m6Am sequesters PCF11 to suppress premature termination and drive neuroblastoma differentiation.

N6,2'-O-dimethyladenosine (m6Am) is an abundant mRNA modification that impacts multiple diseases, but its function remains controversial because the m6Am reader is unknown. Using quantitative proteomics, we identified transcriptional terminator premature cleavage factor II (PCF11) as a m6Am-specific reader in human cells. Direct quantification of mature versus nascent RNAs reveals that m6Am does not regulate mRNA stability but promotes nascent transcription. Mechanistically, m6Am functions by sequestering PCF11 away from proximal RNA polymerase II (RNA Pol II). This suppresses PCF11 from dissociating RNA Pol II near transcription start sites, thereby promoting full-length transcription of m6Am-modified RNAs. m6Am's unique relationship with PCF11 means m6Am function is enhanced when PCF11 is reduced, which occurs during all-trans-retinoic-acid (ATRA)-induced neuroblastoma-differentiation therapy. Here, m6Am promotes expression of ATF3, which represses neuroblastoma biomarker MYCN. Depleting m6Am suppresses MYCN repression in ATRA-treated neuroblastoma and maintains their tumor-stem-like properties. Collectively, we characterize m6Am as an anti-terminator RNA modification that suppresses premature termination and modulates neuroblastoma's therapeutic response.

Life expectancy and geographic variation in mortality: an observational comparison study of six high-income Anglophone countries.

OBJECTIVE: To compare life expectancy levels and within-country geographic variation in life expectancy across six high-income Anglophone countries between 1990 and 2018. DESIGN: Demographic analysis using aggregated mortality data. SETTING: Six high-income Anglophone countries (USA, UK, Canada, Australia, Ireland and New Zealand), by sex, including an analysis of subnational geographic inequality in mortality within each country. POPULATION: Data come from the Human Mortality Database, the WHO Mortality Database and the vital statistics agencies of six high-income Anglophone countries. MAIN OUTCOME MEASURES: Life expectancy at birth and age 65; age and cause of death contributions to life expectancy differences between countries; index of dissimilarity for within-country geographic variation in mortality. RESULTS: Among six high-income Anglophone countries, Australia is the clear best performer in life expectancy at birth, leading its peer countries by 1.26-3.95 years for women and by 0.97-4.88 years for men in 2018. While Australians experience lower mortality across the age range, most of their life expectancy advantage accrues between ages 45 and 84. Australia performs particularly well in terms of mortality from external causes (including drug- and alcohol-related deaths), screenable/treatable cancers, cardiovascular disease and influenza/pneumonia and other respiratory diseases compared with other countries. Considering life expectancy differences across geographic regions within each country, Australia tends to experience the lowest levels of inequality, while Ireland, New Zealand and the USA tend to experience the highest levels. CONCLUSIONS: Australia has achieved the highest life expectancy among Anglophone countries and tends to rank well in international comparisons of life expectancy overall. It serves as a potential model for lower-performing countries to follow to reduce premature mortality and inequalities in life expectancy.

A PRMT5-ZNF326 axis mediates innate immune activation upon replication stress.

DNA replication stress (RS) is a widespread phenomenon in carcinogenesis, causing genomic instability and extensive chromatin alterations. DNA damage leads to activation of innate immune signaling, but little is known about transcriptional regulators mediating such signaling upon RS. Using a chemical screen, we identified protein arginine methyltransferase 5 (PRMT5) as a key mediator of RS-dependent induction of interferon-stimulated genes (ISGs). This response is also associated with reactivation of endogenous retroviruses (ERVs). Using quantitative mass spectrometry, we identify proteins with PRMT5-dependent symmetric dimethylarginine (SDMA) modification induced upon RS. Among these, we show that PRMT5 targets and modulates the activity of ZNF326, a zinc finger protein essential for ISG response. Our data demonstrate a role for PRMT5-mediated SDMA in the context of RS-induced transcriptional induction, affecting physiological homeostasis and cancer therapy.

A Review of Existing and New Treatments for the Management of Hand Eczema.

