Diabetes drug metformin partly works by altering gut bacteria

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  Metformin produced significant alterations in relative abundance of over 80 bacterial strains. – Photo: Evangelyn Alocilja laboratory, Michigan State University

One more study has shown that metformin that is commonly prescribed to people with Type-2 diabetes alters the gut bacteria. While earlier studies have shown a difference in the gut bacteria population with metformin intake, the latest study published in Nature Medicine finds the altered gut bacteria mediates some of therapeutic benefits of the drug.

To test the therapeutic benefits arising from altered gut bacteria, a team led by Fredrik Backhed from the University of Gothenburg, Sweden and José Manuel Fernàndez-Real at the University of Girona, Spain carried out a four-month-long trial involving 40 volunteers who were recently diagnosed with Type-2 diabetes and had not started treatment. The volunteers where randomly assigned to get either metformin (22 volunteers) or a placebo (18 people). A subset of the control group (13 people) was given metformin six months after the start of the study.

Both the groups consumed a calorie-restricted diet for the entire duration of the study. Neither the volunteers nor the researchers knew who received the medication and who got a placebo.

Owing to calorie-restricted diet, the body-mass index of subjects in both the groups reduced. As expected, the groups that received metformin witnessed a significant decrease in HbA1c and fasting blood glucose levels.

But what came as a surprise was that at the end of four months, metformin was found to have “strong effects” on the gut microbiome. Whole-genome sequencing of faecal samples revealed that only one bacterial strain was altered in subjects receiving placebo while significant alterations in relative abundance of over 80 bacterial strains were seen in the group receiving the medicine.

The drug appeared to promote the growth of Akkermansia muciniphila and Bifidobacterium adolescentis strains of bacteria. Even in in vitro studies, metformin promoted the growth of B. adolescentis. The team found no direct correlation between HbA1c level and A. muciniphila.

To further test the effect of changed gut bacteria in promoting therapeutic effects, the researchers transferred faecal samples from treated participants to germ-free mice that were fed high-fat diet for a week before stool transfer and during 18 days of colonisation. There was no reduction in body weight in mice but improvements in glucose tolerance were seen in animals that received the transplant. But improved glucose metabolism was not seen in all mice that got the stool transplant — only two of the three animals showed improvement. The large differences in the gut bacteria in humans might have resulted in the key bacterial species not being transplanted into mice, the researchers write.

Metformin-induced alterations in microbially-regulated metabolites were seen and this suggests that the metabolites might be “partly responsible for the stronger glucose-lowering effect”. But it “cannot be concluded that the major mechanism of action of metformin is through the microbiota”, they write.

Indian researchers use a novel approach to kill TB bacteria

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The compound isolated from Shayla tree does not directly target the TB bacteria but modulates the immune system to kill the bacteria.

A team of Indian researchers has been able to achieve 100-fold reduction in TB bacterial load in lungs of mice after 60 days of treatment using bergenin — a phytochemical isolated from tender leaves of sakhua or shala tree (Shorea robusta). Unlike the regularly used antibiotic drugs that target the TB bacteria, the bergenin compound modulates the immune system to kill the bacteria found inside the macrophages (a type of white blood cells). The results were published in the journal Frontiers in Cellular and Infection Microbiology.

“Our studies show that the bergenin compound can be used to clear the bacteria, and when used in combination with other TB drugs can produce good results,” says Gobardhan Das from the Special Centre for Molecular Medicine at Jawaharlal Nehru University (JNU), a corresponding author of the paper. “Since the compound does not target the bacteria directly but modulates the immune system to kill the bacteria, it can be used against drug-resistant TB too.”

The researchers undertook several studies to understand the mode of action of the compound. The compound was unable to directly kill TB bacteria when treated with the compound. However, in the case of in vitro studies, the compound was able to kill the bacteria found inside infected cells. In mice infected with TB and treated with the compound, there was significant reduction in the bacterial load in the lungs. Unlike in the case of in vitro studies, in mice the compound was found to activate not only the macrophages but also other cell types (T cells) that led to effective killing of the bacteria. A significant reduction in the number of granulomatic lesions was seen in animals treated with the compound. Also, the bacterial load was 100-fold lower in mice treated with the compound compared with controls (animals that were not treated with bergenin). “These findings strongly suggest that the immune response enhanced by the compound is able to increase the capacity to clear the TB bacteria,” Prof. Das says.

