Genetic and clinical determinants of abdominal aortic diameter: genome-wide association studies, exome array data and Mendelian randomization study.

Progressive dilation of the infrarenal aortic diameter is a consequence of the ageing process and is considered the main determinant of abdominal aortic aneurysm (AAA). We aimed to investigate the genetic and clinical determinants of abdominal aortic diameter (AAD). We conducted a meta-analysis of genome-wide association studies in 10 cohorts (n = 13 542) imputed to the 1000 Genome Project reference panel including 12 815 subjects in the discovery phase and 727 subjects [Partners Biobank cohort 1 (PBIO)] as replication. Maximum anterior-posterior diameter of the infrarenal aorta was used as AAD. We also included exome array data (n = 14 480) from seven epidemiologic studies. Single-variant and gene-based associations were done using SeqMeta package. A Mendelian randomization analysis was applied to investigate the causal effect of a number of clinical risk factors on AAD. In genome-wide association study (GWAS) on AAD, rs74448815 in the intronic region of LDLRAD4 reached genome-wide significance (beta = -0.02, SE = 0.004, P-value = 2.10 × 10-8). The association replicated in the PBIO1 cohort (P-value = 8.19 × 10-4). In exome-array single-variant analysis (P-value threshold = 9 × 10-7), the lowest P-value was found for rs239259 located in SLC22A20 (beta = 0.007, P-value = 1.2 × 10-5). In the gene-based analysis (P-value threshold = 1.85 × 10-6), PCSK5 showed an association with AAD (P-value = 8.03 × 10-7). Furthermore, in Mendelian randomization analyses, we found evidence for genetic association of pulse pressure (beta = -0.003, P-value = 0.02), triglycerides (beta = -0.16, P-value = 0.008) and height (beta = 0.03, P-value < 0.0001), known risk factors for AAA, consistent with a causal association with AAD. Our findings point to new biology as well as highlighting gene regions in mechanisms that have previously been implicated in the genetics of other vascular diseases.

Using ex vivo bioengineered lungs to model pathologies and screening therapeutics: A proof-of-concept study.

Respiratory diseases, claim over eight million lives annually. However, the transition from preclinical to clinical phases in research studies is often hindered, partly due to inadequate representation of preclinical models in clinical trials. To address this, we conducted a proof-of-concept study using an ex vivo model to identify lung pathologies and to screen therapeutics in a humanized rodent model. We extracted and decellularized mouse heart-lung tissues using a detergent-based technique. The lungs were then seeded and cultured with human cell lines (BEAS-2B, A549, and Calu3) for 6-10 days, representing healthy lungs, cancerous states, and congenital pathologies, respectively. By manipulating cultural conditions and leveraging the unique characteristics of the cell lines, we successfully modeled various pathologies, including advanced-stage solid tumors and the primary phase of SARS-CoV-2 infection. Validation was conducted through histology, immunofluorescence staining, and pathology analysis. Additionally, our study involved pathological screening of the efficacy and impact of key anti-neoplastic therapeutics (Cisplatin and Wogonin) in cancer models. The results highlight the versatility and strength of the ex vivo model in representing crucial lung pathologies and screening therapeutics during the preclinical phase. This approach holds promise for bridging the gap between preclinical and clinical research, aiding in the development of effective treatments for respiratory diseases, including lung cancer.

Generation of infant- and pediatric-derived urinary induced pluripotent stem cells competent to form kidney organoids.

