Multifaceted roles of CCAR family proteins in the DNA damage response and cancer.

The cell cycle apoptosis regulator (CCAR) family of proteins consists of two proteins, CCAR1 and CCAR2, that play a variety of roles in cellular physiology and pathology. These multidomain proteins are able to perform multiple interactions and functions, playing roles in processes such as stress responses, metabolism, and the DNA damage response. The evolutionary conservation of CCAR family proteins allows their study in model organisms such as Caenorhabditis elegans, where a role for CCAR in aging was revealed. This review particularly highlights the multifaceted roles of CCAR family proteins and their implications in the DNA damage response and in cancer biology.

Exosome-mediated RNAi of PAK4 prolongs survival of pancreatic cancer mouse model after loco-regional treatment.

With a dismal survival rate, pancreatic cancer (PC) remains one of the most aggressive and devastating malignancies, predominantly due to the absence of a valid biomarker for diagnosis and limited therapeutic options for advanced diseases. Exosomes (Exo) as cell-derived vesicles, are widely used as natural nanocarriers for drug delivery. P21-activated kinase 4 (PAK4) is oncogenic when overexpressed, promoting cell survival, migration and anchorage-independent growth. Herein we validated PAK4 as a therapeutic target in an in vivo PC tumour mouse model using Exo-mediated RNAi following intra-tumoural administration. PC derived Exo were firstly isolated by ultracentrifugation on sucrose cushion and characterised for their surface marker expression, size, number, purity and morphology. SiRNA was encapsulated into Exo via electroporation and dual uptake of Exo and siRNA was investigated by flow cytometry and confocal microscopy. In vitro siPAK4 silencing in PC cells following uptake was assessed by flow cytometry, western blotting, and in vitro scratch assay. In vivo efficacy (tumour growth delay and mouse survival) of siPAK4 was evaluated in PC bearing NSG mouse model. Ex vivo tumours were examined using Haematoxylin and eosin (H&E) staining and immunohistochemistry. Results showed high quality PC-derived PANC-1 Exo were obtained. SiRNA was incorporated in Exo with 16.5% encapsulation efficiency. In vitro imaging confirmed Exo and siRNA co-localisation in cells. PAK4 knockdown was successful with 30 nM Exo-siPAK4 at 24 h post incubation in vitro. Intra-tumoural administration of Exo-siPAK4 (0.03 mg/kg siPAK4 and 6.1 × 1011 Exo, each dose, two doses) reduced PC tumour growth in vivo and enhanced mice survival (p < 0.001), with minimal toxicity observed compared to polyethylenimine (PEI) used as a commercial transfection reagent. H&E staining of tumours showed significant tissue apoptosis in siPAK4 treated groups. PAK4 knockdown prolongs survival of PC-bearing mice suggesting its potential as a new therapeutic target for PC. PANC-1 Exo demonstrated comparable efficacy but safer profile than PEI as in vivo RNAi transfection reagent.

Identification, pathogenicity and histopathology of Colletotrichum sansevieriae causing anthracnose of Sansevieria trifasciata in Malaysia.

AIMS: To characterize causal pathogen of Sansevieria trifasciata anthracnose through morphology and molecular analysis; to evaluate the host range of the pathogen; and to explicate the infection process by the pathogen histopathologically. METHODS AND RESULTS: Symptomatic leaves of S. trifasciata were collected from five states in Malaysia. The causal pathogen was isolated and identified for the first time in Malaysia as C. sansevieriae based on morphological and multi-gene phylogenetic analyses using ITS, TUB2 and GAPDH sequences. Pathogenicity tests were conducted on different hosts. Colletotrichum sansevieriae was not pathogenic towards S. cylindrica, S. masoniana, Furcraea foetida, Chlorophytum comosum, Aloe vera and Gasteria carinata, confirming the exceptionally high host specificity for a species of Colletotrichum. Histopathology was performed using light microscope and scanning electron microscopy to study the infection process of C. sansevieriae on S. trifasciata. Colonization of host leaves by the pathogen was observed 2 days after inoculation. CONCLUSIONS: Colletotrichum sansevieriae caused anthracnose of S. trifasciata in Malaysia. It is a host-specific pathogen and colonized the host intracellularly. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of C. sansevieriae causing anthracnose of S. trifasciata in Malaysia. The host range test and understanding of the infection process will provide better understanding of the host-pathogen relationship and beneficial for effective disease management.

