Decrease of MtDNA copy number affects mitochondrial function and involves in the pathological consequences of ischaemic stroke

The mtDNA copy number can affect the function of mitochondria and play an important role in the development of diseases. However, there are few studies on the mechanism of mtDNA copy number variation and its effects in IS. The specific mechanism of mtDNA copy number variation is still unclear. In this study, mtDNA copy number of 101 IS patients and 101 normal controls were detected by qRT‐PCR, the effect of D‐loop variation on mtDNA copy number of IS patients was explored. Then, a TFAM gene KD‐OE PC12 cell model was constructed to explore the effect of mtDNA copy number variation on mitochondrial function. The results showed that the mtDNA copy number level of the IS group was significantly lower than that of the normal control group (p < 0.05). The relative expression of TFAM gene mRNA in the cells of the OGD/R treatment group was significantly lower than that of the control group (p < 0.05). In addition, after TFAM gene knockdown and over‐expression plasmids were transfected into HEK 293T cells, mtDNA copy number and ATP production level of Sh‐TFAM transfection group was significantly decreased (p < 0.05), while mtDNA copy number and ATP production level of OE‐TFAM transfected group were significantly higher than that of blank control group and OE‐ctrl negative control group (p < 0.01). Our study demonstrated that mitochondrial D‐loop mutation and TFAM gene dysfunction can cause the decrease of mtDNA copy number, thus affecting the mitochondrial metabolism and function of nerve cells, participating in the pathological damage mechanism of IS.     

Leukocyte Mitochondrial DNA Copy Number Is Associated with Chronic Obstructive Pulmonary Disease

Background

Oxidative stress is known to be involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). Evidence suggests that leukocytes mitochondria DNA (mtDNA) is susceptible to undergo mutations, insertions, or depletion in response to reactive oxidative stress (ROS). We hypothesize that mtDNA copy number is associated with the development of COPD.

Methodology/Principal Findings

Relative mtDNA copy number was measured by a quantitative real-time PCR assay using DNA extracted from peripheral leukocytes. MtDNA copy number of peripheral leukocytes in the COPD group (n = 86) is significantly decreased compared with non-smoker group (n = 77) (250.3± 21.5 VS. 464.2± 49.9, P<0.001). MtDNA copy number in the COPD group was less than that in the healthy smoking group, but P value nearly achieved significance (250.3± 21.5 VS. 404.0± 76.7, P = 0.08) MtDNA copy number has no significance with age, gender, body mass index, current smoking, and pack-years in COPD group, healthy smoker group and no smoker group, respectively. Serum glutathione level in the COPD group is significantly decreased compared with healthy smoker and non-smoker groups (4.5± 1.3 VS. 6.2± 1.9 and 4.5± 1.3 VS. 7.1±1.1 mU/mL; P<0.001 respectively). Pearson correlation test shows a significant liner correlation between mtDNA copy number and serum glutathione level (R = 0.2, P = 0.009).

Conclusions/Significance

COPD is associated with decreased leukocyte mtDNA copy number and serum glutathione. COPD is a regulatory disorder of leukocytes mitochondria. However, further studies are needed to determine the real mechanisms about the gene and the function of mitochondria.     

Leukocyte Mitochondrial DNA Copy Number in Blood Is Not Associated with Major Depressive Disorder in Young Adults

Background

Major depressive disorder (MDD) is the leading cause of disability worldwide, and has significant genetic predisposition. Mitochondria may have a role in MDD and so mitochondrial DNA (mtDNA) has been suggested as a possible biomarker for this disease. We aimed to test whether the mtDNA copy number of peripheral blood leukocytes is related to MDD in young adults.

Methods

A case-control study was conducted with 210 MDD patients and 217 healthy controls (HC). The mtDNA copy number was measured by quantitative polymerase chain reaction (qPCR) method. Depression severity was assessed by the Hamilton-17 Depression Rating Scale (HDRS-17).

Results

We found no significant differences in mtDNA copy number between MDD patients and HC, though the power analysis showed that our sample size has enough power to detect the difference. There were also no significant correlations between mtDNA copy number and the clinical characteristics (such as age, age of onset, episodes, Hamilton Depression Rating Scale (HDRS) score and Global Assessment of Function Scale (GAF) score) in MDD patients.

