Synthesis and studies of new purines/pyrimidine derivatives as multi-targeted agents involving various receptor sites in the immune system.

Pro-inflammation, which is developed due to the increased production of cytokines, mainly interleukin-6 (IL-6), during the working of immune system pathways, becomes a major concern these days for many researchers. So, it is desired to design, screen, and synthesize new molecules with multi-parametric features showing their efficacy for Toll-like receptors (TLRs) and inhibiting the disease-causing receptor sites like viral infections, cancers, etc. along with controlling inflammation, fever, and other side effects during such pathways. Further, looking at the literature, curcumin a multi-targeted agent is showing its efficiency toward various receptor sites involved in many diseases as mentioned above. This fascinated us to build up new molecules which behave like curcumin with minimum side effects. In silico studies, involving ADMET studies, toxicological data, and docking analyses, of newly synthesized compounds (3-5) along with tautomers of curcumin i.e., (1-2), and some reported compounds like 9 and 10 have been studied in detail. Great emphasis has been made on analyzing binding energies, protein-ligand structural interactions, stabilization of newly synthesized molecules against various selected receptor sites using such computational tools. Compound 3 is the most efficient multifunctional agent, which has shown its potential toward most of the receptor sites in docking analysis. It has also responded well in Molecular dynamics (MD) simulation toward 5ZLN, 4RJ3, 4YO9, 4YOJ, and 1I1R sites. Finally, studies were extended to understand in vitro anti-inflammatory activity for particularly compound 3 in comparison to diclofenac and curcumin, which signifies the efficiency of compound 3.

Switching of newly synthesized linker-based derivatives of non-steroidal anti-inflammatory drugs toward anti-inflammatory and anticancer activity.

A new series of linker-based derivatives of non-steroidal anti-inflammatory drugs were designed and synthesized. All the compounds were well characterized with the help of various spectroscopic techniques such as FT-IR, 1H NMR, 13C NMR, and HRMS. The main emphasis of this paper is to understand the switching of the most promising compounds 8 and 10 towards anti-inflammatory and anticancer activity in terms of in-silico and in-vitro studies in detail. During the molecular docking study, compounds 8 and 10 demonstrated the importance of hetero atoms as well as the perfect alignment of a compound in the binding pocket of a target site, which may affect their bioactivity. Here, the presence of 1,3­dicarbonyl interactions with ASN 351 in compound 8 (not found in compound 10) may be responsible for its better inhibitory activity against the COX-2 target site. On the other hand, a slight increase in the potency of compound 10 towards anticancer activity may be due to the instantaneous participation of the OH group and carbonyl group to give conventional hydrogen bonds towards THR 149 amino acid residue, which was missing in compound 8. Molecular dynamics simulation was also performed for compounds 10 and 8 toward COX-2 and HER-2 protein sites. Further, compounds 8 and 10 were subjected to in-vitro COX-2 inhibition and cytotoxicity assay and the results obtained were in accordance with the in-silico study. Thus, compound 8 become more potent towards COX-2 inhibition with IC50 value of 48.51 µg/ml and compound 10 showed good bioactivity toward cytotoxic activity with IC50 value of 93.03 µg/ml.

Evolutionarily ancient BAH-PHD protein mediates Polycomb silencing.

Methylation of histone H3 lysine 27 (H3K27) is widely recognized as a transcriptionally repressive chromatin modification but the mechanism of repression remains unclear. We devised and implemented a forward genetic scheme to identify factors required for H3K27 methylation-mediated silencing in the filamentous fungus Neurospora crassa and identified a bromo-adjacent homology (BAH)-plant homeodomain (PHD)-containing protein, EPR-1 (effector of polycomb repression 1; NCU07505). EPR-1 associates with H3K27-methylated chromatin, and loss of EPR-1 de-represses H3K27-methylated genes without loss of H3K27 methylation. EPR-1 is not fungal-specific; orthologs of EPR-1 are present in a diverse array of eukaryotic lineages, suggesting an ancestral EPR-1 was a component of a primitive Polycomb repression pathway.

Nanoparticle-Shielded dsRNA Delivery for Enhancing RNAi Efficiency in Cotton Spotted Bollworm Earias vittella (Lepidoptera: Nolidae).

