Improving Access to Eye Care in Rural Communities: PocDoc's Web-Based Visual Acuity Screening Tool.

Objective: Visual acuity (VA) testing is crucial for early intervention in cases of visual impairment, especially in rural health care. This study aimed to determine the potential of a web-based VA test (PocDoc) in addressing the unique health care needs of rural areas through the comparison in its effectiveness against the conventional VA test in identifying visual impairment among an Indian rural population. Methods: Prospective comparative study conducted in December 2022 at a tertiary referral eye care center in central India. We evaluated all patients with the PocDoc VA tests using three device types, and the conventional VA test. Bland-Altman plot (BAP) compared PocDoc and conventional VA tests. Fisher's exact tests evaluated associations between categorical parameters. Kruskal-Wallis tests followed by post hoc Dunn's tests identified association between categorical parameters and numerical parameters. Results: We evaluated 428 patients (792 measurements of VA) with mean age 36.7 (±23.3) years. PocDoc resulted in slightly worse VA scores (mean logMAR: 0.345) than conventional (mean logMAR: 0.315). Correlation coefficient between the conventional and PocDoc logMAR VA values was rho = 0.845 and rho2 = 0.7133 (p = 6.617 × 10-215; adjusted p = 2.205 × 10-214). Most data points fell within the interchangeable range of ±0.32 on BAP. Difference between the two methods increased with higher logMAR values, indicating poorer agreement for worse VA scores. Conclusions: Identifying and addressing the unique health care needs of rural populations is critical, including access to appropriate and effective VA testing methods. Validating and improving VA testing methods can ensure early intervention and improve the quality of life for individuals with visual impairment.

Cell-type-specific tumour sensitivity identified with a bromodomain targeting PROTAC in adenoid cystic carcinoma.

Salivary gland adenoid cystic carcinoma (ACC) is a rare malignancy with limited treatment options. The development of novel therapies is hindered by a lack of preclinical models. We have generated ACC patient-derived xenograft (PDX) lines that retain the physical and genetic properties of the original tumours, including the presence of the common MYB::NFIB or MYBL1::NFIB translocations. We have developed the conditions for the generation of both 2D and 3D tumour organoid patient-derived ACC models that retain MYB expression and can be used for drug studies. Using these models, we show in vitro and in vivo sensitivity of ACC cells to the bromodomain degrader, dBET6. Molecular studies show a decrease in BRD4 and MYB protein levels and target gene expression with treatment. The most prominent effect of dBET6 on tumours in vivo was a change in the relative composition of ACC cell types expressing either myoepithelial or ductal markers. We show that dBET6 inhibits the progenitor function of ACC cells, particularly in the myoepithelial marker-expressing population, revealing a cell-type-specific sensitivity. These studies uncover a novel mechanistic effect of bromodomain inhibitors on tumours and highlight the need to impact both cell-type populations for more effective treatments in ACC patients. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Designing, synthesis and evaluation of derived analogues of selected small molecule non-peptidic inhibitors against serotype BoNT/ F.

Botulinum neurotoxins are lethal Biowarfare categorized in group A of selected agents, by CDC USA. The unavailability of counter-measures against these neurotoxins has been a matter of extensive research. The 8-hydroxyquinoline (8-HQ) scaffold is established privileged compound and its potential as drug candidate against BoNTs is recently being explored. We have reported 8-HQ compounds NSC1014 and NSC1011 as potential small molecule inhibitors against BoNT/F. In the present study, analogues of NSC84087 and NSC1014 were designed, synthesized and studied for their inhibitory role against BoNT/F intoxication through in silico study, in vitro and in-vivo assays. ∼25 in-house synthesized small molecule inhibitors were evaluated against rBoNT/F light chain through fluorescence thermal shift (FTS) assay and then further assessed through endopeptidase assay. The binding affinity analysis was done through surface plasmon resonance (SPR) based Proteon™ XPR 36 system. Finally, the in-vivo efficacy of these compounds was evaluated in mice model. Analogues C87.9, C87.10 and C87.12 of compound NSC84087 and C14.10, C14.11 and C14.13 of NSC1014 showed promising results through FTS assay and endopeptidase assay. SPR based protein-small molecule interaction studies showed KD values in sub-micromolar range signifying high affinity interaction. The IC50 of C14.10 was found to be the lowest of 3.016 ± 0.798 µM as determined through endopeptidase assay. Finally, efficacy of selected molecules was evaluated in mice, C14.10 and C14.13 protected 40% animals against 4X LD50 and extended survival time up to 200% at 10X LD50. The present study thus proposes the emergence of NSC84087 and NSC1014 analogues as lead compound against BoNT/F.

