Rad54 and Mus81 cooperation promotes DNA damage repair and restrains chromosome missegregation.

Rad54 and Mus81 mammalian proteins physically interact and are important for the homologous recombination DNA repair pathway; however, their functional interactions in vivo are poorly defined. Here, we show that combinatorial loss of Rad54 and Mus81 results in hypersensitivity to DNA-damaging agents, defects on both the homologous recombination and non-homologous DNA end joining repair pathways and reduced fertility. We also observed that while Mus81 deficiency diminished the cleavage of common fragile sites, very strikingly, Rad54 loss impaired this cleavage to even a greater extent. The inefficient repair of DNA double-strand breaks (DSBs) in Rad54(-/-)Mus81(-/-) cells was accompanied by elevated levels of chromosome missegregation and cell death. Perhaps as a consequence, tumor incidence in Rad54(-/-)Mus81(-/-) mice remained comparable to that in Mus81(-/-) mice. Our study highlights the importance of the cooperation between Rad54 and Mus81 for mediating DNA DSB repair and restraining chromosome missegregation.

Blood-brain barrier: an impediment to neuropharmaceuticals.

The blood-brain barrier (BBB) serves as a highly selective barrier separating the central nervous system from the systemic circulation. Although contributing to neurological health, the BBB restricts the ability of drugs to reach their site of action and thus presents a major challenge to the treatment of neurological disorders. Advances in our understanding of the complexity of the BBB have fostered development of novel pharmacometric models and drug delivery strategies to better predict and improve therapeutic access.

Unsafe abortion after legalisation in Nepal: a cross-sectional study of women presenting to hospitals.

OBJECTIVE: To investigate abortion practices of Nepali women requiring postabortion care. DESIGN: Cross-sectional study. SETTING: Four tertiary-care hospitals in urban and rural Nepal. SAMPLE: A total of 527 women presenting with complications from induced abortion in 2010. METHODS: Women completed questionnaires on their awareness of the legal status of abortion and their abortion-seeking experiences. The method of induction and whether the abortion was obtained from an uncertified source was documented. Multivariable logistic regression was used to identify associated factors. MAIN OUTCOME MEASURES: Induction method; uncertified abortion source. RESULTS: In all, 234 (44%) women were aware that abortion was legal in Nepal. Medically induced abortion was used by 359 (68%) women and, of these, 343 (89%) took unsafe, ineffective or unknown substances. Compared with women undergoing surgical abortion, women who had medical abortion were more likely to have obtained information from pharmacists (161/359, 45% versus 11/168, 7%, adjusted odds ratio [aOR] 8.1, 95% confidence interval 4.1-16.0) and to have informed no one about the abortion (28/359, 8% versus 3/168, 2%, aOR 5.5, 95% CI 1.1-26.9). Overall, 291 (81%) medical abortions and 50 (30%) surgical abortions were obtained from uncertified sources; these women were less likely to know that abortion was legal (122/341, 36% versus 112/186, 60%, aOR 0.4, 95% CI 0.2-0.7) and more likely to choose a method because it was available nearby (209/341, 61% versus 62/186, 33%, aOR 2.5, 95% CI 1.5-4.3), compared with women accessing certified sources. CONCLUSIONS: Among women presenting to hospitals in Nepal with complications following induced abortion of pregnancy, the majority had undergone medically induced abortions using unknown substances acquired from uncertified sources. Women using medications and those accessing uncertified providers were less aware that abortion is now legal in Nepal. These findings highlight the need for continued improvements in the provision and awareness of abortion services in Nepal.

Caspase-3 deficiency reveals a physiologic role for Smac/DIABLO in regulating programmed cell death.

