Fully automated counting of DNA damage foci in tumor cell culture: A matter of cell separation.

Analysis and quantification of residual, unrepaired DNA double-strand breaks by detecting damage-associated γH2AX or 53BP1 foci is a promising approach to evaluate radiosensitivity or radiosensitization in tumor cells. Manual foci quantification by eye is well-established but unsatisfactory due to inconsistent foci numbers between different observers, lack of information about foci size and intensity and the time-consuming scoring process. Therefore, automated foci counting is an important goal. Several software solutions for automated foci counting in separately acquired fluorescence microscopy images have been established. The AKLIDES NUK technology by Medipan combines automated microscopy and image processing/ counting, enabling affordable high throughput foci analysis as a routine application. Using this machine, automated foci counting is well established for lymphocytes but has not yet been reported for adherent tumor cells with their irregularly shaped nuclei and heterogeneous foci textures. Here we aimed to use the AKLIDES NUK system for adherent tumor cells growing in clusters. We identified cell separation as a critical step to ensure fast and reliable automated nuclei detection. We validated our protocol for the fully automated quantification of (i) the IR-dose dependent increase and (ii) the ATM as well as PARP inhibitor-induced radiosensitization. Collectively, with this protocol the AKLIDES NUK system facilitates cost effective, fast and high throughput quantitative fluorescence microscopic analysis of DNA damage induced foci such as γH2AX and 53BP1 in adherent tumor cells.

Loss of PTEN-assisted G2/M checkpoint impedes homologous recombination repair and enhances radio-curability and PARP inhibitor treatment response in prostate cancer.

Here we report that PTEN contributes to DNA double-strand break (DSB) repair via homologous recombination (HR), as evidenced by (i) inhibition of HR in a reporter plasmid assay, (ii) enhanced sensitivity to mitomycin-C or olaparib and (iii) reduced RAD51 loading at IR-induced DSBs upon PTEN knockdown. No association was observed between PTEN-status and RAD51 expression either in-vitro or in-vivo in a tissue microarray of 1500 PTEN-deficient prostate cancer (PC) samples. PTEN depletion and sustained activation of AKT sequestered CHK1 in the cytoplasm, thus impairing the G2/M-checkpoint after irradiation. Consistently, AKT inhibition recovered the G2/M-checkpoint and restored HR efficiency in PTEN-depleted cells. We show that, although PTEN loss correlates with a worse prognosis, it may predict for improved response of PC patients to radiotherapy. Further, we provide evidence for the use of PTEN as a biomarker for predicting the response to PARP inhibitors as radiosensitizing agents in prostate cancer. Collectively, these data implicate PTEN in maintaining genomic stability by delaying G2/M-phase progression of damaged cells, thus allowing time for DSB repair by HR. Furthermore, we identify PTEN-status in PC as a putative predictor of (i) radiotherapy response and (ii) response to treatment with PARP inhibitor alone or combined with radiotherapy.

Physician-initiated prospective Italian Registry of carotid stenting with the C-Guard mesh-stent: the IRON-Guard registry. Rationale and design.

According to the World Health Organization, every year, 5 million peoples die for stroke and another 5 million are permanently disabled. Although there are many causes of acute stroke, a common treatable cause of acute stroke is atheromatous narrowing at the carotid bifurcation. Carotid endarterectomy is still the standard of car, even if carotid artery stenting (CAS) has become an effective, less invasive alterantive. Unfortunately, CAS procedure is not yet perfect; regardless the use of an embolic protection device (EPD), percutaneous treatment has been correlated with a risk of cerebral ischemic events related to distal embolization. The objective of the IRON-Guard Registry is to evaluate the clinical outcome of treatment by means of stenting with the C-Guard (InspireMD, Boston, MA, USA) in subjects requiring CAS due to significant extracranial carotid artery stenosis with a physician-initiated, Italian, prospective, multicenter, single-arm study. A total of 200 enrolled subjects divided over different centers are planned to be enrolled. CAS will performed by implanting of C-Guard stent. Procedure will be performed according to the physician's standard of care. Standard procedures will be followed based on the Instructions for Use, for the C-Guard device of Inspire. The primary endpoint of this study is the 30-day rate of major adverse events (MAE), defined as the cumulative incidence of any periprocedural (≤30 days postprocedure) death, stroke or myocardial infarction. Secondary endpoints are rate of late ipsilateral stroke (31 through 365 days), system technical success, device malfunctions, major adverse events (MAEs), serious device-related and procedure-related adverse events, target lesion revascularization, and in-stent restenosis rates.

