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Clamp loaders are pentameric AAA+ assemblies that use ATP to open and close circular DNA sliding clamps around DNA. Clamp loaders show homology in all organisms, from bacteria to human. The eukaryotic PCNA clamp is loaded onto 3 ' primed DNA by the replication factor C (RFC) hetero-pentameric clamp loader. Eukaryotes also have three alternative RFC-like clamp loaders (RLCs) in which the Rfc1 subunit is substituted by another protein. One of these is the yeast Rad24-RFC (Rad17-RFC in human) that loads a 9-1-1 heterotrimer clamp onto a recessed 5 ' end of DNA. Recent structural studies of Rad24-RFC have discovered an unexpected 5 ' DNA binding site on the outside of the clamp loader and reveal how a 5 ' end can be utilized for loading the 9-1-1 clamp onto DNA. In light of these results, new studies reveal that RFC also contains a 5 ' DNA binding site, which functions in gap repair. These studies also reveal many new features of clamp loaders. As reviewed herein, these recent studies together have transformed our view of the clamp loader mechanism.

Background and Aims Lack of tractable immunocompetent animal models amenable to robust experimental challenge impedes vaccine efforts for HCV. Infection with rodent hepacivirus from Rattus norvegicus (RHV-rn1) in rats shares HCV-defining characteristics, including liver tropism, chronicity, and pathology. RHV in vitro cultivation would facilitate genetic studies on particle production, host factor interactions, and evaluation of antibody neutralization guiding HCV vaccine approaches. Approach and Results We report an infectious reverse genetic cell culture system for RHV-rn1 using highly permissive rat hepatoma cells and adaptive mutations in the E2, NS4B, and NS5A viral proteins. Cell culture-derived RHV-rn1 particles (RHVcc) share hallmark biophysical characteristics of HCV and are infectious in mice and rats. Culture adaptive mutations attenuated RHVcc in immunocompetent rats, and the mutations reverted following prolonged infection, but not in severe combined immunodeficiency (SCID) mice, suggesting that adaptive immune pressure is a primary driver of reversion. Accordingly, sera from RHVcc-infected SCID mice or the early acute phase of immunocompetent mice and rats were infectious in culture. We further established an in vitro RHVcc neutralization assay, and observed neutralizing activity of rat sera specifically from the chronic phase of infection. Finally, we found that scavenger receptor class B type I promoted RHV-rn1 entry in vitro and in vivo. Conclusions The RHV-rn1 infectious cell culture system enables studies of humoral immune responses against hepacivirus infection. Moreover, recapitulation of the entire RHV-rn1 infectious cycle in cell culture will facilitate reverse genetic studies and the exploration of tropism and virus-host interactions.

Hidradenitis suppurativa (HS) is a chronic, progressive inflammatory skin disease that is often recalcitrant to multiple treatments. In determining biologic candidacy for patients with HS, we propose a paradigm shift away from Hurley staging and towards consideration of other variables. Biologics represent a long-term treatment option for HS that may serve as a disease-modifying agent. These medications are typically initiated in patients with moderate to severe disease, which, based on inclusion criteria in clinical trials, is often defined as Hurley stage II or III disease, at which point irreversible tissue damage has already occurred. In real-world clinical settings, these considerations include treatments tried and failed, predicted disease trajectory, disease characteristics beyond lesion type, impact of disease on patients' functional status and quality of life, and patient comorbidities, venturing away from the limitations of Hurley stage designations. Future clinical trials may benefit from inclusion of recalcitrant Hurley stage I patients, which may then re-shape treatment guidelines and insurance coverage and improve patient access to biologic treatments.

Low-income, minority seniors face high rates of hypertension that increase cardiovascular risk. Senior centers offer services, including congregate meals, that can be a valuable platform to reach older adults in underserved communities. We implemented two evidence-based interventions not previously tested in this setting: DASH-aligned congregate meals and Self-Measured Blood Pressure (SMBP), to lower blood pressure (BP) at two senior centers serving low-income, racially diverse communities. The study enrolled congregate meal program participants, provided training and support for SMPB, and nutrition and BP education. DASH-aligned meals delivered 40% (lunch) or 70% (breakfast and lunch) of DASH requirements/day. Primary outcomes were change in BP, and BP control, at Month 1. Implementation data collected included client characteristics, menu fidelity, meal attendance, SMBP adherence, meal satisfaction, input from partner organizations and stakeholders, effort, and food costs. We used the RE-AIM framework to analyze implementation. Study Reach included 94 older, racially diverse participants reflecting neighborhood characteristics. Effectiveness: change in systolic BP at Month 1 trended towards significance (-4 mmHg, p = 0.07); change in SMBP reached significance at Month 6 (-6.9 mmHg, p = 0.004). We leveraged existing community-academic partnerships, leading to Adoption at both target sites. The COVID pandemic interrupted Implementation and Maintenance and may have attenuated BP effectiveness. DASH meals served were largely aligned with planned menus. Meal attendance remained consistent; meal satisfaction was high. Food costs increased by 10%. This RE-AIM analysis highlights the acceptability, feasibility, and fidelity of this DASH/SMBP health intervention to lower BP at senior centers. It encourages future research and offers important lessons for organizations delivering services to older adults and addressing cardiovascular risk among vulnerable populations.

