DSS1 restrains BRCA2's engagement with dsDNA for homologous recombination, replication fork protection, and R-loop homeostasis.

DSS1, essential for BRCA2-RAD51 dependent homologous recombination (HR), associates with the helical domain (HD) and OB fold 1 (OB1) of the BRCA2 DSS1/DNA-binding domain (DBD) which is frequently targeted by cancer-associated pathogenic variants. Herein, we reveal robust ss/dsDNA binding abilities in HD-OB1 subdomains and find that DSS1 shuts down HD-OB1's DNA binding to enable ssDNA targeting of the BRCA2-RAD51 complex. We show that C-terminal helix mutations of DSS1, including the cancer-associated R57Q mutation, disrupt this DSS1 regulation and permit dsDNA binding of HD-OB1/BRCA2-DBD. Importantly, these DSS1 mutations impair BRCA2/RAD51 ssDNA loading and focus formation and cause decreased HR efficiency, destabilization of stalled forks and R-loop accumulation, and hypersensitize cells to DNA-damaging agents. We propose that DSS1 restrains the intrinsic dsDNA binding of BRCA2-DBD to ensure BRCA2/RAD51 targeting to ssDNA, thereby promoting optimal execution of HR, and potentially replication fork protection and R-loop suppression.

Isradipine therapy in Cacna1dIle772Met/+ mice ameliorates primary aldosteronism and neurologic abnormalities.

Somatic gain-of-function mutations in the L-type calcium channel CaV1.3 (CACNA1D gene) cause adrenal aldosterone-producing adenomas and micronodules. De novo germline mutations are found in a syndrome of primary aldosteronism, seizures, and neurologic abnormalities (PASNA) as well as in autism spectrum disorder. Using CRISPR/Cas9, we here generated mice with a Cacna1d gain-of-function mutation found in both adenomas and PASNA syndrome (Cacna1dIle772Met/+). These mice show reduced body weight and increased mortality from weaning to approximately 100 days of age. Male mice do not breed, likely due to neuromotor impairment, and the offspring of female mice die perinatally, likely due to lack of maternal care. Mice generated by in vitro fertilization showed elevated intracellular calcium in the aldosterone-producing zona glomerulosa, an elevated aldosterone/renin ratio, and persistently elevated serum aldosterone on a high-salt diet as signs of primary aldosteronism. Anesthesia with ketamine and xylazine induced tonic-clonic seizures. Neurologic abnormalities included hyperlocomotion, impaired performance in the rotarod test, impaired nest building, and slight changes in social behavior. Intracellular calcium in the zona glomerulosa, aldosterone levels, and rotarod performance responded to treatment with the calcium channel blocker isradipine, with implications for the therapy of patients with aldosterone-producing lesions and with PASNA syndrome.

Mental Health Screening in Pediatric Primary Care: Factors Associated With Screening Completion and Elevated Scores.

This study aimed to examine predictors of complete and elevated youth mental health screens. Parents of 4- to 11-year-old children completed the Strengths and Difficulties Questionnaire (SDQ) during a routine, universal mental health screening initiative in primary care. Bivariate logistic regressions were run to examine associations between independent (visit age, sex, race/ethnicity, language, insurance, and guardian) and dependent variables (screening completion and elevated SDQ score). Parents of younger and Spanish-speaking (vs English-speaking) children were less likely to have a complete SDQ screen. Among those with complete SDQ screens, older children, male children, those with public or no insurance, and those who had a mother (vs father) complete the screener were more likely to have an elevated score. Understanding patterns of screening completion rates and predictors of elevated screens provides valuable information to improve resource mapping and planning. Findings can inform mental health screening implementation and optimization within primary care.

Pediatric Provider Experiences with Implementation of Routine Mental Health Screening.