The levels of nitric oxide and a cytokine (TNF-alpha) were found to be enhanced. “We found the bergenin compound was selectively enhancing the frequency of interferon-gamma and interleukin-17-producing T cells in the TB infected animals,” says Dhiraj K. Singh from ICGEB and a co-author of the paper. Interferon-gamma promotes bacteria-killing nitric oxide inside macrophages thus promoting the generation of protective immune responses against TB bacteria.

Previous studies have shown that T helper 1 (Th1) cells play a key role in protecting the host against TB bacteria, while Th2 cells oppose the protection offered by Th1 cells. “There is a dynamic balance between the Th1 and Th2,” says Ved P. Dwivedi from ICGEB and the first author of the paper. “While TB bacteria prevents Th1 response and facilitates Th2 response, the bergenin compound promotes the expression of Th1 and Th17 responses.”

Beats conventional drugs

The compound has been shown to heal wounds faster than conventional drugs. Dr. Debprasad Chattopadhyay, Director of the ICMR-National Institute of Traditional Medicine (ICMR-NITM) in Belgaum, Karanataka, and the other corresponding author of the paper, had isolated the compound. He had seen tribals using the leaves of shala tree for wound-healing.

“The stage is now set to test many more Ayurvedic and plant-derived natural products for their potency against pathogenic diseases,” says Dr. Anand Ranganathan from the Special Centre for Molecular Medicine at JNU and one of the authors of the paper.

Prof. Das with the help of ICMR-NITM plans to carry out further tests in larger animals. If used in combination with other TB drugs the compound can shorten the duration of treatment and prevent the emergence of drug-resistance, the authors write.

Published in The Hindu on May 19, 2017

Euro VI diesel emission norms can avert nearly 174,000 premature deaths

pollution

Under the current diesel emission norms, there is a wide gap between on-road NOx emissions and certification limits.

Despite tighter nitrogen oxides (NOx) emission norms for diesel cars, buses and trucks in several countries, the actual amount of NOx emitted by diesel vehicles is far more during on-road driving conditions than under laboratory testing carried out at the time of certification. As a result, the excess NOx emitted over certification limits caused nearly 38,000 premature deaths in 2015 in the European Union, China and India; India alone had 9,400 deaths due to excess NOx emissions. Over and above the deaths caused by excess NOx emissions, increased air pollution from diesel NOx caused 107,600 premature deaths globally in 2015.

Diesel vehicles in the 11 major markets (Australia, Brazil, Canada, China, the EU, India, Japan, Mexico, Russia, South Korea, and the U.S.) emitted 13.2 million tonnes of NOx under on-road driving conditions, which is 4.6 million tonnes more than the vehicles’ performance under official laboratory testing. Compared with certification testing, the average on-road NOx emission is 2.3 times higher for light-duty diesel vehicles and 1.45 times the limit for heavy-duty diesel vehicles. Diesel vehicles sold in the 11 markets account for about 80% of global sales.

Adopting and enforcing the stricter Euro VI emission norms could “nearly eliminate” on-road diesel-related NOx emissions and avoid nearly 174,000 premature deaths in 2040, says a study published in Nature on May 15. NOx is a key contributor to outdoor air pollution in the forms of ground-level ozone and fine particulate matter of less than 2.5 micrometre size (PM2.5).

Under the current Euro IV diesel emission norms, there is a wide gap between on-road NOx emissions and certification limits. The excess NOx emissions coming from diesel vehicles gained worldwide attention when 11 million Volkswagen vehicles that contained defeat devices that controlled emissions only at the time of emission testing became known. But what is less known is that the current certification procedure adopted for diesel cars, buses and trucks “legally permits higher” vehicle emissions under normal driving conditions than the certification limits.

Euro VI emission norms have in-service testing, in-use emission monitoring, expanded driving conditions and independent verification.

Heavy diesel vehicles accounted for 86% of on-road emissions and over 75% of excess on-road diesel NOx emissions in 2015, about 90% of which is from China and India, the EU, Brazil and the U.S. In the case of diesel cars, the on-road emissions were 130% more than the certification limits.

