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Rapid and precise diagnostic techniques are essential for identifying foodborne pathogens, including Bacillus cereus ( B. cereus ), which poses significant challenges to food safety. Traditional detection methods are limited by long incubation times and high costs. In this context, gold nanoparticle (AuNP)-based lateral flow assays (LFAs) are emerging as valuable tools for rapid screening. However, the use of antibodies in LFAs faces challenges, including complex production processes, ethical concerns, or variability. Here, we address these challenges by proposing an innovative approach using bacteriophage-derived proteins for pathogen detection on LFAs. We used the engineered endolysin cell-wall-binding domain (CBD) and distal tail proteins (Dit) from bacteriophages that specifically target B. cereus . The protein-binding properties, essential for the formation of efficient capture and detection biointerfaces in LFAs, were extensively characterized from the microstructural to the LFA device level. Machine-learning models leverage knowledge of the protein sequence to predict advantageous protein orientations on the nitrocellulose membrane and AuNPs. The study of the biointerface binding quantified the degree of attachment of AuNPs to bacteria, providing, for the first time, a microscopic model of the number of AuNPs binding to bacteria. It highlighted the binding of up to one hundred 40 nm AuNPs per bacterium in conditions mimicking LFAs. Eventually, phage proteins were demonstrated as efficient bioreceptors in a straightforward LFA prototype combining the two proteins, providing a rapid colorimetric response within 15 min upon the detection of 10 5 B. cereus cells. Recombinantly produced phage binding proteins present an opportunity to generate a customizable library of proteins with precise binding capabilities, offering a cost-effective and ethical alternative to antibodies. This study enhances our understanding of phage protein biointerfaces, laying the groundwork for their utilization as efficient bioreceptors in LFAs and rapid point-of-care diagnostic assays, thus potentially strengthening public health measures.

Background: Staphylococcus aureus is a significant foodborne pathogen, commonly detected in milk and meat products. Conventional detection methods have limited sensitivity and specificity, which are time-consuming and susceptible to background interference from complex samples, and cannot effectively distinguish live and dead bacteria., Results: Herein, we developed a novel adenosine triphosphate (ATP) bioluminescence method coupled with magnetic separation, which is based on phage-encoded endolysin LysSA163-CBD (CBD 163) for rapid and specific detection of viable Staphylococcus aureus. The expressed protein (CBD 163) was derived from the phage LSA2301 and was successfully expressed in Escherichia coli BL21 following an induction period of 4 h at 37 °C, with a molecular weight approximating 40 kDa. The optimal conditions for CBD-magnetic beads (cMBs) to capture S. aureus cells were determined to be 100 μL/mL cMBs at 25 °C for 30 min. The viable S. aureus cells were disrupted by the Cetyl trimethyl ammonium bromide (CTAB) to release intracellular ATP. Then, the ATP reacted with the firefly luciferase and D-Luciferin-based bioluminescence (BL) reagents solution to generate intensive BL signal. The CBD-magnetic separation-ATP bioluminescence (cMS-BL) assay was able to quickly detect viable S. aureus via ATP bioluminescence in 45 min, with a detection range from 5 × 10 3 to 5 × 10 7  CFU/mL and limit of detection (LOD) of 190 CFU/mL. Additionally, the cMS-BL method exhibited high specificity and anti-interference ability, which has been successfully applied to quantify S. aureus cells in crayfish-tail, chicken, and skim milk., Significance and Novelty: These results demonstrate the potential of CBD 163 as a versatile and robust biorecognition element for rapid and specific detection of viable S. aureus in food matrices. The proposed phage-derived bacteria-binding proteins-based protocol for BL detection shows various advantages, including high sensitivity, simple operation, and the capability to distinguish live bacteria, providing a strategy for designing high-quality biorecognition element toward foodborne pathogens., 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: A hydrolytic enzyme called protease from microbial sources has expansive applications in leather as well as tannery industries and thus leaves no choice but to discover a potential candidate for efficient yield of protease with eccentric characteristics., Methods and Results: In the current study, using skim milk agar medium, three bacterial strains (Pro SS14, Pro NTL1, Pro SM) were identified for protease production out of thirty tannery waste samples. The bacterial isolates were identified through morphological, biochemical and molecular basis and the highest protease producer (134 U/mL) having the paramount protein content (8.75%) with the greatest biomass (absorbance 1.7 at 600 nm) was further bioinformatically identified as Bacillus subtilis Pro NTL1. Moreover, the cultural conditions-pH, temperature, incubation period and salinity were tested to get the optimized condition for the three isolates where Bacillus subtilis Pro NTL1 gave best growth at pH 9 demonstrating alkaline protease producing capability, at 45 °C for 72 h proving its thermotolerance characteristics and at 3% NaCl concentration declaring its halotolerance property. Furthermore, following Kirby-Bauer agar disc diffusion technique, the antimicrobial susceptibility pattern was investigated showing Bacillus subtilis Pro NTL1 was resistant to ampicillin, penicillin, sulfafurazole and sensitive to amoxicillin, gentamycin, cefotaxime, kanamycin, imipenem, tetracycline. Additionally, Bacillus subtilis Pro NTL1 showed antagonistic activity against three pathogens named Bacillus cereus, Bacillus megaterium and Escherichia coli., Conclusions: Thus, these findings suggest that Bacillus subtilis Pro NTL1 is a newly isolated strain with unique features which might help to contribute in several biotechnological purposes with significant industrial benefits., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Fibroblast activation protein-α (FAP) has emerged as a promising target in the field of radiopharmaceuticals due to its selective expression in cancer-associated fibroblasts (CAFs) and other pathological conditions involving fibrosis and inflammation. Recent advancements have focused on developing FAP-specific radioligands for diagnostic imaging and targeted radionuclide therapy. This perspective summarized the latest progress in FAP radiopharmaceutical development, highlighting novel radioligands, preclinical evaluations, and potential clinical applications. Additionally, we analyzed the advantages and existing problems of targeted FAP radiopharmaceuticals, and discussed the key breakthrough directions of this target, so as to improve the development and conversion of FAP-targeted radiopharmaceuticals., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Zehui Wu reports financial support was provided by the National Natural Science Foundation of China (82372018 and 81701753). Zehui Wu reports financial support was provided by the Beijing Natural Science Foundation (7222299). Zehui Wu reports a relationship with Capital Medical University that includes: 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 Masson SAS. All rights reserved.)

Orchestrated action of peptidoglycan (PG) synthetases and hydrolases is vital for bacterial growth and viability. Although the function of several PG synthetases and hydrolases is well understood, the function, regulation, and mechanism of action of PG hydrolases characterised as lysostaphin-like endopeptidases have remained elusive. Many of these M23 family members can hydrolyse glycyl-glycine peptide bonds and show lytic activity against Staphylococcus aureus whose PG contains a pentaglycine bridge, but their exact substrate specificity and hydrolysed bonds are still vaguely determined. In this work, we have employed NMR spectroscopy to study both the substrate specificity and the bond cleavage of the bactericide lysostaphin and the S. aureus PG hydrolase LytM. Yet, we provide substrate-level evidence for the functional role of these enzymes. Indeed, our results show that the substrate specificities of these structurally highly homologous enzymes are similar, but unlike observed earlier both LytM and lysostaphin prefer the D-Ala-Gly cross-linked part of mature peptidoglycan. However, we show that while lysostaphin is genuinely a glycyl-glycine hydrolase, LytM can also act as a D-alanyl-glycine endopeptidase., Competing Interests: LA, SV, CT, MJ, IP, HT, PP No competing interests declared, (© 2024, Antenucci et al.)

