G protein-coupled receptor signaling in human ductal pancreatic cancer cells: neurotensin responsiveness and mitogenic stimulation

Neuropeptides and their corresponding G protein-coupled receptors (GPCRs) are increasingly implicated in the autocrine/paracrine stimulation of growth of human cancers. We report that neurotensin induced rapid Ca2+ mobilization from intracellular stores followed by Ca2+ influx in five human ductal pancreatic cancer cell lines: HPAF-II, Capan-1, Capan-2, PANC-1, and MIA PaCa-2. In addition, most cell lines exhibited Ca2+ responses to multiple neuropeptides including bombesin, bradykinin, cholecystokinin, and vasopressin and to bioactive lipids, including lysophosphatidic acid (LPA), that also act via GPCRs. The well-differentiated line HPAF-II responded to at least seven independent GPCR agonists. The concentrations of neurotensin required to induce half-maximal effects (EC50) in HPAF-II and PANC-1 cells were 5 and 8nM, respectively. Digital fluorescence image analysis to measure Ca2+ responses in single cells revealed that 90% or more of HPAF-II and PANC-1 cells responded to 10nM neurotensin. Addition of neurotensin to PANC-1 cells also induced rapid and dose-dependent extracellular-regulated protein kinase (ERK-1 and ERK-2) activation and subsequently, stimulated DNA synthesis. The signaling complexity of GPCRs uncovered by these studies reveals a new aspect in the biology of human pancreatic cancer and could offer the basis for new approaches to the treatment of this disease.     

Biomedical Data Commons (BMDC) prioritizes B-lymphocyte non-coding genetic variants in Type 1 Diabetes

The repurposing of biomedical data is inhibited by its fragmented and multi-formatted nature that requires redundant investment of time and resources by data scientists. This is particularly true for Type 1 Diabetes (T1D), one of the most intensely studied common childhood diseases. Intense investigation of the contribution of pancreatic β-islet and T-lymphocytes in T1D has been made. However, genetic contributions from B-lymphocytes, which are known to play a role in a subset of T1D patients, remain relatively understudied. We have addressed this issue through the creation of Biomedical Data Commons (BMDC), a knowledge graph that integrates data from multiple sources into a single queryable format. This increases the speed of analysis by multiple orders of magnitude. We develop a pipeline using B-lymphocyte multi-dimensional epigenome and connectome data and deploy BMDC to assess genetic variants in the context of Type 1 Diabetes (T1D). Pipeline-identified variants are primarily common, non-coding, poorly conserved, and are of unknown clinical significance. While variants and their chromatin connectivity are cell-type specific, they are associated with well-studied disease genes in T-lymphocytes. Candidates include established variants in the HLA-DQB1 and HLA-DRB1 and IL2RA loci that have previously been demonstrated to protect against T1D in humans and mice providing validation for this method. Others are included in the well-established T1D GRS2 genetic risk scoring method. More intriguingly, other prioritized variants are completely novel and form the basis for future mechanistic and clinical validation studies The BMDC community-based platform can be expanded and repurposed to increase the accessibility, reproducibility, and productivity of biomedical information for diverse applications including the prioritization of cell type-specific disease alleles from complex phenotypes.     

Metabolomic analysis elucidates how shade conditions ameliorate the deleterious effects of greening (Huanglongbing) disease in citrus

Under tropical and subtropical environments, citrus leaves are exposed to excess sunlight, inducing photoinhibition. Huanglongbing (HLB, citrus greening), a devastating phloem-limited disease putatively caused by Candidatus Liberibacter asiaticus, exacerbates this challenge with additional photosynthetic loss and excessive starch accumulation. A combined metabolomics and physiological approach was used to elucidate whether shade alleviates the deleterious effects of HLB in field-grown citrus trees, and to understand the underlying metabolic mechanisms related to shade-induced morpho-physiological changes in citrus. Using metabolite profiling and multinomial logistic regression, we identified pivotal metabolites altered in response to shade. A core metabolic network associated with shade conditions was identified through pathway enrichment analysis and metabolite mapping. We measured physio-biochemical responses and growth and yield characteristics. With these, the relationships between metabolic network and the variables measured above were investigated. We found that moderate-shade alleviates sink limitation by preventing excessive starch accumulation and increasing foliar sucrose levels. Increased growth and fruit yield in shaded compared with non-shaded trees were associated with increased photosystem II efficiency and leaf carbon fixation pathway metabolites. Our study also shows that, in HLB-affected trees under shade, the signaling of plant hormones (auxins and cytokinins) and nitrogen supply were downregulated with reducing new shoot production likely due to diminished needs of cell damage repair and tissue regeneration under shade. Overall, our findings provide the first glimpse of the complex dynamics between cellular metabolites and leaf physiological functions in citrus HLB pathosystem under shade, and reveal the mechanistic basis of how shade ameliorates HLB disease.     

