A pathway leading to a cation-binding pocket determines the selectivity of the NhaP2 antiporter in Vibrio cholerae1.

The Vc-NhaP2 antiporter from Vibrio cholerae exchanges H+ for K+ or Na+ but not for the smaller Li+. The molecular basis of this unusual selectivity remains unknown. Phyre2 and Rosetta software were used to generate a structural model of the Vc-NhaP2. The obtained model suggested that a cluster of residues from different transmembrane segments (TMSs) forms a putative cation-binding pocket in the middle of the membrane: D133 and T132 from TMS V together with D162 and E157 of TMS VI. The model also suggested that L257, G258, and N259 from TMS IX together with T276, D273, Q280, and Y251 from TMS X as well as L289 and L342 from TMS XII form a transmembrane pathway for translocated ions with a built-in filter determining cation selectivity. Alanine-scanning mutagenesis of the identified residues verified the model by showing that structural modifications of the pathway resulted in altered cation selectivity and transport activity. In particular, L257A, G258A, Q280A, and Y251A variants gained Li+/H+ antiport capacity that was absent in the nonmutated antiporter. T276A, D273A, and L289A variants exclusively exchanged K+ for H+, while a L342A variant mediated Na+/H+ exchange only, thus maintaining strict alkali cation selectivity.

Improving health equity for ethnic minority women in Thai Nguyen, Vietnam: qualitative results from an mHealth intervention targeting maternal and infant health service access.

Background: Ethnic minority women (EMW) in Vietnam experience disproportionately high infant and maternal mortality rates due to low social status, poverty and remoteness from health centres. This project piloted and evaluated a low-cost mobile health (mHealth) intervention called mMom utilizing behaviour change communication (BCC) to improve access to maternal, newborn and child health (MNCH) services and health equity among EMW living in remote areas. Methods: The mMom intervention built an integrated mHealth platform which sent timely MNCH information and BCC text messages to participants, and engaged health workers towards increasing their interaction and building demand for quality natal care. Mid-term and final qualitative evaluations were conducted to assess the intervention's acceptability and impact. Results: In evaluations, all participants expressed satisfaction with the quality, timeliness and convenience of the messages, and health workers reported increased efficiency and quality of care. The use of BCC increased care-seeking from EMW and strengthened relationships with health providers. Conclusion: The mMom project demonstrated the acceptability of mHealth in a remote Vietnamese region with a high proportion of disadvantaged EMW. The messages promoted increased contact between participants and health providers, which holds potential to address the marginalization of EMW from the health system. Keywords: behaviour change communication, eHealth, ethnic minorities, health equity, mHealth, MNCH, mobile health, Vietnam.

Treatment of inflammatory arthritis via targeting of tristetraprolin, a master regulator of pro-inflammatory gene expression.

OBJECTIVES: Tristetraprolin (TTP), a negative regulator of many pro-inflammatory genes, is strongly expressed in rheumatoid synovial cells. The mitogen-activated protein kinase (MAPK) p38 pathway mediates the inactivation of TTP via phosphorylation of two serine residues. We wished to test the hypothesis that these phosphorylations contribute to the development of inflammatory arthritis, and that, conversely, joint inflammation may be inhibited by promoting the dephosphorylation and activation of TTP. METHODS: The expression of TTP and its relationship with MAPK p38 activity were examined in non-inflamed and rheumatoid arthritis (RA) synovial tissue. Experimental arthritis was induced in a genetically modified mouse strain, in which endogenous TTP cannot be phosphorylated and inactivated. In vitro and in vivo experiments were performed to test anti-inflammatory effects of compounds that activate the protein phosphatase 2A (PP2A) and promote dephosphorylation of TTP. RESULTS: TTP expression was significantly higher in RA than non-inflamed synovium, detected in macrophages, vascular endothelial cells and some fibroblasts and co-localised with MAPK p38 activation. Substitution of TTP phosphorylation sites conferred dramatic protection against inflammatory arthritis in mice. Two distinct PP2A agonists also reduced inflammation and prevented bone erosion. In vitro anti-inflammatory effects of PP2A agonism were mediated by TTP activation. CONCLUSIONS: The phosphorylation state of TTP is a critical determinant of inflammatory responses, and a tractable target for novel anti-inflammatory treatments.

