The Surprising Diversity of UV-Induced Mutations.

Ultraviolet (UV) light is the most pervasive environmental mutagen and the primary cause of skin cancer. Genome sequencing of melanomas and other skin cancers has revealed that the vast majority of somatic mutations in these tumors are cytosine-to-thymine (C>T) substitutions in dipyrimidine sequences, which, together with tandem CC>TT substitutions, comprise the canonical UV mutation "signature". These mutation classes are caused by DNA damage directly induced by UV absorption, namely cyclobutane pyrimidine dimers (CPDs) or 6-4 pyrimidine-pyrimidone photoproducts (6-4PP), which form between neighboring pyrimidine bases. However, many of the key driver mutations in melanoma do not fit this mutation signature, but instead are caused by T>A, T>C, C>A, or AC>TT substitutions, frequently occurring in non-dipyrimidine sequence contexts. This article describes recent studies indicating that UV light causes a more diverse spectrum of mutations than previously appreciated, including many of the mutation classes observed in melanoma driver mutations. Potential mechanisms for these diverse mutation signatures are discussed, including UV-induced pyrimidine-purine photoproducts and indirect DNA damage induced by UVA light. Finally, the article reviews recent findings indicating that human DNA polymerase eta normally suppresses these non-canonical UV mutation classes, which can potentially explain why canonical C>T substitutions predominate in human skin cancers.

Genome-wide maps of UVA and UVB mutagenesis in yeast reveal distinct causative lesions and mutational strand asymmetries.

Ultraviolet (UV) light primarily causes C > T substitutions in lesion-forming dipyrimidine sequences. However, many of the key driver mutations in melanoma do not fit this canonical UV signature, but are instead caused by T > A, T > C, or C > A substitutions. To what extent exposure to the UVB or UVA spectrum of sunlight can induce these noncanonical mutation classes, and the molecular mechanism involved is unclear. Here, we repeatedly exposed wild-type or repair-deficient yeast (Saccharomyces cerevisiae) to UVB or UVA light and characterized the resulting mutations by whole genome sequencing. Our data indicate that UVB induces C > T and T > C substitutions in dipyrimidines, and T > A substitutions that are often associated with thymine-adenine (TA) sequences. All of these mutation classes are induced in nucleotide excision repair-deficient cells and show transcriptional strand asymmetry, suggesting they are caused by helix-distorting UV photoproducts. In contrast, UVA exposure induces orders of magnitude fewer mutations with a distinct mutation spectrum. UVA-induced mutations are elevated in Ogg1-deficient cells, and the resulting spectrum consists almost entirely of C > A/G > T mutations, indicating they are likely derived from oxidative guanine lesions. These mutations show replication asymmetry, with elevated G > T mutations on the leading strand, suggesting there is a strand bias in the removal or bypass of guanine lesions during replication. Finally, we develop a mutation reporter to show that UVA induces a G > T reversion mutation in yeast that mimics the oncogenic NRAS Q61K mutation in melanoma. Taken together, these findings indicate that UVA and UVB exposure can induce many of the noncanonical mutation classes that cause driver mutations in melanoma.

Contributions of replicative and translesion DNA polymerases to mutagenic bypass of canonical and atypical UV photoproducts.

UV exposure induces a mutation signature of C > T substitutions at dipyrimidines in skin cancers. We recently identified additional UV-induced AC > TT and A > T substitutions that could respectively cause BRAF V600K and V600E oncogenic mutations. The mutagenic bypass mechanism past these atypical lesions, however, is unknown. Here, we whole genome sequenced UV-irradiated yeast and used reversion reporters to delineate the roles of replicative and translesion DNA polymerases in mutagenic bypass of UV-lesions. Our data indicates that yeast DNA polymerase eta (pol η) has varied impact on UV-induced mutations: protecting against C > T substitutions, promoting T > C and AC > TT substitutions, and not impacting A > T substitutions. Surprisingly, deletion rad30Δ increased novel UV-induced C > A substitutions at CA dinucleotides. In contrast, DNA polymerases zeta (pol ζ) and epsilon (pol ε) participated in AC > TT and A > T mutations. These results uncover lesion-specific accurate and mutagenic bypass of UV lesions, which likely contribute to key driver mutations in melanoma.

