Mutations in DARS2 result in global dysregulation of mRNA metabolism and splicing.

Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) is a rare neurological disorder caused by the mutations in the DARS2 gene, which encodes the mitochondrial aspartyl-tRNA synthetase. The objective of this study was to understand the impact of DARS2 mutations on cell processes through evaluation of LBSL patient stem cell derived cerebral organoids and neurons. We generated human cerebral organoids (hCOs) from induced pluripotent stem cells (iPSCs) of seven LBSL patients and three healthy controls using an unguided protocol. Single cells from 70-day-old hCOs were subjected to SMART-seq2 sequencing and bioinformatic analysis to acquire high-resolution gene and transcript expression datasets. Global gene expression analysis demonstrated dysregulation of a number of genes involved in mRNA metabolism and splicing processes within LBSL hCOs. Importantly, there were distinct and divergent gene expression profiles based on the nature of the DARS2 mutation. At the transcript level, pervasive differential transcript usage and differential spliced exon events that are involved in protein translation and metabolism were identified in LBSL hCOs. Single-cell analysis of DARS2 (exon 3) showed that some LBSL cells exclusively express transcripts lacking exon 3, indicating that not all LBSL cells can benefit from the "leaky" nature common to splice site mutations. At the gene- and transcript-level, we uncovered that dysregulated RNA splicing, protein translation and metabolism may underlie at least some of the pathophysiological mechanisms in LBSL. To confirm hCO findings, iPSC-derived neurons (iNs) were generated by overexpressing Neurogenin 2 using lentiviral vector to study neuronal growth, splicing of DARS2 exon 3 and DARS2 protein expression. Live cell imaging revealed neuronal growth defects of LBSL iNs, which was consistent with the finding of downregulated expression of genes related to neuronal differentiation in LBSL hCOs. DARS2 protein was downregulated in iNs compared to iPSCs, caused by increased exclusion of exon 3. The scope and complexity of our data imply that DARS2 is potentially involved in transcription regulation beyond its canonical role of aminoacylation. Nevertheless, our work highlights transcript-level dysregulation as a critical, and relatively unexplored, mechanism linking genetic data with neurodegenerative disorders.

Prognostic value of plasma circulating tumor DNA fraction across four common cancer types: a real-world outcomes study.

BACKGROUND: Genomic analysis of circulating tumor DNA (ctDNA) is increasingly incorporated into the clinical management of patients with advanced cancer. Beyond tumor profiling, ctDNA analysis also can enable calculation of circulating tumor fraction (TF), which has previously been found to be prognostic. While most prognostic models in metastatic cancer are tumor type specific and require significant patient-level data, quantification of TF in ctDNA has the potential to serve as a pragmatic, tumor-agnostic prognostic tool. PATIENTS AND METHODS: This study utilized a cohort of patients in a nationwide de-identified clinico-genomic database with metastatic castration-resistant prostate cancer (mCRPC), metastatic breast cancer (mBC), advanced non-small-cell lung cancer (aNSCLC), or metastatic colorectal cancer (mCRC) undergoing liquid biopsy testing as part of routine care. TF was calculated based on single-nucleotide polymorphism aneuploidy across the genome. Clinical, disease, laboratory, and treatment data were captured from the electronic health record. Overall survival (OS) was evaluated by TF level while controlling for relevant covariables. RESULTS: A total of 1725 patients were included: 198 mCRPC, 402 mBC, 902 aNSCLC, and 223 mCRC. TF ≥10% was highly correlated with OS in univariable analyses for all cancer types: mCRPC [hazard ratio (HR) 3.3, 95% confidence interval (CI) 2.04-5.34, P < 0.001], mBC (HR 2.4, 95% CI 1.71-3.37, P < 0.001), aNSCLC (HR 1.68, 95% CI 1.34-2.1, P < 0.001), and mCRC (HR 2.11, 95% CI 1.39-3.2, P < 0.001). Multivariable assessments of TF had similar point estimates and CIs, suggesting a consistent and independent association with survival. Exploratory analysis showed that TF remained consistently prognostic across a wide range of cutpoints. CONCLUSIONS: Plasma ctDNA TF is a pragmatic, independent prognostic biomarker across four advanced cancers with potential to guide clinical conversations around expected treatment outcomes. With further prospective validation, ctDNA TF could be incorporated into care paradigms to enable precision escalation and de-escalation of cancer therapy based on patient-level tumor biology.

