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36386637

We studied the effect of recombinant human interleukin-1 beta (IL-1) and recombinant human tumor necrosis factor alpha/cachectin (TNF) on glucose kinetics in healthy rats by means of a primed constant infusion of D-(6-3H)glucose and D-[U-14C]glucose. During the isotope (6-hour) and monokine (4-hour) infusion, plasma levels of glucagon and insulin were determined and correlated with changes in glucose metabolism. The rates of glucose appearance (Ra) and disappearance (Rd) were elevated only with IL-1 and were associated with an increase in glucagon and a concomitant decrease in the ratio of insulin to glucagon. Plasma glucose concentration was increased early after IL-1 administration and coincided with the peak in the Ra. The augmentation of the metabolic clearance rate (MCR) and percent of flux oxidized by IL-1 suggest that this monokine induces the utilization of glucose as a substrate. TNF administration failed to modify the Ra or Rd, percent of flux oxidized, or MCR. TNF-treated rats increased the percent of glucose recycling, but not the total rate of glucose production. The results of this experiment suggest that endogenous macrophage products participate in the diverse alterations of carbohydrate metabolism seen during injury and/or infection.

Effect of interleukin-1 and tumor necrosis factor/cachectin on glucose turnover in the rat.

36398420

The purpose of this study was to determine the lineage progression of human and murine very small embryonic-like (HuVSEL or MuVSEL) cells in vitro and in vivo. In vitro, HuVSEL and MuVSEL cells differentiated into cells of all three embryonic germ layers. HuVSEL cells produced robust mineralized tissue of human origin compared with controls in calvarial defects. Immunohistochemistry demonstrated that the HuVSEL cells gave rise to neurons, adipocytes, chondrocytes, and osteoblasts within the calvarial defects. MuVSEL cells were also able to differentiate into similar lineages. First round serial transplants of MuVSEL cells into irradiated osseous sites demonstrated that ∼60% of the cells maintained their VSEL cell phenotype while other cells differentiated into multiple tissues at 3 months. Secondary transplants did not identify donor VSEL cells, suggesting limited self renewal but did demonstrate VSEL cell derivatives in situ for up to 1 year. At no point were teratomas identified. These studies show that VSEL cells produce multiple cellular structures in vivo and in vitro and lay the foundation for future cell-based regenerative therapies for osseous, neural, and connective tissue disorders.

Human and murine very small embryonic-like cells represent multipotent tissue progenitors, in vitro and in vivo.

36399107

The tumor suppressor gene p16 (CDKN2/MTS-1/INK4A) can be inactivated by multiple genetic mechanisms. We analyzed 29 invasive primary head and neck squamous cell carcinomas (HNSCC) for p16 inactivation with immunohistochemistry utilizing a new monoclonal antibody (mAb), DCS-50. p16 staining of the primary lesions was correlated with genetic analysis including: (a) detailed microsatellite analysis of markers at the p16 locus to detect homozygous deletion; (b) sequence analysis of p16; and (c) Southern blot analysis to determine the methylation status of the 5' CpG island of p16. Twenty-four of 29 (83%) head and neck squamous cell carcinoma tumors displayed an absence of p16 nuclear staining using immunohistochemistry. Of these 24 tumors, we found that 16 (67%) harbored homozygous deletions, 5 (21%) were methylated, 1 displayed a rearrangement at the p16 locus, and 1 displayed a frameshift mutation in exon 1. These data suggest that: (a) inactivation of the p16 tumor suppressor gene is a frequent event in squamous cell carcinomas of the head and neck; (b) p16 is inactivated by several distinct and exclusive events including homozygous deletion, point mutation, and promoter methylation; and (c) immunohistochemical analysis for expression of the p16 gene product is an accurate and relatively simple method for evaluating p16 gene inactivation.

High frequency of p16 (CDKN2/MTS-1/INK4A) inactivation in head and neck squamous cell carcinoma.

36399109

Recent studies by our group and others demonstrated a required and conserved role of Stim in store-operated Ca(2+) influx and Ca(2+) release-activated Ca(2+) (CRAC) channel activity. By using an unbiased genome-wide RNA interference screen in Drosophila S2 cells, we now identify 75 hits that strongly inhibited Ca(2+) influx upon store emptying by thapsigargin. Among these hits are 11 predicted transmembrane proteins, including Stim, and one, olf186-F, that upon RNA interference-mediated knockdown exhibited a profound reduction of thapsigargin-evoked Ca(2+) entry and CRAC current, and upon overexpression a 3-fold augmentation of CRAC current. CRAC currents were further increased to 8-fold higher than control and developed more rapidly when olf186-F was cotransfected with Stim. olf186-F is a member of a highly conserved family of four-transmembrane spanning proteins with homologs from Caenorhabditis elegans to human. The endoplasmic reticulum (ER) Ca(2+) pump sarco-/ER calcium ATPase (SERCA) and the single transmembrane-soluble N-ethylmaleimide-sensitive (NSF) attachment receptor (SNARE) protein Syntaxin5 also were required for CRAC channel activity, consistent with a signaling pathway in which Stim senses Ca(2+) depletion within the ER, translocates to the plasma membrane, and interacts with olf186-F to trigger CRAC channel activity.

Genome-wide RNAi screen of Ca(2+) influx identifies genes that regulate Ca(2+) release-activated Ca(2+) channel activity.

36432234

Wedelolactone, a plant coumestan, was shown to act as anti-cancer agent for breast and prostate carcinomas in vitro and in vivo targeting multiple cellular proteins including androgen receptors, 5-lipoxygenase and topoisomerase IIα. It is cytotoxic to breast, prostate, pituitary and myeloma cancer cell lines in vitro at μM concentrations. In this study, however, a novel biological activity of nM dose of wedelolactone was demonstrated. Wedelolactone acts as agonist of estrogen receptors (ER) α and β as demonstrated by transactivation of estrogen response element (ERE) in cells transiently expressing either ERα or ERβ and by molecular docking of this coumestan into ligand binding pocket of both ERα and ERβ. In breast cancer cells, wedelolactone stimulates growth of estrogen receptor-positive cells, expression of estrogen-responsive genes and activates rapid non-genomic estrogen signalling. All these effects can be inhibited by pretreatment with pure ER antagonist ICI 182,780 and they are not observed in ER-negative breast cancer cells. We conclude that wedelolactone acts as phytoestrogen in breast cancer cells by stimulating ER genomic and non-genomic signalling pathways.

Wedelolactone induces growth of breast cancer cells by stimulation of estrogen receptor signalling.

36444198

Blood monocytes are well-characterized precursors for macrophages and dendritic cells. Subsets of human monocytes with differential representation in various disease states are well known. In contrast, mouse monocyte subsets have been characterized minimally. In this study we identify three subpopulations of mouse monocytes that can be distinguished by differential expression of Ly-6C, CD43, CD11c, MBR, and CD62L. The subsets share the characteristics of extensive phagocytosis, similar expression of M-CSF receptor (CD115), and development into macrophages upon M-CSF stimulation. By eliminating blood monocytes with dichloromethylene-bisphosphonate-loaded liposomes and monitoring their repopulation, we showed a developmental relationship between the subsets. Monocytes were maximally depleted 18 h after liposome application and subsequently reappeared in the circulation. These cells were exclusively of the Ly-6C(high) subset, resembling bone marrow monocytes. Serial flow cytometric analyses of newly released Ly-6C(high) monocytes showed that Ly-6C expression on these cells was down-regulated while in circulation. Under inflammatory conditions elicited either by acute infection with Listeria monocytogenes or chronic infection with Leishmania major, there was a significant increase in immature Ly-6C(high) monocytes, resembling the inflammatory left shift of granulocytes. In addition, acute peritoneal inflammation recruited preferentially Ly-6C(med-high) monocytes. Taken together, these data identify distinct subpopulations of mouse blood monocytes that differ in maturation stage and capacity to become recruited to inflammatory sites.

Subpopulations of mouse blood monocytes differ in maturation stage and inflammatory response.

36450001

Proteogenomics has emerged as a field at the junction of genomics and proteomics. It is a loose collection of technologies that allow the search of tandem mass spectra against genomic databases to identify and characterize protein-coding genes. Proteogenomic peptides provide invaluable information for gene annotation, which is difficult or impossible to ascertain using standard annotation methods. Examples include confirmation of translation, reading-frame determination, identification of gene and exon boundaries, evidence for post-translational processing, identification of splice-forms including alternative splicing, and also, prediction of completely novel genes. For proteogenomics to deliver on its promise, however, it must overcome a number of technological hurdles, including speed and accuracy of peptide identification, construction and search of specialized databases, correction of sampling bias, and others. This article reviews the state of the art of the field, focusing on the current successes, and the role of computation in overcoming these challenges. We describe how technological and algorithmic advances have already enabled large-scale proteogenomic studies in many model organisms, including arabidopsis, yeast, fly, and human. We also provide a preview of the field going forward, describing early efforts in tackling the problems of complex gene structures, searching against genomes of related species, and immunoglobulin gene reconstruction.

Proteogenomics to discover the full coding content of genomes: a computational perspective.

36464673

We show that, in vitro, Ca2+-dependent protein kinase C (PKC) phosphorylates recombinant murine p53 protein on several residues contained within a conserved basic region of 25 amino acids, located in the C-terminal part of the protein. Accordingly, synthetic p53-(357-381)-peptide is phosphorylated by PKC at multiple Ser and Thr residues, including Ser360, Thr365, Ser370 and Thr377. We also establish that p53-(357-381)-peptide at micromolar concentrations has the ability to stimulate sequence-specific DNA binding by p53. That stimulation is lost upon phosphorylation by PKC. To further characterise the mechanisms that regulate PKC-dependent phosphorylation of p53-(357-381)-peptide, the phosphorylation of recombinant p53 and p53-(357-381)-peptide by PKC were compared. The results suggest that phosphorylation of full-length p53 on the C-terminal PKC sites is highly dependent on the accessibility of the phosphorylation sites and that a domain on p53 distinct from p53-(357-381)-peptide is involved in binding PKC. Accordingly, we have identified a conserved 27-amino-acid peptide, p53-(320-346)-peptide, within the C-terminal region of p53 and adjacent to residues 357-381 that interacts with PKC in vitro. The interaction between p53-(320-346)-peptide and PKC inhibits PKC autophosphorylation and the phosphorylation of substrates, including p53-(357-381)-peptide, neurogranin and histone H1. Conventional Ca2+-dependent PKC alpha, beta and gamma and the catalytic fragment of PKC (PKM) were nearly equally susceptible to inhibition by p53-(320-346)-peptide. The Ca2+-independent PKC delta was much less sensitive to inhibition. The significance of these findings for understanding the in vivo phosphorylation of p53 by PKC are discussed.

The in vitro phosphorylation of p53 by calcium-dependent protein kinase C--characterization of a protein-kinase-C-binding site on p53.

36480032

The ocular microenvironment is both immunosuppressive and anti-inflammatory in nature. Pigment epithelial (PE) cells isolated from the eye possess the ability to suppress the T cell receptor-dependent activation of T cells and the induction of regulatory T cells in vitro. This property is dependent on the cells’ capacity to produce cell-surface and soluble inhibitory molecules, for example CD86 (B7-2), transforming growth factor (TGF)-β, thrombospondin-1, programmed cell death 1 ligand 1 (PD-L1/B7-H1), and cytotoxic T lymphocyte-associated antigen 2α. Cultured ocular PE cells from the iris, ciliary body, and retina can individually suppress T-cell activation via mechanisms that partially overlap. Moreover, PE-derived regulatory T cells acquire functions that play a role in establishing immune regulation in the eye. Multiple strategies are employed within the eye to control immune-mediated inflammation. This phenomenon is known as immune privilege and is instrumental in helping to prevent extensive damage to bystander cells that would otherwise lead to blindness. This review focuses on the immunosuppressive property and role of ocular PE cells in immune privileged sites.

Role of ocular pigment epithelial cells in immune privilege

36497180

Salmonellosis poses a health problem of large proportions in the United States. Annually, it accounts for more than 40,000 reported cases, 500 deaths, and financial costs well in excess of $50 million. Antimicrobial resistance is increasing in Salmonella strains, a finding that has important public health implications. Although the chain of transmission of the bacteria is often complex, combined epidemiologic and laboratory studies with the use of new methods in molecular biology make it possible to trace antimicrobial-resistant salmonellae to their primary source--foods of animal origin. These studies suggest that the antimicrobial drugs to which food animals are exposed provide selective pressure that leads to the appearance and persistence of resistant strains.

Drug-resistant Salmonella in the United States: an epidemiologic perspective.

36540079

Deamidation of N-terminal Gln by Nt(Q)-amidase, an N-terminal amidohydrolase, is a part of the N-end rule pathway of protein degradation. We detected the activity of Nt(Q)-amidase, termed Ntaq1, in mouse tissues, purified Ntaq1 from bovine brains, identified its gene, and began analyzing this enzyme. Ntaq1 is highly conserved among animals, plants, and some fungi, but its sequence is dissimilar to sequences of other amidases. An earlier mutant in the Drosophila Cg8253 gene that we show here to encode Nt(Q)-amidase has defective long-term memory. Other studies identified protein ligands of the uncharacterized human C8orf32 protein that we show here to be the Ntaq1 Nt(Q)-amidase. Remarkably, "high-throughput" studies have recently solved the crystal structure of C8orf32 (Ntaq1). Our site-directed mutagenesis of Ntaq1 and its crystal structure indicate that the active site and catalytic mechanism of Nt(Q)-amidase are similar to those of transglutaminases.

Glutamine-specific N-terminal amidase, a component of the N-end rule pathway.

36547290

IL-6 is an immunoregulatory cytokine with multiple functions in hemopoiesis, proliferation, and tumorigenesis. IL-6 triggers phosphorylation, dimerization, and nuclear translocation of STAT3, which binds to target promoters and activates transcription. Brahma-related gene 1 (BRG1), the enzymatic engine of the yeast-mating type-switching and sucrose-nonfermenting chromatin-remodeling complex, is essential for recruitment of STAT1 or STAT1/STAT2-containing complexes to IFN targets. We hypothesized that BRG1 might also be required for STAT3 recruitment. In this study, we show that induction of a subset of human IL-6-responsive genes is BRG1 dependent. BRG1 is constitutively present at these targets and is required for STAT3 recruitment, downstream histone modifications, and IL-6-induced chromatin remodeling. IL-6-induced recruitment of STAT3 to the IFN regulatory factor 1 promoter and subsequent mRNA synthesis is BRG1 dependent, even though IFN-gamma-mediated STAT1 recruitment to this locus is BRG1 independent. BRG1 also increased basal expression of IFN-induced transmembrane protein 3 and IFN-gamma-induced protein 16, and the basal chromatin accessibility at the promoter of IFN regulatory factor 1. The effect on basal expression was STAT3 independent, as revealed by small interfering RNA knockdown. Together with prior observations, these data reveal that BRG1 has a broad role in mediating STAT accessibility at multiple cytokine-responsive promoters and exposes promoter specific differences in both the effect of BRG1 on basal chromatin accessibility and on access of different STAT proteins to the same target.

Brahma-related gene 1-dependent STAT3 recruitment at IL-6-inducible genes.