Inhibitor of apoptosis protein (IAP)-binding proteins such as Grim, Reaper and HID have been shown to exert a critical role in regulating caspase activity in species such as D. Melanogaster. However, a comparable role for the mammalian homologue of second mitochondrial-derived activator of caspase/direct IAP-binding protein with low pI (Smac/DIABLO) has yet to be clearly established in vivo. Despite tremendous interest in recent years in the use of so-called Smac mimetics to enhance chemotherapeutic potency, our understanding of the true physiologic nature of Smac/DIABLO in regulating programmed cell death (PCD) remains elusive. In order to critically evaluate the role of Smac/DIABLO in regulating mammalian PCD, deficiency of caspase-3 was used as a sensitizing mutation in order to reduce aggregate levels of executioner caspase activity. We observe that combinatorial deletion of Diablo and Casp3, but neither alone, results in perinatal lethality in mice. Consistent with this, examination of both intrinsic and extrinsic forms of PCD in lines of murine embryonic fibroblasts demonstrate that loss of Smac/DIABLO alters both caspase-dependent and caspase-independent intrinsic PCD. Comparative small interfering RNA inhibition studies of X-linked inhibitor of apoptosis, cellular inhibitor of apoptosis (cIAP)-1, cIAP-2, caspase-6 and -7 in both wild-type and Casp3/Diablo DKO mouse embryonic fibroblast lineages, supports a model in which Smac/DIABLO acts to enhance the early phase executioner caspase activity through the modulation of inhibitory interactions between specific IAP family members and executioner caspases-3 and -7.

EphB2 and EphA4 receptors regulate formation of the principal inter-hemispheric tracts of the mammalian forebrain.

Previously, we have demonstrated that EphB2 activity is required for proper development of the posterior branch of the anterior commissure (ACpp) within the mammalian forebrain. In the present study, using magnetic resonance imaging (MRI), immunohistochemistry, and in vivo stereotactic fluorescence tracing of EphB2, B3, A4 and combinatorial Eph receptor mutants, we have developed a detailed three-dimensional model of how EphB-class receptors interact to regulate commissural formation within the forebrain. The results demonstrate that EphB2 and EphA4 each regulate distinct aspects of axon guidance within the ACpp. Specifically, while EphB2 is required to retard ACpp axons from projecting aberrantly into the ventral forebrain, EphA4 is required to restrict axons from entering the anterior branch of the anterior commissure (ACpa). Together, EphB2 and EphA4 act synergistically to prevent a subpopulation of axons within the anterior branch of the AC from mis-projecting caudally. Analysis of EphA4 null mice using high resolution MRI reveals for the first time that, in addition to errors in midline guidance, loss of EphA4 results in aberrant lateral and ventral displacement of the ACpa tract. In addition, tracing studies in alpha-chimerin null mice reveal that EphA4-mediated effects are not regulated through this pathway. Taken together, the results demonstrate that each of the principal guidance decisions within both anterior and posterior tracts of the anterior commissure can be accounted for by the individual and combinatorial actions of EphB2/A4 receptors.

Excitatory tonus is required for the survival of granule cell precursors during postnatal development within the cerebellum.

In addition to protective effects within the adult central nervous system (CNS), in vivo application of N-methyl-d-aspartate inhibitors such as (+) MK-801 have been shown to induce neurodegeneration in neonatal rats over a specific developmental period. We have systematically mapped the nature and extent of MK-801-induced neurodegeneration throughout the neonatal murine brain in order to genetically dissect the mechanism of these effects. Highest levels of MK-801-induced neurodegeneration are seen in the cerebellar external germinal layer; while mature neurons of the internal granule layer are unaffected by MK-801 treatment. Examination of external germinal layer neurons by electron microscopy, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) and bromodeoxyuridine (BrdU) labeling, and caspase-3 activation demonstrate that these neurons die through the process of programmed cell death soon after they exit from the cell cycle. Significantly, ablation of caspase-3 activity completely inhibited the MK-801-induced (and developmental) programmed cell death of external germinal layer neurons. Similar to caspase-3, inactivation of muscarinic acetylcholine receptors in vivo using scopolamine inhibited MK-801-induced programmed cell death. By contrast, the GABAergic agonist diazepam, either alone or in combination with MK-801, enhanced programmed cell death within external germinal layer neurons. These data demonstrate that, in vivo, cerebellar granule neurons undergo a dramatic change in intracellular signaling in response to molecules present in the local cellular milieu during their first 24 h following exit from the cell cycle.

