Obesity Drives STAT-1-Dependent NASH and STAT-3-Dependent HCC.

Obesity is a major driver of cancer, especially hepatocellular carcinoma (HCC). The prevailing view is that non-alcoholic steatohepatitis (NASH) and fibrosis or cirrhosis are required for HCC in obesity. Here, we report that NASH and fibrosis and HCC in obesity can be dissociated. We show that the oxidative hepatic environment in obesity inactivates the STAT-1 and STAT-3 phosphatase T cell protein tyrosine phosphatase (TCPTP) and increases STAT-1 and STAT-3 signaling. TCPTP deletion in hepatocytes promoted T cell recruitment and ensuing NASH and fibrosis as well as HCC in obese C57BL/6 mice that normally do not develop NASH and fibrosis or HCC. Attenuating the enhanced STAT-1 signaling prevented T cell recruitment and NASH and fibrosis but did not prevent HCC. By contrast, correcting STAT-3 signaling prevented HCC without affecting NASH and fibrosis. TCPTP-deletion in hepatocytes also markedly accelerated HCC in mice treated with a chemical carcinogen that promotes HCC without NASH and fibrosis. Our studies reveal how obesity-associated hepatic oxidative stress can independently contribute to the pathogenesis of NASH, fibrosis, and HCC.

Dysregulation of Grainyhead-like 3 expression causes widespread developmental defects.

BACKGROUND: The gene encoding the transcription factor, Grainyhead-like 3 (Grhl3), plays critical roles in mammalian development and homeostasis. Grhl3-null embryos exhibit thoraco-lumbo-sacral spina bifida and soft-tissue syndactyly. Additional studies reveal that these embryos also exhibit an epidermal proliferation/differentiation imbalance. This manifests as skin barrier defects resulting in peri-natal lethality and defective wound repair. Despite these extensive analyses of Grhl3 loss-of-function models, the consequences of gain-of-function of this gene have been difficult to achieve. RESULTS: In this study, we generated a novel mouse model that expresses Grhl3 from a transgene integrated in the Rosa26 locus on an endogenous Grhl3-null background. Expression of the transgene rescues both the neurulation and skin barrier defects of the knockout mice, allowing survival into adulthood. Despite this, the mice are not normal, exhibiting a range of phenotypes attributable to dysregulated Grhl3 expression. In mice homozygous for the transgene, we observe a severe Shaker-Waltzer phenotype associated with hearing impairment. Micro-CT scanning of the inner ear revealed profound structural alterations underlying these phenotypes. In addition, these mice exhibit other developmental anomalies including hair loss, digit defects, and epidermal dysmorphogenesis. CONCLUSION: Taken together, these findings indicate that diverse developmental processes display low tolerance to dysregulation of Grhl3.

Potential of 3-octanone as a lure and kill agent for control of the Brown garden snail.

The brown garden snail (Cornu aspersum) is a major agricultural pest, causing damage to a wide range of economically important crops. Withdrawal or restricted use of pollutant molluscicides like metaldehyde has prompted a search for more benign control products. This study investigated the response of snails to 3-octanone; a volatile organic compound (VOCs) produced by the insect pathogenic fungus Metarhizium brunneum. Concentrations of 1 - 1000 ppm of 3-octanone were first assessed in laboratory choice assays to determine behavioural response. Repellent activity was found at 1000 ppm whereas attractance was found for the lower concentrations of 1, 10 and 100 ppm. These three concentrations of 3-octanone were carried forward in field evaluations to assess potential for use in "lure and kill" strategies. The highest concentration (100 ppm) was the most attractive to the snails but also the most lethal. Even at the lowest concentration this compound proved toxic effects making 3-octanone an excellent candidate for the development as a snail attractant and molluscicide.

Inhibition of retinoic acid signaling impairs cranial and spinal neural tube closure in mice lacking the Grainyhead-like 3 transcription factor.

