Degradation of MEPE, DMP1, and Release of SIBLING ASARM-Peptides (Minhibins): ASARM-Peptide(s) Are Directly Responsible for Defective Mineralization in HYP

Mutations in PHEX (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) and DMP1 (dentin matrix protein 1) result in X-linked hypophosphatemic rickets (HYP) and autosomal-recessive hypophosphatemic-rickets (ARHR), respectively. Specific binding of PHEX to matrix extracellular phosphoglycoprotein (MEPE) regulates the release of small protease-resistant MEPE peptides [acidic serine- and aspartate-rich MEPE-associated motif (ASARM) peptides]. ASARM peptides are potent inhibitors of mineralization (minhibins) that also occur in DMP1 [MEPE-related small integrin-binding ligand, N-linked glycoprotein (SIBLING) protein]. It is not known whether these peptides are directly responsible for the mineralization defect. We therefore used a bone marrow stromal cell (BMSC) coculture model, ASARM peptides, anti-ASARM antibodies, and a small synthetic PHEX peptide (SPR4; 4.2 kDa) to examine this. Surface plasmon resonance (SPR) and two-dimensional 1H/15N nuclear magnetic resonance demonstrated specific binding of SPR4 peptide to ASARM peptide. When cultured individually for 21 d, HYP BMSCs displayed reduced mineralization compared with wild type (WT) (−87%, P < 0.05). When cocultured, both HYP and WT cells failed to mineralize. However, cocultures (HYP and WT) or monocultures of HYP BMSCs treated with SPR4 peptide or anti-ASARM neutralizing antibodies mineralized normally. WT BMSCs treated with ASARM peptide also failed to mineralize properly without SPR4 peptide or anti-ASARM neutralizing antibodies. ASARM peptide treatment decreased PHEX mRNA and protein (−80%, P < 0.05) and SPR4 peptide cotreatment reversed this by binding ASARM peptide. SPR4 peptide also reversed ASARM peptide-mediated changes in expression of key osteoclast and osteoblast differentiation genes. Western blots of HYP calvariae and BMSCs revealed massive degradation of both MEPE and DMP1 protein compared with the WT. We conclude that degradation of MEPE and DMP-1 and release of ASARM peptides are chiefly responsible for the HYP mineralization defect and changes in osteoblast-osteoclast differentiation.We acknowledge the very kind gift of pure sPHEX by Dr. Philippe Crine (Department of Biochemistry, University of Montreal, and Enobia Pharma). Also, we acknowledge the anti-DMP1 antibodies generously donated by Dr. Larry Fisher, National Institute of Dental and Craniofacial Research, Bethesda, MD. Address all correspondence and requests for reprints to: Peter S. N. Rowe, Department of Internal Medicine, Division of Nephrology and Hypertension, The Kidney Institute, MS 3018, 3901 Rainbow Boulevard, Kansas City, Kansas 66160. E-mail: [email protected]. We acknowledge the generous financial support from the National Institutes of Health to P.S.N.R. (RO-1 AR51598-01; National Institute of Arthritis and Musculoskeletal Diseases). Also, the SPR experiments were performed in the UTHSCSA Center for Macromolecular Interactions, which is supported by grants from the National Cancer Institute (CA54174) and UTHSCSA Executive Research Committee Research fund

