Computational design of mechanically coupled axle-rotor protein assemblies.

Natural molecular machines contain protein components that undergo motion relative to each other. Designing such mechanically constrained nanoscale protein architectures with internal degrees of freedom is an outstanding challenge for computational protein design. Here we explore the de novo construction of protein machinery from designed axle and rotor components with internal cyclic or dihedral symmetry. We find that the axle-rotor systems assemble in vitro and in vivo as designed. Using cryo-electron microscopy, we find that these systems populate conformationally variable relative orientations reflecting the symmetry of the coupled components and the computationally designed interface energy landscape. These mechanical systems with internal degrees of freedom are a step toward the design of genetically encodable nanomachines.

Unilateral vestibular schwannoma and meningiomas in a patient with PIK3CA-related segmental overgrowth: Co-occurrence of mosaicism for 2 rare disorders.

A 28-year-old female with PIK3CA-related segmental overgrowth presented with headaches. She also had a unilateral vestibular schwannoma (VS), as well as 3 small (<2 cm) meningiomas, which according to the Manchester consensus diagnostic criteria for neurofibromatosis 2 (NF2) is sufficient for a clinical diagnosis. Analysis of blood revealed a mosaic PIK3CA c.2740G>A (p.Gly914Arg) mutation, confirming the diagnosis of PIK3CA-related overgrowth, but no mutations in NF2 were detected. Although VS has not previously been reported in PIK3CA-related segmental overgrowth, meningiomas have, raising the question of whether this patient's VS and meningiomas represent coincidental NF2 or phenotypic extension of her overgrowth syndrome. Genetic analysis of the VS revealed a heterozygous NF2 mutation c.784C>T (p.Arg262Ter) and loss of a portion of 22q, including NF2, SMARCB1, and LZTR1 genes. These results suggest that the patient has 2 different mosaic disorders, NF2 and PIK3CA-related overgrowth. The PIK3CA mutation was also present in the VS. Confirmation of the clinical diagnosis of mosaic NF2 in this patient has implications for monitoring and highlights the possibility of co-occurrence of mosaicism for multiple rare disorders in a single patient.

c-Met-induced epithelial carcinogenesis is initiated by the serine protease matriptase.

The progression and negative outcome of a variety of human carcinomas are intimately associated with aberrant activity of the c-Met oncogene. The underlying cause of this dysregulation, however, remains a subject of discussion, as the majority of cancer patients do not present with activating mutations in c-Met receptor itself. In this study, we show that the oncogenic protease matriptase is ubiquitously co-expressed with the c-Met in human squamous cell carcinomas and amplifies migratory and proliferative responses of primary epithelial cells to the cognate ligand for c-Met, pro-hepatocyte growth factor/scatter factor (proHGF/SF), through c-Met and Gab1 signaling. Furthermore, the selective genetic ablation of c-Met from matriptase-expressing keratinocytes completely negates the oncogenic potential of matriptase. In addition, matriptase-dependent carcinoma formation could be blocked by the pharmacological inhibition of the Akt-mammalian target of Rapamycin (mTor) pathway. Our data identify matriptase as an initiator of c-Met-Akt-mTor-dependent signaling axis in tumors and reveal mTor activation as an essential component of matriptase/c-Met-induced carcinogenesis. The study provides a specific example of how epithelial transformation can be promoted by epigenetic acquisition of the capacity to convert a widely available paracrine growth factor precursor to its signaling competent state.

Bacillus cereus induces permeability of an in vitro blood-retina barrier.

Most Bacillus cereus toxin production is controlled by the quorum-sensing-dependent, pleiotropic global regulator plcR, which contributes to the organism's virulence in the eye. The purpose of this study was to analyze the effects of B. cereus infection and plcR-regulated toxins on the barrier function of retinal pigment epithelium (RPE) cells, the primary cells of the blood-retina barrier. Human ARPE-19 cells were apically inoculated with wild-type or quorum-sensing-deficient B. cereus, and cytotoxicity was analyzed. plcR-regulated toxins were not required for B. cereus-induced RPE cytotoxicity, but these toxins did increase the rate of cell death, primarily by necrosis. B. cereus infection of polarized RPE cell monolayers resulted in increased barrier permeability, independent of plcR-regulated toxins. Loss of both occludin and ZO-1 expression occurred by 8 h postinfection, but alterations in tight junctions appeared to precede cytotoxicity. Of the several proinflammatory cytokines analyzed, only interleukin-6 was produced in response to B. cereus infection. These results demonstrate the deleterious effects of B. cereus infection on RPE barrier function and suggest that plcR-regulated toxins may not contribute significantly to RPE barrier permeability during infection.

