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Abstract Background Polymyalgia Rheumatica (PMR) is the commonest inflammatory condition seen in older patients in primary care. To date, however, research has been focused on secondary care cohorts rather than primary care where many patients are exclusively managed. This two year prospective inception cohort study of PMR patients will enable us to understand the full spectrum of this condition. Methods Patients diagnosed with PMR in primary care will be identified via Read codes and mailed a series of postal questionnaires over a two-year period to assess their levels of pain, stiffness and functioning, as well as medication usage and other health-related and socio-demographic characteristics. In addition, participants will be asked for permission to link their survey data to their general practice electronic medical record and to national mortality and cancer registers. Discussion This will be the first large-scale, prospective, observational cohort of PMR patients in primary care. The combination of survey data with medical records and national registers will allow for a full investigation of the natural history and prognosis of this condition in the primary care setting, in which the majority of patients are treated, but where little research on the treatment and outcome of consultation has been undertaken. This will provide information that may lead to improved primary care management of PMR.

Abstract Background There is limited evidence for the clinical and cost effectiveness of occupational therapy (OT) approaches in the management of hand osteoarthritis (OA). Joint protection and hand exercises have been proposed by European guidelines, however the clinical and cost effectiveness of each intervention is unknown. This multicentre two-by-two factorial randomised controlled trial aims to address the following questions: • Is joint protection delivered by an OT more effective in reducing hand pain and disability than no joint protection in people with hand OA in primary care? • Are hand exercises delivered by an OT more effective in reducing hand pain and disability than no hand exercises in people with hand OA in primary care? • Which of the four management approaches explored within the study (leaflet and advice, joint protection, hand exercise, or joint protection and hand exercise combined) provides the most cost-effective use of health care resources Methods/Design Participants aged 50 years and over registered at three general practices in North Staffordshire and Cheshire will be mailed a health survey questionnaire (estimated mailing sample n = 9,500). Those fulfilling the eligibility criteria on the health survey questionnaire will be invited to attend a clinical assessment to assess for the presence of hand or thumb base OA using the ACR criteria. Eligible participants will be randomised to one of four groups: leaflet and advice; joint protection (looking after your joints); hand exercises; or joint protection and hand exercises combined (estimated n = 252). The primary outcome measure will be the OARSI/OMERACT responder criteria combining hand pain and disability (measured using the AUSCAN) and global improvement, 6 months post-randomisation. Secondary outcomes will also be collected for example pain, functional limitation and quality of life. Outcomes will be collected at baseline and 3, 6 and 12 months post-randomisation. The main analysis will be on an intention to treat basis and will assess the clinical and cost effectiveness of joint protection and hand exercises for managing hand OA. Discussion The findings will improve the cost-effective evidence based management of hand OA. Trial registration identifier: ISRCTN33870549

Competing Interests: Declaration of Competing Interest The authors regularly advise vaccine and preventive antibody developers and manufacturers. The views expressed here are those of the authors.

Context: Children with medical complexity (CMC) are at risk for adverse outcomes after discharge. Difficulties with comprehension of and adherence to discharge instructions contribute to these errors. Comprehensive reviews of patient-, caregiver-, provider-, and system-level characteristics and interventions associated with discharge instruction comprehension and adherence for CMC are lacking., Objective: To systematically review the literature related to factors associated with comprehension of and adherence to discharge instructions for CMC., Data Sources: PubMed/Medline, Embase, Cochrane Central Register of Controlled Trials, PsycInfo, Cumulative Index to Nursing and Allied Health Literature, Web of Science (database initiation until March 2023), and OAIster (gray literature) were searched., Study Selection: Original studies examining caregiver comprehension of and adherence to discharge instructions for CMC (Patient Medical Complexity Algorithm) were evaluated., Data Extraction: Two authors independently screened titles/abstracts and reviewed full-text articles. Two authors extracted data related to study characteristics, methodology, subjects, and results., Results: Fifty-one studies were included. More than half were qualitative or mixed methods studies. Few interventional studies examined objective outcomes. More than half of studies examined instructions for equipment (eg, tracheostomies). Common issues related to access, care coordination, and stress/anxiety. Facilitators included accounting for family context and using health literacy-informed strategies., Limitations: No randomized trials met inclusion criteria. Several groups (eg, oncologic diagnoses, NICU patients) were not examined in this review., Conclusions: Multiple factors affect comprehension of and adherence to discharge instructions for CMC. Several areas (eg, appointments, feeding tubes) were understudied. Future work should focus on design of interventions to optimize transitions., (Copyright © 2023 by the American Academy of Pediatrics.)

