Functions of multiple exonucleases are essential for cell viability, DNA repair and homologous recombination in recD mutants of Escherichia coli

Heterotrimeric RecBCD enzyme unwinds and resects a DNA duplex containing blunt double-stranded ends and directs loading of the strand-exchange protein RecA onto the unwound 3'-ending strand, thereby initiating the majority of recombination in wild-type Escherichia coli. When the enzyme lacks its RecD subunit, the resulting RecBC enzyme, active in recD mutants, is recombination proficient although it has only helicase and RecA loading activity and is not a nuclease. However, E. coli encodes for several other exonucleases that digest double-stranded and single-stranded DNA and thus might act in consort with the RecBC enzyme to efficiently promote recombination reactions. To test this hypothesis, I inactivated multiple exonucleases (i.e., exonuclease I, exonuclease X, exonuclease VII, RecJ, and SbcCD) in recD derivatives of the wild-type and nuclease-deficient recB1067 strain and assessed the ability of the resultant mutants to maintain cell viability and to promote DNA repair and homologous recombination. A complex pattern of overlapping and sometimes competing activities of multiple exonucleases in recD mutants was thus revealed. These exonucleases were shown to be essential for cell viability, DNA repair (of UV- and gamma-induced lesions), and homologous recombination (during Hfr conjugation and P1 transduction), which are dependent on the RecBC enzyme. A model for donor DNA processing in recD transconjugants and transductants was proposed.     

Exonuclease requirements for recombination of lambda-phage in recD mutants of Escherichia coli

Recombination of lambda red gam phage in recD mutants is unaffected by inactivation of RecJ exonuclease. Since nucleases play redundant roles in E. coli, we inactivated several exonucleases in a recD mutant and discovered that 5'-3' exonuclease activity of RecJ and exonuclease VII is essential for lambda-recombination, whereas exonucleases of 3'-5' polarity are dispensable. The implications of the presented data on current models for recombination initiation in E. coli are discussed.     

Therapeutic efficacy of 5% NaCl hypertonic solution in patients with bullous keratopathy

A clinical trial was undertaken to evaluate the efficacy of hypertonic solution (5% NaCl) in patients who have bullous keratopathy (BK). The aim of the study was to define the stage of the disease and the thickness of cornea in micrometers, which would be the threshold for therapeutic approach. This was a prospective study on 70 eyes of 55 patients. Patients were divided in two groups at the beginning of the study. The first group (n=33 eyes) included patients with initial stage of BK: only stromal component of corneal oedema was present. The second group (n=37 eyes) included patients with advanced stage of BK: the epithelial component of the disease with bullae on the corneal surface had already developed. Visual acuity, central and peripheral thickness of cornea and morphology of the disease was recorded before therapy, 7 days and 4 weeks after administration of hypertonic solution. Our results shown that the efficacy of hypertonic solution correlates with the severity of clinical picture in patients with BK. When 5% NaCl hypertonic solution was applied in the early stage of the disease, when only stromal component of corneal oedema was presented, visual acuity and pachymetry readings were significantly improved. The threshold pachymerty measurement of corneal thickness justifying the application of hypertonic solution was 613-694 microm (in the central corneal area), and 633-728 microm (at corneal periphery). It seems reasonable to apply hypertonic solution to the patients who have BK and whose pachymetric values are below mentioned range. In terminal stages of BK, when superficial bullae (epithelial component) had already developed, treatment with NaCl was not effective and patients had to be submitted to penetrating keratoplasty.     

Laparoscopic abdominal cysts fenestration using harmonic scalpel

The use of ultrasonically activated scalpel for tissue cutting and coagulation is a potential replacement for electrosurgery, which can be related to different complications. Its working principle is to transform the electric power into the mechanical longitudinal movement of the working part of the instrument, by a piezoelectric transducer situated in the hand piece. Between October 2000 and June 2004, six patients with abdominal cysts were treated by laparoscopy, using the harmonic scalpel. The average age was 40.8 (ranging from 15-60) years. Laparoscopic abdominal cyst fenestration was performed in five patients, and laparoscopic cholecystectomy and abdominal cyst fenestration were done in one patient during the same operation. The average duration of the operation was 40 (ranging from 25-70) minutes and hospital stay was 2.8 (ranging from 1-5) days. Laparoscopic abdominal cyst fenestration using the harmonic scalpel is a safe and successful operation, with good results including all the advantages of the minimally invasive surgery.     

Local recurrence of primary non-ampullary adenocarcinoma of duodenum after surgical treatment--a case report and a literature review

Worldwide there is no general attitude on optimal surgical procedure in treatment of primary non-ampullary adenocarcinoma of duodenum, especially for early stage of duodenal cancer. Some authors prefer local excision and segmental resection while others rather perform duodenopancreatic resection, even in the case of early stage of duodenal cancer with aim to avoid tumor recurrence. In this paper we present a rare clinical course of a 60-year-old male patient with an endoscopically and pathohistologically proven early stage duodenal cancer that was treated by wide local excision. Three years after operation, control endoscopy showed "flat" polyp in the duodenum and radical duodenopancreatic resection was performed. Pathohistological examination of resected specimen showed cancer that had spread throughout the duodenal wall with metastases in the regional lymph nodes. According to our findings and literature review we gave some direction concerning the optimal diagnostic and surgical procedure for this rare tumor.     

