Scale-up of the Internet-based Professional Learning to help teachers promote Activity in Youth (iPLAY) intervention: a hybrid type 3 implementation-effectiveness trial.

BACKGROUND: Whole-of-school programs have demonstrated success in improving student physical activity levels, but few have progressed beyond efficacy testing to implementation at-scale. The purpose of our study was to evaluate the scale-up of the 'Internet-based Professional Learning to help teachers promote Activity in Youth' (iPLAY) intervention in primary schools using the RE-AIM framework. METHODS: We conducted a type 3 hybrid implementation-effectiveness study and collected data between April 2016 and June 2021, in New South Wales (NSW), Australia. RE-AIM was operationalised as: (i) Reach: Number and representativeness of students exposed to iPLAY; (ii) Effectiveness: Impact of iPLAY in a sub-sample of students (n = 5,959); (iii) Adoption: Number and representativeness of schools that received iPLAY; (iv) Implementation: Extent to which the three curricular and three non-curricular components of iPLAY were delivered as intended; (v) Maintenance: Extent to which iPLAY was sustained in schools. We conducted 43 semi-structured interviews with teachers (n = 14), leaders (n = 19), and principals (n = 10) from 18 schools (11 from urban and 7 from rural locations) to determine program maintenance. RESULTS: Reach: iPLAY reached ~ 31,000 students from a variety of socio-economic strata (35% of students were in the bottom quartile, almost half in the middle two quartiles, and 20% in the top quartile). EFFECTIVENESS: We observed small positive intervention effects for enjoyment of PE/sport (0.12 units, 95% CI: 0.05 to 0.20, d = 0.17), perceptions of need support from teachers (0.26 units, 95% CI: 0.16 to 0.53, d = 0.40), physical activity participation (0.28 units, 95% CI: 0.10 to 0.47, d = 0.14), and subjective well-being (0.82 units, 95% CI: 0.32 to 1.32, d = 0.12) at 24-months. Adoption: 115 schools received iPLAY. IMPLEMENTATION: Most schools implemented the curricular (59%) and non-curricular (55%) strategies as intended. Maintenance: Based on our qualitative data, changes in teacher practices and school culture resulting from iPLAY were sustained. CONCLUSIONS: iPLAY had extensive reach and adoption in NSW primary schools. Most of the schools implemented iPLAY as intended and effectiveness data suggest the positive effects observed in our cluster RCT were sustained when the intervention was delivered at-scale. TRIAL REGISTRATION: ACTRN12621001132831.

[The evolution of child psychiatry emergencies: Results and reflections from a Nancy University Hospital study]. / Réflexion sur l'évolution à 10 ans des urgences pédopsychiatriques, CHRU-Nancy.

