Changes in plasma copeptin, the c-terminal portion of arginine vasopressin during water deprivation and excess in healthy subjects.

Abstract:
CONTEXT The measurement of arginine vasopressin (AVP) is often cumbersome because it is unstable with a short half-life time. AVP is derived from a larger precursor peptide along with the more stable peptide copeptin. Copeptin is the C-terminal part of provasopressin and has been shown to be a useful tool to indicate AVP concentration in critically ill patients.
 OBJECTIVE The objective of the study was to evaluate the clinical usefulness of copeptin as a new marker in disordered states of blood volume and plasma osmolality.
 DESIGN AND SETTING This was a prospective observational study in a university hospital.
 PARTICIPANTS AND MAIN OUTCOME MEASURES Three techniques with respective control studies were used in 24 healthy adults to produce changes in plasma osmolality and/or volume: 1) a 28-h water deprivation, 2) a 17-h hypertonic saline infusion combined with thirsting, and 3) a hypotonic saline infusion with iv desmopressin administration during free water intake.
 RESULTS Water deprivation produced a weight loss of 1.7 kg, an increase in plasma osmolality to 294.8 +/- 4.3 mosmol/kg, and an increase of copeptin from 4.6 +/- 1.7 pmol/liter to 9.2 +/- 5.2 pmol/liter (P < 0.0001). During hypertonic saline infusion and thirsting with a raise of plasma osmolality to 296.1 +/- 3.4 mosmol/kg, copeptin increased from 4.9 +/- 3.0 pmol/liter to 19.9 +/- 4.8 pmol/liter (P < 0.0001). Conversely, during hypotonic saline infusion, plasma osmolality decreased to 271.3 +/- 4.1 mosmol/kg, and copeptin decreased from 6.2 +/- 2.4 pmol/liter to 2.4 +/- 2.1 pmol/liter (P < 0.01).
 CONCLUSION Copeptin shows identical changes during disordered water states as previously shown for AVP. It might be a reliable marker of AVP secretion and substitute for the measurement of circulating AVP levels in clinical routine.


Propagation and recovery of intact, infectious Epstein-Barr virus from prokaryotic to human cells.

Abstract: With current techniques, genetic alterations of herpesviruses are difficult to perform, mostly because of the large size of their genomes. To solve this problem, we have designed a system that allows the cloning of any gamma-herpesvirus in Escherichia coli onto an F factor-derived plasmid. Immortalized B cell lines were readily established with recombinant Epstein-Barr virus (EBV), demonstrating that the F factor-cloned EBV genome has all the characteristics of wild-type EBV. Because any genetic modification is possible in E. coli, this experimental approach opens the way to the genetic analysis of all EBV functions. Moreover, it is now feasible to generate attenuated EBV strains in vitro such that vaccine strains can be designed. Because we incorporated the genes for hygromycin resistance and green fluorescent protein onto the E. coli cloned EBV genome, the still open question of the EBV target cells other than B lymphocytes will be addressed.


Chemosensitivity profile assay of circulating cancer cells: prognostic and predictive value in epithelial tumors.

Abstract: The prognostic value associated with the detection of circulating tumor cells (CTCs) in metastatic breast cancer by the CellSearch technology raise additional issues regarding the biological value of this information. We postulated that a drug-resistance profile of CTCs may predict response to chemotherapy in cancer patients and therefore could be used for patient selection. One hundred 5 patients with diagnosis of carcinoma were enrolled in a prospective trial. CTCs were isolated from peripheral blood, and positive samples were evaluated for the expression of a panel of genes involved in anticancer drugs resistance. The drug-resistance profile was correlated with disease-free survival (DFS; patients in adjuvant setting) and time to progression (TTP; metastatic patients) in a 24-months follow-up. Objective response correlation was a secondary end point. Fifty-one percent of patients were found positive for CTCs while all blood samples from healthy donors were negative. The drug-resistance profile correlates with DFS and TTP (p < 0.001 in both). Sensitivity of the test: able to predict treatment response in 98% of patients. Specificity of the test: 100%; no sample from healthy subject was positive for the presence of CTCs. Positive and negative predictive values were found to be 96.5 and 100%, respectively. We identified a drug-resistance profile of CTCs, which is predictive of response to chemotherapy, independent of tumor type and stage of disease. This approach may represent a first step toward the individualization of chemotherapy in cancer patients.


IFI16 and cGAS cooperate in the activation of STING during DNA sensing in human keratinocytes

Abstract: Many human cells can sense the presence of exogenous DNA during infection though the cytosolic DNA receptor cyclic GMP-AMP synthase (cGAS), which produces the second messenger cyclic GMP-AMP (cGAMP). Other putative DNA receptors have been described, but whether their functions are redundant, tissue-specific or integrated in the cGAS-cGAMP pathway is unclear. Here we show that interferon-γ inducible protein 16 (IFI16) cooperates with cGAS during DNA sensing in human keratinocytes, as both cGAS and IFI16 are required for the full activation of an innate immune response to exogenous DNA and DNA viruses. IFI16 is also required for the cGAMP-induced activation of STING, and interacts with STING to promote STING phosphorylation and translocation. We propose that the two DNA sensors IFI16 and cGAS cooperate to prevent the spurious activation of the type I interferon response.


Oncostatic effects of the indole melatonin and expression of its cytosolic and nuclear receptors in cultured human melanoma cell lines.

Abstract: Melatonin has been shown to have oncostatic effects on malignant melanoma in vitro and in vivo. We studied the growth suppressive effects of melatonin over a wide range of concentrations in four melanoma cell lines (SBCE2, WM-98, WM-164 and SKMEL-188) representative for different growth stages and phenotype. Melanoma cells were incubated with melatonin 10(-12)-10(-3) M, and proliferation and clonogenicity was assessed at 12 h and 14 days, respectively. We also determined the expression of cytosolic quinone oxidoreductases NQO1, NQO2 (known as MT3 receptor) and nuclear receptor RORalpha by RT-PCR. Melatonin at pharmacological concentrations (10(-3)-10(-7) M) suppressed proliferation in all melanoma cell lines. In SKMEL-188 cells cultured in serum-free media, melatonin at low concentrations (10(-12)-10(-10) M) also slightly attenuated the proliferation. The effects of pharmacological doses of melatonin were confirmed in the clonogenic assay. Expression of NQO1 was detected in all cell lines, whereas NQO2 and nuclear receptor RORalpha including its isoform RORalpha4 were present only in SBCE2, WM-164 and WM-98. Thus, melatonin differentially suppressed proliferation in melanoma cell lines of different behaviour. The intensity of the oncostatic response to melatonin could be related to the cell-line specific pattern of melatonin cellular receptors and cytosolic binding protein expression.


Lysosomal localization of TRPML3 depends on TRPML2 and the mucolipidosis-associated protein TRPML1.

Abstract: Mucolipidosis type IV is an autosomal recessive lysosomal storage disorder characterized by severe neurodegeneration, achlorhydria, and visual impairments such as corneal opacity and strabismus. The disease arises due to mutations in a group 2 transient receptor potential (TRP)-related cation channel, TRPML1. Mammals encode two additional TRPML proteins named TRPML2 and TRPML3. Information regarding the propensity of these proteins to multimerize, their subcellular distribution and mechanisms that regulate their trafficking are limited. Here we demonstrate that TRPMLs interact to form homo- and heteromultimers. Moreover, the presence of either TRPML1 or TRPML2 specifically influences the spatial distribution of TRPML3. TRPML1 and TRPML2 homomultimers are lysosomal proteins, whereas TRPML3 homomultimers are in the endoplasmic reticulum. However, TRPML3 localizes to lysosomes when coexpressed with either TRPML1 or TRPML2 and is comparably mislocalized when lysosomal targeting of TRPML1 and TRPML2 is disrupted. Conversely, TRPML3 does not cause retention of TRPML1 or TRPML2 in the endoplasmic reticulum. These data demonstrate that there is a hierarchy controlling the subcellular distributions of the TRPMLs such that TRPML1 and TRPML2 dictate the localization of TRPML3 and not vice versa.


Evolution of metastasis revealed by mutational landscapes of chemically induced skin cancers

Abstract: Human tumors show a high level of genetic heterogeneity, but the processes that influence the timing and route of metastatic dissemination of the subclones are unknown. Here we have used whole-exome sequencing of 103 matched benign, malignant and metastatic skin tumors from genetically heterogeneous mice to demonstrate that most metastases disseminate synchronously from the primary tumor, supporting parallel rather than linear evolution as the predominant model of metastasis. Shared mutations between primary carcinomas and their matched metastases have the distinct A-to-T signature of the initiating carcinogen dimethylbenzanthracene, but non-shared mutations are primarily G-to-T, a signature associated with oxidative stress. The existence of carcinomas that either did or did not metastasize in the same host animal suggests that there are tumor-intrinsic factors that influence metastatic seeding. We also demonstrate the importance of germline polymorphisms in determining allele-specific mutations, and we identify somatic genetic alterations that are specifically related to initiation of carcinogenesis by Hras or Kras mutations. Mouse tumors that mimic the genetic heterogeneity of human cancers can aid our understanding of the clonal evolution of metastasis and provide a realistic model for the testing of novel therapies.


A simple two-step protocol for the purification of human pancreatic beta cells

Abstract: Isolated pure human beta cells would be helpful for a number of research purposes. However, lack of beta cell-specific surface antigens has been a major problem. We aimed to develop a simple method for human beta cell isolation based on the initial elimination of ductal cells by their expression of carbohydrate antigen 19-9 (CA19-9), followed by positive selection of beta cells by their expression of polysialic acid–neural cell adhesion molecule (PSA-NCAM). Cell type-specific expression of CA19-9, NCAM and PSA-NCAM was studied in sections of adult human pancreas and in cultured primary endocrine and exocrine cells. Dispersed human islet cells were purified in two steps, after 4 days of suspension culture, by binding to magnetic microbeads coupled to antibodies against CA19-9 and PSA-NCAM. NCAM expression was detected in ducts and islets in the human pancreas. In contrast, PSA-NCAM immunoreactivity was detected only in islets. PSA-NCAM staining in dispersed cells revealed that the marker is expressed in all endocrine cell types, but not in duct cells. Purification of dispersed islet cells using PSA-NCAM microbeads alone did not completely eliminate contaminating duct cells. However, elimination of the duct cells by CA19-9 microbeads followed by positive sorting of the PSA-NCAM-positive cells in five consecutive islet preparations resulted in 90 to 98% pure endocrine cells, of which 89 to 97% were beta cells. We describe a simple and reproducible method for purification of viable human pancreatic beta cells devoid of exocrine acini and ducts.


