Tumor tissue-specific bacterial biomarker panel for colorectal cancer: Bacteroides massiliensis, Alistipes species, Alistipes onderdonkii, Bifidobacterium pseudocatenulatum, Corynebacterium appendicis.

Human microbiome studies have shown diversity to exist among different ethnic populations. However, studies pertaining to the microbial composition of CRC among the Indian population have not been well explored. We aimed to decipher the microbial signature in tumor tissues from North Indian CRC patients. Next-generation sequencing of tumor and adjacent tissue-derived bacterial 16S rRNA V3-V4 hypervariable regions was performed to investigate the abundance of specific microbes. The expression profile analysis deciphered a decreased diversity among the tumor-associated microbial communities. At the phyla level, Proteobacteria was differentially expressed in CRC tissues than adjacent normal. Further, DeSeq2 normalization identified 4 out of 79 distinct species (p < 0.005) only in CRC, Bacteroides massiliensis, Alistipes onderdonkii, Bifidobacterium pseudocatenulatum, and Corynebacterium appendicis. Thus, the findings suggest that microbial signatures can be used as putative biomarkers in diagnosis, prognosis and treatment management of CRC.

A Comparative Study of the Diagnostic and Prognostic Utility of Soluble Urokinase-type Plasminogen Activator Receptor and Procalcitonin in Patients with Sepsis and Systemic Inflammation Response Syndrome.

INTRODUCTION: Differentiation between sepsis and systemic inflammation response syndrome (SIRS) remains a diagnostic challenge for clinicians as both may have similar clinical presentation. A quick and accurate diagnostic tool that can discriminate between these two conditions would aid in appropriate therapeutic decision-making. This prospective study was conducted to evaluate the diagnostic and prognostic utility of soluble urokinase-type plasminogen activator receptor (suPAR) and procalcitonin (PCT) in sepsis and SIRS patients. MATERIALS AND METHODS: Eighty-eight patients were enrolled, of which 29 were SIRS and 59 were sepsis patients. The levels of suPAR and PCT were measured on the day of admission (day 1), day 3, and day 7. RESULTS: The levels of suPAR and PCT were significantly higher (p = 0.05 and p < 0.001, respectively) in sepsis group as compared to the SIRS group. The soluble urokinase-type plasminogen activator receptor was a better diagnostic tool in predicting sepsis over PCT [area under curve (AUC) 0.89 vs 0.82] on day 1. The best cutoff for suPAR was 5.58 pg/mL [96% sensitivity and 90% negative predictive value (NPV)] and the best cut-off for PCT was 1.96 ng/mL (93.1% sensitivity and 80% NPV). However, PCT had better prognostic trends (p = 0.006) to identify nonsurvivors in sepsis group. CONCLUSION: Our findings suggest that both suPAR and PCT can be used as potential test tools to differentiate between SIRS and sepsis. Procalcitonin showed significant prognostic trends to identify nonsurvivors. The soluble urokinase-type plasminogen activator receptor showed better diagnostic potential than PCT on day 1. CLINICAL SIGNIFICANCE: Both suPAR and PCT can be used as surrogate biomarkers to distinguish sepsis from SIRS. Procalcitonin showing a significant prognostic trend to identify nonsurvivors can help the clinicians to take relevant clinical decisions. Also, the use of biomarkers like PCT and suPAR could reduce the inappropriate use of antibiotics in noninfective SIRS. HOW TO CITE THIS ARTICLE: Sharma A, Ray S, Mamidipalli R, Kakar A, Chugh P, Jain R, et al. A Comparative Study of the Diagnostic and Prognostic Utility of Soluble Urokinase-type Plasminogen Activator Receptor and Procalcitonin in Patients with Sepsis and Systemic Inflammation Response Syndrome. Indian J Crit Care Med 2020;24(4):245-251.

Potentials of "stem cell-therapy" in pancreatic cancer: An update.

