Spinal degeneration and lumbar multifidus muscle quality may independently affect clinical outcomes in patients conservatively managed for low back or leg pain.

Few non-surgical, longitudinal studies have evaluated the relations between spinal degeneration, lumbar multifidus muscle (LMM) quality, and clinical outcomes. None have assessed the potential mediating role of the LMM between degenerative pathology and 12-month clinical outcomes. This prospective cohort study used baseline and 12-month follow-up data from 569 patients conservatively managed for low back or back-related leg pain to estimate the effects of aggregate degenerative lumbar MRI findings and LMM quality on 12-month low back and leg pain intensity (0-10) and disability (0-23) outcomes, and explored the mediating role of LMM quality between degenerative findings and 12-month clinical outcomes. Adjusted mixed effects generalized linear models separately estimated the effect of aggregate spinal pathology and LMM quality. Mediation models estimated the direct and indirect effects of pathology on leg pain, and pathology and LMM quality on leg pain, respectively. Multivariable analysis identified a leg pain rating change of 0.99 [0.14; 1.84] (unstandardized beta coefficients [95% CI]) in the presence of ≥ 4 pathologies, and a disability rating change of - 0.65 [- 0.14; - 1.16] for each 10% increase in muscle quality, but no effect on back pain intensity. Muscle quality had a non-significant mediating role (13.4%) between pathology and leg pain intensity. The number of different pathologies present demonstrated a small effect on 12-month leg pain intensity outcomes, while higher LMM quality had a direct effect on 12-month disability ratings but no mediating effect between pathology and leg pain. The relations between degenerative pathology, LMM quality, and pain-related outcomes appear complex and may include independent pathways.

Frequency and indication of non-musculoskeletal examinations: a cross-sectional survey of Quebec chiropractors.

BACKGROUND: Approximately 1% of low back pain is estimated to be caused by serious systemic diseases, including cancer, infection, or abdominal aortic dissection. This study aimed to determine the frequency of execution of non-MSK physical examination procedures among Quebec chiropractors and to identify the clinical context that prompts them to use these physical examination procedures. METHODS: Cross-sectional survey containing 44 questions administered to a random sample of Quebec chiropractors using a succession of online, postal and phone questionnaires. The 4-part survey questionnaire contained six demographic questions, 28 single-choice questions to determine the frequency of execution of non-MSK physical examination procedures, seven short clinical vignettes for which the respondents had to select the non-MSK examinations that would be required, and two questions inquiring about the proportion of new patients for which participants' felt non-MSK examinations were necessary and whether appropriate assessments were performed. The questionnaire was pilot tested, and feedback received integrated prior to administration. We conducted descriptive statistics, Pearson correlations, and an ANOVA. RESULTS: The survey was completed by 182 chiropractors (response rate: 36.4%). The most commonly non-musculoskeletal examination performed daily were blood pressure (12.1%) and cranial nerves (4.9%). The most common tests never performed were oxygen saturation (68.7%), cardiac auscultation (69.2%), tibio-brachial index (71.4%), breast (86.8%), rectal (96.7%), testicular (95.6%), and vaginal (99.9%) exams. Female chiropractors and Quebec University in Trois-Rivières graduates reported that a significantly higher proportion of their new patients required a non-musculoskeletal physical examination compared to male participants (37.2% vs 28.3%) or Canadian Memorial Chiropractic College graduates (33.9% vs 19.9%). Reason for not performing a physical examination included the belief that another healthcare professional was better positioned to perform and/or interpret the related tests (76.4%). CONCLUSIONS: Vital signs and cranial nerve examinations were the most frequency performed non-musculoskeletal examinations reported by chiropractors. Apart from the genitourinary exam almost never performed, most participants chose non-musculoskeletal examinations deemed appropriate for the patient's presentation.

Understanding patient preferences for student clinician attire: a cross-sectional study of a student chiropractic clinic in Australia.

