Rewiring cancer drivers to activate apoptosis.

Genes that drive the proliferation, survival, invasion and metastasis of malignant cells have been identified for many human cancers1-4. Independent studies have identified cell death pathways that eliminate cells for the good of the organism5,6. The coexistence of cell death pathways with driver mutations suggests that the cancer driver could be rewired to activate cell death using chemical inducers of proximity (CIPs). Here we describe a new class of molecules called transcriptional/epigenetic CIPs (TCIPs) that recruit the endogenous cancer driver, or a downstream transcription factor, to the promoters of cell death genes, thereby activating their expression. We focused on diffuse large B cell lymphoma, in which the transcription factor B cell lymphoma 6 (BCL6) is deregulated7. BCL6 binds to the promoters of cell death genes and epigenetically suppresses their expression8. We produced TCIPs by covalently linking small molecules that bind BCL6 to those that bind to transcriptional activators that contribute to the oncogenic program, such as BRD4. The most potent molecule, TCIP1, increases binding of BRD4 by 50% over genomic BCL6-binding sites to produce transcriptional elongation at pro-apoptotic target genes within 15 min, while reducing binding of BRD4 over enhancers by only 10%, reflecting a gain-of-function mechanism. TCIP1 kills diffuse large B cell lymphoma cell lines, including chemotherapy-resistant, TP53-mutant lines, at EC50 of 1-10 nM in 72 h and exhibits cell-specific and tissue-specific effects, capturing the combinatorial specificity inherent to transcription. The TCIP concept also has therapeutic applications in regulating the expression of genes for regenerative medicine and developmental disorders.

Role for Caspase-8 in the Release of IL-1ß and Active Caspase-1 from Viable Human Monocytes during Toxoplasma gondii Infection.

Monocytes are actively recruited to sites of infection and produce the potent proinflammatory cytokine IL-1ß. We previously showed that IL-1ß release during Toxoplasma gondii infection of primary human monocytes requires the NLRP3 inflammasome and caspase-1 but is independent of gasdermin D and pyroptosis. To investigate mechanisms of IL-1ß release, we generated caspase-1, -4, -5, or -8 knockout (KO) THP-1 monocytic cells. Genetic ablation of caspase-1 or -8, but not caspase-4 or -5, decreased IL-1ß release during T. gondii infection without affecting cell death. In contrast, TNF-α and IL-6 secretion were unperturbed in caspase-8 KO cells during T. gondii infection. Dual pharmacological inhibition of caspase-8 and RIPK1 in primary monocytes also decreased IL-1ß release without affecting cell viability or parasite infection. Caspase-8 was also required for the release of active caspase-1 from T. gondii-infected cells and for IL-1ß release during infection with the related apicomplexan parasite Neospora caninum. Surprisingly, caspase-8 deficiency did not impair synthesis or cleavage of pro-IL-1ß, but resulted in the retention of mature IL-1ß within cells. Generation of gasdermin E KO and ATG7 KO THP-1 cells revealed that the release of IL-1ß was not dependent on gasdermin E or ATG7. Collectively, our data indicate that during T. gondii Infection of human monocytes, caspase-8 functions in a novel gasdermin-independent mechanism controlling IL-1ß release from viable cells. This study expands on the molecular pathways that promote IL-1ß in human immune cells and provides evidence of a role for caspase-8 in the mechanism of IL-1ß release during infection.

Leveraging gene co-regulation to identify gene sets enriched for disease heritability.

Identifying gene sets that are associated to disease can provide valuable biological knowledge, but a fundamental challenge of gene set analyses of GWAS data is linking disease-associated SNPs to genes. Transcriptome-wide association studies (TWASs) detect associations between the genetically predicted expression of a gene and disease risk, thus implicating candidate disease genes. However, causal disease genes at TWAS-associated loci generally remain unknown due to gene co-regulation, which leads to correlations across genes in predicted expression. We developed a method, gene co-regulation score (GCSC) regression, to identify gene sets that are enriched for disease heritability explained by predicted expression. GCSC regresses TWAS chi-square statistics on gene co-regulation scores reflecting correlations in predicted gene expression; a gene set is enriched for heritability if genes with high co-regulation to the set have higher TWAS chi-square statistics than genes with low co-regulation to the set, beyond what is expected based on co-regulation to all genes. We verified via simulations that GCSC is well calibrated and well powered. We applied GCSC to gene expression data from GTEx (48 tissues) and GWAS summary statistics for 43 independent diseases and complex traits analyzing a broad set of biological pathways and specifically expressed gene sets. We identified many enriched sets, recapitulating known biology. For Alzheimer disease, we detected evidence of an immune basis, and specifically a role for antigen presentation, in analyses of both biological pathways and specifically expressed gene sets. Our results highlight the advantages of leveraging gene co-regulation within the TWAS framework to identify enriched gene sets.