Synaptogenesis is essential for circuit development; however, it is unknown whether it is critical for the establishment and performance of goal-directed voluntary behaviors. Here, we show that operant conditioning via lever-press for food reward training in mice induces excitatory synapse formation onto a subset of anterior cingulate cortex neurons projecting to the dorsomedial striatum (ACC →DMS ). Training-induced synaptogenesis is controlled by the Gabapentin/Thrombospondin receptor α2δ-1, which is an essential neuronal protein for proper intracortical excitatory synaptogenesis. Using germline and conditional knockout mice, we found that deletion of α2δ-1 in the adult ACC →DMS circuit diminishes training-induced excitatory synaptogenesis. Surprisingly, this manipulation does not impact learning but results in a significant increase in effort exertion without affecting sensitivity to reward value or changing contingencies. Bidirectional optogenetic manipulation of ACC →DMS neurons rescues or phenocopies the behaviors of the α2δ-1 cKO mice, highlighting the importance of synaptogenesis within this cortico-striatal circuit in regulating effort exertion., (© 2023. Springer Nature Limited.)

Purpose: To determine the allegation, precipitating medical issue, and outcome of telephone triage focused malpractice litigation among ophthalmologists., Methods: The WestLaw Edge database was reviewed using terms pertaining to ophthalmology and telemedicine. The search ranged from 4/7/30 to 1/25/22., Results: Of the 510 lawsuits, 3.5% (18/510) met inclusion criteria. 94.5% (17/18) alleged delays in evaluation and/or treatment. 61.1% (11/18) alleged incorrect diagnoses, 38.9% (7/18) claimed improper discussion of risks or informed consent, and 5.6% (1/18) alleged delayed referrals. The precipitating medical issues included retinal detachment in 33.3% (6/18) of cases, post-procedure and post-trauma endophthalmitis in 33.3% (6/18) of cases, ocular trauma without endophthalmitis in 22.2% (4/18) of cases, and bilateral acute retinal necrosis and allergic reactions each accounting for 5.6% (1/18) of cases., Conclusion: Telephone triage creates potential malpractice litigation. Delay in in-person clinical evaluation and alleged failure to inform patients of possible irreversible vision loss may lead to potential malpractice litigation. We suggest offering the option of same day in person evaluation and informing the patient how delay may lead to irreversible vision loss.

Competing Interests: The authors have no relevant financial information or potential conflicts to disclose.

Background: Chikungunya is an arbovirus, transmitted by Aedes mosquitoes, which emerged in the Americas in 2013 and spread rapidly to almost every country on this continent. In Brazil, where the first cases were detected in 2014, it currently has reached all regions of this country and more than 900,000 cases were reported. The clinical spectrum of chikungunya ranges from an acute self-limiting form to disabling chronic forms. The purpose of this study was to estimate the seroprevalence of chikungunya infection in a large Brazilian city and investigate the association between viral circulation and living condition., Methodology/principal Findings: We conducted a population-based ecological study in selected Sentinel Areas (SA) through household interviews and a serologic survey in 2016/2017. The sample was of 1,981 individuals randomly selected. The CHIKV seroprevalence was 22.1% (17.1 IgG, 2.3 IgM, and 1.4 IgG and IgM) and varied between SA from 2.0% to 70.5%. The seroprevalence was significantly lower in SA with high living conditions compared to SA with low living condition. There was a positive association between CHIKV seroprevalence and population density (r = 0.2389; p = 0.02033)., Conclusions/significance: The seroprevalence in this city was 2.6 times lower than the 57% observed in a study conducted in the epicentre of the CHIKV epidemic of this same urban centre. So, the herd immunity in this general population, after four years of circulation of this agent is relatively low. It indicates that CHIKV transmission may persist in that city, either in endemic form or in the form of a new epidemic, because the vector infestation is persistent. Besides, the significantly lower seroprevalences in SA of higher Living Condition suggest that beyond the surveillance of the disease, vector control and specific actions of basic sanitation, the reduction of the incidence of this infection also depends on the improvement of the general living conditions of the population., Competing Interests: The authors have declared that no competing interests exist.

