ABSTRACT
Sequence verification of plasmid DNA is critical for many cloning and molecular biology workflows. To leverage high-throughput sequencing, several methods have been developed that add a unique DNA barcode to individual samples prior to pooling and sequencing. However, these methods require an individual plasmid extraction and/or in vitro barcoding reaction for each sample processed, limiting throughput and adding cost. Here, we develop an arrayed in vivo plasmid barcoding platform that enables pooled plasmid extraction and library preparation for Oxford Nanopore sequencing. This method has a high accuracy and recovery rate, and greatly increases throughput and reduces cost relative to other plasmid barcoding methods or Sanger sequencing. We use in vivo barcoding to sequence verify >45 000 plasmids and show that the method can be used to transform error-containing dispersed plasmid pools into sequence-perfect arrays or well-balanced pools. In vivo barcoding does not require any specialized equipment beyond a low-overhead Oxford Nanopore sequencer, enabling most labs to flexibly process hundreds to thousands of plasmids in parallel.
Subject(s)
Gene Library , High-Throughput Nucleotide Sequencing , Plasmids , Plasmids/genetics , High-Throughput Nucleotide Sequencing/methods , Sequence Analysis, DNA/methods , DNA/genetics , DNA Barcoding, Taxonomic/methods , Nanopore Sequencing/methodsABSTRACT
This study is to explore the proportion of significant liver histopathology in hepatitis B e antigen (HBeAg)-negative chronic hepatitis B virus (HBV)-infected patients with normal alanine aminotransferase (ALT) and investigate noninvasive indicators for predicting significant liver histopathology. A total of 201 HBeAg-negative chronic HBV-infected patients with normal ALT who underwent liver biopsy were involved in this study. Significant liver histological changes were defined as necroinflammation grade ≥2 (G ≥ 2) and/or fibrosis stage ≥2 (S ≥ 2). The results showed that 42.3% (85/201) and 45.8% (92/201) of the HBeAg-negative patients with normal ALT have significant liver necroinflammation (G ≥ 2) and fibrosis (S ≥ 2), respectively. High normal ALT (>22 U/L), high level of serum HBV DNA (>3.42 log IU/mL), and low level of prealbumin (PA) (<170 mg/L) were independent predictors for significant liver necroinflammation, and the predictive value of the combined indicators was 0.750 (P < 0.001), while high normal ALT (>24 U/L) and high level of FIB-4 (>1.53) were independent predictors for significant liver fibrosis, and the predictive value of the combined indicators was 0.740 (P < 0.001). In conclusion, more than 40% of HBeAg-negative patients with normal ALT have significant liver histopathology and require immediate antiviral treatment. ALT, PA, HBV DNA, and FIB-4 can independently predict significant liver inflammation and fibrosis for HBeAg-negative patients with normal ALT. Lowering the treatment threshold of ALT may benefit the HBeAg-negative chronic HBV-infected patients. IMPORTANCE: Hepatitis B e antigen (HBeAg)-negative chronic hepatitis B virus (HBV)-infected patients with normal alanine aminotransferase (ALT) were supposed to have a low risk of progression to cirrhosis or hepatocellular carcinoma, and it was recommended to regularly follow up or undergo liver biopsy to assess liver histopathology according to the major international guidelines. However, this study indicates that a considerable number of HBeAg-negative chronic HBV-infected patients with normal ALT have significant liver histopathology and require immediate antiviral treatment. Besides, several clinical commonly used noninvasive indicators were found that can be used to predict significant liver histopathology; thereby liver biopsy might be avoided for HBeAg-negative chronic HBV-infected patients with normal ALT.