Temporal correlation between postreperfusion complement deposition and severe primary graft dysfunction in lung allografts.

Growing evidence implicates complement in the pathogenesis of primary graft dysfunction (PGD). We hypothesized that early complement activation postreperfusion could predispose to severe PGD grade 3 (PGD-3) at 72 hours, which is associated with worst posttransplant outcomes. Consecutive lung transplant patients (n = 253) from January 2018 through June 2023 underwent timed open allograft biopsies at the end of cold ischemia (internal control) and 30 minutes postreperfusion. PGD-3 at 72 hours occurred in 14% (35/253) of patients; 17% (44/253) revealed positive C4d staining on postreperfusion allograft biopsy, and no biopsy-related complications were encountered. Significantly more patients with PGD-3 at 72 hours had positive C4d staining at 30 minutes postreperfusion compared with those without (51% vs 12%, P < .001). Conversely, patients with positive C4d staining were significantly more likely to develop PGD-3 at 72 hours (41% vs 8%, P < .001) and experienced worse long-term outcomes. In multivariate logistic regression, positive C4d staining remained highly predictive of PGD-3 (odds ratio 7.92, 95% confidence interval 2.97-21.1, P < .001). Hence, early complement deposition in allografts is highly predictive of PGD-3 at 72 hours. Our data support future studies to evaluate the role of complement inhibition in patients with early postreperfusion complement activation to mitigate PGD and improve transplant outcomes.

Chemical Localization With Robotic Bronchoscopy: Can It Aid Resection of Subsolid Lung Nodules?

INTRODUCTION: Subsolid nodules or those located deep in lung parenchyma are difficult to localize using minimally invasive thoracic surgery. While image-guided percutaneous needle localization has been performed, it is inconvenient and has potential complications. In this study, the role of chemical localization using robotic bronchoscopy to facilitate resection was evaluated. METHODS: Consecutive patients undergoing surgical resection for lung nodules between 8/2019-3/2022 were included. Patients with subsolid lung nodules, or small nodules deep in lung parenchyma that were deemed difficult to localize, were chemically localized (CL) using robotic bronchoscopy before resection. Clinico-demographic data were obtained retrospectively using a prospectively maintained database. RESULTS: Localization of lung nodules before resection was performed in 139 patients while 110 patients were not localized. Daily activity score was higher for localized patients. Nodules in the localized group were smaller (P < 0.001) and had similar solid:ground glass ratio. In the localized group, larger margins were observed, and no re-resection of the parenchymal margin was required. Twenty patients in the non-localized group required re-resection intraoperatively due to close pathological margins or inability to locate the nodule in the resected specimen. Operative time was a median of 10-15 min longer for localized patients, P < 0.001. Length of stay was shorter in the localized group (P < 0.05). CONCLUSIONS: Chemical localization of lung nodules using robotic bronchoscopy appears to be a safe and effective method of identifying the location of nodules with small size and less density and aids increased tumor margins intraoperatively.

Concomitant Biceps Tenodesis Does Not Compromise Arthroscopic Rotator Cuff Repair Outcomes.

