Multi-parallel qPCR provides increased sensitivity and diagnostic breadth for gastrointestinal parasites of humans: field-based inferences on the impact of mass deworming

BACKGROUND: Although chronic morbidity in humans from soil transmitted helminth (STH) infections can be reduced by anthelmintic treatment, inconsistent diagnostic tools make it difficult to reliably measure the impact of deworming programs and often miss light helminth infections. METHODS: Cryopreserved stool samples from 796 people (aged 2-81 years) in four villages in Bungoma County, western Kenya, were assessed using multi-parallel qPCR for 8 parasites and compared to point-of-contact assessments of the same stools by the 2-stool 2-slide Kato-Katz (KK) method. All subjects were treated with albendazole and all Ascaris lumbricoides expelled post-treatment were collected. Three months later, samples from 633 of these people were re-assessed by both qPCR and KK, re-treated with albendazole and the expelled worms collected. RESULTS: Baseline prevalence by qPCR (n = 796) was 17 % for A. lumbricoides, 18 % for Necator americanus, 41 % for Giardia lamblia and 15% for Entamoeba histolytica. The prevalence was <1% for Trichuris trichiura, Ancylostoma duodenale, Strongyloides stercoralis and Cryptosporidium parvum. The sensitivity of qPCR was 98% for A. lumbricoides and N. americanus, whereas KK sensitivity was 70% and 32%, respectively. Furthermore, qPCR detected infections with T. trichiura and S. stercoralis that were missed by KK, and infections with G. lamblia and E. histolytica that cannot be detected by KK. Infection intensities measured by qPCR and by KK were correlated for A. lumbricoides (r = 0.83, p < 0.0001) and N. americanus (r = 0.55, p < 0.0001). The number of A. lumbricoides worms expelled was correlated (p < 0.0001) with both the KK (r = 0.63) and qPCR intensity measurements (r = 0.60). CONCLUSIONS: KK may be an inadequate tool for stool-based surveillance in areas where hookworm or Strongyloides are common or where intensity of helminth infection is low after repeated rounds of chemotherapy. Because deworming programs need to distinguish between populations where parasitic infection is controlled and those where further treatment is required, multi-parallel qPCR (or similar high throughput molecular diagnostics) may provide new and important diagnostic information

Amyloid Plaques Beyond Aβ: A Survey of the Diverse Modulators of Amyloid Aggregation

Aggregation of the amyloid-β (Aβ) peptide is strongly correlated with Alzheimer’s disease (AD). Recent research has improved our understanding of the kinetics of amyloid fibril assembly and revealed new details regarding different stages in plaque formation. Presently, interest is turning toward studying this process in a holistic context, focusing on cellular components which interact with the Aβ peptide at various junctures during aggregation, from monomer to cross-β amyloid fibrils. However, even in isolation, a multitude of factors including protein purity, pH, salt content, and agitation affect Aβ fibril formation and deposition, often producing complicated and conflicting results. The failure of numerous inhibitors in clinical trials for AD suggests that a detailed examination of the complex interactions that occur during plaque formation, including binding of carbohydrates, lipids, nucleic acids, and metal ions, is important for understanding the diversity of manifestations of the disease. Unraveling how a variety of key macromolecular modulators interact with the Aβ peptide and change its aggregation properties may provide opportunities for developing therapies. Since no protein acts in isolation, the interplay of these diverse molecules may differentiate disease onset, progression, and severity, and thus are worth careful consideration

