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Subarachnoid hemorrhage (SAH) can be associated with neurological deficits and has profound consequences for mortality and morbidity. Cerebral vasospasm (CVS) and delayed cerebral ischemia affect neurological outcomes in SAH patients, but their mechanisms are not fully understood, and effective treatments are limited. Here, we report that urotensin II receptor UT plays a pivotal role in both early events and delayed mechanisms following SAH in male mice. Few days post-SAH, UT expression is triggered by blood or hemoglobin in the leptomeningeal compartment. UT contributes to perimeningeal glia limitans astrocyte reactivity, microvascular alterations and neuroinflammation independent of CNS-associated macrophages (CAMs). Later, CAM-dependent vascular inflammation and subsequent CVS develop, leading to cognitive dysfunction. In an SAH model using humanized UT h+ / h+ male mice, we show that post-SAH CVS and behavioral deficits, mediated by UT through Gq/PLC/Ca 2+ signaling, are prevented by UT antagonists. These results highlight the potential of targeting UT pathways to reduce early meningeal response and delayed cerebral ischemia in SAH patients., (© 2024. The Author(s).)

GPR56, an adhesion G-protein coupled receptor (aGPCRs) with constitutive and ligand-promoted activity, is involved in many physiological and pathological processes. Whether the receptor's constitutive or ligand-promoted activation occur through the same molecular mechanism, and whether different activation modes lead to functional selectivity between G proteins is unknown. Here we show that GPR56 constitutively activates both G12 and G13. Unlike constitutive activation and activation with 3-a-acetoxydihydrodeoxygedunin (3αDOG), stimulation with an antibody, 10C7, directed against GPR56's extracellular domain (ECD) led to an activation that favors G13 over G12. An autoproteolytically deficient mutant, GPR56-T383A, was also activated by 10C7 indicating that the tethered agonist (TA) exposed through autocatalytic cleavage, is not required for this activation modality. In contrast, this proteolysis-resistant mutant could not be activated by 3αDOG indicating different modes of activation by the two ligands. We show that an N-terminal truncated GPR56 construct (GPR56-Δ1-385) is devoid of constitutive activity but was activated by 3αDOG. Similarly to 3αDOG, 10C7 promoted the recruitment of b-arrestin-2 but GPR56 internalization was β-arrestin independent. Despite the slow activation mode of 10C7 that favors G13 over G12, it efficiently activated the downstream Rho pathway in BT-20 breast cancer cells. These data show that different GPR56 ligands have different modes of activation yielding differential G protein selectivity but converging on the activation of the Rho pathway both in heterologous expressions system and in cancer cells endogenously expressing the receptor. 10C7 is therefore an interesting tool to study both the processes underlying GPR56 activity and its role in cancer cells.

GPR56, an adhesion G-protein coupled receptor (aGPCRs) with constitutive and ligand-promoted activity, is involved in many physiological and pathological processes. Whether the receptor's constitutive or ligand-promoted activation occur through the same molecular mechanism, and whether different activation modes lead to functional selectivity between G proteins is unknown. Here we show that GPR56 constitutively activates both G12 and G13. Unlike constitutive activation and activation with 3-α-acetoxydihydrodeoxygedunin (3αDOG), stimulation with an antibody, 10C7, directed against GPR56's extracellular domain (ECD) led to an activation that favors G13 over G12. An autoproteolytically deficient mutant, GPR56-T383A, was also activated by 10C7 indicating that the tethered agonist (TA) exposed through autocatalytic cleavage, is not required for this activation modality. In contrast, this proteolysis-resistant mutant could not be activated by 3αDOG indicating different modes of activation by the two ligands. We show that an N-terminal truncated GPR56 construct (GPR56-Δ1-385) is devoid of constitutive activity but was activated by 3αDOG. Similarly to 3αDOG, 10C7 promoted the recruitment of β-arrestin-2 but GPR56 internalization was β-arrestin independent. Despite the slow activation mode of 10C7 that favors G13 over G12, it efficiently activated the downstream Rho pathway in BT-20 breast cancer cells. These data show that different GPR56 ligands have different modes of activation yielding differential G protein selectivity but converging on the activation of the Rho pathway both in heterologous expressions system and in cancer cells endogenously expressing the receptor. 10C7 is therefore an interesting tool to study both the processes underlying GPR56 activity and its role in cancer cells., (© 2024. The Author(s).)

The United States Centers for Disease Control and the National Institute of Diabetic, Digestive and Kidney Disorders now estimates that there are now over 34.2 million patients with diabetes in the United Sates, or over 10.2% of our population. Diabetic foot pathology leads to over 150,000 lower extremity amputations yearly in the United States alone. Many of these infections are initiated from externally applied shearing forces applied to deformities of the lesser toes., Competing Interests: Disclosure I have no relevant disclosures for this work., (Copyright © 2023 Elsevier Inc. All rights reserved.)

TMPRSS13, a member of the Type II Transmembrane Serine Proteases (TTSP) family, is involved in cancer progression and in cell entry of respiratory viruses. To date, no inhibitors have been specifically developed toward this protease. In this study, a chemical library of 65 ketobenzothiazole-based peptidomimetic molecules was screened against a proteolytically active form of recombinant TMPRSS13 to identify novel inhibitors. Following an initial round of screening, subsequent synthesis of additional derivatives supported by molecular modelling, uncovered important molecular determinants involved in TMPRSS13 inhibition. One inhibitor, N-0430, achieved low nanomolar affinity towards TMPRSS13 activity in a cellular context. Using a SARS-CoV-2 pseudovirus cell entry model, we further show the ability of N-0430 to block TMPRSS13-dependent entry of the pseudovirus. The identified peptidomimetic inhibitors and the molecular insights of their potency gained from this study will aid in the development of specific TMPRSS13 inhibitors.

TMPRSS6 is a potential therapeutic target for the treatment of iron overload due to its role in regulating levels of hepcidin. Although potent TMPRSS6 inhibitors have been previously developed, their lack of specificity requires optimization to avoid potential side effects before pursuing preclinical development with in vivo models. Here, using computer-aided drug design based on a TMPRSS6 homology model, we reveal that the S2 position of TMPRSS6 offers a potential avenue to achieve selectivity against other members of the TTSP family. Accordingly, we synthesized novel peptidomimetic molecules containing lipophilic amino acids at the P2 position to exploit this unexplored pocket. This enabled us to identify TMPRSS6-selective small molecules with low nanomolar affinity. Finally, pharmacokinetic parameters were determined, and a compound was found to be potent in cellulo toward its primary target while retaining TTSP-subtype selectivity and showing no signs of alteration in in vitro TEER experiments.

