Multiphase comparative study for WHO/ISUP nuclear grading diagnostic model based on enhanced CT images of clear cell renal cell carcinoma.

To compare and analyze the diagnostic value of different enhancement stages in distinguishing low and high nuclear grade clear cell renal cell carcinoma (ccRCC) based on enhanced computed tomography (CT) images by building machine learning classifiers. A total of 51 patients (Dateset1, including 41 low-grade and 10 high-grade) and 27 patients (Independent Dateset2, including 16 low-grade and 11 high-grade) with pathologically proven ccRCC were enrolled in this retrospective study. Radiomic features were extracted from the corticomedullary phase (CMP), nephrographic phase (NP), and excretory phase (EP) CT images, and selected using the recursive feature elimination cross-validation (RFECV) algorithm, the group differences were assessed using T-test and Mann-Whitney U test for continuous variables. The support vector machine (SVM), random forest (RF), XGBoost (XGB), VGG11, ResNet18, and GoogLeNet classifiers are established to distinguish low-grade and high-grade ccRCC. The classifiers based on CT images of NP (Dateset1, RF: AUC = 0.82 ± 0.05, ResNet18: AUC = 0.81 ± 0.02; Dateset2, XGB: AUC = 0.95 ± 0.02, ResNet18: AUC = 0.87 ± 0.07) obtained the best performance and robustness in distinguishing low-grade and high-grade ccRCC, while the EP-based classifier performance in poorer results. The CT images of enhanced phase NP had the best performance in diagnosing low and high nuclear grade ccRCC. Firstorder_Kurtosis and firstorder_90Percentile feature play a vital role in the classification task.

Megalin blockade with cilastatin ameliorates multiple wasp sting-induced acute kidney injury in rats.

Cilastatin has been shown to prevent various drug-induced nephrotoxicities and confer renoprotection in a mouse model of glycerol-mediated rhabdomyolysis-induced acute kidney injury (AKI). The present study aimed to investigate whether cilastatin attenuates wasp sting-induced AKI in rats. Male Wistar rats were divided into the control, cilastatin, AKI, and AKI + cilastatin groups. Nephrotoxicity was assessed using renal function, rhabdomyolysis (creatine kinase, CK) and intravascular hemolysis (lactate dehydrogenase, LDH) markers, and histological changes. In addition, tubular injury biomarkers, apoptosis, oxidative stress markers, complement C3 expression, and urine and blood myoglobin levels were examined. Compared with the control or cilastatin group, the AKI group showed significant histological damage, increased levels of CK, LDH, and creatinine, and increased mRNA expression of tubular injury biomarkers. Cilastatin ameliorated wasp venom-induced kidney injury by attenuating oxidative stress and apoptosis. Cilastatin also reduced C3 expression in the renal tubular cells. In addition, cilastatin reduced serum myoglobin levels and increased urine myoglobin concentrations. Therefore, megalin blockade with cilastatin attenuates wasp venom-induced AKI owing to its antioxidative and antiapoptotic properties.

Phospholipase A2 inhibitor varespladib prevents wasp sting-induced nephrotoxicity in rats.

This study aimed to clarify whether varespladib, a phospholipase A2 (PLA2) inhibitor, can be used as a therapeutic agent for wasp sting-induced acute kidney injury (AKI). Rats were divided into control, AKI, and AKI + varespladib groups. The AKI model was established by subcutaneously injecting wasp venom at five different sites in rats. Varespladib treatment showed a significant inhibitory effect on wasp venom PLA2in vitro and in vivo. Moreover, we observed that varespladib decreased the levels of rhabdomyolysis and hemolysis markers compared with that in the AKI group. Histopathological changes in the kidney decreased significantly, and rat serum creatinine levels were reduced after varespladib administration. The significantly regulated genes in the kidney of the AKI group were mostly involved in inflammatory response pathway, and the administration of varespladib remarkably attenuated the expression of these genes. Therefore, varespladib inhibited wasp sting-induced functional and pathological damage to the kidneys. We propose that the PLA2 inhibitor varespladib protects the kidney tissue in a wasp sting-induced AKI model by inhibiting PLA2 activity.

A Closed-Tube Nested Quantitative PCR Assay for Rapid Detection of Intron 22 Inversions in the Factor VIII Gene.

