A novel phosphorylation site involved in dissociating RAF kinase from the scaffolding protein 14-3-3 and disrupting RAF dimerization.

Rapidly accelerated fibrosarcoma (ARAF, BRAF, CRAF) kinase is central to the MAPK pathway (RAS-RAF-MEK-ERK). Inactive RAF kinase is believed to be monomeric, autoinhibited, and cytosolic, while activated RAF is recruited to the membrane via RAS-GTP, leading to the relief of autoinhibition, phosphorylation of key regulatory sites, and dimerization of RAF protomers. Although it is well known that active and inactive BRAF have differential phosphorylation sites that play a crucial role in regulating BRAF, key details are still missing. In this study, we report the characterization of a novel phosphorylation site, BRAFS732 (equivalent in CRAFS624), located in proximity to the C-terminus binding motif for the 14-3-3 scaffolding protein. At the C terminus, 14-3-3 binds to BRAFpS729 (CRAFpS621) and enhances RAF dimerization. We conducted mutational analysis of BRAFS732A/E and CRAFS624A/E and revealed that the phosphomimetic S→E mutant decreases 14-3-3 association and RAF dimerization. In normal cell signaling, dimerized RAF phosphorylates MEK1/2, which is observed in the phospho-deficient S→A mutant. Our results suggest that phosphorylation and dephosphorylation of this site fine-tune the association of 14-3-3 and RAF dimerization, ultimately impacting MEK phosphorylation. We further characterized the BRAF homodimer and BRAF:CRAF heterodimer and identified a correlation between phosphorylation of this site with drug sensitivity. Our work reveals a novel negative regulatory role for phosphorylation of BRAFS732 and CRAFS624 in decreasing 14-3-3 association, dimerization, and MEK phosphorylation. These findings provide insight into the regulation of the MAPK pathway and may have implications for cancers driven by mutations in the pathway.

Cross-streamline migration and near-wall depletion of elastic fibers in micro-channel flows.

The complex dynamics of elastic fibers in viscous fluids are central to many biological and industrial systems. Fluid-structure interactions underlying these dynamics govern the shape and transport of flexible fibers, and understanding these interactions can help tune flow properties in applications such as microfluidic separation, printing and clogging. In this work, we use slender-body theory to study micromechanical dynamics that arise from the coupling between the elastic backbone of a fiber and the local straining flow that contributes to filament flipping and cross-streamline migration. The resulting transverse drift is unbiased in either direction in simple shear flow. However, a non-uniform shear rate results in bias towards regions of high shear, which we connect to the shape transitions during flips. We discover a depletion layer that forms near the boundaries of pressure-driven channel flow due to the competition between such a cross-streamline drift and steric exclusion from the walls. Finally, we develop scaling laws for the curvature of filaments during flip events, demonstrating the origin of the drift bias in non-uniform flows, and confirm this behavior from our simulations. Put together, these results shed light on the role of a local and dominant coupling between elasticity and viscous resistance in dictating long-term dynamics and transport of elastic fibers in confined flows.

TBP facilitates RNA Polymerase I transcription following mitosis.

The TATA-box binding protein (TBP) is the sole transcription factor common in the initiation complexes of the three major eukaryotic RNA Polymerases (Pol I, II and III). Although TBP is central to transcription by the three RNA Pols in various species, the emergence of TBP paralogs throughout evolution has expanded the complexity in transcription initiation. Furthermore, recent studies have emerged that questioned the centrality of TBP in mammalian cells, particularly in Pol II transcription, but the role of TBP and its paralogs in Pol I transcription remains to be re-evaluated. In this report, we show that in murine embryonic stem cells TBP localizes onto Pol I promoters, whereas the TBP paralog TRF2 only weakly associates to the Spacer Promoter of rDNA, suggesting that it may not be able to replace TBP for Pol I transcription. Importantly, acute TBP depletion does not fully disrupt Pol I occupancy or activity on ribosomal RNA genes, but TBP binding in mitosis leads to efficient Pol I reactivation following cell division. These findings provide a more nuanced role for TBP in Pol I transcription in murine embryonic stem cells.

A neuroendocrine pathway modulating osmotic stress in Drosophila.

