Performance of a modified hybrid functional in the simultaneous description of stoichiometric and reduced TiO2 polymorphs.
Conventional density functionals with either the local density approximation (LDA) or the generalized gradient approximation (GGA) form of the exchange-correlation potential fail to describe the electronic structure of a large number of metal oxides. Both the LDA and the GGA grossly underestimate the band gaps of these materials which severely affect the description of oxygen vacancy point defect states in reduced samples. To find a pragmatic approach to simultaneously and accurately describe the atomic and electronic structures of the most common TiO2 polymorphs, we explore the effect of the percentage of exact, non-local, Fock exchange on the electronic structure of stoichiometric rutile and anatase. From these results, a modified hybrid functional is proposed to properly describe the atomic structures, formation enthalpies and electronic structures of rutile and anatase and, at the same time, the results of reduced samples are also in good agreement with the available experimental results. The present approach can be safely used to accurately describe numerous TiO2 based materials containing defects or realistic nanoparticles for which the required large unit cells or system sizes hinder the use of GW related techniques.


Factors associated with tocolytic hospitalizations in Taiwan: evidence from a population-based and longitudinal study from 1997 to 2004.
The use of tocolytic hospitalization in antenatal care is controversial and worthy of more research. We investigated individual, institutional, and area factors that affect the use of tocolytic hospitalizations in Taiwan where fertility has rapidly declined. Longitudinal data from the 1996 to 2004 National Health Insurance Research Database in Taiwan were used to identify tocolytic hospitalizations. The probit model was used to estimate factors associated with tocolytic hospitalizations. The decline in fertility was significantly associated with the probability of tocolytic hospitalizations. Several physician and institutional factors-including physician's age, hospital ownership, accreditation status, bed size, and teaching status-were also significantly correlated to the dependent variables. The provision of inpatient tocolysis is influenced not only by clinical considerations but also by physician, institutional, and area factors unrelated to clinical need. Fertility declines in Taiwan may have led obstetricians/gynecologists to provide more tocolysis to make up for their lost income. If the explanation is further validated, reimbursement policies may need to be reviewed to correct for overuse of inpatient tocolysis. The correlation could also be explained by the increasing use of artificial reproductive technologies and higher social value of newborns. In addition, the physician and institutional variations observed in the study indicate potential misuse of inpatient tocolysis that warrant further investigation.


Pediatric blunt splenic trauma: a comprehensive review.
Abdominal trauma is a leading cause of death in children older than 1 year of age. The spleen is the most common organ injured following blunt abdominal trauma. Pediatric trauma patients present unique clinical challenges as compared to adults, including different mechanisms of injury, physiologic responses, and indications for operative versus nonoperative management. Splenic salvage techniques and nonoperative approaches are preferred to splenectomy in order to decrease perioperative risks, transfusion needs, duration/cost of hospitalization, and risk of overwhelming postsplenectomy infection. Early and accurate detection of splenic injury is critical in both adults and children; however, while imaging findings guide management in adults, hemodynamic stability is the primary determinant in pediatric patients. After initial diagnosis, the primary role of imaging in pediatric patients is to determine the level and duration of care. We present a comprehensive literature review regarding the mechanism of injury, imaging, management, and complications of traumatic splenic injury in pediatric patients. Multiple patients are presented with an emphasis on the American Association for the Surgery of Trauma organ injury grading system. Clinical practice guidelines from the American Pediatric Surgical Association are discussed and compared with our experience at a large community hospital, with recommendations for future practice guidelines.


Variability of intensive care admission decisions for the very elderly.
Although increasing numbers of very elderly patients are requiring intensive care, few large sample studies have investigated ICU admission of very elderly patients. Data on pre triage by physicians from other specialities is limited. This observational cohort study aims at examining inter-hospital variability of ICU admission rates and its association with patients' outcomes. All patients over 80 years possibly qualifying for ICU admission who presented to the emergency departments (ED) of 15 hospitals in the Paris (France) area during a one-year period were prospectively included in the study. Main outcome measures were ICU eligibility, as assessed by the ED and ICU physicians; in-hospital mortality; and vital and functional status 6 months after the ED visit. 2646 patients (median age 86; interquartile range 83-91) were included in the study. 94% of participants completed follow-up (n = 2495). 12.4% (n = 329) of participants were deemed eligible for ICU admission by ED physicians and intensivists. The overall in-hospital and 6-month mortality rates were respectively 27.2% (n = 717) and 50.7% (n = 1264). At six months, 57.5% (n = 1433) of patients had died or had a functional deterioration. Rates of patients deemed eligible for ICU admission ranged from 5.6% to 38.8% across the participating centers, and this variability persisted after adjustment for patients' characteristics. Despite this variability, we found no association between level of ICU eligibility and either in-hospital death or six-month death or functional deterioration. In France, the likelihood that a very elderly person will be admitted to an ICU varies widely from one hospital to another. Influence of intensive care admission on patients' outcome remains unclear. ClinicalTrials.gov NCT00912600.


Investigation of the role of human breast milk in caries development.
The objective of this study was to determine the caries-related risk associated with human breast milk (HBM). First, the plaque pH of 18 children (12-24 months) was monitored before and after a five-minute feeding with HBM to determine the pH drop and minimum pH obtained. Second, Streptococcus sobrinus 6715 was cultured for 3 hr in HBM, and the increase in the number of colony forming units (cfus) and the culture pH was measured. Third, HBM was incubated for 24 hr with powdered enamel to determine the solubility of mineral in the absence of bacteria. Fourth, HBM was mixed with acid to determine the buffering capabilities. Finally, enamel windows were created on extracted premolar crowns (N = 33) that were colonized with Mutans Streptococci and incubated with HBM. Caries was assessed visually and radiographically for 12 weeks. One- and two-way ANOVAs of these five assays demonstrated that HBM did not cause a significant drop in plaque pH when compared to rinsing with water; HBM supported moderate bacterial growth; calcium and phosphate were actually deposited onto enamel powder after incubation with HBM; the buffer capacity of HBM was very poor; and HBM alone did not cause enamel decalcification even after 12 weeks exposure. However, when supplemented with 10% sucrose, HBM caused dentinal caries in 3.2 weeks. It is concluded that human breast milk is not cariogenic.


The Impact of Special Operations Medics and Corpsmen on Military Medical Student Training: A Qualitative Study.
Operation Gunpowder is a high-fidelity military medical field practicum conducted by the Uniformed Services University of Health Sciences, Bethesda, MD. During this multi-day combat simulation, Special Operations Medics and Corpsmen teach military medical students how to treat patients in an austere, resource-limited environment. To investigate the effectiveness of this teaching model, our research team used a qualitative phenomenological design to explore medical students' experiences being taught by Special Operations Medics and Corpsmen during Operation Gunpowder. We found two themes regarding the medical students' personal and professional development: an increased understanding of medics' skills and capabilities and the realization of their future roles as educators and leaders. Our study suggests that the use of Special Operations Corpsmen and Medics in medical student training is a valuable model for both military and civilian medical education and training.


CD31+ cells represent highly angiogenic and vasculogenic cells in bone marrow: novel role of nonendothelial CD31+ cells in neovascularization and their therapeutic effects on ischemic vascular disease.
Bone marrow (BM) cells play an important role in physiological and therapeutic neovascularization. However, it remains unclear whether any specific uncultured BM cell populations have higher angiogenic and vasculogenic activities. Moreover, there has been controversy regarding the vasculogenic ability of BM cells. Preliminary flow cytometric analysis showed that CD31, traditionally a marker for endothelial cells, is expressed in certain nonendothelial BM mononuclear cells in both human and mouse. Based on the conserved CD31 expression in the axis of hematopoietic stem/progenitor cells (HSC/HPCs) to endothelial cells, we further sought to determine the comprehensive vasculogenic and angiogenic characteristics of human and mouse BM-derived CD31(+) cells. Flow cytometric analysis demonstrated that all CD31(+) cells derived from BM were CD45(+) and expressed markers for both HSC/HPCs and endothelial cells. Comprehensive gene expression analyses revealed that BM-CD31(+) cells expressed higher levels of angiogenic genes than CD31(-) cells. Endothelial progenitor cells, as well as HSC/HPCs, were almost exclusively confined to the CD31(+) cell fraction, and culture of CD31(+) cells under defined conditions gave rise to endothelial cells. Finally, injection of CD31(+) cells into ischemic hindlimb repaired ischemia, increased expression of angiogenic and chemoattractive factors, and, in part, directly contributed to vasculogenesis, as demonstrated by both 3D confocal microscopy and flow cytometry. These data indicate that BM-CD31(+) cells represent highly angiogenic and vasculogenic cells and can be a novel and highly promising source of cells for cell therapy to treat ischemic cardiovascular diseases.


Neural induction in Xenopus requires inhibition of Wnt-beta-catenin signaling.
Canonical Wnt signals have been implicated in multiple events during early embryogenesis, including primary axis formation, neural crest induction, and A-P patterning of the neural plate. The mechanisms by which Wnt signals can direct distinct fates in cell types that are closely linked both temporally and spatially remains poorly understood. However, recent work has suggested that the downstream transcriptional mediators of this pathway, Lef/Tcf family DNA binding proteins, may confer distinct outcomes on these signals in some cellular contexts. In this study, we first examined whether inhibitory mutants of XTcf3 and XLef1 might block distinct Wnt-dependent signaling events during the diversification of cell fates in the early embryonic ectoderm. We found that a Wnt-unresponsive mutant of XTcf3 potently blocks neural crest formation, whereas an analogous mutant of XLef1 does not, and that the difference in activity mapped to the C-terminus of the proteins. Significantly, the inhibitory XTcf3 mutant also blocked expression of markers of anterior-most cell types, including cement gland and sensory placodes, indicating that Wnt signals are required for rostral as well as caudal ectodermal fates. Unexpectedly, we also found that blocking canonical Wnt signals in the ectoderm, using the inhibitory XTcf3 mutant or by other means, dramatically expanded the size of the neural plate, as evidenced by the increased expression of early pan-neural markers such as Sox3 and Nrp1. Conversely, we find that upregulation of canonical Wnt signals interferes with the induction of the neural plate, and this activity can be separated experimentally from Wnt-mediated neural crest induction. Together these findings provide important and novel insights into the role of canonical Wnt signals during the patterning of vertebrate ectoderm and indicate that Wnt inhibition plays a central role in the process of neural induction.