Evolutionary History of Rabies in Ghana

Rabies virus (RABV) is enzootic throughout Africa, with the domestic dog (Canis familiaris) being the principal vector. Dog rabies is estimated to cause 24,000 human deaths per year in Africa, however, this estimate is still considered to be conservative. Two sub-Saharan African RABV lineages have been detected in West Africa. Lineage 2 is present throughout West Africa, whereas Africa 1a dominates in northern and eastern Africa, but has been detected in Nigeria and Gabon, and Africa 1b was previously absent from West Africa. We confirmed the presence of RABV in a cohort of 76 brain samples obtained from rabid animals in Ghana collected over an eighteen-month period (2007–2009). Phylogenetic analysis of the sequences obtained confirmed all viruses to be RABV, belonging to lineages previously detected in sub-Saharan Africa. However, unlike earlier reported studies that suggested a single lineage (Africa 2) circulates in West Africa, we identified viruses belonging to the Africa 2 lineage and both Africa 1 (a and b) sub-lineages. Phylogeographic Bayesian Markov chain Monte Carlo analysis of a 405 bp fragment of the RABV nucleoprotein gene from the 76 new sequences derived from Ghanaian animals suggest that within the Africa 2 lineage three clades co-circulate with their origins in other West African countries. Africa 1a is probably a western extension of a clade circulating in central Africa and the Africa 1b virus a probable recent introduction from eastern Africa. We also developed and tested a novel reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of RABV in African laboratories. This RT-LAMP was shown to detect both Africa 1 and 2 viruses, including its adaptation to a lateral flow device format for product visualization. These data suggest that RABV epidemiology is more complex than previously thought in West Africa and that there have been repeated introductions of RABV into Ghana. This analysis highlights the potential problems of individual developing nations implementing rabies control programmes in the absence of a regional programme

Reconciling the biogeography of an invader through recent and historic genetic patterns: the case of topmouth gudgeon Pseudorasbora parva

© 2018 The Author(s) The genetic variability and population structure of introduced species in their native range are potentially important determinants of their invasion success, yet data on native populations are often poorly represented in relevant studies. Consequently, to determine the contribution of genetic structuring in the native range of topmouth gudgeon Pseudorasbora parva to their high invasion success in Europe, we used a dataset comprising of 19 native and 11 non-native populations. A total of 666 samples were analysed at 9 polymorphic microsatellite loci and sequenced for 597 bp of mitochondrial DNA. The analysis revealed three distinct lineages in the native range, of which two haplogroups were prevalent in China (100%), with a general split around the Qinling Mountains. Dating of both haplogroups closely matched past geological events. More recently, its distribution has been influenced by fish movements in aquaculture, resulting in gene flow between previously separated populations in Northern and Southern China. Their phylogeography in Europe indicate as few as two introductions events and two dispersal routes. Microsatellite data revealed native populations had higher genetic diversity than those in the invasive range, a contrast to previous studies on P. parva. This study confirms the importance of extensive sampling in both the native and non-native range of invasive species in evaluating the influence of genetic variability on invasion success

Whole-genome sequencing for prediction of Mycobacterium tuberculosis drug susceptibility and resistance : a retrospective cohort study

BACKGROUND : Diagnosing drug-resistance remains an obstacle to the elimination of tuberculosis. Phenotypic drugsusceptibility testing is slow and expensive, and commercial genotypic assays screen only common resistancedetermining mutations. We used whole-genome sequencing to characterise common and rare mutations predicting drug resistance, or consistency with susceptibility, for all fi rst-line and second-line drugs for tuberculosis. METHODS : Between Sept 1, 2010, and Dec 1, 2013, we sequenced a training set of 2099 Mycobacterium tuberculosis genomes. For 23 candidate genes identifi ed from the drug-resistance scientifi c literature, we algorithmically characterised genetic mutations as not conferring resistance (benign), resistance determinants, or uncharacterised. We then assessed the ability of these characterisations to predict phenotypic drug-susceptibility testing for an independent validation set of 1552 genomes. We sought mutations under similar selection pressure to those characterised as resistance determinants outside candidate genes to account for residual phenotypic resistance. FINDINGS : We characterised 120 training-set mutations as resistance determining, and 772 as benign. With these mutations, we could predict 89·2% of the validation-set phenotypes with a mean 92·3% sensitivity (95% CI 90·7–93·7) and 98·4% specifi city (98·1–98·7). 10·8% of validation-set phenotypes could not be predicted because uncharacterised mutations were present. With an in-silico comparison, characterised resistance determinants had higher sensitivity than the mutations from three line-probe assays (85·1% vs 81·6%). No additional resistance determinants were identifi ed among mutations under selection pressure in non-candidate genes. INTERPRETATION : A broad catalogue of genetic mutations enable data from whole-genome sequencing to be used clinically to predict drug resistance, drug susceptibility, or to identify drug phenotypes that cannot yet be genetically predicted. This approach could be integrated into routine diagnostic workfl ows, phasing out phenotypic drugsusceptibility testing while reporting drug resistance early.Wellcome Trust, National Institute of Health Research, Medical Research Council, and the European Union.http://www.thelancet.com/infectionhb201

