Reduced expression of neurofibromin in the soft tissue tumours obtained from patients with neurofibromatosis type I.
1. We analysed the expression of neurofibromin mRNAs, encoded by the gene responsible for neurofibromatosis type 1, and of neurofibromin protein in nine soft tissue tumours by S1 nuclease mapping and Western blot analyses. Four tumours were obtained from patients with neurofibromatosis type 1, comprising two neurofibromas, one fibrolipoma and one malignant schwannoma, and five neurogenic tumours were obtained from non-neurofibromatosis type 1 patients. 2. All tumours, except for a malignant schwannoma, similarly expressed three species of mRNA encoding neurofibromin, an isoform with the insertion of 21 amino acids in the domain related to ras GTPase-activating protein, and an N-terminal isoform lacking this domain. 3. Western blot analysis demonstrated deficiency of neurofibromin in the tumours derived from three out of the four neurofibromatosis type 1 patients: a fibrolipoma, a malignant schwannoma and a neurofibroma. In contrast, reduction in neurofibromin was not detected in the five tumours obtained from non-neurofibromatosis type 1 patients. Furthermore, the expression of ras GTPase-activating protein was detected in all nine tumours examined. 4. The undetectable or reduced level of neurofibromin in the tumours obtained from neurofibromatosis type 1 patients suggests that this deficiency is closely related to their tumourigenesis.


Primitive chain network simulations of probe rheology.
Probe rheology experiments, in which the dynamics of a small amount of probe chains dissolved in immobile matrix chains is discussed, have been performed for the development of molecular theories for entangled polymer dynamics. Although probe chain dynamics in probe rheology is considered hypothetically as single chain dynamics in fixed tube-shaped confinement, it has not been fully elucidated. For instance, the end-to-end relaxation of probe chains is slower than that for monodisperse melts, unlike the conventional molecular theories. In this study, the viscoelastic and dielectric relaxations of probe chains were calculated by primitive chain network simulations. The simulations semi-quantitatively reproduced the dielectric relaxation, which reflects the effect of constraint release on the end-to-end relaxation. Fair agreement was also obtained for the viscoelastic relaxation time. However, the viscoelastic relaxation intensity was underestimated, possibly due to some flaws in the model for the inter-chain cross-correlations between probe and matrix chains.


Primary Membranous Glomerulonephritis: The Role of Serum and Urine Biomarkers in Patient Management.
The detection of phospholipase A2 receptor (PLA2R) and thrombospondin domain containing 7A THSD7A among primary membranous glomerulonephritis (MGN) patients transformed the diagnosis, treatment monitoring, and prognosis. Anti-PLA2R can be detected in 70-90% of primary MGN patients while anti-THSD7A in 2-3% of anti-PLA2R negative primary MGN patients depending on the technique used. Serum and urine samples are less invasive and non-invasive, respectively, and thus can detect the presence of anti-PLA2R and anti-THSD7A with higher sensitivity and specificity, which is significant in patient monitoring and prognosis. It is better than exposing patients to a frequent biopsy, which is an invasive procedure. Different techniques of detection of PLA2R and THSD7A in patients' urine and sera were reviewed to provide newer and alternative techniques. We proposed the use of biomarkers (PLA2R and THSD7A) in the diagnosis, treatment decision, and follow-up of patients with primary MGN. In addition, other prognostic renal biomarkers like retinol binding protein (RBP) and beta-2 microglobulin were reviewed to detect the progression of renal damage for early intervention.


Placenta-derived exosomes and syncytiotrophoblast microparticles and their role in human reproduction: immune modulation for pregnancy success.
The syncytiotrophoblast (STB) of human placenta constitutively produces and secretes extracellular vesicles of different size, morphology and function that enter the maternal circulation, and participate in the maternal-fetal cross-talk during pregnancy. Syncytiotrophoblast-derived microvesicles/microparticles (STBM) are larger microvesicles (0.2-2 μm) shed by the apical plasma membrane of the STB as a result of cell activation and turnover. Simultaneously with the STBM shedding, the STB produces and secretes exosomes--nanosized (30-100/150 nm) membrane-bound microvesicles that originate from the endosomal compartment. They convey cell-cell contact 'by proxy' transporting signals/packages of information between donor and recipient cells locally or/and at a distance. STBM and exosomes, delivered directly in the maternal blood surrounding the chorionic villi of the placenta, have contrasting biological functions. While the exosomes are immunosuppressive down regulating maternal immunity in pluripotent ways, the main effects of STBM on the maternal immune system are pro-inflammatory, immune activating, and pro-coagulant. Since both STBM and exosomes are present in the maternal circulation throughout normal pregnancy logical questions are what is the net effect of these vesicles on the maternal immune system and is this effect beneficial or detrimental to pregnancy. In this review, the current knowledge about placenta-derived extracellular vesicles with a main focus on exosomes is summarized and discussed. In a concluding remark, a hypothetical proposal on how STBM and exosomes might interact in pregnancy is discussed and a way to evaluate this interaction is suggested.


Electrolyte tuning of surfactant interfacial behavior for enhanced density-based separations of single-walled carbon nanotubes.
We study the interfacial behavior between the straight-chain alkyl surfactant sodium dodecyl sulfate (SDS) and single-walled carbon nanotubes (SWNTs) as a function of added electrolytes, including NaCl. We observe an increase in photoluminescence intensity and narrowing of spectral line widths with electrolyte addition, indicating a change in SDS aggregation number that leads to a pronounced volume change in the nanotube/SDS composite structure. By tuning the interfacial dynamics through NaCl addition and temperature change, we demonstrate that this volume change can be used to yield diameter-dependent separation of metallic and semiconducting SWNTs, without the use of any additional cosurfactant, through density gradient ultracentrifugation. The diameter-dependent fractionation follows the intrinsic relation expected for the density of unfunctionalized nanotubes, indicating a simple amplification of these inherent density differences as the mechanism for salt enhanced separations. Isolation of enriched metallic and semiconducting fractions further illustrates that the surface aggregation characteristics of SDS on metallic SWNTs are different from that on the semiconducting chiralities. These experiments illustrate the governing behavior of surface phenomena and interfacial forces on the diameter-dependent fractionation of SWNTs and point to new routes for enhancing existing separations strategies.


T cell responses to an HLA-A2-restricted adipophilin peptide correlate with BMI in patients with atherosclerosis.
Atherosclerosis is an inflammatory disease causing a vast array of cardiovascular diseases. Adipophilin has been reported to be highly expressed in atherosclerotic lesions. This study investigated the possible existence of auto-reactive T cells against an HLA-A02-restricted adipophilin-derived peptide as well as peptides from Epstein-barr virus (EBV), Cytomegalovirus (CMV) and influenza (Flu) virus in patients with atherosclerosis. HLA-A02 expression on peripheral blood mononuclear cells (PBMCs) was examined by flow cytometry. PBMCs from HLA-A02 individuals were stimulated with adipophilin, CMV, EBV, and Flu peptides at a concentration of 10 µM. Interferon (IFN)-γ production was evaluated in the culture supernatant using a commercial ELISA test. The levels of IFN-γ production against an HLA-A02-restricted adipophilin peptide and peptides from CMV, EBV, and Flu revealed no statistically significant differences between patients and healthy controls. However, we found a positive correlation between IFN-γ production against adipophilin and Body mass index (BMI) of patients (R = 0.8, P = 0.003), whereas no significant correlation was found in healthy controls (R = -0.267, P = 0.378). No correlation between BMI and IFN-γ production against CMV, EBV, or Flu peptides was found. Atherosclerotic patients with higher BMIs might have greater numbers of T cells against adipophilin that is highly expressed in atherosclerotic plaques. Therefore, autoimmune reactions may have a greater role in the development of atherosclerosis in individuals with higher BMI.


Doppler ultrasound and tibial tuberosity maturation status predicts pain in adolescent male athletes with Osgood-Schlatter's disease: a case series with comparison group and clinical interpretation.
The pathogenesis of the Osgood-Schlatter's disease (OSD) is still debated. The fragmentation of the ossification centre has been questioned as a definitive sign of OSD and has been seen as a normal development of the anterior tibial tubercle (ATT). It is unknown if such changes are present in the presumed pathological tendon insertion seen in OSD, nor the relation of Doppler-positive changes to pain on clinical examination. A prospective analysis was carried out on 20 consecutive symptomatic male athletes (13.9 years±1.3) and a comparison group of asymptomatic subjects. All underwent a comparative clinical assessment and ultrasound with colour Doppler scan on both knees. Subjective pain was recorded with a visual analogue scale (VAS) during provocative manoeuvres: palpation, resisted contraction and single leg squat. Positive Doppler US (within the distal end of the patellar tendon) was associated with higher pain on palpation (47±24.5 vs 18±11.4, p<0.01) and resisted static contraction (59±20.2 vs 27±12.5, p<0.001) compared with Doppler-negative subjects. No Doppler activity was found in the comparison group. VAS for palpation and resisted contraction of the athletes graded as stage 2 (51.1±22.0 and 60.0±21.2) were significantly higher than stage 3 (17.8±12.0 and 18.9±16.9) and stage 4 (15.0±7.1 and 25.0±7.1; p<0.01). More painful OSD is associated with the presence of neo-vessels. This may be linked with a particular stage of ATT maturation and applied compressive forces. A Doppler ultrasound scan adds practical information to develop the care plan of the patient.


Growth factor-activated stem cell circuits and stromal signals cooperatively accelerate non-integrated iPSC reprogramming of human myeloid progenitors.
Nonviral conversion of skin or blood cells into clinically useful human induced pluripotent stem cells (hiPSC) occurs in only rare fractions (~0.001%-0.5%) of donor cells transfected with non-integrating reprogramming factors. Pluripotency induction of developmentally immature stem-progenitors is generally more efficient than differentiated somatic cell targets. However, the nature of augmented progenitor reprogramming remains obscure, and its potential has not been fully explored for improving the extremely slow pace of non-integrated reprogramming. Here, we report highly optimized four-factor reprogramming of lineage-committed cord blood (CB) myeloid progenitors with bulk efficiencies of ~50% in purified episome-expressing cells. Lineage-committed CD33(+)CD45(+)CD34(-) myeloid cells and not primitive hematopoietic stem-progenitors were the main targets of a rapid and nearly complete non-integrated reprogramming. The efficient conversion of mature myeloid populations into NANOG(+)TRA-1-81(+) hiPSC was mediated by synergies between hematopoietic growth factor (GF), stromal activation signals, and episomal Yamanaka factor expression. Using a modular bioinformatics approach, we demonstrated that efficient myeloid reprogramming correlated not to increased proliferation or endogenous Core factor expressions, but to poised expression of GF-activated transcriptional circuits that commonly regulate plasticity in both hematopoietic progenitors and embryonic stem cells (ESC). Factor-driven conversion of myeloid progenitors to a high-fidelity pluripotent state was further accelerated by soluble and contact-dependent stromal signals that included an implied and unexpected role for Toll receptor-NFκB signaling. These data provide a paradigm for understanding the augmented reprogramming capacity of somatic progenitors, and reveal that efficient induced pluripotency in other cell types may also require extrinsic activation of a molecular framework that commonly regulates self-renewal and differentiation in both hematopoietic progenitors and ESC.


