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Nitrogen (N) is one of the primary macronutrients required for crop growth and yield. This nutrient is especially limiting wheat yields in the dry and low fertile agro-ecologies having low N in the root zone soil strata. Moreover, majority of farmers in India and South Asia are small to marginal with meagre capacity to invest in costly nitrogen fertilizers. Therefore, there is an immense need to identify lines that use nitrogen efficiently. A set of 50 diverse wheat genotypes consisting of indigenous germplasm lines (05), cultivars released for commercial cultivation (23) and selected elite lines from CIMMYT nurseries (22) were evaluated in an alpha-lattice design with two replications, a six-rowed plot of 2.5m length for 24 agro morphological, physiological and NUE related traits during two consecutive crop seasons in an N-depleted precision field under two different N levels of 50%-N50 (T1) and 100%-N100 (T2) of recommended N, i.e., 100 kg/ha. Analysis of variance revealed significant genetic variation among genotypes for all the traits studied. About 11.36% yield reduction was observed at reduced N levels. Significant correlations among NUE traits and yield component traits were observed which indicated pivotal role of N remobilization to the grain in enhancing yield levels. Among N-insensitive genotypes identified based on their yielding ability at low N levels, UASBW13356, UASBW13358, UASBW13354, UASBW13357 and KRL1-4 showed their inherent genotypic plasticity toward N application. The genotypes with more yield and high to moderate NUtE can be used as parents for the breeding of N efficient genotypes for marginal agro-ecologies. Low N tolerant genotypes identified from the current investigation may be further utilized in the identification of genomic regions responsible for NUE and its deployment in wheat breeding programs. The comprehensive data of 24 traits under different nitrogen levels for diverse genotypes from India and global sources (mainly CIMMYT) should be useful for supporting breeding for NUE and thus will be of great help for small and marginal farmers in India and South Asia.

U2 snRNP is an essential component of the spliceosome. It is responsible for branch point recognition in the spliceosome A-complex via base-pairing of U2 snRNA with an intron to form the branch helix. Small molecule inhibitors target the SF3B component of the U2 snRNP and interfere with A-complex formation during spliceosome assembly. We previously found that the first SF3B inhibited-complex is less stable than A-complex and hypothesized that SF3B inhibitors interfere with U2 snRNA secondary structure changes required to form the branch helix. Using RNA chemical modifiers, we probed U2 snRNA structure in A-complex and SF3B inhibited splicing complexes. The reactivity pattern for U2 snRNA in the SF3B inhibited-complex is indistinguishable from that of A-complex, suggesting that they have the same secondary structure conformation, including the branch helix. This observation suggests SF3B inhibited-complex instability does not stem from an alternate RNA conformation and instead points to the inhibitors interfering with protein component interactions that normally stabilize U2 snRNP's association with an intron. In addition, we probed U2 snRNA in the free U2 snRNP in the presence of SF3B inhibitor and again saw no differences. However, increased protection of nucleotides upstream of Stem I in the absence of SF3A and SF3B proteins suggests a change of secondary structure at the very 5' end of U2 snRNA. Chemical probing of synthetic U2 snRNA in the absence of proteins results in similar protections and predicts a previously uncharacterized extension of Stem I. Because this stem must be disrupted for SF3A and SF3B proteins to stably join the snRNP, the structure has the potential to influence snRNP assembly and recycling after spliceosome disassembly.

ObjectiveTo evaluate the efficacy of corneal neurotisation using sural nerve graft coaptation of the contralateral supratrochlear nerve in unilateral neurotrophic keratopathy and corneal anesthesia. Corneal neuralization has emerged as a potential option in the treatment of neurotropic keratopathy, however not free from the predicament. We evaluated the long-term outcome of corneal neurotisation in the treatment of unresponsive unilateral neurotropic keratopathy using surgical variations to mimic and expedient the surgical procedure.MethodsA Prospective interventional study involving patients with unilateral neurotrophic keratopathy (NK) who did not respond to medical measures was conducted. The study parameters evaluated were best-corrected visual acuity improvement, ocular surface evaluation parameters [tear break-up time (TBUT), Schirmer's 1, and ocular surface staining scores (corneal and conjunctival staining)], central corneal sensation (Cochet Bonnet esthesiometer), sub-basal nerve fiber length (SBNFL), and sub-basal nerve fiber density (SBNFD) determined by central confocal microscopy at recruitment and during follow-up at 1-month, 3-month, 6-month, 9-month and 12-month respectively, following corneal neurotization.ResultsEleven eyes of 11 patients with unilateral neurotrophic keratopathy (NK) who underwent corneal neurotisation were studied. The mean follow-up was 10.09±2.31months (range, 6-12). Mean best corrected visual acuity in log MAR at baseline, 1.35±0.52 improved significantly to 1.06±0.76 (P = 0.012) at 3 months and continued to 0.55±0.60 (P = 0.027) at 12 months. There was a significant reduction in NK grade severity and improvement in the ocular surface as early as 1 month, and central corneal sensations (P = 0.024) as soon as 3 months. Mean corneal SBNF improved from 3.12±1.84 mm/mm2 to 4.49±1.88 at 1 month (P = 0.008), 13.31±3.61 mm/mm2 (P = 0.028) at 12 months. Mean central corneal SBNFD evident at 6 months was 1.83±2.54no/mm2 (P = 0.018) and 4.90±3.12no/mm2 (P = 0.028) at 12 months.ConclusionThis study substantiates the routine practice of corneal neurotisation by simplifying the intricacies observed during the procedure.

Schizophrenia spectrum disorders (SSDs) are associated with significant functional impairments, disability, and low rates of personal recovery, along with tremendous economic costs linked primarily to lost productivity and premature mortality. Efforts to delineate the contributors to disability in SSDs have highlighted prominent roles for a diverse range of symptoms, physical health conditions, substance use disorders, neurobiological changes, and social factors. These findings have provided valuable advances in knowledge and helped define broad patterns of illness and outcomes across SSDs. Unsurprisingly, there have also been conflicting findings for many of these determinants that reflect the heterogeneous population of individuals with SSDs and the challenges of conceptualizing and treating SSDs as a unitary categorical construct. Presently it is not possible to identify the functional course on an individual level that would enable a personalized approach to treatment to alter the individual's functional trajectory and mitigate the ensuing disability they would otherwise experience. To address this ongoing challenge, this study aims to conduct a longitudinal multimodal investigation of a large cohort of individuals with SSDs in order to establish discrete trajectories of personal recovery, disability, and community functioning, as well as the antecedents and predictors of these trajectories. This investigation will also provide the foundation for the co-design and testing of personalized interventions that alter these functional trajectories and improve outcomes for people with SSDs.

