Identification of critical residues of subunit H in its interaction with subunit E of the A-ATP synthase from Methanocaldococcus jannaschii

The boomerang-like H subunit of A(1)A(0) ATP synthase forms one of the peripheral stalks connecting the A(1) and A(0) sections. Structural analyses of the N-terminal part (H1-47) of subunit H of the A(1)A(0) ATP synthase from Methanocaldococcus jannaschii have been performed by NMR spectroscopy. Our initial NMR structural calculations for H1-47 indicate that amino acid residues 7-44 fold into a single alpha-helical structure. Using the purified N- (E1-100) and C-terminal domains (E101-206) of subunit E, NMR titration experiments revealed that the N-terminal residues Met1-6, Lys10, Glu11, Ala15, Val20 and Glu24 of H1-47 interact specifically with the N-terminal domain E1-100 of subunit E. A more detailed picture regarding the residues of E1-100 involved in this association was obtained by titration studies using the N-terminal peptides E1-20, E21-40 and E41-60. These data indicate that the N-terminal tail E41-60 interacts with the N-terminal amino acids of H1-47, and this has been confirmed by fluorescence correlation spectroscopy results. Analysis of (1)H-(15)N heteronuclear single quantum coherence (HSQC) spectra of the central stalk subunit F in the presence and absence of E101-206 show no obvious interaction between the C-terminal domain of E and subunit F. The data presented provide, for the first time, structural insights into the interaction of subunits E and H, and their arrangement within A(1)A(0) ATP synthase.     

Expression of AMPA receptor subunits at synapses in laminae I–III of the rodent spinal dorsal horn

Neuropathic pain may arise following peripheral nerve injury though the molecular mechanisms associated with this are unclear. We used proteomic profiling to examine changes in protein expression associated with the formation of hyper-excitable neuromas derived from rodent saphenous nerves. A two-dimensional difference gel electrophoresis (2D-DIGE) profiling strategy was employed to examine protein expression changes between developing neuromas and normal nerves in whole tissue lysates. We found around 200 proteins which displayed a >1.75-fold change in expression between neuroma and normal nerve and identified 55 of these proteins using mass spectrometry. We also used immunoblotting to examine the expression of low-abundance ion channels Nav1.3, Nav1.8 and calcium channel α2δ-1 subunit in this model, since they have previously been implicated in neuronal hyperexcitability associated with neuropathic pain. Finally, S³⁵methionine in vitro labelling of neuroma and control samples was used to demonstrate local protein synthesis of neuron-specific genes. A number of cytoskeletal proteins, enzymes and proteins associated with oxidative stress were up-regulated in neuromas, whilst overall levels of voltage-gated ion channel proteins were unaffected. We conclude that altered mRNA levels reported in the somata of damaged DRG neurons do not necessarily reflect levels of altered proteins in hyper-excitable damaged nerve endings. An altered repertoire of protein expression, local protein synthesis and topological re-arrangements of ion channels may all play important roles in neuroma hyper-excitability.     

Somatic embryogenesis in Abutilon indicum (L.) Sweet and assessment of genetic homogeneity using SCoT markers

This study describes an efficient plant regeneration protocol for Abutilon indicum via somatic embryogenesis from 2,4-dichlorophenoxyacetic acid (2,4-D)-induced leaf-derived callus on MS medium, fortified with 13.32 μM 6-benzyladenine (BA), 2.68 μM α-naphthalene acetic acid (NAA), 200 mgl^?1-activated charcoal, and 11.54 μM ascorbic acid. This combination produced the highest (15.5 ± 0.7) number of somatic embryos after four weeks of culture. Further, the embryogenic calli were transferred to MS medium supplemented with 13.32 μM BA, 1.44 μM gibberellic acid (GA₃), and 3% (w/v) sucrose and showed highest rate of germination (76.3 ± 7.0%). The germinated somatic embryos showed maximum plantlet conversion (62.6 ± 1.90%) on ½ MS medium supplemented with 4.92 μM indole-3-butyric acid and 6.0% sucrose (w/v). The highest frequency of secondary somatic embryogenesis (34.4 ± 0.82) was observed on ½ MS medium, supplemented with 133 μM FeSO₄·7H₂O, 74 μM ethylene diamine tetraacetic acid disodium dihydrate (disodium EDTA), and 15% polyethylene glycol-4000 (PEG-4000) after three weeks of subculture. Scanning electron microscopy observations also substantiated the development of primary and secondary somatic embryos from embryogenic calli. Start codon targeted polymorphism (SCoT) marker analysis of 214 somatic embryo-derived plantlets amplified 167 numbers of bands ranging from 230 to 2125 bp. The homogeneous banding pattern confirmed the genetic uniformity of this sample of somatic embryo-derived plantlets as compared with the donor plant.     