Environmental factors challenge the physiological homeostasis in animals, thereby evoking stress responses. Various mechanisms have evolved to counter stress at the organism level, including regulation by neuropeptides. In recent years, much progress has been made on the mechanisms and neuropeptides that regulate responses to metabolic/nutritional stress, as well as those involved in countering osmotic and ionic stresses. Here, we identified a peptidergic pathway that links these types of regulatory functions. We uncover the neuropeptide Corazonin (Crz), previously implicated in responses to metabolic stress, as a neuroendocrine factor that inhibits the release of a diuretic hormone, CAPA, and thereby modulates the tolerance to osmotic and ionic stress. Both knockdown of Crz and acute injections of Crz peptide impact desiccation tolerance and recovery from chill-coma. Mapping of the Crz receptor (CrzR) expression identified three pairs of Capa-expressing neurons (Va neurons) in the ventral nerve cord that mediate these effects of Crz. We show that Crz acts to restore water/ion homeostasis by inhibiting release of CAPA neuropeptides via inhibition of cAMP production in Va neurons. Knockdown of CrzR in Va neurons affects CAPA signaling, and consequently increases tolerance for desiccation, ionic stress and starvation, but delays chill-coma recovery. Optogenetic activation of Va neurons stimulates excretion and simultaneous activation of Crz and CAPA-expressing neurons reduces this response, supporting the inhibitory action of Crz. Thus, Crz inhibits Va neurons to maintain osmotic and ionic homeostasis, which in turn affects stress tolerance. Earlier work demonstrated that systemic Crz signaling restores nutrient levels by promoting food search and feeding. Here we additionally propose that Crz signaling also ensures osmotic homeostasis by inhibiting release of CAPA neuropeptides and suppressing diuresis. Thus, Crz ameliorates stress-associated physiology through systemic modulation of both peptidergic neurosecretory cells and the fat body in Drosophila.

A very unusual wide QRS tachycardia: VT or SVT?

INTRODUCTION: A 13 old girl presented with recurrent wide QRS tachycardia since she was 4. METHODS: An electrophysiologic study was performed. RESULTS: The electrophysiologic study showed that QRS complexes identical to those of the tachycardia could be elicited with premature atrial extrastimuli but with a shorter atrioventricular (AV) delay when the QRS was wide compared with narrow QRS complexes. The tachycardia was ablated at 9 o'clock on the tricuspid annulus demonstrating the presence of an atriofascicular fiber. CONCLUSION: We believe that this atypical behavior can be explained by AV nodal like longitudinal dissociation of a slowly conducting accessory pathway.

Novel series of tunable µOR modulators with enhanced brain penetration for the treatment of opioid use disorder, pain and neuropsychiatric indications.

Structural optimization of a previously reported agonist of µOR, PZM21 is described resulting in the discovery of a novel series of amides with at least 4-folds enhanced CNS penetration in rat. Furthermore, these efforts yielded compounds with varying levels of efficacy on the receptor ranging from high efficacy agonists such as compound 20 to antagonists, such as 24. The correlation between in vitro activation of µOR and relative activity in models of analgesia for these compounds is discussed. The compelling results obtained in these studies demonstrate the potential utility of these newly discovered compounds in the treatment of pain and opioid use disorder.

Ratio of carbon dioxide veno-arterial difference to oxygen arterial-venous difference is not associated with lactate decrease after fluid bolus in critically ill patients with hyperlactatemia: results from a prospective observational study.

