Evaluation of a vancomycin dosing nomogram in achieving high target trough concentrations in Taiwanese patients.
The use of a vancomycin dosing nomogram is an alternative and more cost-effective method to conventional dosing; it reliably allows the achievement of trough vancomycin serum concentrations of 5-15 mg/l, with a successful clinical response. Recent guidelines have further recommended that the trough concentration be maintained at 15-20mg/l for complicated infections. However, to date no published nomogram has been constructed to achieve the optimal trough of 15-20mg/l in an Asian population. This study aimed to develop two vancomycin nomograms for the achievement of trough concentrations of 5-15 mg/l and 15-20mg/l in the Taiwanese population, and to ensure the clinical efficacy and safety of such nomograms. The estimated concentrations and the real concentrations in our patient population were compared between six pharmacokinetic models to see which was the most precise. As the Ambrose method was the best at predicting the trough, this was used to create two nomograms, one for a target trough at 5-15 mg/l and the other for a target trough at 15-20mg/l. We then evaluated the nomograms by analyzing the number of patients with the target vancomycin trough concentration, clinical and microbiological outcomes, and safety. More patients who had dosing according to the nomogram had a vancomycin trough concentration within the desired target range than patients who had conventional dosing (65.1% vs. 32.1%, p = 0.001). These patients also had a higher rate of 'cure' as the clinical response (35.7% vs. 27.1%) and 'eradication' as the microbiological response (46.4% vs. 29.2%), and a lower rate of nephrotoxicity (14.3% vs. 22.9%). For the patients with a complicated infection, more had a trough between 15 and 20mg/l when vancomycin was dosed with the nomogram than when dosed conventionally (41.2% vs. 12.1%, p = 0.019). We found that when dosing vancomycin with these nomograms, patients tended to have vancomycin trough concentrations within the target range and also to have a better outcome with regard to clinical efficacy and the safety profile.


Orexin receptors in the hippocampal dentate gyrus modulated the restraint stress-induced analgesia in the animal model of chronic pain.
Previous studies have shown that stressful stimuli induced an adaptive response of reduced nociception, known as stress-induced analgesia (SIA). Since orexin neuropeptides are involved in pain modulation, and orexin neurons, primarily located in the lateral hypothalamus (LH), project to various hippocampal regions, such as the dentate gyrus (DG), the current study aimed to examine the role of orexin receptors within the DG region in the restraint SIA in the animal model of chronic pain. One hundred-thirty adult male Wistar rats (230-250 g) were unilaterally implanted with a cannula above the DG region. Animals were given SB334867 or TCS OX2 29 (1, 3, 10, and 30 nmol, 0.5 µl/rat) into the DG region as orexin-1 receptor (OX1r) and orexin-2 receptor (OX2r) antagonists, respectively, five min before exposure to a 3-hour restraint stress (RS) period. Animals were then undergone the formalin test to assess pain-related behaviors as the animal model of chronic pain. The results showed that RS produces an analgesic response during the early and late phases of the formalin test. However, intra-DG microinjection of OX1r and OX2r antagonists attenuated the restraint SIA. OX2r antagonist was more potent than OX1r antagonist in the early phase of the formalin test, while OX1r antagonist was little more effective in the late phase. Predominantly, it could be concluded that the orexinergic system in the DG region might act as a potential endogenous pain control system and a novel target for treating stress-related disorders.


Can text messages reach the parts other process measures cannot reach: an evaluation of a behavior change intervention delivered by mobile phone?
Process evaluation is essential in developing, piloting and evaluating complex interventions. This often involves observation of intervention delivery and interviews with study participants. Mobile telephone interventions involve no face to face contact, making conventional process evaluation difficult. This study assesses the utility of novel techniques for process evaluation involving no face to face contact. Text messages were delivered to 34 disadvantaged men as part of a feasibility study of a brief alcohol intervention. Process evaluation focused on delivery of the text messages and responses received from study participants. The computerized delivery system captured data on receipt of the messages. The text messages, delivered over 28 days, included nine which asked questions. Responses to these questions served as one technique for process evaluation by ascertaining the nature of engagement with the study and with steps on the causal chain to behavior change. A total of 646 SMS text messages were sent to participants. Of these, 613 messages (95%) were recorded as delivered to participants' telephones. 88% of participants responded to messages that asked questions. There was little attenuation in responses to the questions across the intervention period. Content analysis of the responses revealed that participants engaged with text messages, thought deeply about their content and provided carefully considered personal responses to the questions. Socially disadvantaged men, a hard to reach population, engaged in a meaningful way over a sustained period with an interactive intervention delivered by text message. The novel process measures used in the study are unobtrusive, low cost and collect real-time data on all participants. They assessed the fidelity of delivery of the intervention and monitored retention in the study. They measured levels of engagement and identified participants' reactions to components of the intervention. These methods provide a valuable addition to conventional process evaluation techniques.


Oral LPS Dosing Induces Local Immunological Changes in the Pancreatic Lymph Nodes in Mice.
Lacking the initial contact between the immune system and microbial-associated molecular patterns (MAMPs), such as lipopolysaccharides (LPS), early in life, may be regarded as one of the causal factors of the increasing global increase in the incidence of autoimmune diseases, such as type 1 diabetes (T1D). Previously, a reduced incidence of T1D accompanied by dramatically increased abundances of both the mucin-metabolising bacterium Akkermansia muciniphila, and LPS-carrying Proteobacteria was observed, when vancomycin was given to pups of nonobese diabetic (NOD) mice. While the T1D incidence reducing effect of A. muciniphila has been shown in further studies, little is known as to whether the increased abundance of LPS-carrying bacteria also has a protective effect. Therefore, we fed NOD pups with Eschericia coli LPS orally from birth to weaning, which decreased the gene expressions of TNFα, IL-10, IL-6, IFNγ, IL-1β, IL-2, IL-4, and FoxP3 in the pancreatic lymph nodes, while the same gene expression profile in the spleen was unaffected. However, no significant difference in the incidence of T1D, gut microbiota composition, or ileum expression of the genetic markers of gut permeability, Claudin8, Occludin, Zonulin-1 (Tjp1), Claudin15, Muc1, and Muc2 were observed in relation to LPS ingestion. It is, therefore, concluded that early life oral E. coli LPS has an impact on the local immune response, which, however, did not influence T1D incidence in NOD mice later in life.


Accumulating mitochondrial DNA mutations drive premature hematopoietic aging phenotypes distinct from physiological stem cell aging.
Somatic stem cells mediate tissue maintenance for the lifetime of an organism. Despite the well-established longevity that is a prerequisite for such function, accumulating data argue for compromised stem cell function with age. Identifying the mechanisms underlying age-dependent stem cell dysfunction is therefore key to understanding the aging process. Here, using a model carrying a proofreading-defective mitochondrial DNA polymerase, we demonstrate hematopoietic defects reminiscent of premature HSC aging, including anemia, lymphopenia, and myeloid lineage skewing. However, in contrast to physiological stem cell aging, rapidly accumulating mitochondrial DNA mutations had little functional effect on the hematopoietic stem cell pool, and instead caused distinct differentiation blocks and/or disappearance of downstream progenitors. These results show that intact mitochondrial function is required for appropriate multilineage stem cell differentiation, but argue against mitochondrial DNA mutations per se being a primary driver of somatic stem cell aging.


Novel ciliate lipases for enzyme replacement during exocrine pancreatic insufficiency.
Exocrine pancreatic insufficiency caused by inflammation or pancreatic tumors results in nutrient malfunction by a lack of digestive enzymes and neutralization compounds. Despite satisfactory clinical results with current enzyme therapies, a normalization of fat absorption in patients is rare. An individualized therapy is required that includes high dosage of enzymatic units, usage of enteric coating, and addition of gastric proton pump inhibitors. The key goal to improve this therapy is to identify digestive enzymes with high activity and stability in the gastrointestinal tract. We cloned and analyzed three novel ciliate lipases derived from Tetrahymena thermophila. Using highly precise pH-STAT-titration and colorimetric methods, we determined stability and lipolytic activity under physiological conditions in comparison with commercially available porcine and fungal digestive enzyme preparations. We measured from pH 2.0 to 9.0, with different bile salts concentrations, and substrates such as olive oil and fat derived from pig diet. Ciliate lipases CL-120, CL-130, and CL-230 showed activities up to 220-fold higher than Creon, pancreatin standard, and rizolipase Nortase within a pH range from pH 2.0 to 9.0. They are highly active in the presence of bile salts and complex pig diet substrate, and more stable after incubation in human gastric juice compared with porcine pancreatic lipase and rizolipase. The newly cloned and characterized lipases fulfilled all requirements for high activity under physiological conditions. These novel enzymes are therefore promising candidates for an improved enzyme replacement therapy for exocrine pancreatic insufficiency.


A randomized trial of an early measles vaccine at 4½ months of age in Guinea-Bissau: sex-differential immunological effects.
After measles vaccine (MV), all-cause mortality is reduced more than can be explained by the prevention of measles, especially in females. We aimed to study the biological mechanisms underlying the observed non-specific and sex-differential effects of MV on mortality. Within a large randomised trial of MV at 4.5 months of age blood samples were obtained before and six weeks after randomisation to early MV or no early MV. We measured concentrations of cytokines and soluble receptors from plasma (interleukin-1 receptor agonist (IL-1Ra), IL-6, IL-8, IL-10, tumor necrosis factor (TNF)-α, monocyte chemoattractant protein (MCP)-1, soluble urokinase-type plasminogen activator receptor), and secreted cytokines (interferon-γ, TNF-α, IL-5, IL-10, IL-13, IL-17) after in vitro challenge with innate agonists and recall antigens. We analysed the effect of MV in multiple imputation regression, overall and stratified by sex. The majority of the infants had previously been enrolled in a randomised trial of neonatal vitamin A. Post hoc we explored the potential effect modification by neonatal vitamin A. Overall, MV versus no MV was associated with higher plasma MCP-1 levels, but the effect was only significant among females. Additionally, MV was associated with increased plasma IL-1Ra. MV had significantly positive effects on plasma IL-1Ra and IL-8 levels in females, but not in males. These effects were strongest in vitamin A supplemented infants. Vitamin A shifted the effect of MV in a pro-inflammatory direction. In this explorative study we found indications of sex-differential effects of MV on several of the plasma biomarkers investigated; in particular MV increased levels in females, most strongly in vitamin A recipients. The findings support that sex and micronutrient supplementation should be taken into account when analysing vaccine effects. clinicaltrials.gov number NCT 00168545.


