A comparative account of phototoxicity of anthracene and pyrene in the tadpoles of the anuran amphibian Fejervarya limnocharis using multiple toxicological end points.

Anthracene (Anth) and pyrene (Pyr), two of the priority polycyclic aromatic hydrocarbons (PAHs), being lipophilic in nature, not only accumulate in animals, but also settle in the sediment of water bodies leading to continuous exposure for animals. Anth and Pyr when exposed to sunlight can be photoactivated and have harmful effects on aquatic organisms. A comparative analysis was carried out to assess the acute, sub-chronic, genetic and biochemical toxicity of Anth and Pyr in F. limnocharis tadpoles following short exposures to sunlight on a daily basis. In the bioaccumulation studies, it was found that both Anth and Pyr accumulated in the tadpole tissues in a concentration and time dependent manner. The LC50 values for Anth (under 15 min of daily sunlight exposure) were found to be 2.87, 2.59, 2.28, 1.80 mg/L at 24, 48, 72 and 96 h of the exposures. The corresponding LC50 values for Pyr were 1.03, 0.80, 0.62, 0.42 mg/L. Sublethal exposure of Anth and Pyr affected the survivality, time to metamorphosis as well as morphometric parameters under sunlight exposure. In the genotoxicity assessment studies, particularly the micronucleus test and comet assay, it was found that Pyr led to a higher incidence of micronucleus formation and DNA damage in comparison to Anth. The exposure to PAHs resulted in significant changes in the activity of antioxidant-mediated protective response, specifically the SOD activity, which varied between the groups treated with Anth and Pyr. On the other hand, Pyr treated group showed a higher level of GSH as compared to Anth treated groups. Moreover, the elevation in MDA level in the Anth and Pyr treated groups suggests an increase in lipid peroxidation. Future research should focus on understanding the ecotoxicological risk faced by anuran amphibia due to PAHs that frequently occur in aquatic environments and developing strategies to mitigate these risks.

Structural Basis for Norovirus Inhibition by Human Milk Oligosaccharides.

Histo-blood group antigens (HBGAs) are important binding factors for norovirus infections. We show that two human milk oligosaccharides, 2'-fucosyllactose (2'FL) and 3-fucosyllactose (3FL), could block norovirus from binding to surrogate HBGA samples. We found that 2'FL and 3FL bound at the equivalent HBGA pockets on the norovirus capsid using X-ray crystallography. Our data revealed that 2'FL and 3FL structurally mimic HBGAs. These results suggest that 2'FL and 3FL might act as naturally occurring decoys in humans.

Human noroviruses' fondness for histo-blood group antigens.

UNLABELLED: Human noroviruses are the dominant cause of outbreaks of gastroenteritis around the world. Human noroviruses interact with the polymorphic human histo-blood group antigens (HBGAs), and this interaction is thought to be important for infection. Indeed, synthetic HBGAs or HBGA-expressing enteric bacteria were shown to enhance norovirus infection in B cells. A number of studies have found a possible relationship between HBGA type and norovirus susceptibility. The genogroup II, genotype 4 (GII.4) noroviruses are the dominant cluster, evolve every other year, and are thought to modify their binding interactions with different HBGA types. Here we show high-resolution X-ray crystal structures of the capsid protruding (P) domains from epidemic GII.4 variants from 2004, 2006, and 2012, cocrystallized with a panel of HBGA types (H type 2, Lewis Y, Lewis B, Lewis A, Lewis X, A type, and B type). Many of the HBGA binding interactions were found to be complex, involving capsid loop movements, alternative HBGA conformations, and HBGA rotations. We showed that a loop (residues 391 to 395) was elegantly repositioned to allow for Lewis Y binding. This loop was also slightly shifted to provide direct hydrogen- and water-mediated bonds with Lewis B. We considered that the flexible loop modulated Lewis HBGA binding. The GII.4 noroviruses have dominated outbreaks over the past decade, which may be explained by their exquisite HBGA binding mechanisms, their fondness for Lewis HBGAs, and their temporal amino acid modifications. IMPORTANCE: Our data provide a comprehensive picture of GII.4 P domain and HBGA binding interactions. The exceptionally high resolutions of our X-ray crystal structures allowed us to accurately recognize novel GII.4 P domain interactions with numerous HBGA types. We showed that the GII.4 P domain-HBGA interactions involved complex binding mechanisms that were not previously observed in norovirus structural studies. Many of the GII.4 P domain-HBGA interactions we identified were negative in earlier enzyme-linked immunosorbent assay (ELISA)-based studies. Altogether, our data show that the GII.4 norovirus P domains can accommodate numerous HBGA types.