Computational modelling of biological processes poses multiple challenges in each stage of the modelling exercise. Some significant challenges include identifiability, precisely estimating parameters from limited data, informative experiments and anisotropic sensitivity in the parameter space. One of these challenges' crucial but inconspicuous sources is the possible presence of large regions in the parameter space over which model predictions are nearly identical. This property, known as sloppiness, has been reasonably well-addressed in the past decade, studying its possible impacts and remedies. However, certain critical unanswered questions concerning sloppiness, particularly related to its quantification and practical implications in various stages of system identification, still prevail. In this work, we systematically examine sloppiness at a fundamental level and formalise two new theoretical definitions of sloppiness. Using the proposed definitions, we establish a mathematical relationship between the parameter estimates' precision and sloppiness in linear predictors. Further, we develop a novel computational method and a visual tool to assess the goodness of a model around a point in parameter space by identifying local structural identifiability and sloppiness and finding the most sensitive and least sensitive parameters for non-infinitesimal perturbations. We demonstrate the working of our method in benchmark systems biology models of various complexities. The pharmacokinetic HIV infection model analysis identified a new set of biologically relevant parameters that can be used to control the free virus in an active HIV infection.

Modeling and prediction of traffic systems is a challenging task due to the complex interactions within the system. Identification of significant regressors and using them to improve travel time predictions is a concept of interest. In previous studies, such regressors were identified offline and were static in nature. In this study, an iterative joint clustering and prediction approach is proposed to accurately predict spatiotemporal patterns in travel time. The clustering module is tied to the prediction module, and a prediction model is trained on each cluster. The combined clustering and prediction are then iterated until a chosen metric is optimized. This orients clusters of data towards prediction while enabling model development on subsets of travel time data with similar prediction complexity. The clusters created using the joint clustering and prediction approach confirmed to the real-world traffic scenario, forming clusters of high travel time at busy intersections and bus stops across the study stretch and forming clusters of low travel time in the sub-urban areas of the city. Further, a comparison of the developed framework with base methods demonstrated a decrease in prediction errors by at least 22.83%. This indicates that creating clusters of data that are sensitive to the quality of predictions using the joint clustering and prediction framework improves the accuracy of travel time predictions. The study also proposes criteria for choosing the best predictions when cluster-based predictions are used.

Infection with infectious hypodermal and hematopoietic necrosis virus (IHHNV) is a crustacean disease that caused large-scale mortality in Penaeus stylirostris, deformity and growth retardation in Penaeus vannamei and Penaeus monodon. We surveyed the presence of IHHNV in three major shrimp-producing regions in Ecuador, namely Guayas, El Oro, and Esmeralda. The data show that IHHNV is endemic (3.3-100% prevalence) to shrimp farms in these regions. The whole genome sequences of representative circulating IHHNV genotypes in Ecuador and Peru showed that these genotypes formed a separate cluster within the Type II genotypes and were divergent from other geographical isolates of IHHNV originating in Asia, Africa, Australia, and Brazil. In experimental bioassays using specific pathogen-free (SPF) P. vannamei, P. monodon, and P. stylirostris and representative IHHNV isolates from Ecuador and Peru, the virus did not cause any mortality or induce clinical signs in any of the three penaeid species. Although IHHNV-specific Cowdry type A inclusion bodies were histologically detected in experimentally challenged P. vannamei and P. monodon and confirmed by in situ hybridization, no such inclusions were observed in P. stylirostris. Moreover, P. vannamei had the highest viral load, followed by P. monodon and P. stylirostris. Based on IHHNV surveillance data, we conclude that the currently farmed P. vannamei lines in Ecuador are tolerant to circulating IHHNV genotypes. The genome sequence and experimental bioassay data showed that, although the currently circulating genotypes are infectious, they do not induce clinical lesions in the three commercially important penaeid species. These findings suggest a potentially evolving virus-host relationship where circulating genotypes of IHHNV co-exist in equilibrium with P. vannamei raised in Peru and Ecuador.

BackgroundThe use of neo-adjuvant chemotherapy in treating osteosarcoma has improved patients' average 5 year survival rate from 20% to 70% in the past 30 years. However, for patients who progress after chemotherapy, its effectiveness diminishes due to the emergence of multi-drug resistance (MDR) after prolonged therapy.Methodology/principal findingsIn order to overcome both the dose-limiting side effects of conventional chemotherapeutic agents and the therapeutic failure resulting from MDR, we designed and evaluated a novel drug delivery system for MDR1 siRNA delivery. Novel biocompatible, lipid-modified dextran-based polymeric nanoparticles were used as the platform for MDR1 siRNA delivery; and the efficacy of combination therapy with this system was evaluated. In this study, multi-drug resistant osteosarcoma cell lines (KHOS(R2) and U-2OS(R2)) were treated with the MDR1 siRNA nanocarriers and MDR1 protein (P-gp) expression, drug retention, and immunofluoresence were analyzed. Combination therapy of the MDR1 siRNA loaded nanocarriers with increasing concentrations of doxorubicin was also analyzed. We observed that MDR1 siRNA loaded dextran nanoparticles efficiently suppresses P-gp expression in the drug resistant osteosarcoma cell lines. The results also demonstrated that this approach may be capable of reversing drug resistance by increasing the amount of drug accumulation in MDR cell lines.Conclusions/significanceLipid-modified dextran-based polymeric nanoparticles are a promising platform for siRNA delivery. Nanocarriers loaded with MDR1 siRNA are a potential treatment strategy for reversing MDR in osteosarcoma.

Vascular smooth muscle cell (VSMC) phenotypic modulation is characterized by the downregulation of SMC actin cytoskeleton proteins. Our published study shows that depletion of SM22α (aka SM22, Transgelin, an actin cytoskeleton binding protein) promotes inflammation in SMCs by activating NF-κB signal pathways both in cultured VSMCs and in response to vascular injury. The goal of this study is to investigate the underlying molecular mechanisms whereby SM22 suppresses NF-κB signaling pathways under inflammatory condition. NF-κB inducing kinase (Nik, aka MAP3K14, activated by the LTβR) is a key upstream regulator of NF-κB signal pathways. Here, we show that SM22 overexpression suppresses the expression of NIK and its downstream NF-κB canonical and noncanonical signal pathways in a VSMC line treated with a LTβR agonist. SM22 regulates NIK expression at both transcriptional and the proteasome-mediated post-translational levels in VSMCs depending on the culture condition. By qPCR, chromatin immunoprecipitation and luciferase assays, we found that Nik is a transcription target of serum response factor (SRF). Although SM22 is known to be expressed in the cytoplasm, we found that SM22 is also expressed in the nucleus where SM22 interacts with SRF to inhibit the transcription of Nik and prototypical SRF regulated genes including c-fos and Egr3. Moreover, carotid injury increases NIK expression in Sm22-/- mice, which is partially relieved by adenovirally transduced SM22. These findings reveal for the first time that SM22 is expressed in the nucleus in addition to the cytoplasm of VSMCs to regulate the transcription of Nik and its downstream proinflammatory NF-kB signal pathways as a modulator of SRF during vascular inflammation.

