LED-Fluorescence Microscopy for Diagnosis of Pulmonary Tuberculosis under Programmatic Conditions in India

Background

Light-emitting diode fluorescence microscopy (LED-FM) has been shown to be more sensitive than conventional bright field microscopy using Ziehl-Neelsen (ZN) stain in detecting sputum smear positive tuberculosis in controlled laboratory conditions. In 2012, Auramine O staining based LED-FM replaced conventional ZN microscopy in 200 designated microscopy centres (DMC) of medical colleges operating in collaboration with India’s Revised National Tuberculosis Control Programme. We aimed to assess the impact of introduction of LED-FM services on sputum smear positive case detection under program conditions.

Methods

This was a before and after comparison study. In 15 randomly selected medical college DMCs, all presumptive TB patients who underwent sputum smear examination in the years 2011 (before LED-FM) and 2012 (after LED-FM) were compared. An additional 15 comparable DMCs that implemented conventional ZN sputum smear microscopy were also selected for comparison between 2011 and 2012.

Results

The proportion of presumptive TB patients (PTP)found sputum smear positive increased by 30%- from 13.6% (3432/25159) in 2011 to 17.8% (4706/26426) in 2012 (P value <0.01) in the sites that implemented LED-FM microscopy, whereas in DMCs where the ZN staining procedure is followed the proportion of sputum smear positive had remained unchanged (13.0%versus 12.6%;P value0.31).

Conclusion

Use of LED-FM significantly increased the proportion of smear positive cases among presumptive TB patients under routine program conditions in high workload laboratories. The study provides operational evidence needed to scale-up the use of LED-FM in similar settings in India and beyond.     

When size does matter: organelle size influences the properties of transport mediated by molecular motors

Background

Organelle transport is driven by the action of molecular motors. In this work, we studied the dynamics of organelles of different sizes with the aim of understanding the complex relation between organelle motion and microenvironment.

Methods

We used single particle tracking to obtain trajectories of melanosomes (pigmented organelles in Xenopus laevis melanophores). In response to certain hormones, melanosomes disperse in the cytoplasm or aggregate in the perinuclear region by the combined action of microtubule and actin motors.

Results and conclusions

Melanosome trajectories followed an anomalous diffusion model in which the anomalous diffusion exponent (α) provided information regarding the trajectories' topography and thus of the processes causing it. During aggregation, the directionality of big organelles was higher than that of small organelles and did not depend on the presence of either actin or intermediate filaments (IF). Depolymerization of IF significantly reduced α values of small organelles during aggregation but slightly affect their directionality during dispersion.

General significance

Our results could be interpreted considering that the number of copies of active motors increases with organelle size. Transport of big organelles was not influenced by actin or IF during aggregation showing that these organelles are moved processively by the collective action of dynein motors. Also, we found that intermediate filaments enhance the directionality of small organelles suggesting that this network keeps organelles close to the tracks allowing their efficient reattachment. The higher directionality of small organelles during dispersion could be explained considering the better performance of kinesin-2 vs. dynein at the single molecule level.     

Endothelin regulates neural crest deployment and fate to form great vessels through Dlx5/Dlx6-independent mechanisms

Endothelin-1 (Edn1), originally identified as a vasoconstrictor peptide, is involved in the development of cranial/cardiac neural crest-derived tissues and organs. In craniofacial development, Edn1 binds to Endothelin type-A receptor (Ednra) to induce homeobox genes Dlx5/Dlx6 and determines the mandibular identity in the first pharyngeal arch. However, it remains unsolved whether this pathway is also critical for pharyngeal arch artery development to form thoracic arteries. Here, we show that the Edn1/Ednra signaling is involved in pharyngeal artery development by controlling the fate of neural crest cells through a Dlx5/Dlx6-independent mechanism. Edn1 and Ednra knock-out mice demonstrate abnormalities in pharyngeal arch artery patterning, which include persistent first and second pharyngeal arteries, resulting in additional branches from common carotid arteries. Neural crest cell labeling with Wnt1-Cre transgene and immunostaining for smooth muscle cell markers revealed that neural crest cells abnormally differentiate into smooth muscle cells at the first and second pharyngeal arteries of Ednra knock-out embryos. By contrast, Dlx5/Dlx6 knockout little affect the development of pharyngeal arch arteries and coronary arteries, the latter of which is also contributed by neural crest cells through an Edn-dependent mechanism. These findings indicate that the Edn1/Ednra signaling regulates neural crest differentiation to ensure the proper patterning of pharyngeal arch arteries, which is independent of the regional identification of the pharyngeal arches along the dorsoventral axis mediated by Dlx5/Dlx6.     

High Beta-Palmitate Fat Controls the Intestinal Inflammatory Response and Limits Intestinal Damage in Mucin Muc2 Deficient Mice

Background

Palmitic-acid esterified to the sn-1,3 positions of the glycerol backbone (alpha, alpha’-palmitate), the predominant palmitate conformation in regular infant formula fat, is poorly absorbed and might cause abdominal discomfort. In contrast, palmitic-acid esterified to the sn-2 position (beta-palmitate), the main palmitate conformation in human milk fat, is well absorbed. The aim of the present study was to examine the influence of high alpha, alpha’-palmitate fat (HAPF) diet and high beta-palmitate fat (HBPF) diet on colitis development in Muc2 deficient (Muc2^−/−) mice, a well-described animal model for spontaneous enterocolitis due to the lack of a protective mucus layer.

