Pancreatic tumor cells with mutant K-ras suppress ERK activity by MEK-dependent induction of MAP kinase phosphatase-2.

Abstract: Activating mutations within the K-ras gene occur in a high percentage of human pancreatic carcinomas. We reported previously that the presence of oncogenic, activated K-ras in human pancreatic carcinoma cell lines did not result in constitutive activation of the extracellular signal-regulated kinases (ERK1 and ERK2). In the present study, we further characterized the ERK signaling pathway in pancreatic tumor cell lines in order to determine whether the ERK pathway is subject to a compensatory downregulation. We found that the attenuation of serum-induced ERK activation was not due to a delay in the kinetics of ERK phosphorylation. Treatment with the tyrosine phosphatase inhibitor orthovanadate increased the level of ERK phosphorylation, implicating a vanadate-sensitive tyrosine phosphatase in the negative regulation of ERK. Furthermore, expression of a dual specificity phosphatase capable of inactivating ERK known as mitogen-activated protein (MAP) kinase phosphatase-2 (MKP-2) was elevated in most of the pancreatic tumor cell lines and correlated with the presence of active MAP kinase kinase (MEK). Taken together, these results suggest that pancreatic tumor cells expressing oncogenic K-ras compensate, in part, by upregulating the expression of MKP-2 to repress the ERK signaling pathway.


E2F3 is a mediator of DNA damage-induced apoptosis.

Abstract: The E2F transcription factors have emerged as critical apoptotic effectors. Herein we report that the E2F family member E2F3a can be induced by DNA damage through transcriptional and posttranslational mechanisms. We demonstrate that the posttranslational induction of human E2F3a is dependent on the checkpoint kinases. Moreover, we show that human E2F3a is a substrate for the checkpoint kinases (chk kinases) and that mutation of the chk phosphorylation site eliminates the DNA damage inducibility of the protein. Furthermore, we demonstrate that E2F1 and E2F2 are transcriptionally induced by DNA damage in an E2f3-dependent manner. Finally, using both in vitro and in vivo approaches, we establish that E2f3 is required for DNA damage-induced apoptosis. Thus, our data reveal the novel ability of E2f3 to function as a master regulator of the DNA damage response.


miR-294/miR-302 promotes proliferation, suppresses G1-S restriction point, and inhibits ESC differentiation through separable mechanisms.

Abstract: The miR-294 and miR-302 microRNAs promote the abbreviated G1 phase of the embryonic stem cell (ESC) cell cycle and suppress differentiation induced by let-7. Here, we evaluated the role of the retinoblastoma (Rb) family proteins in these settings. Under normal growth conditions, miR-294 promoted the rapid G1-S transition independent of the Rb family. In contrast, miR-294 suppressed the further accumulation of cells in G1 in response to nutrient deprivation and cell-cell contact in an Rb-dependent fashion. We uncovered five additional miRNAs (miR-26a, miR-99b, miR-193, miR-199a-5p, and miR-218) that silenced ESC self-renewal in the absence of other miRNAs, all of which were antagonized by miR-294 and miR-302. Four of the six differentiation-inducing miRNAs induced an Rb-dependent G1 accumulation. However, all six still silenced self-renewal in the absence of the Rb proteins. These results show that the miR-294/miR-302 family acts through Rb-dependent and -independent pathways to regulate the G1 restriction point and the silencing of self-renewal, respectively.


Bacteriophage latent-period evolution as a response to resource availability.

Abstract: Bacteriophages (phages) modify microbial communities by lysing hosts, transferring genetic material, and effecting lysogenic conversion. To understand how natural communities are affected it is important to develop predictive models. Here we consider how variation between models--in eclipse period, latent period, adsorption constant, burst size, the handling of differences in host quantity and host quality, and in modeling strategy--can affect predictions. First we compare two published models of phage growth, which differ primarily in terms of how they model the kinetics of phage adsorption; one is a computer simulation and the other is an explicit calculation. At higher host quantities (approximately 10(8) cells/ml), both models closely predict experimentally determined phage population growth rates. At lower host quantities (10(7) cells/ml), the computer simulation continues to closely predict phage growth rates, but the explicit model does not. Next we concentrate on predictions of latent-period optima. A latent-period optimum is the latent period that maximizes the population growth of a specific phage growing in the presence of a specific quantity and quality of host cells. Both models predict similar latent-period optima at higher host densities (e.g., 17 min at 10(8) cells/ml). At lower host densities, however, the computer simulation predicts latent-period optima that are much shorter than those suggested by explicit calculations (e.g., 90 versus 1,250 min at 10(5) cells/ml). Finally, we consider the impact of host quality on phage latent-period evolution. By taking care to differentiate latent-period phenotypic plasticity from latent-period evolution, we argue that the impact of host quality on phage latent-period evolution may be relatively small.


Adult T-cell leukemia/lymphoma in London: clinical experience of 21 cases.

Abstract: Adult T-cell leukemia/lymphoma (ATLL) is uncommon in the United Kingdom and has so far been restricted to people of Afro-Caribbean extraction. Between 1981 and 1995, 21 cases presented to 2 inner London teaching hospitals where 17% of the population are of Afro-Caribbean origin. Clinical presentations were similar to those of the disease in HTLV-I-endemic areas. Major responses (CR + PR) were obtained in 10/16 assessable patients (63%) treated with combination chemotherapy. However, median survival was only 5.5 months. Disease progression and opportunistic infection were the major causes of treatment failure and death. Three patients (14%) relapsed in the central nervous system (CNS). Our cases confirm the profound immunosuppression in ATLL. The poor prognosis of acute and lymphoma types of ATLL highlight the need for new approaches to treatment such as zidovudine and alpha-interferon, incorporating prophylaxis against CNS disease and opportunistic infections.


Poverty, Hunger, Education, and Residential Status Impact Survival in HIV

Abstract: Despite combination antiretroviral therapy (ART), HIV infected people have higher mortality than non-infected. Lower socioeconomic status (SES) predicts higher mortality in many chronic illnesses but data in people with HIV is limited. We evaluated 878 HIV infected individuals followed from 1995 to 2005. Cox proportional hazards for all-cause mortality were estimated for SES measures and other factors. Mixed effects analyses examined how SES impacts factors predicting death. The 200 who died were older, had lower CD4 counts, and higher viral loads (VL). Age, transmission category, education, albumin, CD4 counts, VL, hunger, and poverty predicted death in univariate analyses; age, CD4 counts, albumin, VL, and poverty in the multivariable model. Mixed models showed associations between (1) CD4 counts with education and hunger; (2) albumin with education, homelessness, and poverty; and (3) VL with education and hunger. SES contributes to mortality in HIV infected persons directly and indirectly, and should be a target of health policy in this population.


Concentrations of tenofovir and emtricitabine in saliva: implications for preexposure prophylaxis of oral HIV acquisition.

Abstract: To prevent acquisition of HIV through oral sex, drugs used for preexposure prophylaxis (Prep) need to diffuse in saliva. We measured tenofovir (TFV) and emtricitabine (FTC) concentrations simultaneously in the plasma and saliva of 41 HIV-infected patients under stable antiretroviral treatment. Mean ratios of saliva/plasma concentration were 3% (±4%) and 86.9% (±124%) for TFV and FTC, respectively. Tenofovir disoproxil fumarate (TDF) should be used in combination with FTC to prevent oral acquisition of HIV.


Increased lean body mass as an early indicator of olanzapine-induced weight gain in healthy men.

Abstract: One of the primary limitations of many psychiatric medications is weight gain, the mechanism of which remains to be fully elucidated. We conducted a 2-week double-blind placebo-controlled study on weight gain with olanzapine, which is frequently but unpredictably associated with this side effect, to address the possible mechanisms of weight gain independent of changes in the psychiatric condition for which it is prescribed. Healthy male volunteers were randomly assigned to olanzapine (5 mg/day for 7 days, then 10 mg/day for 7 days) or a matching placebo. Of the 24 participants, 19 completed the study (olanzapine, n=13; placebo, n=6). Body weight, glucose, triglyceride, total cholesterol, lipid, leptin, insulin, and aldosterone levels, resting metabolic rate, body composition, physical activity, and 24-h dietary intake were assessed. A significant increase in weight as well as triglyceride, insulin, and leptin levels were found in the olanzapine group as a whole. In participants receiving olanzapine who actually gained weight (n=8), lean but not fat mass increased, as did insulin, fasting glucose, total cholesterol, low-density lipoprotein, and non-high-density lipoprotein levels, whereas aldosterone levels decreased. There were no significant metabolic or endocrine changes in participants receiving placebo or in those receiving olanzapine who did not gain weight. Early metabolic changes appear to be independent of accumulation of fat.


QTc interval length and QT dispersion as predictors of mortality in patients with non-insulin-dependent diabetes.

Abstract: Patients with non-insulin-dependent diabetes (NIDDM) are at independent risk of cardiovascular death. The reason is only partially understood. The aim of our study was therefore to evaluate the impact of corrected QT interval length (QTc) and QT dispersion (QT-disp) on mortality in a cohort of 324 Caucasian NIDDM patients. A resting 12-lead ECG was recorded at baseline. Maximum (QT-max) and minimum QT (QT-min) intervals were measured, and QT-max was corrected for heart rate (QTc-max). QT-disp was defined as the difference between QT-max and QT-min. QTc-max was 454 (376-671) ms(1/2) (median (range)) and QT-disp 61 (0-240) ms. Prolonged QTc interval (PQTc), defined as QTc-max > 440 ms(1/2), was present in 67% of the patients and prolonged QT-disp (PQT-disp), defined as QT-disp > 50 ms, was present in 51%. During the 9-year follow-up period, 100 patients died (52 from cardiovascular diseases). Thirty-seven percent of the patients with PQTc died compared with 17% with normal QTc interval (p<0.001). The Cox proportional hazard model, including putative risk factors at baseline, revealed the following independent predictors of all cause mortality; QTc-max (p<0.05), age (p<0.0001), albuminuria (p<0.01), retinopathy (p<0.01), HbA1c (p<0.05), insulin treatment (p<0.01), total cholesterol (p<0.01), serum creatinine (p<0.05) and presence of cardiac heart disease based on Minnesota coded ECG (p<0.001). Whereas QT-disp was not a predictor, QTc-max interval was an independent predictor of cardiovascular mortality. Our study showed a high prevalence of QTc and QT-disp abnormalities and indicated that QTc-max but not QT-disp is an independent predictor of all cause and cardiovascular mortality in NIDDM patients.


Recombinant human tumor necrosis factor administered as a five-day continuous infusion in cancer patients: phase I toxicity and effects on lipid metabolism.

