Developmental programming mediated by complementary roles of imprinted Grb10 in mother and pup.

Developmental programming links growth in early life with health status in adulthood. Although environmental factors such as maternal diet can influence the growth and adult health status of offspring, the genetic influences on this process are poorly understood. Using the mouse as a model, we identify the imprinted gene Grb10 as a mediator of nutrient supply and demand in the postnatal period. The combined actions of Grb10 expressed in the mother, controlling supply, and Grb10 expressed in the offspring, controlling demand, jointly regulate offspring growth. Furthermore, Grb10 determines the proportions of lean and fat tissue during development, thereby influencing energy homeostasis in the adult. Most strikingly, we show that the development of normal lean/fat proportions depends on the combined effects of Grb10 expressed in the mother, which has the greater effect on offspring adiposity, and Grb10 expressed in the offspring, which influences lean mass. These distinct functions of Grb10 in mother and pup act complementarily, which is consistent with a coadaptation model of imprinting evolution, a model predicted but for which there is limited experimental evidence. In addition, our findings identify Grb10 as a key genetic component of developmental programming, and highlight the need for a better understanding of mother-offspring interactions at the genetic level in predicting adult disease risk.

Latent TGF-ß binding protein 3 identifies a second heart field in zebrafish.

The four-chambered mammalian heart develops from two fields of cardiac progenitor cells distinguished by their spatiotemporal patterns of differentiation and contributions to the definitive heart. The first heart field differentiates earlier in lateral plate mesoderm, generates the linear heart tube and ultimately gives rise to the left ventricle. The second heart field (SHF) differentiates later in pharyngeal mesoderm, elongates the heart tube, and gives rise to the outflow tract and much of the right ventricle. Because hearts in lower vertebrates contain a rudimentary outflow tract but not a right ventricle, the existence and function of SHF-like cells in these species has remained a topic of speculation. Here we provide direct evidence from Cre/Lox-mediated lineage tracing and loss-of-function studies in zebrafish, a lower vertebrate with a single ventricle, that latent TGF-ß binding protein 3 (ltbp3) transcripts mark a field of cardiac progenitor cells with defining characteristics of the anterior SHF in mammals. Specifically, ltbp3(+) cells differentiate in pharyngeal mesoderm after formation of the heart tube, elongate the heart tube at the outflow pole, and give rise to three cardiovascular lineages in the outflow tract and myocardium in the distal ventricle. In addition to expressing Ltbp3, a protein that regulates the bioavailability of TGF-ß ligands, zebrafish SHF cells co-express nkx2.5, an evolutionarily conserved marker of cardiac progenitor cells in both fields. Embryos devoid of ltbp3 lack the same cardiac structures derived from ltbp3(+) cells due to compromised progenitor proliferation. Furthermore, small-molecule inhibition of TGF-ß signalling phenocopies the ltbp3-morphant phenotype whereas expression of a constitutively active TGF-ß type I receptor rescues it. Taken together, our findings uncover a requirement for ltbp3-TGF-ß signalling during zebrafish SHF development, a process that serves to enlarge the single ventricular chamber in this species.

Vitamin E and memantine in Alzheimer's disease: clinical trial methods and baseline data.