INTRODUCTION: Mental illness is one of the most common causes of disability, morbidity and mortality in childhood. According to the scientific literature, the prevalence of mental health disorders is an estimated 10% to 20% in the USA and similar results are found in France. Although primordial, outpatient care often appears insufficient with inequalities in its geographical distribution and its accessibility. These past decades have been marked by an increase in consultations for mental disorders in pediatric emergency departments. Is this trend indicative of a "defect" in the healthcare organization? Identifying the root causes of this inflation in psychiatric consultations seems of paramount importance in the improvement of healthcare policies. In France and worldwide, only a few studies deal with this subject. That is why we proposed to observe the evolution of the number of consultations for mental health reasons in the pediatric emergency department of Nancy University Hospital and to detail their characteristics. MATERIALS AND METHODS: Ancillary comparative and retrospective study (2003-2013) on minors having received a child psychiatry consultation within the pediatric emergency department of Nancy University Hospital. RESULTS: The number of consultations for mental health reasons increased by 119% (97 in 2003; 212 in 2013), while consultations for pediatrics reasons remained stable over the period studied. Consultations mainly dealt with females representing 55.6% of consultations in 2003 and 63.7% in 2013. Mean age of consultants was stable: 13.9 years (standard deviation=3.3 years) in 2003; 14.1 (2.5) years in 2013. Family structure witnessed a three-fold increase in the single-parent model. Regarding consultation motives, behavioral disorders were significantly more represented in 2013: 27.7% (RR=1.7; 95% CI 1.0-2.8; P<0.05) versus 16.5%. As far as diagnosis is concerned (ICD-10), emotional and behavioral disorders increased to 35.9% from 12.6% (RR=2.8; IC95% 1.6-5.1; P=0.0001). CONCLUSIONS: In France, as well as in other western countries, the number of visits in pediatric emergency departments for mental health reasons more than doubled over a 10-year span. This growth mostly concerned externalizing disorders as a motive for consultation. Causes for this increase are multifactorial and closely related to the prevalence of psychiatric disorders in children. Some studies showed that economic factors played a major role on mental illness during such a downturn as the financial crisis of 2007-2008. Unemployment caused by economic crises can weaken pediatric caregivers and therefore their patients. Evolution of family structure and value also explains this trend. These past decades, the two-parent model, relevant till the 1960s, has evolved to a point where single parents are more quickly overwhelmed. Family values are now focused on consensus rather than duty and hedonism has become a central value. Women are more involved in the working world which became for all a performance field. Several studies have shown that social settings where competitiveness is the norm breed externalized disorders in children by advocating short-term efficiency. Moreover, the widespread use of screens in households as well as early exposure impact the psychomotor development, decrease the amount of sleep and may be responsible for the occurrence of many psychiatric disorders. There are some epidemiological reasons too. In 1971, Omran introduced a concept called "epidemiological transition" explaining how mental health issues appeared in the limelight through to the decline of infectious and cardiovascular diseases. This phenomenon has already occurred in western countries which could explain the increase in the prevalence of psychiatric disorders. In Africa, there is evidence it may have already started. Beyond all these considerations, the increase in consultations for mental disorders in pediatric emergency departments can be explained by a change in care consumption habits. Going straight to the local emergency department, accessible on a 24/7 basis, is easier than waiting for an outpatient appointment and is also free for the have-nots lacking proper insurance coverage. Scarce resources in ambulatory care may also explain the increased recourse to emergency services. Several reports have shown a lack of child psychiatrists and their uneven geographical distribution. For example, in the US only a third of children with mental disorders receive proper care, a lack which doubled between 1997 and 2010. Despite the reason for this trend, it is important to propose a better fitting of the healthcare system to the population needs, and to improve prevention and early identification. All these changes require further collective reflection.

Testing Critical Slowing Down as a Bifurcation Indicator in a Low-Dissipation Dynamical System.

We study a two-dimensional low-dissipation nonautonomous dynamical system, with a control parameter that is swept linearly in time across a transcritical bifurcation. We investigate the relaxation time of a perturbation applied to a variable of the system and we show that critical slowing down may occur at a parameter value well above the bifurcation point. We test experimentally the occurrence of critical slowing down by applying a perturbation to the accessible control parameter and we find that this perturbation leaves the system behavior unaltered, thus providing no useful information on the occurrence of critical slowing down. The theoretical analysis reveals the reasons why these tests fail in predicting an incoming bifurcation.

[Cervicofacial nontuberculous mycobacteria in children: Clinical, microbiological and therapeutic features. A retrospective study and literature review]. / Adénites cervico-faciales à mycobactéries atypiques de l'enfant : caractéristiques cliniques, microbiologiques et thérapeutiques. Étude rétrospective et revue de la littérature.