Objectives: Previous studies have investigated the role of clinical attire in establishing patient-held perceptions of professionalism and knowledgeability across various healthcare settings. This study aimed to understand patients' preferences for chiropractic student attire. Methods: Three hundred and twenty patients were recruited from a university chiropractic clinic and asked to complete an online questionnaire. The patients' preferences for five different attires were rated and calculated as the composite score of five domains (knowledgeable, trustworthy, caring, professional, and comfortable). Results: While 71.9% of participants indicated that how students dress was important to them, most (63.4%) disagreed that wearing a white coat was essential for chiropractic student clinicians. The most preferred form of attire was the current clinic shirt. Conclusion: The attire worn by chiropractic student clinicians at a single institution was found to be an influential attribute. Student chiropractic clinicians should dress professionally to make a good first impression. This study provided some guidance with the ongoing debate around students' dress code.

Objectif: Des études antérieures ont examiné le rôle de la tenue vestimentaire en clinique dans l'établissement des perceptions des patients quant au professionnalisme et à la compétence dans divers environnements de soins de santé. Cette étude visait à comprendre les préférences des patients en matière de tenue vestimentaire des étudiants en chiropratique. Méthodologie: Trois cent vingt patients ont été recrutés dans une clinique chiropratique universitaire et invités à remplir un questionnaire en ligne. Les préférences des patients pour cinq tenues différentes ont été évaluées et calculées en tant que score composite de cinq domaines (bien informé, digne de confiance, attentionné, professionnel et confortable). Résultats: Si 71,9 % des participants ont indiqué que la tenue vestimentaire des étudiants était importante pour eux, la plupart (63,4 %) n'étaient pas d'accord avec le fait que le port d'une blouse blanche était essentiel pour les étudiants cliniciens en chiropratique. La tenue vestimentaire la plus appréciée était la chemise de clinique actuelle. Conclusion: La tenue vestimentaire des étudiants cliniciens en chiropratique d'un même établissement s'est révélée être un attribut influent. Les étudiants en chiropratique doivent s'habiller de manière professionnelle pour faire une bonne première impression. Cette étude a permis d'éclairer le débat en cours sur le code vestimentaire des étudiants.

The impact of pregnancy on women with adolescent idiopathic scoliosis: a scoping review.

INTRODUCTION: Adolescent idiopathic scoliosis is the most common spinal deformity encountered in adolescents and larger curves are more prevalent in girls. For females with scoliosis, women's health issues are of particular concern, especially pregnancy. The aim of this review was to summarise the best available evidence to determine the influence of pregnancy on scoliosis-related outcomes in women with scoliosis and whether scoliosis affects maternal-health outcomes, differentiating between patients who have been managed conservatively and/or surgically. EVIDENCE ACQUISITION: A search was conducted using CINAHL, Scopus, Cochrane Database, MEDLINE, and EMBASE from inception to May 2023 to identify relevant articles in any language. The scoping review followed the PRISMA-ScR guidelines. Studies were eligible if they included pregnant women (primiparous or multiparous) with a diagnosis of scoliosis of unknown aetiology. The results were summarized by outcomes, including pregnancy and scoliosis-related outcomes and type of management. EVIDENCE SYNTHESIS: Our comprehensive search strategy identified 6872 articles, of which 50 articles were eligible for this review. Back pain appears to be more prevalent in this population during pregnancy and associated with the major curve and the decrease of lumbar lordosis. There have been reports of failed attempted spinal anaesthesia among patients with instrumented scoliosis correction and minor complications related to epidural anaesthesia at a higher rate compared to non-instrumented patients and healthy controls, however successful spinal analgesia can be achieved in patients with instrumented scoliosis correction. Overall, the caesarean section rate was similar in scoliosis patients compared to controls without scoliosis and to national averages. Curve progression occurs in some but not all patients during pregnancy, and this phenomenon occurs irrespective of the treatment received. CONCLUSIONS: Higher-quality prospective longitudinal research is needed to understand the relationship between pregnancy and adolescent idiopathic scoliosis. Further, the patient's perspective, concerns and fears surrounding pregnancy with scoliosis are yet to be explored. Exploring the impact of pregnancy on women with adolescent idiopathic scoliosis would have clinically relevant outcomes and could help provide pertinent answers to patients and healthcare workers and help guide future research.