Subject(s)
Hepatitis B, Chronic , Humans , Hepatitis B, Chronic/diagnosis , Hepatitis B, Chronic/drug therapy , Hepatitis B e Antigens/therapeutic use , Alanine Transaminase , DNA, Viral , Liver Cirrhosis/diagnosis , Liver Cirrhosis/pathology , Fibrosis , Biomarkers , Antiviral Agents/therapeutic useABSTRACT
A begomovirus isolated from whiteflies (Bemisia tabaci) and tomato, sweet potato in China was found to be representative of a distinct begomovirus species, for which the name tomato yellow leaf curl Chuxiong virus (TYLCCxV) is proposed. The results of genomic identification and sequence comparison showed that TYLCCxV shares the highest complete nucleotide sequence identity (88.3%) with croton yellow vein mosaic virus (CroYVMV), and may have originated from the recombination between synedrella leaf curl virus (SyLCV) and squash leaf curl Yunnan virus (SLCuYV). Agrobacterium-mediated inoculation showed that TYLCCxV is highly infectious for a range of plant species, producing upward leaf curling, leaf crumpling, chlorosis, distortion, and stunt symptoms in Solanum lycopersicum plants. The results of Southern blot indicated that TYLCCxV is capable of efficiently replicating two heterologous betasatellites. The inoculation of PVX::C4 on Nicotiana benthamiana induced upward leaf curling and stem elongation symptoms, suggesting that TYLCCxV C4 functions as a symptom determinant. TYLCCxV V2 is an important virulence factor that induces downward leaf curling symptoms, elicits systemic necrosis, and suppresses local and systemic GFP silencing in co-agroinfiltrated N. benthamiana and transgenic 16c plants. Considering the multifunctional virulence proteins V2 and C4, the possibility of TYLCCxV causing devastating epidemics on tomato in China is discussed.
Subject(s)
Begomovirus , Hemiptera , Solanum lycopersicum , Animals , RNA Interference , Begomovirus/genetics , Plant Diseases , ChinaABSTRACT
Sequence verification of plasmid DNA is critical for many cloning and molecular biology workflows. To leverage high-throughput sequencing, several methods have been developed that add a unique DNA barcode to individual samples prior to pooling and sequencing. However, these methods require an individual plasmid extraction and/or in vitro barcoding reaction for each sample processed, limiting throughput and adding cost. Here, we develop an arrayed in vivo plasmid barcoding platform that enables pooled plasmid extraction and library preparation for Oxford Nanopore sequencing. This method has a high accuracy and recovery rate, and greatly increases throughput and reduces cost relative to other plasmid barcoding methods or Sanger sequencing. We use in vivo barcoding to sequence verify >45,000 plasmids and show that the method can be used to transform error-containing dispersed plasmid pools into sequence-perfect arrays or well-balanced pools. In vivo barcoding does not require any specialized equipment beyond a low-overhead Oxford Nanopore sequencer, enabling most labs to flexibly process hundreds to thousands of plasmids in parallel.
ABSTRACT
Hypocotyls undergo different morphogenesis in light and dark conditions, with cortical microtubules being reoriented in response to light to coordinate cell growth status. Kinesins are microtubule-based motor proteins that are mostly responsible for transporting organelles and vesicles, although some can also regulate microtubule organization; however, it is currently not known whether they are involved in microtubule reorientation and hypocotyl elongation. In this study, we found that ARMADILLO REPEAT KINESIN 2 (ARK2) negatively regulated the hypocotyl elongation of Arabidopsis. The hypocotyl cells of plants with the ark2 null allele were longer than those of the wild type and had relatively more transversely arranged cortical microtubules. In addition, ARK2 co-localized with cortical microtubules and facilitated the light-induced reorientation of the cortical microtubule arrays. Interestingly, the ARK2 protein is stable in the light and degraded through the 26S proteasome pathway in the dark. Furthermore, we determined that ARK2 could interact with the E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), which contributed to down-regulation of ARK2 in darkness that might benefit hypocotyl growth in the dark.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Armadillo Domain Proteins , Arabidopsis/growth & development , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Armadillo Domain Proteins/metabolism , Hypocotyl , Kinesins/genetics , Kinesins/metabolism , Light , Microtubules/metabolismABSTRACT
Microtubule dynamics and organization are important for plant cell morphogenesis and development. The microtubule-based motor protein kinesins are mainly responsible for the transport of some organelles and vesicles, although several have also been shown to regulate microtubule organization. The ARMADILLO REPEAT KINESIN (ARK) family is a plant-specific motor protein subfamily that consists of three members (ARK1, ARK2, and ARK3) in Arabidopsis thaliana. ARK2 has been shown to participate in root epidermal cell morphogenesis. However, whether and how ARK2 associates with microtubules needs further elucidation. Here, we demonstrated that ARK2 co-localizes with microtubules and facilitates microtubule bundling in vitro and in vivo. Pharmacological assays and microtubule dynamics analyses indicated that ARK2 stabilizes cortical microtubules. Live-cell imaging revealed that ARK2 moves along cortical microtubules in a processive mode and localizes both at the plus-end and the sidewall of microtubules. ARK2 therefore tracks and stabilizes the growing plus-ends of microtubules, which facilitates the formation of parallel microtubule bundles.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Microtubules/metabolism , Kinesins/genetics , Kinesins/metabolism , Microtubule-Associated Proteins/metabolismABSTRACT
Objective: Exploring the risk factors of conjunctival squamous cell carcinoma (CSCC) and establishing a prognostic model. Methods: Information on patients with CSCC was extracted from the SEER database, conducting a retrospective study. 650 patients with CSCC were finally included in the model. Descriptive analysis was performed by Chi-square test and T-test. The risk factors of CSCC were explored by COX multivariate analysis, and the corresponding prognostic model was established as a result. Results: The all-cause mortality rate of CSCC was 38.3%, and the risk factors were age (HR = 1.077), sex (HR = 0.691), grade (HR = 7.857), laterality (HR = 1.403), N (HR = 7.195), M (HR = 0.217), and surgery (HR = 1.618), all P < 0.05. The new model had C index and area under curve ROC (AUC) value greater than 0.7. Calibration curve, Net Reclassification Index (NRI), Integrated Discrimination Improvement (IDI), and Decision Curve Analysis (DCA) indicate the new model has better predictive performance than the American Joint Committee on Cancer (AJCC-TNM). Conclusions: Compared with the clinical guidance of AJCC (TNM) for patients with CSCC, the established model exhibits good performance and can provide guidance for clinical decision-making.
