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The human complement pathway plays a pivotal role in immune defence, homeostasis, and autoimmunity regulation, and complement-based therapeutics have emerged as promising interventions, with both antagonistic and agonistic approaches being explored. The classical pathway of complement is initiated when the C1 complex binds to hexameric antibody platforms. Recent structural data revealed that C1 binds to small, homogeneous interfaces at the periphery of the antibody platforms. Here, we have developed a novel strategy for complement activation using macrocyclic peptides designed to mimic the interface between antibodies and the C1 complex. In vitro selection utilizing the RaPID system identified a cyclic peptide (cL3) that binds to the C1 complex via the globular head domains of C1q. Notably, when immobilized on surfaces, cL3 effectively recruits C1 from human serum, activates C1s proteases, and induces lysis of cell-mimetic lipid membranes. This represents the first instance of a peptide capable of activating complement by binding C1 when immobilized. Further characterization and synthesis of deletion mutants revealed a critical cycle size of cL3 essential for C1 binding and efficient complement activation. Importantly, cL3 also demonstrated the ability to inhibit complement-mediated lysis without affecting C1 binding, highlighting its potential as a therapeutic modality to prevent complement-dependent cytotoxicity whilst promoting cellular phagocytosis and cell clearance. In summary, this study introduces the concept of " Peptactins " - peptide-based activators of complement - and underscores the potential of macrocyclic peptides for complement modulation, offering potential advantages over traditional biologicals in terms of size, production, and administration., Competing Interests: SMWRH, LA, ALB and THS are co-authors on a patent describing the development and use of peptactins. SAKJ declares no competing interests., (This journal is © The Royal Society of Chemistry.)

The correlation between TNM staging and histology variations in a sample of patients who underwent neoadjuvant chemotherapy demonstrates a positive impact on both increasing conservative surgery and achieving pCR, resulting in better outcomes in terms of disease-free survival (DFS) and the risk of relapse. Benefits have also been highlighted in terms of cosmetic outcomes, postoperative complications, and psychological benefits. However, the overall outcomes must be evaluated according to the subtype and individual characteristics of the patients.

Internet-based cognitive behavioral therapy (iCBT) offers a scalable, cost-effective, accessible, and low-threshold form of psychotherapy. Recent advancements explored the use of conversational agents such as chatbots and voice assistants to enhance the delivery of iCBT. These agents can deliver iCBT-based exercises, recognize and track emotional states, assess therapy progress, convey empathy, and potentially predict long-term therapy outcome. However, existing systems predominantly utilize categorical approaches for emotional modeling, which can oversimplify the complexity of human emotional states. To address this, we developed a transformer-based model for dimensional text-based emotion recognition, fine-tuned with a novel, comprehensive dimensional emotion dataset comprising 75,503 samples. This model significantly outperforms existing state-of-the-art models in detecting the dimensions of valence, arousal, and dominance, achieving a Pearson correlation coefficient of r  = 0.90, r  = 0.77, and r  = 0.64, respectively. Furthermore, a feasibility study involving 20 participants confirmed the model's technical effectiveness and its usability, acceptance, and empathic understanding in a conversational agent-based iCBT setting, marking a substantial improvement in personalized and effective therapy experiences., Competing Interests: The authors declare there are no competing interests., (©2024 Striegl et al.)

Purpose: A complication of using silicone oil as an intraocular endotamponade is its adhesion to intraocular lenses (IOLs). Forward light scattering is a measure to quantify the optical disturbance caused by adherent oil droplets. We tested the straylight caused by silicone oil adhesion to different IOLs and examined whether an approved cleaning solution, F4H5, reverses the induced straylight., Design: An experimental study., Methods: Two hydrophobic acrylic IOL models and 1 hydrophilic model with a hydrophobic surface (n = 8 per model: 24 lenses) had straylight measured before contact with silicone oils, providing a baseline for subsequent testing: 12 lenses with lighter-than-water silicone oil (Siluron 2000) and 12 with heavier-than-water oil (Densiron 68). The final measurement was performed after cleansing with F4H5 when we used scanning electron and light microscopy to detect surface changes., Results: Straylight was majorly increased in IOLs with adherent silicone oil (baseline vs adherent oil median 3.1 [2.1, 3.9] and 39.7 [22.7, 87.8] deg 2 /sr, respectively; P < .001). No difference was seen between heavier- and lighter-than-water silicone oils. Between IOL types, induced straylight varied significantly, with 1 hydrophobic model reaching the highest average straylight. F4H5 significantly reduced straylight values in all IOL types (median 9.4 [5.4, 13.8] deg 2 /sr). The microscopy revealed surface changes on the IOLs even after cleaning., Conclusions: Silicone oil adhesion to IOLs can induce amounts of straylight known to cause severe optical disturbance. F4H5 cleansing solution reversed straylight values to only slightly increased values. We found no difference in straylight formation between the lighter- and heavier-than-water silicone oils., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Recently, OTULIN haploinsufficiency was linked to enhanced susceptibility to Staphylococcus aureus infections accompanied by local necrosis and systemic inflammation. The pathogenesis observed in haploinsufficient patients differs from the hyperinflammation seen in classical OTULIN-related autoinflammatory syndrome (ORAS) patients and is characterized by increased susceptibility of dermal fibroblasts to S. aureus alpha toxin-inflicted cytotoxic damage. Immunological abnormalities were not observed in OTULIN haploinsufficient patients, suggesting a non-hematopoietic basis. In this research report, we investigated an Otulin +/- mouse model after in vivo provocation with lipopolysaccharide (LPS) to explore the potential role of hematopoietic-driven inflammation in OTULIN haploinsufficiency. We observed a hyperinflammatory signature in LPS-provoked Otulin +/- mice, which was driven by CD64 + monocytes and macrophages. Bone marrow-derived macrophages (BMDMs) of Otulin +/- mice demonstrated higher proinflammatory cytokine secretion after in vitro stimulation with LPS or polyinosinic:polycytidylic acid (Poly(I:C)). Our experiments in full and mixed bone marrow chimeric mice suggest that, in contrast to humans, the observed inflammation was mainly driven by the hematopoietic compartment with cell-extrinsic effects likely contributing to inflammatory outcomes. Using an OTULIN haploinsufficient mouse model, we validated the role of OTULIN in the regulation of environmentally directed inflammation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Staels, Bücken, De Vuyst, Willemsen, Van Nieuwenhove, Gerbaux, Neumann, Malviya, Van Meerbeeck, Haughton, Seldeslachts, Gouwy, Martinod, Vande Velde, Proost, Yshii, Schlenner, Schrijvers, Liston and Humblet-Baron.)