BACKGROUND: Alzheimer's disease (AD) has been associated with both oxidative stress and excessive glutamate activity. A clinical trial was designed to compare the effectiveness of (i) alpha-tocopherol, a vitamin E antioxidant; (ii) memantine (Namenda), an N-methyl-D-aspartate antagonist; (iii) their combination; and (iv) placebo in delaying clinical progression in AD. METHODS: The Veterans Affairs Cooperative Studies Program initiated a multicenter, randomized, double-blind, placebo-controlled trial in August 2007, with enrollment through March 2012 and follow-up continuing through September 2012. Participants with mild-to-moderate AD who were taking an acetylcholinesterase inhibitor were assigned randomly to 2000 IU/day of alpha-tocopherol, 20 mg/day memantine, 2000 IU/day alpha-tocopherol plus 20 mg/day memantine, or placebo. The primary outcome for the study is the Alzheimer's Disease Cooperative Study/Activities of Daily Living Inventory. Secondary outcome measures include the Mini-Mental State Examination; the Alzheimer's Disease Assessment Scale, cognitive portion; the Dependence Scale; the Neuropsychiatric Inventory; and the Caregiver Activity Survey. Patient follow-up ranged from 6 months to 4 years. RESULTS: A total of 613 participants were randomized. The majority of the patients were male (97%) and white (86%), with a mean age of 79 years. The mean Alzheimer's Disease Cooperative Study/Activities of Daily Living Inventory score at entry was 57 and the mean Mini-Mental State Examination score at entry was 21. CONCLUSION: This large multicenter trial will address the unanswered question of the long-term safety and effectiveness of alpha-tocopherol, memantine, and their combination in patients with mild-to-moderate AD taking an acetylcholinesterase inhibitor. The results are expected in early 2013.

Comparative genomics identifies genes mediating cardiotoxicity in the embryonic zebrafish heart.

Retinoic acid (RA) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) activate distinct ligand-dependent transcription factors, and both cause cardiac malformation and heart failure in zebrafish embryos. We hypothesized that they cause this response by hyperactivating a common set of genes critical for heart development. To test this, we used microarrays to measure transcript changes in hearts isolated from zebrafish embryos 1, 2, 4, and 12 h after exposure to 1 muM RA. We used hierarchical clustering to compare the transcriptional responses produced in the embryonic heart by RA and TCDD. We could identify no early responses in common between the two agents. However, at 12 h both treatments produced a dramatic downregulation of a common cluster of cell cycle progression genes, which we term the cell cycle gene cluster. This was associated with a halt in heart growth. These results suggest that RA and TCDD ultimately trigger a common transcriptional response associated with heart failure, but not through the direct activation of a common set of genes. Among the genes rapidly induced by RA was Nr2F5, a member of the COUP-TF family of transcriptional repressors. We found that induction of Nr2F5 was both necessary and sufficient for the cardiotoxic response to RA.

Heart malformation is an early response to TCDD in embryonic zebrafish.

The zebrafish (Danio rerio) has become an attractive vertebrate model for studying developmental processes, and is emerging as a model system for studying the mechanisms by which toxic compounds perturb normal development. When exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) shortly after fertilization, zebrafish embryos exhibit pericardial edema and reduced blood flow by 72 h post fertilization (hpf). To better understand the progression of dioxin toxicity in zebrafish, we have examined the effects of TCDD on heart development. At 72 hpf, TCDD-treated embryos exhibited altered looping, with the atria positioned distinctly posterior to the ventricles, contrary to the looping of control hearts, where the two chambers lied side by side. Moreover, the ventricles in dioxin-exposed hearts became more compact, and the atria elongated in comparison to controls. These defects are not secondary to pericardial edema because they were observed when edema formation was suppressed with osmotic support. In addition to morphological changes, TCDD produced functional deficits in the developing hearts, including blood regurgitation and a striking ventricular standstill that became prevalent by 120 hpf. We also assessed the effect of TCDD on the heart size using stereological measurements, which demonstrated significant reduction in heart tissue volume at 72 hpf. Perhaps our most significant finding was a decrease in the total number of cardiomyocytes in TCDD-exposed embryos by 48 hpf, one day prior to observable effects on peripheral blood flow. We conclude that the developing heart is an important target for TCDD in zebrafish.

Zebrafish and cardiac toxicology.

Model systems are a mainstay in toxicological research. Zebrafish are rapidly becoming an important model organism for studying vertebrate development. The advantages of zebrafish: short reproductive cycle, production of numerous transparent, synchronously developing embryos, low cost, and standardization make zebrafish an attractive model for toxicologists as well. The use of these fish to study heart development has moved forward very rapidly, laying the groundwork for studying the effects of chemicals on cardiac development and function. Here we describe approaches that can be used to study cardiac toxicity in developing zebrafish, focusing on examples where zebrafish embryos have been especially useful in understanding the impact of specific toxicants on heart development and function.

