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Aims: Alteplase is a cornerstone thrombolytic agent in clinical practice but presents a potential bleeding risk. Stroke patients need pre-screening to exclude haemorrhagic stroke before using alteplase. In this study, we develop a new thrombolytic agent citPA5, characterized by an enhanced safety profile and minimal bleeding tendency., Methods and Results: A clot lysis agent, named citPA5, is developed based on rtPA with point mutations to completely suppress its proteolytic activity in the absence of fibrin. In the presence of fibrin, citPA5 exhibited significantly higher fibrinolytic activity (a 15.8-fold increase of kcat/Km). Furthermore, citPA5 showed resistance to endogenous fibrinolysis inhibitor, PAI-1, resulting in enhanced potency. In a series of safety evaluation experiments, including thrombelastography assay, mice tail bleeding assay, and a murine intracerebral haemorrhage (ICH) model, citPA5 did not cause systemic bleeding or worsen ICH compared with alteplase. This highlights the low risk of bleeding associated with citPA5. Finally, we found that citPA5 effectively improved cerebral blood flow and reduced infarct volume in a carotid embolism-induced stroke model., Conclusion: This clot lysis agent, citPA5, not only exhibits a low risk of bleeding but also demonstrates highly effective thrombolysis capabilities. As a result, citPA5 shows great potential for administration prior to the classification of stroke types, making it possible for use in ambulances at the onset of stroke when symptoms are identified. The findings presented in this study also suggest that this strategy could be applied to develop a new generation of fibrinolytic drugs that offer greater safety and specificity in targeting fibrin., Competing Interests: Conflict of interest: none declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Because of the high similarity in structure and sequence, it is challenging to distinguish the S1 pocket among serine proteases, primarily due to the only variability at residue 190 (A190 and S190). Peptide or protein-based inhibitors typically target the negatively charged S1 pocket using lysine or arginine as the P1 residue, yet neither discriminates between the two S1 pocket variants. This study introduces two arginine analogues, L-4-guanidinophenylalanine (12) and L-3-(N-amidino-4-piperidyl)alanine (16), as novel P1 residues in peptide inhibitors. 16 notably enhances affinities across all tested proteases, whereas 12 specifically improved affinities towards proteases possessing S190 in the S1 pocket. By crystallography and molecular dynamics simulations, we discovered a novel mechanism involving a water exchange channel at the bottom of the S1 pocket, modulated by the variation of residue 190. Additionally, the specificity of 12 towards the S190-presenting S1 pocket is dependent on this water channel. This study not only introduces novel P1 residues to engineer inhibitory potency and specificity of peptide inhibitors targeting serine proteases, but also unveils a water-mediated molecular mechanism of targeting serine proteases., 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 Inc. All rights reserved.)

In the type III CRISPR system, cyclic oligoadenylate (cOA) molecules act as second messengers, activating various promiscuous ancillary nucleases that indiscriminately degrade host and viral DNA/RNA. Conversely, ring nucleases, by specifically cleaving cOA molecules, function as off-switches to protect host cells from dormancy or death, and allow viruses to counteract immune responses. The fusion protein Csx1-Crn2, combining host ribonuclease with viral ring nuclease, represents a unique self-limiting ribonuclease family. Here, we describe the structures of Csx1-Crn2 from the organism of Marinitoga sp., in both its full-length and truncated forms, as well as in complex with cA4. We show that Csx1-Crn2 operates as a homo-tetramer, a configuration crucial for preserving the structural integrity of the HEPN domain and ensuring effective ssRNA cleavage. The binding of cA4 to the CARF domain triggers significant conformational changes across the CARF, HTH, and into the HEPN domains, leading the two R-X4-6-H motifs to form a composite catalytic site. Intriguingly, an acetate ion was found to bind at this composite site by mimicking the scissile phosphate. Further molecular docking analysis reveals that the HEPN domain can accommodate a single ssRNA molecule involving both R-X4-6-H motifs, underscoring the importance of HEPN domain dimerization for its activation., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

ABC transporters are a highly conserved membrane protein class that promote the transport of substances across membranes. Under drought conditions, insects primarily regulate the content of cuticular hydrocarbon (CHC) to retain water and prevent evaporative loss. Involvement of ABC transporter protein G (ABCG) subfamily genes in insect CHC transport has been relatively understudied. In this study, we demonstrated that ABCG4 gene in Acyrthosiphon pisum (ApABCG4) is involved in CHC transport and affects drought tolerance by regulating CHC accumulation. ApABCG4 is strongly expressed in the abdominal cuticle and embryonic stages of A. pisum. Effective silencing of ApABCG4 was achieved using RNAi, and the silencing duration was analyzed. ApABCG4 silencing resulted in a significant decrease in the total and component contents of the CHC and cuticular waxy coatings of A. pisum. Nevertheless, the internal hydrocarbon content remained unchanged. The lack of cuticular hydrocarbons significantly reduced the drought tolerance of A. pisum, shortening its survival time under drought stress. Drought stress caused significant upregulation of ApABCG4. Molecular docking showed that ApABCG4 has a high binding affinity for nine n-alkanes of CHC through electrostatic interactions. These results indicate that ApABCG4 is a novel RNAi target with key applications in aphid biological control., Competing Interests: Declaration of competing interest No conflict of interest., (Copyright © 2024. Published by Elsevier B.V.)

Objective: To expand the clinical phenotype and mutation spectrum of familial mesial temporal lobe epilepsy (FMTLE) and provide a new perspective for exploring the pathological mechanisms of epilepsy caused by leucine-rich glioma inactivated 1 (LGI1) variants., Methods: We reported clinical data from two families with FMTLE and screened patients for variants in the LGI1 gene using Whole-exome sequencing and Sanger sequencing. The clinical features of FMTLE were analysed. The pathogenicity of the causative loci was assessed according to the American College of Medical Genetics and Genomics guidelines, and potential pathogenic mechanisms were predicted through multiple bioinformatics and molecular dynamics software., Results: We identified two novel LGI1 truncating variants within two large families with FMTLE: LGI1 (c.1174C>T, p.Q392X) and LGI1 (c.703C>T, p.Q235X). Compared to previous reports, we found that focal to bilateral tonic-clonic seizures are a common type of seizure in FMTLE. The clinical phenotypes of patients with FMTLE caused by LGI1 variants were relatively mild, and all patients responded well to valproic acid. Bioinformatics analyses and molecular dynamics simulations showed that protein structure and interactions were considerably weakened or damaged as a result of both variants., Conclusion: This study presents the first report identifying LGI1 as a potential novel pathogenic gene within FMTLE families, thereby broadening the mutation spectrum associated with FMTLE. The findings of this study offer novel insights and avenues for understanding the intricate molecular mechanisms underlying LGI1 variants and their correlations with patient phenotypes. This study proposes the possibility of familial focal epilepsy syndromes overlapping., Competing Interests: Declaration of competing interest None of the authors has any conflict of interest to disclose. We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this report is consistent with those guidelines., (Copyright © 2024 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.)