36558211

OBJECTIVES To explore the different characteristics of high and low fat consumers, in particular their macronutrient intake and body mass index. DESIGN Reanalysis of data from the Dietary and Nutritional Survey of British Adults. Comparisons were made between groups defined as high and low fat consumers on the basis of 7-day weighed food records considered to be valid for energy intake. Individuals were classified in two ways according to the percentage energy from fat (FAT%) and the absolute amount of fat consumed (FATg). The criteria for classification of the high FAT% being > 45% (high fat) and < or = 35% (low fat). For the FATg group the threshold for the high fat group was > 138 g/day (men) and > 102 g/day (women), and for the low fat group < 85 g/day (men) and < 70 g/day (women). SETTING Dietary data was collected from private households in Great Britain between 1986 and 1987. SUBJECTS From the total population of 2197, individuals who were slimming, ill or had an EI: BMR of < 1.2 were excluded in order to use data which was most likely to represent habitual energy intakes. From the remaining 1240 subjects, 10.8% of this sample (6.1% of the total population) were classified as low fat consumers (76 men and 58 women) and 15.4% high fat (8.7% of the total population, 93 men and 98 women). MAIN OUTCOME MEASURES Macronutrient consumption and body mass index (BMI). RESULTS 30% of the subjects changed fat group classification when the criteria of defining high and low fat groups altered from FAT% to FATg. Nutrient intakes differed according to definition of the groups. The high fat FATg group ate significantly more of all nutrients than the low fat FATg group. However, this was not seen for the FAT% analysis, with the high fat group eating more fat and less carbohydrate. The average BMI tended to be higher in the high fat than the low fat groups, particularly in the FATg analysis. However, the high fat group contained a wide range of BMIs. Further exploration of BMI in the high fat groups, showed that age (an 11-year difference) was the only variable to distinguish individuals in the top and bottom quartiles of BMI. CONCLUSIONS High and low fat consumers differ according to a number of variables, and this is affected by how these groups are defined (FAT% or FATg). High fat consumers tend to have a higher BMI than low fat consumers, but not all high fat consumers are overweight or obese.

High and low fat consumers, their macronutrient intake and body mass index: further analysis of the National Diet and Nutrition Survey of British Adults.

36606083

Many fundamental aspects of DNA replication, such as the exact locations where DNA synthesis is initiated and terminated, how frequently origins are used, and how fork progression is influenced by transcription, are poorly understood. Via the deep sequencing of Okazaki fragments, we comprehensively document replication fork directionality throughout the S. cerevisiae genome, which permits the systematic analysis of initiation, origin efficiency, fork progression, and termination. We show that leading-strand initiation preferentially occurs within a nucleosome-free region at replication origins. Using a strain in which late origins can be induced to fire early, we show that replication termination is a largely passive phenomenon that does not rely on cis-acting sequences or replication fork pausing. The replication profile is predominantly determined by the kinetics of origin firing, allowing us to reconstruct chromosome-wide timing profiles from an asynchronous culture.

Quantitative, genome-wide analysis of eukaryotic replication initiation and termination.

36618603

The differentiation in vitro of murine embryonic stem cells to embryoid bodies mimics events that occur in vivo shortly before and after embryonic implantation. We have used this system, together with differential cDNA cloning, to identify genes the expression of which is regulated during early embryogenesis. Here we describe the isolation of several such cDNA clones, one of which corresponds to the gene H19. This gene is activated in extraembryonic cell types at the time of implantation, suggesting that it may play a role at this stage of development, and is subsequently expressed in all of the cells of the mid-gestation embryo with the striking exception of most of those of the developing central and peripheral nervous systems. After birth, expression of this gene ceases or is dramatically reduced in all tissues.

The murine H19 gene is activated during embryonic stem cell differentiation in vitro and at the time of implantation in the developing embryo.

36623997

In wild-type budding yeast strains, the proteins encoded by SIR3, SIR4 and RAP1 co-localize with telomeric DNA in a limited number of foci in interphase nuclei. Immunostaining of Sir2p shows that in addition to a punctate staining that coincides with Rap1 foci, Sir2p localizes to a subdomain of the nucleolus. The presence of Sir2p at both the spacer of the rDNA repeat and at telomeres is confirmed by formaldehyde cross-linking and immunoprecipitation with anti-Sir2p antibodies. In strains lacking Sir4p, Sir3p becomes concentrated in the nucleolus, by a pathway requiring SIR2 and UTH4, a gene that regulates life span in yeast. The unexpected nucleolar localization of Sir2p and Sir3p correlates with observed effects of sir mutations on rDNA stability and yeast longevity, defining a new site of action for silent information regulatory factors.

Localization of Sir2p: the nucleolus as a compartment for silent information regulators.

36637129

Reprogramming of somatic cells into pluripotency stem cell state has opened new opportunities in cell replacement therapy and disease modeling in a number of neurological disorders. It still remains unknown, however, to what degree the grafted human-induced pluripotent stem cells (hiPSCs) differentiate into a functional neuronal phenotype and if they integrate into the host circuitry. Here, we present a detailed characterization of the functional properties and synaptic integration of hiPSC-derived neurons grafted in an in vitro model of hyperexcitable epileptic tissue, namely organotypic hippocampal slice cultures (OHSCs), and in adult rats in vivo. The hiPSCs were first differentiated into long-term self-renewing neuroepithelial stem (lt-NES) cells, which are known to form primarily GABAergic neurons. When differentiated in OHSCs for 6 weeks, lt-NES cell-derived neurons displayed neuronal properties such as tetrodotoxin-sensitive sodium currents and action potentials (APs), as well as both spontaneous and evoked postsynaptic currents, indicating functional afferent synaptic inputs. The grafted cells had a distinct electrophysiological profile compared to host cells in the OHSCs with higher input resistance, lower resting membrane potential, and APs with lower amplitude and longer duration. To investigate the origin of synaptic afferents to the grafted lt-NES cell-derived neurons, the host neurons were transduced with Channelrhodopsin-2 (ChR2) and optogenetically activated by blue light. Simultaneous recordings of synaptic currents in grafted lt-NES cell-derived neurons using whole-cell patch-clamp technique at 6 weeks after grafting revealed limited synaptic connections from host neurons. Longer differentiation times, up to 24 weeks after grafting in vivo, revealed more mature intrinsic properties and extensive synaptic afferents from host neurons to the lt-NES cell-derived neurons, suggesting that these cells require extended time for differentiation/maturation and synaptogenesis. However, even at this later time point, the grafted cells maintained a higher input resistance. These data indicate that grafted lt-NES cell-derived neurons receive ample afferent input from the host brain. Since the lt-NES cells used in this study show a strong propensity for GABAergic differentiation, the host-to-graft synaptic afferents may facilitate inhibitory neurotransmitter release, and normalize hyperexcitable neuronal networks in brain diseases, for example, such as epilepsy.

Optogenetics reveal delayed afferent synaptogenesis on grafted human-induced pluripotent stem cell-derived neural progenitors.

36642096

BACKGROUND Type 1 diabetes mellitus is a chronic autoimmune disease caused by the pathogenic action of T lymphocytes on insulin-producing beta cells. Previous clinical studies have shown that continuous immune suppression temporarily slows the loss of insulin production. Preclinical studies suggested that a monoclonal antibody against CD3 could reverse hyperglycemia at presentation and induce tolerance to recurrent disease. METHODS We studied the effects of a nonactivating humanized monoclonal antibody against CD3--hOKT3gamma1(Ala-Ala)--on the loss of insulin production in patients with type 1 diabetes mellitus. Within 6 weeks after diagnosis, 24 patients were randomly assigned to receive either a single 14-day course of treatment with the monoclonal antibody or no antibody and were studied during the first year of disease. RESULTS Treatment with the monoclonal antibody maintained or improved insulin production after one year in 9 of the 12 patients in the treatment group, whereas only 2 of the 12 controls had a sustained response (P=0.01). The treatment effect on insulin responses lasted for at least 12 months after diagnosis. Glycosylated hemoglobin levels and insulin doses were also reduced in the monoclonal-antibody group. No severe side effects occurred, and the most common side effects were fever, rash, and anemia. Clinical responses were associated with a change in the ratio of CD4+ T cells to CD8+ T cells 30 and 90 days after treatment. CONCLUSIONS Treatment with hOKT3gamma1(Ala-Ala) mitigates the deterioration in insulin production and improves metabolic control during the first year of type 1 diabetes mellitus in the majority of patients. The mechanism of action of the anti-CD3 monoclonal antibody may involve direct effects on pathogenic T cells, the induction of populations of regulatory cells, or both.

Anti-CD3 monoclonal antibody in new-onset type 1 diabetes mellitus.

36651210

Embryonic stem cells have the ability to remain undifferentiated and proliferate indefinitely in vitro while maintaining the potential to differentiate into derivatives of all three embryonic germ layers. These cells have, therefore, potential for in vitro differentiation studies, gene function, and so on. The aim of this study was to produce a human embryonic stem cell line. An inner cell mass of a human blastocyst was separated and cultured on mouse embryonic fibroblasts in embryonic stem cell medium with related additives. The established line was evaluated by morphology; passaging; freezing and thawing; alkaline phosphatase; Oct-4 expression; anti-surface markers including Tra-1-60 and Tra-1-81; and karyotype and spontaneous differentiation. Differentiated cardiomyocytes and neurons were evaluated by transmission electron microscopy and immunocytochemistry. Here, we report the derivation of a new embryonic stem cell line (Royan H1) from a human blastocyst that remains undifferentiated in morphology during continuous passaging for more than 30 passages, maintains a normal XX karyotype, is viable after freezing and thawing, and expresses alkaline phosphatase, Oct-4, Tra-1-60, and Tra-1-81. These cells remain undifferentiated when grown on mouse embryonic fibroblast feeder layers in the presence or absence of recombinant human leukemia inhibitory factor. Royan H1 cells can differentiate in vitro in the absence of feeder cells and can produce embryoid bodies that can further differentiate into beating cardiomyocytes as well as neurons. These results define Royan H1 cells as a new human embryonic stem cell line.

Establishment and in vitro differentiation of a new embryonic stem cell line from human blastocyst.

36653415

Cancer cells consume glucose and secrete lactate in culture. It is unknown whether lactate contributes to energy metabolism in living tumors. We previously reported that human non-small-cell lung cancers (NSCLCs) oxidize glucose in the tricarboxylic acid (TCA) cycle. Here, we show that lactate is also a TCA cycle carbon source for NSCLC. In human NSCLC, evidence of lactate utilization was most apparent in tumors with high 18fluorodeoxyglucose uptake and aggressive oncological behavior. Infusing human NSCLC patients with 13C-lactate revealed extensive labeling of TCA cycle metabolites. In mice, deleting monocarboxylate transporter-1 (MCT1) from tumor cells eliminated lactate-dependent metabolite labeling, confirming tumor-cell-autonomous lactate uptake. Strikingly, directly comparing lactate and glucose metabolism in vivo indicated that lactate's contribution to the TCA cycle predominates. The data indicate that tumors, including bona fide human NSCLC, can use lactate as a fuel in vivo.

Lactate Metabolism in Human Lung Tumors

36654066

Methionine is converted by the transmethylation/transsulfuration pathway to homocysteine which may exert atherogenic effects by several mechanisms, including lipid peroxidation. Therefore, the excessive dietary methionine may induce the development of atherosclerosis. To test this hypothesis, plasma and aortic thiobarbituric acid reactive substances (TBARS), as well as activities of aortic and erythrocyte superoxide dismutase (SOD), catalase and selenium-dependent glutathione peroxidase (GPX) were measured in rabbits fed a diet enriched with 0.3% methionine for 6 or 9 months. Histological examinations of aortas also were performed. Feeding rabbits a methionine-enriched diet for 6 or 9 months resulted in significant increases in plasma and aortic TBARS levels and aortic antioxidant enzyme activities. However, a decrease in plasma antioxidant activity (AOA) was observed. In erythrocytes, SOD activity increased, catalase remained normal and GPX decreased in the treated animals. Histological examination of aortas showed typical atherosclerotic changes, such as intimal thickening, deposition of cholesterol, and calcification in methionine-fed rabbits. These results confirm that high-methionine diet may induce atherosclerosis in rabbits and indicate disturbances in lipid peroxidation and antioxidant processes as possible mechanisms of its atherogenic influence.

Increased lipid peroxidation as a mechanism of methionine-induced atherosclerosis in rabbits.

36708463

A major question is whether genes encoded on the sex chromosomes act directly in non-gonadal tissues to cause sex differences in development or function, or whether all sex differences in somatic tissues are induced by gonadal secretions. As part of this question we asked whether mouse X-Y homologous gene pairs are expressed in brain in a sex-specific fashion. Using RT-PCR and northern blot analysis, we assessed mRNA expression in brain of eight Y-linked genes as well as their X-linked homologues, at three ages: 13.5 days post coitum, the day of birth (P1) and adult. Transcripts of six Y genes were expressed at one or more ages: Usp9y, Ube1y, Smcy, Eif2s3y, Uty and Dby. Their expression also occurred in XY female brain, and therefore does not require testicular secretions. Six X-linked homologues (Usp9x, Ube1x, Smcx, Eif2s3x, Utx and Dbx) were also expressed in brain, and in adulthood all of these transcripts were expressed at significantly higher levels in brains of females than in brains of males, irrespective of their X-inactivation status. For five of these gene pairs, the expression of the Y-linked homologue in males was not sufficient to compensate for the female bias in X gene expression. Three X-Y gene pairs, Usp9x/y, Ube1x/y and Eif2s3x/y, appeared to be differentially regulated (expressed in brain in a different age- or tissue-dependent pattern), and hence may not be functionally equivalent. These sex differences in X-Y gene expression suggest several mechanisms by which these genes may participate in sex differences in brain development and function.

Sex differences in sex chromosome gene expression in mouse brain.

36713289

An increasing number of human diseases are recognized to result from recurrent DNA rearrangements involving unstable genomic regions. These are termed genomic disorders, in which the clinical phenotype is a consequence of abnormal dosage of gene(s) located within the rearranged genomic fragments. Both inter- and intrachromosomal rearrangements are facilitated by the presence of region-specific low-copy repeats (LCRs) and result from nonallelic homologous recombination (NAHR) between paralogous genomic segments. LCRs usually span approximately 10-400 kb of genomic DNA, share >or= 97% sequence identity, and provide the substrates for homologous recombination, thus predisposing the region to rearrangements. Moreover, it has been suggested that higher order genomic architecture involving LCRs plays a significant role in karyotypic evolution accompanying primate speciation.

Genome architecture, rearrangements and genomic disorders.

36721932

OBJECTIVE Nociceptive and neuropathic components both contribute to pain. Since these components require different pain management strategies, correct pain diagnosis before and during treatment is highly desirable. As low back pain (LBP) patients constitute an important subgroup of chronic pain patients, we addressed the following issues: (i) to establish a simple, validated screening tool to detect neuropathic pain (NeP) components in chronic LBP patients, (ii) to determine the prevalence of neuropathic pain components in LBP in a large-scale survey, and (iii) to determine whether LBP patients with an NeP component suffer from worse, or different, co-morbidities. METHODS In co-operation with the German Research Network on Neuropathic Pain we developed and validated the painDETECT questionnaire (PD-Q) in a prospective, multicentre study and subsequently applied it to approximately 8000 LBP patients. RESULTS The PD-Q is a reliable screening tool with high sensitivity, specificity and positive predictive accuracy; these were 84% in a palm-top computerised version and 85%, 80% and 83%, respectively, in a corresponding pencil-and-paper questionnaire. In an unselected cohort of chronic LBP patients, 37% were found to have predominantly neuropathic pain. Patients with NeP showed higher ratings of pain intensity, with more (and more severe) co-morbidities such as depression, panic/anxiety and sleep disorders. This also affected functionality and use of health-care resources. On the basis of given prevalence of LBP in the general population, we calculated that 14.5% of all female and 11.4% of all male Germans suffer from LBP with a predominant neuropathic pain component. CONCLUSION Simple, patient-based, easy-to-use screening questionnaires can determine the prevalence of neuropathic pain components both in individual LBP patients and in heterogeneous cohorts of such patients. Since NeP correlates with more intense pain, more severe co-morbidity and poorer quality of life, accurate diagnosis is a milestone in choosing appropriate therapy.

painDETECT: a new screening questionnaire to identify neuropathic components in patients with back pain.