Microscopic hematuria is a risk factor of incident chronic kidney disease in the Korean general population: a community-based prospective cohort study.

BACKGROUND: Although asymptomatic microscopic hematuria (MH) is a common finding in clinical practice, its long-term outcome remains unknown. AIM: This study evaluated the clinical implication of MH in the general population using a large-scale long-term longitudinal cohort database. METHODS: This study included 8719 participants from the Korean Genome and Epidemiology Study between 2001 and 2014. MH was defined as ≥5 red blood cells per high-power field in random urinalysis without evidence of pyuria. The primary study outcome measure was incident chronic kidney disease (CKD), defined as estimated glomerular filtration rate <60 ml min-1⋅1.73⋅m-2. RESULTS: During a median follow-up of 11.7 years, CKD occurred in 677 (7.8%) subjects. In Cox regression after adjustment for multiple confounders, subjects with MH had a significantly higher risk of incident CKD than those without [hazard ratio (HR) 1.45, 95% confidence interval (CI) 1.12-1.87; P = 0.005]. Isolated MH without proteinuria was also a risk factor of incident CKD (HR 1.37, 95% CI 1.04-1.79; P = 0.023) and the risk was further increased in MH with concomitant proteinuria (HR 5.41, 95% CI 2.54-11.49; P < 0.001). In propensity score matching analysis after excluding subjects with proteinuria, multi-variable stratified Cox regression analysis revealed that subjects with isolated MH had a significantly higher risk of incident CKD than those without (HR 1.83, 95% CI 1.14-2.94; P = 0.012). CONCLUSION: The presence of MH is associated with an increased risk of incident CKD in the general population. Therefore, attentive follow-up is warranted in persons with MH for early detection of CKD.

Management of paediatric high-grade blunt renal trauma: a 10-year single-centre UK experience.

OBJECTIVE: To report the management and outcome of paediatric patients sustaining high-grade blunt renal trauma. PATIENTS AND METHODS: Medical records were examined for all American Association for the Surgery of Trauma (AAST) grade III-V blunt renal trauma cases admitted to a paediatric trauma centre from 2005 to 2015. Data collected and analysed included: demographics, imaging modalities, management, length of hospital stay (LOS), complications, and follow-up outcomes. RESULTS: In all, 18 children (12 boys, six girls) with mean (range) age 11 (4-15) years were included. According to the AAST grading criteria, 39% (seven of 18) of the patients had grade III, 50% (nine of 18) grade IV, and 11% (two of 18) grade V injuries; 44% (eight of 18) had concomitant injuries. Most of the patients were managed conservatively (89%, 16 of 18), although two of the 16 subsequently needed JJ-stent insertion during inpatient stay for symptomatic urinoma(s). In all, 11% (two of 18) of the patients required interventional radiology service(s), involving selective embolisation for life-threatening renal tract haemorrhage. Blood transfusion for renal injury exclusively was required in 11% (two of 18) of the patients. In all, 89% (16 of 18) of the patients had at least one follow-up imaging study before hospital discharge; most (13 of 16) had ultrasonography and three required computed tomography. The median (range) LOS was 11 (4-31) days. In all, 17% (three of 18) of the patients required hospital re-admission within 30 days for complications and all required interventional procedures: JJ stent for urinoma (one), embolisation of renal arterio-venous fistula (one), and embolisation for a post-traumatic pseudoaneurysm (one). Overall, the median (range) follow-up was 6 (2-60) months. In all, 78% (14 of 18) of the patients had dimercaptosuccinic acid studies, with 11 showing reductions in renal function (range 3-44%). CONCLUSIONS: This study supports a care pathway strategy advocating conservative management of high-grade renal injuries in children. However, patients may experience a relative decline in renal function with higher grade injuries indicating the need for monitoring and follow-up.

