RNF4 prevents genomic instability caused by chronic DNA under-replication.

Eukaryotic genome stability is maintained by a complex and diverse set of molecular processes. One class of enzymes that promotes proper DNA repair, replication and cell cycle progression comprises small ubiquitin-like modifier (SUMO)-targeted E3 ligases, or STUbLs. Previously, we reported a role for the budding yeast STUbL synthetically lethal with sgs1 (Slx) 5/8 in preventing G2/M-phase arrest in a minichromosome maintenance protein 10 (Mcm10)-deficient model of replication stress. Here, we extend these studies to human cells, examining the requirement for the human STUbL RING finger protein 4 (RNF4) in MCM10 mutant cancer cells. We find that MCM10 and RNF4 independently promote origin firing but regulate DNA synthesis epistatically and, unlike in yeast, the negative genetic interaction between RNF4 and MCM10 causes cells to accumulate in G1-phase. When MCM10 is deficient, RNF4 prevents excessive DNA under-replication at hard-to-replicate regions that results in large DNA copy number alterations and severely reduced viability. Overall, our findings highlight that STUbLs participate in species-specific mechanisms to maintain genome stability, and that human RNF4 is required for origin activation in the presence of chronic replication stress.

A critical threshold of MCM10 is required to maintain genome stability during differentiation of induced pluripotent stem cells into natural killer cells.

Natural killer (NK) cell deficiency (NKD) is a rare disease in which NK cell function is reduced, leaving affected individuals susceptible to repeated viral infections and cancer. Recently, a patient with NKD was identified carrying compound heterozygous variants of MCM10 (minichromosome maintenance protein 10), an essential gene required for DNA replication, that caused a significant decrease in the amount of functional MCM10. NKD in this patient presented as loss of functionally mature late-stage NK cells. To understand how MCM10 deficiency affects NK cell development, we generated MCM10 heterozygous (MCM10+/-) induced pluripotent stem cell (iPSC) lines. Analyses of these cell lines demonstrated that MCM10 was haploinsufficient, similar to results in other human cell lines. Reduced levels of MCM10 in mutant iPSCs was associated with impaired clonogenic survival and increased genomic instability, including micronuclei formation and telomere erosion. The severity of these phenotypes correlated with the extent of MCM10 depletion. Significantly, MCM10+/- iPSCs displayed defects in NK cell differentiation, exhibiting reduced yields of hematopoietic stem cells (HSCs). Although MCM10+/- HSCs were able to give rise to lymphoid progenitors, these did not generate mature NK cells. The lack of mature NK cells coincided with telomere erosion, suggesting that NKD caused by these MCM10 variants arose from the accumulation of genomic instability including degradation of chromosome ends.

Discovery and Anti-Inflammatory Activity of a Cyanobacterial Fatty Acid Targeting the Keap1/Nrf2 Pathway.

The monounsaturated fatty acid 7(E)-9-keto-hexadec-7-enoic acid (1) and three structurally related analogues with different oxidation states and degrees of unsaturation (2-4) were discovered from a marine benthic cyanobacterial mat collected from Delta Shoal, Florida Keys. Their structures were elucidated using NMR spectroscopy and mass spectrometry. The structure of 1 contained an α,ß-unsaturated carbonyl system, a key motif required for the activation of the Keap1/Nrf2-ARE pathway that is involved in the activation of antioxidant and phase II detoxification enzymes. Compounds 1-4 were screened in ARE-luciferase reporter gene assay using stably transfected HEK293 cells, and only 1 significantly induced Nrf2 activity at 32 and 10 µM, whereas 2-4 were inactive. As there is crosstalk between inflammation and oxidative stress, subsequent biological studies were focused on 1 to investigate its anti-inflammatory potential. Compound 1 induced Nqo1, a well-known target gene of Nrf2, and suppressed iNos transcript levels, which translated into reduced levels of nitric oxide in LPS-activated mouse macrophage RAW264.7 cells, a more relevant model for inflammation. RNA sequencing was performed to capture the effects of 1 on a global level and identified additional canonical pathways and upstream regulators involved in inflammation and immune response, particularly those related to multiple sclerosis. A targeted survey of marine cyanobacterial samples from other geographic locations, including Guam, suggested the widespread occurrence of 1. Furthermore, the previous isolation of 1 from marine diatoms and green algae implied a potentially important ecological role across marine algal eukaryotes and prokaryotes. The previous isolation from sea lettuce raises the possibility of dietary intervention to attenuate inflammation and related disease progression.

