The Class II Phosphatidylinositol 3 kinase C2β Is Required for the Activation of the K+ Channel KCa3.1 and CD4 T-Cells

The Ca²⁺-activated K⁺ channel KCa3.1 is required for Ca²⁺ influx and the subsequent activation of T-cells. We previously showed that nucleoside diphosphate kinase beta (NDPK-B), a mammalian histidine kinase, directly phosphorylates and activates KCa3.1 and is required for the activation of human CD4 T lymphocytes. We now show that the class II phosphatidylinositol 3 kinase C2β (PI3K-C2β) is activated by the T-cell receptor (TCR) and functions upstream of NDPK-B to activate KCa3.1 channel activity. Decreased expression of PI3K-C2β by siRNA in human CD4 T-cells resulted in inhibition of KCa3.1 channel activity. The inhibition was due to decreased phosphatidylinositol 3-phosphate [PI(3)P] because dialyzing PI3K-C2β siRNA-treated T-cells with PI(3)P rescued KCa3.1 channel activity. Moreover, overexpression of PI3K-C2β in KCa3.1-transfected Jurkat T-cells led to increased TCR-stimulated activation of KCa3.1 and Ca²⁺ influx, whereas silencing of PI3K-C2β inhibited both responses. Using total internal reflection fluorescence microscopy and planar lipid bilayers, we found that PI3K-C2β colocalized with Zap70 and the TCR in peripheral microclusters in the immunological synapse. This is the first demonstration that a class II PI3K plays a critical role in T-cell activation.     

Does Genetic Diversity Predict Health in Humans?

Genetic diversity, especially at genes important for immune functioning within the Major Histocompatibility Complex (MHC), has been associated with fitness-related traits, including disease resistance, in many species. Recently, genetic diversity has been associated with mate preferences in humans. Here we asked whether these preferences are adaptive in terms of obtaining healthier mates. We investigated whether genetic diversity (heterozygosity and standardized mean d²) at MHC and nonMHC microsatellite loci, predicted health in 153 individuals. Individuals with greater allelic diversity (d²) at nonMHC loci and at one MHC locus, linked to HLA-DRB1, reported fewer symptoms over a four-month period than individuals with lower d². In contrast, there were no associations between MHC or nonMHC heterozygosity and health. NonMHC-d² has previously been found to predict male preferences for female faces. Thus, the current findings suggest that nonMHC diversity may play a role in both natural and sexual selection acting on human populations.     

Gib2, a novel Gbeta-like/RACK1 homolog, functions as a Gbeta subunit in cAMP signaling and is essential in Cryptococcus neoformans

Canonical G proteins are heterotrimeric, consisting of alpha, beta, and gamma subunits. Despite multiple Galpha subunits functioning in fungi, only a single Gbeta subunit per species has been identified, suggesting that non-conventional G protein signaling exists in this diverse group of eukaryotic organisms. Using the Galpha subunit Gpa1 that functions in cAMP signaling as bait in a two-hybrid screen, we have identified a novel Gbeta-like/RACK1 protein homolog, Gib2, from the human pathogenic fungus Cryptococcus neoformans. Gib2 contains a seven WD-40 repeat motif and is predicted to form a seven-bladed beta propeller structure characteristic of beta transducins. Gib2 is also shown to interact, respectively, with two Ggamma subunit homologs, Gpg1 and Gpg2, similar to the conventional Gbeta subunit Gpb1. In contrast to Gpb1 whose overexpression promotes mating response, overproduction of Gib2 suppresses defects of gpa1 mutation in both melanization and capsule formation, the phenotypes regulated by cAMP signaling and associated with virulence. Furthermore, depletion of Gib2 by antisense suppression results in a severe growth defect, suggesting that Gib2 is essential. Finally, Gib2 is shown to also physically interact with a downstream target of Gpa1-cAMP signaling, Smg1, and the protein kinase C homolog Pkc1, indicating that Gib2 is also a multifunctional RACK1-like protein.     

