Enhanced prime editing systems by manipulating cellular determinants of editing outcomes.

While prime editing enables precise sequence changes in DNA, cellular determinants of prime editing remain poorly understood. Using pooled CRISPRi screens, we discovered that DNA mismatch repair (MMR) impedes prime editing and promotes undesired indel byproducts. We developed PE4 and PE5 prime editing systems in which transient expression of an engineered MMR-inhibiting protein enhances the efficiency of substitution, small insertion, and small deletion prime edits by an average 7.7-fold and 2.0-fold compared to PE2 and PE3 systems, respectively, while improving edit/indel ratios by 3.4-fold in MMR-proficient cell types. Strategic installation of silent mutations near the intended edit can enhance prime editing outcomes by evading MMR. Prime editor protein optimization resulted in a PEmax architecture that enhances editing efficacy by 2.8-fold on average in HeLa cells. These findings enrich our understanding of prime editing and establish prime editing systems that show substantial improvement across 191 edits in seven mammalian cell types.

An aberrant immune-epithelial progenitor niche drives viral lung sequelae.

The long-term physiological consequences of respiratory viral infections, particularly in the aftermath of the COVID-19 pandemic-termed post-acute sequelae of SARS-CoV-2 (PASC)-are rapidly evolving into a major public health concern1-3. While the cellular and molecular aetiologies of these sequelae are poorly defined, increasing evidence implicates abnormal immune responses3-6 and/or impaired organ recovery7-9 after infection. However, the precise mechanisms that link these processes in the context of PASC remain unclear. Here, with insights from three cohorts of patients with respiratory PASC, we established a mouse model of post-viral lung disease and identified an aberrant immune-epithelial progenitor niche unique to fibroproliferation in respiratory PASC. Using spatial transcriptomics and imaging, we found a central role for lung-resident CD8+ T cell-macrophage interactions in impairing alveolar regeneration and driving fibrotic sequelae after acute viral pneumonia. Specifically, IFNγ and TNF derived from CD8+ T cells stimulated local macrophages to chronically release IL-1ß, resulting in the long-term maintenance of dysplastic epithelial progenitors and lung fibrosis. Notably, therapeutic neutralization of IFNγ + TNF or IL-1ß markedly improved alveolar regeneration and pulmonary function. In contrast to other approaches, which require early intervention10, we highlight therapeutic strategies to rescue fibrotic disease after the resolution of acute disease, addressing a current unmet need in the clinical management of PASC and post-viral disease.

Prime editing for precise and highly versatile genome manipulation.

Programmable gene-editing tools have transformed the life sciences and have shown potential for the treatment of genetic disease. Among the CRISPR-Cas technologies that can currently make targeted DNA changes in mammalian cells, prime editors offer an unusual combination of versatility, specificity and precision. Prime editors do not require double-strand DNA breaks and can make virtually any substitution, small insertion and small deletion within the DNA of living cells. Prime editing minimally requires a programmable nickase fused to a polymerase enzyme, and an extended guide RNA that both specifies the target site and templates the desired genome edit. In this Review, we summarize prime editing strategies to generate programmed genomic changes, highlight their limitations and recent developments that circumvent some of these bottlenecks, and discuss applications and future directions.

In vivo HSC prime editing rescues sickle cell disease in a mouse model.

Sickle cell disease (SCD) is a monogenic disease caused by a nucleotide mutation in the ß-globin gene. Current gene therapy studies are mainly focused on lentiviral vector-mediated gene addition or CRISPR/Cas9-mediated fetal globin reactivation, leaving the root cause unfixed. We developed a vectorized prime editing system that can directly repair the SCD mutation in hematopoietic stem cells (HSCs) in vivo in a SCD mouse model (CD46/Townes mice). Our approach involved a single intravenous injection of a nonintegrating, prime editor-expressing viral vector into mobilized CD46/Townes mice and low-dose drug selection in vivo. This procedure resulted in the correction of ∼40% of ßS alleles in HSCs. On average, 43% of sickle hemoglobin was replaced by adult hemoglobin, thereby greatly mitigating the SCD phenotypes. Transplantation in secondary recipients demonstrated that long-term repopulating HSCs were edited. Highly efficient target site editing was achieved with minimal generation of insertions and deletions and no detectable off-target editing. Because of its simplicity and portability, our in vivo prime editing approach has the potential for application in resource-poor countries where SCD is prevalent.

