Expression and processing of the Hepatitis E virus ORF1 nonstructural polyprotein

BACKGROUND: The ORF1 of hepatitis E virus (HEV) encodes a nonstructural polyprotein of ~186 kDa that has putative domains for four enzymes: a methyltransferase, a papain-like cysteine protease, a RNA helicase and a RNA dependent RNA polymerase. In the absence of a culture system for HEV, the ORF1 expressed using bacterial and mammalian expression systems has shown an ~186 kDa protein, but no processing of the polyprotein has been observed. Based on these observations, it was proposed that the ORF1 polyprotein does not undergo processing into functional units. We have studied ORF1 polyprotein expression and processing through a baculovirus expression vector system because of the high level expression and post-translational modification abilities of this system. RESULTS: The baculovirus expressed ORF1 polyprotein was processed into smaller fragments that could be detected using antibodies directed against tags engineered at both ends. Processing of this ~192 kDa tagged ORF1 polyprotein and accumulation of lower molecular weight species took place in a time-dependent manner. This processing was inhibited by E-64d, a cell-permeable cysteine protease inhibitor. MALDI-TOF analysis of a 35 kDa processed fragment revealed 9 peptide sequences that matched the HEV methyltransferase (MeT), the first putative domain of the ORF1 polyprotein. Antibodies to the MeT region also revealed an ORF1 processing pattern identical to that observed for the N-terminal tag. CONCLUSION: When expressed through baculovirus, the ORF1 polyprotein of HEV was processed into smaller proteins that correlated with their proposed functional domains. Though the involvement of non-cysteine protease(s) could not be be ruled out, this processing mainly depended upon a cysteine protease

Prevalence of Smartphone Addiction and its Relation with Depression among School-going Adolescents

Background: Smartphone addiction among adolescents is an increasingly recognized problem worldwide. It affects the psychological well-being of an individual. Aim and objective: The current study aimed to assess smartphone addiction’s prevalence and its relation to depression among adolescents. Methods: This cross-sectional study was conducted among 400 school-going adolescents. Smartphone Addiction Scale - Short version (SAS-SV) and Patient Health Questionnaire (PHQ-9) were used to assess the prevalence of smartphone addiction and depression. Data were analyzed using Epi info software for windows (CDC, Atlanta). Statistical significance was set at p < 0.05. Results: The mean age of study participants was 14.4 years (SD=1.5 years). The prevalence of smartphone addiction was 23%, while depression was present among 45% of the study participants. Comparatively higher duration of smartphone use was significantly associated with smartphone addiction. Depression was significantly higher among smartphone addicts (77.2%) as compared to their counterparts (35.4%). Conclusion and Recommendation: The smartphone usage of adolescents, if not monitored, could lead to its addiction and thus increase the risk of depression among them. To prevent smartphone addiction, limiting children’s screen time is recommended. In this regard, parents can play a pivotal role by becoming responsible digital role models for their children

Bombyx mori nucleopolyhedrovirus: molecular biology and biotechnological applications for large-scale synthesis of recombinant proteins

Bombyx mori nucleopolyhedrosis virus (BmNPV), a natural pathogen of great economic significance in sericulture, has been exploited to generate recombinant baculoviruses harbouring genes of choice and achieve high level expression of cloned foreign genes. The BmNPV-based expression system offers the advantage of using the larval host rather than the insectderived cell lines for economic large-scale synthesis of biomolecules. Since commercial rearing of silkworms is routinely practised, a part of the silkworm stocks can be diverted to produce biomolecules other than silk and the more sophisticated tissue culture methodologies can be dispensed with on an industrial scale. Nonetheless, the BmNPV-based expression considerably lags behind the AcMNPV system in vogue today, in terms of the choice of expression-optimized cloning vectors and easy recombinant generation kits that are commercially available. This review provides a brief outline of the current state of knowledge on the basic biology and genomics of BmNPV and how the virus has been made use of to produce biomolecules through its larval host, the mulberry silkworm. Since hyper-transcription from the viral very late gene promoters forms the basis of high level expression through recombinant baculoviruses, some aspects of the viral gene transcription and the role of late gene expression factors have also been included in the review

Real-Time Information Access in Urban Environments: A User Interaction Study Using the Real-Time Information Test

