Perspectives and Experiences of Public Safety Personnel Engaged in a Peer-Led Workplace Reintegration Program Post Critical Incident or Operational Stress Injury: A Qualitative Thematic Analysis.

INTRODUCTION: Public safety personnel (PSP) experience operational stress injuries (OSIs), which can put them at increased risk of experiencing mental health and functional challenges. Such challenges can result in PSP needing to take time away from the workplace. An unsuccessful workplace reintegration process may contribute to further personal challenges for PSP and their families as well as staffing shortages that adversely affect PSP organizations. The Canadian Workplace Reintegration Program (RP) has seen a global scale and spread in recent years. However, there remains a lack of evidence-based literature on this topic and the RP specifically. The current qualitative study was designed to explore the perspectives of PSP who had engaged in a Workplace RP due to experiencing a potentially psychologically injurious event or OSI. METHODS: A qualitative thematic analysis analyzed interview data from 26 PSP who completed the RP. The researchers identified five themes: (1) the impact of stigma on service engagement; (2) the importance of short-term critical incident (STCI) program; (3) strengths of RP; (4) barriers and areas of improvement for the RP; and (5) support outside the RP. DISCUSSION: Preliminary results were favorable, but further research is needed to address the effectiveness, efficacy, and utility of the RP. CONCLUSION: By addressing workplace reintegration through innovation and research, future initiatives and RP iterations can provide the best possible service and support to PSP and their communities.

Global prevalence of musculoskeletal pain in rural and urban populations. A systematic review with meta-analysis. Musculoskeletal pain in rural and urban populations.

INTRODUCTION: To systematically compare the global prevalence of musculoskeletal pain and care-seeking in rural and urban populations. METHODS: A systematic review with meta-analysis of observational studies reporting a direct comparison of rural and urban populations was conducted worldwide and included back, knee, hip, shoulder, neck pain and a broad diagnosis of 'musculoskeletal pain'. A search strategy combining terms related to 'prevalence', 'musculoskeletal pain' and 'rural' was used on the following databases: MEDLINE, Embase, CINAHL, Scopus, and rural and remote health from their inception to 1 June 2022. Random-effects meta-analysis was used to pool the data. Results were presented as odds ratios (OR) along with 95% confidence intervals (95% CI). RESULTS: A total of 42 studies from 24 countries were included with a total population of 489 439 participants. The quality scores for the included studies, using the modified Newcastle Ottawa Scale tool, showed an average score of 0.78/1, which represents an overall good quality. The pooled analysis showed statistically greater odds of hip (OR = 1.62, 95% CI = 1.23-2.15), shoulder (OR = 1.42, 95% CI = 1.06-1.90) and overall musculoskeletal pain (OR = 1.26, 95% CI = 1.08-1.47) in rural populations compared to urban populations. Although the odds of seeking treatment were higher in rural populations this relationship was not statistically significant (OR = 0.76, 95% CI = 0.55-1.03). CONCLUSION: Very low-certainty evidence suggests that musculoskeletal, hip and shoulder pain are more prevalent in rural than urban areas, although neck, back and knee pain, along with care-seeking, showed no significant difference between these populations. Strategies aimed to reduce the burden of musculoskeletal pain should consider the specific needs and limited access to quality evidence-based care for musculoskeletal pain of rural populations.

Neurexin 3 Regulates Synaptic Connections Between Central Amygdala Neurons and Excitable Cells of the Lateral Parabrachial Nucleus in Rats with Varicella Zoster Induced Orofacial Pain.

