Developmental and adult phenotyping directly from mutant embryonic stem cells

Tetraploid embryo complementation assay has shown that mouse ES cells alone are capable of supporting embryonic development and adult life of mice. Newly established F(1) hybrid ES cells allow the production of ES cell-derived animals at a high enough efficiency to directly make ES cell-based genetics feasible. Here we report the establishment and characterization of 12 new F(1) hybrid ES cell lines and the use of one of the best (G4) in a gain- and loss-of-function genetic study, where the in vivo phenotypes were assessed directly from ES cell-derived embryos. We found the generation of G4 ES cell-derived animals to be very efficient. Furthermore, even after two consecutive rounds of genetic modifications, the majority of transgenic lines retained the original potential of the parental lines; with 10-40% of chimeras producing ES cell-derived animals/embryos. Using these genetically altered ES cells, this success rate, in most cases, permitted the derivation of a sufficient number of mutants for initial phenotypic analyses only a few weeks after the establishment of the cell lines. Although the experimental design has to take into account a moderate level of uncontrolled damage on ES cell lines, our proof-of-principle experiment provides useful data to assist future designs harnessing the power of this technology to accelerate our understanding of gene function.     

IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 degrees C. International Federation of Clinical Chemistry and Laboratory Medicine. Part 4. Reference procedure for the measurement of catalytic concentration of alanine aminotransferase.

This paper is the fourth in a series dealing with reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 degrees C and the certification of reference preparations. Other parts deal with: Part 1. The Concept of Reference Procedures for the Measurement of Catalytic Activity Concentrations of Enzymes; Part 2. Reference Procedure for the Measurement of Catalytic Concentration of Creatine Kinase; Part 3. Reference Procedure for the Measurement of Catalytic Concentration of Lactate Dehydrogenase; Part 5. Reference Procedure for the Measurement of Catalytic Concentration of Aspartate Aminotransferase; Part 6. Reference Procedure for the Measurement of Catalytic Concentration of Gamma-Glutamyltransferase; Part 7. Certification of Four Reference Materials for the Determination of Enzymatic Activity of Gamma-Glutamyltransferase, Lactate Dehydrogenase, Alanine Aminotransferase and Creatine Kinase at 37 degrees C. A document describing the determination of preliminary upper reference limits is also in preparation. The procedure described here is deduced from the previously described 30 degrees C IFCC reference method. Differences are tabulated and commented on in Appendix 2.     

Neutrophils do not mediate the pathophysiological sequelae of Cryptosporidium parvum infection in neonatal piglets

Cryptosporidium parvum is a minimally invasive protozoal pathogen of intestinal epithelium that results in villus atrophy, mucosal lipid peroxidation, diarrhea, and diminished barrier function. Influx of neutrophils is a consistent feature of human and animal cryptosporidiosis, and yet their contribution to the pathological sequelae of infection has not been investigated. Accordingly, we used an established neonatal piglet model of C. parvum infection to examine the role of neutrophils in disease pathogenesis by inhibiting their recruitment and activation in vivo using a monoclonal anti-CD18 antibody. Infected piglets were treated daily with anti-CD18 or isotype control immunoglobulin G and euthanized at peak infection, at which time neutrophil infiltrates, lipid peroxidation, severity of infection, and intestinal barrier function were quantified. C. parvum infection resulted in a significant increase in mucosal neutrophil myeloperoxidase activity that was prevented by treatment of piglets with anti-CD18 antibody. Neutrophil recruitment was dependent on mucosal superoxide formation (prevented by treatment of infected piglets with superoxide dismutase). Neutrophils did not contribute to peroxynitrite formation or peroxidative injury of C. parvum-infected mucosa and had no impact on the severity of epithelial infection, villus atrophy, or diarrhea. The presence of neutrophils in C. parvum-infected mucosa was associated with enhanced barrier function that could not be attributed to mucosal elaboration of prostaglandins or stimulation of their synthesis. These studies are the first to demonstrate that neutrophilic inflammation arising in response to infection by a noninvasive epithelial pathogen results in physiologic rather than pathological effects in vivo.     