In recent times, cell-therapies like T-activated cells, dendritic cells and natural killer cells have shown increasing promise in treating cancers as evidenced by both animal and human studies in the literature. In addition, stem cells are also being considered as potent anti-cancer agents since they act through multi-pronged approaches (chemokines, cytokines, paracrine action). In this review, we have attempted to discuss the inferences of studies that have used different sub-types of stem cells namely mesenchymal stem cells (MSCs), hematopoietic stem cells (HSCs) and neural stem cells (NSCs) in in-vitro/in-vivo mice and/or human studies as a treatment modality for pancreatic cancer. Pancreatic cancers are diagnosed in late/metastatic stages hence limiting its progress to partial/disease-free status. Recent literature supports evidences of stem cell therapy in pancreatic cancer with promising results; yet their impact remains inconclusive due to limited studies in human subjects. With reference to the treatment options for pancreatic cancer, the most studied sub-type of stem cells was HSCs as evident from the available clinical trials. The suggested mechanism of the HSC-transplantation is presumably via the graft-versus-tumor effect that elicits an anti-tumor immune response activated by the T-cell repertoires. On the other hand, the property of MSCs like tropism, migration to tumor site and activation of host immune cells by its secretome, appear to be able to regulate pancreatic tumor microenvironment. Further, drug delivery potential could be mediated via engineered MSCs to enhance the bioavailability of drug/prodrug at tumor site. Conclusively, stem cells have shown great potentials as next-generation therapeutic options.

Role of Homocysteine in Cognitive Impairement and Alzheimer's Disease.

A high circulating concentration of the non proteinogenic amino acid homocysteine has been implicated as a risk factor for Alzheimer's Disease and its prodromal stage, mild cognitive impairement. Furthermore, hyperhomocysteinaemia has been directly attributed to a deficiency in vitamins B12, folate, and B6. Several studies have demonstrated decrease in progression of mild cognitive impairement to Alzheimer's Disease, and some have even shown an improvement in cognition after vitamin supplements with B12 and folate. Plausible mechanisms linking hyperhomocysteinaemia to Alzheimer's and cognitive impairement have been hypothesized and demonstrated in hyperhomocysteinemic mice models. However, some studies have not elucidated any benefit of vitamin supplements in subjects with cognitive impairment. Hence, multicentric clinical studies need to be conducted to substantiate the mechanisms of neuronal degeneration due to hyperhomocysteinaemia and to demonstrate the beneficial effect of folate, B6 and B12 supplements on cognition.

Can mesenchymal stem cell therapy be the interim management of COVID-19?

COVID-19 pandemic has accounted for ~ 4.3 million confirmed cases and ~ 292,000 deaths (till 12th May, 2020) across the globe since its outbreak. Several anti-viral drugs such as RNA dependent RNA polymerase inhibitors (remdesivir, favipiravir, ribavirin), protease inhibitors (lopinavir, ritonavir) and drugs targeting endocytic pathway (hydroxychloroquine) are being evaluated for COVID-19 but standard therapeutics yet not available. Severe health deterioration in critically ill patients is characterized by pulmonary edema, severe respiratory distress, cytokine storm and septic shock. To combat cytokine storm, immune-therapy targeting IL-1, IL-2, IL-6 and TNFα are being evaluated and one of the promising immune-modulator is the mesenchymal stem cells (MSCs) that can surmount the severity of COVID-19 infections. Recent studies have shown that MSC-therapy significantly dampens the cytokine storm in critically ill COVID-19 patients. This communication endows with the insight of stem cell therapy and summarizes the recent studies on COVID-19 patients.

Diversity of edible insects in a Natural World Heritage Site of India: entomophagy attitudes and implications for food security in the region.

Insects not only play a significant role in the ecological process of nature but since pre-historic times have also formed a part of the human diet. With a still growing population and skewed demographic structures across most societies of the world, their role as nutrient-rich food has been increasingly advocated by researchers and policymakers globally. In this study, we examine the edible insect diversity and entomophagy attitudes of ethnic people in Manas National Park, a UNESCO Natural World Heritage Site, located in Assam (India). The study involved a field investigation through which the pattern of entomophagy and the attitude towards insect-eating was studied. Following this, we examined the edible insect diversity and abundance at different sampling points. A total of 22 species of edible insects belonging to fifteen families and eight orders were recorded from different habitat types. Out of these 22 species, Orthopterans showed a maximum number of eight species followed by Hymenoptera (four), Hemiptera (three), Lepidoptera (two), Blattodea (two) and one species each from Coleoptera, Odonata, and Mantodea. Dominance, diversity, and equitability indices were computed along with the relative abundance of the insects concerning four habitat types. Aspects of the economic significance of entomophagy were also observed during the field investigation. To manage insects in the interest of food security, more attention should be given to sustainable collecting and rearing methods emphasizing their economic, nutritional, and ecological advantages.

