Analysis of the genome and transcriptome of Cryptococcus neoformans var. grubii reveals complex RNA expression and microevolution leading to virulence attenuation.

Cryptococcus neoformans is a pathogenic basidiomycetous yeast responsible for more than 600,000 deaths each year. It occurs as two serotypes (A and D) representing two varieties (i.e. grubii and neoformans, respectively). Here, we sequenced the genome and performed an RNA-Seq-based analysis of the C. neoformans var. grubii transcriptome structure. We determined the chromosomal locations, analyzed the sequence/structural features of the centromeres, and identified origins of replication. The genome was annotated based on automated and manual curation. More than 40,000 introns populating more than 99% of the expressed genes were identified. Although most of these introns are located in the coding DNA sequences (CDS), over 2,000 introns in the untranslated regions (UTRs) were also identified. Poly(A)-containing reads were employed to locate the polyadenylation sites of more than 80% of the genes. Examination of the sequences around these sites revealed a new poly(A)-site-associated motif (AUGHAH). In addition, 1,197 miscRNAs were identified. These miscRNAs can be spliced and/or polyadenylated, but do not appear to have obvious coding capacities. Finally, this genome sequence enabled a comparative analysis of strain H99 variants obtained after laboratory passage. The spectrum of mutations identified provides insights into the genetics underlying the micro-evolution of a laboratory strain, and identifies mutations involved in stress responses, mating efficiency, and virulence

The Bandim TBscore – reliability, further development, and evaluation of potential uses

Background: The tuberculosis (TB) case detection rate has stagnated at 60% due to disorganized case finding and insensitivity of sputum smear microscopy. Of the identified TB cases, 4% die while being treated, monitored with tools that insufficiently predict failure/mortality. Objective: To explore the TBscore, a recently proposed clinical severity measure for pulmonary TB (PTB) patients, and to refine, validate, and investigate its place in case finding. Design: The TBscore's inter-observer agreement was assessed and compared to the Karnofsky Performance Score (KPS) (paper I). The TBscore's variables underlying constructs were assessed, sorting out unrelated items, proposing a more easily assessable TBscoreII, which was validated internally and externally (paper II). Finally, TBscore and TBscoreII's place in PTB-screening was examined in paper III. Results: The inter-observer variability when grading PTB patients into severity classes was moderate for both TBscore (κ W=0.52, 95% CI 0.46–0.56) and KPS (κ W=0.49, 95% CI 0.33–0.65). KPS was influenced by HIV status, whereas TBscore was unaffected by it. In paper II, proposed TBscoreII was validated internally, in Guinea-Bissau, and externally, in Ethiopia. In both settings, a failure to bring down the score by ≥25% from baseline to 2 months of treatment predicted subsequent failure (p=0.007). Finally, in paper III, TBscore and TBscoreII were assessed in health-care-seeking adults and found to be higher in PTB-diagnosed patients, 4.9 (95% CI 4.6–5.2) and 3.9 (95% CI 3.8–4.0), respectively, versus patients not diagnosed with PTB, 3.0 (95% CI 2.7–3.2) and 2.4 (95% CI 2.3–2.5), respectively. Had we referred only patients with cough >2 weeks to sputum smear, we would have missed 32.1% of the smear confirmed cases in our cohort. A TBscoreII>=2 missed 8.6%. Conclusions: TBscore and TBscoreII are useful monitoring tools for PTB patients on treatment, as they could fill the void which currently exists in risk grading of patients. They may also have a role in PTB screening; however, this requires our findings to be repeated elsewhere

A unification of probabilistic choice within a design-based model of reversible computation

We see reversible computing as a generalisation of sequential computation obtained by revoking the law of the excluded miracle. Our execution language includes naked guarded commands and non-deterministic choice. Choices which lead to miraculous continuations invoke reverse computation, and non-deterministic choice plays the rôle of provisional choice within a backtracking context. We require probabilistic choice for symmetry breaking and sampling large search spaces, but must formulate it differently from previous approaches to obtain the required interactions between probabilistic choice and non-deterministic choice and between probabilistic choice and feasibility. Our formulation allows us to derive the post-distributions which characterise a program, and we use these to construct a relational model. We consider refinement as containment of convex closures within distribution space, qualified with additional conditions to avoid over-refinement. We link the non-probabilistic and probabilistic versions of the model with a Galois connection and show that classical designs are a retract of our probabilistic designs. We consider the interaction between probabilistic and non-deterministic choice and find the same initially counter-intuitive results that have been noted by other investigators. We provide an alternative formulation, within the same model, of oblivious non-determinism, which allows all non-deterministic choices to be moved to the start of a computation. We consider the interaction between probabilistic choice and feasibility that is required to match an operational interpretation in which infeasible commands provoke reverse execution, and we present a small case study to show how the interaction between probabilistic choice and feasibility can be exploited in a practical program. All programming structures described here are supported by our implementation platform, the Reversible Virtual Machine, whose development has accompanied our theoretical investigations

