The next generation of ocular pathogen detection

Metagenomic next-generation sequencing is a powerful method for pathogen detection that combines advanced genome sequencing technology with cutting-edge bioinformatics to analyze microbial populations. Metagenomic next-generation sequencing has the potential to identify uncommon, unculturable, and even previously unidentified pathogens from a clinical isolate. Of particular interest to ophthalmology, this robust data extraction can occur from very small volume clinical samples. Here we discuss the opportunities and limitations of this technique and their current and future application to ophthalmic diagnostics

Predictors of measles vaccination coverage among children 6-59 months of age in the Democratic Republic of the Congo.

BackgroundMeasles is a significant contributor to child mortality in the Democratic Republic of the Congo (DRC), despite routine immunization programs and supplementary immunization activities (SIA). Further, national immunization coverage levels may hide disparities among certain groups of children, making effective measles control even more challenging. This study describes measles vaccination coverage and reporting methods and identifies predictors of vaccination among children participating in the 2013-2014 DRC Demographic and Health Survey (DHS).MethodsWe examined vaccination coverage of 6947 children aged 6-59 months. A multivariate logistic regression model was used to identify predictors of vaccination among children reporting vaccination via dated card in order to identify least reached children. We also assessed spatial distribution of vaccination report type by rural versus urban residence.ResultsUrban children with educated mothers were more likely to be vaccinated (OR = 4.1, 95% CI: 1.6, 10.7) versus children of mothers with no education, as were children in wealthier rural families (OR = 2.9, 95% CI: 1.9, 4.4). At the provincial level, urban areas more frequently reported vaccination via dated card than rural areas.ConclusionsResults indicate that, while the overall coverage level of 70% is too low, socioeconomic and geographic disparities also exist which could make some children even less likely to be vaccinated. Dated records of measles vaccination must be increased, and groups of children with the greatest need should be targeted. As access to routine vaccination services is limited in DRC, identifying and targeting under-reached children should be a strategic means of increasing country-wide effective measles control

Neuronal deletion of GSK3beta increases microtubule speed in the growth cone and enhances axon regeneration via CRMP-2 and independently of MAP1B and CLASP2

BACKGROUND: In the adult central nervous system, axonal regeneration is abortive. Regulators of microtubule dynamics have emerged as attractive targets to promote axonal growth following injury as microtubule organization is pivotal for growth cone formation. In this study, we used conditioned neurons with high regenerative capacity to further dissect cytoskeletal mechanisms that might be involved in the gain of intrinsic axon growth capacity. RESULTS: Following a phospho-site broad signaling pathway screen, we found that in conditioned neurons with high regenerative capacity, decreased glycogen synthase kinase 3β (GSK3β) activity and increased microtubule growth speed in the growth cone were present. To investigate the importance of GSK3β regulation during axonal regeneration in vivo, we used three genetic mouse models with high, intermediate or no GSK3β activity in neurons. Following spinal cord injury, reduced GSK3β levels or complete neuronal deletion of GSK3β led to increased growth cone microtubule growth speed and promoted axon regeneration. While several microtubule-interacting proteins are GSK3β substrates, phospho-mimetic collapsin response mediator protein 2 (T/D-CRMP-2) was sufficient to decrease microtubule growth speed and neurite outgrowth of conditioned neurons and of GSK3β-depleted neurons, prevailing over the effect of decreased levels of phosphorylated microtubule-associated protein 1B (MAP1B) and through a mechanism unrelated to decreased levels of phosphorylated cytoplasmic linker associated protein 2 (CLASP2). In addition, phospho-resistant T/A-CRMP-2 counteracted the inhibitory myelin effect on neurite growth, further supporting the GSK3β-CRMP-2 relevance during axon regeneration. CONCLUSIONS: Our work shows that increased microtubule growth speed in the growth cone is present in conditions of increased axonal growth, and is achieved following inactivation of the GSK3β-CRMP-2 pathway, enhancing axon regeneration through the glial scar. In this context, our results support that a precise control of microtubule dynamics, specifically in the growth cone, is required to optimize axon regrowth