The spotted bollworm Earias vittella (Lepidoptera: Nolidae) is a polyphagous pest with enormous economic significance, primarily affecting cotton and okra. However, the lack of gene sequence information on this pest has a significant constraint on molecular investigations and the formulation of superior pest management strategies. An RNA-seq-based transcriptome study was conducted to alleviate such limitations, and de novo assembly was performed to obtain transcript sequences of this pest. Reference gene identification across E. vittella developmental stages and RNAi treatments were conducted using its sequence information, which resulted in identifying transcription elongation factor (TEF), V-type proton ATPase (V-ATPase), and Glyceraldehyde -3-phosphate dehydrogenase (GAPDH) as the most suitable reference genes for normalization in RT-qPCR-based gene expression studies. The present study also identified important developmental, RNAi pathway, and RNAi target genes and performed life-stage developmental expression analysis using RT-qPCR to select the optimal targets for RNAi. We found that naked dsRNA degradation in the E. vittella hemolymph is the primary reason for poor RNAi. A total of six genes including Juvenile hormone methyl transferase (JHAMT), Chitin synthase (CHS), Aminopeptidase (AMN), Cadherin (CAD), Alpha-amylase (AMY), and V-type proton ATPase (V-ATPase) were selected and knocked down significantly with three different nanoparticles encapsulated dsRNA conjugates, i.e., Chitosan-dsRNA, carbon quantum dots-dsRNA (CQD-dsRNA), and Lipofectamine-dsRNA conjugate. These results demonstrate that feeding nanoparticle-shielded dsRNA silences target genes and suggests that nanoparticle-based RNAi can efficiently manage this pest.

Transcriptome analysis unravels RNAi pathways genes and putative expansion of CYP450 gene family in cotton leafhopper Amrasca biguttula (Ishida).

Cotton Leafhopper, Amrasca biguttula is an important pest of cotton and okra in the Indian subcontinent. Presently limited genomic/transcriptomic information is available for this insect in any of open source databases. The present study reports the first assembled and annotated de novo transcriptome of cotton leafhopper. Out of 75,551 transcripts, 39,613 CDS (Coding Sequence) were predicted with 35,282 showing positive blast hits with NCBI nr database. The Gene ontology (GO) analysis annotated 7431 CDS  with KEGG pathway categorizing these CDS into 22 different functional groups. The majority of CDS were annotated in signal transduction and transport catabolism pathways. The sequence data was screened for RNAi pathway genes and presence of 37 transcripts associated with this process confirmed the existence of robust RNAi machinery. The role of core RNAi machinery genes (Dicer-2, Ago-2, Piwi and Staufen) has been validated through dsRNA feeding studies. The data resource has also been used to identify potential RNAi targets and genes associated with insecticide detoxification specifically CYP 450 family. The current study provides a useful sequence resource which can be used to initiate molecular studies in this insect with emphasis on insecticide resistance, RNAi and functional genomics.

RNA sequencing, selection of reference genes and demonstration of feeding RNAi in Thrips tabaci (Lind.) (Thysanoptera: Thripidae).

BACKGROUND: Thrips tabaci is a severe pest of onion and cotton. Due to lack of information on its genome or transcriptome, not much is known about this insect at the molecular level. To initiate molecular studies in this insect, RNA was sequenced; de novo transcriptome assembly and analysis were performed. The RNAseq data was used to identify reference and RNAi pathway genes in this insect. Additionally, feeding RNAi was demonstrated in T. tabaci for the first time. RESULTS: From the assembled transcriptome, 27,836 coding sequence (CDS) with an average size of 1236 bp per CDS were identified. About 85.4% of CDS identified showed positive Blast hits. The homologs of most of the core RNAi machinery genes were identified in this transcriptome. To select reference genes for reverse-transcriptase real-time quantitative PCR (RT-qPCR) experiments, 14 housekeeping genes were identified in the transcriptome and their expression was analyzed by (RT-qPCR). UbiCE in adult, 28s in nymphs and SOD under starvation stress were identified as the most stable reference genes for RT-qPCR. Feeding dsSNF7 and dsAQP caused 16.4- and 14.47-fold reduction in SNF7 and AQP mRNA levels respectively, when compared to their levels in dsGFP fed control insects. Feeding dsSNF7 or dsAQP also caused 62 and 72% mortality in T. tabaci. Interestingly, simultaneous feeding of dsRNAs targeting SNF7 or AQP and one of the RNAi pathway genes (Dicer-2/Aubergine/Staufen) resulted in a significant reduction in RNAi of target genes. These data suggest the existence of robust RNAi machinery in T. tabaci. CONCLUSION: The current research is the first report of the assembled, analyzed and annotated RNAseq resource for T. tabaci, which may be used for future molecular studies in this insect. Reference genes validated across stages and starvation stress provides first-hand information on stable genes in T. tabaci. The information on RNAi machinery genes and significant knockdown of the target gene through dsRNA feeding in synthetic diet confirms the presence of efficient RNAi in this insect. These data provide a solid foundation for further research on developing RNAi as a method to manage this pest.