Search-and-replace genome editing without double-strand breaks or donor DNA.

Most genetic variants that contribute to disease1 are challenging to correct efficiently and without excess byproducts2-5. Here we describe prime editing, a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase, programmed with a prime editing guide RNA (pegRNA) that both specifies the target site and encodes the desired edit. We performed more than 175 edits in human cells, including targeted insertions, deletions, and all 12 types of point mutation, without requiring double-strand breaks or donor DNA templates. We used prime editing in human cells to correct, efficiently and with few byproducts, the primary genetic causes of sickle cell disease (requiring a transversion in HBB) and Tay-Sachs disease (requiring a deletion in HEXA); to install a protective transversion in PRNP; and to insert various tags and epitopes precisely into target loci. Four human cell lines and primary post-mitotic mouse cortical neurons support prime editing with varying efficiencies. Prime editing shows higher or similar efficiency and fewer byproducts than homology-directed repair, has complementary strengths and weaknesses compared to base editing, and induces much lower off-target editing than Cas9 nuclease at known Cas9 off-target sites. Prime editing substantially expands the scope and capabilities of genome editing, and in principle could correct up to 89% of known genetic variants associated with human diseases.

Differentiation of Prior SARS-CoV-2 Infection and Postacute Sequelae by Standard Clinical Laboratory Measurements in the RECOVER Cohort.

BACKGROUND: There are currently no validated clinical biomarkers of postacute sequelae of SARS-CoV-2 infection (PASC). OBJECTIVE: To investigate clinical laboratory markers of SARS-CoV-2 and PASC. DESIGN: Propensity score-weighted linear regression models were fitted to evaluate differences in mean laboratory measures by prior infection and PASC index (≥12 vs. 0). (ClinicalTrials.gov: NCT05172024). SETTING: 83 enrolling sites. PARTICIPANTS: RECOVER-Adult cohort participants with or without SARS-CoV-2 infection with a study visit and laboratory measures 6 months after the index date (or at enrollment if >6 months after the index date). Participants were excluded if the 6-month visit occurred within 30 days of reinfection. MEASUREMENTS: Participants completed questionnaires and standard clinical laboratory tests. RESULTS: Among 10 094 participants, 8746 had prior SARS-CoV-2 infection, 1348 were uninfected, 1880 had a PASC index of 12 or higher, and 3351 had a PASC index of zero. After propensity score adjustment, participants with prior infection had a lower mean platelet count (265.9 × 109 cells/L [95% CI, 264.5 to 267.4 × 109 cells/L]) than participants without known prior infection (275.2 × 109 cells/L [CI, 268.5 to 282.0 × 109 cells/L]), as well as higher mean hemoglobin A1c (HbA1c) level (5.58% [CI, 5.56% to 5.60%] vs. 5.46% [CI, 5.40% to 5.51%]) and urinary albumin-creatinine ratio (81.9 mg/g [CI, 67.5 to 96.2 mg/g] vs. 43.0 mg/g [CI, 25.4 to 60.6 mg/g]), although differences were of modest clinical significance. The difference in HbA1c levels was attenuated after participants with preexisting diabetes were excluded. Among participants with prior infection, no meaningful differences in mean laboratory values were found between those with a PASC index of 12 or higher and those with a PASC index of zero. LIMITATION: Whether differences in laboratory markers represent consequences of or risk factors for SARS-CoV-2 infection could not be determined. CONCLUSION: Overall, no evidence was found that any of the 25 routine clinical laboratory values assessed in this study could serve as a clinically useful biomarker of PASC. PRIMARY FUNDING SOURCE: National Institutes of Health.

Effect of Neutralizing Monoclonal Antibody Treatment on Early Trajectories of Virologic and Immunologic Biomarkers in Patients Hospitalized With COVID-19.

