New onset diabetes after kidney transplantation is associated with increased mortality-A retrospective cohort study.

OBJECTIVE: Clinical outcomes in individuals with new onset diabetes after transplantation (NODAT) and the optimal treatment for this complication are poorly characterized. This study was intended to better define these issues. METHODS: Patients who underwent kidney transplantation and did not have diabetes prior to transplantation were included in the study. Clinical outcomes were compared between those who developed NODAT and those who did not. In those who developed NODAT, oral therapy was compared with insulin based therapy. RESULTS: A total of 266 kidney transplant recipients were included, of which 71 (27%) developed NODAT during the time of the follow-up. Using Cox multivariate analysis adjusted for age and gender, hazard ratio for overall mortality among patients with NODAT versus those without NODAT was 2.69 (95% CI 1.04-7.01). Among patients who developed NODAT, 29 patients (40%) were treated with an insulin-based regimen. At the end of follow-up, no difference was found in mean HbA1c, and therapy regimen was not associated with greater mortality. CONCLUSIONS: New onset diabetes in kidney transplanted patients is associated with increased mortality compared with kidney transplanted patients without NODAT.

Predicting Clostridium difficile infection in diabetic patients and the effect of metformin therapy: a retrospective, case-control study.

Data on risk factors for Clostridium difficile infection (CDI) in diabetic patients are scarce. Recently, it has been shown that metformin increases the Bacteroidetes/Firmicutes ratio; therefore, it may yield a protective effect against CDI. We aimed to assess risk factors for CDI in diabetic patients beyond antibiotic treatment, and to determine the impact of metformin therapy on the development of CDI in these patients. In this retrospective, case-control study, all consecutive CDI diabetic patients, from January 2009 to December 2013, were included and compared to consecutive diabetic patients without CDI, hospitalized during the same period and in the same departments. Of 7,670 patients tested for C. difficile toxins, 486 were diabetics. Of them, 150 (30.8 %) were positive for C. difficile toxins and 336 (69.1 %) were negative. On multivariate analysis, metformin treatment was associated with a significant reduction in CDI [odds ratio (OR) = 0.58; 95 % confidence interval (CI), 0.37-0.93; p = 0.023], while heart failure was associated with significantly higher rates of CDI (OR = 1.654; 95 % CI, 1.007-2.716; p = 0.047), together with poor functional status, previous hospitalization, and abdominal surgery. Our findings suggest that, in diabetic patients, in addition to the well-recognized risk factors, heart failure is an additional risk factor for CDI, while metformin treatment seems to have a protective effect against the development of CDI. The exact mechanisms underlying this protective effect remain to be fully understood.

Loss-of-function mutations in a calcium-channel alpha1-subunit gene in Xp11.23 cause incomplete X-linked congenital stationary night blindness.

X-linked congenital stationary night blindness (CSNB) is a recessive non-progressive retinal disorder characterized by night blindness, decreased visual acuity, myopia, nystagmus and strabismus. Two distinct clinical entities of X-linked CSNB have been proposed. Patients with complete CSNB show moderate to severe myopia, undetectable rod function and a normal cone response, whereas patients with incomplete CSNB show moderate myopia to hyperopia and subnormal but measurable rod and cone function. The electrophysiological and psychophysical features of these clinical entities suggest a defect in retinal neurotransmission. The apparent clinical heterogeneity in X-linked CSNB reflects the recently described genetic heterogeneity in which the locus for complete CSNB (CSNB1) was mapped to Xp11.4, and the locus for incomplete CSNB (CSNB2) was refined within Xp11.23 (ref. 5). A novel retina-specific gene mapping to the CSNB2 minimal region was characterized and found to have similarity to voltage-gated L-type calcium channel alpha1-subunit genes. Mutation analysis of this new alpha1-subunit gene, CACNA1F, in 20 families with incomplete CSNB revealed six different mutations that are all predicted to cause premature protein truncation. These findings establish that loss-of-function mutations in CACNA1F cause incomplete CSNB, making this disorder an example of a human channelopathy of the retina.

