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Background: Coronavirus disease 2019 (COVID-19) has caused a global pandemic with a high mortality rate and infecting people worldwide. The COVID-19 vaccines that are currently in development or already approved are expected to provide at least some protection against the emerging variants of the virus but the mutations may reduce the efficacy of the existing vaccines. Purified phytochemicals from medicinal plants provide a helpful framework for discovering new therapeutic leads as they have long been employed in traditional medicine to treat many disorders. Objective: The objectives of the study are to exploit the anti-HIV bioactive compounds against SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) through molecular docking studies and perform the Absorption, Distribution, Metabolism, Excretion and Toxicity (ADMET) properties of potential compounds. Methods: Molecular docking was performed to study the interaction of ligands with the target sites of RdRp protein (PDB: 6M71) using AutoDock Vina. The ADMET properties of potential compounds were predicted using the pkCSM platform. Results: A total of 151 phytochemicals derived from the medicinal plants with recognized antiviral activity and 18 anti-HIV drugs were virtually screened against COVID-19 viral RdRp to identify putative inhibitors that facilitate the development of potential anti-COVID-19 drug candidates. The computational studies identified 34 compounds and three drugs inhibiting viral RdRp with binding energies ranging from -10.2 to -8.5 kcal/ mol. Among these, five compounds, namely Michellamine B, Quercetin 3-O-(2'',6''-digalloyl)-beta-D-galactopyranoside, Corilagin, Hypericin, and 1,2,3,4,6-Penta-O-galloyl-beta-D-glucose residues bound efficiently with the binding site of RdRp. Besides, Lopinavir, Maraviroc, and Remdesivir drugs also inhibited SARS-CoV-2 polymerase. In addition, the ADMET properties of top potential compounds were also predicted in comparison to the drugs. Conclusion: The present study suggested that these potential drug candidates can be further subjected to in vitro and in vivo studies that may help develop effective anti-COVID-19 drugs.

Maize is susceptible to colonization by Aspergillus flavus, which is the major aflatoxins producing fungus. Aflatoxins are fungal secondary metabolites that have been reported to adversely affect normal cell metabolism and seed germination. In this study, 106 maize seed samples were sampled across 13 states throughout India, and were screened for incidences of fungal infection. Among the seed samples collected across India nearly all maize seed samples were found to be infected with Aspergillus. All morphologically confirmed isolates belonging to Aspergillus section Flavi were subjected to various aflatoxin, biochemical, physiological and genotype assays, as well as phylogenetic analyses. Among 60 isolates tested, eight were non-aflatoxigenic in nature as determined by icELISA, and seven of those eight isolates showed absence of one or more aflatoxin cluster genes in PCR assay. The morphological, physiological and biochemical characterizations failed to show variation based on their toxigenicity. The phenetic and molecular genetic diversity could be observed based on RAPD and ISSR analyses and little isolate diversity could be determined based on geography or aflatoxigenicity. An inferred ITS sequence phylogeny segregated isolates into only two lineage groups: a large group I (including Ia and Ib) and small group II contained A. flavus isolates closely related to A. parasiticus. The current study highlights inherent diverse A. flavus infections, exhibiting a range of aflatoxin concentrations, that are present in maize seeds and they are important because of the potential threat to agricultural production and consumption.

Plants display extraordinary ability to revive tissues and organs lost or damaged in injury. This is evident from the root tip restoration and classical experiments in stem demonstrating re-establishment of vascular continuity. While recent studies have begun to unravel the mechanistic understanding of tissue restoration in response to injury in underground plant organs, the molecular mechanisms of the same in aerial organs remain to be ventured deeper. Here, we discuss the possibility of unearthing the regulatory mechanism that can confer universal regeneration potential to plant body and further provide a comprehensive understanding of how tissue and organ regeneration gets triggered in response to mechanical injury and later gets terminated after re-patterning and regaining the appropriate size.

The main focus of this review is to discuss the current advancement in nano-metallic caused phytotoxicity on living organisms and current challenges in crops. Nanostructured materials provide new tools in agriculture to boost sustainable food production, but the main concern is that large-scale production and release of nanomaterials (NMs) into the ecosystem is a rising threat to the surrounding environment that is an urgent challenge to be addressed. The usage of NMs directly influences the transport pathways within plants, which directly relates to their stimulatory/ inhibitory effects. Because of the unregulated nanoparticles (NMs) exposure to soil, they are adsorbed at the root surface, followed by uptake and inter/intracellular mobility within the plant tissue, while the aerial exposure is taken up by foliage, mostly through cuticles, hydathodes, stigma, stomata, and trichomes, but the actual mode of NMs absorption into plants is still unclear. NMs-plant interactions may have stimulatory or inhibitory effects throughout their life cycle depending on their composition, size, concentration, and plant species. Although many publications on NMs interactions with plants have been reported, the knowledge on their uptake, translocation, and bioaccumulation is still a question to be addressed by the scientific community. One of the critical aspects that must be discovered and understood is detecting NMs in soil and the uptake mechanism in plants. Therefore, the nanopollution in plants has yet to be completely understood regarding its impact on plant health, making it yet another artificial environmental influence of unknown long-term consequences. The present review summarizes the uptake, translocation, and bioaccumulation of NMs in plants, focusing on their inhibitory effects and mechanisms involved within plants.

