Chemokine receptor inhibition by AMD3100 is strictly confined to CXCR4

This study was undertaken to demonstrate the unique specificity of the chemokine receptor CXCR4 antagonist AMD3100. Calcium flux assays with selected chemokine/cell combinations, affording distinct chemokine receptor specificities, revealed no interaction of AMD3100 with any of the chemokine receptors CXCR1 through CXCR3, or CCR1 through CCR9. In contrast, AMD3100 potently inhibited CXCR4-mediated calcium signaling and chemotaxis in a concentration-dependent manner in different cell types. Also, AMD3100 inhibited stromal cell-derived factor (SDF)-1-induced endocytosis of CXCR4, but did not affect phorbol ester-induced receptor internalization. Importantly, AMD3100 by itself was unable to elicit intracellular calcium fluxes, to induce chemotaxis, or to trigger CXCR4 internalization, indicating that the compound does not act as a CXCR4 agonist. Specific small-molecule CXCR4 antagonists such as AMD3100 may play an important role in the treatment of human immunodeficiency virus infections and many other pathological processes that are dependent on SDF-1/CXCR4 interactions (e.g. rheumatoid arthritis, atherosclerosis, asthma and breast cancer metastasis).     

Genomic organisation and chromosomal assignment of ODF2 (outer dense fiber 2), encoding the main component of sperm tail outer dense fibers and a centrosomal scaffold protein

ODF2 (outer dense fiber 2) was first described as the main protein component of the sperm tail cytoskeleton, the outer dense fibers, but was shown recently to be a component of the centrosomal scaffold in chicken. In mouse two related ODF2 cDNA clones were isolated which have been suggested to be most likely the result of alternative splicing. We show here the exon/intron organisation of mouse ODF2 and demonstrate that alternative splicing results in related cDNA sequences and most likely explains, at least partially, the highly complex protein pattern detected on Western blots. ODF2 was mapped to rat chromosome 3 and more specifically by FISH analysis at bands 3q11-->3q12. In addition, we demonstrate that ODF2 is indeed a component of the centrosome and the mitotic spindle poles in mammals.     

Purification of the fengycin synthetase multienzyme system from Bacillus subtilis b213

The purification of the multienzyme system producing the lipodecapeptide fengycin in Bacillus subtilis b213 was investigated. By gel filtration of a cell free extract of this organism three enzyme fractions were obtained from which five multifunctional components of fengycin synthetase were separated by high resolution anion-exchange FPLC procedures. These proteins were characterized by their thioester formation activities with ¹⁴C-labeled substrate amino acids and by N-terminal sequencing. Correlation of these data with the DNA sequences of the pps (fen) operons in three B. subtilis strains provided detailed knowledge on the structural and functional organization of fengycin synthetase.     

Frustration‐guided motion planning reveals conformational transitions in proteins

Proteins exist as conformational ensembles, exchanging between substates to perform their function. Advances in experimental techniques yield unprecedented access to structural snapshots of their conformational landscape. However, computationally modeling how proteins use collective motions to transition between substates is challenging owing to a rugged landscape and large energy barriers. Here, we present a new, robotics‐inspired motion planning procedure called dCC‐RRT that navigates the rugged landscape between substates by introducing dynamic, interatomic constraints to modulate frustration. The constraints balance non‐native contacts and flexibility, and instantaneously redirect the motion towards sterically favorable conformations. On a test set of eight proteins determined in two conformations separated by, on average, 7.5 Å root mean square deviation (RMSD), our pathways reduced the Cα atom RMSD to the goal conformation by 78%, outperforming peer methods. We then applied dCC‐RRT to examine how collective, small‐scale motions of four side‐chains in the active site of cyclophilin A propagate through the protein. dCC‐RRT uncovered a spatially contiguous network of residues linked by steric interactions and collective motion connecting the active site to a recently proposed, non‐canonical capsid binding site 25 Å away, rationalizing NMR and multi‐temperature crystallography experiments. In all, dCC‐RRT can reveal detailed, all‐atom molecular mechanisms for small and large amplitude motions. Source code and binaries are freely available at https://github.com/ExcitedStates/KGS/ .     

Quantification of Campylobacter Species Cross-Contamination during Handling of Contaminated Fresh Chicken Parts in Kitchens

Numerous outbreak investigations and case-control studies for campylobacteriosis have provided evidence that handling Campylobacter-contaminated chicken products is a risk factor for infection and illness. There is currently extremely limited quantitative data on the levels of Campylobacter cross-contamination in the kitchen, hindering risk assessments for the pathogen commodity combination of Campylobacter and chicken meat. An exposure assessment needs to quantify the transfer of the bacteria from chicken to hands and the kitchen environment and from there onto ready-to-eat foods. We simulated some typical situations in kitchens and quantified the Campylobacter transfer from naturally contaminated chicken parts most commonly used in Germany. One scenario simulated the seasoning of five chicken legs and the reuse of the same plate for cooked meat. In another, five chicken breast filets were cut into small slices on a wooden board where, without intermediate cleaning, a cucumber was sliced. We also investigated the transfer of the pathogen from chicken via hands to a bread roll. The numbers of Campylobacter present on the surfaces of the chicken parts, hands, utensils, and ready-to-eat foods were detected by using Preston enrichment and colony counting after surface plating on Karmali agar. The mean transfer rates from legs and filets to hands were 2.9 and 3.8%. The transfer from legs to the plate (0.3%) was significantly smaller (P < 0.01) than the percentage transferred from filets to the cutting board and knife (1.1%). Average transfer rates from hands or kitchen utensils to ready-to-eat foods ranged from 2.9 to 27.5%.     