Creating and testing a questionnaire to predict immediate and strong positive responders to spinal manipulative therapy for non-specific low back pain. A pilot study.

BACKGROUND: Many chiropractors use spinal manipulative techniques (SMT) to treat spinal pain. A recent Delphi study posited 18 items across five domains as predictors of patients experiencing non-specific low back pain most likely to experience a strong and immediate positive response to SMT. We sought to create a 'pen and paper' questionnaire that would measure these items and then pilot its use in a clinical setting to determine its 'usability' for a larger study. Knowing this information would inform a more efficacious use of SMT. METHOD: Of the 18 items identified in the Delphi study, 13 were deemed historical in nature and readily provided by the chiropractor and patient. A literature search revealed reliable and valid measures for two more items. The remaining three items were generated by creating descriptive questions matched to an appropriate Likert scale. A panel of six chiropractors who had used SMT for at least 7 years when treating non-specific low back pain was formed to evaluate the items for clarity and relevance. Ten Western Australian chiropractors were then recruited to pilot the questionnaire on ten consecutive patients with non-specific low back pain where SMT was used from March to June 2020. Ethics approval was obtained from Murdoch University. RESULTS: COVID-19 restrictions impacted on practitioner recruitment and delayed the data collection. Of the intended 100 participants, only 63 could be recruited over a 3-month period from seven chiropractors. Time constraints forced the closure of the data collection. The measures of all predictor items demonstrated ceiling effects. Feedback from open-ended practitioner questions was minimal, suggesting an ease of use. CONCLUSION: The length of time and level of participation required to collect the calculated sample size was inadequate and suggested that incentivization may be required for a larger investigation. Significant ceiling effects were found and suggested that participants did so because of a positive bias toward chiropractic care and the use of SMT. The questionnaires in this pilot study require alternative measures and further validation before use in a larger study.

Lumbar multifidus muscle morphology is associated with low back-related pain duration, disability, and leg pain: A cross-sectional study in secondary care.

BACKGROUND: Systematic reviews and studies exploring associations between morphologic change of paraspinal muscles and low back pain or related outcomes such as disability, radiculopathy, and physical workload, have reported conflicting results. This study explores the associations between lumbar multifidus muscle quality and clinical outcomes relating to low back pain. METHODS: Cross-sectional study of spinal clinic outpatients presenting with a primary complaint of low back and/or leg symptoms. Univariable and multivariable regression models were used to investigate associations between MRI-based multifidus muscle cross-sectional area at L4 and L5 and clinical outcomes for low back pain, leg pain, disability, restricted motion, and strenuous nature of work. Results were reported with ß-coefficients, odds ratios (OR), or incidence rate ratios (IRR) and their corresponding 95% confidence intervals, based on a 10% difference in muscle quality for each clinical variable. Multivariable analyses were adjusted for age, sex, and BMI. RESULTS: 875 patients [487 females; mean (SD) age: 43.6 (10.2) years] were included. In the multivariable analyses, muscle quality was significantly associated with disability (0-23 scale) [ß: -0.74, 95% CI: -1.14, -0.34], leg pain intensity (0-10 scale) [ß: -0.25, 95% CI: -0.46, -0.03], and current pain duration of more than 12 months [OR: 1.27, 95% CI: 1.03, 1.55]. No associations were found for low back pain intensity, morning stiffness, painful active range of motion, or work nature. CONCLUSIONS: Patients with higher lumbar multifidus muscle quality reported lower levels of low back pain-related disability and leg pain intensity, indicating that muscle quality may play a role in the etiology of lumbar spine disorders. However, the clinical importance of these associations is uncertain due to the low magnitude of identified associations. Future longitudinal studies are needed to understand the effect of lumbar multifidus muscle quality on lumbar-related pain and disability.