Switch of substrate specificity of hyperthermophilic acylaminoacyl peptidase by combination of protein and solvent engineering

The inherent evolvability of promiscuous enzymes endows them with great potential to be artificially evolved for novel functions. Previously, we succeeded in transforming a promiscuous acylaminoacyl peptidase (apAAP) from the hyperthermophilic archaeon Aeropyrum pernix K1 into a specific carboxylesterase by making a single mutation. In order to fulfill the urgent requirement of thermostable lipolytic enzymes, in this paper we describe how the substrate preference of apAAP can be further changed from p-nitrophenyl caprylate (pNP-C8) to p-nitrophenyl laurate (pNP-C12) by protein and solvent engineering. After one round of directed evolution and subsequent saturation mutagenesis at selected residues in the active site, three variants with enhanced activity towards pNP-C12 were identified. Additionally, a combined mutant W474V/F488G/R526V/T560W was generated, which had the highest catalytic efficiency (k_cat/K_m) for pNP-C12, about 71-fold higher than the wild type. Its activity was further increased by solvent engineering, resulting in an activity enhancement of 280-fold compared with the wild type in the presence of 30% DMSO. The structural basis for the improved activity was studied by substrate docking and molecular dynamics simulation. It was revealed that W474V and F488G mutations caused a significant change in the geometry of the active center, which may facilitate binding and subsequent hydrolysis of bulky substrates. In conclusion, the combination of protein and solvent engineering may be an effective approach to improve the activities of promiscuous enzymes and could be used to create naturally rare hyperthermophilic enzymes.     

HLA-DR+ CD38+ CD4+ T lymphocytes have elevated CCR5 expression and produce the majority of R5-tropic HIV-1 RNA in vivo

Percentages of activated T cells correlate with HIV-1 disease progression, but the underlying mechanisms are not fully understood. We hypothesized that HLA-DR(+) CD38(+) (DR(+) 38(+)) CD4(+) T cells produce the majority of HIV-1 due to elevated expression of CCR5 and CXCR4. In phytohemagglutinin (PHA)-stimulated CD8-depleted peripheral blood mononuclear cells (PBMC) infected with HIV-1 green fluorescent protein (GFP) reporter viruses, DR(-) 38(+) T cells constituted the majority of CCR5 (R5)-tropic (median, 62%) and CXCR4 (X4)-tropic HIV-1-producing cells (median, 61%), although cell surface CCR5 and CXCR4 were not elevated in this subset of cells. In lymph nodes from untreated individuals infected with R5-tropic HIV-1, percentages of CCR5(+) cells were elevated in DR(+) 38(+) CD4(+) T cells (median, 36.4%) compared to other CD4(+) T-cell subsets (median values of 5.7% for DR(-) 38(-) cells, 19.4% for DR(+) 38(-) cells, and 7.6% for DR(-) 38(+) cells; n = 18; P < 0.001). In sorted CD8(-) lymph node T cells, median HIV-1 RNA copies/10(5) cells was higher for DR(+) 38(+) cells (1.8 × 10(6)) than for DR(-) 38(-) (0.007 × 10(6)), DR(-) 38(+) (0.064 × 10(6)), and DR(+) 38(-) (0.18 × 10(6)) subsets (n = 8; P < 0.001 for all). After adjusting for percentages of subsets, a median of 87% of viral RNA was harbored by DR(+) 38(+) cells. Percentages of CCR5(+) CD4(+) T cells and concentrations of CCR5 molecules among subsets predicted HIV-1 RNA levels among CD8(-) DR/38 subsets (P < 0.001 for both). Median HIV-1 DNA copies/10(5) cells was higher in DR(+) 38(+) cells (5,360) than in the DR(-) 38(-) (906), DR(-) 38(+) (814), and DR(+) 38(-) (1,984) subsets (n = 7; P ≤ 0.031). Thus, DR(+) 38(+) CD4(+) T cells in lymph nodes have elevated CCR5 expression, are highly susceptible to infection with R5-tropic virus, and produce the majority of R5-tropic HIV-1. PBMC assays failed to recapitulate in vivo findings, suggesting limited utility. Strategies to reduce numbers of DR(+) 38(+) CD4(+) T cells may substantially inhibit HIV-1 replication.     