BACKGROUND: Human induced pluripotent stem cells (iPSCs) are a promising tool to investigate pathogenic mechanisms underlying human genetic conditions, such as congenital anomalies of the kidney and urinary tract (CAKUT). Currently, iPSC-based research in pediatrics is limited by the invasiveness of cell collection. METHODS: Urine cells (UCs) were isolated from pediatric urine specimens, including bag collections, and reprogrammed using episomal vectors into urinary iPSCs (UiPSCs). Following iPSC-quality assessment, human kidney organoids were generated. RESULTS: UCs were isolated from 71% (12/17) of single, remnant urine samples obtained in an outpatient setting (patients 1 month-17 years, volumes 10-75 ml). Three independent UCs were reprogrammed to UiPSCs with early episome loss, confirmed pluripotency and normal karyotyping. Subsequently, these UiPSCs were successfully differentiated into kidney organoids, closely resembling organoids generated from control fibroblast-derived iPSCs. Importantly, under research conditions with immediate sample processing, UC isolation was successful 100% for target pediatric CAKUT patients and controls (11/11) after at most two urine collections. CONCLUSIONS: Urine in small volumes or collected in bags is a reliable source for reprogrammable somatic cells that can be utilized to generate kidney organoids. This constitutes an attractive approach for patient-specific iPSC research involving infants and children with wide applicability and a low threshold for participation.

Divergent reprogramming routes lead to alternative stem-cell states.

Pluripotency is defined by the ability of a cell to differentiate to the derivatives of all the three embryonic germ layers: ectoderm, mesoderm and endoderm. Pluripotent cells can be captured via the archetypal derivation of embryonic stem cells or via somatic cell reprogramming. Somatic cells are induced to acquire a pluripotent stem cell (iPSC) state through the forced expression of key transcription factors, and in the mouse these cells can fulfil the strictest of all developmental assays for pluripotent cells by generating completely iPSC-derived embryos and mice. However, it is not known whether there are additional classes of pluripotent cells, or what the spectrum of reprogrammed phenotypes encompasses. Here we explore alternative outcomes of somatic reprogramming by fully characterizing reprogrammed cells independent of preconceived definitions of iPSC states. We demonstrate that by maintaining elevated reprogramming factor expression levels, mouse embryonic fibroblasts go through unique epigenetic modifications to arrive at a stable, Nanog-positive, alternative pluripotent state. In doing so, we prove that the pluripotent spectrum can encompass multiple, unique cell states.

Genome-wide characterization of the routes to pluripotency.

Somatic cell reprogramming to a pluripotent state continues to challenge many of our assumptions about cellular specification, and despite major efforts, we lack a complete molecular characterization of the reprograming process. To address this gap in knowledge, we generated extensive transcriptomic, epigenomic and proteomic data sets describing the reprogramming routes leading from mouse embryonic fibroblasts to induced pluripotency. Through integrative analysis, we reveal that cells transition through distinct gene expression and epigenetic signatures and bifurcate towards reprogramming transgene-dependent and -independent stable pluripotent states. Early transcriptional events, driven by high levels of reprogramming transcription factor expression, are associated with widespread loss of histone H3 lysine 27 (H3K27me3) trimethylation, representing a general opening of the chromatin state. Maintenance of high transgene levels leads to re-acquisition of H3K27me3 and a stable pluripotent state that is alternative to the embryonic stem cell (ESC)-like fate. Lowering transgene levels at an intermediate phase, however, guides the process to the acquisition of ESC-like chromatin and DNA methylation signature. Our data provide a comprehensive molecular description of the reprogramming routes and is accessible through the Project Grandiose portal at http://www.stemformatics.org.

Myocardial bridging is associated with exercise-induced ventricular arrhythmia and increases in QT dispersion.

