Search

Waterfowl serve as indicators of ecosystem health and represent a pathway of contaminant exposure for hunters who consume them. In the northeast Atlantic Flyway, data on baseline contaminant loads in waterfowl are lacking. We assessed five species of commonly harvested (and consumed) waterfowl for mercury, polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans, polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs), and per- and polyfluoroalkyl substances (PFAS). We found that Canada geese (Branta canadensis) and wood ducks (Aix sponsa) had lower contaminant burdens than mallards (Anas platyrhynchos), American green-winged teal (Anas carolinensis), and American black ducks (Anas rubripes). Regardless of species, all samples contained detectable levels of PCBs and at least one OCP and PFAS. Mercury and OCPs posed limited non-cancer risks to human consumers who follow current waterfowl consumption advisories. Based on a probabilistic risk assessment, lower consumption rates were required to reduce risks from PCBs compared to other contaminants. Protective human consumption advisories depend in part on the level of allowable cancer risk and whether contaminants were considered toxicologically to act individually or collectively. Accordingly, revisions to consumption advisories to protect public hunter health given these new data hinge upon risk management decisions. These data can be used to update waterfowl consumption advisories in the northeast Atlantic Flyway and inform future research into the health effects of legacy and contemporary contaminants on the sustainability of waterfowl populations., 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 © 2025. Published by Elsevier B.V.)

Background: Total knee arthroplasty (TKA) is one of the most common orthopaedic procedures and the number of patients which undergo TKA will continue to rise in the coming years. Consecutively, the number of necessary revision surgeries will increase. One of the main reasons for revision surgery is aseptic loosening because of a so-called stress-shielding effect. Typically, revision of a primary TKA is done from a bicondylar surface replacement to a stem-anchored prosthesis, which, due to higher stress-shielding, have a shorter survival time than non-stem-anchored systems. Similar to endoprosthetic treatment in pediatric tumor orthopedics, non-ingrown cementless stems can be used. The study aim was to investigate whether this concept can also be applied to reduce stress-shielding in the tibial metaphysis after revision TKA in adults., Methods: Six tibial biomechanical bone with stemmed tibial TKA components were implanted using surface cementing and a floating-embedded stem or classic full cementing. After implantation, axial force was applied in such a way that the same load was generated as during walking. Two high-resolution cameras and illumination spots were used to record changes on the bone surface circumferentially in three regions of interest and from three different views., Results: With regard to the fixation method, a significant difference could be demonstrated in the metaphyseal and in the middle region around the stem (p < 0.001). At the tip of the stems, the reduction of strain energy density showed a stress shielding reduction for the floating-stemmed models in two of three views (ventromedial p = 0.002, lateral p = 0.398, and dorsal: p = 0.027)., Conclusions: In revision surgery after TKA, the use of floating-embedded, uncemented stems without bony ingrowth shows significant reduction of metaphyseal stress-shielding within the proximal tibia. This technique could be a viable alternative to prevent early aseptic loosening and should be examined in future in-vivo studies., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Abnormal mechanical loading is one of the major risk factors for articular cartilage degeneration. Engineered mesenchymal stromal cell (MSC)-derived cartilage holds great promise for cell-based cartilage repair. However, physiological loading protocols were shown to reduce matrix synthesis of MSC-derived neocartilage in vitro and the regulators of this undesired mechanoresponse remain poorly understood. Parathyroid hormone-related protein (PTHrP) is involved in cartilage development and can affect extracellular matrix (ECM) production during MSC chondrogenesis opposingly, depending on a continuous or transient exposure. PTHrP is induced by various mechanical cues in multiple tissues and species; but whether PTHrP is regulated in response to loading of human engineered neocartilage and may affect matrix synthesis in a positive or negative manner is unknown. The aim of this study was to investigate whether dynamic loading adjusts PTHrP-signaling in human MSC-derived neocartilage and whether it regulates matrix synthesis and other factors involved in the MSC mechanoresponse. Interestingly, MSC-derived chondrocytes significantly upregulated PTHrP mRNA (PTHLH) expression along with its second messenger cAMP in response to loading in our custom-built bioreactor. Exogenous PTHrP(1-34) induced the expression of known mechanoresponse genes (FOS, FOSB, BMP6) and significantly decreased glycosaminoglycan (GAG) and collagen synthesis similar to loading. The adenylate-cyclase inhibitor MDL-12,330A rescued the load-mediated decrease in GAG synthesis, indicating a direct involvement of cAMP-signaling in the reduction of ECM production. According to COL2A1-corrected hypertrophy-associated marker expression, load and PTHrP treatment shared the ability to reduce expression of MEF2C and PTH1R. In conclusion, the data demonstrate a significant mechanoinduction of PTHLH and a negative contribution of the PTHrP-cAMP signaling axis to GAG synthesis in MSC-derived chondrocytes after loading. To improve ECM synthesis and the mechanocompetence of load-exposed neocartilage, inhibition of PTHrP activity should be considered for MSC-based cartilage regeneration strategies., (© 2024 Wiley Periodicals LLC.)

