Development of a Proteomic Workflow for the Identification of Heparan Sulphate Proteoglycan-Binding Substrates of ADAM17.

Ectodomain shedding, which is the proteolytic release of transmembrane proteins from the cell surface, is crucial for cell-to-cell communication and other biological processes. The metalloproteinase ADAM17 mediates ectodomain shedding of over 50 transmembrane proteins ranging from cytokines and growth factors, such as TNF and EGFR ligands, to signalling receptors and adhesion molecules. Yet, the ADAM17 sheddome is only partly defined and biological functions of the protease have not been fully characterized. Some ADAM17 substrates (e.g., HB-EGF) are known to bind to heparan sulphate proteoglycans (HSPG), and we hypothesised that such substrates would be under-represented in traditional secretome analyses, due to their binding to cell surface or pericellular HSPGs. Thus, to identify novel HSPG-binding ADAM17 substrates, we developed a proteomic workflow that involves addition of heparin to solubilize HSPG-binding proteins from the cell layer, thereby allowing their mass spectrometry detection by heparin-treated secretome (HEP-SEC) analysis. Applying this methodology to murine embryonic fibroblasts stimulated with an ADAM17 activator enabled us to identify 47 transmembrane proteins that were shed in response to ADAM17 activation. This included known HSPG-binding ADAM17 substrates (i.e., HB-EGF, CX3CL1) and 14 novel HSPG-binding putative ADAM17 substrates. Two of these, MHC-I and IL1RL1, were validated as ADAM17 substrates by immunoblotting.

Developments in Transfusion Medicine: Pulmonary Transfusion Reactions and Novel Blood Cell Labeling Techniques.

Staying updated on advancements in transfusion medicine is crucial, especially in critical care and perioperative setting, where timely and accurate transfusions can be lifesaving therapeutic interventions. This narrative review explores the landscape of transfusion-related adverse events, focusing on pulmonary transfusion reactions such as transfusion-associated circulatory overload (TACO) and transfusion-related acute lung injury (TRALI). TACO and TRALI are the leading causes of transfusion-related morbidity and mortality; however, specific treatments are lacking. Understanding the current incidence, diagnostic criteria, pathogenesis, treatment, and prevention strategies can equip clinicians to help reduce the incidence of these life-threatening complications. The review discusses emerging pathogenic mechanisms, including the possible role of inflammation in TACO and the mechanisms of reverse TRALI and therapeutic targets for TACO and TRALI, emphasizing the need for further research to uncover preventive and treatment modalities. Despite advancements, significant gaps remain in our understanding of what occurs during transfusions, highlighting the necessity for improved monitoring methods. To address this, the review also presents novel blood cell labeling techniques in transfusion medicine used for improving monitoring, quality assessment, and as a consequence, potentially reducing transfusion-related complications. This article aims to provide an update for anesthesiologists, critical care specialists, and transfusion medicine professionals regarding recent advancements and developments in the field of transfusion medicine.

A new silicon phthalocyanine dye induces pyroptosis in prostate cancer cells during photoimmunotherapy.

Photoimmunotherapy (PIT) combines the specificity of antibodies with the cytotoxicity of light activatable photosensitizers (PS) and is a promising new cancer therapy. We designed and synthesized, in a highly convergent manner, the silicon phthalocyanine dye WB692-CB2, which is novel for being the first light-activatable PS that can be directly conjugated via a maleimide linker to cysteines. In the present study we conjugated WB692-CB2 to a humanized antibody with engineered cysteines in the heavy chains that specifically targets the prostate-specific membrane antigen (PSMA). The resulting antibody dye conjugate revealed high affinity and specificity towards PSMA-expressing prostate cancer cells and induced cell death after irradiation with red light. Treated cells exhibited morphological characteristics associated with pyroptosis. Mechanistic studies revealed the generation of reactive oxygen species, triggering a cascade of intracellular events involving lipid peroxidation, caspase-1 activation, gasdermin D cleavage and membrane rupture followed by release of pro-inflammatory cellular contents. In first in vivo experiments, PIT with our antibody dye conjugate led to a significant reduction of tumor growth and enhanced overall survival in mice bearing subcutaneous prostate tumor xenografts. Our study highlights the future potential of the new phthalocyanine dye WB692-CB2 as PS for the fluorescence-based detection and PIT of cancer, including local prostate tumor lesions, and systemic activation of anti-tumor immune responses by the induction of pyroptosis.