Hydrodynamic profile of young swimmers: changes over a competitive season.

The aim of this study was to analyze the changes in the hydrodynamic profile of young swimmers over a competitive season and to compare the variations according to a well-designed training periodization. Twenty-five swimmers (13 boys and 12 girls) were evaluated in (a) October (M1); (b) March (M2); and (c) June (M3). Inertial and anthropometrical measures included body mass, swimmer's added water mass, height, and trunk transverse surface area. Swimming efficiency was estimated by the speed fluctuation, stroke index, and approximate entropy. Active drag was estimated with the velocity perturbation method and the passive drag with the gliding decay method. Hydrodynamic dimensionless numbers (Froude and Reynolds numbers) and hull velocity (i.e., speed at Froude number = 0.42) were also calculated. No variable presented a significant gender effect. Anthropometrics and inertial parameters plus dimensionless numbers increased over time. Swimming efficiency improved between M1 and M3. There was a trend for both passive and active drag increase from M1 to M2, but being lower at M3 than at M1. Intra-individual changes between evaluation moments suggest high between- and within-subject variations. Therefore, hydrodynamic changes over a season occur in a non-linear fashion way, where the interplay between growth and training periodization explain the unique path flow selected by each young swimmer.

Multi-length or 24 cm ureteric stent? A multicentre randomised comparison of stent-related symptoms using a validated questionnaire.

UNLABELLED: WHAT'S KNOWN ON THE SUBJECT? AND WHAT DOES THE STUDY ADD?: One of the suggested factors for stent-related symptoms is that excess distal intravesical stent mass may cause bladder irritation. There is a lack of studies investigating this in a randomised controlled fashion using a validated questionnaire. This study compared two of the most commonly used length of stents (a 30 cm multi-length vs a 24 cm long stent) and showed no significance difference in stent-related symptoms in patients with either of these stents. OBJECTIVE: To investigate whether excessive redundant intravesical stent component contributes to the severity of stent-related symptoms in patients with a ureteric stent. We compared stent-related symptoms in patients who had either a standard 24 cm or multi-length ureteric stent. PATIENTS AND METHODS: In all, 162 patients with upper urinary tract calculi requiring ureteric stent insertion were randomised to receive either a 6 F × 24 cm Contour(TM) or multi-length 6 F × 22-30 cm Contour VL(TM) stent. Patients were requested to complete the validated Bristol Ureteric Stent Symptom Questionnaire (USSQ) at 1 and 4 weeks after stent insertion and 4 weeks after removal. The mean scores for each domain of the USSQ for both groups were compared using the Student's t-test. Any adverse events, e.g. stent migration, early removal of stent due to stent-related symptoms and failure of stent insertion, were also recorded. RESULTS: In all, 153 patients who had successful stent insertion were requested to complete the USSQ and 74% of patients returned at least the week 1 questionnaire. At 1 and 4 weeks with the stent in situ, comparison of the mean scores showed no significant difference in urinary symptoms, pain, general health, work performance, sexual dysfunction and number of days patients stayed in bed or reduced their routine activities. Three (2%) patients had their stent removed early due to stent-related symptoms and five (3%) had failed stent insertion. CONCLUSIONS: This study did not find any difference in symptoms between the 24 cm or multi-length Contour stents. However, the study was not powered to detect small differences particularly for the pain symptom domain. Stents should only be used sparingly and the stent dwell-time should be minimised.

G-CSF induces a potentially tolerant gene and immunophenotype profile in T cells in vivo.

G-CSF can induce functional immune tolerance in man. In this study, purified T cells from G-CSF-mobilized peripheral blood stem cell (PBSC) donors were analysed by gene expression profiling and immunophenotyping. Results suggested a predominantly immune tolerant profile with upregulation of genes related to Th2 and Treg cells, downregulation of genes associated with Th1 cells, cytotoxicity, antigen presentation and graft-versus-host disease (GVHD) and overexpression of negative regulators of Th17 differentiation. Immunophenotyping revealed that during G-CSF exposure donors had reduced levels of T cells with a Th17 phenotype (CD4+IL-17A+CCR6+IL-23R+), more than three times lower compared to normal controls. G-CSF also led to increased levels of CD4+CD25highCD45RO+ Treg cells. Furthermore, mRNA levels of RORgammat, a Th17-specific transcription factor, decreased in T cells isolated from G-CSF-mobilized PBSC harvests. Th17 cells have been implicated in autoimmune diseases and GVHD pathophysiology. Our study is the first to report the effect of G-CSF on the Th17 subpopulation.