Introduction: Herpes Zoster in humans is the result of varicella zoster virus (VZV) infection. Injecting rats with varicella zoster virus produces pain similar to herpes zoster "shingles" pain in humans. . In a previous study, orofacial pain was induced by injecting the whisker pad of male rats with VZV and the pain response increased after attenuating neurexin 3 (Nrxn3) expression in the central amygdala. Neurons descend from the central amygdala to the lateral parabrachial nucleus and orofacial pain signals ascend to the lateral parabrachial nucleus. GABAergic neurons within the central amygdala regulate pain by inhibiting activity within the lateral parabrachial nucleus. Attenuating Nrxn3 expression in the central amygdala increased GABA release in the lateral parabrachial nucleus suggesting Nrxn3 controls pain by regulating GABA release. Nrxn3 can also control synaptic connections between neurons, and we hypothesized that Nrxn3 knockdown in the central amygdala would reduce the number of GABAergic synaptic connections in the lateral parabrachial nucleus and increase VZV associated pain. Methods: To test this idea, the number of synaptic connections between GABAergic cells of the central amygdala and excitatory or dynorphin positive neurons within the lateral parabrachial nucleus were quantitated after infusion of a virus expressing synaptophysin. Synaptophysin is a synaptic vesicle protein that labels neuronal synaptic connections. These connections were measured in rats with and without whisker pad injection of VZV and knockdown of Nrxn3 within the central amygdala. Orofacial pain was measured using a place escape avoidance paradigm. Results: GABAergic synaptic connections were reduced in the lateral parabrachial nucleus after Nrxn3 knockdown. Rats with a reduction in the number of connections had an increase in VZV associated orofacial pain. Immunostaining with the pain marker prodynorphin indicated that the reduction in GABAergic connections was primarily associated with prodynorphin positive neurons. Discussion: The results suggest Nrxn3 reduces VZV associated orofacial pain, in part, by enhancing synaptic connections between GABA cells of the central amygdala and pain neurons within the lateral parabrachial nucleus.

Promoting public engagement in interdisciplinary biological systems education by leveraging American sports-inspired bracket contests on social media and web.

The complexity of modern biology poses challenges in fostering interdisciplinary understanding, particularly between practicing scientists and the public. Furthermore, scientists often lack formal training in science communication, despite various motivations to engage the public. The science literacy of the public in the biological sciences can also vary across socio-economic and cultural backgrounds. Leveraging popular culture and informal learning practices to promote active learning offers promising avenues to enhance public understanding of biological systems. Organized sports hold collective recognition across various communities and cultures, serving as a means to bring people together. Notably, the NCAA March Madness event holds widespread national and international popularity, presenting an opportunity to laterally apply this concept to promote science communication within STEM and biology education. An educational social media and web-based contest tool was developed integrating NCAA-inspired brackets with animal biological systems concepts. The tool featured tournament-style matchups based on animal biological systems, interesting animal facts, and a voting system, all housed within a user-friendly interface. To encourage regular user access to the tool, graphic designs were developed for all social media posts to aid in visual recruitment to the voting website. Based on online metrics, the use of social media garnered repeat users across both the public and educators. The latter noted the tool's simplicity and informative content. Application of this social media and web-based bracket contest tool, which leverages informal settings for active learning for use in biology education, can foster science communication to engage audiences, improve comprehension, and promote interdisciplinary biology education.

Implementation of a Multimodal Enhanced Recovery Protocol in Ambulatory Anorectal Surgery: A Randomized Trial.