Background: There is growing concern about individuals reported to suffer repeat COVID-19 disease episodes, these in a small number of cases characterised as de novo infections with distinct sequences, indicative of insufficient protective immunity even in the short term., Methods: Observational case series and case-control studies reporting 33 cases of recurrent, symptomatic, qRT-PCR positive COVID-19. Recurrent disease was defined as symptomatic recurrence after symptom-free clinical recovery, with release from isolation >14 days from the beginning of symptoms confirmed by qRT-PCR. The case control study-design compared this group of patients with a control group of 62 patients randomly selected from the same COVID-19 database., Results: Of 33 recurrent COVID-19 patients, 26 were female and 30 were HCW. Mean time to recurrence was 50.5 days which was associated with being a HCW (OR 36.4 (p <0.0001)), and blood type A (OR 4.8 (p = 0.002)). SARS-CoV-2 antibodies were signifcantly lower in recurrent patients after initial COVID-19  (2.4 ± 0.610; p<0.0001) and after recurrence (6.4 ± 11.34; p = 0.007).  Virus genome sequencing identified reinfection by a different isolate in one patient., Conclusions: This is the first detailed case series showing COVID-19 recurrence with qRT-PCR positivity. For one individual detection of phylogenetically distinct genomic sequences in the first and second episodes confirmed bona fide renfection, but in most cases the data do not formally distinguish between reinfection and re-emergence of a chronic infection reservoir. These episodes were significantly associated with reduced Ab response during initial disease and argue the need for ongoing vigilance without an assumption of protection after a first episode., Competing Interests: Declaration of Competing Interests All authors report there are no conflicts of interest., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Introduction: We performed an epidemiological surveillance of the Chikungunya (CHIKV) lineages in Bahia after the 2014 East/Central/South African (ECSA) genotype outbreak., Methods: Reverse-transcription polymerase chain reaction (RT-PCR), viral isolation, and phylogenetic analyses were conducted on serum samples from 605 patients with CHIKV-like symptoms during 2014-2018., Results: Of the 605 samples, 167 were CHIKV-positive. Viral isolation was achieved for 20 samples; their phylogenetic analysis (E2 protein) revealed the presence of ECSA lineage and reinforced the phylogenetic relationship between ECSA and Indian Ocean lineages., Conclusions: The genomic surveillance of CHIKV showed that only ECSA lineage circulated in Bahia since the 2014 outbreak.

Background: Hepatocyte-like cells (iHEPs) generated by transcription factor-mediated direct reprogramming of somatic cells have been studied as potential cell sources for the development of novel therapies targeting liver diseases. The mechanisms involved in direct reprogramming, stability after long-term in vitro expansion, and safety profile of reprogrammed cells in different experimental models, however, still require further investigation., Methods: iHEPs were generated by forced expression of Foxa2/Hnf4a in mouse mesenchymal stromal cells and characterized their phenotype stability by in vitro and in vivo analyses., Results: The iHEPs expressed mixed hepatocyte and liver progenitor cell markers, were highly proliferative, and presented metabolic activities in functional assays. A progressive loss of hepatic phenotype, however, was observed after several passages, leading to an increase in alpha-SMA + fibroblast-like cells, which could be distinguished and sorted from iHEPs by differential mitochondrial content. The resulting purified iHEPs proliferated, maintained liver progenitor cell markers, and, upon stimulation with lineage maturation media, increased expression of either biliary or hepatocyte markers. In vivo functionality was assessed in independent pre-clinical mouse models. Minimal engraftment was observed following transplantation in mice with acute acetaminophen-induced liver injury. In contrast, upon transplantation in a transgenic mouse model presenting host hepatocyte senescence, widespread engraftment and uncontrolled proliferation of iHEPs was observed, forming islands of epithelial-like cells, adipocyte-like cells, or cells presenting both morphologies., Conclusion: The results have significant implications for cell reprogramming, suggesting that iHEPs generated by Foxa2/Hnf4a expression have an unstable phenotype and depend on transgene expression for maintenance of hepatocyte-like characteristics, showing a tendency to return to the mesenchymal phenotype of origin and a compromised safety profile.

Oropouche virus (OROV) is a negative-sense, single-stranded RNA arbovirus transmitted to humans by the midge Culicoides paraenesis, causing Oropouche fever. Reports of its outbreak in Brazil have so far been restricted to the Central-Northern region of the country. However, its incidence is underestimated, mainly due to its clinical similarities with other arbovirus diseases, including dengue (DENV), chikungunya (CHIKV), and zika (ZIKV), and the lack of specific diagnostic tests. Here, we report for the first time, the detection of OROV in saliva and urine samples, and cases of autochthone OROV infections in Salvador Metropolitan region, Bahia, a Northeastern capital in the coast of Brazil. Serum, saliva, and urine samples negative for DENV, CHIKV, and ZIKV were tested for OROV using a reverse transcription nested polymerase chain reaction (RT-nested-PCR) protocol, and 2 serum, 2 saliva, and 1 urine samples were positive. This report shows the need for an efficient surveillance system for controlling the spread of this virus, and suggests the use of saliva and urine as alternative samples for OROV detection in the absence of serum samples.