Genotypic characterization of 'inferred' rifampin mutations in GenoType MTBDRplus assay and its association with phenotypic susceptibility testing of Mycobacterium tuberculosis.

In GenoType MTBDRplus assay [line probe assay (LPA)], when Mycobacterium tuberculosis (M. tuberculosis) sample DNA fails to hybridize to at least 1 rpoB wild-type probe and any mutation probe, it is inferred as rifampin (RIF)-resistant. In this study, we sought to identify such 'inferred' mutations in M. tuberculosis isolates (n = 203) by rpoB gene sequencing and determined their association with phenotypic resistance. D516Y, H526N, L511P mutations were associated with both phenotypically sensitive (59%, n = 38/64) and resistant (23.7%, n = 33/139) antimicrobial susceptibility testing (AST) results, whereas S531W mutation was associated with only RIF-resistant isolates (33%, n = 46/139). These results demonstrated that, at standard drug concentrations, some 'inferred' mutations may be missed by RIF-AST (phenotypically sensitive). The use of LPA permits identification of these RIF-resistant isolates, and incorporation of additional mutation probes (e.g., S531W) could further increase LPA specificity. Further studies are needed to establish the significance of the type of 'inferred' mutation with clinical/treatment outcomes.

Biophysical Characterization and Drug Delivery Potential of Exosomes from Human Wharton's Jelly-Derived Mesenchymal Stem Cells.

Cell-derived exosomes (30-200 nm) as biological "nanocarriers" have attracted a great deal of interest for therapeutic applications due to their ability to internalize in in vivo biological systems (i.e., cells). Although they can be harvested from various sources including stem cells, yet an appropriate isolation and characterization protocol to obtain "pure" exosomal population is needed. For potential clinical applications, understanding the functional ability of exosomes and their purity, that is, free from microvesicles, apoptotic bodies, and protein aggregates, is a pre-requisite. To achieve high purity and yield of exosomes from human Wharton's jelly-derived mesenchymal stem cells (hWJ-MSCs) in the size range of 30-200 nm, we have performed and compared three isolation procedures: ultracentrifugation (UC), sucrose cushion (SC), and commercially available reagent (CR). The isolated exosomes were characterized using nanoparticle tracking analysis (NTA), field emission scanning electron microscopy (FESEM), and atomic force microscopy (AFM). Furthermore, to understand the therapeutic potential of the hWJ-MSC-derived exosomes (hWJ-ME) to target pancreatic tumor cells, the internalization efficacy has been evaluated on the MiaPaCa-2 cell lines using confocal microscopy and flow cytometry. The NTA results showed sucrose cushion to be an optimal method for exosome isolation with high purity (86.8%), as compared to UC (40.5%; p = 0.050) and CR (38%; p = 0.050). Optical analysis by FESEM and AFM revealed that SC-isolated exosomes presented a spherical morphology, whereas UC- and CR-isolated exosomes exhibited an uneven morphology. Furthermore, the data from confocal images and flow cytometry showed that hWJ-ME were internalized by MiaPaCa-2, demonstrating the feasibility of exosomes as a "potential nanocarrier". Thus, our study suggests that a combination of NTA (yield), AFM (dimensions), and FESEM (morphology and topography) could provide sensitive biophysical characterization of hWJ-ME. In the future, enriched exosomes could be used as a delivery vehicle to transport target-specific drugs or gene-silencing constructs to tumors.

DNA vaccine for rabies: relevance of the trans-membrane domain of the glycoprotein in generating an antibody response.

Various studies have demonstrated the potential of immunization with DNA vaccines encoding the rabies virus glycoprotein (RV-G) to elicit humoral responses. In the present study, we have designed four constructs using a VR1020 vector, wherein the RV-G ectodomain has been cloned without the signal sequence (SS) and the trans-membrane domain (TD) (rGVR), without the SS but with the TD (rGVRt), with the SS but without the TD (rGVRs) and with the SS and the TD (rGVRst), under the control of a cytomegalovirus (CMV) promoter, and downstream of the tissue plasminogen activator (TPA) signal sequence. In addition, RV-G has been expressed as a His6 tag fusion protein, both in Escherichia coli as well as in baculovirus expression systems. Using a prime-boost strategy, BALB/cJ mice administered with the rGVRt construct either in saline (intramuscularly) or adsorbed onto gold microcarriers (delivered intradermally by gene gun) generated the highest rabies virus neutralizing antibody (RVNA) titers. Inclusion of the SS, in addition to the TD (rGVRst), led to a significant decrease in RVNA titers, compared to the rGVRt construct. The DNA vaccine construct lacking both the SS and the TD domain and the vaccine having only the SS generated lower antibody responses, compared to the rGVRt construct. After priming with DNA vaccine, boosting with both E. coli- as well as baculovirus-expressed rRV-G led to an increase in the RVNA titers. The present results demonstrate that a DNA vaccine encoding the full-length sequence of the ectodomain plus TD of the mature native RV-G is capable of expressing an 'ideal' immunogen to produce RVNA titers.