Isocitrate dehydrogenase 1 R132C mutation occurs exclusively in microsatellite stable colorectal cancers with the CpG island methylator phenotype

The CpG Island Methylator Phenotype (CIMP) is fundamental to an important subset of colorectal cancer; however, its cause is unknown. CIMP is associated with microsatellite instability but is also found in BRAF mutant microsatellite stable cancers that are associated with poor prognosis. The isocitrate dehydrogenase 1 (IDH1) gene causes CIMP in glioma due to an activating mutation that produces the 2-hydroxyglutarate oncometabolite. We therefore examined IDH1 alteration as a potential cause of CIMP in colorectal cancer. The IDH1 mutational hotspot was screened in 86 CIMP-positive and 80 CIMP-negative cancers. The entire coding sequence was examined in 81 CIMP-positive colorectal cancers. Forty-seven cancers varying by CIMP-status and IDH1 mutation status were examined using Illumina 450K DNA methylation microarrays. The R132C IDH1 mutation was detected in 4/166 cancers. All IDH1 mutations were in CIMP cancers that were BRAF mutant and microsatellite stable (4/45, 8.9%). Unsupervised hierarchical cluster analysis identified an IDH1 mutation-like methylation signature in approximately half of the CIMP-positive cancers. IDH1 mutation appears to cause CIMP in a small proportion of BRAF mutant, microsatellite stable colorectal cancers. This study provides a precedent that a single gene mutation may cause CIMP in colorectal cancer, and that this will be associated with a specific epigenetic signature and clinicopathological features.     

双源CT冠状动脉血管成像诊断心肌桥的价值探讨

目的：研究双源CT冠状动脉血管成像诊断心肌桥的临床价值。方法：选择260例具有典型心前区不适的患者进行双源CT冠脉血管成像检查,观察其发生部位,测量其长度和深度并进行分析。结果：260例受检患者中,62例共70段存在心肌桥,检出率达20.76%,高于文献报道的检出率18.2%。所有心肌桥均发生于左前降支,其中近段17段（24.4%）,中段43段（61.4%）,远段10段（14.2%）。心肌桥平均长度为15.8±6.4mm,深度为1.4±0.85mm。结论：双源CT冠状动脉血管成像因其便捷无创,不受心率严格限制且价格低廉可作为心肌桥筛查的理想检查手段。     

MR弥散加权成像对鉴别诊断良恶性椎体压缩性骨折的临床价值研究

目的：探讨MR弥散加权成像（DWI）鉴别诊断良恶性椎体压缩性骨折的临床价值。方法：对57例经临床或病理证实的椎体良恶性压缩性骨折患者行矢状位T1M、T2WI、T2WI／FS及DWI扫描,研究其在常规序列和DWI序列上的表现,将常规MR序列和DWI序列检出率进行比较,测量正常椎体及病变椎体的表观弥散系数（ADC）值,并进行统计学分析。结果：（1）MR常规序列和DWI序列（b=500s／mm2）表现：良性椎体压缩性骨折呈长T1长或等T2改变,T2WI／FS呈高信号,DWI可以呈高信号、等信号及低信号;恶性椎体压缩性骨折呈长T1长T2信号,大部分病灶T2WUFS及DWI呈高信号,少数变现为低信号;（2）MR常规序列和DWI序列（b=500s／mm2）病灶检出率的比较：T1WI、T2WI／FS及DWI序列病灶检出率均高于T2WI序列,其间的差别有显著性意义（P〈0．01）,T1WI、T2WI／FS及DWI序列病灶检出率之间无显著性差异（P〉0．01）;（3）ADC值比较：在DWI（b=500s／mm2）上,良性组ADC值为（2．03±0．83）×10^3mm^2/s,恶性组ADC值为（1．37±0．75）×10^-3mm^2/s,正常组ADC值为（0．36±0．21）×10^-3mm^2／s,成像条件相同时,良性组高于恶性组,两组间有明显的统计学意义（P〈0．05）。结论：DWI可较好的反映椎体的弥散特征,ADC值作为量化指标可对良恶性椎体压缩性骨折进行可靠鉴别。     

