Expanding the limits of computer-assisted sperm analysis through the development of open software

Computer assisted sperm analysis (CASA) systems can reduce errors occurring in manual analysis. However, commercial CASA systems are frequently not applicable at the forefront of challenging research endeavors. The development of open source software may offer important solutions for researchers working in related areas. Here, we present an example of this, with the development of three new modules for the OpenCASA software (hosted at Github). The first is theChemotactic Sperm Accumulation Module, a powerful tool for studying sperm chemotactic behavior, analyzing the sperm accumulation in the direct vicinity of the stimuli. This module was validated by comparing fish sperm accumulation, with or without the influence of an attractant. The analysis clearly indicated cell accumulation in the treatment group, while the distribution of sperm was random in the control group. The second is the Sperm Functionality Module, based on the ability to recognize five sperm subpopulations according to their fluorescence patterns associated with the plasma membrane and acrosomal status. The last module is the Sperm Concentration Module, which expands the utilities of OpenCASA. These last two modules were validated, using bull sperm, by comparing them with visual counting by an observer. A high level of correlation was achieved in almost all the data, and a good agreement between both methods was obtained. With these newly developed modules, OpenCASA is consolidated as a powerful free and open-source tool that allowsdifferent aspects of sperm quality to be evaluated, with many potential applications for researchers

DNA sonication inverse PCR for genome scale analysis of uncharacterized flanking sequences

1. There are few available tools to comprehensively and economically identify uncharacterized flanking regions that are not extremely labour intensive and which exploit the advantages of emerging long‐read sequencing platforms. 2. We describe SIP; a sonication‐based inverse PCR high‐throughput sequencing strategy to investigate uncharacterized flanking region sequences, including those flanking mobile DNA. SIP combines unbiased fragmentation by sonication and target enrichment by coupling outward facing PCR priming with long‐read sequencing technologies. 3. We demonstrate the effectiveness of SIP by determining retroviral integrations which are high copy and challenging to characterize. We further describe SIP's workflow, examine retroviral (proviral) enrichment and characterize viral structural variants identified. When SIP was coupled with long‐read sequencing using the PacBio RS II platform, proviral integration was extensively characterized at high sequence depth per integration. By interrogating the sequence data, we were also able to test several intrinsic factors including SIP's propensity to form chimeric sequences and adapter ligation efficiencies. 4. SIP is an adaption of a traditional molecular biology technique that can be used to characterize any unknown genomic flanking sequence or to extend any sequence for which only minimal sequence information is available. SIP can be applied broadly to study complex biological systems such as mobile genetic elements with high throughput

Estudo clínico duplo cego comparando praziquantel com oxamniquine

Com objetivo de se compararem a tolerabilidade e eficácia do praziquantel e oxamniquine, procedeu-se a um estudo prospectivo duplo-cego envolvendo 120 pacientes com esquistossomose intestinal ou hepatintestinal. Os pacientes foram randomizados em dois grupos. Um foi tratado com praziquantel, na dose de 55 mg/kg de peso, o outro com oxamniquine, 15 mg/kg de peso, sempre administrados em dose única por via oral. O diagnóstico e seguimento parasitológicos basearam-se ho exame de fazes peio método de Kate Katz. Em 73 de 77 casos negativos após tratamento, executaram-se biópsias retais. Efeitos colaterais, principalmente tontura, sonolência, dores abdominais, cefaléia, náuseas e diarréia foram observados em 87% dos casos. Sua incidência, intensidade e duração foram semelhantes em ambos os grupos, mas a dor abdominal foi significativamente mais freqüente após praziquantel, havendo maior tendência para tontura intensa após oxamniquine. Observou-se aumento significante de alamina-aminotransferase e gama-glutamiltransferase após oxamniquine e de bilirrubina total após praziquantel. Um total de 48 pacientes tratados com praziquantel e 46 com oxamniquine completaram os exames de controle até o sexto mês. As percentages de cura foram de 79,2% e de 84,8% respectivamente, diferença não significativa. Os pacientes não curados mostraram redução média do número de ovos de 93,5% e de 84,1%, diferença não significativa. Cinco pacientes retratados com praziquantel curaram-se, mas somente um de três retratados com oxamniquine. Estes resultados mostram, que ambas as drogas-apesar de diferentes propriedades farmacológicas provocam reações colaterais semelhantes e apresentam eficácia terapêutica comparável.A double-blind clinical trial involving 120 patients with chronic schistosomiasis was carried out to compare the tolerability and efficacy of praziquantel and oxamniquine. The patients were randomly allocated into two groups. One was treated with praziquantel, 55 mg/kg of body weight CBWT), and the other one with oxamniquine, 15mg/kg bwt, administered in a single oral dose. The diagnosis and the parasitological follow-up was based on stool examinations by quantitative Kato-Katz method and on rectal biopsies. Side-effects mainly dizziness, sleepness, abdominal distress, headache, nausea and diarrhea were observed in 87% of the cases. Their incidence, intensity and duration were similar for both drugs but abdominal pain was significantly more frequent after praziquantel intake and severe dizziness was more commonly reported after oxamniquine. A significant increase of alanine-aminotransferase and y-glutamyltransferase was found with the latter drug and of total bilirubin with the former one. A total of 48 patients treated with praziquantel and 46 with oxamniquine completed with negative findings the required three post-treatment parasitological controls three slides of each stool sample on the first, third and sixth month. The achieved cure rates were 79.2% and 84.8%, respectively, a difference without statistical significance. The non-cured cases showed a mean reduction in the number of eggs per gram of feces of 93.5% after praziquantel and of 84.1% after oxamniquine. This diference also was not significant. Five patients retreated with praziquantel were cured but only one out of three treated a second time with oxamniquine. These findings show that both drugs despite their different chemical structures, pharmacological properties and mechanisms-of-action induce similar side-effects as well as a comparable therapeutical efficacy, in agreement with the results reported from analogous investigations