Reconfigurable micro-mould for the manufacture of truly 3D polymer microfluidic devices

Organised by: Cranfield UniversityThis paper concerns the concept, the design and the manufacturing steps for the fabrication of a precision mould for micro-injection moulding of truly three dimensional microfluidic devices. The mould was designed using the concept of replaceable cavities to enable the flexible development of the complex microfluidic device and to reduce machining time and therefore costs during the prototyping, testing and subsequent production phase. The precision machining technique used for the cavity manufacture was micromilling.Mori Seiki – The Machine Tool Compan

Study of blood flow behavior in microchannels

Microfluidic (also known as lab-on-a-chip) devices offer the capability ofmanipulating very low volumes of fluids (of the order of micro litres) for severalapplications including medical diagnostics. This property makes microfluidicdevices very attractive when the fluid, such as blood, has a limited supply becausethe patients cannot easily and frequently provide a large sample. This is typically thecase for aged, diseased patients that do require frequent sampling during acute careor of older people that have the option of being treated and cared for at home [1].Prototype lab-on-a-chip devices for medical diagnostics comprise a number ofelements which separately perform different functions within the system. Activitywithin the research community is focusing on the better integration of devicefunctionalities with the long term goal of creating fully integrated, portable,affordable clinical devices. However, engineering these solutions for the largevolume production of lab-on-a-chip devices requires design rules which are not yetentirely available.This paper describes the results obtained from a set of experiments run to drawgeneric design rules for the manufacture of a cells/plasma micro separator [2]. Thecells/plasma micro separator was selected for investigation because it is a strategicelement required in the preparation of blood samples for many different analyticaldevices. The experiments focused on the study of the behaviour of whole bloodpassing through micro constrictions which are required for enhancing the separationeffect [3].The test microfluidic device was an aluminium specimen designed andmanufactured to incorporate micro constrictions of different width and length. The metallic aluminium test device was designed for manufacturing by micromilling anddiamond cutting processes in view of applying these techniques to the manufactureof micro-moulds for the high-volume production of plastic microfluidic devices viamicro-injection moulding.The widths of the constrictions were 23, 53 and 93µm and the lengths were 300 and700µm. The blood flow pattern and the level of haemolysis generated in the wholeblood were determined for flow rates between 0.2 and 1 ml/min. Initial resultssuggested that the above conditions generate a stable flow and do not cause bloodhaemolysis following passage through the narrow constrictions. This result impliesthat constrictions as narrow as 23 µm and as long as 700µm can be safely used inblood microfluidic devices under appropriate flow conditions without the risk ofdamaging the blood compon

Morphine potentiates neuropathogenesis of SIV infection in rhesus macaques

Despite the advent of antiretroviral therapy, complications of HIV-1 infection with concurrent drug abuse are an emerging problem. Opiates are well-known to modulate immune responses by preventing the development of cell-mediated immune responses. Their effect on the pathogenesis of HIV-1 infection however, remains controversial. Using the Simian Immunodeficiency Virus/ macaque model of HIV pathogenesis, we sought to explore the impact of morphine on disease progression and pathogenesis. Sixteen Rhesus macaques were divided into 2 groups; 4 were administered saline and twelve others morphine routinely. Both groups of animals were then inoculated with SIVmacR71/17E and followed longitudinally for disease pathogenesis. The morphine group (M+V) exhibited a trend towards higher mortality rates and retardation in weight gain compared to the virus alone group. Interestingly, a subset of M+V animals succumbed to disease within weeks post-infection. These rapid progressors also exhibited a higher incidence of other end-organ pathologies. Despite the higher numbers of circulating CD4+ and CD8+T cells in the M+V group, CD4:CD8 ratios between the groups remained unchanged. Plasma and CSF viral load in the M+V group was at least a log higher than the control group. Similarly there was a trend toward increased virus build-up in the brains of M+V animals compared with controls. A novel finding of this study was the increased influx of infected monocyte/macrophages in the brains of M+V animals