CHIP-mediated degradation and DNA damage-dependent stabilization regulate base excision repair proteins.

Base excision repair (BER) is the major pathway for processing of simple lesions in DNA, including single-strand breaks, base damage, and base loss. The scaffold protein XRCC1, DNA polymerase beta, and DNA ligase IIIalpha play pivotal roles in BER. Although all these enzymes are essential for development, their cellular levels must be tightly regulated because increased amounts of BER enzymes lead to elevated mutagenesis and genetic instability and are frequently found in cancer cells. Here we report that BER enzyme levels are linked to and controlled by the level of DNA lesions. We demonstrate that stability of BER enzymes increases after formation of a repair complex on damaged DNA and that proteins not involved in a repair complex are ubiquitylated by the E3 ubiquitin ligase CHIP and subsequently rapidly degraded. These data identify a molecular mechanism controlling cellular levels of BER enzymes and correspondingly the efficiency and capacity of BER.

Safety of supervised trainee-performed laparoscopic surgery for inflammatory bowel disease.

PURPOSE: There is a significant risk of surgical resection during the lifetime of an inflammatory bowel disease (IBD) patient: laparoscopic surgery has been increasingly applied to the management of IBD with short and long-term advantages. The aim of this study is to demonstrate that laparoscopic surgery for IBD, performed by a surgical trainee under the supervision of an experienced trainer, is feasible and safe. METHODS: All surgical procedures were sub-divided in six critical steps in order to define the procedure as supervised trainee performed (STP) when the trainer was present unscrubbed in the theatre or assisting and trainer performed (TNER) when the trainer performed two or more critical steps of the procedure. Included were all patients undergoing laparoscopic resection for IBD between January 2009 and December 2013. Thirty-day mortality and morbidity were the primary outcomes. Reoperations and rehospitalizations within 30 days of discharge were recorded prospectively and were the secondary outcomes together with conversion rate and length of hospital stay. RESULTS: One hundred fifty-one patients were included: 77 (50.99%) STP and 74 (49.01%) TNER. No deaths occurred, and 30-day morbidity was 27.15% with no differences between the groups. Operating time was longer in the STP (166.6 ± 53.31 vs 130.4 ± 49.15). Five patients (2 vs 3) required reoperation (3.31%), while 13 patients (8.6%) required readmission. CONCLUSIONS: Laparoscopic surgery for IBD performed by a supervised trainee is safe compared to trainers performed procedures despite a longer operating time. Randomized clinical trials are needed to confirm these preliminary results and to investigate long-term outcomes.

Ubiquitin ligase ARF-BP1/Mule modulates base excision repair.

Base excision repair (BER) is the major cellular pathway involved in removal of endogenous/spontaneous DNA lesions. Here, we study the mechanism that controls the steady-state levels of BER enzymes in human cells. By fractionating human cell extract, we purified the E3 ubiquitin ligase Mule (ARF-BP1/HectH9) as an enzyme that can ubiquitylate DNA polymerase beta (Pol beta), the major BER DNA polymerase. We identified lysines 41, 61 and 81 as the major sites of modification and show that replacement of these lysines to arginines leads to increased protein stability. We further show that the cellular levels of Pol beta and its ubiquitylated derivative are modulated by Mule and ARF and siRNA knockdown of Mule leads to accumulation of Pol beta and increased DNA repair. Our findings provide a novel mechanism regulating steady-state levels of BER proteins.

Abdominal wall reconstruction using biosynthetic absorbable mesh in high-risk complex ventral hernia.