Structural Evolution of the Emerging 2014-2015 GII.17 Noroviruses.

Recent reports suggest that human genogroup II genotype 17 (GII.17) noroviruses are increasing in prevalence. We analyzed the evolutionary changes of three GII.17 capsid protruding (P) domains. We found that the GII.17 P domains had little cross-reactivity with antisera raised against the dominant GII.4 strains. X-ray structural analysis of GII.17 P domains from 2002 to 2014 and 2015 suggested that surface-exposed substitutions on the uppermost part of the P domain might have generated a novel 2014-2015 GII.17 variant.

Toxic effects of fenvalerate in tadpoles of Fejervarya limnocharis: a multiparametric study.

Pyrethroids are among the most widely used insecticides. Fenvalerate (FEN), a synthetic pyrethroid, is frequently used in domestic and agricultural settings to control insects which ultimately find its way into the aquatic ecosystems. The larval stages of amphibians, which are experiencing a rapid population decline, are spent in aquatic habitats, thus making them vulnerable to FEN exposure. The potential toxic effects of pyrethoids in general and FEN in particular are not well understood. The present study was carried out to assess the toxicity of FEN in tadpoles of Fejervarya limnocharis. FEN at different concentrations (0, 4, 5, 6, 7, and 8 mg/L) induced substantial lethal effects. The estimated LC50 values were 8.54, 6.73, 5.44, and 4.44 mg/L at 24, 48, 72, and 96 h respectively. Exposure to environmentally relevant sub-lethal concentrations delayed metamorphosis and reduced survivality. FEN was found to be genotoxic in erythrocyte micronucleus and comet assay. Further, sub-lethal concentrations of FEN adversely affected the antioxidant defense mechanism of the exposed individuals with parallel increase oxidative damage to membrane lipids. The swimming behavior in the form of startle response, swirl response, and total movements was decreased with a concomitant decrease in AChE activity. In addition, FEN exhibited significant cardiotoxicity by decreasing the cardiac rate of the exposed individuals. The present findings clearly indicate that FEN can cause significant toxicity to the tadpoles of F. limnocharis affecting their survival and fitness in the natural environment.

Crystal structures explain functional differences in the two actin depolymerization factors of the malaria parasite.

Apicomplexan parasites, such as the malaria-causing Plasmodium, utilize an actin-based motor for motility and host cell invasion. The actin filaments of these parasites are unusually short, and actin polymerization is under strict control of a small set of regulatory proteins, which are poorly conserved with their mammalian orthologs. Actin depolymerization factors (ADFs) are among the most important actin regulators, affecting the rates of filament turnover in a multifaceted manner. Plasmodium has two ADFs that display low sequence homology with each other and with the higher eukaryotic family members. Here, we show that ADF2, like canonical ADF proteins but unlike ADF1, binds to both globular and filamentous actin, severing filaments and inducing nucleotide exchange on the actin monomer. The crystal structure of Plasmodium ADF1 shows major differences from the ADF consensus, explaining the lack of F-actin binding. Plasmodium ADF2 structurally resembles the canonical members of the ADF/cofilin family.

A comprehensive review on current COVID-19 detection methods: From lab care to point of care diagnosis.