Determination and application of immunodominant regions of SARS coronavirus spike and nucleocapsid proteins recognized by sera from different animal species

Knowledge of immunodominant regions in major viral antigens is important for rational design of effective vaccines and diagnostic tests. Although there have been many reports of such work done for SARS-CoV, these were mainly focused on the immune responses of humans and mice. In this study, we aim to search for and compare immunodominant regions of the spike (S) and nucleocapsid (N) proteins which are recognized by sera from different animal species, including mouse, rat, rabbit, civet, pig and horse. Twelve overlapping recombinant protein fragments were produced in Escherichia coli, six each for the S and N proteins, which covered the entire coding region of the two proteins. Using a membrane-strip based Western blot approach, the reactivity of each antigen fragment against a panel of animal sera was determined. Immunodominant regions containing linear epitopes, which reacted with sera from all the species tested, were identified for both proteins. The S3 fragment (aa 402-622) and the N4 fragment (aa 220-336) were the most immunodominant among the six S and N fragments, respectively. Antibodies raised against the S3 fragment were able to block the binding of a panel of S-specific monoclonal antibodies (mAb) to SARS-CoV in ELISA, further demonstrating the immunodominance of this region. Based on these findings, one-step competition ELISAs were established which were able to detect SARS-CoV antibodies from human and at least seven different animal species. Considering that a large number of animal species are known to be susceptible to SARS-CoV, these assays will be a useful tool to trace the origin and transmission of SARS-CoV and to minimise the risk of animal-to-human transmission.     

Fertility desires and the feasibility of contraception counseling among genital fistula patients in eastern Democratic Republic of the Congo

Objective

To determine the fertility and contraceptive desires of genital fistula patients in eastern Democratic Republic of the Congo (DRC) and to evaluate the impact of contraceptive counseling and its effect on contraceptive knowledge and use.

Methods

Group contraceptive counseling was offered to fistula patients at HEAL Africa Hospital between February and May 2010. Fertility desires and contraceptive knowledge were assessed via verbally administered questionnaires before and after counseling, and use of modern contraceptive methods was tracked.

Results

Of the 61 participants, 22/34 (64.7%) of those who desired children wanted to wait at least 1 year after repair before attempting pregnancy. Overall, 31/58 (53.4%) women had heard of birth control, although only 15 (24.6%) knew any specific methods, and none had ever used contraception. After counseling, all participants could recall 1 or more methods. Of the 25 participants discharged over the subsequent 3 months, 5 (20.0%) and 3 additional fistula patients selected a modern method of contraception.

Conclusion

Desire for contraception and birth spacing among women with fistula is significant. Basic group contraception counseling and access are feasible and lead to increased contraceptive knowledge and use.     

Efficacy of Emodepside plus Praziquantel Tablets (Profender? Tablets for Dogs) against Mature and Immature Infections with Toxocara canis and Toxascaris leonina in Dogs

The efficacy of emodepside plus praziquantel tablets (Profender? tablets for dogs) against mature adult, immature adult and larval stages of Toxocara canis and Toxascaris leonina was evaluated in ten randomised, blinded and placebo-controlled dose confirmation studies in naturally or experimentally infected dogs. The tablets were used at the proposed minimum dose of 1 mg emodepside and 5 mg praziquantel per kg body weight. Efficacy was calculated based on worm counts after necropsy. Five studies demonstrated >99% efficacy against mature adult, >92% efficacy against immature adult, >98% efficacy against L4 and >94% efficacy against L3 larval stages of T. canis. Another five studies demonstrated >99% efficacy against mature and immature adult and >95% efficacy against L4 larval stages of T. leonina. No side effects of the treatment were observed. Emodepside plus praziquantel tablets thus provide a comprehensive new treatment option for ascarid infections in the dog.     

