A Modified Delphi Study for Curricular Content of Simulation-Based Medical Education for Pediatric Residency Programs.

OBJECTIVE: We sought to establish core knowledge topics and skills that are important to teach pediatric residents using simulation-based medical education (SBME). METHODS: We conducted a modified Delphi process with experts in pediatric SBME. Content items were adapted from the American Board of Pediatrics certifying exam content and curricular components from pediatric entrustable professional activities (EPAs). In round 1, participants rated 158 items using a four-point Likert scale of importance to teach through simulation in pediatric residency. A priori, we defined consensus for item inclusion as ≥70% rated the item as extremely important and exclusion as ≥70% rated the item not important. Criteria for stopping the process included reaching consensus to include and/or exclude all items, with a maximum of three rounds. RESULTS: A total of 59 participants, representing 46 programs and 25 states participated in the study. Response rates for the three rounds were 92%, 86% and 90%, respectively. The final list includes 112 curricular content items deemed by our experts as important to teach through simulation in pediatric residency. Seventeen procedures were included. Nine of the seventeen EPAs had at least one content item that experts considered important to teach through simulation as compared to other modalities. CONCLUSIONS: Using consensus methodology, we identified the curricular items important to teach pediatric residents using SBME. Next steps are to design a simulation curriculum to encompass this content.

Scaling Access to Contraception for Youth in Urban Slums: The Challenge Initiative's Systems-Based Multi-Pronged Strategy for Youth-Friendly Cities.

INTRODUCTION: More than half of all adolescents globally live in Asia, with India having the largest adolescent population in the world at 253 million. In sub-Saharan Africa, adolescents make up the greatest proportion of the population, with 23% of the population aged 10-19. And these numbers are predicted to grow rapidly-particularly in urban areas as rural youth migrate to cities for economic opportunities. While adolescents and youth are subject to high sexual and reproductive health risks, few efforts have been documented for addressing these in urban settings, especially in poor settlements. METHODS: The Challenge Initiative (TCI) is a demand-driven, family planning platform for sustainable scale and impact that lets city governments-in particular urban slums-lead implementation. It is currently active in 11 countries in Africa and Asia. In June 2018, TCI heightened its focus on adolescent and youth sexual and reproductive health (AYSRH) for youth living in urban slums. It now supports 39 city governments. TCI dedicates technical and program support to married (including first-time parents) and unmarried youth ages 15-24 years. Using an innovative coaching model and an online learning platform (TCI University), TCI supports city governments as they implement AYSRH interventions to accelerate the impact of TCI's model for rapid scale. RESULTS: TCI has been assessing the performance of cities implementing its AYSRH approaches using its RAISE tool and has found considerable improvement over two rounds of assessments through TCI coaching and support for adaptation of its high-impact interventions between the first and second round. CONCLUSIONS: TCI's AYSRH approach scaled rapidly to 39 cities and multiple urban slums since 2018, using its evidence-based interventions and coaching model. In the context of universal health coverage, TCI has supported segmented demand generation and improved access to quality and affordable contraceptive as well as youth-friendly health services. It provides a menu of interventions for cities to implement for youth-including such approaches as public-private partnerships with pharmacies and quality assurance using quick checklists-along with an innovative coaching model. This approach has facilitated greater access to contraceptive methods of choice for youth.

CRISPR-Cas9 In Vivo Gene Editing for Transthyretin Amyloidosis.

