Genome Wide Conditional Mouse Knockout Resources.

The International Knockout Mouse Consortium (IKMC) developed high throughput gene trapping and gene targeting pipelines that produced mostly conditional mutations of more than 18,500 genes in C57BL/6N mouse embryonic stem (ES) cells which have been archived and are freely available to the research community as a frozen resource. From this unprecedented resource more than 6,000 mutant mouse strains have been produced by the IKMC and mostly the International Mouse Phenotyping Consortium (IMPC). In addition, a cre-driver resource was established including 250 inducible cre-driver mouse strains in a C57BL/6 background. Complementing the cre-driver resource, a collection of comprising 27 cre-driver rAAVs has also been produced. The resources can be easily accessed at the IKMC/IMPC web portal (www.mousephenotype.org). The IKMC/IMPC resource is a standardized reference library of mouse models with defined genetic backgrounds that enables the analysis of gene-disease associations in mice of different genetic makeup and should therefore have a major impact on biomedical research.

Treating depression with botulinum toxin: a pooled analysis of randomized controlled trials.

INTRODUCTION: Botulinum toxin A (BTA) injection into the glabellar region is currently being studied as a treatment for major depressive disorder (MDD). Here we explore efficacy data of this novel approach in a pooled analysis. METHODS: A literature search revealed 3 RCTs on this topic. Individual patient data and clinical end points shared by these 3 trials were pooled and analyzed as one study (n=134) using multiple regression models with random effects. RESULTS: In the pooled sample, the BTA (n=59) and the placebo group (n=75) did not differ in the baseline variables. Efficacy outcomes revealed BTA superiority over placebo: Improvement in the Hamilton Depression Rating Scale or Montgomery-Asberg Depression Rating Scale 6 weeks after baseline was 45.7% for BTA vs. 14.6% for placebo (p<0.0001), corresponding to a BTA response rate of 54.2% (vs. 10.7%) and a BTA remission rate of 30.5% (vs. 6.7%). DISCUSSION: Equalling the status of a meta-analysis, this study increases evidence that a single treatment of BTA into the glabellar region can reduce symptoms of MDD. Further studies are needed to better understand how BTA exerts its mood-lifting effect.

Localized Igf-1 transgene expression sustains hypertrophy and regeneration in senescent skeletal muscle.

Aging skeletal muscles suffer a steady decline in mass and functional performance, and compromised muscle integrity as fibrotic invasions replace contractile tissue, accompanied by a characteristic loss in the fastest, most powerful muscle fibers. The same programmed deficits in muscle structure and function are found in numerous neurodegenerative syndromes and disease-related cachexia. We have generated a model of persistent, functional myocyte hypertrophy using a tissue-restricted transgene encoding a locally acting isoform of insulin-like growth factor-1 that is expressed in skeletal muscle (mIgf-1). Transgenic embryos developed normally, and postnatal increases in muscle mass and strength were not accompanied by the additional pathological changes seen in other Igf-1 transgenic models. Expression of GATA-2, a transcription factor normally undetected in skeletal muscle, marked hypertrophic myocytes that escaped age-related muscle atrophy and retained the proliferative response to muscle injury characteristic of younger animals. The preservation of muscle architecture and age-independent regenerative capacity through localized mIgf-1 transgene expression suggests clinical strategies for the treatment of age or disease-related muscle frailty.

Antioxidant amelioration of dilated cardiomyopathy caused by conditional deletion of NEMO/IKKgamma in cardiomyocytes.

RATIONALE: Insight into the function of nuclear factor (NF)-kappaB in the adult heart has been hampered by the embryonic lethality of constitutive NF-kappaB inactivation. OBJECTIVE: The goal of the present study was therefore to gain insights into the role of NF-kappaB pathway specifically in mouse cardiomyocytes by conditional deletion of the NF-kappaB essential modulator (NEMO). METHODS AND RESULTS: Using a Cre/loxP system, we disrupted the Nemo gene in a cardiomyocyte-specific manner in the heart, which simulated gene expression changes underlying human heart failure and caused adult-onset dilated cardiomyopathy accompanied by inflammation and apoptosis. Pressure overload challenges of NEMO-deficient young hearts precociously induced the functional decrements that develop spontaneously in older knockout animals. Moreover, oxidative stress in NEMO-deficient cardiomyocytes is a critical pathological component that can be attenuated with antioxidant diet in vivo. CONCLUSIONS: These results reveal an essential physiological role for NEMO-mediated signaling in the adult heart to maintain cardiac function in response to age-related or mechanical challenges, in part through modulation of oxidative stress.