Sulfotransferase gene copy number variation: pharmacogenetics and function.

Pharmacogenetics is the study of the role of inheritance in variation to drug response. Drug response phenotypes can vary from adverse drug reactions at one end of the spectrum to equally serious lack of the desired effect of drug therapy at the other. Many of the current important examples of pharmacogenetics involve inherited variation in drug metabolism. Sulfate conjugation catalyzed by cytosolic sulfotransferase (SULT) enzymes, particularly SULT1A1, is a major pathway for drug metabolism in humans. Pharmacogenetic studies of SULT1A1 began over a quarter of a century ago and have advanced from biochemical genetic experiments to include cDNA and gene cloning, gene resequencing, and functional studies of the effects of single nucleotide polymorphisms (SNPs). SNP genotyping, in turn, led to the discovery of functionally important copy number variations (CNVs) in the SULT1A1 gene. This review will briefly describe the evolution of our understanding of SULT1A1 pharmacogenetics and CNV, as well as challenges involved in utilizing both SNP and CNV data in an attempt to predict SULT1A1 function. SULT1A1 represents one example of the potential importance of CNV for the evolving disciplines of pharmacogenetics and pharmacogenomics.

Risk factors for urinary complications after renal transplantation.

Urinary complications are common following renal transplantation. The aim of this study is to evaluate the risk factors associated with renal transplant urinary complications. We collected data on 1698 consecutive renal transplants patients. The association of donor, transplant and recipient characteristics with urinary complications was assessed by univariable and multivariable Cox proportional hazards models, fitted to analyze time-to-event outcomes of urinary complications and graft failure. Urinary complications were observed in 105 (6.2%) recipients, with a 2.8% ureteral stricture rate, a 1.7% rate of leak and stricture, and a 1.6% rate of urine leaks. Seventy percent of these complications were definitively managed with a percutaneous intervention. Independent risk factors for a urinary complication included: male recipient, African American recipient, and the "U"-stitch technique. Ureteral stricture was an independent risk factor for graft loss, while urinary leak was not. Laparoscopic donor technique (compared to open living donor nephrectomy) was not associated with more urinary complications. Our data suggest that several patient characteristics are associated with an increased risk of a urinary complication. The U-stitch technique should not be used for the ureteral anastomosis.

Hot topic: using a stearoyl-CoA desaturase transgene to alter milk fatty acid composition.

Stearoyl-CoA desaturase enzyme converts specific medium- and long-chain saturated fatty acids to their monounsaturated form. Transgenic goats expressing a bovine beta-lactoglobulin promoter-rat stearoyl-CoA desaturase cDNA construct in mammary gland epithelial cells were produced by pronuclear microinjection. The fatty acid composition of milk from 4 female transgenic founders was analyzed on d 7, 14, and 30 of their first lactation. In 2 animals, the expression of the transgene changed the overall fatty acid composition of the resulting milk fat to a less saturated and more monounsaturated fatty acid profile at d 7 of lactation; however, this effect diminished by d 30. In addition, one animal had an increased proportion of the rumen-derived monounsaturated fatty acid C18:1 trans11 converted by stearoyl-CoA desaturase to the conjugated linoleic acid isomer C18:2 cis9 trans11. Milk that has higher proportions of monounsaturated fatty acids and conjugated linoleic acid may have benefits for human cardiovascular health.

Evidence of increased substrate availability to in vitro-derived bovine foetuses and association with accelerated conceptus growth.

Changes in placental development have been associated with foetal abnormalities after in vitro embryo manipulations. This study was designed to investigate bovine conceptus development and substrate levels in plasma and fluids in in vivo- and in vitro-produced (IVP) concepti and neonates. In vivo-produced and IVP embryos were derived by established embryo production procedures. Pregnant animals from both groups were slaughtered on days 90 or 180 of gestation, or allowed to go to term. Conceptus and neonatal physical traits were recorded; foetal, maternal and neonatal blood, and foetal fluids were collected for the determination of blood and fluid chemistry, and glucose, fructose and lactate concentrations. Placental transcripts for specific glucose transporters were determined by quantitative RT-PCR. No significant differences in uterine and conceptus traits were observed between groups on day 90. On day 180, larger uterine, placental and foetal weights, and an increase in placental gross surface area (SA) in IVP pregnancies were associated with increased glucose and fructose accumulation in foetal plasma and associated fluids, with no differences in the expression of components of the glucose transporter system. Therefore, the enlarged placental SA in IVP pregnancies suggests an increase in substrate uptake and transport capacity. Newborn IVP calves displayed higher birth weights and plasma fructose concentrations soon after birth, findings which appeared to be associated with clinical and metabolic distress. Our results indicated larger concepti and increased placental fructogenic capacity in mid- to late IVP pregnancies, features which appeared to be associated with an enhanced substrate supply, potentially glucose, to the conceptus.

