Analysis of protein expression in zebrafish during gonad differentiation by targeted proteomics

The molecular mechanisms governing sex determination and differentiation in the zebrafish (Danio rerio) are not fully understood. To gain more insights into the function of specific genes in these complex processes, the expression of multiple candidates needs to be assessed, preferably on the protein level. Here, we developed a targeted proteomics method based on selected reaction monitoring (SRM) to study the candidate sex-related proteins in zebrafish which were selected based on a global proteomics analysis of adult gonads and representational difference analysis of male and female DNA, as well as on published information on zebrafish and other vertebrates. We employed the developed SRM protocols to acquire time-resolved protein expression profiles during the gonad differentiation period in vas::EGFP transgenic zebrafish. Evidence on protein expression was obtained for the first time for several candidate genes previously studied only on the mRNA level or suggested by bioinformatic predictions. Tuba1b (tubulin alpha 1b), initially included in the study as one of the potential housekeeping proteins, was found to be preferentially expressed in the adult testis with nearly absent expression in the ovary. The revealed changes in protein expression patterns associated with gonad differentiation suggest that several of the examined proteins, especially Ilf2 and Ilf3 (interleukin enhancer-binding factors 2 and 3), Raldh3 (retinaldehyde dehydrogenase type 3), Zgc:195027 (low density lipoprotein-related receptor protein 3) and Sept5a (septin 5a), may play a specific role in the sexual differentiation in zebrafish.

On the road through the Bolivian Amazon: a multi-level land governance analysis of deforestation

Previous studies have shown that collective property rights offer higher flexibility than individual property and improve sustainable community-based forest management. Our case study, carried out in the Beni department of Bolivia, does not contradict this assertion, but shows that collective rights have been granted in areas where ecological contexts and market facilities were less favourable to intensive land use. Previous experiences suggest investigating political processes in order to understand the criteria according to which access rights were distributed. Based on remote sensing and on a multi-level land governance framework, our research confirms that land placed under collective rights, compared to individual property, is less affected by deforestation among Andean settlements. However, analysis of the historical process of land distribution in the area shows that the distribution of property rights is the result of a political process based on economic, spatial, and environmental strategies that are defined by multiple stakeholders. Collective titles were established in the more remote areas and distributed to communities with lower productive potentialities. Land rights are thus a secondary factor of forest cover change which results from diverse political compromises based on population distribution, accessibility, environmental perceptions, and expected production or extraction incomes.

An examination of the Gonorrhea Cases & Places Study: An analysis of the Theory of Gender and Power, Situational/Environmental Variables Theory, and Sexual Script Theory as it relates to risky sexual behavior in African American adults

Objective. The purpose of the study is to provide a holistic depiction of behavioral & environmental factors contributing to risky sexual behaviors among predominantly high school educated, low-income African Americans residing in urban areas of Houston, TX utilizing the Theory of Gender and Power, Situational/Environmental Variables Theory, and Sexual Script Theory. Methods. A cross-sectional study was conducted via questionnaires among 215 Houston area residents, 149 were women and 66 were male. Measures used to assess behaviors of the population included a history of homelessness, use of crack/cocaine among several other illicit drugs, the type of sexual partner, age of participant, age of most recent sex partner, whether or not participants sought health care in the last 12 months, knowledge of partner's other sexual activities, symptoms of depression, and places where partner's were met. In an effort to determine risk of sexual encounters, a risk index employing the variables used to assess condom use was created categorizing sexual encounters as unsafe or safe. Results. Variables meeting the significance level of p<.15 for the bivariate analysis of each theory were entered into a binary logistic regression analysis. The block for each theory was significant, suggesting that the grouping assignments of each variable by theory were significantly associated with unsafe sexual behaviors. Within the regression analysis, variables such as sex for drugs/money, low income, and crack use demonstrated an effect size of ≥ ± 1, indicating that these variables had a significant effect on unsafe sexual behavioral practices. Conclusions. Variables assessing behavior and environment demonstrated a significant effect when categorized by relation to designated theories.

Bifurcation and singularity analysis of a molecular network for the induction of long-term memory.

