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.     

The C. elegans Male Exercises Directional Control during Mating through Cholinergic Regulation of Sex-Shared Command Interneurons

Background

Mating behaviors in simple invertebrate model organisms represent tractable paradigms for understanding the neural bases of sex-specific behaviors, decision-making and sensorimotor integration. However, there are few examples where such neural circuits have been defined at high resolution or interrogated.

Methodology/Principal Findings

Here we exploit the simplicity of the nematode Caenorhabditis elegans to define the neural circuits underlying the male’s decision to initiate mating in response to contact with a mate. Mate contact is sensed by male-specific sensilla of the tail, the rays, which subsequently induce and guide a contact-based search of the hermaphrodite’s surface for the vulva (the vulva search). Atypically, search locomotion has a backward directional bias so its implementation requires overcoming an intrinsic bias for forward movement, set by activity of the sex-shared locomotory system. Using optogenetics, cell-specific ablation- and mutant behavioral analyses, we show that the male makes this shift by manipulating the activity of command cells within this sex-shared locomotory system. The rays control the command interneurons through the male-specific, decision-making interneuron PVY and its auxiliary cell PVX. Unlike many sex-shared pathways, PVY/PVX regulate the command cells via cholinergic, rather than glutamatergic transmission, a feature that likely contributes to response specificity and coordinates directional movement with other cholinergic-dependent motor behaviors of the mating sequence. PVY/PVX preferentially activate the backward, and not forward, command cells because of a bias in synaptic inputs and the distribution of key cholinergic receptors (encoded by the genes acr-18, acr-16 and unc-29) in favor of the backward command cells.

Conclusion/Significance

Our interrogation of male neural circuits reveals that a sex-specific response to the opposite sex is conferred by a male-specific pathway that renders subordinate, sex-shared motor programs responsive to mate cues. Circuit modifications of these types may make prominent contributions to natural variations in behavior that ultimately bring about speciation.     

Cardiac Fibroblast-Dependent Extracellular Matrix Accumulation Is Associated with Diastolic Stiffness in Type 2 Diabetes

Cardiovascular complications are a leading cause of death in patients with type 2 diabetes mellitus (T2DM). Diastolic dysfunction is one of the earliest manifestations of diabetes-induced changes in left ventricular (LV) function, and results from a reduced rate of relaxation and increased stiffness. The mechanisms responsible for increased stiffness are not completely understood. Chronic hyperglycemia, advanced glycation endproducts (AGEs), and increased levels of proinflammatory and profibrotic cytokines are molecular pathways known to be involved in regulating extracellular matrix (ECM) synthesis and accumulation resulting in increased LV diastolic stiffness. Experiments were conducted using a genetically-induced mouse model of T2DM generated by a point mutation in the leptin receptor resulting in nonfunctional leptin receptors (db/db murine model). This study correlated changes in LV ECM and stiffness with alterations in basal activation of signaling cascades and expression of profibrotic markers within primary cultures of cardiac fibroblasts from diabetic (db/db) mice with nondiabetic (db/wt) littermates as controls. Primary cultures of cardiac fibrobroblasts were maintained in 25 mM glucose (hyperglycemic-HG; diabetic db/db) media or 5 mM glucose (normoglycemic-NG, nondiabetic db/wt) media. The cells then underwent a 24-hour exposure to their opposite (NG; diabetic db/db) media or 5 mM glucose (HG, nondiabetic db/wt) media. Protein analysis demonstrated significantly increased expression of type I collagen, TIMP-2, TGF-β, PAI-1 and RAGE in diabetic db/db cells as compared to nondiabetic db/wt, independent of glucose media concentration. This pattern of protein expression was associated with increased LV collagen accumulation, myocardial stiffness and LV diastolic dysfunction. Isolated diabetic db/db fibroblasts were phenotypically distinct from nondiabetic db/wt fibroblasts and exhibited a profibrotic phenotype in normoglycemic conditions.     

A Randomized Controlled Pilot Study of Home-Based Step Training in Older People Using Videogame Technology

Background

Stepping impairments are associated with physical and cognitive decline in older adults and increased fall risk. Exercise interventions can reduce fall risk, but adherence is often low. A new exergame involving step training may provide an enjoyable exercise alternative for preventing falls in older people.

