Mouse models of NADK2 deficiency analyzed for metabolic and gene expression changes to elucidate pathophysiology.

NADK2 encodes the mitochondrial form of nicotinamide adenine dinucleotide (NAD) kinase, which phosphorylates NAD. Rare recessive mutations in human NADK2 are associated with a syndromic neurological mitochondrial disease that includes metabolic changes, such as hyperlysinemia and 2,4 dienoyl CoA reductase (DECR) deficiency. However, the full pathophysiology resulting from NADK2 deficiency is not known. Here, we describe two chemically induced mouse mutations in Nadk2-S326L and S330P-which cause severe neuromuscular disease and shorten lifespan. The S330P allele was characterized in detail and shown to have marked denervation of neuromuscular junctions by 5 weeks of age and muscle atrophy by 11 weeks of age. Cerebellar Purkinje cells also showed progressive degeneration in this model. Transcriptome profiling on brain and muscle was performed at early and late disease stages. In addition, metabolomic profiling was performed on the brain, muscle, liver and spinal cord at the same ages and on plasma at 5 weeks. Combined transcriptomic and metabolomic analyses identified hyperlysinemia, DECR deficiency and generalized metabolic dysfunction in Nadk2 mutant mice, indicating relevance to the human disease. We compared findings from the Nadk model to equivalent RNA sequencing and metabolomic datasets from a mouse model of infantile neuroaxonal dystrophy, caused by recessive mutations in Pla2g6. This enabled us to identify disrupted biological processes that are common between these mouse models of neurological disease, as well as those processes that are gene-specific. These findings improve our understanding of the pathophysiology of neuromuscular diseases and describe mouse models that will be useful for future preclinical studies.

From admissions to licensure: education data associations from a multi-centre undergraduate medical education collaboration.

This paper reports the findings of a Canada based multi-institutional study designed to investigate the relationships between admissions criteria, in-program assessments, and performance on licensing exams. The study's objective is to provide valuable insights for improving educational practices across different institutions. Data were gathered from six medical schools: McMaster University, the Northern Ontario School of Medicine University, Queen's University, University of Ottawa, University of Toronto, and Western University. The dataset includes graduates who undertook the Medical Council of Canada Qualifying Examination Part 1 (MCCQE1) between 2015 and 2017. The data were categorized into five distinct sections: demographic information as well as four matrices: admissions, course performance, objective structured clinical examination (OSCE), and clerkship performance. Common and unique variables were identified through an extensive consensus-building process. Hierarchical linear regression and a manual stepwise variable selection approach were used for analysis. Analyses were performed on data set encompassing graduates of all six medical schools as well as on individual data sets from each school. For the combined data set the final model estimated 32% of the variance in performance on licensing exams, highlighting variables such as Age at Admission, Sex, Biomedical Knowledge, the first post-clerkship OSCE, and a clerkship theta score. Individual school analysis explained 41-60% of the variance in MCCQE1 outcomes, with comparable variables to the analysis from of the combined data set identified as significant independent variables. Therefore, strongly emphasising the need for variety of high-quality assessment on the educational continuum. This study underscores the importance of sharing data to enable educational insights. This study also had its challenges when it came to the access and aggregation of data. As such we advocate for the establishment of a common framework for multi-institutional educational research, facilitating studies and evaluations across diverse institutions. This study demonstrates the scientific potential of collaborative data analysis in enhancing educational outcomes. It offers a deeper understanding of the factors influencing performance on licensure exams and emphasizes the need for addressing data gaps to advance multi-institutional research for educational improvements.

Structural and Biochemical Characterization of Spa47 Provides Mechanistic Insight into Type III Secretion System ATPase Activation and Shigella Virulence Regulation.