SARS-CoV-2 Omicron subvariants with increased transmissibility and immune evasion are spreading globally with alarming persistence. Whether the mutations and evolution of spike (S) Omicron subvariants alter the viral hijacking of human TMPRSS2 for viral entry remains to be elucidated. This is particularly important to investigate because of the large number and diversity of mutations of S Omicron subvariants reported since the emergence of BA.1. Here we report that human TMPRSS2 is a molecular determinant of viral entry for all the Omicron clinical isolates tested in human lung cells, including ancestral Omicron subvariants (BA.1, BA.2, BA.5), contemporary Omicron subvariants (BQ.1.1, XBB.1.5, EG.5.1) and currently circulating Omicron BA.2.86. First, we used a co-transfection assay to demonstrate the endoproteolytic cleavage by TMPRSS2 of spike Omicron subvariants. Second, we found that N-0385, a highly potent TMPRSS2 inhibitor, is a robust entry inhibitor of virus-like particles harbouring the S protein of Omicron subvariants. Third, we show that N-0385 exhibits nanomolar broad-spectrum antiviral activity against live Omicron subvariants in human Calu-3 lung cells and primary patient-derived bronchial epithelial cells. Interestingly, we found that N-0385 is 10-20 times more potent than the repositioned TMPRSS2 inhibitor, camostat, against BA.5, EG.5.1, and BA.2.86. We further found that N-0385 shows broad synergistic activity with clinically approved direct-acting antivirals (DAAs), i.e., remdesivir and nirmatrelvir, against Omicron subvariants, demonstrating the potential therapeutic benefits of a multi-targeted treatment based on N-0385 and DAAs., Competing Interests: Declaration of competing interest PLB and RL are inventors on patent applications (US9365853B2 and US10988505B2) that cover matriptase and other type II transmembrane serine protease inhibitors for treating and preventing viral infections, respiratory disorders, inflammatory disorders, pain disorders, tissue disorders, hyperproliferative disorders, and disorders associated with iron overload. The remaining authors declare that they have no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Critical sized bone defects in the ankle are becoming increasingly more common in patients undergoing limb reconstruction with tibiotalocalcaneal arthrodesis. Bulk allografts have not fared well over time. There have been scattered preliminary reports using custom spinal cages or 3D-printed Titanium Implants to address the critical bony defect; however, the cost of these devices is prohibitive in many clinical practice settings. The purpose of this investigation is to report the preliminary experience using a commercially available Trabecular Metal (Zimmer-Biomet) tibial metaphyseal cone combined with a retrograde locked intramedullary nail to address this challenging problem. Eight consecutive patients underwent tibiotalocalcaneal arthrodesis using a commercially available Trabecular Metal tibial metaphyseal cone combined with a retrograde locked intramedullary nail. Five developed bone loss secondary to neuropathic (Charcot) bony resorption and 3 underwent surgery for failed total ankle arthroplasty. All 8 patients eventually achieved clinical and radiographic healing and were able to ambulate with standard footwear. One patient developed a postoperative wound infection at the site of calcaneal locking screws, which resolved with debridement and parenteral antibiotic therapy. Critical bone defects about the ankle have successfully addressed with custom 3D titanium implants. This small series suggests that similar clinical outcomes can be achieved with the use of a commercially available porous tantalum metaphyseal spacer borrowed from our arthroplasty colleagues, combined with the use of a retrograde locked intramedullary nail. Levels of Evidence : Level 4: Retrospective case series., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

The carriage of Salmonella in pigs is a major concern for the agri-food industry and for global healthcare systems. Humans could develop salmonellosis when consuming contaminated pig products. On the other hand, some Salmonella serotypes could cause disease in swine, leading to economic losses on farms. The purpose of the present study was to characterize the anti- Salmonella activity of a novel Bacillus -based probiotic using a bioreactor containing a piglet-derived intestinal microbiota. Two methods of probiotic administration were tested: a single daily and a continuous dose. Salmonella enumeration was performed using selective agar at T24h, T48h, T72h, T96h and T120h. The DNA was extracted from bioreactor samples to perform microbiome profiling by targeted 16S rRNA gene sequencing on Illumina Miseq. The quantification of short-chain fatty acids (SCFAs) was also assessed at T120h. The probiotic decreased Salmonella counts at T96 for the daily dose and at T120 for the continuous one. Both probiotic doses affected the alpha and beta diversity of the piglet-derived microbiota ( p < 0.05). A decrease in acetate concentration and an increase in propionate proportion were observed in the continuous condition. In conclusion, the tested Bacillus -based product showed a potential to modulate microbiota and reduce Salmonella colonization in a piglet-derived intestinal microbiota and could therefore be used in vivo.

Objective: To identify facilitators and barriers to older adults' participation in telehealth interventions for primary prevention and health promotion., Methods: Relevant articles were searched using keywords in Embase and MEDLINE. Study characteristics, type of telehealth interventions and technology involved, as well as facilitators and barriers to their use, were extracted from selected articles. The Unified Theory of Acceptance and Use of Technology 2 (UTAUT2) model was used to organise data., Results: A total of 24 articles (pertaining to 20 studies) were included. Nine facilitators and 11 barriers influencing the participation in telehealth interventions for primary prevention and health promotion among older adults were identified. The most recurrent facilitators were related to the individual's performance expectancy and effort expectancy, as well as the presence of a social dimension associated with the intervention (i.e. having a good relationship with the other participants in the program). The two most prevalent barriers were also related to effort expectancy and performance expectancy, followed by barriers related to the inherent characteristics of the technology and older adults' health condition. Experience, age and gender were also found to moderate technology use and acceptance., Conclusions: This rapid review highlights the importance of adopting a holistic perspective when designing telehealth interventions aimed at preventive and health promotion purposes among older adults., (© 2023 The Authors. Australasian Journal on Ageing published by John Wiley & Sons Australia, Ltd on behalf of AJA Inc’.)

Total ankle arthroplasty is a topic that has recently gained increasing interest, largely due to the improved outcomes, which have been demonstrated by short- and mid-term research studies on the newer, third-generation implant designs. The purpose of this review is to provide an updated assessment of the quality of outcomes research on total ankle arthroplasty., Competing Interests: Disclosure The authors of this article have no disclosures to report., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Effective perioperative pain control following knee arthroscopy allows patients to reduce narcotic intake, avoid side effects of these medications, and recover more quickly. Adductor canal nerve blockade (ACB) and intra-articular injection of local anesthetic have been described as adjuvant treatments for postoperative pain control following surgery of the knee. This study directly compares the effect of each of these treatment modalities. Patients undergoing knee arthroscopy were blinded and randomized to receive either an ACB ( n  = 60) or intra-articular injection of local anesthetic (IAB, n  = 64). Outcome measures included patient reported visual analog scale (VAS) scores at 1, 2, 4, 8, 16, 24, 36, 48 hours and 1 week and total narcotic consumption at 12, 24, and 48 hours postoperatively. Student's t -tests were used to compare unadjusted VAS scores at each time point and use of postoperative pain medication between treatment groups. Adjusted VAS scores were estimated in a multivariable general linear model with interaction of time and treatment group and other relevant covariates. There were no statistically significant differences between the two groups in terms of gender, age, body mass index, and insurance type. ACB patients had significantly higher pain scores than IAB patients at hours 1 and 2 (hour 1: 4.02 [2.99] vs. 2.59 [3.00], p  = 0.009; hour 2: 3.12 [2.44] vs. 2.17 [2.62], p  = 0.040). ACB patients had higher pain scores than IAB patients up to hour 16, though hours 4 to 16 were not significantly different. Adjusted covariate analyses demonstrate an additional statistically significant reduction in pain score in the IAB group at hour 4. There were no differences in narcotic consumption. Intraoperative local anesthetic and regional ACB each provides adequate pain control following knee arthroscopy, and intraoperative local anesthetic may provide enhanced pain control for up to 4 hours postoperatively. LEVEL OF EVIDENCE: : Level 1 evidence, randomized control trial., Competing Interests: D.E. is a paid consultant for Stryker Corporation. No other disclosures., (Thieme. All rights reserved.)

Human influenza viruses cause acute respiratory symptoms that can lead to death. Due to the emergence of antiviral drug-resistant strains, there is an urgent requirement for novel antiviral agents and innovative therapeutic strategies. Using the peptidomimetic ketobenzothiazole protease inhibitor RQAR-Kbt (IN-1, aka N-0100) as a starting point, we report how substituting P2 and P4 positions with natural and unnatural amino acids can modulate the inhibition potency toward matriptase, a prototypical type II transmembrane serine protease (TTSP) that acts as a priming protease for influenza viruses. We also introduced modifications of the peptidomimetics N-terminal groups, leading to significant improvements (from μM to nM, 60 times more potent than IN-1) in their ability to inhibit the replication of influenza H1N1 virus in the Calu-3 cell line derived from human lungs. The selectivity towards other proteases has been evaluated and explained using molecular modeling with a crystal structure recently obtained by our group. By targeting host cell TTSPs as a therapeutic approach, it may be possible to overcome the high mutational rate of influenza viruses and consequently prevent potential drug resistance., (© 2023 The Authors. ChemMedChem published by Wiley-VCH GmbH.)