Cerebrospinal fluid biomarkers in clinical subtypes of early-onset Alzheimer's disease.
Accurate diagnosis of sporadic early-onset Alzheimer's disease (EOAD) can be challenging, and cerebrospinal fluid (CSF) biomarkers may assist in this process. We compared CSF indices between three EOAD subtypes: amnestic, logopenic progressive aphasia (LPA), and posterior cortical atrophy (PCA). We identified 21 amnestic EOAD, 20 LPA, and 12 PCA patients with CSF data, which included amyloid β1-42 (Aβ42), total tau (t-tau), phospho-tau181 (p-tau), and Aβ42/t-tau index (ATI) levels. Aβ42 and ATI levels were similar across groups, but t-tau and p-tau levels were significantly lower in PCA patients. The Aβ42 and ATI data confirm the commonality of the Aβ pathology in EOAD. The lower tau indices in PCA patients may reflect differences in the distribution of neurofibrillary tangles or rates of neurodegeneration.


The Association between IGF-1 Polymorphisms, IGF-1 Serum Levels, and Cognitive Functions in Healthy Adults: The Amsterdam Growth and Health Longitudinal Study.
Several studies have demonstrated an association between polymorphisms in the insulin-like growth factor-1 (IGF-1) gene and IGF-1 serum levels. IGF-1 levels have been associated with cognitive functioning in older persons and growth hormone deficient patients. The present study investigates whether IGF-1 polymorphisms, IGF-1 levels, and cognition are interconnected in healthy adults. Data of 277 participants (mean age: 42.4 years) of the Amsterdam Growth and Health Longitudinal Study on IGF-1 promoter polymorphisms, IGF-1 serum level, spatial working memory (SWM), paired associate learning (PAL), and IQ tests were analyzed. (M)ANOVAs were applied to confirm the associations between IGF-1 polymorphisms and IGF-1 levels and between IGF-1 levels and cognition. Three groups were distinguished based on specific IGF-1 polymorphism alleles: a homozygote 192 bp/192 bp genotype, a heterozygote 192 bp/x genotype, and a noncarrier x/x genotype. Although different IGF-1 levels were found for the three genotypes, performance on all cognitive tasks and IQ measures was similar. Despite the associations between IGF-1 polymorphisms and IGF-1 levels, no association was found between cognition and IGF-1 levels. It seems that IGF-1 does not play a role in the cognitive performance of healthy middle-aged adults. Possible, IGF-1 fulfills a more developmental and protective role in cognition which becomes apparent during childhood, old-age, or disease.


Agonism of CD11b reprograms innate immunity to sensitize pancreatic cancer to immunotherapies.
Although checkpoint immunotherapies have revolutionized the treatment of cancer, not all tumor types have seen substantial benefit. Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy in which very limited responses to immunotherapy have been observed. Extensive immunosuppressive myeloid cell infiltration in PDAC tissues has been postulated as a major mechanism of resistance to immunotherapy. Strategies concomitantly targeting monocyte or granulocyte trafficking or macrophage survival, in combination with checkpoint immunotherapies, have shown promise in preclinical studies, and these studies have transitioned into ongoing clinical trials for the treatment of pancreatic and other cancer types. However, compensatory actions by untargeted monocytes, granulocytes, and/or tissue resident macrophages may limit the therapeutic efficacy of such strategies. CD11b/CD18 is an integrin molecule that is highly expressed on the cell surface of these myeloid cell subsets and plays an important role in their trafficking and cellular functions in inflamed tissues. Here, we demonstrate that the partial activation of CD11b by a small-molecule agonist (ADH-503) leads to the repolarization of tumor-associated macrophages, reduction in the number of tumor-infiltrating immunosuppressive myeloid cells, and enhanced dendritic cell responses. These actions, in turn, improve antitumor T cell immunity and render checkpoint inhibitors effective in previously unresponsive PDAC models. These data demonstrate that molecular agonism of CD11b reprograms immunosuppressive myeloid cell responses and potentially bypasses the limitations of current clinical strategies to overcome resistance to immunotherapy.


Origin Specific Genomic Selection: A Simple Process To Optimize the Favorable Contribution of Parents to Progeny.
Modern crop breeding is in constant demand for new genetic diversity as part of the arms race with genetic gain. The elite gene pool has limited genetic variation and breeders are trying to introduce novelty from unadapted germplasm, landraces and wild relatives. For polygenic traits, currently available approaches to introgression are not ideal, as there is a demonstrable bias against exotic alleles during selection. Here, we propose a partitioned form of genomic selection, called Origin Specific Genomic Selection (OSGS), where we identify and target selection on favorable exotic alleles. Briefly, within a population derived from a bi-parental cross, we isolate alleles originating from the elite and exotic parents, which then allows us to separate out the predicted marker effects based on the allele origins. We validated the usefulness of OSGS using two nested association mapping (NAM) datasets: barley NAM (elite-exotic) and maize NAM (elite-elite), as well as by computer simulation. Our results suggest that OSGS works well in its goal to increase the contribution of favorable exotic alleles in bi-parental crosses, and it is possible to extend the approach to broader multi-parental populations.


Annotation of the zebrafish genome through an integrated transcriptomic and proteomic analysis.
Accurate annotation of protein-coding genes is one of the primary tasks upon the completion of whole genome sequencing of any organism. In this study, we used an integrated transcriptomic and proteomic strategy to validate and improve the existing zebrafish genome annotation. We undertook high-resolution mass-spectrometry-based proteomic profiling of 10 adult organs, whole adult fish body, and two developmental stages of zebrafish (SAT line), in addition to transcriptomic profiling of six organs. More than 7,000 proteins were identified from proteomic analyses, and ∼ 69,000 high-confidence transcripts were assembled from the RNA sequencing data. Approximately 15% of the transcripts mapped to intergenic regions, the majority of which are likely long non-coding RNAs. These high-quality transcriptomic and proteomic data were used to manually reannotate the zebrafish genome. We report the identification of 157 novel protein-coding genes. In addition, our data led to modification of existing gene structures including novel exons, changes in exon coordinates, changes in frame of translation, translation in annotated UTRs, and joining of genes. Finally, we discovered four instances of genome assembly errors that were supported by both proteomic and transcriptomic data. Our study shows how an integrative analysis of the transcriptome and the proteome can extend our understanding of even well-annotated genomes.


Gradual shutdown of virus production resulting in latency is the norm during the chronic phase of human immunodeficiency virus replication and differential rates and mechanisms of shutdown are determined by viral sequences.
Most CD4+ lymphocytes in lymph nodes of both asymptomatic HIV-1-infected individuals and AIDS patients are nonproductively or latently infected. It is not clear how these cells come about because infection of resting lymphocytes results in abortive infection and infection of activated lymphocytes results in productive infection. The frequency and mechanisms underlying nonproductive or latent HIV infections of normal CD4+ lymphocytes largely remain unexplored, and because HIV latency has principally been studied in latently infected cell clones of established cell lines, it is not even clear how often this type of infection occurs in cell lines. We demonstrate herein that chronic HIV replication in populations of normal phytohemagglutinin-stimulated peripheral blood CD4(+)-enriched lymphocytes, as well as an established T-cell line (CEM), gradually shuts down in the vast majority of cells. The nonproducing cells in these cultures still harbored HIV provirus, and HIV could be reactivated in CEM cells by treatment with phorbol ester, showing that this was latent infection. Thus, HIV's life cycle should probably be considered as consisting of two phases an acute exponential rise in production of virus progeny which levels at some peak, followed by a gradual decline of progeny production during the chronic phase leading to viral latency. Temporal analyses of the steady-state levels of viral mRNAs in populations of chronically infected CEM cells as virus production declined revealed the two mechanisms of HIV latency which have previously been described in the OM-10.1 and U1 or ACH-2 latently infected cell clones (i.e., apparent overall shutdown of HIV transcription and "blocked early-stage latency" involving enhanced splicing of viral pre-mRNAs) However, which mechanism was employed, as well as the rate of shutdown, depended on the virus strain.


Dysphagia following combined anterior-posterior cervical spine surgeries.
This study was undertaken to evaluate the incidence of and risk factors associated with the development of dysphagia following same-day combined anterior-posterior cervical spine surgeries. The records of 30 consecutive patients who underwent same-day combined anterior-posterior cervical spine surgery were reviewed. The presence of dysphagia was assessed by a formalized screening protocol using history/clinical presentation and a bedside swallowing test, followed by formal evaluation by speech and language pathologists and/or fiberoptic endoscopic evaluation of swallowing/modified barium swallow when necessary. Age, sex, previous cervical surgeries, diagnoses, duration of procedure, specific vertebral levels and number of levels operated on, degree of sagittal curve correction, use of anterior plate, estimated blood loss, use of recombinant human bone morphogenetic protein-2 (rhBMP-2), and length of hospital stay following procedures were analyzed. In the immediate postoperative period, 13 patients (43.3%) developed dysphagia. Outpatient follow-up data were available for 11 patients with dysphagia, and within this subset, all cases of dysphagia resolved subjectively within 12 months following surgery. The mean numbers of anterior levels surgically treated in patients with and without dysphagia were 5.1 and 4.0, respectively (p = 0.004). All patients (100%) with dysphagia had an anterior procedure that extended above C-4, compared with 58.8% of patients without dysphagia (p = 0.010). Patients with dysphagia had significantly greater mean correction of C2-7 lordosis than patients without dysphagia (p = 0.020). The postoperative sagittal occiput-C2 angle and the change in this angle were not significantly associated with the occurrence of dysphagia (p = 0.530 and p = 0.711, respectively). Patients with postoperative dysphagia had significantly longer hospital stays than those who did not develop dysphagia (p = 0.004). No other significant difference between the dysphagia and no-dysphagia groups was identified; differences with respect to history of previous anterior cervical surgery (p = 0.141), use of an anterior plate (p = 0.613), and mean length of anterior cervical operative time (p = 0.541) were not significant. The incidence of dysphagia following combined anterior-posterior cervical surgery in this study was comparable to that of previous reports. The risk factors for dysphagia that were identified in this study were increased number of anterior levels exposed, anterior surgery that extended above C-4, and increased surgical correction of C2-7 lordosis.