Prediction of symptomatic improvement after exposure-based treatment for irritable bowel syndrome.
Several studies show that psychological treatments relieve symptoms for patients suffering from irritable bowel syndrome (IBS). However, there are no consistent findings that show what patient characteristics make a psychological treatment more or less likely to result in improvement. We have previously conducted a study of a newly developed internet-delivered cognitive behavioral therapy (ICBT) that emphasized exposure to IBS symptoms and IBS-related situations and reduced symptom-related avoidance. The study showed that the treatment led to improvement in IBS symptoms compared to a waiting list and that treatment gains were maintained over a 15-18 month follow-up period. The aim of the present study was to investigate several possible predictors of short- and long-term treatment outcome in terms of symptom improvement, based on data collected in the previously conducted treatment trial. Demographics, comorbid psychological distress, IBS-related fear and avoidance behaviors, and IBS-related disability were investigated as predictors of treatment outcome in the sample consisting of 79 participants diagnosed with IBS who had undergone 10 weeks of ICBT. Predictors that were significantly correlated with symptom levels at post-treatment and follow-up were entered into multiple regression analyses that controlled for pre-treatment symptom levels. There were measures within each domain, i.e., comorbid psychological distress, IBS-related fear and avoidance behaviors, and IBS-related disability, with the exception of demographic data, that were correlated with the symptom levels at post-treatment and follow-up. However, when these were entered into a multiple regression analyses that controlled for pre-treatment levels, none remained a significant predictor of the post-treatment and follow-up symptomatic status. The study did not find any individual characteristics that made patients more or less likely to respond to the exposure-based ICBT. The finding that comorbid psychological distress did not predict outcome is in accordance with previous studies. Reliable predictors for response to any type of psychological treatment for IBS remain to be established.


Fatal Candida septic shock during systemic chemotherapy in lung cancer patient receiving corticosteroid replacement therapy for hypopituitarism: a case report.
Invasive candidiasis has increased as nosocomial infection recently in cancer patients who receive systemic chemotherapy, and the timely risk assessment for developing such specific infection is crucial. Especially in those concomitantly with hypopituitarism, febrile neutropenia with candidiasis can cause severe stress and lead potentially to sudden fatal outcome when the temporal steroid coverage for the adrenal insufficiency is not fully administered. We report a 72-year-old male case diagnosed as non-small-cell lung cancer, Stage IIIA. He had received a steroid replacement therapy for the prior history of hypophysectomy due to pituitary adenoma with hydrocortisone of 3.3 mg/day, equivalent to prednisolone of 0.8 mg/day. This very small dosage of steroid was hardly supposed to weaken his immune system, but rather potentially led to an inappropriate supplementation of his adrenal function, assuming that the serum sodium and chlorine levels decreased. On Day 6 of second cycle of chemotherapy with carboplatin and paclitaxel, he developed sudden febrile neutropenia, septic shock and ileus, leading to death. After his death, the venous blood culture on Day 7 detected Candida albicans. Autopsy findings showed a massive necrotizing enterocolitis with extensive Candida invasion into submucous tissue. In conclusion, this case may suggest that (i) immediate initiation of antifungal therapy soon after the careful risk assessment of Candida infection and (ii) adequate administration of both basal steroid replacement therapy and temporal steroid coverage for febrile neutropenia might have improved his fatal outcome.


Structure and heterogeneity of the a sequences of human herpesvirus 6 strain variants U1102 and Z29 and identification of human telomeric repeat sequences at the genomic termini.
The unit-length genome of human herpesvirus 6 (HHV-6) consists of a single unique component (U) bounded by direct repeats DRL and DRR and forms head-to-tail concatemers during productive infection. cis-elements which mediate cleavage and packaging of progeny virions (a sequences) are found at the termini of all herpesvirus genomes. In HHV-6, DRL and DRR are identical and a sequences may therefore also occur at the U-DR junctions to give the arrangement aDRLa-U-aDRRa. We have sequenced the genomic termini, the U-DRR junction, and the DRR.DRL junction of HHV-6 strain variants U1102 and Z29. A (GGGTTA)n motif identical to the human telomeric repeat sequence (TRS) was found adjacent to, but did not form, the termini of both strain variants. The DRL terminus and U-DRR junction contained sequences closely related to that of the well-conserved herpesvirus packaging signal Cn-Gn-Nn-Gn (pac-1), followed by tandem arrays of TRSs separated by single copies of a hexanucleotide repeat. HHV-6 strain U1102 contained repeat sequences not found in HHV-6 Z29. In contrast, the DRR terminus of both variants contained a simple tandem array of TRSs and a close homolog of a herpesvirus pac-2 signal (GCn-Tn-GCn). The DRR.DRL junction was formed by simple head-to-tail linkage of the termini, yielding an intact cleavage signal, pac-2.x.pac-1, where x is the putative cleavage site. The left end of DR was the site of intrastrain size heterogeneity which mapped to the putative a sequences. These findings suggest that TRSs form part of the a sequence of HHV-6 and that the arrangement of a sequences in the genome can be represented as aDRLa-U-a-DRRa.


Cost-effectiveness of an advance notification letter to increase colorectal cancer screening.
The aim of this study is to evaluate the cost-effectiveness of a patient-direct mailed advance notification letter on participants of a National Bowel Cancer Screening Program (NBCSP) in Australia, which was launched in August 2006 and offers free fecal occult blood testing to all Australians turning 50, 55, or 65 years of age in any given year. This study followed a hypothetical cohort of 50-year-old, 55-year-old, and 65-year-old patients undergoing fecal occult blood test (FOBT) screening through a decision analytic Markov model. The intervention compared two strategies: (i) advance letter, NBCSP, and FOBT compared with (ii) NBCSP and FOBT. The main outcome measures were life-years gained (LYG), quality-adjusted life-years (QALYs) gained and incremental cost-effectiveness ratio. An advance notification screening letter would yield an additional 54 per 100,000 colorectal cancer deaths avoided compared with no letter. The estimated cost-effectiveness was $3,976 per LYG and $6,976 per QALY gained. An advance notification letter in the NBCSP may have a significant impact on LYG and cancer deaths avoided. It is cost-effective and offers a feasible strategy that could be rolled out across other screening program at an acceptable cost.


Blockade of mitogen-activated protein kinase cascade signaling in interleukin 6-independent multiple myeloma cells.
Interleukin 6 (IL-6) is a growth factor for multiple myeloma (MM) cells, yet not all MM cell lines or patient cells require IL-6 for their growth. It is well known that IL-6 activates the signal transducers and activators of transcription (stat) 1-stat3 heterodimer, stat3 homodimer, and Ras-dependent mitogen-activated protein kinase (MAPK) cascades in multiple cell systems. We have shown previously that the MAPK pathway is an important pathway for IL-6-mediated MM cell growth. In this study, we delineate the pattern of upstream MAPK cascade activation in IL-6-responsive B9 cells and in IL-6-nonresponsive U266, OCI-My5, and RPMI8226 MM cells to define sites of blockade of this pathway associated with loss of responsiveness to IL-6. In B9 cells, IL-6 triggered the following in sequence: gp130 phosphorylation, gp130-to-protein tyrosine phosphatase 1D (PTP1D) binding, PTP1D phosphorylation, PTP1D complex formation with Grb2-Son of sevenless 1 (Sos1), and Sos1 phosphorylation. gp130 phosphorylation, gp130-to-PTP1D binding, PTP1D phosphorylation, and PTP1D-to-Grb2 binding are also induced by IL-6 in all IL-6-independent MM cell lines studied. However, Grb2 is not associated with Sos1, and neither Grb2-to-Sos1 binding nor Sos1 phosphorylation is triggered by IL-6 in OCI-My5 MM cells. On the other hand, Grb2 and Sos1 are associated constitutively in U266 and RPMI8226 MM cells, but phosphorylation of Sos1 is not induced by IL-6. These data suggest that lack of Sos1 activation is associated with loss of IL-6 responsiveness in MM cell lines that grow independently of IL-6.


Whole-Exome Sequencing Analyses of Inflammatory Bowel Disease-Associated Colorectal Cancers.
A long duration of inflammatory bowel disease (IBD) increases the risk for colorectal cancer. Mutation analysis of limited numbers of genes has indicated that colorectal tumors that develop in patients with IBD differ from those of patients without IBD. We performed whole-exome sequencing analyses to characterize the genetic landscape of these tumors. We collected colorectal tumor and non-neoplastic tissues from 31 patients with IBD and colorectal cancer (15 with ulcerative colitis, 14 with Crohn's disease, and 2 with indeterminate colitis) and performed whole-exome sequencing analyses of the microdissected tumor and matched nontumor tissues. We identified somatic alterations by comparing matched specimens. The prevalence of mutations in sporadic colorectal tumors was obtained from previously published exome-sequencing studies. Two specimens had somatic mutations in the DNA proofreading or mismatch repair genes POLE, MLH1, and MSH6 and the tumor cells had a hypermutable phenotype. The remaining tumors had, on average, 71 alterations per sample. TP53 was the most commonly mutated gene, with prevalence similar to that of sporadic colorectal tumors (63% of cases). However, tumors from the patients with IBD had a different mutation spectrum. APC and KRAS were mutated at significantly lower rates in tumors from patients with IBD than in sporadic colorectal tumors (13% and 20% of cases, respectively). Several genes were mutated more frequently or uniquely in tumors from patients with IBD, including SOX9 and EP300 (which encode proteins in the WNT pathway), NRG1 (which encodes an ERBB ligand), and IL16 (which encodes a cytokine). Our study also revealed recurrent mutations in components of the Rho and Rac GTPase network, indicating a role for noncanonical WNT signaling in development of colorectal tumors in patients with IBD. Colorectal tumors that develop in patients with IBD have distinct genetic features from sporadic colorectal tumors. These findings could be used to develop disease-specific markers for diagnosis and treatment of patients with IBD and colorectal cancer.


