Identification and analysis of putative tRNA genes in baculovirus genomes.

Transfer RNAs (tRNAs) genes are both coded for and arranged along some viral genomes representing the entire virosphere and seem to play different biological functions during infection, other than transferring the correct amino acid to a growing peptide chain. Baculovirus genome description and annotation has focused mostly on protein-coding genes, microRNA, and homologous regions. Here we carried out a large-scale in silico search for putative tRNA genes in baculovirus genomes. Ninety-six of 257 baculovirus genomes analyzed was found to contain at least one putative tRNA gene. We found great diversity in primary and secondary structure, in location within the genome, in intron presence and size, and in anti-codon identity. In some cases, genes of tRNA-containing genomes were found to have a bias for the codons specified by the tRNAs present in such genomes. Moreover, analysis revealed that most of the putative tRNA genes possessed conserved motifs for tRNA type 2 promoters, including the A-box and B-box motifs with few mismatches from the eukaryotic canonical motifs. From publicly available small RNA deep sequencing datasets of baculovirus-infected insect cells, we found evidence that a putative Autographa californica multiple nucleopolyhedrovirus Gln-tRNA gene was transcribed and modified with the addition of the non-templated 3'-CCA tail found at the end of all tRNAs. Further research is needed to determine the expression and functionality of these viral tRNAs.

A therapeutic combination of two small molecule toxin inhibitors provides broad preclinical efficacy against viper snakebite.

Snakebite is a medical emergency causing high mortality and morbidity in rural tropical communities that typically experience delayed access to unaffordable therapeutics. Viperid snakes are responsible for the majority of envenomings, but extensive interspecific variation in venom composition dictates that different antivenom treatments are used in different parts of the world, resulting in clinical and financial snakebite management challenges. Here, we show that a number of repurposed Phase 2-approved small molecules are capable of broadly neutralizing distinct viper venom bioactivities in vitro by inhibiting different enzymatic toxin families. Furthermore, using murine in vivo models of envenoming, we demonstrate that a single dose of a rationally-selected dual inhibitor combination consisting of marimastat and varespladib prevents murine lethality caused by venom from the most medically-important vipers of Africa, South Asia and Central America. Our findings support the translation of combinations of repurposed small molecule-based toxin inhibitors as broad-spectrum therapeutics for snakebite.

Survival of thermotolerant coliforms in municipal biosolids after application in tropical soil cultivated with Eucalyptus.

The increasing production of biosolids in urban areas has been led to a search for alternative disposal avenues of this residue, which is rich in organic matter and nutrients. Agricultural land application of biosolids, motivated by its fertilizing power, is a widespread practice in many countries, but there are safety and regulatory concerns regarding the presence of pathogens in Class B biosolids. In addition, the scarcity of studies under tropical climate conditions raises questions that impede the agricultural use of this residue in some regions. The objective of this study was to evaluate the survival of thermotolerant coliforms over 12 months (52 weeks) after biosolids application on the surface of a Quartzipsamment neosol cultivated with Eucalyptus. Two different biosolids were studied: those generated by a biological treatment system with complete mixing aeration ponds followed by decantation ponds (Treatment A) and biosolids from a biological treatment system with conventional activated biosolids reactors (Treatment B), both delineated in randomized blocks with four replicates. After application on the forest soil, we estimated an average survival time of 54 weeks for thermotolerant coliforms present in Treatment A biosolids and 93 weeks in Treatment B biosolids. Thermotolerant coliforms persist much longer under tropical climate conditions in Brazil than in comparable studies under temperate climate conditions. This reaffirms the need to carry out studies covering the full range of moisture and temperature regimes in which biosolids are applied as fertilizer.