OBJECTIVE: Despite the utility of universal screening, most pediatric providers rarely use mental health (MH) screening tools. As such, provider descriptions of their experiences with universal screening are limited. The goal of this study was to describe barriers to, and facilitators of, universal MH screening implementation, the perceived impact of such screening, impressions of a screening-focused quality improvement (QI) Learning Collaborative, and lessons learned. METHOD: We invited primary care clinicians participating in a large-scale QI Learning Collaborative on MH screening (n = 107) to complete postproject interviews. Interviews were transcribed and analyzed using constant comparative qualitative analysis, an inductive, iterative process. RESULTS: Eleven interviews were completed and analyzed. Practice sites included academic health centers, a private practice, and a federally qualified health center. Providers described the positive impact of screening (increased identification of MH concerns) and barriers and facilitators of screening at the practice level (clinic and leadership buy-in and electronic medical record integration), the provider level (provider beliefs about the importance of screening), and the patient level (parent literacy). Challenges of linking families with care after screening included lack of adequate referrals, long wait lists, limited bilingual providers, insurance gaps, and inadequate feedback loops. Access to on-site MH clinicians and participation in the Learning Collaborative were described as beneficial. CONCLUSION: Findings elucidate how universal MH screening can be sustainably integrated into real-world primary care settings and may facilitate the uptake of American Academy of Pediatrics recommendations for best practices in screening for MH concerns.

Mesenchymal stem cell­derived extracellular vesicles promote the in vitro proliferation and migration of breast cancer cells through the activation of the ERK pathway.

Mesenchymal stem cells (MSCs) have been demonstrated to be involved in tumor progression and the modulation of the tumor microenvironment, partly through their secretome. Extracellular vesicles (EVs) are membranous nanovesicles secreted by multiple types of cells and have been demonstrated to mediate intercellular communication in both physiological and pathological conditions. However, numerous questions still remain regarding the underlying mechanisms and functional consequences of these interactions. The purpose of this study was to investigate the effects of human umbilical cord mesenchymal stem cell­derived EVs (hUC­MSC­EVs) on the proliferation, migration and invasion of human breast cancer cells. We successfully generated and identified hUC­MSCs and hUC­MSC­EVs which were used in this study. The results revealed that treatment of the MDA­MB­231 and MCF­7 human breast cancer cells with medium containing hUC­MSC­EVs significantly enhanced the proliferation, migration and invasion of the cells in vitro. Treatment of the cells with medium containing hUC­MSC­EVs also reduced E­cadherin expression and increased N­cadherin expression, thus promoting the epithelial­mesenchymal transition (EMT) of the breast cancer cells. Treatment of the breast cancer cells with extracellular signal­regulated kinase (ERK) inhibitor prior to the interaction with hUC­MSC­EVs significantly reversed the enhanced proliferation, migration and invasion, as well as the EMT of the breast cancer cells induced by the hUC­MSC­EVs. On the whole, these data indicate that hUC­MSC­EVs promote the invasive and migratory potential of breast cancer cells through the induction of EMT via the ERK pathway, leading to malignant tumor progression and metastasis. Taken together, the findings of this study suggest that targeting pathways to reverse EMT may lead to the development of novel therapeutic approaches with which to combat breast cancer.

Electronic and Steric Tuning of Catalytic H2 Evolution by Cobalt Complexes with Pentadentate Polypyridyl-Amine Ligands.