Inflammation and tissue fibrosis co-exist and are causally linked to organ dysfunction 1,2 . However, the molecular mechanisms driving immune-fibroblast cell communication in human cardiac disease remain unexplored and there are at present no approved treatments that directly target cardiac fibrosis 3,4 . Here we performed multiomic single-cell gene expression, epitope mapping and chromatin accessibility profiling in 45 healthy donor, acutely infarcted and chronically failing human hearts. We identified a disease-associated fibroblast trajectory that diverged into distinct populations reminiscent of myofibroblasts and matrifibrocytes, the latter expressing fibroblast activator protein (FAP) and periostin (POSTN). Genetic lineage tracing of FAP + fibroblasts in vivo showed that they contribute to the POSTN lineage but not the myofibroblast lineage. We assessed the applicability of experimental systems to model cardiac fibroblasts and demonstrated that three different in vivo mouse models of cardiac injury were superior compared with cultured human heart and dermal fibroblasts in recapitulating the human disease phenotype. Ligand-receptor analysis and spatial transcriptomics predicted that interactions between C-C chemokine receptor type 2 (CCR2) macrophages and fibroblasts mediated by interleukin-1β (IL-1β) signalling drove the emergence of FAP/POSTN fibroblasts within spatially defined niches. In vivo, we deleted the IL-1 receptor on fibroblasts and the IL-1β ligand in CCR2 + monocytes and macrophages, and inhibited IL-1β signalling using a monoclonal antibody, and showed reduced FAP/POSTN fibroblasts, diminished myocardial fibrosis and improved cardiac function. These findings highlight the broader therapeutic potential of targeting inflammation to treat tissue fibrosis and preserve organ function., Competing Interests: Competing interests X.L., T.Y., S.Y.S., A.F., M.F., X.Z., S.J., C.-M.L. and B.A. are or were employed by Amgen. The other authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Chronic hypertension leads to injury and fibrosis in major organs. Fibroblast activation protein (FAP) is one of key molecules in tissue fibrosis, and 68 Ga-labeled FAP inhibitor-46 (FAPI46) PET is a recently developed method for evaluating FAP. The aim of this study was to evaluate FAP expression and fibrosis in a hypertension model and to test the feasibility of 68 Ga-FAPI46 PET in hypertension. Methods: Hypertension was induced in mice by angiotensin II infusion for 4 wk. 68 Ga-FAPI46 biodistribution studies and PET scanning were conducted at 1, 2, and 4 wk after hypertension modeling, and uptake in the major organs was measured. The FAP expression and fibrosis formation of the heart and kidney tissues were analyzed and compared with 68 Ga-FAPI46 uptake. Subgroups of the hypertension model underwent angiotensin receptor blocker administration and high-dose FAPI46 blocking, for comparison. As a preliminary human study, 68 Ga-FAPI46 PET images of lung cancer patients were analyzed and compared between hypertension and control groups. Results: Uptake of 68 Ga-FAPI46 in the heart and kidneys was significantly higher in the hypertension group than in the sham group as early as week 1 and decreased after week 2. The uptake was specifically blocked in the high-dose blocking study. Immunohistochemistry also revealed FAP expression in both heart and kidney tissues. However, overt fibrosis was observed in the heart, whereas it was absent from the kidneys. The angiotensin receptor blocker-treated group showed lower uptake in the heart and kidneys than did the hypertension group. In the pilot human study, renal uptake of 68 Ga-FAPI46 significantly differed between the hypertension and control groups. Conclusion: In hypertension, FAP expression is increased in the heart and kidneys from the early phases and decreases over time. FAP expression appears to represent fibrosis activity preceding or underlying fibrotic tissue formation. 68 Ga-FAPI46 PET has potential as an effective imaging method for evaluating FAP expression in progressive fibrosis by hypertension., (© 2024 by the Society of Nuclear Medicine and Molecular Imaging.)

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma and is associated with a poor prognosis. Data from the Gene Expression Profiling Interactive Analysis (GEPIA) database revealed dysregulated expression of several ubiquitin-specific proteases (USPs) in DLBCL tissues (DLBCL vs. non-DLBCL = 47 vs. 337), including USP19 (log 2 fold change = 1.17, P < 0.05). USP19 is closely linked to tumorigenesis, but its role in DLBCL progression remains largely unknown. Here, we investigated the role of USP19 in DLBCL development. Genetic manipulation of USP19 using adenovirus-based vectors was performed in two DLBCL cell lines, SUDHL4 and DB cells. The results showed that USP19 knockdown suppressed the proliferation, anchorage-independent growth and xenograft tumor formation of DLBCL cells and arrested the cell cycle at the G1 stage. In parallel, DLBCL cells overexpressing USP19 acquired a more malignant phenotype. Next, to explore USP19 interactors, we performed co-immunoprecipitation/liquid chromatography-mass spectrometry and identified potential interacting proteins. Among them, Parkinson disease protein 7 (PARK7), a member of the peptidase C56 family known to be involved in carcinogenesis, was further validated to bind with and be stabilized by USP19. Additionally, we found that USP19 induced PARK7 deubiquitylation in both DLBCL cell lines, and PARK7 acted as a downstream effector of USP19 in regulating the growth of DLBCL cells. Collectively, USP19 exerts a tumor-promoting role in DLBCL through interacting with and stabilizing PARK7., (© 2024 Federation of European Biochemical Societies.)

Spinal cord injury (SCI) is a significant health concern, as it presently has no effective treatment in the clinical setting. Inflammation is a key player in the pathophysiological process of SCI, with a number of studies evidencing that the inhibition of the NF-κB signaling pathway may impede the inflammatory response and improve SCI. OTULIN, as a de-ubiquitination enzyme, the most notable is its anti-inflammatory effect. OTULIN can inhibit the NF-κB signaling pathway to suppress the inflammatory reaction via de-ubiquitination. In addition, OTULIN may promote vascular regeneration through the Wnt/β-catenin pathway in the wake of SCI. In this review, we analyze the structure and physiological function of OTULIN, along with both NF-κB and Wnt/β-catenin signaling pathways. Furthermore, we examine the significant role of OTULIN in SCI through its impairment of the NF-κB signaling pathway, which could open the possibility of it being a novel interventional target for the condition., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Cells utilize diverse organelles to maintain homeostasis and to respond to extracellular stimuli. Recently, multifaceted aspects of organelle stress caused by various factors have been emerging. The endosome is an essential organelle, functioning as the central hub for membrane trafficking in cooperation with the ubiquitin system. However, knowledge regarding endosomal stress, which refers to organelle stress of the endosome, is currently limited. We recently revealed ubiquitin-mediated endosomal stress of early endosomes (EEs) and its responsive signaling pathways. These findings shed light on the relevance of ubiquitin-mediated endosomal stress to physiological and pathological processes. Here, we present a hypothesis that ubiquitin-mediated endosomal stress may have significant roles in biological contexts and that ubiquitin-specific protease 8 is a key regulator of ubiquitin clearance from EEs., (© 2024 The Author(s). BioEssays published by Wiley Periodicals LLC.)

This work aims to investigate the utility of positron emission tomography/computed tomography (PET/CT) with fibroblast activation protein inhibitors (FAPI) in urological neoplasms, including prostate cancer, urothelial carcinoma, and renal cell carcinoma. Although the available data are very preliminary, FAPI PET showed potential for detecting primary prostate cancer with low prostate-specific membrane antigen expression, while prostate-specific membrane antigen PET/CT outperformed FAPI PET/CT in detecting biochemical recurrence. In urothelial carcinoma, FAPI PET/CT demonstrated increased detection rates compared with deoxy-2-[18F]fluoro-D-glucose PET/CT, in particular in small lymph node metastases, whose identification is still an unmet clinical need. Limited data are available for renal cell carcinoma. In conclusion, FAPI PET emerges as a promising imaging modality for urological neoplasms, in particular bladder cancer. Further research is warranted to establish its role in guiding therapeutic decisions and as a potential novel theranostic agent., (© 2024. The Author(s).)

Cholangiocarcinoma (CCA) is a hepatobiliary carcinoma with uncontrolled cell proliferation, poor prognosis, and high mortality. The ovarian tumor structural domain (OTU) containing protein 6B (OTUD6B) belongs to the OTU deubiquitin family and is vital in tumor development. However, its expression and biological function in CCA remain unknown. The expression of OTUD6B in CCA was analyzed using TIMER2.0, UALCAN, and GEO databases. MTT, clonal formation assay, immunofluorescence staining, immunohistochemistry staining, and flow cytometry examined the regulation of OTUD6B on cell proliferation, cycle, and apoptosis. The effects of OTUD6B on tumor volume and weight were assessed using the xenograft tumor model. The activities of PTK2 and STAT3 were detected by western blot and CO-IP. The biological database identified that OTUD6B was upregulated in CCA. In CCA cells, OTUD6B knockdown reduced CCA cell proliferation and promoted apoptosis. Cell cycle analysis indicated that the cycle stopped at the G0/G1 phase after OTU6B downregulation. Furthermore, OTUD6B knockdown resulted in a decrease in tumor volume and weight in xenograft tumor models. Mechanistically, OTUD6B is involved in the deubiquitination of PTK2. PTK2 further affected the phosphorylation of STAT3 thereby regulating the CCA process. Our study demonstrates that OTUD6B knockdown participates in the ubiquitination of PTK2 and phosphorylation of STAT3 to alleviate the process of CCA. These results suggest that OTUD6B may be a potential new strategy for CCA treatment., (© 2024 International Federation of Cell Biology.)