Observed psychopathology in offspring of parents with major depressive disorder, bipolar disorder and schizophrenia.

BACKGROUND: Children of parents with mood and psychotic disorders are at elevated risk for a range of behavioral and emotional problems. However, as the usual reporter of psychopathology in children is the parent, reports of early problems in children of parents with mood and psychotic disorders may be biased by the parents' own experience of mental illness and their mental state. METHODS: Independent observers rated psychopathology using the Test Observation Form in 378 children and youth between the ages of 4 and 24 (mean = 11.01, s.d. = 4.40) who had a parent with major depressive disorder, bipolar disorder, schizophrenia, or no history of mood and psychotic disorders. RESULTS: Observed attentional problems were elevated in offspring of parents with major depressive disorder, bipolar disorder and schizophrenia (effect sizes ranging between 0.31 and 0.56). Oppositional behavior and language/thought problems showed variable degrees of elevation (effect sizes 0.17 to 0.57) across the three high-risk groups, with the greatest difficulties observed in offspring of parents with bipolar disorder. Observed anxiety was increased in offspring of parents with major depressive disorder and bipolar disorder (effect sizes 0.19 and 0.25 respectively) but not in offspring of parents with schizophrenia. CONCLUSIONS: Our results suggest that externalizing problems and cognitive and language difficulties may represent a general manifestation of familial risk for mood and psychotic disorders, while anxiety may be a specific marker of liability for mood disorders. Observer assessment may improve early identification of risk and selection of youth who may benefit from targeted prevention.

Translational challenges in advancing regenerative therapy for treating neurological disorders using nanotechnology.

The focus of regenerative therapies is to replace or enrich diseased or injured cells and tissue in an attempt to replenish the local environment and function, while slowing or halting further degeneration. Targeting neurological diseases specifically is difficult, due to the complex nature of the central nervous system, including the difficulty of bypassing the brain's natural defense systems. While cell-based regenerative therapies show promise in select tissues, preclinical and clinical studies have been largely unable to transfer these successes to the brain. Advancements in nanotechnologies have provided new methods of central nervous system access, drug and cell delivery, as well as new systems of cell maintenance and support that may bridge the gap between regenerative therapies and the brain. In this review, we discuss current regenerative therapies for neurological diseases, nanotechnology as nanocarriers, and the technical, manufacturing, and regulatory challenges that arise from inception to formulation of nanoparticle-regenerative therapies.

The role of therapy and service animals in the lives of persons with disabilities.

Over the past 20 years, a great deal of attention has been paid to the psychological and physiological impact that animals have on the lives of people with disabilities. The focus of this paper is to demystify the mechanisms that are affected as a consequence of these interactions as well as to describe the value of various types of therapeutic working animals. For human-animal interactions to become more legitimised, more rigorous empirical research needs to be undertaken to demonstrate the value of such interactions as well as the need for changes in the public policy impacting them.

Depuis une vingtaine d'années, l'impact psychologique et physiologique des animaux sur la qualité de vie des personnes handicapées suscite un grand intérêt. Le présent article vise à démystifier les mécanismes touchés par les effets de ces interactions tout en décrivant l'apport concret des différents types d'animaux thérapeutiques. Afin que les interactions humains­animaux gagnent en légitimité, il convient de mener des recherches empiriques rigoureuses afin de démontrer l'importance de ces interactions ainsi que la nécessité de modifier les politiques publiques les concernant.

En el curso de los últimos 20 años se ha venido prestando gran atención a los efectos, a la vez psicológicos y fisiológicos, que los animales ejercen en la vida de las personas con discapacidad. El autor se centra en desentrañar los mecanismos que se ven influidos por estas interacciones y en describir la utilidad que revisten diversos tipos de animales de trabajo terapéutico. Para conferir mayor legitimidad a la interacción de las personas con los animales es preciso llevar a cabo investigaciones empíricas más rigurosas, que sirvan para demostrar el interés de este tipo de relaciones y la necesidad de modificar las políticas públicas que repercuten en ellas.

Examining predictors of help-seeking behaviours in patients with mood and anxiety symptoms.

