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Normally, von Willebrand factor (VWF) remains inactive unless its A1A2 domains undergo a shear stress-triggered conformational change. We demonstrated the capacity of a recombinant A2 domain of VWF to bind and to affect fibrin formation, altering the fibrin clot structure. The data indicated that VWF contains an additional binding site for fibrin in the A2 domain that plays a role in the incorporation of VWF to the polymerizing fibrin. This study is to examine the hypothesis that active plasma VWF directly influence fibrin polymerization and the structure of fibrin clots. The study used healthy and type 3 von Willebrand disease (VWD) plasma, purified plasma VWF, fibrin polymerization assays, confocal microscopy and scanning electron microscopy. The exposed A2 domain in active VWF harbors additional binding sites for fibrinogen, and significantly potentiates fibrin formation (Pâ<â0.02). Antibody against the A2 domain of VWF significantly decreased the initial rate of change of fibrin formation (Pâ<â0.002). Clot analyses revealed a significant difference in porosity between normal and type 3 VWD plasma (Pâ<â0.008), further supported by scanning electron microscopy, which demonstrated thicker fibrin fibers in the presence of plasma VWF (Pâ<â0.0003). Confocal immunofluorescence microscopy showed punctate VWF staining along fibrin fibrils, providing visual evidence of the integration of plasma VWF into the fibrin matrix. The study with type 3 VWD plasma supports the hypothesis that plasma VWF directly influences fibrin polymerization and clot structure. In addition, a conformational change in the A1A2 domains exposes a hidden fibrin(ogen) binding site, indicating that plasma VWF determines the fibrin clot structure.
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Fibrina , Factor de von Willebrand , Factor de von Willebrand/metabolismo , Humanos , Fibrina/metabolismo , Fibrina/ultraestructura , Enfermedad de von Willebrand Tipo 3/sangre , Sitios de Unión , Microscopía Electrónica de Rastreo/métodosRESUMEN
BACKGROUND: Low back pain (LBP) is a common public health problem resulting in workforce loss. OBJECTIVE: This study aims to evaluate the LBP status and its affecting factors among drivers in a city in southeast Turkey. METHODS: This cross-sectional questionnaire survey study was conducted among 323 drivers. The chi-square test and logistic regression analysis were used to analyze the data. RESULTS: The mean age of the drivers was 41.7±11.5 years (min: 19, max: 70), and 83.9% were married, and all were men. LBP was found in 59.4% of drivers. It was significantly higher in drivers with poor socioeconomic status, dissatisfied with their life, having a chronic illness, physically inactive, having sleep disorders, exposed to bad road conditions, prolonged vibration, high physical- psychological workload, and a family history of LBP (pâ< â0.05). There was no significant association between age, education level, and BMI with LBP (pâ> â0.05). CONCLUSION: There is limited study on this subject in Turkey. Further studies can raise awareness about this issue and create an educational plan.
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Integrin αIIbß3 is the key receptor regulating platelet retraction and accumulation and a proven drug-target for antithrombotic therapies. Here we resolve the cryo-EM structures of the full-length αIIbß3, which covers three distinct states along the activation pathway. Firstly, we obtain the αIIbß3 structure at 3 Å resolution in the inactive state, revealing the overall topology of the heterodimer with the transmembrane (TM) helices and the ligand-binding domain tucked in a specific angle proximity to the TM region. After the addition of a Mn2+ agonist, we resolve two coexisting structures representing two new states between inactive and active state. Our structures show conformational changes of the αIIbß3 activating trajectory and a unique twisting of the integrin legs, which is required for platelets accumulation. Our structure provides direct structural evidence for how the lower legs are involved in full-length integrin activation mechanisms and offers a new strategy to target the αIIbß3 lower leg.
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Microscopía por Crioelectrón , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria , Humanos , Sitios de Unión , Manganeso/metabolismo , Manganeso/química , Modelos Moleculares , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/química , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Integrina beta3/química , Integrina alfa2/químicaRESUMEN
In this issue of Structure, Chataigner et al. reveal that Contactin-2's homotypic interaction, a glycosylation-dependent process, generates a broad conformational landscape. This structural plasticity, driven by conformational equilibria and sugar coating, facilitates adaptation to diverse ligands and environmental conditions, highlighting its dynamic role in neuronal function.