Topical bromfenac as adjunctive treatment with intravitreal ranibizumab for diabetic macular edema.

PURPOSE: To determine the safety and efficacy of adding topical bromfenac 0.09% in the treatment of diabetic macular edema. METHODS: Seventy patients (70 eyes) with center involved diabetic macular edema with macular thickness (300-500 µm) were included. Patients were divided randomly into two groups: 35 eyes in each group. Both groups were treated with intravitreal ranibizumab monthly for three consecutive months. Bromfenac 0.09% eye drops twice daily was added to the treatment of study group for six months from commencement of treatment. The efficacy of topical bromfenac was evaluated by comparing both groups through follow-up period as regards to visual acuity, central and average thickness and the need for re-injection. RESULTS: Patients treated with topical bromfenac in addition to intravitreal ranibizumab revealed significant improvement in visual acuity, more reduction in central and average macular thickness and less tendency to need reinjection compared to those treated with ranibizumab alone (p 0.013, p 0.010 and p 0.022, respectively). No side effects was encountered with the use of topical bromfenac. CONCLUSION: Topical bromfenac 0.09% twice a day could enhance and sustain the efficacy of intravitreal ranibizumab in the treatment of diabetic macular edema without increasing the incidence of corneal side effects.

Biglycan regulates bone development and regeneration.

Endochondral bone development and regeneration relies on activation and proliferation of periosteum derived-cells (PDCs). Biglycan (Bgn), a small proteoglycan found in extracellular matrix, is known to be expressed in bone and cartilage, however little is known about its influence during bone development. Here we link biglycan with osteoblast maturation starting during embryonic development that later affects bone integrity and strength. Biglycan gene deletion reduced the inflammatory response after fracture, leading to impaired periosteal expansion and callus formation. Using a novel 3D scaffold with PDCs, we found that biglycan could be important for the cartilage phase preceding bone formation. The absence of biglycan led to accelerated bone development with high levels of osteopontin, which appeared to be detrimental to the structural integrity of the bone. Collectively, our study identifies biglycan as an influencing factor in PDCs activation during bone development and bone regeneration after fracture.

Vitamin D and lactoferrin attenuate stress-induced colitis in Wistar rats via enhancing AMPK expression with inhibiting mTOR-STAT3 signaling and modulating autophagy.

Irritable bowel syndrome (IBS) is a global gastrointestinal disorder closely related to psychological stress exposure and local colonic inflammation. Herein, we investigated the effect of wrap-restraint stress (WRS) on rat behavior, on adenosine monophosphate-activated protein kinase-mammalian/mechanistic target of rapamycin-signal transducer and activator of transcription 3 (AMPK-mTOR-STAT3) signaling, and autophagy in colonic mucosa. The impact of chronic administration of vitamin D3 and lactoferrin was compared. Twenty-four male Wistar rats were randomly divided into four groups. Chronic WRS protocol was applied as a rodent model of IBS. Group I: naïve animals, Group II: WRS animals, Group III: WRS-exposed and treated with vitamin D3 (500 IU/kg/day), and Group IV: WRS-exposed and treated with lactoferrin (300 mg/kg/day). In this study, we found that chronic administration of each of vitamin D3 and lactoferrin resulted in a significant increase in social interaction test, interleukin-10, AMPK, optical density of LC3B, goblet cell count and marked decrease in serum cortisol level, STAT3, inflammatory cell count, and optical density of mTOR in comparison to the WRS rats. Our findings suggest that both vitamin D3 and Lactoferrin could augment colonic autophagy through enhanced AMPK expression and inhibition of mTOR-STAT3 signaling, which offers practical insights into their clinical use in the prevention and therapy of IBS. However, lactoferrin intake as a nutritional supplement could be more helpful for stress-induced colitis treatment than vitamin D3.

The Role of Type VI Collagen in Alveolar Bone.