BACKGROUND: A myocardial bridge (MB) has been associated with ventricular arrhythmia and sudden death during exercise. QT dispersion (QTd) is a measure of abnormal repolarization and may predict ventricular arrhythmia. We investigated the frequency of ventricular arrhythmias during exercise and the QTd at rest and after exercise, in patients with an MB compared to a normal cohort. METHODS: We studied the rest and stress ECG tracings of patients with an MB suspected by focal septal buckling on exercise echocardiography (EE) (Echo-MB group, N = 510), those with an MB confirmed by another examination (MB group, N = 110), and healthy controls (Control group, N = 198). RESULTS: The frequency of exercise-induced premature ventricular contractions (PVCs) was significantly higher in the Echo-MB and MB groups compared with the Control group (both p < .001). In all, 25 patients (4.9%) in the Echo-MB group, seven patients (6.4%) in the MB group and no patients in the Control group had exercise-induced non-sustained ventricular tachycardia (NSVT). There was no difference in the baseline QTd between the groups. In the Echo-MB and MB groups, QTd postexercise increased significantly when compared with baseline (both p < .001). Patients with NSVT had a higher frequency of male gender and an even greater increase in QTd with exercise compared with the non-NSVT group. DISCUSSION: There is an increased frequency of exercise-induced PVCs and NSVT in patients with MBs. Exercise significantly increases QTd in MB patients, with an even greater increase in QTd in MB patients with NSVT. Exercise in MB patients results in ventricular arrhythmias and abnormalities in repolarization.

Producing DFT/MM enzyme reaction trajectories from SCC-DFTB/MM driving forces to probe the underlying electronics of a glycosyltransferase reaction.

The SCC-DFTB/MIO/CHARMM free energy surface for a glycosyltransferase, TcTS, is benchmarked against a DFT/MM reaction trajectory using the same CHARMM MM force field ported to the NWChem package. The popular B3LYP functional, against which the MIO parameter set was parameterized is used to optimize TS structures and run DFT reaction dynamics. A novel approach was used to generate reaction forces from a SCC-DFTB/MIO/CHARMM reaction surface to drive B3LYP/6-31G/MM and B3LYP/6-31G(d)/MM reaction trajectories. Although TS structures compare favorably, differences stemming primarily from a minimal basis set approximation prevented a successful 6-31G(d) FEARCF reaction dynamics trajectory. None the less, the dynamic evolution of the B3LYP/6-31G/MM-computed electron density provided an opportunity to perform NBO analysis along the reaction trajectory. Here, we illustrate that a successful ab initio reaction trajectory is computationally accessible when the underlying potential energy function of the semi-empirical method used to produce driving forces is sufficiently close to the ab initio potential. © 2017 Wiley Periodicals, Inc.

AML cells have low spare reserve capacity in their respiratory chain that renders them susceptible to oxidative metabolic stress.

Mitochondrial respiration is a crucial component of cellular metabolism that can become dysregulated in cancer. Compared with normal hematopoietic cells, acute myeloid leukemia (AML) cells and patient samples have higher mitochondrial mass, without a concomitant increase in respiratory chain complex activity. Hence these cells have a lower spare reserve capacity in the respiratory chain and are more susceptible to oxidative stress. We therefore tested the effects of increasing the electron flux through the respiratory chain as a strategy to induce oxidative stress and cell death preferentially in AML cells. Treatment with the fatty acid palmitate induced oxidative stress and cell death in AML cells, and it suppressed tumor burden in leukemic cell lines and primary patient sample xenografts in the absence of overt toxicity to normal cells and organs. These data highlight a unique metabolic vulnerability in AML, and identify a new therapeutic strategy that targets abnormal oxidative metabolism in this malignancy.

Myocardial Bridges on Coronary Computed Tomography Angiography　- Correlation With Intravascular Ultrasound and Fractional Flow Reserve.

