bio

September 28, 2015, 7:58 pm

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i am bio student and i want to do biotech engineering plz tell me the scope in india and colleges and after that placement with salary

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Update needed on Home-page

September 30, 2015, 6:52 am

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Hello Sunil Nagpal,

When we open the home page of 'http://www.biotechnologyforums.com/' , the 'Overview' section which shows the 'Newest' posts needs to be updated with date column. The other sections have date and time columns associated with them.
The reason is that it becomes difficult for users to keep track of the last post which they had read/visited earlier. Like a newspaper where we can (read/keep track of) all the articles posted on a day.

Workshop: Science, Ethics, and Governance Considerations for Gene Drive Research

October 6, 2015, 8:17 am

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Join the National Academies of Sciences, Engineering, and Medicine Committee on Gene Drive Research in Non-Human Organisms: Recommendations for Responsible Conduct   for a FREE workshop and webinar series to explore the science, ethics and governance of gene drive research. These events are part of the Committee’s information-gathering process as it prepares its consensus report, expected to be released in 2016, and will feature presentations and Q&A sessions with the leading researchers and decision-makers in the field. For more information, please visit the study website at http://nas-sites.org/gene-drives/.

Gene drives are systems to ensure that a gene modification will be inherited by an organism’s offspring, effectively driving modifications throughout a population from one generation to the next. Gene drives are not a new concept, but have recently gained prominence with the development of targeted genome editing with CRISPR/Cas9 endonucleases. This powerful approach is quite different from past approaches to genetically engineer organisms, where the spread of modified genes is not a goal, and mechanisms to prevent their spread are sometimes used.

Workshop: Science, Ethics, and Governance Considerations for Gene Drive Research
Monday, October 28, 2015, 8:00 a.m-6:00 p.m. EDT
2101 Constitution Ave NW, Washington, DC 20418 and webcast
FREE

 
Join this workshop on October 28 to discuss the state of the science of gene drive research, responsible conduct and ethics, perspectives on opportunities and limitations in low and middle income countries, and scales of governance (see preliminary agenda here). Researchers will share the capabilities and tradeoffs of gene drive techniques, the ethics of research on emerging technologies, and mechanisms for the governance of advances in biotechnology. Please register to attend.


Gene Drives Webinar Series
 
Tune in as the committee invites speakers to review the state of the science and scientific approaches to reduce risk of spread to wild populations; to discuss oversight mechanisms for responsible conduct of research; and to assess ethical, social, and legal concerns.


Gene Drive Organisms
Thursday, October 15, 10am – 12:00pm EST
Click Here to Register to attend

This webinar will focus on the latest gene drive research in different organisms, including mosquitoes and rodents, with topics including:

• The Landscape of Gene Drive Research in Different Organisms – Fred Gould, Professor of Entomology at North Carolina State University
  
• Gene Drives in Mosquitoes for Disease Vector Control – Zachary Adelman, Associate Professor of Entomology at Virginia Tech
  
• Gene Drives in Rodents for Invasive Species Control –  John Godwin, Associate Professor of Biology at North Carolina State University
  

Current Status and Next Directions For Basic Research on Gene Drives

Wednesday, October 21, 3:00-4:00pm EDT
Click Here to Register to attend

Join this webinar to review the current status of gene drive research and learn about the next directions for the field with speakers Ethan Bier, Professor of Biological Sciences, and Valentino Gantz, Post Doctoral Researcher, both of the University of California San Diego


Implications of Gene Drive Research on Biosecurity
November 19, 10:00am – 11:45am  EDT
Click here to Register to attend

Explore the implications of gene drive research on biosecurity with this webinar, featuring presentations on:

• General Considerations for Biosecurity– Edward You, Federal Bureau of Investigations
  
• Potential for the Use of Gene Drives in Entomological Warfare— Amesh Adalja, University of Pittsburg Medical Center
  
• Implications of Gene Drives for Agricultural Security - Jacqueline Fletcher, Oklahoma State
  

Get updates on this study by signing up for email notifications: http://nas-sites.org/gene-drives/signup/

Understanding the Basics of Tuberculosis

October 8, 2015, 10:45 am

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Tuberculosis (or TB) is said to infect around one-third of the world’s population.  Although it is one of the most widespread transmittable diseases, it manages to go undetected in most of the cases, resulting in fatal consequences.

 It primarily affects the lungs (pulmonary) as well as some other parts of the body (extrapulmonary), and causes damage to the critical organs, disturbing their functions. 

The fact that the diagnosis can get tricky despite of the latest medical advances is what makes this disease dangerous. There has been repeated campaigning done by the governments for quick identification and treatment of TB but challenges continue to remain.

Here is a detailed look at the way this disease affects the human body, the diagnostics presently used as well as which research areas should be focused on for a better future.


Which organism causes TB?  


The Mycobacterium sp.
Mycobacterium tuberculosis affects the humans.

      

 Source: http://www.bio.davidson.edu

It is a rod shaped, aerobic bacillus and has a lipid rich thick cell wall (mainly mycolic acid) which is mostly responsible for its high resistance to the immunity system and medical treatments. It infects intracellularly.


How does the infection spread?

It spreads through the air when a person inhales the aerosol droplets of respiratory fluids released by an affected person’s cough or sneeze.


What are some common symptoms?

• Persistent coughing for many weeks.
  
• Coughing out blood or sputum is a strong symptom.
  
• Sweating at night during sleep.
  
• Weight loss.
  
• Sudden occurrences of high fever.
  
• Fatigue.
  
• Chest pain.
  
• No appetite for food.
  
• Getting chills.
  

Who are prone to TB?

• People exposed to affected patients or to people who are unaware that they have the disease.
  
• People with poor or compromised immunity.
  
• People suffering from malnutrition.
  
• People with HIV or any other disease which affects the immunity system.
  
• People who smoke or suffer from silicosis, diabetes show increased susceptibility to TB.
  
• People who have weakened immunity system due to cancer treatment.
  
• In many patients, a protein which fights the infection, maybe expressed below the normal level because of a hereditary or acquired mutation in its gene.
  

How does M. tuberculosis cause the infection?

• The bacteria reach lungs and enter pulmonary alveoli.
  

• They invade the endosomes of alveolar macrophages and start replicating.
  

• Macrophages identify the pathogens and start phagocytosis but the phagolysosome formation is inhibited by bacteria. Also, the cell wall helps in protection, in the cases when phagolysosome is formed. M. Tuberculosis blocks certain molecules like autoantigens as well, but continues getting access to nutrients for its growth. Hence, the multiplication continues.
  

Formation of granulomatous infection by Delayed Type Hypersensitivity

• CD4+, TH1 T cells secrete cytokines which induce macrophages, T lymphocytes, B lymphocytes, and fibroblasts to aggregate and form granulomas by surrounding the infected macrophages.
  
• Fusions happen when new macrophages attack the infected ones and multinucleated cells form, resulting in palpable nodules.
  
• The tubercle sequesters the infected macrophage and prevents the infection from spreading further.
  
• The immune system gets suppressed as macrophages and dendritic cells present in the granuloma cannot present antigens to the lymphocytes. 
  
• Necrosis happens in the centre of the tubercles because of high amount of lysosomal enzymes. 
  
• Also, the huge number of activated macrophages release lytic enzymes which destroy surrounding healthy cells. This results in circular regions of necrotic tissues which get calcified as the lesions heal. These can then be seen on X-rays.
  
• In many cases, increased lytic acid secretion may cause the lesion or granuloma to rupture, thus releasing the bacteria into blood or lymph vessels, which is dangerous.
  

                                     

                                              Source: http://www.classconnection.s3.amazonaws.com

How is TB diagnosed?


Here are some methods used to identify a TB patient:

•  Chest X-ray: The doctor generally prescribes a Chest PA to check the presence of any calcified lesions and cavities in the lungs. Fibrotic scars, nodules, cloudy appearance of the lung airspace, enlarged lymph nodes in hila, pleural effusion etc. are some of the common findings if the person is affected by TB. 
  

                                            
                                                           Source: http://www.scielo.br
    

•  Sputum culture: Sputum is the coughed up mixture of saliva and mucus of the respiratory tract and may even contain blood.  This would contain the bacteria if the person is infected. Sputum is collected and sent for stain test. The Ziehl-Neelsen stain or auramine-rhodamine staining, can be used to identify M. Tuberculosis.
  

          PCR is followed to confirm the presence of M. Tuberculosis.

•  Mantoux test : A standard dose of Tuberculin units is intradermally injected into the forearm skin of a person and after around 2- 3 days, the forearm is observed. If there is a hard raised area which may cause pain or give an uneasy feeling, the diameter of the area is recorded. If the area is significant, the person is said to have been infected with the M. Tuberculosis because of which his/her immunity system has mounted a hypersensitive defence to the bacteria. The mount is there as the infected person had already been producing sensitised TH1 cells specific for the injected antigen.
  
          
           Source: http://www.tti.library.tcu.edu.tw
  
  

          This test may result in false positive results, mainly because BCG may have been administered earlier to the patient which causes the
           mount to appear.
          Hence, the test not reliable. It is always used in conjunction with any of the above tests.
          Another test known as the Heaf test is no longer in use.

•  The Antibody from Lymphocyte Secretions or ALS Assay method is used to detect active TB rapidly. The plasma B cells release antibody in response to TB antigens. So, an extracted sample of PBMC is cultured and the supernatant is tested against BCG by ELISA to diagnose TB. 
  

• Scientists have developed the T-SPOT.TB ELISPOT Interferon-γ (interferon-gamma) release assays (IGRAs) in which the property of M. tuberculosis antigens to stimulate the host cells to produce interferon gamma is exploited. Large scale use is yet to happen though it is already in use in many developed countries.
  

•  The health parameters of the patient are checked, like any instances of persistent coughing, sputum production, fever, chills, drop in weight etc.  A full blood count test is also recommended. It is advised to contact the doctor when the regular antibiotic medicines don’t seem to heel a persistent respiratory illness.
  

• Although these are the most used tests, there are many other methods followed as well and the tests conducted vary from country to country. 
  

How to prevent TB?

There has not been any guaranteed method to prevent the infection. However, the following are commonly suggested:

•  Bacillus Calmette-Guérin (BCG): It is a vaccine prepared from an attenuated strain of Mycobacetrium bovis and is intermediately effective against TB infection for around 15 years. The effectiveness varies due to geographical locations, genetic difference between populations etc. It helps in preventing the highly serious forms of the disease like childhood tuberculous meningitis and miliary disease.
  
  
•  Adequate public awareness should be raised by the health officials.
  
  
•  Close contact with high risk people and patients should be avoided, if possible.
  
  
•  Patients should minimise their human contact till they are well set on the path of recovery. They should wear masks to prevent others from catching the disease.
  

Is TB curable? If so, what are the popularly used medicines to treat it?


TB is curable if the patient completes the full course (generally 6 to 9 months) of antibiotics, as prescribed by the doctor. This is also followed up with regular check ups.
The drugs prescribed depend on the location of the TB infection.

These medicines are commonly recommended: (the dose strength and drugs vary with time)

• Some first line drugs are: Isoniazid, Rifampicin, Ethambutol, Pyrazinamide etc.
  

Here’s a diagram explaining how these drugs work.


                                

                                                                                Source: http://www.niaid.nih.gov
  

•  Multi drug resistant TB happens when the bacteria is resistant to the first line drugs, mainly Isoniazid and Rifampicin. MDR-TB can happen in a previously unaffected person or after ineffective treatment of a TB patient with first line drugs. This happens due to mutating bacteria, drug efflux systems, secretion of drug inactivating enzymes etc. In many cases, the ineffective drug is identified and suitable second line drugs are given for further treatment.
  

         Second line drugs classes in case the first line doesn’t work: Aminoglycosides, Thioamides, Peptides etc.

         Examples: Kanamycin, Viomycin, Amikacin, Ciproflaxin, Moxifloxacin etc.

         These are more expensive and can take around 2 years for the course to complete.


Here’s a diagram explaining how these drugs work.

                                        

                                      Source: http://www.niaid.nih.gov

• There are third line drugs as well but their efficacy hasn’t been proved yet.
  

What are the focus areas for research regarding combating TB?