Hand eczema is a chronic condition that affects an estimated 14.5% of the general population. It has severe quality of life ramifications in those that struggle with it, including days missed from work or school, productivity loss and impaired work functioning. For years, the standard of care included topical moisturizing creams, topical steroids and more recently systemic agents. As new therapeutic targets emerge and recent advances are being developed, it is now more possible than ever that hand eczema can be managed via the underlying mechanisms. A review of the literature was conducted to identify current treatment options for hand eczema and chronic hand eczema. The terms 'hand eczema', 'hand dermatitis' were used to search PubMed, CENTRAL and Embase. To identify new therapies still undergoing investigation, we used the terms 'hand eczema', 'hand dermatitis', 'atopic dermatitis', and 'vesicular eczema of hands and/or feet' to search Clinicaltrials.gov for all studies until December 2022. There were 56 ongoing clinical trials identified for pharmacological treatments for hand eczema on Clinicaltrials.gov from 2000 - 2022, with 16 that are new or ongoing. These included studies for dupilumab, ruxolitinib, delgocitinib (LEO124249), gusacitinib (ASN002), AFX 5931, and roflumilast (ARQ-252). Two major classes of drugs emerging for the treatment of hand eczema include IL-4/IL-13 inhibitors and JAK inhibitors. With the increase in efficacy seen with these new drugs, we are also noting improved adverse effect profiles, making them attractive options to add to a clinician's management toolbox for patients with hand eczema.

Exploring the Quality of Narrative Feedback Provided to Residents During Ambulatory Patient Care in Medicine and Surgery.

OBJECTIVES: The transition to competency-based medical education (CBME) has increased the volume of residents' assessment data; however, the quality of the narrative feedback is yet to be used as feedback-on-feedback for faculty. Our objectives were (1) to explore and compare the quality and content of narrative feedback provided to residents in medicine and surgery during ambulatory patient care and (2) to use the Deliberately Developmental Organization framework to identify strengths, weaknesses, and opportunities to improve quality of feedback within CBME. METHODS: We conducted a mixed convergent methods study with residents from the Departments of Surgery (DoS; n = 7) and Medicine (DoM; n = 9) at Queen's University. We used thematic analysis and the Quality of Assessment for Learning (QuAL) tool to analyze the content and quality of narrative feedback documented in entrustable professional activities (EPAs) assessments for ambulatory care. We also examined the association between the basis of assessment, time to provide feedback, and the quality of narrative feedback. RESULTS: Forty-one EPA assessments were included in the analysis. Three major themes arose from thematic analysis: Communication, Diagnostics/Management, and Next Steps. Quality of the narrative feedback varied; 46% had sufficient evidence about residents' performance; 39% provided a suggestion for improvement; and 11% provided a connection between the suggestion and the evidence. There were significant differences between DoM and DoS in quality of feedback scores for evidence (2.1 [1.3] vs. 1.3 [1.1]; p < 0.01) and connection (0.4 [0.5] vs. 0.1 [0.3]; p = 0.04) domains of the QuAL tool. Feedback quality was not associated with the basis of assessment or time taken to provide feedback. CONCLUSION: The quality of the narrative feedback provided to residents during ambulatory patient care was variable with the greatest gap in providing connections between suggestions and evidence about residents' performance. There is a need for ongoing faculty development to improve the quality of narrative feedback provided to residents.

Smoking and the widening inequality in life expectancy between metropolitan and nonmetropolitan areas of the United States.

Background: Geographic inequality in US mortality has increased rapidly over the last 25 years, particularly between metropolitan and nonmetropolitan areas. These gaps are sizeable and rival life expectancy differences between the US and other high-income countries. This study determines the contribution of smoking, a key contributor to premature mortality in the US, to geographic inequality in mortality over the past quarter century. Methods: We used death certificate and census data covering the entire US population aged 50+ between Jan 1, 1990 and Dec 31, 2019. We categorized counties into 40 geographic areas cross-classified by region and metropolitan category. We estimated life expectancy at age 50 and the index of dissimilarity for mortality, a measure of inequality in mortality, with and without smoking for these areas in 1990-1992 and 2017-2019. We estimated the changes in life expectancy levels and percent change in inequality in mortality due to smoking between these periods. Results: We find that the gap in life expectany between metros and nonmetros increased by 2.17 years for men and 2.77 years for women. Changes in smoking-related deaths are responsible for 19% and 22% of those increases, respectively. Among the 40 geographic areas, increases in life expectancy driven by changes in smoking ranged from 0.91 to 2.34 years for men while, for women, smoking-related changes ranged from a 0.61-year decline to a 0.45-year improvement. The most favorable trends in years of life lost to smoking tended to be concentrated in large central metros in the South and Midwest, while the least favorable trends occurred in nonmetros in these same regions. Smoking contributed to increases in mortality inequality for men aged 70+, with the contribution ranging from 8 to 24%, and for women aged 50-84, ranging from 14 to 44%. Conclusions: Mortality attributable to smoking is declining fastest in large cities and coastal areas and more slowly in nonmetropolitan areas of the US. Increasing geographic inequalities in mortality are partly due to these geographic divergences in smoking patterns over the past several decades. Policies addressing smoking in non-metropolitan areas may reduce geographic inequality in mortality and contribute to future gains in life expectancy.