BACKGROUND: Transthyretin amyloidosis, also called ATTR amyloidosis, is a life-threatening disease characterized by progressive accumulation of misfolded transthyretin (TTR) protein in tissues, predominantly the nerves and heart. NTLA-2001 is an in vivo gene-editing therapeutic agent that is designed to treat ATTR amyloidosis by reducing the concentration of TTR in serum. It is based on the clustered regularly interspaced short palindromic repeats and associated Cas9 endonuclease (CRISPR-Cas9) system and comprises a lipid nanoparticle encapsulating messenger RNA for Cas9 protein and a single guide RNA targeting TTR. METHODS: After conducting preclinical in vitro and in vivo studies, we evaluated the safety and pharmacodynamic effects of single escalating doses of NTLA-2001 in six patients with hereditary ATTR amyloidosis with polyneuropathy, three in each of the two initial dose groups (0.1 mg per kilogram and 0.3 mg per kilogram), within an ongoing phase 1 clinical study. RESULTS: Preclinical studies showed durable knockout of TTR after a single dose. Serial assessments of safety during the first 28 days after infusion in patients revealed few adverse events, and those that did occur were mild in grade. Dose-dependent pharmacodynamic effects were observed. At day 28, the mean reduction from baseline in serum TTR protein concentration was 52% (range, 47 to 56) in the group that received a dose of 0.1 mg per kilogram and was 87% (range, 80 to 96) in the group that received a dose of 0.3 mg per kilogram. CONCLUSIONS: In a small group of patients with hereditary ATTR amyloidosis with polyneuropathy, administration of NTLA-2001 was associated with only mild adverse events and led to decreases in serum TTR protein concentrations through targeted knockout of TTR. (Funded by Intellia Therapeutics and Regeneron Pharmaceuticals; ClinicalTrials.gov number, NCT04601051.).

Sympathetic Regulation of the NCC (Sodium Chloride Cotransporter) in Dahl Salt-Sensitive Hypertension.

Increased sympathoexcitation and renal sodium retention during high salt intake are hallmarks of the salt sensitivity of blood pressure. The mechanism(s) by which excessive sympathetic nervous system release of norepinephrine influences renal sodium reabsorption is unclear. However, studies demonstrate that norepinephrine can stimulate the activity of the NCC (sodium chloride cotransporter) and promote the development of SSH (salt-sensitive hypertension). The adrenergic signaling pathways governing NCC activity remain a significant source of controversy with opposing studies suggesting a central role of upstream α1- and ß-adrenoceptors in the canonical regulatory pathway involving WNKs (with-no-lysine kinases), SPAK (STE20/SPS1-related proline alanine-rich kinase), and OxSR1 (oxidative stress response 1). In our previous study, α1-adrenoceptor antagonism in norepinephrine-infused male Sprague-Dawley rats prevented the development of norepinephrine-evoked SSH in part by suppressing NCC activity and expression. In these studies, we used selective adrenoceptor antagonism in male Dahl salt-sensitive rats to test the hypothesis that norepinephrine-mediated activation of the NCC in Dahl SSH occurs via an α1-adrenoceptor dependent pathway. A high-salt diet evoked significant increases in NCC activity, expression, and phosphorylation in Dahl salt-sensitive rats that developed SSH. Increases were associated with a dysfunctional WNK1/4 dynamic and a failure to suppress SPAK/OxSR1 activity. α1-adrenoceptor antagonism initiated before high-salt intake or following the establishment of SSH attenuated blood pressure in part by suppressing NCC activity, expression, and phosphorylation. Collectively, our findings support the existence of a norepinephrine-activated α1-adrenoceptor gated pathway that relies on WNK/SPAK/OxSR1 signaling to regulate NCC activity in SSH.

Sympathetic regulation of NCC in norepinephrine-evoked salt-sensitive hypertension in Sprague-Dawley rats.

Salt sensitivity of blood pressure is characterized by inappropriate sympathoexcitation and renal Na+ reabsorption during high salt intake. In salt-resistant animal models, exogenous norepinephrine (NE) infusion promotes salt-sensitive hypertension and prevents dietary Na+-evoked suppression of the Na+-Cl- cotransporter (NCC). Studies of the adrenergic signaling pathways that modulate NCC activity during NE infusion have yielded conflicting results implicating α1- and/or ß-adrenoceptors and a downstream kinase network that phosphorylates and activates NCC, including with no lysine kinases (WNKs), STE20/SPS1-related proline-alanine-rich kinase (SPAK), and oxidative stress response 1 (OxSR1). In the present study, we used selective adrenoceptor antagonism in NE-infused male Sprague-Dawley rats to investigate the differential roles of α1- and ß-adrenoceptors in sympathetically mediated NCC regulation. NE infusion evoked salt-sensitive hypertension and prevented dietary Na+-evoked suppression of NCC mRNA, protein expression, phosphorylation, and in vivo activity. Impaired NCC suppression during high salt intake in NE-infused rats was paralleled by impaired suppression of WNK1 and OxSR1 expression and SPAK/OxSR1 phosphorylation and a failure to increase WNK4 expression. Antagonism of α1-adrenoceptors before high salt intake or after the establishment of salt-sensitive hypertension restored dietary Na+-evoked suppression of NCC, resulted in downregulation of WNK4, SPAK, and OxSR1, and abolished the salt-sensitive component of hypertension. In contrast, ß-adrenoceptor antagonism attenuated NE-evoked hypertension independently of dietary Na+ intake and did not restore high salt-evoked suppression of NCC. These findings suggest that a selective, reversible, α1-adenoceptor-gated WNK/SPAK/OxSR1 NE-activated signaling pathway prevents dietary Na+-evoked NCC suppression, promoting the development and maintenance of salt-sensitive hypertension.