Desiderata for digital consent in genomic research.
Herein, we describe the characterization of a Digital Consent (DC) System to support current ethical-legal issues associated with challenges posed by informed consent for genomic research. A potential solution to support ongoing interaction with patients and allow control over how their data and samples are being used in genomic research can be Digital Consent based. But there are other challenges that need to be addressed, such as incidental findings when analyzing the results of genomic tests (not expected). This paper addresses security and privacy recommendations for the development of precision medicine, and the interoperability references of Health Information Standardization Organizations such as HL7 and IHE, as well as recent research in the field of ethics in Genomic Medicine. As a result of this work, ten key features that need to be further explored have been identified in order to support the realization of DC in Genomic Research.


Expression of the aryl hydrocarbon receptor in growth plate cartilage and the impact of its local modulation on longitudinal bone growth.
Although dioxin has been reported to impair bone growth in both humans and animals, the underlying mechanisms have not been clarified. We conducted this study to rule out if dioxin may directly target the growth plate, via local modulation of the aryl hydrocarbon receptor (AhR). Initial studies in rare tissue samples of the human growth plate confirmed that the AhR protein is widely expressed in growth plate cartilage. To explore the local role of the AhR, mechanistic studies were performed in a well-established model of cultured fetal rat metatarsal bones. The longitudinal growth of these bones was monitored while being exposed to AhR modulators. The AhR agonist, 2,3,7,8-tetrachlorodibenzo-p-dioxin, did not affect bone growth at any concentrations tested (1 pM-10 nM). In contrast, the AhR antagonist, alpha-naphthoflavone, suppressed bone growth and increased chondrocyte apoptosis, although only at a high, potentially cytotoxic concentration (50 µM). We conclude that although the AhR is widely expressed in the growth plate, bone growth is not modulated when locally activated, and therefore, dioxin-induced growth failure is likely mediated through systemic rather than local actions.


Adult neurotrophic factor-secreting stem cells: a potential novel therapy for neurodegenerative diseases.
Stem cell-based therapy is a promising approach for the treatment of neurodegenerative disease. In our laboratory, a novel protocol has been developed to induce bone marrow-derived mesenchymal stem cells into neurotrophic factor-secreting cells. These cells produce and secrete factors such as BDNF (brain-derived neurotrophic factor) and GDNF (glial-derived neurotrophic factor). To evaluate the migratory capacity and efficacy of NTF-SC in animal models of Parkinson's disease and Huntington's disease. MSCs underwent two-phase medium-based induction. An efficacy study was conducted on the 6-hydroxydopamine-induced lesion, a rat model for Parkinson's disease. Cells were transplanted on the day of 6-OHDA administration, and amphetamine-induced rotations were measured as a primary behavioral index. In a second experiment, migratory behavior was examined by transplanting cells a distance from a quinolinic acid-induced striatal lesion, a rat model for Huntington's disease. Migration, in vivo, was monitored using longitudinal magnetic resonance imaging scans followed by histology. NTF-SCs attenuated amphetamine-induced rotations by 45%. HPLC analysis demonstrated a marked decrease in dopamine depletion, post-cellular treatment. Moreover, histological assessments revealed that the engrafted cells migrated and acted to regenerate the damaged striatal dopaminergic nerve terminal network. In a preliminary work on an animal model for Huntington's disease, we demonstrated by high resolution MR images and correlating histology that induced cells migrated along the internal capsule towards the QA-induced lesion. The induced MSCs are a potential therapy for neurodegenerative diseases, due both to their NTF secretion and their ability to migrate towards the diseased tissue.


Examining the Role of Autonomy Support, Goal Setting, and Care Coordination Quality on HIV PrEP Adherence in Black Men Who Have Sex with Men: HPTN 073.
Autonomy support is a concept that is derived from self-determination theory. Autonomy refers to the freedom to act as one chooses. The current study aimed to examine if autonomy support was associated with dried blood spot validated pre-exposure prophylaxis (PrEP) adherence, and whether the association was mediated by PrEP adherence goal setting and progress toward PrEP adherence goals. Our sample was drawn from Black men who have sex with men (MSM) from across three cities (Chapel Hill, NC; Los Angeles, CA; and Washington, DC) in the United States between February 2013 and September 2014. We used logistic regression to evaluate associations between study variables and path analysis to test mediation effects. Participants were, on average, 28 [standard deviation (SD) = 1.12] years old and 25% were unemployed. We found that MSM who experienced high autonomy support were more likely to adhere to PrEP [odds ratio (OR) = 1.17; 95% confidence interval: 1.00-1.38]. MSM who set PrEP adherence goals were more likely to adhere to PrEP. Moreover, MSM who reported making progress toward their goals were also more likely to adhere to PrEP. Finally, client perception of coordination quality enhanced the magnitude of the association between goal setting and goal progress and the effect size of goal progress on PrEP adherence. Autonomy support, goal setting, goal monitoring/evaluation, and care coordination quality influenced PrEP adherence among Black MSM. Our findings indicate that while it is important to set goals for PrEP adherence, goal setting may need to be accompanied by progress monitoring to achieve the maximal effect.


Nerve atrophy in trigeminal neuralgia due to neurovascular compression and its association with surgical outcomes after microvascular decompression.
Idiopathic trigeminal neuralgia (TN) is caused by neurovascular compression and is often related to morphological changes in the trigeminal nerve. The aim of this study was to quantitatively measure atrophic changes of trigeminal nerves in patients with TN, and to further investigate whether nerve atrophy affected the efficacy of microvascular decompression (MVD). We conducted a prospective case-control study of 60 consecutive patients with TN and 30 sex- and age-matched healthy controls. All subjects underwent high-resolution three-dimensional MRI. The volume of the cisternal segment of trigeminal nerves was measured and compared using 3D Slicer software. Patients with TN underwent primary MVD and regular follow-up for at least 2 years. Associations of nerve atrophy with patient characteristics and operative outcomes were analyzed. The mean volume of the affected trigeminal nerve was significantly reduced in comparison to that of the nonaffected side (65.8 ± 21.1 versus 77.9 ± 19.3 mm3, P = 0.001) and controls (65.8 ± 21.1 versus 74.7 ± 16.5 mm3, P = 0.003). Fifty-two patients (86.7%) achieved complete pain relief without medication immediately after surgery, and 77.6% of patients were complete pain relief at the 2-year follow-up. The Spearman correlation test showed that there was a positive correlation (r = 0.46, P = 0.018) between the degree of trigeminal nerve indentation and nerve atrophy. In multivariate logistic regression analysis, two factors, indentation on nerve root (OR = 2.968, P = 0.022) and degree of nerve atrophy (OR = 1.18, P = 0.035), were associated with the long-term outcome. TN is associated with atrophy on the affected nerve. Furthermore, greater nerve atrophy is associated with more severe trigeminal nerve indentation and better long-term outcome following MVD.


Cytotoxic drug penetration studies in multicellular tumour spheroids.
1. The three-dimensional structure of human lung tumour spheroids conferred a degree of resistance to the anthracyclines adriamycin, 4'-deoxydoxorubicin, daunomycin and daunomycin-low density lipoprotein complex in comparison with cells grown as a monolayer, as assessed by delayed growth and clonogenic cell survival. 2. 4'-Deoxydoxorubicin induced a longer growth delay and greater clonogenic cell kill than adriamycin in spheroids, although it was no more cytotoxic in monolayer. 3. Fluorescent microscopy demonstrated that the more lipophilic analogues partitioned into the spheroid more rapidly and to a greater degree than adriamycin. 4. The spheroid model demonstrated that penetration is an important aspect of resistance to anthracycline drugs, and this approach may represent a better in vitro system for testing lipophilic analogues of cytotoxic drugs.


The Relationship Between Adult Attachment Orientation and Mindfulness: a Systematic Review and Meta-analysis.
Mindfulness can be measured as an individual trait, which varies between individuals. In recent years, research has investigated the overlap between trait mindfulness and attachment. The aim of the present review and meta-analysis was to investigate the current evidence linking adult attachment dimensions to trait mindfulness dimensions, and to quantitatively synthesize these findings using meta-analyses. A systematic literature search was conducted using five scientific databases of which, upon review, 33 articles met inclusion criteria. Inclusion criteria were peer-reviewed journals and dissertations published in English that relied on quantitative methods using reliable and validated self-report measures where study participants were aged 16 years and older. Random-effects model meta-analytic procedures were used to investigate the relationship between both constructs. Cross-sectional studies found significant negative correlations between adult attachment insecurity, on either dimension (anxiety or avoidance) and both total mindfulness score and all five sub-dimensions of mindfulness (act with awareness, observe, describe, non-reacting, and non-judging), with the exception of a non-significant positive correlation between attachment anxiety and observe. The effect size of the relationships ranged from small to medium. The overall mean effect sizes were moderate (anxiety, r+ = .34; avoidance, r+ = -.28), with both attachment dimensions associated with lower levels of total mindfulness. Results are discussed in relation to theory and research. Implications for future research include the need to utilize longitudinal design to address causality and mechanisms of the relationship between these constructs.


The food and nutrient intakes of the Tarahumara Indians of Mexico.
A nutritional survey of 372 semiacculturated Tarahumara Indians in the Sierra Madre Occidental Mountains of Mexico was carried out to determine the composition of their diet and its nutritional adequacy. Dietary histories from 174 adults and 198 children were obtained by interviews and field observations during 1973 and 1974. The histories for the children were calculated in part from the menus of six boarding church schools. Nutrient calculations of daily intake were based upon food composition tables and some actual analyses of Tarahumara foods. The protein intake was ample, at 87 g, and generously met the FAO/WHO recommendations for daily intake of essential amino acids. Fat contributed only 12% of total calories, its composition being 2% saturated and 5% polyunsaturated with a P/S ratio of 2. The mean dietary cholesterol intake was very low, less than 100 mg/day, and the plant sterol intake was high, over 400 mg/day. Carbohydrate comprised 75 to 80% of total calories, mostly from starch. Only 6% of total calories were derived from simple sugars. The crude fiber intake was high, 18 to 21 g/day. Salt consumption was moderately low, 5 to 8 g/day. The daily intakes of calcium, iron, vitamin A, ascorbic acid, thiamin niacin, riboflavin, and vitamin B6 exceeded or approximated the FAO/WHO recommendations. Thus, the simple diet of the Tarahumara Indians, composed primarily of beans and corn, provided a high intake of complex carbohydrate and was low in fat and cholesterol. Their diet was found to be generally of high nutritional quality and would, by all criteria, be considered antiatherogenic.