Background: Endothelial hyperpermeability-induced vascular dysfunction is a prevalent and significant characteristic in critical illnesses such as sepsis and other conditions marked by acute systemic inflammation. Platelet endothelial cell adhesion molecule-1 (PECAM-1) and Tie2 serve as transmembrane receptors within endothelial cells (ECs), playing pivotal roles not only in maintaining EC-EC junctions but also in influencing vasculogenesis, vessel homeostasis, and vascular remodeling., Objectives: At present, the molecular basis of the PECAM-1-Tie2 interaction remains inadequately elucidated. In the study, recombinant soluble PECAM-1 (sPECAM-1) and Tie2 (sTie2) were expressed by Drosophila S2 and HEK293 expression systems, respectively. The interactions between sPECAM-1 and sTie2 were investigated using the Surface Plasmon Resonance (SPR) and size-exclusion chromatography methods. An immunofluorescence assay was used to detect the binding of sPECAM-1 and sTie2 on endothelial cells., Results: PECAM-1 was found to bind with sTie2 in a sodium and pH-dependent manner as confirmed by the ELISA, the D5-D6 domains of PECAM-1 might play a crucial role in binding with sTie2. Surface Plasmon Resonance (SPR) results showed that the full length of sPECAM-1 has the strongest binding affinity (K D  = 48.4 nM) with sTie2, compared to sPECAM-1-D1-D4 and sPECAM-1-D1-D2. This result is consistent with that in the ELISA. In addition, size-exclusion chromatography demonstrated that sPECAM-1, sTie2, and Ang1 can form a ternary complex., Conclusion: In this study, we determined that sPECAM-1 binds to sTie2 in a pH and sodium-dependent manner. The full length of sPECAM-1 has the strongest binding affinity, and the D5-D6 domains in sPECAM-1 play a crucial role in the interaction between sPECAM-1 and sTie2., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Glucose and fatty acid overload-induced glucolipid toxicity of pancreatic β-cells is associated with the development of diabetes. Endoplasmic reticulum stress (ERS) plays an essential role in this process. Ghrelin, a peptide secreted by the pancreas, negatively correlates with oxidative stress. The study aimed to investigate ghrelin's role in glycolipid-induced β-cell dysfunction and its possible mechanism., Methods: Mouse insulinoma β-cell, NIT-1 cells, were stimulated with high fat and high glucose to induce glucolipid toxicity. High fat and high glucose-induced NIT-1 cells were treated with acylated ghrelin (AG) or [d-Lys3]-growth hormone releasing peptide (GHRP)-6. Flow cytometry and Cell Counting Kit-8 (CCK-8) assay were performed to assess apoptosis and cell viability. The protein expression related to apoptosis, inositol-requiring kinase 1 (IRE1)/c-Jun N-terminal kinase (JNK) signaling, and ERS were investigated using western blot. Enzyme-linked immunosorbent assay (ELISA) was adopted to examine insulin's synthesis and secretion levels., Results: Ghrelin treatment improved cell viability while inhibiting cell glucolipotoxicity-induced NIT-1 cell apoptosis. Ghrelin can promote the synthesis and secretion of insulin in NIT-1 cells. Mechanistically, ghrelin attenuates ERS and inhibits the IRE1/JNK signaling pathway in NIT-1 cells induced by glucolipotoxicity., Conclusion: Ghrelin improves β-cellular dysfunction induced by glucolipotoxicity by inhibiting the IRE1/JNK pathway induced by ERS. It could be an effective treatment for β-cellular dysfunction.

The urokinase-type plasminogen activator receptor (uPAR) emerges as a key target for anti-metastasis owing to its pivotal role in facilitating the invasive and migratory processes of cancer cells. Recently, we identified the uPAR-targeting anti-metastatic ability of diltiazem (22), a commonly used antihypertensive agent. Fine-tuning the chemical structures of known hits represents a vital branch of drug development. To develop novel anti-metastatic drugs, we performed an interface-driven structural evolution strategy on 22. The uPAR-targeting and anti-cancer abilities of this antihypertensive drug wereidentified by us recently. Based on in silico strategy, including extensive molecular dynamics (MD) simulations, hierarchical binding free energy predictions, and ADMET profilings, we designed, synthesized, and identified three new diltiazem derivatives (221-8, 221-57, and 221-68) as uPAR inhibitors. Indeed, all of these three derivatives exhibited uPAR-depending inhibitory activity against PC-3 cell line invasion at micromolar level. Particularly, derivatives 221-68 and 221-8 showed enhanced uPAR-dependent inhibitory activity against the tumor cell invasion compared to the original compound. Microsecond timesclae MD simulations demonstrated the optimized moiety of 221-68 and 221-8 forming more comprehensive interactions with the uPAR, highlighting the reasonability of our strategy. This work introduces three novel uPAR inhibitors, which not only pave the way for the development of effective anti-metastatic therapeutics, but also emphasize the efficacy and robustness of an in silico-based lead compound optimization strategy in drug design., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Both overt and indolent inflammatory insults in heart transplantation can accelerate pathologic cardiac remodeling, but there are few tools for monitoring the speed and severity of remodeling over time. To address this need, we developed an automated computational pathology system to measure pathologic remodeling in transplant biopsy samples in a large, retrospective cohort of n=2167 digitized heart transplant biopsy slides. Biopsy images were analyzed to identify the pathologic stromal changes associated with future allograft loss or advanced allograft vasculopathy. Biopsy images were then analyzed to assess which historical allo-inflammatory events drive progression of these pathologic stromal changes over time in serial biopsy samples. The top-5 features of pathologic stromal remodeling most strongly associated with adverse outcomes were also strongly associated with histories of both overt and indolent inflammatory events. Our findings identify previously unappreciated subgroups of higher- and lower-risk transplant patients, and highlight the translational potential of digital pathology analysis., Competing Interests: Conflicts of Interest: None declared.

Human heavy chain ferritin (HFn) protein cage has been explored as a nanocarrier for targeted anticancer drug delivery. Here, we introduced a matrix metalloproteinases (MMPs)-cleavable sequence into the DE loop of HFn, creating an MMP-responsive variant, MR-HFn, for localized and extracellular drug release. The crystal structure of MR-HFn revealed that the addition of the MMPs recognition sequence did not affect the self-assembly of HFn but presented a surface-exposed loop susceptible to MMPs cleavage. Biochemical analysis indicated that this engineered protein cage is responsive to MMPs, enabling the targeted release of encapsulated drugs. To evaluate the therapeutic potential of this engineered protein cage, monosubstituted β-carboxy phthalocyanine zinc (CPZ), a type of photosensitizer, was loaded inside this protein cage. The prepared CPZ@MR-HFn showed higher uptake and stronger phototoxicity in MMPs overexpressed tumor cells, as well as enhanced penetration into multicellular tumor spheroids compared with its counterpart CPZ@HFn in vitro. In vivo, CPZ@MR-HFn displayed a higher tumor inhibitory rate than CPZ@HFn under illumination. These results indicated that MR-HFn is a promising nanocarrier for anticancer drug delivery and the MMP-responsive strategy here can also be adapted for other stimuli., Competing Interests: Declaration of competing interest The authors declare no conflict of interest in this work., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Lung cancer ranks among the primary contributors to cancer-related fatalities on a global scale. Multiple research investigations have demonstrated that there exists a dysbiosis within the intestinal bacteria and short-chain fatty acids (SCFAs) is linked with immune responses in lung cancer. Qingfei mixture (QFM) has been widely used in treating lung cancer, yet the active ingredients and roles of the QFM on immune responses by targeting gut microbiota remain to be elucidated. The chemical constituents of QFM were qualitatively examined by UPLC/Q-TOF-MS. Additionally, we evaluated the therapeutic impact of the organic substance QFM on lung cancer, aiming to elucidate its mechanisms for improving the tumor-immune microenvironment. Herein, we constructed a Lewis lung carcinoma (LLC)-bearing mice model with QFM treatment to observe tumor growth and immune cell changes. Then, the feces were collected and a combinatory study using metagenomes, non-targeted metabonomics, and targeted metabonomics of SCFAs was performed. In vitro experiments have been conducted to estimate the roles of acetate and sodium propionate in CD8 + T cells. Furthermore, we treated tumor-bearing mice with QFM, QFM + MHY1485 (an mTOR activator), and QFM + an antibiotic mixture (ABX) to explore the potential therapeutic benefit of regulation of the tumor microenvironment. A total of 96 compounds were obtained from QFM by UPLC/Q-TOF-MS. Besides, the findings demonstrated that QFM exhibited significant efficacy against lung cancer, manifesting in reduced tumor growth and improved immune responses. In investigating its mechanisms, we integrated gut microbiota sequencing and fecal metabolomics, revealing that QFM effectively restored disruptions in gut microbiota and SCFAs in mice with lung cancer. QFM, acetate, or sodium propionate contributed to the up-regulation of IFN-γ, Gzms-B, perforin, IL-17, IL-6, IL-12, TNF-α expressions and decreased HDAC and IL-10 levels in vitro and in vivo . Moreover, MHY1485 and ABX weakened the effects of QFM on immunomodulation. Collectively, these results suggest that QFM may facilitate immune responses in the LLC-bearing mice via regulating the gut microbiota-derived SCFAs at least partially through targeting the mTOR signaling pathway., Competing Interests: 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., (© 2024 The Author(s).)