From the bottom of the heart: anteroposterior decisions in cardiac muscle differentiation.

Recently, studies on specification of axes in the developing embryo have focused on the heart, which is the first functional organ to form and probably responds to common cues controlling positional information in surrounding tissues. The early differentiation of heart cells affords an opportunity to link the acquisition of regional identity with the signals underlying terminal differentiation. In the past year, a wealth of information on these signals has emerged, elucidating the general pathways controlling body axes in the context of the developing heart.

Epidemiological and genetic characteristics of norovirus outbreaks in long-term care facilities, 2003-2006.

To identify the epidemiological and genetic characteristics of norovirus (NoV) outbreaks and estimate the impact of NoV infections in an older population, we analysed epidemiological and laboratory data collected using standardized methods from long-term care facilities (LTCFs) during 2003-2006. Faecal specimens were tested for NoV by real-time reverse transcriptase-polymerase chain reaction. NoV strains were genotyped by sequencing. Of the 234 acute gastroenteritis (AGE) outbreaks reported, 163 (70%) were caused by NoV. The annual attack rate of outbreak-associated NoV infection in LTCF residents was 4%, with a case-hospitalization rate of 3·1% and a case-fatality rate of 0·5%. GII.4 strains accounted for 84% of NoV outbreaks. Median duration of illness was longer for GII.4 infections than non-GII.4 infections (33 vs. 24 h, P<0·001). Emerging GII.4 strains (Hunter/2004, Minerva/2006b, Terneuzen/2006a) gradually replaced the previously dominant strain (Farmington Hills/2002) during 2004-2006. NoV GII.4 strains are now associated with the majority of AGE outbreaks in LTCFs and prolonged illness in Oregon.

High-throughput applicable genomic sex typing of chicken by TaqMan real-time quantitative polymerase chain reaction.

Genetic sex typing of vertebrate animals is an essential technique for research on reproductive phenomena such as sex determination of embryonic tissues. Polymerase chain reaction amplification of genomic DNA segments in the Z and W sex chromosomes has been widely used as a standard laboratory method to determine genetic sex of the chicken (Gallus gallus domesticus). However, conventional protocols for PCR determination of avian sex typically involve tedious steps of genomic DNA isolation, which often require relatively large amounts of tissue samples, and the purity of genomic DNA specimens significantly affects PCR efficiency. Moreover, detection of sex chromosome-specific PCR products by gel electrophoresis is prone to misjudgment caused by amplification of contaminating genomic DNA segments derived from tissue or DNA samples as well as previously generated PCR products. Thus, the credibility of genetic sex typing by conventional PCR-based methods that measure the relative amounts of the end product DNA amplicons critically depends on several experimental steps that are potentially vulnerable to errors. Here, we describe an optimized protocol of chicken genetic sex typing by TaqMan real-time quantitative PCR amplification of markers on the sex chromosomes. This TaqMan sex typing method accurately quantifies relative amounts of the Z and W sex chromosome markers directly from only 0.5 to 2 microL of total blood lysate without nucleic acid purification. The real-time amplification curves of the quantitative PCR reaction readily distinguished truly homozygous (ZZ) and heterozygous (ZW) sex chromosomes from contamination of the sex chromosomal DNA, ensuring highly credible sex determination. Thus, the TaqMan typing of chicken genetic sex has several advantageous features for high-throughput operation compared with conventional methods.

Proliferation precedes differentiation in IGF-I-stimulated myogenesis.