In silico functional and structural characterization revealed virulent proteins of Francisella tularensis strain SCHU4.

Francisella tularensis is a pathogenic, aerobic gram-negative coccobacillus bacterium. It is the causative agent of tularemia, a rare infectious disease that can attack skin, lungs, eyes, and lymph nodes. The genome of F. tularensis has been sequenced, and ~16% of the proteome is still uncharacterized. Characterizations of these proteins are essential to find new drug targets for better therapeutics. In silico characterization of proteins has become an extremely important approach to determine the functionality of proteins as experimental functional elucidation is unable to keep pace with the current growth of the sequence database. Initially, we have annotated 577 Hypothetical Proteins (HPs) of F. tularensis strain SCHU4 with seven bioinformatics tools which characterized them based on the family, domain and motif. Out of 577 HPs, 119 HPs were annotated by five or more tools and are further screened to predict their virulence properties, subcellular localization, transmembrane helices as well as physicochemical parameters. VirulentPred predicted 66 HPs out of 119 as virulent. These virulent proteins were annotated to find the interacting partner using STRING, and proteins with high confidence interaction scores were used to predict their 3D structures using Phyre2. The three virulent proteins Q5NH99 (phosphoserine phosphatase), Q5NG42 (Cystathionine beta-synthase) and Q5NG83 (Rrf2-type helix turn helix domain) were predicted to involve in modulation of cytoskeletal and innate immunity of host, H2S (hydrogen sulfide) based antibiotic tolerance and nitrite and iron metabolism of bacteria. The above predicted virulent proteins can serve as novel drug targets in the era of antibiotic resistance.

The Mutational Concordance of Fixed Formalin Paraffin Embedded and Fresh Frozen Gastro-Oesophageal Tumours Using Whole Exome Sequencing.

1. BACKGROUND: The application of massively parallel sequencing has led to the identification of aberrant druggable pathways and somatic mutations within therapeutically relevant genes in gastro-oesophageal cancer. Given the widespread use of formalin-fixed paraffin-embedded (FFPE) samples in the study of this disease, it would be beneficial, especially for the purposes of biomarker evaluation, to assess the concordance between comprehensive exome-wide sequencing data from archival FFPE samples originating from a prospective clinical study and those derived from fresh-frozen material. 2. METHODS: We analysed whole-exome sequencing data to define the mutational concordance of 16 matched fresh-frozen and FFPE gastro-oesophageal tumours (N = 32) from a prospective clinical study. We assessed DNA integrity prior to sequencing and then identified coding mutations in genes that have previously been implicated in other cancers. In addition, we calculated the mutant-allele heterogeneity (MATH) for these samples. 3. RESULTS: Although there was increased degradation of DNA in FFPE samples compared with frozen samples, sequencing data from only two FFPE samples failed to reach an adequate mapping quality threshold. Using a filtering threshold of mutant read counts of at least ten and a minimum of 5% variant allele frequency (VAF) we found that there was a high median mutational concordance of 97% (range 80.1-98.68%) between fresh-frozen and FFPE gastro-oesophageal tumour-derived exomes. However, the majority of FFPE tumours had higher mutant-allele heterogeneity (MATH) scores when compared with corresponding frozen tumours (p < 0.001), suggesting that FFPE-based exome sequencing is likely to over-represent tumour heterogeneity in FFPE samples compared to fresh-frozen samples. Furthermore, we identified coding mutations in 120 cancer-related genes, including those associated with chromatin remodelling and Wnt/ß-catenin and Receptor Tyrosine Kinase signalling. 4. CONCLUSIONS: These data suggest that comprehensive genomic data can be generated from exome sequencing of selected DNA samples extracted from archival FFPE gastro-oesophageal tumour tissues within the context of prospective clinical trials.