In this study, "Real-Time Information Access in Urban Environments: A User Interaction Study Using the Real-Time Information Test," participant data revealed a diverse group with an average age of 31, a balanced gender distribution, varying education levels (40% Bachelor's, 20% Master's, 40% PhD), and an average of 6 years of experience with urban navigation. The findings of the Real-Time Information Test (RTIT) showed an average job completion time of 140 seconds and a low average error count of 1.2, demonstrating competency in interacting with real-time information systems. Furthermore, the User Satisfaction Survey found an average of 8.4 overall satisfaction ratings, 8.4 user-friendliness ratings, and 7.8 information accuracy ratings, indicating excellent user experiences. These results highlight user variety, increases in job efficiency and accuracy, and high user satisfaction, all of which contribute to a comprehensive knowledge of real-time information access in urban contexts, with implications for system advancements and urban planning

Representação de componentes compartilhados na via de floração e via de desenvolvimento do chicote de carvão em cana-de-açúcar

Sugarcane is one of the most valuable industrial and agricultural cash crops, currently being grown in more than 100 countries across the globe, and also contributes significantly to the Brazilian economy. Sugarcane smut disease caused by basidiomycete biotrophic fungi Sporisorium scitamineum is a prominent threat for sugarcane, costing up to 80% of the yield, and in turn, have a major economic impact. Therefore, unraveling components of molecular crosstalk between smut fungi and sugarcane is imperative to find a solution to manage this disease and protect the livelihoods of farmers worldwide. This work aimed to contribute to this cause by understanding the correlation of smut fungi and the flowering pathway in sugarcane. In the first chapter, the current understanding of the flowering pathway in the model plant Arabidopsis thaliana and the correlation between smut fungi with flowering pathways was reviewed. Smut fungi are biotrophic in nature and generally infect grass species, and some of the smut fungi species have been demonstrated to modulate the floral structures of the host plants and interfere with the flowering pathway to survive and reproduce. Since Sugarcane is also a grass species, we hypothesized the existence of a similar mechanism in the sugarcane smut fungi pathosystem. This is why we embarked on this journey to understand this correlation by studying sugarcane- smut interaction at a molecular level by specifically focusing on the components of the flowering pathway. In the second chapter, we used RNASeq to generate transcriptional profiling of smut-resistant (SP80-3280) and smut-susceptible (IAC66-6) genotypes 48 hours after inoculation with smut fungi and also constructed co-expression networks to study the effect of smut fungus infection on the expression levels of putative orthologs of flowering genes in sugarcane. Our data unraveled that the smut fungus elicits an antagonistic expression pattern of flowering genes in two sugarcane genotypes with contrasting levels of smut tolerance. Transcriptional profiling and co-expression networks of the smut resistant genotype suggest the repression of flowering pathway, and on the contrary, the data from the smut-susceptible genotype suggest that smut fungus induces the activation of flowering pathway. Our results also indicated the potential epigenetic regulation at play in the sugarcane-smut pathosystem. Plants, in general, accelerate the floral transitioning process under stress conditions to reproduce before it succumbs to the stress, and our results specifically from the susceptible genotype suggest that potentially, a similar mechanism exists in sugarcane as well. In the third chapter, based on these results, we conducted an exploratory study to evaluate the influence of a known flowering repressor, ethephon, in sugarcane-smut interaction at a physiological and molecular level. No physiological impact on the smut disease progression or whip developmental process was observed due to ethephon treatment in time-points analyzed. The effect of ethephon on candidate flowering genes varied depending on the genotype and the developmental stage. Ethephon seems to potentially have the ability to modulate the expression behavior of candidate flowering genes independently of the fungus as all the candidate flowering genes tested had lower expression levels in ethephon treated samples compared to the untreated ones in the susceptible genotype. For the first time, this study has provided an in-depth analysis of correlation of flowering-related genes in the sugarcane-smut pathosystem. It encourages the idea that the