Integrating gene expression and GO classification for PCA by preclustering

Background  

Gene expression data can be analyzed by summarizing groups of individual gene expression profiles based on GO annotation information. The mean expression profile per group can then be used to identify interesting GO categories in relation to the experimental settings. However, the expression profiles present in GO classes are often heterogeneous, i.e., there are several different expression profiles within one class. As a result, important experimental findings can be obscured because the summarizing profile does not seem to be of interest. We propose to tackle this problem by finding homogeneous subclasses within GO categories: preclustering.     

Phosphorylation of p38 by GRK2 at the docking groove unveils a novel mechanism for inactivating p38MAPK

p38 Mitogen-activated protein kinases (MAPK) are a family of Ser/Thr kinases that regulate important cellular processes such as stress responses, differentiation, and cell-cycle control . Activation of MAPK is achieved through a linear signaling cascade in which upstream kinases (MAPKKs) dually phosphorylate MAPKs at a conserved 3-amino-acid motif (Thr-X-Tyr) . G-protein-coupled receptor kinases (GRKs) are known to selectively phosphorylate G-protein-coupled receptors (GPCRs) and thus trigger desensitization . We report that GRK2 is a novel inactivating kinase of p38MAPK. p38 associates with GRK2 endogenously and is phosphorylated by GRK2 at Thr-123, a residue located at its docking groove. Mimicking phosphorylation at this site impairs the binding and activation of p38 by MKK6 and diminishes the capacity of p38 to bind and phosphorylate its substrates. Accordingly, p38 activation is decreased or increased when cellular GRK2 levels are enhanced or reduced, respectively. Changes in GRK2 levels and activity can modify p38-dependent processes such as differentiation of preadipocytic cells and LPS-induced cytokine release, enhanced in macrophages from GRK2(+/-) mice. Phosphorylation of p38 at a region key for its interaction with different partners uncovers a new mechanism for the regulation of this important family of kinases.     

Receptor specificity and functional comparison of recombinant sea bass (Dicentrarchus labrax) gonadotropins (FSH and LH) produced in different host systems

Different yields, biopotency, and in vivo pharmacokinetics are obtained for recombinant sea bass gonadoltropins depending on the production system and DNA construct, but they show specific activation of their corresponding receptors. Gonadotropins (GTHs) are glycoprotein hormones that play a major role in the regulation of gonadal functions. Recently, we succeeded in isolating the native sea bass Fsh from sea bass pituitaries, but to ensure the availability of bioactive GTHs and no cross-contamination with other related glycoproteins, recombinant sea bass GTHs were produced using two expression systems-insect and mammalian cells-and different constructs that yielded tethered or noncovalently bound dimers. Their production levels, binding specificity to their homologous cognate receptors, and bioactivity were investigated and compared. Both expression systems were successful in the generation of bioactive recombinant GTHs, but insect Sf9 cells yielded higher amounts of recombinant proteins than mammalian Chinese Hamster Ovary (CHO) stable clones. All recombinant GTHs activated their cognate receptors without cross-ligand binding and were able to stimulate sea bass gonadal steroidogenesis in vitro, although with different biopotencies. To assess their use for in vivo applications, their half-life in sea bass plasma was evaluated. Sf9-GTHs had a lower in vivo stability compared with CHO-GTHs due to their rapid clearance from the blood circulation. Cell-dependent glycosylation could be contributing to the final in vivo stability and biopotency of these recombinant glycoproteins. In conclusion, both insect and mammalian expression systems produced bioactive sea bass recombinant gonadotropins, although with particular features useful for different proposes (e.g., antibody production or in vivo studies, respectively).     

Sex-sorted bovine spermatozoa and DNA damage: II. Dynamic features

This study examined the dynamic response of Spermatozoa DNA Fragmentation after sex selection in bulls using a MoFlo^® SX (Beckman Coulter, Miami FL) spermatozoa sorter. The dynamic response of spermatozoa DNA fragmentation refers to the changing values of SDF, i.e., rate of SDF (rSDF), when analyzed periodically over a set incubation time at 37 °C. A dynamic assessment of SDF using non-sorted and sex-sorted spermatozoa samples during 72 h of incubation at 37 °C was performed. Results showed a reduced DNA longevity in sex-sorted frozen-thawed spermatozoa, with spermatozoa DNA damage appearing between 24 h and 48 h. The baseline SDF level was higher in conventional frozen-thawed than in sex-sorted frozen-thawed spermatozoa samples; while the reverse occurred for the rSDF. The afore-mentioned result produced a crossover point between both dynamic tendencies of SDF for sex-sorted versus conventional samples. We defined this crossover point as the Crossover Positioning Time (CPT) or the time (in hours) where both curves crossover after a period of spermatozoa incubation at 37 °C. The point at which the CPT occurs could be used as an indicator of the rSDF for individual bulls after X- and Y-chromosome bearing spermatozoa selection. CPT values produced a window of SDF ranging between 24 h and 48 h in the present experiment. It is proposed that higher values for CPT are indicative of bulls presenting chromatin that is more resistant to the external stressors affecting spermatozoa DNA after spermatozoa sorting.     