Pittosporum eriocarpum Royle, a medicinally important taxon, is endemic to Uttarakhand region of Himalaya. It has become endangered due to over-collection and the loss of habitats. As raising plants through seeds in this plant is problematic, a reliable protocol for micropropagation using nodal explants has been developed. High shoot regeneration (95%) occurred in MS medium augmented with BA 0.4mg/l in combination IBA 0.6mg/l. In vitro regenerated shoots were rooted in MS medium supplemented with three auxins, of which 0.6 mg/l indole butyric acid proved to be the best for rooting (90%) with maximum number of roots per shoot. Thereafter, rooted plants were hardened and nearly 73% of rooted shoots were successfully acclimatized and established in the field. Start codon targeted (SCoT), inter simple sequence repeats (ISSR) and random amplified polymorphic DNA (RAPD) markers were used to validate the genetic homogeneity amongst nine in vitro raised plantlets with mother plant. DNA fingerprints of in vitro regenerated plantlets displayed monomorphic bands similar to mother plant, indicating homogeneity among the micropropagated plants with donor mother plant. The similarity values were calculated based on SCoT, ISSR and RAPD profiles which ranged from 0.89 to 1.00, 0.91 to 1.00 and 0.95 to 1.00 respectively. The dendrograms generated through Unweighted Pair Group Method with arithmetic mean (UPGMA) analysis revealed 97% similarity amongst micropropagated plants with donor mother plant, thus confirming genetic homogeneity of micropropagated clones. This is the first report on micropropagation and genetic homogeneity assessment of P. eriocarpum. The protocol would be useful for the conservation and large scale production of P. eriocarpum to meet the demand for medicinal formulations and also for the re-introduction of in vitro grown plants in the suitable natural habitats to restore the populations.

Using morphological, histological, and TEM analyses of the cranium, we provide a detailed description of bone and suture growth in zebrafish. Based on expression patterns and localization, we identified osteoblasts at different degrees of maturation. Our data confirm that, unlike in humans, zebrafish cranial sutures maintain lifelong patency to sustain skull growth. The cranial vault develops in a coordinated manner resulting in a structure that protects the brain. The zebrafish cranial roof parallels that of higher vertebrates and contains five major bones: one pair of frontal bones, one pair of parietal bones, and the supraoccipital bone. Parietal and frontal bones are formed by intramembranous ossification within a layer of mesenchyme positioned between the dermal mesenchyme and meninges surrounding the brain. The supraoccipital bone has an endochondral origin. Cranial bones are separated by connective tissue with a distinctive architecture of osteogenic cells and collagen fibrils. Here we show RNA in situ hybridization for col1a1a, col2a1a, col10a1, bglap/osteocalcin, fgfr1a, fgfr1b, fgfr2, fgfr3, foxq1, twist2, twist3, runx2a, runx2b, sp7/osterix, and spp1/ osteopontin, indicating that the expression of genes involved in suture development in mammals is preserved in zebrafish. We also present methods for examining the cranium and its sutures, which permit the study of the mechanisms involved in suture patency as well as their pathological obliteration. The model we develop has implications for the study of human disorders, including craniosynostosis, which affects 1 in 2,500 live births.

PURPOSE:To formulate hydroxypropyl methylcellulose-stabilized self-emulsifying solid dispersible carriers of noscapine to enhance oral bioavailability. METHODS:Formulation of noscapine (Nos) self-emulsifying solid dispersible microparticles (SESDs) was afforded by emulsification using an optimized formula of Labrafil M1944, Tween-80, and Labrasol followed by spray-drying with hydroxypropyl methylcellulose (HPMC), with and without mannosamine (Mann-Nos_SESDs and Nos_SESDs respectively); self-microemulsifying liquid dispersions (SMEDDs) with and without mannosamine (Mann-Nos_SMEDDs and Nos_SMEDDs respectively) were also prepared. SMEDDs and SESDs were characterized for size, polydispersity, surface charge, entrapment efficiency, in vitro permeability, in vitro release kinetics, and oral pharmacokinetics in Sprague-Dawley rats (10 mg/kg p.o). The antitumor efficacy of Mann-Nos_SESDs on the basis of chemosensitization to cisplatin (2.0 mg/kg, i.v.) was investigated in a chemorefractory lung tumor Nu/Nu mouse model up to a maximal oral dose of 300 mg/kg. RESULTS:The oil/surfactant/co-surfactant mixture of Labrafil M1944, Tween-80, and Labrasol optimized at weight ratios of 62.8:9.30:27.90% produced stable self-microemulsifying dispersions (SMEDDs) at a SMEDD to water ratio of 1-3:7-9 parts by weight. SMEDDs had hydrodynamic diameters between 231 and 246 nm; surface charges ranged from -16.50 to -18.7 mV; and entrapment efficiencies were between 32 and 35%. SESDs ranged in size between 5.84 and 6.60 μm with surface charges from -10.62 to -12.40 mV and entrapment efficiencies of 30.96±4.66 and 32.05±3.72% (Nos_SESDs and Mann-Nos_SESDs respectively). Mann-Nos_SESDs exhibited saturating uptake across Caco-2 monolayers (Papp = 4.94±0.18 × 10(-6) cm/s), with controlled release of 50% of Nos in 6 hr at pH 6.8 following Higuchi kinetics. Mann-Nos_ SESDs was 40% more bioavailable compared to Nos_SESDs; and was effective in sensitizing H1650 SP cells to Cisplatin in vitro and in an orthotopic lung tumor model of H1650 SP origin. CONCLUSIONS:Mannosylated noscapine self-emulsifying solid dispersions (Mann-Nos_SESDs) are bioavailable and potentiate the antineoplastic effect of cisplatin-based chemotherapy in cisplatin-resistant NSCLC.