PURPOSE: To compare outcomes of patients who underwent rotator cuff repair (RCR) with concomitant biceps tenodesis with those who underwent an isolated RCR. METHODS: Exclusion criteria included previous ipsilateral shoulder surgery, irreparable rotator cuff tears, rotator cuff arthropathy, calcific tendinitis, adhesive capsulitis requiring a capsular release, or advanced osteoarthritis of the glenohumeral joint. Patients were indicated for biceps tenodesis if they had any degree of tendon tearing, moderate-to-severe tenosynovitis, instability, or a significant degenerative SLAP tear. Primary outcome measures included American Shoulder and Elbow Surgeons score, Simple Shoulder Test, EuroQoL 5-Dimension 5-Level visual analog scale, EuroQoL 5-Dimension 5-Level, and a site-specific questionnaire, which focused on surgical expectations, satisfaction, and complications. Multivariate analysis of variance to analyze descriptive statistics and determine significant differences between the patient groups for subjective and objective outcome measures were performed. RESULTS: There were no significant differences for pain/visual analog scale (0.34 ± 0.09 vs 0.47 ± 0.09, P = .31), American Shoulder and Elbow Surgeons score (96.69 ± 0.87 vs 94.44 ± 0.91, P = .07), and Simple Shoulder Test (11.42 ± 0.17 vs 10.95 ± 0.18, P = .06) between the RCR with concomitant biceps tenodesis and isolated RCR at a minimum of 2 years' postoperatively. This is despite the RCR with concomitant biceps tenodesis group having significantly larger rotator cuff tears (4.25 ± 0.30 cm2 vs 2.80 ± 0.32 cm2, P = .001) than the isolated RCR group. CONCLUSIONS: This study revealed that concomitant biceps tenodesis does not compromise outcomes when compared with an isolated RCR at 2-year follow-up, despite this group having larger rotator cuff tears. LEVEL OF EVIDENCE: Level III, retrospective case study.

Age-dependent transcriptomic profiles of leukocytes in pediatric population.

Immunity at birth is considered immature. Following birth, our immune function is considered to grow and reach maturation over time. To obtain granular information of leukocyte functions and transcriptomic profiles in pediatric cohort, we examined leukocyte profiles in infants, preschool and school children using single cell RNA sequencing of their peripheral blood mononuclear cells (PBMCs). Monocytes and natural killer (NK) cells showed immaturity in infants. Their innate and adaptive immunity was developed by preschool age. Adaptive immune cells showed different maturation patterns. CD4, CD8 naïve T cells and plasma cells continued to mature untill school age. In CD8 naïve T cells, innate immunity was upregulated in infants, in support of our knowledge that they manifests more innate cell-like phenotype soon after birth. Many signaling pathways have been differentially up- and/or down-regulated in infants, preschool and school children. Their contribution to the development of the immune system needs to be delineated.

Breast cancer treatment and ovarian function.

The aim of this study was to provide an update on ovarian function and the mechanisms of gonadal damage after exposure to chemotherapy in breast cancer survivors. The alkylating agents are toxic to both primordial and growing follicles. However, anti-metabolite drugs are more likely to destroy preantral and antral follicles. Younger patients are more likely to have a higher ovarian reserve, and therefore, more likely to retain some residual ovarian function after exposure to gonadotoxic regimens. However, there can be significant variability in ovarian reserve among patients of the same age. Furthermore, patients with critically diminished ovarian reserve may continue to menstruate regularly. Therefore age and menstrual status are not reliable indicators of good ovarian reserve and might give a false sense of security and result in an adverse outcome if the patient is consulted without considering more reliable quantitative markers of ovarian reserve (antral follicle count and anti-Müllerian hormone) and fertility preservation is not pursued. In contrast to well-documented ovarian toxicity of older chemotherapy regimens, data for newer taxane-containing protocols have only accumulated in the last decade and data are still very limited regarding the impact of targeted therapies on ovarian function.

Rural-Urban Disparities in Receipt of Surgery for Potentially Resectable Non-Small Cell Lung Cancer.

INTRODUCTION: Access to cancer care, especially surgery, is limited in rural areas. However, the specific reasons rural patient populations do not receive surgery for non-small cell lung cancer (NSCLC) is unknown. We investigated geographic disparities in reasons for failure to receive guideline-indicated surgical treatment for patients with potentially resectable NSCLC. METHODS: The National Cancer Database was used to identify patients with clinical stage I-IIIA (N0-N1) NSCLC between 2004 and 2018. Patients from rural areas were compared to urban areas, and the reason for nonreceipt of surgery was evaluated. Adjusted odds of (1) primary nonsurgical management, (2) surgery being deemed contraindicated due to risk, (3) surgery being recommended but not performed, and (4) overall failure to receive surgery were determined. RESULTS: The study included 324,785 patients with NSCLC with 42,361 (13.0%) from rural areas. Overall, 62.4% of patients from urban areas and 58.8% of patients from rural areas underwent surgery (P < 0.001). Patients from rural areas had increased odds of (1) being recommended primary nonsurgical management (adjusted odds ratio [aOR]: 1.14, 95% confidence interval [CI]: 1.05-1.23), (2) surgery being deemed contraindicated due to risk (aOR: 1.19, 95% CI: 1.07-1.33), (3) surgery being recommended but not performed (aOR: 1.13, 95% CI: 1.01-1.26), and (4) overall failure to receive surgery (aOR: 1.21, 95% CI: 1.13-1.29; all P < 0.001). CONCLUSIONS: There are geographic disparities in the management of NSCLC. Rural patient populations are more likely to fail to undergo surgery for potentially resectable disease for every reason examined.

