Doublesex homolog is sex-specifically spliced and governs the sexual differentiation process in the whitefly Bemisia tabaci AsiaII-1.

The silverleaf whitefly Bemisia tabaci is one of the most destructive of crop pests globally. In Northern India cotton is predominately infested by the Asia II-1 species of B. tabaci. Though B. tabaci exhibits unique haplodiploidy in its reproductive behavior, to date very little is known regarding its sex determination mechanism. Here, an in-depth characterization of the AsiaII-1 doublesex (Btdsx) gene, which has been implicated in sex determination in B. tabaci, indicates the inclusion of six exons and five introns. The pre-mRNA is shown to sex-specifically splice, producing four male isoforms and one female isoform. These BtDsx proteins share common DNA binding (OD1) domains whereas they differ at their C-termini. RT-qPCR analysis revealed a significantly higher expression of Btdsx in female adults compared to that in male adults and earlier developmental stages. Functional characterization of Btdsx through RNA interference (RNAi) resulted in a significant reduction in its expression in both sexes. Btdsx knockdown concomitantly resulted in up-regulation of the expression of vitellogenin (vg) and vitellogenin receptor (vgr) genes in males and their down-regulation in females. Btdsx knockdown followed by mating resulted in reduced fecundity and percent egg hatching; however, no impact was observed on the female: male ratios in the progeny obtained from knockdown parents.

Identification and validation of stage-specific reference genes for gene expression analysis in Callosobruchus maculatus (Coleoptera: Bruchidae).

In light of a number of recent studies highlighting the increasing research interest in bruchids, it is crucial to validate suitable reference genes that could be used in quantitative gene expression studies. Callosobruchus maculatus is a serious pest of stored grains and field legumes in which reference genes have not been assessed and validated to date. The present study aimed to identify and validate reference genes in different developmental stages of C. maculatus shortlisted from commonly used reference genes such as VATPase, TRIP12, TBP, TF11D, ACTIN, GST, ANNEXIN, PTCD3, RPL32, and ß -Tub in various insects. Dedicated algorithms like GeNorm, NormFinder, and BestKeeper were used to analyze the stability of these candidate genes, which revealed GST for third instar, ANNEXIN and PTCD3 for the fourth instar, TF11D and VATPase for male pupa, RPL32 and ß-tub for female pupa, ß-tub and TBP for adult male and VATPase and GST for adult females as suitable reference genes for expression studies in C. maculatus. The final comprehensive ranking using RefFinder identified GST and TBP as the best reference genes for all the developmental stages of C. maculatus. To the best of our knowledge, this is the first report which evaluates and validates stable reference genes in C. maculatus. The information of stage-specific gene expression, generated in this study will be useful for future molecular, physiological, and biochemical studies on C. maculatus and other closely related bruchids.

Role of Peroral Endoscopic Myotomy in Advanced Achalasia Cardia With Sigmoid and/or Megaesophagus: A Systematic Review and Metanalysis.

BACKGROUND/AIMS: Sigmoid esophagus and/or megaesophagus are considered as an advanced stage in the natural history of achalasia cardia. The role of peroral endoscopic myotomy (POEM) in these subset of patients is emerging. We performed a systematic review and metanalysis to study the efficacy of POEM in advanced achalasia cardia with sigmoid and megaesophagus. METHODS: A literature search in PubMed and Embase was done from inception till August 3, 2021 to look for studies reporting exclusively on the role of POEM in advanced achalasia cardia with sigmoid and/or megaesophagus. The random effect method with inverse variance approach was used for the computation of pooled prevalence. For 2 groups' analysis of continuous outcome standardized mean difference was used as the summary measure. RESULTS: Eleven studies with 428 patients were included for analysis. The pooled technical and clinical success was 98.27% (95% CI, 96.19-99.22; I2 = 0%) and 89.38% (95% CI, 84.49-92.86; I2 = 26%) and on subgroup analysis into sigmoid and megaesophagus it was (98.06% [95% CI, 95.41-99.19; I2 = 0%], 98.47% [95% CI, 92.72-99.69; I2 = 0%] and 87.92% [95% CI, 80.68-92.70; I2 = 37%], 88.36% [95% CI, 62.62-97.17; I2 = 77%]) respectively. The clinical success at < 1 year and 1-3 year follow-up was 89.37% (95% CI, 82.82-93.61; I2 = 0%) and 88.66% (95% CI, 81.65-91.22; I2 = 46%) respectively. There was a significant reduction in the post-POEM scores with standardized mean difference for Eckardt score (4.81), for integrated relaxation pressure at 4 seconds (1.93), and for lower esophageal sphincter pressure (2.06). CONCLUSIONS: POEM is an effective modality of treatment even in the subset of patients of advanced achalasia cardia with sigmoid and megaesophagus.