Conclusion

Our study suggests that leukocyte mtDNA copy number is unlikely to contribute to MDD, but it doesn’t mean that we can exclude the possibility of involvement of mitochondria in the disease. Further studies are required to elucidate whether mtDNA can be a biomarker of MDD.     

Oxidants and antioxidants in myocardial infarction (MI): Investigation of ischemia modified albumin,malondialdehyde, superoxide dismutase and catalase in individuals diagnosed with ST elevated myocardial infarction (STEMI) and non-STEMI (NSTEMI)

BackgroundCoronary ischemia can lead to myocardial damage and necrosis. The pathogenesis of cardiovascular diseases often includes increased oxidative stress and decreased antioxidant defense. The study aimed to assess levels of ischemia modified albumin (IMA), malondialdehyde acid (MDA), superoxide dismutase (SOD), and catalase in individuals diagnosed with ST elevated myocardial infarction (STEMI) and non-STEMI.MethodsThe present study prospectively included 50 STEMI patients, 55 NSTEMI patients, and 55 healthy subjects. Only patients who were recently diagnosed with STEMI or NSTEMI were included in this study. IMA, MDA, SOD, and catalase activities were measured spectrophotometrically. Significant coronary artery lesions were determined by angiography.ResultsPatients with ACS had significantly greater IMA and MDA values than the healthy controls (p<0.001). Besides, patients with STEMI had IMA levels that were significantly greater than those of the patients with NSTEMI (p<0.001), while the reverse was true for MDA levels (p<0.001). The healthy controls had the highest levels of SOD and catalase levels, followed by patients with STEMI and patients with NSTEMI, respectively (p<0.001). There was a significant negative correlation among MDA and SOD with catalase levels (r = -0.771 p<0.001 MDA vs catalase; r = -0.821 p<0.001 SOD vs catalase).ConclusionsData obtained in this study reveals that compared to healthy controls, STEMI and NSTEMI patients had increased levels of MDA and IMA and decreased levels of SOD and catalase.     

Peripheral Blood Mitochondrial DNA as a Biomarker of Cerebral Mitochondrial Dysfunction following Traumatic Brain Injury in a Porcine Model

Background

Traumatic brain injury (TBI) has been shown to activate the peripheral innate immune system and systemic inflammatory response, possibly through the central release of damage associated molecular patterns (DAMPs). Our main purpose was to gain an initial understanding of the peripheral mitochondrial response following TBI, and how this response could be utilized to determine cerebral mitochondrial bioenergetics. We hypothesized that TBI would increase peripheral whole blood relative mtDNA copy number, and that these alterations would be associated with cerebral mitochondrial bioenergetics triggered by TBI.

Methodology

Blood samples were obtained before, 6 h after, and 25 h after focal (controlled cortical impact injury: CCI) and diffuse (rapid non-impact rotational injury: RNR) TBI. PCR primers, unique to mtDNA, were identified by aligning segments of nuclear DNA (nDNA) to mtDNA, normalizing values to nuclear 16S rRNA, for a relative mtDNA copy number. Three unique mtDNA regions were selected, and PCR primers were designed within those regions, limited to 25-30 base pairs to further ensure sequence specificity, and measured utilizing qRT-PCR.

Results

Mean relative mtDNA copy numbers increased significantly at 6 and 25 hrs after following both focal and diffuse traumatic brain injury. Specifically, the mean relative mtDNA copy number from three mitochondrial-specific regions pre-injury was 0.84 ± 0.05. At 6 and 25 h after diffuse non-impact TBI, mean mtDNA copy number was significantly higher: 2.07 ± 0.19 (P < 0.0001) and 2.37 ± 0.42 (P < 0.001), respectively. Following focal impact TBI, relative mtDNA copy number was also significantly higher, 1.35 ± 0.12 (P < 0.0001) at 25 hours. Alterations in mitochondrial respiration in the hippocampus and cortex post-TBI correlated with changes in the relative mtDNA copy number measured in peripheral blood.

