Novel PATL2 variants cause female infertility with oocyte maturation defect.

PURPOSE: Oocyte maturation defect (OOMD) is a rare cause of in vitro fertilization failure characterized by the production of immature oocytes. Compound heterozygous or homozygous PATL2 mutations have been associated with oocyte arrest at the germinal vesicle (GV), metaphase I (MI), and metaphase II (MII) stages, as well as morphological changes. METHODS: In this study, we recruited three OOMD cases and conducted a comprehensive multiplatform laboratory investigation. RESULTS: Whole exome sequence (WES) revealed four diagnostic variants in PATL2, nonsense mutation c.709C > T (p.R237*) and frameshift mutation c.1486_1487delinsT (p.A496Sfs*4) were novel mutations that have not been reported previously. Furthermore, the pathogenicity of these variants was predicted using in silico analysis, which indicated detrimental effects. Molecular dynamic analysis suggested that the A496S variant disrupted the hydrophobic segment, leading to structural changes that affected the overall protein folding and stability. Additionally, biochemical and molecular experiments were conducted on cells transfected with wild-type (WT) or mutant PATL2 (p.R237* and p.A496Sfs*4) plasmid vectors. CONCLUSIONS: The results demonstrated that PATL2A496Sfs*4 and PATL2R237* had impacts on protein size and expression level. Interestingly, expression levels of specific genes involved in oocyte maturation and early embryonic development were found to be simultaneously deregulated. The findings in our study expand the variation spectrum of the PATL2 gene, provide solid evidence for counseling on future pregnancies in affected families, strongly support the application of in the diagnosis of OOMD, and contribute to the understanding of PATL2 function.

Both epilepsy and anti-seizure medications affect bone metabolism in children with self-limited epilepsy with centrotemporal spikes.

OBJECTIVE: Bone metabolism can be influenced by a range of factors. We selected children with self-limited epilepsy with centrotemporal spikes (SeLECTS) and lifestyles similar to those of healthy children to control for the confounding factors that may influence bone metabolism. We aimed to identify the specific effects of epilepsy and/or anti-seizure medications (ASMs) on bone metabolism. METHODS: Patients with SeLECTS were divided into an untreated group and a monotherapy group, and the third group was a healthy control group. We determined the levels of various biochemical markers of bone metabolism, including procollagen type I nitrogenous propeptide (PINP), alkaline phosphatase (ALP), osteocalcin (OC), collagen type I cross-linked C-telopeptide (CTX), calcium, magnesium, phosphorus, parathyroid hormone (PTH), and vitamin D3 (VD3 ). RESULTS: A total of 1487 patients (from 19 centers) were diagnosed with SeLECTS; 1032 were analyzed, including 117 patients who did not receive any ASMs (untreated group), 643 patients who received only one ASM (monotherapy group), and 272 children in the healthy control group. Except for VD3 , other bone metabolism of the three groups were different (p < .001). Bone metabolism was significantly lower in the untreated group than the healthy control group (p < .05). There were significant differences between the monotherapy and healthy control group in the level of many markers. However, when comparing the monotherapy and untreated groups, the results were different; oxcarbazepine, levetiracetam, and topiramate had no significant effect on bone metabolism. Phosphorus and magnesium were significantly lower in the valproic acid group than the untreated group (adjusted p < .05, Cliff's delta .282-.768). CTX was significantly higher in the lamotrigine group than in the untreated group (adjusted p = .012, Cliff's delta = .316). SIGNIFICANCE: Epilepsy can affect many aspects of bone metabolism. After controlling epilepsy and other confounders that affect bone metabolism, we found that the effects of ASMs on bone metabolism differed. Oxcarbazepine, levetiracetam, and topiramate did not affect bone metabolism, and lamotrigine corrected some of the abnormal markers of bone metabolism in patients with epilepsy.

The predictive value of selected serum microRNAs for acute GVHD by TaqMan MicroRNA arrays.

