SUMOylation of PPARγ by Rosiglitazone Prevents LPS-Induced NCoR Degradation Mediating Down Regulation of Chemokines Expression in Renal Proximal Tubular Cells

Rosiglitazone (RGL), a synthetic agonist for peroxisome proliferator activated receptor γ (PPARγ), exhibits a potent anti-inflammatory activity by attenuating local infiltration of neutrophils and monocytes in the renal interstitium. To evaluate the mechanisms that account for inhibiting inflammatory cells infiltration, we investigated the effect of RGL on chemokines secretion and nuclear factor-kappa B (NF-κB) activation in human renal proximal tubular cells (PTCs). We demonstrated that RGL significantly inhibited lipopolysaccharide (LPS)-induced interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) production in a dose-dependent manner, without appreciable cytotoxicity. Chromatin immunoprecipitation (ChIP) assays clearly revealed that, RGL inhibited p65 binding to IL-8/MCP-1 gene promoters in LPS-stimulated PTCs. Interestingly, further experiments showed RGL reversed LPS-induced nuclear receptor corepressor (NCoR) degradation. In addition, knockdown of protein inhibitor of activated STAT1 (PIAS1), an indispensable small ubiquitin-like modifier (SUMO) ligase, abrogated the effects of RGL on antagonizing LPS-induced IL-8/MCP-1 overexpression and NCoR degradation. These findings suggest that, RGL activates PPARγ SUMOylation, inhibiting NCoR degradation and NF-κB activation in LPS-stimulated PTCs, which in turn decrease chemokines expression. The results unveil a new mechanism triggered by RGL for prevention of tubular inflammatory injury.     

A Novel PRPF31 Mutation in a Large Chinese Family with Autosomal Dominant Retinitis Pigmentosa and Macular Degeneration

Purpose

This study was intended to identify the disease causing genes in a large Chinese family with autosomal dominant retinitis pigmentosa and macular degeneration.

Methods

A genome scan analysis was conducted in this family for disease gene preliminary mapping. Snapshot analysis of selected SNPs for two-point LOD score analysis for candidate gene filter. Candidate gene PRPF31 whole exons'' sequencing was executed to identify mutations.

Results

A novel nonsense mutation caused by an insertion was found in PRPF31 gene. All the 19 RP patients in 1085 family are carrying this heterozygous nonsense mutation. The nonsense mutation is in PRPF31 gene exon9 at chr19:54629961-54629961, inserting nucleotide “A” that generates the coding protein frame shift from p.307 and early termination at p.322 in the snoRNA binding domain (NOP domain).

Conclusion

This report is the first to associate PRPF31 gene''s nonsense mutation and adRP and JMD. Our findings revealed that PRPF31 can lead to different clinical phenotypes in the same family, resulting either in adRP or syndrome of adRP and JMD. We believe our identification of the novel “A” insertion mutation in exon9 at chr19:54629961-54629961 in PRPF31 can provide further genetic evidence for clinical test for adRP and JMD.     

Mutation Inactivation of Nijmegen Breakage Syndrome Gene (NBS1) in Hepatocellular Carcinoma and Intrahepatic Cholangiocarcinoma

Nijmegen breakage syndrome (NBS) with NBS1 germ-line mutation is a human autosomal recessive disease characterized by genomic instability and enhanced cancer predisposition. The NBS1 gene codes for a protein, Nbs1(p95/Nibrin), involved in the processing/repair of DNA double-strand breaks. Hepatocellular carcinoma (HCC) is a complex and heterogeneous tumor with several genomic alterations. Recent studies have shown that heterozygous NBS1 mice exhibited a higher incidence of HCC than did wild-type mice. The objective of the present study is to assess whether NBS1 mutations play a role in the pathogenesis of human primary liver cancer, including HBV-associated HCC and intrahepatic cholangiocarcinoma (ICC). Eight missense NBS1 mutations were identified in six of 64 (9.4%) HCCs and two of 18 (11.1%) ICCs, whereas only one synonymous mutation was found in 89 control cases of cirrhosis and chronic hepatitis B. Analysis of the functional consequences of the identified NBS1 mutations in Mre11-binding domain showed loss of nuclear localization of Nbs1 partner Mre11, one of the hallmarks for Nbs1 deficiency, in one HCC and two ICCs with NBS1 mutations. Moreover, seven of the eight tumors with NBS1 mutations had at least one genetic alteration in the TP53 pathway, including TP53 mutation, MDM2 amplification, p14ARF homozygous deletion and promoter methylation, implying a synergistic effect of Nbs1 disruption and p53 inactivation. Our findings provide novel insight on the molecular pathogenesis of primary liver cancer characterized by mutation inactivation of NBS1, a DNA repair associated gene.     