Triple-negative breast cancer (TNBC) is the deadliest form of breast cancer with limited treatment options. The persistence of highly tumorigenic CD44-expressing subpopulation referred to as cancer stem cells (CSCs), endowed with the self-renewal capacity, has been associated with therapeutic resistance, hence clinical relapses. To mitigate these undesired events, targeted immunotherapies using antibody-photoconjugate (APC) or antibody-drug conjugate (ADC), were developed to specifically release cytotoxic payloads within targeted cells overexpressing cognate antigen receptors. Therefore, an αCD44(scFv)-SNAP-tag antibody fusion protein was engineered through genetic fusion of a single-chain antibody fragment (scFv) to a SNAPf-tag fusion protein, capable of self-conjugating with benzylguanine-modified light-sensitive near-infrared (NIR) phthalocyanine dye IRDye700DX (BG-IR700) or the small molecule toxin auristatin-F (BG-AURIF). Binding of the αCD44(scFv)-SNAPf-IR700 photoimmunoconjugate to antigen-positive cells was demonstrated by confocal microscopy and flow cytometry. By switching to NIR irradiation, CD44-expressing TNBC was selectively killed through induced phototoxic activities. Likewise, the αCD44(scFv)-SNAPf-AURIF immunoconjugate was able to selectively accumulate within targeted cells and significantly reduced cell viability through antimitotic activities at nano- to micromolar drug concentrations. This study provides an in vitro proof-of-concept for a future strategy to selectively destroy light-accessible superficial CD44-expressing TNBC tumors and their metastatic lesions which are inaccessible to therapeutic light.

Background: In vitro chondrogenesis of mesenchymal stromal cells (MSCs) driven by the essential chondro-inducer transforming growth factor (TGF)-β is instable and yields undesired hypertrophic cartilage predisposed to bone formation in vivo. TGF-β can non-canonically activate bone morphogenetic protein-associated ALK1/2/3 receptors. These have been accused of driving hypertrophic MSC misdifferentiation, but data remained conflicting. We here tested the antihypertrophic capacity of two highly specific ALK1/2/3 inhibitors - compound A (CompA) and LDN-212854 (LDN21) - in order to reveal potential prohypertrophic contributions of these BMP/non-canonical TGF-β receptors during MSC in vitro chondrogenesis., Methods: Standard chondrogenic pellet cultures of human bone marrow-derived MSCs were treated with TGF-β and CompA (500 nM) or LDN21 (500 nM). Daily 6-hour pulses of parathyroid hormone-related peptide (PTHrP[1-34], 2.5 nM, from day 7) served as potent antihypertrophic control treatment. Day 28 samples were subcutaneously implanted into immunodeficient mice., Results: All groups underwent strong chondrogenesis, but GAG/DNA deposition and ACAN expression were slightly but significantly reduced by ALK inhibition compared to solvent controls along with a mild decrease of the hypertrophy markers IHH-, SPP1-mRNA, and Alkaline phosphatase (ALP) activity. When corrected for the degree of chondrogenesis (COL2A1 expression), only pulsed PTHrP but not ALK1/2/3 inhibition qualified as antihypertrophic treatment. In vivo, all subcutaneous cartilaginous implants mineralized within 8 weeks, but PTHrP pretreated samples formed less bone and attracted significantly less haematopoietic marrow than ALK1/2/3 inhibitor groups., Conclusions: Overall, our data show that BMP-ALK1/2/3 inhibition cannot program mesenchymal stromal cells toward stable chondrogenesis. BMP-ALK1/2/3 signalling is no driver of hypertrophic MSC misdifferentiation and BMP receptor induction is not an adverse prohypertrophic side effect of TGF-β that leads to endochondral MSC misdifferentiation. Instead, the prohypertrophic network comprises misregulated PTHrP/hedgehog signalling and WNT activity, and a potential contribution of TGF-β-ALK4/5-mediated SMAD1/5/9 signalling should be further investigated to decide about its postulated prohypertrophic activity. This will help to successfully engineer cartilage replacement tissues from MSCs in vitro and translate these into clinical cartilage regenerative therapies., (© 2024. The Author(s).)