Cryptic diversity within the harmful dinoflagellate Akashiwo sanguinea in coastal Chinese waters is related to differentiated ecological niches.
Blooms of the harmful dinoflagellate Akashiwo sanguinea are responsible for the mass mortality of fish and invertebrates in coastal waters. This cosmopolitan species includes several genetically differentiated clades. Four clonal cultures were established by isolating single cells from Xiamen Harbour (the East China Sea) for morphological and genetic analyses. The cultures displayed identical morphology but were genetically different, thus revealing presence of cryptic diversity in the study area. New details of the apical structure complex of Akashiwo sanguinea were also found. To investigate whether the observed cryptic diversity was related to environmental differentiation, 634 cells were obtained from seasonal water samples collected from 2008 to 2012. These cells were sequenced by single-cell PCR. For comparison with Chinese material, additional large subunit ribosomal DNA sequences were obtained for three established strains from Malaysian and French waters. To examine potential ecological differentiation of the distinct genotypes, growth responses of the studied strains were tested under laboratory conditions at temperatures of 12°C to 33°C. These experiments showed four distinct ribotypes of A. sanguinea globally, with the ribotypes A and B co-occuring in Xiamen Harbour. Ribotype A of A. sanguinea was present year-round in Xiamen Harbour, but it only bloomed in the winter and spring, thus corresponding to the winter type. In contrast, A. sanguinea ribotype B bloomed only in the summer, corresponding to the summer type. This differentiation supports the temperature optimum conditions that were established for these two ribotypes in the laboratory. Ribotype A grew better at lower temperatures compared to ribotype B which preferred higher temperatures. These findings support the idea that various ribotypes of A. sanguinea correspond to distinct ecotypes and allopatric speciation occurred in different climatic regions followed by dispersal.


Keratinocyte-secreted laminin 5 can function as a transient receptor for human papillomaviruses by binding virions and transferring them to adjacent cells.
Human papillomaviruses (HPVs) replicate only in the terminally differentiating epithelium of the skin and mucosa. While infection of basal keratinocytes is considered a requirement for permissive infection, it remains unclear whether virions can specifically target basal cells for adsorption and uptake following epithelial wounding. We present evidence that HPV binds specifically to laminin 5 (LN5), a component of the extracellular matrix (ECM) secreted by migrating and basal keratinocytes. HPV type 11 capsids colocalized with LN5 in the ECM secreted by vaginal keratinocytes. Binding of both virions and virus-like particles to purified LN5 and to the LN5-rich ECM secreted by cultured keratinocytes was effectively blocked by pretreatment with anti-LN5 antibodies. HPV capsid binding to human cervical mucosa sections included the basement membrane which contains LN5. Cultured keratinocytes expressing alpha6 integrin, a transmembrane protein known to bind LN5, were readily infected by virions preadsorbed to LN5-containing substrates, whereas mutant keratinocytes lacking alpha6 integrin were relatively resistant to infection via this route. These findings suggest a model of natural HPV infection in which proliferating keratinocytes expressing alpha6 integrin at the site of epithelial wounding might be targeted by virions adsorbed transiently to LN5 secreted by migrating keratinocytes.


Zolpidem induces paradoxical metabolic and vascular changes in a patient with PVS.
Zolpidem is a non-benzodiazepine drug used for the therapy of insomnia, which has selectivity for stimulating the effect of GABA-A receptors. Recently, a paradoxical arousing effect of zolpidem in patients with severe brain damage has been repeatedly reported. A placebo-controlled magnetic resonance study was conducted to evaluate its effect on BOLD and metabolites spectral signals in a patient with severe brain injuries and an age-matched healthy volunteer. A multi-modal analysis was used to assess aspects in the pharmacologically-induced changes in the resting-state brain metabolism. A significantly increased BOLD signal was transiently localized in the left frontal cortices, bilateral anterior cingulated areas, left thalamus and right head of the caudate nucleus. The healthy subject showed a deactivation of the frontal, parietal and temporal cortices. BOLD signal changes were found to significantly correlate with concentrations of extravascular metabolites in the left frontal cortex. It is discussed that, when zolpidem attaches to modified GABA receptors of neurodormant brain cells, brain activation is induced. This might explain the significant correlations of BOLD signal changes and proton-MRS metabolites in this patient after zolpidem. It was concluded that proton-MRS and BOLD signal assessment could be used to study zolpidem-induced metabolic modulation in a resting state.


Increased epidermal growth factor receptor expression in metaplastic bronchial epithelium.
Epidermal growth factor receptor (EGFr) is expressed in human bronchial epithelial cells, and non-small cell lung cancers express increased EGFr. Squamous metaplasia of the bronchial epithelium occurs in chronic smokers and is considered an early premalignant change. In this study, EGFr expression was examined in biopsies of histologically normal and metaplastic bronchial tissues obtained from 69 smokers who were enrolled in a randomized placebo-controlled chemoprevention trial. This trial tested the effects of 6 months of treatment with 13-cis retinoic acid (13cRA) on bronchial metaplasia. EGFr expression was examined as a marker of bronchial metaplasia and response to 13cRA treatment. In bronchial biopsies obtained from patients in this study, EGFr expression was higher in metaplastic biopsies than in normal biopsies (P = 0.02). Smoking cessation during treatment correlated with reduced metaplasia (P < 0.001) and EGFr expression (P = 0.02), but multivariate analysis suggested that this effect of smoking cessation on EGFr expression was dependent upon reversal of bronchial metaplasia. 13cRA treatment did not alter EGFr expression (P = 0.23). Baseline EGFr expression levels in metaplastic biopsies did not predict metaplasia reversal. This study demonstrated that increased EGFr expression is a biomarker of bronchial metaplasia, but it did not support the hypothesis that EGFr is a biomarker of retinoid response in lung cancer chemoprevention trials.


Comparison of LDL-cholesterol direct measurement with the estimate using the Friedewald formula in a sample of 10,664 patients.
To compare direct measurement of LDL-cholesterol (LDL-C) determined by a homogeneous method with LDL-cholesterol estimation determined by the Friedewald formula in a large heterogeneous population. The measurements of total cholesterol (TC) and triglycerides (TG) were performed using traditional enzymatic methods. The measurements of HDL-C and LDL-C were performed using direct methods with no precipitation, and the estimation of the LDL-C fraction was calculated using the Friedewald formula. On linear regression analysis, the 2 methods had extremely significant correlation coefficients (P < 0.001). However, the Friedewald formula had a positive bias in regard to the direct method, more pronounced with TC levels > 201 mg/dL. This positive bias also occurred in regard to TG levels < or =150 mg/dL. No bias was observed between the methods for TG levels ranging from 151 to 200 mg/dL and from 201 to 300 mg/dL. On the other hand, for TG levels ranging from 301 to 400 mg/dL, this bias of the Friedewald formula became negative. The Friedewald formula did not have a homogeneous performance for estimating LDL-C levels in samples with different TG levels as compared with that of the direct method, what could launch doubts on patients classification on the risk of developing coronary artery disease.


Permanent cerebrospinal fluid diversion in subarachnoid hemorrhage: Influence of physician practice style.
Acute hydrocephalus (HCP) after aneurysmal subarachnoid hemorrhage (SAH) often persists. Our previous study described factors that singly and combined in a formula correlate with permanent CSF diversion. We now aimed to determine whether the same parameters are applicable at an institution with different HCP management practice. We reviewed records of 181 consecutive patients who presented with SAH and received an external ventricular drain (EVD) for acute HCP. After exclusion and inclusion criteria were met, 71 patients were analyzed. Data included admission Fisher and Hunt and Hess grades, aneurysm location, treatment modality, ventricle size, CSF cell counts and protein levels, length of stay (LOS) in the hospital, and the presence of craniectomy. Outcome measures were: (1) initial EVD challenge outcome; (2) shunting within 3 months; and (3) LOS. Shunting correlated with Hunt and Hess grade, CSF protein, and the presence of craniectomy. The formula derived in our previous study demonstrated a weaker correlation with initial EVD challenge failure. Several parameters that correlated with shunting in the previous study were instead associated with LOS in this study. The decision to shunt depends on management choices in the context of a disease process that may improve over time. Based on the treatment strategy, the shunting rate may be lowered but LOS increased. Markers of disease severity in patients with HCP after SAH correlate with both shunt placement and LOS. This is the first study to directly evaluate the effect of different practice styles on the shunting rate. Differences in HCP management practices should inform the design of prospective studies.


Attenuated Toxoplasma gondii Stimulates Immunity to Pancreatic Cancer by Manipulation of Myeloid Cell Populations.
Suppressive myeloid cells represent a significant barrier to the generation of productive antitumor immune responses to many solid tumors. Eliminating or reprogramming suppressive myeloid cells to abrogate tumor-associated immune suppression is a promising therapeutic approach. We asked whether treatment of established aggressive disseminated pancreatic cancer with the immunotherapeutic attenuated Toxoplasma gondii vaccine strain CPS would trigger tumor-associated myeloid cells to generate therapeutic antitumor immune responses. CPS treatment significantly decreased tumor-associated macrophages and markedly increased dendritic cell infiltration of the pancreatic tumor microenvironment. Tumor-resident macrophages and dendritic cells, particularly cells actively invaded by CPS, increased expression of costimulatory molecules CD80 and CD86 and concomitantly boosted their production of IL12. CPS treatment increased CD4(+) and CD8(+) T-cell infiltration into the tumor microenvironment, activated tumor-resident T cells, and increased IFNγ production by T-cell populations. CPS treatment provided a significant therapeutic benefit in pancreatic tumor-bearing mice. This therapeutic benefit depended on IL12 and IFNγ production, MyD88 signaling, and CD8(+) T-cell populations. Although CD4(+) T cells exhibited activated effector phenotypes and produced IFNγ, CD4(+) T cells as well as natural killer cells were not required for the therapeutic benefit. In addition, CD8(+) T cells isolated from CPS-treated tumor-bearing mice produced IFNγ after re-exposure to pancreatic tumor antigen, suggesting this immunotherapeutic treatment stimulated tumor cell antigen-specific CD8(+) T-cell responses. This work highlights the potency and immunotherapeutic efficacy of CPS treatment and demonstrates the significance of targeting tumor-associated myeloid cells as a mechanism to stimulate more effective immunity to pancreatic cancer.


The PB1 and the ZZ domain of the autophagy receptor p62/SQSTM1 regulate the interaction of p62/SQSTM1 with the autophagosome protein LC3B.
Autophagy is a highly conserved cellular process that allows degradation of large macromolecules. p62/SQSTM1 is a key adaptor protein that interacts both with material to be degraded and with LC3 at the autophagosome, enabling degradation of cargos such as protein aggregates, lipid droplets and damaged organelles by selective autophagy. Dysregulation of autophagy contributes to the pathogenesis of many diseases. In this study we investigated if the interaction of p62/SQSTM1 with LC3B could be regulated. We purified full-length p62/SQSTM1 and established an in vitro assay that measures the interaction with LC3B. We used the assay to determine the role of the different domains of p62/SQSTM1 in the interaction with LC3B. We identified a mechanism of regulation of p62/SQSTM1 where the ZZ and the PB1 domains regulate the exposure of the LIR-sequence to enable or inhibit the interaction with LC3B. A mutation to mimic the phosphorylation of a site on the ZZ domain leads to increased interaction with LC3B. Also, a small compound that binds to the ZZ domain enhances interaction with LC3B. Dysregulation of these mechanisms in p62/SQSTM1 could have implications for diseases where autophagy is affected. In conclusion, our study highlights the regulated nature of p62/SQSTM1 and its ability to modulate the interaction with LC3B through a LIR-sequence Accessibility Mechanism (LAM). Furthermore, our findings suggest the potential for pharmacological modulation of the exposure of LIR, paving the way for future therapeutic strategies. This article is protected by copyright. All rights reserved.