Conclusions

Alterations in peripheral blood relative mtDNA copy numbers may be a novel biosignature of cerebral mitochondrial bioenergetics with exciting translational potential for non-invasive diagnostic and interventional studies.     

Prevalence of Myopia and Its Risk Factors in Urban School Children in Delhi: The North India Myopia Study (NIM Study)

PurposeAssess prevalence of myopia and identify associated risk factors in urban school children.MethodsThis was a cross-sectional study screening children for sub-normal vision and refractive errors in Delhi. Vision was tested by trained health workers using ETDRS charts. Risk factor questionnaire was filled for children with vision <6/9.5, wearing spectacles and for a subset (10%) of randomly selected children with normal vision. All children with vision <6/9.5 underwent cycloplegic refraction. The prevalence of myopia <-0.5 diopters was assessed. Association of risk factors and prevalence of myopia was analyzed for children with myopia and randomly selected non myopic children and adjusted odds ratio values for all risk factors were estimated.ResultsA total number of 9884 children were screened with mean age of 11.6 + 2.2 years and 66.8% boys. Prevalence of myopia was 13.1% with only 320 children (24.7%) wearing appropriate spectacles. Mean myopic spherical error was -1.86 + 1.4 diopters. Prevalence of myopia was higher in private schools compared to government schools (p<0.001), in girls vs. boys (p = 0.004) and among older (> 11 years) children (p<0.001). There was a positive association of myopia with studying in private schools vs. government schools (p<0.001), positive family history (p< 0.001) and higher socio-economic status (p = 0.037). Positive association of presence of myopia was observed with children studying/reading > 5 hours per day (p < 0.001), watching television > 2 hours / day (p < 0.001) and with playing computer/video/mobile games (p < 0.001). An inverse association with outdoor activities/playing was observed with children playing > 2 hours in a day.ConclusionMyopia is a major health problem in Indian school children. It is important to identify modifiable risk factors associated with its development and try to develop cost effective intervention strategies.     

Peripheral Blood Mitochondrial DNA Copy Number Is Associated with Prostate Cancer Risk and Tumor Burden

Alterations of mitochondrial DNA (mtDNA) have been associated with the risk of a number of human cancers; however, the relationship between mtDNA copy number in peripheral blood leukocytes (PBLs) and the risk of prostate cancer (PCa) has not been investigated. In a case-control study of 196 PCa patients and 196 age-paired healthy controls in a Chinese Han population, the association between mtDNA copy number in PBLs and PCa risk was evaluated. The relative mtDNA copy number was measured using quantitative real-time PCR; samples from three cases and two controls could not be assayed, leaving 193 cases and 194 controls for analysis. PCa patients had significantly higher mtDNA copy numbers than controls (medians 0.91 and 0.82, respectively; P<0.001). Dichotomized at the median value of mtDNA copy number in the controls, high mtDNA copy number was significantly associated with an increased risk of PCa (adjusted odds ratio  = 1.85, 95% confidence interval: 1.21–2.83). A significant dose-response relationship was observed between mtDNA copy number and risk of PCa in quartile analysis (P _trend = 0.011). Clinicopathological analysis showed that high mtDNA copy numbers in PCa patients were significantly associated with high Gleason score and advanced tumor stage, but not serum prostate-specific antigen level (P = 0.002, 0.012 and 0.544, respectively). These findings of the present study indicate that increased mtDNA copy number in PBLs is significantly associated with an increased risk of PCa and may be a reflection of tumor burden.     

A Multicenter,Randomized, Controlled Trial of Osteopathic Manipulative Treatment on Preterms

BackgroundDespite some preliminary evidence, it is still largely unknown whether osteopathic manipulative treatment improves preterm clinical outcomes.ResultsA total of 695 newborns were randomly assigned to either the study group (n= 352) or the control group (n=343). A statistical significant difference was observed between the two groups for the primary outcome (13.8 and 17.5 days for the study and control group respectively, p<0.001, effect size: 0.31). Multivariate analysis showed a reduction of the length of stay of 3.9 days (95% CI -5.5 to -2.3, p<0.001). Furthermore, there were significant reductions with treatment as compared to usual care in cost (difference between study and control group: 1,586.01€; 95% CI 1,087.18 to 6,277.28; p<0.001) but not in daily weight gain. There were no complications associated to the intervention.ConclusionsOsteopathic treatment reduced significantly the number of days of hospitalization and is cost-effective on a large cohort of preterm infants.     