Norepinephrine-evoked salt-sensitive hypertension requires impaired renal sodium chloride cotransporter activity in Sprague-Dawley rats.

Recent studies have implicated a role of norepinephrine (NE) in the activation of the sodium chloride cotransporter (NCC) to drive the development of salt-sensitive hypertension. However, the interaction between NE and increased salt intake on blood pressure remains to be fully elucidated. This study examined the impact of a continuous NE infusion on sodium homeostasis and blood pressure in conscious Sprague-Dawley rats challenged with a normal (NS; 0.6% NaCl) or high-salt (HS; 8% NaCl) diet for 14 days. Naïve and saline-infused Sprague-Dawley rats remained normotensive when placed on HS and exhibited dietary sodium-evoked suppression of peak natriuresis to hydrochlorothiazide. NE infusion resulted in the development of hypertension, which was exacerbated by HS, demonstrating the development of the salt sensitivity of blood pressure [MAP (mmHg) NE+NS: 151 ± 3 vs. NE+HS: 172 ± 4; P < 0.05]. In these salt-sensitive animals, increased NE prevented dietary sodium-evoked suppression of peak natriuresis to hydrochlorothiazide, suggesting impaired NCC activity contributes to the development of salt sensitivity [peak natriuresis to hydrochlorothiazide (µeq/min) Naïve+NS: 9.4 ± 0.2 vs. Naïve+HS: 7 ± 0.1; P < 0.05; NE+NS: 11.1 ± 1.1; NE+HS: 10.8 ± 0.4). NE infusion did not alter NCC expression in animals maintained on NS; however, dietary sodium-evoked suppression of NCC expression was prevented in animals challenged with NE. Chronic NCC antagonism abolished the salt-sensitive component of NE-mediated hypertension, while chronic ANG II type 1 receptor antagonism significantly attenuated NE-evoked hypertension without restoring NCC function. These data demonstrate that increased levels of NE prevent dietary sodium-evoked suppression of the NCC, via an ANG II-independent mechanism, to stimulate the development of salt-sensitive hypertension.

Severe and enduring mental health problems within an established substance misuse treatment partnership.

Aims and method The study reports findings of an investigation into the presence of severe and enduring mental health problems within the four statutory and non-statutory teams of an established substance misuse treatment partnership. Results Of a total of 772 clients in the four teams surveyed, 69 (8.9%) were identified as having severe and enduring mental health problems and problem substance use in the past 12 months. Alcohol was the most prevalent substance used by this predominantly male group. Different rates were found across the four teams, with higher numbers in the non-statutory teams. The clients displayed significant levels of self-harm and suicide risk and were responsible for 131 acute service contacts over the past 12 months. Clinical implications Clients with severe and enduring mental health problems engaged with substance misuse services display high levels of complex need. It is important to identify the best and most effective service response to this group.

A pilot study to assess the feasibility and impact of a brief motivational intervention on problem drug and alcohol use in adult mental health inpatient units: study protocol for a randomized controlled trial.