Involvement of ATM in homologous recombination after end resection and RAD51 nucleofilament formation.

Ataxia-telangiectasia mutated (ATM) is needed for the initiation of the double-strand break (DSB) repair by homologous recombination (HR). ATM triggers DSB end resection by stimulating the nucleolytic activity of CtIP and MRE11 to generate 3'-ssDNA overhangs, followed by RPA loading and RAD51 nucleofilament formation. Here we show for the first time that ATM is also needed for later steps in HR after RAD51 nucleofilament formation. Inhibition of ATM after completion of end resection did not affect RAD51 nucleofilament formation, but resulted in HR deficiency as evidenced by (i) an increase in the number of residual RAD51/γH2AX foci in both S and G2 cells, (ii) the decrease in HR efficiency as detected by HR repair substrate (pGC), (iii) a reduced SCE rate and (iv) the radiosensitization of cells by PARP inhibition. This newly described role for ATM was found to be dispensable in heterochromatin-associated DSB repair, as KAP1-depletion did not alleviate the HR-deficiency when ATM was inhibited after end resection. Moreover, we demonstrated that ATR can partly compensate for the deficiency in early, but not in later, steps of HR upon ATM inhibition. Taken together, we describe here for the first time that ATM is needed not only for the initiation but also for the completion of HR.

An αIIb mutation in patients with Glanzmann thrombasthenia located in the N-terminus of blade 1 of the ß-propeller (Asn2Asp) disrupts a calcium binding site in blade 6.

BACKGROUND: Studies of Glanzmann thrombasthenia (GT)-causing mutations has generated invaluable information on the formation and function of integrin αIIbß(3). OBJECTIVE: To characterize the mutation in four siblings of an Israeli Arab family affected by GT, and to analyze the relationships between the mutant protein structure and its function using artificial mutations. METHODS AND RESULTS: Sequencing disclosed a new A97G transversion in the αIIb gene predicting Asn2Asp substitution at blade 1 of the ß-propeller. Alignment with other integrin α subunits revealed that Asn2 is highly conserved. No surface expression of αIIbß(3) was found in patients' platelets and baby hamster kidney (BHK) cells transfected with mutated αIIb and WT ß(3). Although the αIIbß(3) was formed, the mutation impaired its intracellular trafficking. Molecular dynamics simulations and modeling of the αIIbß(3) crystal indicated that the Asn2Asp mutation disrupts a hydrogen bond between Asn2 and Leu366 of a calcium binding domain in blade 6, thereby impairing calcium binding that is essential for intracellular trafficking of αIIbß(3). Substitution of Asn2 to uncharged Ala or Gln partially decreased αIIbß(3) surface expression, while substitution by negatively or positively charged residues completely abolished surface expression. Unlike αIIbß(3), αVß(3) harboring the Asn2Asp mutation was surface expressed by transfected BHK cells, which is consistent with the known lower sensitivity of αVß(3) to calcium chelation compared with αIIbß(3). CONCLUSION: The new GT causing mutation highlights the importance of calcium binding domains in the ß-propeller for intracellular trafficking of αIIbß(3). The mechanism by which the mutation exerts its deleterious effect was elucidated by molecular dynamics.

Prevalence of plasmid-mediated quinolone resistance determinants in Enterobacteriaceae from Tunisia.

The spread of plasmid-mediated quinolone resistance determinants (qnr-like determinants, aac(6')-Ib-cr and qepA genes) was evaluated in a collection of 281 nalidixic acid-resistant enterobacterial isolates recovered between September 2005 and December 2007 at the Sahloul Hospital, Sousse, Tunisia. Sixteen percent of those isolates carried qnr genes encoding the QnrB1, QnrB2, QnrA6 or QnrS1 determinants. Most qnr-positive isolates were extended-spectrum ß-lactamase (ESBL) producers, being predominantly of the CTX-M-15 type, but also of the SHV-28 and SHV-12 types. The qnr genes were located on plasmids with a size in the range 55-150 kb. The qnrB2 gene was associated with sul1-type integron structures and the qnrB1 gene was associated with orf1005, whereas the genetic environment of qnrA6 was unknown. In two isolates, the qnrS1 gene was located downstream of an ISEcl2 element on plasmids that often carried the narrow-spectrum ß-lactamase gene bla(LAP-2); qepA and aac(6')-Ib-cr were not detected. The present study highlights the wide spread of Qnr-like determinants in Tunisia, with an association with the ESBL CTX-M-15 in human clinical isolates.