Innate and adaptive resistance to RNAi: a major challenge and hurdle to the development of double stranded RNA-based pesticides.

RNA interference (RNAi) is a post-transcriptional gene silencing process where short interfering RNAs degrade targeted mRNA. Exploration of gene function through reverse genetics is the major achievement of RNAi discovery. Besides, RNAi can be used as a potential strategy for the control of insect pests. This has led to the idea of developing RNAi-based pesticides. Differential RNAi efficiency in the different insect orders is the biggest biological obstacle in developing RNAi-based pesticides. dsRNA stability, the sensitivity of core RNAi machinery, uptake of dsRNA and amplification and spreading of the RNAi signal are the key factors responsible for RNAi efficiency in insects. This review discusses the physiological and adaptive factors responsible for reduced RNAi in insects that pose a major challenge in developing dsRNA- based pesticides.

Impact of Different Patterns of Organ Failure on Mortality in Acute Necrotizing Pancreatitis.

OBJECTIVES: Organ failure (OF) and infected necrosis (IN) are the most important predictors of mortality in necrotizing acute pancreatitis (AP). We studied the relationship between timing (onset and duration) and patterns of OF with mortality and the impact of IN on mortality. METHODS: Consecutive patients with necrotizing AP between January 2017 and February 2020 were analyzed retrospectively for OF and its impact on outcome. Organ failure was divided as single OF, simultaneous multiple OF (SiMOF) and sequential multiple OF (SeMOF). Mortality was compared for timing of onset, total duration and patterns of OF. RESULTS: Among 300 patients with necrotizing AP, 174 (58%) had OF. Mortality was not associated with onset of OF (P = 0.683) but with duration of OF (P = 0.006). Mortalities for single OF, SiMOF, and SeMOF were 11.8%, 30.4%, and 69.2% respectively (P < 0.001). On Cox proportional hazard analysis, adjusted hazard ratio of risk of mortality for OF with IN versus IN, SiMOF versus single OF and SeMOF versus single OF was 3.183, 2.878, and 8.956, respectively (P = 0.023, <0.030, and <0.001, respectively). CONCLUSIONS: Duration of OF was associated with increased mortality and SeMOF had worse outcome than single OF and SiMOF.

Hepatic tuberculosis: myriad of hues.

INTRODUCTION: Gastrointestinal tuberculosis is an important form of extrapulmonary tuberculosis but liver involvement is uncommon. Hepatic tuberculosis can manifest in variable manners including as a space occupying lesion, diffuse infiltration by granulomas (granulomatous hepatitis) and as liver abscess. Hepatic tuberculosis is usually associated with lesions elsewhere but the diagnosis is often not considered because of rarity. CASE REPORT: We report two cases of hepatic involvement by tuberculosis: one in the form of liver abscess and the other having multiple hypodense hepatic lesions along with peritoneal involvement. The presence of ascites, visceral scalloping and multiple hypodense lesions resulted in a diagnostic confusion with disseminated malignancy but GeneXpert Ultra (Cepheid, USA) helped in making the diagnosis. DISCUSSION: Hepatic tuberculosis should be considered in the differential diagnosis of hepatic space occupying lesions and abscesses. This is especially important in TB endemic regions and in patients where there is involvement of other organs especially the lungs. CONCLUSIONS: Newer microbiological modalities including GeneXpert Ultra could help in making a diagnosis of hepatic tuberculosis.

