromosome sets come together to form tetrad), chromosomes line up with homologs, crossing over
  - Metaphase: tetrads line up at metaphase plate, random alignment
  - Anaphase: tetrad separation, formation at opposite poles, homologs separate with their centromeres intact
  - Telophase: nuclear membrane forms, two haploid daughter cells form
- Meiosis II
  - Prophase: chromosomes condense
  - Metaphase: chromosomes line up single file, not pairs, on the metaphase plate
  - Anaphase: chromosomes split at centromere
  - Telophase: nuclear membrane forms and 4 total haploid cells are produced
- Genetic variation
  - Crossing over: homologous chromosomes swap genetic material
  - Independent assortment: homologous chromosomes line up randomly
  - Random fertilization: random sperm and random egg interact
- Gametogenesis
  - Spermatogenesis: sperm production
  - Oogenesis: egg cells production (¼ of them degenerate)
Fundamentals of Heredity
- Traits: expressed characteristics
- Gene: “chunk” of DNA that codes for a specific trait
- Homologous chromosomes: two copies of a gene
- Alleles: copies of chromosome may differ bc of crossing over
- Homozygous/Heterozygous: identical/different
- Phenotype: physical representation of genotype
- Generations
  - Parent or P1
  - Filial or F1
  - F2
- Law of dominance: one trait masks the other one
  - Complete: one trait completely covers the other one
  - Incomplete: traits are both expressed
  - Codominance: traits combine
- Law of segregation (Mendel): each gamete gets one copy of a gene
- Law of independent assortment (Mendel): traits segregate independently from one another
- Locus: location of gene on chromosome
- Linked genes: located on the same chromosome, loci less than 50 cM apart
- Gene maps and linkage maps
- Nondisjunction: inability of chromosomes to separate (ex down syndrome)
- Polygenic: many genes influence one phenotype
- Pleiotropic: one gene influences many phenotypes
- Epistasis: one gene affects another gene
- Mitochondrial and chloroplast DNA is inherited maternally
Diseases/Disorders
- Genetic:
  - Tay-Sachs: can’t break down specific lipid in brain
  - Sickle cell anemia: misshapen RBCs
  - Color blindness
  - Hemophilia: lack of clotting factors
- Chromosomal:
  - Turner: only one X chromosome
  - Klinefelter: XXY chromosomes
  - Down syndrome (trisomy 21): nondisjunction
Crosses
- Sex-linked stuff
- Blood type
- Barr bodies: in women, two X chromosomes; different chromosomes expressed in different parts of the body, thus creating two different phenotype expressions in different places

Calculations

Pedigree/Punnett Square
Recombination stuff
- Recombination rate = # of recombinable offspring/ total offspring (times 100) units: map units

Relevant Experiments

Mendel

6) Gene Expression and Regulation

Content

DNA and RNA Structure
- Prokaryotic organisms typically have circular chromosomes
- Plasmids = extrachromosomal circular DNA molecules
- Purines (G, A) are double-ringed while pyrimidines (C, T, U) have single ring
- Types of RNA:
  - mRNA - (mature) messenger RNA (polypeptide production)
  - tRNA - transfer RNA (polypeptide production)
  - rRNA - ribosomal RNA (polypeptide production)
  - snRNA - small nuclear RNA (bound to snRNPs - small nuclear ribonucleoproteins)
  - miRNA - microRNA (regulatory)
DNA Replication
- Steps:
  - Helicase opens up the DNA at the replication fork.
  - Single-strand binding proteins coat the DNA around the replication fork to prevent rewinding of the DNA.
  - Topoisomerase works at the region ahead of the replication fork to prevent supercoiling.
  - Primase synthesizes RNA primers complementary to the DNA strand.
  - DNA polymerase III extends the primers, adding on to the 3' end, to make the bulk of the new DNA.
  - RNA primers are removed and replaced with DNA by DNA polymerase I.
  - The gaps between DNA fragments are sealed by DNA ligase.
Protein Synthesis
- 61 codons code for amino acids, 3 code as STOP - UAA, UAG, UGA - 64 total
- Transcription Steps:
  - RNA polymerase binds to promoter (before gene) and separate the DNA strands
  - RNA polymerase fashions a complementary RNA strand from a DNA strand
  - Coding strand is same as RNA being made, template strand is complementary
  - Terminator on gene releases the RNA polymerase
- RNA Processing Steps (Eukaryotes):
  - 5’ cap and 3’ (poly-A tail, poly A polymerase) tail is added to strand (guanyl transferase)
  - Splicing of the RNA occurs in which introns are removed and exons are added by spliceosome
  - Cap/tail adds stability, splicing makes the correct sequence (“gibberish”)
- Translation Steps:
  - Initiation complex is the set up of a ribosome around the beginning of an mRNA fragment
  - tRNA binds to codon, amino acid is linked to other amino acid
  - mRNA is shifted over one codon (5’ to 3’)
  - Stop codon releases mRNA
Gene Expression
- Translation of mRNA to a polypeptide occurs on ribosomes in the cytoplasm as well as rough ER
- Translation of the mRNA occurs during transcription in prokaryotes
- Genetic info in retroviruses is an exception to normal laws: RNA to DNA is possible with reverse transcriptase, which allows the virus to integrate into the host’s DNA
- Regulatory sequences = stretches of DNA that interact with regulatory proteins to control transcription
- Epigenetic changes can affect expression via mods of DNA or histones
- Observable cell differentiation results from the expression of genes for tissue-specific proteins
- Induction of transcription factors during dev results in gene expression
- Prokaryotes: operons transcribed in a single mRNA molecule, inducible system
- Eukaryotes: groups of genes may be influenced by the same transcription factors to coordinate expression
- Promoters = DNA sequences that RNA polymerase can latch onto to initiate
- Negative regulators inhibit gene expression by binding to DNA and blocking transcription
- Acetylation (add acetyl groups)- more loosely wound/ less tightly coiled/compressed
- Methylation of DNA (add methyl groups) - less transcription- more tightly wound
Mutation and Genetic Variation
- Disruptions in genes (mutations) change phenotypes
- Mutations can be +/-/neutral based on their effects that are conferred by the protein formed - environmental context
- Errors in DNA replication or repair as well as external factors such as radiation or chemical exposure cause them
- Mutations are the primary source of genetic variation
- Horizontal acquisition in prokaryotes - transformation (uptake of naked DNA), transduction (viral DNA transmission), conjugation (cell-cell DNA transfer), and transposition (DNA moved within/between molecules) - increase variation
- Related viruses can (re)combine genetic material in the same host cell
- Types of mutations: frameshift, deletion, insertion
Genetic Engineering
- Electrophoresis separates molecules by size and charge
- PCR magnifies DNA fragments
- Bacterial transformation introduces DNA into bacterial cells
Operons
- Almost always prokaryotic
- Promoter region has operator in it
- Structural genes follow promoter
- Terminator ends operon
- Regulatory protein is active repressor
- Active repressor can be inactivated
- Enhancer: remote gene that require activators
- RNAi: interference with miRNA
- Anabolic pathways are normally on and catabolic pathways are normally off

Calculations

Transformation efficiency (colonies/DNA)
Numbers of base pairs (fragment lengths)
Cutting enzymes in a plasmid or something (finding the lengths of each section)