BACKGROUND: Few studies report outcomes for enhanced recovery pathways in ambulatory anorectal surgery. We hypothesize that an ambulatory anorectal enhanced recovery pathway with multimodal analgesia can reduce postoperative opioid use. OBJECTIVE: To compare postoperative opioid use in patients undergoing ambulatory anorectal surgery who receive multimodal analgesia vs. standard of care without multimodal analgesia. DESIGN: A prospective randomized trial of patients undergoing elective anal fistula or hemorrhoid surgery from September 2018 to May 2022. SETTING: Urban teaching hospital. PATIENTS: Adults aged 18 to 70 undergoing elective anal fistula or hemorrhoid surgery from September 2018 to May 2022. INTERVENTION: Multimodal enhanced recovery pathway including pre- and postoperative non-opioid analgesia with oral acetaminophen, gabapentin and ketolorac. MAIN OUTCOME MEASURES: Primary endpoint was oral opioid use during the first postoperative week. Secondary endpoints included maximum pain and nausea scores, adverse events and emergency room or hospital admissions during the first 30 days postoperatively. RESULTS: Of the 109 enrolled patients, 20 were lost to follow-up. The remaining 89 patients had a median age of 38 (range, 20-67) years and included 41 (46%) females. There were no significant differences between the enhanced recovery protocol (Arm E) and non-enhanced recovery protocol (Arm NE) groups in terms of preoperative and surgical characteristics. The study primary endpoint, oral MME use during the first week, was significantly higher among patients in the NE arm (79 mg; range, 0-600) than patients in the E arm (8 mg; range, 0-390) (p = 0.002). On subgroup analysis, both fistula and hemorrhoid surgery patients assigned to the NE arm took significantly higher oral MME in the first week than patients in the E arm. There was no significant difference in secondary endpoints. LIMITATIONS: Patients and providers were not blinded. Our findings are limited to hemorrhoid and fistula surgery and may not be applicable to other anorectal procedures. CONCLUSIONS: Enhanced recovery protocols including multimodal analgesia should be used in elective anal fistula and hemorrhoid surgery to decrease postoperative opioid use. See Video Abstract . TRIAL REGISTRATION NUMBER: ClinicalTrials.gov ID NCT03738904.

Microbial signatures and continuum in endometrial cancer and benign patients.

BACKGROUND: Endometrial cancer is a multifactorial disease with inflammatory, metabolic and potentially microbial cues involved in disease pathogenesis. The endometrial cancer microbiome has been poorly characterised so far and studies have often overestimated bacterial biomass due to lack of integration of appropriate contamination controls. There is also a scarcity of evidence on the functionality of microbial microenvironments in endometrial cancer. This work addresses that knowledge gap by interrogating the genuine, contamination-free microbial signatures in the female genital tract and rectum of women with endometrial cancer and the mechanistic role of microbiome on carcinogenic processes. RESULTS: Here we sampled different regions of the reproductive tract (vagina, cervix, endometrium, fallopian tubes and ovaries) and rectum of 61 patients (37 endometrial cancer; 24 benign controls). We performed 16S rRNA gene sequencing of the V1-V2 hypervariable regions and qPCR of the 16S rRNA gene to qualitatively and quantitatively assess microbial communities and used 3D benign and endometrial cancer organoids to evaluate the effect of microbial products of L. crispatus, which was found depleted in endometrial cancer patients following primary analysis, on endometrial cell proliferation and inflammation. We found that the upper genital tract of a subset of women with and without endometrial cancer harbour microbiota quantitatively and compositionally distinguishable from background contaminants. Endometrial cancer was associated with reduced cervicovaginal and rectal bacterial load together with depletion of Lactobacillus species relative abundance, including L. crispatus, increased bacterial diversity and enrichment of Porphyromonas, Prevotella, Peptoniphilus and Anaerococcus in the lower genital tract and endometrium. Treatment of benign and malignant endometrial organoids with L. crispatus conditioned media exerted an anti-proliferative effect at high concentrations but had minimal impact on cytokine and chemokine profiles. CONCLUSIONS: Our findings provide evidence that the upper female reproductive tract of some women contains detectable levels of bacteria, the composition of which is associated with endometrial cancer. Whether this is a cause or consequence of cancer pathophysiology and what is the functional significance of this finding remain to be elucidated to guide future screening tools and microbiome-based therapeutics. Video Abstract.

Association between miRNAs in serum at 10-14 gestational weeks and spontaneous preterm delivery.

