Advances in TERS (tip-enhanced Raman scattering) for biochemical applications

TERS (tip-enhanced Raman scattering) provides exceptional spatial resolution without any need for labelling and has become a versatile tool for biochemical analysis. Two examples will be highlighted here. On the one hand, TERS measurements on a single mitochondrion are discussed, monitoring the oxidation state of the central iron ion of cytochrome c, leading towards a single protein characterization scheme in a natural environment. On the other hand, a novel approach of single molecule analysis is discussed, again based on TERS experiments on DNA and RNA, further highlighting the resolution capabilities of this method.     

Multiple sclerosis - remyelination failure as a cause of disease progression

Multiple sclerosis (MS) is the most frequent demyelinating disease of the central nervous system (CNS) that affects worldwide about 2.5 million people. The morphological correlates of the disease are multiple lesions in brain and spinal cord which are characterized by demyelination, inflammation, gliosis and axonal damage. The underlying cause for the permanent neurological deficits in MS patients is axonal loss. Demyelinated axons are prone to damage due to the lack of trophic support by myelin sheaths and oligodendrocytes, as well as the increased vulnerability to immune mediated attacks. Remyelination occurs, but especially in chronic lesions is frequently limited to a small rim at the lesion border. Current treatment strategies are based on anti-inflammatory or immunomodulatory drugs and have the potential to reduce the numbers of newly evolving lesions, although as yet no treatment strategy exists to influence or prevent the progressive disease phase. Therefore, the development of neuroprotective treatment options, such as the promotion of endogenous remyelination is an attractive strategy. A prerequisite for the development of such new treatments is the understanding of the mechanisms leading to remyelination and the reasons for insufficient endogenous repair in chronic MS. This review will therefore provide an overview of the current concepts regarding remyelination in the rodent and human CNS. We will also summarize a selected number of inhibitory pathways and non-disease related factors which may contribute to remyelination failure in chronic MS.     

Signatures of balancing selection are maintained at disease resistance loci following mating system evolution and a population bottleneck in the genus Capsella

ABSTRACT: BACKGROUND: Population bottlenecks can lead to a loss of variation at disease resistance loci, which could have important consequences for the ability of populations to adapt to pathogen pressure. Alternatively, current or past balancing selection could maintain high diversity, creating a strong heterogeneity in the retention of polymorphism across the genome of bottlenecked populations. We sequenced part of the LRR region of 9 NBS-LRR disease resistance genes in the outcrossing Capsella grandiflora and the recently derived, bottlenecked selfing species Capsella rubella, and compared levels and patterns of nucleotide diversity and divergence with genome-wide reference loci. RESULTS: In strong contrast with reference loci, average diversity at resistance loci was comparable between C. rubella and C. grandiflora, primarily due to two loci with highly elevated diversity indicative of past or present balancing selection. Average between-species differentiation was also reduced at the set of R-genes compared with reference loci, which is consistent with the maintenance of ancestral polymorphism. CONCLUSIONS: Historical or ongoing balancing selection on plant disease resistance genes is a likely contributor to the retention of ancestral polymorphism in some regions of the bottlenecked Capella rubella genome.     

Association between Clean Delivery Kit Use,Clean Delivery Practices,and Neonatal Survival: Pooled Analysis of Data from Three Sites in South Asia

Background

Sepsis accounts for up to 15% of an estimated 3.3 million annual neonatal deaths globally. We used data collected from the control arms of three previously conducted cluster-randomised controlled trials in rural Bangladesh, India, and Nepal to examine the association between clean delivery kit use or clean delivery practices and neonatal mortality among home births.