Lazarus's gate: challenges and potential of epigenetic reprogramming of somatic cells.

The past year has seen tremendous advances in epigenetic reprogramming of somatic cells. Direct genesis of pluripotent stem cells, in contrast to earlier somatic cell nuclear transfer (SCNT) techniques, removes significant ethical and regulatory concerns regarding the utilization of human oocytes and zygotes, and represents a significant step toward the development of nonxenogeneic production methods. While significant technical hurdles remain, this and related technologies are enabling new approaches toward clinical treatments, basic research and diagnostics, and drug evaluation.

Differential sensitivity of skeletal and fusimotor neurons to Bcl-2-mediated apoptosis during neuromuscular development.

Proper development of the nervous system requires that a carefully controlled balance be maintained between both proliferation and neuronal survival. The process of programmed cell death is believed to play a key role in regulating levels of neuronal survival, in large part through the action of antiapoptotic proteins, such as Bcl-2. Consistent with this, Bcl-2 has been shown to be a key regulator of apoptotic signaling in post-mitotic neurons. However, we still know remarkably little regarding the role that Bcl-2 plays in regulating the survival of specific motor neuron populations. In the present study, we have examined somatic motor neurons of the lumbar spinal cord, and branchiomotor neurons of the facial nucleus in bcl-2-null mice to determine the differential dependence among motor neuron populations with respect to Bcl-2-mediated survival. Examination of neuronal and axon number, axonal area, and the distribution of axonal loss in bcl-2-null mice demonstrates that, in contrast to the great majority of alpha motor neurons, gamma motor neurons exhibit a unique dependence upon bcl-2 for survival. These results demonstrate, for the first time, the connection between Bcl-2 expression, motor neuron survival, and the establishment of different motor populations.

Reducing maternal mortality due to elective abortion: Potential impact of misoprostol in low-resource settings.

Over 99% of deaths due to abortion occur in developing countries. Maternal deaths due to abortion are preventable. Increasing the use of misoprostol for elective abortion could have a notable impact on maternal mortality due to abortion. As a test of this hypothesis, this study estimated the reduction in maternal deaths due to abortion in Africa, Asia and Latin America. The estimates were adjusted to changes in assumptions, yielding different possible scenarios of low and high estimates. This simple modeling exercise demonstrated that increased use of misoprostol, an option for pregnancy termination already available to many women in developing countries, could significantly reduce mortality due to abortion. Empirical testing of the hypothesis with data collected from developing countries could help to inform and improve the use of misoprostol in those settings.

Forward genetic screen of mouse reveals dominant missense mutation in the P/Q-type voltage-dependent calcium channel, CACNA1A.

Dominant mutations of the P/Q-type Ca(2+) channel (CACNA1A) underlie several human neurological disorders, including episodic ataxia type 2, familial hemiplegic migraine 1 (FHM1) and spinocerebellar ataxia 6, but have not been found previously in the mouse. Here we report the first dominant ataxic mouse model of Cacna1a mutation. This Wobbly mutant allele of Cacna1a was identified in an ethylnitrosourea (ENU) mutagenesis dominant behavioral screen. Heterozygotes exhibit ataxia from 3 weeks of age and have a normal life span. Homozygotes have a righting reflex defect from postnatal day 8 and later develop severe ataxia and die prematurely. Both heterozygotes and homozygotes exhibit cerebellar atrophy with focal reduction of the molecular layer. No obvious loss of Purkinje cells or decrease in size of the granule cell layer was observed. Real-time polymerase chain reaction revealed altered expression levels of Cacna1g, Calb2 and Th in Wobbly cerebella, but Cacna1a messenger RNA and protein levels were unchanged. Positional cloning revealed that Wobbly mice have a missense mutation leading to an arginine to leucine (R1255L) substitution, resulting in neutralization of a positively charged amino acid in repeat III of voltage sensor segment S4. The dominance of the Wobbly mutation more closely resembles patterns of CACNA1A mutation in humans than previously described mouse recessive mutants (tottering, leaner, rolling Nagoya and rocker). Positive-charge neutralization in S4 has also been shown to underlie several cases of human dominant FHM1 with ataxia. The Wobbly mutant thus highlights the importance of the voltage sensor and provides a starting point to unravel the neuropathological mechanisms of this disease.