Inactivation of CDK/pRb Pathway Normalizes Survival Pattern of Lymphoblasts Expressing the FTLD-Progranulin Mutation c.709-1G>A

8 figuras, 2 tablasBackground Mutations in the progranulin (PGRN) gene, leading to haploinsufficiency, cause familial frontotemporal lobar degeneration (FTLD-TDP), although the pathogenic mechanism of PGRN deficit is largely unknown. Allelic loss of PGRN was previously shown to increase the activity of cyclin-dependent kinase (CDK) CDK6/pRb pathway in lymphoblasts expressing the c.709-1G>A PGRN mutation. Since members of the CDK family appear to play a role in neurodegenerative disorders and in apoptotic death of neurons subjected to various insults, we investigated the role of CDK6/pRb in cell survival/death mechanisms following serum deprivation. Methodology/Principal Findings We performed a comparative study of cell viability after serum withdrawal of established lymphoblastoid cell lines from control and carriers of c.709-1G>A PGRN mutation, asymptomatic and FTLD-TDP diagnosed individuals. Our results suggest that the CDK6/pRb pathway is enhanced in the c.709-1G>A bearing lymphoblasts. Apparently, this feature allows PGRN-deficient cells to escape from serum withdrawal-induced apoptosis by decreasing the activity of executive caspases and lowering the dissipation of mitochondrial membrane potential and the release of cytochrome c from the mitochondria. Inhibitors of CDK6 expression levels like sodium butyrate or the CDK6 activity such as PD332991 were able to restore the vulnerability of lymphoblasts from FTLD-TDP patients to trophic factor withdrawal. Conclusion/Significance The use of PGRN-deficient lymphoblasts from FTLD-TDP patients may be a useful model to investigate cell biochemical aspects of this disease. It is suggested that CDK6 could be potentially a therapeutic target for the treatment of the FTLD-TDPThis work has been supported by grants from Ministry of Education and Science (SAF2007-61701, SAF2010-15700, SAF2011-28603), Fundación Eugenio Rodríguez Pascual, and Basque Government (Saiotek program 2008–2009). NE holds a fellowship of the JAE predoctoral program of the CSICPeer reviewe

Adaptation and conservation insights from the koala genome

The koala, the only extant species of the marsupial family Phascolarctidae, is classified as ‘vulnerable’ due to habitat loss and widespread disease. We sequenced the koala genome, producing a complete and contiguous marsupial reference genome, including centromeres. We reveal that the koala’s ability to detoxify eucalypt foliage may be due to expansions within a cytochrome P450 gene family, and its ability to smell, taste and moderate ingestion of plant secondary metabolites may be due to expansions in the vomeronasal and taste receptors. We characterized novel lactation proteins that protect young in the pouch and annotated immune genes important for response to chlamydial disease. Historical demography showed a substantial population crash coincident with the decline of Australian megafauna, while contemporary populations had biogeographic boundaries and increased inbreeding in populations affected by historic translocations. We identified genetically diverse populations that require habitat corridors and instituting of translocation programs to aid the koala’s survival in the wild

Effects on short term outcome of non-invasive ventilation use in the emergency department to treat patients with acute heart failure: A propensity score-based analysis of the EAHFE Registry