HOX genes promote cell proliferation and are potential therapeutic targets in adrenocortical tumours.

BACKGROUND: Understanding the pathways that drive adrenocortical carcinoma (ACC) is essential to the development of more effective therapies. This study investigates the role of the transcription factor HOXB9 and other HOX factors in ACC and its treatment. METHODS: We used transgenic mouse models to determine the role of Hoxb9 in adrenal tumour development. Patient transcriptomic data was analysed for the expression of HOX genes and their association with disease. Drug response studies on various adrenocortical models were done to establish novel therapeutic options. RESULTS: Our human ACC dataset analyses showed high expression of HOXB9, and other HOX factors, are associated with poorer prognosis. Transgenic overexpression of Hoxb9 in the adrenal cortex of mice with activated Ctnnb1 led to larger adrenal tumours. This phenotype was preferentially observed in male mice and was characterised by more proliferating cells and an increase in the expression of cell cycle genes, including Ccne1. Adrenal tumour cells were found to be dependent on HOX function for survival and were sensitive to a specific peptide inhibitor. CONCLUSIONS: These studies show Hoxb9 can promote adrenal tumour progression in a sex-dependent manner and have identified HOX factors as potential drug targets, leading to novel therapeutic approaches in ACC.

Characterization of immune response induced against catalytic domain of botulinum neurotoxin type E.

Botulinum neurotoxins (BoNTs) represent a family of bacterial toxins responsible for neuroparalytic disease 'botulism' in human and animals. Their potential use as biological weapon led to their classification in category 'A' biowarfare agent by Centers for Disease Control and Prevention (CDC), USA. In present study, gene encoding full length catalytic domain of BoNT/E-LC was cloned, expressed and protein was purified using Ni-NTA chromatography. Humoral immune response was confirmed by Ig isotyping and cell-mediated immunity by cytokine profiling and intracellular staining for enumeration of IFN-γ secreting CD4+ and CD8+ T cells. Increased antibody titer with the predominance of IgG subtype was observed. An interaction between antibodies produced against rBoNT/E-LC was established that showed the specificity against BoNT/E in SPR assay. Animal protection with rBoNT/E-LC was conferred through both humoral and cellular immune responses. These findings were supported by cytokine profiling and flow cytometric analysis. Splenocytes stimulated with rBoNT/E-LC showed a 3.27 and 2.8 times increase in the IFN-γ secreting CD4+ and CD8+ T cells, respectively; in immunized group (P < 0.05). Protection against BoNT/E challenge tended to relate with increase in the percentage of rBoNT/E-LC specific IL-2 in the splenocytes supernatant (P = 0.034) and with IFN-γ-producing CD4+ T cell responses (P = 0.045). We have immunologically evaluated catalytically active rBoNT/E-LC. Our results provide valuable investigational report for immunoprophylactic role of catalytic domain of BoNT/E.

Targeted 8-hydroxyquinoline fragment based small molecule drug discovery against neglected botulinum neurotoxin type F.

