Theoretical volume profiles as a tool for probing transition states: folding kinetics.

The mechanism by which conformational changes, particularly folding and unfolding, occur in proteins and other biopolymers has been widely discussed in the literature. Molecular dynamics (MD) simulations of protein folding present a formidable challenge since these conformational changes occur on a time scale much longer than what can be afforded at the current level of computational technology. Transition state (TS) theory offers a more economic description of kinetic properties of a reaction system by relating them to the properties of the TS, or for flexible systems, the TS ensemble (TSE). The application of TS theory to protein folding is limited by ambiguity in the definition of the TSE for this process. We propose to identify the TSE for conformational changes in flexible systems by comparison of its experimentally determined volumetric property, known as the volume of activation, to the structure-specific volume profile of the process calculated using MD. We illustrate this approach by its successful application to unfolding of a model chain system.

Molecular dynamics calculation of molecular volumes and volumes of activation.

Experimentally, the effects of pressure on reaction rates are described by their pressure derivatives, known as volumes of activation. Transition state theory directly links activation volumes to partial molar volumes of reactants and transition states. We discuss a molecular dynamics method for the accurate calculation of molecular volumes, within which the volumes of molecular species are obtained as a difference between the volumes of pure solvent and solvent with a single molecule inserted. The volumes thus obtained depend on the molecular geometry, the strength and type of the solute-solvent interactions, as well as temperature and pressure. The partial molar volumes calculated using this approach agree well with experimental data. Since this method can also be applied to transition state species, it allows for quantitative analysis of experimental volumes of activation in terms of structural parameters of the corresponding transition states. The efficiency of the approach is illustrated by calculation of volumes of activation for three nonpolar reactions in nonpolar solvents. The results agree well with the experimental data.

A faint type of supernova from a white dwarf with a helium-rich companion.

Supernovae are thought to arise from two different physical processes. The cores of massive, short-lived stars undergo gravitational core collapse and typically eject a few solar masses during their explosion. These are thought to appear as type Ib/c and type II supernovae, and are associated with young stellar populations. In contrast, the thermonuclear detonation of a carbon-oxygen white dwarf, whose mass approaches the Chandrasekhar limit, is thought to produce type Ia supernovae. Such supernovae are observed in both young and old stellar environments. Here we report a faint type Ib supernova, SN 2005E, in the halo of the nearby isolated galaxy, NGC 1032. The 'old' environment near the supernova location, and the very low derived ejected mass ( approximately 0.3 solar masses), argue strongly against a core-collapse origin. Spectroscopic observations and analysis reveal high ejecta velocities, dominated by helium-burning products, probably excluding this as a subluminous or a regular type Ia supernova. We conclude that it arises from a low-mass, old progenitor, likely to have been a helium-accreting white dwarf in a binary. The ejecta contain more calcium than observed in other types of supernovae and probably large amounts of radioactive (44)Ti.

Preimplantation genetic diagnosis for BRCA1/2--a novel clinical experience.

OBJECTIVE: To describe our 2-year experience with preimplantation genetic diagnosis (PGD) for carriers of mutations in the genes BRCA1 and BRCA2, the dilemmas incurred and the lessons learned. METHODS: We collected data on those carriers of BRCA1/2 mutations who applied for PGD counseling and who decided to proceed. We describe the PGD procedures that were conducted and their outcome. RESULTS: Ten carriers of BRCA1/2 mutations applied for PGD counseling, seven were healthy, and three were BC survivors. Eight women needed in vitro fertilization (IVF) because of coexisting infertility. After counseling, six opted for the procedure and five of them underwent PGD for the BRCA mutation. In one of these PGD, fluorescence in situ hybridization (FISH) analysis for chromosomes 21, X and Y was also performed. Three women conceived, each in the first treatment attempt. One of them gave birth to twins, the second to a singleton and the third is currently pregnant. During the pregnancies, dilemmas concerning PGD confirmation were discussed. CONCLUSIONS: PGD is an acceptable reproductive option for BRCA mutation carriers, especially for those who require IVF due to fertility problems. Discussion of this option should be carried out with sensitivity, taking into account the age of the woman, her health, fertility status and emotional state. Confirmatory prenatal diagnosis may not always be encouraged.

Determinants of pancreatic beta-cell regeneration.

Recent studies have revealed a surprising plasticity of pancreatic beta-cell mass. beta-cell mass is now recognized to increase and decrease in response to physiological demand, for example during pregnancy and in insulin-resistant states. Moreover, we and others have shown that mice recover spontaneously from diabetes induced by killing of 70-80% of beta-cells, by beta-cell regeneration. The major cellular source for new beta-cells following specific ablation, as well as during normal homeostatic maintenance of adult beta-cells, is proliferation of differentiated beta-cells. More recently, it was shown that one form of severe pancreatic injury, ligation of the main pancreatic duct, activates a population of embryonic-type endocrine progenitor cells, which can differentiate into new beta-cells. The molecular triggers for enhanced beta-cell proliferation during recovery from diabetes and for activation of embryonic-type endocrine progenitors remain unknown and represent key challenges for future research. Taken together, recent data suggest that regenerative therapy for diabetes may be a realistic goal.

