TWO DIFFERENT SPECIES OF EUGLENA,E. GENICULATA AND E. MYXOCYLINDRACEA (EUGLENOPHYCEAE), ARE VIRTUALLY GENETICALLY AND MORPHOLOGICALLY IDENTICAL1

We investigated the similarity of a single Euglena myxocylindracea strain, isolated originally by Bold and MacEntee, to several Euglena geniculata strains on both morphological and DNA levels. We found the three DNA stretches, consisting of fragments coding for the parts of cytoplasmic and chloroplast small subunit rRNA, and the internal transcribed spacer (ITS2) of cytoplasmic rDNA, with the combined length of 4332 nucleotides, are identical in E. myxocylindracea and E. geniculata, strain SAG 1224‐4b. Morphological differences between E. myxocylindracea and any E. geniculata strain examined were well within the range of E. geniculata variability as well. The only difference behind the distinction of E. myxocylindracea from E. geniculata is the presence of the second chloroplast in the latter. However, we were able to induce the appearance of the second chloroplast in the cells of E. myxocylindracea and its disappearance in the cells of E. geniculata by changing the composition of the culture media. We therefore conclude that E. myxocylindracea Bold and MacEntee should be regarded as an environmental form of E. geniculata Dujardin. For the first time the morphology of E. geniculata chloroplasts was shown as revealed by confocal laser microscopy.     

New evidence that nuclear import of endogenous beta-catenin is LEF-1 dependent, while LEF-1 independent import of exogenous beta-catenin leads to nuclear abnormalities

The once accepted idea that LEF-1 transports beta-catenin into nuclei has recently been challenged by experiments using exogenous beta-catenin. Here, we investigated the effects of beta-catenin and LEF-1 on nuclear import of beta-catenin using different combinations of exogenous and endogenous molecules over longer lengths of time than previously studied. Nuclear beta-catenin is not detectable in corneal fibroblasts and epithelia or NIH 3T3 and MDCK cells. In LEF-1 transfections, we show that the B-box of LEF-1 is required to move cytoplasmic endogenous beta-catenin into the nuclei of such cells, proving that LEF-1 does transport endogenous beta-catenin into nuclei. Moreover, transfection of uveal melanoma cells with B-box deficient LEF-1 inhibits nuclear import of beta-catenin by endogenous LEF-1. However, the movement of overexpressed exogenous beta-catenin into nuclei is unaffected by the presence or absence of LEF-1 and forms abnormal nuclear aggregates that are a prelude to subsequent apoptosis. We conclude that nuclear transport of exogenous beta-catenin independently of LEF-1 has questionable physiological significance.     

Mammalian NHE2 Na(+)/H+ exchanger mediates efflux of potassium upon heterologous expression in yeast

Na(+)/H+exchangers form a broad family of transporters that mediate opposing fluxes of alkali metal cations and protons across cell membranes. They play multiple roles in different organisms (protection from toxic cations, regulation of cell volume or pH). Rat NHE2 exchanger was expressed in a Saccharomyces cerevisiae mutant strain lacking its own exporters of alkali metal cations. Though most of the overexpressed NHE2 remained entrapped in the secretory pathway, part of it reached the plasma membrane and mediated K+ efflux from the yeast. We demonstrate for the first time that a mammalian Na(+)/H+ exchanger transports alkali metal cations in yeast in the opposite direction than in mammalian cells, and that the substrate specificity of the rat NHE2 exchanger is limited only to potassium cations upon expression in yeast cells.     

Prevalence,antimicrobial resistance profile,and characterization of multi-drug resistant bacteria from various infected wounds in North Egypt