Abstract: Recombinant human tumor necrosis factor (rH-TNF) is a cytotoxic monokine with pleiotropic effects. A phase I trial of rH-TNF was initiated using a five-day continuous intravenous (IV) infusion repeated every 28 days. Thirty-eight courses of therapy were administered to 19 patients. The starting dose was 5 X 10(4) U/m2/d, with escalations to 1.0 X 10(5), 2.0 X 10(5), 2.4 X 10(5), and 3.0 X 10(5) U/m2/d. Systemic side effects, including fever, chills, hypotension, fatigue, anorexia, and headaches, were mild and self-limiting. At the maximum tolerated dose of 3.0 X 10(5) U/m2/d, dose-limiting hematologic toxicity was manifested by transient thrombocytopenia and leukopenia. Elevated bilirubin levels were also seen at the higher dose levels. Lipoprotein analysis demonstrated that the five-day treatment with rH-TNF was associated with decreases in high-density lipoproteins, as well as increases in triglycerides and very-low-density lipoproteins. Pharmacokinetic studies using an enzyme-linked immunosorbent assay (ELISA) test indicated plasma rH-TNF levels less than 0.2 U/mL. The recommended phase II dose of rH-TNF administered as a five-day continuous infusion is 2.4 X 10(5) U/m2/d.


Stress-dependent regulation of FOXO transcription factors by the SIRT1 deacetylase.

Abstract: The Sir2 deacetylase modulates organismal life-span in various species. However, the molecular mechanisms by which Sir2 increases longevity are largely unknown. We show that in mammalian cells, the Sir2 homolog SIRT1 appears to control the cellular response to stress by regulating the FOXO family of Forkhead transcription factors, a family of proteins that function as sensors of the insulin signaling pathway and as regulators of organismal longevity. SIRT1 and the FOXO transcription factor FOXO3 formed a complex in cells in response to oxidative stress, and SIRT1 deacetylated FOXO3 in vitro and within cells. SIRT1 had a dual effect on FOXO3 function: SIRT1 increased FOXO3's ability to induce cell cycle arrest and resistance to oxidative stress but inhibited FOXO3's ability to induce cell death. Thus, one way in which members of the Sir2 family of proteins may increase organismal longevity is by tipping FOXO-dependent responses away from apoptosis and toward stress resistance.


TLR-Mediated Innate Production of IFN-γ by CD8+ T Cells Is Independent of Glycolysis.

Abstract: CD8(+) T cells can respond to unrelated infections in an Ag-independent manner. This rapid innate-like immune response allows Ag-experienced T cells to alert other immune cell types to pathogenic intruders. In this study, we show that murine CD8(+) T cells can sense TLR2 and TLR7 ligands, resulting in rapid production of IFN-γ but not of TNF-α and IL-2. Importantly, Ag-experienced T cells activated by TLR ligands produce sufficient IFN-γ to augment the activation of macrophages. In contrast to Ag-specific reactivation, TLR-dependent production of IFN-γ by CD8(+) T cells relies exclusively on newly synthesized transcripts without inducing mRNA stability. Furthermore, transcription of IFN-γ upon TLR triggering depends on the activation of PI3K and serine-threonine kinase Akt, and protein synthesis relies on the activation of the mechanistic target of rapamycin. We next investigated which energy source drives the TLR-induced production of IFN-γ. Although Ag-specific cytokine production requires a glycolytic switch for optimal cytokine release, glucose availability does not alter the rate of IFN-γ production upon TLR-mediated activation. Rather, mitochondrial respiration provides sufficient energy for TLR-induced IFN-γ production. To our knowledge, this is the first report describing that TLR-mediated bystander activation elicits a helper phenotype of CD8(+) T cells. It induces a short boost of IFN-γ production that leads to a significant but limited activation of Ag-experienced CD8(+) T cells. This activation suffices to prime macrophages but keeps T cell responses limited to unrelated infections.


Recommendations for the presurgical psychosocial evaluation of bariatric surgery patients.

Abstract: Psychosocial factors have significant potential to affect long-term outcomes of bariatric surgery, including emotional adjustment, adherence to the recommended postoperative lifestyle regimen, weight loss outcomes, and co-morbidity improvement and or resolution. Thus, it is recommended that bariatric behavioral health clinicians with specialized knowledge and experience be involved in the evaluation and care of patients both before and after surgery. The evaluating clinician plays a number of important roles in the multidisciplinary treatment of the bariatric patient. Central among these is the role of identifying factors that may pose challenges to optimal surgical outcome and providing recommendations to the patient and bariatric team on how to address these issues. This document outlines recommendations for the psychosocial evaluation of bariatric surgery patients, appropriate qualifications of those conducting these evaluations, communication of evaluation results and suggested treatment plan, and the extension of behavioral healthcare of the bariatric patient to the entire span of the surgical and postsurgical process.


The epidemiology of trachoma in Eastern Equatoria and Upper Nile States, southern Sudan.

Abstract:
OBJECTIVE Limited surveys and anecdotal data indicate that trachoma is endemic in the states of Eastern Equatoria and Upper Nile in southern Sudan. However, its magnitude and geographical distribution are largely unknown. We conducted surveys to ascertain the prevalence and geographical distribution of trachoma, and to identify targets for control interventions.
 METHODS Population-based cross-sectional surveys were conducted in nine sites in southern Sudan between September 2001 and June 2004. Two-stage random cluster sampling with probability proportional to size was used to select the sample. Trachoma grading was done using the WHO simplified grading system.
 FINDINGS A total of 17 016 persons were examined, a response rate of 86.1% of the enumerated population. Prevalence of signs of active trachoma in children aged 1-9 years was: TF=53.7% (95% confidence interval (CI)=52.1-55.3); TI=42.7% (95% CI=41.2-44.2); TF and/or TI=64.1% (95% CI=62.5-65.5). Prevalence of trichiasis (TT) in children aged less than 15 years was 1.2% (95% CI=0.9-1.4), while TT prevalence in persons aged 15 years and above was 9.2% (95% CI=8.6-9.9). Women were more likely to have trichiasis compared to men (odds ratio (OR)=1.57; 95% CI=1.34-1.84). Tentative extrapolation to the states of Eastern Equatoria and Upper Nile estimates that there is a backlog of 178,250 (lower and upper bounds=156,027-205,995) persons requiring surgery and the entire population, estimated to be over 3.9 million, is in need of the SAFE strategy to control blinding trachoma.
 CONCLUSION Trachoma is a public health problem in all nine of the study sites surveyed. The unusually high prevalence of active trachoma and TT in children points to the severity of the problem. There is urgent need to implement trachoma control interventions in trachoma endemic regions of southern Sudan.


A single residue determines the cooperative binding property of a primosomal DNA replication protein, PriB, to single-stranded DNA.

Abstract: PriB is a primosomal protein required for re-initiation of replication in bacteria. We characterized and compared the DNA-binding properties of PriB from Salmonella enterica serovar Typhimurium LT2 (StPriB) and Escherichia coli (EcPriB). Only one residue of EcPriB, V6, was different in StPriB (replaced by A6). Previous structural information revealed that this residue is located on the putative dimer-dimer interface of PriB and is not involved in single-stranded DNA (ssDNA) binding. The cooperative binding mechanism of StPriB to DNA is, however, very different from that of EcPriB. Unlike EcPriB, which forms a single complex with ssDNAs of various lengths, StPriB forms two or more distinct complexes. Based on these results, as well as information on structure, binding modes for forming a stable complex of PriB with ssDNA of 25 nucleotides (nt), (EcPriB)25, and (StPriB)25 are proposed.


Genomic organization and chromosomal location of the human dsRNA adenosine deaminase gene: the enzyme for glutamate-activated ion channel RNA editing.

Abstract: The structure of the human gene encoding the double-stranded RNA (dsRNA) adenosine deaminase (DRADA) was characterized. This nuclear localized enzyme is involved in the RNA editing required for the expression of certain subtypes of glutamate-gated ion channel subunits. The DRADA gene span 30 kb pairs and harbors 15 exons. The transcription of the DRADA gene driven by the putative promoter region, which contains no typical TATA or CCAAT box-like sequences, is initiated at multiple sites, 164 to 216 nucleotides upstream of the translation initiation codon. The three dsRNA binding motifs (DRBM), 70 amino acid residues long, are each encoded by two exons plus an intervening sequence that interrupts the motif at the identical amino acid position. This finding is consistent with the notion that the dsRNA binding domains may be composed of two separate functional subdomains. Fluorescent in situ hybridization localized the DRADA gene on the long arm chromosome 1, region q21. The gene structure and sequence information reported in this study will facilitate the investigation of involvement of DRADA in hereditary diseases that may be the result of malfunction of glutamate-gated ion channels.


"We charge them; otherwise we cannot run the hospital" front line workers, clients and health financing policy implementation gaps in Ghana.

Abstract:
OBJECTIVES This paper examines policy implementation gaps of user fees plus exemptions and health insurance in providing financial access to primary clinical care for children under five in Ghana.
 METHODS Methods included analysis of routine data, focus group discussions, in-depth interviews, and administration of a structured questionnaire.
 RESULTS Providers modified exemptions policy implementation arrangements, sometimes giving partial or no exemptions. Clients who knew or suspected exemption entitlements failed to request them because of fear of negative reactions from providers. Providers attributed their modification of implementation arrangements and negative reactions to the threat posed to the financial viability of their institutions by reimbursement uncertainty and delays. At the time of the study insurance coverage was low and frontline workers were not noticeably modifying implementation arrangements. However, the underlying goal conflicts, resource scarcity, conditions of work and relationships between frontline workers and clients that fueled the exemptions policy implementation gaps were unchanged. The potential for the health insurance policy to stumble over implementation gaps as happened with the exemptions policy therefore remained.
 CONCLUSIONS Policies that do not take into account the incentives for frontline worker adherence and align them better with policy objectives may experience implementation gaps.


EML4-ALK translocation predicts better outcome in lung adenocarcinoma patients with wild-type EGFR.

Abstract:
INTRODUCTION The echinoderm microtubule-associated protein like 4-anaplastic lymphoma kinase (EML4-ALK) fusion represents a novel target in a subset of non-small cell lung cancer, especially adenocarcinoma. EML4-ALK fusion is mutually exclusive with epidermal growth factor receptor (EGFR) mutations. To understand the impact of EML4-ALK on the prognosis of non-small cell lung cancer, we examined EML4-ALK fusion in lung adenocarcinoma from patients with wild-type EGFR and analyzed their clinical treatment outcomes.
 METHODS Lung adenocarcinoma patients with malignant pleural effusions having wild-type EGFR and measurable target lesions were enrolled for EML4-ALK analysis by reverse transcription-polymerase chain reaction and direct sequencing. Demographic data, EML4-ALK status, and survival data were analyzed. We also performed fluorescence in situ hybridization on some available tumor samples to validate the PCR result. In addition, K-ras mutation was analyzed for patients without EML4-ALK fusion genes.
 RESULTS A total of 116 patients with wild-type EGFR sequencing results had complete clinical data for analysis. No patients received ALK inhibitor therapy. There were 39 patients (34%) with the EML4-ALK fusion gene. The concordance rate between reverse transcription-polymerase chain reaction and fluorescence in situ hybridization was 85%. The K-ras mutation rate for patients without EML4-ALK fusion gene was 6.5%. By multivariate analysis, patients who had better performance status (p < 0.001) and EML4-ALK translocation (p = 0.017) had longer overall survival. Comparing patients with tumors harboring variant 1 with those harboring nonvariant 1 EML4-ALK fusion genes, there were no significant differences in clinical factors and survival outcome.
 CONCLUSION For lung adenocarcinoma patients with wild-type EGFR, EML4-ALK translocation is associated with longer overall survival.