36749390

Purpose To propose a semiquantitative dual fluorescein angiography (FA) and indocyanine green angiography (ICGA) scoring system for uveitis that would assist in the follow-up of disease progression and monitoring response to treatment. Methods The scoring system was based on the FA scoring systems, the standardized ICGA protocol, and schematic interpretation of ICGA findings in posterior uveitis that have been previously published. We assigned scores to the fluorescein and ICG angiographic signs that represent ongoing inflammatory process in the posterior segment. We rated each angiographic sign according to the impact it has on our appreciation of active intraocular inflammation. In order to permit direct comparison between FA and ICGA, we multiplied the total ICGA score by a coefficient of 2 to adjust to the total score of FA. Results A total maximum score of 40 was assigned to the FA signs, including optic disc hyperfluorescence, macular edema, retinal vascular staining and/or leakage, capillary leakage, retinal capillary nonperfusion, neovascularization of the optic disc, neovascularization elsewhere, pinpoint leaks, and retinal staining and/or subretinal pooling. A total maximum score of 20 was assigned to the ICGA signs, including early stromal vessel hyperfluorescence, choroidal vasculitis, dark dots or areas (excluding atrophy), and optic disc hyperfluorescence. Conclusion The combined fluorescein and ICG angiographic scoring system proposed herein may help estimate the magnitude of retinal versus choroidal inflammation, monitor disease progression and response to treatment, and provide comparable data for clinical studies. The applicability of the proposed system needs to be tested in clinical settings, and intra- and interobserver variations need to be determined.

Scoring of dual fluorescein and ICG inflammatory angiographic signs for the grading of posterior segment inflammation (dual fluorescein and ICG angiographic scoring system for uveitis)

36799998

Acute kidney injury (AKI) is a complex disorder comprising several etiological factors and occurring in multiple settings. The disorder has a variety of clinical manifestations that range from minimal elevation in serum creatinine level to anuric renal failure. We describe the formation of a multidisciplinary collaborative network focused on AKI. This Acute Kidney Injury Network has proposed uniform standards for diagnosing and classifying AKI. These proposed standards will need to be validated in future studies, a process that will be facilitated by the Acute Kidney Injury Network, which offers a forum that encourages acquisition of knowledge to improve patient outcomes.

Improving outcomes of acute kidney injury: report of an initiative

36816310

Sorting signals for cargo selection into coated vesicles are usually in the form of short linear motifs. Three motifs for clathrin-mediated endocytosis have been identified: YXXPhi, [D/E]XXXL[L/I] and FXNPXY. To search for new endocytic motifs, we made a library of CD8 chimeras with random sequences in their cytoplasmic tails, and used a novel fluorescence-activated cell sorting (FACS)-based assay to select for endocytosed constructs. Out of the five tails that were most efficiently internalized, only one was found to contain a conventional motif. Two contain dileucine-like sequences that appear to be variations on the [D/E]XXXL[L/I] motif. Another contains a novel internalization signal, YXXXPhiN, which is able to function in cells expressing a mutant mu2 that cannot bind YXXPhi, indicating that it is not a variation on the YXXPhi motif. Similar sequences are present in endogenous proteins, including a functional YXXXPhiN (in addition to a classical YXXPhi) in cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Thus, the repertoire of endocytic motifs is more extensive than the three well-characterized sorting signals.

A Screen for Endocytic Motifs

36830715

Hypertrophic scarring and poor intrinsic axon growth capacity constitute major obstacles for spinal cord repair. These processes are tightly regulated by microtubule dynamics. Here, moderate microtubule stabilization decreased scar formation after spinal cord injury in rodents through various cellular mechanisms, including dampening of transforming growth factor-β signaling. It prevented accumulation of chondroitin sulfate proteoglycans and rendered the lesion site permissive for axon regeneration of growth-competent sensory neurons. Microtubule stabilization also promoted growth of central nervous system axons of the Raphe-spinal tract and led to functional improvement. Thus, microtubule stabilization reduces fibrotic scarring and enhances the capacity of axons to grow.

Microtubule stabilization reduces scarring and causes axon regeneration after spinal cord injury.

36831892

Considerable energetic investment is devoted to altering large stretches of chromatin adjacent to DNA double strand breaks (DSBs). Immediately ensuing DSB formation, a myriad of histone modifications are elicited to create a platform for inducible and modular assembly of DNA repair protein complexes in the vicinity of the DNA lesion. This complex signaling network is critical to repair DNA damage and communicate with cellular processes that occur in cis and in trans to the genomic lesion. Failure to properly execute DNA damage inducible chromatin changes is associated with developmental abnormalities, immunodeficiency, and malignancy in humans and in genetically engineered mouse models. This review will discuss current knowledge of DNA damage responsive histone changes that occur in mammalian cells, highlighting their involvement in the maintenance of genome integrity.

Histone tails: Directing the chromatin response to DNA damage.

36838958

Uncoupling protein 1 (Ucp1), which is localized in the mitochondrial inner membrane of mammalian brown adipose tissue (BAT), generates heat by uncoupling oxidative phosphorylation. Upon cold exposure or nutritional abundance, sympathetic neurons stimulate BAT to express Ucp1 to induce energy dissipation and thermogenesis. Accordingly, increased Ucp1 expression reduces obesity in mice and is correlated with leanness in humans. Despite this significance, there is currently a limited understanding of how Ucp1 expression is physiologically regulated at the molecular level. Here, we describe the involvement of Sestrin2 and reactive oxygen species (ROS) in regulation of Ucp1 expression. Transgenic overexpression of Sestrin2 in adipose tissues inhibited both basal and cold-induced Ucp1 expression in interscapular BAT, culminating in decreased thermogenesis and increased fat accumulation. Endogenous Sestrin2 is also important for suppressing Ucp1 expression because BAT from Sestrin2(-/-) mice exhibited a highly elevated level of Ucp1 expression. The redox-inactive mutant of Sestrin2 was incapable of regulating Ucp1 expression, suggesting that Sestrin2 inhibits Ucp1 expression primarily through reducing ROS accumulation. Consistently, ROS-suppressing antioxidant chemicals, such as butylated hydroxyanisole and N-acetylcysteine, inhibited cold- or cAMP-induced Ucp1 expression as well. p38 MAPK, a signaling mediator required for cAMP-induced Ucp1 expression, was inhibited by either Sestrin2 overexpression or antioxidant treatments. Taken together, these results suggest that Sestrin2 and antioxidants inhibit Ucp1 expression through suppressing ROS-mediated p38 MAPK activation, implying a critical role of ROS in proper BAT metabolism.

Sestrin2 inhibits uncoupling protein 1 expression through suppressing reactive oxygen species.

36855703

XPG is a member of the FEN-1 structure-specific endonuclease family. It has 3'-junction cutting activity on bubble substrates and makes the 3'-incision in the human dual incision (excision nuclease) repair system. To investigate the precise role of XPG in nucleotide excision repair, we mutagenized two amino acid residues thought to be involved in DNA binding and catalysis, overproduced the mutant proteins using a baculovirus/insect cell system, and purified and characterized the mutant proteins. The mutation D77A had a modest effect on junction cutting and excision activity and gave rise to uncoupled 5'-incision by mammalian cell-free extracts. The D812A mutation completely abolished the junction cutting and 3'-incision activities of XPG, but the excision nuclease reconstituted with XPG (D812A) carried out normal 5'-incision at the 23rd-24th phosphodiester bonds 5' to a (6-4) photoproduct without producing any 3'-incision. It is concluded that Asp-812 is an active site residue of XPG and that in addition to making the 3'-incision, the physical presence of XPG in the protein-DNA complex is required non-catalytically for subsequent 5'-incision by XPF-ERCC1.

The non-catalytic function of XPG protein during dual incision in human nucleotide excision repair.

36860856

Computer techniques readily extract from the brainwaves an orderly sequence of brain potentials locked in time to sound stimuli. The potentials that appear 8 to 80 msec after the stimulus resemble 3 or 4 cycles of a 40-Hz sine wave; we show here that these waves combined to form a single, stable, composite wave when the sounds are repeated at rates around 40 per sec. This phenomenon, the 40-Hz event-related potential (ERP), displays several properties of theoretical and practical interest. First, it reportedly disappears with surgical anesthesia, and it resembles similar phenomena in the visual and olfactory system, facts which suggest that adequate processing of sensory information may require cyclical brain events in the 30- to 50-Hz range. Second, latency and amplitude measurements on the 40-Hz ERP indicate it may contain useful information on the number and basilar membrane location of the auditory nerve fibers a given tone excites. Third, the response is present at sound intensities very close to normal adult thresholds for the audiometric frequencies, a fact that could have application in clinical hearing testing.

A 40-Hz auditory potential recorded from the human scalp.

36889513

Bile acids regulate the transcription of genes that control cholesterol homeostasis through molecular mechanisms that are poorly understood. Physiological concentrations of free and conjugated chenodeoxycholic acid, lithocholic acid, and deoxycholic acid activated the farnesoid X receptor (FXR; NR1H4), an orphan nuclear receptor. As ligands, these bile acids and their conjugates modulated interaction of FXR with a peptide derived from steroid receptor coactivator 1. These results provide evidence for a nuclear bile acid signaling pathway that may regulate cholesterol homeostasis.

Bile acids: natural ligands for an orphan nuclear receptor.

36904081

The yeast ribosomal protein gene RPL32 of Saccharomyces cerevisiae is of particular interest for two reasons: 1) it is adjacent to another ribosomal protein gene, RP29, whose divergent transcription may be driven from the same control sequences, and 2) it appears that the splicing of its transcript is regulated by the product of the gene, ribosomal protein in L32. RPL32 has been analyzed in detail. It is essential for cell growth. Its sequence predicts L32 to be a protein of 105 amino acids, somewhat basic near the NH2 terminus, rather acidic near the COOH terminus, and homologous to ribosomal protein L30 of mammals. The reading frame has been confirmed by partial NH2-terminal analysis of L32. The nucleotide sequence also predicts an intron of 230 nucleotides, which begins with the unusual sequence GTCAGT and ends 40 nucleotides downstream of the consensus sequence TAC-TAAC. The intron has been confirmed by determination of the sequence of a cDNA clone. Transcription initiates 58 nucleotides upstream of the AUG initiation codon, and the polyadenylation site occurs 100 nucleotides downstream of the termination codon. Regulation of the transcription of ribosomal protein genes has been linked to two related consensus sequences. Analysis of the intergenic region between RP29 and RPL32 reveals three copies of these sequences. A deletion removing all three sequences reduces synthesis of a L32-LacZ fusion protein by more than 90%. Some residual activity, however, remains.

The yeast ribosomal protein L32 and its gene.

36921186

Female human induced pluripotent stem cell (hiPSC) lines exhibit variability in X-inactivation status. The majority of hiPSC lines maintain one transcriptionally active X (Xa) and one inactive X (Xi) chromosome from donor cells. However, at low frequency, hiPSC lines with two Xas are produced, suggesting that epigenetic alterations of the Xi occur sporadically during reprogramming. We show here that X-inactivation status in female hiPSC lines depends on derivation conditions. hiPSC lines generated by the Kyoto method (retroviral or episomal reprogramming), which uses leukemia inhibitory factor (LIF)-expressing SNL feeders, frequently had two Xas. Early passage Xa/Xi hiPSC lines generated on non-SNL feeders were converted into Xa/Xa hiPSC lines after several passages on SNL feeders, and supplementation with recombinant LIF caused reactivation of some of X-linked genes. Thus, feeders are a significant factor affecting X-inactivation status. The efficient production of Xa/Xa hiPSC lines provides unprecedented opportunities to understand human X-reactivation and -inactivation.

Derivation conditions impact X-inactivation status in female human induced pluripotent stem cells.

36950726

Several research groups have recently reported on markedly reduced levels of 5-hydroxymethylcytosine (5hmC) in human breast, liver, lung, pancreatic, colon, prostate, brain, and myeloid cancers. We studied benign compound nevi (BCN, n=17), dysplastic compound nevi (DCN, n=15), superficial spreading melanomas [SSM, stratified in <1 mm (n=19) and >4 mm (n=18) Breslow tumor thickness], and cutaneous metastatic disease (CMD, n=24). Immunohistochemistry included specific antibodies against 5hmC, 5-methylcytosine (5mC), and ten-eleven translocation 2 protein (TET2). Immunohistological scoring showed significantly (P<0.0001) higher median 5hmC levels in BCN and DCN than in thin SSM, thick SSM, and CMD. 5mC immunoreactivity did not differ significantly (P=0.15) between nevi and melanoma. The intensity of TET2 expression was predominantly weak but was found to be significantly (P<0.0001) more often in nevi than in thin SSM, thick SSM, and CMD. We have shown that 5hmC levels and TET2 expression are significantly reduced in advanced melanomas compared with nevi and thin melanomas. It is suggested that 5hmC and TET2 possibly play an important role in the epigenetic regulation of melanoma development and progression.

Loss of 5-hydroxymethylcytosine and ten-eleven translocation 2 protein expression in malignant melanoma.

36960449

BACKGROUND Knowledge gaps have contributed to considerable variation among international dietary recommendations for vitamin D. OBJECTIVE We aimed to establish the distribution of dietary vitamin D required to maintain serum 25-hydroxyvitamin D [25(OH)D] concentrations above several proposed cutoffs (ie, 25, 37.5, 50, and 80 nmol/L) during wintertime after adjustment for the effect of summer sunshine exposure and diet. DESIGN A randomized, placebo-controlled, double-blind 22-wk intervention study was conducted in men and women aged 20-40 y (n = 238) by using different supplemental doses (0, 5, 10, and 15 microg/d) of vitamin D(3) throughout the winter. Serum 25(OH)D concentrations were measured by using enzyme-linked immunoassay at baseline (October 2006) and endpoint (March 2007). RESULTS There were clear dose-related increments (P < 0.0001) in serum 25(OH)D with increasing supplemental vitamin D(3). The slope of the relation between vitamin D intake and serum 25(OH)D was 1.96 nmol x L(-1) x microg(-1) intake. The vitamin D intake that maintained serum 25(OH)D concentrations of >25 nmol/L in 97.5% of the sample was 8.7 microg/d. This intake ranged from 7.2 microg/d in those who enjoyed sunshine exposure, 8.8 microg/d in those who sometimes had sun exposure, and 12.3 microg/d in those who avoided sunshine. Vitamin D intakes required to maintain serum 25(OH)D concentrations of >37.5, >50, and >80 nmol/L in 97.5% of the sample were 19.9, 28.0, and 41.1 microg/d, respectively. CONCLUSION The range of vitamin D intakes required to ensure maintenance of wintertime vitamin D status [as defined by incremental cutoffs of serum 25(OH)D] in the vast majority (>97.5%) of 20-40-y-old adults, considering a variety of sun exposure preferences, is between 7.2 and 41.1 microg/d.

Estimation of the dietary requirement for vitamin D in healthy adults.

36962270

We have modified an Escherichia coli vector expressing 66-kDa HIV-1 reverse transcriptase (p66) so that it simultaneously expresses this and the pol-coded protease. The twin expression cassette yields high quantities of both reverse transcriptase and protease; however, under these conditions, 50% of the over-expressed p66 reverse transcriptase is processed, resulting in accumulation of large quantities of p66/p51 enzyme. Furthermore, addition of a poly(histidine) affinity label at the amino terminus of the reverse-transcriptase-coding sequence (His-p66) permits a simple, rapid purification of milligram quantities of either p66 or p66/p51 enzyme from a crude lysate by metal chelate affinity chromatography. Purified His-p66 and His-p66/His-p51 reverse transcriptase exhibit both reverse transcriptase and RNase H activity. Purification by metal chelate chromatography of a p66/p51 enzyme wherein only the p66 component is labelled strengthens the argument for the existence of a heterodimer.

