Improved screening of fall risk using free-living based accelerometer data.

Falls are one of the most costly population health issues. Screening of older adults for fall risks can allow for earlier interventions and ultimately lead to better outcomes and reduced public health spending. This work proposes a solution to limitations in existing fall screening techniques by utilizing a hip-based accelerometer worn in free-living conditions. The work proposes techniques to extract fall risk features from periods of free-living ambulatory activity. Analysis of the proposed techniques is conducted and compared with existing screening methods using Functional Tests and Lab-based Gait Analysis. 1705 Older Adults from Umea (Sweden) were assessed. Data consisted of 1 Week of hip worn accelerometer data, gait measurements and performance metrics for 3 functional tests. Retrospective and Prospective fall data were also recorded based on the incidence of falls occurring 12 months before and after the study commencing respectively. Machine learning based experiments show accelerometer based measures perform best when predicting falls. Prospective falls had a sensitivity and specificity of 0.61 and 0.66 respectively while retrospective falls had a sensitivity and specificity of 0.61 and 0.68 respectively.

Proteasome inhibition for treatment of leishmaniasis, Chagas disease and sleeping sickness.

Chagas disease, leishmaniasis and sleeping sickness affect 20 million people worldwide and lead to more than 50,000 deaths annually. The diseases are caused by infection with the kinetoplastid parasites Trypanosoma cruzi, Leishmania spp. and Trypanosoma brucei spp., respectively. These parasites have similar biology and genomic sequence, suggesting that all three diseases could be cured with drugs that modulate the activity of a conserved parasite target. However, no such molecular targets or broad spectrum drugs have been identified to date. Here we describe a selective inhibitor of the kinetoplastid proteasome (GNF6702) with unprecedented in vivo efficacy, which cleared parasites from mice in all three models of infection. GNF6702 inhibits the kinetoplastid proteasome through a non-competitive mechanism, does not inhibit the mammalian proteasome or growth of mammalian cells, and is well-tolerated in mice. Our data provide genetic and chemical validation of the parasite proteasome as a promising therapeutic target for treatment of kinetoplastid infections, and underscore the possibility of developing a single class of drugs for these neglected diseases.

Spontaneous Selection of Cryptosporidium Drug Resistance in a Calf Model of Infection.

The intestinal protozoan Cryptosporidium is a leading cause of diarrheal disease and mortality in young children. There is currently no fully effective treatment for cryptosporidiosis, which has stimulated interest in anticryptosporidial development over the last ∼10 years, with numerous lead compounds identified, including several tRNA synthetase inhibitors. Here, we report the results of a dairy calf efficacy trial of the methionyl-tRNA (Cryptosporidium parvum MetRS [CpMetRS]) synthetase inhibitor 2093 and the spontaneous emergence of drug resistance. Dairy calves experimentally infected with Cryptosporidium parvum initially improved with 2093 treatment, but parasite shedding resumed in two of three calves on treatment day 5. Parasites shed by each recrudescent calf had different amino acid-altering mutations in the gene encoding CpMetRS (CpMetRS), yielding either an aspartate 243-to-glutamate (D243E) or a threonine 246-to-isoleucine (T246I) mutation. Transgenic parasites engineered to have either the D243E or T246I CpMetRS mutation using CRISPR/Cas9 grew normally but were highly 2093 resistant; the D243E and T246I mutant-expressing parasites, respectively, had 2093 half-maximal effective concentrations (EC50s) that were 613- and 128-fold that of transgenic parasites with wild-type CpMetRS. In studies using recombinant enzymes, the D243E and T246I mutations shifted the 2093 IC50 >170-fold. Structural modeling of CpMetRS based on an inhibitor-bound Trypanosoma brucei MetRS crystal structure suggested that the resistance mutations reposition nearby hydrophobic residues, interfering with compound binding while minimally impacting substrate binding. This is the first report of naturally emerging Cryptosporidium drug resistance, highlighting the need to address the potential for anticryptosporidial resistance and establish strategies to limit its occurrence.

Methionyl-tRNA synthetase inhibitor has potent in vivo activity in a novel Giardia lamblia luciferase murine infection model.