Crystal structures of Giardia lamblia guanine phosphoribosyltransferase at 1.75 A(,).
Giardia lamblia, the protozoan parasite responsible for giardiasis, requires purine salvage from its host for RNA and DNA synthesis. G. lamblia expresses an unusual purine phosphoribosyltransferase with a high specificity for guanine (GPRTase). The enzyme's sequence significantly diverges from those of related enzymes in other organisms. The transition state analogue immucillinGP is a powerful inhibitor of HGXPRTase from malaria [Li, C. M., et al. (1999) Nat. Struct. Biol. 6, 582-587] and is also a 10 nM inhibitor of G. lamblia GPRTase. Cocrystallization of GPRTase with immucillinGP led unexpectedly to a GPRTase.immucillinG binary complex with an open catalytic site loop. Diffusion of ligands into preformed crystals gave a GPRTase.immucillinGP.Mg(2+).pyrophosphate complex in which the open loop is stabilized by crystal contacts. G. lamblia GPRTase exhibits substantial structural differences from known purine phosphoribosyltransferases at positions remote from the catalytic site, but conserves most contacts to the bound inhibitor. The filled catalytic site with an open catalytic loop provides insight into ligand binding. One active site Mg(2+) ion is chelated to pyrophosphate, but the other is chelated to two conserved catalytic site carboxylates, suggesting a role for these amino acids. This arrangement of Mg(2+) and pyrophosphate has not been reported in purine phosphoribosyltransferases. ImmucillinG in the binary complex is anchored by its 9-deazaguanine group, and the iminoribitol is disordered. No Mg(2+) or pyrophosphate is detected; thus, the 5'-phosphoryl group is needed to immobilize the iminoribitol prior to magnesium pyrophosphate binding. Filling the catalytic site involves (1) binding the purine ring, (2) anchoring the 5'-phosphate to fix the ribosyl group, (3) binding the first Mg(2+) to Asp125 and Glu126 carboxyl groups and binding Mg(2+).pyrophosphate, and (4) closing the catalytic site loop and formation of bound (Mg(2+))(2). pyrophosphate prior to catalysis. Guanine specificity is provided by two peptide carbonyl oxygens hydrogen-bonded to the exocyclic amino group and a weak interaction to O6. Transition state formation involves N7 protonation by Asp129 acting as the general acid.


Systemic distribution of blood flow during T-2 toxin induced shock in swine.
Three groups of swine (6 per group) were used to determine hemodynamic and blood flow alterations induced by T-2 toxin. Two groups were dosed at 0.6 or 2.4 mg/kg T-2 toxin, and one group served as a vehicle control (70% ethanol). Organ blood flow was determined at 0 hr (predosing) and at 90-min intervals for 6 hr postdosing using 15-micron diameter radionuclide labeled microspheres injected into the left atrium. Hemodynamic parameters were obtained at the same time points. The infusion of T-2 toxin resulted in reductions in cardiac output. This trend appeared to reverse itself in the low dose animals after 3 hr, whereas in the high dose group, cardiac output continued to decline. Mean aortic pressure (MAP) declined in a dose dependent fashion which tended to parallel the reduction observed in cardiac output. Heart rate was increased in both groups treated with T-2 toxin. Blood flow, to the brain, heart, and kidneys decreased following exposure to the toxin. The relative percentage of cardiac output received by these organs, however, was maintained despite the drop in blood flow. Pancreatic and splenic blood flows were the most severely compromised as a result of T-2 toxicosis. Consequently, the percentage of cardiac output going to the pancreas and spleen was dramatically reduced. Adrenal, hepatic, and total gastrointestinal blood flows increased or did not change from control values. As a result, the percentage of cardiac output supplying these organs increased.


Surgical Site Infection Reduction by the Solutions for Patient Safety Hospital Engagement Network.
Surgical site infections (SSIs) negatively affect patients and the health care system. National standards for SSI prevention do not exist in pediatric settings. We sought to reduce SSI-related harm by implementing a prevention bundle through the Solutions for Patient Safety (SPS) national hospital engagement network. Our study period was January 2011 to December 2013. We formed a national workgroup of content and quality improvement experts. We focused on 3 procedure types at high risk for SSIs: cardiothoracic, neurosurgical shunt, and spinal fusion surgeries. We used the Model for Improvement methodology and the Centers for Disease Control and Prevention SSI definition. After literature review and consultation with experts, we distributed a recommended bundle among network partners. Institutions were permitted to adopt all or part of the bundle and reported local bundle adherence and SSI rates monthly. Our learning network used webinars, discussion boards, targeted leader messaging, and in-person learning sessions. Recommended bundle elements encompassed proper preoperative bathing, intraoperative skin antisepsis, and antibiotic delivery. Within 6 months, the network achieved 96.7% reliability among institutions reporting adherence data. A 21% reduction in SSI rate was reported across network hospitals, from a mean baseline rate of 2.5 SSIs per 100 procedures to a mean rate of 1.8 SSIs per 100 procedures. The reduced rate was sustained for 15 months. Adoption of a SSI prevention bundle with concomitant reliability measurement reduced the network SSI rate. Linking reliability measurement to standardization at an institutional level may lead to safer care.


A randomized controlled trial of event-specific prevention strategies for reducing problematic drinking associated with 21st birthday celebrations.
While research has documented heavy drinking practices and associated negative consequences of college students turning 21, few studies have examined prevention efforts aimed at reducing high-risk drinking during 21st birthday celebrations. The present study evaluated the comparative efficacy of a general prevention effort (i.e., Brief Alcohol Screening and Intervention for College Students, or BASICS) and event-specific prevention in reducing 21st birthday drinking and related negative consequences. Furthermore, this study evaluated inclusion of peers in interventions and mode of intervention delivery (i.e., in-person vs. via the Web). Participants included 599 college students (46% male): men who intended to consume at least 5 drinks and women who intended to consume at least 4 drinks on their 21st birthday. After completing a screening/baseline assessment approximately 1 week before turning 21, participants were randomly assigned to 1 of 6 conditions: 21st birthday in-person BASICS, 21st birthday web BASICS, 21st birthday in-person BASICS plus friend intervention, 21st birthday web BASICS plus friend intervention, BASICS, or an attention control. A follow-up assessment was completed approximately 1 week after students' birthdays. Results indicated a significant intervention effect for BASICS in reducing blood alcohol content reached and number of negative consequences experienced. All 3 in-person interventions reduced negative consequences experienced. Results for the web-based interventions varied by drinking outcome and whether a friend was included. Overall, results provide support for both general intervention and ESP approaches across modalities for reducing extreme drinking and negative consequences associated with turning 21. These results suggest there are several promising options for campuses seeking to reduce both use and negative consequences associated with 21st birthday celebrations.


Multicystic dysplastic kidney: four-year evaluation.
Multicystic dysplastic kidney is a relatively common developmental abnormality in infants and children. Additional abnormalities like vesicoureteral reflux, ureteropelvic junction obstruction, and ureterovesical junction obstruction may accompany multicystic dysplastic kidney. In this article, we reviewed the pathogenesis, diagnosis, associated urinary tract anomalies and results of management of multicystic dysplastic kidney in the light of the literature. We retrospectively assessed 20 children with unilateral multicystic dysplastic kidney between January 2005 and December 2009. Mean duration of follow-up was 35 ± 8.7 months. All children with multicystic dysplastic kidney underwent nephrectomy. Patient characteristics, the pathology findings, associated urinary tract anomalies and results of management were reviewed. Abdominal ultrasound, voiding cystourethrography and renal scintigraphy revealed vesicoureteral reflux in 3 (15%) children, ureteropelvic junction obstruction in 3 (15%) children, and a duplex system in the contralateral kidney in 1 (5%) child. Blood pressure values, serum urea, creatinine and urinalysis were within normal range in all children during follow-up. A conservative approach to children with multicystic dysplastic kidney has been advocated, but surgical removal is also another management modality on the basis of risk of hypertension, mass effect, potential for malignant change, and cost of repeated ultrasound examination.


Twenty amino acids at the C-terminus of PA-X are associated with increased influenza A virus replication and pathogenicity.
The PA-X protein, arising from ribosomal frameshift during PA translation, was recently discovered in influenza A virus (IAV). The C-terminal domain 'X' of PA-X proteins in IAVs can be classified as full-length (61 aa) or truncated (41 aa). In the main, avian influenza viruses express full-length PA-X proteins, whilst 2009 pandemic H1N1 (pH1N1) influenza viruses harbour truncated PA proteins. The truncated form lacks aa 232-252 of the full-length PA-X protein. The significance of PA-X length in virus function remains unclear. To address this issue, we constructed a set of contemporary influenza viruses (pH1N1, avian H5N1 and H9N2) with full and truncated PA-X by reverse genetics to compare their replication and host pathogenicity. All full-length PA-X viruses in human A549 cells conferred 10- to 100-fold increase in viral replication and 5-8% increase in apoptosis relative to corresponding truncated PA-X viruses. Full-length PA-X viruses were more virulent and caused more severe inflammatory responses in mice. Furthermore, aa 233-252 at the C terminus of PA-X strongly suppressed co-transfected gene expression by ∼ 50%, suggesting that these terminal 20 aa could play a role in enhancing viral replication and contribute to virulence.