Rapid purification of homodimer and heterodimer HIV-1 reverse transcriptase by metal chelate affinity chromatography.

36991551

Abstract Some cocoas and chocolates are rich in ()epicatechin and its related oligomers, the procyanidins. Fractions of these compounds, isolated from the seeds of Theobroma cacao, caused dosedependent inhibition of isolated rabbit 15-lipoxygenase-1 with the larger oligomers being more active; the decamer fraction revealed an IC 50 of 0.8 M. Among the monomeric flavanols, epigallocatechin gallate (IC 50 = 4 M) and epicatechin gallate (5 M) were more potent than ()epicatechin (IC50 = 60 M). ()Epicatechin and procyanidin nonamer also inhibited the formation of 15-hydroxyeicosatetraenoic acid from arachidonic acid in rabbit smooth muscle cells transfected with human 15-lipoxygenase-1. In contrast, inhibition of the lipoxygenase pathway in J774A.1 cells transfected with porcine leukocytetype 12- lipoxygenase (another representative of the 12/15- lipoxygenase family) was only observed upon sonication of the cells, suggesting a membrane barrier for flavanols in these cells. Moreover, epicatechin (IC50 approx. 15 M) and the procyanidin decamer inhibited recombinant human platelet 12-lipoxygenase. These observations suggest general lipoxygenase inhibitory potency of flavanols and procyanidins that may contribute to their putative beneficial effects on the cardiovascular system in man. Thus, they may provide a plausible explanation for recent literature reports indicating that procyanidins decrease the leukotriene/prostacyclin ratio in humans and human aortic endothelial cells.

Polyphenols of Cocoa: Inhibition of Mammalian 15-Lipoxygenase

37029185

Although evaluation of the treatment of congestive heart failure is usually based on objective clinical outcomes, patient self-assessment is increasingly recognized as an important component of evaluation. A study was designed to measure the quality of life of 134 patients with symptoms of advanced heart failure who were being evaluated for possible heart transplantation. The patients' quality of life was assessed using a mix of subjective and objective measures, including functional status, physical symptoms, emotional state, and psychosocial adaptation. There was no significant relationship between patients' cardiac ejection fraction and any quality-of-life measures; however, the results of a 6-minute walking test, New York Heart Association classification, and self-reported functional status were all significantly correlated with psychosocial adjustment. Self-reported functional status, depression, and hostility accounted for 43% of the variance in total psychosocial adjustment to illness. These findings support the inclusion of quality of life as an outcome measure in any evaluation of treatment efficacy and suggest that interventions to improve the quality of life of patients with advanced heart failure need to be targeted at reducing depression and hostility and increasing daily activity levels.

Quality of life in patients with advanced heart failure.

37037012

This study was designed to determine whether cardiac vagal afferents exert an inhibitory influence on increases in regional vascular resistance during exercise and to determine whether endurance exercise training enhances the inhibitory influence of cardiac vagal afferents. We measured changes in regional vascular resistance in 12 rabbits at rest and during running at 12.6 m/min, 20% grade, before and after reversible denervation of cardiac afferents (intrapericardial procainamide HCl, 2%). In addition, these procedures were repeated in five of these rabbits following an 8-wk endurance exercise training program. Because intrapericardial injections of procainamide anesthetize both the efferent as well as the afferent innervation to the heart, it was necessary to determine the effects of blocking the efferent innervation on the regulation of regional vascular resistance during exercise. Rabbits were instrumented with Doppler ultrasonic flow probes around the renal (R), mesenteric (M), ascending, and terminal aortic (TA) arteries. Catheters were positioned in the central ear artery and vein and pericardial sac. Mean arterial pressure, heart rate, cardiac output, R, M, TA, and systemic (S) resistances were determined. Exercise changed R (+37 +/- 4%), M (+88 +/- 9%), TA (-62 +/- 6%), and S (-34 +/- 3) resistances. Subsequent cardiac efferent blockade alone had no significant effect on regional vascular resistance during exercise. Combined efferent and afferent blockade resulted in significant increases in R (+62 +/- 6%) and M resistance (+134 +/- 13%) but did not alter TA (-51 +/- 4%) or S (-27 +/- 2%) resistance during exercise. Exercise training significantly enhanced the inhibitory influence of cardiac afferents on R and M regional vascular resistance.(ABSTRACT TRUNCATED AT 250 WORDS)

Regional vascular resistance during exercise: role of cardiac afferents and exercise training.

37065914

BACKGROUND AND PURPOSE Soluble corin was decreased in coronary heart disease. Given the connections between cardiac dysfunction and stroke, circulating corin might be a candidate marker of stroke risk. However, the association between circulating corin and stroke has not yet been studied in humans. Here, we aimed to examine the association in patients wtith stroke and community-based healthy controls. METHODS Four hundred eighty-one patients with ischemic stroke, 116 patients with hemorrhagic stroke, and 2498 healthy controls were studied. Serum soluble corin and some conventional risk factors of stroke were examined. Because circulating corin was reported to be varied between men and women, the association between serum soluble corin and stroke was evaluated in men and women, respectively. RESULTS Patients with ischemic and hemorrhagic stroke had a significantly lower level of serum soluble corin than healthy controls in men and women (all P values, <0.05). In multivariate analysis, men in the lowest quartile of serum soluble corin were more likely to have ischemic (odds ratio [OR], 4.90; 95% confidence interval, 2.99-8.03) and hemorrhagic (OR, 17.57; 95% confidence interval, 4.85-63.71) stroke than men in the highest quartile. Women in the lowest quartile of serum soluble corin were also more likely to have ischemic (OR, 3.10; 95% confidence interval, 1.76-5.44) and hemorrhagic (OR, 8.54; 95% confidence interval, 2.35-31.02) stroke than women in the highest quartile. ORs of ischemic and hemorrhagic stroke were significantly increased with the decreasing levels of serum soluble corin in men and women (all P values for trend, <0.001). CONCLUSIONS Serum soluble corin was decreased in patients with stroke compared with healthy controls. Our findings raise the possibility that serum soluble corin may have a pathogenic role in stroke.

Serum Soluble Corin is Decreased in Stroke.

37118634

BACKGROUND Umbilical cord infection (omphalitis) is a risk factor for neonatal sepsis and mortality in low-resource settings where home deliveries are common. We aimed to assess the effect of umbilical-cord cleansing with 4% chlorhexidine (CHX) solution, with or without handwashing with antiseptic soap, on the incidence of omphalitis and neonatal mortality. METHODS We did a two-by-two factorial, cluster-randomised trial in Dadu, a rural area of Sindh province, Pakistan. Clusters were defined as the population covered by a functional traditional birth attendant (TBA), and were randomly allocated to one of four groups (groups A to D) with a computer-generated random number sequence. Implementation and data collection teams were masked to allocation. Liveborn infants delivered by participating TBAs who received birth kits were eligible for enrolment in the study. One intervention comprised birth kits containing 4% CHX solution for application to the cord at birth by TBAs and once daily by family members for up to 14 days along with soap and educational messages promoting handwashing. One intervention was CHX solution only and another was handwashing only. Standard dry cord care was promoted in the control group. The primary outcomes were incidence of neonatal omphalitis and neonatal mortality. The trial is registered with ClinicalTrials.gov, number NCT00682006. FINDINGS 187 clusters were randomly allocated to one of the four study groups. Of 9741 newborn babies delivered by participating TBAs, factorial analysis indicated a reduction in risk of omphalitis with CHX application (risk ratio [RR]=0·58, 95% CI 0·41-0·82; p=0·002) but no evidence of an effect of handwashing (RR=0·83, 0·61-1·13; p=0·24). We recorded strong evidence of a reduction in neonatal mortality in neonates who received CHX cleansing (RR=0·62, 95 % CI 0·45-0·85; p=0·003) but no evidence of an effect of handwashing promotion on neonatal mortality (RR=1·08, 0·79-1·48; p=0·62). We recorded no serious adverse events. INTERPRETATION Application of 4% CHX to the umbilical cord was effective in reducing the risk of omphalitis and neonatal mortality in rural Pakistan. Provision of CHX in birth kits might be a useful strategy for the prevention of neonatal mortality in high-mortality settings. FUNDING The United States Agency for International Development.

Topical application of chlorhexidine to neonatal umbilical cords for prevention of omphalitis and neonatal mortality in a rural district of Pakistan: a community-based, cluster-randomised trial.

37138639

The IKK kinase complex is the core element of the NF-kappaB cascade. It is essentially made of two kinases (IKKalpha and IKKbeta) and a regulatory subunit, NEMO/IKKgamma. Additional components may exist, transiently or permanently, but their characterization is still unsure. In addition, it has been shown that two separate NF-kappaB pathways exist, depending on the activating signal and the cell type, the canonical (depending on IKKbeta and NEMO) and the noncanonical pathway (depending solely on IKKalpha). The main question, which is still only partially answered, is to understand how an NF-kappaB activating signal leads to the activation of the kinase subunits, allowing them to phosphorylate their targets and eventually induce nuclear translocation of the NF-kappaB dimers. I will review here the genetic, biochemical, and structural data accumulated during the last 10 yr regarding the function of the three IKK subunits.

The IKK complex, a central regulator of NF-kappaB activation.

37156349

Independent mobility in early childhood has been associated with the development of various cognitive and psychosocial skills. However, children with physical disabilities are not always able to move independently and may be at risk for delays in these areas. Early provision of powered mobility can offer young children an opportunity for independent mobility. Despite this, there is little information to help determine when a young child has the cognitive skills necessary to operate a powered wheelchair safely. This current research aims to identify these skills. A cognitive assessment battery and a wheelchair mobility training and assessment program were developed. Twenty-six children with physical disabilities between the ages of 20 and 36 months were evaluated on the cognitive assessment and participated in the wheelchair training and assessment program. A stepwise regression analysis was used to determine which of the cognitive skills predicted wheelchair mobility performance. The cognitive domains of spatial relations and problem solving were found to be significant and accounted for 57% of the variance in wheelchair skills. Developmental cut-off points on these scales as they relate to wheelchair skills are presented and clinical applications are discussed.

Cognitive predictors of young children's readiness for powered mobility.

37164306

A key event in the mechanism of mouse embryonic stem cell (mESC) pluripotency is phosphorylation, dimerisation and translocation to the nucleus of the signal transducer and activator of transcription3, Stat3. We used RNAi to suppress the levels of the co-chaperone Hsp70/Hsp90 organising protein (Hop) in an mESC line. Hop knockdown caused 68% depletion in Stat3 mRNA levels, decreased soluble pYStat3 levels, and led to an extranuclear accumulation of Stat3. The major binding partner of Hop, Hsp90, co-localised with a small non-nuclear fraction of Stat3 in mESCs, and both Stat3 and Hop co-precipitated with Hsp90. Hop knockdown did not affect Nanog and Oct4 protein levels; however, Nanog mRNA levels were decreased. We found that in the absence of Hop, mESCs lost their pluripotent ability to form embryoid bodies with a basement membrane. These data suggest that Hop facilitates the phosphorylation and nuclear translocation of Stat3, implying a role for the Hsp70/Hsp90 chaperone heterocomplex machinery in pluripotency signalling.

Knockdown of the co-chaperone Hop promotes extranuclear accumulation of Stat3 in mouse embryonic stem cells.

37182501

Two mechanisms account for generation of the human antibody repertoire; V(D)J recombination during the early stages of B-cell development in the bone marrow and somatic mutation of immunoglobulin genes in mature B cells responding to antigen in the periphery. V(D)J recombination produces diversity by random joining of gene segments and somatic mutation by introducing random point mutations. Both are required to attain the degree of antigen receptor diversification that is necessary for immune protection: defects in either mechanism are associated with increased susceptibility to infection. However, the downside of producing enormous random diversity in the antibody repertoire is the generation of autoantibodies. To prevent autoimmunity B cells expressing autoantibodies are regulated by strict mechanisms that either modify the specificity of autoantibodies or the fate of cells expressing such antibodies. Abnormalities in B-cell self-tolerance are associated with a large number of autoimmune diseases, but the precise nature of the defects is less well defined. Here we summarize recent data on the self-reactive B-cell repertoire in healthy humans and in patients with autoimmunity.

B-cell self-tolerance in humans.

37204802

Jumonji domain-containing 6 (JMJD6) is a member of the Jumonji C domain-containing family of proteins. Compared to other members of the family, the cellular activity of JMJD6 is still not clearly defined and its biological function is still largely unexplored. Here we report that JMJD6 is physically associated with the tumor suppressor p53. We demonstrated that JMJD6 acts as an α-ketoglutarate- and Fe(II)-dependent lysyl hydroxylase to catalyze p53 hydroxylation. We found that p53 indeed exists as a hydroxylated protein in vivo and that the hydroxylation occurs mainly on lysine 382 of p53. We showed that JMJD6 antagonizes p53 acetylation, promotes the association of p53 with its negative regulator MDMX, and represses transcriptional activity of p53. Depletion of JMJD6 enhances p53 transcriptional activity, arrests cells in the G1 phase, promotes cell apoptosis, and sensitizes cells to DNA damaging agent-induced cell death. Importantly, knockdown of JMJD6 represses p53-dependent colon cell proliferation and tumorigenesis in vivo, and significantly, the expression of JMJD6 is markedly up-regulated in various types of human cancer especially in colon cancer, and high nuclear JMJD6 protein is strongly correlated with aggressive clinical behaviors of colon adenocarcinomas. Our results reveal a novel posttranslational modification for p53 and support the pursuit of JMJD6 as a potential biomarker for colon cancer aggressiveness and a potential target for colon cancer intervention.

JMJD6 Promotes Colon Carcinogenesis through Negative Regulation of p53 by Hydroxylation

37205685

Malaria resistant to chloroquine has now been confirmed in more than 40 countries. The drug was introduced in 1934, but was not in large-scale use until the early 1950s. Anecdotal reports suggest that resistance emerged as early as 1957 both in Colombia and along the then Cambodia-Thailand border area. But by 1960, resistance in these areas was confirmed - and may represent two separate events. Resistance spread rapidly, with a new focus of resistance confirmed in East Africa by 1977. Chloroquine resistance represents a severe problem both for prophylaxis and treatment of malaria. In this aricle, David Payne traces the spread of resistance and discusses some of its implications.

Spread of chloroquine resistance in Plasmodium falciparum.

37205759

The Apolipoprotein (Apo) family is implicated in lipid metabolism. There are five types of Apo: Apoa, Apob, Apoc, Apod, and Apoe. Apoe has been demonstrated to play a central role in lipoprotein metabolism and to be essential for efficient receptor-mediated plasma clearance of chylomicron remnants and VLDL remnant particles by the liver. Apoe-deficient (Apoe(-/-)) mice develop atherosclerotic plaques spontaneously, followed by obesity. In this study, we investigated whether lipid deposition caused by Apoe knockout affects reproduction in female mice. The results demonstrated that Apoe(-/-) mice were severely hypercholesterolemic, with their cholesterol metabolism disordered, and lipid accumulating in the ovaries causing the ovaries to be heavier compared with the WT counterparts. In addition, estrogen and progesterone decreased significantly at D 100. Quantitative PCR analysis demonstrated that at D 100 the expression of cytochromeP450 aromatase (Cyp19a1), 3β-hydroxysteroid dehydrogenase (Hsd3b), mechanistic target of rapamycin (Mtor), and nuclear factor-κB (Nfkb) decreased significantly, while that of BCL2-associated agonist of cell death (Bad) and tuberous sclerosis complex 2 (Tsc2) increased significantly in the Apoe(-/-) mice. However, there was no difference in the fertility rates of the Apoe(-/-) and WT mice; that is, obesity induced by Apoe knockout has no significant effect on reproduction. However, the deletion of Apoe increased the number of ovarian follicles and the ratio of ovarian follicle atresia and apoptosis. We believe that this work will augment our understanding of the role of Apoe in reproduction.