A Formal Synthesis of (+)-Hannokinol Using a Chiral Horner-Wittig Reagent.

Herein, we report a concise and efficient formal synthesis of (+)-hannokinol. Key to this new strategy is the use of a chiral Horner-Wittig reagent, readily available from 2-deoxy-D-ribose, to introduce the chiral 1,3-diol motif.

Rational correction of pathogenic conformational defects in HTRA1.

Loss-of-function mutations in the homotrimeric serine protease HTRA1 cause cerebral vasculopathy. Here, we establish independent approaches to achieve the functional correction of trimer assembly defects. Focusing on the prototypical R274Q mutation, we identify an HTRA1 variant that promotes trimer formation thus restoring enzymatic activity in vitro. Genetic experiments in Htra1R274Q mice further demonstrate that expression of this protein-based corrector in trans is sufficient to stabilize HtrA1-R274Q and restore the proteomic signature of the brain vasculature. An alternative approach employs supramolecular chemical ligands that shift the monomer-trimer equilibrium towards proteolytically active trimers. Moreover, we identify a peptidic ligand that activates HTRA1 monomers. Our findings open perspectives for tailored protein repair strategies.

Cleavage site-directed antibodies reveal the prion protein in humans is shed by ADAM10 at Y226 and associates with misfolded protein deposits in neurodegenerative diseases.

Proteolytic cell surface release ('shedding') of the prion protein (PrP), a broadly expressed GPI-anchored glycoprotein, by the metalloprotease ADAM10 impacts on neurodegenerative and other diseases in animal and in vitro models. Recent studies employing the latter also suggest shed PrP (sPrP) to be a ligand in intercellular communication and critically involved in PrP-associated physiological tasks. Although expectedly an evolutionary conserved event, and while soluble forms of PrP are present in human tissues and body fluids, for the human body neither proteolytic PrP shedding and its cleavage site nor involvement of ADAM10 or the biological relevance of this process have been demonstrated thus far. In this study, cleavage site prediction and generation (plus detailed characterization) of sPrP-specific antibodies enabled us to identify PrP cleaved at tyrosin 226 as the physiological and apparently strictly ADAM10-dependent shed form in humans. Using cell lines, neural stem cells and brain organoids, we show that shedding of human PrP can be stimulated by PrP-binding ligands without targeting the protease, which may open novel therapeutic perspectives. Site-specific antibodies directed against human sPrP also detect the shed form in brains of cattle, sheep and deer, hence in all most relevant species naturally affected by fatal and transmissible prion diseases. In human and animal prion diseases, but also in patients with Alzheimer`s disease, sPrP relocalizes from a physiological diffuse tissue pattern to intimately associate with extracellular aggregated deposits of misfolded proteins characteristic for the respective pathological condition. Findings and research tools presented here will accelerate novel insight into the roles of PrP shedding (as a process) and sPrP (as a released factor) in neurodegeneration and beyond.

Synthesis of the monomeric counterpart of Marinomycin A and B.

The synthesis of polyketide natural products has been a captivating pursuit in organic chemistry, with a particular focus on selectively introducing 1,3-polyol units. Among these natural products, Marinomycins A-D have garnered substantial interest due to their exceptional structural features and potent cytotoxicity. In this paper, we present a novel approach for synthesising the monomeric counterparts of Marinomycin A and B. Our method employs a previously established iterative cycle in conjunction with a standardised polyketide building block. Through this strategy, we showcase a promising pathway towards total and partial syntheses of these intriguing natural products.

A dynamic in vitro model of Down syndrome neurogenesis with trisomy 21 gene dosage correction.