BACKGROUND: Methionyl-tRNA synthetase (MetRS) inhibitors are under investigation for the treatment of intestinal infections caused by Giardia lamblia. OBJECTIVES: To properly analyse the therapeutic potential of the MetRS inhibitor 1717, experimental tools including a robust cell-based assay and a murine model of infection were developed based on novel strains of G. lamblia that employ luciferase reporter systems to quantify viable parasites. METHODS: Systematic screening of Giardia-specific promoters and luciferase variants led to the development of a strain expressing the click beetle green luciferase. Further modifying this strain to express NanoLuc created a dual reporter strain capable of quantifying parasites in both the trophozoite and cyst stages. These strains were used to develop a high-throughput cell assay and a mouse infection model. A library of MetRS inhibitors was screened in the cell assay and Compound-1717 was tested for efficacy in the mouse infection model. RESULTS: Cell viability in in vitro compound screens was quantified via bioluminescence readouts while infection loads in mice were monitored with non-invasive whole-animal imaging and faecal analysis. Compound-1717 was effective in clearing mice of Giardia infection in 3 days at varying doses, which was supported by data from enzymatic and phenotypic cell assays. CONCLUSIONS: The new in vitro and in vivo assays based on luciferase expression by engineered G. lamblia strains are useful for the discovery and development of new therapeutics for giardiasis. MetRS inhibitors, as validated by Compound-1717, have promising anti-giardiasis properties that merit further study as alternative therapeutics.

Imaging of the post-treatment neck.

Post-treatment imaging of the neck is complex. It is important to have an understanding of the expected treatment related appearances as well as the possible complications. Common findings after radiation therapy include generalised soft-tissue oedema and thickening of the skin and platysma muscle. There are a number of complications of radiation that may be seen on imaging, including osteoradionecrosis, chondronecrosis, and accelerated atherosclerosis. Surgical procedures are variable depending on the primary tumour site and extent. The use of flap reconstructions can further complicate the imaging appearances. Any new nodule of enhancement or bone/cartilage erosion should raise concern for tumour recurrence. It is also important to assess for nodal recurrence. A standardised approach to reporting may help to increase accuracy and guide treatment decisions.

Optimization of Methionyl tRNA-Synthetase Inhibitors for Treatment of Cryptosporidium Infection.

Cryptosporidiosis is one of the leading causes of moderate to severe diarrhea in children in low-resource settings. The therapeutic options for cryptosporidiosis are limited to one drug, nitazoxanide, which unfortunately has poor activity in the most needy populations of malnourished children and HIV-infected persons. We describe here the discovery and early optimization of a class of imidazopyridine-containing compounds with potential for treating Cryptosporidium infections. The compounds target the Cryptosporidium methionyl-tRNA synthetase (MetRS), an enzyme that is essential for protein synthesis. The most potent compounds inhibited the enzyme with Ki values in the low picomolar range. Cryptosporidium cells in culture were potently inhibited with 50% effective concentrations as low as 7 nM and >1,000-fold selectivity over mammalian cells. A parasite persistence assay indicates that the compounds act by a parasiticidal mechanism. Several compounds were demonstrated to control infection in two murine models of cryptosporidiosis without evidence of toxicity. Pharmacological and physicochemical characteristics of compounds were investigated to determine properties that were associated with higher efficacy. The results indicate that MetRS inhibitors are excellent candidates for development for anticryptosporidiosis therapy.

Evidence for changing nerve growth factor signalling mechanisms during development, maturation and ageing in the rat molar pulp.

AIM: To examine rat molar pulp innervation and identify complex cellular signalling systems involving nerve growth factor (NGF) and its p75 receptors (NGFR) at different stages of development, maturation and ageing. METHODOLOGY: Decalcified mandibular first molar mesial cusps from Wistar rats of ages 0 day; 1, 2, 3, 4, 6, 9, 12 and 24 weeks (n = 5 per group) were sectioned (10 µm) and incubated with antibodies for NGF, NGFR, calcitonin gene-related peptide (CGRP) and neurofilament. Nerve densities in worn and intact regions of 3- to 24-week-old rats were compared by anova, Bonferroni and t-tests. RESULTS: During odontogenesis, differences in NGF and NGFR expression were observed, with no evidence of nerve fibres, suggesting a signalling mechanism controlling cellular differentiation and dentine formation. Tooth wear in 4-week rats was associated with reduced NGF expression and significantly decreased CGRP axons within affected odontoblast regions. The underlying subodontoblasts started expressing NGF which continued until 9 weeks. This may promote a significant increase in CGRP nerve density in affected regions. Nerve density in intact odontoblast regions increased gradually and reached significant levels in 12-week rats. Reduction in nerve densities within worn and intact regions of cusps was observed at 24 weeks. CONCLUSIONS: Age-related changes and responses to tooth wear may be controlled by the NGF signalling mechanism, with roles in odontoblast/subodontoblast communication and control of sensory innervation at different stages of tooth development, maturation and ageing. Greater understanding of cellular and nerve regulation in the injured pulp may promote therapeutic strategies for pulp survival.

