Overexpression of the lens epithelium-derived growth factor/p75 integrase binding domain inhibits human immunodeficiency virus replication

We initially identified lens epithelium-derived growth factor/p75 (LEDGF/p75) as a binding partner of human immunodeficiency virus type 1 (HIV-1) integrase. To investigate the role of LEDGF/p75 in HIV replication and its potential as a new antiviral target, we stably overexpressed two different fragments containing the integrase binding domain (IBD) of LEDGF/p75 fused to enhanced green fluorescent protein (eGFP). HIV-1 replication was severely inhibited by overexpression of the eGFP-IBD fusion proteins, while no inhibition was observed in cell lines overexpressing the interaction-deficient D366A mutant. Quantitative PCR pinpointed the block to the integration step, whereas nuclear import was not affected. Competition of the IBD fusion proteins with endogenous LEDGF/p75 for binding to integrase led to a potent defect in HIV-1 replication in both HeLaP4- and MT-4-derived cell lines. A previously described diketo acid-resistant HIV-1 strain remained fully susceptible to inhibition, suggesting that this strategy will also work in patients who harbor strains resistant to the current experimental integrase inhibitors. These data support LEDGF/p75 as an important cofactor for HIV replication and provide proof of concept for the LEDGF/p75-integrase interaction as a novel target for treating HIV-1 infection.     

Applicability of the WHO Maternal Near Miss Criteria in a Low-Resource Setting

Background

Maternal near misses are increasingly used to study quality of obstetric care. Inclusion criteria for the identification of near misses are diverse and studies not comparable. WHO developed universal near miss inclusion criteria in 2009 and these criteria have been validated in Brazil and Canada.

Objectives

To validate and refine the WHO near miss criteria in a low-resource setting.

Methods

A prospective cross-sectional study was performed in a rural referral hospital in Tanzania. From November 2009 until November 2011, all cases of maternal death (MD) and maternal near miss (MNM) were included. For identification of MNM, a local modification of the WHO near miss criteria was used, because most laboratory-based and some management-based criteria could not be applied in this setting. Disease-based criteria were added as they reflect severe maternal morbidity. In the absence of a gold standard for identification of MNM, the clinical WHO criteria were validated for identification of MD.

Results

32 MD and 216 MNM were identified using the locally adapted near miss criteria; case fatality rate (CFR) was 12.9%. WHO near miss criteria identified only 60 MNM (CFR 35.6%). All clinical criteria, 25% of the laboratory-based criteria and 50% of the management-based criteria could be applied. The threshold of five units of blood for identification of MNM led to underreporting of MNM. Clinical criteria showed specificity of 99.5% (95%CI: 99.4%–99.7%) and sensitivity of 100% (95%CI: 91.1%–100%). Some inclusion criteria did not contribute to the identification of cases and therefore may be eligible for removal.

Conclusion

The applicability of the WHO near miss criteria depends on the local context, e.g. level of health care. The clinical criteria showed good validity. Lowering the threshold for blood transfusion from five to two units in settings without blood bank and addition of disease-based criteria in low-resource settings is recommended.     

Projecting terrestrial biodiversity intactness with GLOBIO 4

Scenario‐based biodiversity modelling is a powerful approach to evaluate how possible future socio‐economic developments may affect biodiversity. Here, we evaluated the changes in terrestrial biodiversity intactness, expressed by the mean species abundance (MSA) metric, resulting from three of the shared socio‐economic pathways (SSPs) combined with different levels of climate change (according to representative concentration pathways [RCPs]): a future oriented towards sustainability (SSP1xRCP2.6), a future determined by a politically divided world (SSP3xRCP6.0) and a future with continued global dependency on fossil fuels (SSP5xRCP8.5). To this end, we first updated the GLOBIO model, which now runs at a spatial resolution of 10 arc‐seconds (~300 m), contains new modules for downscaling land use and for quantifying impacts of hunting in the tropics, and updated modules to quantify impacts of climate change, land use, habitat fragmentation and nitrogen pollution. We then used the updated model to project terrestrial biodiversity intactness from 2015 to 2050 as a function of land use and climate changes corresponding with the selected scenarios. We estimated a global area‐weighted mean MSA of 0.56 for 2015. Biodiversity intactness declined in all three scenarios, yet the decline was smaller in the sustainability scenario (?0.02) than the regional rivalry and fossil‐fuelled development scenarios (?0.06 and ?0.05 respectively). We further found considerable variation in projected biodiversity change among different world regions, with large future losses particularly for sub‐Saharan Africa. In some scenario‐region combinations, we projected future biodiversity recovery due to reduced demands for agricultural land, yet this recovery was counteracted by increased impacts of other pressures (notably climate change and road disturbance). Effective measures to halt or reverse the decline of terrestrial biodiversity should not only reduce land demand (e.g. by increasing agricultural productivity and dietary changes) but also focus on reducing or mitigating the impacts of other pressures.     