The perceptions of social responsibility for community resilience to flooding: the impact of past experience, age, gender and ethnicity

Community resilience to flooding depends, to a large extent, on the participation of community members to take more responsibility for enhancing their own resilience. The perception of social responsibility (SR) which is argued to be one of the antecedents influencing individual’s willingness to undertake resilient behaviours can significantly contribute to community resilience through individual and collective actions. Understanding of factors influencing the perceptions of SR of individuals within community might help with developing strategies to increase the perceptions of SR. This research explores perceptions of SR in relation to flooding for householders and local businesses and establishes their relationships with experience of flooding and demographic factors of age, gender and ethnicity. The data were obtained via a questionnaire survey of three communities in Birmingham and one community in South East London, UK, three with experience of flooding and one without. A total of 414 responses were received and used in the multiple regression analysis. The analysis identified ‘experience of flooding’, ‘age’ and ‘South Asian’ ethnic group as significant variables, suggesting that older individuals from South Asian ethnic groups with previous experience of flooding are likely to be more socially responsible than others without these attributes

How have ART treatment programmes changed the patterns of excess mortality in people living with HIV? Estimates from four countries in East and Southern Africa

Background: Substantial falls in the mortality of people living with HIV (PLWH) have been observed since the introduction of antiretroviral therapy (ART) in sub-Saharan Africa. However, access and uptake of ART have been variable in many countries. We report the excess deaths observed in PLWH before and after the introduction of ART. We use data from five longitudinal studies in Malawi, South Africa, Tanzania, and Uganda, members of the network for Analysing Longitudinal Population-based HIV/AIDS data on Africa (ALPHA). Methods: Individual data from five demographic surveillance sites that conduct HIV testing were used to estimate mortality attributable to HIV, calculated as the difference between the mortality rates in PLWH and HIV-negative people. Excess deaths in PLWH were standardized for age and sex differences and summarized over periods before and after ART became generally available. An exponential regression model was used to explore differences in the impact of ART over the different sites. Results: 127,585 adults across the five sites contributed a total of 487,242 person years. Before the introduction of ART, HIV-attributable mortality ranged from 45 to 88 deaths per 1,000 person years. Following ART availability, this reduced to 14–46 deaths per 1,000 person years. Exponential regression modeling showed a reduction of more than 50% (HR =0.43, 95% CI: 0.32–0.58), compared to the period before ART was available, in mortality at ages 15–54 across all five sites. Discussion: Excess mortality in adults living with HIV has reduced by over 50% in five communities in sub-Saharan Africa since the advent of ART. However, mortality rates in adults living with HIV are still 10 times higher than in HIV-negative people, indicating that substantial improvements can be made to reduce mortality further. This analysis shows differences in the impact across the sites, and contrasts with developed countries where mortality among PLWH on ART can be similar to that of the general population. Further research is urgently needed to establish why the different impacts on mortality were observed and how the care and treatment programmes in these countries can be more effective in reducing mortality further

RNase L Mediated Protection from Virus Induced Demyelination

IFN-α/β plays a critical role in limiting viral spread, restricting viral tropism and protecting mice from neurotropic coronavirus infection. However, the IFN-α/β dependent mechanisms underlying innate anti-viral functions within the CNS are poorly understood. The role of RNase L in viral encephalomyelitis was explored based on its functions in inhibiting translation, inducing apoptosis, and propagating the IFN-α/β pathway through RNA degradation intermediates. Infection of RNase L deficient (RL−/−) mice with a sub-lethal, demyelinating mouse hepatitis virus variant revealed that the majority of mice succumbed to infection by day 12 p.i. However, RNase L deficiency did not affect overall control of infectious virus, or diminish IFN-α/β expression in the CNS. Furthermore, increased morbidity and mortality could not be attributed to altered proinflammatory signals or composition of cells infiltrating the CNS. The unique phenotype of infected RL−/− mice was rather manifested in earlier onset and increased severity of demyelination and axonal damage in brain stem and spinal cord without evidence for enhanced neuronal infection. Increased tissue damage coincided with sustained brain stem infection, foci of microglia infection in grey matter, and increased apoptotic cells. These data demonstrate a novel protective role for RNase L in viral induced CNS encephalomyelitis, which is not reflected in overall viral control or propagation of IFN-α/β mediated signals. Protective function is rather associated with cell type specific and regional restriction of viral replication in grey matter and ameliorated neurodegeneration and demyelination

Rapid Diagnosis of Tuberculosis with the Xpert MTB/RIF Assay in High Burden Countries: A Cost-Effectiveness Analysis