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Contactina 2 , Contactinas , Azúcares , Contactina 2/química , Contactina 2/fisiología , Contactinas/química , Contactinas/fisiología , GlicosilaciónRESUMEN
PURPOSE: The study aimed to determine body image and levels after surgery in cornea transplant patients. MATERIALS AND METHODS: The population of this cross-sectional study was composed of 383 patients presented to the Eye Bank unit of a University Hospital after corneal transplantation. Sample size of 193 patients was calculated with 0.5 power, a margin of error of 5%, representing 95% of the universe. The data were collected through face-to-face interviews with the patients by the researcher and the study was completed with 178 patients in September-November 2022. The data were collected using a Patient Information Form, the Body Image Scale, and the General Self-Efficacy Scale. Parametric tests, Pearson Correlation, Student's t-test, and One-Way Analysis of Variance tests were performed were used in the data analysis. RESULTS: It was determined that the mean Body Image Scale score of the transplant patients participating in the study was 159.41⯱â¯36.99 and the mean Self-Efficacy Scale score was 30.37⯱â¯8.31. When the comparison of the mean scores was examined, the difference between the mean scores of gender, marital status, occupation, and body image scale was statistically significant (pâ¯<â¯.05), while the difference between the self-efficacy mean scores was not statistically significant (pâ¯>â¯.05). There was a positive, moderately strong significant relationship between body image and the self-efficacy of the patients (pâ¯<â¯.01) (râ¯=â¯.57). CONCLUSION: It was found that the patient's body image and self-efficacy levels were high, and self-efficacy increased as the body image increased.
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Imagen Corporal , Trasplante de Córnea , Humanos , Autoeficacia , Estudios Transversales , Hospitales UniversitariosRESUMEN
BACKGROUND: Patients with intermediate and high-risk oropharyngeal cancer (OPC) have poorer response to standard treatment and poorer overall survival compared to low-risk OPC. CompARE is designed to test alternative approaches to intensified treatment for these patients to improve survival. METHODS: CompARE is a pragmatic phase III, open-label, multicenter randomised controlled trial with an adaptive multi-arm, multi-stage design and an integrated QuinteT Recruitment Intervention. Eligible OPC patients include those with human papillomavirus (HPV) negative, T1-T4, N1-N3 or T3-4, N0, or HPV positive N3, T4, or current smokers (or ≥ 10 pack years previous smoking history) with T1-T4, N2b-N3. CompARE was originally designed with four arms (one control [arm 1] and three experimental: arm 2-induction chemotherapy followed by arm 1; arm 3-dose-escalated radiotherapy plus concomitant cisplatin; and arm 4-resection of primary followed by arm 1). The three original experimental arms have been closed to recruitment and a further experimental arm opened (arm 5-induction durvalumab followed by arm 1 and then adjuvant durvalumab). Currently recruiting are arm 1 (control): standard treatment of 3-weekly cisplatin 100 mg/m2 or weekly 40 mg/m2 with intensity-modulated radiotherapy using 70 Gy in 35 fractions ± neck dissection determined by clinical and radiological assessment 3 months post-treatment, and arm 5 (intervention): one cycle of induction durvalumab 1500 mg followed by standard treatment then durvalumab 1500 mg every 4 weeks for a total of 6 months. The definitive and interim primary outcome measures are overall survival time and event-free survival (EFS) time, respectively. Secondary outcome measures include quality of life, toxicity, swallowing outcomes, feeding tube incidence, surgical complication rates, and cost-effectiveness. The design anticipates that after approximately 7 years, 84 required events will have occurred to enable analysis of the definitive primary outcome measure for this comparison. Planned interim futility analyses using EFS will also be performed. DISCUSSION: CompARE is designed to be efficient and cost-effective in response to new data, emerging new treatments or difficulties, with the aim of bringing new treatment options for these patients. TRIAL REGISTRATION: ISRCTN ISRCTN41478539 . Registered on 29 April 2015.
