Depressive symptoms during the transition to adolescence: Left hippocampal volume as a marker of social context sensitivity.

The transition to adolescence is a critical period for mental health development. Socio-experiential environments play an important role in the emergence of depressive symptoms with some adolescents showing more sensitivity to social contexts than others. Drawing on recent developmental neuroscience advances, we examined whether hippocampal volume amplifies social context effects in the transition to adolescence. We analyzed 2-y longitudinal data from the Adolescent Brain Cognitive Development (ABCD®) study in a diverse sample of 11,832 youth (mean age: 9.914 y; range: 8.917 to 11.083 y; 47.8% girls) from 21 sites across the United States. Socio-experiential environments (i.e., family conflict, primary caregiver's depressive symptoms, parental warmth, peer victimization, and prosocial school environment), hippocampal volume, and a wide range of demographic characteristics were measured at baseline. Youth's symptoms of major depressive disorder were assessed at both baseline and 2 y later. Multilevel mixed-effects linear regression analyses showed that negative social environments (i.e., family conflict, primary caregiver's depressive symptoms, and peer victimization) and the absence of positive social environments (i.e., parental warmth and prosocial school environment) predicted greater increases in youth's depressive symptoms over 2 y. Importantly, left hippocampal volume amplified social context effects such that youth with larger left hippocampal volume experienced greater increases in depressive symptoms in more negative and less positive social environments. Consistent with brain-environment interaction models of mental health, these findings underscore the importance of families, peers, and schools in the development of depression during the transition to adolescence and show how neural structure amplifies social context sensitivity.

Variant-to-function mapping of late-onset Alzheimer's disease GWAS signals in human microglial cell models implicates RTFDC1 at the CASS4 locus.

Late-onset Alzheimer's disease (LOAD) research has principally focused on neurons over the years due to their known role in the production of amyloid beta plaques and neurofibrillary tangles. In contrast, recent genomic studies of LOAD have implicated microglia as culprits of the prolonged inflammation exacerbating the neurodegeneration observed in patient brains. Indeed, recent LOAD genome-wide association studies (GWAS) have reported multiple loci near genes related to microglial function, including TREM2 , ABI3 , and CR1 . However, GWAS alone cannot pinpoint underlying causal variants or effector genes at such loci, as most signals reside in non-coding regions of the genome and could presumably confer their influence frequently via long-range regulatory interactions. We elected to carry out a combination of ATAC-seq and high-resolution promoter-focused Capture-C in two human microglial cell models (iPSC-derived microglia and HMC3) in order to physically map interactions between LOAD GWAS-implicated candidate causal variants and their corresponding putative effector genes. Notably, we observed consistent evidence that rs6024870 at the GWAS CASS4 locus contacted the promoter of nearby gene, RTFDC1 . We subsequently observed a directionallly consistent decrease in RTFDC1 expression with the the protective minor A allele of rs6024870 via both luciferase assays in HMC3 cells and expression studies in primary human microglia. Through CRISPR-Cas9-mediated deletion of the putative regulatory region harboring rs6024870 in HMC3 cells, we observed increased pro-inflammatory cytokine secretion and decreased DNA double strand break repair related, at least in part, to RTFDC1 expression levels. Our variant-to-function approach therefore reveals that the rs6024870-harboring regulatory element at the LOAD ' CASS4' GWAS locus influences both microglial inflammatory capacity and DNA damage resolution, along with cumulative evidence implicating RTFDC1 as a novel candidate effector gene.

Development of a Tremor Detection Algorithm for Use in an Academic Movement Disorders Center.

Tremor, defined as an "involuntary, rhythmic, oscillatory movement of a body part", is a key feature of many neurological conditions including Parkinson's disease and essential tremor. Clinical assessment continues to be performed by visual observation with quantification on clinical scales. Methodologies for objectively quantifying tremor are promising but remain non-standardized across centers. Our center performs full-body behavioral testing with 3D motion capture for clinical and research purposes in patients with Parkinson's disease, essential tremor, and other conditions. The objective of this study was to assess the ability of several candidate processing pipelines to identify the presence or absence of tremor in kinematic data from patients with confirmed movement disorders and compare them to expert ratings from movement disorders specialists. We curated a database of 2272 separate kinematic data recordings from our center, each of which was contemporaneously annotated as tremor present or absent by a movement physician. We compared the ability of six separate processing pipelines to recreate clinician ratings based on F1 score, in addition to accuracy, precision, and recall. The performance across algorithms was generally comparable. The average F1 score was 0.84±0.02 (mean ± SD; range 0.81-0.87). The second highest performing algorithm (cross-validated F1=0.87) was a hybrid that used engineered features adapted from an algorithm in longstanding clinical use with a modern Support Vector Machine classifier. Taken together, our results suggest the potential to update legacy clinical decision support systems to incorporate modern machine learning classifiers to create better-performing tools.