GNB3, eNOS,and Mitochondrial DNA Polymorphisms Correlate to Natural Longevity in a Xinjiang Uygur Population

Background

In centenarian populations, application of the positive biology approach (examination of positive phenotypes in aging) has revealed that mitochondrial DNA (mtDNA) mutation accumulation may be linked to human longevity; however, the role of guanine nucleotide-binding protein (G protein) abnormalities modulated by G-protein beta-3 (GNB3) and nitrate (NO2) production associated with endothelial nitric oxide synthase (eNOS), commonly appearing in age-related diseases, remains undetermined.

Objective

The association between the mtDNA 5178A/C, mtDNA 10398A/G, GNB3 C825T, and eNOS polymorphisms and longevity in a Uygur population (Xinjiang region, China) were investigated.

Methods

A total of 275 experimental subjects aged ≥100 or with 4 generations currently living were screened for inclusion in the centenarian (>100 years) and nonagenarian groups (90–100 years), and 112 65–70 year old control subjects were selected. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to examine mtDNA 5178A/C, mtDNA 10398A/G, GNB3 C825T, and eNOS. Associations between polymorphic loci, genotypes, and longevity were analyzed.

Results

165 included subjects (M∶F = 107∶58; mean age = 97±3 years; mean age 100–113 years) were assigned to the centenarian (M∶F = 46/19; n = 65) and nonagenarian groups (M∶F = 61/39; n = 100). Associations between mtDNA C5178A and A10398G polymorphisms with longevity in the centenarian group with mtDNA genotype frequencies 5178A and 10398G were 66.79% and 36.8%.

Conclusions

Applying the overwhelming longevity observed in Uygur populations, these findings demonstrate that mtDNA 5178A/C and 10398A/G, GNB3 C825T, and eNOS polymorphisms are useful as a genetic basis for longevity.     

Validity and Reliability of Using a Self-Lavaging Device for Cytology and HPV Testing for Cervical Cancer Screening: Findings from a Pilot Study

Self-sampling could increase cervical cancer screening uptake. While methods have been identified for human papillomavirus (HPV) testing, to date, self-sampling has not provided adequate specimens for cytology. We piloted the validity and reliability of using a self-lavaging device for cervical cytology and HPV testing. We enrolled 198 women in New York City in 2008–2009 from three ambulatory clinics where they received cervical cancer screening. All were asked to use the Delphi Screener™ to self-lavage 1–3 months after clinician-collected index cytological smear (100 normal; 98 abnormal). Women with abnormal cytology results from either specimen underwent colposcopy; 10 women with normal results from both specimens also underwent colposcopy. We calculated sensitivity of self-collected cytology to detect histologically confirmed high grade lesions (cervical intraepithelial neoplasia, CIN, 2+); specificity for histology-negative (CIN 1 or lower), paired cytology negative, or a third cytology negative; and kappa for paired results. One hundred and ninety-seven (99.5%) women self-collected a lavage. Seventy-five percent had moderate to excellent cellularity, two specimens were unsatisfactory for cytology. Seven of 167 (4%) women with definitive results had CIN2+; one had normal and six abnormal cytology results with the self-lavage (sensitivity = 86%, 95% Confidence Interval, CI: 42, 100). The kappa for paired cytology was low (0.36; 95% CI: 0.25, 0.47) primarily due to clinician specimens with atypical squamous cells of undetermined significance (ASC-US) and low grade squamous intraepithelial lesion (LSIL) coded as normal using Screener specimens. However, three cases of HSIL were coded as ASC-US and one as normal using Screener specimens. Seventy-three women had paired high-risk HPV tests with a kappa of 0.66 (95% CI: 0.49, 0.84). Based on these preliminary findings, a larger study to estimate the performance of the Screener for co-testing cytology and HPV or for HPV testing with cytology triage is warranted.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00702208","term_id":"NCT00702208"}}NCT00702208     