A major challenge in prevention and early treatment of organ fibrosis is the lack of valuable tools to assess the evolving profibrotic maladaptive repair after injury in vivo in a non-invasive way. Here, using acute kidney injury (AKI) as an example, we tested the utility of fibroblast activation protein (FAP) imaging for dynamic assessment of maladaptive repair after injury. The temporospatial pattern of kidney FAP expression after injury was first characterized. Single-cell RNA sequencing and immunostaining analysis of patient biopsies were combined to show that FAP was specifically upregulated in kidney fibroblasts after AKI and was associated with fibroblast activation and chronic kidney disease (CKD) progression. This was corroborated in AKI mouse models, where a sustained and exaggerated kidney FAP upregulation was coupled to persistent fibroblast activation and a fibrotic outcome, linking kidney FAP level to post-insult maladaptive repair. Furthermore, using positron emission tomography (PET)/CT scanning with FAP-inhibitor tracers ([ 18 F]FAPI-42, [ 18 F]FAPT) targeting FAP, we demonstrated the feasibility of non-invasively tracking of maladaptive repair evolution toward kidney fibrosis. Importantly, a sustained increase in kidney [ 18 F]FAPT (less hepatobiliary metabolized than [ 18 F]FAPI-42) uptake reflected persistent kidney upregulation of FAP and characterized maladaptive repair after AKI. Kidney [ 18 F]FAPT uptake at hour 2-day 7 correlated with kidney fibrosis 14 days after AKI. Similar changes in [ 18 F]FAPI-42 PET/CT imaging were observed in patients with AKI and CKD progression. Thus, persistent kidney FAP upregulation after AKI was associated with maladaptive repair and a fibrotic outcome. Hence, FAP-specific PET/CT imaging enables dynamic visualization of maladaptive repair after AKI and prediction of kidney fibrosis within a clinically actionable window., (Copyright © 2024 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Enzymes are integral to numerous industrial processes, with a growing global demand for various enzyme types. Protease enzymes, in particular, have proven to be cost-effective, stable, and compatible alternatives to traditional chemical processes in both industrial and environmental applications. In this study, an alkaline protease-producing strain of Aeromonas spp. was isolated from a wastewater treatment plant in Iran. The protease production was confirmed by culturing the strain on casein agar medium. The bacterium was identified through morphological, biochemical, and 16 S rRNA sequencing analyses. The optimal culture medium for bacterial growth and enzyme production was obtained using peptone, salt, yeast extract, galactose, and CaCl₂ at an initial pH of 8. Maximum protease production was achieved after 20 h of incubation at 40 °C. To partially purify the enzyme, the supernatant of the bacterial culture medium was first centrifuged, and the enzyme was precipitated using ammonium sulfate, followed by dialysis. Zymography revealed the production of one type of protease during bacterial growth. The partially purified protease exhibited optimal activity at pH 8.5 and maximum stability at pH 9. The optimum temperature for maximum enzyme activity was observed at 50 °C, with 100% residual activity retained for 1 h at 0 °C. The effect of metal ions on enzyme activity was assessed, revealing that KCl induced the most significant effects (p < 0.0001) on enzyme activity. Chemical amino acid modifiers and inhibitors, such as EDTA, DEPSI, and IAA, did not exhibit significant inhibition. In contrast, PMSF and HNBB significantly (p < 0.0001) reduced enzyme activity, suggesting that the enzyme could be classified as a serine protease. The protease also demonstrated high stability in the presence of 2% SDS, showing no signs inactivation. The alkaline pH optimum, thermal stability, and resistance to SDS exhibited by the protease produced by the Aeromonas strain are particularly promising characteristics that warrant further investigation. Based on preliminary tests and the enzyme's characteristics, this protease can be recommended for various applications, pending further studies., (© 2024. The Author(s).)

Triple-negative breast cancer (TNBC) presents limited therapeutic options and is associated with poor prognosis. Early detection and the development of novel therapeutic agents are therefore imperative. Fibroblast activation protein (FAP) is a membrane protein expressed on cancer-associated fibroblasts (CAFs) that plays an essential role in TNBC proliferation, migration, and invasion. Consequently, it is hypothesized that the Astatine ( 211 At)-labeled FAP inhibitor (FAPI) selectively exerts anti-tumor effects through alpha-particle emission. In this study, we aimed to assess its theranostic capabilities by integrating [ 18 F]FAPI-74 PET imaging with targeted alpha therapy using [ 211 At]FAPI1 in TNBC models. Mice xenografts were established by transplanting MDA-MB-231 and HT1080 cells (control). As a parallel diagnostic method, [ 18 F]FAPI-74 was administered for PET imaging to validate FAP expression. A single dose of [ 211 At]FAPI1 (1.04 ± 0.10 MBq) was administered to evaluate the therapeutic efficacy. [ 18 F]FAPI-74 exhibited high accumulation in MDA-MB-231 xenografts, and FAP expression was pathologically confirmed via immunostaining. The group that received [ 211 At]FAPI1 (n = 11) demonstrated a significantly enhanced anti-tumor effect compared with the control group (n = 7) ( p = 0.002). In conclusion, [ 18 F]FAPI-74 PET imaging was successfully used to diagnose FAP expression, and as [ 211 At]FAPI1 showed promising therapeutic efficacy in TNBC models, it is expected to be a viable therapeutic option.

Background: Osteoarthritis (OA) is a common joint disease that leads to chronic pain and functional limitations. Recent research has revealed soluble fibroblast activation protein (FAP) secreted from OA synovium could degrade type II collagen (Col2) in cartilage to promote the progression of OA. This study aimed to reveal the role of FAP from chondrocytes in OA and develop a novel lipid nanoparticle (LNP)-FAP siRNA delivery system for OA treatment., Methods: The expression of FAP in the cartilage of knee OA patients was investigated using [68 Ga]Ga-FAPI-04 PET in vivo and immunofluorescence, western blotting, and RT-qPCR in vitro. Cell senescence was determined by senescence-associated β-galactosidase (SA-β-Gal) assay after FAP overexpressing or knockdown in chondrocytes. An OA model with chondrocyte-specific FAP knockout mice was applied to investigate the role of FAP in chondrocyte senescence and OA development. The therapeutic effects of lipid nanoparticle (LNP) @FAP siRNA on cartilage degeneration were evaluated in the rat OA model., Results: Our study found that higher [68 Ga]Ga-FAPI-04 uptake was detected in knee OA patients by PET/CT scan. FAP mRNA and protein levels were highly expressed in OA-damaged cartilage. Moreover, we found that overexpression of FAP promotes chondrocyte senescence, and the genetic knockout of FAP in chondrocytes alleviates OA. Knockdown FAP by siRNA could alleviate chondrocyte senescence and suppress the NF-κB pathway to reduce the senescence-associated secretory phenotype (SASP). In the rat model of OA, intraarticular injection of LNP@FAP siRNA can reduce senescent cells and ameliorate cartilage destruction., Conclusion: FAP-positive chondrocytes play a significant role in the pathogenesis of OA. Targeting these cells selectively has the potential to mitigate the progression of the disease. Our study provides valuable insights into the intraarticular injection of LNP@FAP siRNA as a promising strategy for the treatment of OA., (© 2024. The Author(s).)

Circadian clocks rely on transcriptional/translational feedback loops involving clock genes and their corresponding proteins. While the primary oscillations originate from gene expression, the precise control of clock protein stability plays a pivotal role in establishing the 24-hour circadian rhythms. Most clock proteins are degraded through the ubiquitin/26S proteasome pathway, yet the enzymes responsible for ubiquitination and deubiquitination remain poorly characterised. We identified a missense allele (ubp12-3, S327F) of the UBP12 gene/protein in Arabidopsis. Despite ubp12-3 exhibited a short period phenotype similar to that of a loss-of-function allele, molecular analysis indicated elevated protease activity in ubp12-3. We demonstrated that early flowering of ubp12 mutants is a result of the shortened circadian period rather than a direct alteration of UBP12 function. Analysis of protease activity of non-phosphorylatable (S327A, S327F) and phosphomimetic (S327D) derivatives in bacteria suggested that phosphorylation of serine 327 inhibits UBP12 enzymatic activity, which could explain the over-functioning of S327F in vivo. We showed that phosphomimetic mutations of the conserved serine in the Neurospora and human orthologues reduced ubiquitin cleavage activity suggesting that not only the primary structures of UBP12-like enzymes are phylogenetically conserved across a wide range of species, but also the molecular mechanisms governing their enzymatic activity., (© 2024. The Author(s).)