Chikungunya virus (CHIKV) has never been detected in human breast milk. This is a brief report of CHIKV infection in a breastfeeding woman of a 3-month-old baby. The mother's CHIKV-RT PCR was positive in serum, urine and milk. The baby's CHIKV serology and reverse transcription polimerase chain reaction (RT-PCR) were negative. The detection of CHIKV in milk raises clinical and epidemiologic questions.

Chronic Chagas disease cardiomyopathy, caused by Trypanosoma cruzi infection, is a major cause of heart failure in Latin America. Galectin-3 (Gal-3) has been linked to cardiac remodeling and poor prognosis in heart failure of different etiologies. Herein, we investigated the involvement of Gal-3 in the disease pathogenesis and its role as a target for disease intervention. Gal-3 expression in mouse hearts was evaluated during T. cruzi infection by confocal microscopy and flow cytometry analysis, showing a high expression in macrophages, T cells, and fibroblasts. In vitro studies using Gal-3 knockdown in cardiac fibroblasts demonstrated that Gal-3 regulates cell survival, proliferation, and type I collagen synthesis. In vivo blockade of Gal-3 with N-acetyl-d-lactosamine in T. cruzi-infected mice led to a significant reduction of cardiac fibrosis and inflammation in the heart. Moreover, a modulation in the expression of proinflammatory genes in the heart was observed. Finally, histological analysis in human heart samples obtained from subjects with Chagas disease who underwent heart transplantation showed the expression of Gal-3 in areas of inflammation, similar to the mouse model. Our results indicate that Gal-3 plays a role in the pathogenesis of experimental chronic Chagas disease, favoring inflammation and fibrogenesis. Moreover, by demonstrating Gal-3 expression in human hearts, our finding reinforces that this protein could be a novel target for drug development for Chagas cardiomyopathy., (Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Introduction: New therapeutic options are necessary for patients with chronic Chagas disease, a leading cause of heart failure in Latin American countries. Stem cell therapy focused on improving cardiac function is a promising approach for treating heart disease. Here, we evaluated the therapeutic effects of cardiac mesenchymal stem cells (CMSCs) in a mouse model of chronic Chagas disease., Methods: CMSCs were isolated from green fluorescent protein (GFP) transgenic C57BL/6 mouse hearts and tested for adipogenic, osteogenic, chondrogenic, endothelial, and cardiogenic differentiation potentials evaluated by histochemical and immunofluorescence techniques. A lymphoproliferation assay was performed to evaluate the immunomodulatory activity of CMSCs. To investigate the therapeutic potential of CMSCs, C57BL/6 mice chronically infected with Trypanosoma cruzi were treated with 106 CMSCs or saline (control) by echocardiography-guided injection into the left ventricle wall. All animals were submitted to cardiac histopathological and immunofluorescence analysis in heart sections from chagasic mice. Analysis by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was performed in the heart to evaluate the expression of cytokines involved in the inflammatory response., Results: CMSCs demonstrated adipogenic, osteogenic, and chondrogenic differentiation potentials. Moreover, these cells expressed endothelial cell and cardiomyocyte features upon defined stimulation culture conditions and displayed immunosuppressive activity in vitro. After intramyocardial injection, GFP+ CMSCs were observed in heart sections of chagasic mice one week later; however, no observed GFP+ cells co-expressed troponin T or connexin-43. Histopathological analysis revealed that CMSC-treated mice had a significantly decreased number of inflammatory cells, but no reduction in fibrotic area, two months after treatment. Analysis by qRT-PCR demonstrated that cell therapy significantly decreased tumor necrosis factor-alpha expression and increased transforming growth factor-beta in heart samples., Conclusions: We conclude that the CMSCs exert a protective effect in chronic chagasic cardiomyopathy primarily through immunomodulation.