Strategy for a globally coordinated response to a priority neglected tropical disease: snakebite envenoming

In one of his final essays, statesman and former United Nations secretary general Kofi Annan said, ‘Snakebite is the most important tropical disease you’ve never heard of’. Mr. Annan firmly believed that victims of snakebite envenoming should be recognised and afforded greater efforts at improved prevention, treatment, and rehabilitation. During the last years of his life, he advocated strongly for the World Health Organisation (WHO) and the global community to give greater priority to this disease of poverty and its victims. Snakebite envenoming (SBE) affects as many as 2.7 million people every year, most of whom live in some of the world’s most remote, poorly developed, and politically marginalised tropical communities. With annual mortality of 81,000 to 138,000 and 400,000 surviving victims suffering permanent physical and psychological disabilities, SBE is a disease in urgent need of attention. Like many diseases of poverty, SBE has failed to attract requisite public health policy inclusion and investment for driving sustainable efforts to reduce the medical and societal burden. This is largely due to the demographics of the affected populations and their lack of political voice.

Analgesic effect of morphine and tramadol in standard toxicity assays in mice injected with venom of the snake Bothrops asper.

Routine laboratory animal tests necessary to assess the toxicity of snake venoms and the preclinical neutralizing ability of antivenoms and other inhibitory substances induce significant pain and distress. This has prompted initiatives to introduce the routine use of analgesia. In this study, the analgesic effect of morphine and tramadol was assessed in tests assessing the lethal, hemorrhagic, myotoxic and edema-forming activities of the venom of the viperid snake Bothrops asper. The Mouse Grimace Scale (MGS) and mouse-exploration activity were used to assess pain and its inhibition by the analgesics. Results demonstrate that tests assessing lethality and myotoxicity induce higher levels of pain than assays quantifying hemorrhagic and edema-forming activities. Our observations also indicate that pretreatment of mice with both analgesics, at the doses used, were similarly effective in reducing the MGS magnitude and increase mouse-exploration activity after the administration of B. asper venom. Moreover, the analgesic effect of both drugs was more evident in the myotoxic and lethality assays. Combined with previous observations showing that these analgesics do not alter the extent of toxic effects induced by B. asper venom, our results strongly indicate that the use of analgesia (using either morphine or tramadol) should be considered in the routine assessment of venom toxicity and antivenom efficacy.

Snakebite envenoming.

Snakebite envenoming is a neglected tropical disease that kills >100,000 people and maims >400,000 people every year. Impoverished populations living in the rural tropics are particularly vulnerable; snakebite envenoming perpetuates the cycle of poverty. Snake venoms are complex mixtures of proteins that exert a wide range of toxic actions. The high variability in snake venom composition is responsible for the various clinical manifestations in envenomings, ranging from local tissue damage to potentially life-threatening systemic effects. Intravenous administration of antivenom is the only specific treatment to counteract envenoming. Analgesics, ventilator support, fluid therapy, haemodialysis and antibiotic therapy are also used. Novel therapeutic alternatives based on recombinant antibody technologies and new toxin inhibitors are being explored. Confronting snakebite envenoming at a global level demands the implementation of an integrated intervention strategy involving the WHO, the research community, antivenom manufacturers, regulatory agencies, national and regional health authorities, professional health organizations, international funding agencies, advocacy groups and civil society institutions.

Freeze-dried EchiTAb+ICP antivenom formulated with sucrose is more resistant to thermal stress than the liquid formulation stabilized with sorbitol.

EchiTAb + ICP is a pan-African antivenom used for the treatment of snakebite envenomation in rural sub-Saharan African communities, where the cold chain can be difficult to maintain. To develop a formulation of EchiTAb + ICP that can be distributed and stored without refrigeration, we submitted three different formulations of EchiTAb + ICP: control (i.e. liquid antivenom formulated without stabilizer), liquid antivenom stabilized with sorbitol, and freeze-dried antivenom formulated with sucrose, to an accelerated stability study (i.e. 38 ± 2 °C and 75% relative humidity for 6 months). We analyzed changes in color, residual humidity, reconstitution time (for freeze-dried preparation), pH, osmolality, total protein concentration, antibody monomers content, turbidity, bacterial endotoxins, and pre-clinical neutralizing efficacy of the lethal effect of Echis ocellatus venom at 0, 3 and 6 months. In the control formulation, instability was evidenced by the development of a yellow coloration and an increment in aggregation and turbidity, without change in its neutralizing activity. The sorbitol-stabilized formulation did not develop marked aggregation or turbidity, but instability was evidenced by the development of yellow coloration and a drop in the neutralizing potency. The freeze-dried formulation maintained its neutralizing potency and did not show marked signs of instability, thus indicating that freeze-drying could confer EchiTAb + ICP with improved thermal stability required for distribution and storage at room temperature in sub-Saharan Africa.