Neglect as an undefined and overlooked aspect of medical student mistreatment: A systematic review of the literature.

PURPOSE: Although the mistreatment of medical students is a well-researched topic, the scope of mistreatment often leaves out neglect, a subtype for which there is no accepted definition based in the published literature. This review sought to summarize the existing data on the prevalence and descriptors of neglect, identify strategies seen in the literature designed to improve it, and offer a synthesized definition of this phenomenon to guide future research. METHODS: Following PRISMA guidelines, a relevant systematic literature search from 2000 to April 2021 was performed to identify literature on neglect in clinical settings within American medical schools. RESULTS: Neglect, a poorly defined phenomenon in medical education related to the suboptimal learning environment, is often excluded from research on medical student mistreatment. Neglect is a barrier to a successful learning environment, yet a paucity of data and the heterogeneous nature of the present literature render it difficult to estimate its true prevalence. Studies that include neglect frequently assess it solely as the result of identity discrimination or stated career interests. Recent interventions include promoting longitudinal relationships between students and clinical faculty and establishing teaching expectations. CONCLUSIONS: Neglect is the mistreatment of medical students by the medical care team via a lack of meaningful inclusion in the clinical environment such that it has a notable negative impact on learning and student well-being, regardless of intentionality. An established definition that is grounded in the literature is required to create a common point of reference and understand its true prevalence, its associated variables, and the best mitigation strategies, as well as to guide future research, which should examine neglect independently and as a consequence of personal and professional identities.

Systematic Literature Review of Text Messaging Interventions to Promote Medication Adherence Among People With Serious Mental Illness.

OBJECTIVE: Mobile health tools are feasible options to encourage behavior change among patients with serious mental illness. Mobile health tools vary widely, both in platforms used and content delivered. This literature review assessed the use of text messaging interventions to promote medication adherence among patients with serious mental illness. METHODS: A systematic literature review using PRISMA guidelines examined short message service (SMS) text messaging interventions promoting medication adherence to people with a serious mental illness diagnosis. Databases included PubMed, Cochrane, CINAHL, and PsycINFO. Data extraction included demographic information, participant diagnoses, intervention components, medication class, adherence measures, research design, and study outcomes. Study quality was also assessed. RESULTS: Of 114 full-text articles screened, 10 articles were selected from nine unique interventions (N=937 people with serious mental illness). Study durations ranged from 30 days to 18 months, with frequency of SMS ranging from twice weekly to 12 times daily. Of the nine unique trials, most reported using an automated server to deliver SMS messages (N=7), two-way SMS capabilities (N=6), customized message content or timing (N=7), and additional components (e.g., provider contact, educational content, and monetary rewards) (N=7). Seven of the 10 articles reported statistically significant improvement in medication adherence and in at least one clinical outcome. CONCLUSIONS: Evidence to date indicates that text messaging interventions are feasible and appear to improve medication adherence and clinical outcomes among patients with serious mental illness. Future research should assess implementation approaches and how to scale up efforts in nonresearch settings.

Active esophageal cooling for the prevention of thermal injury during atrial fibrillation ablation: a randomized controlled pilot study.