Conditions under which skeletal myoblasts are cultured in vitro are critical to growth and differentiation of these cells into mature skeletal myofibers. We examined several culture conditions that promoted human skeletal myoblast (HSkM) culture and examined the effect of microRNAs and mechanical stimulation on differentiation. Culture conditions for HSkM are different from those that enable rapid C2C12 myoblast differentiation. Culture on a growth factor-reduced Matrigel (GFR-MG)-coated surface in 2% equine serum-supplemented differentiation medium to promote HSkM differentiation under static conditions was compared with culture conditions used for C2C12 cell differentiation. Such conditions led to a >20-fold increase in myogenic miR-1, miR-133a, and miR-206 expression, a >2-fold increase in myogenic transcription factor Mef-2C expression, and an increase in sarcomeric α-actinin protein. Imposing ±10% cyclic stretch at 0.5 Hz for 1 h followed by 5 h of rest over 2 wk produced a >20% increase in miR-1, miR-133a, and miR-206 expression in 8% equine serum and a >35% decrease in 2% equine serum relative to static conditions. HSkM differentiation was accelerated in vitro by inhibition of proliferation-promoting miR-133a: immunofluorescence for sarcomeric α-actinin exhibited accelerated development of striations compared with the corresponding negative control, and Western blotting showed 30% more α-actinin at day 6 postdifferentiation. This study showed that 100 μg/ml GFR-MG coating and 2% equine serum-supplemented differentiation medium enhanced HSkM differentiation and myogenic miR expression and that addition of antisense miR-133a alone can accelerate primary human skeletal muscle differentiation in vitro.

Status epilepticus (SE) is a severe clinical manifestation of epilepsy associated with intense neuronal loss and inflammation, two key factors involved in the pathophysiology of temporal lobe epilepsy. Bone marrow mononuclear cells (BMMC) attenuated the consequences of pilocarpine-induced SE, including neuronal loss, in addition to frequency and duration of seizures. Here we investigated the effects of BMMC transplanted early after the onset of SE in mice, as well as the involvement of soluble factors produced by BMMC in the effects of the cell therapy. Mice were injected with pilocarpine for SE induction and randomized into three groups: transplanted intravenously with 1 × 10(7) BMMC isolated from GFP transgenic mice, injected with BMMC lysate, and saline-treated controls. Cell tracking, neuronal counting in hippocampal subfields and cytokine analysis in the serum and brain were performed. BMMC were found in the brain 4 h following transplantation and their numbers progressively decreased until 24 h following transplantation. A reduction in hippocampal neuronal loss after SE was found in mice treated with live BMMC and BMMC lysate when compared to saline-treated, SE-induced mice. Moreover, the expression of inflammatory cytokines IL-1β, TNF-α, IL-6 was decreased after injection of live BMMC and to a lesser extent, of BMMC lysate, when compared to SE-induced controls. In contrast, IL-10 expression was increased. Analysis of markers for microglia activation demonstrated a reduction of the expression of genes related to type 1-activation. BMMC transplantation promotes neuroprotection and mediates anti-inflammatory effects following SE in mice, possibly through the secretion of soluble factors.

Genipin, the multipotent ingredient in Gardenia jasmenoides fruit extract (GFE), may be an effective candidate for treatment following stroke or traumatic brain injury (TBI). Secondary injury includes damage mediated by reactive oxygen species (ROS) and reactive nitrogen species (RNS), which can alter the biological function of key cellular structures and eventually lead to cell death. In this work, we studied the neuroprotective potential of genipin against damage stemming from ROS and RNS production in organotypic hippocampal slice cultures (OHSC), as well as its potential as a direct free radical scavenger. A 50 µM dose of genipin provided significant protection against tert-butyl hydroperoxide (tBHP), a damaging organic peroxide. This dosage of genipin significantly reduced cell death at 48 h compared to vehicle control (0.1% DMSO) when administered 0, 1, 6, and 24 h after addition of tBHP. Similarly, genipin significantly reduced cell death at 48 h when administered 0, 1, 2, and 6h after addition of rotenone, which generates reactive oxygen species via a more physiologically relevant mechanism. Furthermore, genipin significantly reduced both cell death and nitrite levels at 24 h caused by S-nitroso-N-acetylpenicillamine (SNAP), a direct nitric oxide (NO) donor, and successfully quenched 1,1-Diphenyl-2-picryl-hydrazyl (DPPH), a stable free radical, suggesting that genipin may act as a direct free radical scavenger. Our encouraging findings suggest that genipin should be tested in animal models of CNS injury with a significant component of ROS- and RNS-mediated damage, such as TBI and stroke, to assess its in vivo efficacy., (© 2013 Published by Elsevier B.V.)