Mutation of a nuclear receptor gene, NR2E3, causes enhanced S cone syndrome, a disorder of retinal cell fate.

Hereditary human retinal degenerative diseases usually affect the mature photoreceptor topography by reducing the number of cells through apoptosis, resulting in loss of visual function. Only one inherited retinal disease, the enhanced S-cone syndrome (ESCS), manifests a gain in function of photoreceptors. ESCS is an autosomal recessive retinopathy in which patients have an increased sensitivity to blue light; perception of blue light is mediated by what is normally the least populous cone photoreceptor subtype, the S (short wavelength, blue) cones. People with ESCS also suffer visual loss, with night blindness occurring from early in life, varying degrees of L (long, red)- and M (middle, green)-cone vision, and retinal degeneration. The altered ratio of S- to L/M-cone photoreceptor sensitivity in ESCS may be due to abnormal cone cell fate determination during retinal development. In 94% of a cohort of ESCS probands we found mutations in NR2E3 (also known as PNR), which encodes a retinal nuclear receptor recently discovered to be a ligand-dependent transcription factor. Expression of NR2E3 was limited to the outer nuclear layer of the human retina. Our results suggest that NR2E3 has a role in determining photoreceptor phenotype during human retinogenesis.

Characterization of gap junction proteins in the bladder of Cx43 mutant mouse models of oculodentodigital dysplasia.

Oculodentodigital dysplasia (ODDD) is a rare developmental disease resulting from germline mutations in the GJA1 gene that encodes the gap junction protein connexin43 (Cx43). In addition to the classical ODDD symptoms that affect the eyes, teeth, bone and digits, in some cases ODDD patients have reported bladder impairments. Thus, we chose to characterize the bladder in mutant mouse models of ODDD that harbor two distinct Cx43 mutations, G60S and I130T. Histological assessment revealed no difference in bladder detrusor wall thickness in mutant compared to littermate control mice. The overall localization of Cx43 in the lamina propria and detrusor also appeared to be similar in the bladders of mutant mice with the exception that the G60S mice had more instances of intracellular Cx43. However, both mutant mouse lines exhibited a significant reduction in the phosphorylated P1 and P2 isoforms of Cx43, while only the I130T mice exhibited a reduction in total Cx43 levels. Interestingly, Cx26 levels and distribution were not altered in mutant mice as it was localized to intracellular compartments and restricted to the basal cell layers of the urothelium. Our studies suggest that these two distinct genetically modified mouse models of ODDD probably mimic patients who lack bladder defects or other factors, such as aging or co-morbidities, are necessary to reveal a bladder phenotype.

Hedonic perception of odors in children aged 5-8 years is similar across 18 countries: Preliminary data.

OBJECTIVE: Olfactory preference emerges very early in life, and the sense of smell in children rapidly develops until the second decade of life. It is still unclear whether hedonic perception of odors is shared in children inhabiting different regions of the globe. METHODS: Five-hundred ten healthy children (N = 510; ngirls = 256; nboys = 254) aged from 5 to 8 years from 18 countries rated the pleasantness of 17 odors. RESULTS: The hedonic perception of odors in children aged between 5 and 8 years was rather consistent across 18 countries and mainly driven by the qualities of an odor and the overall ability of children to label odorants. CONCLUSION: Conclusions from this study, being a secondary analysis, are limited to the presented set of odors that were initially selected for the development of U-Sniff test and present null findings for the cross-cultural variability in hedonic perception of odors across 18 countries. These two major issues should be addressed in the future to either contradict or replicate the results presented herewith. This research lays fundament for posing further research questions about the developmental aspects of hedonic perception of odors and opens a new door for investigating cross-cultural differences in chemosensory perception of children.

Optical gain in single tensile-strained germanium photonic wire.