Multi-messenger observations of a binary neutron star merger

On 2017 August 17 a binary neutron star coalescence candidate (later designated GW170817) with merger time 12:41:04 UTC was observed through gravitational waves by the Advanced LIGO and Advanced Virgo detectors. The Fermi Gamma-ray Burst Monitor independently detected a gamma-ray burst (GRB 170817A) with a time delay of ~1.7 s with respect to the merger time. From the gravitational-wave signal, the source was initially localized to a sky region of 31 deg2 at a luminosity distance of 40+8-8 Mpc and with component masses consistent with neutron stars. The component masses were later measured to be in the range 0.86 to 2.26 Mo. An extensive observing campaign was launched across the electromagnetic spectrum leading to the discovery of a bright optical transient (SSS17a, now with the IAU identification of AT 2017gfo) in NGC 4993 (at ~40 Mpc) less than 11 hours after the merger by the One- Meter, Two Hemisphere (1M2H) team using the 1 m Swope Telescope. The optical transient was independently detected by multiple teams within an hour. Subsequent observations targeted the object and its environment. Early ultraviolet observations revealed a blue transient that faded within 48 hours. Optical and infrared observations showed a redward evolution over ~10 days. Following early non-detections, X-ray and radio emission were discovered at the transient’s position ~9 and ~16 days, respectively, after the merger. Both the X-ray and radio emission likely arise from a physical process that is distinct from the one that generates the UV/optical/near-infrared emission. No ultra-high-energy gamma-rays and no neutrino candidates consistent with the source were found in follow-up searches. These observations support the hypothesis that GW170817 was produced by the merger of two neutron stars in NGC4993 followed by a short gamma-ray burst (GRB 170817A) and a kilonova/macronova powered by the radioactive decay of r-process nuclei synthesized in the ejecta

Expression of Bmi-1 is a prognostic marker in bladder cancer

<p>Abstract</p> <p>Background</p> <p>The molecular mechanisms of the development and progression of bladder cancer are poorly understood. The objective of this study was to analyze the expression of Bmi-1 protein and its clinical significance in human bladder cancer.</p> <p>Methods</p> <p>We examined the expression of Bmi-1 mRNA and Bmi-1 protein by RT-PCR and Western blot, respectively in 14 paired bladder cancers and the adjacent normal tissues. The expression of Bmi-1 protein in 137 specimens of bladder cancer and 30 specimens of adjacent normal bladder tissue was determined by immunohistochemistry. Statistical analyses were applied to test the relationship between expression of Bmi-1, and clinicopathologic features and prognosis.</p> <p>Results</p> <p>Expression of Bmi-1 mRNA and protein was higher in bladder cancers than in the adjacent normal tissues in 14 paired samples (<it>P </it>< 0.01). By immunohistochemical examination, five of 30 adjacent normal bladder specimens (16.7%) versus 75 of 137 bladder cancers (54.3%) showed Bmi-1 protein expression (<it>P </it>< 0.05). Bmi-1 protein expression was intense in 20.6%, 54.3%, and 78.8% of tumors of histopathological stages G1, G2, and G3, respectively (<it>P </it>< 0.05). Expression of Bmi-1 protein was greater in invasive bladder cancers than in superficial bladder cancers (81.5% versus 32.5%, <it>P </it>< 0.05). In invasive bladder cancers, the expression of Bmi-1 protein in progression-free cancers was similar to that of cancers that have progressed (80.0% versus 82.4%, <it>P </it>> 0.5). In superficial bladder cancers, the expression of Bmi-1 protein in recurrent cases was higher than in recurrence-free cases (62.5% versus 13.7%, <it>P </it>< 0.05). Bmi-1 expression was positively correlated with tumor classification and TNM stage (<it>P </it>< 0.05), but not with tumor number (<it>P </it>> 0.05). Five-year survival in the group with higher Bmi-1 expression was 50.8%, while it was 78.5% in the group with lower Bmi-1 expression (<it>P </it>< 0.05). Patients with higher Bmi-1 expression had shorter survival time, whereas patients with lower Bmi-1 expression had longer survival time (<it>P </it>< 0.05).</p> <p>Conclusion</p> <p>Expression of Bmi-1 was greater in bladder cancers than in the adjacent normal tissues. The examination of Bmi-1 protein expression is potentially valuable in prognostic evaluation of bladder cancer.</p

TRAIL death receptors DR4 and DR5 mediate cerebral microvascular endothelial cell apoptosis induced by oligomeric Alzheimer's Aβ

