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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.
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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.
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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.
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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.
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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.
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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.
Subject(s)
Hypertension, Pulmonary , Animals , Hypertension, Pulmonary/etiology , Hypertrophy, Right Ventricular/etiology , Hypoxia/complications , Lung , Male , Pulmonary Artery , RatsABSTRACT
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.
Subject(s)
Gastroesophageal Reflux/drug therapy , Gastroesophageal Reflux/microbiology , Gastrointestinal Microbiome/drug effects , Proton Pump Inhibitors/therapeutic use , Adult , Aged , Bacteria/genetics , Bacteria/isolation & purification , Feces/microbiology , Female , Gastric Mucosa/microbiology , Humans , Male , Microbiota , Middle Aged , RNA, Ribosomal, 16S/geneticsABSTRACT
cDNA coding a prolyl aminopeptidase (PAP) was cloned from Aspergillus oryzae and over expressed in Bacillus subtilis with a 6×His tag in N-terminus. The recombinant prolyl aminopeptidase was secreted to extracellular by adding 2 mM CaCl2 and 5% D-sorbitol in TB medium; the enzyme activity in fermented supernatant increased from 7.2 to 41.5 U mL-1. It has been purified 4.3-fold through Ni-chelating affinity chromatography with a recovery of 47.3%. The purified enzyme is stable below 50 °C and within pH 6-11, and with the highest activity at pH 7.5 and 50 °C. Several kinds of salt can activate enzyme activity in a certain concentration and the relative activity was 127.02% even when the concentration of NaCl reached 4.36 M. It cleaved N-terminal Pro residues from many peptides but shown different hydrolysis rates for various Pro-X dipeptides or peptides which are of different lengths. It combined with alkaline protease and leucine aminopeptidase to hydrolyze casein, many free amino acid especially proline and small peptide of hydrolysate increased significantly.
Subject(s)
Aminopeptidases/genetics , Aminopeptidases/metabolism , Aspergillus oryzae/enzymology , Bacillus subtilis/genetics , Protein Engineering/methods , Amino Acid Sequence , Aminopeptidases/isolation & purification , Aspergillus oryzae/genetics , Bacillus subtilis/cytology , Caseins/metabolism , DNA, Recombinant/genetics , Extracellular Space/drug effects , Extracellular Space/genetics , Gene Expression , Hydrogen-Ion Concentration , Hydrolysis , Substrate Specificity , TemperatureABSTRACT
OBJECTIVES: The aim of this study was to develop a clinical risk model that is predictive of in-hospital mortality in elderly patients hospitalized with acute heart failure (AHF). METHODS: 2486 patients who were 60 years and older from intensive care units of Cardiology Department in the hospital were analyzed. Independent risk factors for in-hospital mortality were obtained by binary logistic regression and then used to establish the risk prediction score system (RPSS). The area under the curve (AUC) of receiver operator characteristic and C-statistic test were adopted to assess the performance of RPSS and to compare with previous get with the guidelines-heart failure (GWTG-HF). RESULTS: By binary logistic regression analysis, heart rate (OR: 1.043, 95% CI: 1.030-1.057, P < 0.001), left ventricular ejection fraction (OR: 0.918, 95% CI: 0.833-0.966, P < 0.001), pH value (OR: 0.001, 95% CI: 0.000-0.002, P < 0.001), renal dysfunction (OR: 0.120, 95% CI: 0.066-0.220, P < 0.001) and NT-pro BNP (OR: 3.463, 95% CI: 1.870-6.413, P < 0.001) were independent risk factors of in-hospital mortality for elderly AHF patients. Additionally, RPSS, which was composed of all the above-mentioned parameters, provided a better risk prediction than GWTG-THF (AUC: 0.873 vs. 0.818, P = 0.016). CONCLUSIONS: Our risk prediction model, RPSS, provided a good prediction for in-hospital mortality in elderly patients with AHF.
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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.
