Deep Whole-Genome Sequencing of 100 Southeast Asian Malays

Whole-genome sequencing across multiple samples in a population provides an unprecedented opportunity for comprehensively characterizing the polymorphic variants in the population. Although the 1000 Genomes Project (1KGP) has offered brief insights into the value of population-level sequencing, the low coverage has compromised the ability to confidently detect rare and low-frequency variants. In addition, the composition of populations in the 1KGP is not complete, despite the fact that the study design has been extended to more than 2,500 samples from more than 20 population groups. The Malays are one of the Austronesian groups predominantly present in Southeast Asia and Oceania, and the Singapore Sequencing Malay Project (SSMP) aims to perform deep whole-genome sequencing of 100 healthy Malays. By sequencing at a minimum of 30× coverage, we have illustrated the higher sensitivity at detecting low-frequency and rare variants and the ability to investigate the presence of hotspots of functional mutations. Compared to the low-pass sequencing in the 1KGP, the deeper coverage allows more functional variants to be identified for each person. A comparison of the fidelity of genotype imputation of Malays indicated that a population-specific reference panel, such as the SSMP, outperforms a cosmopolitan panel with larger number of individuals for common SNPs. For lower-frequency (<5%) markers, a larger number of individuals might have to be whole-genome sequenced so that the accuracy currently afforded by the 1KGP can be achieved. The SSMP data are expected to be the benchmark for evaluating the value of deep population-level sequencing versus low-pass sequencing, especially in populations that are poorly represented in population-genetics studies.     

A comparison of the palaeolimnology of Peipsi and Võrtsjärv: connected shallow lakes in north-eastern Europe for the twentieth century, especially in relation to eutrophication progression and water-level fluctuations

We applied a multi-proxy palaeolimnological approach to provide insights into the natural variability and human-mediated trends of two interconnected temperate large shallow lakes, Peipsi and Võrtsjärv, during the twentieth century. The history of the lakes was assessed on the basis of age-related changes in the sediment main constituents (water, organic matter and carbonate), sub-fossil pigments, diatom assemblages and organic matter dissolved in pore water. The temporal changes in the palaeodata indicate an increase of the in-lake biological production in both lakes from about the 1960s, suggesting enhanced nutrient inputs. In subsequent decades, the gradual increase of autochthonous organic matter becomes more obvious, indicating progressive eutrophication of the lakes. Palaeolimnological indicators from the sediment record of Lake Peipsi indicate a slight recession of the lake’s eutrophication in the 1990s but not for Lake Võrtsjärv. The results of the study also suggest that after the lakes became eutrophied, the climatically induced water-level fluctuations ceased to be the main driver determining the abundance of phytoplankton. Responses of the lakes to human-induced impacts are better recorded in the sediments of Lake Peipsi than in those of Lake Võrtsjärv, which is shallower of the two and where the wave-induced resuspension of deposits markedly smooths or erases the signals of environmental changes. The results of the investigation expand the knowledge on how large shallow lakes respond to human-mediated and natural perturbations, including those in the lake catchment areas and the capability of the lakes to store the chronology and sequence of these changes.     

Stress-Induced Visceral Hypersensitivity in Maternally Separated Rats Can Be Reversed by Peripherally Restricted Histamine-1-Receptor Antagonists

Background

The histamine-1 receptor (H1R) antagonist ketotifen increased the threshold of discomfort in hypersensitive IBS patients. The use of peripherally restricted and more selective H1R antagonists may further improve treatment possibilities. We examined the use of fexofenadine and ebastine to reverse post-stress visceral hypersensitivity in maternally separated rats.

Methods

The visceromotor response to colonic distension was assessed in adult maternally separated and nonhandled rats pre- and 24 hours post water avoidance. Subsequently rats were treated with vehicle alone or different dosages of fexofenadine (1.8 and 18 mg/kg) or ebastine (0.1 and 1.0 mg/kg) and re-evaluated. Colonic tissue was collected to assess relative RMCP-2 and occludin expression levels by Western blot and histamine-1 receptor by RT-qPCR. β-hexosaminidase release by RBL-2H3 cells was used to establish possible mast cell stabilizing properties of the antagonists.

Key results

Water avoidance only induced enhanced response to distension in maternally separated rats. This response was reversed by 1.8 and 18 mg/kg fexofenadine. Reversal was also obtained by 1.0 but not 0.1 mg/kg ebastine. RMCP-2 expression levels were comparable in these two ebastine treatment groups but occludin was significantly higher in 1.0 mg/kg treated rats. There were no differences in histamine-1 receptor expression between nonhandled and maternally separated rats. Fexofenadine but not ebastine showed mast cell stabilizing quality.