Administration of recombinant tPA (rtPA, or trade name Alteplase®) is an FDA-approved therapy for acute ischemic stroke (AIS), but poses the risk of hemorrhagic complications. Recombinant tPA can be rapidly inactivated by the endogenous inhibitor, plasminogen activator inhibitor 1 (PAI-1). In this work, we study a novel treatment approach that combines a PAI-1 inhibitor, PAItrap4, with a reduced dose of rtPA to address the hemorrhagic concern of rtPA. PAItrap4 is a highly specific and very potent protein-based inhibitor of PAI-1, comprising of a variant of uPA serine protease domain, human serum albumin, and a cyclic RGD peptide. PAItrap4 efficiently targets and inhibits PAI-1 on activated platelets, and also possesses a long half-life in vivo. Our results demonstrate that PAItrap4 effectively counteracts the inhibitory effects of PAI-1 on rtPA, preserving rtPA activity based on amidolytic and clot lysis assays. In an in vivo murine stroke model, PAItrap4, together with low-dose rtPA, enhances the blood perfusion in the stroke-affected areas, reduces infarct size, and promotes neurological recovery in mice. Importantly, such treatment does not increase the amount of cerebral hemorrhage, thus reducing the risk of cerebral hemorrhage. In addition, PAItrap4 does not compromise the normal blood coagulation function in mice, demonstrating its safety as a therapeutic agent. These findings highlight this combination therapy as a promising alternative for the treatment of ischemic stroke, offering improved safety and efficacy., Competing Interests: Declaration of competing interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled: Specific inhibition on PAI-1 reduces the dose of Alteplase for ischemic stroke treatment., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Mycobacterium tuberculosis (Mtb) is a crucial and highly destructive intracellular pathogen responsible for causing tuberculosis (TB). The emergence and dissemination of multi-drug resistant Mtb has further aggravated the TB crisis, leading to high mortality. Mtb FadD2 is a fatty acyl-coenzyme A (CoA) synthetase that modifies the cell envelope and plays an important role in reducing Mtb susceptibility to pyrazinoic acid (POA). However, the functional mechanism of Mtb FadD2 remains poorly understood. Here, we successfully expressed, purified and obtained monomeric FadD2 by using buffer (500 mM NaCl, 20 mM Tris-HCl, pH7.4 and 5 % glycerol). Palmitate was found to be the optimal substrate for FadD2. Fatty acyl-CoA synthetase activity reached maximum at 450 μM palmitate, and the K m value was 318.2 μM for palmitate. The results of mutation experiments indicated the critical role of T370 and K551 in the enzymatic activity of FadD2. Our work provides a guideline and concept for the development of novel drugs against Mtb., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Cardiac allograft rejection is the leading cause of early graft failure and is a major focus of postheart transplant patient care. While histological grading of endomyocardial biopsy samples remains the diagnostic standard for acute rejection, this standard has limited diagnostic accuracy. Discordance between biopsy rejection grade and patient clinical trajectory frequently leads to both overtreatment of indolent processes and delayed treatment of aggressive ones, spurring the need to investigate the adequacy of the current histological criteria for assessing clinically important rejection outcomes., Methods: N=2900 endomyocardial biopsy images were assigned a rejection grade label (high versus low grade) and a clinical trajectory label (evident versus silent rejection). Using an image analysis approach, n=370 quantitative morphology features describing the lymphocytes and stroma were extracted from each slide. Two models were constructed to compare the subset of features associated with rejection grades versus those associated with clinical trajectories. A proof-of-principle machine learning pipeline-the cardiac allograft rejection evaluator-was then developed to test the feasibility of identifying the clinical severity of a rejection event., Results: The histopathologic findings associated with conventional rejection grades differ substantially from those associated with clinically evident allograft injury. Quantitative assessment of a small set of well-defined morphological features can be leveraged to more accurately reflect the severity of rejection compared with that achieved by the International Society of Heart and Lung Transplantation grades., Conclusions: Conventional endomyocardial samples contain morphological information that enables accurate identification of clinically evident rejection events, and this information is incompletely captured by the current, guideline-endorsed, rejection grading criteria., Competing Interests: Disclosures Dr Feldman is an equity holder and has technology licensed to both Elucid Bioimaging and Inspirata Inc. Dr Feldman is a scientific advisory consultant for Inspirata Inc and is on its scientific advisory board. Dr Feldman is also a consultant for Phillips Healthcare, XFIN, and Verbio. Dr Margulies holds research grants from Amgen and serves as a scientific consultant/advisory board member of Bristol Myers Squibb. Dr Madabhushi is an equity holder in Elucid Bioimaging, Inspirata Inc, and Picture Health. He has served as a scientific advisory board member of Picture Health, SimbioSys, and Aiforia Inc. He also has sponsored research agreements with AstraZeneca, Bristol Myers Squibb, Boehringer Ingelheim, and Eli Lilly. His technology has been licensed to Elucid Bioimaging and Picture Health. He is also involved in 3 different National Institutes of Health R01 grants with Inspirata Inc.

Cancer remains one of the most pressing challenges to global healthcare, exerting a significant impact on patient life expectancy. Cancer metastasis is a critical determinant of the lethality and treatment resistance of cancer. The urokinase-type plasminogen activator receptor (uPAR) shows great potential as a target for anticancer and antimetastatic therapies. In this work, we aimed to identify potential uPAR inhibitors by structural dynamics-based virtual screenings against a natural product library on four representative apo-uPAR structural models recently derived from long-timescale molecular dynamics (MD) simulations. Fifteen potential inhibitors ( NP1 - NP15 ) were initially identified through molecular docking, consensus scoring, and visual inspection. Subsequently, we employed MD-based molecular mechanics-generalized Born surface area (MM-GBSA) calculations to evaluate their binding affinities to uPAR. Structural dynamics analyses further indicated that all of the top 6 compounds exhibited stable binding to uPAR and interacted with the critical residues in the binding interface between uPAR and its endogenous ligand uPA, suggesting their potential as uPAR inhibitors by interrupting the uPAR-uPA interaction. We finally predicted the ADMET properties of these compounds. The natural products NP5 , NP12 , and NP14 with better binding affinities to uPAR than the uPAR inhibitors previously discovered by us were proven to be potentially orally active in humans. This work offers potential uPAR inhibitors that may contribute to the development of novel effective anticancer and antimetastatic therapeutics.Communicated by Ramaswamy H. Sarma.

Aim: N6-methyladenosine (m6A) RNA methylation exerts a regulatory effect on endometrioid ovarian cancer (EOC), but the specific m6A regulator genes in EOC remain to be explored. This study investigated that sulforaphene (Sul) is implicated in EOC development by regulating methyltransferase-like 3 (METTL3)., Methods: The dysregulated m6A RNA methylation genes in EOC were determined by methylated RNA immunoprecipitation (MeRIP-seq) and RNA sequencing. The roles of METTL3 and/or Sul on viability, proliferative ability, cell cycle, and apoptosis of EOC cells were determined by MTT, colony formation, flow cytometry, and TUNEL staining assay, respectively. The expression of METTL3 and apoptosis-related proteins in EOC cells was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot assays., Results: Five m6A RNA methylation regulators (METTL3, ELF3, IGF2BP2, FTO, and METTL14) were differentially expressed in EOC, among which METTL3 had the highest expression level. Silencing METTL3 reduced the clonal expansion and viability of EOC cells, and caused the cells to arrest in the G0/G1 phase. This also promoted apoptosis in the EOC cells and activated the FAS/FADD and mitochondrial apoptosis pathways. In contrast, overexpressing METTL3 had the opposite effect. Sul, in a dose-dependent manner, reduced the viability of EOC cells but promoted their apoptosis. Sul also increased the levels of IGF2BP2 and FAS, while decreasing the levels of KRT8 and METTL3. Furthermore, Sul was able to reverse the effects of METTL3 overexpression on EOC cells., Conclusions: Sul could suppress cell proliferation and promote apoptosis of EOC cells by inhibiting the METTL3 to activate the FAS/FADD and apoptosis-associated pathways., Competing Interests: The authors declare there are no competing interests., (©2023 Yu et al.)