Withdrawal reflexes of the mollusk Aplysia exhibit sensitization, a simple form of long-term memory (LTM). Sensitization is due, in part, to long-term facilitation (LTF) of sensorimotor neuron synapses. LTF is induced by the modulatory actions of serotonin (5-HT). Pettigrew et al. developed a computational model of the nonlinear intracellular signaling and gene network that underlies the induction of 5-HT-induced LTF. The model simulated empirical observations that repeated applications of 5-HT induce persistent activation of protein kinase A (PKA) and that this persistent activation requires a suprathreshold exposure of 5-HT. This study extends the analysis of the Pettigrew model by applying bifurcation analysis, singularity theory, and numerical simulation. Using singularity theory, classification diagrams of parameter space were constructed, identifying regions with qualitatively different steady-state behaviors. The graphical representation of these regions illustrates the robustness of these regions to changes in model parameters. Because persistent protein kinase A (PKA) activity correlates with Aplysia LTM, the analysis focuses on a positive feedback loop in the model that tends to maintain PKA activity. In this loop, PKA phosphorylates a transcription factor (TF-1), thereby increasing the expression of an ubiquitin hydrolase (Ap-Uch). Ap-Uch then acts to increase PKA activity, closing the loop. This positive feedback loop manifests multiple, coexisting steady states, or multiplicity, which provides a mechanism for a bistable switch in PKA activity. After the removal of 5-HT, the PKA activity either returns to its basal level (reversible switch) or remains at a high level (irreversible switch). Such an irreversible switch might be a mechanism that contributes to the persistence of LTM. The classification diagrams also identify parameters and processes that might be manipulated, perhaps pharmacologically, to enhance the induction of memory. Rational drug design, to affect complex processes such as memory formation, can benefit from this type of analysis.

Occupational therapy articles in serial publications: an analysis of sources.

This study was designed to locate and document serial literature on occupational therapy published since 1900. Emphasis is placed on finding articles on occupational therapy or by occupational therapists from sources other than those normally associated with the professional journals. Multiple sources were used including print indexes, online databases, occupational therapy bibliographies, and tables of contents or yearly indexes. Almost 7,000 articles were identified, not including those published in foreign journals. Occupational therapy publications have increased steadily since 1900, with the most rapid increase during the 1970s and 1980s when five new occupational therapy journals were initiated. Suggestions for formulating search strategies are included.

Analysis of clonality and antibiotic resistance among early clinical isolates of Enterococcus faecium in the United States.

BACKGROUND: The Enterococcus faecium genogroup, referred to as clonal complex 17 (CC17), seems to possess multiple determinants that increase its ability to survive and cause disease in nosocomial environments. METHODS: Using 53 clinical and geographically diverse US E. faecium isolates dating from 1971 to 1994, we determined the multilocus sequence type; the presence of 16 putative virulence genes (hyl(Efm), esp(Efm), and fms genes); resistance to ampicillin (AMP) and vancomycin (VAN); and high-level resistance to gentamicin and streptomycin. RESULTS: Overall, 16 different sequence types (STs), mostly CC17 isolates, were identified in 9 different regions of the United States. The earliest CC17 isolates were part of an outbreak that occurred in 1982 in Richmond, Virginia. The characteristics of CC17 isolates included increases in resistance to AMP, the presence of hyl(Efm) and esp(Efm), emergence of resistance to VAN, and the presence of at least 13 of 14 fms genes. Eight of 41 of the early isolates with resistance to AMP, however, were not in CC17. CONCLUSIONS: Although not all early US AMP isolates were clonally related, E. faecium CC17 isolates have been circulating in the United States since at least 1982 and appear to have progressively acquired additional virulence and antibiotic resistance determinants, perhaps explaining the recent success of this species in the hospital environment.

Identification and phenotypic characterization of a second collagen adhesin, Scm, and genome-based identification and analysis of 13 other predicted MSCRAMMs, including four distinct pilus loci, in Enterococcus faecium.

