Application of the Milan System of reporting salivary gland cytopathology and assessing its use as a risk stratification tool.

INTRODUCTION: The Milan System for Reporting Salivary Gland Cytopathology (MSRSGC) was proposed by American Society of Cytopathology and International Academy of Cytology to standardize reporting system ensuring uniformity and better communication between clinicians and cytopathologists. AIMS AND OBJECTIVES: 1. To categorize salivary lesions as per MSRSGC. 2. To establish diagnostic accuracy of MSRSGC and calculate risk of malignancy for each diagnostic category. MATERIALS AND METHODS: An ambispective study was conducted over 5 years including 45 FNAC cases of salivary lesions. The cases were categorized according to Milan System, and diagnostic accuracy of MSRSGC was established. Risk of malignancy for each category was calculated. RESULTS: The patients' age ranged from 13 to 77 years with maximum swellings in parotid (53.3%) followed by submandibular (15.6%) and submental (2.2%). Histopathological follow-up was done in all cases. The number of cases in each category was as follows: category I: 03 (6.7%), category II: 06 (13.3%), category III: 4 (8.9%), category IVA: 25 (55.6%), category IVB: 04 (8.9%), category V: 02 (4.4%), and category VI: 01 (2.2%). The calculated ROM was as follows: category I: 0%, category II: 0%, category III: 0% category IVA: 8.0%, category IVB: 25%, category V: 50%, and category VI: 100%. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy with (without) indeterminate categories for malignancy were 88.9%(96.9), 66.7%(50.0), 94.1%(94.1), 50.0%(66.7), and 85.7%(91.9), respectively. CONCLUSION: MSRSGC is an effective and standardized reporting system for categorization and risk stratification of salivary swellings which helps in enhancing patient care.

Superficial CD34 Positive Fibroblastic Tumor: A Rare Entity With Cytological and Histopathological Correlation.

Superficial CD34-positive fibroblastic tumor (SPFT) is an extremely rare neoplasm of borderline (intermediate) category. To the best of our knowledge, less than 40 cases have been reported in the English literature. It is imperative to understand and emphasize its cytological features as fine needle aspiration cytology (FNAC) is still considered a first line of investigation in such cases in many countries including India. We present a case of a young male aged 27 years who presented to the General Surgery OPD with a history of slow-growing mass over right thigh for 7 years. FNAC and subsequent histopathological examination revealed a diagnosis of SPFT.

Effective gene silencing using type I-E CRISPR system in the multiploid, radiation-resistant bacterium Deinococcus radiodurans.

The extremely radiation-resistant bacterium, Deinococcus radiodurans, is a microbe of importance, both, for studying stress tolerance mechanisms and as a chassis for industrial biotechnology. However, the molecular tools available for use in this organism continue to be limiting, with its multiploid genome presenting an additional challenge. In view of this, the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas tools provide a large repertoire of applications for gene manipulation. We show the utility of the type I-E Cascade system for knocking down gene expression in this organism. A single-vector system was designed for the expression of the Cascade components as well as the crRNA. The type I-E Cascade system was better tolerated than the type II-A dCas9 system in D. radiodurans. An assayable acid phosphatase gene, phoN integrated into the genome of this organism could be knocked down to 10% of its activity using the Cascade system. Cascade-based knockdown of ssb, a gene important for radiation resistance resulted in poor recovery post-irradiation. Targeting the Radiation and Desiccation Response Motif (RDRM), upstream of the ssb, prevented de-repression of its expression upon radiation exposure. In addition to this, multi-locus targeting was demonstrated on the deinococcal genome, by knocking down both phoN and ssb expression simultaneously. The programmable CRISPR interference tool developed in this study will facilitate the study of essential genes, hypothetical genes, and cis-elements involved in radiation response as well as enable metabolic engineering in this organism. Further, the tool can be extended for implementing high-throughput approaches in such studies. IMPORTANCE Deinococcus radiodurans is a microbe that exhibits a very high degree of radiation resistance. In addition, it is also identified as an organism of industrial importance. We report the development of a gene-knockdown system in this organism by engineering a type I-E clustered regularly interspaced short palindromic repeat (CRISPR)-Cascade system. We used this system to silence an assayable acid phosphatase gene, phoN to 10% of its activity. The study further shows the application of the Cascade system to target an essential gene ssb, that caused poor recovery from radiation. We demonstrate the utility of CRISPR-Cascade to study the role of a regulatory cis-element in radiation response as well as for multi-gene silencing. This easy-to-implement CRISPR interference system would provide an effective tool for better understanding of complex phenomena such as radiation response in D. radiodurans and may also enhance the potential of this microbe for industrial application.

