DoUBLing up: ubiquitin and ubiquitin-like proteases in genome stability.

Maintaining stability of the genome requires dedicated DNA repair and signalling processes that are essential for the faithful duplication and propagation of chromosomes. These DNA damage response (DDR) mechanisms counteract the potentially mutagenic impact of daily genotoxic stresses from both exogenous and endogenous sources. Inherent to these DNA repair pathways is the activity of protein factors that instigate repair processes in response to DNA lesions. The regulation, coordination, and orchestration of these DDR factors is carried out, in a large part, by post-translational modifications, such as phosphorylation, ubiquitylation, and modification with ubiquitin-like proteins (UBLs). The importance of ubiquitylation and UBLylation with SUMO in DNA repair is well established, with the modified targets and downstream signalling consequences relatively well characterised. However, the role of dedicated erasers for ubiquitin and UBLs, known as deubiquitylases (DUBs) and ubiquitin-like proteases (ULPs) respectively, in genome stability is less well established, particularly for emerging UBLs such as ISG15 and UFM1. In this review, we provide an overview of the known regulatory roles and mechanisms of DUBs and ULPs involved in genome stability pathways. Expanding our understanding of the molecular agents and mechanisms underlying the removal of ubiquitin and UBL modifications will be fundamental for progressing our knowledge of the DDR and likely provide new therapeutic avenues for relevant human diseases, such as cancer.

Dispase/collagenase cocktail allows for coisolation of satellite cells and fibroadipogenic progenitors from human skeletal muscle.

Satellite cells (SCs) and fibroadipogenic progenitors (FAPs) are progenitor populations found in muscle that form new myofibers postinjury. Muscle development, regeneration, and tissue-engineering experiments require robust progenitor populations, yet their isolation and expansion are difficult given their scarcity in muscle, limited muscle biopsy sizes in humans, and lack of methodological detail in the literature. Here, we investigated whether a dispase and collagenase type 1 and 2 cocktail could allow dual isolation of SCs and FAPs, enabling significantly increased yield from human skeletal muscle. Postdissociation, we found that single cells could be sorted into CD56 + CD31-CD45- (SC) and CD56-CD31-CD45- (FAP) cell populations, expanded in culture, and characterized for lineage-specific marker expression and differentiation capacity; we obtained ∼10% SCs and ∼40% FAPs, with yields twofold better than what is reported in current literature. SCs were PAX7+ and retained CD56 expression and myogenic fusion potential after multiple passages, expanding up to 1012 cells. Conversely, FAPs expressed CD140a and differentiated into either fibroblasts or adipocytes upon induction. This study demonstrates robust isolation of both SCs and FAPs from the same muscle sample with SC recovery more than two times higher than previously reported, which could enable translational studies for muscle injuries.NEW & NOTEWORTHY We demonstrated that a dispase/collagenase cocktail allows for simultaneous isolation of SCs and FAPs with 2× higher SC yield compared with other studies. We provide a thorough characterization of SC and FAP in vitro expansion that other studies have not reported. Following our dissociation, SCs and FAPs were able to expand by up to 1012 cells before reaching senescence and maintained differentiation capacity in vitro demonstrating their efficacy for clinical translation for muscle injury.

Advances in Small Molecular Agents against Oral Cancer.

Oral cancer is a common malignancy with a high mortality rate. Although surgery is the best treatment option for patients with cancer, this approach is ineffective for advanced metastases. Molecular agents are irreplaceable in preventing and treating distant metastases. This review aims to summarise the molecular agents used for the treatment of oral cancer in the last decade and describe their sources and curative effects. These agents are classified into phenols, isothiocyanates, anthraquinones, statins, flavonoids, terpenoids, and steroids. The mechanisms of action of these agents include regulating the expression of cell signalling pathways and related proteases to affect the proliferation, autophagy, migration, apoptosis, and other biological aspects of oral cancer cells. This paper may serve as a reference for subsequent studies on the treatment of oral cancer.

