Snai1 regulates cell lineage allocation and stem cell maintenance in the mouse intestinal epithelium

Snail family members regulate epithelial‐to‐mesenchymal transition (EMT) during invasion of intestinal tumours, but their role in normal intestinal homeostasis is unknown. Studies in breast and skin epithelia indicate that Snail proteins promote an undifferentiated state. Here, we demonstrate that conditional knockout of Snai1 in the intestinal epithelium results in apoptotic loss of crypt base columnar stem cells and bias towards differentiation of secretory lineages. In vitro organoid cultures derived from Snai1 conditional knockout mice also undergo apoptosis when Snai1 is deleted. Conversely, ectopic expression of Snai1 in the intestinal epithelium in vivo results in the expansion of the crypt base columnar cell pool and a decrease in secretory enteroendocrine and Paneth cells. Following conditional deletion of Snai1, the intestinal epithelium fails to produce a proliferative response following radiation‐induced damage indicating a fundamental requirement for Snai1 in epithelial regeneration. These results demonstrate that Snai1 is required for regulation of lineage choice, maintenance of CBC stem cells and regeneration of the intestinal epithelium following damage.     

BCL::MP‐fold: Membrane protein structure prediction guided by EPR restraints

For many membrane proteins, the determination of their topology remains a challenge for methods like X‐ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. Electron paramagnetic resonance (EPR) spectroscopy has evolved as an alternative technique to study structure and dynamics of membrane proteins. The present study demonstrates the feasibility of membrane protein topology determination using limited EPR distance and accessibility measurements. The BCL::MP‐Fold (BioChemical Library membrane protein fold) algorithm assembles secondary structure elements (SSEs) in the membrane using a Monte Carlo Metropolis (MCM) approach. Sampled models are evaluated using knowledge‐based potential functions and agreement with the EPR data and a knowledge‐based energy function. Twenty‐nine membrane proteins of up to 696 residues are used to test the algorithm. The RMSD100 value of the most accurate model is better than 8 Å for 27, better than 6 Å for 22, and better than 4 Å for 15 of the 29 proteins, demonstrating the algorithms' ability to sample the native topology. The average enrichment could be improved from 1.3 to 2.5, showing the improved discrimination power by using EPR data. Proteins 2015; 83:1947–1962. © 2015 Wiley Periodicals, Inc     

RANTES/CCL5 and risk for coronary events: results from the MONICA/KORA Augsburg case-cohort, Athero-Express and CARDIoGRAM studies

Background

The chemokine RANTES (regulated on activation, normal T-cell expressed and secreted)/CCL5 is involved in the pathogenesis of cardiovascular disease in mice, whereas less is known in humans. We hypothesised that its relevance for atherosclerosis should be reflected by associations between CCL5 gene variants, RANTES serum concentrations and protein levels in atherosclerotic plaques and risk for coronary events.

Methods and Findings

We conducted a case-cohort study within the population-based MONICA/KORA Augsburg studies. Baseline RANTES serum levels were measured in 363 individuals with incident coronary events and 1,908 non-cases (mean follow-up: 10.2±4.8 years). Cox proportional hazard models adjusting for age, sex, body mass index, metabolic factors and lifestyle factors revealed no significant association between RANTES and incident coronary events (HR [95% CI] for increasing RANTES tertiles 1.0, 1.03 [0.75–1.42] and 1.11 [0.81–1.54]). None of six CCL5 single nucleotide polymorphisms and no common haplotype showed significant associations with coronary events. Also in the CARDIoGRAM study (>22,000 cases, >60,000 controls), none of these CCL5 SNPs was significantly associated with coronary artery disease. In the prospective Athero-Express biobank study, RANTES plaque levels were measured in 606 atherosclerotic lesions from patients who underwent carotid endarterectomy. RANTES content in atherosclerotic plaques was positively associated with macrophage infiltration and inversely associated with plaque calcification. However, there was no significant association between RANTES content in plaques and risk for coronary events (mean follow-up 2.8±0.8 years).

Conclusions

High RANTES plaque levels were associated with an unstable plaque phenotype. However, the absence of associations between (i) RANTES serum levels, (ii) CCL5 genotypes and (iii) RANTES content in carotid plaques and either coronary artery disease or incident coronary events in our cohorts suggests that RANTES may not be a novel coronary risk biomarker. However, the potential relevance of RANTES levels in platelet-poor plasma needs to be investigated in further studies.     

A Variant In the Abo Gene Explains the Variation in Soluble E-Selectin Levels—Results from Dense Genotyping in Two Independent Populations

Background

Elevated soluble (s) E-selectin levels have been associated with various cardiovascular diseases. Recently, genetic variants in the ABO blood group have been related to E-selectin levels in a small cohort of patients with type 1 diabetes. We evaluated whether this association is reproducible in two large samples of Caucasians.

Methodology/ Principal Findings

Data of the present study was drawn from the population-based MONICA/KORA Augsburg study (n = 1,482) and the patients-based LURIC study (n = 1,546). A high-density genotyping array (50K IBC Chip) containing single-nucleotide polymorphisms (SNPs) from E-selectin candidate genes selected on known biology of E-selectin metabolism, mouse genetic studies, and human genetic association studies, was used for genotyping. Linear regression analyses with adjustment for age and sex (and survey in KORA) were applied to assess associations between gene variants and sE-selectin concentrations. A number of 12 SNPs (in KORA) and 13 SNPs (in LURIC), all from the ABO blood group gene, were significantly associated with the log-transformed concentration of E-selectin. The strongest association was observed for rs651007 with a change of log-transformed sE-selectin per one copy of the minor allele of −0.37 ng/ml (p = 1.87×10⁻¹⁰³) in KORA and −0.35 ng/ml (p = 5.11×10⁻⁸⁴) in LURIC. Inclusion of rs651007 increased the explained sE-selectin variance by 0.256 in KORA and 0.213 in LURIC. All SNPs had minor allele frequencies above 20% showing a substantial gene variation.

