Anti-CD99 Antibody Therapy Triggers Macrophage-Dependent Ewing Cell Death In Vitro and Myeloid Cell Recruitment In Vivo.

BACKGROUND: Ewing sarcoma is a rare tumor of the bone or soft tissues characterized by diffuse membranous staining for CD99. As this tumor remains incurable in the metastatic, relapsed, and refractory settings, we explored the downstream immune implications of targeting CD99. METHODS: We discovered a human anti-CD99 antibody (NOA2) by phagemid panning and investigated NOA2 immune cell-mediated cytotoxicity in vitro and in vivo focusing on the myeloid cell compartment, given that M2 macrophages are present in human tumors and associated with a poor prognosis. RESULTS: NOA2 is capable of inducing immune effector cell-mediated Ewing death in vitro via engagement of macrophages. Mice with metastatic Ewing tumors, treated with NOA2, experience tumor growth arrest and an associated increase in intratumoral macrophages. Further, incubation of macrophages and Ewing cells with NOA2, in conjunction with anti-PILRα antibody blockade in vitro, results in the reactivation of previously dormant macrophages possibly due to interrupted binding of Ewing CD99 to macrophage PILRα. CONCLUSIONS: These studies are the first to demonstrate the role of human immune effector cells in anti-CD99-mediated Ewing tumor death. We propose that the engagement of CD99 by NOA2 results in the recruitment of intratumoral macrophages. In addition, interruption of the CD99:PILRα checkpoint axis may be a relevant therapeutic approach to activate tumor-associated macrophages.

MFSD7c functions as a transporter of choline at the blood-brain barrier.

Mutations in the orphan transporter MFSD7c (also known as Flvcr2), are linked to Fowler syndrome. Here, we used Mfsd7c knockout (Mfsd7c-/-) mice and cell-based assays to reveal that MFSD7c is a choline transporter at the blood-brain barrier (BBB). We performed comprehensive metabolomics analysis and detected differential changes of metabolites in the brains and livers of Mfsd7c-/-embryos. Particularly, we found that choline-related metabolites were altered in the brains but not in the livers of Mfsd7c-/- embryos. Thus, we hypothesized that MFSD7c regulates the level of choline in the brain. Indeed, expression of human MFSD7c in cells significantly increased choline uptake. Interestingly, we showed that choline uptake by MFSD7c is greatly increased by choline-metabolizing enzymes, leading us to demonstrate that MFSD7c is a facilitative transporter of choline. Furthermore, single-cell patch clamp analysis showed that the import of choline by MFSD7c is electrogenic. Choline transport function of MFSD7c was shown to be conserved in vertebrates, but not in yeasts. We demonstrated that human MFSD7c is a functional ortholog of HNM1, the yeast choline importer. We also showed that several missense mutations identified in patients exhibiting Fowler syndrome had abolished or reduced choline transport activity. Mice lacking Mfsd7c in endothelial cells of the central nervous system suppressed the import of exogenous choline from blood but unexpectedly had increased choline levels in the brain. Stable-isotope tracing study revealed that MFSD7c was required for exporting choline derived from lysophosphatidylcholine in the brain. Collectively, our work identifies MFSD7c as a choline exporter at the BBB and provides a foundation for future work to reveal the disease mechanisms of Fowler syndrome.

Comparison of success and cost after retrieval of two inferior vena cava filters.

