Mitotracker Green Is a P-Glycoprotein Substrate

P-glycoprotein has a widespread expression on normal tissues. The protein has also been strongly associated with the multidrug resistance phenotype (MDR) on tumor cells. The employment of flow cytometry and confocal microscopy has contributed to the discovery and application of new particular fluorescent dyes. Nevertheless, several studies are being performed in different cellular types neglecting the expression/activity of MDR proteins. Because many fluorochromes have been reported as P-glycoprotein substrates, an especial attention must be given to the properties of new dyes in the presence of MDR proteins. Flow cytometric analyzes of Mitotracker Green (MTG) fluorescence profile were performed in a human erythroleukemic cell line and its resistant counterpart. In this report we demonstrated that MTG, a probe used to evaluate the mitochondrial mass, is a P-glycoprotein substrate and its staining profile is dependent on the activity of this protein. In vitro studies on a human erythroleukemic cell line and its resistant counterpart revealed that MDR modulators (Cyclosporin A, Verapamil, and Trifluoperazine) alter the MTG fluorescence pattern on a resistant cell line. The findings suggest that attention should be given to the expression of P-glycoprotein when performing an evaluation of mitochondria properties with MTG.     

Process of Making Three-dimensional Microstructures using Vaporization of a Sacrificial Component

Vascular structures in natural systems are able to provide high mass transport through high surface areas and optimized structure. Few synthetic material fabrication techniques are able to mimic the complexity of these structures while maintaining scalability. The Vaporization of a Sacrificial Component (VaSC) process is able to do so. This process uses sacrificial fibers as a template to form hollow, cylindrical microchannels embedded within a matrix. Tin (II) oxalate (SnOx) is embedded within poly(lactic) acid (PLA) fibers which facilitates the use of this process. The SnOx catalyzes the depolymerization of the PLA fibers at lower temperatures. The lactic acid monomers are gaseous at these temperatures and can be removed from the embedded matrix at temperatures that do not damage the matrix. Here we show a method for aligning these fibers using micromachined plates and a tensioning device to create complex patterns of three-dimensionally arrayed microchannels. The process allows the exploration of virtually any arrangement of fiber topologies and structures.     

Testing hypotheses driving genetic structure in the cooperatively breeding Brown‐headed Nuthatch Sitta pusilla

Habitat fragmentation has often been implicated in the decline of many species. For habitat specialists and/or sedentary species, loss of habitat can result in population isolation and lead to negative genetic effects. However, factors other than fragmentation can often be important and also need to be considered when assessing the genetic structure of a species. We genotyped individuals from 13 populations of the cooperatively breeding Brown‐headed Nuthatch Sitta pusilla in Florida to test three alternative hypotheses regarding the effects that habitat fragmentation might have on genetic structure. A map of potential habitat developed from recent satellite imagery suggested that Brown‐headed Nuthatch populations in southern Florida occupied smaller and more isolated habitat patches (i.e. were more fragmented) than populations in northern Florida. We also genotyped individuals from a small, isolated Brown‐headed Nuthatch population on Grand Bahama Island. We found that populations associated with more fragmented habitat in southern Florida had lower allelic richness than populations in northern Florida (P = 0.02), although there were no differences in heterozygosity. Although pairwise estimates of F_ST were low overall, values among southern populations were generally higher than northern populations. Population assignment tests identified K = 3 clusters corresponding to a northern cluster, a southern cluster and a unique population in southeast Florida; using sampling localities as prior information revealed K = 7 clusters, with greater structure only among southern Florida populations. The Bahamas population showed moderate to high differentiation compared with Florida populations. Overall, our results suggest that fragmentation could affect gene flow in Brown‐headed Nuthatch populations and is likely to become more pronounced over time.     

