Recent Advances in the Detection of Food Toxins Using Mass Spectrometry.

Edibles are the only source of nutrients and energy for humans. However, ingredients of edibles have undergone many physicochemical changes during preparation and storage. Aging, hydrolysis, oxidation, and rancidity are some of the major changes that not only change the native flavor, texture, and taste of food but also destroy the nutritive value and jeopardize public health. The major reasons for the production of harmful metabolites, chemicals, and toxins are poor processing, inappropriate storage, and microbial spoilage, which are lethal to consumers. In addition, the emergence of new pollutants has intensified the need for advanced and rapid food analysis techniques to detect such toxins. The issue with the detection of toxins in food samples is the nonvolatile nature and absence of detectable chromophores; hence, normal conventional techniques need additional derivatization. Mass spectrometry (MS) offers high sensitivity, selectivity, and capability to handle complex mixtures, making it an ideal analytical technique for the identification and quantification of food toxins. Recent technological advancements, such as high-resolution MS and tandem mass spectrometry (MS/MS), have significantly improved sensitivity, enabling the detection of food toxins at ultralow levels. Moreover, the emergence of ambient ionization techniques has facilitated rapid in situ analysis of samples with lower time and resources. Despite numerous advantages, the widespread adoption of MS in routine food safety monitoring faces certain challenges such as instrument cost, complexity, data analysis, and standardization of methods. Nevertheless, the continuous advancements in MS-technology and its integration with complementary techniques hold promising prospects for revolutionizing food safety monitoring. This review discusses the application of MS in detecting various food toxins including mycotoxins, marine biotoxins, and plant-derived toxins. It also explores the implementation of untargeted approaches, such as metabolomics and proteomics, for the discovery of novel and emerging food toxins, enhancing our understanding of potential hazards in the food supply chain.

Modeling and monitoring the effects of three partly conserved Ile residues in the dimerization domain of a Mip-like virulence factor from Escherichia coli.

FKBP22, an Escherichia coli-made peptidyl-prolyl cis-trans isomerase, has shown considerable homology with Mip-like virulence factors. While the C-terminal domain of this enzyme is used for executing catalytic function and binding inhibitor, the N-terminal domain is employed for its dimerization. To precisely determine the underlying factors of FKBP22 dimerization, its structural model, developed using a suitable template, was carefully inspected. The data show that the dimeric FKBP22, like dimeric Mip proteins, has a V-like shape. Further, it dimerizes using 40 amino acid residues including Ile 9, Ile 17, Ile 42, and Ile 65. All of the above Ile residues except Ile 9 are partly conserved in the Mip-like proteins. To confirm the roles of the partly conserved Ile residues, three FKBP22 mutants, constructed by substituting them with an Ala residue, were studied as well. The results together indicate that Ile 65 has little role in maintaining the dimeric state or enzymatic activity of FKBP22. Conversely, both Ile 17 and Ile 42 are essential for preserving the structure, enzymatic activity, and dimerization ability of FKBP22. Ile 42 in particular looks more essential to FKBP22. However, none of these two Ile residues is required for binding the cognate inhibitor. Additional computational studies also indicated the change of V-shape and the dimeric state of FKBP22 due to the Ala substitution at position 42. The ways Ile 17 and Ile 42 protect the structure, function, and dimerization of FKBP22 have been discussed at length.Communicated by Ramaswamy H. Sarma.

In silico analyses of Wnt1 nsSNPs reveal structurally destabilizing variants, altered interactions with Frizzled receptors and its deregulation in tumorigenesis.

