I find it interesting and reassuring that medicine is now turning to trying to fix our own defense systems in order to cure certain diseases rather than giving a strong drug (or a combination of drugs) that will kill the “bad” cells along with many of the good ones, and therefore making us weak and often experiencing side effects, for which there is a need for more drugs, and so on….

One of these defense or “balance maintenance” systems that has gotten a lot of attention recently is the huge and immensely varied bacterial “cosmos” we have in our guts called “microbiome” (see my previous post on this subject).

But the subject of this post is immunotherapy, a result of research conducted in the “cancer immunology” field, which is a type of cancer treatment designed to boost the body's natural defenses to attack and eliminate tumors. In order for our immune system to defend us from viruses and bacteria, it first has to “recognize” parts of these organisms. The parts that are recognized are “antigens” which are “seen” by our “antibodies”; these antigens are usually located on the surface of the invading organisms. A helpful way to think about antigen-antibody interactions is that of a key and its lock, where the antigen is the key that fits in the antibody lock as shown in the figure where the antibody is bound to the cell surface antigen. Cancer cells on the other hand are our own cells, although abnormal. They are not necessarily recognized by our immune system, and they then proliferate rapidly to form tumors due to specific mutations in these cells that make them divide in an uncontrolled and faster way compared to normal cells in the same parts of our bodies (tissues).

There are different types of immunotherapies being used or developed to treat specific cancers. They essentially consist of helping the immune system recognize these cancer cells and strengthen the immune response to hopefully ensure tumor reduction and/or elimination.

One type of immunotherapy currently used consists of blocking the ability of certain immune “checkpoint” proteins that normally keep immune responses at a low level. Some tumors can use these proteins to suppress immune responses against them. This checkpoint blocking immunotherapy results in an increased ability to destroy cancer cells. Several such inhibitors have been approved by the FDA (see table below) for advanced melanoma treatment, and others are in development.

Therapeutic antibodies is another type of immunotherapy, consisting of antibodies that are made in the laboratory to destroy cancer cells. Antibody–drug conjugates (ADCs) have been approved by the FDA for treatment of different cancers (see table below). The drug that is linked to the antibody is usually a toxic substance or poison that enters and kills the tumor cell after this one is recognized by the antibody part of the antibody-drug “hybrid”. Other therapeutic antibodies that are not conjugated to a toxic substance make cancer cells commit suicide (apoptosis) when they bind to them via different mechanisms, also triggering destruction of tumor cells by immune action known as “cytotoxicity”. There are also FDA-approved antibodies in this category (see table below) as well as some non-antibody immune conjugates linked to cancer-killing drugs which act in the same way.

A very promising experimental form of immunotherapy called adoptive cell transfer (ACT) has been applied in small clinical trials on patients with very advanced cancers, mainly blood cancers. Some patients have shown complete remission lasting several years. This approach is based on extraction of patient’s cells that are involved in immune recognition of cancer cells, then these cells are grown to large amounts, “activated” to attack tumor cells or sometimes they are genetically modified to do so, and finally they are infused back into the patient so they can do their anti-cancer job.

Another immunotherapy approach is the use of cancer treatment vaccines which are usually made from a patient’s own tumor cells, or immune cells which are manipulated/activated in the laboratory to react against tumor cells and then given back to the patient. One such vaccine has been FDA-approved for treatment of metastatic prostate cancer in men (although it does not cure the cancer, it has been shown to extend the life of patients by several months; see table below) and other candidate vaccines are being tested to treat forms of brain, breast, and lung cancer.

Table: Immunotherapies for which there are FDA-approved drugs

Do you think that when you walk around or exercise, you are carrying with you only humans cells with your own DNA? Think again. The way we view microbes, especially those that live on and within our bodies, has changed recently to a much more positive perspective. The number of microbes (mostly bacteria, but also viruses, parasites and fungi), tiny microscopic organisms with their own DNA living in each of our bodies has been estimated recently to be as high as 3-fold the number of human cells that contain our own DNA (both in the order of trillions). If we took all of them together this mass would weigh an estimated 2.5-3 pounds - a little over a kilogram. These bacteria (and their bacterial DNA which is present in higher quantities than our own when we look at the number of genes!) are called collectively “microbiome”.

Microbiomes are symbiotic microorganism communities that live within us, and some people view them even as another body organ. In the gut, they may colonize intestinal walls in the form of biofilms (check out my post on biofilms if you want to know more about them). There are microbiomes that colonize different parts of our bodies, thus we have a skin microbiome, an oral microbiome and the most important, abundant, and studied so far, the gut microbiome. Microbiomes  are dynamic, and can change over time for example when we move to a new location with different weather, food, environment and living conditions. Each person’s microbiome changes as we grow older, when we move to a different place with different weather, food and environmental and living conditions. They have been shown to affect/be affected by our diet, our birth (whether natural or by Cesarean section), antibiotics we take, anti-cancer therapies we receive, our immune system and many others. Our microbiomes start changing and evolving from the time we are born. Babies acquire a variety of microbe species from the mother when they pass through the birth canal during natural birth, and also from breast milk. It seems that in general the wider variety you get of these creatures, the better it is for your health, and the lack of variety in gut microbiomes for example may result in food sensitivities and allergies.

With technological advances both in science and health/medicine fields and as a result of increased collaboration between researchers and clinicians in different fields and institutions nationally and internationally, an explosion of new discoveries generates quick expanding grounds for new medical applications. The study of the human microbiome and how it affects our health is an emerging and exciting field. The most studied to date, and by far the most dense and abundant and diverse in number of bacterial species is the gut microbiome. These bacteria help us process and extract nutrients from the food we eat as they contain in their genomes genes that encode enzymes that are needed for these process that we (humans) lack, they also produce some good vitamins and prevent the growth of harmful microbes. Correlations have been demonstrated between some human diseases and the presence/absence of specific gut microbial species or species proportions in gut microbiomes, including inflammatory bowel disease, obesity, mood disorders (anxiety, stress and depression), allergy and asthma, colon cancer and others. Autoimmune diseases such as diabetes, rheumatoid arthritis, muscular dystrophy, multiple sclerosis, and fibromyalgia are associated with microbiome dysfunction.

One recent application of microbiomes is the use of “fecal transplants” (yes, feces from one person administered to another) to cure certain conditions such as  infections with Clostridium difficile, which cause severe diarrhea and other unpleasant gastrointestinal symptoms. This bacterium kills tens of thousands  annually, in part due to its antibiotic resistance occurring in some infections (there is an older post in this blog on this topic, look for “drug-resistant infections”) which makes antibiotics not effective and these would additionally kill good bacteria. A new treatment being used with some success is to administer sick patients diluted feces from a healthy individual (usually a family member) to provide an entire set of good bacteria that can colonize the lower intestine and keep the C. difficile infection under control. As unpleasant as this may sound, this “fecal transplant” technique is working in a majority of cases, especially recurring infections, and new delivery routes especially oral capsules are being explored - currently the options are via colonoscopy, enemas or through a nasogastric tube.

What can we do to try to keep our gut microbiome in good shape and with an ample variety of the “good” bacteria that make us healthy? Besides eating healthy and not taking unnecessary antibiotics that destroy it, we can add pre and probiotics, which help make it stronger or reconstitute it when affected by medications. Probiotics (found in special yogurt labeled as such, aged cheeses and fermented products such as  sauerkraut, tempeh and kimchi) contain good bacteria that help control growth of harmful bacteria, while prebiotics are not alive microorganisms but carbohydrates/fiber that cannot be digested by the human body and instead are food for probiotic bacteria. Prebiotics are found in whole grains, bananas, onions, garlic, honey and artichokes.

