Transgenic mice for in vivo epigenome editing with CRISPR-based systems.

CRISPR-Cas9 technologies have dramatically increased the ease of targeting DNA sequences in the genomes of living systems. The fusion of chromatin-modifying domains to nuclease-deactivated Cas9 (dCas9) has enabled targeted epigenome editing in both cultured cells and animal models. However, delivering large dCas9 fusion proteins to target cells and tissues is an obstacle to the widespread adoption of these tools for in vivo studies. Here, we describe the generation and characterization of two conditional transgenic mouse lines for epigenome editing, Rosa26:LSL-dCas9-p300 for gene activation and Rosa26:LSL-dCas9-KRAB for gene repression. By targeting the guide RNAs to transcriptional start sites or distal enhancer elements, we demonstrate regulation of target genes and corresponding changes to epigenetic states and downstream phenotypes in the brain and liver in vivo, and in T cells and fibroblasts ex vivo. These mouse lines are convenient and valuable tools for facile, temporally controlled, and tissue-restricted epigenome editing and manipulation of gene expression in vivo.

Full-length dystrophin restoration via targeted exon integration by AAV-CRISPR in a humanized mouse model of Duchenne muscular dystrophy.

Targeted gene-editing strategies have emerged as promising therapeutic approaches for the permanent treatment of inherited genetic diseases. However, precise gene correction and insertion approaches using homology-directed repair are still limited by low efficiencies. Consequently, many gene-editing strategies have focused on removal or disruption, rather than repair, of genomic DNA. In contrast, homology-independent targeted integration (HITI) has been reported to effectively insert DNA sequences at targeted genomic loci. This approach could be particularly useful for restoring full-length sequences of genes affected by a spectrum of mutations that are also too large to deliver by conventional adeno-associated virus (AAV) vectors. Here, we utilize an AAV-based, HITI-mediated approach for correction of full-length dystrophin expression in a humanized mouse model of Duchenne muscular dystrophy (DMD). We co-deliver CRISPR-Cas9 and a donor DNA sequence to insert the missing human exon 52 into its corresponding position within the DMD gene and achieve full-length dystrophin correction in skeletal and cardiac muscle. Additionally, as a proof-of-concept strategy to correct genetic mutations characterized by diverse patient mutations, we deliver a superexon donor encoding the last 28 exons of the DMD gene as a therapeutic strategy to restore full-length dystrophin in >20% of the DMD patient population. This work highlights the potential of HITI-mediated gene correction for diverse DMD mutations and advances genome editing toward realizing the promise of full-length gene restoration to treat genetic disease.

Variability in Antimicrobial Prescribing Across Five Ambulatory Settings Within a Large Integrated Health System.

Background: Lack of access to timely, detailed antibiotic use data has limited ambulatory antibiotic stewardship efforts. Antibiotic utilization is tracked across ambulatory care sites and emergency departments (ED) within a large integrated health system. Methods: This is a retrospective cohort analysis from June 1, 2019 to May 31, 2020 comparing antibiotic prescribing for all patients with ICD-10 diagnosis codes for cystitis, otitis media, pharyngitis, sinusitis, and upper respiratory tract infections (URTIs) among five ambulatory care departments across northeast Ohio and southeast Florida locations: ED, Urgent Care (UC), On-Demand Telehealth (TEL), Pediatrics (PED), and Primary Care (PC). Results: A total of 261,947 encounters were included (ED:56,766, UC:92,749, TEL:8,783, PED:29,151, PC:74,498) for the treatment of cystitis (30,932), otitis media (22,094), pharyngitis (59,964), sinusitis (53,693), or URTI (95,264). The population was 63% female with a median age of 34.2 years [12.8-56.3]. A total of 17% of patients had documented penicillin allergies and 18% of patients with pharyngitis received Group A Streptococcus (GAS) testing. Antibiotics were prescribed in 44% of encounters (ED:21,746 [38%], UC:45,652 [49%], TEL:4,622 [53%], PED:10,909 [37%], PC:33,547 [45%]; P < 0.001). Guideline concordant antibiotics were prescribed in 65% of encounters (ED:14,338 [66%], UC:31,532 [69%], TEL:3,869 [84%], PED:8,212 [75%], PC:17,263 [51%]; P < 0.001). Conclusions: Observed rates of antibiotic and guideline concordant antibiotic prescribing were similar to national published rates of antibiotic prescribing in the ambulatory setting. The variability in antibiotic prescribing demonstrates opportunities for targeted outpatient stewardship efforts. Timely antibiotic tracking tools can facilitate ambulatory antimicrobial stewardship activities.

"Your Family is Always With You": Perceptions of Parental Relationships Among Hispanic/Latinx Young Adults Pursuing STEM Careers.