FOXG1 targets BMP repressors and cell cycle inhibitors in human neural progenitor cells.

FOXG1 is a critical transcription factor in human brain where loss-of-function mutations cause a severe neurodevelopmental disorder, while increased FOXG1 expression is frequently observed in glioblastoma. FOXG1 is an inhibitor of cell patterning and an activator of cell proliferation in chordate model organisms but different mechanisms have been proposed as to how this occurs. To identify genomic targets of FOXG1 in human neural progenitor cells (NPCs), we engineered a cleavable reporter construct in endogenous FOXG1 and performed chromatin immunoprecipitation (ChIP) sequencing. We also performed deep RNA sequencing of NPCs from two females with loss-of-function mutations in FOXG1 and their healthy biological mothers. Integrative analyses of RNA and ChIP sequencing data showed that cell cycle regulation and Bone Morphogenic Protein (BMP) repression gene ontology categories were over-represented as FOXG1 targets. Using engineered brain cell lines, we show that FOXG1 specifically activates SMAD7 and represses CDKN1B. Activation of SMAD7 which inhibits BMP signaling may be one way that FOXG1 patterns the forebrain, while repression of cell cycle regulators such as CDKN1B may be one way that FOXG1 expands the NPC pool to ensure proper brain size. Our data reveal novel mechanisms on how FOXG1 may control forebrain patterning and cell proliferation in human brain development.

Exploring 1st- and 2nd-year chiropractic students' willingness and attitudes toward peer physical examination.

OBJECTIVE: This study aimed to explore chiropractic students' perceptions and attitudes about the appropriateness of peer physical examination as a teaching tool and their willingness and comfort with it. METHODS: A modified version of a validated questionnaire was used. First- and 2nd-year chiropractic students at Murdoch University were approached during their practical sessions. The responses were analyzed using descriptive statistics reporting frequencies and percentages. Comparison between classes, age, and sex was evaluated by cross-tabulation. RESULTS: A total of 184 questionnaires were completed with a response rate of 76.6%. Our results demonstrated that most students were comfortable with and willing to participate in peer physical examination as well as trusted it as an appropriate part of their training and a valuable learning experience. Nevertheless, a small percentage were uncomfortable with peer physical examination and regarded it as an unprofessional activity. In addition, it was revealed that younger females (≤20 years) reported feeling unnecessarily exposed and therefore significantly less comfortable with peer physical examination. They were also less comfortable when examined in the inguinal area by a student of the opposite sex. CONCLUSION: Although peer physical examination appears to be a very popular training tool, it still has a few areas of concern that need to be investigated and addressed to improve students' attitude, perception, and comfort with this teaching technique. Further studies could investigate how other factors such as religious beliefs contribute toward students' perception and attitudes regarding peer physical examination.

Reliability and validity of physical examination tests for the assessment of ankle instability.