Isolation and partial characterization of an unusual human immunodeficiency retrovirus from two persons of west-central African origin.

Abstract: An unusual human retrovirus was isolated from two patients with persistent generalized lymphadenopathy who originate from West-Central Africa and are currently residing in Belgium. Although the virus shared a number of the same biological and morphological properties as human immunodeficiency retrovirus type 1 (HIV-1) and HIV-2, significant antigenic differences could be demonstrated. Several of the viral proteins also differed in molecular weight from the corresponding HIV-1 and HIV-2 proteins. Partial chemical cleavage of the most highly conserved viral proteins resulted in patterns which differed from those of HIV-1 and HIV-2. Furthermore, nucleic acid hybridization experiments were capable of discriminating between the virus types. Sequence analysis of the viral U3 region revealed a unique enhancer organization not found in other immunodeficiency viruses. The data indicated that the new isolate is more closely related to HIV-1 than to HIV-2 but clearly differs in a number of important respects.


Familial recurrence-pattern analysis of nonsyndromic isolated cleft palate--a Danish Registry study.

Abstract: The finding of an association between genetic variation at the transforming growth-factor alpha (TGFA) locus and nonsyndromic isolated cleft palate (CP) represents a potentially important breakthrough in our understanding of this condition. The present study was undertaken to assess the feasibility of detecting linkage to putative CP-susceptibility loci, such as TGFA. To this end, the familial recurrence pattern for CP was evaluated to determine the most likely mode of inheritance for this condition. The study took advantage of the high ascertainment and uniform registration of CP in Denmark. In addition, the study utilized estimates of familial recurrence that were obtained by register linkage and, hence, were not subject to either recall bias or the potentially biasing influence of nonresponders. The recurrence risks for first-, second-, and third-degree relatives of 1,364 nonsyndromic CP probands were estimated to be 2.74% (72/2,628), 0.28% (3/1,068), and 0.00% (0/360), respectively. These estimates are close to published estimates based on questionnaire and interview data. The population prevalence for nonsyndromic CP was, however, found to be considerable higher than usually reported (0.058% [1,456/2,523,023]). Analyses of these and previously published data, using the method presented by Risch, indicated that major-locus or additive multilocus inheritance of CP is unlikely. The familial recurrence pattern was, however, consistent with CP being determined by several interacting loci. Under such a model, a single locus accounting for more than a sixfold increase in the risk to first-degree relatives of CP probands is unlikely, whereas a single locus accounting for a threefold increase provided a good fit to the data. Such a locus could be detected in a realistic sample of affected sib pairs.


Klebsiella aerogenes urease gene cluster: sequence of ureD and demonstration that four accessory genes (ureD, ureE, ureF, and ureG) are involved in nickel metallocenter biosynthesis.

Abstract: The region located immediately upstream from the Klebsiella aerogenes urease structural genes was sequenced and shown to possess an open reading frame capable of encoding a 29.8-kDa peptide. Deletions were generated in this gene, denoted ureD, and in each of the genes (ureE, ureF, and ureG) located immediately downstream of the three structural genes. Transformation of the mutated plasmids into Escherichia coli resulted in high levels of urease expression, but the enzyme was inactive (deletions in ureD, ureF, or ureG) or only partially active (deletions in ureE). Ureases were purified from the recombinant cells and shown to be identical to control enzyme when analyzed by gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis; however, in every case the activity levels correlated to nickel contents as analyzed by atomic absorption analysis. UreD, UreE, UreF, and UreG peptides were tentatively identified by gel electrophoretic comparison of mutant and control cell extracts, by in vivo expression of separately cloned genes, or by in vitro transcription-translation analyses; the assignments were confirmed for UreE and UreG by amino-terminal sequencing. The latter peptides (apparent M(r)s, 23,900 and 28,500) were present at high levels comparable to those of the urease subunits, whereas the amounts of UreF (apparent M(r), 27,000) and UreD (apparent M(r), 29,300) were greatly reduced, perhaps because of the lack of good ribosome binding sites in the regions upstream of these open reading frames. These results demonstrate that all four accessory genes are necessary for the functional incorporation of the urease metallocenter.


Disturbances in the positioning, proliferation and apoptosis of neural progenitors contribute to subcortical band heterotopia formation.

Abstract: Cortical malformations are commonly associated with intractable epilepsy and other developmental disorders. Our studies utilize the tish rat, a spontaneously occurring genetic model of subcortical band heterotopia (SBH) associated with epilepsy, to evaluate the developmental events underlying SBH formation in the neocortex. Our results demonstrate that Pax6(+) and Tbr2(+) progenitors are mislocalized in tish(+/-) and tish(-/-)- neocortex throughout neurogenesis. In addition, mislocalized tish(-/-) progenitors possess a longer cell cycle than wild type or normally-positioned tish(-/-) progenitors, owing to a lengthened G(2)+M+G(1) time. This mislocalization is not associated with adherens junction breakdown or loss of radial glial polarity in the ventricular zone (VZ), as assessed by immunohistochemistry against phalloidin (to identify F-actin), aPKC-λ and Par3. However, vimentin immunohistochemistry indicates that the radial glial scaffold is disrupted in the region of the tish(-/-) heterotopia. Moreover, lineage tracing experiments using in utero electroporation in tish(-/-) neocortex demonstrate that mislocalized progenitors do not retain contact with the ventricular surface and that ventricular/subventricular zone (SVZ) progenitors produce neurons that migrate into both the heterotopia and cortical plate (CP). Taken together, these findings define a series of developmental errors contributing to SBH formation that differs fundamentally from a primary error in neuronal migration.


Continuous glucose monitoring in pregnant women with type 1 diabetes (CONCEPTT): a multicentre international randomised controlled trial

Abstract:
BACKGROUND Pregnant women with type 1 diabetes are a high-risk population who are recommended to strive for optimal glucose control, but neonatal outcomes attributed to maternal hyperglycaemia remain suboptimal. Our aim was to examine the effectiveness of continuous glucose monitoring (CGM) on maternal glucose control and obstetric and neonatal health outcomes.
 METHODS In this multicentre, open-label, randomised controlled trial, we recruited women aged 18-40 years with type 1 diabetes for a minimum of 12 months who were receiving intensive insulin therapy. Participants were pregnant (≤13 weeks and 6 days' gestation) or planning pregnancy from 31 hospitals in Canada, England, Scotland, Spain, Italy, Ireland, and the USA. We ran two trials in parallel for pregnant participants and for participants planning pregnancy. In both trials, participants were randomly assigned to either CGM in addition to capillary glucose monitoring or capillary glucose monitoring alone. Randomisation was stratified by insulin delivery (pump or injections) and baseline glycated haemoglobin (HbA1c). The primary outcome was change in HbA1c from randomisation to 34 weeks' gestation in pregnant women and to 24 weeks or conception in women planning pregnancy, and was assessed in all randomised participants with baseline assessments. Secondary outcomes included obstetric and neonatal health outcomes, assessed with all available data without imputation. This trial is registered with ClinicalTrials.gov, number NCT01788527.
 FINDINGS Between March 25, 2013, and March 22, 2016, we randomly assigned 325 women (215 pregnant, 110 planning pregnancy) to capillary glucose monitoring with CGM (108 pregnant and 53 planning pregnancy) or without (107 pregnant and 57 planning pregnancy). We found a small difference in HbA1c in pregnant women using CGM (mean difference -0·19%; 95% CI -0·34 to -0·03; p=0·0207). Pregnant CGM users spent more time in target (68% vs 61%; p=0·0034) and less time hyperglycaemic (27% vs 32%; p=0·0279) than did pregnant control participants, with comparable severe hypoglycaemia episodes (18 CGM and 21 control) and time spent hypoglycaemic (3% vs 4%; p=0·10). Neonatal health outcomes were significantly improved, with lower incidence of large for gestational age (odds ratio 0·51, 95% CI 0·28 to 0·90; p=0·0210), fewer neonatal intensive care admissions lasting more than 24 h (0·48; 0·26 to 0·86; p=0·0157), fewer incidences of neonatal hypoglycaemia (0·45; 0·22 to 0·89; p=0·0250), and 1-day shorter length of hospital stay (p=0·0091). We found no apparent benefit of CGM in women planning pregnancy. Adverse events occurred in 51 (48%) of CGM participants and 43 (40%) of control participants in the pregnancy trial, and in 12 (27%) of CGM participants and 21 (37%) of control participants in the planning pregnancy trial. Serious adverse events occurred in 13 (6%) participants in the pregnancy trial (eight [7%] CGM, five [5%] control) and in three (3%) participants in the planning pregnancy trial (two [4%] CGM and one [2%] control). The most common adverse events were skin reactions occurring in 49 (48%) of 103 CGM participants and eight (8%) of 104 control participants during pregnancy and in 23 (44%) of 52 CGM participants and five (9%) of 57 control participants in the planning pregnancy trial. The most common serious adverse events were gastrointestinal (nausea and vomiting in four participants during pregnancy and three participants planning pregnancy).
 INTERPRETATION Use of CGM during pregnancy in patients with type 1 diabetes is associated with improved neonatal outcomes, which are likely to be attributed to reduced exposure to maternal hyperglycaemia. CGM should be offered to all pregnant women with type 1 diabetes using intensive insulin therapy. This study is the first to indicate potential for improvements in non-glycaemic health outcomes from CGM use.
 FUNDING Juvenile Diabetes Research Foundation, Canadian Clinical Trials Network, and National Institute for Health Research.


Interleukin-12 and -23 Control Plasticity of CD127(+) Group 1 and Group 3 Innate Lymphoid Cells in the Intestinal Lamina Propria.

Abstract: Human group 1 ILCs consist of at least three phenotypically distinct subsets, including NK cells, CD127(+) ILC1, and intraepithelial CD103(+) ILC1. In inflamed intestinal tissues from Crohn's disease patients, numbers of CD127(+) ILC1 increased at the cost of ILC3. Here we found that differentiation of ILC3 to CD127(+) ILC1 is reversible in vitro and in vivo. CD127(+) ILC1 differentiated to ILC3 in the presence of interleukin-2 (IL-2), IL-23, and IL-1β dependent on the transcription factor RORγt, and this process was enhanced in the presence of retinoic acid. Furthermore, we observed in resection specimen from Crohn's disease patients a higher proportion of CD14(+) dendritic cells (DC), which in vitro promoted polarization from ILC3 to CD127(+) ILC1. In contrast, CD14(-) DCs promoted differentiation from CD127(+) ILC1 toward ILC3. These observations suggest that environmental cues determine the composition, function, and phenotype of CD127(+) ILC1 and ILC3 in the gut.