Excess gene dosage from chromosome 21 (chr21) causes Down syndrome (DS), spanning developmental and acute phenotypes in terminal cell types. Which phenotypes remain amenable to intervention after development is unknown. To address this question in a model of DS neurogenesis, we derived trisomy 21 (T21) human induced pluripotent stem cells (iPSCs) alongside, otherwise, isogenic euploid controls from mosaic DS fibroblasts and equipped one chr21 copy with an inducible XIST transgene. Monoallelic chr21 silencing by XIST is near-complete and irreversible in iPSCs. Differential expression reveals that T21 neural lineages and iPSCs share suppressed translation and mitochondrial pathways and activate cellular stress responses. When XIST is induced before the neural progenitor stage, T21 dosage correction suppresses a pronounced skew toward astrogenesis in neural differentiation. Because our transgene remains inducible in postmitotic T21 neurons and astrocytes, we demonstrate that XIST efficiently represses genes even after terminal differentiation, which will empower exploration of cell type-specific T21 phenotypes that remain responsive to chr21 dosage.

Activation of the MAPK network provides a survival advantage during the course of COVID-19-induced sepsis: a real-world evidence analysis of a multicenter COVID-19 Sepsis Cohort.

PURPOSE: There is evidence that lower activity of the RAF/MEK/ERK network is associated with positive outcomes in mild and moderate courses of COVID-19. The effect of this cascade in COVID-19 sepsis is still undetermined. Therefore, we tested the hypothesis that activity of the RAF/MEK/ERK network in COVID-19-induced sepsis is associated with an impact on 30-day survival. METHODS: We used biomaterial from 81 prospectively recruited patients from the multicentric CovidDataNet.NRW-study cohort (German clinical trial registry: DRKS00026184) with their collected medical history, vital signs, laboratory parameters, microbiological findings and patient outcome. ERK activity was measured by evaluating ERK phosphorylation using a Proximity Ligation Assay. RESULTS: An increased ERK activity at 4 days after diagnosis of COVID-19-induced sepsis was associated with a more than threefold increased chance of survival in an adjusted Cox regression model. ERK activity was independent of other confounders such as Charlson Comorbidity Index or SOFA score (HR 0.28, 95% CI 0.10-0.84, p = 0.02). CONCLUSION: High activity of the RAF/MEK/ERK network during the course of COVID-19 sepsis is a protective factor and may indicate recovery of the immune system. Further studies are needed to confirm these results.

The Alzheimer's disease-linked protease BACE2 cleaves VEGFR3 and modulates its signaling.

The ß-secretase ß-site APP cleaving enzyme (BACE1) is a central drug target for Alzheimer's disease. Clinically tested, BACE1-directed inhibitors also block the homologous protease BACE2. Yet little is known about physiological BACE2 substrates and functions in vivo. Here, we identify BACE2 as the protease shedding the lymphangiogenic vascular endothelial growth factor receptor 3 (VEGFR3). Inactivation of BACE2, but not BACE1, inhibited shedding of VEGFR3 from primary human lymphatic endothelial cells (LECs) and reduced release of the shed, soluble VEGFR3 (sVEGFR3) ectodomain into the blood of mice, nonhuman primates, and humans. Functionally, BACE2 inactivation increased full-length VEGFR3 and enhanced VEGFR3 signaling in LECs and also in vivo in zebrafish, where enhanced migration of LECs was observed. Thus, this study identifies BACE2 as a modulator of lymphangiogenic VEGFR3 signaling and demonstrates the utility of sVEGFR3 as a pharmacodynamic plasma marker for BACE2 activity in vivo, a prerequisite for developing BACE1-selective inhibitors for safer prevention of Alzheimer's disease.

Influence of E-cadherin deficiency on the dendrite morphology of Langerhans cells.

The expression of E-cadherin on Langerhans cells (LC) is required for adequate dendrite intercalation between epidermal keratinocytes. Upon disruption of epidermal homeostasis by tape stripping, E-cadherin competent LC extend dendrites reaching up to the epidermal surface, while E-cad deficient LC lack this ability.

PLGA nanocapsules as a delivery system for a recombinant LRP-based therapeutic.