Many studies have been conducted to elucidate the role of Type VI collagen in muscle and tendon, however, its role in oral tissues remains unclear. In this study, an α2(VI) deficient mouse (Col6α2-KO) model was used to examine the role of Type VI collagen in oral tissues. Tissue volume and mineral density were measured in oral tissues by µCT. Proteome analysis was performed using protein extracted from alveolar bone. In addition, alveolar bone was evaluated with a periodontitis induced model. µCT analysis showed the Col6α2-KO mice had less volume of alveolar bone, dentin and dental pulp, while the width of periodontal ligament (PDL) was greater than WT. The mineral density in alveolar bone and dentin were elevated in Col6α2-KO mice compared with WT. Our proteome analysis showed significant changes in proteins related to ECM organization and elevation of proteins associated with biomineralization in the Col6α2-KO mice. In induced periodontitis, Col6α2-KO mice had greater alveolar bone loss compared with WT. In conclusion, Type VI collagen has a role in controlling biomineralization in alveolar bone and that changes in the ECM of alveolar bone could be associated with greater bone loss due to periodontitis.

Type VI Collagen Regulates Endochondral Ossification in the Temporomandibular Joint.

For many years there has been a keen interest in developing regenerative treatment for temporomandibular joint-osteoarthritis (TMJ-OA). Currently, there is no consensus treatment due to the limited self-healing ability of articular cartilage and lack of understanding of the complex mechanisms regulating cartilage development in the TMJ. Endochondral ossification, the process of subchondral bone formation through chondrocyte differentiation, is critical for TMJ growth and development, and is tightly regulated by the composition of the extracellular matrix (ECM). Type VI collagen is a highly expressed ECM component in the TMJ cartilage, yet its specific functions are largely unknown. In this study, we investigated α2(VI)-deficient (Col6a2-knockout [KO]) mice, which are unable to secret or incorporate type VI collagen into their ECM. Compared with wild-type (WT) mice, the TMJ condyles of Col6a2-KO mice exhibit decreased bone volume/tissue volume (BV/TV) and a larger bone marrow space, suggesting the α2(VI)-deficient condyles have a failure in endochondral ossification. Differentiating chondrocytes are the main source of bone cells during endochondral ossification. Our study shows there is an increased number of chondrocytes in the proliferative zone and decreased Col10-expressing chondrocytes in Col6a2-KO cartilage, all pointing to abnormal chondrocyte differentiation and maturation. In addition, RNA sequencing (RNAseq) analysis identified distinct gene expression profiles related to cell cycle and ECM organization that were altered in the mutant condyles. These data also suggest that bone morphogenetic protein 2 (BMP2) activity was deregulated during chondrocyte differentiation. Immunohistochemical analysis indicated an upregulation of Col2 and Acan expression in Col6a2-KO cartilage. Moreover, the expression of pSmad1/5/8 and Runx2 was decreased in the Col6a2-KO cartilage compared with WT controls. Taken together, our data indicate that type VI collagen expressed in the TMJ cartilage is important for endochondral ossification, possibly by modulating the ECM and altering/disrupting signaling pathways important for TMJ chondrocyte differentiation. Published 2022. This article is a U.S. Government work and is in the public domain in the USA. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

The relationship between obstetrical interventions and the increase in U.S. preterm births, 2014-2019.

We examined the relationship between obstetrical intervention and preterm birth in the United States between 2014 and 2019. This observational study analyzed 2014-2019 US birth data to assess changes in preterm birth, cesarean delivery, induction of labor, and associated risks. Logistic regression modeled the odds of preterm obstetrical intervention (no labor cesarean or induction) after risk adjustment. The percentage of singleton preterm births in the United States increased by 9.4% from 2014-2019. The percent of singleton, preterm births delivered by cesarean increased by 6.0%, while the percent with induction of labor increased by 39.1%. The percentage of singleton preterm births where obstetrical intervention (no labor cesarean or induction) potentially impacted the gestational age at delivery increased from 47.6% in 2014 to 54.9% in 2019. Preterm interventions were 13% more likely overall in 2019 compared to 2014 and 17% more likely among late preterm births, after controlling for demographic and medical risk factors. Compared to non-Hispanic White women, Non-Hispanic Black women had a higher risk of preterm obstetric interventions. Preterm infants have higher morbidity and mortality rates than term infants, thus any increase in the preterm birth rate is concerning. A renewed effort to understand the trends in preterm interventions is needed to ensure that obstetrical interventions are evidence-based and are limited to those cases where they optimize outcomes for both mothers and babies.

United States community births increased by 20% from 2019 to 2020.