Labs

Gel Electrophoresis Lab
- Phosphates in DNA make it negative (even though it’s an acid!), so it moves to positive terminal on the board
- Smaller DNA is quicc, compare it to a standard to calculate approx. lengths
Bacterial Transformation Lab
- Purpose of sugar: arabinose is a promoter which controls the GFP in transformed cells, turns it on, also green under UV
- Purpose of flipping upside down: condensation forms but doesn’t drip down
- Purpose of heat shock: increases bacterial uptake of foreign DNA
- Plasmids have GFP (green fluorescent protein) and ampicillin resistance genes
- Calcium solution puts holes in bacteria to allow for uptake of plasmids
PCR Lab
- DNA + primers + nucleotides + DNA polymerase in a specialized PCR tube in a thermal cycler
- Primers bind to DNA before it can repair itself, DNA polymerase binds to the primers and begins replication
- After 30 cycles, there are billions of target sequences

Relevant Experiments

Avery: harmful + harmless bacteria in mice, experimented with proteins vs DNA of bacteria
Griffith: Avery’s w/o DNA vs protein
Hershey and Chase: radioactively labeled DNA and protein
Melson and Stahl: isotopic nitrogen in bacteria, looked for cons/semi/dispersive DNA
Beadle and Tatum: changed medium’s amino acid components to find that a metabolic pathway was responsible for turning specific proteins into other proteins, “one gene one enzyme”
Nirenberg: discovered codon table

7) Natural Selection

Scientific Theory: no refuting evidence (observation + experimentation), time, explain a brand/extensive range of phenomena
Theory of Natural Selection
- Definition
  - Not all offspring (in a population) will survive
  - Variation among individuals in a population
  - Some variations were more favourable than others in a particular environment
  - Those with more favourable variations were more likely to survive and reproduce.
  - These favourable variations were passed on and increased in frequency over time.
Types of Selection:
- Directional selection: one phenotype favored at one of the extremes of the normal distribution
  - ”Weeds out” one phenotype
  - Ony can happen if a favored allele is already present
- Stabilizing Selection: Organisms within a population are eliminated with extreme traits
  - Favors “average” or medium traits
  - Ex. big head causes a difficult delivery; small had causes health deficits
- Disruptive Selection: favors both extremes and selects against common traits
  - Ex. sexual selection (seems like directional but it’s not because it only affects one sex, if graph is only males then directional)
Competition for limited resources results in differential survival, favourable phenotypes are more likely to survive and produce more offspring, thus passing traits to subsequent generations.
- Biotic and abiotic environments can be more or less stable/fluctuating, and this affects the rate and direction of evolution
  - Convergent evolution occurs when similar selective pressures result in similar phenotypic adaptations in different populations or species.
  - Divergent evolution: groups from common ancestor evolve, homology
  - Different genetic variations can be selected in each generation.
  - Environments change and apply selective pressures to populations.
- Evolutionary fitness is measured by reproductive success.
- Natural selection acts on phenotypic variations in populations.
  - Some phenotypic variations significantly increase or decrease the fitness of the organism in particular environments.
- Through artificial selection, humans affect variation in other species.
  - Humans choose to cause artificial selection with specific traits, accidental selection caused by humans is not artificial
- Random occurrences
  - Mutation
  - Genetic drift - change in existing allele frequency
  - Migration
- Reduction of genetic variation within a given population can increase the differences between populations of the same species.
- Conditions for a population or an allele to be in Hardy-Weinberg equilibrium are
  - Large population size
  - Absence of migration
  - No net mutations
  - Random mating
  - Absence of selection
- Changes in allele frequencies provide evidence for the occurrence of evolution in a population.
- Small populations are more susceptible to random environmental impact than large populations.
- Gene flow: transference of genes/alleles between populations
Speciation: one species splits off into multiple species
- Sympatric (living together i.e. disruption) Allopatric (physically separate, i.e. founder effect) Parapatric (habitats overlapping)
  - Polyploidy (autopolyploidy), sexual selection
- Species: group of populations whose members can interbreed and produce healthy, fertile offspring but can’t breed with other species (ex. a horse and donkey can produce a mule but a mule is nonviable, so it doesn’t qualify)
  - Morphological definition: body shape and structural characteristics define a species
  - Ecological species definition: way populations interact with their environments define a species
  - Phylogenetic species definition: smallest group that shares a common ancestor is a species
- Prezygotic barriers: barriers to reproduction before zygote is formed
  - Geographical error: two organisms are in different areas
  - Behavioural error (i.e. mating rituals aren’t the same)
  - Mechanical error: “the pieces don’t fit together”
  - Temporal error (i.e. one organism comes out at night while the other comes out in the day)
  - Zygotic/Gametic isolation: sperm and egg don’t physically meet
- Postzygotic barriers: barriers to reproduction after zygote is formed
  - Hybrid viability: developmental errors of offspring
  - Hybrid fertility: organism is sterilized
  - Hybrid breakdown: offspring over generations aren’t healthy
- Hybrid zone: region in which members of different species meet and mate
  - Reinforcement: hybrids less fit than parents, die off, strength prezygotic barriers
  - Fusion: two species may merge into one population
  - Stability: stable hybrid zones mean hybrids are more fit than parents, thus creating a stable population, but can be selected against in hybrid zones as well
- Punctuated equilibria: long periods of no or little change evolutionarily punctuated by short periods of large change, gradualism is just slow evolution
- Evidence of evolution
  - Paleontology (Fossils)
  - Comparative Anatomy
  - Embryology: embryos look the same as they grow
  - Biogeography: distribution of flora and fauna in the environment (pangea!)
  - Biochemical: DNA and proteins and stuff, also glycolysis
- Phylogenetic trees
  - Monophyletic: common ancestor and all descendants
  - Polyphyletic: descendants with different ancestors
  - Paraphyletic: leaving specifies out of group
- Out group: basal taxon, doesn’t have traits others do
- Cline: graded variation within species (i.e. different stem heights based on altitude)
- Anagenesis: one species turning into another species
- Cladogenesis: one species turning into multiple species
- Taxon: classification/grouping
- Clade: group of species with common ancestor
- Horizontal gene transfer: genes thrown between bacteria
- Shared derived characters: unique to specific group
- Shared primitive/ancestral characters: not unique to a specific group but is shared within group
Origins of life
- Stages
  - Inorganic formation of organic monomers (miller-urey experiment)
  - Inorganic formation of organic polymers (catalytic surfaces like hot rock or sand)
  - Protobionts and compartmentalization (liposomes, micelles)
  - DNA evolution (RNA functions as enzyme)
- Shared evolutionary characteristics across all domains
  - Membranes
  - Cell comm.
  - Gene to protein
  - DNA
  - Proteins
- Extant = not extinct
- Highly conserved genes = low rates of mutation in history due to criticalness (like electron transport chain)
- Molecular clock: dating evolution using DNA evidence
- Extinction causes niches for species to fill
- Eukaryotes all have common ancestor (shown by membrane-bound organelles, linear chromosomes, and introns)

Calculations

Hardy-Weinberg
- p + q = 1
- p^2 + 2pq +q^2 = 1
Chi Squared

Labs

Artificial Selection Lab
- Trichrome trait hairs
- Anthocyanin for second trait (purple stems)
- Function of the purple pigment?
- Function of trichome hairs?
BLAST Lab
- Putting nucleotides into a database outputs similar genes

Relevant Experiments

Darwin
Lamarck
Miller-Urey
- Slapped some water, methane, ammonia, and hydrogen is some flasks and simulated early earth with heat and stuff and it made some amino acids.

Structure of bacteria

Introduction:

The agents of human infectious disease bacteria
belong to five major groups of organisms ,and these are fungi, bacteria, protozoa, helminths,and viruses.
Bacteria is present in the prokaryote kingdom.
Depending upon the shape, bacteria are classified into three basic groups, these are cocci, bacilli, spirochetes.
The size of bacteria varies from about 0.2 to 5 μm.

https://preview.redd.it/m0gotfqfpl551.jpg?width=1920&format=pjpg&auto=webp&s=9a47dd9a0d17cc6653114c84a1f85d9f83a24b9f
The size of the smallest bacteria (Mycoplasma) and the largest viruses (poxviruses) are almost same.The longest bacteria rods are the size of some yeasts and human RBC.