Breaking the drug-resistant TB transmission cycle important

GeneXpert

Nearly two months after the Health Ministry set a highly ambitious target of working towards elimination of tuberculosis by 2025, a study published in The Lancet Infectious Diseases indicates that India’s TB crisis is all set to snowball by 2040 when one in 10 cases could be drug-resistant. What is even more alarming is that the increased number of drug-resistant cases — both multidrug-resistant TB (resistant to more than one of the first-line drugs) and extensively drug-resistant TB (additionally resistant to fluoroquinolones and at least one of the second-line injectable drugs) — will come from direct transmission from infected people to others rather than by strains acquiring resistance to TB drugs during treatment due to inappropriate treatment or discontinuation of treatment midway. The study found that “most incident” MDR cases are “not caused” by acquired drug resistance, and that acquired drug resistance will become a “decreasing cause” of drug-resistant TB. The increased availability of drugs to fight drug-sensitive TB has led to the emergence of MDR-TB strains. With increasing number of MDR-TB cases, there has been a shift in the way people get infected with drug-resistant TB — from strains acquiring drug resistance during treatment to direct transmission of MDR-TB strains from an infected person. The same trend is seen in the case of XDR-TB too. As a result, in high MDR-TB burden countries such as India, improved treatment outcomes in people might only reduce and not eliminate drug-resistant TB. Till 2015, only about 93,000 people with MDR-TB have been diagnosed and put on treatment.

The study, based on a mathematical model to forecast how TB is likely to progress in the four most-affected countries (Russia, the Philippines and South Africa, India), suggests that the number of new MDR-TB cases in a year in India will touch 12.4% by 2040, up from 7.9% in 2000. In the case of XDR-TB, the incident cases will rise to 8.9%, up from 0.9% in 2000. In 2015, the four countries accounted for about 40% (more than 230,000) of all drug-resistant TB cases in the world. Besides increasing the number of people who are diagnosed early and successfully treated, India’s TB control programme has come up with enhanced interventions to break the transmission cycle of the bacteria in the community. One of the ways this can be achieved is by carrying out immediate screening of all family members of a patient who has been diagnosed with the disease. Contact screening of family members and preventive treatment of all children below the age of five years who have not developed TB disease are already a part of the Revised National Tuberculosis Control Program (RNTCP) but is rarely done. Another important strategy that has to be adopted is making drug susceptibility testing universal and mandatory. Developing more accurate, cheaper and effective diagnostic tests and improved treatment regimens that are less expensive and of shorter duration will also go a long way in winning the war against the disease.

Published in The Hindu on May 12, 2017

Preterm babies don’t gain growth by early initiation of complementary food

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Babies started early on complementary food tend to suffer more due to diarrhoea and lower tract infections.

Babies born preterm (before 37 completed weeks of gestation) have a higher energy requirement than babies born full term and therefore fail to gain weight adequately. Parents of preterm babies and doctors alike are not sure whether breast milk or formula milk alone will meet the energy requirements after the first four months and whether preterm babies should be started on complementary food. While normal babies are given solids and semi-solids only from six months of age, early initiation of complementary food, which has a higher calorie density, in preterm babies appears to be a good idea to meet their energy needs and improve their growth (weight and length).

Till recently there was little evidence of whether earlier introduction of complementary feeding (prior to six months of corrected age) would improve growth of preterm babies.

No gain in growth

A study published a few days ago in The Lancet Global Health has found an answer to this vexatious issue — early initiation of complementary feeding in preterm babies born before 34 weeks of gestation does not improve growth.

Doctors from the All India Institute of Medical Sciences (AIIMS), Safdarjung Hospital and Kasturba Hospital, all in New Delhi, enrolled 403 babies born before 34 weeks of gestation and randomly assigned them to two groups — one in which they were started on complementary feeding at four months of corrected age and the other group of babies where complementary feeding was initiated at six months of corrected age. The corrected age refers to age that is corrected for the period of prematurity — for a baby born at 32 weeks of gestation, which is approximately two months earlier than the normal gestation period, the corrected age is 10 months at the end of one year of birth.

Complementary feeding was standardised in both the groups in terms of frequency, consistency, type of food, preparing food hygienically, and ways of feeding. Complementary foods were given in addition to breastfeeding/other milk feeding.

“Even though one group of babies was started on complementary feeding at an earlier age of four months of corrected age, there was no difference in growth compared with babies who were started on complementary feeding at six months of corrected age,” says Dr. Ramesh Agarwal from the Department of Paediatrics at AIIMS, one of the corresponding authors of the paper.