Renal Transplant Patients Have a Lower Risk of Complications and Mortalities After Total Knee Arthroplasty Compared to Those on Hemodialysis: A Large National Database Study.

BACKGROUND: Total knee arthroplasty (TKA) in end-stage renal disease is associated with complications. Controversy exists whether elective TKA should be performed while patients are on hemodialysis (HD) or following renal transplant (RT). This study compares TKA outcomes in HD versus RT patients. METHODS: A national database was retrospectively reviewed using International Classification of Diseases codes to identify HD and RT patients who underwent primary TKA from 2010 to 2018. Demographics, comorbidities, and hospital factors were compared using Wald and Chi-squared tests. The primary outcome was in-hospital mortalities while secondary outcomes included quality outcomes and medical/surgical complications. Multivariate regressions were used to determine independent associations. Significance was determined with a 2-tailed P value of .05. There were 13,611 patients who underwent TKA (61.1 HD and 38.9% RT). Patients who had RT were younger, had fewer comorbidities, and more likely to have private insurance. RESULTS: The RT patients had a lower rate of mortality (odds ratio (OR) 0.23, P < .01)), complications (OR 0.63, P < .01), cardiopulmonary complications (OR 0.44, P = .02), sepsis (OR 0.22, P < .001), and blood transfusion (OR 0.35, P < .001) during the index hospitalization. This cohort was also found to have decreased length of stay (-2.0 days, P < .001), non-home discharge (OR 0.57, P < .001), and hospital cost (-$5,300, P < .001). Patients who had RT had a lower rate of readmission (OR 0.54, P < .001), periprosthetic joint infection (OR 0.50, P < .01), and surgical site infection (OR 0.37, P < .001) within 90 days. CONCLUSION: These findings suggest that HD patients are a high-risk population in TKA compared to RT patients and warrant stringent perioperative monitoring.

Genes with High Network Connectivity Are Enriched for Disease Heritability.

Recent studies have highlighted the role of gene networks in disease biology. To formally assess this, we constructed a broad set of pathway, network, and pathway+network annotations and applied stratified LD score regression to 42 diseases and complex traits (average N = 323K) to identify enriched annotations. First, we analyzed 18,119 biological pathways. We identified 156 pathway-trait pairs whose disease enrichment was statistically significant (FDR < 5%) after conditioning on all genes and 75 known functional annotations (from the baseline-LD model), a stringent step that greatly reduced the number of pathways detected; most significant pathway-trait pairs were previously unreported. Next, for each of four published gene networks, we constructed probabilistic annotations based on network connectivity. For each gene network, the network connectivity annotation was strongly significantly enriched. Surprisingly, the enrichments were fully explained by excess overlap between network annotations and regulatory annotations from the baseline-LD model, validating the informativeness of the baseline-LD model and emphasizing the importance of accounting for regulatory annotations in gene network analyses. Finally, for each of the 156 enriched pathway-trait pairs, for each of the four gene networks, we constructed pathway+network annotations by annotating genes with high network connectivity to the input pathway. For each gene network, these pathway+network annotations were strongly significantly enriched for the corresponding traits. Once again, the enrichments were largely explained by the baseline-LD model. In conclusion, gene network connectivity is highly informative for disease architectures, but the information in gene networks may be subsumed by regulatory annotations, emphasizing the importance of accounting for known annotations.

Center Volume Impacts Readmissions and Mortality after Congenital Cardiac Surgery.