• Researchers are trying to find new cell wall proteins which can be sufficient to trigger an immunity response as well as provide a longer protection in comparison to the BCG vaccine. There are many new vaccines like MVA85A which are undergoing trials.
  
  
• Tuberculosis Necrotizing Toxin was discovered recently and is the only known toxin of Mycobacterium tuberculosis. It kills and escapes from the macrophages by hydrolysing NAD+. This is a major discovery and can pave the future of TB drug research.
  

If you prefer videos, here is a fun way to learn about  TB
 
                                                         




There are many institutions advocating TB research and creating awareness about this disease which can often be a silent killer by going undetected.
The TB patients often hesitate in going to the doctors. Many people think that it cannot be cured. Many don’t follow the entire medicine course. Clearly, better awareness amongst people and quick diagnostics with effective drugs are the needs of this hour.

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Websites and Books referred to :  

• Immunology- Kuby
  
•  en.wikipedia.org
  
•  www.niaid.nih.gov   
  

Any mistakes pointed out will be appreciated.
I have tried to keep it simple for both the general public and the students. If the bacterial infection or any other part is not understood, feel free to contact me via the reply box. It is hoped that the article was informative.
Any suggestions are highly welcomed. Thank you for going through this post.

Meat from vegetable

October 10, 2015, 4:17 am

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Hello. I have an idea. We need gene modify some fruit or vegetable to make it meat. I mean, gene modify this vegetable to make

inside it animal protein. In this case we will have cheap meat which we can grow on garden and we won't kill animals.

What do you think? Is it possible?

anti-HCP Antibodies

October 10, 2015, 4:44 am

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When using polyclonal antibodies to detect an array of host cell proteins what changes to that polyclonal antibody require a recharacterization. More specifically would the following situation warrant a recharacterization: if the non specific binding of the polyclonal antibody is high and, in attempts to lower the background, the antibody is run over a column (that does not contain that antigens of interest).

Pulmonary embolism (PE)

October 11, 2015, 1:09 pm

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Pulmonary Embolism

 

 
Pulmonary embolism is the blockage of a pulmonary artery or one of its branches by a blood-born blood clot or foreign material.

Examples of foreign materials : fat, air bubbles and or a tumor
 
Causes of Pulmonary embolism are :

1-Genetic blood clotting disorder
2-fracture happened to leg or hip
3-Cancer : The chance of diagnosing a malignancy is increased
for around 2 years after an episode of thromboembolic disease; usually, these are cancers of advanced stage with a consequently poor prognosis.23,24 Especially in patients with idiopathic thromboembolic disease, the existence of cancer is very probable and should therefore
be checked for thoroughly.(1)

4-Surgery : Local trauma and orthopaedic operations, especially
in the region of the pelvis, hips, thighs and knees, cause damage to the venous wall endothelium. It is believed that surgery predisposes to PE, for an interval of more than a month post operation. It has been
found that 25% of cases of PE occur 15-30 days after surgery, and 15% after the 30th day. The 18th postoperative day has the highest degree of risk. (2)
 
5-Smoking

6-Hormone Therapy : An important meta-analysis of 12 studies determined that the relative risk of thromboembolic disease in

women under hormone replacement therapy in the post-menopausal period is 2.1, with higher values (3.5) during the first year of treatment.19 An interesting randomized, placebo-controlled, prospective study found that the risk of PE in post-menopausal women taking a combination of oestrogen and progesterone was about twice that in the control group.20 The incidence of PE has also been investigated in relation to the use of raloxifen and tamoxifen for the prevention and treatment of breast cancer. The rate of occurrence of PE in recently published studies was found to be 2.5 to 3 times higher than in control groups. (3)

7-Thrombophilia
 
In one fifth of cases, genetic predisposition is the main cause of PE, although one of the classical risk factors from Virchow’s triad may also be present. The doctor should suspect genetic predisposition when there is:
a) a strong family history of thromboembolic disease;
b) thrombosis in unusual anatomical sites (upper body
or upper limbs, when there is no central line catheter);
c) repeated thrombosis with no known risk factors; d)
thrombosis occurring at a young age; e) resistance to usual anticoagulant therapy.
 
It has been known for a long time that a lack of protein C, protein S, and antithrombin III is associated with an extremely high risk of thromboembolic disease. However, these genetic abnormalities are only identified in 5% of patients with DVT.27 In an even smaller percentage of these patients, insufficiency of the fibrinolytic system (hypoplasminogenaemia, abnormal plasminogen, insufficiency of plasminogentPA activator) and insufficiency of factor XII may be encountered. Relatively recently, a mutation of factor V has been found (replacement of arginin with glutamin in position 506 on factor V) which is known as factor V Leiden. This hereditary abnormality is encountered in a high proportion of the general population with heterozygous dominant form (3-4%) and is responsible for 20% of cases of DVT.27 Factor V Leiden increases coagulability, causing resistance to activated protein C.28,29 Even though by itself factor V Leiden exerts only a mild thrombogenic effect, increasing coagulability by 2-3 times, the knowledge of its existence is extremely important in circumstances that increase resistance to activated protein C, such as the use of contraceptive tablets or pregnancy. The use of contraceptives in combination with factor V Leiden increases the likelihood of thromboembolic disease by 35 times.30 In conditions of increased probability of thromboembolic disease—prolonged immobility, postoperative period, etc.—it is essential to intensify preventive treatment (4)
 
 
8-Contraceptives
 
Third generation contraceptives, which contain newer progesterones, appear to be free of side effects such as acne and piliation. However, they have been implicated in actions related to the coagulation mechanism, such as resistance to activated protein C; there is thus an increased risk of thromboembolism, indeed even greater than for second generation contraceptives. 15 Advanced age and the smoking habit increase the likelihood of complications among contraceptive
users.16 Despite the increased risk of thromboembolism, the chance of a fatal episode of PE remains small. (5)
 



What is pulmonary embolism ?

As we all know that lungs are responsible for breathing process .Lungs consist of alveoli that are responsible for oxygen and carbon dioxide exchange and these alveoli are separated from each other by elastic membranes. Very small blood vessels and capillaries run within these membranes between the alveoli and do the gas exchange
 
As we know, arteries carry the oxygenated blood to the heart to be pumped to all the body while veins carry the deoxygenated blood to the heart and pumped through pulmonary artery to lungs to be oxygenated again. If a thrombus formed and was broken off and entered one of the body veins, it is now in the circulating system and it enters the pulmonary artery and block it, so it will cause 2 problems here, 1-it will prevent the gas exchange 2-Decrease the blood supply to lung cells which will cause death to these cells and cause what is called lung infarction.
Pulmonary embolism is one of the life threating diseases and must be taken care of when the patient comes to any clinic because of chest pain, but pulmonary embolism isn't only caused by blood clots, but also caused by other materials I mentioned above as :
1-   Fat embolism which is derived from broken femur
2-  Tumor cells from cancer
 
The risk of pulmonary thrombosis is the same as vein thrombosis and they are 3 risks known as Virchow's triad
1-prolonged immobilization or alterations in normal blood flow (stasis)
2-increased clotting potential of the blood
3-Damage to the walls of the veins.
 
Prolonged immobilization is caused by prolonged setting in the car or spending a lot of time in hospital while increased clotting potential of the blood is due to medication like estrogen intake or birth control bills or cancer or smoking or increased blood cells level in the blood
 
Signs and symptoms

1-Chest pain : which is described as sharp and get sharper while talking a deep breath
2-Cough that may produce bloody sputum
3-Shortness of breath
The patient may show stable rate of respiration, heart rate and blood pressure but often have elevated heart rate .
Most common sign of pulmonary embolism are :
1-Increased hear rate that is called tachycardia
2-Increased respiratory rate
3-Bluish discoloration of mucus membrane and skin due to decreased oxygen level in the blood
As a result of progression, the heart and respiratory rate increase to compensate the unreached oxygen to body organs , and in case of large thrombus it will lead to complete blockage of the artery preventing blood from entering the lung and as a result no reaches the left side of the lung which will lead to sudden death and collapse
About 25% of patients will pulmonary embolism die suddenly from sudden collapse, breathing stoppage and cardiac arrest without prior symptoms.
 
Diagnosis

Sometimes it is hard to detect the pulmonary embolism in a patient specially if the patient already suffer from high blood pressure or heart rate, so when the patient feels chest pain or shortage in breathing he\she must seek a doctor  .
 
PERC Rule for Pulmonary Embolus
 
Being able to assess a patient and determine the risk for pulmonary embolus is very useful, since many patients have chest pain and shortness of breath when seen in an emergency department or urgent care facility.
The PERC rule suggests that in low risk patients, if the answer is no to the following questions, that the risk of pulmonary embolus is very low (less than 2%) and no further evaluation for pulmonary embolism is necessary or required:
·        Age greater than 50
·        Heart rate greater than 100
·        Oxygen saturation on room air less than 95%
·        Previous history of venous thromboembolism
·        Trauma or surgery within the last 4 weeks
·        Coughing up blood
·        Exogenous estrogen prescription
·            Unilateral leg swelling  
 


Basic test 
 
1-CT : To see across your lung



2-CBC : complete blood count
3-electrocardiography (ECG): This test measures your heart’s electrical activity.
4-Chest X-ray



and 


X-ray of pulmonary embolism due due to foreign body blockage like gun shot (missile pulmonary embolous) 

5-Pulmonary angiogram : This test involves making a small incision so your doctor can guide specialized tools through your veins. Your doctor will inject a special dye so that the blood vessels of the lung can be seen
 
6-MRi
 
 
Treatment
 
Treatment of pulmonary embolism depends on the size and location of the clot.
 
1-Anti coagulants :
 
a-Heparin : is given by injection and it is initially used because they start working immediately
 

b-Warfarin : If you're diagnosed with a pulmonary embolism, you'll usually start taking warfarin tablets after you've have the initial injections of heparin.It is usually taken for 3 months . Warfarin can cause a wide range of side effects, including:

1-bleeding problems

2-headaches

3-diarrhoea

4-nausea and vomiting 

5-jaundice

 

2-Surgery may be necessary to remove problematic clots like :

a- vein filter: the doctor will use thin wire and to put a small filter in your inferior vena cava. It prevents traveling of blood clot in your veins from your leg to your lung

b- clot removal: a thin tube called catheter suctions large clots from your artery .(it isn't not usually effective due to difficulty faced)

c- open surgery: It is used in emergency situation in case of shock or clots can't be removed by any medications

 

Prevention:

 

1-Take a break from setting

2-Drink plenty of fluids :water prevents dehydration which is a causing factor of clot but avoid drinking alcohol

3-Physical activity

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
References:
 
(1) Prandoni P, Lensing AW, Buller HR, et al: Deep-vein thrombosis
and the incidence of subsequent symptomatic cancer. N
Engl J Med 1992; 327: 1128-1133.
 
(2) Huber O, Bounameaux H, Borst F, et al: Postoperative pulmonary
embolism after hospital discharge: an underestimated
risk. Arch Surg 1992; 127: 310-313.
(3) Cummings SR, Eckert S, Krueger KA, et al: The effect of
raloxifene on risk of breast cancer in postmenopausal women:
results from the MORE randomized trial. JAMA 1999;
281: 2189-2197.
Cuzick J, Forbes J, Edwards R, et al: First results from the
International Breast Cancer Intervention Study (IBIS-
(4) Ryan DH, Crowther MA, Ginsberg JS, et al: Relation of factor
V Leiden genotype to risk for acute deep venous thrombosis
after joint replacement therapy. Ann Intern Med 1998;
128: 270-276.
Zakynthinos E, Pantazopoulos K: Acute mechanical mitral
valve thrombosis early after neurosurgery: is factor V Leiden
mutation contributing? Int J Cardiol 2004; 96: 487-488.
 
(5) Parkin L, Skegg DC, Wilson M, et al: Oral contraceptives and
fatal pulmonary embolism. Lancet 2000; 355: 2133-2134.
 
 
 

Human Genome Project

October 12, 2015, 12:35 am

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What are the new features of Human Genome Project???how this differ from basic genome project? What are the benefits of Human Genome Project?

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Any relations between cell differentiation and cell division?