Transitioning to virtual ambulatory care during the COVID-19 pandemic: a qualitative study of faculty and resident physician perspectives.

BACKGROUND: The COVID-19 pandemic resulted in a rapid shift from in-person to virtual care delivery for many medical specialties across Canada. The purpose of this study was to explore the lived experiences of resident physicians and faculty related to teaching, learning and assessment during ambulatory virtual care encounters within the competency-based medical education model. METHODS: In this qualitative phenomenological study, we recruited resident physicians (postgraduate year [PGY] 1-5 trainees) and faculty from the Departments of Surgery and Medicine at Queen's University, Ontario, via purposive sampling. Participants were not required to have exposure to virtual care. Interviews were conducted from September 2020 to March 2021 by 1 researcher, and 2 researchers conducted focus groups via Zoom to explore participants' experiences with the transition to virtual care. These were audio-recorded and transcribed verbatim; qualitative data were analyzed thematically. RESULTS: There were 18 male and 19 female participants; 20 were resident physicians and 17 were faculty; 19 were from the Department of Surgery and 18 from the Department of Medicine. All faculty participants had participated in virtual care during ambulatory care; 2 PGY-1 residents in surgery had not actively participated in virtual care, although they had participated in clinics where faculty were using virtual care. The mean age of faculty participants was 38 (standard deviation [SD] 8.6) years, and the mean age of resident physicians was 29 (SD 5.4) years. Overall, 28 interviews and 4 focus groups (range 2-3 participants per group) were conducted, and 4 themes emerged: teaching and learning, assessment, logistical considerations, and suggestions. Barriers to teaching included the lack of direct observations and teaching time, and barriers to assessment included an absence of specific Entrustable Professional Activities (EPAs) and feedback focused on virtual care-related competencies. Logistical challenges included lack of technological infrastructure, insufficient private office space and administrative burdens. Both resident physicians and faculty did not foresee virtual care limiting resident physicians' ability to progress within competency-based medical education. Benefits of virtual care included increased accessibility to patients for follow-up visits, for disclosing patients' results and for out-of-town visits. Suggestions included faculty development, improved access to technology and space, educational guidelines for conducting virtual care encounters, and development of virtual care-specific competencies and EPAs. INTERPRETATION: In the postgraduate program we studied, virtual care imposed substantial barriers on teaching, learning and assessment during the first year of the COVID-19 pandemic. Adapting to new circumstances such as virtual care with suggestions from resident physicians and faculty may help to ensure the continuity of postgraduate medical education throughout the COVID-19 pandemic.

Clonally expanded CD8 T cells characterize amyotrophic lateral sclerosis-4.

Amyotrophic lateral sclerosis (ALS) is a heterogenous neurodegenerative disorder that affects motor neurons and voluntary muscle control1. ALS heterogeneity includes the age of manifestation, the rate of progression and the anatomical sites of symptom onset. Disease-causing mutations in specific genes have been identified and define different subtypes of ALS1. Although several ALS-associated genes have been shown to affect immune functions2, whether specific immune features account for ALS heterogeneity is poorly understood. Amyotrophic lateral sclerosis-4 (ALS4) is characterized by juvenile onset and slow progression3. Patients with ALS4 show motor difficulties by the time that they are in their thirties, and most of them require devices to assist with walking by their fifties. ALS4 is caused by mutations in the senataxin gene (SETX). Here, using Setx knock-in mice that carry the ALS4-causative L389S mutation, we describe an immunological signature that consists of clonally expanded, terminally differentiated effector memory (TEMRA) CD8 T cells in the central nervous system and the blood of knock-in mice. Increased frequencies of antigen-specific CD8 T cells in knock-in mice mirror the progression of motor neuron disease and correlate with anti-glioma immunity. Furthermore, bone marrow transplantation experiments indicate that the immune system has a key role in ALS4 neurodegeneration. In patients with ALS4, clonally expanded TEMRA CD8 T cells circulate in the peripheral blood. Our results provide evidence of an antigen-specific CD8 T cell response in ALS4, which could be used to unravel disease mechanisms and as a potential biomarker of disease state.