Orbits, Spirals, and Trapped States: Dynamics of a Phoretic Janus Particle in a Radial Concentration Gradient.

A long-standing goal in colloidal active matter is to understand how gradients in fuel concentration influence the motion of phoretic Janus particles. Here, we present a theoretical description of the motion of a spherical phoretic Janus particle in the presence of a radial gradient of the chemical solute driving self-propulsion. Radial gradients are a geometry relevant to many scenarios in active matter systems and naturally arise due to the presence of a point source or sink of fuel. We derive an analytical solution for the Janus particle's velocity and quantify the influence of the radial concentration gradient on the particle's trajectory. Compared to a phoretic Janus particle in a linear gradient in fuel concentration, we uncover a much richer set of dynamic behaviors including circular orbits and trapped stationary states. We identify the ratio of the phoretic mobilities between the two domains of the Janus particle as a central quantity in tuning their dynamics. Our results provide a path for developing optimum protocols for tuning the dynamics of phoretic Janus particles and mixing fluid at the microscale. In addition, this work suggests a method for quantifying the surface properties of phoretic Janus particles, which have proven to be challenging to probe experimentally.

3D chromatin-based variant-to-gene maps across 57 human cell types reveal the cellular and genetic architecture of autoimmune disease susceptibility.

A portion of the genetic basis for many common autoimmune disorders has been uncovered by genome-wide association studies (GWAS), but GWAS do not reveal causal variants, effector genes, or the cell types impacted by disease-associated variation. We have generated 3D genomic datasets consisting of promoter-focused Capture-C, Hi-C, ATAC-seq, and RNA-seq and integrated these data with GWAS of 16 autoimmune traits to physically map disease-associated variants to the effector genes they likely regulate in 57 human cell types. These 3D maps of gene cis-regulatory architecture are highly powered to identify the cell types most likely impacted by disease-associated genetic variation compared to 1D genomic features, and tend to implicate different effector genes than eQTL approaches in the same cell types. Most of the variants implicated by these cis-regulatory architectures are highly trait-specific, but nearly half of the target genes connected to these variants are shared across multiple autoimmune disorders in multiple cell types, suggesting a high level of genetic diversity and complexity among autoimmune diseases that nonetheless converge at the level of target gene and cell type. Substantial effector gene sharing led to the common enrichment of similar biological networks across disease and cell types. However, trait-specific pathways representing potential areas for disease-specific intervention were identified. To test this, we pharmacologically validated squalene synthase, a cholesterol biosynthetic enzyme encoded by the FDFT1 gene implicated by our approach in MS and SLE, as a novel immunomodulatory drug target controlling inflammatory cytokine production by human T cells. These data represent a comprehensive resource for basic discovery of gene cis-regulatory mechanisms, and the analyses reported reveal mechanisms by which autoimmune-associated variants act to regulate gene expression, function, and pathology across multiple, distinct tissues and cell types.

Prevalence and clinical features of cervical dystonia in Parkinson's disease.

