Transcriptional dynamics during Rhodococcus erythropolis infection with phage WC1.

BACKGROUND: Belonging to the Actinobacteria phylum, members of the Rhodococcus genus thrive in soil, water, and even intracellularly. While most species are non-pathogenic, several cause respiratory disease in animals and, more rarely, in humans. Over 100 phages that infect Rhodococcus species have been isolated but despite their importance for Rhodococcus ecology and biotechnology applications, little is known regarding the molecular genetic interactions between phage and host during infection. To address this need, we report RNA-Seq analysis of a novel Rhodococcus erythopolis phage, WC1, analyzing both the phage and host transcriptome at various stages throughout the infection process. RESULTS: By five minutes post-infection WC1 showed upregulation of a CAS-4 family exonuclease, putative immunity repressor, an anti-restriction protein, while the host showed strong upregulation of DNA replication, SOS repair, and ribosomal protein genes. By 30 min post-infection, WC1 DNA synthesis genes were strongly upregulated while the host showed increased expression of transcriptional and translational machinery and downregulation of genes involved in carbon, energy, and lipid metabolism pathways. By 60 min WC1 strongly upregulated structural genes while the host showed a dramatic disruption of metal ion homeostasis. There was significant expression of both host and phage non-coding genes at all time points. While host gene expression declined over the course of infection, our results indicate that phage may exert more selective control, preserving the host's regulatory mechanisms to create an environment conducive for virion production. CONCLUSIONS: The Rhodococcus genus is well recognized for its ability to synthesize valuable compounds, particularly steroids, as well as its capacity to degrade a wide range of harmful environmental pollutants. A detailed understanding of these phage-host interactions and gene expression is not only essential for understanding the ecology of this important genus, but will also facilitate development of phage-mediated strategies for bioremediation as well as biocontrol in industrial processes and biomedical applications. Given the current lack of detailed global gene expression studies on any Rhodococcus species, our study addresses a pressing need to identify tools and genes, such as F6 and rpf, that can enhance the capacity of Rhodococcus species for bioremediation, biosynthesis and pathogen control.

Xenopus embryos show a compensatory response following perturbation of the Notch signaling pathway.

As an essential feature of development, robustness ensures that embryos attain a consistent phenotype despite genetic and environmental variation. The growing number of examples demonstrating that embryos can mount a compensatory response to germline mutations in key developmental genes has heightened interest in the phenomenon of embryonic robustness. While considerable progress has been made in elucidating genetic compensation in response to germline mutations, the diversity, mechanisms, and limitations of embryonic robustness remain unclear. In this work, we have examined whether Xenopus laevis embryos are able to compensate for perturbations of the Notch signaling pathway induced by RNA injection constructs that either upregulate or inhibit this signaling pathway. Consistent with earlier studies, we found that at neurula stages, hyperactivation of the Notch pathway inhibited neural differentiation while inhibition of Notch signaling increases premature differentiation as assayed by neural beta tubulin expression. However, surprisingly, by hatching stages, embryos begin to compensate for these perturbations, and by swimming tadpole stages most embryos exhibited normal neuronal gene expression. Using cell proliferation and TUNEL assays, we show that the compensatory response is, in part, mediated by modulating levels of cell proliferation and apoptosis. This work provides an additional model for addressing the mechanisms of embryonic robustness and of genetic compensation.

Seasonal Variations and Health Risk Assessment of Trace Elements in Seti River Basin, Gandaki Province, Nepal.

To explore the seasonal variability and associated health risks of dissolved trace elements (TEs), a total of 45 water samples (15 samples in each season) were collected from the Seti River Basin during pre-monsoon, monsoon, and post-monsoon seasons. A total of 15 TEs were analysed and the results revealed that Arsenic (As), Chromium (Cr), and Nickel (Ni) were primarily controlled by lithogenic sources, whereas, Cadmium (Cd), Cobalt (Co), Cesium (Cs), Cupper (Cu), Manganese (Mn), Lead (Pb), and Zinc (Zn) were derived from both anthropic and geogenic sources. Additionally, the concentration of Zn was markedly high, especially during the post-monsoon season nearby the landfill site, indicating the anthropic signatures. The hazard index and cancer index were found to be < 1.00 and 10-6-10-4, respectively indicating minimum health impacts. The water quality index was < 50 throughout the sampling periods, with better water quality during the monsoon. This study could help to formulate the necessary policies for the sustainability of the Himalayan River Basins.