Recent studies have demonstrated that mesenchymal stem cells (MSCs) combined with CD34+ hematopoietic/stem progenitor cells (HSPCs) can function as surrogate urinary bladder cells to synergistically promote multi-faceted bladder tissue regeneration. However, the molecular pathways governing these events are unknown. The pleiotropic effects of Wnt5a and Cyr61 are known to affect aspects of hematopoiesis, angiogenesis, and muscle and nerve regeneration. Within this study, the effects of Cyr61 and Wnt5a on bladder tissue regeneration were evaluated by grafting scaffolds containing modified human bone marrow derived MSCs. These cell lines were engineered to independently over-express Wnt5a or Cyr61, or to exhibit reduced expression of Cyr61 within the context of a nude rat bladder augmentation model. At 4 weeks post-surgery, data demonstrated increased vessel number (~250 vs ~109 vessels/mm2) and bladder smooth muscle content (~42% vs ~36%) in Cyr61OX (over-expressing) vs Cyr61KD (knock-down) groups. Muscle content decreased to ~25% at 10 weeks in Cyr61KD groups. Wnt5aOX resulted in high numbers of vessels and muscle content (~206 vessels/mm2 and ~51%, respectively) at 4 weeks. Over-expressing cell constructs resulted in peripheral nerve regeneration while Cyr61KD animals were devoid of peripheral nerve regeneration at 4 weeks. At 10 weeks post-grafting, peripheral nerve regeneration was at a minimal level for both Cyr61OX and Wnt5aOX cell lines. Blood vessel and bladder functionality were evident at both time-points in all animals. Results from this study indicate that MSC-based Cyr61OX and Wnt5aOX cell lines play pivotal roles with regards to increasing the levels of functional vasculature, influencing muscle regeneration, and the regeneration of peripheral nerves in a model of bladder augmentation. Wnt5aOX constructs closely approximated the outcomes previously observed with the co-transplantation of MSCs with CD34+ HSPCs and may be specifically targeted as an alternate means to achieve functional bladder regeneration.

We investigated the streptomycin-induced stress response in Salmonella enterica serovars with a laser optical sensor, BARDOT (bacterial rapid detection using optical scattering technology). Initially, the top 20 S. enterica serovars were screened for their response to streptomycin at 100 μg/mL. All, but four S. enterica serovars were resistant to streptomycin. The MIC of streptomycin-sensitive serovars (Enteritidis, Muenchen, Mississippi, and Schwarzengrund) varied from 12.5 to 50 μg/mL, while streptomycin-resistant serovar (Typhimurium) from 125-250 μg/mL. Two streptomycin-sensitive serovars (Enteritidis and Mississippi) were grown on brain heart infusion (BHI) agar plates containing sub-inhibitory concentration of streptomycin (1.25-5 μg/mL) and a streptomycin-resistant serovar (Typhimurium) was grown on BHI containing 25-50 μg/mL of streptomycin and the colonies (1.2 ± 0.1 mm diameter) were scanned using BARDOT. Data show substantial qualitative and quantitative differences in the colony scatter patterns of Salmonella grown in the presence of streptomycin than the colonies grown in absence of antibiotic. Mass-spectrometry identified overexpression of chaperonin GroEL, which possibly contributed to the observed differences in the colony scatter patterns. Quantitative RT-PCR and immunoassay confirmed streptomycin-induced GroEL expression while, aminoglycoside adenylyltransferase (aadA), aminoglycoside efflux pump (aep), multidrug resistance subunit acrA, and ribosomal protein S12 (rpsL), involved in streptomycin resistance, were unaltered. The study highlights suitability of the BARDOT as a non-invasive, label-free tool for investigating stress response in Salmonella in conjunction with the molecular and immunoassay methods.

Mutations in the autosomal genes TMPRSS3, TMC1, USHIC, CDH23 and TMIE are known to cause hereditary hearing loss. To study the contribution of these genes to autosomal recessive, non-syndromic hearing loss (ARNSHL) in India, we examined 374 families with the disorder to identify potential mutations. We found four mutations in TMPRSS3, eight in TMC1, ten in USHIC, eight in CDH23 and three in TMIE. Of the 33 potentially pathogenic variants identified in these genes, 23 were new and the remaining have been previously reported. Collectively, mutations in these five genes contribute to about one-tenth of ARNSHL among the families examined. New mutations detected in this study extend the allelic heterogeneity of the genes and provide several additional variants for structure-function correlation studies. These findings have implications for early DNA-based detection of deafness and genetic counseling of affected families in the Indian subcontinent.

Cell-autonomous immunity to the bacterial pathogen Chlamydia trachomatis and the protozoan pathogen Toxoplasma gondii is controlled by two families of Interferon (IFN)-inducible GTPases: Immunity Related GTPases (IRGs) and Guanylate binding proteins (Gbps). Members of these two GTPase families associate with pathogen-containing vacuoles (PVs) and solicit antimicrobial resistance pathways specifically to the intracellular site of infection. The proper delivery of IRG and Gbp proteins to PVs requires the autophagy factor Atg5. Atg5 is part of a protein complex that facilitates the transfer of the ubiquitin-like protein Atg8 from the E2-like conjugation enzyme Atg3 to the lipid phosphatidylethanolamine. Here, we show that Atg3 expression, similar to Atg5 expression, is required for IRG and Gbp proteins to dock to PVs. We further demonstrate that expression of a dominant-active, GTP-locked IRG protein variant rescues the PV targeting defect of Atg3- and Atg5-deficient cells, suggesting a possible role for Atg proteins in the activation of IRG proteins. Lastly, we show that IFN-induced cell-autonomous resistance to C. trachomatis infections in mouse cells depends not only on Atg5 and IRG proteins, as previously demonstrated, but also requires the expression of Atg3 and Gbp proteins. These findings provide a foundation for a better understanding of IRG- and Gbp-dependent cell-autonomous resistance and its regulation by Atg proteins.

