Chromatin context-dependent regulation and epigenetic manipulation of prime editing.

We set out to exhaustively characterize the impact of the cis-chromatin environment on prime editing, a precise genome engineering tool. Using a highly sensitive method for mapping the genomic locations of randomly integrated reporters, we discover massive position effects, exemplified by editing efficiencies ranging from ∼0% to 94% for an identical target site and edit. Position effects on prime editing efficiency are well predicted by chromatin marks, e.g., positively by H3K79me2 and negatively by H3K9me3. Next, we developed a multiplex perturbational framework to assess the interaction of trans-acting factors with the cis-chromatin environment on editing outcomes. Applying this framework to DNA repair factors, we identify HLTF as a context-dependent repressor of prime editing. Finally, several lines of evidence suggest that active transcriptional elongation enhances prime editing. Consistent with this, we show we can robustly decrease or increase the efficiency of prime editing by preceding it with CRISPR-mediated silencing or activation, respectively.

Tumor Interferon Signaling Regulates a Multigenic Resistance Program to Immune Checkpoint Blockade.

Therapeutic blocking of the PD1 pathway results in significant tumor responses, but resistance is common. We demonstrate that prolonged interferon signaling orchestrates PDL1-dependent and PDL1-independent resistance to immune checkpoint blockade (ICB) and to combinations such as radiation plus anti-CTLA4. Persistent type II interferon signaling allows tumors to acquire STAT1-related epigenomic changes and augments expression of interferon-stimulated genes and ligands for multiple T cell inhibitory receptors. Both type I and II interferons maintain this resistance program. Crippling the program genetically or pharmacologically interferes with multiple inhibitory pathways and expands distinct T cell populations with improved function despite expressing markers of severe exhaustion. Consequently, tumors resistant to multi-agent ICB are rendered responsive to ICB monotherapy. Finally, we observe that biomarkers for interferon-driven resistance associate with clinical progression after anti-PD1 therapy. Thus, the duration of tumor interferon signaling augments adaptive resistance and inhibition of the interferon response bypasses requirements for combinatorial ICB therapies.

Global absence and targeting of protective immune states in severe COVID-19.

Although infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has pleiotropic and systemic effects in some individuals1-3, many others experience milder symptoms. Here, to gain a more comprehensive understanding of the distinction between severe and mild phenotypes in the pathology of coronavirus disease 2019 (COVID-19) and its origins, we performed a whole-blood-preserving single-cell analysis protocol to integrate contributions from all major immune cell types of the blood-including neutrophils, monocytes, platelets, lymphocytes and the contents of the serum. Patients with mild COVID-19 exhibit a coordinated pattern of expression of interferon-stimulated genes (ISGs)3 across every cell population, whereas these ISG-expressing cells are systemically absent in patients with severe disease. Paradoxically, individuals with severe COVID-19 produce very high titres of anti-SARS-CoV-2 antibodies and have a lower viral load compared to individuals with mild disease. Examination of the serum from patients with severe COVID-19 shows that these patients uniquely produce antibodies that functionally block the production of the ISG-expressing cells associated with mild disease, by activating conserved signalling circuits that dampen cellular responses to interferons. Overzealous antibody responses pit the immune system against itself in many patients with COVID-19, and perhaps also in individuals with other viral infections. Our findings reveal potential targets for immunotherapies in patients with severe COVID-19 to re-engage viral defence.

SCITO-seq: single-cell combinatorial indexed cytometry sequencing.

The development of DNA-barcoded antibodies to tag cell surface molecules has enabled the use of droplet-based single-cell sequencing (dsc-seq) to profile protein abundances from thousands of cells simultaneously. As compared to flow and mass cytometry, the high per cell cost of current dsc-seq-based workflows precludes their use in clinical applications and large-scale pooled screens. Here, we introduce SCITO-seq, a workflow that uses splint oligonucleotides (oligos) to enable combinatorially indexed dsc-seq of DNA-barcoded antibodies from over 105 cells per reaction using commercial microfluidics. By encoding sample barcodes into splint oligos, we demonstrate that multiplexed SCITO-seq produces reproducible estimates of cellular composition and surface protein expression comparable to those from mass cytometry. We further demonstrate two modified splint oligo designs that extend SCITO-seq to achieve compatibility with commercial DNA-barcoded antibodies and simultaneous expression profiling of the transcriptome and surface proteins from the same cell. These results demonstrate SCITO-seq as a flexible and ultra-high-throughput platform for sequencing-based single-cell protein and multimodal profiling.

