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

With 18 million tonnes, a remote island turns into plastic junkyard

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Plastic debris on Henderson Island in Pacific Ocean

The beaches of Henderson Island, an uninhabited island in the South Pacific Ocean about 5,000 km away from the nearest population centre, are heavily littered with plastic waste. The beaches have an estimated 38 million plastic debris items weighing 17.6 tonnes.

The largest of the four islands of the Pitcairn Island group, Henderson Island is a Unesco World Heritage Listed site. Since it is uninhabited, its ecology is largely untouched by humans.

With 671 plastic items per sq metre on the surface of the beaches, the island has the highest density of plastic waste reported from anywhere in the world. And the amount of plastic waste on the island is ever growing with about 27 new plastic items per metre getting accumulated on a daily basis; in the North Beach of the Island alone, about 3,570 items get deposited daily. The results were published in the journal Proceedings of the National Academy of Sciences.

crab-OptimizedIn 2015, the researchers enumerated over 53,000 plastic items and arrived at an estimate of 37 million items littered on the beach. And alarmingly, even the 37 million plastic items may be an underestimation.  The reason: the team could not sample plastic waste buried below 10 cm from the surface and particles below 2 mm size and those found in the cliff areas and rocky coastline were not sampled.

With plastic waste disintegrating, smaller items were predominant, with microplastic accounting for 62% of items found in the Henderson Island.

The Henderson Island is located ion the western boundary of the South Pacific Gyre, a known plastic-accumulation zone for debris carried from South America (27%) or deposited by fishing boats.

“The plastic waste creates a physical barrier and contributes to a reduction in the number of sea turtles laying attempts, lower density of shoreline invertebrate communities and increased hazard of entanglement of coastal-nesting seabirds,” they write.

“Research has shown that more than 200 species are known to be at risk from eating plastic, and 55 per cent of the world’s seabirds, including two species found on Henderson Island, are at risk from marine debris,” Dr Jennifer Lavers from the University of Tasmania, Australia and the first author of the paper says in a release.

With the 17.6 tonnes of plastic waste found on the island accounting for only about 2 seconds of global production of plastic, the amount of waste that would get accumulated even in remote islands is bound to increases and further impact the exceptional natural beauty and biodiversity of these islands.

When dogs become the biggest predators of livestock

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Dogs were responsible for 64% of livestock deaths in the Trans-Himalayan region, much more than snow leopards. – Photo: Kesang Chunit

Dogs might be man’s best friends but they also turn out to be livestock’s biggest predators, at least in the Trans Himalayas. In the Upper Spiti landscape of the Trans-Himalayan region of India, of the 340 animals killed by predators in 2013 across 25 villages, free-ranging dogs (which move about freely in the landscape) were responsible for nearly 64% of livestock deaths, much more than snow leopards (that killed about 29%).

Even the livestock deaths attributed to wolves might indeed be attributed to dogs as there are very few wolves in the area.

While dogs predominantly killed small-bodied livestock (sheep and goats) and a few medium-sized animals such as donkeys, snow leopards killed larger animals such as horses and yaks. Even from the financial point of view, dogs caused more economic loss per year to people than snow leopards.

Sheep and goats did not display the same kind of anti-predator response towards dogs as they would do to wild predatory animals.The results of a study based on an interview survey were published in the journal Ambio. The study was jointly carried out by Ashoka Trust for Research in Ecology and the Environment (ATREE), Bengaluru, and Nature Conservation Foundation.

Livestock size

Chandrima Home from ATREE and the first author of the paper wanted to test if livestock depredation was due to abundance of dogs in a place or if it was livestock population that determined predation. “We found dogs responded strongly to livestock abundance and support the prey abundance hypothesis,” says Ms. Home.

The main reason why dogs turned out to be bigger predators than snow leopard could partly be explained by the naivety exhibited by livestock and familiarity of the predators (dogs). As a result, the sheep and goats did not display the same kind of anti-predator response towards dogs as they would do to wild predatory animals. That explains why sheep and goats accounted for 80% of the kills by dogs.