Laser-assisted derivation of human embryonic stem cell lines from IVF embryos after preimplantation genetic diagnosis.

BACKGROUND: Human embryonic stem cells (hESCs) suitable for future transplantation therapy should preferably be developed in an animal-free system. Our objective was to develop a laser-based system for the isolation of the inner cell mass (ICM) that can develop into hESC lines, thereby circumventing immunosurgery that utilizes animal products. METHODS: Hatching was assisted by micromanipulation techniques through a laser-drilled orifice in the zona pellucida of 13 abnormal preimplantation genetic diagnosed blastocysts. ICMs were dissected from the trophectoderm by a laser beam and plated on feeders to derive hESC lines. RESULTS: eight ICMs were isolated from nine hatched blastocysts and gave rise to three hESC lines affected by myotonic dystrophy type 1, hemophilia A and a carrier of cystic fibrosis 405 + 1G > A mutation. Five blastocysts that collapsed during assisted hatching or ICM dissection were plated whole, giving rise to an additional line affected by fragile X. All cell lines expressed markers of pluripotent stem cells and differentiated in vitro and in vivo into the three germ layers. CONCLUSIONS: These hESC lines can serve as an important model of the genetic disorders that they carry. Laser-assisted isolation of the ICMs may be applied for the derivation of new hESC lines in a xeno-free system for future clinical applications.

Using performance measures to identify plans of action to improve care.

BACKGROUND: Health care organizations dedicate enormous time and resources collecting data to measure the performance of physicians, hospitals, and other medical facilities. These measures may reflect outcomes, processes of care, patient perceptions of the quality of care, and resource utilization and cost. However, less thought is given to how the performance data should be used to improve care. The data must be translated into clinically relevant terms that assess the decisions of the clinical staff and the functioning of the systems that support the delivery of care. The processes of care are identified through record review, analysis of the system of care delivery, and patient interview, and are then further assessed to determine the underlying causes. EXAMPLES: Examples, drawn from case studies, are provided to illustrate how to identify and address components of care requiring improvement. DISCUSSION: Physician behavior is an important component of care in all performance measures. Modification of some patterns of behavior, including those of nursing and other support staff, may be needed to reduce some types of error. For this reason it is important to involve physicians in the process of discovering root causes. When the root cause involves the medical care system, an interdisciplinary approach will be needed. This may involve administrators, nurses, pharmacists, home care and discharge planners, and office personnel. One recommended approach to QI is to identify system errors and then design changes in the system to reduce that type of error. CONCLUSION: Performance measures must be translated into the components(s) of care that are implicated in the measure. Once this component has been identified as the reason behind the suboptimal measure, its root cause should be used to structure the most effective intervention.

Risk factors for adolescent substance abuse.

Reviews research on risk factors for adolescent substance use disorders (SUD) and discusses possible relationships between SUDs and learning disabilities (LD). Individual level factors (genetic, biologic, other familial, and psychiatric) emerge as very important in the risk equation, as well as the interaction between individual risk and environmental conditions. Commonalities between SUD risk and LD include prenatal substance exposure, family history of SUD, conduct disorder, social skills deficits, and academic failure; however, further research is needed to establish whether individuals with LD face a specific risk for SUDs, and if so, what the nature of that risk might be.

Child psychopathology risk factors for drug abuse: overview.

Introduces the special section on Child Psychopathology Risk Factors for Substance Use Disorders. This article summarizes important principles, the current literature, contributions to this section, and issues for future research. Psychopathological conditions are strongly associated with substance use disorders, and some childhood psychopathological conditions may constitute precursors to this comorbidity. Conduct disorder constitutes a strong risk factor for substance use disorders, and bipolar disorder, although more rare, may also constitute a significant risk. Data for other child psychiatric conditions are mixed or lacking; however, important subgroups may be at risk and merit further attention. Underlying characteristics, such as temperament and self-regulation, merit further study as possible explanatory variables. Such studies hold the key for targeting and improving preventive and therapeutic interventions.

Genetic testing of breast and ovarian cancer patients: clinical characteristics and hormonal risk modifiers.

