Structure of the C-terminal half of UvrC reveals an RNase H endonuclease domain with an Argonaute-like catalytic triad

Removal and repair of DNA damage by the nucleotide excision repair pathway requires two sequential incision reactions, which are achieved by the endonuclease UvrC in eubacteria. Here, we describe the crystal structure of the C-terminal half of UvrC, which contains the catalytic domain responsible for 5' incision and a helix-hairpin-helix-domain that is implicated in DNA binding. Surprisingly, the 5' catalytic domain shares structural homology with RNase H despite the lack of sequence homology and contains an uncommon DDH triad. The structure also reveals two highly conserved patches on the surface of the protein, which are not related to the active site. Mutations of residues in one of these patches led to the inability of the enzyme to bind DNA and severely compromised both incision reactions. Based on our results, we suggest a model of how UvrC forms a productive protein-DNA complex to excise the damage from DNA.     

Functions and oxidative stress status of leukocytes in patients with nephrotic syndrome

This study was conducted to establish the functions and oxidative stress status in leukocytes of adult patients with nephrotic syndrome. Thirty adult patients with nephrotic syndrome and 32 controls were included. Phagocytosis ability, the killing ability of the micro-organism phagosited of polymorphonuclear leukocytes (PMNL) and monocytes, along with oxidative stress parameters of PMNLs were assessed. There was no statistically significant difference in phagocytosis function of PMNLs and monocytes of patients when compared to those of controls. PMNL burst activities of the patient and control groups also showed no difference; however, the monocyte burst activities of patients were significant (p = 0.012). The glutathione peroxidase (GSH-Px) activities in PMNLs of the patients with nephrotic syndrome were significantly higher (p = 0.026) when compared to those of controls. In comparison with those of the control subjects, the patients had also higher selenium levels in their PMNLs (p < 0.001). Although PMNL malonyldialdehyde (MDA) levels of the patients seem to be higher than those of controls, the difference had no statistical significance (p = 0.071). Conclusively, in the patients with nephrotic syndrome, PMNLs appear to be exposed to an oxidative stress as indicated by their increased GSH-Px activities and selenium content. However, PMNLs in nephrotic syndrome patients seem to be coping with the insulting oxidative stress, as suggested by their near-normal MDA productions. Furthermore, these data suggest that nephrotic syndrome appears not to have an influence on phagocytosis and killing abilities of granulocytes and monocytes as long as these cells can overcome the oxidative stress to which they are exposed in this disease.     

Biodegradation kinetics of 2,4-dichlorophenol by acclimated mixed cultures

The biodegradation kinetics of 2,4-dichlorophenol (2,4-DCP) by culture (Culture M) acclimated to mixture of 4-chlorophenol (4-CP) and 2,4-DCP and the culture (Culture 4) acclimated to 4-CP only were investigated in aerobic batch reactors. Also, pure strains isolated from mixed cultures were searched for their ability towards the biodegradation of 2,4-DCP. Culture 4 was able to completely degrade 2,4-DCP up to 80 mg/L within 30 h and removal efficiency dropped to 21% upon increasing initial concentration to 108.8 mg/L. When the Culture M was used, complete degradation of 2,4-DCP in the range of 12.5-104.4 mg/L was attained. A linear relationship between time required for complete degradation and initial 2,4-DCP concentrations was observed for both mixed cultures. It was observed that the Haldane equation can be used to predict specific degradation rate (SDR) (R(2)>0.99) as a function of initial 2,4-DCP concentrations and it adequately describes 2,4-DCP concentration profiles. Both of the mixed cultures settled well, which is important to maintain good removal efficiency for longer periods of time for real full-scale applications. Although the pure strains isolated from mixed cultures were found to have higher SDR of 2,4-DCP compared to mixed cultures, they did not settle well under quiescent conditions.     

Akt Links Insulin Signaling to Albumin Endocytosis in Proximal Tubule Epithelial Cells