Structure of bacteria:

Cell wall of the bacteria:

This the outermost component which is present in all the bacteria (except Mycoplasma species).

The significance of this complex bacterial cell wall are to protect the protoblasts from external noxae, which then help to withstand and to maintain the osmotic pressure gradient between the cell interior and the extracellular environment, to give the cell it's outer form and to facilitate communication with its surrounding.
Some bacteria have surface features external to the cell wall, such as capsule, flagella and pili.
The cell wall is located external to the cytoplasmic membrane and is made up of peptidoglycan.

Cytoplasmic membrane:

The cytoplasmic membrane is present just inside the peptidoglycan layer of the cell wall.It is composed of phospholipid bilayer similar in microscopic appearance to that in eukaryotic cells.

Cytoplasm of bacteria:

When observed through the electron microscope it is seen that the cytoplasm of the bacteria has two distinct areas , these are,

An amorphous matrix, in which ribosomes, nutrient granules, metabolites and plasmids are present.
An inner, nucleoid region which is composed of DNA.

Ribosomes:

The ribosomes of the bacteria are the place, where protein synthesis takes place, like the eukaryotic cells. But the chemical composition and the size of the bacterial are different from that of eukaryotic ribosomes.
The size of bacterial ribosomes is 70S , and contains 50S and 30S subunits, but the size of the eukaryotic ribosomes are 80S , and it contains 60S and 40S subunits.

Granules:

Nucleoid:

This is the area of the cytoplasm in the bacteria in which DNA is located.The DNA which is present in the prokaryotes ,are circular molecule,and which has a molecular weight of about 2 × 10^9 and also contains about 2000 genes.There is resemblance to the eukaryotic nucleus becuse in the nucleoid nuclear membrane, nucleolus ,mitotic spindle, histone are not present.
There is no introns present in the bacterial DNA.

Plasmids:

Plasmids are extra chromosomal, double-stranded , circular DNA molecules that are capable of replicating independently of the bacterial chromosome.Although plasmids are usually extra chromosomal, they can be integrated into the bacterial chromosome.In both gram positive and gram negative bacteria plasmids occur, and several different types of plasmid can exist in one cell these are
1.Transmissible plasmids

Neurotransmissible plasmids.

Transposons:

Transposons are prices of DNA , that move fastly from one site to another either within or between the DNAs of bacteria, plasmids and bacteriophages.They are nicknamed as ''jumping genes'' because of their unusual ability to move.Transposons can code for drug resistance enzymes, toxins or a variety of metabolic enzymes and can either cause mutations in the gene into which they insert or alert the expression of nearby genes.There is four identifiable domains present in the transposons

Structure out side the cell wall:

Capsule:

It is a gelatinous layer present surrounding the entire bacterium.The capsule is composed of polysaccharide (except in the anthrax bacillus).The capsule is important because of it's four important role
1.It is determinant of virulence of many bacteria since it limits the ability of phagocytes to engulf the bacteria.
2.By using antiserum against the capsular polysaccharides , specific identification of an organisms can be made.
3.In certain vaccines capsular polysaccharides are used as antigens , because of their role in eliciting protective antibodies.
4.In the adherence of bacteria to the human tissues, capsule has an important role, which is an important primary step in causing infections.

Flagella:

Flagella are long, whip-like appendages that help in the movement of bacteria toward nutrient and other attractants,a process which is known as chemotaxis.The long filament, which acts as a propeller,is composed of many subunits of a single protein,flagellin.
Flagellated bacteria have a characteristic number and location of flagella.
Flagella are medially important for two reasons
1.Some species of motile bacteria are common causes of urinary tract infections.
2.Some species of bacteria are identified in the clinical laboratory by the use of specific antibodies against flagellar proteins.

Pili:

Pili are hair like filaments that extend from the cell surface.They are shorter and straighter than flagella,and are made up of pilin.
Pili have two main role:
1.They regulate the attachment of bacteria with the specific receptor present on the human cell surface.
2.A specialized kind of pilus,the sex pilus, forms the attachment between the male and the female bacteria during conjugation.

Glycocalyx(Slime layer) of bacteria:

It is a polysaccharide coating that is secreted by many bacteria. The glycocalyx is an important component of biofilms.
The glycocalyx mediates adherence of certain bacteria.

Bacterial spores:

These highly resistance structures are formed in response to adverse conditions by two genera of medially important gram positive rods: the genus Bacillus and the genus Clostridium.