Lipopolysaccharide (LPS, endotoxin) is ubiquitous and represents a harmful contaminant of pharmaceutical compounds, recombinant biologicals and drug products. The pyrogen can induce severe immune responses and pathology in vitro and in vivo. Health authorities require strict control of endotoxin in parenteral drugs. However, for research and pre-clinical compound analysis, endotoxin testing is not a required quality control, which may cause potential drawbacks in the translational pipeline. Endotoxin testing is usually performed by the Limulus amebocyte lysate (LAL) assay, which is hampered by the so-called low endotoxin recovery (LER) effect when certain drug formulations are tested. A comprehensive study including structural, biophysical, and biological analyses was conducted to identify LER root cause for phosphate- and polysorbate-containing parenteral drug products. LPS in water showed extended ribbon-like aggregate structures. In placebo (formulation buffer without drug) and in drug product (drug in formulation buffer), a reaggregation of LPS into a network of interlinked micelles with hidden head group charges, and a strong reduction of the negative surface potential was observed. The non-accessibility of the LPS backbone has a direct impact leading (i) to a loss of activation of the LAL-cascade, (ii) reduced activation of the TLR4/MD-2 receptor system, and (iii) increased survival in a mouse model of endotoxemia. These data provide a structure-based explanation of the LER-underlying mechanisms. A human whole blood assay is shown to resolve LER and detect the pyrogenic activity of endotoxin with high sensitivity. This may open new test options to improve quality control in drug development and drug safety., Competing Interests: Declaration of Competing Interest KB is holding the patent for the peptide L-Pep19–2.5 Aspidasept® and is the CSO of Brandenburg Antiinfektiva GmbH, Borstel, Germany. FVW is employed at Roche-Genentech. WC is employed at BioAgilytix Europe GmbH. The authors declare that they have no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Purpose: Triple-negative breast cancer (TNBC) is phenotypic of breast tumors lacking expression of the estrogen receptor (ER), the progesterone receptor (PgR), and the human epidermal growth factor receptor 2 (HER2). The paucity of well-defined molecular targets in TNBC, coupled with the increasing burden of breast cancer-related mortality, emphasizes the need to develop targeted diagnostics and therapeutics. While antibody-drug conjugates (ADCs) have emerged as revolutionary tools in the selective delivery of drugs to malignant cells, their widespread clinical use has been hampered by traditional strategies which often give rise to heterogeneous mixtures of ADC products., Methods: Utilizing SNAP-tag technology as a cutting-edge site-specific conjugation method, a chondroitin sulfate proteoglycan 4 (CSPG4)-targeting ADC was engineered, encompassing a single-chain antibody fragment (scFv) conjugated to auristatin F (AURIF) via a click chemistry strategy., Results: After showcasing the self-labeling potential of the SNAP-tag component, surface binding and internalization of the fluorescently labeled product were demonstrated on CSPG4-positive TNBC cell lines through confocal microscopy and flow cytometry. The cell-killing ability of the novel AURIF-based recombinant ADC was illustrated by the induction of a 50% reduction in cell viability at nanomolar to micromolar concentrations on target cell lines., Conclusion: This research underscores the applicability of SNAP-tag in the unambiguous generation of homogeneous and pharmaceutically relevant immunoconjugates that could potentially be instrumental in the management of a daunting disease like TNBC., (© 2023. The Author(s).)