Attention has recently been drawn to Enterococcus faecium because of an increasing number of nosocomial infections caused by this species and its resistance to multiple antibacterial agents. However, relatively little is known about the pathogenic determinants of this organism. We have previously identified a cell-wall-anchored collagen adhesin, Acm, produced by some isolates of E. faecium, and a secreted antigen, SagA, exhibiting broad-spectrum binding to extracellular matrix proteins. Here, we analysed the draft genome of strain TX0016 for potential microbial surface components recognizing adhesive matrix molecules (MSCRAMMs). Genome-based bioinformatics identified 22 predicted cell-wall-anchored E. faecium surface proteins (Fms), of which 15 (including Acm) had characteristics typical of MSCRAMMs, including predicted folding into a modular architecture with multiple immunoglobulin-like domains. Functional characterization of one [Fms10; redesignated second collagen adhesin of E. faecium (Scm)] revealed that recombinant Scm(65) (A- and B-domains) and Scm(36) (A-domain) bound to collagen type V efficiently in a concentration-dependent manner, bound considerably less to collagen type I and fibrinogen, and differed from Acm in their binding specificities to collagen types IV and V. Results from far-UV circular dichroism measurements of recombinant Scm(36) and of Acm(37) indicated that these proteins were rich in beta-sheets, supporting our folding predictions. Whole-cell ELISA and FACS analyses unambiguously demonstrated surface expression of Scm in most E. faecium isolates. Strikingly, 11 of the 15 predicted MSCRAMMs clustered in four loci, each with a class C sortase gene; nine of these showed similarity to Enterococcus faecalis Ebp pilus subunits and also contained motifs essential for pilus assembly. Antibodies against one of the predicted major pilus proteins, Fms9 (redesignated EbpC(fm)), detected a 'ladder' pattern of high-molecular-mass protein bands in a Western blot analysis of cell surface extracts from E. faecium, suggesting that EbpC(fm) is polymerized into a pilus structure. Further analysis of the transcripts of the corresponding gene cluster indicated that fms1 (ebpA(fm)), fms5 (ebpB(fm)) and ebpC(fm) are co-transcribed, a result consistent with those for pilus-encoding gene clusters of other Gram-positive bacteria. All 15 genes occurred frequently in 30 clinically derived diverse E. faecium isolates tested. The common occurrence of MSCRAMM- and pilus-encoding genes and the presence of a second collagen-binding protein may have important implications for our understanding of this emerging pathogen.

{\it Hox\/} genes and specification of regional identity in the axial skeleton: Analysis of the individual and combined mutant phenotypes of the paralogous group 4 murine {\it Hox\/} genes; {\it hoxa\/}-4, {\it hoxb\/}-4 and {\it hoxd\/}-4

The Hox gene products are transcription factors involved in specifying regional identity along the anteroposterior body axis. In Drosophila, where these genes are known as HOM-C (Homeotic-complex) genes and where they have been most extensively studied, they are expressed in restricted domains along the anteroposterior axis with different anterior limits. Genetic analysis of a large number of gain- and loss-of-function alleles of these genes has revealed that these genes are important in specifying segmental identity at their anterior limits of expression. Furthermore, there is a functional dominance of posterior genes over anterior genes, such that posterior genes can dominantly specify their developmental programs in spite of the expression of more anterior genes in the same segment. In the mouse, there are four clusters of HOM-C genes, called Hox genes. Thus, there may be up to four genes, called paralogs, that are more highly homologous to each other and to their Drosophila homolog than they are to the other mouse Hox genes. The single mutants for two paralogous genes, hoxa-4 and hoxd-4, presented in this dissertation, are similar to several other mouse Hox mutants in that they show partial, incompletely penetrant homeotic transformations of vertebrae at their anterior limit of expression. These mutants were then bred with hoxb-4 mutants (Ramirez-Solis, et al. 1993) to generate the three possible double mutant combinations as well as the triple mutant. The skeletal phenotypes of these group 4 Hox compound mutants displayed clear alterations in regional identity, such that a nearly complete transformation towards the morphology of the first cervical vertebra occurs. These results suggest a certain degree of functional redundancy among paralogous genes in specifying regional identity. Furthermore, there was a remarkable dose-dependent increase in the number of vertebrae transformed to a first cervical vertebra identity, including the second through the fifth cervical vertebrae in the triple mutant. Thus, these genes are required in a larger anteroposterior domain than is revealed by the single mutant phenotypes alone, such that multiple mutations in these genes result in transformations of vertebrae that are not at their anterior limit of expression. ^