sugarcane smut fungus has the same modus operandi as other smut fungi species to interfere with flowering pathways for its own growth and reproduction purposes. This is the first study in the direction of depicting the shared components in flowering and smut whip development pathways and should encourage more detailed studies in future to unravel all the components in these two distinct genetic pathways. This information will help in finding better management solutions for the smut disease in sugarcane.A cana-de-açúcar é uma das culturas de rendimento industrial e agrícola mais valiosas, sendo cultivada atualmente em mais de 100 países em todo o mundo, e também contribui significativamente para a economia brasileira. A doença do carvão da cana, causada pelo fungo biotrófico basidiomiceto Sporisorium scitamineum, é uma ameaça proeminente para a cana-de-açúcar, custando até 80% do rendimento e, por sua vez, tem grande impacto econômico. Portanto, desvendar os componentes do crosstalk molecular entre os fungos de carvão e a cana-de-açúcar é imperativo para encontrar uma solução para gerenciar essa doença e proteger os meios de subsistência dos agricultores em todo o mundo. Este trabalho teve como objetivo contribuir para estes estudos, entendendo a correlação dos fungos de carvão e a via de floração da cana-de-açúcar. No primeiro capítulo, a compreensão atual da via de floração na planta modelo Arabidopsis thaliana e a correlação dos fungos de carvão com a via de floração foi revisada. Os fungos de carvão são biotróficos por natureza e geralmente infectam espécies de gramíneas, sendo que algumas das espécies modulam as estruturas florais das plantas hospedeiras e interferem na via de floração para sobreviver e se reproduzir. Como a cana-de-açúcar também é uma espécie de gramínea, hipotetizamos a existência de um mecanismo semelhante no patossistema do fungo de carvão da cana-de-açúcar. Desta forma o objetivo geral deste trabalho é entender essa correlação estudando a interação cana-carvão em nível molecular, focando especificamente nos componentes da via de floração. No segundo capítulo, usamos RNASeq para gerar perfis transcricionais de genótipos resistentes (SP80- 3280) e suscetíveis ao carvão (IAC66-6) 48 horas após a inoculação com o fungo de carvão e utilizando redes de co-expressão estudamos o efeito da infecção do fungo em níveis de expressão de ortólogos putativos de genes de floração em cana-de- açúcar. Nossos dados revelaram que os fungos de carvão provocam um padrão de expressão antagônico de genes de floração em dois genótipos de cana-de-açúcar com níveis contrastantes de tolerância à doença. O perfil transcricional e as redes de co-expressão do genótipo resistente sugerem a repressão da via de floração e, ao contrário, os dados do genótipo suscetível ao carvão sugerem que o fungo induz a ativação da via de floração. Nossos resultados também indicaram a potencial regulação epigenética no patossistema da cana-de-açúcar. As plantas em geral aceleram o processo de transição floral sob condições de estresse para se reproduzir antes de sucumbirem ao estresse, e nossos resultados especificamente para o genótipo suscetível sugerem que potencialmente, um mecanismo semelhante também existe na cana-de-açúcar. No terceiro capítulo, com base nesses resultados, realizamos um estudo exploratório para avaliar a influência de um conhecido repressor de floração, o etefon, na interação cana-carvão em nível fisiológico e molecular. Nenhum impacto fisiológico na progressão da doença do carvão ou no processo de desenvolvimento do chicote foi observado devido ao tratamento com ethephon nos momentos avaliados. O efeito do ethephon nos genes candidatos à floração varia de acordo com o genótipo e o estádio de desenvolvimento, e o ethephon parece potencialmente ter a capacidade de modular o comportamento de expressão dos genes candidatos à floração independentemente do fungo, já que todos os genes candidatos à floração testados apresentaram menor expressão nas amostras tratadas com ethephon em comparação com as não tratadas no genótipo suscetível. Este estudo forneceu, pela primeira vez, uma análise aprofundada da correlação de genes relacionados ao florescimento no patossistema cana-de-açúcar e incentiva a ideia de que os fungos de carvão têm o mesmo modus operandi que outras espécies de fungos do carvão para interferir na via de floração para sua própria finalidade de crescimento e reprodução. Este é o primeiro estudo na direção de descrever os componentes compartilhados nas vias de desenvolvimento da floração e do chicote e deve encorajar estudos mais detalhados no futuro para desvendar todos os componentes nessas duas vias genéticas distintas. Essas informações ajudarão a encontrar melhores soluções de manejo para a doença do carvão na cana-de-açúcar