Do older patients receive adequate stroke care? An experience of a neurovascular clinic.

BACKGROUND: National guidelines and government directives have adopted policies for urgent assessment of patients with a transient ischaemic attack or minor stroke not admitted to hospital. The risk of recurrent stroke increases substantially with age, as does the potential benefit of secondary prevention. In order to develop effective strategies for older patients, it is important to identify how stroke care is currently provided for this patient group. METHODS: Between 2004 and 2006, older patients (>75 years) referred to a neurovascular clinic were compared with younger patients (< or =75 years). Sociodemographic details, clinical features, resource use and secondary prevention in a neurovascular clinic were collected. RESULTS: Of 379 patients referred to the clinic, 129 (34%) were given a non-stroke diagnosis. Of the remaining 250 patients, 149 (60%) were < or =75 years. Median time from symptom onset to clinic appointment was similar for the two groups (24 (IQR 15-42) vs 24 (IQR 14-43) days; p = 0.58). Older patients were more likely to be in atrial fibrillation (10.1% vs 22.8%, p<0.001) and have lacunar stroke (34.7% vs 22.1%; p = 0.04). CT rates were similar in the two groups (27.8% vs 80.0%, p = 0.75). Scans were performed more quickly in younger patients (p<0.01). MRI scan rates were higher in younger patients (26% vs 4%, p<0.01), as was carotid Doppler imaging (92% vs 77%, p<0.01). There were no differences in prescribed secondary preventive treatments. Older patients experienced less delay for carotid endarterectomy (49 vs 90 days, p<0.01). Younger patients were more likely to be given advice on weight reduction (30.2% vs 12.9%, p<0.01) and diet (46.3% vs 31.7%, p = 0.02) than older patients. CONCLUSIONS: Older patients were less likely to receive diagnostic investigations and lifestyle modification advice than younger patients. Guidelines need to be adopted to ensure prompt evidence-based stroke care in the outpatient setting.

Establishment of Withania somnifera hairy root cultures for the production of withanolide A.

Withania sominifera (Indian ginseng) was transformed by Agrobacterium rhizogenes. Explants from seedling roots, stems, hypocotyls, cotyledonary nodal segments, cotyledons and young leaves were inoculated with A. rhizogenes strain R1601. Hairy (transformed) roots were induced from cotyledons and leaf explants. The transgenic status of hairy roots was confirmed by polymerase chain reaction using nptII and rolB specific primers and, subsequently, by Southern analysis for the presence of nptII and rolB genes in the genomes of transformed roots. Four clones of hairy roots were established; these differed in their morphology. The doubling time of faster growing cultures was 8-14 d with a fivefold increase in biomass after 28 d compared with cultured, non-transformed seedling roots. MS-based liquid medium was superior for the growth of transformed roots compared with other culture media evaluated (SH, LS and N6), with MS-based medium supplemented with 40 g/L sucrose being optimal for biomass production. Cultured hairy roots synthesized withanolide A, a steroidal lactone of medicinal and therapeutic value. The concentration of withanolide A in transformed roots (157.4 microg/g dry weight) was 2.7-fold more than in non-transformed cultured roots (57.9 microg/g dry weight).

A novel class of phosphonate nucleosides. 9-[(1-phosphonomethoxycyclopropyl)methyl]guanine as a potent and selective anti-HBV agent.