Mercury (Hg) inputs have particularly impacted the northeastern United States due to its proximity to anthropogenic emissions sources and abundant habitats that efficiently convert inorganic Hg into methylmercury. Intensive research and monitoring efforts over the past 50 years in New York State, USA, have informed the assessment of the extent and impacts of Hg exposure on fishes and wildlife. By synthesizing Hg data statewide, this study quantified temporal trends of Hg exposure, spatiotemporal patterns of risk, the role that habitat and Hg deposition play in producing spatial patterns of Hg exposure in fish and other wildlife, and the effectiveness of current monitoring approaches in describing Hg trends. Most temporal trends were stable, but we found significant declines in Hg exposure over time in some long-sampled fish. The Adirondack Mountains and Long Island showed the greatest number of aquatic and terrestrial species with elevated Hg concentrations, reflecting an unequal distribution of exposure risk to fauna across the state. Persistent hotspots were detected for aquatic species in central New York and the Adirondack Mountains. Elevated Hg concentrations were associated with open water, forests, and rural, developed habitats for aquatic species, and open water and forested habitats for terrestrial species. Areas of consistently elevated Hg were found in areas driven by atmospheric and local Hg inputs, and habitat played a significant role in translating those inputs into biotic exposure. Continued long-term monitoring will be important in evaluating how these patterns continue to change in the face of changing land cover, climate, and Hg emissions., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Elaborate bioreactor cultivation or expensive growth factor supplementation can enhance extracellular matrix production in engineered neocartilage to provide sufficient mechanical resistance. We here investigated whether raising extracellular calcium levels in chondrogenic cultures to physiologically relevant levels would provide a simple and inexpensive alternative to enhance cartilage neogenesis from human articular chondrocytes (AC) or bone marrow-derived mesenchymal stromal cells (BMSC). Interestingly, AC and BMSC-derived chondrocytes showed an opposite response to a calcium increase from 1.8 mM to 8 mM by which glycosaminoglycan (GAG) and collagen type II production were elevated during BMSC chondrogenesis but depressed in AC, leading to two-fold higher GAG/DNA values in BMSC-based neocartilage compared to the AC group. According to control treatments with Mg 2+ or sucrose, these effects were specific for CaCl 2 rather than divalent cations or osmolarity. Importantly, undesired pro-hypertrophic traits were not stimulated by calcium treatment. Specific induction of PTHrP mRNA and protein by 8.0mM calcium only in AC, along with negative effects of recombinant PTHrP 1-34 on cartilage matrix production, suggested that the PTHrP pathway contributed to the detrimental effects in AC-based neocartilage. Altogether, raising extracellular calcium levels was discovered as a novel, simple and inexpensive stimulator for BMSC-based cartilage neogenesis without the need for special bioreactors, whereas such conditions should be avoided for AC.

Aims: To assess low concentration hydrogen peroxide (LCHP) (H2O2) vapor dispersed with a home humidifier for its ability to decontaminate vehicle interiors contaminated with Bacillus anthracis surrogate Bacillus atrophaeus spores., Methods and Results: Efficacy of a vaporized 3% H2O2 solution was evaluated for liquid volumes, on/off vehicle heating, ventilation, and air conditioning (HVAC) system operations, and temperatures that ranged from 5 to 27°C. Survival of the spores was assessed by quantification of remaining viable spores with efficacy quantified in terms of mean log10 reduction. Decontamination efficacy after the 6-day dwell time increased when the 3% H2O2 liquid volume was doubled, increasing from 4-of-10 to 10-of-10 nondetects (zero colonies counted using standard dilution and filter plating) inside the vehicle cabin. Recirculating cabin air through the HVAC system during decontamination decreased efficacy to 6-of-10 non-detects. While no 6-log10 reduction in viable spores was observed on the cabin filter with the cabin filter kept in place, a 6-log10 reduction was achieved after its removal and placement in the cabin during treatment., Conclusions: Results from this study allow for informed decisions on the use of LCHP vapor as an effective decontamination approach for vehicle interiors., (Published by Oxford University Press on behalf of Applied Microbiology International 2023.)