Currently, the diagnosis of acute graft-versus-host disease (aGVHD) is mainly based on clinical symptoms and biopsy results. This study was designed to further explore new no noninvasive biomarkers for aGVHD prediction/diagnosis. We profiled miRNAs in serum pools from patients with aGVHD (grades II-IV) (n = 9) and non-aGVHD controls (n = 9) by real-time qPCR-based TaqMan MicroRNA arrays. Then, predictive models were established using related miRNAs (n = 38) and verified by a double-blind trial (n = 54). We found that miR-411 was significantly down regulated when aGVHD developed and recovered when aGVHD was controlled, which demonstrated that miR-411 has potential as an indicator for aGVHD monitoring. We developed and validated a predictive model and a diagnostic model for aGVHD. The predictive model included two miRNAs (miR-26b and miR-374a), which could predict an increased risk for aGVHD 1 or 2 weeks in advance, with an AUC, Positive Predictive Value (PPV), and Negative Predictive Value (NPV) of 0.722, 76.19 %, and 69.70 %, respectively. The diagnostic model included three miRNAs (miR-28-5p, miR-489, and miR-671-3p) with an AUC, PPV, and NPV of 0.841, 85.71 % and 83.33 %, respectively. Our results show that circulating miRNAs (miR-26b and miR-374a, miR-28-5p, miR-489 and miR-671-3p) may serve as biomarkers for the prediction and diagnosis of grades II-IV aGVHD.

A novel mixed integer programming for multi-biomarker panel identification by distinguishing malignant from benign colorectal tumors.

Multi-biomarker panels can capture the nonlinear synergy among biomarkers and they are important to aid in the early diagnosis and ultimately battle complex diseases. However, identification of these multi-biomarker panels from case and control data is challenging. For example, the exhaustive search method is computationally infeasible when the data dimension is high. Here, we propose a novel method, MILP_k, to identify serum-based multi-biomarker panel to distinguish colorectal cancers (CRC) from benign colorectal tumors. Specifically, the multi-biomarker panel detection problem is modeled by a mixed integer programming to maximize the classification accuracy. Then we measured the serum profiling data for 101 CRC patients and 95 benign patients. The 61 biomarkers were analyzed individually and further their combinations by our method. We discovered 4 biomarkers as the optimal small multi-biomarker panel, including known CRC biomarkers CEA and IL-10 as well as novel biomarkers IMA and NSE. This multi-biomarker panel obtains leave-one-out cross-validation (LOOCV) accuracy to 0.7857 by nearest centroid classifier. An independent test of this panel by support vector machine (SVM) with threefold cross validation gets an AUC 0.8438. This greatly improves the predictive accuracy by 20% over the single best biomarker. Further extension of this 4-biomarker panel to a larger 13-biomarker panel improves the LOOCV to 0.8673 with independent AUC 0.8437. Comparison with the exhaustive search method shows that our method dramatically reduces the searching time by 1000-fold. Experiments on the early cancer stage samples reveal two panel of biomarkers and show promising accuracy. The proposed method allows us to select the subset of biomarkers with best accuracy to distinguish case and control samples given the number of selected biomarkers. Both receiver operating characteristic curve and precision-recall curve show our method's consistent performance gain in accuracy. Our method also shows its advantage in capturing synergy among selected biomarkers. The multi-biomarker panel far outperforms the simple combination of best single features. Close investigation of the multi-biomarker panel illustrates that our method possesses the ability to remove redundancy and reveals complementary biomarker combinations. In addition, our method is efficient and can select multi-biomarker panel with more than 5 biomarkers, for which the exhaustive methods fail. In conclusion, we propose a promising model to improve the clinical data interpretability and to serve as a useful tool for other complex disease studies. Our small multi-biomarker panel, CEA, IL-10, IMA, and NSE, may provide insights on the disease status of colorectal diseases. The implementation of our method in MATLAB is available via the website: http://doc.aporc.org/wiki/MILP_k.

[Determination of 20 Trace Elements in the Blood Collection Tubes with ICP-MS].