Study of a synthetic human olfactory receptor 17-4: expression and purification from an inducible mammalian cell line.
In order to begin to study the structural and functional mechanisms of olfactory receptors, methods for milligram-scale purification are required. Here we demonstrate the production and expression of a synthetically engineered human olfactory receptor hOR17-4 gene in a stable tetracycline-inducible mammalian cell line (HEK293S). The olfactory receptor gene was fabricated from scratch using PCR-based gene-assembly, which facilitated codon optimization and attachment of a 9-residue bovine rhodopsin affinity tag for detection and purification. Induction of adherent cultures with tetracycline together with sodium butyrate led to hOR17-4 expression levels of approximately 30 microg per 150 mm tissue culture plate. Fos-choline-based detergents proved highly capable of extracting the receptors, and fos-choline-14 (N-tetradecylphosphocholine) was selected for optimal solubilization and subsequent purification. Analysis by SDS-PAGE revealed both monomeric and dimeric receptor forms, as well as higher MW oligomeric species. A two-step purification method of immunoaffinity and size exclusion chromatography was optimized which enabled 0.13 milligrams of hOR17-4 monomer to be obtained at >90% purity. This high purity of hOR17-4 is not only suitable for secondary structural and functional analyses but also for subsequent crystallization trials. Thus, this system demonstrates the feasibility of purifying milligram quantities of the GPCR membrane protein hOR17-4 for fabrication of olfactory receptor-based bionic sensing device.


Endothelial cell death after ionizing radiation does not impair vascular structure in mouse tumor models.
The effect of radiation therapy on tumor vasculature has long been a subject of debate. Increased oxygenation and perfusion have been documented during radiation therapy. Conversely, apoptosis of endothelial cells in irradiated tumors has been proposed as a major contributor to tumor control. To examine these contradictions, we use multiphoton microscopy in two murine tumor models: MC38, a highly vascularized, and B16F10, a moderately vascularized model, grown in transgenic mice with tdTomato-labeled endothelium before and after a single (15 Gy) or fractionated (5 × 3 Gy) dose of radiation. Unexpectedly, even these high doses lead to little structural change of the perfused vasculature. Conversely, non-perfused vessels and blind ends are substantially impaired after radiation accompanied by apoptosis and reduced proliferation of their endothelium. RNAseq analysis of tumor endothelial cells confirms the modification of gene expression in apoptotic and cell cycle regulation pathways after irradiation. Therefore, we conclude that apoptosis of tumor endothelial cells after radiation does not impair vascular structure.


Dopant assisted-atmospheric pressure photoionization (DA-APPI) liquid chromatography-mass spectrometry for the quantification of 27-hydroxycholesterol in plasma.
27-Hydroxycholesterol (27OH-Chol) is of potential diagnostic interest due to its role in maintaining whole-body cholesterol homeostasis. Dopant assisted-atmospheric pressure photoionization (DA-APPI) has improved the sensitivity of 27OH-Chol analysis, in comparison to the published LC-APCI-MS method, allowing quantification from a very low amount of sample (<or=50 microL plasma). The method was validated for quantification from 50 microL and 15 microL plasma, with the limit of quantification (LOQ) of 10 ng/mL and 40 ng/mL plasma, respectively. A further advantage is that no prior derivatization was needed, unlike the LC-ESI-MS or the standard GC-MS method. The method validation also resulted in good linearity and recovery of 91-106%. The within-day and between-day coefficient of variation were less than 15% while giving the accuracy of 90.9-113.4%. This report summarizes the effects of some critical parameters on the ionization of 27OH-Chol using APPI, as well as the validation of the method. The sensitivity achieved with DA-APPI broadens the usefulness of the LC-MS method in clinical applications.


The aromaticity of dicupra[10]annulenes.
An extensive theoretical investigation of the electronic structure of a tested fair model dicupra[10]annulene compound, based on the analysis of atom-pair delocalization indices, Bader's molecular graph, the inspection of the canonical molecular orbitals, the z components of their Nuclear Independent Chemical Shifts, NICS(0)zz, and the normalized Giambiagi multicenter delocalization indices, concludes that the perimeter aromaticity of the dicupra[10]annulene ring is consistent with both 10 and 14 π-electron Hückel aromatic 10-membered rings. In either case, the 10-membered ring encloses two 6 π-electron aromatic inner rings, hinged at the Cu-Cu bond. This work demonstrates that the aromaticity of dicupra[10]annulenes closely resembles that of naphthalene. Hence, they are best regarded as metalla-polyacenes, which could make the building blocks of extended structures such as metalated nanotubes.


Attenuation of pathogenicity of fowl plague virus by recombination with other influenza A viruses nonpathogenic for fowl: nonexculsive dependence of pathogenicity on hemagglutinin and neuraminidase of the virus.
A number of antigenic hybrids of influenza A viruses were produced possessing either the hemagglutinin or the neuraminidase of fowl plague virus and the corresponding antigen derived from another influenza A virus. Other recombinants were obtained carrying both surface antigens of fowl plague virus but differing from the parent in certain biological properties. None of the recombinants isolated were pathogenic for adult chickens. Most recombinants obtained after crosses between reciprocal recombinants carrying both fowl plague virus surface antigens were also apathogenic for chickens. Using the same parent recombinants for double infection some of the progeny "back-recombinants" were pathogenic, whereas others were not. From these results it is concluded that the surface components do not by themselves determine the pathogenicity of influenza A viruses.


Protective effects of Nigella sativa oil and thymoquinone against toxicity induced by the anticancer drug cyclophosphamide.
Constituents of the Nigella sativa seed are reported to possess potent antioxidant effects. Treatment with anticancer drugs such as cyclophosphamide (CTX) is associated with significant toxicity due to over-production of reactive oxygen species, resulting in increased levels of oxidative stress. The aim of this study is to test whether or not N. sativa L oil (NSO) or its active ingredient, thymoquinone (TQ), can reduce CTX-induced toxicity. Male albino rats were treated with intraperitoneal administration of phosphate buffered saline (PBS) or 200 mg/Kg CTX followed by intragastric administration of NSO or TQ on alternate days for 12 days. Administration of NSO and TQ was initiated 6 h before or after CTX injection. Twenty-four hours after the last NSO and TQ treatment, blood and liver were harvested to analyse toxicity-related parameters. Treatment with CTX induced significant toxicity as shown by decrease in haemoglobin concentration and increases in blood sugar levels, activities of liver enzymes, bilirubin, urea, creatinine, lipids (triglyceride, cholesterol and low-density lipoprotein (LDL)-cholesterol) and lipid peroxidation in the liver. Treatment with NSO or TQ induced significant reduction in overall toxicity. The antitoxic effects of NSO and TQ were associated with induction of antioxidant mechanisms. These results suggest that administration of NSO or TQ can lower CTX-induced toxicity as shown by an up-regulation of antioxidant mechanisms, indicating a potential clinical application for these agents to minimise the toxic effects of treatment with anticancer drugs.


Effect of histamine on proliferation of normal human adult lung fibroblasts.
Fibrotic lung tissue shows increased connective tissue deposition and fibroblast proliferation and in addition a substantial increase in mast cell numbers in and around the fibrotic area. To elucidate the question of whether products of mast cells affect the proliferative behaviour of structural cells in the lung and thereby contribute to fibrogenesis, the effect of histamine, a prominent mast cell derived mediator, on the in vitro proliferation of primary cultures of normal adult human lung fibroblasts was studied. Histamine enhanced fibroblast proliferation in a dose dependent manner, with an optimum effect at a physiological concentration of 10(-7) mol/l. This effect occurred when cells were exposed to histamine at restricted times during cell growth and was shown to depend in part on the stage of the cell cycle reached by the fibroblasts. The histamine induced proliferation was mediated through an H2 histamine receptor on the fibroblast, being inhibited by cimetidine, an H2 antagonist, and not by pyrilamine maleate, an antagonist of the H1 receptor. Mast cell products such as histamine may interact with and promote the increased fibroblast proliferation found in pulmonary fibrosis.


Phase-Dependent Suppression of Beta Oscillations in Parkinson's Disease Patients.
Synchronized oscillations within and between brain areas facilitate normal processing, but are often amplified in disease. A prominent example is the abnormally sustained beta-frequency (∼20 Hz) oscillations recorded from the cortex and subthalamic nucleus of Parkinson's disease patients. Computational modeling suggests that the amplitude of such oscillations could be modulated by applying stimulation at a specific phase. Such a strategy would allow selective targeting of the oscillation, with relatively little effect on other activity parameters. Here, activity was recorded from 10 awake, parkinsonian patients (6 male, 4 female human subjects) undergoing functional neurosurgery. We demonstrate that stimulation arriving on a particular patient-specific phase of the beta oscillation over consecutive cycles could suppress the amplitude of this pathophysiological activity by up to 40%, while amplification effects were relatively weak. Suppressive effects were accompanied by a reduction in the rhythmic output of subthalamic nucleus (STN) neurons and synchronization with the mesial cortex. While stimulation could alter the spiking pattern of STN neurons, there was no net effect on firing rate, suggesting that reduced beta synchrony was a result of alterations to the relative timing of spiking activity, rather than an overall change in excitability. Together, these results identify a novel intrinsic property of cortico-basal ganglia synchrony that suggests the phase of ongoing neural oscillations could be a viable and effective control signal for the treatment of Parkinson's disease. This work has potential implications for other brain diseases with exaggerated neuronal synchronization and for probing the function of rhythmic activity in the healthy brain.SIGNIFICANCE STATEMENT In Parkinson's disease (PD), movement impairment is correlated with exaggerated beta frequency oscillations in the cerebral cortex and subthalamic nucleus (STN). Using a novel method of stimulation in PD patients undergoing neurosurgery, we demonstrate that STN beta oscillations can be suppressed when consecutive electrical pulses arrive at a specific phase of the oscillation. This effect is likely because of interrupting the timing of neuronal activity rather than excitability, as stimulation altered the firing pattern of STN spiking without changing overall rate. These findings show the potential of oscillation phase as an input for "closed-loop" stimulation, which could provide a valuable neuromodulation strategy for the treatment of brain disorders and for elucidating the role of neuronal oscillations in the healthy brain.