Ascorbic-acid transporter Slc23a1 is essential for vitamin C transport into the brain and for perinatal survival

Abstract: The only proven requirement for ascorbic acid (vitamin C) is in preventing scurvy, presumably because it is a cofactor for hydroxylases required for post-translational modifications that stabilize collagen. We have created mice deficient in the mouse ortholog (solute carrier family 23 member 1 or Slc23a1) of a rat ascorbic-acid transporter, Svct2 (ref. 4). Cultured embryonic fibroblasts from homozygous Slc23a1−/− mice had less than 5% of normal ascorbic-acid uptake. Ascorbic-acid levels were undetectable or markedly reduced in the blood and tissues of Slc23a1−/− mice. Prenatal supplementation of pregnant females did not elevate blood ascorbic acid in Slc23a1−/− fetuses, suggesting Slc23a1 is important in placental ascorbic-acid transport. Slc23a1−/− mice died within a few minutes of birth with respiratory failure and intraparenchymal brain hemorrhage. Lungs showed no postnatal expansion but had normal surfactant protein B levels. Brain hemorrhage was unlikely to be simply a form of scurvy since Slc23a1−/− mice showed no hemorrhage in any other tissues and their skin had normal skin 4-hydroxyproline levels despite low ascorbic-acid content. We conclude that Slc23a1 is required for transport of ascorbic acid into many tissues and across the placenta. Deficiency of the transporter is lethal in newborn mice, thereby revealing a previously unrecognized requirement for ascorbic acid in the perinatal period.


MVP-mediated exosomal sorting of miR-193a promotes colon cancer progression

Abstract: Exosomes are emerging mediators of intercellular communication; whether the release of exosomes has an effect on the exosome donor cells in addition to the recipient cells has not been investigated to any extent. Here, we examine different exosomal miRNA expression profiles in primary mouse colon tumour, liver metastasis of colon cancer and naive colon tissues. In more advanced disease, higher levels of tumour suppressor miRNAs are encapsulated in the exosomes. miR-193a interacts with major vault protein (MVP). Knockout of MVP leads to miR-193a accumulation in the exosomal donor cells instead of exosomes, inhibiting tumour progression. Furthermore, miR-193a causes cell cycle G1 arrest and cell proliferation repression through targeting of Caprin1, which upregulates Ccnd2 and c-Myc. Human colon cancer patients with more advanced disease show higher levels of circulating exosomal miR-193a. In summary, our data demonstrate that MVP-mediated selective sorting of tumour suppressor miRNA into exosomes promotes tumour progression.


Casual blood pressure and neurocognitive function in children with chronic kidney disease: a report of the children with chronic kidney disease cohort study.

Abstract:
BACKGROUND AND OBJECTIVES Children with chronic kidney disease (CKD) are at risk for cognitive dysfunction, and over half have hypertension. Data on the potential contribution of hypertension to CKD-associated neurocognitive deficits in children are limited. Our objective was to determine whether children with CKD and elevated BP (EBP) had decreased performance on neurocognitive testing compared with children with CKD and normal BP.
 DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS This was a cross-sectional analysis of the relation between auscultatory BP and neurocognitive test performance in children 6 to 17 years enrolled in the Chronic Kidney Disease in Children (CKiD) project.
 RESULTS Of 383 subjects, 132 (34%) had EBP (systolic BP and/or diastolic BP ≥90(th) percentile). Subjects with EBP had lower mean (SD) scores on Wechsler Abbreviated Scales of Intelligence (WASI) Performance IQ than those with normal BP (normal BP versus EBP, 96.1 (16.7) versus 92.4 (14.9), P = 0.03) and WASI Full Scale IQ (97.0 (16.2) versus 93.4 (16.5), P = 0.04). BP index (subject's BP/95(th) percentile BP) correlated inversely with Performance IQ score (systolic, r = -0.13, P = 0.01; diastolic, r = -0.19, P < 0.001). On multivariate analysis, the association between lower Performance IQ score and increased BP remained significant after controlling for demographic and disease-related variables (EBP, β = -3.7, 95% confidence interval [CI]: -7.3 to -0.06; systolic BP index, β = -1.16 to 95% CI: -2.1, -0.21; diastolic BP index, β = -1.17, 95% CI: -1.8 to -0.55).
 CONCLUSIONS Higher BP was independently associated with decreased WASI Performance IQ scores in children with mild-to-moderate CKD.


MicroRNA-192 in diabetic kidney glomeruli and its function in TGF-beta-induced collagen expression via inhibition of E-box repressors.

Abstract: Key features of diabetic nephropathy (DN) include the accumulation of extracellular matrix proteins such as collagen 1-alpha 1 and -2 (Col1a1 and -2). Transforming growth factor beta1 (TGF-beta), a key regulator of these extracellular matrix genes, is increased in mesangial cells (MC) in DN. By microarray profiling, we noted that TGF-beta increased Col1a2 mRNA in mouse MC (MMC) but also decreased mRNA levels of an E-box repressor, deltaEF1. TGF-beta treatment or short hairpin RNAs targeting deltaEF1 increased enhancer activity of upstream E-box elements in the Col1a2 gene. TGF-beta also decreased the expression of Smad-interacting protein 1 (SIP1), another E-box repressor similar to deltaEF1. Interestingly, we noted that SIP1 is a target of microRNA-192 (miR-192), a key miR highly expressed in the kidney. miR-192 levels also were increased by TGF-beta in MMC. TGF-beta treatment or transfection with miR-192 decreased endogenous SIP1 expression as well as reporter activity of a SIP1 3' UTR-containing luciferase construct in MMC. Conversely, a miR-192 inhibitor enhanced the luciferase activity, confirming SIP1 to be a miR-192 target. Furthermore, miR-192 synergized with deltaEF1 short hairpin RNAs to increase Col1a2 E-box-luc activity. Importantly, the in vivo relevance was noted by the observation that miR-192 levels were enhanced significantly in glomeruli isolated from streptozotocin-injected diabetic mice as well as diabetic db/db mice relative to corresponding nondiabetic controls, in parallel with increased TGF-beta and Col1a2 levels. These results uncover a role for miRs in the kidney and DN in controlling TGF-beta-induced Col1a2 expression by down-regulating E-box repressors.


Activation of PAR(2) receptors sensitizes primary afferents and causes leukocyte rolling and adherence in the rat knee joint.

Abstract:
BACKGROUND AND PURPOSE The PAR(2) receptors are involved in chronic arthritis by mechanisms that are as yet unclear. Here, we examined PAR(2) activation in the rat knee joint.    EXPERIMENTAL APPROACH PAR(2) in rat knee joint dorsal root ganglia (DRG) cells at L3-L5, retrogradely labelled with Fluoro-gold (FG) were demonstrated immunohistochemically. Electrophysiological recordings from knee joint nerve fibres in urethane anaesthetized Wistar rats assessed the effects of stimulating joint PAR(2) with its activating peptide, 2-furoyl-LIGRLO-NH(2) (1-100 nmol·100 μL(-1) , via close intra-arterial injection). Fibre firing rate was recorded during joint rotations before and 15 min after administration of PAR(2) activating peptide or control peptide. Leukocyte kinetics in the synovial vasculature upon PAR(2) activation were followed by intravital microscopy for 60 min after perfusion of 2-furoyl-LIGRLO-NH(2) or control peptide. Roles for transient receptor potential vanilloid-1 (TRPV1) or neurokinin-1 (NK(1) ) receptors in the PAR(2) responses were assessed using the selective antagonists, SB366791 and RP67580 respectively.    KEY RESULTS PAR(2) were expressed in 59 ± 5% of FG-positive DRG cells; 100 nmol 2-furoyl-LIGRLO-NH(2) increased joint fibre firing rate during normal and noxious rotation, maximal at 3 min (normal; 110 ± 43%, noxious; 90 ± 31%). 2-Furoyl-LIGRLO-NH(2) also significantly increased leukocyte rolling and adhesion over 60 min. All these effects were blocked by pre-treatment with SB366791 and RP67580 (P < 0.05 compared with 2-furoyl-LIGRLO-NH(2) alone).
 CONCLUSIONS AND IMPLICATIONS PAR(2) receptors play an acute inflammatory role in the knee joint via TRPV1- and NK(1) -dependent mechanisms involving both PAR(2) -mediated neuronal sensitization and leukocyte trafficking.


Polyphosphate kinase 1, a central node in the stress response network of Mycobacterium tuberculosis, connects the two-component systems MprAB and SenX3-RegX3 and the extracytoplasmic function sigma factor, sigma E.

Abstract: Polyphosphate (poly P) metabolism regulates the stress response in mycobacteria. Here we describe the regulatory architecture of a signal transduction system involving the two-component system (TCS) SenX3-RegX3, the extracytoplasmic function sigma factor sigma E (SigE) and the poly P-synthesizing enzyme polyphosphate kinase 1 (PPK1). The ppk1 promoter of Mycobacterium tuberculosis is activated under phosphate starvation. This is attenuated upon deletion of an imperfect palindrome likely representing a binding site for the response regulator RegX3, a component of the two-component system SenX3-RegX3 that responds to phosphate starvation. Binding of phosphorylated RegX3 to this site was confirmed by electrophoretic mobility shift assay. The activity of the ppk1 promoter was abrogated upon deletion of a putative SigE binding site. Pull-down of SigE from M. tuberculosis lysates of phosphate-starved cells with a biotinylated DNA harbouring the SigE binding site confirmed the likely binding of SigE to the ppk1 promoter. In vitro transcription corroborated the involvement of SigE in ppk1 transcription. Finally, the overexpression of RseA (anti-SigE) attenuated ppk1 expression under phosphate starvation, supporting the role of SigE in ppk1 transcription. The regulatory elements identified in ppk1 transcription in this study, combined with our earlier observation that PPK1 is itself capable of regulating sigE expression via the MprAB TCS, suggest the presence of multiple positive-feedback loops in this signalling circuit. In combination with the sequestering effect of RseA, we hypothesize that this architecture could be linked to bistability in the system that, in turn, could be a key element of persistence in M. tuberculosis.


Natural history of asymptomatic unruptured cerebral aneurysms evaluated at CT angiography: growth and rupture incidence and correlation with epidemiologic risk factors.

Abstract:
PURPOSE To characterize the relationship between aneurysm size and epidemiologic risk factors with growth and rupture by using computed tomographic (CT) angiography.    MATERIALS AND METHODS In this HIPAA-compliant, institutional review board approved study, patients with known asymptomatic unruptured intracerebral aneurysms were followed up longitudinally with CT angiographic examinations. Growth was defined as an increase in one or more dimensions above the measurement error, and at least 5% volume by using the ABC/2 method. Associations of epidemiologic factors with aneurysm growth and rupture were analyzed by using logistic regression analysis. Intra- and interobserver agreement coefficients for dimension, volume, and growth were evaluated by using the Pearson correlation coefficient and difference of means with 95% confidence intervals, the agreement statistic, and the McNemar χ(2).
 RESULTS Patients (n = 165) with aneurysms (n = 258) had a mean follow-up time of 2.24 years from time of diagnosis. Forty-six of 258 (18%) aneurysms in 38 patients grew larger. Spontaneous rupture occurred in four of 228 (1.8%) intradural aneurysms of average size (6.2 mm). Risk of aneurysm rupture per patient-year was 2.4% (95% CI: 0.5%, 7.12%) with growth and 0.2% (95% CI: 0.006%, 1.22%) without growth (P = .034). There was a 12-fold higher risk of rupture for growing aneurysms (P < .002), with high intra- and interobserver correlation coefficients for size, volume, and growth. Tobacco smoking (3.806, one degree of freedom; P < .015,) and initial size (5.895, two degrees of freedom; P < .051) were independent covariates, predicting 78.4% of growing aneurysms.
 CONCLUSION These results support imaging follow-up of all patients with aneurysms, including those whose aneurysms are smaller than the current 7-mm treatment threshold. Aneurysm growth, size, and smoking were associated with increased rupture risk.