Development of a high resolution three-dimensional surgical atlas of the murine head for strains 129S1/SvImJ and C57Bl/6J using magnetic resonance imaging and micro-computed tomography.

The mouse has emerged as a major experimental model system for examining the functional properties of the mammalian CNS; both during development and following CNS injury. Histologic procedures currently used to determine the relative position of structures within the CNS are presently limited in their ability to take full advantage of this system for surgical and morphometric procedures. We present here the first three-dimensional interactive digital atlas of the murine brain and skull for two genetically important strains of mice; 129S1/SvImJ and C57Bl/6J. The final resolution of these digital atlases is 54 micro m(3). These representations of the murine brain and skull, in conjunction with our development of a new, more dynamic master coordinate system, provide improved accuracy with respect to targeting CNS structures during surgery compared with previous systems. The interactive three-dimensional nature of these atlases also provide users with stereotactic information necessary to perform accurate "off-axis" surgical procedures, as is commonly required for experiments such as in vivo micro-electroporation. In addition, three-dimensional analysis of the brain and skull shape in C57Bl, 129Sv, CD1, and additional murine strains, suggests that a stereotactic coordinate system based upon the lambda and rostral confluence of the sinuses at the sagittal midline, provides improved accuracy compared with the traditional lambda-bregma landmark system. These findings demonstrate the utility of developing highly accurate and robust three-dimensional representations of the murine brain and skull, in which experimental outputs can be directly compared using a unified coordinate system. The aim of these studies is to enhance comparative morphometric analyses and stereotactic surgical procedures in mice.

A three-dimensional MRI atlas of the mouse brain with estimates of the average and variability.

Although there is growing interest in finding mouse models of human disease, no technique for quickly and quantitatively determining anatomical mutants currently exists. Magnetic resonance imaging (MRI) is ideally suited to probe fine structures in mice. This technology is three-dimensional, non-destructive and rapid compared to histopathology; hence MRI scientists have been able to create detailed three-dimensional images of 60 mum resolution or better. The data is digital which lends itself to sophisticated image processing algorithms. Here we show a variational MRI atlas constructed from nine excised brains of 8 week old 129S1/SvImJ male mice. This new type of atlas is comprised of an unbiased average brain--created from alignment of the individual brains--and the mathematical descriptors of anatomical variation across the individuals. We found that the majority of internal points in the individuals never varied more than 117 microm from equivalent points in the atlas. A three-dimensional annotation of the average image was performed and used to estimate the mean and standard deviation of volumes in a variety of structures across the individual brains; these volumes never differed by more than 5%. Our results indicate that variational atlases of inbred strains represent a well-defined basis against which mutant outliers can be readily compared.

Physician gender effects on preventive screening and counseling: an analysis of male and female patients' health care experiences.