Introduction: Surgical smoke generated through the use of electrical surgical devices poses a risk to the surgeon, medical personnel in the operating room, and the patient by exposing them to environmentally hazardous particulate matter. Previous investigation has shown that surgical smoke leads to an increased risk of pulmonary conditions, circulatory disorders, and irritation of the eyes, nose, and throat. Transmission of infectious disease can occur through inhalation of viral particles, and the presence of carcinogens are also of major concern. The deleterious effects of surgical smoke are well documented in several subspecialties, namely dermatology and general surgery, but there has been little discussion on the topic amongst orthopedic surgeons., Methods: A non-systematic review of the literature was completed with the aim of identifying the major categories of adverse health effects associated with surgical smoke inhalation and offering recommendations to reduce these hazards in the orthopedic surgical community., Results: Three primary categories of risk associated with surgical smoke inhalation were identified: inflammation, viral/bacterial transmission, and carcinogenicity. In addition, strategies for mitigating risk and best practice recommendations were explored., Conclusion: Surgical smoke is an under-recognized occupational hazard within the orthopedic surgery literature. There are several strategies which can be employed to reduce risk. Further investigation is needed to understand the long-term impact of these risks, as well as what can be done to improve the practicality and compliance with protective measures., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Antivirals with broad coronavirus activity are important for treating high-risk individuals exposed to the constantly evolving SARS-CoV-2 variants of concern (VOCs) as well as emerging drug-resistant variants. We developed and characterized a novel class of active-site-directed 3-chymotrypsin-like protease (3CLpro) inhibitors ( C2-C5a ). Our lead direct-acting antiviral (DAA), C5a , is a non-covalent, non-peptide with a dissociation constant of 170 nM against recombinant SARS-CoV-2 3CLpro. The compounds C2-C5a exhibit broad-spectrum activity against Omicron subvariants (BA.5, BQ.1.1, and XBB.1.5) and seasonal human coronavirus-229E infection in human cells. Notably, C5a has median effective concentrations of 30-50 nM against BQ.1.1 and XBB.1.5 in two different human cell lines. X-ray crystallography has confirmed the unique binding modes of C2-C5a to the 3CLpro, which can limit virus cross-resistance to emerging Paxlovid-resistant variants. We tested the effect of C5a with two of our newly discovered host-directed antivirals (HDAs): N-0385, a TMPRSS2 inhibitor, and bafilomycin D (BafD), a human vacuolar H + -ATPase [V-ATPase] inhibitor. We demonstrated a synergistic action of C5a in combination with N-0385 and BafD against Omicron BA.5 infection in human Calu-3 lung cells. Our findings underscore that a SARS-CoV-2 multi-targeted treatment for circulating Omicron subvariants based on DAAs ( C5a ) and HDAs (N-0385 or BafD) can lead to therapeutic benefits by enhancing treatment efficacy. Furthermore, the high-resolution structures of SARS-CoV-2 3CLpro in complex with C2-C5a will facilitate future rational optimization of our novel broad-spectrum active-site-directed 3C-like protease inhibitors.

BACKGROUND : Cold snare polypectomy (CSP) is increasingly used for polypectomy and is recommended as the first-line modality for small (< 10 mm) polyps. This study aimed to evaluate incomplete resection rates (IRRs) when using CSP for colorectal polyps of 4-20 mm. METHODS : Adults (45-80 years) undergoing screening, surveillance, or diagnostic colonoscopy and CSP by one of nine endoscopists were included. The primary outcome was the IRR for colorectal polyps of 4-20 mm, defined as the presence of polyp tissue in marginal biopsies after resection of serrated polyps or adenomas. Secondary outcomes included the IRR for serrated polyps, ease of resection, and complications. RESULTS:  413 patients were included (mean age 63; 48 % women) and 182 polyps sized 4-20 mm were detected and removed by CSP. CSP required conversion to hot snare resection in < 1 % of polyps of < 10 mm and 44 % of polyps sized 10-20 mm. The IRRs for polyps < 10 mm and ≥ 10 mm were 18 % and 21 %. The IRR was higher for serrated polyps (26 %) compared with adenomas (16 %). The IRR was higher for flat (IIa) polyps (odds ratio [OR] 2.9, 95 %CI 1.1-7.4); and when resection was judged as difficult (OR 4.2, 95 %CI 1.5-12.1), piecemeal resection was performed (OR 6.6, 95 %CI 2.0-22.0), or visible residual polyp was present after the initial resection (OR 5.4, 95 %CI 2.0-14.9). Polyp location, use of a dedicated cold snare, and submucosal injection were not associated with incomplete resection. Intraprocedural bleeding requiring endoscopic intervention occurred in 4.7 %. CONCLUSIONS : CSP for polyps of 4-9 mm is safe and feasible; however, for lesions ≥ 10 mm, CSP failure occurs frequently, and the IRR remains high even after technical success. Incomplete resection was associated with flat polyps, visual residual polyp, piecemeal resection, and difficult polypectomies., Competing Interests: D. von Renteln is supported by a “Fonds de Recherche du Québec Santé” career development award and has received research funding from ERBE, Ventage, Pendopharm, and Pentax; he is a consultant for Boston Scientific and Pendopharm.R. Djinbachian, F. Benard, A. Barkun, M. Bouin, S. Bouchard, E. Deslandres, B. Panzini, S.Sidani, R. Leduc, and B.C. Jobse declare that they have no conflicts of interest relevant to this paper., (Thieme. All rights reserved.)

The purpose of this study was to describe the social isolation of older adults in the Côte-des-Neiges neighbourhood (Montreal, Canada) from the perspectives of older adults and community stakeholders. To do so, a descriptive qualitative study was conducted, involving community-dwelling older adults and a variety of key stakeholders from the neighbourhood. Seven focus groups were held, with a total of 37 participants. Focus group transcripts were analyzed using the approach of Miles, Huberman, and Saldaña. Participants reported that social isolation of older adults is characterized by gaps in social interactions (scarcity of social interactions, lack of social support, and unsatisfying relationships) as well as by low social participation that can be depicted in three ways: (1) exclusion by society, (2) self-restriction of participation, and (3) low eagerness to socialize. This study highlights that there is a diversity in how social isolation of older adults manifests itself. It can be the result of a deliberate choice (or not), as well as being desired (or not). These aspects of the phenomenon of social isolation of older adults are still not well described. However, they offer relevant avenues for rethinking approaches to intervention development.

Nanotechnology has enabled the development of innovative therapeutics, diagnostics, and drug delivery systems. Nanoparticles (NPs) can influence gene expression, protein synthesis, cell cycle, metabolism, and other subcellular processes. While conventional methods have limitations in characterizing responses to NPs, omics approaches can analyze complete sets of molecular entities that change upon exposure to NPs. This review discusses key omics approaches, namely transcriptomics, proteomics, metabolomics, lipidomics and multi-omics, applied to the assessment of biological responses to NPs. Fundamental concepts and analytical methods used for each approach are presented, as well as good practices for omics experiments. Bioinformatics tools are essential to analyze, interpret and visualize large omics data, and to correlate observations in different molecular layers. The authors envision that conducting interdisciplinary multi-omics analyses in future nanomedicine studies will reveal integrated cell responses to NPs at different omics levels, and the incorporation of omics into the evaluation of targeted delivery, efficacy, and safety will improve the development of nanomedicine therapies., Competing Interests: Declaration of Competing Interest Rene P. Zahedi is CEO of MRM Proteomics, Inc. The rest of 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 © 2023 Elsevier B.V. All rights reserved.)