A search for the presence of the enteroviral capsid protein VP1 in pancreases of patients with Type 1 (insulin-dependent) diabetes and pancreases and hearts of infants who died of coxsackieviral myocarditis

Abstract: Using an antiserum raised to a recombinant coxsackie virus B3 capsid protein, VP1, an immunocytochemical technique was developed which was capable of detecting the presence of all coxsackie B viruses in formalin fixed paraffin embedded infected tissue culture cells. This technique was tested on autopsy heart and pancreas from 21 patients who were thought to have died of acute coxsackievirus B myocarditis. Cardiac myocytes were positive for the VP1 protein in 12 of 20 cases where the heart was available for study. Insulitis was present in the pancreas in seven of these cases and in all seven islet endocrine cells containing VP1 were found. VP1 was only rarely found in exocrine pancreas. In heart and pancreas, cells shown to contain VP1 usually showed signs of necrosis. Autopsy pancreases from 88 patients who had died at clinical presentation of Type 1 (insulin-dependent) diabetes mellitus showed no evidence of the presence of VP1. The continuing destruction of insulin-secreting B cells seen at the time of death in the diabetic pancreas is unlikely to be due to a direct cytopathic effect of a coxsackie B virus. However, this study does not exclude the possibility that a persistent infection of B cells by a defective enterovirus may result in their destruction by an autoimmune mechanism.


Direct reprogramming of mouse fibroblasts to cardiomyocyte-like cells using Yamanaka factors on engineered poly(ethylene glycol) (PEG) hydrogels.

Abstract: Direct reprogramming strategies enable rapid conversion of somatic cells to cardiomyocytes or cardiomyocyte-like cells without going through the pluripotent state. A recently described protocol couples Yamanaka factor induction with pluripotency inhibition followed by BMP4 treatment to achieve rapid reprogramming of mouse fibroblasts to beating cardiomyocyte-like cells. The original study was performed using Matrigel-coated tissue culture polystyrene (TCPS), a stiff material that also non-specifically adsorbs serum proteins. Protein adsorption-resistant poly(ethylene glycol) (PEG) materials can be covalently modified to present precise concentrations of adhesion proteins or peptides without the unintended effects of non-specifically adsorbed proteins. Here, we describe an improved protocol that incorporates custom-engineered materials. We first reproduced the Efe et al. protocol on Matrigel-coated TCPS (the original material), reprogramming adult mouse tail-tip mouse fibroblasts (TTF) and mouse embryonic fibroblasts (MEF) to cardiomyocyte-like cells that demonstrated striated sarcomeric α-actinin staining, spontaneous calcium transients, and visible beating. We then designed poly(ethylene glycol) culture substrates to promote MEF adhesion via laminin and RGD-binding integrins. PEG hydrogels improved proliferation and reprogramming efficiency (evidenced by beating patch number and area, gene expression, and flow cytometry), yielding almost twice the number of sarcomeric α-actinin positive cardiomyocyte-like cells as the originally described substrate. These results illustrate that cellular reprogramming may be enhanced using custom-engineered materials.


Loss of fibronectin from the aged stem cell niche affects the regenerative capacity of skeletal muscle in mice

Abstract: Age-related changes in the niche have long been postulated to impair the function of somatic stem cells. Here we demonstrate that the aged stem cell niche in skeletal muscle contains substantially reduced levels of fibronectin (FN), leading to detrimental consequences for the function and maintenance of muscle stem cells (MuSCs). Deletion of the gene encoding FN from young regenerating muscles replicates the aging phenotype and leads to a loss of MuSC numbers. By using an extracellular matrix (ECM) library screen and pathway profiling, we characterize FN as a preferred adhesion substrate for MuSCs and demonstrate that integrin-mediated signaling through focal adhesion kinase and the p38 mitogen-activated protein kinase pathway is strongly de-regulated in MuSCs from aged mice because of insufficient attachment to the niche. Reconstitution of FN levels in the aged niche remobilizes stem cells and restores youth-like muscle regeneration. Taken together, we identify the loss of stem cell adhesion to FN in the niche ECM as a previously unknown aging mechanism.


Why do patients with multimorbidity in England report worse experiences in primary care? Evidence from the General Practice Patient Survey

Abstract:
OBJECTIVES To describe and explain the primary care experiences of people with multiple long-term conditions in England.
 DESIGN AND METHODS Using questionnaire data from 906,578 responders to the English 2012 General Practice Patient Survey, we describe the primary care experiences of patients with long-term conditions, including 583,143 patients who reported one or more long-term conditions. We employed mixed effect logistic regressions to analyse data on six items covering three care domains (access, continuity and communication) and a single item on overall primary care experience. We controlled for sociodemographic characteristics, and for general practice using a random effect, and further, controlled for, and explored the importance of, health-related quality of life measured using the EuroQoL (EQ-5D) scale.
 RESULTS Most patients with long-term conditions report a positive experience of care at their general practice (after adjusting for sociodemographic characteristics and general practice, range 74.0-93.1% reporting positive experience of care across seven questions) with only modest variation by type of condition. For all three domains of patient experience, an increasing number of comorbid conditions is associated with a reducing percentage of patients reporting a positive experience of care. For example, compared with respondents with no long-term condition, the OR for reporting a positive experience is 0.83 (95% CI 0.80 to 0.87) for respondents with four or more long-term conditions. However, this relationship is no longer observed after adjusting for health-related quality of life (OR (95% CI) single condition=1.23 (1.21 to 1.26); four or more conditions=1.31 (1.25 to 1.37)), with pain making the greatest difference among five quality of life variables included in the analysis.
 CONCLUSIONS Patients with multiple long-term conditions more frequently report worse experiences in primary care. However, patient-centred measures of health-related quality of life, especially pain, are more important than the number of conditions in explaining why patients with multiple long-term conditions report worse experiences of care.


The rice FISH BONE gene encodes a tryptophan aminotransferase, which affects pleiotropic auxin-related processes.

Abstract: Auxin is a fundamental plant hormone and its localization within organs plays pivotal roles in plant growth and development. Analysis of many Arabidopsis mutants that were defective in auxin biosynthesis revealed that the indole-3-pyruvic acid (IPA) pathway, catalyzed by the TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA) and YUCCA (YUC) families, is the major biosynthetic pathway of indole-3-acetic acid (IAA). In contrast, little information is known about the molecular mechanisms of auxin biosynthesis in rice. In this study, we identified a auxin-related rice mutant, fish bone (fib). FIB encodes an orthologue of TAA genes and loss of FIB function resulted in pleiotropic abnormal phenotypes, such as small leaves with large lamina joint angles, abnormal vascular development, small panicles, abnormal organ identity and defects in root development, together with a reduction in internal IAA levels. Moreover, we found that auxin sensitivity and polar transport activity were altered in the fib mutant. From these results, we suggest that FIB plays a pivotal role in IAA biosynthesis in rice and that auxin biosynthesis, transport and sensitivity are closely interrelated.


Successful mouse cloning of an outbred strain by trichostatin A treatment after somatic nuclear transfer.

Abstract: Although the somatic cloning technique has been used for numerous applications and basic research of reprogramming in various species, extremely low success rates have plagued this technique for a decade. Further in mice, the "clonable" strains have been limited to mainly hybrid F1 strains such as B6D2F1. Recently, we established a new efficient cloning technique using trichostatin A (TSA) which leads to a 2-5 fold increase in success rates for mouse cloning of B6D2F1 cumulus cells. To further test the validity of this TSA cloning technique, we tried to clone the adult ICR mouse, an outbred strain, which has never been directly cloned before. Only when TSA was used did we obtain both male and female cloned mice from cumulus and fibroblast cells of adult ICR mice with 4-5% success rates, which is comparable to 5-7% of B6D2F1. Thus, the TSA treatment is the first cloning technique to allow us to successfully clone outbred mice, demonstrating that this technique not only improves the success rates of cloning from hybrid strains, but also enables mouse cloning from normally "unclonable" strains.


Mortality from causes amenable and non-amenable to medical care: the experience of eastern Europe.

Abstract:
OBJECTIVE To investigate comparative national trends in mortality from conditions amenable to timely, appropriate medical care and from those considered not to be amenable to such care.
 DESIGN Analysis of trends in direct age standardised mortality from the 1950s to 1987.
 SETTING Four eastern European nations (Hungary, Czechoslovakia, Poland, the German Democratic Republic) and two western European (the Federal Republic of Germany and England and Wales) and two North American nations (United States and Canada).    SUBJECTS The total populations of the relevant countries during the period examined.
 MAIN OUTCOME MEASURES Proportional changes over time in age standardised mortality. Mortality from amenable and non-amenable causes was restricted to the age group 0-64.
 RESULTS A divergence in the trends for all cause mortality between eastern Europe and the western nations occurred in about 1970, when the rates in western countries steadily declined but those in eastern Europe remained fairly static. In the age group 0-64 mortality from causes considered amenable to medical care fell less quickly in eastern Europe than in the West, particularly after 1970. In the same age group, mortality from non-amenable causes rose in eastern European countries from the late 1960s compared with substantial declines in such mortality in the West.
 CONCLUSIONS Non-amenable causes of death seem to be the principal, but not exclusive, reason for lack of improvement in trends in all cause mortality in eastern Europe from 1970. The agenda for action in eastern Europe should give priority to a healthier lifestyle and improvement of the environment though not neglect enhancements in the quality and efficiency of direct health services.


Dynamic regulation of heterochromatin function via phosphorylation of HP1-family proteins.

Abstract: Heterochromatin is characterized by methylation of histone H3 at lysine 9, which is recognized by well-conserved HP1-family proteins. Heterochromatin participates in various chromosome functions, which include transcriptional gene silencing and sister-chromatid cohesion. These heterochromatic functions are carried out by various effector proteins that associate with HP1-family proteins; however, the regulation of this association with the effectors is not well understood. Recently, we showed that phosphorylation of the fission-yeast HP1 homolog Swi6 regulates the association of the transcriptional regulators differentially and changes the transcriptional activity of heterochromatin, without affecting sister-chromatid cohesion. This study, together with another study performed using other systems, indicates that phosphorylation of HP1/Swi6 provides a dynamic pathway for the differential regulation of heterochromatin in response to inter- and intracellular signals.


Modeling neurogenesis impairment in Down syndrome with induced pluripotent stem cells from Trisomy 21 amniotic fluid cells.