Evidence‐based selection of theories for designing behaviour change interventions: Using methods based on theoretical construct domains to understand clinicians' blood transfusion behaviour

Background Many theories of behaviour are potentially relevant to predictive and intervention studies but most studies investigate a narrow range of theories. Michie et al. (2005) agreed 12 ‘theoretical domains’ from 33 theories that explain behaviour change. They developed a ‘Theoretical Domains Interview’ (TDI) for identifying relevant domains for specific clinical behaviours, but the framework has not been used for selecting theories for predictive studies. It was used here to investigate clinicians' transfusion behaviour in intensive care units (ICU). Evidence suggests that red blood cells transfusion could be reduced for some patients without reducing quality of care. Objectives (1) To identify the domains relevant to transfusion practice in ICUs and neonatal intensive care units (NICUs), using the TDI. (2) To use the identified domains to select appropriate theories for a study predicting transfusion behaviour. Methods An adapted TDI about managing a patient with borderline haemoglobin by watching and waiting instead of transfusing red blood cells was used to conduct semi‐structured, one‐to‐one interviews with 18 intensive care consultants and neonatologists across the UK. Results Relevant theoretical domains were: knowledge, beliefs about capabilities, beliefs about consequences, social influences, behavioural regulation. Further analysis at the construct level resulted in selection of seven theoretical approaches relevant to this context: Knowledge‐Attitude‐Behaviour Model, Theory of Planned Behaviour, Social Cognitive Theory, Operant Learning Theory, Control Theory, Normative Model of Work Team Effectiveness and Action Planning Approaches. Conclusions This study illustrated, the use of the TDI to identify relevant domains in a complex area of inpatient care. This approach is potentially valuable for selecting theories relevant to predictive studies and resulted in greater breadth of potential explanations than would be achieved if a single theoretical model had been adopted.     

Surgery for perforated colorectal malignancy in an Asian population: an institution’s experience over 5 years

Introduction  

Perforated colorectal malignancy is associated with numerous peri-operative complications and dismal long-term survival. The study aimed to review the outcome and factors predicting peri-operative complications and long-term survival of patients who underwent surgery for perforated colorectal malignancy.     

Heart rate and reinforcement sensitivity in ADHD

BACKGROUND: Both theoretical and clinical accounts of attention-deficit/hyperactivity disorder (ADHD) implicate a dysfunctional reinforcement system. This study investigated heart rate parameters in response to feedback associated with reward and response cost in ADHD children and controls aged 8 to 12. METHODS: Heart rate responses (HRRs) following feedback and heart rate variability (HRV) in the low frequency band (.04-.08 Hz), a measure of mental effort, were calculated during a time production paradigm. Performance was coupled to monetary gain, loss or feedback-only in a cross-over design. RESULTS: Children with ADHD exhibited smaller HRRs to feedback compared to controls. HRV of children with ADHD decreased when performance was coupled to reward or response cost compared to feedback-only. HRV of controls was similar across conditions. CONCLUSIONS: Children with ADHD were characterised by (a) possible abnormalities in feedback monitoring and (b) motivational deficits, when no external reinforcement is present.     

Do harsh and positive parenting predict parent reports of deceitful‐callous behavior in early childhood?

Background: The relationship between parenting and the development of antisocial behavior in children is well established. However, evidence for associations between dimensions of parenting and callous‐unemotional (CU) traits is mixed. As CU traits appear critical to understanding a subgroup of youth with antisocial behavior, more research addressing the link between early parenting and CU traits is needed. Methods: The current study investigated longitudinal predictions between measures of harsh and positive parenting, and early CU behavior. Data from mother‐child dyads (N = 731; 49% female) were collected from a multi‐ethnic, high‐risk sample with young children, and included self‐reported and multi‐method observed parenting. CU behavior was assessed using a previously validated measure of deceitful‐callous behavior ( Hyde et al., 2011 ). Results: Results suggest that dimensions of harsh parenting, but not positive parenting, contribute to the development of child deceitful‐callous behavior. Nevertheless, deceitful‐callous behavior showed strong stability over time and the effects of harsh parenting, especially observed harshness, were modest. Conclusions: The current findings have implications for developmental psychopathology and early interventions for antisocial behavior. The results also raise a number of issues about measuring emerging CU behavior in very young children, including the interrelation between parent perceptions and reports of child behavior, parent reactions, and the subsequent development of severe antisocial behavior.