Hispanic/Latinx young adults remain significantly underrepresented in science, technology, engineering, and mathematics (STEM) fields, yet the role families play in these young adults' trajectories in STEM is still underexamined. The purpose of this study was to examine the relational supports and constraints that Hispanic/Latinx college students in STEM majors experienced with their parents as they moved through college and transitioned into their first year of graduate school or full-time employment. Two rounds of interviews were conducted with 18 Hispanic/Latinx young adults who were part of an undergraduate STEM program at a Hispanic-majority university. Most of the study participants reported benefiting from immense emotional support from their parents; however, this emotional support was often simultaneously coupled with home-school value conflicts and a dynamic we call "conversational constraints." Results from this study point to important interventions involving family that might improve the rates of participation of Hispanic/Latinx students from a range of socioeconomic backgrounds in STEM fields.

Older Adults in the United States and COVID-19: A Qualitative Study of Perceptions, Finances, Coping, and Emotions.

Introduction: Older adults have the poorest coronavirus (COVID-19) prognosis with the highest risk of death due to complications, making their COVID-19 experiences particularly important. Guided by the stress-appraisal-coping theoretical model, we sought to understand COVID-related perceptions and behaviors of older adults residing in the United States. Materials and Methods: We used convenience sampling to recruit persons with the following inclusion criteria: Aged ≥ 65 years, English fluency, and U.S. residency. Semi structured in-depth interviews were conducted remotely and audio recorded between April 25, 2020 and May 7, 2020. Interviews were professionally transcribed with a final study sample of 43. A low-inference qualitative descriptive design was used to provide a situated understanding of participants' life experiences using their naturalistic expressions. Results: The mean age of participants was 72.4 ± 6.7. Slightly over half were female (55.8%), 90.6% were White, and 18.6% lived alone. The largest percentages of participants resided in a rural area (27.9%) or small city (25.6%). We identified four themes, including (1) risk perception, (2) financial impact, (3) coping, and (4) emotions. Most participants were aware of their greater risk for poor COVID-19 outcomes but many did not believe in their increased risk. Financial circumstances because of the pandemic varied with largely no financial impacts, while others reported negative impacts and a few reported positive impacts. Coping was problem- and emotion-focused. Problem-focused coping included precautionary efforts and emotion-focused coping included creating daily structure, pursuing new and/or creative activities, connecting with others in new ways, and minimizing news media exposure. Overall, emotional health was negatively affected by the pandemic although some participants reported positive emotional experiences. Conclusions: Perceiving themselves as high risk for COVID-19 complications, older adults used precautionary measures to protect themselves from contracting the virus. The precautionary measures included social isolation, which can negatively affect mental health. Older adults will need to be resourceful and draw on existing resources to cope, such as engaging in creative activities and new strategies to connect with others. Our findings underscore the importance of the preservation of mental health during extended periods of isolation by taking advantage of low-to-no-cost existing resources.

The Emerging STEM Paths and Science Identities of Hispanic/Latinx College Students: Examining the Impact of Multiple Undergraduate Research Experiences.

This study reports findings from 19 interviews with Hispanic/Latinx students participating in a university-wide, multiyear program designed to retain students from underrepresented backgrounds in science, technology, engineering, and mathematics (STEM) at a Hispanic-serving institution. We focus on the impact that having multiple opportunities to engage in faculty-mentored, cocurricular undergraduate research experiences (UREs) had on students' STEM paths in college and the cultivation of their science identities. In addition to professional and psychosocial benefits, our findings suggest that having the opportunity to spend multiple summers in UREs at partnering institutions away from home helped to strengthen Hispanic/Latinx students' comfort levels with being away from their families and helped them recognize the broad range of opportunities available to them for graduate school.

Physician-to-Physician Electronic Consultation: A Tool for the Pediatric Infectious Diseases Specialist to Document Encounters and Quantify Effort.

E-consults replace "curbside" interactions, facilitate provider-specialist communication, document within the medical record, and track relative value units (RVUs). Pediatric infectious diseases (PID) E-consults commonly relate to vaccines, exposures, diagnoses, and treatments. The documented RVU effort of 197 consecutive PID E-consults was equivalent to 70 level 4 new outpatient consults.

Navigating Social Relationships with Mentors and Peers: Comfort and Belonging among Men and Women in STEM Summer Research Programs.