Diabetes mellitus (DM) has become an epidemic, causing a significant decline in quality of life of individuals due to its multisystem involvement. Kidney is an important target organ in DM accounting for the majority of patients requiring renal replacement therapy at dialysis units. Microalbuminuria (MA) has been a valuable tool to predict end-organ damage in DM but its low sensitivity has driven research efforts to seek other alternatives. Albumin is taken up by albumin receptors, megalin and cubilin in the proximal tubule epithelial cells. We demonstrated that insulin at physiological concentrations induce albumin endocytosis through activation of protein kinase B (Akt) in proximal tubule epithelial cells. Inhibition of Akt by a phosphorylation deficient construct abrogated insulin induced albumin endocytosis suggesting a role for Akt in insulin-induced albumin endocytosis. Furthermore we demonstrated a novel interaction between Akt substrate 160kDa (AS160) and cytoplasmic tail of megalin. Mice with type 1 DM (T1D) displayed decreased Akt, megalin, cubilin and AS160 expression in their kidneys in association with urinary cubilin shedding preceding significant MA. Patients with T1D who have developed MA in the EDC (The Pittsburgh Epidemiology of Diabetes Complications) study demonstrated urinary cubilin shedding prior to development of MA. We hypothesize that perturbed insulin-Akt cascade in DM leads to alterations in trafficking of megalin and cubilin, which results in urinary cubilin shedding as a prelude to MA in early diabetic nephropathy. We propose that utilization of urinary cubilin shedding, as a urinary biomarker, will allow us to detect and intervene in diabetic nephropathy (DN) at an earlier stage.     

Lack of a time-dependent effect of melatonin on radiation-induced apoptosis in cultured rat lymphocytes

Ionizing radiation is widely used for the treatment of solid tumors and it is thought to act by directly targeting tumor clonogens, also known as stem cells. Apoptosis is a genetically programmed mechanism of cell death often characterized by internucleosomal DNA cleavage. Although it has been previously shown that lymphocytes readily undergo apoptosis in patients receiving anticancer drugs or treatment with ionizing radiation, this is the first study to investigate the influence of radiotherapy and melatonin on apoptosis in rat lymphocytes at two different times of the day. Melatonin, a free radical scavenger, is an endogenous neurohormone predominantly synthesized in and secreted by the pineal gland. It has been shown that melatonin inhibits apoptosis in normal cells but it increases the rate of apoptosis in various cancer cells. Therefore, in the present study, the effect of melatonin on apoptosis in cultured lymphocytes was studied after total body irradiation (TBI) was given to rats in the morning (1 HALO) or evening (13 HALO) with morphological and DNA fragmentation analysis. Two-way analysis of variance (ANOVA) revealed that radiation increased the rate of apoptosis in rat lymphocytes after TBI, and melatonin treatment did not reduce the rate of apoptosis after TBI at either time point. We conclude that the lack of an effect of melatonin on the apoptosis rate in rat lymphocytes might be due to the dose-dependent effect of melatonin, the time course of apoptosis investigated, or the cell type in which apoptosis was examined.     

Stobadine inhibits doxorubicin-induced apoptosis through a caspase-9 dependent pathway in P815 mastocytoma cells

Doxorubicin (DOXO), a widely used chemotherapeutic agent, induces apoptosis in transformed and non-transformed cells. The apoptotic effect of DOXO has been linked to the generation of reactive oxygen species (ROS). Antioxidants may be effective in the prevention of DOX-induced apoptosis. In the present study we investigated the effects of stobadine, a pyridoindole antioxidant in a DOXO-induced apoptosis model of P815 cells by flow cytometric analyses and by measuring caspase-3 and caspase-9 activities. Pretreating cells with stobadine significantly increased cell viability and decreased apoptosis rate. Inhibition in apoptosis was observed at maximum levels following treatment of cells with 10(-7)M stobadine as evident from flow cytometric analyses. The antiapoptotic effect of stobadine was further confirmed by inhibition of caspase-3 and caspase-9 activities. We found that the antioxidative effects of stobadine were comparable to the effects of a well known antioxidant, N-acetyl l-cysteine (NAC).     

LX-8000R and UriSed 2 fully automated urine analyzers comparison to manual microscopic examination

BackgroundUrinalysis has an important place in evaluating kidney and urinary tract infections. Automated urine analyzers enhance productivity and turnover in laboratories and economize time and labor required for analysis. In the present study, we evaluated and compared analytic and diagnostic performance of UriSed2 with LX-8000R, which is a novel image-based automated urine sediment analyzer.MethodsA total of 178 urine samples sent to our laboratory were evaluated by the two urine analyzers and standard manual microscopy. Precision and comparison studies were done in accordance with CLSI guidelines.ResultsSensitivity assessment revealed similar outcomes with both UriSed2 and LX-8000R devices for erythrocyte count (RBC), whereas UriSed2 device yielded higher outcomes for leukocyte count (WBC) and epithelial cells (EPI) than LX-8000R analyzer. Specificity of UriSed2 for WBC and RBC was higher than that of LX-8000R device. According to Gamma statistics, both urine analyzers showed perfect consistency for WBC, RBC and EPI cell counts. Manuel microscopy revealed statistically significant correlation between LX-8000R and UriSed2 in terms of WBC and RBC. Manual evaluation by Bland-Altman analysis demonstrated lower WBC and RBC values and higher EPI as compared to both UriSed2 and LX-8000R devices. As the result of Passing-Bablok regression analysis, both devices were found to be inconsistent with manual microscopy.ConclusionsWe think that evaluation of automated urine analyzers will be more meaningful when they are evaluated together with urine samples and patient clinical findings in addition to comparing with manual microscopy.     