Enhancing the Early Differential Diagnosis of Plateau Iris and Pupillary Block Using A-Scan Ultrasonography

PurposeTo distinguish the frequently misdiagnosed plateau iris eyes from pupillary block group and normal group, we compared the ocular biometrical parameters of them by A-scan ultrasongraphy.MethodsIn total, we retrospectively reviewed general characteristics and ocular findings including ocular biometric measurements of 71 normal, 39 plateau iris, and 83 pupillary block eyes.ResultsThe normal controls, plateau iris group and pupillary block group were significantly different in age, but not in gender. The anterior chamber depth tended to decrease and the lens thickness tended to increase from normal to plateau iris to pupillary block eyes. Compared to those of plateau iris group, the pupillary block group had significantly shallower anterior chamber depth (2.90mm vs. 2.33mm; p<0.001), thicker lens (4.77mm vs. 5.11mm; p<0.001), shorter axial length (23.16mm vs. 22.63mm; p<0.001), smaller relative lens position (2.28 vs. 2.16; p<0.001) and larger lens/axial length factor (2.06 vs. 2.26; p<0.001). However, when comparing plateau iris and normal eyes, only axial length and lens/axial length factor were significantly different (23.16 vs. 23.54; p<0.05 and 2.06 vs. 1.96; p<0.05).ConclusionsMeasured by A-scan ultrasonography, the ocular biometrics of plateau iris were significantly different from those of pupillary block eyes. However, our A-scan ultrasongraphy generally found no significant biometric differences between plateau iris and normal eyes. These findings suggest that while A-scan ultrasonography might be used as a practical tool for differentiating plateau iris and papillary block eyes, a more meticulous gonioscopy and other assessments may be necessary to distinguish plateau iris from normal eyes.     

Mitochondrial Genome Analysis of Primary Open Angle Glaucoma Patients

Primary open angle glaucoma (POAG) is a multi-factorial optic disc neuropathy characterized by accelerating damage of the retinal ganglion cells and atrophy of the optic nerve head. The vulnerability of the optic nerve damage leading to POAG has been postulated to result from oxidative stress and mitochondrial dysfunction. In this study, we investigated the possible involvement of the mitochondrial genomic variants in 101 patients and 71 controls by direct sequencing of the entire mitochondrial genome. The number of variable positions in the mtDNA with respect to the revised Cambridge Reference Sequence (rCRS), have been designated “Segregating Sites”. The segregating sites present only in the patients or controls have been designated “Unique Segregating Sites (USS)”. The population mutation rate (θ = 4N_eμ) as estimated by Watterson’s θ (θ_w), considering only the USS, was significantly higher among the patients (p = 9.8×10⁻¹⁵) compared to controls. The difference in θ_w and the number of USS were more pronounced when restricted to the coding region (p<1.31×10⁻²¹ and p = 0.006607, respectively). Further analysis of the region revealed non-synonymous variations were significantly higher in Complex I among the patients (p = 0.0053). Similar trends were retained when USS was considered only within complex I (frequency 0.49 vs 0.31 with p<0.0001 and mutation rate p-value <1.49×10⁻⁴³) and ND5 within its gene cluster (frequency 0.47 vs 0.23 with p<0.0001 and mutation rate p-value <4.42×10⁻⁴⁷). ND5 is involved in the proton pumping mechanism. Incidentally, glaucomatous trabecular meshwork cells have been reported to be more sensitive to inhibition of complex I activity. Thus mutations in ND5, expected to inhibit complex I activity, could lead to generation of oxidative stress and favor glaucomatous condition.     

