Complement Receptor 3 Forms a Compact High-Affinity Complex with iC3b.

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.

Expression and Characterization of Intein-Cyclized Trimer of Staphylococcus aureus Protein A Domain Z.

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 ℃ 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.

Comparison and ensemble of 2D and 3D approaches for COVID-19 detection in CT images.

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.

Neutralizing Aptamers Block S/RBD-ACE2 Interactions and Prevent Host Cell Infection.

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 ≈7 nM) and also blocked interaction of S/RBD with ACE2 receptors (IC50 ≈5 nM). Importantly, aptamers were able to neutralize S protein-expressing viral particles and prevent host cell infection, suggesting a promising COVID-19 therapy strategy.

Radiotherapy plus cisplatin or cetuximab in low-risk human papillomavirus-positive oropharyngeal cancer (De-ESCALaTE HPV): an open-label randomised controlled phase 3 trial.

BACKGROUND: The incidence of human papillomavirus (HPV)-positive oropharyngeal cancer, a disease affecting younger patients, is rapidly increasing. Cetuximab, an epidermal growth factor receptor inhibitor, has been proposed for treatment de-escalation in this setting to reduce the toxicity of standard cisplatin treatment, but no randomised evidence exists for the efficacy of this strategy. METHODS: We did an open-label randomised controlled phase 3 trial at 32 head and neck treatment centres in Ireland, the Netherlands, and the UK, in patients aged 18 years or older with HPV-positive low-risk oropharyngeal cancer (non-smokers or lifetime smokers with a smoking history of <10 pack-years). Eligible patients were randomly assigned (1:1) to receive, in addition to radiotherapy (70 Gy in 35 fractions), either intravenous cisplatin (100 mg/m2 on days 1, 22, and 43 of radiotherapy) or intravenous cetuximab (400 mg/m2 loading dose followed by seven weekly infusions of 250 mg/m2). The primary outcome was overall severe (grade 3-5) toxicity events at 24 months from the end of treatment. The primary outcome was assessed by intention-to-treat and per-protocol analyses. This trial is registered with the ISRCTN registry, number ISRCTN33522080. FINDINGS: Between Nov 12, 2012, and Oct 1, 2016, 334 patients were recruited (166 in the cisplatin group and 168 in the cetuximab group). Overall (acute and late) severe (grade 3-5) toxicity did not differ significantly between treatment groups at 24 months (mean number of events per patient 4·8 [95% CI 4·2-5·4] with cisplatin vs 4·8 [4·2-5·4] with cetuximab; p=0·98). At 24 months, overall all-grade toxicity did not differ significantly either (mean number of events per patient 29·2 [95% CI 27·3-31·0] with cisplatin vs 30·1 [28·3-31·9] with cetuximab; p=0·49). However, there was a significant difference between cisplatin and cetuximab in 2-year overall survival (97·5% vs 89·4%, hazard ratio 5·0 [95% CI 1·7-14·7]; p=0·001) and 2-year recurrence (6·0% vs 16·1%, 3·4 [1·6-7·2]; p=0·0007). INTERPRETATION: Compared with the standard cisplatin regimen, cetuximab showed no benefit in terms of reduced toxicity, but instead showed significant detriment in terms of tumour control. Cisplatin and radiotherapy should be used as the standard of care for HPV-positive low-risk patients who are able to tolerate cisplatin. FUNDING: Cancer Research UK.

Ligand- and cation-induced structural alterations of the leukocyte integrin LFA-1.

In αI integrins, including leukocyte function-associated antigen 1 (LFA-1), ligand-binding function is delegated to the αI domain, requiring extra steps in the relay of signals that activate ligand binding and coordinate it with cytoplasmic signals. Crystal structures reveal great variation in orientation between the αI domain and the remainder of the integrin head. Here, we investigated the mechanisms involved in signal relay to the αI domain, including whether binding of the ligand intercellular adhesion molecule-1 (ICAM-1) to the αI domain is linked to headpiece opening and engenders a preferred αI domain orientation. Using small-angle X-ray scattering and negative-stain EM, we define structures of ICAM-1, LFA-1, and their complex, and the effect of activation by Mn2+ Headpiece opening was substantially stabilized by substitution of Mg2+ with Mn2+ and became complete upon ICAM-1 addition. These agents stabilized αI-headpiece orientation, resulting in a well-defined orientation of ICAM-1 such that its tandem Ig-like domains pointed in the opposite direction from the ß-subunit leg of LFA-1. Mutations in the integrin ßI domain α1/α1' helix stabilizing either the open or the closed ßI-domain conformation indicated that α1/α1' helix movements are linked to ICAM-1 binding by the αI domain and to the extended-open conformation of the ectodomain. The LFA-1-ICAM-1 orientation described here with ICAM-1 pointing anti-parallel to the LFA-1 ß-subunit leg is the same orientation that would be stabilized by tensile force transmitted between the ligand and the actin cytoskeleton and is consistent with the cytoskeletal force model of integrin activation.