Zinc oxide nanoparticles have become one of the most popular metal oxide nanoparticles and recently emerged as a promising potential candidate in the fields of optical, electrical, food packaging, and biomedical applications due to their biocompatibility, low toxicity, and low cost. They have a role in cell apoptosis, as they trigger excessive reactive oxygen species (ROS) formation and release zinc ions (Zn2+) that induce cell death. The zinc oxide nanoparticles synthesized using the plant extracts appear to be simple, safer, sustainable, and more environmentally friendly compared to the physical and chemical routes. These biosynthesized nanoparticles possess strong biological activities and are in use for various biological applications in several industries. Initially, the present review discusses the synthesis and recent advances of zinc oxide nanoparticles from plant sources (such as leaves, stems, bark, roots, rhizomes, fruits, flowers, and seeds) and their biomedical applications (such as antimicrobial, antioxidant, antidiabetic, anticancer, anti-inflammatory, photocatalytic, wound healing, and drug delivery), followed by their mechanisms of action involved in detail. This review also covers the drug delivery application of plant-mediated zinc oxide nanoparticles, focusing on the drug-loading mechanism, stimuli-responsive controlled release, and therapeutic effect. Finally, the future direction of these synthesized zinc oxide nanoparticles’ research and applications are discussed.

Rhizobacteria from pearl millet were screened to produce 1-aminocyclopropane-1-carboxylate (ACC) deaminase and to evaluate its role in alleviating drought stress. Amongst 96 isolates, 28 were positive for ACC deaminase production, with MMR04 offering maximum activity of 2196.23 nmol of α-ketobutyrate produced mg–1 of protein h–1. The ACC deaminase producing rhizobacteria with multiple beneficial properties along with root colonization and non-pathogenic were selected [Bacillus amyloliquefaciens (MMR04), Bacillus subtilis (MMR18) and Stenotrophomonas maltophilia (MMR36)] to confirm the presence of ACC deaminase gene. A significant enhancement in seed germination (91.75%) and seedling vigor (1213.73) was noted upon seed treatment with MMR04 and hence further evaluated for its ability to induce drought stress. The seed treatment with MMR04 improved plant growth parameters and total chlorophyll and RWC in plants grown under severe drought stress (G5) conditions compared to control plants. In addition, MMR04 seed treatment enhanced proline, APX and SOD activity while decreased the MDA content up to 2.3 fold compared to untreated plants (G5). Gene expression studies revealed a significant decrease of 3.3 and 1.8 fold in the relative expression of drought-responsive (DREB-1E) and ethylene-responsive factor (ERF-1B) marker genes, respectively and an increase of 2.2 and 2.9 fold in the relative expression of APX1 and SOD1, respectively in MMR04 treated plants grown under G5 conditions over control. The results confirmed that ACC deaminase producing B. amyloliquefaciens MMR04 could defend the pearl millet plants against drought stress through an antioxidative system, thereby warranting its application in drought stress management.

Rhizosphere-resident fungi that are helpful to plants are generally termed as ‘plant growth promoting fungi’ (PGPF). These fungi are one of the chief sources of the biotic inducers known to give their host plants numerous advantages, and they play a vital role in sustainable agriculture. Today’s biggest challenge is to satisfy the rising demand for crop protection and crop yield without harming the natural ecosystem. Nowadays, PGPF has become an eco-friendly way to improve crop yield by enhancing seed germination, shoot and root growth, chlorophyll production, and fruit yield, etc., either directly or indirectly. The mode of action of these PGPF includes the solubilization and mineralization of the essential micro- and macronutrients needed by plants to regulate the balance for various plant processes. PGPF produce defense-related enzymes, defensive/volatile compounds, and phytohormones that control pathogenic microbes’ growth, thereby assisting the plants in facing various biotic and abiotic stresses. Therefore, this review presents a holistic view of PGPF as efficient natural biofertilizers to improve crop plants’ growth and resistance.

The study was undertaken to investigate the antioxidant, genotoxic, and cytotoxic potentialities of phyto-fabricated zinc oxide nanoparticles (ZnO-NPs) from Ipomoea obscura (L.) Ker Gawl. aqueous leaf extract. The UV-visible spectral analysis of the ZnO-NPs showed an absorption peak at 304 nm with a bandgap energy of 3.54 eV, which are characteristics of zinc nanoparticles. Moreover, the particles were of nano-size (~24.26 nm) with 88.11% purity and were agglomerated as observed through Scanning Electron Microscopy (SEM). The phyto-fabricated ZnO-NPs offered radical scavenging activity (RSA) in a dose-dependent manner with an IC50 of 0.45 mg mL−1. In addition, the genotoxicity studies of ZnO-NPs carried out on onion root tips revealed that the particles were able to significantly inhibit the cell division at the mitotic stage with a mitotic index of 39.49%. Further, the cytotoxic studies on HT-29 cells showed that the phyto-fabricated ZnO-NPs could arrest the cell division as early as in the G0/G1 phase (with 92.14%) with 73.14% cells showing early apoptotic symptoms after 24 h of incubation. The results of the study affirm the ability of phyto-fabricated ZnO-NPs from aqueous leaf extract of I. obscura is beneficial in the cytotoxic application.