High‐throughput CRISPRi phenotyping identifies new essential genes in Streptococcus pneumoniae

Genome‐wide screens have discovered a large set of essential genes in the opportunistic human pathogen Streptococcus pneumoniae. However, the functions of many essential genes are still unknown, hampering vaccine development and drug discovery. Based on results from transposon sequencing (Tn‐seq), we refined the list of essential genes in S. pneumoniae serotype 2 strain D39. Next, we created a knockdown library targeting 348 potentially essential genes by CRISPR interference (CRISPRi) and show a growth phenotype for 254 of them (73%). Using high‐content microscopy screening, we searched for essential genes of unknown function with clear phenotypes in cell morphology upon CRISPRi‐based depletion. We show that SPD_1416 and SPD_1417 (renamed to MurT and GatD, respectively) are essential for peptidoglycan synthesis, and that SPD_1198 and SPD_1197 (renamed to TarP and TarQ, respectively) are responsible for the polymerization of teichoic acid (TA) precursors. This knowledge enabled us to reconstruct the unique pneumococcal TA biosynthetic pathway. CRISPRi was also employed to unravel the role of the essential Clp‐proteolytic system in regulation of competence development, and we show that ClpX is the essential ATPase responsible for ClpP‐dependent repression of competence. The CRISPRi library provides a valuable tool for characterization of pneumococcal genes and pathways and revealed several promising antibiotic targets.     

O-acetylation of cryptococcal capsular glucuronoxylomannan is essential for interference with neutrophil migration

The capsular polysaccharide glucuronoxylomannan (GXM) of Cryptococcus neoformans has been shown to interfere with neutrophil migration. Although several receptors have been implied to mediate this process, the structural perspectives are unknown. Here, we assess the contribution of 6-O-acetylation and xylose substitution of the (1-->3)-alpha-d-mannan backbone of GXM, the variable structural features of GXM, to the interference with neutrophil migration. We compare chemically deacetylated GXM and acetyl- or xylose-deficient GXM from genetically modified strains with wild-type GXM in their ability to inhibit the different phases of neutrophil migration. Additionally, we verify the effects of de-O-acetylation on neutrophil migration in vivo. De-O-acetylation caused a dramatic reduction of the inhibitory capacity of GXM in the in vitro assays for neutrophil chemokinesis, rolling on E-selectin and firm adhesion to endothelium. Genetic removal of xylose only marginally reduced the ability of GXM to reduce firm adhesion. In vivo, chemical deacetylation of GXM significantly reduced its ability to interfere with neutrophil recruitment in a model of myocardial ischemia (65% reduction vs a nonsignificant reduction in tissue myeloperoxidase, respectively). Our findings indicate that 6-O-acetylated mannose of GXM is a crucial motive for the inhibition of neutrophil recruitment.     

Estimating Body Composition in Adolescent Sprint Athletes: Comparison of Different Methods in a 3 Years Longitudinal Design

A recommended field method to assess body composition in adolescent sprint athletes is currently lacking. Existing methods developed for non-athletic adolescents were not longitudinally validated and do not take maturation status into account. This longitudinal study compared two field methods, i.e., a Bio Impedance Analysis (BIA) and a skinfold based equation, with underwater densitometry to track body fat percentage relative to years from age at peak height velocity in adolescent sprint athletes. In this study, adolescent sprint athletes (34 girls, 35 boys) were measured every 6 months during 3 years (age at start = 14.8 ± 1.5yrs in girls and 14.7 ± 1.9yrs in boys). Body fat percentage was estimated in 3 different ways: 1) using BIA with the TANITA TBF 410; 2) using a skinfold based equation; 3) using underwater densitometry which was considered as the reference method. Height for age since birth was used to estimate age at peak height velocity. Cross-sectional analyses were performed using repeated measures ANOVA and Pearson correlations between measurement methods at each occasion. Data were analyzed longitudinally using a multilevel cross-classified model with the PROC Mixed procedure. In boys, compared to underwater densitometry, the skinfold based formula revealed comparable values for body fatness during the study period whereas BIA showed a different pattern leading to an overestimation of body fatness starting from 4 years after age at peak height velocity. In girls, both the skinfold based formula and BIA overestimated body fatness across the whole range of years from peak height velocity. The skinfold based method appears to give an acceptable estimation of body composition during growth as compared to underwater densitometry in male adolescent sprinters. In girls, caution is warranted when interpreting estimations of body fatness by both BIA and a skinfold based formula since both methods tend to give an overestimation.     

Ant Queen Egg-Marking Signals: Matching Deceptive Laboratory Simplicity with Natural Complexity

Background

Experiments under controlled laboratory conditions can produce decisive evidence for testing biological hypotheses, provided they are representative of the more complex natural conditions. However, whether this requirement is fulfilled is seldom tested explicitly. Here we provide a lab/field comparison to investigate the identity of an egg-marking signal of ant queens. Our study was based on ant workers resolving conflict over male production by destroying each other''s eggs, but leaving queen eggs unharmed. For this, the workers need a proximate cue to discriminate between the two egg types. Earlier correlative evidence indicated that, in the ant Pachycondyla inversa, the hydrocarbon 3,11-dimethylheptacosane (3,11-diMeC₂₇) is more abundant on the surface of queen-laid eggs.

Methodology

We first tested the hypothesis that 3,11-diMeC₂₇ functions as a queen egg-marking pheromone using laboratory-maintained colonies. We treated worker-laid eggs with synthetic 3,11-diMeC₂₇ and found that they were significantly more accepted than sham-treated worker-laid eggs. However, we repeated the experiment with freshly collected field colonies and observed no effect of treating worker-laid eggs with 3,11-diMeC₂₇, showing that this compound by itself is not the natural queen egg-marking pheromone. We subsequently investigated the overall differences of entire chemical profiles of eggs, and found that queen-laid eggs in field colonies are more distinct from worker-laid eggs than in lab colonies, have more variation in profiles, and have an excess of longer-chain hydrocarbons.

Conclusions

Our results suggest that queen egg-marking signals are significantly affected by transfer to the laboratory, and that this change is possibly connected to reduced queen fertility as predicted by honest signaling theory. This change is reflected in the worker egg policing response under field and laboratory conditions.