A Kalman-Filter Based Approach to Identification of Time-Varying Gene Regulatory Networks

Motivation

Conventional identification methods for gene regulatory networks (GRNs) have overwhelmingly adopted static topology models, which remains unchanged over time to represent the underlying molecular interactions of a biological system. However, GRNs are dynamic in response to physiological and environmental changes. Although there is a rich literature in modeling static or temporally invariant networks, how to systematically recover these temporally changing networks remains a major and significant pressing challenge. The purpose of this study is to suggest a two-step strategy that recovers time-varying GRNs.

Results

It is suggested in this paper to utilize a switching auto-regressive model to describe the dynamics of time-varying GRNs, and a two-step strategy is proposed to recover the structure of time-varying GRNs. In the first step, the change points are detected by a Kalman-filter based method. The observed time series are divided into several segments using these detection results; and each time series segment belonging to two successive demarcating change points is associated with an individual static regulatory network. In the second step, conditional network structure identification methods are used to reconstruct the topology for each time interval. This two-step strategy efficiently decouples the change point detection problem and the topology inference problem. Simulation results show that the proposed strategy can detect the change points precisely and recover each individual topology structure effectively. Moreover, computation results with the developmental data of Drosophila Melanogaster show that the proposed change point detection procedure is also able to work effectively in real world applications and the change point estimation accuracy exceeds other existing approaches, which means the suggested strategy may also be helpful in solving actual GRN reconstruction problem.     

Mesenchymal Stem Cell Transplantation Enhancement in Myocardial Infarction Rat Model under Ultrasound Combined with Nitric Oxide Microbubbles

Objective

This study evaluated the effects of ultrasound combined with the homemade nitric oxide (NO) micro-bubble destruction on the in vitro proliferation, apoptosis, and migration of mesenchymal stem cells (MSCs). Furthermore, we studied whether or not irradiation of the NO micro-bubble combined with bone-marrow derived MSC infusion had a better effect on treating myocardial infarction. The possible mechanism of MSC delivery into the infarcted myocardium was also investigated.

Methods

The murine bone marrow-derived MSCs were isolated, cultured, irradiated, and combined with different concentrations of NO microbubbles. MTT proliferation assay, annexin V-FITC apoptosis detection, migration assay, and RT-PCR were performed 24 h after the irradiation. The NO micro-bubbles was a intravenously injected, followed by the infusion of MSCs, which were labeled by CM-Dil. Myocardium was harvested 48 h later and the distribution of MSCs was observed by laser scanning confocal microscope after frozen sectioning. Echocardiography, histological examination, RT-PCR, and western blotting were performed four weeks after the cell transplantation.

Results

Ultrasound combined with 1:70 NO micro-bubbles had no significant impact on the proliferation or apoptosis of MSCs. Transwell chamber findings demonstrated that MSCs migrated more efficiently in group that underwent ultrasound combined with 1:70 NO micro-bubbles. The Real-time PCR results indicated that the expression of CXCR4 was much higher in the group undergoing ultrasound combined with 1:70 NO micro-bubbles. The normalized fluorescence intensity greatly increased in the group of US+NO micro-bubbles and the cardiac function was also markedly improved. Immunohistochemical staining showed that the capillary density was much greater in the group of US+NO micro-bubbles as compared to that of the other groups. RT-PCR and western blotting also revealed a higher SDF-1 and VEGF expression in the group of US+NO micro-bubbles.