Nanotechnology has revolutionized with lots of applications in the medicinal field to prevent, detect and treat several biological problems including disease and infections. The plant virus nanoparticles (VNPs) and virus-like nanoparticles (VLPs) obtained through viral nanotechnology have become a versatile platform in several fields such as in obtaining high selectivity and specificity, optics and biosensing, drug delivery and targeting, nanocatalysis, next-generation nanoelectronics. The capsid proteins in plant viruses aid in the production of novel nanomaterials, also they can self-assemble and form well-organized icosahedral viruses with altered coat protein subunits, interior and exterior size properties. The virus interior is particularly used to protect the sensitive compounds and encapsulate them, while their exterior can be utilized to coat small molecules in a precised manner. These properties of viruses including their biocompatibility nature have led to the development of VNPs/VLPs to achieve targeted drug delivery. Plant viruses are natural immunogenic and thus they are altered to use as vaccines against various pathogens. In this chapter, we discuss their applications and role in nanoparticle synthesis to create an effective and alternate way related to both medicine and nanotechnology disciplines.

The versatile regeneration capability of leaves enable even a detached Arabidopsis leaf to yield two kinds of regenerative responses namely, wound healing at the cut end in form of callus formation or de novo root regeneration (DNRR). Using various experimental approaches, we show that the factor favouring DNRR over callus formation seems to be a mechanical cue, possibly touch, at the cut end of the detached leaf. Here, we show that the forced expression of a PLETHORA transcription factor can bypass the need for touch to initiate DNRR. Our findings provide a genetic frame-work for touch dependant DNRR and suggest that a core PLT transcription regulatory module acts in response to mechano-sensing stimuli.

Recurring damage to the aerial organs of plants necessitates their prompt repair, particularly their vasculature. While vascular regeneration assays for aerial plant parts such as the stem and inflorescence stalk are well established, those for leaf vasculature remain unexplored. Recently, we established a new vascular regeneration assay in growing leaves and discovered the underlying molecular mechanism. Here, we describe the detailed stepwise method for the incision and regeneration assay used to study leaf vascular regeneration. By using a combination of micro-surgical perturbations, brightfield microscopy, and other experimental approaches, we further show that the age of the leaf as well as the position and size of the injury determine the overall success rate of regeneration. This easy-to-master vascular regeneration assay is an efficient and rapid method to study the mechanism of vascular regeneration in growing leaves. The assay can be readily combined with cellular and molecular biology techniques.

BackgroundRecurring damage to aerial organs of plants necessitates their prompt repair, particularly their vasculature. While vascular regeneration assay in aerial plant parts such as stem and inflorescence stalk are well established, those on leaf vasculature remained unexplored. Recently we established a new vascular regeneration assay in growing leaf and discovered the underlying molecular mechanism.ResultsHere we describe the detailed stepwise method of incision and the regeneration assay used for studying the leaf vascular regeneration. By using a combination of micro-surgical perturbations, brightfield microscopy and other experimental approaches, our new findings show that the regeneration efficiency decreases with aging of the leaf, and increases with the nearness of the wound towards the proximal end of the leaf.ConclusionThis easy-to-master vascular regeneration assay is an efficient and rapid method to study the mechanism of vascular regeneration in growing leaves. It can be readily adapted for other plant species and can be combined with cellular and molecular biology techniques.

In agriculture, eco-friendly management practices worldwide are required to enhance food security under the changing agro-climatic environmental conditions with avoiding the use of harmful chemicals. Plant growth promoting rhizobacteria (PGPR) is a group of soil microbes closely associated with roots and capable of improving plant growth. 1-Aminocyclopropane-1-carboxylic acid (ACC) deaminase producing PGPR (natural or genetically engineered) in various crop plants are considered as the foreseeable future significant environmental remediation approach to equilibrate plant ethylene levels under various abiotic stresses. The objective of the present review was to highlight the role of ACC deaminase producing rhizobacteria as potential alternatives for the management of abiotic stress in plants. Recently, PGPR has attracted the attention of many researchers for the development of biofertilizers as an eco-friendly approach. However, the selection of potential PGPR strain is a critical factor, and the plant responses to environmental conditions often vary depending upon the bacterial strain, plant genotypes, experimental sites, and season time. Extensive studies have been suggested that the use of PGPR could have emerged as a promising and compelling alternative method for the sustainability of agriculture. Hence, the present review provides an insight on the application of ACC deaminase producing rhizobacteria as an alternative towards growing abiotic stress competence in crop plants and its mechanism involved during the induction process. The review also highlights the research gap in the usage of native rhizobacteria with a potential to produce ACC deaminase as an important strategy to overcome abiotic stress tolerance in crop plants in the near future.

Aerial organs of plants, being highly prone to local injuries, require tissue restoration to ensure their survival. However, knowledge of the underlying mechanism is sparse. In this study, we mimicked natural injuries in growing leaves and stems to study the reunion between mechanically disconnected tissues. We show that PLETHORA (PLT) and AINTEGUMENTA (ANT) genes, which encode stem cell-promoting factors, are activated and contribute to vascular regeneration in response to these injuries. PLT proteins bind to and activate the CUC2 promoter. PLT proteins and CUC2 regulate the transcription of the local auxin biosynthesis gene YUC4 in a coherent feed-forward loop, and this process is necessary to drive vascular regeneration. In the absence of this PLT-mediated regeneration response, leaf ground tissue cells can neither acquire the early vascular identity marker ATHB8, nor properly polarise auxin transporters to specify new venation paths. The PLT-CUC2 module is required for vascular regeneration, but is dispensable for midvein formation in leaves. We reveal the mechanisms of vascular regeneration in plants and distinguish between the wound-repair ability of the tissue and its formation during normal development.