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Neoplasias Orofaríngeas , Infecciones por Papillomavirus , Humanos , Cisplatino/efectos adversos , Calidad de Vida , Resultado del Tratamiento , Neoplasias Orofaríngeas/terapia , Ensayos Clínicos Controlados Aleatorios como Asunto , Estudios Multicéntricos como Asunto , Ensayos Clínicos Fase III como AsuntoRESUMEN
PURPOSE: Tumor hypoxia is an adverse prognostic factor in head and neck squamous cell carcinoma (HNSCC). We assessed whether patients with hypoxic HNSCC benefited from the addition of nimorazole to definitive intensity modulated radiation therapy (IMRT). METHODS AND MATERIALS: NIMRAD was a phase 3, multicenter, placebo-controlled, double-anonymized trial of patients with HNSCC unsuitable for concurrent platinum chemotherapy or cetuximab with definitive IMRT (NCT01950689). Patients were randomized 1:1 to receive IMRT (65 Gy in 30 fractions over 6 weeks) plus nimorazole (1.2 g/m2 daily, before IMRT) or placebo. The primary endpoint was freedom from locoregional progression (FFLRP) in patients with hypoxic tumors, defined as greater than or equal to the median tumor hypoxia score of the first 50 patients analyzed (≥0.079), using a validated 26-gene signature. The planned sample size was 340 patients, allowing for signature generation in 85% and an assumed hazard ratio (HR) of 0.50 for nimorazole effectiveness in the hypoxic group and requiring 66 locoregional failures to have 80% power in a 2-tail log-rank test at the 5% significance level. RESULTS: Three hundred thirty-eight patients were randomized by 19 centers in the United Kingdom from May 2014 to May 2019, with a median follow-up of 3.1 years (95% CI, 2.9-3.4). Hypoxia scores were available for 286 (85%). The median patient age was 73 years (range, 44-88; IQR, 70-76). There were 36 (25.9%) locoregional failures in the hypoxic group, in which nimorazole + IMRT did not improve FFLRP (adjusted HR, 0.72; 95% CI, 0.36-1.44; P = .35) or overall survival (adjusted HR, 0.96; 95% CI, 0.53-1.72; P = .88) compared with placebo + IMRT. Similarly, nimorazole + IMRT did not improve FFLRP or overall survival in the whole population. In total (N = 338), 73% of patients allocated nimorazole adhered to the drug for ≥50% of IMRT fractions. Nimorazole + IMRT caused more acute nausea compared with placebo + IMRT (Common Terminology Criteria for Adverse Events version 4.0 G1+2: 56.6% vs 42.4%, G3: 10.1% vs 5.3%, respectively; P < .05). CONCLUSIONS: Addition of the hypoxia modifier nimorazole to IMRT for locally advanced HNSCC in older and less fit patients did not improve locoregional control or survival.
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PURPOSE: Lumican is a major extracellular matrix (ECM) component in the cornea that is upregulated after injury and promotes corneal wound healing. We have recently shown that peptides designed based on the 13 C-terminal amino acids of lumican (LumC13 and LumC13C-A) are able to recapitulate the effects of lumican on promoting corneal wound healing. Herein we used computational chemistry to develop peptide mimetics derived from LumC13C-A with increased stability and half-life that are biologically active and non-toxic, thereby promoting corneal wound healing with increased pharmacological potential. METHODS: Different peptides staples were rationalized using LumC13C-A sequence by computational chemistry, docked to TGFßRI and the interface binding energies compared. Lowest scoring peptides were synthesized, and the toxicity of peptides tested using CCK8-based cell viability assay. The efficacy of the stapled peptides at promoting corneal wound healing was tested using a proliferation assay, an in vitro scratch assay using human corneal epithelial cells and an in vivo murine corneal debridement wound healing model. RESULTS: Binding free energies were calculated using MMGBSA algorithm, and peptides LumC13C and LumC13S5 displayed superior binding to ALK5 compared to the non-stapled peptide LumC13C-A. The presence of the hydrocarbon staple in LumC13C enhances the stability of the α-helical conformation, thereby facilitating more optimal interactions with the ALK5 receptor. The stapled peptides do not present cytotoxic effects on human corneal epithelial cells at a 300 nM concentration. Similar to lumican and LumC13C-A, both C13C and LumC13S5 significantly promote corneal wound healing both in vitro and in vivo. CONCLUSIONS: Highly stable and non-toxic stapled peptides designed based on LumC13, significantly promote corneal wound healing. As a proof of principle, our data shows that more stable and pharmacologically relevant peptides can be designed based on endogenous peptide sequences for treating various corneal pathologies.
