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Abstract The Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is an epidemic, zoonotically emerging pathogen initially reported in Saudi Arabia in 2012. MERS-CoV has the potential to mutate or recombine with other coronaviruses, thus acquiring the ability to efficiently spread among humans and become pandemic. Its high mortality rate of up to 35% and the absence of effective targeted therapies call for the development of antiviral drugs for this pathogen. Since the beginning of the SARS-CoV-2 pandemic, extensive research has focused on identifying protease inhibitors for the treatment of SARS-CoV-2. Our intention was therefore to assess whether these protease inhibitors are viable options for combating MERS-CoV. To that end, we used previously established protease assays to quantify inhibition of SARS-CoV-2, MERS-CoV and other main proteases. Nirmatrelvir inhibited several of these proteases, whereas ensitrelvir was less broadly active. To simulate nirmatrelvir’s clinical use against MERS-CoV and subsequent resistance development, we applied a safe, surrogate virus-based system. Using the surrogate virus, we previously selected hallmark mutations of SARS-CoV-2-Mpro, such as T21I, M49L, S144A, E166A/K/V and L167F. In the current study, we selected a pool of MERS-CoV-Mpro mutants, characterized the resistance and modelled the steric effect of catalytic site mutants S142G, S142R, S147Y and A171S.

Nirmatrelvir was the first protease inhibitor specifically developed against the SARS-CoV-2 main protease (3CLpro/Mpro) and licensed for clinical use. As SARS-CoV-2 continues to spread, variants resistant to nirmatrelvir and other currently available treatments are likely to arise. This study aimed to identify and characterize mutations that confer resistance to nirmatrelvir. To safely generate Mpro resistance mutations, we passaged a previously developed, chimeric vesicular stomatitis virus (VSV-Mpro) with increasing, yet suboptimal concentrations of nirmatrelvir. Using Wuhan-1 and Omicron Mpro variants, we selected a large set of mutants. Some mutations are frequently present in GISAID, suggesting their relevance in SARS-CoV-2. The resistance phenotype of a subset of mutations was characterized against clinically available protease inhibitors (nirmatrelvir and ensitrelvir) with cell-based, biochemical and SARS-CoV-2 replicon assays. Moreover, we showed the putative molecular mechanism of resistance based on in silico molecular modelling. These findings have implications on the development of future generation Mpro inhibitors, will help to understand SARS-CoV-2 protease inhibitor resistance mechanisms and show the relevance of specific mutations, thereby informing treatment decisions.

Protein kinases act as central molecular switches in the control of cellular functions. Alterations in the regulation and function of protein kinases may provoke diseases including cancer. In this study we investigate the conformational states of such disease-associated kinases using the high sensitivity of the kinase conformation (KinCon) reporter system. We first track BRAF kinase activity conformational changes upon melanoma drug binding. Second, we also use the KinCon reporter technology to examine the impact of regulatory protein interactions on LKB1 kinase tumor suppressor functions. Third, we explore the conformational dynamics of RIP kinases in response to TNF pathway activation and small molecule interactions. Finally, we show that CDK4/6 interactions with regulatory proteins alter conformations which remain unaffected in the presence of clinically applied inhibitors. Apart from its predictive value, the KinCon technology helps to identify cellular factors that impact drug efficacies. The understanding of the structural dynamics of full-length protein kinases when interacting with small molecule inhibitors or regulatory proteins is crucial for designing more effective therapeutic strategies.

This work studied the microstructure and mechanical properties of Ti-6Al-4V in situ alloyed with 3 wt% Cr by laser powder bed fusion (LPBF). Specimens with a relative density of 99.14 ± 0.11% were produced, showing keyhole and lack of fusion pores. Due to incomplete mixing of the components during melting, chemical inhomogeneities were observed in the solidified material. The addition of Cr promoted thermal supercooling during solidification and induced a reduction in the primary β grain size in the longitudinal direction and a weakening of the otherwise strong ⟨100⟩β texture, both typical issues for Ti-6Al-4V produced by LPBF. The primary β at first transformed martensitically to α’, but by preheating the substrate plate to 500 °C and cyclically reheating the material by melting subsequent layers, in situ martensite decomposition was achieved, resulting in a fine lamellar α + β microstructure. In addition, the B19 phase was detected in the β matrix, presumably caused by Fe impurities in the Cr powder feedstock. Specimens exhibited a hardness of 402 ± 18 HV10, and an excellent ultimate tensile strength of 1450 ± 22 MPa at an elongation at break of 4.5 ± 0.2%.