“The small-bodied livestock numbers are reducing and the large-bodied livestock is showing an increasing trend,” she says. With continued predation of livestock, there has also been a decline in the population of sheep and goat during the last five years. One village has stopped keeping small-bodied livestock since 2013 due to increased frequency of depredation by dogs. There have also been instances when dogs have killed calves of larger-bodies animals. Such attacks may increase in future as the number of sheep and goats keep reducing.

According to the paper, compensation is paid only for livestock killed by wild animals and not by dogs.

Of the 25 villages studied, two villages generated a huge volume of daily organic waste leading to an increase in the number of dogs. Totally, the researchers identified about 570 dogs in the 25 villages. “But only a subset of dogs predate on livestock and these dogs move from one village to another,” Ms. Home clarifies.

According to a 2017 paper in the journal Biological Conservation, domestic dogs have contributed to 11 vertebrate extinctions and are a known or potential threat to at least 188 threatened species worldwide.

Published in The Hindu on May 13, 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.

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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

IIT Madras team produces white light using pomegranate, turmeric extract

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Dr. Vikram Singh produced white light by irradiating the pomegranate extract and carbon nanoparticles made from pomegranate extract with UV light.

Dr. Vikram Singh, former research scholar in the Department of Chemistry, IIT Madras won the BIRAC Gandhian Young Technological Innovation (GYTI) Award 2017 for his work on producing white light emission using natural extracts.

Dr. Singh and Prof. Ashok Mishra from the Department of Chemistry, IIT Madras used a mixture of two natural extracts — red pomegranate and turmeric — to produce white light emission. The researchers used a simple and environment-friendly procedure to extract dyes from pomegranate and turmeric.

While polyphenols and anthocyanins present in red pomegranate emit at blue and orange-red regions of the wavelength respectively, curcumin from turmeric emits at the green region of the wavelength. White light emission is produced when red, blue and green mix together. This is probably the first time white light emission has been generated using low-cost, edible natural dyes. The results were published in the journal Scientific Reports.

“We had to mix the two extracts in a particular ratio to get white light,” says Dr. Singh, the first author of the paper; he is currently at Lucknow’s CSIR-Central Drug Research Institute (CDRI). By changing the concentration of the two extracts the researchers were able to get different colour temperatures (tunability).

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White light produced by irradiating UV at 380 nm.

“When we mix the two extracts and irradiate it with UV radiation at 380 nm, we observed energy transfer (FRET mechanism) taking place from polyphenols to curcumin to anthocyanins, which helps to get perfect white light emission,” says Dr. Singh. For FRET mechanism to take place there must be spectral overlap between a donor and an acceptor.

In this case, there is a perfect overlap of emission of polyphenols with absorption by curcumin so the energy from polyphenols is transferred to curcumin. Since there is also a perfect overlap of emission of curcumin with absorption by anthocyanin, the energy of curcumin is transferred to anthocyanin.

As a result of this energy transfer from one dye to the other, when the extract is irradiated with UV light at 380 nm (blue region of the wavelength), the polyphenols emit in the blue region of the wavelength and transfers its energy to curcumin. The excited curcumin emits in the green region of the wavelength and transfers its energy to anthocyanin, which emits light in the red region of the wavelength.

“Because of the energy transfer, even if you excite in the blue wavelength we were able to get appropriate intensity distribution across the visual wavelength,” says Prof. Mishra, who is the corresponding author of the paper.

White light sans turmeric  

Taking the work further, the duo produced carbon nanoparticles using pomegranate and to their surprise it was producing fairly green emission. So instead of using turmeric to get green wavelength, the researchers used carbon nanoparticles made from pomegranate extract. “We could get white emission, though it is not as white as when we used turmeric. It’s slightly bluish but well within the white zone,” says Prof. Mishra. “It is attractive to use a single source to create white light emission.”

The principle by which the pomegranate extract and carbon nanoparticles made from the extract is the same as in the case when pomegranate and turmeric extracts were used. The results were published in the Journal of Materials Chemistry C.

Though this natural mixture of dye can be used in a wide variety of applications such as tunable laser, LEDs, white light display, much work needs to be done in terms of photostability and chemical stability before it becomes ready for translation. Biosystems have an inherent tendency to breakdown and so this has to be addressed.

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