OBJECTIVE: The objective of this study was to help guide inferior vena cava (IVC) filter choices by better understanding the retrieval characteristics, complications, and total costs between two commonly used IVC filters. METHODS: All patients who underwent retrieval or attempted retrieval of Denali (Bard Peripheral Vascular) or Option (Argon Medical Devices) IVC filters were identified between March 2016 and October 2021 at a single tertiary care center. Those with imaging studies that permitted evaluation of filter placement, presence or degree of tilt, and/or hooking of the filter into the IVC wall were included in the present study. Filter retrieval success, number of attempts, use of advanced techniques, and fluoroscopy and procedural times were recorded and compared between the two filters. RESULTS: A total of 87 patients presented for retrieval of 52 Denali and 35 Option Elite filters during the study period. Denali filters were more likely to be successfully retrieved at the first attempt (94% vs 77%; P = .019). The procedural and fluoroscopy times were shorter for Denali filters (29 minutes vs 63 minutes [P < .001] and 7 minutes vs 25 minutes [P < .001], respectively). Denali filters were less likely to be significantly tilted (≥15○) at retrieval (12% vs 29%; P < .001) or to have the filter hook embedded in the IVC wall (6% vs 40%; P < .001). Tilting of the filter of ≥15○ had no significant effects on the retrieval success rate (no tilt or tilt <15○ vs tilt of ≥15○: 98% vs 100%; P = .58). In contrast, filter hook penetration into the IVC wall significantly reduced successful recovery (41% vs 99%; P < .001). CONCLUSIONS: The findings from this study suggest that although the filter designs are similar, a benefit exists in the ease of retrievability of the Denali over the Option filter. We found that tilting and hooking of the filter in the IVC wall occurred significantly more with the Option filter. These factors likely made retrieval more difficult and contributed to the longer procedure and fluoroscopy times.

Establishing chromosomal design-build-test-learn through a synthetic chromosome and its combinatorial reconfiguration.

Chromosome-level design-build-test-learn cycles (chrDBTLs) allow systematic combinatorial reconfiguration of chromosomes with ease. Here, we established chrDBTL with a redesigned synthetic Saccharomyces cerevisiae chromosome XV, synXV. We designed and built synXV to harbor strategically inserted features, modified elements, and synonymously recoded genes throughout the chromosome. Based on the recoded chromosome, we developed a method to enable chrDBTL: CRISPR-Cas9-mediated mitotic recombination with endoreduplication (CRIMiRE). CRIMiRE allowed the creation of customized wild-type/synthetic combinations, accelerating genotype-phenotype mapping and synthetic chromosome redesign. We also leveraged synXV as a "build-to-learn" model organism for translation studies by ribosome profiling. We conducted a locus-to-locus comparison of ribosome occupancy between synXV and the wild-type chromosome, providing insight into the effects of codon changes and redesigned features on translation dynamics in vivo. Overall, we established synXV as a versatile reconfigurable system that advances chrDBTL for understanding biological mechanisms and engineering strains.

A spotlight on global collaboration in the Sc2.0 yeast consortium.

The Synthetic Yeast Genome Project (Sc2.0) is an international collaboration that aims to create and optimize synthetic versions of each Saccharomyces cerevisiae chromosome, with the ultimate goal of assembling a yeast organism with a synthetic with design features facilitating applications in synthetic biology and engineering projects. The consortium research groups are global, and, here, we highlight the work of the China-based Sc2.0 researchers and their thoughts on the future of Sc2.0 and synthetic biology.

Tea Tree Essential Oil Kills Escherichia coli and Staphylococcus epidermidis Persisters.

Persister cells are a small subpopulation of non-growing bacteria within a population that can survive long exposures to antibiotic treatment. Following antibiotic removal, persister cells can regrow and populate, playing a key role in the chronic reoccurrence of bacterial infections. The development of new molecules and methods to kill bacterial persisters is critical. Essential oils and other natural products have long been studied for their antimicrobial effects. Here, we studied the effectiveness of tea tree essential oil (TTO), a common component in many commercial care products, against Escherichia coli and Staphylococcus epidermidis persister cells. Using biphasic kill curve assays, we found that concentrations of 0.5% and 1.0% TTO for E. coli and S. epidermidis, respectively, completely eradicated persister cells over a period of 24 h, with the component terpinen-4-ol responsible for most of the killing. Using a colorimetric assay, it was determined that the TTO exhibited its anti-persister effects through a membrane disruption mechanism.

The variable conversion of neutralizing anti-SARS-CoV-2 single-chain antibodies to IgG provides insight into RBD epitope accessibility.