ERAAP synergizes with MHC class I molecules to make the final cut in the antigenic peptide precursors in the endoplasmic reticulum.

Abstract: The major histocompatibility complex class I molecules display peptides (pMHC I) on the cell surface for immune surveillance by CD8(+) T cells. These peptides are generated by proteolysis of intracellular polypeptides by the proteasome in the cytoplasm and then in the endoplasmic reticulum (ER) by the ER aminopeptidase associated with antigen processing (ERAAP). To define the unknown mechanism of ERAAP function in vivo, we analyzed naturally processed peptides in cells with or without appropriate MHC I and ERAAP. In the absence of MHC I, ERAAP degraded the antigenic precursors in the ER. However, MHC I molecules could bind proteolytic intermediates and were essential for generation of the final peptide by ERAAP. Thus, ERAAP synergizes with MHC I to generate the final pMHC I repertoire.


Estimates of animal and plant protein intake in US adults: results from the Third National Health and Nutrition Examination Survey, 1988-1991.

Abstract:
OBJECTIVE To describe the sources of protein intake in a sample of the US adult population and among subgroups defined by race-ethnicity, age, and gender.
 DESIGN The Third National Health and Nutrition Examination Survey, 1988-1991, is a stratified random sample of the total civilian noninstitutionalized population, drawn from the 50 United States and the District of Columbia. For all foods consumed by the participants, based on a 24-hour dietary recall, protein sources and the contribution of each protein type to the total protein intake were determined.    SUBJECTS Adult participants in the third National Health and Nutrition Examination Survey (n = 7,924).    STATISTICAL ANALYSES Weighted total, age-specific, and age-adjusted mean protein intakes were calculated using SAS and WesVarPC. Statistical differences were determined by 2-tailed t tests.
 RESULTS The main protein source in the American diet is animal protein (69%). Meat, fish, and poultry protein combined contributed the most to animal protein (42%), followed by dairy protein (20%). Grains (18%) contributed the most to plant protein consumption. Women consumed a lower percentage of beef (14%) and pork (7%) protein than did men (18% and 9%, respectively). Women also consumed a higher percentage of poultry (13%), dairy (22%), and fruit and vegetable (11%) protein than did men (11%, 19%, and 9%, respectively). Blacks reported eating a higher percentage of poultry (18%) and pork (11%) protein and a lower percent of dairy protein (14%) than did whites (12%, 7%, and 22%, respectively) and Mexican-Americans (11%, 8%, and 17%, respectively). Mexican-Americans consumed a higher percentage of legume (7%) and egg (7%) protein than did whites (4% and 4%, respectively) and blacks (4% and 5%, respectively). Whites consumed a higher percentage of grain protein (19%) than did blacks (16%) and Mexican-Americans (15%).
 CONCLUSIONS These results show that, although the percentage of total energy from protein may be similar among race-ethnicities and between men and women, their sources of protein are different. These differences should be taken into account when providing nutrition education for specific populations.


CYP18A1, a key enzyme of Drosophila steroid hormone inactivation, is essential for metamorphosis.

Abstract: Ecdysteroids are steroid hormones, which coordinate major developmental transitions in insects. Both the rises and falls in circulating levels of active hormones are important for coordinating molting and metamorphosis, making both ecdysteroid biosynthesis and inactivation of physiological relevance. We demonstrate that Drosophila melanogaster Cyp18a1 encodes a cytochrome P450 enzyme (CYP) with 26-hydroxylase activity, a prominent step in ecdysteroid catabolism. A clear ortholog of Cyp18a1 exists in most insects and crustaceans. When Cyp18a1 is transfected in Drosophila S2 cells, extensive conversion of 20-hydroxyecdysone (20E) into 20-hydroxyecdysonoic acid is observed. This is a multi-step process, which involves the formation of 20,26-dihydroxyecdysone as an intermediate. In Drosophila larvae, Cyp18a1 is expressed in many target tissues of 20E. We examined the consequences of Cyp18a1 inactivation on Drosophila development. Null alleles generated by excision of a P element and RNAi knockdown of Cyp18a1 both result in pupal lethality, possibly as a consequence of impaired ecdysteroid degradation. Our data suggest that the inactivation of 20E is essential for proper development and that CYP18A1 is a key enzyme in this process.


The sialyltransferase ST3GAL6 influences homing and survival in multiple myeloma.

Abstract: Glycosylation is a stepwise procedure of covalent attachment of oligosaccharide chains to proteins or lipids, and alterations in this process, especially increased sialylation, have been associated with malignant transformation and metastasis. The role of altered sialylation in multiple myeloma (MM) cell trafficking has not been previously investigated. In the present study we identified high expression of β-galactoside α-2,3-sialyltransferase, ST3GAL6, in MM cell lines and patients. This gene plays a key role in selectin ligand synthesis in humans through the generation of functional sialyl Lewis X. In MRC IX patients, high expression of this gene is associated with inferior overall survival. In this study we demonstrate that knockdown of ST3GAL6 results in a significant reduction in levels of α-2,3-linked sialic acid on the surface of MM cells with an associated significant reduction in adhesion to MM bone marrow stromal cells and fibronectin along with reduced transendothelial migration in vitro. In support of our in vitro findings, we demonstrate significantly reduced homing and engraftment of ST3GAL6 knockdown MM cells to the bone marrow niche in vivo, along with decreased tumor burden and prolonged survival. This study points to the importance of altered glycosylation, particularly sialylation, in MM cell adhesion and migration.


High-Density Lipoproteins Exert Pro-inflammatory Effects on Macrophages via Passive Cholesterol Depletion and PKC-NF-κB/STAT1-IRF1 Signaling.

Abstract: Membrane cholesterol modulates a variety of cell signaling pathways and functions. While cholesterol depletion by high-density lipoproteins (HDLs) has potent anti-inflammatory effects in various cell types, its effects on inflammatory responses in macrophages remain elusive. Here we show overt pro-inflammatory effects of HDL-mediated passive cholesterol depletion and lipid raft disruption in murine and human primary macrophages in vitro. These pro-inflammatory effects were confirmed in vivo in peritoneal macrophages from apoA-I transgenic mice, which have elevated HDL levels. In line with these findings, the innate immune responses required for clearance of P. aeruginosa bacterial infection in lung were compromised in mice with low HDL levels. Expression analysis, ChIP-PCR, and combinatorial pharmacological and genetic intervention studies unveiled that both native and reconstituted HDL enhance Toll-like-receptor-induced signaling by activating a PKC-NF-κB/STAT1-IRF1 axis, leading to increased inflammatory cytokine expression. HDL's pro-inflammatory activity supports proper functioning of macrophage immune responses.


Hypoxia of the renal medulla--its implications for disease.

Abstract: In land mammals, a major task of the kidney is to reabsorb water to allow survival in a dry environment. Water conservation is enhanced by the renal medulla, which concentrates the urine to a level up to four times the osmolality of plasma. To produce this unique gradient of osmolality, the medulla has a countercurrent system of vessels and tubules that dictates active reabsorption of sodium in a milieu poor in oxygen (Figure 1).1 In this review, we describe how hypoxia of the medulla may relate to susceptibility to acute and chronic renal injury. The Renal Medullary Concentrating Mechanism as . . .


Uncoupling Mitochondrial Respiration for Diabesity.

Abstract: Until recently, the mechanism of adaptive thermogenesis was ascribed to the expression of uncoupling protein 1 (UCP1) in brown and beige adipocytes. UCP1 is known to catalyze a proton leak of the inner mitochondrial membrane, resulting in uncoupled oxidative metabolism with no production of adenosine triphosphate and increased energy expenditure. Thus increasing brown and beige adipose tissue with augmented UCP1 expression is a viable target for obesity-related disorders. Recent work demonstrates an UCP1-independent pathway to uncouple mitochondrial respiration. A secreted enzyme, PM20D1, enriched in UCP1+ adipocytes, exhibits catalytic and hydrolytic activity to reversibly form N-acyl amino acids. N-acyl amino acids act as endogenous uncouplers of mitochondrial respiration at physiological concentrations. Administration of PM20D1 or its products, N-acyl amino acids, to diet-induced obese mice improves glucose tolerance by increasing energy expenditure. In short-term studies, treated animals exhibit no toxicity while experiencing 10% weight loss primarily of adipose tissue. Further study of this metabolic pathway may identify novel therapies for diabesity, the disease state associated with diabetes and obesity.


A functional polymorphism in the prodynorphin gene promotor is associated with temporal lobe epilepsy.

Abstract: The prodynorphin gene (PDYN) encoding the anticonvulsant peptide dynorphin is a strong candidate for a seizure suppressor gene and thus a possible modulator of susceptibility to temporal lobe epilepsy. We performed a case control association study in 155 patients with nonlesional temporal lobe epilepsy and 202 controls and found that PDYN promotor low-expression L-alleles confer an increased risk for temporal lobe epilepsy in patients with a family history for seizures. Irrespective of the familial background, L-homozygotes display a higher risk for secondarily generalized seizures and status epilepticus.


Cardiovascular and renal benefits of dry bean and soybean intake.

Abstract: Dry beans and soybeans are nutrient-dense, fiber-rich, and are high-quality sources of protein. Protective and therapeutic effects of both dry bean and soybean intake have been documented. Studies show that dry bean intake has the potential to decrease serum cholesterol concentrations, improve many aspects of the diabetic state, and provide metabolic benefits that aid in weight control. Soybeans are a unique source of the isoflavones genistein and diadzein, which have numerous biological functions. Soybeans and soyfoods potentially have multifaceted health-promoting effects, including cholesterol reduction, improved vascular health, preserved bone mineral density, and reduction of menopausal symptoms. Soy appears to have salutary effects on renal function, although these effects are not well understood. Whereas populations consuming high intakes of soy have lower prevalences of certain cancers, definitive experimental data are insufficient to clarify a protective role of soy. The availability of legume products and resources is increasing, incorporating dry beans and soyfoods into the diet can be practical and enjoyable. With the shift toward a more plant-based diet, dry beans and soy will be potent tools in the treatment and prevention of chronic disease.


NuA4 lysine acetyltransferase Esa1 is targeted to coding regions and stimulates transcription elongation with Gcn5.

Abstract: NuA4, the major H4 lysine acetyltransferase (KAT) complex in Saccharomyces cerevisiae, is recruited to promoters and stimulates transcription initiation. NuA4 subunits contain domains that bind methylated histones, suggesting that histone methylation should target NuA4 to coding sequences during transcription elongation. We show that NuA4 is cotranscriptionally recruited, dependent on its physical association with elongating polymerase II (Pol II) phosphorylated on the C-terminal domain by cyclin-dependent kinase 7/Kin28, but independently of subunits (Eaf1 and Tra1) required for NuA4 recruitment to promoters. Whereas histone methylation by Set1 and Set2 is dispensable for NuA4's interaction with Pol II and targeting to some coding regions, it stimulates NuA4-histone interaction and H4 acetylation in vivo. The NuA4 KAT, Esa1, mediates increased H4 acetylation and enhanced RSC occupancy and histone eviction in coding sequences and stimulates the rate of transcription elongation. Esa1 cooperates with the H3 KAT in SAGA, Gcn5, to enhance these functions. Our findings delineate a pathway for acetylation-mediated nucleosome remodeling and eviction in coding sequences that stimulates transcription elongation by Pol II in vivo.


