Single Cell Analysis of Lymph Node Tissue from HIV-1 Infected Patients Reveals that the Majority of CD4+ T-cells Contain One HIV-1 DNA Molecule

Genetic recombination contributes to the diversity of human immunodeficiency virus (HIV-1). Productive HIV-1 recombination is, however, dependent on both the number of HIV-1 genomes per infected cell and the genetic relationship between these viral genomes. A detailed analysis of the number of proviruses and their genetic relationship in infected cells isolated from peripheral blood and tissue compartments is therefore important for understanding HIV-1 recombination, genetic diversity and the dynamics of HIV-1 infection. To address these issues, we used a previously developed single-cell sequencing technique to quantify and genetically characterize individual HIV-1 DNA molecules from single cells in lymph node tissue and peripheral blood. Analysis of memory and naïve CD4⁺ T cells from paired lymph node and peripheral blood samples from five untreated chronically infected patients revealed that the majority of these HIV-1-infected cells (>90%) contain only one copy of HIV-1 DNA, implying a limited potential for productive recombination in virus produced by these cells in these two compartments. Phylogenetic analysis revealed genetic similarity of HIV-1 DNA in memory and naïve CD4⁺ T-cells from lymph node, peripheral blood and HIV-1 RNA from plasma, implying exchange of virus and/or infected cells between these compartments in untreated chronic infection.     

Human Spermatogenic Failure Purges Deleterious Mutation Load from the Autosomes and Both Sex Chromosomes,including the Gene DMRT1

Gonadal failure, along with early pregnancy loss and perinatal death, may be an important filter that limits the propagation of harmful mutations in the human population. We hypothesized that men with spermatogenic impairment, a disease with unknown genetic architecture and a common cause of male infertility, are enriched for rare deleterious mutations compared to men with normal spermatogenesis. After assaying genomewide SNPs and CNVs in 323 Caucasian men with idiopathic spermatogenic impairment and more than 1,100 controls, we estimate that each rare autosomal deletion detected in our study multiplicatively changes a man''s risk of disease by 10% (OR 1.10 [1.04–1.16], p<2×10⁻³), rare X-linked CNVs by 29%, (OR 1.29 [1.11–1.50], p<1×10⁻³), and rare Y-linked duplications by 88% (OR 1.88 [1.13–3.13], p<0.03). By contrasting the properties of our case-specific CNVs with those of CNV callsets from cases of autism, schizophrenia, bipolar disorder, and intellectual disability, we propose that the CNV burden in spermatogenic impairment is distinct from the burden of large, dominant mutations described for neurodevelopmental disorders. We identified two patients with deletions of DMRT1, a gene on chromosome 9p24.3 orthologous to the putative sex determination locus of the avian ZW chromosome system. In an independent sample of Han Chinese men, we identified 3 more DMRT1 deletions in 979 cases of idiopathic azoospermia and none in 1,734 controls, and found none in an additional 4,519 controls from public databases. The combined results indicate that DMRT1 loss-of-function mutations are a risk factor and potential genetic cause of human spermatogenic failure (frequency of 0.38% in 1306 cases and 0% in 7,754 controls, p = 6.2×10⁻⁵). Our study identifies other recurrent CNVs as potential causes of idiopathic azoospermia and generates hypotheses for directing future studies on the genetic basis of male infertility and IVF outcomes.     

The Brazilian Preference: Cesarean Delivery among Immigrants in Portugal

Objective

To evaluate how the country of origin affects the probability of being delivered by cesarean section when giving birth at public Portuguese hospitals.

Study Design

Women delivered of a singleton birth (n = 8228), recruited from five public level III maternities (April 2005–August 2006) during the procedure of assembling a birth cohort, were classified according to the country of origin and her migration status as Portuguese (n = 7908), non-Portuguese European (n = 84), African (n = 77) and Brazilian (n = 159). A Poisson model was used to evaluate the association between country of birth and cesarean section that was measured by adjusted prevalence ratio (PR) and respective 95% confidence intervals (95%CI).