Subject(s)
Graft vs Host Disease/diagnosis , Hematopoietic Stem Cell Transplantation/adverse effects , MicroRNAs/blood , Acute Disease , Adolescent , Adult , Allografts , Area Under Curve , Biomarkers , Double-Blind Method , Female , Graft vs Host Disease/blood , Graft vs Host Disease/genetics , Humans , Kaplan-Meier Estimate , Male , Middle Aged , Models, Biological , Predictive Value of Tests , ROC Curve , Real-Time Polymerase Chain Reaction , Single-Blind Method , Tumor Necrosis Factor-alpha/analysis , Young AdultABSTRACT
AIM: Joint biomarker panel takes advantage of coherence across multiple level datasets and holds the promise to improve disease diagnosis accuracy. METHODS: We collected 101 colorectal cancer and 95 benign samples, measured the molecular concentrations by serum assays and mass spectra, and developed LPGLO (Linear Programming based on Group Lasso Optimization) to identify the joint biomarker panel. RESULTS: A joint biomarker panel, with ten serum biomarkers and six mass spectra peaks, achieves LOOCV accuracy 0.8724, which is better than the optimal panels identified from separate datasets (LOOCV = 0.7551 for mass spectra only or 0.8265 for serum assay only) and the simply merged dataset (LOOCV = 0.8622). CONCLUSION: LPGLO is useful to identify joint biomarker panel to help tumor diagnosis.
Subject(s)
Biomarkers, Tumor/blood , Colorectal Neoplasms/diagnosis , Models, Theoretical , Colon/metabolism , Colon/pathology , Colorectal Neoplasms/pathology , Humans , Logistic Models , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Support Vector MachineABSTRACT
AIM: To investigate the relationship of serum levels of polyunsaturated fatty acid (PUFA) with kinds of cytokines in colorectal cancer (CRC). METHODS: Serum samples of 100 CRC patients were collected. The concentration of total n-3 PUFA which included C18:3 n-3, C20:5 n-3, C22:5 n-3, C22:6 n-3 and the total n-6 PUFA included C18:2 n-6, C18:3 n-6, C20:3 n-6, C20:4 n-6, and C22:5 n-6 were detected on GC-2010 Plus Gas Chromatograph with a OmegawaxTM 250 column. Cytokines were detected by MagPlexTM-C microspheres. P values for the trend were estimated by creating a continuous variable using the median value within quartiles. RESULTS: Interleukin-6 (IL-6) showed significantly positive association with the C20:4 n-6 (P for trend = 0.004). Interferon gamma (IFN-γ) showed significant positive association with the C22:5 n-3 (P for trend = 0.035). IL-8 and matrix metalloproteinase-9 (MMP-9) showed significant inverse association with the C22:6 n-3 (P for trend = 0.049, and 0.021). MMP-2 showed significant inverse association with the C20:5 n-3 (P for trend = 0.008). MMP-7 showed significantly positive association with the ratio of n-6 PUFA and n-3 PUFA (P for trend = 0.008). MMP-7 also showed significantly inverse association with the ratio of C20:4 n-6 and (n-6 PUFA + n-3 PUFA) (P for trend = 0.024). IL-10 (P for trend = 0.023) and IL-6 (P for trend = 0.036) showed significantly positive association with the ratio of C20:4 n-6 and C20:5 n-3. CONCLUSION: Our data suggested that serum levels of PUFA is related to the inflammation of CRC, and also play different role in regulation of immune response.
Subject(s)
Biomarkers, Tumor/blood , Colorectal Neoplasms/blood , Cytokines/blood , Fatty Acids, Omega-3/blood , Fatty Acids, Omega-6/blood , Inflammation Mediators/blood , HumansABSTRACT
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.
Subject(s)
Spectrum Analysis , Humans , Reference Values , Trace ElementsABSTRACT
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.