Long Non-Coding RNAs in Insects.

Only a small subset of all the transcribed RNAs are used as a template for protein translation, whereas RNA molecules that are not translated play a very important role as regulatory non-coding RNAs (ncRNAs). Besides traditionally known RNAs (ribosomal and transfer RNAs), ncRNAs also include small non-coding RNAs (sncRNAs) and long non-coding RNAs (lncRNAs). The lncRNAs, which were initially thought to be junk, have gained a great deal attention because of their regulatory roles in diverse biological processes in animals and plants. Insects are the most abundant and diverse group of animals on this planet. Recent studies have demonstrated the role of lncRNAs in almost all aspects of insect development, reproduction, and genetic plasticity. In this review, we describe the function and molecular mechanisms of the mode of action of different insect lncRNAs discovered up to date.

Genome-editing approaches and applications: a brief review on CRISPR technology and its role in cancer.

The development of genome-editing technologies in 1970s has discerned a new beginning in the field of science. Out of different genome-editing approaches such as Zing-finger nucleases, TALENs, and meganucleases, clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR/Cas9) is a recent and versatile technology that has the ability of making changes to the genome of different organisms with high specificity. Cancer is a complex process that is characterized by multiple genetic and epigenetic changes resulting in abnormal cell growth and proliferation. As cancer is one of the leading causes of deaths worldwide, a large number of studies are done to understand the molecular mechanisms underlying the development of cancer. Because of its high efficiency and specificity, CRISPR/Cas9 has emerged as a novel and powerful tool in the field of cancer research. CRISPR/Cas9 has the potential to accelerate cancer research by dissecting tumorigenesis process, generating animal and cellular models, and identify drug targets for chemotherapeutic approaches. However, despite having tremendous potential, there are certain challenges associated with CRISPR/Cas9 such as safe delivery to the target, potential off-target effects and its efficacy which needs to be addressed prior to its clinical application. In this review, we give a gist of different genome-editing technologies with a special focus on CRISPR/Cas9 development, its mechanism of action and its applications, especially in different type of cancers. We also highlight the importance of CRISPR/Cas9 in generating animal models of different cancers. Finally, we present an overview of the clinical trials and discuss the challenges associated with translating CRISPR/Cas9 in clinical use.

Coronavirus disease (COVID-19) and the liver: a comprehensive systematic review and meta-analysis.

BACKGROUND: Liver function derangements have been reported in coronavirus disease (COVID-19), but reported rates are variable. METHODS: We searched PubMed and Embase with terms COVID and SARS-COV-2 from December 1, 2019 till April 5, 2020. We estimated overall prevalence, stratified prevalence based on severity, estimated risk ratio (RR), and estimated standardized mean difference (SMD) of liver function parameters in severe as compared to non-severe COVID. Random effect method utilizing inverse variance approach was used for pooling the data. RESULTS: In all, 128 studies were included. The most frequent abnormalities were hypoalbuminemia [61.27% (48.24-72.87)], elevations of gamma-glutamyl transferase (GGT) [27.94% (18.22-40.27)], alanine aminotransferase (ALT) [23.28% (19.92-27.01)], and aspartate aminotransferase (AST) [23.41% (18.84-28.70)]. Furthermore, the relative risk of these abnormalities was higher in the patients with severe COVID-19 when compared to non-severe disease [Hypoalbuminemia-2.65 (1.38-5.07); GGT-2.31 (1.6-3.33); ALT-1.76 (1.44-2.15); AST-2.30 (1.82-2.90)]. The SMD of hypoalbuminemia, GGT, ALT, and AST elevation in severe as compared to non-severe were - 1.05 (- 1.27 to - 0.83), 0.76 (0.40-1.12), 0.42 (0.27-0.56), and 0.69 (0.52-0.86), respectively. The pooled prevalence and RR of chronic liver disease as a comorbidity was 2.64% (1.73-4) and 1.69 (1.05-2.73) respectively. CONCLUSION: The most frequent abnormality in liver functions was hypoalbuminemia followed by derangements in gamma-glutamyl transferase and aminotransferases, and these abnormalities were more frequent in severe disease. The systematic review was, however, limited by heterogeneity in definitions of severity and liver function derangements. Graphical depiction of the summary of meta-analytic findings a) pooled prevalence of abnormalities b) Risk ratio of abnormality in severe versus non-severe COVID-19 c) standardized mean difference (SMD) between severe and non-severe group and d) pooled prevalence for parameters based on severity stratification for bilirubin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), albumin, globulin and acute hepatic injury (AHI) . Also estimates for overall/total liver disease (TLD) and chronic liver disease (CLD) amongst COVID-19 patients are depicted in a, b, d. For d) In addition to severity stratification, Overall (all studies for a particular estimate) and combined (only those studies which reported severity) estimates are provided.