A multicomponent strategy to improve the availability of antivenom for treating snakebite envenoming.

Snakebite envenoming is a common but neglected public health problem, particularly in impoverished rural regions of sub-Saharan Africa, Asia and Latin America. The only validated treatment for this condition is passive immunotherapy with safe and effective animal-derived antivenoms. However, there is a long-lasting crisis in the availability of these life-saving medications, particularly in sub-Saharan Africa and parts of Asia. We herein advocate a multicomponent strategy to substantially improve the availability of safe and effective antivenoms at the global level. This strategy is based on: (i) preparing validated collections of representative venom pools from the most medically dangerous snakes in high-risk regions of the world; (ii) strengthening the capacity of national antivenom manufacturing and quality control laboratories and their regulatory authorities and establishing new facilities in developing countries through technology transfer, as an integral part of efforts to develop their biological products industry; (iii) getting established laboratories to generate antivenoms for various regions of the world; and (iv) getting governments and relevant organizations to give snakebite envenoming due recognition within national and international public health policy frameworks. These ways of making antivenom available should be complemented by actions to improve health information systems, the accessibility of antivenoms, the training of medical and nursing staff, and community-based education. Such a multicomponent strategy involving stakeholders on many levels could help consolidate sustainable improvements in antivenom availability worldwide.

L'envenimation par morsure de serpent est un problème de santé publique fréquent, mais négligé, en particulier dans les régions rurales pauvres de l'Afrique subsaharienne, de l'Asie et de l'Amérique latine. Le seul traitement validé pour soigner cet état est l'immunothérapie passive avec des sérums antivenimeux d'origine animale sûrs et efficaces. Cependant, une crise durable limite actuellement la disponibilité de ces médicaments vitaux, surtout en Afrique subsaharienne et dans certaines parties de l'Asie. Nous préconisons ici une stratégie à composants multiples pour améliorer considérablement la disponibilité des sérums antivenimeux sûrs et efficaces à l'échelle mondiale. Cette stratégie repose sur: (i) la préparation de collections validées de groupes représentatifs de venins prélevés sur les serpents les plus dangereux sur le plan médical dans les régions à haut risque du monde; (ii) le renforcement de la capacité de production nationale des sérums antivenimeux, des laboratoires de contrôle qualité et de leurs organismes de réglementation, et la création de nouvelles installations dans les pays en développement par transfert de technologies, en tant que partie intégrante de la stratégie de développement de leur industrie de produits biologiques; (iii) la production par les laboratoires déjà établis de sérums antivenimeux pour les différentes régions du monde; et (iv) la reconnaissance officielle par les gouvernements et les organisations compétentes de l'envenimation par morsure de serpent dans le cadre des politiques de santé publique nationales et internationales. Ces façons de rendre disponibles les sérums antivenimeux devraient être complétées par des actions visant à améliorer les systèmes d'informations sanitaires, l'accessibilité des sérums antivenimeux, la formation du personnel médical et infirmier et les programmes communautaires d'éducation. Une telle stratégie à composants multiples impliquant des acteurs à différents niveaux pourrait contribuer à consolider les améliorations durables en matière de disponibilité des sérums antivenimeux dans le monde entier.