Aryl hydrocarbon receptor 2 mediates 2,3,7,8-tetrachlorodibenzo-p-dioxin developmental toxicity in zebrafish.

In order to use the zebrafish as a model vertebrate to investigate the developmental toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), it is essential to know whether one or both forms of the zebrafish aryl hydrocarbon receptor (AHR), zfAHR1 or zfAHR2, mediate toxicity. To determine the role of zfAHR2, an antisense morpholino approach was used to knock down translation of the protein. No effect of the zfahr2 morpholino (zfahr2-MO) was seen on normal development in embryos not treated with TCDD. Injection of embryos at the 1-2 cell stage with zfahr2-MO decreased TCDD-induced transcription of zfCYP1A mRNA until 96 h post fertilization (hpf), and immuno-histochemical detection of zfCYP1A protein in embryos at 72 hpf revealed a dramatic decrease in expression. The zfahr2-MO completely protected embryos from TCDD-induced edema and anemia and provided protection against TCDD-induced reductions in peripheral blood flow initially; however, a slight reduction in blood flow was observed at later times when the morpholino was no longer effective. Due to persistence of TCDD and decreasing effectiveness of the zfahr2-MO over time, the morpholino provided only transient protection against TCDD-induced inhibition of chondrogenesis of the lower jaw, and no protection against an effect of TCDD that was initiated late in development, blockade of swimbladder inflation. The zfahr2-MO did not protect embryos from TCDD-induced mortality but did produce a 48 h delay in its onset. Endpoints of TCDD developmental toxicity manifested in zfahr2 morphants at late stages of development, beyond 144 hpf, were clearly different from TCDD-exposed embryos injected with a control morpholino. Most strikingly, zfahr2 morphants exposed to TCDD never developed edema. Taken together, these results demonstrate that zfAHR2 mediates several endpoints of TCDD developmental toxicity in zebrafish.

Diagnostic and procedural imaging curricula in physical therapist professional degree programs.

STUDY DESIGN: Descriptive survey. OBJECTIVE: To describe the status of diagnostic and procedural imaging curricula within United States physical therapist professional degree programs. BACKGROUND: As patient direct access to physical therapy services increases, the ability to refer patients directly for diagnostic imaging could promote more efficient delivery of care. Appropriate patient referral is contingent on physical therapists having the requisite knowledge base and skills. While evidence describing imaging competence of physical therapists with advanced training in military institutions exists, evidence is lacking for other physical therapists, including new graduates of physical therapist professional degree programs. METHODS: Faculty members teaching imaging at 206 United States physical therapist professional degree programs recognized by the Commission on Accreditation in Physical Therapy Education were recruited via e-mail correspondence. An e-mail attachment included the survey on which faculty reported imaging curricula and faculty qualifications, attitudes, and experiences. RESULTS: Faculty from 155 (75.2%) programs responded to the survey, with imaging being included in the curriculum of 152 programs. Content was integrated by required standalone courses or clinical science track courses, and/or through elective courses. The average reported estimate of imaging contact hours was 24.4 hours (range, 2-75 hours). Emphasis was on the musculoskeletal system, including 76.3% of the required standalone course content. Student competence was assessed in 147 (96.7%) programs, primarily by written (66.7%) and practical (19.7%) examinations. Faculty rated student competence on a scale of 1 (not competent) to 5 (competent), with ratings ranging from a high of 4.0 (identifying normal anatomy on plain-film radiography) to a low of 1.9 (identifying common tissue pathological processes/injuries on ultrasound). CONCLUSION: While a majority of programs reported including imaging curricula, variability was noted in all curricular aspects. These results may serve as a benchmark for faculty to assess existing curricula, allow for further development of imaging curricula, and provide a benchmark for the profession regarding current level of training for recent graduates of entry-level physical therapist professional degree programs.