Mucopolysaccharidosis type I (MPS I) is an inherited lysosomal disease caused by lowered activity of the enzyme alpha-L-iduronidase (IDUA). Current therapeutic options show limited efficacy and do not treat some important aspects of the disease. Therefore, it may be advantageous to identify strategies that could improve the efficacy of existing treatments. Pharmacological chaperones are small molecules that protect proteins from degradation, and their use in combination with enzyme replacement therapy (ERT) has been proposed as an alternative therapeutic strategy. Using the SEE-Tx® proprietary computational drug discovery platform, a new allosteric ligand binding cavity in IDUA was identified distal from the active site. Virtual high-throughput screening of approximately 5 million compounds using the SEE-Tx® docking platform identified a subset of small molecules that bound to the druggable cavity and functioned as novel allosteric chaperones of IDUA. Experimental validation by differential scanning fluorimetry showed an overall hit rate of 11.4%. Biophysical studies showed that one exemplary hit molecule GT-01803 bound to (Kd = 22 μM) and stabilized recombinant human IDUA (rhIDUA) in a dose-dependent manner. Co-administration of rhIDUA and GT-01803 increased IDUA activity in patient-derived fibroblasts. Preliminary in vivo studies have shown that GT-01803 improved the pharmacokinetic (PK) profile of rhIDUA, increasing plasma levels in a dose-dependent manner. Furthermore, GT-01803 also increased IDUA enzymatic activity in bone marrow tissue, which benefits least from standard ERT. Oral bioavailability of GT-01803 was found to be good (50%). Overall, the discovery and validation of a novel allosteric chaperone for rhIDUA presents a promising strategy to enhance the efficacy of existing treatments for MPS I. The compound's ability to increase rhIDUA activity in patient-derived fibroblasts and its good oral bioavailability underscore its potential as a potent adjunct to ERT, particularly for addressing aspects of the disease less responsive to standard treatment., Competing Interests: Elena Cubero, Ana Ruano, Aida Delgado, Xavier Barril, Ana Trapero, Manolo Bellotto, Beatriz Calvo-Flores Guzmán, Natalia Pérez-Carmona and Ana Maria Garcia-Collazo are employees of Gain Therapeutics Sucursal en España or GT Gain Therapeutics SA. Sara Morales, Roberto Maj, past employees, and Lorenzo Leoni, scientific adviser, declare no conflicts of interest., (Copyright: © 2024 Cubero et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Complement activation protects against infection but also contributes to pathological mechanisms in a range of clinical conditions such as autoimmune diseases and transplant rejection. Complement-inhibitory drugs, either approved or in development, usually act systemically, thereby increasing the risk for infections. We therefore envisioned a novel class of bispecific antibodies (bsAbs) which are capable of site-directed complement inhibition by bringing endogenous complement regulators in the vicinity of defined cell surface antigens. Here, we analyzed a comprehensive set of obligate bsAbs designed to crosslink a specific target with either complement regulator factor H (FH) or C4b-binding protein (C4BP). The bsAbs were assessed for their capacity to inhibit complement activation and cell lysis in an antigen-targeted manner. We observed that the bsAbs inhibited classical, lectin, and alternative pathway complement activation in which sufficient endogenous serum FH and C4BP could be recruited to achieve local inhibition. Importantly, the bsAbs effectively protected antigen-positive liposomes, erythrocytes, and human leukocytes from complement-mediated lysis. In conclusion, localized complement inhibition by bsAbs capable of recruiting endogenous human complement regulators (such as FH or C4BP) to cell surfaces potentially provides a novel therapeutic approach for the targeted treatment of complement-mediated diseases., Competing Interests: Author PWHIP was employed by Gyes BV. FSB, PWHIP, and LAT are listed as inventors on a patent application regarding the application of targeted complement inhibition. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Wang, van de Bovenkamp, Dijkstra, Abendstein, Borggreven, Pool, Zuijderduijn, Gstöttner, Gelderman, Damelang, Vidarsson, Blom, Domínguez-Vega, Parren, Sharp and Trouw.)

This work introduces a new sustainable alternative of powdered activated carbon (PAC) - magnetically harvestable and reusable after regeneration via inductive heating - for the adsorptive removal of organic micropollutants (OMP) from secondary wastewater effluents. For this purpose, two commercial PACs - lignite "L" (1187 m 2 /g) and coconut "C"-based (1524 m 2 /g) - were modified with magnetic iron oxide following two different synthesis approaches: infiltration ("infiltr") and surface deposition ("depos") route. The resulting magnetic powdered activated carbons (mPAC) and their precursor PACs were fully characterized before application. The iron oxide content of the modified "L" and "C" samples was ∼30 % and ∼20 %, respectively. Iron oxide gives the PAC beneficial magnetic properties for easy magnetic separation and simultaneously acts as an inductively heatable agent for the carbon regeneration. The infiltrated samples displayed better inductive heating performance and regeneration than their deposited counterparts. Tests with real wastewater showed fast adsorption kinetics of the organic load following the pseudo-second-order kinetic model. Adsorption isotherms were compliant with the Freundlich isotherm model. Sample "L-infiltr" had the best overall adsorption performance throughout 5 reuse cycles when intermediately inductively regenerated (<3 % drop in organics removal per cycle with intermediate regeneration vs. ∼10 % drop per cycle without regeneration). The treated supernatant was additionally tested for 31 representative organic micropollutants and their transformation products (pharmaceuticals, personal care products, industrial chemicals, etc.), where 26 OMPs had consistently high removal (>85 %) throughout 5 cycles with intermediate regeneration and for 28 OMPs the total adsorption efficiency dropped by <5 % after 5 cycles., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

The classical complement pathway is activated by antigen-bound IgG antibodies. Monomeric IgG must oligomerize to activate complement via the hexameric C1q complex, and hexamerizing mutants of IgG appear as promising therapeutic candidates. However, structural data have shown that it is not necessary to bind all six C1q arms to initiate complement, revealing a symmetry mismatch between C1 and the hexameric IgG complex that has not been adequately explained. Here, we use DNA nanotechnology to produce specific nanostructures to template antigens and thereby spatially control IgG valency. These DNA-nanotemplated IgG complexes can activate complement on cell-mimetic lipid membranes, which enabled us to determine the effect of IgG valency on complement activation without the requirement to mutate antibodies. We investigated this using biophysical assays together with 3D cryo-electron tomography. Our data revealed the importance of interantigen distance on antibody-mediated complement activation, and that the cleavage of complement component C4 by the C1 complex is proportional to the number of ideally spaced antigens. Increased IgG valency also translated to better terminal pathway activation and membrane attack complex formation. Together, these data provide insights into how nanopatterning antigen-antibody complexes influence the activation of the C1 complex and suggest routes to modulate complement activation by antibody engineering. Furthermore, to our knowledge, this is the first time DNA nanotechnology has been used to study the activation of the complement system.

Background: Volumetric Absorptive Microsampling (VAMS) is emerging as a valuable technique in the collection of dried biological specimens, offering a potential alternative to traditional sampling methods. The objective of this study was to assess the suitability of 30 μL VAMS for the measurement of endogenous steroid hormones., Methods: A novel LC-MS/MS method was developed for the quantification of 18 analytes in VAMS samples, including main endogenous free steroids and phase II metabolites of androgens. The method underwent validation in accordance with ISO/IEC 17025:2017 and World Anti-Doping Agency (WADA) requirements. Subsequently, it was applied to authentic VAMS samples obtained from 20 healthy volunteers to assess the stability of target analytes under varying storage conditions., Results: The validation protocol assessed method's selectivity, matrix effect, extraction recovery, quantitative performance, carry-over and robustness. The analysis of authentic samples demonstrated the satisfactory stability of monitored steroids in VAMS stored at room temperature, 4 °C, -20 °C and -80 °C for up to 100 days and subjected to up to 3 freezing-thawing cycles., Conclusions: The validated LC-MS/MS method demonstrated its suitability for the measurement of steroids in dried blood VAMS. The observed stability of steroidal compounds suggests promising prospects for future applications of VAMS, both in anti-doping contexts and clinical research., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Basic psychological need crafting assumes that need-based experiences are enhanced through intentional behavior and thought changes. Despite its known benefits outside of the work context, need crafting instruments designed for this context, and the implications of need crafting for employee functioning, remain underexplored. Thus, this study set out to adapt and validate the need crafting scale (NCS) among employees ( n = 229). Results supported the construct, discriminant validity, and criterion validity of the NCS. The research also revealed that, through need crafting, employees reported enhanced experiences related to their needs, which led to higher work effort and engagement and a reduced desire to leave their jobs. Additionally, the different types of need crafting had differential direct effects on employee functioning, supporting a more nuanced understanding of the construct. As the first of its kind, the study underpins the relevance and generalizability of the NCS and need crafting in the workplace.

Hydrogen sulfide (H 2 S) has been proposed to protect bacteria from antibiotics, pointing to H 2 S-producing enzymes as possible targets for the development of antibiotic adjuvants. Here, MIC assays performed with Pseudomonas aeruginosa mutants producing altered H 2 S levels demonstrate that H 2 S does not affect antibiotic resistance in this bacterium. Moreover, correlation analyses in a large collection of P. aeruginosa cystic fibrosis isolates argue against the protective role of H 2 S from antibiotic activity during chronic lung infection., Competing Interests: The authors declare no conflict of interest.