Without a doubt, the current global pandemic affects all walks of our life. It affected almost every age group all over the world with a disease named COVID-19, declared as a global pandemic by WHO in early 2020. Due to the high transmission and moderate mortality rate of this virus, it is also regarded as the panic-zone virus. This potentially deadly virus has pointed up the significance of COVID-19 research. Due to the rapid transmission of COVID-19, early detection is very crucial. Presently, there are different conventional techniques are available for coronavirus detection like CT-scan, PCR, Sequencing, CRISPR, ELISA, LFA, LAMP. The urgent need for rapid, accurate, and cost-effective detection and the requirement to cut off shortcomings of traditional detection methods, make scientists realize to advance new technologies. Biosensors are one of the reliable platforms for accurate, early diagnosis. In this article, we have pointed recent diagnosis approaches for COVID-19. The review includes basic virology of SARS-CoV-2 mainly clinical and pathological features. We have also briefly discussed different types of biosensors, their working principles, and current advancement for COVID-19 detection and prevention.

Elevated Expression of the RAGE Variant-V in SCLC Mitigates the Effect of Chemotherapeutic Drugs.

Small cell lung carcinoma (SCLC) is a highly aggressive malignancy with a very high mortality rate. A prominent part of this is because these carcinomas are refractory to chemotherapies, such as etoposide or cisplatin, making effective treatment almost impossible. Here, we report that elevated expression of the RAGE variant-V in SCLC promotes homology-directed DNA DSBs repair when challenged with anti-cancer drugs. This variant exclusively localizes to the nucleus, interacts with members of the double-strand break (DSB) repair machinery and thus promotes the recruitment of DSBs repair factors at the site of damage. Increased expression of this variant thus, promotes timely DNA repair. Congruently, the tumor cells expressing high levels of variant-V can tolerate chemotherapeutic drug treatment better than the RAGE depleted cells. Our findings reveal a yet undisclosed role of the RAGE variant-V in the homology-directed DNA repair. This variant thus can be a potential target to be considered for future therapeutic approaches in advanced SSLC.

Crystallization and preliminary structural characterization of the two actin-depolymerization factors of the malaria parasite.

The malaria parasite Plasmodium depends on its actin-based motor system for motility and host-cell invasion. Actin-depolymerization factors are important regulatory proteins that affect the rate of actin turnover. Plasmodium has two actin-depolymerization factors which seem to have different functions and display low sequence homology to the higher eukaryotic family members. Plasmodium actin-depolymerization factors 1 and 2 have been crystallized. The crystals diffracted X-rays to maximum resolutions of 2.0 and 2.1 A and belonged to space groups P3(1)21 or P3(2)21, with unit-cell parameters a = b = 68.8, c = 76.0 A, and P2(1)2(1)2, with unit-cell parameters a = 111.6, b = 57.9, c = 40.5 A, respectively, indicating the presence of one or two molecules per asymmetric unit in both cases.

Biophysical characterization of fibroblast growth factor homologous factor-1b (FHF-1b): sodium dodecyl sulfate promotes two state folding.

The current article describes the biophysical characterization and folding studies of fibroblast growth factor homologous factor-1b (FHF-1b) in comparison with acidic fibroblast growth factor (FGF-1). Our data indicates that FHF-1 is significantly more stable than FGF-1. The folding mechanism of these two proteins seems to be different although they share high degree of sequence and structural similarity. FHF-1 unfolds through stable intermediate state while unfolding of FGF-1 is two-state. Interestingly, low concentration of sodium dodecyl sulfate (SDS) drives the folding pathway of FHF-1b to two-state.

Structural analysis of bovine norovirus protruding domain.

We determined a structure of a bovine (genogroup III, GIII) norovirus capsid protruding (P) domain using X-ray crystallography. The bovine P domain was reminiscent of other norovirus genogroups (GI, GII, GIV, and GV), but closely matched the human GI P domain. We also identified a monoclonal antibody that was capable of binding the five different (GI-GV) P domains. Our data suggests that genetically diverse noroviruses still contain common epitopes.

Structural Constraints on Human Norovirus Binding to Histo-Blood Group Antigens.