Eat, play, learn well-a novel approach to co-production and analysis grid for environments linked to obesity to engage local communities in a child healthy weight action plan.

OBJECTIVES: To describe the process of combining Analysis Grid for Environments Linked to Obesity (ANGELO) with community engagement, qualitative and co-production methods to promote local strategies around child healthy weight (CHW) and to highlight steps taken to engage local people in developing a community CHW action plan around two school communities in Dundee, Scotland. STUDY DESIGN: The Eat, Play, Learn Well (Learn Well) approach applied an action-oriented research approach, using qualitative methods. METHODS: Focus group discussions (FGDs), a co-production approach, and ANGELO were linked by applying a novel three-step process. FGDs were recorded by scribe and following face-to-face interview's key themes were identified using a novel, predefined five-step process, and ANGELO grids were populated. Prioritization events allowed local people to rank most important health statements, with community conversations offering further insights to help create a local CHW action plan. RESULTS: Three FGDs were conducted with parents (n = 24) and two with workers (n = 15). Eighty-seven attended a prioritization event at school B (41 adults), 59 attended at school A (35 adults), where each school community chose its top four priorities from 11 health statements developed. Two further community conversations then took place and led to the creation of a CHW action plan with five overarching themes. CONCLUSIONS: The Learn Well test approach helped gain important insights into local environments linked to obesity and production of a pragmatic, step-by-step process suitable for real-life public health practice that can enable local people to identify key early intervention and prevention priorities, in a tangible way.

Development of Methionyl-tRNA Synthetase Inhibitors as Antibiotics for Gram-Positive Bacterial Infections.

Antibiotic-resistant bacteria are widespread and pose a growing threat to human health. New antibiotics acting by novel mechanisms of action are needed to address this challenge. The bacterial methionyl-tRNA synthetase (MetRS) enzyme is essential for protein synthesis, and the type found in Gram-positive bacteria is substantially different from its counterpart found in the mammalian cytoplasm. Both previously published and new selective inhibitors were shown to be highly active against Gram-positive bacteria with MICs of ≤1.3 µg/ml against Staphylococcus, Enterococcus, and Streptococcus strains. Incorporation of radioactive precursors demonstrated that the mechanism of activity was due to the inhibition of protein synthesis. Little activity against Gram-negative bacteria was observed, consistent with the fact that Gram-negative bacterial species contain a different type of MetRS enzyme. The ratio of the MIC to the minimum bactericidal concentration (MBC) was consistent with a bacteriostatic mechanism. The level of protein binding of the compounds was high (>95%), and this translated to a substantial increase in MICs when the compounds were tested in the presence of serum. Despite this, the compounds were very active when they were tested in a Staphylococcus aureus murine thigh infection model. Compounds 1717 and 2144, given by oral gavage, resulted in 3- to 4-log decreases in the bacterial load compared to that in vehicle-treated mice, which was comparable to the results observed with the comparator drugs, vancomycin and linezolid. In summary, the research describes MetRS inhibitors with oral bioavailability that represent a class of compounds acting by a novel mechanism with excellent potential for clinical development.

Variants at the OCA2/HERC2 locus affect time to first cutaneous squamous cell carcinoma in solid organ transplant recipients collected using two different study designs.