Relatively little is known about individual characteristics that factor into the decision to seek help for mood and anxiety symptoms. The current study was undertaken in order to examine factors that predict the likelihood of seeking help for mood and anxiety symptoms amongst a clinical population. Patients (N = 278) referred to a tertiary care clinic in Toronto, Canada were asked about their help-seeking behaviours (HSB) through initial intake assessments and self-administered questionnaires, including the Beck Anxiety Inventory, Anxiety Sensitivity Index, Intolerance of Uncertainty Scale, and Beck Depression Inventory-II. Correlates of anxiety and depression were examined to determine whether they could predict HSB amongst individuals with Generalized Anxiety Disorder with or without comorbid Major Depressive Disorder, as well as Panic Disorder and Social Anxiety Disorder. Psychiatric diagnoses were then examined to determine whether comorbidity and demographic factors impacted HSB. Results indicated that there were significant differences in anxiety and depression correlating mainly with anxiety sensitivity, as a predictor of HSB, and that there is a complex relationship between disorder type and demographic variables. The implications of these findings and suggested targeted interventions are discussed.

Genetic testing for BRCA1 and BRCA2 in the Province of Ontario.

In 2001, genetic testing for BRCA1 and BRCA2 was introduced in Ontario, for women at high-risk of breast or ovarian cancer. To date over 30,000 individuals have been tested throughout Ontario. Testing was offered to all Ontario residents who were eligible under any of 13 criteria. We report the results of tests conducted at Mount Sinai Hospital from 2007 to 2014. A total of 4726 individuals were tested, 764 (16.2%) were found to carry a pathogenic variant (mutation). Among 3684 women and men who underwent testing without a known familial BRCA mutation, 331 (9.0%) were found to carry a mutation. Among 1042 women and men tested for a known family mutation, 433 (41.6%) were positive. There were 603 female mutation carriers, of these, 303 were affected with breast or ovarian cancer (50%) and 16 with another cancer (2.3%). Of 284 unaffected female carriers, 242 (85%) were tested for a known family mutation and 42 (15%) were the first person in the family to be tested. By placing greater emphasis on recruiting unaffected female relatives of known mutation carriers for testing, greater than one-half of newly identified carriers will be unaffected.

Specific systemic lupus erythematosus disease manifestations in the six months prior to conception are associated with similar disease manifestations during pregnancy.

Past studies have focused on aggregate lupus disease activity during pregnancy and have produced conflicting results. Our study evaluated lupus activity based on involvement of five specific organ systems during the six months prior to conception and during pregnancy. We assessed 147 pregnancies among 113 women followed at Brigham and Women's Lupus Center, 1990-2013. Organ-specific activity included hematologic disorder, nephritis, skin disease, arthritis, and serositis. We hypothesized that the presence of organ-specific activity six months prior to conception would increase the risk for that same type of activity during pregnancy. Our study population was 68% white; 100% had a positive ANA and 30% had a history of nephritis. Among women with organ-specific lupus activity during the six months before conception, the crude odds for the same type of activity during pregnancy was 7.7- to 32.5-fold higher compared to women without that type of activity immediately before conception. An adjusted logistic regression model also indicated significantly higher odds of organ-specific activity during pregnancy if that type of activity were present six months before conception. Approaching lupus based on specific organ systems may be a useful way for women and their physicians to consider the potential risk for disease activity during pregnancy.

Development of SLE among "potential SLE" patients seen in consultation: long-term follow-up.