Subject(s)
Biomarkers, Tumor/blood , Colorectal Neoplasms/blood , Early Detection of Cancer , Neoplasms/blood , Adult , Aged , Aged, 80 and over , Colorectal Neoplasms/pathology , Female , Humans , Male , Middle AgedABSTRACT
Small non-coding microRNAs (miRNAs) are involved in cancer development and progression, and serum profiles of cervical cancer patients may be useful for identifying novel miRNAs. We performed deep sequencing on serum pools of cervical cancer patients and healthy controls with 3 replicates and constructed a small RNA library. We used MIREAP to predict novel miRNAs and identified 2 putative novel miRNAs between serum pools of cervical cancer patients and healthy controls after filtering out pseudo-pre-miRNAs using Triplet-SVM analysis. The 2 putative novel miRNAs were validated by real time PCR and were significantly decreased in cervical cancer patients compared with healthy controls. One novel miRNA had an area under curve (AUC) of 0.921 (95% CI: 0.883, 0.959) with a sensitivity of 85.7% and a specificity of 88.2% when discriminating between cervical cancer patients and healthy controls. Our results suggest that characterizing serum profiles of cervical cancers by Solexa sequencing may be a good method for identifying novel miRNAs and that the validated novel miRNAs described here may be cervical cancer-associated biomarkers.
Subject(s)
MicroRNAs/blood , MicroRNAs/genetics , Uterine Cervical Neoplasms/blood , Uterine Cervical Neoplasms/genetics , Base Sequence , Biomarkers, Tumor/blood , Case-Control Studies , Female , High-Throughput Nucleotide Sequencing/methods , Humans , Middle Aged , Molecular Sequence DataABSTRACT
A strain that exhibited intracellular proline-specific aminopeptidase (PAP) activity was isolated from soy sauce koji and identified as Aspergillus oryzae JN-412. The gene coding PAP was cloned and efficiently expressed in Escherichia coli BL21 in a biologically active form. The highest specific activity reached 52.28 U mg(-1) at optimum cultivation conditions. The recombinant enzyme was purified 3.3-fold to homogeneity with a recovery of 36.7% from cell-free extract using Ni-affinity column chromatography. It appeared as a single protein band on SDS-PAGE with molecular mass of approximately 50 kDa. The purified enzyme exhibited the highest activity at 60 °C and pH 7.5. The enzyme activity was inhibited by PMSF and ions like Zn2+ and Cu2+. DTT, ß-mercaptoethanol, EDTA, and ions like Co2+, Mg2+, Mn2+, and Ca2+ had no influence on enzyme activity, whereas Ni2+ enhanced the enzyme activity. By using collagen as a substrate, the purified recombinant prolyl aminopeptidase contributed to the hydrolysis of collagen when used in combination with neutral protease, and free amino acids in collagen hydrolysates was significantly increased.