BACKGROUND: Anecdotal and emerging evidence suggested that the 2020 COVID-19 pandemic may have influenced women's attitudes toward community birth. Our purpose was to examine trends in community births from 2019 to 2020, and the risk profile of these births. METHODS: Recently released 2020 birth certificate data were compared with prior years' data to analyze trends in community births by socio-demographic and medical characteristics. RESULTS: In 2020, there were 71 870 community births in the United States, including 45 646 home births and 21 884 birth center births. Community births increased by 19.5% from 2019 to 2020. Planned home births increased by 23.3%, while birth center births increased by 13.2%. Increases occurred in every US state, and for all racial and ethnic groups, particularly non-Hispanic Black mothers (29.7%), although not all increases were statistically significant. In 2020, 1 of every 50 births in the United States was a community birth (2.0%). Women with planned home and birth center births were less likely than women with hospital births to have several characteristics associated with poor pregnancy outcomes, including teen births, smoking during pregnancy, obesity, and preterm, low birthweight, and multiple births. More than two-thirds of planned home births were self-paid, compared with one-third of birth center and just 3% of hospital births. CONCLUSIONS: It is to the great credit of United States midwives working in home and birth center settings that they were able to substantially expand their services during a worldwide pandemic without compromising standards in triaging women to optimal settings for safe birth.

Coordinate roles for collagen VI and biglycan in regulating tendon collagen fibril structure and function.

Tendon is a vital musculoskeletal tissue that is prone to degeneration. Proper tendon maintenance requires complex interactions between extracellular matrix components that remain poorly understood. Collagen VI and biglycan are two matrix molecules that localize pericellularly within tendon and are critical regulators of tissue properties. While evidence suggests that collagen VI and biglycan interact within the tendon matrix, the relationship between the two molecules and its impact on tendon function remains unknown. We sought to elucidate potential coordinate roles of collagen VI and biglycan within tendon by defining tendon properties in knockout models of collagen VI, biglycan, or both molecules. We first demonstrated co-expression and co-localization of collagen VI and biglycan within the healing tendon, providing further evidence of cooperation between the two molecules during nascent tendon matrix formation. Deficiency in collagen VI and/or biglycan led to significant reductions in collagen fibril size and tendon mechanical properties. However, collagen VI-null tendons displayed larger reductions in fibril size and mechanics than seen in biglycan-null tendons. Interestingly, knockout of both molecules resulted in similar properties to collagen VI knockout alone. These results indicate distinct and non-additive roles for collagen VI and biglycan within tendon. This work provides better understanding of regulatory interactions between two critical tendon matrix molecules.

Development of a Symptom-Focused Model to Guide the Prescribing of Antipsychotics in Children and Adolescents: Results of the First Phase of the Safer Use of Antipsychotics in Youth (SUAY) Clinical Trial.

OBJECTIVE: To develop a new approach to prescribing guidelines as part of a pragmatic trial, Safer Use of Antipsychotics in Youth (SUAY; ClinicalTrials.gov Identifier: NCT03448575), which supports prescribers in delivering high-quality mental health care to youths. METHOD: A nominal group technique was used to identify first- to nth-line treatments for target symptoms and potential diagnoses. The panel included US pediatricians, child and adolescent psychiatrists, and psychopharmacology experts. Meeting materials included information about Medicaid review programs, systematic reviews, prescribing guidelines, and a description of the pragmatic trial. Afterward, a series of 4 webinar discussions were held to achieve consensus on recommendations. RESULTS: The panel unanimously agreed that the guideline should focus on target symptoms rather than diagnoses. Guidance included recommendations for first- to nth-line treatment of target mental health symptoms, environmental factors to be addressed, possible underlying diagnoses that should first be considered and ruled out, and general considerations for pharmacological and therapeutic treatments. CONCLUSION: Prescribing guidelines are often ignored because they do not incorporate the real-world availability of first-line psychosocial treatments, comorbid conditions, and clinical complexity. Our approach addresses some of these concerns. If the approach proves successful in our ongoing pragmatic trial, Safer Use of Antipsychotics in Youth (SUAY), it may serve as a model to state Medicaid programs and health systems to support clinicians in delivering high-quality mental health care to youths. CLINICAL TRIAL REGISTRATION INFORMATION: Safer Use of Antipsychotics in Youth; http://clinicaltrials.gov/; NCT03448575.