BACKGROUND: High ratio of the carbon dioxide veno-arterial difference to the oxygen arterial-venous difference (PvaCO2/CavO2) is associated with fluid bolus (FB) induced increase in oxygen consumption (VO2). This study investigated whether PvaCO2/CavO2 was associated with decreases in blood-lactate levels FB in critically ill patients with hyperlactatemia. METHODS: This prospective observational study examined adult patients in the intensive care unit (ICU) with lactate levels > 1.5 mmol/L who received FBs. Blood-lactate levels were measured before and after FB under unchanged metabolic, respiratory, and hemodynamic conditions. The primary outcome was blood-lactate levels after FB. Significant decreases in blood-lactate levels were considered as blood-lactate levels < 1.5 mmol/L or a decrease of more than 10% compared to baseline. RESULTS: The study enrolled 40 critically ill patients, and their median concentration of blood lactate was 2.6 [IQR:1.9 - 3.8] mmol/L. There were 27 (68%) patients with PvaCO2/CavO2 ≥ 1.4 mmHg/ml, and 10 of them had an increase in oxygen consumption (dVO2) ≥ 15% after FB, while 13 (32%) patients had PvaCO2/CavO2 < 1.4 mmHg/ml before FB, and none of them had dVO2 ≥ 15% after FB. FB increased the cardiac index in patients with high and low preinfusion PvaCO2/CavO2 (13.4% [IQR: 8.3 - 20.2] vs. 8.8% [IQR: 2.9 - 17.4], p = 0.34). Baseline PvaCO2/CavO2 was not found to be associated with a decrease in blood lactate after FB (OR: 0.88 [95% CI: 0.39 - 1.98], p = 0.76). A positive correlation was observed between changes in blood lactate and baseline PvaCO2/CavO2 (r = 0.35, p = 0.02). CONCLUSIONS: In critically ill patients with hyperlactatemia, PvaCO2/CavO2 before FB cannot be used to predict decreases in blood-lactate levels after FB. Increased PvaCO2/CavO2 is associated with less decrease in blood-lactate levels.

Trajectories of psychological distress and social integration in newly resettled refugees: findings from the Building a New Life in Australia longitudinal study.

PURPOSE: Refugees are at greater risk of mental illness due to stressors encountered post-resettlement. However, few longitudinal studies have examined the within-person effects of these stressors, especially with respect to social integration. This study aims to examine what factors are associated with psychological distress in a longitudinal sample of refugees resettled in Australia. METHODS: This study used data from three Waves of the Building a New Life in Australia study, collected between 2013 and 2018. The eligible sample included 1881 adult respondents, clustered in 1175 households. We conducted multilevel mixed-effects growth modelling incorporating time-variant and time-invariant covariates with psychological distress, using the Kessler Psychological Distress Scale (K6). RESULTS: Rates of high psychological distress increased across the 5-year follow-up period. Social integration stressors (e.g. discrimination, lower sense of belonging, loneliness, lower English proficiency) were associated with higher levels of psychological distress over time. Refugees reporting loneliness not only had a greater risk of elevated psychological distress at each time point, but the difference in risk increased over each time point. Refugees who were exposed to traumatic events, older, female and of Middle Eastern background were also more likely to report higher levels of psychological distress over time. CONCLUSIONS: These findings highlight the importance of identifying refugees who may encounter difficulties with social integration in the early years of resettlement. Newly arrived refugees may benefit from longer-term resettlement programmes that address post-migratory stressors, particularly with regards to loneliness, to reduce the prevalence of elevated psychological distress during the early years of resettlement.

Protein complex stoichiometry and expression dynamics of transcription factors modulate stem cell division.

Stem cells divide and differentiate to form all of the specialized cell types in a multicellular organism. In the Arabidopsis root, stem cells are maintained in an undifferentiated state by a less mitotically active population of cells called the quiescent center (QC). Determining how the QC regulates the surrounding stem cell initials, or what makes the QC fundamentally different from the actively dividing initials, is important for understanding how stem cell divisions are maintained. Here we gained insight into the differences between the QC and the cortex endodermis initials (CEI) by studying the mobile transcription factor SHORTROOT (SHR) and its binding partner SCARECROW (SCR). We constructed an ordinary differential equation model of SHR and SCR in the QC and CEI which incorporated the stoichiometry of the SHR-SCR complex as well as upstream transcriptional regulation of SHR and SCR. Our model prediction, coupled with experimental validation, showed that high levels of the SHR-SCR complex are associated with more CEI division but less QC division. Furthermore, our model prediction allowed us to propose the putative upstream SHR regulators SEUSS and WUSCHEL-RELATED HOMEOBOX 5 and to experimentally validate their roles in QC and CEI division. In addition, our model established the timing of QC and CEI division and suggests that SHR repression of QC division depends on formation of the SHR homodimer. Thus, our results support that SHR-SCR protein complex stoichiometry and regulation of SHR transcription modulate the division timing of two different specialized cell types in the root stem cell niche.

Epigenetic factor siRNA screen during primary KSHV infection identifies novel host restriction factors for the lytic cycle of KSHV.