October 12, 2015, 1:13 am

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Any relations between cell differentiation and cell division? I do not know whether the cells division is needed when comes to cell differentiation. Anyone knows? Thanks.

looking for reviewers

October 14, 2015, 3:05 am

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Greeting
i am new author , I am looking for reviewers for my paper submission with title: (Study the Influence of Nitrogen on Renin Production by Fungi Rhizomucor miehei using Solid –State Fermentation )


i dont know persons to review my research ,could any body here tell me if he can review my paper ,he should have email on institute website (not @hotmail or @yahoo ,ie @ institute.org)

thanks

HIV-AIDS : One of the Biggest Global Threats of this Century

October 14, 2015, 5:59 am

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One Day in the Doctor’s Clinic:  

Muhammad Ali is a poor truck driver. He has to travel months in truck, spending sleepless nights away from his family. But for last few weeks he cannot go to his work. He is suffering from flu and some other symptoms like sore on throat, rash, muscle and joint aches and pains. In spite of taking proper medications as per the doctor, no improvement occurred. Finally the doctor advised Ali to have a test, a test of HIV-AIDS and this is the day when he is supposed to have the confidential test report.
Doctor : "…Mr. Ali, as per the test reports, I am sorry to say , you are HIV positive….
Ali : (Breaks into tears) …doctor please recheck the reports…
Doctor: Sorry . I am helpless, all the test reports confirm you are HIV+ .
Ali : Doctor, AIDS doesn't have any medicine right? If my family and friends come to know about this, they will not even talk to me, they will leave me. I have heard AIDS spreads by shaking hand also, how can I live such a cursed life, doctor? Better you give me poison ! (with breaking voice and tears)
Doctor: Wait my dear, don’t be hopeless. You are not alone. There are millions of people in the world like you and I have told you are HIV+, but never told you are having AIDS. Yes, it doesn't have proper medicine to cure completely, but it can be controlled. You are lucky that you have got to know about this at very early stage. Now if you take the drug properly, you ‘ll have a normal life span, and one more thing, it is not an infectious disease, so if your friends shake hands with you, take lunch there is no way of transmission until you involve in a physical relationship or your blood gets in contact with the other…"

HIV & AIDS :  

Yes, Ali is not alone. There are around 37 million HIV+ around the world. But a person with HIV+ is not affected with AIDS? It is true indeed. HIV stands for Human Immunodeficiency Virus which is a retrovirus. Among different HIV, HIV-1 is the strain that affects the Human (Homo sapiens) . It breaks the immune system that actually protects a person from different diseases.  Suppose a country say India has its own defence mechanism to protect its people. Now if someway the border security force are killed and all the army base camps are destroyed by any external forces, all the terrorist groups and other neighbouring countries those want to damage will be happy to attack. Same way when HIV breaks down the immune system by destroying the CD4+ lymphocyte cells, all the microorganisms that healthy individuals can harbour with no illness, will cause serious disease in such impaired immunity. A simple fever may also be fatal. This is called AIDS i.e. Acquired Immunodeficiency Syndrome, a syndrome where the body becomes immunodeficient to protect a person. That means when HIV infection is at the very beginning, if one can control the virus population, he can still protect himself from developing AIDS.

In 2014 1.2 million people died of AIDS. So HIV is the cause of the resulting disease AIDS. But the difference with other viral disease with HIV is that once a person gets affected with HIV-1, he will carry it until the last day of his life. The only way to control this is to take regular Anti-Retroviral Therapy (ART) as prescribed by the doctor. AIDS is the extreme stage of HIV-1 infection when the body is no longer capable of defending the opportunistic diseases. The normal CD4+ T cell in a healthy person varies from 500 cells to 1600 cells per mm3 of blood. But when this number falls below 200 cells/mm3 one is supposed to develop AIDS.  

[Table-1: Created by Author, SOURCE: CDC guidelines for AIDS diagnosis, 1993 revision. Note: All categories coloured in Red is indicator of AIDS ]

Origin of HIV :

By using gene sequence evidences and other Phylogenetic analysis scientists have concluded that the present version of HIV (Human Immunodeficiency Virus-1) is a mutated form of SIV ( Simian Immunodeficiency Virus) which is found in the chimpanzee of Western Africa. It is thought that when ancient Africans hunted these chimpanzees the SIV entered to human body by direct contact of blood. With time it mutated in the human body and got spread through out the world.

Molecular Mechanism of HIV-1 Infection in Human :

Before going into the molecular mechanism of HIV-1 infection let us have a look on the HIV-1 genome organization. Many information about the life cycle of HIV-1 have been obtained by the in vitro studies of HIV-1 infected mammalian cells. HIV-1 is a retrovirus meaning it is having RNA as its genetic material. In this ribonucleotide sequence, there are 9 genes namely
•gag :The gene product of gag are p17, p24, p9, p7 . These proteins are responsible for making the nucleocapsid of the offspring virus.
•pol : The gene products are p64( having Reverse Transcriptase and RNase activity), p51(having reverse transcriptase activity), p10 (protease activity that cleaves the gag precursor) and p32 (with Integrase activity).
•env : The resulting protein of this gene is gp 41 and gp 120. The gp 120 appears to play the most vital role to bind and interact with the CD4+ in T-cells while the gp 41 helps  in the fusion process.

Among other six genes tat, rev and nef encode for regulatory proteins, vif and vpu code for proteins essential for virion maturation and the last one vpr encodes for a weak transcriptional activator.

[Fig-1 : A. HIV-1 Structure. B. The HIV-1 gene organization. Created by Author. Source: Kuby Immunology 6th ed.]

Now let us consider the steps of HIV-1 infection in Human:

1) In the immune system both the T helper cell subpopulation and the macrophages play major role in defending the invading microorganisms. In case of HIV-1 , the transmembrane protein gp-120 on the surface of the virus can only interact with the CD4+ of the T helper cell as well as the monocytes. But, this interaction can’t turn to a successful attachment until a co-receptor helps. For T helper cell and macrophage it is CXCR4 and CCR5 respectively.
2) After the successful attachment the fusogenic domain of gp 41 and the G-protein coupled co-receptor (CXCR4 or CCR5) on the target cell enables the fusion of the plasma membrane with the virus capsid. Then the viral genome along with the enzyme machinery enter the cell keeping the coat outside.
3) After getting into the cell the reverse transcriptase makes RNA-cDNA hybrid from the information of ssRNA template. Next the original ssRNA template is degraded by ribonuclease and 2nd DNA strand is made to form dsDNA.
4) Then the viral ds DNA is translocated to the host cell nucleus and integrated into the genome by the integrase enzyme forming a Provirus stage.
5) The viral transcription factors now stimulate the host cell transcription and make ssRNA followed by the translation to make necessary proteins.
6) Next the ssRNA assembles with the proteins including gp 41 and gp 120 and buds out forming viral coat. Finally they release bursting the host cell.

This way the main two warrior of the immune system starts dying and the whole body becomes susceptible to opportunistic infections leading to AIDS.

[Fig-2 Life Cycle of HIV Source:graphs.net]

The Symptoms :  

From the day of exposure to HIV-1 the symptoms differ in various patients with time. In many patients no usual symptoms appear and HIV remains undetected for several years until it ultimately develops to AIDS. That is why HIV can be only confirmed by testing an individual. There are mainly three phases of developing AIDS.

1) Acute Stage:These are the symptoms appearing due to the natural response of the body to the infection within the 3-4 weeks of exposure and often referred to Acute Retroviral Syndrome. The symptoms include
• Flu with fever
• Rash
• Sore on the throat
• Headache
• Pain in the Muscle and the joints
2) Chronic Phase: After the acute phase which continues from few weeks to months, in the Chronic phase as the body tries to defend the HIV and able to do so to some extent mostly no or mild symptoms occurs. This lasts for about 10years. This is the stage where if the Anti Retroviral Therapy (ART) is taken a person can live his normal life span without developing AIDS.
3) AIDS : If no ART is taken HIV-1 keeps replicating and increasing their population by destroying the body’s natural defence system throughout the chronic phase. When they become successful in doing so in around 9-10years, the opportunistic diseases starts attacking, and actual symptoms of AIDS appears. Some of the symptoms are given below:
• Rapid loss of weight
• High fever with night sweats
• Extreme tiredness
• Swelling of the lymph glands in the armpits, groin, or neck
• Diarrhea mostly lasts for few 1-2 weeks
• mouth or genitals sores
• Pneumonia
• neurologic disorders like depression and memory loss.

Some very common opportunistic diseases at this stage are :
i) Candidiasis (caused by Candida albicans) : Sores in the mouth (thrush) appears.
ii) Vulvovaginal yeast infection that does not respond to treatment.
iii) Coccidioidomycosis (caused by a fungus called Coccidioides immitis): High fever.
iv) Cryptococcosis (caused by Cryptococcus neoformans) : This fungus enters through the lungs and cause pneumonia.
v) Tuberculosis (caused by Mycobacterium tuberculosis).

The Modes of Transmission of HIV-1:

There are many misconceptions among people about the transmission of HIV-1 from the patients to normal individual. It needs direct contact with at least one of the body fluids such as Blood, Semen, rectal fluids, pre-seminal fluids, vaginal fluids or breast milk.
Here is the list of possible modes of transmission:

[Fig-3: The Modes of HIV Transmission: Source: Aids.gov]

A) Most Common Route:
• Sexual relationship between a HIV+ individual with an unaffected person is the most common way of HIV as reported till date. Among different sexual behaviour it is found that Anal sex is much more risker than the vaginal sex. In US most of the reported cases of HIV+ are the homosexual men.
• Also having multiple sex partner having high risk of transmitting HIV.

[Fig-4: Different modes of HIV Transmission in US as per CDC 2010 data. Created by Author. Source: Source: Centers for Disease Control and Prevention. "HIV Infections Attributed to Male-to-Male Sexual Contact — Metropolitan Statistical Areas, United States and Puerto Rico, 2010" MMWR 61 (47): 962-966.]

Here is a well documented video that will explain different modes of HIV transmission including some preventive measures that will be discussed later in this article.

B) Possible other routes:  
• An infected mother can transmit HIV to her child during her pregnancy.
• By using syringe/needle that is already used by any HIV+ individual.
• At the time of blood transfusion or organ-transplantation.
• Any direct contact with the blood of the patient.

C) Impossible ways: Here is the misconceptions people have about the transmission of HIV. Genuinely speaking HIV is a virus and it cannot sustain outside the host cell environment. Even in semen it can hardly be viable 30sec outside the body. So, the following can never cause HIV-AIDS:
• Air or Water
• Mosquito bites or any kind of insects.
• Shaking hands or hugging a HIV+ individual
• Sharing Lunch-dinner in the same plate, this transmits LOVE not HIV.

How Do I  Know, if  I am Having HIV? Simple, Get Tested :

As already told before no symptom is enough to ensure the presence of HIV in an individual because from the acute phase to the AIDS symptoms vary significantly, sometimes it can disappear for years. So, the only way is to get diagnosed.  The most common way to test HIV is to detect the anti-HIV antibody present in the serum of the patients after months of the infection has occurred. When the body starts to make anti HIV antibody, the person is said to be Seroconverted or seropositive for HIV-1. The following graph shows the serologic profile of a patient over time.

[Fig-5: Serologic profile of HIV infection showing three stages in the infection process. Source: Kuby Immunology , 6th Ed. ]

There are separate modes of diagnosing HIV infection. Some of those are discussed below.

1) Antibody screening test (Immunoassay): Most common test for antibody detection is ELISA (Enzyme Linked Immunosorbant Assay). It is having a sensitivity of 0.0001-0.01 microgram of antibody/ml of sample. The procedure of detecting HIV is shown in the image below.

[Fig-6:  HIV Detection by ELISA Essay , SOURCE: http://tube.medchrome.com/2011/11/elisa-...-detection]

Though it is a very sensitive assay, sometimes it may give false negative results. As the body needs some time to develop antibody against the antigen from the day of infection. So, if the result comes negative in spite of having symptoms, the patient must have repeated test as the window period (the time between one gets exposed to HIV but no test can detect presence of HIV )vary from patient to patient.  As this is done as a part of screen test, some other tests (like Western Blot) needs to be done to ensure the infection once the ELISA test shows  positive result.