Assessing Sarcocornia as a Salt Substitute: Effects on Lipid Profile and Gelatinase Activity.

Sodium, although essential for life, is a key factor in changes in vascular function and cardiovascular disease when consumed in excess. Sarcocornia spp., a halophyte plant with many nutritional benefits, presents itself as a promising substitute for the consumption of purified salt. Matrix metalloproteinases (MMPs) 2 and 9 are widely studied due to their action in physiological processes and as biomarkers at the diagnostic level due to their increased expression in inflammatory processes. This study aimed to evaluate whether replacing salt with Sarcocornia perennis (S. perennis) powder in healthy young people leads to an improvement in biochemical profiles and the attenuation of MMP-2 and MMP-9 activity. In the present study, 30 participants were randomized into a control group that consumed salt and an intervention group that replaced salt with powdered S. perennis. The evaluation of the biochemical parameters was carried out by the spectrophotometry method, and the evaluation of MMP activity was carried out by zymography. A significant decrease was observed in the intervention group in total cholesterol, high-density lipoprotein cholesterol (HDL-c), and creatinine (p-value ≤ 0.05), along with lower but not significantly different mean values of triglycerides. Regarding MMP activity after the intervention, a lower mean value was observed for MMP-9 activity, with there being higher mean values for MMP-2 activity, both with p-values ≥ 0.05. The results confirmed that the consumption of S. perennis is a beneficial choice for health regarding the lipid profile. The evaluation of MMP activity indicated the potential of S. perennis in the regulation of MMP-9 activity in healthy individuals, along with the need for the further study of these proteases in individuals with pathologies.

Reducing Immunoreactivity of Gluten Peptides by Probiotic Lactic Acid Bacteria for Dietary Management of Gluten-Related Diseases.

Immunoreactive gluten peptides that are not digested by peptidases produced by humans can trigger celiac disease, allergy and non-celiac gluten hypersensitivity. The aim of this study was to evaluate the ability of selected probiotic strains to hydrolyze immunoreactive gliadin peptides and to identify peptidase-encoding genes in the genomes of the most efficient strains. Residual gliadin immunoreactivity was measured after one- or two-step hydrolysis using commercial enzymes and bacterial peptidase preparations by G12 and R5 immunoenzymatic assays. Peptidase preparations from Lacticaseibacillus casei LC130, Lacticaseibacillus paracasei LPC100 and Streptococcus thermophilus ST250 strains significantly reduced the immunoreactivity of gliadin peptides, including 33-mer, and this effect was markedly higher when a mixture of these strains was used. In silico genome analyses of L. casei LC130 and L. paracasei LPC100 revealed the presence of genes encoding peptidases with the potential to hydrolyze bonds in proline-rich peptides. This suggests that L. casei LC130, L. paracasei LPC100 and S. thermophilus ST250, especially when used as a mixture, have the ability to hydrolyze immunoreactive gliadin peptides and could be administered to patients on a restricted gluten-free diet to help treat gluten-related diseases.

Early recovery of proteasome activity in cells pulse-treated with proteasome inhibitors is independent of DDI2.

Rapid recovery of proteasome activity may contribute to intrinsic and acquired resistance to FDA-approved proteasome inhibitors. Previous studies have demonstrated that the expression of proteasome genes in cells treated with sub-lethal concentrations of proteasome inhibitors is upregulated by the transcription factor Nrf1 (NFE2L1), which is activated by a DDI2 protease. Here, we demonstrate that the recovery of proteasome activity is DDI2-independent and occurs before transcription of proteasomal genes is upregulated but requires protein translation. Thus, mammalian cells possess an additional DDI2 and transcription-independent pathway for the rapid recovery of proteasome activity after proteasome inhibition.

Antimicrobial peptide thanatin fused endolysin PA90 (Tha-PA90) for the control of Acinetobacter baumannii infection in mouse model.