Chikungunya is a virus transmitted by the same daytime-biting mosquitoes that deliver dengue virus into humans, Aedes aegypti and Aedes albopictus, and it also results in similar symptoms compared to dengue, including fever of over 39°C/102°F. Symptoms usually appear 3-7 days after an infected mosquito bites the person. The word “chinkungunya” means "that which bends up” in Makonde language in Tanzania/Mozambique. Because symptoms are similar to dengue, and the virus is often present in the same areas, it is difficult sometimes to diagnose correctly or differentiate between these two viral diseases. Fever, joint pain and skin rash are more common and intense in chikungunya than dengue cases; bleeding is common in dengue hemorrhagic fever as well as a drop in platelets count, and not a symptom of chikungunya. There are no vaccines or specific medicines available for either of these two diseases, only their symptoms are treated with medication. Unlike dengue there is no bleeding in chikungunya. Other symptoms are headache, vomiting, nausea, weakness and muscle pain.

Although this virus has been reported in Africa, Asia and Europe in the past causing outbreaks, the fist reports of cases caused by local transmission (meaning local mosquitoes infected and spreading the virus to humans, as opposed to imported cases by travelers from endemic areas) in the American continent are very recent, just as of December 2013 in the Caribbean. This map from the CDC (available at http://www.cdc.gov/chikungunya/geo/index.html) shows countries and territories where chikungunya local transmission cases have been reported as of September 23, 2014

I currently live in Brazil, where a very interesting strategy is being implemented to combat mosquito transmission of the dengue virus. A team of researchers led by Luciano Moreira infected mosquitoes with an intracellular bacteria called Wolbachia which as a result can not carry the dengue virus. Wolbachia is actually a naturally-occurring bacteria in many insects. The team is planning to release thousands of infected mosquitoes a month for the next four months, the first batch this month in Rio de Janeiro. The hope is that these mosquitoes will reproduce and become the dominant mosquito in Brazil, reducing cases of dengue in humans.

Wolbachia infection in a male mosquito who fertilises the eggs of a female without the bacteria, results in the production of eggs which do not hatch . If both male and female, or only the female mosquito are infected wit Wolbachia, all future generations of mosquito will carry this bacteria. This is illustrated in the figure below from http://www.eliminatedengue.com/our-research/wolbachia. Thus, after a while mosquitoes with Wolbachia become predominant without release of more mosquitoes being necessary.





Some studies show that these Wolbachia-infected mosquitoes also do not carry the chikungunya virus, so this strategy may end up reducing the incidence of both viral fever diseases in Brazil and other places where this Wolbachia-infected mosquito release strategy may be implemented- currently this program is also taking place in Australia, Vietnam and Indonesia.

It is usually easier to argue for biology than for other sciences that studying it as children will be useful to us later in life. It is a more obvious and less abstract connection with Nature and living beings. Still, when I started graduate school in "molecular biology" it became abstract, and trying to explain what exactly I spent more than 5 years of my life studying and working on in one single laboratory was tricky at times. Nowadays though, we read about "genes", "DNA" and "mutations" in newspapers and websites written for lay audiences. The important applications in some fields, especially medicine, that molecular biology and genetics offer are now more widely appreciated.

We all know that there is genetics in our lives, even intuitively when we see resemblances with our parents and grandparents, mostly physical in nature but also in character and personality sometimes. We have also been aware for centuries that if there is a disease in our family tree, chances are we will get it too. In simple terms, there is a "defective" gene running in the family which is being inherited with a certain probability. In fact, the "defect" is an alteration of the "normal" copy of the gene - a mutation or group of mutations in a gene that encodes a protein product with a specific role (for more information on these topics see “genetics and mutations” on my homepage, and the blog entry on PCR and its applications). These mutations result in an abnormal form (or absence, or decreased amount) of the protein product that this gene encodes, which, in the right context (the cells or tissue where this protein is relevant or "expressed") can lead to diseases such as cancer. What molecular biology has provided us with are tools, which become more efficient every year, to determine which specific mutations are linked to certain diseases. Once this is determined, the test becomes generally available (although this may be restricted to those who can afford it when insurance does not cover it) to people at risk. This risk, once again, is usually defined based on family history. Below is a table showing some diseases for which the mutations in the gene(s) affected are well or partially known.

There is a genetic risk associated with certain diseases.  For diseases like cancer, additional elements such as environmental factors, stress, smoking and life style also influence the risk of developing disease. Long-term research conducted on patients (and sometimes their families) suffering specific diseases has led to genetic testing being now available for people either suffering symptoms or at potential risk of developing symptoms later in life, as well as those who may be just “carriers” for the (in this case, recessive) mutations conferring the risk and who can transmit the risk to their progeny by passing the “defective” gene on. These tests are performed on a sample from the patient (usually blood) and use PCR-based DNA sequencing or related techniques to detect a specific gene that is known to be associated with the disease. The results will indicate whether the patient’s gene contains mutations that are associated with the disease, and genetic counseling may be recommended to discuss exact risk. In some cases, when the patient presents symptoms, genetic testing is done to confirm the diagnosis. For "dominant" mutations such as the ones that result in Huntington's disease (a severe neurodegenerative illness that becomes apparent most often in mid-life) inheriting one copy of the mutated gene is enough to predict an almost 100% of developing Huntington's  at some point in life.

For people who suspect genetic risk in their families, they may want to be tested with their partners before trying to get pregnant. There is also a risk associated with certain ethnic backgrounds. For example, as I am about 75% Ashkenazi Jew, I got tested for a panel of possible mutations which are associated with pretty severe disorders that usually result in progressive degeneration and early death. Most of these mutations are of very high frequency in this population (about 1:100 in some cases or higher) compared to almost absent in other backgrounds. Both parents must be carriers of the “defective” (or mutated) gene for the disease for a child to get it though, so a positive test for an adult means only that he/she is a carrier. If both parents are carriers, there is a 25% chance of having a sick child (who would inherit one defective gene from each parent). This child also has a 50% chance of being a carrier (inheriting one defective copy from either parent) and a 25% chance of not inheriting any of the mutated genes, as shown in the top panel of the figure below. Another scenario (lower figure) shows the risk of passing a carrier gene to your child when only one of the parents is a carrier (50%).

Different life ‘happenings’ can restrict the access to information we have from our ancestors- generations moving abroad because of war or persecution (and subsequently losing touch with older generations and relatives), being adopted, or having parents dying at a young age. When you are a female and you lose your mother early in life,  as you grow older you wonder about female-specific life processes and how these were (or would have been) for her. Especially when going through events such as having your first period (menarche), getting pregnant and having children, and later on, menopause. These events can also be genetically determined to a certain extent in terms of (for example) how old we are when they happen and which ‘symptoms’ are associated with them. The risk of developing thyroid disease may also have a strong genetic component.

Malaria is a disease prevalent in tropical and sub tropical parts of the 3 continents that cover these areas (America, Africa and Asia).  Although efforts towards treatment, prevention and elimination in different regions have resulted in a substantial decrease in morbidity and mortality, malaria still kills about 2000 people per day, most of them being children in Africa.

After a person is bitten by an infected mosquito, the incubation period ranges from 1-4 weeks. The first symptoms are usually very similar to the flu: aches and pains, fever, headache. After a few days, the typical malaria symptoms occur: chills followed firstly by a high fever for a few hours, and then by profuse sweating. In between these episodes, the patient may feel well. Because the initial symptoms overlap with those of other diseases (both tropical which often include fever, as well as non exclusively tropical like the flu), malaria tends to be overdiagnosed in malaria endemic areas (underdeveloped, tropical countries mostly), and in more developed, temperate areas where it is not prevalent (patients may be travelers returning from regions where malaria is endemic) it could be underdiagnosed as medical doctors are not used to thinking of the possibility of people being exposed to certain tropical diseases.

Malaria is a complicated disease at many levels. For example, there are different parasite species that can infect humans. The two main parasites in the genus Plasmodium that cause malaria in humans are P. falciparum and P. vivax. In Africa, the main species is P. falciparum, and in Asia it is P. vivax, athough in Asia patients are found infected with either and also with both (mixed  infection) species. The life cycle of Plasmodium comprises different stages in the two animals hosts (human and mosquito- see figure below), and this becomes a major problem when treating the disease, as anti-malarial drugs, when administered to humans, may not kill all parasite stages. P. vivax can further complicate the cycle by producing dormant stages (hypnozoites) in the liver that 'reactivate' several weeks, months or years later.

