Replication Fork Progression during Re-replication Requires the DNA Damage Checkpoint and Double-Strand Break Repair

Replication origins are under tight regulation to ensure activation occurs only once per cell cycle. Origin re-firing in a single S phase leads to the generation of DNA double-strand breaks (DSBs) and activation of the DNA damage checkpoint. If the checkpoint is blocked, cells enter mitosis with partially re-replicated DNA that generates chromosome breaks and fusions. These types of chromosomal aberrations are common in numerous human cancers, suggesting that re-replication events contribute to cancer progression. It was proposed that fork instability and DSBs formed during re-replication are the result of head-to-tail collisions and collapse of adjacent replication forks. However, previously studied systems lack the resolution to determine whether the observed DSBs are generated at sites of fork collisions. Here, we utilize the Drosophila ovarian follicle cells, which exhibit re-replication under precise developmental control, to model the consequences of re-replication at actively elongating forks. Re-replication occurs from specific replication origins at six genomic loci, termed Drosophila amplicons in follicle cells (DAFCs). Precise developmental timing of DAFC origin firing permits identification of forks at defined points after origin initiation. Here, we show that DAFC re-replication causes fork instability and generates DSBs at sites of potential fork collisions. Immunofluorescence and ChIP-seq demonstrate the DSB marker γH2Av is enriched at elongating forks. Fork progression is reduced in the absence of DNA damage checkpoint components and nonhomologous end-joining (NHEJ), but not homologous recombination. NHEJ appears to continually repair forks during re-replication to maintain elongation.National Institutes of Health (U.S.) (Grant GM57940)Massachusetts Institute of Technology. School of Science (Fellowship in Cancer Research

Maternal Perceptions of Mealtimes: Comparison of Children with Autism Spectrum Disorder and Children with Typical Development

Background: This study examined mealtime techniques reported by mothers of preschool children with Autism Spectrum Disorder (ASD) and mothers of children with typical development (TD). The mothers’ perceived levels of success and sources of information for mealtime techniques were also reported. Method: The participants were 24 mothers of children with ASD (ASD group) and 24 mothers of children with typical development (TD group) between 3 and 6 years of age. The Background Information Survey and the Mealtime Techniques Interview were administered. Results: The ASD group used significantly more techniques in categories of food appearances, restrictive diets, and vitamin/supplement therapy. The TD group used significantly more techniques in the categories of etiquette and negative consequences. Both groups rated techniques similarly with no significant difference between the perceived rate of success for each category. Finally, 91% of mealtime techniques for both groups were parent generated with few from professionals. Conclusion: The results showed that many of the mothers in both groups used similar mealtime techniques and most implemented techniques that were self-generated with generally moderate perception of success. Occupational therapists should collaboratively work with families to increase mealtime success by recommending interventions that are individualized and family centered

Ensuring Equitable Application of Interventions to Vulnerable Subpopulations in the Kentucky Consortium for Accountable Health Communities (KC-AHC)

Introduction: The Centers for Medicare and Medicaid Services (CMS) has funded the Accountable Health Communities (AHC) model to test whether systematically identifying and addressing the health-related social needs (HRSNs) of individuals would impact healthcare utilization and total cost of care for Medicare and Medicaid beneficiaries. Toward this effort, AHCs implement screening, referral, and community navigation services in their local areas. There are 28 CMS-funded AHCs nationwide, including the Kentucky Consortium for Accountable Health Communities (KC-AHC). Purpsoe: This study aims to assess the equity of KC-AHC model activities in three vulnerable sub-populations: dual enrollees, disabled individuals, and women. Methods: Twenty-eight primary care clinical sites across 19 healthcare organizations administered (in-person or telephonic) the AHC screening instrument from August 2018 to April 2021. Every six months, social needs positivity rates, navigation eligibility, service opted-in rates and delivery data were monitored among dual enrollees, disabled persons, and women. Subpopulations were compared to their comparisons (for example, non-dual enrollees) and to available benchmarked data. Results: All proportions of subpopulation in screened beneficiaries approximated or exceeded regional benchmarks. While needs among groups fluctuated over time, most reflected positivity rates in excess of comparisons: (1) rates among females ranged from 29.6% to 36.1%, but tended to narrow (relative to males) over time; (2) disabled individuals’ positivity rate ranged from 27.8% to 36.1% but also lessened over time compared with non-disabled counterparts; and (3) positive rates among the dually-enrolled ranged from 34.7% to 42.4%, with the disparity to non-dual enrollees remaining relatively stable. Rates of opt-in and receipt of navigation in dual enrollees and women did not show disparities. There was a persistent gap in opt-in rates between disabled and non-disabled beneficiaries, though one was not identified in receipt. Implications: Results suggest that the KC-AHC adequately screened dual enrollees, disabled individuals, and women during model implementation. The AHC Model may have helped to narrow gaps in social needs between sub-populations and comparison groups, with beneficiaries becoming better connected to community services