Plants can repair wounds throughout their body when they suffer injuries. The molecular mechanisms underlying this universal regeneration potential, and how regulation of tissue regeneration compares with its formation during development, remains unknown. Here, we reveal an essential role for members of the PLETHORA(PLT)/ AINTEGUMENTA(ANT) gene family in activating innate responses to injuries of the form that growing aerial parts of the plant naturally encounter. Strikingly, PLT act through the embryonic symmetry determinant CUP-SHAPED COTYLEDON2(CUC2) but not through root or shoot stem cell regulators in this process. Wound repair and vascular regeneration in damaged aerial organs growing in a normal developmental context were severely impaired in plt/ant mutant combinations as well as in cuc2 mutant. We find that the PLT-CUC2 module-dependent control of local hormonal environment and cell polarisation drives vascular regeneration in damaged aerial organs. We further provide several lines of evidence for PLT-CUC2 driven mechanism of wound repair and finally, rescue the regeneration defects by reconstituting endogenous local hormone production in the mutants. Interestingly, the necessity of PLT-CUC2 module specifically for vascular regeneration, but not vascular tissue development, distinguishes tissue’s ability to repair wound from its developmental program. This study reveals the essential mechanism of wound repair across the plant body and discerns the regulation of tissue regeneration from its formation during development.

There is a critical unmet need for reliable markers of disease and disease course in mild cognitive impairment (MCI) and early Alzheimer's disease (AD). The growing appreciation of the importance of inflammation in early AD has focused attention on inflammatory biomarkers in cerebrospinal fluid or plasma; however, non-specific inflammation markers have disappointed to date. We have adopted a targeted approach, centered on an inflammatory pathway already implicated in the disease. Complement, a core system in innate immune defense and potent driver of inflammation, has been implicated in pathogenesis of AD based on a confluence of genetic, histochemical, and model data. Numerous studies have suggested that measurement of individual complement proteins or activation products in cerebrospinal fluid or plasma is useful in diagnosis, prediction, or stratification, but few have been replicated. Here we apply a novel multiplex assay to measure five complement proteins and four activation products in plasma from donors with MCI, AD, and controls. Only one complement analyte, clusterin, differed significantly between control and AD plasma (controls, 295 mg/l; AD, 388 mg/l: p - 5). A model combining clusterin with relevant co-variables was highly predictive of disease. Three analytes (clusterin, factor I, terminal complement complex) were significantly different between MCI individuals who had converted to dementia one year later compared to non-converters; a model combining these three analytes with informative co-variables was highly predictive of conversion. The data confirm the relevance of complement biomarkers in MCI and AD and build the case for using multi-parameter models for disease prediction and stratification.

While in the movie Deadpool it is possible for a human to recreate an arm from scratch, in reality plants can even surpass that. Not only can they regenerate lost parts, but also the whole plant body can be reborn from a few existing cells. Despite the decades old realization that plant cells possess the ability to regenerate a complete shoot and root system, it is only now that the underlying mechanisms are being unraveled. De novo plant regeneration involves the initiation of regenerative mass, acquisition of the pluripotent state, reconstitution of stem cells and assembly of regulatory interactions. Recent studies have furthered our understanding on the making of a complete plant system in the absence of embryonic positional cues. We review the recent studies probing the molecular mechanisms of de novo plant regeneration in response to external inductive cues and our current knowledge of direct reprogramming of root to shoot and vice versa. We further discuss how de novo regeneration can be exploited to meet the demands of green culture industries and to serve as a general model to address the fundamental questions of regeneration across the plant kingdom.

Thirty-four Aspergillus flavus isolates were recovered from sorghum seeds sampled across five states in India. Our study included (1) species confirmation through PCR assay, (2) quantification of total aflatoxin concentrations by the indirect competitive-ELISA (ic-ELISA) method, and (3) analysis of molecular diversity among the A. flavus isolates using β-tubulin, ITS, and ISSR markers. Among the isolates studied, 28 were found to be positive for the production of aflatoxins. ITS and β-tubulin phylogenetic analysis segregated the A. flavus sample population into two major groups or clades with little to no subdivision based on geography. In contrast, ISSR analysis also separated the A. flavus isolates into two main clusters, showing a distance of 0.0–0.5, with one cluster exhibiting a high level of diversity though no geographic or chemotype subdivision could be observed. The majority of sampled A. flavus isolates were highly toxigenic, and also highly diversified in terms of toxin-producing potential in-vitro. Genetic diversity among the sorghum isolates of A. flavus further warrants the development of appropriate farming management practices as well as improved aflatoxin detection measures in India.

