A Combination of CD28 (rs1980422) and IRF5 (rs10488631) Polymorphisms Is Associated with Seropositivity in Rheumatoid Arthritis: A Case Control Study

IntroductionThe aim of the study was to analyse genetic architecture of RA by utilizing multiparametric statistical methods such as linear discriminant analysis (LDA) and redundancy analysis (RDA).MethodsA total of 1393 volunteers, 499 patients with RA and 894 healthy controls were included in the study. The presence of shared epitope (SE) in HLA-DRB1 and 11 SNPs (PTPN22 C/T (rs2476601), STAT4 G/T (rs7574865), CTLA4 A/G (rs3087243), TRAF1/C5 A/G (rs3761847), IRF5 T/C (rs10488631), TNFAIP3 C/T (rs5029937), AFF3 A/T (rs11676922), PADI4 C/T (rs2240340), CD28 T/C (rs1980422), CSK G/A (rs34933034) and FCGR3A A/C (rs396991), rheumatoid factor (RF), anti–citrullinated protein antibodies (ACPA) and clinical status was analysed using the LDA and RDA.ResultsHLA-DRB1, PTPN22, STAT4, IRF5 and PADI4 significantly discriminated between RA patients and healthy controls in LDA. The correlation between RA diagnosis and the explanatory variables in the model was 0.328 (Trace = 0.107; F = 13.715; P = 0.0002). The risk variants of IRF5 and CD28 genes were found to be common determinants for seropositivity in RDA, while positivity of RF alone was associated with the CTLA4 risk variant in heterozygous form. The correlation between serologic status and genetic determinants on the 1st ordinal axis was 0.468, and 0.145 on the 2nd one (Trace = 0.179; F = 6.135; P = 0.001). The risk alleles in AFF3 gene together with the presence of ACPA were associated with higher clinical severity of RA.ConclusionsThe association among multiple risk variants related to T cell receptor signalling with seropositivity may play an important role in distinct clinical phenotypes of RA. Our study demonstrates that multiparametric analyses represent a powerful tool for investigation of mutual relationships of potential risk factors in complex diseases such as RA.     

Reference Charts for Fetal Cerebellar Vermis Height: A Prospective Cross-Sectional Study of 10605 Fetuses

Objective

To establish reference charts for fetal cerebellar vermis height in an unselected population.

Methods

A prospective cross-sectional study between September 2009 and December 2014 was carried out at ALTAMEDICA Fetal–Maternal Medical Centre, Rome, Italy. Of 25203 fetal biometric measurements, 12167 (48%) measurements of the cerebellar vermis were available. After excluding 1562 (12.8%) measurements, a total of 10605 (87.2%) fetuses were considered and analyzed once only. Parametric and nonparametric quantile regression models were used for the statistical analysis. In order to evaluate the robustness of the proposed reference charts regarding various distributional assumptions on the ultrasound measurements at hand, we compared the gestational age-specific reference curves we produced through the statistical methods used. Normal mean height based on parametric and nonparametric methods were defined for each week of gestation and the regression equation expressing the height of the cerebellar vermis as a function of gestational age was calculated. Finally the correlation between dimension/gestation was measured.

Results

The mean height of the cerebellar vermis was 12.7mm (SD, 1.6mm; 95% confidence interval, 12.7–12.8mm). The regression equation expressing the height of the CV as a function of the gestational age was: height (mm) = -4.85+0.78 x gestational age. The correlation between dimension/gestation was expressed by the coefficient r = 0.87.

Conclusion

This is the first prospective cross-sectional study on fetal cerebellar vermis biometry with such a large sample size reported in literature. It is a detailed statistical survey and contains new centile-based reference charts for fetal height of cerebellar vermis measurements.     

Single Nucleotide Polymorphisms in IL17A and IL6 Are Associated with Decreased Risk for Pulmonary Tuberculosis in Southern Brazilian Population

In Mycobacterium tuberculosis (MTB) infection, the complex interaction of host immune system and the mycobacteria is associated with levels of cytokines production that play a major role in determining the outcome of the disease. Several single-nucleotide polymorphisms (SNPs) in cytokine genes have been associated with tuberculosis (TB) outcome. The aim of this study was to evaluate the association between previously reported SNPs IL2–330 T>G (rs2069762); IL4–590 C>T (rs2243250); IL6–174 G>C (rs1800795); IL10–592 A>C (rs1800872); IL10–1082 G>A (rs1800896); IL17A -692 C>T (rs8193036); IL17A -197 G>A (rs2275913); TNF -238 G>A (rs361525); TNF -308 G>A (rs1800629) and IFNG +874 T>A (rs2430561) and pulmonary TB (PTB) susceptibility. We conducted a case-control study in individuals from Southern Brazil who were recruited between February 2012 and October 2013 in a high incidence TB city. We performed a multiplex genotyping assay in 191 patients with PTB and 175 healthy subjects. Our results suggest a decreased risk for PTB development associated with the IL17A -197A allele (OR = 0.29; p = 0.04), AA genotype (OR = 0.12; p = 0.04) and A carrier (AG/AA) (OR = 0.29; p = 0.004) and IL6 -174C carrier (CC/CG) (OR = 0.46; p = 0.04). We could not properly analyze IL17A -692 C>T (rs8193036) and IFNG +874T>A due to genotypic inconsistencies and found no evidence of association for the IL2, IL4, IL10 and TNF polymorphisms and PTB. In conclusion, our results show a protective effect of IL17 and IL6 polymorphisms on PTB outcome in Southern Brazilian population.     