Like many Gram-negative pathogens, Shigella rely on a complex type III secretion system (T3SS) to inject effector proteins into host cells, take over host functions, and ultimately establish infection. Despite these critical roles, the energetics and regulatory mechanisms controlling the T3SS and pathogen virulence remain largely unclear. In this study, we present a series of high resolution crystal structures of Spa47 and use the structures to model an activated Spa47 oligomer, finding that ATP hydrolysis may be supported by specific side chain contributions from adjacent protomers within the complex. Follow-up mutagenesis experiments targeting the predicted active site residues validate the oligomeric model and determined that each of the tested residues are essential for Spa47 ATPase activity, although they are not directly responsible for stable oligomer formation. Although N-terminal domain truncation was necessary for crystal formation, it resulted in strictly monomeric Spa47 that is unable to hydrolyze ATP, despite maintaining the canonical ATPase core structure and active site residues. Coupled with studies of ATPase inactive full-length Spa47 point mutants, we find that Spa47 oligomerization and ATP hydrolysis are needed for complete T3SS apparatus formation, a proper translocator secretion profile, and Shigella virulence. This work represents the first structure-function characterization of Spa47, uniquely complementing the multitude of included Shigella T3SS phenotype assays and providing a more complete understanding of T3SS ATPase-mediated pathogen virulence. Additionally, these findings provide a strong platform for follow-up studies evaluating regulation of Spa47 oligomerization in vivo as a much needed means of treating and perhaps preventing shigellosis.

Visible-Light-Driven Valorization of Biomass Intermediates Integrated with H2 Production Catalyzed by Ultrathin Ni/CdS Nanosheets.

Photocatalytic upgrading of crucial biomass-derived intermediate chemicals (i.e., furfural alcohol, 5-hydroxymethylfurfural (HMF)) to value-added products (aldehydes and acids) was carried out on ultrathin CdS nanosheets (thickness ∼1 nm) decorated with nickel (Ni/CdS). More importantly, simultaneous H2 production was realized upon visible light irradiation under ambient conditions utilizing these biomass intermediates as proton sources. The remarkable difference in the rates of transformation of furfural alcohol and HMF to their corresponding aldehydes in neutral water was observed and investigated. Aided by theoretical computation, it was rationalized that the slightly stronger binding affinity of the aldehyde group in HMF to Ni/CdS resulted in the lower transformation of HMF to 2,5-diformylfuran compared to that of furfural alcohol to furfural. Nevertheless, photocatalytic oxidation of furfural alcohol and HMF under alkaline conditions led to complete transformation to the respective carboxylates with concomitant production of H2.

Novel axon projection after stress and degeneration in the Dscam mutant retina.

The Down syndrome cell adhesion molecule gene (Dscam) is required for normal dendrite patterning and promotes developmental cell death in the mouse retina. Loss-of-function studies indicate that Dscam is required for refinement of retinal ganglion cell (RGC) axons in the lateral geniculate nucleus, and in this study we report and describe a requirement for Dscam in the maintenance of RGC axon projections within the retina. Mouse Dscam loss of function phenotypes related to retinal ganglion cell axon outgrowth and targeting have not been previously reported, despite the abundance of axon phenotypes reported in Drosophila Dscam1 loss and gain of function models. Analysis of the Dscam deficient retina was performed by immunohistochemistry and Western blot analysis during postnatal development of the retina. Conditional targeting of Dscam and Jun was performed to identify factors underlying axon-remodeling phenotypes. A subset of RGC axons were observed to project and branch extensively within the Dscam mutant retina after eye opening. Axon remodeling was preceded by histological signs of RGC stress. These included neurofilament accumulation, axon swelling, axon blebbing and activation of JUN, JNK and AKT. Novel and extensive projection of RGC axons within the retina was observed after upregulation of these markers, and novel axon projections were maintained to at least one year of age. Further analysis of retinas in which Dscam was conditionally targeted with Brn3b or Pax6α Cre indicated that axon stress and remodeling could occur in the absence of hydrocephalus, which frequently occurs in Dscam mutant mice. Analysis of mice mutant for the cell death gene Bax, which executes much of Dscam dependent cell death, identified a similar axon misprojection phenotype. Deleting Jun and Dscam resulted in increased axon remodeling compared to Dscam or Bax mutants. Retinal ganglion cells have a very limited capacity to regenerate after damage in the adult retina, compared to the extensive projections made in the embryo. In this study we find that DSCAM and JUN limit ectopic growth of RGC axons, thereby identifying these proteins as targets for promoting axon regeneration and reconnection.

Metabolic regulation of stem cell function.

Stem cell function is regulated by intrinsic mechanisms, such as transcriptional and epigenetic regulators, as well as extrinsic mechanisms, such as short-range signals from the niche and long-range humoral signals. Interactions between these regulatory mechanisms and cellular metabolism are just beginning to be identified. In multiple systems, differentiation is accompanied by changes in glycolysis, oxidative phosphorylation and the levels of reactive oxygen species. Indeed, metabolic pathways regulate proliferation and differentiation by regulating energy production and the generation of substrates for biosynthetic pathways. Some metabolic pathways appear to function differently in stem cells as compared with restricted progenitors and differentiated cells. They also appear to influence stem cell function by regulating signal transduction, epigenetic marks and oxidative stress. Studies to date illustrate the importance of metabolism in the regulation of stem cell function and suggest complex cross-regulation likely exists between metabolism and other stem cell regulatory mechanisms.