2) Western Blot Test : This is a test by which the different proteins of the HIV from the infected sample is separated by polyacraylamide gel elctrophoresis.  Generally blood sample is taken for study. When antibody for both the gag and env is detected by the ELISA or other antibody detecting assays, doctor confirms HIV infection after getting positive result in Western Blot.

[Fig-7 Western Blot Pattern for HIV Test . Source: http://www.bio-rad.com ]

3) Viral Load test : Viral load is the amount of viral particle present per ml of blood in the HIV+ individual. This is detected any of the following procedure: RT-PCR , Branched chain DNA(bDNA) method or the nucleic acid sequence based amplification method.
This is generally done when someone is diagnosed  positive for HIV infection. This is because of the regular monitoring of viral load from the base value keeps the doctor on track how the patient is responding to the Anti Retroviral Therapy (ART).

4) CD4 Count: Along with Viral load test constant monitoring of CD4 count is also done. CD4 count id the number of CD4+ lymphocytes present per ml of blood. As the viral load and CD4 count is inversely proportional , i.e. if the no of virus particle increases definitely the no of CD4 lymphocytes will be destroyed and the immune system will get weaker with time. So both keeps the doctor on track.

5) Antibody Differentiation test: This test is done to ensure that the HIV infection is due to  HIV-1 or HIV-2 .  

6) Rapid Test : These test are very quick and reliable for screening. These tests use fluid samples like blood, oral or vaginal fluid  and gets the result within 20 mins. Presently a number of  Rapid HIV test kits are approved by FDA. Below is a very short video on Rapid HIV Testing.

7) Home-bases test:
• Home Access HIV-1 Test System : This is an anonymous test. Here the patient will take the kit and after blood sampling send it to the FDA approved laboratory. The report can be obtained in the next 3-4 business days.
• OraQuick In-Home HIV Test : This kit gives very quick result at home.

How to face this Global Threat?--The Prevention:

To prevent any disease, the first thing to be considered is to spread awareness about the disease. Based on the modes of transmission of HIV-1 , here are some few measures to prevent it.

1) Sexually transmission:
i) Take precautions like using condom (for both male and female).
ii) Don’t live a polygamy life and be honest to your partner.
iii) Live a sexually disciplined life.

2) Blood or other modes :
i) Screening blood samples. This must be done by the blood banks.
ii) Always use fresh needles or syringes.

In case of mother to child , testing should be done at the time of pregnancy. If the result is positive proper medication and advice should be taken from the doctor.

Above of all , if the report is positive for an individual , appropriate measures must be taken along with the full course of the Anti Retro Viral therapy as prescribed by the doctor.

Combat with HIV - The Anti Retroviral Therapy (ART)  :

A number of therapeutic agents have been developed to treat the HIV in the infected patient. All these anti retroviral drugs mainly target at different stages of HIV life cycle. Here is a short video featuring how HIV infects an individual and the machinery that can be blocked to reverse the effects in an infected person.

So, as shown in the video, the main targets of the Anti-Retroviral drug fall under four categories:

1) Blocking Reverse transcriptase action by Nucleoside and Non-nucleoside analogue: These are actually the nucleoside analogues that mimic the naturally occurring nucleosides. If these are incorporated into the growing chain of cDNA made by reverse transcriptase, there will be a chain termination and no functional cDNA will be made. Thereby stopping the lifecycle. Example of such FDA approved drugs are Zidovudine, or AZT (Azidothymidine).

2) Inhibitor of Protease : After the proteins are synthesized in the host cell , these need to be cleaved for making functional virus particle by the enzyme called Protease. These class of drugs inhibit the protease activity and thereby preventing the formation of new mature virion. Examples for such drugs are Nelfinavir (Viracept) , Indinavir (Crixivan) etc.

3) Inhibitor of Integrase : Scientists have developed drugs that can inhibit the integrase activity so that the viral dsDNA cannot integrate to the host cell genome. Currently FDA approved available drug for Integrase inhibitor is Isentress (generic name raltegravir) and Tivicay (generic name Dolutegravir). Researchers are still  working on in this topic.

4) Fusion Inhibitors-Blocking the attachment of HIV to the Host cell : Research is going on to develop therapeutic agents to target both gp 41 and CXCR 4 / CCR5 and stopping them from activating the G-coupled receptor signalling pathway. Currently FDA approved available drug for Fusion inhibitor is Fuzeon (generic name enfuvirtide, T-20).

Though the mode of action of these drugs are different, one single drug is not effective to treat HIV. For this reason often in ART, a combination of the nucleoside analogues and protease inhibitors are used. This is designated as HAART (Highly Active Anti-Retroviral Therapy). The main disadvantage of using all these drugs is most are not  HIV-1 specific. So, they once enter the host cell can equally affect the host cell machinery also and thus killing the host cell. Another point is that there are number of side effects seen in the patients taking ART.

A Look into the Current Research Scenario:

All the research going on different parts of the world focus on three aspects of combating HIV-1.

1)  Developing more effective Anti-Retroviral Therapy : This is the traditional approach followed by various scientist since HIV epidemic. Till date there are six lines of anti-retroviral drug developed in total. Introduction of HAART (highly active anti-retroviral therapy) significantly reduced the mortality rate of HIV patients. Scientists of the The Aaron Diamond AIDS Research Center, New York under The Rockefeller University are committed to find effective ways to target HIV and improve the efficiency of ART.  

[Fig-8 : Changes in survival of people infected with HIV as more efficient ART is introduced with time. Source: Lohse et al (2007), "Survival of persons with and without HIV infection in Denmark, 1995-2005."]

2)Eradicating the HIV Reservoirs : It is true that HIV cannot be cured. Once a person gets affected he continues to carry it until his death. It doesn’t matter how much efficient and effective ART you take. ART can help you only to lower down the viral load i.e. the number of virus in your body by blocking specific part of the HIV life cycle. But it cannot make it to zero. Here the term comes called HIV Reservoirs. As we have seen the after getting into the host cell the cDNA made by the reverse transcriptase enzyme gets integrated to the host genome. Now if new virus particle manufacturing is disturbed by ART, still HIV is hidden with it’s genomic information with in the infected blood cells. That is why after several years of taking ART course when one discontinues it, soon within few weeks the HIV symptoms reappear. So if the hidden HIV reservoirs can be eradicated fully only then a person can be cured. But this is not successful yet. The leading research on this aspect is being done by AIDS Research Alliance of America, Los Angeles, CA. They are trying to develop Prostratin , a strategy by which the HIV reservoirs can be eradicated. (A detailed update to this novel approach will be posted soon )

3) Developing HIV Vaccine : It is pretty sure that developing a vaccine is the only way to have a permanent cure of HIV. So far in human history most of the viral infectious diseases like small pox , polio are defeated by developing novel vaccine. But the problem for HIV is that vaccination can be done only when it is found that an individual is fully cured of HIV infection which is not reported yet. More over HIV is an infection not a disease. So the development of memory cells and the natural vaccination phenomena don’t fit to this. But, ground breaking research is going on in various laboratories of the world. The International AIDS Vaccine Initiative, New York is one of the leading labs with high progress of developing the vaccine. (Why developing a HIV vaccine is such a challange? An update on this is coming soon )

4) Microbicides : Other aspects are developing some strategies like Microbicides which are gels, foams or creams that can be applied topically inside the vagina to prevent sexually transmission of HIV . National Institute of Allergy and Infectious Diseases under the National Institute of Health , US are doing some extraordinary research in this field.

A messege to the reader:

Dear reader , 1st December is coming soon. Like every year we all ‘ll observe this day by different events , campaign, slogan etc. to raise awareness and to support the HIV affected people. But what about the rest 364 days? In those days what most of us do is to maintain a safe distance to a person with HIV+. I don’t know if you have a prior knowledge about HIV-AIDS or not, but after reading this I hope you must have some clear ideas of it . Now I wish that you can help others to understand about this global threat and clear the myths-misconceptions about HIV-AIDS among the people around you. Every day about 5,600 people contract HIV which means approximately 230 every hour. There are many Muhammad Ali-s around us. We cannot cure them, we cannot make them HIV free. But we can make them smile for the rest of their life. It is obvious that we cannot help everyone but everyone can help someone. Spending time to give mental support to these helpless people is what we all can do. We can share our knowledge to make people aware about HIV transmission so that they have a disciplined sex life. All these will not make you a HIV+ but definitely make you a good human being. Spread Love, spread Happiness not HIV  

For More information and Current research You can visit:

1) The American Foundation for AIDS Research, New York (http://www.amfar.org/)
2) CDC - Centers for Disease Control and Prevention, (http://www.cdc.gov/hiv/)
3) Aids.gov (https://www.aids.gov/hiv-aids-basics/)
4) U.S Food and Drug Administration (http://www.fda.gov )
5) National institute of allergy and infectious diseases (http://www.niaid.nih.gov/topics/hivaids/...fault.aspx)
6) The International AIDS Vaccine Initiative, New York (https://www.iavi.org)
7) Aids Info (https://aidsinfo.nih.gov/clinical-trials)

Career Opportunities in San Diego AKA Biotech Beach

October 14, 2015, 6:00 pm

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Hello Job Seekers,



I am writing this thread for those who are interested in career opportunities in the Biotech, Pharmaceutical and Medical Device industry. I am a Scientific Recruiter in the San Diego region that has multiple openings that range from entry level top Senior Staff Scientist. My office is centrally located and my door is open to any candidate with recent and relevant work experience with any of the hard sciences used in the science industry. Contact me regarding any questions on the job openings at hand. 


Thanks, 

Ezra Chavarin
echavari@aerotek.com
https://www.linkedin.com/in/ezrachavarin

Microbial Biosynthesis of Contraceptive Hormones

October 14, 2015, 7:50 pm

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Please note:
All figures and schemes referenced throughout the following text are shown in the PDF attached to this thread. Additionally, all references, which are indicated by numbers in [brackets] (sorry, I could not figure out how to make them superscripts), are given at the end of the aforementioned PDF. I genuinely hope you enjoy the read; I guarantee that you will learn something new before finishing.

Introduction

History of hormonal contraception. The term ‘hormonal contraception’ refers to birth control methods in which steroid hormones are the active pharmaceutical ingredients (API). There are two types of hormonal contraception: progestogen-only, in which one steroid hormone (specifically, one belonging to the progestogen class, hence the name) is used, and combined, in which two hormones are used. In 1940, the chemist Russell Marker helped develop the first progestogen-only drug by extracting the phytosterol diosgenin from barbasco, a wild Mexican yam, and converting it into progesterone. However, because progesterone is destroyed by the digestive system when taken orally, it was only available in injection form; thus, a chemical analog that could be more easily and conveniently delivered was sought. In 1951, Carl Djerassi’s lab chemically synthesized norethisterone, the first highly orally active progestin, which was eight times more potent than the progesterone synthesized by the body. Subsequently, an isomer of norethisterone was used as an API in Enovid, the first combined oral contraceptive pill (COCP), which was approved by the FDA in 1960.[1]

Steroid pharmaceutical industry. Steroids represent one of the largest sectors of the worldwide pharmaceutical industry. To date, there are over 300 approved steroid-based drugs to treat a large variety of health complications, ranging from certain cancers to central nervous system and metabolic disorders. In 2007, the global market of the steroid pharmaceutical industry was around $10 billion, and it continues to consistently increase each year. Currently, only the antibiotic sector is larger.[2]

Steroids and steroid derivatives. The primary characteristic structural feature of steroids is an arrangement of four fused rings—three cyclohexanes (rings A, B, and C) and one cyclopentane ring (ring D)—that are comprised of 17 carbon atoms bonded together. This carbon skeleton is called gonane (Figure 1a), and it forms the core of all steroid molecules. However, because the oxidation states of the rings can differ and various functional groups can be attached to the four-ring core, steroids are a diverse group of compounds with many possible derivatives.[3]