G protein-coupled receptors engage both G proteins and β-arrestins, and their coupling can be biased by ligands and mutations. Here, to resolve structural elements and mechanisms underlying effector coupling to the angiotensin II (AngII) type 1 receptor (AT1R), we combined alanine scanning mutagenesis of the entire sequence of the receptor with pharmacological profiling of Gα q and β-arrestin engagement to mutant receptors and molecular dynamics simulations. We showed that Gα q coupling to AT1R involved a large number of residues spread across the receptor, whereas fewer structural regions of the receptor contributed to β-arrestin coupling regulation. Residue stretches in transmembrane domain 4 conferred β-arrestin bias and represented an important structural element in AT1R for functional selectivity. Furthermore, we identified allosteric small-molecule binding sites that were enclosed by communities of residues that produced biased signaling when mutated. Last, we showed that allosteric communication within AT1R emanating from the Gα q coupling site spread beyond the orthosteric AngII-binding site and across different regions of the receptor, including currently unresolved structural regions. Our findings reveal structural elements and mechanisms within AT1R that bias Gα q and β-arrestin coupling and that could be harnessed to design biased receptors for research purposes and to develop allosteric modulators.

Diabetics are a highly comorbid population with an elevated risk profile when undergoing surgery. Proper preparation and management of modifiable risk factors can optimize outcomes in diabetics. A multidisciplinary approach to preoperative optimization, including surgeons, primary care providers, and anesthesiologists, ensures diabetic patients receive comprehensive evaluation before elective surgery. Orthopedic surgeons must understand preoperative optimization goals as they pertain to nutrition, glycemic control, and cardiovascular disease in diabetic patients., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a global public health crisis. The reduced efficacy of therapeutic monoclonal antibodies against emerging SARS-CoV-2 variants of concern (VOCs), such as omicron BA.5 subvariants, has underlined the need to explore a novel spectrum of antivirals that are effective against existing and evolving SARS-CoV-2 VOCs. To address the need for novel therapeutic options, we applied cell-based high-content screening to a library of natural products (NPs) obtained from plants, fungi, bacteria, and marine sponges, which represent a considerable diversity of chemical scaffolds. The antiviral effect of 373 NPs was evaluated using the mNeonGreen (mNG) reporter SARS-CoV-2 virus in a lung epithelial cell line (Calu-3). The screening identified 26 NPs with half-maximal effective concentrations (EC 50 ) below 50 μM against mNG-SARS-CoV-2; 16 of these had EC 50 values below 10 μM and three NPs (holyrine A, alotaketal C, and bafilomycin D) had EC 50 values in the nanomolar range. We demonstrated the pan-SARS-CoV-2 activity of these three lead antivirals against SARS-CoV-2 highly transmissible Omicron subvariants (BA.5, BA.2 and BA.1) and highly pathogenic Delta VOCs in human Calu-3 lung cells. Notably, holyrine A, alotaketal C, and bafilomycin D, are potent nanomolar inhibitors of SARS-CoV-2 Omicron subvariants BA.5 and BA.2. The pan-SARS-CoV-2 activity of alotaketal C [protein kinase C (PKC) activator] and bafilomycin D (V-ATPase inhibitor) suggest that these two NPs are acting as host-directed antivirals (HDAs). Future research should explore whether PKC regulation impacts human susceptibility to and the severity of SARS-CoV-2 infection, and it should confirm the important role of human V-ATPase in the VOC lifecycle. Interestingly, we observed a synergistic action of bafilomycin D and N-0385 (a highly potent inhibitor of human TMPRSS2 protease) against Omicron subvariant BA.2 in human Calu-3 lung cells, which suggests that these two highly potent HDAs are targeting two different mechanisms of SARS-CoV-2 entry. Overall, our study provides insight into the potential of NPs with highly diverse chemical structures as valuable inspirational starting points for developing pan-SARS-CoV-2 therapeutics and for unravelling potential host factors and pathways regulating SARS-CoV-2 VOC infection including emerging omicron BA.5 subvariants., 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 © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

TMPRSS6 is a type II transmembrane serine protease involved in iron homeostasis expressed as 4 isoforms in humans. TMPRSS6 isoform 2 downregulates hepcidin production by cleaving hemojuvelin and other surface proteins of hepatocytes. The functions of catalytically impaired isoforms 3 and 4 are still unknown. Here we demonstrate that TMPRSS6 isoforms 3 and 4 reduce the proteolytic activity of isoform 2 and uncover the ability of isoforms to interact. Moreover, we identified 49 potential protein partners common to TMPRSS6 isoforms, including TfR1, known to be involved in iron regulation. By co-expressing TMPRSS6 and TfR1, we show that TfR1 is cleaved and shed from the cell surface. Further, we demonstrate that TMPRSS6 isoforms 3 and 4 behave as dominant negative., Competing Interests: The authors have declared that no competing interests exist.

Latrophilin-3 (Lphn3; also known as ADGRL3) is a member of the adhesion G Protein Coupled Receptor subfamily, which participates in the stabilization and maintenance of neuronal networks by mediating intercellular adhesion through heterophilic interactions with transmembrane ligands. Polymorphisms modifying the Lphn3 gene are associated with attention-deficit/hyperactivity disorder (ADHD) in children and its persistence into adulthood. How these genetic alterations affect receptor function remains unknown. Here, we conducted the functional validation of distinct ADHD-related Lphn3 variants bearing mutations in the receptor's adhesion motif-containing extracellular region. We found that all variants tested disrupted the ability of Lphn3 to stabilize intercellular adhesion in a manner that was distinct between ligands classes, but which did not depend on ligand-receptor interaction parameters, thus pointing to altered intrinsic receptor signaling properties. Using G protein signaling biosensors, we determined that Lphn3 couples to Gαi1, Gαi2, Gαs, Gαq, and Gα13. However, all ADHD-related receptor variants consistently lacked intrinsic as well as ligand-dependent Gα13 coupling efficiency while maintaining unaltered coupling to Gαi, Gαs, and Gαq. Consistent with these alterations, actin remodeling functions as well as actin-relevant RhoA signaling normally displayed by the constitutively active Lphn3 receptor were impeded by select receptor variants, thus supporting additional signaling defects. Taken together, our data point to Gα13 selective signaling impairments as representing a disease-relevant pathogenicity pathway that can be inherited through Lphn3 gene polymorphisms. This study highlights the intricate interplay between Lphn3 GPCR functions and the actin cytoskeleton in modulating neurodevelopmental cues related to ADHD etiology., (© 2022. The Author(s).)

The COVID-19 pandemic caused by the SARS-CoV-2 virus remains a global public health crisis. Although widespread vaccination campaigns are underway, their efficacy is reduced owing to emerging variants of concern 1,2 . Development of host-directed therapeutics and prophylactics could limit such resistance and offer urgently needed protection against variants of concern 3,4 . Attractive pharmacological targets to impede viral entry include type-II transmembrane serine proteases (TTSPs) such as TMPRSS2; these proteases cleave the viral spike protein to expose the fusion peptide for cell entry, and thus have an essential role in the virus lifecycle 5,6 . Here we identify and characterize a small-molecule compound, N-0385, which exhibits low nanomolar potency and a selectivity index of higher than 10 6 in inhibiting SARS-CoV-2 infection in human lung cells and in donor-derived colonoids 7 . In Calu-3 cells it inhibits the entry of the SARS-CoV-2 variants of concern B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.617.2 (Delta). Notably, in the K18-human ACE2 transgenic mouse model of severe COVID-19, we found that N-0385 affords a high level of prophylactic and therapeutic benefit after multiple administrations or even after a single administration. Together, our findings show that TTSP-mediated proteolytic maturation of the spike protein is critical for SARS-CoV-2 infection in vivo, and suggest that N-0385 provides an effective early treatment option against COVID-19 and emerging SARS-CoV-2 variants of concern., (© 2022. The Author(s).)