Zero-field slow relaxation of magnetization in cobalt(ii) single-ion magnets: suppression of quantum tunneling of magnetization by tailoring the intermolecular magnetic coupling.
The correlation between magnetic relaxation dynamics and the alignment of single-ion magnets (SIMs) in a crystal was investigated using four analogous cobalt(ii) complexes with unique hydrogen-bond networks. The hydrogen-bonding interactions in the crystals resulted in a relatively short intermolecular Co⋯Co distance, which led to non-zero intermolecular magnetic coupling. All the complexes with a Co⋯Co distance shorter than 6.5 Å exhibited zero-field slow magnetic relaxation as weak magnetic interactions split the ground ±Ms levels and suppressed quantum tunneling of magnetization (QTM). In particular, antiferromagnetically coupled one-dimensional chain SIM networks effectively suppressed QTM when the two intrachain Co⋯Co distances were non-equivalent. However, when the two distances in a chain were equivalent and each molecular symmetry axis aligned parallell within the chain, QTM suppression was insufficient because magnetic coupling from the adjacent molecules was virtually cancelled. Partial substitution of the CoII ion with the diamagnetic ZnII ion up to 33% for this complex resulted in complete QTM suppression in the absence of an external field. These results show that the manipulation of intermolecular distances and alignments is effective for suppressing undesired QTM events in SIMs.


Dynamics of Nanodroplets on Vibrating Surfaces.
We report the results of molecular dynamics investigations into the behavior of nanoscale water droplets on surfaces subjected to cyclic-frequency normal vibration. Our results show, for the first time, a range of vibration-induced phenomena, including the existence of the following different regimes: evaporation, droplet oscillation, and droplet lift-off. We also describe the effect of different surface wettabilities on evaporation. The outcomes of this work can be utilized in the design of future nanoengineered technologies that employ surface/bulk acoustic waves, such as water-based cooling systems for high-heat-generating processor chips, by tuning the vibration frequency and amplitude, as well as the surface wettability, to obtain the desired performance.


Identification of serum analytes and metabolites associated with aerobic capacity.
Studies aimed at identifying serum markers of cellular metabolism (biomarkers) that are associated at baseline with aerobic capacity (VO₂max) in young, healthy individuals have yet to be reported. Therefore, the goal of the present study was to use the standard chemistry screen and untargeted mass spectrometry (MS)-based metabolomic profiling to identify significant associations between baseline levels of serum analytes or metabolites with VO₂max (77 subjects, age range 18-35 years). Use of multivariable linear regression identified three analytes (standard chemistry screen) and twenty-three metabolites (MS-based metabolomics) containing significant, sex-adjusted associations with VO₂max. In addition, fourteen metabolites were found to contain sex-specific associations with aerobic capacity. Subsequent stepwise multivariable linear regression identified the combination of SGOT, 4-ethylphenylsulfate, tryptophan, γ-tocopherol, and α-hydroxyisovalerate as overall, sex-adjusted baseline predictors of VO₂max (adjusted R(2) = 0.66). However, the results of the stepwise model were found to be sensitive to outliers; therefore, random forest (RF) regression was performed. Use of RF regression identified a combination of seven covariates that explained 57.6 % of the variability inherent in VO₂max. Furthermore, inclusion of significant analytes, metabolites and sex-specific metabolites into a stepwise regression model identified the combination of five metabolites in males and seven metabolites in females as being able to explain 80 and 58 % of the variability inherent in VO₂max, respectively. In conclusion, the evidence presented in the current report is the first attempt to identify baseline serum biomarkers that are significantly associated with VO₂max in young, healthy adult humans.


Preventing the return of fear in humans using reconsolidation update mechanisms.
Recent research on changing fears has examined targeting reconsolidation. During reconsolidation, stored information is rendered labile after being retrieved. Pharmacological manipulations at this stage result in an inability to retrieve the memories at later times, suggesting that they are erased or persistently inhibited. Unfortunately, the use of these pharmacological manipulations in humans can be problematic. Here we introduce a non-invasive technique to target the reconsolidation of fear memories in humans. We provide evidence that old fear memories can be updated with non-fearful information provided during the reconsolidation window. As a consequence, fear responses are no longer expressed, an effect that lasted at least a year and was selective only to reactivated memories without affecting others. These findings demonstrate the adaptive role of reconsolidation as a window of opportunity to rewrite emotional memories, and suggest a non-invasive technique that can be used safely in humans to prevent the return of fear.


Complex cooperative networks from evolutionary preferential attachment.
In spite of its relevance to the origin of complex networks, the interplay between form and function and its role during network formation remains largely unexplored. While recent studies introduce dynamics by considering rewiring processes of a pre-existent network, we study network growth and formation by proposing an evolutionary preferential attachment model, its main feature being that the capacity of a node to attract new links depends on a dynamical variable governed in turn by the node interactions. As a specific example, we focus on the problem of the emergence of cooperation by analyzing the formation of a social network with interactions given by the Prisoner's Dilemma. The resulting networks show many features of real systems, such as scale-free degree distributions, cooperative behavior and hierarchical clustering. Interestingly, results such as the cooperators being located mostly on nodes of intermediate degree are very different from the observations of cooperative behavior on static networks. The evolutionary preferential attachment mechanism points to an evolutionary origin of scale-free networks and may help understand similar feedback problems in the dynamics of complex networks by appropriately choosing the game describing the interaction of nodes.


Tolbutamide reverses membrane hyperpolarisation induced by activation of D2 receptors and GABAB receptors in isolated substantia nigra neurones.
A high density of binding sites for sulphonylureas is found in the substantia nigra. These binding sites may be linked to ATP-regulated K-channels (K-ATP channels) as sulphonylureas selectively inhibit these channels in pancreatic beta-cells. We have studied the effect of the sulphonylurea tolbutamide on the electrical properties of freshly isolated neurones from guinea-pig substantia nigra, using the perforated patch technique. In spontaneously firing cells, both the D2 agonist quinpirole and the GABAB agonist baclofen abolished firing, hyperpolarised the cell and increased the whole-cell conductance. Tolbutamide reversed all three effects, but was without effect in the absence of quinpirole or baclofen. We suggest that D2 and GABAB receptors activate a K-channel that is blocked by tolbutamide.


Human activity patterns and skeletal metric indicators in the upper limb.
This study investigates skeletal metric traits of long bones of upper limb and the relationship between these traits and human activity in males of a recent, well-documented skeletal sample of Italian population from the Frassetto collection (Department of Experimental Evolutionary Biology, University of Bologna). The study analyzes the impact of some human activities on the skeleton, taking into account the possibility of an assessment of functional stresses caused by these activities on the basis of metric characteristics. The data consist of measurements of linear and angular bone traits, obtained by traditional and new instruments. With the purpose to find out the best indicators of occupational stress among the measured traits and indices, univariate and multivariate statistical analyses were carried out. Then the results obtained previously were used to analyze a sample with unknown occupation. The efficacy of metric stress indicators is discussed.


Induction of UDP-glucuronosyltransferase 2B15 gene expression by the major active metabolites of tamoxifen, 4-hydroxytamoxifen and endoxifen, in breast cancer cells.
We previously reported upregulation of UGT2B15 by 17β-estradiol in breast cancer MCF7 cells via binding of the estrogen receptor α (ERα) to an estrogen response unit (ERU) in the proximal UGT2B15 promoter. In the present study, we show that this ERα-mediated upregulation was significantly reduced by two ER antagonists (fulvestrant and raloxifene) but was not affected by a third ER antagonist, 4-hydroxytamoxifen (4-OHTAM), a major active tamoxifen (TAM) metabolite. Furthermore, we found that, similar to 17β-estradiol, 4-OHTAM and endoxifen (another major active TAM metabolite) elevated UGT2B15 mRNA levels, and that this stimulation was significantly abrogated by fulvestrant. Further experiments using 4-OHTAM revealed a critical role for ERα in this regulation. Specifically; knockdown of ERα expression by anti-ERα small interfering RNA reduced the 4-OHTAM-mediated induction of UGT2B15 expression; 4-OHTAM activated the wild-type but not the ERU-mutated UGT2B15 promoter; and chromatin immunoprecipitation assays showed increased ERα occupancy at the UGT2B15 ERU in MCF7 cells upon exposure to 4-OHTAM. Together, these data indicate that both 17β-estradiol and the antiestrogen 4-OHTAM upregulate UGT2B15 in MCF7 cells via the same ERα-signaling pathway. This is consistent with previous observations that both 17β-estradiol and TAM upregulate a common set of genes in MCF7 cells via the ER-signaling pathway. As 4-OHTAM is a UGT2B15 substrate, the upregulation of UGT2B15 by 4-OHTAM in target breast cancer cells is likely to enhance local metabolism and inactivation of 4-OHTAM within the tumor. This represents a potential mechanism that may reduce TAM therapeutic efficacy or even contribute to the development of acquired TAM resistance.


Thermal lens evolution and compensation in a high power KGW Raman laser.
The transient thermal lens in a high-average power double metal tungstate Raman laser has been investigated. An external cavity potassium gadolinium tungstate (KGW) laser designed for second-Stokes output was burst-pumped with up to 46 W of average power at a pulse repetition rate of 38 kHz. At low duty-cycle, the laser generated up to 18 W of on-time average Raman power with a conversion efficiency of 40%. At high duty cycle, efficiency is reduced and the near-field beam profile expands in the X1' crystal direction over a period of tens of milliseconds. The evolution of the spatial beam properties occurs in response to the development of a highly astigmatic thermal lens with fast-axis susceptibility of approximately -1.7 m-1 per watt of Raman output power. We show that the likely cause for astigmatism is primarily photo-elastic in origin. Beam circularization was achieved by incorporating an intracavity convex cylindrical lens.


EFFECTS OF "SWIM WITH THE TURTLES" TOURIST ATTRACTIONS ON GREEN SEA TURTLE (CHELONIA MYDAS) HEALTH IN BARBADOS, WEST INDIES.
Along the West Coast of Barbados a unique relationship has developed between endangered green sea turtles (Chelonia mydas) and humans. Fishermen began inadvertently provisioning these foraging turtles with fish offal discarded from their boats. Although initially an indirect supplementation, this activity became a popular attraction for visitors. Subsequently, demand for this activity increased, and direct supplementation or provisioning with food began. Food items offered included raw whole fish (typically a mixture of false herring [Harengula clupeola] and pilchard [Harengula humeralis]), filleted fish, and lesser amounts of processed food such as hot dogs, chicken, bread, or various other leftovers. Alterations in behavior and growth rates as a result of the provisioning have been documented in this population. The purpose of this study was to determine how tourism-based human interactions are affecting the overall health of this foraging population and to determine what potential health risks these interactions may create for sea turtles. Juvenile green sea turtles (n=29) were captured from four sites off the coast of Barbados, West Indies, and categorized into a group that received supplemental feeding as part of a tour (n=11) or an unsupplemented group (n=18) that consisted of individuals that were captured at sites that did not provide supplemental feeding. Following capture, a general health assessment of each animal was conducted. This included weight and morphometric measurements, a systematic physical examination, determination of body condition score and body condition index, epibiota assessment and quantification, and clinical pathology including hematologic and biochemical testing and nutritional assessments. The supplemented group was found to have changes to body condition, vitamin, mineral, hematologic, and biochemical values. Based on these results, recommendations were made to decrease negative behaviors and health impacts for turtles as a result of this provisioning.