Purpose

To assess the feasibility and safety of unsupervised, home-based step pad training and determine the effectiveness of this intervention on stepping performance and associated fall risk in older people.

Design

Single-blinded two-arm randomized controlled trial comparing step pad training with control (no-intervention).

Setting/Participants

Thirty-seven older adults residing in independent-living units of a retirement village in Sydney, Australia.

Intervention

Intervention group (IG) participants were provided with a computerized step pad system connected to their TVs and played a step game as often as they liked (with a recommended dose of 2–3 sessions per week for 15–20 minutes each) for eight weeks. In addition, IG participants were asked to complete a choice stepping reaction time (CSRT) task once each week.

Main Outcome Measures

CSRT, the Physiological Profile Assessment (PPA), neuropsychological and functional mobility measures were assessed at baseline and eight week follow-up.

Results

Thirty-two participants completed the study (86.5%). IG participants played a median 2.75 sessions/week and no adverse events were reported. Compared to the control group, the IG significantly improved their CSRT (F_31,1 = 18.203, p<.001), PPA composite scores (F_31,1 = 12.706, p = 0.001), as well as the postural sway (F_31,1 = 4.226, p = 0.049) and contrast sensitivity (F_31,1 = 4.415, p = 0.044) PPA sub-component scores. In addition, the IG improved significantly in their dual-task ability as assessed by a timed up and go test/verbal fluency task (F_31,1 = 4.226, p = 0.049).

Conclusions

Step pad training can be safely undertaken at home to improve physical and cognitive parameters of fall risk in older people without major cognitive and physical impairments.

Trial Registration

Australian New Zealand Clinical Trials Registry ACTRN12611001081909.     

Real-time detection of DNA topological changes with a fluorescently labeled cruciform

Topoisomerases are essential cellular enzymes that maintain the appropriate topological status of DNA and are the targets of several antibiotic and chemotherapeutic agents. High-throughput (HT) analysis is desirable to identify new topoisomerase inhibitors, but standard in vitro assays for DNA topology, such as gel electrophoresis, are time-consuming and are not amenable to HT analysis. We have exploited the observation that closed-circular DNA containing an inverted repeat can release the free energy stored in negatively supercoiled DNA by extruding the repeat as a cruciform. We inserted an inverted repeat containing a fluorophore-quencher pair into a plasmid to enable real-time monitoring of plasmid supercoiling by a bacterial topoisomerase, Escherichia coli gyrase. This substrate produces a fluorescent signal caused by the extrusion of the cruciform and separation of the labels as gyrase progressively underwinds the DNA. Subsequent relaxation by a eukaryotic topoisomerase, human topo IIα, causes reintegration of the cruciform and quenching of fluorescence. We used this approach to develop a HT screen for inhibitors of gyrase supercoiling. This work demonstrates that fluorescently labeled cruciforms are useful as general real-time indicators of changes in DNA topology that can be used to monitor the activity of DNA-dependent motor proteins.     

Submillisecond Elastic Recoil Reveals Molecular Origins of Fibrin Fiber Mechanics

Fibrin fibers form the structural scaffold of blood clots. Thus, their mechanical properties are of central importance to understanding hemostasis and thrombotic disease. Recent studies have revealed that fibrin fibers are elastomeric despite their high degree of molecular ordering. These results have inspired a variety of molecular models for fibrin’s elasticity, ranging from reversible protein unfolding to rubber-like elasticity. An important property that has not been explored is the timescale of elastic recoil, a parameter that is critical for fibrin’s mechanical function and places a temporal constraint on molecular models of fiber elasticity. Using high-frame-rate imaging and atomic force microscopy-based nanomanipulation, we measured the recoil dynamics of individual fibrin fibers and found that the recoil was orders of magnitude faster than anticipated from models involving protein refolding. We also performed steered discrete molecular-dynamics simulations to investigate the molecular origins of the observed recoil. Our results point to the unstructured αC regions of the otherwise structured fibrin molecule as being responsible for the elastic recoil of the fibers.     