Refinement of the MHC Risk Map in a Scandinavian Primary Sclerosing Cholangitis Population

Background

Genetic variants within the major histocompatibility complex (MHC) represent the strongest genetic susceptibility factors for primary sclerosing cholangitis (PSC). Identifying the causal variants within this genetic complex represents a major challenge due to strong linkage disequilibrium and an overall high physical density of candidate variants. We aimed to refine the MHC association in a geographically restricted PSC patient panel.

Methodology/Principal Findings

A total of 365 PSC cases and 368 healthy controls of Scandinavian ancestry were included in the study. We incorporated data from HLA typing (HLA-A, -B, -C, -DRB3, -DRB1, -DQB1) and single nucleotide polymorphisms across the MHC (n = 18,644; genotyped and imputed) alongside previously suggested PSC risk determinants in the MHC, i.e. amino acid variation of DRβ, a MICA microsatellite polymorphism and HLA-C and HLA-B according to their ligand properties for killer immunoglobulin-like receptors. Breakdowns of the association signal by unconditional and conditional logistic regression analyses demarcated multiple PSC associated MHC haplotypes, and for eight of these classical HLA class I and II alleles represented the strongest association. A novel independent risk locus was detected near NOTCH4 in the HLA class III region, tagged by rs116212904 (odds ratio [95% confidence interval] = 2.32 [1.80, 3.00], P = 1.35×10⁻¹¹).

Conclusions/Significance

Our study shows that classical HLA class I and II alleles, predominantly at HLA-B and HLA-DRB1, are the main risk factors for PSC in the MHC. In addition, the present assessments demonstrated for the first time an association near NOTCH4 in the HLA class III region.     

Sulfonates as terminal electron acceptors for growth of sulfite-reducing bacteria (Desulfitobacterium spp.) and sulfate-reducing bacteria: effects of inhibitors of sulfidogenesis.

This study demonstrates the ability of Desulfitobacterium spp. to utilize aliphatic sulfonates as terminal electron acceptors (TEA) for growth. Isethionate (2-hydroxyethanesulfonate) reduction by Desulfitobacterium hafniense resulted in acetate as well as sulfide accumulation in accordance with the expectation that the carbon portion of isethionate was oxidized to acetate and the sulfur was reduced to sulfide. The presence of a polypeptide, approximately 97 kDa, was evident in isethionate-grown cells of Desulfitobacterium hafniense, Desulfitobacterium sp. strain PCE 1, and the two sulfate-reducing bacteria (SRB)-Desulfovibrio desulfuricans IC1 (T. J. Lie, J. R. Leadbetter, and E. R. Leadbetter, Geomicrobiol. J. 15:135-149, 1998) and Desulfomicrobium norvegicum; this polypeptide was not detected when these bacteria were grown on TEA other than isethionate, suggesting involvement in its metabolism. The sulfate analogs molybdate and tungstate, effective in inhibiting sulfate reduction by SRB, were examined for their effects on sulfonate reduction. Molybdate effectively inhibited sulfonate reduction by strain IC1 and selectively inhibited isethionate (but not cysteate) reduction by Desulfitobacterium dehalogenans and Desulfitobacterium sp. strain PCE 1. Desulfitobacterium hafniense, however, grew with both isethionate and cysteate in the presence of molybdate. In contrast, tungstate only partially inhibited sulfonate reduction by both SRB and Desulfitobacterium spp. Similarly, another inhibitor of sulfate reduction, 1,8-dihydroxyanthraquinone, effectively inhibited sulfate reduction by SRB but only partially inhibited sulfonate reduction by both SRB and Desulfitobacterium hafniense.