BACKGROUND: Biosynthetic mesh may represent an improvement on biological and large pore synthetic meshes for high-risk complex ventral hernia repair. This study aimed to evaluate the performance of polyglycolic acid (PGA):trimethylene carbonate (TMC) biosynthetic mesh for reinforcement of the midline fascial closure in a single-stage repair of complex ventral hernias in high-risk patients. METHODS: A retrospective review was undertaken for patients who underwent a planned open single-stage complex ventral hernia repair with a single unit of PGA:TMC biosynthetic mesh between May 2013 and August 2017. Data on outcome variables were recorded and quality of life assessed using the Short Form-12 (SF-12) instrument. RESULTS: Overall, 56 patients underwent abdominal wall reconstruction for complex ventral hernias. All meshes were placed in the retrorectus position. Some 39% underwent component separation. The majority of patients (86%, n = 48) had high risk (grade 2 or 3) hernias according to the Ventral Hernia Working Group classification. Overall hernia recurrence rate was 3.6% (n = 2). Postoperative surgical site infection occurred in 26.8% (n = 15). Median follow-up by clinical examination was 6 months (range 4–17). Median telephone follow-up was 21 months (range 4–54). Pre- and post-treatment SF-12 quality of life assessments demonstrated significant improvements in both the physical and mental components. CONCLUSION: This study reports a large series of abdominal wall reconstructions using biosynthetic mesh in complex ventral hernia. The findings indicate promising early outcome data associated with use of biosynthetic mesh. Larger well-controlled studies with longer follow-up are needed for confirmation of these findings.

Meta-analysis and trial sequential analysis of three-port vs four-port laparoscopic cholecystectomy (level 1 evidence).

To compare the outcomes of three-port and four-port laparoscopic cholecystectomy. In compliance with PRISMA statement standards, electronic databases were searched to identify all comparative studies investigating outcomes of three-port vs four-port laparoscopic cholecystectomy. Two techniques were compared using direct comparison meta-analysis model. The risks of type 1 or type 2 error in the meta-analysis model were assessed using trial sequential analysis model. The certainty of evidence was assessed using GRADE system. Random effects modelling was applied to calculate pooled outcome data. Analysis of 2524 patients from 17 studies showed that both techniques were comparable in terms of operative time (MD:- 0.13, P = 0.88), conversion to open operation (OR:0.80, P = 0.43), gallbladder perforation (OR: 1.43, P = 0.13), bleeding from gallbladder bed (OR:0.81, P = 0.34), bile duct injury (RD: 0.00, P = 0.97), iatrogenic visceral injury (RD: - 0.00, P = 0.81), bile or stone spillage (OR:1.67, P = 0.08), port site infection (OR: 0.90, P = 0.76), port site hernia (RD: 0.00, P = 0.89), port site haematoma (RD: - 0.01, P = 0.23), port site seroma (RD: 0.00, P = 1.00), and need for reoperation (RD: - 0.00, P = 0.94). However, the three-port technique was associated with lower VAS pain score at 12 h (MD: - 0.66, P < 0.00001) and 24 h (MD: - 0.54, P < 0.00001) postoperatively, shorter length of hospital stay (MD:-0.09, P = 0.41), and shorter time to return to normal activities (MD: - 0.79, P = 0.02). Trial sequential analysis confirmed that the meta-analysis was conclusive with no significant risks of type 1 or type 2 error. Robust evidence (level 1 with high certainty) suggests that in an elective setting with uncomplicated cholelithiasis as indication for cholecystectomy, three-port laparoscopic cholecystectomy is comparable with the four-port technique in terms of procedural and morbidity outcomes and may be associated with less postoperative pain, shorter length of hospital stay and shorter time to return to normal activities.

Effect of aprotinin on renal dysfunction in patients undergoing on-pump and off-pump cardiac surgery: a retrospective observational study.