Differences in sleep disturbance parameters between oncology outpatients and their family caregivers.

PURPOSE: This study compared the occurrence rates for and severity ratings of sleep disturbance in patient-family caregiver (FC) dyads. PATIENTS AND METHODS: In total, 102 dyads were recruited from two radiation therapy (RT) departments. Patients and their FCs completed the Pittsburgh Sleep Quality Index (PSQI) and the General Sleep Disturbance Scale (GSDS) and wore wrist actigraphs to obtain subjective and objective measures of the occurrence and severity of sleep disturbance at the initiation of RT. Match paired t tests were used to evaluate for dyadic differences. RESULTS: No differences were found in the occurrence of clinically significant levels of sleep disturbance between patients and their FCs that ranged between 40% and 50% using subjective and objective measures. Few differences were found in the severity of any of the sleep-wake parameters between patients and FCs using both the subjective and objective measures of sleep disturbance. CONCLUSION: The findings from this study suggest that patients with cancer and their FCs experience similar levels of sleep disturbance and that both groups could benefit from interventions that aim to promote restful sleep. In addition to routine and systematic assessment of sleep disturbance by oncology clinicians, interventions are needed that take into account the specific needs of the patient and the FC as well as the potential for partners' sleep patterns to influence one another.

Understanding dioxin developmental toxicity using the zebrafish model.

Zebrafish (Danio rerio) have advantages over mammals as an animal model for investigating developmental toxicity. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (dioxin, TCDD), a persistent global contaminant, is the most comprehensively studied developmental toxicant in zebrafish. The hallmark responses of TCDD developmental toxicity manifested in zebrafish larvae include edema, anemia, hemorrhage, and ischemia associated with arrested growth and development. Heart and vasculature development and function are severely impaired, and jaw malformations occur secondary to inhibited chondrogenesis. The swim bladder fails to inflate, and the switch from embryonic to adult erythropoiesis is blocked. This profile of developmental toxicity responses, commonly referred to as "blue sac syndrome" because the edematous yolk sac appears blue, is observed in the larval form of all freshwater fish species exposed to TCDD at the embryonic stage of development. Components of the aryl hydrocarbon receptor/aryl hydrocarbon receptor nuclear translocator (AHR/ARNT) signaling pathway in zebrafish have been identified and functionally characterized. Their role in mediating TCDD toxicity has been determined using morpholinos to specifically knockdown the translation of zfAHR1, zfAHR2, zfARNT1, and zfARNT2 mRNAs, respectively, and a line of zfARNT2 null mutant zebrafish has provided further insight. These studies have shown that zfAHR2 and zfARNT1 mediate TCDD developmental toxicity. In addition, the growing use of molecular and genomic tools for research on zebrafish have led to advances in our understanding of the mechanism of TCDD developmental toxicity at the molecular level, including the recent finding that toxicity is not mediated by increased cytochrome P4501A (zfCYP1A) expression.

Aryl hydrocarbon receptor activation produces heart-specific transcriptional and toxic responses in developing zebrafish.

Proper regulation of the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor, is required for normal vertebrate cardiovascular development. AHR hyperactivation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during zebrafish (Danio rerio) development results in altered heart morphology and function, culminating in death. To identify genes that may cause cardiac toxicity, we analyzed the transcriptional response to TCDD in zebrafish hearts. Zebrafish larvae were exposed to TCDD for 1 h at 72 h after fertilization (hpf), and the hearts were extracted for microarray analysis at 1, 2, 4, and 12 h after exposure (73, 74, 76, and 84 h postfertilization). The remaining body tissue was also collected at each time for comparison. TCDD rapidly induced expression in 42 genes within 1 to2hof exposure. These genes function in xenobiotic metabolism, proliferation, heart contractility, and pathways that regulate heart development. Furthermore, these expression changes preceded signs of cardiovascular toxicity, characterized by decreased stroke volume, peripheral blood flow, and a halt in heart growth. This identifies strong candidates for important AHR target genes. It is noteworthy that the TCDD-induced transcriptional response in the hearts of zebrafish larvae was substantially different from that induced in the rest of the body tissues. One of the biggest differences included a cluster of genes that were down-regulated 12 h after exposure in heart tissue, but not in the body samples. More than 70% of the transcripts in this heart-specific cluster promote cellular growth and proliferation. Thus, the developing heart stands out as being responsive to TCDD at both the level of toxicity and gene expression.