Wnt1 is the first mammalian Wnt gene, which is discovered as proto-oncogene and in human the gene is located on the chromosome 12q13. Mutations in Wnt1 are reported to be associated with various cancers and other human diseases. The structural and functional consequences of most of the non-synonymous SNPs (nsSNPs), present in the human Wnt1 gene, are not known. In the present work, extensive bioinformatics analyses are used to screen 292 nsSNPs of Wnt1 for predicting pathogenic and harmless polymorphisms. We have identified 10 highly deleterious nsSNPs among which 7 are located within the highly conserved areas. These 10 nsSNPs are also predicted to affect the post-translational modifications of Wnt1. Further, structure based stability analyses of these 10 highly deleterious nsSNPs revealed 8 variants as highly destabilizing. These 8 highly destabilizing variants were shown to have high BC score and high RMSIP score from normal mode analyses. Based on the deformation energies, obtained from the normal mode analyses, variants like G169A, G169S, G331R and G331S were found to be unstable. Molecular Dynamics (MD) simulations revealed structural stability and fluctuation of WT Wnt1 and its prioritized variants. RMSD remained fluctuating mostly between 4 and 5 Å and occasionally between 3.5 and 5.5 Å ranges. RMSF in the CTD region (residues 330-360) of the binding pocket were lower compared to that of WT. Studying the impacts of nsSNPs on the binding interface of Wnt1 and seven Frizzled receptors have predicted substitutions which can stabilize or destabilize the binding interface. We have found that Wnt1 and FZD8-CRD is the best docked complex in our study. MD simulation based analyses of wild type Wnt1-FZD8-CRD complex and the 8 prioritized variants revealed that RMSF was higher in the unstructured regions and RMSD remained fluctuating in the region of 5 Å ± 1 Å. We have also observed differential Wnt1 gene expression pattern in normal, tumor and metastatic conditions across different tissues. Wnt1 gene expression was significantly higher in metastatic tissues of lungs, colon and skin; and was significantly lower in metastatic tissues of breast, esophagus and kidney. We have also found that Wnt1 deregulation is associated with survival outcome in patients with gastric and breast cancer. Furthermore, these computationally screened highly deleterious nsSNPs of Wnt1 can be analyzed in population based genetic studies and may help understand the Wnt1 associated diseases.

Remote communication between unstructured and structured regions of Bcl-2 tunes its ligand binding capacity: Mechanistic insights.

Bcl-2, the prototypic, anti-apoptotic member of Bcl-2 family possesses a long Intrinsically Disordered Region (IDR) of more than sixty amino acid residues. In spite of a number of experimental evidences on the influence of IDR to regulate the function of the protein, the molecular basis is not yet established. The present work with ~8µs conformational sampling of Bcl-2, using molecular dynamics in all atom description, offers a molecular mechanistic insight into the communication between the IDR and the structured region. The results indicate a highly significant role of the IDR in controlling the movements of the atoms which form the primary binding site. Although the influence of the IDR of the wild type Bcl-2 on its structured region, especially on the binding site, seems to be ordinary, but the hidden pathway of communication gets elucidated as the perturbation due to single-site phosphorylation on S70 works as a marker of the path; the contrast brought by the data obtained after truncating the IDR highlights it further. In wild type Bcl-2, apparently there is no direct communication between the IDR and binding site. But in the phosphorylated system, a communication channel between the IDR and the binding site has been established, as evidenced from the network analysis, which results in an increased correlation between the binding pocket residues and that redistributs the sampling of conformations of the system; one of the major consequences of these changes is witnessed in its enhanced affinity for binding with its partner Bax.

Oligotrophy vs. copiotrophy in an alkaline and saline habitat of Lonar Lake.