Methods

Muc2^−/− mice received AIN-93G reference diet, HAPF diet or HBPF diet for 5 weeks after weaning. Clinical symptoms, intestinal morphology and inflammation in the distal colon were analyzed.

Results

Both HBPF diet and AIN-93G diet limited the extent of intestinal erosions and morphological damage in Muc2^−/− mice compared with HAPF diet. In addition, the immunosuppressive regulatory T (Treg) cell response as demonstrated by the up-regulation of Foxp3, Tgfb1 and Ebi3 gene expression levels was enhanced by HBPF diet compared with AIN-93G and HAPF diets. HBPF diet also increased the gene expression of Pparg and enzymatic antioxidants (Sod1, Sod3 and Gpx1), genes all reported to be involved in promoting an immunosuppressive Treg cell response and to protect against colitis.

Conclusions

This study shows for the first time that HBPF diet limits the intestinal mucosal damage and controls the inflammatory response in Muc2^−/− mice by inducing an immunosuppressive Treg cell response.     

Environmental DNA from Residual Saliva for Efficient Noninvasive Genetic Monitoring of Brown Bears (Ursus arctos)

Noninvasive genetic sampling is an important tool in wildlife ecology and management, typically relying on hair snaring or scat sampling techniques, but hair snaring is labor and cost intensive, and scats yield relatively low quality DNA. New approaches utilizing environmental DNA (eDNA) may provide supplementary, cost-effective tools for noninvasive genetic sampling. We tested whether eDNA from residual saliva on partially-consumed Pacific salmon (Oncorhynchus spp.) carcasses might yield suitable DNA quality for noninvasive monitoring of brown bears (Ursus arctos). We compared the efficiency of monitoring brown bear populations using both fecal DNA and salivary eDNA collected from partially-consumed salmon carcasses in Southeast Alaska. We swabbed a range of tissue types from 156 partially-consumed salmon carcasses from a midseason run of lakeshore-spawning sockeye (O. nerka) and a late season run of stream-spawning chum (O. keta) salmon in 2014. We also swabbed a total of 272 scats from the same locations. Saliva swabs collected from the braincases of salmon had the best amplification rate, followed by swabs taken from individual bite holes. Saliva collected from salmon carcasses identified unique individuals more quickly and required much less labor to locate than scat samples. Salmon carcass swabbing is a promising method to aid in efficient and affordable monitoring of bear populations, and suggests that the swabbing of food remains or consumed baits from other animals may be an additional cost-effective and valuable tool in the study of the ecology and population biology of many elusive and/or wide-ranging species.     

Growth kinetics and mathematical modeling of Synechocystis sp. PCC 6803 under flashing light

In photobioreactors and natural systems, microalgae are subjected to rapidly changing light intensities (LI) due to light attenuation and mixing. A controlled way to study the effect of rapidly changing LI is to subject cultures to flashing light. In this study, series of flashing-light experiments were conducted using Synechocystis sp. PCC6803 with constant overall average LI of approximately 84 μmol·m⁻²·s⁻¹ and relative times in the light and dark varied. The results were also compared with simulated results using a mathematical model including an absorbed pool of light energy, photoacclimation, and photoinhibition. With equal time in light and dark, the specific growth rate (μ) systematically decreased with increasing light duration, and µ decreased further when the ratio of light to dark was decreased. The model captured both trends with the mechanistic explanation that when the light duration was very short the changes in the pool of absorbed LI were smoothed out across the light and dark periods, whereas longer durations caused the biomass to experience discrete light and dark conditions that lead to reduced light absorption, more energy loss to nonphotochemical quenching, and more photodamage. These growth effects were accentuated as the ratio of light to dark decreased.     

Genes,germs, and schizophrenia: an evolutionary perspective

Literature on schizophrenia and other mental illnesses has emphasized the compatibility of evidence with genetic causation without adequately considering alternative hypotheses of disease causation. Although some studies from the mid-20th century reported associations between certain pathogens and schizophrenia, only recently has the possibility of infectious causation of schizophrenia again become an active focus of research. Infectious causation of schizophrenia is still, however, generally regarded as less well demonstrated than genetic causation. This article evaluates the evidence that has been used to support genetic and infectious causation. Our consideration of infectious causation focuses on the protozoan Toxoplasma gondii but also assesses other pathogens that may contribute to the development of some of the illnesses currently categorized as schizophrenia. Although evidence generally accepted as demonstrating genetic causation can be readily explained by hypotheses of infectious causation, some of the evidence implicating infectious causation cannot be similarly explained by genetic causation. This asymmetry indicates that a scientific approach to the causation of schizophrenia needs to put a greater emphasis on tests that distinguish hypotheses of genetic causation from those of infectious causation.