Neuroblastomas (NBs) are a clinically heterogeneous group of extra cranial pediatric tumors. Patients with high-risk, metastatic NBs have a long-term survival rate of below 40%, and are often resistant to current therapeutic modalities. Due to toxic side effects associated with radiation and chemotherapies, development of new agents is warranted to overcome resistance and effectively treat this disease in clinic. CARP-1 functional mimetics (CFMs) are an emerging class of small molecule compounds that inhibit growth of diverse cancer cell types. Here we investigated NB inhibitory potential of CFMs and the molecular mechanisms involved. CFM-1, -4, and -5 inhibited NB cell growth, in vitro, independent of their p53 and MYCN status. CFM-4 and -5 induced apoptosis in NB cells in part by activating pro-apoptotic stress-activated kinases (SAPKs) p38 and JNK, stimulating CARP-1 expression and cleavage of PARP1, while promoting loss of the oncogenes C and N-myc as well as mitotic cyclin B1. Treatments of NB cells with CFM-4 or -5 also resulted in loss of Inhibitory κB (IκB) α and β proteins. Micro-RNA profiling revealed upregulation of XIAP-targeting miR513a-3p in CFM-4-treated NB, mesothelioma, and breast cancer cells. Moreover, exposure of NB and breast cancer cells to CFM-4 or -5 resulted in diminished expression of anti-apoptotic XIAP1, cIAP1, and Survivin proteins. Expression of anti-miR513a-5p or miR513a-5p mimic, however, interfered with or enhanced, respectively, the breast cancer cell growth inhibition by CFM-4. CFMs also impacted biological properties of the NB cells by blocking their abilities to migrate, form colonies in suspension, and invade through the matrix-coated membranes. Our studies indicate anti-NB properties of CFM-4 and 5, and suggest that these CFMs and/or their future analogs have potential as anti-NB agents.

Dithiocarbamate compound Disulfiram (DSF) that binds with copper and functions as an inhibitor of aldehyde dehydrogenase is a Food and Drug Administration approved agent for treatment of alcoholism. Copper complexed DSF (DSF-Cu) also possesses anti-tumor and chemosensitizing properties; however, its molecular mechanisms of action remain unclear. Here we investigated malignant pleural mesothelioma (MPM) suppressive effects of DSF-Cu and the molecular mechanisms involved. DSF-Cu inhibited growth of the murine as well as human MPM cells in part by increasing levels of ubiquitinated proteins. DSF-Cu exposure stimulated apoptosis in MPM cells that involved activation of stress-activated protein kinases (SAPKs) p38 and JNK1/2, caspase-3, and cleavage of poly-(ADP-ribose)-polymerase, as well as increased expression of sulfatase 1 and apoptosis transducing CARP-1/CCAR1 protein. Gene-array based analyses revealed that DSF-Cu suppressed cell growth and metastasis-promoting genes including matrix metallopeptidase 3 and 10. DSF inhibited MPM cell growth and survival by upregulating cell cycle inhibitor p27Kip1, IGFBP7, and inhibitors of NF-κB such as ABIN 1 and 2 and Inhibitory κB (IκB)α and β proteins. DSF-Cu promoted cleavage of vimentin, as well as serine-phosphorylation and lysine-63 linked ubiquitination of podoplanin. Administration of 50 mg/kg DSF-Cu by daily i.p injections inhibited growth of murine MPM cell-derived tumors in vivo. Although podoplanin expression often correlates with metastatic disease and poor prognosis, phosphorylation of serines in cytoplasmic domain of podoplanin has recently been shown to interfere with cellular motility and migration signaling. Post-translational modification of podoplanin and cleavage of vimentin by DSF-Cu underscore a metastasis inhibitory property of this agent and together with our in vivo studies underscore its potential as an anti-MPM agent.

Medulloblastomas (MBs) constitute an aggressive class of intracranial pediatric tumors. Current multimodality treatments for MBs include surgery, ionizing radiation, and chemotherapy. Toxic side effects of therapies coupled with high incidence of recurrence and the metastatic spread warrant development of more effective, less toxic therapies for this disease. CARP-1/CCAR1 is a peri-nuclear phospho-protein that is a co-activator of the cell cycle regulatory anaphase promoting complex/cyclosome (APC/C) E3 ligase. CARP-1 functional mimetics (CFMs) are a novel class of small molecule compounds that interfere with CARP-1 binding with APC/C subunit APC-2, and suppress growth of a variety of cancer cells in part by promoting apoptosis. Here we investigated MB growth inhibitory potential of the CFMs and found that CFM-4 inhibits growth of MB cells in part by inducing CARP-1 expression, promoting PARP cleavage, activating pro-apoptotic stress-activated protein kinases (SAPK) p38 and JNK, and apoptosis. Gene-array-based analysis of the CFM-4-treated Daoy MB cells indicated down-regulation of a number of key cell growth and metastasis-promoting genes including cell motility regulating small GTP binding protein p21Rac1, and extracellular matrix metallopeptidase (MMP)-10. Moreover, CFM-4 treatment stimulated expression of a number of molecules such as neurotrophin (NTF)3, and NF-κB signaling inhibitors ABIN1 and 2 proteins. Overexpression of NTF3 resulted in reduced MB cell viability while knock-down of NTF3 interfered with CFM-4-dependent loss of viability. CFMs also attenuated biological properties of the MB cells by blocking their abilities to migrate, form colonies in suspension, and invade through the matrix-coated membranes. Together our data support anti-MB properties of CFM-4, and provide a proof-of-concept basis for further development of CFMs as potential anti-cancer agents for MBs.

OBJECTIVES: This study was conducted to evaluate the performance of World Health Organisation (WHO) verbal autopsy tool in determining major causes of neonatal deaths. METHODS: From a tertiary care hospital and a government multispecialty hospital, the attending paediatricians ascertained a clinical cause of death for 371 neonatal deaths. Trained field workers conducted verbal autopsy (VA) interviews. Two independent paediatricians, who had no access to the clinical information, assigned cause of death as per verbal autopsy. Analysis was based on 313 cases in which both clinical diagnosis and VA diagnosis was obtained. FINDINGS: As per the clinical diagnosis, four most common causes of neonatal deaths were sepsis (29.1%), preterm birth (27.8%), birth asphyxia (27.2%), and congenital anomalies (11.5%). Cause specific mortality fractions by VA diagnosis were statistically similar to those obtained by clinical diagnosis except for birth asphyxia (16.3%). Diagnostic accuracy of verbal autopsy diagnosis against clinical diagnosis ranged from 78% to 92% in ascertaining different underlying causes of death. Area under the Receiver-Operator Characteristics curve (95% confidence interval) was 0.75 (0.69-0.80) for sepsis, 0.74 (0.68-0.80) for preterm birth, 0.73 (0.65-0.82) for congenital anomaly and 0.70 (0.64-0.75) for birth asphyxia. Kappa for all four causes was moderate (0.46-0.55). INTERPRETATION: The WHO verbal autopsy tools can provide reasonably good estimates of predominant causes of neonatal deaths in countries where neonatal mortality is high. Caution is required to interpret cause specific mortality fraction (CSMF) for birth asphyxia by VA because it is likely to be an underestimate.