Impact of choice of feeding tubes on long-term swallow function following chemoradiotherapy for oropharyngeal carcinoma.

Background: Prior reports have raised concerns that a prophylactic gastrostomy may be detrimental to long-term swallow function. This study evaluates patient-reported swallow function following chemoradiotherapy for oropharyngeal carcinoma in relation to the use of a prophylactic gastrostomy or nasogastric (NG) tube as required. Material and methods: The MD Anderson Dysphagia Inventory (MDADI) was posted to 204 disease-free patients at least 2 years following chemoradiotherapy for oropharyngeal carcinoma between 2010 and 2014. Results: Overall, 181/204 (89%) patients returned questionnaire at a median of 34 months post-treatment. 97/181 (54%) and 84/181 (46%) were managed with an approach of a prophylactic gastrostomy or NG tube as required, respectively. A prophylactic gastrostomy was associated with higher rates of enteral feeding (92% vs. 58%, p < .001), lower median percentage weight loss (7.0% vs. 9.4%, p < .001), increased duration of enteral feed (median 3.3 vs. 1.1 months, p < .001). There was no significant difference in patient-reported swallow function measured by MDADI summary scores and subscales for patients managed with an approach of prophylactic gastrostomy or NG as required. Duration of enteral feed correlated negatively with composite MDADI scores. A subgroup of 116/181 (64%) patients were documented as having been offered a choice of enteral feeding approach and therefore can be considered to represent clinical equipoise; there were no significant differences in MDADI scores according to route. Conclusions: Despite concern regarding the use of a prophylactic gastrostomy in prior studies, the approaches of using a prophylactic gastrostomy or an NG tube as required to support patients during/after chemoradiotherapy for oropharyngeal carcinoma were associated with similar long-term swallow outcomes.

Ultrasound-guided dorsal penile nerve block vs neurostimulator-guided pudendal nerve block in children undergoing hypospadias surgery: A prospective, randomized, double-blinded trial.

BACKGROUND AND AIMS: Hypospadias is a common congenital malformation in pediatric patients. Surgical repair of this malformation is a painful procedure and has long-term effects. Pudendal and penile nerve blocks are commonly preferred techniques for maintaining postoperative analgesia. However, the conventional landmark-based penile block technique involves numerous potential complications and provides a shorter analgesic period compared to the pudendal block. A promising ultrasound-guided dorsal penile nerve block was recently described. We aimed to compare the analgesic effectiveness of ultrasound-guided penile nerve block with that of neurostimulator-guided pudendal nerve block. METHOD: Thirty-three patients aged 1-7 years were included in this prospective, double-blinded, randomized controlled trial. Patients were divided into two groups and received either ultrasound-guided dorsal penile nerve block or neurostimulator-guided pudendal nerve block. All blocks were performed by the same two anesthesiologists, and the same surgeons performed the surgical procedures. The Face, Legs, Activity, Cry, and Consolability (FLACC) scale was used for postoperative pain management. The primary outcome of the study was time to first analgesic requirement. Secondary outcomes were FLACC scores at different time points, and types and cumulative doses of analgesic drugs. RESULTS: Dorsal penile nerve block provided longer analgesia than pudendal nerve block (32.29 ± 5.47 hours and 21.13 ± 3.53 hours, respectively; differences in mean: 11.16, 95% CI: 7.873-14.465) (P < .001). FLACC scores at the time of first analgesic requirement were significantly lower in dorsal penile nerve block group than pudendal nerve block group (median [IQR]: 2 [2-2.5] and 3 [3-5], respectively; differences in median: -1, 95% CI: -1.851 to -0.149) (P < .001). CONCLUSION: Ultrasound-guided dorsal penile nerve block provided a longer analgesic period and reduced opioid consumption compared to neurostimulator-guided pudendal nerve block.

Leukocyte integrin αLß2 headpiece structures: The αI domain, the pocket for the internal ligand, and concerted movements of its loops.

