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Aims: This study investigates the activity of the broad-spectrum bacteriocin nisin against a large panel of Gram-negative bacterial isolates, including relevant plant, animal, and human pathogens. The aim is to generate supportive evidence towards the use/inclusion of bacteriocin-based therapeutics and open avenues for their continued development. Methods and Results: Nisin inhibitory activity was screened against a panel of 575 strains of Gram-negative bacteria, encompassing 17 genera. Nisin inhibition was observed in 309 out of 575 strains, challenging the prevailing belief that nisin lacks effectiveness against Gram-negative bacteria. The genera Acinetobacter, Helicobacter, Erwinia, and Xanthomonas exhibited particularly high nisin sensitivity. Conclusions: The findings of this study highlight the promising potential of nisin as a therapeutic agent for several key Gram-negative plant, animal, and human pathogens. These results challenge the prevailing notion that nisin is less effective or ineffective against Gram-negative pathogens when compared to Gram-positive pathogens and support future pursuits of nisin as a complementary therapy to existing antibiotics. Significance and Impact of Study: This research supports further exploration of nisin as a promising therapeutic agent for numerous human, animal, and plant health applications, offering a complementary tool for infection control in the face of multidrug-resistant bacteria.

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Cerebral malaria is the deadliest complication that can arise from Plasmodium infection. CD8 T-cell engagement of brain vasculature is a putative mechanism of neuropathology in cerebral malaria. To define contributions of brain endothelial cell major histocompatibility complex (MHC) class I antigen-presentation to CD8 T cells in establishing cerebral malaria pathology, we developed novel H-2Kb LoxP and H-2Db LoxP mice crossed with Cdh5-Cre mice to achieve targeted deletion of discrete class I molecules, specifically from brain endothelium. This strategy allowed us to avoid off-target effects on iron homeostasis and class I-like molecules, which are known to perturb Plasmodium infection. This is the first endothelial-specific ablation of individual class-I molecules enabling us to interrogate these molecular interactions. In these studies, we interrogated human and mouse transcriptomics data to compare antigen presentation capacity during cerebral malaria. Using the Plasmodium berghei ANKA model of experimental cerebral malaria (ECM), we observed that H-2Kb and H-2Db class I molecules regulate distinct patterns of disease onset, CD8 T-cell infiltration, targeted cell death and regional blood-brain barrier disruption. Strikingly, ablation of either molecule from brain endothelial cells resulted in reduced CD8 T-cell activation, attenuated T-cell interaction with brain vasculature, lessened targeted cell death, preserved blood-brain barrier integrity and prevention of ECM and the death of the animal. We were able to show that these events were brain-specific through the use of parabiosis and created the novel technique of dual small animal MRI to simultaneously scan conjoined parabionts during infection. These data demonstrate that interactions of CD8 T cells with discrete MHC class I molecules on brain endothelium differentially regulate development of ECM neuropathology. Therefore, targeting MHC class I interactions therapeutically may hold potential for treatment of cases of severe malaria.

Asunto(s)

Malaria Cerebral , Ratones , Humanos , Animales , Malaria Cerebral/patología , Malaria Cerebral/prevención & control , Células Endoteliales/patología , Encéfalo/patología , Barrera Hematoencefálica/patología , Linfocitos T CD8-positivos , Endotelio/patología , Ratones Endogámicos C57BL , Modelos Animales de Enfermedad

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INTRODUCTION: Thyroidectomy provides definitive treatment for autoimmune thyroid disease (AITD) often resulting in improved quality of life. Historically, patients with AITD undergoing thyroidectomy have increased rates of postoperative hypoparathyroidism and recurrent laryngeal nerve palsy. We investigated the outcomes of preoperative medications in patients with AITD undergoing thyroidectomy. METHODS: We performed a retrospective analysis of patients who underwent thyroidectomy for AITD at a single institution from 2015 to 2021. Surgical outcomes and perioperative laboratory values were analyzed by type of AITD and type of preoperative medical treatment: none, saturated solution of potassium iodide (SSKI), corticosteroids, or both SSKI and corticosteroids. RESULTS: A total of 123 patients underwent thyroidectomy for AITD and were included in analysis: 50 received no preoperative medications, 40 received SSKI, 20 received corticosteroids, and 13 received both. Seventy-six patients had Graves' disease and 47 had Hashimoto's thyroiditis. There were no significant differences in blood loss, operative time, wound complications, hematoma, or recurrent laryngeal nerve injury for patients treated with preoperative corticosteroids compared to those who were not. Patients who received corticosteroids and patients with Graves' disease more commonly had at least one instance of hypocalcemia postoperatively (P < 0.01, P = 0.01), although only on postoperative day 1 was mean calcium < 8.5 mg/dL. There was no difference in rate of transient or permanent hypoparathyroidism. CONCLUSIONS: Patients who received corticosteroids preoperatively had no increased risk of complications. They did have mildly lower calcium levels in the early postoperative period, although no difference in hypoparathyroidism. Further exploration is warranted to investigate the impact of preoperative corticosteroids on operative difficulty, quality of life, and autoantibody clearance.