Conclusions/ Significance

Our findings in two independent samples indicate that the genetic variants at the ABO locus affect sE-selectin levels. Since distinct genome-wide association studies linked the ABO gene with myocardial infarction (MI) in the presence of coronary atherosclerosis and with coronary artery disease, these findings may not only enhance our understanding of adhesion molecule biology, but may also provide a focus for several novel research avenues.     

A PCR-based high-throughput screen with multiround sample pooling: application to somatic cell gene targeting

Here, we describe a method of systematic PCR screening with multiround sample pooling for the isolation of rare PCR-positive samples. As an example, we have applied this protocol to the recovery of gene-targeted clones in human somatic cells comprising only 0.02-0.17% of cells transduced with targeting vectors. Initially, cells infected with targeting vectors are seeded and grown in fourteen 96-well tissue culture plates. Samples are then collected from these plates and subjected to two rounds of pooling to yield twelve 'superpools' used for an initial PCR. After identifying PCR-positive samples, de-pooling is carried out with successive rounds of PCR screening, using samples of decreasing complexity. Single-cell cloning is subsequently performed to isolate gene-targeted clones. The entire protocol can be completed in 4-8 weeks depending on the proliferative capacity of the cell line.     

Suprachiasmatic nuclei may regulate the rhythm of leptin hormone release in Syrian hamsters (Mesocricetus auratus)

The suprachiasmatic nuclei (SCN) generate the circadian rhythm of many hormones. The hormone leptin is a metabolic signal that informs the brain about fat and energy stores of the body. We investigated whether the rhythm of leptin hormone release in Syrian hamsters is directly controlled by the SCN. Three experiments were performed: in the first, hamsters were SCN-lesioned; in the second, hamsters were exposed to different feeding regimes; and in the third, hamsters were adrenalectomized and implanted with cortisol capsules to maintain constant glucocorticoid release. Blood samples were collected before and after the experiments at different clock times and examined for leptin levels by enzyme-linked immunosorbant assay (ELISA). Different feeding regimes and constant glucocorticoid release did not alter the rhythm of leptin release; whereas, SCN lesions abolished the rhythm. The results of the present study suggest the rhythm in leptin release in Syrian hamsters may be controlled by the SCN.     

Limb lengths of primary school children in a city from western region of Turkey

Anthropometry has been used for the assessment of growth at different ages. Among the anthropometric measurements, weight, height, arm circumference, and lower and upper limb lengths are of the most important criteria showing the development of children in school age. The aim of the present study was to measure the lengths of upper (arm, forearm, hand) and lower (thigh, leg, foot) limbs of children studying in primary schools of Aydin, a city in the western region of Turkey, and to assess the differences according to the gender (female, male) and living areas (urban, rural). In different age groups, many differences were observed when compared for gender and area. Differences were also seen when compared with the other studies done in different part of Turkey. The data was the first one for the region and it might be useful for further regional studies or for national comparisons. More studies designed with bigger sample sizes that cover many cities belonging to the same region of the country are needed. In addition to cross-sectional studies, longitudinal studies may give more useful knowledge for understanding the growth of children.     

Structural insights into the first incision reaction during nucleotide excision repair

Nucleotide excision repair is a highly conserved DNA repair mechanism present in all kingdoms of life. The incision reaction is a critical step for damage removal and is accomplished by the UvrC protein in eubacteria. No structural information is so far available for the 3' incision reaction. Here we report the crystal structure of the N-terminal catalytic domain of UvrC at 1.5 A resolution, which catalyzes the 3' incision reaction and shares homology with the catalytic domain of the GIY-YIG family of intron-encoded homing endonucleases. The structure reveals a patch of highly conserved residues surrounding a catalytic magnesium-water cluster, suggesting that the metal binding site is an essential feature of UvrC and all GIY-YIG endonuclease domains. Structural and biochemical data strongly suggest that the N-terminal endonuclease domain of UvrC utilizes a novel one-metal mechanism to cleave the phosphodiester bond.     

Factors influencing the phytoplankton steady state assemblages in a drinking-water reservoir (Ömerli reservoir, Istanbul)

In situ growth of heterotrophic nanoflagellates (HNF) in Lake Donghu, a eutrophic shallow lake in mainland China, was studied from January 1999 to March 2000 using a modified Weisse protocol. The study results indicated that the growth rates of HNF showed pronounced seasonal variation (–0.37–1.25 d^–1), reaching the maximum during spring to early summer. When the water temperature was higher than 25.5°C, HNF growth was inversely proportional to water temperature. There was an effect by bacterial abundance and autotrophic picoplankton on HNF growth that depended on location. HNF biomass was the highest in late spring, and the HNF production ranged from –2.25 to 35.45 mg l^–1 d^–1 with mean of 3.17 mg l^–1d^–1. When considered in the context of biomass and production data for zooplankton in Lake Donghu, it was evident that HNF contributed significantly to the total zooplankton production in Lake Donghu. These in situ studies indicate that temperature and food supply are the major determinants of HNF abundance and productivity.