Methicillin-resistant Staphylococcus spp. present challenges in clinical and veterinary settings because effective antimicrobial agents are limited. Phage-encoded peptidoglycan-degrading enzyme, endolysin, is expected to be a novel antimicrobial agent. The enzymatic activity has recently been shown to be influenced by the linker between functional domains in the enzyme. S6&#95;ORF93 (ORF93) is one of the endolysins derived from previously isolated Staphylococcus giant phage S6. The ORF93 was speculated to have a catalytic and peptidoglycan-binding domain with a long linker. In this study, we examined the influence of linker shortening on the characteristics of ORF93. We produce wild-type ORF93 and the linker deletion mutants using an Escherichia coli expression system. These mutants were designated as ORF93-Δ05, ORF93-Δ10, ORF93-Δ15, and ORF93-Δ20, from which 5, 10, 15, and 20 amino acids were removed from the linker, respectively. Except for the ORF93-Δ20, ORF93 and its mutants were expressed as soluble proteins. Moreover, ORF93-Δ15 showed the highest yield and bacteriolytic activity, while the antimicrobial spectrum was homologous. The cold storage experiment showed a slight effect by the linker deletion. According to our results and other studies, linker investigations are crucial in endolysin development., Competing Interests: The authors declare that no competing interests exist., (Copyright: © 2024 Munetomo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Emergence of antibiotic resistance in pathogenic Mycobacterium tuberculosis ( Mtb ) has elevated tuberculosis to a serious global threat, necessitating alternate solutions for its eradication. D29 mycobacteriophage can infect and kill several mycobacterial species including Mtb . It encodes an endolysin LysA to hydrolyze host bacteria peptidoglycan for progeny release. We previously showed that out of the two catalytically active domains of LysA [N-terminal domain (NTD) and lysozyme-like domain], NTD, when ectopically expressed in Mycobacterium smegmatis ( Msm ), is able to kill the bacterium nearly as efficiently as full-length LysA. Here, we dissected the functioning of NTD to develop it as a phage-derived small molecule anti-mycobacterial therapeutic. We performed a large-scale site-directed mutagenesis of the conserved residues in NTD and examined its structure, stability, and function using molecular dynamic simulations coupled with biophysical and biochemical experiments. Our data show that NTD functions as a putative cysteine peptidase with a catalytic triad composed of Cys41, His112, and Glu137, acting as nucleophile, base, and acid, respectively, and showing characteristics similar to the NlpC/P60 family of cysteine peptidases. Additionally, our peptidoglycan hydrolysis assays suggested that NTD hydrolyzes only mycobacterial peptidoglycan and does not act on Gram-positive and Gram-negative bacterial peptidoglycans. More importantly, the combined activity of exogenously added NTD and sub-lethal doses of anti-mycobacterial drugs kills Msm in vitro and exhibits disruption of pre-formed mycobacterial biofilm. We additionally show that NTD treatment increases the permeability of antibiotics in Msm , which reduces the minimum inhibitory concentration of the antibiotics. Collectively, we present NTD as a promising phage-derived therapeutic against mycobacteria.IMPORTANCEMycobacteriophages are the viruses that use mycobacteria as host for their progeny production and, in the process, kill them. Mycobacteriophages are, therefore, considered as promising alternatives to antibiotics for killing pathogenic Mycobacterium tuberculosis . The endolysin LysA produced by mycobacteriophage D29 plays an important role in host cell lysis and virion release. Our work presented here highlights the functioning of LysA's N-terminal catalytic domain (NTD) in order to develop it as phage-derived small molecule therapeutics. We show that combined treatment of exogenously added NTD and sub-lethal doses of anti-mycobacterial drugs kills M. smegmatis , shows synergism by reducing the minimum inhibitory concentration of these antibiotics, and exhibits disruption of pre-formed mature biofilm. These outcomes and our detailed biochemical and biophysical dissection of the protein further pave the way toward engineering and development of NTD as a promising therapeutic against mycobacterial infections such as tuberculosis., Competing Interests: The authors declare no conflict of interest.

Appropriate repair of damaged DNA and the suppression of DNA damage responses at telomeres are essential to preserve genome stability. DNA damage response (DDR) signaling consists of cascades of kinase-driven phosphorylation events, fine-tuned by proteolytic and regulatory ubiquitination. It is not fully understood how crosstalk between these two major classes of post-translational modifications impact DNA repair at deprotected telomeres. Hence, we performed a functional genetic screen to search for ubiquitin system factors that promote KAP1 S824 phosphorylation, a robust DDR marker at deprotected telomeres. We identified that the OTU family deubiquitinase (DUB) OTUD5 promotes KAP1 S824 phosphorylation by facilitating ATM activation, through stabilization of the ubiquitin ligase UBR5 that is required for DNA damage-induced ATM activity. Loss of OTUD5 impairs KAP1 S824 phosphorylation, which suppresses end-joining mediated DNA repair at deprotected telomeres and at DNA breaks in heterochromatin. Moreover, we identified an unexpected role for the heterochromatin factor KAP1 in suppressing DNA repair at telomeres. Altogether our work reveals an important role for OTUD5 and KAP1 in relaying DDR-dependent kinase signaling to the control of DNA repair at telomeres and heterochromatin., (© 2024. The Author(s).)

Fibroblast activation protein (FAP) is specifically expressed on cancer-associated fibroblasts in over 90% of tumors and is considered a promising target for cancer theranostics. Here, we developed a novel tracer, DOTA-FAPT, and labeled it with gallium-68 and lutetium-177 as a theranostic pair. [ 68 Ga]Ga/[ 177 Lu]Lu-FAPT exhibited high stability and hydrophilicity, as well as strong affinity to the FAP target. Micro-PET/CT imaging revealed that [ 68 Ga]Ga-FAPT exhibited significantly increased uptake in tumors and extended retention in A549-FAP and U87MG tumor xenografts as compared to [ 68 Ga]Ga-FAPI-04, demonstrating favorable pharmacokinetic characteristics in vivo. Therapeutic studies showed that [ 177 Lu]Lu-FAPT had higher tumor accumulation compared to [ 177 Lu]Lu-FAPI-04, leading to stronger tumor growth inhibition. The first-in-human evaluation also revealed that [ 68 Ga]Ga-FAPT has good in vivo distribution and superior diagnostic efficacy on primary and lymph node metastases in a patient with lung cancer. Our encouraging results suggest that 68 Ga/ 177 Lu-labeled DOTA-FAPT is a theranostic pair with broad application prospect.

Mitochondria provide cells with energy and regulate the cellular metabolism. Almost all mitochondrial proteins are nuclear-encoded, translated on ribosomes in the cytoplasm, and subsequently transferred to the different subcellular compartments of mitochondria. Here, we developed OptoMitoImport, an optogenetic tool to control the import of proteins into the mitochondrial matrix via the presequence pathway on demand. OptoMitoImport is based on a two-step process: first, light-induced cleavage by a TEV protease cuts off a plasma membrane-anchored fusion construct in close proximity to a mitochondrial targeting sequence; second, the mitochondrial targeting sequence preceding the protein of interest recruits to the outer mitochondrial membrane and imports the protein fused to it into mitochondria. Upon reaching the mitochondrial matrix, the matrix processing peptidase cuts off the mitochondrial targeting sequence and releases the protein of interest. OptoMitoImport is available as a two-plasmid system as well as a P2A peptide or IRES sequence-based bicistronic system. Fluorescence studies demonstrate the release of the plasma membrane-anchored protein of interest through light-induced TEV protease cleavage and its localization to mitochondria. Cell fractionation experiments confirm the presence of the peptidase-cleaved protein of interest in the mitochondrial fraction. The processed product is protected from proteinase K treatment. Depletion of the membrane potential across the inner mitochondria membrane prevents the mitochondrial protein import, indicating an import of the protein of interest by the presequence pathway. These data demonstrate the functionality of OptoMitoImport as a generic system with which to control the post-translational mitochondrial import of proteins via the presequence pathway.

This study aims to develop an efficient chitin-based purification system, leveraging a novel design where the target proteins, superfolding green fluorescent protein (sfGFP) and Thermus antranikianii trehalose synthase (TaTS), fused with a chitin-binding domain (ChBD) from Bacillus circulans WL-12 chitinase A1 and a tobacco etch virus protease (TEVp) cleavage site. This configuration allows for the effective immobilization of the target proteins on chitin beads, facilitating the removal of endogenous proteins. A mutant TEVp, H-TEVS219V-ChBD, fused with the His-tag and ChBD, is employed to cleave the target proteins from the chitin beads specifically. Subsequently, fresh chitin beads are added for adsorption to remove H-TEVS219V-ChBD in the solution, thereby significantly improving the purity of the target protein. Our results confirm that this system can efficiently and specifically purify and recover sfGFP and TaTS, achieving electrophoretic-grade purity exceeding 90%. This system holds significant potential for industrial production and other applications.