Vascular deposition of amyloid-β (Aβ) in sporadic and familial Alzheimer's disease, through poorly understood molecular mechanisms, leads to focal ischemia, alterations in cerebral blood flow, and cerebral micro-/macro-hemorrhages, significantly contributing to cognitive impairment. Here, we show that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptors DR4 and DR5 specifically mediate oligomeric Aβ induction of extrinsic apoptotic pathways in human microvascular cerebral endothelial cells with activation of both caspase-8 and caspase-9. The caspase-8 inhibitor cellular FLICE-like inhibitory protein (cFLIP) is downregulated, and mitochondrial paths are engaged through BH3-interacting domain death agonist (Bid) cleavage. Upregulation of DR4 and DR5 and colocalization with Aβ at the cell membrane suggests their involvement as initiators of the apoptotic machinery. Direct binding assays using receptor chimeras confirm the specific interaction of oligomeric Aβ with DR4 and DR5 whereas apoptosis protection achieved through RNA silencing of both receptors highlights their active role in downstream apoptotic pathways unveiling new targets for therapeutic intervention

Loss of the SxxSS Motif in a Human T-Cell Factor-4 Isoform Confers Hypoxia Resistance to Liver Cancer: An Oncogenic Switch in Wnt Signaling

PURPOSE: Aberrantly activated Wnt/β-catenin signaling is important in hepatocellular carcinoma (HCC) development. Downstream gene expressions involving the Wnt/β-catenin cascade occur through T-cell factor (TCF) proteins. Here, we show the oncogenic potential of human TCF-4 isoforms based on the expression of a single conserved SxxSS motif. METHODS: We investigated the TCF-4J and K isoform pair characterized by the presence (K) or absence (J) of the SxxSS motif. The mRNA expression profiles were examined in 47 pairs of human HCCs and adjacent non-cancerous liver tissues by RT-PCR. Proliferation, sphere assays and immunoblot analysis were performed under normoxia and hypoxia conditions. The ability of HCC cells overexpressing TCF-4J (J cells) and K (K cells) to grow as solid tumors in nude mice was explored. RESULTS: TCF-4J expression was significantly upregulated in HCC tumors compared to corresponding peritumor and normal liver and was preferentially expressed in poorly differentiated HCCs. In contrast, TCF-4K was downregulated in those same HCC tumors. TCF-4J-overexpressing HCC cells (J cells) revealed a survival advantage under hypoxic conditions, high proliferation rate and formation of aggregates/spheres compared to overexpression of TCF-4K (K cells). The hypoxic J cells had high expression levels of HIF-2α and EGFR as possible mechanisms to promote tumorigenesis. Increased stability of HIF-2α under hypoxia in J cells was associated with a decreased level of von Hippel-Lindau (VHL) protein, a known E3 ligase for HIF-αs. In a xenograft model, the J cells rapidly developed tumors compared to K cells. Tumor tissues derived from J cells exhibited high expression levels of HIF-2α and EGFR compared to the slow developing and small K cell derived tumors. CONCLUSIONS: Our results suggest that the specific TCF-4J isoform, which lacks a regulatory SxxSS motif, has robust tumor-initiating potential under hypoxic conditions

Coexisting Cyclic Parthenogens Comprise a Holocene Species Flock in Eubosmina

Background: Mixed breeding systems with extended clonal phases and weak sexual recruitment are widespread in nature but often thought to impede the formation of discrete evolutionary clusters. Thus, cyclic parthenogens, such as cladocerans and rotifers, could be predisposed to ‘‘species problems’ ’ and a lack of discrete species. However, species flocks have been proposed for one cladoceran group, Eubosmina, where putative species are sympatric, and there is a detailed paleolimnological record indicating a Holocene age. These factors make the Eubosmina system suitable for testing the hypotheses that extended clonal phases and weak sexual recruitment inhibit speciation. Although common garden experiments have revealed a genetic component to the morphotypic variation, the evolutionary significance of the morphotypes remains controversial. Methodology/Principal Findings: In the present study, we tested the hypothesis of a single polymorphic species (i.e., mixing occurs but selection maintains genes for morphology) in four northern European lakes where the morphotypes coexist. Our evidence is based on nuclear DNA sequence, mitochondrial DNA sequence, and morphometric analysis of coexisting morphotypes. We found significant genetic differentiation, genealogical exclusivity, and morphometric differentiation for coexisting morphotypes. Conclusions: We conclude that the studied morphotypes represent a group of young species undergoing speciation wit