BACKGROUND: Nontuberculous mycobacteria (NTM) is a common cause of lymphadenitis. A rise in incidence has been reported. Our main aim was to describe the clinical features, microbiological aspects and treatment of the disease. METHODS: We conducted a retrospective, monocentric study between January 2008 and December 2017 (University Hospital of Nantes). INCLUSION CRITERIA: age<18 years, 1 positive lymph node specimen with identification of the species in culture, head-and-neck localization. RESULTS: Forty-nine patients were enrolled from 2008 to 2017. Median age was 28 months (range: 6-141 months). Median time to confirmation of diagnosis was 2.1 months (range: 0.7-6 months). The sites encountered were mandibular (45%), cervical (33%), and parotid (16%). The main clinical signs were a tender nodule (70%), purplish nodule (59%) or painless nodule (83%), without fever (88%). The species identified were: Mycobacterium avium (n=26), M. lentiflavum (n=13), M. intracellulare (n=7), M. malmoense (n=2) and M. scrofulaceum (n=1). Antibiotic treatment was frequent (77% of cases). DISCUSSION: This study is the second largest French cohort of NTM lymphadenitis in children with microbiological confirmation. The most frequent presentation was a tender, purplish, and painless mandibular nodule. The predominant species was M. avium. M. lentiflavum, which emerged during our study, does not figure in any European studies before 2014 but appears in the most recent studies. The effects of discontinuation of mandatory BCG immunization in France in NMT is not statistically demonstrable here due to lack of relevant data prior to 2007. CONCLUSION: A possible diagnosis of NTM lymphadenitis should not be overlooked in children presenting painless, purplish, cervicofacial tumefaction.

[Interest and difficulties in setting up pharmaceutical reconciliations for patients with dressings for complex wounds]. / Intérêts et difficultés de mise en place de conciliations pharmaceutiques pour les patients porteurs de pansements pour plaies complexes.

OBJECTIVES: The management of wounds is a commonplace activity in a patient's healthcare pathway. The involvement of the pharmacist in the management of complex dressings can help strengthen the continuity of this care thanks to the pharmaceutical reconciliation. The objective of this study was to improve the quality of information transmitted between the city and the hospital regarding complex wound dressings. METHODS: This is a prospective study consisting of two groups in three services (medicine, diabetology, vascular surgery): the control group corresponded to a classic patient care and in the intervention group, the pharmacists performed dressing reconciliations. A follow-up of the patients after coming back home was realized with healthcare professionals of city involved. RESULTS: Twenty patients were included in the control group and 19 in the intervention group. Entry conciliation has improved the quality and quantity of information on wounds transmitted between the city and the hospital. Exit conciliation has increased from 60 to 100% wound and dressing output prescriptions. One hundred percent of nurses surveyed were satisfied with the patients care. CONCLUSIONS: Reconciliation would improve information transmitted between the city and the hospital and avoid a break in the continuity of complex wounds cares. However, the time dedicated and the adhesion of the care services were difficulties encountered. This study is the first highlighting the interest of medical device reconciliation and could allow reconciliation extension toward other medical devices.

Clinical value of circulating endothelial cells and of soluble CD146 levels in patients undergoing surgery for non-small cell lung cancer.

BACKGROUND: Previous studies indicate that endothelial injury, as demonstrated by the presence of circulating endothelial cells (CECs), may predict clinical outcome in cancer patients. In addition, soluble CD146 (sCD146) may reflect activation of angiogenesis. However, no study has investigated their combined clinical value in patients undergoing resection for non-small cell lung cancer (NSCLC). METHODS: Data were collected from preoperative blood samples from 74 patients who underwent resection for NSCLC. Circulating endothelial cells were defined, using the CellSearch Assay, as CD146+CD105+CD45-DAPI+. In parallel, sCD146 was quantified using an ELISA immunoassay. These experiments were also performed on a group of 20 patients with small-cell lung cancer, 60 healthy individuals and 23 patients with chronic obstructive pulmonary disease. RESULTS: The CEC count and the plasma level of sCD146 were significantly higher in NSCLC patients than in the sub-groups of controls (P<0.001). Moreover, an increased CEC count was associated with higher levels of sCD146 (P=0.010). Both high CEC count and high sCD146 plasma level at baseline significantly correlated with shorter progression-free survival (P<0.001, respectively) and overall survival (P=0.005; P=0.009) of NSCLC patients. CONCLUSIONS: The present study provides supportive evidence to show that both a high CEC count and a high sCD146 level at baseline correlate with poor prognosis and may be useful for the prediction of clinical outcome in patients undergoing surgery for NSCLC.

The Intersection of Growth Mindset and Accreditation in Pharmacy Education.