Anodic cyclization reactions: the total synthesis of alliacol a.
An anodic cyclization-Friedel Crafts alkylation strategy has been used to rapidly assemble the core ring system of alliacol A and to complete a formal total synthesis of the natural product. The anodic cyclization reaction was used to effect the coupling of a nucleophilic furan ring to the normally nucleophilic carbon of a silyl enol ether. The substrate for this initial cyclization reaction contained all of the carbons needed for completing the total synthesis. The electrolysis proceeded in high yield and could be accomplished with the use of a 6 V lantern battery.


Helicobacter pylori cagA, iceA and vacA genotypes in patients with gastric cancer in Taiwan.
Helicobacter pylori (H pylori ) has been linked to chronic gastritis, peptic ulcer, gastric cancer and MALT-lymphoma. The link of genotypes of H pylori to gastric cancer remains controversial. The aim of this study was to investigate the H pylori vacA alleles, cagA and iceA in patients with gastric cancer in Taiwan. Patients with gastric cancer, peptic ulcer and chronic gastritis were enrolled in this study. We obtained biopsy specimens from the stomach at least 2 cm away from the tumor margin in patients with gastric cancer, and from the antrum of stomach in patients with peptic ulcer or chronic gastritis. DNA extraction and polymerase chain reaction were used to detect the presence or absence of cagA and to assess the polymorphism of vacA and iceA. A total of 168 patients (gastric ulcer: 77, duodenal ulcer: 66, and chronic gastritis: 25) were found to have positive PCR results of the biopsy specimens from patients with peptic ulcer and chronic gastritis. We found positive cagA (139/168, 83%), m2 (84/168, 50%) and iceA1 (125/168, 74%) strains in the majority of patients. In patients with gastric cancer, the vacA s1a and s1c subtypes were less commonly found than those in non-cancer patients (35/66 vs 127/168, P = 0.0001 for s1a and 13/66 vs 93/168, P<0.0001 for s1c). In the middle region, the m1T strain in patients with gastric cancer was more than that of non-cancer patients (23/66 vs 33/168, P = 0.02). In Taiwan, H pylori with positive vacA s1a, cagA and iceA1 strains are found in the majority of patients with gastric cancer or non-cancer patients. In patients with gastric cancer, the vacA s1a and s1c subtypes are less and m1T is more than in patients with peptic ulcer and chronic gastritis.


Influence of Process Parameters on the Tribological Behavior of PEO Coatings on CP-Titanium 4+ Alloys for Biomedical Applications.
Wear resistant ceramic coatings were generated on novel commercially pure titanium grade 4+ alloys by the plasma electrolytic oxidation technique (PEO) in an aluminate and zirconia containing electrolyte. The coatings were obtained adopting a full regular two-level factorial design of experiments (DoE) varying the PEO process parameters current density, repetition rate and duty cycle. The generated coatings were characterized with respect to its wear resistance and mechanical properties by reciprocal ball-on-flat tests and nanoindentation measurements. Thickness, morphology and phase formation of the PEO coatings was analyzed by scanning electron microscopy (SEM/EDS) and X-ray diffraction. XRD results indicate the formation of crystalline aluminium titanate (TiAl2O5) as well as t-ZrO2 and alumina leading to an increase in hardness and wear resistance of the PEO coatings. Evaluation of the DoE's parameter interaction shows that the main effects for generating wear resistant coatings are current density and repetition rate. In particular, the formation of mechanically stable and adhesive corundum and zirconia containing coatings with increasing current density and frequency turned out to be responsible for the improvement of the tribological properties. Overall, the PEO processing significantly improves the wear resistance of the CP titanium base alloy.


MicrobPad MD: microbial pathogen diagnostic methods database.
Medical pathogens induce infections, illnesses and sometimes serious medical conditions in the infected hosts. Diagnosis of these pathogens is important for proper treatment and investigation of pathogenesis processes. Molecular techniques have been developed for facilitating accurate, sensitive and low-cost diagnosis of these pathogens. Based on these techniques, diagnostic devices have been developed for a number of pathogens. More devices are needed for comprehensive coverage of medical pathogens. To facilitate the development of these devices, a database with integrated information about diagnostic methods, targets, and primers/probes for the known bacterial, fungal and viral pathogens is needed. We developed the microbial pathogen diagnostic methods database MicrobPad MD (http://bidd.nus.edu.sg/group/MicrobPad/MicrobPad.asp or http://pha-bidd.nus.edu.sg/group/MicrobPad/MicrobPad.asp) to provide comprehensive information about the molecular diagnostic techniques, targets, primers/probes, detection procedures and conditions, and tested diagnostic accuracies and limit of diagnosis for 314 bacterial, fungal and viral species from 61 genera. While available, additional information such as pathogen strains and hosts, tissue distribution or habitats, cultivation methods, biochemical characteristics, virulence factors, morphology, diseases, symptoms, treatment and prevention methods are provided. Our Database covers 242 gene targets, 700 primers/probes, 340 virulence factors, and 261 diseases. Cross-links to the NCBI genome and SwissProt/UniProt databases are provided.


gamma-Aminobutyric acidA receptor heterogeneity in rat central nervous system: studies with clonazepam and other benzodiazepine ligands.
The properties of [3H]clonazepam, [3H]diazepam and [3H]zolpidem (N,N,6[trimethyl-2-(4-methyl-phenyl)imidazo[1,2-a]pyridine-3-acetamide hemitratrate) binding to synaptic membranes of cerebellum, cortex, olfactory bulb, striatum and spinal cord of rat were compared to the binding properties of [3H]flunitrazepam, [3H]flumazenil and [3H]midazolam. In the cerebellar, cortical and olfactory bulb membranes, the density of high-affinity binding sites of all these tritiated benzodiazepine (BZ) ligands is almost identical. In contrast, in the striatum, the density of [3H]clonazepam and [3H]zolpidem binding sites is approximately 60 and 30%, respectively, of the density of [3H]diazepam, [3H]flunitrazepam or [3H]flumazenil sites. In spinal cord membranes, the number of high-affinity binding sites of [3H]clonazepam and [3H]zolpidem is less than 20% of the number of binding sites for [3H]diazepam, [3H]flunitrazepam, [3H]flumazenil and [3H]midazolam. Moreover, the displacement of [3H]flunitrazepam from spinal cord membranes by clonazepam and zolpidem was characterized by high IC50 values and Hill slopes significantly less than 1. Because [3H]BZ ligand binding in the spinal cord is enhanced by gamma-aminobutyric acid (GABA), these data suggest that different regions of the rat central nervous system may contain different GABA-BZ receptor subtypes. The different pharmacological properties of clonazepam, diazepam and zolpidem (i.e., regarding their ability to enhance bicuculline seizure threshold, to decrease locomotor activity, to induce ataxia or to elicit anticonflict action) further support the concept that in the rat central nervous system preferential occupancy of heterogeneous GABAA receptors by these drugs can be related to their effects on behavior.


Role of myosin phosphatase isoforms in cGMP-mediated smooth muscle relaxation.
In vitro experiments showing the activation of the myosin phosphatase via heterophilic leucine zipper interactions between its targeting subunit (MYPT1) and cGMP-dependent protein kinase I suggested a pathway for smooth muscle relaxation (Surks, H. K., Mochizuki, N., Kasai, Y., Georgescu, S. P., Tang, K. M., Ito, M., Lincoln, T. M., and Mendelsohn, M. E. (1999) Science 286, 1583-1587). The relationship between MYPT1 isoform expression and smooth muscle responses to cGMP signaling in vivo has not been explored. MYPT1 isoforms that contain or lack a C-terminal leucine zipper are generated in birds and mammals by cassette-type alternative splicing of a 31-nucleotide exon. The avian and mammalian C-terminal isoforms are highly conserved and expressed in a tissue-specific fashion. In the mature chicken the tonic contracting aorta and phasic contracting gizzard exclusively express the leucine zipper positive and negative MYPT1 isoforms, respectively. Expression of the MYPT1 isoforms is also developmentally regulated in the gizzard, which switches from leucine zipper positive to negative isoforms around the time of hatching. This switch coincides with the development in the gizzard of a cGMP-resistant phenotype, i.e. inability to dephosphorylate myosin and relax in response to 8-bromo-cGMP after calcium activation. Furthermore, association of cGMP-dependent protein kinase I with MYPT1 is detected by immunoprecipitation only in the tissue that expresses the leucine zipper positive isoform of MYPT1. These results suggest that the regulated splicing of MYPT1 is an important determinant of smooth muscle phenotypic diversity and the variability in the response of smooth muscles to the calcium desensitizing effect of cGMP signaling.


Group-level patterns emerge from individual speed as revealed by an extremely social robotic fish.
Understanding the emergence of collective behaviour has long been a key research focus in the natural sciences. Besides the fundamental role of social interaction rules, a combination of theoretical and empirical work indicates individual speed may be a key process that drives the collective behaviour of animal groups. Socially induced changes in speed by interacting animals make it difficult to isolate the effects of individual speed on group-level behaviours. Here, we tackled this issue by pairing guppies with a biomimetic robot. We used a closed-loop tracking and feedback system to let a robotic fish naturally interact with a live partner in real time, and programmed it to strongly copy and follow its partner's movements while lacking any preferred movement speed or directionality of its own. We show that individual differences in guppies' movement speed were highly repeatable and in turn shaped key collective patterns: a higher individual speed resulted in stronger leadership, lower cohesion, higher alignment and better temporal coordination of the pairs. By combining the strengths of individual-based models and observational work with state-of-the-art robotics, we provide novel evidence that individual speed is a key, fundamental process in the emergence of collective behaviour.


Identification of MYO4, a second class V myosin gene in yeast.
We have isolated a fourth myosin gene (MYO4) in yeast (Saccharomyces cerevisiae). MYO4 encodes a approximately 170 kDa (1471 amino acid) class V myosin, using the classification devised by Cheney et al. (1993a; Cell Motil. Cytoskel. 24, 215-223); the motor domain is followed by a neck region containing six putative calmodulin-binding sites and a tail with a short potential 'coiled-coil' domain. A comparison with other myosins in GenBank reveals that Myo4 protein is most closely related to the yeast Myo2 protein, another class V myosin. Deletion of MYO4 produces no detectable phenotype, either alone or in conjunction with mutations in myo2 or other myosin genes, the actin gene, or secretory genes. However, overexpression of MYO4 or MYO2 results in several morphological abnormalities, including the formation of short strings of unseparated cells in diploid strains, or clusters of cells in haploid strains. Alterations of MYO4 or MYO2 indicate that neither the motor domains nor tails of these myosins are required to confer the overexpression phenotype, whereas the neck region may be required. Although this phenotype is similar to that seen upon MYO1 deletion, we provide evidence that the overexpression of Myo4p or Myo2p is not simply interfering with Myo1p function.


