D-Galactose Decreases MitoNEET Levels in Liver Cancer Cells: Impact on Cellular Bioenergetics

Galactose is a simple sugar that at supraphysiological concentrations accelerates aging and age-related complications, which lead to impaired mitochondrial functions. MitoNEET is a small mitochondrial membrane protein with a molecular mass of 12.2 kDa that functions in diabetes, iron metabolism, regulation of oxidative phosphorylation (OXPHOS) and reactive oxygen species (ROS) homeostasis. MitoNEET knockdown increases cellular respiration rates and ROS levels similar to galactose treatment. Pioglitazone, an antidiabetic drug, binds to mitoNEET and ameliorates galactose toxicity. Cellular mitoNEET levels, exposure of cells to galactose medium, and pioglitazone treatment directly influence cellular respiration. To elucidate the role of mitoNEET in galactose induced toxicity (aging), we measured cellular mitoNEET levels using immunoblotting technique in galactose and pioglitazone treated human liver cancer cells (HepG2), as well as isolated synaptosomes from old and young mice. We also monitored liver cancer cell bioenergetics, using respirometry and calorimetry, of galactose and pioglitazone treated cells. Immunoblotting revealed treatment with galactose reduced endogenous mitoNEET levels and those of a chimeric mitoNEET protein fused to a green fluorescent protein (mNT-GFP), under control of the human cytomegalovirus (CMV) promotor. Furthermore, to determine whether mitoNEET mediates pioglitazone ameliorated galactose toxicity, cells were treated with pioglitazone. Pioglitazone rescued galactose reduced mitoNEET levels in a dose dependent manner. A concentration of 60 μM pioglitazone reduced cellular mitoNEET levels compared to controls but not mNT-GFP levels. However, pioglitazone at lower concentrations partially restored mitoNEET levels observed with galactose treatment. Since the synthesis of endogenous mitoNEET and mNT-GFP are regulated by different promoters, D-galactose treatment likely increased degradation rates of mitoNEET. Moreover, unlike galactose treatment in HepG2 cells, immunoblotting revealed higher mitoNEET levels in synaptosomes isolated from aged mice compared to their younger counterparts. We hypothesized that galactose and pioglitazone interact with mitoNEET and alter cellular bioenergetics. Acute treatment with pioglitazone significantly reduced complex I respiration in HepG2 and HepG2-mNT-GFP cells independent of treatment with galactose. Surprisingly, galactose treated HepG2-mNT-GFP cells were less sensitive to pioglitazone treatment. HepG2 cells treated with galactose and HepG2-mNT-GFP cells independent of treatment showed higher OXPHOS activity compared to controls in absence of galactose, but HepG2-mNTGFP cells did not respond to galactose treatment. Unlike respiration, galactose treatment significantly reduced cellular heat flow in HepG2 and HepG2-mNT-GFP cells measured via calorimetry. However, HepG2-mNT-GFP and pioglitazone treated cells showed higher heat dissipation compared to control HepG2 cells without galactose. Our results show that mitoNEET overexpression increases respiration rates and overall energy transduction in HepG2 cells but does not impact the response to pioglitazone treatment

Effect of altitude and shade on production and physical attributes of Coffee in Gulmi, Syangja and Palpa districts of Nepal

Coffee (Coffea spps.) is the second most traded commodity in the world after raw oil. Coffee is grown in mid hills of Nepal from an altitude of 700masl to 1500masl under different shade management practices. Nepalese coffee farmers grow coffee in a traditional way with almost zero application of inorganic fertilizers, pesticides and hence Nepalese coffee is popular as organic coffee or specialty coffee in the world. A study was carried out in three Coffee potent adjoining districts of Nepal: Gulmi, Syangja and Palpa. Ripe coffee cherries were harvested from every 200m altitude from 700masl to 1500masl under shade management and without shade management practices. Different physical attributes such as 1000 cherry weight, wet parchment weight, dry parchment weight, green beans weight, defected beans, and green bean diameter were observed. Production from each altitude level was recorded and highest production (7.04 kg per plant) was obtained from an altitude of 900-1100masl . The highest 1000 cherry weight (1297.17g) and the highest green bean weight (450.33 g) were obtained from 900-1100masl. Under no shade management, number of defected beans were 98 per 1000 beans whereas it was 64 under shade. The interaction of altitude and shade management practice had significant effect upon production (P=0.035), 1000 cherry weight (P<0.001), dry parchment weight (P=0.049) and green bean weight (P<0.05). Coffee produced at an altitude of 900-1100masl under shade management practice were found to have higher production and of better quality with fewer defected beans whereas that produced at extreme lower of 700-900masl and extreme higher altitude of  1300-1500masl were found to have lower production and poor quality