Cartilage microbial DNA patterns have been recently characterized in osteoarthritis (OA). The objectives of this study were to evaluate the gut origins of cartilage microbial DNA, to characterize cartilage microbial changes with age, obesity, and OA in mice, and correlate these to gut microbiome changes. We used 16S rRNA sequencing performed longitudinally on articular knee cartilage from germ-free (GF) mice following oral microbiome inoculation and cartilage and cecal samples from young and old wild-type mice with/without high-fat diet-induced obesity (HFD) and with/without OA induced by destabilization of the medial meniscus (DMM) to evaluate gut and cartilage microbiota. Microbial diversity was assessed, groups compared, and functional metagenomic profiles reconstructed. Findings were confirmed in an independent cohort by clade-specific qPCR. We found that cartilage microbial patterns developed at 48 h and later timepoints following oral microbiome inoculation of GF mice. Alpha diversity was increased in SPF mouse cartilage samples with age (P = 0.013), HFD (P = 5.6E-4), and OA (P = 0.029) but decreased in cecal samples with age (P = 0.014) and HFD (P = 1.5E-9). Numerous clades were altered with aging, HFD, and OA, including increases in Verrucomicrobia in both cartilage and cecal samples. Functional analysis suggested changes in dihydroorotase, glutamate-5-semialdehyde dehydrogenase, glutamate-5-kinase, and phosphoribosylamine-glycine ligase, in both cecum and cartilage, with aging, HFD, and OA. In conclusion, cartilage microbial DNA patterns develop rapidly after the introduction of a gut microbiome and change in concert with the gut microbiome during aging, HFD, and OA in mice. DMM-induced OA causes shifts in both cartilage and cecal microbiome patterns independent of other factors., (© 2023. The Author(s).)

Guidelines are important tools to guide the diagnosis and treatment of patients to improve the decision-making process of health professionals. They are periodically updated according to new evidence. Four new Guidelines in 2021, 2022 and 2023 referred to pediatric pacing and defibrillation. There are some relevant changes in permanent pacing. In patients with atrioventricular block, the heart rate limit in which pacemaker implantation is recommended was decreased to reduce too-early device implantation. However, it was underlined that the heart rate criterion is not absolute, as signs or symptoms of hemodynamically not tolerated bradycardia may even occur at higher rates. In sinus node dysfunction, symptomatic bradycardia is the most relevant recommendation for pacing. Physiological pacing is increasingly used and recommended when the amount of ventricular pacing is presumed to be high. New recommendations suggest that loop recorders may guide the management of inherited arrhythmia syndromes and may be useful for severe but not frequent palpitations. Regarding defibrillator implantation, the main changes are in primary prevention recommendations. In hypertrophic cardiomyopathy, pediatric risk calculators have been included in the Guidelines. In dilated cardiomyopathy, due to the rarity of sudden cardiac death in pediatric age, low ejection fraction criteria were demoted to class II. In long QT syndrome, new criteria included severely prolonged QTc with different limits according to genotype, and some specific mutations. In arrhythmogenic cardiomyopathy, hemodynamically tolerated ventricular tachycardia and arrhythmic syncope were downgraded to class II recommendation. In conclusion, these new Guidelines aim to assess all aspects of cardiac implantable electronic devices and improve treatment strategies.

Metformin is a highly effective medication for managing type 2 diabetes mellitus. Recent studies have shown that it has significant therapeutic benefits in various organ systems, particularly the liver. Although the effects of metformin on metabolic dysfunction-associated steatotic liver disease and metabolic dysfunction-associated steatohepatitis are still being debated, it has positive effects on cirrhosis and anti-tumoral properties, which can help prevent the development of hepatocellular carcinoma. Furthermore, it has been proven to improve insulin resistance and dyslipidaemia, commonly associated with liver diseases. While more studies are needed to fully determine the safety and effectiveness of metformin use in liver diseases, the results are highly promising. Indeed, metformin has a terrific potential for extending its full therapeutic properties beyond its traditional use in managing diabetes.

Excessive fibrous capsule formation around silicone mammary implants (SMI) involves immune reactions to silicone. Capsular fibrosis, a common SMI complication linked to host responses, worsens with specific implant topographies. Our study with 10 patients investigated intra- and inter-individually, reduced surface roughness effects on disease progression, wound responses, chronic inflammation, and capsular composition. The results illuminate the significant impact of surface roughness on acute inflammatory responses, fibrinogen accumulation, and the subsequent fibrotic cascade. The reduction of surface roughness to an average roughness of 4 μm emerges as a promising approach for mitigating detrimental immune reactions, promoting healthy wound healing, and curbing excessive fibrosis. The identified proteins adhering to rougher surfaces shed light on potential mediators of pro-inflammatory and pro-fibrotic processes, further emphasizing the need for meticulous consideration of surface design. The composition of the implant capsule and the discovery of intracapsular HSP60 expression highlight the intricate web of stress responses and immune activation that can impact long-term tissue outcomes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Schoberleitner, Faserl, Tripp, Pechriggl, Sigl, Brunner, Zelger, Hermann-Kleiter, Baier, Steinkellner, Sarg, Egle, Brunner and Wolfram.)

Breast cancer is the most common cancer in women globally, often necessitating mastectomy and subsequent breast reconstruction. Silicone mammary implants (SMIs) play a pivotal role in breast reconstruction, yet their interaction with the host immune system and microbiome remains poorly understood. This study investigates the impact of SMI surface topography on host antimicrobial responses, wound proteome dynamics, and microbial colonization. Biological samples were collected from ten human patients undergoing breast reconstruction with SMIs. Mass spectrometry profiles were analyzed for acute and chronic wound proteomes, revealing a nuanced interplay between topography and antimicrobial response proteins. 16S rRNA sequencing assessed microbiome dynamics, unveiling topography-specific variations in microbial composition. Surface topography alterations influenced wound proteome composition. Microbiome analysis revealed heightened diversity around rougher SMIs, emphasizing topography-dependent microbial invasion. In vitro experiments confirmed staphylococcal adhesion, growth, and biofilm formation on SMI surfaces, with increased texture correlating positively with bacterial colonization. This comprehensive investigation highlights the intricate interplay between SMI topography, wound proteome dynamics, and microbial transmission. The findings contribute to understanding host-microbe interactions on SMI surfaces, essential for optimizing clinical applications and minimizing complications in breast reconstruction.

Aims: Antimicrobial resistance is a global threat to human health, and Acinetobacter baumannii is a paradigmatic example of how rapidly bacteria become resistant to clinically relevant antimicrobials. The emergence of multidrug-resistant A. baumannii strains has forced the revival of colistin as a last-resort drug, suddenly leading to the emergence of colistin resistance. We investigated the genetic and molecular basis of colistin resistance in A. baumannii, and the mechanisms implicated in its regulation and dissemination., Methods: Comparative genomic analysis was combined with genetic, biochemical, and phenotypic assays to characterize Φ19606, an A. baumannii temperate bacteriophage that carries a colistin resistance gene., Results: Ф19606 was detected in 41% of 523 A. baumannii complete genomes and demonstrated to act as a mobile vehicle of the colistin resistance gene eptA1, encoding a functional lipid A phosphoethanolamine transferase. The eptA1 gene is coregulated with its chromosomal homolog pmrC via the PmrAB two-component system and confers colistin resistance when induced by low calcium and magnesium levels. Resistance selection assays showed that the eptA1-harbouring phage Ф19606 promotes the emergence of spontaneous colistin-resistant mutants., Conclusions: Φ19606 is an unprecedented example of a self-transmissible phage vector implicated in the dissemination of colistin resistance., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The outer membrane (OM) is an essential structure of Gram-negative bacteria that provides mechanical strength and protection from large and/or hydrophobic toxic molecules, including many antibiotics. The OM is composed of glycerophospholipids (GPLs) and lipopolysaccharide (LPS) in the inner and outer leaflets, respectively, and hosts integral β-barrel proteins and lipoproteins. While the systems responsible for translocation and insertion of LPS and OM proteins have been elucidated, the mechanism(s) mediating transport of GPLs from the inner membrane to the OM has remained elusive for decades. Very recently, studies performed in Escherichia coli proposed a role in this process for AsmA-like proteins that are predicted to share structural features with eukaryotic lipid transporters. In this study, we provide the first systematic investigation of AsmA-like proteins in a bacterium other than E. coli , the opportunistic human pathogen Pseudomonas aeruginosa . Bioinformatic analyses revealed that P. aeruginosa possesses seven AsmA-like proteins. Deletion of asmA -like genes in many different combinations, coupled with conditional mutagenesis, revealed that four AsmA-like proteins are redundantly essential for growth and OM integrity in P. aeruginosa , including a novel AsmA-like protein (PA4735) that is not present in E. coli . Cells depleted of AsmA-like proteins showed severe defects in the OM permeability barrier that were partially rescued by lowering the synthesis or transport of LPS. Since fine balancing of GPL and LPS levels is crucial for OM integrity, this evidence supports the role of AsmA-like proteins in GPL transport toward the OM., Importance: Given the importance of the outer membrane (OM) for viability and antibiotic resistance in Gram-negative bacteria, in the last decades, several studies have focused on the characterization of the systems involved in OM biogenesis, which have also been explored as targets for antibacterial drug development. However, the mechanism mediating translocation of glycerophospholipids (GPLs) to the OM remained unknown until recent studies provided evidence that AsmA-like proteins could be responsible for this process. Here, we demonstrate for the first time that AsmA-like proteins are essential and redundant for growth and OM integrity in a Gram-negative bacterium other than the model organism Escherichia coli and demonstrate that the human pathogen Pseudomonas aeruginosa has an additional essential AsmA-like protein that is not present in E. coli , thus expanding the range of AsmA-like proteins that play key functions in Gram-negative bacteria., Competing Interests: The authors declare no conflict of interest.