BACKGROUND: Neutralizing monoclonal antibodies (nmAbs) failed to show clear benefit for hospitalized patients with coronavirus disease 2019 (COVID-19). Dynamics of virologic and immunologic biomarkers remain poorly understood. METHODS: Participants enrolled in the Therapeutics for Inpatients with COVID-19 trials were randomized to nmAb versus placebo. Longitudinal differences between treatment and placebo groups in levels of plasma nucleocapsid antigen (N-Ag), anti-nucleocapsid antibody, C-reactive protein, interleukin-6, and D-dimer at enrollment, day 1, 3, and 5 were estimated using linear mixed models. A 7-point pulmonary ordinal scale assessed at day 5 was compared using proportional odds models. RESULTS: Analysis included 2149 participants enrolled between August 2020 and September 2021. Treatment resulted in 20% lower levels of plasma N-Ag compared with placebo (95% confidence interval, 12%-27%; P < .001), and a steeper rate of decline through the first 5 days (P < .001). The treatment difference did not vary between subgroups, and no difference was observed in trajectories of other biomarkers or the day 5 pulmonary ordinal scale. CONCLUSIONS: Our study suggests that nmAb has an antiviral effect assessed by plasma N-Ag among hospitalized patients with COVID-19, with no blunting of the endogenous anti-nucleocapsid antibody response. No effect on systemic inflammation or day 5 clinical status was observed. CLINICAL TRIALS REGISTRATION: NCT04501978.

Interstitial Macrophages Mediate Efferocytosis of Alveolar Epithelium during Influenza Infection.

Efferocytosis is a process whereby apoptotic cells are cleared to maintain tissue homeostasis. In the lungs, efferocytosis has been implicated in several acute and chronic inflammatory diseases. A long-standing method to study efferocytosis in vivo is to instill apoptotic cells into the lungs to evaluate macrophage uptake. However, this approach provides nonphysiologic levels of cells to the airspaces, where there is preferential access to the alveolar macrophages. To circumvent this limitation, we developed a new method to study efferocytosis of damaged alveolar type 2 (AT2) epithelial cells in vivo. A reporter mouse that expresses TdTomato in AT2 epithelial cells was injured with influenza (strain PR8) to induce apoptosis of AT2 cells. We were able to identify macrophages that acquire red fluorescence after influenza injury, indicating efferocytosis of AT2 cells. Furthermore, evaluation of macrophage populations led to the surprising finding that lung interstitial macrophages were the primary efferocyte in vivo. In summary, we present a novel finding that the interstitial macrophage, not the alveolar macrophage, primarily mediates clearance of AT2 cells in the lungs after influenza infection. Our method of studying efferocytosis provides a more physiologic approach in evaluating the spatiotemporal dynamics of apoptotic cell clearance in vivo and opens new avenues to study the mechanisms by which efferocytosis regulates inflammation.

SARS-CoV-2 Neutralizing Antibody LY-CoV555 in Outpatients with Covid-19.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (Covid-19), which is most frequently mild yet can be severe and life-threatening. Virus-neutralizing monoclonal antibodies are predicted to reduce viral load, ameliorate symptoms, and prevent hospitalization. METHODS: In this ongoing phase 2 trial involving outpatients with recently diagnosed mild or moderate Covid-19, we randomly assigned 452 patients to receive a single intravenous infusion of neutralizing antibody LY-CoV555 in one of three doses (700 mg, 2800 mg, or 7000 mg) or placebo and evaluated the quantitative virologic end points and clinical outcomes. The primary outcome was the change from baseline in the viral load at day 11. The results of a preplanned interim analysis as of September 5, 2020, are reported here. RESULTS: At the time of the interim analysis, the observed mean decrease from baseline in the log viral load for the entire population was -3.81, for an elimination of more than 99.97% of viral RNA. For patients who received the 2800-mg dose of LY-CoV555, the difference from placebo in the decrease from baseline was -0.53 (95% confidence interval [CI], -0.98 to -0.08; P = 0.02), for a viral load that was lower by a factor of 3.4. Smaller differences from placebo in the change from baseline were observed among the patients who received the 700-mg dose (-0.20; 95% CI, -0.66 to 0.25; P = 0.38) or the 7000-mg dose (0.09; 95% CI, -0.37 to 0.55; P = 0.70). On days 2 to 6, the patients who received LY-CoV555 had a slightly lower severity of symptoms than those who received placebo. The percentage of patients who had a Covid-19-related hospitalization or visit to an emergency department was 1.6% in the LY-CoV555 group and 6.3% in the placebo group. CONCLUSIONS: In this interim analysis of a phase 2 trial, one of three doses of neutralizing antibody LY-CoV555 appeared to accelerate the natural decline in viral load over time, whereas the other doses had not by day 11. (Funded by Eli Lilly; BLAZE-1 ClinicalTrials.gov number, NCT04427501.).

Bamlanivimab plus Etesevimab in Mild or Moderate Covid-19.