Objective: To assess the effects of non-invasive ventilation (NIV) in emergency department (ED) patients with acute heart failure (AHF) on short term outcomes. Methods: Patients from the EAHFE Registry (a multicenter, observational, multipurpose, cohort-designed database including consecutive AHF patients in 41 Spanish EDs) were grouped based on NIV treatment (NIV+ and NIV–groups). Using propensity score (PS) methodology, we identified two subgroups of patients matched by 38 covariates and compared regarding 30-day survival (primary outcome). Interaction was investigated for age, sex, ischemic cardiomyopathy, chronic obstructive pulmonary disease, AHF precipitated by an acute coronary syndrome (ACS), AHF classified as hypertensive or acute pulmonary edema (APE), and systolic blood pressure (SBP). Secondary outcomes were intensive care unit (ICU) admission; mechanical ventilation; in-hospital, 3-day and 7-day mortality; and prolonged hospitalization (>7 days). Results: Of 11, 152 patients from the EAHFE (age (SD): 80 (10) years; 55.5% women), 718 (6.4%) were NIV+ and had a higher 30-day mortality (HR = 2.229; 95%CI = 1.861–2.670) (p 85 years, p < 0.001), AHF associated with ACS (p = 0.045), and SBP < 100 mmHg (p < 0.001). No significant differences were found in the secondary endpoints except for more prolonged hospitalizations in NIV+ patients (OR = 1.445; 95%CI = 1.122–1.862) (p = 0.004). Conclusion: The use of NIV to treat AHF in ED is not associated with improved mortality outcomes and should be cautious in old patients and those with ACS and hypotension

Influence of the length of hospitalisation in post-discharge outcomes in patients with acute heart failure: Results of the LOHRCA study

Objective: To investigate the relationship between length of hospitalisation (LOH) and post-discharge outcomes in acute heart failure (AHF) patients and to ascertain whether there are different patterns according to department of initial hospitalisation. Methods: Consecutive AHF patients hospitalised in 41 Spanish centres were grouped based on the LOH (15 days). Outcomes were defined as 90-day post-discharge all-cause mortality, AHF readmissions, and the combination of both. Hazard ratios (HRs), adjusted by chronic conditions and severity of decompensation, were calculated for groups with LOH >6 days vs. LOH <6 days (reference), and stratified by hospitalisation in cardiology, internal medicine, geriatrics, or short-stay units. Results: We included 8563 patients (mean age: 80 (SD = 10) years, 55.5% women), with a median LOH of 7 days (IQR 4–11): 2934 (34.3%) had a LOH 15 days. The 90-day post-discharge mortality was 11.4%, readmission 32.2%, and combined endpoint 37.4%. Mortality was increased by 36.5% (95%CI = 13.0–64.9) when LOH was 11–15 days, and by 72.0% (95%CI = 42.6–107.5) when >15 days. Conversely, no differences were found in readmission risk, and the combined endpoint only increased 21.6% (95%CI = 8.4–36.4) for LOH >15 days. Stratified analysis by hospitalisation departments rendered similar post-discharge outcomes, with all exhibiting increased mortality for LOH >15 days and no significant increments in readmission risk. Conclusions: Short hospitalisations are not associated with worse outcomes. While post-discharge readmissions are not affected by LOH, mortality risk increases as the LOH lengthens. These findings were similar across hospitalisation departments

Clinical phenotypes of acute heart failure based on signs and symptoms of perfusion and congestion at emergency department presentation and their relationship with patient management and outcomes

Objective To compare the clinical characteristics and outcomes of patients with acute heart failure (AHF) according to clinical profiles based on congestion and perfusion determined in the emergency department (ED). Methods and results Overall, 11 261 unselected AHF patients from 41 Spanish EDs were classified according to perfusion (normoperfusion = warm; hypoperfusion = cold) and congestion (not = dry; yes = wet). Baseline and decompensation characteristics were recorded as were the main wards to which patients were admitted. The primary outcome was 1-year all-cause mortality; secondary outcomes were need for hospitalisation during the index AHF event, in-hospital all-cause mortality, prolonged hospitalisation, 7-day post-discharge ED revisit for AHF and 30-day post-discharge rehospitalisation for AHF. A total of 8558 patients (76.0%) were warm+ wet, 1929 (17.1%) cold+ wet, 675 (6.0%) warm+ dry, and 99 (0.9%) cold+ dry; hypoperfused (cold) patients were more frequently admitted to intensive care units and geriatrics departments, and warm+ wet patients were discharged home without admission. The four phenotypes differed in most of the baseline and decompensation characteristics. The 1-year mortality was 30.8%, and compared to warm+ dry, the adjusted hazard ratios were significantly increased for cold+ wet (1.660; 95% confidence interval 1.400-1.968) and cold+ dry (1.672; 95% confidence interval 1.189-2.351). Hypoperfused (cold) phenotypes also showed higher rates of index episode hospitalisation and in-hospital mortality, while congestive (wet) phenotypes had a higher risk of prolonged hospitalisation but decreased risk of rehospitalisation. No differences were observed among phenotypes in ED revisit risk. Conclusions Bedside clinical evaluation of congestion and perfusion of AHF patients upon ED arrival and classification according to phenotypic profiles proposed by the latest European Society of Cardiology guidelines provide useful complementary information and help to rapidly predict patient outcomes shortly after ED patient arrival