Functional analysis of histones H3 and H4 in Tup1 repression and in GCN5 activation

Tup1 forms a complex with Ssn6 in yeast. Ssn6-Tup1 complex is recruited via direct interactions with specific DNA binding proteins to a specific promoter region and mediates repression of several sets of genes including a-cell specific genes (asg) in $\alpha$ cells. It has been shown that repression of asgs also requires histone H4 and that Tup1 can directly interact with H3 and H4 in vitro. To address whether histone H3 is required for the repression of asgs, I have examined the effect of H3 and H4 mutations on the expression of a $\alpha$2-controlled LacZ reporter. Assay of $\beta$-glactosidase shows that mutations in either H3 or H4 cause a weak derepression of the reporter gene. Some double mutations result in a stronger derepression, while others do not. The H3 N-terminal deletion also leads to a slightly decreased expression of the reporter gene in $\alpha$ cells. Our data suggest that the N-termini of both H3 and H4 are cooperatively involved in the repression of a-cell specific genes in $\alpha$ cells, possibly through their interaction with Tup1.^ GCN5 was originally identified as a transcriptional regulator required to activate a subset of genes in yeast. Recently, it has been shown that GCN5 encodes the catalytic subunit of a nuclear histone acetyltransferase, providing the first direct link between histone acetylation and gene transcription. Recombinant Gcn5p (rGcn5p) exhibits a limited substrate specificity in vitro. However, neither the specificity of this enzyme in vivo nor the importance of particular acetylated residues to transcription or cell growth are well defined. In order to define the sites of histone acetylation mediated by Gcn5p in vivo and assess the significance of histone acetylation, more than 30 yeast strains have been constructed to bear specific H3 and/or H4 mutations in the presence or absence of GCN5 function. Our genetic data suggest that Gcn5p may have additional targets in vivo that are not identified as the targets of rGcn5p by previous studies. Western analysis using antibodies specifically recognizing particular acetylated isoforms of H3 and H4 led us to conclude that Gcn5p is necessary for full acetylation of multiple sites in both H3 and H4 in vivo. Consistent with these observations, rGcn5p still acetylates histones H3 and H4 bearing mutations either in H3 K14 or H4 K8,16, sites previously identified as the targets of acetylation by rGcn5p in H3 and H4. Our data also demonstrated that Gcn5p-mediated acetylation events are important for normal progression of the cell cycle and for transcriptional activation. Furthermore, a critical overall level of acetylation is essential for cell viability. ^

The Application of Comorbidity Indices to Predict Early Postoperative Outcomes After Laparoscopic Roux-en-Y Gastric Bypass: A Nationwide Comparative Analysis of Over 70,000 Cases

BACKGROUND: Patients undergoing laparoscopic Roux-en-Y gastric bypass (LRYGB) often have substantial comorbidities, which must be taken into account to appropriately assess expected postoperative outcomes. The Charlson/Deyo and Elixhauser indices are widely used comorbidity measures, both of which also have revised algorithms based on enhanced ICD-9-CM coding. It is currently unclear which of the existing comorbidity measures best predicts early postoperative outcomes following LRYGB. METHODS: Using the Nationwide Inpatient Sample, patients 18 years or older undergoing LRYGB for obesity between 2001 and 2008 were identified. Comorbidities were assessed according to the original and enhanced Charlson/Deyo and Elixhauser indices. Using multivariate logistic regression, the following early postoperative outcomes were assessed: overall postoperative complications, length of hospital stay, and conversion to open surgery. Model performance for the four comorbidity indices was assessed and compared using C-statistics and the Akaike's information criterion (AIC). RESULTS: A total of 70,287 patients were included. Mean age was 43.1 years (SD, 10.8), 81.6 % were female and 60.3 % were White. Both the original and enhanced Elixhauser indices modestly outperformed the Charlson/Deyo in predicting the surgical outcomes. All four models had similar C-statistics, but the original Elixhauser index was associated with the smallest AIC for all of the surgical outcomes. CONCLUSIONS: The original Elixhauser index is the best predictor of early postoperative outcomes in our cohort of patients undergoing LRYGB. However, differences between the Charlson/Deyo and Elixhauser indices are modest, and each of these indices provides clinically relevant insight for predicting early postoperative outcomes in this high-risk patient population.