BACKGROUND: Myocardial bridges (MB) are commonly seen on coronary CT angiography (CCTA) in asymptomatic individuals, but in patients with recurrent typical angina symptoms, yet no obstructive coronary artery disease (CAD), evaluation of their potential hemodynamic significance is clinically relevant. The aim of this study was to compare CCTA to invasive coronary angiography (ICA), including intravascular ultrasound (IVUS), to confirm MB morphology and estimate their functional significance in symptomatic patients.Methods and Results:We retrospectively identified 59 patients from our clinical databases between 2009 and 2014 in whom the suspicion for MB was raised by symptoms of recurrent typical angina in the absence of significant obstructive CAD on ICA. All patients underwent CCTA, ICA and IVUS. MB length and depth by CCTA agreed well with length (0.6±23.7 mm) and depth (CT coverage) as seen on IVUS. The product of CT length and depth (CT coverage), (MB muscle index (MMI)), ≥31 predicted an abnormal diastolic fractional flow reserve (dFFR) ≤0.76 with a sensitivity and specificity of 74% and 62% respectively (area under the curve=0.722). CONCLUSIONS: In patients with recurrent symptoms of typical angina yet no obstructive CAD, clinicians should consider dynamic ischemia from an MB in the differential diagnosis. The product of length and depth (i.e., MMI) by CCTA may provide some non-invasive insight into the hemodynamic significance of a myocardial bridge, as compared with invasive assessment with dFFR.

Management of Suspected Fluid Balance Issues in Participants of Wilderness Endurance Events.

Dehydration and exercise-associated hyponatremia (EAH) are both relatively common conditions during wilderness endurance events. Whereas dehydration is treated with fluids, EAH is appropriately managed with fluid restriction and a sodium bolus but can worsen with isotonic or hypotonic fluids. Therefore, caution is recommended in the provision of postevent rehydration in environments where EAH is a potential consideration because accurate field assessment of hydration status can be challenging, and measurement of blood sodium concentration is rarely possible in the wilderness. Dehydration management with oral rehydration is generally adequate and preferred to intravenous rehydration, which should be reserved for athletes with sustained orthostasis or inability to tolerate oral fluid ingestion after some rest. In situations where intravenous hydration is initiated without known blood sodium concentration or hydration status, an intravenous concentrated sodium solution should be available in the event of acute neurological deterioration consistent with the development of EAH encephalopathy.

Is it Appropriate for Patients to be Discharged at the Scene by Paramedics?

BACKGROUND: Outcomes of patients who are discharged at the scene by paramedics are not fully understood. OBJECTIVE: We aimed to describe the risk of re-presentation and/or death in prehospital patients discharged at the scene. METHODS: We conducted a retrospective cohort study using linked ambulance, emergency department (ED), and death data. We compared outcomes in patients who were discharged at the scene by paramedics with those who were transported to ED by paramedics and then discharged from ED between January 1 and December 31, 2013 in metropolitan Perth, Western Australia. Occurrences of subsequent ambulance requests, ED attendance, hospital admission and death were compared between those discharged at the scene and those discharged from ED. RESULTS: There were 47,330 patients during the study period, of whom 19,732 and 27,598 patients were discharged at the scene and from ED, respectively. Compared to those discharged from ED, those discharged at the scene were more likely to subsequently: request an ambulance (6.1% vs. 1.8%, adjusted odds ratio [adj OR] 3.4; 95% confidence interval [CI] 3.0-3.9), attend ED (4.6% vs. 1.4%, adj OR 3.3; 95% CI 2.8-3.8), be admitted to hospital (3.3% vs. 0.8%, adj OR 4.2; 95% CI 3.4-5.1). Those discharged at the scene tended towards an increased likelihood of death (0.2% vs. 0.1%, adj OR 1.8; 95% CI 0.99-3.2) within 24 hours of discharge compared to those discharged from ED. CONCLUSION: Patients attended by paramedics who were discharged at the scene had more subsequent events than those who were transported to and discharged from ED. Further consideration needs to be given to who is suitable to be discharged at the scene by paramedics.

Exercise-associated hyponatremic encephalopathy in an endurance open water swimmer.

Exercise-associated hyponatremia and its more serious form, known as exercise-associated hyponatremic encephalopathy, are recognized as some of the most important medical problems seen in a variety of different forms of endurance exercise. We describe a case of exercise-associated hyponatremic encephalopathy presenting as altered conscious state and seizures in a woman who had completed a 20-km open ocean swim. Her serum sodium measured approximately 1 hour after her seizure was 119 mmol/L on point-of-care testing. With ongoing critical care support and the use of hypertonic saline, she was able to be extubated the next day, neurologically intact, and ultimately was discharged from hospital without neurological sequelae. This case emphasizes both the importance of considering exercise-associated hyponatremic encephalopathy as a cause of neurological impairment in all athletes and the pivotal role of hypertonic saline in the treatment of this condition.