Conclusions

Our results indicate that the peripherally restricted 2^nd generation H1-receptor antagonists fexofenadine and ebastine are capable of reversing post stress visceral hypersensitivity in rat. These data justify future IBS patient trials with these well tolerated compounds.     

Complications of Antiretroviral Therapy Initiation in Hospitalised Patients with HIV-Associated Tuberculosis

Background

HIV-associated tuberculosis is a common coinfection in Sub-Saharan Africa, which causes high morbidity and mortality. A sub-set of HIV-associated tuberculosis patients require prolonged hospital admission, during which antiretroviral therapy initiation may be required. The aim of this study was to document the causes of clinical deterioration of hospitalised patients with HIV-associated tuberculosis starting antiretroviral therapy in order to inform healthcare practice in low- to middle-income countries.

Methods

Prospective, observational cohort study of adult inpatients with HIV-associated tuberculosis starting antiretroviral therapy in a dedicated tuberculosis hospital in Cape Town, South Africa. Causes of clinical deterioration and outcome were recorded in the first 12 weeks of antiretroviral therapy. Patients with rifampicin-resistant tuberculosis were excluded.

Results

Between May 2009 and November 2010, 112 patients (60% female), with a median age of 32 years were enrolled. At baseline the median CD4 count was 55 cells/mm³ (IQR 31–106) and HIV viral load 5.6 log copies/mL. All patients had significant comorbidity: 82% were bed-bound, 65% had disseminated tuberculosis and 27% had central nervous system tuberculosis. Seventy six patients (68%) developed 144 clinical events after starting antiretroviral therapy. TB-IRIS, hospital-acquired infections and significant drug toxicities occurred in 42%, 20.5% and 15% of patients respectively. A new opportunistic disease occurred in 15% of patients and a thromboembolic event in 8%. Mortality during the 12 week period was 10.6%.

Conclusions

High rates of TB-IRIS, hospital-acquired infections and drug toxicities complicate the course of patients with HIV-associated tuberculosis starting antiretroviral therapy in hospital. Despite the high morbidity, mortality was relatively low. Careful clinical management and adequate resources are needed in hospitalised HIV-TB patients in the 1^st three months following ART initiation.     

The advantage of laser‐capture microdissection over whole tissue analysis in proteomic profiling studies

Laser‐capture microdissection (LCM) offers a reliable cell population enrichment tool and has been successfully coupled to MS analysis. Despite this, most proteomic studies employ whole tissue lysate (WTL) analysis in the discovery of disease biomarkers and in profiling analyses. Furthermore, the influence of tissue heterogeneity in WTL analysis, nor its impact in biomarker discovery studies have been completely elucidated. In order to address this, we compared previously obtained high resolution MS data from a cohort of 38 breast cancer tissues, of which both LCM enriched tumor epithelial cells and WTL samples were analyzed. Label‐free quantification (LFQ) analysis through MaxQuant software showed a significantly higher number of identified and quantified proteins in LCM enriched samples (3404) compared to WTLs (2837). Furthermore, WTL samples displayed a higher amount of missing data compared to LCM both at peptide and protein levels (p‐value < 0.001). 2D analysis on co‐expressed proteins revealed discrepant expression of immune system and lipid metabolisms related proteins between LCM and WTL samples. We hereby show that LCM better dissected the biology of breast tumor epithelial cells, possibly due to lower interference from surrounding tissues and highly abundant proteins. All data have been deposited in the ProteomeXchange with the dataset identifier PXD002381 ( http://proteomecentral.proteomexchange.org/dataset/PXD002381 ).     

Algal Diet of Small-Bodied Crustacean Zooplankton in a Cyanobacteria-Dominated Eutrophic Lake

Small-bodied cladocerans and cyclopoid copepods are becoming increasingly dominant over large crustacean zooplankton in eutrophic waters where they often coexist with cyanobacterial blooms. However, relatively little is known about their algal diet preferences. We studied grazing selectivity of small crustaceans (the cyclopoid copepods Mesocyclops leuckarti, Thermocyclops oithonoides, Cyclops kolensis, and the cladocerans Daphnia cucullata, Chydorus sphaericus, Bosmina spp.) by liquid chromatographic analyses of phytoplankton marker pigments in the shallow, highly eutrophic Lake Võrtsjärv (Estonia) during a seasonal cycle. Copepods (mainly C. kolensis) preferably consumed cryptophytes (identified by the marker pigment alloxanthin in gut contents) during colder periods, while they preferred small non-filamentous diatoms and green algae (identified mainly by diatoxanthin and lutein, respectively) from May to September. All studied cladoceran species showed highest selectivity towards colonial cyanobacteria (identified by canthaxanthin). For small C. sphaericus, commonly occuring in the pelagic zone of eutrophic lakes, colonial cyanobacteria can be their major food source, supporting their coexistence with cyanobacterial blooms. Pigments characteristic of filamentous cyanobacteria and diatoms (zeaxanthin and fucoxanthin, respectively), algae dominating in Võrtsjärv, were also found in the grazers’ diet but were generally avoided by the crustaceans commonly dominating the zooplankton assemblage. Together these results suggest that the co-occurring small-bodied cyclopoid and cladoceran species have markedly different algal diets and that the cladocera represent the main trophic link transferring cyanobacterial carbon to the food web in a highly eutrophic lake.     

