Efficient Recombinase-Mediated Cassette Exchange in hPSCs to Study the Hepatocyte Lineage Reveals AAVS1 Locus-Mediated Transgene Inhibition

Tools for rapid and efficient transgenesis in "safe harbor" loci in an isogenic context remain important to exploit the possibilities of human pluripotent stem cells (hPSCs). We created hPSC master cell lines suitable for FLPe recombinase-mediated cassette exchange (RMCE) in the AAVS1 locus that allow generation of transgenic lines within 15 days with 100% efficiency and without random integrations. Using RMCE, we successfully incorporated several transgenes useful for lineage identification, cell toxicity studies, and gene overexpression to study the hepatocyte lineage. However, we observed unexpected and variable transgene expression inhibition in vitro, due to DNA methylation and other unknown mechanisms, both in undifferentiated hESC and differentiating hepatocytes. Therefore, the AAVS1 locus cannot be considered a universally safe harbor locus for reliable transgene expression in vitro, and using it for transgenesis in hPSC will require careful assessment of the function of individual transgenes

What is influencing the phenotype of the common homozygous polymerase-γ mutation p.Ala467Thr?

Polymerase-γ (POLG) is a major human disease gene and may account for up to 25% of all mitochondrial diseases in the UK and in Italy. To date, >150 different pathogenic mutations have been described in POLG. Some mutations behave as both dominant and recessive alleles, but an autosomal recessive inheritance pattern is much more common. The most frequently detected pathogenic POLG mutation in the Caucasian population is c.1399G>A leading to a p.Ala467Thr missense mutation in the linker domain of the protein. Although many patients are homozygous for this mutation, clinical presentation is highly variable, ranging from childhood-onset Alpers-Huttenlocher syndrome to adult-onset sensory ataxic neuropathy dysarthria and ophthalmoparesis. The reasons for this are not clear, but familial clustering of phenotypes suggests that modifying factors may influence the clinical manifestation. In this study, we collected clinical, histological and biochemical data from 68 patients carrying the homozygous p.Ala467Thr mutation from eight diagnostic centres in Europe and the USA. We performed DNA analysis in 44 of these patients to search for a genetic modifier within POLG and flanking regions potentially involved in the regulation of gene expression, and extended our analysis to other genes affecting mitochondrial DNA maintenance (POLG2, PEO1 and ANT1). The clinical presentation included almost the entire phenotypic spectrum of all known POLG mutations. Interestingly, the clinical presentation was similar in siblings, implying a genetic basis for the phenotypic variability amongst homozygotes. However, the p.Ala467Thr allele was present on a shared haplotype in each affected individual, and there was no correlation between the clinical presentation and genetic variants in any of the analysed nuclear genes. Patients with mitochondrial DNA haplogroup U developed epilepsy significantly less frequently than patients with any other mitochondrial DNA haplotype. Epilepsy was reported significantly more frequently in females than in males, and also showed an association with one of the chromosomal markers defining the POLG haplotype. In conclusion, our clinical results show that the homozygous p.Ala467Thr POLG mutation does not cause discrete phenotypes, as previously suggested, but rather there is a continuum of clinical symptoms. Our results suggest that the mitochondrial DNA background plays an important role in modifying the disease phenotype but nuclear modifiers, epigenetic and environmental factors may also influence the severity of disease

Improvement of lower extremity electrodiagnostic findings following a trial of spinal manipulation and motion-based therapy

BACKGROUND: Lumbar disc herniation is a problem frequently encountered in manual medicine. While manual therapy has shown reasonable success in symptomatic management of these cases, little information is known how manual therapy may affect the structure and function of the lumbar disc itself. In cases where lumbar disc herniation is accompanied by radicular symptoms, electrodiagnostic testing has been used to provide objective clinical information on nerve function. This report examines the treatment rendered for a patient with lower extremity neurological deficit, as diagnosed on electrodiagnostic testing. Patient was treated using spinal manipulation and exercises performed on a Pettibon Wobble Chair™, using electrodiagnostic testing as the primary outcome assessment. CASE PRESENTATION: An elderly male patient presented to a private spine clinic with right-sided foot drop. He had been prescribed an ankle-foot orthosis for this condition. All sensory, motor, and reflex findings in the right leg and foot were absent. This was validated on prior electromyography and nerve conduction velocity testing, performed by a board certified neurologist. Patient was treated using spinal manipulation twice-weekly and wobble chair exercises three times daily for 90 days total. Following this treatment, the patient was referred for follow-up electrodiagnostic studies. Significant improvements were made in these studies as well as self-rated daily function. CONCLUSION: Motion-based therapies, as part of a comprehensive rehabilitation program, may contribute to the restoration of daily function and the reversal of neurological insult as detected by electrodiagnostic testing. Electrodiagnostic testing may be a useful clinical tool to evaluate the progress of chiropractic patients with lumbar disc herniation and radicular pain syndromes