A machine learning algorithm has identified an antibiotic that kills E. coli and many other disease-causing bacteria, including some strains that are resistant to all known antibiotics. To test it, mice were infected on purpose with A. baumannii and C. difficile and the antibiotic cleared the mice of both infections.
"The computer model, which can screen more than a hundred million chemical compounds in a matter of days, is designed to pick out potential antibiotics that kill bacteria using different mechanisms than those of existing drugs."
"The researchers also identified several other promising antibiotic candidates, which they plan to test further. They believe the model could also be used to design new drugs, based on what it has learned about chemical structures that enable drugs to kill bacteria."
"The machine learning model can explore, in silico, large chemical spaces that can be prohibitively expensive for traditional experimental approaches."
"Over the past few decades, very few new antibiotics have been developed, and most of those newly approved antibiotics are slightly different variants of existing drugs." "We're facing a growing crisis around antibiotic resistance, and this situation is being generated by both an increasing number of pathogens becoming resistant to existing antibiotics, and an anemic pipeline in the biotech and pharmaceutical industries for new antibiotics."
"The researchers designed their model to look for chemical features that make molecules effective at killing E. coli. To do so, they trained the model on about 2,500 molecules, including about 1,700 FDA-approved drugs and a set of 800 natural products with diverse structures and a wide range of bioactivities."
"Once the model was trained, the researchers tested it on the Broad Institute's Drug Repurposing Hub, a library of about 6,000 compounds. The model picked out one molecule that was predicted to have strong antibacterial activity and had a chemical structure different from any existing antibiotics. Using a different machine-learning model, the researchers also showed that this molecule would likely have low toxicity to human cells."
"This molecule, which the researchers decided to call halicin, after the fictional artificial intelligence system from '2001: A Space Odyssey,' has been previously investigated as possible diabetes drug. The researchers tested it against dozens of bacterial strains isolated from patients and grown in lab dishes, and found that it was able to kill many that are resistant to treatment, including Clostridium difficile, Acinetobacter baumannii, and Mycobacterium tuberculosis. The drug worked against every species that they tested, with the exception of Pseudomonas aeruginosa, a difficult-to-treat lung pathogen."
"Preliminary studies suggest that halicin kills bacteria by disrupting their ability to maintain an electrochemical gradient across their cell membranes. This gradient is necessary, among other functions, to produce ATP (molecules that cells use to store energy), so if the gradient breaks down, the cells die. This type of killing mechanism could be difficult for bacteria to develop resistance to, the researchers say."
"The researchers found that E. coli did not develop any resistance to halicin during a 30-day treatment period. In contrast, the bacteria started to develop resistance to the antibiotic ciprofloxacin within one to three days, and after 30 days, the bacteria were about 200 times more resistant to ciprofloxacin than they were at the beginning of the experiment."
The way the system works is, they developed a "directed message passing neural network", open sourced as "Chemprop", that learns to predict molecular properties directly from the graph structure of the molecule, where atoms are represented as nodes and bonds are represented as edges. For every molecule, the molecular graph corresponding to each compound's simplified molecular-input line-entry system (SMILES) string was reconstructed, and the set of atoms and bonds determined using an open-source package called RDKit. From this a feature vector describing each atom and bond was computed, with the number of bonds for each atom, formal charge, chirality, number of bonded hydrogens, hybridization, aromaticity, atomic mass, bond type for each bond (single/double/triple/aromatic), conjugation, ring membership, and stereochemistry. "Aromatic" refers to rings of bonds. "Conjugation" refers to those chemistry diagrams you see where they look like alternating single and double (or sometimes triple) bonds -- what's going on here is the molecule has connected p orbitals with electrons that move around. "Stereochemistry" refers to the fact that molecules with the same formula can form different "stereoisomers", which have different 3D arrangements that are mirror images of each other.
From here, and the reason the system is called "directed message passing", the model applies a series of message passing steps where it aggregates information from neighboring atoms and bonds to build an understanding of local chemistry. "On each step of message passing, each bond's featurization is updated by summing the featurization of neighboring bonds, concatenating the current bond's featurization with the sum, and then applying a single neural network layer with non-linear activation. After a fixed number of message-passing steps, the learned featurizations across the molecule are summed to produce a single featurization for the whole molecule. Finally, this featurization is fed through a feed-forward neural network that outputs a prediction of the property of interest. Since the property of interest in our application was the binary classification of whether a molecule inhibits the growth of E. coli, the model is trained to output a number between 0 and 1, which represents its prediction about whether the input molecule is growth inhibitory."
The system has additional optimizations including 200 additional molecule-level features computed with RDKit to overcome the problem that the message passing paradigm works for local chemistry, it does not do well with global molecular features, and this is especially true the larger the molecule gets and the larger the number of message-passing hops involved.
They used a Bayesian hyperparameter optimization system, which optimized such things as the number of hidden and feed-forward layers in the neural network and the amount of dropout (a regularization technique) involved.
On top of that they used ensembling, which in this case involved independently training several copies of the same model and combining their output. They used an ensemble of 20 models.
The training set was 2,335 molecules, with 120 of them having "growth inhibitory" effects against E. coli.
Once trained, the system was set loose on the Drug Repurposing Hub library, which was 6,111 molecules, the WuXi anti-tuberculosis library, which was 9,997 molecules, and parts of the ZINC15 database thought to contain likely antibiotic molecules, which was 107,349,233 molecules.
A final set of 6,820 compounds was found, and further reduced using the scikit-learn random forest and support vector machine classifiers.
To predict the toxicity of the molecules, they retrained Chemprop on a different training set, called the ClinTox dataset. This dataset has 1,478 molecules with clinical trial toxicity and FDA approval status. Once this model was made it was used to test the toxicity of the candidate antibiotic molecules.
At that point they hit the lab and started growing E. coli on 96 flat-bottomed assay plates. 63 molecules were tested. The chemical they named halicin did the best and went on to further testing against other bacteria and in mice.
http://news.mit.edu/2020/artificial-intelligence-identifies-new-antibiotic-0220

SPOILER WARNING for the latest chapters of the Manga
In chapter 120 of TPN we are finally given some insight into the origins of the demons and their need to eat humans to survive. There were a couple of different ways that the manga could have gone with this and I was intrigued that they used a more scientific approach discussing evolution via vertical and horizontal gene transfer.

Vertical Gene Transfer

Evolution, as I have covered before, is how an organism slowly changes over time as it acquires traits that help it better survive in its environment. The new traits arise from random mutations and any new trait that helps the organism survive and reproduce is going to become more frequent in the population as organisms with that trait have more offspring that also have the trait. For example, when a new mutation grants an insect resistance to a pesticide, the insects that have resistance increase in number while the ones that don’t die off. This continues until almost all of the insects are resistant to that particular pesticide, as was the case with flies and DDT. Now what I just described is actually vertical gene transfer or the passing of genetic traits from parent to offspring. FYI- vertical gene transfer is not usually used to refer to this process as it is normally referred to as evolution or natural selection.

Horizontal Gene Transfer

Horizontal gene transfer, on the other hand, works a bit differently and instead of passing genetic material from parent to offspring, the genetic material is passed to another individual in the same generation. Additionally, the information is being passed between organisms that are different species. It most common in bacteria and happens via the transfer of plasmids, which I discussed in my post on the quirks of My Hero Academia. To recap, plasmids are extra chromosomal segments of DNA that can be transferred between bacteria and some single celled organisms like yeast.
The plasmids are not necessary for the bacteria to survive, but they often grant additional traits like antibiotic resistance. The plasmids can be transferred from one organism to another via a process called conjugation. In conjugation the plasmid reproduces and is then transferred to another bacteria via an extension of the cell called a pilus.
Now by this point you are probably thinking this is all nice and good but the demons of The Promised Neverland evolved by eating other animals and absorbing their genes and not by reproducing or sharing plasmids with them. As it turns out, bacteria can absorb plasmids and genetic material from the environment around them, as was discovered by the transformation experiments of Fredrick Griffith that showed that you could turn a harmless bacterium into a pathogenic one via the remains of dead pathogenic bacteria.
Oswald Avery expanded on Griffith’s research on transformation proving that it was DNA passing between the dead and live bacteria that was causing the transformation. This of course fits with the demon’s evolving through absorbing the DNA of other organisms. However, by this point I think we can also assume that demons are eukaryotic organisms, given that they for all intents and purposes appear to be large complex multicellular organisms. So how does gene transfer work in eukaryotic organisms?

Endosymbiotic Theory

If we go by what Norman says in the manga that the demons might have started as bacteria, the first step in their evolution via horizontal gene transfer would be by absorbing plasmids of other bacteria. But bacteria are small single celled organisms that do not form large complex organisms like the current demons we see in the manga. So how did they become the demons we see in the manga?
As it turns out, horizontal gene transfer played a role in the evolution of eukaryotic cells in the real world. If you know anything about eukaryotic cells, you know that the DNA is contained inside the nucleus and the cell contains structures called organelles. Organelles can be thought of as miniature organs that perform a particular function for a cell like our own organs do for our bodies. There are a number of organelles, but there are only 2 that concern us today and they are the mitochondria and the chloroplast. This is because they both contain their own DNA and replicate separately from the rest of the cell. These two organelles are proof of something called endosymbiotic theory.
In a simplified form endosymbiotic theory is the theory that eukaryotic cells arose when a large bacterium ate another smaller bacterium, and instead of digesting it let it live inside of itself. The smaller cell now lived on inside of the larger one granting it new traits like the ability to perform photosynthesis (chloroplast) or more efficiently breakdown sugar (mitochondria).
It also fits nicely with Norman’s explanation of demon evolution via eating other organisms. However, their evolution didn’t stop there, as horizontal gene transfer allowed them to continue to evolve.

Horizontal Gene Transfer in Multicellular Organisms

Now this is were we get to some very new and interesting aspects of biology. Studies have shown that horizontal gene transfer has happened between various organisms such as:
Bacteria to fungi, plants, and animals
Insects to fungus and round worms
Human to protozoan
Scientists know that these transfers have occurred but the how and why has yet to be determined; it is suspected that DNA transposons are involved. Transposons are segments of DNA that do not like to stay where they are in an organism’s genome and have a tendency to jump around. Think of it like a page in a book that keeps jumping around the book. Lastly, all of this transferring is thought to play a role in the evolution of numerous species, but it is only just now being investigated. In fact, up to 3% of the human genome could be made of transposons. So modern science does still match up with Norman’s explanation of horizontal gene transfer playing a role in the evolution of demons.