BACKGROUND: This study addresses the urgent need for infection control agents driven by the rise of drug-resistant pathogens such as Acinetobacter baumannii. Our primary aim was to develop and assess a novel endolysin, Tha-PA90, designed to combat these challenges. METHODS: Tha-PA90 incorporates an antimicrobial peptide (AMP) called thanatin at its N-terminus, enhancing bacterial outer membrane permeability and reducing host immune responses. PA90 was selected as the endolysin component. The antibacterial activity of the purified Tha-PA90 was evaluated using an in vitro colony-forming unit (CFU) reduction assay and a membrane permeability test. A549 cells were utilized to measure the penetration into the cytosol and the cytotoxicity of Tha-PA90. Finally, infection control was monitored in A. baumannii infected mice following the intraperitoneal administration of Tha-PA90. RESULTS: Tha-PA90 demonstrated remarkable in vitro efficacy, completely eradicating A. baumannii strains, even drug-resistant variants, at a low concentration of 0.5 µM. Notably, it outperformed thanatin, achieving only a < 3-log reduction at 4 µM. Tha-PA90 exhibited 2-3 times higher membrane permeability than a PA90 and thanatin mixture or PA90 alone. Tha-PA90 was found within A549 cells' cytosol with no discernible cytotoxic effects. Furthermore, Tha-PA90 administration extended the lifespan of A. baumannii-infected mice, reducing bacterial loads in major organs by up to 3 logs. Additionally, it decreased proinflammatory cytokine levels (TNF-α and IL-6), reducing the risk of sepsis from rapid bacterial lysis. Our findings indicate that Tha-PA90 is a promising solution for combating drug-resistant A. baumannii. Its enhanced efficacy, low cytotoxicity, and reduction of proinflammatory responses render it a potential candidate for infection control. CONCLUSIONS: This study underscores the significance of engineered endolysins in addressing the pressing challenge of drug-resistant pathogens and offers insights into improved infection management strategies.

Engineering strategies and challenges of endolysin as an antibacterial agent against Gram-negative bacteria.

Bacteriophage endolysin is a novel antibacterial agent that has attracted much attention in the prevention and control of drug-resistant bacteria due to its unique mechanism of hydrolysing peptidoglycans. Although endolysin exhibits excellent bactericidal effects on Gram-positive bacteria, the presence of the outer membrane of Gram-negative bacteria makes it difficult to lyse them extracellularly, thus limiting their application field. To enhance the extracellular activity of endolysin and facilitate its crossing through the outer membrane of Gram-negative bacteria, researchers have adopted physical, chemical, and molecular methods. This review summarizes the characterization of endolysin targeting Gram-negative bacteria, strategies for endolysin modification, and the challenges and future of engineering endolysin against Gram-negative bacteria in clinical applications, to promote the application of endolysin in the prevention and control of Gram-negative bacteria.

Serratia montpellierensis sp. nov., Isolated from Laboratory-Reared Parasitic Wasps Psyttalia lounsburyii Silvestri and Psyttalia ponerophaga Silvestri (Hymenoptera: Braconidae).

Two strains of bacteria, PsyLou2AT and PsyPon4B, were isolated from adult braconid wasps Psyttalia lounsburyii and Psyttalia ponerophaga, respectively. These laboratory-reared wasps were investigated as agents for biological control of the olive fruit fly, Bactrocera oleae. Analysis of 16S rRNA genes of the two isolates demonstrated that they were highly related and belonged to the genus Serratia. Genomic sequencing of these isolates revealed genomes of 5,152,551 bp and 5,154,385 bp for PsyLou2AT and PsyPon4B, respectively, and both genomes had a mol% G+C content of 59.6%. Phylogenetic analyses using BLAST-based average nucleotide identity (ANIb), and digital DNA-DNA hybridization methods indicated that PsyLou2AT was most closely related to Serratia nevei S15T, producing ANIb and dDDH values of 96.11% and 70.2%, respectively. Since these values were literally on the species cutoff threshold, additional S. nevei genome assemblies were analyzed using ANIb and dDDH calculations. This revealed that among assemblies that were clearly identifiable as S. nevei, S. nevei S15T was the most closely related to PsyLou2AT, and that a majority of assemblies produced dDDH values of 68.3-68.7% relative to PsyLou2AT. Additionally, PsyLou2AT differed biochemically from S. nevei S15T in that it produced positive Voges Proskauer tests, produced protease, lacked arginine dihydrolase, and did not utilize D-lactose. Hence, PsyLou2AT represents a novel taxon within the Serratia, for which we propose the name Serratia montpellierensis sp. nov. The type strain is PsyLou2AT (=LMG 32817T =NRRL B-65689T).

