MicroBlogs

17-Aug-2021

What If… Cells Could Talk? : New technique to program Memories into bacterial

cells by DNA Rewriting
By: Pratibha Gautam (MSc student)

According to a recent study conducted at MIT by a team of biological engineers, a new technique has been devised which can enable us to efficiently edit bacterial genomes such that we can control cellular phenotypes efficiently by dynamically changing information stored in the genomes. This technique has been named HiSCRIBE (High-Efficiency SCRIBE), which is believed to achieve up to 100% mutation homogeneity without any selection pressure. This approach deals with the permanent inscription of spatial and temporal information into bacterial cells for generations which can be retrieved by DNA sequencing methods. Thus, one can lock a particular DNA sequence as a memory within one bacterial which is bound to be passed onto further generations of those bacteria through this technique. The best part is that this method is not limited to specified laboratory conditions but it can be employed in natural community which means it gives an advantage of in-situ applications. This technique is based on the use of a retro-element to mediate DNA editing without the requirement of any target-specific element or selection pressure. This technique is demonstrated to be efficient, scarless and it is independent of the use of the Cis-Regulatory element which is an integral part of the CRISPR/Cas mediated genetic engineering system. It can target microbial genomes within complex communities such as the human microbiome for editing, activating, and/or silencing genes living in a particular bacterial species within its natural ecosystem. It is employed for mapping of cellular structure and interactions into the bacterial memory in the form of DNA sequences. Also, it helps in keeping a record of cellular evolution. This method is likely to be used in the future for studying the evolutionary dynamics and interactions between bacterial populations. The research has been conducted by Fahim Farzadfard while Timothy Lu, Nava Gharaei, and Robert Citorik are the co-authors of the paper.

Researchers at MIT have worked for several years to use DNA as a storehouse of the memory of cellular events. In 2014, Lu and Farzadfard were able to develop a way to genetically engineer E. coli as a genomic tape recorder that stored the long-term memories of Chemical Exposure events. This was achieved by the synthesis of a reverse transcriptase enzyme called “Retron” by the cells. This enzyme was able to produce a single-stranded DNA when expressed inside a cell. Along with retron, another enzyme i.e., recombinase was engineered into cells to “write” specific ssDNA sequences into the targeted site by Beta mediated Recombination. The production of DNA depends upon a stimulus which may either be activation by a specific molecule or external factors such as Light or Biological Reagents. After DNA synthesis, Recombinase comes into the picture when it inserts the DNA at a preplanned genome site. This technique is called SCRIBE (Synthetic Cellular Recorders Integrating Biological Events). Using this technique we can easily convert the transcriptional signals into DNA memory. But, it had a major limitation of low writing efficiency of only 1 in 10,000 cells per generation. This was because of the inherent defensive property of E. coli cells to resist accumulation and integration of single-stranded DNA into their genomes by the introduction of Double-stranded DNA Breaks. In the 2014 Study, Lu stated that this technique can perform long-term recordings of a cell’s environment as well. For example, if we let some cells be alone in water in a specific area for a week and then collect it. Their DNA sequencing could reveal exposure to certain bacteria or Toxins in the water. The HiSCRIBE technique is an updated version of the SCRIBE technique which came in 2014. It was observed that HiSCRIBE can be introduced to cells via various methods like transduction and conjugation. This may be used for continuously improving a specific phenotype in the cell.

The present 2021 study aims at boosting writing efficiency. This was achieved by performing some modifications in cellular genetic makeup. The first step was to disable Exonucleases (E.g., xseA, recJ, and xonA) which were responsible for breaking the ssDNA. Also, specific genes involved in Mismatch Repair System (E.g., ΔmutS) were being knocked out that were supposed to prevent the integration of ssDNA into the bacterial genome and interfere with ssDNA stability. It was also found that the optimum length of the homology arm between the HiSCRIBE generated ssDNA template and Target is about 35 bps. Nearly Universal incorporation of Genetic modifications was achieved by this method which paved way for an exceptional and efficient genome editing tool that too without the need of any selection. According to Farzadfard, this approach could help in upcoming evolutionary engineering or studies by allowing a replay of evolution over and over. 

HiSCRIBE can be used as a source of persistent intracellular recombinogenic oligonucleotides which can be introduced even by low-efficiency delivery methods and bypass the need for multiple recombination rounds and prevent off-target mutations. The efficiency was proved by a screenable plating assay through which it was observed that after acquiring the HiSCRIBE plasmid, about 99% of the transformed cells executed the DNA editing in deliberated manner and about 100% editing efficiency was observed over course of 2 days for about 60 generations.

In an application of this technique, a galactose degrading enzyme gene was engineered in E. coli DH5αPRO cells, which overexpress lacI and tetR that was growing in a mixed culture along with several other bacterial species. With the help of intracellularly expressed ssDNA, there was a reversion of two stop codons to their wild types within the KanR (Kanamycin Resistance) cassette. Here, IPTG was used as an inducer while the Retron Cassette was placed on a synthetic operon expressed from a plasmid. Thus, the plasmid was assessed in a population by measuring recombination frequency in the presence or absence of IPTG.

These improvements allowed the utilization of technology in situations where the previous techniques could not be used. The researchers also showed that SCRIBE could record the duration as well as the intensity of exposure into a specific molecule. But, the newly developed HiSCRIBE system is advantageous within the fact that we are able to trace those exposures as well as any other additional event that occurred due to the exposure, such as the interaction between cells. 