INTRODUCTION: Preterm delivery (PTD) is the leading cause of death in children under 5 years of age. Cervical shortening detected by ultrasound can be used to predict PTD, but prediction is not perfect, and complementary diagnostic markers are needed. Recently, specific plasma microribonucleic acid (miRNAs) detected in early second trimester were shown to be associated with spontaneous PTD in high-risk women with a singleton pregnancy. The aim of this study was to explore to what extent these miRNAs are associated with spontaneous PTD and cervical length in a general population. MATERIAL AND METHODS: This study is a nested case-control study within the CERVIX study. The CERVIX study evaluated the ability of cervical length screening with transvaginal ultrasound to identify women at risk of PTD. In the present study, women who delivered spontaneously <34 weeks (n = 61) were compared with a control group of women who delivered at full term (39 + 0 to 40 + 6 gestational weeks, n = 205). Archived serum samples were analyzed with RT-qPCR for miRNA expression levels of let-7a-5p, miR-150-5p, miR-15b-5p, miR-185-5p, miR-191-5p, miR-19b-3p, miR-23a-3p, miR-374a-5p, and miR-93-5p. The mean relative expression was compared between the groups. Sub-analyses were performed for women delivering <32, <30, and <28 weeks versus the full-term group. RESULTS: The analyzed miRNAs were not significantly differentially expressed in women delivering <34 weeks compared to those delivering at full term. MiR-191-5p and miR-93-5p were significantly overexpressed in women who delivered <32 weeks, and further increase in fold change was observed with decreasing gestational age at delivery. The level of miR-15b-5p was significantly higher in women delivering at <30 weeks compared to those delivering at full term. CONCLUSIONS: Our study shows that overexpression of miR-93-5p, miR-15b-5p, and miR-191-5p in serum at early gestation is associated with spontaneous PTD in a general population. Further research is needed to evaluate the potential of these miRNAs as future biomarkers for spontaneous PTD, as well as their pathophysiological role in spontaneous PTD.

Reduced Likelihood of Hospitalization with the JN.1 or HV.1 SARS-CoV-2 Variants Compared to the EG.5 Variant.

Within a multi-state viral genomic surveillance program, we evaluated whether proportions of SARS-CoV-2 infections attributed to the JN.1 variant and to XBB-lineage variants (including HV.1 and EG.5) differed between inpatient and outpatient care settings during periods of cocirculation. Both JN.1 and HV.1 were less likely than EG.5 to account for infections among inpatients versus outpatients (aOR=0.60 [95% CI: 0.43-0.84; p=0.003] and aOR=0.35 [95% CI: 0.21-0.58; p<0.001], respectively). JN.1 and HV.1 variants may be associated with a lower risk of severe illness. The severity of COVID-19 may have attenuated as predominant circulating SARS-CoV-2 lineages shifted from EG.5 to HV.1 to JN.1.

Canonical and Noncanonical Functions of the BH3 Domain Protein Bid in Apoptosis, Oncogenesis, Cancer Therapeutics, and Aging.

Effective cancer therapy with limited adverse effects is a major challenge in the medical field. This is especially complicated by the development of acquired chemoresistance. Understanding the mechanisms that underlie these processes remains a major effort in cancer research. In this review, we focus on the dual role that Bid protein plays in apoptotic cell death via the mitochondrial pathway, in oncogenesis and in cancer therapeutics. The BH3 domain in Bid and the anti-apoptotic mitochondrial proteins (Bcl-2, Bcl-XL, mitochondrial ATR) it associates with at the outer mitochondrial membrane provides us with a viable target in cancer therapy. We will discuss the roles of Bid, mitochondrial ATR, and other anti-apoptotic proteins in intrinsic apoptosis, exploring how their interaction sustains cellular viability despite the initiation of upstream death signals. The unexpected upregulation of this Bid protein in cancer cells can also be instrumental in explaining the mechanisms behind acquired chemoresistance. The stable protein associations at the mitochondria between tBid and anti-apoptotic mitochondrial ATR play a crucial role in maintaining the viability of cancer cells, suggesting a novel mechanism to induce cancer cell apoptosis by freeing tBid from the ATR associations at mitochondria.