ATP-hydrolysis-coupled actin polymerization is a fundamental mechanism of cellular force generation(1-3). In turn, force(4,5) and actin filament (F-actin) nucleotide state(6) regulate actin dynamics by tuning F-actin's engagement of actin-binding proteins through mechanisms that are unclear. Here we show that the nucleotide state of actin modulates F-actin structural transitions evoked by bending forces. Cryo-electron microscopy structures of ADP-F-actin and ADP-P-i-F-actin with sufficient resolution to visualize bound solvent reveal intersubunit interfaces bridged by water molecules that could mediate filament lattice flexibility. Despite extensive ordered solvent differences in the nucleotide cleft, these structures feature nearly identical lattices and essentially indistinguishable protein backbone conformations that are unlikely to be discriminable by actin-binding proteins. We next introduce a machine-learning-enabled pipeline for reconstructing bent filaments, enabling us to visualize both continuous structural variability and side-chain-level detail. Bent F-actin structures reveal rearrangements at intersubunit interfaces characterized by substantial alterations of helical twist and deformations in individual protomers, transitions that are distinct in ADP-F-actin and ADP-P-i-F-actin. This suggests that phosphate rigidifies actin subunits to alter the bending structural landscape of F-actin. As bending forces evoke nucleotide-state dependent conformational transitions of sufficient magnitude to be detected by actin-binding proteins, we propose that actin nucleotide state can serve as a co-regulator of F-actin mechanical regulation.

Vocal activity and signal characteristics of mammals are driven by several factors that result in both stability and plasticity over multiple time scales. All three extant species of manatee communicate with several calls that are especially important for maintaining contact between cows and calves. Determining if calf calls differ across manatee species will provide insights into the evolution of species-specific acoustic communication traits. We investigated the interspecific differences in the vocalizations of calves of Amazonian manatees (Trichechus inunguis) and the two subspecies of the West Indian manatee (T. manatus). Vocalizations of individual calves were recorded in rehabilitation centers in Brazil, Puerto Rico, the United States, and Mexico. The acoustic structure of calls produced by manatee calves varied between species and with body size. Amazonian manatee calves produced shorter calls with multiple notes at higher frequency while West Indian calves produced modulated calls that were lower in frequency and longer in duration. Smaller West Indian calves produced frequency modulated, hill-shaped calls that flattened with an increase in body length. Our results provide evidence for divergence in the ontogeny of vocalizations across T. manatus and T. inunguis and suggest variation in body size contributed to the evolution of differences in the characteristics of their calls.

Nerves are a component of the tumor microenvironment contributing to cancer pro-gression, but the role of cells from nerves in facilitating cancer invasion remains poorly understood. Here we show that Schwann cells (SC) activated by cancer cells collectively function as tumor-activated Schwann cell tracks (TAST) that promote cancer cell migration and invasion. Nonmyeli-nating SCs form TASTs and have cell gene expression signatures that correlate with diminished survival in patients with pancreatic ductal adenocarcinoma. In TASTs, dynamic SCs form tracks that serve as cancer pathways and apply forces on cancer cells to enhance cancer motility. These SCs are activated by c-Jun, analogous to their reprogramming during nerve repair. This study reveals a mechanism of cancer cell inva-sion that co-opts a wound repair process and exploits the ability of SCs to collectively organize into tracks. These findings establish a novel paradigm of how cancer cells spread and reveal therapeutic opportunities. SIGNIFICANCE: How the tumor microenvironment participates in pancreatic cancer progression is not fully understood. Here, we show that SCs are activated by cancer cells and collectively organize into tracks that dynamically enable cancer invasion in a c-Jun-dependent manner.

Vaccines targeting HIV-1's gp160 spike protein are stymied by high viral mutation rates and structural chicanery. gp160's membrane-proximal external region (MPER) is the target of naturally arising broadly neutralizing antibodies (bnAbs), yet MPER-based vaccines fail to generate bnAbs. Here, nanodisc-embedded spike protein was investigated by cryo-electron microscopy and molecular-dynamics simulations, revealing spontaneous ectodomain tilting that creates vulnerability for HIV-1. While each MPER protomer radiates centrally towards the three-fold axis contributing to a membrane-associated tripod structure that is occluded in the upright spike, tilting provides access to the opposing MPER. Structures of spike proteins with bound 4E10 bnAb Fabs reveal that the antibody binds exposed MPER, thereby altering MPER dynamics, modifying average ectodomain tilt, and imposing strain on the viral membrane and the spike's transmembrane segments, resulting in the abrogation of membrane fusion and informing future vaccine development. The membrane-proximal external region of HIV-1 spike protein is a broadly neutralizing antibody target. Here, cryo-electron microscopy and molecular dynamics reveal spontaneous ectodomain tilting, a vulnerability exploitable for vaccine design.