When trying to eliminate malaria from specific regions, coordinated efforts including vector control have to be deployed to reduce the mosquito population and hence transmission. These mosquito control measures are mainly insecticide-treated mosquito nets and indoor residual spraying, which can be very effective in preventing malaria among children in endemic areas.

Plasmodium Malaria parasites life cycle. The life cycle of the Plasmodium parasite that causes human malaria is complicated and involves two hosts, humans and the Anopheles mosquito. Only the female mosquito feeds on blood, so only ‘her’ can transmit malaria. When she-moquito bites a person, it can inject Plasmodium stages called ‘sporozoites’ into the blood. Sporozoites travel through the bloodstream to the liver, mature into ‘merozoites’, and eventually infect the human red blood cells (RBCs), where they again reproduce and create many more merozoites, until a mosquito takes a blood meal from an infected human containing the parasite stages ‘gamectocytes’ which derive from merozoites in the human blood. These parasites then reach the mosquito's stomach and eventually invade the mosquito salivary glands. When this mosquito bites a human, these sporozoites complete and repeat the complex Plasmodium life cycle.  The characteristic periodic fevers seen in malaria patients are precipitated by synchronous parasite development and RBCs rupture, which releases new merozoites, malaria antigens and toxic metabolites. Figure from:      http://medilinks.blogspot.com/2010/11/malaria-malaria-is-caused-by-parasite.html

A disease to cure another disease: malariatherapy (malaria inoculation to cure neurosyphilis)

Believe it or not, in the early 20th century, before penicillin was discovered and available to cure many diseases including neurosyphilis, doctors would give patients malaria on purpose by injecting blood with parasites from ill patients, which proved to result in remission from neurosyphilis in many cases. After a few fever episodes, the patient will be given quinine, the effective antimalarial drug at the time. Empirical observations had revealed that some of these (by then considered terminal) patients would get better and even go into remission after experiencing infections that elicited high fevers in the patient. Neurosyphilis is an advanced syphilis stage which attacks the nervous system, causing paralysis and psychosis, and which may have been an important factor in the rising number of patients in asylums in the earlier 20th century. Julius Wagner-Jauregg won the 1927 Nobel prize for his discovery of the therapeutic value of malaria inoculation in the treatment of neurosyphilis.

Antimalarial drug resistance: a never-ending problem?

If there is a disease that has consistently shown through several decades of different treatments that these, especially when given as mono therapy, can lead to development of drug resistance, this is malaria (for details on how drug resistance arises in infectious organisms, see my blog post “drug-resistant infections”). Different drugs have been successfully used (when initially introduced) to treat and prevent malaria, with each gradually losing its effectiveness with time as drug resistance emerged. Quinine was the first such drug, introduced in the 17th century as an antimalarial (which is debatable as there is some evidence that Peruvian Quechua Indians were aware of its anti-fever properties long before). As a curious note, tonic water contains large amounts of quinine, and this could be the reason behind its popular use in drinks such as gin and tonic in British colonies.  Quinine potency began fading in the 1940s, and it was replaced with chloroquine. Once evidence of emergence of drug resistance to chloroquine was revealed in the late 1950s and early 1960s, a new combination treatment, sulfadoxine-pyrimethamine (SP) was introduced and used for two decades until it was partially replaced by mefloquine due to SP resistance reported. Resistance to mefloquine appeared in the 1980s.

Today, largely due to research led by Professor Nicholas (Nick) White, WHO recommends artemisinin-based combination therapy (ACT) as the main malaria treatment option. ACT consists of a combination of drugs containing one artemisinin derivative and a slower-acting partner, such as SP or mefloquine. Artemisinin, also known as Qinghaosu, is isolated from the plant Artemisia annua, sweet wormwood, a herb employed in Chinese traditional medicine. There are different ACTs, the exact composition of each depending on the geographic area and associated drug resistance patterns. The reason for this combo is to prevent drug resistance: the artemisinin derivative acts faster on the parasites, killing most of them while the older, slower drugs remain longer in the patient, killing off remaining parasites.  There are however, reports of artemisinin resistance emerging on the Thai-Cambodian border. Fear of artemisinin resistance spreading is currently prompting intense research in this area as well as efforts towards malaria elimination to prevent resistance spreading. Containment of resistance early is crucial, as otherwise it could spread through West Asia towards Africa, and once in Africa it would have devastating effects- about 85% of worldwide malaria cases are due to P. falciparum infection, and the vast majority of these cases occur in Africa.

Primaquine and G6PD deficiency

Yesterday, a review on the safety of yet another antimalarial (an old and very effective one) primaquine, which belongs to the 8-aminoquinolines group of drugs is out as a WHO (World Health Organization) document and is downloadable from this link:

http://www.who.int/malaria/publications/atoz/9789241506977/en/

We wrote this review almost 2 years ago to aid evaluate the safety of primaquine when given as a single dose to prevent transmission to the mosquito of the malaria-causing parasite P. falciparum, which results in falciparum malaria and is the main cause of malaria in Africa. Primaquine can be administered in addition to ACT treatment to prevent transmission of the P. falciparum gametocyte stages from the human host to the mosquito vector (P. falciparum gametocyte picture below). This is the current WHO recommendation. However, because there is a risk of hemolytic anemia (when RBCs ‘lyse’ or break up) resulting from primaquine ingestion in individuals who are glucose 6-phosphate dehydrogenase (G6PD) deficient, primaquine use is not as widespread as it could be in malaria endemic areas. There is now evidence that a lower dose of primaquine, which is still effective in killing gametocytes, is safe when given to G6PD deficient individuals, and this is the most recent WHO recommendation.

G6PD deficiency is the most common, hereditary enzyme deficiency affecting approximately 400 million people worldwide. It occurs mainly in malaria-endemic regions, and it is thought to provide some protection against malaria. G6PD deficiency discovery was in fact possible when studies in the USA in the 1950s involving volunteers who received antimalarials in different doses and regimens showed ‘sensitivity’ to primaquine. However, many different agents can trigger hemolytic anemia in G6PD people besides antimalarials (see figure below). 

For example, in the Mediterranean region, where a specific G6PD deficiency variant is present, favism is well known. All patients with favism are G6PD deficient, some of them can suffer from acute hemolytic anemia after eating them



Fava beans. Figure from:
http://www.wisegeek.com/what-is-a-g6pd-deficiency.htm

If you have never been amazed at evolution of a given species, think of coevolution of two species. In addition to these, picture coevolution of a parasite and its two different hosts, for a total of THREE species adapting to coexist one inside the other …. think of malaria: a parasite adapted to living inside two different hosts, undergoing differentiation into different stages in each of these as part of a life cycle which involves sexual as well as asexual reproduction in different organs or cell types within each host. Then add to this complexity the fact that malaria has been an important factor in the prevalence of certain hemopathies (RBC defects) in humans such as G6PD deficiency, α-thalassemia and sickle-cell disease. These genetic (inherited) conditions are more frequent in malaria endemic regions because they confer certain degree of ‘protection’ against malaria. Why? Because malaria parasites invade and grow and divide inside RBCs, so mutations in humans which affect these cells and make them ‘defective’ in a way that parasites can not invade/grow as efficiently, result in individuals who are more ‘resistant’ to malaria infection. The selective pressure in this case could be viewed as similar to emergence of drug resistance in a pathogenic organism. It is always the TARGET of the drug (inside the pathogen) or of the parasite (in humans- RBCs in the case of malaria) that is mutated to make this target now immune and able to avoid or be more resistant to the threat (threat = the drug for the pathogen, and the malaria parasite for the RBC). And, since I am a geneticist, I love that the basis of these mechanisms is that the gene that encodes the protein which is the target is now ‘mutated’ at the DNA level to result in a ‘defective’ protein that is only selected by the threatening agent pressure. This results in the survival of the fittest, as Darwin would say. And we could also cite Einsteinian relativity, as the fitness of the mutated protein is only higher when the selective pressure is there, often once this is gone (presence of the drug or malaria) the non-mutated gene should show higher fitness instead.