El envenenamiento por mordedura de serpiente es un problema de salud pública común pero desatendido, especialmente en las regiones rurales más pobres de África subsahariana, Asia y América Latina. El único tratamiento reconocido contra estas mordeduras es la inmunoterapia pasiva con sueros antiofídicos de origen animal seguros y eficaces. Sin embargo, la disponibilidad de estos medicamentos esenciales para salvar vidas lleva mucho tiempo en crisis, en particular en África subsahariana y en algunas zonas de Asia. En el presente documento, abogamos por una estrategia multicomponente para mejorar de forma sustancial la disponibilidad de sueros antiofídicos seguros y eficaces en todo el mundo. La estrategia se basa en: (i) preparar colecciones reconocidas de sueros antiofídicos representativos de las serpientes más peligrosas en zonas de alto riesgo del mundo; (ii) reforzar la capacidad nacional de producción de sueros antiofídicos y la calidad de los laboratorios de control y sus autoridades normativas, así como crear instalaciones nuevas en los países en desarrollo por medio de la transferencia de tecnología como parte integral de los esfuerzos por desarrollar su industria de productos biológicos; (iii) conseguir que los laboratorios consolidados fabriquen sueros antiofídicos para varias regiones del mundo; y (iv) conseguir que los gobiernos y las organizaciones pertinentes otorguen al envenenamiento por mordedura de serpiente el reconocimiento debido dentro del marco de las políticas nacionales e internacionales de salud pública. Estas tareas dirigidas a facilitar el suero antiofídico deben complementarse con acciones para mejorar los sistemas de información sobre la salud, la accesibilidad de los antiofídicos, la formación del personal médico y de enfermería, y la educación comunitaria. Una estrategia multicomponente de ese tipo, que incluye a los interesados a varios niveles, podría ayudar a consolidar mejoras sostenibles en la disponibilidad de antiofídicos en todo el mundo.

Infant malnutrition predicts conduct problems in adolescents.

OBJECTIVES: The purpose of this study was to compare the prevalence of conduct problems in a well-documented sample of Barbadian adolescents malnourished as infants and a demographic comparison group and to determine the extent to which cognitive impairment and environmental factors account for this association. METHODS: Behavioral symptoms were assessed using a 76-item self-report scale in 56 Barbadian youth (11-17 years of age) with histories of protein-energy malnutrition (PEM) limited to the first year of life and 60 healthy classmates. Group comparisons were carried out by longitudinal and cross-sectional multiple regression analyses at 3 time points in childhood and adolescence. RESULTS: Self-reported conduct problems were more prevalent among previously malnourished youth (P < 0.01). Childhood IQ and home environmental circumstances partially mediated the association with malnutrition. Teacher-reported classroom behaviors at earlier ages were significantly correlated with youth conduct problems, confirming the continuity of conduct problems through childhood and adolescence. DISCUSSION: Self-reported conduct problems are elevated in children and adolescents with histories of early childhood malnutrition. Later vulnerability to increased conduct problems appears to be mediated by the more proximal neurobehavioral effects of the malnutrition on cognitive function and by adverse conditions in the early home environment.

Cognitive impairment as a mediator in the developmental pathway from infant malnutrition to adolescent depressive symptoms in Barbadian youth.

OBJECTIVE: Depressive symptoms are elevated in adolescents who experienced significant malnutrition early in life. Early malnutrition can also have a significant impact on cognitive functioning, presumably because of the adverse impact of the malnutrition on the very young brain. In the context of a developmental cascade model, we tested the hypothesis that the association between early malnutrition and adolescent depressive symptoms is mediated by the cognitive impairment that ensues from the malnutrition. METHODS: We evaluated Barbadian youth (N = 57) hospitalized for moderate to severe protein-energy malnutrition in the first year of life and healthy controls (N = 60) longitudinally. The primary hypothesis was tested by multiple regression models. RESULTS: After adjusting for covariates, early malnutrition predicted both cognitive functioning in childhood (IQ, p < .001; attention problems, p < .01; Common Entrance Examination, p < .01; and adolescent depressive symptoms, p < .05). Childhood cognitive functioning mediated the association between early malnutrition and depressive symptoms in adolescence (p < .001). Maternal depressive symptoms were a significant but independent predictor of adolescent depressive symptoms (p < .05). CONCLUSIONS: Cognitive compromise in childhood accounts indirectly for elevated depressive symptoms in previously malnourished adolescents, consistent with a developmental cascade model. The direct link between malnutrition and depressive symptoms in adolescence is small.

Genetic and biological variation among nucleopolyhedrovirus isolates from the fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae).