OBJECTIVES: To describe existing growth mindset literature within pharmacy and health care education, describe how a growth mindset can be beneficial in the accreditation process, and propose potential ways to promote a growth mindset in faculty, preceptors, students, and staff within pharmacy education. FINDINGS: To help pharmacy learners develop a growth mindset, existing literature emphasizes the need for a shift toward and aligning assessment with a growth mindset, helping to create self-directed adaptive learners, leading to health care providers who can adjust their practice to tackle expected and unexpected challenges throughout their careers. Strategies to create a culture of growth mindset identified include training faculty and learners on growth mindset and developing new assessments that track a learner's growth. Recommendations for pharmacy educators include encouraging educators to assess their own growth mindset and use a variety of teaching methods and provide feedback on learner effort that encourages the process of learning rather than focusing on individual attributes, traits, and results. SUMMARY: Growth mindset intersects with accreditation standards for both professional degree programs and providers of continuing pharmacy education. Continuing professional development process is one way to encourage faculty, staff, and students to develop a growth mindset. While a growth mindset can have many positive impacts on pharmacy accreditation, it is essential to recognize that achieving and maintaining accreditation is a multifaceted process involving numerous factors. A growth mindset can positively influence pharmacy education accreditation by fostering a culture of continuous improvement, innovation, resilience, student-centeredness, data-driven decision-making, collaboration, and effective leadership.

Determinants of beta-lactam PK/PD target attainment in critically ill patients: A single center retrospective study.

PURPOSE: We aimed to identify factors associated with achieving target BL plasma concentrations and describe real world data for therapeutic drug monitoring (TDM). METHODS: A retrospective single center study was conducted. We collected data from patients admitted to ICU with at least one BL TDM. We assessed the proportion of patients attaining the recommended plasma concentrations (i.e 100%fT > 4 to 8 MIC). Univariate and multivariate analyses was performed to identify the determinants of BL target attainment. RESULTS: 156 patients were included. At the first dosing, 34% achieved target BL plasma concentrations, 50% were overdosed, and 16% were underdosed. Median time for 1st TDM were 4 (SD = 2.9) days. Multivariate analysis revealed that CKD-EPI estimated glomerular filtration rate (OR = 1.02; CI [1.01; 1.03]; p < 0.0001) and total body weight (OR = 1.03; CI [1.01; 1.04]; p = 0.0048) were the main determinant of BL target attainment. Conversely, Continuous Renal Replacement Therapy (OR = 0.28; CI [0.09; 0.89]; p = 0.0318) and meropenem use (OR = 0.31; CI [0.14; 0.69]; p = 0.0041) were identified as risk factors for overdosing. No factor was associated with underdosing. CONCLUSION: Achieving target BL plasma concentrations remains challenging in ICUs. Identifying predictive factors of BL target attainment would favor implementing rapid dosing optimization strategies in both under and overdosing high risk patients.

Auditory performance after cochlear reimplantation.

AIM: The aim of this study was to evaluate the impact of cochlear reimplantation (CR) on hearing performance in children and adults with severe to profound hearing loss. MATERIAL AND METHODS: Retrospective observational study. OBJECTIVES: The main objective of this study was to determine whether there was a difference in hearing performance before and after CR. Secondary objectives were to analyze reasons for CR; to assess correlations between auditory performance and complete electrode reinsertion during CR, age, gender, explantation-to-CR interval, and interval between first implantation and CR; and to assess difference in APCEI score and the French evaluation protocol for implanted patients before and after CR. RESULTS: Comparison of speech perception scores before and after explantation-reimplantation showed no significant difference (P>0.005) at 1 year or at 2 years after CR. In 80% of cases, reimplantation was due to hard implant failure. In other cases, it was undertaken for soft failure (diminished performance but no abnormalities on integrity testing) (8%), medical reasons (6%), or undetermined reasons (6%). There was no significant correlation between auditory performance at 1 or 2 years and complete or incomplete reinsertion of electrodes, age, gender, explantation-to-CR interval, or interval between first implantation and CR (P>0.005). For the adult subgroup, the French evaluation protocol scores did not differ after reimplantation (P=0.62). Likewise, for the child sub-group, APCEI and CAP results did not deteriorate after reimplantation. CONCLUSION: Reimplantation had no negative impact on hearing and speech perception, but provided performance equivalent to or better than after initial implantation.