Every now and again, people (mostly women) point out and discuss possible reasons for the fact that in many science fields women are, still, very underrepresented - of course this is true not only in science ...

We have gone a long way, historically, from not even having the right to attend higher education institutes to being able to study and train in these fields, but are still far from where we would like to be.  A piece in the NYTimes last year by Eileen Pollack centered mostly on this subject within the physics and math disciplines is available here:

http://www.nytimes.com/2013/10/06/magazine/why-are-there-still-so-few-women-in-science.html?emc=eta1

I could add to the numerous cases and studies mentioned in the above article, based on my personal experience in molecular biology over almost 20 years in different laboratories and several US research institutions, examples from my research field. It is very uncommon to find males, for example, working in laboratories led by female PIs (Principal Investigators = heads of laboratories) unless these are well established and funded researchers. In contrast, male PIs attract both genders.

For women who become mothers during their PhDs or postdoctoral jobs, professional survival depends a great deal on whether the PI has a family and is a caregiver for his/her kids or not (or has an understanding of what this means). Many of us opt to dedicate some time to family not just because we feel this the right thing to do, but because in some environments the work hours required are incompatible with being home when we need (and want) to be. If our spouses have jobs which bring a higher income and demand longer hours (including traveling), this decision becomes a no-brainer, especially with the current economy, as staying home will be cheaper than working and hiring a full time nanny or paying for daycare. Other women, after spending ≥ 5 years of full time laboratory research work, quit this type of research after obtaining their PhDs to take alternative jobs (still science-related) which pay better and/or offer more family-friendly hours.

To focus on a more positive note but also to remark on how much more effort women sometimes have to make to “make it” as scientists, let's briefly look at  amazing discoveries by a few women in science in this post dedicated to them. Those working about a century ago often had to teach and train themselves, including even setting up laboratories at home. Another common circumstance was often not having children. I am not going to address the ongoing discussion in modern times centered on whether women can or can not make it professionally and be successful while having a family that includes children. I think it should be obvious to many that when possible, this requires a lot of sacrifice from family members and extra hard work compared to the equivalent situation for a man- of course, as always, generalizations come with exceptions and there are examples of great men who have supported women scientists (and non scientists!) and who are also great care givers at home.

Rita Levi-Montalcini  (1909-2012 .... 103 years old!)

Had children? No

Discoveries: NGF (Nerve Growth Factor)

She asked her father “permission to engage in a professional career” when she was 20 years old. In 1936 she graduated from medical school summa cum laude in Medicine and Surgery.

In 1940 in Turin, she decided to build a small research setup in her bedroom, which she moved to a country cottage where the family moved after Turin was bombed in 1941. In the Fall of 1943, when Italy was invaded by the Germans the family fled to Florence to live underground. In August of 1944, when the Germans were forced to leave Florence she was hired as a medical doctor to treat war refugees. After the war ended in 1945 she returned with her family to Turin to resume academic positions at the University. In the Fall of 1947, she was invited by Professor Viktor Hamburger in St. Louis to do research, and she stayed until retirement in 1977 dividing her time between Italy and the US.

In 1986, Levi-Montalcini and collaborator Stanley Cohen received the Nobel Prize in Medicine.

Background: Italian, neuroscientist

Research conducted in: Italy, USA

Marie Sklodowska-Curie (1867-1934)

Had children? Two daughters- one of them, Irene Joliot-Curie was awarded the Nobel Prize in chemistry in 1935 for their discovery of artificial radioactivity shared with her husband Frederic Joliot-Curie.

Discoveries: Pioneering research on radioactivity: a theory and techniques for isolating radioactive isotopes, discovery of polonium (named after her native country Poland) and radium.

She was the first woman to win a Nobel Prize (she won two: 1903 in Physics, shared with her husband Pierre Curie and Henri Becquerel, and 1911 in Chemistry). First female professor at the University of Paris. She died in 1934 in France from aplastic anemia developed by her work exposure to radiation; during her working years there was no awareness that exposure to radiation could be extremely harmful so she did not use any protection. Her papers are still radioactive so people who want to take a look have to use protection.

Background: Polish, physicist and chemist

Research conducted in: France

Rosalind Elsie Franklyn (1920-1958)


Had children? No, died at 38 from ovarian cancer

Discoveries: Watson and Crick’s “discovery” and model of the double helical structure of DNA reported in 1953 (and for which they shared the  they shared the Nobel Prize in Physiology or Medicine with Wilkins in 1962, after her death) was based in part on her X-ray diffraction images and accompanying interpretation


Background: British (UK), X-ray crystallographer, physical chemist

Research conducted in: UK

Barbara McClintock (1902-1992)

Had children? No, never married either

Discoveries: Nobel Prize in Physiology or Medicine in 1983 for mobile genetic elements (transposons) which she discovered in maize and reported in the early 1950s but the relevance of which in other organisms was not recognized until molecular biology unveiled their existence in the 1070s. Her original observations, based on experiments she carried out on her own, demonstrated that hereditary information was not as stable as previously thought, and her pointing to “jumping genes” was not received with the attention they deserved.  If you want a more detailed explanation of her discoveries, you can check out her own at a Cold Spring Harbor site, the institute where she conducted most of her research:

http://old.weedtowonder.org/mcclintock/transposition.html

Background: USA, geneticist

Research conducted in: USA

Elizabeth Blackburn (born 1948)


Had children?
One son

Discoveries: Telomerase, the enzyme that lengthens telomeres (see my blog post on telomeres for more info) for which she won the Nobel Prize in Physiology or Medicine in 2009, shared with Carol Greider and Jack Szostak.

Background: Australian-USA, molecular biologist

Research conducted in: USA

I have always been a bit shocked at how many people (and I'm not referring to very young ones in particular!) are unaware of their blood type, especially in the US.

Knowing your blood type, and that of your relatives, and especially your partner's and children's is very important not only in case of emergencies for which blood transfusions are needed, but also, as it was my case, to prevent a maternal reaction from an Rh- mother such as myself towards an Rh+ fetus.

Before blood types were known, when blood was supposed to be the same for everybody, fatal blood transfusions occurred. The Austrian biologist and physician Karl Landsteiner discovered the ABO human blood groups in 1901 and proposed their classification and transfusion-compatible groups, all this the body of work for which he received in 1930 the Nobel Prize in Physiology or Medicine. Later on his work with Alexander Wiener led to the identification of the Rhesus (Rh) factor in 1937  which was first discovered in Rhesus monkeys, where the name comes from.

Red blood cells (RBCs), the blood component which transports oxygen and gives blood its red color (as opposed to white blood cells which are also in blood and are an important part of the immune system that fights infection) also carry "antigens" on their cell surface, exposed towards the outside of the cell and facing the liquid (plasma) in which "antibodies" may be found. Specific antigens are bound by specific antibodies. The latter are usually made by our bodies in response to a "foreign" substance (viruses and bacteria are good examples) and by binding to them at specific spots (antigenic) they can signal and trigger an immune response in which complex mechanisms bring about specialized cells that can "clean up" our bodies from infectious agents. The antigens present on the surface of our RBCs that determine our blood type are: A and B antigens (from the ABO group, group O has no antigens present) and the Rh factor.

Our blood type is determined by the antigens our RBCs present (A, B, none or both) in combination with the absence or presence of the additional Rh factor. Result: 8 possible different blood types. This is schematically shown in the figure below.

Since I am a geneticist, it is relevant to point out that our blood type is based on genes we carry on our DNA, which are inherited from our parents and encode for these different antigens. This is why, before the era of DNA testing for things such as paternity, one could use blood type as an indication of whether or not a child could or not be the biological descendant of a specific father. This is not as precise (by far!) as DNA testing, but could rule out paternity in certain cases (for example, an AB child of an A mother can not have an O or A father, it would have to be a B or AB type that provided the "B" gene copy for the child) although not show with 100% certainty that the individual is indeed the father, as it is the case of DNA testing.