High Fat Diet Accelerates Pathogenesis of Murine Crohn’s Disease-Like Ileitis Independently of Obesity

Background

Obesity has been associated with a more severe disease course in inflammatory bowel disease (IBD) and epidemiological data identified dietary fats but not obesity as risk factors for the development of IBD. Crohn’s disease is one of the two major IBD phenotypes and mostly affects the terminal ileum. Despite recent observations that high fat diets (HFD) impair intestinal barrier functions and drive pathobiont selection relevant for chronic inflammation in the colon, mechanisms of high fat diets in the pathogenesis of Crohn’s disease are not known. The aim of this study was to characterize the effect of HFD on the development of chronic ileal inflammation in a murine model of Crohn’s disease-like ileitis.

Methods

TNF^ΔARE/WT mice and wildtype C57BL/6 littermates were fed a HFD compared to control diet for different durations. Intestinal pathology and metabolic parameters (glucose tolerance, mesenteric tissue characteristics) were assessed. Intestinal barrier integrity was characterized at different levels including polyethylene glycol (PEG) translocation, endotoxin in portal vein plasma and cellular markers of barrier function. Inflammatory activation of epithelial cells as well as immune cell infiltration into ileal tissue were determined and related to luminal factors.

Results

HFD aggravated ileal inflammation but did not induce significant overweight or typical metabolic disorders in TNF^ΔARE/WT. Expression of the tight junction protein Occludin was markedly reduced in the ileal epithelium of HFD mice independently of inflammation, and translocation of endotoxin was increased. Epithelial cells showed enhanced expression of inflammation-related activation markers, along with enhanced luminal factors-driven recruitment of dendritic cells and Th17-biased lymphocyte infiltration into the lamina propria.

Conclusions

HFD feeding, independently of obesity, accelerated disease onset of small intestinal inflammation in Crohn’s disease-relevant mouse model through mechanisms that involve increased intestinal permeability and altered luminal factors, leading to enhanced dendritic cell recruitment and promoted Th17 immune responses.     

Identifying common prognostic factors in genomic cancer studies: A novel index for censored outcomes

Background  

With the growing number of public repositories for high-throughput genomic data, it is of great interest to combine the results produced by independent research groups. Such a combination allows the identification of common genomic factors across multiple cancer types and provides new insights into the disease process. In the framework of the proportional hazards model, classical procedures, which consist of ranking genes according to the estimated hazard ratio or the p-value obtained from a test statistic of no association between survival and gene expression level, are not suitable for gene selection across multiple genomic datasets with different sample sizes. We propose a novel index for identifying genes with a common effect across heterogeneous genomic studies designed to remain stable whatever the sample size and which has a straightforward interpretation in terms of the percentage of separability between patients according to their survival times and gene expression measurements.     

Observations of neutral buoyancy in diapausing copepods Calanoides acutus during Antarctic winter

The herbivorous Antarctic copepod Calanoides acutus overwinters inactively in a resting stage (diapause) at depths below 500 m. It is assumed that during diapause C. acutus is neutrally buoyant in order to retain energy reserves otherwise depleted by swimming activities. However, so far, no experimental observations on its buoyancy have been reported and our knowledge of buoyancy regulation mechanisms is incomplete. In the present study, species-specific differences in buoyancy were assessed visually. Observations were made of specimens from the diapausing cohort of C. acutus and compared to another herbivorous copepod Calanus propinquus, which overwinters actively feeding in the upper water layers. Freshly caught copepods were anaesthetized in a 3-amino-benzoic acid ethyl ester (MS222) in seawater solution in order to exclude the influence of swimming movements on buoyancy control. It was shown that C. propinquus was negatively buoyant, whereas diapausing C. acutus remained neutrally buoyant. This is the first record that neutral buoyancy in diapausing copepods is maintained by the biochemical body composition without the additional need of swimming movements.     

Pluripotency deficit in clones overcome by clone-clone aggregation: epigenetic complementation?

Abnormal gene expression patterns in somatic cell clones and their attrition in utero are commonly considered a consequence of errors in nuclear reprogramming. We observe that mouse clone blastocysts have less than half the normal cell number, and that higher cell number correlates with correct expression of Oct4, a gene essential for peri-implantation development and embryonic pluripotency. To increase the cell number, we aggregated genetically identical clones at the 4-cell stage. Clone-clone aggregates did not form more blastocysts, but the majority expressed Oct4 normally and had higher rates of fetal and postnatal development. Fertilized blastocysts with low cell numbers, induced by removal of two blastomeres at the 4-cell stage, did not exhibit abnormal Oct4 expression, indicating that improved gene expression and post-implantation development of clone-clone aggregates is not a consequence of increased cell number. Rather, we propose that complementation of non-cell-autonomous defects of genetically identical, but epigenetically different, embryos results in improved gene expression in clone-clone aggregates.