Development of Language and Culture

Now this is where Norman’s explanation starts to go off the rails a bit, as I read that last part stating the demons developed language and culture as a direct result of eating humans and not as a result of their improved mental capacities via gene transfer. It is as if they gained the knowledge as a direct result of eating humans. FYI- while we are on the subject, what about the other intelligent animal species like gorillas or dolphins, as they too would have influenced the demon’s intelligence and development?
By this point you are all probably saying well, duh, you can’t absorb another organism’s knowledge by eating it……
Well, this is where it gets interesting. In the 1950’s and 60’s a scientist by the name of Dr. James V. McConnell was investigating memory using flatworms.
His experiment was rather simple, in that he would train a flatworm to go through a maze and then cut up the worm into tiny pieces feeding it to other non-trained flatworms. The non-trained flatworms would then be able to go through the maze like a trained worm. Does this mean the demons of The Promised Neverland are really just mutated flatworms?
No, because scientists could never reproduce the study, and the original study was shown to have a number of flaws. FYI- Some more recent studies have shown that flatworms do have some interesting quirks when it comes to memory. First off, let me inform you that flatworms have an amazing ability to regenerate and that includes having their head cut off. In this case trained flatworms had their heads removed and yet still remembered the training even after the brain was completely regrown. Whether this means that their body has some other way of retaining memory outside the brain or that the study was flawed in some way is still open to debate.

That’s not how it works

Honestly, I don’t know what else to say to this part other than that’s not how biology and genetics works. You can’t lose a genetic trait once you have it, even if it is a plasmid. According to Norman the demons will lose their human intelligence, becoming wild, after not eating humans for 6 months.
Oh, and an organism whose genetic code can change that much would have a lot of problems with cancer and other cell growth disorders. FYI- yes, I know that some viruses can mutate very quickly, but viruses are not considered to be living things, as they lack cells, among other things.

Plot Point

Now if you were like me when you read chapter 120 you were thinking, wait a minute, something doesn’t add up. Well it looks like Emma and Ray thought the same thing too as of chapter 123.
They also come up with several explanations as to why Sung-joo and Musica are able to retain human form and intelligence without eating humans. We will have to wait until their talk with Norman to find out the answer to that question.

Conclusion

The Promised Neverland has been one of my favorites for a while now, with its mix of action, adventure, and mystery. The latest chapters have been no different and it’s refreshing to see a manga use new discoveries in science as part of the plot. I do realize that it does get some things wrong, but I’d like to think I wasn’t the only one who went to go look up more about the topic. As much as I liked the use of biology in chapter 120 of The Promised Neverland, I am going to have to call it busted.
For now, anyway, because it looks like there is still more to be revealed about the biology of demons. This is because of the way they seemed to gain knowledge and intelligence. The manga also downplays standard evolution a bit too much for my liking. Then there is the matter of the fact that the demons will revert to wild animals after not eating humans for 6 months, which I do realize is questionable right now, so I will revisit the topic in the future as the manga reveals more information.

Bacteria are able to synthesise complex macromolecules from a variety of natural building blocks. It is now shown that they can also be utilized to produce entirely abiotic polymers from readily available synthetic precursors.

Abstract

The ability to harness cellular redox processes for abiotic synthesis might allow the preparation of engineered hybrid living systems. Towards this goal we describe a new bacteria‐mediated iron‐catalysed reversible deactivation radical polymerisation (RDRP), with a range of metal‐chelating agents and monomers that can be used under ambient conditions with a bacterial redox initiation step to generate polymers. Cupriavidus metallidurans, Escherichia coli, and Clostridium sporogenes species were chosen for their redox enzyme systems and evaluated for their ability to induce polymer formation. Parameters including cell and catalyst concentration, initiator species, and monomer type were investigated. Water‐soluble synthetic polymers were produced in the presence of the bacteria with full preservation of cell viability. This method provides a means by which bacterial redox systems can be exploited to generate “unnatural” polymers in the presence of “host” cells, thus setting up the possibility of making natural–synthetic hybrid structures and conjugates.
https://ift.tt/2SMA6Tt

Abstract

This is a preliminary characterisation of a bacteriocin, BacL49 produced by Lactococcus lactis ssp. lactis. This bacteriocin is significant due to its activity against Streptococcus iniae, a bacterial pathogen causing severe economic losses in the global aquaculture of various fish. Spot-on-lawn and microtitre plate assays were used to test antagonistic activity of the bacteriocin. BacL49 is heat and pH stable (100 °C for 60min, pH 2.5-9.5), and sensitive to proteinase K, a-chymotrypsin, trypsin and papain. BacL49 has a bactericidal mode of action and is produced during late log phase growth. BacL49 exhibits a broad activity spectrum against S. iniae, antagonising 93.75% (45/48) of S. iniae isolates collected from a variety of hosts and environments. The apparent molecular masses of the active protein components determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis are 5 and 54kDa. Molecular analyses were performed to locate the genetic determinants of BacL49. PCR of chromosomal DNA successfully amplified the structural gene encoding the precursor of nisin. Subsequent analysis of nucleotide sequences of the PCR products revealed it to be identical to the nis Z structural gene of nisin Z. There is a paucity of reports examining the inhibition of S. iniae by a lactococcal bacteriocin or even L. lactis as an aquacultural probiotic. This is one of the first studies to identify nisin genes in a strain of L. lactis exhibiting activity against S. iniae. BacL49 is a candidate biocontrol agent for mitigation of this important fish pathogen.
Keywords: Bacteriocin; Lactococcus lactis; Probiotic; Streptococcus iniae; Nisin Z
Go to