Neural tube closure is a dynamic morphogenic event in early embryonic development. Perturbations of this process through either environmental or genetic factors induce the severe congenital malformations known collectively as neural tube defects (NTDs). Deficiencies in maternal folate intake have long been associated with NTDs, as have mutations in critical neurulation genes that include the Grainyhead-like 3 (Grhl3) gene. Mice lacking this gene exhibit fully penetrant thoraco-lumbo-sacral spina bifida and a low incidence of exencephaly. Previous studies have shown that exposure of pregnant mice carrying hypomorphic Grhl3 alleles to exogenous retinoic acid (RA) increases the incidence and severity of NTDs in their offspring. Here, we demonstrate that inhibition of RA signaling using a high affinity pan-RA receptor antagonist administered to pregnant mice at E7.5 induces fully penetrant exencephaly and more severe spina bifida in Grhl3-null mice. Later administration, although prior to neural tube closure has no effect. Similarly, blockade of RA in the context of reduced expression of Grhl2, a related gene known to induce NTDs, has no effect. Taken together, these findings provide new insights into the complexities of the interplay between RA signaling and Grhl3-induced neurulation.

Telomere length de novo assembly of all 7 chromosomes and mitogenome sequencing of the model entomopathogenic fungus, Metarhizium brunneum, by means of a novel assembly pipeline.

BACKGROUND: More accurate and complete reference genomes have improved understanding of gene function, biology, and evolutionary mechanisms. Hybrid genome assembly approaches leverage benefits of both long, relatively error-prone reads from third-generation sequencing technologies and short, accurate reads from second-generation sequencing technologies, to produce more accurate and contiguous de novo genome assemblies in comparison to using either technology independently. In this study, we present a novel hybrid assembly pipeline that allowed for both mitogenome de novo assembly and telomere length de novo assembly of all 7 chromosomes of the model entomopathogenic fungus, Metarhizium brunneum. RESULTS: The improved assembly allowed for better ab initio gene prediction and a more BUSCO complete proteome set has been generated in comparison to the eight current NCBI reference Metarhizium spp. genomes. Remarkably, we note that including the mitogenome in ab initio gene prediction training improved overall gene prediction. The assembly was further validated by comparing contig assembly agreement across various assemblers, assessing the assembly performance of each tool. Genomic synteny and orthologous protein clusters were compared between Metarhizium brunneum and three other Hypocreales species with complete genomes, identifying core proteins, and listing orthologous protein clusters shared uniquely between the two entomopathogenic fungal species, so as to further facilitate the understanding of molecular mechanisms underpinning fungal-insect pathogenesis. CONCLUSIONS: The novel assembly pipeline may be used for other haploid fungal species, facilitating the need to produce high-quality reference fungal genomes, leading to better understanding of fungal genomic evolution, chromosome structuring and gene regulation.

Grainyhead-like transcription factors: guardians of the skin barrier.

There has been selective pressure to maintain a skin barrier since terrestrial animals evolved 360 million years ago. These animals acquired an unique integumentary system with a keratinized, stratified, squamous epithelium surface barrier. The barrier protects against dehydration and entry of microbes and toxins. The skin barrier centres on the stratum corneum layer of the epidermis and consists of cornified envelopes cemented by the intercorneocyte lipid matrix. Multiple components of the barrier undergo cross-linking by transglutaminase (TGM) enzymes, while keratins provide additional mechanical strength. Cellular tight junctions also are crucial for barrier integrity. The grainyhead-like (GRHL) transcription factors regulate the formation and maintenance of the integument in diverse species. GRHL3 is essential for formation of the skin barrier during embryonic development, whereas GRHL1 maintains the skin barrier postnatally. This is achieved by transactivation of Tgm1 and Tgm5, respectively. In addition to its barrier function, GRHL3 plays key roles in wound repair and as an epidermal tumour suppressor. In its former role, GRHL3 activates the planar cell polarity signalling pathway to mediate wound healing by providing directional migration cues. In squamous epithelium, GRHL3 regulates the balance between proliferation and differentiation, and its loss induces squamous cell carcinoma (SCC). In the skin, this is mediated through increased expression of MIR21, which reduces the expression levels of GRHL3 and its direct target, PTEN, leading to activation of the PI3K-AKT signalling pathway. These data position the GRHL family as master regulators of epidermal homeostasis across a vast gulf of evolutionary history.