Mycobacterium tuberculosis ( Mtb ) is an important and harmful intracellular pathogen that is responsible for the cause of tuberculosis (TB). Mtb capsular polysaccharides can misdirect the host's immune response pathways, resulting in additional challenges in TB treatment. These capsule polysaccharides are biosynthesized by stealth proteins, including CpsY. The structure and functional mechanism of Mtb CpsY are not completely delineated. Here, we reported the crystal structure of CpsY 201-520 at 1.64 Å. CpsY 201-520 comprises three β-sheets with five α-helices on one side and three on the other. Four conserved regions (CR1-CR4) are located near and at the base of its catalytic cavity, and three spacer segments (S1-S3) surround the catalytic cavity. Site-directed mutagenesis demonstrated the strict conservation of R419 at CR3 and S1-S3 in regulating the phosphotransferase activity of CpsY 201-520 . In addition, deletion of S2 or S3 (∆S2 or ∆S3) dramatically increased the activity compared to the wild-type (WT) CpsY 201-520 . Results from molecular dynamics (MD) simulations showed that S2 and S3 are highly flexible. Our study provides new insights for the development of new vaccines and targeted immunotherapy against Mtb.

The excessive use of quaternary ammonium compounds (QACs) following the COVID-19 pandemic has raised substantial concerns regarding their biosafety. Overuse of QACs has been associated with chronic biological adverse effects, including genotoxicity or carcinogenicity. In particular, inadvertent intravascular administration or oral ingestion of QACs can lead to fatal acute toxicity. To enhance the biosafety and antimicrobial efficacy of QACs, this study reports a new series of QACs, termed as PACs, with the alkyl chain of benzalkonium substituted by a phthalocyanine moiety. Firstly, the rigid phthalocyanine moiety enhances the selectivity of QACs to bacteria over human cells and reduces alkyl chain's entropic penalty of binding to bacterial membranes. Furthermore, phthalocyanine neutralizes hemolysis and cytotoxicity of QACs by binding with albumin in plasma. Our experimental results demonstrate that PACs inherit the optical properties of phthalocyanine and validate the broad-spectrum antibacterial activity of PACs in vitro. Moreover, the intravascular administration of the most potent PAC, PAC1a, significantly reduced bacterial burden and ameliorated inflammation level in a bacteria-induced septic mouse model. This study presents a new strategy to improve the antimicrobial efficacy and biosafety of QACs, thus expanding their range of applications to the treatment of systemic infections., 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 B.V. All rights reserved.)

Photodynamic therapy (PDT) is recognized as a powerful method to inactivate cells. However, the photosensitizer (PS), a key component of PDT, has suffered from undesired photobleaching. Photobleaching reduces reactive oxygen species (ROS) yields, leading to the compromise of and even the loss of the photodynamic effect of the PS. Therefore, much effort has been devoted to minimizing photobleaching in order to ensure that there is no loss of photodynamic efficacy. Here, we report that a type of PS aggregate showed neither photobleaching nor photodynamic action. Upon direct contact with bacteria, the PS aggregate was found to fall apart into PS monomers and thus possessed photodynamic inactivation against bacteria. Interestingly, the disassembly of the bound PS aggregate in the presence of bacteria was intensified by illumination, generating more PS monomers and leading to an enhanced antibacterial photodynamic effect. This demonstrated that on a bacterial surface, the PS aggregate photo-inactivated bacteria via PS monomer during irradiation, where the photodynamic efficiency was retained without photobleaching. Further mechanistic studies showed that PS monomers disrupted bacterial membranes and affected the expression of genes related to cell wall synthesis, bacterial membrane integrity, and oxidative stress. The results obtained here are applicable to other types of PSs in PDT.

Diltiazem and glibenclamide are commonly used hypotensive and antidiabetic drugs. This study reports the discovery of the potential antitumor and antimetastatic effects of these two drugs using a structural dynamics-driven virtual screening targeting urokinase receptor (uPAR). Owing to uPAR's high flexibility, currently resolved crystal structures of uPAR, all in ligand-bound states, provide limited representations of its physiological conformation. To improve the accuracy of screening, we performed a long-timescale molecular dynamics simulation and obtained the representative conformations of apo-uPAR as the targets for our screening. Experimentally, we demonstrated that diltiazem and glibenclamide bound uPAR with K D values in the micromolar range. In addition, both compounds effectively suppressed tumor growth and metastasis in a uPAR-dependent manner in vitro and in vivo . This work not only provides two potent uPAR inhibitors but also reports a proof-of-concept study on the potential off-label antitumor and antimetastatic uses of diltiazem and glibenclamide.

Recombinant tissue-type plasminogen activator (rtPA, or Alteplase) is the first approved thrombolytic drug for acute ischemic stroke, but suffers from a short half-life and poor resistance to plasminogen activator inhibitor (PAI-1), limiting its clinical use. The development of novel thrombolytic agents with improved benefit/risk balance has always been of great significance. In this study, we identified a mutant of serine protease domain of tPA (named ΔtPA A146V ) capable of escaping the inhibition by endogenous PAI-1 with 66-fold increased resistance compared to the wild type tPA. Based on this mutant, we generated a triple fusion ΔtPA (TriF-ΔtPA) containing albumin and fibrin binding peptide(FBP). The fusion with albumin effectively prolonged the plasma half-life of ΔtPA in mice to 144 min, which is much longer than ΔtPA and did not affect its thrombolytic activity. Furthermore, FBP rendered fibrin specificity of the fusion protein, giving a dissociation constant of ∼ 25 ± 0.9 μM. In a novel murine carotid embolism-induced stroke (CES) model, i.v. administration of TriF-ΔtPA promoted vascular recanalization, reduced infarct volume, and mitigated neurobehavioral deficits more significantly compared to ΔtPA-HSA or Alteplase, showing little bleeding risk. Together, this long-acting PAI-1-resistant thrombolytic agent holds great potential for clinical applications., 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. Published by Elsevier B.V.)

Rutin is a flavonoid that exists in plants and in commonly consumed foods. In recent years, rutin has been demonstrated to have anti-thrombotic efficacy through its inhibition of protein disulfide isomerase. However, the low aqueous solubility and high dose limit the therapeutic applications of rutin. In this study, we found that the chelation of zinc ions increased rutin aqueous solubility by 4-fold. More importantly, the thus-formed rutin:Zn complex inhibited PDI activity more potently than rutin itself. In a murine model with electric current-induced arterial thrombosis, the rutin:Zn complex slowed mouse arterial occlusion compared to rutin without increasing bleeding risk. Thus, the zinc chelation not only improved rutin aqueous solubility but achieved stronger inhibition of PDI. Furthermore, zinc chelation of a selected list of flavonoids containing the adjacent keto and phenoxy groups also increased their inhibition of PDI. Hence, our study provides a strategy to promote flavonoids' anti-thrombotic properties., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Urokinase plasminogen activator receptor (uPAR) is a key participant in extracellular proteolysis, tissue remodeling and cell motility. uPAR overexpresses in most solid tumors and several hematologic malignancies, but has low levels on normal tissues, thus is advocated as a molecular target for cancer therapy. One of the obstacles for the evaluation of uPAR targeting agents in preclinical study is the species specificity, where targeting agents for human uPAR  usually not bind to murine uPAR. Here, we develop a targeting agent that binds to both murine and human uPAR. This targeting agent is genetically fused to human serum albumin, a commonly used drug carrier, and the final construct is named as uPAR targeting carrier (uPARTC). uPARTC binds specifically to uPAR-overexpressing 293T/huPAR and 293T/muPAR as demonstrated by flow cytometry. A cytotoxic compound, celastrol, is embedded into uPARTC non-covalently. The resulting macromolecular complex show effective proliferation inhibition on both murine and human uPAR overexpressing cells, and exhibit potent antitumor efficacy on hepatoma H22-bearing mice. This work demonstrates that uPARTC is a promising tumor targeting drug carrier, which address the species-specificity challenge of uPAR targeting agents and can be used to load other cytotoxic compounds., 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. Published by Elsevier B.V.)