Quantitative Metaproteomics and Activity-based Protein Profiling of Patient Fecal Microbiome Identifies Host and Microbial Serine-type Endopeptidase Activity Associated With Ulcerative Colitis

The gut microbiota plays an important yet incompletely understood role in the induction and propagation of ulcerative colitis (UC). Organism-level efforts to identify UC-associated microbes have revealed the importance of community structure, but less is known about the molecular effectors of disease. We performed 16S rRNA gene sequencing in parallel with label-free data-dependent LC-MS/MS proteomics to characterize the stool microbiomes of healthy (n = 8) and UC (n = 10) patients. Comparisons of taxonomic composition between techniques revealed major differences in community structure partially attributable to the additional detection of host, fungal, viral, and food peptides by metaproteomics. Differential expression analysis of metaproteomic data identified 176 significantly enriched protein groups between healthy and UC patients. Gene ontology analysis revealed several enriched functions with serine-type endopeptidase activity overrepresented in UC patients. Using a biotinylated fluorophosphonate probe and streptavidin-based enrichment, we show that serine endopeptidases are active in patient fecal samples and that additional putative serine hydrolases are detectable by this approach compared with unenriched profiling. Finally, as metaproteomic databases expand, they are expected to asymptotically approach completeness. Using ComPIL and de novo peptide sequencing, we estimate the size of the probable peptide space unidentified (“dark peptidome”) by our large database approach to establish a rough benchmark for database sufficiency. Despite high variability inherent in patient samples, our analysis yielded a catalog of differentially enriched proteins between healthy and UC fecal proteomes. This catalog provides a clinically relevant jumping-off point for further molecular-level studies aimed at identifying the microbial underpinnings of UC.     

Baseline serum levels of cardiac biomarkers in patients with stable coronary artery disease

Stable coronary artery disease (CAD) can cause repetitive reversible myocardial ischaemia, and it seems to be possible that reversibly injured myocardium releases small amounts of soluble cytoplasmic proteins. Hence, the aim was to evaluate the effect of stable CAD on baseline serum levels of cardiac biomarkers. We studied 68 consecutive outpatients referred for gated myocardial perfusion imaging. Before a treadmill exercise test, blood samples for measurement of creatine kinase (CK), CK-myocardial band (CK-MB) mass, myoglobin, aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) were collected. Normal perfusion patterns were detected in 29 (43%) patients (group 1) and perfusion defects were detected in 39 (57%) patients (group 2). Baseline serum levels of biomarkers except CK were significantly higher in group 2 (p=0.001). Stable CAD increases baseline levels of CK-MB mass, myoglobin, AST and LDH in the serum and this increase is related to the extent and severity of the perfusion defect and to some extent the ejection fraction of the left ventricle.     

Frequent overexpression of CADM1/IGSF4 in lung adenocarcinoma

We reported comprehensive screening for antigens (Ags) overexpressed on various carcinomas via isolation of human monoclonal antibodies (mAbs) that may be therapeutic in a previous paper (Proc. Natl. Acad. Sci. USA 105, 7287-7292, 2008). Twenty-one distinct Ags highly expressed on several carcinomas were identified and 356 mAbs with unique sequences turned out to bind to one of the 21 Ags. Among them CADM1/IGSF4 which had been originally referred to as tumor suppressor lung cancer 1 (TSLC1) was included. Therefore we examined the expression of CADM1 in lung cancers in this study. Eight different anti CADM1 mAbs were used for immunohistochemical analysis of 29 fresh lung cancer specimens. Staining patterns were categorized to six groups based on the extent of positive staining and the localization of stained portions. While overexpression of CADM1 was observed on the cell surface of adenocarcinomas at a high frequency, around 60%, positive stainings were rarely observed on that of other lung carcinomas including squamous cell carcinomas. Moreover, some clones among the eight mAbs gave different staining patterns from those by the other clones against the same fresh specimen, suggesting presence of variant forms of CADM1 differentiated by mAbs.     

Transferability of microsatellite markers from Eucalyptus spp. to Eugenia dysenterica (Myrtaceae family)

Cagaita (Eugenia dysenterica) is a widespread plant found in the Brazilian cerrado. Its fruit is used for popular consumption and for industrial purposes. A battery of 346 primer pairs developed for Eucalyptus spp. was tested on cagaita. Only 10 primer pairs were found to be transferable between the two species. Using a polyacrilamide gel, an average of 10.4 alleles per locus was detected, in a sample of 116 individuals from 10 natural populations of cagaita. Seven polymorphic loci allowed estimation of genetic parameters, including expected average heterozigosity H_E = 0.442, diversity among populations, R_ST = 0.268 and gene flow Nm = 0.680.     