A Novel Open Tubular Capillary Electrochromatographic Method for Differentiating the DNA Interaction Affinity of Environmental Contaminants

The interaction of chemicals with DNA may lead to genotoxicity, mutation or carcinogenicity. A simple open tubular capillary electrochromatographic method is proposed to rapidly assess the interaction affinity of three environmental contaminants (1,4-phenylenediamine, pyridine and 2,4-diaminotoluene) to DNA by measuring their retention in the capillaries coated with DNA probes. DNA oligonucleotide probes were immobilized on the inner wall of a fused silica capillary that was first derivatized with 3-(aminopropyl)-triethoxysilane (APTES). The difference in retention times and factors was considered as the difference in interaction affinity of the contaminants to the DNA probes. The interaction of the contaminants with both double-stranded (dsDNA) and single-stranded DNA (ssDNA) coatings was compared. Retention factors of 1,4-phenylenediamine, pyridine and 2,4-diaminotoluene in the capillary coated with ssDNA probe were 0.29, 0.42, and 0.44, respectively. A similar trend was observed in the capillary coated with dsDNA, indicating that 2,4-diaminotoluene has the highest affinity among the three contaminants. The relative standard deviation (RSD) for the retention factors was in the range of 0.05–0.69% (n = 3). The results demonstrated that the developed technique could be applied for preliminary screening purpose to provide DNA interaction affinity information of various environmental contaminants.     

Disposable orbitally shaken TubeSpin bioreactor 600 for Sf9 cell cultivation in suspension

Disposable orbitally shaken TubeSpin bioreactor 600 tubes (TS600s) were recently developed for the bench-scale cultivation of animal cells in suspension. Here we compared batch cultures of Sf9 insect cells in TS600s, spinner flasks, and shake flasks. Superior cell growth was observed in TS600s and shake flasks as compared with spinner flasks, and more favorable oxygen-enriched cell culture conditions were observed in TS600s as compared with either spinner or shake flasks. The results demonstrated the suitability of TS600s as a disposable vessel for the cultivation of Sf9 cells in suspension.     

Massive excretion of calcium oxalate from late prepupal salivary glands of Drosophila melanogaster demonstrates active nephridial‐like anion transport

The Drosophila salivary glands (SGs) were well known for the puffing patterns of their polytene chromosomes and so became a tissue of choice to study sequential gene activation by the steroid hormone ecdysone. One well‐documented function of these glands is to produce a secretory glue, which is released during pupariation to fix the freshly formed puparia to the substrate. Over the past two decades SGs have been used to address specific aspects of developmentally‐regulated programmed cell death (PCD) as it was thought that they are doomed for histolysis and after pupariation are just awaiting their fate. More recently, however, we have shown that for the first 3–4 h after pupariation SGs undergo tremendous endocytosis and vacuolation followed by vacuole neutralization and membrane consolidation. Furthermore, from 8 to 10 h after puparium formation (APF) SGs display massive apocrine secretion of a diverse set of cellular proteins. Here, we show that during the period from 11 to 12 h APF, the prepupal glands are very active in calcium oxalate (CaOx) extrusion that resembles renal or nephridial excretory activity. We provide genetic evidence that Prestin, a Drosophila homologue of the mammalian electrogenic anion exchange carrier SLC26A5, is responsible for the instantaneous production of CaOx by the late prepupal SGs. Its positive regulation by the protein kinases encoded by fray and wnk lead to increased production of CaOx. The formation of CaOx appears to be dependent on the cooperation between Prestin and the vATPase complex as treatment with bafilomycin A₁ or concanamycin A abolishes the production of detectable CaOx. These data demonstrate that prepupal SGs remain fully viable, physiologically active and engaged in various cellular activities at least until early pupal period, that is, until moments prior to the execution of PCD.     