 Two types of steroid derivatives are sterols and steroid hormones. Sterols, or “steroid alcohols,” are a steroid subgroup characterized by the presence of a hydroxyl group at position three of ring A (Figure 1b). They are found naturally in plants (phytosterols), animals (zoosterols), and fungi. One well known zoosterol that is vital to animal cell membrane structure is cholesterol, a precursor to numerous vitamins and steroid hormones. Steroid hormones, according to the particular receptor to which they bind, can be grouped into five distinct classes: estrogens, progestogens, androgens, glucocorticoids, and mineralocorticoids. Steroid hormones are signalling molecules that are found naturally in humans, assisting with metabolism, immune functions, and the development of sexual characteristics.[3]
Estrogens and progestogens. Estrogens are a class of steroid hormones that are characterized by an estrane skeleton made up of 18 carbons. Although they are found naturally in both sexes, estrogens are significantly more prevalent in females than males; they are the major female sex hormones and are produced primarily in the ovaries. The three major estrogens that naturally occur in women are estrone, estradiol, and estriol (Figure 2a). Estriol is released during pregnancy and is the least potent of the three. Estrone is the least prevalent of the three, being released only during menopause. Because it is released throughout the reproductive years of women, estradiol is the most prevalent of the three. Furthermore, it is also the most potent, being around 80 times more potent than estriol.[4]
Progestogens, which are named after their pro-gestational functions, are a class of steroid hormones characterized by a pregnane skeleton made up of 21 carbon atoms. Because they precede other steroids in the first step of the steroidogenic pathway, certain progestogens are the precursors to all other steroids—thus, all steroid-producing tissues must be capable of producing progestogens. The major naturally occurring progestogen is progesterone (Figure 2b). Synthetic progestogens are called progestins.[5]

“The Patch.” Ethinyl estradiol (EE) and norelgestromin, an estrogen and progestogen, respectively, are the APIs in the transdermal combined contraceptive patch (matrix-type) Ortho Evra®, known simply as “the Patch”. Its primary mechanism of action is ovulation prevention, but the Patch also inhibits sperm penetration through the cervix by increasing the amount of and viscosity of cervical mucus. Covering an area of 20 cm2, the Patch is applied once-weekly for three consecutive weeks (21 days), followed by a patch-free week. Each Ortho Evra® patch contains 6 mg norelgestromin and 0.75 mg EE and delivers 150 µg norelgestromin and 20 µg EE daily to the systemic circulation for seven full days.[6]

 

Microbial steroid biotransformation

Because of the numerous widespread medicinal applications of steroids, pharmaceutical companies and scientists alike are continually looking for better, more efficient methods (chemical and biosynthetic) to mass-produce steroids. Microbial conversion (or transformation) is one biosynthetic method that has been used to industrially produce steroids for many decades. Steroid biotransformation is achieved through hemisynthesis that mainly starts with β-sitosterol (or diosgenin and other phytosterols) and involves a varying number of sophisticated chemical and microbial bioconversion steps.[2,7]

Advantages. One major advantage of microbial steroid conversion is that functionalization (namely hydroxylation) can be performed both regio- and stereospecifically—thus, conversions can be made at certain sites on the sterol that would otherwise be unavailable using chemical reactions. Additionally, even under relatively mild conditions, several reactions can be completed in one step which is not feasible in chemical-based approaches. Furthermore, metabolic pathways can be constructed in specific sequences in the newly generated strain. Lastly, biosynthetic pathways are, in general, more ecologically friendly than chemical syntheses.[8]

Needs and issues. The overarching need in this area of pharmaceutical research and development is cost-efficient, economical processes to produce steroids. Because many chemical reactions are economical, the number of steroid biotransformations that can compete with chemical reactions on a cost basis on an industrial scale is limited. The primary issue with microbial steroid conversion is the low aqueous solubility of steroids,  resulting in poor availability of substrate to whole-cell biocatalysts. Biotransformation in organic media has been developed to help circumvent this issue; however, the high toxicity of organic solvents to cells is a major limiting factor of this approach. Other limiting factors that are of concern include: the formation of side products; yield variations due to biological variations; undesirable degradation of the steroid product by whole cells; and low selectivity of whole-cell biocatalysts due to the inhibition effect. The advantages and disadvantages of the use of biosynthesis for the production of two APIs, EE and norelgestromin, have been considered and are discussed below.[8]

 

Ethinyl Estradiol

Use as an API. Ethinyl estradiol belongs to the estrogen class of steroid hormones, and it is a commonly used API in various hormonal contraceptives. Coupled with a progestin, EE is an API in both COCPs (i.e.,Ortho-Cyc) and transdermal contraceptive patches (i.e., Ortho-Evra®), and its primary mechanism of action is to prevent ovulation; this is achieved by inhibiting follicle-stimulating hormone (FSH) from being released.[7] The main difference between COCPs and contraceptives patches is the varying pharmacokinetic properties of EE.

The range of daily EE dosage delivered by Ortho-Evra® (~20 µg) is comparable to that of the COCP Ortho-Cyclen® (~35 µg),[7] but drug delivery via the Patch is much more consistent over a given time period. The average steady state concentration of EE and the area under its time-vs.-concentration curve are approximately 60% higher in women using the Patch than in those using Ortho-Cyclen®; however, the peak concentration of EE is 25% lower in women who use the Patch (Figure 3[1]).[6] The adverse effects of these differences are not yet known, but increased estrogen exposure has been shown to increase the risk of certain health complications such as venous thromboembolisms. Additionally, when delivered via the Patch, EE can stay in the blood for up to ten full days, suggesting that a patient could wait up to two days to apply a new patch after removing one and still be protected.[7]

Synthesis of ethinyl estradiol. EE can be synthesized via both chemical and biosynthetic pathways. The chemical process, requires numerous reaction steps and toxic chemicals and is quite burdensome and inefficient. Conversely, a biosynthesis of EE can be performed in three relatively straightforward steps that are explained below. The first two steps of this synthesis are carried out using biological catalysts, and the final step is a straightforward chemical reaction.
Bioconversion of phytosterols to androstenedione. The first step of the synthesis of EE is converting phytosterols to androstenedione (AD), an important precursor to many steroid-based drugs. Peréz et al. tested three different soybean oil samples containing various proportions of three different phytosterols:  stigmasterol, β-sitosterol, and campesterol (Figure 4). Increased concentration of β-sitosterol appears to correlate with the higher yield of AD (Figure 5). The final optimized reaction is shown in Scheme 1.[9] After the discovery of Mycobacterium, this method became widely used in industry because of its low cost and ease of transformation into AD.[10]
Mycobacterium MB3683 was chosen because it gave the highest yield of AD and the lowest yield of ADD. These cells were grown in NB medium for 48 hours, at 30°C, and with shaking at 200 rpm. The cultures were grown to 10 % (v/v), and placed in either 50 mL NB or MS media containing the phytosterols. The media were prepared with a phytosterol concentration of 1 mg mL-1. After the 5 days of incubation at the same temperature and shaking, the cultures were autoclaved and the product concentrations were tested.[9]

As shown in Scheme 1, the yield of AD was 65%, while the yield of the side product was only 2%. Based on the data presented in Figure 5 for yield of AD from the various soybean oils tested, it appears that the β-sitosterol concentration is weakly correlated to higher AD yield. VN-3 had a lower concentration of β-sitosterol and resulted in a lower percent yield of AD as compared to VN-1. The paper suggests that this could be due to a better bioaccessibility to substrates of mycobacterial cells or that a stigmasterol regulating mechanism in the enzyme 1,2 steroid dehydrogenase could be at work.[9]

Malaviya et al.[10] present a mechanism for the biotransformation of β-sitosterol to AD by the Mycobacterium. The side chain cleavage, which is the main step, requires the regeneration of cofactors such as NAD+ and FAD. This process starts by hydroxylating the C27, which is then oxidized to a carbonyl group. Then the C28 is carboxylated.[10] The numbering convention can be seen in Figure 6. The presence of sitosterol helps to induce the two enzymatic reactions. The first of these is catalyzed by three enzymes, while the second is dependent on the dissolved CO2 concentration. In Mycobacterium, side chain cleavage of β-sitosterol can be induced by propionate or by propinyl-SCoA. Dissolved CO2 (1%) affects the yield of AD positively, possibly because excess aeration changes the way the cell metabolizes the substrate. Through the cleavage of one sitosterol, 3 molecules of propionyl-SCoA, 3 molecules of FADH2 , 3 molecules of NADH, and one molecule of acetic acid were created. These products can then be used for the production of ATP. If the sitosterol is broken down completely, 18 molecules of NADH and 7 molecules of FADH2 are created, which means 80 molecules of ATP can be produced from one molecule of β-sitosterol. This presents a challenge for the biological catalysis of this cleavage because it is energetically favorable for the cell to entirely break down the molecule.[10]

The chemical synthesis goes through multiple reaction steps making it unfavorable when compared to the biosynthetic step. The main challenges are cleaving the side chain of the C17 and dealing with the very sensitive steroid ring structure. This, combined with the harmful and toxic reagents like pyridine, means a very lengthy, costly, and low yield, making the biosynthetic route the more adequate method.[10]  However, there are still problems with the biosynthetic pathway and areas for improvement, for example, the problems with the conversion of sitosterols to AD are the degradation of the steroid nucleus and inhibition of the side chain degradation by the reaction products.

Some potential solutions proposed by Malaviya et al.[10] include inhibiting the 9-α-hydroxylase and screening for the improvement of the microorganism to give a higher yield of AD. In order to maintain the steroid nucleus of AD, the action of 9-α-hydroxylase and 3-ketosteroid-1(2)-dehydrogenase must be blocked. The activation of 3-ketosteroid-1(2)-dehydrogenase triggers the formation of a double bond between the C1 and the C2, leading to the formation of ADD. The activation of 9-α-hydroxylase leads to the addition of a hydroxyl group on C9 forming 9-α-hydroxy-4-androstene-3,17-dione. Inhibition of these enzymes would help to boost yields, but can be lethal to the cells. 9-α-hydroxylase is a monooxygenase that is important to the electron transport chain and contains proteins that require Fe2+; therefore, a good way to inhibit this enzyme is to chelate Fe2+, using a chemical like 8-hydroxyquinolone. Also in the commercial arena, scientist are trying to screen and improve the bacteria used. This improvement can be achieved by developing strains which are less sensitive to phytosterol toxicity or by mutagenic treatment that increases the efficiency with which the bacteria cleaves the side chain.[10] 

There is also the major problem of low solubility of the phytosterols in aqueous media, which is the factor that makes this the bottleneck of EE production. Malaviya et al. [10] propose five major ways to get around this problem. These included: biotransformation in two-phase systems, bio-transformation in cloud point systems, biotransformation using immobilized biocatalysts, and microemulsions and liposomes as an alternative biotransformation system. However, these methods have proven inadequate for application industry as of 2008.[10] While research is being conducted to find solutions to the issues with biosynthesis, this process is used in industry because it is a better way to make AD than the chemical method.

Androstenedione (AD) to estrone. The next step of the synthesis is the conversion of the AD formed in the previous section to estrone (Scheme 2).  To accomplish this, the A-ring has to be aromatized, the C19 has to be removed, and the carbonyl group at position 3 has to be oxidized. This step currently is not done biosynthetically in industry, but we propose a biosynthetic method to form estrone biosynthetically using P450arom, a human aromatase.

Currently, the P450arom-expression plasmids have to be constructed. Kagawa et al. [11] describe a method to accomplish this. In brief, the GroES/GroEL expression plasmid pGro12 was obtained from an outside source and the gene of interest was spliced in using site directed mutagenesis. Kagawa et al. also describe a process for the preparation of P450arom, it is summarized below. In order to prepare the P450arom, E.Coli DH5α cells with the expression plasmids were incubated overnight in 5 ml TB with 100 µg mL-1 ampicillin at 37°C. These cultures (2 mL) were diluted into 250 mL TB media in 3 L culture flask and incubated for 4 hours at 37°C.[11] IPTG, gamma-aminolevulinic acid, and arabinose for induction of molecular chaperones GroES/GroEL were added to the culture. Then, the culture was incubated for 28 hours at 28°C.[11] Cells were harvested by centrifugation and lysed, then centrifuged in order to separate supernatant. This solution was purified to extract the P450arom. The yield for this reaction was 13.4 nmol P450 (mg protein)-1.[11]

This P450arom was then added directly to the AD solution. First, the P450arom would oxidize C19, and then activate the concerted elimination of C19. The C19 is eliminated as formic acid, and the 1-β and 2-β hydrogen atoms are eliminated from the A-ring.[11]  The third step that oxidizes the carboxyl group is unclear, but it results in an estrone. We propose doing this step of the synthesis in a fermentation setup. While this change would introduce new issues, such as diffusion and solubility limitations, it could potentially decrease both capital and operating costs by eliminating the protein purification steps. However, both methods would need to be analyzed to ensure that the most economical process is used.

