title: Downstream of Mutant KRAS, the Transcription Regulator YAP Is Essential for Neoplastic Progression to Pancreatic Ductal Adenocarcinoma

text: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with poor survival rates and frequently carries oncogenic KRAS mutation. However, KRAS has thus far not been a viable therapeutic target. We found that the abundance of YAP mRNA, which encodes Yes-associated protein (YAP), a protein regulated by the Hippo pathway during tissue development and homeostasis, was increased in human PDAC tissue compared with that in normal pancreatic epithelia. In genetically engineered KrasG12D and KrasG12D:Trp53R172H mouse models, pancreas-specific deletion of Yap halted the progression of early neoplastic lesions to PDAC without affecting normal pancreatic development and endocrine function. Although Yap was dispensable for acinar to ductal metaplasia (ADM), an initial step in the progression to PDAC, Yap was critically required for the proliferation of mutant Kras or Kras:Trp53 neoplastic pancreatic ductal cells in culture and for their growth and progression to invasive PDAC in mice. Yap functioned as a critical transcriptional switch downstream of the oncogenic KRAS–mitogen-activated protein kinase (MAPK) pathway, promoting the expression of genes encoding secretory factors that cumulatively sustained neoplastic proliferation, a tumorigenic stromal response in the tumor microenvironment, and PDAC progression in Kras and Kras:Trp53 mutant pancreas tissue. Together, our findings identified Yap as a critical oncogenic KRAS effector and a promising therapeutic target for PDAC and possibly other types of KRAS-mutant cancers.


title: Rho, Rac and Cdc42 regulate actin organization and cell adhesion in macrophages.

text: Rho family proteins are known to regulate actin organization in fibroblasts, but their functions in cells of haematopoietic origin have not been studied in detail. Bac1.2F5 cells are a colony-stimulating factor-1 (CSF-1)-dependent murine macrophage cell line; CSF-1 stimulates their proliferation and motility, and acts as a chemoattractant. CSF-1 rapidly induced actin reorganization in Bac1 cells: it stimulated the formation of filopodia, lamellipodia and membrane ruffles at the plasma membrane, as well as the appearance of fine actin cables within the cell interior. Microinjection of constitutively activated (V12)Rac1 stimulated lamellipodium formation and membrane ruffling. The dominant inhibitory Rac mutant, N17Rac1, inhibited CSF-1-induced lamellipodium formation, and also induced cell rounding. V12Cdc42 induced the formation of long filopodia, while the dominant inhibitory mutant N17Cdc42 prevented CSF-1-induced formation of filopodia but not lamellipodia. V14RhoA stimulated actin cable assembly and cell contraction, while the Rho inhibitor, C3 transferase, induced the loss of actin cables. Bac1 cells had cell-to-substratum adhesion sites containing beta1 integrin, pp125FAK, paxillin, vinculin, and tyrosine phosphorylated proteins. These 'focal complexes' were present in growing and CSF-1-starved cells, but were disassembled in cells injected with N17Cdc42 or N17Rac1. Interestingly, beta1 integrin did not disperse until long after focal phosphotyrosine and vinculin staining had disappeared. We conclude that in Bac1 macrophages Cdc42, Rac and Rho regulate the formation of distinct actin filament-based structures, and that Cdc42 and Rac are also required for the assembly of adhesion sites to the extracellular matrix.


title: Visualizing the function and fate of neutrophils in sterile injury and repair

text: Neutrophils have been implicated as harmful cells in a variety of inappropriate inflammatory conditions where they injure the host, leading to the death of the neutrophils and their subsequent phagocytosis by monocytes and macrophages. Here we show that in a fully repairing sterile thermal hepatic injury, neutrophils also penetrate the injury site and perform the critical tasks of dismantling injured vessels and creating channels for new vascular regrowth. Upon completion of these tasks, they neither die at the injury site nor are phagocytosed. Instead, many of these neutrophils reenter the vasculature and have a preprogrammed journey that entails a sojourn in the lungs to up-regulate CXCR4 (C-X-C motif chemokine receptor 4) before entering the bone marrow, where they undergo apoptosis.


title: The effect of mass screening on incidence and mortality of squamous and adenocarcinoma of cervix uteri.

text: OBJECTIVE To describe the efficacy of the Finnish mass screening program for cervical squamous carcinoma and adenocarcinoma, as reflected by changes of incidence and mortality rate. METHODS Cervical cancer incidence and mortality data were obtained from the Finnish Cancer Registry. Data were available from the year 1953, when the registry was established. The nationwide mass screening program in Finland was started in the mid-1960s. A centralized organization administers this program. Women age 30-60 years are notified for screening every 5 years. RESULTS The mean incidence of cervical carcinoma in the early 1960s was 15.4 per 10(5) woman-years. In 1991, it was only 2.7 per 10(5) woman-years. The mortality rate has decreased in the same proportion since the mass screening program. In the early 1960s, the mortality was 6.6 and in 1991 1.4 per 10(5) woman-years. However, the decrease of the incidence is seen almost exclusively in squamous cell carcinomas. The mortality caused by adenocarcinoma has decreased in screened birth cohorts, but the incidence rate has remained the same. CONCLUSIONS The Finnish mass screening program has been effective and its continuation is of utmost importance. In the future more attention should be given to glandular cell atypias in cervical smears. Thus, it might be possible to decrease the incidence of cervical adenocarcinoma.


