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Avian Flu Research Controversy

Harsh Patel
Introduction
Avian influenza (AI), also known as Bird Flu, is an infectious disease caused by various strains of AI virus. It majorly affects wild water fowl for example geese and ducks, however it can cause large-scale outbreak among domestic poultry resulting into significant economic loss1. Majority of AI viruses do not cross the species barrier and infect humans, nonetheless, two strains – namely A (H5N1) and A (H7N9) – have exhibited zoonotic potential, causing serious illness and even deaths in people. Moreover, human infections caused by these two strains of virus have been linked with high mortality rates. WHO has received 650 case-reports of A (H5N1) infections since 2003 from 15 different countries, 60% of which proved fatal2. A(H7N9) strain of AI virus have infected 130 humans in China since March 2013, causing 43 deaths3. Most human cases of AI infections to date are believed to be caused by exposure to infected birds, either dead or live. The virus is not known to transmit from person to person so far. However, natural mutations in the virus may enable it to cross over and spread among humans4. With its high mortality potential, increased transmissibility will render it sever threat to public health1. Moreover, wide-spread outbreaks in poultry severely debilitate local as well as international trade.
Two groups, one based in US led by Dr. Yoshiharo Kawaoka at University of Wisconsin Madison and another based in Netherland led by Dr. Ron A. M. Fouchier at Erasmus Medical Center, have been specialized in avian influenza research. Their recent experiments aimed to create novel strain of AI virus with enhanced transmissibility in ferrets triggered intense controversy5,6. Both scientific and general community expressed their concerns regarding utility and handling of super-mutant viruses.
‘Gain-of-Function’ (GOF) Experiments
Research groups engaged in creating mutant AI virus strains argues that in order to assess the pandemic potential of natural virus completely, further investigation is required which may involve ‘gain-of-function’ experiments3. These experiments are aimed to identify mutations which can enhance immunogenicity, host adaptation ability, drug resistance, transmissibility, and pathogenicity of the natural virus. Due to its close resemblance with human infection, ferret infection model is commonly used among influenza research community. Studies on these mammals have shown that relatively small set of mutations in H5N1 virus enables its respiratory transmission. Such genetic changes, if acquired by naturally circulating virus could result in worst outcome for human population. The proponents of these experiments propose that knowledge gained from genetically engineered virus research can help identifying set of the mutations to look for during the epidemic, and designing vaccines and pharmaceuticals in advance which can counteract the viral resistance. They also claim that controversy surrounding these experiments has increased dialogue on the matters of biosafety and biosecurity, and raised public awareness in this field3.
Concerns
Experiments involving genetically modified viruses have stirred up numerous concerns not just among the general public but also among the research community. Genetically engineered viruses resulting from GOF experiments often regarded as ‘Potential Pandemic Pathogens’ (PPPs) due to its potential for enhanced transmission and substantial virulence. Because of its novel characteristics, current human population is likely to have limited immunity against them7. Some public health experts have expressed their fear that accidental or deliberate release of these PPPs can lead to man-made epidemic. They also remains skeptical regarding benefits of GOF experiments. Suitability of ferret infection model have been questioned by some scientists who argues that the strategy disregards the phenomena called epistasis, which states that phenotype resulting from any mutation largely depends on its interaction with genetic background of different species8,9. Regarding the vaccine claims, the opponents of these studies offers that in-depth molecular mechanism of transmission is not necessary for vaccine development. They further suggests that efforts on improving and stockpiling existing universal influenza vaccines alongside efficient large-scale production would be more worthwhile than mass-producing assorted vaccines which targets limited number of antigens with genetic variations10.
Ethical considerations
Due to its potential for being misused, experiments involving PPPs are usually regarded in context of ‘dual use research of concern’ (DURC)10. Because of its pandemic potency, access to this knowledge and/or pathogens into wrong hands pauses significant danger to public health. According to sixth point of Nuremberg Code, “The degree of risk to be taken should never exceed that determined by the humanitarian importance of the problem to be solved by the experiment.”11 Seventy four national academies of science have also expressed that “Scientists have an obligation to do no harm. They should always take into a consideration the reasonable consequences of their own activities”12. Both the guidelines emphasize consideration and evaluation of long-term risk to general population. Moreover, second point of Nuremberg Code states, “The experiment should be such as to yield fruitful results for the good of society, unprocurable by other methods or means of study, and not random and unnecessary in nature.”11 The opponents of GOF experiments have put forward several strategies to investigate nature of influenza infections which do not require creation of mutant viruses. Going forward without thorough consideration of these experimental strategies would disregard basic concern of biosafety.
Regulatory guidelines and policies
Soon after confronted by the controversy, research groups working on mutant AI viruses voluntarily declared 60-day moratorium on their experiments to allow government and other regulatory agencies to form required framework. Meanwhile, WHO gathered a panel of experts in the field of avian influenza research and public health for technical consultation, and issued several seminal suggestions for moving forward4. US Department of Health and Human Services (HHS) have delineated the framework for new review procedure for research proposals involving highly pathogenic avian influenza (HPAI) viruses. Scope of this framework includes but not limited to reviewing research proposals for making funding decisions, evaluating potential for significant scientific and public health benefit, and assessing biosafety and biosecurity risks involved13. Centers for Disease Control and Prevention (CDC) also carried out review of required biosafety measures for such experiments and issued recommendations for risk-assessment14.