BACKGROUND: Severe endoscopically detected esophageal thermal lesions (EDELs) have been associated with higher risk of progression to atrio-esophageal fistula (AEF) following radiofrequency catheter ablation (RFCA) of atrial fibrillation (AF). We sought to evaluate safety and feasibility of active esophageal cooling using the Attune Medical Esophageal Heat Transfer Device (EnsoETM) to limit frequency or severity of EDELs. OBJECTIVE: We sought To evaluate safety and feasibility of active esophageal cooling using the Attune Medical Esophageal Heat Transfer Device (EnsoETM) to limit frequency or severity of EDELs METHODS: Consecutive patients undergoing first-time RFCA were randomized in a 1:1 fashion to esophageal cooling (device group) or standard temperature monitoring (control group). Ablation on the posterior wall was performed with a maximum power of 30W for up to 20s. All patients underwent EGD within 48 h. Endoscopy findings were classified as 1, erythema-mild injury; 2, superficial ulceration-moderate injury; 3, deep ulceration-significant injury; and 4, fistula/perforation. Severe EDELs were defined as grade 3 or 4 lesions. RESULTS: Forty-four patients completed the study (22 device group, 22 control group). Adjunctive posterior wall isolation was performed more frequently in the device group (11/22, 50% vs. 4/22, 18%). EDELs were detected in 5/22 (23%) control group patients, with mild or moderate injury in 2/5 patients (40%) and severe thermal injury in 3/5 patients (60%). In the device group, EDELs were detected in 8/22 (36%) patients, with mild or moderate injury in 7/8 (87%) patients and severe thermal injury in 1/8 (12%) patients. There was no acute perforation or AEF during follow-up. CONCLUSIONS: Active esophageal cooling may reduce the occurrence of severe EDELs. A larger randomized study is warranted to further evaluate the benefit of this strategy.

Repetition Priming in Treatment of Anomia.

PURPOSE: Repetition priming has been suggested as a method for targeting implicit processes in anomia treatment. Prior studies have used masked priming for this purpose. This study extends that work with visible primes, a more clinically feasible approach. METHOD: This study used a single-subject design across three participants with aphasia. Treatment involved repeated exposure to identity primes (trained condition) or sham primes (untrained condition) paired with pictures. Analyses assessed acquisition effects for trained items and untrained items that were seen during the training period, generalization to untrained items that had not been seen, and generalization to broader language skills, immediately and 3 months post-treatment. RESULTS: All participants improved in naming trained items immediately after treatment, with greater improvements for trained than for untrained items. All participants maintained some degree of improvement on trained items 3 months post-treatment, although the degree differed across participants. Inconsistent generalization occurred to unexposed items. Improvements were noted in some areas of broader language ability, although these varied. CONCLUSIONS: These data suggest a repetition priming treatment paradigm may increase naming accuracy for individuals with anomia and may benefit other aspects of language. Participant factors may have influenced response to treatment. Directions for future investigation are discussed.

Alcohol use in Tanzanians with chronic psychotic disorders and poor medication adherence.

BACKGROUND: The burden of chronic psychotic disorders (CPDs) in sub-Saharan Africa (SSA) is significant. Poorly medically adherent patients are more likely to have worse outcomes and require more resources. However, factors impacting effective treatment of CPD in this population are unclear. AIM: Examine the relationship between alcohol use and disease management and compare alcohol risk stratification between the Alcohol Use Disorders Identification Test (AUDIT) and Alcohol, Smoking and Substance Involvement Screening Test (ASSIST) in poorly medication adherent Tanzanians with CPD. SETTING: Muhimbili National Hospital and ambulatory clinics in Dar es Salaam, Tanzania. METHODS: 100 Tanzanians with CPDs and suboptimal medication adherence were dichotomized into low and moderate-to-high risk alcohol use based on AUDIT scores and compared regarding medication attitudes, adherence and psychiatric symptoms. Patients completed the ASSIST for comparison to AUDIT risk stratification. RESULTS: Moderate-to-high risk alcohol users had worse medication attitudes (p < 0.01), medication adherence (previous week, p = 0.01; previous month, p < 0.001), and psychiatric symptoms (p = 0.03). They were younger, predominately male and more likely to have a family history of alcohol abuse. A logistic regression analysis found age, gender and family history of abuse as significant predictors of hazardous alcohol use (p = 0.02, 0.02, < 0.01, respectively). Risk stratification between AUDIT and ASSIST aligned in 85% of participants. CONCLUSION: Alcohol use is an important consideration in treating poorly adherent Tanzanians with CPD. The ASSIST was comparable to the AUDIT in stratifying risky alcohol use with the additional benefit of screening for other substances.