Abstract: Down syndrome (DS), or Trisomy 21 (T21) syndrome, one of the most common chromosomal abnormalities, is caused by an extra duplication of chromosome 21. In studies of neuron development, experimental models based on human cells are considered to be the most desired and accurate for basic research. The generation of diseased induced pluripotetn stem (iPS) cell is a critical step in understanding the developmental stages of complex neuronal diseases. Here, we generated human DS iPS cell lines from second trimester amniotic fluid (AF) cells with T21 by co-expressing Yamanaka factors through lentiviral delivery and subsequently differentiated them into neuronal progenitor cells (NPCs) for further analyses. T21 AF-iPS cells were characterized for the expression of pluripotent markers and for their ability to differentiate into all three germ layers by forming embryoid bodies in vitro and teratomas in vivo. The T21 AF-iPS cells maintained their unique pattern of chromosomal karyotypes: three pairs of chromosome 21. The level of amyloid precursor protein was significantly increased in NPCs derived from T21 AF-iPS cells compared with NPCs from normal AF-iPS cells. The expression levels of miR-155 and miR-802 in T21 AF-iPS-NPCs were highly elevated in the presence of low expression of MeCP2. We observed that T21 iPS-NPCs generated fewer neurons compared with controls. T21 iPS-NPCs exhibit developmental defects during neurogenesis. Our findings suggest that T21 AF-iPS cells serve as a good source to further elucidate the impairment neurogenesis of DS and the onset of Alzheimer's disease.


MYC/MIZ1-dependent gene repression inversely coordinates the circadian clock with cell cycle and proliferation

Abstract: The circadian clock and the cell cycle are major cellular systems that organize global physiology in temporal fashion. It seems conceivable that the potentially conflicting programs are coordinated. We show here that overexpression of MYC in U2OS cells attenuates the clock and conversely promotes cell proliferation while downregulation of MYC strengthens the clock and reduces proliferation. Inhibition of the circadian clock is crucially dependent on the formation of repressive complexes of MYC with MIZ1 and subsequent downregulation of the core clock genes BMAL1 (ARNTL), CLOCK and NPAS2. We show furthermore that BMAL1 expression levels correlate inversely with MYC levels in 102 human lymphomas. Our data suggest that MYC acts as a master coordinator that inversely modulates the impact of cell cycle and circadian clock on gene expression.


Controlling nuclear receptors: the circular logic of cofactor cycles

Abstract: Nuclear receptors regulate many biologically important processes in development and homeostasis by their bimodal function as repressors and activators of gene transcription. A finely tuned modulation of the transcriptional activities of nuclear receptors is crucial for determining highly specific and diversified programmes of gene expression. Recent studies have provided insights into the molecular mechanisms that are required to switch between repression and activation functions, the combinatorial roles of the multiple cofactor complexes that are required for mediating transcriptional regulation, and the central question of how several different signalling pathways can be integrated at the nuclear level to achieve specific profiles of gene expression.


Reprogramming fibroblasts into bipotential hepatic stem cells by defined factors.

Abstract: Recent studies have demonstrated direct reprogramming of fibroblasts into a range of somatic cell types, but to date stem or progenitor cells have only been reprogrammed for the blood and neuronal lineages. We previously reported generation of induced hepatocyte-like (iHep) cells by transduction of Gata4, Hnf1α, and Foxa3 in p19 Arf null mouse embryonic fibroblasts (MEFs). Here, we show that Hnf1β and Foxa3, liver organogenesis transcription factors, are sufficient to reprogram MEFs into induced hepatic stem cells (iHepSCs). iHepSCs can be stably expanded in vitro and possess the potential of bidirectional differentiation into both hepatocytic and cholangiocytic lineages. In the injured liver of fumarylacetoacetate hydrolase (Fah)-deficient mice, repopulating iHepSCs become hepatocyte-like cells. They also engraft as cholangiocytes into bile ducts of mice with DDC-induced bile ductular injury. Lineage conversion into bipotential expandable iHepSCs provides a strategy to enable efficient derivation of both hepatocytes and cholangiocytes for use in disease modeling and tissue engineering.


Spatial genome organization: contrasting views from chromosome conformation capture and fluorescence in situ hybridization.

Abstract: Although important for gene regulation, most studies of genome organization use either fluorescence in situ hybridization (FISH) or chromosome conformation capture (3C) methods. FISH directly visualizes the spatial relationship of sequences but is usually applied to a few loci at a time. The frequency at which sequences are ligated together by formaldehyde cross-linking can be measured genome-wide by 3C methods, with higher frequencies thought to reflect shorter distances. FISH and 3C should therefore give the same views of genome organization, but this has not been tested extensively. We investigated the murine HoxD locus with 3C carbon copy (5C) and FISH in different developmental and activity states and in the presence or absence of epigenetic regulators. We identified situations in which the two data sets are concordant but found other conditions under which chromatin topographies extrapolated from 5C or FISH data are not compatible. We suggest that products captured by 3C do not always reflect spatial proximity, with ligation occurring between sequences located hundreds of nanometers apart, influenced by nuclear environment and chromatin composition. We conclude that results obtained at high resolution with either 3C methods or FISH alone must be interpreted with caution and that views about genome organization should be validated by independent methods.


Development and evaluation of a self-administered computerized 24-h dietary recall method for adolescents in Europe

Abstract: Objective:To describe the development of a European computerized 24-h dietary recall method for adolescents, and to investigate the feasibility of self-administration (self report) by comparison with administration by a dietician (interview).Methods:Two hundred and thirty-six adolescents (mean age 14.6 years (s.d.=1.7)) of eight European cities completed the 24-h recall (Young Adolescents Nutrition Assessment on Computer (YANA-C)) twice (once by self-report and once by interview).Results:A small but significant underestimate in energy (61 (s.e.=31) kcal) and fat (4.2 (s.e.=1.7) g) intake was found in the self-reports in comparison with the interviews; no significant differences were found for the intake of carbohydrates, proteins, fibre, calcium, iron and ascorbic acid. Spearman's correlations were highly significant for all nutrients and energy ranging between 0.86 and 0.91. Agreement in categorizing the respondents as consumers and non-consumers for the 29 food groups was high (kappa statistics ⩾0.73). Percentage omissions were on average 3.7%; percentage intrusions: 2.0%. Spearman's correlations between both modes were high for all food groups, for the total sample (⩾0.76) as well as for the consumers only (⩾0.72). Analysing the consumer only, on an average 54% of the consumed amounts were exactly the same; nevertheless, only for one group ‘rice and pasta’ a significant difference in consumption was found. Conclusion:Adaptation, translation and standardization of YANA-C make it possible to assess the dietary intake of adolescents in a broad international context. In general, good agreement between the administration modes was found, the latter offering significant potential for large-scale surveys where the amount of resources to gather data is limited.


JTK_CYCLE: an efficient nonparametric algorithm for detecting rhythmic components in genome-scale data sets.

Abstract: Circadian rhythms are oscillations of physiology, behavior, and metabolism that have period lengths near 24 hours. In several model organisms and humans, circadian clock genes have been characterized and found to be transcription factors. Because of this, researchers have used microarrays to characterize global regulation of gene expression and algorithmic approaches to detect cycling. This article presents a new algorithm, JTK_CYCLE, designed to efficiently identify and characterize cycling variables in large data sets. Compared with COSOPT and the Fisher's G test, two commonly used methods for detecting cycling transcripts, JTK_CYCLE distinguishes between rhythmic and nonrhythmic transcripts more reliably and efficiently. JTK_CYCLE's increased resistance to outliers results in considerably greater sensitivity and specificity. Moreover, JTK_CYCLE accurately measures the period, phase, and amplitude of cycling transcripts, facilitating downstream analyses. Finally, JTK_CYCLE is several orders of magnitude faster than COSOPT, making it ideal for large-scale data sets. JTK_CYCLE was used to analyze legacy data sets including NIH3T3 cells, which have comparatively low amplitude oscillations. JTK_CYCLE's improved power led to the identification of a novel cluster of RNA-interacting genes whose abundance is under clear circadian regulation. These data suggest that JTK_CYCLE is an ideal tool for identifying and characterizing oscillations in genome-scale data sets.


Homogeneously dispersed multimetal oxygen-evolving catalysts

Abstract: Earth-abundant first-row (3d) transition metal–based catalysts have been developed for the oxygen-evolution reaction (OER); however, they operate at overpotentials substantially above thermodynamic requirements. Density functional theory suggested that non-3d high-valency metals such as tungsten can modulate 3d metal oxides, providing near-optimal adsorption energies for OER intermediates. We developed a room-temperature synthesis to produce gelled oxyhydroxides materials with an atomically homogeneous metal distribution. These gelled FeCoW oxyhydroxides exhibit the lowest overpotential (191 millivolts) reported at 10 milliamperes per square centimeter in alkaline electrolyte. The catalyst shows no evidence of degradation after more than 500 hours of operation. X-ray absorption and computational studies reveal a synergistic interplay between tungsten, iron, and cobalt in producing a favorable local coordination environment and electronic structure that enhance the energetics for OER.


Recovery of supraspinal control of stepping via indirect propriospinal relay connections after spinal cord injury

Abstract: Spinal cord injuries (SCIs) in humans and experimental animals are often associated with varying degrees of spontaneous functional recovery during the first months after injury. Such recovery is widely attributed to axons spared from injury that descend from the brain and bypass incomplete lesions, but its mechanisms are uncertain. To investigate the neural basis of spontaneous recovery, we used kinematic, physiological and anatomical analyses to evaluate mice with various combinations of spatially and temporally separated lateral hemisections with or without the excitotoxic ablation of intrinsic spinal cord neurons. We show that propriospinal relay connections that bypass one or more injury sites are able to mediate spontaneous functional recovery and supraspinal control of stepping, even when there has been essentially total and irreversible interruption of long descending supraspinal pathways in mice. Our findings show that pronounced functional recovery can occur after severe SCI without the maintenance or regeneration of direct projections from the brain past the lesion and can be mediated by the reorganization of descending and propriospinal connections. Targeting interventions toward augmenting the remodeling of relay connections may provide new therapeutic strategies to bypass lesions and restore function after SCI and in other conditions such as stroke and multiple sclerosis.


Microbiome-driven allergic lung inflammation is ameliorated by short-chain fatty acids

Abstract: The mammalian gastrointestinal tract harbors a microbial community with metabolic activity critical for host health, including metabolites that can modulate effector functions of immune cells. Mice treated with vancomycin have an altered microbiome and metabolite profile, exhibit exacerbated T helper type 2 cell (Th2) responses, and are more susceptible to allergic lung inflammation. Here we show that dietary supplementation with short-chain fatty acids (SCFAs) ameliorates this enhanced asthma susceptibility by modulating the activity of T cells and dendritic cells (DCs). Dysbiotic mice treated with SCFAs have fewer interleukin-4 (IL4)-producing CD4+ T cells and decreased levels of circulating immunoglobulin E (IgE). In addition, DCs exposed to SCFAs activate T cells less robustly, are less motile in response to CCL19 in vitro, and exhibit a dampened ability to transport inhaled allergens to lung draining nodes. Our data thus demonstrate that gut dysbiosis can exacerbate allergic lung inflammation through both T cell- and DC-dependent mechanisms that are inhibited by SCFAs.