A case of diffuse panbronchiolitis caused by Mycoplasma amphoriforme.

Mycoplasma amphoriforme is a novel specie which was discovered in 2003 and associated with congenital immune deficiency. It has been described as an underlying cause of bronchopneumonia. There is limited description of the in vitro sensitivities. In this article, we present the first description of M. amphoriforme as the causative agent of diffuse panbronchiolitis in a patient with X-linked hypogammaglobulinema and bronchiectasis, with symptoms improved by treatment with azithromycin. We also describe the difficulty obtaining this organism through routine culture and the need to consider culture independent methods of recovery when the suspicion is high.

Adjudicating Mild Cognitive Impairment Due to Alzheimer's Disease as a Novel Endpoint Event in the TOMMORROW Prevention Clinical Trial.

BACKGROUND: The onset of mild cognitive impairment (MCI) is an essential outcome in Alzheimer's disease (AD) prevention trials and a compelling milestone for clinically meaningful change. Determining MCI, however, may be variable and subject to disagreement. Adjudication procedures may improve the reliability of these determinations. We report the performance of an adjudication committee for an AD prevention trial. METHODS: The TOMMORROW prevention trial selected cognitively normal participants at increased genetic risk for AD and randomized them to low-dose pioglitazone or placebo treatment. When adjudication criteria were triggered, a participant's clinical information was randomly assigned to a three-member panel of a six-member independent adjudication committee. Determination of whether or not a participant reached MCI due to AD or AD dementia proceeded through up to three review stages - independent review, collaborative review, and full committee review - requiring a unanimous decision and ratification by the chair. RESULTS: Of 3494 participants randomized, the committee adjudicated on 648 cases from 386 participants, resulting in 96 primary endpoint events. Most participants had cases that were adjudicated once (n = 235, 60.9%); the rest had cases that were adjudicated multiple times. Cases were evenly distributed among the eight possible three-member panels. Most adjudicated cases (485/648, 74.8%) were decided within the independent review (stage 1); 14.0% required broader collaborative review (stage 2), and 11.1% needed full committee discussion (stage 3). The primary endpoint event decision rate was 39/485 (8.0%) for stage 1, 29/91 (31.9%) for stage 2, and 28/72 (38.9%) for stage 3. Agreement between the primary event outcomes supported by investigators' clinical diagnoses and the decisions of the adjudication committee increased from 50% to approximately 93% (after around 100 cases) before settling at 80-90% for the remainder of the study. CONCLUSIONS: The adjudication process was designed to provide independent, consistent determinations of the trial endpoints. These outcomes demonstrated the extent of uncertainty among trial investigators and agreement between adjudicators when the transition to MCI due to AD was prospectively assessed. These methods may inform clinical endpoint determination in future AD secondary prevention studies. Reliable, accurate assessment of clinical events is critical for prevention trials and may mean the difference between success and failure.

From Silos to Solidarity: Case Study of a Patient-Centered, Integrative Approach to Opioid Tapering and Chronic Pain Mitigation in a Multidisciplinary AIDS Clinic.

BACKGROUND: People with HIV (PWH) are at a disproportionate risk for experiencing both chronic pain and opioid use disorder (OUD). Prescription opioid tapering is typically addressed within the "silo model" of medical care, whereby attention is focused solely on opioid addiction rather than also addressing chronic pain management, and limited communication occurs between patient and providers. OBJECTIVE: This descriptive case study examined an integrative, collaborative care model consisting of Provider, Physical Therapist (PT), and Patient aimed at decreasing chronic pain and opioid use within a multidisciplinary HIV/AIDS clinic. METHOD: A physical-therapy based model of chronic pain mitigation and physician-driven opioid tapering was implemented. The Provider, PT, and Patient worked collaboratively to address physiological pain, pain coping skills and opioid tapering. A patient case example was used to illustrate the implementation of the model for a future, larger study in the same patient population. RESULTS: This model was feasible in this case example in terms of clinic workflow and acceptability to both the Patient and Providers in this clinic. After the intervention, the Patient's pain was fully eliminated, and he had ceased all opioid use. CONCLUSION: Results of this case study suggest that utilizing an integrative, patient-centered approach to both chronic pain management and opioid tapering may be feasible within the context of a multidisciplinary HIV/AIDS clinic. Generalizability is limited by case study model; however, this gives insight into the value of a collaborative alternative compared to a "silo" model of opioid tapering and chronic pain management in preparation for a larger study.