Subject(s)
Bone Neoplasms , Breast Neoplasms , Carcinoma, Squamous Cell , Head and Neck Neoplasms , Humans , Female , Prognosis , Retrospective Studies , Neoplasm Staging , Carcinoma, Squamous Cell/pathology , Squamous Cell Carcinoma of Head and Neck/pathology , Risk Factors , Bone Neoplasms/pathology , Breast Neoplasms/pathology , Head and Neck Neoplasms/pathologyABSTRACT
Type 2 diabetes mellitus (T2DM) is a strong indicator of late stent thrombosis (LST). Platelet bioenergetic dysfunction, although critical to the pathogenesis of diabetic macrovascular complications, remains uncharacterized in T2DM patients who developed LST. Here, we explored the mechanistic link between the alterations in platelet bioenergetics and LST in the setting of T2DM. Platelet bioenergetics, metabolomics, and their interactomes were analyzed in a nested case-control study including 15 T2DM patients who developed LST and 15 matched T2DM patients who did not develop LST (non-LST). Overall, we identified a bioenergetic alteration in T2DM patients with LST characterized by an imbalanced NAD+/NADH redox state resulting from deficient mitochondrial complex I (NADH: ubiquinone oxidoreductase) activity, which led to reduced ATP-linked and maximal mitochondrial respiration, increased glycolytic flux, and platelet hyperactivation compared with non-LST patients. Congruently, platelets from LST patients exhibited downregulation of tricarboxylic acid cycle and NAD+ biosynthetic pathways as well as upregulation of the proximal glycolytic pathway, a metabolomic change that was primarily attributed to compromised mitochondrial respiration rather than increased glycolytic flux as evidenced by the integrative analysis of bioenergetics and metabolomics. Importantly, both bioenergetic and metabolomic aberrancies in LST platelets could be recapitulated ex vivo by exposing the non-LST platelets to a low dose of rotenone, a complex I inhibitor. In contrast, normalization of the NAD+/NADH redox state, either by increasing NAD+ biosynthesis or by inhibiting NAD+ consumption, was able to improve mitochondrial respiration, inhibit mitochondrial oxidant generation, and consequently attenuate platelet aggregation in both LST platelets and non-LST platelets pretreated with low-dose rotenone. These data, for the first time, delineate the specific patterns of bioenergetic and metabolomic alterations for T2DM patients who suffer from LST, and establish the deficiency of complex I-derived NAD+ as a potential pathogenic mechanism in platelet abnormalities.