Impacts of AlaAT3 transgenic poplar on rhizosphere soil chemical properties, enzyme activity, bacterial community, and metabolites under two nitrogen conditions.

Poplar stands as one of the primary afforestation trees globally. We successfully generated transgenic poplar trees characterized by enhanced biomass under identical nutrient conditions, through the overexpression of the pivotal nitrogen assimilation gene, pxAlaAT3. An environmental risk assessment was conducted for investigate the potential changes in rhizosphere soil associated with these overexpressing lines (OL). The results show that acid phosphatase activity was significantly altered under ammonium in OL compared to the wild-type control (WT), and a similar difference was observed for protease under nitrate. 16SrDNA sequencing indicated no significant divergence in rhizosphere soil microbial community diversity between WT and OL. Metabolomics analysis revealed that the OL caused minimal alterations in the metabolites of the rhizosphere soil, posing no potential harm to the environment. With these findings in mind, we anticipate that overexpressed plants will not adversely impact the surrounding soil environment.

Preparation of Healthy Single Neuron or Astrocyte Suspension from Adult Mouse Brain for RNA-seq.

Single-cell RNA sequencing (scRNA-seq) has been widely applied in neuroscience research, enabling the investigation of cellular heterogeneity at the transcriptional level, the characterization of rare cell types, and the detailed analysis of the stochastic nature of gene expression. Isolation of single nerve cells in good health, especially from the adult rodent brain, is the most difficult and critical process for scRNA-seq. Here, we describe methods to optimize protease digestion of brain slices, which enable yield of millions of cells in good health from the adult brain.

Linking the protease activity to the nematicidal action of edible mushroom.

Biological control using edible mushrooms as natural enemies is a sustainable alternative for pest management. Despite the well-established literature on toxins and secondary metabolites produced by these fungi in the biochemical control of nematodes, the nematicidal activity of proteases from different Pleurotus species is yet to be investigated. Therefore, this study aimed to correlate protease to the nematicidal activity of different mushrooms, Pleurotus sp., P. ostreatus (SB), P. ostreatus (Pearl), and P. djamor. For such a purpose, we performed motility assays of Panagrellus sp. at different time intervals, 6, 12, and 24 h for each of the mushrooms. In addition, the protease activity was measured using different pH (5, 7, and 9) and fermentation time intervals (45 and 75 days). Furthermore, we also evaluated the effect of this cell-free extract on Panagrellus sp. In response to these experiments, all edible mushrooms showed a reduction over 82% for the nematode-feeding activity (p < 0.01). The cell-free crude extract of each of the fungi studied showed nematocidal activity (p < 0.01). For the 45-day fermentation, P. djamor exhibited statistical significance (p < 0.01) compared with the others, reaching a reduction percentage of 73%. For the 75-day fermentation, Pleurotus sp. and P. ostreatus (Pearl) showed significant differences compared with the other fungi (p < 0.01), with reduction percentages of 64 and 62%, respectively. Herein, protease activity was associated with the nematicidal action of different Pleurotus species in controlling Panagrellus sp.

USP19 regulates DNA methylation damage repair and confers temozolomide resistance through MGMT stabilization.

