Prediction of human protein interactome of dengue virus non-structural protein 5 (NS5) and its downstream immunological implications.

The non-structural protein 5 (NS5) is the most conserved protein among flaviviruses, a family that includes the dengue virus. It functions both as an RNA-dependent RNA polymerase and an RNA-methyltransferase and is therefore essential for the replication of viral RNA. The discovery that dengue virus NS5 protein (DENV-NS5) can also localize to the nucleus has resulted in renewed interest in its potential roles at the host-virus interface. In this study, we have used two complementary computational approaches in parallel - one based on linear motifs (ELM) and another based on tertiary structure of the protein (DALI) - to predict the host proteins that DENV-NS5 might interact with. Of the 42 human proteins predicted by both these methods, 34 are novel. Pathway analysis of these 42 human proteins shows that they are involved in key host cellular processes related to cell cycle regulation, proliferation, protein degradation, apoptosis, and immune responses. A focused analysis of transcription factors that directly interact with the predicted DENV-NS5 interacting proteins was performed, followed by the identification of downstream genes that are differentially expressed after dengue infection using previously published RNA-seq data. Our study provides unique insights into the DENV-NS5 interaction network and delineates mechanisms whereby DENV-NS5 could impact the host-virus interface. The novel interactors identified in this study could be potentially targeted by NS5 to modulate the host cellular environment in general, and the immune response in particular, thereby extending the role of DENV-NS5 beyond its known enzymatic functions. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03569-0.

Dengue Virus Non-Structural Protein 5 as a Versatile, Multi-Functional Effector in Host-Pathogen Interactions.

Dengue is emerging as one of the most prevalent mosquito-borne viral diseases of humans. The 11kb RNA genome of the dengue virus encodes three structural proteins (envelope, pre-membrane, capsid) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5), all of which are translated as a single polyprotein that is subsequently cleaved by viral and host cellular proteases at specific sites. Non-structural protein 5 (NS5) is the largest of the non-structural proteins, functioning as both an RNA-dependent RNA polymerase (RdRp) that replicates the viral RNA and an RNA methyltransferase enzyme (MTase) that protects the viral genome by RNA capping, facilitating polyprotein translation. Within the human host, NS5 interacts with several proteins such as those in the JAK-STAT pathway, thereby interfering with anti-viral interferon signalling. This mini-review presents annotated, consolidated lists of known and potential NS5 interactors in the human host as determined by experimental and computational approaches respectively. The most significant protein interactors and the biological pathways they participate in are also highlighted and their implications discussed, along with the specific serotype of dengue virus as appropriate. This information can potentially stimulate and inform further research efforts towards providing an integrative understanding of the mechanisms by which NS5 manipulates the human-virus interface in general and the innate and adaptive immune responses in particular.

The Role of Constructive Neutral Evolution in the Development of Complexity from Symbioses: A Microbe-Centric View.

Symbiogenesis presents the biologist with very different explanatory issues compared to the lineal and selectionist view of evolution based on individual entities, whether genes, organisms or species. A key question is how the co-existence of two or more partners in close association during a given generation can ultimately be stabilized enough to be transmitted to the next, how the ensuing complexity is maintained and how this arrangement impacts the reproductive fitness of the collective over evolutionary time. In this chapter, we highlight some observations gleaned from the microbial world that could shed light on this problem if viewed within the framework of constructive neutral evolution.

A Pediatric Case of Accidental Eucalyptus Oil Poisoning from New Delhi, India: Emergency Measures, Historical Context, and Implications for Practice.

Eucalyptus oil (EO) and EO containing products are readily available worldwide over the counter as topical nasal decongestants, rubefacients, anti-pyretics, and anti-inflammatory agents. However, EO is poisonous when orally ingested, or otherwise internally administered, resulting in serious outcomes such as seizures, vomiting, drowsiness, and even death. In this case report, we describe emergency measures adopted in a suspected case of EO ingestion by a 17-month-old female infant. It was found that stomach washes with normal saline followed by the oral administration of ranitidine to prevent vomiting restored and maintained normalcy over a 24-hour period. We situate our experience within the Indian context and recommend that pediatricians and healthcare workers routinely and explicitly warn parents about the serious consequences of the incorrect usage of substances containing essential oils in general and EO in particular.

Prokaryotic horizontal gene transfer within the human holobiont: ecological-evolutionary inferences, implications and possibilities.