To investigate the contamination of blood collection tubes, 20 trace elements (Al, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Mo, Cd, Sn, Sb, Ba, W, Hg, Tl, Pb) in 13 different types of blood collection tube were studied with ICP-MS method. The lixivium of H(2)O and 10% HNO(3) were measured with ICP-MS, and then the contamination coming from the blood collection tube is specified. According to the concentration range of human blood, plasma and serum from recently published literature, this report presents a detailed analysis of capable trace elements for each blood collection tube. The results showed that, tube No.1 is capable to analyze 18 trace elements in the human serum; tube No.6 is capable to analyze 15 trace elements in the human plasma; tube No. 13 is capable to analyze 17 trace elements in the human blood. But we still should be aware that, the elements Sb and W in tube No.1, the elements V, Cr, Ni, and Sb in tube No.6, and the elements Al, Sb and W in tube No.13, are in the same magnitude of the normal trace element concentration range in the human serum, plasma and blood. They might affect the testing results. The serum collected from the same volunteer by tube No.1 and tube No.3 were compared here, the results show that, almost each trace element concentration of human serum from tube No.1 is lower than from tube No.3, especially for elements Al, V, Cr, Mn, As, Sn, and Sb. The results indicate that the blood collection tubes show great impact on determination of trace element.

A thermo-stable lysine aminopeptidase from Pseudomonas aeruginosa: Isolation, purification, characterization, and sequence analysis.

Pseudomonas aeruginosa NJ-814, isolated from garden soil, produced an extracellular aminopeptidase that was purified using ammonium sulfate precipitation and ion exchange chromatography. The purity was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the Mr value of the enzyme was estimated to be 55 kDa. The purified enzyme shows maximum activity at pH 9.0 and 80 °C. It exhibits high thermo-stability. Half of the activity can remain after incubation at 80 °C for 119 min. It is stable within pH range of 7.5-10.5. It is strongly activated by Co(2+) and inhibited by Fe(2+) , Cu(2+) , Ni(2+) , Zn(2+) , and ethylene diamine tetraacetic acid (EDTA). The specificity of the enzyme was investigated. Within several aminoacyl-p-nitroanilines (AA-pNA), Lys-pNA is proven to be the optimal substrate. The Michaelis-Menten constant (Km ) of the enzyme for Lys-pNA and Leu-pNA were 2.32 and 9.41 mM, respectively. Peptide map fingerprinting shows that the sequence of the enzyme is highly similar to aminopeptidase Y from P. aeruginosa 18A. It can be speculated that this enzyme is a Zn(2+) -dependent enzyme and contains two zinc ions in its active site.

hTERT rs2736098 genetic variants and susceptibility of hepatocellular carcinoma in the Chinese population: a case-control study.

BACKGROUND: The human telomerase reverse transcriptase (hTERT) gene encodes the catalytic subunit of telomerase, which mediates pleiotropic effects, including the regulation of senescence and proliferation and plays an important role in carcinogenesis. This study attempts to clarify the genetic predisposition to hepatocellular carcinoma (HCC), focusing on the hTERT gene rs2736098 polymorphism. METHOD: Four hundred patients with HCC and 400 non-cancer controls were genotyped to elucidate the potential association between hTERT rs2736098 polymorphism and HCC risks. RESULTS: Compared with the controls, the patients with HCC had a lower frequency of G/G genotype (33.3% vs 44.3%, P=0.001) and a higher frequency of G/A (51.5% vs 39.5%, P=0.001). Allele genotypic frequencies in the patients differed from those of the controls (P=0.040). The data of this study rs2736098[A] allele contributed significantly to HCC risk in female patients (OR=1.78, 95% CI, 1.17-2.72, P=0.007), patients with HCV infection (OR=2.89, 95% CI, 1.08-7.70, P=0.031), non-drinker patients (OR=1.32, 95% CI, 1.06-1.65, P=0.015), and patients not affected by HBV (OR=1.77, 95% CI, 1.30-2.40, P<0.001). CONCLUSIONS: rs2736098[A] may be an independent hereditary parameter in HCC, but some risk factors would cover up the association by more powerful hepatocarcinogenesis. These results are important guidance for further studies in detecting HCC-associated single nucleotide polymorphisms.

Associations between Borg's rating of perceived exertion and changes in urinary organic acid metabolites after outdoor weight-bearing hiking.

