Ex vivo generation of a functional and regenerative wound epithelium from axolotl (Ambystoma mexicanum) skin

Urodele amphibians (salamanders) are unique among adult vertebrates in their ability to regenerate structurally complete and fully functional limbs. Regeneration is a stepwise process that requires interactions between keratinocytes, nerves and fibroblasts. The formation of a wound epithelium covering the amputation site is an early and necessary event in the process but the molecular mechanisms that underlie the role of the wound epithelium in regeneration remain unclear. We have developed an ex vivo model that recapitulates many features of in vivo wound healing. The model comprises a circular explant of axolotl (Ambystoma mexicanum) limb skin with a central circular, full thickness wound. Re‐epithelialization of the wound area is rapid (typically <11 h) and is dependent on metalloproteinase activity. The ex vivo wound epithelium is viable, responds to neuronal signals and is able to participate in ectopic blastema formation and limb regeneration. This ex vivo model provides a reproducible and tractable system in which to study the cellular and molecular events that underlie wound healing and regeneration.     

Structure and evolution of the genomes ofsorghum bicolor andZea mays

Cloned maize genes and random maize genomic fragments were used to construct a genetic map of sorghum and to compare the structure of the maize and sorghum genomes. Most (266/280) of the maize DNA fragments hybridized to sorghum DNA and 145 of them detected polymorphisms. The segregation of 111 markers was analyzed in 55 F₂ progeny. A genetic map was generated with 96 loci arranged in 15 linkage groups spanning 709 map units. Comparative genetic mapping of sorghum and maize is complicated by the fact that many loci are duplicated, often making the identification of orthologous sequences ambiguous. Relative map positions of probes which detect only a single locus in both species indicated that multiple rearrangements have occurred since their divergence, but that many chromosomal segments have conserved synteny. Some sorghum linkage groups were found to be composed of sequences that detect loci on two different maize chromosomes. The two maize chromosomes to which these loci mapped were generally those which commonly share duplicated sequences. Evolutionary models and implications are discussed.     

The in vitro survival of human monosomies and trisomies as embryonic stem cells

Chromosomal aneuploidies are responsible for severe human genetic diseases. Aiming at creating models for such disorders, we have generated human embryonic stem cell (hESC) lines from pre-implantation genetic screened (PGS) embryos. The overall analysis of more than 400 aneuploid PGS embryos showed a similar risk of occurrence of monosomy or trisomy for any specific chromosome. However, the generation of hESCs from these embryos revealed a clear bias against monosomies in autosomes. Moreover, only specific trisomies showed a high chance of survival as hESC lines, enabling us to present another categorization of human aneuploidies. Our data suggest that chromosomal haploinsufficiency leads to lethality at very early stages of human development.     

Lipid production and fatty acid composition of selected strains belonging to Mucorales

The lipid accumulation, fatty acid composition and γ-linolenic acid (GLA) production by 28 strains belonging to Mucorales were investigated. The lipid content varied from 5 to 30% on dry biomass and the percentage of γ-linolenic acid in total intracellular lipid was in a range from 2.5 to 15.4% (w/w). The best yield of γ-linolenic acid (expressed as mg GLA per 1 g biomass) was found for Mucor mucedo CCF – 1384 (28.4) and Cunninghamella echinulata CCF – 103 (25.1).     

Antibiotic selection pressure and macrolide resistance in nasopharyngeal Streptococcus pneumoniae: a cluster-randomized clinical trial

Background

It is widely thought that widespread antibiotic use selects for community antibiotic resistance, though this has been difficult to prove in the setting of a community-randomized clinical trial. In this study, we used a randomized clinical trial design to assess whether macrolide resistance was higher in communities treated with mass azithromycin for trachoma, compared to untreated control communities.

Methods and Findings

In a cluster-randomized trial for trachoma control in Ethiopia, 12 communities were randomized to receive mass azithromycin treatment of children aged 1–10 years at months 0, 3, 6, and 9. Twelve control communities were randomized to receive no antibiotic treatments until the conclusion of the study. Nasopharyngeal swabs were collected from randomly selected children in the treated group at baseline and month 12, and in the control group at month 12. Antibiotic susceptibility testing was performed on Streptococcus pneumoniae isolated from the swabs using Etest strips. In the treated group, the mean prevalence of azithromycin resistance among all monitored children increased from 3.6% (95% confidence interval [CI] 0.8%–8.9%) at baseline, to 46.9% (37.5%–57.5%) at month 12 (p = 0.003). In control communities, azithromycin resistance was 9.2% (95% CI 6.7%–13.3%) at month 12, significantly lower than the treated group (p<0.0001). Penicillin resistance was identified in 0.8% (95% CI 0%–4.2%) of isolates in the control group at 1 year, and in no isolates in the children-treated group at baseline or 1 year.