Intranasal nanoemulsion vaccine confers long‐lasting immunomodulation and sustained unresponsiveness in a murine model of milk allergy

BackgroundImmunotherapy for food allergy requires prolonged treatment protocols and, in most cases, does not lead to durable modulation of the allergic immune response. We have demonstrated an intranasal (IN) nanoemulsion adjuvant that redirects allergen‐specific Th2 responses toward Th1 and Th17 immunity, and protects from allergen challenge after only 2‐4 monthly administrations. Here, we investigate the ability of this technology to provide long‐term modulation of allergy in a murine model of cow’s milk allergy.MethodsSix weeks after sensitization to bovine casein, mice received four, monthly IN immunizations with nanoemulsion formulated with casein. Protection from casein challenge was assessed at 4 and 16 weeks after the final vaccine administration.ResultsThe NE vaccine significantly blunted the physiological responses to allergen challenge, and this effect persisted for at least 16 weeks. The protection from challenge was associated with the suppression of casein‐specific Th2 immunity and induced Th1 and Th17 cytokines as well as induction of IL‐10. Of interest, while immunized animals showed significantly decreased Th2 cytokine responses, cow’s milk‐specific IgE remained elevated in the serum at levels associated with reactivity in control sensitized animals. Protection was associated with suppressed mast cell activation and markedly reduced mast cell infiltration into the small intestine.ConclusionThe sustained unresponsiveness of at least 16 weeks after vaccination suggests that the nanoemulsion vaccine alters the allergic phenotype in a persistent manner different from traditional desensitization, and this leads to long‐term suppressive effects on allergic disease without eliminating serum IgE.This study evaluates the ability of an intranasal nanoemulsion‐based vaccine to induce long‐term modulation of allergic reactions in a mouse model of cow’s milk allergy. Intranasal immunization with nanoemulsion adjuvant suppresses Th2 responses and anaphylaxis. The sustained unresponsiveness of at least 16 weeks after vaccination suggests that the nanoemulsion vaccine alters the allergic phenotype.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154959/1/all14064_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154959/2/all14064.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154959/3/all14064-sup-0003-FigS3.pd

Association of Previous Measles Infection With Markers of Acute Infectious Disease Among 9- to 59-Month-Old Children in the Democratic Republic of the Congo.

BackgroundTransient immunosuppression and increased susceptibility to other infections after measles infection is well known, but recent studies have suggested the occurrence of an "immune amnesia" that could have long-term immunosuppressive effects.MethodsWe examined the association between past measles infection and acute episodes of fever, cough, and diarrhea among 2350 children aged 9 to 59 months whose mothers were selected for interview in the 2013-2014 Democratic Republic of the Congo (DRC) Demographic and Health Survey (DHS). Classification of children who had had measles was completed using maternal recall and measles immunoglobulin G serostatus obtained via dried-blood-spot analysis with a multiplex immunoassay. The association with time since measles infection and fever, cough, and diarrhea outcomes was also examined.ResultsThe odds of fever in the previous 2 weeks were 1.80 (95% confidence interval [CI], 1.25-2.60) among children for whom measles was reported compared to children with no history of measles. Measles vaccination demonstrated a protective association against selected clinical markers of acute infectious diseases.ConclusionOur results suggest that measles might have a long-term effect on selected clinical markers of acute infectious diseases among children aged 9 to 59 months in the DRC. These findings support the immune-amnesia hypothesis suggested by others and underscore the need for continued evaluation and improvement of the DRC's measles vaccination program

Interactions between subunits of Saccharomyces cerevisiae RNase MRP support a conserved eukaryotic RNase P/MRP architecture