Genetics of atrial fibrillation.

PURPOSE OF REVIEW: Atrial fibrillation is the most common cardiac arrhythmia worldwide. There is considerable interest in better understanding the molecular genetics and biology of atrial fibrillation to inform the development of new therapies and improve clinical management. This review summarizes recent advances in our understanding of the genetic basis of atrial fibrillation and new efforts to utilize genetics to inform clinical management. RECENT FINDINGS: Genome-wide association studies in diverse populations have increased the number of genetic loci associated with atrial fibrillation and its specific subtypes. Large-scale biobanks with deep phenotyping have provided invaluable data to study the impact of both common and rare variants on atrial fibrillation, susceptibility, and prognosis. Polygenic risk scores help improve individual atrial fibrillation risk stratification and prognostication. SUMMARY: Our understanding of atrial fibrillation genetics is rapidly improving with larger and more diverse genome-wide association studies. Translating genetic discoveries into molecular pathways and new therapeutic targets remains a bottleneck in the development of new therapies for atrial fibrillation. Genetic risk scores have shown early promise in improving atrial fibrillation risk stratification; however, their broader utility for the general population remains unclear.

Can inflammation predict social media use? Linking a biological marker of systemic inflammation with social media use among college students and middle-aged adults.

Drawing on recent evidence that inflammation may promote social affiliative motivation, the present research proposes a novel perspective that inflammation may be associated with more social media use. In a cross-sectional analysis of a nationally representative sample, Study 1 (N = 863) found a positive association between C-reactive protein (CRP), a biomarker of systemic inflammation, and the amount of social media use by middle-aged adults. Study 2 (N = 228) showed that among college students CRP was prospectively associated with more social media use 6 weeks later. Providing stronger evidence of the directionality of this effect, Study 3 (N = 171) showed that in college students CRP predicted increased social media use in the subsequent week even after controlling for current week's use. Additionally, in exploratory analyses of CRP and different types of social media use in the same week, CRP was only associated with using social media for social interaction and not for other purposes (e.g., entertainment). The present research sheds light on the social effects of inflammation and highlights potential benefits of using social media as a context for studying the impact of inflammation on social motivation and behavior.

Feasibility study of student-led fall prevention care management: Reducing fall risks in assisted living facilities.

Falls are common in Assisted Living Facilities (ALFs). We evaluated the feasibility, acceptability, and preliminary impact of student-led Fall Prevention Care Management (FPCM) on reducing fall risks in ALFs. Residents who were age ≥65, had a fall in the previous year Or considered high fall risk at the facility, and who had a MoCA cognition score>15 were enrolled. The FPCM interventions were semi-structured to facilitate students' learning while addressing participants' unique fall risks. Twenty-five older adults in the U.S. completed the study (recruitment rate: 55%; retention rate: 64%). Participants rated the study as 87.16 (100 = excellent), and likelihood to recommend the study to others was 80.85 (100 = most likely). Participants were 84% female, mean age 88.6 years old. Fall risks such as fear of falling decreased from 16.05 to 15.12 (p = .022), fall prevention behaviors increased from 2.94 to 3.07 (p = .048), and the level of confidence to prevent falls increased from 63.38 to 78.35 (p = .015). Students commonly provided education and coaching on fall prevention strategies, and addressed emotional and behavioral aspects of fall prevention. With improvement with recruitment and retention, student-led FPCM intervention is a promising approach for fall prevention in ALF.

Cohort and nested case-control study of cutaneous squamous cell carcinoma in solid organ transplant recipients, by medication.