Lysophosphatidylinositol causes neurite retraction via GPR55, G13 and RhoA in PC12 cells.
GPR55 was recently identified as a putative receptor for certain cannabinoids, and lysophosphatidylinositol (LPI). Recently, the role of cannabinoids as GPR55 agonists has been disputed by a number of reports, in part, because studies investigating GPR55 often utilized overexpression systems, such as the GPR55-overexpressing HEK293 cells, which make it difficult to deduce the physiological role of endogenous GPR55. In the present study, we found that PC12 cells, a neural model cell line, express endogenous GPR55, and by using these cells, we were able to examine the role of endogenous GPR55. Although GPR55 mRNA and protein were expressed in PC12 cells, neither CB(1) nor CB(2) mRNA was expressed in these cells. GPR55 was predominantly localized on the plasma membrane in undifferentiated PC12 cells. However, GPR55 was also localized in the growth cones or the ruffled border in differentiated PC12 cells, suggesting a potential role for GPR55 in the regulation of neurite elongation. LPI increased intracellular Ca(2+) concentration and RhoA activity, and induced ERK1/2 phosphorylation, whereas endogenous and synthetic cannabinoids did not, thereby suggesting that cannabinoids are not GPR55 agonists. LPI also caused neurite retraction in a time-dependent manner accompanied by the loss of neurofilament light chain and redistribution of actin in PC12 cells differentiated by NGF. This LPI-induced neurite retraction was found to be G(q)-independent and G(13)-dependent. Furthermore, inactivation of RhoA function via C3 toxin and GPR55 siRNA knockdown prevented LPI-induced neurite retraction. These results suggest that LPI, and not cannabinoids, causes neurite retraction in differentiated PC12 cells via a GPR55, G(13) and RhoA signaling pathway.


[Efficacy on depression in breast cancer treated with acupuncture and auricular acupressure].
To compare the efficacy difference in treatment of depression in breast cancer between the combined therapy of acupuncture and auricular acupressure and western medication. Sixty patients were randomized into an observation group and a control group, 30 cases in each one. In the observation group, the combined therapy of acupuncture and auricular acupressure was adopted. The main acupoints of acupuncture were Hegu (LI 4), Tai-chong (LR 3), Baihui (GV 20), Zusanli (ST 36), Qihai (CV 6), etc. The supplementary acupoints were combined according to the syndrome differentiation. The treatment was given once every day, 5 treatments a week, at the interval of 2 days among weeks. The auricular acupressure was applied to gan (CO12, liver), pi (CO13, spleen), neifenmi (CO18, endocrine), etc., once every 4 days, on each side in one treatment. In the control group, fluoxetine hydrochloride capsules were prescribed for oral administration, 20 mg, once a day. The Hamilton depression rating scale (HAMD) was used to assess the disease severity and efficacy before treatment, in 4 and 8 weeks of treatment separately. HAMD factor changes were observed before treatment and at the end of the 8th week. The Asberg antidepressants scale (SERS) was applied to safety assessment. The total effective rate was 86.7% (26/30) in the observation group, better than 63.3% (19/30) in the control group (P<0.05). At the end of the 4th and 8th weeks, HAMD scores were all reduced apparently in the two groups (all P<0.01). At the end of the 8th week, the scores of the HAMD factor 1 (anxiety/somatic system), factor 5 (retardation) and factor 6 (sleep disturbance) were all reduced as compared with those before treatment in the two groups (all P<0.01); the results in the observation group were better than those in the control group (P<0.05, P<0.01). SERS score in the observation group was lower obviously than that in the control group (P<0.01). The combined therapy of acupuncture and auricular acupressure achieves the antidepression effect in treatment of depression in breast cancer and has less side effects and high safety. The efficacy is superior to fluoxetine hydrochloride capsules.


Recurrence of high-grade squamous intraepithelial neoplasia in neovagina: case report and review of the literature.
Recurrent carcinoma in situ in neovagina is rare, and the optimal modality of treatment is unclear. A 33-year-old multiparous woman was referred for vulvar intraepithelial neoplasia, vaginal intraepithelial neoplasia, and cervical intraepithelial neoplasia, underwent skinning vulvectomy with perianal excision, total vaginectomy, vaginal hysterectomy, and vaginal reconstruction with split-thickness skin graft. Ten years after initial surgery, the recurrence as a high-grade intraepithelial neoplasia in the upper one third of neovagina was detected. For that reason, the upper one third of vaginectomy with at least 5-mm tumor-free border and vaginal reconstruction with split-thickness skin graft were performed. She has attended her regular follow-up for 3 years with no evidence of disease. All patients with vaginoplasty should undergo regular follow-up. This report is the seventh such report in English literature of patients previously treated for in situ carcinoma who later developed recurrence in the graft.


Mechanisms of diastolic dysfunction in heart failure.
Abnormalities of diastole are common to most forms of congestive heart failure (HF). Diastolic function is broadly defined as the ability of the heart to fill adequately and at normal pressure to charge the ventricular pump for each subsequent contraction. It is determined by both active and passive processes occurring at the level of the myocyte, extracellular matrix, and left ventricular chamber. Forces extrinsic to the myocardium-such as the influence of right heart filling, pericardial and extracardiac constraints, and cardiac preload and afterload also contribute. Nearly half of patients with HF have apparently preserved systolic function, and this has focused attention on diastolic dysfunction as a dominant contributor to symptoms, sparking interest for understanding and treating diastolic abnormalities. This review focuses on the mechanisms determining normal and pathologic cardiac relaxation and distensibility and highlights how these abnormalities may be therapeutically targeted to improve diastolic function in human HF.


Hydraulic contacts controlling water flow across porous grains.
Water flow between porous grains varies widely depending on the water distribution in contacts between grains. The hydraulic behavior of contacts varies from highly conductive when water fills the contacts to a bottleneck to flow as water pressure drops and contact asperities rapidly drain. Such changes greatly impact the hydraulic conductivity of porous grain packs such as aggregated soil. The dominant driving force of water flow across contacts is capillarity, often quantified relative to gravity and viscous forces using the capillary and Bond numbers. For fast water infiltration, viscous forces dominate. For simplicity we modeled the water distribution between spherical porous grains whose surfaces are covered by spherical bumps of much smaller radii. We provide experimental evidence obtained by neutron radiography and synchrotron-based x-ray tomographic microscopy documenting transitions in the flow behavior across contacts.


Entropic Forces Drive Cellular Contact Guidance.
Contact guidance-the widely known phenomenon of cell alignment induced by anisotropic environmental features-is an essential step in the organization of adherent cells, but the mechanisms by which cells achieve this orientational ordering remain unclear. Here, we seeded myofibroblasts on substrates micropatterned with stripes of fibronectin and observed that contact guidance emerges at stripe widths much greater than the cell size. To understand the origins of this surprising observation, we combined morphometric analysis of cells and their subcellular components with a, to our knowledge, novel statistical framework for modeling nonthermal fluctuations of living cells. This modeling framework is shown to predict not only the trends but also the statistical variability of a wide range of biological observables, including cell (and nucleus) shapes, sizes, and orientations, as well as stress-fiber arrangements within the cells with remarkable fidelity with a single set of cell parameters. By comparing observations and theory, we identified two regimes of contact guidance: 1) guidance on stripe widths smaller than the cell size (w ≤ 160 μm), which is accompanied by biochemical changes within the cells, including increasing stress-fiber polarization and cell elongation; and 2) entropic guidance on larger stripe widths, which is governed by fluctuations in the cell morphology. Overall, our findings suggest an entropy-mediated mechanism for contact guidance associated with the tendency of cells to maximize their morphological entropy through shape fluctuations.


Changes of diet and dominant intestinal microbes in farmland frogs.
Agricultural activities inevitably result in anthropogenic interference with natural habitats. The diet and the gut microbiota of farmland wildlife can be altered due to the changes in food webs within agricultural ecosystems. In this work, we compared the diet and intestinal microbiota of the frog Fejervarya limnocharis in natural and farmland habitats in order to understand how custom farming affects the health of in vivo microbial ecosystems. The occurrence, abundance, and the numbers of prey categories of stomach content were significantly different between the frogs inhabiting natural and farmland habitats. In addition, differences in the abundance, species richness, and alpha-diversity of intestinal microbial communities were also statistically significant. The microbial composition, and particularly the composition of dominant microbes living in intestines, indicated that the land use practices might be one of factors affecting the gut microbial community composition. Although the first three dominant microbial phyla Bacteroidetes, Firmicutes, and Proteobacteria found in the intestines of frogs were classified as generalists among habitats, the most dominant gut bacterial phylum Bacteroidetes in natural environments was replaced by the microbial phylum Firmicutes in farmland frogs. Increased intestinal microbial richness of the farmland frogs, which is mostly contributed by numerous microbial species of Proteobacteria, Actinobacteria, Acidobacteria, and Planctomycetes, not only reflects the possible shifts in microbial community composition through the alteration of external ecosystem, but also indicates the higher risk of invasion by disease-related microbes. This study indicates that anthropogenic activities, such as the custom farming, have not only affected the food resources of frogs, but also influenced the health and in vivo microbial ecosystem of wildlife.


The use of capillary electrophoresis for studying the in vitro glucuronidation of 7-hydroxycoumarin.
An assay utilizing CE, has been developed for studying the in vitro metabolism of 7-hydroxycoumarin to 7-hydroxycoumarin-glucuronide. A reaction mixture containing a crude preparation of bovine uridine diphosphate (UDP) glucuronyl transferase (UDPGT) and the substrates uridine diphosphate glucuronic acid (UDPGA) and 7-hydroxycoumarin was prepared. Aliquots were removed from the reaction mixture at specific time intervals and analyzed on a 57-cm untreated silica capillary for the presence of 7-hydroxycoumarin-glucuronide. Samples were electrophoresed in a 100 mM phosphate/11 mM deoxycholic acid (sodium salt)/acetonitrile electrolyte buffer, pH 7.0, at 30 kV, with detection at 320 nm. The method allowed the determination of 7-hydroxycoumarin-glucuronide without sample cleanup, with a limit of detection of 2 micrograms/mL, and a linear detection range of 0 microgram/mL to 100 micrograms/mL. The 7-hydroxycoumarin-glucuronide, concentrations were calculated from calibration curves of standards prepared in enzyme solution. The initial rate of production of 7-hydroxycoumarin-glucuronide, in the first 70 min, was 3.1 +/- 0.13 nmol/mL/min/mg protein. The method was fast and reliable with percentage relative standard deviations for concentration of 7-hydroxycoumarin-glucuronide, over time, of less than 10%.