Il y a eu une pression de sélection pour maintenir la barrière cutanée depuis l'évolution des animaux terrestres pendant 360 millions d'années. Ces animaux ont acquis un système tégumentaire unique avec un épithélium squameux, stratifié, kératinisé comme barrière de surface. La barrière protège contre la déshydratation et l'entée de microbes et de toxines. La barrière cutanée est centrée sur la couche du stratum corneum de l'épiderme et consiste en des enveloppes cimentées par une matrice lipidique intercornéocytaire. Les composants multiples de la barrière subissent des remaniements par les enzymes transglutaminases (TGM) tandis que la kératine fournit un soutien mécanique supplémentaire. Les jonctions serrées cellulaires jouent aussi un rôle crucial pour l'intégrité de barrière. Les facteurs de transcriptions GRHL (grainyhead-like) régulent la formation et le maintien du tégument dans différentes espèces. GRHL3 est essentielle pour la formation de la barrière cutanée au cours du développement embryonnaire tandis que GRHL1 maintient la barrière cutanée après la naissance. Ceci est permis respectivement par transactivation de Tgm1 et Tgm5. En plus de cette fonction barrière, GRHL3 joue un rôle clé dans la cicatrisation et en tant que suppresseur de tumeur épidermique. Dans ses rôles principaux, GRHL3 active la voie de signal de polarité cellulaire plane pour soutenir la cicatrisation en fournissant des repaires directionnels de migration. Dans les épithéliums squameux, GRHL3 régule la balance entre prolifération et différentiation, et sa perte induit le carcinome épidermoïde (SCC). Dans la peau ceci est médié par une augmentation de l'expression de MIR21, qui réduit le niveau d'expression de GRHL3 et sa cible directe, PTEN, menant à l'activation de la voie de signal PI3K-AKT. Ces données positionnent la famille GRHL comme régulatrice majeure de l'homéostasie épidermique à travers le vaste gouffre de l'histoire de l'évolution.

Ha habido una presión selectiva para mantener una barrera cutánea desde que los animales terrestres evolucionaron hace 360 ​​millones de años. Estos animales adquirieron un sistema tegumentario único con una barrera superficial de epitelio escamoso estratificado queratinizado. La barrera protege contra la deshidratación y la entrada de microbios y toxinas. La barrera cutánea se centra en la capa de estrato córneo de la epidermis y consta de membranas cornificadas cementadas por una matriz lipídica intercorneocitaria. Múltiples componentes de la barrera se unen por la actividad de enzimas transglutaminasas (TGM), mientras que las queratinas proporcionan resistencia mecánica adicional. Las uniones celulares estrechas también son cruciales para la integridad de la barrera. Los factores de transcripción similares a grainyhead (cabeza granulada) (GRHL) regulan la formación y mantenimiento del tegumento en diversas especies. GRHL3 es esencial para la formación de la barrera cutánea durante el desarrollo embrionario, mientras que GRHL1 mantiene la barrera cutánea postnatal. Esto se logra mediante la transactivación de Tgm1 y Tgm5, respectivamente. Además de su función de barrera, GRHL3 juega un papel clave en la reparación de heridas y como supresor de tumores epidérmicos. En su función de cicatrización, GRHL3 activa la vía de señalización de la polaridad celular plana para mediar en la cicatrización de heridas proporcionando señales de migración direccional. En el epitelio escamoso, GRHL3 regula el equilibrio entre la proliferación y la diferenciación, y su pérdida induce el carcinoma de células escamosas (SCC). En la piel, esto está mediado por una mayor expresión de MIR21, que reduce los niveles de expresión de GRHL3 y su sustrato directo, PTEN, lo que lleva a la activación de la vía de señal intracelular PI3K-AKT. Estos datos colocan la familia de factores de transcripción GRHL como reguladores críticos de la homeostasis epidérmica a través de una extensa historia evolutiva.