Conclusions

NO micro-bubbles could be used in the cell transplantation, which efficiently promoted the MSC homing into the infarcted myocardium.     

Risk Factors for Poor Treatment Outcomes in Patients with MDR-TB and XDR-TB in China: Retrospective Multi-Center Investigation

Background

The treatment of patients with MDR- and XDR-TB is usually more complex, toxic and costly and less effective than treatment of other forms of TB. However, there is little information available on risk factors for poor outcomes in patients with MDR- and XDR-TB in China.

Methodology/Principal Findings

We retrospectively analyzed the clinical records of HIV-negative TB Patients with culture-proven MDR- or XDR-TB who were registered from July 2006 to June 2011 at five large-scale Tuberculosis Specialized Hospitals in China. Among 1662 HIV-seronegative TB cases which were culture-positive for M. tuberculosis complex and had positive sputum-smear microscopy results, 965 cases (58.1%) were DR-TB, and 586 cases (35.3%) were classified as having MDR-TB, accounting for 60.7% of DR-TB. 169 cases (10.2%) were XDR-TB, accounting for 17.5% of DR-TB, 28.8% of MDR-TB. The MDR-TB patients were divided into XDR-TB group (n=169) and other MDR-TB group (non-XDR MDR-TB) (n=417). In total, 240 patients (40.95%) had treatment success, and 346 (59.05%) had poor treatment outcomes. The treatment success rate in other MDR-TB group was 52.2%, significantly higher than that in the XDR-TB group (13%, P<0.001). In multivariate logistic regression analysis, poor outcomes were associated with duration of previous anti-TB treatment of more than one year (OR, 0.077; 95% CI, 0.011-0.499, P<0.001), a BMI less than 18.5 kg/m² (OR, 2.185; 95% CI, 1.372-3.478, P<0.001), XDR (OR, 13.368; 95% CI, 6.745-26.497, P<0.001), retreatment (OR, 0.171; 95% CI, 0.093-0.314, P<0.001), diabetes (OR, 0.305; 95% CI, 0.140-0.663, P=0.003), tumor (OR, 0.095; 95% CI, 0.011-0.795, P=0.03), decreased albumin (OR, 0.181; 95% CI, 0.118-0.295, P<0.001), cavitation (OR, 0.175; 95% CI, 0.108-0.286, P<0.001).

Conclusions/Significance

The patients with MDR-TB and XDR-TB have poor treatment outcomes in China.The presence of extensive drug resistance, low BMI, hypoalbuminemia, comorbidity, cavitary disease and previous anti-TB treatment are independent prognostic factors for poor outcome in patients with MDR-TB.     

Improvements of the Surgical Technique on the Established Mouse Model of Orthotopic Single Lung Transplantation

Background

A wide range of knockout and transgenic murine models for the study of nonimmune and immune mechanisms in lung transplants are available nowadays, but the microsurgical techniques are difficult to learn. We describe methods to simplify techniques and facilitate learning.

Methods

Traditional procedures were implemented to perform lung transplants in 30 cases (group 1). Improved techniques which included cuff without tail, broadening of the cuff diameter for bronchus, establishment of one tunnel between three structures, innovative technology of the vascular anastomosis and placement of the chest tube post-operation were used to perform lung transplants in 30 cases (group 2).

Results

The improved techniques considerably shorten operative times (96.75±6.16 min and 85.32±6.98 min in groups 1 and 2, respectively). The survival rates in the recipient animals were 86.7% and 96.7% in groups 1 and 2, respectively. Chest X-rays and macroscopic changes of transplanted recipients showed that grafts were well inflated on postoperative day 30. There was no significant difference of the arterial oxygen tension (PaO₂) between two groups (115.9±7.11 mm Hg and 116.3±6.87 mm Hg in groups 1 and 2, respectively). Histologically, no lung injury was seen in grafts.

Conclusions

We described the modified procedures of orthotopic left lung transplants in mice, which could shorten operative time and increase survival rate.