Ultrafast folding of alpha3D: a de novo designed three-helix bundle protein

Here, we describe the folding/unfolding kinetics of alpha3D, a small designed three-helix bundle. Both IR temperature jump and ultrafast fluorescence mixing methods reveal a single-exponential process consistent with a minimal folding time of 3.2 +/- 1.2 micros (at approximately 50 degrees C), indicating that a protein can fold on the 1- to 5-micros time scale. Furthermore, the single-exponential nature of the relaxation indicates that the prefactor for transition state (TS)-folding models is probably >or=1 (micros)-1 for a protein of this size and topology. Molecular dynamics simulations and IR spectroscopy provide a molecular rationale for the rapid, single-exponential folding of this protein. alpha3D shows a significant bias toward local helical structure in the thermally denatured state. The molecular dynamics-simulated TS ensemble is highly heterogeneous and dynamic, allowing access to the TS via multiple pathways.     

Transcatheter electrosurgery in bipolar or monopolar modes

• Transcatheter electrosurgery has emerging value in a range of other new procedures that require traversing tissue (transcaval access, transcatheter Glenn Shunt) or slicing tissue (LAMPOON slicing of the mitral valve and BASILICA slicing of the aortic valve).
• This is the first report of bipolar radiofrequency wires used to cross lesions in humans, reported here in seven re‐entry CTO cases.
• The bipolar configuration may provide directionality to charge without need for wire alignment and advancement, but is theoretically disadvantageous for tissue “cutting” because of problems with charge concentration.

     

Phosphorylation of cytosolic proteins by resting and activated human neutrophils

A study was conducted on the phosphorylation of proteins in the neutrophil cytosol in response to phorbol myristate acetate (PMA) and N- formyl-methionyl-leucyl-phenylalanine (fMLP). Autoradiography of gel electrophoretograms prepared from neutrophils incubated with 32Pi in the presence and absence of the activators showed nine proteins whose state of phosphorylation was affected by neutrophil activation. 32P was gained by eight of these proteins and was lost by the ninth. For all but one of these proteins, the change in the extent of labeling appeared to reach completion by one to two minutes. It was possible to quantitate the changes in 32P content of three of the nine proteins. One of these was the 20-kD protein that lost label when the neutrophils were activated. Quantitation showed that over half the 32P present in this protein in the resting state was gone within 0.2 minutes after activation. The other two were proteins weighing 11 and 69 kD. The phosphorylation characteristics of these two proteins differed, depending on whether activation had been carried out with PMA or fMLP. These differences in protein phosphorylation support other evidence suggesting that PMA and fMLP do not activate neutrophils by identical biochemical pathways. Differences in phosphorylation between resting and activated cells were not affected by dibutyryl cyclic guanosine monophosphate (cGMP), dibutyryl cyclic adenosine monophosphate (cAMP), theophylline, aspirin, hydrocortisone, or colchicine. The differences were abolished, however, by 30 mumol/L trifluoperazine. This finding is consistent with the hypothesis that the calcium/calmodulin system plays a biochemical role in the activation of neutrophils.     

弓形虫培养上清体外对CD4+CD25+T细胞的影响

目的探讨体外培养条件下弓形虫培养上清对CD4+CD25+T细胞的影响。方法用弓形虫培养上清和自小鼠脾脏分离的CD4+CD25+T细胞共同孵育，Annexin-v染色法检测CD4+CD25+T细胞的凋亡情况，淋巴细胞增殖实验检测CD4+CD25+T细胞对CD4+CD25-T细胞的抑制功能。结果  用弓形虫培养上清孵育10 h的小鼠脾脏CD4+CD25+T细胞有(36. 90±0.36)%出现凋亡，和RPMI-1640孵育组相比，凋亡率上升了（13. 60士2.15）%。与RPMI-1640孵育组相比，用弓形虫培养上清处理5、10 h的小鼠脾脏CD4+CD2 5+T细胞对CD4+CD25 -T细胞的抑制功能下降(P<0.01)。结论  弓形虫培养上清中可能存在某些成分能够诱导CD4+CD25+T细胞出现凋亡，并使CD4+CD25+T缅胞对CD4+CD25-T细胞的抑制功能下降。     

Speech Perception with Noise Vocoding and Background Noise: An EEG and Behavioral Study

This study explored the physiological response of the human brain to degraded speech syllables. The degradation was introduced using noise vocoding and/or background noise. The goal was to identify physiological features of auditory-evoked potentials (AEPs) that may explain speech intelligibility. Ten human subjects with normal hearing participated in syllable-detection tasks, while their AEPs were recorded with 32-channel electroencephalography. Subjects were presented with six syllables in the form of consonant-vowel-consonant or vowel-consonant-vowel. Noise vocoding with 22 or 4 frequency channels was applied to the syllables. When examining the peak heights in the AEPs (P1, N1, and P2), vocoding alone showed no consistent effect. P1 was not consistently reduced by background noise, N1 was sometimes reduced by noise, and P2 was almost always highly reduced. Two other physiological metrics were examined: (1) classification accuracy of the syllables based on AEPs, which indicated whether AEPs were distinguishable for different syllables, and (2) cross-condition correlation of AEPs (r_cc) between the clean and degraded speech, which indicated the brain’s ability to extract speech-related features and suppress response to noise. Both metrics decreased with degraded speech quality. We further tested if the two metrics can explain cross-subject variations in their behavioral performance. A significant correlation existed for r_cc, as well as classification based on early AEPs, in the fronto-central areas. Because r_cc indicates similarities between clean and degraded speech, our finding suggests that high speech intelligibility may be a result of the brain’s ability to ignore noise in the sound carrier and/or background.     