OBJECTIVE: To characterize the relationship between surgical volume and postoperative outcomes in congenital heart surgery, we used a national cohort to assess the costs, readmissions, and complications in children undergoing cardiac operations. STUDY DESIGN: The Nationwide Readmissions Database was used to identify pediatric patients (≤18 years) undergoing congenital cardiac surgery from 2010 to 2017. Hospitals were categorized based on deciles and tertiles of annual caseload with high-volume categorized as the highest tertile of volume. Multivariable regression models adjusting for patient and hospital characteristics were used to study the impact of volume on 30-day nonelective readmission, mortality, home discharge, and resource use. RESULTS: Of an estimated 69 448 hospitalizations included for analysis, 56 672 (82%) occurred at high-volume centers. After adjustment for key clinical factors, each decile increase in volume was associated with a 25% relative decrease in the odds of mortality, a 14% decrease in the odds of nonhome discharge, and a 4% relative decrease in the likelihood of 30-day nonelective readmission. After risk adjustment, each incremental increase in volume decile was associated with a one-half-day decrease in the hospital length of stay, but did not alter costs of the index hospitalization. However, after including all readmissions within 30 days of the index discharge, high-volume centers were associated with significantly lower costs compared with low-volume hospitals. CONCLUSIONS: Increased congenital cardiac surgery volume is associated with improved mortality, reduced duration of hospitalization, 30-day readmissions, and resource use. These findings demonstrate the inverse relationship between hospital volume and resource use and may have implications for the centralization of care for congenital cardiac surgery.

Association of Perceived Stress With Incident Heart Failure.

BACKGROUND: The relationship between psychological stress and heart failure (HF) has not been well studied. We sought to assess the relationship between perceived stress and incident HF. METHODS: We used data from the national REasons for Geographic And Racial Differences in Stroke (REGARDS) study, a large prospective biracial cohort study that enrolled community-dwellers aged 45 years and older between 2003 and 2007, with follow-up. We included participants free of suspected prevalent HF who completed the Cohen 4-item Perceived Stress Scale (PSS-4). Our outcome variables were incident HF event, HF with reduced ejection fraction events, and HF with preserved ejection fraction events. We estimated Cox proportional hazard models to determine if PSS-4 quartiles were independently associated with incident HF events, adjusting for sociodemographics, social support, unhealthy behaviors, comorbid conditions, and physiologic parameters. We also tested interactions by baseline statin use, given its anti-inflammatory properties. RESULTS: Among 25,785 participants with a mean age of 64 ± 9.3 years, 55% were female and 40% were Black. Over a median follow-up of 10.1 years, 1109 ± 4.3% experienced an incident HF event. In fully adjusted models, the PSS-4 was not associated with HF or HF with reduced ejection fraction. However, PSS-4 quartiles 2-4 (compared with the lowest quartile) were associated with incident HF with preserved ejection fraction (Q2 hazard ratio 1.37, 95% confidence interval 1.00-1.88; Q3 hazard ratio 1.42, 95% confidence interval 1.03-1.95; Q4 hazard ratio 1.41, 95% confidence interval 1.04-1.92). Notably, this association was attenuated among participants who took a statin at baseline (P for interaction = .07). CONCLUSIONS: Elevated perceived stress was associated with incident HF with preserved ejection fraction but not HF with reduced ejection fraction.

Non-Covalently Associated Streptavidin Multi-Arm Nanohubs Exhibit Mechanical and Thermal Stability in Cross-Linked Protein-Network Materials.

Constructing protein-network materials that exhibit physicochemical and mechanical properties of individual protein constituents requires molecular cross-linkers with specificity and stability. A well-known example involves specific chemical fusion of a four-arm polyethylene glycol (tetra-PEG) to desired proteins with secondary cross-linkers. However, it is necessary to investigate tetra-PEG-like biomolecular cross-linkers that are genetically fused to the proteins, simplifying synthesis by removing additional conjugation and purification steps. Non-covalently, self-associating, streptavidin homotetramer is a viable, biomolecular alternative to tetra-PEG. Here, a multi-arm streptavidin design is characterized as a protein-network material platform using various secondary, biomolecular cross-linkers, such as high-affinity physical (i.e., non-covalent), transient physical, spontaneous chemical (i.e., covalent), or stimuli-induced chemical cross-linkers. Stimuli-induced, chemical cross-linkers fused to multi-arm streptavidin nanohubs provide sufficient diffusion prior to initiating permanent covalent bonds, allowing proper characterization of streptavidin nanohubs. Surprisingly, non-covalently associated streptavidin nanohubs exhibit extreme stability, which translates into material properties that resemble hydrogels formed by chemical bonds even at high temperatures. Therefore, this study not only establishes that the streptavidin nanohub is an ideal multi-arm biopolymer precursor but also provides valuable guidance for designing self-assembling nanostructured molecular networks that can properly harness the extraordinary properties of protein-based building blocks.

National Assessment of Early Discharge After Video-Assisted Thoracoscopic Surgery for Lung Resection.