Tem havido uma pressão seletiva para manter a barreira cutânea desde a evolução dos animais terrestres há 360 milhões de anos. Estes animais adquiriram um sistema tegumentar único com uma barreira de superfície escamosa, estratificada e queratinizada. A barreira protege contra a desidratação e entrada de micróbios e toxinas. A barreira cutânea é centrada na camada do estrato córneo da epiderme e consiste em envelopes cornificados revestidos pela matriz lipídica intercorneocítica. Vários componentes da barreira sofrem ligação cruzada por enzimas transglutaminase (TGM), enquanto as queratinas fornecem resistência mecânica adicional. As junções celulares também são cruciais para a integridade da barreira. Os fatores de transcrição do tipo grainyhead (GRHL) regulam a formação e manutenção do tegumento em diversas espécies. GRHL3 é essencial para a formação da barreira cutânea durante o desenvolvimento embrionário, enquanto GRHL1 mantém a barreira cutânea pós-natal. Isso é obtido pela transativação de Tgm1 e Tgm5, respectivamente. Além de sua função de barreira, GRHL3 desempenha papéis importantes no reparo de feridas e como supressor de tumor epidérmico. Em sua função anterior, GRHL3 ativa a via de sinalização de polaridade celular planar para mediar a cicatrização de feridas, fornecendo pistas de migração direcional. No epitélio escamoso, o GRHL3 regula o equilíbrio entre a proliferação e a diferenciação, e sua perda induz o carcinoma de células escamosas (CCE). Na pele, isso é mediado pelo aumento da expressão de MIR21, que reduz os níveis de expressão de GRHL3 e seu alvo direto, PTEN, levando à ativação da via de sinalização PI3K-AKT. Esses dados posicionam a família GRHL como reguladores mestres da homeostase epidérmica em um vasto abismo da história evolutiva.

Endogenous methicillin resistant staphylococcus aureus as a source of post-operative surgical site infections.

OBJECTIVE: To determine frequency of Endogenous Methicillin Resistant Staphylococcus aureus (MRSA) in pre-operative patients and its frequency in Surgical Site Infections (SSIs) post operatively. METHODS: It was a descriptive cross sectional conducted at Department of Microbiology, Fauji Foundation Hospital Rawalpindi (FFH), Pakistan. Samples were collected from 1st November-31st May 2018. Total 75 samples were collected during the period. Consecutive non-probability sampling technique was utilized. Specimens were collected from nose, axilla and groin of preoperative patients. Methicillin Resistant Staphylococcus aureus was identified if only isolated from these sites. Patients were followed till his/her discharge from the hospital and if they developed infection post operatively, pus specimen from infected site was also collected and identified. RESULTS: Out of 75 specimen preoperatively, 11(14.7%) were identified as endogenous MRSA. From these, 33(44%) developed Surgical site infections (SSIs), among them 19(57%) were MRSA (09 endogenous, 10 exogenous), 7(21.2%) were Escherichia coli, 3(9.1%) were Klebsiella pneumoniae, 3(9.1%) were Enterococcus faecalis and 1(3%) was Methicillin Sensitive Staphylococcus aureus. CONCLUSIONS: The results of this study determined that Endogenous Methicillin Resistant Staphylococcus aureus (MRSA) could be isolated from patients going for surgery if microbiological screening was done at the time of admission. This could prevent patients from Surgical Site Infection Post operatively by these endogenous MRSA. This search and wipe out strategy is able to curtail the events of outbreak, reduce hospital stay and decrease budget of the hospital by providing guidance in choice of empirical therapy for infection.

Another case of mistaken identity? Vaccinia virus in another live Camelpox vaccine.

Camelpox virus is the causative agent of Camelpox, a highly contagious disease of camels. A high passage Camelpox virus strain has previously been reported to contain several genes which more closely resemble Vaccinia, a virus species with no known natural host, encompassing various strains that show high inter-strain genomic variation. In this study, we demonstrate that yet another high passage, live attenuated vaccine, comprising a different strain of Camelpox virus, contains genomic sequences that match a differing strain of Vaccinia virus. These results are discussed in the context of hypotheses put forward to explain the unknown origins of Vaccinia virus, suggesting further studies to elucidate evolutionary trajectories of Orthopoxviruses through passaging.

Metarhizium brunneum Blastospore Pathogenesis in Aedes aegypti Larvae: Attack on Several Fronts Accelerates Mortality.