Objectives: The Murphy Roths Large (MRL)/MpJ 'superhealer' mouse strain is protected from post-traumatic osteoarthritis (OA), although no studies have evaluated the microbiome in the context of this protection. This study characterised microbiome differences between MRL and wild-type mice, evaluated microbiome transplantation and OA and investigated microbiome-associated immunophenotypes., Methods: Cecal material from mixed sex C57BL6/J (B6) or female MRL/MpJ (MRL) was transplanted into B6 and MRL mice, then OA was induced by disruption of the medial meniscus surgery (DMM). In other experiments, transplantation was performed after DMM and transplantation was performed into germ-free mice. Transplanted mice were bred through F2. OARSI, synovitis and osteophyte scores were determined blindly 8 weeks after DMM. 16S microbiome sequencing was performed and metagenomic function was imputed. Immunophenotypes were determined using mass cytometry., Results: MRL-into-B6 transplant prior to DMM showed reduced OA histopathology (OARSI score 70% lower transplant vs B6 control), synovitis (60% reduction) and osteophyte scores (30% reduction) 8 weeks after DMM. When performed 48 hours after DMM, MRL-into-B6 transplant improved OA outcomes but not when performed 1-2 weeks after DMM. Protection was seen in F1 (60% reduction) and F2 progeny (30% reduction). Several cecal microbiome clades were correlated with either better (eg, Lactobacillus, R=-0.32, p=0.02) or worse (eg, Rikenellaceae , R=0.43, p=0.001) OA outcomes. Baseline immunophenotypes associated with MRL-into-B6 transplants and MRL included reduced double-negative T cells and increased CD25+CD4+ T cells., Conclusion: The gut microbiome is responsible in part for OA protection in MRL mice and is transferrable by microbiome transplantation. Transplantation induces resting systemic immunophenotyping changes that correlate with OA protection., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Objective: Females have reduced osteoarthritis (OA) in surgical models. The objective of the current study was to evaluate a sex-linked gut microbiome in the pathogenesis of OA., Methods: We induced OA via destabilization of the medial meniscus surgery in adult male and female C57BL6/J mice with and without opposite-sex microbiome transplantation. Eight weeks later, animals were euthanized, and OA severity, synovitis, and osteophyte scores were determined. Serum lipopolysaccharide was measured chromogenically, and serum cytokines were quantified via multiplex immunoassay. Cecal microbiome profiles were generated using 16S deep sequencing., Results: Males had worse OA histology (3.5x, P = 6 × 10 -7 ), synovitis (2.4x, P = 5 × 10 -4 ), and osteophyte scores (3.7x, P = 3 × 10 -4 ) than females. Male-into-female transplantation worsened all outcomes (histology 1.8x, P = 0.02; synovitis 2.0x, P = 3 × 10 -5 ; osteophyte 2.1x, P = 0.01) compared to females, whereas female-into-male transplantation improved all outcomes except for synovitis (histology 0.53x, P = 2 × 10 -4 ; osteophyte 0.28x, P = 5 × 10 -4 ) compared to males. In the gut microbiome analysis, 44 clades were different in at least one group comparison; 5 clades were correlated with the Osteoarthritis Research Society International score (Lactobacillus R = -0.40, Aldercreutzia R = -0.40, rc4&#95;4 R = -0.55, Sutterella R = -0.37, and Clostridiales R = 0.36). In the cytokine analysis, 10 analytes were different in at least one group comparison; 3 were different in two groups (female and female-into-male transplants vs male comparisons, all reduced in female and female-into-male transplants), including interleukin-12 (0.66x, P = 0.02; 0.66x, P = 0.02, respectively), eotaxin (0.74x, P = 5 × 10 -6 ; 0.57x, P = 0.03), and tumor necrosis factor ⍺ (0.49x, P = 0.03; 0.52x, P = 0.009)., Conclusion: Sex-linked differences in the mouse gut microbiome are associated with OA outcomes, are reversible by opposite-sex microbiome transplantation, and are associated with serum cytokine changes., (© 2023 American College of Rheumatology.)

Vanishing white matter (VWM) is a devastating autosomal recessive leukodystrophy, resulting in neurological deterioration and premature death, and without curative treatment. Pathogenic hypomorphic variants in subunits of the eukaryotic initiation factor 2B (eIF2B) cause VWM. eIF2B is required for regulating the integrated stress response (ISR), a physiological response to cellular stress. In patients' central nervous system, reduced eIF2B activity causes deregulation of the ISR. In VWM mouse models, the extent of ISR deregulation correlates with disease severity. One approach to restoring eIF2B activity is by inhibition of GSK3β, a kinase that phosphorylates eIF2B and reduces its activity. Lithium, an inhibitor of GSK3β, is thus expected to stimulate eIF2B activity and ameliorate VWM symptoms. The effects of lithium were tested in zebrafish and mouse VWM models. Lithium improved motor behavior in homozygous eif2b5 mutant zebrafish. In lithium-treated 2b4 he 2b5 ho mutant mice, a paradoxical increase in some ISR transcripts was found. Furthermore, at the dosage tested, lithium induced significant polydipsia in both healthy controls and 2b4 he 2b5 ho mutant mice and did not increase the expression of other markers of lithium efficacy. In conclusion, lithium is not a drug of choice for further development in VWM based on the limited or lack of efficacy and significant side-effect profile., Competing Interests: JB is on the board of wFluidx, Inc., owns stock in Orchard Therapeutics, and has consulted for Bluebird bio, Calico Life Sciences, Denali Therapeutics, Enzyvant, and Neurogene. MK and TEMA have a patent PCT/NL2018/050293 on guanabenz in VWM pending to VUmc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Witkamp, Oudejans, Hoogterp, Hu-A-Ng, Glaittli, Stevenson, Huijsmans, Abbink, van der Knaap and Bonkowsky.)

The leukodystrophy vanishing white matter (VWM) is characterized by chronic and episodic acute neurological deterioration. Curative treatment is presently unavailable. Pathogenic variants in the genes encoding eukaryotic initiation factor 2B (eIF2B) cause VWM and deregulate the integrated stress response (ISR). Previous studies in VWM mouse models showed that several ISR-targeting compounds ameliorate clinical and neuropathological disease hallmarks. It is unclear which ISR components are suitable therapeutic targets. In this study, effects of 4-phenylbutyric acid, tauroursodeoxycholic acid, or pridopidine (PDPD), with ISR targets upstream or downstream of eIF2B, were assessed in VWM mice. In addition, it was found that the composite ataxia score represented motor decline of VWM mice more accurately than the previously used neuroscore. 4-phenylbutyric acid and tauroursodeoxycholic acid did not improve VWM disease hallmarks, whereas PDPD had subtle beneficial effects on motor skills. PDPD alone does not suffice as treatment in VWM mice but may be considered for combination therapy. Also, treatments aimed at ISR components upstream of eIF2B do not improve chronic neurological deterioration; effects on acute episodic decline remain to be investigated., (© 2024 Oudejans et al.)