OBJECTIVE: To study the prevalence and clinical features of cervical dystonia in Parkinson's disease (CD-PD). BACKGROUND: PD features various forms of dystonia, including CD. Yet, the prevalence and clinical features of CD in PD patients are not well-characterized. METHODS: We conducted a single site, prospective study where consecutively evaluated PD patients were examined for the presence of CD to ascertain its prevalence. For each case of CD-PD, a standardized questionnaire assessing demographic and clinical features was completed. Statistical analysis was performed to compare CD-PD characteristics to those of a previously published large idiopathic CD cohort. RESULTS: Of 301 consecutive PD patients evaluated, 28 (9.3 %) had CD, far surpassing estimates of CD prevalence in the general population. This CD-PD cohort was predominantly male (71 %) with a mean age of 70.9 ± 8.1 years. The mean duration of PD was 10.4 ± 6.7 years. In most cases (n = 19, 68 %), CD developed after the onset of PD. Five patients reported dystonia improvements in response to levodopa, while none reported medication-induced worsening. In contrast to CD-PD, those with ICD (n = 209) were on average younger (59.7 ± 10.1) and mostly female (74 %, p < 0.001). In addition, CD-PD was overall less severe as measured by the Global Dystonia Rating Scale (GDRS) (p = 0.002) and featured less head tremor and pain. CONCLUSION: Our findings indicate CD is overrepresented in PD compared to the general population and has clinical features distinct from those of ICD. These results justify larger, more comprehensive studies of CD-PD to better understand its frequency, pathophysiology, clinical characteristics, and associated risk factors.

Seamless site-directed mutagenesis in complex cloned DNA sequences using the RedEx method.

Seamless site-directed mutagenesis is an important technique for studying protein functions, tuning enzyme catalytic activities and modifying genetic elements in multiple rounds because it can insert, delete or substitute nucleotides, DNA segments or even entire genes at the target site without introducing any unwanted change. To facilitate seamless site-directed mutagenesis in large plasmids and bacterial artificial chromosomes (BACs) with repetitive sequences, we recently developed the RedEx strategy. Compared with previous methods, our approach achieves the recovery of correct recombinants with high accuracy by circumventing unwanted recombination between repetitive sequences. RedEx readily yields more than 80% accuracy in seamless DNA insertion and deletion in large multimodular polyketide synthase gene clusters, which are among the most difficult targets due to the large number of repetitive DNA sequences in modules encoding almost identical enzymes. Here we present the RedEx method by describing in detail the seamless site-directed mutagenesis in a BAC vector. Overall, the process includes three parts: (1) insertion of the RedEx cassette containing the desired mutation together with selection-counterselection markers flanked by unique restriction sites and 20-bp overlapping sequences into the target site by recombineering, (2) removal of the selection-counterselection markers in the BAC by restriction digestion and (3) circularization of the linear BAC by exonuclease-mediated in vitro DNA annealing. This protocol can be performed within 3 weeks and will enable researchers with DNA cloning experience to master seamless site-directed mutagenesis to accelerate their research.

Shared and unique 3D genomic features of substance use disorders across multiple cell types.

Recent genome-wide association studies (GWAS) have revealed shared genetic components among alcohol, opioid, tobacco and cannabis use disorders. However, the extent of the underlying shared causal variants and effector genes, along with their cellular context, remain unclear. We leveraged our existing 3D genomic datasets comprising high-resolution promoter-focused Capture-C/Hi-C, ATAC-seq and RNA-seq across >50 diverse human cell types to focus on genomic regions that coincide with GWAS loci. Using stratified LD regression, we determined the proportion of genomewide SNP heritability attributable to the features assayed across our cell types by integrating recent GWAS summary statistics for the relevant traits: alcohol use disorder (AUD), tobacco use disorder (TUD), opioid use disorder (OUD) and cannabis use disorder (CanUD). Statistically significant enrichments (P<0.05) were observed in 14 specific cell types, with heritability reaching 9.2-fold for iPSC-derived cortical neurons and neural progenitors, confirming that they are crucial cell types for further functional exploration. Additionally, several pancreatic cell types, notably pancreatic beta cells, showed enrichment for TUD, with heritability enrichments up to 4.8-fold, suggesting genomic overlap with metabolic processes. Further investigation revealed significant positive genetic correlations between T2D with both TUD and CanUD (FDR<0.05) and a significant negative genetic correlation with AUD. Interestingly, after partitioning the heritability for each cell type's cis-regulatory elements, the correlation between T2D and TUD for pancreatic beta cells was greater (r=0.2) than the global genetic correlation value. Our study provides new genomic insights into substance use disorders and implicates cell types where functional follow-up studies could reveal causal variant-gene mechanisms underpinning these disorders.

Severe sexual abuse in childhood and altered neurophysiological response to reward in female adults.

