PIK3R1 Mutations Cause Syndromic Insulin Resistance with Lipoatrophy

Short stature, hyperextensibility of joints and/or inguinal hernia, ocular depression, Rieger anomaly, and teething delay (SHORT) syndrome is a developmental disorder with an unknown genetic cause and hallmarks that include insulin resistance and lack of subcutaneous fat. We ascertained two unrelated individuals with SHORT syndrome, hypothesized that the observed phenotype was most likely due to de novo mutations in the same gene, and performed whole-exome sequencing in the two probands and their unaffected parents. We then confirmed our initial observations in four other subjects with SHORT syndrome from three families, as well as 14 unrelated subjects presenting with syndromic insulin resistance and/or generalized lipoatrophy associated with dysmorphic features and growth retardation. Overall, we identified in nine affected individuals from eight families de novo or inherited PIK3R1 mutations, including a mutational hotspot (c.1945C>T [p.Arg649Trp]) present in four families. PIK3R1 encodes the p85α, p55α, and p50α regulatory subunits of class IA phosphatidylinositol 3 kinases (PI3Ks), which are known to play a key role in insulin signaling. Functional data from fibroblasts derived from individuals with PIK3R1 mutations showed severe insulin resistance for both proximal and distal PI3K-dependent signaling. Our findings extend the genetic causes of severe insulin-resistance syndromes and provide important information with respect to the function of PIK3R1 in normal development and its role in human diseases, including growth delay, Rieger anomaly and other ocular affections, insulin resistance, diabetes, paucity of fat, and ovarian cysts.     

Colistin heteroresistance in Enterobacter cloacae is regulated by PhoPQ‐dependent 4‐amino‐4‐deoxy‐l‐arabinose addition to lipid A

The Enterobacter cloacae complex (ECC) consists of closely related bacteria commonly associated with the human microbiota. ECC are increasingly isolated from healthcare‐associated infections, demonstrating that these Enterobacteriaceae are emerging nosocomial pathogens. ECC can rapidly acquire multidrug resistance to conventional antibiotics. Cationic antimicrobial peptides (CAMPs) have served as therapeutic alternatives because they target the highly conserved lipid A component of the Gram‐negative outer membrane. Many Enterobacteriaceae fortify their outer membrane with cationic amine‐containing moieties to prevent CAMP binding, which can lead to cell lysis. The PmrAB two‐component system (TCS) directly activates 4‐amino‐4‐deoxy‐l ‐arabinose (l ‐Ara4N) biosynthesis to result in cationic amine moiety addition to lipid A in many Enterobacteriaceae such as E. coli and Salmonella. In contrast, PmrAB is dispensable for CAMP resistance in E. cloacae. Interestingly, some ECC clusters exhibit colistin heteroresistance, where a subpopulation of cells exhibit clinically significant resistance levels compared to the majority population. We demonstrate that E. cloacae lipid A is modified with l ‐Ara4N to induce CAMP heteroresistance and the regulatory mechanism is independent of the PmrAB_Ecl TCS. Instead, PhoP_Ecl binds to the arnB_Ecl promoter to induce l ‐Ara4N biosynthesis and PmrAB‐independent addition to the lipid A disaccharolipid. Therefore, PhoPQ_Ecl contributes to regulation of CAMP heteroresistance in some ECC clusters.     

Neuronal assemblies: single cortical neurons are obedient members of a huge orchestra

Spontaneous cortical activity of single neurons is often either dismissed as noise, or is regarded as carrying no functional significance and hence is ignored. Our findings suggest that such concepts should be revised. We explored the coherent population activity of neuronal assemblies in primary sensory area in the absence of a sensory input. Recent advances in real-time optical imaging based on voltage-sensitive dyes (VSDI) have facilitated exploration of population activity and its intimate relationship to the activity of individual cortical neurons. It has been shown by in vivo intracellular recordings that the dye signal measures the sum of the membrane potential changes in all the neuronal elements in the imaged area, emphasizing subthreshold synaptic potentials and dendritic action potentials in neuronal arborizations originating from neurons in all cortical layers whose dendrites reach the superficial cortical layers. Thus, the VSDI has allowed us to image the rather illusive activity in neuronal dendrites that cannot be readily explored by single unit recordings. Surprisingly, we found that the amplitude of this type of ongoing subthreshold activity is of the same order of magnitude as evoked activity. We also found that this ongoing activity exhibited high synchronization over many millimeters of cortex. We then investigated the influence of ongoing activity on the evoked response, and showed that the two interact strongly. Furthermore, we found that cortical states that were previously associated only with evoked activity can actually be observed also in the absence of stimulation, for example, the cortical representation of a given orientation may appear without any visual input. This demonstration suggests that ongoing activity may also play a major role in other cortical function by providing a neuronal substrate for the dependence of sensory information processing on context, behavior, memory and other aspects of cognitive function.     