cAMP-phosphodiesterase 4 (PDE4) inhibitors are currently approved for the treatment of inflammatory diseases. There is interest in expanding the therapeutic application of PDE4 inhibitors to metabolic disorders, as their chronic application induces weight loss in patients and animals and improves glucose handling in mouse models of obesity and diabetes. Unexpectedly, we have found that acute PDE4 inhibitor treatment induces a temporary increase, rather than a decrease, in blood glucose levels in mice. Blood glucose levels in postprandial mice increase rapidly upon drug injection, reaching a maximum after ~45 min, and returning to baseline within ~4 h. This transient blood glucose spike is replicated by several structurally distinct PDE4 inhibitors, suggesting that it is a class effect of PDE4 inhibitors. PDE4 inhibitor treatment does not reduce serum insulin levels, and the subsequent injection of insulin potently reduces PDE4 inhibitor-induced blood glucose levels, suggesting that the glycemic effects of PDE4 inhibition are independent of changes in insulin secretion and/or sensitivity. Conversely, PDE4 inhibitors induce a rapid reduction in skeletal muscle glycogen levels and potently inhibit the uptake of 2-deoxyglucose into muscle tissues. This suggests that reduced glucose uptake into muscle tissue is a significant contributor to the transient glycemic effects of PDE4 inhibitors in mice.

Antibody-drug conjugates (ADCs) are bifunctional molecules combining the targeting potential of monoclonal antibodies with the cancer-killing ability of cytotoxic drugs. This simple yet intelligently designed system directly addresses the lack of specificity encountered with conventional anti-cancer treatment regimes. However, despite their initial success, the generation of clinically sustainable and effective ADCs has been plagued by poor tumor penetration, undefined chemical linkages, unpredictable pharmacokinetic profiles, and heterogeneous mixtures of products. To this end, we generated a SNAP-tag-based fusion protein targeting the epidermal growth factor receptor (EGFR)-a biomarker of aggressive and drug-resistant cancers. Here, we demonstrate the use of a novel click coupling strategy to engineer a benzylguanine (BG)-linker-auristatin F (AuriF) piece that can be covalently tethered to the EGFR-targeting SNAP-tag-based fusion protein in an irreversible 1:1 stoichiometric reaction to form a homogeneous product. Furthermore, using these recombinant ADCs to target EGFR-overexpressing tumor cells, we provide a proof-of-principle for generating biologically active antimitotic therapeutic proteins capable of inducing cell death in a dose-dependent manner, thus alleviating some of the challenges of early ADC development., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

Adipose-derived stromal cells (ASC) are a promising alternative cell source to chondrocytes as well as to bone marrow-derived mesenchymal stromal cells (BMSC) in cartilage tissue engineering and repair. Here we describe ASC isolation from liposuction by-products by collagenase-based tissue digestion combined with cell filtration and followed by monolayer attachment and expansion culture. Quality control requires confirmation of correct surface marker expression and multilineage differentiation potential by a trilineage differentiation assay., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The purpose of this study was to evaluate and compare the decontamination efficacy of liquid formaldehyde solutions for three soil types (sand, loam, and clay) against spores of Bacillus anthracis (B.a.) and Bacillus atrophaeus. Approximately 1 x 108 colony forming units were inoculated into each sample. Through a series of six bench-scale experiments, two concentrations and two volumes of liquid formaldehyde solution were then added to the soil samples and allowed to remain in contact for either 24 or 48 hours. Decontamination efficacy was assessed at either 22° or 10°C with or without lids atop the sample jars. Complete inactivation (no spores recovered from the soil samples, typically providing > 7 log reduction) of B.a. occurred in all soil types in five of the six tests, while complete inactivation of B. atrophaeus was achieved in all soil types for three of the six tests. The results demonstrated a higher probability of complete inactivation of spores for samples that were covered, samples that received the higher volume of formaldehyde, and those contaminated with B.a. Overall, the use of liquid formaldehyde solution (2.5-5%) was highly effective in inactivating entire spore populations (typically > 107 CFU) for both B.a. and B. atrophaeus in the soil matrices studied. Covering the soil after application would allow for less formaldehyde solution to be used without impacting the overall efficacy of the process. The data from this study may aid in the selection of appropriate decontamination parameters when using liquid formaldehyde for soil remediation. The data may also aid in the decision to use B. atrophaeus as a surrogate for B.a. when performing further decontamination studies using liquid formalin solutions., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