Targeting mitotic exit leads to tumor regression in vivo: Modulation by Cdk1, Mastl, and the PP2A/B55α,δ phosphatase.

Abstract: Targeting mitotic exit has been recently proposed as a relevant therapeutic approach against cancer. By using genetically engineered mice, we show that the APC/C cofactor Cdc20 is essential for anaphase onset in vivo in embryonic or adult cells, including progenitor/stem cells. Ablation of Cdc20 results in efficient regression of aggressive tumors, whereas current mitotic drugs display limited effects. Yet, Cdc20 null cells can exit from mitosis upon inactivation of Cdk1 and the kinase Mastl (Greatwall). This mitotic exit depends on the activity of PP2A phosphatase complexes containing B55α or B55δ regulatory subunits. These data illustrate the relevance of critical players of mitotic exit in mammals and their implications in the balance between cell death and mitotic exit in tumor cells.


UV light-induced DNA synthesis arrest in HeLa cells is associated with changes in phosphorylation of human single-stranded DNA-binding protein.

Abstract: We show that DNA replication activity in extracts of human HeLa cells decreases following UV irradiation. Alterations in replication activity in vitro parallel the UV-induced block in cell cycle progression of these cells in culture. UV irradiation also induces specific changes in the pattern of phosphorylation of the 34 kDa subunit of a DNA replication protein, human single-stranded DNA-binding protein (hSSB). The appearance of a hyperphosphorylated form of hSSB correlates with reduced in vitro DNA replication activity in extracts of UV-irradiated cells. Replication activity can be restored to these extracts in vitro by addition of purified hSSB. These results suggest that UV-induced DNA synthesis arrest may be mediated in part through phosphorylation-related alterations in the activity of hSSB, an essential component of the DNA replication apparatus.


Study of infectious intestinal disease in England: rates in the community, presenting to general practice, and reported to national surveillance. The Infectious Intestinal Disease Study Executive.

Abstract:
OBJECTIVE To establish the incidence and aetiology of infectious intestinal disease in the community and presenting to general practitioners. Comparison with incidence and aetiology of cases reaching national laboratory based surveillance.
 DESIGN Population based community cohort incidence study, general practice based incidence studies, and case linkage to national laboratory surveillance.
 SETTING 70 general practices throughout England.
 PARTICIPANTS 459 975 patients served by the practices. Community surveillance of 9776 randomly selected patients.
 MAIN OUTCOME MEASURES Incidence of infectious intestinal disease in community and reported to general practice.
 RESULTS 781 cases were identified in the community cohort, giving an incidence of 19.4/100 person years (95% confidence interval 18.1 to 20.8). 8770 cases presented to general practice (3.3/100 person years (2.94 to 3.75)). One case was reported to national surveillance for every 1.4 laboratory identifications, 6.2 stools sent for laboratory investigation, 23 cases presenting to general practice, and 136 community cases. The ratio of cases in the community to cases reaching national surveillance was lower for bacterial pathogens (salmonella 3.2:1, campylobacter 7.6:1) than for viruses (rotavirus 35:1, small round structured viruses 1562:1). There were many cases for which no organism was identified.
 CONCLUSIONS Infectious intestinal disease occurs in 1 in 5 people each year, of whom 1 in 6 presents to a general practitioner. The proportion of cases not recorded by national laboratory surveillance is large and varies widely by microorganism. Ways of supplementing the national laboratory surveillance system for infectious intestinal diseases should be considered.


A dual function of the CRISPR-Cas system in bacterial antivirus immunity and DNA repair.

Abstract: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and the associated proteins (Cas) comprise a system of adaptive immunity against viruses and plasmids in prokaryotes. Cas1 is a CRISPR-associated protein that is common to all CRISPR-containing prokaryotes but its function remains obscure. Here we show that the purified Cas1 protein of Escherichia coli (YgbT) exhibits nuclease activity against single-stranded and branched DNAs including Holliday junctions, replication forks and 5'-flaps. The crystal structure of YgbT and site-directed mutagenesis have revealed the potential active site. Genome-wide screens show that YgbT physically and genetically interacts with key components of DNA repair systems, including recB, recC and ruvB. Consistent with these findings, the ygbT deletion strain showed increased sensitivity to DNA damage and impaired chromosomal segregation. Similar phenotypes were observed in strains with deletion of CRISPR clusters, suggesting that the function of YgbT in repair involves interaction with the CRISPRs. These results show that YgbT belongs to a novel, structurally distinct family of nucleases acting on branched DNAs and suggest that, in addition to antiviral immunity, at least some components of the CRISPR-Cas system have a function in DNA repair.


Intravenous valproate is well tolerated in unstable patients with status epilepticus.

Abstract: The authors reviewed hospital records of 13 patients with status epilepticus and hypotension who received IV valproate therapy. Most patients were elderly (74.4 +/- 12.5 [SD] years) and received a loading dose of valproate of 25.1 +/- 5.0 mg/kg (range 14.7 to 32.7), at a rate of 36.6 +/- 25.1 mg/min (range 6.3 to 100). There were no significant changes in blood pressure, pulse, or use of vasopressors. The data suggest that valproate loading is well tolerated, even in patients with cardiovascular instability.


Interplays between ATM/Tel1 and ATR/Mec1 in sensing and signaling DNA double-strand breaks.

Abstract: DNA double-strand breaks (DSBs) are highly hazardous for genome integrity because they have the potential to cause mutations, chromosomal rearrangements and genomic instability. The cellular response to DSBs is orchestrated by signal transduction pathways, known as DNA damage checkpoints, which are conserved from yeasts to humans. These pathways can sense DNA damage and transduce this information to specific cellular targets, which in turn regulate cell cycle transitions and DNA repair. The mammalian protein kinases ATM and ATR, as well as their budding yeast corresponding orthologs Tel1 and Mec1, act as master regulators of the checkpoint response to DSBs. Here, we review the early steps of DSB processing and the role of DNA-end structures in activating ATM/Tel1 and ATR/Mec1 in an orderly and reciprocal manner.


Expression of the Norrie disease gene (Ndp) in developing and adult mouse eye, ear, and brain.

Abstract: The Norrie disease gene (Ndp) codes for a secreted protein, Norrin, that activates canonical Wnt signaling by binding to its receptor, Frizzled-4. This signaling system is required for normal vascular development in the retina and for vascular survival in the cochlea. In mammals, the pattern of Ndp expression beyond the retina is poorly defined due to the low abundance of Norrin mRNA and protein. Here, we characterize Ndp expression during mouse development by studying a knock-in mouse that carries the coding sequence of human placental alkaline phosphatase (AP) inserted at the Ndp locus (Ndp(AP)). In the CNS, Ndp(AP) expression is apparent by E10.5 and is dynamic and complex. The anatomically delimited regions of Ndp(AP) expression observed prenatally in the CNS are replaced postnatally by widespread expression in astrocytes in the forebrain and midbrain, Bergman glia in the cerebellum, and Müller glia in the retina. In the developing and adult cochlea, Ndp(AP) expression is closely associated with two densely vascularized regions, the stria vascularis and a capillary plexus between the organ of Corti and the spiral ganglion. These observations suggest the possibility that Norrin may have developmental and/or homeostatic functions beyond the retina and cochlea.


Brain region-specific up-regulation of mouse apolipoprotein E by pharmacological estrogen treatments.

Abstract: Cerebral apolipoprotein E (apoE) has been implicated in neuronal protection and repair. Due to the variable levels and types of estrogen receptors within different brain regions, the effect of estrogen on apoE and the mechanism of this effect may vary within different regions. Ovariectomized female C57BL/6 mice were treated with pharmacological levels of 17 beta-estradiol or placebo for 5 days, resulting in supraphysiological plasma levels of estradiol in the treated mice. ApoE and glial fibrillary acidic protein (GFAP) levels were measured in the cortex, hippocampus and diencephalon. 17 beta-Estradiol up-regulated apoE but not GFAP in the cortex and diencephalon, whereas in the hippocampus, GFAP and apoE were equally up-regulated. Treatment of estrogen receptor (ER) alpha knockout mice with 17 beta-estradiol or treatment of C57BL/6 mice with 17 alpha-estradiol, a poor estrogen receptor agonist, specifically induced apoE in the cortex, but not in the diencephalon. These results indicate that 17 beta-estradiol effects on apoE are either directly or indirectly mediated by ER alpha in the diencephalon, while the effects in the cortex may be mediated by a non-classical mechanism or by ER beta. Measurement of mRNA levels in estrogen versus placebo-treated wild-type mice indicated that the effect of 17 beta-estradiol on apoE was not associated with changes in apoE mRNA levels.


A novel immortalized human endometrial stromal cell line with normal progestational response.

Abstract: Obtaining primary human endometrial stromal cells (HESCs) for in vitro studies is limited by the scarcity of adequate human material and the inability to passage these cells in culture for long periods. Immortalization of these cells would greatly facilitate studies; however, the process of immortalization often results in abnormal karyotypes and aberrant functional characteristics. To meet this need, we have introduced telomerase into cultured HESCs to prevent the normal shortening of telomeres observed in adult somatic cells during mitosis. We have now developed and analyzed a newly immortalized HESC line that contains no clonal chromosomal structural or numerical abnormalities. In addition, when compared with the primary unpassaged parent cells, the new cell line displayed similar biochemical endpoints after treatment with ovarian steroids. Classical decidualization response to estradiol plus medroxyprogesterone acetate were seen in both morphologically, and progestin was seen to induce or regulate the expression of IGF binding protein-1, fibronectin, prolactin, tissue factor, plasminogen activator inhibitor-1, and Fas/Fas ligand. In summary, an immortalized HESC line has been developed that is karyotypically, morphologically, and phenotypically similar to the primary parent cells, and it is a powerful and consistent resource for in vitro work.


Comparison of the intrinsic kinase activity and substrate specificity of c-Abl and Bcr-Abl.

Abstract: We studied the intrinsic tyrosine kinase activity and substrate specificity of c-Abl and Bcr-Abl protein tyrosine kinases (PTKs) using the peptide substrates discovered from a synthetic combinatorial peptide library. Our data indicate that the phosphorylation of these peptides by Bcr-Abl was consistently stronger than that by c-Abl. Bcr-Abl also showed substrate preference towards those peptides with one or more positive charges.


Strong association between respiratory viral infection early after hematopoietic stem cell transplantation and the development of life-threatening acute and chronic alloimmune lung syndromes.