Introduction: DNA methylation is part of the epigenetic regulatory mechanism present in all normal cells. It is tissue-specific and stably maintained throughout development, but often abnormally changed in cancer. Non-small-cell lung carcinoma (NSCLC) is the most deadly type of cancer, involving different tumor subtypes. This heterogeneity is a challenge for correct diagnosis and patient treatment. The stability and specificity make of DNA methylation a very suitable marker for epigenetic phenotyping of tumors., Methods: To identify candidate markers for use in NSCLC diagnosis, we used genomewide DNA methylation maps that we had previously generated by MethylCap and next-generation sequencing and listed the most significant differentially methylated regions (DMRs). The 25 DMRs with highest significance in their methylation scores were selected. The methylation status of these DMRs was investigated in 61 tumors and matching control lung tissues by methylation-specific polymerase chain reaction., Results: We found 12 novel DMRs that showed significant differences between tumor and control lung tissues. We also identified three novel DMRs for each of the two most common NSCLC subtypes, adenocarcinomas and squamous cell carcinomas. We propose a panel of five DMRs, composed of novel and known markers that exhibit high specificity and sensitivity to distinguish tumors from control lung tissues., Conclusion: Novel markers will aid the development of a highly specific epigenetic panel for accurate identification and subtyping of NSCLC tumors.

Background: Non-small cell lung carcinoma (NSCLC) is a complex malignancy that owing to its heterogeneity and poor prognosis poses many challenges to diagnosis, prognosis and patient treatment. DNA methylation is an important mechanism of epigenetic regulation involved in normal development and cancer. It is a very stable and specific modification and therefore in principle a very suitable marker for epigenetic phenotyping of tumors. Here we present a genome-wide DNA methylation analysis of NSCLC samples and paired lung tissues, where we combine MethylCap and next generation sequencing (MethylCap-seq) to provide comprehensive DNA methylation maps of the tumor and paired lung samples. The MethylCap-seq data were validated by bisulfite sequencing and methyl-specific polymerase chain reaction of selected regions., Results: Analysis of the MethylCap-seq data revealed a strong positive correlation between replicate experiments and between paired tumor/lung samples. We identified 57 differentially methylated regions (DMRs) present in all NSCLC tumors analyzed by MethylCap-seq. While hypomethylated DMRs did not correlate to any particular functional category of genes, the hypermethylated DMRs were strongly associated with genes encoding transcriptional regulators. Furthermore, subtelomeric regions and satellite repeats were hypomethylated in the NSCLC samples. We also identified DMRs that were specific to two of the major subtypes of NSCLC, adenocarcinomas and squamous cell carcinomas., Conclusions: Collectively, we provide a resource containing genome-wide DNA methylation maps of NSCLC and their paired lung tissues, and comprehensive lists of known and novel DMRs and associated genes in NSCLC.

Aim: To determine the human Y-chromosome haplogroup backgrounds of intermediate-sized variant alleles displayed by short tandem repeat (STR) loci DYS392, DYS449, and DYS385, and to evaluate the potential of each intermediate variant to elucidate new phylogenetic substructure within the human Y-chromosome haplogroup tree., Methods: Molecular characterization of lineages was achieved using a combination of Y-chromosome haplogroup defining binary polymorphisms and up to 37 short tandem repeat loci. DNA sequencing and median-joining network analyses were used to evaluate Y-chromosome lineages displaying intermediate variant alleles., Results: We show that DYS392.2 occurs on a single haplogroup background, specifically I1*-M253, and likely represents a new phylogenetic subdivision in this European haplogroup. Intermediate variants DYS449.2 and DYS385.2 both occur on multiple haplogroup backgrounds, and when evaluated within specific haplogroup contexts, delineate new phylogenetic substructure, with DYS449.2 being informative within haplogroup A-P97 and DYS385.2 in haplogroups D-M145, E1b1a-M2, and R1b*-M343. Sequence analysis of variant alleles observed within the various haplogroup backgrounds showed that the nature of the intermediate variant differed, confirming the mutations arose independently., Conclusions: Y-chromosome short tandem repeat intermediate variant alleles, while relatively rare, typically occur on multiple haplogroup backgrounds. This distribution indicates that such mutations arise at a rate generally intermediate to those of binary markers and STR loci. As a result, intermediate-sized Y-STR variants can reveal phylogenetic substructure within the Y-chromosome phylogeny not currently detected by either binary or Y-STR markers alone, but only when such variants are evaluated within a haplogroup context.