One of its major applications was the DNA Barcoding experiment where conjugation between two bacterial cells was recorded by their respective DNA Barcode sequences to inform which two cells have interacted with one other. Mapping of cells by this type of method is very important for studying biofilms. It is expected that a similar approach could be used for mammalian cells as well which can help us to trace diseases as well. This can be achieved by Barcoding the viruses that can cross Neural synapses to trace the interaction between neurons and thus Brain Connectome Mapping might be possible.

Another major advantage of this technique lies in the fact that it can target specific bacterial species within a community of various diverse species. This species-selective editing method can be exploited to overcome Antibiotic Resistance by silencing the resistance genes of bacteria.

It was demonstrated that this technique can cause faster evolutionary rates than natural evolution in a synthetic ecosystem of Bacteria and Bacteriophages. They were shown to continuously rewrite their genome segments to evolve autonomously by optimizing the ability to consume Lactose. Several more research years would be required to optimize and use this technique on Mammalian cells but this one study has paved the way for more flexible and specific genome editing projects which may work with a range of stimuli to yield diverse applications for the study of bacterial physiology.

References:

1. Efficient Retroelement-Mediated DNA Writing in Bacteria
2. New method opens the door to efficient genome writing in bacteria

23-Jun-2021

SAY HELLO TO OUR NEW FRIEND! -“Dysomobacter Welbionis”–

An impressive new gut bacterium.
By: Suyagya Jayaswal (MSc student)

Eureka! we found a new friend.

Recently researchers at University of Louvain discovered a new bacterium Dysomobacter welbionis. Dysomo is a Greek word meaning bad smell and Welbionis is the Walloon  organisation that funded this research. The bacteria showed various peculiar characteristics that can have a beneficial impact on human health in many ways.

So, what is new about this bacterium?

It is a gut commensal bacterium that has a strong positive impact on obesity, inflammation, and diabetes. The experimental evidence as published in “Gut” shows that this bacterium can produce butyrate which serves to be a good player in increasing intestinal immunity and fighting the risk associated with colon cancer.

Researchers at UC Louvain that included Emilie Moens de Hase and Tiphaine Le Roy used improved cultivation technique for the detection of the bacterium. Stool sample analysis have shown that about 70% of the human population harbours this bacterium in the gut, however diabetic individual showed a lower level of this bacterium. It is quite surprising that such novel bacterium took so long to be discovered.

‘The Anti-obesity effect’ - The research performed on mouse model depicted that when mice with high fat diet were fed with these freshly cultured bacteria daily for 6 days, no significant  level of fat induced weight gain was observed, whereas high fat diet mice that was not fed this bacterium showed an increase in the weight. Treating mice on a longer basis with this bacterium showed better results.

Generally, High fat diet results in increased lipid content however mice administered with this live bacterium showed reduction in this high fat lipid content and even showed lower brown adipose tissue expansion.

‘The glucose effect’ – Mouse administered with this bacterium holds potential to show higher mitochondrial number and  better glucose tolerance  than those mouse in which live D.welbionsis was not administered. It seems that the beneficial roles of our new bacterial  friend- Dysomobacter welbionis J115 t requires a long list to be illustrated,  with many more properties still to be discovered.

It also has a role to play in ‘Inflammation'- Administration of live bacterium resulted in low macrophage infiltration and inflammation. These studies were done using quantitative PCR.

High fat diet fed mice with D.welbionis showed high citrate synthase activity  than normal mice and hence showed  high mitochondrial content that trigger an increased body temperature, energy expenditure and an improved energy metabolism, However, the exact mechanism of the same still need to be encoded.

Butyrate production by D.welbionis J115t showed the property of increased mitochondrial number in Brown adipose tissue and thermogenesis. Further the bacteria also produce some other bioactive lipids that controls inflammation.

However experimental evidence showed that Pasteurization of this bacterium may result in reduced or no beneficial effect.

So, what is in store for future?

Further research, need to be done by combining D.welbioinis with  Akkermansia to know more  about the effect of both these bacteria on our gut immune as suggested by researchers at UCLouvain. It can now be stated that Dysomobacter welbionis is an efficient bacterium with superior quality that has enormous effect associated with obesity, diabetes, inflammatory diseases, BMI. Daily bacterial doses when administered to mice for more than 6 weeks contributed to reduced cases of high fat diet obesity by acting on adipose tissue metabolism.

References:

1. Dysosmobacter welbionis is a newly isolated human commensal bacterium preventing diet-induced obesity and metabolic disorders in mice
2. A new bacteria, made in Belgium (and UCLouvain)

19-Jun-2021

Bioconcrete: A self healing and eco-friendly alternative
By: Ayushi Singh (MSc student)

Concrete is considered to be the most widely used and inexpensive building material. But despite that there are several disadvantages of using concrete i.e. cracking, corrosion, premature deterioration thereby reducing its life span. As compared to the conventional methods for repairing cracks by using adhesive chemicals, the microbial-crack healing could be a more promising approach.

Microbial-concrete or bio-concrete is basically a microbe-based strategy where concrete is treated with microorganisms in order to repair cracks, which makes it a microbial self-healing long-lasting and ecofriendly alternative with high compressive strength. Bio-concrete is based on the phenomenon of bio-mineralization, where the precipitation of calcium carbonate by bacteria is used to heal the cracks. MICCP (microbially induced calcium carbonate precipitation) is the ability of microorganisms to form calcium carbonate extracellularly via metabolic activities. So, this feature of bacteria allows the self-healing of concrete.