GATA3 transcription factor abrogates Smad4 transcription factor-mediated fascin overexpression, invadopodium formation, and breast cancer cell invasion.

Abstract: Transforming growth factor β (TGFβ) is a potent and context-dependent regulator of tumor progression. TGFβ promotes the lung metastasis of basal-like (but not the luminal-like) breast cancer. Here, we demonstrated that fascin, a pro-metastasis actin bundling protein, was a direct target of the canonical TGFβ-Smad4 signaling pathway in basal-like breast cancer cells. TGFβ and Smad4 induced fascin overexpression by directly binding to a Smad binding element on the fascin promoter. We identified GATA3, a transcription factor crucial for mammary gland morphogenesis and luminal differentiation, as a negative regulator of TGFβ- and Smad4-induced fascin overexpression. When ectopically expressed in basal-like breast cancer cells, GATA-3 abrogated TGFβ- and Smad4-mediated overexpression of fascin and other TGFβ response genes, invadopodium formation, cell migration, and invasion, suggesting suppression of the canonical TGFβ-Smad signaling axis. Mechanistically, GATA3 abrogated the canonical TGFβ-Smad signaling by abolishing interactions between Smad4 and its DNA binding elements, potentially through physical interactions between the N-terminal of GATA3 and Smad3/4 proteins. Our findings provide mechanistic insight into how TGFβ-mediated cell motility and invasiveness are differentially regulated in breast cancer.


Involving family members in cancer care: focus group considerations of patients and oncological providers.

Abstract: Family members are an integral part of a patient's cancer care from the moment the diagnosis is delivered to the conclusion of treatment. Family members bring with them a range of emotional reactions, interpersonal dynamics and expectations for the care the patient receives. This study is part of a multi-institutional project to continue to improve the process of cancer care. In this study, 19 focus groups (11 patient and 8 provider) were conducted concerning issues related to doctor-patient communication in eight cancer centers in the United States. The content of the conversations was analyzed and thematic categories emerged that highlight the various strengths and difficulties associated with family involvement. The focus groups' comments support the need for explicit conversations between professional caregivers, patients and their loved ones, in order to negotiate the expectations and needs of each team member. Implications for clinical practice and strategies for working with family members are offered.


Replication Fork Slowing and Reversal upon DNA Damage Require PCNA Polyubiquitination and ZRANB3 DNA Translocase Activity

Abstract: DNA damage tolerance during eukaryotic replication is orchestrated by PCNA ubiquitination. While monoubiquitination activates mutagenic translesion synthesis, polyubiquitination activates an error-free pathway, elusive in mammals, enabling damage bypass by template switching. Fork reversal is driven in vitro by multiple enzymes, including the DNA translocase ZRANB3, shown to bind polyubiquitinated PCNA. However, whether this interaction promotes fork remodeling and template switching in vivo was unknown. Here we show that damage-induced fork reversal in mammalian cells requires PCNA ubiquitination, UBC13, and K63-linked polyubiquitin chains, previously involved in error-free damage tolerance. Fork reversal in vivo also requires ZRANB3 translocase activity and its interaction with polyubiquitinated PCNA, pinpointing ZRANB3 as a key effector of error-free DNA damage tolerance. Mutations affecting fork reversal also induced unrestrained fork progression and chromosomal breakage, suggesting fork remodeling as a global fork slowing and protection mechanism. Targeting these fork protection systems represents a promising strategy to potentiate cancer chemotherapy.


Decoupling activation and exhaustion of B cells in spontaneous controllers of HIV infection.

Abstract:
OBJECTIVE To define the impact of chronic viremia and associated immune activation on B-cell exhaustion in HIV infection.
 DESIGN Progressive HIV infection is marked by B-cell anergy and exhaustion coupled with dramatic hypergammaglobulinemia. Although both upregulation of CD95 and loss of CD21 have been used as markers of infection-associated B-cell dysfunction, little is known regarding the specific profiles of dysfunctional B cells and whether persistent viral replication and its associated immune activation play a central role in driving B-cell dysfunction.
 METHODS Multiparameter flow cytometry was used to define the profile of dysfunctional B cells. The changes in the expression of CD21 and CD95 were tracked on B-cell subpopulations in patients with differential control of viral replication.
 RESULTS : Although the emergence of exhausted, CD21 tissue-like memory B cells followed similar patterns in both progressors and controllers, the frequency of CD21 activated memory B cells was lower in spontaneous controllers.
 CONCLUSION Our results suggest that the loss of CD21 and the upregulation of CD95 occur as separate events during the development of B-cell dysfunction. The loss of CD21 is a marker of B-cell exhaustion induced in the absence of appreciable viral replication, whereas the upregulation of CD95 is tightly linked to persistent viral replication and its associated immune activation. Thus, these dysfunctional profiles potentially represent two functionally distinct states within the B-cell compartment.


A common variant in BRCA2 is associated with both breast cancer risk and prenatal viability

Abstract: Inherited mutations in the gene BRCA2 predispose carriers to early onset breast cancer, but such mutations account for fewer than 2% of all cases in East Anglia. It is likely that low penetrance alleles explain the greater part of inherited susceptibility to breast cancer; polymorphic variants in strongly predisposing genes, such as BRCA2, are candidates for this role. BRCA2 is thought to be involved in DNA double strand break-repair. Few mice in which Brca2 is truncated survive to birth; of those that do, most are male, smaller than their normal littermates and have high cancer incidence. Here we show that a common human polymorphism (N372H) in exon 10 of BRCA2 confers an increased risk of breast cancer: the HH homozygotes have a 1.31-fold (95% CI, 1.07–1.61) greater risk than the NN group. Moreover, in normal female controls of all ages there is a significant deficiency of homozygotes compared with that expected from Hardy-Weinberg equilibrium, whereas in males there is an excess of homozygotes: the HH group has an estimated fitness of 0.82 in females and 1.38 in males. Therefore, this variant of BRCA2 appears also to affect fetal survival in a sex-dependent manner.


A twin-study of genetic contributions to morningness-eveningness and depression.

Abstract: Circadian rhythms are associated with the preference for sleep-wake timing, also known as morningness-eveningness (ME). Both circadian rhythms and ME are influenced by genetic factors. Studies show an association between eveningness and depression. This study investigates the heritability of ME and whether ME and depression share common genetic influences. Study participants (n = 1237) were from the Vietnam Era Twin Study of Aging, a longitudinal study of aging with a baseline in midlife. Participants received the Morningness-Eveningness Questionnaire (MEQ) and the Center for Epidemiologic Studies Depression (CES-D) Scale as part of an extensive neurocognitive and psychosocial assessment. MEQ correlations between members of twin pairs were 0.41 (95% CI 0.31-0.49) for monozygotic (MZ) twins and 0.28 for dizygotic (DZ) twins (95% CI 0.19-0.41). CES-D correlations were 0.38 (95% CI 0.28-0.46) for MZ twins and 0.24 (95% CI 0.14-0.36) for DZ twins. Greater eveningness (i.e. lower MEQ scores) was significantly related to more depression symptoms (phenotypic correlation = -0.15 (95% CI -0.21 to -0.09). In the best fitting model, the heritability estimates are 0.42 for the MEQ and 0.37 for the CES-D. A significant genetic correlation of -0.21 indicated that ME and depression share a significant amount of their underlying genetic variance. The genetic covariance between ME and depression accounted for 59.1% of the phenotypic correlation. Of the CES-D sub-scales, Depressed Mood and Interpersonal Difficulties were significantly heritable, while only Well-Being had a significant genetic correlation with ME. ME and depression are both heritable (ME 0.42, depression 0.37) and share common genetic factors, suggesting an overlap in etiology and the relevance of circadian rhythms to depression. Further study of this relationship may help elucidate etiological factors in depression and targets for treatment.


The Transcription Factor T-bet Regulates Intestinal Inflammation Mediated by Interleukin-7 Receptor+ Innate Lymphoid Cells

Abstract: Mice lacking the transcription factor T-bet in the innate immune system develop microbiota-dependent colitis. Here, we show that interleukin-17A (IL-17A)-producing IL-7Rα(+) innate lymphoid cells (ILCs) were potent promoters of disease in Tbx21(-/-)Rag2(-/-) ulcerative colitis (TRUC) mice. TNF-α produced by CD103(-)CD11b(+) dendritic cells synergized with IL-23 to drive IL-17A production by ILCs, demonstrating a previously unrecognized layer of cellular crosstalk between dendritic cells and ILCs. We have identified Helicobacter typhlonius as a key disease trigger driving excess TNF-α production and promoting colitis in TRUC mice. Crucially, T-bet also suppressed the expression of IL-7R, a key molecule involved in controlling intestinal ILC homeostasis. The importance of IL-7R signaling in TRUC disease was highlighted by the dramatic reduction in intestinal ILCs and attenuated colitis following IL-7R blockade. Taken together, these data demonstrate the mechanism by which T-bet regulates the complex interplay between mucosal dendritic cells, ILCs, and the intestinal microbiota.


A population-based cohort study to elucidate temporal relationship between schizophrenia and metabolic syndrome (KCIS no. PSY3).

Abstract:
BACKGROUND The bidirectional relationships between metabolic syndrome (MetS) and schizophrenia (SCZ) play a crucial role in clinical treatment of both diseases but such bidirectional causal effects have not been comprehensively elucidated.    AIMS To investigate the influence of MetS on incident SCZ and the opposite direction as well as their predictors for each direction with a population-based cohort sample.
 METHOD We enrolled 76,545 subjects who had participated in a community-based health screening program during 1999-2004. After excluding those with the existing MetS or SCZ at baseline, the two normal prospective cohorts corresponding to each independent variable of MetS or SCZ, respectively, were followed over time to ascertain incident outcome of SCZ and MetS. The crude and adjusted hazard ratios for the effect of the predictor on each incident outcome were estimated after controlling for the possible confounding factors.
 RESULTS The overall incidence rate (per 10(5)person-years) of SCZ was 61.15. The incidence rate in patients with MetS was lower than those without (44.24 versus 64.20), indicating the presence of MetS failed to find an increased risk of developing incident SCZ. However, participants with abnormal waist circumference (WC) were two times (95% CI: 1.37 to 2.93) more likely to yield incident SCZ compared to those with normal WC. In the opposite direction, the incidence of MetS was statistically higher in patients with SCZ than those without SCZ (11.25% vs 7.94%, respectively), suggesting SCZ conferred a higher risk for yielding incident MetS (adjusted hazard ratio=1.89, 95% CI: 1.36, 2.63).
 CONCLUSIONS After examining the bidirectional causal relationships between SCZ and MetS with the theoretically sound and large population-based prospective cohort study, central obesity, one of the individual components of MetS, was corroborated as an independent predictor for incident SCZ. Patients diagnosed with SCZ were at greater risk of having incident cases of MetS. Such significant temporal bidirectional relationships between SCZ and central obesity suggest a reciprocal interaction exits between SCZ and central obesity.