OBJECTIVES: Botulinum neurotoxins are highly potent biological warfare agents. The unavailability of countermeasures against these neurotoxins has been a matter of extensive research. However, no clinical therapeutics has come to existence till date. The 8-hydroxyquinoline (8-HQ) scaffold is established privileged compound and its potential as drug candidate against BoNTs is recently being explored. METHODS: In present work, three course studies were performed involving in silico, in vitro and in vivo cascade to screen 8-HQ small molecule inhibitors against BoNT/F intoxication. ~800 molecules obtained from open repositories were screened in silico and commercially obtained twenty-four 8-HQ derived small molecule inhibitors were evaluated against rBoNT/F light chain through fluorescence thermal shift (FTS) assay. Selected compounds were further evaluated through endopeptidase assay. Further binding affinity analysis was done through surface plasmon resonance (SPR) based Proteon™ XPR 36 system. Finally, the in vivo efficacy of these compounds was evaluated in mice model. RESULTS: Three compounds NSC1011, NSC1014 and NSC84094 were found to be highly inhibitory after screening of 8-HQ compounds through FTS assay and endopeptidase assay. SPR based protein-small molecule interaction studies showed highest affinity binding of NSC1014 (KD: 5.58E-06) with BoNT/F-LC. NSC1011, NSC1014, and NSC84094 displayed IC50 of 30.47 ±â€¯6.24, 14.91 ±â€¯2.49 and 17.39 ±â€¯2.74 µM, respectively, in endopeptidase assay. NSC1011 and NSC1014 displayed marked extension of survival time in mice model. CONCLUSION: NSC1011 and NSC1014 have emerged as promising drug candidate against BoNT/F intoxication displaying higher potential than previously reported compounds.

Identification of Inhibitors against Botulinum Neurotoxins: 8-Hydroxyquinolines Hold Promise.

Botulinum neurotoxins (BoNTs) are the most toxic category A biological warfare agents. There is no therapeutics available for BoNT intoxication yet, necessitating the development of a medical countermeasure against these neurotoxins. The discovery of small molecule-based drugs has revolutionized in the last two decades resulting in the identification of several small molecule inhibitors of BoNTs. However, none progressed to clinical trials. 8-Hydroxyquinolines scaffold-based molecules are important 'privileged structures' that can be exploited as inhibitors of a diverse range of targets. In this review, our study of recent reports suggests the development of 8-hydroxyquinoline derived molecules as a potential drug may be on the horizon.

Correction: Suppression of interferon gene expression overcomes resistance to MEK inhibition in KRAS-mutant colorectal cancer.

A correction to this paper has been published and can be accessed via a link at the top of the paper.

Suppression of interferon gene expression overcomes resistance to MEK inhibition in KRAS-mutant colorectal cancer.

Despite showing clinical activity in BRAF-mutant melanoma, the MEK inhibitor (MEKi) trametinib has failed to show clinical benefit in KRAS-mutant colorectal cancer. To identify mechanisms of resistance to MEKi, we employed a pharmacogenomic analysis of MEKi-sensitive versus MEKi-resistant colorectal cancer cell lines. Strikingly, interferon- and inflammatory-related gene sets were enriched in cell lines exhibiting intrinsic and acquired resistance to MEK inhibition. The bromodomain inhibitor JQ1 suppressed interferon-stimulated gene (ISG) expression and in combination with MEK inhibitors displayed synergistic effects and induced apoptosis in MEKi-resistant colorectal cancer cell lines. ISG expression was confirmed in patient-derived organoid models, which displayed resistance to trametinib and were resensitized by JQ1 co-treatment. In in vivo models of colorectal cancer, combination treatment significantly suppressed tumor growth. Our findings provide a novel explanation for the limited response to MEK inhibitors in KRAS-mutant colorectal cancer, known for its inflammatory nature. Moreover, the high expression of ISGs was associated with significantly reduced survival of colorectal cancer patients. Excitingly, we have identified novel therapeutic opportunities to overcome intrinsic and acquired resistance to MEK inhibition in colorectal cancer.

Biochemical Characterization of In vitro Reconstituted Biologically Active Recombinant Shiga Toxin.