MiR-132 Inhibits Expression of SIRT1 and Induces Pro-inflammatory Processes of Vascular Endothelial Inflammation through Blockade of the SREBP-1c Metabolic Pathway

Purpose

Inflammation participates centrally in all stages of atherosclerosis (AS), which begins with pro-inflammatory processes and inflammatory changes in the endothelium, related to lipid metabolism. MicroRNA (miRNA) inhibition of inflammation related to SIRT1 has been shown to be a promising therapeutic approach for AS. However, the mechanism of action is unknown.

Methods

We investigated whether miRNAs regulate the SIRT1 and its downstream SREBP-lipogenesis-cholesterogenesis metabolic pathway in human umbilical vein endothelial cells (HUVECs). HUVECs were transfected with miR-132 mimics and inhibitors, and then treated with or without tumor necrosis factor α (TNFα). The effects of miR-132 on pro-inflammatory processes, proliferation and apoptosis were assessed.

Results

We identified that the relative 3’ UTR luciferase activities of SIRT1 were significantly decreased in miR-132 transfected HUVECs (0.338?±?0.036) compared to control (P?=?0.000). miR-132 inhibited SIRT1 expression of mRNA level in HUVECs (0.53?±?0.06) (P?SIRT1. mRNA expression and protein levels of SREBP (0.45?±?0.07), fatty acid synthase (FASN) (0.55?±?0.09) and 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR) (0.62?±?0.08) (P?TNF-α, and inhibited its proliferation, viability and migration.

Conclusions

SIRT1 mRNAs are direct targets of miR-132. miR-132 controls lipogenesis and cholesterogenesis in HUVECs by inhibiting SIRT1 and SREBP-1c expression and their downstream regulated genes, including FASN and HMGCR. Inhibition of SIRT1 by miR-132 was associated with lipid metabolism-dependent pro-inflammatory processes in HUVECs. The newly identified miRNA, miR-132 represents a novel targeting mechanism for AS therapy.     

一体化PET/MR结合统计参数图辅助^11C-PIB显像的半定量分析及其临床应用

目的 研究一体化PET/MR结合统计参数图(SPM)辅助^11C-匹兹堡化合物B(PIB)用于β-淀粉样蛋白(Aβ)PET显像半定量分析的准确性,探索其用于认知障碍的诊断及鉴别诊断的可行性.方法 回顾分析2018年1月至2019年9月在华中科技大学同济医学院附属协和医院PET中心进行^11C-PIB PET/MR扫描,临床最终确诊的13例阿尔茨海默病(AD)患者[男4例,女9例;年龄(59.2±5.8)岁]和10例血管性认知障碍(VCD)患者[男9例,女1例;年龄(59.5±11.5)岁].结合三维T1加权成像(3D T1WI)对^11C-PIB PET图像分别进行脑区手动勾画和SPM辅助半自动分割,获得8个关键脑区(大脑白质、纹状体、丘脑、后扣带回、额叶皮质、后顶叶皮质、颞叶外侧皮质和枕叶皮质)与小脑皮质的标准摄取值比值(SUVR).对2种方法所获结果进行Pearson相关分析;采用两独立样本t检验、配对t检验分析数据.结果 AD组与VCD组患者的年龄和简易精神状态检查量表(MMSE)评分[(19.7±4.7)和(21.7±3.8)分]差异均无统计学意义(t值:0.095和1.098,均P>0.05).除丘脑外(r=0.179,P=0.413),分割法和勾画法在其余7个关键脑区获得的SUVR均有良好的相关性(r值:0.678~0.893,均P<0.05).AD组8个关键脑区的SUVR均明显高于VCD组(1.519~2.055与1.105~1.618;t值:2.799~11.582,均P相似文献   

Evidence that the LRRK2 ROC domain Parkinson's disease‐associated mutants A1442P and R1441C exhibit increased intracellular degradation

Mutations in the leucine‐rich repeat kinase 2 (lrrk2) gene are the leading genetic cause of Parkinson's disease (PD). In characterizing the novel ROC domain mutant A1442P, we compared its steady‐state protein levels, propensity to aggregate, and toxicity with the pathogenic R1441C mutant and wild‐type (WT) LRRK2. Mutant (R1441C and A1442P) and WT LRRK2 fused to green fluorescent protein (GFP) and FLAG were transiently expressed in HEK293 cells using plasmid constructs. Western analysis and fluorescence microscopy consistently demonstrated lower mutant LRRK2 protein levels compared with WT. A time‐course expression study using flow cytometry showed that WT LRRK2 expression increased initially but then plateaued by 72 hr. Conversely, R1441C and A1442P mutant expression attained 85% and 74% of WT levels at 24 hr but fell to 68% and 55% of WT levels by 72 hr, respectively. We found that proteasome inhibition markedly increased mutant LRRK2 to levels approaching those of WT. Taken together, our findings reveal increased intracellular degradation for both mutants. Furthermore, the impact of mutant and WT LRRK2 expression on HEK293 cell viability was assessed under normative and oxidative (hydrogen peroxide) conditions and found not to differ. Expression of WT and mutant LRRK2 protein gave rise to intracellular aggregates of similar appearance and cellular localization. In summary, we provide evidence that the novel A1442P mutant and the previously investigated R1441C pathogenic mutant exhibit increased intracellular degradation, a property reportedly demonstrated for the pathogenic LRRK2 kinase domain mutant I2020T. © 2013 Wiley Periodicals, Inc.