Introduction

Streptococcus iniae is one of the most economically important bacterial pathogens causing streptococcosis in fish. Globally, economic damages have been in excess of US$100 million per year [1] with as much as 70% loss in annual production in certain fish cultures [2]. S. iniae causes disease and mortality in at least 30 species of fish in marine, brackish and freshwater environments [3,4]. Outbreaks of S. iniae typically occur in warm-water, cultured situations throughout the world including Australia, Bahrain, Israel, Japan, Korea, Spain, Italy, and the United States [4,5-10]. Epizootic incidents have also been reported in wild populations [11,12], the most notable being the 1999 and 2008 Caribbean fish kills [13,14]. Zoonotic infections of S. iniae, normally caused by percutaneous injuries sustained during raw seafood preparations, have been reported in Canada, China, Hong Kong, Singapore, Taiwan, and the United States [15]. Prevention and treatment of S. iniae in aquaculture remains difficult, particularly with the industry seeking safer alternatives to antibiotics and vaccines. Antibiotics, including erythromycin have previously been successful in treating streptococcus is in fish [16]. Nonetheless, the use of antibiotics in aquaculture is gradually being eschewed due to widespread development of antibiotic resistance in the environment and in cultured fish, which causes consumer concern [16-18]. In some cases, antibiotics are also believed to merely suppress clinical symptoms without eliminating the infection, thereby promoting the development of "carrier fish" [1].
Research devoted to creating effective vaccines for prevention of S. iniae suffered a major setback when a novel serotype of S. iniae caused severe outbreaks in Israel amongst vaccinated rainbow trout (Oncorhynchus mykiss) [19,20]. Since then, commercial vaccines against S. iniae have been marketed, but are very limited geographically [21,22]. Alternative vaccines have yielded successful results but are still undergoing testing [2325]. In spite of this progress, the task of effectively vaccinati Oceanogr Fish Open Access J 3(2): OFOAJ.MS.ID.555610 (2017) each individual can be tedious, expensive, and even stressful for the fish [26]. Vaccinations can be futile when applied to juvenile fish that may not be fully immunocompetent [27]. Additionally, S. iniae is capable of surviving in the aquatic environment without a host [11,28,29] and this was speculated to have been a factor in the evolution of the new serotype by allowing the bacterium to evade the immune response of the vaccinated fish [19]. Furthermore, the outbreak in Israel is evidence of serotype diversity that could enable S. iniae to eventually overcome yet another vaccine. Thus, the quest continues for a long-term solution that can be universally and easily applied throughout the aquaculture industry [25]. Chemical-free "green solutions" appear to be the next era of therapeutics for preventing bacterial epizootics in fish [17]. Probiotics, either in the form of whole cells or cell components such as bacteriocins, are anticipated as being effective replacements for antibiotics and chemotherapeutics to fight infectious diseases [30]. Bacteriocins are a group of proteinaceous molecules that are biologically active against bacteria that are closely related to the bacteriocin producer, while the producer is immune [31-33]. Ubiquitous across all bacterial genera, these ribosomally synthesized peptides confer a selective advantage to their producers [31,32,34]. Bacteriocins are recognized as potentially useful agents in the control of bacterialinfections due to their effectiveness, non-toxicity and relatively cheap production [32,35,36]. Only two published studies on bacteriocins have tested S. iniae as an indicator species [37,38].
An inhibitory substance, identified as bacteriocin BacL49, was found during experimentation with the library of S. iniae and other aquatic bacteria from James Cook University (JCU). The bacteriocin was produced by a strain of Lactococcus lactis ssp. lactis and was observed to inhibit a large spectrum of S. iniae isolates. L. lactis, along with the rest of the lactic acid bacteria (LAB) are considered the most prolific of all the Gram-positive bacteriocinogens [32,39]. A versatile species, variousstrains of L. lactis can produce an assortment of bacteriocins that are predominantly encoded on plasmids [40-43]. Some L. lactis strains produce the bacteriocin nisin, a small (<5kDa), membrane-active member of the Lantibiotic class of LAB bacteriocins capable of antagonistic activity towards a wide range of Gram-positive bacteria [44-46]. Nisin is heat-tolerant (115-121 °C) at low pH levels, making it an ideal preservative in pasteurised and acidic food products [45,46]. Being "generally regarded as safe," nisin has achieved worldwide recognition as a non-toxic food additive in over 50 countries [31,32,45]. As produced by various strains of L. lactis, nisin occurs as natural variants with designations A, Z, Q, and F [47-49]. 106 Unlike most plasmid-encoded bacteriocins produced by L. lactis, nisin is encoded on a conjugative transposon [50-52], a chromosomally- associated segment of DNA with the capacity to repeatedly insert into and mobilise plasmids and genomes [52,53]. Thus, one objective of this study was to identify the genetic determinants of the bacteriocin BacL49 through assessing the prevalence of plasmids in L. lactis subsp. lactis L49 and examining the bacterial chromosomal DNA for nisin genes. This study also provides a brief characterisation of bacteriocin BacL49 and highlights the potential of this substance as a green solution for S. iniae infections in fish.
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Materials And Methods