BACKGROUND: A relatively understudied but growing body of research indicates that individuals with a history of childhood trauma exhibit altered reward processing in adulthood. Research to date has focused on adversity broadly, with studies typically finding evidence of blunted response to rewards in adults with a history of childhood trauma. OBJECTIVE: Given the role of reward processing in risk for psychopathology and the particularly pathogenic nature of sexual abuse (SA), the present study sought to assess whether adults with a history of severe childhood SA exhibit altered neurophysiological response to rewards. PARTICIPANTS AND SETTING: Female adults (N = 105) were included from two study sites that used the same measures of childhood trauma (Childhood Trauma Questionnaire, CTQ), reward processing (Doors Task), and psychopathology (SCID). METHODS: Based on participants' CTQ and SCID responses, three groups were created: Severe SA (n = 36), Clinical Match (with comparable lifetime psychopathology but no-to-minimal SA history; n = 35), and Healthy Controls (n = 34). Group differences in RewP amplitude were assessed. RESULTS: The Severe SA group exhibited larger reward positivity (RewP) amplitude to monetary rewards than the Clinical Match and Healthy Control groups (partial ƞ2 = 0.06, p = .047). This effect remained after covarying for severity of other forms of childhood trauma. CONCLUSIONS: Our study found that severe SA in childhood was related to a heightened response to reward in adulthood. Furthermore, this was not attributable to the severity of other forms of early trauma or comorbid psychopathology. Future studies are needed to identify how heightened reward processing following severe childhood SA may be implicated in the onset and course of psychopathology.

Exploring a mother's engagement in team sport: An application of an extended theory of planned behaviour.

OBJECTIVES: Mothers of young children are at-risk for low physical activity. Organised team sport provides additional social and mental health benefits above that of physical activity. To better understand engagement in team sport, this study aimed to apply the theory of planned behaviour, with the addition of maternal identity and social support. DESIGN: A prospective two-part online study was conducted two weeks apart to collect data from 122 mothers in Australia (M = 32.22, SD = 4.42). METHODS: Two hierarchical multiple regression analyses were conducted to predict intention and engagement in team sports. A moderation was conducted to determine the effect of social support on the intention-behaviour relationship. RESULTS: Results indicated that 65.1 % of the variance in intention was explained, with years since previously played (ß = -0.17), attitudes (ß = 0.20), subjective norms (ß = 0.41), and perceived behavioural control (ß = 0.16) being significant, and level of team sport social support, and maternal identity being non-significant. Additionally, 65.7 % of the variance in behaviour was explained, with perceived behavioural control (ß = 0.28) and intention (ß = 0.50) being significant, and years since previously played and social supportbeing non-significant. Social support moderated the relationship between intention and engagement, such that social support facilitated engagement. CONCLUSIONS: The findings show support for the predictive ability of the theory and mothers within a sporting context. Behaviour change techniques related to strengthening intention and personal agency could be used to inform interventions intending to increase team sports participation.

A natural experiment during lockdown and on-going care-home COVID-19 outbreaks showed a single dose of vaccine reduced hospitalisation and deaths of care-home residents in North West England.

OBJECTIVE: To examine the effectiveness of one dose of the COVID-19 vaccine on care-home residents. STUDY DESIGN: Natural experiment. METHODS: We compared the effectiveness of single doses of Pfizer/BioNTech BNT162b2 (effective at 10 days) and AstraZeneca ChAdOx1 (effective at 14 days) vaccines in vaccinated and control (unvaccinated) homes. Using routine data, all care-homes reporting COVID-19 outbreaks between 11/12/2020 and 12/3/2021 in a sub-region of North West England were included. RESULTS: Of 126 care-homes (4042 residents), with outbreaks, 55 (44%, 1686 residents) reported onset dates before vaccination commenced; 38 (30%, 1304 residents) reported onset < 10 (BNT162b2) and < 14 days (ChAdOx1) after vaccine administration; and 33 (26%, 1052 residents) reported onset > 10 (BNT162b2) and > 14 (ChAdOx1) days after vaccination. Eighty-nine (71%) homes used ChAdOx1 vaccine. A single dose of vaccine before the outbreak onset significantly lowered the risk of symptoms (reduced by 48%), positivity (by 65%), hospitalisation (by 68%), and death (by 81%). Some vaccine effectiveness was also noted in care-homes that received one dose of vaccine within 10-14 days of outbreak onset. The number needed to vaccinate to prevent one resident from COVID-19-related hospitalisation was 34, and death was 17. CONCLUSIONS: This real-world, natural experiment adds to the evidence of COVID-19 vaccine effectiveness from different studies using varying designs. In the context of lockdown's impact on infection rates and on-going care-home outbreaks, a single dose of either ChAdOx1 or BNT162b2 vaccine had a significant impact on reducing COVID-19 related hospitalisation and death in care-home residents. Natural experiments should be used more in public health.