Quantifying the efficacy of an automated facial coding software using videos of parents.

Introduction: This work explores the use of an automated facial coding software - FaceReader - as an alternative and/or complementary method to manual coding. Methods: We used videos of parents (fathers, n = 36; mothers, n = 29) taken from the Avon Longitudinal Study of Parents and Children. The videos-obtained during real-life parent-infant interactions in the home-were coded both manually (using an existing coding scheme) and by FaceReader. We established a correspondence between the manual and automated coding categories - namely Positive, Neutral, Negative, and Surprise - before contingency tables were employed to examine the software's detection rate and quantify the agreement between manual and automated coding. By employing binary logistic regression, we examined the predictive potential of FaceReader outputs in determining manually classified facial expressions. An interaction term was used to investigate the impact of gender on our models, seeking to estimate its influence on the predictive accuracy. Results: We found that the automated facial detection rate was low (25.2% for fathers, 24.6% for mothers) compared to manual coding, and discuss some potential explanations for this (e.g., poor lighting and facial occlusion). Our logistic regression analyses found that Surprise and Positive expressions had strong predictive capabilities, whilst Negative expressions performed poorly. Mothers' faces were more important for predicting Positive and Neutral expressions, whilst fathers' faces were more important in predicting Negative and Surprise expressions. Discussion: We discuss the implications of our findings in the context of future automated facial coding studies, and we emphasise the need to consider gender-specific influences in automated facial coding research.

Acrylic-based culture plate format perfusion device to establish liver endothelial-epithelial interface.

Microphysiological Systems (MPSs) or organs-on-chips, are microfluidic devices used to model human physiology in vitro. Polydimethylsiloxane (PDMS) is the most widely used material for organs-on-chips due to its established fabrication methods and biocompatibility properties. However, non-specific binding of small molecules limits PDMS for drug screening applications. Here, we designed a novel acrylic-based MPS to capture the physiological architecture that is observed universally in tissues across the body: the endothelial-epithelial interface (EEI). To reconstruct the EEI biology, we designed a membrane-based chip that features endothelial cells on the underside of the membrane exposed to mechanical shear from the path of media flow, and epithelial cells on the opposite side of the membrane protected from flow, as they are in vivo. We used a liver model with a hepatic progenitor cell line and human umbilical vein endothelial cells to assess the biological efficacy of the MPS. We computationally modeled the physics that govern the function of perfusion through the MPS. Empirically, efficacy was measured by comparing differentiation of the hepatic progenitor cells between the MPS and 2D culture conditions. We demonstrated that the MPS significantly improved hepatocyte differentiation, increased extracellular protein transport, and raised hepatocyte sensitivity to drug treatment. Our results strongly suggest that physiological perfusion has a profound effect on proper hepatocyte function, and the modular chip design motivates opportunities for future study of multi-organ interactions.

Musculoskeletal case-mix adjustment in a UK primary/community care cohort: Testing musculoskeletal models to make recommendations in this setting.

Benchmarking musculoskeletal (MSK) services is limited by the need to adjust for differences in patient characteristics/case-mix. Without this providers and services cannot be usefully compared. This paper investigates the predictive ability of case-mix adjustment models in a primary/community care cohort. OBJECTIVES: To investigate the predictive ability of two existing MSK case-mix adjustment models and compare to the predictive ability of an evidence informed and statistically informed model. METHOD: A secondary analysis of the 'Subgrouping for Targeted Treatment in Musculoskeletal Conditions' cluster randomised controlled trial data (n = 1211). Stepwise linear regression models were built and compared using available baseline variables. The MSK-HQ was used as the primary functional status outcome. RESULTS: Two existing models were compared (UK National PROMs Model, US FOTO Model) using available variables. Of these models the modified US FOTO model showed the best predictive ability in this cohort predicting 44% of the variation in MSK-HQ outcome, the modified UK National PROMs model predicted 41%. A newly developed evidence informed model (Keele Model 1) performed no better than the existing models, and a statistically informed model (Keele Model 2) gave only an additional 2% increase in model power compared to the modified US FOTO model. CONCLUSION: All models showed strong predictive ability. The modified US FOTO model looks to be best suited to the UK primary/community care cohort of the existing models. This model performed so well that we recommend that this model is used in a UK setting moving forwards rather than development of an alternative UK model.