Long-term survival of nonhuman primate islets implanted in an omental pouch on a biodegradable scaffold.

The aim of this study was to test whether an omental pouch can be used as an alternative site for islet implantation in diabetic monkeys. Here we report the successful engraftment of islets in diabetic cynomolgus monkeys when loaded on a synthetic biodegradable scaffold and placed in an omental pouch. One autologous and five allogeneic diabetic monkey transplants under the cover of steroid-free immune suppression (SFIS) were undertaken. Fasting blood glucose (FBG) and C-peptide (CP), exogenous insulin requirements (EIR), intravenous glucose tolerance test (IVGTT), A1C and histopathology were used to assess islet engraftment and survival. All animals achieved CP levels > 1.0 ng/mL following transplant, a 66-92% posttransplant decrease in EIR and reduced A1C. Following graft removal, CP became negative and histopathological analysis of the explanted grafts demonstrated well-granulated and well-vascularized, insulin-positive islets, surrounded by T-cell subsets and macrophages. Compared to intrahepatic allogeneic islet transplants (n = 20), there was a delayed engraftment for omental pouch recipients but similar levels of CP production were ultimately achieved, with a broad range of IEQ/kg transplanted in both sites. Our results suggest this extrahepatic transplantation site has potential as an alternative site for clinical islet cell transplantation.

Some biochemical properties of Chinese hamster cells sensitive and resistant to actinomycin D.

A graded series of drug-resistant Chinese hamster sublines has been examined for biochemical changes accompanying resistance to actinomycin D. The most highly resistant subline, DC-3F/AD X, is maintained at 10 microg/ml of the antibiotic. It was shown that over 250 times more actinomycin D is required to inhibit RNA synthesis in this subline than in the parental DC-3F line. The DC-3F/AD X subline was also shown to have a somewhat reduced capacity to transport uridine as compared to parental cells. Sensitive cells took up over 50 times more tritiated antibiotic than the most resistant cells, as determined in a 1-h assay. Uptake of actinomycin D was shown to be temperature-dependent in both resistant and sensitive cells and was not influenced by various metabolic inhibitors. Resistance could not be explained by a rapid uptake and release of the antibiotic, as demonstrated in efflux experiments, or by its metabolism. In addition, highly resistant cells which are cross-resistant to puromycin were shown to have a reduced capacity to take up labeled puromycin. These studies provide further evidence indicating that the mechanism of resistance to actinomycin D is reduced permeability to drug and suggesting that cell membrane alteration accounts for resistance to both actinomycin D and puromycin.

Oxygen isotope fractionation in the system dolomite-calcite-carbon dioxide.

Oxygen isotopes were equilibrated between carbon dioxide and calcite at four temperatures in the range 350 degrees to 610 degrees C and between carbon dioxide and dolomite at 350 degrees and 400 degrees C. Carbon of unusual isotopic composition was used as a tracer to demonstrate the nature and extent of the exchange process. Extrapolation of these data at lower temperatures indicates that at 25 degrees C dolomite is enriched in oxygen-18 by 6.8 per mil with respect to calcite. This result indicates that those natural dolomite-calcite assemblages which show very small fractionations were not formed in isotopic equilibrium.

Uranium and Lead Isotopic Stability in a Metamict Zircon under Experimental Hydrothermal Conditions.

Abstract. Disturbance of the uranium-lead isotopic system in a metamict Ceylon zircon has been produced in a 2 molal NaCI solution at 500 degrees C and 1000 bars fluid pressure. Loss of radiogenic lead to the extent of 61 percent in 13 days was the most significant effect. The experimental results support the episodic rather than continuous lead-loss interpretation of natural zircon systems utilized in geochronology.