Evolutionary relationship between the cysteine and histidine rich domains (CHORDs) and Btk-type zinc fingers.

Summary: Cysteine and histidine rich domains (CHORDs), implicated in immunity and disease resistance signaling in plants, and in development and signal transduction in muscles and tumorigenesis in animals, are seen to have a cylindrical three-dimensional structure stabilized by the tetrahedral chelation of two zinc ions. CHORDs are regarded as novel zinc-binding domains and classified independently in Pfam and ECOD. Our sequence and structure analysis reveals that both the zinc-binding sites in CHORD possess a zinc ribbon fold and are likely related to each other by duplication and circular permutation. Interestingly, we also detect an evolutionary relationship between each of the CHORD zinc fingers (ZFs) and the Bruton's tyrosine kinase (Btk)-type ZF of the zinc ribbon fold group. Btk_ZF is found in eukaryotic Tec kinase family proteins that are also implicated in signaling pathways in several lineages of hematopoietic cells involved in mammalian immunity. Our analysis suggests that the unique zinc-stabilized fold seen only in the CHORD and Btk_ZFs likely emerged specifically in eukaryotes to mediate diverse signaling pathways. Supplementary information: Supplementary data are available at Bioinformatics online.

Evolutionary relationships between heme-binding ferredoxin α + ß barrels.

BACKGROUND: The α + ß barrel superfamily of the ferredoxin-like fold consists of a functionally diverse group of evolutionarily related proteins. The barrel architecture of these proteins is formed by either homo-/hetero-dimerization or duplication and fusion of ferredoxin-like domains. Several members of this superfamily bind heme in order to carry out their functions. RESULTS: We analyze the heme-binding sites in these proteins as well as their barrel topologies. Our comparative structural analysis of these heme-binding barrels reveals two distinct modes of packing of the ferredoxin-like domains to constitute the α + ß barrel, which is typified by the Type-1/IsdG-like and Type-2/OxdA-like proteins, respectively. We examine the heme-binding pockets and explore the versatility of the α + ß barrels ability to accommodate heme or heme-related moieties, such as siroheme, in at least three different sites, namely, the mode seen in IsdG/OxdA, Cld/DyP/EfeB/HemQ and siroheme decarboxylase barrels. CONCLUSIONS: Our study offers insights into the plausible evolutionary relationships between the two distinct barrel packing topologies and relate the observed heme-binding sites to these topologies.

An atypical segment swap in the DN and DC domains of the Acr_tran family resistance-nodulation-cell division pump.

Domain/segment swapping is an exchange of equivalent secondary structure element(s) among two or more protein domains resulting in the reconstitution of the original fold while simultaneously causing oligomerization. Here we report an example of the outer membrane factor docking region of the Acr_tran family (PF00873) resistance-nodulation-cell division pump, in which a swapped, misfolded state, of the ferredoxin-like fold of the DN and DC domains, effectuates oligomerization. The atypical segment swap and the associated displacement of a region of the ferredoxin-like fold leads to a topology that is distinct from the original fold. To our knowledge, such segment swaps and associated fold change are rare. This exemplifies the role of functional constraints including oligomerization that determine the interplay between sequence and the three-dimensional structure of proteins.

The Ku-Mar zinc finger: A segment-swapped zinc ribbon in MarR-like transcription regulators related to the Ku bridge.