Characterization of Major Complications of Bridge Plating of Distal Radius Fractures at a Level I Trauma Center.

BACKGROUND: Comminuted, markedly displaced distal radius fractures can cause instability requiring advanced stabilization with dorsal bridge plating. However, published complication rates of bridge plating widely vary. We hypothesize that complications of bridge plating of distal radius fractures are more prevalent than published rates. METHODS: A retrospective review was performed on all patients at an academic level I trauma center treated with a bridge plate for a distal radius fracture from 2014 to 2022. RESULTS: Sixty-five wrists were included in the final analysis: average age 53 years, male 51%, average plate retention 4 months, and average follow-up 6 months. Carpal tunnel release (CTR) was performed at time of primary procedure in 7 (10%) cases. Radial height, radial inclination, dorsal tilt, and ulnar variance were all significantly improved (P < .001). Grip strength, flexion, extension, and supination were significantly limited (P < .03). Twenty-one patients (32%) developed 35 major complications requiring unplanned reoperation, including mechanical hardware-related complication (15%), deep infection (11%), nonunion/delayed union (9%), adhesions (6%), median neuropathy (6%), symptomatic arthritis (5%), and tendon rupture (2%). Plate breakage occurred in 3 patients (5%) and was always localized over the central drill holes of the bridge plate. CONCLUSIONS: Major complications for bridge plating of distal radius fractures were higher at our institution than previously published. Plate breakage should prompt reconsideration of plate design to avoid drill holes over the wrist joint. Signs and symptoms of carpal tunnel syndrome should be carefully assessed at initial presentation, and consideration for concomitant CTR should be strongly considered.

Multicultural Efficacy Beliefs in Higher Education: Examining University Instructors' Burnout and Mental Well-Being.

Canadian universities are experiencing a dramatic increase in enrollment of students from diverse backgrounds. Evidence suggests many educators are not prepared to teach in multicultural contexts. Educators' lack of preparedness to teach in such contexts may lead them to develop burnout, which can negatively impact their mental and professional well-being. However, self-efficacy beliefs may buffer against job burnout and promote mental well-being. Hence, multicultural efficacy is an important factor for teaching in multicultural settings. In this study, we examined the relationships of multicultural efficacy with university instructors' burnout and mental well-being. A total of 158 faculty and sessional instructors were recruited from four prominent higher education institutions in Canada. The results revealed that multicultural efficacy was significantly related to the Personal Accomplishment facet of burnout and mental well-being, even after controlling variance accounted for by demographics, job-related characteristics, teaching self-efficacy, and colour-blind racial attitudes. These findings indicate that domain specific multicultural efficacy and general teaching self-efficacy are distinct constructs. Further, findings may inform the development of training opportunities and diversity-related workshops to enhance university instructors' awareness of diversity, social justice issues, and multicultural efficacy to better equip them for instruction in multicultural classrooms.

Leaching of antibiotic resistance genes and microbial assemblages following poultry litter applications in karst and non-karst landscapes.

Antibiotic resistance is increasingly recognized as a critical challenge affecting human, animal, and environmental health. Yet, environmental dynamics and transport of antibiotic resistance genes (ARGs) and microbial communities in karst and non-karst leachate following poultry litter land applications are not well understood. This study investigates impacts of broiler poultry litter application on the proliferation of ARGs (tetW, qnrS, ermB, sulI, and blaCTX-M-32), class 1 integron (intI1 i), and alterations in microbial communities (16S rRNA) within karst derived soils, which are crucial and under-researched systems in the global hydrological cycle, and non-karst landscapes. Using large, intact soil columns (45 cm diam. × 100 cm depth) from karst and non-karst landscapes, the role of preferential flow and ARG transport in leachate was enumerated following surface application of poultry litter and simulated rain events. This research demonstrated that in poultry litter amended karst soils, ARG (i.e., ermB and tetW) abundance in leachate increased 1.5 times compared to non-karst systems (p < 0.05), highlighting the influence of geological factors on ARG proliferation. Notably, microbial communities in karst soil leachate exhibited increased diversity and abundance, suggesting a potential linkage between microbial composition and ARG presence. Further, our correlation and network analyses identified relationships between leachate ARGs, microbial taxa, and physicochemical properties, underscoring the complex interplay in these environmentally sensitive areas. These findings illuminate the critical role of karst systems in shaping ARG abundance and pollutant dispersal and microbial community dynamics, thus emphasizing the need for landscape-specific approaches in managing ARG dissemination to the environment. This study provides a deeper understanding of hydrogeological ARG dynamics but also lays the groundwork for future research and strategies to mitigate ARG dissemination through targeted manure applications across agricultural landscapes.

