Agonist-dependent phosphorylation of the formyl peptide receptor is regulated by the membrane proximal region of the cytoplasmic tail

Formyl peptide receptor (FPR) is a chemoattractant G protein-coupled receptor (GPCR) involved in the innate immune response against bacteria. Receptor activation is terminated by receptor phosphorylation of two serine- and threonine-rich regions located in the distal half of the cytoplasmic tail. In this study we show that introduction of an amino acid with a bulky side chain (leucine or glutamine) adjacent to a single leucine, L320, in the membrane-proximal half of the cytoplasmic tail, significantly enhanced receptor phosphorylation, beta-arrestin1/2 translocation, and receptor endocytosis, without affecting G(i)-mediated ERK1/2 activation and release of intracellular calcium. In addition, the point mutations resulted in diminished susceptibility to trypsin, suggesting a conformation different from that of wild type FPR. Alignment of the FPR sequence with the rhodopsin sequence showed that L320 resides immediately C-terminal of an amphipathic region that in rhodopsin forms helix 8. Deletion of seven amino acids (Delta309-315) from the predicted helix 8 of FPR (G307-S319) caused reduced cell signaling as well as defects in receptor phosphorylation, beta-arrestin1/2 translocation and endocytosis. Thus, the amino acid content in the N-terminal half of the cytoplasmic tail influences the structure and desensitization of FPR.     

A Defined and Xeno-Free Culture Method Enabling the Establishment of Clinical-Grade Human Embryonic,Induced Pluripotent and Adipose Stem Cells

Background

The growth of stem cells in in vitro conditions requires optimal balance between signals mediating cell survival, proliferation, and self-renewal. For clinical application of stem cells, the use of completely defined conditions and elimination of all animal-derived materials from the establishment, culture, and differentiation processes is desirable.

Methodology/Principal Findings

Here, we report the development of a fully defined xeno-free medium (RegES), capable of supporting the expansion of human embryonic stem cells (hESC), induced pluripotent stem cells (iPSC) and adipose stem cells (ASC). We describe the use of the xeno-free medium in the derivation and long-term (>80 passages) culture of three pluripotent karyotypically normal hESC lines: Regea 06/015, Regea 07/046, and Regea 08/013. Cardiomyocytes and neural cells differentiated from these cells exhibit features characteristic to these cell types. The same formulation of the xeno-free medium is capable of supporting the undifferentiated growth of iPSCs on human feeder cells. The characteristics of the pluripotent hESC and iPSC lines are comparable to lines derived and cultured in standard undefined culture conditions. In the culture of ASCs, the xeno-free medium provided significantly higher proliferation rates than ASCs cultured in medium containing allogeneic human serum (HS), while maintaining the differentiation potential and characteristic surface marker expression profile of ASCs, although significant differences in the surface marker expression of ASCs cultured in HS and RegES media were revealed.

Conclusion/Significance

Our results demonstrate that human ESCs, iPSCs and ASCs can be maintained in the same defined xeno-free medium formulation for a prolonged period of time while maintaining their characteristics, demonstrating the applicability of the simplified xeno-free medium formulation for the production of clinical-grade stem cells. The basic xeno-free formulation described herein has the potential to be further optimized for specific applications relating to establishment, expansion and differentiation of various stem cell types.     

Tumor necrosis factor alpha promotes the expression of immunosuppressive proteins and enhances the cell growth in a human bone marrow-derived stem cell culture

Mesenchymal stem cells (MSCs) are widely used in experimental treatments for various conditions that involve normal tissue regeneration via inflammatory repair. It is known that MSCs can secrete multiple soluble factors and suppress inflammation. Even though the effect of MSCs on inflammation has been extensively studied, the effect of inflammation on MSCs is poorly understood. One of the major cytokines released at the site of inflammation is tumor necrosis factor alpha (TNF-α) which is known to induce MSC invasion and proliferation. Therefore, we wanted to test the effects of TNF-α exposure on MSCs derived from human bone marrow. We found, as expected, that cell proliferation was significantly enhanced during TNF-α exposure. However, according to the cell surface marker analysis, the intensity of several antigens in the minimum criteria panel for MSCs proposed by International Society of Cellular Therapy (ISCT) was decreased dramatically, and in certain cases, the criteria for MSCs were not fulfilled. In addition, TNF-α exposure resulted in a significant but transient increase in human leukocyte antigen and CD54 expression. Additional proteomic analysis by two-dimensional difference gel electrophoresis and mass spectrometry revealed three proteins whose expression levels decreased and 8 proteins whose expression levels increased significantly during TNF-α exposure. The majority of these proteins could be linked to immunosuppressive and signalling pathways. These results strongly support reactive and immunosuppressive activation of MSCs during TNF-α exposure, which might influence MSC differentiation stage and capacity.     