ABSTRACT
BACKGROUND: Outcome prediction tools for patients with type 2 diabetes mellitus (T2DM) undergoing percutaneous coronary intervention (PCI) are lacking. Here, we developed a machine learning-based metabolite classifier for predicting 1-year major adverse cardiovascular events (MACEs) after PCI among patients with T2DM. METHODS: Serum metabolomic profiling was performed in a nested case-control study of 108 matched pairs of patients with T2DM occurring and not occurring MACEs at 1 year after PCI, then the matched pairs were 1:1 assigned into the discovery and internal validation sets. External validation was conducted using targeted metabolite analyses in an independent prospective cohort of 301 patients with T2DM receiving PCI. The function of candidate metabolites was explored in high glucose-cultured human aortic smooth muscle cells (HASMCs). RESULTS: Overall, serum metabolome profiles differed between diabetic patients with and without 1-year MACEs after PCI. Through VSURF, a machine learning approach for feature selection, we identified the 6 most important metabolic predictors, which mainly targeted the nicotinamide adenine dinucleotide (NAD+) metabolism. The 6-metabolite model based on random forest and XGBoost algorithms yielded an area under the curve (AUC) of ≥ 0.90 for predicting MACEs in both discovery and internal validation sets. External validation of the 6-metabolite classifier also showed good accuracy in predicting MACEs (AUC 0.94, 95% CI 0.91-0.97) and target lesion failure (AUC 0.89, 95% CI 0.83-0.95). In vitro, there were significant impacts of altering NAD+ biosynthesis on bioenergetic profiles, inflammation and proliferation of HASMCs. CONCLUSION: The 6-metabolite model may help for noninvasive prediction of 1-year MACEs following PCI among patients with T2DM.
Subject(s)
Diabetes Mellitus, Type 2 , Percutaneous Coronary Intervention , Case-Control Studies , Diabetes Mellitus, Type 2/diagnosis , Diabetes Mellitus, Type 2/etiology , Humans , NAD , Percutaneous Coronary Intervention/adverse effects , Prospective StudiesABSTRACT
Vegetation restoration is an essential approach to re-establish the ecological balance in subalpine areas. Changes in vegetation cover represent, to some extent, vegetation growth trends and are the consequence of a complex of different natural factors and human activities. Microtopography influences vegetation growth by affecting the amount of heat and moisture reaching the ground, a role that is more pronounced in subalpine areas. However, little research is concerned with the characteristics and dynamics of vegetation restoration in different microtopography types. The respective importance of the factors driving vegetation changes in subalpine areas is also not clear yet. We used linear regression and the Hurst exponent to analyze the trends in vegetation restoration and sustainability in different microtopography types since 2000, based on Fractional Vegetation Cover (FVC) and identified potential driving factors of vegetation change and their importance by using Geographical Detector. The results show that: (1) The FVC in the region under study has shown an up-trend since 2000, and the rate of increase is 0.26/year (P = 0.028). It would be going from improvement to degradation, continuous decrease or continuous significant decrease in 47.48% of the region, in the future. (2) The mean FVC is in the following order: lower slope (cool), lower slope, lower slope (warm), valley, upper slope (warm), upper slope, valley (narrow), upper slope (cool), cliff, mountain/divide, peak/ridge (warm), peak/ridge, peak/ridge (cool). The lower slope is the microtopographic type with the best vegetation cover, and ridge peak is the most difficult to be afforested. (3) The main factors affecting vegetation restoration in subalpine areas are aspect, microtopographic type, and soil taxonomy great groups. The interaction between multiple factors has a much stronger effect on vegetation cover than single factors, with the effect of temperatures and aspects having the most significant impact on the vegetation cover changes. Natural factors have a greater impact on vegetation restoration than human factors in the study area. The results of this research can contribute a better understanding of the influence of different drivers on the change of vegetation cover, and provide appropriate references and recommendations for vegetation restoration and sustainable development in typical logging areas in subalpine areas.
Subject(s)
Ecosystem , Soil , Humans , Tibet , Temperature , Cold TemperatureABSTRACT
Identification of adaptive targets in experimental evolution typically relies on extensive replication and genetic reconstruction. An alternative approach is to directly assay all mutations in an evolved clone by generating pools of segregants that contain random combinations of evolved mutations. Here, we apply this method to 6 Saccharomyces cerevisiae clones isolated from 4 diploid populations that were clonally evolved for 2,000 generations in rich glucose medium. Each clone contains 17-26 mutations relative to the ancestor. We derived intermediate genotypes between the founder and the evolved clones by bulk mating sporulated cultures of the evolved clones to a barcoded haploid version of the ancestor. We competed the resulting barcoded diploids en masse and quantified fitness in the experimental and alternative environments by barcode sequencing. We estimated average fitness effects of evolved mutations using barcode-based fitness assays and whole-genome sequencing for a subset of segregants. In contrast to our previous work with haploid evolved clones, we find that diploids carry fewer beneficial mutations, with modest fitness effects (up to 5.4%) in the environment in which they arose. In agreement with theoretical expectations, reconstruction experiments show that all mutations with a detectable fitness effect manifest some degree of dominance over the ancestral allele, and most are overdominant. Genotypes with lower fitness effects in alternative environments allowed us to identify conditions that drive adaptation in our system.