Abstract: Alloimmune lung syndromes (allo-LS), including idiopathic pneumonia syndrome, bronchiolitis obliterans syndrome, and bronchiolitis obliterans organizing pneumonia, are severe complications after hematopoietic stem cell transplantation (HSCT). In our cohort of 110 pediatric patients, 30 had allo-LS (27.3%), 18 with idiopathic pneumonia syndrome and 12 with bronchiolitis obliterans syndrome. Multivariate analysis showed that respiratory viral infection early after HSCT is an important predictor for the development of allo-LS (P <.0001). This was true for all viruses tested. In multivariate analysis, allo-LS was the only predictor for higher mortality (P = .04). Paradoxically, prolonged administration of immunosuppressive agents because of acute graft-versus-host disease had a protective effect on the development of allo-LS (P = .004). We hypothesize that early infection of the respiratory tract with a common cold virus makes the lungs a target for alloimmunity.


Hepatitis C virus blocks interferon effector function by inducing protein kinase R phosphorylation.

Abstract: Hepatitis C virus (HCV) is a single-stranded RNA virus encoding a single polyprotein whose translation is driven by an internal ribosome entry site (IRES). HCV infection strongly induces antiviral interferon-stimulated gene (ISG) expression in the liver, yet it persists, suggesting that HCV can block ISG effector function. We now show that HCV infection triggers phosphorylation and activation of the RNA-dependent protein kinase PKR, which inhibits eukaryotic translation initiation factor eIF2 alpha and attenuates ISG protein expression despite normal ISG mRNA induction. ISG protein induction is restored and the antiviral effects of interferon are enhanced when PKR expression is suppressed in interferon-treated infected cells. Whereas host protein translation, including antiviral ISGs, is suppressed by activated PKR, HCV IRES-dependent translation is not. These results suggest that the ability of HCV to activate PKR may, paradoxically, be advantageous for the virus during an IFN response by preferentially suppressing the translation of ISGs.


Adult c-Kit(+) progenitor cells are necessary for maintenance and regeneration of olfactory neurons.

Abstract: The olfactory epithelium houses chemosensory neurons, which transmit odor information from the nose to the brain. In adult mammals, the olfactory epithelium is a uniquely robust neuroproliferative zone, with the ability to replenish its neuronal and non-neuronal populations due to the presence of germinal basal cells. The stem and progenitor cells of these germinal layers, and their regulatory mechanisms, remain incompletely defined. Here we show that progenitor cells expressing c-Kit, a receptor tyrosine kinase marking stem cells in a variety of embryonic tissues, are required for maintenance of the adult neuroepithelium. Mouse genetic fate-mapping analyses show that embryonically, a c-Kit(+) population contributes to olfactory neurogenesis. In adults under conditions of normal turnover, there is relatively sparse c-Kit(+) progenitor cell (ckPC) activity. However, after experimentally induced neuroepithelial injury, ckPCs are activated such that they reconstitute the neuronal population. There are also occasional non-neuronal cells found to arise from ckPCs. Moreover, the selective depletion of the ckPC population, utilizing temporally controlled targeted diphtheria toxin A expression, results in failure of neurogenesis after experimental injury. Analysis of this model indicates that most ckPCs reside among the globose basal cell populations and act downstream of horizontal basal cells, which can serve as stem cells. Identification of the requirement for olfactory c-Kit-expressing progenitors in olfactory maintenance provides new insight into the mechanisms involved in adult olfactory neurogenesis. Additionally, we define an important and previously unrecognized site of adult c-Kit activity.


An Oct4-Centered Protein Interaction Network in Embryonic Stem Cells

Abstract: Transcription factors, such as Oct4, are critical for establishing and maintaining pluripotent cell identity. Whereas the genomic locations of several pluripotency transcription factors have been reported, the spectrum of their interaction partners is underexplored. Here, we use an improved affinity protocol to purify Oct4-interacting proteins from mouse embryonic stem cells (ESCs). Subsequent purification of Oct4 partners Sall4, Tcfcp2l1, Dax1, and Esrrb resulted in an Oct4 interactome of 166 proteins, including transcription factors and chromatin-modifying complexes with documented roles in self-renewal, but also many factors not previously associated with the ESC network. We find that Esrrb associated with the basal transcription machinery and also detect interactions between transcription factors and components of the TGF-beta, Notch, and Wnt signaling pathways. Acute depletion of Oct4 reduced binding of Tcfcp2l1, Dax1, and Esrrb to several target genes. In conclusion, our purification protocol allowed us to bring greater definition to the circuitry controlling pluripotent cell identity.


Crystal structure, biochemical and cellular activities demonstrate separate functions of MTH1 and MTH2

Abstract: Deregulated redox metabolism in cancer leads to oxidative damage to cellular components including deoxyribonucleoside triphosphates (dNTPs). Targeting dNTP pool sanitizing enzymes, such as MTH1, is a highly promising anticancer strategy. The MTH2 protein, known as NUDT15, is described as the second human homologue of bacterial MutT with 8-oxo-dGTPase activity. We present the first NUDT15 crystal structure and demonstrate that NUDT15 prefers other nucleotide substrates over 8-oxo-dGTP. Key structural features are identified that explain different substrate preferences for NUDT15 and MTH1. We find that depletion of NUDT15 has no effect on incorporation of 8-oxo-dGTP into DNA and does not impact cancer cell survival in cell lines tested. NUDT17 and NUDT18 were also profiled and found to have far less activity than MTH1 against oxidized nucleotides. We show that NUDT15 is not a biologically relevant 8-oxo-dGTPase, and that MTH1 is the most prominent sanitizer of the cellular dNTP pool known to date.


De novo expression of Trpm4 initiates secondary hemorrhage in spinal cord injury

Abstract: The role of transient receptor potential M4 (Trpm4), an unusual member of the Trp family of ion channels, is poorly understood. Using rodent models of spinal cord injury, we studied involvement of Trpm4 in the progressive expansion of secondary hemorrhage associated with capillary fragmentation, the most destructive mechanism of secondary injury in the central nervous system. Trpm4 mRNA and protein were abundantly upregulated in capillaries preceding their fragmentation and formation of petechial hemorrhages. Trpm4 expression in vitro rendered COS-7 cells highly susceptible to oncotic swelling and oncotic death following ATP depletion. After spinal cord injury, in vivo gene suppression in rats treated with Trpm4 antisense or in Trpm4−/− mice preserved capillary structural integrity, eliminated secondary hemorrhage, yielded a threefold to fivefold reduction in lesion volume and produced a substantial improvement in neurological function. To our knowledge, this is the first example of a Trp channel that must undergo de novo expression for manifestation of central nervous system pathology.


Differential expression of bcl-2 in intestinal epithelia. Correlation with attenuation of apoptosis in colonic crypts and the incidence of colonic neoplasia.

Abstract: The cell-positional incidence of both spontaneous and damage-induced apoptosis of epithelial cells was assessed in longitudinal sections of the crypts of small intestine and colon of BDF1 mice. This was compared, using immunohistochemistry, with the pattern of expression of bcl-2, a suppressor of apoptosis. In the small intestine, apoptosis was maximal around cell position 4 from the base of the crypt; this closely corresponds to the position considered to contain the stem cells. In the colon, however, apoptosis was not confined to the area considered to harbour the stem cells (position 1 and 2). Instead, apoptosis was attenuated and distributed along the length of the crypt. Some cells at the base of murine colonic crypts expressed bcl-2 protein, whereas bcl-2 was absent in the crypts of the small intestine. Most pertinently, bcl-2 was absent from small intestinal crypt cells at positions 4-5 (the stem cell region). The importance of the expression of bcl-2 to the attenuation of apoptosis in stem cells was confirmed by analysis of the levels of both spontaneous and induced apoptosis in homozygously bcl-2 null C57BL/6 mice: in colonic crypts the level of spontaneous apoptosis rose significantly, and selectively at the base of the crypt, in comparison with crypts from wild-type animals. In contrast, there was no rise in spontaneous apoptosis in the small intestinal crypts from the bcl-2 null animals. Analysis of sections of human colon and small intestine also showed that expression of bcl-2 was confined to the base of the colonic crypt. The attenuation of apoptosis by bcl-2 in the region of the stem cells of the colonic crypts may dispose these to neoplastic transformation. Indeed, analysis of human carcinomas revealed expression of bcl-2, which in some samples was reciprocal with the expression of p53.


Dishevelled Activates JNK and Discriminates between JNK Pathways in Planar Polarity and wingless Signaling

Abstract: Frizzled family proteins have been described as receptors of Wnt signaling molecules. In Drosophila, the two known Frizzled proteins are associated with distinct developmental processes. Genesis of epithelial planar polarity requires Frizzled, whereas Dfz2 affects morphogenesis by wingless-mediated signaling. Dishevelled is required in both signaling pathways. Here, we use genetic and overexpression assays to show that Dishevelled activates JNK cascades. Rescue analysis reveals different protein domain requirements in Dishevelled for the two pathways; the C-terminal DEP domain is essential to rescue planar polarity defects and induce JNK signaling. Furthermore, the planar polarity-specific dsh1 allele is mutated in the DEP domain. Our results indicate that different Wnt/Fz signals activate distinct intracellular pathways, and Dishevelled discriminates among them by distinct domain interactions.


Genome-wide lineage-specific transcriptional networks underscore Ikaros-dependent lymphoid priming in hematopoietic stem cells.

Abstract: The mechanisms regulating lineage potential during early hematopoiesis were investigated. First, a cascade of lineage-affiliated gene expression signatures, primed in hematopoietic stem cells (HSCs) and differentially propagated in lineage-restricted progenitors, was identified. Lymphoid transcripts were primed as early as the HSC, together with myeloid and erythroid transcripts. Although this multilineage priming was resolved upon subsequent lineage restrictions, an unexpected cosegregation of lymphoid and myeloid gene expression and potential past a nominal myeloid restriction point was identified. Finally, we demonstrated that whereas the zinc finger DNA-binding factor Ikaros was required for induction of lymphoid lineage priming in the HSC, it was also necessary for repression of genetic programs compatible with self-renewal and multipotency downstream of the HSC. Taken together, our studies provide new insight into the priming and restriction of lineage potentials during early hematopoiesis and identify Ikaros as a key bivalent regulator of this process.


Lessons from the past: managing insecticide resistance in malaria control and eradication programmes.

Abstract: The distribution of insecticide-treated bednets to help combat the burden of malaria in sub-Saharan Africa has accelerated in the past 5 years. Additionally, many countries are also considering, or have already begun, indoor residual spraying campaigns. These are positive developments, since vector control has repeatedly proven to be an effective means of reducing malaria transmission. However, the sustainability of these insecticide-based interventions relies on the continuing susceptibility of the anopheles vectors to the limited number of available insecticides. Continual monitoring for early signs of insecticide resistance and the adoption of carefully considered resistance management strategies are therefore required. Regrettably, this essential monitoring component is frequently given a low priority in the push to meet ambitious coverage targets. We outline the key requirements for establishing an insecticide resistance surveillance system and urge all those involved in malaria vector control, either directly or as facilitators, to ensure that these measures are incorporated into control programmes. Failure to act now will inevitably lead to a future breakdown in disease control and jeopardise hopes of eradicating this major public-health problem.