Gender disparities in science, technology, engineering, and mathematics (STEM) are well documented, yet little is known about how women and men students establish social relationships with research mentors and peers and how that shapes their experiences in STEM. We conducted a series of interviews with 17 undergraduate students from a Hispanic majority institution regarding their participation in STEM-focused summer research programs at nine universities. Differences in levels of comfort in relationships were present when comparing men and women. Women students expressed comfort in relationships with mentors who provided psychosocial mentoring, were available to answer questions, and were of the same gender; they expressed some social discomfort in informal interactions with mentors. Men students felt comfortable with mentors who provided limited guidance, little psychosocial mentoring, and opportunities for informal interactions. In terms of peer relationships, women sought out the confidence of a few similar peers, while men were comfortable with a wide variety of peers. Men's greater comfort with social relationships seemed to reflect their affinity with the masculine-dominated culture of STEM. For women, cultivating safe spaces through relationships with supportive peers and working with same-gender faculty mentors seemed to mitigate some of the discomforting aspects of their STEM research experiences.

The conundrum of social class: Disparities in publishing among STEM students in undergraduate research programs at a Hispanic majority institution.

Research on the science, technology, engineering, and math (STEM) student development pipeline has largely ignored social class and instead examined inequalities based on gender and race. We investigate the role of social class in undergraduate student research publications. Data come from a sample of 213 undergraduate research participants majoring in STEM at a Hispanic-majority institution. Based on generalized estimating equations that adjust for student demographics, research confidence, mentoring experiences, duration/number of research experiences, and clustering by major, we find that higher income students and continuing-generation students (vs. first-generation students) were significantly more likely to publish. Continuing-generation students had an even greater likelihood of publishing than first-generation students as students accrued more research confidence, spent more hours/week with faculty mentors, and conducted research for more months. Results suggest that undergraduate research programs designed to enhance diversity may help close some gaps (e.g., gender) but inadvertently reproduce class inequalities.

Factors Influencing Student Gains from Undergraduate Research Experiences at a Hispanic-Serving Institution.

Undergraduate research experiences (UREs) confer many benefits to students, including improved self-confidence, better communication skills, and an increased likelihood of pursuing science careers. Additionally, UREs may be particularly important for racial/ethnic minority students who are underrepresented in the science workforce. We examined factors hypothetically relevant to underrepresented minority student gains from UREs at a Hispanic-serving institution, such as mentoring quality, family income, being Latino/a, and caring for dependents. Data came from a 2013 survey of University of Texas at El Paso students engaged in 10 URE programs (n = 227). Using generalized linear models (GzLMs) and adjusting for known covariates, we found that students who reported receiving higher-quality mentorship, spending more hours caring for dependents, and receiving more programmatic resources experienced significantly greater gains from their URE in all three areas we examined (i.e., thinking and working like a scientist, personal gains, and gains in skills). In two of three areas, duration of the URE was positive and significant. Being Latino/a was positive and significant only in the model predicting personal gains. Across the three models, quality of mentorship was the most important correlate of gains. This suggests that providing training to faculty mentors involved in UREs may improve student outcomes and increase program efficacy.

Functional analysis of fibronectin isoforms in chondrogenesis: Full-length recombinant mesenchymal fibronectin reduces spreading and promotes condensation and chondrogenesis of limb mesenchymal cells.

Fibronectin (FN), a large dimeric glycoprotein, functions primarily as a connecting molecule in the extracellular matrices of tissues by mediating both cell-matrix and matrix-matrix interactions. All members of the FN family are products of a single FN gene; heterogeneity arises from the alternative splicing of at least three regions (IIIB, IIIA, and V) during processing of a common primary transcript. During chick embryonic limb chondrogenesis, FN structure changes from B+A+ in precartilage mesenchyme to B+A- in differentiated cartilage, and exon IIIA has been shown to be necessary for the process of mesenchymal cellular condensation, a requisite event that precedes overt expression of chondrocyte phenotype. This study aims to investigate the mechanistic action of the FN isoforms in mesenchymal chondrogenesis and, in particular, to identify the specific cellular function in mesenchymal condensation mediated by the mesenchymal (B+A+) FN isoform. Full-length cDNAs corresponding to four splice variants (B+A+, B+A-, B-A+, B-A-) of FN were constructed, and expressed the corresponding proteins using a baculovirus expression vector system. Cell adhesion assays with purified proteins showed that, although the relative levels of cell attachment were approximately the same, chick limb-bud mesenchymal cells spread up to 40 % less on mesenchymal (B+A+) FN than on cartilage (B+A-) FN, (B-A+) FN, or plasma (B-A-) FN. Cellular condensation and chondrogenic differentiation were also promoted in high-density micromass cultures of limb mesenchymal cells plated onto B+A+ FN. These observations suggest that the process of mesenchymal condensation is mediated at least in part by the enhanced ability of chondrogenic mesenchymal cells to migrate and aggregate as a consequence of residing in and interacting with mesenchymal FN. Our findings are consistent with and provide a mechanistic basis for previous observations that rounding of limb mesenchymal cells precedes the onset of chondrogenesis.