IMPORTANCE Bariatric surgery is the most effective treatment for severe obesity; yet it is unclear whether the long-term safety and comparative effectiveness of these operations differ across racial and ethnic groups. OBJECTIVE To compare outcomes of Roux-en-Y gastric bypass (RYGB) and sleeve gastrectomy (SG) across racial and ethnic groups in the National Patient-Centered Clinical Research Network (PCORnet) Bariatric Study. DESIGN, SETTING, AND PARTICIPANTS Thiswas a retrospective, observational, comparative effectiveness cohort study that comprised 25 health care systems in the PCORnet Bariatric Study. Patients were adults and adolescents aged 12 to 79 years who underwent a primary (first nonrevisional) RYGB or SG operation between January 1, 2005, and September 30, 2015, at participating health systems. Patient race and ethnicity included Black, Hispanic, White, other, and unrecorded. Data were analyzed from July 1, 2021, to January 17, 2022. EXPOSURE RYGB or SG. OUTCOMES Percentage total weight loss (%TWL); type 2 diabetes remission, relapse, and change in hemoglobin A1c (HbA1c) level; and postsurgical safety and utilization outcomes (operations, interventions, revisions/conversions, endoscopy, hospitalizations, mortality, 30-day major adverse events) at 1, 3, and 5 years after surgery. RESULTS A total of 36 871 patients (mean [SE] age, 45.0 [11.7] years; 29 746 female patients [81%]) were included in the weight analysis. Patients identified with the following race and ethnic categories: 6891 Black (19%), 8756 Hispanic (24%), 19 645 White (53%), 826 other (2%), and 783 unrecorded (2%). Weight loss and mean reductions in HbA(1c) level were larger for RYGB than SG in all years for Black, Hispanic, and White patients (difference in 5-year weight loss: Black, -7.6%; 95% CI, -8.0 to -7.1; P <.001; Hispanic, -6.2%; 95% CI, -6.6 to -5.9; P <.001; White, -5.9%; 95% CI, -6.3 to -5.7; P <.001; difference in change in year 5 HbA1c level: Black, -0.29; 95% CI, -0.51 to -0.08; P =.009; Hispanic, -0.45; 95% CI, -0.61 to -0.29; P <.001; and White, -0.25; 95% CI, -0.40 to -0.11; P =.001.) The magnitude of these differences was small among racial and ethnic groups (1%-3% of %TWL). Black and Hispanic patients had higher risk of hospitalization when they had RYGB compared with SG (hazard ratio [HR], 1.45; 95% CI, 1.17-1.79; P =.001 and 1.48; 95% CI, 1.22-1.79; P <.001, respectively). Hispanic patients had greater risk of all-cause mortality (HR, 2.41; 95% CI, 1.24-4.70; P =.01) and higher odds of a 30-day major adverse event (odds ratio, 1.92; 95% CI, 1.38-2.68; P <.001) for RYGB compared with SG. There was no interaction between race and ethnicity and operation type for diabetes remission and relapse. CONCLUSIONS AND RELEVANCE Variability of the comparative effectiveness of operations for %TWL and HbA(1c) level across race and ethnicity was clinically small; however, differences in safety and utilization outcomes were clinically and statistically significant for Black and Hispanic patients who had RYGB compared with SG. These findings can inform shared decision-making regarding bariatric operation choice for different racial and ethnic groups of patients.

Recently, screens for mediators of resistance to FLT3 and ABL kinase inhibitors in leukemia resulted in the discovery of LZTR1 as an adapter of a Cullin-3 RING E3 ubiquitin ligase complex responsible for the degradation of RAS GTPases. In parallel, dysregulated LZTR1 expression via aberrant splicing and mutations was identifi ed in clonal hematopoietic condi-tions. Here we identify that loss of LZTR1, or leukemia-associated mutants in the LZTR1 substrate and RAS GTPase RIT1 that escape degradation, drives hematopoietic stem cell (HSC) expansion and leuke-mia in vivo . Although RIT1 stabilization was suffi cient to drive hematopoietic transformation, transfor-mation mediated by LZTR1 loss required MRAS. Proteolysis targeting chimeras (PROTAC) against RAS or reduction of GTP-loaded RAS overcomes LZTR1 loss-mediated resistance to FLT3 inhibitors. These data reveal proteolysis of noncanonical RAS proteins as novel regulators of HSC self-renewal, defi ne the function of RIT1 and LZTR1 mutations in leukemia, and identify means to overcome drug resistance due to LZTR1 downregulation.SIGNIFICANCE: Here we identify that impairing proteolysis of the noncanonical RAS GTPases RIT1 and MRAS via LZTR1 downregulation or leukemia-associated mutations stabilizing RIT1 enhances MAP kinase activation and drives leukemogenesis. Reducing the abundance of GTP-bound KRAS and NRAS overcomes the resistance to FLT3 kinase inhibitors associated with LZTR1 downregulation in leukemia.