Asunto(s)

Enfermedad de Graves , Enfermedad de Hashimoto , Hipoparatiroidismo , Humanos , Tiroidectomía/efectos adversos , Tiroidectomía/métodos , Yoduro de Potasio/uso terapéutico , Estudios Retrospectivos , Calcio , Calidad de Vida , Complicaciones Posoperatorias/epidemiología , Complicaciones Posoperatorias/etiología , Complicaciones Posoperatorias/tratamiento farmacológico , Enfermedad de Graves/cirugía , Enfermedad de Hashimoto/cirugía , Hipoparatiroidismo/etiología , Corticoesteroides/efectos adversos

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BACKGROUND: Although outpatient thyroidectomy has become common, few large-scale studies have examined post-thyroidectomy emergency department use, readmission, and encounters not resulting in readmission, known as "treat-and-release" encounters. We evaluated post-outpatient thyroidectomy emergency department use and readmission and characterized associated factors. METHODS: Using the Healthcare Cost and Utilization Project databases, we identified adult outpatient (same-day or <24-hour discharge) thyroidectomies performed in Florida, Maryland, and New York from 2016 to 2017. We identified the procedures linked with emergency department treat-and-release encounters and readmissions within 30 days postoperatively and the factors associated with post-thyroidectomy emergency department use and readmission. RESULTS: Of the 17,046 patients who underwent outpatient thyroidectomy at 374 facilities, 7.5% had emergency department treat-and-release encounters and 2.3% readmissions. The most common reasons for emergency department treat-and-release encounters (9.9%) and readmissions (22.2%) were hypocalcemia-related diagnoses. Greater odds of treat-and-release were associated with identifying as non-Hispanic Black (adjusted odds ratio: 1.5, 95% confidence interval: 1.3-1.8) or Hispanic race/ethnicity (adjusted odds ratio: 1.4, 95% CI: 1.1-1.6), having Medicaid insurance (adjusted odds ratio: 2.7, 95% CI: 2.3-3.2), and living in non-metropolitan areas (adjusted odds ratio: 1.6, 95% CI: 1.1-2.2). We observed no associations between these factors and the odds of readmission. CONCLUSION: Emergency department use after outpatient thyroidectomy is common. Racial, ethnic, socioeconomic, and geographic disparities are associated with treat-and-release encounters but not readmissions. Standardization of perioperative care pathways, focusing on identifying and addressing specific issues in vulnerable populations, could improve care, reduce disparities, and improve patient experience by avoiding unnecessary emergency department visits after outpatient thyroidectomy.

Asunto(s)

Pacientes Ambulatorios , Tiroidectomía , Adulto , Estados Unidos/epidemiología , Humanos , Tiroidectomía/efectos adversos , Medicaid , Florida/epidemiología , Servicio de Urgencia en Hospital , Readmisión del Paciente , Estudios Retrospectivos

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Chalcones and their derivatives are a privileged scaffold in medicinal chemistry, demonstrating numerous biological activities. These molecules have shown significant potential toward the development of novel cancer therapies. While much is known about modification to the chalcone aryl rings, little is known about conformations of the bridge between the aryl rings. Here we report the synthesis and biological evaluation of a series of molecules with flexible and rigid bridge conformations. Crystal structures of a select group of molecules were determined. Flexibility in the chalcone bridge containing the enone moiety was determined to be important for activity. Screening in three distinct cancer cell lines showed significant differences in the activity between the flexible and rigid conformations. Crystal structures suggest an increase in bond rotation and weakened π-bonding in the flexible chalcone bridge, which may contribute to the stronger anti-proliferative activity.