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Lesiones de la Cornea , Epitelio Corneal , Humanos , Animales , Ratones , Lumican/metabolismo , Lumican/farmacología , Córnea/patología , Lesiones de la Cornea/metabolismo , Cicatrización de Heridas , Péptidos/farmacología , Péptidos/metabolismo , Epitelio Corneal/metabolismoRESUMEN
Integrin αIIbß3 is the key receptor regulating platelet retraction and accumulation, thus pivotal for hemostasis, and arterial thrombosis as well as a proven drug-target for antithrombotic therapies. Here we resolve the cryoEM structures of the intact full-length αIIbß3, which covers three distinct states along the activation pathway. Here, we resolve intact αIIbß3 structure at 3Å resolution, revealing the overall topology of the heterodimer with the transmembrane (TM) helices and the head region ligand-binding domain tucked in a specific angle proximity to the TM region. In response to the addition of an Mn2+ agonist, we resolved two coexisting states, "intermediate" and "pre-active". Our structures show conformational changes of the intact αIIbß3 activating trajectory, as well as a unique twisting of the lower integrin legs representing intermediate state (TM region at a twisting conformation) integrin and a coexisting pre-active state (bent and opening in leg), which is required for inducing the transitioning platelets to accumulate. Our structure provides for the first time direct structural evidence for the lower legs' involvement in full-length integrin activation mechanisms. Additionally, our structure offers a new strategy to target the αIIbß3 lower leg allosterically instead of modulating the affinity of the αIIbß3 head region.
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BACKGROUND: Fibrinogen is a soluble, multisubunit, and multidomain dimeric protein, which, upon its proteolytic cleavage by thrombin, is converted to insoluble fibrin, initiating polymerization that substantially contributes to clot growth. Fibrinogen contains numerous, transiently accessible "cryptic" epitopes for hemostatic and immunologic proteins, suggesting that fibrinogen exhibits conformational flexibility, which may play functional roles in its temporal and spatial interactions. Hitherto, there have been limited integrative approaches characterizing the solution structure and internal flexibility of fibrinogen. METHODS: Here, utilizing a multipronged, biophysical approach involving 2 solution-based techniques, temperature-dependent hydrogen-deuterium exchange mass spectrometry and small angle X-ray scattering, corroborated by negative stain electron microscopy, we present a holistic, conformationally dynamic model of human fibrinogen in solution. RESULTS: Our data reveal 4 major and distinct conformations of fibrinogen accommodated by a high degree of internal protein flexibility along its central scaffold. We propose that the fibrinogen structure in the solution consists of a complex, conformational landscape with multiple local minima. This is further supported by the location of numerous point mutations that are linked to dysfibrinogenemia and posttranslational modifications, residing near the identified fibrinogen flexions. CONCLUSION: This work provides a molecular basis for the structural "dynamism" of fibrinogen that is expected to influence the broad swath of its functionally diverse macromolecular interactions and fine-tune the structural and mechanical properties of blood clots.
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Productos de Degradación de Fibrina-Fibrinógeno , Trombosis , Humanos , Fibrina/química , Fibrinógeno/metabolismo , Conformación MolecularRESUMEN
Staphylococcus aureus protein A (SpA) is an IgG Fc-binding virulence factor that is widely used in antibody purification and as a scaffold to develop affinity molecules. A cyclized SpA Z domain could offer exopeptidase resistance, reduced chromatographic ligand leaching after single-site endopeptidase cleavage, and enhanced IgG binding properties by preorganization, potentially reducing conformational entropy loss upon binding. In this work, a Z domain trimer (Z3) was cyclized using protein intein splicing. Interactions of cyclic and linear Z3 with human IgG1 were characterized by differential scanning fluorimetry (DSF), surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC). DSF showed a 5 â increase in IgG1 melting temperature when bound by each Z3 variant. SPR showed the dissociation constants of linear and cyclized Z3 with IgG1 to be 2.9 nM and 3.3 nM, respectively. ITC gave association enthalpies for linear and cyclic Z3 with IgG1 of -33.0 kcal/mol and -32.7 kcal/mol, and -T∆S of association 21.2 kcal/mol and 21.6 kcal/mol, respectively. The compact cyclic Z3 protein contains 2 functional binding sites and exhibits carboxypeptidase Y-resistance. The results suggest cyclization as a potential approach toward more stable SpA-based affinity ligands, and this analysis may advance our understanding of protein engineering for ligand and drug development.