OBJECTIVE: To elucidate the relationship between USP19 and O(6)-methylguanine-DNA methyltransferase (MGMT) after temozolomide treatment in glioblastoma (GBM) patients with chemotherapy resistance. METHODS: Screening the deubiquitinase pannel and identifying the deubiquitinase directly interacts with and deubiquitination MGMT. Deubiquitination assay to confirm USP19 deubiquitinates MGMT. The colony formation and tumor growth study in xenograft assess USP19 affects the GBM sensitive to TMZ was performed by T98G, LN18, U251, and U87 cell lines. Immunohistochemistry staining and survival analysis were performed to explore how USP19 is correlated to MGMT in GBM clinical management. RESULTS: USP19 removes the ubiquitination of MGMT to facilitate the DNA methylation damage repair. Depletion of USP19 results in the glioblastoma cell sensitivity to temozolomide, which can be rescued by overexpressing MGMT. USP19 is overexpressed in glioblastoma patient samples, which positively correlates with the level of MGMT protein and poor prognosis in these patients. CONCLUSION: The regulation of MGMT ubiquitination by USP19 plays a critical role in DNA methylation damage repair and GBM patients' temozolomide chemotherapy response.

Bacteriophages as a potential substitute for antibiotics: A comprehensive review.

Over the years, the administration of antibiotics for the purpose of addressing bacterial infections has become increasingly challenging due to the increased prevalence of antimicrobial resistance exhibited by various strains of bacteria. Multidrug-resistant (MDR) bacterial species are rising due to the unavailability of novel antibiotics, leading to higher mortality rates. With these conditions, there is a need for alternatives in which phage therapy has made promising results. Phage-derived endolysins, phage cocktails, and bioengineered phages are effective and have antimicrobial properties against MDR and extensively drug-resistant strains. Despite these, it has been observed that phages can give antimicrobial activity to more than one bacterial species. Thus, phage cocktail against resistant strains provides broad spectrum treatment and magnitude of effectivity, which is many folds higher than antibiotics. Many commercially available endolysins such as Staphefekt SA.100, Exebacase (CF-301), and N-Rephasin®SAL200 are used in biofilm penetration and treating plant diseases. The role of CMP1 phage endolysin in transgenic tomato plants in preventing Clavibacter michiganensis infection and the effectiveness of phage in protecting Atlantic salmon from vibriosis have been reported. Furthermore, phage-derived endolysin therapy, such as TSPphg phage exogenous treatment, can aid in disrupting cell walls, leading to bacterial cell lysis. As animals in aquaculture and slaughterhouses are highly susceptible to bacterial infections, effective phage therapy instead of antibiotics can help treat poultry animals, preserve them, and facilitate disease-free trade. Using bioengineered phages and phage cocktails enhances the effectiveness by providing a broad spectrum of phages and target specificity. Research is currently being conducted on clinical trials to confirm the efficacy of engineered phages and phage cocktails in humans. Although obtaining commercial approval may be time-consuming, it will be beneficial in the postantibiotic era. This review provides an overview of the significance of phage therapy as a potential alternative to antibiotics in combating resistant bacterial strains and its application to various fields and emphasizes the importance of safeguarding and ensuring treatment efficacy.

OTUD5 promotes the growth of hepatocellular carcinoma by deubiquitinating and stabilizing SLC38A1.