Establishment of viral latency is not only essential for lifelong Kaposi's sarcoma-associated herpesvirus (KSHV) infection, but it is also a prerequisite of viral tumorigenesis. The latent viral DNA has a complex chromatin structure, which is established in a stepwise manner regulated by host epigenetic factors during de novo infection. However, despite the importance of viral latency in KSHV pathogenesis, we still have limited information about the repertoire of epigenetic factors that are critical for the establishment and maintenance of KSHV latency. Therefore, the goal of this study was to identify host epigenetic factors that suppress lytic KSHV genes during primary viral infection, which would indicate their role in latency establishment. We performed an siRNA screen targeting 392 host epigenetic factors during primary infection and analyzed which ones affect the expression of the viral replication and transcription activator (RTA) and/or the latency-associated nuclear antigen (LANA), which are viral genes essential for lytic replication and latency, respectively. As a result, we identified the Nucleosome Remodeling and Deacetylase (NuRD) complex, Tip60 and Tip60-associated co-repressors, and the histone demethylase KDM2B as repressors of KSHV lytic genes during both de novo infection and the maintenance of viral latency. Furthermore, we showed that KDM2B rapidly binds to the incoming viral DNA as early as 8 hpi, and can limit the enrichment of activating histone marks on the RTA promoter favoring the downregulation of RTA expression even prior to the polycomb proteins-regulated heterochromatin establishment on the viral genome. Strikingly, KDM2B can also suppress viral gene expression and replication during lytic infection of primary gingival epithelial cells, revealing that KDM2B can act as a host restriction factor of the lytic cycle of KSHV during both latent and lytic infections in multiple different cell types.

TCR Repertoires of Thymic Conventional and Regulatory T Cells: Identification and Characterization of Both Unique and Shared TCR Sequences.

Thymic regulatory T cells (tTreg) are critical in the maintenance of normal T cell immunity and tolerance. The role of TCR in tTreg selection remains incompletely understood. In this study, we assessed TCRα and TCRß sequences of mouse tTreg and thymic conventional CD4+ T cells (Tconv) by high-throughput sequencing. We identified αß TCR sequences that were unique to either tTreg or Tconv and found that these were distinct as recognized by machine learning algorithm and by preferentially used amino acid trimers in αß CDR3 of tTreg. In addition, a proportion of αß TCR sequences expressed by tTreg were also found in Tconv, and machine learning classified the great majority of these shared αß TCR sequences as characteristic of Tconv and not tTreg. These findings identify two populations of tTreg, one in which the regulatory T cell fate is associated with unique properties of the TCR and another with TCR properties characteristic of Tconv for which tTreg fate is determined by factors beyond TCR sequence.

Prevalence of common mental disorders in adult Syrian refugees resettled in high income Western countries: a systematic review and meta-analysis.

BACKGROUND: The immense social upheaval and ongoing humanitarian crisis created by the 2011 war in Syria has forced millions of civilians to flee their homeland, many of whom seek refugee status in Western nations. Whilst it is known that the prevalence of mental illness is higher within refugee populations, this systematic review and meta-analysis aims to pool the prevalence rates of common mental disorders (namely posttraumatic stress disorder, depression and generalized anxiety disorder) in adult Syrian refugees resettled in high income Western countries. METHODS: Seven electronic databases (Medline, PsychInfo, CINAHL, PTSDpubs, SCOPUS, PubMed and Embase) were searched up to the 31st of December 2020. Using pre-determined inclusion and exclusion criteria, relevant articles were screened by title and abstract, and later by full text. A meta-analysis was used to estimate the prevalence rates for each mental illness. RESULTS: Eleven studies met the eligibility criteria for the systematic review. Nine of these studies had a low-moderate risk of bias and were included in the meta-analysis. Of the 4873 refugees included in the meta-analysis, the total pooled prevalence rate of having any of the three mental disorders was 33% (CI 95%, 27-40%), 40% for anxiety (CI 95%, 31-50%), 31% for depression (CI 95%, 20-44%) and 31% for PTSD (CI 95%, 22-41%). A meta-regression revealed that the total pooled prevalence rate for having any of the three mental disorders was not influenced by age, host country, duration in host country, educational or marital status. CONCLUSIONS: Despite significant study heterogeneity, the prevalence rates of common mental disorders in adult Syrian refugees resettled in high-income Western countries are significantly higher than reported rates in the general population.