Long-term follow-up after biliary stent placement for postcholecystectomy bile duct strictures: a multicenter study.

BACKGROUND AND STUDY AIMS: Endoscopic stenting is a recognized treatment of postcholecystectomy biliary strictures. Large multicenter reports of its long-term efficacy are lacking. Our aim was to analyze the long-term outcomes after stenting in this patient population, based on a large experience from several centers in France. METHODS: Members of the French Society of Digestive Endoscopy were asked to identify patients treated for a common bile duct postcholecystectomy stricture. Patients with successful stenting and follow-up after removal of stent(s) were subsequently included and analyzed. Main outcome measures were long-term success of endoscopic stenting and related predictors for recurrence (after one stenting period) or failure (at the end of follow-up). RESULTS: A total of 96 patients were eligible for inclusion. The mean number of stents inserted at the same time was 1.9±0.89 (range 1-4). Stent-related morbidity was 22.9% (n=22). The median duration of stenting was 12 months (range 2-96 months). After a mean follow-up of 6.4±3.8 years (range 0-20.3 years) the overall success rate was 66.7% (n=64) after one period of stenting and 82.3% (n=79) after additional treatments. The mean time to recurrence was 19.7±36.6 months. The most significant independent predictor of both recurrence and failure was a pathological cholangiography at the time of stent removal. CONCLUSION: Endoscopic stenting helps to avoid surgery in more than 80% of patients bearing postcholecystectomy common bile duct strictures. However, a persistent anomaly on cholangiography at the time of stent removal is a strong predictor of recurrence and may lead to consideration of surgery.

Protection of chimpanzees from high-dose heterologous HIV-1 challenge by DNA vaccination.

Novel approaches for the generation of more effective vaccines for HIV-1 are of significant importance. In this report we analyze the immunogenicity and efficacy of an HIV-1 DNA vaccine encoding env, rev and gag/pol in a chimpanzee model system. The immunized animals developed specific cellular and humoral immune responses. Animals were challenged with a heterologous chimpanzee titered stock of HIV-1 SF2 virus and followed for 48 weeks after challenge. Polymerase chain reaction coupled with reverse transcription (RT-PCR) results indicated infection in the control animal, whereas those animals vaccinated with the DNA constructs were protected from the establishment of infection. These studies serve as an important benchmark for the use of DNA vaccine technology for the production of protective immune responses.

AAVrh.10-mediated genetic delivery of bevacizumab to the pleura to provide local anti-VEGF to suppress growth of metastatic lung tumors.

Vascular endothelial growth factor (VEGF) produced by tumor cells has a central role in stimulating angiogenesis required for tumor growth. Humanized monoclonal anti-VEGF antibody (bevacizumab, Avastin), approved as a treatment for non-squamous, non-small cell lung cancer, requires administration every 3 weeks. We hypothesized that an intrapleural administration of an adeno-associated virus (AAV) vector expressing an anti-VEGF-A antibody equivalent of bevacizumab would result in sustained anti-VEGF-A localized expression within the lung and suppress metastatic tumor growth. The AAV vector AAVrh.10alphaVEGF encodes the light chain and heavy chain complementary DNAs of monoclonal antibody A.4.6.1, a murine antibody that specifically recognizes human VEGF-A with the same antigen-binding site as bevacizumab. A metastatic lung tumor model was established in severe combined immunodeficient mice by intravenous administration of human DU145 prostate carcinoma cells. Intrapleural administration of AAVrh.10alphaVEGF directed long-term expression of the anti-human VEGF-A antibody in lung, as shown by sustained, high-level anti-human VEGF titers in lung epithelial lining fluid for 40 weeks, which was the duration of the study. In the AAVrh.10alphaVEGF-treated animals, tumor growth was significantly suppressed (P<0.05), the numbers of blood vessels and mitotic nuclei in the tumor was decreased (P<0.05) and there was increased survival (P<0.05). Thus, intrapleural administration of an AAVrh.10 vector, encoding the murine monoclonal antibody equivalent of bevacizumab, effectively suppresses the growth of metastatic lung tumors, suggesting AAV-mediated gene transfer to the pleura to deliver bevacizumab locally to the lung as a novel alternative platform to conventional monoclonal antibody therapy.