A leading theory for ovarian carcinogenesis proposes that inflammation associated with incessant ovulation is a driver of oncogenesis. Consistent with this theory, nonsteroidal anti-inflammatory drugs (NSAIDs) exert promising chemopreventive activity for ovarian cancer. Unfortunately, toxicity is associated with long-term use of NSAIDs due to their cyclooxygenase (COX) inhibitory activity. Previous studies suggest the antineoplastic activity of NSAIDs is COX independent, and rather may be exerted through phosphodiesterase (PDE) inhibition. PDEs represent a unique chemopreventive target for ovarian cancer given that ovulation is regulated by cyclic nucleotide signaling. Here we evaluate PDE10A as a novel therapeutic target for ovarian cancer. Analysis of The Cancer Genome Atlas (TCGA) ovarian tumors revealed PDE10A overexpression was associated with significantly worse overall survival for patients. PDE10A expression also positively correlated with the upregulation of oncogenic and inflammatory signaling pathways. Using small molecule inhibitors, Pf-2545920 and a novel NSAID-derived PDE10A inhibitor, MCI-030, we show that PDE10A inhibition leads to decreased ovarian cancer cell growth and induces cell cycle arrest and apoptosis. We demonstrate these pro-apoptotic properties occur through PKA and PKG signaling by using specific inhibitors to block their activity. PDE10A genetic knockout in ovarian cancer cells through CRISP/Cas9 editing lead to decreased cell proliferation, colony formation, migration and invasion, and in vivo tumor growth. We also demonstrate that PDE10A inhibition leads to decreased Wnt-induced β-catenin nuclear translocation, as well as decreased EGF-mediated activation of RAS/MAPK and AKT pathways in ovarian cancer cells. These findings implicate PDE10A as novel target for ovarian cancer chemoprevention and treatment., (© 2022. The Author(s).)

The analysis of blood samples from mice treated with the PDE4 inhibitor Roflumilast revealed an unexpected reduction in serum potassium levels, while sodium and chloride levels were unaffected. Treatment with several structurally distinct PAN-PDE4 inhibitors, including Roflumilast, Rolipram, RS25344, and YM976 dose-dependently reduced serum potassium levels, indicating the effect is a class-characteristic property. PDE4 inhibition also induces hypothermia and hypokinesia in mice. However, while general anesthesia abrogates these effects of PDE4 inhibitors, potassium levels decrease to similar extents in both awake as well as in fully anesthetized mice. This suggests that the hypokalemic effects of PDE4 inhibitors occur independently of hypothermia and hypokinesia. PDE4 inhibition reduces serum potassium within 15 min of treatment, consistent with a rapid transcellular shift of potassium. Catecholamines promote the uptake of potassium into the cell via increased cAMP signaling. PDE4 appears to modulate these adrenoceptor-mediated effects, as PDE4 inhibition has no additional effects on serum potassium in the presence of saturating doses of the β-adrenoceptor agonist Isoprenaline or the α 2 -blocker Yohimbine, and is partially blocked by pre-treatment with the β-blocker Propranolol. Together, these data suggest that PDE4 inhibitors reduce serum potassium levels by modulating the adrenergic regulation of cellular potassium uptake.