BACKGROUND: Developing methods to monitor exercise load and evaluate body fatigue and muscle injury over time in hiking training remains a key problem to be solved. A widely used psycho-physical tool to assess the subjective perception of effort during exercise is Borg's rating of perceived exertion (BRPE) scale. Data on the relationships and validity of the BRPE compared to objectively assessed metabolic criteria are still lacking, especially urinary organic acid concentrations. AIM: To verify whether the BRPE scale could be used in the prescription of outdoor hiking with weight-bearing and reveal the relationship between the BRPE scale and urinary physiological measures. METHODS: Eighty-nine healthy men (average age: 22 years) were enrolled in a 40 km (6 h) hiking training exercise with a 20 kg load. After training, the BRPE scale (6-20) was completed. All participants were divided into three groups according to the rating of the BRPE scale. Urine samples were collected before and after training. Urinary myoglobin levels were measured immediately using the fluorescent immunoassay method. The remaining urine was subpacked and frozen for the subsequent detection of urinary organic acids using gas chromatography and mass spectrometry. RESULTS: The contents of organic acids and myoglobin in urine were significantly increased after participants hiked 40 km (6 h) with a 20 kg load. Only orthogonal partial least-squares discrimination analysis performed well in separating the group with a BRPE score of 6-12 from the group with a BRPE score of 13-20. Significant differences in the urine levels of several organic acids were observed between the two groups, and the heatmap also presented different metabolic profiles based on BRPE. According to the standard of a variable importance in the projection > 1, fold change > 1.5 and P < 0.05, 19 different metabolites of urinary organic acids were screened and enriched in pathways mainly including the citrate cycle (tricarboxylic acid cycle) and alanine, aspartate and glucose metabolism. CONCLUSION: The BRPE scale identified significantly different urinary organic acid profiles between the higher and lower BRPE value groups, and, thus, could be used to monitor body fatigue in individuals participating in long-distance outdoor hiking with weight bearing.

Identification of a novel mutation in the FGF10 gene in a Chinese family with obvious congenital lacrimal duct dysplasia in lacrimo-auriculo-dento-digital syndrome.

AIM: To identify the pathogenic gene variant in a family with lacrimo-auriculo-dento-digital syndrome [LADD (MIM 149730)] showing congenital lacrimal duct dysplasia as the main clinical manifestation and lay the foundation for future research on the pathogenic gene. METHODS: Ophthalmological examinations, including slit-lamp biomicroscopy and lacrimal duct probing, and computed tomography dacryocystography (CT-DCG) were performed for all participants. The family pedigree was drawn, genetic features were analyzed, and the genomic DNA of the subjects was extracted. Pathogenic genes were screened via whole exome sequencing (WES) and confirmed using Sanger sequencing. RESULTS: Six patients belonged to this three-generation family, and their clinical manifestations included congenital nasolacrimal duct obstruction, congenital absence of lacrimal puncta and canaliculi, lacrimal fistulae, and limb deformities. This pattern indicates autosomal dominant inheritance. Diagnosis was based on the clinical characteristics of LADD syndrome, which presented in all the patients in this family. A novel frameshift mutation in the FGF10 gene (NM_004465.1), c.234dupC (p.Trp79Leus*15), was identified in all patients via WES. The variant was confirmed by Sanger sequencing and classified as a "pathogenic mutation" according to the American College of Medical Genetics and Genomics (ACMG) variant interpretation guidelines. CONCLUSION: A novel frameshift mutation in the FGF10 gene is found in all patients. This finding helps this family with LADD syndrome receiving a more accurate clinical diagnosis and genetic counseling by extending the mutation range of the FGF10 gene.

Genes expression profiling of peripheral blood cells of patients with hepatocellular carcinoma.