Aedes aegypti is the vector of a wide range of diseases (e.g. yellow fever, dengue, Chikungunya and Zika) which impact on over half the world's population. Entomopathogenic fungi such as Metarhizium anisopliae and Beauveria bassiana have been found to be highly efficacious in killing mosquito larvae but only now are the underlying mechanisms for pathogenesis being elucidated. Recently it was shown that conidia of M. anisopliae caused stress induced mortality in Ae. aegypti larvae, a different mode of pathogenicity to that normally seen in terrestrial hosts. Blastospores constitute a different form of inoculum produced by this fungus when cultured in liquid media and although blastospores are generally considered to be more virulent than conidia no evidence has been presented to explain why. In our study, using a range of biochemical, molecular and microscopy methods, the infection process of Metarhizium brunneum (formerly M. anisopliae) ARSEF 4556 blastospores was investigated. It appears that the blastospores, unlike conidia, readily adhere to and penetrate mosquito larval cuticle. The blastospores are readily ingested by the larvae but unlike the conidia are able infect the insect through the gut and rapidly invade the haemocoel. The fact that pathogenicity related genes were upregulated in blastospores exposed to larvae prior to invasion, suggests the fungus was detecting host derived cues. Similarly, immune and defence genes were upregulated in the host prior to infection suggesting mosquitoes were also able to detect pathogen-derived cues. The hydrophilic blastospores produce copious mucilage, which probably facilitates adhesion to the host but do not appear to depend on production of Pr1, a cuticle degrading subtilisin protease, for penetration since protease inhibitors did not significantly alter blastospore virulence. The fact the blastospores have multiple routes of entry (cuticle and gut) may explain why this form of the inoculum killed Ae. aegypti larvae in a relatively short time (12-24hrs), significantly quicker than when larvae were exposed to conidia. This study shows that selecting the appropriate form of inoculum is important for efficacious control of disease vectors such as Ae. aegypti.

Combined use of the entomopathogenic fungus, Metarhizium brunneum, and the mosquito predator, Toxorhynchites brevipalpis, for control of mosquito larvae: Is this a risky biocontrol strategy?

Mosquitoes transmit several diseases, which are of global significance (malaria, dengue, yellow fever, Zika). The geographic range of mosquitoes is increasing due to climate change, tourism and trade. Both conidial and blastospore formulations of the entomopathogenic fungus, Metarhizium brunneum ARSEF 4556, are being investigated as mosquito larvicides. However, concerns have been raised over possible non-target impacts to arthropod mosquito predators such as larvae of Toxorhynchites brevipalpis which feed on larvae of mosquito vector species. Laboratory-based, small container bioassays showed, that T. bevipalpis larvae are susceptible to relatively high concentrations (i.e. ≥107 spores ml-1) of inoculum with blastospores being significantly more virulent than conidia. At lower concentrations (e.g. <107 spores ml-1), it appears that M. brunneum complements T. brevipalpis resulting in higher control than if either agent was used alone. At a concentration of 105 spores ml-1, the LT50 of for conidia and blastospores alone was 5.64 days (95% CI: 4.79-6.49 days) and 3.89 days (95% CI: 3.53-4.25 days), respectively. In combination with T. brevipalpis, this was reduced to 3.15 days (95% CI: 2.82-3.48 days) and 2.82 days (95% CI: 2.55-3.08 days). Here, combined treatment with the fungus and predator was beneficial but weaker than additive. At 107 and 108 blastospores ml-1, mosquito larval mortality was mostly due to the fungal pathogen when the predator was combined with blastospores. However, with conidia, the effects of combined treatment were additive/synergistic at these high concentrations. Optimisation of fungal concentration and formulation will reduce: (1) risk to the predator and (2) application rates and costs of M. brunneum for control of mosquito larvae.

Loss of cargo binding in the human myosin VI deafness mutant (R1166X) leads to increased actin filament binding.

Mutations in myosin VI have been associated with autosomal-recessive (DFNB37) and autosomal-dominant (DFNA22) deafness in humans. Here, we characterise an myosin VI nonsense mutation (R1166X) that was identified in a family with hereditary hearing loss in Pakistan. This mutation leads to the deletion of the C-terminal 120 amino acids of the myosin VI cargo-binding domain, which includes the WWY-binding motif for the adaptor proteins LMTK2, Tom1 as well as Dab2. Interestingly, compromising myosin VI vesicle-binding ability by expressing myosin VI with the R1166X mutation or with single point mutations in the adaptor-binding sites leads to increased F-actin binding of this myosin in vitro and in vivo As our results highlight the importance of cargo attachment for regulating actin binding to the motor domain, we perform a detailed characterisation of adaptor protein binding and identify single amino acids within myosin VI required for binding to cargo adaptors. We not only show that the adaptor proteins can directly interact with the cargo-binding tail of myosin VI, but our in vitro studies also suggest that multiple adaptor proteins can bind simultaneously to non-overlapping sites in the myosin VI tail. In conclusion, our characterisation of the human myosin VI deafness mutant (R1166X) suggests that defects in cargo binding may leave myosin VI in a primed/activated state with an increased actin-binding ability.