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Antineoplásicos , Chalcona , Chalconas , Chalcona/farmacología , Chalcona/química , Chalconas/química , Relación Estructura-Actividad , Antineoplásicos/química

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OBJECTIVE: To report the short-term and long-term outcomes of dogs that underwent the modified closed and traditional closed anal sacculectomy procedures for the treatment of anal sac neoplasia. ANIMALS: 90 client-owned dogs. Methods: The medical records of 2 tertiary referral hospitals were reviewed to identify dogs that underwent anal sacculectomy for treatment of anal sac neoplasia between January 2016 and December 2020. Data collected included signalment and preoperative diagnostic findings. The occurrence of intraoperative and postoperative complications, short-term outcomes, and long-term outcomes were also collected. Descriptive statistics were calculated to summarize dog signalment information, and recurrence, metastasis, and survival proportions were compared between techniques using Fisher exact tests. RESULTS: 35 and 55 dogs, respectively, underwent the modified or traditional closed anal sacculectomy procedure. Minor postoperative complications that resolved with minimal intervention occurred in 5 of 35 (14.3%) modified approach dogs and 12 of 55 (21.8%) traditional approach dogs. Tumor recurrence was confirmed in 8 of 35 (22.9%) modified and 8 of 55 (26.4%) traditional approach dogs and was suspected in 3 of 35 (8.6%) and 6 of 55 (13.2%; P = .68), respectively. Confirmed metastatic disease was identified in 8 of 35 (22.9%) and 14 of 53 (26.4%) modified and traditional approach dogs, respectively, and was suspected in 4 of 35 (11.4%) and 7 of 53 (13.2%). Sixty-three (70%) dogs survived to study conclusion. CLINICAL RELEVANCE: No benefits in complication rate or local recurrence were identified in dogs following the modified approach as opposed to the traditional closed anal sacculectomy technique.

Asunto(s)

Sacos Anales , Neoplasias del Ano , Enfermedades de los Perros , Humanos , Perros , Animales , Sacos Anales/cirugía , Sacos Anales/patología , Recurrencia Local de Neoplasia/veterinaria , Neoplasias del Ano/patología , Neoplasias del Ano/veterinaria , Registros Médicos , Complicaciones Posoperatorias/veterinaria , Enfermedades de los Perros/cirugía , Enfermedades de los Perros/patología , Estudios Retrospectivos

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Respiratory syncytial virus (RSV) remains a leading cause of hospitalizations and death for young children and adults over 65. The worldwide impact of RSV has prioritized the search for an RSV vaccine, with most targeting the critical fusion (F) protein. However, questions remain about the mechanism of RSV entry and RSV F triggering and fusion promotion. This review highlights these questions, specifically those surrounding a cleaved 27 amino acids long peptide within F, p27.

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CD1 is an antigen presenting glycoprotein homologous to MHC I; however, CD1 proteins present lipid rather than peptide antigen. CD1 proteins are well established to present lipid antigens of Mycobacterium tuberculosis (Mtb) to T cells, but understanding the role of CD1-restricted immunity in vivo in response to Mtb infection has been limited by availability of animal models naturally expressing the CD1 proteins implicated in human response: CD1a, CD1b and CD1c. Guinea pigs, in contrast to other rodent models, express four CD1b orthologs, and here we utilize the guinea pig to establish the kinetics of gene and protein expression of CD1b orthologs, as well as the Mtb lipid-antigen and CD1b-restricted immune response at the tissue level over the course of Mtb infection. Our results indicate transient upregulation of CD1b expression during the effector phase of adaptive immunity that wanes with disease chronicity. Gene expression indicates that upregulation of CD1b is the result of transcriptional induction across all CD1b orthologs. We show high CD1b3 expression on B cells, and identify CD1b3 as the predominant CD1b ortholog in pulmonary granuloma lesions. We identify ex vivo cytotoxic activity directed against CD1b that closely paralleled the kinetic changes in CD1b expression in Mtb infected lung and spleen. This study confirms that CD1b expression is modulated by Mtb infection in lung and spleen, leading to pulmonary and extrapulmonary CD1b-restricted immunity as a component of the antigen-specific response to Mtb infection.