Telomerase activity is directly affected by the laminin receptor precursor (LRP) protein, a highly conserved nonintegrin transmembrane receptor, which has been shown to have therapeutic effects in ageing, and age-related diseases. Recently, it has been found that overexpression of LRP-FLAG, by plasmid transfection, leads to a significant increase in telomerase activity in cell culture models. This may indicate that upregulation of LRP can be used to treat various age-related diseases. However, transfection is not a viable treatment strategy for patients. Therefore, we present a nanoencapsulated protein-based drug synthesised using poly(lactic-co-glycolic acid) (PLGA) nanocapsules for delivery of the 37 kDa LRP protein therapeutic. PLGA nanocapsules were synthesised using the double emulsification-solvent evaporation technique. Different purification methods, including filtration and centrifugation, were tested to ensure that the nanocapsules were within the optimal size range, and the BCA assay was used to determine encapsulation efficiency. The completed drug was tested in a HEK-293 cell culture model, to investigate the effect on cell viability, LRP protein levels and telomerase activity. A significant increase in total LRP protein levels with a concomitant increase in cell viability and telomerase activity was observed. Due to the observed increase in telomerase activity, this approach could represent a safer alternative to plasmid transfection for the treatment of age-related diseases.

Prevalence and prognostic relevance of invasive fungal disease during veno-arterial ECMO: A retrospective single-center study.

PURPOSE: To assess the prevalence and relevance of invasive fungal disease (IFD) during veno-arterial (V-A) extracorporeal membrane oxygenation (ECMO). METHODS: Retrospective analysis from January 2013 to November 2023 of adult V-A ECMO cases at a German University Hospital. Parameters relating to IFD, demographics, length of stay (LoS), days on ECMO and mechanical ventilation, prognostic scores and survival were assessed. Multivariable logistic regression analyses with IFD and death as dependent variables were performed. Outcome was assessed after propensity score matching IFD-patients to non-IFD-controls. RESULTS: 421 patients received V-A ECMO. 392 patients with full electronic datasets were included. The prevalence of IFD, invasive candidiasis and probable invasive pulmonary aspergillosis was 4.6%, 3.8% and 1.0%. Severity of acute disease, pre-existing moderate-to-severe renal disease and continuous kidney replacement therapy were predictive of IFD. In-hospital mortality (94% (17/18) compared to 67% (252/374) in non-IFD patients (p = 0.0156)) was predicted by female sex, SOFA score at admission, SAVE score and IFD (for IFD: OR: 8.31; CI: 1.60-153.18; p: 0.044). There was no difference in outcome after matching IFD-cases to non-IFD-controls. CONCLUSIONS: IFD are detected in about one in 20 patients on V-A ECMO, indicating mortality >90%. However, IFD do not contribute to prognosis in this population.

iRhom2 regulates ectodomain shedding and surface expression of the major histocompatibility complex (MHC) class I.

Proteolytic release of transmembrane proteins from the cell surface, the so called ectodomain shedding, is a key process in inflammation. Inactive rhomboid 2 (iRhom2) plays a crucial role in this context, in that it guides maturation and function of the sheddase ADAM17 (a disintegrin and metalloproteinase 17) in immune cells, and, ultimately, its ability to release inflammatory mediators such as tumor necrosis factor α (TNFα). Yet, the macrophage sheddome of iRhom2/ADAM17, which is the collection of substrates that are released by the proteolytic complex, is only partly known. In this study, we applied high-resolution proteomics to murine and human iRhom2-deficient macrophages for a systematic identification of substrates, and therefore functions, of the iRhom2/ADAM17 proteolytic complex. We found that iRhom2 loss suppressed the release of a group of transmembrane proteins, including known (e.g. CSF1R) and putative novel ADAM17 substrates. In the latter group, shedding of major histocompatibility complex class I molecules (MHC-I) was consistently reduced in both murine and human macrophages when iRhom2 was ablated. Intriguingly, it emerged that in addition to its shedding, iRhom2 could also control surface expression of MHC-I by an undefined mechanism. We have demonstrated the biological significance of this process by using an in vitro model of CD8+ T-cell (CTL) activation. In this model, iRhom2 loss and consequent reduction of MHC-I expression on the cell surface of an Epstein-Barr virus (EBV)-transformed lymphoblastoid cell line dampened activation of autologous CTLs and their cell-mediated cytotoxicity. Taken together, this study uncovers a new role for iRhom2 in controlling cell surface levels of MHC-I by a dual mechanism that involves regulation of their surface expression and ectodomain shedding.

The biological controls of soil carbon accumulation following wildfire and harvest in boreal forests: A review.