Mindfulness in the Clinic Waiting Room May Decrease Pain: Results from Three Pilot Randomized Controlled Trials.

Introduction: Mindfulness interventions can improve a broad range of patient outcomes, but traditional mindfulness-based interventions are time and resource intensive. Emerging evidence indicates brief, single-session mindfulness interventions can also improve patient outcomes, and brief mindfulness interventions can be embedded into medical care pathways with minimal disruption. However, the direct impact of a brief mindfulness intervention on patients' pain while waiting in the clinic waiting room remains unexamined. Objective: A series of three, pilot, randomized controlled trials (RCTs) were conducted to examine the impact of a brief, audio-recorded, mindfulness intervention on patients' pain in the clinic waiting room. Method: Study 1 examined an 8-min mindfulness recording delivered before a provider visit; Study 2 examined a 5-min mindfulness recording after a provider visit; and Study 3 examined a 4-min mindfulness recording before a provider visit. Time- and attention-matched control conditions were used in each study. Studies 1 and 2 were conducted in an academic cancer hospital. Study 3 was conducted at a walk-in orthopedic clinic. Pain intensity was measured in each of the three studies. Anxiety and depression symptoms were measured in Studies 2 and 3. Pain unpleasantness was measured in Study 3. Results: A brief (i.e., 4- to 8-min), audio-recorded mindfulness intervention decreased patients' pain intensity in the clinic waiting room, whether delivered before (Study 1 Cohen's d=1.01, Study 3 Cohen's d=0.39) or after (Study 2 Cohen's d=0.89) a provider visit. Mindfulness had a significant effect on anxiety symptoms in both studies in which it was measured. No effect on depression symptoms was observed. Conclusions: Results from these three pilot RCTs indicate brief, audio-recorded, mindfulness interventions may be capable of quickly decreasing clinical symptoms. As such, embedding brief, audio-recorded, mindfulness interventions in clinic waiting rooms may have the potential to improve patient outcomes. The continued investigation of this intervention approach is needed. Clinical Trial Registrations: NCT04477278 and NCT06099964.

Senataxin deficiency disrupts proteostasis through nucleolar ncRNA-driven protein aggregation.

Senataxin is an evolutionarily conserved RNA-DNA helicase involved in DNA repair and transcription termination that is associated with human neurodegenerative disorders. Here, we investigated whether Senataxin loss affects protein homeostasis based on previous work showing R-loop-driven accumulation of DNA damage and protein aggregates in human cells. We find that Senataxin loss results in the accumulation of insoluble proteins, including many factors known to be prone to aggregation in neurodegenerative disorders. These aggregates are located primarily in the nucleolus and are promoted by upregulation of non-coding RNAs expressed from the intergenic spacer region of ribosomal DNA. We also map sites of R-loop accumulation in human cells lacking Senataxin and find higher RNA-DNA hybrids within the ribosomal DNA, peri-centromeric regions, and other intergenic sites but not at annotated protein-coding genes. These findings indicate that Senataxin loss affects the solubility of the proteome through the regulation of transcription-dependent lesions in the nucleus and the nucleolus.