Endometrial Cancer: Analysing Patterns of Recurrence and Real-Life Outcome Data Using the 2020 ESGO-ESTRO-ESP Risk Stratification System.

AIMS: To evaluate patterns of recurrence and explore the prognostic differences between the 2018 FIGO staging system and the 2020 ESGO-ESTRO-ESP risk stratification system of endometrial cancer with an emphasis on early-stage disease. BACKGROUND: The incidence of endometrial cancer has risen by around 60% since the 90's. It is projected that by 2035 endometrial cancer will be the sixth most common cause of cancer-related death amongst females. METHODS: This was a retrospective cohort study which included patients treated between 2010 and 2017. Primary endpoints were overall survival (OS) and recurrence-free survival (RFS). Kaplan-Meyer survival analysis was used to assess OS and RFS across different risk groups. Cox proportional hazards regression was used to evaluate prognostic risk factors implicated in recurrence. Different recurrence patterns across the subgroups were analysed with Pearson's chi-square test. RESULTS: The study included 692 patients with a recurrence rate of 14.9%. The median time to recurrence was 17.1 months (IQR:8.8-28.4). The mean OS varied between 97.2 months in the low-risk group to 63.1 months in the high-risk group (p < 0.001). Mean RFS was 96.1 in the low-risk group and 58.9 in the high-risk group (p < 0.001). RFS was predicted by the following factors; high risk group (OR=3.87; p = 0.041), LVSI (OR=2.54, p = 0.005), carcinosarcoma (OR=2.20, p = 0.021) and serous subtype (OR=1.91, p = 0.01). Logistic regression was used to evaluate risk factors for loco-regional and distant recurrence. Patients in the low-risk group were less likely to have distant recurrence (OR=0.08, p = 0.004). Similarly, negative LVSI and Grade 1 cancers were associated with decreased risk of distant recurrence (OR=0.34, p = 0.006 and OR=0.33, p = 0.007, respectively). There were no significant risk factors for loco-regional recurrence. CONCLUSIONS: The 2020 ESGO-ESTRO-ESP risk stratification provides accurate estimates of recurrence risk and survival. Those treated in line with current guidance have significantly better outcomes.

Deutetrabenazine Provides Long-Term Benefit for Tardive Dyskinesia Regardless of Underlying Condition and Dopamine Receptor Antagonist Use: A Post Hoc Analysis of the 3-Year, Open-Label Extension Study.

BACKGROUND: Deutetrabenazine is approved for adults with tardive dyskinesia (TD). Data based on underlying psychiatric condition and baseline dopamine receptor antagonist (DRA) use are limited. METHODS: Patients with TD who completed parent studies ARM-TD or AIM-TD were eligible for the 3-year, open-label extension study (RIM-TD; NCT02198794). In RIM-TD, deutetrabenazine was titrated based on dyskinesia control and tolerability. In this post hoc analysis of RIM-TD, total motor Abnormal Involuntary Movement Scale (AIMS) score and adverse events (AEs) were analyzed by underlying condition and DRA use at parent study baseline. RESULTS: Of 343 patients enrolled in RIM-TD, 336 were included in the analysis by underlying condition, and 337 were included in the analysis by DRA use. One hundred eighty-nine of 205 (92%) patients with psychotic disorders (schizophrenia/schizoaffective disorder) and 65 of 131 (50%) with mood and other disorders (depression/bipolar disorder/other) were receiving a DRA. Mean (SE) deutetrabenazine doses at week 145 were 40.4 (1.13), 38.5 (1.21), 39.9 (1.00), and 38.5 (1.48) mg/d for patients with psychotic disorders, those with mood and other disorders, and those receiving DRAs or not, respectively. Mean (SD) changes in total motor AIMS score from this study baseline to week 145 were -6.3 (4.53), -7.1 (4.92), -6.1 (4.42), and -7.5 (5.19). Exposure-adjusted incidence rates (number of AEs/patient-years) of AEs were similar across groups: any (1.02, 1.71, 1.08, 1.97), serious (0.10, 0.12, 0.10, 0.12), and leading to discontinuation (0.07, 0.05, 0.06, 0.05). CONCLUSIONS: Long-term deutetrabenazine provided clinically meaningful improvements in TD-related movements, with a favorable benefit-risk profile, regardless of underlying condition or DRA use.