Chronic obstructive pulmonary disease and asthma-associated Proteobacteria, but not commensal Prevotella spp., promote Toll-like receptor 2-independent lung inflammation and pathology.

Recent studies of healthy human airways have revealed colonization by a distinct commensal bacterial microbiota containing Gram-negative Prevotella spp. However, the immunological properties of these bacteria in the respiratory system remain unknown. Here we compare the innate respiratory immune response to three Gram-negative commensal Prevotella strains (Prevotella melaninogenica, Prevotella nanceiensis and Prevotella salivae) and three Gram-negative pathogenic Proteobacteria known to colonize lungs of patients with chronic obstructive pulmonary disease (COPD) and asthma (Haemophilus influenzae B, non-typeable Haemophilus influenzae and Moraxella catarrhalis). The commensal Prevotella spp. and pathogenic Proteobacteria were found to exhibit intrinsic differences in innate inflammatory capacities on murine lung cells in vitro. In vivo in mice, non-typeable H. influenzae induced severe Toll-like receptor 2 (TLR2)-independent COPD-like inflammation characterized by predominant airway neutrophilia, expression of a neutrophilic cytokine/chemokine profile in lung tissue, and lung immunopathology. In comparison, P. nanceiensis induced a diminished neutrophilic airway inflammation and no detectable lung pathology. Interestingly, the inflammatory airway response to the Gram-negative bacteria P. nanceiensis was completely TLR2-dependent. These findings demonstrate weak inflammatory properties of Gram-negative airway commensal Prevotella spp. that may make colonization by these bacteria tolerable by the respiratory immune system.

Innate responses to putative ancestral hosts: is the attraction of Western flower thrips to pine pollen a result of relict olfactory receptors?

Pollinophagy is widely documented in the order Thysanoptera, with representative individuals from six of the nine divergent families known to feed on pollen. Various pollens of the genus Pinus increase the development time, fecundity, longevity, and settling preference of Western Flower Thrips (WFT), Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae). Certain species of flower thrips discriminate among pollen types, but no studies have elucidated the olfactory cues that play a role in their pollen preferences. In this study, the volatile organic compounds emitted by pollens of the genus Pinus were elucidated. Various chemicals from pollen headspace elicited electrophysiological responses from WFT antennae. The compound (S)-(-)-verbenone, identified in pollen headspace, attracted WFT in a 4-arm olfactometer. This compound has potential for use in integrated pest management programs against the pest. We present the hypothesis that this polyphagous insect may have retained ancestral 'relict' olfactory receptors through the course of evolution, to explain this attraction to pine pollen. This attraction has allowed the insect to find and exploit an unusual nutrient source that significantly increases its fitness. The study demonstrates how fossil record analysis and subsequent evolutionary knowledge can aid in explaining possibilities as to why some insects sense and respond to chemicals that would otherwise seem peculiar to their ecology, allowing insight into the evolutionary forces that may shape insect olfactory systems over time.

Development and laboratory validation of a plant-derived repellent blend, effective against Aedes aegypti [Diptera: Culicidae], Anopheles gambiae [Diptera: Culicidae] and Culex quinquefasciatus [Diptera: Culicidae].