Electroporation (EP) of mRNA into human cells is a broadly applicable method to transiently express proteins of choice in a variety of different cell types. We have spent more than two decades to optimize and adapt this method, first for antigen-loading of dendritic cells (DCs) and subsequently for T cells, B cells, bulk PBMCs, and several cell lines. In this regard, antigens were introduced, processed, and presented in context of MHC class I and II. Next to that, functional proteins like adhesion receptors, T-cell receptors (TCRs), chimeric antigen receptors (CARs), constitutively active signal transducers (i.e. caIKK), and others were successfully expressed. We have also established this protocol under full GMP compliance as part of a manufacturing license to produce mRNA-electroporated DCs and mRNA-electroporated T cells for therapeutic applications in clinical trials. Therefore, we here want to share our universal mRNA electroporation protocol and the experience we have gathered with this method. The advantages of the transfection method presented here are: (1) easy adaptation to different cell types; (2) scalability from 10 6 to approximately 10 8 cells per shot; (3) high transfection efficiency (80-99%); (4) homogenous protein expression; (5) GMP compliance if the EP is performed in a class A clean room; and (6) no transgene integration into the genome. The provided protocol involves: OptiMEM ® as EP medium, a square-wave pulse with 500 V, and 4 mm cuvettes. To adapt the protocol to differently sized cells, simply the pulse time has to be altered. Thus, we share an overview of proven electroporation settings (including recovery media), which we have established for various cell types. Next to the basic protocol, we also provide an extensive list of hints and tricks, which, in our opinion, are of great value for everyone who intends to use this transfection technique., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Proteolytic activity plays an essential role in Pseudomonas aeruginosa adaptation and survival in challenging environments, including the infection site. Here, a short review of the eight known proteases secreted by P. aeruginosa and of the methods used to detect their activity is provided. In addition, three simple and handy methods routinely used in our laboratory to detect proteases are described in detail. In particular, the skim milk plate assay and the azocasein assay are useful for the detection of whole proteases activity in colony-growing and cell-free culture supernatants, respectively. Conversely, the Elastin Congo-red assay allows detecting the activity of the LasB elastase, the major protease secreted by P. aeruginosa, in cell-free culture supernatants., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

In Pseudomonas aeruginosa relevant features including virulence and biofilm formation are controlled by quorum sensing (QS), a cell density-dependent intercellular communication system based on the production and response to signal molecules. P. aeruginosa has evolved chemically distinct compounds employed as QS signal molecules (QSSMs) that can be detected and quantified through rapid, sensitive, and low-cost methods based on whole-cell biosensors. Here, we present a series of protocols based on whole-cell biosensors for qualitative and quantitative analysis of QSSMs produced by P. aeruginosa. These protocols can be used to investigate the impact of environmental conditions, genetic modifications, or quorum quenching agents on the production of QSSMs in P. aeruginosa., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Importance: Single-cell analyses can reveal that despite experiencing identical physico-chemical conditions, individual bacterial cells within a monoclonal population may exhibit variations in gene expression. Such phenotypic heterogeneity has been described for several aspects of bacterial physiology, including QS activation. This study demonstrates that the transition of non-quorate cells to the quorate state is a graded process that does not occur at a specific cell density and that subpopulations of non-quorate cells also persist at high cell density. Here, we provide a mechanistic explanation for this phenomenon, showing that a negative feedback regulatory loop integrated into the las system has a pivotal role in promoting cell-to-cell variation in the QS activation state and in limiting the transition of non-quorate cells to the quorate state in P. aeruginosa ., Competing Interests: The authors declare no conflict of interest.

Autoantibodies directed against complement component C1q are commonly associated with autoimmune diseases, especially systemic lupus erythematosus. Importantly, these anti-C1q autoantibodies are specific for ligand-bound, solid-phase C1q and do not bind to fluid-phase C1q. In patients with anti-C1q, C1q levels are in the normal range, and the autoantibodies are thus not depleting. To study these human anti-C1q autoantibodies at the molecular level, we isolated C1q-reactive B cells and recombinantly produced nine monoclonal antibodies (mAbs) from four different healthy individuals. The isolated mAbs were of the IgG isotype, contained extensively mutated variable domains, and showed high affinity to the collagen-like region of C1q. The anti-C1q mAbs exclusively bound solid-phase C1q in complex with its natural ligands, including immobilized or antigen-bound IgG, IgM or CRP, and necrotic cells. Competition experiments reveal that at least 2 epitopes, also targeted by anti-C1q antibodies in sera from SLE patients, are recognized. Electron microscopy with hexameric IgG-C1q immune complexes demonstrated that multiple mAbs can interact with a single C1q molecule and identified the region of C1q targeted by these mAbs. The opsonization of immune complexes with anti-C1q greatly enhanced Fc-receptor-mediated phagocytosis but did not increase complement activation. We conclude that human anti-C1q autoantibodies specifically bind neo-epitopes on solid-phase C1q, which results in an increase in Fc-receptor-mediated effector functions that may potentially contribute to autoimmune disease immunopathology., Competing Interests: Competing interests statement: D.J.D. and L.A.T. are coinventors on a patent application describing antibodies against complement protein C1q.

Introduction: Microbes are increasingly (re)considered for environmental assessments because they are powerful indicators for the health of ecosystems. The complexity of microbial communities necessitates powerful novel tools to derive conclusions for environmental decision-makers, and machine learning is a promising option in that context. While amplicon sequencing is typically applied to assess microbial communities, metagenomics and total RNA sequencing (herein summarized as omics-based methods) can provide a more holistic picture of microbial biodiversity at sufficient sequencing depths. Despite this advantage, amplicon sequencing and omics-based methods have not yet been compared for taxonomy-based environmental assessments with machine learning., Methods: In this study, we applied 16S and ITS-2 sequencing, metagenomics, and total RNA sequencing to samples from a stream mesocosm experiment that investigated the impacts of two aquatic stressors, insecticide and increased fine sediment deposition, on stream biodiversity. We processed the data using similarity clustering and denoising (only applicable to amplicon sequencing) as well as multiple taxonomic levels, data types, feature selection, and machine learning algorithms and evaluated the stressor prediction performance of each generated model for a total of 1,536 evaluated combinations of taxonomic datasets and data-processing methods., Results: Sequencing and data-processing methods had a substantial impact on stressor prediction. While omics-based methods detected a higher diversity of taxa than amplicon sequencing, 16S sequencing outperformed all other sequencing methods in terms of stressor prediction based on the Matthews Correlation Coefficient. However, even the highest observed performance for 16S sequencing was still only moderate. Omics-based methods performed poorly overall, but this was likely due to insufficient sequencing depth. Data types had no impact on performance while feature selection significantly improved performance for omics-based methods but not for amplicon sequencing., Discussion: We conclude that amplicon sequencing might be a better candidate for machine-learning-based environmental stressor prediction than omics-based methods, but the latter require further research at higher sequencing depths to confirm this conclusion. More sampling could improve stressor prediction performance, and while this was not possible in the context of our study, thousands of sampling sites are monitored for routine environmental assessments, providing an ideal framework to further refine the approach for possible implementation in environmental diagnostics., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Hempel, Buchner, Mack, Brasseur, Tulpan, Leese and Steinke.)

Background: Finding the appropriate endovascular revascularization strategy for patients with peripheral artery disease and a popliteal artery lesion remains particulary challenging. Data regarding predictors for a beneficial outcome are scarce. Patients and methods: All endovascular procedures of popliteal artery lesions (n=227) performed in 197 patients between February 2009 and May 2018 at our institution were retrospectively analyzed. Hemodynamically relevant restenosis represented the primary endpoint. Results: The overall technical success rate was 98% and yielded 99% for stenoses (n=145) and 97% for occlusions (n=82). In a median follow-up of 10 months, the overall rate of restenosis was 23%. After 1 and 2 years, the primary patency rates were 76% and 55% and the secondary patency rate was 100%, respectively. The estimated probability of restenosis was significantly higher in stented lesions (stent vs. no stent; 36.0% vs. 19.1%; p=0.030). Multivariate analysis identified stent implantation (hazard ratio: 2.4; overall P=0.010) and diabetes (hazard ratio 2.0; P=0.023) as significant predictors for the development of restenosis. Conclusions: Endovascular therapy for popliteal artery disease was associated with high technical success rates and accompanied with a promising mid-term outcome, particularly in lesions treated with balloon angioplasty alone.