BACKGROUND: Variants at the oculocutaneous albinism 2 (OCA2)/HECT and RLD domain containing E3 ubiquitin protein ligase 2 (HERC2) locus have been associated with pigmentation phenotypes and risk of developing several types of skin cancer. OBJECTIVES: To evaluate OCA2/HERC2 locus variants for their impact on time to develop cutaneous squamous cell carcinoma (cSCC) in organ transplant recipients (OTRs) who are at elevated risk of developing cSCC. METHODS: Participants were solid OTRs ascertained from two centres (n = 125 and 261) with an average of 13·1 years of follow-up post-transplant. DNA was available for genotyping for all participants, in addition to medical records and questionnaire data. The Ohio State University study had a case-control design with prospective follow-up, and the University of California San Francisco study was a national cross-sectional survey with retrospective chart review. RESULTS: OCA2 variants rs12913832 and rs916977 were significantly associated with time to first cSCC post-transplant. OTRs homozygous for the brown-eye alleles of rs916977 (GG) and rs12913832 (AA) had significant delays of time to first cSCC post-transplant compared with individuals homozygous for the blue-eye alleles (hazard ratio 0·34, P < 0·001 and hazard ratio 0·54, P = 0·012, respectively). Both variants were highly associated with eye colour in the combined studies (P < 0·001). CONCLUSIONS: This study is the first to show an association between OCA2/HERC2 variants and time to first cSCC post-transplant. This may impact dermatological screening recommendations for high-risk populations.

Optimization of a binding fragment targeting the "enlarged methionine pocket" leads to potent Trypanosoma brucei methionyl-tRNA synthetase inhibitors.

Potent inhibitors of Trypanosoma brucei methionyl-tRNA synthetase were previously designed using a structure-guided approach. Compounds 1 and 2 were the most active compounds in the cyclic and linear linker series, respectively. To further improve cellular potency, SAR investigation of a binding fragment targeting the "enlarged methionine pocket" (EMP) was performed. The optimization led to the identification of a 6,8-dichloro-tetrahydroquinoline ring as a favorable fragment to bind the EMP. Replacement of 3,5-dichloro-benzyl group (the EMP binding fragment) of inhibitor 2 using this tetrahydroquinoline fragment resulted in compound 13, that exhibited an EC50 of 4nM.

Discovery of N-(2-aminoethyl)-N-benzyloxyphenyl benzamides: New potent Trypanosoma brucei inhibitors.

A phenotypic screen of a compound library for antiparasitic activity on Trypanosoma brucei, the causative agent of Human African Trypanosomiasis (HAT), led to the identification of N-(2-aminoethyl)-N-phenyl benzamides as a starting point for hit-to-lead medicinal chemistry. Eighty two analogues were prepared, which led to the identification of a set of highly potent N-(2-aminoethyl)-N-benzyloxyphenyl benzamides with the most potent compound 73 having an in vitro EC50=0.001µM. The compounds displayed drug-like properties when tested in a number of in vitro assays. Compound 73 was orally bioavailable and displayed good plasma and brain exposure in mice, cured 2 out of 3 mice infected with Trypanosoma brucei in acute model when dosed orally at 50mg/kg once per day for 4days. Given its potent antiparasitic properties and its ease of synthesis, compound 73 represents a potential lead for the development of drug to treat Human African Trypanosomiasis.

A binding hotspot in Trypanosoma cruzi histidyl-tRNA synthetase revealed by fragment-based crystallographic cocktail screens.

American trypanosomiasis, commonly known as Chagas disease, is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. The chronic form of the infection often causes debilitating morbidity and mortality. However, the current treatment for the disease is typically inadequate owing to drug toxicity and poor efficacy, necessitating a continual effort to discover and develop new antiparasitic therapeutic agents. The structure of T. cruzi histidyl-tRNA synthetase (HisRS), a validated drug target, has previously been reported. Based on this structure and those of human cytosolic HisRS, opportunities for the development of specific inhibitors were identified. Here, efforts are reported to identify small molecules that bind to T. cruzi HisRS through fragment-based crystallographic screening in order to arrive at chemical starting points for the development of specific inhibitors. T. cruzi HisRS was soaked into 68 different cocktails from the Medical Structural Genomics of Pathogenic Protozoa (MSGPP) fragment library and diffraction data were collected to identify bound fragments after soaking. A total of 15 fragments were identified, all bound to the same site on the protein, revealing a fragment-binding hotspot adjacent to the ATP-binding pocket. On the basis of the initial hits, the design of reactive fragments targeting the hotspot which would be simultaneously covalently linked to a cysteine residue present only in trypanosomatid HisRS was initiated. Inhibition of T. cruzi HisRS was observed with the resultant reactive fragments and the anticipated binding mode was confirmed crystallographically. These results form a platform for the development of future generations of selective inhibitors for trypanosomatid HisRS.