Awareness and Use of the Sickle Cell Disease Toolbox by Primary Care Providers in North Carolina.

BACKGROUND: Sickle cell disease (SCD) is a complex chronic blood disorder characterized by severe disease complications ideally managed by both hematologists and primary care providers (PCP's). PCP's report knowledge gaps and discomfort with SCD management. Our team developed and a decision support tool for SCD management (SCD Toolbox) based on the National Heart, Lung, and Blood Institute's SCD guidelines. We surveyed PCPs in North Carolina (NC) prior to formal dissemination to determine current co-management practices, assess toolbox acceptability, use, format preferences, and understand which algorithms would be most helpful. METHOD: A 23-item baseline needs assessment survey was disseminated to PCPs throughout NC. RESULTS: A total of 63 medical providers responded to the survey and of these respondents, 64% reported caring for 1 to 10 patients with SCD. Only 39% of PCPs reported regular communication with an SCD specialist. Providers reported the highest level of awareness of the pediatric and adult health maintenance tools (41% and 39% respectively) and highest use of the pediatric (26%) and adult (28%) health maintenance tools. Respondents also expressed a desire to have access to multiple toolbox formats (37%) (website, mobile app and/or paper). LIMITATIONS: The use of a convenience sample and low survey response are study limitations which hinder generalizability. CONCLUSIONS: PCPs rarely co-managed with a specialist, had low awareness and use of SCD toolbox, and requested multiple formats for the toolbox.

Racial and Ethnic Disparities in Maternal Mortality in the United States Using Enhanced Vital Records, 2016‒2017.

Objectives. To better understand racial and ethnic disparities in US maternal mortality. Methods. We analyzed 2016-2017 vital statistics mortality data with cause-of-death literals (actual words written on the death certificate) added. We created a subset of confirmed maternal deaths that had pregnancy mentions in the cause-of-death literals. Primary cause of death was identified and recoded using cause-of-death literals. We examined racial and ethnic disparities both overall and by primary cause. Results. The maternal mortality rate for non-Hispanic Black women was 3.55 times that for non-Hispanic White women. Leading causes of maternal death for non-Hispanic Black women were eclampsia and preeclampsia and postpartum cardiomyopathy with rates 5 times those for non-Hispanic White women. Non-Hispanic Black maternal mortality rates from obstetric embolism and obstetric hemorrhage were 2.3 to 2.6 times those for non-Hispanic White women. Together, these 4 causes accounted for 59% of the non-Hispanic Black‒non-Hispanic White maternal mortality disparity. Conclusions. The prominence of cardiovascular-related conditions among the leading causes of confirmed maternal death, particularly for non-Hispanic Black women, necessitates increased vigilance for cardiovascular problems during the pregnant and postpartum period. Many of these deaths are preventable.

Causes contributing to the excess maternal mortality risk for women 35 and over, United States, 2016-2017.

To better understand age-related disparities in US maternal mortality, we analyzed 2016-2017 vital statistics mortality data with cause-of-death literal text (actual words written on the death certificate) added. We created a subset of confirmed maternal deaths which had pregnancy mentions in the cause-of-death literals. Primary cause of death was identified and recoded using cause-of-death literals. Age-related disparities were examined both overall and by primary cause. Compared to women <35, the 2016-2017 US maternal mortality rate was twice as high for women aged 35-39, four times higher for women aged 40-44, and 11 times higher for women aged 45-54 years. Obstetric hemorrhage was the leading cause of death for women aged 35+ with rates 4 times higher than for women <35, followed by postpartum cardiomyopathy with a 3-fold greater risk. Obstetric embolism, eclampsia/preeclampsia, and Other complications of obstetric surgery and procedures each had a two-fold greater risk of death for women aged 35+. Together these 5 causes of death accounted for 70.9% of the elevated maternal mortality risk for women aged 35+. The excess maternal mortality risk for women aged 35+ was focused among a few causes of death and much of this excess mortality is preventable. Early detection and treatment, as well as continued care during the postpartum year is critical to preventing these deaths. The Alliance for Innovation on Maternal Health has promulgated patient safety bundles with specific interventions that health care systems can adopt in an effort to prevent these deaths.

Inhibition of stromal biglycan promotes normalization of the tumor microenvironment and enhances chemotherapeutic efficacy.