The insulin-like growth factors (IGFs) have dramatic and complex effects on the growth of many tissues and have been implicated in both the proliferation and differentiation of skeletal muscle cells. A detailed analysis of gene expression was performed in L6E9 myoblast cultures treated with IGF-I to dissect the early events leading to the stimulation of myogenic differentiation by this growth factor. A time course of transcript accumulation in confluent L6E9 myoblasts treated with defined media containing IGF-I revealed an initial transient decrease in myogenic factors, accompanied by an increase in cell cycle markers and cell proliferation. This pattern was reversed at later time points, when the subsequent activation of myogenic factors resulted in a net increase in structural gene expression and larger myotubes. The data presented here support the hypothesis that IGF-I activates proliferation first, and subsequently stimulates events leading to the expression of muscle-specific genes in myogenic cell cultures.

Regulation of a muscle-specific transgene by retinoic acid.

Retinoic acid (RA) has been shown to have variable effects on myogenic differentiation in cell culture. The application of RA on primary cultures of embryonic somites, limb buds, and neonatal limbs inhibited myogenic differentiation in a dose-dependent way as indicated by the repression of: (a) myotube formation, (b) myosin heavy chain protein accumulation, (c) myosin light chain (MLC) 1/3, alpha sk-actin and myogenic factor transcript expression. Expression of retinoic acid receptors (RAR) was also affected by RA treatment, specifically RAR gamma transcripts were induced. To further understand the pleiotropic action of RA on myogenesis, we took advantage of two muscle-specific transgene markers which consisted of CAT reporter genes driven by regulatory elements either from the myosin light chain 1/3 locus (MLC-CAT) or the alpha-skeletal actin gene (alpha sk actin-CAT). RA inhibited MLC-CAT transgene but not alpha sk actin-CAT transgene expression in primary cultures from these mice. Analysis of MLC-CAT expression in transgenic mouse primary cultures and in stably transfected C2C12 cells demonstrated that repression of MLC-CAT activity by RA was dependent upon diffusible factors in chick embryo extract. We hypothesize that during development, the pleiotropic effects of RA on myogenesis do not depend solely on the distribution and concentration of RA itself, but are also influenced by extracellular signals in the embryonic environment.

BK viral enhancer element and a human cellular homolog.

Comparison of two closely related primate papovaviruses, simian virus 40 (SV40) and human BK virus (BKV), reveals that the only region of extensive divergence, the tandem sequences adjacent to the origins of DNA replication, is responsible in SV40 for enhancing early gene expression. This study demonstrates a similar enhancer function for the analogous repeated region in BKV. The dissimilarity in sequence of the BKV and SV40 enhancer elements suggests that they may have been acquired since SV40 and BKV diverged. A locus cloned from the human genome homologous to the BKV tandem repeats has been shown to function as low level enhancer element in mammalian cells. These data support the hypothesis that viral enhancer sequences may be evolutionarily related to host cell sequences.

Maturation of the myogenic program is induced by postmitotic expression of insulin-like growth factor I.

The molecular mechanisms underlying myogenic induction by insulin-like growth factor I (IGF-I) are distinct from its proliferative effects on myoblasts. To determine the postmitotic role of IGF-I on muscle cell differentiation, we derived L6E9 muscle cell lines carrying a stably transfected rat IGF-I gene under the control of a myosin light chain (MLC) promoter-enhancer cassette. Expression of MLC-IGF-I exclusively in differentiated L6E9 myotubes, which express the embryonic form of myosin heavy chain (MyHC) and no endogenous IGF-I, resulted in pronounced myotube hypertrophy, accompanied by activation of the neonatal MyHC isoform. The hypertrophic myotubes dramatically increased expression of myogenin, muscle creatine kinase, beta-enolase, and IGF binding protein 5 and activated the myocyte enhancer factor 2C gene which is normally silent in this cell line. MLC-IGF-I induction in differentiated L6E9 cells also increased the expression of a transiently transfected LacZ reporter driven by the myogenin promoter, demonstrating activation of the differentiation program at the transcriptional level. Nuclear reorganization, accumulation of skeletal actin protein, and an increased expression of beta1D integrin were also observed. Inhibition of the phosphatidyl inositol (PI) 3-kinase intermediate in IGF-I-mediated signal transduction confirmed that the PI 3-kinase pathway is required only at early stages for IGF-I-mediated hypertrophy and neonatal MyHC induction in these cells. Expression of IGF-I in postmitotic muscle may therefore play an important role in the maturation of the myogenic program.

