Rapid identification of invasive fungal species using sensitive universal primers-based PCR and restriction endonuclease digestions coupled with high-resolution melting analysis

BackgoundConventional diagnosis of invasive fungal disease from blood cultures is often notoriously delayed and inadequately sensitive. We aimed to develop a universal primers-based polymerase chain reaction (PCR) assay and restriction fragment length polymorphisms (RFLP) for rapid identification of invasive fungal disease (IFD).MethodsWe evaluated 16 clinical fungal species using a combination of PCR assays with 3 different restriction endonucleases targeting various internal transcribed spacer (ITS) regions and high resolution melting analysis (HRMA). Serial samples from 75 patients suspected to have IFD were analyzed for clinical verification.ResultsWe have designed a universal PCR capable of amplifying a portion of the 18S rRNA gene of 16 clinically important fungal species. The restriction patterns of most PCR products generated by EcoRI or double digested by ClaI and AvaI were different, except Aspergillus niger and Aspergillus flavus had a similar pattern, and Aspergillus fumigatus and Aspergillus terreus had a similar pattern. All these species had a unique melting curve shape using the HRMA. Both HRMA and universal PCR had adequate sensitivity, and all sixteen reference fungal species can be clearly distinguished by the universal PCR-RFLP-HRMA assay. With a reference library of 176 clinically relevant fungal strains, and 75 clinical samples from patients with suspicious IFD were tested, our assay identified 100% and 61.1% of isolates from the reference library and clinical samples, respectively.ConclusionsUniversal PCR and RFLP coupled with HRMA could be a highly discriminative and useful molecular diagnostic that could enhance the current diagnostic, treatment, and surveillance methods of invasive fungal disease.     

A case report of cutaneous melanocytoma with CRTC1‐TRIM11 fusion: Is CMCT distinct from clear cell sarcoma of soft tissue?

Pathological diagnosis of dermal melanocytic tumors is often problematic owing to histological resemblance. Recently, cutaneous melanocytoma with CRTC1‐TRIM11 (CMCT) was added to this category. However, only six cases have been reported so far. We herein present a case of a 77‐year‐old Japanese man with CMCT. The patient presented a nodule in the right thigh and underwent surgical resection. Histological examination indicated a well‐demarcated 6 × 5 mm‐sized tumor nodule in the dermis and subcutis. The tumor was amelanotic, consisting of uniform nests and fascicles of spindled, or epithelioid cells. The melanocytic nature was evident by immunohistochemistry. The CRTC1‐TRIM11 fusion was detected by TRIM11 immunostaining, chromogenic in situ hybridization, and RT‐PCR/direct sequencing. He has been free from the tumor for 1 year after additional resection. The main differential diagnosis of CMCT includes primary and metastatic dermal malignant melanomas (MM) and dermal/subcutaneous clear cell sarcoma (CCS). Additionally, histological overlap with paraganglioma‐like dermal melanocytic tumor was considered. Although some investigators argue that CMCT is a variant of CCS, we think it should be separated from CCS, and subcutaneous/dermal CCS should be confined to tumors with EWSR1‐ATF1/ CREB1 fusion. However, longer follow‐up and more case studies are needed for revealing the true prognosis of CMCT.     

RNA-Seq analysis in an avian model of maternal phenylketonuria

Cardiac malformations (CVMs) are a leading cause of infant morbidity and mortality. CVMs are particularly prevalent when the developing fetus is exposed to high levels of phenylalanine in-utero in mothers with Phenylketonuria. Yet, elucidating the underlying molecular mechanism leading to CVMs has proven difficult. In this study we used RNA-Seq to investigate an avian model of MPKU and establish differential gene expression (DEG) characteristics of the early developmental stages HH10, 12, and 14. In total, we identified 633 significantly differentially expressed genes across stages HH10, 12, and 14. As expected, functional annotation of significant DEGs identified associations seen in clinical phenotypes of MPKU including CVMs, congenital heart defects, craniofacial anomalies, central nervous system defects, and growth anomalies. Additionally, there was an overrepresentation of genes involved in cardiac muscle contraction, adrenergic signaling in cardiomyocytes, migration, proliferation, metabolism, and cell survival. Strikingly, we identified significant changes in expression with multiple genes involved in Retinoic Acid (RA) metabolism and downstream targets. Using qRTPCR, we validated these findings and identified a total of 42 genes within the RA pathway that are differentially expressed. Here, we report the first elucidation of the molecular mechanisms of cardiovascular malformations in MPKU conducted at early developmental timepoints. We provide evidence suggesting a link between PHE exposure and the alteration of RA pathway. These results are promising and offer novel findings associated with congenital heart defects in MPKU.     

Prevalence of hepatitis E virus infection in multiple transfused Brazilian patients with thalassemia and sickle cell disease

Hepatitis E virus (HEV) is a leading cause of acute hepatitis worldwide. The virus is acquired by fecal-oral route; however, it can also be transmitted by blood transfusion. The objective of the study was to examine anti-HEV immunoglobulin G and HEV RNA prevalence in multiple transfused patients with thalassemia and sickle cell disease (SCD), and in blood donors. The HEV seroprevalence in the patients was 13% (20% in thalassemics; 7.7% in SCD), and 11% in blood donors. No positive result for HEV RNA was obtained. This is a pioneer study examining HEV circulation in Brazilian patients with hemoglobinopathies.     