Health and access to care: perspectives of homeless youth in Baltimore City, U.S.A.

Abstract: Homeless youth suffer from high rates of health problems, yet little is known about their perceptions of or context for their own health issues. In this study, a combination of qualitative techniques from participatory rural appraisal and rapid assessment procedures was used to investigate the perceptions of health needs of shelter-based youth in Baltimore, MD in the U.S.A. The most common youth-identified health problems included STDs, HIV/AIDS, pregnancy, depression, drug use and injuries. These correlate well with more objective health status data for the same youth. The youth spoke of environmental safety threats of violence and victimization by adults, as well as racism and sexism in their lives. Youth reported that trusted adult figures such as grandmothers are important sources of health advice. Many homeless youth from less than ideal family situations remain in contact with and continue to seek advice from parents and other family members. Health interventions with urban street youth need to acknowledge the primacy of the social context for these youth, as well as the reality of violence as a daily health threat.


Differential requirement for CARMA1 in agonist-selected T-cell development.

Abstract: Caspase recruitment domain-containing membrane-associated guanylate kinase protein-1 (CARMA1) is a critical component of the NF-kappaB signaling cascade mediated by TCR engagement. In addition to activation of naïve T cells, TCR signaling is important for the development of agonist-selected T-cell subsets such as Treg, NKT cells, and CD8-alpha alpha T cells. However, little is known about the role of CARMA1 in the development of these lineages. Here we show that CARMA1-deficient mice (CARMA1(-/-)) have altered populations of specific subsets of agonist-selected T cells. Specifically, CARMA1(-/-) mice have impaired natural and adaptive Treg development, whereas NKT cell numbers are normal compared with wild-type mice. Interestingly, CD8-alpha alpha T cells, which may also be able to develop through an extrathymic selection pathway, are enriched in the gut of CARMA1(-/-) mice, whereas memory-phenotype CD4(+) T cells (CD62L(low)/CD44(high)) are present at reduced numbers in the periphery. These results indicate that CARMA1 is essential for Treg development, but is not necessary for the development of other agonist-selected T-cell subsets. Overall, these data reveal an important but differential role for CARMA1-mediated TCR signaling in T-cell development.


Simian immunodeficiency viruses of diverse origin can use CXCR4 as a coreceptor for entry into human cells.

Abstract: Primary simian immunodeficiency virus (SIV) isolated from sooty mangabey (SIVsm [n = 6]), stumptail (SIVstm [n = 1]), mandrill (SIVmnd [n = 1]), and African green (SIVagm [n = 1]) primates were examined for their ability to infect human cells and for their coreceptor requirements. All isolates infected human peripheral blood mononuclear cells (PBMCs) from a CCR5(+/+) donor, and seven of eight isolates tested also infected CCR5(-/-) PBMCs. Analysis of coreceptor utilization using GHOST and U87 cell lines revealed that all of the isolates tested used CCR5 and the orphan receptors STRL33 and GPR15. Coreceptors such as CCR2b, CCR3, CCR8, and CX3CR1 were also utilized by some primary SIV isolates. More importantly, we found that CXCR4 was used as a coreceptor by the SIVstm, the SIVagm, and four of the SIVsm isolates in GHOST and U87 cells. These data suggest that primary SIV isolates from diverse primate species can utilize CXCR4 for viral entry, similar to what has been described for human immunodeficiency viruses.


Cloning of rat uncoupling protein-3 and uncoupling protein-2 cDNAs: their gene expression in rats fed high-fat diet.

Abstract: In order to elucidate energy balance in the skeletal muscle, we cloned cDNA of a homologue of uncoupling protein (UCP) from rat skeletal muscle. We also cloned rat UCP-2 cDNA from rat brown adipose tissue (BAT). The UCP cloned from rat skeletal muscle showed 57% and 72% identity with rat UCP-1 and UCP-2. The mRNA was expressed abundantly in the skeletal muscle, moderately in the BAT, and slightly in the white adipose tissue (WAT) with a major band at 2.5 kb and a minor band at 2.8 kb, while the UCP-2 gene expression was widely detected in the whole body with substantial levels in the WAT and with slight levels in the skeletal muscle and BAT. The rat UCP cloned in the present study showed 86% identity with the recently cloned human UCP-3, which was also expressed abundantly in the skeletal muscle with a signal of 2.4 kb. Therefore, the rat UCP was considered to be rat UCP-3. In rats fed high-fat diet the UCP-3 gene expression was augmented 2-fold in the skeletal muscle while UCP-2 mRNA levels were increased significantly (1.6-fold) in the epididymal WAT. Augmented expression of UCPs may provide defense against high-fat induced obesity and impairment of glucose metabolism.


A two-step mechanism for epigenetic specification of centromere identity and function

Abstract: The basic determinant of chromosome inheritance, the centromere, is specified in many eukaryotes by an epigenetic mark. Using gene targeting in human cells and fission yeast, chromatin containing the centromere-specific histone H3 variant CENP-A is demonstrated to be the epigenetic mark that acts through a two-step mechanism to identify, maintain and propagate centromere function indefinitely. Initially, centromere position is replicated and maintained by chromatin assembled with the centromere-targeting domain (CATD) of CENP-A substituted into H3. Subsequently, nucleation of kinetochore assembly onto CATD-containing chromatin is shown to require either the amino- or carboxy-terminal tail of CENP-A for recruitment of inner kinetochore proteins, including stabilizing CENP-B binding to human centromeres or direct recruitment of CENP-C, respectively.


Cancer stem cell theory in gastrointestinal malignancies: recent progress and upcoming challenges

Abstract: A growing body of evidence supports the notion that malignant tumors are heterogeneous and contain diverse subpopulations of cells with unique characteristics including the ability to initiate a tumor and metastasize. This phenomenon might be explained by the so-called cancer stem cell (CSC) theory. Recent technological developments have allowed a deeper understanding and characterization of CSCs. Even though the application of this theory to hematopoietic malignancies and solid tumors holds promise for new ways to treat cancer, it also brings some skepticism. Efficacious therapeutic approaches targeting the CSC population should be explored to overcome therapeutic failure and improve patient outcomes. This review will focus on the intrinsic and extrinsic regulation of CSCs, as well as the development of therapeutic approaches against CSCs, predominantly focusing on gastrointestinal malignancies.


The WUSCHEL-related homeobox gene WOX11 is required to activate shoot-borne crown root development in rice.

Abstract: In rice (Oryza sativa), the shoot-borne crown roots are the major root type and are initiated at lower stem nodes as part of normal plant development. However, the regulatory mechanism of crown root development is poorly understood. In this work, we show that a WUSCHEL-related Homeobox (WOX) gene, WOX11, is involved in the activation of crown root emergence and growth. WOX11 was found to be expressed in emerging crown roots and later in cell division regions of the root meristem. The expression could be induced by exogenous auxin or cytokinin. Loss-of-function mutation or downregulation of the gene reduced the number and the growth rate of crown roots, whereas overexpression of the gene induced precocious crown root growth and dramatically increased the root biomass by producing crown roots at the upper stem nodes and the base of florets. The expressions of auxin- and cytokinin-responsive genes were affected in WOX11 overexpression and RNA interference transgenic plants. Further analysis showed that WOX11 directly repressed RR2, a type-A cytokinin-responsive regulator gene that was found to be expressed in crown root primordia. The results suggest that WOX11 may be an integrator of auxin and cytokinin signaling that feeds into RR2 to regulate cell proliferation during crown root development.


Inducible depletion of satellite cells in adult, sedentary mice impairs muscle regenerative capacity without affecting sarcopenia

Abstract: A key determinant of geriatric frailty is sarcopenia, the age-associated loss of skeletal muscle mass and strength. Although the etiology of sarcopenia is unknown, the correlation during aging between the loss of activity of satellite cells, which are endogenous muscle stem cells, and impaired muscle regenerative capacity has led to the hypothesis that the loss of satellite cell activity is also a cause of sarcopenia. We tested this hypothesis in male sedentary mice by experimentally depleting satellite cells in young adult animals to a degree sufficient to impair regeneration throughout the rest of their lives. A detailed analysis of multiple muscles harvested at various time points during aging in different cohorts of these mice showed that the muscles were of normal size, despite low regenerative capacity, but did have increased fibrosis. These results suggest that lifelong reduction of satellite cells neither accelerated nor exacerbated sarcopenia and that satellite cells did not contribute to the maintenance of muscle size or fiber type composition during aging, but that their loss may contribute to age-related muscle fibrosis.


Findings to date from the ASCUS-LSIL Triage Study (ALTS).

Abstract: Controversy exists in the United States regarding the proper evaluation and management of low-grade squamous intraepithelial lesion (LSIL) and equivocal (atypical squamous cells of undetermined significance [ASCUS, now ASC-US]) cervical cytologic interpretations. To address this issue, the National Cancer Institute initiated the ASCUS-LSIL Triage Study (ALTS). ALTS is a multicenter, randomized clinical trial designed to evaluate 3 alternative methods of management, namely, immediate colposcopy, cytologic follow-up, and triage by human papillomavirus (HPV) DNA testing. This article summarizes the major findings of ALTS that have been published to date. Patients with ASCUS (n = 3488) or LSIL (n = 1572) were randomly assigned to research arms between November 1996 and December 1998, and were monitored for 2 years. The disease outcome was histologic cervical intraepithelial neoplasia (CIN) 3/cancer. The prevalence of oncogenic HPV was too high to permit effective triage of LSIL using HPV DNA testing by Hybrid Capture 2. However, for the women referred with a cytologic interpretation of ASCUS, HPV triage proved useful, with sensitivity equivalent to immediate colposcopy and a halving of colposcopic referrals. Among older women with ASCUS, HPV testing remained sensitive for detecting CIN 3 and cancer, but the referral percentage was dramatically lower compared to younger women. ALTS yielded insight into the performance of cytology and histopathology; experienced pathologists differed significantly in their interpretations of cervical abnormalities, especially histologic CIN 1 and cytologic ASCUS. Nonetheless, it was possible to distinguish a relatively uncommon type of ASCUS, equivocal for high-grade squamous intraepithelial lesion, that has a high positive predictive value for identifying women with underlying high-grade CIN. Many additional analyses are underway.