The Role of Geography in the Assessment of Quality: Evidence from the Medicare Advantage Program

The Affordable Care Act set in motion a renewed emphasis on quality of care evaluation. However, the evaluation strategies of quality by the Centers for Medicare and Medicaid Services do not consider geography when comparisons are made among plans. Using an overall measure of a plan’s quality in the public sector—the Medicare Advantage (MA) star ratings—we explored the impact of geography in these ratings. We identified 2,872 U.S counties in 2010. The geographic factor predicted a larger fraction of the MA ratings’ compared to socio-demographic factors which explained less. Also, after the risk adjustments, almost half of the U.S. states changed their ranked position in the star ratings. Further, lower MA star ratings were identified in the Southeastern region. These findings suggest that the geographic component effect on the ratings is not trivial and should be considered in future adjustments of the metric, which may enhance the transparency, accountability, and importantly level the playing field more effectively when comparing quality across health plans.     

Preferential Secretion of Thymic Stromal Lymphopoietin (TSLP) by Terminally Differentiated Esophageal Epithelial Cells: Relevance to Eosinophilic Esophagitis (EoE)

Eosinophilic esophagitis (EoE) is a chronic Th2 and food antigen-mediated disease characterized by esophageal eosinophilic infiltration. Thymic stromal lymphopoetin (TSLP), an epithelial derived cytokine which bridges innate and Th2-type adaptive immune responses in other allergic conditions, is overexpressed in esophageal biopsies of EoE subjects. However, the triggers of TSLP expression in the esophageal epithelium are unknown. The objective of the current study was to characterize TSLP expression in human esophageal epithelium in EoE in vivo and to determine the role of food antigens upon epithelial TSLP expression in vitro. Using immunohistochemistry (IHC), we localized TSLP in esophageal biopsies of active EoE (≥15 eos/hpf), inactive EoE (<15 eos/hpf) and non-EoE control subjects, and found that TSLP expression was restricted to the differentiated suprabasal layer of the epithelium in actively inflamed EoE biopsies. Consistent with these results in vivo, inducible TSLP protein secretion was higher in CaCl₂ differentiated telomerase-immortalized esophageal epithelial cells (EPC2-hTERT) compared to undifferentiated cells of the basal phenotype, following stimulation with the TLR3 ligand poly(I:C). To determine whether food antigens could directly induce epithelial TSLP secretion, differentiated and undifferentiated primary esophageal epithelial cells from EoE and non-EoE subjects were challenged with food antigens clinically relevant to EoE: Chicken egg ovalbumin (OVA), wheat, and milk proteins beta-lactoglobulin (blg) and beta-casein. Food antigens failed to induce TSLP secretion by undifferentiated cells; in contrast, only OVA induced TSLP secretion in differentiated epithelial cells from both EoE and control cell lines, an effect abolished by budesonide and NF-κb inhibition. Together, our study shows that specific food antigens can trigger innate immune mediated esophageal TSLP secretion, suggesting that esophageal epithelial cells at the barrier surface may play a significant role in the pathogenesis of EoE by regulating TSLP expression.     

CD45 Isoform Profile Identifies Natural Killer (NK) Subsets with Differential Activity

The leucocyte-specific phosphatase CD45 is present in two main isoforms: the large CD45RA and the short CD45RO. We have recently shown that distinctive expression of these isoforms distinguishes natural killer (NK) populations. For example, co-expression of both isoforms identifies in vivo the anti tumor NK cells in hematological cancer patients. Here we show that low CD45 expression associates with less mature, CD56^bright, NK cells. Most NK cells in healthy human donors are CD45RA⁺CD45RO^-. The CD45RA^-RO⁺ phenotype, CD45RO cells, is extremely uncommon in B or NK cells, in contrast to T cells. However, healthy donors possess CD45RA^dimRO^- (CD45RA^dim cells), which show immature markers and are largely expanded in hematopoietic stem cell transplant patients. Blood borne cancer patients also have more CD45RA^dim cells that carry several features of immature NK cells. However, and in opposition to their association to NK cell progenitors, they do not proliferate and show low expression of the transferrin receptor protein 1/CD71, suggesting low metabolic activity. Moreover, CD45RA^dim cells properly respond to in vitro encounter with target cells by degranulating or gaining CD69 expression. In summary, they are quiescent NK cells, with low metabolic status that can, however, respond after encounter with target cells.