Morphoea Profunda Presenting with Atrophic Skin Lesions in a 26 Year Old Female: A Case Report

Morphoea Profunda is a rare variant of Morphoea that presents clinically as a solitary fibrotic plaque. Morphoea Profunda presenting with atrophic lesions has rarely been reported in literature. We report the case of a 26 year old Nepalese lady who presented to us with multiple non-inflammatory atrophic lesions on her body without significant skin induration, pigmentation and texture change. The findings on histopathology confirmed a diagnosis of Morphoea Profunda. Hence, Morphoea Profunda should be considered in the differential diagnosis of anyone presenting with asymptomatic atrophy of the skin

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

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 that are in addition to sensitizing the cells to mitochondrial toxins may occur. As such, the screening of pharmaceutical agents against transformed cells while using galactose must be carefully evaluated. Pioglitazone is used 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 galactosecontaining medium to investigate the role of pioglitazone on cellular bioenergetics employing calorimetry and respirometry. Compared to 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 addition of pioglitazone for cells cultured in glucose, but not for cells cultured in galactose. Our calorimetric data indicate that a reduction in cellular capacity for glycolysis might be the mechanism responsible for the increase in sensitivity to pioglitazone, and likely mitochondrial toxins in general, for cells cultured in galactose. Furthermore, oxygen consumption rates were decreased after addition of pioglitazone to cells grown in glucose, but remained unchanged for cells grown in presence of galactose. Taken together, we demonstrate that pioglitazone induced a reduction in mitochondrial activity that was partially compensated via an increase in glycolysis in the presence of glucose

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

Factors associated with utilization of adolescent-friendly services in Bhaktapur district, Nepal

Background: The status of adolescent sexual and reproductive health (SRH) in Nepal is alarming. Adolescent-friendly services (AFS) were introduced to cater the health needs of adolescents. Optimal utilization of the services with wider accessibility is required to prevent adolescents from adopting life-threatening behaviors that result in poor SRH-related outcomes. Despite the upgrading of health facilities to adolescent-friendly sites, studies reveal low utilization of the service. However, these studies failed to explore the factors influencing the low levels of service utilization in these adolescent-friendly facilities. This study quantified the utilization of AFS and identified factors associated with its utilization among adolescents of Bhaktapur district. Methods: A cross-sectional survey of 362 systematic randomly selected adolescents from four village development committees of Bhaktapur district was conducted, using a self-administered questionnaire. Relationships between utilization of AFS and associated factors were determined by multivariate logistic regression at a level of significance with a p value of less than 0.05 and adjusted odds ratio. Key informant interviews and focus group discussions with adolescents were used to collect qualitative data which were then described using thematic analysis. Result: About a quarter (24.7%) of the respondents had utilized the adolescent-friendly services. Factors positively associated with the utilization of services included adolescents aged 15-19 years, female, heard about AFS, lack of fear of being seen while getting SRH services, lack of shyness about receiving SRH services, and the perceived need for SRH services as soon as illness became apparent. The qualitative findings revealed lack of awareness about the services, socio-cultural barriers, confidentiality, feasible service hours, and the preference for of same-sex service providers as the factors affecting utilization. Conclusion: The utilization of adolescent-friendly services was very low in Bhaktapur district. Most of the adolescents were unaware of the existence of the AFS which emphasizes the need to focus on the increasing awareness of SRH and AFS by the government in coordination with local schools, clubs, etc. Creating an enabling environment in the service delivery sites, and ensuring privacy and confidentiality, as well as ensuring same-sex service providers and feasible service hours to adolescents, could increase the service utilization

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

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