Mosquitoes of the genera Aedes, Anopheles and Culex vector a wide range of pathogens seriously affecting humans and livestock on a global scale. Over-reliance on insecticides and repellents has driven research into alternative, naturally-derived compounds to fulfil the same objectives. Steam distilled extracts of four plants with strong, yet attractive, volatile profiles were initially assessed for repellency in a dual-port olfactometer using Aedes aegypti as the model species. Picea sitchensis was found to be the most repellent, proving comparable to leading products when applied at 100% (p = 1.000). Key components of conifer-derived volatile profiles were then screened via electroantennography before those components eliciting an electrophysiological response were assayed individually in the olfactometer; according to WHO protocol. The most promising 5 were selected for reductive analyses to produce an optimised semiochemical blend. This combination, and a further two variations of the blend, were then progressed to a multi-species analysis using the BG-test whereby bite-attempt frequency on hands was assessed under different repellent treatments; assays were compared between Aedes aegypti, Anopheles gambiae and Culex quinquefasciatus. Efficacy was found against all three species, although it was found that Ae. aegypti was the most susceptible to the repellent, with An. gambiae being the least. Here, a novel, naturally-derived blend is presented with weak spatial repellency, as confirmed in laboratory assays. Further work will be required to assess the full extent of the potential of the products, both in terms of field application and species screening; however, the success of the products developed demonstrate that plant metabolites have great capacity for use in the repellent sector; both to improve upon known compounds and to reduce the usage of toxic products currently on the market.

Novo plant-based mosquito repellent shows promise for exclusion of Aedes mosquitoes from "window" entry.

Mosquitoes threaten over half of the world's population through vectored diseases such as malaria, zika, yellow fever, dengue, and chikungunya. Mosquitoes have a highly developed olfactory system attuned to chemotaxis relating to host-seeking, mating, and oviposition behavior. In this study, we aimed to determine the spatial efficacy of 2 plant-based repellent blends (Blend3 and Blend4 that had previously been found to successfully repel Aedes, Anopheles and Culex mosquitoes in wind tunnel assays) in excluding Aedes aegypti from the window entry. A new cage system was developed for parallel "no-choice" and "choice" olfactometric assays. In the no-choice trial, Blends 3 and 4, as well as commercial products (N, N-diethyl-3-methylbenzamide, p-menthane-3,8-diol [PMD], 3-(N-n-butyl-N-acetyl)-amino-propionic acid ethyl ester, and 2-(2-hydroxyethyl)-1-methylpropylstyrene 1-piperidine carboxylate), were adsorbed into filter papers of different sizes and placed in a window created between 2 attached bug dorms. Then, the number of mosquitoes entering the window was counted through a 6-min period. In choice olfactometric assays, Blends 3, 4, and PMD were adsorbed into filter paper and the number of mosquitoes moving away from Blend 3 and PMD were compared. No-choice assays showed that Blend3 (P < 0.001) and Blend4 (P = 0.0012) were more repellent than the best commercial product PMD. Additionally, while Blend 4 was significantly more repellent than Blend 3 (P = 0.012) in the choice assay, overall, these 2 blends show promise as new repellents for the spatial exclusion of Aedes aegypti from window entry alone or as part of a "push-pull'' strategy.

Comparative genomics of Metarhizium brunneum strains V275 and ARSEF 4556: unraveling intraspecies diversity.

Entomopathogenic fungi belonging to the Order Hypocreales are renowned for their ability to infect and kill insect hosts, while their endophytic mode of life and the beneficial rhizosphere effects on plant hosts have only been recently recognized. Understanding the molecular mechanisms underlying their different lifestyles could optimize their potential as both biocontrol and biofertilizer agents, as well as the wider appreciation of niche plasticity in fungal ecology. This study describes the comprehensive whole genome sequencing and analysis of one of the most effective entomopathogenic and endophytic EPF strains, Metarhizium brunneum V275 (commercially known as Lalguard Met52), achieved through Nanopore and Illumina reads. Comparative genomics for exploring intraspecies variability and analyses of key gene sets were conducted with a second effective EPF strain, M. brunneum ARSEF 4556. The search for strain- or species-specific genes was extended to M. brunneum strain ARSEF 3297 and other species of genus Metarhizium, to identify molecular mechanisms and putative key genome adaptations associated with mode of life differences. Genome size differed significantly, with M. brunneum V275 having the largest genome amongst M. brunneum strains sequenced to date. Genome analyses revealed an abundance of plant-degrading enzymes, plant colonization-associated genes, and intriguing intraspecies variations regarding their predicted secondary metabolic compounds and the number and localization of Transposable Elements. The potential significance of the differences found between closely related endophytic and entomopathogenic fungi, regarding plant growth-promoting and entomopathogenic abilities, are discussed, enhancing our understanding of their diverse functionalities and putative applications in agriculture and ecology.

Single-cell transcriptomics reveals the cellular identity of a novel progenitor population crucial for murine neural tube closure.