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OBJECTIVES: To evaluate suturing skills of veterinary students using 3 common performance assessments (PAs) and to compare findings to data obtained by an electromyographic armband. SAMPLE: 16 second-year veterinary students. PROCEDURES: Students performed 4 suturing tasks on synthetic tissue models 1, 3, and 5 weeks after a surgical skills course. Digital videos were scored by 4 expert surgeons using 3 PAs (an Objective Structured Clinical Examination [OSCE]- style surgical binary checklist, an Objective Structured Assessment of Technical Skill [OSATS] checklist, and a surgical Global Rating Scale [GRS]). Surface electromyography (sEMG) data collected from the dominant forearm were input to machine learning algorithms. Performance assessment scores were compared between experts and correlated to task completion times and sEMG data. Inter-rater reliability was calculated using the intraclass correlation coefficient (ICC). Inter-rater agreement was calculated using percent agreement with varying levels of tolerance. RESULTS: Reliability was moderate for the OSCE and OSATS checklists and poor for the GRS. Agreement was achieved for the checklists when moderate tolerance was applied but remained poor for the GRS. sEMG signals did not correlate well with checklist scores or task times, but features extracted from signals permitted task differentiation by routine statistical comparison and correct task classification using machine learning algorithms. CLINICAL RELEVANCE: Reliability and agreement of an OSCE-style checklist, OSATS checklist, and surgical GRS assessment were insufficient to characterize suturing skills of veterinary students. To avoid subjectivity associated with PA by raters, further study of kinematics and EMG data is warranted in the surgical skills evaluation of veterinary students.

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Inteligencia Artificial , Educación en Veterinaria , Animales , Reproducibilidad de los Resultados

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Mycobacterium tuberculosis (M.tb) has infected one-quarter of the world's population and led to the deaths of 1.6 million individuals in 2021 according to estimates from the World Health Organization. The rise in prevalence of multidrug-resistant and extensively drug-resistant M.tb strains coupled with insufficient therapies to treat such strains has motivated the development of more effective treatments and/or delivery modalities. Bedaquiline, a diarylquinoline antimycobacterial agent, effectively targets mycobacterial ATP synthase but may lead to systemic complications upon oral delivery. Targeted delivery of bedaquiline to the lungs represents an alternative strategy to harness the sterilizing benefits of the drug against M.tb while mitigating off-target side effects. Two pulmonary delivery modalities were developed herein, including dry powder inhalation and liquid instillation. Despite bedaquiline's poor water solubility, spray drying was performed in predominantly aqueous conditions (≥80%) to avoid a closed-loop, inert system. Aerosols of spray-dried bedaquiline with L-leucine excipient outperformed spray-dried bedaquiline alone, demonstrating superior fine particle fraction metrics (~89% of the emitted dose below <5 µm), suitable for inhalation therapies. Furthermore, the use of a 2-hydroxypropyl-ß-cyclodextrin excipient allowed a molecular dispersion of bedaquiline in an aqueous solution for liquid instillation. Both delivery modalities were successfully administered to Hartley guinea pigs for pharmacokinetic analysis and were well-tolerated by the animals. Intrapulmonary liquid delivery of bedaquiline led to adequate serum absorption and appropriate peak serum concentrations of the drug. The liquid formulation was superior in systemic uptake compared to the powder formulation. The predominant route via which M.tb bacilli enter the body is aerosol droplets that are deposited onto airway surfaces. For this reason, we believe that further studies should focus on inhalation or intrapulmonary therapies that target the site of entry and primary site of infection for M.tb.

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OBJECTIVE: To evaluate the effects of ala vestibuloplasty on cardiopulmonary and lifestyle-related parameters in brachycephalic (BC) cats. STUDY DESIGN: Prospective cohort. ANIMALS: Client-owned BC cats (n = 19). METHODS: Cats were assessed preoperatively by airway computed tomography (CT), endoscopy, contrast echocardiography, cardiac biomarkers, and structured owner questionnaire. Ala vestibuloplasty was performed bilaterally, and blood values, imaging, and owner questionnaire responses were re-evaluated 8-20 weeks postoperatively. RESULTS: Cats were presented for predominantly respiratory clinical signs attributable to brachycephaly. Preoperatively, all cats had stenotic nares, prolonged normalized pulmonary transit time (nPTT) (mean 5.43 ± 1.10 s), and a hyperattenuating pulmonary pattern. No complications occurred following surgery. Postoperatively, nPTT (mean 3.89 ± 0.74 s, p < .001) and frequencies of sneezing (p = .002), snoring (p = .006), open-mouth breathing (p = .0004), and nasal discharge (p = .019) were decreased. Cats exhibited increased activity (p = .005), less frequent dyspnea during activity (p < .001), longer duration of activity before becoming dyspneic (p = .002), faster recovery from activity (p < .001), and decreased respiratory noise (p < .001). Median questionnaire scores improved from preoperative to postoperative (p < .001). CONCLUSION: Anatomic, echocardiographic, and CT changes were common in this cohort of clinically affected BC cats. Pulmonary blood flow and respiratory function were improved after surgery. CLINICAL SIGNIFICANCE: Stenotic nares are the predominant airway abnormality in BC cats. Ala vestibuloplasty is a safe procedure that improves cardiac and CT abnormalities and respiratory and other clinical signs in BC cats.