Purpose: Residual glandular tissue (RGT) after risk reducing mastectomy (RRME) is associated with a risk of developing breast cancer for women with a familial predisposition. We aim to examine various surgery-related variables to make risk more easily assessable and to aid in decision-making., Materials and Methods: Pre- and postoperative breast MRI scans from 2006 to 2021 of patients with proven pathogenic mutation were included. The postoperative remaining skin flap was recorded using distance measurements at 8 equally distributed clockwise points and retromamillary. Each breast was volumetrized, as well as existing RGT. Patient-related covariates were further recorded and their influence on RGT was investigated uni- and multivariately., Results: 81 patients (49 with BRCA1, 24 with BRCA2, 9 with other mutations), who were on average 39 years old, had 117 breasts analyzed. The mean follow-up was 71 months. In multivariate analysis, the independent variable skin flap thickness had a positive effect (p ≤ 0.01), while surgeon experience negatively affected RGT (p ≤ 0.05). The incision type was found to impact RGT as well, with nipple-sparing mastectomy (NSM) with inframammary fold incision leading to more RGT (p ≤ 0.01 - p ≤ 0.05), and skin-sparing mastectomy (SSM) with an inverted T incision leading to less (p ≤ 0.01)., Conclusion: Different surgical variables have an impact on postoperative RGT, which is an important tool to quantify the risk of developing breast cancer after RRME. In order to effectively consider these variables in future preoperative/intraoperative management, they must be carefully taken into account., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (© 2023 Published by Elsevier Ltd.)

Spray-drying is a popular and well-known "drying tool" for engineers. This perspective highlights that, beyond this application, spray-drying is a very interesting and powerful tool for materials chemists to enable the design of multifunctional and hybrid materials. Upon spray-drying, the confined space of a liquid droplet is narrowed down, and its ingredients are forced together upon "falling dry." As  detailed in this article, this enables the following material formation strategies either individually or even in combination: nanoparticles and/or molecules can be assembled; precipitation reactions as well as chemical syntheses can be performed; and templated materials can be designed. Beyond this, fragile moieties can be processed, or "precursor materials" be prepared. Post-treatment of spray-dried objects eventually enables the next level in the design of complex materials. Using spray-drying to design (particulate) materials comes with many advantages-but also with many challenges-all of which are outlined here. It is believed that multifunctional, hybrid materials, made via spray-drying, enable very unique property combinations that are particularly highly promising in myriad applications-of which catalysis, diagnostics, purification, storage, and information are highlighted., (© 2023 The Authors. Advanced Materials published by Wiley-VCH GmbH.)

Introduction: The increase in bacterial strains resistant to conventional antibiotics is an alarming problem for human health and could lead to pandemics in the future. Among bacterial pathogens responsible for a large variety of severe infections there is Pseudomonas aeruginosa . Therefore, there is an urgent need for new molecules with antimicrobial activity or that can act as adjuvants of antibiotics already in use. In this scenario, antimicrobial peptides (AMPs) hold great promise. Recently, we characterized a frog-skin AMP derived from esculentin-1a, namely Esc(1-21)-1c, endowed with antipseudomonal activity without being cytotoxic to human cells. Methods: The combinatorial effect of the peptide and antibiotics was investigated through the checkerboard assay, differential proteomic and transcriptional analysis. Results: Here, we found that Esc(1-21)-1c can synergistically inhibit the growth of P. aeruginosa cells with three different antibiotics, including tetracycline. We therefore investigated the underlying mechanism implemented by the peptide using a differential proteomic approach. The data revealed a significant decrease in the production of three proteins belonging to the MexAB-OprM efflux pump upon treatment with sub-inhibitory concentration of Esc(1-21)-1c. Down-regulation of these proteins was confirmed by transcriptional analysis and direct measurement of their relative levels in bacterial cells by tandem mass spectrometry analysis in multiple reaction monitoring scan mode. Conclusion: These evidences suggest that treatment with Esc(1-21)-1c in combination with antibiotics would increase the intracellular drug content making bacteria more susceptible to the antibiotic. Overall, these results highlight the importance of characterizing new molecules able to synergize with conventional antibiotics, paving the way for the development of alternative therapeutic strategies based on AMP/antibiotic formulations to counteract the emergence of resistant bacterial strains and increase the use of "old" antibiotics in medical practice., Competing Interests: Author ADS was employed by CEINGE Biotecnologie Avanzate. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Canè, Casciaro, Di Somma, Loffredo, Puglisi, Battaglia, Mellini, Cappiello, Rampioni, Leoni, Amoresano, Duilio and Mangoni.)

Streptogramins are the last line of defense antimicrobials with pristinamycin as a representative substance used as therapeutics against highly resistant pathogenic bacteria. However, the emergence of (multi)drug-resistant pathogens renders these valuable antibiotics useless; making it necessary to derivatize compounds for new compound characteristics, which is often difficult by chemical de novo synthesis due to the complex nature of the molecules. An alternative to substance derivatization is mutasynthesis. Herein, we report about a mutasynthesis approach, targeting the phenylglycine (Phg) residue for substance derivatization, a pivotal component of streptogramin antibiotics. Mutasynthesis with halogenated Phg(-like) derivatives altogether led to the production of two new derivatized natural compounds, as there are 6-chloropristinamycin I and 6-fluoropristinamycin I based on LC-MS/MS analysis. 6-Chloropristinamycin I and 6-fluoropristinamycin I were isolated by preparative HPLC, structurally confirmed using NMR spectroscopy and tested for antimicrobial bioactivity. In a whole-cell biotransformation approach using an engineered E. coli BL21(DE3) pET28- hmo /pACYC- bcd-gdh strain, Phg derivatives were generated fermentatively. Supplementation with the E. coli biotransformation fermentation broth containing 4-fluorophenylglycine to the pristinamycin mutasynthesis strain resulted in the production of 6-fluoropristinamycin I, demonstrating an advanced level of mutasynthesis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (This journal is © The Royal Society of Chemistry.)

Background: Opacification through calcification of hydrophilic acrylic intraocular lenses is a serious complication of cataract surgery, which usually results in explantation of the lens. In the process of calcification, the intraocular lens material plays a crucial role: calcification only occurs in hydrophilic acrylic lenses. Hydrophobic acrylic lenses show no crystal formation within the polymer. Hydrophilic acrylic lenses from some manufacturers have hydrophobic surface properties. The question arises as to what influence these surface properties have on the risk of calcification., Objective: The present study investigated whether the hydrophobic surface properties of hydrophilic acrylic lenses can prevent calcification., Material and Methods: Using an electrophoretic in vitro model of calcification, two hydrophilic lenses with hydrophobic surface properties were compared to two hydrophilic lenses and a hydrophobic negative control to determine the risk of calcification. The lenses were then analyzed by optical microscopy, Alizarin Red and Von Kossa staining, scanning electron microscopy (SEM) and energy dispersive X‑ray spectroscopy (EDX)., Results: All four hydrophilic lens models showed calcification within the polymer. No difference was found between the hydrophilic lenses and the hydrophilic lenses with hydrophobic surface properties in terms of crystal formation. The hydrophobic negative control showed no calcification., Conclusion: The investigation conducted in this study under standardized conditions could show that hydrophobic surface properties of hydrophilic acrylic lenses do not protect against calcium phosphate crystal formation within the polymer. There also is a risk of calcification in these lens models., (© 2023. The Author(s).)