INTRODUCTION: Clinicians rely on certain physical examination tests to diagnose and potentially grade ankle sprains and ankle instability. Diagnostic error and inaccurate prognosis may have important repercussions for clinical decision-making and patient outcomes. Therefore, it is important to recognize the diagnostic value of orthopaedic tests through understanding the reliability and validity of these tests. OBJECTIVE: To systematically review and report evidence on the reliability and validity of orthopaedic tests for the diagnosis of ankle sprains and instability. METHODS: PubMed, CINAHL, Scopus, and Cochrane databases were searched from inception to December 2021. In addition, the reference list of included studies, located systematic reviews, and orthopaedic textbooks were searched. All articles reporting reliability or validity of physical examination or orthopaedic tests to diagnose ankle instability or sprains were included. Methodological quality of the reliability and the validity studies was assessed with The Quality Appraisal for Reliability studies checklist and the Quality Assessment of Diagnostic Accuracy Studies-2 respectively. We identified the number of times the orthopaedic test was investigated and the validity and/or reliability of each test. RESULTS: Overall, sixteen studies were included. Three studies assessed reliability, eight assessed validity, and five evaluated both. Overall, fifteen tests were evaluated, none demonstrated robust reliability and validity scores. The anterolateral talar palpation test reported the highest diagnostic accuracy. Further, the anterior drawer test, the anterolateral talar palpation, the reverse anterior lateral drawer test, and palpation of the anterior talofibular ligament reported the highest sensitivity. The highest specificity was attributed to the anterior drawer test, the anterolateral drawer test, the reverse anterior lateral drawer test, tenderness on palpation of the proximal fibular, and the squeeze test. CONCLUSION: Overall, the diagnostic accuracy, reliability, and validity of physical examination tests for the assessment of ankle instability were limited. Physical examination tests should not be used in isolation, but rather in combination with the clinical history to diagnose an ankle sprain. Preliminary evidence suggests that the overall validity of physical examination for the ankle may be better if conducted five days after the injury rather than within 48 h of injury.

Spinal degeneration is associated with lumbar multifidus morphology in secondary care patients with low back or leg pain.

Associations between multifidus muscle morphology and degenerative pathologies have been implied in patients with non-specific low back pain, but it is unknown how these are influenced by pathology severity, number, or distribution. MRI measures of pure multifidus muscle cross-sectional area (CSA) were acquired from 522 patients presenting with low back and/or leg symptoms in an outpatient clinic. We explored cross-sectional associations between the presence, distribution, and/or severity of lumbar degenerative pathologies (individually and in aggregate) and muscle outcomes in multivariable analyses (beta coefficients [95% CI]). We identified associations between lower pure multifidus muscle CSA and disc degeneration (at two or more levels): - 4.51 [- 6.72; - 2.3], Modic 2 changes: - 4.06 [- 6.09; - 2.04], endplate defects: - 2.74 [- 4.58; - 0.91], facet arthrosis: - 4.02 [- 6.26; - 1.78], disc herniations: - 3.66 [- 5.8; - 1.52], and when > 5 pathologies were present: - 6.77 [- 9.76; - 3.77], with the last supporting a potential dose-response relationship between number of spinal pathologies and multifidus morphology. Our findings could hypothetically indicate that these spinal and muscle findings: (1) are part of the same degenerative process, (2) result from prior injury or other common antecedent events, or (3) have a directional relationship. Future longitudinal studies are needed to further examine the complex nature of these relationships.

Kabuki syndrome stem cell models reveal locus specificity of histone methyltransferase 2D (KMT2D/MLL4).

Kabuki syndrome is frequently caused by loss-of-function mutations in one allele of histone 3 lysine 4 (H3K4) methyltransferase KMT2D and is associated with problems in neurological, immunological and skeletal system development. We generated heterozygous KMT2D knockout and Kabuki patient-derived cell models to investigate the role of reduced dosage of KMT2D in stem cells. We discovered chromosomal locus-specific alterations in gene expression, specifically a 110 Kb region containing Synaptotagmin 3 (SYT3), C-Type Lectin Domain Containing 11A (CLEC11A), Chromosome 19 Open Reading Frame 81 (C19ORF81) and SH3 And Multiple Ankyrin Repeat Domains 1 (SHANK1), suggesting locus-specific targeting of KMT2D. Using whole genome histone methylation mapping, we confirmed locus-specific changes in H3K4 methylation patterning coincident with regional decreases in gene expression in Kabuki cell models. Significantly reduced H3K4 peaks aligned with regions of stem cell maps of H3K27 and H3K4 methylation suggesting KMT2D haploinsufficiency impact bivalent enhancers in stem cells. Preparing the genome for subsequent differentiation cues may be of significant importance for Kabuki-related genes. This work provides a new insight into the mechanism of action of an important gene in bone and brain development and may increase our understanding of a specific function of a human disease-relevant H3K4 methyltransferase family member.