Update on zona pellucida glycoproteins based contraceptive vaccine.

Zona pellucida (ZP) glycoproteins, due to their critical role in mammalian fertilization, have been proposed as candidate immunogens for development of a contraceptive vaccine. Active immunization studies in a variety of animal species, employing either native or recombinant zona proteins, has established their contraceptive potential. Hence, ZP glycoprotein-based contraceptive vaccines have a very good potential for controlling wild life population. To make it a realistic proposition, additional research inputs are required to develop new potent adjuvants and novel practical strategies for vaccine delivery. The observed ovarian dysfunction, often associated with immunization by ZP glycoproteins, is one of the major obstacles for their application in the control of human population. Ongoing studies to delineate epitopes of ZP glycoproteins that will generate an immune response capable of inhibiting fertility without any untoward effects on ovarian functions will help in determining their feasibility for human use.

Zona pellucida glycoproteins based immunocontraceptive vaccines: strategies for development and their applications.

The mammalian oocyte is surrounded by an extra-cellular matrix, the zona pellucida (ZP), composed of three major glycoproteins (ZP1, ZP2 and ZP3). The ZP glycoproteins, by virtue of their tissue specificity and critical role during mammalian fertilization, have emerged as potential candidate antigens for the development of an immunocontraceptive vaccine. Molecular characterization of ZP glycoproteins from several species, reveals a variable degree of homology among the deduced primary amino acid sequences, which provided an opportunity to undertake active immunization studies in heterologous animal models. Active immunization of various animal species with either native ZP glycoproteins or those obtained by recombinant DNA technology led to the inhibition of fertility. Thus ZP glycoproteins based immunocontraceptive vaccines offer an attractive proposition for controlling wild life population. To make it a practical proposition, additional research inputs are required to optimize and devise novel strategies for vaccine delivery. Observed ovarian dysfunction, often associated with immunization by ZP glycoproteins is one of the major stumbling blocks for their use in humans. Ongoing studies to delineate appropriate B cell epitopes of ZP glycoproteins that are devoid of oophoritogenic T-cell epitopes, which will inhibit fertility without concomitant oophoritis, will be critical to determine their feasibility for human use.

DNA vaccine encoding chimeric protein encompassing epitopes of human ZP3 and ZP4: immunogenicity and characterization of antibodies.

Immunization with zona pellucida (ZP) glycoproteins leads to curtailment of fertility often associated with ovarian dysfunction. To avoid ovarian dysfunction, synthetic peptides corresponding to ZP glycoproteins have been proposed as candidate immunogens. In the present study, plasmid DNA encoding a human ZP glycoprotein-3 (ZP3) epitope corresponding to amino acid (aa) residues 334-343 and a human ZP glycoprotein-4 (ZP4) epitope corresponding to aa residues 251-273 separated by a triglycine spacer was constructed using the mammalian expression vector, VR1020. The plasmid DNA construct expressed both human ZP3 and ZP4 epitopes, as revealed by transient transfection of COS-1 (African green monkey, kidney) mammalian cells. Active immunization of female BALB/cJ mice with the DNA vaccine led to generation of antibodies reactive with baculovirus-expressed recombinant human ZP3, ZP4 and ZP3((334-343aa))-GGG-ZP4((251-273aa)) synthetic peptide in an ELISA as well as T cell responses. Antibodies generated by the DNA vaccine also recognized native ZP. The immune sera significantly inhibited (p<0.005) the binding of FITC-labeled ZP3 to capacitated human sperm, whereas no inhibition in the binding of FITC-labeled ZP4 was observed. However, a significant decrease in acrosomal exocytosis mediated by both recombinant human ZP3 (p<0.005) and ZP4 (p<0.005) was observed in presence of the immune sera. These studies demonstrate that a DNA vaccine can be designed to elicit antibodies against small epitopes of ZP glycoproteins.