Monoclonal antibody (mAb) therapies have rapidly become a powerful class of therapeutics with applications covering a diverse range of clinical indications. Though most widely used for the treatment of cancer, mAbs are also playing an increasing role in the defense of viral infections, most recently with palivizumab for prevention and treatment of severe RSV infections in neonatal and pediatric populations. In addition, during the COVID-19 pandemic, mAbs provided a bridge to the rollout of vaccines; however, their continued role as a therapeutic option for those at greatest risk of severe disease has become limited due to the emergence of neutralization resistant Omicron variants. Although there are many techniques for the identification of mAbs, including single B cell cloning and immunization of genetically engineered mice, the low cost, rapid throughput and technological simplicity of antibody phage display has led to its widespread adoption in mAb discovery efforts. Here we used our 27-billion-member naïve single-chain antibody (scFv) phage library to identify a panel of neutralizing anti-SARS-CoV-2 scFvs targeting diverse epitopes on the receptor binding domain (RBD). Although typically a routine process, we found that upon conversion to IgG, a number of our most potent clones failed to maintain their neutralization potency. Kinetic measurements confirmed similar affinity to the RBD; however, mechanistic studies provide evidence that the loss of neutralization is a result of structural limitations likely arising from initial choice of panning antigen. Thus this work highlights a risk of scFv-phage panning to mAb conversion and the importance of initial antigen selection.

Evidence-based opioid prescribing guidelines after lung resection: a prospective, multicenter analysis.

Background: Opioid prescribing guidelines have significantly decreased overprescribing and post-discharge use after cardiac surgery; however, limited recommendations exist for general thoracic surgery patients, a similarly high-risk population. We examined opioid prescribing and patient-reported use to develop evidence-based, opioid prescribing guidelines after lung cancer resection. Methods: This prospective, statewide, quality improvement study was conducted between January 2020 to March 2021 and included patients undergoing surgical resection of a primary lung cancer across 11 institutions. Patient-reported outcomes at 1-month follow-up were linked with clinical data and Society of Thoracic Surgery (STS) database records to characterize prescribing patterns and post-discharge use. The primary outcome was quantity of opioid used after discharge; secondary outcomes included quantity of opioid prescribed at discharge and patient-reported pain scores. Opioid quantities are reported in number of 5-mg oxycodone tablets (mean ± standard deviation). Results: Of the 602 patients identified, 429 met inclusion criteria. Questionnaire response rate was 65.0%. At discharge, 83.4% of patients were provided a prescription for opioids of mean size 20.5±13.1 pills, while patients reported using 8.2±13.0 pills after discharge (P<0.001), including 43.7% who used none. Those not taking opioids on the calendar day prior to discharge (32.4%) used fewer pills (4.4±8.1 vs. 11.7±14.9, P<0.001). Refill rate was 21.5% for patients provided a prescription at discharge, while 12.5% of patients not prescribed opioids at discharge required a new prescription before follow-up. Pain scores were 2.4±2.5 for incision site and 3.0±2.8 for overall pain (scale 0-10). Conclusions: Patient-reported post-discharge opioid use, surgical approach, and in-hospital opioid use before discharge should be used to inform prescribing recommendations after lung resection.

Monolithically integrated 112 Gbps PAM4 optical transmitter and receiver in a 45†nm CMOS-silicon photonics process.

We demonstrate a transmitter and receiver in a silicon photonics platform for O-band optical communication that monolithically incorporates a modulator driver, traveling-wave Mach-Zehnder modulator, control circuitry, photodetector, and transimpedance amplifier (TIA) in the GlobalFoundries Fotonix (45SPCLO) platform. The transmitter and receiver show an open 112 Gbps PAM4 eye at a 4.3 pJ/bit energy efficiency, not including the laser. Extensive use of gain-peaking enables our modulator driver and TIA to achieve the high bandwidths needed in the 45 nm CMOS-silicon photonics process. Our results suggest an alternative to the frequent approach of bump-bonding BiCMOS drivers and TIAs to silicon photonics.

An allosteric pan-TEAD inhibitor blocks oncogenic YAP/TAZ signaling and overcomes KRAS G12C inhibitor resistance.