A randomized, placebo-controlled trial of diindolylmethane for breast cancer biomarker modulation in patients taking tamoxifen

Abstract: Diindolylmethane (DIM), a bioactive metabolite of indole-3-carbinol found in cruciferous vegetables, has proposed cancer chemoprevention activity in the breast. There is limited evidence of clinically relevant activity of DIM or long-term safety data of its regular use. A randomized, double-blind, placebo-controlled trial was conducted to determine the activity and safety of combined use of BioResponse DIM® (BR-DIM) with tamoxifen. Women prescribed tamoxifen (n = 130) were randomly assigned oral BR-DIM at 150 mg twice daily or placebo, for 12 months. The primary study endpoint was change in urinary 2/16α-hydroxyestrone (2/16α-OHE1) ratio. Changes in 4-hydroxyestrone (4-OHE1), serum estrogens, sex hormone-binding globulin (SHBG), breast density, and tamoxifen metabolites were assessed. Ninety-eight women (51 placebo, 47 DIM) completed intervention; compliance with treatment was >91%. BR-DIM increased the 2/16α-OHE1 ratio (+3.2 [0.8, 8.4]) compared to placebo (−0.7 [−1.7, 0.8], P < 0.001). Serum SHBG increased with BR-DIM compared to placebo (+25 ± 22 and +1.1 ± 19 nmol/L, respectively). No change in breast density measured by mammography or by MRI was observed. Plasma tamoxifen metabolites (endoxifen, 4-OH tamoxifen, and N-desmethyl-tamoxifen) were reduced in women receiving BR-DIM versus placebo (P < 0.001). Minimal adverse events were reported and did not differ by treatment arm. In patients taking tamoxifen for breast cancer, daily BR-DIM promoted favorable changes in estrogen metabolism and circulating levels of SHBG. Further research is warranted to determine whether BR-DIM associated decreases in tamoxifen metabolites, including effects on endoxifen levels, attenuates the clinical benefit of tamoxifen. Trial Registration: ClinicalTrials.gov NCT01391689.


Intracellular coenzymes as natural biomarkers for metabolic activities and mitochondrial anomalies.

Abstract: Mitochondria play a pivotal role in energy metabolism, programmed cell death and oxidative stress. Mutated mitochondrial DNA in diseased cells compromises the structure of key enzyme complexes and, therefore, mitochondrial function, which leads to a myriad of health-related conditions such as cancer, neurodegenerative diseases, diabetes and aging. Early detection of mitochondrial and metabolic anomalies is an essential step towards effective diagnoses and therapeutic intervention. Reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) play important roles in a wide range of cellular oxidation-reduction reactions. Importantly, NADH and FAD are naturally fluorescent, which allows noninvasive imaging of metabolic activities of living cells and tissues. Furthermore, NADH and FAD autofluorescence, which can be excited using distinct wavelengths for complementary imaging methods and is sensitive to protein binding and local environment. This article highlights recent developments concerning intracellular NADH and FAD as potential biomarkers for metabolic and mitochondrial activities.


Biochemical differences in the alphabeta T cell receptor.CD3 surface complex between CD8+ and CD4+ human mature T lymphocytes.

Abstract: We have reported the existence of biochemical and conformational differences in the alphabeta T cell receptor (TCR) complex between CD4(+) and CD8(+) CD3gamma-deficient (gamma(-)) mature T cells. In the present study, we have furthered our understanding and extended the observations to primary T lymphocytes from normal (gamma(+)) individuals. Surface TCR.CD3 components from CD4(+) gamma(-) T cells, other than CD3gamma, were detectable and similar in size to CD4(+) gamma(+) controls. Their native TCR.CD3 complex was also similar to CD4(+) gamma(+) controls, except for an alphabeta(deltaepsilon)(2)zeta(2) instead of an alphabetagammaepsilondeltaepsilonzeta(2) stoichiometry. In contrast, the surface TCRalpha, TCRbeta, and CD3delta chains of CD8(+) gamma(-) T cells did not possess their usual sizes. Using confocal immunofluorescence, TCRalpha was hardly detectable in CD8(+) gamma(-) T cells. Blue native gels (BN-PAGE) demonstrated the existence of a heterogeneous population of TCR.CD3 in these cells. Using primary peripheral blood T lymphocytes from normal (gamma(+)) donors, we performed a broad epitopic scan. In contrast to all other TCR.CD3-specific monoclonal antibodies, RW2-8C8 stained CD8(+) better than it did CD4(+) T cells, and the difference was dependent on glycosylation of the TCR.CD3 complex but independent of T cell activation or differentiation. RW2-8C8 staining of CD8(+) T cells was shown to be more dependent on lipid raft integrity than that of CD4(+) T cells. Finally, immunoprecipitation studies on purified primary CD4(+) and CD8(+) T cells revealed the existence of TCR glycosylation differences between the two. Collectively, these results are consistent with the existence of conformational or topological lineage-specific differences in the TCR.CD3 from CD4(+) and CD8(+) wild type T cells. The differences may be relevant for cis interactions during antigen recognition and signal transduction.


Estrogen receptor-alpha promotes alternative macrophage activation during cutaneous repair.

Abstract: Efficient local monocyte/macrophage recruitment is critical for tissue repair. Recruited macrophages are polarized toward classical (proinflammatory) or alternative (prohealing) activation in response to cytokines, with tight temporal regulation crucial for efficient wound repair. Estrogen acts as a potent anti-inflammatory regulator of cutaneous healing. However, an understanding of estrogen/estrogen receptor (ER) contribution to macrophage polarization and subsequent local effects on wound healing is lacking. Here we identify, to our knowledge previously unreported, a role whereby estrogen receptor α (ERα) signaling preferentially polarizes macrophages from a range of sources to an alternative phenotype. Cell-specific ER ablation studies confirm an in vivo role for inflammatory cell ERα, but not ERβ, in poor healing associated with an altered cytokine profile and fewer alternatively activated macrophages. Furthermore, we reveal intrinsic changes in ERα-deficient macrophages, which are unable to respond to alternative activation signals in vitro. Collectively, our data reveal that inflammatory cell-expressed ERα promotes alternative macrophage polarization, which is beneficial for timely healing. Given the diverse physiological roles of ERs, these findings will likely be of relevance to many pathologies involving excessive inflammation.


Disturbance in cerebral spinal fluid sphingolipid content is associated with memory impairment in subjects infected with the human immunodeficiency virus

Abstract: Despite widespread use of antiretroviral therapies to control replication of the human immunodeficiency virus (HIV), dysfunctions of cognition that are collectively termed HIV-associated neurocognitive disorders (HAND) still occur in approximately 50% of those infected by the virus. Currently there is not a biomarker that can identify HIV-infected people who are at risk for the development of HAND. Previous studies have identified particular sphingolipid species that are dysregulated in HAND, but the neurocognitive correlates of these biochemical findings are not currently understood. To address this question, we compared cerebrospinal fluid (CSF) levels of sphingomyelin, ceramide, and sterol species with performance on standard neurological tests designed to assess the function of multiple cognitive and motor domains in HIV-infected subjects. We found that sphingomyelin:ceramide ratios for acyl chain lengths of C16∶0, C18∶0, C22∶0, and C24∶0 were associated with worse performance on several indices of memory. The most striking finding was for the acyl chain of C18∶0 that consistently associatedwith performance onmultiple tests of memory. These findings suggest that the sphingomyelin:ceramide ratio for C18∶0 may be a reasonable surrogate marker for memory dysfunction in HIV-infected subjects.


Tumor-Induced IL-6 Reprograms Host Metabolism to Suppress Anti-tumor Immunity

Abstract: In patients with cancer, the wasting syndrome, cachexia, is associated with caloric deficiency. Here, we describe tumor-induced alterations of the host metabolic response to caloric deficiency that cause intratumoral immune suppression. In pre-cachectic mice with transplanted colorectal cancer or autochthonous pancreatic ductal adenocarcinoma (PDA), we find that IL-6 reduces the hepatic ketogenic potential through suppression of PPARalpha, the transcriptional master regulator of ketogenesis. When these mice are challenged with caloric deficiency, the resulting relative hypoketonemia triggers a marked rise in glucocorticoid levels. Multiple intratumoral immune pathways are suppressed by this hormonal stress response. Moreover, administering corticosterone to elevate plasma corticosterone to a level that is lower than that occurring in cachectic mice abolishes the response of mouse PDA to an immunotherapy that has advanced to clinical trials. Therefore, tumor-induced IL-6 impairs the ketogenic response to reduced caloric intake, resulting in a systemic metabolic stress response that blocks anti-cancer immunotherapy.


Tracking adipogenesis during white adipose tissue development, expansion and regeneration

Abstract: White adipose tissue displays high plasticity. We developed a system for the inducible, permanent labeling of mature adipocytes that we called the AdipoChaser mouse. We monitored adipogenesis during development, high-fat diet (HFD) feeding and cold exposure. During cold-induced 'browning' of subcutaneous fat, most 'beige' adipocytes stem from de novo–differentiated adipocytes. During HFD feeding, epididymal fat initiates adipogenesis after 4 weeks, whereas subcutaneous fat undergoes hypertrophy for a period of up to 12 weeks. Gonadal fat develops postnatally, whereas subcutaneous fat develops between embryonic days 14 and 18. Our results highlight the extensive differences in adipogenic potential in various fat depots.


Blockade of adrenoreceptors inhibits the splenic response to stroke.

Abstract: Recent studies have highlighted the involvement of the peripheral immune system in delayed cellular degeneration after stroke. In the permanent middle cerebral artery occlusion (MCAO) model of stroke, the spleen decreases in size. This reduction occurs through the release of splenic immune cells. Systemic treatment with human umbilical cord blood cells (HUCBC) 24 h post-stroke blocks the reduction in spleen size while significantly reducing infarct volume. Splenectomy 2 weeks prior to MCAO also reduces infarct volume, further demonstrating the detrimental role of this organ in stroke-induced neurodegeneration. Activation of the sympathetic nervous system after MCAO results in elevated catecholamine levels both at the level of the spleen, through direct splenic innervation, and throughout the systemic circulation upon release from the adrenal medulla. These catecholamines bind to splenic alpha and beta adrenoreceptors. This study examines whether catecholamines regulate the splenic response to stroke. Male Sprague-Dawley rats either underwent splenic denervation 2 weeks prior to MCAO or received injections of carvedilol, a pan adrenergic receptor blocker, prazosin, an alpha1 receptor blocker, or propranolol, a beta receptor blocker. Denervation was confirmed by reduced splenic expression of tyrosine hydroxylase. Denervation prior to MCAO did not alter infarct volume or spleen size. Propranolol treatment also had no effects on these outcomes. Treatment with either prazosin or carvedilol prevented the reduction in spleen size, yet only carvedilol significantly reduced infarct volume (p < 0.05). These results demonstrate that circulating blood borne catecholamines regulate the splenic response to stroke through the activation of both alpha and beta adrenergic receptors.