HCC (hepatocellular carcinoma) is often diagnosed at an advanced stage with poor prognosis. Peripheral blood may be useful in cancer classification, and therefore we investigated the gene expression found by Affymetrix HG-U133 Plus2.0 microarray, with samples from nine HCC patients and five healthy NC (normal controls). A total of 726 probe sets showed significant differences based on the criteria of P<0.05 and absolute fold change >2. The genes were related to many biological functions, including immune response, transcription regulation and metabolism processes. Ten genes [IL-8 (interleukin 8), GOS2 (G0 /G1 switch gene 2), CXCR4 (CXC chemokine receptor 4), FOS, RPS24 (40S ribosomal protein S24), HAP90AA1, PFDN5, RPL27, GZMA and PFN1] showing significant differences were confirmed by real-time PCR in 54 HCC patients and 56 healthy NC. Seven genes [IL-8, GOS2, CXCR4, FOS, RPS24, HSP90AA1 (heat shock protein 90AA1) and PFN1] showed significant difference both in RT-PCR (reverse transcription-PCR) and microarray. Expression of IL-8 and FOS proteins was up-regulated in HCC compared with healthy controls. A gene signature in peripheral blood which can distinguish HCC patients and healthy controls may have been identified.

The identification of miR-375 as a potential biomarker in distal gastric adenocarcinoma.

MicroRNAs (miRNAs) are endogenous small noncoding RNAs that negatively regulate gene expression at the posttranscriptional level and play an important role in carcinogenesis. Herein, we characterized the global expression of miRNA in distal gastric adenocarcinomas and determined if circulating miRNAs could be used as biomarkers for distal gastric adenocarcinoma. We used a microarray screening system to detect dysregulated miRNAs in distal gastric adenocarcinoma tissues. The expression of a subset of five aberrantly expressed miRNAs (miR-375, -196b, -204, -18b, and -93) were further quantified in an independent set of clinical samples of distal gastric adenocarcinoma by real-time quantitative RT-PCR (rt-qRT-PCR). We also used rt-qRT-PCR to investigate the expression levels of putative miRNA biomarkers in serum and tumor cell lines. In our study, the expression of a subset of microRNAs was altered in distal gastric adenocarcinoma compared to normal tissue, miR-375 was significantly downregulated in distal gastric adenocarcinoma tissues, to a level that was significantly lower than cardia adenocarcinoma (p < 0.05). The circulating serum levels of miR-375 in patients who had distal gastric adenocarcinoma were also much lower than normal controls (p < 0.001). As a biomarker, miR-375 yielded a receiver operating characteristic area under the curve of 0.835. The specificity and sensitivity was 80% and 85%, respectively, in the discrimination of distal gastric adenocarcinoma from control, at a normalized cutoff of 0.218. The expression of miR-375 was downregulated both in distal gastric adenocarcinoma tissues and serum of patients with distal gastric adenocarcinoma. These data suggest miR-375 is a potential biomarker for distal gastric adenocarcinoma.

Genome-wide methylation profiling of early colorectal cancer using an Illumina Infinium Methylation EPIC BeadChip.

BACKGROUND: DNA methylation is a part of epigenetic modification, that is closely related to the growth and development of colorectal cancer (CRC). Specific methylated genes and methylated diagnostic models of tumors have become current research focuses. The methylation status of circulating DNA in plasma might serve as a potential biomarker for CRC. AIM: To investigate genome-wide methylation pattern in early CRC using the Illumina Infinium Human Methylation 850K BeadChip. METHODS: The 850K Methylation BeadChip was used to analyze the genome-wide methylation status of early CRC patients (n = 5) and colorectal adenoma patients (n = 5). Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enrichment analyses were performed on the selected differentially methylated sites to further discover candidate methylation biomarkers in plasma. RESULTS: A total of 1865 methylated CpG sites with significant differences were detected, including 676 hypermethylated sites and 1189 hypomethylated sites. The distribution of these sites covered from the 1st to 22nd chromosomes and are mainly distributed on the gene body and gene promoter region. GO and KEGG enrichment analysis showed that the functions of these genes were related to biological regulation, molecular binding, transcription factor activity and signal transduction pathway. CONCLUSION: The study demonstrated that the Illumina Infinium Human Methylation 850K BeadChip can be used to investigate genome-wide methylation status of plasma DNA in early CRC and colorectal adenoma patients.