Boreal forests are frequently subjected to disturbances, including wildfire and clear-cutting. While these disturbances can cause soil carbon (C) losses, the long-term accumulation dynamics of soil C stocks during subsequent stand development is controlled by biological processes related to the balance of net primary production (NPP) and outputs via heterotrophic respiration and leaching, many of which remain poorly understood. We review the biological processes suggested to influence soil C accumulation in boreal forests. Our review indicates that median C accumulation rates following wildfire and clear-cutting are similar (0.15 and 0.20 Mg ha-1 year-1, respectively), however, variation between studies is extremely high. Further, while many individual studies show linear increases in soil C stocks through time after disturbance, there are indications that C stock recovery is fastest early to mid-succession (e.g. 15-80 years) and then slows as forests mature (e.g. >100 years). We indicate that the rapid build-up of soil C in younger stands appears not only driven by higher plant production, but also by a high rate of mycorrhizal hyphal production, and mycorrhizal suppression of saprotrophs. As stands mature, the balance between reductions in plant and mycorrhizal production, increasing plant litter recalcitrance, and ectomycorrhizal decomposers and saprotrophs have been highlighted as key controls on soil C accumulation rates. While some of these controls appear well understood (e.g. temporal patterns in NPP, changes in aboveground litter quality), many others remain research frontiers. Notably, very little data exists describing and comparing successional patterns of root production, mycorrhizal functional traits, mycorrhizal-saprotroph interactions, or C outputs via heterotrophic respiration and dissolved organic C following different disturbances. We argue that these less frequently described controls require attention, as they will be key not only for understanding ecosystem C balances, but also for representing these dynamics more accurately in soil organic C and Earth system models.

Radiological follow-up of cemento-osseous dysplasia on cone-beam computed tomography.

This study investigated the natural course of cemento-osseous dysplasia (COD) on cone-beam computed tomography (CBCT). Retrospectively, 104 CBCT scans from 36 patients (mean age, 44.5 years; 33 female and three male) with mandibular COD (10 florid, seven focal, 19 periapical) were included, based upon clinico-radiological features, without complications such as infection and related surgery. Changes in maximum diameter and morphology (lytic, mixed lytic-sclerotic, sclerotic) were evaluated in 83 lesions, with a mean follow-up of 28.3 months. The occurrence of a diameter increase was assessed by time-to-event analysis; interreader agreement for diameter and morphological evaluation by intraclass correlation coefficient and weighted κ statistics, respectively. Fifteen of 83 (18.1%) lesions (eight florid, one focal, six periapical) in 10 patients increased in diameter; 12 of 83 (14.5%) lesions (five florid, seven periapical) in 11 patients changed morphologically. The median period until a diameter increase was longest (120 months) for periapical COD, and shortest (66 months) for florid COD (p = 0.023). There was high reader agreement (ICC = 0.891; weighted κ = 0.901). In conclusion, CBCT is an effective tool with which to follow-up COD. If any, the natural progress in uncomplicated COD is prolonged, which underlines its non-surgical character and aids in its long-term management.

Delayed Meniscus Repair Lowers the Functional Outcome of Primary ACL Reconstruction.

BACKGROUND: Our purpose was to evaluate whether the time of intervention and the type of meniscus surgery (repair vs. partial meniscectomy) play a role in managing anterior cruciate ligament (ACL) reconstructions with concurrent meniscus pathologies. METHODS: We performed a prospective cohort study which differentiated between early and late ACL reconstructions with a cut-off at 3 months. Patients were re-evaluated after 2 years. RESULTS: Thirty-nine patients received an operation between 2-12 weeks after the injury, and thirty patients received the surgery between 13-28 weeks after trauma. The strongest negative predictive factor of the International Knee Documentation Committee subjective knee form in a hierarchical regression model was older age (ß = -0.49 per year; 95% CI [-0.91; -0.07]; p = 0.022; partial R2 = 0.08)). The strongest positive predictive factor was a higher preoperative Tegner score (ß = 3.6; 95% CI [0.13; 7.1]; p = 0.042; partial R2 = 0.07) and an interaction between meniscus repair surgery and the time of intervention (ß = 27; 95% CI [1.6; 52]; p = 0.037; partial R2 = 0.07), revealing a clinical meaningful difference as to whether meniscus repairs were performed within 12 weeks after trauma or were delayed. There was no difference whether partial meniscectomy was performed early or delayed. CONCLUSIONS: Surgical timing plays a crucial role when surgeons opt for a meniscus repair rather than for a meniscectomy.