Calcium Activity Dynamics Correlate with Neuronal Phenotype at a Single Cell Level and in a Threshold-Dependent Manner.

Calcium is a ubiquitous signaling molecule that plays a vital role in many physiological processes. Recent work has shown that calcium activity is especially critical in vertebrate neural development. Here, we investigated if calcium activity and neuronal phenotype are correlated only on a population level or on the level of single cells. Using Xenopus primary cell culture in which individual cells can be unambiguously identified and associated with a molecular phenotype, we correlated calcium activity with neuronal phenotype on the single-cell level. This analysis revealed that, at the neural plate stage, a high frequency of low-amplitude spiking activity correlates with an excitatory, glutamatergic phenotype, while high-amplitude spiking activity correlates with an inhibitory, GABAergic phenotype. Surprisingly, we also found that high-frequency, low-amplitude spiking activity correlates with neural progenitor cells and that differentiating cells exhibit higher spike amplitude. Additional methods of analysis suggested that differentiating marker tubb2b-expressing cells exhibit relatively persistent and predictable calcium activity compared to the irregular activity of neural progenitor cells. Our study highlights the value of using a range of thresholds for analyzing calcium activity data and underscores the importance of employing multiple methods to characterize the often irregular, complex patterns of calcium activity during early neural development.

Calcium Signaling in Vertebrate Development and Its Role in Disease.

Accumulating evidence over the past three decades suggests that altered calcium signaling during development may be a major driving force for adult pathophysiological events. Well over a hundred human genes encode proteins that are specifically dedicated to calcium homeostasis and calcium signaling, and the majority of these are expressed during embryonic development. Recent advances in molecular techniques have identified impaired calcium signaling during development due to either mutations or dysregulation of these proteins. This impaired signaling has been implicated in various human diseases ranging from cardiac malformations to epilepsy. Although the molecular basis of these and other diseases have been well studied in adult systems, the potential developmental origins of such diseases are less well characterized. In this review, we will discuss the recent evidence that examines different patterns of calcium activity during early development, as well as potential medical conditions associated with its dysregulation. Studies performed using various model organisms, including zebrafish, Xenopus, and mouse, have underscored the critical role of calcium activity in infertility, abortive pregnancy, developmental defects, and a range of diseases which manifest later in life. Understanding the underlying mechanisms by which calcium regulates these diverse developmental processes remains a challenge; however, this knowledge will potentially enable calcium signaling to be used as a therapeutic target in regenerative and personalized medicine.

Modulation of cellular energetics by galactose and pioglitazone.

The Warburg effect is ameliorated by culturing transformed cells in the presence of galactose instead of glucose as the primary carbon source. However, metabolic consequences may occur in addition to sensitizing the cells to mitochondrial toxins. The screening of pharmaceutical agents against transformed cells while using galactose must therefore be carefully evaluated. Pioglitazone is employed in clinical applications to treat type-2 diabetes but clearly has other off-target effects. Human hepatocellular carcinoma cells (HepG2) were cultured in glucose or galactose-containing medium to investigate the role of pioglitazone on cellular bioenergetics by calorimetry and respirometry. Compared with cells cultured in 10 mM glucose, HepG2 cells cultured in the presence of 10 mM galactose showed decreased metabolic activity as measured by cellular heat flow. Interestingly, cellular heat flow increased after the addition of pioglitazone for cells cultured in glucose, but not for cells cultured in galactose. Our calorimetric data indicated that a reduction in cellular capacity for glycolysis was the mechanism responsible for the increase in sensitivity to pioglitazone, and possibly to mitochondrial toxins in general, for cells cultured in galactose. Furthermore, oxygen consumption rates were decreased after the addition of pioglitazone to cells grown in glucose but remained unchanged for cells grown in the presence of galactose. We have demonstrated that pioglitazone induces a reduction in mitochondrial activity that is partially compensated via an increase in glycolysis in the presence of glucose.

Youth Suicide in Nepal: A Growing Concern.

In Nepal, suicidal behaviour among adolescents is a growing concern, and the country has been in the grip of a major mental health disaster for many years. The issue must be addressed immediately, and there must be urgent and concerted action by state institutions, healthcare practitioners, and society. Official statistics show the increased rates of completed suicide, whereas those cases that attempted suicide and survived the event remain unaccounted for. The fortunate suicide victims, who were timely rescued, resuscitated, and survived; in the recent years as witnessed at a tertiary hospital in the western region of Nepal, depict a grim picture of suicidality among youths, particularly among teenagers and young adults. The authors recognize the urgent need to tackle and address the problem of youth suicide in the country. Keywords: Cultural factors; mental health crisis; Nepal; risk-taking behaviour; suicide prevention.