Methylation of the tyrosine hydroxylase gene is dysregulated by cocaine dependence in the human striatum.

Cocaine dependence is a chronic, relapsing disorder caused by lasting changes in the brain. Animal studies have identified cocaine-related alterations in striatal DNA methylation; however, it is unclear how methylation is related to cocaine dependence in humans. We generated methylomic profiles of the nucleus accumbens using human postmortem brains from a cohort of individuals with cocaine dependence and healthy controls (n = 25 per group). We found hypermethylation in a cluster of CpGs within the gene body of tyrosine hydroxylase (TH), containing a putative binding site for the early growth response 1 (EGR1) transcription factor, which is hypermethylated in the caudate nucleus of cocaine-dependent individuals. We replicated this finding and found it to be specific to striatal neuronal nuclei. Furthermore, this locus demonstrates enhancer activity which is attenuated by methylation and enhanced by EGR1 overexpression. These results suggest that cocaine dependence alters the epigenetic regulation of dopaminergic signaling genes.

Association between the expression of lncRNA BASP-AS1 and volume of right hippocampal tail moderated by episode duration in major depressive disorder: a CAN-BIND 1 report.

The pathophysiology of major depressive disorder (MDD) encompasses an array of changes at molecular and neurobiological levels. As chronic stress promotes neurotoxicity there are alterations in the expression of genes and gene-regulatory molecules. The hippocampus is particularly sensitive to the effects of stress and its posterior volumes can deliver clinically valuable information about the outcomes of antidepressant treatment. In the present work, we analyzed individuals with MDD (N = 201) and healthy controls (HC = 104), as part of the CAN-BIND-1 study. We used magnetic resonance imaging (MRI) to measure hippocampal volumes, evaluated gene expression with RNA sequencing, and assessed DNA methylation with the (Infinium MethylationEpic Beadchip), in order to investigate the association between hippocampal volume and both RNA expression and DNA methylation. We identified 60 RNAs which were differentially expressed between groups. Of these, 21 displayed differential methylation, and seven displayed a correlation between methylation and expression. We found a negative association between expression of Brain Abundant Membrane Attached Signal Protein 1 antisense 1 RNA (BASP1-AS1) and right hippocampal tail volume in the MDD group (ß = -0.218, p = 0.021). There was a moderating effect of the duration of the current episode on the association between the expression of BASP1-AS1 and right hippocampal tail volume in the MDD group (ß = -0.48, 95% C.I. [-0.80, -0.16]. t = -2.95 p = 0.004). In conclusion, we found that overexpression of BASP1-AS1 was correlated with DNA methylation, and was negatively associated with right tail hippocampal volume in MDD.

Treatment-emergent and trajectory-based peripheral gene expression markers of antidepressant response.

Identifying biomarkers of antidepressant response may advance personalized treatment of major depressive disorder (MDD). We aimed to identify longitudinal changes in gene expression associated with response to antidepressants in a sample of MDD patients treated with escitalopram. Patients (N = 153) from the CAN-BIND-1 cohort were treated for 8 weeks, and depressive symptoms were assessed using the Montgomery-Åsberg Depression Rating Scale at 0, 2, 4, 6, and 8 weeks. We identified three groups of patients according to response status: early responders (22.9%), later responders (32.0%), and nonresponders (45.1%). RNA sequencing was performed in blood obtained at weeks 0, 2, and 8. RNA expression was modeled using growth models, and differences in the longitudinal changes in expression according to response were investigated using multiple regression models. The expression of RNAs related to response was investigated in the brains of depressed individuals, as well as in neuronal cells in vitro. We identified four RNAs (CERCAM, DARS-AS1, FAM228B, HBEGF) whose change over time was independently associated with a response status. For all except HBEGF, responders showed higher expression over time, compared to nonresponders. While the change in all RNAs differentiated early responders from nonresponders, changes in DARS-AS1 and HBEGF also differentiated later responders from nonresponders. Additionally, HBEGF was downregulated in the brains of depressed individuals, and increased in response to escitalopram treatment in vitro. In conclusion, using longitudinal assessments of gene expression, we provide insights into biological processes involved in the intermediate stages of escitalopram response, highlighting several genes with potential utility as biomarkers of antidepressant response.