According to a study which was recently conducted, six alkaliphilic bacteria viz. Bacillus subtilis, Brevibacillus sp., P. dendritiformis, B. methylotrophicus, B. licheniformis, S. maltophilia were isolated from the sub-surface mica mine and were mixed with cement mortar, and their compressive strengths were investigated. An improvement was observed in the compressive strength of mortar and healing ability of cracks but out of all the six isolates, Bacillus subtilis showed highest compressive strength at a concentration of 10^4 cells/ml as compared to other five isolates. The filling of cracks by calcium carbonate precipitate was observed using scanning electron microscopy (SEM).

Another approach can be by using GMOs. For example: Genetically modified version of Bacillus subtilis known as ‘BacillaFilla’ contain cells that germinate at pH of concrete, and upon germination they descend down the crack and by using mechanism of quorum sensing sufficient cells are accumulated that triggers the production of a mix of CaCO3 and ‘bacterial glue’, this combines with bacterial cells to fill the cracks. Other method for concrete healing can be done via carrying out phenotypic mutations in Sporosarcina pasteurii using UV irradiation that showed an increased urease activity in mutants that increases the calcium carbonate precipitation.

Therefore, these are certain ways by which microbes can heal the cracks in concrete. The application of MICCP has a potential to enhance the permeability properties as well as the mechanical properties of cementitious materials. But there are certain limitations that are to be considered before successful commercialization of this method. For example, there is a lesser bacterial precipitation deep in cracks as compared to crack surface because of less availability of oxygen in deep cracks. So, significant improvements and research are needed for successful application at commercial scale.

References:

1. Subsurface Endospore-Forming Bacteria Possess Bio-Sealant Properties
2. Microbial healing of cracks in concrete: A review.

19-Jun-2021

Acquaintance turned foe: Transition of Staphylococcus epidermidis to an infectious Pathogen
By: Aman Gupta (MSc student)

The bacterium called Staphylococcus epidermidisis is principally an innocuous commensal microbe that is part of the normal skin flora of humans. Moreover, it had been noticed that some strains of S.epidermidisis poses risk of infections involving indwelling foreign devices and surgical wounds. It is responsible for the bacteremia in immunocompromised individual whose treatment poses arduous. Thus it is in the list of hospital acquired infection. These bacteria are found to be resistant to antibiotic methicillin, therefore they are on the list of most worrisome bacterial infections in the hospital. For example it has been known for more than fifteen years that S. epidermidis ST 23 is a deadly pathogen, and scientists have been puzzled about how such an innocuous bacteria can transforms itself into a deadly pathogen. Now researchers have uncovered some facts that can explain this transition.

Scientists have now pinpointed the gene cluster called tarIJLM which could permits this bacteria to alter structures of their cell walls that assist Staphylococci to attach more freely to endothelial cells so that they could persist in blood to become a pathogen. Moreover, these alterations also permit the spread of methicillin resistance from Staphylococcus epidermidis to other species of the same genera which are also potential pathogen such as Staphylococcus aureus.

The cell wall of all the gram-positive bacteria is made of teichoic acids, and same is true for the members of genera Staphylococci too. During an investigation by researchers, it was observed that wall teichoic acids (poly-glycerolphosphate) which is an attribute of commensal species are also present in certain infectious strains of S.epidermidis. Apart from that, it also posses additional gene cluster which is known for the synthesis of Staphylococcus aureus-type wall teichoic acid called poly-ribitol phosphate (RboP). The presence of wall teichoic acid of S.aureus in S.epidermidis renders them less invasive towards the skin and mucous membranes and provides them the opportunity to penetrate the endothelial tissue of their human host.

It has been known that gene transfer in between the bacterial species is common which are mediated by the bacteriophage. Additionally, these gene transfer are species-specific due to the limitation of bacterial structure specificity for bacteriophage to bind. Thus, the gene transfer between S.aureus and S.epidermidis seems impossible because of their morphologically different cell wall structures. However as soon as  S.epidermidis starts expressing the Staphylococcus aureus-type wall teichoic acid, it makes it possible to transfer genetic element to transform pathogenic S.aureus to even more threatening methicillin-resistant S.aureus.

As stated by Prof. Andreas Peschel who is part of Cluster of Excellence CMFI and Professor at DZIF, these breakthrough findings lay down the foundation to develop better treatment or vaccinations against the deadly pathogen like S. epidermidis ST 23 which is in the list of HA-MRSE (healthcare-associated methicillin-resistant S. epidermidis).

These findings were published in the journal Nature Microbiology and research was carried out jointly by researchers from Cluster of excellence “Controlling Microbes to Fight Infections” (CMFI) of the University of Tübingen whose primary goal is in the development of a new strategy for controlling infections; and German Center for Infection Research (DZIF) with the help of certain other researchers from different Universities around the globe.