Source and identification of BacL49 producer. A lawn of S. iniae AS-04-1524#1 (JCU isolate S42; Table 1) was observed as a mixed culture, with one distinct colony type antagonizing the growth of the other. Both isolates were identified using a PCR assay for the lactate oxidase (lctO) gene of S. iniae using the primer combination LOX-1/LOX-2 [54], and a 16S rDNA PCR assay using universal primers 27F/1492R. PCR products were cleaned and sequenced by Macrogen Inc. (Korea). The 16S PCR assay returned a sequence 99% identical to Lactococcus lactis ssp. lactis in BLAST. According to sequencing, the antagonizing bacterium was identified as L. lactis L49, originally isolated from a moribund sleepy cod (Oxyeleotris lineolatus) at JCU. The inhibitory substance produced by L. lactis L49 was designated as BacL49.
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*Altered TAAHL accession number. Changes were made for unknown reasons by previous researchers; it is likely these isolates are from the correct animal accession but the final number has been altered (ie. -9 and -11). **Invalid TAAHL accession number.
Bacterial growth and isolation of BacL49. Unless otherwise stated, bacteria were propagated aerobically at 28 °C in heart infusion broth (HIB) or agar (HIA) produced by the addition of 1.5% technical agar to HIB. Bacterial lawns were made by seeding HIA plates with 1ml overnight growth of bacteria, removing excess culture and allowing lawns to dry at room temperature. To produce cell-free supernatant (cfs) containing BacL49, L. lactis L49 was grown aerobically at 28 °C for 10-12h, then centrifuged at 4300g for 5min and filtered through 0.45|im. Aliquots of cfs were stored at 4 °C.
135 Assays for antagonistic activity. The activity of L. lactis L49 against 48 isolates of S. iniae (Table 1) was determined by an altered deferred antagonism method [55]. Briefly, an overnight L. lactis L49 culture was streaked in a single line on an HIA plate with a sterile loop. Bacterial isolates and a control of uninoculated broth were then streaked with sterile loops in parallel at right angles to L49. Zones of inhibition were measured after incubation for 16h. For interest, streak plates were made to determine the activity of L. lactis L49 against B antibiotic resistant human pathogens including E. coli B597, E. coli 53e, community acquired methicillin-resistant Staphylococcus aureus and Streptococcus pneumoniae. For these human isolates, one plate was incubated at 37 °C with CO2 and the other was incubated aerobically at 37 °C to ensure sufficient growth of the pathogens. Zones of inhibition were measured after 24h.
146 Antagonistic activity of BacL49 was detected qualitatively on solid media using a spot-on-lawn assay [56]. Antagonistic activity was quantified in liquid media using a modified microtitre plate assay [57]. The indicator culture was grown to an optical density of approximately 0.2 measured at 540nm, and added to two-fold dilutions of the growth medium or cfs (treated or untreated) in duplicate 96-well round bottom plates. Plates were incubated aerobically for 3h at 28 °C and the optical density measured. Antagonistic activity was defined as the reciprocal of the dilution causing 50% growth inhibition (determined by optical density) relative to the control culture without cfs (AU = arbitrary units). S. iniae S23 was used as the indicator strain for all spot-on-lawn and microtitre plate assays due to its high level of sensitivity to BacL49.
Effect of heat, pH and enzymes on BacL49 activity. To determine the heat stability of BacL49 activity, cfs samples (pH 5) were heated at 100 °C for 10, 20, 30 and 60min. Samples were cooled to room temperature before spot-on-lawn and microtitre plate assays. To determine the effect of pH on BacL49 activity, cfs samples were adjusted to pH levels between 1.5 and 9.5 using 1N NaOH or HCl. Samples were incubated with agitation for 2h, then readjusted to pH 5 (the pH level of untreated cfs following incubation) before spot-on-lawn and microtitre plate assays. To determine the effect of different enzymes on BacL49 activity, cfs samples were adjusted to pH 7.0 and treated to a final concentration of 2mg ml-1 with the following enzymes: proteinase K (40 units mg-1), a-chymotrypsin (59.3 units mg- 1), trypsin (2.6 units mg-1), pepsin, papain (19 units mg-1), and catalase (1340 units mg-1). Samples were incubated at 37 °C with agitation for 2h, then at 100 °C for 5min to deactivate enzymes. Remaining antagonistic activity was measured using the spot- on-lawn assay. An untreated cfs sample was used as a control in all assays.
170 Kinetics of production and activity. The kinetics of BacL49 production by L. lactis L49 were investigated by measuring the growth of the bacterium and activity of BacL49 produced over the same period. Overnight growth of L. lactis L49 was added to HIB (3.75% volume) and the culture was incubated with agitation. The optical density of the culture was measured at 600nm every hour until 12h, then periodically to 28h. At each reading, 1ml culture was removed to produce cfs, which was stored at 4 °C. Antagonistic activity was measured using a spot- on-lawn assay following the collection of all cfs samples.
The activity of BacL49 was determined as either bacteriostatic or bactericidal by measuring the growth of indicator strain S. iniae S23 after the addition of BacL49. Early log-phase growth of the indicator strain was distributed into triplicate 10ml aliquots. One ml of cfs was added to two indicator cultures and HIB was added to the remaining control culture. Optical density was measured at 600nm over 24h. To measure cell viability in a treated culture, mid log-phase growth of the indicator strain was distributed into duplicate 10ml aliquots. One ml cfs was added to one aliquot and HIB was added to the control. Optical density measurements at 600nm were taken over time and 10-fold serial dilutions were grown on HIA to measure cell forming units (cfu) of the indicator strain in both cultures at 48h.
Protein purification. L. lactis L49 was grown overnight in HIB previously filtered through Millipore type HA filters (to remove unwanted proteins from the media). A portion of the cfs produced from this culture was treated with ammonium sulphate in two stages. For the primary precipitation, saturated ammonium sulphate solution was slowly added to 100ml cfs while stirring to 60% saturation at room temperature. The solution was agitated 12h at 4 °C, then centrifuged at 4500g for 30min at 4 °C. The precipitate was resuspended in 10ml sterile distilled water. A small portion was removed and stored at 4 °C for the activity assay. Saturated ammonium sulphate solution was added to the remaining primary solution while stirring to 80% saturation at RT. This solution was agitated 12h at 4 °C, then centrifuged at 4500g for 30min at 4 °C. The secondary precipitate was resuspended in 1ml (1% original volume) sterile distilled water and stored at 4 °C. The activity of the ammonium sulphate precipitated (primary and secondary) proteins was tested with a microtitre plate assay.
Approximate molecular size of BacL49 was determined by tris-tricine sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE). Aliquots of the partially purified protein samples and untreated cfs were prepared with TruSep tricine SDS sample buffer and run on 16% tris-tricine gels with a low MW prestained protein ladder (Fermentas, Australia) at a constant 150V. Gels were silver stained using a Silver Stain Plus staining kit (BioRad, Australia) according to the manufacturer's protocol 208.
Proteins separated by SDS PAGE were eluted from the gel using a modified protocol from Busarcevic et al. [58]. Following agitation at room temperature overnight, the eluted samples were centrifuged at 10,000g for 5min and the supernatants concentrated by vacuum at 33 °C for 20min. Two controls were used: one elution made with a slice of gel from an unused well lane (containing no protein) and one with elution buffer only. Eluted samples were assayed for antimicrobial activity with a spot-on-lawn assay.
DNA purification. Isolation of plasmid DNA was carried out using the Wizard® Plus SV Minipreps DNA Purification System (Promega, Australia). Low-copy number plasmids were assumed for all strains. Genomic DNA isolations were carried out using the High Pure PCR Prep Kit (Roche, Australia) for the detection of nisin structural genes. DNA isolation procedures were performed according manufacturer specifications with the addition of a lysozyme step for Gram-positive bacteria. Negative control preparations contained no bacterial culture.
DNA products were resolved by electrophoresis on agarose gels stained with GelRedTM and then visualised under UV light. Since L. lactis strains often harbour a wide size-range (2kb to about 100kb) of plasmids (Teuber and Geis, 2006), plasmid DNA products were visualised using two different DNA ladders. These included the 1kb GeneRulerTM (Fermentas, Australia) used with 1% (w/v) agarose gels at 90V and the Lambda Mix Marker 19 (Fermentas, Australia) used with 0.5% (w/v) agarose gels at 40V.
PCR analysis of the nisin gene. Oligonucleotide primers (Macrogen, Korea) were designed using the NCBI-ORF Finder and OLIGO 7 primer analysis software (Table 2) to target the nis A structural gene. Genomic DNA samples were prepared for PCR using GoTaq® Green master mix (Promega, Australia). As L. lactis L53 was found to contain a plasmid (results not shown), this isolate was included in the procedure. For negative controls, a tube of reaction mix containing indicator strain S. iniae S23 and one without sample DNA were included for the nisin structural gene protocol, and for the PCR protocol, respectively. DNA was amplified in a thermocycler (Eppendorf, Australia) set for denaturation at 94 °C, annealing at 55 °C and extension at 72 °C. PCR products were visualised on 2% (w/v) agarose gels at 120V, along with a 50bp GeneRulerTM (Fermentas, Australia). Sequence information (Macrogen, Korea) of the PCR products was analysed using Sequencher® 5.0 software. The resulting consensus sequences were compared to DNA and protein sequences contained in the National Center for Biotechnology Information (NCBI) database via BLAST search of "highly similar" sequences.
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Results

L. lactis L49 produced antagonistic activity against 93.75% (45/48) of S. iniae isolates in the JCU library (Figure 1). L. lactis L49 failed to inhibit the growth of isolates S32, S39, S47 and the human pathogens tested. Antagonistic activity of BacL49 remained following 60min at 100 °C and over a broad pH range (pH 2.5-9.5), though activity was weaker at pH levels higher than 5.5 (Table 3). The cfs also showed antagonistic activity against the indicator after exposure to pepsin and catalase, but lost activity when exposed to proteinase K, a-chymotrypsin, trypsin and papain. L. lactis L49 began producing BacL49 at the end of the log growth phase (Figure 2). BacL49 production reached a maximum at early stationary phase, but began to drop following600min growth and continued to decrease to the end of 24h.
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Activity measured as present (+) or absent (-) for enzyme tests. Absence antagonistic activity (-) correlates to senstivity (s) to the enzyme tested, and antagonism of the indicator (+) correlates to resistance (r) to the enzyme tested; Cfs: Cell Free Supernatant.
A Growth of the indicator strain was not only inhibited following the addition of BacL49, but the optical density of the culture continued to drop steadily over time without recovery, while the control culture grew normally to a high optical density. The cell viability of the treated indicator culture dropped 10fold in the first 30min following the addition of BacL49 and continued to drop over 70min while the control culture cfu increased 10-fold.
Ammonium sulphate precipitation successfully concentrated the bacteriocin (Table 3). The primary precipitate showed 8 times the activity (in AU) of the untreated cfs and the secondary precipitate showed at least 64 times the activity of the untreated cfs (1/512 was the highest dilution made of the substances tested in the microtitre assay).
Tris-tricine SDS PAGE allowed separation of the low molecular weight proteins, and multiple protein bands were clarified after silver staining of the gel (Figure 3). Following elution of proteins from the tris-tricine gel slices, only minor antagonistic activity was produced on the indicator strain by the small molecular weight band (5kDa) but strong antagonism was produced by the large molecular weight (54kDa) band.
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Following plasmid extraction, DNA samples of L49 showed faint bands that were too unremarkable to be denoted as plasmids (Figure 4). Based on this data, further plasmid experimentation was not pursued. PCR for the nisin structural gene resulted in products just over 100bp for isolates L49 and L53 (Figure 5). No bands were detected for S23 or the control. Sequence analysis of the L49 PCR product yielded a consensus sequence of the nisin structural gene (Figure 6) that was 99% homologous to the nis A structural gene and 100% homologous to the nisZ structural gene (Table 4). The protein BLAST search showed 100% homology with the Nisin Z precursor, followed by that of nisins F and A, and then nisin Q (Table 4). A deduced amino acid sequence of BacL49 was also obtained from the BLAST search, which showed that BacL49 was identical to nisin Z.
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Discussion