Identifying the Volcanic Eruption Depicted in a Neolithic Painting at ?atalh?yük,Central Anatolia,Turkey

A mural excavated at the Neolithic Çatalhöyük site (Central Anatolia, Turkey) has been interpreted as the oldest known map. Dating to ∼6600 BCE, it putatively depicts an explosive summit eruption of the Hasan Dağı twin-peaks volcano located ∼130 km northeast of Çatalhöyük, and a birds-eye view of a town plan in the foreground. This interpretation, however, has remained controversial not least because independent evidence for a contemporaneous explosive volcanic eruption of Hasan Dağı has been lacking. Here, we document the presence of andesitic pumice veneer on the summit of Hasan Dağı, which we dated using (U-Th)/He zircon geochronology. The (U-Th)/He zircon eruption age of 8.97±0.64 ka (or 6960±640 BCE; uncertainties 2σ) overlaps closely with ¹⁴C ages for cultural strata at Çatalhöyük, including level VII containing the “map” mural. A second pumice sample from a surficial deposit near the base of Hasan Dağı records an older explosive eruption at 28.9±1.5 ka. U-Th zircon crystallization ages in both samples range from near-eruption to secular equilibrium (>380 ka). Collectively, our results reveal protracted intrusive activity at Hasan Dağı punctuated by explosive venting, and provide the first radiometric ages for a Holocene explosive eruption which was most likely witnessed by humans in the area. Geologic and geochronologic lines of evidence thus support previous interpretations that residents of Çatalhöyük artistically represented an explosive eruption of Hasan Dağı volcano. The magmatic longevity recorded by quasi-continuous zircon crystallization coupled with new evidence for late-Pleistocene and Holocene explosive eruptions implicates Hasan Dağı as a potential volcanic hazard.     

The effects of NOS3 Glu298Asp variant on colorectal cancer risk and progression in Turkish population

Endothelial nitric oxide synthase (eNOS), coded by the gene NOS3, may play an important role in uncontrollable cellular growth in several cancer types. Our study was performed to test the association between Glu298Asp polymorphisms in the NOS3 gene and colorectal cancer risk and progression. In this study, NOS3 Glu298Asp polymorphism was genotyped in 84 patients with colorectal cancer and 99 healthy subjects using polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) analysis. There were significant differences in the distribution of NOS3 genotypes and frequencies of the alleles between colorectal cancer patients and controls (P = 0.016, P = 0.006, respectively). The increased frequency of NOS3 Glu298Asp homozygotes genotypes in patients who had advanced tumour stage was statistically significant (P = 0.042). Our findings have suggested that NOS3 Glu298Asp polymorphism might be associated with the risk and progression of colorectal cancer in Turkish population.     

Standard abdominal wound edge protection with surgical dressings vs coverage with a sterile circular polyethylene drape for prevention of surgical site infections (BaFO): study protocol for a randomized controlled trial

ABSTRACT: BACKGROUND: Postoperative surgical site infections cause substantial morbidity, prolonged hospitalization, costs and even mortality and remain one of the most frequent surgical complications. Approximately 14% to 30% of all patients undergoing elective open abdominal surgery are affected and methods to reduce surgical site infection rates warrant further investigation and evaluation in randomized controlled trials. METHODS: To investigate whether the application of a circular plastic wound protector reduces the rate of surgical site infections in general and visceral surgical patients that undergo midline or transverse laparotomy by 50%. BaFO is a randomized, controlled, patient-blinded and observer-blinded multicenter clinical trial with two parallel surgical groups. The primary outcome measure will be the rate of surgical site infections within 45 days postoperative assessed according to the definition of the Center for Disease Control. Statistical analysis of the primary endpoint will be based on the intention-to-treat population. The global level of significance is set at 5% (2 sided) and sample size (n = 258 per group) is determined to assure a power of 80% with a planned interim analysis for the primary endpoint after the inclusion of 340 patients. DISCUSSION: The BaFO trial will explore if the rate of surgical site infections can be reduced by a single, simple, inexpensive intervention in patients undergoing open elective abdominal surgery. Its pragmatic design guarantees high external validity and clinical relevance. Trial registration http://www.clinicaltrials.gov NCT01181206. Date of registration: 11 August 2010; date of first patient randomized: 8 September 2010.