Asunto(s)

Enfermedades de los Gatos , Craneosinostosis , Animales , Gatos/cirugía , Estudios Prospectivos , Vestibuloplastia/veterinaria , Craneosinostosis/cirugía , Craneosinostosis/veterinaria , Pulmón , Respiración , Enfermedades de los Gatos/cirugía

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OBJECTIVE: To determine the influence of brachycephaly on respiratory, gastrointestinal, sleep, and activity-related parameters in cats. STUDY DESIGN: Prospective questionnaire-based study. ANIMALS: A total of 194 BC and 1003 non-BC cats. METHODS: Owners completed an online questionnaire regarding respiratory, gastrointestinal, sleep, and activity-related parameters. Response options were scored, and individual scores summed to give a total clinical severity score for each cat. RESULTS: Brachycephalic cats had more frequent snoring (odds ratio [OR] 6.89; 95% confidence interval [CI]: 5.06-9.41), sneezing (OR 6.52; CI: 4.75-8.98), nasal discharge (OR 8.26; 95% CI 5.77-11.85), coughing (OR 1.75; CI: 1.17-2.59), and dyspnea (OR 5.32; CI: 3.42-8.28); shorter activity before becoming dyspneic (OR 2.71; CI: 1.93-3.79), slower recovery from activity (OR 3.17; CI: 2.19-4.57), lower activity levels (OR 2.16; CI: 1.59-2.95), and increased respiratory noise (OR 6.68; CI: 4.71-9.52); and more hypersalivation (OR 2.50; CI: 1.47-4.16), halitosis (OR 1.40; CI: 1.00-1.95), and difficulty chewing (OR 5.19; CI: 3.65-7.38). Median clinical severity scores were higher for BC cats than non-BC cats (p < .0001). CONCLUSIONS: Brachycephalic cats (BC) were at risk for respiratory, gastrointestinal, and activity-related symptoms compared to non-BC cats. CLINICAL RELEVANCE: Some BC cats exhibit clinically relevant symptoms and behaviors as reported by owners. Medical or surgical interventions may improve these symptoms and warrant investigation.

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Enfermedades de los Gatos , Craneosinostosis , Gatos , Animales , Estudios Prospectivos , Craneosinostosis/veterinaria

RESUMEN

A dog underwent lung lobectomy for removal of a mass. Histopathology was consistent with narrow resection of a mast cell tumor. Postoperative pneumothorax was successfully treated using autologous blood pleurodesis. Progression of disease despite adjunctive treatment with several chemotherapetutic agents and radiation therapy resulted in euthanasia approximately 4 months postoperatively.

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Since the first sequencing of the human genome, associated sequencing costs have dramatically lowered, leading to an explosion of genomic data. This valuable data should in theory be of huge benefit to the global community, although unfortunately the benefits of these advances have not been widely distributed. Much of today's clinical-genomic data is siloed and inaccessible in adherence with strict governance and privacy policies, with more than 97% of hospital data going unused, according to one reference. Despite these challenges, there are promising efforts to make clinical-genomic data accessible and useful without compromising security. Specifically, federated data platforms are emerging as key resources to facilitate secure data sharing without having to physically move the data from outside of its organizational or jurisdictional boundaries. In this perspective, we summarize the overarching progress in establishing federated data platforms, and highlight critical considerations on how they should be managed to ensure patient and public trust. These platforms are enabling global collaboration and improving representation of underrepresented groups, since sequencing efforts have not prioritized diverse population representation until recently. Federated data platforms, when combined with advances in no-code technology, can be accessible to the diverse end-users that make up the genomics workforce, and we discuss potential strategies to develop sustainable business models so that the platforms can continue to enable research long term. Although these platforms must be carefully managed to ensure appropriate and ethical use, they are democratizing access and insights to clinical-genomic data that will progress research and enable impactful therapeutic findings.