Throughout history, blood types have been associated with certain personality types, as well as special diets that are supposed to be good for individuals of a given blood type. There is no real scientific evidence to support this, although there are studies showing higher or lower risk for people with specific blood types to suffer from certain diseases or cancers.

So once you know your blood type, meaning you know what antigens your RBCs present, you also know which antibodies your blood could make in the presence of the antigens your RBCs do not present on their surface. This is shown in the figure below.

Both the presence of antigens that determine our blood type, and the possible presence of antibodies in the liquid part of our blood (plasma) AGAINST antigens that our RBCs do not have (because they are recognized as foreign substances once they enter your bodies) determine our blood type compatibility, which groups we can donate to and which ones we can receive blood from. In the table below you can find your blood type alongside possible donors and recipients.

I am Rh-negative. For a woman, especially for a pregnant woman, this becomes a worry if the father of the baby is Rh-positive. It is not a big risk for the first baby, but it is for later ones after the mother has been exposed ("sensitized") to the baby's Rh-positive blood during pregnancy and/or delivery. In subsequent pregnancies, these women can develop an anti-Rh immune response against the Rh-positive blood of their fetuses. Her anti-Rh antibodies can cross the placenta to destroy the fetal Rh-positive cells which can result in anemia possibly leading to jaundice, heart failure, or organ enlargement and may require Rh-negative blood transfusions. Many women had stillborn babies after having 1 or 2 healthy babies before the Rh factor was known. Also fatality or very bad adverse reactions occurred when Rh-negative people received blood from Rh-positive donors (again, not the first time, but after being "sensitized").

Nowadays, pregnant Rh-negative women like me are given something called a "RhoGAM" shot at different times during pregnancy (as well as after any procedure that involves risk of exposure to Rh-positive fetal blood such as amniocentesis and also after a miscarriage or any bleeding) and right after delivery if the baby is confirmed to be Rh-positive. This preparation (shot) which is given intramuscularly, can actually prevent the so called "Rhesus disease" or "hemolytic disease of the newborn". RhoGAM is made from donated plasma from Rh-negative people who have been exposed to Rh-positive blood and have therefore produced anti-Rh antibodies (RhoGAM is also called Rh immune globulin) after being sensitized. One such donor is an amazing Australian man who has given blood over 1000 times and is estimated to have saved over two million unborn babies- see him in a 2 min video at:
http://www.youtube.com/watch?v=OMe4WSzmFnQ

The anti-Rh antibodies delivered with the RhoGAM shot will destroy any RBCs containing Rh-positive antigens in the maternal blood and thus prevent any adverse reaction. A shock for me was to find out that every single time I got the RhoGAM shot (which was not cheap!) it was NOT covered by my health insurance, which covered everything else related to pregnancy and delivery. As I live now in Asia, it is good to be aware that Rh-negative blood is very rare (about 1%) among Asians, people of African-American descent and native Americans, whereas it is over 15% for Caucasians and over 20% in particular for for Basque people.

So..... if you still don't know which type your blood is, you better go find out :-)

As discussed in this blog before, the explosive development of molecular biology techniques has resulted in amazing applications especially in medicine-related fields. "Sequencing" people's whole genomes (meaning all 23 pairs of chromosomes from one individual- see karyotype figure in my homepage) is now a service offered at a much lower cost  with results available much faster. There is, however, an important consideration related to which part of the body the sample should come from, as it has become evident from a variety of data that the DNA sequence is not necessarily the same in all cells (tissues) from the same individual. For a nice overview of the ways in which these genome "chimeras" can originate sometimes in humans, a great read is the recent article by Carl Zimmer's "DNA double take" available here:
http://www.nytimes.com/2013/09/17/science/dna-double-take.html?pagewanted=all&_r=0

Chimeric "clones" can arise at different stages of development in either men or women due to mutations in cells which give rise to groups of descendant cells containing these mutations, or as cancer tissues which themselves contain mutated sequences compared to healthy tissues in the same person. There are at least 2 additional ways in which (only) women can think of themselves as chimeras: a combination of different types of cells rather than just the one we started with as a zygote. Although a more precise term would be micro-chimeras, as the group of cells inside some tissues that are different is quite small compared to the rest. One of these two women-specific chimera processes happens to all women, and the second only to those that have been pregnant:

1) X chromosome inactivation (It may be useful for this section to refer to the karyotype figure on my homepage):  In females, one of the two X chromosomes in each cell has to be "inactivated" (and in this process, once again, specifically modified histones and other epigenetic factors are involved :-)  so only one of them is "expressed" in each cell. This process is a form of what is known as "dosage compensation" and it happens in many animal species to keep the amount of proteins made from X chromosomes the same in males and females. Genetically, one X chromosome in each female embryo came from the mother, and the other one from the father, so X inactivation results in either of these being inactivated in different cells. In males, the sex chromosome pair consists of one X (coming from the mother) and one Y (coming from the father), thus there is no need for X inactivation.  X inactivation in females occurs early in development and in a random fashion. The inactive X chromosome suffers "condensation" and is visible under the microscope as a very dense smaller mass called a "Barr body". To put it simply, this condensation into a Barr body makes the genes in the inactive X chromosome inaccessible to proteins that would otherwise be responsible for these genes' expression into active proteins. Once a particular X chromosome is inactivated, the same one will be inactivated in all its daughter cells. However, at the initial stages one cell and its neighbor may have opposite X chromosomes inactivated, resulting in a "mosaic". 

A classic visual example of these mosaics are female cats that are heterozygous for a coat color gene located on the X chromosome (X-linked). Heterozygote means the gene sequences on each of the two chromosomes in the pair (in this case, XX) are not the same, there are 2 different "alleles", one coming from the mother (located on the maternal chromosome X) and the other from the father (on the paternal X chromosome). This coat gene encodes a protein which results in orange hair color when there is one of these  alleles present, or non-color when the alternative allele is (in which case the color may be black or white or a variation depending on additional genes located on other chromosomes that we are not focusing on here). Females with one allele for orange color and one for non-orange are tortoise shell and calico cats, visual mosaics of orange and other colors all over their coats. Each blotch contains only  cells coming from a single cell in the embryo after X inactivation. For all of you cat lovers out there (myself included) you can now spot a female cat when you see an orange mosaic such as the one in the picture below (from wikipedia). Males will never be orange  mosaics- they will show either all orange or no orange in their coats.

Fetal growth inside a woman's body offers possible stem cells circulating in the blood and resulting in fetal-maternal transfer into different tissues. Fetal blood contains a variety of stem cell types and during pregnancy; these "progenitor" cells circulate within maternal blood. Stem or progenitor cells have the capability of originating specific tissue cell types including blood, skin, liver and even heart. Fetal cells with this regenerative potential have been found in brain, liver, kidney, and lung injuries and are called fetal microchimeric cells. Initially, the big question was whether these microchimeric cells (fetal in origin but later on integrating into maternal tissues) are related to causing the injury or alternatively, they are targeted there to help the repair process by generating new healthy tissue where needed. Women with pregnancy-associated heart failure recover better compared to others with the same condition. In a study conducted in mice using an engineered GFP protein to mark fetal cells (see GFP section on my homepage), heart attacks were induced in pregnant mice and two weeks later, fetal cells were shown as 2% of the maternal heart and even formed blood vessels (see bright green GFP parts in the microscopy figure below from this study).

Bacteria can cause infections inside bigger organisms or live in the environment without causing infection. Either way, it has become apparent only recently that the main form of microbial growth in nature is as biofilms. In the past, in microbiology research different bacteria were usually grown in liquid cultures (or on agar plates) where antibiotic susceptibility was assayed. When grown in liquid medium, bacterial cells are homogeneous in shape, separated from each other and live in a "planktonic" manner. However, most microorganisms exist primarily by attaching to and growing upon other surfaces, both inert and alive which range from plants and soil, water systems and pipes, medical devices including catheters, heart valves and IUDs to animal tissues such as tooth enamel, heart valves, lungs, urogenital surfaces, the middle ear and many others.