9-[1-(Phosphonomethoxycyclopropyl)methyl]guanine (PMCG, 1), representative of a novel class of phosphonate nucleosides, blocks HBV replication with excellent potency (EC(50) = 0.5 microM) in a primary culture of HepG2 2.2.15 cells. It exhibits no significant cytotoxicity in several human cell lines up to 1.0 mM. It does not inhibit replication of human immunodeficiency virus (HIV-1) or herpes simplex virus (HSV-1) at 30 microM. Many purine base analogues of 1 also exhibit inhibitory activity against HBV, but at 30 microM, pyrimidine analogues do not. 1 is 4 times more potent than 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA), which was used as a positive control (EC(50) = 2.0 microM). The characteristic cyclopropyl moiety at the 2'-position of 1 was prepared by titanium-mediated Kulinkovich cyclopropanation. 1 was modified to give the orally available drug candidate, PMCDG Dipivoxil (2). Compound 2 exhibited excellent efficacy when administered at 5 mg per kg per day in a study with woodchucks infected with woodchuck hepatitis B virus (WHBV). Drug candidate 2 has successfully completed phase I clinical trials and is currently undergoing phase II clinical studies for evaluation of efficacy.

Particle phase acidity and oligomer formation in secondary organic aerosol.

A series of controlled laboratory experiments are carried out in dual Teflon chambers to examine the presence of oligomers in secondary organic aerosols (SOA) from hydrocarbon ozonolysis as well as to explore the effect of particle phase acidity on SOA formation. In all seven hydrocarbon systems studied (i.e., alpha-pinene, cyclohexene, 1-methyl cyclopentene, cycloheptene, 1-methyl cyclohexene, cyclooctene, and terpinolene), oligomers with MW from 250 to 1600 are present in the SOA formed, both in the absence and presence of seed particles and regardless of the seed particle acidity. These oligomers are comparable to, and in some cases, exceed the low molecular weight species (MW < 250) in ion intensities in the ion trap mass spectra, suggesting they may comprise a substantial fraction of the total aerosol mass. It is possible that oligomers are widely present in atmospheric organic aerosols, formed through acid- or base-catalyzed heterogeneous reactions. In addition, as the seed particle acidity increases, larger oligomers are formed more abundantly in the SOA; consequently, the overall SOA yield also increases. This explicit effect of particle phase acidity on the composition and yield of SOA may have important climatic consequences and need to be considered in relevant models.

P38 MAPK pathway is involved in the urokinase plasminogen activator expression in human gastric SNU-638 cells.

Overexpression of urokinase plasminogen activator (uPA) is known to correlate closely with tumor cell invasion and metastasis. In gastric cancer, however, the mechanism for induction of uPA remains to be elucidated. In this study, we investigated the intracellular signaling for uPA expression in human gastric carcinoma cells (AGS, SNU-1, SNU-5, and SNU-638). SNU-638 cells which expressed a high level of uPA was found to be highly invasive on a matrigel, while AGS, SNU-1, and SNU-5 cells with low levels of uPA expression were only slightly invasive. SNU-638 cells showed a much higher P38 MAPK activity than the 3 other cell lines. However, there was no significant difference in the activities of P44/42 MAPK (Erk-1/2), JNK and Akt among the above cell lines. Treatment of SNU-638 cells with SB203580, a specific P38 MAPK inhibitor, reduced both the promoter activity and mRNA expression of uPA. Expression of a vector encoding a mutated-type P38alpha MAPK resulted in decrease in the uPA promoter activity in SNU-638 cells. These results suggest that P38 MAPK signaling pathway is important for uPA expression in gastric SNU-638 cells by enhancing the promoter activity of uPA.

Outcomes of primary image-guided drainage of parapneumonic effusions in children.

OBJECTIVE: To assess the outcome of image-guided needle aspiration when compared with image-guided percutaneous catheter drainage in the management of parapneumonic effusions in children. METHODS: A retrospective chart review was conducted of the medical records, microbiology, and radiology reports of 67 children who presented with parapneumonic effusions and underwent primary image-guided drainage between April 1, 1995, and April 1, 2000. RESULTS: Thirty-four patients had aspiration only, and 33 patients had pigtail catheters placed. The 2 drainage methods had similar median length of stay and complication rates. The reintervention rate in this study was 27% (18 patients). Children who underwent primary aspiration without catheter placement had significantly higher rates of reintervention. Method of drainage, pH lower than 7.2, and loculation of the fluid collection were independent predictors of reintervention. A low glucose level was an additive predictor of reintervention when the pH was low. CONCLUSIONS: Aspiration and catheter drainage of parapneumonic effusions had similar complication rates and lengths of stay, but children who underwent primary aspiration had significantly higher reintervention rates, particularly when pH and glucose levels were low. Therefore, primary catheter placement for parapneumonic effusions should be considered in children who undergo diagnostic thoracentesis. The decision regarding tube placement could be facilitated by the on-site availability of a pH meter and a glucometer.