2,3,7,8-Tetrachlorodibenzo-p-dioxin activation of the aryl hydrocarbon receptor/aryl hydrocarbon receptor nuclear translocator pathway causes developmental toxicity through a CYP1A-independent mechanism in zebrafish.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that dimerizes with ARNT to mediate responses to compounds such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). TCDD and other AHR agonists cause toxic responses in early life stages of fish, including the zebrafish, Danio rerio. The most well characterized target gene for the AHR/aryl hydrocarbon receptor nuclear translocator (ARNT) dimer is a cytochrome P450, CYP1A. Induction of CYP1A by TCDD has been correlated with certain toxic responses in developing zebrafish and has been postulated to mediate these responses. To determine whether CYP1A is the important downstream effector enzyme for the AHR/ARNT pathway, we used morpholino oligonucleotides (MOs) to block induction of CYP1A in response to TCDD in zebrafish embryos. Although the zfcyp1a-MO effectively prevented CYP1A up-regulation, it did not prevent the signs of developmental toxicity, including pericardial edema, slowed blood flow, craniofacial malformation, and defects in erythropoiesis. We conclude that the important target for the AHR/ARNT pathway in developing zebrafish exposed to TCDD is not zfcyp1a. This suggests an alternative model in which TCDD-activated AHR/ARNT disrupts the normal process of growth and development by altering programs of gene expression or function.

Immortalization of human uterine leiomyoma and myometrial cell lines after induction of telomerase activity: molecular and phenotypic characteristics.

In vitro model systems for studying uterine leiomyomas are limited in that human-derived leiomyoma cells grow poorly in culture compared with normal myometrial cells and begin to senesce early, at approximately passage 10 in our studies. To our knowledge, a good in vitro human-derived cell culturing system for leiomyomas does not exist. In an attempt to fill this void, we have immortalized a uterine leiomyoma cell line by inducing telomerase activity, which allows cells to bypass their normal programmed senescence. Telomerase activity was induced by infecting the target (uterine leiomyoma and normal myometrial) cells with a retroviral vector containing hTERT, the gene for the catalytic subunit of telomerase. Subsequent analysis by RT-PCR and the telomeric repeat amplification protocol assay confirmed expression of the inserted gene and induction of telomerase activity in leiomyoma and myometrial cells. Analysis of cells for estrogen receptor-alpha and progesterone receptor proteins by Western blotting showed no change in expression of these proteins between the immortalized and parental leiomyoma and myometrial cells. Both immortalized and parental myometrial and leiomyoma cells expressed the smooth muscle-specific cytoskeletal protein alpha-actin and were negative for mutant p53 protein as evidenced by immunocytochemical staining. The immortalized leiomyoma and myometrial cells showed no anchorage-independent growth, with the exception of a small subpopulation of immortalized leiomyoma cells at a higher passage that did form two to three small colonies (per 50,000 cells) in soft agar. None of the immortalized cells were tumorigenic in nude mice. In conclusion, our data show the successful insertion of the hTERT gene into leiomyoma and myometrial cells and the immortalization of these cell lines without phenotypic alteration from the parental cell types (up to 200 population doublings). These cells should help to advance research in understanding the molecular pathways involved in the conversion of a normal myometrial cell to a leiomyoma cell and the mechanisms responsible for the growth of uterine leiomyomas. Answers to these questions will undoubtedly lead to the development of more effective treatment and intervention regimens for clinical cases of uterine leiomyoma.