Brain activity during reappraisal and associations with psychotherapy response in social anxiety and major depression: a randomized trial.

BACKGROUND: Cognitive behavioral therapy (CBT) is an effective treatment for patients with social anxiety disorder (SAD) or major depressive disorder (MDD), yet there is variability in clinical improvement. Though prior research suggests pre-treatment engagement of brain regions supporting cognitive reappraisal (e.g. dorsolateral prefrontal cortex [dlPFC]) foretells CBT response in SAD, it remains unknown if this extends to MDD or is specific to CBT. The current study examined associations between pre-treatment neural activity during reappraisal and clinical improvement in patients with SAD or MDD following a trial of CBT or supportive therapy (ST), a common-factors comparator arm. METHODS: Participants were 75 treatment-seeking patients with SAD (n = 34) or MDD (n = 41) randomized to CBT (n = 40) or ST (n = 35). Before randomization, patients completed a cognitive reappraisal task during functional magnetic resonance imaging. Additionally, patients completed clinician-administered symptom measures and a self-report cognitive reappraisal measure before treatment and every 2 weeks throughout treatment. RESULTS: Results indicated that pre-treatment neural activity during reappraisal differentially predicted CBT and ST response. Specifically, greater trajectories of symptom improvement throughout treatment were associated with less ventrolateral prefrontal cortex (vlPFC) activity for CBT patients, but more vlPFC activity for ST patients. Also, less baseline dlPFC activity corresponded with greater trajectories of self-reported reappraisal improvement, regardless of treatment arm. CONCLUSIONS: If replicated, findings suggest individual differences in brain response during reappraisal may be transdiagnostically associated with treatment-dependent improvement in symptom severity, but improvement in subjective reappraisal following psychotherapy, more broadly.

The effects of family support and smartphone-derived homestay on daily mood and depression among sexual and gender minority adolescents.

Sexual and gender minority (SGM) adolescents are at elevated risk for depression. This risk is especially pronounced among adolescents whose home environment is unsupportive or nonaffirming, as these adolescents may face familial rejection due to their identity. Therefore, it is critical to better understand the mechanisms underlying this risk by probing temporally sensitive associations between negative mood and time spent in potentially hostile home environments. The current study included adolescents (N = 141; 43% SGM; 13-18 years old), oversampled for depression history, who completed clinical interviews assessing lifetime psychiatric history and depression severity as well as self-report measures of social support. Participants also installed an app on their personal smartphones, which assessed their daily mood and geolocation-determined mobility patterns over a 6-month follow-up period. Over the 6-month follow-up period, SGM adolescents reported elevated depression severity and lower daily mood relative to non-SGM youth. Interestingly, SGM adolescents who reported low family support experienced lower daily mood than non-SGM adolescents, particularly on days when they spent more time at home. Current findings reinforce evidence for disparities in depression severity among SGM adolescents and highlight family support as a key factor. Specifically, more time spent in home environments with low family support was associated with worse mood among SGM adolescents. These results underscore the need for clinical interventions to support SGM youth, particularly interventions that focus on familial relationships and social support within the home environment. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

CRISPR-Cas13b-mediated suppression of HBV replication and protein expression.