In the present investigation, seven rhizobacteria and nine Trichoderma spp. were evaluated to suppress seed-borne mycotoxigenic fungi (Aspergillus flavus and Fusarium verticillioides) and mycotoxin (aflatoxin and fumonisin) and to improve planting value of maize. Under in vitro conditions, these beneficial microorganisms suppressed the growth of A. flavus and F. verticillioides to various extents. Bacillus sp. (Bsp 3/aM), Pseudomonas putida (Has 1/c), Trichoderma asperellum (M5) and T. asperellum (T2) exhibited the greatest antagonistic effect on seed-borne mycotoxigenic fungi, and subsequently reduced mycotoxin concentrations in seeds. Under greenhouse conditions, these four biocontrol strains were also found to increase root length, shoot length, % germination, vigour index, fresh weight and dry weight of seedlings. Considering their overall performances, strains Bsp 3/aM, Has 1/c, M5 and T2 were selected for field studies as microbial talcum formulations. Among the tested microbial formulations, strain Bsp 3/aM significantly increased yield by 9.4% and 6.2% over the control in two maize cultivars Hema and Pearl, respectively. Increased plant growth and yield was also correlated with nutrient uptake in both the tested cultivars. All microbial formulation recorded significantly (p ≤ 0.05) reduced A. flavus infection and aflatoxin contamination in harvested seeds. But, none of the microbial formulations were found significant (p ≤ 0.05) in reducing F. verticilliodes incidence and fumonisin contamination. Our findings indicate that these microbial antagonists indirectly improve host health by suppressing seed-borne incidence of mycotoxigenic fungi and directly by facilitating nutrient uptake, thereby revealing their potential as both biofertilisers and biopesticides for maize production.

Seed-borne fungi such as Fusarium verticillioides and Aspergillus flavus are capable of causing several diseases to maize (Zea mays L.) and produce mycotoxins, which can have adverse effects on animal health and productivity. In the present study, efficacy of 100 extracts of 20 different plant species were tested for their antimicrobial potential against toxigenic Fusarium verticillioides and Aspergillus flavus. Among these, extracts of Toddalia asiatica, Decalepis hamiltonii, Vitex negundo and Psoralea corylifolia reduced A. flavus and F. verticillioides incidence significantly. Hydropriming with T. asiatica plant extract increased seed germination and seedling vigour significantly; however, D. hamiltonii and V. negundo did not show any improvements in seed quality parameters tested. In turn, P. corylifolia treated seeds showed potent antimicrobial potential but reduced seed germination and seedling vigour. The present study revealed that hydropriming of maize seeds with some plant extracts can be a pote...

Muscle contraction is a highly fine-tuned process that requires the precise and timely construction of large protein sub-assemblies to form sarcomeres. Mutations in many genes encoding constituent proteins of this macromolecular machine result in defective functioning of the muscle tissue. However, the pathways underlying muscle degeneration, and manifestation of myopathy phenotypes are not well understood. In this study, we explored transcriptional alterations that ensue from the absence of the two major muscle proteins - myosin and actin - using the Drosophila indirect flight muscles. Our aim was to understand how the muscle tissue responds as a whole to the absence of either of the major scaffold proteins, whether the responses are generic to the tissue; or unique to the thick versus thin filament systems. Our results indicated that muscles respond by altering gene transcriptional levels in multiple systems active in muscle remodelling, protein degradation and heat shock responses. However, there were some responses that were filament-specific signatures of muscle degeneration, like immune responses, metabolic alterations and alterations in expression of muscle structural genes and mitochondrial ribosomal genes. These general and filament-specific changes in gene expression may be of relevance to human myopathies.

In order to establish the current scenario of aflatoxigenic fungal infection and aflatoxin contamination in sorghum seeds across India, 58 seed samples were collected from different agro-climatic regions. Among these, 67.2% samples were infected with Aspergillus spp. and 28% were found contaminated with aflatoxins ranging from 0.0 to 130 μg kg−1. Greenhouse studies revealed no correlation between incidence of Aspergillus flavus and aflatoxin content, and its effect on seed quality parameters. Among the 37 A. flavus strains isolated, six were non-aflatoxigenic when analysed through cultural, TLC and ic-ELISA. Seed treatment with biocontrol agents (antagonistic Rhizobacteria and Trichoderma) suppressed the growth of A. flavus under laboratory and significantly enhanced seed quality variables under greenhouse conditions to a various extent. Field trials with selected biocontrol agents showed that talcum powder formulations of Pseudomonas putida Has-1/c, Bacillus spp. 3/a, Trichoderma asperellum M5 and T. asp...

BACKGROUND Fusarium spp. are not only pathogenic to plants but are also known as toxin producers that negatively affect animal and human health. The identification of Fusarium spp. remains one of the most critical issues in fungal taxonomy. In this study, different strains of Fusarium spp. were isolated from sorghum seed samples and identified at the molecular level by tef-1α gene amplification. A multiplex polymerase chain reaction (mPCR) assay was developed to differentiate toxigenic and non-toxigenic Fusarium spp. by designing a primer for the Fum21 gene along with the Fum1 and Fum8 genes. A competitive direct enzyme-linked immunosorbent assay (CD-ELISA) was employed to assess the fumonisin-producing ability of Fusarium spp. Phylogenetic analyses were performed using partial sequences of tef-1α and inter-simple sequence repeat (ISSR) markers of different Fusarium spp. RESULTS All 27 isolates of Fusarium spp. were positive for the tef-1α gene and revealed the presence of F. verticillioides, F. thapsina and F. cf. incarnatum-equiseti complex. The standardized mPCR assay distinguished toxigenic and non-toxigenic F. verticillioides. Further, mPCR fumonisin-positive F. verticillioides isolates were also positive by CD-ELISA. The tef-1α gene sequence was found to be useful in revealing intraspecific polymorphism to some extent. ISSR markers revealed a high level of polymorphism among different isolates of Fusarium spp., and the dendrogram of ISSR analyses grouped the 27 isolates into two major clusters. CONCLUSION The present method provided rapid and reliable detection of fumonisin-producing Fusarium spp. The mPCR assay could be an alternative strategy to current conventional mycotoxin analytical techniques and a reliable tool for high-throughput monitoring of major mycotoxin-producing fungi during the processing steps of food and feed commodities. © 2013 Society of Chemical Industry