BACKGROUND: Aprotinin is used during cardiac surgery for its blood-saving effects. However, reports suggest a possible association between use of this drug and increased renal dysfunction and mortality. We investigated the effect of aprotinin on renal dysfunction in cardiac surgery, considering the cofactors on-pump versus off-pump surgery and co-medication with angiotensin-converting enzyme (ACE) inhibitors. METHODS: Our analysis included 9875 patients undergoing on-pump or off-pump cardiac surgery from Jan 1, 2000, to Sept 30, 2007. Of these patients, 9106 were included in the retrospective observational study analysis. With propensity-adjusted, multivariate staged logistic regression, we analysed separately the incidence of renal dysfunction in patients receiving aprotinin, tranexamic acid, or no antifibrinolytic treatment in the presence or absence of preoperative ACE inhibitor treatment, for both on-pump and off-pump surgical techniques. FINDINGS: In 5434 patients undergoing on-pump cardiac surgery, the odds ratio (OR) between aprotinin and an increased risk of renal dysfunction without ACE inhibitor was 1.81 (95% CI 0.79-4.13, p=0.162) and with ACE inhibitor 1.73 (0.56-5.32, p=0.342). In the 848 patients taking ACE inhibitors and undergoing off-pump cardiac surgery, aprotinin was associated with a greater than two-fold increase in the risk of renal dysfunction after off-pump cardiac surgery (OR 2.87 [1.25-6.58], p=0.013). INTERPRETATION: Our results have shown that aprotinin seems to be safe during on-pump cardiac surgery. However, the combination of aprotinin and ACE inhibitors during off-pump cardiac surgery is associated with a significant risk of postoperative renal dysfunction.

Multistage selection strategies: simulating the effects on adverse impact and expected performance for various predictor combinations.

Examination of the trade-off between mean performance and adverse impact has received empirical attention for single-stage selection strategies; however, research for multistage selection strategies is almost nonexistent. The authors used Monte Carlo simulation to explore the trade-off between expected mean performance and minority hiring in multistage selection strategies and to identify those strategies most effective in balancing the trade-off. In total, 43 different multistage selection strategies were modeled; they reflected combinations of predictors with a wide range of validity, subgroup differences, and predictor intercorrelations. These selection models were examined across a variety of net and stage-specific selection ratios. Though it was still the case that an increase in minority hiring was associated with a decrease in predicted performance for many scenarios, the current results revealed that certain multistage strategies are much more effective than others for managing the performance and adverse impact trade-offs. The current study identified several multistage strategies that are clearly more desirable than those strategies previously suggested in the literature for practitioners who seek a practical selection system that will yield a high-performing and highly representative workforce.

Work stressors, role-based performance, and the moderating influence of organizational support.

As a test of the 2-dimensional model of work stressors, the present study proposed differential relationships between challenge stressors and hindrance stressors and role-based performance, which were expected to be moderated by organizational support. In a sample of 215 employees across 61 offices of a state agency, the authors obtained a positive relationship between challenge stressors and role-based performance and a negative relationship between hindrance stressors and role-based performance. In addition, organizational support moderated the relationship between challenge stressors and role-based performance but did not moderate the relationship between hindrance stressors and role-based performance. This suggests that organizations would benefit from increasing challenges in the workplace as long as they are supportive of employees and removing hindrances. Further implications for organizational theory and practice are discussed. (PsycINFO Database Record (c) 2009 APA, all rights reserved).

Mapping and conformational characterization of the DNA-binding region of the breast cancer susceptibility protein BRCA1.

The breast cancer susceptibility gene, BRCA1, encodes a large nuclear phosphoprotein, the major isoform of which is 1863 amino acids in size. Structure-function studies have been largely restricted to the only two domains identified by homology searches: the RING (really interesting new gene) and BRCT (BRCA1 C-terminus) domains. However, we have recently reported the identification of a large central soluble region of BRCA1 (residues 230-534) that binds specifically to four-way junction DNA, a property that potentially facilitates its role in the repair of DNA lesions by homologous recombination. We have now used a combination of limited proteolysis and extension cloning to identify more accurately the DNA-binding region of BRCA1. Limited trypsinolysis of BRCA1-(230-534) resulted in the production of a soluble domain identified as residues 230-339. However, after cloning, expression and purification of this region, studies revealed that it was unable to bind to four-way junctions, suggesting that the DNA-binding activity, in part, resides within residues 340-534. A series of fragments extending from residue 340 were produced, and each was tested for its ability to bind to four-way junction DNA in gel retardation assays. In these experiments, residues 340-554 of BRCA1 were identified as the minimal DNA-binding region. We then went on to characterize the conformation of this region using CD spectroscopy and analytical centrifugation.

Tragedy of the commons in the chemostat.