MYC‐driven inhibition of the glutamate‐cysteine ligase promotes glutathione depletion in liver cancer

How MYC reprograms metabolism in primary tumors remains poorly understood. Using integrated gene expression and metabolite profiling, we identify six pathways that are coordinately deregulated in primary MYC‐driven liver tumors: glutathione metabolism; glycine, serine, and threonine metabolism; aminoacyl‐tRNA biosynthesis; cysteine and methionine metabolism; ABC transporters; and mineral absorption. We then focus our attention on glutathione (GSH) and glutathione disulfide (GSSG), as they are markedly decreased in MYC‐driven tumors. We find that fewer glutamine‐derived carbons are incorporated into GSH in tumor tissue relative to non‐tumor tissue. Expression of GCLC, the rate‐limiting enzyme of GSH synthesis, is attenuated by the MYC‐induced microRNA miR‐18a. Inhibition of miR‐18a in vivo leads to increased GCLC protein expression and GSH abundance in tumor tissue. Finally, MYC‐driven liver tumors exhibit increased sensitivity to acute oxidative stress. In summary, MYC‐dependent attenuation of GCLC by miR‐18a contributes to GSH depletion in vivo, and low GSH corresponds with increased sensitivity to oxidative stress in tumors. Our results identify new metabolic pathways deregulated in primary MYC tumors and implicate a role for MYC in regulating a major antioxidant pathway downstream of glutamine.     

GHOST: global hepatitis outbreak and surveillance technology

Background

Hepatitis C is a major public health problem in the United States and worldwide. Outbreaks of hepatitis C virus (HCV) infections associated with unsafe injection practices, drug diversion, and other exposures to blood are difficult to detect and investigate. Effective HCV outbreak investigation requires comprehensive surveillance and robust case investigation. We previously developed and validated a methodology for the rapid and cost-effective identification of HCV transmission clusters. Global Hepatitis Outbreak and Surveillance Technology (GHOST) is a cloud-based system enabling users, regardless of computational expertise, to analyze and visualize transmission clusters in an independent, accurate and reproducible way.

Results

We present and explore performance of several GHOST implemented algorithms using next-generation sequencing data experimentally obtained from hypervariable region 1 of genetically related and unrelated HCV strains. GHOST processes data from an entire MiSeq run in approximately 3 h. A panel of seven specimens was used for preparation of six repeats of MiSeq libraries. Testing sequence data from these libraries by GHOST showed a consistent transmission linkage detection, testifying to high reproducibility of the system. Lack of linkage among genetically unrelated HCV strains and constant detection of genetic linkage between HCV strains from known transmission pairs and from follow-up specimens at different levels of MiSeq-read sampling indicate high specificity and sensitivity of GHOST in accurate detection of HCV transmission.

Conclusions

GHOST enables automatic extraction of timely and relevant public health information suitable for guiding effective intervention measures. It is designed as a virtual diagnostic system intended for use in molecular surveillance and outbreak investigations rather than in research. The system produces accurate and reproducible information on HCV transmission clusters for all users, irrespective of their level of bioinformatics expertise. Improvement in molecular detection capacity will contribute to increasing the rate of transmission detection, thus providing opportunity for rapid, accurate and effective response to outbreaks of hepatitis C. Although GHOST was originally developed for hepatitis C surveillance, its modular structure is readily applicable to other infectious diseases. Worldwide availability of GHOST for the detection of HCV transmissions will foster deeper involvement of public health researchers and practitioners in hepatitis C outbreak investigation.