Human norovirus interacts with the polymorphic human histo-blood group antigens (HBGAs), and this interaction is thought to be important for infection. The genogroup II genotype 4 (GII.4) noroviruses are the dominant cluster, evolve every other year, and are thought to modify their binding interactions with different HBGA types. Most human noroviruses bind HBGAs, while some strains were found to have minimal or no HBGA interactions. Here, we explain some possible structural constraints for several noroviruses that were found to bind poorly to HBGAs by using X-ray crystallography. We showed that one aspartic acid was flexible or positioned away from the fucose moiety of the HBGAs and this likely hindered binding, although other fucose-interacting residues were perfectly oriented. Interestingly, a neighboring loop also appeared to influence the loop hosting the aspartic acid. These new findings might explain why some human noroviruses bound HBGAs poorly, although further studies are required.

Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography.

The norovirus capsid is composed of a single major structural protein, termed VP1. VP1 is subdivided into a shell (S) domain and a protruding (P) domain. The S domain forms a contiguous scaffold around the viral RNA, whereas the P domain forms viral spikes on the S domain and contains determinants for antigenicity and host-cell interactions. The P domain binds carbohydrate structures, i.e., histo-blood group antigens, which are thought to be important for norovirus infections. In this protocol, we describe a method for producing high quality norovirus P domains in high yields. These proteins can then be used for X-ray crystallography and ELISA in order to study antigenicity and host-cell interactions. The P domain is firstly cloned into an expression vector and then expressed in bacteria. The protein is purified using three steps that involve immobilized metal-ion affinity chromatography and size exclusion chromatography. In principle, it is possible to clone, express, purify, and crystallize proteins in less than four weeks, which makes this protocol a rapid system for analyzing newly emerging norovirus strains.

Structural analysis of a feline norovirus protruding domain.

Norovirus infects different animals, including humans, mice, dogs, and cats. Here, we show an X-ray crystal structure of a feline GIV.2 norovirus capsid-protruding (P) domain to 2.35Å resolution. The feline GIV.2 P domain was reminiscent of human norovirus P domains, except for a novel P2 subdomain α-helix and an extended P1 subdomain interface loop. These new structural features likely obstructed histo-blood group antigens, which are attachment factors for human norovirus, from binding at the equivalent sites on the feline GIV.2 P domain. Additionally, an ELISA showed that the feline GIV.2 was antigenically distinct from a human GII.10 norovirus.

Rational approaches for drug designing against leishmaniasis.

Leishmaniasis has been ignored for many years mainly because it plagues remote and poor areas. However, recently, it has drawn attention of several investigators, and active research is going on for antileishmanial drug discovery. The current available drugs have high failure rates and significant side effects. Recently, liposomal preparations of amphotericin B are available and have proved to be a better drug, but they are very expensive. Miltefosine is one of the few orally administered drugs that are effective against Leishmania. However, it has exhibited teratogenicity, hence, should not be administered to pregnant women. Thus, the search for novel and improved antileishmanial drugs continue. A rational approach to design and develop new antileishmanials can be to identify several metabolic and biochemical differences between host and parasite that can be exploited as drug target. Moreover, many natural products also have significant antileishmanial activity and are yet to be exploited. In the current review, we aim to bring together various drug targets of Leishmania, recent development in the field, future prospects, and hope in the area.

In silico studies on tryparedoxin peroxidase of Leishmania infantum: structural aspects.

Tryparedoxin peroxidase (TryP) is a key enzyme of the trypanothione-dependent metabolism for removal of oxidative stress in leishmania. These enzymes function as antioxidants through their peroxidase and peroxynitrite reductase activities. Inhibitors of this enzyme are presumed to be antilesihmania drugs and structural studies are prerequisite of rational drug design. We have constructed three dimensional structure of TryP of Leishmania infantum using comparative modeling. Structural analysis reveals several interesting features. Moreover, it shows remarkable structural difference with human host glutathione peroxidase, an enzyme involved in similar function and TryP from Leishmania major.

Modeled structure of trypanothione reductase of Leishmania infantum.

Trypanothione reductase is an important target enzyme for structure-based drug design against Leishmania. We used homology modeling to construct a three-dimensional structure of the trypanothione reductase (TR) of Leishmania infantum. The structure shows acceptable Ramachandran statistics and a remarkably different active site from glutathione reductase(GR). Thus, a specific inhibitor against TR can be designed without interfering with host (human) GR activity.