Hematopoietic stem cell transplantation in utero produces sheep-goat chimeras.

Both allogeneic and xenogeneic hematopoietic chimera models have been developed, including fetal sheep models that demonstrated high levels of stable, multilineage engraftment created by in utero hematopoietic stem cell transplantation. The aim of this study was to test the efficacy of in utero transplantation to create xenogeneic sheep-goat hematopoietic chimeras. Fetal liver cells and T-cell-depleted adult bone marrow were tested as sources of hematopoietic stem cells. Donor cells were injected intraperitoneally into 130 recipient fetuses between 49 and 62 days of gestation. Groups 1 and 2 received crude fetal liver cell preparations. Group 3 received fetal liver cells that were incubated overnight in a phytohemagglutinin-stimulated lymphocyte-conditioned medium (PHA-LCM). In Group 4, hematopoietic stem cells were concentrated by using additional density separations. Group 5 fetal recipients received low-density, T-cell-depleted adult bone marrow cells. In Group 1, fetuses were accessed via hysterotomy. Hematopoietic stem cells were injected into Groups 2, 3, 4, and 5 without cutting through the uterine wall. Fetal survival in the five groups ranged from 56 to 100%. The percentage of chimeras from injected fetuses ranged from 43 to 92% by FACS and PCR analyses; however, levels of chimerism were low (<1%). The highest rates of chimerism were found among recipients of low-density fetal liver cells. Despite the pre-immunocompetent status of the fetal recipients and the genetic similarities between sheep and goats, high levels of engraftment were not observed. The consistently low levels of chimerism observed in this study, as well as the poor results recently reported by others using these procedures, indicate that significant barriers exist to transplanting hematopoietic stem cells in utero.

Evidence against humoral immune attack as the cause of sheep-goat interspecies and hybrid pregnancy failure in the doe.

The failure of interspecies and hybrid pregnancies between the domestic sheep (Ovis aries) and goat (Capra hircus) is not completely understood. The sheep-goat hematopoietic chimera is a unique model for studying the role of the maternal immune response in failure of interspecies and hybrid pregnancies between these species. Hematopoietic chimeras were created by in utero transplantation of sheep fetal liver cells into goat fetuses. The resulting chimeric females were recipients of sheep demi-embryos genetically identical to their sheep cells and/or were bred to a ram to create a hybrid pregnancy. Pregnancy sera were analyzed for the presence of anti-species antibodies (Ab) using a lymphocyte microcytotoxicity assay. None of the concepti survived to term. Gross and histological evaluations of two interspecies sheep concepti revealed abnormal placentome formation. The humoral immune response of several hematopoietic chimeras to the challenging concepti differed from control animals. We observed delayed onset of Ab production, low and absent titers, and persistent Ab titers with delayed fetal death. Ultrasonography typically revealed normal fetal development associated with high volumes of placental fluids and retarded placentome development. We conclude that fetal death was associated with abnormal placental development that was not the result of maternal humoral immune attack.

Fetal leukocyte trafficking as a stimulus for the production of maternal antibodies in the goat.

The production of antibodies during pregnancy or after parturition is a natural occurrence in many mammalian species. Fetal cells have been detected in the peripheral blood of women and mice and are thought to be the immune stimulus for antibody production. The aim of this study was to investigate if the production of maternal anti-fetal antibodies during ruminant pregnancy is the result of fetal leukocyte trafficking across the placenta. Maternal pregnancy serum was collected from 94 does whose fetuses received sheep hematopoietic stem cells via in utero transplantation at 49 to 62 d of gestation. Serum samples were collected before surgery and at weekly intervals throughout gestation. A lymphocyte microcytotoxicity assay was used to screen the serum samples from does that carried chimeric fetuses to term (n = 75). Of these 75 does, 28 parous does had presurgery serum that contained alloreactive antibodies. Nine of the 75 does had nonreactive presurgery serum, but they produced alloreactive antibody titers during gestation. Xenoreactive antibodies were detected in the pregnancy sera from 2 of the 75 does tested. Hemolytic assays confirmed the species-specificity of the xenoreactive serum from these 2 does. In view of the fact that hematopoietic cells were the only source of anti-sheep antibody stimulation in this model, we propose that fetal leukocyte trafficking does take place across the caprine placenta.

Can methodological features account for patient-treatment matching findings in the alcohol field?