Some health risks

On the other hand, the study indicates that early initiation of complementary feeding had some negative fallout. “There were more hospitalisations in the group that started on complementary feeding at four months of corrected age,” he says. Though overall hospital admission in both the groups was low, babies in the four-month group were at increased risk of hospital admission due to diarrhoea and lower respiratory tract infections. “There could be several reasons for this increased risk, including potential contamination of complementary foods due to inadequate hygiene or having less breast milk,” he says.

“Our study shows that there is no difference in growth whether complementary feeding is started at four or six months of corrected age. But there are more infections when complementary feeding is started earlier. So it is advisable that complementary feeding is started only at six months of corrected age in preterm babies less than 34 weeks of gestation,” says Dr. Agarwal. However, studying the difference in growth and not infection was the primary objective of the study.

Published in The Hindu on May 7, 2017

Indian researchers reverse multidrug resistance in E. coli

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(From left) Dr. Saurabh Mishra, Dr. Amit Singh and Prashant Shukla of IISc have been able to make drug-resistant E. coli become sensitive to antibiotics by inhibiting hydrogen sulphide synthesis.

Indian researchers have unravelled the mechanism by which hydrogen sulphide (H2S) gas produced by bacteria protects them from antibiotics and plays a key role in helping bacteria develop drug resistance. And by blocking/disabling the enzyme that triggers the biosynthesis of hydrogen sulphide in bacteria, the researchers from Bengaluru’s Indian Institute of Science (IISc) and Indian Institute of Science Education and Research (IISER) Pune have been able to reverse antibiotic resistance in E. coli bacteria; E. coli bacteria were isolated from patients suffering from urinary tract infection. The results were published in the journal Chemical Science.

Antibiotics kill by increasing the levels of reactive oxygen species (oxidative stress) inside bacterial cells. So any mechanism that detoxifies or counters reactive oxygen species generated by antibiotics will reduce the efficacy of antibiotics. “Hydrogen sulphide does this to nullify the effect of antibiotics,” says Dr. Amit Singh from the Department of Microbiology and Cell Biology at IISc and one of the corresponding authors of the paper. “When bacteria face reactive oxygen species a protective mechanism in the bacteria kicks in and more hydrogen sulphide is produced.” Hydrogen sulphide successfully counters reactive oxygen species and reduces the efficacy of antibiotics.

There was nearly 50% reduction in drug-resistance when hydrogen sulphide production was blocked.The researchers carried out simple experiments to establish this. They first ascertained that regardless of the mode of action of antibiotics, the drugs uniformly induce reactive oxygen species formation inside E. coli bacteria. Then to test if increased levels of hydrogen sulphide gas inside bacteria counter reactive oxygen species produced upon treatment with antibiotics, a small molecule that produces hydrogen sulphide in a controlled manner inside the bacteria was used. “Hydrogen sulphide  released by the molecule was able to counter reactive oxygen species and reduce the ability of antibiotics to kill bacteria,” says Dr. Singh.

Harinath-Optimized

Prof. Harinath Chakrapani’s team at IISER Pune synthesised the small molecule.

The small molecule was synthesised by a team led by Prof. Harinath Chakrapani from the Department of Chemistry, IISER, Pune; he is one of the corresponding authors of the paper. “We designed the small molecule keeping in mind that synthesis should be easy, efficiency in producing hydrogen sulfide should be high and the molecule should release hydrogen sulfide only inside bacteria and not mammalian cells,” says Vinayak S. Khodade from the Department of Chemistry, IISER, Pune and one of the authors of the paper who contributed equally like the first author. The researchers were able to selectively increase hydrogen sulphide levels inside a wide variety of bacteria.

To reconfirm hydrogen sulphide’s role in countering reactive oxygen species, the team took multidrug-resistant, pathogenic strains of E. coli from patients suffering from urinary tract infection and measured the hydrogen sulphide levels in these strains. “We found the drug-resistant strains were naturally producing more hydrogen sulphide compared with drug-sensitive E. coli,” says Prashant Shukla from the Department of Microbiology and Cell Biology at IISc and the first author of the paper. So the team used a chemical compound that inhibits an enzyme responsible for hydrogen sulphide production. “There was nearly 50% reduction in drug-resistance when hydrogen sulphide production was blocked,” Dr. Singh says.

“Bacteria that are genetically resistant to antibiotics actually become sensitive to antibiotics when hydrogen sulphide synthesis is inhibited,” says Prof. Chakrapani. The multidrug-resistant E. coli regained its ability to survive antibiotics when hydrogen sulphide was once again supplied by introducing the small molecule synthesised by Prof. Chakrapani.