Obesity occurring in apolipoprotein E-knockout mice has mild effects on fertility.

37207226

The heart has both the greatest caloric needs and the most robust oxidation of fatty acids (FAs). Under pathological conditions such as obesity and type 2 diabetes, cardiac uptake and oxidation are not balanced and hearts accumulate lipid potentially leading to cardiac lipotoxicity. We will first review the pathways utilized by the heart to acquire FAs from the circulation and to store triglyceride intracellularly. Then we will describe mouse models in which excess lipid accumulation causes heart dysfunction and experiments performed to alleviate this toxicity. Finally, the known relationships between heart lipid metabolism and dysfunction in humans will be summarized.

Lipid metabolism and toxicity in the heart.

37248570

After a lapse of almost 40 years, malaria eradication is back on the global health agenda. Inspired by the Gates Malaria Forum in October 2007,1,2 key organizations are starting to debate the pros and cons of redefining eradication as an explicit goal of malaria control efforts. Attempts to eliminate malaria in southern Africa3 and Pacific Island states,4 and WHO’s Global Malaria Programme agenda and field manual for malaria elimination,5,6 foreshadow this movement towards another global attempt at eradication. When marking 60 years of WHO’s commitment to fighting malaria, we must ask what has been achieved, but also what can we learn from the past. We now know so much more about the biology of parasite-host responses, the determinants of endemicity and transmission dynamics, the social, economic and cultural implications of malaria at household, community and national levels, and the demands made upon health systems in endemic countries. We do not yet know how to synthesize and integrate this knowledge to achieve elimination in different settings. Regional malaria elimination campaigns were first conducted in the late 1940s, preparing the ground for the Global Malaria Eradication Program in 1955. This campaign succeeded in eliminating malaria from Europe, North America, the Caribbean and parts of Asia and South-Central America.7 But no major success occurred in sub-Saharan Africa, which accounts for 80% of today’s burden of malaria.8 When the aspiration of global eradication was abandoned in 1969, the main reasons for failure were technical challenges of executing the strategy especially in Africa. The post-eradication era from 1969 to 1991 focused on technical issues, and research and development for new tools, leading to advances in drug and vaccine development, vector control and insecticide-treated nets. These decades also brought a better understanding of the social, economic and cultural dimensions of malaria. There was little global support provided specifically for malaria control in the newly independent states of Africa that were struggling to establish broad-based health systems and primary health care. By 1992, the combination of a worsening malaria situation and promising technical developments led to renewed global focus on malaria control. The Roll Back Malaria initiative, launched by WHO in 1998, led to the Abuja Declaration in 2000, which defined progressive intervention coverage targets for control designed to eliminate malaria as a public health problem, while emphasizing that this could only be achieved through vastly strengthened local health systems.9 Increased resources through the Global Fund to Fight AIDS, Tuberculosis and Malaria, the World Bank’s Booster Program, the US President’s Malaria Initiative and many others has meant that this page is finally beginning to turn as intervention coverage is rising.10 It is against this background that we hear this call for elimination/eradication. The challenges remain formidable. We all know that elimination in Africa is not possible with current tools. But efforts must focus beyond simply developing better tools, to include how existing and future tools can be strategically combined for maximum synergistic effectiveness when integrated into different health and social systems prevailing in endemic areas. Aiming at elimination and eradication further implies the need for effective surveillance strategies to monitor progress (again a challenge for health systems). This in turn requires a better understanding of malaria transmission heterogeneity in a globalized world with rapidly changing dynamics in environment, climate, migration and transnational cooperation. Maintaining long-term momentum in the face of success in regional elimination while waiting to achieve final eradication will be a major challenge. Shrinking the map by starting on the malaria margins with the “easy-to-eliminate” settings will boost morale initially but may bring marginal benefits to such areas at the expense of those where the burden of malaria is highest. Any strategic plan – and here we learn again from the past – needs to be a synchronous global effort, locally adapted in all endemic areas. Although we lack sufficient knowledge, systems and tools to eradicate malaria today, we do have a window of political will and financial resources to refocus on the goal of effective control through universal coverage of appropriate interventions. The prerequisites for a successful start are: (i) a process of inclusive discourse to agree on global vision, goals and strategy; and (ii) a global plan for all endemic areas describing how, where and when we move from control towards elimination. What must distinguish the new era, especially in Africa, is a real rather than rhetorical emphasis on health systems. ■

Malaria eradication back on the table.

37248765

N-acetyl-dinaline (CI-994) is an investigational anti-cancer drug which inhibits histone deacetylases. We evaluated the interaction between CI-994 and conventional chemotherapeutics used in acute myeloid leukemia (AML) in a rat model for AML and Brown Norway rat acute myelocytic leukemia (BNML). In vitro, CI-994 in combination with cytarabine (ara-C), daunorubicin and mitoxantrone, resulted in moderate synergism. In vivo, higher dosages of CI-994 induced complete remissions. CI-994/ara-C was very active against BNML. The combinations of CI-994/daunorubicin and CI-994/mitoxantrone were also active against BNML. This study demonstrates favorable in vitro and in vivo interactions between CI-994 and conventional anti-cancer agents used for the treatment of AML.

CI-994 (N-acetyl-dinaline) in combination with conventional anti-cancer agents is effective against acute myeloid leukemia in vitro and in vivo.

37249641

Genome function, replication, integrity, and propagation rely on the dynamic structural organization of chromosomes during the cell cycle. Genome folding in interphase provides regulatory segmentation for appropriate transcriptional control, facilitates ordered genome replication, and contributes to genome integrity by limiting illegitimate recombination. Here, we review recent high-resolution chromosome conformation capture and functional studies that have informed models of the spatial and regulatory compartmentalization of mammalian genomes, and discuss mechanistic models for how CTCF and cohesin control the functional architecture of mammalian chromosomes.

CTCF and Cohesin in Genome Folding and Transcriptional Gene Regulation.

37256966

Melatonin modulates a wide array of physiological events with pleiotropic effects on the immune system. While the relevance of specific melatonin membrane receptors has been well established for several biological functions, retinoic acid-related orphan receptor alpha (RORα) has been suggested as a mediator of nuclear melatonin signalling by results obtained from pharmacological approaches. However, a melatonin-mediated downstream effect cannot be ruled out, and further evidence is needed to support a direct interaction between melatonin and RORα. Here, we show that RORα is mainly located in human Jurkat T-cell nucleus, and it is co-immunoprecipitated with melatonin. Moreover, immunocytochemistry studies confirmed the co-localization of melatonin and RORα. Melatonin promoted a time-dependent decrease in nuclear RORα levels, suggesting a role in the RORα transcriptional activity. Interestingly, RORα acts as a molecular switch implicated in the mutually exclusive generation of Th17 and Treg cells, both involved in the harm/protection balance of immune conditions such as autoimmunity or acute transplant rejection. Therefore, the identification of melatonin as a natural modulator of RORα gives it a tremendous therapeutic potential for a variety of clinical disorders.

Melatonin synthesized by T lymphocytes as a ligand of the retinoic acid-related orphan receptor.

37269418

The cytokine and antibody response to Trichuris trichiura infection was determined for 96 persons living in an area where the parasite is highly endemic and infection exhibits a convex age intensity profile. In response to stimulation with T. trichiura antigen, a small proportion of the study group produced interleukin (IL)-4 (7%), IL-9 (5%), and IL-13 (17%). A larger proportion produced IL-10 (97%), tumor necrosis factor (TNF)-alpha (93%), and interferon (IFN)-gamma (32%). The levels of TNF-alpha (P =.016) and IFN-gamma (P =.012) significantly increased with age, suggesting a switch to a more chronic infection phenotype. The predominant parasite-specific antibodies produced were IgG1, IgG4, IgA, and IgE. Unlike the IgG subclasses and IgA, parasite-specific IgE correlated negatively with infection intensity, as defined by egg output (P =.008), and positively with host age (P =.010). These findings suggest a mixed cytokine response in trichuriasis and an IgE-associated level of protection.

Age- and infection intensity-dependent cytokine and antibody production in human trichuriasis: the importance of IgE.

37296667

In order to improve boar sperm quality during frozen-thawed process, the influence of the presence of trehalose on success of cryopreservation of boar sperm were investigated. We evaluated freeze-thawing tolerance of boar spermatozoa in a base cooling extender with the addition of different trehalose concentrations (0, 25, 50, 100 and 200mmol/l), and tried to determine the optimum concentration of trehalose. We chose sperm motility, acrosome integrity, membrane integrity and cryocapacitation as parameters to evaluate cryopreservation capacity of boar spermatozoa. We obtained the best results for 100mmol/l trehalose-supplemented extenders, with values of 49.89% for motility, 66.52% for acrosome integrity and 44.61% for membrane integrity, while freeze-thawing tolerance was diminished significantly for 200mmol/l of trehalose. Before and after capacitation, the CTC score for semen diluted by extender containing 100mmol/l trehalose was 3.68% and 43.82%, respectively. In conclusion, trehalose could confer a greater cryoprotective capacity to boar spermatozoa. Trehalose-supplementation with 100mmol/l concentration in basic extender could significantly improve sperm motility, membrane integrity and acrosome integrity parameters, and reduce boar spermatozoa cryocapacitation during the cryopreservation process.

The cryoprotective effect of trehalose supplementation on boar spermatozoa quality.

37297740

Polyploidy, recognized by multiple copies of the haploid chromosome number, has been described in plants, insects, and in mammalian cells such as, the platelet precursors, the megakaryocytes. Several of these cell types reach high ploidy via a different cell cycle. Megakaryocytes undergo an endomitotic cell cycle, which consists of an S phase interrupted by a gap, during which the cells enter mitosis but skip anaphase B and cytokinesis. Here, we review the mechanisms that lead to this cell cycle and to polyploidy in megakaryocytes, while also comparing them to those described for other systems in which high ploidy is achieved. Overall, polyploidy is associated with an orchestrated change in expression of several genes, of which, some may be a result of high ploidy and hence a determinant of a new cell physiology, while others are inducers of polyploidization. Future studies will aim to further explore these two groups of genes.

Roads to polyploidy: the megakaryocyte example.

37311371

The memory T cell pool functions as a dynamic repository of antigen-experienced T lymphocytes that accumulate over the lifetime of the individual. Recent studies indicate that memory T lymphocytes contain distinct populations of central memory (TCM) and effector memory (TEM) cells characterized by distinct homing capacity and effector function. This review addresses the heterogeneity of TCM and TEM, their differentiation stages, and the current models for their generation and maintenance in humans and mice.

Central memory and effector memory T cell subsets: function, generation, and maintenance.

37328025

Cells cope with blockage of replication fork progression in a manner that allows DNA synthesis to be completed and genomic instability minimized. Models for resolution of blocked replication involve fork regression to form Holliday junction structures. The human RecQ helicases WRN and BLM (deficient in Werner and Bloom syndromes, respectively) are critical for maintaining genomic stability and thought to function in accurate resolution of replication blockage. Consistent with this notion, WRN and BLM localize to sites of blocked replication after certain DNA-damaging treatments and exhibit enhanced activity on replication and recombination intermediates. Here we examine the actions of WRN and BLM on a special Holliday junction substrate reflective of a regressed replication fork. Our results demonstrate that, in reactions requiring ATP hydrolysis, both WRN and BLM convert this Holliday junction substrate primarily to a four-stranded replication fork structure, suggesting they target the Holliday junction to initiate branch migration. In agreement, the Holliday junction binding protein RuvA inhibits the WRN- and BLM-mediated conversion reactions. Importantly, this conversion product is suitable for replication with its leading daughter strand readily extended by DNA polymerases. Furthermore, binding to and conversion of this Holliday junction are optimal at low MgCl(2) concentrations, suggesting that WRN and BLM preferentially act on the square planar (open) conformation of Holliday junctions. Our findings suggest that, subsequent to fork regression events, WRN and/or BLM could re-establish functional replication forks to help overcome fork blockage. Such a function is highly consistent with phenotypes associated with WRN- and BLM-deficient cells.

The Werner and Bloom syndrome proteins help resolve replication blockage by converting (regressed) holliday junctions to functional replication forks.

37336085

PURPOSE We assessed the nephroprotective effects of montelukast sodium and N-acetylcysteine on secondary renal damage due to unilateral ureteral obstruction in a rat model. MATERIALS AND METHODS In this study 30 Wistar albino male rats were randomized into 3 groups, including placebo, N-acetylcysteine and montelukast sodium. Three rats served as the control group. The left ureter of the rats was sutured with 4-zero polyglactin sutures. Medications were given 3 days before obstruction and continued for 15 days. Dimercaptosuccinic acid renal scintigraphy was performed before obstruction and on day 15. Rats were sacrificed on day 15 and histopathological examinations were done. We biochemically assessed oxidative stress markers (myeloperoxidase and malondialdehyde), sulfhydryl and total nitrite for lipid peroxidation, oxidative protein damage and antioxidant levels, respectively. RESULTS On pathological examination inflammation and tubular epithelial damage in the N-acetylcysteine and montelukast sodium groups were less than in the placebo group (p <0.05). No difference was seen in normal kidneys. Myeloperoxidase, malondialdehyde and total nitrite levels in the N-acetylcysteine group, and myeloperoxidase and malondialdehyde levels in the montelukast sodium group were lower than in the placebo group (p <0.05). No statistical difference was seen in sulfhydryl levels (p >0.05) or among the N-acetylcysteine, montelukast sodium and placebo groups on scintigraphy (p >0.05). No pathological, chemical and scintigraphic differences were seen among the N-acetylcysteine, montelukast sodium and sham treated groups (p >0.05). CONCLUSIONS N-acetylcysteine and montelukast sodium have a protective effect against obstructive damage of the kidney. However, further investigations are needed.

Do Montelukast Sodium and N-Acetylcysteine Have a Nephroprotective Effect on Unilateral Ureteral Obstruction? A Placebo Controlled Trial in a Rat Model.

37362689

The bulk of ATP consumed by various cellular processes in higher eukaryotes is normally produced by five multimeric protein complexes (I-V) embedded within the inner mitochondrial membrane, in a process known as oxidative phosphorylation (OXPHOS). Maintenance of energy homeostasis under most physiological conditions is therefore contingent upon the ability of OXPHOS to meet cellular changes in bioenergetic demand, with a chronic failure to do so being a frequent cause of human disease. With the exception of Complex II, the structural subunits of OXPHOS complexes are encoded by both the nuclear and the mitochondrial genomes. The physical separation of the two genomes necessitates that the expression of the 13 mitochondrially encoded polypeptides be co-ordinated with that of relevant nuclear-encoded partners in order to assemble functional holoenzyme complexes. Complex biogenesis is a highly ordered process, and several nuclear-encoded factors that function at distinct stages in the assembly of individual OXPHOS complexes have been identified.

Oxidative phosphorylation: synthesis of mitochondrially encoded proteins and assembly of individual structural subunits into functional holoenzyme complexes.