OBJECTIVE: Despite enthusiasm for the potential of matching patients to alcohol treatments to improve outcomes, consistent findings have not emerged. This review considers the extent to which methodological factors may account for the pattern of findings from research on Patient x Alcohol Treatment interactions. METHOD: We focused on 55 studies that compared more than one type of alcohol treatment and included formal statistical tests for interactions. We examined four predictors of the number of significant interactions found in the 55 studies: (1) the number of statistical tests for interactions conducted, (2) the average number of participants, (3) whether or not participants were randomized to treatment and (4) the proportion of tested interactions that were hypothesis- or rationale-driven, as opposed to exploratory. RESULTS: Only the number of statistical tests for interactions predicted the number of patient-treatment interactions identified per study (zero-order r = 0.47; r2 = 0.22). A substantial number of tests for interactions (43) was conducted, on average, per study. Only a minority of the studies (33%) included enough participants to have a reasonable probability (0.80) of identifying a medium-sized matching effect. CONCLUSIONS: Drawing general conclusions regarding matching patients to alcohol treatments is hampered because Type I error has contributed to the matches identified, studies in this area are often underpowered, and the combinations of patient and treatment variables that have been tested are few relative to the numerous possible combinations. To be productive, future research will need to focus on patients at the extremes of matching dimensions and on distinct treatments. (J Stud. Alcohol 62: 62-73, 2001)

Successful pregnancy in goats carrying their genetically identical conceptus.

Mammalian pregnancies are naturally allogeneic, but syngeneic pregnancies have been carried to term in laboratory animal species. The need for maternal immune recognition during mammalian pregnancy is still unclear. Allogeneic pregnancies are protected from maternal immune attack by the nature of the trophoblast and its interactions with maternal tissues at the maternal-fetal interface. Syngeneic pregnancy models and the success of pregnancies in immunosuppressed mice challenge the necessity of a maternal immune response in mammals. This study was designed to investigate if outbred, domestic sheep and goats can successfully establish and maintain a syngeneic pregnancy. Embryo splitting and cryopreservation techniques were used to enable sheep and goat demi-embryos to be transferred to genetically identical females. Allogeneic pregnancies were established from the transfer of demi-embryos subjected to the same manipulations to assess demi-embryo survival and pregnancy rates under conventional immune compatibility conditions. Syngeneic pregnancies were established and carried to term in goats (2/11) but not in sheep (0/24). Microsatellite and DNA fingerprinting analyses confirmed that each kid was a genetically identical twin to the female that carried it to term. Our results demonstrated that genetic disparity is not required for the establishment and maintenance of pregnancy in goats, but our results were inconclusive for sheep.

Transdisciplinary work with psychology from a population health perspective: an illustration.

One of the important implications of a population health perspective in public health is an increase in the need for transdisciplinary ways of working. The Community Health Research Unit (CHRU) is presented as an example of an environment where psychology and psychologists work with other disciplines to conduct applied research in population health. Research activities were examined to identify how the disciplines collaborate and to provide evidence of successful interdisciplinary and transdisciplinary approaches which incorporate health psychology. The strengths and challenges of multi-, inter-, and transdisciplinary approaches were examined through a poll of CHRU members. Further, members' views about the contributions of psychology to their work were gathered. Issues of working with different disciplines in a transdisciplinary approach are highlighted and future directions are suggested.

A model for building collective capacity in community-based programs: the Elderly in Need Project.

As the focus of health promotion moves from individuals to organizations, communities and broader social policy, the models that guide public health program planning and development need reexamination. Public health nurses are gaining experience in strengthening and supporting the ability of communities to grow and change. This study aimed to illuminate the process. Data, gathered as part of an action research project to develop individual and community-based strategies to decrease isolation in frail older adults, provided a rich source of qualitative data. Analysis was directed toward identifying the factors and processes of capacity-building. The emerging model was clarified and partially validated with academics and practitioners from health promotion programs across the age span. Four stages of building collective capacity were identified: identifying common ground, working cooperatively, working in partnership, and working across the community. At each stage, processes of relationship building, project management and capacity-building resulted in stage specific products. A model of building collective capacity, grounded in community health practice and supported by the literature provides a base for developing practice indicators, and practice guidelines which will strengthen the ability to reach health goals.

Identifying older people in need using action research.