Immediate loading of complete-arch fixed prostheses for edentulous maxillae after flapless guided implant placement: a 1-year prospective clinical study.
The purpose of this study was to evaluate prospectively the clinical effectiveness of immediate loading of implants with complete-arch fixed prostheses in edentulous maxillae after the implants were placed with flapless guided surgery. Patients with edentulous maxillae who visited a single dental clinic during the recruitment period (April 2011 to May 2012) were selected by consecutive sampling and predetermined selection criteria. Four to six implants were placed in each patient with flapless guided surgery. The outcome measures were insertion torque, surgical time, postoperative pain (100-mm visual analog scale), marginal bone level change, implant survival, and oral health-related quality of life. Patients were followed for up to 1 year. Fifty patients received 290 implants. All implants were immediately loaded 2 hours after placement with prefabricated fixed provisional prostheses. Two patients dropped out of the study. Hence, analyses were conducted for 278 implants in 48 patients (26 men, 22 women; mean age 56.0 ± 8.3 years; age range, 34 to 74 years). The average insertion torque was 57.7 ± 16.6 Ncm, the average surgical time was 22.9 ± 4.7 minutes, and the average score for postoperative pain was 14.7 ± 9.3. One year after immediate loading, the implant survival rate was 98.6%. Four implants showed mobility early after implant placement and were lost. Mean marginal bone level changes were -0.32 ± 0.43 mm. Scores on the Oral Health Impact Profile-54 (Japanese version) improved significantly after prosthetic treatment. The results of this study indicate that this treatment method for edentulous maxillae is predictable, with a high implant survival rate and a clinically reasonable improvement in oral health-related quality of life.


Platelet serotonin transporter and 5-HT2A receptor binding in adolescents with eating disorders.
The pathogenetic involvement of the serotonergic system in eating disorders is an established finding. Conclusions from platelet studies are based on results from investigations of subjects with a mean age of 20 years or more. The aim was to investigate whether previous findings in adults are valid also for adolescents who are examined within a relatively short interval after the onset of the eating disorder. [(3)H]paroxetine binding to the platelet serotonin transporter and [(3)H]lysergic acid diethylamide ([(3)H]LSD) binding to the 5-HT2A receptor was studied in 15 female adolescents with eating disorders (11 with anorexia nervosa and 4 with clearly anorectic eating behaviour not fulfilling the criteria for anorexia nervosa) and 32 controls. The patients revealed a higher density of serotonin transporters and a lower density of 5-HT2A receptors compared with healthy controls of the same age (775 ± 165 vs. 614 ± 111 fmol/mg protein (p = 0.003) for [(3)H]paroxetine binding and 215 ± 59 vs. 314 ± 151 fmol/mg protein (p = 0.005) for [(3)H]LSD binding). The findings of increased density of platelet serotonin transporters and reduced density of 5-HT2A receptors differ from previous results in older patients. The lower patient age and the short duration of disease in the present study, possibly in conjunction with variations in stress-related psychological and biological factors, may have caused these differences. Although the present findings contradict prevailing evidence, they add further information concerning the nature of serotonergic involvement in eating disorders and indicate that demographic and course-related factors might influence the regulation of the serotonin system in these disorders.


Accumulation of persistent organic pollutants in consumers of eel from polluted rivers compared to marketable eel.
Globally, many river sediments are seriously contaminated with persistent organic pollutants (POPs) known to accumulate in aquatic food. In the Netherlands, toxicological risks of human exposure to dioxins and dioxin-like compounds led to a ban on eel fishing in the Rhine-Meuse delta. The aim of this study is to investigate differences in serum POP levels in consumers of eel from high-polluted areas and consumers of eel from low-polluted areas or aquaculture. In total 80 Dutch men were included, aged 40-70 years, with a habitual eel consumption of at least one portion (150 g) per month. Total levels of dioxins and dioxin-like compounds were measured in serum of all participants with the DR CALUX bioassay, validated with GC-MS. For a subgroup of 38 participants extensive POP measurements were performed. We revealed that consumption of eel from polluted rivers resulted in 2.5 and up to 10 times increased levels of dioxins and polychlorinated biphenyls (PCBs) respectively compared to controls. The highest PCB levels were detected for PCB 153, with a median level of 896 ng/g lipid and a maximum level of 5000 ng/g lipid in the high-exposed group. Furthermore, hydroxylated PCB metabolites (OH-PCBs: sum of 4-OH-CB107, 4-OH-CB146, 4'-OH-CB172, and 4-OH-CB187) were 8 times higher in men who consumed eel from polluted areas, and detected at levels (median 4.5 ng/g ww) reported to cause adverse health effects. Also, the majority of the perfluoroalkyl substances (PFASs) were significantly higher in consumers of eel from pullulated areas. In conclusion, this study is the first to reveal that (past) consumption of eel from polluted rivers resulted in high body burdens of dioxins, PCBs, OH-PCBs and PFASs. We confirmed the predictions made in a former risk assessment, and the high levels of dioxins and dioxin-like compounds as well as the OH-PCBs are of health concern.


Modulation of deregulated chaperone-mediated autophagy by a phosphopeptide.
The P140 peptide, a 21-mer linear peptide (sequence 131-151) generated from the spliceosomal SNRNP70/U1-70K protein, contains a phosphoserine residue at position 140. It significantly ameliorates clinical manifestations in autoimmune patients with systemic lupus erythematosus and enhances survival in MRL/lpr lupus-prone mice. Previous studies showed that after P140 treatment, there is an accumulation of autophagy markers sequestosome 1/p62 and MAP1LC3-II in MRL/lpr B cells, consistent with a downregulation of autophagic flux. We now identify chaperone-mediated autophagy (CMA) as a target of P140 and demonstrate that its inhibitory effect on CMA is likely tied to its ability to alter the composition of HSPA8/HSC70 heterocomplexes. As in the case of HSPA8, expression of the limiting CMA component LAMP2A, which is increased in MRL/lpr B cells, is downregulated after P140 treatment. We also show that P140, but not the unphosphorylated peptide, uses the clathrin-dependent endo-lysosomal pathway to enter into MRL/lpr B lymphocytes and accumulates in the lysosomal lumen where it may directly hamper lysosomal HSPA8 chaperoning functions, and also destabilize LAMP2A in lysosomes as a result of its effect on HSP90AA1. This dual effect may interfere with the endogenous autoantigen processing and loading to major histocompatibility complex class II molecules and as a consequence, lead to lower activation of autoreactive T cells. These results shed light on mechanisms by which P140 can modulate lupus disease and exert its tolerogenic activity in patients. The unique selective inhibitory effect of the P140 peptide on CMA may be harnessed in other pathological conditions in which reduction of CMA activity would be desired.


Cost effectiveness of a pharmacist-led information technology intervention for reducing rates of clinically important errors in medicines management in general practices (PINCER).
We recently showed that a pharmacist-led information technology-based intervention (PINCER) was significantly more effective in reducing medication errors in general practices than providing simple feedback on errors, with cost per error avoided at £79 (US$131). We aimed to estimate cost effectiveness of the PINCER intervention by combining effectiveness in error reduction and intervention costs with the effect of the individual errors on patient outcomes and healthcare costs, to estimate the effect on costs and QALYs. We developed Markov models for each of six medication errors targeted by PINCER. Clinical event probability, treatment pathway, resource use and costs were extracted from literature and costing tariffs. A composite probabilistic model combined patient-level error models with practice-level error rates and intervention costs from the trial. Cost per extra QALY and cost-effectiveness acceptability curves were generated from the perspective of NHS England, with a 5-year time horizon. The PINCER intervention generated £2,679 less cost and 0.81 more QALYs per practice [incremental cost-effectiveness ratio (ICER): -£3,037 per QALY] in the deterministic analysis. In the probabilistic analysis, PINCER generated 0.001 extra QALYs per practice compared with simple feedback, at £4.20 less per practice. Despite this extremely small set of differences in costs and outcomes, PINCER dominated simple feedback with a mean ICER of -£3,936 (standard error £2,970). At a ceiling 'willingness-to-pay' of £20,000/QALY, PINCER reaches 59 % probability of being cost effective. PINCER produced marginal health gain at slightly reduced overall cost. Results are uncertain due to the poor quality of data to inform the effect of avoiding errors.


The prevalence of anti-Helicobacter (Campylobacter) pylori antibodies in patients and healthy blood donors.
An enzyme linked immunosorbent assay (ELISA) with a sonicated suspension of Helicobacter (Campylobacter) pylori as antigen was used to detect anti-H. pylori antibodies in 517 patients without dyspepsia or peptic ulcer symptoms and 401 healthy blood donors. The criterion of seropositivity was determined from a receiver operating curve computed with the values of optical densities of 48 sera from dyspeptic patients with proven helicobacter-associated gastritis and 16 sera from dyspeptic patients with normal antral mucosa and no microbiological or histological evidence of H. pylori infection. The 227 (44%) seropositive persons amongst the patient group appeared to be significantly higher than the 142 (35%) sera with antibodies in the blood donors tested (p less than 0.03), even when adjustment was made for increasing age. We conclude that the prevalence of antibodies against H. pylori increases with age and that although antibodies are more prevalent in patients attending a hospital than in healthy blood donors, seropositivity suggestive of current or past infection can be found in one third of a randomly chosen population of blood donors.


Brain-derived neurotrophic factor amplifies neurotransmitter responses and promotes synaptic communication in the enteric nervous system.
Besides its role in neuronal growth and differentiation, brain-derived neurotrophic factor (BDNF) has been implicated in the control of peristalsis where it serves to enhance gastrointestinal motility. To unravel the cellular mechanisms governing BDNF's effect on motility. Studies were performed in primary myenteric neuron cultures and whole-mount preparations derived from guinea pig ileum. Expression of BDNF and its tropomyosin-related kinase B (TrkB) receptor was assessed by immunohistochemistry. Intracellular Ca(2+) concentration ([Ca(2+)](i)) changes in myenteric neurons were monitored using Fluo-4, and neurotransmitter release kinetics at enteric synapses were evaluated with FM1-43 imaging. Immunohistochemistry revealed the presence of BDNF and TrkB in mucosa, submucosal plexus and myenteric ganglia. Primary cultures also expressed BDNF and TrkB and were used to study the physiological effects of BDNF. None of the neurons studied displayed a [Ca(2+)](i) change when challenged with BDNF. However, BDNF exposure caused an enhancement of Ca(2+) transients induced by serotonin and substance P, which was reversed by the Trk receptor blocker K-252a (0.1 microM). BDNF exposure also resulted in an amplification of spontaneous network activity which was reflected in an increased number of synaptic vesicle clusters. Furthermore, BDNF treatment facilitated FM1-43-labelled vesicle destaining in enteric terminals during field stimulation. The findings demonstrate that BDNF is able to enhance rather than directly activate enteric nervous system signalling. Therefore, the promotion of motility by BDNF seems to result from its potent modulating role on enteric neuronal activity and synaptic communication.