Water, hydrogen, deuterium, carbon, carbon-13, and oxygen-18 content of selected lunar material.

The water content of the breccia is 150 to 455 ppm, with a deltaD from-580 to -870 per mil. Hydrogen gas content is 40 to 53 ppm with a deltaD of -830 to -970 per mil. The CO(2) is 290 to 418 ppm with delta (13)C = + 2.3 to + 5.1 per mil and delta(18)O = 14.2 to 19.1 per mil. Non-CO(2) carbon is 22 to 100 ppm, delta(13)C = -6.4 to -23.2 per mil. Lunar dust is 810 ppm H(2)O (D = 80 ppm) and 188 ppm total carbon(delta(13)C = -17.6 per mil). The (18)O analyses of whole rocks range from 5.8 to 6.2 per mil. The temperature of crystallization of type B rocks is 1100 degrees to 1300 degrees C, based on the oxygen isotope fractionation between coexisting plagioclase and ilmenite.

Socio-demographic and geographic analysis of overweight and obesity in Canadian adults using the Canadian Community Health Survey (2005).

Using the 2005 Canadian Community Health Survey, this study examined how overweight and obesity in Canadian adults are distributed across socio-demographic and geographic groupings. Overweight and obesity prevalence were modeled against socio-demographic indicators using Poisson regression and were assessed geographically using choropleth maps. The Gini coefficient was used to assess the distribution of prevalence across risk groups. The potential impacts of high risk versus population-based prevention approaches on the population prevalence of obesity were also examined. Of adults aged 25 to 64 years, 17% were obese and 53% were overweight or obese, with the highest proportions observed in older age groups, among those who were physically inactive, white or non-immigrant, with low educational levels, and living in the prairie and east coast regions. Recalculation of obesity rates under the different prevention scenarios demonstrated that population-based approaches could achieve a four-fold greater decrease in obesity cases than high risk approaches, highlighting the need for broader population strategies for obesity prevention in Canada.

Mechanisms of transcription factor deregulation in lymphoid cell transformation.

The most frequent targets of genetic alterations in human lymphoid leukemias are transcription factor genes with essential functions in blood cell development. TAL1, LYL1, HOX11 and other transcription factors essential for normal hematopoiesis are often misexpressed in the thymus in T-cell acute lymphoblastic leukemia (T-ALL), leading to differentiation arrest and cell transformation. Recent advances in the ability to assess DNA copy number have led to the discovery that the MYB transcription factor oncogene is tandemly duplicated in T-ALL. The NOTCH1 gene, which is essential for key embryonic cell-fate decisions in multicellular organisms, was found to be activated by mutation in a large percentage of T-ALL patients. The gene encoding the FBW7 protein ubiquitin ligase, which regulates the turnover of the intracellular form of NOTCH (ICN), is also mutated in T-ALL, resulting in stabilization of the ICN and activation of the NOTCH signaling pathway. In mature B-lineage ALL and Burkitt lymphoma, the MYC transcription factor oncogene is overexpressed due to translocation into the IG locus. PAX5, a transcription factor essential for B-lineage commitment, is inactivated in 32% of cases of B-progenitor ALL. Translocations resulting in oncogenic fusion transcription factors also occur frequently in this form of ALL. The most frequent transcription factor chimeric fusion, TEL-AML1, is an initiating event in B-progenitor ALL that acts by repressing transcription. Therefore, deregulated transcription and its consequent effects on key developmental pathways play a major role in the molecular pathogenesis of lymphoid malignancy. Once the full complement of cooperating mutations in transformed B- and T-progenitor cells is known, and the deregulated downstream pathways have been elucidated, it will be possible to identify vulnerable components and to target them with small-molecule inhibitors.

Epstein-Barr virus-encoded EBNA1 regulates cellular gene transcription and modulates the STAT1 and TGFbeta signaling pathways.