INTRODUCTION: Video-assisted thoracoscopic surgery (VATS) techniques permit shorter postoperative length of stay (LOS). However, it remains unknown whether earlier discharge increases the risk of adverse postoperative events. We examined whether shorter LOS following elective VATS lung resection was associated with increased rates of readmission or postoperative complications. METHODS: Patients who underwent elective thoracoscopic segmentectomy, lobectomy, or bilobectomy for lung neoplasms from 2011 to 2018 were identified in the American College of Surgeons National Surgical Quality Improvement Program (ACS NSQIP) dataset. Postoperative LOS was treated as an ordinal variable. The examined outcomes were 30-d readmission and 30-d postdischarge death or serious morbidity (DSM). Multivariable logistic regression models evaluated the association of LOS with outcomes. The most common readmission diagnoses were identified for each operation. RESULTS: Among 14,418 patients, 12,410 (86.1%) underwent lobectomy, 1764 (12.2%) underwent segmentectomy, and 244 (1.7%) underwent bilobectomy. The median LOS was 3 d for patients undergoing lobectomy (IQR 2-5) and segmentectomy (IQR 2-4), and 4 d for bilobectomy (IQR 3-6). Readmission rates varied with admission time and ranged from 5.0% for patients with LOS ≤1 d to 8.5% for LOS ≥5 d. The most common readmission diagnoses were pneumothorax (19.0%) and wound complications (13.4%). Each one-day increase in LOS was associated with an increased risk of readmission (OR 1.10, 95% CI 1.04-1.17, P < 0.001). No association was seen between earlier discharge and DSM (OR 1.08, 95% CI 0.99-1.18, P = 0.070). CONCLUSIONS: Early discharge following VATS lung resection is not associated with increased rates of readmission or postoperative complications among patients undergoing surgery for cancer, and may safely be considered for selected patients with uncomplicated postoperative recovery.

Rural-urban survival disparities for patients with surgically treated lung cancer.

BACKGROUND: Nonsmall-cell lung cancer (NSCLC) is a common diagnosis among patients living in rural areas and small towns who face unique challenges accessing care. We examined differences in survival for surgically treated rural and small-town patients compared to those from urban and metropolitan areas. METHODS: The National Cancer Database was used to identify surgically treated NSCLC patients from 2004 to 2016. Patients from rural/small-town counties were compared to urban/metro counties. Differences in patient clinical, sociodemographic, hospital, and travel characteristics were described. Survival differences were examined with Kaplan-Meier curves and Cox proportional hazards models. RESULTS: The study included 366 373 surgically treated NSCLC patients with 12.4% (n = 45 304) categorized as rural/small-town. Rural/small-town patients traveled farther for treatment and were from areas characterized by lower income and education(all p < 0.001). Survival probabilities for rural/small-town patients were worse at 1 year (85% vs. 87%), 5 years (48% vs. 54%), and 10 years (26% vs. 31%) (p < 0.001). Travel distance >100 miles (hazard ratio [HR] = 1.11, 95% confidence interval [CI]: 1.07-1.16, vs. <25 miles) and living in a rural/small-town county (HR = 1.04, 95% CI: 1.01-1.07) were associated with increased risk for death. CONCLUSIONS: Rural and small-town patients with surgically treated NSCLC had worse survival outcomes compared to urban and metropolitan patients.

Defining synonymous codon compression schemes by genome recoding.

Synthetic recoding of genomes, to remove targeted sense codons, may facilitate the encoded cellular synthesis of unnatural polymers by orthogonal translation systems. However, our limited understanding of allowed synonymous codon substitutions, and the absence of methods that enable the stepwise replacement of the Escherichia coli genome with long synthetic DNA and provide feedback on allowed and disallowed design features in synthetic genomes, have restricted progress towards this goal. Here we endow E. coli with a system for efficient, programmable replacement of genomic DNA with long (>100-kb) synthetic DNA, through the in vivo excision of double-stranded DNA from an episomal replicon by CRISPR/Cas9, coupled to lambda-red-mediated recombination and simultaneous positive and negative selection. We iterate the approach, providing a basis for stepwise whole-genome replacement. We attempt systematic recoding in an essential operon using eight synonymous recoding schemes. Each scheme systematically replaces target codons with defined synonyms and is compatible with codon reassignment. Our results define allowed and disallowed synonymous recoding schemes, and enable the identification and repair of recoding at idiosyncratic positions in the genome.