BACKGROUND: Knowledge is needed about the risk of cutaneous squamous cell carcinoma (cSCC) in solid organ transplant recipients (SOTRs) using contemporary immunosuppressive regimens. OBJECTIVE: Evaluate the risk of cSCC in relation to medications used by SOTRs. METHODS: The cohort and nest case-control study included 3308 SOTRs and 65,883 persons without transplantation during 2009-2019. Incident cSCC was identified from pathology data, and medications were identified from pharmacy data. Adjusted hazard ratios and 95% confidence intervals (CIs) were estimated using Cox proportional hazards analysis, with voriconazole examined as a time-dependent variable. RESULTS: The annual incidence of cSCC was 1.69% in SOTRs and 0.30% in persons without transplantation. The adjusted hazard ratio of cSCC associated with lung transplant was 14.83 (95% CI, 9.85-22.33) for lung and 6.53-10.69 for other organs. Risk in Latinx persons was higher than in other non-White groups. Among lung recipients, the hazard ratio was 1.14 for each month of voriconazole use (95% CI, 1.04-1.26). Azathioprine use for ≥7 months, relating to mycophenolate mofetil intolerance, was associated with a 4.22-fold increased risk of cSCC (95% CI, 1.90-9.40). Belatacept and other immunsuppressive medications were not associated with risk. LIMITATION: The number of events was somewhat small. CONCLUSIONS: The knowledge of risks and benefits in diverse patients can translate to improvements in care.

Evaluation of Cisplatin-Induced Pathology in the Larval Zebrafish Lateral Line.

Cisplatin is an effective anticancer agent, but also causes permanent hearing loss by damaging hair cells-the sensory receptors essential for hearing. There is an urgent clinical need to protect cochlear hair cells in patients undergoing cisplatin chemotherapy. The zebrafish lateral line organ contains hair cells and has been frequently used in studies to screen for otoprotective compounds. However, these studies have employed a wide range of cisplatin dosages and exposure times. We therefore performed a comprehensive evaluation of cisplatin ototoxicity in the zebrafish lateral line with the goal of producing a standardized, clinically relevant protocol for future studies. To define the dose- and time-response patterns of cisplatin-induced hair-cell death, we treated 6-day-old larvae for 2 h in 50 µM-1 mM cisplatin and allowed them to recover. We observed delayed hair cell death, which peaked at 4-8 h post-exposure. Cisplatin also activated a robust inflammatory response, as determined by macrophage recruitment and phagocytosis of hair cells. However, selective depletion of macrophages did not affect hair cell loss. We also examined the effect of cisplatin treatment on fish behavior and found that cisplatin-induced lateral line injury measurably impaired rheotaxis. Finally, we examined the function of remaining hair cells that appeared resistant to cisplatin treatment. We observed significantly reduced uptake of the cationic dye FM1-43 in these cells relative to untreated controls, indicating that surviving hair cells may be functionally impaired. Cumulatively, these results indicate that relatively brief exposures to cisplatin can produce hair cell damage and delayed hair cell death. Our observations provide guidance on standardizing methods for the use of the zebrafish model in studies of cisplatin ototoxicity.

The influence of threat on the efficiency of goal-directed attentional control.

Anxiety has consistently been found to potentiate attentional capture by physically salient stimuli, which could be due to enhanced distractor processing, impaired goal-directed attention, or both. At the same time, a recent study demonstrated that a threat manipulation reduces attentional capture by reward-associated stimuli, suggesting that anxiety does not increase distractibility or, otherwise, interfere with the control of attention generally. Here, we experimentally induced anxiety via threat-of-shock in the adaptive choice visual search task to examine whether the experience of threat influences goal-directed attentional control. Participants chose to search through one of two task-relevant colors on each trial, where searching through the less abundant color would be optimal for maximizing performance. Performance was evaluated with and without the threat of unpredictable electric shock. Under threat, participants were more optimal in their visual search and missed fewer targets. Performance improvements were demonstrated on trials that the optimal target color switched, demonstrating that threat is beneficial in adapting to changing attentional demands. Our findings demonstrate that threat can facilitate the efficiency of goal-directed attentional control and are at odds with an antagonistic relationship between anxiety and the control of attention.