Relationship between onset-to-door time and door-to-thrombolysis time: a pooled analysis of 10 dedicated stroke centers

BACKGROUND AND PURPOSE Inverse relationship between onset-to-door time (ODT) and door-to-needle time (DNT) in stroke thrombolysis was reported from various registries. We analyzed this relationship and other determinants of DNT in dedicated stroke centers. METHODS Prospectively collected data of consecutive ischemic stroke patients from 10 centers who received IV thrombolysis within 4.5 hours from symptom onset were merged (n=7106). DNT was analyzed as a function of demographic and prehospital variables using regression analyses, and change over time was considered. RESULTS In 6348 eligible patients with known treatment delays, median DNT was 42 minutes and kept decreasing steeply every year (P<0.001). Median DNT of 55 minutes was observed in patients with ODT ≤30 minutes, whereas it declined for patients presenting within the last 30 minutes of the 3-hour time window (median, 33 minutes) and of the 4.5-hour time window (20 minutes). For ODT within the first 30 minutes of the extended time window (181-210 minutes), DNT increased to 42 minutes. DNT was stable for ODT for 30 to 150 minutes (40-45 minutes). We found a weak inverse overall correlation between ODT and DNT (R(2)=-0.12; P<0.001), but it was strong in patients treated between 3 and 4.5 hours (R(2)=-0.75; P<0.001). ODT was independently inversely associated with DNT (P<0.001) in regression analysis. Octogenarians and women tended to have longer DNT. CONCLUSIONS DNT was decreasing steeply over the last years in dedicated stroke centers; however, significant oscillations of in-hospital treatment delays occurred at both ends of the time window. This suggests that further improvements can be achieved, particularly in the elderly.

A Comparative Analysis of Beaconless Opportunistic Routing Protocols for Video Dissemination over Flying Ad-Hoc Networks

A reliable and robust routing service for Flying Ad-Hoc Networks (FANETs) must be able to adapt to topology changes, and also to recover the quality level of the delivered multiple video flows under dynamic network topologies. The user experience on watching live videos must also be satisfactory even in scenarios with network congestion, buffer overflow, and packet loss ratio, as experienced in many FANET multimedia applications. In this paper, we perform a comparative simulation study to assess the robustness, reliability, and quality level of videos transmitted via well-known beaconless opportunistic routing protocols. Simulation results shows that our developed protocol XLinGO achieves multimedia dissemination with Quality of Experience (QoE) support and robustness in a multi-hop, multi-flow, and mobile networks, as required in many multimedia FANET scenarios.

Analysis of caesarean section rates over time in a single Swiss centre using a ten-group classification system.

OBJECTIVE Caesarean section (CS) rates have risen over the past two decades. The aim of this observational study was to identify time-dependent variations in CS and vaginal delivery rates over a period of 11 years. METHOD All deliveries (13,701 deliveries during the period 1999-2009) at the University Women's Hospital Bern were analysed using an internationally standardised and approved ten-group classification system. Caesarean sections on maternal request (CSMR) were evaluated separately. RESULTS We detected an overall CS rate of 36.63% and an increase in the CS rate over time (p <0.001). Low-risk profile groups were the two largest populations and displayed low CS rates, with significantly decreasing relative size over time. The relative size of groups with induced labour increased significantly, but this did not have an impact on the overall CS rate. Pregnancies complicated by breech position, multiple pregnancies and abnormal lies did not have an impact on overall CS rate. The biggest contributor to a high CS rate was preterm delivery and the existence of a uterine scar from a previous CS. CSMR was 1.45% and did not have an impact on the overall CS rate. CONCLUSION The observational study identified wide variations in caesarean section and vaginal delivery rates across the groups over time, and a shift towards high-risk populations was noted. The biggest contributors to high CS rates were identified; namely, previous uterine scar and preterm delivery. Interventions aiming to reduce CS rates are planned.