The ubiquity of horizontal gene transfer in the living world, especially among prokaryotes, raises interesting and important scientific questions regarding its effects on the human holobiont i.e., the human and its resident bacterial communities considered together as a unit of selection. Specifically, it would be interesting to determine how particular gene transfer events have influenced holobiont phenotypes in particular ecological niches and, conversely, how specific holobiont phenotypes have influenced gene transfer events. In this synthetic review, we list some notable and recent discoveries of horizontal gene transfer among the prokaryotic component of the human microbiota, and analyze their potential impact on the holobiont from an ecological-evolutionary viewpoint. Finally, the human-Helicobacter pylori association is presented as an illustration of these considerations, followed by a delineation of unresolved questions and avenues for future research.

On the Tacit Aspects of Science Pedagogy in Higher Education.

In this article, we examine the concept of tacit knowledge and its implications for science education. We suggest that the history of scientific ideas and the personal nature of learning imply that higher education in scientific fields, wherein the generation of new knowledge, insights and understanding is paramount, would greatly benefit by acknowledging the irreducible role of the non-formal and the incidental in scientific innovation and advances.

Osmoregulation in Saccharomyces cerevisiae via mechanisms other than the high-osmolarity glycerol pathway.

The response of Saccharomyces cerevisiae to osmotic stress, whether arising from environmental conditions or physiological processes, has been intensively studied in the last two decades. The well-known high-osmolarity glycerol (HOG) signalling pathway that is induced in response to osmotic stress interacts with other signalling pathways such as the cell wall integrity and the target of rapamycin pathways. Osmotic balance is also maintained by the regulated opening and closing of channel proteins in both the cell membrane and intracellular organelles such as the vacuole. Additionally, environmental stresses, including osmotic shock, induce intracellular calcium signalling. Thus, adaptation to environmental stresses in general, and osmotic stress in particular, is dependent on the concerted action of components of multiple interacting pathways. In this review, we describe some of the major mechanisms and molecules involved in osmoregulation via pathways other than the high-osmolarity glycerol pathway and their known interactions with one another that have been discovered over the last two decades.

The Chromosomal parDE2 Toxin-Antitoxin System of Mycobacterium tuberculosis H37Rv: Genetic and Functional Characterization.

Mycobacterium tuberculosis H37Rv escapes host-generated stresses by entering a dormant persistent state. Activation of toxin-antitoxin modules is one of the mechanisms known to trigger such a state with low metabolic activity. M. tuberculosis harbors a large number of TA systems mostly located within discernible genomic islands. We have investigated the parDE2 operon of M. tuberculosis H37Rv encoding MParE2 toxin and MParD2 antitoxin proteins. The parDE2 locus was transcriptionally active from growth phase till late stationary phase in M. tuberculosis. A functional promoter located upstream of parD2 GTG start-site was identified by 5'-RACE and lacZ reporter assay. The MParD2 protein transcriptionally regulated the P parDE2 promoter by interacting through Arg16 and Ser15 residues located in the N-terminus. In Escherichia coli, ectopic expression of MParE2 inhibited growth in early stages, with a drastic reduction in colony forming units. Live-dead analysis revealed that the reduction was not due to cell death alone but due to formation of viable but non-culturable cells (VBNCs) also. The toxic activity of the protein, identified in the C-terminal residues Glu98 and Arg102, was neutralized by the antitoxin MParD2, both in vivo and in vitro. MParE2 inhibited mycobacterial DNA gyrase and interacted with the GyrB subunit without affecting its ATPase activity. Introduction of parE2 gene in the heterologous M. smegmatis host prevented growth and colony formation by the transformed cells. An M. smegmatis strain containing the parDE2 operon also switched to a non-culturable phenotype in response to oxidative stress. Loss in colony-forming ability of a major part of the MParE2 expressing cells suggests its potential role in dormancy, a cellular strategy for adaptation to environmental stresses. Our study has laid the foundation for future investigations to explore the physiological significance of parDE2 operon in mycobacterial pathogenesis.

The Role of DNA Restriction-Modification Systems in the Biology of Bacillus anthracis.

Restriction-modification (R-M) systems are widespread among prokaryotes and, depending on their type, may be viewed as selfish genetic elements that persist as toxin-antitoxin modules, or as cellular defense systems against phage infection that confer a selective advantage to the host bacterium. Studies in the last decade have made it amply clear that these two options do not exhaust the list of possible biological roles for R-M systems. Their presence in a cell may also have a bearing on other processes such as horizontal gene transfer and gene regulation. From genome sequencing and experimental data, we know that Bacillus anthracis encodes at least three methylation-dependent (typeIV) restriction endonucleases (RE), and an orphan DNA methyltransferase. In this article, we first present an outline of our current knowledge of R-M systems in B. anthracis. Based on available DNA sequence data, and on our current understanding of the functions of similar genes in other systems, we conclude with hypotheses on the possible roles of the three REs and the orphan DNA methyltransferase.

