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Russia's return to Middle East and North Africa has become more evident since it intervened in Syria in 2015 at the request of Bashar al-Assad. Debates regarding Moscow's attitude have [...]

We examine de facto autonomy across regulatory agencies and policy sectors. Yet not much is known whether, how and why de facto autonomy could vary across policy areas within the same policy sector. This article demonstrates the existence of such variation and suggests that this variation depends on the interplay between stakeholders' diverging (or overlapping) policy preferences, deficient (or superior) organizational policy capacity, and institutional arrangements leading to enabled (or constrained) de facto autonomy. Relying on elite interviews and secondary resources, this study builds on an illustrative study on bank regulation in Turkey in the post‐GFC period and presents a nuanced understanding of de facto autonomy: a multi‐policy area approach to de facto autonomy that allows us to examine variation in de facto autonomy across policy areas, the determinants of the variation, and whether de facto autonomy is constrained or enabled, which structures the regulatory policy process. [ABSTRACT FROM AUTHOR]

LiFe1−xEuxPO4 (x = 0–0.1) samples were successfully fabricated by glass–ceramics technique quenching from high temperature. Thermal properties were analyzed by DTA analysis. Crystallization activation energy and Avrami parameters were calculated depending on the Eu content and heating rates. The XRD analysis shows that there are two-phases of LiFePO4 and Li3Fe2P3O12 which compete with Eu-doping in the samples and it is found that the x = 0.02 sample has the lowest impurity phases. The battery cells exhibit similar cycling voltammetry (CV) data with the undoped LiFePO4 battery cells. The charging/discharging cycles measurements were done for C/2-rate at room conditions and the best capacity value for C/2-rate was obtained as 149.4 mAh/g for x = 0.02 Eu-doped sample. Although the best capacity was obtained for x = 0.02, the best capacity fade was obtained for x = 0.03 Eu-doped sample. C-rate measurements provided that x = 0.02 cells have the promising results for the battery applications. [ABSTRACT FROM AUTHOR]

This study investigates the structural and photoluminescence (PL) characteristics of Tb 3+ -incorporated K 3 Y(BO 2 ) 6 (KYBO) phosphors synthesized via a microwave-assisted sol-gel technique. X-ray diffraction (XRD) and Rietveld refinement confirmed the formation of a pure hexagonal phase, with lattice expansion due to Tb³⁺ doping. PL studies revealed strong green emissions centered at 541 nm, attributed to the ⁵D₄ → ⁷F₅ transitions of Tb³⁺ ions, with the highest intensity observed at 5 wt% Tb³⁺. A decrease in emission was observed at higher concentrations due to concentration quenching. Temperature-dependent PL measurements revealed reverse thermal quenching enhancing PL intensity. Chromaticity analysis based on CIE 1931 coordinates showed stable green emission across all concentrations, with a maximum color purity of 89.74% observed for the KYBO:3 wt% Tb³⁺ sample. The results, along with reverse thermal quenching behavior observed between 470K and 550K, suggest that these phosphors exhibit excellent potential for lighting and display technologies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