OBJECTIVE: To identify factors associated with development of systemic lupus erythematosus (SLE) among patients evaluated at a tertiary care Lupus Center for potential SLE. METHODS: We identified patients first seen at the Brigham and Women's Hospital Lupus Center between 1 January 1992 and 31 December 2012 and thought to have potential SLE by a board-certified rheumatologist. All had 1-3 SLE ACR criteria at initial visit and > 2 follow-up visits ≥ 3 months apart. We reviewed medical records through 15 May 2013 for: SLE signs and symptoms, autoimmune serologies, prescriptions and diagnoses by board-certified rheumatologists. Bivariable analyses and multivariable logistic regression models were used to identify independent predictors of developing SLE. RESULTS: Two hundred and sixty four patients met inclusion criteria. At initial visit, mean age was 39.2 (SD 12.4) years, 94% were female and 67% white. Mean number of SLE ACR criteria was 2.7 (SD 1.0) and 88% were antinuclear antibody (ANA) positive at initial consultation. Mean follow-up time was 6.3 (SD 4.3) years and 67% were prescribed hydroxychloroquine in follow-up. At most recent visit, 56 (21%) had been diagnosed with SLE; 47 (18%) were thought not to have SLE and 161 (61%) were still considered to have potential SLE. In multivariable regression models, oral ulcers (OR 2.40, 95% CI 1.03-5.58), anti-dsDNA (OR 2.59, 95% CI 1.25-5.35) and baseline proteinuria or cellular casts (OR 16.20, 95% CI 1.63-161.02) were independent predictors of developing SLE. The most common other final diagnoses included fibromyalgia, Sjögren's syndrome, mixed connective tissue disease and cutaneous lupus. CONCLUSION: Among patients with potential SLE at initial consultation, 21% were diagnosed with definite SLE within 6.3 years. Oral ulcers, anti-dsDNA and proteinuria or cellular casts were independent predictors of developing definite SLE. A better means of accurately identifying those who will develop SLE among those presenting with potential disease is necessary.

[Anthropological approach to current parental perceptions of children's seizures]. / Approche anthropologique des représentations parentales actuelles des convulsions chez l'enfant.

UNLABELLED: Pediatric seizures are a common symptom, especially when associated with fever. This phenomenon is still shocking and traumatic for parents. The study analyzed current parental perceptions of seizures in order to improve the quality of management, care, and explanations provided to families at our emergency unit. METHODS: Using an anthropological approach, we analyzed 28 interviews of 37 parents whose child was admitted to our pediatric emergency unit between November 2007 and August 2008 due to a first seizure. RESULTS: The parental experience of the crisis was marked by upsetting memories of a "scary"-looking body and the perception of imminent death. Parental interpretations of the pathophysiology of the event were often wrong; very few mentioned the possibility of its cerebral origin, leading to inappropriate rescue attempts (e.g., giving CPR). The meaning attributed by parents to the word "seizure" and "epilepsy" usually referred to an exact clinical description of the phenomenon, but many admitted being unfamiliar with the term or at least its origin. Many studies have found the expectation of imminent death as well as inappropriate behaviors. This is the first study to consider interpretations expressed by parents around the convulsive phenomenon and to confirm a low level of knowledge of the symptom. Some historical interpretations persisted (e.g., the influence of excessive mood, anger, menstruation, demonic possession). CONCLUSION: Understanding and integrating these parental interpretations seems essential to improving care for families who first experience this symptom. This study motivated the implementation of a special educational workshop on seizures in 2010.

Retrospective study of 108 foals with septic osteomyelitis.

OBJECTIVE: To determine the clinical characteristics, short-term outcome and future athletic performance of foals with septic osteomyelitis. DESIGN: Retrospective clinical study of 108 Thoroughbred foals with radiographic evidence of bone infection that were presented at the Scone Veterinary Hospital between August 1995 and December 2001. Medical records were reviewed and information concerning signalment, the clinical, laboratory and radiographic findings, treatment and outcome was obtained. Racing records were obtained and evaluated for surviving foals that had reached racing age. RESULTS: Mean age of foals at initial evaluation was 39 days (range 1-180 days); 21 foals had multiple radiographic bone lesions (19.4%), and 76 had concurrent septic arthritis (70.4%). The most frequently affected bones were the femur, tibia and distal phalanx. In total, 87 foals were discharged from the hospital (80.6%), 79 survived long-term to reach racing age and 52 raced (65.8%). Overall, 48% (52/108) of the foals treated for osteomyelitis raced. Foals less than 30 days of age at the time of diagnosis, critically ill foals and those with multiple bones or joints affected were significantly less likely to be discharged from hospital. Multiple septic joints, but not multiple bone involvement, had an unfavourable prognosis for racing. CONCLUSIONS: The prognosis for survival of foals with septic osteomyelitis or osteitis is favourable. Multiple bone or joint involvement is an important short-term prognostic indicator; however, the involvement of multiple joints, but not multiple infected bones, is associated with an unfavourable prognosis for racing.

Alveolar macrophage activation and an emphysema-like phenotype in adiponectin-deficient mice.