Biofilm Development in Gram-Positive and Gram-Negative Bacteria

Biofilms are the communities of microorganisms, especially bacteria attached to a biotic or abiotic surface. These biofilms live in a self-sustained matrix and produce different substances called extracellular polymeric substances (EPS) which are responsible for the pathogenicity of a number of bacteria such as Pseudomonas aeruginosa, Staphylococcus aureus, Vibrio cholerae, Klebsiella pneumoniae, Escherichia coli, etc. These EPS substance makes it difficult to eradicate the biofilm present on the surface. Biofilm formation is a five-step process. Biofilms can be monospecies or multispecies. In biofilms, cells communicate via Quorum Sensing (QS). QS is the regulation of gene expression in bacteria with respect to changes in cell population density. In QS, bacteria produce various signaling molecules called Auto-inducers (AI). AI concentration increases as the bacterial population increases. Bacteria respond to these AIs results in an alteration of gene expression, which results in the release of various virulence factors. QS involves a two-component signaling process which is different for both Gram-positive and Gram-negative bacteria. QS and EPS make the bacteria resistant to various antibiotics, which make the eradication difficult and hence requires more effective treatment. This article discusses the biofilm structure, phenomenon of biofilm formation, signaling, and pathogenicity to highlight the understanding of processes involved in biofilm formation

Recombinant Bombyx mori nucleopolyhedrovirus harboring green fluorescent protein

The gene encoding the green fluorescent protein (gfp) under the control of the highly expressed Autographa californica nucleopolyhedrovirus (AcMNPV)-polyhedrin promoter has been introduced into the polyhedrin (polh) locus of Bombyx mori nucleopolyhedrovirus (BmNPV) by homologous recombination. The insect host larvae and the cultured cells infected with This recombinant virus (vBmGFP) showed high levels of expression of gfp. The larval tissues permissive to virus multiplication could be readily visualized using the tagged recombinant virus, thus providing a direct approach to study the progress of virus infection or its control in the animal host. The highly expressed recombinant protein, GFP, could be easily solubilized from fat bodies. Thus, the caterpillar-based expression could serve as an economic alternative method for the large-scale production of recombinant proteins, even when they are nonsecretory in nature. Further, if the recombinant vBmGFP is used as a parent in generating other recombinants, conversion of the fluorescent plaques to colorless plaques serves as an easy means for screening recombinants. Such a method is especially helpful for BmNPV-recombinant selections in the absence of the other simplified techniques as are available for the prototype baculovirus AcMNPV system

Hepatitis E Virus Cysteine Protease Has Papain Like Properties Validated by in silico Modeling and Cell-Free Inhibition Assays

Hepatitis E virus (HEV) has emerged as a global health concern during the last decade. In spite of a high mortality rate in pregnant women with fulminant hepatitis, no antiviral drugs or licensed vaccine is available in India. HEV-protease is a pivotal enzyme responsible for ORF1 polyprotein processing leading to cleavage of the non-structural enzymes involved in virus replication. HEV-protease region encoding 432–592 amino acids of Genotype-1 was amplified, expressed in Sf21 cells and purified in its native form. The recombinant enzyme was biochemically characterized using SDS-PAGE, Western blotting and Immunofluorescence. The enzyme activity and the inhibition studies were conducted using Zymography, FTC-casein based protease assay and ORF1 polyprotein digestion. To conduct ORF1 digestion assay, the polyprotein, natural substrate of HEV-protease, was expressed in E. coli and purified. Cleavage of 186 kDa ORF1 polyprotein by the recombinant HEV-protease lead to appearance of non-structural proteins viz. Methyltransferase, Protease, Helicase and RNA dependent RNA polymerase which were confirmed through immunoblotting using antibodies generated against specific epitopes of the enzymes. FTC-casein substrate was used for kinetic studies to determine Km and Vmax of the enzyme and also the effect of different metal ions and other protease inhibitors. A 95% inhibition was observed with E-64 which was validated through in silico analysis. The correlation coefficient between inhibition and docking score of Inhibitors was found to have a significant value of r2 = 0.75. The predicted 3D model showed two domain architecture structures similar to Papain like cysteine protease though they differed in arrangements of alpha helices and beta sheets. Hence, we propose that HEV-protease has characteristics of “Papain-like cysteine protease,” as determined through structural homology, active site residues and class-specific inhibition. However, conclusive nature of the enzyme remains to be established