30-day and one-year readmission rate in 11,270 patients with surgical treatment for proximal femoral fractures across Austria.

Aims: Patients with proximal femoral fractures (PFFs) are often multimorbid, thus unplanned readmissions following surgery are common. We therefore aimed to analyze 30-day and one-year readmission rates, reasons for, and factors associated with, readmission risk in a cohort of patients with surgically treated PFFs across Austria. Methods: Data from 11,270 patients with PFFs, treated surgically (osteosyntheses, n = 6,435; endoprostheses, n = 4,835) at Austrian hospitals within a one-year period (January to December 2021) was retrieved from the Leistungsorientierte Krankenanstaltenfinanzierung (Achievement-Oriented Hospital Financing). The 30-day and one-year readmission rates were reported. Readmission risk for any complication, as well as general medicine-, internal medicine-, and surgery/injury-associated complications, and factors associated with readmissions, were investigated. Results: The 30-day and one-year readmission rates due to any complication were 15% and 47%, respectively. The 30-day readmission rate (p = 0.001) was higher in endoprosthesis than osteosynthesis patients; this was not the case for the one-year readmission rate (p = 0.138). Internal medicine- (n = 2,273 (20%)) and surgery/injury-associated complications (n = 1,612 (14%)) were the most common reason for one-year readmission. Regardless of the surgical procedure, male sex was significantly associated with higher readmission risk due to any, as well as internal medicine-associated, complication. Advanced age was significantly associated with higher readmission risk after osteosynthesis. In both cohorts, treatment at mid-sized hospitals was significantly associated with lower readmission risk due to any complication, while prolonged length of stay was associated with higher one-year readmission risks due to any complication, as well as internal-medicine associated complications. Conclusion: Future health policy decisions in Austria should focus on optimization of perioperative and post-discharge management of this vulnerable patient population.

Assessing SOFA score trajectories in sepsis using machine learning: A pragmatic approach to improve the accuracy of mortality prediction.

INTRODUCTION: An increasing amount of longitudinal health data is available on critically ill septic patients in the age of digital medicine, including daily sequential organ failure assessment (SOFA) score measurements. Thus, the assessment in sepsis focuses increasingly on the evaluation of the individual disease's trajectory. Machine learning (ML) algorithms may provide a promising approach here to improve the evaluation of daily SOFA score dynamics. We tested whether ML algorithms can outperform the conventional ΔSOFA score regarding the accuracy of 30-day mortality prediction. METHODS: We used the multicentric SepsisDataNet.NRW study cohort that prospectively enrolled 252 sepsis patients between 03/2018 and 09/2019 for training ML algorithms, i.e. support vector machine (SVM) with polynomial kernel and artificial neural network (aNN). We used the Amsterdam UMC database covering 1,790 sepsis patients for external and independent validation. RESULTS: Both SVM (AUC 0.84; 95% CI: 0.71-0.96) and aNN (AUC 0.82; 95% CI: 0.69-0.95) assessing the SOFA scores of the first seven days led to a more accurate prognosis of 30-day mortality compared to the ΔSOFA score between day 1 and 7 (AUC 0.73; 95% CI: 0.65-0.80; p = 0.02 and p = 0.05, respectively). These differences were even more prominent the shorter the time interval considered. Using the SOFA scores of day 1 to 3 SVM (AUC 0.82; 95% CI: 0.68 0.95) and aNN (AUC 0.80; 95% CI: 0.660.93) led to a more accurate prognosis of 30-day mortality compared to the ΔSOFA score (AUC 0.66; 95% CI: 0.58-0.74; p < 0.01 and p < 0.01, respectively). Strikingly, all these findings could be confirmed in the independent external validation cohort. CONCLUSIONS: The ML-based algorithms using daily SOFA scores markedly improved the accuracy of mortality compared to the conventional ΔSOFA score. Therefore, this approach could provide a promising and automated approach to assess the individual disease trajectory in sepsis. These findings reflect the potential of incorporating ML algorithms as robust and generalizable support tools on intensive care units.