Multi-drug resistant (MDR) bacteria associated with wounds are extremely escalating. This study aims to survey different wounds in Alexandria hospitals, North Egypt, to explore the prevalence and characteristics of MDR bacteria for future utilization in antibacterial wound dressing designs. Among various bacterial isolates, we determined 22 MDR bacteria could resist different classes of antibiotics. The collected samples exhibited the prevalence of mono-bacterial infections (60%), while 40% included poly-bacterial species due to previous antibiotic administration. Moreover, Gram-negative bacteria showed dominance with a ratio of 63.6%, while Gram-positive bacteria reported 36.4%. Subsequently, the five most virulent bacteria were identified following the molecular approach by 16S rRNA and physiological properties using the VITEK 2 automated system. They were deposited in GenBank as Staphylococcus haemolyticus MST1 ({"type":"entrez-nucleotide","attrs":{"text":"KY550377","term_id":"1304487819","term_text":"KY550377"}}KY550377), Pseudomonas aeruginosa MST2 ({"type":"entrez-nucleotide","attrs":{"text":"KY550378","term_id":"1304487820","term_text":"KY550378"}}KY550378), Klebsiella pneumoniae MST3 ({"type":"entrez-nucleotide","attrs":{"text":"KY550379","term_id":"1304487821","term_text":"KY550379"}}KY550379), Escherichia coli MST4 ({"type":"entrez-nucleotide","attrs":{"text":"KY550380","term_id":"1304487822","term_text":"KY550380"}}KY550380), and Escherichia coli MST5 ({"type":"entrez-nucleotide","attrs":{"text":"KY550381","term_id":"1304487823","term_text":"KY550381"}}KY550381). In terms of isolation source, S. haemolyticus MST1 was isolated from a traumatic wound, while P. aeruginosa MST2 and E. coli MST4 were procured from hernia surgical wounds, and K. pneumoniae MST3 and E. coli MST5 were obtained from diabetic foot ulcers. Antibiotic sensitivity tests exposed that K. pneumoniae MST3, E. coli MST4, and E. coli MST5 are extended-spectrum β-lactamases (ESBLs) bacteria. Moreover, S. haemolyticus MST1 belongs to the methicillin-resistant coagulase-negative staphylococcus (MRCoNS), whereas P. aeruginosa MST2 exhibited resistance to common empirical bactericidal antibiotics. Overall, the study provides new insights into the prevalent MDR bacteria in Egypt for further use as specific models in formulating antibacterial wound dressings.     

Mechanism of proteasome gate modulation by assembly chaperones Pba1 and Pba2

The active sites of the proteasome are housed within its central core particle (CP), a barrel-shaped chamber of four stacked heptameric rings, and access of substrates to the CP interior is mediated by gates at either axial end. These gates are constitutively closed and may be opened by the regulatory particle (RP), which binds the CP and facilitates substrate degradation. We recently showed that the heterodimeric CP assembly chaperones Pba1/2 also mediate gate opening through an unexpected structural arrangement that facilitates the insertion of the N terminus of Pba1 into the CP interior; however, the full mechanism of Pba1/2-mediated gate opening is unclear. Here, we report a detailed analysis of CP gate modulation by Pba1/2. The clustering of key residues at the interface between neighboring α-subunits is a critical feature of RP-mediated gate opening, and we find that Pba1/2 recapitulate this strategy. Unlike RP, which inserts at six α-subunit interfaces, Pba1/2 insert at only two α-subunit interfaces. Nevertheless, Pba1/2 are able to regulate six of the seven interfacial clusters, largely through direct interactions. The N terminus of Pba1 also physically interacts with the center of the gate, disrupting the intersubunit contacts that maintain the closed state. This novel mechanism of gate modulation appears to be unique to Pba1/2 and therefore likely occurs only during proteasome assembly. Our data suggest that release of Pba1/2 at the conclusion of assembly is what allows the nascent CP to assume its mature gate conformation, which is primarily closed, until activated by RP.     

shASPP2 H22稳转肝癌细胞系的构建及ASPP2敲低对H22移植瘤小鼠血管生成的影响

摘要 目的：构建小鼠shASPP2 H22稳转肝癌细胞系,观察ASPP2敲低对血管生成的影响。方法：针对小鼠ASPP2基因设计了3个不同的shRNA干扰序列（Y18421,Y18422,Y18423）及1个对照序列（GL427NC2）,采用双酶切（Age Ⅰ和EcoR Ⅰ）及质粒连接构建重组质粒,使用菌落PCR和测序比对进行鉴定;使用293T细胞将各重组质粒包装慢病毒并测定滴度;将 shASPP2和对照慢病毒质粒转染H22细胞,采用流式细胞术测定转染效率;采用qRT-PCR、Western Blot法观察shASPP2慢病毒对H22细胞ASPP2的干扰效果;采用CCK8法观察ASPP2敲低对H22细胞增殖的影响;采用Western Blot法观察ASPP2敲低对H22细胞及上清VEGF表达和分泌的影响;采用细胞注射法建立小鼠ASPP2敲低H22细胞皮下移植瘤模型,游标卡尺法观察肿瘤体积大小,采用活体激光共聚焦观察肿瘤血管生成情况,采用Western Blot法观察肿瘤组织VEGF的表达。结果：双酶切、菌落PCR和测序鉴定结果表示各重组质粒构建成功;各重组质粒经慢病毒包装后,测定显示Y18421、Y18422、Y18423和GL427NC2慢病毒质粒的滴度分别为3.40×10⁸ TU/mL、4.08×10⁸ TU/mL、5.49×10⁸ TU/mL和1.7×10⁹ TU/mL;Y18421、Y18422、Y18423及GL427NC2慢病毒质粒转染效率分别为：86.2 %、69.6 %、60.8 %和76.9 %。与GL427NC2 H22细胞相比,Y18421 H22细胞的ASPP2 mRNA及蛋白的表达明显降低（P<0.01,P<0.05）;Y18421细胞在培养24,48,72 h后增殖速率显著增加（P<0.0001,P<0.001,P<0.01）;Y18421细胞及上清的VEGF表达显著升高（P<0.001,P<0.01,P<0.05）。与GL427NC2 细胞移植瘤相比,Y18421细胞移植瘤体积明显增大（P<0.05）,总血管长度显著增加（P<0.05）,VEGF蛋白的表达明显上调（P<0.05）。结论：小鼠shASPP2 H22稳转肝癌细胞系构建成功,ASPP2敲低可能通过上调VEGF的表达促进小鼠H22细胞移植瘤血管生成。     