Cytomegalovirus infection reduced CD70 expression, signaling and expansion of viral specific memory CD8+ T cells in healthy human adults.

BACKGROUND: Cytomegalovirus (CMV) infection leads to effector memory CD8+ T cell expansion and is associated with immune dysfunction in older adults. However, the molecular alterations of CMV-specific CD8+ T cells in CMV infected healthy young and middle-aged adults has not been fully characterized. RESULTS: We compared CD8+ T cells specific for a CMV epitope (pp65495-503, NLV) and an influenza A virus (IAV) epitope (M158-66, GIL) from the same young and middle-aged healthy adults with serum positive for anti-CMV IgG. Compared to the IAV-specific CD8+ T cells, CMV-specific CD8+ T cells contained more differentiated effector memory (TEM and TEMRA) cells. Isolated CMV-specific central memory (TCM) but not naïve (TN) cells had a significant reduced activation-induced expansion in vitro compared to their IAV-specific counterparts. Furthermore, we found that CD70 expression was reduced in CMV-specific CD28+CD8+ TCM and that CD70+ TCM had better expansion in vitro than did CD70- TCM. Mechanistically, we showed that CD70 directly enhanced MAPK phosphorylation and CMV-specific CD8+ TCM cells had a reduced MAPK signaling upon activation. Lastly, we showed that age did not exacerbate reduced CD70 expression in CMV- specific CD8+ TCM cells. CONCLUSION: Our findings showed that CMV infection causes mild expansion of CMV-NLV-specific CD8+ T cells, reduced CD70 expression and signaling, and proliferation of CMV-NLV-specific CD8+ TCM cells in young and middle-aged healthy adults and revealed an age-independent and CMV infection-specific impact on CD8+ memory T cells.

Partial Extraction Therapy: A Review of Human Clinical Studies.

Partial extraction therapy (PET) is a collective concept encompassing a group of surgical techniques including socket shield, root membrane, proximal shield, pontic shield, and root submergence. PET uses the patient's own root structure to maintain blood supply derived from the periodontal ligament complex to preserve the periodontium and peri-implant tissues during restorative and implant therapy. This review aims to summarize the current knowledge regarding PET techniques and present a comprehensive evaluation of human clinical studies in the literature. Two independent reviewers conducted electronic and manual searches until January 1, 2021, in the following electronic bibliographic databases: PubMed, EMBASE, and Dentistry & Oral Sciences Source. Gray literature was searched to identify additional candidates for potential inclusion. Articles were screened by a group of 4 reviewers using the Covidence software and synthesized. A systematic search of the literature yielded 5714 results. Sixty-four articles were selected for full-text assessment, of which 42 eligible studies were included in the review. Twelve studies were added to the synthesis after a manual search of the reference lists. A total of 54 studies were examined in this review. In sum, PET techniques offer several clinical advantages: (1) preservation of buccal bone postextraction and limitation of alveolar ridge resorption, (2) mitigation of the need for invasive ridge augmentation procedures, and (3) soft-tissue dimensional stability and high esthetic outcomes. Further randomized clinical studies with larger sample sizes are needed to improve the understanding of the long-term clinical outcomes of PET.

Cardiac resynchronization therapy pacemaker implant in a patient with ARTO mitral device in situ.

The ARTO device is a percutaneous device for functional mitral regurgitation composed of a transseptal anchor and a T-bar sitting in the coronary sinus which reduce the minor axis of the mitral valve. We present a case showing the technical feasibility of an LV lead implant in patients with an ARTO device in situ.

Outcomes of primary versus revisional robotically assisted laparoscopic Roux-en-Y gastric bypass: a multicenter analysis of ten-year experience.