Developing a core outcome set for community and primary care musculoskeletal services: A consensus approach.

BACKGROUND: At present there is no core outcome set (COS) for use in community and primary care Musculoskeletal (MSK) services across the UK. Services are therefore collecting different MSK outcomes and metrics in different ways and at different times. Standardising MSK data collection is essential in order for fair and impactful benchmarking to improve the quality of care delivered to the millions of patients presenting each year with MSK disorders. OBJECTIVE: To gain consensus on a proposed set of metrics that could be used to develop a COS for use in routine practice in community and primary care MSK services in the UK and to make recommendations to inform a future national MSK audit. METHODS: A consensus process involving researchers, healthcare professionals and patients. Previous research generated an initial list of proposed metrics. This proposal was then taken to wider stakeholder consensus via an online survey designed for both healthcare professionals and MSK service users. RESULTS: 199 respondents completed the survey, 166 healthcare professionals and 33 service users (25/33 eligible to answer all items within the survey). Metrics that reached strong consensus were; age, pain site, comorbidities, duration of symptoms, work status, work absence, work absence duration. No Patient Reported Outcome Measures (PROMs) met strong consensus and all Patient Reported Experience Measures (PREMs) other than timeliness/convenience met strong consensus criteria. CONCLUSION: 7 baseline factors and 9 PREM domains reached strong consensus. The MSK-HQ PROM was the highest rated outcome measure so was also recommended for inclusion in an MSK COS.

Resilience as a communal concept: Understanding adolescent resilience in the context of the Syrian refugee crisis in Bar Elias, Lebanon.

BACKGROUND: The conflict in Syria has led to the displacement of 1.5 million refugees into the neighboring country of Lebanon, with a majority that have yet to return to their homeland. Syrian adolescents in the town of Bar Elias in the Bekaa Valley, Lebanon have lived and grown in the face of resource-limited environments, restricted movement, and a longing for return. Resilience is manifested in the adaptation to such circumstances through close supportive relationships, social engagement, employment, and religion. There is a communal aspect to resilience that is important to the adolescent refugee experience and to the efforts supporting these communities. METHODS: Fifteen one-to-one interviews and two focus groups, with a total of eighteen Syrian adolescents, were analyzed using an inductive thematic analysis informed by grounded theory principles. Participants were recruited through partnering non-governmental organizations (NGOs) in the area, and ethical approval was granted through UCL and the American University in Beirut (AUB). RESULTS: Syrian adolescents highlighted supportive relationships, communal activities and spaces, memories of home, employment, and shared environments as integral elements to their personal adaptation. Methods of resilience involved social cohesion and establishing stability for one's family and close community. Adaptation to the present is intertwined with facing the consequences of displacement in this new context and maintaining aspirations for a bright future. Engaging with the environments they share and help create is an important facet of resilience and occurs through group gatherings , hobbies, and online communication. Additionally, inner strength can be derived from religious activities and empowers individual processing. CONCLUSION: This study illuminates the elements and mechanisms embodied in these adolescents' communities and relationships that allow for adaptation to life in Bar Elias. These factors strengthen their approach to overcome social barriers and practice resilience. These communal aspects of the adolescents' lives also connect to their memories of home, current environment, and future aspirations.

The integrated stress response contributes to tRNA synthetase-associated peripheral neuropathy.

Dominant mutations in ubiquitously expressed transfer RNA (tRNA) synthetase genes cause axonal peripheral neuropathy, accounting for at least six forms of Charcot-Marie-Tooth (CMT) disease. Genetic evidence in mouse and Drosophila models suggests a gain-of-function mechanism. In this study, we used in vivo, cell type­specific transcriptional and translational profiling to show that mutant tRNA synthetases activate the integrated stress response (ISR) through the sensor kinase GCN2 (general control nonderepressible 2). The chronic activation of the ISR contributed to the pathophysiology, and genetic deletion or pharmacological inhibition of Gcn2 alleviated the peripheral neuropathy. The activation of GCN2 suggests that the aberrant activity of the mutant tRNA synthetases is still related to translation and that inhibiting GCN2 or the ISR may represent a therapeutic strategy in CMT.