Progression of age related maculopathy in phakic versus pseudophakic eyes

Age-related maculopathy (ARM) is one of the leading causes of central visual acuity loss in older western population. Many factors are responsible for the fast development of ARM. One of this is significant increases of optical radiations through artificial lens after removal of the catarctous lens. The aim of this study was to compare progression of ARM in phakic and pseudophakic patients and to calculate the possibility ofpseudophakia as a risk factor for faster progression of ARM. Medical records of 76 patients, older than 60 years (32 male and 44 female) with early forms of ARM were randomly evaluated. They had undergone cataract removal by phacoemulsification with intraocular lens implantation from January 2002 to December 2006 at the Department of Ophthalmology, Rijeka University Hospital, Croatia. Patients were examined two weeks after the surgery and followed up for two years. The control group consisted of 48 patients (21 males and 27 females) with also early forms of ARM, older than 60 years, examined at the Policlinic Department from January 2006 to December 2006 and followed up at least for two years without any cataract surgery. Comparing progression of ARM in these two groups, a total of 19 patients (25%) in pseudophakic group showed progression to late forms of ARM, but only 6 patients (12.5 %) in the control group developed these aggressive ARM forms. More aggressive forms of ARM in pseudophakic group indicate that pseudophakia should be considered as a risk factor for development of ARM.     

The role of air quality in perception of health of the local population

The research aim was to investigate and establish the perception of health among population groups with different number of industrial polluters in their living environment. Namely, the Kostrena living area (3250 inhabitants) is situated near big industrial pollutant producers: INA oil refinery Urinj, thermo power plant Rijeka, shipyard "Viktor Lenac", while in Crikvenica living area such pollutant producers do not exist. In this research 146 subjects from Kostrena participated (74 men and 72 women with age average 40.8, SD 9.2 years), and 157 subjects from Crikvenica area (79 men and 78 women with age average 39.4, SD 10.1 years). The perception of health was measured by the means of SF-36 questionnaire variables. Lung function (FEV1, FVC, FEV25/75, PEF), cardiovascular function (systolic and diastolic arterial blood pressure, heart rate were also measured, including body proportion measures by the means of waste and hip circumference and silhouette test, in order to establish the eventual objective physical health parameter difference among examined population groups. Subjects from Kostrena perceived their general health, mental health, and vitality worse than subjects from Crikvenica. Group from Kostrena felt themself more limited in their roles due to emotional problems, perceiving more pain than group from Crikvenica. On the opposite site, social functioning of subjects from Kostrena was better in comparison to Crikvenica group. No signifficant differences between groups were found in physical functioning and in role limitation due to physical problems. In addition, there were no significant differences between groups in objective physical health parameters, such as pulmonary function, arterial blood pressure, pulse, and waist to hip ratio. Subjects using sensoric systems and the mental cognition about harmful environmental factors, perceive their health worse if there are present industrial resources in their environment, even when concentration of the pollutants are within legal ranges.     

Global transcriptional profiles of beating clusters derived from human induced pluripotent stem cells and embryonic stem cells are highly similar

Background

The C-terminal Eps15 homology domain-containing protein 1 (EHD1) is ubiquitously expressed and regulates the endocytic trafficking and recycling of membrane components and several transmembrane receptors. To elucidate the function of EHD1 in mammalian development, we generated Ehd1 ^-/- mice using a Cre/loxP system.

Results

Both male and female Ehd1 ^-/- mice survived at sub-Mendelian ratios. A proportion of Ehd1 ^-/- mice were viable and showed smaller size at birth, which continued into adulthood. Ehd1 ^-/- adult males were infertile and displayed decreased testis size, whereas Ehd1 ^-/- females were fertile. In situ hybridization and immunohistochemistry of developing wildtype mouse testes revealed EHD1 expression in most cells of the seminiferous epithelia. Histopathology revealed abnormal spermatogenesis in the seminiferous tubules and the absence of mature spermatozoa in the epididymides of Ehd1 ^-/- males. Seminiferous tubules showed disruption of the normal spermatogenic cycle with abnormal acrosomal development on round spermatids, clumping of acrosomes, misaligned spermatids and the absence of normal elongated spermatids in Ehd1 ^-/- males. Light and electron microscopy analyses indicated that elongated spermatids were abnormally phagocytosed by Sertoli cells in Ehd1 ^-/- mice.

Conclusions

Contrary to a previous report, these results demonstrate an important role for EHD1 in pre- and post-natal development with a specific role in spermatogenesis.     

The MicroArray Quality Control (MAQC) project shows inter- and intraplatform reproducibility of gene expression measurements

Over the last decade, the introduction of microarray technology has had a profound impact on gene expression research. The publication of studies with dissimilar or altogether contradictory results, obtained using different microarray platforms to analyze identical RNA samples, has raised concerns about the reliability of this technology. The MicroArray Quality Control (MAQC) project was initiated to address these concerns, as well as other performance and data analysis issues. Expression data on four titration pools from two distinct reference RNA samples were generated at multiple test sites using a variety of microarray-based and alternative technology platforms. Here we describe the experimental design and probe mapping efforts behind the MAQC project. We show intraplatform consistency across test sites as well as a high level of interplatform concordance in terms of genes identified as differentially expressed. This study provides a resource that represents an important first step toward establishing a framework for the use of microarrays in clinical and regulatory settings.