Neuron-derived extracellular vesicles enriched from plasma show altered size and miRNA cargo as a function of antidepressant drug response.

Previous work has demonstrated that microRNAs (miRNAs) change as a function of antidepressant treatment (ADT) response. However, it is unclear how representative these peripherally detected miRNA changes are to those occurring in the brain. This study aimed to use peripherally extracted neuron-derived extracellular vesicles (NDEV) to circumvent these limitations and investigate neuronal miRNA changes associated with antidepressant response. Samples were collected at two time points (baseline and after 8 weeks of follow-up) from depressed patients who responded (N = 20) and did not respond (N = 20) to escitalopram treatment, as well as controls (N = 20). Total extracellular vesicles (EVs) were extracted from plasma, and then further enriched for NDEV by immunoprecipitation with L1CAM. EVs and NDEVs were characterized, and NDEV miRNA cargo was extracted and sequenced. Subsequently, studies in cell lines and postmortem tissue were conducted. Characterization of NDEVs revealed that they were smaller than other EVs isolated from plasma (p < 0.0001), had brain-specific neuronal markers, and contained miRNAs enriched for brain functions (p < 0.0001) Furthermore, NDEVs from depressed patients were smaller than controls (p < 0.05), and NDEV size increased with ADT response (p < 0.01). Finally, changes in NDEV cargo, specifically changes in miR-21-5p, miR-30d-5p, and miR-486-5p together (p < 0.01), were associated with ADT response. Targets of these three miRNAs were altered in brain tissue from depressed individuals (p < 0.05). Together, this study indicates that changes in peripherally isolated NDEV can act as both a clinically accessible and informative biomarker of ADT response specifically through size and cargo.

Integrative DNA Methylation and Gene Expression Analysis in the Prefrontal Cortex of Mexicans Who Died by Suicide.

BACKGROUND: Suicide represents a major health concern, especially in developing countries. While many demographic risk factors have been proposed, the underlying molecular pathology of suicide remains poorly understood. A body of evidence suggests that aberrant DNA methylation and expression is involved. In this study, we examined DNA methylation profiles and concordant gene expression changes in the prefrontal cortex of Mexicans who died by suicide. METHODS: In collaboration with the coroner's office in Mexico City, brain samples of males who died by suicide (n = 35) and age-matched sudden death controls (n = 13) were collected. DNA and RNA were extracted from prefrontal cortex tissue and analyzed with the Infinium Methylation480k and the HumanHT-12 v4 Expression Beadchips, respectively. RESULTS: We report evidence of altered DNA methylation profiles at 4430 genomic regions together with 622 genes characterized by differential expression in cases vs controls. Seventy genes were found to have concordant methylation and expression changes. Metacore-enriched analysis identified 10 genes with biological relevance to psychiatric phenotypes and suicide (ADCY9, CRH, NFATC4, ABCC8, HMGA1, KAT2A, EPHA2, TRRAP, CD22, and CBLN1) and highlighted the association that ADCY9 has with various pathways, including signal transduction regulated by the cAMP-responsive element modulator, neurophysiological process regulated by the corticotrophin-releasing hormone, and synaptic plasticity. We therefore went on to validate the observed hypomethylation of ADCY9 in cases vs control through targeted bisulfite sequencing. CONCLUSION: Our study represents the first, to our knowledge, analysis of DNA methylation and gene expression associated with suicide in a Mexican population using postmortem brain, providing novel insights for convergent molecular alterations associated with suicide.