Novel insights into the mechanisms mediating the local antihypertrophic effects of cardiac atrial natriuretic peptide: role of cGMP-dependent protein kinase and RGS2

Cardiac atrial natriuretic peptide (ANP) locally counteracts cardiac hypertrophy via the guanylyl cyclase-A (GC-A) receptor and cGMP production, but the downstream signalling pathways are unknown. Here, we examined the influence of ANP on β-adrenergic versus Angiotensin II (Ang II)-dependent (Gs vs. Gαq mediated) modulation of Ca2+i-handling in cardiomyocytes and of hypertrophy in intact hearts. L-type Ca2+ currents and Ca2+i transients in adult isolated murine ventricular myocytes were studied by voltage-clamp recordings and fluorescence microscopy. ANP suppressed Ang II-stimulated Ca2+ currents and transients, but had no effect on isoproterenol stimulation. Ang II suppression by ANP was abolished in cardiomyocytes of mice deficient in GC-A, in cyclic GMP-dependent protein kinase I (PKG I) or in the regulator of G protein signalling (RGS) 2, a target of PKG I. Cardiac hypertrophy in response to exogenous Ang II was significantly exacerbated in mice with conditional, cardiomyocyte-restricted GC-A deletion (CM GC-A KO). This was concomitant to increased activation of the Ca2+/calmodulin-dependent prohypertrophic signal transducer CaMKII. In contrast, β-adrenoreceptor-induced hypertrophy was not enhanced in CM GC-A KO mice. Lastly, while the stimulatory effects of Ang II on Ca2+-handling were absent in myocytes of mice deficient in TRPC3/TRPC6, the effects of isoproterenol were unchanged. Our data demonstrate a direct myocardial role for ANP/GC-A/cGMP to antagonize the Ca2+i-dependent hypertrophic growth response to Ang II, but not to β-adrenergic stimulation. The selectivity of this interaction is determined by PKG I and RGS2-dependent modulation of Ang II/AT1 signalling. Furthermore, they strengthen published observations in neonatal cardiomyocytes showing that TRPC3/TRPC6 channels are essential for Ang II, but not for β-adrenergic Ca2+i-stimulation in adult myocytes

Serum big endothelin-1 as a biomarker in oral squamous cell carcinoma patients: an analytical study

Detection of abnormally elevated levels of molecules in patients with oral cancer may be useful in early diagnosis. These markers can be included in current Histopathology grading and in TNM staging systems of Oral Squamous Cell Carcinoma (OSCC) to make it more efficient. Several pro-angiogenic molecules have been assessed for the same reason. Endothelin-1 (ET-1) is a vasoactive peptide associated with the development and spread of many solid tumors, including Squamous Cell Carcinoma (SCC), but its utility in OSCC has not been confirmed. Objective This study aims to evaluate the role of the serum big ET-1 as a biomarker of OSCC, by correlating it with the clinical staging and the histopathological grading. Material and Methods Serum levels of big ET-1 measured by the sandwich Enzyme-Linked Immunosorbent Assay (ELISA) in 40 OSCC cases were compared with the levels from the control group using independent t-test. Clinical stages and histopathological grades of OSCC cases were compared in relation to their mean levels of serum big ET-1, one using the Analysis of Variance (ANOVA) test and the other the independent t-test, respectively. The significance of the mean difference between the groups was evaluated by Tukey&#8217;s multiple comparison test. All statistical analyses were performed on GraphPad statistical software version 5.0. Results By comparing the mean of the big ET-1 concentrations of cases and controls, the independent t-test revealed significant higher big ET-1 concentration of OSCC cases when compared to controls (p<0.0001). Tukey&#8217;s multiple comparison test also revealed statistically significant difference among all OSCC stages in relation to the mean levels of serum big ET-1. However, the mean of the big ET-1 concentrations of cases of grade I and of grade II did not differ statistically (p=0.729). Conclusion Serum big ET-1 levels may be useful as a diagnostic tool in OSCC and as an adjunct to OSCC staging. However, its use as a prognostic marker warrants larger prospective studies