BACKGROUND: An inversion of intron 22 in the Factor VIII gene (Inv22) is the causative mutation for 45% of severe hemophilia A cases. Available methods for molecular diagnosis of Inv22 are generally tedious and not ideal for routine clinical use. METHODS: We report here a new method using a single closed-tube nested quantitative PCR (CN-qPCR) for rapid detection of Inv22. This method combines a 12-cycle long-distance PCR (LD-PCR) amplifying the int22h regions, followed by a duplex qPCR targeting two specific regions close to the int22h regions. All reagents were added to a single PCR mixture for the closed-tube assay. Sequential LD-PCR and qPCR was achieved by designing primers at substantially different melting temperatures and optimizing PCR conditions. RESULTS: Seventy-nine male hemophilia A patients of different disease severity were tested by both the CN-qPCR assay and the standard LD-PCR assay. CN-qPCR successfully made calls for all samples, whereas LD-PCR failed in eight samples. For the 71 samples where both methods made calls, the concordance was 100%. Inv22 was detected in 17 out of the 79 samples. Additionally, CN-qPCR achieved clear separation for 10 female carriers and 10 non-Inv22 females, suggesting the assay may also be useful for molecular diagnosis of female carriers. CONCLUSIONS: This new CN-qPCR method may provide a convenient and accurate F8 Inv22 test suitable for clinical use.

A Two-Step Strategy for the Rapid Enrichment of Nitrosocosmicus-Like Ammonia-Oxidizing Thaumarchaea.

Ammonia-oxidizing archaea (AOA) are widely distributed on the earth and play a significant role in the global nitrogen cycle. Although dozens of AOA strains were obtained in the last 13 years, it is still necessary to obtain more AOA strains for the entire exploration of their ecology, physiology, and underlying biochemistry in different environments. In this study, we designed a two-step strategy for the rapid enrichment of Nitrosocosmicus-like AOA from soils. Firstly, combination of kanamycin and ampicillin was chosen as the selective stress for bacteria and quartz sands were used as the attachment of AOA cells during the first step cultivation; only after 40-75 days cultivation, AOA enrichments with abundance >20% were obtained. Secondly, combination of ciprofloxacin and azithromycin was chosen as the selective stress for the following cultivation; it is able to penetrate the biofilms and kill the bacterial cells inside the aggregate, contributing to the AOA enrichments reached high abundances (90%) only after one-time cultivation. Basing on this strategy, three AOA strains were obtained from agricultural soils only after 90-150 days cultivation. Phylogenetic analysis suggested these AOA belong to the soil group I.1b Thaumarchaeota and are closely related to the genus Nitrosocosmicus. In general, AOA enrichment or isolation is very difficult and time-consuming (an average of 2-3 years). Here, we provide a new strategy for the rapid enrichment of high abundance of Nitrosocosmicus-like AOA from soil, which gives a new solution to the AOA enrichment and cultivation in a short period.

Insight into the diversity of antibiotic resistance genes in the intestinal bacteria of shrimp Penaeus vannamei by culture-dependent and independent approaches.

Antibiotic resistance genes (ARGs) that distributed in antibiotic resistant bacteria (ARBs) are widespread in aquaculture and have great threats to the aquatic organism as well as to human. However, our understanding about the risk of ARGs to the health of aquatic organism is still limited. In the present study, we got a deep insight into the diversity of ARGs in the intestinal bacteria of shrimp by culture-dependent and independent approaches. Results of the PCR-based detection and culture-dependent analysis indicated that the tetracycline, sulfadiazine, quinolone and erythromycin resistance genes were prevalent in the commercial shrimps that bought from aquatic markets or supermarket. The culture-independent plasmid metagenomic analysis identified 62 different ARGs, which were classified into 21 types, with abundances ranging from 13 to 1418 ppm. The analysis suggested that most of the ARGs come from the plasmids originating from Vibrio (accounted for 2.8-51%) and Aeromonas (accounted for 16-55%), and the Vibrio group was concluded to be the main bacterial pathogen that probably resulted in the shrimp disease. Accordingly, the plasmid metagenomic that focuses on the mobile genetic elements has great potential on the identification of ARGs in complex environments.

Mutational and phenotypic spectrum of phenylalanine hydroxylase deficiency in Zhejiang Province, China.