Taspase 1 is a unique protease not only pivotal for embryonic development but also implicated in leukemias and solid tumors. As such, this enzyme is a promising while still challenging therapeutic target, and with its protein structure featuring a flexible loop preceding the active site a versatile model system for drug development. Supramolecular ligands provide a promising complementary approach to traditional small-molecule inhibitors. Recently, the multivalent arrangement of molecular tweezers allowed the successful targeting of Taspase 1's surface loop. With this study we now want to take the next logic step und utilize functional linker systems that not only allow the implementation of novel properties but also engage in protein surface binding. Consequently, we chose two different linker types differing from the original divalent assembly: a backbone with aggregation-induced emission (AIE) properties to enable monitoring of binding and a calix[4]arene scaffold initially pre-positioning the supramolecular binding units. With a series of four AIE-equipped ligands with stepwise increased valency we demonstrated that the functionalized AIE linkers approach ligand binding affinities in the nanomolar range and allow efficient proteolytic inhibition of Taspase 1. Moreover, implementation of the calix[4]arene backbone further enhanced the ligands' inhibitory potential, pointing to a specific linker contribution., (© 2024 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Human periodontal ligament cells (hPDLCs) contain multipotent postnatal stem cells that can differentiate into PDL fibroblasts, osteoblasts, and cementoblasts. Interaction between the extracellular environment and stem cells is an important factor for differentiation into other progenitor cells. To identify cell surface molecules that induce PDL fibroblastic differentiation, we developed a series of monoclonal antibodies against membrane/ECM molecules. One of these antibodies, an anti-PDL25 antibody, recognizes approximately a 100 kDa protein, and this antigenic molecule accumulates in the periodontal ligament region of tooth roots. By mass spectrometric analysis, we found that the antigenic molecule recognized by the anti-PDL25 antibody is fibroblast activation protein α (FAPα). The expression level of FAPα/PDL25 increased in TGF-β1-induced PDL fibroblasts, and this protein was localized in the cell boundaries and elongated processes of the fibroblastic cells. Ectopic expression of FAPα induced fibroblastic differentiation. In contrast, expression of representative markers for PDL differentiation was decreased by knock down and antibody blocking of FAPα/PDL25. Inhibition of dipeptidyl peptidase activity by a potent FAPα inhibitor dramatically inhibited PDL fibroblastic marker expression but did not affect in cell proliferation and migration., 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 The Authors. Published by Elsevier Inc. All rights reserved.)

Ubiquitin-specific proteases (USPs) are a family of multi-domain deubiquitinases (DUBs) with variable architectures, some containing regulatory auxiliary domains. Among the USP family, all occurrences of intramolecular regulation presently known are autoactivating. USP8 remains the sole exception as its putative WW-like domain, conserved only in vertebrate orthologs, is autoinhibitory. Here, we present a comprehensive structure-function analysis describing the autoinhibition of USP8 and provide evidence of the physical interaction between the WW-like and catalytic domains. The solution structure of full-length USP8 reveals an extended, monomeric conformation. Coupled with DUB assays, the WW-like domain is confirmed to be the minimal autoinhibitory unit. Strikingly, autoinhibition is only observed with the WW-like domain in cis and depends on the length of the linker tethering it to the catalytic domain. Modeling of the WW:CD complex structure and mutagenesis of interface residues suggests a novel binding site in the S1 pocket. To investigate the interplay between phosphorylation and USP8 autoinhibition, we identify AMP-activated protein kinase as a highly selective modifier of S718 in the 14-3-3 binding motif. We show that 14-3-3γ binding to phosphorylated USP8 potentiates autoinhibition in a WW-like domain-dependent manner by stabilizing an autoinhibited conformation. These findings provide mechanistic details on the autoregulation of USP8 and shed light on its evolutionary significance., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

A vaccine is needed to combat the Chlamydia epidemic. Replication-deficient viral vectors are safe and induce antigen-specific T-cell memory. We tested the ability of intramuscular immunization with modified vaccinia Ankara (MVA) virus or chimpanzee adenovirus (ChAd) expressing chlamydial outer membrane protein (OmcB) or the secreted protein, chlamydial protease-like activating factor (CPAF), to enhance T-cell immunity and protection in mice previously infected with plasmid-deficient Chlamydia muridarum CM972 and elicit protection in naïve mice. MVA.OmcB or MVA.CPAF increased antigen-specific T cells in CM972-immune mice ∼150 and 50-fold, respectively, but failed to improve bacterial clearance. ChAd.OmcB/MVA.OmcB prime-boost immunization of naïve mice elicited a cluster of differentiation (CD) 8-dominant T-cell response dominated by cluster of differentiation (CD)8 T cells that failed to protect. ChAd.CPAF/ChAd.CPAF prime-boost also induced a CD8-dominant response with a marginal reduction in burden. Challenge of ChAd.CPAF-immunized mice genetically deficient in CD4 or CD8 T cells showed that protection was entirely CD4-dependent. CD4-deficient mice had prolonged infection, whereas CD8-deficient mice had higher frequencies of CPAF-specific CD4 T cells, earlier clearance, and reduced burden than wild-type controls. These data reinforce the essential nature of the CD4 T-cell response in protection from chlamydial genital infection in mice and the need for vaccine platforms that drive CD4-dominant responses., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Polysaccharide polymers are increasingly being used as chaperon-like macromolecules in assisting protein folding of unfolded protein molecules. They interact with unfolded or partially folded proteins in a charge and conformation specific manner that results in the formation of stable protein-polysaccharide complexes. In most of the cases, the complex formation of protein-polysaccharide is driven via non-covalent interactions that have found to endorse the activity of proteins. T4L (18.7 kDa) and T7L (17 kDa) endolysins belong to the hydrolase and amidase class of peptidoglycan degrading enzymes. Both T4L and T7L exist in partially folded forms and are devoid of lytic activity at low pH conditions. In the current study, we assessed the binding of alginate with T4L and T7L at pH 7 and 3 using variety of biophysical and biochemical techniques. Spectroscopic studies revealed differential structural modulations of partially folded T4L and T7L upon their interaction with alginate. Further, the complex formation of alginate with partially folded T4L/T7L was confirmed by ITC and STEM. Additionally, the formed complexes of alginate with both T4L/T7L PF endolysins were found to be chemically and enzymatically stable. Moreover, such complexes were also marked with differential enhancement in their lytic activities at acidic pH conditions. This implied the potency of alginate as an excellent choice of matrix to preserve the structural and functional integrity of partially folded forms of T4L and T7L at highly acidic conditions., Competing Interests: Declaration of interest statement 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.)

Bacterial resistance to antibiotics is a significant challenge that is associated with increased morbidity and mortality. Gram-negative bacteria are particularly problematic due to an outer membrane (OM). Current alternatives to antibiotics include antimicrobial peptides or proteins and multifunctional systems such as dendrimers. Antimicrobial proteins such as lysins can degrade the bacterial cell wall, whereas dendrimers can permeabilize the OM, enhancing the activity of endolysins against gram-negative bacteria. In this study, we present a three-stage action of endolysin combined with two different carbosilane (CBS) silver metallodendrimers, in which the periphery is modified with N-heterocyclic carbene (NHC) ligands coordinating a silver atom. The different NHC ligands contained hydrophobic methyl or N-donor pyridyl moieties. The effects of these endolysin/dendrimer combinations are based on OM permeabilization, peptidoglycan degradation, and reactive oxygen species production. The results showed that CBS possess a permeabilization effect (first action), significantly reduced bacterial growth at higher concentrations alone and in the presence of endolysin, increased ROS production (second action), and led to bacterial cell damage (third action). The complex formed between the CHAP domain of endolysin and a CBS silver metallodendrimer, with a triple mechanism of action, may represent an excellent alternative to other antimicrobials with only one resistance mechanism., 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 The Authors. Published by Elsevier B.V. All rights reserved.)

Low-grade serous ovarian carcinoma (LGSC) is a rare and lethal subtype of ovarian cancer. LGSC is pathologically, biologically, and clinically distinct from the more common high-grade serous ovarian carcinoma (HGSC). LGSC arises from serous borderline ovarian tumours (SBTs). The mechanism of transformation for SBTs to LGSC remains poorly understood. To better understand the biology of LGSC, we performed whole proteome profiling of formalin-fixed, paraffin-embedded tissue blocks of LGSC (n = 11), HGSC (n = 19), and SBTs (n = 26). We identified that the whole proteome is able to distinguish between histotypes of the ovarian epithelial tumours. Proteins associated with the tumour microenvironment were differentially expressed between LGSC and SBTs. Fibroblast activation protein (FAP), a protein expressed in cancer-associated fibroblasts, is the most differentially abundant protein in LGSC compared with SBT. Multiplex immunohistochemistry (IHC) for immune markers (CD20, CD79a, CD3, CD8, and CD68) was performed to determine the presence of B cells, T cells, and macrophages. The LGSC FAP + stroma was associated with greater abundance of Tregs and M2 macrophages, features not present in SBTs. Our proteomics cohort reveals that there are changes in the tumour microenvironment in LGSC compared with its putative precursor lesion, SBT. These changes suggest that the tumour microenvironment provides a supportive environment for LGSC tumourigenesis and progression. Thus, targeting the tumour microenvironment of LGSC may be a viable therapeutic strategy. © 2024 The Pathological Society of Great Britain and Ireland., (© 2024 The Pathological Society of Great Britain and Ireland.)