Deciphering the Calcium Code: A Review of Calcium Activity Analysis Methods Employed to Identify Meaningful Activity in Early Neural Development.

The intracellular and intercellular flux of calcium ions represents an ancient and universal mode of signaling that regulates an extensive array of cellular processes. Evidence for the central role of calcium signaling includes various techniques that allow the visualization of calcium activity in living cells. While extensively investigated in mature cells, calcium activity is equally important in developing cells, particularly the embryonic nervous system where it has been implicated in a wide variety array of determinative events. However, unlike in mature cells, where the calcium dynamics display regular, predictable patterns, calcium activity in developing systems is far more sporadic, irregular, and diverse. This renders the ability to assess calcium activity in a consistent manner extremely challenging, challenges reflected in the diversity of methods employed to analyze calcium activity in neural development. Here we review the wide array of calcium detection and analysis methods used across studies, limiting the extent to which they can be comparatively analyzed. The goal is to provide investigators not only with an overview of calcium activity analysis techniques currently available, but also to offer suggestions for future work and standardization to enable informative comparative evaluations of this fundamental and important process in neural development.

High Suicide Rates among Nepalese Population: Need for Action.

Suicide is a global public health problem and the burden has remained stable for last decades. The age standardized suicide rate was 9.77 per 100,000 in 2019 with males and older population being more affected. Based on age, the highest suicide rate was observed in individuals of age 80 years with suicide rate of 64.9 per 100,000 in male, 18.2 per 100,000 in female and 37.4 per 100,000 in both sexes in this age group. In all age group, the suicide rates are higher among males compared to females. Universal, selective and indicative preventive strategies should be implemented to reduce the burden of suicide in Nepal. Keywords: Nepal; prevention; suicide.

Environmental Determinants of Malaria Prevalence and the Adaptation Strategies in Western Nepal.

BACKGROUND: Current literatures seem devoted only on relating climate change with malaria. Overarching all possible environmental determinants of malaria prevalence addressed by scanty literature in Nepal is found apposite research at this moment. This study aims to explore the environmental determinants of malaria prevalence in western Nepal. METHODS: Cross-sectional data collected from community people were used to identify the environmental determinants of malaria prevalence in western Nepal. Probit and logistic regressions are used for identifying determinants. RESULTS: The results reveal that environmental variables: winter temperature (aOR: 2.14 [95% CI: 1.00-4.56]), flooding (aOR: 2.45 [CI: 1.28-4.69]), heat waves (aOR: 3.14 [CI: 1.16-8.46]) and decreasing river water level (aOR: 0.25 [CI: 0.13-0.47]) are found major factors to influence malaria prevalence in western Nepal. Besides, pipeline drinking water (aOR: 0.37 [0.17-0.81]), transportation facility (aOR: 1.18 [1.07-1.32]) and awareness programs (aOR: 2.62 [0.03-6.65]) are exigent social issues to influence malaria prevalence in Nepal. To be protected from disease induced by environmental problems, households have used extra season specific clothes, iron nets and mosquito nets, use of insecticide in cleaning toilet and so on. CONCLUSIONS: Adaptation mechanism against these environmental issues together with promoting pipeline drinking water, transportation facility and awareness programs are the important in malaria control in Nepal. Government initiation with incentivized adaptation mechanism for the protection of environment with caring household attributes possibly help control malaria in western Nepal.

Ordinance on the Safety and Security of Health Workers and Health Institutions in Nepal: A Critical Analysis.

Violence against health workers has been considered a common issue throughout the world. The protection of health workers in low and middle-income countries such as Nepal has not been considered a serious issue by the government. But due to the surge of COVID-19 pandemic and increasing violence against health workers, commendable steps have been taken by the Government of Nepal to protect the safety and security of health workers and health institutions in Nepal. However, the question mark on effective implementation of the ordinance exits suggesting for the appropriate action from concerned authorities and strong collaboration among these sectors. Keywords: Health worker; Nepal; ordinance; safety.

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis.