A phase I study of axitinib (AG-013736) in combination with bevacizumab plus chemotherapy or chemotherapy alone in patients with metastatic colorectal cancer and other solid tumors.

BACKGROUND: Axitinib and bevacizumab are targeted therapies against the vascular endothelial growth factor pathway. METHODS: Patients with previously treated solid tumors received axitinib (starting dose 5 mg twice daily) combined with FOLFOX plus bevacizumab (1, 2, or 5 mg/kg, cohorts 1-3, respectively), FOLFIRI (cohort 4), or FOLFOX (cohort 5). Safety and pharmacokinetics were assessed. RESULTS: Thirty patients were enrolled (n = 16, 8, and 6 for cohorts 1-3, 4, and 5, respectively). Plasma concentrations and pharmacokinetic (PK) parameters were similar when drugs were administered alone and in various combinations. Most treatment-emergent adverse events (AEs) were mild to moderate and clinically manageable (most common: nausea, fatigue, diarrhea, anorexia, hypertension). Two of the four patients receiving axitinib with FOLFOX plus 5 mg/kg bevacizumab experienced dose-limiting toxicity (DLT) of inability to resume treatment for 14 days following treatment interruption (associated AE: hypertension); the maximum tolerated dose of bevacizumab in this combination was 2 mg/kg. No DLTs occurred with axitinib plus FOLFIRI or FOLFOX. Ten patients had RECIST-confirmed partial tumor responses (objective response rate: 33.3%). CONCLUSION: Axitinib is well tolerated in combination with FOLFOX, FOLFIRI, or FOLFOX plus 2 mg/kg bevacizumab. PK interactions appear to be absent.

ADVIRC is caused by distinct mutations in BEST1 that alter pre-mRNA splicing.

Autosomal dominant vitreoretinochoroidopathy (ADVIRC), a retinal dystrophy often associated with glaucoma and cataract, forms part of a phenotypic spectrum of 'bestrophinopathies'. It has been shown previously that ADVIRC results from BEST1 mutations that cause exon skipping and lead to the production of shortened and internally deleted isoforms. This study describes a novel ADVIRC mutation and show that it disrupts an exonic splice enhancer (ESE) site, altering the binding of a splicing-associated SR protein. As with previous ADVIRC mutations, the novel c.704T-->C mutation in exon 6 altered normal splicing in an ex vivo splicing assay. Both this and another exon 6 ADVIRC-causing mutation (c.707G-->A) either weakened or abolished splicing in an ESE-dependent splice assay compared with a nearby exon 6 mutation associated with Best disease (c.703G-->C). Gel shift assays were undertaken with RNA oligonucleotides encompassing the ADVIRC and Best disease mutations with four of the most commonly investigated SR proteins. Although SC35, SRp40 and SRp55 proteins all bound to the wild-type and mutated sequences with similar intensities, there was increased binding of ASF/SF2 to the two ADVIRC-mutated sequences compared with the wild-type or Best disease-mutated sequences. The exon skipping seen for these two exon 6 ADVIRC mutations and their affinity for ASF/SF2 suggests that the region encompassing these mutations may form part of a CERES (composite exonic regulatory elements of splicing) site.

Emergency Medical Services: a resource for victims of domestic violence?

BACKGROUND: Domestic violence (DV), also known as intimate partner violence (IPV), is one of the leading causes of serious injury among women of childbearing age. As first responders on the scene during DV calls where personal injuries have occurred, Emergency Medical Services (EMS) could routinely identify, report and assist victims of violence. Yet, little is known of the prevalence of DV calls in EMS practice, Emergency Medical Technicians' (EMT) knowledge and comfort in responding to such calls, or how they care for victims. METHOD: The objectives of this study were to assess EMTs' knowledge of and experience with providing care to victims of DV in the province of Ontario, Canada. Data were gathered through an online, short-answer survey. Survey data were analysed using basic frequency displays, and descriptive statistics are reported. RESULTS: Almost 500 EMTs participated in this study, the vast majority of whom (90%) attended at least one DV call in the preceding year, with 65% attending between 10 and 20 DV calls. The majority of respondents (84.5%) wished for more education and training on the issue. CONCLUSION: EMTs have frequent contact with victims of DV yet have received little education about the issue. The majority of those surveyed would like specific education and training on DV.

Resistance carrying plasmid in a traumatic wound.