Innate immune recognition is the first line of host defense against invading microorganisms. It is a based on the detection, by pattern recognition receptors (PRRs), of invariant molecular signatures that are unique to microorganisms. TLR2 is a PRR that plays a major role in the detection of Gram-positive bacteria by recognizing cell envelope lipid-linked polymers, also called macroamphiphiles, such as lipoproteins, lipoteichoic acids and mycobacterial lipoglycans. These microbe-associated molecular patterns (MAMPs) display a structure based on a lipid anchor, being either an acylated cysteine, a glycosylated diacylglycerol or a mannosyl-phosphatidylinositol respectively, and having in common a diacylglyceryl moiety. A fourth class of macroamphiphile, namely lipoglycans, whose lipid anchor is made, as for lipoteichoic acids, of a glycosylated diacylglycerol unit rather than a mannosyl-phosphatidylinositol, is found in Gram-positive bacteria and produced by certain Actinobacteria, including Micrococcus luteus, Stomatococcus mucilaginosus and Corynebacterium glutamicum. We report here that these alternative lipoglycans are also recognized by TLR2 and that they stimulate TLR2-dependant cytokine production, including IL-8, TNF-α and IL-6, and cell surface co-stimulatory molecule CD40 expression by a human macrophage cell line. However, they differ by their co-receptor requirement and the magnitude of the innate immune response they elicit. M. luteus and S. mucilaginosus lipoglycans require TLR1 for recognition by TLR2 and induce stronger responses than C. glutamicum lipoglycan, sensing of which by TLR2 is dependent on TLR6. These results expand the repertoire of MAMPs recognized by TLR2 to lipoglycans based on a glycosylated diacylglycerol lipid anchor and reinforce the paradigm that macroamphiphiles based on such an anchor, including lipoteichoic acids and alternative lipoglycans, induce TLR2-dependant innate immune responses.

The methyltransferase enzyme (MTase), which catalyzes the transfer of a methyl group from S-adenosyl-methionine (AdoMet) to viral RNA, and generates S-adenosyl-homocysteine (AdoHcy) as a by-product, is essential for the life cycle of many significant human pathogen flaviviruses. Here we investigated inhibition of the flavivirus MTase by several AdoHcy-derivatives. Unexpectedly we found that AdoHcy itself barely inhibits the flavivirus MTase activities, even at high concentrations. AdoHcy was also shown to not inhibit virus growth in cell-culture. Binding studies confirmed that AdoHcy has a much lower binding affinity for the MTase than either the AdoMet co-factor, or the natural AdoMet analog inhibitor sinefungin (SIN). While AdoMet is a positively charged molecule, SIN is similar to AdoHcy in being uncharged, and only has an additional amine group that can make extra electrostatic contacts with the MTase. Molecular Mechanics Poisson-Boltzmann Sovation Area analysis on AdoHcy and SIN binding to the MTase suggests that the stronger binding of SIN may not be directly due to interactions of this amine group, but due to distributed differences in SIN binding resulting from its presence. The results suggest that better MTase inhibitors could be designed by using SIN as a scaffold rather than AdoHcy.

Metastatic melanoma remains one of the most invasive and highly drug resistant cancers. The over expression of anti-apoptotic protein Mcl-1 has been associated with inferior survival, poor prognosis and chemoresistance of malignant melanoma. A BH3 mimetic, ABT-737, has demonstrated efficacy in several forms of cancers. However, the efficacy of ABT-737 depends on Mcl-1. Because the over expression of Mcl-1 is frequently observed in melanoma, specifically targeting of Mcl-1 may overcome the resistance of ABT-737. In this study, we investigated the effects of Maritoclax, a novel Mcl-1-selective inhibitor, alone and in combination with ABT-737, on the survival of human melanoma cells.For cell viability assessment we performed MTT assay. Apoptosis was determined using western blot and flow cytometric analysis.The treatment of Maritoclax reduced the cell viability of melanoma cells with an IC50 of between 2.2-5.0 µM. Further, treatment of melanoma cells with Maritoclax showed significant decrease in Mcl-1 expression. We found that Maritoclax was able to induce apoptosis in melanoma cells in a caspase-dependent manner. Moreover, Maritoclax induced Mcl-1 degradation via the proteasome system, which was associated with its pro-apoptotic activity. We also found that Maritoclax treatment increased mitochondrial translocation of Bim and Bmf. Importantly, Maritoclax markedly enhanced the efficacy of ABT-737 against melanoma cells in both two- and three-dimensional spheroids.Taken together, these results suggest that targeting of Mcl-1 by Maritoclax may represent a new therapeutic strategy for melanoma treatment that warrants further investigation as a single therapy or in combination with other agents such as Bcl-2 inhibitors.

Live-attenuated SIV vaccines (LAVs) have been the most effective to date in preventing or partially controlling infection by wild-type SIV in non-human primate models of HIV-1 transmission to women acting by mechanisms of protection that are not well understood. To gain insights into mechanisms of protection by LAVs that could aid development of effective vaccines to prevent HIV-1 transmission to women, we used in situ tetramer staining to determine whether increased densities or changes in the local distribution of SIV-specific CD8 T cells correlated with the maturation of SIVΔnef vaccine-induced protection prior to and after intra-vaginal challenge with wild-type SIVmac251. We evaluated the immunodominant Mamu-A1*001:01/Gag (CM9) and Mamu-A1*001:01/Tat (SL8) epitope response in genital and lymphoid tissues, and found that tetramer+ cells were present at all time points examined. In the cervical vaginal tissues, most tetramer+ cells were distributed diffusely throughout the lamina propria or co-localized with other CD8 T cells within lymphoid aggregates. The distribution and densities of the tetramer+ cells at the portal of entry did not correlate with the maturation of protection or change after challenge. Given these findings, we discuss the possibility that changes in other aspects of the immune system, including the quality of the resident population of virus-specific effector CD8 T cells could contribute to maturation of protection, as well as the potential for vaccine strategies that further increase the size and quality of this effector population to prevent HIV-1 transmission.

