Luteinizing hormone-releasing hormone stimulates arachidonic acid release in rat granulosa cells

The effect of LHRH on arachidonic acid release was studied in rat granulosa cells in primary culture. In cells prelabeled with [3H]arachidonic acid, LHRH caused an increase in the level of [3H]arachidonic acid released in the culture medium, to 125-150% of control levels at the end of a 60-min incubation period. In subsequent time-course and dose-response experiments, a significant effect on [3H]arachidonic acid release could be observed as early as 15 min after LHRH addition, and the lowest effective dose was 10(-8) M LHRH. Addition of LH, FSH, prostaglandin F2 alpha, or (Bu)2cAMP was without effect. Likewise, an agonistic LHRH analog (LHRHa, 10(-8) M) also markedly stimulated [3H]arachidonic acid from cultured granulosa cells, and the effects of both LHRH and LHRHa were blocked by concomitant presence of a potent LHRH antagonist. In addition to [3H]arachidonic acid release in the culture medium, the effect of LHRH on the level of radiolabel present in cellular phospholipids was also examined. In granulosa cells prelabeled with [3H] arachidonic acid, LHRH significantly depleted the level of radioactivity previously incorporated into cellular phosphatidylinositol, as early as 5 min after its addition, to 85% of control levels. The level of radiolabel found in other major phospholipids such as phosphatidylserine/phosphatidylcholine and phosphatidylethanolamine, as well as the intracellular level of unesterified [3H]arachidonic acid, were not significantly affected by LHRH. The effect of LHRH on [3H]arachidonic acid release from prelabeled phospholipids as well as the LHRH-induced loss of radioactivity previously incorporated into phosphatidylinositol could be reversed by verapamil, suggesting a possible calcium dependency. Taken together, these data support the notion that arachidonic acid liberation from phospholipids may be associated with the mechanism of action of LHRH on ovarian cells.     

Expression dynamics of NADPH oxidases during early zebrafish development

Nicotinamide dinucleotide phosphate oxidases (NOX) control various cellular signaling cascades. In the nervous system, there is recent evidence that NOX‐derived reactive oxygen species (ROS) regulate neurite outgrowth, regeneration, and stem cell proliferation; however, a comprehensive NOX gene expression analysis is missing for all major model systems. Zebrafish embryos provide an excellent model system to study neurodevelopment and regeneration because they develop quickly and are well suited for in vivo imaging and molecular approaches. Although the sequences of five NOX genes (nox1, nox2/cybb, nox4, nox5, and duox) have been identified in the zebrafish genome, nothing is known about their expression pattern. Here, we used quantitative polymerase chain reaction combined with in situ hybridization to develop a catalog of nox1, nox2/cybb, nox5, and duox expression in zebrafish during early nervous system development from 12 to 48 hours post fertilization. We found that expression levels of nox1, nox5, and duox are dynamic during the first 2 days of development, whereas nox2/cybb levels remain remarkably stable. By sectioning in situ hybridized embryos, we found a pattern of broad and overlapping NOX isoform expression at 1 and 1.5 days post fertilization. After 2 days of development, a few brain regions displayed increased NOX expression levels. Collectively, these results represent the first comprehensive analysis of NOX gene expression in the zebrafish and will provide a basis for future studies aimed at determining the functions of NOX enzymes in neurodevelopment and regeneration. J. Comp. Neurol. 524:2130–2141, 2016. © 2015 Wiley Periodicals, Inc.     

No Association between Dietary Vitamin K Intake and Fracture Risk in Chinese Community-Dwelling Older Men and Women: A Prospective Study

Data on the association between dietary vitamin K intake and fracture risk are limited among Chinese. This study examined such an association in community-dwelling elderly in Hong Kong. We present data from 2,944 subjects (1,605 men, 1,339 women) who participated in a prospective cohort study. Baseline dietary intakes of energy, protein, calcium, vitamin D, and vitamin K were assessed using a food-frequency questionnaire. Data on incident hip fracture and nonvertebral fracture during a median of 6.9 follow-up years were collected from a hospital database. Cox regression analyses were performed with adjustments for age, education attainment, smoking status, alcohol use, body mass index, hip bone mineral density, physical activity, use of calcium supplement, and energy-adjusted nutrient intakes. There were 29 (1.8 %) men and 19 (1.4 %) women with incident hip fractures and 97 (6.0 %) men and 88 (6.6 %) women with nonvertebral fractures. The median (interquartile range) of dietary vitamin K intake was 241.8 (157.5–360.8) and 238.9 (162.4–343.6) μg/day in men and women, respectively. Similar dietary vitamin K intakes were observed between subjects with hip or nonvertebral fractures and subjects without hip or nonvertebral fractures. In both men and women, dietary vitamin K intake was not associated with fracture risks at all measured sites in either crude or adjusted models. In Chinese community-dwelling elderly, hip or nonvertebral fracture risk was not associated with dietary vitamin K intake. The high dietary vitamin K intake of the studied group may have limited the ability to detect the association between vitamin K intake and fracture risk.     

A novel multimodal platform for assessing surgical technical skills

BackgroundEstablished methods for assessing surgical performance face limitations. Global rating scales and procedure-specific checklists are resource intensive and rely on expert opinions. Alternatives that use technology to track hand movements, such as magnetic and optical tracking systems, are generally expensive and ill suited to the surgical environment.MethodsThe authors present a new platform that integrates a novel, low-cost optical tracking system, magnetic tracking technology and a videographic recording system to quantify surgical performance synchronously across all modalities. The validity of this platform was tested by examining its ability to differentiate between the performance of expert and novice participants on a basic surgical task.ResultsEach modality was able to differentiate between expert and novice participants, and metrics were well correlated across modalities.ConclusionsThe authors have developed a platform for assessing surgical performance. It can operate in the absence of expert raters and has the potential to provide immediate feedback to trainees.     