Two putative oxidative-stress sensor proteins from Pseudomonas aeruginosa, PA1607 and PA1374, belong to the MarR family of transcription regulators and possess a unique mode of dimerization. In these proteins, in addition to the α-helices involved in dimerization, inter-subunit contacts are strengthened by additional C-terminal ß-strands. Using sequence and structure analysis we show that these ß-strands constitute a novel segment-swapped zinc ribbon domain. We detect the presence of the zinc ribbon domain in MarR proteins from many bacterial homologs. While the metal-chelating residues of the zinc ribbons are absent in most members of this family, we could however identify several species of Proteobacteria, Actinobacteria and Firmicutes that possess intact zinc-chelating sites. Conservation pattern of metal-chelating residues together with the extensive structural resemblance to zinc ribbons, in particular to the bridge-region of the dsDNA break repair protein Ku, suggests that the C-terminal ß-rich region of these proteins is a zinc ribbon. Sequence analysis also supports a distant evolutionary connection between the zinc ribbons of the MarR and Ku families. However, unlike Ku where the segment-swapped zinc ribbons play a role in DNA-binding and obligate dimerization, their primary role in MarR appears to be in dimerization and strengthening of inter-subunit contacts.

The insertion domain 1 of class IIA dimeric glycyl-tRNA synthetase is a rubredoxin-like zinc ribbon.

The insertion domain 1 (ID1) of class IIA dimeric glycyl-tRNA synthetase (α2GRS) is an appended domain in the core catalytic region of the enzyme. ID1 has been shown to play a role in tRNA aminoacylation, mediating interaction with the acceptor arm of tRNA and diadenosine tetraphosphate (Ap4A) synthesis. Mutations in α2GRS, including those in the ID1 region, have been implicated in distal hereditary motor neuropathy-V (dHMN-V) and Charcot-Marie-Tooth (CMT) disease. Through sequence and structure based evolutionary analysis, we show that ID1 of α2GRS is a rubredoxin-like zinc ribbon domain. The zinc-chelating cysteines of ID1 are well conserved in all archaeal versions of the enzyme and also in several eukaryotes, which most likely have acquired them via horizontal gene transfer from bacteria; but in all other eukaryotes, the zinc-chelating residues are not preserved. ID1 from bacteria display a selective preservation of zinc-binding residues, ranging from complete conservation to complete loss. The ID1 from different organisms harbor variable-sized non-conserved insertions between the two zinc-binding half-sites of the zinc ribbon. Three of the previously identified CMT-associated mutations in α2GRS, viz., human D146N, mouse C157R and human S211F, are located in the zinc ribbon region of ID1. Interestingly, human Asp146 which is implicated in the synthesis of Ap4A, a molecule known to act during neuronal transmission, has also been reported to be mutated in dHMN-V, suggesting a possible link between hereditary motor neuropathy and Ap4A synthesis.

Plausible Drug Targets in the Streptococcus mutans Quorum Sensing Pathways to Combat Dental Biofilms and Associated Risks.

Streptococcus mutans, a Gram positive facultative anaerobe, is one among the approximately seven hundred bacterial species to exist in human buccal cavity and cause dental caries. Quorum sensing (QS) is a cell-density dependent communication process that respond to the inter/intra-species signals and elicit responses to show behavioral changes in the bacteria to an aggressive forms. In accordance to this phenomenon, the S. mutans also harbors a Competing Stimulating Peptide (CSP)-mediated quorum sensing, ComCDE (Two-component regulatory system) to regulate several virulence-associated traits that includes the formation of the oral biofilm (dental plaque), genetic competence and acidogenicity. The QS-mediated response of S. mutans adherence on tooth surface (dental plaque) imparts antibiotic resistance to the bacterium and further progresses to lead a chronic state, known as periodontitis. In recent years, the oral streptococci, S. mutans are not only recognized for its cariogenic potential but also well known to worsen the infective endocarditis due to its inherent ability to colonize and form biofilm on heart valves. The review significantly appreciate the increasing complexity of the CSP-mediated quorum-sensing pathway with a special emphasis to identify the plausible drug targets within the system for the development of anti-quorum drugs to control biofilm formation and associated risks.

Repurposing TRASH: emergence of the enzyme organomercurial lyase from a non-catalytic zinc finger scaffold.