The toxic effect of thioacetamide on rat liver in vitro.
Thioacetamide (TAA) is a hepatotoxin frequently used for experimental purposes which produces centrilobular necrosis after a single dose administration. In spite of the fact that oxidative stress seems to play a very important role in the mechanism of TAA-induced injury, the effect of TAA on hepatocytes in primary culture with respect to the influence on mitochondria has yet to be verified. Hepatocytes were incubated for 24h in a medium containing TAA (0-70 mmol/l). Glutathione content (GSH/GSSG), reactive oxygen species and malondialdehyde formation were assessed as markers of cell redox state. Toxicity was determined by lactate dehydrogenase leakage and WST-1 assay. The functional capacity of hepatocytes was evaluated from albumin and urea production. Mitochondrial metabolism was assessed by measuring mitochondrial membrane potential and oxygen consumption. Our results show that a profound decrease in the GSH level in hepatocytes precedes a sharp rise in endogenous ROS production. ROS production correlates with an increase in lipoperoxidation. Mitochondria are affected by TAA secondarily as a consequence of oxidative stress. Oxidation of the NADH-dependent substrates of respiratory Complex I is significantly more sensitive to the toxic action of TAA than oxidation of the flavoprotein-dependent substrate of Complex II. Mitochondria can also maintain their membrane potential better when they utilize succinate as a respiratory substrate. It appears that GSH should be depleted below a certain critical level in order to cause a marked increase in lipid peroxidation. Mitochondrial injury can then occur and cell death develops.


Ambiguous base pairing of the purine analogue 1-(2-deoxy-beta-D-ribofuranosyl)-imidazole-4-carboxamide during PCR.
In principle the hydrogen bonding capacities of 1-(2-deoxy-beta-D-ribofuranosyl)-imidazole-4-carboxamide (dY), and its N-propyl derivative (dYPr), allow them to pair to all four deoxynucleosides. Their triphosphate derivatives (dYTP and dYPrTP) are preferentially incorporated as dATP analogues in a PCR reaction. However, once incorporated into a DNA template their ambiguous hydrogen bonding potential gave rise to misincorporation at frequencies of approximately 3 x 10(-2) per base per amplification. Most of the substitutions were transitions resulting from rotation about the carboxamide bond when part of the template. Between 11-15% of transversions were noted implying rotation of purine or imidazole moieties about the glycosidic bond. As part of a DNA template, dYPr behaved in the same way as dY, despite its propyl moiety. These deoxyimidazole derivatives are among the most radical departures from the canonical bases used so far as substrates in PCR and could be used to generate mutant gene libraries.


Updating Darwin: Information and entropy drive the evolution of life.
The evolution of species, according to Darwin, is driven by struggle - by competition between variant autonomous individuals for survival of the fittest and reproductive advantage; the outcome of this struggle for survival is natural selection. The Neo-Darwinians reframed natural selection in terms of DNA: inherited genotypes directly encode expressed phenotypes; a fit phenotype means a fit genotype - thus the evolution of species is the evolution of selfish, reproducing individual genotypes. Four general characteristics of advanced forms of life are not easily explained by this Neo-Darwinian paradigm: 1) Dependence on cooperation rather than on struggle, manifested by the microbiome, ecosystems and altruism; 2) The pursuit of diversity rather than optimal fitness, manifested by sexual reproduction; 3) Life's investment in programmed death, rather then in open-ended survival; and 4) The acceleration of complexity, despite its intrinsic fragility. Here I discuss two mechanisms that can resolve these paradoxical features; both mechanisms arise from viewing life as the evolution of information. Information has two inevitable outcomes; it increases by autocatalyis and it is destroyed by entropy. On the one hand, the autocalalysis of information inexorably drives the evolution of complexity, irrespective of its fragility. On the other hand, only those strategic arrangements that accommodate the destructive forces of entropy survive - cooperation, diversification, and programmed death result from the entropic selection of evolving species. Physical principles of information and entropy thus fashion the evolution of life.


The clinical, immunological and microbiological impact of the 10-valent pneumococcal-Protein D conjugate vaccine in children with recurrent protracted bacterial bronchitis, chronic suppurative lung disease and bronchiectasis: A multi-centre, double-blind, randomised controlled trial.
We aimed to determine the efficacy of the 10-valent pneumococcal-Haemophilus influenzae protein D conjugate vaccine (PHiD-CV) in children aged 18-months to <18-years with recurrent protracted bacterial bronchitis (rPBB), chronic suppurative lung disease (CSLD) or bronchiectasis. In a multi-centre, double-blind randomised controlled trial, children received two doses, 2-months apart of the 10vPHiD-CV or quadrivalent meningococcal-ACYW135 conjugate vaccine. Active surveillance for acute exacerbations, respiratory symptoms and antibiotic use was undertaken through to 12-months after the second vaccine dose (clinical cohort only). Serum, saliva and nasopharyngeal swabs were collected to measure immunological and microbiological effects (immunology cohort). Between December 2012 and August 2015, 62 children were enrolled onto the clinical protocol (1 excluded from clinical analyses due to unblinding), while 74 contributed to the immunology cohort (overall mean age = 6.8-years (standard deviation = 3.7), 42 (56.8%) male). The absolute risk difference comparing the 10vPHiD-CV group (n = 31 children) to the MenACYW135 group (n = 30 children) for acute exacerbations was -0.5 exacerbations/100-weeks at risk (95% confidence interval (CI) -2.0, 0.9). Compared to the MenACYW135 group, children who received the 10vPHiD-CV were less likely to have respiratory symptoms in each fortnight of surveillance (incidence density ratio (IDR) 0.82, 95%CI 0.61, 1.10) and required fewer short-course (<14-days duration) antibiotics (IDR 0.81, 95% CI 0.61, 1.09). The vaccine was immunogenic and no serious adverse events related to the vaccine were reported. In conclusion, 10vPHiD-CV might have a future role in managing children with rPBB, CSLD and bronchiectasis, but larger multicentre trials are needed to confirm or refute findings from this preliminary study.


Hippocampal gene expression induced by cold swim stress depends on sex and handling.
Stress-related disorders such as PTSD and depression are more prevalent in women than men. One reason for such discordance may be that brain regions involved in stress responses are more sensitive to stress in females. Here, we compared the effects of acute stress on gene transcription in the hippocampus of female and male mice, and also examined the involvement of two key stress-related hormones, corticosterone and corticotropin releasing hormone (Crh). Using quantitative reverse transcription polymerase chain reaction (RT-qPCR), we measured gene expression of Fos, Per1 and Sgk1 45 min after exposure to brief cold swim stress. Stress induced a stronger increase in Fos and Per1 expression in females than males. The handling control procedure increased Fos in both sexes, but occluded the effects of stress in males. Further, handling increased Per1 only in males. Sgk1 was insensitive to handling, and increased in response to stress similarly in males and females. The transcriptional changes observed after swim stress were not mimicked by corticosterone injections, and the stress-induced increase in Fos, Per1 and Sgk1 could neither be prevented by pharmacologically blocking glucocorticoid receptor (GR) nor by blocking Crh receptor 1 (Crhr1) before stress exposure. Finally, we demonstrate that the effects are stressor-specific, as the expression of target genes could not be increased by brief restraint stress in either sex. In summary, we find strong effects of acute swim stress on hippocampal gene expression, complex interactions between handling and sex, and a remarkably unique response pattern for each gene. Overall, females respond to a cold swim challenge with stronger hippocampal gene transcription than males, independent of two classic mediators of the stress response, corticosterone and Crh. These findings may have important implications for understanding the higher vulnerability of women to certain stress-related neuropsychiatric diseases.


Desire for female sterilization among women wishing to limit births in rural Rakai, Uganda.
Uganda has an unmet need for family planning of 34% and a total fertility rate of 6.2. We assessed the desire for female sterilization among sexually active women who wanted to stop childbearing in rural Rakai district, Uganda. 7192 sexually active women enrolled in a community cohort between 2002 and 2008 were asked about fertility intentions. Those stating that they did not want another child (limiters) were asked whether they would be willing to accept female sterilization, if available. Trends in desire for sterilization were determined by chi-square test for trend, and Modified Poisson regression was used to estimate prevalence rate ratios and 95% confidence intervals of the associations between desire for sterilization and socio-demographic characteristics and HIV status. From 2002 to 2008, the proportion of limiters dropped (from 47.2% to 43.7%; p<.01). Use of pills and injectables among limiters significantly increased, 38.9% to 50.3% (p<.0001), while use of intrauterine devices and implants declined from 3.3% to 1.7% (p<0.001). The desire for sterilization significantly increased from 54.2% to 63.1% (p<0.0001), and this was consistently higher among the HIV-positive (63.6-70.9%, p<0.01) than HIV-negative women (53.3-61.2%, p<0.0001). Factors significantly associated with the desire for sterilization included higher number of living children (>=3), being HIV-infected and having received HIV counseling and testing. There is latent and growing desire for sterilization in this population. Our findings suggest a need to increase permanent contraception services for women who want to limit childbearing in this setting. A large unmet need for permanent female contraception services exists in Uganda. Efforts to increase the method mix by increasing permanent contraception services could reduce fertility rates and undesired births.


Maternal exposure to a high-magnitude earthquake during pregnancy influences pre-reading skills in early childhood.
Exposure to an adverse prenatal environment can influence fetal development and result in long-lasting changes in the offspring. However, the association between maternal exposure to stressful events during pregnancy and the achievement of pre-reading skills in the offspring is unknown. Here we examined the association between prenatal exposure to the Chilean high-magnitude earthquake that occurred on February 27th, 2010 and the development of early reading precursors skills (listening comprehension, print knowledge, alphabet knowledge, vocabulary, and phonological awareness) in children at kindergarten age. This multilevel retrospective cohort study including 3280 children, of whom 2415 were unexposed and 865 were prenatally exposed to the earthquake shows substantial evidence that maternal exposure to an unambiguously stressful event resulted in impaired pre-reading skills and that a higher detrimental effect was observed in those children who had been exposed to the earthquake during the first trimester of gestation. In addition, females were more significantly affected by the exposure to the earthquake than their male peers in alphabet knowledge; contrarily, males were more affected than females in print knowledge skills. These findings suggest that early intervention programs for pregnant women and/or children exposed to prenatal stress may be effective strategies to overcome impaired pre-reading skills in children.