Subject(s)
Diploidy , Saccharomyces cerevisiae , Adaptation, Physiological/genetics , Genetic Fitness , Haploidy , Mutation , Saccharomyces cerevisiae/geneticsABSTRACT
BACKGROUND: Hyperhomocysteinemia (HHcy) is an independent risk factor for cardiovascular disease. Total serum homocysteine (tHcy) status varies greatly with ethnicity and gender. Here, we studied the tHcy status by investigating concentration of tHcy and calculating prevalence of HHcy according to different age groups and genders. METHODS: This is a cross-sectional study of 10,258 participants (7,248 males and 3,010 females) above 19 years old from Henan Province, northern China. tHcy levels were determined enzymatically. HHcy was defined as a tHcy level higher than 15 µmol/L. RESULTS: In the whole population, the median value of tHcy was 13.56 (11.50, 16.50) µmol/L, and the HHcy prevalence was 34.61%. Males had much higher tHcy levels than females: 14.51 (12.58, 17.71) µmol/L vs. 11.23 (9.75, 12.97) µmol/L, p < 0.001. Also, males had much higher HHcy prevalence than females (44.33% vs. 11.20%, p < 0.001, OR = 6.33, 95% CI: 5.59 - 7.14). HHcy prevalence and tHcy levels increased greatly for both genders above 60 years old. CONCLUSIONS: Our results demonstrated that prevalence of HHcy is very high in northern China. Implementation of tHcy-lowering strategies is needed.
Subject(s)
Homocysteine , Hyperhomocysteinemia , Adult , China/epidemiology , Cross-Sectional Studies , Female , Humans , Hyperhomocysteinemia/diagnosis , Hyperhomocysteinemia/epidemiology , Male , Middle Aged , Prevalence , Risk Factors , Young AdultABSTRACT
OBJECTIVE: To verify the differences in serum levels of urea, creatinine, and uric acid (UA) between pregnant and nonpregnant women and establish specific reference intervals of serum urea, creatinine, and UA for pregnant women, and thus help for the detection of kidney disease in pregnancy. METHODS: Based on the selection criteria, 1312 apparently healthy pregnant women and 1301 nonpregnant women were enrolled in this study. The levels of serum urea, creatinine, and UA were compared between the pregnant and nonpregnant women. The differences in the 3 indicators among different age groups and trimesters in pregnant women were studied. Finally, reference intervals were established by nonparametric methods according to the recommendation of Clinical and Laboratory Standards Institute guideline C28-A3. RESULTS: Compared with nonpregnant women, pregnant women had a significantly lower level of serum urea, creatinine, and UA (all P <.01), and no significant age-related differences in the 3 indicators were observed among the pregnant women (P >.05). However, the levels of these indicators were significantly different among the 3 trimesters (all P <.01 or P =.01). Accordingly, trimester-specific reference intervals of serum urea (1.6-4.4 mmol/L; 1.6-4.2 mmol/L; 1.6-4.4 mmol/L), creatinine (36-68 µmol/L; 34-66 µmol/L; 36-68 µmol/L), and UA (122-297 µmol/L; 129-327 µmol/L; 147-376 µmol/L) for trimesters 1, 2, and 3, respectively, were established. CONCLUSION: These newly established reference intervals will be valuable for the detection and monitoring of kidney disease in pregnancy.
Subject(s)
Pregnant Women , Uric Acid , China , Creatinine , Female , Humans , Pregnancy , UreaABSTRACT
To characterize how protein-protein interaction (PPI) networks change, we quantified the relative PPI abundance of 1.6 million protein pairs in the yeast Saccharomyces cerevisiae across nine growth conditions, with replication, for a total of 44 million measurements. Our multi-condition screen identified 13,764 pairwise PPIs, a threefold increase over PPIs identified in one condition. A few 'immutable' PPIs are present across all conditions, while most 'mutable' PPIs are rarely observed. Immutable PPIs aggregate into highly connected 'core' network modules, with most network remodeling occurring within a loosely connected 'accessory' module. Mutable PPIs are less likely to co-express, co-localize, and be explained by simple mass action kinetics, and more likely to contain proteins with intrinsically disordered regions, implying that environment-dependent association and binding is critical to cellular adaptation. Our results show that protein interactomes are larger than previously thought and contain highly dynamic regions that reorganize to drive or respond to cellular changes.