Conclusions

This cluster-randomized clinical trial demonstrated that compared to untreated control communities, nasopharyngeal pneumococcal resistance to macrolides was significantly higher in communities randomized to intensive azithromycin treatment. Mass azithromycin distributions were given more frequently than currently recommended by the World Health Organization''s trachoma program. Azithromycin use in this setting did not select for resistance to penicillins, which remain the drug of choice for pneumococcal infections.

Trial registration

www.ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00322972","term_id":"NCT00322972"}}NCT00322972 Please see later in the article for the Editors'' Summary     

Virologic Response to Tipranavir-Ritonavir or Darunavir-Ritonavir Based Regimens in Antiretroviral Therapy Experienced HIV-1 Patients: A Meta-Analysis and Meta-Regression of Randomized Controlled Clinical Trials

Background

The development of tipranavir and darunavir, second generation non-peptidic HIV protease inhibitors, with marked improved resistance profiles, has opened a new perspective on the treatment of antiretroviral therapy (ART) experienced HIV patients with poor viral load control. The aim of this study was to determine the virologic response in ART experienced patients to tipranavir-ritonavir and darunavir-ritonavir based regimens.

Methods and Findings

A computer based literature search was conducted in the databases of HINARI (Health InterNetwork Access to Research Initiative), Medline and Cochrane library. Meta-analysis was performed by including randomized controlled studies that were conducted in ART experienced patients with plasma viral load above 1,000 copies HIV RNA/ml. The odds ratios and 95% confidence intervals (CI) for viral loads of <50 copies and <400 copies HIV RNA/ml at the end of the intervention were determined by the random effects model. Meta-regression, sensitivity analysis and funnel plots were done. The number of HIV-1 patients who were on either a tipranavir-ritonavir or darunavir-ritonavir based regimen and achieved viral load less than 50 copies HIV RNA/ml was significantly higher (overall OR = 3.4; 95% CI, 2.61– 4.52) than the number of HIV-1 patients who were on investigator selected boosted comparator HIV-1 protease inhibitors (CPIs-ritonavir). Similarly, the number of patients with viral load less than 400 copies HIV RNA/ml was significantly higher in either the tipranavir-ritonavir or darunavir-ritonavir based regimen treated group (overall OR = 3.0; 95% CI, 2.15 – 4.11). Meta-regression showed that the viral load reduction was independent of baseline viral load, baseline CD4 count and duration of tipranavir-ritonavir or darunavir-ritonavir based regimen.

Conclusions

Tipranavir and darunavir based regimens were more effective in patients who were ART experienced and had poor viral load control. Further studies are required to determine their consistent viral load suppression effect as the duration of treatment is more prolonged.     

Homozygosity mapping identifies a novel GIPC3 mutation causing congenital nonsyndromic hearing loss in a Saudi family

Hearing loss is one of the most common sensory disorders in humans and has a genetic cause in 50% of the cases. Our recent studies indicate that nonsyndromic hearing loss (NSHL) in the Saudi Arabian population is genetically heterogeneous and is not caused by mutations in GJB2 and GJB6, the most common genes for deafness in various populations worldwide. Identification of the causative gene/mutation in affected families is difficult due to extreme genetic heterogeneity and lack of phenotypic variability. We utilized an SNP array-based whole-genome homozygosity mapping approach in search of the causative gene, for the phenotype in a consanguineous Saudi family, with five affected individuals presenting severe to profound congenital NSHL. A single shared block of homozygosity was identified on chromosome 19p13.3 encompassing GIPC3, a recently identified hearing loss gene. Subsequently, a novel mutation c.122 C>A (p.T41K) in GIPC3 was found. This is the first report of GIPC3 mutation in a Saudi family. The presence of the GIPC3 mutations in only one of 100 Saudi families with congenital NSHL suggests that it appears to be a rare cause of familial or sporadic deafness in this population.     

Risk factors for ocular chlamydia after three mass azithromycin distributions

Background

An important component of the World Health Organization''s comprehensive trachoma elimination strategy is the provision of repeated annual mass azithromycin distributions, which are directed at reducing the burden of ocular chlamydia. Knowledge of characteristics associated with infection after mass antibiotic treatments could allow trachoma programs to focus resources to those most likely to be infected with ocular chlamydia.

