Blog Science Advances - LV Version

Welcome to Science Advances - LV Version. Here, I will be sharing more about clinically important scientific discoveries that I read from research articles and will share my views on them.

 

Topic: RNA Interference, What is it?
Source: https://www.ncbi.nlm.nih.gov/probe/docs/techrnai/

Introduction
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is a conserved biological response to double-stranded RNA that mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes. This natural mechanism for sequence-specific gene silencing promises to revolutionize experimental biology and may have important practical applications in functional genomics, therapeutic intervention, agriculture and other areas.

Endogenous Triggers of RNAi Pathway

Endogenous triggers of RNAi pathway include foreign DNA or double-stranded RNA (dsRNA) of viral origin, aberrant transcripts from repetitive sequences in the genome such as transposons, and pre-microRNA (miRNA). In plants, RNAi forms the basis of virus-induced gene silencing (VIGS), suggesting an important role in pathogen resistance. A possible mechanism underlying the regulation of endogenous genes by the RNAi machinery was suggested from studies of C. elegans. In mammalian cells long (>30nt) double-stranded RNAs usually cause Interferon response.

A Simplified Model of the RNAi Pathway

A simplified model for the RNAi pathway is based on two steps, each involving ribonuclease enzyme. In the first step, the trigger RNA (either dsRNA or miRNA primary transcript) is processed into an short, interfering RNA (siRNA) by the RNase II enzymes Dicer and Drosha. In the second step, siRNAs are loaded into the effector complex RNA-induced silencing complex (RISC). The siRNA is unwound during RISC assembly and the single-stranded RNA hybridizes with mRNA target. Gene silencing is a result of nucleolytic degradation of the targeted mRNA by the RNase H enzyme Argonaute (Slicer). If the siRNA/mRNA duplex contains mismatches the mRNA is not cleaved. Rather, gene silencing is a result of translational inhibition.

RNAi in Experiments and Therapeutics - Mechanism of Action

RNAi can be triggered experimentally by exogenous introduction of dsRNA or constructs which express shRNAs. The high degrees of efficiency and specificity are the main advantages of RNAi. Consequently, RNAi is used in functional genomics (systematic analysis of loss-of-function phenotypes induced by RNAi triggers) and developing therapies for the treatment of viral infection, dominant disorders, neurological disorders, and many types of cancers (in vivo inactivation of gene products linked to human disease progression and pathology).

 

Topic: Gene Mapping. How does it work?

Many diseases are linked to genetics. For example, the most common form of cystic fibrosis comes from the CFTR∆F508 mutation. This means that the CFTR has a deletion of 3 nucleotides, leading to the phenylalanine in the 508th position being removed. Cancer is also a very complicated disease that causes deaths globally, with such deaths being more than AIDS, malaria and tuberculosis combined.

Thomas Hunt Morgan, in which the genetic mapping distance unit, the centimorgan (cM) is named after, was working on D. melanogaster when he realized that for certain dihybrid crosses, there was a statistically significant deviation from what would be predicted by Mendelian genetics. He observed that the parental phenotypes were present in large numbers while the non-parental (recombinant) phenotypes was present in much smaller numbers. Hence, he introduced the concept of genetic linkage, with 1cM (~1 million base pairs in humans) distance corresponding to 1% of recombination (due to crossing over in meiosis I).


Since the discovery of restriction enzymes and the polymerase chain reaction (PCR), geneticists and molecular biologists have been developing techniques to map the human genome in hopes of identifying suspects in crime scenes, genetic screening for human diseases and looking for biomarkers in human cancers, amongst other applications. This gives rise to restriction fragment length polymorphism (RFLP) analysis, whereby we can analyze the presence of mutations based on restriction digest fragments.