Chandipura virus changes cellular miRNome in human microglial cells.

Chandipura virus (CHPV) is a neurotropic virus, known to cause encephalitis in humans. The microRNAs (miRNA/miR) play an important role in the pathogenesis of viral infection. The present study is focused on the role of miRNAs during CHPV (strain 1653514) infection in human microglial cells. The deep sequencing of CHPV-infected human microglial cells identified a total of 12 differentially expressed miRNA (DEMs). To elucidate the role of DEMs, the target gene prediction, Gene Ontology term (GO Term), pathway enrichment analysis, and miRNA-messenger RNA (mRNA) interaction network analysis was performed. The GO terms and pathway enrichment analysis provided 146 enriched genes; which were involved in interferon response, cytokine and chemokine signaling. Further, the WGCNA (weighted gene coexpression network analysis) of the enriched genes were discretely categorized into three modules (blue, brown, and turquoise). The hub genes in the blue module may correlate to CHPV induced neuroinflammation. Altogether, the miRNA-mRNA interaction network and WGCNA study revealed the following pairs, hsa-miR-542-3p and FAF1, hsa-miR-92a-1-5p and MYD88, and hsa-miR-3187-3p and TNFRSF21, which may contribute to neuroinflammation during CHPV infection in human microglial cells.

Heterologous expression of cyanobacterial PCS confers augmented arsenic and cadmium stress tolerance and higher artemisinin in Artemisia annua hairy roots.

The present study provides the first report of heterologous expression of phytochelatin synthase from Anabaena PCC 7120 (anaPCS) into the hairy roots of Artemisia annua. Transformed hairy roots of A. annua expressing anaPCS gene showed better tolerance to heavy metals, viz., arsenic (As) and cadmium (Cd) owing to 143 and 191% more As- and Cd-accumulation, respectively, as compared to normal roots with a bioconcentration factor (BCF) of 9.7 and 21.1 for As and Cd, respectively. Under As and Cd stresses, transformed hairy roots possessed significantly higher amounts of phytochelatins and thiols probably due to the presence of both AaPCS (Artemisia annua PCS) and anaPCS. In addition, artemisinin synthesis was also induced in transformed hairy roots under heavy metals stresses. In-silico analysis revealed the presence of conserved motifs in both AaPCS and anaPCS sequences as well as structural modelling of PCS functional domain was conducted. Interaction of AaPCS and anaPCS proteins with CdCl2 and sodium arsenate gene ontology analysis gave insights to anaPCS functioning in transformed hairy roots of A. annua. The study provides transformed hairy roots of A. annua as an efficient tool for effective phytoremediation with added advantages of artemisinin extraction from hairy roots used for phytoremediation. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11816-021-00682-5.

Chikungunya virus modulates the miRNA expression patterns in human synovial fibroblasts.

Chikungunya virus (CHIKV) is an alphavirus transmitted by mosquitoes. CHIKV infection leads to polyarthritis and polyarthralgia among patients. The synovial fibroblasts are the primary target for CHIKV. The microRNAs (miRNAs) are the small endogenous noncoding RNAs which posttranscriptionally regulate the expression of genes by binding to their target messenger RNAs (mRNAs) through their 3'-untranslated regions. The miRNAs are the key regulators for various pathological processes including viral infection, cancer, cardiovascular disease, and neurodegeneration. This study was designed to dissect out the roles of miRNAs during CHIKV (Ross Strain E1: A226V) infection in primary human synovial fibroblasts. The miRNA microarray profiling was performed on the primary human synovial fibroblasts infected by CHIKV. The gene target prediction analysis, enrichment, and network analysis were performed by various bioinformatics analyses. The subset of 26 differentially expressed microRNAs (DEMs) were identified through microarray profiling and were further screened for gene predictions, Gene Ontology, pathway enrichment, and miRNA-mRNA network using various bioinformatics tools. The bioinformatics analysis indicates the role of DEMs by suppressing the immune response which may contribute to CHIKV persistence in human primary synovial fibroblasts. Our study provides the plausible roles of DEMs, miRNAs, and mRNA interactions and pathways involved in the molecular pathogenesis of CHIKV.