Transient receptor potential canonical (TRPC) channels are membrane proteins involved in regulating Ca 2+ homeostasis, and whose functions are modulated by G protein-coupled receptors (GPCR). In this study, we developed bioluminescent resonance energy transfer (BRET) biosensors to better study channel conformational changes following receptor activation. For this study, two intramolecular biosensors, GFP10-TRPC7-RLucII and RLucII-TRPC7-GFP10, were constructed and were assessed following the activation of various GPCRs. We first transiently expressed receptors and the biosensors in HEK293 cells, and BRET levels were measured following agonist stimulation of GPCRs. The activation of GPCRs that engage Gα q led to a Gα q -dependent BRET response of the functional TRPC7 biosensor. Focusing on the Angiotensin II type-1 receptor (AT 1 R), GFP10-TRPC7-RLucII was tested in rat neonatal cardiac fibroblasts, expressing endogenous AT 1 R and TRPC7. We detected similar BRET responses in these cells, thus validating the use of the biosensor in physiological conditions. Taken together, our results suggest that activation of Gα q -coupled receptors induce conformational changes in a novel and functional TRPC7 BRET biosensor.

Background: The prevalence of back pain in the pediatric population is increasing, and the workup of these patients presents a clinical challenge. Many cases are selflimited, but failure to diagnose a pathology that requires clinical intervention can carry severe repercussions. Magnetic resonance imaging (MRI) carries a high cost to the patient and health care system, and may even require procedural sedation in the pediatric population. The aim of this study was to develop a scoring system based on pediatric patient factors to help determine when an MRI will change clinical management., Methods: This is a retrospective cohort analysis of consecutive pediatric patients who presented to clinic with a chief complaint of back pain between 2010 and 2018 at single orthopaedic surgery practice. Comprehensive demographic and presentation variables were collected. A predictive model of factors that influence whether MRI results in a change in management was then generated using cross-validation least absolute shrinkage and selection operator logistic regression analysis., Results: A total of 729 patients were included, with a mean age of 15.1 years (range: 3 to 20 y). Of these, 344 (47.2%) had an MRI. A predictive model was generated, with nocturnal symptoms (5 points), neurological deficit (10 points), age (0.7 points per year), lumbar pain (2 points), sudden onset of pain (3.25 points), and leg pain (3.75 points) identified as significant predictors. A combined score of greater than 9.5 points for a given patient is highly suggestive that an MRI will result in a change in clinical management (specificity: 0.93; positive predictive value: 0.92)., Conclusions: A predictive model was generated to help determine when ordering an MRI may result in a change in clinical management for workup of back pain in the pediatric population. The main factors included the presence of a neurological deficit, nocturnal symptoms, sudden onset, leg pain, lumbar pain, and age. Care providers can use these findings to better determine if and when an MRI might be appropriate., Level of Evidence: Level III-diagnostic study., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Delayed graft function (DGF) of a kidney transplant results in increased cost and complexity of management. For clinical care or a DGF trial, it would be ideal to accurately predict individual DGF risk and provide preemptive treatment. A calculator developed by Irish et al has been useful for predicting population but not individual risk., Methods: We analyzed the Irish calculator (IC) in the DeKAF prospective cohort (incidence of DGF = 20.4%) and investigated potential improvements., Results: We found that the predictive performance of the calculator in those meeting Irish inclusion criteria was comparable with that reported by Irish et al. For cohorts excluded by Irish: (a) in pump-perfused kidneys, the IC overestimated DGF risk; (b) in simultaneous pancreas kidney transplants, the DGF risk was exceptionally low. For all 3 cohorts, there was considerable overlap in IC scores between those with and those without DGF. Using a modified definition of DGF-excluding those with single dialysis in the first 24 h posttransplant-we found that the calculator had similar performance as with the traditional DGF definition. Studying whether DGF prediction could be improved, we found that recipient cardiovascular disease was strongly associated with DGF even after accounting for IC-predicted risk., Conclusions: The IC can be a useful population guide for predicting DGF in the population for which it was intended but has limited scope in expanded populations (SPK, pump) and for individual risk prediction. DGF risk prediction can be improved by inclusion of recipient cardiovascular disease., Competing Interests: R.B.M. was supported in part by the UAB-UCSD O’Brien Core Center for Acute Kidney Injury Research (NIH P30-DK079337) (5UO1DK115997) and Department of Veterans Affairs (5-IO1-BX003272). The other authors declare no conflicts of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Background ApoAI (apolipoproteins AI) and apoAII (apolipoprotein AII) are structural and functional proteins of high-density lipoproteins (HDL) which undergo post-translational modifications at specific residues, creating distinct proteoforms. While specific post-translational modifications have been reported to alter apolipoprotein function, the full spectrum of apoAI and apoAII proteoforms and their associations with cardiometabolic phenotype remains unknown. Herein, we comprehensively characterize apoAI and apoAII proteoforms detectable in serum and their post-translational modifications and quantify their associations with cardiometabolic health indices. Methods and Results Using top-down proteomics (mass-spectrometric analysis of intact proteins), we analyzed paired serum samples from 150 CARDIA (Coronary Artery Risk Development in Young Adults) study participants from year 20 and 25 exams. Measuring 15 apoAI and 9 apoAII proteoforms, 6 of which carried novel post-translational modifications, we quantified associations between percent proteoform abundance and key cardiometabolic indices. Canonical (unmodified) apoAI had inverse associations with HDL cholesterol and HDL-cholesterol efflux, and positive associations with obesity indices (body mass index, waist circumference), and triglycerides, whereas glycated apoAI showed positive associations with serum glucose and diabetes mellitus. Fatty-acid‒modified ApoAI proteoforms had positive associations with HDL cholesterol and efflux, and inverse associations with obesity indices and triglycerides. Truncated and dimerized proteoforms of apoAII were associated with HDL cholesterol (positively) and obesity indices (inversely). Several proteoforms had no significant associations with phenotype. Conclusions Associations between apoAI and AII and cardiometabolic indices are proteoform-specific. These results provide "proof-of-concept" that precise chemical characterization of human apolipoproteins will yield improved insights into the complex pathways through which proteins signify and mediate health and disease.

The custom-fabricated total contact cast is commonly used in the treatment of diabetic foot disorders. This resource-consuming treatment option has been associated with iatrogenic morbidity as well as the need for urgent cast removal and inspection of the underlying limb when potential problems arise. Over a 10-year period, 381 diabetic patients had 2265 total contact cast applications by certified orthopaedic technologists, in a university orthopaedic practice, under the supervision of university faculty. Patients were stratified by glycemic control based on hemoglobin A1c levels, and obesity based on body mass index (BMI). Complications were grouped as (1) development of a new ulcer or wound, (2) new or increasing odor or drainage, (3) wound infection, (4) gangrene, (5) newly identified osteomyelitis, and (6) pain or discomfort necessitating cast change or removal. At least 1 complication was observed in 159 of 381 patients. The odds of experiencing a cast-related event for patients with a BMI greater than 30 kg/m 2 was 1.55 times greater than patients with a BMI less than 25 kg/m 2 . As compared to patients with good glycemic control, the odds of experiencing a cast-associated complication was 1.27 times greater in patients with moderate glycemic control and 1.48 times greater in patients with poor glycemic control. The total contact cast is commonly used in the treatment of diabetic foot morbidity. Treatment-associated morbidity may well be greater than previously appreciated. Complications are more likely in patients who have poor glycemic control and are morbidly obese. This information will hopefully stimulate interest in developing commercially available nonrigid alternatives that retain the attributes of the resource-consuming rigid device, with the potential advantage of avoiding the associated morbidity. Levels of Evidence : Level IV, retrospective chart review.