The Epstein-Barr virus (EBV)-encoded EBNA1 protein is expressed in all virus-associated tumors where it plays an essential role in the maintenance, replication and transcription of the EBV genome. Transcriptional profiling of EBNA1-expressing carcinoma cells demonstrated that EBNA1 also influences the expression of a range of cellular genes including those involved in translation, transcription and cell signaling. Of particular interest was the ability of EBNA1 to enhance expression of STAT1 and sensitize cells to interferon-induced STAT1 activation with resultant enhancement of major histocompatibility complex expression. A negative effect of EBNA1 on the expression of TGFbeta1-responsive betaig-h3 and PAI-1 genes was confirmed at the protein level in EBV-infected carcinoma cells. This effect resulted from the ability of EBNA1 to repress TGFbeta1-induced transcription via a reduction in the interaction of SMAD2 with SMAD4. More detailed analysis revealed that EBNA1 induces a lower steady-state level of SMAD2 protein as a consequence of increased protein turnover. These data show that EBNA1 can influence cellular gene transcription resulting in effects that may contribute to the development of EBV-associated tumors.

EUS and clinical characteristics of cystic pancreatic neuroendocrine tumors.

Pancreatic neuroendocrine tumors (PNETs) may rarely appear as cystic or mixed solid-cystic masses. The endoscopic ultrasound (EUS) morphology and cyst fluid characteristics of these tumors are not well clarified. We retrospectively identified nine adult patients with nine single cystic pancreatic neuroendocrine tumors (CNETs). These nine included 0.67 % of the 1344 patients with pancreatic cystic lesions and 9.5 % of the 95 confirmed PNETs evaluated over the 12-year study period. At presentation, four patients were asymptomatic and five had known acute pancreatitis (n = 2), MEN-1 syndrome with hypoglycemia (n = 1), and abdominal pain (n = 2). Median maximal tumor diameter was 26 mm (range 20 - 64 mm). EUS morphology was mixed solid and cystic (n = 4) or cystic alone (n = 5). Cytology from EUS-fine-needle aspiration (FNA) (median 2 passes; range 1 - 6) demonstrated a PNET, and immunocytochemistry was confirmatory in all patients. Cyst fluid carcinoembryonic antigen (CEA) (n = 4) and amylase (n = 5) ranged from 0.1 to 1.8 ng/ml (normal 0 - 2.5 ng/ml) and 72 to 1838 U/L (normal 25 - 161 U/L), respectively. Six patients underwent surgery, and the preoperative diagnosis was confirmed in all.

MAPK p38 regulates inflammatory gene expression via tristetraprolin: Doing good by stealth.

Tristetraprolin (TTP) is an RNA-destabilizing protein that exerts profound anti-inflammatory effects by inhibiting the expression of tumour necrosis factor and many other inflammatory mediators. The mitogen-activated protein kinase (MAPK) p38 signaling pathway controls the strength and duration of inflammatory responses by regulating both the expression and function of TTP. The kinase MK2 (MAPK activated kinase 2) is activated by MAPK p38, and in turn phosphorylates TTP at two critical serine residues. One consequence of these phosphorylations is the protection of TTP from proteasome-mediated degradation. Another consequence is the loss of mRNA destabilizing activity. The control of TTP expression and function by the MAPK p38 pathway provides an elegant mechanism for coupling the on and off phases of inflammatory responses, and dictating the precise kinetics of expression of individual inflammatory mediators.

Inactivation of lysosomal proteases by oxidized low density lipoprotein is partially responsible for its poor degradation by mouse peritoneal macrophages.

Deficient processing of apo B in oxidized LDL (ox-LDL) by macrophage lysosomal proteases has been documented and attributed to modifications in apo B. We have investigated whether direct inactivation of lysosomal proteases by ox-LDL could also be responsible for this deficient degradation. When mouse peritoneal macrophages (MPM) were preincubated for 21 h at 37 degrees C with ox-LDL, LDL, or vortex-aggregated LDL, only ox-LDL inhibited the subsequent degradation of 125I-labeled forms of the above lipoproteins. Uptake of labeled lipoproteins was not appreciably affected by preincubation with ox-LDL, suggesting that the inhibition was at the level of lysosomal degradation. Thiol protease activity of cell extracts at pH 4.0, was reduced in MPM preincubated with ox-LDL relative to cells preincubated with LDL or medium alone. Extracts from untreated MPM, or mixtures of cathepsin B and D, showed a reduced ability to degrade 125I-LDL at pH 4.5 and reduced cathepsin B activity, after incubation with ox-LDL relative to incubation with LDL. Thus, the reduced degradation of lipoproteins in MPM pretreated with ox-LDL could be due to direct inactivation of the lysosomal protease, cathepsin B.