Simple and economic colloidal centrifugation protocols may be incorporated into the clinical equine sperm processing procedure

For many years in human assisted-reproduction procedures there have been special protocols to prepare and improve sperm quality. Colloidal centrifugation (CC) is a useful technique that has been proved to enhance semen quality by selection of the best spermatozoa for different species. Its use is recommended to improve fertility of subfertile stallions but current CC protocols are clinically complicated in the equine sperm processing technique due to economic and technical difficulties. The aim of this study was to determine the optimal processing procedures to adapt the use of a CC product (EquiPure?) in the equine reproduction industry. A total of nineteen ejaculates were collected from 10 Purebred Spanish Horses (P.R.E horses) using a Missouri artificial vagina. Gel-free semen aliquots were analyzed prior to treatment (control). Semen was subjected to one of six CC protocols with EquiPure? and centrifuged samples were statistically evaluated by ANOVA and Duncan tests (p<0.05) for sperm quality and recovery rate. We obtained higher values by colloidal centrifugation in LIN, STR and BCF variables and DNA fragmentation index trended to be lower in most of the CC protocols. The studied protocols were shown to be as efficient in improving equine sperm quality as the current commercial EquiPure?, with the added advantage of being much more economical and simple to use. According to these results it seems to be possible to incorporate single layer and or high colloidal centrifugation volume protocols what would make them simple, economic and clinically viable for the equine sperm processing procedure.     

First case of erysipelas in a free-ranging bottlenose dolphin (Tursiops truncatus) stranded in the Mediterranean Sea

An adult female bottlenose dolphin (Tursiops truncatus) stranded alive and subsequently died several minutes later on the Mediterranean coast of Spain on 14 July 2010. Clinical examination revealed foam through the blowhole and rales upon lung auscultation. On venipuncture, the blood was abnormally dense and dark. Hematological and biochemical abnormalities included dehydration, leukocytosis (48 600 leukocytes microl(-1)) characterized by neutrophilia (48 200 neutrophils microl(-1)), and elevated bilirubin (4.38 mg dl(-1)), alanine aminotransferase (382.3 U l(-1)), aspartate aminotransferase (1449.3 U l(-1)), lactate dehydrogenase (1631.3 U l(-1)), and creatine kinase (404.7 U l(-1)). The most relevant findings of the gross examination were rhomboid-shaped skin lesions, stable froth in the trachea, pulmonary congestion, abnormally thick and rough pleura with adhesions, edematous and congestive superficial cervical and tracheobronchial lymph nodes, red-tinged urine, and severe brain congestion. Histopathology of the kidney, lung, skin, and brain revealed multisystemic intravascular bacterial emboli. Samples of skin, brain, and lung were cultured on Columbia blood agar under both aerobic and anaerobic conditions, and pure and heavy bacterial cultures were obtained from skin and brain samples. The microorganism isolated was Gram-positive, catalase-negative, facultatively anaerobic, and rod-shaped. The isolates were identified as Erysipelothrix rhusiopathiae by the API Coryne biochemical system. Based on the gross and microscopic findings, a diagnosis of acute E. rhusiopathiae septicemia was made. To the best of our knowledge, this is the first report of E. rhusiopathiae septicemia in a free-ranging bottlenose dolphin.     

Cardiotrophin-1 promotes a high survival rate in rabbits with lethal fulminant hepatitis of viral origin

Rabbit hemorrhagic disease virus (RHDV) causes lethal fulminant hepatitis closely resembling acute liver failure (ALF) in humans. In this study, we investigated whether cardiotrophin-1 (CT-1), a cytokine with hepatoprotective properties, could attenuate liver damage and prolong survival in virus-induced ALF. Twenty-four rabbits were infected with 2 × 10(4) hemagglutination units of RHDV. Twelve received five doses of CT-1 (100 μg/kg) starting at 12 h postinfection (hpi) (the first three doses every 6 h and then two additional doses at 48 and 72 hpi), while the rest received saline. The animals were analyzed for survival, serum biochemistry, and viral load. Another cohort (n = 22) was infected and treated similarly, but animals were sacrificed at 30 and 36 hpi to analyze liver histology, viral load, and the expression of factors implicated in liver damage and repair. All infected rabbits that received saline died by 60 hpi, while 67% of the CT-1-treated animals survived until the end of the study. Treated animals showed improved liver function and histology, while the viral loads were similar. In the livers of CT-1-treated rabbits we observed reduction of oxidative stress, diminished PARP1/2 and JNK activation, and decreased inflammatory reaction, as reflected by reduced expression of tumor necrosis factor alpha, interleukin-1β, Toll-like receptor 4, VCAM-1, and MMP-9. In addition, CT-1-treated rabbits exhibited marked upregulation of TIMP-1 and increased expression of cytoprotective and proregenerative growth factors, including platelet-derived growth factor B, epidermal growth factor, platelet-derived growth factor receptor β, and c-Met. In conclusion, in a lethal form of acute viral hepatitis, CT-1 increases animal survival by attenuating inflammation and activating cytoprotective mechanisms, thus representing a promising therapy for ALF of viral origin.     

Cork stoppers industry: defining appropriate mould colonization

AIMS: The main aims of this work were the study of cork slabs moulds colonization and the evaluation of the moulds diversity during cork processing steps, in different cork stoppers factories. Simultaneously, it was envisaged to perform an evaluation of the air quality. METHODS AND RESULTS: Moulds were isolated and identified from cork slabs and cork samples in four cork stoppers factories. The identification was based on morphological characters and microscopic observation of the reproductive structures. Airborne spore dispersion was assessed using a two stage Andersen sampler. It was observed that Chrysonilia sitophila was always present on cork slabs during the maturing period, but mould diversity appeared to be associated to the different factory configurations and processing steps. CONCLUSIONS: Spatial separation of the different steps of the process, including physical separation of the maturation step, is essential to guarantee high air quality and appropriate cork slabs colonization, i.e. C. sitophila dominance. The sorting and cutting of the edges of cork slabs after boiling and before the maturing step is also recommended. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is very important for the cork stopper industry as it gives clear indications on how to keep high quality manufacturing standards and how to avoid occupational health problems.