Chemical cross-linking and mass spectrometry for protein structural modeling

The growth of gene and protein sequence information is currently so rapid that three-dimensional structural information is lacking for the overwhelming majority of known proteins. In this review, efforts towards rapid and sensitive methods for protein structural characterization are described, complementing existing technologies. Based on chemical cross-linking and offering the analytical speed and sensitivity of mass spectrometry these methodologies are thought to contribute valuable tools towards future high throughput protein structure elucidation.     

颈性眩晕中医证型与经颅超声脑动脉血流检测结果的相关性分析

摘要 目的：分析颈性眩晕中医证型与经颅超声脑动脉血流检测结果的相关性。方法：选取2021年5月-2023年5月收治颈性眩晕患者作为研究对象,根据不同中医证型分为痰湿中阻组、肝阳上亢组、肝肾阴虚组和气血亏虚组,每组各纳入20例;并另选取健康体检患者30例作为对照组,均给予多普勒超声检查。分析不同中医证型者与对照组者多普勒超声检查特征与脑动脉血流变化[左右椎动脉、基底动脉及大脑中动脉收缩期峰值血流速度（VS）、平均血流速度(Vm)、舒张期峰值血流速度(Vd)及搏动指数(PI)]。结果：痰湿中阻组、肝肾阴虚组和气血亏虚组左右椎动脉、基底动脉及大脑中动脉VS均低于对照组（P＜0.05）;肝阳上亢组左右椎动脉、基底动脉及大脑中动脉VS均高于对照组（P＜0.05）;不同中医证型组间VS比较,肝阳上亢组＞痰湿中阻组＞肝肾阴虚组＞气血亏虚组;痰湿中阻组左右椎动脉、基底动脉均高于对照组（P＜0.05）,大脑中动脉Vm均低于对照组（P＜0.05）;肝阳上亢组左右椎动脉、基底动脉及大脑中动脉Vm均高于对照组（P＜0.05）;肝肾阴虚组左右椎动脉、基底动脉及大脑中动脉Vm均低于对照组（P＜0.05）;气血亏虚组大脑中动脉Vm均低于对照组（P＜0.05）,左右椎动脉、基底动脉Vm和对照组无显著性差异（P>0.05）;不同中医证型组间Vm比较,肝阳上亢组＞痰湿中阻组＞气血亏虚组＞肝肾阴虚组;痰湿中阻组左右椎动脉、基底动脉Vd均低于对照组（P＜0.05）,大脑中动脉Vd均高于对照组（P＜0.05）;肝阳上亢组左右椎动脉、基底动脉及大脑中动脉Vd均低于对照组（P＜0.05）;肝肾阴虚组左右椎动脉及大脑中动脉Vd均低于对照组（P＜0.05）,基底动脉Vd和对照组无差异（P＞0.05）;气血亏虚组左右椎动脉Vd均低于对照组（P＜0.05）,基底动脉Vd和对照组无差异（P＞0.05）,大脑中动脉Vd均高于对照组（P＜0.05）;不同中医证型组间Vd比较,气血亏虚组＞痰湿中阻组＞肝肾阴虚组＞肝阳上亢组;痰湿中阻组和气血亏虚组左右椎动脉、基底动脉及大脑中动脉PI均低于对照组（P＜0.05）;肝阳上亢组和肝肾阴虚组左右椎动脉、基底动脉及大脑中动脉PI均高于对照组（P＜0.05）;不同中医证型组间PI比较,肝阳上亢组＞肝肾阴虚组＞痰湿中阻组＞气血亏虚组。结论：不同中医证型的眩晕患者会出现不同程度脑动脉血流动力学异常,且不同组间存在差异,通过经颅多普勒超声检查,可以对眩晕中医证型提供参考价值。