The Association between Mycobacterium Tuberculosis Genotype and Drug Resistance in Peru

BackgroundThe comparison of Mycobacterium tuberculosis bacterial genotypes with phenotypic, demographic, geospatial and clinical data improves our understanding of how strain lineage influences the development of drug-resistance and the spread of tuberculosis.MethodsTo investigate the association of Mycobacterium tuberculosis bacterial genotype with drug-resistance. Drug susceptibility testing together with genotyping using both 15-loci MIRU-typing and spoligotyping, was performed on 2,139 culture positive isolates, each from a different patient in Lima, Peru. Demographic, geospatial and socio-economic data were collected using questionnaires, global positioning equipment and the latest national census.ResultsThe Latin American Mediterranean (LAM) clade (OR 2.4, p<0.001) was significantly associated with drug-resistance and alone accounted for more than half of all drug resistance in the region. Previously treated patients, prisoners and genetically clustered cases were also significantly associated with drug-resistance (OR''s 2.5, 2.4 and 1.8, p<0.001, p<0.05, p<0.001 respectively).ConclusionsTuberculosis disease caused by the LAM clade was more likely to be drug resistant independent of important clinical, genetic and socio-economic confounding factors. Explanations for this include; the preferential co-evolution of LAM strains in a Latin American population, a LAM strain bacterial genetic background that favors drug-resistance or the "founder effect" from pre-existing LAM strains disproportionately exposed to drugs.     

Chronic Kidney Disease Is Characterized by “Double Trouble” Higher Pulse Pressure plus Night-Time Systolic Blood Pressure and More Severe Cardiac Damage

Background

Hypertension plays a key role in chronic kidney disease (CKD), but CKD itself affects the blood pressure (BP) profile. The aim of this study was to assess the association of BP profile with CKD and the presence of cardiac organ damage.

Methods

We studied 1805 patients, referred to our Hypertension Centre, in whom ABPM, blood tests, and echocardiography were clinically indicated. The glomerular filtration rate was estimated (eGFR) using the MDRD equation and CKD was defined as eGFR<60 mL/min/1.73 m². Cardiac organ damage was evaluated by echocardiography.

Results

Among patients with CKD there were higher systolic blood pressure (SBP) during the night-time, greater prevalence of non-dippers (OR: 1.8) and increased pulse pressure (PP) during 24-hour period, daytime and night-time (all p<0.001). Patients with CKD had a greater LVM/h^2.7 index, and a higher prevalence of left ventricular hypertrophy and diastolic dysfunction (all p<0.001). Nocturnal SBP and PP correlated more strongly with cardiac organ damage (p<0.001). Patients with CKD had a greater Treatment Intensity Score (p<0.001) in the absence of a significantly greater BP control.

Conclusions

CKD patients have an altered night-time pressure profile and higher PP that translate into a more severe cardiac organ damage. In spite of a greater intensity of treatment in most patients with CKD, BP control was similar to patients without CKD. Our findings indicate the need of a better antihypertensive therapy in CKD, better selected drugs, dosages and posology to provide optimal coverage of 24 hours and night-time BP.     

Reactive oxygen species regulate DNA copy number in isolated yeast mitochondria by triggering recombination-mediated replication

Mitochondrial DNA (mtDNA) encodes proteins that are essential for cellular ATP production. Reactive oxygen species (ROS) are respiratory byproducts that damage mtDNA and other cellular components. In Saccharomyces cerevisiae, the oxidized base excision-repair enzyme Ntg1 introduces a double-stranded break (DSB) at the mtDNA replication origin ori5; this DSB initiates the rolling-circle mtDNA replication mediated by the homologous DNA pairing protein Mhr1. Thus, ROS may play a role in the regulation of mtDNA copy number. Here, we show that the treatment of isolated mitochondria with low concentrations of hydrogen peroxide increased mtDNA copy number in an Ntg1- and Mhr1-dependent manner. This treatment elevated the DSB levels at ori5 of hypersuppressive [rho^–] mtDNA only if Ntg1 was active. In vitro Ntg1-treatment of hypersuppressive [rho^–] mtDNA extracted from hydrogen peroxide-treated mitochondria revealed increased oxidative modifications at ori5 loci. We also observed that purified Ntg1 created breaks in single-stranded DNA harboring oxidized bases, and that ori5 loci have single-stranded character. Furthermore, chronic low levels of hydrogen peroxide increased in vivo mtDNA copy number. We therefore propose that ROS act as a regulator of mtDNA copy number, acting through the Mhr1-dependent initiation of rolling-circle replication promoted by Ntg1-induced DSB in the single-stranded regions at ori5.     