BACKGROUND & AIMS: New antiviral approaches that target multiple aspects of the HBV replication cycle to improve rates of functional cure are urgently required. HBV RNA represents a novel therapeutic target. Here, we programmed CRISPR-Cas13b endonuclease to specifically target the HBV pregenomic RNA and viral mRNAs in a novel approach to reduce HBV replication and protein expression. METHODS: Cas13b CRISPR RNAs (crRNAs) were designed to target multiple regions of HBV pregenomic RNA. Mammalian cells transfected with replication competent wild-type HBV DNA of different genotypes, a HBV-expressing stable cell line, a HBV infection model and a hepatitis B surface antigen (HBsAg)-expressing stable cell line were transfected with PspCas13b-BFP (blue fluorescent protein) and crRNA plasmids, and the impact on HBV replication and protein expression was measured. Wild-type HBV DNA, PspCas13b-BFP and crRNA plasmids were simultaneously hydrodynamically injected into mice, and serum HBsAg was measured. PspCas13b mRNA and crRNA were also delivered to a HBsAg-expressing stable cell line via lipid nanoparticles and the impact on secreted HBsAg determined. RESULTS: Our HBV-targeting crRNAs strongly suppressed HBV replication and protein expression in mammalian cells by up to 96% (p <0.0001). HBV protein expression was also reduced in a HBV-expressing stable cell line and in the HBV infection model. CRISPR-Cas13b crRNAs reduced HBsAg expression by 50% (p <0.0001) in vivo. Lipid nanoparticle-encapsulated PspCas13b mRNA reduced secreted HBsAg by 87% (p = 0.0168) in a HBsAg-expressing stable cell line. CONCLUSIONS: Together, these results show that CRISPR-Cas13b can be programmed to specifically target and degrade HBV RNAs to reduce HBV replication and protein expression, demonstrating its potential as a novel therapeutic option for chronic HBV infection. IMPACT AND IMPLICATIONS: Owing to the limitations of current antiviral therapies for hepatitis B, there is an urgent need for new treatments that target multiple aspects of the HBV replication cycle to improve rates of functional cure. Here, we present CRISPR-Cas13b as a novel strategy to target HBV replication and protein expression, paving the way for its development as a potential new treatment option for patients living with chronic hepatitis B.

Morphological and molecular preservation through universal preparation of fresh-frozen tissue samples for multimodal imaging workflows.

The landscape of tissue-based imaging modalities is constantly and rapidly evolving. While formalin-fixed, paraffin-embedded material is still useful for histological imaging, the fixation process irreversibly changes the molecular composition of the sample. Therefore, many imaging approaches require fresh-frozen material to get meaningful results. This is particularly true for molecular imaging techniques such as mass spectrometry imaging, which are widely used to probe the spatial arrangement of the tissue metabolome. As high-quality fresh-frozen tissues are limited in their availability, any sample preparation workflow they are subjected to needs to ensure morphological and molecular preservation of the tissues and be compatible with as many of the established and emerging imaging techniques as possible to obtain the maximum possible insights from the tissues. Here we describe a universal sample preparation workflow, from the initial step of freezing the tissues to the cold embedding in a new hydroxypropyl methylcellulose/polyvinylpyrrolidone-enriched hydrogel and the generation of thin tissue sections for analysis. Moreover, we highlight the optimized storage conditions that limit molecular and morphological degradation of the sections. The protocol is compatible with human and plant tissues and can be easily adapted for the preparation of alternative sample formats (e.g., three-dimensional cell cultures). The integrated workflow is universally compatible with histological tissue analysis, mass spectrometry imaging and imaging mass cytometry, as well as spatial proteomic, genomic and transcriptomic tissue analysis. The protocol can be completed within 4 h and requires minimal prior experience in the preparation of tissue samples for multimodal imaging experiments.

Variant-to-function analysis of the childhood obesity chr12q13 locus implicates rs7132908 as a causal variant within the 3' UTR of FAIM2.

The ch12q13 locus is among the most significant childhood obesity loci identified in genome-wide association studies. This locus resides in a non-coding region within FAIM2; thus, the underlying causal variant(s) presumably influence disease susceptibility via cis-regulation. We implicated rs7132908 as a putative causal variant by leveraging our in-house 3D genomic data and public domain datasets. Using a luciferase reporter assay, we observed allele-specific cis-regulatory activity of the immediate region harboring rs7132908. We generated isogenic human embryonic stem cell lines homozygous for either rs7132908 allele to assess changes in gene expression and chromatin accessibility throughout a differentiation to hypothalamic neurons, a key cell type known to regulate feeding behavior. The rs7132908 obesity risk allele influenced expression of FAIM2 and other genes and decreased the proportion of neurons produced by differentiation. We have functionally validated rs7132908 as a causal obesity variant that temporally regulates nearby effector genes and influences neurodevelopment and survival.