Rice endosperm is cost‐effective for the production of recombinant griffithsin with potent activity against HIV

Protein microbicides containing neutralizing antibodies and antiviral lectins may help to reduce the rate of infection with human immunodeficiency virus (HIV) if it is possible to manufacture the components in large quantities at a cost affordable in HIV‐endemic regions such as sub‐Saharan Africa. We expressed the antiviral lectin griffithsin (GRFT), which shows potent neutralizing activity against HIV, in the endosperm of transgenic rice plants (Oryza sativa), to determine whether rice can be used to produce inexpensive GRFT as a microbicide ingredient. The yield of ^OSGRFT in the best‐performing plants was 223 μg/g dry seed weight. We also established a one‐step purification protocol, achieving a recovery of 74% and a purity of 80%, which potentially could be developed into a larger‐scale process to facilitate inexpensive downstream processing. ^OSGRFT bound to HIV glycans with similar efficiency to GRFT produced in Escherichia coli. Whole‐cell assays using purified ^OSGRFT and infectivity assays using crude extracts of transgenic rice endosperm confirmed that both crude and pure ^OSGRFT showed potent activity against HIV and the crude extracts were not toxic towards human cell lines, suggesting they could be administered as a microbicide with only minimal processing. A freedom‐to‐operate analysis confirmed that GRFT produced in rice is suitable for commercial development, and an economic evaluation suggested that 1.8 kg/ha of pure GRFT could be produced from rice seeds. Our data therefore indicate that rice could be developed as an inexpensive production platform for GRFT as a microbicide component.     

Early initiation of breastfeeding: a systematic literature review of factors and barriers in South Asia

Background

Early or timely initiation of breastfeeding is crucial in preventing newborn deaths and influences childhood nutrition however remains low in South Asia and the factors and barriers warrant greater consideration for improved action. This review synthesises the evidence on factors and barriers to initiation of breastfeeding within 1 h of birth in South Asia encompassing Afghanistan, Bangladesh, Bhutan, India, Maldives, Nepal, Pakistan and Sri Lanka.

Methods

Studies published between 1990 and 2013 were systematically reviewed through identification in Academic Search Complete, CINAHL, Global Health, MEDLINE and Scopus databases. Twenty-five studies meeting inclusion criteria were included for review. Structured thematic analysis based on leading frameworks was undertaken to understand factors and barriers.

Results

Factors at geographical, socioeconomic, individual, and health-specific levels, such as residence, education, occupation, income, mother’s age and newborn’s gender, and ill health of mother and newborn at delivery, affect early or timely breastfeeding initiation in South Asia. Reported barriers impact through influence on acceptability by traditional feeding practices, priests’ advice, prelacteal feeding and discarding colostrum, mother-in-law’s opinion; availability and accessibility through lack of information, low access to media and health services, and misperception, support and milk insufficiency, involvement of mothers in decision making.

Conclusions

Whilst some barriers manifest similarly across the region some factors are context-specific thus tailored interventions are imperative. Initiatives halting factors and directed towards contextual barriers are required for greater impact on newborn survival and improved nutrition in the South Asia region.

     

Vertical Migration and Mortality of Marine Benthos in Dredged Material: A Synthesis

This account describes the comparative response of four species of benthic invertebrates to burial in terms of vertical migration and mortality, and provides a synthesis of studies and recommendations upon which to assess future impacts. The species featured were the bivalve Mercenaries mercenaria, the amphipod crustacean Parahaustorius longimerus, and the polychaetes Scoloplos fragilis and Nereis succinea. There was evidence of synergistic effects on burrowing activity and mortality with changes in time of burial, sediment depth, sediment type and temperature. In sediment with silt-clay, N. succinea was the most resistant species followed by M. mercenaria, S. fragilis and P. longimerus. In sediment without silt-clay the order of percent mortality was reversed. Studies of surface water chemistry and sediment geochemistry showed that dissolved oxygen decreased significantly and ammonia and sulfide increased significantly between the surface and below 2.0 cm within a 15-day period. Based on these results and physiological tolerances from the literature it was concluded that M. mercenaria and N. succinea would be relatively resistant to chemical effects of spoil disposal, whereas S. fragilis and P. longimerus would be less resistant to such effects. Vertical migration of benthic invertebrates through dredge disposal can be a viable mechanism of recolonization under certain conditions. Some effects of burial of benthos can be predicted based on morphology, behavior and physiology. These biological features were discussed with examples dealing with molluscs, crustaceans, and polychaetes. Finally, recommendations were made concerning the type of studies to provide additional data to aid management agencies in decision making about future dredging and disposal practices.