On the Mechanism of Self-Assembly by a Hydrogel-Forming Peptide.

Self-assembling peptide-based hydrogels are a class of tunable soft materials that have been shown to be highly useful for a number of biomedical applications. The dynamic formation of the supramolecular fibrils that compose these materials has heretofore remained poorly characterized. A better understanding of this process would provide important insights into the behavior of these systems and could aid in the rational design of new peptide hydrogels. Here, we report the determination of the microscopic steps that underpin the self-assembly of a hydrogel-forming peptide, SgI37-49. Using theoretical models of linear polymerization to analyze the kinetic self-assembly data, we show that SgI37-49 fibril formation is driven by fibril-catalyzed secondary nucleation and that all the microscopic processes involved in SgI37-49 self-assembly display an enzyme-like saturation behavior. Moreover, this analysis allows us to quantify the rates of the underlying processes at different peptide concentrations and to calculate the time evolution of these reaction rates over the time course of self-assembly. We demonstrate here a new mechanistic approach for the study of self-assembling hydrogel-forming peptides, which is complementary to commonly used materials science characterization techniques.

Fructose-1,6-bisphosphatase opposes renal carcinoma progression.

Clear cell renal cell carcinoma (ccRCC), the most common form of kidney cancer, is characterized by elevated glycogen levels and fat deposition. These consistent metabolic alterations are associated with normoxic stabilization of hypoxia-inducible factors (HIFs) secondary to von Hippel-Lindau (VHL) mutations that occur in over 90% of ccRCC tumours. However, kidney-specific VHL deletion in mice fails to elicit ccRCC-specific metabolic phenotypes and tumour formation, suggesting that additional mechanisms are essential. Recent large-scale sequencing analyses revealed the loss of several chromatin remodelling enzymes in a subset of ccRCC (these included polybromo-1, SET domain containing 2 and BRCA1-associated protein-1, among others), indicating that epigenetic perturbations are probably important contributors to the natural history of this disease. Here we used an integrative approach comprising pan-metabolomic profiling and metabolic gene set analysis and determined that the gluconeogenic enzyme fructose-1,6-bisphosphatase 1 (FBP1) is uniformly depleted in over six hundred ccRCC tumours examined. Notably, the human FBP1 locus resides on chromosome 9q22, the loss of which is associated with poor prognosis for ccRCC patients. Our data further indicate that FBP1 inhibits ccRCC progression through two distinct mechanisms. First, FBP1 antagonizes glycolytic flux in renal tubular epithelial cells, the presumptive ccRCC cell of origin, thereby inhibiting a potential Warburg effect. Second, in pVHL (the protein encoded by the VHL gene)-deficient ccRCC cells, FBP1 restrains cell proliferation, glycolysis and the pentose phosphate pathway in a catalytic-activity-independent manner, by inhibiting nuclear HIF function via direct interaction with the HIF inhibitory domain. This unique dual function of the FBP1 protein explains its ubiquitous loss in ccRCC, distinguishing FBP1 from previously identified tumour suppressors that are not consistently mutated in all tumours.

Pharmacist experiences and perspectives with Oregon's Death with Dignity Act.

BACKGROUND: Medical aid-in-dying (AID) is increasingly available in the United States. Despite their substantial role in the medication use process, pharmacists' involvement in medical AID has been overlooked. OBJECTIVE: To describe pharmacist attitudes toward medical AID and experiences with Oregon's Death with Dignity Act (DWDA). METHODS: Qualitative analysis of Oregon-licensed pharmacists with professional interactions involving Oregon's DWDA. Data were collected through semistructured focus groups and analyzed with immersion-crystallization methods. RESULTS: Sixteen Oregon pharmacists participated in this study. The participants were almost evenly divided between males and females, who varied in age, years of pharmacy experience, and the number of DWDA encounters. Of these, 14 pharmacists agreed to participate in the DWDA process while 2 declined. Three themes emerged. First, pharmacists identified logistical challenges that negatively affected their ability to assist patients seeking medical AID. Second, pharmacists described the content and other patient counseling considerations for DWDA prescriptions. Third, pharmacists discussed how values and preferences informed their decisions related to medical AID requests. CONCLUSION: Pharmacists' involvement in medical AID has been largely focused on medication dispensing and patient counseling, and medical AID prescriptions raise unique challenges. Their decisions to participate were frequently tied to support for patient autonomy, although more research is needed to capture the diversity of attitudes, perspectives, and experiences related to their involvement with medical AID, particularly for those who decline to participate. There is a need to develop educational materials and other resources to assist pharmacists in navigating medical AID requests.