Dissecting cardosin B trafficking pathways in heterologous systems.
In cardoon pistils, while cardosin A is detected in the vacuoles of stigmatic papillae, cardosin B accumulates in the extracellular matrix of the transmitting tissue. Given cardosins' high homology and yet different cellular localisation, cardosins represent a potentially useful model to understand and study the structural and functional plasticity of plant secretory pathways. The vacuolar targeting of cardosin A was replicated in heterologous species so the targeting of cardosin B was examined in these systems. Inducible expression in transgenic Arabidopsis and transient expression in tobacco epidermal cells were used in parallel to study cardosin B intracellular trafficking and localisation. Cardosin B was successfully expressed in both systems where it accumulated mainly in the vacuole but it was also detected in the cell wall. The glycosylation pattern of cardosin B in these systems was in accordance with that observed in cardoon high-mannose-type glycans, suggesting that either the glycans are inaccessible to the Golgi processing enzymes due to cardosin B conformation or the protein leaves the Golgi in an early step before Golgi-modifying enzymes are able to modify the glycans. Concerning cardosin B trafficking pathway, it is transported through the Golgi in a RAB-D2a-dependent route, and is delivered to the vacuole via the prevacuolar compartment in a RAB-F2b-dependent pathway. Since cardosin B is secreted in cardoon pistils, its localisation in the vacuoles in cardoon ovary and in heterologous systems, suggests that the differential targeting of cardosins A and B in cardoon pistils results principally from differences in the cells in which these two proteins are expressed.


Epidemiology of colorectal cancer in Turkey: A cross-sectional disease registry study (A Turkish Oncology Group trial).
This study aimed to determine the epidemiological characteristics of colorectal cancer in Turkey. In this multicenter, prospective, and cross-sectional registry study, data for 968 patients with colorectal cancer from 21 centers in 7 geographic regions were analyzed. Diagnosis was colon cancer in 662 (68.4%) and rectum cancer in 306 (31.6%) patients. In total, 60.9% of patients was male; mean age was 58.9±12.6 years. Among patients, 15.0% was drinking alcohol, 17.5% was smoking, 1.5% had familial history of polyposis, 15.0% had diabetes mellitus, 1.0% had inflammatory bowel disease. Fruit and vegetable consumption was low (<3 times/week) in 35.5% and red meat consumption was high (≥3 times/week) in 47.4% of the patients. Median time-to diagnosis was 3.0 months and 4.0 months for patients with colon and rectum cancer, respectively. Mean body mass index was >25 in all group of patients. Distal rectum (61.3%) and sigmoid colon (36.8%) were the most common locations of cancer, for rectum and colon respectively. In total, 85.6% of patients were operated; 25.8% had emergency surgery. Low anterior resection rate was 64.2% in rectum cancer. In majority (89.8%) of the patients with rectum cancer who received preoperative treatment, conventional chemo-radiotherapy regimen was given. pTNM staging at diagnosis showed that stage III and IV patients were in majority (35.9% and 29.7%, respectively). Colon cancer is more frequent than rectum cancer in Turkey. Colorectal cancer patients are diagnosed at later stages. Most of the cases were operated. Interregional differences for risk factors are worthwhile for evaluation in future trials.


Sclerostin deficient mice rapidly heal bone defects by activating β-catenin and increasing intramembranous ossification.
We investigated the influence of the osteocyte protein, sclerostin, on fracture healing by examining the dynamics and mechanisms of repair of single-cortex, stabilized femoral defects in sclerostin knockout (Sost(-/-); KO) and sclerostin wild-type (Sost(+/+); WT) mice. Fourteen days following generation of bone defects, Sost KO mice had significantly more bone in the healing defect than WT mice. The increase in regenerating bone was due to an increase in the thickness of trabecularized spicules, osteoblast numbers and surfaces within the defect. Enhanced healing of bone defects in Sost KO mice was associated with significantly more activated β-catenin expression than observed in WT mice. The findings were similar to those observed in Axin2(-/-) mice, in which β-catenin signaling is known to be enhanced to facilitate bone regeneration. Taken together, these data indicate that enhanced β-catenin signaling is present in Sost(-/-) mice that demonstrate accelerated healing of bone defects, suggesting that modulation of β-catenin signaling in bone could be used to promote fracture repair.


The Association Between Long-Term Bisphosphonate Use and the Risk of Fracture Among Women Aged 50 or Older with Osteoporosis.
Osteoporosis is a prevalent disease, and bisphosphonates can effectively reduce the risk of osteoporotic fractures. However, the association between the length of the medication treatment and the risk of fractures remains unclear. The purpose of this study was to evaluate the association between long-term bisphosphonate use (treatment duration ≥5 years) and the risk of fractures among women with osteoporosis aged 50 or older. We conducted a retrospective cohort study by using the 2001-2011 National Health Insurance Research Database in Taiwan. We included women who were 50 years or older, who had a diagnosis of osteoporosis, and who were newly initiating oral bisphosphonates between January 1, 2002 and December 31, 2003. The index date was the date of the first dispensing of oral bisphosphonate during the enrollment period. Women were considered to be using bisphosphonates until they had a gap in supply of more than 3 months. We classified bisphosphonate use as long term (≥5 years) and regular (<5 years) based on its length of use. The dependent variable was the time to the first observed clinical fracture. Cox-proportional hazard regression models were used to evaluate the association between long-term bisphosphonate use and the risk of fractures. The study included 1342 women with a mean age of 71 years. Of them, 83 (6.2%) were long-term bisphosphonate users. A total of 185 (13.8%) had a fracture. After adjustments, long-term bisphosphonate use was not associated with a lower risk of fractures than was regular bisphosphonate use (adjusted hazard ratio: 1.49, 95% CI: 0.91-2.45). This study found no evidence of a lower risk of fractures to be associated with long-term bisphosphonate use among women aged 50 or older with osteoporosis in Taiwan. Orthopedists as well as other healthcare providers should be aware of the limited benefits of long-term bisphosphonate use.


The diagnostic challenge of euthyroid hyperthyroxinemia.
In euthyroid hyperthyroxinemia high levels of thyroxine (T4) may be either transient or persistent, associated with high, normal, or low levels of tri-iodothyronine (T3). Euthyroid hyperthyroxinemia may occur: as a response to abnormal plasma binding (thyroxine binding globulin, albumin, prealbumin, or autoantibodies), because of hormone resistance, after exposure to drugs such as amiodarone, cholecystographic contrast agents, or propranolol, during acute psychiatric illness or stress, and in hyperemesis gravidarum. In some instances the cause of persistent hyperthyroxinemia still remains obscure. No single investigation (including free hormone measurement and the response of thyrotropin to its releasing hormone) can distinguish all of these entities from true hyperthyroidism. Hence, re-evaluation in cases of diagnostic uncertainty should begin with clinical reassessment. Techniques are now available to identify easily some causes of euthyroid hyperthyroxinemia, allowing us to recognise patients and relatives who are at risk of inappropriate treatment. Because measurement of serum T4 remains the key investigation for diagnosis of thyroid dysfunction, it is important to appreciate the full range of conditions that compromise its specificity.


New MAPS for misfolded proteins.
Clearing misfolded proteins from the cytoplasm is essential to maintain cellular homeostasis. Now, a parallel clearance system is described that uses the deubiquitylase USP19 to enable secretion of misfolded cytoplasmic proteins when conventional proteasomal degradation is compromised. Misfolding-associated protein secretion (MAPS) has important implications for protein quality control and prion-like transmission.


Cardiac Resynchronization Therapy Outcomes in Type 2 Diabetic Patients: Role of MicroRNA Changes.
Heart failure (HF) and type 2 diabetes mellitus (T2DM) are two growing and related diseases in general population and particularly in elderly people. In selected patients affected by HF and severe dysfunction of left ventricle ejection fraction (LVEF), with left bundle brunch block, the cardiac resynchronization therapy with a defibrillator (CRT) is the treatment of choice to improve symptoms, NYHA class, and quality of life. CRT effects are related to alterations in genes and microRNAs (miRs) expression, which regulate cardiac processes involved in cardiac apoptosis, cardiac fibrosis, cardiac hypertrophy and angiogenesis, and membrane channel ionic currents. Different studies have shown a different prognosis in T2DM patients and T2DM elderly patients treated by CRT-D. We reviewed the literature data on CRT-D effect on adult and elderly patients with T2DM as compared with nondiabetic patients.


Comparative behavioral pharmacology of cocaine and the selective dopamine uptake inhibitor RTI-113 in the squirrel monkey.
The behavioral effects of 3beta-(4-chlorophenyl)tropane-2beta-carboxylic acid phenyl ester hydrochloride (RTI-113; 0.03-1.0 mg/kg), a selective dopamine uptake inhibitor, were compared with those of cocaine (0.03-3.0 mg/kg) and 1-(2-[bis(4-fluorophenyl)methoxy]ethyl)-4-(3-phenylpropyl)piperazine dihydrochloride (GBR 12909; 0.03-3.0 mg/kg) in squirrel monkeys. Intermediate doses of each drug produced significant increases in response rate maintained by a fixed-interval (FI) 300-s schedule of stimulus termination, but RTI-113 was less effective than cocaine or GBR 12909. The order of potency for increasing response rate was RTI-113 >/= cocaine > GBR 12909. In drug time course determinations, RTI-113 and GBR 12909 had longer durations of action than cocaine. RTI-113 substituted completely for cocaine in subjects trained to discriminate cocaine and saline under a two-lever drug-discrimination procedure maintained by food delivery. RTI-113 also reliably maintained self-administration behavior in subjects trained under a second-order FI 900-s schedule of i.v. cocaine delivery. Pretreatment with RTI-113 significantly decreased responding for cocaine at the highest pretreatment dose, but RTI-113 had similar effects on responding maintained by a second-order FI 900-s schedule of stimulus termination. The results indicate that the behavioral pharmacology of RTI-113 is similar to that of cocaine, further implicating a prominent role for dopamine uptake inhibition in the behavioral effects of cocaine. Its longer duration of action in conjunction with less pronounced behavioral-stimulant effects are desirable properties for a substitute pharmacotherapy for cocaine abuse. RTI-113 effectively decreased cocaine self-administration behavior, although its direct rate-altering effects may have contributed to the interactions obtained.