High-resolution crystal structures of the headpiece of lymphocyte function-associated antigen-1 (integrin αLß2) reveal how the αI domain interacts with its platform formed by the α-subunit ß-propeller and ß-subunit ßI domains. The αLß2 structures compared with αXß2 structures show that the αI domain, tethered through its N-linker and a disulfide to a stable ß-ribbon pillar near the center of the platform, can undergo remarkable pivoting and tilting motions that appear buffered by N-glycan decorations that differ between αL and αX subunits. Rerefined ß2 integrin structures reveal details including pyroglutamic acid at the ß2 N terminus and bending within the EGF1 domain. Allostery is relayed to the αI domain by an internal ligand that binds to a pocket at the interface between the ß-propeller and ßI domains. Marked differences between the αL and αX subunit ß-propeller domains concentrate near the binding pocket and αI domain interfaces. Remarkably, movement in allostery in the ßI domain of specificity determining loop 1 (SDL1) causes concerted movement of SDL2 and thereby tightens the binding pocket for the internal ligand.

Structural and cellular mechanisms of peptidyl-prolyl isomerase Pin1-mediated enhancement of Tissue Factor gene expression, protein half-life, and pro-coagulant activity.

Tissue Factor is a cell-surface glycoprotein expressed in various cells of the vasculature and is the principal regulator of the blood coagulation cascade and hemostasis. Notably, aberrant expression of Tissue Factor is associated with cardiovascular pathologies such as atherosclerosis and thrombosis. Here, we sought to identify factors that regulate Tissue Factor gene expression and activity. Tissue Factor gene expression is regulated by various transcription factors, including activating protein-1 and nuclear factor-κ B. The peptidyl-prolyl isomerase Pin1 is known to modulate the activity of these two transcription factors, and we now show that Pin1 augments Tissue Factor gene expression in both vascular smooth muscle cells and activated endothelial cells via activating protein-1 and nuclear factor-κ B signaling. Furthermore, the cytoplasmic domain of Tissue Factor contains a well-conserved phospho-Ser258-Pro259 amino-acid motif recognized by Pin1. Using co-immunoprecipitation and solution nuclear magnetic resonance spectroscopy, we show that the WW-domain of Pin1 directly binds the cytoplasmic domain of Tissue Factor. This interaction occurs via the phospho-Ser258-Pro259 sequence in the Tissue Factor cytoplasmic domain and results in increased protein half-life and pro-coagulant activity. Taken together, our results establish Pin1 as an upstream regulator of Tissue Factor-mediated coagulation, thereby opening up new avenues for research into the use of specific Pin1 inhibitors for the treatment of diseases characterized by pathological coagulation, such as thrombosis and atherosclerosis.

Modelling of a Hall Effect-Based Current Sensor with an Open Core Magnetic Concentrator.

The present paper deals with the modelling of a Hall effect current sensor with open core magnetic concentrator. 3D magnetic field modelling is carried out using the finite element method (FEM) and Comsol Multiphysics software. Two rectangular core constructions are considered. Different geometric parameters of the magnetic concentrator are varied and their influence on the sensor characteristic is studied, with the aim of reducing the dependence on the output signal on the distance to the conductor. Of the studied parameters, core window length leads to the most significant change in the sensor characteristic. Future work can include the optimization of the sensor construction.

Activated ß2 Integrins Restrict Neutrophil Recruitment during Murine Acute Pseudomonal Pneumonia.

Rapid neutrophil recruitment is critical for the efficient clearance of bacterial pathogens from the lungs. Although ß2 integrins and their activation are required for neutrophil recruitment from postcapillary venules of the systemic circulation into inflamed tissues, the involvement of integrins in neutrophil recruitment in response to respiratory infection varies among bacterial pathogens. For stimuli eliciting ß2 integrin-dependent neutrophil influx, including Pseudomonas aeruginosa, it remains unclear whether the activation of ß2 integrins is an essential step in this process. In the current study, we analyze neutrophil trafficking within the lungs of mice infected with Pseudomonas aeruginosa and evaluate the role of ß2 integrin activation through genetic deletion of talin-1 or Kindlin-3 or by pharmacological inhibition of high-affinity ß2 integrins using a small molecule allosteric antagonist. We observe that attenuation of high-affinity ß2 integrins leads to an enhancement of neutrophil emigration into lung interstitium and airspaces. Neutrophil effector functions, including the production of reactive oxygen species and the phagocytosis of bacteria, are only partially dependent on high-affinity ß2 integrins. These results reveal a mechanism by which activated ß2 integrins limit neutrophil entry into the lung tissue and airspaces during acute pseudomonal pneumonia and suggest potential strategies for modulating neutrophil-mediated host defense.