Arsenic trioxide (ATO) mediates PML-RARA (promyelocytic leukemia–retinoic acid receptor-α) oncoprotein degradation via the proteasome pathway and this degradation appears to be critical for achieving cure in acute promyeloytic leukemia (APL). We have previously demonstrated significant micro-environment-mediated drug resistance (EMDR) to ATO in APL. Here we demonstrate that this EMDR could be effectively overcome by combining a proteasome inhibitor (bortezomib) with ATO. A synergistic effect on combining these two agents in vitro was noted in both ATO-sensitive and ATO-resistant APL cell lines. The mechanism of this synergy involved downregulation of the nuclear factor-κB pathway, increase in unfolded protein response (UPR) and an increase in reactive oxygen species generation in the malignant cell. We also noted that PML-RARA oncoprotein is effectively cleared with this combination in spite of proteasome inhibition by bortezomib, and that this clearance is mediated through a p62-dependent autophagy pathway. We further demonstrated that proteasome inhibition along with ATO had an additive effect in inducing autophagy. The beneficial effect of this combination was further validated in an animal model and in an on-going clinical trial. This study raises the potential of a non-myelotoxic proteasome inhibitor replacing anthracyclines in the management of high-risk and relapsed APL.

Antisense transcription is pervasive among biological systems and one of the products of antisense transcription is natural antisense transcripts (NATs). Emerging evidences suggest that they are key regulators of gene expression. With the discovery of NATs in Plasmodium falciparum, it has been suggested that these might also be playing regulatory roles in this parasite. However, all the reports describing the diversity of NATs have come from parasites in culture condition except for a recent study published by us. In order to explore the in vivo diversity of NATs in P. falciparum clinical isolates, we performed a whole genome expression profiling using a strand-specific 244K microarray that contains probes for both sense and antisense transcripts. In this report, we describe the experimental procedure and analysis thereof of the microarray data published recently in Gene Expression Omnibus (GEO) under accession number GSE44921. This published data provide a wealth of information about the prevalence of NATs in P. falciparum clinical isolates from patients with diverse malaria related disease conditions. Supplementary information about the description and interpretation of the data can be found in a recent publication by Subudhi et al. in Experimental Parasitology (2014).

Natural antisense transcripts (NATs) have been detected in many organisms and shown to regulate gene expression. Similarly, NATs have also been observed in malaria parasites with most studies focused on Plasmodium falciparum. There were no reports on the presence of NATs in Plasmodium vivax, which has also been shown to cause severe malaria like P. falciparum, until a recent study published by us. To identify in vivo prevalence of antisense transcripts in P. vivax clinical isolates, we performed whole genome expression profiling using a custom designed strand-specific microarray that contains probes for both sense and antisense strands. Here we describe the experimental methods and analysis of the microarray data available in Gene Expression Omnibus (GEO) under GSE45165. Our data provides a resource for exploring the presence of antisense transcripts in P. vivax isolated from patients showing varying clinical symptoms. Related information about the description and interpretation of the data can be found in a recent publication by Boopathi and colleagues in Infection, Genetics and Evolution 2013.

Background: There are several techniques to analyse copy number variation in both research and clinical settings, such as whole genome amplification (sWGA), SNP arrays and one of the most commonly used techniques, array based comparative genomic hybridization (aCGH). In the latter, copy number comparison is obtained between differentially labelled target and reference DNAs by measuring ratio of fluorescence intensity of probes indicating loss or gain in the chromosomal region. Methods: Here we carry out a comparative analysis between two Plasmodium falciparum parasite isolates (Pf-isolate-2 and Pf-isolate-1) causing malaria using array CGH. The array contains approximately 418,577, 60mer custom-designed probes with an average probe spacing of 56 bp. The significant major variations (amplifications and deletions) copy number variations (CNV) in Pf-isolate-2 (Pf-2) in comparison with Pf-isolate-1 (Pf-1), are reported. Results: CNVs have been seen in all the chromosomes in Pf-2, most of the deletions have been seen mostly in sub-telomeric and telomeric regions of the chromosomes that comprises of variant surface antigen family genes. Apart from the subtelomeric regions other parts of the chromosomes have also shown CNVs. Novel variations , like continuous amplification of 28kb region (249817-278491) of chromosome-8, which covers for 3 genes two of which codes for conserved Plasmodium proteins with unknown function (MAL8P1.139, PF08_0122) and tRNA pseudouridine synthase, putative (PF08_0123). Amplifications in regions harboring genes like GTP cyclohydrolase I (GCH-1, PFL1155W) and ribosomal protein, L24, putative (PFL1150C) of chromosome 12 were seen. Conclusion: Other than known variations reported earlier, some novel variations have also been seen in the chromosomes of Pf-2. This is an experimental case study reporting major amplifications and deletions in Pf-isolate-2 in comparison with Pf-isolate-1 using a tiling array based comparative genomic hybridization approach.