Resequencing and clinical associations of the 9p21.3 region: a comprehensive investigation in the Framingham heart study.

BACKGROUND: 9p21.3 is among the most strongly replicated regions for cardiovascular disease. There are few reports of sequencing the associated 9p21.3 interval. We set out to sequence the 9p21.3 region followed by a comprehensive study of genetic associations with clinical and subclinical cardiovascular disease and its risk factors, as well as with copy number variation and gene expression, in the Framingham Heart Study (FHS). METHODS AND RESULTS: We sequenced 281 individuals (94 with myocardial infarction, 94 with high coronary artery calcium levels, and 93 control subjects free of elevated coronary artery calcium or myocardial infarction), followed by genotyping and association in >7000 additional FHS individuals. We assessed genetic associations with clinical and subclinical cardiovascular disease, risk factor phenotypes, and gene expression levels of the protein-coding genes CDKN2A and CDKN2B and the noncoding gene ANRIL in freshly harvested leukocytes and platelets. Within this large sample, we found strong associations of 9p21.3 variants with increased risk for myocardial infarction, higher coronary artery calcium levels, and larger abdominal aorta diameters and no evidence for association with traditional cardiovascular disease risk factors. No common protein-coding variation, variants in splice donor or acceptor sites, or copy number variation events were observed. By contrast, strong associations were observed between genetic variants and gene expression, particularly for a short isoform of ANRIL and for CDKN2B. CONCLUSIONS: Our thorough genomic characterization of 9p21.3 suggests common variants likely account for observed disease associations and provides further support for the hypothesis that complex regulatory variation affecting ANRIL and CDKN2B gene expression may contribute to increased risk for clinically apparent and subclinical coronary artery disease and aortic disease.

The transfer of host MHC class I protein protects donor cells from NK cell and macrophage-mediated rejection during hematopoietic stem cell transplantation and engraftment in mice.

Human hematopoietic stem cell engraftment has been studied extensively using xenograft transplant models with immunocompromised mice. It is standard practice to incorporate mouse models, such as the limiting dilution assay, to accurately assess the number of repopulating stem cells in bone marrow or umbilical cord blood collections or to confirm the long-term repopulating ability of cultured hematopoietic stem cells. In a previous study using a standard NOD/SCID mouse model to assess human hematopoietic stem cell engraftment we observed that all human cells had mouse MHC class I protein on their surface, suggesting that this is a mechanism adopted by the cells to evade host immune surveillance. To determine whether this was a xenograft phenomenon we studied host MHC transfer in an intraspecies mouse model and observed similar results. The transfer of MHC class I proteins has implications for antigen presentation and immune modulation. In this report, we used a standard mouse model of bone marrow transplantation to demonstrate that surface protein transfer between cells plays an important role in protecting donor hematopoietic cells from NK cell and macrophage-mediated rejection. The transfer of intact MHC class I antigens from host cells to transplanted donor cells confers a self identity on these otherwise foreign cells. This gives them the ability to evade detection by the host NK cells and macrophages. Once full donor chimerism is established, transplanted cells no longer require host MHC class I protein transfer to survive.

The need for salt: does a relationship exist between cystic fibrosis and exercise-associated hyponatremia?