含微乳头结构肺腺癌CT重建征象特征及上皮间质转化相关基因表达

目的 探讨含微乳头结构（MPP）肺腺癌具有的CT重建征象特征,上皮间质转化（EMT）相关分子表达情况以及它们之间的关系。 方法 选取含MPP侵袭性肺腺癌（IAC）37例、不含MPP 80例及其对应的癌旁组织,比较临床病理特征、CT重建征象;应用Real-time PCR及Western blotting检测EMT有关标识分子表达变化情况。 结果 含MPP组淋巴结转移比例较高、毛刺、胸膜凹陷征、实性及分叶比例较高（均P<0.001）。 含MPP组E-钙黏蛋白（cadherin）及β-连环蛋白（β-catenin）表达均下调（P<0.05）,N-cadherins、波形蛋白（vimentin）、Snail及转化生长因子（TGF）-β表达均上调（P<0.05）。有毛刺组E cadherin及β-catenin表达下调者所占比例较高,而vimentin表达上调者所占比例较高（均P<0.05）;实性结节组E-cadherin表达下调者所占比例较高,而N-cadherin及vimentin表达上调者所占比例较高（均P<0.05）。 结论 含MPP浸润性肺腺癌具有特征性CT重建征象,发生了EMT改变。其CT重建征象与EMT相关分子表达存在联系。     

二氢杨梅素对绒毛膜癌细胞增殖和迁移能力的影响

目的 探讨二氢杨梅素（DMY）对绒毛膜癌（绒癌）JEG-3及JAR细胞增殖和迁移能力的影响。 方法 MTT法检测不同浓度的二氢杨梅素(0 mg/L, 20 mg/L, 40 mg/L, 60 mg/L, 80 mg/L)作用一定时间后,对绒癌JEG-3和JAR细胞增殖能力的影响;细胞划痕实验和Transwell法检测不同浓度二氢杨梅素(0 mg/L, 40 mg/L, 60 mg/L, 80 mg/L)分别作用绒癌JEG-3细胞及JAR细胞一定时间后,对其迁移能力的影响;Real-time PCR和Western blotting方法分别检测不同浓度二氢杨梅素(0 mg/L, 40 mg/L, 60 mg/L, 80 mg/L)作用绒癌JEG-3及JAR细胞后,基质金属蛋白酶2（MMP-2）mRNA和蛋白表达水平的影响。 结果 不同浓度二氢杨梅素作用绒癌JEG-3和JAR细胞24 h和48 h后,随着二氢杨梅素浓度增加,对JEG-3和JAR细胞增殖抑制作用增强（P＜0.05）。二氢杨梅素作用绒癌JEG-3及JAR细胞后,显著抑制细胞迁移能力,且具有浓度依赖性（P＜0.05）。不同浓度二氢杨梅素作用JEG-3和JAR细胞后,MMP-2 的mRNA和蛋白表达水平明显下降（P＜0.05）。 结论 二氢杨梅素能够抑制JEG-3及JAR细胞的增殖能力且具有浓度依赖性,同时二氢杨梅素可能通过下调绒癌JEG-3及JAR细胞中MMP-2 mRNA和蛋白的表达,抑制绒癌细胞的侵袭迁移。     

瑞舒伐他汀预处理对缺血再灌注大鼠大脑中动脉血管平滑肌细胞炎症因子的影响及其机制

目的 探讨瑞舒伐他汀预处理对大鼠局灶性脑缺血再灌注后大脑中动脉血管平滑肌细胞（VSMCs）中炎性细胞因子白细胞介素（IL）-1β、IL-6、肿瘤坏死因子ɑ（TNF-α）表达的影响以及可能的机制。 方法 36只健康成年SD大鼠,雌雄不限,随机分为假手术组,局灶性脑缺血再灌注组,瑞舒伐他汀预处理组,每组12只。大脑中动脉缺血2 h再灌注24 h后, Real-time PCR及Western blotting法检测大鼠大脑中动脉VSMCs中IL-1β、IL-6和TNF-α以及核因子κB（NF-κB）mRNA和蛋白的表达。 结果 再灌注24 h后,模型组VSMCs IL-1β、IL-6 以及TNF-αmRNA和蛋白的表达明显增加,而给予瑞舒伐他汀预处理后,可明显抑制大脑中动脉VSMCs中IL-1β、IL-6和TNF-α mRNA和蛋白的高表达,同时也可发现,VSMCs中NF-κB mRNA和蛋白的表达也明显减少。 结论 瑞舒伐他汀预处理可有效抑制大脑中动脉VSMCs中IL-1β、IL-6和TNF-α mRNA和蛋白的表达,从而减轻缺血再灌注后炎症反应对脑组织的进一步损伤作用。瑞舒伐他汀预处理对IL-1β、IL-6及TNF-α的作用可能与其减少VSMCs中NF-κB的表达有关。