Affinity maturation of an anti-V antigen IgG expressed in situ through adenovirus gene delivery confers enhanced protection against Yersinia pestis challenge.

Genetic transfer of neutralizing antibodies (Abs) has been shown to confer strong and persistent protection against bacterial and viral infectious agents. Although it is well established that for many exogenous neutralizing Abs increased antigen affinity correlates with protection, the effect of antigen affinity on Abs produced in situ after adenoviral gene transfer has not been examined. The mouse IgG2b monoclonal Ab, 2C12.4, recognizes the Yersinia pestis type III secretion apparatus protein, LcrV (V antigen), and confers protection in mice when administered as an IgG intraperitoneally or after genetic immunization with engineered, replication-defective serotype 5 human adenovirus (Ad). The 2C12.4 Ab was expressed as a single-chain variable fragment (scFv) in Escherichia coli and was shown to display an equilibrium dissociation constant (K(D))=3.5 nM by surface plasmon resonance analysis. The 2C12.4 scFv was subjected to random mutagenesis, and variants with increased affinity were isolated by flow cytometry using the anchored periplasmic expression bacterial display system. After a single round of mutagenesis, variants displaying up to 35-fold lower K(D) values (H8, K(D)=100 pM) were isolated. The variable domains of the H8 scFv were used to replace those of the parental 2C12.4 IgG encoded in the Ad vector, AdalphaV, giving rise to AdalphaV.H8. The two adenoviral vectors resulted in similar titers of anti-V antigen Abs 3 days after immunization, with 10(9), 10(10) or 10(11) particle units (pu). After intranasal challenge with 363 LD(50) (lethal dose, 50%) of Y. pestis CO92, 54% of the mice immunized with 10(10) pu of AdalphaV.H8 survived through the 14 day end point compared with only 15% survivors for the group immunized with AdalphaV expressing the lower-affinity 2C12.4 (P<0.04; AdalphaV versus AdalphaV.H8). These results indicate that affinity maturation of a neutralizing Ab delivered by genetic transfer may confer increased protection not only for Y. pestis challenge but also possibly for other pathogens.

[Congenital cyst and fistula of the face and neck]. / Kystes et fistules congénitaux de la face et du cou.

The authors propose an update on cervicofacial congenital cysts and fistulas' symptomatology. Embryological data, epidemiology and clinical manifestations are described. A reminder of the therapeutic principles is proposed as well as the evolution of these congenital pathologies, which may or may not involve the branchial system. branchial.

Reduced progression of bone erosion in cytomegalovirus seropositive rheumatoid arthritis patients.

BACKGROUND: Human cytomegalovirus (HCMV) seropositivity has been associated with higher inflammation during rheumatoid arthritis (RA). However, no data are available on the impact of HCMV seropositivity on bone erosion progression during RA. METHODS: We selected 487 individuals of ESPOIR cohort who fulfilled the 2010 ACR/EULAR criteria for RA. HCMV serology for these patients was determined using Architect CMV IgG assay. Baseline and 1-year central X-ray reading using modified Total Sharp Score (mTSS), Erosion Sharp Score, and joint space narrowing Sharp score were used to quantify structural damage progression. We performed univariate and multivariate analyses to investigate the association between HCMV status and bone erosion progression. RESULTS: We analyzed 273 HCMV seropositive (HCMV+) and 214 HCMV seronegative (HCMV-) RA patients. At inclusion, HCMV+ patients were less frequently ACPA+ (49.8% versus 58.9%, p < 0.0465) and had a higher DAS28-ESR (5.55 ± 1.24 versus 5.20 ± 1.14, p < 0.0013) in comparison with HCMV-. At 1 year, bone erosion progression (delta erosion Sharp score > 1 point) was lower in HCMV+ patients (16.1% versus 25.2%, p = 0.0128) in comparison with HCMV-. HCMV+ status remained independently associated with lower bone erosion progression in multivariate analysis. CONCLUSIONS: Our findings suggest that, independently of other confounding factors, HCMV seropositivity is associated with a lower progression of bone erosion during RA.