Variability in the clinical practice of maintaining the patency of peripheral intravenous catheters.
To establish variations in clinical practice associated with maintaining the patency of peripheral intravenous catheters (PIC) and to determine to what extent such clinical practice falls within the limits of the available scientific evidence, based on a random sample Spanish public hospitals. A cross-sectional, descriptive study was carried out in non-psychiatric public hospitals and their associated medical and surgical units. Cluster (hospitals), stratified (hospital size), and single level (all units) sampling was applied. A questionnaire was mailed to all of the units involved. A sample of 341 valid questionnaires was analysed (response rate 54.5%). Only one praxis-modality was carried out in the majority of units. Intermittent flushing and heparin saline versus normal saline was the most frequent modality employed, over those of continuous flushing and non-heparinised saline. There was a high degree of variation in the quantity of heparin administered: 81.7% when flushing was carried out with heparinised saline and 48.2% when it was conducted with concentrated heparin. About 40% of this variation was associated with the hospital in question, rather than with the unit. The clinical practice fell within the limits of available scientific evidence in fewer than half of the units studied. There was a high degree of variability in the practice of maintaining PIC patency. A significant part of this variation was attributable to the hospital in which the practice was carried out. Moreover, most of this practice was carried out beyond the limits of available scientific evidence.


Suppressor of cytokine signaling-3 is a biomechanical stress-inducible gene that suppresses gp130-mediated cardiac myocyte hypertrophy and survival pathways.
The gp130 cytokine receptor activates a cardiomyocyte survival pathway during the transition to heart failure following the biomechanical stress of pressure overload. Although gp130 activation is observed transiently during transverse aortic constriction (TAC), its mechanism of inactivation is largely unknown in cardiomyocytes. We show here that suppressor of cytokine signaling 3 (SOCS3), an intrinsic inhibitor of JAK, shows biphasic induction in response to TAC. The induction of SOCS3 was closely correlated with STAT3 phosphorylation, as well as the activation of an embryonic gene program, suggesting that cardiac gp130-JAK signaling is precisely controlled by this endogenous suppressor. In addition to its cytoprotective action, gp130-dependent signaling induces cardiomyocyte hypertrophy. Adenovirus-mediated gene transfer of SOCS3 to ventricular cardiomyocytes completely suppressed both hypertrophy and antiapoptotic phenotypes induced by leukemia inhibitory factor (LIF). To our knowledge, this is the first clear evidence that these two separate cardiomyocyte phenotypes induced by gp130 activation lie downstream of JAK. Three independent signaling pathways, STAT3, MEK1-ERK1/2, and AKT activation, that are coinduced by LIF stimulation were completely suppressed by SOCS3 overexpression. We conclude that SOCS3 is a mechanical stress-inducible gene in cardiac muscle cells and that it directly modulates stress-induced gp130 cytokine receptor signaling as the key molecular switch for a negative feedback circuit for both myocyte hypertrophy and survival.


The participation of community members on medical institutional review boards.
The goal of this study was to describe the contributions of community members (unaffiliated members) who serve on institutional review boards (IRBs) at large medical research centers and to compare their contributions to those of other IRB members. We observed and audiotaped 17 panel meetings attended by community members and interviewed 15 community members, as well as 152 other members and staff. The authors coded transcripts of the panel meetings and reviewed the interviews of the community members. Community members played a lesser role as designated reviewers than other members. They were infrequently primary reviewers and expressed hesitation about the role. As secondary or tertiary reviewers, they were less active participants than other members in those roles. Community members were more likely to focus on issues related to confidentiality when reviewing an application than other reviewers. When they were not designated reviewers, however, they played a markedly greater role and their discussion focused more on consent disclosures than other reviewers. They did not appear to represent the community so much as to provide a nonscientific view of the protocol and the consent form.


Concurrent diagnostic validity of a structured psychiatric interview.
In order to estimate the concurrent validity of a structured psychiatric interview, we compared interview diagnoses obtained for 101 psychiatric inpatients to those recorded in the same patients' hospital charts. For most diagnoses considered, concordance was found to be high. For those in which concordance was low, we examined the reasons for the diagnostic discrepancy. Diagnostic errors that were judged to have occurred on the basis of the structural interview often seemed to have resulted from a lack of longitudinal clinical observation. However, more errors were judged to have occurred in the hospital charts, apparently because of physician oversight. We conclude that the concurrent validity of this structured interview is high and that such examinations might be useful not only for research but also for the routine initial evaluation of psychiatric patients.


Intratympanic dexamethasone treatment for control of subjective idiopathic tinnitus: our clinical experience.
In this report, we summarize our clinical experience with intratympanic dexamethasone treatment (IDT) for control of tinnitus. From March 2000 through February 2001, we observed 54 patients (23 women, 31 men; mean age, 49.6 +/- 7.2 years; range, 24-71 years) suffering from subjective idiopathic tinnitus (SIT). After common audiological tests had been performed; all patients underwent specific topodiagnostic tests to verify the cochlear SIT genesis. The 50 subjects with positive results from a furosemide test and negative results from caraverine and carbamazepine tests were selected for the IDT, consisting of transtympanic perfusion of 4 mg dexamethasone to the round window via the middle ear. The treatment was repeated three times daily for 3 consecutive months. Its short-term effects were evaluated 2 weeks after the last perfusion. In 17 of 50 of these patients (34%), the SIT disappeared; 20 of the 50 (40%) reported a significant decrease of the symptom; and the remaining 13 of the 50 (26%) did not experience any improvement. Therefore, we believe that IDT represents an effective drug delivery system for SIT control, as long as the condition arises from inner ear disorders only and treatment occurs within 3 months of symptom onset.


Right and left ventricular myocardial perfusion reserves correlate with right ventricular function and pulmonary hemodynamics in patients with pulmonary arterial hypertension.
To evaluate the relationships of right ventricular (RV) and left ventricular (LV) myocardial perfusion reserves with ventricular function and pulmonary hemodynamics in patients with pulmonary arterial hypertension (PAH) by using adenosine stress perfusion cardiac magnetic resonance (MR) imaging. This HIPAA-compliant study was institutional review board approved. Twenty-five patients known or suspected to have PAH underwent right heart catheterization and adenosine stress MR imaging on the same day. Sixteen matched healthy control subjects underwent cardiac MR imaging only. RV and LV perfusion values at rest and at adenosine-induced stress were calculated by using the Fermi function model. The MR imaging-derived RV and LV functional data were calculated by using dedicated software. Statistical testing included Kruskal-Wallis tests for continuous data, Spearman rank correlation tests, and multiple linear regression analyses. Seventeen of the 25 patients had PAH: 11 with scleroderma-associated PAH, and six with idiopathic PAH. The remaining eight patients had scleroderma without PAH. The myocardial perfusion reserve indexes (MPRIs) in the PAH group (median RV MPRI, 1.7 [25th-75th percentile range, 1.3-2.0]; median LV MPRI, 1.8 [25th-75th percentile range, 1.6-2.1]) were significantly lower than those in the scleroderma non-PAH (median RV MPRI, 2.5 [25th-75th percentile range, 1.8-3.9] [P = .03]; median LV MPRI, 4.1 [25th-75th percentile range, 2.6-4.8] [P = .0003]) and control (median RV MPRI, 2.9 [25th-75th percentile range, 2.6-3.6] [P < .01]; median LV MPRI, 3.6 [25th-75th percentile range, 2.7-4.1] [P < .01]) groups. There were significant correlations between biventricular MPRI and both mean pulmonary arterial pressure (mPAP) (RV MPRI: ρ = -0.59, Bonferroni P = .036; LV MPRI: ρ = -0.79, Bonferroni P < .002) and RV stroke work index (RV MPRI: ρ = -0.63, Bonferroni P = .01; LV MPRI: ρ = -0.75, Bonferroni P < .002). In linear regression analysis, mPAP and RV ejection fraction were independent predictors of RV MPRI. mPAP was an independent predictor of LV MPRI. Biventricular vasoreactivity is significantly reduced with PAH and inversely correlated with RV workload and ejection fraction, suggesting that reduced myocardial perfusion reserve may contribute to RV dysfunction in patients with PAH.


Histamine-releasing factor enhances food allergy.
Food allergy occurs due to IgE- and mast cell-dependent intestinal inflammation. Previously, we showed that histamine-releasing factor (HRF), a multifunctional protein secreted during allergy, interacts with a subset of IgE molecules and that the HRF dimer activates mast cells in an HRF-reactive IgE-dependent manner. In this study, we investigated whether HRF plays any role in food allergy. Specifically, we determined that prophylactic and therapeutic administration of HRF inhibitors that block HRF-IgE interactions reduces the incidence of diarrhea and mastocytosis in a murine model of food allergy. Food allergy-associated intestinal inflammation was accompanied by increased secretion of the HRF dimer into the intestine in response to proinflammatory, Th2, and epithelial-derived cytokines and HRF-reactive IgE levels at the elicitation phase. Consistent with these data, patients with egg allergy had higher blood levels of HRF-reactive IgE compared with individuals that were not hypersensitive. Successful oral immunotherapy in egg-allergy patients and food-allergic mice reduced HRF-reactive IgE levels, thereby suggesting a pathological role for HRF in food allergy. Together, these results suggest that antigen and HRF dimer amplify intestinal inflammation by synergistically activating mast cells and indicate that HRF has potential as a therapeutic target in food allergy.