An ageing population has implications for community-based health promotion and disease prevention. There is concern about older people who do not fit into existing programmes and services yet need minimal support to maintain independence. A study was designed to develop approaches to gain access to this hard to reach population, assess needs and design and test interventions to integrate them into the community. The study, informed by theories of health promotion and social support, used action research methods. Participant observation documented in field notes, together with case notes and clinical assessments, provided a rich source of qualitative and quantitative data. This article discusses the needs assessment. Over a 3-year period, public health nurses linked with community groups in a predominantly francophone, urban community to identify the target group. Key characteristics of the target group included limitations with instrumental activities of daily living and low levels of social support combined with stressful life situations that challenged adaptation. Three patterns of inadequate support were identified.

Stress-induced cortisol, mood, and fat distribution in men.

OBJECTIVE: A previous study in our laboratory (Moyer et al., Obes Res. 1994;2:255-62 found that, in response to uncontrollable laboratory stress, women with a high waist-to-hip ratio (WHR) had higher cortisol reactivity, poorer coping skills, and lower anger responses than women with low WHR. We aimed to compare high WHR men's stress responses to these women. RESEARCH METHODS AND PROCEDURES: The current study examined cortisol reactivity and psychological data of 27 healthy high WHR men exposed to the same laboratory challenges as the women from our previous study. Men's data are discussed in relation to that of the high and low WHR women. RESULTS: Men responded to the stress with increases in both cortisol and blood pressure. In comparison with the high and low WHR women, men had significantly higher total cortisol on the stress day. However, when comparing a sub-sample of men and women matched in WHR's, differences in cortisol secretion were greatly diminished and no longer significant. In addition, men had higher desire for control than both high and low WHR women, and lower mood reactivity than low WHR women. Despite the lower mood reactivity of high WHR groups, the high mood reactors among the high WHR women, and to a lesser extent, men, tended to have higher cortisol reactivity. DISCUSSION: These results suggest that the psychological differences and greater exposure to cortisol observed among the high WHR men and women may have played a role in contributing to their greater abdominal fat depots.

Decisions about prenatal testing for chromosomal disorders: perceptions of a diverse group of pregnant women.

We conducted a study to elucidate factors influencing women's decisions regarding prenatal genetic screening for and diagnosis of chromosomal disorders and to learn about their experiences with these tests and with the medical system. Using focus group interviews and questionnaire assessments, we obtained detailed impressions of a diverse group of 75 pregnant women. Participants varied with respect to race/ethnicity, religious background, and reproductive history, as well as in their decisions about use of prenatal screening and diagnostic testing. Substantial variation surfaced in attitudes toward testing. Factors influencing women's views included available resources, feelings about having a child with Down syndrome, moral beliefs, family and social influences, perceptions of one's own health, the difficulty of becoming pregnant, and willingness to put the fetus at elevated miscarriage risk. Such findings indicate that age-based policies regarding access to prenatal diagnoses that, among other reasons, are based on the balance of risks between bearing a child with a chromosomal abnormality versus procedure-related loss are incompatible with the range of concerns that women bring to this decision and the weight individual women may assign to the outcomes.

Progestin implants can rescue demi-embryo pregnancies in goats: a case study.

Survival after transfer of demi-embryos (i.e., half-embryos produced by embryo splitting) to recipients usually is lower than survival after transfer of intact embryos. Reduced survival after demi-embryo transfer could be due to loss of viability after splitting, failure of a viable demi-embryo to prevent corpus luteum (CL) regression in the recipient female, or a combination of factors. From a retrospective analysis of pregnancy and embryo survival rates after demi-embryo transfer in sheep and goats, we report the rescue of caprine demi-embryo pregnancies in which CL regression occurred at the end of diestrus despite the presence of a viable conceptus in the uterus with progestin implants. Day 5 or 6 morulae and blastocysts were flushed from superovulated ewes and does and split into demi-embryos of approximately equal halves. Demi-embryos were either transferred fresh to synchronized recipients of the homologous species or frozen in liquid nitrogen. Approximately half of the recipient does and ewes were treated with norgestomet implants on Day 10 of the embryo transfer cycle and again 2 wk later. Serum collected on Day 25 from recipients with implants was assayed for progesterone to determine if a CL of pregnancy had been maintained. Pregnancy was diagnosed by ultrasonography on Day 35 of gestation. Corpus luteum regression occurred despite the presence of a viable conceptus in the uterus in 6 of 55 progestin-treated caprine demi-embryo recipients and in 0 of 66 ovine demi-embryo recipients. Five of the caprine pregnancies were maintained to term with norgestomet implants and produced 5 live kids. The sixth fetus, which was carried by a progestin implant-treated 8-mo-old doeling, died at approximately 50 d of gestation. These results suggest that, at least in goats, some demi-embryos may provide inadequate signaling for maternal recognition of pregnancy, and such pregnancies can be rescued with progestin treatment to the doe.