Shiga toxin-producing Escherichia coli O100:H⁻: stx2e in drinking water contaminated by waste water in Finland

In November 2007, 450 m³ of treated wastewater leaked into the drinking water distribution system contaminating the drinking water of over 10,000 inhabitants of Nokia, Southern Finland. Nearly 1,000 people visited the health centre because of gastroenteritis during the following 5 weeks. A wide range of enteric pathogens was found in the patients. The authors used the 16-plex PCR to investigate whether the five major diarrheagenic Escherichia coli pathotypes (EPEC, ETEC, STEC, EIEC or EAEC) were present in the contaminated drinking water and in the patients’ stool samples. The contaminated drinking water was positive for genes characteristic of various E. coli pathotypes: pic, invE, hlyA, ent, escV, eae, aggR, stx ₂ , estIa and astA. These genes, except stx ₂ , hlyA and invE, were also detected in the stool samples of the patients linked to this outbreak. A sorbitol positive, streptomycin resistant STEC strain was isolated from the drinking water, and belonged to the serotype O100:H ^– , produced Stx2 toxin (titre 1:8 by reversed-passive latex agglutination method), and carried the genes stx _2e, estIa and irp2.     

Novel Microbiological and Spatial Statistical Methods to Improve Strength of Epidemiological Evidence in a Community-Wide Waterborne Outbreak

Failures in the drinking water distribution system cause gastrointestinal outbreaks with multiple pathogens. A water distribution pipe breakage caused a community-wide waterborne outbreak in Vuorela, Finland, July 2012. We investigated this outbreak with advanced epidemiological and microbiological methods. A total of 473/2931 inhabitants (16%) responded to a web-based questionnaire. Water and patient samples were subjected to analysis of multiple microbial targets, molecular typing and microbial community analysis. Spatial analysis on the water distribution network was done and we applied a spatial logistic regression model. The course of the illness was mild. Drinking untreated tap water from the defined outbreak area was significantly associated with illness (RR 5.6, 95% CI 1.9–16.4) increasing in a dose response manner. The closer a person lived to the water distribution breakage point, the higher the risk of becoming ill. Sapovirus, enterovirus, single Campylobacter jejuni and EHEC O157:H7 findings as well as virulence genes for EPEC, EAEC and EHEC pathogroups were detected by molecular or culture methods from the faecal samples of the patients. EPEC, EAEC and EHEC virulence genes and faecal indicator bacteria were also detected in water samples. Microbial community sequencing of contaminated tap water revealed abundance of Arcobacter species. The polyphasic approach improved the understanding of the source of the infections, and aided to define the extent and magnitude of this outbreak.     

Association of Serum 25-Hydroxyvitamin D with Lifestyle Factors and Metabolic and Cardiovascular Disease Markers: Population-Based Cross-Sectional Study (FIN-D2D)

Objectives

Low serum 25-hydroxyvitamin D (25OHD) level has been associated with an increased risk of several chronic diseases. Our aim was to determine lifestyle and clinical factors that are associated with 25OHD level and to investigate connection of 25OHD level with metabolic and cardiovascular disease markers.

Design

In total, 2868 Finnish men and women aged 45–74 years participated in FIN-D2D population-based health survey in 2007. Participants that had a serum sample available (98.4%; n = 2822) were included in this study. 25OHD was measured with chemiluminescent microparticle immunoassay method.

Results

The mean 25OHD level was 58.2 nmol/l in men (n = 1348) and 57.1 nmol/l in women (n = 1474). Mean 25OHD level was lower in the younger age groups than in the older ones (p<0.0001 both in men and women). This study confirmed that low physical activity (p<0.0001 both in men and women), smoking (p = 0.0002 in men and p = 0.03 in women) and high BMI (p<0.0001 in women) are factors that independently associate with low 25OHD level. Of the metabolic and cardiovascular disease markers high triglyceride concentration (p = 0.02 in men and p = 0.001 in women) and high apolipoprotein B/apolipoprotein A1 ratio (p = 0.04 in men and p = 0.03 in women) were independently associated with low 25OHD level.

Conclusions

Higher age did not predict lower 25OHD level in this study population of aged 45–74 years which may derive from a healthy life-style of “active pensioners”. Low physical activity and smoking came up as independent lifestyle factors associated with low 25OHD level. Defining the molecular mechanisms behind the associations of 25OHD with low physical activity and smoking are important objective in future studies. The association of 25OHD with BMI, high triglyceride concentration and apolipoprotein B/apolipoprotein A1 ratio may be related to the role of vitamin D in inflammation, but more detailed studies are needed.     

Comparison of solution-based exome capture methods for next generation sequencing

Background

Techniques enabling targeted re-sequencing of the protein coding sequences of the human genome on next generation sequencing instruments are of great interest. We conducted a systematic comparison of the solution-based exome capture kits provided by Agilent and Roche NimbleGen. A control DNA sample was captured with all four capture methods and prepared for Illumina GAII sequencing. Sequence data from additional samples prepared with the same protocols were also used in the comparison.

Results

We developed a bioinformatics pipeline for quality control, short read alignment, variant identification and annotation of the sequence data. In our analysis, a larger percentage of the high quality reads from the NimbleGen captures than from the Agilent captures aligned to the capture target regions. High GC content of the target sequence was associated with poor capture success in all exome enrichment methods. Comparison of mean allele balances for heterozygous variants indicated a tendency to have more reference bases than variant bases in the heterozygous variant positions within the target regions in all methods. There was virtually no difference in the genotype concordance compared to genotypes derived from SNP arrays. A minimum of 11× coverage was required to make a heterozygote genotype call with 99% accuracy when compared to common SNPs on genome-wide association arrays.

Conclusions

Libraries captured with NimbleGen kits aligned more accurately to the target regions. The updated NimbleGen kit most efficiently covered the exome with a minimum coverage of 20×, yet none of the kits captured all the Consensus Coding Sequence annotated exons.