Structural modifications of molecular cobalt catalysts have provided important insights into the structure-function relationship for the hydrogen evolution reaction. We have shown that replacement of equatorial pyridines with more basic and conjugate isoquinoline groups of a pentadentate ligand results in lower overpotential and higher catalytic activity for electro- and photolytic H2 production in aqueous solutions. To fully understand the electronic and steric effects of the axial group that lies trans to the proposed cobalt hydride intermediate, isoquinoline groups were introduced in two new pentadentate ligands, N, N-bis(2-pyridinylmethyl)[3-(2-pyridinyl)isoquinoline)]-1-methanamine (DPA-1-MPI) and N, N-bis(2-pyridinylmethyl)[1-(2-pyridinyl)-isoquinoline)]-3-methanamine (DPA-3-MPI). Despite a slight structural difference of the introduced isoquinoline group, the resulting cobalt complexes display drastic changes in their electro- and photochemical properties. There are positive shifts of 290 and 260 mV, respectively, for the CoII/CoI and CoIII-H/CoII-H couples from [Co(DPA-1-MPI)(H2O)](PF6)3 to [Co(DPA-3-MPI)(H2O)](PF6)3, with the former being ∼32 times as active as the latter in photocatalytic H2 production. Density functional theory (DFT) calculations show that the protonation of CoI to yield the CoIII-H species is energetically more favorable for [Co(DPA-1-MPI)(H2O)](PF6)3 than that of [Co(DPA-3-MPI)(H2O)](PF6)3. Both experimental results and DFT computations suggest that the presence of a planar conjugate bipyridyl unit or its isoquinoline derivative is a key feature for stabilizing low valent CoI species toward proton binding. The incorporation of an electron-donating group trans to the proposed Co-H species also facilitates proton binding and H-H bond formation, which is proposed to occur by the heterolytic coupling of CoII-H species. The overall catalytic H2 evolution is presented as the modified electron transfer (E)-proton transfer (C)-electron transfer (E)-proton transfer (C) (mod-ECEC) pathway. This study provides important new insight into the electronic and steric factors controlling catalytic H2 production by Co complexes with pentadentate ligands.

Mental Health Screening Quality Improvement Learning Collaborative in Pediatric Primary Care.

BACKGROUND: In the United States, up to 20% of children experience a mental health (MH) disorder in a given year, many of whom remain untreated. Routine screening during annual well visits is 1 strategy providers can use to identify concerns early and facilitate appropriate intervention. However, many barriers exist to the effective implementation of such screening. METHODS: A 15-month quality improvement learning collaborative was designed and implemented to improve screening practices in primary care. Participating practices completed a survey at 3 time points to assess preparedness and ability to promote and support MH issues. Monthly chart reviews were performed to assess the rates of screening at well visits, documentation of screening results, and appropriate coding practices. RESULTS: Ten practices (including 107 providers) were active participants for the duration of the project. Screening rates increased from 1% at baseline to 74% by the end of the project. For the 1 practice for which more comprehensive data were available, these screening rates were sustained over time. Documentation of results and appropriate billing for reimbursement mirrored the improvement seen in screening rates. CONCLUSIONS: The learning collaborative model can improve MH screening practices in pediatric primary care, an important first step toward early identification of children with concerns. More information is needed about the burden placed on practices and providers to implement these changes. Future research will be needed to determine if improved identification leads to improved access to care and outcomes.

Behavioral Health Integration in Health Care Settings: Lessons Learned from a Pediatric Hospital Primary Care System.

Behavioral health integration within primary care has been evolving, but literature traditionally focuses on smaller scale efforts. We detail how behavioral health has been integrated across a large, urban pediatric hospital system's six primary care clinics (serving over 35,000 children annually and insured predominately through Medicaid) and discuss strategies for success in sustaining and expanding efforts to achieve effective integration of behavioral health into primary care. In a time span of 3 years, the clinics have implemented routine, universal behavioral health screening at well child visits, participated in a 15-month behavioral health screening quality improvement learning collaborative, and integrated the work of psychologists and psychiatrists. Additional work remains to be done in improving family engagement, further expanding services, and ensuring sustainability.

Immunogenicity of native or pegylated E. coli and Erwinia asparaginases assessed by ELISA and surface plasmon resonance (SPR-biacore) assays of IgG antibodies (Ab) in sera from patients with acute lymphoblastic leukemia (ALL).