Overt or concealed accessory pathways are the anatomic substrates of ventricular preexcitation (VP), Wolff-Parkinson-White syndrome (WPW) and paroxysmal supraventricular tachycardia (PSVT). These arrhythmias are commonly observed in pediatric age. PSVT may occur at any age, from fetus to adulthood, and its symptoms range from none to syncope or heart failure. VP too can range from no symptoms to sudden cardiac death. Therefore, these arrhythmias frequently need risk stratification, electrophysiologic study, drug or ablation treatment. In this review of the literature, recommendations are given for diagnosis and treatment of fetal and pediatric age (≤12 years) WPW, VP, PSVT, and criteria for sport participation., (Copyright © 2023 Italian Federation of Cardiology - I.F.C. All rights reserved.)

In the field of nuclear medicine, the β + -emitting 43 Sc and β - -emitting 47 Sc are promising candidates in cancer diagnosis and targeted radionuclide therapy (TRT) due to their favorable decay schema and shared pharmacokinetics as a true theranostic pair. Additionally, scandium is a group-3 transition metal (like 177 Lu) and exhibits affinity for DOTA-based chelators, which have been studied in depth, making the barrier to implementation lower for 43/47 Sc than for other proposed true theranostics. Before 43/47 Sc can see widespread pre-clinical evaluation, however, an accessible production methodology must be established and each isotope's radiolabeling and animal imaging capabilities studied with a widely utilized tracer. As such, a simple means of converting an 18 MeV biomedical cyclotron to support solid targets and produce 43 Sc via the 42 Ca(d,n) 43 Sc reaction has been devised, exhibiting reasonable yields. The Nat Ti(γ,p) 47 Sc reaction is also investigated along with the successful implementation of chemical separation and purification methods for 43/47 Sc. The conjugation of 43/47 Sc with PSMA-617 at specific activities of up to 8.94 MBq/nmol and the subsequent imaging of LNCaP-ENZaR tumor xenografts in mouse models with both 43/47 Sc-PSMA-617 are also presented.

The efficacy of the late-evening snack (LES) has been extensively studied due to the impact of the longest intermeal duration occurring at night in patients with cirrhosis. While actual clinical guidelines on nutrition in chronic liver disease recommend an LES, no specific nutritional compositions have been reported by the European Association for the Study of the Liver (EASL) and the European Society for Clinical Nutrition and Metabolism (ESPEN). Late-evening snacks vary greatly among studies, including natural foods and/or nutritional supplements, yet oral supplements still need to fully meet the LES's nutritional composition. In addition, many hepatologists need to gain experience in nutritional approaches and have access to registered dieticians who can help them manage patients with liver disease. Therefore, this review study aims to summarise evidence regarding using LESs and the mechanisms behind long starvation in patients with cirrhosis. It also provides a practical nutritional guide with several LES options based on common natural foods tailored to special patients' nutritional requirements and geographical backgrounds. In preventing accelerated starvation and related protein malnutrition and sarcopenia in patients with cirrhosis, the nutritional composition of LESs is essential. The proper and straightforward application of the LES's rational nutrition is an advantage to cirrhotic patients and should be carried out by healthcare professionals to enhance the overall liver function and nutritional status of patients with cirrhosis.

Opacification of intraocular lenses (IOLs) due to material changes is a serious complication that can compromise the good visual outcomes of uncomplicated cataract surgery. In hydrophobic acrylic IOLs, opacification can result from glistening formation, while in hydrophilic acrylic IOLs, there is a risk of calcification due to the formation of calcium phosphates within the polymer. Over time, various methods have been developed to investigate calcification in hydrophilic acrylic IOLs. The aim of this article is to provide an overview of standard histological staining and models used to simulate IOL calcification. Histological staining can be used to detect calcification and assess the extent of crystal formation. The development of in vivo and in vitro replication models has helped to identify the underlying pathomechanisms of calcification. In vivo models are suitable for assessing the biocompatibility of IOL materials. Bioreactors as an in vitro model can be used to investigate the kinetics of crystal formation within the polymer. The replication of IOL calcification under standardized conditions using electrophoresis allows for the comparison of different lens materials with respect to the risk of calcification. The combination of different analytical and replication methods can be used in the future to further investigate the pathomechanisms of calcium phosphate crystal formation and the influence of risk factors. This may help to prevent calcification of hydrophilic acrylic IOLs and associated explantation and complications., Competing Interests: Erklärung zu finanziellen Interessen Forschungsförderung erhalten: ja, von einer anderen Institution (Pharma-oder Medizintechnikfirma usw.); Honorar/geldwerten Vorteil für Referententätigkeit erhalten: ja, von einer anderen Institution (Pharma-oder Medizintechnikfirma usw.); Bezahlter Berater/interner Schulungsreferent/Gehaltsempfänger: nein; Patent/Geschäftsanteile/Aktien (Autor/Partner, Ehepartner, Kinder) an im Bereich der Medizin aktiven Firma: nein; Patent/Geschäftsanteile/Aktien (Autor/Partner, Ehepartner, Kinder) an zu Sponsoren dieser Fortbildung bzw. durch die Fortbildung in ihren Geschäftsinteressen berührten Firma: nein. Erklärung zu nichtfinanziellen Interessen Die Autorinnen/Autoren geben an, dass kein Interessenkonflikt besteht./ Declaration of financial interests Received research funding: yes, from another institution (pharmaceutical or medical device company, etc.); Received honorarium/monetary benefit for speaking engagements: yes, from another institution (pharmaceutical or medical device company, etc.); Paid consultant/internal training speaker/salary recipient: no; Patent/business shares/shares (author/partner, children) in company active in the field of medicine: no; Paid consultant/internal training speaker/salary recipient: no; Patent/business shares/shares (author/partner, spouse, children) in company active in the field of medicine: no; Patent/business shares/shares (author/partner, spouse, children) in company sponsor of this training or company whose business interests are affected by the training: no. Declaration of non-financial interests The authors declare that there is no conflict of interest., (Thieme. All rights reserved.)

Purpose: FOXP3 deficiency results in severe multisystem autoimmunity in both mice and humans, driven by the absence of functional regulatory T cells. Patients typically present with early and severe autoimmune polyendocrinopathy, dermatitis, and severe inflammation of the gut, leading to villous atrophy and ultimately malabsorption, wasting, and failure to thrive. In the absence of successful treatment, FOXP3-deficient patients usually die within the first 2 years of life. Hematopoietic stem cell transplantation provides a curative option but first requires adequate control over the inflammatory condition. Due to the rarity of the condition, no clinical trials have been conducted, with widely unstandardized therapeutic approaches. We sought to compare the efficacy of lead therapeutic candidates rapamycin, anti-CD4 antibody, and CTLA4-Ig in controlling the physiological and immunological manifestations of Foxp3 deficiency in mice., Method: We generated Foxp3-deficient mice and an appropriate clinical scoring system to enable direct comparison of lead therapeutic candidates rapamycin, nondepleting anti-CD4 antibody, and CTLA4-Ig., Results: We found distinct immunosuppressive profiles induced by each treatment, leading to unique protective combinations over distinct clinical manifestations. CTLA4-Ig provided superior breadth of protective outcomes, including highly efficient protection during the transplantation process., Conclusion: These results highlight the mechanistic diversity of pathogenic pathways initiated by regulatory T cell loss and suggest CTLA4-Ig as a potentially superior therapeutic option for FOXP3-deficient patients., (© 2023. The Author(s).)

Brain oedema is a life-threatening complication of various neurological conditions. Understanding molecular mechanisms of brain volume regulation is critical for therapy development. Unique insight comes from monogenic diseases characterized by chronic brain oedema, of which megalencephalic leukoencephalopathy with subcortical cysts (MLC) is the prototype. Variants in MLC1 or GLIALCAM, encoding proteins involved in astrocyte volume regulation, are the main causes of MLC. In some patients, the genetic cause remains unknown. We performed genetic studies to identify novel gene variants in MLC patients, diagnosed by clinical and MRI features, without MLC1 or GLIALCAM variants. We determined subcellular localization of the related novel proteins in cells and in human brain tissue. We investigated functional consequences of the newly identified variants on volume regulation pathways using cell volume measurements, biochemical analysis and electrophysiology. We identified a novel homozygous variant in AQP4, encoding the water channel aquaporin-4, in two siblings, and two de novo heterozygous variants in GPRC5B, encoding the orphan G protein-coupled receptor GPRC5B, in three unrelated patients. The AQP4 variant disrupts membrane localization and thereby channel function. GPRC5B, like MLC1, GlialCAM and aquaporin-4, is expressed in astrocyte endfeet in human brain. Cell volume regulation is disrupted in GPRC5B patient-derived lymphoblasts. GPRC5B functionally interacts with ion channels involved in astrocyte volume regulation. In conclusion, we identify aquaporin-4 and GPRC5B as old and new players in genetic brain oedema. Our findings shed light on the protein complex involved in astrocyte volume regulation and identify GPRC5B as novel potentially druggable target for treating brain oedema., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Guarantors of Brain.)