Neural tube closure in vertebrates is achieved through a highly dynamic and coordinated series of morphogenic events involving neuroepithelium, surface ectoderm, and neural plate border. Failure of this process in the caudal region causes spina bifida. Grainyhead-like 3 (GRHL3) is an indispensable transcription factor for neural tube closure as constitutive inactivation of the Grhl3 gene in mice leads to fully penetrant spina bifida. Here, through single-cell transcriptomics we show that at E8.5, the time-point preceding mouse neural tube closure, co-expression of Grhl3, Tfap2a, and Tfap2c defines a previously unrecognised progenitor population of surface ectoderm integral for neural tube closure. Deletion of Grhl3 expression in this cell population using a Tfap2a-Cre transgene recapitulates the spina bifida observed in Grhl3-null animals. Moreover, conditional inactivation of Tfap2c expression in Grhl3-expressing neural plate border cells also induces spina bifida. These findings indicate that a specific neural plate border cellular cohort is required for the early-stage neurulation.

Identification of the odorant binding proteins of Western Flower Thrips (Frankliniella occidentalis), characterization and binding analysis of FoccOBP3 with molecular modelling, molecular dynamics simulations and a confirmatory field trial.

Olfactory systems are indispensable for insects as they, including Western Flower Thrips (Frankliniella occidentalis), use olfactory cues for ovipositing and feeding. F. occidentalis use odorant binding proteins (OBPs) to transport semiochemicals to odorant receptors to induce a behavioural response from the sensillum lymph of the insect's antennae. This study identifies four OBPs of F. occidentalis and analyses their expression at three stages of growth: larvae, adult males and adult females. Further, it investigates the presence of conserved motifs and their phylogenetic relationship to other insect species. Moreover, FoccOBP3 was in silico characterized to analyse its structure along with molecular docking and molecular dynamics simulations to understand its binding with semiochemicals of F. occidentalis. Molecular docking revealed the interactions of methyl isonicotinate, p-anisaldehyde and (S)-(-)-verbenone with FoccOBP3. Moreover, molecular dynamics simulations showed bonding stability of these ligands with FoccOBP3, and field trials validated that Lurem TR (commercial product) and p-anisaldehyde had greater attraction as compared to (S)-(-)-verbenone, given the compound's binding with FoccOBP3. The current study helps in understanding the tertiary structure and interaction of FoccOBP3 with lures using computational and field data and will help in the identification of novel lures of insects in the future, given the importance of binding with OBPs.Communicated by Ramaswamy H. Sarma.

Entomopathogenic Fungi in Biological Plant Protection: The Machinery of Multicomponent System Interactions.

Plant protection faces a growing number of challenges, partly stemming from intensification of plant cultivation to ensure food security for a rapidly growing global population [...].

Evaluation of Fungal Volatile Organic Compounds for Control the Plant Parasitic Nematode Meloidogyne incognita.

Plant parasitic nematodes are a serious threat to crop production worldwide and their control is extremely challenging. Fungal volatile organic compounds (VOCs) provide an ecofriendly alternative to synthetic nematicides, many of which have been withdrawn due to the risks they pose to humans and the environment. This study investigated the biocidal properties of two fungal VOCs, 1-Octen-3-ol and 3-Octanone, against the widespread root-knot nematode Meloidogyne incognita. Both VOCs proved to be highly toxic to the infective second-stage juveniles (J2) and inhibited hatching. Toxicity was dependent on the dose and period of exposure. The LD50 of 1-Octen-3-ol and 3-Octanone was 3.2 and 4.6 µL, respectively. The LT50 of 1-Octen-3-ol and 3-Octanone was 71.2 and 147.1 min, respectively. Both VOCs were highly toxic but 1-Octen-3-ol was more effective than 3-Octanone. Exposure of M. incognita egg-masses for 48 h at two doses (0.8 and 3.2 µL) of these VOCs showed that 1-Octen-3-ol had significantly greater nematicidal activity (100%) than 3-Octanone (14.7%) and the nematicide metham sodium (6.1%). High levels of reactive oxygen species detected in J2 exposed to 1-Octen-3-ol and 3-Octanone suggest oxidative stress was one factor contributing to mortality and needs to be investigated further.