Diuretics in acute kidney injury.

BACKGROUND: In an acute care setting, diuretics are often prescribed to maintain or increase urine output in patients presenting with acute kidney injury (AKI). The rationale behind giving diuretics is that they may protect the kidney from ischemic injury by maintaining a nonoliguric state. There have been many studies both supporting and criticizing diuretic use in AKI for improving overall patient outcomes. METHODS: A systematic review of the literature was conducted to evaluate the role of diuretics including osmotics, loop diuretics, and nesiritide in modifying AKI. RESULTS: There was no evidence to suggest that the use of loop diuretics in AKI reduces mortality, the need for dialysis, the number of dialysis sessions, or length of Intensive Care Unit/hospital stay or that it increases the recovery of renal function. There is no benefit for the use of mannitol as an osmotic diuretic over hydration in rhabdomyolysis. In contrast, mannitol was found to cause more harm and to induce nephropathy. Nesiritide did not improve renal function in patients with decompensated heart failure and mild chronic renal insufficiency. Nesiritide may be effective in the prevention of AKI when applied in lower doses for a prolonged period of time in patients with mild to moderate renal insufficiency. CONCLUSIONS: Diuretics have been shown to be ineffective in the prevention of AKI or for improving outcomes once AKI occurs. At best, diuretics can help decrease symptoms of pulmonary edema secondary to volume overload.

[Characterization of the resistance mechanism to beta-lactams in Acinetobacter baumannii strains isolated in the university hospital Sahloul in Tunisia (2005)]. / Caractérisation des mécanismes enzymatiques de résistance aux beta-lactamines chez des souches de Acinetobacter baumannii isolées à l'hôpital universitaire Sahloul, Sousse en Tunisie (2005).

OBJECTIVE: The bacterial multiresistance to beta-lactams and imipenem is an emergent feature in the university hospital Sahloul in Tunisia. This study was conducted to elucidate natural and acquired mechanism of resistance to beta-lactams in strains of Acinetobacter baumannii isolated in different wards of the hospital. MATERIALS AND METHODS: A specimen of 26 clinical strains of Acinetobacter baumannii was studied. beta-lactamases characterization was done by isoelectric focusing on gel of crude enzymatic extract, phenotypic tests for detection of extended spectrum beta-lactamases (ESBL) and metallo-beta-lactamases (MBL) and finally by amplification (PCR) and sequencing of genes encoding naturally occurring AmpC, the insertion sequence ISAbaI and oxacillinase with carbapenemase activity. Study of clonality of strains was performed by analysis of genomic DNA digested by the restriction enzyme ApaI and separated by pulsed field gel electrophoresis (PFGE). RESULTS: The isoelectric focusing on gel revealed two bands of beta-lactamase activity with a pI upper than 8. None ESBL or MBL was detected. PCR for AmpC, ISAbaI and OXA-69 were positive for all studied strains. The sequencing of PCR products show high identity (99-100%) with genes described previously. PFGE analysis has demonstrated clonality of studied strains. CONCLUSION: Resistance to beta-lactams including imipenem is associated to the hyper production of the AmpC enzyme and expression of OXA-69. Those enzymatic mechanisms are associated with the natural low permeability to beta-lactams which characterize Acinetobacter baumannii strains. High clonal relationship of studied strains proved by PFGE analysis has shown the necessity of implementation of strict hygienic rules and rational antibiotic usage.

[Clinical and epidemiological characterization of infections due to imipenem resistant Acinetobacter baumannii at the university hospital Sahloul, Tunisia]. / Caractérisation clinico-épidémiologique des infections à Acinetobacter baumannii résistant à l'imipénème au CHU Sahloul, Tunisie.

OBJECTIVE: to characterize epidemiological and clinical features related to the multi-drug Acinetobacter baumannii infections in the university hospital Sahloul in Tunisia. MATERIAL AND METHODS: retrospective study including twenty-four imipenem resistant Acinetobacter baumannii isolated from twenty patients hospitalized in different wards of the hospital. Study of clinical features related to the infection by multi-drug Acinetobacter baumannii, bacterial identification by classical identification scheme, antibiotic susceptibilities were determined by the disk diffusion method; genotyping was performed by arbitrarily-primed PCR. RESULTS: the most incriminated ward was the intensive care unit with a high prevalence of septicaemia. All studied strains were multi-drug to all beta-lactams tested. Genotyping has shown the clonality of studied strains. Features incriminated in the acquisition of infection were essentially immunodeficiency, invasive manoeuvring and antibiotherapy. CONCLUSION: multidrug Acinetobacter baumannii is increasingly isolated in our hospital. Rational use of antibiotics and rigorous application of hygienic rules could contribute to limit dissemination of such strains.