Effects of Intermittent Normoxia on Chronic Hypoxic Pulmonary Hypertension and Right Ventricular Hypertrophy in Rats.

Liu, Chunlei, Xu Chen, Ge Guo, Xiang Xu, Xin Li, Qingxia Wei, Yanying Shen, Hanlu Li, Jianxiu Hao, Ya Ping Tian, and Kunlun He. Effects of intermittent normoxia on chronic hypoxic pulmonary hypertension and right ventricular hypertrophy in rats. High Alt Med Biol. 22: 184-192, 2021. Background: Individuals with chronically low arterial oxygen tension owing to high altitude develop elevated rates of pulmonary hypertension (PH) and right ventricular (RV) hypertrophy. However, the effects of the frequency and duration of normoxic exposure on PH and RV hypertrophy have not been adequately assessed; thus, we aimed to analyze the same. Materials and Methods: PH and RV hypertrophy were induced in 60 rats using a hypobaric chamber. Of these 60 rats, every 10 were exposed to normoxic conditions for 30 minutes once (1T/D), three times (3T/D), or five times daily (5T/D), or for one 150-minute recovery daily (1LT/D). Furthermore, 10 rats were housed in a normoxic environment, and another 10 were subjected to continuous hypoxia. After 4 weeks, hemodynamic measurements were recorded, and the hearts were harvested for pathomorphological observations. Results: Average pulmonary arterial pressures (PAP) of control rats and those exposed to hypobaric hypoxia were 14.1 and 32.3 mmHg, respectively. After 30 minutes of exposure to normoxia 3T/D, 5T/D, or 1LT/D, PAP values were reduced to 27.1, 27.9, or 26.8 mmHg, respectively. Four weeks of hypoxic exposure elevated the RV/heart weight (HW) ratios, while exposure to normoxia 3T/D, 5T/D, and 1LT/D significantly reduced RV/HW. In addition, exposure to normoxia 3T/D, 5T/D, 1LT/D reduced the percentage wall thickness of the pulmonary artery as well as the hypertrophy indices of atrial natriuretic peptide, brain natriuretic peptide, and myosin heavy chain 7 (MYH-7). Conclusions: Thirty-minute exposure to normoxic conditions of 3T/D, 5T/D, or 1LT/D effectively ameliorates PH and RV thickening.

Exploring the molecular mechanisms underlying the potentiation of exogenous growth hormone on alcohol-induced fatty liver diseases in mice.

BACKGROUND: Growth hormone (GH) is an essential regulator of intrahepatic lipid metabolism by activating multiple complex hepatic signaling cascades. Here, we examined whether chronic exogenous GH administration (via gene therapy) could ameliorate liver steatosis in animal models of alcoholic fatty liver disease (AFLD) and explored the underlying molecular mechanisms. METHODS: Male C57BL/6J mice were fed either an alcohol or a control liquid diet with or without GH therapy for 6 weeks. Biochemical parameters, liver histology, oxidative stress markers, and serum high molecular weight (HMW) adiponectin were measured. Quantitative real-time PCR and western blotting were also conducted to determine the underlying molecular mechanism. RESULTS: Serum HMW adiponectin levels were significantly higher in the GH1-treated control group than in the control group (3.98 ± 0.71 µg/mL vs. 3.07 ± 0.55 µg/mL; P < 0.001). GH1 therapy reversed HMW adiponectin levels to the normal levels in the alcohol-fed group. Alcohol feeding significantly reduced hepatic adipoR2 mRNA expression compared with that in the control group (0.71 ± 0.17 vs. 1.03 ± 0.19; P < 0.001), which was reversed by GH therapy. GH1 therapy also significantly increased the relative mRNA (1.98 ± 0.15 vs. 0.98 ± 0.15) and protein levels of SIRT1 (2.18 ± 0.37 vs. 0.99 ± 0.17) in the alcohol-fed group compared with those in the control group (both, P < 0.001). The alcohol diet decreased the phosphorylated and total protein levels of hepatic AMP-activated kinase-α (AMPKα) (phosphorylated protein: 0.40 ± 0.14 vs. 1.00 ± 0.12; total protein: 0.32 ± 0.12 vs. 1.00 ± 0.14; both, P < 0.001) and peroxisome proliferator activated receptor-α (PPARα) (phosphorylated protein: 0.30 ± 0.09 vs. 1.00 ± 0.09; total protein: 0.27 ± 0.10 vs. 1.00 ± 0.13; both, P < 0.001), which were restored by GH1 therapy. GH therapy also decreased the severity of fatty liver in alcohol-fed mice. CONCLUSIONS: GH therapy had positive effects on AFLD and may offer a promising approach to prevent or treat AFLD. These beneficial effects of GH on AFLD were achieved through the activation of the hepatic adiponectin-SIRT1-AMPK and PPARα-AMPK signaling systems.