Restricted Access:
Another important aspect of this issue is access to the knowledge on how to create PPPs by selective mutations of already deadly virus. Experts feared that despite high security storage of mutant viruses, published methodology will be sufficient to generate PPPs for those who have intent to harm. National Science Advisory Board for Biosecurity (NSABB) intervened in this situation and recommended that detailed methodology be excluded from the original manuscripts. While some scientists welcomed the move, the other group was disappointed for depriving responsible avian influenza research community from the useful knowledge. In its technical recommendations, WHO notes that there is no practical mechanism available that allows release of such information to limited audience. Moreover, it would not be too difficult for the expert scientists in the field to figure out the omitted information since there was no novel methodology was utilized4.
Personal verdict
Taking quantifiable risk associated with these experiments into consideration, I would suggest that meticulous and objective risk-benefit assessment should be executed before planning and conducting such experiments. Alternative experimental strategies such as studying infectious nature and genetic variability of field-isolates, and focusing on biophysical interaction resulting from interaction of multiple sites among viral proteins, rather than single amino acid substitutions should be pursued to the maximum possible extent to avoid unnecessary risk. If working out through these considerations needs more time, I would definitely sign on the moratorium.
References
http://www.who.int/mediacentre/factsheets/avian_influenza/en/
http://www.flu.gov/about_the_flu/h5n1/
Fouchier, Ron AM, and Yoshihiro Kawaoka. “Avian flu: Gain-of-function experiments on H7N9.”Nature500.7461 (2013): 150-151.
http://www.who.int/influenza/human_animal_interface/mtg_report_h5n1.pdf?ua=1
Imai, Masaki, et al. “Experimental adaptation of an influenza H5 HA confers respiratory droplet transmission to a reassortant H5 HA/H1N1 virus in ferrets.”Nature486.7403 (2012): 420-428.
Herfst, Sander, et al. “Airborne transmission of influenza A/H5N1 virus between ferrets.”Science336.6088 (2012): 1534-1541.
Lipsitch, Marc. “Avian influenza: ferret H7N9 flu model questioned.”Nature 501.7465 (2013): 33-33.
Gong, Lizhi Ian, Marc A. Suchard, and Jesse D. Bloom. “Stability-mediated epistasis constrains the evolution of an influenza protein.”Elife2 (2013).
Kryazhimskiy, Sergey, et al. “Prevalence of epistasis in the evolution of influenza A surface proteins.”PLoS genetics7.2 (2011): e1001301.
Lipsitch, Marc, and Alison P. Galvani. “Ethical Alternatives to Experiments with Novel Potential Pandemic Pathogens.”PLoS medicine11.5 (2014): e1001646.
“Trials of War Criminals before the Nuremberg Military Tribunals under Control Council Law No. 10”, Vol. 2, pp. 181-182. Washington, D.C.: U.S. Government Printing Office, 1949
Interacademy Panel on International Issues (2005) IAP statement on biosecurity. Trieste (Italy): Interacademy Panel on International Issues
http://www.phe.gov/s3/dualuse/Documents/funding-hpai-h5n1.pdf
http://www.cdc.gov/flu/avianflu/h7n9/risk-assessment.htm

Effect of Inhibitor on Pancreatic Lipase Activity

Lipase is an enzyme that the body uses to break down fats in food into fatty acids and glycerol so that they can be absorbed in the micro villi in small intestine into the bloodstream. Lipase is primarily produced in the pancreas but is also in the mouth and stomach. Most people produce enough pancreatic lipase, but people with cystic fibrosis, Crohn’s disease, and celiac disease may not have enough lipase to get the nutrition they need from food. [1] Similar like other enzymes, lipases help to regulate biochemical reactions in human body. Many of those reactions might happen without the enzyme but at very slow rate. However, if an enzyme is present reaction is faster and more efficient. [2] Role of lipase is represented on figure 1 bellow [3] :
“One of the many enzymes pancreatic juice contains is lipase. As a result of the alkalinity of the bile salts, the pH of the duodenum is approximately 7.0, which is also the optimum pH for pancreatic lipase. Having been fully digested in the duodenum, the lipid-soluble fatty acids and glycerol diffuse through the phospholipid bilayer of the plasma membranes making up the epithelial cells of the small intestine. Milk is a white liquid composed mostly of water (87.3%), with small amounts of fats (3.9%), and non-fat solids such as proteins and lactose (8.8). Milk contains more fat than most liquids and a majority of these lipids are classed as triglycerides which therefore makes milk a suitable liquid to be used for this experiment. Using solid fat such as lard would be impractical because the enzyme lipase would only be able to bind with lipids on the surface of the lard, meaning there would be an extremely slow reaction rate. The globules of fat found in the milk gives the lipids a larger surface area and provides more ‘surfaces’ that the lipase enzyme can bind to.” [4]
Three kinds of tea, oolong, green and black, have been widely used for their purported health properties from ancient times all over the world, especially to prevent obesity and lipid metabolism. [5] In vitro studies with green tea extracts containing 25% of catechins have shown its capacity (in conditions similar to physiological ones) to significantly inhibit the gastric lipase, and in a lower extent also the pancreatic lipase. Thus, the lipolysis of long-chain triglycerides is reduced in a 37%. [6]
In this experiment, we will investigate the effect of green tea extract (inhibitor) on the pancreatic lipase activity in milk (Alpsko mleko with 3.3% of fat). Therefore our research question will be:
How do different amounts (0.5mL, 1mL, 2.5mL, 5mL, 10mL) of green tea (Camellia sinensis) extracts which can act as lipase inhibitor, affect the rate of reaction catalysed by pancreatic lipase?