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Inteínas , Staphylococcus aureus , Humanos , Inteínas/genética , Ligandos , Termodinámica , Inmunoglobulina G , Calorimetría/métodos , Unión ProteicaRESUMEN
Allosteric activation and silencing of leukocyte ß2-integrins transpire through cation-dependent structural changes, which mediate integrin biosynthesis and recycling, and are essential to designing leukocyte-specific drugs. Stepwise addition of Mg2+ reveals two mutually coupled events for the αXß2 ligand-binding domain-the αX I-domain-corresponding to allostery establishment and affinity maturation. Electrostatic alterations in the Mg2+-binding site establish long-range couplings, leading to both pH- and Mg2+-occupancy-dependent biphasic stability change in the αX I-domain fold. The ligand-binding sensorgrams show composite affinity events for the αX I-domain accounting for the multiplicity of the αX I-domain conformational states existing in the solution. On cell surfaces, increasing Mg2+ concentration enhanced adhesiveness of αXß2. This work highlights how intrinsically flexible pH- and cation-sensitive architecture endows a unique dynamic continuum to the αI-domain structure on the intact integrin, thereby revealing the importance of allostery establishment and affinity maturation in both extracellular and intracellular integrin events.
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Integrina alfaXbeta2 , Cationes Bivalentes , Integrina alfaXbeta2/química , Integrina alfaXbeta2/metabolismo , Ligandos , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
Detecting COVID-19 in computed tomography (CT) or radiography images has been proposed as a supplement to the RT-PCR test. We compare slice-based (2D) and volume-based (3D) approaches to this problem and propose a deep learning ensemble, called IST-CovNet, combining the best 2D and 3D systems with novel preprocessing and attention modules and the use of a bidirectional Long Short-Term Memory model for combining slice-level decisions. The proposed ensemble obtains 90.80% accuracy and 0.95 AUC score overall on the newly collected IST-C dataset in detecting COVID-19 among normal controls and other types of lung pathologies; and 93.69% accuracy and 0.99 AUC score on the publicly available MosMedData dataset that consists of COVID-19 scans and normal controls only. The system also obtains state-of-art results (90.16% accuracy and 0.94 AUC) on the COVID-CT-MD dataset which is only used for testing. The system is deployed at Istanbul University Cerrahpasa School of Medicine where it is used to automatically screen CT scans of patients, while waiting for RT-PCR tests or radiologist evaluation.
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BACKGROUND: While the coronavirus (COVID-19) pandemic creates fear and anxiety on the students' academic achievement, the risk of an infectious disease may negatively affect education by reducing the concentration ability of students. Thus, it is essential to evaluate the knowledge, behaviors, anxiety levels, and hygiene status of students. OBJECTIVE: This study aimed to investigate the knowledge, behavior change, anxiety, and hygiene status of university students about COVID-19. METHODS: The investigation was conducted with students of three vocational schools of health services located in different provinces of Turkey (nâ=â1055). Data collected by an online survey consisted of knowledge questions about COVID-19, items about behavior change, the hygiene behavior scale (HBS), and the Generalized Anxiety Disorder 7 (GAD-7) scale. RESULTS: The knowledge scores of women were significantly higher than the scores of men. Of the students, 59.6%showed positive hygiene behaviors, and 31.5%had anxiety. Female students' total HDC scale score was significantly lower than that of men, and the total HDC scale score of those who received hand hygiene education was significantly lower than that of participants who did not receive training, which shows a positive hygiene behavior. The total GAD-7 scale score of women was significantly higher than that of men. More than half of the students showed positive hygiene behaviors, and about a third had anxiety. CONCLUSIONS: Based on the study's results, psychological support and training should be provided to students.