Hydrocarbons Are Essential for Optimal Cell Size,Division, and Growth of Cyanobacteria

Cyanobacteria are intricately organized, incorporating an array of internal thylakoid membranes, the site of photosynthesis, into cells no larger than other bacteria. They also synthesize C15-C19 alkanes and alkenes, which results in substantial production of hydrocarbons in the environment. All sequenced cyanobacteria encode hydrocarbon biosynthesis pathways, suggesting an important, undefined physiological role for these compounds. Here, we demonstrate that hydrocarbon-deficient mutants of Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803 exhibit significant phenotypic differences from wild type, including enlarged cell size, reduced growth, and increased division defects. Photosynthetic rates were similar between strains, although a minor reduction in energy transfer between the soluble light harvesting phycobilisome complex and membrane-bound photosystems was observed. Hydrocarbons were shown to accumulate in thylakoid and cytoplasmic membranes. Modeling of membranes suggests these compounds aggregate in the center of the lipid bilayer, potentially promoting membrane flexibility and facilitating curvature. In vivo measurements confirmed that Synechococcus sp. PCC 7002 mutants lacking hydrocarbons exhibit reduced thylakoid membrane curvature compared to wild type. We propose that hydrocarbons may have a role in inducing the flexibility in membranes required for optimal cell division, size, and growth, and efficient association of soluble and membrane bound proteins. The recent identification of C15-C17 alkanes and alkenes in microalgal species suggests hydrocarbons may serve a similar function in a broad range of photosynthetic organisms.Cyanobacteria (oxygenic photosynthetic bacteria) are found in nearly every environment on Earth and are major contributors to global carbon and nitrogen fixation (Galloway et al., 2004; Zwirglmaier et al., 2008). They are distinguished among prokaryotes in containing multiple internal thylakoid membranes, the site of photosynthesis, and a large protein compartment, the carboxysome, involved in carbon fixation. Despite these extra features, cyanobacteria can be as small as 0.6 µm in diameter (Raven, 1998).All cyanobacteria with sequenced genomes encode the pathway for the biosynthesis of hydrocarbons, implying an important, although as-yet-undefined, role for these compounds (Lea-Smith et al., 2015). The major forms are C15-C19 alkanes and alkenes, which can be synthesized from fatty acyl-acyl-carrier proteins (ACPs) by one or other of two separate pathways (Fig. 1; Schirmer et al., 2010; Mendez-Perez et al., 2011). The majority of species produce alkanes and alkenes via acyl-ACP reductase (FAR) and aldehyde deformylating oxygenase (FAD; Schirmer et al., 2010; Li et al., 2012; Coates et al., 2014; Lea-Smith et al., 2015). Cyanobacterial species lacking the FAR/FAD pathway synthesize alkenes via olefin synthase (Ols; Mendez-Perez et al., 2011; Coates et al., 2014; Lea-Smith et al., 2015). This suggests that hydrocarbons produced by either pathway serve a similar role in the cell. Homologs of FAR/FAD or Ols are not present in other bacteria or plant and algal species. However, C15-C17 alkanes and alkenes, synthesized by an alternate, uncharacterized pathway, were recently detected in a range of green microalgae, including Chlamydomonas reinhardtii, Chlorella variabilis NC64A, and several Nannochloropsis species (Sorigué et al., 2016). In C. reinhardtii, hydrocarbons were primarily localized to the chloroplast, which originated in evolution from a cyanobacterium that was engulfed by a host organism (Howe et al., 2008). Hydrocarbons may therefore have a similar role in cyanobacteria, some green microalgae species, and possibly a broader range of photosynthetic organisms.Open in a separate windowFigure 1.Hydrocarbon biosynthesis is encoded in all sequenced cyanobacteria. Detailed are the two hydrocarbon biosynthetic pathways, indicated in blue and red, respectively, in cyanobacteria. The number of species encoding the enzymes in each pathway is indicated.Hydrocarbons act as antidesiccants, waterproofing agents, and signaling molecules in insects (Howard and Blomquist, 2005) and prevent water loss, ensure pollen viability, and influence pathogen interactions in plants (Kosma et al., 2009; Bourdenx et al., 2011). However, the function of hydrocarbons in cyanobacteria has not been determined. Characterization of cyanobacterial hydrocarbon biosynthesis pathways has provided the basis for investigating synthetic microbial biofuel systems, which may be a renewable substitute for fossil fuels (Schirmer et al., 2010; Choi and Lee, 2013; Howard et al., 2013). However, secretion of long-chain hydrocarbons from the cell into the medium, which is likely essential for commercially viable production, has not been observed in the absence of a membrane solubilization agent (Schirmer et al., 2010; Tan et al., 2011). Cyanobacterial hydrocarbons also have a significant environmental role. Due to the abundance of cyanobacteria in the environment, hydrocarbon production is considerable, with hundreds of millions of tons released into the ocean per annum following cell death (Lea-Smith et al., 2015). This production may be sufficient to sustain populations of hydrocarbon-degrading bacteria, which can then play an important role in consuming anthropogenic oil spills (Lea-Smith et al., 2015).Here, we investigated the cellular location and role of hydrocarbons in both spherical Synechocystis sp. PCC 6803 (Synechocystis) and rod-shaped Synechococcus sp. PCC 7002 (Synechococcus) cells. We developed a model of the cyanobacterial membrane, which indicated that hydrocarbons aggregate in the middle of the lipid bilayer and, when present at levels observed in cells, lead to membrane swelling associated with pools of hydrocarbon. This suggested that alkanes may facilitate membrane curvature. In vivo measurements of Synechococcus thylakoid membrane conformation are consistent with this model.