Adiponectin is an adipocyte-derived collectin that acts on a wide range of tissues including liver, brain, heart, and vascular endothelium. To date, little is known about the actions of adiponectin in the lung. Herein, we demonstrate that adiponectin is present in lung lining fluid and that adiponectin deficiency leads to increases in proinflammatory mediators and an emphysema-like phenotype in the mouse lung. Alveolar macrophages from adiponectin-deficient mice spontaneously display increased production of tumor necrosis factor-alpha (TNF-alpha) and matrix metalloproteinase (MMP-12) activity. Consistent with these observations, we found that pretreatment of alveolar macrophages with adiponectin leads to TNF-alpha and MMP-12 suppression. Together, our findings show that adiponectin leads to macrophage suppression in the lung and suggest that adiponectin-deficient states may contribute to the pathogenesis of inflammatory lung conditions such as emphysema.

Long-term potentiation in the rat dentate gyrus is associated with enhanced Arc/Arg3.1 protein expression in spines, dendrites and glia.

Electron microscopic immunocytochemical methods were used to determine the localization, subcellular distribution and expression of activity-regulated cytoskeletal protein (Arc/Arg3.1) in dentate gyrus after unilateral induction of long-term potentiation (LTP) in the perforant pathway of anaesthetized rats. At 2 h post-induction, immunoreaction product was visible in the dentate gyrus in both the granule cell and molecular layers. Arc expression was higher in the potentiated than the unstimulated contralateral hemisphere. Single-section electron microscopy analysis in unstimulated tissue and in tissue prepared 2 and 4 h after LTP induction showed Arc immunoreactivity (Arc-IR) in dendrites, dendritic spines and glia. Arc-IR was associated with synaptic and non-synaptic plasma membrane apposed to axon terminals and with cytoplasmic organelles, including the cytoskeleton. Arc-IR was also present in neuronal perikarya and there was occasional labelling of nuclei and axons. At 2 h post-LTP induction, there were significant increases in Arc-IR within the granule cell and molecular layers of the dentate gyrus and particularly within the middle molecular layer relative to the inner and outer molecular layers. This increase in Arc expression 2 h after LTP induction was blocked by the N-methyl-D-aspartate receptor antagonist (RS)-3-2-carboxypiperazin-4-yl-propyl-1-phosphonic acid. In animals killed 4 h after LTP induction, Arc expression had declined and differences between the potentiated and unpotentiated hemispheres were no longer significant. Our data provide ultrastructural evidence for a transient LTP-associated increase in the expression of Arc protein in the middle molecular layer of the dentate gyrus, with preferential targeting to dendrites, dendritic spines and glia.

Derivation of lung epithelium from bone marrow cells.

Recent studies demonstrate the capacity of BM-derived cells to engraft as differentiated cells of a variety of organs, including lung. In this paper, we review the current state-of-the-art in this area. We also summarize our work demonstrating that cultured adherent marrow cells can serve as progenitors of lung alveolar epithelium.

Extracellular Ca2+ depletion contributes to fast activity-dependent modulation of synaptic transmission in the brain.

Synaptic activation is associated with rapid changes in intracellular Ca(2+), while the extracellular Ca(2+) level is generally assumed to be constant. Here, using a novel optical method to measure changes in extracellular Ca(2+) at high spatial and temporal resolution, we find that brief trains of synaptic transmission in hippocampal area CA1 induce transient depletion of extracellular Ca(2+). We show that this depletion, which depends on postsynaptic NMDA receptor activation, decreases the Ca(2+) available to enter individual presynaptic boutons of CA3 pyramidal cells. This in turn reduces the probability of consecutive synaptic releases at CA3-CA1 synapses and therefore contributes to short-term paired-pulse depression of minimal responses. This activity-dependent depletion of extracellular Ca(2+) represents a novel form of fast retrograde synaptic signaling that can modulate glutamatergic information transfer in the brain.

Canadian survey of clinical status at dialysis initiation 1998-1999: a multicenter prospective survey.