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COVID-19 , Ansiedad , Trastornos de Ansiedad , Estudios Transversales , Femenino , Servicios de Salud , Humanos , Higiene , Masculino , SARS-CoV-2 , Instituciones Académicas , Estudiantes , TurquíaRESUMEN
BACKGROUND: The onset of the COVID-19 pandemic necessitated rapid changes to the practice of head and neck oncology in UK. There was a delay between the onset of the pandemic and the release of guidelines from cancer societies and networks, leading to a variable response of individual centres. This survey was conducted to assess the pre-Covid-19 pandemic standard of practice for head and neck oncology patients and the treatment modifications introduced during the first wave of the pandemic in UK. METHODOLOGY: The UK National Cancer Research Institute (NCRI) Head and Neck Clinical Studies Group initiated a multi-centre survey using questionnaire to investigate the effect on feeding tube practice, radiotherapy (RT) fractionation and volumes, use of chemotherapy in the neo-adjuvant, concurrent and palliative setting, the use of immunotherapy in the palliative setting, access to radiology and histopathology services, and availability of surgical procedures. RESULTS: 30 centres were approached across UK; 23 (76.7%) centres responded and were included in the survey. There were differences in the standard practices in feeding tube policy, RT dose and fractionation as well as concurrent chemotherapy use. 21 (91%) participating centres had at least one treatment modification. 15 (65%) centres initiated a change in radical RT; changing to either a hypofractionation or acceleration schedule. For post-operative RT 10 centres (43.5%) changed to a hypofractionation schedule. 12 (52.2%) centres stopped neo-adjuvant chemotherapy for all patients; 13 (56.5%) centres followed selective omission of chemotherapy in concurrent chemo-radiotherapy patients, 17 (73.9%) centres changed first-line chemotherapy treatment to pembrolizumab (following NHS England's interim guidance) and 8 (34.8%) centres stopped the treatment early or offered delays for patients that have been already on systemic treatment. The majority of centres did not have significant changes associated with surgery, radiology, histopathology and dental screening. CONCLUSION: There are variations in the standard of practice and treatment modifications for head and neck cancer patients during Covid-19 pandemic. A timely initiative is required to form a consensus on head and neck cancer management in the UK and other countries.
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BACKGROUND: Radical (chemo)radiotherapy offers potentially curative treatment for patients with locally advanced laryngeal or hypopharyngeal cancer. We aimed to show that dose-escalated intensity-modulated radiotherapy (DE-IMRT) improved locoregional control. METHODS: We performed a phase III open-label randomised controlled trial in patients with laryngeal or hypopharyngeal cancer (AJCC III-IVa/b, TNM 7). Patients were randomised (1:1) to DE-IMRT or standard dose IMRT (ST-IMRT) using a minimisation algorithm, balancing for centre, tumour site, nodal status and chemotherapy use. DE-IMRT was 67.2 gray (Gy) in 28 fractions (f) to the primary tumour and 56Gy/28f to at-risk nodes; ST-IMRT was 65Gy/30f to primary tumour and 54Gy/30f to at-risk nodes. Suitable patients received 2 cycles of concomitant cisplatin and up to 3 cycles of platinum-based induction chemotherapy. The primary end-point was time to locoregional failure analysed by intention-to-treat analysis using competing risk methodology. FINDINGS: Between February 2011 and October 2015, 276 patients (138 ST-IMRT; 138 DE-IMRT) were randomised. A preplanned interim futility analysis met the criterion for early closure. After a median follow-up of 47.9 months (interquartile range 37.5-60.5), there were locoregional failures in 38 of 138 (27.5%) ST-IMRT patients and 42 of 138 (30.4%) DE-IMRT patients; an adjusted subhazard ratio of 1.16 (95% confidence interval: 0.74-1.83, p = 0.519) indicated no evidence of benefit with DE-IMRT. Acute grade 2 pharyngeal mucositis was reported more frequently with DE-IMRT than with ST-IMRT (42% vs. 32%). No differences in grade ≥3 acute or late toxicity rates were seen. CONCLUSION: DE-IMRT did not improve locoregional control in patients with laryngeal or hypopharyngeal cancer. The trial is registered: ISRCTN01483375.
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Neoplasias Hipofaríngeas/radioterapia , Neoplasias Laríngeas/radioterapia , Anciano , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
The receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 spike (S) protein plays a central role in mediating the first step of virus infection to cause disease: virus binding to angiotensin-converting enzyme 2 (ACE2) receptors on human host cells. Therefore, S/RBD is an ideal target for blocking and neutralization therapies to prevent and treat coronavirus disease 2019 (COVID-19). Using a target-based selection approach, we developed oligonucleotide aptamers containing a conserved sequence motif that specifically targets S/RBD. Synthetic aptamers had high binding affinity for S/RBD-coated virus mimics (K D≈7â nM) and also blocked interaction of S/RBD with ACE2 receptors (IC50≈5â nM). Importantly, aptamers were able to neutralize S protein-expressing viral particles and prevent host cell infection, suggesting a promising COVID-19 therapy strategy.