An 11-year-old child with cerebral palsy required hospital admission for respiratory distress following administration of high-frequency chest wall compression (HFCWC). The child had severe neurologic impairment and an ineffective cough effort. HFCWC is effective in treating cystic fibrosis, but its use has not been widely studied in other diagnoses. Although highly effective in mobilizing mucus, HFCWC does not assist in removing airway secretions. In the absence of an effective cough, additional devices or techniques may be required.

It is accepted that resistance of Plasmodium falciparum to chloroquine (CQ) is caused primarily by mutations in the pfcrt gene. However, a consensus has not yet been reached on the mechanism by which resistance is achieved. CQ-resistant (CQR) parasite lines accumulate less CQ than do CQ-sensitive (CQS) parasites. The CQR phenotype is complex with a component of reduced energy-dependent CQ uptake and an additional component that resembles energy-dependent CQ efflux. Here we show that the required energy input is in the form of the proton electrochemical gradient across the digestive vacuole (DV) membrane. Collapsing the DV proton gradient (or starving the parasites of glucose) results in similar levels of CQ accumulation in CQS and CQR lines. Under these conditions the accumulation of CQ is stimulated in CQR parasite lines but is reduced in CQS lines. Energy deprivation has no effect on the rate of CQ efflux from CQR lines implying that mutant PfCRT does not function as an efflux pump or active carrier. Using pfcrt-modified parasite lines we show that the entire CQ susceptibility phenotype is switched by the single K76T amino acid change in PfCRT. The efflux of CQ in CQR lines is not directly coupled to the energy supply, consistent with a model in which mutant PfCRT functions as a gated channel or pore, allowing charged CQ species to leak out of the DV.

The challenge of chronic pulmonary disease in children with neurodisability is presented for the primary care physician. Physical assessment and diagnostic tests for evaluation of chronic lung disease are reviewed. Specific respiratory management plans for these children are designed to stabilize chronic pulmonary disease and prevent acute illness with goals of maintaining family routine and stress reduction of every day living. Management and therapy options for mechanical ventilatory support due to eminent ventilatory failure are presented.

Chloroquine resistance (CQR) in Plasmodium falciparum is associated with mutations in the digestive vacuole transmembrane protein PfCRT. However, the contribution of individual pfcrt mutations has not been clarified and other genes have been postulated to play a substantial role. Using allelic exchange, we show that removal of the single PfCRT amino-acid change K76T from resistant strains leads to wild-type levels of CQ susceptibility, increased binding of CQ to its target ferriprotoporphyrin IX in the digestive vacuole and loss of verapamil reversibility of CQ and quinine resistance. Our data also indicate that PfCRT mutations preceding residue 76 modulate the degree of verapamil reversibility in CQ-resistant lines. The K76T mutation accounts for earlier observations that CQR can be overcome by subtly altering the CQ side-chain length. Together, these findings establish PfCRT K76T as a critical component of CQR and suggest that CQ access to ferriprotoporphyrin IX is determined by drug-protein interactions involving this mutant residue.

The number of adolescents who attempt or complete suicide is increasing. Risk factors range from mental disorders, to problems at school, family problems, or difficulties in establishing relationships. A further important, and too often underestimated, risk factor for adolescent suicide is the presence of suicidal behavior in the adolescent's family. We investigated 184 high school adolescents in a region in Slovenia with a high suicide rate (30/100,000/year). They were questioned by means of an anonymous questionnaire about the presence of suicidal behavior in their relatives and about the presence of suicidal thoughts, plans, and acts in themselves. The results revealed that 13% of the adolescents studied had a relative who had attempted suicide and a further 9% of the adolescents had lost a relative due to suicide. About half of all females and almost a third of males had had suicidal thoughts (differences between sexes were statistically significant: chi2 = 6.13; p < .01). Attempted suicide among relatives was positively correlated with the presence of suicidal plans among adolescents (phi = 0.15; p < .05). This correlation proved to be even stronger and statistically more significant in men when we split the sample by gender. All variables (suicidal thoughts, suicidal plans, and suicide attempts) in the adolescent males positively correlated with attempted suicide among their relatives ((phi = 0.28, p < .01; phi) = 0.26, p < .05; phi = 0.34, p < .01; respectively). Our results speak in favor of a higher risk of suicidal behavior among adolescents with suicidal behavior in their families.