37405449

The family of an elderly man with Barrett's esophagus was examined for gastroesophageal reflux and development of Barrett's esophagus. All five living children have gastroesophageal reflux or esophagitis, or both, and three have unequivocal Barrett's esophagus. Two third-generation descendents have gastroesophageal reflux. This pattern suggests autosomal dominant transmission of the gastroesophageal reflux trait. The family also has a high prevalence of cancer, which may represent the cancer family syndrome.

Familial gastroesophageal reflux and development of Barrett's esophagus.

37424881

OBJECTIVE Folate and vitamin B12 are two vital regulators in the metabolic process of homocysteine, which is a risk factor of atherothrombotic events. Low folate intake or low plasma folate concentration is associated with increased stroke risk. Previous randomized controlled trials presented discordant findings in the effect of folic acid supplementation-based homocysteine lowering on stroke risk. The aim of the present review was to perform a meta-analysis of relevant randomized controlled trials to check the how different folate fortification status might affect the effects of folic acid supplementation in lowering homocysteine and reducing stroke risk. DESIGN Relevant randomized controlled trials were identified through formal literature search. Homocysteine reduction was compared in subgroups stratified by folate fortification status. Relative risks with 95 % confidence intervals were used as a measure to assess the association between folic acid supplementation and stroke risk. SETTING The meta-analysis included fourteen randomized controlled trials, SUBJECTS A total of 39 420 patients. RESULTS Homocysteine reductions were 26·99 (sd 1·91) %, 18·38 (sd 3·82) % and 21·30 (sd 1·98) %, respectively, in the subgroups without folate fortification, with folate fortification and with partial folate fortification. Significant difference was observed between the subgroups with folate fortification and without folate fortification (P=0·05). The relative risk of stroke was 0·88 (95 % CI 0·77, 1·00, P=0·05) in the subgroup without folate fortification, 0·94 (95 % CI 0·58, 1·54, P=0·82) in the subgroup with folate fortification and 0·91 (95 % CI 0·82, 1·01, P=0·09) in the subgroup with partial folate fortification. CONCLUSIONS Folic acid supplementation might have a modest benefit on stroke prevention in regions without folate fortification.

The effect of folate fortification on folic acid-based homocysteine-lowering intervention and stroke risk: a meta-analysis.

37437064

Mesenchymal stem cells (MSCs) display substantial cell-to-cell variation. This heterogeneity manifests among donors, among tissue sources, and within cell populations. Such pervasive variability complicates the use of MSCs in regenerative applications and may limit their therapeutic efficacy. Most conventional assays measure MSC properties in bulk and, as a consequence, mask this cell-to-cell variation. Recent studies have identified extensive variability amongst and within clonal MSC populations, in dimensions including functional differentiation capacity, molecular state (e.g. epigenetic, transcriptomic, and proteomic status), and biophysical properties. While the origins of these variations remain to be elucidated, potential mechanisms include in vivo micro-anatomical heterogeneity, epigenetic bistability, and transcriptional fluctuations. Emerging tools for single cell analysis of MSC gene and protein expression may yield further insight into the mechanisms and implications of single cell variation amongst these cells, and ultimately improve the clinical utility of MSCs in tissue engineering and regenerative medicine applications. This review outlines the dimensions across which MSC heterogeneity is present, defines some of the known mechanisms that govern this heterogeneity, and highlights emerging technologies that may further refine our understanding and improve our clinical application of this unique cell type.

On the origin and impact of mesenchymal stem cell heterogeneity: new insights and emerging tools for single cell analysis.

37438296

Age-dependent decline in skeletal muscle function leads to several inherited and acquired muscular disorders in elderly individuals. The levels of microRNAs (miRNAs) could be altered during muscle maintenance and repair. We therefore performed a comprehensive investigation for miRNAs from five different periods of bovine skeletal muscle development using next-generation small RNA sequencing. In total, 511 miRNAs, including one putatively novel miRNA, were identified. Thirty-six miRNAs were differentially expressed between prenatal and postnatal stages of muscle development including several myomiRs (miR-1, miR-206 and let-7 families). Compared with miRNA expression between different muscle tissues, 14 miRNAs were up-regulated and 22 miRNAs were down-regulated in the muscle of postnatal stage. In addition, a novel miRNA was predicted and submitted to the miRBase database as bta-mir-10020. A dual luciferase reporter assay was used to demonstrate that bta-mir-10020 directly targeted the 3'-UTR of the bovine ANGPT1 gene. The overexpression of bta-mir-10020 significantly decreased the DsRed fluorescence in the wild-type expression cassette compared to the mutant type. Using three computational approaches - miranda, pita and rnahybrid - these differentially expressed miRNAs were also predicted to target 3609 bovine genes. Disease and biological function analyses and the KEGG pathway analysis revealed that these targets were statistically enriched in functionality for muscle growth and disease. Our miRNA expression analysis findings from different states of muscle development and aging significantly expand the repertoire of bovine miRNAs now shown to be expressed in muscle and could contribute to further studies on growth and developmental disorders in this tissue type.

Altered microRNA expression in bovine skeletal muscle with age.

37444589

Although 13 years have passed since identification of human immunodeficiency virus-1 (HIV-1) as the cause of AIDS, we do not yet know how HIV kills its primary target, the T cell that carries the CD4 antigen. We and others have shown an increase in the percentage of apoptotic cells among circulating CD4+ (and CD8+) T cells of HIV-seropositive individuals and an increase in frequency of apoptosis with disease progression. However, it is not known if this apoptosis occurs in infected or uninfected T cells. We show here, using in situ labelling of lymph nodes from HIV-infected children and SIV-infected macaques, that apoptosis occurs predominantly in bystander cells and not in the productively infected cells themselves. These data have implications for pathogenesis and therapy, namely, arguing that rational drug therapy may involve combination agents targeting viral replication in infected cells and apoptosis of uninfected cells.

Apoptosis occurs predominantly in bystander cells and not in productively infected cells of HIV- and SIV-infected lymph nodes

37450671

The protein component of Alzheimer's disease amyloid [neurofibrillary tangles (NFT), amyloid plaque core and congophilic angiopathy] is an aggregated polypeptide with a subunit mass of 4 kd (the A4 monomer). Based on the degree of N-terminal heterogeneity, the amyloid is first deposited in the neuron, and later in the extracellular space. Using antisera raised against synthetic peptides, we show that the N terminus of A4 (residues 1-11) contains an epitope for neurofibrillary tangles, and the inner region of the molecule (residues 11-23) contains an epitope for plaque cores and vascular amyloid. The non-protein component of the amyloid (aluminum silicate) may form the basis for the deposition or amplification (possible self-replication) of the aggregated amyloid protein. The amyloid of Alzheimer's disease is similar in subunit size, composition but not sequence to the scrapie-associated fibril and its constituent polypeptides. The sequence and composition of NFT are not homologous to those of any of the known components of normal neurofilaments.

Neuronal origin of a cerebral amyloid: neurofibrillary tangles of Alzheimer's disease contain the same protein as the amyloid of plaque cores and blood vessels.

37480103

CONTEXT During pregnancy, serum levels of estrogen, progesterone, and other hormones are markedly higher than during other periods of life. Pregnancy hormones primarily are produced in the placenta, and signs of placental impairment may serve as indirect markers of hormone exposures during pregnancy. During pregnancy, these markers have been inconsistently associated with subsequent risk of breast cancer in the mother. OBJECTIVE To examine associations between indirect markers of hormonal exposures, such as placental weight and other pregnancy characteristics, and maternal risk of developing breast cancer. DESIGN AND SETTING Population-based cohort study using data from the Swedish Birth Register, the Swedish Cancer Register, the Swedish Cause of Death Register, and the Swedish Register of Population and Population Changes. PARTICIPANTS Women included in the Sweden Birth Register who delivered singletons between 1982 and 1989, with complete information on date of birth and gestational age. Women were followed up until the occurrence of breast cancer, death, or end of follow-up (December 31, 2001). Cox proportional hazards models were used to estimate associations between hormone exposures and risks of breast cancer. MAIN OUTCOME MEASURE Incidence of invasive breast cancer. RESULTS Of 314,019 women in the cohort, 2216 (0.7%) developed breast cancer during the follow-up through 2001, of whom 2100 (95%) were diagnosed before age 50 years. Compared with women who had placentas weighing less than 500 g in 2 consecutive pregnancies, the risk of breast cancer was increased among women whose placentas weighed between 500 and 699 g in their first pregnancy and at least 700 g in their second pregnancy (or vice versa) (adjusted hazard ratio, 1.82; 95% confidence interval [CI], 1.07-3.08), and the corresponding risk was doubled among women whose placentas weighed at least 700 g in both pregnancies (adjusted hazard ratio, 2.05; 95% CI, 1.15-3.64). A high birth weight (> or =4000 g) in 2 successive births was associated with an increased risk of breast cancer before but not after adjusting for placental weight and other covariates (adjusted hazard ratio, 1.10; 95% CI, 0.76-1.59). CONCLUSIONS Placental weight is positively associated with maternal risk of breast cancer. These results further support the hypothesis that pregnancy hormones are important modifiers of subsequent maternal breast cancer risk.

Pregnancy characteristics and maternal risk of breast cancer.

37488367

OBJECTIVE To investigate heritability and continuum versus categorical approaches to attention-deficit hyperactivity disorder (ADHD), using a large-scale twin sample. METHOD A cohort of 1,938 families with twins and siblings aged 4 to 12 years, recruited from the Australian National Health and Medical Research Council Twin Registry, was assessed for ADHD using a DSM-III-R-based maternal rating scale. Probandwise concordance rates and correlations in monozygotic and dizygotic twins and siblings were calculated, and heritability was examined using the De Fries and Fulker regression technique. RESULTS There was a narrow (additive) heritability of 0.75 to 0.91 which was robust across familial relationships (twin, sibling, and twin-sibling) and across definitions of ADHD as part of a continuum or as a disorder with various symptom cutoffs. There was no evidence for nonadditive genetic variation or for shared family environmental effects. CONCLUSIONS These findings suggest that ADHD is best viewed as the extreme of a behavior that varies genetically throughout the entire population rather than as a disorder with discrete determinants. This has implications for the classification of ADHD and for the identification of genes for this behavior, as well as implications for diagnosis and treatment.

Attention-deficit hyperactivity disorder: a category or a continuum? Genetic analysis of a large-scale twin study.

37549932

Resistance to apoptosis, often achieved by the overexpression of antiapoptotic proteins, is common and perhaps required in the genesis of cancer. However, it remains uncertain whether apoptotic defects are essential for tumor maintenance. To test this, we generated mice expressing a conditional BCL-2 gene and constitutive c-myc that develop lymphoblastic leukemia. Eliminating BCL-2 yielded rapid loss of leukemic cells and significantly prolonged survival, formally validating BCL-2 as a rational target for cancer therapy. Loss of this single molecule resulted in cell death, despite or perhaps attributable to the presence of other oncogenic events. This suggests a generalizable model in which aberrations inherent to cancer generate tonic death signals that would otherwise kill the cell if not opposed by a requisite apoptotic defect(s).

Antiapoptotic BCL-2 is required for maintenance of a model leukemia.

37562370

OBJECTIVE The infrapatellar fat pad (IPFP) has been identified as a source of anterior knee pain. Fibrosis and marked inflammatory infiltrate in the IPFP of patients with arthritis of the knee and reduction in pain post knee replacement in patients following resection of the IPFP have been observed. We have investigated changes in the IPFP of rats undergoing the monoiodoacetate (MIA) model of degenerative joint disease, a model that exhibits some histopathological similarities to osteoarthritis (OA). METHODS Rats were injected intra-articularly with MIA and the development of weight bearing asymmetry was followed for 21 days as compared to vehicle-injected animals. In addition, IPFPs were removed from both ipsilateral and contralateral joints. Both inflammatory infiltrate and histopathological changes were analysed. RESULTS MIA injection caused marked weight bearing asymmetry. Ipsilateral IPFP wet weights were significantly increased on days 1 and 3 in MIA-treated animals. MIA treatment also resulted in significant increases in IPFP total white blood cells and monocytes on days 1, 3, and 7 and neutrophils on days 1 and 3. This was supported by histopathological findings at early time points which progressed to adipocyte necrosis, IPFP fibrosis, patellar cartilage and subchondral bone necrosis with synovial hyperplasia at later timepoints. CONCLUSIONS The current study clearly demonstrated that marked inflammatory changes in the IPFP occur during the early stage of the MIA model of OA which may contribute to the pain observed at this early stage. The role of the IPFP in later stages of the model needs to be further explored.

Cellular and histopathological changes in the infrapatellar fat pad in the monoiodoacetate model of osteoarthritis pain.

37578311

Although essentially unknown as a therapeutic concept as recently as a decade ago, it is difficult now to open a cardiology journal, attend a cardiology meeting, or scan a newspaper without being caught up in the excitement generated by the thought that angiogenesis therapy may soon have a major impact on the treatment of patients with atherosclerotic lesions obstructing arteries that supply the myocardium or legs. Potent therapeutic interventions, however, are rarely free of at least the potential for causing harmful effects. Angiogenesis therapy is no exception. Despite this and despite the fact that the only reasonably powered, randomized, double-blind clinical studies to date have failed to demonstrate primary end-point efficacy,1 thoughtful consideration of the serious side effects that might accompany any therapeutic benefit has been largely missing from scientific communications. For the lay media, a switch from this unbounded enthusiasm to profound skepticism occurred recently after a report of the tragic death of a young man caused by injection of large amounts of an adenoviral vector into the hepatic artery (unrelated to angiogenesis therapy).2 For the scientific community, however, there has been a general lack of in-depth discussion of the potential dangers inherent in angiogenesis interventions. Such a discussion is appropriate not only because of the event cited above but also because considerable mechanistic data are available that actually permit us to identify specific side effects that we might anticipate as potential complications of angiogenesis therapy. The following therefore is a discussion of potential complications based predominantly on our knowledge of the underlying biological activities of 2 of the most potent angiogenic cytokines, vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF). Angiogenic agents are thought to have the potential to induce neovascularization in many different tissues. This becomes important in that under clinical circumstances it …

Angiogenesis therapy: amidst the hype, the neglected potential for serious side effects.

37583120

OBJECTIVE Obesity and being overweight during adulthood have been consistently linked to increased risk for development of dementia later in life, especially Alzheimer's disease. They have also been associated with cognitive dysfunction and brain structural alterations in otherwise healthy adults. Although proton magnetic resonance spectroscopy may distinguish between neuronal and glial components of the brain and may point to neurobiological mechanisms underlying brain atrophy and cognitive changes, no spectroscopic studies have yet assessed the relationships between adiposity and brain metabolites. METHODS We have utilized magnetic resonance imaging and proton magnetic resonance spectroscopic imaging data from 50 healthy middle-aged participants (mean age, 41.7 +/- 8.5 years; 17 women), who were scanned as control subjects for another study. RESULTS After adjustment for age and sex, greater body mass indices (BMIs) correlated with: (1) lower concentrations of N-acetylaspartate (spectroscopic marker of neuronal viability) in frontal (p = 0.001), parietal (p = 0.006), and temporal (p = 0.008) white matter; (2) lower N-acetylaspartate in frontal gray matter (p = 0.01); and (3) lower concentrations of choline-containing metabolites (associated with membrane metabolism) in frontal white matter (p = 0.05). INTERPRETATION These results suggest that increased BMI at midlife is associated with neuronal and/or myelin abnormalities, primarily in the frontal lobe. Because white matter in the frontal lobes is more prone to the effects of aging than in other lobes, our results may reflect accelerated aging in individuals with high levels of adiposity. Thus, greater BMI may increase the odds of developing an age-related disease, such as Alzheimer's disease.