Just to give a quick case study on sickle cell anemia, HbA, the normal hemoglobin in our blood has the sequence …CTCCTGAGGAG…, while the sickle cell hemoglobin, HbS, has the sequence …CTCCTGTGGAG…. What we could do is to run a PCR experiment (which we will not go into detail) and do a restriction digest with XhoI (sequence: C|CTGAG). As we can see, there will be two fragments upon digestion for HbA but one upon digestion for HbS (based on the abovementioned sequences). By running the samples after digestion on an agarose (or polyacrylamide) gel, we can see the DNA bands and tell if the person is afflicted, normal or a carrier. A variation of such a test could be done as follows:


Genetic maps have been used successfully to find the gene responsible for relatively rare, single-gene inherited disorders such as cystic fibrosis and Duchenne muscular dystrophy. Genetic maps are also useful in guiding scientists to the many genes that are believed to play a role in the development of more common disorders such as asthma, heart disease, diabetes, cancer, and psychiatric conditions. However, the field of epigenetics has also been crucial in helping to identify whether the expression of certain genes is high or low, and thus their implication in disease.

References:
http://www.biology-pages.info/R/RFLPs.html
https://www.genome.gov/10000715/genetic-mapping-fact-sheet/

 

Topic: Cancer Immunotherapy
Cancer is a health epidemic that kills more than AIDS, malaria and tuberculosis combined, globally. As such, novel therapies have been designed to target cancer cells. There has been a lot of papers regarding immunotherapy that have been published recently, aiming to use the patient's immune system against the cancer. It promises things such as less severe side effects and more effective treatment of the cancer.

Immunotherapy works by programming the dendritic cells of the immune system to recognize the cancer cells as foreign bodies, which leads the immune system to spring an attack on the cancer cells.

It all started when the sarcoma of a patient diagnosed as incurable reduced until it disappeared after being infected by a bacteria of the Streptococcus genus. William Coley, a surgeon from the New York Cancer Hospital (now called Memorial Sloan Kettering Cancer Center), thought that the patient’s immune system had reacted not only against the infection, but also against the cancer, and to test it, he infected one of his own patients with the same kind of bacteria. He recovered only a few weeks later.

Spurred on by success, over the following years Coley infected several more patients, trying different combinations. Although he continued to harvest success stories, the procedure only worked on occasions and nobody knew what caused it to succeed or fail. Moreover, it was not so effective on other tumors. Radiotherapy and chemotherapy, which are much more docile and lend themselves to pre-established protocol, were championed as the cancer-fighting weapons of choice.

But his idea was never totally abandoned. In recent decades, various methods for treating cancer via the immune system have been tested: with specific antibodies, cytokines – molecules released during the defensive reaction – and therapeutic vaccinations against the tumor. However, with the exception of certain antibodies, few significant results have been found and there is only one vaccination approved to treat prostate cancer, which has limited effectiveness.



In June 2013, during the yearly meeting of the American Society of Clinical Oncology (ASCO) held in Chicago, Ribas and a colleague brought two studies to light, which were immediately published in the New England Journal of Medicine. Both are Phase 1 trials, small, initial studies using various doses to test the safety of a drug, not to establish their actual effectiveness. Both were performed on patients with advanced melanoma resistant to treatment with very short life expectancy.

The study led by Ribas included 135 patients who were treated with lambrolizumab, an antibody directed against PD-1. This molecule is an Achilles’ heel in the defences that protect us against cancer, T lymphocytes (or T cells), which destroy tumor cells. When the PD-1 in lymphocytes joins to its complementary PD-L1, located on the surface of the cancer cell, a cascade of reactions occurs, which finally renders the lymphocytes incapable of performing their role. The defences are left powerless against the tumor, which can thus hide away from its constant surveillance.

This is where the lambrolizumab comes into action. The antibody’s mission is to prevent this harmful union, which enables the defences to release their safety brake, recognize the tumor as foreign once more and attack it. There is a change in the paradigm: The cancer is not attacked directly; rather, the immune system’s army is released to battle with all its artillery.

Overall, 38% of patients treated this way responded significantly to the treatment, and this percentage rose for those who received the highest doses. And, although not enough time has passed yet to draw any conclusions, Ribas explains to SINC that, in light of the results, “a lasting response can be invoked in the immune system, although we would surely have to administer long-term treatment in order to achieve this.”

This lasting effect is key. Much personalised medicine is based on targeted therapies that block a particular aspect of each tumor, but in many cases the tumor reoccurs as it adapts to the treatment. In a way, this type of immunotherapy, which recruits a much more versatile army, able to recognize numerous enemies, enables cells with memory to be generated, which are retrained to attack the tumor.