Background: Airway management in Duchenne patients can be challenging. We present a case of an anticipated difficult airway in a 24-year-old Duchenne patient that was managed by planning different suitable strategies based on the unanticipated difficult airway algorithm of the Difficult Airway Society (DAS). Case presentation: The patient initially presented with appendicitis, requiring a laparoscopic appendectomy within 6 h. Due to the underlying condition and a known difficult airway, we anticipated potential airway problems and successfully managed the “can ventilate but cannot intubate” situation using the algorithm. The difficult airway was attributed to reduced mandibular mobility, limited inclination or reclination, a large tongue, prominent incisors, and a posteriorly positioned epiglottis. Despite thorough preparation and team briefing, we experienced three failed intubation attempts. Considering limited nighttime resources, the urgency of the surgery, the need for a tube for laparoscopy, and the risk of exacerbating airway issues, we made the decision to awaken the patient and wait for a second attempt after the epiglottis swelling had subsided. We used reversible, short-acting agents for induction, enabling us to continue with the algorithm within the allotted timeframe. In a second stage, we successfully performed fiberoptic-guided intubation via a supraglottic airway device using the Aintree intubation catheter, utilizing more favorable resources. Conclusions: For a patient with Duchenne muscular dystrophy and a difficult airway, advanced expertise is critical. Detailed anesthesia planning, clear team communication, and the use of reversible, short-acting agents are crucial. Adherence to the Difficult Airway Society guidelines is essential for safe airway management.

Abstract We report an Osprey-based computational protocol to prospectively identify oncogenic mutations that act via disruption of molecular interactions. It is applicable to analyse both protein-protein and protein-DNA interfaces and it is validated on a dataset of clinically relevant mutations. In addition, it is used to predict previously uncharacterised patient mutations in CDK6 and p16 genes, which are experimentally confirmed to impair complex formation.

Key messages Immediate detection and management of traumatic coagulopathy improves outcomes of severely injured patients. This guideline follows management of the severe trauma patient in chronological order, with a focus on prevention of possible exsanguination. These structured recommendations support measures that prioritise the optimisation of resources for the benefit of bleeding control based on scientific evidence. Empirical management should not be implemented unless no method of monitoring bleeding and coagulation is available. Optimal organisation of the resuscitation team for the bleeding trauma patient includes implementation of these guidelines.

The swift and automated design of antennas remains a challenging aspect in research due to the specific design needs for individual applications. Alterations in resonance frequency or boundary conditions necessitate time-consuming re-designs. Though the application of evolutionary optimization and generative methods in general to antenna design has seen success, it has been mostly restricted to two-dimensional structures. In this work, we present an approach for designing three-dimensional antennas using a genetic algorithm coupled with a region-growing algorithm - to ensure manufacturability - implemented in Matlab manufactured via laser powder bed fusion (LPBF). As a simulation tool for optimization CST is used. The antenna has been optimized in a completely automated manner and was produced using the metal 3D printing technology LPBF and aluminium based AlSi10Mg powder. The presented concept, which builds upon previous two-dimensional techniques, allows for significant flexibility in design, adapting to changing boundary conditions, and avoiding the geometric restrictions seen in prior methods. The optimized antenna has a size of $3.01 \text {cm} \times 3.43 \text {cm} \times 1.67 \text {cm}$ and was measured in an anechoic chamber. According to measurements a minimum reflection coefficient of $\mathrm {-19.95\,\, \text {dB}}$ at $\mathrm {2.462~ \text {G} \text { Hz} }$ and a bandwidth of $\mathrm {308.8~ \text {M} \text { Hz} }$ are observed. CST simulation results predict an efficiency of $\mathrm {98.91~\%}$ and the maximum antenna gain is measured at $\mathrm {2.45~ \text {G} \text { Hz} }$ to be $\mathrm {3.27~ \text {dB} i}$ . Simulations made with CST and Ansys HFSS and measurements are in excellent agreement with a deviation of the resonance frequency of only $\mathrm {0.13~\%}$ , thus further establishing genetic algorithms as a highly viable option for the design of novel antenna structures.