BACKGROUND: Patients with underlying medical conditions are at increased risk for severe coronavirus disease 2019 (Covid-19). Whereas vaccine-derived immunity develops over time, neutralizing monoclonal-antibody treatment provides immediate, passive immunity and may limit disease progression and complications. METHODS: In this phase 3 trial, we randomly assigned, in a 1:1 ratio, a cohort of ambulatory patients with mild or moderate Covid-19 who were at high risk for progression to severe disease to receive a single intravenous infusion of either a neutralizing monoclonal-antibody combination agent (2800 mg of bamlanivimab and 2800 mg of etesevimab, administered together) or placebo within 3 days after a laboratory diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The primary outcome was the overall clinical status of the patients, defined as Covid-19-related hospitalization or death from any cause by day 29. RESULTS: A total of 1035 patients underwent randomization and received an infusion of bamlanivimab-etesevimab or placebo. The mean (±SD) age of the patients was 53.8±16.8 years, and 52.0% were adolescent girls or women. By day 29, a total of 11 of 518 patients (2.1%) in the bamlanivimab-etesevimab group had a Covid-19-related hospitalization or death from any cause, as compared with 36 of 517 patients (7.0%) in the placebo group (absolute risk difference, -4.8 percentage points; 95% confidence interval [CI], -7.4 to -2.3; relative risk difference, 70%; P<0.001). No deaths occurred in the bamlanivimab-etesevimab group; in the placebo group, 10 deaths occurred, 9 of which were designated by the trial investigators as Covid-19-related. At day 7, a greater reduction from baseline in the log viral load was observed among patients who received bamlanivimab plus etesevimab than among those who received placebo (difference from placebo in the change from baseline, -1.20; 95% CI, -1.46 to -0.94; P<0.001). CONCLUSIONS: Among high-risk ambulatory patients, bamlanivimab plus etesevimab led to a lower incidence of Covid-19-related hospitalization and death than did placebo and accelerated the decline in the SARS-CoV-2 viral load. (Funded by Eli Lilly; BLAZE-1 ClinicalTrials.gov number, NCT04427501.).

Multidimensional Pattern Recognition in High-Resolution 2D and 3D Spectra of Gas-Phase Molecules.

ConspectusWhen molecules transition from the condensed phase to the gas phase, their spectra undergo a dramatic transformation as well; each peak in a condensed-phase spectrum can yield thousands of peaks in the gas phase because the molecules are free to rotate and those rotational motions are quantized. These gas-phase spectra contain a wealth of detailed information about molecular structure and behavior, but peak densities are often so high that congestion obscures the patterns needed to assign peaks and extract molecular constants. This Account describes how coherent multidimensional techniques not only reduce peak densities and congestion in gas-phase spectra but also create multidimensional patterns that are easy to recognize and interpret. First, all peaks with the same vibrational quantum numbers form rotational patterns such as X's, double parabolas, and asterisks. These rotational patterns are composed of basic units and can provide immediate information about the molecule's structure, behavior, and rotational selection rules. Second, groups of these rotational patterns can be arranged into vibrational patterns that form arrays of rectangles or parallelograms. These vibrational patterns can be used to determine wave-mixing processes and measure vibrational constants. Coherent multidimensional spectroscopy therefore automatically separates vibrational and rotational information and then sorts peaks by vibrational and rotational quantum number. Furthermore, if the sample is composed of a mixture, then these patterns can also sort peaks by species, and higher-dimensional techniques can even provide the ability to select a species in the mixture. These techniques have successfully produced highly patterned 2D and 3D spectra for samples that otherwise generate patternless spectra such as isotopologue mixtures and vibronically perturbed molecules such as NO2.High densities of states can lead to congestion and perturbations that make it difficult to accurately assign peaks using the information that is traditionally available from 1D spectra: a peak's intensity and its frequency. Coherent 2D and 3D techniques are well-suited for dealing with and learning from perturbations because the coordinate of each peak in multidimensional space includes multiple frequency values. Accurate assignments are possible when peaks in 2D or 3D spectra that are perturbed along one frequency axis are unperturbed along an orthogonal frequency axis. Furthermore, patterns often repeat in adjacent rows or columns, so regions that are less congested can be used to resolve or identify key peaks or patterns in regions that are severely congested. Perturbations can make the spacings within multidimensional rotational and vibrational patterns slightly irregular, but these automatically generated patterns remain easy to recognize and analyze.This Account describes three high-resolution coherent multidimensional spectroscopy techniques, the types of patterns they can produce, and how information can be extracted from these patterns. This work is being conducted at Spelman College, a historically Black college for women where all of the students are undergraduates. The resulting techniques are not only highly effective for dealing with some of the most congested, perturbed, and challenging spectroscopic systems, but they are relatively easy to use, moderate in price to set up, and quick to run.