Ribonuclease MRP is an endonuclease, related to RNase P, which functions in eukaryotic pre-rRNA processing. In Saccharomyces cerevisiae, RNase MRP comprises an RNA subunit and ten proteins. To improve our understanding of subunit roles and enzyme architecture, we have examined protein-protein and protein–RNA interactions in vitro, complementing existing yeast two-hybrid data. In total, 31 direct protein–protein interactions were identified, each protein interacting with at least three others. Furthermore, seven proteins self-interact, four strongly, pointing to subunit multiplicity in the holoenzyme. Six protein subunits interact directly with MRP RNA and four with pre-rRNA. A comparative analysis with existing data for the yeast and human RNase P/MRP systems enables confident identification of Pop1p, Pop4p and Rpp1p as subunits that lie at the enzyme core, with probable addition of Pop5p and Pop3p. Rmp1p is confirmed as an integral subunit, presumably associating preferentially with RNase MRP, rather than RNase P, via interactions with Snm1p and MRP RNA. Snm1p and Rmp1p may act together to assist enzyme specificity, though roles in substrate binding are also indicated for Pop4p and Pop6p. The results provide further evidence of a conserved eukaryotic RNase P/MRP architecture and provide a strong basis for studies of enzyme assembly and subunit function

Skeletal muscle ACC2 S212 phosphorylation is not required for the control of fatty acid oxidation during exercise

During submaximal exercise fatty acids are a predominant energy source for muscle contractions. An important regulator of fatty acid oxidation is acetyl‐CoA carboxylase (ACC), which exists as two isoforms (ACC1 and ACC2) with ACC2 predominating in skeletal muscle. Both ACC isoforms regulate malonyl‐CoA production, an allosteric inhibitor of carnitine palmitoyltransferase 1 (CPT‐1); the primary enzyme controlling fatty acyl‐CoA flux into mitochondria for oxidation. AMP‐activated protein kinase (AMPK) is a sensor of cellular energy status that is activated during exercise or by pharmacological agents such as metformin and AICAR. In resting muscle the activation of AMPK with AICAR leads to increased phosphorylation of ACC (S79 on ACC1 and S221 on ACC2), which reduces ACC activity and malonyl‐CoA; effects associated with increased fatty acid oxidation. However, whether this pathway is vital for regulating skeletal muscle fatty acid oxidation during conditions of increased metabolic flux such as exercise/muscle contractions remains unknown. To examine this we characterized mice lacking AMPK phosphorylation sites on ACC2 (S212 in mice/S221 in humans‐ACC2‐knock‐in [ACC2‐KI]) or both ACC1 (S79) and ACC2 (S212) (ACC double knock‐in [ACCD‐KI]) during submaximal treadmill exercise and/or ex vivo muscle contractions. We find that surprisingly, ACC2‐KI mice had normal exercise capacity and whole‐body fatty acid oxidation during treadmill running despite elevated muscle ACC2 activity and malonyl‐CoA. Similar results were observed in ACCD‐KI mice. Fatty acid oxidation was also maintained in muscles from ACC2‐KI mice contracted ex vivo. These findings indicate that pathways independent of ACC phosphorylation are important for regulating skeletal muscle fatty acid oxidation during exercise/muscle contractions

Flatworm-specific transcriptional regulators promote the specification of tegumental progenitors in Schistosoma mansoni

Schistosomes infect more than 200 million people. These parasitic flatworms rely on a syncytial outer coat called the tegument to survive within the vasculature of their host. Although the tegument is pivotal for their survival, little is known about maintenance of this tissue during the decades schistosomes survive in the bloodstream. Here, we demonstrate that the tegument relies on stem cells (neoblasts) to specify fusogenic progenitors that replace tegumental cells lost to turnover. Molecular characterization of neoblasts and tegumental progenitors led to the discovery of two flatworm-specific zinc finger proteins that are essential for tegumental cell specification. These proteins are homologous to a protein essential for neoblast-driven epidermal maintenance in free-living flatworms. Therefore, we speculate that related parasites (i.e., tapeworms and flukes) employ similar strategies to control tegumental maintenance. Since parasitic flatworms infect every vertebrate species, understanding neoblast-driven tegumental maintenance could identify broad-spectrum therapeutics to fight diseases caused by these parasites