Subject(s)
Protein Interaction Maps , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , EnvironmentABSTRACT
OBJECTIVE: Experimental evidence suggests a close link between PARP (poly[ADP-ribose] polymerase) activation and diabetic endothelial dysfunction. Here, we tested whether PARP activity in circulating leukocytes was associated with coronary artery disease (CAD) among patients with type 2 diabetes mellitus (T2DM). Approach and Results: We performed observational and bidirectional Mendelian randomization studies of 3149 Chinese individuals with T2DM who underwent coronary angiography, with leukocyte PARP activity, 16 tag single-nucleotide polymorphisms in PARP1 and PARP2, and 17 CAD risk single-nucleotide polymorphisms analyzed. Of 3149 participants, 1180 who further received percutaneous coronary intervention were prospectively followed for 1 year to track major adverse cardiovascular and cerebrovascular events. Overall, greater PARP activity was cross-sectionally associated with an odds ratio of 1.23 for obstructive CAD, and prospectively with a hazard ratio of 1.34 for 1-year major adverse cardiovascular and cerebrovascular events after percutaneous coronary intervention (both P<0.001). Using a genetic score of 5 screened single-nucleotide polymorphisms in PARP1 and PARP2 as the instrumental variable, genetically predicted elevation in PARP activity showed a causal association with obstructive CAD (odds ratio=1.35, P<0.001). In contrast, the genetic risk of CAD had no significant effect on PARP activity. Ex vivo and in vitro cultures of human monocytes showed that rs747657, as the lead single-nucleotide polymorphism strongly associated with PARP activity, caused the differential binding of transcription factor GATA2 (GATA-binding protein 2) to an intronic regulatory region in PARP1, thus modulating PARP1 expression and PARP activity. CONCLUSIONS: Greater PARP activity may have causal roles in the development of obstructive CAD among patients with diabetes mellitus.
Subject(s)
Coronary Artery Disease/enzymology , Coronary Stenosis/enzymology , Diabetes Mellitus, Type 2/enzymology , Leukocytes/enzymology , Poly (ADP-Ribose) Polymerase-1/blood , Poly(ADP-ribose) Polymerases/blood , Aged , China , Coronary Artery Disease/blood , Coronary Artery Disease/genetics , Coronary Artery Disease/therapy , Coronary Stenosis/blood , Coronary Stenosis/genetics , Coronary Stenosis/therapy , Cross-Sectional Studies , Diabetes Mellitus, Type 2/blood , Diabetes Mellitus, Type 2/genetics , Female , Genetic Predisposition to Disease , Humans , Male , Mendelian Randomization Analysis , Middle Aged , Percutaneous Coronary Intervention/adverse effects , Phenotype , Poly (ADP-Ribose) Polymerase-1/genetics , Poly(ADP-ribose) Polymerases/genetics , Polymorphism, Single Nucleotide , Prospective Studies , Risk Assessment , Risk Factors , THP-1 Cells , Treatment OutcomeABSTRACT
BACKGROUND & AIMS: The 4977-bp mitochondrial deletion (mtDNA4977 deletion), as a hallmark of mitochondrial oxidative damage, may play an important role in coronary artery disease (CAD), but its interaction with folate deficiency among diabetic patients is largely unknown. We aimed to explore the joint association of leukocyte mtDNA4977 deletion and serum folate status with obstructive CAD in Chinese adults with type 2 diabetes. METHODS: We cross-sectionally analyzed the angiographic data of 2017 diabetic patients without B-vitamin supplementation. Of the 2017 participants, 756 who received percutaneous coronary intervention (PCI) completed prospective follow-up of one year. In vitro, we explored the mediation effects of mitochondrial reactive oxygen species (mtROS) in folic acid (FA)-deficient human aortic smooth muscle cells (HASMCs) under hyperglycemic conditions. RESULTS: Cross-sectionally, the multivariate odds ratios (ORs) for obstructive CAD were 1.41 (95% CI: 1.29-1.55) for greater mtDNA4977 deletion, and 1.15 (95% CI: 1.05-1.25) for lower folate levels. Particularly, the combination of high mtDNA4977 deletion (top tertile) and folate deficiency (serum folate < 6 ng/mL) was associated with more than 2-fold increased odds of having obstructive CAD and higher degrees of coronary stenosis. Prospectively, the hazard ratio for all-cause death at 1-year after PCI was up to 2.37 (95% CI: 1.21-4.63) for folate-deficient participants in the top tertile of mtDNA4977 deletion. In HASMCs, the adverse effects of FA deficiency were aggravated by induction of mtROS, and attenuated by scavenging of mtROS. CONCLUSIONS: The risk of obstructive CAD may be greatly increased by the interaction between greater mtDNA4977 deletion and folate deficiency among diabetic patients.