With the aim of addressing the difficult problem of biodegradable organic nitrogen in biochemical effluent of a printing and dyeing industrial park, the combined ozonation-sequencing batch biofilm reactor (O 3 -SBBR) process was used for advanced treatment. The influencing factors and degradation kinetics were analyzed; quenching experiments were carried out; and the types of free radicals, succinate dehydrogenase activity, and denitrification function genes were determined. The results showed that the suitable ozonation condition was pH 8.0-8.5, O 3 concentration was approximately 35.0 mg·L -1 , O 3 dosage was approximately 100.0 mg·L -1 , and reaction time was 90.0-120.0 min. Organic nitrogen in the biochemical effluent by ozonation conformed to the pseudo first-order kinetic model, and the maximum rate constant k was 0.01035 min -1 (experimental conditions:pH 8.0, ozone dosage 150.0 mg·L -1 , and ozone concentration 35.0 mg·L -1 ). Ozonation significantly improved the denitrification performance of the sequencing biofilm batch reactor (SBBR), and the denitrification efficiency increased from 19.8% (SBBR) to 32.9% (O 3 -SBBR). Ozonation could convert organic nitrogen and organic substances with strong toxicity and difficult biological utilization into small molecular substances with low toxicity and biodegradability. The abundance of functional genes ( nirS, nirK , and nor ) in the O 3 -SBBR combined process was significantly higher than that in the single SBBR, which further confirmed that ozonation could improve the nitrogen removal performance of SBBR. The operation cost of the combined process was 0.74-1.07 yuan·m -3 , with good technical economy. This study provided a basis for the application of the O 3 -SBBR combined process in the advanced treatment of biochemical effluent in printing and dyeing industrial parks.

Triple-negative breast cancer (TNBC) is highly aggressive and causes a higher proportion of metastatic cases. However, therapies directed to specific molecular targets have rarely achieved clinically meaningful improvements in the outcome of TNBC therapy. A urokinase-type plasminogen activator (uPA), one of the best-validated biomarkers of breast cancer, is an extracellular proteolytic serine protease involved in many pathological and physiological processes, including tumor cell invasion and metastasis. Nafamostat mesylate (NM) is a synthetic compound that inhibits various serine proteases and has been used as a therapeutic agent for the treatment of TNBC. Nevertheless, NM has poor specificity for serine proteases and is easy be hydrolyzed; moreover, the inhibitory mechanism of TNBC therapy is unclear. In this study, we combine NM with a macromolecular drug delivery vehicle, mouse amino-terminal fragment of urokinase-human serum albumin (mATF-HSA), to form a complex (mATF-HSA:NM) using the dilution-incubation-purification method. mATF specifically targets uPAR overexpressed on the surface of TNBC cells; moreover, HSA prevents NM from being hydrolyzed by numerous serine proteases. mATF-HSA:NM showed stronger inhibitory effects on the proliferation and metastasis of TNBC in vitro and in vivo without significant cytotoxicity on normal cells and tissues. In addition, we demonstrated that NM mediates metastasis of TNBC cells through inhibition of uPA using a stable uPA knockdown cell line (MDA-MB231 shuPA). Overall, we have developed a macromolecular complex targeted to treat high uPAR-expressing tumor types, and mATF-HSA can potentially be used to load other types of drugs with tumor-targeting specificity for mouse tumor models and is a promising tool to study tumor biology in mouse tumor models.

Quaternary ammonium compounds (QACs) are inexpensive and readily available disinfectants, and have been widely used, especially since the COVID-19 outbreak. The toxicity of QACs to humans has raised increasing concerns in recent years. Here, a new type of QACs was synthesized by replacing the alkyl chain with zinc phthalocyanine (ZnPc), which consists of a large aromatic ring and is hydrophobic in nature, similar to the alkyl chain of QACs. Three ZnPc-containing disinfectants were synthesized and fully characterized. These compounds showed 15-16 fold higher antimicrobial effect against Gram-negative bacteria than the well-known QACs with half-maximal inhibitory (IC 50 ) values of 1.43 μM, 2.70 μM, and 1.31 μM, respectively. With the assistance of 680 nm light, compounds 4 and 6 had much higher bactericidal toxicities at nanomolar concentrations. Compound 6 had a bactericidal efficacy of close to 6 logs (99.9999% kill rate) at 1 μM to Gram-positive bacteria, including MRSA, under light illumination. Besides, these compounds were safe for mammalian cells. In a mouse model, compound 6 was effective in healing wound infection. Importantly, compound 6 was easily degraded at working concentrations under sunlight illumination, and is environmentally friendly. Thus, compound 6 is a novel and promising disinfectant., Competing Interests: 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., (© 2022 Elsevier Ltd. All rights reserved.)

Thrombosis occurs in both macrovasculature and microvasculature, causing various cardio-cerebral vascular diseases. The lack of effective and safe antithrombotic drugs leads to a public health crisis. Mounting evidence suggests that protein disulfide isomerase (PDI) plays a critical role in the initial stage of thrombus formation, motivating the research of the feasibility of PDI inhibitors as novel anti-thrombotics. Rutin, one of the most potent PDI inhibitors, was reported to suppress platelet aggregation and thrombosis in animal models, but further studies and clinical translation were restricted due to its low aqueous solubility and oral bioavailability. In this work, we fabricated rutin-loaded lipid-based nano-formulation (NanoR) and characterized their physical-chemical properties, release profiles, pharmacokinetic process, and pharmacodynamic function against thrombosis in macrovessels and microvessels. NanoR provided increased solubility and dissolution of rutin to achieve earlier T max and higher C max than the sodium salt of rutin (NaR) after oral gavage. Ex vivo studies demonstrated that NanoR significantly inhibited thrombin generation and clot formation in the plasma of mice. Importantly, such effect was reversed by exogenous recombinant PDI, demonstrating the specificity of the NanoR. In direct current-induced arterial thrombosis model and ferric chloride-induced microvascular thrombosis model, NanoR exhibited greatly enhanced antithrombotic activity compared with NaR. NanoR also showed good safety performance according to tail bleeding assay, global coagulation tests, and histological analysis. Overall, our current results indicated that NanoR offers a promising antithrombotic treatment with potential for clinical translation.

The species-area relationship (SAR) and its mechanisms regarding microbes are not as clear as those of plants and animals; this may result from the impact of sampling effects and the confusion between SAR and distance attenuation. We hypothesize that we can find more accurate microbial SAR curve, after removing these two factors. In this study, 27 leaves of three horticultural plants were selected as island models, and microbial biodiversity assessment was done with HTS (high-throughput sequencing). The separate and small systems using leaves as islands allow us to conduct a comprehensive survey of the microbial biodiversity of the leaves, without disturbance from sampling effects and distance attenuation effects. Interestingly, we did not find microbial SAR in those 27 leaves (also not found in evergreen trees Magnolia grandiflora and Eriobotrya japonica), but we did find significant microbial SAR in deciduous tree Ficus altissima. No significant differences were found between the different trees at the alpha diversity level of microbial biodiversity, but quite different on beta diversity. The results of beta diversity partition showed that F. altissima had the highest similarity of the microbial community among the leaves compared to those of M. grandiflora and E. japonica. Since leaf genesis in deciduous plants is more simultaneous than in evergreen plants; the result suggested that inconsistent historical background of leaf islands may mask microbial SAR. Thus, intensive sampling and consistent historical background are important for understanding microbial SAR., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Air pollution caused by bacteria and viruses has posed a serious threat to public health. Commercial air purifiers based on dense fibrous filters can remove particulate matter, including airborne pathogens, but do not kill them efficiently. Here, we developed a double-grafted antibacterial fiber material for the high-efficiency capture and inactivation of airborne microorganisms. Tetracarboxyl phthalocyanine zinc, a photosensitizer, was first grafted onto the polyester (PET) fiber, followed by coating with chitosan on the surface of PET fiber to make a double-grafted fiber material. Under the irradiation of light with a specific wavelength (680 nm), double-grafted fiber materials killed up to 99.99% of Gram-positive bacteria and Gram-negative bacteria and had a significant antibacterial effect on drug-resistant bacteria. The double-grafted PET fiber showed broad-spectrum antibacterial activities and was capable to inactivate drug-resistant bacteria. Notably, in filtration experiments for airborne bacteria, this double-grafted PET fiber demonstrated a high bacteria capture efficiency (95.68%) better than the untreated PET fiber (64.87%). Besides, the double-grafted PET fiber was capable of efficiently killing airborne bacteria. This work provides a new idea for the development of air filtration materials that can efficiently kill airborne pathogen and has good biosafety.