The other study was led by Jedd Wolchok from the Memorial Sloan Kettering Cancer Center in New York (would you remember Coley?). In this case they treated 53 patients with two different antibodies: nivolumab, against PD-1; and ipilimumab, against CTLA-4, another molecule implicated in inhibiting the immune system, whose use for melanoma has been approved since 2011.

The results were very similar to those of the previous study: 40% of patients responded to the treatment, a percentage that rose to 53% when administering the combination of doses that turned out to be the most effective. However, the side effects were notably greater as a consequence of autoimmune reactions. The immune system, now ‘freed’, attacked the patient’s own tissues.

Immunotherapy brings about great potential in targeting cancers in this day and age, and we look forward to great revolutionary progress in this field in the next couple of years.

Reference: http://www.agenciasinc.es/en/Report...er-is-the-scientific-breakthrough-of-the-year

 

Topic: Flaviviruses
You might have heard the term flavivirus recently due to the outbreak of Zika virus in Central and South America. Zika, along with West Nile virus, dengue, yellow fever and Japanese encephalitis, belongs to this family of virus – of which many are threats to public health.

Flaviviruses are defined by the shape and size of the virus particle (which is extremely small and not visible by the naked eye but requires a high powered electron microscope). They are able to replicate and spread within both insects and mammals, and they infect humans and domesticated animals.

Flaviviruses are arboviruses, which means they are spread via infected arthropod vectors such as ticks and mosquitoes. Some flaviviruses (such as West Nile) exist in a bird-mosquito cycle and infections in humans are typically incidental and a “dead-end” for the virus. This means it cannot be transmitted to a new mosquito. However, yellow fever, dengue and Zika exist predominantly in a human-mosquito cycle. These viruses grow very well in the human body and therefore allow the re-infection of mosquitoes.

The geographical location of flaviviruses is determined primarily by the distribution of the mosquito or tick vector. For the most part, they are confined to tropical and sub-tropical regions, particularly Southeast Asia and South America. However, Australia has two native flaviviruses – Murray Valley encephalitis and its own strain of West Nile called “kunjin”. Australia also has epidemic episodes of dengue occurring in far North Queensland.

The Aedes aegypti mosquitoes responsible for spreading Zika and yellow fever have been shown to be able to adapt in high-density urban areas, which means it is important to find methods to contain flaviviruses. The urbanization of the ever-growing human population and the impact of climate change are increasing the population at risk of contracting flavivirus infections. A flavivirus is transmitted via the bite of an infected tick or mosquito. It enters the bloodstream and invades and infects cells called monocytes in the immune system. The virus is then transported to lymph nodes and targets organs within the body, where different flaviviruses cause different symptoms. It is shown that the NS2B-NS3 protease is one of the key enzymes in the viral replication cycle.

Symptoms generally take seven days to appear and can last for an additional seven days. Some flaviviruses, such as West Nile, can enter the brain and induce encephalitis, whereas yellow fever infects the liver, dengue can cause shock and haemorrhage within the body, and Zika induces joint and muscle pain upon infection. It is not currently understood why these symptoms occur, but research is being conducted to try to uncover how the viruses affect the body.

Currently vaccines are available for some flaviviruses – including yellow fever, Japanese encephalitis and tick-borne encephalitis virus. A vaccine for dengue was recently licensed for use in Brazil, the Philippines and Mexico. The development of a dengue vaccine has been challenging due to the different variations of the virus. Being exposed to one type potentially worsens subsequent infections with another type of the same virus. To avoid this complication, the current vaccine trials have included all four dengue variations in their formulations. A recent advancement in controlling flaviviruses is with the use of a bacterium called Wolbachia. Mosquitoes that harbor this bacteria are completely resistant to subsequent flavivirus infection, and the bacteria can infect and remain persistent within mosquito populations. If flaviviruses can’t establish infection within the vector host, this limits its maintenance in the environment. The Doherty Institute, along with Monash and Oxford University, is researching implementation of this form of biological control.

References:
http://theconversation.com/zika-dengue-yellow-fever-what-are-flaviviruses-53969
http://science.sciencemag.org/content/early/2016/12/07/science.aai9309
http://apps.who.int/iris/bitstream/10665/163997/1/dbv28p58.pdf