Relationship between miR-335 and sterol levels after in vitro hypoxia-ischemia of primary brain cells
Namood-E Sahar, Zeljka Korade, and Eric Peeples
Background/Purpose: Cholesterol homeostasis is vital for synaptogenesis and myelination during fetal and neonatal brain development. Both brain and plasma sterol levels have been shown to be associated with outcomes in adult stroke, and animal studies have suggested brain and plasma sterol changes in neonatal hypoxic-ischemic brain injury. MicroRNA-335 (miR-335) has been associated with brain sterol metabolism and our preliminary data showed that sterol metabolism is dysregulated in the brain after hypoxic-ischemic injury in a mouse model. This study sought to describe the effect of oxygen-glucose deprivation in vitro on individual brain cell populations and to validate the associations between sterol levels and miR-335 levels. Lastly, we assessed the feasibility of transfecting miR-335 mimics or miRNA antagonists (antagomiRs) in these brain cell cultures to set up future experiments to alter miR-335 levels for neuroprotection.
Methods: Primary cells were isolated from embryonic day 18 CD1 mouse brains. Cells were maintained in DMEM for mixed glial culture and Neurobasal media with B27 for neurons. Once astrocytes were confluent, microglia were separated from the mixed glial culture by shaking to provide pure cell populations. Each of the three cell types underwent oxygen and glucose deprivation (OGD: glucose/pyruvate/B27-free media, 1% O2, 5% CO2) for 4 hours followed by replacement of glucose/pyruvate/B27 and resumption of normoxia. At 24 hours after OGD, cells (n=12 wells OGD and 12 normoxia) were washed, counted, and sterols analyzed by LC-MS/MS, normalized to the number of cells/well. Additional cells underwent measurement of miR-335 expression using quantitative PCR and/or transfection with miR-335 mimic or antagomiR.
Results: Although lanosterol is increased after OGD in neurons, desmosterol and cholesterol levels were decreased. In microglia and astrocytes, cholesterol levels were lower than in neurons but increased after OGD. MiR-335 expression in neurons and astrocytes were inverse to cholesterol level changes, though this association was not seen in microglia. Lastly, red fluorescent protein-labelled miR-335 mimic was visualized in both neurons and astrocytes after transfection and miR-335 expression changes were seen after transfection in astrocytes.
Conclusions: Sterol levels are altered after OGD and may be associated with OGD changes. Transfection of miR-335 mimic and antagomiR is feasible and future studies using these tools will allow for better understanding of the effects of neonatal HIBI on sterol levels. This approach could allow for identification of targets to aid in developing therapeutics.
Expanding the phenotype of HNRNPU-related disorders to include brief, resolved, unexplained events (BRUE)
Jonah Scheffler, Kristen P. Fishler, and Lois J. Starr
hnRNP-U deficiency is caused by pathogenic variants in HNRNPU, which encodes the heterogeneous nuclear ribonucleoprotein U (HNRNPU), a highly conserved protein responsible for assisting spliceosomes in mediating transcription and alternative splicing activity. HnRNPs are responsible for the regulation of translation at the presynaptic sites as well as the transportation of stabilized mRNAs along the axonal cytoskeleton. Here, we report a 2-year-old-male with a HNRNPU variant with a new presentation of apparent recurrent apneic spells with an underlying epileptic origin. These were described as apnea followed by desaturation and tachycardia in the 180's-200 range prior to resolution of symptoms. He also had autistiform behaviors, hypotonia, global developmental delay, heart defects, and unique facial features. The anesthetist professional parents describe multiple BRUE. At 26 months, he presented to the hospital with hypotonia and unique facial features, global developmental delay, autistiform behaviors, dyspraxia with cognitive disability and a change in mental status. On physical exam, the proband had telecanthus, a broad nasal bridge, short palpebral fissures, mild nevus flammeus changes on his face, a single right palmar crease, and a modified single crease on the left. EKG showed a sinus rhythm with intermittent 1st degree AV block, blocked premature atrial contractions, left axis deviation, right bundle branch block, and an ejection fraction of 67%. Echocardiography re-identified an atrial septal defect. Brain MRI showed a T2/FLAIR hyperintense signal in the white matter of the parietal lobes, left greater than right. EEG identified generalized slowing indicative of a mild nonspecific encephalopathy. History of episodes were determined to be consistent with partial onset seizures with eye opening, deviation, and tachycardia with apnea and medical treatment ensued.