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological processes. In particular, appropriate regulation of calcium activity patterns during embryogenesis is necessary for many aspects of vertebrate neural development, including proper neural tube closure, synaptogenesis, and neurotransmitter phenotype specification. While the observation that calcium activity patterns can differ in both frequency and amplitude suggests a compelling mechanism by which these fluxes might transmit encoded signals to downstream effectors and regulate gene expression, existing population-level approaches have lacked the precision necessary to further explore this possibility. Furthermore, these approaches limit studies of the role of cell-cell interactions by precluding the ability to assay the state of neuronal determination in the absence of cell-cell contact. Therefore, we have established an experimental workflow that pairs time-lapse calcium imaging of dissociated neuronal explants with a fluorescence in situ hybridization assay, allowing the unambiguous correlation of calcium activity pattern with molecular phenotype on a single-cell level. We were successfully able to use this approach to distinguish and characterize specific calcium activity patterns associated with differentiating neural cells and neural progenitor cells, respectively; beyond this, however, the experimental framework described in this article could be readily adapted to investigate correlations between any time-series activity profile and expression of a gene or genes of interest.

Expression of trpv channels during Xenopus laevis embryogenesis.

Transient receptor potential (TRP) cation channel genes code for an extensive family of conserved proteins responsible for a variety of physiological processes, including sensory perception, ion homeostasis, and chemical signal transduction. The TRP superfamily consists of seven subgroups, one of which is the transient receptor potential vanilloid (trpv) channel family. While trpv channels are relatively well studied in adult vertebrate organisms given their role in functions such as nociception, thermoregulation, and osmotic regulation in mature tissues and organ systems, relatively little is known regarding their function during embryonic development. Although there are some reports of the expression of specific trpv channels at particular stages in various organisms, there is currently no comprehensive analysis of trpv channels during embryogenesis. Here, performing in situ hybridization, we examined the spatiotemporal expression of trpv channel mRNA during early Xenopus laevis embryogenesis. Trpv channels exhibited unique patterns of embryonic expression at distinct locations including the trigeminal ganglia, spinal cord, cement gland, otic vesicle, optic vesicle, nasal placode, notochord, tailbud, proctodeum, branchial arches, epithelium, somite and the animal pole during early development. We have also observed the colocalization of trpv channels at the animal pole (trpv 2/4), trigeminal ganglia (trpv 1/2), and epithelium (trpv 5/6). These localization patterns suggest that trpv channels may play diverse roles during early embryonic development.

The genetic insulator RiboJ increases expression of insulated genes.

A primary objective of synthetic biology is the construction of genetic circuits with behaviors that can be predicted based on the properties of the constituent genetic parts from which they are built. However a significant issue in the construction of synthetic genetic circuits is a phenomenon known as context dependence in which the behavior of a given part changes depending on the choice of adjacent or nearby parts. Interactions between parts compromise the modularity of the circuit, impeding the implementation of predictable genetic constructs. To address this issue, investigators have devised genetic insulators that prevent these unintended context-dependent interactions between neighboring parts. One of the most commonly used insulators in bacterial systems is the self-cleaving ribozyme RiboJ. Despite its utility as an insulator, there has been no systematic quantitative assessment of the effect of RiboJ on the expression level of downstream genetic parts. Here, we characterized the impact of insulation with RiboJ on expression of a reporter gene driven by a promoter from a library of 24 frequently employed constitutive promoters in an Escherichia coli model system. We show that, depending on the strength of the promoter, insulation with RiboJ increased protein abundance between twofold and tenfold and increased transcript abundance by an average of twofold. This result demonstrates that genetic insulators in E. coli can impact the expression of downstream genes, information that is essential for the design of predictable genetic circuits and constructs.

Genome Sequences of 19 Rhodococcus erythropolis Cluster CA Phages.

We report the complete genome sequences of 19 cluster CA bacteriophages isolated from environmental samples using Rhodococcus erythropolis as a host. All of the phages are Siphoviridae, have similar genome lengths (46,314 to 46,985 bp) and G+C contents (58.5 to 58.8%), and share nucleotide sequence similarity.

Genome Sequences of Mycobacteriophages Amgine, Amohnition, Bella96, Cain, DarthP, Hammy, Krueger, LastHope, Peanam, PhelpsODU, Phrank, SirPhilip, Slimphazie, and Unicorn.

We report the genome sequences of 14 cluster K mycobacteriophages isolated using Mycobacterium smegmatis mc²155 as host. Four are closely related to subcluster K1 phages, and 10 are members of subcluster K6. The phage genomes span considerable sequence diversity, including multiple types of integrases and integration sites.