A safe and convenient pseudovirus-based inhibition assay to detect neutralizing antibodies and screen for viral entry inhibitors against the novel human coronavirus MERS-CoV.
Evidence points to the emergence of a novel human coronavirus, Middle East respiratory syndrome coronavirus (MERS-CoV), which causes a severe acute respiratory syndrome (SARS)-like disease. In response, the development of effective vaccines and therapeutics remains a clinical priority. To accomplish this, it is necessary to evaluate neutralizing antibodies and screen for MERS-CoV entry inhibitors. In this study, we produced a pseudovirus bearing the full-length spike (S) protein of MERS-CoV in the Env-defective, luciferase-expressing HIV-1 backbone. We then established a pseudovirus-based inhibition assay to detect neutralizing antibodies and anti-MERS-CoV entry inhibitors. Our results demonstrated that the generated MERS-CoV pseudovirus allows for single-cycle infection of a variety of cells expressing dipeptidyl peptidase-4 (DPP4), the confirmed receptor for MERS-CoV. Consistent with the results from a live MERS-CoV-based inhibition assay, the antisera of mice vaccinated with a recombinant protein containing receptor-binding domain (RBD, residues 377-662) of MERS-CoV S fused with Fc of human IgG exhibited neutralizing antibody response against infection of MERS-CoV pseudovirus. Furthermore, one small molecule HIV entry inhibitor targeting gp41 (ADS-J1) and the 3-hydroxyphthalic anhydride-modified human serum albumin (HP-HSA) could significantly inhibit MERS-CoV pseudovirus infection. Taken together, the established MERS-CoV inhibition assay is a safe and convenient pseudovirus-based alternative to BSL-3 live-virus restrictions and can be used to rapidly screen MERS-CoV entry inhibitors, as well as evaluate vaccine-induced neutralizing antibodies against the highly pathogenic MERS-CoV.


The use of E-cigarettes as a risk factor for oral potentially malignant disorders and oral cancer: a rapid review of clinical evidence.
The popularity of e-cigarettes has increased rapidly in the last decade, particularly among teens and young adults, being advertised as a less harmful alternative to conventional tobacco products. However, in vitro and in vivo studies have evidenced a variable quantity of potentially harmful components and some recognized carcinogens which may cause DNA damage in oral cells. Additionally, evidence suggests that e-cigarettes may play active roles in the pathogenesis of other malignancies, such as lung and bladder cancers. Therefore, this rapid review aimed to assess the available clinical evidence about using e-cigarettes as a risk factor for oral potentially malignant disorders (OPMD) and oral cancer. A systematic search for English language articles published was performed in PubMed (MEDLINE), Embase, Scopus, and Web of Science. After the study selection process, the authors included twelve clinical studies about OPMD and oral cancer risk in e-cigarette users. The main findings showed the presence of carcinogenic compounds in saliva and morphologic changes, DNA damage, and molecular pathways related to carcinogenesis in the oral cells of e-cigarette users. However, results were inconsistent compared to tobacco smokers and control groups. the current clinical evidence on this topic is limited and insufficient to support using e-cigarettes as a risk factor for OPMD and oral cancer. Nevertheless, dental care professionals should advise patients responsibly about the potentially harmful effects of e-cigarettes on the oral mucosa cells. Future long-term and well-designed clinical studies are needed.


Enhanced cell disruption strategy in the release of recombinant hepatitis B surface antigen from Pichia pastoris using response surface methodology.
Cell disruption strategies by high pressure homogenizer for the release of recombinant Hepatitis B surface antigen (HBsAg) from Pichia pastoris expression cells were optimized using response surface methodology (RSM) based on the central composite design (CCD). The factors studied include number of passes, biomass concentration and pulse pressure. Polynomial models were used to correlate the above mentioned factors to project the cell disruption capability and specific protein release of HBsAg from P. pastoris cells. The proposed cell disruption strategy consisted of a number of passes set at 20 times, biomass concentration of 7.70 g/L of dry cell weight (DCW) and pulse pressure at 1,029 bar. The optimized cell disruption strategy was shown to increase cell disruption efficiency by 2-fold and 4-fold for specific protein release of HBsAg when compared to glass bead method yielding 75.68% cell disruption rate (CDR) and HBsAg concentration of 29.20 mg/L respectively. The model equation generated from RSM on cell disruption of P. pastoris was found adequate to determine the significant factors and its interactions among the process variables and the optimum conditions in releasing HBsAg when validated against a glass bead cell disruption method. The findings from the study can open up a promising strategy for better recovery of HBsAg recombinant protein during downstream processing.


Genomic Analyses of >3,100 Nasopharyngeal Pneumococci Revealed Significant Differences Between Pneumococci Recovered in Four Different Geographical Regions.
Understanding the structure of a bacterial population is essential in order to understand bacterial evolution. Estimating the core genome (those genes common to all, or nearly all, strains of a species) is a key component of such analyses. The size and composition of the core genome varies by dataset, but we hypothesized that the variation between different collections of the same bacterial species would be minimal. To investigate this, we analyzed the genome sequences of 3,118 pneumococci recovered from healthy individuals in Reykjavik (Iceland), Southampton (United Kingdom), Boston (United States), and Maela (Thailand). The analyses revealed a "supercore" genome (genes shared by all 3,118 pneumococci) of 558 genes, although an additional 354 core genes were shared by pneumococci from Reykjavik, Southampton, and Boston. Overall, the size and composition of the core and pan-genomes among pneumococci recovered in Reykjavik, Southampton, and Boston were similar. Maela pneumococci were distinctly different in that they had a smaller core genome and larger pan-genome. The pan-genome of Maela pneumococci contained several >25 Kb sequence regions (flanked by pneumococcal genes) that were homologous to genomic regions found in other bacterial species. Overall, our work revealed that some subsets of the global pneumococcal population are highly heterogeneous, and our hypothesis was rejected. This is an important finding in terms of understanding genetic variation among pneumococci and is also an essential point of consideration before generalizing the findings from a single dataset to the wider pneumococcal population.


Protocol for determining the diagnostic validity of physical examination maneuvers for shoulder pathology.
Shoulder complaints are the third most common musculoskeletal problem in the general population. There are an abundance of physical examination maneuvers for diagnosing shoulder pathology. The validity of these maneuvers has not been adequately addressed. We propose a large Phase III study to investigate the accuracy of these tests in an orthopaedic setting. We will recruit consecutive new shoulder patients who are referred to two tertiary orthopaedic clinics. We will select which physical examination tests to include using a modified Delphi process. The physician will take a thorough history from the patient and indicate their certainty about each possible diagnosis (certain the diagnosis is absent, present or requires further testing). The clinician will only perform the physical examination maneuvers for diagnoses where uncertainty remains. We will consider arthroscopy the reference standard for patients who undergo surgery within 8 months of physical examination and magnetic resonance imaging with arthrogram for patients who do not. We will calculate the sensitivity, specificity and positive and negative likelihood ratios and investigate whether combinations of the top tests provide stronger predictions of the presence or absence of disease. There are several considerations when performing a diagnostic study to ensure that the results are applicable in a clinical setting. These include, 1) including a representative sample, 2) selecting an appropriate reference standard, 3) avoiding verification bias, 4) blinding the interpreters of the physical examination tests to the interpretation of the gold standard and, 5) blinding the interpreters of the gold standard to the interpretation of the physical examination tests. The results of this study will inform clinicians of which tests, or combination of tests, successfully reduce diagnostic uncertainty, which tests are misleading and how physical examination may affect the magnitude of the confidence the clinician feels about their diagnosis. The results of this study may reduce the number of costly and invasive imaging studies (MRI, CT or arthrography) that are requisitioned when uncertainty about diagnosis remains following history and physical exam. We also hope to reduce the variability between specialists in which maneuvers are used during physical examination and how they are used, all of which will assist in improving consistency of care between centres.


A highly active bagasse-derived solid acid catalyst with properties suitable for production of biodiesel.
A novel bagasse-based solid acid catalyst was successfully prepared through sulfonation of incompletely carbonized bagasse. A range of conditions for producing the catalyst were investigated, and the optimized catalyst, produced under carbonization at 648 K for 0.5 h and sulfonation at 423 K for 15 h, showed excellent catalytic activity and resulted in around 95 % yield of methyl oleate. Its activity was not only substantially greater than that of niobic acid and Amberlyst-15, but also comparable to or superior to that of catalysts made from pure starch or glucose, respectively. Additionally, the bagasse-derived catalyst could be repeatedly employed for at least eight cycles and still retained around 90 % of its original activity, exhibiting excellent operational stability. Furthermore, the catalyst efficiently converted waste cooking oils with 38.6 wt % free fatty acids into biodiesel and afforded a high yield of about 93.8 % within 12 h. These results clearly show that the bagasse-derived catalyst is economic, eco-friendly, and promising for biodiesel production from low-cost feedstocks and may find wide applications.


Factors predicting the therapeutic response to infliximab during maintenance therapy in Japanese patients with Crohn's disease.
Since anti-tumor necrosis factor (TNF)-α agents (TNF-α inhibitors) induce both clinical response and remission in patients with moderate to severe inflammatory bowel disease (IBD), the use of anti-TNF therapies has fundamentally changed the approach to treatment for patients with IBD. Infliximab (IFX) is a TNF-α inhibitor approved for the induction and remission of Crohn's disease (CD). However, even among patients who initially demonstrate a clinical response to IFX therapy, secondary loss of response occurs, although the reason remains unknown. We therefore investigated predictive factors associated with the response to IFX in long-term maintenance treatment in Japanese CD patients. Eight types of single-nucleotide polymorphisms (SNPs) were investigated using the real-time PCR method, and patient characteristics were collected from the electronic medical records. The Crohn's Disease Activity Index criteria were used as the response to IFX therapy. The observation period was 1 year after IFX had been administered for more than 1 year. Associations between the IFX response and patient characteristics were evaluated using the multivariate logistic regression model. We studied 121 unrelated adult Japanese with CD treated for more than 1 year with IFX as outpatients at Keio University Hospital from November 1, 2014 to November 30, 2015. Among them, 71 were classified as in remisson. In multivariate analysis, patients with the TNF-α 857C>T C/C genotype, shorter disease duration, without double dosing, and combination treatment with an immunomodulator had higher remisson rates than those with the C/T or T/T genotype, longer disease duration, with double dosing, and no combination treatment with an immunomodulator. The response to IFX in Japanese CD patients may therefore be predicted by these 4 characteristics in actual clinical practice.