Differentiating mesenchymal stromal cells (MSCs) into articular chondrocytes (ACs) for application in clinical cartilage regeneration requires a profound understanding of signaling pathways regulating stem cell chondrogenesis and hypertrophic degeneration. Classifying endochondral signals into drivers of chondrogenic speed versus hypertrophy, we here focused on insulin/insulin-like growth factor 1 (IGF1)-induced phosphoinositide 3-kinase (PI3K)/AKT signaling. Aware of its proliferative function during early but not late MSC chondrogenesis, we aimed to unravel the late pro-chondrogenic versus pro-hypertrophic PI3K/AKT role. PI3K/AKT activity in human MSC and AC chondrogenic 3D cultures was assessed via Western blot detection of phosphorylated AKT. The effects of PI3K inhibition with LY294002 on chondrogenesis and hypertrophy were assessed via histology, qPCR, the quantification of proteoglycans, and alkaline phosphatase activity. Being repressed by ACs, PI3K/AKT activity transiently rose in differentiating MSCs independent of TGFβ or endogenous BMP/WNT activity and climaxed around day 21. PI3K/AKT inhibition from day 21 on equally reduced chondrocyte and hypertrophy markers. Proving important for TGFβ-induced SMAD2 phosphorylation and SOX9 accumulation, PI3K/AKT activity was here identified as a required stage-dependent driver of chondrogenic speed but not of hypertrophy. Thus, future attempts to improve MSC chondrogenesis will depend on the adequate stimulation and upregulation of PI3K/AKT activity to generate high-quality cartilage from human MSCs.

Purpose: The burden of surgical disease in children from low and middle-income countries (LMICs) is becoming more recognized as significant and undertreated.  We recently reviewed our health system's experience with providing quaternary-level surgical care to children from LMICs through a partnership with World Pediatric Project (WPP), a not-for-profit organization., Methods: A retrospective review was performed of all WPP-sponsored patients who received surgical care at our children's hospital from LMICs in the Caribbean and Central America from July 2000 to August 2018., Results: Two hundred and fifty-five patients (average age: 5.9 ± 5.3 years; range: <1-18 years) from 14 countries received a total of 371 moderately to significantly complex operations from 10 pediatric surgical subspecialties, with cardiac, neurosurgery, craniofacial and general/thoracic surgical subspecialties being the most common. The average length of hospital stay was 10.7 ± 18.9 days.  All patients had the opportunity to follow-up with local providers and/or visiting WPP-sponsored surgical teams. 227 patients (93.8%) were seen by WPP providers or released to an in-country physician partnering with WPP. There were 21 (8.2%) total, minor and major, postoperative complications.  Five deaths (2.0%) occurred at our institution and 7 from disease progression, after returning to their home country., Conclusions: Providing complex surgical care to LMIC children in the US may help address a significant global burden.  This care can be provided by multiple subspecialists with excellent outcomes, good follow-up, and low complication and mortality rates.  Having a supportive health care system, volunteer surgeons, and an organization that manages logistics and provides financial support is essential., Type of Study: Clinical research, retrospective review LEVEL OF EVIDENCE: Level IV., Competing Interests: Declaration of Competing Interest None of the authors listed have declared conflict of interest., (Copyright © 2022. Published by Elsevier Inc.)

Background: Fully functional regeneration of skeletal defects by multipotent progenitor cells requires that differentiating cells gain the specific mechano-competence needed in the target tissue. Using cartilage neogenesis as an example, we asked whether proper phenotypic differentiation of mesenchymal stromal cells (MSC) into chondrocytes in vitro will install the adequate biological mechano-competence of native articular chondrocytes (AC)., Methods: The mechano-competence of human MSC- and AC-derived neocartilage was compared during differentiation for up to 35 days. The neocartilage layer was subjected to physiologic dynamic loading in a custom-designed bioreactor and assayed for mechano-sensitive gene and pathway activation, extracellular matrix (ECM) synthesis by radiolabel incorporation, nitric oxide (NO) and prostaglandin E 2 (PGE 2 ) production. Input from different pathways was tested by application of agonists or antagonists., Results: MSC and AC formed neocartilage of similar proteoglycan content with a hardness close to native tissue. Mechano-stimulation on day 21 and 35 induced a similar upregulation of mechano-response genes, ERK phosphorylation, NO production and PGE 2 release in both groups, indicating an overall similar transduction of external mechanical signals. However, while AC maintained or enhanced proteoglycan synthesis after loading dependent on tissue maturity, ECM synthesis was always significantly disturbed by loading in MSC-derived neocartilage. This was accompanied by significantly higher COX2 and BMP2 background expression, > 100-fold higher PGE 2 production and a weaker SOX9 stimulation in response to loading in MSC-derived neocartilage. Anabolic BMP-pathway activity was not rate limiting for ECM synthesis after loading in both groups. However, NFκB activation mimicked the negative loading effects and enhanced PGE 2 production while inhibition of catabolic NFκB signaling rescued the load-induced negative effects on ECM synthesis in MSC-derived neocartilage., Conclusions: MSC-derived chondrocytes showed a higher vulnerability to be disturbed by loading despite proper differentiation and did not acquire an AC-like mechano-competence to cope with the mechanical stress of a physiologic loading protocol. Managing catabolic NFκB influences was one important adaptation to install a mechano-resistance closer to AC-derived neocartilage. This new knowledge asks for a more functional adaptation of MSC chondrogenesis, novel pharmacologic co-treatment strategies for MSC-based clinical cartilage repair strategies and may aid a more rational design of physical rehabilitation therapy after AC- versus MSC-based surgical cartilage intervention., (© 2022. The Author(s).)