Amodiaquine (AQ) (2) is a 4-aminoquinoline antimalarial that can cause adverse side effects including agranulocytosis and liver damage. The observed drug toxicity is believed to involve the formation of an electrophilic metabolite, amodiaquine quinoneimine (AQQI), which can bind to cellular macromolecules and initiate hypersensitivity reactions. We proposed that interchange of the 3' hydroxyl and the 4' Mannich side-chain function of amodiaquine would provide a new series of analogues that cannot form toxic quinoneimine metabolites via cytochrome P450-mediated metabolism. By a simple two-step procedure, 10 isomeric amodiaquine analogues were prepared and subsequently examined against the chloroquine resistant K1 and sensitive HB3 strains of Plasmodium falciparum in vitro. Several analogues displayed potent antimalarial activity against both strains. On the basis of the results of in vitro testing, isoquine (ISQ1 (3a)) (IC(50) = 6.01 nM +/- 8.0 versus K1 strain), the direct isomer of amodiaquine, was selected for in vivo antimalarial assessment. The potent in vitro antimalarial activity of isoquine was translated into excellent oral in vivo ED(50) activity of 1.6 and 3.7 mg/kg against the P. yoelii NS strain compared to 7.9 and 7.4 mg/kg for amodiaquine. Subsequent metabolism studies in the rat model demonstrated that isoquine does not undergo in vivo bioactivation, as evidenced by the complete lack of glutathione metabolites in bile. In sharp contrast to amodiaquine, isoquine (and Phase I metabolites) undergoes clearance by Phase II glucuronidation. On the basis of these promising initial studies, isoquine (ISQ1 (3a)) represents a new second generation lead worthy of further investigation as a cost-effective and potentially safer alternative to amodiaquine.

The maintenance of acidic pH in the digestive vacuole of the malaria parasite is thought to be crucial to the digestion of host cell haemoglobin and the subsequent process of heme detoxification. It may also be important in the mode of action of chloroquine and in the mechanism of resistance to the drug. Obtaining a definitive measurement of digestive vacuole pH has been surprisingly difficult. Some of the techniques for the measurement of pH in acid vesicles are outlined here along with some key aspects that are specific to malaria parasites. The use of acridine orange and dextran-tagged dyes as probes for the measurement of digestive vacuole pH has proved problematic, yet some surprising findings have emerged from work with these compounds.

We reviewed the records of 20 liver transplant patients who underwent 28 procedures [endoscopic retrograde cholangiopancreatography (ERCP)] to rule out biliary obstruction, treat bile leaks, dilate and/or stent strictures, or remove stones and debris. Three patients (two with abnormal T-tube cholangiograms and one with hyperbilirubinemia) underwent ERCP to rule out obstruction. Therapeutic ERCP (sphincterotomy with balloon dilatation or stone extraction) was successful in 16 of 17 patients, including seven of nine in whom there was resolution of bile leaks without the use of stents or surgery. Mild pancreatitis occurring in one patient was the only complication experienced that was related to ERCP. We conclude that ERCP is a safe and important modality in the medical management of biliary tract complications after orthotopic liver transplantation.

Perfusion of hearts with nitro blue tetrazolium resulted in gross formazan staining of intact myocardium, while infarcted zones and scars were unstained or were poorly stained. Subsequent perfusion with 3.7% formaldehyde solution provided a rigid specimen with well-preserved anatomical structures and landmarks suitable for detailed correlation of three-dimensional infarct distribution with clinical data. Specimens are particularly amenable to further gross dissection of mapping of lesions from interval slices, or both.

This research study of 13,878 youths indicates that religion is not by itself a very important predictor of youth substance use. It is, however, more strongly related to alcohol use than drug use. Also, fundamentalist religious groups have the lowest percentages of substance use in general, while the more liberal types of religious groups have the lowest percentages of heavy substance use. Of the six dimensions of religion used in the study to predict youth substance use, importance of religion to the subject was the most important, with church membership second, and the fundamentalism-liberalism scale of religious groups third.

Spectrograms from 2200 to 20 A of optical emissions produced by a simple lamp using fast (3-10-keV) electron impact on tungsten and tantalum targets are presented. The lamp dissipated up to 5 kW/cm(2) on the target surface, simultaneously cleaning the surface and generating bremsstrahlung in the soft x-ray region with 3-keV electron impact and longer wavelength transition radiation at the higher energy. In both the bremsstrahlung and transition radiation modes, the lamp emissions were of sufficient strength and reproducibility to qualify the two mechanisms as potential processes for use in a secondary standard light source. Calculated spectral distributions of transition radiation for normally incident electrons on tungsten, tantalum, and aluminum targets, with a viewing direction 45 degrees from the normal, are also shown.