Generation of neutralizing activity against human immunodeficiency virus type 1 in serum by antibody gene transfer.
Although several human immunodeficiency virus (HIV) vaccine approaches have elicited meaningful antigen-specific T-cell responses in animal models, no single vaccine candidate has engendered antibodies that broadly neutralize primary isolates of HIV type 1 (HIV-1). Thus, there remains a significant gap in the design of HIV vaccines. To address this issue, we exploited the existence of rare human monoclonal antibodies that have been isolated from HIV-infected individuals. Such antibodies neutralize a wide array of HIV-1 field isolates and have been shown to be effective in vivo. However, practical considerations preclude the use of antibody preparations as a prophylactic passive immunization strategy in large populations. Our concept calls for an antibody gene of choice to be transferred to muscle where the antibody molecule is synthesized and distributed to the circulatory system. In these experiments, we used a recombinant adeno-associated virus (rAAV) vector to deliver the gene for the human antibody IgG1b12 to mouse muscle. Significant levels of HIV-neutralizing activity were found in the sera of mice for over 6 months after a single intramuscular administration of the rAAV vector. This approach allows for predetermination of antibody affinity and specificity prior to "immunization" and avoids the need for an active humoral immune response against the HIV envelope protein.


Initial motivations for alcohol treatment: relations with patient characteristics, treatment involvement, and dropout.
This study examines (a) the relation of initial treatment motivations to alcoholics' involvement in outpatient treatment and dropout and (b) the relations among patient characteristics, severity, alcohol expectancies, motivation, and treatment retention. A treatment motivation questionnaire (TMQ) was developed to assess both internalized and external motivations for treatment, as well as confidence in the treatment and orientation towards interpersonal help seeking. In Study 1, the TMQ was administered to 109 outpatients entering an alcoholism clinic. Based on these data the scale was revised and was administered to a subsequent sample of 98 subjects seeking treatment. Information about demographic variables, measures of substance use, alcohol expectancies, and psychiatric severity was also gathered. Eight weeks after intake, outcome was evaluated through attendance records and clinician ratings. Results revealed that internalized motivation was associated with greater patient involvement and retention in treatment. Subjects high in both internalized and external motivation demonstrated the best attendance and treatment retention while those low in internalized motivation showed the poorest treatment retention while those low in internalized motivation showed the poorest treatment response, regardless of the level of external motivation. Problem severity was also related to a greater degree of internalized motivation. The importance of initial motivations in understanding treatment response and dropout is discussed.


A comparison of in vitro methods for assessing the potency of therapeutic antisera against the venom of the coral snake Micrurus nigrocinctus.
Therapeutic antisera against Micrurus nigrocinctus venom were tested for protection against lethality, as well as for inhibition of the nicotinic acetylcholine receptor (AchR)-binding and neutralization of phospholipase A2 (PLA2) activities of the homologous venom. Protection against venom lethality did not correlate with inhibition of AchR-binding activity, whereas there was a significant correlation between antisera potency and inhibition of PLA2 activity (r = 0.82, n = 10, P < 0.02). Inhibition of PLA2 activity could be useful in assessing the protective efficacy of M. nigrocinctus antisera during antivenom production. Micrurus nigrocinctus nigrocinctus venom proteins were fractionated by cation-exchange chromatography on Mono S FPLC and fractions assayed for lethality, AchR-binding and PLA2 activities. Antisera were titrated by enzyme-linked immunoassay (ELISA) against a crude M. n. nigrocinctus venom, two FPLC lethal fractions containing AchR-binding activity, and two toxins purified from M. n. nigrocinctus venom. No correlation was found between protective efficacy and the ELISA titer against any of these antigens. Compared to other elapid venoms that contain few toxins as major components, M. n. nigrocinctus venom appears to be more complex and its lethal effect is likely to be due to the combined effect of several neurotoxins.


Genomic and transcriptomic correlates of Richter transformation in chronic lymphocytic leukemia.
The transformation of chronic lymphocytic leukemia (CLL) to high-grade B-cell lymphoma is known as Richter syndrome (RS), a rare event with dismal prognosis. In this study, we conducted whole-genome sequencing (WGS) of paired circulating CLL (PB-CLL) and RS biopsies (tissue-RS) from 17 patients recruited into a clinical trial (CHOP-O). We found that tissue-RS was enriched for mutations in poor-risk CLL drivers and genes in the DNA damage response (DDR) pathway. In addition, we identified genomic aberrations not previously implicated in RS, including the protein tyrosine phosphatase receptor (PTPRD) and tumor necrosis factor receptor-associated factor 3 (TRAF3). In the noncoding genome, we discovered activation-induced cytidine deaminase-related and unrelated kataegis in tissue-RS affecting regulatory regions of key immune-regulatory genes. These include BTG2, CXCR4, NFATC1, PAX5, NOTCH-1, SLC44A5, FCRL3, SELL, TNIP2, and TRIM13. Furthermore, differences between the global mutation signatures of pairs of PB-CLL and tissue-RS samples implicate DDR as the dominant mechanism driving transformation. Pathway-based clonal deconvolution analysis showed that genes in the MAPK and DDR pathways demonstrate high clonal-expansion probability. Direct comparison of nodal-CLL and tissue-RS pairs from an independent cohort confirmed differential expression of the same pathways by RNA expression profiling. Our integrated analysis of WGS and RNA expression data significantly extends previous targeted approaches, which were limited by the lack of germline samples, and it facilitates the identification of novel genomic correlates implicated in RS transformation, which could be targeted therapeutically. Our results inform the future selection of investigative agents for a UK clinical platform study. This trial was registered at www.clinicaltrials.gov as #NCT03899337.


Post-treatment de-phosphorylation of p53 correlates with dasatinib responsiveness in malignant melanoma.
Dasatinib (Sprycel) was developed as a tyrosine kinase inhibitor targeting Bcr-Abl and the family of Src kinases. Dasatinib is commonly used for the treatment of acute lymphoblastic and chronic myelogenous leukemia. Previous clinical studies in melanoma returned inconclusive results and suggested that patients respond highly heterogeneously to dasatinib as single agent or in combination with standard-of-care chemotherapeutic dacarbazine. Reliable biomarkers to predict dasatinib responsiveness in melanoma have not yet been developed. Here, we collected comprehensive in vitro data from experimentally well-controlled conditions to study the effect of dasatinib, alone and in combination with dacarbazine, on cell proliferation and cell survival. Sixteen treatment conditions, covering therapeutically relevant concentrations ranges of both drugs, were tested in 12 melanoma cell lines with diverse mutational backgrounds. Melanoma cell lines responded heterogeneously and, importantly, dasatinib and dacarbazine did not synergize in suppressing proliferation or inducing cell death. Since dasatinib is a promiscuous kinase inhibitor, possibly affecting multiple disease-relevant pathways, we also determined if basal phospho-protein amounts and treatment-induced changes in phospho-protein levels are indicative of dasatinib responsiveness. We found that treatment-induced de-phosphorylation of p53 correlates with dasatinib responsiveness in malignant melanoma. Loss of p53 phosphorylation might be an interesting candidate for a kinetic marker of dasatinib responsiveness in melanoma, pending more comprehensive validation in future studies.


The catabolite control protein CcpA controls ammonium assimilation in Bacillus subtilis.
Carbon catabolite repression of several catabolic operons in Bacillus subtilis is mediated by the repressor CcpA. An inactivation of the ccpA gene has two distinct phenotypes: (i) catabolite repression of catabolic operons is lost and (ii) the growth of bacteria on minimal medium is severely impaired. We have analyzed the physiological properties of a ccpA mutant strain and show that the ccpA mutation does not affect sugar transport. We have isolated extragenic suppressors of ccpA that suppress the growth defect (sgd mutants). Catabolite repression of beta-xylosidase synthesis was, however, not restored suggesting that the suppressor mutations allow differentiation between the phenotypes of the ccpA mutant. A close inspection of the growth requirements of the ccpA mutant revealed the inability of the mutant to utilize inorganic ammonium as a single source of nitrogen. An intact ccpA gene was found to be required for expression of the gltAB operon encoding glutamate synthase. This enzyme is necessary for the assimilation of ammonium. In a sgd mutant, gltAB operon expression was no longer dependent on ccpA, suggesting that the poor expression of the gltAB operon is involved in the growth defect of the ccpA mutant.


Antimutagenic and Synergistic Cytotoxic Effect of Cisplatin and Honey Bee Venom on 4T1 Invasive Mammary Carcinoma Cell Line.
Honey bee venom (HBV) has various biological activities such as the inhibitory effect on several types of cancer. Cisplatin is an old and potent drug to treat most of the cancers. Our aim in the present study was to determine antimutagenic and cytotoxic effects of HBV on mammary carcinoma, exclusively and in combination with cisplatin. In this study, 4T1 cell line was cultured in RPMI-1640 with 10% fetal bovine serum (FBS), at 37°C in humidified CO2 incubator. The cell viabilities were examined by the MTT assay. Also, HBV was screened for its antimutagenic activity via the Ames test. The results were assessed by SPSS software version 19 and one-way ANOVA method considering p < 0.05 level of significance. The results showed that 6 mg/ml of HBV, 20 μg/ml of cisplatin, and 6 mg/ml HBV with 10 μg/ml cisplatin could induce approximately 50% of 4T1 cell death. The concentration 7 mg/ml of HBV with of 62.76% inhibitory rate showed the highest antimutagenic activity in comparison with other treatment groups. The MTT assay demonstrated that HBV and cisplatin could cause cell death in a dose-dependent manner. The cytotoxic effect of cisplatin also promoted by HBV. Ames test outcomes indicated that HBV could act as a significant mutagenic agent. The antimutagenic activity of HBV was increased considerably in the presence of S9 mix. Therefore, our findings have revealed that HBV can enhance the cytotoxic effect of cisplatin drug and its cancer-preventing effects.


Flux distributions in anaerobic, glucose-limited continuous cultures of Saccharomyces cerevisiae.
A stoichiometric model describing the anaerobic metabolism of Saccharomyces cerevisiae during growth on a defined medium was derived. The model was used to calculate intracellular fluxes based on measurements of the uptake of substrates from the medium, the secretion of products from the cells, and of the rate of biomass formation. Furthermore, measurements of the biomass composition and of the activity of key enzymes were used in the calculations. The stoichiometric network consists of 37 pathway reactions involving 43 compounds of which 13 were measured (acetate, CO2, ethanol, glucose, glycerol, NH4+, pyruvate, succinate, carbohydrates, DNA, lipids, proteins and RNA). The model was used to calculate the production rates of malate and fumarate and the ethanol measurement was used to validate the model. All rate measurements were performed on glucose-limited continuous cultures in a high-performance bioreactor. Carbon balances closed within 98%. The calculations comprised flux distributions at specific growth rates of 0.10 and 0.30 h-1. The fluxes through reactions located around important branch points of the metabolism were compared, i.e. the split between the pentose phosphate and the Embden-Meyerhoff-Parnas pathways. Also the model was used to show the probable existence of a redox shunt across the inner mitochondrial membrane consisting of the reactions catalysed by the mitochondrial and the cytosolic alcohol dehydrogenase. Finally it was concluded that cytosolic isocitrate dehydrogenase is probably not present during growth on glucose. The importance of basing the flux analysis on accurate measurements was demonstrated through a sensitivity analysis. It was found that the accuracy of the measurements of CO2, ethanol, glucose, glycerol and protein was critical for the correct calculation of the flux distribution.