References:

1. Staphylococcus epidermidis clones express Staphylococcus aureus-type wall teichoic acid to shift from a commensal to pathogen lifestyle
2. From harmless skin bacteria to dreaded pathogens
3. Staphylococcus: From Harmless Skin Bacteria to Deadly Pathogen

18-Jun-2021

POSTBIOTICS: The Inanimate Biotherapeutics
By: Sreyashi Nath (MSc student)

Today’s biotherapeutic world possesses no boundaries and is continuously approaches to discover newer ways to achieve better well-being goals. After the quite recent exploration of probiotics and prebiotics, a new concept of “Postbiotics” has been indirectly unfolded in various research-based findings and commercial industries. However, in many instances, it has been confusingly misunderstood with other related terms such as heat-killed probiotics, paraprobiotics, parapsychobiotics, ghost probiotics, metabiotics, bacterial lysates, and so on. Consequently, a master panel of the International Scientific Association for Probiotics and Prebiotics (ISAPP) was summoned to propose a universal definition of postbiotic, and to set up its promising foundation for future expansion.

In contrast to the previously established definition of probiotics as “live beneficial microorganisms”; prebiotics are defined as “substrates to be selectively exploited by resident microflora”; and paraprobiotics as “functionally non-viable microbial cells or crude cell extracts” which are known to offer health benefits to the human and animal user when given in an ample concentration, the panel proposed the best-fit definition of postbiotics as “preparation of inanimate microorganisms and/or their components that confers a health benefit on the host”. The term ‘Inanimate’ confers the fact that live microbial cells have been killed off unless intending the loss of health-benefit functions. Therefore, the formulation method of a specific postbiotic preparation plays a key role in determining the beneficial effects. The panel clarifies that the anticipation of microbial end product metabolites or vaccine will not be entertained under the definition of postbiotics unless accompanied by inanimate and defined microbial cell biomass and also suggested categorizing purified microbial-derived components under specific chemical class rather as a postbiotic such as butyrate and lactic acid. Thus, one may define postbiotics as inactivated microbial cell counterparts, along with or without metabolites that have potential health-benefiting attributes.

A well-defined criterion has been made to qualify a preparation as postbiotic: “(1) Molecular perspective of the progenitor microorganisms to ensure accurate identification and screening of potential genes for safety concern; (2) Detailed account of the inactivation procedure and the base matrix; (3) Confirmation of inactivation; (4) Verification of health benefits analysis in the host through controlled and high-quality clinical trial; (5) Comprehensive picture of all the ingredient of the postbiotic formulation; and (6) Clinical safety evidence of postbiotic preparation in the selected host for the deliberate use.”

The inactivation of live microbial biomass (i.e., cessation of microbial replication) is not optimized by thermal processing as they may affect the nutritional value, sensory attribute, flavor, and immunomodulatory characteristics of postbiotic strains. Rather, non-thermal processing such as electric field, high pressure, ionizing radiation, heating through a magnetic field, ultrasonnication, high-voltage electrical discharge, and spray dryings are usually preferred to procure stable and safe postbiotic preparations. However, it is critical to check if the extent and type of treatment are not compromising the desired health benefits of specific postbiotic preparations.

The desired health effects do not essentially require the intact cells; rather the preserved microbial components such as pili, cell wall parts, or other structures having retained activity of effector molecules and excluded live microbial toxicity. In terms of their health benefit attributes, five mechanisms of action have also been proposed by the panel: “(1) Modification of the resident microflora population; (2) Improved roles of epithelial barrier; (3) Modification of local and systemic immunologic reactions; (4) Modification of systemic metabolous reactions; and (5) Nervous system-mediated systemic signaling.”
The pre-clinical analysis of postbiotics in adults is reported to exhibit next general antimicrobials, on-point anti-inflammatory and immunomodulating responses, eradication of gut and upper respiratory tract infections, enhanced immunization, relief from stress, sensation, and pain. However limited data have been obtained regarding postbiotic benefits in pediatric settings.

If compared to live biotherapeutics such as probiotic, postbiotics have certain potential advantages such as innate stability to deal with harsh industrial processes and long-term storage conditions; intellectual property protection of microbial strain used to derive the postbiotic preparation as it limits its further isolation; and safer health benefits in terms of effective but killed microbial counterparts. Whereas probiotics have evidential drawbacks such as septicemia in compromised individuals, poor clinical safety data, emerging antibiotic resistance, virulence gene transfer, short shelf-life, cumbersome production and maintenance, disruption of healthy gut microflora, risk of opportunistic infection, ambiguous translocation to blood or tissue, inflammatory responses, and so on. Therefore, postbiotics can promisingly replace the probiotics as an alternative health-promoting bioactive component having safer intended use and substantiated health benefits to deal with diverse pathological conditions. However, in real clinical settings, a robust risk-benefit analysis is strongly recommended before treating any acute and chronic diseases.

References:
1. The International Scientific Association of Probiotics and Prebiotics (ISAPP) consensus statement on the definition and scope of postbiotics
2. Postbiotics-parabiotics: the new horizons in microbial biotherapy and functional foods
3. Balancing the risks and rewards of live biotherapeutics

12-Apr-2021

Possibilities of farming in space: Novel Bacterial Strains may Support Plant Growth
By: Pratibha Gautam (MSc student)

With the increase and frequent space exploration, space has become a new field of human activity. One such activity is growing plants in space. Although, as of now, astronauts have to rely on dried, canned foods for their survival, since growing fresh vegetables remains a challenge due to extreme environmental constraints. However, a study published in a recent issue of Frontiers of Microbiology claims that it might be possible in future to grow fresh food in space.

