Manipulation of cell cycle progression can counteract the apparent loss of correction frequency following oligonucleotide-directed gene repair

BACKGROUND: Single-stranded oligonucleotides (ssODN) are used routinely to direct specific base alterations within mammalian genomes that result in the restoration of a functional gene. Despite success with the technique, recent studies have revealed that following repair events, correction frequencies decrease as a function of time, possibly due to a sustained activation of damage response signals in corrected cells that lead to a selective stalling. In this study, we use thymidine to slow down the replication rate to enhance repair frequency and to maintain substantial levels of correction over time. RESULTS: First, we utilized thymidine to arrest cells in G1 and released the cells into S phase, at which point specific ssODNs direct the highest level of correction. Next, we devised a protocol in which cells are maintained in thymidine following the repair reaction, in which the replication is slowed in both corrected and non-corrected cells and the initial correction frequency is retained. We also present evidence that cells enter a senescence state upon prolonged treatment with thymidine but this passage can be avoided by removing thymidine at 48 hours. CONCLUSION: Taken together, we believe that thymidine may be used in a therapeutic fashion to enable the maintenance of high levels of treated cells bearing repaired genes

Applying spatial regression to evaluate risk factors for microbiological contamination of urban groundwater sources in Juba, South Sudan

This study developed methodology for statistically assessing groundwater contamination mechanisms. It focused on microbial water pollution in low-income regions. Risk factors for faecal contamination of groundwater-fed drinking-water sources were evaluated in a case study in Juba, South Sudan. The study was based on counts of thermotolerant coliforms in water samples from 129 sources, collected by the humanitarian aid organisation M,decins Sans FrontiSres in 2010. The factors included hydrogeological settings, land use and socio-economic characteristics. The results showed that the residuals of a conventional probit regression model had a significant positive spatial autocorrelation (Moran\u27s I = 3.05, I-stat = 9.28); therefore, a spatial model was developed that had better goodness-of-fit to the observations. The most significant factor in this model (p-value 0.005) was the distance from a water source to the nearest Tukul area, an area with informal settlements that lack sanitation services. It is thus recommended that future remediation and monitoring efforts in the city be concentrated in such low-income regions. The spatial model differed from the conventional approach: in contrast with the latter case, lowland topography was not significant at the 5% level, as the p-value was 0.074 in the spatial model and 0.040 in the traditional model. This study showed that statistical risk-factor assessments of groundwater contamination need to consider spatial interactions when the water sources are located close to each other. Future studies might further investigate the cut-off distance that reflects spatial autocorrelation. Particularly, these results advise research on urban groundwater quality

Circulating alpha1-antitrypsin in the general population: Determinants and association with lung function

BACKGROUND: Severe alpha1-antitrypsin (AAT) deficiency associated with low AAT blood concentrations is an established genetic COPD risk factor. Less is known about the respiratory health impact of variation in AAT serum concentrations in the general population. We cross-sectionally investigated correlates of circulating AAT concentrations and its association with FEV1. METHODS: In 5187 adults (2669 females) with high-sensitive c-reactive protein (CRP) levels < or = 10 mg/l from the population-based Swiss SAPALDIA cohort, blood was collected at the time of follow-up examination for measuring serum AAT and CRP. RESULTS: Female gender, hormone intake, systolic blood pressure, age in men and in postmenopausal women, as well as active and passive smoking were positively, whereas alcohol intake and BMI inversely correlated with serum AAT levels, independent of CRP adjustment. We observed an inverse association of AAT with FEV1 in the total study population (p < 0.001), that disappeared after adjustment for CRP (p = 0.28). In addition, the AAT and FEV1 association was modified by gender, menopausal status in women, and smoking. CONCLUSION: The results of this population-based study reflect a complex interrelationship between tobacco exposure, gender related factors, circulating AAT, systemic inflammatory status and lung function

Immunoblot analysis of the seroreactivity to recombinant Borrelia burgdorferi sensu lato antigens, including VlsE, in the long-term course of treated patients with Erythema migrans