Neurobehavioral complications of type I diabetes. Examination of possible risk factors.
There is increasing interest in the possibility that diabetes mellitus may be associated with a series of neurobehavioral, or neuropsychological, changes; i.e., learning, memory, problem solving, mental and motor speed, and eye-hand coordination may sometimes be disrupted in diabetic children and adults, and this disruption may be a consequence of certain disease-related variables. To date, four neurobehavioral risk factors have been identified. For children and adolescents, the most potent risk factors appear to be age at diagnosis and the occurrence of schoolroom-related problems. Children who develop diabetes early in life are more likely to show serious cognitive impairments on virtually all measures. In addition, diabetic children with school attendance problems tend to score lower than expected on verbal IQ tests and school achievement tests. For adults, the most carefully studied risk factor is degree of metabolic control. Adults in poor control are far more likely to manifest subtle changes on measures of mental efficiency. This may be particularly evident on tasks that require the development and deployment of sophisticated information-processing strategies. A fourth variable--severe episodes of hypoglycemia--is known to disrupt functioning in diabetic subjects of any age, although it has not yet been studied systematically in large-scale studies. Indeed, because virtually all extant studies have been based on limited neurobehavioral and biomedical assessments of relatively few volunteer subjects, this list of risk factors must be considered tentative at best.


Reducing the risk of pandemic influenza in Aboriginal communities.
Aboriginal people are particularly vulnerable to pandemic influenza A, H1N109. This was first recognized in the First Nations of Canada. There have been calls for close planning with Aboriginal people to manage these risks. This article describes the process and findings from preliminary community consultations into reducing influenza risk, including pandemic H1N1(09) swine influenza, in Aboriginal communities in the Hunter New England area of northern New South Wales, Australia. Consultation was conducted with 6 Aboriginal communities in response to the rapidly evolving pandemic and was designed to further develop shared understanding between health services and Aboriginal communities about appropriate and culturally safe ways to reduce the influenza risk in communities. Agreed risk mitigation measures identified in partnership are being introduced throughout Hunter New England area. Five theme areas were identified that posed particular challenges to limiting the negative impact of pandemic influenza; and a number of potential solutions emerged from focus group discussions: (1) local resource person: local identified 'go to' people are heard and trusted, but need to have an understanding of H1N109; (2) clear communication: information must be presented simply, clearly and demonstrating respect for local culture; (3) access to health services: sick people need to know where to get help and how to get there without infecting others; (4) households and funerals: infection control messages should be aligned with the reality of life in Aboriginal communities, and the importance of attending family and cultural gatherings; (5) social and community support issues: Aboriginal people need to have a say in how support is provided. Influenza pandemics are a serious threat to the health and social functioning of Aboriginal communities. Measures to reduce the risk of influenza in communities must be developed with the communities to maximise their acceptance. The process of engagement and ongoing respectful negotiations with communities is critical to developing culturally appropriate pandemic mitigation and management strategies.


Guidance on Evaluating Options for Representing Clinical Data within Health Information Systems.
The health information system PlunketPlus is a clinical initiative of Plunket (the Royal New Zealand Plunket Society) with a goal of further improving the health outcomes for children in New Zealand. The success of PlunketPlus depends heavily on how data is represented within the system. The purpose of the study described in this paper was to use PlunketPlus as a case study to inform the development of guidance on evaluating options for representing clinical data within health information systems, with a particular focus on automating existing informational processes. It has been possible to take some of the lessons learned to inform the development of initial more generic guidance that might be applicable across a range of domains. This paper concludes with a description of how Plunket applied the guidance as part of the development of PlunketPlus.


Attention to form or surface properties modulates different regions of human occipitotemporal cortex.
We carried out 2 functional magnetic resonance imaging experiments to investigate the cortical mechanisms underlying the contribution of form and surface properties to object recognition. In experiment 1, participants performed same-different judgments in separate blocks of trials on pairs of unfamiliar "nonsense" objects on the basis of their form, surface properties (i.e., both color and texture), or orientation. Attention to form activated the lateral occipital (LO) area, whereas attention to surface properties activated the collateral sulcus (CoS) and the inferior occipital gyrus (IOG). In experiment 2, participants were required to make same-different judgments on the basis of texture, color, or form. Again attention to form activated area LO, whereas attention to texture activated regions in the IOG and the CoS, as well as regions in the lingual sulcus and the inferior temporal sulcus. Within these last 4 regions, activation associated with texture was higher than activation associated with color. No color-specific cortical areas were identified in these regions, although parts of V1 and the cuneus yielded higher activation for color as opposed to texture. These results suggest that there are separate form and surface-property pathways in extrastriate cortex. The extraction of information about an object's color seems to occur relatively early in visual analysis as compared with the extraction of surface texture, perhaps because the latter requires more complex computations.


RC time constant of single lung equals that of both lungs together: a study in chronic thromboembolic pulmonary hypertension.
The product of resistance, R, and compliance, C (RC time), of the entire pulmonary circulation is constant. It is unknown if this constancy holds for individual lungs. We determined R and C in individual lungs in chronic thromboembolic pulmonary hypertension (CTEPH) patients where resistances differ between both lungs. Also, the contribution of the proximal pulmonary arteries (PA) to total lung compliance was assessed. Patients (n=23) were referred for the evaluation of CTEPH. Pressure was measured by right heart catheterization and flows in the main, left, and right PA by magnetic resonance imaging. Total, left, and right lung resistances were calculated as mean pressure divided by mean flow. Total, left, and right lung compliances were assessed by the pulse pressure method. Proximal compliances were derived from cross-sectional area change DeltaA and systolic-diastolic pressure difference DeltaP (DeltaA/DeltaP) in main, left, and right PA, multiplied by vessel length. The lung with the lowest blood flow was defined "low flow" (LF), the contralateral lung "high flow" (HF). Total resistance was 0.57+/-0.28 mmHg.s(-1).ml(-1), and resistances of LF and HF lungs were 1.57+/-0.2 vs. 1.00+/-0.1 mmHg.s(-1).ml(-1), respectively, P<0.0001. Total compliance was 1.22+/-1.1 ml/mmHg, and compliances of LF and HF lung were 0.47+/-0.11 and 0.62+/-0.12 ml/mmHg, respectively, P=0.01. Total RC time was 0.49+/-0.2 s, and RC times for the LF and HF lung were 0.45+/-0.2 and 0.45+/-0.1 s, respectively, not different. Proximal arterial compliance, given by the sum of main, right, and left PA compliances, was only 19% of total lung compliance. The RC time of a single lung equals that of both lungs together, and pulmonary arterial compliance comes largely from the distal vasculature.


Mkk4 is a negative regulator of the transforming growth factor beta 1 signaling associated with atrial remodeling and arrhythmogenesis with age.
Atrial fibrillation (AF), often associated with structural, fibrotic change in cardiac tissues involving regulatory signaling mediators, becomes increasingly common with age. In the present study, we explored the role of mitogen-activated protein kinase kinase 4 (Mkk4), a critical component of the stress-activated mitogen-activated protein kinase family, in age-associated AF. We developed a novel mouse model with a selective inactivation of atrial cardiomyocyte Mkk4 (Mkk4(ACKO)). We characterized and compared electrophysiological, histological, and molecular features of young (3- to 4-month), adult (6-month), and old (1-year) Mkk4(ACKO) mice with age-matched control littermates (Mkk4(F/F)). Aging Mkk4(ACKO) mice were more susceptible to atrial tachyarrhythmias than the corresponding Mkk4(F/F) mice, showing characteristic slow and dispersed atrial conduction, for which modeling studies demonstrated potential arrhythmic effects. These differences paralleled increased interstitial fibrosis, upregulated transforming growth factor beta 1 (TGF-β1) signaling and dysregulation of matrix metalloproteinases in Mkk4(ACKO), compared to Mkk4(F/F), atria. Mkk4 inactivation increased the sensitivity of cultured cardiomyocytes to angiotensin II-induced activation of TGF-β1 signaling. This, in turn, enhanced expression of profibrotic molecules in cultured cardiac fibroblasts, suggesting cross-talk between these two cell types in profibrotic signaling. Finally, human atrial tissues in AF showed a Mkk4 downregulation associated with increased production of profibrotic molecules, compared to findings in tissue from control subjects in sinus rhythm. These findings together demonstrate, for the first time, that Mkk4 is a negative regulator of the TGF-β1 signaling associated with atrial remodeling and arrhythmogenesis with age, establishing Mkk4 as a new potential therapeutic target for treating AF.


A quantitative magnetic resonance imaging study of changes in brain morphology from infancy to late adulthood.
To model in vivo the dynamic interrelations of head size, gray matter, white matter, and cerebrospinal fluid (CSF) volumes from infancy to old age using magnetic resonance imaging (MRI). Cross-sectional, between-subjects using an age-regression model. A Veterans Affairs medical center and community hospitals. There were 88 male and female subjects aged 3 months to 30 years whose clinical MRI film had been read as normal and 73 healthy male volunteers aged 21 to 70 years who had an MRI performed specifically for this study. These MRI data were quantified using a semiautomated computer technique for segmenting images into gray matter, white matter, and CSF compartments. The cortex was defined geometrically as the outer 45% on each analyzed slice, and the volumes of cortical white matter, gray matter, and CSF were computed. Subcortical (ventricular) CSF volume was computed for the inner 55% of each analyzed slice. In the younger sample, intracranial volume increased by about 300 mL from 3 months to 10 years. The same patterns of change in volume of each compartment across the age range were seen in both sexes: cortical gray matter volume peaked around age 4 years and decreased thereafter; cortical white matter volume increased steadily until about age 20 years; cortical and ventricular CSF volumes remained constant. In the older sample, brain volumes were statistically adjusted for normal variation in head size through a regression procedure and revealed the following pattern: cortical gray matter volume decreased curvilin-early, showing an average volume loss of 0.7 mL/y, while cortical white matter volume remained constant during the five decades; complementary to the cortical gray matter decrease, cortical CSF volume increased by 0.6 mL/y and ventricular volumes increased by 0.3 mL/y. These patterns of growth and change seen in vivo with MRI are largely consistent with neuropathological studies, as well as animal models of development, and may reflect neuronal progressive and regressive processes, including cell growth, myelination, cell death, and atrophy.