AIMS: The current growth in end-stage kidney disease populations has led to increased efforts to understand the impact of status at dialysis initiation on long-term outcomes. Our main objective was to improve the understanding of current Canadian nephrology practice between October 1998 and December 1999. METHODS: Fifteen nephrology centers in 7 provinces participated in a prospective data collection survey. The main outcome of interest was the clinical status at dialysis initiation determined by: residual kidney function, preparedness for chronic dialysis as measured by presence or absence of permanent peritoneal or hemodialysis access, hemoglobin and serum albumin. Uremic symptoms at dialysis initiation were also recorded, however, in some cases these symptom data were obtained retrospectively. RESULTS: Data on 251 patients during 1-month periods were collected. Patients commenced dialysis at mean calculated creatinine clearance levels of approximately 10 ml/min, with an average of 3 symptoms. 35% of patients starting dialysis had been known to nephrologists for less than 3 months. These patients are more likely to commence without permanent access and with lower hemoglobin and albumin levels. Even of those known to nephrologists, only 66% had permanent access in place. CONCLUSIONS: Patients commencing dialysis in Canada appear to be doing so in relative concordance with published guidelines with respect to timing of initiation. Despite an increased awareness of kidney disease, a substantial number of patients continues to commence dialysis without previous care by a nephrologist. Of those who are seen by nephrologists, clinical and laboratory parameters are suboptimal according to current guidelines. This survey serves as an important baseline for future comparisons after the implementation of educational strategies for referring physicians and nephrologists.

Perineuronal nets in the rhesus monkey and human basal forebrain including basal ganglia.

Perineuronal nets of extracellular matrix have been shown to characterize the microenvironment of individual neurons and the chemoarchitecture of brain regions such as basal forebrain nuclei. Previous work has also demonstrated that neurons in the human cerebral cortex ensheathed by perineuronal nets rarely undergo cytoskeletal changes in Alzheimer's disease, suggesting a neuroprotective effect of extracellular matrix components. It is not known, however, whether or not perineuronal nets are absent in the microenvironment of the cholinergic basal forebrain neurons that are involved early in the cascade of neurodegeneration in humans. Therefore, the present study was undertaken to examine the distribution patterns of perineuronal nets in the basal forebrain of the higher primates, rhesus monkey and human. Cytochemical staining was performed with the lectin Wisteria floribunda agglutinin and a polyclonal antibody to core proteins of chondroitin sulfate proteoglycans in the perfusion-fixed tissue of rhesus monkeys. In human brains, perineuronal nets were only stained with the immunoreaction for chondroitin sulfate proteoglycans. The results showed similar characteristics in distribution patterns of perineuronal nets in the medial septum, the diagonal band of Broca, the basal nucleus of Meynert (Ch1-Ch4), the lateral septum, the caudate-putamen, and the globus pallidus in both species. Double-labelling revealed that the vast majority of cholinergic neurons, labelled either with antibodies to choline acetyltransferase or the low-affinity neurotrophin receptor p75(NTR), were not ensheathed by perineuronal nets. A small subpopulation of net-associated neurons in close proximity to or intermingled with cholinergic neurons of the Ch1-Ch4 cell groups was found to be immunoreactive for parvalbumin. In the caudate-putamen, a large number of the parvalbumin-positive neurons were surrounded by perineuronal nets, whereas in the external and internal segments of the globus pallidus the coincidence of both markers was nearly complete. The study demonstrates that perineuronal nets of extracellular matrix are associated with different types of non-cholinergic neurons in the primate basal forebrain. The absence of nets around cholinergic basal forebrain neurons may be related to their slow modulatory activity but may also contribute to their susceptibility to degeneration in Alzheimer's disease.

Bone marrow-derived cells as progenitors of lung alveolar epithelium.

We assessed the capacity of plastic-adherent cultured bone marrow cells to serve as precursors of differentiated parenchymal cells of the lung. By intravenously delivering lacZ-labeled cells into wild-type recipient mice after bleomycin-induced lung injury, we detected marrow-derived cells engrafted in recipient lung parenchyma as cells with the morphological and molecular phenotype of type I pneumocytes of the alveolar epithelium. At no time after marrow cell injection, did we detect any engraftment as type II pneumocytes. In addition, we found that cultured and fresh aspirates of bone marrow cells can express the type I pneumocyte markers, T1alpha and aquaporin-5. These observations challenge the current belief that adult alveolar type I epithelial cells invariably arise from local precursor cells and raise the possibility of using injected marrow-derived cells for therapy of lung diseases characterized by extensive alveolar damage.