Transmission of time varying magnetic field through body tissue

Based on the principles of interaction of electromagnetic waves with semi-infinite media, a simplified model is developed for the study of biological tissue — EM wave interaction problems. The four common types of biological tissues viz, bone, blood, alveolar and muscle tissue, each in turn has been considered. Expression for the transmission loss of the incident EM waves have been derived and numerically evaluated. The results indicate that there is a frequency dependence of the transmission loss and variations with tissue type exist and that losses become significant above 30 MHz. Attenuation for muscular tissue is maximum at frequencies of about 200 MHz whereas the corresponding frequency for lung tissue is nearly the double. Suitable experiments have been designed and performed to verify the salient results of the model and a fair degree of agreement has been found between the experimental results and theoretical predictions. Possibilities for the improvement of the model and future applications of the model have been outlined.     

Distribution and Determinants of Cytomegalovirus Induced End Organ Disease/s among People Living with HIV/AIDS in a Poor Resource Setting: Observation from India

BackgroundIn India, despite well-established anti-retroviral treatment programs, Cytomegalovirus (CMV) infection-related end-organ diseases (EODs) still remain a major concern resulting in exacerbation of morbidity and mortality among HIV/AIDS patients. A prospective study was designed to understand the distribution and prognosis of CMV associated EODs and to determine a standardized cut-off value for serum CMV viral load associated with the development of EODs amongst HIV/AIDS subjects.MethodsIn a cohort of 400 late-diagnosed HAART naïve HIV/AIDS subjects attending anti-retroviral centers of Kolkata during 2008–2014, the median duration of follow-up was 560 days, and at least 3 visits subsequent to the baseline were mandatory for eligibility. HIV-1 and CMV viral load were estimated by performing Real-Time Polymerase Chain Reactions (PCR).ResultsAmong subjects, 40.5% (162/400) had CMV EODs which were more common at lower CD4 counts. Poor prognosis and higher death rate were associated with a low CD4 count and increased HIV-1 and CMV viral loads. Subjects having higher CD4 count responded better to therapy [for CD4 = 60–100: Risk Ratio:RR = 1.48 (95% Confidence Interval: 95%CI = 1.18–1.82) and for CD4 = 30–59: RR = 1.64 (95%CI = 1.18–2.27)]. The cut off value of the serum CMV viral load (expressed as log10DNA/ml serum) associated with the development of EODs and disseminated CMV EODs was determined as 5.4 (p<0.0001) and 6.4 (p<0.0001) respectively. These cut offs were found to have satisfactorily high sensitivity, specificity, positive and negative predictive values.ConclusionPrognosis of CMV EOD was poor as indicated by higher death rates among subjects with lower CD4 count, and specific cut-off values were found to have useful potential for identification and treatment of CMV infected HIV/AIDS patients in due time to avoid CMV EODs among HIV/AIDS subjects. Targeted intervention programs seemed to be required urgently to make these cut-offs operational in order to minimize the burden of CMV EOD in this vulnerable population.     

PKDL—A Silent Parasite Pool for Transmission of Leishmaniasis in Kala-azar Endemic Areas of Malda District,West Bengal,India