OBJECTIVE: To isolate and identify antibiotic-resistant bacteria from the exudate of a complex wound and determine if antibiotic resistance genes are chromosomal or plasmid borne. METHOD: Antibiotic resistant bacteria from wound exudate of a single clinical sample were selected on agar media with ampicillin. A single colony was further screened for resistance to kanamycin by antibiotic-supplemented agar and to other antibiotics by an automated Phoenix instrument. Identification of the isolate was carried out by biochemical profiling and by 16S rDNA analysis. RESULTS: Approximately 51% of total bacteria in the wound exudate with identical colony morphotype were resistant to 100 microg/ml of ampicillin. A single colony from this population also demonstrated resistance to 50 microg/ml of kanamycin on kanamycin-supplemented agar. Further antimicrobial sensitivity testing by the Phoenix instrument indicated resistance to inhibitory concentrations of amoxicillin-clavulanate, ampicillin-sulbactam, cefazolin, gentamicin, nitrofurantoin, tobramycin, and trimethoprim-sulfamethoxazole. Biochemical and 16S rDNA analysis identified this bacterial isolate as a member of genus Enterobacter. A plasmid preparation from this isolate successfully transferred ampicillin and kanamycin resistance to E. coli competent cells. E. coli transformants displayed two resistance phenotypes and the plasmids from these transformants displayed two different restriction type patterns, with one correlating to ampicillin and kanamycin resistance and the other only to ampicillin resistance. CONCLUSION: A multiple antibiotic-resistant Enterobacter spp. from the wound fluid of a clinical sample was found to carry an antibiotic-resistant plasmid in a closely related species E. coli. The presence of antibiotic resistance plasmid in Enterobacteria that are part of the normal microbial flora of the human gut and skin could lead to the spread of resistance phenotype and emergence of antibiotic resistant pathogens. This study suggests normal human microbial fl ora could be a potential reservoir for resistance genes.

Dominant negative effects by inactive Spa47 mutants inhibit T3SS function and Shigella virulence.

Type three secretion systems (T3SS) are complex nano-machines that evolved to inject bacterial effector proteins directly into the cytoplasm of eukaryotic cells. Many high-priority human pathogens rely on one or more T3SSs to cause disease and evade host immune responses, underscoring the need to better understand the mechanisms through which T3SSs function and their role(s) in supporting pathogen virulence. We recently identified the Shigella protein Spa47 as an oligomerization-activated T3SS ATPase that fuels the T3SS and supports overall Shigella virulence. Here, we provide both in vitro and in vivo characterization of Spa47 oligomerization and activation in the presence and absence of engineered ATPase-inactive Spa47 mutants. The findings describe mechanistic details of Spa47-catalyzed ATP hydrolysis and uncover critical distinctions between oligomerization mechanisms capable of supporting ATP hydrolysis in vitro and those that support T3SS function in vivo. Concentration-dependent ATPase kinetics and experiments combining wild-type and engineered ATPase inactive Spa47 mutants found that monomeric Spa47 species isolated from recombinant preparations exhibit low-level ATPase activity by forming short-lived oligomers with active site contributions from at least two protomers. In contrast, isolated Spa47 oligomers exhibit enhanced ATP hydrolysis rates that likely result from multiple preformed active sites within the oligomeric complex, as is predicted to occur within the context of the type three secretion system injectisome. High-resolution fluorescence microscopy, T3SS activity, and virulence phenotype analyses of Shigella strains co-expressing wild-type Spa47 and the ATPase inactive Spa47 mutants demonstrate that the N-terminus of Spa47, not ATPase activity, is responsible for incorporation into the injectisome where the mutant strains exhibit a dominant negative effect on T3SS function and Shigella virulence. Together, the findings presented here help to close a significant gap in our understanding of how T3SS ATPases are activated and define restraints with respect to how ATP hydrolysis is ultimately coupled to T3SS function in vivo.

Phase II study of continuous daily sunitinib dosing in patients with previously treated advanced non-small cell lung cancer.