Immunogenicity of zona pellucida glycoprotein-3 and spermatozoa YLP(12) peptides presented on Johnson grass mosaic virus-like particles.

For safer and effective immunocontraception, zona (ZP3) and spermatozoa specific (YLP(12)) peptides have been presented on virus-like particles (VLPs) derived from Johnson grass mosaic virus coat protein. Immunization of FvB/cJ female mice with VLPs presenting YLP(12)-ZP3 fusion peptide and a physical mixture of VLPs presenting either YLP(12) or ZP3 epitope led to generation of specific antibody responses and a significant reduction in litters born per mice (p<0.005). Significant curtailment of fertility was also observed in animals immunized with adjuvnated ZP3 and YLP(12) synthetic peptides. These results suggest that VLPs can be used to present gamete epitopes for immunocontraception.

Feasibility and challenges in the development of immunocontraceptive vaccine based on zona pellucida glycoproteins.

The zona pellucida (ZP) glycoproteins play a crucial role during fertilization and thus are considered as important target antigens for the development of immunocontraceptive vaccines aiming to inhibit fertility at a pre-fertilization stage. In order to evaluate the immunocontraceptive potential of ZP glycoproteins, bonnet monkey (Macaca radiata) ZP2, ZP3 and ZP4 have been cloned and expressed using either E. coli or baculovirus expression systems. Active immunization studies with the recombinant ZP glycoproteins in female baboons (Papio anubis) and bonnet monkeys revealed curtailment of fertility. In order to minimize the ovarian pathology, synthetic peptides corresponding to B cell epitopes that are devoid of 'oophoritogenic' T cell epitopes were designed and their in vitro immunocontraceptive potential explored. There are several issues that need to be addressed before ZP glycoproteins based immunocontraceptive vaccines become feasible for use in humans. Nonetheless, the utility of such a vaccine is imminent for controlling wild life population. In this direction, active immunization of female non-descript dogs with recombinant canine ZP3 conjugated to diphtheria toxoid led to curtailment of fertility. Further, canine ZP3 has also been expressed in insect cells as a fusion protein with rabies virus glycoprotein G (RV-G), an antigen that is involved in providing protection against rabies. The immunogenicity of such a recombinant protein and its potential to curtail fertility was explored both in female mice and dogs. Simultaneously, DNA vaccine encoding canine ZP3 and RV-G have been made and evaluated for their immunogenicity. The results obtained so far, current shortcomings and the possible ways to circumvent these have been discussed in the present manuscript.

Antibodies generated in response to plasmid DNA encoding zona pellucida glycoprotein-B inhibit in vitro human sperm-egg binding.

To investigate the immunogenicity of plasmid DNA encoding bonnet monkey (Macaca radiata) zona pellucida (ZP) glycoprotein-B (bmZPB), the cDNA corresponding to bmZPB, excluding the N-terminal signal sequence and C-terminus transmembrane-like domain, was cloned in mammalian expression vector VR1020 downstream of tissue plasminogen activator signal sequence under cytomegalovirus promoter (VRbmZPB). In vitro transfection of COS-1, COS-7, CHO, HEK-293, and UM-449 mammalian cells with VRbmZPB plasmid DNA led to the expression of bmZPB. Expression of bmZPB in transfected cells was cytosolic. Flow cytometry analysis of COS-1 cells transfected with VRbmZPB revealed that approximately 15% cells expressed bmZPB. The expressed bmZPB has an apparent molecular weight of 57 kDa. Immunization of male BALB/cJ mice with VRbmZPB plasmid DNA in saline as compared to VR1020 immunized group, elicited significant antibodies against E. coli expressed recombinant bmZPB as evaluated in ELISA. The antibodies generated by VRbmZPB plasmid DNA recognized bonnet monkey as well as human ZP. The immune sera obtained from mice immunized with VRbmZPB plasmid DNA also inhibited, in vitro, the binding of spermatozoa to the ZP in the hemizona assay. These studies, for the first time, demonstrate the feasibility of DNA vaccine to generate antibodies against ZP that recognize native protein and inhibit human sperm-oocyte binding.