MLH1 mediates PARP-dependent cell death in response to the methylating agent N-methyl-N-nitrosourea.

BACKGROUND: Methylating agents such as N-methyl-N-nitrosourea (MNU) can cause cell cycle arrest and death either via caspase-dependent apoptosis or via a poly(ADP-ribose) polymerase (PARP)-dependent form of apoptosis. We wished to investigate the possible role of MLH1 in signalling cell death through PARP. METHODS: Fibroblasts are particularly dependent on a PARP-mediated cell death response to methylating agents. We used hTERT-immortalised normal human fibroblasts (WT) to generate isogenic MLH1-depleted cells, confirmed by quantitative PCR and western blotting. Drug resistance was measured by clonogenic and cell viability assays and effects on the cell cycle by cell sorting. Damage signalling was additionally investigated using immunostaining. RESULTS: MLH1-depleted cells were more resistant to MNU, as expected. Despite having an intact G(2)/M checkpoint, the WT cells did not initially undergo cell cycle arrest but instead triggered cell death directly by PARP overactivation and nuclear translocation of apoptosis-inducing factor (AIF). The MLH1-depleted cells showed defects in this pathway, with decreased staining for phosphorylated H2AX, altered PARP activity and reduced AIF translocation. Inhibitors of PARP, but not of caspases, blocked AIF translocation and greatly decreased short-term cell death in both WT and MLH1-depleted cells. This MLH1-dependent response to MNU was not blocked by inhibitors of ATM/ATR or p53. CONCLUSION: These novel data indicate an important role for MLH1 in signalling PARP-dependent cell death in response to the methylating agent MNU.

Normal FGF23 levels in adult idiopathic phosphate diabetes.

Fibroblast growth factor 23 (FGF23), a recently discovered phosphaturic substance playing a key role in genetic and oncogenic phosphate diabetes, is involved in the physiological regulation of phosphate metabolism. Moderate idiopathic phosphate diabetes (IPD) leading to male osteoporosis and diffuse pain resembling fibromyalgia has been described. The aim of our study was to define the role of FGF23 in the mechanism of IPD. The study concerned 29 patients with IPD, mean age 53 +/- 11 years, of whom 72% were men. Fifteen subjects without bone disease and with normal serum phosphate and calcium levels were used as controls. Phosphate diabetes was confirmed by phosphate reabsorption level <85% and phosphate reabsorption threshold (TmPO4/GFR) <0.83. Known causes of phosphate diabetes were excluded. Fasting level of FGF23, serum phosphate, 1-25(OH)2D3, and parathyroid hormone were measured in patients and compared with FGF23 and serum phosphate in healthy controls. Spinal and hip bone mineral density (BMD) were measured by osteodensitometry. Sixteen of 29 patients had diffuse pain, 10 had osteoporosis according to the World Health Organization criteria, and 11 had osteopenia. Serum phosphate was significantly lower in patients than in controls, but FGF23 levels did not differ. Compared to patients with normal bone status, patients with osteopenia and osteoporosis had significantly decreased FGF23 levels, whereas serum phosphate was identical in the two groups. In all patients, serum phosphate and FGF23 were positively correlated and FGF23 and 1-25(OH)2D3 were negatively correlated. FGF23 seems not be a cause of IPD, and the FGF23/phosphate/1-25(OH)2D3 axis appeared to be functional.

Structural integrity of hepatocyte tight junctions.

