Performance of Three LED-Based Fluorescence Microscopy Systems for Detection of Tuberculosis in Uganda

BACKGROUND: Direct smear microscopy using Ziehl-Neelsen (ZN) staining is the mainstay of tuberculosis (TB) diagnosis in most high burden countries, but is limited by low sensitivity in routine practice, particularly in high human immunodeficiency virus (HIV) prevalence settings. METHODS: We compared the performance of three commercial light emitting diode (LED)-based microscopy systems (Primostar™ iLED, Lumin™ and AFTER®) for fluorescent detection of Mycobacterium tuberculosis with ZN microscopy on slides prepared from sputum of TB suspects. Examination time for LED-based fluorescent microscopy (LED FM) and ZN slides was also compared, and a qualitative user appraisal of the LED FM systems was carried out. RESULTS: LED FM was between 5.6 and 9.4% more sensitive than ZN microscopy, although the difference was not statistically significant. There was no significant difference in the sensitivity or specificity of the three LED FM systems, although the specificity of Fraen AFTER was somewhat lower than the other LED FM methods. Examination time for LED FM was 2 and 4 times less than for ZN microscopy. LED FM was highly acceptable to Ugandan technologists, although differences in operational performance of the three systems were reported. CONCLUSIONS: LED FM compares favourably with ZN microscopy, with equivalent specificity and a modest increase in sensitivity. Screening of slides was substantially quicker using LED FM than ZN, and LED FM was rated highly by laboratory technologists. Available commercial systems have different operational characteristics which should be considered prior to programmatic implementation

Autoluminescent Plants

Prospects of obtaining plants glowing in the dark have captivated the imagination of scientists and layman alike. While light emission has been developed into a useful marker of gene expression, bioluminescence in plants remained dependent on externally supplied substrate. Evolutionary conservation of the prokaryotic gene expression machinery enabled expression of the six genes of the lux operon in chloroplasts yielding plants that are capable of autonomous light emission. This work demonstrates that complex metabolic pathways of prokaryotes can be reconstructed and function in plant chloroplasts and that transplastomic plants can emit light that is visible by naked eye

Inhibitor of apoptosis proteins, NAIP, cIAP1 and cIAP2 expression during macrophage differentiation and M1/M2 polarization

Monocytes and macrophages constitute the first line of defense of the immune system against external pathogens. Macrophages have a highly plastic phenotype depending on environmental conditions; the extremes of this phenotypic spectrum are a pro-inflammatory defensive role (M1 phenotype) and an anti-inflammatory tissue-repair one (M2 phenotype). The Inhibitor of Apoptosis (IAP) proteins have important roles in the regulation of several cellular processes, including innate and adaptive immunity. In this study we have analyzed the differential expression of the IAPs, NAIP, cIAP1 and cIAP2, during macrophage differentiation and polarization into M1 or M2. In polarized THP-1 cells and primary human macrophages, NAIP is abundantly expressed in M2 macrophages, while cIAP1 and cIAP2 show an inverse pattern of expression in polarized macrophages, with elevated expression levels of cIAP1 in M2 and cIAP2 preferentially expressed in M1. Interestingly, treatment with the IAP antagonist SMC-LCL161, induced the upregulation of NAIP in M2, the downregulation of cIAP1 in M1 and M2 and an induction of cIAP2 in M1 macrophages.This work was supported by Universidad de Granada, Plan Propio 2015;#P3B: FAM, VMC (http://investigacion.ugr.es/pages/planpropio/2015/ resoluciones/p3b_def_28072015); Universidad de Granada CEI BioTic;#CAEP2-84: VMC (http:// biotic.ugr.es/pages/resolucionprovisional enseaanzapractica22demayo/!); and Canadian nstitutes of Health Research;#231421, #318176, #361847: STB, ECL, RK (http://www.cihr-irsc.gc. ca/e/193.html). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Restoring brain function after stroke - bridging the gap between animals and humans

Stroke is the leading cause of complex adult disability in the world. Recovery from stroke is often incomplete, which leaves many people dependent on others for their care. The improvement of long-term outcomes should, therefore, be a clinical and research priority. As a result of advances in our understanding of the biological mechanisms involved in recovery and repair after stroke, therapeutic opportunities to promote recovery through manipulation of poststroke plasticity have never been greater. This work has almost exclusively been carried out in preclinical animal models of stroke with little translation into human studies. The challenge ahead is to develop a mechanistic understanding of recovery from stroke in humans. Advances in neuroimaging techniques now enable us to reconcile behavioural accounts of recovery with molecular and cellular changes. Consequently, clinical trials can be designed in a stratified manner that takes into account when an intervention should be delivered and who is most likely to benefit. This approach is expected to lead to a substantial change in how restorative therapeutic strategies are delivered in patients after stroke

Physical Analyses of E. coli Heteroduplex Recombination Products In Vivo: On the Prevalence of 5′ and 3′ Patches