This study was carried out by Researchers from NASA’s Jet Propulsion Laboratory (JPL) and University of Hyderabad (UoH). The discovery of four bacterial strains isolated from different locations on International Space Station (ISS) served as the basis of this study. One was discovered on a dining table; another on an overhead panel in a research area used to study low gravity; the third in the Cupola observatory. The fourth species, which was already known of, was found on an old air-purifying filter, which had been returned to Earth. Space explorers have always used ISS as a test bed for surveying microbial growth. Thus, with help of this discovery, Researchers suggested that these microbes may help in creating the ‘fuel’ to help plants withstand extreme situations in space. These strains belong to the family of Methylobacteriaceae which are typically found in soil and freshwater.

One of the bacteria was identified as Methylorubrum rhodesianum, while the other three were novel species. Biochemical testing revealed that these three were Gram-negative, rod-shaped, catalase-positive, oxidase-positive, motile bacteria. The 16S rRNA sequencing shows < 99.4 sequence similarity of these species with Methylobacterium indicum indicating that they might belong to the same clade. M. indicum is known to support plant life by nitrogen fixation, phosphate solubilization, abiotic stress tolerance, plant growth promotion and biocontrol activity against plant pathogens.

Scientists believe the bacteria will be helpful to the growth of plants in space, and act as a unique tool to support longer and healthy survival of future astronauts in space. Out of the three novel strains, one was named Methylobacterium ajmalii, in honour of the renowned Indian biodiversity scientist, Dr. Ajmal Khan (former professor at Annamalai University). The genome analysis of these strains reports the presence of certain plant growth promoting genes. Although being novel, these bacteria have originated from Earth itself and transported along with cargo spacecrafts to ISS, as stated by Christine Moissl-Eichinger.

The pioneers of this study are NASA researchers Dr. Kasthuri Venkateswaran (Senior research scientist at NASA’s Jet Propulsion Laboratory), Dr Nitin Kumar Singh (Planetary protection Engineer at JPL) and C.C. Wang (WorldQuant Initiative for Quantitative Prediction) and Prof. Appa Rao Podile from the University of Hyderabad (UoH) with expertise on plant growth promoting bacteria and plant microbiome and Dr. Ramprasad (CSIR-pool scientist). Along with them, other researchers from University of Southern California, Cornell University and University of Hyderabad were also involved with the study.

It has been suggested by the lead authors of the study that these strains might possess some “biotechnologically useful genetic determinants that may help growing plants in extreme places where resources are minimal”. However, further experimentation in microgravity environment is required to support this study.

References:

1. Methylobacterium ajmalii sp. nov., Isolated From the International Space Station
2. Farming in space may be possible, says study
3. NASA discovers three microbes that may help grow plants on Mars
4. New bacteria lurking on ISS no space oddity, says scientist

22-Feb-2021

Disinfection of Super-resistome by Super-wrapped killer

By: Sreyashi Nath (MSc student)

“All That Is Born Must Die”, and the saying is going to get true even for the expected to be immortal superbugs. The powerful antibiotic-resistant bacteria that have failed most of the antibiotics ever since made, now get trapped by the wrapped mind of nanotechnology and chemistry. A group of brilliant minds has found the solution for full-proof waste-water treatment, even to relieve from the pool of antibiotic-resistant bacteria (ARB) as well as extracellular antibiotic-resistant genes (eARGs) by using microspheres wrapped with nitrogen-doped reduced graphene oxide (NRGO).  

Antibiotic resistome (i.e., ARB as well as the eARGs) has hijacked a wide boundary of the environment and it is a critical threat for mankind to have them in our drinking water and surrounding environment even after applying the specialized waste-water treatment methods. Photoelectrocatalytic treatments have proven to be effective in destroying both ARB as well as eARGs from aqueous solutions. It has the potential to produce various reactive oxygen species (ROS) that can degrade the essential biomolecules such as lipid, protein, and nucleic acid to eradicate all kinds of microbial pollutants from waste-water.

The photocatalytic ability can be further enhanced by increasing the specific surface area using 2D photoactive nanosheets. Here then it has been proposed to use photocatalytic microspheres surrounded with a thin layer of NRGO shell i.e., NRGO wrapped Bi2O2CO3 Microspheres (NGWM), which has the potential to improve bacterial adhesion near photocatalytic sites due to enhanced ROS generation. It provides less negative zeta potential as well as decreased electrostatic repulsion, which increases the likelihood of catalyst-bacteria clash via hydrophobic interactions. It also facilitates the immediate arrest of eARGs released from cell lysis. Hence, resulted in a kind of disinfection that can completely clean the secondary effluent from waste-water plant. 

It has provided various kinds of chemistry-based advantages over the conventional photocatalytic methods such as shorter distance between bacteria and photocatalytic surface which doesn’t let the releasing eARGs escape the attack of microspheres, the shell even increased the photocatalytic affinity towards the antibiotic-resistant plasmids by various covalent bonds, and also the shell mediates increased in shelf-life of the microsphere by preventing photo corrosion from irradiation. These NGWM can be reused and remain stable even after 10 cycles.  

As compared to methods earlier were adopted i.e., the use of nanosheet assembled wrapped microsphere in reduced graphene oxide without nitrogen doping, the results were not that hit. It able to achieve remarkable ROS generation but increased cell lysis increases the escape and spread of eARGs into the environment. It was due to weak adhesion between the bacterial contaminant and the microspheres. However, when nitrogen doping was employed, it reduces the structural defects within the assembled nanosphere plane thus improving the interaction between the photocatalytic microsphere and the wrapping agent NRGO. It also intensifies the efficiency of electron transfer due to the presence of nitrogen atoms and consequently providing enhanced ROS generation. Overall, it can be concluded that wrapping by NRGO enhanced the trapping efficiency of this photocatalysis-induced disinfection. 