Fibroblast activation protein-α (FAP) plays a crucial role in various physiological and pathological processes, making it a key target for cancer diagnostics and therapeutics. However, in vivo detection of FAP activity with fluorogenic probes remains challenging due to the rapid diffusion and clearance of fluorescent products from the target. Herein, we developed a self-immobilizing near-infrared (NIR) fluorogenic probe, Hcy-CF 2 H-PG, by introducing a difluoromethyl group to FAP substrate-caged NIR fluorophore. Upon selective activation by FAP, the fluorescence of Hcy-CF 2 H-PG was triggered, followed by the covalent labelling of FAP. Hcy-CF 2 H-PG demonstrated significantly improved sensitivity, selectivity, and long-lasting labelling capacity for FAP both in vitro and in vivo, compared to that of non-immobilized probes. This represents a noteworthy advancement in FAP detection and cancer diagnostics within complex physiological systems., 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.)

Objective: There is a lack of effective biomarkers for predicting the distant metastasis in nasopharyngeal carcinoma (NPC). We aimed to explore the expression of FAP + Cancer-associated fibroblasts (CAFs) derived CXCL1 in NPC and its predictive values for distant metastasis and correlation with PD-L1 expression., Materials and Methods: A total of 345 patients with locoregionally advanced NPC were retrospectively enrolled (the training cohort: the validation cohort = 160:185). Co-expression of CXCL1 and FAP and the expression of PD-L1 were detected by multi-immunofluorescence staining and immunohistochemistry, respectively. The primary end-point was distant metastasis-free survival (DMFS). The Kaplan-Meier method was used to calculate the survival. The Cox proportional hazards model was used to assess prognostic risk factors., Results: A novel CXCL1 + &#95;FAP + phenotype in CAFs was identified in NPC and then used to divide patients into low and high risk groups. Both in the training cohort and validation cohort, patients in the high risk group had poorer DMFS, overall survival (OS), progression-free survival (PFS) and locoregional relapse-free survival (LRFS) than patients in the low risk group. Multivariate analysis revealed CXCL1 + &#95;FAP + phenotype was an independent prognostic factor for DMFS, OS, PFS and LRFS. Further results showed patients in the high risk group had higher PD-L1 expression than those in the low risk group., Conclusion: Our study showed CXCL1 + &#95;FAP + phenotype in CAFs could effectively classified locoregionally advanced NPC patients into different risk groups for distant metastasis and might be a potential biomarker for anti-PD-1/PD-L1 immunotherapy., 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.)

Tobacco Etch virus (TEV) protease is one of the most common tools for removing fusion tags, but no study has shown that TEV can be expressed at high levels in the GRAS host strain Bacillus subtilis and purified for further application. In this study, the fusion protein BsLysSN-TEV C/S-His-TEV consisting of a fusion tag, N-terminal domain of a lysyl-tRNA synthetase (BsLysSN) coded by B. subtilis lysS gene, placed at the N-terminus followed by an endoprotease TEV cleavage site and then the expression of this fusion protein in the cytoplasm of B. subtilis was investigated. The SDS-PAGE and Western-blot analysis demonstrated that His-TEV was overexpressed under the induction of IPTG. This result infers that His-TEV protease showed promising activity in the B. subtilis cytoplasm by the cleavage of the fusion protein. These cleavage products could be purified using the Ni-NTA column, which effectively cleaved the purified recombinant protein substrate, which can be applied in the protein purification process to remove the fusion tag. Significantly, since both His-TEV protease and the fusion recombinant protein substrate are expressed in the endotoxin-free host strain, the tag removal and purified product should be theoretically endotoxin-free, which could be a promising approach for producing therapeutic proteins and also for other relevant biomedical applications., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Stimuli-responsive nanomaterials show promise in eradicating Staphylococcus aureus biofilm from implants. Peptidoglycan hydrolases (PGHs) are cationic antimicrobials that can be bioengineered to improve the targeting of persisters and drug-resistant bacteria. However, these molecules can be degraded before reaching the target and/or present limited efficacy against biofilm. Therefore, there is an urgent need to improve their potency. Herein, PGH-polyphosphate nanoparticles (PGH-PP NPs) are formed by ionotropic gelation between cationic PGHs and anionic polyphosphate, with the aim of protecting PHGs and delivering them at the target site triggered by alkaline phosphatase (AP) from S. aureus biofilm. Optimized conditions for obtaining M23-PP NPs and GH15-PP NPs are presented. Size, zeta potential, and transmission electron microscopy imaging confirm the nanoscale size. The system demonstrates outstanding performance, as evidenced by a dramatic reduction in PGHs' minimum inhibitory concentration and minimum bactericidal concentration, together with protection against proteolytic effects, storage stability, and cytotoxicity towards the Caco-2 and HeLa cell lines. Time-kill experiments show the great potential of these negatively charged delivery systems in overcoming the staphylococcal biofilm barrier. Efficacy under conditions inhibiting AP proves the enzyme-triggered delivery of PGHs. The enzyme-responsive PGH-PP NPs significantly enhance the effectiveness of PGHs against bacteria residing in biofilm, offering a promising strategy for eradicating S. aureus biofilm.

Synthesis and maturation of Okazaki Fragments is an incessant and highly efficient metabolic process completing the synthesis of the lagging strands at replication forks during S phase. Accurate Okazaki fragment maturation (OFM) is crucial to maintain genome integrity and, therefore, cell survival in all living organisms. In eukaryotes, OFM involves the consecutive action of DNA polymerase Pol ∂, 5' Flap endonuclease Fen1 and DNA ligase I, and constitutes the best example of a sequential process coordinated by the sliding clamp PCNA. For OFM to occur efficiently, cooperation of these enzymes with PCNA must be highly regulated. Here, we present evidence of a role for the K164-PCNA-deubiquitylase Ubp10 in the maturation of Okazaki fragments in the budding yeast Saccharomyces cerevisiae. We show that Ubp10 associates with lagging-strand DNA synthesis machineries on replicating chromatin to ensure timely ligation of Okazaki fragments by promoting PCNA dissociation from chromatin requiring lysine 164 deubiquitylation., (© 2024. The Author(s).)

Fibroblast activation protein (FAP) has recently gained significant attention as a promising tumor biomarker for both diagnosis and therapeutic applications. A series of radiopharmaceuticals based on fibroblast activation protein inhibitors (FAPIs) have been developed and translated into the clinic. Though some of them such as radiolabeled FAPI-04 probes have achieved favorable in vivo imaging performance, further improvement is still highly desired for obtaining radiopharmaceuticals with a high theranostics potential. In this study, we innovatively designed an FAPI ligand SMIC-3002 by changing the core quinoline motif of FAPI-04 to the quinolinium scaffold. The engineered molecule was further radiolabeled with 68 Ga to generate a positron emission tomography (PET) probe, [ 68 Ga]Ga-SMIC-3002, which was then evaluated in vitro and in vivo. [ 68 Ga]Ga-SMIC-3002 demonstrated high in vitro stability, nanomolar affinity for FAP (8 nM for protein, 23 nM for U87MG cells), and specific uptake in FAP-expressing tumors, with a tumor/muscle ratio of 19.1 and a tumor uptake of 1.48 ± 0.03 ID/g% at 0.5 h in U87MG tumor-bearing mice. In summary, the quinolinium scaffold can be successfully used for the development of the FAP-targeted tracer. [ 68 Ga]Ga-SMIC-3002 not only shows high potential for clinical translation but also offers insights into designing a new generation of FAPI tracers.

Molecular imaging holds the potential for noninvasive and accurate grading of liver fibrosis. It is limited by the lack of biomarkers that strongly correlate with liver fibrosis grade. Here, we discover the grading potential of fibroblast activation protein alpha (FAPα) for liver fibrosis through transcriptional analysis and biological assays on clinical liver samples. The protein and mRNA expression of FAPα are linearly correlated with fibrosis grade (R 2  = 0.89 and 0.91, respectively). A FAPα-responsive MRI molecular nanoprobe is prepared for quantitatively grading liver fibrosis. The nanoprobe is composed of superparamagnetic amorphous iron nanoparticles (AFeNPs) and paramagnetic gadoteric acid (Gd-DOTA) connected by FAPα-responsive peptide chains (ASGPAGPA). As liver fibrosis worsens, the increased FAPα cut off more ASGPAGPA, restoring a higher T 1 -MRI signal of Gd-DOTA. Otherwise, the signal remains quenched due to the distance-dependent magnetic resonance tuning (MRET) effect between AFeNPs and Gd-DOTA. The nanoprobe identifies F1, F2, F3, and F4 fibrosis, with area under the curve of 99.8%, 66.7%, 70.4%, and 96.3% in patients' samples, respectively. This strategy exhibits potential in utilizing molecular imaging for the early detection and grading of liver fibrosis in the clinic., (© 2024. The Author(s).)