Perinatal Oral Health Education and Compliance with the First Dental Visit.

Purpose: The purpose of this study was to determine which mode of education given to mothers of newborns resulted in the greatest compliance for establishing an age one dental visit and identify reasons why mothers do not bring their child for their first dental visit.
Methods: Several modes of education presented information to mothers: (1) written literature provided by a nurse; (2) a nurse verbally presenting and providing literature; (3) a resident verbally presenting and providing literature; and (4) control-no education. The chi-square test of homogeneity was utilized to determine if there was a difference in compliance to make an age one dental appointment.
Results: The retained sample consisted of 277 children evaluated between 2014 and 2018. There was no statistical difference between the educational methods regarding compliance to make an age one dental visit (P >0.05). There was a significant difference between marital status (P =0.0005), with 95 percent of single mothers likely to schedule the visit.
Conclusion: No particular maternal educational method resulted in greater compliance with scheduling an age one dental visit. Single mothers were more likely to schedule the visit. The primary reason for not visiting was a lack of primary care provider recommendations.

Interactional Synchrony and Its Association with Social and Communication Ability in Children With and Without Autism Spectrum Disorder.

Social partners tend to coordinate their behaviors in time. This "interactional synchrony" is associated with a host of positive social outcomes, making it ripe for study in autism spectrum disorder (ASD). Twenty children with ASD and 17 typically developing (TD) children participated in conversations with familiar and unfamiliar adults. Conversations were rated for movement synchrony and verbal synchrony, and mothers completed measures regarding children's everyday social and communication skills. Children with ASD exhibited less interactional synchrony, with familiar and unfamiliar partners, than TD peers. Beyond group-level differences, interactional synchrony negatively correlated with autism symptom severity, and predicted dimensional scores on established social and communication measures. Results suggest that disrupted interactional synchrony may be associated with impaired social functioning in ASD.

Site-specific glycosylation of Ebola virus glycoprotein by human polypeptide GalNAc-transferase 1 induces cell adhesion defects.

The surface glycoprotein (GP) of Ebola virus causes many of the virus's pathogenic effects, including a dramatic loss of endothelial cell adhesion associated with widespread hemorrhaging during infection. Although the GP-mediated deadhesion depends on its extracellular mucin-like domain, it is unknown whether any, or all, of this domain's densely clustered O-glycosylation sites are required. It is also unknown whether any of the 20 distinct polypeptide GalNAc-transferases (ppGalNAc-Ts) that initiate mucin-type O-glycosylation in human cells are functionally required. Here, using HEK293 cell lines lacking specific glycosylation enzymes, we demonstrate that GP requires extended O-glycans to exert its deadhesion effect. We also identified ppGalNAc-T1 as largely required for the GP-mediated adhesion defects. Despite its profound effect on GP function, the absence of ppGalNAc-T1 only modestly reduced the O-glycan mass of GP, indicating that even small changes in the bulky glycodomain can cause loss of GP function. Indeed, protein-mapping studies identified a small segment of the mucin-like domain critical for function and revealed that mutation of five glycan acceptor sites within this segment are sufficient to abrogate GP function. Together, these results argue against a mechanism of Ebola GP-induced cell detachment that depends solely on ectodomain bulkiness and identify a single host-derived glycosylation enzyme, ppGalNAc-T1, as a potential target for therapeutic intervention against Ebola virus disease.

Nonstructural Protein σ1s Is Required for Optimal Reovirus Protein Expression.