Methods and Findings

Hierarchical, logistic regression models were used to explore the association between neonatal mortality and clean delivery kit use or clean delivery practices in 19,754 home births, controlling for confounders common to all study sites. We tested the association between kit use and neonatal mortality using a pooled dataset from all three sites and separately for each site. We then examined the association between individual clean delivery practices addressed in the contents of the kit (boiled blade and thread, plastic sheet, gloves, hand washing, and appropriate cord care) and neonatal mortality. Finally, we examined the combined association between mortality and four specific clean delivery practices (boiled blade and thread, hand washing, and plastic sheet). Using the pooled dataset, we found that kit use was associated with a relative reduction in neonatal mortality (adjusted odds ratio 0.52, 95% CI 0.39–0.68). While use of a clean delivery kit was not always accompanied by clean delivery practices, using a plastic sheet during delivery, a boiled blade to cut the cord, a boiled thread to tie the cord, and antiseptic to clean the umbilicus were each significantly associated with relative reductions in mortality, independently of kit use. Each additional clean delivery practice used was associated with a 16% relative reduction in neonatal mortality (odds ratio 0.84, 95% CI 0.77–0.92).

Conclusions

The appropriate use of a clean delivery kit or clean delivery practices is associated with relative reductions in neonatal mortality among home births in underserved, rural populations. Please see later in the article for the Editors'' Summary     

A sensitive and specific blocking ELISA for the detection of rabbit calicivirus RCV-A1 antibodies

ABSTRACT: BACKGROUND: Antibodies to non-pathogenic rabbit caliciviruses (RCVs) cross-react in serological tests for rabbit hemorrhagic disease virus (RHDV) and vice versa, making epidemiological studies very difficult where both viruses occur. It is important to understand the distribution and interaction of the two viruses because the highly pathogenic RHDV has been used as a biocontrol agent for wild rabbits in Australia and New Zealand for the past 17 years. The presence of the benign RCV Australia 1 (RCV-A1) is considered a key factor for the failure of RHDV mediated rabbit control in cooler, wetter areas of Australia. RESULTS: A highly sensitive and specific blocking ELISA was developed for the detection of RCV-A1 antibodies. When sera from rabbits with a known infection history for either RCV-A1 or RHDV were tested, this assay showed 100% sensitivity and no cross-reactivity with RHDV sera (100% specificity). CONCLUSIONS: This new ELISA not only allows the detection of RCV-A1 at a population level, but also permits the serological status of individual rabbits to be determined more reliably than previously described methods. This robust and simple to perform assay is therefore the tool of choice for studying RCV-A1 epidemiology in Australian wild rabbit populations.     

Comprehensive Exploration of the Effects of miRNA SNPs on Monocyte Gene Expression

We aimed to assess whether pri-miRNA SNPs (miSNPs) could influence monocyte gene expression, either through marginal association or by interacting with polymorphisms located in 3''UTR regions (3utrSNPs). We then conducted a genome-wide search for marginal miSNPs effects and pairwise miSNPs × 3utrSNPs interactions in a sample of 1,467 individuals for which genome-wide monocyte expression and genotype data were available. Statistical associations that survived multiple testing correction were tested for replication in an independent sample of 758 individuals with both monocyte gene expression and genotype data. In both studies, the hsa-mir-1279 rs1463335 was found to modulate in cis the expression of LYZ and in trans the expression of CNTN6, CTRC, COPZ2, KRT9, LRRFIP1, NOD1, PCDHA6, ST5 and TRAF3IP2 genes, supporting the role of hsa-mir-1279 as a regulator of several genes in monocytes. In addition, we identified two robust miSNPs × 3utrSNPs interactions, one involving HLA-DPB1 rs1042448 and hsa-mir-219-1 rs107822, the second the H1F0 rs1894644 and hsa-mir-659 rs5750504, modulating the expression of the associated genes.As some of the aforementioned genes have previously been reported to reside at disease-associated loci, our findings provide novel arguments supporting the hypothesis that the genetic variability of miRNAs could also contribute to the susceptibility to human diseases.     