Hence, this novel approach of NGWM-based photocatalytic treatment has the potential to minimize the widespread antibiotic resistance from sewage-water plants.

References:

1. Jiang, Q., Yin, H., Li, G., Liu, H., An, T., Wong, P. K., & Zhao, H. (2017). Elimination of antibiotic-resistance bacterium and its associated/dissociative bla TEM-1 and aac (3)-II antibiotic-resistance genes in aqueous system via photo electrocatalytic process. Water Research, 125, 219–226. doi: 10.1016/j.watres.2017.08.050

2. Li, D., Yu, P., Zhou, X., Kim, J.-H., Zhang, Y., & Alvarez, P. J. J. (2020). Hierarchical Bi2O2CO3 wrapped with modified graphene oxide for adsorption-enhanced photocatalytic inactivation of antibiotic resistant bacteria and resistance genes. Water Research, 116157. doi: 10.1016/j.watres.2020.116157

22-Aug-2020

Florida Mosquitoes
By: Vishal Dashora (PhD student)

On 18th August 2020, the much debated issue of releasing genetically modified mosquitoes in Florida was finally drawn to conclusion. The Florida Keys Mosquito Control District (FKMCD) finally approved the release of 750 million GM mosquitoes over a two year period around the Florida Keys area. This issue has been a hot topic of discussion for the last five years. Various environmental groups have condemned this solution claiming that people of Florida were being subjected to a “JURASSIC PARK EXPERIMENT”. Let’s start by looking at the problem.

The Sunshine state is home to 80 known species of mosquitoes. Every year fatal diseases like Dengue, Zika, Encephalitis and Malaria find their way into the communities of Florida affecting hundreds of local people as well as travelers. The local authorities have tried to contain the situation by creating awareness in population about the possible areas where mosquitoes can breed and about what insecticides can be used to control the population of these menaces. The high temperature and high humidity of the Florida states creates an ideal breeding ground for mosquitoes.

As a prospective solution, a UK-based biotech company called Oxitec created a genetically modified strain of Aedes aegypti named OX5034 which they proposed to release in the affected regions of Florida. The male mutant mosquitoes released will hopefully mate with female wild mosquitoes (female Aedes mosquitoes are the primary carriers of diseases) and will pass on a lethal gene to the female offsprings. The female offsprings are engineered to die at a larval stage due to the absence of Tetracycline.

The idea is to release millions of male GM Aedes mosquitoes over a period of time to reduce the population of female Aedes.  In early 2020, the company was bought by a US venture and along with support of Bill and Melinda Gates foundation, Oxitec has carried out trials for OX5034 in Brazil. The company has faced considerable problems in implementing this solution in Florida. Outrage from multiple environmental groups along with protests from community has delayed the trials.

The USEPA considers GM insects as bio-pesticides and due to unspecified laws and regulations for bio-pesticides, the GM mosquitoes have not been classified as risk free solutions. According to some reports, a small percentage of female mosquitoes didn’t die as expected and continued to proliferate. This may be because of the Tetracycline present in septic tanks, pet foods and animal manure. One major concern is the increase in number of other species of Aedes mosquitoes as the population of Aedes aegypti will go down which can result in the spread of new unknown diseases.

Despite all the concerns around it, the local officials have approved the release of 750 million mosquitoes into Florida state by 2021 and it remains to be seen how this will plan out during the ongoing COVID-19 pandemic. One can only hope for the best.

References:

1.Florida mosquitoes: 750 million genetically modified insects to be released – BBC news
2.Mosquitoes in Florida
3.Genetically engineered mosquitoes – Coming soon to Florida and Texas? – Friends of The Earth
4.Florida Keys releasing genetically modified mosquitoes to fight illness – Tampa Bay times

10-Jun-2020

The situation of Post-kala-azar dermal leishmaniasis (PKDL) in Indian subcontinent

By: Vishal Dashora (PhD student)

Visceral Leishmaniasis (VL), also known as kala azar, results from the infection with Leishmania donovani or Leishmania infantum. It is characterized by symptoms like splenomegaly, irregular fever, anemia and weight loss. VL is endemic in countries like India, Brazil and Sudan where it generally affects the poorest of the poor. In India, VL is mostly contained in Bihar (90% of the cases), West Bengal and eastern Uttar Pradesh. Several programs are underway to limit VL incidence rates like KalaCORE (2019) and Kala Azar Elimination program (2005).

Recently, Post-kala-azar dermal leishmaniasis or PKDL has been recorded in India at an increasing trend. PKDL is an intermediate complication of VL, which occurs either during or after the treatment of VL. Drugs like Miltefosine can decrease the parasitical load and cure the VL symptoms but the re-emergence of parasites is very likely.

The clinical features of PKDL include skin rash consisting of macules, papules or nodules in an otherwise healthy individual. PKDL is mostly associated with L. donovani infections and occurs in 5-20% of VL patients. Although the patients are cured of splenomegaly and are no longer under-nourished, the social stigma present with a dermal disease is still very much present. Psychological studies have shown significant decline in mental health, social functioning, and general health in PKDL patients.