We have investigated the optical properties of tensile-strained germanium photonic wires. The photonic wires patterned by electron beam lithography (50 µm long, 1 µm wide and 500 nm thick) are obtained by growing a n-doped germanium film on a GaAs substrate. Tensile strain is transferred in the germanium layer using a Si3N4 stressor. Tensile strain around 0.4% achieved by the technique corresponds to an optical recombination of tensile-strained germanium involving light hole band around 1690 nm at room temperature. We show that the waveguided emission associated with a single tensile-strained germanium wire increases superlinearly as a function of the illuminated length. A 20% decrease of the spectral broadening is observed as the pump intensity is increased. All these features are signatures of optical gain. A 80 cm⁻¹ modal optical gain is derived from the variable strip length method. This value is accounted for by the calculated gain material value using a 30 band k · p formalism. These germanium wires represent potential building blocks for integration of nanoscale optical sources on silicon.

Predicting the pathogenicity of RPE65 mutations.

To assist in distinguishing disease-causing mutations from nonpathogenic polymorphisms, we developed an objective algorithm to calculate an "estimate of pathogenic probability" (EPP) based on the prevalence of a specific variation, its segregation within families, and its predicted effects on protein structure. Eleven missense variations in the RPE65 gene were evaluated in patients with Leber congenital amaurosis (LCA) using the EPP algorithm. The accuracy of the EPP algorithm was evaluated using a cell-culture assay of RPE65-isomerase activity The variations were engineered into plasmids containing a human RPE65 cDNA and the retinoid isomerase activity of each variant was determined in cultured cells. The EPP algorithm predicted eight substitution mutations to be disease-causing variants. The isomerase catalytic activities of these RPE65 variants were all less than 6% of wild-type. In contrast, the EPP algorithm predicted the other three substitutions to be non-disease-causing, with isomerase activities of 68%, 127%, and 110% of wild-type, respectively. We observed complete concordance between the predicted pathogenicities of missense variations in the RPE65 gene and retinoid isomerase activities measured in a functional assay. These results suggest that the EPP algorithm may be useful to evaluate the pathogenicity of missense variations in other disease genes where functional assays are not available.

Molecular characterization and functional expression of the human cardiac gap junction channel.

Gap junctions permit the passage of ions and chemical mediators from cell to cell. To identify the molecular genetic basis for this coupling in the human heart, we have isolated clones from a human fetal cardiac cDNA library which encode the full-length human cardiac gap junction (HCGJ) mRNA. The predicted amino acid sequence is homologous to the rat cardiac gap junction protein, connexin43 (Beyer, E. D., D. Paul, and D. A. Goodenough. 1987. J. Cell Biol. 105:2621-2629), differing by 9 of 382 amino acids. HCGJ mRNA is detected as early as fetal week 15 and persists in adult human cardiac samples. Genomic DNA analysis suggests the presence of two highly homologous HCGJ loci, only one of which is functional. Stable transfection of the HCGJ cDNA into SKHep1 cells, a human hepatoma line which is communication deficient, leads to the formation of functional channels. Junctional conductance in pairs of transfectants containing 10 copies of the HCGJ sequence is high (approximately 20 nS). Single channel currents are detectable in this expression system and correspond to conductances of approximately 60 pS. These first measurements of the HCGJ channel are similar to the junctional conductance recorded between pairs of rat or guinea pig cardiocytes.

Discovery of intense gamma-ray flashes of atmospheric origin.

Detectors aboard the Compton Gamma Ray Observatory have observed an unexplained terrestrial phenomenon: brief, intense flashes of gamma rays. These flashes must originate in the atmosphere at altitudes above at least 30 kilometers in order to escape atmospheric absorption and reach the orbiting detectors. At least a dozen such events have been detected over the past 2 years. The photon spectra from the events are very hard (peaking in the high-energy portion of the spectrum) and are consistent with bremsstrahlung emission from energetic (million-electron volt) electrons. The most likely origin of these high-energy electrons, although speculative at this time, is a rare type of high-altitude electrical discharge above thunderstorm regions.