Objective: We evaluated whether immunoblotting is capable of substantiating the posttreatment clinical assessment of patients with erythema migrans ( EM), the hallmark of early Lyme borreliosis. Methods: In 50 patients, seroreactivity to different antigens of Borrelia burgdorferi sensu lato was analyzed by a recombinant immunoblot test (IB) in consecutive serum samples from a minimum follow-up period of 1 year. Antigens in the IgG test were decorin- binding protein A, internal fragment of p41 (p41i), outer surface protein C (OspC), p39, variable major protein-like sequence expressed (VlsE), p58 and p100; those in the IgM test were p41i, OspC and p39. Immune responses were correlated with clinical and treatment-related parameters. Results: Positive IB results were found in 50% before, in 57% directly after therapy and in 44% by the end of the follow-up for the IgG class, and in 36, 43 and 12% for the IgM class. In acute and convalescence phase sera, VlsE was most immunogenic on IgG testing 60 and 70%), and p41i (46 and 57%) and OspC (40 and 57%) for the IgM class. By the end of the follow-up, only the anti-p41i lgM response was significantly decreased to 24%. Conclusions: No correlation was found between IB results and treatment-related parameters. Thus, immunoblotting does not add to the clinical assessment of EM patients after treatment. Copyright (c) 2008 S. Karger AG, Basel

Relationship of circulating hyaluronic Acid levels to disease control in asthma and asthmatic pregnancy.

Uncontrolled asthma is a risk factor for pregnancy-related complications. Hyaluronic acid (HA), a potential peripheral blood marker of tissue fibrosis in various diseases, promotes eosinophil survival and plays a role in asthmatic airway inflammation as well as in physiological processes necessary to maintain normal pregnancy; however the level of circulating HA in asthma and asthmatic pregnancy is unknown. We investigated HA levels in asthmatic patients (N = 52; asthmatic pregnant (AP) N = 16; asthmatic non-pregnant (ANP) N = 36) and tested their relationship to asthma control. Serum HA level was lower in AP than in ANP patients (27 [24.7-31.55] vs. 37.4 [30.1-66.55] ng/mL, p = 0.006); the difference attenuated to a trend after its adjustment for patients' age (p = 0.056). HA levels and airway resistance were positively (r = 0.467, p = 0.004), HA levels and Asthma Control Test (ACT) total score inversely (r = -0.437, p = 0.01) associated in ANP patients; these relationships remained significant even after their adjustments for age. The potential value of HA in the determination of asthma control was analyzed using ROC analysis which revealed that HA values discriminate patients with ACT total score >/=20 (controlled patients) and <20 (uncontrolled patients) with a 0.826 efficacy (AUC, 95% CI: 0.69-0.97, p = 0.001) when 37.4 ng/mL is used as cut-off value in ANP group, and with 0.78 efficacy (AUC, 95% CI: 0.65-0.92, p = 0.0009) in the whole asthmatic cohort. In conclusion circulating HA might be a marker of asthma control, as it correlates with airway resistance and has good sensitivity in the detection of impaired asthma control. Decrease of HA level in pregnancy may be the consequence of pregnancy induced immune tolerance

Biologic markers of risk in nipple aspirate fluid are associated with residual cancer and tumour size