A PCR-based method was used to identify and distinguish among 40 uncharacterized nucleopolyhedrovirus (NPV) isolates from larvae of the moth Spodoptera frugiperda that were part of an insect virus collection. Phylogenetic analysis was carried out with sequences amplified from two strongly conserved loci (polh and lef-8) from the 40 isolates in the collection and from eight previously studied S. frugiperda NPV (SfMNPV) isolates. To further distinguish these isolates, analysis was also carried out with sequences from two less-conserved loci, hr4 and hr5. Phylogenetic inference from the sequence data could distinguish among several of the individual isolates and between different groups of isolates from Georgia (USA) and Colombia, South America. A stronger degree of bootstrap support for the phylogenetic trees was obtained with the hr4 and hr5 homologous repeat sequences. Sequencing and phylogenetic analysis detected a relatively high degree of larva-to-larva sequence divergence occurring among isolates of SfMNPV collected from the same field in Missouri, USA. Restriction endonuclease analysis of viral DNA from larvae infected with five isolates from Georgia, Missouri, Louisiana, Florida (USA), and Colombia allowed for further comparison with other previously reported isolates of SfMNPV. Bioassays with these five geographically distinct isolates detected minor differences in virulence. This study highlights the use of PCR to rapidly distinguish and characterize large numbers of historical baculovirus isolates from the same host using minimal quantities of material, and the use of sequences from homologous repeat regions to distinguish closely related isolates of the same NPV species.

Purification and functional characterisation of rhinocerase, a novel serine protease from the venom of Bitis gabonica rhinoceros

BACKGROUND: Serine proteases are a major component of viper venoms and are thought to disrupt several distinct elements of the blood coagulation system of envenomed victims. A detailed understanding of the functions of these enzymes is important both for acquiring a fuller understanding of the pathology of envenoming and because these venom proteins have shown potential in treating blood coagulation disorders. METHODOLOGY/PRINCIPAL FINDINGS: In this study a novel, highly abundant serine protease, which we have named rhinocerase, has been isolated and characterised from the venom of Bitis gabonica rhinoceros using liquid phase isoelectric focusing and gel filtration. Like many viper venom serine proteases, this enzyme is glycosylated; the estimated molecular mass of the native enzyme is approximately 36 kDa, which reduces to 31 kDa after deglycosylation. The partial amino acid sequence shows similarity to other viper venom serine proteases, but is clearly distinct from the sequence of the only other sequenced serine protease from Bitis gabonica. Other viper venom serine proteases have been shown to exert distinct biological effects, and our preliminary functional characterization of rhinocerase suggest it to be multifunctional. It is capable of degrading alpha and beta chains of fibrinogen, dissolving plasma clots and of hydrolysing a kallikrein substrate. CONCLUSIONS/SIGNIFICANCE: A novel multifunctional viper venom serine protease has been isolated and characterised. The activities of the enzyme are consistent with the known in vivo effects of Bitis gabonica envenoming, including bleeding disorders, clotting disorders and hypotension. This study will form the basis for future research to understand the mechanisms of serine protease action, and examine the potential for rhinocerase to be used clinically to reduce the risk of human haemostatic disorders such as heart attacks and strokes.

Preclinical assessment of the efficacy of a new antivenom (EchiTAb-Plus-ICP) for the treatment of viper envenoming in sub-Saharan Africa.

A preclinical assessment was performed on the neutralizing efficacy of a whole IgG polyspecific antivenom (EchiTAb-Plus-ICP), designed for the treatment of snakebite envenomings in Nigeria. It was generated by immunizing horses with the venoms of Echis ocellatus, Bitis arietans and Naja nigricollis, the most medically important species in Nigeria. Antivenom was tested against the venoms of E. ocellatus, Echis leucogaster, Echis pyramidum leakeyi, B. arietans, Bitis gabonica, Bitis rhinoceros and Bitis nasicornis. The neutralization of the venom toxins responsible for the lethal, hemorrhagic, coagulant and local necrotizing activities was assessed, since these are the most significant effects that characterize envenoming by these species. Echis sp venoms exerted lethal, hemorrhagic, coagulant and necrotizing effects, whereas the Bitis sp venoms tested induced lethality, hemorrhage and necrosis, but were devoid of coagulant activity. The antivenom was effective in the neutralization of all effects tested in all venoms. Highest neutralization was achieved against the venoms of E. ocellatus and B. arietans, and the lowest neutralizing potency was against the venom of B. nasicornis, a species that has a low clinical relevance. It is concluded that EchiTAb-Plus-ICP, whilst specifically designed for Nigeria, has a good preclinical neutralizing profile against homologous and heterologous viperid venoms from other sub-Saharan African locations. It therefore constitutes a promising therapeutic option for the treatment of snakebite envenoming in this region.