The myosin light chain enhancer and the skeletal actin promoter share a binding site for factors involved in muscle-specific gene expression.

The myosin light chain (MLC) 1/3 enhancer (MLC enhancer), identified at the 3' end of the skeletal MLC1/3 locus, contains a sequence motif that is homologous to a protein-binding site of the skeletal muscle alpha-actin promoter. Gel shift, competition, and footprint assays demonstrated that a CArG motif in the MLC enhancer binds the proteins MAPF1 and MAPF2, previously identified as factors interacting with the muscle regulatory element of the skeletal alpha-actin promoter. Transient transfection assays with constructs containing the chloramphenicol acetyltransferase reporter gene demonstrated that a 115-bp subfragment of the MLC enhancer is able to exert promoter activity when provided with a silent nonmuscle TATA box. A point mutation at the MAPF1/2-binding site interferes with factor binding and abolishes the promoter activity of the 115-bp fragment. The observation that an oligonucleotide encompassing the MAPF1/2 site of the MLC enhancer alone cannot serve as a promoter element suggests that additional factor-binding sites are necessary for this function. The finding that MAPF1 and MAPF2 recognize similar sequence motifs in two muscle genes, simultaneously activated during muscle differentiation, implies that these factors may have a role in coordinating the activation of contractile protein gene expression during myogenesis.

Distinct gene expression patterns in skeletal and cardiac muscle are dependent on common regulatory sequences in the MLC1/3 locus.

The myosin light-chain 1/3 locus (MLC1/3) is regulated by two promoters and a downstream enhancer element which produce two protein isoforms in fast skeletal muscle at distinct stages of mouse embryogenesis. We have analyzed the expression of transcripts from the internal MLC3 promoter and determined that it is also expressed in the atria of the heart. Expression from the MLC3 promoter in these striated muscle lineages is differentially regulated during development. In transgenic mice, the MLC3 promoter is responsible for cardiac-specific reporter gene expression while the downstream enhancer augments expression in skeletal muscle. Examination of the methylation status of endogenous and transgenic promoter and enhancer elements indicates that the internal promoter is not regulated in a manner similar to that of the MLC1 promoter or the downstream enhancer. A GATA protein consensus sequence in the proximal MLC3 promoter but not the MLC1 promoter binds with high affinity to GATA-4, a cardiac muscle- and gut-specific transcription factor. Mutation of either the MEF2 or GATA motifs in the MLC3 promoter attenuates its activity in both heart and skeletal muscles, demonstrating that MLC3 expression in these two diverse muscle types is dependent on common regulatory elements.

Cardiomyopathy in Irx4-deficient mice is preceded by abnormal ventricular gene expression.

To define the role of Irx4, a member of the Iroquois family of homeobox transcription factors in mammalian heart development and function, we disrupted the murine Irx4 gene. Cardiac morphology in Irx4-deficient mice (designated Irx4(Delta ex2/Delta ex2)) was normal during embryogenesis and in early postnatal life. Adult Irx4(Delta ex2/Delta ex2) mice developed a cardiomyopathy characterized by cardiac hypertrophy and impaired contractile function. Prior to the development of cardiomyopathy, Irx4(Delta ex2/Delta ex2) hearts had abnormal ventricular gene expression: Irx4-deficient embryos exhibited reduced ventricular expression of the basic helix-loop-helix transcription factor eHand (Hand1), increased Irx2 expression, and ventricular induction of an atrial chamber-specific transgene. In neonatal hearts, ventricular expression of atrial natriuretic factor and alpha-skeletal actin was markedly increased. Several weeks subsequent to these changes in embryonic and neonatal gene expression, increased expression of hypertrophic markers BNP and beta-myosin heavy chain accompanied adult-onset cardiac hypertrophy. Cardiac expression of Irx1, Irx2, and Irx5 may partially compensate for loss of Irx4 function. We conclude that Irx4 is not sufficient for ventricular chamber formation but is required for the establishment of some components of a ventricle-specific gene expression program. In the absence of genes under the control of Irx4, ventricular function deteriorates and cardiomyopathy ensues.