T helper 2 cytokines inhibit autophagic control of intracellular Mycobacterium tuberculosis.

Abstract: Autophagy is a recently recognized immune effector mechanism against intracellular pathogens. The role of autophagy in innate immunity has been well established, but the extent of its regulation by the adaptive immune response is less well understood. The T helper 1 (Th1) cell cytokine IFN-gamma induces autophagy in macrophages to eliminate Mycobacterium tuberculosis. Here, we report that Th2 cytokines affect autophagy in macrophages and their ability to control intracellular M. tuberculosis. IL-4 and IL-13 abrogated autophagy and autophagy-mediated killing of intracellular mycobacteria in murine and human macrophages. Inhibition of starvation-induced autophagy by IL-4 and IL-13 was dependent on Akt signaling, whereas the inhibition of IFN-gamma-induced autophagy was Akt independent and signal transducer and activator of transcription 6 (STAT6) dependent. These findings establish a mechanism through which Th1-Th2 polarization differentially affects the immune control of intracellular pathogens.


Lipid-based nutrient supplement increases the birth size of infants of primiparous women in Ghana.

Abstract:
BACKGROUND The International Lipid-Based Nutrient Supplements Project developed a small-quantity (20 g/d) lipid-based nutrient supplement (LNS) for pregnant and lactating women.
 OBJECTIVE We evaluated the effects of prenatal LNS supplementation on fetal growth.
 DESIGN In a community-based, partially double-blind, individually randomized controlled trial, 1320 women ≤20 wk pregnant received 60 mg Fe/400 μg folic acid (IFA), or 1-2 Recommended Dietary Allowances of 18 micronutrients, including 20 mg Fe (MMN), or LNS with the same micronutrients as the MMN group, plus 4 minerals and macronutrients contributing 118 kcal (LNS) daily until delivery. Fetal growth was compared across groups by using intention-to-treat analysis. The primary outcome was birth length.
 RESULTS This analysis included 1057 women (IFA = 349, MMN = 354, LNS = 354). Groups did not differ significantly in mean birth length, length-for-age z score (LAZ), head circumference, or percentage low birth length but differed in mean birth weight (P = 0.044), weight-for-age z score (WAZ; P = 0.046), and BMI-for-age z score (BMIZ; P = 0.040), with a trend toward differences in low birth weight (P = 0.069). In pairwise comparisons, the LNS group had greater mean birth weight (+85 g; P = 0.040), WAZ (+0.19; P = 0.045), and BMIZ (+0.21; P = 0.035) and a lower risk of low birth weight (RR: 0.61, 95% CI: 0.39, 0.96; P = 0.032) than did the IFA group. The other group differences were not significant. The effect of intervention was modified by mother's parity, age, height, baseline hemoglobin, household food insecurity, and child sex, with parity being the most consistent modifier. Among primiparous women (IFA = 131; MMN = 110; LNS = 128), the LNS group had greater mean birth length (+0.91 cm; P = 0.001), LAZ (+0.47; P = 0.001), weight (+237 g; P < 0.001), WAZ (+0.56; P < 0.001), BMIZ (+0.52; P < 0.001), head circumference (0.50 cm; P = 0.017), and head circumference-for-age z score (+0.40; P = 0.022) than did the IFA group; similar differences were found when comparing the LNS and MMN groups among primiparous women, and no group differences were found among multiparous women.
 CONCLUSION Prenatal LNS supplementation can improve fetal growth among vulnerable women in Ghana, particularly primiparous women. This trial was registered at clinicaltrials.gov as NCT00970866.


Complex formation of SMAP/KAP3, a KIF3A/B ATPase motor-associated protein, with a human chromosome-associated polypeptide.

Abstract: We have recently isolated SMAP (Smg GDS-associated protein; Smg GDS: small G protein GDP dissociation stimulator) as a novel Smg GDS-associated protein, which has Armadillo repeats and is phosphorylated by Src tyrosine kinase. SMAP is a human counterpart of mouse KAP3 (kinesin superfamily-associated protein) that is associated with mouse KIF3A/B (a kinesin superfamily protein), which functions as a microtubule-based ATPase motor for organelle transport. We isolated here a SMAP-interacting protein from a human brain cDNA library, identified it to be a human homolog of Xenopus XCAP-E (Xenopus chromosome-associated polypeptide), a subunit of condensins that regulate the assembly and structural maintenance of mitotic chromosomes, and named it HCAP (Human chromosome-associated polypeptide). Tissue and subcellular distribution analyses indicated that HCAP was ubiquitously expressed and highly concentrated in the nuclear fraction, where SMAP and KIF3B were also present. SMAP was extracted as a ternary complex with HCAP and KIF3B from the nuclear fraction in the presence of Mg-ATP. The results suggest that SMAP/KAP3 serves as a linker between HCAP and KIF3A/B in the nucleus, and that SMAP/KAP3 plays a role in the interaction of chromosomes with an ATPase motor protein.


Surveillance for foodborne-disease outbreaks--United States, 1993-1997.

Abstract:
PROBLEM/CONDITION Since 1973, CDC has maintained a collaborative surveillance program for collection and periodic reporting of data on the occurrence and causes of foodborne-disease outbreaks (FBDOs) in the United States.    REPORTING PERIOD COVERED This summary reviews data from January 1993 through December 1997.    DESCRIPTION OF SYSTEM The Foodborne-Disease Outbreak Surveillance System reviews data concerning FBDOs, defined as the occurrence of two or more cases of a similar illness resulting from the ingestion of a common food. State and local public health departments have primary responsibility for identifying and investigating FBDOs. State, local, and territorial health departments use a standard form to report these outbreaks to CDC.
 RESULTS During 1993-1997, a total of 2,751 outbreaks of foodborne disease were reported (489 in 1993, 653 in 1994, 628 in 1995, 477 in 1996, and 504 in 1997). These outbreaks caused a reported 86,058 persons to become ill. Among outbreaks for which the etiology was determined, bacterial pathogens caused the largest percentage of outbreaks (75%) and the largest percentage of cases (86%). Salmonella serotype Enteritidis accounted for the largest number of outbreaks, cases, and deaths; most of these outbreaks were attributed to eating eggs. Chemical agents caused 17% of outbreaks and 1% of cases; viruses, 6% of outbreaks and 8% of cases; and parasites, 2% of outbreaks and 5% of cases.
 INTERPRETATION The annual number of FBDOs reported to CDC did not change substantially during this period or from previous years. During this reporting period, S. Enteritidis continued to be a major cause of illness and death. In addition, multistate outbreaks caused by contaminated produce and outbreaks caused by Escherichia coli O157:H7 remained prominent.    ACTIONS TAKEN Current methods to detect FBDOs are improving, and several changes to improve the ease and timeliness of reporting FBDO data are occurring (e.g., a revised form to simplify FBDO reporting by state health departments and electronic reporting methods). State and local health departments continue to investigate and report FBDOs as part of efforts to better understand and define the epidemiology of foodborne disease in the United States. At the regional and national levels, surveillance data provide an indication of the etiologic agents, vehicles of transmission, and contributing factors associated with FBDOs and help direct public health actions to reduce illness and death caused by FBDOs.


Metformin Normalizes Type 2 Diabetes-Induced Decrease in Cell Proliferation and Neuroblast Differentiation in the Rat Dentate Gyrus

Abstract: In this study, we observed the effects of metformin, one of the most widely prescribed drugs for the treatment of type 2 diabetes, on cell proliferation and neuroblast differentiation in the subgranular zone of the hippocampal dentate gyrus (SZDG) in Zucker diabetic fatty (ZDF) rats, which are a model for type 2 diabetes. For this, metformin was administered orally once a day to 14-week-old ZDF rats for 2 weeks and the animals were sacrificed at 16 weeks of age. During this period, blood glucose levels were higher in the vehicle-treated ZDF rats than in the Zucker lean control (ZLC) rats. Metformin treatment significantly decreased the blood glucose levels from 15.5 weeks of age. In the SZDG, Ki67 (a marker for cell proliferation)- and doublecortin (DCX, a marker for differentiated neuroblasts)-immunoreactive cells were much lower in the vehicle-treated ZDF rats than in the ZLC rats. In the metformin-treated ZDF group, Ki67- and DCX-immunoreactive cells were significantly increased in the SZDG compared to those in the vehicle-treated ZDF group. These results suggest that diabetes significantly reduces cell proliferation and neuroblast differentiation in the SZDG and that metformin treatment normalizes the reduction of cell proliferation and neuroblast differentiation in the SZDG in diabetic rats.


The prevalence of congenital malformations is still higher in pregnant women with pregestational diabetes despite near-normal HbA1c: a literature review.

Abstract:
AIMS/HYPOTHESIS We assessed the association between congenital malformations and maternal hyperglycemia in pregnant women with pregestational (type 1 or type 2) diabetes and investigated if the rate of congenital malformations was similar in women with near-normal glycemic control compared to the background population. We also assessed the association between congenital malformations and maternal hyperglycemia in pregnant women with pregestational diabetes with special focus on women with near-normal HbA1c in early pregnancy.    MATERIALS AND METHODS This is a literature review based on an electronic literature search of the databases PubMed, Cochrane, Embase and Web of Science conducted in July 2017 using the search terms diabetes, pregnancy, HbA1c or glycemic control and congenital anomaly or congenital anomaly. We included original papers in English published after 1997 with data on congenital malformations and HbA1c in at least 250 women with pregestational diabetes.
 RESULTS Nine papers with in total 6225 women with type 1 diabetes and 2334 women with type 2 diabetes were included. The prevalence of congenital malformations was 6.4% in women with type 1 diabetes and 4.3% in women with type 2 diabetes and for the combined group of women with pregestational diabetes, the relative risk compared to the background population was 3.2. In women with HbA1c < 53 mmol/mol (7.0%) in early pregnancy or HbA1c 53-64 mmol/mol (7.0-8.0%) the prevalence of congenital malformations was 4.3 and 3.7%, respectively, with a relative risk of 2.2 and 1.9, respectively.
 CONCLUSIONS In pregnant women with pregestational diabetes the prevalence of congenital abnormalities was threefold higher in women with pregestational diabetes compared to the background population. However, HbA1c below 53 mmol/mol (7.0%) in early pregnancy was also associated with a two times increased risk of congenital malformations compared to the background population.


Local and systemic insulin resistance resulting from hepatic activation of IKK-beta and NF-kappaB.

Abstract: We show that NF-kappaB and transcriptional targets are activated in liver by obesity and high-fat diet (HFD). We have matched this state of chronic, subacute 'inflammation' by low-level activation of NF-kappaB in the liver of transgenic mice, designated LIKK, by selectively expressing constitutively active IKK-b in hepatocytes. These mice exhibit a type 2 diabetes phenotype, characterized by hyperglycemia, profound hepatic insulin resistance, and moderate systemic insulin resistance, including effects in muscle. The hepatic production of proinflammatory cytokines, including IL-6, IL-1beta and TNF-alpha, was increased in LIKK mice to a similar extent as induced by HFD in in wild-type mice. Parallel increases were observed in cytokine signaling in liver and mucscle of LIKK mice. Insulin resistance was improved by systemic neutralization of IL-6 or salicylate inhibition of IKK-beta. Hepatic expression of the IkappaBalpha superrepressor (LISR) reversed the phenotype of both LIKK mice and wild-type mice fed an HFD. These findings indicate that lipid accumulation in the liver leads to subacute hepatic 'inflammation' through NF-kappaB activation and downstream cytokine production. This causes insulin resistance both locally in liver and systemically.