It is estimated in the US that 80% of all microbial infections involve biofilms, which are the main form of growth especially in chronic infections. Below some of the most studied microbes that cause human infections and can grow as biofilms:

Soon after bacterial (and fungal) biofilm formation began to be studied in research laboratories it became evident that these forms of existence were MUCH more resistant to antibiotics that their equivalents grown in liquid media - sometimes up to 1000 times more amount of antibiotic was needed to result in the same killing or growth inhibition activity in biofilms compared to liquid cultures (these assays as performed in vitro in the lab). This is an important concept because bacteria were classically grown in liquid cultures before and used for  standard antibiotic resistance assays. For more on drug-resistant infections, see my previous blog post on that subject.

Biofilm formation is a process whereby microorganisms irreversibly attach to and grow on a surface and form a community. The cells that start the process are planktonic, but they undergo transformations both morphologically and in gene expression patterns which result in their growth as a biofilm. They also produce extracellular polymers that facilitate attachment and matrix formation. The biolfilm is a complex semi-multicellular structure which may consist of different types of cells, with internal channels for water and nutrients circulation and some differentiated cells called "persisters" which are dormant cells that survive after most of the biofilm has been destroyed by the action of antibiotics. Most antibiotics base their action on targeting actively growing cells, which persisters are not. Thus, tese cells could be an important target for biofilm-related antibiotic resistance drug development.

Within  biofilms, cells can communicate and cooperate with each other through "quorum sensing" processes which occur via the secretion of signalling molecules in a population-dependent manner and allow them to sense their proximity. Cells within biofilms are much more densely packed than their planktonic counterparts, a circumstance that has been shown to result in a higher degree of gene transfer between cells. In summary, biofilms are a form of microbial growth very different from and much more complex than planktonic cells from the same species:

As I write this blog, I realize that pretty much all topics covered so far have boomed in the last 2 decades or so, during which I've been lucky enough to work in labs researching them: histones, epigenetics, telomeres, biofilms ...  As with other recent technological advances, this science research explosion in certain fields is due in part to the development, availability and constant improvement of laboratory techniques and analysis software that allow researchers to further investigate processes that were out of reach before. Without exception though, it has been mainly the use of genetic tools, especially mutants, that has proved the most enlightening when elucidating the mechanisms responsible for the phenomena studied. As research progressed and results became available, these fields have become increasingly relevant to medicine and public health.

For a short 6 min video on description of biofilms and medical relevance by researchers, see: http://youtu.be/lpI4WCM_9pM

Telomeres, as it happened with histones, were looked at as having more of a structural role as chromosome ends than a more active one in terms of regulating cell aging and cancer. A connection between telomere length and these cellular outcomes has become recently more evident.

Telomeres refer to the ends of linear chromosomes (from Greek: telos = end, meros = part) consisting of little caps that contain no genes but specific repetitive DNA sequences as shown in the figure below on the left -a hexanucleotide sequence that goes on and on... They can be visualized under the fluorescence microscope by using appropriate staining techniques, shown on the microscopy photo below on the right.

These chromatin-related fields (histones, telomeres) have grown and attracted attention from other fields such as medicine, and in turn money has become easier to get for labs working on these subjects although the global economy and science research funding situation make it still hard for many researchers to fund their labs. In the early '90s there were general "chromatin" or "nucleus" meetings with a few dozen people attending from the labs working on many different topics, each of them now a huge field on its own. A few years later meetings became more focused, with several hundred people or more attending just for histones (or a specific aspect within the chromatin field) or telomeres. The '90s was also the decade during which the first whole organisms were "sequenced" for the first time. I remember attending the 1994 "yeast meeting" in Seattle where it was announced that a collaborative effort between different labs in the US and Europe was about to yield the whole genome sequence of the budding yeast Saccharomyces cerevisiae. This yeast (my favorite! check my home page) was the first eukaryotic organism to be sequenced- the total amount of DNA was about 12.5 million base pairs of DNA and it took several years. Later on other organisms were sequenced, including other eukaryotes, different bacterial species, mouse and eventually human. The human genome is about 3.3 billion base pairs of DNA and nowadays you can pay money to have your whole individual genome sequenced in a relatively very short time by specialized companies, and due to high competition, the prices are going down. But as usual, I'm diverging from the topic here, which is telomeres...

Telomeres have been shown to be very important in maintaining chromosome integrity. The mechanism by which these last bits of DNA on either chromosome end are replicated and maintained at a certain length is extremely complex, requiring a group of specialized RNA and proteins together to form the enzyme telomerase, responsible for lengthening telomeres. Telomerase was discovered and studied in different organisms for the first time in the '90s. The telomerase complex includes a "reverse transcriptase" enzyme (for details on these enzymes check my previous post on PCR) called TERT for TElomere Reverse Transcriptase.

Because "DNA breaks" are very bad for the cell (these can be induced by chemical agents, UV and other types of radiation, etc) and have to be sensed and repaired by specialized cellular processes and enzymes, telomeres (which are essentially pieces of DNA exposed at the tips of chromosomes) have to be protected by what is called "capping" so they are not "repaired" and sometimes fused with other chromosome ends in an attempt to fix what the cell might wrongly see as a DNA break. There are proteins bound to telomeres that block access to them, called "shelterins" during times of the cell cycle in which they might be seen as broken chromosomes.

Because of the way telomere replication occurs, they get shorter and shorter with each cell division, until they reach a critical shortness resulting in chromosome instability, cell senescence and eventually cell death. This mechanism is sometimes referred to as a cellular "clock" that regulates how many times a cell should divide before dying.

In several organisms where the correlation between cell age and telomere length has been evaluated, it has been shown that in general older cells have shorter telomeres, although this is not the case in every species or in all tissues from the same organism. One problem with these observations is that in general the way telomere length is analyzed involves extracting DNA containing telomeres from a lot of cells, therefore the measurement is an average length from the whole population. As shown in the figure below, in old individuals the accumulation of a proportion of senescent cells with critically short telomeres compromise tissue function and regeneration, contributing to aging and associated diseases. A better indicator is the measurement of the proportion of cells with very short telomeres in a population rather than the average telomere length of the same population.

Figure modified from the review by Vera and Blasco (2012) “Beyond average: potential for measurement of short telomeres”, Aging, 4:379-392

Aging of course is a topic that everyone is interested in, especially when anti-aging possibilities arise. When telomere length and telomerase were studied further, some commercial products appeared in the market such as  supplements and anti-aging creams claiming development of new formulas containing agents that could sow down aging based on counteracting the short telomeres effect or providing telomerase, such as a $1,500 skin cream (http://tmagazine.blogs.nytimes.com/2010/04/22/miracle-worker/).

In 2009, the Nobel Prize in Physiology or Medicine went to Elizabeth Blackburn, Carol Greider and Jack Szostak for their studies on telomeres. A short interview with Elizabeth Blackburn on new and exciting applied research on telomeres if found at:
http://www.scientificamerican.com/article.cfm?id=blackburn-elizabeth-telomeres-anecdotes-from-nobel-prize-winner

A malignant, cancer cell which divides in an unregulated manner, usually has levels of telomerase activity higher than equivalent cells form the same tissue which are not malignant (these cells in fact have low or no detectable levels of telomerase unless they are stem or progenitor cells). This has been shown to be the case not for early forms of cancer but more advanced ones; telomerase can be detected in approximately 90% of all malignant tumors which makes it a highly attractive therapeutic target. These cells are thought to activate telomerase to lengthen the otherwise critically short telomeres which would induce senescence and death. The presence of active telomerase confers the immortality intrinsic to cancer cells which can divide uncontrollably.

Whenever researchers find something that makes cancer cells be what they are, the immediate reaction is to look for ways to eliminate or inhibit this something. For example if it is a protein or enzyme such as telomerase, one can "screen" for inhibitors in vitro in the lab first, then move on to animal models and eventually test a possible cancer therapy in humans in clinical trials. However, there is a 10% of cancers which can extend telomeres by using a telomerase-independent mechanism for which this treatment would not work, and of course there is the concern that the target is actually an enzyme that should be active in stem cells in non-cancerous tissues.

