The rat SCO responsiveness to prolonged water deprivation: implication of Reissner's fiber and serotonin system

The osmotic stress is a potent stimulus that can trigger several peripheral as well as central impairments. The brain is a vulnerable target of the osmotic stress and particularly circumventricular organs (CVOs) regarding their strategic localization as sensory organs of biochemical changes in the blood and cerebrospinal fluid circulations. The subcommissural organ (SCO) is a CVO which releases doubly in the CSF and blood circulation a glycoprotein called Reissner's fiber (RF) that has been associated to several functions including electrolyte and water balances. The present work was aimed on the assessment of the secretory activity of the SCO and its serotoninergic innervation following 2 weeks of total water restriction in Wistar rat. Using the immunohistochemistry of RF and serotonin (5HT), our data showed a significant overall reduction of RF immunoreactivity within both ependymal and hypendymal cells of the SCO of dehydrated rats compared to their corresponding controls, this decrease was concomitant with an enhancement of fibers 5HT immunoreactivity in the SCO as well as in the classical ependyma and in the dorsal raphe nucleus (DRN), constituting the origin of this innervation. The present findings support the possible involvement of the SCO in the response to prolonged water deprivation by decreasing its secretory materials which may result from either a direct peripheral hormonal control and/or the consequence of the enhanced 5HT innervation of the SCO.     

Development and Clinical Evaluation of Clotrimazole–β-Cyclodextrin Eyedrops for the Treatment of Fungal Keratitis

Fungal keratitis is a serious corneal disease that may result in loss of vision. There are limited treatment options available in Iraqi eye hospitals which might be the main reason behind the poor prognosis of many cases. The purpose of this study was to prepare and pharmaceutically evaluate clotrimazole–β-cyclodextrin (CTZ–β-CD) eyedrops then clinically assess its therapeutic efficacy on fungal keratitis compared with extemporaneous amphotericin B eyedrops (0.5% w/v). A CTZ–β-CD ophthalmic solution was prepared and evaluated by various physicochemical, microbiological, and biological tests. The prepared formula was stable in 0.05 M phosphate buffer pH 7.0 at 40 ± 2°C and 75 ± 5% RH for a period of 6 months. Light has no significant effect on the formula’s stability. The CTZ–β-CD eyedrops efficiently complied with the isotonicity, sterility, and antimicrobiological preservative effectiveness tests. Results of the clinical study revealed that 20 (80%) patients showed a favorable response to the CTZ–β-CD eyedrops, while 16 patients (64%) exhibited a favorable response to amphotericin B (P > 0.05). The mean course of treatment was significantly (P < 0.05) less in the CTZ treatment group than in the amphotericin group (21.5 ± 5.2 vs. 28.3 ± 6.4 days, respectively). The CTZ formulation was significantly (P < 0.05) more effective in the management of severe cases and also against Candida sp. than amphotericin B. There was no significant difference (P < 0.05) between both therapies against filamentous fungi. The CTZ–β-CD formulation can be used alternatively to other ophthalmic antimycotic treatment options in developing countries where stability, cost, or efficacy is a limiting factor.Key words: clotrimazole, β-cyclodextrin, eyedrops, fungal keratitis, Iraq     

Development of automated imaging and analysis for zebrafish chemical screens.

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK pathway in zebrafish embryos. The zebrafish embryo is an ideal system for in vivo high-content chemical screens. The 1-day old embryo is approximately 1mm in diameter and can be easily arrayed into 96-well plates, a standard format for high throughput screening. During the first day of development, embryos are transparent with most of the major organs present, thus enabling visualization of tissue formation during embryogenesis. The complete automation of zebrafish chemical screens is still a challenge, however, particularly in the development of automated image acquisition and analysis. We previously generated a transgenic reporter line that expresses green fluorescent protein (GFP) under the control of FGF activity and demonstrated their utility in chemical screens ¹. To establish methodology for high throughput whole organism screens, we developed a system for automated imaging and analysis of zebrafish embryos at 24-48 hours post fertilization (hpf) in 96-well plates ². In this video we highlight the procedures for arraying transgenic embryos into multiwell plates at 24hpf and the addition of a small molecule (BCI) that hyperactivates FGF signaling ³. The plates are incubated for 6 hours followed by the addition of tricaine to anesthetize larvae prior to automated imaging on a Molecular Devices ImageXpress Ultra laser scanning confocal HCS reader. Images are processed by Definiens Developer software using a Cognition Network Technology algorithm that we developed to detect and quantify expression of GFP in the heads of transgenic embryos. In this example we highlight the ability of the algorithm to measure dose-dependent effects of BCI on GFP reporter gene expression in treated embryos.     

Old and new questions about cholinesterases

Cholinesterases have been intensively studied for a long time, but still offer many fascinating and fundamental questions regarding their evolution, activity, biosynthesis, folding, post-translational modifications, association with structural proteins (ColQ, PRiMA and maybe others), export or degradation. They constitute an excellent model to study these processes, particularly because of the sensitivity and specificity of enzymic assays. In addition, a number of provocative ideas concerning their proposed non-conventional, or non-catalytic functions deserve to be further documented.     