Summary: Prospective predictions of drug-resistant protein mutants could improve the design of therapeutics less prone to resistance. Here, we describe RESISTOR, an algorithm that uses structure- and sequence-based criteria to predict resistance mutations. We demonstrate the process of using RESISTOR to predict ERK2 mutants likely to arise in melanoma ablating the efficacy of the ERK1/2 inhibitor SCH779284. RESISTOR is included in the free and open-source computational protein design software OSPREY.For complete details on the use and execution of this protocol, please refer to Guerin et al..1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Introduction: Data on the perioperative bleeding risk associated with elevated plasma levels of direct factor Xa inhibitors (FXa inhibitors) are limited. This study examines perioperative red blood cell (RBC) loss in patients undergoing urgent surgery with a residual FXa inhibitor level exceeding 100 mcg/L and without preoperative FXa inhibitor reversal. Methods: This retrospective analysis includes data from 32 patients who underwent urgent noncardiac surgery between 2018 and 2022. This study aims to analyze perioperative RBC loss in patients undergoing urgent surgery with a residual FXa inhibitor level exceeding 100 mcg/L and without preoperative FXa inhibitor antidote‐based reversal or unspecific treatment with 4‐factor prothrombin complex concentrate (PCC). All patients were managed using a watch‐and‐wait strategy. Results: The last determination of FXa inhibitor plasma concentration prior to surgery showed a median of 245 mcg/L (IQR 144–345), with a median time interval of 3.8 h (IQR 2.4–7.2) before incision. Median RBC loss during surgery was 49 mL (IQR 0–253), 189 mL (IQR 104–217) until POD1 and 254 mL (IQR 58–265) until POD3. Only one patient required intraoperative treatment with 4‐factor‐PCC and none required reversal with andexanet alfa. Linear regression models found no significant influence of FXa inhibitor plasma levels on intraoperative RBC loss. Rivaroxaban was associated with higher RBC loss until postoperative Day 1 compared with apixaban. No thromboembolic events were observed. Conclusion: Despite markedly elevated plasma concentrations of residual direct FXa inhibitors, perioperative RBC loss was limited in patients undergoing urgent noncardiac surgery. The intraoperative watch‐and‐wait strategy with selective intraoperative FXa inhibitor reversal or treatment only when required appears to be an appropriate approach. [ABSTRACT FROM AUTHOR]

The choice of appropriate processing parameters in laser powder bed fusion is firmly established in the state-of-the-art additive manufacturing community. However, optimisation of scanning strategy would result in improved material properties. Here, the optimal scanning strategy for fatigue-loaded high-performance aluminium alloys, such as Scalmalloy®, was investigated. This study demonstrates how to reduce uncontrolled interactions of the laser with the distinct weld plume, created by highly volatile alloying elements such as Mg. Tensile and fatigue testing were used to assess the structural integrity of specimens, in which different welding modes had been used. It is shown that a combination of: scan vector angle restriction; reduction of the scan vector length; and laser spot adjustments reduce the overall defect size and improves the build quality in Scalmalloy®. A bimodal microstructure with outstanding mechanical properties was observed: an ultimate tensile strength of 524 MPa was achieved with 17 % elongation at fracture. In order to evaluate the influence of the defect size, fatigue tests were performed at a stress ratio of R=0.1. Under optimal processing conditions, fatigue strengths of up to 360MPa at 3·107 cycles were obtained, significantly outperforming both conventionally and additively produced aluminium alloys.