We present a proof of principle for the phenomenon of the tragedy of the commons that is at the center of many theories on the evolution of cooperation. Whereas the tragedy is commonly set in a game theoretical context, and attributed to an underlying Prisoner's Dilemma, we take an alternative approach based on basic mechanistic principles of species growth that does not rely on the specification of payoffs which may be difficult to determine in practice. We establish the tragedy in the context of a general chemostat model with two species, the cooperator and the cheater. Both species have the same growth rate function and yield constant, but the cooperator allocates a portion of the nutrient uptake towards the production of a public good -the "Commons" in the Tragedy- which is needed to digest the externally supplied nutrient. The cheater on the other hand does not produce this enzyme, and allocates all nutrient uptake towards its own growth. We prove that when the cheater is present initially, both the cooperator and the cheater will eventually go extinct, hereby confirming the occurrence of the tragedy. We also show that without the cheater, the cooperator can survive indefinitely, provided that at least a low level of public good or processed nutrient is available initially. Our results provide a predictive framework for the analysis of cooperator-cheater dynamics in a powerful model system of experimental evolution.

Update on late relapse of germ cell tumor: a clinical and molecular analysis.

PURPOSE: Analysis of patients with late relapse (LR) of germ cell tumor (GCT) with reports on clinical characteristics, outcomes, and molecular and cytogenetic features. PATIENTS AND METHODS: Eighty-three patients evaluated at Indiana University from 1993 through 2000 for relapse of GCT more than 2 years from initial therapy were reviewed. Available specimens were investigated for expression of the transcription regulator FoxD3 and apurinic/apyrimidinic endonuclease and the presence of chromosome 12 abnormalities. RESULTS: Median interval from initial presentation to LR was 85 months. Forty-three of 49 LR patients who underwent surgery were rendered disease free (NED), and 20 (46.5%) remain continuously NED. Thirty-two patients received chemotherapy, but only six (18.8%) obtained a complete remission. Five of these patients remain continuously NED after chemotherapy alone, including three who were chemotherapy naïve. Eighteen of these 32 patients were successfully rendered NED by postchemotherapy surgery, and 12 remain continuously NED. Two patients continue on observation with no treatment for their LR. Overall, 69 of the 81 treated patients (85.2%) ultimately achieved an NED state, and 38 (46.9%) remain continuously NED with median follow-up from LR therapy of 24.5 months (range, 1 to 83 months), whereas nine other patients are currently NED after therapy for subsequent relapses. Because of the small numbers of specimens tested, we were unable to draw any definitive conclusions from the molecular and cytogenetic analyses. CONCLUSION: GCT patients require lifetime follow-up. At the time of LR, surgical resection alone remains our preferred therapy.

Successful Management of Jejunal Perforation in Burkitt's Lymphoma: A Case Report.

Burkitt's lymphoma (BL) is rare, rapidly growing, and highly aggressive. Urgent commencement of chemotherapy is vital to prevent complications and promote a favourable prognosis. Any factor causing a delay in the initiation of chemotherapy will affect prognosis significantly. Intestinal perforation is a well-known complication with devastating consequences. It inevitably leads to a delay in the initiation of chemotherapy. There are few reports in the literature that discuss this complication. Furthermore, there are no reports of patients that have survived intestinal perforation occurring prior to the commencement of chemotherapy. We present a case of a 55-year-old male who survived perforation of advanced sporadic BL of the jejunum occurring prior to the commencement of chemotherapy. Critical aspects of the patients care are discussed.

CK2 phosphorylation of XRCC1 facilitates dissociation from DNA and single-strand break formation during base excision repair.

CK2 phosphorylates the scaffold protein XRCC1, which is required for efficient DNA single-strand break (SSB) repair. Here, we express an XRCC1 protein (XRCC1(ckm)) that cannot be phosphorylated by CK2 in XRCC1 mutated EM9 cells and show that the role of this post-translational modification gives distinct phenotypes in SSB repair and base excision repair (BER). Interestingly, we find that fewer SSBs are formed during BER after treatment with the alkylating agent dimethyl sulfate (DMS) in EM9 cells expressing XRCC1(ckm) (CKM cells) or following inhibition with the CK2 inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT). We also show that XRCC1(ckm) protein has a higher affinity for DNA than wild type XRCC1 protein and resides in an immobile fraction on DNA, in particular after damage. We propose a model whereby the increased affinity for DNA sequesters XRCC1(ckm) and the repair enzymes associated with it, at the repair site, which retards kinetics of BER. In conclusion, our results indicate that phosphorylation of XRCC1 by CK2 facilitates the BER incision step, likely by promoting dissociation from DNA.