BACKGROUND: Homologous recombination in Escherichia coli creates patches (non-crossovers) or splices (half crossovers), each of which may have associated heteroduplex DNA. Heteroduplex patches have recombinant DNA in one strand of the duplex, with parental flanking markers. Which DNA strand is exchanged in heteroduplex patches reflects the molecular mechanism of recombination. Several models for the mechanism of E. coli RecBCD-mediated recombinational double-strand-end (DSE) repair specify that only the 3'-ending strand invades the homologous DNA, forming heteroduplex in that strand. There is, however, in vivo evidence that patches are found in both strands. METHODOLOGY/PRINCIPLE FINDINGS: This paper re-examines heteroduplex-patch-strand polarity using phage lambda and the lambdadv plasmid as DNA substrates recombined via the E. coli RecBCD system in vivo. These DNAs are mutant for lambda recombination functions, including orf and rap, which were functional in previous studies. Heteroduplexes are isolated, separated on polyacrylamide gels, and quantified using Southern blots for heteroduplex analysis. This method reveals that heteroduplexes are still found in either 5' or 3' DNA strands in approximately equal amounts, even in the absence of orf and rap. Also observed is an independence of the RuvC Holliday-junction endonuclease on patch formation, and a slight but statistically significant alteration of patch polarity by recD mutation. CONCLUSIONS/SIGNIFICANCE: These results indicate that orf and rap did not contribute to the presence of patches, and imply that patches occurring in both DNA strands reflects the molecular mechanism of recombination in E. coli. Most importantly, the lack of a requirement for RuvC implies that endonucleolytic resolution of Holliday junctions is not necessary for heteroduplex-patch formation, contrary to predictions of all of the major previous models. This implies that patches are not an alternative resolution of the same intermediate that produces splices, and do not bear on models for splice formation. We consider two mechanisms that use DNA replication instead of endonucleolytic resolution for formation of heteroduplex patches in either DNA strand: synthesis-dependent-strand annealing and a strand-assimilation mechanism

Bilateral salpingo-oophorectomy versus GnRH analogue in the adjuvant treatment of premenopausal breast cancer patients : cost-effectiveness evaluation of breast cancer outcome, ovarian cancer prevention and treatment

BACKGROUND AND OBJECTIVE: There is no available evidence to recommend gonadotropin-releasing hormone (GnRH) analogue-based ovarian suppression versus bilateral salpingo-oophorectomy (BSO) in the adjuvant treatment of early breast cancer, since the two approaches are considered equivalent in terms of oncologic outcome. The role of surgical ovarian ablation has been revitalized based on the advances of minimally invasive surgery, and a better understanding of clinical and molecular basis of hereditary breast/ovarian cancer syndromes. The aim of this study is to analyze the cost-effectiveness of laparoscopic BSO and GnRH analogue administration in patients aged 40-49 years with hormone-sensitive breast cancer. METHODS: A probabilistic decision tree model was developed to evaluate costs and outcomes of ovarian ablation through laparoscopic BSO, or ovarian suppression through monthly injections of GnRH analogue. Results were expressed as incremental costs per quality-adjusted life years (QALYs) gained. RESULTS: Laparoscopic BSO strategy was associated with a lower mean total cost per patient than GnRH treatment, and considering the difference in terms of QALYs, the incremental effectiveness did not demonstrate a notable difference between the two approaches. From the National Health Service perspective, and for a time horizon of 5 years, laparoscopic BSO was the dominant option compared to GnRH treatment; laparoscopic BSO was less expensive than GnRH, €2385 [95% confidence interval (CI) = 2044, 2753] vs €7093 (95% CI = 3409, 12,105), respectively, and more effective. CONCLUSION: Surgical ovarian ablation is more cost-effective than GnRH administration in the adjuvant treatment of hormone-sensitive breast cancer patients aged 40-49 years, and the advantage of preventing ovarian cancer through laparoscopic BSO should be considered

Lipid accumulation and dendritic cell dysfunction in cancer

Professional antigen presenting cells, dendritic cells (DC) are responsible for initiation and maintenance of immune responses. Here, we report that a substantial proportion of DCs in tumor-bearing mice and cancer patients have increased levels of triglycerides. Lipid accumulation in DCs was caused by increased uptake of extracellular lipids due to up-regulation of scavenger receptor A. DCs with high lipid content were not able to effectively stimulate allogeneic T cells or present tumor-associated antigens. DCs with high and normal lipid levels did not differ in expression of MHC and co-stimulatory molecules. However, lipid-laden DCs had reduced capacity to process antigens. Pharmacological normalization of lipid levels in DCs with an inhibitor of acetyl-CoA carboxylase restored the functional activity of DCs and substantially enhanced the effects of a cancer vaccine. These findings support the regulation of immune responses in cancer by manipulation of lipid levels in DCs