Results

The cesarean section rate varied from 32.1% in non-Portuguese European to 48.4% in Brazilian women (p = 0.008). After adjustment for potential confounders and compared to Portuguese women as a reference, Brazilian women presented significantly higher prevalence of cesarean section (PR = 1.26; 95%CI: 1.08–1.47). The effect was more evident among multiparous women (PR = 1.39; 95%CI: 1.12–1.73) and it was observed when cesarean section was performed either before labor (PR = 1.43; 95%CI: 0.99–2.06) or during labor (PR = 1.30; 95%CI: 1.07–1.58).

Conclusions

The rate of cesarean section was significantly higher among Brazilian women and it was independent of the presence of any known risk factors or usual clinical indications, suggesting that cultural background influences the mode of delivery overcoming the expected standard of care and outcomes in public health services.     

The Bacterial Protein Azurin Impairs Invasion and FAK/Src Signaling in P-Cadherin-Overexpressing Breast Cancer Cell Models

P-cadherin overexpression occurs in about 30% of all breast carcinomas, being a poor prognostic factor for breast cancer patients. In a cellular background of wild-type E-cadherin, we have previously shown that its expression promotes invasion, motility and migration of breast cancer cells due to the induced secretion of metalloproteases (MMPs) to the extracellular medium and to the concomitant shedding of a pro-invasive soluble form of this protein (sP-cad). Azurin is secreted by Pseudomonas aeruginosa and induces in vitro and in vivo cytotoxicity after its preferential penetration in human cancer cells relative to normal cells. Three different breast cancer cell lines, MCF-7/AZ.Mock, MCF-7/AZ.Pcad and SUM149 were treated with sub-killing doses of azurin. Invasion of these cells was measured using Matrigel Invasion Assays and MTT assays were performed to determine cell viability upon treatment and the effects on cadherins expression was determined by Western blot and Immunofluorescence. Gelatin Zymography was used to determine activity of MMP2 in the conditioned media of azurin treated and untreated cells and the phosphorylation levels of intracellular signaling proteins were determined by Western blot. The invasive phenotype of these breast cancer cells was significantly reduced by azurin. Azurin (50–100 µM) also caused a specific decrease on P-cadherin protein levels from 30–50% in MCF-7/AZ.Pcad and SUM149 breast cancer cell lines, but the levels of E-cadherin remain unaltered. More, the levels of sP-cad and the activity of MMP2 were reduced in the extracellular media of azurin treated cells and we also observed a decrease in the phosphorylation levels of both FAK and Src proteins. Our data show that azurin specifically targets P-cadherin, not E-cadherin, abrogating P-cadherin-mediated invasive effects and signaling. Therefore, azurin could possibly be considered a therapeutic tool to treat poor-prognosis breast carcinomas overexpressing P-cadherin in a wild type E-cadherin context.     

The Use of Bayesian Latent Class Cluster Models to Classify Patterns of Cognitive Performance in Healthy Ageing

The main focus of this study is to illustrate the applicability of latent class analysis in the assessment of cognitive performance profiles during ageing. Principal component analysis (PCA) was used to detect main cognitive dimensions (based on the neurocognitive test variables) and Bayesian latent class analysis (LCA) models (without constraints) were used to explore patterns of cognitive performance among community-dwelling older individuals. Gender, age and number of school years were explored as variables. Three cognitive dimensions were identified: general cognition (MMSE), memory (MEM) and executive (EXEC) function. Based on these, three latent classes of cognitive performance profiles (LC1 to LC3) were identified among the older adults. These classes corresponded to stronger to weaker performance patterns (LC1>LC2>LC3) across all dimensions; each latent class denoted the same hierarchy in the proportion of males, age and number of school years. Bayesian LCA provided a powerful tool to explore cognitive typologies among healthy cognitive agers.     