Targeting Tanycytes: Balance between Efficiency and Specificity.
Tanycytes are peculiar ependymoglial cells lining the bottom and the lateral wall of the third ventricle. For a decade, the utilization of molecular genetic approaches allowed us to make important discoveries about their diverse physiological functions. Here, I review the current methods used to target tanycytes, focusing on their specificity, their efficiency, their limitations, as well as their potential future improvements.


Surface glycosaminoglycans and calcium distribution in 3T3 cells.
Resting 3T3 cells have relatively more sulphated glycosaminoglycans and Ca2+ in their cell coat than do growing or SV40-virus-transformed cells. It is suggested that the different Ca2+-binding capacity controls cellular activities by affecting the distribution of Ca2+ between the intra- and ecto-cellular compartments.


Ontogeny and phylogeny: molecular signatures of selection, constraint, and temporal pleiotropy in the development of Drosophila.
Karl Ernst Von Baer noted that species tend to show greater morphological divergence in later stages of development when compared to earlier stages. Darwin originally interpreted these observations via a selectionist framework, suggesting that divergence should be greatest during ontogenic stages in which organisms experienced varying 'conditions of existence' and opportunity for differential selection. Modern hypotheses have focused on the notion that genes and structures involved in early development will be under stronger purifying selection due to the deleterious pleiotropic effects of mutations propagating over the course of ontogeny, also known as the developmental constraint hypothesis. Using developmental stage-specific expressed sequence tag (EST) libraries, we tested the 2 hypotheses by comparing the rates of evolution of 7,180 genes obtained from 6 species of the Drosophila melanogaster group with respect to ontogeny, and sex and reproduction-related functions in gonadal tissues. Supporting morphological observations, we found evidence of a pattern of increasing mean evolutionary rate in genes that are expressed in subsequent stages of development. Furthermore, supporting expectations that early expressed genes are constrained in divergence, we found that embryo stage genes are involved in a higher mean number of interactions as compared to later stages. We noted that the accelerated divergence of genes in the adult stage is explained by those expressed specifically in the male gonads, whose divergence is driven by positive selection. In addition, accelerated gonadal gene divergence occurs only in the adult stage, suggesting that the effects of selection are observed primarily at the stages during which they are expected occur. Finally, we also found a significant correlation between temporal specificity of gene expression and evolutionary rate, supporting expectations that genes with ubiquitous expression are under stronger constraint. Taken together, these results support both the developmental constraint hypothesis limiting the divergence of early expressed developmentally important genes, leading to a gradient of divergence rates over ontogeny (embryonic < larval/pupal < adult), as well as Darwin's 'selection opportunity' hypothesis leading to increased divergence in adults, particularly in the case of reproductive tissues. We suggest that a constraint early/opportunity late model best explains divergence over ontogeny.


Genome-wide association study of prostate cancer identifies a second risk locus at 8q24.
Recently, common variants on human chromosome 8q24 were found to be associated with prostate cancer risk. While conducting a genome-wide association study in the Cancer Genetic Markers of Susceptibility project with 550,000 SNPs in a nested case-control study (1,172 cases and 1,157 controls of European origin), we identified a new association at 8q24 with an independent effect on prostate cancer susceptibility. The most significant signal is 70 kb centromeric to the previously reported SNP, rs1447295, but shows little evidence of linkage disequilibrium with it. A combined analysis with four additional studies (total: 4,296 cases and 4,299 controls) confirms association with prostate cancer for rs6983267 in the centromeric locus (P = 9.42 x 10(-13); heterozygote odds ratio (OR): 1.26, 95% confidence interval (c.i.): 1.13-1.41; homozygote OR: 1.58, 95% c.i.: 1.40-1.78). Each SNP remained significant in a joint analysis after adjusting for the other (rs1447295 P = 1.41 x 10(-11); rs6983267 P = 6.62 x 10(-10)). These observations, combined with compelling evidence for a recombination hotspot between the two markers, indicate the presence of at least two independent loci within 8q24 that contribute to prostate cancer in men of European ancestry. We estimate that the population attributable risk of the new locus, marked by rs6983267, is higher than the locus marked by rs1447295 (21% versus 9%).


Template-directed pausing in in vitro DNA synthesis by DNA polymerase a from Drosophila melanogaster embryos.
The activity of Drosophila melanogaster DNA polymerase alpha on DNA-primed single-stranded DNA templates has been examined. The DNA templates contain a 1471-nucleotide sequence from the heavy-strand origin region of mouse mtDNA inserted into the single-stranded bacteriophage vector M13Gori1. Preferred sites for pausing of in vitro DNA synthesis have been mapped within the cloned mtDNA insert and in the G4 cDNA strand origin which is contained within the vector DNA. Analysis of nascent DNA strands from replicative intermediates has revealed that pause sites are discrete and lie both at the positions of predicted stable dyads and in regions lacking the potential for formation of such structures. The patterns of kinetic pause sites observed for Escherichia coli DNA polymerase III holoenzyme is qualitatively similar to that found for DNA polymerase alpha. A subset of the observed kinetic pause signals are recognized by E. coli DNA polymerase I under similar conditions.


Expression of human skin-specific genes defined by transcriptomics and antibody-based profiling.
To increase our understanding of skin, it is important to define the molecular constituents of the cell types and epidermal layers that signify normal skin. We have combined a genome-wide transcriptomics analysis, using deep sequencing of mRNA from skin biopsies, with immunohistochemistry-based protein profiling to characterize the landscape of gene and protein expression in normal human skin. The transcriptomics and protein expression data of skin were compared to 26 (RNA) and 44 (protein) other normal tissue types. All 20,050 putative protein-coding genes were classified into categories based on patterns of expression. We found that 417 genes showed elevated expression in skin, with 106 genes expressed at least five-fold higher than that in other tissues. The 106 genes categorized as skin enriched encoded for well-known proteins involved in epidermal differentiation and proteins with unknown functions and expression patterns in skin, including the C1orf68 protein, which showed the highest relative enrichment in skin. In conclusion, we have applied a genome-wide analysis to identify the human skin-specific proteome and map the precise localization of the corresponding proteins in different compartments of the skin, to facilitate further functional studies to explore the molecular repertoire of normal skin and to identify biomarkers related to various skin diseases.


The brain-specific Beta4 subunit downregulates BK channel cell surface expression.
The large-conductance K(+) channel (BK channel) can control neural excitability, and enhanced channel currents facilitate high firing rates in cortical neurons. The brain-specific auxiliary subunit β4 alters channel Ca(++)- and voltage-sensitivity, and β4 knock-out animals exhibit spontaneous seizures. Here we investigate β4's effect on BK channel trafficking to the plasma membrane. Using a novel genetic tag to track the cellular location of the pore-forming BKα subunit in living cells, we find that β4 expression profoundly reduces surface localization of BK channels via a C-terminal ER retention sequence. In hippocampal CA3 neurons from C57BL/6 mice with endogenously high β4 expression, whole-cell BK channel currents display none of the characteristic properties of BKα+β4 channels observed in heterologous cells. Finally, β4 knock-out animals exhibit a 2.5-fold increase in whole-cell BK channel current, indicating that β4 also regulates current magnitude in vivo. Thus, we propose that a major function of the brain-specific β4 subunit in CA3 neurons is control of surface trafficking.


Interactions between spore morphogenetic mutations affect cell types, sporulation, and pathogenesis in Magnaporthe grisea.
We have previously defined four single-gene mutations, con1, con2, con4, and con7, that control various stages of spore morphogenesis in the rice blast fungus. To delineate the developmental pathway of spore morphogenesis, we investigated the interactions among these morphogenetic genes by generating strains with double mutations via transformation-mediated gene disruption. Plasmids containing portions of the inactivated CON4 and CON7 genes were introduced into strains harboring single mutation to produce double mutants. Interaction between con1 and con4 resulted in reduced vegetative growth and suppression of sporulation. Interaction between con1 and con7 suppressed spore production but not vegetative growth. The con2/con4 and con4/con7 double mutants produced hybrid spore types with characteristic features of both parental mutants. The con2/con7 mutant produced con2 type spores, indicating that con2 is epistatic to con7 in spore morphogenesis. The epistatic relationship, however, reversed when traits related to pathogenesis were considered. Double mutants harboring the con7 mutation could not form appressoria or colonize plant tissue, indicating that con7 is epistatic to con2 and con4 in appressorium formation and pathogenesis. Thus, morphogenetic genes interact at multiple levels leading to different epistatic relationships in the pathways of spore morphogenesis, appressorium formation, and pathogenesis.


Two anatomically distinct niches regulate stem cell activity.
The niche microenvironment controls stem cell number, fate, and behavior. The bone marrow, intestine, and skin are organs with highly regenerative potential, and all produce a large number of mature cells daily. Here, focusing on adult stem cells in these organs, we compare the structures and cellular components of their niches and the factors they produce. We then define the niche as a functional unit for stem cell regulation. For example, the niche possibly maintains quiescence and regulates fate in stem cells. Moreover, we discuss our hypothesis that many stem cell types are regulated by both specialized and nonspecialized niches, although hematopoietic stem cells, as an exception, are regulated by a nonspecialized niche only. The specialized niche is composed of 1 or a few types of cells lying on the basement membrane in the epithelium. The nonspecialized niche is composed of various types of cells widely distributed in mesenchymal tissues. We propose that the specialized niche plays a role in local regulation of stem cells, whereas the nonspecialized niche plays a role in relatively broad regional or systemic regulation. Further work will verify this dual-niche model to understand mechanisms underlying stem cell regulation.