Reovirus nonstructural protein σ1s is required for the establishment of viremia and hematogenous viral dissemination. However, the function of σ1s during the reovirus replication cycle is not known. In this study, we found that σ1s was required for efficient reovirus replication in simian virus 40 (SV40)-immortalized endothelial cells (SVECs), mouse embryonic fibroblasts, human umbilical vein endothelial cells (HUVECs), and T84 human colonic epithelial cells. In each of these cell lines, wild-type reovirus produced substantially higher viral titers than a σ1s-deficient mutant. The σ1s protein was not required for early events in reovirus infection, as evidenced by the fact that no difference in infectivity between the wild-type and σ1s-null viruses was observed. However, the wild-type virus produced markedly higher viral protein levels than the σ1s-deficient strain. The disparity in viral replication did not result from differences in viral transcription or protein stability. We further found that the σ1s protein was dispensable for cell killing and the induction of type I interferon responses. In the absence of σ1s, viral factory (VF) maturation was impaired but sufficient to support low levels of reovirus replication. Together, our results indicate that σ1s is not absolutely essential for viral protein production but rather potentiates reovirus protein expression to facilitate reovirus replication. Our findings suggest that σ1s enables hematogenous reovirus dissemination by promoting efficient viral protein synthesis, and thereby reovirus replication, in cells that are required for reovirus spread to the blood.IMPORTANCE Hematogenous dissemination is a critical step in the pathogenesis of many viruses. For reovirus, nonstructural protein σ1s is required for viral spread via the blood. However, the mechanism by which σ1s promotes reovirus dissemination is unknown. In this study, we identified σ1s as a viral mediator of reovirus protein expression. We found several cultured cell lines in which σ1s is required for efficient reovirus replication. In these cells, wild-type virus produced substantially higher levels of viral protein than a σ1s-deficient mutant. The σ1s protein was not required for viral mRNA transcription or viral protein stability. Since reduced levels of viral protein were synthesized in the absence of σ1s, the maturation of viral factories was impaired, and significantly fewer viral progeny were produced. Taken together, our findings indicate that σ1s is required for optimal reovirus protein production, and thereby viral replication, in cells required for hematogenous reovirus dissemination.

Serotype-Specific Killing of Large Cell Carcinoma Cells by Reovirus.

Reovirus is under development as a therapeutic for numerous types of cancer. In contrast to other oncolytic viruses, the safety and efficacy of reovirus have not been improved through genetic manipulation. Here, we tested the oncolytic capacity of recombinant strains (rs) of prototype reovirus laboratory strains T1L and T3D (rsT1L and rsT3D, respectively) in a panel of non-small cell lung cancer (NSCLC) cell lines. We found that rsT1L was markedly more cytolytic than rsT3D in the large cell carcinoma cell lines tested, whereas killing of adenocarcinoma cell lines was comparable between rsT1L and rsT3D. Importantly, non-recombinant T1L and T3D phenocopied the kinetics and magnitude of cell death induced by recombinant strains. We identified gene segments L2, L3, and M1 as viral determinants of strain-specific differences cell killing of the large cell carcinoma cell lines. Together, these results indicate that recombinant reoviruses recapitulate the cell killing properties of non-recombinant, tissue culture-passaged strains. These studies provide a baseline for the use of reverse genetics with the specific objective of engineering more effective reovirus oncolytics. This work raises the possibility that type 1 reoviruses may have the capacity to serve as more effective oncolytics than type 3 reoviruses in some tumor types.

The anti-inflammatory CASPASE-12 gene does not influence SLE phenotype in African-Americans.

In the vast majority of human populations, the gene encoding CASPASE-12 (CASP12) has a premature termination codon that precludes the production of protein. However, approximately 20% of persons of recent African descent have a single nucleotide polymorphism (#rs497116; A->G) that turns the stop codon into one encoding Arg. The subsequent functional allele is a risk factor for sepsis as it uniquely downregulates inflammatory cytokines in African-Americans (AA). To determine if CASP12 could be protective for systemic lupus erythematosus (SLE) in AA, we genotyped AA SLE patients and controls. There was a weak association between CASP12 genotype with the absence of anti-dsDNA autoantibodies in SLE patients. No effect was seen upon serum interleukin-1 beta levels, nor was any other protective effect noted for the CASP12 genotype, whether upon association with SLE, or any of the 11 American College of Rheumatology classification criteria. CASP12 genotype thus does not influence the phenotype of SLE in AA.