For the eradication of VL in India, the focus has been on the elimination of transmission sources and reservoirs of the parasite. PKDL patients pose a great risk to this campaign as they can still transmit the parasite. The accurate diagnosis of PKDL is still a challenge because of the symptomatic resemblance to that of vitiligo and leprosy. Delayed or faulty diagnosis may lead to silent reservoirs of L. donovani which can frustrate the elimination efforts. Considerable research is directed towards making efficient and accurate diagnostic kits for correct identification of PKDL.

Some reports have suggested the occurrence of PKDL in patients with no history of VL. In such cases, the dermal lesions can be easily mistaken for something else. Hence, the complete treatment of VL and efficient diagnosis of PKDL is necessary to control the incidence rates of PKDL in Indian subcontinent.

References:

1. Zijlstra EE (2019) Biomarkers in Post-kala-azar Dermal Leishmaniasis. Front. Cell. Infect. Microbiol. 9:228. doi: 10.3389/fcimb.2019.00228
2. Trindade et al. BMC Infectious Diseases (2015) 15:543 DOI 10.1186/s12879-015-1260
3. Topno RK, Rabi Das VN, Kumar M, Madhukar M, Pandey K, Verma N, et al. (2020) Advanced case of PKDL due to delayed treatment: A rare case report. PLoS Negl Trop Dis
4. Zijlstra Parasites & Vectors (2016) 9:464 DOI 10.1186/s13071-016-1721-
5. Jaiswal P, Datta S, Sardar B, Chaudhuri SJ, Maji D, Ghosh M, et al. (2018) Glycoproteins in circulating immune complexes are biomarkers of patients with Indian PKDL: A study from endemic districts of West Bengal, India. PLoS ONE 13(2):e0192302.
6. Duthie MS, Goto Y, Ghosh P, Mondal D. Impact of sequelae of visceral leishmaniasis and their contribution to ongoing transmission of Leishmania donovani. Pathog Dis. 2019;77(6):ftz057. doi:10.1093/femspd/ftz057
7. Le Rutte, E. A., Zijlstra, E. E., & de Vlas, S. J. (2019). Post-Kala-Azar Dermal Leishmaniasis as a Reservoir for Visceral Leishmaniasis Transmission. Trends in parasitology, 35(8), 590–592. https://doi.org/10.1016/j.pt.2019.06.007

15-Feb-2020

Supra-Kingdom of Life

By: Shubhi Khare (MSc student)

A scientist is never off duty and this was shown by graduate student at Dalhousie University Yana Eglit. She was on a hike with her friends in Canadian woods from where she picked up the dirt and soaked in water. After few days she checked to see if the water had revived any dormant microbes. She found two different types of hemimastigote in the soil, one of which was an entirely new species, and team named it Hemimastix kukwesjijk.

The hemimastigote were first identified in 19th century, but remain mystery due to the inability of scientist to figure out the kingdom of these creatures. Hemimastigote are about two hundredth of mm in length. They run quickly using dozen of flagella on their surface. Unlike other microorganisms they move their flagella in random directions rather in a coordinated manner. They belong to domain of eukaryotes. They represent 'known unknown’ protist lineages- moderately well described groups, but their position on tree of life is unknown because they are difficult to culture in labs and sequence. An advance genetic analysis done by a team at Dalhousie University shows that they more different from other organism and they represent a new “Major Branch” of evolutionary tree sequence and they represent their own distinct lineage apart from other super groups.Their DNA is present in the form of chromosomes within a distinct nucleus. The organisms have no common ancestors with any other living thing in the last billion years and they are described as “Voracious Little Ogres”.

The discoveries like these will more profoundly tell us about the steadily growing number of taxonomic addition. We will keep uncovering not new species or classes but entirely new kingdom of life and these kind of data will reshape the tree of life because many more branches will be discovered.

Further reading:
1. Quanta Magazine
2. CBC News

14-Feb-2020

Newly found Bacteriophage is a little too big

By: Tanishqua Pawar (MSc student)

Earth’s ecosystem is no less than a box full of surprises. UC Berkeley researchers unraveled a new mystery about earth’s microbiomes by recently discovering 351 different phages which have genome size of around 200 kbp which is way too larger than the genome sizes of some of the already known bacteria, which ranges from 130 kbp to 14 Mbp. These huge phages are 15 times larger than the usual phage genome size i.e. 50 kbp.
Among these newly discovered phages, one of them have a genome size of around 735,000 base-pairs, which is nearly 15 times larger than the usual phage genome, making it the largest phage genome in the world as of now.

Because of the huge size of the genome, the 351 megaphages were thus divided into 10 groups, which were named to pay homage to the authors that contributed in the publishing of the paper by naming them after the languages spoken in their native countries. Mahaphage (Sanskrit), Kabirphage, Dakhmphage and Jabbarphage (Arabic); Kyodaiphage (Japanese); Biggiephage (Australian), Whopperphage (American); Judaphage (Chinese), Enormephage (French); and Kaempephage (Danish), these were the names given as per the words for “Big” in their respective languages.

The researchers and collaborators did commendable work by searching the grounds of 30 different environments, ranging from the guts of premature infants and pregnant women to a Tibetan hot spring, a South African bioreactor, hospital rooms, oceans, lakes and deep underground. The huge phage’s DNA can make a protein which is an analogous of a Cas9 protein named as Cas∅(phi) which is a part of gene-editing tool. CRISPR –Cas9 is a revolutionary tool developed by Jennifer Doudna of UC Berkeley and her European colleague, Emmanuelle Charpentier.