BACKGROUND: Shiga toxins comprise a family of related proteins produced by bacteria Shigella dysenteriae and some strains of Escherichia coli that cause severe clinical manifestations. Severe Shiga toxin intoxication results in Haemolytic-Uremic Syndrome (HUS), up to 50% of HUS patients manifest some degree of renal failure and ~10% of such cases develop permanent renal failure or death. OBJECTIVE: In present research work production of biologically active rStx from non-toxic rStxA and rStxB subunits were established that can be used in many biomedical applications. METHODS: Purification of Shiga toxin from bacteria is a multistep time consuming process resulting in low yield. To overcome this problem, the rStxA and rStxB protein were separately cloned and expressed in E. coli host and purified through affinity chromatography. GST pull-down assay was performed for interaction study between rStxA and pentameric rStxB. The affinity between A and B subunits of reconstituted recombinant Shiga toxin (AB5) was determined by SPR. The biological activity of the toxin was confirmed in Vero cells and mouse lethality assay. RESULTS: The yield of GST-StxA and His6X-StxB obtained after affinity chromatography was estimated to 2 and 5 mg/l, respectively. Samples analyzed in pull down assay revealed two bands of ~58 kDa (rStxA) and ~7.7 kDa (rStxB) on SDS-PAGE. Affinity was confirmed through SPR with KD of 0.85 pM. This rStx produced from 1:5 molar ratio found to be cytotoxic in Vero cell line and resulted lethality in mouse. CONCLUSIONS: Large scale production of rStx using the method can facilitate screening and evaluation of small molecule inhibitors for therapeutics development.

FFPE breast tumour blocks provide reliable sources of both germline and malignant DNA for investigation of genetic determinants of individual tumour responses to treatment.

BACKGROUND: Bio-banked formalin-fixed paraffin-embedded (FFPE) tissues provide an excellent opportunity for translational genomic research. Historically matched blood has not always been collected as a source of germline DNA. This project aimed to establish if normal FFPE breast tissue could be used as an alternative to blood. METHODS: Exome sequencing was carried out on matched tumour tissue, normal breast tissue and blood on five patients in the START trial. Retrieved samples had been archived at different centres for at least 13 years. Following tissue macro-dissection and DNA extraction, targeted exome capture was performed using SureSelect Human All Exome v5 reagents (Agilent). Illumina paired-end libraries were prepared from the captured target regions and sequenced on a HiSeq2500 (Illumina) acquiring 2 × 75 bp reads. Somatic variants were called using the MuTect software analysis tool and copy number abnormalities (CNA) were identified using CNVkit. Targeted sequencing and droplet digital PCR were used to validate somatic variants and CNA, respectively. RESULTS: Overlap of somatic variants and CNA called on tumour versus blood and tumour versus normal breast tissue was good. Agreement in somatic variant calling ranged from 76.9 to 93.6%. Variants with an allele frequency lower than 10% were more difficult to validate irrespective of the type of germline DNA used. Pearson's correlation coefficients for paired comparisons of CNA using blood or normal tissue as reference ranged from 0.70 to 0.94. CONCLUSIONS: There is good correlation between the somatic mutations and CNA called using archived blood or normal breast tissue as germline reference material.

Capture Hi-C identifies putative target genes at 33 breast cancer risk loci.

Genome-wide association studies (GWAS) have identified approximately 100 breast cancer risk loci. Translating these findings into a greater understanding of the mechanisms that influence disease risk requires identification of the genes or non-coding RNAs that mediate these associations. Here, we use Capture Hi-C (CHi-C) to annotate 63 loci; we identify 110 putative target genes at 33 loci. To assess the support for these target genes in other data sources we test for associations between levels of expression and SNP genotype (eQTLs), disease-specific survival (DSS), and compare them with somatically mutated cancer genes. 22 putative target genes are eQTLs, 32 are associated with DSS and 14 are somatically mutated in breast, or other, cancers. Identifying the target genes at GWAS risk loci will lead to a greater understanding of the mechanisms that influence breast cancer risk and prognosis.

High level expression and immunochemical characterization of botulinum neurotoxin type F light chain.