Promoter-specific IGF2 imprinting status and its plasticity during human liver development.
IGF2 has been shown to be expressed preferentially from the paternally derived allele, although the maternal allele can be found active during both prenatal and postnatal development as well as in neoplastic tumours in humans. We addressed here whether or not the biallelic expression patterns that can be seen during postnatal human liver development reflected a coordinated change in the activities of the four promoters of human IGF2. We show here that the P2, P3 and P4 promoters, but not the P1 promoter, display monoallelic activity in embryonic, neonatal and younger infant liver specimens. The P2, P3 and P4 promoters can, however, be found active either monoallelically or biallelically or even monoallelically on opposite parental alleles in older infant and adult liver specimens. In contrast, H19, which is closely linked to IGF2, is monoallelically expressed in all postnatal liver samples analysed. We conclude that the functional imprinting status of IGF2 during postnatal liver development appears to be promoter/enhancer-specific and either partly or completely independent of H19.


A Literature Review and Framework Proposal for Halitosis Assessment in Cigarette Smokers and Alternative Nicotine-Delivery Products Users.
Halitosis is a health condition which counts cigarette smoking (CS) among its major risk factors. Cigarette smoke can cause an imbalance in the oral bacterial community, leading to several oral diseases and conditions, including intraoral halitosis. Although the best approach to decrease smoking-related health risks is quitting smoking, this is not feasible for many smokers. Switching to potentially reduced-risk products, like electronic vapor products (EVP) or heated tobacco products (HTP), may help improve the conditions associated with CS. To date, there have been few systematic studies on the effects of CS on halitosis and none have assessed the effects of EVP and HTP use. Self-assessment studies have shown large limitations owing to the lack of reliability in the participants' judgment. This has compelled the scientific community to develop a strategy for meaningful assessment of these new products in comparison with cigarettes. Here, we compiled a review of the existing literature on CS and halitosis and propose a 3-layer approach that combines the use of the most advanced breath analysis techniques and multi-omics analysis to define the interactions between oral bacterial species and their role in halitosis both in vitro and in vivo. Such an approach will allow us to compare the effects of different nicotine-delivery products on oral bacteria and quantify their impact on halitosis. Defining the impact of alternative nicotine-delivery products on intraoral halitosis and its associated bacteria will help the scientific community advance a step further toward understanding the safety of these products and their potentiall risks for consumers.


Prediction of Therapy Response and Prognosis in Leukemias by Flow Cytometric MDR Assays.
Multidrug resistance (MDR) is an unwanted phenomenon, that may cause therapy failure in several neoplasms including hematological malignancies. The purpose of any type of laboratory MDR assay is to reliably identify such patients and to provide useful data to clinicians with a relatively short turnaround time. MDR can be multicausal and several previous data identified a group of transmembrane proteins - the ATP-binding casette (ABC) proteins - that may be involved in MDR in various hematological malignancies. The prototype of these proteins is the P-glycoprotein (Pgp, MDR1, ABCB1) that is a seven-membrane spanning transmembrane protein capable of extruding several cytotoxic drugs that are of key importance in the treatment of hematological disorders. Similarly other ABC proteins - Multidrug resistance associated protein 1 (ABCC1) and breast cancer resistance protein (ABCG2) are both capable of pumping out cytotoxic drugs. Here, we present flow cytometric methods to identify MDR proteins by antigen and activity assays. The advantage of flow technology is the short turnaround time and its relative easiness compared to nucleic acid based technologies. However, for the activity assays, it should be noted, that these functional tests require live cells, thus adequate results can only be provided if the specimen transport can be completed within 6 hours of sample collection. Identification of MDR proteins provides prognostic information and may modulate therapy, thus signifies a clinically useful information in the evaluation of patients with leukemias.


Membrane behavior of exocytic vesicles. II. Fate of the trichocyst membranes in Paramecium after induced trichocyst discharge.
A specific exocytic process, the discharge of spindle trichocyts of Paramecium caudatum, was examined by means of the electron microscope. This exocytosis is induced by an electric shock simultaneously in nearly all of the trichocysts (ca. 6,000-8,000) of a single cell. Single paramecia were subjected to the shock and then fixed at defined times after the shock so that the temporal sequence of the pattern of changes of the trichocyst membranes after exocytosis could be studied. The trichocyst vacuoles fuse with the plasma membrane only for the length of time required for expulsion to take place. After exocytosis, the membrane of the vacuole does not become incorporated into the plasma membrane; rather, the collapsed vacuole is pinched off and breaks up within the cytoplasm. The membrane vesiculates into small units which can no longer be distinguished from vesicles of the same dimensions that exist normally within the cell's cytoplasm. The entire process is completed within 5-10 min. These results differ from the incorporation of mucocyst membranes into the plasma membrane as proposed for Tetrahymena.


Profound deficits in hippocampal synaptic plasticity after traumatic brain injury and seizure is ameliorated by prophylactic levetiracetam.
To determine the precise effects of post-traumatic seizure activity on hippocampal processes, we induced seizures at various intervals after traumatic brain injury (TBI) and analyzed plasticity at CA1 Schaffer collateral synapses. Rats were initially separated into two groups; one exposed solely to fluid percussion injury (FPI) at 2 Psi and the other only receiving kainic acid (KA)-induced seizures without FPI. Electrophysiological (ePhys) studies including paired-pulse stimulation for short-term presynaptic plasticity and long-term potentiation (LTP) of CA1 Schaffer collateral synapses of the hippocampus for post-synaptic function survey were followed at post-event 1 hour, 3 and 7 days respectively. Additional rats were exposed to three seizures at weekly intervals starting 1 week or 2 weeks after TBI and compared with seizures without TBI, TBI without seizures, and uninjured animals. An additional group placed under the same control variables were treated with levetiracetam prior to seizure induction. The ePhys studies related to post-TBI induced seizures were also followed in these additional groups. Seizures affected the short- and long-term synaptic plasticity of the hippocampal CA3-CA1 pathway. FPI itself suppressed LTP and field excitatory post synaptic potentials (fEPSP) in the CA1 Schaffer collateral synapses; KA-induced seizures that followed FPI further suppressed synaptic plasticity. The impairments in both short-term presynaptic and long-term plasticity were worse in the rats in which early post-TBI seizures were induced than those in which later post-TBI seizures were induced. Finally, prophylactic infusion of levetiracetam for one week after FPI reduced the synaptic plasticity deficits in early post-TBI seizure animals. Our data indicates that synaptic plasticity (i.e., both presynaptic and postsynaptic) suppression occurs in TBI followed by a seizure and that the interval between the TBI and seizure is an important factor in the severity of the resulting deficits. Furthermore, the infusion of prophylactic levetiracetam could partially reverse the suppression of synaptic plasticity.


Stimulation of the posterior cord predicts successful infraclavicular block.
Infraclavicular (IC) block is often performed by localizing one cord within the brachial plexus sheath and placing all the local anesthetic solution at that location. We hypothesized that posterior cord stimulation would be associated with a greater likelihood of IC block success. We enrolled 369 patients scheduled for surgery to the lower arm or hand in a prospective, nonrandomized observational trial. All underwent IC blocks using a standard technique, and the cord stimulated immediately before drug injection was recorded. Motor and sensory functioning were evaluated 15 min after injection. Compared with stimulation of either the lateral or medial cord, stimulation of the posterior cord was associated with rapid onset of motor block in significantly more nerves, as well as a decreased likelihood of block failure (motor and sensory block inadequate to perform surgery). Failure rates were 5.8% for posterior cord, 28.3% for lateral (P < 0.05), and 15.4% for medial (P < 0.05). The differences were highly significant when adjusted for multiple possible confounders, such as gender, body mass index, location of the incision, and level of training of the individual performing the block (P < 0.001, lateral versus posterior; P = 0.003, medial versus posterior). A low failure rate was also predicted by stimulation of more than one cord simultaneously (P < 0.05). We conclude that injection after locating the posterior cord or multiple cords predicts successful IC block.


Cellular responses in the skin of the gilthead sea bream Sparus aurata L. and the sea bass Dicentrarchus labrax (L.) exposed to high ammonia.
Adult gilthead sea bream Sparus aurata and sea bass Dicentrarchus labrax were exposed for 24 and 48 h, respectively, to two concentrations of ammonia each (mean values of 3·34 and 13·10 mg l(-1) TA-N in S. aurata; 2·99 and 11·90 mg l(-1) TA-N in D. labrax). Light microscopy and computerized morphometry were used to evaluate ammonia-induced alterations in skin structure during exposure and following recovery in normal water. In S. aurata, ammonia exposure induced a concentration-dependent increase in the number (hyperplasia) of neutral mucous cells (mc), with peak values at 24 h recovery after exposure. An increase in the dispersion of melanosomes in skin melanocytes was also observed in the dermis and occasionally in the epidermis of S. aurata, with peak values at 24 h of ammonia exposure. Exposure of D. labrax to ammonia had, likewise, concentration-dependent effects on mucous secretion. Of the two types of mc in this species, there was an increase in the number of the neutral mc and a reduction in the much more numerous acid mc, with peak values at 24 and 48 h, respectively, of ammonia exposure. The more intense mucous secretion in D. labrax compared to S. aurata could be related to the lower tolerance to ammonia in D. labrax, as reported elsewhere. Finally, the increase in melanosome dispersion was less evident in D. labrax, due to highly variable control values. These morphological alterations to the skin could be useful indicators of non-specific stress in cultured fishes.


Food allergy in children: results of a standardized protocol for oral desensitization.
Food allergy in children is still an unresolved problem that merits investigation, particularly when the food is fundamental for the child's growth. Reports in the literature that deal with the possibility of a desensitizing treatment are sporadic and often inconsistent, and no standardized protocols are yet available. In this paper we propose a standardized oral desensitization program for food allergy in children. The treatment was carried out in 14 cases with allergy to food (milk in 6 cases, egg in 5, fish in 2 and apple in 1 case). The control group consisted of 10 age and sex matched allergic subjects (5 to milk, 4 to egg and 1 to fish), who underwent a strict elimination diet regimen. Compliance to treatment was satisfactory, since 12 out of the 14 treated cases (85.7%) completed the program. Treatment was successful in 100% of the cases that completed the program: all the treated patients are now able to tolerate any food with no untoward effects or need for preventive drugs. The proposed standardized oral desensitization treatment may represent a safe and convenient alternative in the management of food-allergic subjects.