Background: The COVID-19 pandemic has changed the way orthopaedics programs are educating and recruiting residents and applicants. With an increased focus on online and virtual programming, there has been an uptick in social media usage by orthopaedics residencies as a means of communicating with applicants. This study investigated the growth in utilization of social media platforms by residency programs since the beginning of the COVID-19 pandemic., Methods: Instagram and Twitter were queried for each orthopaedic surgery residency program. It was determined if each program with a corresponding social media account was created before or after March 1, 2020. The number of posts per month were tabulated for accounts that existed prior to March 1, 2020., Results: 187 orthopaedic surgery residency programs were identified using the AAMC ERAS database. Of these programs, 74 (41.6%) were found to have an Instagram profile, and 50 (26.7%) were found to have a Twitter page. Of the 74 Instagram profiles, 45 were created after March 1, 2020, representing a 155% increase. Of the 50 Twitter pages, 15 were created after March 1, 2020, representing a 43% increase. Instagram accounts that were active before the pandemic had a 96% increase in the number of posts per month, on average, after March 1, 2020., Conclusion: Over one-third of programs are utilizing social media for recruitment purposes. There has been an 155% increase in Instagram and 43% increase in Twitter usage by residency programs since March 1, 2020. Instagram accounts created prior to the pandemic also demonstrated a near doubling of increased utilization after March. This represents a new, cost-effective way to connect with applicants in a time when in-person interactions are limited. Level of Evidence: III., Competing Interests: Disclosures: The authors report no potential conflicts of interest related to this study., (Copyright © The Iowa Orthopaedic Journal 2021.)

Introduction: Type II transmembrane serine proteases (TTSPs) of the human respiratory tract generate high interest owing to their ability, among other roles, to cleave surface proteins of respiratory viruses. This step is critical in the viral invasion of coronaviruses, including SARS-CoV-2 responsible for COVID-19, but also influenza viruses and reoviruses. Accordingly, these cell surface enzymes constitute appealing therapeutic targets to develop host-based therapeutics against respiratory viral diseases. Additionally, their deregulated levels or activity has been described in non-viral diseases such as fibrosis, cancer, and osteoarthritis, making them potential targets in these indications., Areas Covered: Areas covered: This review includes WIPO-listed patents reporting small molecules and peptide-based inhibitors of type II transmembrane serine proteases of the respiratory tract., Expert Opinion: Expert opinion: Several TTSPs of the respiratory tract represent attractive pharmacological targets in the treatment of respiratory infectious diseases (notably COVID-19 and influenza), but also against idiopathic pulmonary fibrosis and lung cancer. The current emphasis is primarily on TMPRSS2, matriptase, and hepsin, yet other TTSPs await validation. Compounds listed herein are predominantly peptidomimetic inhibitors, some with covalent reversible mechanisms of action and high potencies. Their selectivity profile, however, are often only partially characterized. Preclinical data are promising and warrant further advancement in the above diseases.

Shoulder arthroplasty is an effective treatment option for patients with symptomatic shoulder arthritis and rotator cuff arthropathy. Although there have been reports of variations in complication rates according to insurance type, few studies have examined the effect of payer status on functional outcomes. Patients who underwent elective shoulder arthroplasty performed by a single fellowship-trained surgeon and had a minimum of 1 year of follow-up were queried. Patient characteristics were compared across insurance types. Each patient completed the American Shoulder and Elbow Surgeons (ASES) questionnaire preoperatively and postoperatively. A generalized linear mixed model was specified to predict ASES score at 1 year and included preoperative ASES score as an adjustment variable. A total of 84 patients underwent 91 procedures. Before surgery, ASES score differed by insurance type (P=.014), with lower scores in the Medicaid cohort compared with the private insurance cohort (20.4 vs 38.8, P=.009). After controlling for baseline ASES score, postoperative ASES score at 1-year follow-up differed by insurance type (P<.001). Patients with private insurance had better ASES scores (85.6) than patients with Medicaid (55.2) (P<.001) and workers' compensation (57.1) (P=.028). Patients with Medicare (80.6) had better ASES scores at follow-up compared with those with Medicaid (P<.001). Patients with Medicaid are at risk for significantly lower postoperative functional outcome scores after shoulder arthroplasty compared with patients with private insurance and Medicare. In this study, patients with Medicaid had lower preoperative ASES scores compared with other groups. These observed differences are likely multifactorial and should be acknowledged when counseling patients. [Orthopedics. 2020;43(6):e523-e528.]., (Copyright 2020, SLACK Incorporated.)

The current work focuses on the production of glucose oxidase (GOD) in sterilized biosolid (BS) slurries containing BS and municipal wastewater effluent. Various parameters were optimized for maximizing the GOD production and the effects of biostimulation on GOD production was investigated by adding synthetic media components. The studies on inoculum characteristics at an inoculum age of 72 h and inoculum size of 20% (w/v) produced high GOD activities of around 6012 U/L in 25% (dw/v) BS media. Further, the effect of ultrasonication time was determined to release BS-bound GOD in order to maximize enzymes recovery. Using 1000 U/L of the BS-based GOD for 0.55 M glucose oxidation produced the maximum H 2 O 2 concentration of 216 ppm. The produced H 2 O 2 was utilized for bio-Fenton based advanced oxidation process for the partial removal of 15 pharmaceutically active compounds., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: About half of late kidney allograft losses are attributed to death with function (DWF), a poorly characterized outcome. An ongoing question is whether DWF is a consequence of chronic allograft dysfunction. Using the prospective Long-term Deterioration of Kidney Allograft Function study database, we sought to better define the impact, phenotype, and clinical course of DWF in the current era., Methods: Three thousand five hundred eighty-seven kidney recipients with functional grafts at 90 days post-transplant were followed prospectively for a median of 5.2 years., Results: Characteristics at transplantation in those with DWF (N = 350, 9.8%) differed from those who otherwise lost their grafts (death-censored graft failure [DC-GF], N = 295, 8.2%) or maintained function (N = 2942, 82.0%); DWF patients were older, sicker, and had been on dialysis longer, with more preexisting cardiovascular disease, whereas DC-GF patients experienced more early rejection, more acute rejection after 90 days, and a clinically significant decrease in kidney function before graft failure. In contrast, the clinical course after transplantation in DWF patients did not differ before death from those who maintained function throughout., Conclusions: DWF and DC-GF in kidney transplant recipients represent differing clinical phenotypes occurring in distinct patient populations. Reducing the impact of DWF requires better definition of causes and clinical course and then trials of therapies to improve outcomes. Composite endpoints in clinical trials that group DWF and DC-GF together may obscure important clinical findings.

The amyloid beta peptide (Aβ) is derived from the amyloid precursor protein (APP) by secretase processing. APP is also cleaved by numerous other proteases, such as the type II transmembrane serine protease matriptase, with consequences on the production of Aβ. Because the APP homolog protein amyloid-like protein 1 (APLP1) shares similarities with APP, we sought to determine if matriptase also plays a role in its processing. Here, we demonstrate that matriptase directly interacts with APLP1 and that APLP1 is cleaved in cellulo by matriptase in its E1 ectodomains at arginine 124. Replacing Arg124 with Ala abolished APLP1 processing by matriptase. Using a bioluminescence resonance energy transfer (BRET) assay we found that matriptase reduces APLP1 homodimeric interactions. This study identifies matriptase as the first protease cleaving APLP1 in its dimerization domain, potentially altering the multiple functions associated with dimer formation.