In vitro anti-proliferative activities of ellagic acid

The potential cytotoxic and anti-proliferative activities of ellagic acid (a naturally occurring bioactive compound in berries, grapes, and nuts) was evaluated using human umbilical vein endothelial cells (HUVEC), normal human lung fibroblast cells HEL 299, Caco-2 colon, MCF-7 breast, Hs 578T breast, and DU 145 human prostatic cancer cells. Ellagic acid at concentration in the range 10-100 micromol/L did not affect the viability of normal fibroblast cells during a 24-hour incubation. An increase in adenosine triphosphate (ATP) bioluminescence of approximately 18-21% was observed in normal cells incubated with ellagic acid. In contrast, ellagic acid at 1-100 micromol/L dose-dependently inhibited HUVEC tube formation and proliferation on a reconstituted extracellular matrix and showed strong anti-proliferative activity against the colon, breast, and prostatic cancer cell lines investigated. The most sensitive cells were the Caco-2, and the most resistant were the breast cancer cells. Ellagic acid induced cancer cell death by apoptosis as shown by the microscopic examination of cell gross morphology. Ellagic acid induced reduced cancer cell viability as shown by decreased ATP levels of the cancer cells. After 24 hours incubation of 100 micromol/L of ellagic acid with Caco-2, MCF-7, Hs 578T, and DU 145 cancer cells, ellagic acid suppressed fetal bovine serum (FBS) stimulation of cell migration. The apoptosis induction was accompanied by a decreased in the levels of pro-matrix metalloproteinase-2 (pro-MMP-2 or gelatinase A), pro-matrix metalloproteinase-9 (pro-MMP-9 or gelatinase B), and vascular endothelial growth factor (VEGF(165)) in conditioned media. The results suggest that ellagic acid expressed a selective cytotoxicity and anti-proliferative activity, and induced apoptosis in Caco-2, MCF-7, Hs 578T, and DU 145 cancer cells without any toxic effect on the viability of normal human lung fibroblast cells. It was also observed that the mechanism of apoptosis induction in ellagic acid-treated cancer cells was associated with decreased ATP production, which is crucial for the viability of cancer cells.     

Gene expression profiles during short‐term heat stress in the red sea coral Stylophora pistillata

During the past several decades, corals worldwide have been affected by severe bleaching events leading to wide‐spread coral mortality triggered by global warming. The symbiotic Red Sea coral Stylophora pistillata from the Gulf of Eilat is considered an opportunistic ‘r’ strategist. It can thrive in relatively unstable environments and is considered a stress‐tolerant species. Here, we used a S. pistillata custom microarray to examine gene expression patterns and cellular pathways during short‐term (13‐day) heat stress. The results allowed us to identify a two‐step reaction to heat stress, which intensified significantly as the temperature was raised to a 32 °C threshold, beyond which, coping strategies failed at 34 °C. We identified potential ‘early warning genes’ and ‘severe heat‐related genes’. Our findings suggest that during short‐term heat stress, S. pistillata may divert cellular energy into mechanisms such as the ER‐unfolded protein response (UPR) and ER‐associated degradation (ERAD) at the expense of growth and biomineralization processes in an effort to survive and subsequently recover from the stress. We suggest a mechanistic theory for the heat stress responses that may explain the success of some species which can thrive under a wider range of temperatures relative to others.     

Antibody light chain variable domains and their biophysically improved versions for human immunotherapy

We set out to gain deeper insight into the potential of antibody light chain variable domains (V_Ls) as immunotherapeutics. To this end, we generated a naïve human V_L phage display library and, by using a method previously shown to select for non-aggregating antibody heavy chain variable domains (V_Hs), we isolated a diversity of V_L domains by panning the library against B cell super-antigen protein L. Eight domains representing different germline origins were shown to be non-aggregating at concentrations as high as 450 µM, indicating V_L repertoires are a rich source of non-aggregating domains. In addition, the V_Ls demonstrated high expression yields in E. coli, protein L binding and high reversibility of thermal unfolding. A side-by-side comparison with a set of non-aggregating human V_Hs revealed that the V_Ls had similar overall profiles with respect to melting temperature (T_m), reversibility of thermal unfolding and resistance to gastrointestinal proteases. Successful engineering of a non-canonical disulfide linkage in the core of V_Ls did not compromise the non-aggregation state or protein L binding properties. Furthermore, the introduced disulfide bond significantly increased their T_ms, by 5.5–17.5 °C, and pepsin resistance, although it somewhat reduced expression yields and subtly changed the structure of V_Ls. Human V_Ls and engineered versions may make suitable therapeutics due to their desirable biophysical features. The disulfide linkage-engineered V_Ls may be the preferred therapeutic format because of their higher stability, especially for oral therapy applications that necessitate high resistance to the stomach’s acidic pH and pepsin.     

Phospholipase D protects ECV304 cells against TNFalpha-induced apoptosis

Tumor necrosis factor alpha (TNFalpha), a pleiotropic cytokine, activates both apoptotic and pro-survival signals depending on the cell model. Using ECV304 cells, which can be made TNFalpha-sensitive by cycloheximide (CHX) co-treatment, we evaluated the potential roles of ceramide and phospholipase D (PLD) in TNFalpha-induced apoptosis. TNFalpha/CHX induced a robust increase in ceramide levels after 16 h of treatment when cell death was maximal. PLD activity was increased at early time point (1h) whereas both PLD activity and PLD1 protein were strongly decreased after 24h. TNFalpha/CHX-induced cell death was significantly lowered by exogenous bacterial PLD and phoshatidic acid, and in cells overexpressing PLD1. Conversely, cells depleted in PLD proteins by small interference RNA (siRNA) treatment exhibited higher susceptibility to apoptosis. These results show that PLD exerts a protective role against TNFalpha-induced cell death.