Botulinum neurotoxins (BoNTs) are the most toxic biological substances known. Their potential use as biological warfare agent results in their classification as category A biowarfare agent by Centers for Disease Control and Prevention (CDC), USA. Presently, there are no approved detection system and pharmacological treatments for BoNT intoxication. Although a toxoid vaccine is available for immuno-prophylaxis, vaccines cannot reverse the effect of pre-translocated toxin. Direct handling of the live BoNTs for developing detection and therapeutics may pose fatal danger. This concern was addressed by purifying the recombinant catalytically active light chain of BoNT/F. BoNT/F-LC gene was amplified from the genomic DNA using specifically designed primers and expressed in Escherichia coli. Expression and purification profile were optimized under different conditions for biologically active light chain production. Specific polyclonal antibodies generated against type F illustrates in vivo neutralization in mice and rabbit. These antibodies play key role in conceiving the development of high throughput SPR based detection system which is a highly precise label free technique for protein interaction analysis. The presented work is first of its kind, signifying the production of highly stable and active rBoNT/F-LC and its immunochemical characterization. The study aids in paving the path towards developing a persistent detection system as well as in presenting comprehended scheme for in vitro small molecule therapeutics analysis.

Profiling of red pigment produced by Streptomyces sp. JAR6 and its bioactivity.

Actinomycetes strain was isolated from leaf litter soil sample and was identified as Streptomyces sp. by conventional and molecular approaches. The biologically active compound responsible for antimicrobial and anticancer activity of the strain JAR6 was elucidated by solid state fermentation followed by subsequent chromatographic and spectroscopic analysis. Extraction, purification and structural confirmation of red pigment metabolite viz undecylprodigiosin were established on the basis of spectroscopic studies and comparing the data from the literature. The biologically active compound was tested against Gram-positive and Gram-negative clinical isolates and its minimum inhibitory concentration was recorded. The antimicrobial activity of undecylprodigiosin is more prominent against Salmonella sp., Proteus mirabilis, Shigella sp. and Enterococcus sp. whereas, it was less effective against Staphylococcus aureus, Klebsiella pneumonia and Escherichia coli. The anticancer activity of undecylprodigiosin was tested against HeLa cell lines and it exhibited commendable cytotoxicity effect with IC50 value of 145 µg/ml. The present investigation reveals that undecylprodigiosin produced by Streptomyces strain JAR6 is a potent bioactive metabolite with effective pharmaceutical properties.

In Vitro Antimicrobial Potential of the Lichen Parmotrema sp. Extracts against Various Pathogens.

OBJECTIVE(S): The ongoing increasing antibiotic resistance is one of the biggest challenges faced by global public health. The perennial need for new antimicrobials against a background of increasing antibiotic resistance in pathogenic and opportunistic microorganisms obliges the scientific community to constantly develop new drugs and antimicrobial agents. Lichens are known prolific sources of natural antimicrobial drugs and biologically active natural products. This study was aimed to explore in vitro antimicrobial activity of lichen Parmotrema sp. MATERIAL AND METHODS: The methanol and aqueous extracts of lichen Parmotrema sp. was extracted using Soxhlet extractor. Antibiotic assessment of methanol and aqueous extracts was done against eight bacterial (Escherichia coli, Staphylococcus aureus, Proteus mirabilis, Salmonella sp., Shigella sp., Enterococci faecalis, Pseudomonas aeruginosa, Klebsiella pneumoniae,) clinical pathogens and five plant pathogenic fungal strains (Aspergillus terreus strain JAS1, Scedosporium sp. JAS1, Ganoderma sp. JAS4, Candida tropicalis and Fusarium sp.) by Kirby-Bauer method. RESULTS: The methanol lichen Parmotrema sp. extract inhibited all the test organisms. The highest antibacterial activity was found against Pseudomonas aeruginosa and Staphylococcus aureus. The weakest activity was manifested in Salmonella sp. and Scedosporium sp. JAS1. Strong antifungal effect was found against Ganoderma sp. JAS4 and Fusarium sp. The aqueous lichen Parmotrema sp. extract revealed neither antibacterial nor antifungal activity. CONCLUSION: The present study shows that tested lichen Parmotrema sp. extracts demonstrated a strong antimicrobial effect. That suggests the active components from methanol extracts of the investigated lichen Parmotrema sp. can be used as natural antimicrobial agent against pathogens.

A Biological Approach to the Synthesis of Silver Nanoparticles with Streptomyces sp JAR1 and its Antimicrobial Activity.