The mercury resistance pathway enzyme organomercurial lyase (MerB) catalyzes the conversion of organomercurials to ionic mercury (Hg(2+)). Here, we provide evidence for the emergence of this enzyme from a TRASH-like, non-enzymatic, treble-clef zinc finger ancestor by domain duplication and fusion. Surprisingly, the structure-stabilizing metal-binding core of the treble-clef appears to have been repurposed in evolution to serve a catalytic role. Novel enzymatic functions are believed to have evolved from ancestral generalist catalytic scaffolds or from already specialized enzymes with catalytic promiscuity. The emergence of MerB from a zinc finger ancestor serves as a rare example of how a novel enzyme may emerge from a non-catalytic scaffold with a related binding function.

Gene expression profiling of 49 human tumor xenografts from in vitro culture through multiple in vivo passages--strategies for data mining in support of therapeutic studies.

BACKGROUND: Development of cancer therapeutics partially depends upon selection of appropriate animal models. Therefore, improvements to model selection are beneficial. RESULTS: Forty-nine human tumor xenografts at in vivo passages 1, 4 and 10 were subjected to cDNA microarray analysis yielding a dataset of 823 Affymetrix HG-U133 Plus 2.0 arrays. To illustrate mining strategies supporting therapeutic studies, transcript expression was determined: 1) relative to other models, 2) with successive in vivo passage, and 3) during the in vitro to in vivo transition. Ranking models according to relative transcript expression in vivo has the potential to improve initial model selection. For example, combining p53 tumor expression data with mutational status could guide selection of tumors for therapeutic studies of agents where p53 status purportedly affects efficacy (e.g., MK-1775). The utility of monitoring changes in gene expression with extended in vivo tumor passages was illustrated by focused studies of drug resistance mediators and receptor tyrosine kinases. Noteworthy observations included a significant decline in HCT-15 colon xenograft ABCB1 transporter expression and increased expression of the kinase KIT in A549 with serial passage. These trends predict sensitivity to agents such as paclitaxel (ABCB1 substrate) and imatinib (c-KIT inhibitor) would be altered with extended passage. Given that gene expression results indicated some models undergo profound changes with in vivo passage, a general metric of stability was generated so models could be ranked accordingly. Lastly, changes occurring during transition from in vitro to in vivo growth may have important consequences for therapeutic studies since targets identified in vitro could be over- or under-represented when tumor cells adapt to in vivo growth. A comprehensive list of mouse transcripts capable of cross-hybridizing with human probe sets on the HG-U133 Plus 2.0 array was generated. Removal of the murine artifacts followed by pairwise analysis of in vitro cells with respective passage 1 xenografts and GO analysis illustrates the complex interplay that each model has with the host microenvironment. CONCLUSIONS: This study provides strategies to aid selection of xenograft models for therapeutic studies. These data highlight the dynamic nature of xenograft models and emphasize the importance of maintaining passage consistency throughout experiments.

Investigating multifaceted action of ibuprofen derivatives towards cox isozymes and interleukin-6 (IL-6) site correlating with various target sites.

The multifaceted action of new ibuprofen analogs has been investigated against inflammation, neurological and pro-inflammation factors. On the basis of ADMET (absorption, distribution, metabolism, excretion, and toxicity) analysis, molecular docking as well as molecular dynamics simulation, compound 3 was thought to have good anti-inflammatory activity. As the presence of structural interactions such as conventional hydrogen bonds and electrostatic interactions through the nitrogen atoms of the linker in compound 3 gave strong evidence of its potency. The major finding of the current work is that the presence of appropriate number of hetero atoms (NH, OH) in a compound makes it more efficient than the number of labile groups (i.e., hydroxyl groups). Additionally, the position of hetero atoms in a compound and orientation also play a vital role in its efficacy. It was also screened for in vitro anti-inflammatory activity by membrane stability method, where it has shown 90.8% protection of RBC hemolysis. Thus, compound 3 with effective structural features may have good anti-inflammatory activity.Communicated by Ramaswamy H. Sarma.

Early Predictors of Ventilator Associated Pneumonia in Preterm Neonates Admitted in a Special Newborn Care Unit.