[Evaluation of peruvian money test in screening of cognitive impairment among older adults].
Objectives To evaluate the Peruvian adaptation of the money test (Eurotest) for identifying cognitive impairment among >60-year-old adults. Materials and methods This is a phase I study of diagnostic test, with a convenience sampling and calculation of the test´s sensitivity and specificity, based on a pretest prevalence of 50%. The criteria of the Diagnostic and Statistical Manual of Mental Disorders (DSM IV) and Global Deterioration Scale (GDS) were used for the operational definition of patients with cognitive impairment. Receiver operating characteristic (ROC) curve analysis was used to identify the optimal cut-off value. Results The study evaluated 42 cases and 42 controls; there was no significant difference between age (77.88 ± 6.01 years vs. 6.49 76.14 ± years) and years of education (13.69 ± 3.70 years vs. 8.17 ± 4.71 years). The Peruvian version of the Eurotest has a sensitivity of 90.5% and specificity of 83.3% with cut-off value of 24. Conclusions The Peruvian adapted version of the Eurotest, called prueba de la moneda peruana could be useful in screening for cognitive impairment among older adults.


The natural history of multiple sclerosis: a geographically based study. 2. Predictive value of the early clinical course.
Controversy exists regarding the predictive value of the early clinical course of multiple sclerosis (MS). Three parameters often considered are the attack rate, the first interattack interval and the rate at which disability develops in the early years of the disease. We have recorded the time to reach successive levels of disability defined by the Kurtzke Disability Status Scale (DSS) in 1,099 MS patients followed at University Hospital, London, Canada between 1972 and 1984. Our population is particularly suitable because of its size, the high degree of ascertainment of cases in the community, and the regular follow-up provided. Life table analysis was used to compare survival in patients stratified according to the above three parameters using DSS 6 as end point. Significant differences were evident in the survival distributions. Despite the extensive interindividual variation in the rate at which disability developed, the early course of MS may be useful in determining the relative risk of rapid progression.


Gene selection for classification of microarray data based on the Bayes error.
ABSTRACT: BACKGROUND: With DNA microarray data, selecting a compact subset of discriminative genes from thousands of genes is a critical step for accurate classification of phenotypes for, e.g., disease diagnosis. Several widely used gene selection methods often select top-ranked genes according to their individual discriminative power in classifying samples into distinct categories, without considering correlations among genes. A limitation of these gene selection methods is that they may result in gene sets with some redundancy and yield an unnecessary large number of candidate genes for classification analyses. Some latest studies show that incorporating gene to gene correlations into gene selection can remove redundant genes and improve classification accuracy. RESULTS: In this study, we propose a new method, Based Bayes error Filter (BBF), to select relevant genes and remove redundant genes in classification analyses of microarray data. The effectiveness and accuracy of this method is demonstrated through analyses of five publicly available microarray datasets. The results show that our gene selection method is capable of achieving better accuracies than previous studies, while being able to effectively select relevant genes, remove redundant genes and obtain efficient and small gene sets for sample classification purposes. CONCLUSION: The proposed method can effectively identify a compact set of genes with high classification accuracy. This study also indicates that application of the Bayes error is a feasible and effective wayfor removing redundant genes in gene selection.


Machine learning approach for prediction of hearing preservation in vestibular schwannoma surgery.
In vestibular schwannoma patients with functional hearing status, surgical resection while preserving the hearing is feasible. Hearing levels, tumor size, and location of the tumor have been known to be candidates of predictors. We used a machine learning approach to predict hearing outcomes in vestibular schwannoma patients who underwent hearing preservation surgery: middle cranial fossa, or retrosigmoid approach. After reviewing the medical records of 52 patients with a pathologically confirmed vestibular schwannoma, we included 50 patient's records in the study. Hearing preservation was regarded as positive if the postoperative hearing was within serviceable hearing (50/50 rule). The categorical variable included the surgical approach, and the continuous variable covered audiometric and vestibular function tests, and the largest diameter of the tumor. Four different algorithms were lined up for comparison of accuracy: support vector machine(SVM), gradient boosting machine(GBM), deep neural network(DNN), and diffuse random forest(DRF). The average accuracy of predicting hearing preservation ranged from 62% (SVM) to 90% (DNN). The current study is the first to incorporate machine learning methodology into a prediction of successful hearing preservation surgery. Although a larger population may be needed for better generalization, this study could aid the surgeon's decision to perform a hearing preservation approach for vestibular schwannoma surgery.


In-Hospital Macro-, Meso-, and Micro-Drivers and Interventions for Antibiotic Use and Resistance: A Rapid Evidence Synthesis of Data from Canada and Other OECD Countries.
Hospitals continue to face challenges in reducing incorrect antibiotic use due to social and cultural factors at the level of the health system, the care facility, the provider, and the patient. The objective of this paper is to highlight the social and cultural drivers of antimicrobial use and resistance and targeted interventions for secondary and tertiary care settings in Canada and other OECD countries. This paper is an extension of the synthesis conducted for the Public Health Agency of Canada's 2019 Spotlight Report: Preserving Antibiotics Now and Into the Future. We conducted a systematic review with a few modifications to meet rapid timelines. We conducted a search in Ovid MEDLINE and McMaster University's evidence databases for systematic reviews and then for individual Canadian studies. To cast a wider net, we searched OECD organization websites and screened reference lists from systematic reviews. We synthesized the evidence narratively and categorized the evidence into macro-, meso-, and microlevel. A total of 70 studies were (a) from OCED countries and summarized evidence of potential sociocultural antimicrobial resistance and use barriers or facilitators and/or interventions addressing these challenges; (b) systematic reviews with 50% of included studies that are situated in secondary and tertiary settings; and (c) published in Canada's two official languages, English and French. We found that hospital structures and policies may influence antibiotic utilization and variations in antimicrobial management. Microlevel factors may sway inappropriate prescribing among clinicians. The amount and type of antibiotics used may affect resistance rates. Interventions were mainly comprised of antibiotic stewardship and training that modify clinician behavior and that educate patients and carers. This evidence synthesis illustrates the various drivers of, and interventions for, antimicrobial use and resistance at the macro-, meso-, and microlevel in secondary and tertiary settings. We demonstrate that upstream drivers may lead to downstream events that influence antimicrobial resistance.


Standardizing contact investigation protocols.
The State of Alabama Department of Public Health Division of Tuberculosis Control. To standardize contact investigation protocols and implement an intervention to increase TB field worker adherence to the protocols with the goal of promoting efficiency and effectiveness in contact investigations. A process evaluation of existing data collection and management systems and protocols was performed. Standardized protocols and an intervention to increase TB field worker adherence to the protocols were created and pilot tested. These were then implemented and formative evaluation data were collected. The process evaluation revealed considerable variance among field workers with regard to protocols and definitions of variables related to contact investigations. Protocols were standardized and an intervention targeted at TB field workers was developed. The intervention consisted of a training workshop and the development of a computer-based contact investigation module. This was successfully implemented throughout the state. To perform effective contact investigations and conduct studies to improve the effectiveness of these investigations, TB control programs must pay careful attention to precisely defining variables and concepts related to the contact investigation. Furthermore, protocols must be standardized and resources devoted to training of TB field workers to ensure adherence to protocols.


Mapping urban and peri-urban breeding habitats of Aedes mosquitoes using a fuzzy analytical hierarchical process based on climatic and physical parameters.
The spread of dengue fever depends mainly on the availability of favourable breeding sites for its mosquito vectors around human dwellings. To investigate if the various factors influencing breeding habitats can be mapped from space, dengue indices, such as the container index, the house index and the Breteau index, were calculated from Ministry of Public health data collected three times annually in Phitsanulok, Thailand between 2009 and 2011. The most influential factors were found to be temperature, humidity, rainfall, population density, elevation and land cover. Models were worked out using parameters mostly derived from freely available satellite images and fuzzy logic software with parameter synchronisation and a predication algorithm based on data mining and the Decision Tree method. The models developed were found to be sufficiently flexible to accommodate additional parameters and sampling data that might improve prediction of favourable breeding hotspots. The algorithm applied can not only be used for the prediction of near real-time scenarios with respect to dengue, but can also be applied for monitoring other diseases influenced by environmental and climatic factors. The multi-criteria model presented is a cost-effective way of identifying outbreak hotspots and early warning systems lend themselves for development based on this strategy. The proposed approach demonstrates the successful utilisation of remotely sensed images to map mosquito breeding habitats.


Implication of human N-alpha-acetyltransferase 5 in cellular proliferation and carcinogenesis.
The N-alpha-acetyltransferase NatB, composed in Saccharomyces cerevisiae by the Nat3p and Mdm20p subunits, is an important factor for yeast growth and resistance to several stress agents. However, the expression and functional role of the mammalian counterpart has not yet been analysed. Here, we report the identification of Nat3p human homologue (hNAT5/hNAT3) and the characterization of its biological function. We found that hNAT5/hNAT3 silencing in HeLa cells results in inhibition of cell proliferation and increased sensitivity to the pro-apoptotic agent MG132. Moreover, inhibition of hNAT5/hNAT3 expression induces p53 activation and upregulation of the antiproliferative protein p21(WAF1/CIP1). The changes of the cellular transcriptome after hNAT5/hNAT3 knockdown confirmed the involvement of this protein in cell growth and survival processes. Among the genes differentially expressed, we observed upregulation of several p53-dependent antiproliferative and pro-apoptotic genes. In the c-myc transgenic mice, which is a model of inducible hepatocarcinoma, we found that hNAT5/hNAT3 was upregulated when the tumour was induced. In accordance with this observation, we noticed increased hNAT5/hNAT3 protein level in neoplastic versus non-neoplastic tissue in a high proportion of patients with hepatocellular carcinoma. Consequently, our results suggest that hNAT5/hNAT3 is required for cellular proliferation and can be implicated in tumour growth.


An examination of early neural and cognitive alterations in hippocampal-spatial function of ghrelin receptor-deficient rats.
Ghrelin, a hormone implicated in the regulation of feeding and energy balance, has also been associated with neural function underlying learning and memory. These effects are thought to be mediated by ghrelin targeting receptors at extra hypothalamic sites such as the hippocampus. Exogenous ghrelin administration increases dendritic spine density in the hippocampal CA1 region and neurogenesis in the dentate gyrus (DG), while improving memory in rats. In the present study, we sought to determine whether rats lacking the ghrelin receptor would show early neural or cognitive decline measured via hippocampal integrity (spine density and neurogenesis) and spatial learning and memory. As such, we used young and middle-aged adult rats with mutations to the gene encoding for the ghrelin receptor (GHS-R KO) and wildtype (WT) littermates to determine differences in performance on hippocampal-dependent tasks (the water maze and radial arm maze). In addition, we examined the hippocampal dentate gyrus of these rats for differences in dendritic spine density and cell proliferation (doublecortin). Overall, results demonstrated that spine density and doublecortin staining in the dentate gyrus of the young GHS-R KO group was similar to that seen in middle-aged groups (both KO and WT) and lower than the young WT group. Middle-aged GHS-R KO and WT groups showed deficits on the radial arm maze food-motivated task but not the water maze task. These data suggest that impaired ghrelin signaling leads to an early onset decrement in hippocampal structural integrity that may manifest in non- spatial-related behavioral deficits.