IgG3 is unique among the IgG subclasses due to its extended hinge, allotypic diversity and enhanced effector functions, including highly efficient pathogen neutralisation and complement activation. It is also underrepresented as an immunotherapeutic candidate, partly due to a lack of structural information. Here, we use cryoEM to solve structures of antigen-bound IgG3 alone and in complex with complement components. These structures reveal a propensity for IgG3-Fab clustering, which is possible due to the IgG3-specific flexible upper hinge region and may maximise pathogen neutralisation by forming high-density antibody arrays. IgG3 forms elevated hexameric Fc platforms that extend above the protein corona to maximise binding to receptors and the complement C1 complex, which here adopts a unique protease conformation that may precede C1 activation. Mass spectrometry reveals that C1 deposits C4b directly onto specific IgG3 residues proximal to the Fab domains. Structural analysis shows this to be caused by the height of the C1-IgG3 complex. Together, these data provide structural insights into the role of the unique IgG3 extended hinge, which will aid the development and design of upcoming immunotherapeutics based on IgG3., (© 2023. The Author(s).)

Background: Mechanical central venous catheter (CVC) placement complications are mostly malposition or iatrogenic pneumothorax. Verification of catheter position by chest X-ray (CXR) is usually performed postoperatively., Objectives: This prospective observational study assessed the diagnostic accuracy of peri-operative ultrasound and a 'bubble test' to detect malposition and pneumothorax., Method: Sixty-one patients undergoing peri-operative CVC placement were included. An ultrasound protocol was used to directly visualise the CVC, perform the 'bubble test' and assess for the presence of pneumothorax. The time from agitated saline injection to visualisation of microbubbles in the right atrium was evaluated to determine the correct position of the CVC. The time required to perform the ultrasound assessment was compared to that of conducting the CXR., Results: Chest X-ray identified 12 (19.7%) malpositions while ultrasound identified 8 (13.1%). Ultrasound showed a sensitivity of 0.85 (95% confidence interval [CI]: 0.72 to 0.93) and a specificity of 0.5 (95% CI: 0.16 to 0.84). The positive and negative predictive values were 0.92 (95% CI: 0.80 to 0.98) and 0.33 (95% CI: 0.10 to 0.65), respectively. No pneumothorax was identified on ultrasound and CXR. The median time for ultrasound assessment was significantly shorter at 4 min (interquartile range [IQR]: 3-6 min), compared to performing a CXR that required a median time of 29 min (IQR: 18-56 min) ( p < 0.0001)., Conclusion: This study showed that ultrasound produced a high sensitivity and moderate specificity in detecting CVC malposition., Contribution: Ultrasound can improve efficiency when used as a rapid bedside screening test to detect CVC malposition., Competing Interests: The authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article., (© 2023. The Authors.)

Background Typically defined as a thromboinflammatory disease, ischemic stroke features early and delayed inflammatory responses, which determine the extent of ischemia-related brain damage. T and natural killer cells have been implicated in neuronal cytotoxicity and inflammation, but the precise mechanisms of immune cell-mediated stroke progression remain poorly understood. The activating immunoreceptor NKG2D is expressed on both natural killer and T cells and may be critically involved. Methods and Results An anti-NKG2D blocking antibody alleviated stroke outcome in terms of infarct volume and functional deficits, coinciding with reduced immune cell infiltration into the brain and improved survival in the animal model of cerebral ischemia. Using transgenic knockout models devoid of certain immune cell types and immunodeficient mice supplemented with different immune cell subsets, we dissected the functional contribution of NKG2D signaling by different NKG2D-expressing cells in stroke pathophysiology. The observed effect of NKG2D signaling in stroke progression was shown to be predominantly mediated by natural killer and CD8 + T cells. Transfer of T cells with monovariant T-cell receptors into immunodeficient mice with and without pharmacological blockade of NKG2D revealed activation of CD8 + T cells irrespective of antigen specificity. Detection of the NKG2D receptor and its ligands in brain samples of patients with stroke strengthens the relevance of preclinical observations in human disease. Conclusions Our findings provide a mechanistic insight into NKG2D-dependent natural killer- and T-cell-mediated effects in stroke pathophysiology.

Liver transplantation (LT) is a complex surgical procedure requiring thorough pre- and post-operative planning and care. The nutritional status of the patient before, during, and after LT is crucial to surgical success and long-term prognosis. This review aims to assess nutritional status assessment and management before, during, and after LT, with a focus on patients who have undergone bariatric surgery. We performed a comprehensive topic search on MEDLINE, Ovid, In-Process, Cochrane Library, EMBASE, and PubMed up to March 2023. It identifies key factors influencing the nutritional status of liver transplant patients, such as pre-existing malnutrition, the type and severity of liver disease, comorbidities, and immunosuppressive medications. The review highlights the importance of pre-operative nutritional assessment and intervention, close nutritional status monitoring, individualised nutrition care plans, and ongoing nutritional support and monitoring after LT. The review concludes by examining the effect of bariatric surgery on the nutritional status of liver transplant recipients. The review offers valuable insights into the challenges and opportunities for optimising nutritional status before, during, and after LT.

Background: Urinary tract infection (UTI) represents one of the most common infectious diseases and a major cause of antibiotic prescription in children. To prevent recurrent infections and long-term complications, low-dose continuous antibiotic prophylaxis (CAP) has been used. However, the efficacy of CAP is controversial. The aim of this document was to develop updated guidelines on the efficacy and safety of CAP to prevent pediatric UTIs. Methods: A panel of experts on pediatric infectious diseases, pediatric nephrology, pediatric urology, and primary care was asked clinical questions concerning the role of CAP in preventing UTIs in children. Overall, 15 clinical questions were addressed, and the search strategy included accessing electronic databases and a manual search of gray literature published in the last 25 years. After data extraction and narrative synthesis of results, recommendations were developed using the Grading of Recommendations, Assessment, Development, and Evaluations (GRADE) methodology. Results: The use of CAP is not recommended in children with a previous UTI, with recurrent UTIs, with vesicoureteral reflux (VUR) of any grade, with isolated hydronephrosis, and with neurogenic bladder. CAP is suggested in children with significant obstructive uropathies until surgical correction. Close surveillance based on early diagnosis of UTI episodes and prompt antibiotic therapy is proposed for conditions in which CAP is not recommended. Conclusions: Our systematic review shows that CAP plays a limited role in preventing recurrences of UTI in children and has no effect on its complications. On the other hand, the emergence of new antimicrobial resistances is a proven risk.

Autoimmune limbic encephalitis (ALE) presents with new-onset mesial temporal lobe seizures, progressive memory disturbance, and other behavioral and cognitive changes. CD8 T cells are considered to play a key role in those cases where autoantibodies (ABs) target intracellular antigens or no ABs were found. Assessment of such patients presents a clinical challenge, and novel noninvasive imaging biomarkers are urgently needed. Here, we demonstrate that visualization of the translocator protein (TSPO) with [ 18 F]DPA-714-PET-MRI reveals pronounced microglia activation and reactive gliosis in the hippocampus and amygdala of patients suspected with CD8 T cell ALE, which correlates with FLAIR-MRI and EEG alterations. Back-translation into a preclinical mouse model of neuronal antigen-specific CD8 T cell-mediated ALE allowed us to corroborate our preliminary clinical findings. These translational data underline the potential of [ 18 F]DPA-714-PET-MRI as a clinical molecular imaging method for the direct assessment of innate immunity in CD8 T cell-mediated ALE.