The Hippo pathway is a key growth control pathway that is conserved across species. The downstream effectors of the Hippo pathway, YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif), are frequently activated in cancers to drive proliferation and survival. Based on the premise that sustained interactions between YAP/TAZ and TEADs (transcriptional enhanced associate domain) are central to their transcriptional activities, we discovered a potent small-molecule inhibitor (SMI), GNE-7883, that allosterically blocks the interactions between YAP/TAZ and all human TEAD paralogs through binding to the TEAD lipid pocket. GNE-7883 effectively reduces chromatin accessibility specifically at TEAD motifs, suppresses cell proliferation in a variety of cell line models and achieves strong antitumor efficacy in vivo. Furthermore, we uncovered that GNE-7883 effectively overcomes both intrinsic and acquired resistance to KRAS (Kirsten rat sarcoma viral oncogene homolog) G12C inhibitors in diverse preclinical models through the inhibition of YAP/TAZ activation. Taken together, this work demonstrates the activities of TEAD SMIs in YAP/TAZ-dependent cancers and highlights their potential broad applications in precision oncology and therapy resistance.

Isolation of full-length IgG antibodies from combinatorial libraries expressed in the cytoplasm of Escherichia coli.

Here we describe a facile and robust genetic selection for isolating full-length IgG antibodies from combinatorial libraries expressed in the cytoplasm of redox-engineered Escherichia coli cells. The method is based on the transport of a bifunctional substrate comprised of an antigen fused to chloramphenicol acetyltransferase, which allows positive selection of bacterial cells co-expressing cytoplasmic IgGs called cyclonals that specifically capture the chimeric antigen and sequester the antibiotic resistance marker in the cytoplasm. The utility of this approach is first demonstrated by isolating affinity-matured cyclonal variants that specifically bind their cognate antigen, the leucine zipper domain of a yeast transcriptional activator, with subnanomolar affinities, which represent a ~20-fold improvement over the parental IgG. We then use the genetic assay to discover antigen-specific cyclonals from a naïve human antibody repertoire, leading to the identification of lead IgG candidates with affinity and specificity for an influenza hemagglutinin-derived peptide antigen.

Allele-Specific Gene Regulation, Phenotypes, and Therapeutic Vulnerabilities in Estrogen Receptor Alpha-Mutant Endometrial Cancer.

Activating estrogen receptor alpha (ER; also known as ESR1) mutations are present in primary endometrial and metastatic breast cancers, promoting estrogen-independent activation of the receptor. Functional characterizations in breast cancer have established unique molecular and phenotypic consequences of the receptor, yet the impact of ER mutations in endometrial cancer has not been fully explored. In this study, we used CRISPR-Cas9 to model the clinically prevalent ER-Y537S mutation and compared results with ER-D538G to discover allele-specific differences between ER mutations in endometrial cancer. We found that constitutive activity of mutant ER resulted in changes in the expression of thousands of genes, stemming from combined alterations to ER binding and chromatin accessibility. The unique gene expression programs resulted in ER-mutant cells developing increased cancer-associated phenotypes, including migration, invasion, anchorage-independent growth, and growth in vivo. To uncover potential treatment strategies, we identified ER-associated proteins via Rapid Immunoprecipitation and Mass Spectrometry of Endogenous Proteins and interrogated two candidates, CDK9 and NCOA3. Inhibition of these regulatory proteins resulted in decreased growth and migration, representing potential novel treatment strategies for ER-mutant endometrial cancer. IMPLICATIONS: This study provides insight into mutant ER activity in endometrial cancer and identifies potential therapies for women with ER-mutant endometrial cancer.

Engineering Escherichia coli for diagnosis and management of hyperuricemia.