Lipids help double-stranded RNA in endosomal escape and improve RNA interference in the fall armyworm, Spodoptera frugiperda.

RNA interference (RNAi) is a valuable method for understanding the gene function and holds great potential for insect pest management. While RNAi is efficient and systemic in coleopteran insects, RNAi is inefficient in lepidopteran insects. In this study, we explored the possibility of improving RNAi in the fall armyworm (FAW), Spodoptera frugiperda cells by formulating dsRNA with Cellfectin II (CFII) transfection reagent. The CFII formulated dsRNA was protected from degradation by endonucleases present in Sf9 cells conditioned medium, hemolymph and midgut lumen contents collected from the FAW larvae. Lipid formulated dsRNA also showed reduced accumulation in the endosomes of Sf9 cells and FAW tissues. Exposing Sf9 cells and tissues to CFII formulated dsRNA caused a significant knockdown of endogenous genes. CFII formulated dsIAP fed to FAW larvae induced knockdown of iap gene, growth retardation and mortality. Processing of dsRNA into siRNA was detected in Sf9 cells and Spodoptera frugiperda larvae treated with CFII conjugated 32 P-UTP labeled dsGFP. Overall, the present study concluded that delivering dsRNA formulated with CFII transfection reagent helps dsRNA escapes from the endosomal accumulation and improved RNAi efficiency in the FAW cells and tissues.

Comparative analysis of double-stranded RNA degradation and processing in insects.

RNA interference (RNAi) based methods are being developed for pest management. A few products for control of coleopteran pests are expected to be commercialized soon. However, variability in RNAi efficiency among insects is preventing the widespread use of this technology. In this study, we conducted research to identify reasons for variability in RNAi efficiency among thirty-seven (37) insects belonging to five orders. Studies on double-stranded RNA (dsRNA) degradation by dsRNases and processing of labeled dsRNA to siRNA showed that both dsRNA degradation and processing are variable among insects belonging to different orders as well as among different insect species within the same order. We identified homologs of key RNAi genes in the genomes of some of these insects and studied their domain architecture. These data suggest that dsRNA digestion by dsRNases and its processing to siRNAs in the cells are among the major factors contributing to differential RNAi efficiency reported among insects.

RNA interference in the Colorado potato beetle, Leptinotarsa decemlineata: Identification of key contributors.