Halogenated triarylboranes: synthesis, properties and applications in catalysis.
Halogenated triarylboranes (BAr3) have been known for decades, however it has only been since the surge of interest in main group catalysis that their application as strong Lewis acid catalysts has been recognised. This review aims to look past the popular tris(pentafluorophenyl)borane [B(C6F5)3] to the other halogenated triarylboranes, to give a greater breadth of understanding as to how tuning the Lewis acidity of BAr3 by modifications of the aryl rings can lead to improved reactivity. In this review, a discussion on Lewis acidity determination of boranes is given, the synthesis of these boranes is discussed, and examples of how they are being used for catalysis and frustrated Lewis pair (FLP) chemistry are explained.


Follow-Up of Metal-on-Metal Hip Arthroplasty Patients Is Currently Not Evidence Based or Cost Effective.
Over one-million patients worldwide have received metal-on-metal (MoM) hip arthroplasties with a significant proportion requiring revision surgery in the short-term for adverse reaction to metal debris (ARMD). Worldwide authorities have subsequently issued follow-up guidance for MoM hip patients. This article compares follow-up guidelines for MoM hips published by five worldwide authorities, analyses these protocols in relation to published evidence, and assesses the financial implications of these guidelines. A number of major differences exist between authorities regarding patient follow-up, with vast cost differences between protocols (£84 to £988/patient/year for stemmed MoM hips and £0 to £988/patient/year for hip resurfacing). Current worldwide guidance is neither evidence-based nor financially sustainable with most protocols lacking the sensitivity to detect asymptomatic ARMD lesions.


A topographical method for the development of neural networks for artificial brain evolution.
Developmental neural networks, which are constructed according to developmental rules (i.e., genes), have the potential to be differentiated into heteromorphic neural structures capable of performing various kinds of activities. The fact that the biological neural architectures are found to be highly repetitive, layered, and topographically organized has important consequences for neural development methods. The purpose of this article is to propose a neural development method that can construct topographical neural connections, that is, a topographical development method, to facilitate fast and efficient development. This is achieved by arborizing neural connections on a developmental tree that rarely produces dead connections. Modular gene expression and corresponding modular networks have an important role in a gradual evolutionary process. Gene expression for modular networks is also proposed here as a way to reduce the probability of fatal mutants created through gene alteration. The corresponding evolutionary experiment shows that various neural structures--layered, repetitive, modular, and complex ones like those in the biological brain--can be constructed and easily observed. It also demonstrates that due to the efficiency of the proposed method, large neural networks can be easily managed, thereby making it suitable for long duration evolutionary experiments.


Tar DNA binding protein-43 (TDP-43) associates with stress granules: analysis of cultured cells and pathological brain tissue.
Tar DNA Binding Protein-43 (TDP-43) is a principle component of inclusions in many cases of frontotemporal lobar degeneration (FTLD-U) and amyotrophic lateral sclerosis (ALS). TDP-43 resides predominantly in the nucleus, but in affected areas of ALS and FTLD-U central nervous system, TDP-43 is aberrantly processed and forms cytoplasmic inclusions. The mechanisms governing TDP-43 inclusion formation are poorly understood. Increasing evidence indicates that TDP-43 regulates mRNA metabolism by interacting with mRNA binding proteins that are known to associate with RNA granules. Here we show that TDP-43 can be induced to form inclusions in cell culture and that most TDP-43 inclusions co-localize with SGs. SGs are cytoplasmic RNA granules that consist of mixed protein-RNA complexes. Under stressful conditions SGs are generated by the reversible aggregation of prion-like proteins, such as TIA-1, to regulate mRNA metabolism and protein translation. We also show that disease-linked mutations in TDP-43 increased TDP-43 inclusion formation in response to stressful stimuli. Biochemical studies demonstrated that the increased TDP-43 inclusion formation is associated with accumulation of TDP-43 detergent insoluble complexes. TDP-43 associates with SG by interacting with SG proteins, such as TIA-1, via direct protein-protein interactions, as well as RNA-dependent interactions. The signaling pathway that regulates SGs formation also modulates TDP-43 inclusion formation. We observed that inclusion formation mediated by WT or mutant TDP-43 can be suppressed by treatment with translational inhibitors that suppress or reverse SG formation. Finally, using Sudan black to quench endogenous autofluorescence, we also demonstrate that TDP-43 positive-inclusions in pathological CNS tissue co-localize with multiple protein markers of stress granules, including TIA-1 and eIF3. These data provide support for accumulating evidence that TDP-43 participates in the SG pathway.


Mapping Exposure to Multi-Pollutants Using Environmental Biomonitors-A Multi-Exposure Index.
Atmosphere is a major pathway for transport and deposition of pollutants in the environment. In industrial areas, organic compounds are released or formed as by-products, such as polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/F's). Inorganic chemical elements, including lead and arsenic, are also part of the pollutants mixture, and even in low concentrations may potentially be toxic and carcinogenic. However, assessing the spatial pattern of their deposition is difficult due to high spatial and temporal heterogeneity. Lichens have been used as biomonitors of atmospheric deposition, because these organisms encompass greater spatial detail than air monitoring stations and provide an integration of overall pollution. Based upon the ability of lichens to concentrate pollutants such as PCDD/F and chemical elements, the main objectives of this study were to develop a new semi-quantitative multi-pollutant toxicity exposure index (TEQ-like), derived from risk estimates, in an attempt to correlate several atmospheric pollutants to human exposure levels. The actual pollutant concentrations were measured in the environment, from biomonitors (organisms that integrate multi-pollutants), enabling interpolation and mapping of contaminant deposition within the region. Thus, the TEQ-like index provides a spatial representation not from absolute accumulation of the different pollutants, but from the accumulation weighted by their relative risk. The assessment of environmental human exposure to multi-pollutants through atmospheric deposition may be applied to industries to improve mitigation processes or to health stakeholders to target populations for a comprehensive risk assessment, epidemiological studies, and health recommendations.


Mooren-type hepatitis C virus-associated corneal ulceration.
Two patients with bilateral Mooren-type ulcers had underlying chronic hepatitis C virus (HCV) infection. Both patients also had chronic, pruritic dermatitis, which in one patient was diagnosed as hidradenitis suppurativa. Serum from the first patient and serum, conjunctiva, and liver from the second patient were analyzed for HCV genomic RNA using the reverse transcriptase-polymerase chain reaction. Serum anti-HCV antibodies were monitored with a commercially available second-generation test. Liver and conjunctival biopsies were evaluated histopathologically. Liver biopsy showed severe hepatitis in the first patient, but normal liver tissue in the second. Hepatitis C virus genomic RNA was detected in the serum of both patients. In the first patient, the virus was detected 4 months after completion of interferon alfa-2b treatment for chronic active hepatitis. In the second patient, HCV genomic RNA was detected in serum, but not in conjunctiva or liver tissue. Hepatitis C virus could not be detected in the serum of the second patient after 2 weeks of interferon alfa-2b treatment. Both patients had serum anti-HCV antibodies. In case 1, there was a marked improvement in the corneal disease during and after 6 months of interferon alfa-2b treatment for chronic active hepatitis that paralleled a return of serum liver enzyme levels to the normal range. In the second patient, the corneal disease improved after 6 weeks of interferon alfa-2b treatment, but abruptly worsened when the patient discontinued therapy. The corneal disease improved again after interferon alfa-2b was reinstituted. Chronic HCV virus infection is associated with Mooren-type peripheral ulcerative keratitis. All patients with Mooren-type ulcers should be tested for evidence of HCV infection in consultation with a liver specialist. Even when improvement is obtained with interferon alfa-2b treatment, however, continued follow-up is important because relapse is common and repeat treatment may be effective.


Data management and functional annotation of the Korean reference plasma proteome.
As human plasma is clinically valuable, reference data from healthy donors can be a useful source for serological biomarker studies. To make a reliable protein catalog of the Korean plasma proteome, various experimental methods, such as 1-D HPLC, 2-D LC, and narrow ranged 2-DE prior to MALDI-TOF and LC-MS/MS, were used to identify unique plasma proteins in this population. To compile candidates with high confidence, two different search engines were used to select proteins with a false discovery rate of less than or equal to 1%. From this rigorous selection process, we initially identified 494 distinct Korean plasma proteins. After multilevel stepwise filtrations with stringent, identification parameters were applied to acquire plasma protein list with the maximum confidence; a total 185 distinct plasma proteins were identified and integrated into our Korean human plasma proteome project database along with several bioinformatics analysis results, including gene ontology, biological pathways, tissue expression, and disease association. This is the first publicly available single ethnic group-specific plasma proteome database (http://proteomix.org/khppp/).


Characterization of pathogenic monoclonal autoantibodies derived from muscle-specific kinase myasthenia gravis patients.
Myasthenia gravis (MG) is a chronic autoimmune disorder characterized by muscle weakness and caused by pathogenic autoantibodies that bind to membrane proteins at the neuromuscular junction. Most patients have autoantibodies against the acetylcholine receptor (AChR), but a subset of patients have autoantibodies against muscle-specific tyrosine kinase (MuSK) instead. MuSK is an essential component of the pathway responsible for synaptic differentiation, which is activated by nerve-released agrin. Through binding MuSK, serum-derived autoantibodies inhibit agrin-induced MuSK autophosphorylation, impair clustering of AChRs, and block neuromuscular transmission. We sought to establish individual MuSK autoantibody clones so that the autoimmune mechanisms could be better understood. We isolated MuSK autoantibody-expressing B cells from 6 MuSK MG patients using a fluorescently tagged MuSK antigen multimer, then generated a panel of human monoclonal autoantibodies (mAbs) from these cells. Here we focused on 3 highly specific mAbs that bound quantitatively to MuSK in solution, to MuSK-expressing HEK cells, and at mouse neuromuscular junctions, where they colocalized with AChRs. These 3 IgG isotype mAbs (2 IgG4 and 1 IgG3 subclass) recognized the Ig-like domain 2 of MuSK. The mAbs inhibited AChR clustering, but intriguingly, they enhanced rather than inhibited MuSK phosphorylation, which suggests an alternative mechanism for inhibiting AChR clustering.