Healthy people with nature in mind

This is the final version of the article. Available from the publisher via the DOI in this record.BACKGROUND: The global disease burden resulting from climate change is likely to be substantial and will put further strain on public health systems that are already struggling to cope with demand. An up- stream solution, that of preventing climate change and associated adverse health effects, is a promising approach, which would create win-win-situations where both the environment and human health benefit. One such solution would be to apply methods of behaviour change to prompt pro-environmentalism, which in turn benefits health and wellbeing. DISCUSSION: Based on evidence from the behavioural sciences, we suggest that, like many social behaviours, pro- environmental behaviour can be automatically induced by internal or external stimuli. A potential trigger for such automatic pro-environmental behaviour would be natural environments themselves. Previous research has demonstrated that natural environments evoke specific psychological and physiological reactions, as demonstrated by self-reports, epidemiological studies, brain imaging techniques, and various biomarkers. This suggests that exposure to natural environments could have automatic behavioural effects, potentially in a pro-environmental direction, mediated by physiological reactions. Providing access and fostering exposure to natural environments could then serve as a public health tool, together with other measures, by mitigating climate change and achieving sustainable health in sustainable ecosystems. However, before such actions are implemented basic research is required to elucidate the mechanisms involved, and applied investigations are needed to explore real world impacts and effect magnitudes. As environmental research is still not sufficiently integrated within medical or public health studies there is an urgent need to promote interdisciplinary methods and investigations in this critical field. Health risks posed by anthropogenic climate change are large, unevenly distributed, and unpredictable. To ameliorate negative impacts, pro-environmental behaviours should be fostered. Potentially this could be achieved automatically through exposure to favourable natural environments, with an opportunity for cost-efficient nature-based solutions that provide benefits for both the environment and public health

Hemiarthroplasty versus angle-stable locking compression plate osteosynthesis in the treatment of three- and four-part fractures of the proximal humerus in the elderly: design of a randomized controlled trial

ABSTRACT: BACKGROUND: The optimal surgical management of dislocated three- and four-part fractures of the proximal humerus in elderly patients remains unclear. Most used techniques are hemiarthroplasty and angle-stable locking compression plate osteosynthesis. In the current literature there is no evidence available presenting superior results between hemiarthroplasty and angle-stable locking compression plate osteosynthesis in terms of speed of recovery, pain, patient satisfaction, functional outcome, quality of life or complications. METHODS/DESIGN: A randomized controlled multicenter trial will be conducted. Patients older than 60 years of age with a dislocated three- or four-part fracture of the proximal humerus as diagnosed by X-rays and CT-scans will be included. Exclusion criteria are a fracture older than 14 days, multiple comorbidity, multitrauma, a pathological fracture, previous surgery on the injured shoulder, severely deranged function caused by a previous disease, "head-split" proximal humerus fracture and unwillingness or inability to follow instructions. Participants will be randomized between surgical treatment with hemiarthroplasty and angle-stable locking compression plate osteosynthesis. Measurements will take place preoperatively and 3 months, 6 months, 9 months, 12 months and 24 months postoperatively. Primary outcome measure is speed of recovery of functional capacity of the affected upper limb using the Disabilities of Arm, Shoulder and Hand score (DASH). Secondary outcome measures are pain, patient satisfaction, shoulder function, quality of life, radiological evaluation and complications. Data will be analyzed on an intention-to-treat basis, using univariate and multivariate analyses. DISCUSSION: Both hemiarthroplasty and angle-stable locking compression plate osteosynthesis are used in the current treatment of dislocated three-and four-part fractures of the proximal humerus. There is a lack of level-1 studies comparing these two most-used surgical treatment options. This randomized controlled multicenter trial has been designed to determine which surgical treatment option provides the fastest recovery of functional capacity of the affected upper limb, and will provide better outcomes in pain, satisfaction, shoulder function, quality of life, radiological evaluation and complications. TRIAL REGISTRATION NUMBER: The trial is registered in the Netherlands Trial Registry (NTR2461)

Executive function does not predict coping with symptoms in stable patients with a diagnosis of schizophrenia

<p>Abstract</p> <p>Background</p> <p>Associations between coping with and control over psychotic symptoms were examined using the Maastricht Assessment of Coping Strategies-24, testing the hypothesis that the cognitive domain of executive functioning predicted quality and quantity of coping.</p> <p>Methods</p> <p>MACS-24 was administered to 32 individuals with a diagnosis of schizophrenia. For each of 24 symptoms, experience of distress, type of coping and the resulting degree of perceived control were assessed. Coping types were reduced to two contrasting coping categories: symptomatic coping (SC) and non-symptomatic coping (NSC; combining active problem solving, passive illness behaviour, active problem avoiding, and passive problem avoiding). Cognitive functioning was assessed using the GIT (Groninger Intelligence Test), the Zoo map (BADS: Behavioural Assessment of Dysexecutive function), Stroop-test and Trail making.</p> <p>Results</p> <p>Cognitive function was not associated with frequency of coping, nor did cognitive function differentially predict SC or NSC. Cognitive function similarly was not associated with symptom distress or level of perceived control over the symptom.</p> <p>Conclusion</p> <p>There was no evidence that cognitive function predicts quantity or quality of coping with symptoms in people with a diagnosis of schizophrenia. Variation in the realm of emotion regulation and social cognition may be more predictive of coping with psychotic symptoms.</p