Mutations in the cone-rod homeobox gene are associated with the cone-rod dystrophy photoreceptor degeneration.

Crx is a novel paired-like homeodomain protein that is expressed predominantly in retinal photoreceptors and pinealocytes. Its gene has been mapped to chromosome 19q13.3, the site of a disease locus for autosomal dominant cone-rod dystrophy (CORDII). Analysis of the proband from a family with autosomal dominant CORD revealed an Arg41Trp substitution in the third residue of the CRX homeodomain. The sequence change cosegregated with the disease phenotype and was not detected in 247 normal controls. Recombinant CRX homeodomain containing the Arg41Trp substitution showed decreased DNA binding activity. Analysis of another 169 CORD probands identified three additional CRX sequence variations (Arg41Gln, Val242Met, and a 4 bp deletion in codons 196/7) that were not found among the controls. This data suggests that mutations in the CRX gene are associated with photoreceptor degeneration and that the Crx protein is necessary for the maintenance of normal cone and rod function.

Cyclic AMP regulates the HERG K(+) channel by dual pathways.

Lethal cardiac arrhythmias are a hallmark of the hereditary Long QT syndrome (LQTS), a disease produced by mutations of cardiac ion channels [1]. Often these arrhythmias are stress-induced, suggesting a relationship between beta-adrenergic activation of adenylate cyclase and cAMP-dependent alteration of one or more of the ion channels involved in LQTS. Second messengers modulate ion channel activity either by direct interaction or through intermediary kinases and phosphatases. Here we show that the second messenger cAMP regulates the K(+) channel mutated in the LQT2 form of LQTS, HERG [2], both directly and indirectly. Activation of cAMP-dependent protein kinase (PKA) causes phosphorylation of HERG accompanied by a rapid reduction in current amplitude, acceleration of voltage-dependent deactivation, and depolarizing shift in voltage-dependent activation. In a parallel pathway, cAMP directly binds to the HERG protein with the opposing effect of a hyperpolarizing shift in voltage-dependent activation. The summation of cAMP-mediated effects is a net diminution of the effective current, but when HERG is complexed with with the K(+) channel accessory proteins MiRP1 or minK, the stimulatory effects of cAMP are favored. These findings provide a direct link between stress and arrhythmia by a unique mechanism where a single second messenger exerts complex regulation of an ion channel via two distinct pathways.

Regulated expression of foreign genes in vivo after germline transfer.

Tight transcriptional control of foreign genes introduced into the germline of transgenic mice would be of great experimental value in studies of gene function. To develop a system in which the spatial and temporal expression of candidate genes implicated in cardiac development or function could be tightly controlled in vivo, we have generated transgenic mice expressing a tetracycline-controlled transactivator (tTA) under the control of a rat alpha myosin heavy chain promoter (MHC alpha-tTA mice), as well as mice harboring a candidate target gene implicated in the control of differentiation, Id1 (tet-Id1 mice). No expression of the target transgene was detected in any tissues of hemizygous tet-Id1 mice. Genetic crosses with MHC alpha-tTA mice resulted in transactivation of the Id1 transgene, but expression was restricted to heart, where tTA was expressed. Furthermore, transactivation of the target gene was tightly and reversibly controlled by systemic therapy with tetracycline, both in utero and postnatally. These studies demonstrate the feasibility of such a binary approach for tightly controlling the timing and extent of expression of transgenes in vivo. This approach should be generally useful for the ectopic expression of candidate genes in selected tissues during delineated developmental stages.

Tetracycline-regulated cardiac gene expression in vivo.