Cutaneous manifestations of COVID-19: a systematic review and analysis of individual patient-level data.

Distinctive patterns in the cutaneous manifestations of COVID-19 have been recently reported. We conducted a systematic review to identify case reports and case series characterizing cutaneous manifestations of confirmed COVID-19. Key demographic and clinical data from each case were extracted and analyzed. The primary outcome measure was risk factor analysis of skin related outcomes for severe COVID-19 disease. Seventy-one case reports and series comprising 144 cases of cutaneous involvement in COVID-19 were included. The most frequently occurring morphologies were: morbilliform (30.6%), varicelliform (18.8%), urticarial (13.2%), chilblains-like (12.5%), and acro-ischemic (9%). The median age of patients was 51 years (mean: 45.9, range: 0 to 91). Patients with chilblains-like eruptions had lower frequencies of extracutaneous COVID-19 symptoms (5/18, 27.8%, P<0.05) and were less likely to have severe COVID-19 disease (2/18, 11%, 95% CI 1.4% to 34.7%, P=0.02). Patients with livedoid and acro-ischemic morphologies had severe COVID-19 more frequently than those with other morphologies (17/21, 81%, 95% CI 58.0% to 94.5%, P<0.0001). The most frequently observed cutaneous manifestations of COVID-19 (morbilliform, varicelliform, and urticarial) are well-described patterns of viral exanthems. However, chilblains-like, livedoid, and acro-ischemic morphologies are not traditionally associated with viral infections and were significantly associated with severity of COVID-19 disease.

Highly Sensitive Micropatterned Interdigitated Electrodes for Enhancing the Concentration Effect Based on Dielectrophoresis.

The concentration effect of dielectrophoresis (DEP) enables detection of biomolecules with high sensitivity. In this study, microstructures were patterned between the interdigitated microelectrodes (IMEs) to increase the concentration effect of DEP. The microstructures increased the electric field gradient ( ∇ | E 2 | ) between the IMEs to approximately 6.61-fold higher than in the bare IMEs with a gap of 10 µm, resulting in a decreased optimal voltage to concentrate amyloid beta 42 (Aß42, from 0.8 Vpp to 0.5 Vpp) and tau-441 (from 0.9 Vpp to 0.6 Vpp) between the IMEs. Due to the concentration effect of DEP, the impedance change in the optimal condition was higher than the values in the reference condition at 2.64-fold in Aß42 detection and at 1.59-fold in tau-441 detection. This concentration effect of DEP was also verified by counting the number of gold (Au) particles which conjugated with the secondary antibody. Finally, an enhanced concentration effect in the patterned IMEs was verified by measuring the impedance change depending on the concentration of Aß42 and tau-441. Our results suggest that microstructures increase the concentration effect of DEP, leading to enhanced sensitivity of the IMEs.

HIF modulation of Wnt signaling regulates skeletal myogenesis in vivo.