BACKGROUND: Studies have documented that patients of female physicians receive higher levels of preventive services. However, most studies include patients of only one gender, examine mainly gender-specific screening services, and do not examine patient education and counseling. OBJECTIVES: This study tests both physician- and patient-gender effects on screening and counseling services received in the past year and considers effects of gender-matched patient-physician pairs. RESEARCH DESIGN: Multivariate analyses are conducted to assess direct and interactive (physician x patient) gender effects and to control for important covariates. SUBJECTS: Data are from the 1998 Commonwealth Fund Survey of Women's Health, a nationally representative sample of U.S. adults. The analytic sample includes 1,661 men and 1,288 women ages 18 and over. MEASURES: Dependent variables are measures of patient-reported screening and counseling services received, including gender-specific and gender-nonspecific services and counseling on general health habits and sensitive topics. RESULTS: Female physician gender is associated with a greater likelihood of receiving preventive counseling for both male and female patients. For female patients, there is an increased likelihood of receiving more gender-specific screening (OR = 1.36, P <0.05) and counseling (OR = 1.40, P <0.05). These analyses provide no evidence that gender-matched physician-patient pairs provide an additional preventive care benefit beyond the main effect of female physician gender. CONCLUSIONS: Female physician gender influences the provision of both screening and counseling services. These influences may reflect physicians' practice and communication styles as well as patients' preferences and expectations.

Gender and patient satisfaction in managed care plans: analysis of the 1999 HEDIS/CAHPS 2.0H Adult Survey.

This paper investigates gender differences in satisfaction, and in the variables associated with satisfaction, using the Consumer Assessment of Health Plans Study (CAHPS) adult questionnaire administered by the National Committee for Quality Assurance (NCQA) as part of HEDIS 1999. Data represent 97,873 men and women enrolled in 206 commercial managed care plans nationwide. Mean plan-level gender differences in satisfaction measures are small, with no consistent pattern of one gender being more satisfied than the other. Controlling for health plan, member, utilization, and selected HEDIS performance indicators, health plan characteristics account for the largest proportion of variance explained in satisfaction. Not-for-profit status and lower turnover of primary care providers are stronger determinants of women's than men's satisfaction. We conclude that it can be useful to analyze CAHPS scores by gender to identify areas for quality improvement in women's health care.

Managed care and women's health: access, preventive services, and satisfaction.

This paper examines insured women's access to health care, receipt of preventive services, and satisfaction with care by the types of health plans in which they are enrolled. Three types of plans are compared: managed care (HMOs and PPOs), fee-for-service with utilization controls, and traditional fee-for-service. For women who have been enrolled in their plans for at least one year, we find the same or better access to care in managed care plans as compared with other plans; receipt of more gender-specific clinical preventive services in managed care plans, but no differences among types of plans for non-gender-specific preventive services or counseling services; and lower satisfaction with care in managed care plans. The implications for practice and policy are discussed.

The receptor tyrosine kinase EphB2 regulates NMDA-dependent synaptic function.

Members of the Eph family of receptor tyrosine kinases control many aspects of cellular interactions during development, including axon guidance. Here, we demonstrate that EphB2 also regulates postnatal synaptic function in the mammalian CNS. Mice lacking the EphB2 intracellular kinase domain showed wild-type levels of LTP, whereas mice lacking the entire EphB2 receptor had reduced LTP at hippocampal CA1 and dentate gyrus synapses. Synaptic NMDA-mediated current was reduced in dentate granule neurons in EphB2 null mice, as was synaptically localized NR1 as revealed by immunogold localization. Finally, we show that EphB2 is upregulated in hippocampal pyramidal neurons in vitro and in vivo by stimuli known to induce changes in synaptic structure. Together, these data demonstrate that EphB2 plays an important role in regulating synaptic function.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) deficiency results in exencephaly and is required for apoptosis within the developing CNS.

Tumor necrosis factor receptor-associated factors (TRAFs) are adaptor proteins important in mediating intracellular signaling. We report here that targeted deletion of traf6 greatly increases the frequency of failure of neural tube closure and exencephaly in traf6 (-/-) mice. The penetrance of this defect is influenced by genetic background. Neural tube fusion requires the coordination of several biological processes, including cell migration invoked by contact-dependent signaling, cell proliferation, and programmed cell death (PCD). To gain greater insight into the role of TRAF6 in these processes, neural development and migration within the CNS of traf6 (-/-) mice and controls were assessed through temporal examination of a number of immunohistochemical markers. In addition, relative levels of cellular proliferation and PCD were examined throughout embryonic development using bromodeoxyuridine (BrdU) and in situ terminal deoxynucleotidyl transferase-mediated dUTP biotinylated nick end labeling (TUNEL), respectively. The data suggest that loss of TRAF6 does not significantly alter the level of cellular proliferation or the pattern of neural differentiation per se, but rather regulates the level of PCD within specific regions of the developing CNS. Substantial reductions in TUNEL were observed within the ventral diencephalon and mesencephalon in exencephalic traf6 (-/-) embryos. Our results demonstrate a novel and prominent role for TRAF6 in the regional control of PCD within the developing CNS.