Insights into the structure-activity relationship of the anticancer compound ZJ-101: A role played by the amide moiety.

ZJ-101, a structurally simplified analog of marine natural product superstolide A, was previously designed and synthesized in our laboratory. In the present study four new analogs of ZJ-101 were designed and synthesized to investigate the structure-activity relationship of the acetamide moiety of the molecule. The biological evaluation showed that the amide moiety is important for the molecule's anticancer activity. Replacing the amide with other functional groups such as a sulfonamide group, a carbamate group, and a urea group resulted in the decrease in anticancer activity.

Thermochemistry of Gas-Phase Thermal Oxidation of C2 to C8 Perfluorinated Sulfonic Acids with Extrapolation to C16.

New ideal-gas thermochemistry Cp°(T), H°(T), S°(T), and G°(T) are predicted for 53 species involved in the thermal destruction of perfluorinated sulfonic acids (PFSAs) ranging from C2 to C8 in perfluorinated alkyl chain length. Species were selected by considering both the pyrolytic and oxidative pathways of PFSA destruction. After the sulfur-containing moieties are removed, subsequent reactions largely involve species from a prior set of thermochemistry for the thermal destruction of perfluorinated carboxylic acids (Ram et al., J. Phys. Chem. A, 2024, 128, 7, 1313-1326). Enthalpies of formation at 0 K are computed using a new isogyric reaction scheme. Rigid-rotor harmonic-oscillator partition functions were calculated over a 200-2500 K temperature range using rovibrational properties at G4 (≤C3S1 species) and M06-2X-D3(0)/def2-QZVPP (≥C4S1 species), employing the 1D hindered rotor approximation to correct for torsional modes. Seven-coefficient NASA polynomial fits are reported in standardized formats. Bond dissociation energies and important reaction equilibria are examined to provide insights into the reactivity of potentially persistent species. Extrapolated NASA polynomials are also systematically predicted for 126 species larger than C8/C8S1 in size, allowing reasonably accurate estimates of thermochemistry without the need for expensive electronic structure calculations.

Perturbation of the insomnia WDR90 genome-wide association studies locus pinpoints rs3752495 as a causal variant influencing distal expression of neighboring gene, PIG-Q.

Although genome-wide association studies (GWAS) have identified loci for sleep-related traits, they do not directly uncover the underlying causal variants and corresponding effector genes. The majority of such variants reside in non-coding regions and are therefore presumed to impact cis-regulatory elements. Our previously reported 'variant-to-gene mapping' effort in human induced pluripotent stem cell (iPSC)-derived neural progenitor cells (NPCs), combined with validation in both Drosophila and zebrafish, implicated phosphatidyl inositol glycan (PIG)-Q as a functionally relevant gene at the insomnia "WDR90" GWAS locus. However, importantly that effort did not characterize the corresponding underlying causal variant. Specifically, our previous 3D genomic datasets nominated a shortlist of three neighboring single nucleotide polymorphisms (SNPs) in strong linkage disequilibrium within an intronic enhancer region of WDR90 that contacted the open PIG-Q promoter. We sought to investigate the influence of these SNPs collectively and then individually on PIG-Q modulation to pinpoint the causal "regulatory" variant. Starting with gross level perturbation, deletion of the entire region in NPCs via CRISPR-Cas9 editing and subsequent RNA sequencing revealed expression changes in specific PIG-Q transcripts. Results from individual luciferase reporter assays for each SNP in iPSCs revealed that the region with the rs3752495 risk allele (RA) induced a ~2.5-fold increase in luciferase expression. Importantly, rs3752495 also exhibited an allele-specific effect, with the RA increasing the luciferase expression by ~2-fold versus the non-RA. In conclusion, our variant-to-function approach and in vitro validation implicate rs3752495 as a causal insomnia variant embedded within WDR90 while modulating the expression of the distally located PIG-Q.

Constitutive internalisation of EP2 differentially regulates G protein signalling.