FOXA1/2 depletion drives global reprogramming of differentiation state and metabolism in a human liver cell line and inhibits differentiation of human stem cell-derived hepatic progenitor cells.

FOXA factors are critical members of the developmental gene regulatory network (GRN) composed of master transcription factors (TF) which regulate murine cell fate and metabolism in the gut and liver. How FOXA factors dictate human liver cell fate, differentiation, and simultaneously regulate metabolic pathways is poorly understood. Here, we aimed to determine the role of FOXA2 (and FOXA1 which is believed to compensate for FOXA2) in controlling hepatic differentiation and cell metabolism in a human hepatic cell line (HepG2). siRNA mediated knockdown of FOXA1/2 in HepG2 cells significantly downregulated albumin (p < .05) and GRN TF gene expression (HNF4α, HEX, HNF1ß, TBX3) (p < .05) and significantly upregulated endoderm/gut/hepatic endoderm markers (goosecoid [GSC], FOXA3, and GATA4), gut TF (CDX2), pluripotent TF (NANOG), and neuroectodermal TF (PAX6) (p < .05), all consistent with partial/transient reprograming. shFOXA1/2 targeting resulted in similar findings and demonstrated evidence of reversibility of phenotype. RNA-seq followed by bioinformatic analysis of shFOXA1/2 knockdown HepG2 cells demonstrated 235 significant downregulated genes and 448 upregulated genes, including upregulation of markers for alternate germ layers lineages (cardiac, endothelial, muscle) and neurectoderm (eye, neural). We found widespread downregulation of glycolysis, citric acid cycle, mitochondrial genes, and alterations in lipid metabolism, pentose phosphate pathway, and ketogenesis. Functional metabolic analysis agreed with these findings, demonstrating significantly diminished glycolysis and mitochondrial respiration, with concomitant accumulation of lipid droplets. We hypothesized that FOXA1/2 inhibit the initiation of human liver differentiation in vitro. During human pluripotent stem cells (hPSC)-hepatic differentiation, siRNA knockdown demonstrated de-differentiation and unexpectedly, activation of pluripotency factors and neuroectoderm. shRNA knockdown demonstrated similar results and activation of SOX9 (hepatobiliary). These results demonstrate that FOXA1/2 controls hepatic and developmental GRN, and their knockdown leads to reprogramming of both differentiation and metabolism, with applications in studies of cancer, differentiation, and organogenesis.

Bridge vs Terminal Cyano-coordination in Binuclear Cobalt Porphyrin Dimers: Interplay of Electrons between Metal and Ligand and Spin-Coupling via Bridge.

Three cyano-coordinated cobalt porphyrin dimers were synthesized and thoroughly characterized. The X-ray structure of the complexes reveals that cyanide binds in a terminal fashion in both the anti and trans isomers of ethane- and ethylene-bridged cobalt porphyrin dimers, while in the cis ethylene-bridged dimer, cyanides bind in both terminal and bridging modes. The nonconjugated ethane-bridged complex stabilizes exclusively a diamagnetic metal-centered oxidation of type CoIII(por)(CN)2 both in the solid and in solution. In contrast, the complexes with the conjugated ethylene-bridge contain signatures of both paramagnetic ligand-centered oxidation of the type CoII(por•+)(CN)2 and diamagnetic metal-centered oxidation of type CoIII(por)(CN)2 with the metal-centered oxidized species being the major component in the solid state as observed in XPS, while the ligand-centered oxidized species are present in a significant amount in solution. 1H NMR spectrum in solution displays two set of signals corresponding to the simultaneous presence of both the diamagnetic and paramagnetic species. EPR and magnetic investigation reveal that there is a moderate ferromagnetic coupling between the unpaired electrons of the low-spin CoII center and the porphyrin π-cation radical in CoII(por•+)(CN)2 species as well as an antiferromagnetic coupling between the two CoII(por•+) units through the ethylene and CN bridges.

Robustness of Threshold Collision-Induced Dissociation Simulations for Bond Dissociation Energies.