The significance of discontinuities frequently found in freeze-fracture replicas of the tight junction was evaluated using complementary replicas of hepatocyte junctions from control and bile duct-ligated rats. An extensive analysis of complementary replicas using rotary platinum shadowing indicates that discontinuities in the protoplasmic (P) fracture face do not represent structural breaks in the tight-junctional network. In no case did P-face discontinuities correspond with interruptions in the groove network on the complementary extracellular (E) face. Quantitative analysis of replicas shows that P-face discontinuities result in part from "transfer" of material to the complementary E face (approximately 7% of the junctional length). However, many P-face discontinuities (7-30% of the junctional length) are matched only by a groove on the complementary E face. This finding demonstrates that a significant amount of material can be lost during freeze-fracture. An analysis of junctions from bile duct-ligated rats, which are known to have an increased paracellular permeability, shows comparable transfer and loss of material. However, the number of junctional elements and the tight-junction network density was significantly reduced by bile duct ligation. These observations indicate that discontinuities in tight-junctional elements result during the preparation of freeze-fracture replicas and are not physiologically important features of the junctional barrier. Variation in the number of elements provides the best explanation for observed differences in tight-junction permeability.

Localization of Na+,K+-ATPase alpha-subunit to the sinusoidal and lateral but not canalicular membranes of rat hepatocytes.

Controversy has recently developed over the surface distribution of Na+,K+-ATPase in hepatic parenchymal cells. We have reexamined this issue using several independent techniques. A monoclonal antibody specific for the endodomain of alpha-subunit was used to examine Na+,K+-ATPase distribution at the light and electron microscope levels. When cryostat sections of rat liver were incubated with the monoclonal antibody, followed by either rhodamine or horseradish peroxidase-conjugated goat anti-mouse secondary, fluorescent staining or horseradish peroxidase reaction product was observed at the basolateral surfaces of hepatocytes from the space of Disse to the tight junctions bordering bile canaliculi. No labeling of the canalicular plasma membrane was detected. In contrast, when hepatocytes were dissociated by collagenase digestion, Na+,K+-ATPase alpha-subunit was localized to the entire plasma membrane. Na+,K+-ATPase was quantitated in isolated rat liver plasma membrane fractions by Western blots using a polyclonal antibody against Na+,K+-ATPase alpha-subunit. Plasma membranes from the basolateral domain of hepatocytes possessed essentially all of the cell's estimated Na+,K+-ATPase catalytic activity and contained a 96-kD alpha-subunit band. Canalicular plasma membrane fractions, defined by their enrichment in alkaline phosphatase, 5' nucleotidase, gamma-glutamyl transferase, and leucine aminopeptidase had no detectable Na+,K+-ATPase activity and no alpha-subunit band could be detected in Western blots of these fractions. We conclude that Na+,K+-ATPase is limited to the sinusoidal and lateral domains of hepatocyte plasma membrane in intact liver. This basolateral distribution is consistent with its topology in other ion-transporting epithelia.

Structural and functional polarity of canalicular and basolateral plasma membrane vesicles isolated in high yield from rat liver.

A method has been developed for routine high yield separation of canalicular (cLPM) from basolateral (blLPM) liver plasma membrane vesicles of rat liver. Using a combination of rate zonal floatation (TZ-28 zonal rotor, Sorvall) and high speed centrifugation through discontinuous sucrose gradients, 9-16 mg of cLPM and 15-28 mg of blLPM protein can be isolated in 1 d. cLPM are free of the basolateral markers Na+/K+-ATPase and glucagon-stimulatable adenylate cyclase activities, but are highly enriched with respect to homogenate in the "canalicular marker" enzyme activities leucylnaphthylamidase (48-fold), gamma-glutamyl-transpeptidase (60-fold), 5'-nucleotidase (64-fold), alkaline phosphatase (71-fold), Mg++-ATPase (83-fold), and alkaline phosphodiesterase I (116-fold). In contrast, blLPM are 34-fold enriched in Na+/K+-ATPase activity, exhibit considerable glucagon-stimulatable adenylate cyclase activity, and demonstrate a 4- to 15-fold increase over homogenate in the various "canalicular markers." cLPM have a twofold higher content of sialic acids, cholesterol; and sphingomyelin compared with blLPM. At least three canalicular-(130,000, 100,000, and 58,000 mol wt) and several basolateral-specific protein bands have been detected after SDS PAGE of the two LPM subfractions. Specifically, the immunoglobin A-binding secretory component is restricted to blLPM as demonstrated by immunochemical techniques. These data indicate virtually complete separation of basolateral from canalicular LPM and demonstrate multiple functional and compositional polarity between the two surface domains of hepatocytes.