The prostanoid G protein-coupled receptor (GPCR) EP2 is widely expressed and implicated in endometriosis, osteoporosis, obesity, pre-term labour and cancer. Internalisation and intracellular trafficking are critical for shaping GPCR activity, yet little is known regarding the spatial programming of EP2 signalling and whether this can be exploited pharmacologically. Using three EP2-selective ligands that favour activation of different EP2 pathways, we show that EP2 undergoes limited agonist-driven internalisation but is constitutively internalised via dynamin-dependent, ß-arrestin-independent pathways. EP2 was constitutively trafficked to early and very early endosomes (VEE), which was not altered by ligand activation. APPL1, a key adaptor and regulatory protein of the VEE, did not impact EP2 agonist-mediated cAMP. Internalisation was required for ~70% of the acute butaprost- and AH13205-mediated cAMP signalling, yet PGN9856i, a Gαs-biased agonist, was less dependent on receptor internalisation for its cAMP signalling, particularly in human term pregnant myometrial cells that endogenously express EP2. Inhibition of EP2 internalisation partially reduced calcium signalling activated by butaprost or AH13205 and had no effect on PGE2 secretion. This indicates an agonist-dependent differential spatial requirement for Gαs and Gαq/11 signalling and a role for plasma membrane-initiated Gαq/11-Ca2+-mediated PGE2 secretion. These findings reveal a key role for EP2 constitutive internalisation in its signalling and potential spatial bias in mediating its downstream functions. This, in turn, could highlight important considerations for future selective targeting of EP2 signalling pathways.

Risk Factors for Microscopic Disease Positivity at Ileocolic Resection Margins for Crohn's Disease.

INTRODUCTION: Interest in microscopic margin positivity during surgical resection of medical-refractory Crohn's disease has been renewed with multiple recent studies showing an association between microscopic margin positivity with disease recurrence. Our aim was to determine risk factors for microscopic margin disease positivity following ileocolic resection (ICR). MATERIALS AND METHODS: A prospectively-maintained database of patients with Crohn's disease undergoing ICR at a tertiary-referral center was queried. Margin positivity was defined as the presence of cryptitis, erosion, transmural inflammation with lymphoid aggregates, or architectural distortion at either ileal (proximal) or colonic (distal) margins. RESULTS: Amongst 584 patients, 97 patients had a positive microscopic margin (17%) of which 46% had a positive proximal margin, 17% had a positive distal margin, and 13% had both positive and distal margins. Using multivariable logistic regression analysis, index ICR was associated with less odds of positive margin (odds ratio [OR] 0.46, 95% confidence interval [CI] 0.24-0.89, p=0.02), and granuloma presence was associated with increased odds (OR 2.26, 95% CI 1.23-4.21, p=0.01). CONCLUSION: We found that repeat ileocolic resection and granuloma presence were predictors of microscopic margin disease.

Regulation of transcription patterns, poly(ADP-ribose), and RNA-DNA hybrids by the ATM protein kinase.

The ataxia telangiectasia mutated (ATM) protein kinase is a master regulator of the DNA damage response and also an important sensor of oxidative stress. Analysis of gene expression in ataxia-telangiectasia (A-T) patient brain tissue shows that large-scale transcriptional changes occur in patient cerebellum that correlate with the expression level and guanine-cytosine (GC) content of transcribed genes. In human neuron-like cells in culture, we map locations of poly(ADP-ribose) and RNA-DNA hybrid accumulation genome-wide with ATM inhibition and find that these marks also coincide with high transcription levels, active transcription histone marks, and high GC content. Antioxidant treatment reverses the accumulation of R-loops in transcribed regions, consistent with the central role of reactive oxygen species in promoting these lesions. Based on these results, we postulate that transcription-associated lesions accumulate in ATM-deficient cells and that the single-strand breaks and PARylation at these sites ultimately generate changes in transcription that compromise cerebellum function and lead to neurodegeneration over time in A-T patients.