The rising burden of Alzheimer's disease mortality in rural America.

Since the 1990s, there has been a striking urban-rural divergence in life expectancy within the United States, with metropolitan areas achieving strong life expectancy increases and nonmetropolitan areas experiencing stagnation or actual declines in life expectancy. While Alzheimer's disease and related dementias (ADRD) are likely to pose a particular challenge in nonmetropolitan areas, we know relatively little about the level of ADRD mortality in nonmetropolitan areas, how it has changed over time, and whether it is contributing to metropolitan/nonmetropolitan life expectancy gaps. This study finds that ADRD mortality has risen more rapidly in nonmetropolitan areas than in all other metro areas (large central metros, suburbs, and medium/small cities) between 1999 and 2019. While death rates from ADRD were nearly identical in large central metros and nonmetros in 1999, a clear metro/nonmetro gradient has emerged and widened substantially over the past two decades. Today, nonmetros now experience the highest levels of ADRD mortality, while large central metros have the lowest levels. These metro/nonmetro gaps in ADRD differ substantially by region, with the largest gaps observed in the Middle Atlantic and South Atlantic. The contribution of ADRD to metro/nonmetro differences life expectancy at age 65 is now considerable in many regions, reaching up to 30% for women and 13% for men. In several regions, ADRD's contribution to female life expectancy gaps is on par with or exceeds the contributions of other leading causes of death such as heart disease, cancer, and chronic lower respiratory diseases. The rising burden of Alzheimer's disease mortality is likely to pose a substantial challenge in rural areas of the United States which are aging rapidly, experiencing adverse mortality trends, and increasingly disadvantaged in terms of socioeconomic resources and health care infrastructure.

HNRNPM controls circRNA biogenesis and splicing fidelity to sustain cancer cell fitness.

High spliceosome activity is a dependency for cancer cells, making them more vulnerable to perturbation of the splicing machinery compared to normal cells. To identify splicing factors important for prostate cancer (PCa) fitness, we performed pooled shRNA screens in vitro and in vivo. Our screens identified heterogeneous nuclear ribonucleoprotein M (HNRNPM) as a regulator of PCa cell growth. RNA- and eCLIP-sequencing identified HNRNPM binding to transcripts of key homeostatic genes. HNRNPM binding to its targets prevents aberrant exon inclusion and backsplicing events. In both linear and circular mis-spliced transcripts, HNRNPM preferentially binds to GU-rich elements in long flanking proximal introns. Mimicry of HNRNPM-dependent linear-splicing events using splice-switching-antisense-oligonucleotides was sufficient to inhibit PCa cell growth. This suggests that PCa dependence on HNRNPM is likely a result of mis-splicing of key homeostatic coding and non-coding genes. Our results have further been confirmed in other solid tumors. Taken together, our data reveal a role for HNRNPM in supporting cancer cell fitness. Inhibition of HNRNPM activity is therefore a potential therapeutic strategy in suppressing growth of PCa and other solid tumors.

METTL6 is a tRNA m3C methyltransferase that regulates pluripotency and tumor cell growth.

Recently, covalent modifications of RNA, such as methylation, have emerged as key regulators of all aspects of RNA biology and have been implicated in numerous diseases, for instance, cancer. Here, we undertook a combination of in vitro and in vivo screens to test 78 potential methyltransferases for their roles in hepatocellular carcinoma (HCC) cell proliferation. We identified methyltransferase-like protein 6 (METTL6) as a crucial regulator of tumor cell growth. We show that METTL6 is a bona fide transfer RNA (tRNA) methyltransferase, catalyzing the formation of 3-methylcytidine at C32 of specific serine tRNA isoacceptors. Deletion of Mettl6 in mouse stem cells results in changes in ribosome occupancy and RNA levels, as well as impaired pluripotency. In mice, Mettl6 knockout results in reduced energy expenditure. We reveal a previously unknown pathway in the maintenance of translation efficiency with a role in maintaining stem cell self-renewal, as well as impacting tumor cell growth profoundly.