Phenylalanine hydroxylase deficiency (PAHD), one of the genetic disorders resulting in hyperphenylalaninemia, has a complex phenotype with many variants and genotypes among different populations. Here, we describe the mutational and phenotypic spectrum of PAHD in a cohort of 420 patients from neonatal screening between 1999 and 2016. The observed phenotypes comprised 43.57% classic phenylketonuria, 33.10% mild PKU, and 23.33% mild hyperphenylalaninemia, with an overall PAHD incidence of 1 in 20,445. Genetic testing was performed for 209 patients and 72 variants including seven novel variants were identified. These included two synonymous and five pathogenic nonsynonymous variants (p.S36*, p.T186I, p.L255W, p.F302V and p.R413H). The most common variant among all patients was p.R243Q, followed by p.R241C, p.Y204C, p.R111* and c.442-1G > A. Variants p.R53H and p.F392I occurred only in MHP with 19.3% and 8.0% of the observed alleles respectively. The genotypes p.[R241C];[R243Q], p.[R243Q];[R243Q], and p.[Y204C];[R243Q] were abundant across all PAHD patients. The distributions of the null allele and the three defined genotypes, null/null, null/missense, and missense/missense, were significantly different between the cPKU and mPKU patients. However, no significant differences were found between mPKU and MHP patients, indicating that other modifier factors influence the phenotypic outcome in these patients. The data presented here will provide a valuable tool for improved genetic counseling and management of future cases of PAHD in China.

Overexpression of human mitochondrial alanyl-tRNA synthetase suppresses biochemical defects of the mt-tRNAAla mutation in cybrids.

Mutations of mitochondrial transfer RNAs (mt-tRNAs) play a major role in a wide range of mitochondrial diseases because of the vital role of these molecules in mitochondrial translation. It has previously been reported that the overexpression of mitochondrial aminoacyl tRNA synthetases is effective at partially suppressing the defects resulting from mutations in their cognate mt-tRNAs in cells. Here we report a detailed analysis of the suppressive activities of mitochondrial alanyl-tRNA synthetase (AARS2) on mt-tRNAAla 5655 A>G mutant. Mitochondrial defects in respiration, activity of oxidative phosphorylation complexes, ATP production, mitochondrial superoxide, and membrane potential were consistently rescued in m.5655A>G cybrids upon AARS2 expression. However, AARS2 overexpression did not result in a detectable increase in mutated mt-tRNAAla but caused an increase incharged mt-tRNAAla in mutant cybrids, leading to enhanced mitochondrial translation. This indicated that AARS2 improved the aminoacylation activity in the case of m.5655A>G, rather than having a stabilizing effect on the tRNA structure. The data presented in this paper deepen our understanding of the pathogenesis of mt-tRNA diseases.

Mutation analysis of Leber's hereditary optic neuropathy using a multi-gene panel.

The present study investigates the spectrum and incidence of mitochondrial DNA (mtDNA) mutations associated with Leber's hereditary optic neuropathy (LHON) in a Han population using a multi-gene panel with 46 LHON-associated mutations among 13 mitochondrial genes. A total of 23 mutations were observed in a cohort of 275 patients and 281 control subjects using multi-gene panel analysis. The causative mutations associated with LHON were identified to be m.11778G>A, m.14484T>C, m.3460 G>A, m.3635G>A, m.3866T>C and m.3733G>A, responsible for 70.55% cases in the patient cohort. The secondary mutations in the Chinese LHON population were m.12811T>C, m.11696 G>A, m.3316G>A, m.3394T>C, m.14502T>C, m.3497C>T, m.3571C>T, m.12338T>C, m.14693A>G, m.4216T>C and m.15951A>G, with incidences of 5.09, 4.36, 4.00, 4.00, 4.00, 2.55, 1.82, 1.82, 1.45, 1.09 and 1.09%, respectively. Besides three hotspot genes, MT-ND1, MT-ND4 and MT-ND6, MT-ND5 also had a high incidence of secondary mutations. Those mutations reported as rare causative mutations in a European LHON population, m.3376G>A, m.3700G>A and m.4171C>A, m.10663T>C, m.13051G>A, m.14482C>G/A, m.14495A>G and m.14568C>T were undetected in the present study. The primary and secondary mutations associated with LHON in the present multi-gene panel will advance the current understanding of the clinical phenotype of LHON, and provide useful information for early diagnosis.