Heart regeneration in the salamander relies on macrophage-mediated control of fibroblast activation and the extracellular landscape.

In dramatic contrast to the poor repair outcomes for humans and rodent models such as mice, salamanders and some fish species are able to completely regenerate heart tissue following tissue injury, at any life stage. This capacity for complete cardiac repair provides a template for understanding the process of regeneration and for developing strategies to improve human cardiac repair outcomes. Using a cardiac cryo-injury model we show that heart regeneration is dependent on the innate immune system, as macrophage depletion during early time points post-injury results in regeneration failure. In contrast to the transient extracellular matrix (ECM) that normally accompanies regeneration, this intervention resulted in a permanent, highly cross-linked extracellular matrix scar derived from alternative fibroblast activation and lysyl-oxidase enzyme synthesis. The activation of cardiomyocyte proliferation was not affected by macrophage depletion, indicating that cardiomyocyte replacement is an independent feature of the regenerative process, and is not sufficient to prevent fibrotic progression. These findings highlight the interplay between macrophages and fibroblasts as an important component of cardiac regeneration, and the prevention of fibrosis as a key therapeutic target in the promotion of cardiac repair in mammals.

Primary Care Provider Management of Congenital Hypothyroidism Identified Through Newborn Screening.

OBJECTIVE: To assess Primary Congenital Hypothyroidism (CH) management patterns and feasibility of providing long-term care for patients with CH identified through newborn screening by Primary Care Providers (PCPs) in California and Hawaii. STUDY DESIGN: A survey was mailed to all physicians (N=823) listed as the referral doctor for confirmed patients with CH identified through newborn screening programs in both states between 01/01/2009-12/31/2013. Information was collected on CH management patterns, barriers to providing care, and knowledge on CH treatment. Descriptive statistics and bivariate logistic regression results were reported. RESULTS: 206 PCPs completed the survey. Among these, 78% currently have patients with CH and 91% indicated willingness to provide long-term care to new patients with CH. Among PCPs currently caring for patients with CH, 17% managed CH by themselves with limited assistance from endocrinologists; 63% were involved in managing CH but endocrinologists played a larger role than PCPs; 19% were not involved in CH care. Only 49% of PCPs correctly answered questions regarding recommended follow-up frequencies and 23% knew the correct age for a trial off levothyroxine for suspected transient CH. Top two perceived barriers to providing long-term care included "need guidance or support from endocrinologists" (61%) and "not familiar with CH treatment guidelines" (28%). CONCLUSION: The majority of PCPs surveyed are willing to provide long-term care to patients with CH, but need support from endocrinologists and increased knowledge about current treatment guidelines.

In vivo induction of the lymphokine-activated killer phenomenon: interleukin 2-dependent human non-major histocompatibility complex-restricted cytotoxicity generated in vivo during administration of human recombinant interleukin 2.

The availability of purified human recombinant interleukin 2 (IL-2) has enabled clinical trials to test its in vivo effects. We report here the immunological effects of 7 consecutive days of IL-2 treatment given to 25 patients with cancer in a clinical Phase I study. Peripheral blood lymphocytes obtained from patients following therapy with IL-2 had enhanced proliferative responses to IL-2 and enhanced direct cytotoxic activity on K562 target cells. This lytic activity was further augmented by the addition of IL-2 during the 51Cr release assay. Fresh peripheral blood lymphocytes from some patients who had just completed treatment at the higher IL-2 dose levels were able to kill both the natural killer-resistant Daudi cell line and fresh tumor cells while pretreatment samples and peripheral blood lymphocytes from healthy controls were not. This lytic activity was best detected when IL-2 was present in the in vitro effector assay. These results demonstrate that the administration of IL-2 to patients with cancer induces a population of effector cells able to directly destroy natural killer-resistant target cells, when assayed in the presence of IL-2.