Tight regulation of foreign genes expressed in vivo would facilitate studies of many biologic processes and would be useful for gene transfer-based therapies. To test the ability of a tetracycline-regulated gene expression system to function in vivo, we directly injected chimeric tet repressor-VP16 transactivator expression plasmids and luciferase target genes into the hearts of adult rats. Cardiac luciferase activity increased over two orders of magnitude in response to small changes in input tetracycline-controlled transactivator DNA. Transactivation was repressed to background levels by subtherapeutic concentrations of tetracycline in a dose-dependent manner. Target gene expression could be rapidly and reversibly controlled by manipulating antibiotic administration. This system may be particularly useful for in vivo studies of gene function or gene therapies where the timing or extent of expression are critical variables.

Wnt-1 regulation of connexin43 in cardiac myocytes.

Gap junction channels composed of connexin43 (Cx43) are essential for normal heart formation and function. We studied the potential role of the Wnt family of secreted polypeptides as regulators of Cx43 expression and gap junction channel function in dissociated myocytes and intact hearts. Neonatal rat cardiomyocytes responded to Li(+), which mimics Wnt signaling, by accumulating the effector protein beta-catenin and by inducing Cx43 mRNA and protein markedly. Induction of Cx43 expression was also observed in cardiomyocytes cocultured with Rat-2 fibroblasts or N2A neuroblastoma cells programmed to secrete bioactive Wnt-1. By transfecting a Cx43 promoter-reporter gene construct into cardiomyocytes, we demonstrated that the inductive effect of Wnt signaling was transcriptionally mediated. Enhanced expression of Cx43 increased cardiomyocyte cell coupling, as determined by Lucifer Yellow dye transfer and by calcium wave propagation. Conversely, in a transgenic cardiomyopathic mouse model that exhibits ventricular arrhythmias and gap junctional remodeling, beta-catenin and Cx43 expression were downregulated concordantly. In response to Wnt signaling, the accumulating Cx43 colocalized with beta-catenin in the junctional membrane; moreover, forced expression of Cx43 in cardiomyocytes reduced the transactivation potential of beta-catenin. These findings demonstrate that Wnt signaling is an important modulator of Cx43-dependent intercellular coupling in the heart, and they support the hypothesis that dysregulated signaling contributes to altered impulse propagation and arrhythmia in the myopathic heart.

Expression of protein kinase C beta in the heart causes hypertrophy in adult mice and sudden death in neonates.

Protein kinase C (PKC) activation in the heart has been linked to a hypertrophic phenotype and to processes that influence contractile function. To establish whether PKC activation is sufficient to induce an abnormal phenotype, PKCbeta was conditionally expressed in cardiomyocytes of transgenic mice. Transgene expression in adults caused mild and progressive ventricular hypertrophy associated with impaired diastolic relaxation, whereas expression in newborns caused sudden death associated with marked abnormalities in the regulation of intracellular calcium. Thus, the PKC signaling pathway in cardiocytes has different effects depending on the timing of expression and, in the adult, is sufficient to induce pathologic hypertrophy.

Development of the right ventricular inflow tract and moderator band: a possible morphological and functional explanation for Mahaim tachycardia.

Atriofascicular accessory bundles with AV-node like conduction properties can sustain atrioventricular (AV) re-entrant tachycardia (Mahaim tachycardia). During early embryogenesis, the AV canal is situated above the primitive left ventricle (LV), and a right AV connection has not been achieved yet. We studied the formation of the right ventricular (RV) inflow tract in relation to the developing cardiac conduction system and hypothesized a morphological explanation for functional atriofascicular bypass tracts. Analysis of lacZ-expression during sequential stages of cardiogenesis was performed in CCS-lacZ transgenic mice (E9.5 to 15.5). Embryos were stained for beta-galactosidase activity and the myocardial marker HHF35. At early stages CCS-lacZ expression was observed in a ring surrounding the AV canal, which connected at the inner curvature to the primary fold. The first sign of formation of the (CCS-lacZ negative) RV inlet component was a groove in the CCS-lacZ positive tissue of the primary fold. Outgrowth of the RV inlet tract resulted in division of the primary fold in a septal part, the trabecula septomarginalis and a lateral part, the moderator band, which extended laterally up to the right AV ring. Electrophysiological measurements in embryonic hearts (E15.5) in which the right atrium (RA) and RV were isolated from the left atrium (LA) and LV supported the functionality of this AV-connection via the moderator band, by demonstrating sequential atrial and ventricular activation in both RA/RV and LA/LV preparations. In conclusion, our observations may provide a possible morphological and functional explanation for atriofascicular accessory pathways via the moderator band, underlying Mahaim tachycardia.