Xanthomonas oryzae pv. oryzae (Xoo) is a significant bacterial pathogen responsible for outbreaks of bacterial leaf blight in rice, posing a major threat to rice cultivation worldwide. Effective management of this pathogen is crucial for ensuring rice yield and food security. In this study, we identified and characterized a novel Xoo phage, ZP3, isolated from diseased rice leaves in Zhejiang, China, which may offer new insights into biocontrol strategies against Xoo and contribute to the development of innovative approaches to combat bacterial leaf blight. Transmission electron microscopy indicated that ZP3 had a short, non-contractile tail. Genome sequencing and bioinformatic analysis showed that ZP3 had a double-stranded DNA genome with a length of 44,713 bp, a G + C content of 52.2%, and 59 predicted genes, which was similar to other OP1-type Xoo phages belonging to the genus Xipdecavirus . ZP3's endolysin LysZP was further studied for its bacteriolytic action, and the N -terminal transmembrane domain of LysZP is suggested to be a signal-arrest-release sequence that mediates the translocation of LysZP to the periplasm. Our study contributes to the understanding of phage-Xoo interactions and suggests that phage ZP3 and its endolysin LysZP could be developed into biocontrol agents against this phytopathogen.

Rhomboid proteases are universally conserved and facilitate the proteolysis of peptide bonds within or adjacent to cell membranes. While eukaryotic rhomboid proteases have been demonstrated to harbor unique cellular roles, prokaryotic members have been far less characterized. For the first time, we demonstrate that Vibrio cholerae expresses two active rhomboid proteases that cleave a shared substrate at distinct sites, resulting in differential localization of the processed protein. The rhomboid protease rhombosortase (RssP) was previously found to process a novel C-terminal domain called GlyGly-CTERM, as demonstrated by its effect on the extracellular serine protease VesB during its transport through the V. cholerae cell envelope. Here, we characterize the substrate specificity of RssP and GlpG, the universally conserved bacterial rhomboid proteases. We show that RssP has distinct cleavage specificity from GlpG, and specific residues within the GlyGly-CTERM of VesB target it to RssP over GlpG, allowing for efficient proteolysis. RssP cleaves VesB within its transmembrane domain, whereas GlpG cleaves outside the membrane in a disordered loop that precedes the GlyGly-CTERM. Cleavage of VesB by RssP initially targets VesB to the bacterial cell surface and, subsequently, to outer membrane vesicles, while GlpG cleavage results in secreted, fully soluble VesB. Collectively, this work builds on the molecular understanding of rhomboid proteolysis and provides the basis for additional rhomboid substrate recognition while also demonstrating a unique role of RssP in the maturation of proteins containing a GlyGly-CTERM., Importance: Despite a great deal of insight into the eukaryotic homologs, bacterial rhomboid proteases have been relatively understudied. Our research aims to understand the function of two rhomboid proteases in Vibrio cholerae . This work is significant because it will help us better understand the catalytic mechanism of rhomboid proteases as a whole and assign a specific role to a unique subfamily whose function is to process a subset of effector molecules secreted by V. cholerae and other pathogenic bacteria., Competing Interests: The authors declare no conflict of interest.

Background: Fibroblast activation protein-α (FAP), a type-II transmembrane serine protease, is associated with wound healing, cancer-associated fibroblasts, and chronic fibrosing diseases. However, its expression in deep vein thrombosis (DVT) remains unclear. Therefore, this study investigated FAP expression and localization in DVT., Methods: We performed pathological analyses of the aspirated thrombi of patients with DVT (n = 14), classifying thrombotic areas in terms of fresh, cellular lysis, and organizing reaction components. The organizing reaction included endothelialization and fibroblastic reaction. We immunohistochemically examined FAP-expressed areas and cells, and finally analyzed FAP expression in cultured dermal fibroblasts., Results: All the aspirated thrombi showed a heterogeneous mixture of at least two of the three thrombotic areas. Specifically, 83 % of aspirated thrombi showed fresh and organizing reaction components. Immunohistochemical expression of FAP was restricted to the organizing area. Double immunofluorescence staining showed that FAP in the thrombi was mainly expressed in vimentin-positive or α-smooth muscle actin-positive fibroblasts. Some CD163-positive macrophages expressed FAP. FAP mRNA and protein levels were higher in fibroblasts with low-proliferative activity cultured under 0.1 % fetal bovine serum (FBS) than that under 10 % FBS. Fibroblasts cultured in 10 % FBS showed a significant decrease in FAP mRNA levels following supplementation with hemin, but not with thrombin., Conclusions: The heterogeneous composition of venous thrombi suggests a multistep thrombus formation process in human DVT. Further, fibroblasts or myofibroblasts may express FAP during the organizing process. FAP expression may be higher in fibroblasts with low proliferative activity., 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 The Authors. Published by Elsevier Ltd.. All rights reserved.)

By-products like CO₂ and organic acids, produced during Clostridium botulinum growth, appear to inhibit its development and reduce ATP production. A decrease in ATP production creates an imbalance in the ATP/GTP ratio. GTP activates CodY, which regulates BoNT expression. This toxin is released into the extracellular medium. Its light chains act as a specific endopeptidase, targeting SNARE proteins. The specific amino acids released enter the cells and are metabolized by the Stickland reaction, resulting in the synthesis of ATP. This ATP might then be used by histidine kinases to activate Spo0A, the main regulator initiating sporulation, through phosphorylation., Competing Interests: Declaration of competing interest No conflict of interest., (Copyright © 2024 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Proteases that recognize linear amino acid sequences with high specificity became indispensable tools of recombinant protein technology for the removal of various fusion tags. Due to its stringent sequence specificity, the catalytic domain of the nuclear inclusion cysteine protease of tobacco etch virus (TEV PR) is also a widely applied reagent for enzymatic removal of fusion tags. For this reason, efforts have been made to improve its stability and modify its specificity. For example, P1' autoproteolytic cleavage-resistant mutant (S219V) TEV PR was found not only to be nearly impervious to self-inactivation, but also exhibited greater stability and catalytic efficiency than the wild-type enzyme. An R203G substitution has been reported to further relax the P1' specificity of the enzyme, however, these results were obtained from crude intracellular assays. Until now, there has been no rigorous comparison of the P1' specificity of the S219V and S219V/R203G mutants in vitro, under carefully controlled conditions. Here, we compare the P1' amino acid preferences of these single and double TEV PR mutants. The in vitro analysis was performed by using recombinant protein substrates representing 20 P1' variants of the consensus TENLYFQ*SGT cleavage site, and synthetic oligopeptide substrates were also applied to study a limited set of the most preferred variants. In addition, the enzyme-substrate interactions were analyzed in silico. The results indicate highly similar P1' preferences for both enzymes, many side-chains can be accommodated by the S1' binding sites, but the kinetic assays revealed lower catalytic efficiency for the S219V/R203G than for the S219V mutant., (© 2024 The Authors. Proteins: Structure, Function, and Bioinformatics published by Wiley Periodicals LLC.)

Prolonged exposure to free silica leads to the development of silicosis, wherein activated fibroblasts play a pivotal role in its pathogenesis and progression. Fibroblast Activation Protein (FAP), as a biomarker for activated fibroblasts, its expression pattern and role in key aspects of silicosis pathogenesis remain unclear. This study elucidated the expression pattern and function of FAP through population-based epidemiological investigations, establishment of mouse models of silicosis, and in vitro cellular models. Results indicated a significant elevation of FAP in plasma from silicosis patients and lung tissues from mouse models of silicosis. In the cellular model, we observed a sharp increase in FAP expression early in the differentiation process, which remained high expression. Inhibition of FAP suppressed fibroblast differentiation, while overexpression of FAP produced the opposite effect. Moreover, fibroblast-derived FAP can alter the phenotype and function of neighboring macrophages. In summary, we revealed a high expression pattern of FAP in silicosis and its potential mechanistic role in fibrosis, suggesting FAP as a potential therapeutic target for silicosis., 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.)