Salt replacement is often recommended to prevent exercise-associated hyponatremia (EAH) despite a lack of evidence to support such practice. Exercise-associated hyponatremia is known to be a complex process resulting from the interplay of hydration, arginine vasopressin, and sodium balance. Although evidence suggests overhydration is the dominant pathophysiologic factor in most cases, the contributions of sweat sodium losses remain unclear. A theoretical genetic mechanism producing exuberant sweat sodium loss in athletes is the presence of cystic fibrosis (CF) gene. Individuals with CF develop hypovolemic hyponatremia by sodium loss via sweat through a defective chloride ion transport channel, the CF transmembrane conductance regulator (CFTR). Elevated sweat sodium concentrations in CF single heterozygotes suggest that athletes developing EAH may be CFTR carriers. We targeted the 2010 and 2011 Western States Endurance Run ultramarathon, an event where athletes with EAH regularly present in a hypovolemic state, for a cohort maximizing the potential to document such a relationship. A total of 798 runners started the 2010 (n = 423) and 2011 (n = 375) races. Of the 638 finishers, 373 were screened for EAH by blood draw, 60 (16%) were found to have EAH, and 31 (alpha = 0.05 for n = 9) reported their CF result from a saliva-based genetic testing kit. Neither the 31 EAH-positive athletes nor the 25 EAH-negative comparison cohort athletes tested positive for a CF mutation. This null relationship suggests that CFTR mutations are not associated with the development of EAH and that salt supplementation is unnecessary for such a reason.

Wilderness Medical Society practice guidelines for treatment of exercise-associated hyponatremia: 2014 update.

Exercise-associated hyponatremia (EAH) is defined by a serum or plasma sodium concentration below the normal reference range of 135 mmol/L that occurs during or up to 24 hours after prolonged physical activity. It is reported to occur in individual physical activities or during organized endurance events conducted in austere environments in which medical care is limited and often not available, and patient evacuation to definitive care is often greatly delayed. Rapid recognition and appropriate treatment are essential in the severe form to ensure a positive outcome. Failure in this regard is a recognized cause of event-related fatality. In an effort to produce best practice guidelines for EAH in the austere environment, the Wilderness Medical Society convened an expert panel. The panel was charged with the development of evidence-based guidelines for management of EAH. Recommendations are made regarding the situations when sodium concentration can be assessed in the field and when these values are not known. These recommendations are graded on the basis of the quality of supporting evidence and balance between the benefits and risks/burdens for each parameter according to the methodology stipulated by the American College of Chest Physicians. This is an updated version of the original WMS Practice Guidelines for Treatment of Exercise-Associated Hyponatremia published in Wilderness & Environmental Medicine 2013;24(3):228-240.

Efficient Generation of Pancreatic Progenitor Cells from Induced Pluripotent Stem Cells Derived from a Non-Invasive and Accessible Tissue Source-The Plucked Hair Follicle.

The advent of induced pluripotent stem cell (iPSC) technology has brought about transformative advancements in regenerative medicine, offering novel avenues for disease modeling, drug testing, and cell-based therapies. Patient-specific iPSC-based treatments hold the promise of mitigating immune rejection risks. However, the intricacies and costs of producing autologous therapies present commercial challenges. The hair follicle is a multi-germ layered versatile cell source that can be harvested at any age. It is a rich source of keratinocytes, fibroblasts, multipotent stromal cells, and the newly defined Hair Follicle-Associated Pluripotent Stem Cells (HAP). It can also be obtained non-invasively and transported via regular mail channels, making it the ideal starting material for an autologous biobank. In this study, cryopreserved hair follicle-derived iPSC lines (HF-iPS) were established through integration-free vectors, encompassing a diverse cohort. These genetically stable lines exhibited robust expression of pluripotency markers, and showcased tri-lineage differentiation potential. The HF-iPSCs effectively differentiated into double-positive cKIT+/CXCR4+ definitive endoderm cells and NKX6.1+/PDX1+ pancreatic progenitor cells, affirming their pluripotent attributes. We anticipate that the use of plucked hair follicles as an accessible, non-invasive cell source to obtain patient cells, in conjunction with the use of episomal vectors for reprogramming, will improve the future generation of clinically applicable pancreatic progenitor cells for the treatment of Type I Diabetes.