In this study, an investigation was conducted on the structural and photoluminescence (PL) characteristics of LaAl 2 B 4 O 10 (LAB) phosphors initially incorporated with Dy 3+ and Eu 3+ ions. Subsequently, the impact of varying Eu 3+ concentration while maintaining a constant Dy 3+ concentration was examined. Structural characterization was performed using X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), and energy-dispersive X-ray spectroscopy (EDS). XRD analysis confirmed the effective embedding of both dopants into the hexagonal framework of the LAB. The PL emission spectra revealed characteristic emissions of Dy 3+ (blue and yellow) and Eu 3+ (red) ions. The optimized dopant concentrations of both Dy 3+ and Eu 3+ were observed to be 3 wt%. The dominant mechanism for concentration quenching in doped LAB phosphors was determined to be the electric dipole-dipole interaction. Co-doping with Eu 3+ led to a substantial decrease in Dy 3+ emission intensity (∼0.18-fold) while enhancing Eu 3+ emission intensity (∼3.72-fold). The critical energy transfer distance (R C  = 11.64 Å) and the analysis based on the Dexter theory confirmed that the energy transfer mechanism corresponds to dipole-dipole interaction. The color purities and correlated color temperatures (CCT) were estimated, suggesting the potential of these phosphors for warm white and red lighting applications, respectively. The observed energy transfer and luminescence properties, along with the structural and compositional characterization, highlight the promising potential of LAB:Dy 3+ /Eu 3+ co-doped phosphors for advanced lighting and display technologies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: It is thought that the Fracture Risk Assessment Tool (FRAX) score of the World Health Organization (WHO) determines a 10-year fracture risk. This study aimed to investigate the major osteoporotic fracture (MOF) and hip fracture (HF) values determined with the FRAX score and practicality of the FRAX score in kidney transplant recipients (KTRs)., Methods: This study was conducted with 44 female and 59 male KTRs and 100 subjects in the healthy control group. Bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry. FRAX scores were calculated from baseline information (age, height, weight, BMD of the femur and neck T score, fracture history, glucocorticoid use, smoking status, alcohol consumption, and presence of rheumatoid arthritis)., Results: In KTRs, FRAX score MOF, and FRAX score HF were found to be significantly elevated, whereas the BMD femur T score was determined to be significantly low. No significant relationship was found among the FRAX score MOF and HF and kidney function tests and bone parameters. In the receiver operating characteristic (ROC) analysis, which was performed based on the determination of the FRAX score, the cutoff point was determined as ≥ 3.4 for MOF and ≥ 0.4 for HF., Conclusion: In KTRs, increased FRAX score MOF and HF compared with healthy individuals were determined. FRAX score MOF ≥ 3.4 and HF ≥ 0.4 values indicate high-risk patients for increased fracture risk. The high accuracy rates determined suggest that the use of the FRAX score in KTRs is a valuable method for determining fracture risk., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Beyza Algul Durak reports financial support was provided by Ankara City Hospital Pediatric Nephrology Department. Beyza Algul Durak reports a relationship with Ankara City Hospital Pediatric Nephrology Department that includes: non-financial support. Beyza Algul Durak has a patent pending to Determination of Bone Fracture Risk in Kidney Transplant Recipients with FRAX Score. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Introduction: Dickkopf wingless (Wnt) signaling pathway inhibitor-1 (DKK-1) is a potent antagonist of the WNT canonical signaling pathway. DKK-1 is a substance that exerts anabolic effects on bone and is also involved in vascular cell regulation. The study aimed to determine the relationship of DKK-1 with atherosclerosis as determined by carotid artery intima-media thickness (CA-IMT) and arterial stiffness (AS) as determined by brachial-ankle pulse wave velocity (baPWV) in renal transplant recipients (RTRs)., Methods: A total of 62 (62%) male and 38 (438%) female RTRs with a mean age of 44.22 ± 10.88 years were included in the study. RTRs were compared with 65 healthy individuals. CA-IMT measurement with ultrasonography was used as a marker of atherosclerosis. The presence of AS was detected with the baPWV device., Results: Creatinine, CA-IMT, and baPWV were higher in the RTRs compared to the healthy subjects. No difference was determined between the two groups regarding log10 DKK-1. No difference was noted in the levels of CA-IMT and baPWV in patients with log10 DKK-1 > 3.83 pg/mL compared to patients with ≤3.83 pg/mL. Correlation and multivariate analyses showed no correlation between log10 DKK-1 and CA-IMT and baPWV., Discussion: In RTRs, an increased development of atherosclerosis and AS was observed compared to healthy individuals. There was no difference in DKK-1 between the groups based on improved renal function. DKK-1 was not correlated with atherosclerosis and AS., Competing Interests: Declaration of competing interest No potential conflict of interest was reported by the authors., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Matrix metalloproteinases (MMPs) are significant drivers of many diseases, including cancer, and are established targets for drug development. Tissue inhibitors of metalloproteinases (TIMPs) are endogenous MMP inhibitors and are being pursued for the development of anti-MMP therapeutics. TIMPs possess many attractive properties for drug candidates, such as complete MMP inhibition, low toxicity, low immunogenicity, and high tissue permeability. However, a major challenge with TIMPs is their rapid clearance from the bloodstream due to their small size. This study explores a method for extending the plasma half-life of the N-terminal domain of TIMP2 (N-TIMP2) by appending it with a long, intrinsically unfolded tail containing Pro, Ala, and Thr (PATylation). We designed and produced two PATylated N-TIMP2 constructs with tail lengths of 100 and 200 amino acids (N-TIMP2-PAT 100 and N-TIMP2-PAT 200 ). Both constructs demonstrated higher apparent molecular weights and retained high inhibitory activity against MMP-9. N-TIMP2-PAT 200 significantly increased plasma half-life in mice compared to the non-PATylated variant, enhancing its therapeutic potential. PATylation offers distinct advantages for half-life extension, such as fully genetic encoding, monodispersion, and biodegradability. It can be easily applied to N-TIMP2 variants engineered for high affinity and selectivity toward individual MMPs, creating promising candidates for drug development against MMP-related diseases., Competing Interests: The authors declare no conflicts of interest.