Inhibitors of methionyl-tRNA synthetase have potent activity against Giardia intestinalis trophozoites.

The methionyl-tRNA synthetase (MetRS) is a novel drug target for the protozoan pathogen Giardia intestinalis. This protist contains a single MetRS that is distinct from the human cytoplasmic MetRS. A panel of MetRS inhibitors was tested against recombinant Giardia MetRS, Giardia trophozoites, and mammalian cell lines. The best compounds inhibited trophozoite growth at 500 nM (metronidazole did so at ∼5,000 nM) and had low cytotoxicity against mammalian cells, indicating excellent potential for further development as anti-Giardia drugs.

Parents as the start of the solution: a social marketing approach to understanding triggers and barriers to entering a childhood weight management service.

BACKGROUND: Childhood obesity is a sensitive subject and barriers exist with respect to accessing weight management programmes. Social marketing insight gathering provides an opportunity to understand behaviours and address these challenges. This project gained insight into the views of parents/carers on triggers and barriers to entering a childhood weight management service. METHODS: Participants were identified from the public using marketing recruitment. Four focus groups were conducted with parents of school aged children (n = 27) by an experienced interviewer. Twenty two mothers, three fathers and two grandmothers participated, with half describing their child as overweight. Groups discussed health behaviours; attitudes to health messages and weight issues; and motivations, benefits and barriers with respect to accessing weight management services. Discussions were taped and transcribed. Themes were identified using framework analysis of content matrix data analysis. RESULTS: Participants were aware of healthy lifestyle messages, although the ability to implement these was variable. Triggers to seeking help included bullying, health concerns and inability to participate in school activities. Barriers included feeling a lack of control, desire to avoid conflict and no proven case that weight was a problem. Parents wished to be given information regarding their child's weight by a trusted person. The Internet and word of mouth were identified as methods of recruitment into a weight management service, with a focus on fitness, fun and friendliness and being free-of-charge. CONCLUSIONS: Insight gathering can be used to establish parental/carer opinion regarding engaging in childhood weight management services. A fun, friendly programme that is free of charge appealed to parents. Local community involvement around normalising child weight issues may boost referrals into child healthy weight interventions.

Clinical outcome scores for arthroscopic femoral osteochondroplasty in femoroacetabular impingement: a quantitative systematic review.

BACKGROUND AND AIMS: Femoroacetabular impingement is the abnormal contact of the proximal femur and acetabulum during motion. It causes hip pain and joint degeneration in young patients. This systematic review aims to clarify the clinical effect of arthroscopic femoral osteochondroplasty for cam lesions and to review the available literature for the general medical readership, including providers of primary and secondary care. METHODS AND RESULTS: Electronic databases were searched for studies of arthroscopic femoral osteochondroplasty in primary femoroacetabular impingement. A total of 2618 article titles, 242 abstracts and 33 full text articles were considered. Ultimately nine studies with clinical outcome scores met the inclusion criteria and were included in the qualitative systematic review. Six studies were suitable for meta-analysis using an inverse variance, random effects model (RevMan software). In the nine studies, improvements were seen in Western Ontario and McMaster Universities Osteoarthritis index, Non-arthritic Hip Score and Modified Harris Hip Scores. Across the six studies suitable for meta-analysis (537 patients), a 24-point weighted mean improvement in Non-arthritic hip score was seen. This yielded a large overall effect size of 1.6. CONCLUSION: Arthroscopic femoral osteochondroplasty appears to be a beneficial treatment for primary femoroacetabular impingement, with a large effect size seen across six eligible studies.

Induced resistance to methionyl-tRNA synthetase inhibitors in Trypanosoma brucei is due to overexpression of the target.