BACKGROUND: Substance misuse in those with severe mental health problems is common and associated with poor engagement in treatment and treatment outcomes. Up to 44% of those admitted into psychiatric inpatient facilities have coexisting substance-misuse problems. However, this is not routinely addressed as part of their treatment plan. A mental health admission may present a window of opportunity for inpatients to reevaluate the impact of their substance use. This study will aim to evaluate the effectiveness of a targeted brief motivational intervention in improving engagement in treatment and to assess how feasible and acceptable this intervention is to inpatients and staff as a routine intervention. METHODS/DESIGN: This randomized controlled trial will use concealed randomization; blind, independent assessment of outcome at 3 months; characterization of refusers and dropouts; and be analyzed according to the intention-to-treat principle. After baseline assessments, eligible participants will be randomized either to the Brief Integrated Motivational Intervention plus Treatment As Usual, or Treatment as Usual alone. Eligible participants will be those who are new admissions; >18 years; ICD-10 diagnosis of -schizophrenia or related disorder, bipolar affective disorder, recurrent depressive disorder, and DSM-IV diagnosis of substance abuse or dependence over the last 3 months. The primary outcome is engagement in treatment for substance misuse, and secondary outcomes include readiness to change substance misuse together with a cost-effectiveness analysis. Qualitative interviews with staff and participants will assess the acceptability of the intervention. DISCUSSION: This pilot randomized trial will provide the first robust evidence base for inpatient care of people with severe mental health problems and co-morbid substance misuse and provide the groundwork for confirmatory trials to evaluate a potentially feasible, cost-effective, and easy-to-implement treatment option that may be readily integrated into standard inpatient and community-based care. TRIAL REGISTRATION: ISRCTN43548483 Date of ISRCTN assignation: 4/17/2014.

Pediatric hematopoietic cell transplantation.

Hematopoietic cell transplantation is a potentially curative therapeutic modality for a wide range of pediatric malignant and nonmalignant diseases. Hematopoietic cell transplantation has evolved over time to include more indications for transplantation and broad-spectrum treatment and supportive care strategies. This review will address the process of transplantation, donor and stem cell sources, complications, and transplantable diseases.

Characterization of insulin degrading enzyme and other amyloid-ß degrading proteases in human serum: a role in Alzheimer's disease?

Sporadic Alzheimer's disease (AD) patients have low amyloid-ß peptide (Aß) clearance in the central nervous system. The peripheral Aß clearance may also be important but its role in AD remains unclear. We aimed to study the Aß degrading proteases including insulin degrading enzyme (IDE), angiotensin converting enzyme (ACE) and others in blood. Using the fluorogenic substrate V (a substrate of IDE and other metalloproteases), we showed that human serum degraded the substrate V, and the activity was inhibited by adding increasing dose of Aß. The existence of IDE activity was demonstrated by the inhibition of insulin, amylin, or EDTA, and further confirmed by immunocapture of IDE using monoclonal antibodies. The involvement of ACE was indicated by the ability of the ACE inhibitor, lisinopril, to inhibit the substrate V degradation. To test the variations of substrate V degradation in humans, we used serum samples from a homebound elderly population with cognitive diagnoses. Compared with the elderly who had normal cognition, those with probable AD and amnestic mild cognitive impairment (amnestic MCI) had lower peptidase activities. Probable AD or amnestic MCI as an outcome remained negatively associated with serum substrate V degradation activity after adjusting for the confounders. The elderly with probable AD had lower serum substrate V degradation activity compared with those who had vascular dementia. The blood proteases mediating Aß degradation may be important for the AD pathogenesis. More studies are needed to specify each Aß degrading protease in blood as a useful biomarker and a possible treatment target for AD.

Peroxisome proliferator-activated receptor alpha and alpha/gamma agonists do not cause impairment in renal function in the rat.

BACKGROUND/AIMS: Patients treated with peroxisome proliferator-activated receptor analogs (PPAR) alpha or alpha/gamma may develop a transient and reversible increase in serum creatinine, the mechanism of which remains obscure. This study evaluates whether treatment with either PPAR-alpha or -alpha/gamma analogs, fenofibrate or tesaglitazar, may cause deterioration in renal hemodynamics or exert direct tubular or glomerular nephrotoxic effects in rats. METHODS: Male Sprague-Dawley rats (300-320 g) were treated per os with fenofibrate (300 mg/kg/day), tesaglitazar (1.2 mg/kg/day) or vehicle, for 14 days. Glomerular filtration rate (GFR) and renal blood flow (RBF) were measured by inulin clearance and ultrasonic flowmetry, and cumulative excretion of sodium and creatinine were assessed. Biomarkers of glomerular and tubular injury were measured, including urinary albumin excretion and renal mRNA levels of kidney injury molecule 1 (Kim-1), lipocalin 2 (Lcn2), and osteopontin (Spp1). RESULTS: Fenofibrate and tesaglitazar improved the lipid profile, but caused no detectable decrease in GFR or RBF compared with vehicle-treated rats. Furthermore, the cumulative excretions of sodium and creatinine were not altered by the drugs. Finally, there was no significant difference between drug- and vehicle-treated groups in urinary albumin excretion or in the expression of renal injury biomarkers. CONCLUSIONS: In the rat, no direct nephrotoxic effect or deterioration in renal hemodynamics and function were observed following treatment with fenofibrate or tesaglitazar.