血清Irisin、SOST、H2S与膝骨关节炎合并骨质疏松症患者骨密度、骨代谢标志物的相关性及其预测价值

摘要 目的：探讨血清鸢尾素（Irisin）、骨硬化蛋白（SOST）、硫化氢（H₂S）与膝骨关节炎（KOA）合并骨质疏松症（OP）患者骨密度、骨代谢标志物的相关性,分析Irisin、SOST、H₂S预测KOA合并OP的价值。方法：选取2020年4月至2023年4月我院收治的179例KOA患者,根据是否合并OP将其分为OP组（68例）和非OP组（111例）。检测血清Irisin、SOST、H₂S和骨代谢标志物骨钙素（OC）、骨碱性磷酸酶（BALP）、Ⅰ型原胶原N-端前肽（PINP）、抗酒石酸酸性磷酸酶异体（TRACP5b）水平,股骨颈、腰椎L1～4骨密度。Pearson分析血清Irisin、SOST、H₂S与股骨颈、腰椎L1～4骨密度和血清骨代谢标志物的相关性,受试者工作特征（ROC）曲线分析血清Irisin、SOST、H₂S对KOA合并OP的预测价值。结果：OP组血清Irisin、H₂S、OC、BALP、PINP水平,股骨颈、腰椎L1～4骨密度低于非OP组（P＜0.05）,TRACP、SOST水平高于非OP组（P＜0.05）。OP组血清Irisin、H₂S水平与股骨颈、腰椎L1～4骨密度,血清OC、BALP、PINP水平呈正相关（P＜0.05）,与血清TRACP水平呈负相关（P＜0.05）;SOST水平与股骨颈、腰椎L1～4骨密度,血清OC、BALP、PINP水平呈负相关（P＜0.05）,与血清TRACP水平呈正相关（P＜0.05）。Irisin、SOST、H₂S预测KOA合并OP的曲线下面积为0.784、0.773、0.755,联合预测KOA合并OP的曲线下面积为0.908,高于单独预测。结论：KOA合并OP患者血清Irisin、H₂S水平降低、SOST水平增高,低水平Irisin、H₂S和高水平SOST与骨密度降低、骨代谢异常有关,可用于预测KOA合并OP患者骨代谢异常状态和骨质流失风险。     

甲状腺乳头状癌组织YAP、SATB1、ROR2与临床病理特征和复发的关系研究

摘要 目的：探讨甲状腺乳头状癌组织Yes相关蛋白（YAP）、核基质结合区结合蛋白1（SATB1）、受体酪氨酸激酶样孤儿素受体2（ROR2）与临床病理特征和复发的关系。方法：选择2019年6月至2021年6月广东省中医院收治的198例行手术治疗的甲状腺乳头状癌患者,取手术切除的癌组织和癌旁组织,免疫组化法检测YAP、SATB1、ROR2蛋白表达情况。术后随访2年,统计复发情况,根据复发情况将患者分为复发组和未复发组。比较甲状腺乳头状癌患者不同临床病理特征之间YAP、SATB1、ROR2蛋白表达差异,多因素Cox回归分析影响甲状腺乳头状癌术后复发的因素。结果：癌组织中YAP、SATB1、ROR2阳性表达率高于癌旁组织（P＜0.05）。低度分化、TNM分期Ⅲ期、淋巴结转移患者癌组织中YAP、SATB1、ROR2阳性表达率高于中高度分化、TNM分期Ⅰ～Ⅱ期、无淋巴结转移患者（P＜0.05）。198例患者随访2年失访1例,复发21例,复发率为10.66%。复发组癌组织中YAP、SATB1、ROR2阳性表达率高于未复发组癌组织（P＜0.05）。多因素Cox回归分析显示低分化、TNM分期Ⅲ期、未淋巴结清扫、YAP阳性表达、SATB1阳性表达、ROR2阳性表达比是甲状腺乳头状癌术后复发的危险因素（P＜0.05）。结论：甲状腺乳头状癌组织中YAP、SATB1、ROR2阳性表达率显著增高,YAP、SATB1、ROR2阳性表达与低分化、淋巴结转移、晚TNM分期以及术后复发有关。