Extraction of nuclei from archived postmortem tissues for single-nucleus sequencing applications.

Single-cell and single-nucleus sequencing techniques are a burgeoning field with various biological, biomedical and clinical applications. Numerous high- and low-throughput methods have been developed for sequencing the RNA and DNA content of single cells. However, for all these methods, the key requirement is high-quality input of a single-cell or single-nucleus suspension. Preparing such a suspension is the limiting step when working with fragile, archived tissues of variable quality. This hurdle can prevent such tissues from being extensively investigated with single-cell technologies. We describe a protocol for preparing single-nucleus suspensions within the span of a few hours that reliably works for multiple postmortem and archived tissue types using standard laboratory equipment. The stages of the protocol include tissue preparation and dissociation, nuclei extraction, and nuclei concentration assessment and capture. The protocol is comparable to other published protocols but does not require fluorescence-assisted nuclei sorting (FANS) or ultracentrifugation. The protocol can be carried out by a competent graduate student familiar with basic laboratory techniques and equipment. Moreover, these preparations are compatible with single-nucleus (sn)RNA-seq and assay for transposase-accessible chromatin (ATAC)-seq using the 10X Genomics Chromium system. The protocol reliably results in efficient capture of single nuclei for high-quality snRNA-seq libraries.

Non-CG methylation and multiple histone profiles associate child abuse with immune and small GTPase dysregulation.

Early-life adversity (ELA) is a major predictor of psychopathology, and is thought to increase lifetime risk by epigenetically regulating the genome. Here, focusing on the lateral amygdala, a major brain site for emotional homeostasis, we describe molecular cross-talk among multiple mechanisms of genomic regulation, including 6 histone marks and DNA methylation, and the transcriptome, in subjects with a history of ELA and controls. In the healthy brain tissue, we first uncover interactions between different histone marks and non-CG methylation in the CAC context. Additionally, we find that ELA associates with methylomic changes that are as frequent in the CAC as in the canonical CG context, while these two forms of plasticity occur in sharply distinct genomic regions, features, and chromatin states. Combining these multiple data indicates that immune-related and small GTPase signaling pathways are most consistently impaired in the amygdala of ELA individuals. Overall, this work provides insights into genomic brain regulation as a function of early-life experience.

miR-323a regulates ERBB4 and is involved in depression.

Major depressive disorder (MDD) is a complex and debilitating illness whose etiology remains unclear. Small RNA molecules, such as micro RNAs (miRNAs) have been implicated in MDD, where they display differential expression in the brain and the periphery. In this study, we quantified miRNA expression by small RNA sequencing in the anterior cingulate cortex and habenula of individuals with MDD and psychiatrically-healthy controls. Thirty-two miRNAs showed significantly correlated expression between the two regions (False Discovery Rate < 0.05), of which four, miR-204-5p, miR-320b, miR-323a-3p, and miR-331-3p, displayed upregulated expression in MDD. We assessed the expression of predicted target genes of differentially expressed miRNAs in the brain, and found that the expression of erb-b2 receptor tyrosine kinase 4 (ERBB4), a gene encoding a neuregulin receptor, was downregulated in both regions, and was influenced by miR-323a-3p in vitro. Finally, we assessed the effects of manipulating miRNA expression in the mouse ACC on anxiety- and depressive-like behaviors. Mice in which miR-323-3p was overexpressed or knocked-down displayed increased and decreased emotionality, respectively. Additionally, these mice displayed significantly downregulated and upregulated expression of Erbb4, respectively. Overall, our findings indicate the importance of brain miRNAs in the pathology of MDD, and emphasize the involvement of miR-323a-3p and ERBB4 in this phenotype. Future studies further characterizing miR-323a-3p and neuregulin signaling in depression are warranted.