The biological approach to synthesize metal nanoparticles is an important aspect of current nanotechnology research. Silver nanoparticles have been well-known for their inhibitory and antimicrobial effects. The ever-increasing antibiotic resistance in pathogenic and opportunistic microorganisms is a major threat to the health care industry. In the present investigation, silver nanoparticles have been successfully biosynthesized by Streptomyces sp JAR1. Biosynthesized silver nanoparticles were characterized by means of several analytical techniques including a UV-Visible spectrophotometer, Fourier transform infrared spectroscopy, X-ray diffraction pattern analysis, and atomic force microscopy. An evaluation of the antimicrobial activity of silver nanoparticles (AgNPs) was carried out against clinically important pathogenic microorganisms. The metal nanoparticles were also evaluated for their combined effects with antibiotics against the clinical pathogens. The antibacterial activities of the antibiotics increased in the presence of the biologically synthesized AgNPs against the clinically important pathogens. The highest enhancing effect was observed for erythromycin against the test pathogens.

Transcriptome analyses of primitively eusocial wasps reveal novel insights into the evolution of sociality and the origin of alternative phenotypes.

BACKGROUND: Understanding how alternative phenotypes arise from the same genome is a major challenge in modern biology. Eusociality in insects requires the evolution of two alternative phenotypes - workers, who sacrifice personal reproduction, and queens, who realize that reproduction. Extensive work on honeybees and ants has revealed the molecular basis of derived queen and worker phenotypes in highly eusocial lineages, but we lack equivalent deep-level analyses of wasps and of primitively eusocial species, the latter of which can reveal how phenotypic decoupling first occurs in the early stages of eusocial evolution. RESULTS: We sequenced 20 Gbp of transcriptomes derived from brains of different behavioral castes of the primitively eusocial tropical paper wasp Polistes canadensis. Surprisingly, 75% of the 2,442 genes differentially expressed between phenotypes were novel, having no significant homology with described sequences. Moreover, 90% of these novel genes were significantly upregulated in workers relative to queens. Differential expression of novel genes in the early stages of sociality may be important in facilitating the evolution of worker behavioral complexity in eusocial evolution. We also found surprisingly low correlation in the identity and direction of expression of differentially expressed genes across similar phenotypes in different social lineages, supporting the idea that social evolution in different lineages requires substantial de novo rewiring of molecular pathways. CONCLUSIONS: These genomic resources for aculeate wasps and first transcriptome-wide insights into the origin of castes bring us closer to a more general understanding of eusocial evolution and how phenotypic diversity arises from the same genome.

Pyrosequencing the transcriptome of the greenhouse whitefly, Trialeurodes vaporariorum reveals multiple transcripts encoding insecticide targets and detoxifying enzymes.

BACKGROUND: The whitefly Trialeurodes vaporariorum is an economically important crop pest in temperate regions that has developed resistance to most classes of insecticides. However, the molecular mechanisms underlying resistance have not been characterised and, to date, progress has been hampered by a lack of nucleotide sequence data for this species. Here, we use pyrosequencing on the Roche 454-FLX platform to produce a substantial and annotated EST dataset. This 'unigene set' will form a critical reference point for quantitation of over-expressed messages via digital transcriptomics. RESULTS: Pyrosequencing produced around a million sequencing reads that assembled into 54,748 contigs, with an average length of 965 bp, representing a dramatic expansion of existing cDNA sequences available for T. vaporariorum (only 43 entries in GenBank at the time of this publication). BLAST searching of non-redundant databases returned 20,333 significant matches and those gene families potentially encoding gene products involved in insecticide resistance were manually curated and annotated. These include, enzymes potentially involved in the detoxification of xenobiotics and those encoding the targets of the major chemical classes of insecticides. A total of 57 P450s, 17 GSTs and 27 CCEs were identified along with 30 contigs encoding the target proteins of six different insecticide classes. CONCLUSION: Here, we have developed new transcriptomic resources for T. vaporariorum. These include a substantial and annotated EST dataset that will serve the community studying this important crop pest and will elucidate further the molecular mechanisms underlying insecticide resistance.