The threshold collision-induced dissociation (T-CID) method is the workhorse for gas-phase bond dissociation energy (BDE) measurements. However, T-CID does not measure BDEs directly; instead, BDEs are obtained by fitting simulated data to the experimental data. We previously observed several large discrepancies between the computed and experimental BDEs. To analyze the reliability of the experimental values, we previously reported a study of the dissociation rate models in the simulation. Here, we report a study of the collision simulation part, specifically in the L-CID (ligand CID) program. We show that the BDE values are robust even to intentionally introduced mistakes in the simulations, varying in most cases by less than 3 kcal mol-1. The most significant exception is the collisional energy transfer (CET) simulation, which led to deviations larger than 10 kcal mol-1. However, we found that the BDEs obtained with explicitly simulated CET distributions deviated by only 3 kcal mol-1 from those simulated with the original model. Collectively, our results suggest that the T-CID-derived BDE values are robust and are likely to be accurate.

Evaluation of the Accuracy of the Smart Work Injury Management (SWIM) System to Assist Case Managers in Predicting the Work Disability of Injured Workers.

PURPOSE: Many countries have developed clinical decision-making support tools, such as the smart work injury management (SWIM) system in Hong Kong, to predict rehabilitation paths and address global issues related to work injury disability. This study aims to evaluate the accuracy of SWIM by comparing its predictions on real work injury cases to those made by human case managers, specifically with regard to the duration of sick leave and the percentage of permanent disability. METHODS: The study analyzed a total of 442 work injury cases covering the period from 2012 to 2020, dividing them into non-litigated and litigated cases. The Kruskal-Wallis post hoc test with Bonferroni adjustment was used to evaluate the differences between the actual data, the SWIM predictions, and the estimations made by three case managers. The intra-class correlation coefficient was used to assess the inter-rater reliability of the case managers. RESULTS: The study discovered that the predictions made by the SWIM model and a case manager possessing approximately 4 years of experience in case management exhibited moderate reliability in non-litigated cases. Nevertheless, there was no resemblance between SWIM's predictions regarding the percentage of permanent disability and those made by case managers. CONCLUSION: The findings indicate that SWIM is capable of replicating the sick leave estimations made by a case manager with an estimated 4 years of case management experience, albeit with limitations in generalizability owing to the small sample size of case managers involved in the study. IMPLICATIONS: These findings represent a significant advancement in enhancing the accuracy of CDMS for work injury cases in Hong Kong, signaling progress in the field.

Exploring the protective capabilities developed by hospital nurses to deflect workplace violence: a qualitative study in Taiwan.

BACKGROUND: Workplace violence is a serious safety hazard in the healthcare sector and has attracted much attention worldwide, especially for nursing staff. Equipping nursing personnel with protective capabilities for workplace violence can reduce the risk they face in work settings. This study explored scenarios of common violent patient-nurse conflicts in the workplace of hospital nursing staff and their capabilities for de-escalating such conflicts. METHODS: We used a qualitative content analysis for this study. Qualitative interviews were conducted with 21 nurses in two teaching hospitals in New Taipei City, Taiwan, until data saturation was reached. The data were transcribed, encoded, and analyzed and similar concepts were grouped under the same category. RESULTS: Four categories of workplace violence scenarios common to hospital nursing staff were identified: unreasonable requests, caring for high-risk patients, long waiting times for medical consultation, and close contact when caring for patients. Two protective capabilities were recognized: communication and interpersonal capabilities and problem-solving skills. Each scenario may require a different combination of abilities. CONCLUSION: Our findings suggest that a violence-prevention training program could be designed for various workplace violence scenarios to enhance nurses' abilities to de-escalate workplace violence in hospitals.

Senescence of alveolar epithelial progenitor cells: a critical driver of lung fibrosis.