Metallodrugs are important for anticancer treatments. They bind mainly to human serum albumin (HSA) in blood circulation, greatly modulating their pharmacokinetics and anticancer efficacy. Fatty acid (FA) is one of the most important endogenous ligands of HSA with tight binding to HSA and affecting its conformation. However, the effect of fatty acids on metallodrugs interaction with HSA is unknown. Here we identify the binding sites of a widely used metallodrug, cisplatin, in HSA in the presence or absence of a representative fatty acid, myristate, by X-ray crystallography. Our crystal structures indicate that the sidechain of residue Met548 becomes more exposed to solvent in the presence of fatty acid, and is the main Pt binding site together with Met329 in HSA:Myr:cisplatin ternary structure. An undoubted new Pt binding site is detected at His338 in the presence of fatty acid, and additional two sites are also identified at His146 and His440 + K436. In addition, we revealed the mechanism of cisplatin-induced HSA aggregation, which is due to the crosslinking between Met298 and His510 of two HSA molecules., (Copyright © 2022. Published by Elsevier B.V.)

Protein disulfide isomerase (PDI) is a vital oxidoreductase. Extracellular PDI promotes thrombus formation but does not affect physiological blood hemostasis. Inhibition of extracellular PDI has been demonstrated as a promising strategy for antithrombotic treatment. Herein, we focused on the major substrate binding site, a unique pocket in the PDI b' domain, and identified four natural products binding to PDI by combining virtual screening with tryptophan fluorescence-based assays against a customized natural product library. These hits all directly bound to the PDI-b' domain and inhibited the reductase activity of PDI. Among them, galangin showed the most prominent potency (5.9 μM) against PDI and as a broad-spectrum inhibitor for vascular thiol isomerases. In vivo studies manifested that galangin delayed the time of blood vessel occlusion in an electricity-induced mouse thrombosis model. Molecular docking and dynamics simulation further revealed that the hydroxyl-substituted benzopyrone moiety of galangin deeply inserted into the interface between the PDI-b' substrate-binding pocket and the a' domain. Together, these findings provide a potential antithrombotic drug candidate and demonstrate that the PDI b' domain is a critical domain for inhibitor development. Besides, we also report an innovative high-throughput screening method for the rapid discovery of PDI b' targeted inhibitors.

MUS81 is an important structure-specific endonuclease responsible for the processing of stalled replication forks and recombination intermediates. In human, MUS81 functions by forming complexes with its regulatory subunits EME1 and EME2, playing distinct roles in G2/M and S phases. Although the structures of MUS81-EME1 have been intensively studied, there is no structure information available about MUS81-EME2. Here, we report the crystal structure of MUS81-EME2, which reveals an overall protein fold similar to that of MUS81-EME1 complex. Further biochemical and structural characterization shows that the MUS81-EME1 and MUS81-EME2 complexes are identical in substrate recognition and endonuclease activities in vitro, implying that the distinct cellular roles of the two complexes could arise from temporal controls in cells. Finally, an extensive structure-guided mutagenesis analysis provides implications for the molecular basis of how the MUS81-EME endonucleases recognize various DNA substrates in a structure-selective manner., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Genetic fusion of human serum albumin to peptides is an important strategy to enhance the plasma half-life of the peptide. An inherent challenge of such method is the reduction of specific activity of the cargo peptides upon connecting at N- or C-termini of albumin. Here, we report a finding that residue 363-364 of albumin can be inserted with a peptide while maintaining the peptide activities. We insert a peptide inhibitor into this site, and at the N-terminus of albumin, for comparison. The chimeric protein displays potent inhibition (IC 50 value of 30 nM) to its target (uPAR), but not the N-terminally fused construct. We also study the chimera of HSA with a cyclic peptide inhibitor of murine urokinase-type plasminogen activator grafted at either the internal site or the N-terminus. The internally peptide-grafted protein possesses a much more potent inhibition compared to the N-terminally located fusion (IC 50 value of 32 nM vs 19 μM). We further demonstrate that such internal fusion does not affect albumin expression, secondary structure, and inherent drug binding activity. Thus, this work identifies a versatile insertion point inside albumin for maintaining fusion peptide activity, and opens a new avenue to expand the applications of albumin fusion technology., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Quercetin-3-rutinoside (rutin) is a bioflavonoid that is common in foods. The finding that quercetin-3-rutinoside inhibits protein disulfide isomerase (PDI) and potently blocks thrombosis in vivo has enabled the evaluation of PDI inhibition in multiple animal models of thrombus formation and has prompted clinical studies of PDI inhibition in thrombosis. Nonetheless, how quercetin-3-rutinoside blocks PDI activity remains an unanswered question. Combining NMR spectroscopy, site-directed mutagenesis, and biological assays, we identified H256 as the key residue for PDI interacting with quercetin-3-rutinoside. Quercetin-3-rutinoside inhibited the activity of PDI (WT) but not PDI (H256A). Molecular dynamic simulations indicated that the flavonoid skeleton, but not the rutinoside conjugate, is embedded in the major binding pocket on the b' domain. Among several quercetin-3-rutinoside analogues tested, only compounds with a phenoxyl group at position 7 showed direct binding to PDI, further supporting our molecular model. Studies using purified coagulation factors showed that quercetin-3-rutinoside inhibited the augmenting effects of PDI (WT), but not PDI (H256A), on tissue factor (TF) activity. Quercetin-3-rutinoside also inhibited chemotherapy-induced TF activity enhancement on endothelial cells. Together, our studies show that residue H256 in PDI and the phenoxyl group at position 7 in quercetin-3-rutinoside are essential for inhibition of PDI by quercetin-3-rutinoside. These results provide new insight into the molecular mechanism by which flavonoids block PDI activity.

Direct thrombus imaging contributes to early detection of thrombosis, and animal models with clinical relevance are vital in the development of new thrombolytics. Here, a facile clot-homing strategy is developed based on the finding that blood clot is negatively charged. Positively charged pentalysine moiety is coupled with phthalocyanine-based fluorophore , and its applications in murine thromboembolic models are described. The probe efficiently stains the cryosection of intracranial thrombi retrieved from patients with cardioembolic stroke. In vitro, the fibrin-rich clot is labeled by the probe at sub-nanomolar concentration. The probe-labeled clot is formed into microparticles (1-5 µm) and intravenously injected into mice for pulmonary embolism modeling. In vivo imaging demonstrates fast accumulation and retention of fluorescent clot microparticles in pulmonary vessels. Recombinant tissue-type plasminogen activator (rtPA) administration greatly reduces near-infrared signal in the lungs in a time-dependent manner. This probe is also tested in a stroke model. Middle cerebral artery is occluded by autologous thrombi formed under electric stimulation. In vivo imaging shows that the probe efficiently homes to thrombus at early stage. Hence, this probe has great potential in real-time imaging of thromboembolism in clinically relevant models, promoting bench-to-bedside translation. This clot-homing principle can be used in other applications., (© 2022 Wiley-VCH GmbH.)

ZnO is an important component in skin-protection products and wound-care medicines. However, ZnO's antibacterial activity is moderate. We developed two types of ZnO microparticles loading with phthalocyanine-type photosensitizers (ZnO/PSs) introducing the photodynamic effects. These photosensitive ZnO microparticles exhibited long-term while moderate antimicrobial effects by continuously releasing Zn 2+ ions. The antimicrobial efficacies were remarkably enhanced by triggering the photodynamic antimicrobial effects. Compared to the sole ZnO which showed non-measurable antimicrobial activity at a concentration of 10 mg/L, both ZnO/PSs demonstrated antimicrobial rates ranged 99%-99.99% against Escherichia coli, normal and drug-resistant Staphylococcus aureus. In a dorsal wound infection mouse model, treatment with ZnO/PSs significantly accelerated the wound recovery rates. ZnO/PSs promoted wound healing by a dual effect: 1) the release of Zn 2+ ions from ZnO facilitating tissue remodeling; 2) the photodynamic effect efficiently eliminates pathogens avoiding infection. Notably, ZnO/PSs inherited the high biosafety of ZnO without causing noticeable toxicity against erythrocyte and endothelial cells. This study not only provides a highly safe and efficient antimicrobial ZnO material for skin cares and wound modulations, but also proposes a strategy to functionalize ZnO materials., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Receptor dimerization of urokinase-type plasminogen activator receptor (uPAR) was previously identified at protein level and on cell surface. Recently, a dimeric form of mouse uPAR isoform 2 was proposed to induce kidney disease. Here, we report the crystal structure of human uPAR dimer at 2.96 Å. The structure reveals enormous conformational changes of the dimer compared to the monomeric structure: D1 of uPAR opens up into a large expanded ring that captures a β-hairpin loop of a neighboring uPAR to form an expanded β-sheet, leading to an elongated, highly intertwined dimeric uPAR. Based on the structure, we identify E49P as a mutation promoting dimer formation. The mutation increases receptor binding to the amino terminal fragment of its primary ligand uPA, induces the receptor to distribute to the basal membrane, promotes cell proliferation, and alters cell morphology via β1 integrin signaling. These results reveal the structural basis for uPAR dimerization, its effect on cellular functions, and provide a basis to further study this multifunctional receptor., (© 2022. The Author(s).)