Allele-defined genome of the autopolyploid sugarcane Saccharum spontaneum L.

Modern sugarcanes are polyploid interspecific hybrids, combining high sugar content from Saccharum officinarum with hardiness, disease resistance and ratooning of Saccharum spontaneum. Sequencing of a haploid S. spontaneum, AP85-441, facilitated the assembly of 32 pseudo-chromosomes comprising 8 homologous groups of 4 members each, bearing 35,525 genes with alleles defined. The reduction of basic chromosome number from 10 to 8 in S. spontaneum was caused by fissions of 2 ancestral chromosomes followed by translocations to 4 chromosomes. Surprisingly, 80% of nucleotide binding site-encoding genes associated with disease resistance are located in 4 rearranged chromosomes and 51% of those in rearranged regions. Resequencing of 64 S. spontaneum genomes identified balancing selection in rearranged regions, maintaining their diversity. Introgressed S. spontaneum chromosomes in modern sugarcanes are randomly distributed in AP85-441 genome, indicating random recombination among homologs in different S. spontaneum accessions. The allele-defined Saccharum genome offers new knowledge and resources to accelerate sugarcane improvement.

Improved biotransformation of arsenic by arsenite oxidase - Chitosan nanoparticle conjugates.

Recent developments in the potential use of nanoparticles as carriers of enzyme have attracted great attention. In the present study, arsenite oxidase (AOase) enzyme capable of transforming the more toxic arsenite [As(III)] to the less toxic arsenate [As(V)] was extracted from an arsenic resistant bacterium (Exiguobacterium sp. As-9) and partially purified. Chitosan nanoparticles were prepared on the basis of ionic gelation of chitosan with tripolyphosphate (TPP) anions. The purified AOase was immobilized efficiently by physical adsorption on to chitosan nanoparticles and were characterized for particle size, morphology, zeta potential, AOase loading efficiency and in vitro transformation assay. The chitosan nanoparticles were spherical in shape with the average diameter of 100nm which increased to 294nm upon successful loading of AOase. Under optimized conditions, the loading capacity of the chitosan nanoparticle was determined to be 71% for AOase. Further, immobilization also increased the stability of AOase at varying temperature (4-37°C) and pH (5-10) for a period of 30days with the increased enzymatic activity (159.57Uml-1). It also facilitated increased biotransformation (89%) of As(III) to As(V). A conceptual understanding of biological responses to AOase loaded chitosan nanoparticles is needed for the development of novel methods of drug delivery.

Antioxidant, antimicrobial activity and bioactive compounds of Bergenia ciliata Sternb.: A valuable medicinal herb of Sikkim Himalaya.

Bergenia ciliata Sternb., commonly known as Paashaanbhed, is a well known herb of Sikkim Himalaya with various pharmaceutical properties. However, scientific exploration of B. ciliata, growing in the Sikkim Himalaya, for phytochemicals and pharmacological properties is in infancy. With this view, the present study was undertaken to investigate B. ciliata leaf extracts for antioxidant, antimicrobial activity and bioactive compounds. Three solvents viz., methanol, ethyl acetate and hexane were used for extraction and the respective leaf extracts were analyzed for total phenolic and flavonoid contents along with the antioxidant and antimicrobial activities. Amongst the tested solvents, methanol was found to be the best solvent for extraction with highest total phenolic contents and the lowest IC50 values for the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays. Methanol extract also exhibited effective antimicrobial activity, particularly against bacteria and actinomycetes. Further, high performance liquid chromatography (HPLC) analysis revealed that methanolic extract contains the highest amount of all the three analyzed bioactive compounds viz. bergenin, catechin and gallic acid. The current study suggests that the methanol extract of B. ciliata is a potential source of natural antioxidant and antimicrobial compounds that can be used in food and drug industries.