Body mass index and magnetic resonance markers of brain integrity in adults.

37592824

Sixty-seven patients with temporal lobe epilepsy without circumscribed, potentially epileptogenic lesions, who were studied with intracranial electrodes and who became seizure free following temporal lobectomy were retrospectively evaluated with regard to preoperative scalp electroencephalographic (EEG) findings, neuropsychological test results, neuroimaging findings, results of surgery, and pathology of resected tissue. Interictal scalp EEG showed paroxysmal abnormalities during prolonged monitoring in 64 patients (96%). These were localized in the anterior temporal region in 60 (94%) of these 64 patients. Bilateral independent paroxysmal activity occurred in 42% of the patients and was preponderant over the side of seizure origin in half. Ictal EEG changes were rarely detected at the time of clinical seizure onset, but lateralized buildup of rhythmic seizure activity during the seizure occurred in 80% of patients. In 13%, the scalp EEG seizure buildup was, however, contralateral to the side of seizure origin as subsequently determined by depth EEG and curative surgery. Lateralized postictal slowing, when present, was a very reliable lateralizing finding. Neuropsychological testing provided lateralizing findings concordant with the side of seizure origin in 73% of patients. When neuropsychological testing produced discordant results or nonlateralizing findings, those patients were usually found to have right temporal seizure origin. Intracarotid amobarbital (Amytal) testing demonstrated absent or marginal memory functions on the side of seizure onset in 63% of patients, but 26 patients (37%) had bilaterally intact memory. In those patients who had magnetic resonance imaging, it was very sensitive in detecting subtle medial temporal abnormalities. These abnormalities were present in 23 of 28 magnetic resonance images, and corresponded with mesial temporal sclerosis on pathological examination in all but 2 patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Characteristics of medial temporal lobe epilepsy: II. Interictal and ictal scalp electroencephalography, neuropsychological testing, neuroimaging, surgical results, and pathology.

37608303

Cristae, the organized invaginations of the mitochondrial inner membrane, respond structurally to the energetic demands of the cell. The mechanism by which these dynamic changes are regulated and the consequences thereof are largely unknown. Optic atrophy 1 (OPA1) is the mitochondrial GTPase responsible for inner membrane fusion and maintenance of cristae structure. Here, we report that OPA1 responds dynamically to changes in energetic conditions to regulate cristae structure. This cristae regulation is independent of OPA1's role in mitochondrial fusion, since an OPA1 mutant that can still oligomerize but has no fusion activity was able to maintain cristae structure. Importantly, OPA1 was required for resistance to starvation-induced cell death, for mitochondrial respiration, for growth in galactose media and for maintenance of ATP synthase assembly, independently of its fusion activity. We identified mitochondrial solute carriers (SLC25A) as OPA1 interactors and show that their pharmacological and genetic blockade inhibited OPA1 oligomerization and function. Thus, we propose a novel way in which OPA1 senses energy substrate availability, which modulates its function in the regulation of mitochondrial architecture in a SLC25A protein-dependent manner.

OPA1-dependent cristae modulation is essential for cellular adaptation to metabolic demand.

37619697

BACKGROUND Phenylpropanolamine is commonly found in appetite suppressants and cough or cold remedies. Case reports have linked the use of products containing phenylpropanolamine to hemorrhagic stroke, often after the first use of these products. To study the association, we designed a case-control study. METHODS Men and women 18 to 49 years of age were recruited from 43 U.S. hospitals. Eligibility criteria included the occurrence of a subarachnoid or intracerebral hemorrhage within 30 days before enrollment and the absence of a previously diagnosed brain lesion. Random-digit dialing identified two matched control subjects per patient. RESULTS There were 702 patients and 1376 control subjects. For women, the adjusted odds ratio was 16.58 (95 percent confidence interval, 1.51 to 182.21; P=0.02) for the association between the use of appetite suppressants containing phenylpropanolamine and the risk of a hemorrhagic stroke and 3.13 (95 percent confidence interval, 0.86 to 11.46; P=0.08) for the association with the first use of a product containing phenylpropanolamine. All first uses of phenylpropanolamine involved cough or cold remedies. For men and women combined, the adjusted odds ratio was 1.49 (95 percent confidence interval, 0.84 to 2.64; P=0.17) for the association between the use of a product containing phenylpropanolamine and the risk of a hemorrhagic stroke, 1.23 (95 percent confidence interval, 0.68 to 2.24; P=0.49) for the association with the use of cough or cold remedies that contained phenylpropanolamine, and 15.92 (95 percent confidence interval, 1.38 to 184.13; P=0.03) for the association with the use of appetite suppressants that contained phenylpropanolamine. An analysis in men showed no increased risk of a hemorrhagic stroke in association with the use of cough or cold remedies containing phenylpropanolamine. No men reported the use of appetite suppressants. CONCLUSIONS The results suggest that phenylpropanolamine in appetite suppressants, and possibly in cough and cold remedies, is an independent risk factor for hemorrhagic stroke in women.

Phenylpropanolamine and the risk of hemorrhagic stroke.

37628989

BACKGROUND Confocal laser endomicroscopy (CLE) is rapidly emerging as a valuable tool for gastrointestinal endoscopic imaging. Fluorescent contrast agents are used to optimize imaging with CLE, and intravenous fluorescein is the most widely used contrast agent. Fluorescein is FDA-cleared for diagnostic angiography of the retina. For these indications, the safety profile of fluorescein has been well-documented; however, to date, fluorescein is not cleared for use with CLE. AIMS To estimate the rate of serious and total adverse events attributable to intravenous fluorescein when used for gastrointestinal CLE. METHODS We performed a cross sectional survey of 16 International Academic Medical Centres with active research protocols in CLE that involved intravenous fluorescein. Centres using i.v. fluorescein for CLE who were actively monitored for adverse events were included. RESULTS Sixteen centres performed 2272 gastrointestinal CLE procedures. The most common dose of contrast agent was 2.5-5 mL of 10% sodium fluorescein. No serious adverse events were reported. Mild adverse events occurred in 1.4% of individuals, including nausea/vomiting, transient hypotension without shock, injection site erythema, diffuse rash and mild epigastric pain. The limitation is that only immediate post procedure events were actively monitored. CONCLUSIONS Use of intravenous fluorescein for gastrointestinal CLE appears to be safe with few acute complications.

The safety of intravenous fluorescein for confocal laser endomicroscopy in the gastrointestinal tract.

37641175

A DNA fraction is spontaneously released from living, but not dead or dying, human, other mammalian, avian, amphibian, plant, and prokaryote cells. The spontaneously released DNA fraction has been shown to be (a) present in both actively dividing and nondividing, differentiated cell populations; (b) labile; (c) associated with DNA-dependent RNA or DNA polymerase; (d) associated with an RNA fraction; and to have (e) a lower molecular weight than the typical genetic DNA fraction; and (f) Alu repeat sequences in increased proportions compared to a unique gene in plasma/serum. On the other hand, early autoradiographic and biochemical and quantitative cytochemical and cytophysical studies on DNA permitted the identification of a DNA fraction which was (1) present in both actively dividing and nondividing, differentiated cell populations; (2) labile; and (3) had a lower molecular weight than the typical genetic DNA fraction. This DNA fraction was termed metabolic DNA (m-DNA) and was proposed as possibly forming extra gene copies for the rapid production of m-RNA, to be destroyed subsequently. Therefore, we suggest that the metabolic DNA fraction might represent the precursor to the formation of the spontaneously released DNA fraction.

Metabolic DNA as the origin of spontaneously released DNA?

37643601

Many viruses go through a maturation step in the final stages of assembly before being transmitted to another host. The maturation process of flaviviruses is directed by the proteolytic cleavage of the precursor membrane protein (prM), turning inert virus into infectious particles. We have determined the 2.2 angstrom resolution crystal structure of a recombinant protein in which the dengue virus prM is linked to the envelope glycoprotein E. The structure represents the prM-E heterodimer and fits well into the cryo-electron microscopy density of immature virus at neutral pH. The pr peptide beta-barrel structure covers the fusion loop in E, preventing fusion with host cell membranes. The structure provides a basis for identifying the stages of its pH-directed conformational metamorphosis during maturation, ending with release of pr when budding from the host.

The flavivirus precursor membrane-envelope protein complex: structure and maturation.

37673301

G-protein coupled receptors (GPCRs) represent a large class of cell surface receptors that mediate a multitude of functions. Over the years, a number of GPCRs and ancillary proteins have been shown to be expressed in skeletal muscle. Unlike the case with other muscle tissues like cardiac and vascular smooth muscle cells, there has been little attempt at systematically analyzing GPCRs in skeletal muscle. Here we have compiled all the GPCRs that are expressed in skeletal muscle. In addition, we review the known function of these receptors in both skeletal muscle tissue and in cultured skeletal muscle cells.

Peptide and non-peptide G-protein coupled receptors (GPCRs) in skeletal muscle.

37677954

A new badnavirus was discovered from pagoda trees showing yellow mosaic symptoms on the leaves by high throughput sequencing of small RNAs. The complete genome of this virus was determined to comprise 7424 nucleotides, and the virus shared 40.4-45.1% identity with that of other badnaviruses. The genome encodes five open reading frames (ORFs) on the plus strand, which includes three conserved badnaviral ORFs. These results suggest that this virus is a new member of the genus Badnavirus in the family Caulimoviridae. The virus is tentatively named pagoda yellow mosaic associated virus (PYMAV). Phylogenetic analysis suggested that this virus together with gooseberry vein banding virus (GVBV) and grapevine vein-clearing virus (GVCV) forms a separate group that is distinct two other well characterized badnaviral groups. Additionally, the viral derived small RNA (vsRNA) profile of PYMAV was analyzed and compared with that of viruses within the same family. Results showed that the most abundant PYMAV vsRNAs were 21-nt, whereas other viruses in the same family have a predominance of 22- or 24-nt vsRNA. The percentage of sense PYMAV vsRNA was almost equal to that of antisense vsRNA, whereas vsRNAs of other viruses in the family display preferences toward the sense strand of their genome. Furthermore, PYMAV vsRNAs were symmetrically distributed along the genome with no obvious vsRNA generating hotspots.

Characterization of complete genome and small RNA profile of pagoda yellow mosaic associated virus, a novel badnavirus in China.

37686718

Malignant gliomas, which include glioblastomas and anaplastic astrocytomas, are the most common primary tumors of the brain. Over the past 30 years, the standard treatment for these tumors has evolved to include maximal safe surgical resection, radiation therapy and temozolomide chemotherapy. While the median survival of patients with glioblastomas has improved from 6 months to 14.6 months, these tumors continue to be lethal for the vast majority of patients. There has, however, been recent substantial progress in our mechanistic understanding of tumor development and growth. The translation of these genetic, epigenetic and biochemical findings into therapies that have been tested in clinical trials is the subject of this review.

Experimental approaches for the treatment of malignant gliomas.

37699461

Beta-cell replacement is considered to be the most promising approach for treatment of type 1 diabetes. Its application on a large scale is hindered by a shortage of cells for transplantation. Activation of insulin expression, storage, and regulated secretion in stem/progenitor cells offers novel ways to overcome this shortage. We explored whether fetal human progenitor liver cells (FH) could be induced to differentiate into insulin-producing cells after expression of the pancreatic duodenal homeobox 1 (Pdx1) gene, which is a key regulator of pancreatic development and insulin expression in beta cells. FH cells possess a considerable replication capacity, and this was further extended by introduction of the gene for the catalytic subunit of human telomerase. Immortalized FH cells expressing Pdx1 activated multiple beta-cell genes, produced and stored considerable amounts of insulin, and released insulin in a regulated manner in response to glucose. When transplanted into hyperglycemic immunodeficient mice, the cells restored and maintained euglycemia for prolonged periods. Quantitation of human C-peptide in the mouse serum confirmed that the glycemia was normalized by the transplanted human cells. This approach offers the potential of a novel source of cells for transplantation into patients with type 1 diabetes.

Reversal of hyperglycemia in mice by using human expandable insulin-producing cells differentiated from fetal liver progenitor cells.

37722384

The ability to reprogram somatic cells to induced pluripotent stem cells (iPSCs) offers an opportunity to generate pluripotent patient-specific cell lines that can help model human diseases. These iPSC lines could also be powerful tools for drug discovery and the development of cellular transplantation therapies. Many methods exist for generating iPSC lines but those best suited for use in studying human diseases and developing therapies must be of adequate efficiency to produce iPSCs from samples that may be of limited abundance, capable of reprogramming cells from both skin fibroblasts and blood, and footprint-free. Several reprogramming techniques meet these criteria and can be utilized to derive iPSCs in projects with both basic scientific and therapeutic goals. Combining these reprogramming methods with small molecule modulators of signaling pathways can lead to successful generation of iPSCs from even the most recalcitrant patient-derived somatic cells.

A review of the methods for human iPSC derivation.

37727521

Epstein-Barr virus (EBV)-encoded small RNAs (EBERs) are nonpolyadenylated, untranslated RNAs, exist most abundantly in latently EBV-infected cells, and are expected to show secondary structures with many short stem-loops. Retinoic acid-inducible gene I (RIG-I) is a cytosolic protein that detects viral double-stranded RNA (dsRNA) inside the cell and initiates signaling pathways leading to the induction of protective cellular genes, including type I interferons (IFNs). We investigated whether EBERs were recognized by RIG-I as dsRNA. Transfection of RIG-I plasmid induced IFNs and IFN-stimulated genes (ISGs) in EBV-positive Burkitt's lymphoma (BL) cells, but not in their EBV-negative counterparts or EBER-knockout EBV-infected BL cells. Transfection of EBER plasmid or in vitro-synthesized EBERs induced expression of type I IFNs and ISGs in RIG-I-expressing, EBV-negative BL cells, but not in RIG-I-minus counterparts. EBERs activated RIG-I's substrates, NF-kappaB and IFN regulatory factor 3, which were necessary for type I IFN activation. It was also shown that EBERs co-precipitated with RIG-I. These results indicate that EBERs are recognized by RIG-I and activate signaling to induce type I IFN in EBV-infected cells.

EB virus-encoded RNAs are recognized by RIG-I and activate signaling to induce type I IFN.

37731372

Although uncommon, pregnancy occurs in women on chronic dialysis. In 1980 the incidence of pregnancy in women on dialysis was 0.9%. Studies from 1992 to 2003 indicate that pregnancy occurred in 1-7% of women on chronic dialysis. Half of the infants born to women on chronic dialysis survive. Of importance is that "intensive dialysis" of 16-24 hr/week is associated with improved infant survival. In this article, the incidence, duration, fetal and maternal complications, and outcomes of pregnancy in women on chronic dialysis are reviewed. The management of anemia, hypertension, electrolytes, bone minerals, and acid-base parameters in the pregnant dialysis patient is also summarized. Recommendations regarding the dialysis prescription for the pregnant woman on hemodialysis (HD) or peritoneal dialysis (PD) are also made. The complex and precarious condition of the pregnant woman on dialysis requires close collaboration between the patient, nephrologist, dialysis staff, obstetrician, and neonatologist to maximize the chance of a successful pregnancy.

Pregnancy in dialysis patients: a review of outcomes, complications, and management.