Subject(s)
Aminopeptidases/genetics , Aminopeptidases/metabolism , Aspergillus oryzae/enzymology , Collagen/metabolism , Genetic Engineering/methods , Proline/metabolism , Proteolysis , Amino Acid Sequence , Aminopeptidases/chemistry , Aminopeptidases/isolation & purification , Aspergillus oryzae/genetics , Cloning, Molecular , Escherichia coli/genetics , Gene Expression , Hydrolysis , Models, Molecular , Molecular Sequence Data , Protein Structure, Secondary , Substrate SpecificityABSTRACT
CA125 and CA72-4 are members of a family of high-molecular weight glycosylated proteins and are commonly considered as biomarkers in the diagnosis of ovarian and gastric cancer, respectively. However, recent studies have revealed that these two markers may be of clinical value in the diagnosis of pancreatic cancer. As the availability of data regarding CA72-4 and CA125 in the diagnosis of pancreatic cancer is limited, the aim of this study was to investigate the clinical value of serum tumor markers CA19-9, CA125 and CA72-4 in the diagnosis of pancreatic carcinoma according to logistic regression analysis and receiver operating characteristic (ROC) curves and to investigate the correlation of these markers with tumor TNM stage and location. An immunoradiometric assay was used to measure pre-operative serum CA19-9, CA125 and CA72-4 levels in 75 patients with pancreatic carcinoma and 70 patients with benign pancreatic diseases. The concentrations of serum CA19-9, CA125 and CA72-4 in patients with pancreatic carcinoma were found to be significantly higher (P<0.05) compared with those with benign pancreatic diseases. The combined detection of two serum markers (CA19-9 + CA72-4) yielded a ROC value of 0.895 that was significantly higher compared to others (P<0.05) in distinguishing pancreatic cancer from benign pancreatic diseases. At optimal cut-off, the sensitivity and specificity of combined detection (CA19-9 + CA72-4) were 70.6 and 92.8%, respectively. The concentrations of CA125 and CA19-9 in patients with pancreatic adenocarcinoma were found to be significantly higher (P<0.05) compared with those of pancreatic neuroendocrine carcinoma. In conclusion, the combined detection of CA19-9 and CA72-4) may significantly improve the diagnostic specificity and the serum concentrations of CA125 and CA19-9 are correlated with tumor histological type.
ABSTRACT
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.
Subject(s)
Aminopeptidases/isolation & purification , Aminopeptidases/metabolism , Pseudomonas aeruginosa/enzymology , Amino Acid Sequence , Aminopeptidases/antagonists & inhibitors , Aminopeptidases/chemistry , Enzyme Stability , Hydrogen-Ion Concentration , Kinetics , Metals/pharmacology , Molecular Sequence Data , Soil Microbiology , Substrate Specificity , TemperatureABSTRACT
Urethanase produced by Penicillium variabile was purified through ultrasonication, concentration by polyethylene glycol 20,000, and Superdex G-200 gel filtration chromatography. The molecular weight of urethanase was determined to be around 96 kDa by gel filtration. The purified enzyme showed a single band in SDS-PAGE with the molecular weight of ~13.7 kDa, which suggests that the enzyme has a multimeric structure composed of the same subunits. Peptide map fingerprinting analysis was then carried out by MALDI/TOF-TOF MS. Within the known sequences in NCBI, glucosamine-6-phosphate deaminase and 6-phosphogluconate dehydrogenase get high score as compared with urethanase. Sequence analysis informs that N-terminal sequence of urethanase is GTNTADNDAA. The Minchaelis constant (Km) and maximum reaction rate (Vm) of urethanase are 27.2 mmol/L and 156.25 µmol/L min, respectively.
Subject(s)
Amidohydrolases/isolation & purification , Amidohydrolases/metabolism , Penicillium/enzymology , Amidohydrolases/chemistry , Amino Acid Sequence , Hydrogen-Ion Concentration , Kinetics , Molecular Sequence Data , Molecular Weight , Penicillium/isolation & purification , Sonication , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , TemperatureABSTRACT
Clinical diagnosis of acute graft-versus-host disease (aGVHD) mainly depends on clinical manifestation and tissue biopsies, leading to a delayed diagnosis and treatment for aGVHD patients when the early symptom is insignificant. Our objective was to investigate the possibility of prewarning the risk of aGVHD before and after allogeneic hematopoietic stem cell transplantation (allo-HSCT) by serum protein profiling combined with serum ferritin. The difference in polypeptide expression before and after transplantation had been compared by using CLINPROT technology, and serum ferritin levels have been analyzed simultaneously. Through combining serum ferritin and MS spectral data, the diagnosis sensitivity and specificity of our model for prewarning severe aGVHD (III~IV°aGVHD) before transplant all increased to 90.0%, while after transplant, the sensitivity and specificity are 78.3% and 86.4%. Our joint prewarning model could predict the risk of aGVHD, especially severe aGVHD before and after transplant, which also provides a reliable method to the continuous monitoring of the condition of patients.