Uric acid disequilibrium is implicated in chronic hyperuricemia-related diseases. Long-term monitoring and lowering of serum uric acid levels may be crucial for diagnosis and effective management of these conditions. However, current strategies are not sufficient for accurate diagnosis and successful long-term management of hyperuricemia. Moreover, drug-based therapeutics can cause side effects in patients. The intestinal tract plays an important role in maintaining healthy serum acid levels. Hence, we investigated the engineered human commensal Escherichia coli as a novel method for diagnosis and long-term management of hyperuricemia. To monitor changes in uric acid concentration in the intestinal lumen, we developed a bioreporter using the uric acid responsive synthetic promoter, pucpro, and uric acid binding Bacillus subtilis PucR protein. Results demonstrated that the bioreporter module in commensal E. coli can detect changes in uric acid concentration in a dose-dependent manner. To eliminate the excess uric acid, we designed a uric acid degradation module, which overexpresses an E. coli uric acid transporter and a B. subtilis urate oxidase. Strains engineered with this module degraded all the uric acid (250 µM) found in the environment within 24 h, which is significantly lower (p < 0.001) compared to wild type E. coli. Finally, we designed an in vitro model using human intestinal cell line, Caco-2, which provided a versatile tool to study the uric acid transport and degradation in an environment mimicking the human intestinal tract. Results showed that engineered commensal E. coli reduced (p < 0.01) the apical uric acid concentration by 40.35% compared to wild type E. coli. This study shows that reprogramming E. coli holds promise as a valid alternative synthetic biology therapy to monitor and maintain healthy serum uric acid levels.

Nucleation and dissolution mechanism underlying amyotrophic lateral sclerosis/frontotemporal lobar dementia-linked fused in sarcoma condensates.

Fused in sarcoma (FUS) is a nuclear RNA-binding protein. Mutations in FUS lead to the mislocalization of FUS from the nucleus to the cytosol and formation of pathogenic aggregates in neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia (FTLD), yet with unknown molecular mechanisms. Using mutant and stress conditions, we visualized FUS localization and aggregate formation in cells. We used single-molecule pull-down (SiMPull) to quantify the native oligomerization states of wildtype (WT) and mutant FUS in cells. We demonstrate that the NLS mutants exhibited the highest oligomerization (>3) followed by other FUS mutants (>2) and WT FUS which is primarily monomeric. Strikingly, the mutant FUS oligomers are extremely stable and resistant to treatment by high salt, hexanediol, RNase, and Karyopherin-ß2 and only soluble in GdnHCl and SDS. We propose that the increased oligomerization units of mutant FUS and their high stability may contribute to ALS/FTLD pathogenesis.

Optimal medical management in patients undergoing peripheral vascular interventions for chronic limb-threatening ischemia is associated with improved outcomes.

OBJECTIVE: Optimizing medical management and risk factor modification are underused strategies in patients with chronic limb-threatening ischemia (CLTI), despite evidence of improved outcomes. The Vascular Quality Initiative (VQI) registry is a tool to improve quality of vascular care. In this study, we used the VQI to evaluate trends in medical management in patients with CLTI undergoing peripheral vascular interventions (PVI), and the impact of changes in management on overall survival (OS), amputation-free survival (AFS), and limb salvage (LS). METHODS: Patients undergoing index PVI for CLTI between 2012 and 2016, with ≥24 months of follow-up were identified from the national VQI registry. Patient details including smoking status and medication use, OS, LS, and AFS were analyzed with linear-by-linear association, t test, and logistic regression. RESULTS: There were 12,370 PVI completed in 11,466 patients. There was a significant increase in infrapopliteal interventions (from 29.8% to 39.0%; P < .001) and PVI performed for tissue loss (from 59.1% to 66.5%; P < .001). The percentage of current smokers at time of PVI decreased (from 36.2% to 30.7%; P = .036). At discharge, statins were initiated in 25%, aspirin in 45%, and P2Y12 therapy in 58% of patients not receiving these medications before PVI. Over the course of follow-up, dual antiplatelet therapy (DAPT) (from 41.1% to 48.0%; P < .001), angiotensin-converting enzyme (ACE) inhibitor (from 46.2% to 51.3%; P < .001), and statin (from 70.4% to 77.5%; P < .001) use increased. Combined DAPT, ACE inhibitor and statin use increased from 33.6% to 39.6% (P ≤ .001). Significant improvement in 24-month OS and AFS was noted (OS, from 90.9% to 93.7% [P = .002]: AFS, from 81.2% to 83.1% [P = .046]), but not LS (from 89.6% to 89.0%; P = .83). Combined therapy with P2Y12 inhibitors, statins and ACE inhibitors was an independent predictor of improved OS (hazard ratio, 0.61; 95% confidence interval, 0.39-0.96; P = .034). DAPT was independent predictor of improved LS (hazard ratio, 0.83; 95% confidence interval, 0.79-0.87; P < .007). CONCLUSIONS: Antiplatelet, ACE inhibitor, and statin use increased over the study period and was associated with improved OS and AFS. LS trends did not change significantly over time, possibly owing to the inclusion of patients with a greater disease burden or inadequate medical management. Medical management, although improved, remained far from optimal and represents an area for continued development.