37762357

Cytomegalovirus (CMV) has highly evolved mechanisms for avoiding detection by the host immune system. Recently, in the genomes of human and primate CMV, a novel gene comprising segments of noncontiguous open reading frames was identified and found to have limited predicted homology to endogenous cellular interleukin-10 (IL-10). Here we investigate the biological activities of the CMV IL-10-like gene product and show it to possess potent immunosuppressive properties. Both purified bacterium-derived recombinant CMV IL-10 and CMV IL-10 expressed in supernatants of human cells were found to inhibit proliferation of mitogen-stimulated peripheral blood mononuclear cells (PBMCs), with specific activity comparable to that of recombinant human IL-10. In addition, CMV IL-10 expressed from human cells inhibited cytokine synthesis, as treatment of stimulated PBMCs and monocytes with CMV IL-10 led to a marked decrease in production of proinflammatory cytokines. Finally, CMV IL-10 was observed to decrease cell surface expression of both major histocompatibility complex (MHC) class I and class II molecules, while conversely increasing expression of the nonclassical MHC allele HLA-G. These results demonstrate for the first time that CMV has a biologically active IL-10 homolog that may contribute to immune evasion during virus infection.

Potent immunosuppressive activities of cytomegalovirus-encoded interleukin-10.

37768883

In vivo activation of Klebsiella aerogenes urease, a nickel-containing enzyme, requires the presence of functional UreD, UreF, and UreG accessory proteins and is further facilitated by UreE. These accessory proteins are proposed to be involved in metallocenter assembly (M. H. Lee, S. B. Mulrooney, M. J. Renner, Y. Markowicz, and R. P. Hausinger, J. Bacteriol. 174:4324-4330, 1992). A series of three UreD-urease apoprotein complexes are present in cells that express ureD at high levels, and these complexes are thought to be essential for in vivo activation of the enzyme (I.-S. Park, M. B. Carr, and R. P. Hausinger, Proc. Natl. Acad. Sci. USA 91:3233-3237, 1994). In this study, we describe the effect of accessory gene deletions on urease complex formation. The ureE, ureF, and ureG gene products were found not to be required for formation of the UreD-urease complexes; however, the complexes from the ureF deletion mutant exhibited delayed elution during size exclusion chromatography. Because these last complexes were of typical UreD-urease sizes according to native gel electrophoretic analysis, we propose that UreF alters the conformation of the UreD-urease complexes. The same studies revealed the presence of an additional series of urease apoprotein complexes present only in cells containing ureD, ureF, and ureG, along with the urease subunit genes. These new complexes were shown to contain urease, UreD, UreF, and UreG. We propose that the UreD-UreF-UreG-urease apoprotein complexes represent the activation-competent form of urease apoprotein in the cell.

Evidence for the presence of urease apoprotein complexes containing UreD, UreF, and UreG in cells that are competent for in vivo enzyme activation.

37822406

Derivation of patient-specific human pluripotent stem cells via somatic cell nuclear transfer (SCNT) has the potential for applications in a range of therapeutic contexts. However, successful SCNT with human cells has proved challenging to achieve, and thus far has only been reported with fetal or infant somatic cells. In this study, we describe the application of a recently developed methodology for the generation of human ESCs via SCNT using dermal fibroblasts from 35- and 75-year-old males. Our study therefore demonstrates the applicability of SCNT for adult human cells and supports further investigation of SCNT as a strategy for regenerative medicine.

Human somatic cell nuclear transfer using adult cells.

37895688

In the spleens of young, adult mice there exist naturally occurring killer lymphocytes with specificity for mouse Moloney leukemia cells. The lytic activity was directed against syngeneic or allogeneic Moloney leukemia cells to a similar extent, but was primarily expressed when tested against in vitro grown leukemia cells. Two leukemias of non-Moloney origin were resistant and so was the mastocytoma line P815. Although killer activity varied between different strains of mice, the specificity of lysis was the same as indicated by competition experiments using unlabeled Moloney or other tumor cells as inhibitors in the cytotoxic assays. Capacity to compete and sensitivy to lysis by the killer cells were found to be highly positively correlated. Analysis of the kinetics of the cytotoxic assay revealed a rapid induction of lysis within one to four hours, arguing against any conventional in vitro induction of immune response. No evidence was found of soluble factors playing any role in the cytolytic assay.

"Natural" killer cells in the mouse. I. Cytotoxic cells with specificity for mouse Moloney leukemia cells. Specificity and distribution according to genotype.

37912677

With the acknowledged problems associated with assessment of functioning thyroid mass and hence radiation dose, our policy had been to give 75 MBq iodine-131 at 6-monthly intervals to patients with Graves' disease until they became euthyroid. Since positron emission tomography (PET) has been available at this hospital, the radiation dose to the thyroid has been calculated with an accuracy of ∼20%, the thyroid mass being determined from an iodine-124 PET scan. A dose-response study has been carried out on 65 patients who have received single or cumulative radiation doses of <80 Gy. The results show that patients who receive a low radiation dose (<20 Gy) at their first treatment have a high probability of remaining toxic at 12 months. In contrast, patients who receive higher radiation doses (>40 Gy) at their first treatment have a high probability of control. The probability of becoming euthyroid increases more rapidly with increasing radiation dose than the probability of becoming hypothyroid. Following this dose-response study, a new treatment protocol has been introduced. A 124I PET tracer study prior to 131I therapy will be performed to enable a prescribed thyroid dose of 50 Gy to be delivered to patients with Graves' disease. Further 131I therapy will only be considered if patients are still toxic at 12 months.

Dose-response study on thyrotoxic patients undergoing positron emission tomography and radioiodine therapy

37916361

OBJECTIVE The study of soluble corin in the circulation before hypertensive disorders of pregnancy (HDP) has been limited. Here we aimed to study serum soluble corin in mid pregnancy in patients with HDP and their age- and gestational weeks-matched controls. METHODS Sixty-eight pairs of cases of HDP and controls were studied. Blood samples were obtained in mid pregnancy between 16 and 20 gestational weeks. Serum soluble corin was examined by enzyme-linked immunosorbent assay methods. The relationship between serum soluble corin and HDP was examined using conditional logistic regression models. RESULTS Serum soluble corin in mid pregnancy was increased in cases with HDP compared with controls (median [interquartile range]: 1968 [1644-2332] pg/mL vs. 1700 [1446-2056] pg/mL, p=0.002). Participants were categorized into quartiles of serum soluble corin distributed in controls. Compared with the lowest quartile, participants in the highest quartile had a significantly increased risk for HDP (odds ratio [OR], 4.21; 95% confidence interval [95% CI], 1.31-13.53) after multivariate adjustment. Nevertheless, we did not find a significantly increased risk for participants in the second (OR, 1.75; 95% CI, 0.44-7.02) and third (OR, 2.80; 95% CI, 0.70-11.18) quartiles. Then the first three quartiles were merged as a reference group to calculate the OR of HDP for participants in the highest quartile and we found a significantly increased risk for HDP in individuals in the highest quartile (OR, 2.28, 95% CI, 1.02-5.06). CONCLUSION Increased serum soluble corin in mid pregnancy was associated with an increased risk for HDP. Our findings suggest that increased serum soluble corin in mid pregnancy could be an indicator for HDP.

Increased serum soluble corin in mid pregnancy is associated with hypertensive disorders of pregnancy.

37949139

Dandelions have long been used in herbal medicine for their choleretic, diuretic, antiinflammatory, appetite-stimulating and laxative properties. An antioxidant property can be supposed as a basis of their-therapeutic effects. To understand the mechanism of the drug's action, the effects of natural extracts on a microsomal fraction of rat liver were examined. The extracts diminished the enzymatically induced-lipid peroxidation and reduced the cytochrome c with and without NADPH in a concentration dependent manner.

The in vitro effect of dandelions antioxidants on microsomal lipid peroxidation.

37964706

Ca2+ entry through store-operated Ca2+ channels drives the production of the pro-inflammatory molecule leukotriene C4 (LTC4) from mast cells through a pathway involving Ca2+-dependent protein kinase C, mitogen-activated protein kinases ERK1/2, phospholipase A2, and 5-lipoxygenase. Here we examine whether local Ca2+ influx through store-operated Ca2+ release-activated Ca2+ (CRAC) channels in the plasma membrane stimulates this signaling pathway. Manipulating the amplitude and spatial extent of Ca2+ entry by altering chemical and electrical gradients for Ca2+ influx or changing the Ca2+ buffering of the cytoplasm all impacted on protein kinase C and ERK activation, generation of arachidonic acid and LTC4 secretion, with little change in the bulk cytoplasmic Ca2+ rise. Similar bulk cytoplasmic Ca2+ concentrations were achieved when CRAC channels were activated in 0.25 mm external Ca2+ versus 2 mm Ca2+ and 100 nm La3+, an inhibitor of CRAC channels. However, despite similar bulk cytoplasmic Ca2+, protein kinase C activation and LTC4 secretion were larger in 2 mm Ca2+ and La3+ than in 0.25 mm Ca2+, consistent with the central involvement of a subplasmalemmal Ca2+ rise. The nonreceptor tyrosine kinase Syk coupled CRAC channel opening to protein kinase C and ERK activation. Recombinant TRPC3 channels also activated protein kinase C, suggesting that subplasmalemmal Ca2+ rather than a microdomain exclusive to CRAC channels is the trigger. Hence a subplasmalemmal Ca2+ increase in mast cells is highly versatile in that it triggers cytoplasmic responses through generation of intracellular messengers as well as long distance changes through increased secretion of paracrine signals.

Local Ca2+ influx through Ca2+ release-activated Ca2+ (CRAC) channels stimulates production of an intracellular messenger and an intercellular pro-inflammatory signal.

37969403

New recombinant strains of attenuated Salmonella typhi used as live oral vaccines elicit potent immune responses. This study examined the patterns of cytokine production and proliferation to specific S. typhi antigens in subjects orally immunized with attenuated S. typhi vaccines CVD 906, CVD 908, and CVD 908 expressing the circumsporozoite protein of Plasmodium falciparum. After immunization, sensitized lymphocytes were found in subjects' blood that exhibited significantly increased proliferative responses and interferon-gamma production to purified S. typhi flagella when compared with preimmunization levels. Significant negative correlations were observed between interleukin-4 production and both interferon-gamma production and proliferation to S. typhi flagella. These results demonstrate that oral immunization with attenuated S. typhi strains alone or with those carrying a foreign gene elicits strong systemic cell-mediated immunity to purified S. typhi antigens, including the production of cytokines compatible with T1-type responses.

Cytokine production patterns and lymphoproliferative responses in volunteers orally immunized with attenuated vaccine strains of Salmonella typhi.

38009906

Expression of the INK4b/ARF/INK4a tumor suppressor locus in normal and cancerous cell growth is controlled by methylation of histone H3 at lysine 27 (H3K27me) as directed by the Polycomb group proteins. The antisense noncoding RNA ANRIL of the INK4b/ARF/INK4a locus is also important for expression of the protein-coding genes in cis, but its mechanism has remained elusive. Here we report that chromobox 7 (CBX7) within the polycomb repressive complex 1 binds to ANRIL, and both CBX7 and ANRIL are found at elevated levels in prostate cancer tissues. In concert with H3K27me recognition, binding to RNA contributes to CBX7 function, and disruption of either interaction impacts the ability of CBX7 to repress the INK4b/ARF/INK4a locus and control senescence. Structure-guided analysis reveals the molecular interplay between noncoding RNA and H3K27me as mediated by the conserved chromodomain. Our study suggests a mechanism by which noncoding RNA participates directly in epigenetic transcriptional repression.

Molecular interplay of the noncoding RNA ANRIL and methylated histone H3 lysine 27 by polycomb CBX7 in transcriptional silencing of INK4a.

38023457

Severe quantitative and qualitative brown adipocyte defects are common in obesity. To investigate whether aberrant expression of tumor necrosis factor alpha (TNF-alpha) in obesity is involved in functional brown fat atrophy, we have studied genetically obese (ob/ob) mice with targeted null mutations in the genes encoding the two TNF receptors. The absence of both TNF receptors or p55 receptor alone resulted in a significant reduction in brown adipocyte apoptosis and an increase in beta(3)-adrenoreceptor and uncoupling protein-1 expression in obese mice. Increased numbers of multilocular functionally active brown adipocytes, and improved thermoregulation was also observed in obese animals lacking TNF-alpha function. These results indicate that TNF-alpha plays an important role in multiple aspects of brown adipose tissue biology and mediates the abnormalities that occur at this site in obesity.

Tumor necrosis factor alpha mediates apoptosis of brown adipocytes and defective brown adipocyte function in obesity.

38025907

Nonalcoholic fatty liver disease (NAFLD) is an increasingly prevalent chronic liver disease for which no approved therapies are available. Despite intensive research, the cellular mechanisms that mediate NAFLD pathogenesis and progression are poorly understood. Although obesity, diabetes, insulin resistance, and related metabolic syndrome, all consequences of a Western diet lifestyle, are well-recognized risk factors for NAFLD development, dysregulated bile acid metabolism is emerging as a novel mechanism contributing to NAFLD pathogenesis. Notably, NAFLD patients exhibit a deficiency in fibroblast growth factor 19 (FGF19), an endocrine hormone in the gut-liver axis that controls de novo bile acid synthesis, lipogenesis, and energy homeostasis. Using a mouse model that reproduces the clinical progression of human NAFLD, including the development of simple steatosis, nonalcoholic steatohepatitis (NASH), and advanced "burnt-out" NASH with hepatocellular carcinoma, we demonstrate that FGF19 as well as an engineered nontumorigenic FGF19 analogue, M70, ameliorate bile acid toxicity and lipotoxicity to restore liver health. Mass spectrometry-based lipidomics analysis of livers from mice treated with FGF19 or M70 revealed significant reductions in the levels of toxic lipid species (i.e., diacylglycerols, ceramides and free cholesterol) and an increase in levels of unoxidized cardiolipins, an important component of the inner mitochondrial membrane. Furthermore, treatment with FGF19 or M70 rapidly and profoundly reduced levels of liver enzymes, resolved the histologic features of NASH, and enhanced insulin sensitivity, energy homeostasis, and lipid metabolism. Whereas FGF19 induced hepatocellular carcinoma formation following prolonged exposure in these mice, animals expressing M70 showed no evidence of liver tumorigenesis in this model. Conclusion: We have engineered an FGF19 hormone that is capable of regulating multiple pathways to deliver antisteatotic, anti-inflammatory, and antifibrotic activities and that represents a potentially promising therapeutic for patients with NASH. (Hepatology Communications 2017;1:1024-1042).

Engineered FGF19 eliminates bile acid toxicity and lipotoxicity leading to resolution of steatohepatitis and fibrosis in mice

38028419

White adipose tissue (WAT) secretes adipokines, which critically regulate lipid metabolism. The present study investigated the effects of alcohol on adipokines and the mechanistic link between adipokine dysregulation and alcoholic fatty liver disease. Mice were fed alcohol for 2, 4, or 8 weeks to document changes in adipokines over time. Alcohol exposure reduced WAT mass and body weight in association with hepatic lipid accumulation. The plasma adiponectin concentration was increased at 2 weeks, but declined to normal at 4 and 8 weeks. Alcohol exposure suppressed leptin gene expression in WAT and reduced the plasma leptin concentration at all times measured. There is a highly positive correlation between plasma leptin concentration and WAT mass or body weight. To determine whether leptin deficiency mediates alcohol-induced hepatic lipid dyshomeostasis, mice were fed alcohol for 8 weeks with or without leptin administration for the last 2 weeks. Leptin administration normalized the plasma leptin concentration and reversed alcoholic fatty liver. Alcohol-perturbed genes involved in fatty acid β-oxidation, very low-density lipoprotein secretion, and transcriptional regulation were attenuated by leptin. Leptin also normalized alcohol-reduced phosphorylation levels of signal transducer Stat3 and adenosine monophosphate-activated protein kinase. These data demonstrated for the first time that leptin deficiency in association with WAT mass reduction contributes to the pathogenesis of alcoholic fatty liver disease.

Leptin deficiency contributes to the pathogenesis of alcoholic fatty liver disease in mice.