An essential saccharide binding domain for the mAb 2C7 established for Neisseria gonorrhoeae LOS by ES-MS and MSn

A study of bacterial surface oligosaccharides were investigated among different strains of Neisseria gonorrhoeae to correlate structural features essential for binding to the MAb 2C7. This epitope is widely expressed and conserved in gonococcal isolates, characteristics essential to an effective candidate vaccine antigen. Sample lipooligosaccharides (LOS), was prepared by a modification of the hot phenol-water method from which de-O-acetylated LOS and oligosaccharide (OS) components were analyzed by ES-MS-CID-MS and ES-MSnin a triple quadrupole and an ion trap mass spectrometer, respectively. Previously documented natural heterogeneity was apparent from both LOS and OS preparations which was admixed with fragments induced by hydrazine and mild acid treatment. Natural heterogeneity was limited to phosphorylation and antenni extensions to the alpha-chain. Mild acid hydrolysis to release OS also hydrolyzed the beta(1-->6) glycosidic linkage of lipid A. OS structures were determined by collisional and resonance excitation combined with MS and multistep MSn which provided sequence information from both neutral loss, and nonreducing terminal fragments. A comparison of OS structures, with earlier knowledge of MAb binding, enzyme treatment, and partial acid hydrolysis indicates a generic overlapping domain for 2C7 binding. Reoccurring structural features include a Hepalpha(1-->3)Hepbeta(1-->5)KDO trisaccharide core branched on the nonreducing terminus (Hep-2) with an alpha(1-->2) linked GlcNAc (gamma-chain), and an alpha-linked lactose (beta-chain) residue. From the central heptose (Hep-1), a beta(1-->4) linked lactose (alpha-chain), moiety is required although extensions to this residue appear unnecessary.      

环磷酰胺对雄性大鼠生殖免疫功能的影响

目的观察环磷酰胺对大鼠睾丸及其细胞免疫的影响,探讨抗肿瘤药物在生殖免疫功能中的机制。方法选用16只15周龄SD大鼠,随机分为对照组和实验组,每组8只;实验组腹腔注射环磷酰胺20mg/kg/d,连续5天,用药两个月后,应用HE染色法研究大鼠睾丸远期组织学变化,用原位缺口末端标记法（TUNEL方法）检测生精小管中生殖细胞凋亡,放射免疫法检测血清睾酮（T）、卵泡刺激素（FSH）、黄体生成素（LH）,流式细胞术进行血液T淋巴细胞亚群分析。结果实验组睾丸生精小管直径缩小、间距增宽、生精上皮变薄、生殖细胞层次和数量减少、生精小管腔多未见精子形成,实验组睾丸生精小管直径、面积、生殖细胞数均显著低于对照组（P〈0.01）;实验组与对照组比较生殖细胞凋亡增多,差异显著（P〈0.01）;实验组与对照组比较血清T明显降低,差异显著（P〈0.01）,血清FSH、LH水平两组间差异无显著性;血液T淋巴细胞亚群分析,实验组与对照组比较CD3＋CD4＋、CD4＋/CD8＋明显降低（P〈0.01）,CD3＋CD8＋明显升高（P〈0.01）。结论环磷酰胺对大鼠睾丸远期损害明显,促进生殖细胞凋亡,降低睾酮的分泌,并抑制T淋巴细胞的免疫功能。     

Unraveling nature's networks

A report on the meeting 'Unravelling Nature's Networks: from Microarray and Proteomic Analysis to Systems Biology', Sheffield, UK, 21-22 July 2003.     

Both the stimulation and inhibition of root hair growth induced by extracellular nucleotides in Arabidopsis are mediated by nitric oxide and reactive oxygen species

Root hairs secrete ATP as they grow, and extracellular ATP and ADP can trigger signaling pathways that regulate plant cell growth. In several plant tissues the level of extracellular nucleotides is limited in part by ectoapyrases (ecto-NTPDases), and the growth of these tissues is strongly influenced by their level of ectoapyrase expression. Both chemical inhibition of ectoapyrase activity and suppression of the expression of two ectoapyrase enzymes by RNAi in Arabidopsis resulted in inhibition of root hair growth. As assayed by a dose-response curve, different concentrations of the poorly hydrolysable nucleotides, ATPγS and ADPβS, could either stimulate (at 7.5–25 μM) or inhibit (at ≥ 150 μM) the growth rate of root hairs in less than an hour. Equal amounts of AMPS, used as a control, had no effect on root hair growth. Root hairs of nia1nia2 mutants, which are suppressed in nitric oxide (NO) production, and of atrbohD/F mutants, which are suppressed in the production of H₂O₂, did not show growth responses to applied nucleotides, indicating that the growth changes induced by these nucleotides in wild-type plants were likely transduced via NO and H₂O₂ signals. Consistent with this interpretation, treatment of root hairs with different concentrations of ATPγS induced different accumulations of NO and H₂O₂ in root hair tips. Two mammalian purinoceptor antagonists also blocked the growth responses induced by extracellular nucleotides, suggesting that they were initiated by a receptor-based mechanism.     

Intrinsic Protein Fluorescence Interferes with Detection of Tear Glycoproteins in SDS-Polyacrylamide Gels Using Extrinsic Fluorescent Dyes

Intrinsic protein fluorescence may interfere with the visualization of proteins after SDS-polyacrylamide electrophoresis. In an attempt to analyze tear glycoproteins in gels, we ran tear samples and stained the proteins with a glycoprotein-specific fluorescent dye. The fluorescence detected was not limited to glycoproteins. There was strong intrinsic fluorescence of proteins normally found in tears after soaking the gels in 40% methanol plus 1-10% acetic acid and, to a lesser extent, in methanol or acetic acid alone. Nanograms of proteins gave visible native fluorescence and interfere with extrinsic fluorescent dye detection. Poly-L-lysine, which does not contain intrinsically fluorescent amino acids, did not fluoresce.