We reported our comparative observations on oligotrophs vs. copiotrophs from a hyper-alkaline and hypersaline habitat, Lonar Lake, situated in the Buldhana district of Maharashtra, India. Cell numbers of oligotrophic and copiotrophic microbes from the sediment were enumerated by the three-tube most probable number (MPN) method using an array of nutrient-rich and oligotrophic (≈10-20 mg carbon L-1) media offering simulated natural conditions of pH and salinity. A total of 50 strains from 15 different genera and 30 different species were isolated from the highest positive dilutions of MPN to identify the taxa of oligotrophs and copiotrophic microorganisms dominating in Lonar Lake. We did not get any true oligotrophs due to their adaptation to higher carbon levels during the isolation procedure. On the contrary, several true copiotrophs, which could not adapt and survive on a low-carbon medium, were isolated. It is also observed that changes in medium composition and nutrient level altered the selection of organisms from the same sample. Our data indicate that copiotrophic microorganisms dominate the eutrophic Lonar Lake, which is also supported by the past metagenomics studies from the same site. We also reported that quick depletion of carbon from oligotrophic medium worked as a limiting factor, inducing cell death after 2-3 generations and preventing the development of visible colonies on plates and sufficient optical density in liquid medium. Therefore, a long-term supply of low levels of carbon, followed by isolation on enriched media, can serve as a good strategy in isolation of novel taxa of microorganism, with industrial or environmental importance.

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.

Detection of Nitric Oxide from Chickpea Using DAF Fluorescence and Chemiluminescence Methods.

The free radical nitric oxide (NO) has emerged as an important signal molecule in plants, due to its involvement in various plant growth, development, and stress responses. For elucidating the role of NO, it is very important to precisely determine, localize, and quantify NO levels. Due to a relatively short half-life and its rapid, complex reactivity with other radicals, together with its capacity to diffuse from the source of production, the quantification of NO in whole plants, tissues, organelles, and extracts is notoriously difficult. Hence, it is essential to employ sensitive procedures for precise detection of NO. Currently available methods can fulfill many requirements to precisely determine NO, but each method has several advantages and pitfalls. In this article, we describe a detailed procedure for the measurement of NO by diaminofluorescein (DAF) in cell-permeable forms (DAF-FM-DA). In this method, the tissues are immersed in DAF-FM DA, leading to their diffusion from the plasma membrane to the inside of the cell, where intracellular esterases cleave the ester bonds, leading to DAF-FM release. The resulting DAF-FM reacts with intracellularly generated NO and forms highly fluorescent triazolofluorescein (DAF-FMT), which can be localized and monitored by fluorescence or confocal microscopy, and can also be detected via fluorimetry and flow cytometry. DAF dyes are very popular as they are non-invasive, relatively easy to handle, and commercially available. Another precise and very sensitive method is chemiluminescence detection of NO, where NO reacts with ozone (O3 ), leading to emission of a quantum of light from which NO can be calculated. Using chickpea seedlings, we describe in detail the measurement of NO using DAF-FM-DA and chemiluminescence methods. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Measurement of nitric oxide from chickpea seedlings using DAF-FM DA fluorescence with fluorescence and confocal microscopy Basic Protocol 2: Chemiluminescence detection of nitric oxide from chickpea seedlings.

Anti-microbial, anti-oxidant, and anti-breast cancer properties unraveled in yeast carotenoids produced via cost-effective fermentation technique utilizing waste hydrolysate.

Introduction: Natural carotenoids are well known for their anti-oxidant property and also shown to have antimicrobial and anticancer efficacy. Production of carotenoids from microbial resources mainly from yeast has attracted commercial interest. Breast cancer has the highest incidence among women, and therapy resistance and lack of effective therapeutic strategies are major treatment bottlenecks, particularly for triple-negative subtypes. Yeast carotenoids are recently being evaluated for affordable, non-toxic, natural product-based therapies. In the present study, we have shown an environment-friendly and inexpensive method for carotenoid production from yeasts, utilizing "mandi" wastes, and investigated the biomedical properties of carotenoids, particularly antineoplastic properties. Methods: Vegetable "mandi" waste was used to prepare waste hydrolysate, a culture medium, in which oleaginous red yeast Rhodosporidium sp. was grown. Carotenoid pigments were extracted using the solvent extraction method and analyzed by UV spectroscopy, thin-layer chromatography (TLC), and high-performance liquid chromatography (HPLC). Antimicrobial, antioxidant, and anticancer activities of the extract were evaluated, followed by in silico docking and absorption, distribution, metabolism, and excretion/toxicity (ADME/T) studies. Results: Carotenoid extract was found to be composed of three main pigments-ß-carotene, torulene, and torularhodin. Extract exhibited significant antioxidant, antimicrobial, and anti-breast cancer activities in vitro while being biocompatible. Interestingly, carotenoids have shown better efficacy in triple-negative breast cancer (TNBC) cells than ER+PR+ cells. In silico evaluation predicted binding with breast cancer-specific molecular targets, specifically the three components showed good binding energy toward VEGF receptors and good drug likeliness properties, as well as less toxicity. Discussion: This is the first report on anti-breast cancer activities, particularly targeting TNBC cells by red yeast carotenoids (ß-carotene, torulene, and torularhodin) produced via a sustainable environment-friendly bioprocess utilizing waste hydrolysate.