Conditional expression and signaling of a specifically designed Gi-coupled receptor in transgenic mice.

To control G protein signaling in vivo, we have modified G protein-coupled receptors to respond exclusively to synthetic small molecule agonists and not to their natural agonist(s). These engineered receptors are designated RASSLs (receptor activated solely by a synthetic ligand). A prototype RASSL (Ro1) based on the Gi-coupled K opioid receptor was expressed in transgenic mice under the control of the tetracycline transactivator (tet) system. Activation of Ro1 expressed in the heart decreased heart rate by up to 80%, an expected effect of increased Gi signaling. Maximal heart rate changes occurred in less than 1 min, demonstrating the speed of this inducible signaling system. This Ro1-mediated slowing of heart rate was also subject to desensitization, which lasted more than 24 h. Both the initial effect on heart rate and the desensitization occurred, even though Ro1 is derived from a human opioid receptor not normally involved in heart rate control. In addition, the tet system was used to induce Ro1 expression in hepatocytes and salivary gland, where Gi signaling is known to control physiologic events such as proliferation and secretion. These studies demonstrate that a RASSL can be inducibly expressed in several mouse tissues and used in vivo to activate G protein signaling in a controllable fashion.

Attainment of educational levels in patients with Leber's congenital amaurosis.

PURPOSE: To assess the educational level attained by patients legally blind with Leber's congenital amaurosis (LCA). DESIGN: Cross-sectional assessment. INTERVENTION: None. MAIN OUTCOME MEASURE: Highest educational level attained by 55 patients with LCA. RESULTS: A total of 55 patients with LCA were included in the study. Of the 55, 54 finished high school. In addition, 36 patients (65%) completed a college education and received a bachelor's degree, and 5 additional patients (9%) were recently accepted to college, whereas 3 others (5%) were currently attending college classes. Further, 18 patients were either pursuing (n = 3) or had attained (n = 15) an educational level beyond a bachelor's degree. CONCLUSIONS: Compromised visual function does not preclude the successful attainment of an academic education in patients with LCA who are substantially visually impaired from birth. These data have clinically relevant implications for the parents of children with LCA and for the patients themselves in providing a tone of optimism for their potential of attaining competitive academic achievements.

Conduction slowing and sudden arrhythmic death in mice with cardiac-restricted inactivation of connexin43.

Cardiac arrhythmia is a common and often lethal manifestation of many forms of heart disease. Gap junction remodeling has been postulated to contribute to the increased propensity for arrhythmogenesis in diseased myocardium, although a causative role in vivo remains speculative. By generating mice with cardiac-restricted knockout of connexin43 (Cx43), we have circumvented the perinatal lethal developmental defect associated with germline inactivation of this gap junction channel gene and uncovered an essential role for Cx43 in the maintenance of electrical stability. Mice with cardiac-specific loss of Cx43 have normal heart structure and contractile function, and yet they uniformly (28 of 28 conditional Cx43 knockout mice observed) develop sudden cardiac death from spontaneous ventricular arrhythmias by 2 months of age. Optical mapping of the epicardial electrical activation pattern in Cx43 conditional knockout mice revealed that ventricular conduction velocity was significantly slowed by up to 55% in the transverse direction and 42% in the longitudinal direction, resulting in an increase in anisotropic ratio compared with control littermates (2.1+/-0.13 versus 1.66+/-0.06; P:<0.01). This novel genetic murine model of primary sudden cardiac death defines gap junctional abnormalities as a key molecular feature of the arrhythmogenic substrate.