OBJECTIVES: Carriers of the mutations 185delAG and 5382insC in the BRCA1 gene and 6174delT in the BRCA2 gene have a substantial life-time risk for breast and ovarian cancers (BC and OC). The aim of the study was to identify the clinical features and the hormonal risk modifiers in mutation carriers and the implication in suggested guidelines for treatment decisions in BRCA1/2 carrier patients. STUDY DESIGN: Breast and/or ovarian cancer patients from the Oncology and Cancer Genetic clinics were tested for the three Ashkenazi founder mutations: 87 patients were identified as carriers of one of these mutations. Clinical presentation and age at onset were correlated with the mutations, in patients with bilateral BC or BC and OC, the length of time that elapsed between the diagnosis of the two cancers was recorded. We compared BC and OC patients with regard to ages at menarche, first pregnancy and menopause, number of pregnancies and deliveries, the use of oral contraceptives, hormonal replacement therapy and fertility treatments. RESULTS: The carriers of the three BRCA1/2 Ashkenazi founder mutations did not differ in clinical presentation nor age at onset. Forty-three patients (74.1%) of 58 BC patients were diagnosed between the ages 30 and 50, only four (6.9%) patients were diagnosed after age 60. Of BC patients diagnosed before age 35, 63.6% developed second BC as compared to 25.5% of those diagnosed after age 35. Ovarian cancer was diagnosed after age 45 in 89.7% of the patients, only one patient was diagnosed under the age of 40. Oral contraceptives use was documented in 61.3% of BC patients as compared to 11.8% of OC patients. Other hormonal factors did not differ between the two groups. CONCLUSIONS: The carriers of the three Ashkenazi founder mutations should be considered at the same risk for BC and for OC and treatment options should be the same. Mutation carriers diagnosed with BC before the age of 35 are at a very high risk for developing second breast cancer. Most ovarian cancers in carriers were diagnosed after age 45, and prophylactic oophorectomy should be postponed to the age of 45. Oral contraceptives might elevate the risk of BC in mutation carriers.

Managing quality in hospital practice.

BACKGROUND: While routine clinical decision-making has a substantial effect on quality, most practising physicians do not routinely examine their outcomes. OBJECTIVES: To set up a practical process for identifying problems in hospital practices of primary care physicians, examine their causes, and develop a quality improvement process that intimately involves practising physicians in problem-solving. DESIGN: All hospital admissions to the Primary Care Service were screened over a 14-month period using simple pre-specified criteria. Quality problems were verified by medical record reviews carried out by two physicians. These problems were discussed at monthly meetings of physicians to characterize the problems fully, identify their causes, and document adverse effects on patient outcomes. SETTING: One community hospital. PARTICIPANTS: Primary care physicians from three group practices and four solo practices who admit patients to the Primary Care Service. INTERVENTIONS: Monthly group discussions plus discussions with individual physicians when time did not permit all quality problems to be discussed at group meetings. Certain issues of high sensitivity were also discussed with the individuals rather than in an open forum. OUTCOME MEASURES: Missed or delayed diagnoses, inappropriate treatments, and complications and their root causes. RESULTS: Quality problems were identified in 6% of all admissions. Of these, 60% were missed or delayed diagnoses, 22% were iatrogenic complications and 18% were inappropriate treatments. Root cause analysis suggested that physician behaviors led to 75% of problems; systems problems to 20% and inadequate knowledge to 5%. Process improvements included development of a call-in system to reduce delays in obtaining X-ray reports; implementation of an anticoagulation monitoring system in one group practice; and a protocol of regular feedback of errors in diagnosis to emergency room physicians. Participating physicians reported increased awareness of common errors and greater attention to detail in patient evaluations. CONCLUSIONS: Knowledge of root causes of quality problems is essential for improving quality of care. A simple routine approach to examining adverse outcomes and how care might be improved in the future was set up. Active participation of practising physicians is essential. Other organizations could use this process for routinely reviewing and improving quality.

Adolescent substance abuse: a review of the past 10 years.

OBJECTIVE: To review and synthesize the recent scientific literature on adolescent substance abuse, covering natural history, epidemiology, etiology, comorbidity, assessment, treatment, and prevention, and to highlight areas for future research. METHOD: Studies of adolescent substance abuse were reviewed with the focus on substance abuse and dependence rather than substance use. RESULTS: There has been a sharp recent resurgence in adolescent drug use. Biological factors, including genetic and temperament characteristics, as well as family environment factors, are emerging as important etiological variables. Comorbidity with other psychiatric disorders, particularly with conduct disorder, is frequent and complicates treatment. New assessment instruments are available for clinical and research use. Among treatment modalities, family-based interventions have received the most study. CONCLUSIONS: The past decade has seen growth in the volume and sophistication of research on adolescent substance abuse and in the conceptualization of this problem. Further research is needed, particularly on the significance of comorbid conditions and on individualized and effective treatment approaches.

Cognitive and behavioral deficits associated with parental alcohol use.