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Hendra virus (HeV) is a zoonotic enveloped member of the family Paramyoxviridae. To successfully infect a host cell, HeV utilizes two surface glycoproteins: the attachment (G) protein to bind, and the trimeric fusion (F) protein to merge the viral envelope with the membrane of the host cell. The transmembrane (TM) region of HeV F has been shown to have roles in F protein stability and the overall trimeric association of F. Previously, alanine scanning mutagenesis has been performed on the C-terminal end of the protein, revealing the importance of ß-branched residues in this region. Additionally, residues S490 and Y498 have been demonstrated to be important for F protein endocytosis, needed for the proteolytic processing of F required for fusion. To complete the analysis of the HeV F TM, we performed alanine scanning mutagenesis to explore the residues in the N-terminus of this region (residues 487-506). In addition to confirming the critical roles for S490 and Y498, we demonstrate that mutations at residues M491 and L492 alter F protein function, suggesting a role for these residues in the fusion process.

Asunto(s)

Virus Hendra/genética , Infecciones por Henipavirus/virología , Fusión de Membrana , Proteínas Virales de Fusión/metabolismo , Alanina/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Membrana Celular/metabolismo , Chlorocebus aethiops , Endocitosis , Endosomas/metabolismo , Genes Reporteros , Virus Hendra/fisiología , Humanos , Mutagénesis Sitio-Dirigida , Dominios Proteicos , Estabilidad Proteica , Células Vero , Proteínas Virales de Fusión/genética

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OBJECTIVE: To describe the presentation, work up, and treatment of a giant parathyroid adenoma presenting as hypercalcemic crisis that ultimately weighed 57 g and extended into the mediastinum, requiring hand-assisted thoracoscopic resection. METHODS: The patient is a 68-year-old man with a prior history of parathyroidectomy, who initially presented with a severe hypercalcemia of 16.3 mg/dL and a parathyroid hormone (PTH) level of 2692 pg/mL on routine labs. RESULTS: Diagnostic and staging work up revealed a 7.2-cm mass extending from just superior to the sternal notch into the right posterior mediastinum to the carina, causing esophageal displacement. No evidence of local invasion or distant metastasis was observed on further imaging, and cytology demonstrated hypercellular parathyroid tissue. The PTH level of the aspirate was >5000 pg/mL. The patient subsequently underwent a right hand-assisted video-assisted thoracoscopic resection of the intrathoracic mass. Final pathology identified a 7.0-cm, 57-g parathyroid adenoma, without any pathologic findings suspicious for malignancy. However, the endocrine surgery team plans for annual laboratory assessment to ensure no recurrence. CONCLUSION: Primary hyperparathyroidism is most commonly caused by a single adenoma. However, in the setting of severe hypercalcemia and elevated PTH, one must have a high suspicion for malignancy, and care should be taken to remove the mass en bloc. For extremely large adenomas extending into the mediastinum, a minimally invasive, hand-assisted, thoracoscopic approach is a safe and effective method of resection.

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The trimeric severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (S) is the sole viral protein responsible for both viral binding to a host cell and the membrane fusion event needed for cell entry. In addition to facilitating fusion needed for viral entry, S can also drive cell-cell fusion, a pathogenic effect observed in the lungs of SARS-CoV-2-infected patients. While several studies have investigated S requirements involved in viral particle entry, examination of S stability and factors involved in S cell-cell fusion remain limited. A furin cleavage site at the border between the S1 and S2 subunits (S1/S2) has been identified, along with putative cathepsin L and transmembrane serine protease 2 cleavage sites within S2. We demonstrate that S must be processed at the S1/S2 border in order to mediate cell-cell fusion and that mutations at potential cleavage sites within the S2 subunit alter S processing at the S1/S2 border, thus preventing cell-cell fusion. We also identify residues within the internal fusion peptide and the cytoplasmic tail that modulate S-mediated cell-cell fusion. In addition, we examined S stability and protein cleavage kinetics in a variety of mammalian cell lines, including a bat cell line related to the likely reservoir species for SARS-CoV-2, and provide evidence that proteolytic processing alters the stability of the S trimer. This work therefore offers insight into S stability, proteolytic processing, and factors that mediate S cell-cell fusion, all of which help give a more comprehensive understanding of this high-profile therapeutic target.