BACKGROUND: Postoperative morbidity after laparoscopic bariatric surgery is considered higher for patients undergoing revisional versus primary procedures. The objective of this retrospective cohort study was to compare outcomes between patients undergoing primary versus revisional robotically assisted laparoscopic (RAL) Roux-en-Y gastric bypass (RYGB). METHODS: Data of all patients who underwent RAL primary and revisional RYGB between 2009 and 2019 at two accredited, high-volume bariatric surgery centers-the Memorial Hermann - Texas Medical Center, Houston, TX, and the Tower Health, Reading Hospital, Reading, PA, were analyzed. Primary outcomes were early (< 30 days) and overall postoperative complications. Secondary outcomes included intraoperative complications, operative times, conversions to laparotomy, length of hospital stay, early (< 30 days) postoperative readmissions and deaths. RESULTS: Data of 1072 patients were analyzed, including 806 primary and 266 revisional RAL RYGB procedures. Longer operative times (203 versus 154 min, P < 0.001), increased number of readmissions for oral intolerance (10.5% versus 6.7%, P = 0.046) and higher rate of gastrojejunal stricture (6.4% versus 2.7%, P = 0.013) were found in the revisional group. Gastrointestinal leak rates were 0.2% for the primary versus 1.1% for the revisional group (P = 0.101). Early (< 30 days) reoperations rates were 2.2% for the primary versus 1.1% for the revisional group (P = 0.318). There were no statistically significant differences between groups in overall and severe complication rates. CONCLUSION: Patients undergoing RAL primary and revisional RYGB had comparable overall outcomes, with a non-significant higher early complication rate in the revisional group. Despite the study being underpowered to detect differences in specific complication rates, the morbidity seen in the revisional RYGB group remains markedly below literature reports of revisional laparoscopic RYGB and might suggest a benefit of robotic assistance. Further prospective studies are needed to confirm these results.

COVID-19's impact on private practice and academic dentistry in North America.

The COVID-19 pandemic is a major public health crisis for countries around the world. In response to this global outbreak, the World Health Organization declared a public health emergency of international concern. Dental professionals are especially at high risk of contracting the COVID-19 virus due to the unique nature of dentistry, more specifically, exposure to aerosols and droplets. When it comes to dental emergencies, it was crucial to maintain urgent dental care services operational to help reduce the burden on our healthcare system and hospitals already under pressure. The COVID-19 pandemic has significantly impacted how dentistry is practiced in North America in both the private practice and academic settings. This article shares the perspectives of dentists practicing in private practice and clinician-researchers in academic dental institutions. More specifically, we discuss about measures implemented to minimize risks of disease transmission, challenges in emergency dental care, impact on patients, as well as impact on the professional and personal lives of the dental team during the COVID-19 crisis.

Resilience and Hope Among Yazidi Women Released From ISIS Enslavement.

ABSTRACT: This study aimed to explore the levels of resilience and hope among Yazidi women who survived captivity by Islamic State of Iraq and Syria (ISIS) and to examine its relationship with posttraumatic stress disorder (PTSD), generalized anxiety, and depressive symptoms. In this cross-sectional study, 139 formerly enslaved Yazidi women were assessed. The mean scores of resilience and hope were below the suggested cutoff means (M = 2.47, SD = 0.48, R = 1-5) and (M = 31.6, SD = 11.7, R = 8-64), respectively. Sociodemographic variables were not related to resilience and hope, other than those women who stayed in captivity for more than a 3-year period who reported significantly lower levels of hope (M = 28.36, SD = 11.69). Formerly enslaved Yazidi women who display higher levels of PTSD, generalized anxiety, and depression exhibit significantly lower levels of resilience and hope. Resilience and hope are therefore important concepts to explore in traumatized populations.

Use of Artificial Intelligence in Dentistry: Current Clinical Trends and Research Advances.

The field of artificial intelligence (AI) has experienced spectacular development and growth over the past two decades. With recent progress in digitized data acquisition, machine learning and computing infrastructure, AI applications are expanding into areas that were previously thought to be reserved for human experts. When applied to medicine and dentistry, AI has tremendous potential to improve patient care and revolutionize the health care field. In dentistry, AI is being investigated for a variety of purposes, specifically identification of normal and abnormal structures, diagnosis of diseases and prediction of treatment outcomes. This review describes some current and future applications of AI in dentistry.

Ventricular tachycardia secondary to leadless pacemaker implantation in the setting of myocardial ischaemia.

Leadless pacemakers are increasingly part of the electrophysiology practice and offer an interesting pacing alternative for patients who have a history of CIED infection. We present the case of a patient who developed life-threatening ventricular arrythmias after a leadless pacemaker implant, in the context of previously unrecognised coronary artery disease.