New classes of antiparasitic drugs active against Trypanosoma brucei are needed to combat human African trypanosomiasis. Inhibitors of methionyl-tRNA synthetase (MetRS) have excellent potential to be developed for this purpose (S. Shibata, J. R. Gillespie, A. M. Kelley, A. J. Napuli, Z. Zhang, K. V. Kovzun, R. M. Pefley, J. Lam, F. H. Zucker, W. C. Van Voorhis, E. A. Merritt, W. G. Hol, C. L. Verlinde, E. Fan, and F. S. Buckner, Antimicrob. Agents Chemother. 55:1982-1989, 2011). In order to assess the potential for resistance to develop against this new class of inhibitors, T. brucei cultures were grown in the presence of MetRS inhibitors or comparison drugs. Resistance up to ∼50 times the baseline 50% inhibitory concentration (IC50) was induced against a MetRS inhibitor after ∼120 days. A similar level of resistance to the clinical drug eflornithine was induced after ∼50 days and for pentamidine after ∼80 days. Thus, resistance was induced more slowly against MetRS inhibitors than against clinically used drugs. The parasites resistant to the MetRS inhibitor were shown to overexpress MetRS mRNA by a factor of 35 over the parental strain. Southern analysis indicated that the MetRS gene was amplified in the genome by nearly 8-fold. When injected into mice, the MetRS inhibitor-resistant parasites caused a reduced level of infection, indicating that the changes associated with resistance attenuated their virulence. This finding and the fact that resistance to MetRS inhibitors developed relatively slowly are encouraging for further development of this class of compounds. Published studies on other antitrypanosomal drugs have primarily shown that alterations in membrane transporters were the mechanisms responsible for resistance. This is the first published report of induced drug resistance in the African trypanosome due to overexpression of the target enzyme.

Selective inhibitors of methionyl-tRNA synthetase have potent activity against Trypanosoma brucei Infection in Mice.

Human African trypanosomiasis continues to be an important public health threat in extensive regions of sub-Saharan Africa. Treatment options for infected patients are unsatisfactory due to toxicity, difficult administration regimes, and poor efficacy of available drugs. The aminoacyl-tRNA synthetases were selected as attractive drug targets due to their essential roles in protein synthesis and cell survival. Comparative sequence analysis disclosed differences between the trypanosome and mammalian methionyl-tRNA synthetases (MetRSs) that suggested opportunities for selective inhibition using drug-like molecules. Experiments using RNA interference on the single MetRS of Trypanosoma brucei demonstrated that this gene product was essential for normal cell growth. Small molecules (diaryl diamines) similar to those shown to have potent activity on prokaryotic MetRS enzymes were synthesized and observed to have inhibitory activity on the T. brucei MetRS (50% inhibitory concentration, <50 nM) and on bloodstream forms of T. brucei cultures (50% effective concentration, as low as 4 nM). Twenty-one compounds had a close correlation between enzyme binding/inhibition and T. brucei growth inhibition, indicating that they were likely to be acting on the intended target. The compounds had minimal effects on mammalian cell growth at 20 µM, demonstrating a wide therapeutic index. The most potent compound was tested in the murine model of trypanosomiasis and demonstrated profound parasite suppression and delayed mortality. A homology model of the T. brucei MetRS based on other MetRS structures was used to model binding of the lead diaryl diamine compounds. Future studies will focus on improving the pharmacological properties of the MetRS inhibitors.

On the nature of bladder sensation: the concept of sensory modulation.

AIMS: Going to the toilet is an essential everyday event. Normally, we do not give much thought to the sensations and factors that trigger voiding behavior: we just go. For many people, this apparently simple task is complicated and dominates their life. They have strong sensations and sudden desires to void, often resulting in incontinence. It is therefore important that we understand the origins for this functional change and identify means to alleviate it. METHODS: Literature survey. RESULTS: A considerable body of work has focused on this problem and ideas and concepts on the nature of bladder sensations are embedded in the literature. In this paper we argue the necessity to return to first principles and a re-examination of the problem. We explore the use of focus groups to identify relevant bladder sensation and what triggers 'bladder' behavior. We argue that there are differences in what can be described as 'introspective bladder sensations' and the sensations reported immediately before a void, 'void sensations'. Finally, we propose an alternative model describing how peripheral information generating 'introspective sensations' and 'void sensations' might be different but interrelated sensations. By exploring such ideas and identifying such complexity it is our intention to stimulate debate and generate further research in the field in order to understand better the physiology of bladder sensation and the pathology of increased urge, frequency and incontinence. CONCLUSIONS: Review of the literature on bladder sensation and the established ideas suggests that we might be missing something and the problem of normal and increased sensation and of urgency may be much more complex.