Whole-Genome Sequencing and Concordance Between Antimicrobial Susceptibility Genotypes and Phenotypes of Bacterial Isolates Associated with Bovine Respiratory Disease.

Extended laboratory culture and antimicrobial susceptibility testing timelines hinder rapid species identification and susceptibility profiling of bacterial pathogens associated with bovine respiratory disease, the most prevalent cause of cattle mortality in the United States. Whole-genome sequencing offers a culture-independent alternative to current bacterial identification methods, but requires a library of bacterial reference genomes for comparison. To contribute new bacterial genome assemblies and evaluate genetic diversity and variation in antimicrobial resistance genotypes, whole-genome sequencing was performed on bovine respiratory disease-associated bacterial isolates (Histophilus somni, Mycoplasma bovis, Mannheimia haemolytica, and Pasteurella multocida) from dairy and beef cattle. One hundred genomically distinct assemblies were added to the NCBI database, doubling the available genomic sequences for these four species. Computer-based methods identified 11 predicted antimicrobial resistance genes in three species, with none being detected in M. bovis While computer-based analysis can identify antibiotic resistance genes within whole-genome sequences (genotype), it may not predict the actual antimicrobial resistance observed in a living organism (phenotype). Antimicrobial susceptibility testing on 64 H. somni, M. haemolytica, and P. multocida isolates had an overall concordance rate between genotype and phenotypic resistance to the associated class of antimicrobials of 72.7% (P < 0.001), showing substantial discordance. Concordance rates varied greatly among different antimicrobial, antibiotic resistance gene, and bacterial species combinations. This suggests that antimicrobial susceptibility phenotypes are needed to complement genomically predicted antibiotic resistance gene genotypes to better understand how the presence of antibiotic resistance genes within a given bacterial species could potentially impact optimal bovine respiratory disease treatment and morbidity/mortality outcomes

Coral restoration effectiveness: multiregional snapshots of the long-term responses of coral assemblages to restoration

Coral restoration is rapidly becoming a mainstream strategic reef management response to address dramatic declines in coral cover worldwide. Restoration success can be defined as enhanced reef functions leading to improved ecosystem services, with multiple benefits at socio-ecological scales. However, there is often a mismatch between the objectives of coral restoration programs and the metrics used to assess their effectiveness. In particular, the scales of ecological benefits currently assessed are typically limited in both time and space, often being limited to short-term monitoring of the growth and survival of transplanted corals. In this paper, we explore reef-scale responses of coral assemblages to restoration practices applied in four well-established coral restoration programs. We found that hard coral cover and structural complexity were consistently greater at restored compared to unrestored (degraded) sites. However, patterns in coral diversity, coral recruitment, and coral health among restored, unrestored, and reference sites varied across locations, highlighting differences in methodologies among restoration programs. Altogether, differences in program objectives, methodologies, and the state of nearby coral communities were key drivers of variability in the responses of coral assemblages to restoration. The framework presented here provides guidance to improve qualitative and quantitative assessments of coral restoration efforts and can be applied to further understanding of the role of restoration within resilience-based reef management

DNA Targeting as a Likely Mechanism Underlying the Antibacterial Activity of Synthetic Bis-Indole Antibiotics