BACKGROUND: Biglycan is a proteoglycan found in the extracellular matrix. We have previously shown that biglycan is secreted from tumor endothelial cells and induces tumor angiogenesis and metastasis. However, the function of stroma biglycan in breast cancer is still unclear. METHODS: Biglycan gene analysis and its prognostic values in human breast cancers were based on TCGA data. E0771 breast cancer cells were injected into WT and Bgn KO mice, respectively. RESULTS: Breast cancer patients with high biglycan expression had worse distant metastasis-free survival. Furthermore, biglycan expression was higher in the tumor stromal compartment compared to the epithelial compartment. Knockout of biglycan in the stroma (Bgn KO) in E0771 tumor-bearing mice inhibited metastasis to the lung. Bgn KO also impaired tumor angiogenesis and normalized tumor vasculature by repressing tumor necrosis factor-ɑ/angiopoietin 2 signaling. Moreover, fibrosis was suppressed and CD8+ T cell infiltration was increased in tumor-bearing Bgn KO mice. Furthermore, chemotherapy drug delivery and efficacy were improved in vivo in Bgn KO mice. CONCLUSION: Our results suggest that targeting stromal biglycan may yield a potent and superior anticancer effect in breast cancer.

Dissemination of Evidence-Based Recommendations for Sickle Cell Disease to Primary Care and Emergency Department Providers in North Carolina: A Cost Benefit Analysis.

Background: Sickle cell disease (SCD) is a genetic condition affecting primarily individuals of African descent, who happen to be disproportionately impacted by poverty and who lack access to health care. Individuals with SCD are at high likelihood of high acute care utilization and chronic pain episodes. The multiple complications seen in SCD contribute to significant morbidity and premature mortality, as well as substantial costs to the healthcare system. Objectives: SCD is a complex chronic disease resulting in the need for primary, specialty and emergency care. Many providers do not feel prepared to care for individuals with SCD, despite the existence of evidence-based guidelines. We report the development of a SCD toolbox and the dissemination process to primary care and emergency department (ED) providers in North Carolina (NC). We report the effect of this dissemination on health-care utilization, cost of care, and overall cost-benefit. Methods: The SCD toolbox was adapted from the National Heart, Lung, and Blood Institute recommendations. Toolbox training was provided to quality improvement specialists who then disseminated the toolbox to primary care providers (PCPs) affiliated with the only NC managed care coordination system and ED providers. Tools were made available in paper, online, and in app formats to participating managed care network practices (n=1 800). Medicaid claims data were analyzed for total costs and benefits of the toolbox dissemination for a 24-month pre- and 18-month post-intervention period. Results: There was no statistically significant shift in the number of outpatient specialty visits, ED visits or hospitalizations. There was a small decrease in the number of PCP visits in the post-implementation period. The dissemination resulted in a net cost-savings of $361 414 ($14.03 per-enrollee per-month on average). However, the estimated financial benefit associated with the dissemination of the SCD toolbox was not statistically significant. Conclusions: Although we did not find the expected shift to increased PCP visits and decreased ED visits and hospitalizations, there were many lessons learned.

Atypical UV Photoproducts Induce Non-canonical Mutation Classes Associated with Driver Mutations in Melanoma.

Somatic mutations in skin cancers and other ultraviolet (UV)-exposed cells are typified by C>T and CC>TT substitutions at dipyrimidine sequences; however, many oncogenic "driver" mutations in melanoma do not fit this UV signature. Here, we use genome sequencing to characterize mutations in yeast repeatedly irradiated with UV light. Analysis of ~50,000 UV-induced mutations reveals abundant non-canonical mutations, including T>C, T>A, and AC>TT substitutions. These mutations display transcriptional asymmetry that is modulated by nucleotide excision repair (NER), indicating that they are caused by UV photoproducts. Using a sequencing method called UV DNA endonuclease sequencing (UVDE-seq), we confirm the existence of an atypical thymine-adenine photoproduct likely responsible for UV-induced T>A substitutions. Similar non-canonical mutations are present in skin cancers, which also display transcriptional asymmetry and dependence on NER. These include multiple driver mutations, most prominently the recurrent BRAF V600E and V600K substitutions, suggesting that mutations arising from rare, atypical UV photoproducts may play a role in melanomagenesis.