RNA interference (RNAi) is a useful reverse genetics tool for investigation of gene function as well as for practical applications in many fields including medicine and agriculture. RNAi works very well in coleopteran insects including the Colorado potato beetle (CPB), Leptinotarsa decemlineata. We used a cell line (Lepd-SL1) developed from CPB to identify genes that play key roles in RNAi. We screened 50 genes with potential functions in RNAi by exposing Lepd-SL1 cells to dsRNA targeting one of the potential RNAi pathway genes followed by incubation with dsRNA targeting inhibitor of apoptosis (IAP, silencing of this gene induces apoptosis). Out of 50 genes tested, silencing of 29 genes showed an effect on RNAi. Silencing of five genes (Argonaute-1, Argonaute-2a, Argonaute-2b, Aubergine and V-ATPase 16 kDa subunit 1, Vha16) blocked RNAi suggesting that these genes are essential for functioning of RNAi in Lepd-SL1 cells. Interestingly, Argonaute-1 and Aubergine which are known to function in miRNA and piRNA pathways respectively are also critical to siRNA pathway. Using 32P labeled dsRNA, we showed that these miRNA and piRNA Argonautes but not Argonaute-2 are required for processing of dsRNA to siRNA. Transfection of pIZT/V5 constructs containing these five genes into Sf9 cells (the cells where RNAi does not work well) showed that expression of all genes tested, except the Argonaute-2a, improved RNAi in these cells. Results from Vha16 gene silencing and bafilomycin-A1 treatment suggest that endosomal escape plays an important role in dsRNA-mediated RNAi in Lepd-SL1 cells.

Reduced stability and intracellular transport of dsRNA contribute to poor RNAi response in lepidopteran insects.

RNA interference (RNAi) has become a widely used reverse genetic tool to study gene function in eukaryotic organisms and is being developed as a technology for insect pest management. The efficiency of RNAi varies among organisms. Insects from different orders also display differential efficiency of RNAi, ranging from highly efficient (coleopterans) to very low efficient (lepidopterans). We investigated the reasons for varying RNAi efficiency between lepidopteran and coleopteran cell lines and also between the Colorado potato beetle, Leptinotarsa decemlineata and tobacco budworm, Heliothis virescens. The dsRNA either injected or fed was degraded faster in H. virescens than in L. decemlineata. Both lepidopteran and coleopteran cell lines and tissues efficiently took up the dsRNA. Interestingly, the dsRNA administered to coleopteran cell lines and tissues was taken up and processed to siRNA whereas the dsRNA was taken up by lepidopteran cell lines and tissues but no siRNA was detected in the total RNA isolated from these cell lines and tissues. The data included in this paper showed that the degradation and intracellular transport of dsRNA are the major factors responsible for reduced RNAi efficiency in lepidopteran insects.

Unique features of a global human ectoparasite identified through sequencing of the bed bug genome.

The bed bug, Cimex lectularius, has re-established itself as a ubiquitous human ectoparasite throughout much of the world during the past two decades. This global resurgence is likely linked to increased international travel and commerce in addition to widespread insecticide resistance. Analyses of the C. lectularius sequenced genome (650 Mb) and 14,220 predicted protein-coding genes provide a comprehensive representation of genes that are linked to traumatic insemination, a reduced chemosensory repertoire of genes related to obligate hematophagy, host-symbiont interactions, and several mechanisms of insecticide resistance. In addition, we document the presence of multiple putative lateral gene transfer events. Genome sequencing and annotation establish a solid foundation for future research on mechanisms of insecticide resistance, human-bed bug and symbiont-bed bug associations, and unique features of bed bug biology that contribute to the unprecedented success of C. lectularius as a human ectoparasite.

Production of all female progeny: evidence for the presence of the male sex determination factor on the Y chromosome.

The red flour beetle, Tribolium castaneum, follows an XX (female) and XY (male) sex determination system. Maternal supply of the protein Transformer (Tra) is required for XX insects to follow the female pathway. The nature and source of the signal that regulates male sex determination in XY beetles are not known. Parental RNAi-aided knockdown in expression of tra masculinizes genetic females (XX) that are fertile. The virgin females mated with these masculinized genetic females produced all female progeny. We present the genetic evidence to show that the factor responsible for male sex determination is present on the Y chromosome. These data also suggest that the Y chromosome in T. castaneum is not required for male fertility.