Multimodality imaging with CT, MR and FDG-PET for radiotherapy target volume delineation in oropharyngeal squamous cell carcinoma.

BACKGROUND: This study aimed to quantify the variation in oropharyngeal squamous cell carcinoma gross tumour volume (GTV) delineation between CT, MR and FDG PET-CT imaging. METHODS: A prospective, single centre, pilot study was undertaken where 11 patients with locally advanced oropharyngeal cancers (2 tonsil, 9 base of tongue primaries) underwent pre-treatment, contrast enhanced, FDG PET-CT and MR imaging, all performed in a radiotherapy treatment mask. CT, MR and CT-MR GTVs were contoured by 5 clinicians (2 radiologists and 3 radiation oncologists). A semi-automated segmentation algorithm was used to contour PET GTVs. Volume and positional analyses were undertaken, accounting for inter-observer variation, using linear mixed effects models and contour comparison metrics respectively. RESULTS: Significant differences in mean GTV volume were found between CT (11.9 cm(3)) and CT-MR (14.1 cm(3)), p < 0.006, CT-MR and PET (9.5 cm(3)), p < 0.0009, and MR (12.7 cm(3)) and PET, p < 0.016. Substantial differences in GTV position were found between all modalities with the exception of CT-MR and MR GTVs. A mean of 64 %, 74 % and 77 % of the PET GTVs were included within the CT, MR and CT-MR GTVs respectively. A mean of 57 % of the MR GTVs were included within the CT GTV; conversely a mean of 63 % of the CT GTVs were included within the MR GTV. CT inter-observer variability was found to be significantly higher in terms of position and/or volume than both MR and CT-MR (p < 0.05). Significant differences in GTV volume were found between GTV volumes delineated by radiologists (9.7 cm(3)) and oncologists (14.6 cm(3)) for all modalities (p = 0.001). CONCLUSIONS: The use of different imaging modalities produced significantly different GTVs, with no single imaging technique encompassing all potential GTV regions. The use of MR reduced inter-observer variability. These data suggest delineation based on multimodality imaging has the potential to improve accuracy of GTV definition. TRIAL REGISTRATION: ISRCTN Registry: ISRCTN34165059 . Registered 2nd February 2015.

A continuous flow microfluidic calorimeter: 3-D numerical modeling with aqueous reactants.

A computational analysis of the reacting flow field, species diffusion and heat transfer processes with thermal boundary layer effects in a microchannel reactor with a coflow configuration was performed. Two parallel adjacent streams of aqueous reactants flow along a wide, shallow, enclosed channel in contact with a substrate, which is affixed to a temperature controlled plate. The Fluent computational fluid dynamics package solved the Navier-Stokes, mass transport and energy equations. The energy model, including the enthalpy of reaction as a nonuniform heat source, was validated by calculating the energy balance at several control volumes in the microchannel. Analysis reveals that the temperature is nearly uniform across the channel thickness, in the direction normal to the substrate surface; hence, measurements made by sensors at or near the surface are representative of the average temperature. Additionally, modeling the channel with a glass substrate and a silicone cover shows that heat transfer is predominantly due to the glass substrate. Finally, using the numerical results, we suggest that a microcalorimeter could be based on this configuration, and that temperature sensors such as optical nanohole array sensors could have sufficient spatial resolution to determine enthalpy of reaction.

Interplay between intrinsic flexibility and sugar coating in Contactin-2 homodimerization.

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.

Determination of body image and self-efficacy levels in patients after cornea transplantation.

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 (p < .05), while the difference between the self-efficacy mean scores was not statistically significant (p > .05). There was a positive, moderately strong significant relationship between body image and the self-efficacy of the patients (p < .01) (r = .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.

Evaluation of low back pain status and affecting factors in drivers in Turkey: A cross-sectional study1.

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 (p <  0.05). There was no significant association between age, education level, and BMI with LBP (p >  0.05). CONCLUSION: There is limited study on this subject in Turkey. Further studies can raise awareness about this issue and create an educational plan.

Full-length αIIbß3 cryo-EM structure reveals intact integrin initiate-activation intrinsic architecture.

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.