Allergies, Helicobacter pylori and the continental enigmas.

Helicobacter pylori, a gastric pathogen, is known to be associated with gastric and duodenal ulcers, and is also a strong risk factor for the development of gastric cancer and lymphoma of the mucosal-associated lymphoid tissue. Ordinarily, this should make a strong case for its eradication at par with any other infectious disease. However, the unique biology of H. pylori and the complexity of its interactions with humans, its only known natural host, do not permit the recommendation of unambiguous preventive and therapeutic measures. Moreover, this organism has co-evolved with humans as a practically universal member of the natural gastric microbiota over at least 100,000 years. H. pylori persists for a lifetime in mostly asymptomatic hosts, and causes clinical disease only in a minority of infections. Therefore, its potential contribution to the maintenance of human immune homeostasis, as is the case with the better-studied members of the intestinal microbiota, is certainly worthy of serious investigation. In this paper, we summarize some interesting and often anecdotal data drawn from recent studies, and examine their significance in the context of the hygiene hypothesis. We also examine whether the lower incidence of gastric cancer over large parts of the world in spite of a high prevalence of infection (the Asian and African enigmas) may be re-interpreted in terms of the hygiene hypothesis. Finally, it is suggested that an evolutionary-ecological approach to the study of H. pylori infection may help in the formulation of strategies for the management of this infection. This may well be an infectious disease wherein medical interventions may have to be personalized to ensure optimal outcomes.

Remembering the forest while viewing the trees: evolutionary thinking in the teaching of molecular biology.

Given the centrality of evolutionary theory to the study of biology, we present a strategy for reinforcing its importance by appropriately recontextualizing classic and well-known experiments that are not explicitly linked with evolution in conventional texts. This exercise gives students an appreciation of the applicability of the theory of evolution in diverse contexts, including those where it is not explicitly mentioned.

From bedside to blackboard: the benefits of teaching molecular biology within a medical context.

Courses in molecular biology are part of practically every degree program in medicine and the life sciences. Historically, many basic discoveries in this field have resulted from investigations by doctors into the nature of diseases. This essay suggests that medical educators deliberately incorporate such material, whether historical or contemporaneous, into their molecular and cell biology courses. An example of such usage, an early report of the detection of bacteriophage activity on pathogenic bacteria, is discussed in detail. Such an approach can potentially narrow the perceived gap between "basic" and "applied" science. As medicine is so intimately and obviously linked with human welfare, this also provides an avenue for educators to discuss issues of scientific integrity and ethics within a "pure science" course.

Methylation-dependent DNA restriction in Bacillus anthracis.

Bacillus anthracis, the causative agent of anthrax, is poorly transformed with DNA that is methylated on adenine or cytosine. Here we characterize three genetic loci encoding type IV methylation-dependent restriction enzymes that target DNA containing C5-methylcytosine (m5C). Strains in which these genes were inactivated, either singly or collectively, showed increased transformation by methylated DNA. Additionally, a triple mutant with an ~30-kb genomic deletion could be transformed by DNA obtained from Dam(+)Dcm(+)E. coli, although at a low frequency of ~10(-3) transformants/10(6)cfu. This strain of B. anthracis can potentially serve as a preferred host for shuttle vectors that express recombinant proteins, including proteins to be used in vaccines. The gene(s) responsible for the restriction of m6A-containing DNA in B. anthracis remain unidentified, and we suggest that poor transformation by such DNA could in part be a consequence of the inefficient replication of hemimethylated DNA in B. anthracis.

Cell-associated hemolysis induced by Helicobacter pylori is mediated by phospholipases with mitogen-activated protein kinase-activating properties.

Pathogenic Helicobacter pylori strains can selectively activate epithelial mitogen-activated protein kinase (MAPK) signaling pathways linked with disease. We now demonstrate that H. pylori-induced hemolysis is strain specific and is mediated by phospholipases PldA1 and PldD. Inactivation of PldD inhibited activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), indicating that H. pylori hemolytic phospholipases also harbor MAPK-activating properties.

The treasure of the humble: lessons from baker's yeast.

The study of model organisms is a powerful and proven experimental strategy for understanding biological processes. This paper describes an attempt to utilize advances in yeast molecular biology to enhance student understanding by presenting a more comprehensive view of several interconnected molecular processes in the overall functioning of an organism, and introducing the concept of the inverse problem as it relates to biology.