Cytochrome P450 is a superfamily of membrane-bound hemoprotein that gets involved with the degradation of xenobiotics and internal metabolites. Accumulated body of evidence indicates that phospholipids play a crucial role in determining the enzymatic activity of cytochrome P450 in the microenvironment by modulating its structure during detoxification; however, the structure-function relationship of cytochrome P4501A, a family of enzymes responsible for degrading lipophilic aromatic hydrocarbons, is still not well defined. Inducibility of cytochrome P4501A in cultured catfish hepatocytes in response to carbofuran, a widely used pesticide around the world, was studied earlier in our laboratory. In this present investigation, we observed that treating catfish with carbofuran augmented total phospholipid in the liver. We examined the role of phospholipid on the of cytochrome P4501A-marker enzyme which is known as ethoxyresorufin-O-deethylase (EROD) in the context of structure and function. We purified the carbofuran-induced cytochrome P4501A protein from catfish liver. Subsequently, we examined the enzymatic activity of purified P4501A protein in the presence of phospholipid, and studied how the structure of purified protein was influenced in the phospholipid environment. Membrane phospholipid appeared to accelerate the enzymatic activity of EROD by changing its structural conformation and thus controlling the detoxification of xenobiotics. Our study revealed the missing link of how the cytochrome P450 restores its enzymatic activity by changing its structural conformation in the phospholipid microenvironment.

The medicinal plant Withania somnifera has been used for over centuries in Indian Ayurvedic Medicine to treat a wide spectrum of disorders. Withaferin A (WA), a bioactive compound that is isolated from this plant, has anti-inflammatory, immuno-modulatory, anti-angiogenic, and anti-cancer properties. Here we investigated malignant pleural mesothelioma (MPM) suppressive effects of WA and the molecular mechanisms involved. WA inhibited growth of the murine as well as patient-derived MPM cells in part by decreasing the chymotryptic activity of the proteasome that resulted in increased levels of ubiquitinated proteins and pro-apoptotic proteasome target proteins (p21, Bax, IκBα). WA suppression of MPM growth also involved elevated apoptosis as evidenced by activation of pro-apoptotic p38 stress activated protein kinase (SAPK) and caspase-3, elevated levels of pro-apoptotic Bax protein and cleavage of poly-(ADP-ribose)-polymerase (PARP). Our studies including gene-array based analyses further revealed that WA suppressed a number of cell growth and metastasis-promoting genes including c-myc. WA treatments also stimulated expression of the cell cycle and apoptosis regulatory protein (CARP)-1/CCAR1, a novel transducer of cell growth signaling. Knock-down of CARP-1, on the other hand, interfered with MPM growth inhibitory effects of WA. Intra-peritoneal administration of 5 mg/kg WA daily inhibited growth of murine MPM cell-derived tumors in vivo in part by inhibiting proteasome activity and stimulating apoptosis. Together our in vitro and in vivo studies suggest that WA suppresses MPM growth by targeting multiple pathways that include blockage of proteasome activity and stimulation of apoptosis, and thus holds promise as an anti-MPM agent.

The DNA alkylating agent temozolomide (TMZ) is widely used in the treatment of human malignancies such as glioma and melanoma. On the basis of previous structure-activity studies, we recently synthesized a new TMZ selenium analog by rationally introducing an N-ethylselenocyanate extension to the amide functionality in TMZ structure.This TMZ-Se analog showed a superior cytotoxicity to TMZ in human glioma and melanoma cells and a more potent tumor-inhibiting activity than TMZ in mouse glioma and melanoma xenograft model. TMZ-Se was also effective against a TMZ-resistant glioma cell line. To explore the mechanism underlying the superior antitumor activity of TMZ-Se, we compared the effects of TMZ and TMZ-Se on apoptosis and autophagy. Apoptosis was significantly increased in tumor cells treated with TMZ-Se in comparison to those treated with TMZ. TMZ-Se also triggered greater autophagic response, as compared with TMZ, and suppressing autophagy partly rescued cell death induced by TMZ-Se, indicating that TMZ-Se-triggered autophagy contributed to cell death. Although mRNA level of the key autophagy gene, Beclin 1, was increased, Beclin 1 protein was down-regulated in the cells treated with TMZ-Se. The decrease in Beclin 1 following TMZ-Se treatment were rescued by the calpain inhibitors and the calpain-mediated degradation of Beclin1 had no effect on autophagy but promoted apoptosis in cells treated with TMZ-Se.Our study indicates that incorporation of Se into TMZ can render greater potency to this chemotherapeutic drug.

The objective of the research was to understand the structural determinants governing protein aggregation into inclusion bodies during expression of recombinant proteins in Escherichia coli. Recombinant human growth hormone (hGH) and asparaginase were expressed as inclusion bodies in E.coli and the kinetics of aggregate formation was analyzed in details. Asparaginase inclusion bodies were of smaller size (200 nm) and the size of the aggregates did not increase with induction time. In contrast, the seeding and growth behavior of hGH inclusion bodies were found to be sequential, kinetically stable and the aggregate size increased from 200 to 800 nm with induction time. Human growth hormone inclusion bodies showed higher resistance to denaturants and proteinase K degradation in comparison to those of asparaginase inclusion bodies. Asparaginase inclusion bodies were completely solubilized at 2-3 M urea concentration and could be refolded into active protein, whereas 7 M urea was required for complete solubilization of hGH inclusion bodies. Both hGH and asparaginase inclusion bodies showed binding with amyloid specific dyes. In spite of its low β-sheet content, binding with dyes was more prominent in case of hGH inclusion bodies than that of asparaginase. Arrangements of protein molecules present in the surface as well as in the core of inclusion bodies were similar. Hydrophobic interactions between partially folded amphiphillic and hydrophobic alpha-helices were found to be one of the main determinants of hGH inclusion body formation. Aggregation behavior of the protein molecules decides the nature and properties of inclusion bodies.

BACKGROUND: Listeria monocytogenes, an intracellular foodborne pathogen, infects immunocompromised hosts. The primary route of transmission is through contaminated food. In the gastrointestinal tract, it traverses the epithelial barrier through intracellular or paracellular routes. Strategies to prevent L. monocytogenes entry can potentially minimize infection in high-risk populations. Listeria adhesion protein (LAP) aids L. monocytogenes in crossing epithelial barriers via the paracellular route. The use of recombinant probiotic bacteria expressing LAP would aid targeted clearance of Listeria from the gut and protect high-risk populations from infection. METHODOLOGY/PRINCIPAL FINDINGS: The objective was to investigate the ability of probiotic bacteria or LAP-expressing recombinant probiotic Lactobacillus paracasei (Lbp(LAP)) to prevent L. monocytogenes adhesion, invasion, and transwell-based transepithelial translocation in a Caco-2 cell culture model. Several wild type probiotic bacteria showed strong adhesion to Caco-2 cells but none effectively prevented L. monocytogenes infection. Pre-exposure to Lbp(LAP) for 1, 4, 15, or 24 h significantly (P