Nonsurgical management of hearing loss.

This article presents the salient points involved in age-induced hearing loss (i.e., presbycusis). The general evaluation, screening, diagnosis, and treatment modalities are discussed. Also, advances in hearing aid technology are described.

A monoclonal antibody diagnoses active Fasciola infection in humans.

Monoclonal antibodies (MABs) were produced after fusion of spleen cells of Fasciola antigen immunized BALB/c mice and non secreting murine myloma cells (P3x63 Ag8). Six MAbs, showing the highest reactivity with purified Fasciola antigen, were prepared. All 6 MABs were IgG2 with Kappa light chain. Reactive epitopes recognized by the six MAbs were glycoprotein in nature, and each MAb recognized a single epitope of Fasciola antigen. No cros reactions were observed with Schistosomal AWSA, hydatid Ag and Entamoeba Ag. EITB technique showed a specific diagnostic band at 17.5 kDa for each of the six MAbs. Anti-Fasciola MAb (AD2) was conjugated with peroxidase and was used with anti-rabbit anti-Fasciola polyclonal antibody in sandwich-ELISA to detect circulating Fasciola antigen in serum and urine samples of 57 fascioliasis patients, 51 schistosomiasis patients, 45 patients infected with other parasites and 47 healthy controls. Sensitivity of the assay in detection of circulating Fasciola antigen in sera and urines of Fasciola infected patients was 100%. The specificity of the assay was calculated among patients infected with schistosomiasis and other parasites and was 98% in serum and 97% in urine. A positive correlation was found between levels of circulating Fasciola antigen in serum and urine samples of fascioliasis patients (r = 0.825, p < 0.05).

Evaluation of specific Fasciola antigen in the immunodiagnosis of human fascioliasis in Egypt.

Enzyme-linked immunosorbent assay (ELISA) and enzyme linked immunoelectrotransfer blot technique (EITB) were employed for the detection of circulating Fasciola antibodies in infected human sera using a specific Fasciola antigen, prepared by immunoaffinity purification of homogenates of Fasciola hepatica adult worms. Ninety two individuals diagnosed clinically and parasitologically were classified into: Fascioliasis group (21 patients), schistosomiasis group (21 patients) and subjects harbouring other parasitic infections (50 patients). Eighteen healthy individuals served as normal controls. ELISA was 100% sensitive and 93% specific with 96.5% diagnostic efficacy, whereas EITB was 100% sensitive and specific with 100% diagnostic efficacy. Our data revealed that ELISA can be used as a good screening test while EITB can serve as a confirmatory test for immunodiagnosis of fascioliasis.

Purification and characterization of a specific Fasciola antigen.

Specific Fasciola antigen was prepared from homogenates of Fasciola hepatica adult worms. The homogenate was ultracentrifuged and the supernatant containing crude Fasciola antigen was then passed over a cyanogen bromide activated sepharose 4B column coupled with antiserum against Schistosoma mansoni adult worm surface antigen. The specific, Schistosoma-free Fasciola antigen was tested for its specificity by immunodiffusion. Characterization of the specific Fasciola antigen was done by gradient poly-acrylamide gel electrophoresis and immunoblotting technique. The electrophoresis migration pattern of specific Fasciola antigen, stained with Coomassie blue, showed 7 bands in the 12-54 kDa regions. Using the immunoblotting technique, a batch of positive fascioliasis sera recognized two specific bands at the 33 and 54 kDa regions.

Efficacy of oxfendazole against natural infestations of nematodes and cestodes in sheep in Egypt.

Oxfendazole liquid suspension (Systamex; Wellcome) was administered orally at the dose of 4.5 mg per kg to 800 indigenous Egyptian sheep clinically affected with Dictyocaulus filaria, Moniezia expansa, Haemonchus contortus, Ostertagia circumcincta, Nematodirus spp, Trichostrongylus axei, Cooperia curticei, Trichuris ovis and Oesophagostomum spp. A 100 per cent clearance was recorded for all parasites with the exception of T ovis which were markedly reduced in number.