Overexpression of G protein-coupled receptors (GPCRs) in tumours is widely used to develop GPCR-targeting radioligands for solid tumour imaging in the context of diagnosis and even treatment. The human vasoactive neuropeptide urotensin II (hUII), which shares structural analogies with somatostatin, interacts with a single high affinity GPCR named UT. High expression of UT has been reported in several types of human solid tumours from lung, gut, prostate, or breast, suggesting that UT is a valuable novel target to design radiolabelled hUII analogues for cancer diagnosis. In this study, two original urotensinergic analogues were first conjugated to a DOTA chelator via an aminohexanoic acid (Ahx) hydrocarbon linker and then -hUII and DOTA-urantide, complexed to the radioactive metal indium isotope to successfully lead to radiolabelled DOTA-Ahx-hUII and DOTA-Ahx-urantide. The 111 In-DOTA-hUII in human plasma revealed that only 30% of the radioligand was degraded after a 3-h period. DOTA-hUII and DOTA-urantide exhibited similar binding affinities as native peptides and relayed calcium mobilization in HEK293 cells expressing recombinant human UT. DOTA-hUII, not DOTA-urantide, was able to promote UT internalization in UT-expressing HEK293 cells, thus indicating that radiolabelled 111 In-DOTA-hUII would allow sufficient retention of radioactivity within tumour cells or radiolabelled DOTA-urantide may lead to a persistent binding on UT at the plasma membrane. The potential of these radioligands as candidates to target UT was investigated in adenocarcinoma. We showed that hUII stimulated the migration and proliferation of both human lung A549 and colorectal DLD-1 adenocarcinoma cell lines endogenously expressing UT. In vivo intravenous injection of 111 In-DOTA-hUII in C57BL/6 mice revealed modest organ signals, with important retention in kidney. 111 In-DOTA-hUII or 111 In-DOTA-urantide were also injected in nude mice bearing heterotopic xenografts of lung A549 cells or colorectal DLD-1 cells both expressing UT. The observed significant renal uptake and low tumour/muscle ratio (around 2.5) suggest fast tracer clearance from the organism. Together, DOTA-hUII and DOTA-urantide were successfully radiolabelled with 111 Indium, the first one functioning as a UT agonist and the second one as a UT-biased ligand/antagonist. To allow tumour-specific targeting and prolong body distribution in preclinical models bearing some solid tumours, these radiolabelled urotensinergic analogues should be optimized for being used as potential molecular tools for diagnosis imaging or even treatment tools., Competing Interests: The authors declare no conflict of interest.

Iron overload disorders are characterized by the body's inability to regulate iron absorption and its storage which can lead to organ failures. Accumulated evidence has revealed that hepcidin, the master regulator of iron homeostasis, is negatively modulated by TMPRSS6 (matriptase-2), a liver-specific type II transmembrane serine protease (TTSP). Here, we report that treatment with a peptidomimetic inhibitor affecting TMPRSS6 activity increases hepcidin production in hepatic cells. Moreover, similar effects were observed when using non-peptidic inhibitors obtained through optimization of hits from high-throughput screening. Using HepG2 cells and human primary hepatocytes, we show that TMPRSS6 inhibitors block TMPRSS6-dependent hemojuvelin cleavage and increase HAMP expression and levels of secreted hepcidin., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The signaling mechanisms of the angiotensin II type 2 receptor (AT 2 R), a heptahelical receptor, have not yet been clearly and completely defined. In the present contribution, we set out to identify the molecular determinants involved in AT 2 R activation. Although AT 2 R has not been shown to engage G q/11 , G 12 , G i2 , and β-arrestin (βarr) pathways as does the AT 1 R upon angiotensin II (AngII) stimulation, the atypical positioning of helix VIII in the recently published AT 2 R structure may play a role in the receptor's capacity to couple to downstream effectors. In the AT 2 R structure, helix VIII points inwards and towards intracellular loop 3 (ICL3) to form tertiary interactions with transmembrane domain 6 (TM6), possibly impeding access to signaling effectors. On the other hand, in most class A GPCRs, helix VIII is found to be engaged in tertiary interactions with ICL1 and away from the effector binding site. Upon closer examination of the AT 2 R structure, we found that the residues contained within intracellular loop 1 (ICL1) may be involved in driving this unusual conformation of helix VIII. To explore this hypothesis, we designed a series of AT 1 R/AT 2 R receptor chimeras to validate the roles of ICL1 and helix VIII in AT 2 R signaling. Substituting the AT 1 R ICL1 into AT 2 R led to a mutant receptor that coupled to G i2 . The substitution of the helix VIII and C-terminal domains of AT 2 R into the AT 1 R backbone led to a mutant receptor that retained AT 1 R-like signaling properties. These results suggest that the C-terminal portion of AT 2 R is compatible with canonical GPCR signaling and that ICL1 of AT 2 R is involved in repositioning helix VIII, which impedes engagement of classical GPCR effectors such as G proteins or βarrs., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Breakfast consumption is associated with better diet quality and healthier weights, yet many adolescents miss breakfast. Nationally, 17.1% of students participate in the School Breakfast Program (SBP). Only 10% of high school students participate., Objective: Our aim was to evaluate an environmental intervention to increase SBP participation in high schools., Design: A group randomized trial was carried out from 2012 to 2015., Participants/setting: Ninth- and 10th-grade students enrolled in 16 rural schools in Minnesota (median 387 students) were randomized to intervention or control condition., Intervention: A school-based intervention that included two key components was implemented over a 12-month period. One component focused on increasing SBP participation by increasing student access to school breakfast through changes in school breakfast service practices (eg, serving breakfast from a grab-n-go cart in the atrium; expanding breakfast service times). The other component focused on promoting school breakfast through student-directed marketing campaigns., Main Outcome Measure: Change in school-level participation in the SBP was assessed between baseline (among ninth and tenth graders) and follow-up (among tenth and eleventh graders). School meal and attendance records were used to assess change in school-level participation rates in the SBP., Statistical Analyses: The Wilcoxon test was used for analysis of difference in change in mean SBP participation rate by experimental group., Results: The median change in SBP participation rate between baseline and follow-up was 3% (interquartile range=13.5%) among the eight schools in the intervention group and 0.5% (interquartile range=0.7%) among the eight schools in the control group. This difference in change between groups was statistically significant (Wilcoxon test, P=0.03). The intervention effect increased throughout the intervention period, with change in mean SBP participation rate by the end of the school year reaching 10.3% (95% CI 3.0 to 17.6). However, among the intervention schools, the change in mean SBP participation rates was highly variable (range=-0.8% to 24.8%)., Conclusions: Interventions designed to improve access to the SBP by reducing environmental and social barriers have potential to increase participation among high school students., (Copyright © 2019 Academy of Nutrition and Dietetics. Published by Elsevier Inc. All rights reserved.)

The DeKAF study was developed to better understand the causes of late allograft loss. Preliminary findings from the DeKAF cross-sectional cohort (with follow-up < 20 months) have been published. Herein, we present long-term outcomes in those recipients (mean follow-up ± SD, 6.6 ± 0.7 years). Eligibility included being transplanted prior to October 1, 2005; serum creatinine ≤ 2.0 mg/dL on January 1, 2006; and subsequently developing new-onset graft dysfunction leading to a biopsy. Mean time from transplant to biopsy was 7.5 ± 6.1 years. Histologic findings and DSA were studied in relation to postbiopsy outcomes. Long-term follow-up confirms and expands the preliminary results of each of 3 studies: (1) increasing inflammation in area of atrophy (irrespective of inflammation in nonscarred areas [Banff i]) was associated with increasingly worse postbiopsy death-censored graft survival; (2) hierarchical analysis based on Banff scores defined clusters (entities) that differed in long-term death-censored graft survival; and (3) C4d-/DSA- recipients had significantly better (and C4d+/DSA+ worse) death-censored graft survival than other groups. C4d+/DSA- and C4d-/DSA+ had similar intermediate death-censored graft survival. Clinical and histologic findings at the time of new-onset graft dysfunction define high- vs low-risk groups for long-term death-censored graft survival, even years posttransplant. These findings can help differentiate groups for potential intervention studies., (© 2018 The American Society of Transplantation and the American Society of Transplant Surgeons.)