Outcomes after extracorporeal membrane oxygenation support in COVID-19 and non-COVID-19 patients.

BACKGROUND: Veno-venous extracorporeal membrane oxygenation (V-V ECMO) support is increasingly used in the management of COVID-19-related acute respiratory distress syndrome (ARDS). However, the clinical decision-making to initiate V-V ECMO for severe COVID-19 still remains unclear. In order to determine the optimal timing and patient selection, we investigated the outcomes of both COVID-19 and non-COVID-19 patients undergoing V-V ECMO support. METHODS: Overall, 138 patients were included in this study. Patients were stratified into two cohorts: those with COVID-19 and non-COVID-19 ARDS. RESULTS: The survival in patients with COVID-19 was statistically similar to non-COVID-19 patients (p = .16). However, the COVID-19 group demonstrated higher rates of bleeding (p = .03) and thrombotic complications (p < .001). The duration of V-V ECMO support was longer in COVID-19 patients compared to non-COVID-19 patients (29.0 ± 27.5 vs 15.9 ± 19.6 days, p < .01). Most notably, in contrast to the non-COVID-19 group, we found that COVID-19 patients who had been on a ventilator for longer than 7 days prior to ECMO had 100% mortality without a lung transplant. CONCLUSIONS: These findings suggest that COVID-19-associated ARDS was not associated with a higher post-ECMO mortality than non-COVID-19-associated ARDS patients, despite longer duration of extracorporeal support. Early initiation of V-V ECMO is important for improved ECMO outcomes in COVID-19 ARDS patients. Since late initiation of ECMO was associated with extremely high mortality related to lack of pulmonary recovery, it should be used judiciously or as a bridge to lung transplantation.

The Characterization of Postoperative Mechanical Respiratory Requirement in Neonates and Infants Undergoing Cardiac Surgery on Cardiopulmonary Bypass in a Single Tertiary Institution.

OBJECTIVES: Although neonates and infants undergoing cardiac surgery on cardiopulmonary bypass (CPB) are at high risk of developing perioperative morbidity and mortality, including lung injury, the intraoperative profile of lung injury in this cohort is not well-described. Given that the postoperative course of patients in the pediatric cardiac surgical arena has become increasingly expedited, the objective of this study was to characterize the profiles of postoperative mechanical ventilatory support in neonates and infants undergoing cardiac surgery on CPB and to examine the characteristics of lung mechanics and lung injury in this patient population who are potentially amendable to early postoperative recovery in a single tertiary pediatric institution. DESIGN: A retrospective data analysis of neonates and infants who underwent cardiac surgery on cardiopulmonary bypass. SETTING: A single-center, university teaching hospital. PARTICIPANTS: The study included 328 neonates and infants who underwent cardiac surgery on cardiopulmonary bypass. INTERVENTIONS: A subset of 128 patients were studied: 58 patients undergoing ventricular septal defect (VSD) repair, 36 patients undergoing complete atrioventricular canal (CAVC) repair, and 34 patients undergoing bidirectional Glenn (BDG) shunt surgery. MEASUREMENTS AND MAIN RESULTS: Of the entire cohort, 3.7% experienced in-hospital mortality. Among all surgical procedures, VSD repair (17.7%) was the most common, followed by CAVC repair (11.0%) and BDG shunt surgery (10.4%). Of patients who underwent VSD repair, CAVC repair, and BDG shunt surgery, 65.5%, 41.7%, and 67.6% were off mechanical ventilatory support within 24 hours postoperatively, respectively. In all three of the surgical repairs, lung compliance decreased after CPB compared to pre-CPB phase. Sixty point three percent of patients with VSD repair and 77.8% of patients with CAVC repair showed a PaO2/FIO2 (P/F) ratio of <300 after CPB. Post- CPB P/F ratios of 120 for VSD patients and 100 for CAVC patients were considered as optimal cutoff values to highly predict prolonged (>24 hours) postoperative mechanical ventilatory support. A higher volume of transfused platelets also was associated with postoperative ventilatory support ≥24 hours in patients undergoing VSD repair, CAVC repair, and BDG shunt surgery. CONCLUSIONS: There was a high incidence of lung injury after CPB in neonates and infants, even in surgeries amendable for early recovery. Given that CPB-related factors (CPB duration, crossclamp time) and volume of transfused platelet were significantly associated with prolonged postoperative ventilatory support, the underlying cause of cardiac surgery-related lung injury can be multi-factorial.