Phytoglobin-NO cycle and AOX pathway play a role in anaerobic germination and growth of deepwater rice.

An important and interesting feature of rice is that it can germinate under anoxic conditions. Though several biochemical adaptive mechanisms play an important role in the anaerobic germination of rice but the role of phytoglobin-nitric oxide cycle and alternative oxidase pathway is not known, therefore in this study we investigated the role of these pathways in anaerobic germination. Under anoxic conditions, deepwater rice germinated much higher and rapidly than aerobic condition and the anaerobic germination and growth were much higher in the presence of nitrite. The addition of nitrite stimulated NR activity and NO production. Important components of phytoglobin-NO cycle such as methaemoglobin reductase activity, expression of Phytoglobin1, NIA1 were elevated under anaerobic conditions in the presence of nitrite. The operation of phytoglobin-NO cycle also enhanced anaerobic ATP generation, LDH, ADH activities and in parallel ethylene levels were also enhanced. Interestingly nitrite suppressed the ROS production and lipid peroxidation. The reduction of ROS was accompanied by enhanced expression of mitochondrial alternative oxidase protein and its capacity. Application of AOX inhibitor SHAM inhibited the anoxic growth mediated by nitrite. In addition, nitrite improved the submergence tolerance of seedlings. Our study revealed that nitrite driven phytoglobin-NO cycle and AOX are crucial players in anaerobic germination and growth of deepwater rice.

Biologia Futura: use of biocides during COVID-19-global reshuffling of the microbiota.

Aim The article reviews the current usage of biocides during this lockdown period for sanitizing our living areas due to the pandemic and discusses the pros and cons. Subject COVID-19 spread like wildfire to over 200 countries of the world across all continents. The causative agent, novel coronavirus (SARS-CoV-2) is being counter attacked by a thorough application of disinfectants and sterilants. However, the virus mutated over 30 times during this global pandemic, creating panic and leading to enhanced pathogenicity and consequently to more stringent sanitation measures for controlling it. However, excessive use of different types of biocides for disinfecting surfaces is highly alarming in several cases. Extensive application of biocides affects the microbial flora, leading to an abrupt decrease in the number and diversity of beneficial microbes that may directly affect the functioning of nutrient cycles. Results The increased concentration of biocides in agricultural land via surface water or pond water indirectly affect the soil and water ecosystem, soil aggregation and fertility. This will also lead to the flourishing of resistant strains due to loss of competition from the other species, which fail to persist after prolonged use of biocides. Conclusion It is necessary to realize the environmental impacts of biocides and sterilants. It is the right time to stop their entry into the agricultural ecosystem by following adequate management strategies and complete neutralization.

Isolation and identification of carotenoid-producing yeast and evaluation of antimalarial activity of the extracted carotenoid(s) against P. falciparum.