Isothermal microcalorimetry, a new tool to monitor drug action against Trypanosoma brucei and Plasmodium falciparum

Isothermal microcalorimetry is an established tool to measure heat flow of physical, chemical or biological processes. The metabolism of viable cells produces heat, and if sufficient cells are present, their heat production can be assessed by this method. In this study, we investigated the heat flow of two medically important protozoans, Trypanosoma brucei rhodesiense and Plasmodium falciparum. Heat flow signals obtained for these pathogens allowed us to monitor parasite growth on a real-time basis as the signals correlated with the number of viable cells. To showcase the potential of microcalorimetry for measuring drug action on pathogenic organisms, we tested the method with three antitrypanosomal drugs, melarsoprol, suramin and pentamidine and three antiplasmodial drugs, chloroquine, artemether and dihydroartemisinin, each at two concentrations on the respective parasite. With the real time measurement, inhibition was observed immediately by a reduced heat flow compared to that in untreated control samples. The onset of drug action, the degree of inhibition and the time to death of the parasite culture could conveniently be monitored over several days. Microcalorimetry is a valuable element to be added to the toolbox for drug discovery for protozoal diseases such as human African trypanosomiasis and malaria. The method could probably be adapted to other protozoan parasites, especially those growing extracellularly.     

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents^1,2. We developed a novel nanoinjection/electrophysiology assay that allows the detection of bioactive modulatory effects of compounds on the function of a neuronal circuit that mediates the escape response in Drosophila melanogaster^3,4. Our in vivo assay, which uses the Drosophila Giant Fiber System (GFS, Figure 1) allows screening of different types of compounds, such as small molecules or peptides, and requires only minimal quantities to elicit an effect. In addition, the Drosophila GFS offers a large variety of potential molecular targets on neurons or muscles. The Giant Fibers (GFs) synapse electrically (Gap Junctions) as well as chemically (cholinergic) onto a Peripheral Synapsing Interneuron (PSI) and the Tergo Trochanteral Muscle neuron (TTMn)⁵. The PSI to DLMn (Dorsal Longitudinal Muscle neuron) connection is dependent on Dα7 nicotinic acetylcholine receptors (nAChRs)⁶. Finally, the neuromuscular junctions (NMJ) of the TTMn and the DLMn with the jump (TTM) and flight muscles (DLM) are glutamatergic^7-12. Here, we demonstrate how to inject nanoliter quantities of a compound, while obtaining electrophysiological intracellular recordings from the Giant Fiber System¹³ and how to monitor the effects of the compound on the function of this circuit. We show specificity of the assay with methyllycaconitine citrate (MLA), a nAChR antagonist, which disrupts the PSI to DLMn connection but not the GF to TTMn connection or the function of the NMJ at the jump or flight muscles.Before beginning this video it is critical that you carefully watch and become familiar with the JoVE video titled "Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster " from Augustin et al⁷, as the video presented here is intended as an expansion to this existing technique. Here we use the electrophysiological recordings method and focus in detail only on the addition of the paired nanoinjections and monitoring technique.     

Genome sequence of the homoacetogenic bacterium Holophaga foetida type strain (TMBS4(T))

Holophaga foetida Liesack et al. 1995 is a member of the phylum Acidobacteria and is of interest for its ability to anaerobically degrade aromatic compounds and for its production of volatile sulfur compounds through a unique pathway. The genome of H. foetida strain TMBS4(T) is the first to be sequenced for a representative of the class Holophagae. Here we describe the features of this organism, together with the complete genome sequence (improved high quality draft), and annotation. The 4,127,237 bp long chromosome with its 3,615 protein-coding and 57 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Complete genome sequence of Dehalogenimonas lykanthroporepellens type strain (BL-DC-9(T)) and comparison to "Dehalococcoides" strains

Dehalogenimonas lykanthroporepellens is the type species of the genus Dehalogenimonas, which belongs to a deeply branching lineage within the phylum Chloroflexi. This strictly anaerobic, mesophilic, non spore-forming, Gram-negative staining bacterium was first isolated from chlorinated solvent contaminated groundwater at a Superfund site located near Baton Rouge, Louisiana, USA. D. lykanthroporepellens was of interest for genome sequencing for two reasons: (a) an unusual ability to couple growth with reductive dechlorination of environmentally important polychlorinated aliphatic alkanes and (b) a phylogenetic position that is distant from previously sequenced bacteria. The 1,686,510 bp circular chromosome of strain BL-DC-9(T) contains 1,720 predicted protein coding genes, 47 tRNA genes, a single large subunit rRNA (23S-5S) locus, and a single, orphan, small subunit rRNA (16S) locus.