This study explores the structural and luminescent properties of terbium (Tb³⁺)-doped lanthanum aluminium borate (LaAl₂B₄O₁₀, abbreviated as LAB) phosphors, a novel host lattice for Tb³⁺ doping. LAB:Tb³⁺ phosphors, with varying dopant concentrations, were synthesized using a microwave-assisted combustion synthesis approach and characterized using X-ray diffraction (XRD), Rietveld refinement, and photoluminescence spectroscopy at both room and low temperatures. The structural analysis confirmed the hexagonal crystal structure of LAB and revealed successful incorporation of Tb³⁺ ions without altering the fundamental lattice. Luminescence studies demonstrated that the LAB:Tb³⁺ phosphors show strong green emission primarily attributed to the 5 D 4 → 7 F 5 transition of Tb³⁺. The optimal doping concentration was determined to be 5 wt% Tb³⁺, which provided maximum luminescence efficiency. This concentration also allowed for a critical study of energy transfer mechanisms within the phosphor, revealing dipole-dipole interactions with a critical distance of 9.80 Å between Tb³⁺ ions. Additionally, the CIE chromaticity coordinates of LAB:0.05 Tb³⁺ were precisely determined to be (0.289, 0.4460), indicating the potential for high-quality green emission suitable for solid-state lighting and display technologies. This work not only demonstrates the potential of LAB:Tb 3+ as a highly efficient green luminescent material, but also sheds light on the mechanisms responsible for energy transfer and concentration quenching., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

This study explores the structural and optical qualities of LaCa 4 O(BO 3 ) 3 (LACOB) phosphors doped with Eu 3+ , Dy 3+ , and Tb 3+ using a microwave-assisted sol-gel technique. It uncovers oxygen-related luminescence defects in LACOB, highlighting emission peaks at 489 and 585 nm for Dy 3+ , a distinct sharp peak at 611 nm for Eu 3+ in the red spectrum, and a notable green emission for Tb 3+ due to specific transitions. The photoluminescence (PL) analysis indicates that luminescence is optimized through precise doping, leveraging dipole interactions, and localized resonant energy transfer, which are influenced by dopant concentration and spatial configuration. Temperature studies show emission intensity variations, particularly noticeable below 100 K for Tb 3+ doped samples, demonstrating the nuanced balance between thermal quenching and luminescence efficiency. This temperature dependency, alongside the identified optimal doping conditions, underscores the potential of these materials for advanced photonic applications, offering insights into their thermal behavior and emission mechanisms under different conditions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

An investigation into the luminescent behavior of YCOB (Yttrium Calcium Oxyborate) doped with Eu 3+ and Dy 3+ ions, synthesized via the combustion method, is presented. The study, employing X-ray diffraction (XRD), Fourier-Transform Infrared Spectroscopy (FTIR), and Energy-Dispersive X-ray Spectroscopy (EDS) analyses, confirms the structural integrity and purity of the synthesized nanophosphors. An XRD pattern exhibiting distinct crystalline peaks indicates that the dopant ions were successfully integrated into the YCOB lattice. The photoluminescence (PL) response of YCOB with Eu 3+ and Dy 3+ ions is thoroughly examined, uncovering distinct excitation and emission spectra. In the case of Eu 3+ doping, excitation spectra reveal a significant charge transfer (CT) band at 254 nm, indicative of electron transfer between oxygen and europium ions. This CT transition enhances our understanding of the excitation behavior, with the dominant and Laporte-forbidden 5 D 0 → 7 F 2 transition. Characteristic peaks at 345 nm in the excitation spectra efficiently stimulate YCOB:Dy 3+ when Dy 3+ is used as a dopant. The primary emission peak at 585 nm corresponds to the hypersensitive electric dipole transition 4 F 9/2 - 6 H 13/2 . Concentration quenching phenomena are observed, with a maximum Eu 3+ concentration of 7 wt % attributed to the dipole-quadrupole interaction. Dy 3+ doping, with a maximum concentration of 2 wt % primarily shows multipolar interactions, especially dipole-dipole interactions. The study extends to CIE chromaticity analysis, emphasizing Eu 3+ doping's suitability for white light-emitting diode (WLED) applications and ensuring color stability. Conversely, varying Dy 3+ concentrations do not yield consistent chromaticity coordinates. These findings have significant implications for the development of advanced phosphor materials across diverse applications, offering a roadmap for optimizing their optical performance., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Complete loss-of-function mutations in the PRKN gene are a major cause of early-onset Parkinson's disease (PD). PRKN encodes the Parkin protein, an E3 ubiquitin ligase that works in conjunction with the ubiquitin kinase PINK1 in a distinct quality control pathway to tag damaged mitochondria for autophagic clearance, i.e., mitophagy. According to previous structural investigations, Parkin protein is typically kept in an inactive conformation via several intramolecular, auto-inhibitory interactions. Here, we performed molecular dynamics simulations (MDS) to provide insights into conformational changes occurring during the de-repression of Parkin and the gain of catalytic activity. We analyzed four different Parkin-activating mutations that are predicted to disrupt certain aspects of its auto-inhibition. All four variants showed greater conformational motions compared to wild-type protein, as well as differences in distances between domain interfaces and solvent-accessible surface area, which are thought to play critical roles as Parkin gains catalytic activity. Our findings reveal that the studied variants exert a notable influence on Parkin activation as they alter the opening of its closed inactive structure, a finding that is supported by recent structure- and cell-based studies. These findings not only helped further characterize the hyperactive variants but overall improved our understanding of Parkin's catalytic activity and nominated targets within Parkin's structure for potential therapeutic designs.