As a prevalent medicinal liquor among Chinese people, a type of Chinese herbal spirit from Jing Brand Co., Ltd (CHS-J) is a newly developed health beverage with the health functions of anti-fatigue and immune enhancement. The researchers from the enterprise found that the contents of several components in CHS-J samples have been significantly decreasing during the stated storage period, as detected by the HPLC-UV method, which would make a great challenge for quality control of CHS-J. Furthermore, the chemical stability of CHS-J during the storage period is greatly challenged affected, especially in the environment of high temperature and light exposure. To systematically reveal the unstable components and promote the quality control of CHS-J, the chemical stability of CHS-J during the shelving storage period was characterized by the UPLC/Q-TOFMS-based metabolomics approach. First, the targeted and untargeted metabolomics approaches discovered the significantly changed components in CHS-J samples produced in different years. Furthermore, the accelerated tests of newly produced CHS samples and several authorized standards were conducted to validate the above results and elucidate the possible mechanisms underlying these chemical changes. Moreover, these chemical changes during the storage period had little influence on the anti-fatigue effect of CHS-J samples. These findings will offer new insight into the understanding of the chemical stability of CHS-J and will facilitate the quality control of CHS-J., Competing Interests: ZW, JL, WY, QY, and Y-CL was employed by the company Jing Brand Co.Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Hu, Wang, Liu, Yang, Yang, Liu, You, Chen and Wan.)

Vascular thiol isomerases (VTIs), including PDI, ERp5, ERp57, ERp72, and thioredoxin-related transmembrane protein 1 (TMX1), have important roles in platelet aggregation and thrombosis. Research on VTIs, their substrates in thrombosis, their regulatory mechanisms, and inhibitor development is an emerging and rapidly evolving area in vascular biology. Here, we describe the structures and functions of VTIs, summarize the relationship between the vascular TIs and thrombosis, and focus on the development of VTI inhibitors for antithrombotic applications., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Recombinant tissue-type plasminogen activator (rtPA) is the clot lysis drug approved for clinical use, and is characterised by a short half-life and substantial inactivation by plasminogen activator inhibitor-1 (PAI-1). We previously discovered that a tPA mutation (A419Y) at the protease domain led to enhanced fibrinolysis activity. In the present study, we studied the mechanism of such mutation in enhancing the proteolytic activity, and whether such enhancement persists in reteplase, an United States Food and Drug Administration-approved tPA truncated variant. We constructed and expressed a series of reteplase-based mutants, including rPA G (glycosylated rPA), rPA G -Y (with A419Y mutant at rPA G ), rPA G -A4 (tetra-alanine mutation at 37-loop of rPA G ), and rPA G -A4/Y (with both) and evaluated their plasminogen activation and PAI-1 resistance. Surface plasmon resonance analysis showed that the rPA G had fibrin affinity comparable to full-length tPA. Moreover, rPA G -Y had 8·5-fold higher plasminogen activation and stronger tolerance to PAI-1 compared to rPA G . We also found that the mutations containing tetra-alanine (rPA G -A4 and rPA G -A4/Y) had dramatically reduced plasminogen activation and impaired clot lysis. In a pulmonary embolism murine model, rPA G -Y displayed a more efficient thrombolytic effect than rPA G . These results identified a novel mutant reteplase variant of tPA with increased fibrinolytic activity, laying the foundation for the development of a new potent fibrinolytic agent., (© 2021 British Society for Haematology and John Wiley & Sons Ltd.)

The ongoing pandemic of coronavirus disease 2019 (COVID-19) posed a major challenge to the public health. Currently, no proven antiviral treatment for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection is available. Here we report compounds pentalysine β-carbonylphthalocyanine zinc (ZnPc5K) and chlorin e6 (ce6) potently inhibited the viral infection and replication in vitro with EC 50 values at nanomolar level. These compounds were first identified by screening a panel of photosensitizers for photodynamic viral inactivation. Such viral inactivation strategy is implementable, and has unique advantages, including resistance to virus mutations, affordability compared to the monoclonal antibodies, and lack of long-term toxicity., Competing Interests: 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., (© 2021 Published by Elsevier Ltd.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the viral pathogen causing the coronavirus disease 2019 (COVID-19) global pandemic. No effective treatment for COVID-19 has been established yet. The serine protease transmembrane protease serine 2 (TMPRSS2) is essential for viral spread and pathogenicity by facilitating the entry of SARS-CoV-2 into host cells. The protease inhibitor camostat, an anticoagulant used in the clinic, has potential anti-inflammatory and antiviral activities against COVID-19. However, the potential mechanisms of viral resistance and antiviral activity of camostat are unclear. Herein, we demonstrate high inhibitory potencies of camostat for a panel of serine proteases, indicating that camostat is a broad-spectrum inhibitor of serine proteases. In addition, we determined the crystal structure of camostat in complex with a serine protease (uPA [urokinase-type plasminogen activator]), which reveals that camostat is inserted in the S1 pocket of uPA but is hydrolyzed by uPA, and the cleaved camostat covalently binds to Ser195. We also generated a homology model of the structure of the TMPRSS2 serine protease domain. The model shows that camostat uses the same inhibitory mechanism to inhibit the activity of TMPRSS2, subsequently preventing SARS-CoV-2 spread. IMPORTANCE Serine proteases are a large family of enzymes critical for multiple physiological processes and proven diagnostic and therapeutic targets in several clinical indications. The serine protease transmembrane protease serine 2 (TMPRSS2) was recently found to mediate SARS-CoV-2 entry into the host. Camostat mesylate (FOY 305), a serine protease inhibitor active against TMPRSS2 and used for the treatment of oral squamous cell carcinoma and chronic pancreatitis, inhibits SARS-CoV-2 infection of human lung cells. However, the direct inhibition mechanism of camostat mesylate for TMPRSS2 is unclear. Herein, we demonstrate that camostat uses the same inhibitory mechanism to inhibit the activity of TMPRSS2 as uPA, subsequently preventing SARS-CoV-2 spread.

Endothelial cells that form the inner layers of both blood and lymphatic vessels are important components of the vascular system and are involved in the pathogenesis of vascular and lymphatic diseases. Angiopoietin (Ang)-Tie axis in endothelial cells is the second endothelium-specific ligand-receptor signaling system necessary for embryonic cardiovascular and lymphatic development in addition to the vascular endothelial growth factor receptor pathway. The Ang-Tie axis also maintains vascular homeostasis by regulating postnatal angiogenesis, vessel remodeling, vascular permeability, and inflammation. Therefore, the dysfunction of this system leads to many vascular and lymphatic diseases. In light of the recent advances on the role of the Ang-Tie axis in vascular and lymphatic system-related diseases, this review summarizes the functions of the Ang-Tie axis in inflammation-induced vascular permeability, vascular remodeling, ocular angiogenesis, shear stress response, atherosclerosis, tumor angiogenesis, and metastasis. Moreover, this review summarizes the relevant therapeutic antibodies, recombinant proteins, and small molecular drugs associated with the Ang-Tie axis.