Theoretical insights into the mechanism of selective Peptide bond hydrolysis catalyzed by [Pd(H(2)O)(4)](2+).

In this study, mechanisms for the hydrolysis of the Gly-Pro bond in Gly-Pro-Met and Gly-Pro-His, the Gly-Sar bond in Gly-Sar-Met, and the Gly-Gly bond in the Gly-Gly-Met peptide catalyzed by [Pd(H(2)O)(4)](2+) (I) have been investigated at the DFT level. In all cases, the optimized structure of the active bidentate complex, formed by the reaction of I with the substrate [Pd(H(2)O)(2){(Gly)-(Pro)-(Met-kappaS,kappaN)}](1+) complex for the Gly-Pro-Met peptide, was found to exist in the trans conformation. This structure is in agreement with the experimentally measured TOCSY and ROESY (1)H NMR spectra. After the formation of this complex, the following two mechanisms have been proposed experimentally: (1) external attack mechanism and (2) internal delivery mechanism. The DFT calculations suggest that in the external attack mechanism the calculated barriers are prohibitively high (i.e., 50-70 kcal/mol) for the cleavage of all the peptide bonds, and therefore, this mechanism is ruled out. However, in the internal delivery mechanism, the bidentate complex is first transformed from the trans to the cis conformation. Here, the overall barriers for the hydrolysis of the Gly-Pro-Met, Gly-Pro-His, Gly-Sar-Met, and Gly-Gly-Met peptide bonds are 38.3, 41.4, 39.8, and 39.2 kcal/mol, respectively. These barriers are in much better agreement with the experimentally measured rate constants at pH 2.0 and at 60 degrees C. The substitution of Pd(II) with Pt(II) was found to make a negligibly small difference (0.53 kcal/mol) on the barrier for the cleavage of the Gly-Pro-His bond. These calculations indicate that after the creation of the active bidentate complex in the trans conformation the internal delivery mechanism is the most energetically feasible.

Evaluation of vacuum filter sock surface sample collection method for Bacillus spores from porous and non-porous surfaces.

Vacuum filter socks were evaluated for recovery efficiency of powdered Bacillus atrophaeus spores from two non-porous surfaces, stainless steel and painted wallboard and two porous surfaces, carpet and bare concrete. Two surface coupons were positioned side-by-side and seeded with aerosolized Bacillus atrophaeus spores. One of the surfaces, a stainless steel reference coupon, was sized to fit into a sample vial for direct spore removal, while the other surface, a sample surface coupon, was sized for a vacuum collection application. Deposited spore material was directly removed from the reference coupon surface and cultured for enumeration of colony forming units (CFU), while deposited spore material was collected from the sample coupon using the vacuum filter sock method, extracted by sonication and cultured for enumeration. Recovery efficiency, which is a measure of overall transfer effectiveness from the surface to culture, was calculated as the number of CFU enumerated from the filter sock sample per unit area relative to the number of CFU enumerated from the co-located reference coupon per unit area. The observed mean filter sock recovery efficiency from stainless steel was 0.29 (SD = 0.14, n = 36), from painted wallboard was 0.25 (SD = 0.15, n = 36), from carpet was 0.28 (SD = 0.13, n = 40) and from bare concrete was 0.19 (SD = 0.14, n = 44). Vacuum filter sock recovery quantitative limits of detection were estimated at 105 CFU m(-2) from stainless steel and carpet, 120 CFU m(-2) from painted wallboard and 160 CFU m(-2) from bare concrete. The method recovery efficiency and limits of detection established in this work provide useful guidance for the planning of incident response environmental sampling for biological agents such as Bacillus anthracis.

