GRP78/BiP/HSPA5/Dna K is a universal therapeutic target for human disease

The chaperone GRP78/Dna K is conserved throughout evolution down to prokaryotes. The GRP78 inhibitor OSU‐03012 (AR‐12) interacted with sildenafil (Viagra) or tadalafil (Cialis) to rapidly reduce GRP78 levels in eukaryotes and as a single agent reduce Dna K levels in prokaryotes. Similar data with the drug combination were obtained for: HSP70, HSP90, GRP94, GRP58, HSP27, HSP40 and HSP60. OSU‐03012/sildenafil treatment killed brain cancer stem cells and decreased the expression of: NPC1 and TIM1; LAMP1; and NTCP1, receptors for Ebola/Marburg/Hepatitis A, Lassa fever, and Hepatitis B viruses, respectively. Pre‐treatment with OSU‐03012/sildenafil reduced expression of the coxsakie and adenovirus receptor in parallel with it also reducing the ability of a serotype 5 adenovirus or coxsakie virus B4 to infect and to reproduce. Similar data were obtained using Chikungunya, Mumps, Measles, Rubella, RSV, CMV, and Influenza viruses. OSU‐03012 as a single agent at clinically relevant concentrations killed laboratory generated antibiotic resistant E. coli and clinical isolate multi‐drug resistant N. gonorrhoeae and MRSE which was in bacteria associated with reduced Dna K and Rec A expression. The PDE5 inhibitors sildenafil or tadalafil enhanced OSU‐03012 killing in N. gonorrhoeae and MRSE and low marginally toxic doses of OSU‐03012 could restore bacterial sensitivity in N. gonorrhoeae to multiple antibiotics. Thus, Dna K and bacterial phosphodiesterases are novel antibiotic targets, and inhibition of GRP78 is of therapeutic utility for cancer and also for bacterial and viral infections. J. Cell. Physiol. 230: 1661–1676, 2015. © 2014 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.     

A cryptic genetic boundary in remnant populations of a long‐lived,bird‐pollinated shrub Banksia sphaerocarpa var. caesia (Proteaceae)

Plant species often exhibit genetic structure at multiple spatial scales. Detection of this structure may depend on the sampling strategy used. We intensively sampled a common, naturally patchy Banksia species within a 200 km² region, in order to assess patterns of genetic diversity and structure at multiple spatial scales. In total, 1321 adult shrubs from 37 geographical populations were genotyped using eight highly polymorphic microsatellite markers developed for the species. Genetic structure was detected at three spatial scales. First, we identified a stark and unexpected division of the landscape into two genetic subregions, one to the north‐east and one to the south‐west of the sampling grid. This differentiation was based on sudden, highly structured differences in common allele frequencies, the cause of which is unknown but that may relate to physical and reproductive barriers to gene flow, localised selection, and/or historical processes. Second, we observed genetic differentiation of populations within these subregions, reflecting previously described patterns of restricted pollen flow in this species. Finally, fine‐scale genetic structure, although weak, was observed within some of the populations (mean S_P = 0.01837). When feasible, intensive sampling may uncover cryptic patterns of genetic structure that would otherwise be overlooked when sampling at broader spatial scales. Further studies using a similar sampling strategy may reveal this type of discontinuity to be a feature of other south‐western Australian taxa and has implications for our understanding of evolution in this landscape as well as conservation into the future. © 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, 115 , 241–255.     

Spindle Checkpoint Factors Bub1 and Bub2 Promote DNA Double-Strand Break Repair by Nonhomologous End Joining

The nonhomologous end-joining (NHEJ) pathway is essential for the preservation of genome integrity, as it efficiently repairs DNA double-strand breaks (DSBs). Previous biochemical and genetic investigations have indicated that, despite the importance of this pathway, the entire complement of genes regulating NHEJ remains unknown. To address this, we employed a plasmid-based NHEJ DNA repair screen in budding yeast (Saccharomyces cerevisiae) using 369 putative nonessential DNA repair-related components as queries. Among the newly identified genes associated with NHEJ deficiency upon disruption are two spindle assembly checkpoint kinases, Bub1 and Bub2. Both observation of resulting phenotypes and chromatin immunoprecipitation demonstrated that Bub1 and -2, either alone or in combination with cell cycle regulators, are recruited near the DSB, where phosphorylated Rad53 or H2A accumulates. Large-scale proteomic analysis of Bub kinases phosphorylated in response to DNA damage identified previously unknown kinase substrates on Tel1 S/T-Q sites. Moreover, Bub1 NHEJ function appears to be conserved in mammalian cells. 53BP1, which influences DSB repair by NHEJ, colocalizes with human BUB1 and is recruited to the break sites. Thus, while Bub is not a core component of NHEJ machinery, our data support its dual role in mitotic exit and promotion of NHEJ repair in yeast and mammals.     