title: Role of 5-HT in the regulation of the brain-pituitary-adrenal axis: effects of 5-HT on adrenocortical cells.

text: Serotonin (5-HT) plays a pivotal role in the regulation of the brain-pituitary-adrenal axis. In particular, 5-HT has been shown to control the activity of hypothalamic CRF neurons and pituitary corticotrope cells through activation of 5-HT1A and (or) 5-HT(2A/2C) receptor subtypes. 5-HT, acting through 5-HT2 receptors, can also trigger the renin-angiotensin system by stimulating renin secretion and consequently can enhance aldosterone production. At the adrenal level, 5-HT produced locally stimulates the secretory activity of adrenocortical cells through a paracrine mode of communication. The presence of 5-HT in the adrenal gland has been demonstrated immunohistochemically and biochemically in various species. In the frog, rat, and pig adrenal gland, 5-HT is synthesized by chromaffin cells, while in the mouse adrenal cortex, 5-HT is contained in nerve fibers. In man, 5-HT is present in perivascular mast cells. In vivo and in vitro studies have shown that 5-HT stimulates corticosteroid secretion in various species (including human). The type of receptor involved in the mechanism of action of 5-HT differs between the various species. In frogs and humans, the stimulatory effect of 5-HT on adrenocortical cells is mediated through a 5-HT4 receptor subtype positively coupled to adenylyl cyclase and calcium influx. In the rat, the effect of 5-HT on aldosterone secretion is mediated via activation of 5-HT7 receptors. Clinical studies indicate that 5-HT4 receptor agonists stimulate aldosterone secretion in healthy volunteers and in patients with corticotropic insufficiency and primary hyperaldosteronism. Local serotonergic control of corticosteroid production may be involved in the physiological control of the activity of the adrenal cortex as well as in the pathophysiology of cortisol and aldosterone disorders.


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

text: 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.


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

text: 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.


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

text: 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.


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

text: 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.


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

text: 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.


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

text: 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.


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

text: 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.


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

text: 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.


title: Role of ocular pigment epithelial cells in immune privilege

text: 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.


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

text: 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.


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

text: 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.


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

text: 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.


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

text: 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.


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

text: 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.


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

text: 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.


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

text: 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.


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

text: 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.


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

text: 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.


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

text: 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.


title: Lactate Metabolism in Human Lung Tumors

text: 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.


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

text: 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.


title: Sex differences in sex chromosome gene expression in mouse brain.

text: 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.


title: Genome architecture, rearrangements and genomic disorders.

text: 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.


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

text: 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.


title: 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)

text: 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.


title: Improving outcomes of acute kidney injury: report of an initiative

text: 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.


title: A Screen for Endocytic Motifs

text: 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.


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

text: 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.


title: Histone tails: Directing the chromatin response to DNA damage.

text: 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.


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

text: 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.


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

text: 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.


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

text: 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.


title: Bile acids: natural ligands for an orphan nuclear receptor.

text: 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.


title: The yeast ribosomal protein L32 and its gene.

text: 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.


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

text: 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.


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

text: 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.


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

text: 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.


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

text: 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.


title: Polyphenols of Cocoa: Inhibition of Mammalian 15-Lipoxygenase

text: 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.


title: Quality of life in patients with advanced heart failure.

text: 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.


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

text: 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)


title: Serum Soluble Corin is Decreased in Stroke.

text: 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.


title: 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.

text: 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.


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

text: 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.


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

text: 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.


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

text: 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.


title: B-cell self-tolerance in humans.

text: 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.


title: JMJD6 Promotes Colon Carcinogenesis through Negative Regulation of p53 by Hydroxylation

text: 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.


title: Spread of chloroquine resistance in Plasmodium falciparum.

text: 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.


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

text: 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.


title: Lipid metabolism and toxicity in the heart.

text: 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.


title: Malaria eradication back on the table.

text: 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. ■


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

text: 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.


title: CTCF and Cohesin in Genome Folding and Transcriptional Gene Regulation.

text: 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.


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

text: 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.


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

text: 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.


title: The cryoprotective effect of trehalose supplementation on boar spermatozoa quality.

text: 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.


title: Roads to polyploidy: the megakaryocyte example.

text: 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.


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

text: 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.


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

text: 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.


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

text: 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.


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

text: 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.


title: Familial gastroesophageal reflux and development of Barrett's esophagus.

text: 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.