     

Towards next generation antisense oligonucleotides: mesylphosphoramidate modification improves therapeutic index and duration of effect of gapmer antisense oligonucleotides

The PS modification enhances the nuclease stability and protein binding properties of gapmer antisense oligonucleotides (ASOs) and is one of very few modifications that support RNaseH1 activity. We evaluated the effect of introducing stereorandom and chiral mesyl-phosphoramidate (MsPA) linkages in the DNA gap and flanks of gapmer PS ASOs and characterized the effect of these linkages on RNA-binding, nuclease stability, protein binding, pro-inflammatory profile, antisense activity and toxicity in cells and in mice. We show that all PS linkages in a gapmer ASO can be replaced with MsPA without compromising chemical stability and RNA binding affinity but these designs reduced activity. However, replacing up to 5 PS in the gap with MsPA was well tolerated and replacing specific PS linkages at appropriate locations was able to greatly reduce both immune stimulation and cytotoxicity. The improved nuclease stability of MsPA over PS translated to significant improvement in the duration of ASO action in mice which was comparable to that of enhanced stabilized siRNA designs. Our work highlights the combination of PS and MsPA linkages as a next generation chemical platform for identifying ASO drugs with improved potency and therapeutic index, reduced pro-inflammatory effects and extended duration of effect.     

Systematic review of cost effectiveness studies of telemedicine interventions

ObjectivesTo systematically review cost benefit studies of telemedicine.DesignSystematic review of English language, peer reviewed journal articles.Results557 articles without cost data categorised by topic. 55 articles with data initially categorised by cost variables employed in the study and conclusions. Only 24/55 (44%) studies met quality criteria justifying inclusion in a quality review. 20/24 (83%) restricted to simple cost comparisons. No study used cost utility analysis, the conventional means of establishing the “value for money” that a therapeutic intervention represents. Only 7/24 (29%) studies attempted to explore the level of utilisation that would be needed for telemedicine services to compare favourably with traditionally organised health care. None addressed this question in sufficient detail to adequately answer it. 15/24 (62.5%) of articles reviewed here provided no details of sensitivity analysis, a method all economic analyses should incorporate.ConclusionThere is no good evidence that telemedicine is a cost effective means of delivering health care.

What is already known on this topic

The use of telemedicine has garnered much attention in the past decadeHundreds of articles have been published claiming that telemedicine is cost effectiveHowever, missing from the literature is a synthesis or meta-analysis of these publications

What this study adds

A comprehensive literature search of cost related articles on telemedicine identified more than 600 articles, but only 9% contained any cost benefit dataOnly 4% of these articles met quality criteria justifying inclusion in a formalised quality review, and most of these were small scale, short term, pragmatic evaluations with few generalisable conclusionsThere is little published evidence to confirm whether or not telemedicine is a cost effective alternative to standard healthcare delivery     

Ultrastructure of sperms in Acoela (Acoelomorpha) and its concordance with molecular systematics

Abstract. The sperms of the Acoela, a group of lower worms, are filiform cells with 2 flagella incorporated into the cell body. Their axonemes can variously have 9+2, 9+1, or 9+0 patterns of microtubules; and singlet microtubules in the cell body can be arranged in axial or cortical positions. An analysis of phylogenetic relationships of acoels based on molecular characters (18S rDNA sequence data) showed that these patterns of microtubules, where known, fell into discrete monophyletic groups. To test this hypothesis, we have expanded the database of sperm characters by examining the ultrastructure of a further 10 species representing 4 acoel families. As expected, the Convolutidae fell into 2 unrelated groups: “small‐bodied convolutids”(Convoluta pulchra, Praeconvoluta tigrina, Pseudaphanostoma smithrii) having 9+2 axonemes and cortical microtubules, and “large‐bodied convolutids” (including Wulguru cuspidata) having 9+0 axonemes and axial microtubules. Also, as expected, a member of the Mecynostomidae (Paedomecynostomum bruneum) has 9+1 axonemes and axial microtubules. Members of a family that appears intermediate by molecular characters, the Otocelididae, significantly have a variety of patterns: axonemes with both 9+2 and 9+0 patterns (Notocelis gullmarensis) or just 9+2 (the other species), and either axial (Philocelis brueggemanni), both axial and cortical (N. gullmarensis) microtubules, or microtubules that bend between axial and cortical positions along the length of the sperm (Otocelis sandara). Members of the Dakuidae (Daku woorimensis) also belong to this intermediate group, having 9+2 axonemes and axial microtubules, while in a fifth otocelidid (Stomatricha hochbergi), sperm characters are like those of the “large‐bodied convolutids” (9+0 axonemes and axial microtubules). Characters of sperm morphology generally support the molecular hypothesis of relationships and confirm a suspected polyphyly of the families Convolutidae, Otocelididae, and Actinoposthiidae.