During low light- (LL) induced state transitions in dark-adapted rice (Oryza sativa) leaves, light-harvesting complex (LHC) II become phosphorylated and associate with PSI complexes to form LHCII-PSI-LHCI supercomplexes. When the leaves are subsequently transferred to high light (HL) conditions, phosphorylated LHCII complexes are no longer phosphorylated. Under the HL-induced transition in LHC phosphorylation status, we observed a new green band in the stacking gel of native green-PAGE, which was determined to be LHCII aggregates by immunoblotting and 77K chlorophyll fluorescence analysis. Knockout mutants of protein phosphatase 1 (PPH1) which dephosphorylates LHCII failed to form these LHCII aggregates. In addition, the ability to develop non-photochemical quenching in the PPH1 mutant under HL was less than for wild-type plants. As determined by immunoblotting analysis, LHCII proteins present in LHCII-PSI-LHCI supercomplexes included the Lhcb1 and Lhcb2 proteins. In this study, we provide evidence suggesting that LHCII in the LHCII-PSI-LHCI supercomplexes are dephosphorylated and subsequently form aggregates to dissipate excess light energy under HL conditions. We propose that this LHCII aggregation, involving LHCII L-trimers, is a newly observed photoprotective light-quenching process operating in the early stage of acclimation to HL in rice plants., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Human serum albumin (HSA), a versatile protein carrier for endogenous and exogenous compounds, is a proven macromolecule to form nanoparticles for drug delivery. To render HSA carrier specificity toward tumors, we designed a recombinant HSA protein fused with Kunitz domain 1 (KD1) of hepatocyte growth factor activator inhibitor type 1, which targets to matriptase, a type II transmembrane serine protease overexpressed on tumor cell surface. The carrier was thus named matriptase targeting carrier (MTC). In this study, we showed that MTC displayed the same inhibitory potency as the KD1 againast matriptase, demonstrating the HSA fusion did not affect the KD1 targeting potency. For tumor optical imaging and ablation, MTC was prepared as nanoparticle drug carrier by a novel method via denaturation and refolding to incorporate photosensitizer, CPZ. This matriptase targeting nanoparticles, CPZ:MTC@NPs, showed high specificity and cytotoxicity for matriptase-overexpressing cancer cells in vitro. In tumor-bearing mice, CPZ:MTC@NPs demonstrated selective accumulation and high retention in matriptase-overexpressing tumor. Under illumination, the nanoparticles significantly reduced tumor volumes (79.6%) as compared to saline control. These findings showed that this supramolecular nanocarrier, a new type of tumor targeting self-assembly nanoparticle, had potential as a highly efficient tumor targeting drug carrier for imaging and therapy., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The emergence of antibiotic-resistant bacteria poses a serious challenge to medical practice worldwide. A small peptide with sequence RWRWRW was previously identified as a core antimicrobial peptide with limited antimicrobial spectrum to bacteria, especially Gram-positive bacteria. By conjugating this peptide and its analogs with lipophilic phthalocyanine (Pc), we identified a new antibiotic peptide [PcG 3 K 5 (RW) 3 ]. The peptide demonstrates increased antimicrobial effect to both Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli . In addition, Pc also provides added and potent antimicrobial effect upon red light illumination. The inhibitory efficacy of PcG 3 K 5 (RW) 3 was increased by ~140-fold to nanomolar range upon illumination. Moreover, PcG 3 K 5 (RW) 3 was safe for mammalian cell and promoted wound healing in the mouse infection model. Our work provides a new direction to optimize antimicrobial peptides to enhance antimicrobial efficacy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Zhang, Chen, Jing, Chen, Ullah, Jiang, Zheng, Yuan and Huang.)

Objective: The experience of safe extubation in the operating room (OR) after transcatheter aortic valve implantation (TAVI) procedure remains not well established. The authors conducted this study to assess the effect of OR extubation in comparison with extubation in the intensive care unit (ICU) on the outcomes and cost in patients undergoing transapical-TAVI., Design: A propensity score-matched analysis., Setting: A single major urban teaching and university hospital., Participants: A total of 266 patients undergoing transapical TAVI under general anesthesia between June 2015 and March 2020., Interventions: Propensity matching on pre- and intraoperative variables was used to identify 99 patients undergoing extubation in the OR versus 72 undergoing extubation in the ICU for outcome analysis., Measurements and Main Results: After matching, extubation in the OR showed significant reductions of length of stay (LOS) in ICU (38.8 ± 17.4 v 58.0 ± 70.0 h, difference -19.2, 95% confidence interval [CI] -35.7 to -2.7, p = 0.009) and postoperative LOS in hospital (7.1 ± 3.9 v 10.1 ± 4.6 d, difference -3.0, 95% CI -4.3 to -1.7, p < 0.0001) compared with ICU extubation, but did not significantly affect the composite incidence of any postoperative complications (46.5% [46 of 99] v 52.8% [38 of 72], difference -6.3%, 95% CI -21.5 to 8.9, p = 0.415). Also, extubation in the OR led to significant reduction of total hospital cost compared with extubation in the ICU (¥303.5 ± 17.3 v ¥329.9 ± 52.3 thousand, difference -26.2, 95% CI -38.8 to -13.7, p < 0.0001)., Conclusions: The current study provided evidence that extubation in the OR could be performed safely without increases in morbidity, mortality, or reintubation rate and could provide cost-effective outcome benefits in patients undergoing transapical-TAVI., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Objective: The quality evaluation of herbal products remains a big challenge. Traceable markers are the core concept of the authentication of herbal products. However, the discovery of traceable markers is labor-intensive and time-consuming. The aim of this study is to develop a convenient approach to rapidly screen the traceable markers for herbal product authentication., Methods: Commercial Jing Liqueur and its 22 species of herbal ingredients were analyzed using HPLC-QTOF-MS and GC-MS to characterize nonvolatile and volatile chemicals. The acquired data were imported into MZmine 2 software for mass detection, chromatogram building, deconvolution and alignment. The aligned data were exported into a csv file and then traceable markers were selected using the built-in filter function in Excel. Finally, the traceable markers were identified by searching against online databases or publications, some of which were confirmed by reference standards., Results: A total of 288 chemical features transferred from herbal materials to Jing Liqueur product were rapidly screened out. Among them, 52 markers detected by HPLC-QTOF-MS were annotated, while nine volatile markers detected by GC-MS were annotated. Moreover, 30 of these markers were confirmed by comparing with reference standards. A chemical fingerprint consisting of traceable markers was finally generated to ensure the authentication and quality consistency of Jing Liqueur., Conclusion: A strategy for rapid discovery of traceable markers in herbal products using MZmine 2 software was developed., Competing Interests: 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., (© 2021 Tianjin Press of Chinese Herbal Medicines. Published by ELSEVIER B.V.)

Islet transplantation (IT) is considered the most effective endocrine replacement therapy for diabetes mellitus (DM). Studies have demonstrated that IT can repair testicular structural injury caused by inflammatory and oxidative stress in a diabetic rat model. However, highly effective exogenous antioxidant and anti-inflammatory drugs can achieve this effect. Testicular interstitial fibrosis caused by long-term hyperglycemia is however difficult to reverse or recover. Thus far, there are no effective drugs that prevent or relieve testicular interstitial fibrosis. Therefore, it is necessary to explore the potential benefit of IT on testicular interstitial fibrosis induced by DM and its underlying molecular mechanisms. In the present study, Wistar rats were used to establish a DM model by intraperitoneal injection of streptozotocin. The diabetic models then underwent IT or received insulin treatment after 12 weeks. IT was more effective than insulin treatment in ameliorating diabetic-induced testicular interstitial fibrosis, Leydig cells apoptosis, testosterone deficiency and poor sperm motility. IT and insulin treatment both significantly inhibited the upregulation of TGF-β1 and phosphorylated Smad2 in DM, with IT being more effective than insulin. The present study's findings proved that IT effectively protects diabetic-induced testicular interstitial fibrosis probably by inhibiting the TGF-β1/Smad2 signaling pathway, which offers hope in male patients with DM complicating with testicular interstitial fibrosis.

Seven known species of Tenebrionidae are recorded from Hainan Island, of which six species are recorded from this island for the first time. Also nine new species of Cnodalonini are described from Hainan Is. under the names of Androsus pullus sp. n., Euhemicera hirtipes sp. n., Gnesis amabilis sp. n., Hemicera (Hemicera) viridula sp. n., Plamius laoshei sp. n., Pseudonautes fulgidicollis sp. n., Taichius caeruleopunctatus sp. n., Tetragonomenes sylvestris sp. n. and Tetraphyllus hlaicolus sp. n.