Post Kala-azar Dermal Leishmaniasis (PKDL) is a chronic but not life-threatening disease; patients generally do not demand treatment, deserve much more attention because PKDL is highly relevant in the context of Visceral Leishmaniasis (VL) elimination. There is no standard guideline for diagnosis and treatment for PKDL. A species-specific PCR on slit skin smear demonstrated a sensitivity of 93.8%, but it has not been applied for routine diagnostic purpose. The study was conducted to determine the actual disease burden in an endemic area of Malda district, West Bengal, comparison of the three diagnostic tools for PKDL case detection and pattern of lesion regression after treatment. The prevalence of PKDL was determined by active surveillance and confirmed by PCR based diagnosis. Patients were treated with either sodium stibogluconate (SSG) or oral miltefosine and followed up for two years to observe lesion regression period. Twenty six PKDL cases were detected with a prevalence rate of 27.5% among the antileishmanial antibody positive cases. Among three diagnostic methods used, PCR is highly sensitive (88.46%) for case confirmation. In majority of the cases skin lesions persisted after treatment completion which gradually disappeared during 6–12 months post treatment period. Reappearance of lesions noted in two cases after 1.5 years of miltefosine treatment. A significant number of PKDL patients would remain undiagnosed without active mass surveys. Such surveys are required in other endemic areas to attain the ultimate goal of eliminating Kala-azar. PCR-based method is helpful in confirming diagnosis of PKDL, referral laboratory at district or state level can achieve it. So a well-designed study with higher number of samples is essential to establish when/whether PKDL patients are free from parasite after treatment and to determine which PKDL patients need treatment for longer period.     

Integrative Metabolic Signatures for Hepatic Radiation Injury

Background

Radiation-induced liver disease (RILD) is a dose-limiting factor in curative radiation therapy (RT) for liver cancers, making early detection of radiation-associated liver injury absolutely essential for medical intervention. A metabolomic approach was used to determine metabolic signatures that could serve as biomarkers for early detection of RILD in mice.

Methods

Anesthetized C57BL/6 mice received 0, 10 or 50 Gy Whole Liver Irradiation (WLI) and were contrasted to mice, which received 10 Gy whole body irradiation (WBI). Liver and plasma samples were collected at 24 hours after irradiation. The samples were processed using Gas Chromatography/Mass Spectrometry and Liquid Chromatography/Mass Spectrometry.

Results

Twenty four hours after WLI, 407 metabolites were detected in liver samples while 347 metabolites were detected in plasma. Plasma metabolites associated with 50 Gy WLI included several amino acids, purine and pyrimidine metabolites, microbial metabolites, and most prominently bradykinin and 3-indoxyl-sulfate. Liver metabolites associated with 50 Gy WLI included pentose phosphate, purine, and pyrimidine metabolites in liver. Plasma biomarkers in common between WLI and WBI were enriched in microbial metabolites such as 3 indoxyl sulfate, indole-3-lactic acid, phenyllactic acid, pipecolic acid, hippuric acid, and markers of DNA damage such as 2-deoxyuridine. Metabolites associated with tryptophan and indoles may reflect radiation-induced gut microbiome effects. Predominant liver biomarkers in common between WBI and WLI were amino acids, sugars, TCA metabolites (fumarate), fatty acids (lineolate, n-hexadecanoic acid) and DNA damage markers (uridine).

Conclusions

We identified a set of metabolomic markers that may prove useful as plasma biomarkers of RILD and WBI. Pathway analysis also suggested that the unique metabolic changes observed after liver irradiation was an integrative response of the intestine, liver and kidney.     

I-OmiI and I-OmiII: Two intron-encoded homing endonucleases within the Ophiostoma minus rns gene

The mitochondrial small subunit ribosomal RNA (rns) gene of the ascomycetous fungus Ophiostoma minus [strain WIN(M)371] was found to contain a group IC2 and a group IIB1 intron at positions mS569 and mS952 respectively. Both introns have open reading frames (ORFs) embedded that encode double motif LAGLIDADG homing endonucleases (I-OmiI and I-OmiII respectively). Codon-optimized versions of I-OmiI and I-OmiII were synthesized for overexpression in Escherichia coli. The in vitro characterization of I-OmiII showed that it is a functional homing endonuclease that cleaves the rns target site two nucleotides upstream (sense strand) of the intron insertion site generating 4 nucleotide 3′ overhangs. The endonuclease activity of I-OmiII was tested using linear and circular substrates and cleavage activity was evaluated at various temperatures. The I-OmiI protein was expressed in E. coli, but purification was difficult, thus the endonuclease activity of this protein was tested via in vivo assays. Overall this study showed that there are many native forms of functional homing endonucleases yet to be discovered among fungal mtDNA genomes.