Genetic testing including microarray and an epilepsy panel that identified no genomic dosage anomalies and a de novo nonsense mutation (c.803+2T>C; p. unknown in HNRNPU), classified as pathogenic. The study of hnRNP complexes have gained momentum in neurodegenerative and tumorigenesis disease research. hnRNPs have a key role in mediating transcription, alternative splicing, and translation activity. Recently, Durkin et al, 2020 (PMID: 32319732) reported 21 previously unreported probands; nearly doubling the recorded patient population. Probands in the literature to date have had variable presentation, but usually with hypotonia, global developmental delays, and seizures. This suggests the addition of HNRNPU to all seizure-related diagnostic panels. We would also recommend including the HNRNPU-related disorders in a differential diagnosis of BRUE and recurrent apneic episodes as any underlying clonic activity may be profoundly subtle.
Jenna Scholl, Oladapo Akinmoladun, Amissabah Kanley, Nathan Gollehon, and Jason Burrows
Depression and suicidal ideation have a higher prevalence in medical students when compared to other age-matched populations.1,2 The SARS-CoV2 pandemic has further threatened medical trainee well-being with increased stress and risk of mental illness.3,4
To compare medical student well-being at a single institution before and during the SARS-CoV2 pandemic.
Third year medical students at a single institution voluntarily completed a survey during pediatric clerkship orientation as part of an ongoing, non-pandemic longitudinal wellness study. The survey instrument includes basic demographic information, the validated World Health Organization (WHO) (FIVE) Well-Being Index (WHO-5), and questions about physical activity. Data from mid-academic year students rotating on the pediatric clerkship in the 2019-2020 (two cohorts, pre-pandemic) and 2020-21 (three cohorts, during pandemic) academic years were compared across compositive survey scores and sub-question scores.
Study participation was 81% pre-pandemic (n=34) and 59% during the pandemic (n=39) with no statistically significant differences in age or gender. Mean WHO-5 well-being scores trended up from the pre-pandemic to pandemic groups without reaching statistical significance (16.3 vs 17.5, p=0.13). The mean individual survey question on feeling fresh and rested significantly improved from pre-pandemic to pandemic groups (2.74 vs 3.23, p<0.05). Remaining question mean scores were similar or trended toward improved well-being from pre-pandemic to pandemic groups; cheerful/good spirits (3.62 vs 3.82, p=0.15), calm/relaxed (3.32 vs 3.33, p=0.48), active/vigorous (3.18 vs 3.31, p=0.28), and interest in daily life (3.41 vs 3.79, p=0.08). Based on using the composite WHO-5 survey as a validated tool to screen for depression, there was a trend towards less students screening positive for depression in the pandemic group (26.5% pre-pandemic vs 10.5% pandemic, p=0.07).
From our small sample of third year medical students at a single institution, we find that well-being is stable when comparing matched groups across academic years before and during the SARS-CoV2 pandemic. Interestingly, survey subsection data demonstrates improved subjective feelings of being rested in the pandemic group and suggests lower rates of depression based on screening cutoffs (WHO-5). We suspect this may be related to changes in the educational environment such as transitioning from the clinical environment to virtual rotations, or time away from rotations entirely which may have allowed for increased personal wellness time. Focus groups and further investigation are necessary to identify which factors are altering levels of student well-being.
- Dyrbye LN, Thomas MR, Shanafelt TD. Systematic review of depression, anxiety, and other indicators of psychological distress among U.S. and Canadian medical students. Acad Med J Assoc Am Med Coll. 2006;81(4):354-373. doi:10.1097/00001888-200604000-00009
- Rotenstein LS, Ramos MA, Torre M, et al. Prevalence of Depression, Depressive Symptoms, and Suicidal Ideation Among Medical Students: A Systematic Review and Meta-Analysis. JAMA. 2016;316(21):2214-2236. doi:10.1001/jama.2016.17324
- Kannampallil TG, Goss CW, Evanoff BA, Strickland JR, McAlister RP, Duncan J. Exposure to COVID-19 patients increases physician trainee stress and burnout. PLOS ONE. 2020;15(8):e0237301. https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0237301. Accessed Oct 5, 2020. doi: 10.1371/journal.pone.0237301.
- Wu S, Li Z, Li Z, et al. The mental state and risk factors of chinese medical staff and medical students in early stages of the COVID-19 epidemic. Compr Psychiatry. 2020;102:152202. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7437442/. Accessed Oct 21, 2020. doi: 10.1016/j.comppsych.2020.152202.
"Patchwork Pericardium" is a reflection into what we see and don't see in medicine as a whole picture. From my lens as third-year medical students, I learned how it important it was to be observant to the world around me.
Introduction: Hemophagocytic lymphohistiocytosis (HLH) is a rare syndrome of overwhelming immune activation causing multiorgan dysfunction that may be genetic and/or acquired. Patients with familial HLH often present by one year of age, though most are asymptomatic in the first month of life. We present the case of a neonate with diarrhea and malnutrition who developed signs and symptoms consistent with a clinical diagnosis of HLH.