Characterization of the specific and sustained GH1 expression induced by rAAV2/1 in normal adult male rats.

Our aim was to investigate the long-term effects of intramuscular injection of rAAV2/1-CMV-GH1 viral particles on GH1 expression in normal adult male rats. We found that specific and sustained GH1 expression did not improve muscle exercise performance despite inducing local muscle hypertrophy. Injection of rAAV2/1-CMV-GH1 had some systemic effects on the liver and heart and on lipid metabolism in the healthy rats. Serum levels of hGH (human growth hormone), insulin, glucose and leptin increased significantly, which might induce insulin resistance. The serum concentration of IGF-1 (insulin-like growth factor 1), IGF-BP3 (insulin-like growth factor binding protein 3) and PIIINP (N-terminal propeptide of type III procollagen) markedly increased at 24 weeks after injection of GH1. In conclusion, GH1 expression driven by AAV2/1 in normal animals did not improve muscle strength but did increase muscle mass and may have systemic effects in healthy animals.

A microarray gene analysis of peripheral whole blood in normal adult male rats after long-term GH gene therapy.

The main aims of this study were to determine the effects of GH gene abuse/misuse in normal animals and to discover genes that could be used as candidate biomarkers for the detection of GH gene therapy abuse/misuse in humans. We determined the global gene expression profile of peripheral whole blood from normal adult male rats after long-term GH gene therapy using CapitalBio 27 K Rat Genome Oligo Arrays. Sixty one genes were found to be differentially expressed in GH gene-treated rats 24 weeks after receiving GH gene therapy, at a two-fold higher or lower level compared to the empty vector group (p < 0.05). These genes were mainly associated with angiogenesis, oncogenesis, apoptosis, immune networks, signaling pathways, general metabolism, type I diabetes mellitus, carbon fixation, cell adhesion molecules, and cytokine-cytokine receptor interaction. The results imply that exogenous GH gene expression in normal subjects is likely to induce cellular changes in the metabolism, signal pathways and immunity. A real-time qRT-PCR analysis of a selection of the genes confirmed the microarray data. Eight differently expressed genes were selected as candidate biomarkers from among these 61 genes. These 8 showed five-fold higher or lower expression levels after the GH gene transduction (p < 0.05). They were then validated in real-time PCR experiments using 15 single-treated blood samples and 10 control blood samples. In summary, we detected the gene expression profiles of rat peripheral whole blood after long-term GH gene therapy and screened eight genes as candidate biomarkers based on the microarray data. This will contribute to an increased mechanistic understanding of the effects of chronic GH gene therapy abuse/misuse in normal subjects.