G protein-coupled receptors (GPCRs) are important therapeutic targets that exhibit functional selectivity (biased signaling), in which different ligands or receptor variants elicit distinct downstream signaling. Understanding all the signaling events and biases that contribute to both the beneficial and adverse effects of GPCR stimulation by given ligands is important for drug discovery. Here, we report the design, validation, and use of pathway-selective bioluminescence resonance energy transfer (BRET) biosensors that monitor the engagement and activation of signaling effectors downstream of G proteins, including protein kinase C (PKC), phospholipase C (PLC), p63RhoGEF, and Rho. Combined with G protein and β-arrestin BRET biosensors, our sensors enabled real-time monitoring of GPCR signaling at different levels in downstream pathways in both native and engineered cells. Profiling of the responses to 14 angiotensin II (AngII) type 1 receptor (AT1R) ligands enabled the clustering of compounds into different subfamilies of biased ligands and showed that, in addition to the previously reported functional selectivity between Gα q and β-arrestin, there are also biases among G protein subtypes. We also demonstrated that biases observed at the receptor and G protein levels propagated to downstream signaling pathways and that these biases could occur through the engagement of different G proteins to activate a common effector. We also used these tools to determine how naturally occurring AT1R variants affected signaling bias. This suite of BRET biosensors provides a useful resource for fingerprinting biased ligands and mutant receptors and for dissecting functional selectivity at various levels of GPCR signaling., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

OBJECTIVEAtlantoaxial instability is an important cause of pain and neurological dysfunction in patients with Down syndrome (DS), frequently requiring instrumented fusion of the upper cervical spine. This study provides a quantitative analysis of C2 morphology in DS patients compared with their peers without DS to identify differences that must be considered for the safe placement of instrumentation.METHODSA retrospective chart review identified age-matched patients with and without DS with a CT scan of the cervical spine. Three-dimensional reconstructions of these scans were made with images along the axis of, and perpendicular to, the pars, lamina, facet, and transverse foramen of C2 bilaterally. Two of the authors performed independent measurements of anatomical structures using these images, and the average of the 2 raters' measurements was recorded. Pedicle height and width; pars axis length (the distance from the facet to the anterior vertebral body through the pars); pars rostrocaudal angle (angle of the pars axis length to the endplate of C2); pars axial angle (angle of the pars axis length to the median coronal plane); lamina height, length, and width; lamina angle (angle of the lamina length to the median coronal plane); and transverse foramen posterior distance (the distance from the posterior wall of the transverse foramen to the tangent of the posterior vertebral body) were measured bilaterally. Patients with and without DS were compared using a mixed-effects model accounting for patient height.RESULTSA total of 18 patients with and 20 patients without DS were included in the analysis. The groups were matched based on age and sex. The median height was 147 cm (IQR 142-160 cm) in the DS group and 165 cm (IQR 161-172 cm) in the non-DS group (p < 0.001). After accounting for variations in height, the mean pars rostrocaudal angle was greater (50.86° vs 45.54°, p = 0.004), the mean transverse foramen posterior distance was less (-1.5 mm vs +1.3 mm, p = 0.001), and the mean lamina width was less (6.2 mm vs 7.7 mm, p = 0.038) in patients with DS.CONCLUSIONSPatients with DS had a steeper rostrocaudal trajectory of the pars, a more posteriorly positioned transverse foramen posterior wall, and a narrower lamina compared with age- and sex-matched peers. These variations should be considered during surgical planning, as they may have implications to safe placement of instrumentation.

Angiotensin II (AngII) type 1 receptor (AT 1 R) is a G protein-coupled receptor known for its role in numerous physiological processes and its implication in many vascular diseases. Its functions are mediated through G protein dependent and independent signaling pathways. AT 1 R has several endogenous peptidic agonists, all derived from angiotensinogen, as well as several synthetic ligands known to elicit biased signaling responses. Here, surface plasmon resonance (SPR) was used as a cell-based and label-free technique to quantify, in real time, the response of HEK293 cells stably expressing the human AT 1 R. The goal was to take advantage of the integrative nature of this assay to identify specific signaling pathways in the features of the response profiles generated by numerous endogenous and synthetic ligands of AT 1 R. First, we assessed the contributions of Gq, G12/13, Gi, Gβγ, ERK1/2 and β-arrestins pathways in the cellular responses measured by SPR where Gq, G12/Rho/ROCK together with β-arrestins and ERK1/2 were found to play significant roles. More specifically, we established a major role for G12 in the early events of the AT 1 R-dependent response, which was followed by a robust ERK1/2 component associated to the later phase of the signal. Interestingly, endogenous AT 1 R ligands (AngII, AngIII and AngIV) exhibited distinct responses signatures with a significant increase of the ERK1/2-like components for both AngIII and AngIV, which points toward possibly distinct physiological roles for the later. We also tested AT 1 R biased ligands, all of which affected both the early and later events. Our results support SPR-based integrative cellular assays as a powerful approach to delineate the contribution of specific signaling pathways for a given cell response and reveal response differences associated with ligands with distinct pharmacological properties., (Crown Copyright © 2018. Published by Elsevier Ltd. All rights reserved.)

The neurotensin receptors are attractive targets for the development of new analgesic compounds. They represent potential alternatives or adjuvants to opioids. Herein, we report the structural optimization of our recently reported macrocyclic peptide analogues of NT(8-13). The macrocycle was formed via ring-closing metathesis (RCM) between an ortho allylated tyrosine residue in position 11 and the side chain of alkene-functionalized amino acid in position 8 of NT(8-13). Minute modifications led to significant binding affinity improvement ( K i improved from 5600 to 15 nM) with greatly improved plasma stability compared to NT(8-13). This study also delineates the structural features influencing these parameters. The signaling profiles of the new macrocycles were determined on the NTS1 receptor, and the physiological effects of the two most potent and stable analogues were assessed in vivo using rodent models. Both compounds displayed strong analgesic effects.

TMPRSS6, also known as matriptase-2, is a type II transmembrane serine protease that plays a major role in iron homeostasis by acting as a negative regulator of hepcidin production through cleavage of the BMP co-receptor haemojuvelin. Iron-refractory iron deficiency anaemia (IRIDA), an iron metabolism disorder, is associated with mutations in the TMPRSS6 gene. By analysing RNA-seq data encoding TMPRSS6 isoforms and other proteins involved in hepcidin production, we uncovered significant differences in expression levels between hepatocellular carcinoma (HCC) cell lines and normal human liver samples. Most notably, TMPRSS6 and HAMP expression was found to be much lower in HepG2 and Huh7 cells when compared to human liver samples. Furthermore, we characterized the common TMPRSS6 polymorphism V736A identified in Hep3B cells, the V795I mutation found in HepG2 cells, also associated with IRIDA, and the G603R substitution recently detected in two IRIDA patients. While variant V736A is as active as wild-type TMPRSS6, mutants V795I and G603R displayed significantly reduced proteolytic activity. Our results provide important information about commonly used liver cell models and shed light on the impact of two TMPRSS6 mutations associated with IRIDA.

G protein coupled receptors (GPCRs) produce pleiotropic effects by their capacity to engage numerous signaling pathways once activated. Functional selectivity (also called biased signaling), where specific compounds can bring GPCRs to adopt conformations that enable selective receptor coupling to distinct signaling pathways, continues to be significantly investigated. However, an important but often overlooked aspect of functional selectivity is the capability of ligands such as angiotensin II (AngII) to adopt specific conformations that may preferentially bind to selective GPCRs structures. Understanding both receptor and ligand conformation is of the utmost importance for the design of new drugs targeting GPCRs. In this study, we examined the properties of AngII cyclic analogs to impart biased agonism on the angiotensin type 1 receptor (AT 1 R). Positions 3 and 5 of AngII were substituted for cysteine and homocysteine residues ([Sar 1 Hcy 3,5 ]AngII, [Sar 1 Cys 3 Hcy 5 ]AngII and [Sar 1 Cys 3,5 ]AngII) and the resulting analogs were evaluated for their capacity to activate the Gq/11, G12, Gi2, Gi3, Gz, ERK and β-arrestin (βarr) signaling pathways via AT 1 R. Interestingly, [Sar 1 Hcy 3,5 ]AngII exhibited potency and full efficacy on all pathways tested with the exception of the Gq pathway. Molecular dynamic simulations showed that the energy barrier associated with the insertion of residue Phe 8 of AngII within the hydrophobic core of AT 1 R, associated with Gq/11 activation, is increased with [Sar 1 Hcy 3,5 ]AngII. These results suggest that constraining the movements of molecular determinants within a given ligand by introducing cyclic structures may lead to the generation of novel ligands providing more efficient biased agonism., (Copyright © 2018 Elsevier Inc. All rights reserved.)