Defining variables
As can be seen from the research question, our independent variable will be the amount of green tea extract added to a reaction and the depended variable will be the rate reaction, which will be expressed as change in pH per unit of time.
Controlling variables
VOLUME OF MILK: milk serves as source of substrate that is needed for lipase action. In order to ensure the same amount of substrate (lipids) will be available at different amount of inhibitor, 5mL of milk will be added to the test tube at every trial.
% OF FAT IN MILK: as fats in milk are essential for examined reaction, we will use the milk with the same percent of containing fat for all measurements.
ROOM TEMPERATURE: as also temperature can affect the rate of reaction, all the measurements will be done in the same classroom, away from any heat source to ensure equal conditions for all trials. Record the room temperature several times, during the experiment to make sure, that temperature is not varying.
LIPASE CONCENTRATION: 5% solution of lipase will be prepared and used at all measurements into order to ensure the same enzyme concentration at all trials
APPARATUS USED: because different laboratory equipment have different uncertainties, make sure that you will use the same balance/pipette/micropipette etc. for the measurement of the same reagent.
2 MATERIALS lipase powder from porcine pancreas [7]
milk (Alpsko mleko with 3.5% of fat) [8]
green tea
deionised water
electronic balance (± 0.001g)
pH probe
LoggerPro interphase
computer with LoggerPro software
electronic thermometer (±0.5°C)
pipette (5mL ± 0.006mL)
pipette (10mL ± 0.006mL)
micropipette (1000μL ± 0.0006 μL)
measuring cylinder (200mL ± 0.5mL)
250mL beaker with stopper
volumetric flask (100mL± 0.1mL)
25 test tubes (20mL)
test tube stand
stopwatch on computer (±0.5)
3 PRELIMINARY PROCEDURES Preparation of green tea extract
In order to prepare tea extract, measure 200mL of deionised water using a measuring cylinder and boil it. Then steep 5 tea bags into boiled water in order to obtain concentrated green tea extract. After 8 minutes take the tea bags out and pour the extract into the 250mL beaker. Seal the beaker and let the extract to cool down.
Preparation of 5% porcine pancreas lipase solution
Put the 100mL volumetric flask on the balance and reset it. Then, measure exactly 5.00 grams of lipase from porcine lipase into the volumetric flask. After that, add deionised water to the conical flask up to the margin for 100mL. Seal the volumetric flask and swirl it 5 times.
METHOD Connect LoggerPro interphase and pH probe to computer. Launch LoggerPro software.
Go to “data collection icon” and set the length of measurement to 3 minutes at sampling rate 10 measurements/minute.
Measure 5.00mL of milk into the test tube with a pipette.
Add three drops of phenolphthalein indicator to the reagents mixture. Solution will become of purple/pink colour. Stir the test tube.
Place the test tube into the stand and put the pH probe into it.
With appropriate pipette/micropipette, add 0mL/ 0.5mL/ 1mL/ 2.5mL/ 5mL or10mL of 5% lipase solution into the test tube containing milk and Na2CO3 solution and press “Start measurement” immediately after adding the lipase.
After you will start the measurement observe what will happen with the colour of the solution. During pH measurement, the time will be monitored constantly on your computer screen. Record the time at which pink colour will disappear into table 1.
Make 5 repeats for each amount of lipase added on the same graph.
Repeat the procedure for all amounts of green tea added.
DEVELOPING A METHOD FOR DATA COLLECTION
In LoggerPro, you will obtain 6 graphs with 5 curves representing the change of pH per unit of time. Make linear regression for group of curves at each amount of green tea extract added.
Write down the equations of the linear regression lines and plot only that lines into the separate graph. Comment on any apparent trends that you notice. Explain the relationship between the amounts of the inhibitor (green tea extract) added on the pH. Deduce under which conditions, the rate of examined reaction (ˆ†pH/time) was the fastest and where the slowest. Justify your answer with the explanation from the literature.
If the indicator phenolphthalein changed from pink/purple colour to colourless, what type of biochemical reaction took its place?
Calculate the average times needed for purple colour of the solution to disappear. Plot the graph: volume of the inhibitor added versus the average time needed for purple colour of the solution to disappear and deduce how presence of green tea extract as an inhibitor affect the rate of lipase catalysed reaction
Evaluate the method and suggest possible improvements.

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