Preventive effects of chronic exogenous growth hormone levels on diet-induced hepatic steatosis in rats.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD), which is characterized by hepatic steatosis, can be reversed by early treatment. Several case reports have indicated that the administration of recombinant growth hormone (GH) could improve fatty liver in GH-deficient patients. Here, we investigated whether chronic exogenous GH levels could improve hepatic steatosis induced by a high-fat diet in rats, and explored the underlying mechanisms. RESULTS: High-fat diet-fed rats developed abdominal obesity, fatty liver and insulin resistance. Chronic exogenous GH improved fatty liver, by reversing dyslipidaemia, fat accumulation and insulin resistance. Exogenous GH also reduced serum tumour necrosis factor-alpha (TNF-alpha) levels, and ameliorated hepatic lipid peroxidation and oxidative stress. Hepatic fat deposition was also reduced by exogenous GH levels, as was the expression of adipocyte-derived adipokines (adiponectin, leptin and resistin), which might improve lipid metabolism and hepatic steatosis. Exogenous GH seems to improve fatty liver by reducing fat weight, improving insulin sensitivity and correcting oxidative stress, which may be achieved through phosphorylation or dephosphorylation of a group of signal transducers and activators of hepatic signal transduction pathways. CONCLUSIONS: Chronic exogenous GH has positive effects on fatty liver and may be a potential clinical application in the prevention or reversal of fatty liver. However, chronic secretion of exogenous GH, even at a low level, may increase serum glucose and insulin levels in rats fed a standard diet, and thus increase the risk of insulin resistance.

Effects of Proton Pump Inhibitors on the Gastrointestinal Microbiota in Gastroesophageal Reflux Disease.

Proton pump inhibitors (PPIs) are commonly used to lessen symptoms in patients with gastroesophageal reflux disease (GERD). However, the effects of PPI therapy on the gastrointestinal microbiota in GERD patients remain unclear. We examined the association between the PPI usage and the microbiota present in gastric mucosal and fecal samples from GERD patients and healthy controls (HCs) using 16S rRNA gene sequencing. GERD patients taking PPIs were further divided into short-term and long-term PPI user groups. We showed that PPI administration lowered the relative bacterial diversity of the gastric microbiota in GERD patients. Compared to the non-PPI-user and HC groups, higher abundances of Planococcaceae, Oxalobacteraceae, and Sphingomonadaceae were found in the gastric microbiota from the PPI-user group. In addition, the Methylophilus genus was more highly abundant in the long-term PPI user group than in the short-term PPI-user group. Despite the absence of differences in alpha diversity, there were significant differences in the fecal bacterial composition of between GERD patients taking PPIs and those not taking PPIs. There was a higher abundance of Streptococcaceae, Veillonellaceae, Acidaminococcaceae, Micrococcaceae, and Flavobacteriaceae present in the fecal microbiota from the PPI-user group than those from the non-PPI-user and HC groups. Additionally, a significantly higher abundance of Ruminococcus was found in GERD patients on long-term PPI medication than that on short-term PPI medication. Our study indicates that PPI administration in patients with GERD has a significant effect on the abundance and structure of the gastric mucosal microbiota but only on the composition of the fecal microbiota.

[Expression changes of metabotropic glutamate receptor 5 in neuropathic pain].

OBJECTIVE: To investigate the expression changes of metabotropic glutamate receptor 5 (mGluR5) in neuropathic pain. METHODS: Eighty-four adult male Sprague Dawley rats weighing 180-220 g were randomly divided into 7 groups (n = 12) : control group; S3, S7, and S14 groups: rats received the sham operation, the mechanical pain threshold was measured, and then the rats were decapitated and the ipsilateral lumbar spinal cord dorsal horn and dorsal root ganglion (DRG) samples were obtained on the 3rd, 7th, 14th postoperative day, respectively; C3, C7, and C14 groups: the chronic sciatic nerve constriction (CCI) model was established, the mechanical pain threshold was measured and the samples were obtained on the 3rd, 7th, 14th postoperative day, respectively. The expression level of mGluR5 mRNA and protein in the spinal cord and DRG were measured using the reverse transcriptase polymerase chain reaction and Western blot. RESULTS: In the CCI group, the mechanical pain threshold in each observation day was significantly lower than in the sham operation group (P < 0.05). In the spinal cord, the expressions of mGluR5 mRNA and protein were significantly elevated in the C3 group than in the S3 and the control group (P < 0.05). On the 7th and the 14th postoperative day, no significant difference was found in the expression of mGluR5 mRNA and protein between CCI groups and the sham operation groups or the control group. No change was detected in DRG mRNA or protein. CONCLUSION: mGluR5 is differentially expressed in spinal cord in response to neuropathic pain, which suggests that mGluR5 may be involved in the mechanism of neuropathic pain.