We previously reported the synthesis and biological activity of a series of cationic bis-indoles with potent, broad-spectrum antibacterial properties. Here, we describe mechanism of action studies to test the hypothesis that these compounds bind to DNA and that this target plays an important role in their antibacterial outcome. The results reported here indicate that the bis-indoles bind selectively to DNA at A/T-rich sites, which is correlated with the inhibition of DNA and RNA synthesis in representative Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) organisms. Further, exposure of E. coli and S. aureus to representative bis-indoles resulted in induction of the DNA damage-inducible SOS response. In addition, the bis-indoles were found to be potent inhibitors of cell wall biosynthesis; however, they do not induce the cell wall stress stimulon in S. aureus, suggesting that this pathway is inhibited by an indirect mechanism. In light of these findings, the most likely basis for the observed activities of these compounds is their ability to bind to the minor groove of DNA, resulting in the inhibition of DNA and RNA synthesis and other secondary effects

Enhancing permanganate chemiluminescence detection for the determination of glutathione and glutathione disulfide in biological matrices

Acidic potassium permanganate chemiluminescence enables direct post-column detection of glutathione, but its application to assess the redox state of a wider range of biological fluids and tissues is limited by its sensitivity. Herein we show that the simple on-line addition of an aqueous formaldehyde solution not only enhances the sensitivity of the procedure by two orders of magnitude, but also provides a remarkable improvement in the selectivity of the reagent towards thiols such as glutathione (compared to phenols and amino acids that do not possess a thiol group). This enhanced mode of detection was applied to the determination of glutathione and its corresponding disulfide species in homogenised striatum samples taken from both wild type mice and the R6/1 transgenic mouse model of Huntington\u27s disease, at both 8 and 12 weeks of age. No significant difference was observed between the GSH/GSSG ratios of wild type mice and R6/1 mice at either age group, suggesting that the early disease progression had not significantly altered the intracellular redox environment

Prion 2016 poster abstracts

Until now, the 3-dimensional structure of infectious mammalian prions and how this differs from non-infectious amyloid fibrils remained unknown. Mammalian prions are hypothesized to be fibrillar or amyloid forms of prion protein (PrP), but structures observed to date have not been definitively correlated with infectivity. One of the major challenges has been the production of highly homogeneous material of demonstrable high specific infectivity to allow direct correlation of particle structure with infectivity. We have recently developed novel methods to obtain exceptionally pure preparations of prions from prion-infected murine brain and have shown that pathogenic PrP in these high-titer preparations is assembled into rod-like assemblies (Wenborn et al. 2015. Sci. Rep. 10062). Our preparations contain very high titres of infectious prions which faithfully transmit prion strain-specific phenotypes when inoculated into mice making them eminently suitable for detailed structural analysis. We are now undertaking structural characterization of prion assemblies and comparing these to the structure of non-infectious PrP fibrils generated from recombinant Pr

Care Transitions From Patient and Caregiver Perspectives

PURPOSE: Despite concerted actions to streamline care transitions, the journey from hospital to home remains hazardous for patients and caregivers. Remarkably little is known about the patient and caregiver experience during care transitions, the services they need, or the outcomes they value. The aims of this study were to (1) describe patient and caregiver experiences during care transitions and (2) characterize patient and caregiver desired outcomes of care transitions and the health services associated with them. METHODS: We interviewed 138 patients and 110 family caregivers recruited from 6 health networks across the United States. We conducted 34 homogenous focus groups (103 patients, 65 caregivers) and 80 key informant interviews (35 patients, 45 caregivers). Audio recordings were transcribed and analyzed using principles of grounded theory to identify themes and the relationship between them. RESULTS: Patients and caregivers identified 3 desired outcomes of care transition services: (1) to feel cared for and cared about by medical providers, (2) to have unambiguous accountability from the health care system, and (3) to feel prepared and capable of implementing care plans. Five care transition services or provider behaviors were linked to achieving these outcomes: (1) using empathic language and gestures, (2) anticipating the patient\u27s needs to support self-care at home, (3) collaborative discharge planning, (4) providing actionable information, and (5) providing uninterrupted care with minimal handoffs. CONCLUSIONS: Clear accountability, care continuity, and caring attitudes across the care continuum are important outcomes for patients and caregivers. When these outcomes are achieved, care is perceived as excellent and trustworthy. Otherwise, the care transition is experienced as transactional and unsafe, and leaves patients and caregivers feeling abandoned by the health care system