Subject(s)
Coronary Artery Disease/complications , DNA, Mitochondrial/blood , Diabetes Mellitus, Type 2/complications , Folic Acid Deficiency/complications , Folic Acid/blood , Aged , China , Coronary Artery Disease/blood , Coronary Artery Disease/genetics , Cross-Sectional Studies , Diabetes Mellitus, Type 2/blood , Diabetes Mellitus, Type 2/genetics , Female , Folic Acid Deficiency/blood , Folic Acid Deficiency/genetics , Humans , Leukocytes , Male , Middle Aged , Odds Ratio , Percutaneous Coronary Intervention , Proportional Hazards Models , Prospective StudiesABSTRACT
Mast cells play a significant role in urticaria pathogenesis. It's evidenced that vitamin D has positive impact in chronic spontaneous urticaria (CSU) recently, but underlying mechanisms remain unclear. In this study, isobaric tag for relative and absolute quantification-liquid chromatography-mass spectrometer/mass spectrometer was used to detect the expression of proteins in sera of CSU patients and healthy subjects. Thirty-one differentially expressed proteins were identified, in which vitamin D binding protein (VDBP) was higher in CSU patients than that in healthy subjects after verification. Our results indicated that sera of CSU patients induced the production of vascular endothelial growth factor (VEGF) in mast cells through PI3K/Akt/p38 MAPK/HIF-1α axis in an IgE-depended way, and 25(OH)D3 suppressed the expression of VEGF by inhibiting this signaling pathway axis in this process. Collectively, these results suggest VDBP to be a potential biomarker and propose a potential mechanism of benefit for vitamin D therapy in CSU.
Subject(s)
Chronic Urticaria/drug therapy , Chronic Urticaria/metabolism , Mast Cells/drug effects , Mast Cells/metabolism , Signal Transduction/drug effects , Vascular Endothelial Growth Factors/metabolism , Vitamin D/pharmacology , Adult , Female , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Immunoglobulin E/metabolism , Male , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
BACKGROUND: Little is known about whether mitochondria 8-hydroxy-2'-deoxyguanosine (8-OHdG), a biomarker of mitochondrial DNA (mtDNA) oxidative damage, contributes to the development of coronary artery disease (CAD) in diabetic patients. Here, we explored the associations of mtDNA 8-OHdG in leukocytes with obstructive CAD, coronary stenosis severity, cardiovascular biomarkers, and 1-year adverse outcomes after coronary revascularization in patients with type 2 diabetes mellitus (T2DM). METHODS: In a total of 1920 consecutive patients with T2DM who underwent coronary angiography due to symptoms of angina or angina equivalents, the presence of obstructive CAD, the number of diseased vessels with ≥ 50% stenosis, and modified Gensini score were cross-sectionally evaluated; the level of mtDNA 8-OHdG was quantified by quantitative PCR. Then, 701 of 1920 diabetic patients who further received coronary revascularization completed 1-year prospective follow-up to document major adverse cardiovascular and cerebral events (MACCEs). In vitro experiments were also performed to observe the effects of mtDNA oxidative damage in high glucose-cultured human umbilical vein endothelial cells (HUVECs). RESULTS: Cross-sectionally, greater mtDNA 8-OHdG was associated with increased odds of obstructive CAD (odds ratio [OR] 1.38, 95% CI confidence interval 1.24-1.52), higher degree of coronary stenosis (number of diseased vessels: OR 1.29, 95% CI 1.19-1.41; modified Gensini scores: OR 1.28, 95% CI 1.18-1.39), and higher levels of C-reactive protein (ß 0.18, 95% CI 0.06-0.31) after adjusting for confounders. Sensitivity analyses using propensity score matching yielded similar results. Stratification by smoking status showed that the association between mtDNA 8-OHdG and obstructive CAD was most evident in current smokers (Pinteration < 0.01). Prospectively, the adjusted hazards ratio per 1-SD increase in mtDNA 8-OHdG was 1.59 (95% CI 1.33-1.90) for predicting 1-year MACCEs after revascularization. In HUVECs, exposure to antimycin A, an inducer for mtDNA oxidative damage, led to adverse alterations in markers of mitochondrial and endothelia function. CONCLUSION: Greater mtDNA 8-OHdG in leukocytes may serve as an independent risk factor for CAD in patients with T2DM.