Evaluation of a wipe surface sample method for collection of Bacillus spores from nonporous surfaces.

Polyester-rayon blend wipes were evaluated for efficiency of extraction and recovery of powdered Bacillus atrophaeus spores from stainless steel and painted wallboard surfaces. Method limits of detection were also estimated for both surfaces. The observed mean efficiency of polyester-rayon blend wipe recovery from stainless steel was 0.35 with a standard deviation of +/-0.12, and for painted wallboard it was 0.29 with a standard deviation of +/-0.15. Evaluation of a sonication extraction method for the polyester-rayon blend wipes produced a mean extraction efficiency of 0.93 with a standard deviation of +/-0.09. Wipe recovery quantitative limits of detection were estimated at 90 CFU per unit of stainless steel sample area and 105 CFU per unit of painted wallboard sample area. The method recovery efficiency and limits of detection established in this work provide useful guidance for the planning of incident response environmental sampling following the release of a biological agent such as Bacillus anthracis.

Development of real-time PCR (TaqMan) assays for the detection and quantification of Botrytis cinerea in planta.

Real-time PCR assays based on TaqMan chemistry have been developed for the detection and quantification of Botrytis cinerea, suitable for a wide range of different host plant species. Assays were designed to the beta-tubulin gene, the intergenic spacer (IGS) region of the nuclear ribosomal DNA and also to a previously published, species-specific sequence characterised amplified region (SCAR) marker; the assays were compared to a published method based on SYBR Green I technology. The assays designed to the IGS region and SCAR marker proved to be highly specific for B. cinerea but assays designed to the beta-tubulin gene and the previously published assay designed to the cutinase-A gene both cross-react with B. fabae. The assay designed to the IGS region was the most sensitive and was able to reliably detect and quantify as little as 20 fg of B. cinerea DNA. The method incorporates the detection of a gene from the plant host to compensate for variations in extraction efficiency and size of sample tested. The assays designed were used to follow the progression of infection of B. cinerea in plant material inoculated with spores to the point of symptom induction. They should be ideally suited to investigating infection processes in-planta and could be used to investigate aspects of infection/plant pathogenesis, by B. cinerea and are particularly suited to the detection and quantification of the pathogen prior to the development of symptoms.

Cytokine preconditioning promotes codifferentiation of human fetal liver CD133+ stem cells into angiomyogenic tissue.

BACKGROUND: CD133 (AC133) is a surface antigen that defines a broad population of stem cells, including myogenic and endothelial progenitors. CD133+ cells are rare in adult tissues, and the factors that support their differentiation into mature angiomyogenic cells are not known. These hurdles have hampered the use of CD133+ cells for therapeutic purposes. Because human fetal liver is a rich source of CD133+ cells, we sought to identify the growth factors that promote codifferentiation of these cells into angiogenic and myogenic cells. METHODS AND RESULTS: Human fetal liver CD133+ and CD133- cell subpopulations were cultured with 5'-azacytidine or vascular endothelial growth factor (VEGF165) and/or brain-derived nerve growth factor (BDNF). CD133+ but not CD133- cells from human fetal liver codifferentiated into spindle-shaped cells, as well as flat adherent multinucleated cells capable of spontaneous contractions in culture. The resulting spindle-shaped cells were confirmed to be endothelial cells by immunohistochemistry analysis for von Willebrand factor and by acetylated LDL uptake. Multinucleated cells were characterized as striated muscles by electron microscopy and immunohistochemistry analysis for myosin heavy chain. Presence of VEGF165 and BDNF significantly enhanced angiomyogenesis in vitro. Inoculation of cells derived from CD133+ cells, but not CD133- cells, into the ear pinna of NOD/SCID mice resulted in the formation of cardiomyocytes, as identified by immunostaining with cardiac troponin-T antibody. These cells generated electrical action potentials, detectable by ECG tracing. CONCLUSIONS: CD133 defines a population of human fetal liver cells capable of differentiating into both angiogenic and myogenic cells. Preconditioning of these CD133+ cells with VEGF165 and BDNF enhances the angiomyogenesis. CD133+ fetal liver cells ultimately may be used for therapeutic angiomyogenesis.