The transcriptional regulator Id3 is expressed in cranial sensory placodes during early avian embryonic development.

The chick homologue of the helix-loop-helix gene Id3 was isolated, and its expression pattern was analyzed during early stages of chick development. Chick Id3 is dynamically expressed in the olfactory, lens, and otic placodes. It is also observed in the epiphysis, nephric primordium, stomodeum, dermomyotome, distal branchial arches, dorsolateral hindbrain, foregut endoderm, dorsal spinal cord, and somites.

Id4 expression and its relationship to other Id genes during avian embryonic development.

We present the sequence and expression pattern of chick Id4 and compare its distribution to that of other vertebrate Id genes. At early stages, Id4 expression is discrete, with transcript transiently expressed in subsets of migrating neural crest cells, the dorsal myocardium, the segmental plate mesoderm, and the tail bud. Later, expression is also observed in the telencephalic vesicles and corneal epithelium. Of all the Id genes, Id4 exhibits the most restricted pattern in the developing nervous system, with little expression in the presumptive neural crest or placodes. Id4 appears in the neural tube much later than other Id genes. However, all four Id genes display overlapping patterns in the branchial arches and tail bud.

Temporally and spatially restricted expression of the helix-loop-helix transcriptional regulator Id1 during avian embryogenesis.

We isolated the chick orthologue of the Id1 helix-loop-helix gene and analyzed its expression pattern during early chick embryo development by whole-mount in situ hybridization. The Id1 expression pattern is dynamic and confined to discrete locations including the neural plate border, prospective olfactory placode, hindbrain, mesenchyme of distal branchial arches and adjacent to placodes, and the distal mesoderm of the limb buds.

The sec6/8 complex is located at neurite outgrowth and axonal synapse-assembly domains.

The molecules that specify domains on the neuronal plasma membrane for the delivery and accumulation of vesicles during neurite outgrowth and synapse formation are unknown. We investigated the role of the sec6/8 complex, a set of proteins that specifies vesicle targeting sites in yeast and epithelial cells, in neuronal membrane trafficking. This complex was found in layers of developing rat brain undergoing synaptogenesis. In cultured hippocampal neurons, the sec6/8 complex was present in regions of ongoing membrane addition: the tips of growing neurites, filopodia, and growth cones. In young axons, the sec6/8 complex was also confined to periodic domains of the plasma membrane. The distribution of synaptotagmin, synapsin1, sec6, and FM1-43 labeling in cultured neurons suggested that the plasma membrane localization of the sec6/8 complex preceded the arrival of synaptic markers and was downregulated in mature synapses. We propose that the sec6/8 complex specifies sites for targeting vesicles at domains of neurite outgrowth and potential active zones during synaptogenesis.

Subunit structure of the mammalian exocyst complex.

The exocyst is a protein complex required for the late stages of secretion in yeast. Unlike the SNAREs (SNAP receptors), important secretory proteins that are broadly distributed on the target membrane, the exocyst is specifically located at sites of vesicle fusion. We have isolated cDNAs encoding the rexo70, rsec5, and rsec15 subunits of the mammalian complex. The amino acid sequences encoded by these genes are between 21% and 24% identical to their yeast homologs. All three genes are broadly expressed and multiple transcripts are observed for rexo70 and rsec15. Characterization of cDNAs encoding the 84-kDa subunit of the mammalian complex revealed a novel protein. mAbs were generated to the mammalian rsec6 subunit of the exocyst complex. rsec6 immunoreactivity is found in a punctate distribution at terminals of PC12 cell processes at or near sites of granule exocytosis.