Hybrid Gene Origination Creates Human-Virus Chimeric Proteins during Infection.

RNA viruses are a major human health threat. The life cycles of many highly pathogenic RNA viruses like influenza A virus (IAV) and Lassa virus depends on host mRNA, because viral polymerases cleave 5'-m7G-capped host transcripts to prime viral mRNA synthesis ("cap-snatching"). We hypothesized that start codons within cap-snatched host transcripts could generate chimeric human-viral mRNAs with coding potential. We report the existence of this mechanism of gene origination, which we named "start-snatching." Depending on the reading frame, start-snatching allows the translation of host and viral "untranslated regions" (UTRs) to create N-terminally extended viral proteins or entirely novel polypeptides by genetic overprinting. We show that both types of chimeric proteins are made in IAV-infected cells, generate T cell responses, and contribute to virulence. Our results indicate that during infection with IAV, and likely a multitude of other human, animal and plant viruses, a host-dependent mechanism allows the genesis of hybrid genes.

Rural-Urban Differences in Adult Life Expectancy in Indonesia: A Parametric g-formula-based Decomposition Approach.

BACKGROUND: Evidence on rural-urban differences in adult mortality in low- and middle-income countries (LMICs) is limited and mixed. We examined the size of and factors contributing to rural-urban life expectancy differences among adults in Indonesia, the third most populous LMIC. METHODS: Data come from the 2000, 2007, and 2014/2015 waves of the Indonesian Family Life Survey, a population-representative longitudinal study with mortality follow-up. We used Poisson regression and life tables to estimate rural-urban differences in life expectancy among 18,867 adult respondents ≥30 years. We then used a novel g-formula-based decomposition to quantify the contribution of rural-urban differences in blood pressure (BP), body mass index (BMI), and smoking to life expectancy differences. RESULTS: Compared with urban adults, life expectancy at age 30 was 2.2 (95% confidence interval [CI] = 0.4, 3.9) years higher for rural men and 1.2 (95% CI = -0.4, 2.7) years higher for rural women. Setting the BMI and systolic BP distribution equal in urban and rural adults reduced the urban mortality penalty by 22% for men and 78% for women, with the majority of this reduction coming from the contribution of rural-urban differences in BMI. Smoking did not contribute to the urban mortality penalty for either men or women. CONCLUSIONS: Adult life expectancy is lower in urban than in rural areas in Indonesia and we estimate that this difference is partly related to differences in BMI and systolic BP.

Continuous hemoglobin and plethysmography variability index monitoring can modify blood transfusion practice and is associated with lower mortality.

To determine the effect of implementing an algorithm of fluid and blood administration based on continuous monitoring of hemoglobin (SpHb) and PVI (plethysmography variability index) on mortality and transfusion on a whole hospital scale. This single-center quality program compared transfusion at 48 h and mortality at 30 days and 90 days after surgery between two 11-month periods in 2013 and 2014 during which all the operating and recovery rooms and intensive care units were equipped with SpHb/PVI monitors. The entire team was trained to use monitors and the algorithm. Team members were free to decide whether or not to use devices. Each device was connected to an electronic wireless acquired database to anonymously acquire parameters on-line and identify patients who received the monitoring. All data were available from electronic files. Patients were divided in three groups; 2013 (G1, n = 9285), 2014 without (G2, n = 5856) and with (G3, n = 3575) goal-directed therapy. The influence of age, ASA class, severity and urgency of surgery and use of algorithm on mortality and blood use were analyzed with cox-proportional hazard models. Because in 2015, SpHb/PVI monitors were no longer available, we assessed post-study mortality observed in 2015 to measure the impact of team training to adjust vascular filling on a patient to patient basis. During non-cardiac surgery, blood was more often transfused during surgery in G3 patients as compared to G2 (66.6% vs. 50.7%, p < 0.001) but with fewer blood units per patient. After adjustment, survival analysis showed a lower risk of transfusion at 48 h in G3 [OR 0.79 (0.68-0.93), p = 0.004] but not in G2 [OR 0.90 (0.78-1.04) p = 0.17] as compared to G1. When adjusting to the severity of surgery as covariable, there was 0.5 and 0.7% differences of mortality at day 30 and 90 whether patients had goal directed therapy (GDT). After high risk surgery, the mortality at day 30 is reduced by 4% when using GDT, and 1% after intermediate risk surgery. There was no difference for low risk surgery. G3 Patients had a lower risk of death at 30 days post-surgery [OR 0.67 (0.49-0.92) p = 0.01] but not G2 patients [OR 1.01, (0.78-1.29), p = 0.96]. In 2015, mortality at 30 days and 90 days increased again to similar levels as those of 2013, respectively 2.18 and 3.09%. Monitoring SpHb and PVI integrated in a vascular filling algorithm is associated with earlier transfusion and reduced 30 and 90-day mortality on a whole hospital scale.