Methodology/Principal Findings

We monitored 12 communities in rural Ethiopia that had received 3 annual mass azithromycin treatments as part of a cluster-randomized trial for trachoma. One year after the third treatment, a random sample of children from each village received conjunctival examination for follicular trachomatous inflammation (TF) and intense trachomatous inflammation (TI), conjunctival swabbing for chlamydial RNA and DNA, and a household survey. The primary outcome for this study was RNA evidence of ocular chlamydia, which we detected in 41 of 573 swabbed children (7.2%, 95%CI 2.7–17.8). In multivariate mixed effects logistic regression models, ocular chlamydial RNA was significantly associated with ocular discharge (OR 2.82, 95%CI 1.07–7.42), missing the most recent mass azithromycin treatment (OR 2.49, 95%CI 1.02–6.05), having a sibling with ocular chlamydia (OR 4.44, 95%CI 1.60–12.29), and above-median community population (OR 7.81, 95%CI 1.56–39.09). Ocular chlamydial infection was also independently associated with TF (OR 3.42, 95%CI 1.56–7.49) and TI (OR 5.39, 95%CI 2.43–11.98).

Conclusions/Significance

In areas with highly prevalent trachoma treated with multiple rounds of mass azithromycin, trachoma programs could consider continuing mass azithromycin treatments in households that have missed prior mass antibiotic treatments, in households with clinically active trachoma, and in larger communities.     

Reliability of measurements performed by community-drawn anthropometrists from rural Ethiopia

Background

Undernutrition is an important risk factor for childhood mortality, and remains a major problem facing many developing countries. Millennium Development Goal 1 calls for a reduction in underweight children, implemented through a variety of interventions. To adequately judge the impact of these interventions, it is important to know the reproducibility of the main indicators for undernutrition. In this study, we trained individuals from rural communities in Ethiopia in anthropometry techniques and measured intra- and inter-observer reliability.

Methods and Findings

We trained 6 individuals without prior anthropometry experience to perform weight, height, and middle upper arm circumference (MUAC) measurements. Two anthropometry teams were dispatched to 18 communities in rural Ethiopia and measurements performed on all consenting pre-school children. Anthropometry teams performed a second independent measurement on a convenience sample of children in order to assess intra-anthropometrist reliability. Both teams measured the same children in 2 villages to assess inter-anthropometrist reliability. We calculated several metrics of measurement reproducibility, including the technical error of measurement (TEM) and relative TEM. In total, anthropometry teams performed measurements on 606 pre-school children, 84 of which had repeat measurements performed by the same team, and 89 of which had measurements performed by both teams. Intra-anthropometrist TEM (and relative TEM) were 0.35 cm (0.35%) for height, 0.05 kg (0.39%) for weight, and 0.18 cm (1.27%) for MUAC. Corresponding values for inter-anthropometrist reliability were 0.67 cm (0.75%) for height, 0.09 kg (0.79%) for weight, and 0.22 kg (1.53%) for MUAC. Inter-anthropometrist measurement error was greater for smaller children than for larger children.

Conclusion

Measurements of height and weight were more reproducible than measurements of MUAC and measurements of larger children were more reliable than those for smaller children. Community-drawn anthropometrists can provide reliable measurements that could be used to assess the impact of interventions for childhood undernutrition.     

Performance of local light microscopy and the ParaScreen Pan/Pf rapid diagnostic test to detect malaria in health centers in Northwest Ethiopia

Background

Diagnostic tests are recommended for suspected malaria cases before treatment, but comparative performance of microscopy and rapid diagnostic tests (RDTs) at rural health centers has rarely been studied compared to independent expert microscopy.

Methods

Participants (N = 1997) with presumptive malaria were recruited from ten health centers with a range of transmission intensities in Amhara Regional State, Northwest Ethiopia during October to December 2007. Microscopy and ParaScreen Pan/Pf® RDT were done immediately by health center technicians. Blood slides were re-examined later at a central laboratory by independent expert microscopists.

Results

Of 1,997 febrile patients, 475 (23.8%) were positive by expert microscopists, with 57.7% P.falciparum, 24.6% P.vivax and 17.7% mixed infections. Sensitivity of health center microscopists for any malaria species was >90% in five health centers (four of which had the highest prevalence), >70% in nine centers and 44% in one site with lowest prevalence. Specificity for health center microscopy was very good (>95%) in all centers. For ParaScreen RDT, sensitivity was ≥90% in three centers, ≥70% in six and <60% in four centers. Specificity was ≥90% in all centers except one where it was 85%.

Conclusions

Health center microscopists performed well in nine of the ten health centers; while for ParaScreen RDT they performed well in only six centers. Overall the accuracy of local microscopy exceeded that of RDT for all outcomes. This study supports the introduction of RDTs only if accompanied by appropriate training, frequent supervision and quality control at all levels. Deficiencies in RDT use at some health centers must be rectified before universal replacement of good routine microscopy with RDTs. Maintenance and strengthening of good quality microscopy remains a priority at health center level.