“As a result of our study, we have a found new mechanism to develop a new class of drug candidates that specifically target multidrug-resistant bacteria,” says Prof. Chakrapani. The researchers already have a few inhibitors that seem capable of blocking hydrogen sulfide production. But efforts are on to develop a library of inhibitors to increase the chances of success.

How H2S acts

The researchers identified that E. coli has two modes of respiration involving two different enzymes. The hydrogen sulfide gas produced shuts down E. coli’s aerobic respiration by targeting the main enzyme (cytochrome bo oxidase (CyoA)) responsible for it. E. coli then switches over to an alternative mode of respiration by relying on a different enzyme — cytochrome bd oxidase (Cydb). Besides enabling respiration, the Cydb enzyme detoxifies the reactive oxygen species produced by antibiotics and blunts the action of antibiotics.

“So we found that hydrogen sulfide activates the Cydb enzyme, which, in turn, is responsible for increasing resistance towards antibiotics,” says Dr. Singh. “If we have a drug-like molecule(s) that blocks hydrogen sulfide production and inhibits Cydb enzyme activity then the combination will be highly lethal against multidrug-resistant bacteria.” This combination can also be used along with antibiotics to effectively treat difficult-to-cure bacterial infections.

The link between hydrogen sulfide and Cydb enzyme in the emergence of drug resistance is another key finding of the study.

Published in The Hindu on May 6, 2017

Novel molecule synthesised by Indian researchers shows promise in treating cancer

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The efficiency of Disarib in selectively killing cancer cells was found to be high in all possible systems that Supriya Vartak (left) and Prof. Sathees Raghavan experimentally tested.

A novel small molecule designed and synthesised by Indian researchers has shown promise in targeted killing of cancer cells. The molecule (Disarib) binds to a protein BCL2 and inhibits the protein from suppressing cell death in cancer cells. While BCL2 protein is produced in excess in cancer cells, its expression is almost undetectable in normal cells. Hence, Disarib targets and kills only cancer cells while sparing normal cells.

Inside a cell there is always a balance between proteins that promote cell death (apoptosis) and those that suppress cell death. Since the proteins (BAX and BAK) that promote cell death get bound to BCL2, normal cell death is suppressed and cancer cells are able to live longer.

A team led by Prof. Sathees C. Raghavan at the Department of Biochemistry, Indian Institute of Science (IISc), Bengaluru demonstrated that Disarib was able to disrupt the interaction of BCL2 and apoptosis-causing BAK protein and cause the death of cancer cells.

However, expression of BCL2 is low in certain cancer cell lines such as breast cancer, chronic myelogenous leukemia and cervical cancer. So the Disarib molecule will be ineffective in these cancers.

The efficiency of Disarib to cause cell death and tumour regression was far superior compared with the FDA-approved ABT199.Disarib is the culmination of eight years of collaborative research involving 24 researchers from eight different research groups across various labs.

Superior than FDA-approved molecule

Unlike the FDA-approved BCL2 inhibitor ABT199, the small molecule binds predominantly to a different domain (BH1) of BCL2 and showed better efficiency in killing cancer cells. Also, compared with ABT199 inhibitor, the small molecule synthesised by Prof. Raghavan’s team did not cause any side effects. The results were published in the journal Biochemical Pharmacology.

Earlier studies had shown that once Disarib binds to BCL2, the proteins that promote cell death were able to create holes in the mitochondria leading to death of cancer cells.

“We have experimentally tested Disarib in all possible systems and the efficiency of Disarib in selectively killing cancer cells was high,” says Supriya V. Vartak from the Department of Biochemistry, Indian Institute of Science (IISc) and one of the first authors of the study. Studies were carried out on three animal models for three different cancers — lymphoma, breast adenocarcinoma and ovarian cancer. Similarly, studies were carried out using cancer cells lines.

“In every case, both in animal studies and cancer cell lines, the efficiency of Disarib to cause cell death and tumour regression was far superior compared with ABT199 when same dosage of Disarib and ABT199 was used,” says Prof. Raghavan. “This is why the molecule has to be taken up for further investigation.”

The team has carried out quite a lot of toxicity studies already. Next step will be to test the toxicity and efficacy of the molecule in cancer cells taken from patients, and also test it in combination with known cancer drugs. If results from humanised mouse models are also encouraging then the molecule can be taken up for clinical trials in humans.

Published in The Hindu on May 5, 2017