Impact of a multidisciplinary trauma team on the outcome of acute subdural haematoma

IntroductionTo review the outcome of patients with post-traumatic acute subdural haematoma (ASDH) before and after the establishment of a hospital trauma team at a designated trauma centre.MethodA retrospective analysis was conducted on 82 consecutive patients who underwent surgery for post-traumatic ASDH. The ‘PRE’ and ‘POST’ groups included patients admitted before and after the establishment of a hospital trauma team, respectively.Injury severity was assessed by the admission Glasgow coma score, imaging findings, and the revised trauma score. Clinical outcome measures were the hospital length of stay and the Glasgow outcome score (GOS) upon hospital discharge.ResultsThe overall mortality rate was 53.7%. No significant difference was found between the PRE and POST groups. The mean length of hospital stay was also comparable between the two groups. The functional status of those who survived acute hospital care was significantly better in the POST group. Good outcome (GOS of 4 or 5) was achieved in 66.7% of the survivors in the POST group, compared with 25.0% in the PRE group (p = 0.024).ConclusionPost-traumatic ASDH carried a poor prognosis. The mortality rate and hospital length of stay of patients were not found to be reduced after the establishment of a hospital trauma team. The latter, however, was associated with significantly better functional outcome amongst survivors. Although causality cannot be established due to the multitude of factors which may have affected patient outcome, our findings nonetheless provide further support for the introduction of a multidisciplinary hospital trauma team for the optimal care of trauma patients.     

Toll-like receptor 4 promotes tubular inflammation in diabetic nephropathy

Inflammation contributes to the tubulointerstitial lesions of diabetic nephropathy. Toll-like receptors (TLRs) modulate immune responses and inflammatory diseases, but their role in diabetic nephropathy is not well understood. In this study, we found increased expression of TLR4 but not of TLR2 in the renal tubules of human kidneys with diabetic nephropathy compared with expression of TLR4 and TLR2 in normal kidney and in kidney disease from other causes. The intensity of tubular TLR4 expression correlated directly with interstitial macrophage infiltration and hemoglobin A1c level and inversely with estimated glomerular filtration rate. The tubules also upregulated the endogenous TLR4 ligand high-mobility group box 1 in diabetic nephropathy. In vitro, high glucose induced TLR4 expression via protein kinase C activation in a time- and dose-dependent manner, resulting in upregulation of IL-6 and chemokine (C-C motif) ligand 2 (CCL-2) expression via IκB/NF-κB activation in human proximal tubular epithelial cells. Silencing of TLR4 with small interfering RNA attenuated high glucose-induced IκB/NF-κB activation, inhibited the downstream synthesis of IL-6 and CCL-2, and impaired the ability of conditioned media from high glucose-treated proximal tubule cells to induce transmigration of mononuclear cells. We observed similar effects using a TLR4-neutralizing antibody. Finally, streptozotocin-induced diabetic and uninephrectomized TLR4-deficient mice had significantly less albuminuria, renal dysfunction, renal cortical NF-κB activation, tubular CCL-2 expression, and interstitial macrophage infiltration than wild-type animals. Taken together, these data suggest that a TLR4-mediated pathway may promote tubulointerstitial inflammation in diabetic nephropathy.     

Pseudotyped adeno‐associated viral vector tropism and transduction efficiencies in murine wound healing

Cell specific gene transfer and sustained transgene expression are goals of cutaneous gene therapy for tissue repair and regeneration. Adeno‐associated virus serotype 2 (AAV2/2) mediated gene transfer to the skin results in stable transgene expression in the muscle fascicles of the panniculus carnosus in mice, with minimal gene transfer to the dermal or epidermal elements. We hypothesized that pseudotyped AAV vectors may have a unique and characteristic tropism and transduction efficiency profile for specific cells in the cutaneous wounds. We compared transduction efficiencies of cells in the epidermis, cells in the dermis, and the fascicles of the panniculus carnosus by AAV2/2 and three pseudotyped AAV vectors, AAV2/5, AAV2/7, and AAV2/8 in a murine excisional wound model. AAV2/5 and AAV2/8 result in significantly enhanced transduction of cells both in the epidermis and the dermis compared to AAV2/2. AAV2/5 transduces both the basilar and supra‐basilar keratinocytes. In contrast, AAV2/8 transduces mainly supra‐basilar keratinocytes. Both AAV2/7 and AAV2/8 result in more efficient gene transfer to the muscular panniculus carnosus compared to AAV2/2. The capsid of the different pseudotyped AAV vectors produces distinct tropism and efficiency profiles in the murine wound healing model. Both AAV2/5 and AAV2/8 administration result in significantly enhanced gene transfer. To further characterize cell specific transduction and tropism profiles of the AAV pseudotyped vectors, we performed in vitro experiments using human and mouse primary dermal fibroblasts. Our data demonstrate that pseudotyping strategy confers a differential transduction of dermal fibroblasts, with higher transduction of both human and murine cells by AAV2/5 and AAV2/8 at early and later time points. At later time points, AAV2/2 demonstrates increased transduction. Interestingly, AAV2/8 appears to be more efficacious in transducing human cells as compared to AAV2/5. The pseudotype‐specific pattern of transduction and tropism observed both in vivo and in vitro suggests that choice of AAV vectors should be based on the desired target cell and the timing of transgene expression in wound healing for gene transfer therapy in dermal wounds.