Plasmodial resistance to a variety of plant-based antimalarial drugs has led toward the discovery of more effective antimalarial compounds having chemical or biological origin. Since natural compounds are considered as safer drugs, in this study, yeast strains were identified and compared for the production of carotenoids that are well-known antioxidants and this metabolite was tested for its antiparasitic activity. Plasmodium falciparum 3D7 strain was selected as the target parasite for evaluation of antimalarial activity of yeast carotenoids using in vitro studies. Data were analyzed by FACS (fluorescence-activated cell sorter) and counted via gold standard Giemsa-stained smears. The extracted yeast carotenoids showed a profound inhibitory effect at a concentration of 10-3 µg/µl and 10-4 µg/µl when compared to ß- carotene as control. SYBR Green1 fluorescent dye was used to confirm the decrease in parasitaemia at given range of concentration. Egress assay results suggested that treated parasite remained stalled at schizont stage with constricted morphology and were darkly stained. Non-toxicity of carotenoids on erythrocytes and on human liver hepatocellular carcinoma cells (HepG2 cells) was shown at a given concentration. This report provides strong evidence for antimalarial effects of extracted yeast carotenoids, which can be produced via a sustainable and cost-effective strategy and may be scaled up for industrial application.

Multi-conformation representation of Mpro identifies promising candidates for drug repurposing against COVID-19.

The COVID-19 main protease (Mpro), one of the conserved proteins of the novel coronavirus is crucial for its replication and so is a very lucrative drug target. Till now, there is no drug molecule that has been convincingly identified as the inhibitor of the function of this protein. The current pandemic situation demands a shortcut to quickly reach to a lead compound or a drug, which may not be the best but might serve as an interim solution at least. Following this notion, the present investigation uses virtual screening to find a molecule which is alraedy approved as a drug for some other disease but could be repurposed to inhibit Mpro. The potential of the present method of work to identify such a molecule, which otherwise would have been missed out, lies in the fact that instead of just using the crystallographically identified conformation of the receptor's ligand binding pocket, molecular dynamics generated ensemble of conformations has been used. It implicitly included the possibilities of "induced-fit" and/or "population shift" mechanisms of ligand fitting. As a result, the investigation has not only identified antiviral drugs like ribavirin, ritonavir, etc., but it has also captured a wide variety of drugs for various other diseases like amrubicin, cangrelor, desmopressin, diosmin, etc. as the potent possibilities. Some of these ligands are versatile to form stable interactions with various different conformations of the receptor and therefore have been statistically surfaced in the investigation. Overall the investigation offers a wide range of compounds for further testing to confirm their scopes of applications to combat the COVID-19 pandemic.

Strain Improvement of Native Saccharomyces cerevisiae LN ITCC 8246 Strain Through Protoplast Fusion To Enhance Its Xylose Uptake.

Co-utilization of xylose and glucose and subsequent fermentation using Saccharomyces cerevisiae could enhance ethanol productivity. Directed engineering approaches have met with limited success due to interconnectivity of xylose metabolism with other intrinsic, hidden pathways. Therefore, random approaches like protoplast fusion were used to reprogram unidentified mechanisms. Saccharomyces cerevisiae LN, the best hexose fermenter, was fused with xylose fermenting Pichia stipitis NCIM 3498. Protoplasts prepared using glucanex were fused under electric impulse and fusants were selected using 10% ethanol and cycloheximide (50 ppm) markers. Two fusants, 1a.23 and 1a.30 showing fast growth on xylose and tolerance to 10% ethanol, were selected. Higher extracellular protein expression observed in fusants as compared to parents was corroborated by higher number of bands resolved by two-dimensional analysis. Overexpression of XYL1, XYL2, XKS, and XUT4 in fusants as compared to S. cerevisiae LN as observed by RT-PCR analysis was substantiated by higher specific activities of XR, XDH, and XKS enzymes in fusants. During lignocellulosic hydrolysate fermentation, fusants could utilize glucose faster than the parent P. stipitis NCIM 3498 and xylose consumption in fusants was higher than S. cerevisiae LN.