How serotonin shapes moral judgment and behavior.
Neuroscientists are now discovering how hormones and brain chemicals shape social behavior, opening potential avenues for pharmacological manipulation of ethical values. Here, we review recent studies showing how altering brain chemistry can alter moral judgment and behavior, focusing in particular on the neuromodulator serotonin and its role in shaping values related to harm and fairness. We synthesize previous findings and consider the potential mechanisms through which serotonin could increase the aversion to harming others. We present a process model whereby serotonin influences social behavior by shifting social preferences in the positive direction, enhancing the value people place on others' outcomes. This model may explain previous findings relating serotonin function to prosocial behavior, and makes new predictions regarding how serotonin may influence the neural computation of value in social contexts.


Prenatal and postpartum depression in fathers and its association with maternal depression: a meta-analysis.
It is well established that maternal prenatal and postpartum depression is prevalent and has negative personal, family, and child developmental outcomes. Paternal depression during this period may have similar characteristics, but data are based on an emerging and currently inconsistent literature. To describe point estimates and variability in rates of paternal prenatal and postpartum depression over time and its association with maternal depression. Studies that documented depression in fathers between the first trimester and the first postpartum year were identified through MEDLINE, PsycINFO, EMBASE, Google Scholar, dissertation abstracts, and reference lists for the period between January 1980 and October 2009. Studies that reported identified cases within the selected time frame were included, yielding a total of 43 studies involving 28 004 participants after duplicate reports and data were excluded. Information on rates of paternal and maternal depression, as well as reported paternal-maternal depressive correlations, was extracted independently by 2 raters. Effect sizes were calculated using logits, which were back-transformed and reported as proportions. Random-effects models of event rates were used because of significant heterogeneity. Moderator analyses included timing, measurement method, and study location. Study quality ratings were calculated and used for sensitivity analysis. Publication bias was evaluated with funnel plots and the Egger method. Substantial heterogeneity was observed among rates of paternal depression, with a meta-estimate of 10.4% (95% confidence interval [CI], 8.5%-12.7%). Higher rates of depression were reported during the 3- to 6-month postpartum period (25.6%; 95% CI, 17.3%-36.1%). The correlation between paternal and maternal depression was positive and moderate in size (r = 0.308; 95% CI, 0.228-0.384). No evidence of significant publication bias was detected. Prenatal and postpartum depression was evident in about 10% of men in the reviewed studies and was relatively higher in the 3- to 6-month postpartum period. Paternal depression also showed a moderate positive correlation with maternal depression.


Vitamin C transport in oxidized form across the rat blood-retinal barrier.
To elucidate the mechanisms of vitamin C transport across the blood-retinal barrier (BRB) in vivo and in vitro. METHODS. [(14)C]Dehydroascorbic acid (DHA) and [(14)C]ascorbic acid (AA) transport in the retina across the BRB were examined using in vivo integration plot analysis in rats, and the transport mechanism was characterized using a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2) as an in vitro model of the inner BRB. The apparent influx permeability clearance (K(in)) per gram of retina of [(14)C]DHA and [(14)C]AA was found to be 2.44 x 10(3) microL/(min x g retina) and 65.4 microL/(min x g retina), respectively. In the retina and brain, the K(in) of [(14)C]DHA was approximately 38 times greater than that of [(14)C]AA, whereas there was no major difference in the heart. The K(in) of [(14)C]DHA in the retina was eight times greater than that in the brain. HPLC analysis revealed that most of the vitamin C accumulated in AA form in the retina. These results suggest that vitamin C is mainly transported in DHA form across the BRB and accumulates in AA form in the rat retina. In an in vitro uptake study in TR-iBRB2 cells, the initial uptake rate of [(14)C]DHA was 37 times greater than that of [(14)C]AA, which is in agreement with the results of the in vivo study. [(14)C]DHA uptake by TR-iBRB2 cells took place in an Na(+)-independent and concentration-dependent manner with a K(m) of 93.4 microM. This process was inhibited by substrates and inhibitors of glucose transporters. [(14)C]DHA uptake was inhibited by D-glucose in a concentration-dependent manner with a 50% inhibition concentration of 5.56 mM. Quantitative real-time PCR and immunostaining analyses revealed that expression of GLUT1 and -3 was greater than that of the Na(+)-dependent L-ascorbic acid transporter (SVCT)-2 in TR-iBRB2 cells. Vitamin C is mainly transported across the BRB as DHA mediated through facilitative glucose transporters and accumulates as AA in the rat retina.


Regulation of gene expression in the mammalian eye and its relevance to eye disease.
We used expression quantitative trait locus mapping in the laboratory rat (Rattus norvegicus) to gain a broad perspective of gene regulation in the mammalian eye and to identify genetic variation relevant to human eye disease. Of >31,000 gene probes represented on an Affymetrix expression microarray, 18,976 exhibited sufficient signal for reliable analysis and at least 2-fold variation in expression among 120 F(2) rats generated from an SR/JrHsd x SHRSP intercross. Genome-wide linkage analysis with 399 genetic markers revealed significant linkage with at least one marker for 1,300 probes (alpha = 0.001; estimated empirical false discovery rate = 2%). Both contiguous and noncontiguous loci were found to be important in regulating mammalian eye gene expression. We investigated one locus of each type in greater detail and identified putative transcription-altering variations in both cases. We found an inserted cREL binding sequence in the 5' flanking sequence of the Abca4 gene associated with an increased expression level of that gene, and we found a mutation of the gene encoding thyroid hormone receptor beta2 associated with a decreased expression level of the gene encoding short-wavelength sensitive opsin (Opn1sw). In addition to these positional studies, we performed a pairwise analysis of gene expression to identify genes that are regulated in a coordinated manner and used this approach to validate two previously undescribed genes involved in the human disease Bardet-Biedl syndrome. These data and analytical approaches can be used to facilitate the discovery of additional genes and regulatory elements involved in human eye disease.


Integrated adaptive optics optical coherence tomography and adaptive optics scanning laser ophthalmoscope system for simultaneous cellular resolution in vivo retinal imaging.
We describe an ultrahigh-resolution (UHR) retinal imaging system that combines adaptive optics Fourier-domain optical coherence tomography (AO-OCT) with an adaptive optics scanning laser ophthalmoscope (AO-SLO) to allow simultaneous data acquisition by the two modalities. The AO-SLO subsystem was integrated into the previously described AO-UHR OCT instrument with minimal changes to the latter. This was done in order to ensure optimal performance and image quality of the AO- UHR OCT. In this design both imaging modalities share most of the optical components including a common AO-subsystem and vertical scanner. One of the benefits of combining Fd-OCT with SLO includes automatic co-registration between two acquisition channels for direct comparison between retinal structures imaged by both modalities (e.g., photoreceptor mosaics or microvasculature maps). Because of differences in the detection scheme of the two systems, this dual imaging modality instrument can provide insight into retinal morphology and potentially function, that could not be accessed easily by a single system. In this paper we describe details of the components and parameters of the combined instrument, including incorporation of a novel membrane magnetic deformable mirror with increased stroke and actuator count used as a single wavefront corrector. We also discuss laser safety calculations for this multimodal system. Finally, retinal images acquired in vivo with this system are presented.


High level of vector competence of Aedes aegypti and Aedes albopictus from ten American countries as a crucial factor in the spread of Chikungunya virus.
Chikungunya virus (CHIKV) causes a major public health problem. In 2004, CHIKV began an unprecedented global expansion and has been responsible for epidemics in Africa, Asia, islands in the Indian Ocean region, and surprisingly, in temperate regions, such as Europe. Intriguingly, no local transmission of chikungunya virus (CHIKV) had been reported in the Americas until recently, despite the presence of vectors and annually reported imported cases. Here, we assessed the vector competence of 35 American Aedes aegypti and Aedes albopictus mosquito populations for three CHIKV genotypes. We also compared the number of viral particles of different CHIKV strains in mosquito saliva at two different times postinfection. Primarily, viral dissemination rates were high for all mosquito populations irrespective of the tested CHIKV isolate. In contrast, differences in transmission efficiency (TE) were underlined in populations of both species through the Americas, suggesting the role of salivary glands in selecting CHIKV for highly efficient transmission. Nonetheless, both mosquito species were capable of transmitting all three CHIKV genotypes, and TE reached alarming rates as high as 83.3% and 96.7% in A. aegypti and A. albopictus populations, respectively. A. albopictus better transmitted the epidemic mutant strain CHIKV_0621 of the East-Central-South African (ECSA) genotype than did A. aegypti, whereas the latter species was more capable of transmitting the original ECSA CHIKV_115 strain and also the Asian genotype CHIKV_NC. Therefore, a high risk of establishment and spread of CHIKV throughout the tropical, subtropical, and even temperate regions of the Americas is more real than ever. Until recently, the Americas had never reported chikungunya (CHIK) autochthonous transmission despite its global expansion beginning in 2004. Large regions of the continent are highly infested with Aedes aegypti and Aedes albopictus mosquitoes, and millions of dengue (DEN) cases are annually recorded. Indeed, DEN virus and CHIK virus (CHIKV) share the same vectors. Due to a recent CHIK outbreak affecting Caribbean islands, the need for a Pan-American evaluation of vector competence was compelling as a key parameter in assessing the epidemic risk. We demonstrated for the first time that A. aegypti and A. albopictus populations throughout the continent are highly competent to transmit CHIK irrespective of the viral genotypes tested. The risk of CHIK spreading throughout the tropical, subtropical, and even temperate regions of the Americas is more than ever a reality. In light of our results, local authorities should immediately pursue and reinforce epidemiological and entomological surveillance to avoid a severe epidemic.