Inorganic phosphors, known for their ability to capture energy from various sources and emit visible light, have become essential in the development of advanced lighting and display technologies. This study explores YCa 4 O(BO 3 ) 3 (YCOB) as a potential host material for phosphors, focusing on the luminescent properties of YCOB phosphors doped with Sm 3+ ions. The successful integration of Sm 3+ ions into the YCOB host lattice is confirmed through structural characterization using X-ray diffraction (XRD), Fourier-Transform Infrared Spectroscopy (FTIR), and Energy-Dispersive X-ray Spectroscopy (EDS). Photoluminescence (PL) studies reveal distinct emission spectra with Stark energy level splitting, indicating a cooperative effect between Y 3+ and Sm 3+ ions. Concentration quenching, mainly attributed to dipole-dipole (d-q) interactions, is observed at higher Sm 3+ concentrations. Temperature-dependent PL measurements demonstrate thermal quenching at lower temperatures and increased emission intensity with higher laser power. Thermal quenching is explained by reduced lattice vibrations and electron-phonon interactions, leading to decreased radiative recombination of charge carriers. The CIE chromaticity data position the samples in the orange-red region, emitting vibrant orange-red light. This comprehensive investigation provides insights into the synthesis and luminescent properties of YCOB:Sm 3+ phosphors, highlighting their potential applications in luminescent devices., 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 © 2023 Elsevier Ltd. All rights reserved.)

With more than 5 million fatalities and close to 300 million reported cases, COVID-19 is the first documented pandemic due to a coronavirus that continues to be a major health challenge. Despite being rapid, uncontrollable, and highly infectious in its spread, it also created incentives for technology development and redefined public health needs and research agendas to fast-track innovations to be translated. Breakthroughs in computational biology peaked during the pandemic with renewed attention to making all cutting-edge technology deliver agents to combat the disease. The demand to develop effective treatments yielded surprising collaborations from previously segregated fields of science and technology. The long-standing pharmaceutical industry's aversion to repurposing existing drugs due to a lack of exponential financial gain was overrun by the health crisis and pressures created by front-line researchers and providers. Effective vaccine development even at an unprecedented pace took more than a year to develop and commence trials. Now the emergence of variants and waning protections during the booster shots is resulting in breakthrough infections that continue to strain health care systems. As of now, every protein of SARS-CoV-2 has been structurally characterized and related host pathways have been extensively mapped out. The research community has addressed the druggability of a multitude of possible targets. This has been made possible due to existing technology for virtual computer-assisted drug development as well as new tools and technologies such as artificial intelligence to deliver new leads. Here in this article, we are discussing advances in the drug discovery field related to target-based drug discovery and exploring the implications of known target-specific agents on COVID-19 therapeutic management. The current scenario calls for more personalized medicine efforts and stratifying patient populations early on for their need for different combinations of prognosis-specific therapeutics. We intend to highlight target hotspots and their potential agents, with the ultimate goal of using rational design of new therapeutics to not only end this pandemic but also uncover a generalizable platform for use in future pandemics., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The 2,5-diketopiperazines are a prominent class of bioactive molecules. The nocardioazines are actinomycete natural products that feature a pyrroloindoline diketopiperazine scaffold composed of two D-tryptophan residues functionalized by N- and C-methylation, prenylation, and diannulation. Here we identify and characterize the nocardioazine B biosynthetic pathway from marine Nocardiopsis sp. CMB-M0232 by using heterologous biotransformations, in vitro biochemical assays, and macromolecular modeling. Assembly of the cyclo-L-Trp-L-Trp diketopiperazine precursor is catalyzed by a cyclodipeptide synthase. A separate genomic locus encodes tailoring of this precursor and includes an aspartate/glutamate racemase homolog as an unusual D/L isomerase acting upon diketopiperazine substrates, a phytoene synthase-like prenyltransferase as the catalyst of indole alkaloid diketopiperazine prenylation, and a rare dual function methyltransferase as the catalyst of both N- and C-methylation as the final steps of nocardioazine B biosynthesis. The biosynthetic paradigms revealed herein showcase Nature's molecular ingenuity and lay the foundation for diketopiperazine diversification via biocatalytic approaches., (© 2023. The Author(s).)