Estrone to ethinyl estradiol. The ethinylation process is a very old one that started in the 1930s.[12] This process includes adding ethyne, sodium, and sodium amide to the estrone (Scheme 3). The ethyne will attack the carbonyl carbon causing the oxidation of the carboxyl group forming a hydroxyl group. This leaves us with the final product, EEl. This process is necessary to change the bioavailability of the estradiol.[13]

Norelgestromin

The progestin norelgestromin (Figure 7) is the second API of Ortho Evra®. It is the progestational component of the patch and acts by preventing the release of luteinizing hormone, which is associated with the initiation of ovulation. The molecule is derived from norgestrel, another progestin, and is the active metabolite of norgestimate.[14] In a study by Abrams et al. [7], administration of norelgestromin through a patch was shown to maintain more consistent levels of drug in the body, which helps to ensure the drug is at a therapeutically effective concentration throughout the administration period. The patch was designed to deliver 150 µg day-1, compared to the pill’s 250 µg day-1. The average concentration achieved via oral delivery was found to be 0.75±0.23 ng mL-1, while the steady state concentration delivered by the patch was 0.83±0.21 ng mL-1. The area under the curve was comparable for the two delivery methods as well.[7]

Norelgestromin presents significant challenges for biosynthesis because of the variations between the API and naturally occurring progestogens. The presence of an ethyl group at C13 of the molecule makes this synthesis particularly difficult because no naturally occurring progestogen has this group,[5] and selective methylation of C18 (methyl group on C13) cannot be carried out biologically. Similarly, the addition of the oxime group at C3 and the ethinyl group at C17 must be be done chemically.  As such, norelgestromin is not a good candidate for a microbial based synthesis; however, a synthesis involving a small biological component can be done.

Synthesis of norgestrel. The synthesis of norelgestromin begins with the synthesis of norgestrel, the key intermediate in the synthesis. The proposed synthesis was adapted from Gibbian et al. [14] and Kleeman et al. [13], and is shown in Scheme 4. Beginning with a two-ring structure, 6-methoxy-1-tetralone, the first step is the addition of a vinyl group via a Grignard reaction, which must be carried out in an organic solvent such as THF or diethyl ether. Following this, a Michael addition is carried out with Product I and 2-ethyl-1,3-cyclopentadione in the presence of a base. This step simultaneously performs a base-catalyzed hydrolysis resulting in Product II, which has three of the four rings of the steroid backbone formed.

The next step is the only biologically active step of the synthesis of norelgestromin. Product II is added to a culture of yeast, Saccharomyces uvarum, to stereospecifically reduce one of the ketone functional groups on what will become ring D (see Product III). The enzyme responsible is a keto reductase, and as such the media may need to be doped with NADP+.[15] The reaction has an overall yield of 44-52%.[14] One method of boosting this yield would be isolating the enzyme and performing the reaction independent of a fermentation process. This would help to rid the system of some of the mass transport limitations that exist in whole-cell catalyzed processes.[13,14]

Following the biosynthetic step, acetic anhydride and a strong acid, in this case toluenesulfonic acid, are used to protect the remaining hydroxyl group on ring D and to hydrolyze the ketone group, allowing ring C to form (see Product IV). Unnecessary double bonds are then saturated in the presence of hydrogen and a palladium-carbon catalyst, followed by potassium hydroxide, methanol, lithium, ammonia, and aniline. This step also unprotects the hydroxyl group on ring D and leaves two unsaturated bonds on ring A (see Product V). The addition of the ethinyl group and the oxidation of the ester to a ketone are then carried out to give norgestrel. This is done by addition of a hindered base (Al(O-iPr)3) in MEK, followed by reaction with lithium acetylide, and, finally, addition of a strong base.

Overall, this synthesis consists of fairly simple organic reactions. Again, this is because of the ethyl group at C13. This group is not naturally occurring, so the use of a natural precursor is not feasible for this synthesis. Therefore, a total synthesis beginning with commercially available reagents is necessary. The use of S. uvarum helps in providing in enantiomerically pure product, but does not shorten the overall synthesis as in the synthesis of EE. With this in mind, a biological synthesis of norgestrel does not provide large advantages over a traditional chemical route.

Synthesis of norelgestromin. The norgestrel from the previous synthesis can be transformed into norelgestromin via a three-step synthesis. The synthesis described was developed in the patent by Tuba et al. [16], and is shown in Scheme 5. The only modification that needs to be completed is the addition of the oxime group at C3. In order to ensure that the group is only added to the appropriate location on the molecule, the hydroxyl group must be protected.

This protection is again accomplished with acetic anhydride and a strong acid, in this case hydrochloric acid. Under nitrogen, a suspension of norgestrel with acetic acid (100 mL), acetic anhydride (6 mL), zinc chloride (2 g), and 6.7% hydrochloric acid in acetic acid (1.6 mL) is stirred for 20 min, then water (5 mL) is added to the mixture. After stirring for an additional 15 min, 18% aqueous hydrochloric acid (3 mL) is added, and the mixture is stirred for another 45 min.  At this point, the reaction is complete, and a crude product is isolated by adding ice water (600 mL), filtering, washing with water, and drying. The total reaction time was about 80 minutes.[16]

Following this step, the intermediate, norgestrel acetate, must be purified. This is accomplished by dissolving the product in dichloromethane (100 mL) and stirring with silica gel (10 g). The mixture is stirred for 30 min, and the gel is then filtered off. The solvent is then boiled off. The remaining product is refluxed in isopropyl ether:acetonitrile (9:1 v/v ratio) mixture for 15 min. The mixture is then cooled in ice water to 0°C, precipitating the product, which is then filtered and dried. The mother liquor can be reprocessed using the same purification method to obtain more product. This step has a yield of 15.4 g (90.5%).[1]

The pure product can then undergo oximation to form norgestimate, a reaction that is again carried out under nitrogen. Hydroxylammonium chloride (76 g) is added to a stirred solution of norgestrel acetate (120 g), acetic acid (1200 mL), and anhydrous sodium acetate (90.2 g). The reaction takes 1 hr and must be maintained under 30°C. The resulting mixture is added to water (10 L) and stirred for 30 min, after which the crude precipitated product is filtered off and dried at 40°C.[16]

The crude product is then dissolved in boiling ethanol (2500 mL), clarified with charcoal (12 g), and filtered. The filtrate is concentrated under partial vacuum (below 40°C) to 400 mL, then cooled to 0°C for 3 hr to precipitate the product. The product is then filtered off and washed with two portions of ethanol (125 mL each), and dried under 40°C. The yield of norgestimate was 102 g (81.6%) with a purity of over 99.5%.[16]

The final step of the synthesis is unprotection of the hydroxyl group on ring D. Again under nitrogen, norgestimate (10 g), methanol (100 mL), and sodium hydroxide (3.25 g) are stirred together at 22°C. After approximately 10 min, the temperature rises by about 10°C and a homogeneous mixture is obtained. The mixture is then stirred for 3 hr at 25°C, and added to chilled water (1000 mL). The pH is adjusted with acetic acid (3mL) to 7-7.5, and the suspension is stirred for 20 min more. The product is then filtered, washed with water, and dried over phosphorous pentoxide at 40°C under partial vacuum. The yield of norgestimate was 8.4 g (94.8%) with a purity of 99.9%.[16]

The overall yield for this synthesis was 70.0%, which seems acceptable. However, the amount of solvent used for each step is quite large, especially when considering scale up of the process. Before a scale up is proposed, a pilot scale operation meant to optimize the solvent use would be prudent to minimize operating and capital cost for a commercial process. Some optimization can also be done when incorporating the two processes. For example, the hydroxyl group on ring D is protected and unprotected twice, so protecting the group early on and leaving it protected until the final step of the synthesis.

Challenges of norelgestromin biosynthesis. Norelgestromin is not a good candidate for microbial synthesis, unlike EE, because of the functional groups circled in Figure 8. The ethyl group is again the most challenging of these groups because it is not naturally occurring, and both selective methylation and ethylation are difficult to accomplish. Thus, using a common natural precursor such as AD for both of the syntheses is not practical. The other two problematic reactions are the oximation and the ethinylation, both of which must be done chemically. Thus, the majority of the functional groups on norelgestromin would have to be added chemically, and the backbone itself has to be made with a total synthesis to ensure the presence of the ethyl group.

 

Conclusions

Microbial synthesis provides a myriad of benefits in the manufacture of steroid molecules. While it has limitations caused by mass transport, solubility, etc., microbes are able to perform complicated chemistry, which would often require multiple chemical steps, in one process. A very good example of this is the microbial synthesis of AD from phytosterols, where the microbe removes all of the unnecessary sidechains and leaves the target intermediate product in a single fermentation process. The fact that the biosynthesis of  estrone can be done in two steps and that forming EE only requires one further reactions, makes it an ideal candidate for biosynthesis.

Microbial synthesis does however have severe limitations when the target molecule does not resemble natural compounds well enough as shown with the synthesis of norelgestromin. The presence of the ethyl group at C13 severely limits the potential for biosynthesis of norelgestromin. Because of this group, the backbone need to be totally synthesized from raw materials as we have shown.

Future work in microbial synthesis includes strain improvement,[10] continued work with two-phase reactor systems, development of green solvent systems, and development of novel hydrophobic delivery methods.[8] These goals are driven by the need to continue improving the productivity of various microbial strains in order to make them more competitive compared to traditional chemical syntheses.