We previously demonstrated that nipple aspirate fluid (NAF) can be obtained from virtually all non-Asian women between the ages of 30 and 72. The focus of this report is to (1) determine the association of candidate markers of breast cancer risk in NAF obtained from fresh mastectomy specimens with residual breast carcinoma, and (2) evaluate the association of the markers with breast tumour progression. Nipple aspiration was performed on 97 specimens. Cytology, DNA index (including % hypertetraploid cells), cell cycle parameters (S phase fraction, % cells in G2/M), prostate-specific antigen (PSA), epidermal growth factor (EGF), testosterone, carcinoembryonic antigen (CEA) and prostaglandin D synthase (PGDS) were evaluated in NAF for their association with (1) residual ductal carcinoma in situ (DCIS) or invasive cancer, and (2) pathologic tumour size. NAF was obtained from 99% (96/97) of specimens. Atypical and malignant NAF cytology were significantly associated with residual DCIS or invasive cancer (P = 0.001) and with larger tumours (P = 0.004). One hundred per cent and 88% of subjects with malignant and atypical NAF cytology, respectively, had residual carcinoma. The percentage of cells in G2/M and DNA index were associated both with risk of residual carcinoma (P = 0.01 for each) and larger tumour size (DNA index, P = 0.03; G2/M, P = 0.05), although neither biomarker improved the ability of NAF cytology, to predict residual breast cancer. Higher DNA index was associated with atypical cytology (P = 0.0001). In summary, atypical and malignant NAF cytology are associated with larger tumour size, and are highly predictive of residual carcinoma after needle or excisional biopsy of the breast. © 1999 Cancer Research Campaig

Transcription Initiation Patterns Indicate Divergent Strategies for Gene Regulation at the Chromatin Level

The application of deep sequencing to map 5′ capped transcripts has confirmed the existence of at least two distinct promoter classes in metazoans: “focused” promoters with transcription start sites (TSSs) that occur in a narrowly defined genomic span and “dispersed” promoters with TSSs that are spread over a larger window. Previous studies have explored the presence of genomic features, such as CpG islands and sequence motifs, in these promoter classes, but virtually no studies have directly investigated the relationship with chromatin features. Here, we show that promoter classes are significantly differentiated by nucleosome organization and chromatin structure. Dispersed promoters display higher associations with well-positioned nucleosomes downstream of the TSS and a more clearly defined nucleosome free region upstream, while focused promoters have a less organized nucleosome structure, yet higher presence of RNA polymerase II. These differences extend to histone variants (H2A.Z) and marks (H3K4 methylation), as well as insulator binding (such as CTCF), independent of the expression levels of affected genes. Notably, differences are conserved across mammals and flies, and they provide for a clearer separation of promoter architectures than the presence and absence of CpG islands or the occurrence of stalled RNA polymerase. Computational models support the stronger contribution of chromatin features to the definition of dispersed promoters compared to focused start sites. Our results show that promoter classes defined from 5′ capped transcripts not only reflect differences in the initiation process at the core promoter but also are indicative of divergent transcriptional programs established within gene-proximal nucleosome organization

Immune system deregulation in hypertensive patients chronically RAS suppressed developing albuminuria

Albuminuria development in hypertensive patients is an indicator of higher cardiovascular (CV) risk and renal damage. Chronic renin-angiotensin system (RAS) suppression facilitates blood pressure control but it does not prevent from albuminuria development. We pursued the identification of protein indicators in urine behind albuminuria development in hypertensive patients under RAS suppression. Urine was collected from 100 patients classified in three groups according to albuminuria development: (a) patients with persistent normoalbuminuria; (b) patients developing de novo albuminuria; (c) patients with maintained albuminuria. Quantitative analysis was performed in a first discovery cohort by isobaric labeling methodology. Alterations of proteins of interest were confirmed by target mass spectrometry analysis in an independent cohort. A total of 2416 proteins and 1223 functional categories (coordinated protein responses) were identified. Immune response, adhesion of immune and blood cells, and phagocytosis were found significantly altered in patients with albuminuria compared to normoalbuminuric individuals. The complement system C3 increases, while Annexin A1, CD44, S100A8 and S100A9 proteins showed significant diminishment in their urinary levels when albuminuria is present. This study reveals specific links between immune response and controlled hypertension in patients who develop albuminuria, pointing to potential protein targets for novel and future therapeutic interventions.Sin financiación4.122 JCR (2017) Q1, 12/64 Multidisciplinary Sciences0.809 SJR (2017) Q2, 4/10 OptometryNo data IDR 2017UE