Asunto(s)

COVID-19/virología , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismo , Animales , Fusión Celular , Línea Celular , Chlorocebus aethiops , Humanos , Procesamiento Proteico-Postraduccional , Estabilidad Proteica , SARS-CoV-2/química , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/genética , Acoplamiento Viral , Internalización del Virus

RESUMEN

The SARS-CoV-2 spike protein (S) is the sole viral protein responsible for both viral binding to a host cell and the membrane fusion event needed for cell entry. In addition to facilitating fusion needed for viral entry, S can also drive cell-cell fusion, a pathogenic effect observed in the lungs of SARS-CoV-2 infected patients. While several studies have investigated S requirements involved in viral particle entry, examination of S stability and factors involved in S cell-cell fusion remain limited. We demonstrate that S must be processed at the S1/S2 border in order to mediate cell-cell fusion, and that mutations at potential cleavage sites within the S2 subunit alter S processing at the S1/S2 border, thus preventing cell-cell fusion. We also identify residues within the internal fusion peptide and the cytoplasmic tail that modulate S cell-cell fusion. Additionally, we examine S stability and protein cleavage kinetics in a variety of mammalian cell lines, including a bat cell line related to the likely reservoir species for SARS-CoV-2, and provide evidence that proteolytic processing alters the stability of the S trimer. This work therefore offers insight into S stability, proteolytic processing, and factors that mediate S cell-cell fusion, all of which help give a more comprehensive understanding of this highly sought-after therapeutic target.

RESUMEN

Chordomas are rare spinal tumors addicted to expression of the developmental transcription factor brachyury. In chordomas, brachyury is super-enhancer associated and preferentially downregulated by pharmacologic transcriptional CDK inhibition, leading to cell death. To understand the underlying basis of this sensitivity, we dissect the brachyury transcription regulatory network and compare the consequences of brachyury degradation with transcriptional CDK inhibition. Brachyury defines the chordoma super-enhancer landscape and autoregulates through binding its super-enhancer, and its locus forms a transcriptional condensate. Transcriptional CDK inhibition and brachyury degradation disrupt brachyury autoregulation, leading to loss of its transcriptional condensate and transcriptional program. Compared with transcriptional CDK inhibition, which globally downregulates transcription, leading to cell death, brachyury degradation is much more selective, inducing senescence and sensitizing cells to anti-apoptotic inhibition. These data suggest that brachyury downregulation is a core tenet of transcriptional CDK inhibition and motivates developing strategies to target brachyury and its autoregulatory feedback loop.

Asunto(s)

Biomarcadores de Tumor/genética , Cordoma/genética , Quinasas Ciclina-Dependientes/genética , Proteínas Fetales/genética , Proteínas de Neoplasias/genética , Neoplasias de la Columna Vertebral/genética , Proteínas de Dominio T Box/genética , Secuencia de Bases , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Cordoma/metabolismo , Cordoma/patología , Quinasas Ciclina-Dependientes/metabolismo , Proteínas Fetales/metabolismo , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Células HEK293 , Histonas/genética , Histonas/metabolismo , Humanos , Queratina-18/genética , Queratina-18/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Proteínas de Neoplasias/metabolismo , Proteolisis , Transducción de Señal , Neoplasias de la Columna Vertebral/metabolismo , Neoplasias de la Columna Vertebral/patología , Proteínas de Dominio T Box/metabolismo

RESUMEN

The SARS-CoV-2 spike protein (S) is the sole viral protein responsible for both viral binding to a host cell and the membrane fusion event needed for cell entry. In addition to facilitating fusion needed for viral entry, S can also drive cell-cell fusion, a pathogenic effect observed in the lungs of SARS-CoV-2 infected patients. While several studies have investigated S requirements involved in viral particle entry, examination of S stability and factors involved in S cell-cell fusion remain limited. We demonstrate that S must be processed at the S1/S2 border in order to mediate cell-cell fusion, and that mutations at potential cleavage sites within the S2 subunit alter S processing at the S1/S2 border, thus preventing cell-cell fusion. We also identify residues within the internal fusion peptide and the cytoplasmic tail that modulate S cell-cell fusion. Additionally, we examine S stability and protein cleavage kinetics in a variety of mammalian cell lines, including a bat cell line related to the likely reservoir species for SARS-CoV-2, and provide evidence that proteolytic processing alters the stability of the S trimer. This work therefore offers insight into S stability, proteolytic processing, and factors that mediate S cell-cell fusion, all of which help give a more comprehensive understanding of this highly sought-after therapeutic target.