The huge phages DNA also has a gene for translation process, usually these ribosomal machinery are found in bacteria and archaea but not in viruses.
This discovery might be able to explain how phage genes are involved in antibiotic resistance and pathogenesis in human. It could also explain how the phages are co-existing in the human gut along with archaea and bacteria. Jill Banfield, who is a CZ Biohub investigator and also director of microbial research at the Innovative Genomics Institute (IGI) said, "Some diseases are caused indirectly by phages, because phages move around genes involved in pathogenesis and antibiotic resistance. And the larger the genome, the larger the capacity you have to move around those sorts of genes, and the higher the probability that you will be able to deliver undesirable genes to bacteria in human microbiomes.”

The metagenomic sequencing reveals large sizes of phage genome and its similarity with each other. These phages carry a lot of potential that might help us to find new tools which could be found useful in genetic engineering. It could also become a source of unknown proteins that might find applications in various industries ranging from medical to agricultural.

Further reading:

1. Berkeley News
2. Science Daily

23-Jan-2020

The Wuhan “Seafood market” Coronavirus

By: Vishal Dashora (PhD Scholar)

 Once upon a time in China, there was monster called Nian which would appear in the middle of the night and eat the villagers, especially children. One day all the villagers decided to hide from the beast but one old man, Yanhuang decided to take revenge. With red clothes on his back and firecrackers in his hand, he managed to scare the monster away and since then every year during spring time the Chinese people celebrate the New Calendar year by decorating the country in red and bursting fire crackers to keep Nian away. But this year it seems that the mythical beast has managed to creep its way through.

There were six reported strains of Coronavirus in humans, until the 31st December of 2019, when a novel strain of Coronavirus was identified in the city of Wuhan, China. Out of 27 suspected cases of the “pneumonia of unknown cause”, majority were stallholders in the Wuhan South China Seafood Market. Being the seventh-largest city of China, Wuhan is a hub for import-export of goods as well as people and hence, neighboring countries like Thailand and Singapore began the basic monitoring of the inbound passengers. On 5th January 2020, the health officials ruled out SARS, MERS and bird flu in the confirmed 59 cases and were already starting the quarantine procedures for the patients as well as the initial contacts.

WHO has confirmed that a novel coronavirus has been isolated from one of the patients, and named it as 2019-nCoV. The first death occurred on 9th January 2020 and the victim was a 61 year old man who regularly shopped at the seafood market. By 11th January the genome of this newly isolated menace was submitted in GenBank and travel guidance was issued all over the country. The first case outside of China was that of a 60 year old Wuhan-resident Chinese woman who had flown to Bangkok. The alarm bells finally rang loud on 14th January, when the officials noticed a couple among the listed cases in Wuhan, only one of which had gone to the market recently. This clearly suggested a human-to-human transmission of the virus.

By 20th January several individual cases has been reported in Japan, Thailand, South Korea and even Washington State where a male patient in his 30s is still being kept in isolation after he was diagnosed with the “mystery virus” when returned from Wuhan. With the Chinese New Year approaching on 25th January 2020, the WHO has conducted an emergency meeting on 22nd January to assess the “potential pandemic risk of the outbreak” as the “Year of the Rat” will attract thousands of tourists the Mainland China. Although the genetic sequence of 2019-nCoV is distinct from SARS and MERS, the previously known antivirals are being repurposed due to the lack of newer vaccines and drugs.

As of 23rd January 2020, 17 people have lost their lives to this havoc and where most of the world has already rejoiced in the wake of a new calendar year, the people of China will be celebrating the “Zhōngguó xīnnián” in considerable apprehension.

Further reading:

1.New York times report
2.CDC Media release
3.WHO statement
4.Wikipedia

22-Jan-2020

The Serendipitous Immune T-cell Discovery

By: Juhi Jain (PhD Scholar)

A new Human T Lymphocyte (T cell) has been accidently discovered by the Prof. Andrew Sewell at Cardiff University in the UK which can target and kill most kind of cancer cells and ignore healthy cells. The new TCR is found to have a broad cancer specificity which raised the prospect of “universal” cancer therapy if it pass all the clinical trials. This T cell is equipped with a novel T cell receptor (TCR) named MR1 which they found by performing Genome-wide CRISPR-Cas9 screening. These MR1 having T cell sense the cancer cells and kill it like HLA. In the in vitro study, this new T cell has been shown to kill lung, skin, blood, colon, breast, bone, prostate, ovarian, kidney and cervical cancer cells, while ignoring healthy cells. To test the therapeutic potential of these cells in vivo, the T-cells having MR1 has been injected into mice with human cancer and a human immune system, that resulted in clearance of cancer cells .These T-cells would be the perfect candidate for T-cell therapy as they could provide a “one-size-fits-all” cancer treatment. The Cardiff group further take out the T-cells of melanoma patients and genetically modified them to express this new TCR and then injected back into the patients. These modified T-cells could not only kill the patient’s own cancer cells, but also other patient’s cancer cells in the laboratory, regardless of the patient’s HLA type which is a promising result in the pave of cancer therapy.

 Further reading:

1. BBC New report
2. Original research article in Nature Immunology