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ZOMBIE SYNDROME, THE WORLD MORE RARE DISEASE

October 15, 2015, 8:34 pm

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INTRUTUCTION
Since it became fashionable zombies, we all want to be one. Not that we have become cannibals and a brain has become our favorite food. Is that one becomes attached to the undead and make you want to get something broken and makeup to go out and scare others. Clearly, unless we are way to a costume party, they will call us for fools. But beware, there are those who are really. Crazy? Dead? Both of them…
Although the idea seems straight out of a novel by Stephen King, there is a condition that makes you a living dead. It's called Cotard syndrome and may well be considered the rarest disease in the world. The sufferer believes that its internal vital organs are paralyzed, their intestines do not work, and their heart is not beating, which have no nerves, no blood or brain. Imagine their bodies in a state of putrefaction and looks, it smells and feels as if this were true. Visual hallucinations occur, as seen in a mirror-shaped body; olfactory hallucinations, and have unpleasant odors, to meat in putrefaction, or tactile hallucinations, feeling that has worms sliding over his skin.
In its more advanced stages, the patient defends the idea that he is dead and is even surrounded by dead, putting people close to him in this area. Along with this belief of death, the patient maintains a sense of immortality, as if he had become a "lost soul". Although this is a typical delusion of the worst depressions such as psychotic or delusional, it can be seen in other severe mental illnesses, such as dementia with psychotic symptoms, schizophrenia, psychosis due to medical conditions or toxic.
[img=724x405]file:///C:/Users/Hogar/AppData/Local/Temp/msohtmlclip1/01/clip_image002.jpg[/img]
This syndrome owes its name to Jules Cotard (1840-1889), a French neurologist who first described the condition in 1880 in a case report of a 43 year old. "Mademoiselle X says it has no brain or nerves or chest or stomach, or intestines; it has only skin and bones of a decomposing body. No soul, for her there is no God and the Devil either. He says he has no need to eat to live and can’t die naturally. Only cease to exist forever if burned. The fire be your only salvation. "He described his patient Cotard. Those who suffer from this disease, do not believe that going to die ... "already dead"...
In 1995 he first performed a clinical classification based on the tests. In a retrospective analysis of 100 cases Cotard syndrome is subdivided into three types. A first type includes a form of psychotic depression, characterized by anxiety, melancholy delusions of guilt and auditory hallucinations. A second type, described as Cotard syndrome type I was associated with hypochondriacal delusions and nihilistic, and absence of depressive episodes. The last group was the Cotard syndrome type II, with anxiety, depression, auditory hallucinations, delusions of immortality, nihilistic delusions and suicidal behavior, and prominent features.
Recently, neurologist Steven Laureys of the University of Liege in Belgium, commented on a patient with this syndrome: "I've been analyzing the PET readings for 15 years and have never seen anyone who was standing, interacting with people, a scan result as abnormal. His brain function is similar to that of a person during anesthesia or sleep. See this pattern in someone who is awake is quite unique. "
Scientifically still they continue to study its causes, although neurology currently set to malfunction of the amygdala and other limbic structures of the brain responsible for emotional--those as the most likely answer you reason. However, it is still an unusual disease.
Main symptoms of Cotard's syndrome:
• Depression
•Suicidal thoughts
• Belief that there is no body. It is a delusion the patient believes to be living some real form when only occurs in imagination
• Belief that are running out of blood.
• Negative thoughts
• Belief that are already dead with olfactory delusions they even smell rotting
• Belief that the worms under their skin
• Belief that they are immortal
• Belief that they are breaking
• Belief that have no internal organs.
• Analgesia or no pain
• Self-mutilation
Management and treatment
Specific management of Cotard syndrome is focused mainly on the management of the underlying clinical condition which is part. As noted in patients with affective disorders, antidepressants can be effective; however, due to the presence of delusions, the electroconvulsive therapy has been strongly suggested by some authors and even has come to pose as the treatment of choice.
When the syndrome is associated with a condition of chronic schizophrenic illness, the prognosis worsens. However, in other schizophrenic patients where the onset of symptoms is rapid onset, use of antipsychotic drugs may favor a good answer.
If the syndrome occurs behind any organic entity, the treatment is the condition that has determined. However, if it appears as an initial presentation of a clinical picture of dementia will be very little chance of improvement in the patient. If the syndrome arises, for example, as a result of a secondary confusional state to an organic condition, as the case reported associated with typhoid, it is clear that the treatment of the disease lead to a complete recovery of the patient.
Other measures to be considered in the management of Cotard syndrome is monitoring the patient, in terms of the possibilities of self-harm and special recommendation in the case of patients with depressive symptoms prevalence of suicide and especially once established the treatment, the patient in the recovery phase reaches regain mobility more actively.
Forecast
The Cotard syndrome prognosis is guarded. Full recovery of the patient can arise suddenly and spontaneously, even in the most severe cases way. However, in cases of moderate intensity they have not developed classical clinical presentation and complete recovery can be rapid or gradual. If the condition is due to an underlying organic condition, evolution is closely related to the patient's chances of recovery of the condition being treated. Sometimes, in the presence of depressive symptoms it may happen that a resolution of depressive symptoms is achieved, but remain delusions.
When the picture is part of a schizophrenic illness, the picture is resolved to the extent that other psychotic symptoms also resolved. Similarly, if the symptoms continue for many years, they can then coexist with chronic schizophrenic condition.
In some cases, life for the patient becomes tolerable and shows a double orientation.  It is to say, it is immersed in a fabulous pseudoreality and, in turn, is able to maintain contact with others. Delirium sometimes has become completely encapsulated consulate, and then the individual is able to assume a jovial mood, and engage in philosophical discussions about their own existence or nonexistence.
CASE
[img=502x374]file:///C:/Users/Hogar/AppData/Local/Temp/msohtmlclip1/01/clip_image004.jpg[/img]
Dr. Steven Laureys is in his office at the University of Liege in Belgium, when it receives a call from his secretary. "It's really important to come and talk to this patient," says so hectic. "He's telling me he's dead."
The patient is called Graham, he is 48 years and one day in 2004 he got out of bed with the conviction that he had died. A few months earlier he had attempted suicide by electrocution, putting a light cord in the bathtub, and the episode caused a depression that led to what is known as Cotard syndrome.
"It's hard to explain," he says. "I feel like my brain no longer existed. They insisted doctors the pills would not work because I had no brain. I fried them in the bathtub." His condition became so extreme that Graham came home one day and went to the cemetery to stay. "I just felt I should be there," he explains. "It was the closest I could be death."
The case went to Adam Zeman, of the University of Exeter in the UK, and Steven Laureys of Liege, whom he underwent a brain imaging test to see what was going on in his head. Concrete, performed a positron emission tomography (PET) and what they found left them impressed: the metabolic activity of the cerebral cortex was more like a person in a vegetative state or anesthetized that a person "wakes up".
"Our data," they write in the study published in the journal Cortex, "suggest that the profound alteration of thought and experience, expressed in Cotard syndrome, it reflects a profound alteration in brain regions responsible for the 'core consciousness' ". His hypothesis, in the absence of further studies of the phenomenon is that the reduction of metabolism occurred in Graham experience that altered the world in which he believed did not exist.
And we say "thought" because, as revealed in New Scientist, Graham has recovered thanks to psychotherapy and medication and left to suffer the syndrome. "I do not feel my brain is dead," he confesses. "Things get a little strange sometimes".
What do you think about this disease?
https://www.newscientist.com/article/dn23583-mindscapes-first-interview-with-a-dead-man?full=true
http://id.tudiscovery.com/el-sndrome-del-zombi-la-enfermedad-ms-rara-del-mundo/

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Biological Characterization

October 16, 2015, 8:39 am

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I am working on a project and that requires various biological characterization of the specimen. I am a mechanical engineer so most of the things are just bouncing of my head. Can anyone please explain the following two procedures 

1. Cytotoxicity Test
2. Hemolysis Test

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Scope in Forensic for biotech students

October 16, 2015, 8:57 am

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Dear Sir,

I am currently in my final year of B.Tech Biotechnology. I am preparing for GATE 2016. I am very much interested in a career in forensic after my M.Tech. I have gone through some of the earlier posts where you have mentioned that biotech students have scope in forensic science. I have checked for job opportunities in various websites but the recruiters want people with a forensic background or with some experience in that field. I would like to know how should I proceed further to have a career in forensics? And which IIT I must choose to help me achieve my goal?

I would like to know about it now so that I can have a clear picture of what I should do.

Thanks,
Sirisha

The Future of Viral Diagnostics: One Test For Every Human And Animal-Borne Virus

October 16, 2015, 10:39 pm

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The Future of Viral Diagnostics: 

One Test For Every Human and Animal-borne Virus

The Ebola Virus

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INTRODUCTION:

A September 2015 study published in the American medical journal Genome Research examines a revolutionary new diagnostic method known as “Enhanced Virome Sequencing Using Targeted Sequence Capture,” or ViroCap.

Developed jointly by the McDonnell Genome Institute and the Department of Pediatrics at Washington University School Of Medicine, the test claims to detect nearly all viruses which affect both humans and animals, and with far greater accuracy than standard diagnostic techniques.

This unique synthesis of scope and precision is expected to yield a wide range of health-care benefits for all people, from identifying lethal viruses, treating common infections, perhaps even the discovery of new, previously unknown diseases.

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THE STANDARD MODEL: METAGENOMIC SHOTGUN SEQUENCING


Before looking at ViroCap an investigation of the prevailing technique is required. This way, the reader can better judge how the two systems compare.

The current model used in analyzing viruses is known as Metagenomic Shotgun Sequencing (or MSS), so called because the DNA sequence is “randomly sheared into small pieces” like a shotgun blast (California State University).  It can be described thus: 

Quote: “a relatively unbiased, culture-independent method in which nucleic acid extracted from a sample is sequenced [and] classified based on similarity to reference genomes. This approach allows comprehensive study of the viral component....”

— Genome Research (September 22, 2015)

MSS:  Metagenomic Shotgun Sequencing

Or to put it in simpler terms, the benefits of MSS include:

• Providing vital genetic information
  
• Ability to classify data into groups
  
• Culture-independence (samples do not have to be grown in a lab and can be taken directly from a patient/environment) 
  

MSS is “particularly useful to studies of the human microbiome, or in layman’s terms, all of the bacteria/viruses/fungi that live in our bodies,” explains MassGenomics.org, an American medical blog dedicated to in-depth analysis of the latest research papers.

However, MSS also has its drawbacks.

According to Dan Koboldt, head of the Human Genetics Analysis Group at Washington University's Genome Institute, “Most efforts to chart the human microbiome have focused on bacteria [which have] relatively stable genomes....Viruses, in contrast, are somewhat under-studied. Part of that is due to the small size and highly variable nature of viral genomes.”



The Human Microbiome


Although MSS is well-adapted for studying the human microbiome, the quote above reveals most of these inquiries have focused on bacterial genomes due to their relative simplicity. Viruses, however, are far more complex and volatile, and therefore less suited to the MSS model.

Another significant flaw of MSS and various diagnostic techniques is their lack of precision. Identifying the countless viruses which infect humans can prove difficult, often requiring several unnecessary tests before a correct diagnosis is found. “That’s because current tests aren’t sensitive enough to detect low levels of viral bugs,” say researchers at Washington University, or they're “limited to detecting only those viruses suspected of being responsible for a patient’s illness.”

Despite its advantages, the statement above shows MSS as less than ideal when it comes to pinpointing viruses. Either the test is not keen enough to find more elusive strains, or multiple attempts are required before determining the correct illness. 

This makes the present system of diagnosing viruses a process of elimination. But sometimes patients do not have any time to spare; while their doctors scramble to learn the cause of their sickness the patient may already be dying.

The recent Ebola outbreak is vivid proof of that reality.

According to a September 2015 report from the World Health Organization, the Ebola virus has left over 11,300 dead. If, however, the lethal virus had been correctly diagnosed back in December 2013, that may have prevented it from spreading beyond Guinean borders into neighboring regions and eventually to faraway nations such as Italy, Spain, the United Kingdom and America.






HEALTH-CARE WORKERS TAKING PRECAUTIONS AGAINST EBOLA


All of these facts and statements demonstrate just how flawed our present day system can be for identifying viruses.

Now let's compare it to the ViroCap test. 

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VIROCAP: WHAT IS IT AND HOW DOES IT WORK?


ViroCap is an alternate method used to enrich the genome sequence of a virus. To develop the test, researchers targeted unique stretches of DNA and RNA from every known group of viruses that infect both humans and animals, according to researchers at Washington University.

The creators of ViroCap describe their new technology as: 

Quote: “a comprehensive viral targeted sequence capture panel that could be used to 1) assess all viruses known to infect vertebrate cells and 2) detect divergent viruses. To this end, we created ViroCap, a targeted sequence capture panel that enhances the detection of a
comprehensive set of viruses with vertebrate hosts.”
— Genome Research (September 22, 2015)

In short, ViroCap differs from MSS in that it specifically targets a certain group of viral genes. Unlike a shotgun blast, ViroCap is more akin to a sniper rifle. However, the data it yields is both detailed and comprehensive.

Additionally, the new test is far more ambitious in scope than MSS: its goal is to analyze all viruses that infect vertebrate cells and even variant strains.

Now as to the question of evidence....

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CLINICAL STUDIES 


To prove ViroCap's superiority researchers performed two human clinical studies, comparing MSS against ViroCap's targeted sequence capture to see which was better at detecting DNA and RNA viruses.

(Note: the virus samples used in both experiments had already been tested positive by the virology lab at St. Louis Children's Hospital)


Process


In each study the same data was sequenced: first, without targeted sequence capture (pre-capture), and then by targeted sequence capture using ViroCap (post-capture). 

Experiment 1:

A total of 14 viruses were present in the clinical samples.


Results:

• 10 viruses were found using MSS pre-capture method
  
• 14 viruses were found using ViroCap's targeted sequence capture
  
• Median percent increase of viral reads was 674 (range >13 to 9335)
  
• Median breadth of coverage increased from 2.1% (range 0 to 89.8%) to 83.2% (range 0.8 to 100%)
  

Researchers describe their findings thus:

Quote:

 “Targeted sequence capture resulted in dramatic improvements in all sequence coverage metrics...including number and percent viral reads, breadth and depth of coverage....”
— Genome Research (Sept 22, 2015)

Primary results have so far established ViroCap as a leader in accuracy, having identified 100% or 4 more viruses than MSS with its targeted sequence capture approach.


Experiment 2:


A total of 18 viruses were present in the clinical samples.