The inhibitory substance BacL49 produced by L. lactis L49 has been identified as a bacteriocin due to meeting the criteria of being a biologically active protein moiety with a bactericidal mode of action [33]. BacL49 can be produced in anaerobic conditions and is capable of diffusing through solid media (results not shown), which rules out hydrogen peroxide and phage activity, respectively, as the cause of antagonism. The protein nature of BacL49 was confirmed by its sensitivity to a number of proteases and the mode of action was confirmed as bactericidal by optical density and cell viability experiments.
BacL49 is heat and pH stable, though antagonistic activity was weaker when the bacteriocin was incubated in an alkaline environment. An acidic pH can be necessary for the retention of the cationic properties of bacteriocin peptides, which appear to be crucial for their antagonistic activity [59]. Most L. lactis bacteriocins, including nisin, are heat stable and tolerant to acidic conditions [21,60-62] but many show different enzyme sensitivities. A review of publications describing Nisin Z production by L. lactis strains isolated from various sources found that nisin Z is consistently sensitive to proteinase-K, but varies in sensitivity to trypsin, a-chymotrypsin, and papain. A few published descriptions of nisin exactly matched the protease sensitivity profile of BacL49 [63-65]. Interestingly, the only other described nisin isolated from fish differs from BacL49 in sensitivity to trypsin and a-chymotrypsin [37].
A bactericidal mode of action was confirmed by the reduction over time in optical density and Viable cell count of the BacL49 treated indicator culture. It is known that many antibiotics kill bacteria by targeting lipid II, thus blocking cell wall synthesis and leading to cell lysis by pore formation [44,66,67], and it is possible that BacL49 performs in a similar manner. The bactericidal mode of action of BacL49 not only confirms this substance is a bacteriocin, it indicates that BacL49 could reduce bacterial loads in fish or the environment by destroying S.iniae cells.
Production of antagonistic activity by the two differently sized peptide bands eluted from SDS PAGE gels (54kDa, and 5kDa) suggests that two different inhibitory substances are being concurrently produced by L. lactis L49. The two component lantibiotic lacticin 3147 consists of two 3-4kDa peptides, but both are required for antagonistic activity [68]. This required cooperation is apparently not the case with the two BacL49 peptides, as evidenced by their independent production of antagonism. It is also possible that cleaving of BacL49 is occurring; the large molecular weight peptide may be an unseparated quaternary structure of the small peptide.
The results of the plasmid extraction do not support the hypothesis that L49 contained plasmids. The faint bands observed after electrophoresis were not indicative of a typical plasmid profile. Chromosomal DNA extraction results suggest that L49 possesses the structural gene encoding the nisin precursor. Interestingly, equally strong bands for the structural gene were evident for isolate L53. Whilst this result was not expected due to isolate L53 only inhibiting 30% of S. iniae isolates in the JCU library (results not shown), it was also not surprising since various strains of L. lactis have been observed to produce nisin.
Based on the NCBI BLAST search results, BacL49 is likely nisin Z. The DNA sequence was shown to differ from that of nisin A exactly as stated in Mulders et al. [48], varying at position by a C to A transversion. Comparison of the deduced amino acid sequence with those of the other nisin variants, coupled with the protein BLAST results, substantiates the nucleotide results. Nisin Z is a natural nisin variant that has only been produced by strains of L. lactis, however these strains have been isolated from a variety of sources from different environments.
Producers of nisin Z have previously been derived from dairy [51,69] and vegetables products [70,71]. Recently, it has been shown that they are also associated with mangroves [72], marine fish [37], and now with freshwater fish. The source of the producing strain could have an impact on how and in what context it can be used most effectively.
Based on the results of this study, it is likely that BacL49 is encoded on a conjugative transposon. Like plasmids, some conjugative transposons can possess a very broad host range [73], allowing for the dissemination of various traits (e.g. antibiotic resistance) across different species [53,74,75]. Further research following this study should focus on isolating this mobile genetic element from L49, characterising it, and assessing its potential novelty and uses by genetic manipulation.
Other bacteriocin-producing L. lactis strains have been isolated from freshwater fish in the past [47,60] however these studies tested bacteriocin activity against S. aureus, L. monocytogenes, and other pathogens important in food spoilage and human infection. This study reports similar findings to that in Heo et al. [37], in which a strain of L. lactis subsp. lactis was isolated from the intestine of a marine olive flounder (Paralichthys olivaceus) and was found to inhibit S.iniae during in vitro experiments. Heo et al. [37] examined the in vitro effects of nisin Z against S. iniae by combining it with varying concentrations of NaCl. The authors concluded that the ability of nisin Z to inhibit the growth of S. iniae was synergistically improved when applied in conjunction with NaCl. This is significant for marine- based aquaculture. The producer of BacL49 was isolated from a freshwater fish species, thus it would be interesting to look for any significant differences in activity between BacL49 and nisin Z originating from a marine source BacL49 could have important applications in the aquaculture industry with regard to S. iniae.
The ability to retain its activity through heating processes would allow this bacteriocin to be readily incorporated into commercial fish food. The fact that BacL49 remains active over a broad pH range is also advantageous because S. iniae can establish in a variety of organs and tissues in infected fish. BacL49 could also be added directly into the culture water as a non-toxic means of biological control of S. iniae in the culture environment and be a supplement to vaccination procedures. This environmental treatment could address the issue of S. iniae cells surviving freely in the water and evading the immune responses of vaccinated fish. Differences in activity, such as increased inhibitory specificity, distinguish bacteriocins from classical antibiotics and are another advantage to their use in a cultured environment [76]. This specificity can reduce the risk of non-target bacteria (particularly beneficial ones) being antagonized and minimize the threat of resistance development. However, the broad activity spectrum of BacL49 on different isolates of S. iniae would prove to be an advantage due to the large variation that exists between strains of this pathogen [76,77].
The pathological effects of bacteriocins must be considered before they are used in an in vivo situation, and it would be beneficial to determine whether the bacteriocin is strongly antigenic [33]. However, most bacteriocins are not toxic to animals at effective antimicrobial concentration due to their specificity [78]. Despite the results of this study showing that high concentrations of BacL49 are easy to achieve, bacteriocin delivery or retention in fish tissues could prove difficult in vivo. If this were the case, L. lactis ssp. lactis L49 could be trialed as a probiotic. Great interest has been shown towards LAB as potential probiotics, as they are well-recognised for their bacteriocinogenic capabilities and presence within the normal microbiota of fish (typically the intestine) [30,79]. Not only can they withstand acidic stomach conditions, but they can grow and colonise the intestine of fish [80]. The piscine origins of L49 may contribute to its survivability and efficacy as a potential probiotic, though there are few studies examining L. lactis as a probiotic specifically against fish pathogens. Though L. lactis has not been documented as a fish pathogen and is regularly present in the aquatic environment and the intestinal tract of both freshwater and marine fish [71,81-84], it would be necessary to confirm isolate L49 as non-pathogenic to the species of fish undergoing treatment. The elucidation of the L. lactis genome and the fact that products from the bacteria are generally regarded as safe make the bacterium a unique candidate for genetically engineered live vaccines as well [85].
BacL49 is significant because it displays a broad activity spectrum for S. iniae isolates, implicating it as a new therapeutic or preventative agent for infections caused by this economically important fish pathogen. Nisin has had a long history as a safe food additive in the manufacturing of various foods for human consumption, thus BacL49 may also be integrated with fish food without serious concern over chemical residues. Purified BacL49 should be tested in vivo to determine antigenicity of the substance in fish, and the bactericidal action of the bacteriocin should be studied in depth to identify problems that may arise with bacterial resistance.
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