Intracellular Nucleic Acid Delivery by the Supercharged Dengue Virus Capsid Protein

Supercharged proteins are a recently identified class of proteins that have the ability to efficiently deliver functional macromolecules into mammalian cells. They were first developed as bioengineering products, but were later found in the human proteome. In this work, we show that this class of proteins with unusually high net positive charge is frequently found among viral structural proteins, more specifically among capsid proteins. In particular, the capsid proteins of viruses from the Flaviviridae family have all a very high net charge to molecular weight ratio (> +1.07/kDa), thus qualifying as supercharged proteins. This ubiquity raises the hypothesis that supercharged viral capsid proteins may have biological roles that arise from an intrinsic ability to penetrate cells. Dengue virus capsid protein was selected for a detailed experimental analysis. We showed that this protein is able to deliver functional nucleic acids into mammalian cells. The same result was obtained with two isolated domains of this protein, one of them being able to translocate lipid bilayers independently of endocytic routes. Nucleic acids such as siRNA and plasmids were delivered fully functional into cells. The results raise the possibility that the ability to penetrate cells is part of the native biological functions of some viral capsid proteins.     

Glocal Clinical Registries: Pacemaker Registry Design and Implementation for Global and Local Integration – Methodology and Case Study

Background

The ability to apply standard and interoperable solutions for implementing and managing medical registries as well as aggregate, reproduce, and access data sets from legacy formats and platforms to advanced standard formats and operating systems are crucial for both clinical healthcare and biomedical research settings.

Purpose

Our study describes a reproducible, highly scalable, standard framework for a device registry implementation addressing both local data quality components and global linking problems.

Methods and Results

We developed a device registry framework involving the following steps: (1) Data standards definition and representation of the research workflow, (2) Development of electronic case report forms using REDCap (Research Electronic Data Capture), (3) Data collection according to the clinical research workflow and, (4) Data augmentation by enriching the registry database with local electronic health records, governmental database and linked open data collections, (5) Data quality control and (6) Data dissemination through the registry Web site. Our registry adopted all applicable standardized data elements proposed by American College Cardiology / American Heart Association Clinical Data Standards, as well as variables derived from cardiac devices randomized trials and Clinical Data Interchange Standards Consortium. Local interoperability was performed between REDCap and data derived from Electronic Health Record system. The original data set was also augmented by incorporating the reimbursed values paid by the Brazilian government during a hospitalization for pacemaker implantation. By linking our registry to the open data collection repository Linked Clinical Trials (LinkedCT) we found 130 clinical trials which are potentially correlated with our pacemaker registry.

Conclusion

This study demonstrates how standard and reproducible solutions can be applied in the implementation of medical registries to constitute a re-usable framework. Such approach has the potential to facilitate data integration between healthcare and research settings, also being a useful framework to be used in other biomedical registries.     

Diurnal patterns of growth and transient reserves of sink and source tissues are affected by cold nights in barley

Barley is described to mostly use sucrose for night carbon requirements. To understand how the transient carbon is accumulated and utilized in response to cold, barley plants were grown in a combination of cold days and/or nights. Both daytime and night cold reduced growth. Sucrose was the main carbohydrate supplying growth at night, representing 50–60% of the carbon consumed. Under warm days and nights, starch was the second contributor with 26% and malate the third with 15%. Under cold nights, the contribution of starch was severely reduced, due to an inhibition of its synthesis, including under warm days, and malate was the second contributor to C requirements with 24–28% of the total amount of carbon consumed. We propose that malate plays a critical role as an alternative carbon source to sucrose and starch in barley. Hexoses, malate, and sucrose mobilization and starch accumulation were affected in barley elf3 clock mutants, suggesting a clock regulation of their metabolism, without affecting growth and photosynthesis however. Altogether, our data suggest that the mobilization of sucrose and malate and/or barley growth machinery are sensitive to cold.