Results:

• 11 viruses were found using MSS pre-capture method
  
• 18 viruses were found using ViroCap's targeted sequence capture
  
• Median percent increase of viral reads was 296 (range >56 to 2,722)
  
• Median breadth of coverage increased from 2.0% (range 0 to 99.9%) to 75.6% (range 13.5 to 100%)
  

Once more ViroCap was the superior of MSS.  In Experiment 2, researchers “again found that targeted sequence capture resulted in dramatic improvements in sequencing parameters.”

The evidence is strong: combining data from both experiments, we discover ViroCap identified a total of 32 viruses compared to MSS's 21, an increase of 52%.

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ADDITIONAL EXPERIMENT DATA

• ViroCap can generate complete/nearly complete genome sequences directly from clinical samples without first culturing the virus
  
  
• ViroCap can identify RHINOVIRUSES by type
  
  
• ViroCap can distinguish between HERPESVIRUS 6B and 6A, ADENOVIRUS types A and C, and POLYOMAVIRUSES JC and BK
  
  
• MSS identified INFLUENZA A pre-capture, but ViroCap classified it specifically as type H3N2 post-capture
  
  
• ViroCap also sequenced ENTEROVIRUS D68 genome directly from clinical samples
  
  

RHINOVIRUS

Once again the facts reinforce ViroCap's targeted sequence capture over the conventional MSS model. Not only can it fully map the genome sequences of a virus, but it does so with remarkable precision.

The significance of this last point cannot be overstated:

Quote:

“...while standard testing identified a virus as influenza A, which causes seasonal flu, [ViroCap] indicated that the virus was a particularly harsh subtype called H3N2. Last flu season, H3N2 contributed to some 36,000 deaths in the United States.”

—Washington University (September 29, 2015)

In the case above, standard MSS testing detected the influenza A virus but failed to pinpoint the H3N2 strain. 

The result? 

36,000 deaths last year in the United States alone; 28,800 in England (DailyMail.uk), and over 600 in Canada (Public Health Agency of Canada). 

Media outlets publicized the large death-toll with headlines like “Flu Season Declared An Epidemic Due To Mutating H3N2 Virus, Ineffective Vaccines (International Business Times).”

H3N2 Strain of Influenza A

But as the experiments revealed, ViroCap was able to precisely identify the virus subtype, “And in some patients – particularly young children, older adults...knowing that the H3N2 strain is present may alter treatment (Washington University).” 

ViroCap's ability to collect detailed genetic data may have helped identify the H3N2 strain of Influenza last year, giving doctors vital knowledge of how to fight the virus and lower the immense death-count.  

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VIROCAP—POTENTIAL MEDICAL BENEFITS


Because ViroCap is such a new medical development studies on its real-world use have yet to be published. According to Washington University it may take several years before the test is clinically available.

So, using my own reasoned arguments backed by proven facts, the following is a list of probable (though still theoretical) future benefits ViroCap may bring.


LOWER COST OF HEALTH-CARE


ViroCap's advantage lies in its precision and ability to detect the most elusive viruses, and for this reason I believe it will lower medical bills. “While PCR tests can screen for up to about 20 similar viruses at one time, it seems that ViroCap could theoretically test for virtually any virus,” reports MedicalNewsToday.com.  

Fewer tests mean lower prices. For example, diagnosing Ebola costs roughly $1000 (Mirror Daily) but according to Genome Research, “ViroCap achieves better viral coverage while requiring...fewer total sequence reads. This increased efficiency has the potential to lower sequencing costs.”

Therefore, it is logical to assume that one of ViroCap's potential benefits would be reduced health-care payments from less testing.



IMPROVED LIFE EXPECTANCY


Researchers at Washington University state: “current tests...are limited to detecting only those viruses suspected of being responsible for a patient’s illness.”  

Compare that to ViroCap:

Quote:

 “With this test, you don’t have to know what you’re looking for...It casts a broad net and can efficiently detect viruses that are present at very low levels...especially useful in situations where a diagnosis remains elusive after standard testing...” 

—Washington University (September 29, 2015)

Doctors will no longer be limited to specific tests which they believe may help them identify a patient's illness, or move from one diagnostic tool to another in the hunt for obscure viruses, but can use a single, far more efficient means of finding the correct diagnosis.

And because it “casts a broad net,” ViroCap can reveal diseases no one was searching for but were nonetheless present. Simply put: the new test can save lives by detecting the nearly undetectable, or identify future health risks through early diagnosis.


BETTER PATIENT CARE


Instead of enduring multiple tests which may cause pain or distress (especially among children, the elderly and mentally impaired) ViroCap would cut down the number of procedures to just one, making hospital care less invasive for patients.

Less testing also reduces risk of infection. The World Health Organization states that every year “hundreds of millions of patients are affected by health care associated infections around the world.” 

As for their impact:     

Quote:

 “health care-associated infections create additional suffering and come at a high cost for patients and their families...prolong hospital stays, create long-term disability, increase resistance to antimicrobials, represent a massive additional financial burden for health systems...and cause unnecessary deaths.”

— World Health Organization (May 5, 2011)

Among several leading causes of infection WHO cites “prolonged and inappropriate use of invasive devices.”

The key word is devices—meaning more than one.

HOSPITAL RELATED INFECTIONS ARE EVER-PRESENT

Of course, not all medical equipment is used for diagnostic purposes, but many still are. And if greater frequency of testing increases the chances of infection, ViroCap's single diagnostic capabilities would reduce the odds of contagion, thereby saving lives and money.



BETTER VETERINARY CARE + SAFER HUMAN-ANIMAL INTERACTIONS


For a vast majority of readers ViroCap's most obvious benefit would be improvements to human health, but it's also a blessing for pet owners and animal lovers. As stated previously, “researchers targeted unique stretches of DNA and RNA from every known group of viruses that infect both humans and animals.”

ViroCap has the power to identify all animal-borne viruses, which means owners can ensure their beloved pets receive the best care from veterinarians. Early detection can lead to early treatment, so our pets can live longer and healthier lives.

VIROCAP MAY HELP PETS LIVE LONGER THROUGH ACCURATE DETECTION OF VIRUSES

Furthermore, endangered species will have a better chance of surviving with this new technology. Zoos and animal sanctuaries often have breeding programs for at-risk creatures, and being able to identify and treat viruses quickly (such as the canine distemper virus which killed four rare Bengal tigers last year—theguardian.com) can prolong their lives, increase populations, and eventually free them from the constant threat of extinction.

VIROCAP HAS THE POTENTIAL TO SAVE ENDANGERED SPECIES THROUGH EARLY AND ACCURATE VIRUS DETECTION

Lions, tigers, polar bears and leopards, these rare and majestic creatures may have a brighter future thanks to ViroCap.

Lastly, safer human-animal interaction can be achieved through ViroCap's keen ability to detect viruses. According to livescience.com, “About 60 percent of all human diseases and 75 percent of all emerging infectious diseases are zoonotic,” meaning they're passed from animals to humans.

Incredibly, over half the illnesses which affect people actually originate in the animal kingdom. And we're already familiar with some of them: Ebola, bird flu, West Nile, Hantavirus.

In most cases infection is spread to humans via livestock (livescience.com), but with ViroCap's ability to detect every known animal virus, there is a strong possibility of lowering the chances for transmission.

Therefore interactions with nature become less of a risk to our health.

HOW VIRUSES ARE PASSED FROM ANIMALS TO HUMANS (ZOONOTICS) 

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CONCLUSION


Although ViroCap is still years away from becoming clinically available it holds great potential. Its unique capacity for scanning a wide range of diseases coupled with its knife-like precision make it a vital tool in the identification and treatment of viruses.

Both humans and animals have a lot to gain from its success: lower health-care bills, increased efficiency and diagnostic speed, and quicker treatment, all of which promise longer, healthier lives.


The fact that the inventors of ViroCap are making their technology “publicly available to scientists and clinicians worldwide, for the benefit of patients and research” (Washington University) serves to help all nations, peoples and animals.

---

The following is a link to entire study published in Genome Research:  http://genome.cshlp.org/content/early/20...9.115.long

---

LIST OF SOURCES (In Alphabetical Order):



Brazier, Yvette. (2015). “A single test could detect almost any virus.” Medical News Today. Retrieved from http://www.medicalnewstoday.com/articles/300259.php


Bryner, Jeanna. (2012). “13 Animal-to-Human Diseases Kill 2.2 Million People Each Year.” livescience.com http://www.livescience.com/21426-global-zoonoses-diseases-hotspots.html


Glum, Julia. (2014) “2014-2015 Flu Season Declared An Epidemic Due To Mutating H3N2 Virus, Ineffective Vaccines.” International Business Times. Retrieved from http://www.ibtimes.com/2014-2015-flu-sea...es-1770728


Koboldt, Dan. (2015). “How to Catch a Virus: Targeted Capture for Viral Sequencing.” Mass Genomics. http://massgenomics.org/2015/10/targeted...ncing.html


Moore, Robert. (2015). “MAJOR MEDICAL BREAKTHROUGH: NEW VIRUS TEST KIT MIGHT DETECT ALL VIRUSES.” Mirror Daily. Retrieved from http://www.mirrordaily.com/breakthrough-...ses/24469/


Rogers, Amy. (2008). “Shotgun Sequencing.” California State University. Retrieved from http://www.csus.edu/indiv/r/rogersa/Bio1...hotgun.pdf


Todd N. Wylie, Kristine M. Wylie, Brandi N. Herter, and Gregory A. Storch. (2015). “Enhanced Virome Sequencing Using Targeted Sequence Capture.” Genome Research. Retrieved from http://genome.cshlp.org/content/early/20...9.115.long


(2015) “Influenza surveillance.” Public Health Agency of Canada. Retrieved from http://www.phac-aspc.gc.ca/influenza/ass...g.php#fig2


(2014). “Dog virus threatens India's dwindling tiger population.” The Guardian. Retrieved from http://www.theguardian.com/world/2014/ja...dia-tigers


(2011) “Health care-associated infections FACT SHEET.” World Health Organization. Retrieved from http://www.who.int/gpsc/country_work/gps...eet_en.pdf


(2015) “New test detects all viruses that infect people, animals.” Washington University In St. Louis. https://news.wustl.edu/news/Pages/28833.aspx

First post with a few questions

October 18, 2015, 12:43 pm

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Hello,

I'm currently in the process of completing a self-administered biotechnology course. Since I don't have a professor or other resources like that, I don't really have an authority to ask these questions to. I'm wondering if anyone can help me answer these questions. Answer is good, but if you could show work that would be excellent as I'm trying to study it.



Question 1) You are given the plasmid pNEB193. Sketch what a predicted gel run would look like if one digests the plasmid with the following enzymes:


Ava II (A)
Pvu I (P)
AvaII+Hind III (A+H)

the sketch is supposed to fit a electrophoresis unit, so it would look like:

Ladder           Uncut           A           P           A+H




^
Those 5 would be the wells 


Then, to follow this sketch, I have the following question:


What would happen if the wells were put by the positive pole instead of the negative?
How might the gel look if one used a higher concentration of agarose? Describe the change
How might the gel change if we increased the voltage from 80V to 150V? Describe the change.
What might happen if the gel were to run too long?




I have some answers that I think are correct, but I'm still kind of at a loss, especially with the sketch. I've only ran an electrophoresis unit once.

NUS PhD Applicatioon

October 19, 2015, 3:24 am

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≪ Previous: First post with a few questions

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Could anyone please advise me about my application for PhD in NUS?

This is my profile:
BSc (Pharmaceutical Sciences)(Honours), first class, from University of Bradford. 
Currently pursuing MSc (Health Sciences Management) Northumbria University.
Both my BSc and MSc are UK degrees, but are awarded in Singapore private uni.
I have no work experience and publications. My Gre Score is 325. I have contacted a supervisor in School of Medicine and she allowed me to use her name in application and I m currently working as an intern to learn her projects.

My concern is that because my BSc degree is a 3-yr course, will I be disadvantaged? Because I heard that NUS prefer a 4 yr honours degree.
The Master course is also a bit not related to PhD. Will the admission feel that I am doing something digressive from my passion to do research?
Could you please suggest a chance for me to get in PhD in NUS?

Thanks in advance.

Colors of Biotechnology

October 19, 2015, 3:38 am

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Hi friends,
 What are the colors of Biotechnology?is it 4 color or 5 color?