OBJECTIVE: To review and synthesize the scientific literature on cognitive and behavioral deficits associated with parental alcohol use and to highlight areas for future attention. METHOD: Studies of children of alcoholic parents (generally fathers) and of children prenatally exposed to alcohol were reviewed, focusing on cognitive and behavioral findings. Relevant animal studies were also reviewed. RESULTS: Large numbers of children may be affected by parental alcohol use. Prenatal alcohol exposure is frequently associated with specific cognitive and behavioral deficits. Children of alcoholic fathers also can present with difficulties in learning, language, and temperament. Similarities in the deficits of these two groups were noted. CONCLUSIONS: The problems associated with parental alcohol use merit much more clinical and research attention. Current clinical approaches often fail to recognize the diagnostic and therapeutic significance of this history, and subgroups of alcohol-affected children may confound research studies of other problems. Subtle deficits in learning, language, and self-regulation may be the most developmentally devastating and the least likely to be identified and addressed effectively. This is an important area in which to combine behavior genetic and environmental approaches to understanding development.

The founder mutations 185delAG and 5382insC in BRCA1 and 6174delT in BRCA2 appear in 60% of ovarian cancer and 30% of early-onset breast cancer patients among Ashkenazi women.

The mutations 185delAG, 188del11, and 5382insC in the BRCA1 gene and 6174delT in the BRCA2 gene were analyzed in 199 Ashkenazi and 44 non-Ashkenazi Jewish unrelated patients with breast and/or ovarian cancer. Of the Jewish Ashkenazi women with ovarian cancer, 62% (13/21) had one of the target mutations, as did 30% (13/43) of women with breast cancer alone diagnosed before the age 40 years and 10% (15/141) of those with breast cancer diagnosed after the age 40 years. Age at ovarian cancer diagnosis was not associated with carrier status. Of 99 Ashkenazi patients with no family history of breast and/or ovarian cancer, 10% carried one of the mutations; in two of them the mutation was proved to be paternally transmitted. One non-Ashkenazi Jewish ovarian cancer patient from Iraq carried the 185delAG mutation. Individual mutation frequencies among breast cancer Ashkenazi patients were 6.7% for 185delAG, 2.2% for 5382insC, and 4.5% for 6174delT, among ovarian cancer patients; 185delAG and 6174delT were about equally common (33% and 29%, respectively), but no ovarian cancer patient carried the 5382insC. More mutations responsible for inherited breast and ovarian cancer probably remain to be found in this population, since 79% of high-incidence breast cancer families and 35% of high-incidence breast/ovarian cancer families had none of the three known founder mutations.

Genotype-phenotype correlations of DHP receptor alpha 1-subunit gene mutations causing hypokalemic periodic paralysis.

Hypokalemic periodic paralysis (hypoKPP) is an autosomal dominant or sporadic disorder characterized by periodic, reversible attacks of muscle weakness. Mutations in the skeletal muscle dihydropyridine receptor alpha 1-subunit that functions as a calcium channel (CACNL1A3) cause hypoKPP. We studied a group of 45 hypoKPP probands and demonstrated mutations in 30 of them. When compared with patients in whom CACNL1A3 mutations were not identified, those with mutations had an earlier age of onset and more often had a family history of hypoKPP. To date, three mutations have been identified. The R1239G mutation has only been found in one family. Of the 30 probands with recognized mutations, R528H accounted for 43% and R1239H was seen in 53%. Age of onset and potassium levels during attacks were lower in patients with the R1239H mutation than those with R528H. Cardiac dysrhythmias co-segregated with hypoKPP in one small kindred with the R528H mutation. No mutations were identified in exons of the gene encoding the S4 segments of domains one and three or the cytoplasmic loop between domains two and three. In addition to the 45 hypoKPP probands, an additional 11 probands with clinical variants of hypoKPP (three thyrotoxic hypoKPP and eight Andersen syndrome patients) were examined for CACNL1A3 mutations and none were found.

Online help: cancer patients participate in a computer-mediated support group.

This study investigated a computer mediated support group of six breast cancer patients. For a three-month period, patients used home computers to connect to a computer bulletin board on which they read messages from and posted messages to each other. The patients had no difficulty learning to use the computer and used it an average of one hour a week. The patients discussed their medical conditions, shared personal concerns, and offered support. This online approach provided many features of traditional face-to-face support groups.

Cystic fibrosis heterozygote screening in the Orthodox Community of Ashkenazi Jews: the Dor Yesharim approach and heterozygote frequency.

In the community of the Orthodox Jews most of the marriages are arranged a screening program that is aimed at preventing the marriage of two carriers of autosomal recessive disorders is conducted by the Dor Yesharim organization. A random sample of 6,076 individuals of the Orthodox Jewish Ashkenazi community, were screened for the five mutations common in Ashkenazi patients (delta F508, W1282X, G542X, N1303K, 3849 + 10Kb C-->T). Two hundred thirty-two carriers were identified, giving a heterozygote frequency of 1:26. The relative frequencies of the individual mutations in the general population were comparable to those in the patients.