Developmental stage of transplanted neural progenitor cells influences anatomical and functional outcomes after spinal cord injury in mice.

Neural progenitor cell (NPC) transplantation is a promising therapeutic strategy for replacing lost neurons following spinal cord injury (SCI). However, how graft cellular composition influences regeneration and synaptogenesis of host axon populations, or recovery of motor and sensory functions after SCI, is poorly understood. We transplanted developmentally-restricted spinal cord NPCs, isolated from E11.5-E13.5 mouse embryos, into sites of adult mouse SCI and analyzed graft axon outgrowth, cellular composition, host axon regeneration, and behavior. Earlier-stage grafts exhibited greater axon outgrowth, enrichment for ventral spinal cord interneurons and Group-Z spinal interneurons, and enhanced host 5-HT+ axon regeneration. Later-stage grafts were enriched for late-born dorsal horn interneuronal subtypes and Group-N spinal interneurons, supported more extensive host CGRP+ axon ingrowth, and exacerbated thermal hypersensitivity. Locomotor function was not affected by any type of NPC graft. These findings showcase the role of spinal cord graft cellular composition in determining anatomical and functional outcomes following SCI.

Synthetic biology: Learning the way toward high-precision biological design.

Since its inception, synthetic biology has overcome many technical barriers but is at a crossroads for high-precision biological design. Devising ways to fully utilize big biological data may be the key to achieving greater heights in synthetic biology.

Engineering of a probiotic yeast for the production and secretion of medium-chain fatty acids antagonistic to an opportunistic pathogen Candida albicans.

Candida albicans is an opportunistic pathogen, with its infection as one of the causes of morbidity or mortality. Notably, the probiotic yeast Saccharomyces cerevisiae var. boulardii has shown the potential to fight against Candida infections. In this study, we aimed to engineer a commercial boulardii strain to produce medium-chain fatty acids (MCFAs) with antagonistic effects against C. albicans. First, we identified and characterized a boulardii strain and created its auxotrophic strain Δura3. Next, we constructed and expressed a heterologous MCFA biosynthetic pathway under the control of inducible and constitutive promoters. Aside from examining MCFA production and secretion, we confirmed MCFAs' effects on C. albicans' anti-biofilm and anti-hyphal formations and the immunomodulatory effect of MCFA-containing supernatants on Caco-2 cells. We found that under constitutive promoters, the engineered boulardii strain constitutively produced and secreted a mixture of C6:0, C8:0, and C10:0. The secreted MCFAs then reduced biofilm and hyphal formations in C. albicans SC5314. We also confirmed that MCFAs upregulated the expression of virulence-related genes in SC5314. Furthermore, we found that the constitutively produced MCFAs in the supernatant induced the upregulation of immune response genes in Caco-2 cells co-cultured with SC5314, indicating MCFAs' roles in immunomodulation. Overall, the engineered boulardii strain produced and secreted MCFAs, as well as demonstrated antagonistic effects against C. albicans SC5314 and immune-modulatory effects in Caco-2. To our knowledge, this represents the first study tackling the metabolic engineering of a commercial probiotic yeast strain to constitutively produce and secrete MCFAs showing anti-Candida effects. Our study forms the basis of the potential development of a live biotherapeutics probiotic yeast against Candida infections through metabolic engineering strategies.