Development and Validation of a Test to Monitor Endoscopic Activity in Patients With Crohn's Disease Based on Serum Levels of Proteins.

BACKGROUND & AIMS: Noninvasive tests to measure endoscopic activity in patients with Crohn's disease (CD) have limitations. We aimed to develop a test to identify patients in remission, based on endoscopic analysis, and monitor CD activity based on serum levels of proteins. METHODS: We developed a test to measure 13 proteins in blood (ANG1, ANG2, CRP, SAA1, IL7, EMMPRIN, MMP1, MMP2, MMP3, MMP9, TGFA, CEACAM1, and VCAM1), called the endoscopic healing index [EHI], using samples from 278 patients with CD from a multinational training cohort. We validated the test using 2 independent cohorts of patients with CD: 116 biologic-naive patients with early-stage CD (validation cohort 1) and 195 biologic-exposed patients with chronic CD (validation cohort 2). The ability of the test to identify patients with active disease vs patients in remission (defined as a simple endoscopic score for CD of ≤2 and ≤1 in each segment, or a total CD endoscopic index of severity score <3) was assessed by using area under receiver operating characteristic curve (AUROC) analysis. The diagnostic accuracy of the test was compared with that of measurement of serum C-reactive protein (CRP) and fecal calprotectin. RESULTS: The EHI scores range from 0 to 100 units; higher scores indicate more severe CD activity, based on endoscopy findings. The EHI identified patients in remission with an AUROC of 0.962 in validation cohort 1 (95% confidence interval, 0.942-0.982) and an AUROC of 0.693 in validation cohort 2 (95% confidence interval, 0.619-0.767), regardless of CD location or phenotype. A cutoff value of 20 points identified patients in remission with the highest level of sensitivity (97.1% in validation cohort 1 and 83.2% in validation cohort 2), with specificity values of 69.0% and 36.6%, respectively. A cutoff value of 50 points identified patients in remission with the highest level of specificity (100% in validation cohort 1 and 87.8% in validation cohort 2), with sensitivity values of 37.3% and 30.0%, respectively. The EHI identified patients in remission with a significantly higher AUROC value than the test for CRP (0.876, P < .001 in validation cohort 1 and 0.624, P = .109 in validation cohort 2). In analysis of patients with available FC measurements, the AUROC value for the EHI did not differ significantly from that of measurement of FC (AUROC, 0.950 for EHI vs AUROC, 0.923 for FC; P = .147 in validation cohort 1 and AUROC, 0.803 for EHI vs AUROC, 0.854 for FC; P = .298 in validation cohort 2). CONCLUSIONS: We developed an index called the EHI to identify patients with CD in endoscopic remission based on blood levels of 13 proteins. The EHI identified patients with resolution of endoscopic disease activity, with good overall accuracy, although with variation between the 2 cohorts assessed. The EHI AUROC values were comparable to measurement of FC and higher than measurement of serum CRP. The test might be used in practice to assess endoscopic activity in patients with CD.

Discovery of a chemical probe for PRDM9.

PRDM9 is a PR domain containing protein which trimethylates histone 3 on lysine 4 and 36. Its normal expression is restricted to germ cells and attenuation of its activity results in altered meiotic gene transcription, impairment of double-stranded breaks and pairing between homologous chromosomes. There is growing evidence for a role of aberrant expression of PRDM9 in oncogenesis and genome instability. Here we report the discovery of MRK-740, a potent (IC50: 80 ± 16 nM), selective and cell-active PRDM9 inhibitor (Chemical Probe). MRK-740 binds in the substrate-binding pocket, with unusually extensive interactions with the cofactor S-adenosylmethionine (SAM), conferring SAM-dependent substrate-competitive inhibition. In cells, MRK-740 specifically and directly inhibits H3K4 methylation at endogenous PRDM9 target loci, whereas the closely related inactive control compound, MRK-740-NC, does not. The discovery of MRK-740 as a chemical probe for the PRDM subfamily of methyltransferases highlights the potential for exploiting SAM in targeting SAM-dependent methyltransferases.