MRI Background Parenchymal Enhancement Is Not Associated with Breast Cancer

BackgroundPreviously, a strong positive association between background parenchymal enhancement (BPE) at magnetic resonance imaging (MRI) and breast cancer was reported in high-risk populations. We sought to determine, whether this was also true for non-high-risk patients.Methods540 consecutive patients underwent breast MRI for assessment of breast findings (BI-RADS 0–5, non-high-risk screening (no familial history of breast cancer, no known genetic mutation, no prior chest irradiation, or previous breast cancer diagnosis)) and subsequent histological work-up. For this IRB-approved study, BPE and fibroglandular tissue FGT were retrospectively assessed by two experienced radiologists according to the BI-RADS lexicon. Pearson correlation coefficients were calculated to explore associations between BPE, FGT, age and final diagnosis of breast cancer. Subsequently, multivariate logistic regression analysis, considering covariate colinearities, was performed, using final diagnosis as the target variable and BPE, FGT and age as covariates.ResultsAge showed a moderate negative correlation with FGT (r = -0.43, p<0.001) and a weak negative correlation with BPE (r = -0.28, p<0.001). FGT and BPE correlated moderately (r = 0.35, p<0.001). Final diagnosis of breast cancer displayed very weak negative correlations with FGT (r = -0.09, p = 0.046) and BPE (r = -0.156, p<0.001) and weak positive correlation with age (r = 0.353, p<0.001). On multivariate logistic regression analysis, the only independent covariate for prediction of breast cancer was age (OR 1.032, p<0.001).ConclusionsBased on our data, neither BPE nor FGT independently correlate with breast cancer risk in non-high-risk patients at MRI. Our model retained only age as an independent risk factor for breast cancer in this setting.     

The acute muscular response following a novel form of pulsed direct current stimulation (Neubie) or traditional resistance exercise

Objectives:To examine changes in muscle thickness (MT), soreness (SOR), and isometric torque (ISO) following exercise with pulsed direct current (Neubie) or traditional high-load (TRAD) exercise.Methods:Thirty-two participants had SOR, MT, and ISO measured before, immediately after, and 24 and 48h following TRAD and Neubie. Rating of perceived exertion (RPE) and discomfort were also measured. Results are displayed as means(SD).Results:For MT, there was a condition x time interaction (p<0.001). For Neubie, MT increased pre [3.7(0.7)cm] to post [3.9(0.8) cm, p<0.001] and remained elevated at 24h. For TRAD, MT increased pre [3.7(0.6)cm] to post [4.0 (0.7)cm, p<0.001] and remained up to 48h. Greater values were observed for TRAD post-exercise. For ISO, both conditions decreased up to 48h. TRAD demonstrated a greater change post exercise (p<0.001). For SOR, both conditions increased up to 48h. Neubie demonstrated greater SOR at 48h (p=0.007). RPE was higher for all sets in TRAD [Mean across sets=16.0(1.9) vs. 13.5(2), p<0.001]. Discomfort was higher in all sets for Neubie [Mean across sets=5.8(1.5)vs. 4.5(2.0), p<0.05].Conclusions:Both conditions showed increased SOR, and decreased ISO for up to 48h, with MT increased for up to 24h. MT remained elevated in TRAD at 48h. Neubie training might be effective for individuals who are looking to experience lower RPE responses during exercise.     

Mitochondrial DNA Ligase Is Dispensable for the Viability of Cultured Cells but Essential for mtDNA Maintenance

Multiple lines of evidence support the notion that DNA ligase III (LIG3), the only DNA ligase found in mitochondria, is essential for viability in both whole organisms and in cultured cells. Previous attempts to generate cells devoid of mitochondrial DNA ligase failed. Here, we report, for the first time, the derivation of viable LIG3-deficient mouse embryonic fibroblasts. These cells lack mtDNA and are auxotrophic for uridine and pyruvate, which may explain the apparent lethality of the Lig3 knock-out observed in cultured cells in previous studies. Cells with severely reduced expression of LIG3 maintain normal mtDNA copy number and respiration but show reduced viability in the face of alkylating and oxidative damage, increased mtDNA degradation in response to oxidative damage, and slow recovery from mtDNA depletion. Our findings clarify the cellular role of LIG3 and establish that the loss of viability in LIG3-deficient cells is conditional and secondary to the ρ⁰ phenotype.     