Pulmonary fibrosis comprises a range of chronic interstitial lung diseases (ILDs) that impose a significant burden on patients and public health. Among these, idiopathic pulmonary fibrosis (IPF), a disease of aging, is the most common and most severe form of ILD and is treated largely by lung transplantation. The lack of effective treatments to stop or reverse lung fibrosis-in fact, fibrosis in most organs-has sparked the need to understand causative mechanisms with the goal of identifying critical points for potential therapeutic intervention. Findings from many groups have indicated that repeated injury to the alveolar epithelium-where gas exchange occurs-leads to stem cell exhaustion and impaired alveolar repair that, in turn, triggers the onset and progression of fibrosis. Cellular senescence of alveolar epithelial progenitors is a critical cause of stemness failure. Hence, senescence impairs repair and thus contributes significantly to fibrosis. In this review, we discuss recent evidence indicating that senescence of epithelial progenitor cells impairs alveolar homeostasis and repair creating a profibrotic environment. Moreover, we discuss the impact of senescent alveolar epithelial progenitors, alveolar type 2 (AT2) cells, and AT2-derived transitional epithelial cells in fibrosis. Emerging evidence indicates that transitional epithelial cells are prone to senescence and, hence, are a new player involved in senescence-associated lung fibrosis. Understanding the complex interplay of cell types and cellular regulatory factors contributing to alveolar epithelial progenitor senescence will be crucial to developing targeted therapies to mitigate their downstream profibrotic sequelae and to promote normal alveolar repair.NEW & NOTEWORTHY With an aging population, lung fibrotic diseases are becoming a global health burden. Dysfunctional repair of the alveolar epithelium is a key causative process that initiates lung fibrosis. Normal alveolar regeneration relies on functional progenitor cells; however, the senescence of these cells, which increases with age, hinders their ability to contribute to repair. Here, we discuss studies on the control and consequence of progenitor cell senescence in fibrosis and opportunities for research.

Cryogenic Ion Vibrational Predissociation (CIVP) Spectroscopy of Aryl Cobinamides in the Gas Phase: How Good Are the Calculations for Vitamin B12 Derivatives?

Aryl corrins represent a novel class of designed B12 derivatives with biological properties of "antivitamins B12". In our previous study, we experimentally determined bond strength in a series of aryl-corrins by the threshold collision-induced dissociation experiments (T-CID) and compared the measured bond dissociation energies (BDEs) with those calculated with density functional theory (DFT). We found that the BDEs are modulated by the side chains around the periphery of the corrin unit. Given that aryl cobinamides have many side chains that increase their conformational space and that the question of a specific structure, measured in the gas phase, was important for further evaluation of our T-CID experiment, we proceeded to analyze structural properties of aryl cobinamides using cryogenic ion vibrational predissociation (CIVP) spectroscopy, static DFT, and Born-Oppenheimer molecular dynamic (BOMD) simulations. We found that none of the examined DFT models could reproduce the CIVP spectra convincingly; both "static" DFT calculations and "dynamic" BOMD simulations provide a surprisingly poor representation of the vibrational spectra, specifically of the number, position, and intensity of bands assigned to hydrogen-bonded versus non-hydrogen-bonded NH and OH moieties. We conclude that, for a flexible molecule with ca. 150 atoms, more accurate approaches are needed before definitive conclusions about computed properties, specifically the structure of the ground-state conformer, may be made.

Risk-Adapted Intraoperative Radiation Therapy (IORT) for Breast Cancer: A Novel Analysis.

BACKGROUND: Randomized trials have shown that risk-adapted intraoperative radiation therapy (IORT) after breast-conserving surgery for low-risk breast cancer patients is a safe alternative to whole-breast radiation therapy (WBRT). The risk-adapted strategy allows additional WBRT for predefined high-risk pathologic characteristics discovered on final histopathology. The greater the percentage of patients receiving WBRT, the lower the recurrence rate. The risk-adapted strategy, although important and necessary, can make IORT appear better than it actually is. METHODS: Risk-adapted IORT was used to treat 1600 breast cancers. They were analyzed by the intention-to-treat method and per protocol to better understand the contribution of IORT with and without additional whole-breast treatment. Any ipsilateral breast tumor event was considered a local recurrence. RESULTS: During a median follow-up period of 63 months, local recurrence differed significantly between the patients who received local treatment and those who received whole-breast treatment. For 1393 patients the treatment was local treatment alone. These patients experienced 79 local recurrences and a 5-year local recurrence probability of 5.95 %. For 207 patients with high-risk final histopathology, additional whole-breast treatment was administered. They experienced two local recurrences and a 5-year local recurrence probability of 0.5 % (p = 0.0009). CONCLUSIONS: Whole-breast treatment works well at reducing local recurrence, and it is a totally acceptable and necessary addition to IORT as part of a risk-adapted program. However, the more whole-breast treatment that is given, the more it dilutes the original plan of simplifying local treatment and the less we understand exactly what IORT contributes to local control as a stand-alone treatment.