Left atrial ligation (LAL) of the chick embryonic heart is a model of the hypoplastic left heart syndrome (HLHS) where a purely mechanical intervention without genetic or pharmacological manipulation is employed to initiate cardiac malformation. It is thus a key model for understanding the biomechanical origins of HLHS. However, its myocardial mechanics and subsequent gene expressions are not well-understood. We performed finite element (FE) modeling and single-cell RNA sequencing to address this. 4D high-frequency ultrasound imaging of chick embryonic hearts at HH25 (ED 4.5) were obtained for both LAL and control. Motion tracking was performed to quantify strains. Image-based FE modeling was conducted, using the direction of the smallest strain eigenvector as the orientations of contractions, the Guccione active tension model and a Fung-type transversely isotropic passive stiffness model that was determined via micro-pipette aspiration. Single-cell RNA sequencing of left ventricle (LV) heart tissues was performed for normal and LAL embryos at HH30 (ED 6.5) and differentially expressed genes (DEG) were identified.After LAL, LV thickness increased by 33%, strains in the myofiber direction increased by 42%, while stresses in the myofiber direction decreased by 50%. These were likely related to the reduction in ventricular preload and underloading of the LV due to LAL. RNA-seq data revealed potentially related DEG in myocytes, including mechano-sensing genes (Cadherins, NOTCH1, etc.), myosin contractility genes (MLCK, MLCP, etc.), calcium signaling genes (PI3K, PMCA, etc.), and genes related to fibrosis and fibroelastosis (TGF-β, BMP, etc.). We elucidated the changes to the myocardial biomechanics brought by LAL and the corresponding changes to myocyte gene expressions. These data may be useful in identifying the mechanobiological pathways of HLHS., (© 2023. The Author(s).)

Health-care waste (HCW) may pose a risk to human health and the environment because of its infectious and/or toxic properties. This study was conducted to evaluate the quantity and composition of all the HCW generated by different producers in Antalya, Turkey, using data obtained from two online systems. Accordingly, this study explored the trends in healthcare waste generation (HCWG) between 2010 and 2020 and the impact of COVID-19 on HCWG by comparing the post- and pre-COVID-19 patterns based on the data obtained from 2,029 different producers. The collected data were based on the waste codes reported by the European Commission, were characterised based on the definition of the World Health Organization, and were further analysed according to the healthcare types defined by the Turkish Ministry of Health to characterize HCW. The findings indicate that the main HCW contributor was infectious waste (94.62 %), most of which was generated by hospitals (80 %). This is due to the inclusion of only HCW fractions in this study and to the definition of infectious waste considered. This study indicates that the categorisation into the type of HCSs may be a good option to assess the increase of HCW quantities, in accordance with the service type, size, and the effects of COVID-19. The correlation results for hospitals offering primary HCS revealed a strong relationship between the HCWG rate and the population per year. This approach may help estimate future trends to promote better HCW management practices for the specific cases considered, and it can even be applied to other cities., 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 © 2023 Elsevier Ltd. All rights reserved.)