Identifying Adult Dengue Patients at Low Risk for Clinically Significant Bleeding

Background

Clinically significant bleeding is important for subsequent optimal case management in dengue patients, but most studies have focused on dengue severity as an outcome. Our study objective was to identify differences in admission parameters between patients who developed clinically significant bleeding and those that did not. We sought to develop a model for discriminating between these patients.

Methods

We conducted a retrospective study of 4,383 adults aged >18 years who were hospitalized with dengue infection at Tan Tock Seng Hospital, Singapore from 2005 to 2008. Patients were divided into those with clinically significant bleeding (n = 188), and those without (n = 4,195). Demographic, clinical, and laboratory variables on admission were compared between groups to determine factors associated with clinically significant bleeding during hospitalization.

Results

On admission, female gender (p<0.001); temperature >38°C (p<0.001); nausea/vomiting (p = 0.009) and abdominal pain/tenderness (p = 0.005); lower systolic blood pressure (p<0.001); higher pulse rate (p<0.001); increased absolute neutrophil count (ANC; p<0.001); reduced absolute lymphocyte count (ALC; p<0.001), haematocrit percentage (p<0.001) and platelet count (p = 0.04), and increased prothrombin time (p = 0.003) were significantly associated with clinically significant bleeding on univariate analysis. Multivariate analysis showed that independent variables in the final model were female gender (aOR 2.85; 95% CI: 1.9–4.33); temperature >38°C (aOR 1.81; 95% CI: 1.27–2.61), nausea/vomiting (aOR 1.39; 95% CI: 0.94–2.12), ANC (aOR 1.3; 95% CI: 1.15–1.46), ALC (aOR 0.4; 95% CI: 0.25–0.64), hematocrit percentage (aOR 0.96; 95% CI: 0.92–1.002) and platelet count (aOR 0.993; 95% CI: 0.988–0.998). At the cutoff of -3.919, the model achieved an AUC of 0.758 (sensitivity:0.87, specificity: 0.38, PPV: 0.06, NPV: 0.98).

Conclusion

Clinical risk factors associated with clinically significant bleeding were identified. This model may be useful to complement clinical judgement in triaging adult dengue patients given the dynamic nature of acute dengue, particularly in pre-identifying those less likely to develop clinically significant bleeding.     

Hydroxyethyl Starch (HES 130/0.4) Impairs Intestinal Barrier Integrity and Metabolic Function: Findings from a Mouse Model of the Isolated Perfused Small Intestine

BackgroundThe application of hydroxyethyl starch (HES) for volume resuscitation is controversially discussed and clinical studies have suggested adverse effects of HES substitution, leading to increased patient mortality. Although, the intestine is of high clinical relevance and plays a crucial role in sepsis and inflammation, information about the effects of HES on intestinal function and barrier integrity is very scarce. We therefore evaluated the effects of clinically relevant concentrations of HES on intestinal function and barrier integrity employing an isolated perfused model of the mouse small intestine.MethodsAn isolated perfused model of the mouse small intestine was established and intestines were vascularly perfused with a modified Krebs-Henseleit buffer containing 3% Albumin (N=7) or 3% HES (130/0.4; N=7). Intestinal metabolic function (galactose uptake, lactate-to-pyruvate ratio), edema formation (wet-to-dry weight ratio), morphology (histological and electron microscopical analysis), fluid shifts within the vascular, lymphatic and luminal compartments, as well as endothelial and epithelial barrier permeability (FITC-dextran translocation) were evaluated in both groups.ResultsCompared to the Albumin group, HES perfusion did not significantly change the wet-to-dry weight ratio and lactate-to-pyruvate ratio. However, perfusing the small intestine with 3% HES resulted in a significant loss of vascular fluid (p<0.01), an increased fluid accumulation in the intestinal lumen (p<0.001), an enhanced translocation of FITC-dextran from the vascular to the luminal compartment (p<0.001) and a significantly impaired intestinal galactose uptake (p<0.001). Morphologically, these findings were associated with an aggregation of intracellular vacuoles within the intestinal epithelial cells and enlarged intercellular spaces.ConclusionA vascular perfusion with 3% HES impairs the endothelial and epithelial barrier integrity as well as metabolic function of the small intestine.     