Gender and patient satisfaction with primary care: tuning in to women in quality measurement.

This study analyzes the relationship between patient gender and satisfaction with primary care visits, using 1999 survey data on 1691 women and 760 men making primary care visits at multiple sites affiliated with a large academic health system designated as a National Center of Excellence in Women's Health (COE). The main findings are that in multivariate analyses controlling for patient and visit characteristics, different aspects of the content of primary care visits are important to women and men. Women's overall satisfaction with visits is more dependent than men's on informational content, continuity of care, and multidisciplinarity. Men's overall satisfaction is more dependent on the personal interest shown in them by providers. No differences in satisfaction are found between those seen in sites affiliated with the COE and other primary care sites within the health system that are not core sites of the COE. We conclude that quality improvement and research in women's primary care could benefit from gender analysis of patient satisfaction data and from more gender-sensitive patient satisfaction measures.

The mammalian ShcB and ShcC phosphotyrosine docking proteins function in the maturation of sensory and sympathetic neurons.

Shc proteins possess SH2 and PTB domains and serve a scaffolding function in signaling by a variety of receptor tyrosine kinases. There are three known mammalian Shc genes, of which ShcB and ShcC are primarily expressed in the nervous system. We have generated null mutations in ShcB and ShcC and have obtained mice lacking either ShcB or ShcC or both gene products. ShcB-deficient animals exhibit a loss of peptidergic and nonpeptidergic nociceptive sensory neurons, which is not enhanced by additional loss of ShcC. Mice lacking both ShcB and ShcC exhibit a significant loss of neurons within the superior cervical ganglia, which is not observed in either mutant alone. The results indicate that these Shc family members possess both unique and overlapping functions in regulating neural development and suggest physiological roles for ShcB/ShcC in TrkA signaling.

Neuronal defects and posterior pituitary hypoplasia in mice lacking the receptor tyrosine phosphatase PTPsigma.

The LAR-family protein tyrosine phosphatase sigma (PTPsigma, encoded by the gene Ptprs) consists of a cell adhesion-like extracellular domain composed of immunoglobulin and fibronectin type-III repeats, a single transmembrane domain and two intracellular catalytic domains. It was previously shown to be expressed in neuronal and lung epithelial tissues in a developmentally regulated manner. To study the role of PTPsigma in mouse development, we inactivated Ptprs by gene targeting. All Ptprs+/- mice developed normally, whereas 60% of Ptprs-/- mice died within 48 hours after birth. The surviving Ptprs-/- mice demonstrated stunted growth, developmental delays and severe neurological defects including spastic movements, tremor, ataxic gait, abnormal limb flexion and defective proprioception. Histopathology of brain sections revealed reduction and hypocellularity of the posterior pituitary of Ptprs-/- mice, as well as a reduction of approximately 50-75% in the number of choline acetyl transferase-positive cells in the forebrain. Moreover, peripheral nerve electrophysiological analysis revealed slower conduction velocity in Ptprs-/- mice relative to wild-type or heterozygous animals, associated with an increased proportion of slowly conducting, small-diameter myelinated fibres and relative hypomyelination. By approximately three weeks of age, most remaining Ptprs-/- mice died from a wasting syndrome with atrophic intestinal villi. These results suggest that PTPsigma has a role in neuronal and epithelial development in mice.