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Complement receptor 3 (CR3, also known as Mac-1, integrin αMß2, or CD11b/CD18) is expressed on a subset of myeloid and certain activated lymphoid cells. CR3 is essential for the phagocytosis of complement-opsonized particles such as pathogens and apoptotic or necrotic cells opsonized with the complement fragment iC3b and, to a lesser extent, C3dg. Although the interaction between the iC3b thioester domain and the ligand binding CR3 αM I-domain is structurally and functionally well characterized, the nature of additional CR3-iC3b interactions required for phagocytosis of complement-opsonized objects remains obscure. In this study, we analyzed the interaction between iC3b and the 150-kDa headpiece fragment of the CR3 ectodomain. Surface plasmon resonance experiments demonstrated a 30 nM affinity of the CR3 headpiece for iC3b compared with 515 nM for the iC3b thioester domain, whereas experiments monitoring binding of iC3b to CR3-expressing cells suggested an affinity of 50 nM for the CR3-iC3b interaction. Small angle x-ray scattering analysis revealed that iC3b adopts an extended but preferred conformation in solution. Upon interaction with CR3, iC3b rearranges to form a compact receptor-ligand complex. Overall, the data suggest that the iC3b-CR3 interaction is of high affinity and relies on minor contacts formed between CR3 and regions outside the iC3b thioester domain. Our results rationalize the more efficient phagocytosis elicited by iC3b than by C3dg and pave the way for the development of specific therapeutics for the treatment of inflammatory and neurodegenerative diseases that do not interfere with the recognition of noncomplement CR3 ligands.
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Complemento C3b/inmunología , Antígeno de Macrófago-1/inmunología , HumanosRESUMEN
Membrane proteins represent major drug targets, and the ability to determine their functions, structures, and conformational changes will significantly advance mechanistic approaches to both biotechnology and bioremediation, as well as the fight against pathogenic bacteria. A pertinent example is Mycobacterium tuberculosis (H37Rv), which contains ~4000 protein-coding genes, with almost a thousand having been categorized as 'membrane protein', and a few of which (~1%) have been functionally characterized and structurally modeled. However, the functions and structures of most membrane proteins that are sparsely, or only transiently, expressed, but essential in small phenotypic subpopulations or under stress conditions such as persistence or dormancy, remain unknown. Our deep quantitative proteomics profiles revealed that the hypothetical membrane protein 730 (Hyp730) WP_010079730 (protein ID Mlut_RS11895) from M. luteus is upregulated in dormancy despite a ~5-fold reduction in overall protein diversity. Its H37Rv paralog, Rv1234, showed a similar proteomic signature, but the function of Hyp730-like proteins has never been characterized. Here, we present an extensive proteomic and transcriptomic analysis of Hyp730 and have also characterized its in vitro recombinant expression, purification, refolding, and essentiality as well as its tertiary fold. Our biophysical studies, circular dichroism, and tryptophan fluorescence are in immediate agreement with in-depth in silico 3D-structure prediction, suggesting that Hyp730 is a double-pass membrane-spanning protein. Ablation of Hyp730-expression did not alter M. luteus growth, indicating that Hyp730 is not essential. Structural homology comparisons showed that Hyp730 is highly conserved and non-redundant in G+C rich Actinobacteria and might be involved, under stress conditions, in an energy-saving role in respiration during dormancy.
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Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/genética , Proteínas de la Membrana/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Secuencia de Aminoácidos , Perfilación de la Expresión Génica , Genoma Bacteriano/genética , Infección Latente/genética , Porinas/genética , Porinas/metabolismo , Proteómica/métodos , ARN Mensajero/genética , Espectrometría de Masas en TándemRESUMEN
The receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 spike (S) protein plays a central role in mediating the first step of virus infection to cause disease: virus binding to angiotensin-converting enzyme 2 (ACE2) receptors on human host cells. Therefore, S/RBD is an ideal target for blocking and neutralization therapies to prevent and treat coronavirus disease 2019 (COVID-19). Using a target-based selection approach, we developed oligonucleotide aptamers containing a conserved sequence motif that specifically targets S/RBD. Synthetic aptamers had high binding affinity for S/RBD-coated virus mimics (KD ≈7â nM) and also blocked interaction of S/RBD with ACE2 receptors (IC50 ≈5â nM). Importantly, aptamers were able to neutralize S protein-expressing viral particles and prevent host cell infection, suggesting a promising COVID-19 therapy strategy.