Subject(s)
8-Hydroxy-2'-Deoxyguanosine/blood , Coronary Artery Disease/blood , Coronary Stenosis/blood , DNA Damage , DNA, Mitochondrial/blood , Diabetes Mellitus, Type 2/blood , Leukocytes/metabolism , Aged , Biomarkers/blood , Cells, Cultured , China/epidemiology , Coronary Artery Disease/diagnostic imaging , Coronary Artery Disease/epidemiology , Coronary Artery Disease/therapy , Coronary Stenosis/diagnostic imaging , Coronary Stenosis/epidemiology , Coronary Stenosis/therapy , Cross-Sectional Studies , Diabetes Mellitus, Type 2/diagnosis , Diabetes Mellitus, Type 2/epidemiology , Female , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Male , Middle Aged , Myocardial Revascularization/adverse effects , Oxidative Stress , Prospective Studies , Risk Assessment , Risk Factors , Severity of Illness Index , Time Factors , Treatment OutcomeABSTRACT
OBJECTIVE: There has been little published work about the reference intervals of thromboelastography (TEG®) tests in pregnancy. Our aim was to establish the trimester-specific reference intervals of TEG tests for healthy pregnant women. METHODS: After excluding outliers, a total of 753 apparently healthy pregnant women aged from 19 to 44â¯years including 252 first trimester women, 340 second trimester women and 161 third trimester women were enrolled in our study. Non-fasting venous blood samples were collected. TEG tests were done on kaolin activated samples and processed on TEG 5000 Hemostasis Analyzer. Nonparametric 2.5th-97.5th percentile intervals were used to define the reference intervals. RESULTS: There were significant differences for TEG tests in pregnant women compared with non-pregnant women. The reference intervals for R, K, Angle, MA, Ly30 and CI were 4.1-10.4â¯min, 0.9-3.1â¯min, 53.6-75.9°, 46.1-69.8â¯mm, 0-10.7% and -5.5-2.5 respectively at first trimester; 3.9-9.7â¯min, 0.8-2.4â¯min, 56.7-78.0°, 49.8-72.1â¯mm, 0-9.7% and -3.7-2.9 at second trimester; 3.8-9.0â¯min, 0.8-2.5â¯min, 57.6-79.3°, 49.4-75.9â¯mm, 0-8.8% and -3.0-2.6 at third trimester. CONCLUSIONS: We established trimester-specific reference intervals of TEG® tests for healthy pregnant women. It's critical to accurate assessment of global haemostatic status during pregnancy and in pregnancy complications.
Subject(s)
Kaolin/chemistry , Thrombelastography/methods , Adult , Aged , Asian People , Female , Healthy Volunteers , Humans , Middle Aged , Pregnancy , Pregnancy Trimesters/blood , Reference ValuesABSTRACT
BACKGROUND AND AIMS: We aimed to identify a blood-based gene expression score (GES) to predict the severity of coronary artery stenosis in patients with known or suspected coronary artery disease (CAD) by integrative use of gene network construction, Support Vector Machine (SVM) algorithm, and multi-cohort validation. METHODS: In the discovery phase, a public blood-based microarray dataset of 110 patients with known CAD was analyzed by weighted gene coexpression network analysis and protein-protein interaction network analysis to identify candidate hub genes. In the training set with 151 CAD patients, bioinformatically identified hub genes were experimentally verified by real-time polymerase chain reaction, and statistically filtered with the SVM algorithm to develop a GES. Internal and external validation of GES was performed in patients with suspected CAD from two validation cohorts (nâ¯=â¯209 and 206). RESULTS: The discovery phase screened 15 network-centric hub genes significantly correlated with the Duke CAD Severity Index. In the training cohort, 12 of 15 hub genes were filtered to construct a blood-based GES12, which showed good discrimination for higher modified Gensini scores (AUC: 0.798 and 0.812), higher Sullivan Extent scores (AUC: 0.776 and 0.778), and the presence of obstructive CAD (AUC: 0.834 and 0.792) in two validation cohorts. A nomogram comprising GES12, smoking status, hypertension status, low density lipoprotein cholesterol level, and body mass index further improved performance, with respect to discrimination, risk classification, and clinical utility, for prediction of coronary stenosis severity. CONCLUSIONS: GES12 is useful in predicting the severity of coronary artery stenosis in patients with known or suspected CAD.