A cost-effectiveness study by Frank Cobelens and colleagues reveals that Xpert MTB/RIF is a cost-effective method of tuberculosis diagnosis that is suitable for use in low- and middle-income settings

Stability Properties of Underdominance in Finite Subdivided Populations

In isolated populations underdominance leads to bistable evolutionary dynamics: below a certain mutant allele frequency the wildtype succeeds. Above this point, the potentially underdominant mutant allele fixes. In subdivided populations with gene flow there can be stable states with coexistence of wildtypes and mutants: polymorphism can be maintained because of a migration-selection equilibrium, i.e., selection against rare recent immigrant alleles that tend to be heterozygous. We focus on the stochastic evolutionary dynamics of systems where demographic fluctuations in the coupled populations are the main source of internal noise. We discuss the influence of fitness, migration rate, and the relative sizes of two interacting populations on the mean extinction times of a group of potentially underdominant mutant alleles. We classify realistic initial conditions according to their impact on the stochastic extinction process. Even in small populations, where demographic fluctuations are large, stability properties predicted from deterministic dynamics show remarkable robustness. Fixation of the mutant allele becomes unlikely but the time to its extinction can be long

Neotropical Bats: Estimating Species Diversity with DNA Barcodes

DNA barcoding using the cytochrome c oxidase subunit 1 gene (COI) is frequently employed as an efficient method of species identification in animal life and may also be used to estimate species richness, particularly in understudied faunas. Despite numerous past demonstrations of the efficiency of this technique, few studies have attempted to employ DNA barcoding methodologies on a large geographic scale, particularly within tropical regions. In this study we survey current and potential species diversity using DNA barcodes with a collection of more than 9000 individuals from 163 species of Neotropical bats (order Chiroptera). This represents one of the largest surveys to employ this strategy on any animal group and is certainly the largest to date for land vertebrates. Our analysis documents the utility of this tool over great geographic distances and across extraordinarily diverse habitats. Among the 163 included species 98.8% possessed distinct sets of COI haplotypes making them easily recognizable at this locus. We detected only a single case of shared haplotypes. Intraspecific diversity in the region was high among currently recognized species (mean of 1.38%, range 0–11.79%) with respect to birds, though comparable to other bat assemblages. In 44 of 163 cases, well-supported, distinct intraspecific lineages were identified which may suggest the presence of cryptic species though mean and maximum intraspecific divergence were not good predictors of their presence. In all cases, intraspecific lineages require additional investigation using complementary molecular techniques and additional characters such as morphology and acoustic data. Our analysis provides strong support for the continued assembly of DNA barcoding libraries and ongoing taxonomic investigation of bats

NADPH oxidase-mediated redox signal contributes to lipoteichoic acid-induced MMP-9 upregulation in brain astrocytes

<p>Abstract</p> <p>Background</p> <p>Lipoteichoic acid (LTA) is a component of gram-positive bacterial cell walls and may be elevated in the cerebrospinal fluid of patients suffering from meningitis. Among matrix metalloproteinases (MMPs), MMP-9 has been observed in patients with brain inflammatory diseases and may contribute to the pathology of brain diseases. Moreover, several studies have suggested that increased oxidative stress is implicated in the pathogenesis of brain inflammation and injury. However, the molecular mechanisms underlying LTA-induced redox signal and MMP-9 expression in brain astrocytes remain unclear.</p> <p>Objective</p> <p>Herein we explored whether LTA-induced MMP-9 expression was mediated through redox signals in rat brain astrocytes (RBA-1 cells).</p> <p>Methods</p> <p>Upregulation of MMP-9 by LTA was evaluated by zymographic and RT-PCR analyses. Next, the MMP-9 regulatory pathways were investigated by pretreatment with pharmacological inhibitors or transfection with small interfering RNAs (siRNAs), Western blotting, and chromatin immunoprecipitation (ChIP)-PCR and promoter activity reporter assays. Moreover, we determined the cell functional changes by migration assay.</p> <p>Results</p> <p>These results showed that LTA induced MMP-9 expression via a PKC(α)-dependent pathway. We further demonstrated that PKCα stimulated p47^phox/NADPH oxidase 2 (Nox2)-dependent reactive oxygen species (ROS) generation and then activated the ATF2/AP-1 signals. The activated-ATF2 bound to the AP-1-binding site of MMP-9 promoter, and thereby turned on MMP-9 gene transcription. Additionally, the co-activator p300 also contributed to these responses. Functionally, LTA-induced MMP-9 expression enhanced astrocytic migration.</p> <p>Conclusion</p> <p>These results demonstrated that in RBA-1 cells, activation of ATF2/AP-1 by the PKC(α)-mediated Nox(2)/ROS signals is essential for upregulation of MMP-9 and cell migration enhanced by LTA.</p