As a final remark, just to emphasize how much better it seems for us to have longer rather than short telomeres, numerous studies that have looked at people with different conditions have found that, besides aging and cancer, short telomeres seem to be associated with the following:

* earlier death (Denmark)
* short sleep duration
* phobias, anxiety, depression, stress, schizophrenia
* childhood chronic or serious illness (Finland)
* several diseases

However, as with all correlations found in clinical research, we have to be very cautious when interpreting these, as they don't necessarily indicate a CAUSAL relationship in one particular direction. All these conditions might affect the way our cells divide and replicate their DNA, resulting in more DNA damage accumulated with time, and therefore shorter telomeres could be a downstream effect.

I have worked in science research in both developed and underdeveloped countries. The type of research, infrastructure, resources and money vary greatly between both settings in the expected direction (more of everything in developed settings) but there are fields that have proven productive and successful in less developed countries. These countries tend to be located in tropical settings, and as a consequence one big area of research is tropical diseases such as malaria, dengue, and Chagas disease and others not specifically tropical but with huge numbers of people affected in the tropical areas such as tuberculosis. The access to high numbers of patients, insect vectors that transmit the disease to humans, and often easier and less regulated conditions to work with the infectious agents (biosafety levels 1/2 for tuberculosis for example in underdeveloped labs as opposed to level 3 in more regulated places such as the US or European labs) allow local labs in less developed tropical countries to conduct great research due in part to the abundance of samples. As a consequence, collaborations are established between labs in developed and underdeveloped countries by which samples isolated from patients or insects in less developed areas are sent to developed countries laboratories which can perform more sophisticated analytical assays, animal model experiments (see previous blog entry on this subject if you are interested) and other research that requires more resources. Expensive reagents that come from developed settings' manufacturers are even more expensive when ordered from tropical settings (as there is additional cost for shipping) and these might get stuck at customs and other places before final delivery where they might not be kept at the required low temperature (on ice or dry ice) and therefore lose

Publication of this type of research from less developed settings becomes a tricky issue though. I have experienced first hand how some potentially clinically relevant research can go unpublished when conducted in less developed countries. There are a few factors associated with this reality, which are beginning to be addressed by the global scientific community:

1) One very important determinant of whether important research gets published or not in a high access journal is the very high fees that are required to publish peer reviewed manuscripts in some cases (especially relative to exchange rates and local cost of living in non-USdollar/GBP/Euro economies). This fee goes entirely to the journals (not to the reviewers, which are researchers asked to volunteer for this purpose) and varies depending on the journal status, type of manuscript, number of color figures etc.

2) The time that requires to write a manuscript is another factor to consider when people spend much of the time on the actual research or teaching in research/teaching universities. There is a pressure to publish in some underdeveloped places, but not as high as in developed countries where publishing is required to get promoted and get tenured and grants. The work might get presented in local meetings, or published in local journals which do not have high exposure or be in English language, and usually the results never make it to potentially interested parties in developing diagnostic or treatment kits such as pharmaceutical companies.

3) The language: tropical settings in Latin America, Asia and some places in Africa (francophone countries) would not often present or write their results in English at local meetings. This makes the possible manuscript writing process take much longer and be much more painful for researchers in these areas.

4) In terms of access to information, money is a big factor that restricts journals and publications that underdeveloped settings have access to as they might not have the subscriptions that developed countries' research institutions usually have .... this is in addition to limited internet access, downloading speed and services, and printing capabilities.

Newer journals such as PLOS have surged recently that not only publish high quality peer-reviewed research (valued as such by the scientific community) but are working on 2 important directions to make research more widely available for those interested in reading about it: 1) they are "open access", which means anyone can look at and download any publication (without paying any fees or having to subscribe to the journal) and more importantly for underdeveloped settings' research authors: 2) they have the option to apply for waved or lower fees for publication depending on the country submitting the work, specifically in their "PLOS neglected tropical diseases" (check out link for info for authors in developing countries: http://www.plosntds.org/static/developing;jsessionid=A3E5334790DDEA736A6EA113AF80CEB6).

Open access journals might not be aware of this, but as I writer of reviews on specific research subjects, I look for images to include in our publications (and for this blog!) to use as such or to modify depending on the context, and preferentially use the ones from open access journals without infringing copyright issues - when you want to use materials from other journals, books and websites you usually have to request permission from the publisher, including often paying fees. PLOS and other open access journals only ask that you cite the source, and the authors who publish there agree to these terms.

NOTE: I have no connection with PLOS, and all the statements made here about publishing research from developed versus less developed countries are generalizations (which, as with all generalizations, come with exceptions).

WHAT IS PCR AND WHAT IS SO COOL ABOUT IT?

Undoubtedly, the one single technique that has revolutionized molecular biology was the introduction PCR (polymerase chain reaction). It has numerous applications in many different fields ranging from scientific and medical research to diagnostics, criminology, forensics, archeology and paleontology. In research PCR has several different uses as it has allowed things to be done that were unimaginable before, or just made things technically possible in a much shorter time. Kary Mullis was awarded the Nobel prize in Chemistry in 1993 for PCR improvements that allowed a fast and exponential amplification of pieces of DNA- he has been a bit controversial afterwards regarding his views on other subjects which you can read more about elsewhere on the web.

In essence, PCR is a technique that "amplifies" a piece of DNA, especially if the amount we have to begin with is so small that unless we get more, there is not much we can do with it. The DNA sample to be amplified from is called "template".  To give an idea of how much DNA amplification you can get with PCR, I will use a story about the invention of chess and rice grains that I heard from my dad (who happens to be a mathematician) when I was a little girl. It goes like this: the king of the country where the inventor of chess lived (some versions say he commissioned the guy, who might have been a mathematician, to come up with a fun game) asked him what he'd like for his invention. The man asked the king to get one grain of rice for the first square of the chess board, two for the second one, four on the third one, and so forth, doubling the amount each time. The king, who thought this was a low price, ordered the treasurer to count the rice and give it to the inventor. The treasurer took more than a week to calculate the amount and explained that it would take more than all the rice in the kingdom to reward the inventor. The total number of grains covering the 64 squares of the chess board would be 18,446,744,073,709,551,615.

PCR amplification follows the same exponential growth: let's say we have one molecule (piece) of DNA in the tube to begin with, although usually there are more pieces in the initial sample. After the first PCR cycle we will have a total of 2 identical pieces of DNA after the action of the DNA polymerase enzyme (see details below). We now enter the second cycle, where the polymerase will make 2 more copies from the existing ones to result in a total of 4 .... and so on. Typical PCR reactions are setup with anywhere between 20-60 cycles.

By the 30th cycle there are already more than 1 billion copies per starting template molecule: as shown in the figure below, there will be exactly 2 billion pieces of DNA on the 30th cycle for each template DNA piece, or 2 billion grains of rice on the 30th chess board square.

A very short youtube video that visually explains PCR cycles and amplification potential is found here:
https://www.youtube.com/watch?v=eEcy9k_KsDI

WHAT ARE THE INGREDIENTS AND HOW DOES THE REACTION WORK?

We might want to "sequence" a piece of DNA we isolated from an organism of interest or an animal or person with a disease of interest or from a crime scene or a fossil. Whatever the material is (human or animal tissue or cells (sometimes containing virus or bacteria as the targets we want to amplify), bone remains etc) the first step is to try to get purified DNA, for which there are commercial kits available that PCR laboratories buy routinely. Afterwards, a mix is set up in a tiny plastic tube or well plate containing the DNA and all the reagents necessary for the PCR reaction. Once again, most of these are purchased from specific companies and in this case consist of special buffers, DNA base units or deoxynucleotide triphosphates (dNTPs in 4 different flavors: G, A, C and T) and most importantly, the "enzyme" DNA polymerase which has made all this possible. The most widely used is a heat-stable form isolated from Thermus aquaticus (an organism that lives and functions at very high temperatures) and referred to for short as Taq.