Updates on artemisinin: an insight to mode of actions and strategies for enhanced global production.

Application of traditional Chinese drug, artemisinin, originally derived from Artemisia annua L., in malaria therapy has now been globally accepted. Artemisinin and its derivatives, with their established safety records, form the first line of malaria treatment via artemisinin combination therapies (ACTs). In addition to its antimalarial effects, artemisinin has recently been evaluated in terms of its antitumour, antibacterial, antiviral, antileishmanial, antischistosomiatic, herbicidal and other properties. However, low levels of artemisinin in plants have emerged various conventional, transgenic and nontransgenic approaches for enhanced production of the drug. According to WHO (2014), approximately 3.2 billion people are at risk of this disease. However, unfortunately, artemisinin availability is still facing its short supply. To fulfil artemisinin's global demand, no single method alone is reliable, and there is a need to collectively use conventional and advanced approaches for its higher production. Further, it is the unique structure of artemisinin that makes it a potential drug not only against malaria but to other diseases as well. Execution of its action through multiple mechanisms is probably the reason behind its wide spectrum of action. Unfortunately, due to clues for developing artemisinin resistance in malaria parasites, it has become desirable to explore all possible modes of action of artemisinin so that new generation antimalarial drugs can be developed in future. The present review provides a comprehensive updates on artemisinin modes of action and strategies for enhanced artemisinin production at global level.

Human papilloma virus infection and psoriasis: Did human papilloma virus infection trigger psoriasis?

Psoriasis is an autoimmune chronic inflammatory skin disease known to be triggered by streptococcal and HIV infections. However, human papilloma virus infection (HPV) as a triggering factor for the development of psoriasis has not been reported yet. We, hereby report a case of plaque type with inverse psoriasis which probably could have been triggered by genital warts (HPV infection) and discuss the possible pathomechanisms for their coexistence and its management.

Modulations of physiological responses and possible involvement of defense-related secondary metabolites in acclimation of Artemisia annua L. against short-term UV-B radiation.

MAIN CONCLUSION: UV - B radiation exposure for upto 3 h did not cause direct damage to physiology, but adjusted secondary metabolism and metabolites accumulation as an effective acclimation mechanism to mitigate the adverse effects of radiation. Artemisia annua L. plants were irradiated with UV-B radiation (280-315 nm; 2.8 Wm(-2)) for different short-term (1, 2, 3 and 4 h) durations. UV-B irradiation of 3 h reduced the photosynthetic rate, stomatal conductance and transpiration rate. However, F v/F m, a sensitive indicator of photosynthetic inhibition, remained stable (0.78) upto 3 h, thereafter it declined sharply (0.72). Interestingly, transcript level of LHCB1, PSBA and PSBO genes related to photosystem II (PSII) were induced under UV-B exposure. In addition, genes coding for Rubisco small (RBCS1B) and large (RBCL) subunits were also upregulated upto 3 h. To mitigate the adverse effects of UV-B radiation, plants tremendously induced defense-related secondary metabolites such as antioxidative phenolics, UV-B absorbing flavonoids, anthocyanins and protective terpenes. The GC-MS analysis of essential oils revealed relatively higher production of monoterpenes over sesquiterpenes as well as 1.2-folds higher total oil yield under UV-B radiation. Owing to its diverse biological activities, the altered quantity and quality of essential oil of A. annua may contribute towards improving its therapeutic properties. The results suggest that UV-B irradiation upto 3 h reduced photosynthesis, probably due to stomatal limitations rather than any direct injury to photosynthetic apparatus as evident from stable F v/F m value, upregulated genes and greater accumulation of their corresponding proteins which gauge PSII health, elevated UV-B absorbing compounds and other protective metabolites. Correlation analysis indicates a significant positive correlation of photosynthetic rate with stomatal conductance while a negative correlation with anthocyanin and monoterpene contents under UV-B radiation. The present study provides first hand information regarding photosynthesis, related physiological parameters and essential oil profiling in response to UV-B radiation in A. annua.