Sleep-Disordered Breathing and Periodic Limb Movements in Narcolepsy with Cataplexy: A Systematic Analysis of 35 Consecutive Patients

Background: Disturbed sleep is a core feature of narcolepsy with cataplexy (NC). Few studies have independently assessed sleep-disordered breathing (SDB) and periodic limb movements (PLMs) in non-homogeneous series of patients with and without cataplexy. We systematically assessed both SDB and PLMs in well-defined NC patients. Methods: We analyzed the clinical and polysomnographic features of 35 consecutive NC patients (mean age 40 ± 16 years, 51% males, 23/23 hypocretin-deficient) to assess the prevalence of SDB (apnea-hypopnea index >5) and PLMs (periodic leg movements in sleep (PLMI) >15) together with their impact on nocturnal sleep and daytime sleepiness using the multiple sleep latency test. Results: 11 (31%) and 14 (40%) patients had SDB and PLMs, respectively. SDB was associated with older age (49 ± 16 vs. 35 ± 13 years, p = 0.02), higher BMI (30 ± 5 vs. 27 ± 6, p = 0.05), and a trend towards higher PLMI (25 ± 20 vs. 12 ± 23, p = 0.052), whereas PLMs with older age (50 ± 16 vs. 33 ± 11 years, p = 0.002) and reduced and fragmented sleep (e.g. sleep efficiency of 82 ± 12% vs. 91 ± 6%, p = 0.015; sleep time of 353 ± 66 vs. 395 ± 28, p = 0.010). SDB and PLMs were also mutually associated (p = 0.007), but not correlated to daytime sleepiness. Conclusions: SDB and PLMs are highly prevalent and associated in NC. Nevertheless, SDB and PLMs are rarely severe, suggesting an overall limited effect on clinical manifestations.

Predictors of adverse events among patients undergoing primary percutaneous coronary intervention: insights from a pooled analysis of the COMFORTABLE AMI and EXAMINATION trials

Aims: The aim of this study was to identify predictors of adverse events among patients with ST-elevation myocardial infarction (STEMI) undergoing contemporary primary percutaneous coronary intervention (PCI). Methods and results: Individual data of 2,655 patients from two primary PCI trials (EXAMINATION, N=1,504; COMFORTABLE AMI, N=1,161) with identical endpoint definitions and event adjudication were pooled. Predictors of all-cause death or any reinfarction and definite stent thrombosis (ST) and target lesion revascularisation (TLR) outcomes at one year were identified by multivariable Cox regression analysis. Killip class III or IV was the strongest predictor of all-cause death or any reinfarction (OR 5.11, 95% CI: 2.48-10.52), definite ST (OR 7.74, 95% CI: 2.87-20.93), and TLR (OR 2.88, 95% CI: 1.17-7.06). Impaired left ventricular ejection fraction (OR 4.77, 95% CI: 2.10-10.82), final TIMI flow 0-2 (OR 1.93, 95% CI: 1.05-3.54), arterial hypertension (OR 1.69, 95% CI: 1.11-2.59), age (OR 1.68, 95% CI: 1.41-2.01), and peak CK (OR 1.25, 95% CI: 1.02-1.54) were independent predictors of all-cause death or any reinfarction. Allocation to treatment with DES was an independent predictor of a lower risk of definite ST (OR 0.35, 95% CI: 0.16-0.74) and any TLR (OR 0.34, 95% CI: 0.21-0.54). Conclusions: Killip class remains the strongest predictor of all-cause death or any reinfarction among STEMI patients undergoing primary PCI. DES use independently predicts a lower risk of TLR and definite ST compared with BMS. The COMFORTABLE AMI trial is registered at: http://www.clinicaltrials.gov/ct2/show/NCT00962416. The EXAMINATION trial is registered at: http://www.clinicaltrials.gov/ct2/show/NCT00828087.