Deeper insight into the molecular pathways that orchestrate skeletal myogenesis should enhance our understanding of, and ability to treat, human skeletal muscle disease. It is now widely appreciated that nutrients, such as molecular oxygen (O2), modulate skeletal muscle formation. During early stages of development and regeneration, skeletal muscle progenitors reside in low O2 environments before local blood vessels and differentiated muscle form. Moreover, low O2 availability (hypoxia) impedes progenitor-dependent myogenesis in vitro through multiple mechanisms, including activation of hypoxia inducible factor 1α (HIF1α). However, whether HIF1α regulates skeletal myogenesis in vivo is not known. Here, we explored the role of HIF1α during murine skeletal muscle development and regeneration. Our results demonstrate that HIF1α is dispensable during embryonic and fetal myogenesis. However, HIF1α negatively regulates adult muscle regeneration after ischemic injury, implying that it coordinates adult myogenesis with nutrient availability in vivo. Analyses of Hif1a mutant muscle and Hif1a-depleted muscle progenitors further suggest that HIF1α represses myogenesis through inhibition of canonical Wnt signaling. Our data provide the first evidence that HIF1α regulates skeletal myogenesis in vivo and establish a novel link between HIF and Wnt signaling in this context.

Trading off Aircraft Fuel Burn and NO x Emissions for Optimal Climate Policy.

Aviation emits pollutants that affect the climate, including CO2 and NO x, NO x indirectly so, through the formation of tropospheric ozone and reduction of ambient methane. To improve the fuel performance of engines, combustor temperatures and pressures often increase, increasing NO x emissions. Conversely, combustor modifications to reduce NO x may increase CO2. Hence, a technology trade-off exists, which also translates to a trade-off between short-lived climate forcers and a long-lived greenhouse gas, CO2. Moreover, the NO x-O3-CH4 system responds in a nonlinear manner, according to both aviation emissions and background NO x. A simple climate model was modified to incorporate nonlinearities parametrized from a complex chemistry model. Case studies showed that for a scenario of a 20% reduction in NO x emissions the consequential CO2 penalty of 2% actually increased the total radiative forcing (RF). For a 2% fuel penalty, NO x emissions needed to be reduced by >43% to realize an overall benefit. Conversely, to ensure that the fuel penalty for a 20% NO x emission reduction did not increase overall forcing, a 0.5% increase in CO2 was found to be the "break even" point. The time scales of the climate effects of NO x and CO2 are quite different, necessitating careful analysis of proposed emissions trade-offs.

Auditory structural connectivity in preterm and healthy term infants during the first postnatal year.

Assessing language development in the first postnatal year is difficult, as receptive and expressive skills are rudimentary. Although outward manifestations of change are limited, the auditory language system is thought to undergo critical development at this age, as the foundations are laid for the rapid onset of spoken language in the second and third years. We recruited 11 infants, 7 healthy controls (gestational age = 40.69 ± 0.56; range from 40 to 41.43) and preterm babies (gestational age = 28.04 ± 0.95; range from 27.43 to 29.43) who underwent a Magnetic Resonance Imaging study during the first postnatal year (age at scan = 194.18 ± 97.98). We assessed white matter tracts using diffusion-weighted magnetic resonance imaging with probabilistic tractography. Fractional anisotropy was found to be largely mature even at one month, although there was a little further increase during the first postnatal year in both the acoustic radiation and the direct brainstem-Heschl's pathway.

Biochemical Properties of a Decoy Oligodeoxynucleotide Inhibitor of STAT3 Transcription Factor.

Cyclic STAT3 decoy (CS3D) is a second-generation, double-stranded oligodeoxynucleotide (ODN) that mimics a genomic response element for signal transducer and activator of transcription 3 (STAT3), an oncogenic transcription factor. CS3D competitively inhibits STAT3 binding to target gene promoters, resulting in decreased expression of proteins that promote cellular proliferation and survival. Previous studies have demonstrated antitumor activity of CS3D in preclinical models of solid tumors. However, prior to entering human clinical trials, the efficiency of generating the CS3D molecule and its stability in biological fluids should be determined. CS3D is synthesized as a single-stranded ODN and must have its free ends ligated to generate the final cyclic form. In this study, we report a ligation efficiency of nearly 95 percent. The ligated CS3D demonstrated a half-life of 7.9 h in human serum, indicating adequate stability for intravenous delivery. These results provide requisite biochemical characterization of CS3D that will inform upcoming clinical trials.