Matrix metalloproteinases (MMPs) regulate the degradation of extracellular matrix (ECM) components in biological processes. MMP activity is controlled by natural tissue inhibitors of metalloproteinases (TIMPs) that non-selectively inhibit the function of multiple MMPs via interaction with the MMPs' Zn 2+ -containing catalytic pocket. Recent studies suggest that TIMPs engineered to confer MMP specificity could be exploited for therapeutic purposes, but obtaining specific TIMP-2 inhibitors has proved to be challenging. Here, in an effort to improve MMP specificity, we incorporated the metal-binding non-canonical amino acids (NCAAs), 3,4-dihydroxyphenylalanine (L-DOPA) and (8-hydroxyquinolin-3-yl)alanine (HqAla), into the MMP-inhibitory N-terminal domain of TIMP2 (N-TIMP2) at selected positions that interact with the catalytic Zn 2+ ion (S2, S69, A70, L100) or with a structural Ca 2+ ion (Y36). Evaluation of the inhibitory potency of the NCAA-containing variants towards MMP-2, MMP-9 and MMP-14 in vitro revealed that most showed a significant loss of inhibitory activity towards MMP-14, but not towards MMP-2 and MMP-9, resulting in increased specificity towards the latter proteases. Substitutions at S69 conferred the best improvement in selectivity for both L-DOPA and HqAla variants. Molecular modeling provided an indication of how MMP-2 and MMP-9 are better able to accommodate the bulky NCAA substituents at the intermolecular interface with N-TIMP2. The models also showed that, rather than coordinating to Zn 2+ , the NCAA side chains formed stabilizing polar interactions at the intermolecular interface with MMP-2 and MMP-9. Our findings illustrate how incorporation of NCAAs can be used to probe-and possibly exploit-differential tolerance for substitution within closely related protein-protein complexes as a means to improve specificity., (© 2023. The Author(s).)

Ovarian clear cell carcinoma (OCCC) is an understudied poor prognosis subtype of ovarian cancer lacking in effective targeted therapies. Efforts to define molecular drivers of OCCC malignancy may lead to new therapeutic targets and approaches. Among potential targets are secreted proteases, enzymes which in many cancers serve as key drivers of malignant progression. Here, we found that inhibitors of trypsin-like serine proteases suppressed malignant phenotypes of OCCC cell lines. To identify the proteases responsible for malignancy in OCCC, we employed activity-based protein profiling to directly analyze enzyme activity. We developed an activity-based probe featuring an arginine diphenylphosphonate warhead to detect active serine proteases of trypsin-like specificity and a biotin handle to facilitate affinity purification of labeled proteases. Using this probe, we identified active trypsin-like serine proteases within the complex proteomes secreted by OCCC cell lines, including two proteases in common, tissue plasminogen activator and urokinase-type plasminogen activator. Further interrogation of these proteases showed that both were involved in cancer cell invasion and proliferation of OCCC cells and were also detected in in vivo models of OCCC. We conclude the detection of tissue plasminogen activator and urokinase-type plasminogen activator as catalytically active proteases and significant drivers of the malignant phenotype may point to these enzymes as targets for new therapeutic strategies in OCCC. Our activity-based probe and profiling methodology will also serve as a valuable tool for detection of active trypsin-like serine proteases in models of other cancers and other diseases., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Matrix metalloproteinases (MMPs) have long been known as key drivers in the development and progression of diseases, including cancer and neurodegenerative, cardiovascular, and many other inflammatory and degenerative diseases, making them attractive potential drug targets. Engineering selective inhibitors based upon tissue inhibitors of metalloproteinases (TIMPs), endogenous human proteins that tightly yet nonspecifically bind to the family of MMPs, represents a promising new avenue for therapeutic development. Here, we used a counter-selective screening strategy for directed evolution of yeast-displayed human TIMP-1 to obtain TIMP-1 variants highly selective for the inhibition of MMP-3 in preference over MMP-10. As MMP-3 and MMP-10 are the most similar MMPs in sequence, structure, and function, our results thus clearly demonstrate the capability for engineering full-length TIMP proteins to be highly selective MMP inhibitors. We show using protein crystal structures and models of MMP-3-selective TIMP-1 variants bound to MMP-3 and counter-target MMP-10 how structural alterations within the N-terminal and C-terminal TIMP-1 domains create new favorable and selective interactions with MMP-3 and disrupt unique interactions with MMP-10. While our MMP-3-selective inhibitors may be of interest for future investigation in diseases where this enzyme drives pathology, our platform and screening strategy can be employed for developing selective inhibitors of additional MMPs implicated as therapeutic targets in disease., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)