Proximal Tubule Dysfunction Is Associated with Podocyte Damage Biomarkers Nephrin and Vascular Endothelial Growth Factor in Type 2 Diabetes Mellitus Patients: A Cross-Sectional Study

Background

There is an ongoing debate as to whether early diabetic nephropathy in Type 2 diabetes mellitus may be attributed to the glomerulus or to the proximal tubule. Urinary excretion of nephrin and vascular endothelial growth factor may increase even in the normoalbuminuria stage. In the course of diabetic nephropathy, the proximal tubule may be involved in the uptake of urinary nephrin and vascular endothelial growth factor.

Materials and Methods

Two groups of consecutive Type 2 diabetes mellitus outpatients (38 normo-, 32 microalbuminuric) and 21 healthy subjects were enrolled in a cross-sectional study and evaluated concerning the relation of proximal tubule dysfunction with the podocyte biomarkers excretion, assessed by ELISA methods. The impact of advanced glycation end-products on this relation was also queried.

Results

Urinary alpha₁-microglobulin and kidney injury molecule-1 correlated with urinary albumin:creatinine ratio (R² = 0.269; p<0.001; R² = 0.125; p<0.001), nephrinuria (R² = 0.529; p<0.001; R² = 0.203; p<0.001), urinary vascular endothelial growth factor (R² = 0.709; p<0.001; R² = 0.360; p<0.001), urinary advanced glycation end-products (R² = 0.578; p<0.001; R² = 0.405; p<0.001), serum cystatin C (R² = 0.130; p<0.001; R² = 0.128; p<0.001), and glomerular filtration rate (R² = 0.167; p<0.001; R² = 0.166; p<0.001); nephrinuria and urinary vascular endothelial growth factor correlated with urinary albumin:creatinine ratio (R² = 0.498; p<0.001; R² = 0.227; p<0.001), urinary advanced glycation end-products (R² = 0.251; p<0.001; R² = 0.308; p<0.001), serum cystatin C (R² = 0.157; p<0.001; R² = 0.226; p<0.001), and glomerular filtration rate (R² = 0.087; p = 0.007; R² = 0.218; p<0.001).

Conclusions

In Type 2 diabetes mellitus there is an association of proximal tubule dysfunction with podocyte damage biomarkers, even in the normoalbuminuria stage. This observation suggests a potential role of the proximal tubule in urinary nephrin and urinary vascular endothelial growth factor processing in early diabetic nephropathy, a fact which could be related to advanced glycation end-products intervention. Podocyte damage and proximal tubule dysfunction biomarkers could be validated as a practical approach to the diagnosis of early diabetic nephropathy by further studies on larger cohorts.     

Mitochondrial DNA Copy Number in Peripheral Blood and Melanoma Risk

Mitochondrial DNA (mtDNA) copy number in peripheral blood has been suggested as risk modifier in various types of cancer. However, its influence on melanoma risk is unclear. We evaluated the association between mtDNA copy number in peripheral blood and melanoma risk in 500 melanoma cases and 500 healthy controls from an ongoing melanoma study. The mtDNA copy number was measured using real-time polymerase chain reaction. Overall, mean mtDNA copy number was significantly higher in cases than in controls (1.15 vs 0.99, P<0.001). Increased mtDNA copy number was associated with a 1.45-fold increased risk of melanoma (95% confidence interval: 1.12-1.97). Significant joint effects between mtDNA copy number and variables related to pigmentation and history of sunlight exposure were observed. This study supports an association between increased mtDNA copy number and melanoma risk that is independent on the known melanoma risk factors (pigmentation and history of sunlight exposure).