Postpartum feeding attitudes, maternal depression, and breastfeeding in Barbados.

Maternal feeding attitudes, maternal moods and infant feeding practices during the first 6 months postpartum were assessed in 226 healthy, well-nourished Barbadian mother-infant dyads. Factor analysis of the feeding attitudes questionnaire resulted in six independent factors. The belief that breastfeeding was better than bottle-feeding was associated with higher family income, more information seeking behavior and older maternal age at the time of her first pregnancy. Women who believed that breastfeeding was better at 7 weeks postpartum were also more likely to breastfeed at concurrent and later ages, up to 6 months postpartum. This belief was also associated with less maternal depression at 7 weeks and 6 months. The association between feeding attitudes and actual feeding practices was significant even after correcting for maternal moods and other background variables. Conversely, after controlling for feeding attitudes, maternal mood at 7 weeks was still significantly associated with infant feeding practices at 6 months. Thus, feeding attitudes and maternal moods were closely linked, but each contributed independently and uniquely to different aspects of breastfeeding, especially at 6 months. These findings suggest that early intervention addressing maternal feeding attitudes, may improve the extent of breastfeeding and the health of children in this setting.

Bed-sharing, breastfeeding and maternal moods in Barbados.

Bed-sharing among Barbadian mothers and infants was studied in relationship to maternal and infant characteristics. This prospective study followed 226 healthy, well-nourished mother-infant dyads at birth, 7 weeks, 3 months, and 6 months postpartum. At each age, approximately half of the infants shared the same beds as their mothers. Bed-sharing was associated with demographic characteristics, especially fewer home conveniences, and also maternal characteristics, including less information seeking by the mother and younger maternal age at first pregnancy. Bed-sharing was also associated with lower infant birth weights. Maternal moods were significantly correlated with bed-sharing, such that mothers who reported having more despair and anxiety were also more likely to sleep with their infants. Bed-sharing was also significantly associated with increased breastfeeding at all infant ages, but this relationship was no longer significant once the effects of maternal moods were controlled. This study emphasizes the importance of assessing maternal moods in studies evaluating the risk and benefits of bed-sharing.

Maternal moods predict breast-feeding in Barbados

This study was designed to identify psychosocial variables affecting early infant feeding practices in Barbados. The sample included 93 healthy women and infants born at the Queen Elizabeth Hospital who were extensively evaluated 7 weeks, 3 months, and 6 months after birth. Maternal moods were assessed with the Zung Depression and Anxiety Scales and the General Adjustment and Morale Scale. Feeding practices were evaluated using a questionnaire developed for this population. The prevalence of mild depression in this population was 16 percent at 7 weeks and increased to 19 percent at 6 months, whereas there were very few cases of moderate-to-severe depression. Disadvantaged environmental conditions, including less information-seeking by the mother, lower family income, and poor maternal health, were closely associated with increased symptoms of depression and anxiety in all women. However, significant predictive relationships between mood and feeding practices remained even when the effects of the home environment were controlled. Specifically, depressive symptoms at 7 weeks postpartum predicted a reduced preference for breastfeeding at current and later infant ages. Conversely, feeding practices did not predict maternal moods at later ages. These findings have important implications for public policy dealing with programs promoting breastfeeding. Early interventions designed to treat mild postnatal depression should be instituted early in the postpartum period to improve the chances for successful breastfeeding.(Au)