OBJECTIVES: To determine the utility of microscopic examination and culture of endotracheal aspirate (ETA) in the early diagnosis of ventilator-associated pneumonia (VAP) in preterm neonates. METHODS: We enrolled 80 consecutive neonates (both inborn and out-born) with gestational age of < 37 weeks admitted in Special Newborn Care Unit (SNCU) and requiring mechanical ventilation (MV) for ≥ 48 hours. The diagnosis of VAP was made using the criteria laid down by the Centers for Disease Control (CDC). RESULTS: 47 preterm neonates (58.5%) developed VAP; the overall incidence was 74.7/1000 ventilator-days. The mean (SD) time (hours) to ETA culture was less as compared to diagnosis based on CDC criteria [108.9 (8.00 hrs) vs 132.4 (53.24); P = 0.004] with sensitivity and specificity of 80.8% and 72.7%, respectively. Outborn delivery was the single most important risk factor for VAP. Multidrug resistant (MDR) Klebsiella pneumoniae (63.9%) was the most prevalent organism. CONCLUSIONS: We noticed a very high incidence of VAP among preterm neonates in SNCU. ETA culture can aid in early diagnosis.

Drought stress detection technique for wheat crop using machine learning.

The workflow of this research is based on numerous hypotheses involving the usage of pre-processing methods, wheat canopy segmentation methods, and whether the existing models from the past research can be adapted to classify wheat crop water stress. Hence, to construct an automation model for water stress detection, it was found that pre-processing operations known as total variation with L1 data fidelity term (TV-L1) denoising with a Primal-Dual algorithm and min-max contrast stretching are most useful. For wheat canopy segmentation curve fit based K-means algorithm (Cfit-kmeans) was also validated for the most accurate segmentation using intersection over union metric. For automated water stress detection, rapid prototyping of machine learning models revealed that there is a need only to explore nine models. After extensive grid search-based hyper-parameter tuning of machine learning algorithms and 10 K fold cross validation it was found that out of nine different machine algorithms tested, the random forest algorithm has the highest global diagnostic accuracy of 91.164% and is the most suitable for constructing water stress detection models.

Does the Capsule Method Increase the Accuracy of the Detection of Ger by Scintigraphy Technique?

Background: The scintigraphy technique is the most sensitive test for the detection of gastroesophageal reflux disease (GERD). Scintigraphy techniques employ two methods: the liquid method and the capsule method. Aim: In this prospective study, we are trying to find out the efficiency of the capsule method for gastroesophageal reflux scintigraphy over the liquid method and to determine the ease of execution of the technique and the interpretation of the results. Materials and Methods: A total of 65 symptomatic patients (age range: 7-71 years; mean age: 35.2 years) were included in the study. They were divided into two groups: group A, which included 18 patients who underwent the liquid method and Group B, which included 47 patients who underwent the capsule method. The average administered dose of 99mTc-labeled sulfur colloid was 11.1-18.5 MBq. Results: The results showed that 45 (69.12%) of the 65 patients tested positive for GERD. Furthermore, 15 were positive in the liquid method and 30 in the capsule method. Grade III reflux was seen in 66.67% of patients, and 33.33% of patients with Grade II and I reflux were diagnosed using both methods. Conclusion: Thus, in conclusion, we can say that both liquid and capsule methods are equally sensitive for the detection of low as well as high refluxate volumes.

Derivatized chitosan-oil-in-water nanocapsules for trans-cinnamaldehyde delivery.

trans-Cinnamaldehyde, known for its bacterial anti-quorum sensing activity when applied at sublethal concentrations, has gained traction given its potential use against multidrug resistant bacteria. In this work, trans-cinnamaldehyde-loaded oil-in-water nanocapsules coated with chitosan, N,N,N-trimethyl chitosan chloride, N-(2-(N,N,N-trimethylammoniumyl)acetyl) chitosan chloride or N-(6-(N,N,N-trimethylammoniumyl)hexanoyl)chitosan chloride were obtained. All the formulated nanocapsules showed a Z-average hydrodynamic diameter ~ 160 nm and ζ-potential higher than +40 mV. N,N,N-trimethyl chitosan-coated oil-in-water nanocapsules showed the greatest trans-cinnamaldehyde association efficiency (99.3 ± 7.6) % and total payload release (88.6 ± 22.5) %, while N-(6-(N,N,N-trimethylammoniumyl)hexanoyl)chitosan chloride chitosan-coated oil-in-water nanocapsules were the only formulations stable in phosphate buffer saline PBS (pH 7.4) upon incubation at 37 °C for 24 h. Future work should address the stability of the developed nanocapsules in culture media and their biological performance.

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.