By far the most important variable element of the PCR reaction is one that the researcher has to "design" (sometimes with the help of programs) to make the DNA amplification possible and specific for the target sequence. These are small pieces of synthetic DNA that one can order to be made based on a sequence provided and called "primers". There are usually 2 primers per piece of DNA to be amplified which flank the target sequence, each of them is complementary to one of the 2 strands of DNA in the sequence.

The PCR reaction tube is then placed into a PCR machine or cycler where one can run "programs" which will expose the contents in the tubes to specific cycles of different temperatures that will result in (see figure below): 1) denaturing of the double stranded DNA so that next the primers can 2) anneal to complementary sequences when the temperature is brought down, and in the next step 3) the primers are extended by the DNA polymerase enzyme by using the dNTPs.

Each one of these 3 steps of each PCR cycle lasts close to 1 min, and a whole PCR goes in average for 2-3 hr. The heating up or cooling down that the machine does between steps to reach the target temperature happens in a matter of seconds. Depending on what you are using the PCR for, it is important to include a tube containing a negative control (a sample containing all the reagents except the DNA template containing the target sequence to be amplified) to make sure the DNA product is specific for your template, as well as a positive one (a DNA sample previously shown to be positive for the target sequence), for example when you are using the PCR for diagnostic purposes.

After running the PCR reaction, you can "purify" the DNA products from all the other stuff present in the tube (by using special kits containing resins and buffers that will specifically bind to the DNA and then elute it) and, after checking that the product is the correct size (by "running a gel" with size markers next to the PCR product) analyze it further.

NUMEROUS (and ever-growing!) APPLICATIONS

By playing with some of the ingredients and the conditions of the PCR (mainly temperatures, number of cycles and primer sequences) one can adapt the technique for specific purposes. Initially, it was used to faithfully obtain multiple copies of a specific fragment of DNA to then get it sequenced or use it for cloning into a "plasmid" or "vector" or to introduce into an organism of choice like bacteria, yeast or mice.

We have been discussing DNA amplification. Although the PCR technique is very powerful and sensitive, it might not work when the sample is too small to begin with. When one is trying to detect DNA from a piece of human or animal remains, or an organism like a bacteria or parasite from tissue or blood of patients for whom the level of infection might not be high enough, one alternative is to use as "template" genes that are present in more than one copy per cell, such as the ribosomal RNA (rRNA) genes. These genes are also used for species classification.

PCR can also be used to quantify gene expression when it is adapted to measure RNA levels - in this case, messenger RNA (mRNA) which is what the DNA in our genes is "transcribed" into. The mRNA, once made, travels from the nucleus of the eukaryotic cell into its cytoplasm, crossing the nuclear membrane, and once in the cytoplasm it is "translated' into its protein product which will perform a specific function, for which it might have to travel again, for example, into the nucleus if it happens to be a histone protein. If you have been following this far (some of this was on my "home" page) now you might get a bit confused by what happens in what's called "reverse transcriptase" PCR (RT-PCR). A comparison between what happens inside eukaryotic cells and RT-PCR is shown in the figure below. For RT-PCR, instead of DNA it is the mRNA which is extracted from the animal or plant sample obtained. Afterwards, an "RT" reaction is setup by which all the mRNA molecules present in the sample are "reverse transcribed" into complementary DNA (cDNA) once. This is made possible by using an enzyme that copies the single stranded mRNA into a complementary single stranded DNA and that we do not have in our cells but it is isolated from RNA viruses which use it to copy their RNAs into cDNAs. After the cDNA is made in the RT reaction, we can proceed with PCR to amplify the target sequence.

A fancier and more recent type of PCR is known as "real time" PCR or quantitative PCR (qPCR), for which bigger and more expensive cycling equipment is needed, which comes with special analysis software and requires additional reagents (special dyes, or primers coupled to dyes and quenchers, some called molecular beacons). Real time qPCR measures the concentration of the DNA product as it is being made in the reaction, as opposed to regular PCR in which the end amount of product is what is measured. This allows to compare amounts in different samples (of DNA or RNA after an RT reaction has been done). There are two types of qPCR, absolute and relative, which require specific reaction components and primers to be used. Serial dilutions of an initial sample are usually run, curves are obtained as the reaction progresses and the special software analyses the results in the region where things are supposed to be linear and provides quantification results. This methodology can be used to measure viral or bacterial loads in patients or compare how much a gene is expressed (mRNA is measured then: RT-PCR) under different conditions.

EXAMPLES OF MEDICAL AND PUBLIC HEALTH DIAGNOSIS THAT RELY MORE ON PCR

Most infectious agents detection methods in the past were developed to detect antibodies present in the blood of the infected individual, or alternatively infection can be detected by looking at blood samples under the microscope to see the organism directly using specific staining methods that facilitate visualization. Because the immune response to infection can take a few weeks, antibody (serological) tests can result in negative results when the infection is at an early stage. PCR (and other molecular assays which I haven't mentioned here) diagnosis, also from blood samples (extracted DNA or RNA) of the same infections can detect the causative agents much earlier. However, there is always a difference in the cost associated with molecular assays such as PCR which make them not as available as cheaper detection methods. In the field, especially in tropical areas, rapid diagnostic tests (RDTs) are usually based on detection of antibodies from the blood of the patient suspected to be sick when presenting with symptoms associated with malaria, HIV, tuberculosis, etc.

HIV diagnosis in infants: Some infections that are easily and cheaply detected by antibody tests, like HIV, have moved on towards detecting the viral DNA or RNA directly using PCR. For babies in Africa for example, where HIV incidence is quite high and many pregnant women are HIV-infected, antibody tests cannot provide a definitive diagnosis of HIV infection in their babies because maternal antibodies cross the placenta into the fetal circulation during pregnancy and remain detectable for more than a year after birth. Therefore the test will detect maternal antibodies and could result in a false positive HIV result when the baby might not have the virus, which would then translate into unnecessary treatment. PCR tests can now be done using DNA that comes from a dried blood spot from a filter paper obtained in remote rural areas such as African or Asian villages, which can be transported to a laboratory without a requirement for refrigeration. Once in the lab, PCR diagnostics is performed and the results are sent back to the village.

HPV diagnosis linked to risk of cervical cancer: HPV is the acronym for human papillomavirus, a group of more than 100 different related viruses that are spread during sexual contact in a skin-to-skin manner. During the annual PAP smear women should get at their gynecologist visits in developing countries, the sample extracted, consisting of cervical cells, is examined under the microscope by an expert cytologist. If the cells seen look "abnormal" in morphology (shape) then the next step is to do molecular diagnosis with specific primers to the known HPVs associated with high risk of developing cervical cancer. On a side note, a Gardasil vaccine is now available against HPV for females aged 9 to 26, approved by the FDA in 2006 consisting of a series of 3 shots over a 6-month period against 4 common types of HPV: 6, 11, 16, and 18. Types 6 and 11 or "high risk" are known to cause around 90% of genital warts cases, and types 16 and 18 are associated with up to 70% of cervical cancer cases.  This vaccine is now also available for the prevention of genital warts in males 9 to 26 years of age. A new Cervarix HPV vaccine was approved by the FDA in 2009 that only prevents against HPV 16 and 18.

Risk for specific cancer types: For people with a known family history of certain cancers such as breast or ovarian cancer, or colon cancer, there are also special molecular (PCR-based) tests that can detect whether or not an individual in the family is at risk of developing the cancer given the presence of mutation (s) associated with this risk. For breast and ovarian cancer, these are mutations in the BRCA1 and BRCA2 genes.

Prenatal screening: From a sample containing fetal cells (chorionic villus, which can be obtained after 10 weeks of pregnancy, or amniocentesis after 15 weeks) many genetic conditions can be detected in the fetus, mainly trisomy 21 (Down syndrome) and trisomys 18 and 13- these are evident by the presence of 3 instead of two chromosomes for each of these pairs (a human karyptype figure showing all chromosome pairs is on my home page). As explained in my home page, these are routinely detected by experts using the microscope on samples of cultured amniotic cells, a process that takes weeks. There are PCR assays available to detect the same abnormalities that require just a few hr to process a sample, resulting in quick results and less anxiety for mothers with risk factors such as family history, advanced maternal age etc.