Effects of Thiazolidinediones on Stroke Recovery: A Case-Matched Controlled Study

Abstract: Anti-inflammatory therapy decreases infarct size and enhances stroke recovery. Thiazolidinedione peroxisome proliferator-activated receptor (PPAR)gamma agonists have potent anti-inflammatory and insulin-sensitizing anti-diabetic actions. Thirty stroke patients with type 2 diabetes admitted for acute inpatient stroke rehabilitation receiving pioglitazone or rosiglitazone were matched for age, sex, initial FIMTM score and interval post-stroke with 30 stroke patients with type 2 diabetes not receiving thiazolidinediones. Relevant outcome variables were compared for both groups. The thiazolidinedione treated group showed significantly greater mean improvement in FIMTM score compared to control group (25.6 ± 10.2 SD vs. 19.8 ± 10.5, respectively, P = 0.015). There was no significant difference in length of rehabilitation hospital stay (24.2 ± 7.6 vs. 25.1 ± 7.4 days, P = 0.657) or final discharge destination (home/institution, 19/11 versus 17/13, P = 0.792). Use of thiazolidinediones was associated with enhanced functional recovery in stroke patients with type 2 diabetes.


Crosstalk between tumor and endothelial cells promotes tumor angiogenesis by MAPK activation of Notch signaling.

Abstract: While significant progress has been made in understanding the induction of tumor vasculature by secreted angiogenic factors, little is known regarding contact-dependent signals that promote tumor angiogenesis. Here, we report that the Notch ligand Jagged1 induced by growth factors via mitogen-activating protein kinase (MAPK) in head and neck squamous cell carcinoma (HNSCC) cells triggered Notch activation in neighboring endothelial cells (ECs) and promoted capillary-like sprout formation. Jagged1-expressing HNSCC cells significantly enhanced neovascularization and tumor growth in vivo. Moreover, the level of Jagged1 was significantly correlated with tumor blood vessel content and associated with HNSCC development. Our results elucidate a novel mechanism by which the direct interplay between tumor cells and ECs promotes angiogenesis through MAPK and Notch signaling pathways.


A Sequential splicing mechanism promotes selection of an optimal exon by repositioning a downstream 5' splice site in preprotachykinin pre-mRNA.

Abstract: To explore the structural basis of alternative splicing, we have analyzed the splicing of pre-mRNAs containing an optional exon, E4, from the preprotachykinin gene. This gene encodes substance P and related tachykinin peptides by alternative splicing of a common pre-mRNA. We have shown that alternative splicing of preprotachykinin pre-mRNA occurs by preferential skipping of optional E4. The competing mechanism that incorporates E4 into the final spliced RNA is constrained by an initial block to splicing of the immediate upstream intervening sequence (IVS), IVS3. This block is relieved by sequential splicing, in which the immediate downstream IVS4 is removed first. The structural change resulting from the first splicing event is directly responsible for activation of IVS3 splicing. This structural rearrangement replaces IVS4 sequences with E5 and its adjacent IVS5 sequences. To determine how this structural change promoted IVS3 splicing, we asked what structural change(s) would restore activity of IVS3 splicing-defective mutants. The most significant effect was observed by a 2-nucleotide substitution that converted the 5' splice site of E4 to an exact consensus match, GUAAGU. Exon 5 sequences alone were found not to promote splicing when present in one or multiple copies. However, when a 15-nucleotide segment of IVS5 containing GUAAGU was inserted into a splicing-defective mutant just downstream of the hybrid exon segment E4E5, splicing activity was recovered. Curiously, the 72-nucleotide L2 exon of adenovirus, without its associated 5' splice site, activates splicing when juxtaposed to E4. Models for the activation of splicing by an RNA structural change are discussed.


Melatonin and the circadian regulation of sleep initiation, consolidation, structure, and the sleep EEG.

Abstract: The endogenous circadian rhythm of melatonin, driven by the suprachiasmatic nucleus, exhibits a close association with the endogenous circadian component of the sleep propensity rhythm and the endogenous circadian component of the variation in electroencephalogram (EEG) oscillations such as sleep spindles and slow waves. This association is maintained even when the sleep-wake cycle is desynchronized from the endogenous circadian rhythm of melatonin. Administration of melatonin during the day increases daytime sleep propensity as indexed by both the latency to sleep onset and sleep consolidation. The EEG during daytime sleep after melatonin administration exhibits characteristics reminiscent of the nocturnal sleep EEG, that is, increased sleep spindle activity and reduced slow-wave sleep and slow-wave activity, as detected by quantitative EEG analysis. Administration of higher doses of melatonin (5 mg or more) prior to nocturnal sleep results in an increase in rapid eye movement (REM) sleep. These data demonstrate that melatonin exerts effects on the main characteristics of human sleep, that is, latency to sleep onset, sleep consolidation, slow waves, sleep spindles, and REM sleep. There is a need for further studies using physiological doses and delivery systems that generate physiological plasma melatonin profiles to firmly establish the role of the endogenous circadian rhythm of melatonin in the circadian regulation of sleep.


Alkylating agent melphalan augments the efficacy of adoptive immunotherapy using tumor-specific CD4+ T cells.

Abstract: In recent years, the immune-potentiating effects of some widely used chemotherapeutic agents have been increasingly appreciated. This provides a rationale for combining conventional chemotherapy with immunotherapy strategies to achieve durable therapeutic benefits. Previous studies have implicated the immunomodulatory effects of melphalan, an alkylating agent commonly used to treat multiple myeloma, but the underlying mechanisms remain obscure. In the present study, we investigated the impact of melphalan on endogenous immune cells as well as adoptively transferred tumor-specific CD4(+) T cells in tumor-bearing mice. We showed that melphalan treatment resulted in a rapid burst of inflammatory cytokines and chemokines during the cellular recovery phase after melphalan-induced myelodepletion and leukodepletion. After melphalan treatment, tumor cells exhibited characteristics of immunogenic cell death, including membrane translocation of the endoplasmic reticulum-resident calreticulin and extracellular release of high-mobility group box 1. Additionally, there was enhanced tumor Ag uptake by dendritic cells in the tumor-draining lymph node. Consistent with these immunomodulatory effects, melphalan treatment of tumor-bearing mice led to the activation of the endogenous CD8(+) T cells and, more importantly, effectively drove the clonal expansion and effector differentiation of adoptively transferred tumor-specific CD4(+) T cells. Notably, the combination of melphalan and CD4(+) T cell adoptive cell therapy was more efficacious than either treatment alone in prolonging the survival of mice with advanced B cell lymphomas or colorectal tumors. These findings provide mechanistic insights into melphalan's immunostimulatory effects and demonstrate the therapeutic potential of combining melphalan with adoptive cell therapy utilizing antitumor CD4(+) T cells.


Hitting the target: emerging technologies in the search for kinase substrates.

Abstract: Through phosphorylation, protein kinases can alter the activity, localization, protein association, and stability of their targets. Despite the importance to our understanding of all aspects of cell biology, progress toward identifying bona fide substrates of specific protein kinases has been slow. Traditionally used techniques to identify true kinase substrates, such as genetics, yeast two-hybrid screens, and biochemical purification, are often laborious and unreliable. However, several new approaches have recently been developed and used successfully to identify genuine in vivo substrates of certain protein kinases. These methods include screening for phosphorylation of proteins from phage expression libraries, peptide library screens to determine optimal motifs favored by specific kinases, the use of phospho-motif antibodies, and an approach that uses structurally altered kinases and allele-specific adenosine triphosphate analogs and kinase inhibitors. We describe these approaches and discuss their utility and inherent caveats.


Neurodevelopmental Outcome After a Single Course of Antenatal Steroids in Children Born Preterm: A Systematic Review and Meta-analysis.

Abstract:
OBJECTIVE To systematically review and integrate data on the neurodevelopmental outcome of children after administration of a single course of antenatal corticosteroids for threatened preterm labor.
 DATA SOURCES MEDLINE, Scopus, CENTRAL, and www.clinicaltrials.gov (inception to August 2014) using combinations of the terms "prenatal," "antenatal," "cortico*," "*steroid*," "betamethasone," "dexamethasone," "neurodevelopment*," "*development*," and "follow-up. " We perused the references of the retrieved articles.
 METHODS OF STUDY SELECTION We included randomized and nonrandomized trials reporting on the neurodevelopmental outcomes of children whose mothers were administered a single course of betamethasone or dexamethasone antenatally for threatened preterm birth as opposed to placebo or no treatment.    TABULATION, INTEGRATION, AND RESULTS Summary risk ratio (RR) was calculated for dichotomous data; standardized mean difference was calculated for trials that measured the same outcome but used different methods. Heterogeneity was assessed using the I statistic. Sensitivity and subgroup analyses were planned according to study design, specific steroid, and mean gestational age at birth. A single course of antenatal corticosteroids was associated with reduced risk for cerebral palsy (seven studies; treated: 390 of 5,199, untreated: 146 of 1,379; RR 0.678, 95% confidence interval [CI] 0.564-0.815), psychomotor development index less than 70 (two studies; treated: 783 of 3,049, untreated: 258 of 969; RR 0.829, 95% CI 0.737-0.933), and severe disability (five studies; treated: 1,567 of 4,840, untreated: 475 of 1,211; RR 0.787, 95% CI 0.729-0.850). Steroid treatment increased the rates of intact survival (six studies; treated: 1,082 of 2,013, untreated: 273 of 561; RR 1.186, 95% CI 1.056-1.332). Betamethasone was found to significantly decrease the risk for severe disability and increase the rate of intact survival. Dexamethasone increased the rate of intact survival; however, data for dexametasone and the other planned subgroup analyses were limited (fewer than 1,000 children at most). The major limitations involved inclusion of nonrandomized studies and scarcity of data on finer neurodevelopmental outcomes.
 CONCLUSION A single course of antenatal corticosteroids in women at high risk for preterm birth appears to improve most neurodevelopmental outcomes in offspring born before 34 weeks of gestation.


Prognostic impact of minimal pleural effusion in non-small-cell lung cancer.

Abstract:
PURPOSE Minimal (< 10 mm thick) pleural effusion (PE) may represent an early phase of malignant PE, but its clinical relevance has rarely been studied. Therefore, we examined the proportion of minimal PE in patients with non-small-cell lung cancer (NSCLC) and its impact on survival. We also considered possible accumulation mechanisms in our data set.
 PATIENTS AND METHODS On the basis of PE status from chest computed tomography scans at diagnosis, 2,061 patients were classified into three groups: no PE, minimal PE, and malignant PE. Twenty-one variables associated with four factors-patient, stage migration, tumor, and treatment-were investigated for correlation with survival.
 RESULTS Minimal PE presented in 272 patients (13.2%). Of 2,061 patients, the proportion of each stage was the following: 5.2% stage I, 10.9% stage II, 13.2% stage IIIA, 23.8% stage IIIB, and 13.9% stage IV. Minimal PE correlated significantly with shorter survival time than did no PE (median survival time, 7.7 v 17.7 months; log-rank P < .001), even after full adjustment with all variables (adjusted hazard ratio, 1.40; 95% CI, 1.21 to 1.62). Prognostic impact of minimal PE was higher in early versus advanced stages (Pinteraction = .001). In 237 patients (87.8%) with minimal PE, pleural invasion or attachment as a direct mechanism was observed, and it was an independent factor predicting worse survival (P = .03).
 CONCLUSION Minimal PE is a commonly encountered clinical concern in staging NSCLCs. Its presence is an important prognostic factor of worse survival, especially in early-stage disease.