A total of 106 maize seed samples were collected from different agro-climatic regions of India. Sixty-two Fusarium isolates were recovered, 90% of which were identified as Fusarium verticillioides based on morphological and molecular characters. Use of the tef-1 alpha gene corrected/refined the morphological species identifications of 11 isolates, and confirmed those of the remaining isolates. Genetic diversity among the Fusarium isolates involved multilocus fingerprinting profiles by Inter Simple Sequence Repeats (ISSR) UP-GMA and tef-1 alpha gene phenetic analyses; for which, we observed no significant differences among the isolates based on geographic origin or fumonisin production; most of the subdivision related to species. Genotyping was performed on the F. verticillioides isolates, using 12 primer sets from the fumonisin pathway, to elucidate the molecular basis of fumonisin production or non-production. One fumonisin-negative isolate, UOMMF-16, was unable to amplify nine of the 12 fumonisin cluster genes tested. We also used the CD-ELISA method to confirm fumonisin production for our 62 Fusarium isolates. Only 15 isolates were found to be fumonisin-negative. Interestingly, genotypic characterization revealed six isolates with various gene deletion patterns that also tested positive for the production of fumonisins via CD-ELISA. Our findings confirm the importance of molecular studies for species delimitation, and for observing genetic and phenotypic diversity, among the Fusaria.

Zearalenone (ZEN) is a mycotoxin from Fusarium species commonly found in many food commodities and are known to cause reproductive disorders, genotoxic and immunosuppressive effects. Although many studies have demonstrated the cytotoxic effects of ZEN, the mechanisms by which ZEN mediates its cytotoxic effects appear to differ according to cell type and route of exposure. Meantime, the available information on the neurotoxic effects of ZEN is very much limited. In the present study we evaluated the role of oxidative stress in ZEN mediated neurotoxicity in SH-SY5Y cells and investigated the possible underlying mechanism. ZEN induced ROS formation and elevated levels of MDA, loss of mitochondrial membrane potential (MMP) and increase in DNA damage in a dose dependent manner as assessed by COMET assay and agarose gel electrophoresis. However, there was no DNA damage by plasmid breakage assay at 6, 12 and 24h time points. DAPI staining showed apoptotic nuclei at 12 and 24h. Further, ZEN treated SH-SY5Y cells showed a marked suppressive effect on the neuronal gene expression. Use of an antioxidant N-acetylcysteine (NAC) reversed the toxin-induced generation of ROS and also attenuated loss of MMP. Collectively, these results suggest that ROS is the main upstream signal leading to increased ZEN mediated neurotoxicity in SH-SY5Y cells.

Mechanisms regulating gene expression in malaria parasites are not well understood. Little is known about how the parasite regulates its gene expression during transition from one developmental stage to another and in response to various environmental conditions. Parasites in a diseased host face environments which differ from the static, well adapted in vitro conditions. Parasites thus need to adapt quickly and effectively to these conditions by establishing transcriptional states which are best suited for better survival. With the discovery of natural antisense transcripts (NATs) in this parasite and considering the various proposed mechanisms by which NATs might regulate gene expression, it has been speculated that these might be playing a critical role in gene regulation. We report here the diversity of NATs in this parasite, using isolates taken directly from patients with differing clinical symptoms caused by malaria infection. Using a custom designed strand specific whole genome microarray, a total of 797 NATs targeted against annotated loci have been detected. Out of these, 545 NATs are unique to this study. The majority of NATs were positively correlated with the expression pattern of the sense transcript. However, 96 genes showed a change in sense/antisense ratio on comparison between uncomplicated and complicated disease conditions. The antisense transcripts map to a broad range of biochemical/metabolic pathways, especially pathways pertaining to the central carbon metabolism and stress related pathways. Our data strongly suggests that a large group of NATs detected here are unannotated transcription units antisense to annotated gene models. The results reveal a previously unknown set of NATs that prevails in this parasite, their differential regulation in disease conditions and mapping to functionally well annotated genes. The results detailed here call for studies to deduce the possible mechanism of action of NATs, which would further help in understanding the in vivo pathological adaptations of these parasites.