Harnessing the immunomodulatory activity of cytokines is a focus of therapies targeting inflammatory disease. The interleukin (IL)-1 superfamily contains pro-inflammatory and anti-inflammatory members that help orchestrate the immune response in adaptive and innate immunity. Of these molecules, IL-37 has robust anti-inflammatory activity across a range of disease models through inhibition of pro-inflammatory signaling cascades downstream of tumor necrosis factor, IL-1, and toll-like receptor pathways. We find that IL-37 is unstable with a poor pharmacokinetic and manufacturing profile. Here, we present the engineering of IL-37 from an unstable cytokine into an anti-inflammatory molecule with an excellent therapeutic likeness. We overcame these shortcomings through site-directed mutagenesis, the addition of a non-native disulfide bond, and the engineering of IL-37 as an Fc-fusion protein. Our results provide a platform for preclinical testing of IL-37 Fc-fusion proteins. The engineering approaches undertaken herein will apply to the conversion of similar potent yet short-acting cytokines into therapeutics., Competing Interests: Declaration of interests Monash University, Hudson Institute (J.C.W., M.F.N., C.A.N.-P., and A.M.E.), and F. Hoffmann-La Roche (A.B., F.S., G.T., and L.L.) hold two patent families on IL-37, namely PCT/AU2016/050495 (Monash and Hudson only) and PCT/EP2020/087031 (Monash, Hudson, and Roche). There are no other conflicts of interest for these authors. All other authors declare no competing interests., (Crown Copyright © 2021. Published by Elsevier Ltd. All rights reserved.)

Cyclic adenosine 3',5'-monophosphate (cAMP)-elevating agents, such as β 2 -adrenergic receptor (β 2 -AR) agonists and phosphodiesterase (PDE) inhibitors, remain a mainstay in the treatment of obstructive respiratory diseases, conditions characterized by airway constriction, inflammation, and mucus hypersecretion. However, their clinical use is limited by unwanted side effects because of unrestricted cAMP elevation in the airways and in distant organs. Here, we identified the A-kinase anchoring protein phosphoinositide 3-kinase γ (PI3Kγ) as a critical regulator of a discrete cAMP signaling microdomain activated by β 2 -ARs in airway structural and inflammatory cells. Displacement of the PI3Kγ-anchored pool of protein kinase A (PKA) by an inhaled, cell-permeable, PI3Kγ mimetic peptide (PI3Kγ MP) inhibited a pool of subcortical PDE4B and PDE4D and safely increased cAMP in the lungs, leading to airway smooth muscle relaxation and reduced neutrophil infiltration in a murine model of asthma. In human bronchial epithelial cells, PI3Kγ MP induced unexpected cAMP and PKA elevations restricted to the vicinity of the cystic fibrosis transmembrane conductance regulator (CFTR), the ion channel controlling mucus hydration that is mutated in cystic fibrosis (CF). PI3Kγ MP promoted the phosphorylation of wild-type CFTR on serine-737, triggering channel gating, and rescued the function of F508del-CFTR, the most prevalent CF mutant, by enhancing the effects of existing CFTR modulators. These results unveil PI3Kγ as the regulator of a β 2 -AR/cAMP microdomain central to smooth muscle contraction, immune cell activation, and epithelial fluid secretion in the airways, suggesting the use of a PI3Kγ MP for compartment-restricted, therapeutic cAMP elevation in chronic obstructive respiratory diseases.