Autosomal-Dominant Multiple Pterygium Syndrome Is Caused by Mutations in MYH3

Multiple pterygium syndrome (MPS) is a phenotypically and genetically heterogeneous group of rare Mendelian conditions characterized by multiple pterygia, scoliosis, and congenital contractures of the limbs. MPS typically segregates as an autosomal-recessive disorder, but rare instances of autosomal-dominant transmission have been reported. Whereas several mutations causing recessive MPS have been identified, the genetic basis of dominant MPS remains unknown. We identified four families affected by dominantly transmitted MPS characterized by pterygia, camptodactyly of the hands, vertebral fusions, and scoliosis. Exome sequencing identified predicted protein-altering mutations in embryonic myosin heavy chain (MYH3) in three families. MYH3 mutations underlie distal arthrogryposis types 1, 2A, and 2B, but all mutations reported to date occur in the head and neck domains. In contrast, two of the mutations found to cause MPS in this study occurred in the tail domain. The phenotypic overlap among persons with MPS, coupled with physical findings distinct from other conditions caused by mutations in MYH3, suggests that the developmental mechanism underlying MPS differs from that of other conditions and/or that certain functions of embryonic myosin might be perturbed by disruption of specific residues and/or domains. Moreover, the vertebral fusions in persons with MPS, coupled with evidence of MYH3 expression in bone, suggest that embryonic myosin plays a role in skeletal development.     

Valuing and Sustaining (or Not) the Ability of Volunteer Community Health Workers to Deliver Integrated Community Case Management in Northern Ghana: A Qualitative Study

Background

Within the integrated community case management of childhood illnesses (iCCM) programme, the traditional health promotion and prevention role of community health workers (CHWs) has been expanded to treatment. Understanding both the impact and the implementation experience of this expanded role are important. In evaluating UNICEF’s implementation of iCCM, this qualitative case study explores the implementation experience in Ghana.

Methods and Findings

Data were collected through a rapid appraisal using focus groups and individual interviews during a field visit in May 2013 to Accra and the Northern Region of Ghana. We sought to understand the experience of iCCM from the perspective of locally based UNICEF staff, their partners, researchers, Ghana health services management staff, CHWs and their supervisors, nurses in health facilities and mothers receiving the service. Our analysis of the findings showed that there is an appreciation both by mothers and by facility level staff for the contribution of CHWs. Appreciation was expressed for the localisation of the treatment of childhood illness, thus saving mothers from the effort and expense of having to seek treatment outside of the village. Despite an overall expression of value for the expanded role of CHWs, we also found that there were problems in supporting and sustaining their efforts. The data showed concern around CHWs being unpaid, poorly supervised, regularly out of stock, lacking in essential equipment and remaining outside the formal health system.

Conclusions

Expanding the roles of CHWs is important and can be valuable, but contextual and health system factors threaten the sustainability of iCCM in Ghana. In this and other implementation sites, policymakers and key donors need to take into account historical lessons from the CHW literature, while exploring innovative and sustainable mechanisms to secure the programme as part of a government owned and government led strategy.     

Proteomic Analysis of Disease Stratified Human Pancreas Tissue Indicates Unique Signature of Type 1 Diabetes

Type 1 diabetes (T1D) and type 2 diabetes (T2D) are associated with functional beta cell loss due to ongoing inflammation. Despite shared similarities, T1D is an autoimmune disease with evidence of autoantibody production, as well as a role for exocrine pancreas involvement. Our hypothesis is that differential protein expression occurs in disease stratified pancreas tissues and regulated proteins from endocrine and exocrine tissues are potential markers of disease and potential therapeutic targets. The study objective was to identify novel proteins that distinguish the pancreas from donors with T1D from the pancreas from patients with T2D, or autoantibody positive non-diabetic donors. Detailed quantitative comprehensive proteomic analysis was applied to snap frozen human pancreatic tissue lysates from organ donors without diabetes, with T1D-associated autoantibodies in the absence of diabetes, with T1D, or with T2D. These disease-stratified human pancreas tissues contain exocrine and endocrine tissues (with dysfunctional islets) in the same microenvironment. The expression profiles of several of the proteins were further verified by western blot. We identified protein panels that are significantly and uniquely upregulated in the three disease-stratified pancreas tissues compared to non-disease control tissues. These proteins are involved in inflammation, metabolic regulation, and autoimmunity, all of which are pathways linked to, and likely involved in, T1 and T2 diabetes pathogenesis. Several new proteins were differentially upregulated in prediabetic, T1D, and T2D pancreas. The results identify proteins that could serve as novel prognostic, diagnostic, and therapeutic tools to preserve functional islet mass in Type 1 Diabetes.     