BACKGROUND: Sunitinib malate (SUTENT) has promising single-agent activity given on Schedule 4/2 (4 weeks on treatment followed by 2 weeks off treatment) in advanced non-small cell lung cancer (NSCLC). METHODS: We examined the activity of sunitinib on a continuous daily dosing (CDD) schedule in an open-label, multicentre phase II study in patients with previously treated, advanced NSCLC. Patients > or =18 years with stage IIIB/IV NSCLC after failure with platinum-based chemotherapy, received sunitinib 37.5 mg per day. The primary end point was objective response rate (ORR). Secondary end points included progression-free survival (PFS), overall survival (OS), 1-year survival rate, and safety. RESULTS: Of 47 patients receiving sunitinib, one patient achieved a confirmed partial response (ORR 2.1% (95% confidence interval (CI) 0.1, 11.3)) and 11 (23.4%) had stable disease (SD) > or =8 weeks. Five patients had SD>6 months. Median PFS was 11.9 weeks (95% CI 8.6, 14.1) and median OS was 37.1 weeks (95% CI 31.1, 69.7). The 1-year survival probability was 38.4% (95% CI 24.2, 52.5). Treatment was generally well tolerated. CONCLUSIONS: The safety profile and time-to-event analyses, albeit relatively low response rate of 2%, suggest single-agent sunitinib on a CDD schedule may be a potential therapeutic agent for patients with advanced, refractory NSCLC.

Agrin isoforms with distinct amino termini: differential expression, localization, and function.

The proteoglycan agrin is required for postsynaptic differentiation at the skeletal neuromuscular junction, but is also associated with basal laminae in numerous other tissues, and with the surfaces of some neurons. Little is known about its roles at sites other than the neuromuscular junction, or about how its expression and subcellular localization are regulated in any tissue. Here we demonstrate that the murine agrin gene generates two proteins with different NH(2) termini, and present evidence that these isoforms differ in subcellular localization, tissue distribution, and function. The two isoforms share approximately 1,900 amino acids (aa) of common sequence following unique NH(2) termini of 49 or 150 aa; we therefore call them short NH(2)-terminal (SN) and long NH(2)-terminal (LN) isoforms. In the mouse genome, LN-specific exons are upstream of an SN-specific exon, which is in turn upstream of common exons. LN-agrin is expressed in both neural and nonneural tissues. In spinal cord it is expressed in discrete subsets of cells, including motoneurons. In contrast, SN-agrin is selectively expressed in the nervous system but is widely distributed in many neuronal cell types. Both isoforms are externalized from cells but LN-agrin assembles into basal laminae whereas SN-agrin remains cell associated. Differential expression of the two isoforms appears to be transcriptionally regulated, whereas the unique SN and LN sequences direct their distinct subcellular localizations. Insertion of a "gene trap" construct into the mouse genome between the LN and SN exons abolished expression of LN-agrin with no detectable effect on expression levels of SN-agrin or on SN-agrin bioactivity in vitro. Agrin protein was absent from all basal laminae in mice lacking LN-agrin transcripts. The formation of the neuromuscular junctions was as drastically impaired in these mutants as in mice lacking all forms of agrin. Thus, basal lamina-associated LN-agrin is required for neuromuscular synaptogenesis, whereas cell-associated SN-agrin may play distinct roles in the central nervous system.

The synaptic protein syntaxin1 is required for cellularization of Drosophila embryos.

Syntaxins are membrane proteins involved in vesicle trafficking and are required for the release of neurotransmitter at nerve terminals. The presence of syntaxins on target membranes has been hypothesized to confer specificity to targeting and fusion via interactions with complementary vesicle-associated proteins, the synaptobrevins or VAMPS. We have mutagenized syntaxin1 in Drosophila and have found that it links the mechanism of synaptic transmission to a distinct cell biological process: the cellularization of early embryos. This specialized form of cell division separates the 6,000 nuclei of the syncytial blastoderm into separate cells through the invagination of the surface membrane of the embryo. During this process, syntaxin1 protein is present on the newly forming lateral cell surfaces and invaginating cleavage furrows. This protein is derived both from maternal deposition of mRNA and protein and from early zygotic transcription. To analyze syntaxin1's role in early development, female germ line mosaics mutant for syntaxin1 expression were generated by mitotic recombination to reduce the maternal contribution. Visualizing the actin cytoskeleton and glycosylated surface proteins reveals that embryos with insufficient syntaxin1 have large acellular patches. The patches do not appear until cellularization begins, and the process fails entirely within these regions. These results provide genetic evidence that membrane trafficking is required for the cellularization of the syncytial blastoderm. We propose that the invagination of the surface membrane proceeds by the fusion of intracellular membrane vesicles with the surface. This reaction uses the same syntaxin1 protein as is required for neurotransmitter secretion at synapses. Thus, a single syntaxin can participate in trafficking steps that are functionally as distinct as synaptic transmission and cell division.