Successful Control of a Co-Infection Caused by Candida albicans and Pseudomonas aeruginosa in Keratitis.

INTRODUCTION: Nowadays, the co-infection of different classes of pathogens is a major concern. The objective of this study was to develop a successful therapy for keratitis caused by the co-infection of Candida sp. with Pseudomonas sp, which is difficult to cure. The study is based on a 47 years old male farmer showing redness and watering in the right eye for 15-days. ; Methods: The microbiological examination was performed to isolate the causative organisms, i.e. Pseudomonas aeruginosa and Candida albicans. They were cultured separately along with their co-culture and treated with ciprofloxacin and amphotericin B during the growing stage to predict a definite cure. ; Results: Scanning electron microscope (SEM) results confirmed the inter-specific interaction between the two different types of microorganisms. Amphotericin-B and Ciprofloxacin showed the least MIC value for both organisms in co-culture. ; Conclusion: Treatment with Amphotericin-B and 5% ciprofloxacin effectively hindered the growth of Pseudomonas aeruginosa and Candida albicans, the co-infection of which caused keratitis. This therapy may be successfully implied for such cases of co-infection in the future.

Synergy of melanin and vitamin-D may play a fundamental role in preventing SARS-CoV-2 infections and halt COVID-19 by inactivating furin protease.

Since the birth of Christ, in these 2019 years, the man on earth has never experienced a survival challenge from any acellular protist compared to SARS-CoV-2. No specific drugs yet been approved. The host immunity is the only alternative to prevent and or reduce the infection and mortality rate as well. Here, a novel mechanism of melanin mediated host immunity is proposed having potent biotechnological prospects in health care management of COVID-19. Vitamin D is known to enhance the rate of melanin synthesis; and this may concurrently regulate the expression of furin expression. In silico analyses have revealed that the intermediates of melanin are capable of binding strongly with the active site of furin protease. On the other hand, furin expression is negatively regulated via 1-α-hydroxylase (CYP27B1), that belongs to vitamin-D pathway and controls cellular calcium levels. Here, we have envisaged the availability of biological melanin and elucidated the bio-medical potential. Thus, we propose a possible synergistic application of melanin and the enzyme CYP27B1 (regulates vitamin D biosynthesis) as a novel strategy to prevent viral entry through the inactivation of furin protease and aid in boosting our immunity at the cellular and humoral levels.

Theoretical analysis of bacterial efflux pumps inhibitors: Strategies in-search of competent molecules and develop next.

Multi-drug resistance (MDR) bacteria pose a significant threat to our ability to effectively treat infections due to the development of several antibiotic resistant mechanisms. A major component in the development of the MDR phenotype in MDR bacteria is over expression of different-type of efflux pumps, which actively pump out antibacterial agents and biocides from the periplasm to the outside of the cell. Consequently, bacterial efflux pumps are an important target for developing novel antibacterial treatments. Potent efflux pump inhibitors (EPIs) could be used as adjunctive therapies that would increase the potency of existing antibiotics and decrease the emergence of MDR bacteria. Several potent inhibitors of efflux pumps have been reported which has been summarized here. All the natural and synthetic EPIs were optimized with Gaussian and Avogadro software. The optimized structures were docked with each class of efflux pumps and their bonding parameters were computed. The theoretical analyses were performed with density functional theory (DFT). Overall, computational study revealed a good trend of electrophilicity and ionization potential of the EPIs, the obtained average values are within in the range of 0.001414 AU ± 0.00032 and 0.208821 AU ± 0.015545, respectively. Interestingly, cathinone interacts with most of the efflux pumps among the tested inhibitors. The electrophilicity and ionization potential of cathinone are 0.00198 and 0.2388 AU, respectively. The study opens a new road for designing future-generation target-specific efflux pump inhibitors, as well as one molecule with multiple inhibition abilities.