Effects of hypoglycemia and prolonged fasting on insulin and glucagon gene expression. Studies with in situ hybridization.

In situ hybridization of proinsulin and proglucagon mRNA was performed in rat pancreas to assess prohormone gene expression during various glucopenic conditions. During a 4-d fast mean blood glucose declined by 48 mg/dl; proinsulin mRNA signal density remained normal while proglucagon mRNA signal density more than doubled. At the end of a continuous 12-d insulin infusion blood glucose averaged 53 +/- 12 mg/dl; proinsulin mRNA signal density declined to 30% of controls while proglucagon mRNA signal density more than doubled. In insulinoma-bearing NEDH rats blood glucose averaged 34 +/- 3.5 mg/dl; the proinsulin mRNA signal was virtually undetectable and proglucagon mRNA signal density was more than twice the controls. There was no detectable change in either beta-cell area or islet number in rats subjected to fasting or insulin infusion, but in insulinoma-bearing rats beta cell area was markedly reduced. Thus compensation during 4 d of starvation involves an increase in glucagon gene expression without change in insulin gene expression or beta cell mass. In moderate insulin-induced hypoglycemia glucagon gene expression is increased and insulin gene expression decreased. In more profound insulinoma-induced hypoglycemia, in addition to the foregoing changes in hormone gene expression, there is a profound reduction in the number of insulin-expressing cells.

Sequestration and inhibition of Daxx-mediated transcriptional repression by PML.

PML fuses with retinoic acid receptor alpha (RARalpha) in the t(15;17) translocation that causes acute promyelocytic leukemia (APL). In addition to localizing diffusely throughout the nucleoplasm, PML mainly resides in discrete nuclear structures known as PML oncogenic domains (PODs), which are disrupted in APL and spinocellular ataxia cells. We isolated the Fas-binding protein Daxx as a PML-interacting protein in a yeast two-hybrid screen. Biochemical and immunofluorescence analyses reveal that Daxx is a nuclear protein that interacts and colocalizes with PML in the PODs. Reporter gene assay shows that Daxx drastically represses basal transcription, likely by recruiting histone deacetylases. PML, but not its oncogenic fusion PML-RARalpha, inhibits the repressor function of Daxx. In addition, SUMO-1 modification of PML is required for sequestration of Daxx to the PODs and for efficient inhibition of Daxx-mediated transcriptional repression. Consistently, Daxx is found at condensed chromatin in cells that lack PML. These data suggest that Daxx is a novel nuclear protein bearing transcriptional repressor activity that may be regulated by interaction with PML.

Activating mutations in NOTCH1 in acute myeloid leukemia and lineage switch leukemias.

Activating mutations in NOTCH1 are found in over 50% of human T-cell lymphoblastic leukemias (T-ALLs). Here, we report the analysis for activating NOTCH1 mutations in a large number of acute myeloid leukemia (AML) primary samples and cell lines. We found activating mutations in NOTCH1 in a single M0 primary AML sample, in three (ML1, ML2 and CTV-1) out of 23 AML cell lines and in the diagnostic (myeloid) and relapsed (T-lymphoid) clones in a patient with lineage switch leukemia. Importantly, the ML1 and ML2 AML cell lines are derived from an AML relapse in a patient initially diagnosed with T-ALL. Overall, these results demonstrate that activating mutations in NOTCH1 are mostly restricted to T-ALL and are rare in AMLs. The presence of NOTCH1 mutations in myeloid and T-lymphoid clones in lineage switch leukemias establishes the common clonal origin of the diagnostic and relapse blast populations and suggests a stem cell origin of NOTCH1 mutations during the molecular pathogenesis of these tumors.