An NFκB Activity Calculator to Delineate Signaling Crosstalk: Type I and II Interferons Enhance NFκB via Distinct Mechanisms.

Nuclear factor kappa B (NFκB) is a transcription factor that controls inflammation and cell survival. In clinical histology, elevated NFκB activity is a hallmark of poor prognosis in inflammatory disease and cancer, and may be the result of a combination of diverse micro-environmental constituents. While previous quantitative studies of NFκB focused on its signaling dynamics in single cells, we address here how multiple stimuli may combine to control tissue level NFκB activity. We present a novel, simplified model of NFκB (SiMoN) that functions as an NFκB activity calculator. We demonstrate its utility by exploring how type I and type II interferons modulate NFκB activity in macrophages. Whereas, type I IFNs potentiate NFκB activity by inhibiting translation of IκBα and by elevating viral RNA sensor (RIG-I) expression, type II IFN amplifies NFκB activity by increasing the degradation of free IκB through transcriptional induction of proteasomal cap components (PA28). Both cross-regulatory mechanisms amplify NFκB activation in response to weaker (viral) inducers, while responses to stronger (bacterial or cytokine) inducers remain largely unaffected. Our work demonstrates how the NFκB calculator can reveal distinct mechanisms of crosstalk on NFκB activity in interferon-containing microenvironments.

MBNL1 alternative splicing isoforms play opposing roles in cancer.

The extent of and the oncogenic role played by alternative splicing (AS) in cancer are well documented. Nonetheless, only few studies have attempted to dissect individual gene function at an isoform level. Here, we focus on the AS of splicing factors during prostate cancer progression, as these factors are known to undergo extensive AS and have the potential to affect hundreds of downstream genes. We identified exon 7 (ex7) in the MBNL1 (Muscleblind-like 1) transcript as being the most differentially included exon in cancer, both in cell lines and in patients' samples. In contrast, MBNL1 overall expression was down-regulated, consistently with its described role as a tumor suppressor. This observation holds true in the majority of cancer types analyzed. We first identified components associated to the U2 splicing complex (SF3B1, SF3A1, and PHF5A) as required for efficient ex7 inclusion and we confirmed that this exon is fundamental for MBNL1 protein homodimerization. We next used splice-switching antisense oligonucleotides (AONs) or siRNAs to compare the effect of MBNL1 splicing isoform switching with knockdown. We report that whereas the absence of MBNL1 is tolerated in cancer cells, the expression of isoforms lacking ex7 (MBNL1 Δex7) induces DNA damage and inhibits cell viability and migration, acting as dominant negative proteins. Our data demonstrate the importance of studying gene function at the level of alternative spliced isoforms and support our conclusion that MBNL1 Δex7 proteins are antisurvival factors with a defined tumor suppressive role that cancer cells tend to down-regulate in favor of MBNL +ex7 isoforms.

Contribution of smoking-attributable mortality to life expectancy differences by marital status among Finnish men and women, 1971-2010.

BACKGROUND: Smoking is known to vary by marital status, but little is known about its contribution to marital status differences in longevity. We examined the changing contribution of smoking to mortality differences between married and never married, divorced or widowed Finnish men and women aged 50 years and above in 1971-2010. DATA AND METHODS: The data sets cover all persons permanently living in Finland in the census years 1970, 1975 through 2000 and 2005 with a five-year mortality follow-up. Smoking-attributable mortality was estimated using an indirect method that uses lung cancer mortality as an indicator for the impact of smoking on mortality from all other causes. RESULTS: Life expectancy differences between the married and the other marital status groups increased rapidly over the 40-year study period because of the particularly rapid decline in mortality among married individuals. In 1971-1975 37-48% of life expectancy differences between married and divorced or widowed men were attributable to smoking, and this contribution declined to 11-18% by 2006-2010. Among women, in 1971-1975 up to 16% of life expectancy differences by marital status were due to smoking, and the contribution of smoking increased over time to 10-29% in 2006-2010. CONCLUSIONS: In recent decades smoking has left large but decreasing imprints on marital status differences in longevity between married and previously married men, and small but increasing imprints on these differences among women. Over time the contribution of other factors, such as increasing material disadvantage or alcohol use, may have increased.