Case: A neonate born at 35 5/7 weeks was transferred to a Level IV neonatal intensive care unit at 39 4/7 weeks for failure to thrive. Though feeding adequately and non-dysmorphic, he had persistent diarrhea on multiple formulas and only gained small amounts of weight via total parental nutrition. He was the first child of consanguineous parents of South Asian descent. Evaluation was inconsistent with milk soy protein intolerance and malabsorption syndromes. Very-early-onset inflammatory bowel disease and congenital diarrheas/enteropathies were considered. Intestinal biopsy was concerning for an underlying primary immunodeficiency. Additional studies showed a mild increase in T cells and low IgM. Lymphocyte proliferation to mitogens and expression of FOXP3, XIAP, and SAP by flow cytometry were unremarkable. At age 44 weeks, he acutely became lethargic, with fevers to 40°C and metabolic acidosis, further developing anemia, thrombocytopenia, lymphocytosis, transaminitis, and hepatosplenomegaly. Hypofibrinogenemia, hyperferritinemia (>10,000), and elevated Soluble IL-2R level led to HLH diagnosis. Per HLH-94 protocol, he began dexamethasone and etoposide; he also received emapalumab. Despite treatment, fulminant liver failure with uncontrollable DIC ensued. Life-sustaining treatments were withdrawn and he died at 46 3/7 weeks.
Discussion: Though colitis is not classically a feature of most primary HLH syndromes, it has been described in one subset of familial HLH. However, sequencing of the associated gene (STXBP2) was normal in our patient. Similarly, among EBV-driven HLH, X-linked lymphoproliferative disease and XIAP deficiency are associated with hypogammaglobulinemia and colitis respectively, but expression of these was normal without evidence of EBV. Perforin/granzyme B expression was increased. Whole-exome sequencing (WES) revealed several variants of unknown significance but was ultimately nondiagnostic. While neonates are more likely to have genetic causes for HLH, around 40% of neonates do not have established genetic diagnoses after WES.
Conclusion: Timely recognition of HLH maximizes the potential for effective treatment like hematopoietic stem cell transplantation. Recognition of colitis as a presenting symptom may facilitate this challenging diagnosis. Rapid and inclusive genetic evaluation, including WES, may help identify the underlying etiology and guide treatment for neonatal HLH.
For my best friend, a healthcare hero, who herself spent several weeks in the NICU as a premature baby and is now helping other babies and their families as a NICU nurse.
Because of the importance of pets in children's lives, I submit a black-and-white head and shoulders photographic portrait of Homer, a beloved neighborhood dog from when children were younger. Photographed by me and developed and printed in my own darkroom. Every family in the neighborhood had a copy of this photographic portrait prominently displayed in their homes, and some might remember this photograph from my old office.
Steven V. Yackley and Terence L. Zach
Case Report: A male infant born at 37 weeks gestation. His mother was a 29-year-old, gravida 4 para 3 woman. During her pregnancy she was curious as to the gender of the fetus and requested a non-invasive prenatal test (NIPT) rather than fetal ultrasound. The NIPT returned with a karyotype of XXY. The male infant was born vaginally with a birth weight of 2670 grams. Apgar scores were 8 and 9 at one and five minutes respectively. He was admitted to the newborn intensive care unit secondary to respiratory distress. He had no obvious dysmorphic features. He had normal appearing male genitalia with testes descended bilaterally. Fluorescent in situ hybridization and blood chromosomes confirmed an XXY karyotype. He was discharged from the newborn intensive care unit with follow up by genetic counselors and close monitoring for developmental delays
Background: Klinefelter Syndrome (KS), a genetic disorder caused by the presence of supernumerary sex chromosomes. An additional X chromosome(s) and hypogonadism are the two defining features of KS. The excess of genes from the additional X chromosome drives the pathogenesis of the disease and distinguishing features of the affected individuals. The increase in the prevalence of prenatal testing has led to the earlier recognition of fetal chromosomal abnormalities. Early detection of KS through NIPT has given the family much needed information and allowing for early diagnosis and intervention, to promote the best outcome for the child. This case shows a patient that will benefit from the early diagnosis of a KS to implement early and timely interventions upon delivery and then later in the developing years. The neonate presented with no distinct, characteristic signs of KS, common in many individuals. Two findings, other than the NIPT results, weakly suggest any possible abnormality. Klinefelter neonates report mildly higher incidences of respiratory distress and low birth weight necessitating need for NICU admission. The underwhelming clinical presentation of the neonate shows that if NIPT wasn’t conducted, neither our patient nor his family would have known he was 47,XXY, until much later in life.
IMPLICATIONS: Timely hormonal intervention is possible for our patient because of NIPT. This demonstrates a need of a standard KS screen. With the technology of NIPT advancing and improving in its ability to detect a variety of other conditions outside of the traditional autosomal aneuploidy, it should be considered a technique for screening for clinically silent conditions, such as mild KS phonotypes.
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