XRCC1 phosphorylation by CK2 is required for its stability and efficient DNA repair.

XRCC1 is a scaffold protein that interacts with several DNA repair proteins and plays a critical role in DNA base excision repair (BER). XRCC1 protein is in a tight complex with DNA ligase IIIalpha (Lig III) and this complex is involved in the ligation step of both BER and repair of DNA single strand breaks. The majority of XRCC1 has previously been demonstrated to exist in a phosphorylated form and cells containing mutant XRCC1, that is unable to be phosphorylated, display a reduced rate of single strand break repair. Here, in an unbiased assay, we demonstrate that the cytoplasmic form of the casein kinase 2 (CK2) protein is the major protein kinase activity involved in phosphorylation of XRCC1 in human cell extracts and that XRCC1 phosphorylation is required for XRCC1-Lig III complex stability. We demonstrate that XRCC1-Lig III complex containing mutant XRCC1, in which CK2 phosphorylation sites have been mutated, is unstable. We also find that a knockdown of CK2 by siRNA results in both reduced XRCC1 phosphorylation and stability, which also leads to a reduced amount of Lig III and accumulation of DNA strand breaks. We therefore propose that CK2 plays an important role in DNA repair by contributing to the stability of XRCC1-Lig III complex.

Long-term follow-up of ultraflex metallic stents in benign and malignant central airway obstruction.

BACKGROUND: We report experience with Ultraflex metallic stents (Boston Scientific, Natick, MA) inserted at rigid bronchoscopy under general anesthesia for palliation of benign and malignant upper airway obstruction. METHODS: Notes of all patients treated with Ultraflex stents from 1999 to 2003 were reviewed for symptomatic response, spirometric data, and any complications before discharge home. Long-term outcome was assessed by questionnaires sent to patients' general practitioners. RESULTS: Recruited were 66 patients (12 benign, 54 malignant airway obstructions). Before discharge home, breathlessness improved in 11 of 12 patients with benign obstruction and in 39 of 54 with malignancies. Postoperative complications in 10 patients with malignant obstructions and in 2 patients with benign obstruction were successfully controlled. It was not possible to perform preoperative pulmonary function tests in most of the patients who presented as emergencies. Mean improvement in forced expiratory volume in 1 second was 0.88 liters in 3 patients with benign obstruction and 0.28 liters in 14 patients with malignant obstruction, and mean peak expiratory flow rate improved by 109 L/min and 97 L/min, respectively. General practitioners completed questionnaires for 12 benign patients and 46 of 54 patients with malignancies. At a mean follow-up of 1017 days (range, 46 to 1120 days), 10 of the 12 patients with benign disease were alive and 7 of 46 patients with malignant airway obstruction were alive, with a median survival of 128 days (mean, 361; range, 3 to 1859 days). Most survivors had Medical Research Council grade III breathlessness or better, with few stent-related symptoms. CONCLUSIONS: Ultraflex stents proved safe and effective in prolonged palliation of benign and malignant airways obstruction.

Identification and purification of a soluble region in the breast cancer susceptibility protein BRCA2.

The BRCA2 gene encodes a large multidomain protein of 3418 residues. Studies to elucidate the mechanisms by which BRCA2 prevents tumour formation have been largely restricted by the size of the protein. Based on secondary structure predictions we have cloned regions across the BRCA2 gene and determined the solubility of the proteins they encode. The fragment consisting of amino acids 290-456 BRCA2 was found predominantly in the soluble portion of the cell lysate and was purified by ion exchange and nickel-NTA affinity chromatography. CD spectroscopy revealed secondary structure elements consistent with a folded peptide and limited proteolysis was used to identify a potential novel domain.