Fusarium spp. are not only pathogenic to plants but are also known as toxin producers that negatively affect animal and human health. The identification of Fusarium spp. remains one of the most critical issues in fungal taxonomy. In this study, different strains of Fusarium spp. were isolated from sorghum seed samples and identified at the molecular level by tef-1α gene amplification. A multiplex polymerase chain reaction (mPCR) assay was developed to differentiate toxigenic and non-toxigenic Fusarium spp. by designing a primer for the Fum21 gene along with the Fum1 and Fum8 genes. A competitive direct enzyme-linked immunosorbent assay (CD-ELISA) was employed to assess the fumonisin-producing ability of Fusarium spp. Phylogenetic analyses were performed using partial sequences of tef-1α and inter-simple sequence repeat (ISSR) markers of different Fusarium spp.All 27 isolates of Fusarium spp. were positive for the tef-1α gene and revealed the presence of F. verticillioides, F. thapsina and F. cf. incarnatum-equiseti complex. The standardized mPCR assay distinguished toxigenic and non-toxigenic F. verticillioides. Further, mPCR fumonisin-positive F. verticillioides isolates were also positive by CD-ELISA. The tef-1α gene sequence was found to be useful in revealing intraspecific polymorphism to some extent. ISSR markers revealed a high level of polymorphism among different isolates of Fusarium spp., and the dendrogram of ISSR analyses grouped the 27 isolates into two major clusters.The present method provided rapid and reliable detection of fumonisin-producing Fusarium spp. The mPCR assay could be an alternative strategy to current conventional mycotoxin analytical techniques and a reliable tool for high-throughput monitoring of major mycotoxin-producing fungi during the processing steps of food and feed commodities.

Plasmodium vivax is the most geographically widespread human malaria parasite causing approximately 130-435 million infections annually. It is an economic burden in many parts of the world and poses a public health challenge along with the other Plasmodium sp. The biology of this parasite is less studied and poorly understood, in spite of these facts. Emerging evidence of severe complications due to infections by this parasite provides an impetus to focus research on the same. Investigating the parasite directly from infected patients is the best way to study its biology and pathogenic mechanisms. Gene expression studies of this parasite directly obtained from the patients has provided evidence of gene regulation resulting in varying amount of transcript levels in the different blood stages. The mechanisms regulating gene expression in malaria parasites are not well understood. Discovery of Natural Antisense Transcripts (NATs) in Plasmodium falciparum has suggested that these might play an important role in regulating gene expression. We report here the genome-wide occurrence of NATs in P. vivax parasites from patients with differing clinical symptoms. A total of 1348 NATs against annotated gene loci have been detected using a custom designed microarray with strand specific probes. Majority of NATs identified from this study shows positive correlation with the expression pattern of the sense (S) transcript. Our data also shows condition specific expression patterns of varying S and antisense (AS) transcript levels. Genes with AS transcripts enrich to various biological processes. To our knowledge this is the first report on the presence of NATs from P. vivax obtained from infected patients with different disease complications. The data suggests differential regulation of gene expression in diverse clinical conditions, as shown by differing sense/antisense ratios and would lead to future detailed investigations of gene regulation.

Aim Imprinted genes are known regulators of embryo growth. Studies from our laboratory have demonstrated that treatment of adult male rats with tamoxifen increased post-implantation loss at around midgestation. Expression of insulin like growth factor 2 ( Igf2 ), a paternally expressed imprinted gene was down-regulated in the resorbing embryos obtained at embryonic day 13. Hypomethylation of Igf2-H19 imprint control region was observed in the resorbing embryo sires and spermatozoa obtained from tamoxifen-treated rats thereby suggesting that errors in imprint acquisition during spermatogenesis can result in embryo loss. The present study aims at studying the expression of other imprinted genes, besides Igf2 in the embryos sired by tamoxifen-treated males. Main methods Gene expression profiles of resorbing versus normal embryos were assessed by microarrays. Real time quantitative RT-PCR for six imprinted genes and four genes involved in cell cycle was done to validate gene expression data. The affected pathways and functions were identified in the resorbing embryos and effect on cell cycle was confirmed by flow cytometry. Key findings Aberrant expression of a number of imprinted genes was observed in the resorbing embryos when compared to the normal embryos at embryonic days 11 and 13. Down-regulation of Notch signaling, Wnt signaling and cell cycle pathway was observed in the resorbing embryos. Significance The study suggests that exposure of male germ cells to tamoxifen during adulthood results in aberrant expression of imprinted genes and down-regulation of development associated pathways in the F 1 progeny thereby causing embryo loss.

There is limited data on the clinical, cellular and molecular changes in relapsed acute promyeloytic leukemia (RAPL) in comparison with newly diagnosed cases (NAPL). We undertook a prospective study to compare NAPL and RAPL patients treated with arsenic trioxide (ATO) based regimens. 98 NAPL and 28 RAPL were enrolled in this study. RAPL patients had a significantly lower WBC count and higher platelet count at diagnosis. IC bleeds was significantly lower in RAPL cases (P=0.022). The ability of malignant promyelocytes to concentrate ATO intracellularly and their in-vitro IC50 to ATO was not significantly different between the two groups. Targeted NGS revealed PML B2 domain mutations in 4 (15.38%) of the RAPL subset and none were associated with secondary resistance to ATO. A microarray GEP revealed 1744 genes were 2 fold and above differentially expressed between the two groups. The most prominent differentially regulated pathways were cell adhesion (n=92), cell survival (n=50), immune regulation (n=74) and stem cell regulation (n=51). Consistent with the GEP data, immunophenotyping revealed significantly increased CD34 expression (P=0.001) in RAPL cases and there was in-vitro evidence of significant microenvironment mediated innate resistance (EM-DR) to ATO. Resistance and relapse following treatment with ATO is probably multi-factorial, mutations in PML B2 domain while seen only in RAPL may not be the major clinically relevant cause of subsequent relapses. In RAPL additional factors such as expansion of the leukemia initiating compartment along with EM-DR may contribute significantly to relapse following treatment with ATO based regimens.