Genome-wide maps of histone modifications unwind in vivo chromatin states of the hair follicle lineage.

Abstract: Using mouse skin, where bountiful reservoirs of synchronized hair follicle stem cells (HF-SCs) fuel cycles of regeneration, we explore how adult SCs remodel chromatin in response to activating cues. By profiling global mRNA and chromatin changes in quiescent and activated HF-SCs and their committed, transit-amplifying (TA) progeny, we show that polycomb-group (PcG)-mediated H3K27-trimethylation features prominently in HF-lineage progression by mechanisms distinct from embryonic-SCs. In HF-SCs, PcG represses nonskin lineages and HF differentiation. In TA progeny, nonskin regulators remain PcG-repressed, HF-SC regulators acquire H3K27me3-marks, and HF-lineage regulators lose them. Interestingly, genes poised in embryonic stem cells, active in HF-SCs, and PcG-repressed in TA progeny encode not only key transcription factors, but also signaling regulators. We document their importance in balancing HF-SC quiescence, underscoring the power of chromatin mapping in dissecting SC behavior. Our findings explain how HF-SCs cycle through quiescent and activated states without losing stemness and define roles for PcG-mediated repression in governing a fate switch irreversibly.


Two classes of endogenous small RNAs in Tetrahymena thermophila.

Abstract: Endogenous small RNAs function in RNA interference (RNAi) pathways to guide RNA cleavage, translational repression, or methylation of DNA or chromatin. In Tetrahymena thermophila, developmentally regulated DNA elimination is governed by an RNAi mechanism involving approximately 27-30-nucleotide (nt) RNAs. Here we characterize the sequence features of the approximately 27-30-nt RNAs and a approximately 23-24-nt RNA class representing a second RNAi pathway. The approximately 23-24-nt RNAs accumulate strain-specifically manner and map to the genome in clusters that are antisense to predicted genes. These findings reveal the existence of distinct endogenous RNAi pathways in the unicellular T. thermophila, a complexity previously demonstrated only in multicellular organisms.


The binding of maize DHN1 to lipid vesicles. Gain of structure and lipid specificity.

Abstract: Dehydrins (DHNs; late embryogenesis abundant D-11) are a family of plant proteins induced in response to abiotic stresses such as drought, low temperature, and salinity or during the late stages of embryogenesis. Spectral and thermal properties of these proteins in purified form suggest that they are "intrinsically unstructured. " However, DHNs contain at least one copy of a consensus 15-amino acid sequence, the "K segment," which resembles a class A2 amphipathic alpha-helical, lipid-binding domain found in other proteins such as apolipoproteins and alpha-synuclein. The presence of the K segment raises the question of whether DHNs bind lipids, bilayers, or phospholipid vesicles. Here, we show that maize (Zea mays) DHN DHN1 can bind to lipid vesicles that contain acidic phospholipids. We also observe that DHN1 binds more favorably to vesicles of smaller diameter than to larger vesicles, and that the association of DHN1 with vesicles results in an apparent increase of alpha-helicity of the protein. Therefore, DHNs, and presumably somewhat similar plant stress proteins in the late embryogenesis abundant and cold-regulated classes may undergo function-related conformational changes at the water/membrane interface, perhaps related to the stabilization of vesicles or other endomembrane structures under stress conditions.


Gene expression in human skeletal muscle: alternative normalization method and effect of repeated biopsies

Abstract: The reverse transcriptase-polymerase chain reaction (RT-PCR) method has lately become widely used to determine transcription and mRNA content in rodent and human muscle samples. However, the common use of endogenous controls for correcting for variance in cDNA between samples is not optimal. Specifically, we investigated (1) a new normalization method based on determining the cDNA content by the flourophores PicoGreen and OliGreen, (2) effect of repeated muscle biopsies on mRNA gene expression, and (3) the spatial heterogeneity in mRNA expression across the muscle. Standard curves using oligo standards revealed a high degree of sensitivity and linearity (2.5–45 ng; R 2>0.99) with OliGreen reagent, as was the case for OliGreen analyses with standard curves constructed from serial dilutions of representative RT samples (R 2 >0.99 for a ten times dilution range of a representative reversed transcribed (RT) sample). Likewise, PicoGreen reagent detected the RNA:DNA hybrid content in RT samples with great sensitivity. Standard curves constructed from both double-stranded lambda DNA (1–10 ng) and from serial dilutions of representative RT samples consistently resulted in linearity with R 2 >0.99. The present determination of cDNA content in reversed transcribed human skeletal muscle RNA samples by both PicoGreen and OliGreen analyses suggests that these fluorophores provide a potential alternative normalization procedure for human gene expression studies. In addition, the present study shows that multiple muscle biopsies obtained from the same muscle do not influence the mRNA response induced by an acute exercise bout for any of the genes examined.


Ionizing radiation-induced long-term expression of senescence markers in mice is independent of p53 and immune status.

Abstract: Exposure to IR has been shown to induce the formation of senescence markers, a phenotype that coincides with lifelong delayed repair and regeneration of irradiated tissues. We hypothesized that IR-induced senescence markers could persist long-term in vivo, possibly contributing to the permanent reduction in tissue functionality. Here, we show that mouse tissues exposed to a sublethal dose of IR display persistent (up to 45 weeks, the maximum time analyzed) DNA damage foci and increased p16(INK4a) expression, two hallmarks of cellular senescence and aging. BrdU-labeling experiments revealed that IR-induced damaged cells are preferentially eliminated, at least partially, in a tissue-dependent manner. Unexpectedly, the accumulation of damaged cells was found to occur independent from the DNA damage response modulator p53, and from an intact immune system, as their levels were similar in wild-type and Rag2(-/-) gammaC(-/-) mice, the latter being deficient in T, B, and NK cells. Together, our results provide compelling evidence that exposure to IR induces long-term expression of senescence markers in vivo, an effect that may contribute to the reduced tissue functionality observed in cancer survivors.


Tamoxifen resistance in MCF7 cells promotes EMT-like behaviour and involves modulation of beta-catenin phosphorylation.

Abstract: We have previously demonstrated that, following acquisition of endocrine resistance, breast cancer cells display an altered growth rate together with increased aggressive behaviour in vitro. Since dysfunctional cell-cell adhesive interactions can promote an aggressive phenotype, we investigated the integrity of this protein complex in our breast cancer model of tamoxifen resistance. In culture, tamoxifen-resistant MCF7 (TamR) cells grew as loosely packed colonies with loss of cell-cell junctions and demonstrated altered morphology characteristic of cells undergoing epithelial-to-mesenchymal transition (EMT). Neutralising E-cadherin function promoted the invasion and inhibited the aggregation of endocrine-sensitive MCF7 cells, whilst having little effect on the behaviour of TamR cells. Additionally, TamR cells had increased levels of tyrosine-phosphorylated beta-catenin, whilst serine/threonine-phosphorylated beta-catenin was decreased. These cells also displayed loss of association between beta-catenin and E-cadherin, increased cytoplasmic and nuclear beta-catenin and elevated transcription of beta-catenin target genes known to be involved in tumour progression and EMT. Inhibition of EGFR kinase activity in TamR cells reduced beta-catenin tyrosine phosphorylation, increased beta-catenin-E-cadherin association and promoted cell-cell adhesion. In such treated cells, the association of beta-catenin with Lef-1 and the transcription of c-myc, cyclin-D1, CD44 and COX-2 were also reduced. These results suggest that homotypic adhesion in tamoxifen-resistant breast cancer cells is dysfunctional due to EGFR-driven modulation of the phosphorylation status of beta-catenin and may contribute to an enhanced aggressive phenotype and transition towards a mesenchymal phenotype in vitro.


Expression profiles of Toll-like receptors in non-small cell lung cancer and idiopathic pulmonary fibrosis.

Abstract: Patients with idiopathic pulmonary fibrosis (IPF) have a higher incidence             of lung cancer. The role of Toll-like receptors (TLRs), a key component of the             innate immunity, in interstitial lung diseases (ILDs) and lung cancer pathogenesis             is not clarified. TLR2, TLR3, TLR4, TLR7, TLR8 and TLR9 mRNA expression was quantitatively             measured by real-time reverse transcriptase polymerase chain reaction (RT-PCR)             in bronchoalveolar lavage fluid (BALF) of 16 IPF patients, 16 non-small cell lung             cancer (NSCLC) patients and 9 control subjects. TLR2, TLR3, TLR4 and TLR9 protein             expression was assessed on BALF T-lymphocytes using flow cytometry. TLR3 mRNA             expression was significantly higher in NSCLC compared to IPF (p=0.023) and controls             (p=0.001). TLR7 mRNA expression levels were significantly higher in both NSCLC             and IPF groups compared to controls (p=0.029, p=0.009). TLR9 expression at the             mRNA level was significantly higher in both NSCLC and IPF groups compared to controls             (p=0.01, p=0.001). Finally, TLR2 mRNA expression was significantly higher in IPF             patients compared to controls (p=0.042). Flow cytometry revealed decreased TLR3             and TLR9 expression in IPF patients compared to the NSCLC group (p=0.02, p=0.014)             and decreased TLR9 expression in IPF compared with the controls (p=0.04). TLR2             protein expression was significantly higher in IPF patients compared to NSCLC             (p=0.04). Increased expression of endosomal TLRs in NSCLC patients and elevated             expression of TLR2 in pulmonary fibrosis are the main results of this study. These             results do not provide support for a common TLR pathway hypothesis between NSCLC             and IPF.


A rare mutation in UNC5C predisposes to late-onset Alzheimer's disease and increases neuronal cell death

Abstract: We have identified a rare coding mutation, T835M (rs137875858), in the UNC5C netrin receptor gene that segregated with disease in an autosomal dominant pattern in two families enriched for late-onset Alzheimer's disease and that was associated with disease across four large case-control cohorts (odds ratio = 2.15, Pmeta = 0.0095). T835M alters a conserved residue in the hinge region of UNC5C, and in vitro studies demonstrate that this mutation leads to increased cell death in human HEK293T cells and in rodent neurons. Furthermore, neurons expressing T835M UNC5C are more susceptible to cell death from multiple neurotoxic stimuli, including β-amyloid (Aβ), glutamate and staurosporine. On the basis of these data and the enriched hippocampal expression of UNC5C in the adult nervous system, we propose that one possible mechanism in which T835M UNC5C contributes to the risk of Alzheimer's disease is by increasing susceptibility to neuronal cell death, particularly in vulnerable regions of the Alzheimer's disease brain.