Structural variation in the neurexin-1 (NRXN1) gene increases risk for both autism spectrum disorders (ASD) and schizophrenia. However, the manner in which NRXN1 gene variation may be related to brain morphology to confer risk for ASD or schizophrenia is unknown.53 healthy individuals between 18-59 years of age were genotyped at 11 single nucleotide polymorphisms of the NRXN1 gene. All subjects received structural MRI scans, which were processed to determine cortical gray and white matter lobar volumes, and volumes of striatal and thalamic structures. Each subject's sensorimotor function was also assessed. The general linear model was used to calculate the influence of genetic variation on neural and cognitive phenotypes. Finally, in silico analysis was conducted to assess potential functional relevance of any polymorphisms associated with brain measures. A polymorphism located in the 3' untranslated region of NRXN1 significantly influenced white matter volumes in whole brain and frontal lobes after correcting for total brain volume, age and multiple comparisons. Follow-up in silico analysis revealed that this SNP is a putative microRNA binding site that may be of functional significance in regulating NRXN1 expression. This variant also influenced sensorimotor performance, a neurocognitive function impaired in both ASD and schizophrenia.Our findings demonstrate that the NRXN1 gene, a vulnerability gene for SCZ and ASD, influences brain structure and cognitive function susceptible in both disorders. In conjunction with our in silico results, our findings provide evidence for a neural and cognitive susceptibility mechanism by which the NRXN1 gene confers risk for both schizophrenia and ASD.

To determine the effect of Ultraviolet-A collagen cross-linking with hypo-osmolar and iso-osmolar riboflavin solutions on stromal collagen ultrastructure in normal and keratoconus ex vivo human corneas.Using small-angle X-ray scattering, measurements of collagen D-periodicity, fibril diameter and interfibrillar spacing were made at 1 mm intervals across six normal post-mortem corneas (two above physiological hydration (swollen) and four below (unswollen)) and two post-transplant keratoconus corneal buttons (one swollen; one unswollen), before and after hypo-osmolar cross-linking. The same parameters were measured in three other unswollen normal corneas before and after iso-osmolar cross-linking and in three pairs of swollen normal corneas, in which only the left was cross-linked (with iso-osmolar riboflavin).Hypo-osmolar cross-linking resulted in an increase in corneal hydration in all corneas. In the keratoconus corneas and unswollen normal corneas, this was accompanied by an increase in collagen interfibrillar spacing (p

Listeria adhesion protein (LAP) is a housekeeping bifunctional enzyme consisting of N-terminal acetaldehyde dehydrogenase (ALDH) and C-terminal alcohol dehydrogenase (ADH). It aids Listeria monocytogenes in crossing the epithelial barrier through a paracellular route by interacting with its host receptor, heat shock protein 60 (Hsp60). To gain insight into the binding interaction between LAP and Hsp60, LAP subdomain(s) participating in the Hsp60 interaction were investigated.Using a ModBase structural model, LAP was divided into 4 putative subdomains: the ALDH region contains N1 (Met(1)-Pro(223)) and N2 (Gly(224)-Gly(411)), and the ADH region contains C1 (Gly(412)-Val(648)) and C2 (Pro(649)-Val(866)). Each subdomain was cloned and overexpressed in Escherichia coli and purified. Purified subdomains were used in ligand overlay, immunofluorescence, and bead-based epithelial cell adhesion assays to analyze each domain's affinity toward Hsp60 protein or human ileocecal epithelial HCT-8 cells.The N2 subdomain exhibited the greatest affinity for Hsp60 with a K(D) of 9.50±2.6 nM. The K(D) of full-length LAP (7.2±0.5 nM) to Hsp60 was comparable to the N2 value. Microspheres (1 µm diameter) coated with N2 subdomain showed significantly (P

The use of neo-adjuvant chemotherapy in treating osteosarcoma has improved patients' average 5 year survival rate from 20% to 70% in the past 30 years. However, for patients who progress after chemotherapy, its effectiveness diminishes due to the emergence of multi-drug resistance (MDR) after prolonged therapy.In order to overcome both the dose-limiting side effects of conventional chemotherapeutic agents and the therapeutic failure resulting from MDR, we designed and evaluated a novel drug delivery system for MDR1 siRNA delivery. Novel biocompatible, lipid-modified dextran-based polymeric nanoparticles were used as the platform for MDR1 siRNA delivery; and the efficacy of combination therapy with this system was evaluated. In this study, multi-drug resistant osteosarcoma cell lines (KHOS(R2) and U-2OS(R2)) were treated with the MDR1 siRNA nanocarriers and MDR1 protein (P-gp) expression, drug retention, and immunofluoresence were analyzed. Combination therapy of the MDR1 siRNA loaded nanocarriers with increasing concentrations of doxorubicin was also analyzed. We observed that MDR1 siRNA loaded dextran nanoparticles efficiently suppresses P-gp expression in the drug resistant osteosarcoma cell lines. The results also demonstrated that this approach may be capable of reversing drug resistance by increasing the amount of drug accumulation in MDR cell lines.Lipid-modified dextran-based polymeric nanoparticles are a promising platform for siRNA delivery. Nanocarriers loaded with MDR1 siRNA are a potential treatment strategy for reversing MDR in osteosarcoma.

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is a widely distributed single-stranded DNA parvovirus that has been responsible for major losses in wild and farmed penaeid shrimp populations on the northwestern Pacific coast of Mexico since the early 1990's. IHHNV has been considered a slow-evolving, stable virus because shrimp populations in this region have recovered to pre-epizootic levels, and limited nucleotide variation has been found in a small number of IHHNV isolates studied from this region. To gain insight into IHHNV evolutionary and population dynamics, we analyzed IHHNV capsid protein gene sequences from 89 Penaeus shrimp, along with 14 previously published sequences. Using Bayesian coalescent approaches, we calculated a mean rate of nucleotide substitution for IHHNV that was unexpectedly high (1.39x10(-4) substitutions/site/year) and comparable to that reported for RNA viruses. We found more genetic diversity than previously reported for IHHNV isolates and highly significant subdivision among the viral populations in Mexican waters. Past changes in effective number of infections that we infer from Bayesian skyline plots closely correspond to IHHNV epizootiological historical records. Given the high evolutionary rate and the observed regional isolation of IHHNV in shrimp populations in the Gulf of California, we suggest regular monitoring of wild and farmed shrimp and restriction of shrimp movement as preventative measures for future viral outbreaks.