BACKGROUND: Therapeutic uses of asparaginases (ASNase) have been shown to elicit immune responses resulting in the development of potentially life-threatening human anti-bacterial antibodies (Ab). A robust screening enzyme-linked immunosorbent assay (ELISA) to detect binding Ab(+) against ASNase has been developed and validated for therapeutic monitoring to support clinical trials. Recently, a protein chip bioassay (Biacore) was developed for the Ab of these proteins. These methods were compared. MATERIALS AND METHODS: A Biacore T-100 analyzer using a protein bioassay and an ELISA assay were used to determine the IgG immmuboglobulin Ab against ASNase in sera from 84 acute lymphoblastic leukemia (ALL) patients plus 6 controls (n=121 samples). These samples were characterized for anti-ASNase Ab neutralizing activity. Human E. coli ASNase, pegaspargase and Erwinase proteins were covalently coupled to the carboxy-methylated dextran matrix of a CM5 sensor chip (surface plasmon resonance, SPR). In the course of a nested experimental design, a wide range of human sera from patients who had obvious clinical allergic reactions after either native or pegaspargase treatments were tested. The data were fitted by a parametric logistic equation (+/-95% confidence interval, CI), which ranged from <3.0% to <14%. RESULTS: The specificity of Ab(+) was evaluated using "spiked" human IgG antibodies. Both assays provide near excellent linearity and sensitivity of response (<0.8 to <500 ratio and 1-3000 resonance units [RU]) of anti-ASNase Ab in human sera with low variance. The bioassay method was ten times more sensitive than the ELISA Ab assay. The lowest limit of quantification of Ab(+) ratio for the SPR assay was 0.6 whereas the upper limit of quantification was 3000 RU. The SPR assay results were in excellent accord with both the Ab(-) and the Ab(+). Ab(-) by the ELISA method (<1.003 ratio) was related to a mean RU value of 8.1. Despite the narrow range of ambiguity around the 1.1 Ab(+) ratio values, the majority of the specimens (93.2%) were determined to be Ab(+) by either ELISA or SPR determination. CONCLUSION: The vast majority (81/84 = 96.4%) of the IgG Ab(+) were neutralizing. The SPR Ab determination technique is reliable, accurate and more sensitive than the ELISA method.

Efficacy of DNA vaccination against western equine encephalitis virus infection.

The efficacy of a DNA vaccine against western equine encephalitis (WEE) infection in mice was evaluated. The 26S structural region was expressed, in vitro from an internal T7 promoter using a rabbit reticulysate transcription/translation system; and from a CMV promoter after transfection into Vero cell monolayers. The proteins synthesized were reactive with anti-WEE virus (WEEV) antibodies, both in western blot analysis and histochemical staining, respectively. When the DNA vaccine plasmid, pVHX-6, was administered intraepidermally to mice, followed by challenge in a lethal mouse model, the level of protection obtained ranged from 50 to 100% amongst three strains of WEEV. Preliminary results suggest the protective immunity provided by the DNA vaccine appears to be a cell-mediated immune response, as elevated cytotoxic T lymphocyte activity was detected against the E2 protein in a T-cell proliferation assay. The efficacy results suggest a DNA vaccine may be a promising approach against WEE infection.

Antifungal activity of camptothecin, trifolin, and hyperoside isolated from Camptotheca acuminata.

Leaf spots and root rots are major fungal diseases in Camptotheca acuminata that limit cultivation of the plant for camptothecin (CPT), a promising anticancer and antiviral alkaloid. Bioassays showed that pure CPT and flavonoids (trifolin and hyperoside) isolated from Camptotheca effectively control fungal pathogens in vitro, including Alternaria alternata, Epicoccum nigrum, Pestalotia guepinii, Drechslera sp., and Fusarium avenaceum, although antifungal activity of these compounds in the plant is limited. CPT inhibited mycelial growth by approximately 50% (EC50) at 10-30 microg/mL and fully inhibited growth at 75-125 microg/mL. The flavonoids were less effective than CPT at 50 microg/mL, particularly within 20 days after treatment, but more effective at 100 or 150 microg/mL. CPT, trifolin, and hyperoside may serve as leads for the development of fungicides.