The Impact of Water,Sanitation and Hygiene Interventions to Control Cholera: A Systematic Review

Background and Methods

Cholera remains a significant threat to global public health with an estimated 100,000 deaths per year. Water, sanitation and hygiene (WASH) interventions are frequently employed to control outbreaks though evidence regarding their effectiveness is often missing. This paper presents a systematic literature review investigating the function, use and impact of WASH interventions implemented to control cholera.

Results

The review yielded eighteen studies and of the five studies reporting on health impact, four reported outcomes associated with water treatment at the point of use, and one with the provision of improved water and sanitation infrastructure. Furthermore, whilst the reporting of function and use of interventions has become more common in recent publications, the quality of studies remains low. The majority of papers (>60%) described water quality interventions, with those at the water source focussing on ineffective chlorination of wells, and the remaining being applied at the point of use. Interventions such as filtration, solar disinfection and distribution of chlorine products were implemented but their limitations regarding the need for adherence and correct use were not fully considered. Hand washing and hygiene interventions address several transmission routes but only 22% of the studies attempted to evaluate them and mainly focussed on improving knowledge and uptake of messages but not necessarily translating this into safer practices. The use and maintenance of safe water storage containers was only evaluated once, under-estimating the considerable potential for contamination between collection and use. This problem was confirmed in another study evaluating methods of container disinfection. One study investigated uptake of household disinfection kits which were accepted by the target population. A single study in an endemic setting compared a combination of interventions to improve water and sanitation infrastructure, and the resulting reductions in cholera incidence.

Discussion and Recommendations

This review highlights a focus on particular routes of transmission, and the limited number of interventions tested during outbreaks. There is a distinct gap in knowledge of which interventions are most appropriate for a given context and as such a clear need for more robust impact studies evaluating a wider array of WASH interventions, in order to ensure effective cholera control and the best use of limited resources.     

Patterns of Hepatitis C Virus RNA Levels during Acute Infection: The InC3 Study

Background

Understanding the patterns of HCV RNA levels during acute hepatitis C virus (HCV) infection provides insights into immunopathogenesis and is important for vaccine design. This study evaluated patterns of HCV RNA levels and associated factors among individuals with acute infection.

Methods

Data were from an international collaboration of nine prospective cohorts of acute HCV (InC³ Study). Participants with well-characterized acute HCV infection (detected within three months post-infection and interval between the peak and subsequent HCV RNA levels≤120 days) were categorised by a priori-defined patterns of HCV RNA levels: i) spontaneous clearance, ii) partial viral control with persistence (≥1 log IU/mL decline in HCV RNA levels following peak) and iii) viral plateau with persistence (increase or <1 log IU/mL decline in HCV RNA levels following peak). Factors associated with HCV RNA patterns were assessed using multinomial logistic regression.

Results

Among 643 individuals with acute HCV, 162 with well-characterized acute HCV were identified: spontaneous clearance (32%), partial viral control with persistence (27%), and viral plateau with persistence (41%). HCV RNA levels reached a high viraemic phase within two months following infection, with higher levels in the spontaneous clearance and partial viral control groups, compared to the viral plateau group (median: 6.0, 6.2, 5.3 log IU/mL, respectively; P=0.018). In the two groups with persistence, Interferon lambda 3 (IFNL3) CC genotype was independently associated with partial viral control compared to viral plateau (adjusted odds ratio [AOR]: 2.75; 95%CI: 1.08, 7.02). In the two groups with viral control, female sex was independently associated with spontaneous clearance compared to partial viral control (AOR: 2.86; 95%CI: 1.04, 7.83).

Conclusions

Among individuals with acute HCV, a spectrum of HCV RNA patterns is evident. IFNL3 CC genotype is associated with initial viral control, while female sex is associated with ultimate spontaneous clearance.