Currently, there is an urgent to develop safe and environmentally friendly alternatives to antibiotics for combating Vibrio parahaemolyticus. Endolysins are considered promising antibacterial agents due to their desirable range of action and ability to deal with antibiotic-resistant bacteria. While numerous Vibrio phages have been identified, the research on their endolysins is still in its infancy. In this study, a novel endolysin called LysVPB was cloned and expressed in Pichia pastoris. Phylogenetic analysis revealed that LysVPB bears little resemblance to other known endolysins, highlighting its unique nature. Homology modeling identified a putative calcium-binding site in LysVPB. The recombinant LysVPB achieved a lytic activity of 64.8 U/mL and had a molecular weight of approximately 17 kDa. LysVPB exhibited enhanced efficacy at pH 9.0, with 60 % of its maximum activity observed within the broad pH range of 6.0-10.0. The catalytic efficiency of LysVPB peaked at 30 °C but significantly declined beyond 50 °C. Ba 2+ , Co 2+ , and Cu 2+ showed inhibitory effects on the activity of LysVPB, while Ca 2+ can boost it to 126.8 %. Furthermore, LysVPB exhibited satisfactory efficacy against strains of V. parahaemolyticus. LysVPB is an innovative phage lysin with good characteristics that are specific to certain hosts. The modular nature of LysVPB allows for efficient domain exchange with alternative lysins as antimicrobial components and fusion with antimicrobial peptides. This opens up possibilities for engineering chimeric lysins in a broader range of target hosts with high antimicrobial effectiveness and strong activity under physiological conditions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing interests., (Copyright © 2024. Published by Elsevier Inc.)

Fibroblast activation protein-α (FAPα) is highly expressed in tumor-associated cells and has become one of the most attractive targeting sites in cancer diagnosis and therapy. To ameliorate the rapid metabolism of FAPα inhibitor (FAPI), here, a multifunctional binding agent was introduced to simultaneously achieve 211 At radiolabeling and tumor retention prolongation of corresponding radiolabeled drug. 211 At-APBA-FAPI was successfully synthesized by conjugating 211 At with the designed FAPI carrier in satisfactory radiochemical yield (>60 %). 211 At-APBA-FAPI exhibited excellent in vitro stability, significant tumor affinity and specific killing effect on FAPα-positive U87MG cells. Molecular docking reveals that FAPI decorated with albumin binder can bind with FAPα protein via multiple intermolecular interactions with a considerable binding energy of -9.66 kcal/mol 211 At-APBA-FAPI exhibits good targeting in murine xenograft models, showing obviously longer tumor retention than previously-reported radioastatinated compound. As a result, 211 At-APBA-FAPI presents pronounced therapeutic effect with ignorable normal organs/tissues biotoxicity. All these indicate that introducing a multifunctional binding agent can effectively enhance the availability of FAPI for 211 At conjugation and tumoricidal effect, providing vital hints for the translation of targeted-alpha therapy based on radiolabeled FAPI derivatives., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Enzymatic resolution of ethyl tetrahydrofuroate to produce (S)-2-ethyl tetrahydrofuroate and (R)-2-tetrahydrofuroic acid is a green biomanufacturing strategy. However, enzymatic activity and selectivity are still limiting factors of their industrial applications and development. In previous study, we incidentally found that a Bacillus licheniformis alkaline protease (BLAP), not a lipase, could specifically resolve ethyl tetrahydrofuroate to produce (S)-2-ethyl tetrahydrofuroate and (R)-2-tetrahydrofuroic acid. In this study, the point-saturation-mutation libraries based on the seven amino acid sites (L105, I113, P114, L115, V309, Y310, and M326) were constructed and screened using the molecular docking technology. It was found that activity of the mutant BLAP Y310E reached 182.78 U/mL with high stereoselectivity, 3.14 times higher than that of the wild-type BLAP. Further simulated mutation analysis showed that the Y310E mutation increased the distance from the substrate ligand to the binding pocket from 2.3 Å to 4.5 Å, reducing steric hindrance to the active center. Under the optimal conditions and after 3.5 h of reaction catalyzed by BLAP Y310E , 200 mM ethyl tetrahydrofuroate was converted to (S)-2-ethyl tetrahydrofuroate and (R)-2-tetrahydrofuroic acid with the ee values of 99.9 % and 68.63 %, respectively. The enantiomeric ratio of BLAP Y310E was 105.5, which was 30.23 times higher than that of BLAP. This study advances the comprehension of protease activity and selectivity mechanisms in resolving ester substances and lays a robust foundation for the industrial production of the optically pure (S)-2-ethyl tetrahydrofuroate and (R)-2-tetrahydrofuroic acid via biological enzymatic methods., Competing Interests: Competing interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The pS273R protease of the African swine fever virus (ASFV) is responsible for the processing of the viral polyproteins pp220 and pp62, precursors of the internal capsid of the virus. The protease is essential for a productive viral infection and is an attractive target for antiviral therapy. This work presents a method for the production of pS273R in E. coli cells by fusing the protease with the SlyD chaperone. The chimeric protein pS273R protease, during expression, is formed in a soluble form possessing enzymatic activity. Subsequently, pS273R separates from SlyD through autocatalytic cleavage at the TEV protease site in vivo. This work devised a straightforward protocol for chromatographic purification, resulting in the production of a highly purified viral protease. Additionally, we suggest using a fluorescence method to assess the activity of pS273R. This method is predicated on a shift in the chimeric protein thioredoxin-EGFP's electrophoretic mobility following its protease cleavage. It was shown that thioredoxin-EGFP substrate is effectively and selectively cleaved by the pS273R protease, even in complex protein mixtures such as mammalian cell lysates., 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.)

The foot-and-mouth disease virus (FMDV) Leader proteinase L pro inhibits host mRNA translation and blocks the interferon response which promotes viral survival. L pro is not required for viral replication in vitro but serotype A FMDV lacking L pro has been shown to be attenuated in cattle and pigs. However, it is not known, whether leaderless viruses can cause persistent infection in vivo after simulated natural infection and whether the attenuated phenotype is the same in other serotypes. We have generated an FMDV O/FRA/1/2001 variant lacking most of the L pro coding region (ΔLb). Cattle were inoculated intranasopharyngeally and observed for 35 days to determine if O FRA/1/2001 ΔLb is attenuated during the acute phase of infection and whether it can maintain a persistent infection in the upper respiratory tract. We found that although this leaderless virus can replicate in vitro in different cell lines, it is unable to establish an acute infection with vesicular lesions and viral shedding nor is it able to persistently infect bovine pharyngeal tissues.

Background: Aspergillus oryzae protease can release the opioid peptide β-casomorphin-10 (CM-10, YPFPGPIPNS, 60-69) from A2-type casein. However, not only is the yield of the active peptide low, but the key enzyme involved in processing has yet to be identified., Results: A significant amount of the opioid peptide 60 YPFPGPIPNSLP 71 (CM-12) was produced from the A2-type casein peptide 53 AQTQSLVYPFPGPIPNSLPQNIPPLTQTPV 82 when the active protease in A. oryzae protease extract was fractionated with DEAE-Sepharose. The fractionated enzyme produced CM-12 from bovine A2-type casein but not from bovine A1 casein. A major protein of 34 kDa was purified and identified as an alkaline protease (Alp). Motif prediction of the Alp cleavage site using Multiple EM for Motif Elicitation analysis revealed preferable cleavage at the C-terminal end of Ser-Leu-Xaa for the release of CM-12. A2-type casein hydrolysate by Alp exhibited similar levels of opioid activity to that of synthetic CM-12 in cAMP-Glo assays with μ-opioid receptor-expressing HEK293 cells. These results suggest that CM-12 is a major opioid peptide in the casein hydrolysate., Conclusion: Our findings showed that Alp fractionated from A. oryzae protease extract produced the opioid peptide CM-12 from A2-type casein as a result of preferential cleavage at the C-terminal end of Ser-Leu-Xaa and the removal of coexisting enzymes. Moreover, docking predictions suggested a stable interaction between CM-12 and the 3D structure of Alp. Casein hydrolysate with Alp-containing CM-12 has the potential for use as a bioactive peptide material with opioid activity. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Gastric cancer (GC) is one of the most common cancers worldwide. Dioscin has been shown to have anti-cancer effects in GC. The aim of this study is to explore a novel mechanism of dioscin in repressing GC progression. Cell viability, proliferation, apoptosis and invasion were measured by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry and transwell assays, respectively. Monodansylcadaverine (MDC) staining was used to assess cell autophagy. The expression of transglutaminase-2 (TGM2), ubiquitin-specific peptidase 8 (USP8) and autophagy-related proteins was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. A xenograft tumor model was established to investigate the function of dioscin in vivo. Dioscin inhibited GC cell proliferation and invasion, but induced apoptosis and autophagy. TGM2 was highly expressed in GC, and dioscin suppressed GC progression by decreasing the protein level of TGM2. Furthermore, USP8 positively regulated TGM2 expression, and TGM2 overexpression reversed the inhibitory effect of USP8 knockdown on GC cell progression. USP8 abated the effect of dioscin in GC cells. Dioscin decreased the protein level of TGM2 via regulating USP8. In addition, dioscin restrained GC tumor growth in vivo. Dioscin played an anti-cancer effect in GC by enhancing cancer cell autophagy via regulating the USP8/TGM2 pathway., Competing Interests: Declarations Conflict of interest The authors declare that they have no financial conflict of interest., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)