BACKGROUND: Deubiquitinating enzymes (DUBs) cleave ubiquitin on substrate molecules to maintain protein stability. DUBs reportedly participate in the tumorigenesis and tumour progression of hepatocellular carcinoma (HCC). OTU deubiquitinase 5 (OTUD5), a DUB family member, has been recognized as a critical regulator in bladder cancer, breast cancer and HCC. However, the expression and biological function of OTUD5 in HCC are still controversial. RESULTS: We determined that the expression of OTUD5 was significantly upregulated in HCC tissues. High levels of OTUD5 were also detected in most HCC cell lines. TCGA data analysis demonstrated that high OTUD5 expression indicated poorer overall survival in HCC patients. OTUD5 silencing prominently suppressed HCC cell proliferation, while its overexpression markedly enhanced the proliferation of HCC cells. Mass spectrometry analysis revealed solute carrier family 38 member 1 (SLC38A1) as a candidate downstream target protein of OTUD5. Coimmunoprecipitation analysis confirmed the interaction between OTUD5 and SLC38A1. OTUD5 knockdown reduced and OTUD5 overexpression increased SLC38A1 protein levels in HCC cells. However, OTUD5 alteration had no effect on SLC38A1 mRNA expression. OTUD5 maintained SLC38A1 stability by preventing its ubiquitin-mediated proteasomal degradation. SLC38A1 silencing prominently attenuated the OTUD5-induced increase in HCC cell proliferation. Finally, OTUD5 knockdown markedly suppressed the growth of HCC cells in vivo. CONCLUSIONS: OTUD5 is an oncogene in HCC. OTUD5 contributes to HCC cell proliferation by deubiquitinating and stabilizing SLC38A1. These results may provide a theoretical basis for the development of new anti-HCC drugs.

A de novo dominant-negative variant is associated with OTULIN-related autoinflammatory syndrome.

OTULIN-related autoinflammatory syndrome (ORAS), a severe autoinflammatory disease, is caused by biallelic pathogenic variants of OTULIN, a linear ubiquitin-specific deubiquitinating enzyme. Loss of OTULIN attenuates linear ubiquitination by inhibiting the linear ubiquitin chain assembly complex (LUBAC). Here, we report a patient who harbors two rare heterozygous variants of OTULIN (p.P152L and p.R306Q). We demonstrated accumulation of linear ubiquitin chains upon TNF stimulation and augmented TNF-induced cell death in mesenchymal stem cells differentiated from patient-derived iPS cells, which confirms that the patient has ORAS. However, although the de novo p.R306Q variant exhibits attenuated deubiquitination activity without reducing the amount of OTULIN, the deubiquitination activity of the p.P152L variant inherited from the mother was equivalent to that of the wild-type. Patient-derived MSCs in which the p.P152L variant was replaced with wild-type also exhibited augmented TNF-induced cell death and accumulation of linear chains. The finding that ORAS can be caused by a dominant-negative p.R306Q variant of OTULIN furthers our understanding of disease pathogenesis.

The mechanism of USP43 in the development of tumor: a literature review.

Ubiquitination of the proteins is crucial for governing protein degradation and regulating fundamental cellular processes. Deubiquitinases (DUBs) have emerged as significant regulators of multiple pathways associated with cancer and other diseases, owing to their capacity to remove ubiquitin from target substrates and modulate signaling. Consequently, they represent potential therapeutic targets for cancer and other life-threatening conditions. USP43 belongs to the DUBs family involved in cancer development and progression. This review aims to provide a comprehensive overview of the existing scientific evidence implicating USP43 in cancer development. Additionally, it will investigate potential small-molecule inhibitors that target DUBs that may have the capability to function as anti-cancer medicines.

Genetic interaction mapping reveals functional relationships between peptidoglycan endopeptidases and carboxypeptidases.

Peptidoglycan (PG) is the main component of the bacterial cell wall; it maintains cell shape while protecting the cell from internal osmotic pressure and external environmental challenges. PG synthesis is essential for bacterial growth and survival, and a series of PG modifications are required to allow expansion of the sacculus. Endopeptidases (EPs), for example, cleave the crosslinks between adjacent PG strands to allow the incorporation of newly synthesized PG. EPs are collectively essential for bacterial growth and must likely be carefully regulated to prevent sacculus degradation and cell death. However, EP regulation mechanisms are poorly understood. Here, we used TnSeq to uncover novel EP regulators in Vibrio cholerae. This screen revealed that the carboxypeptidase DacA1 (PBP5) alleviates EP toxicity. dacA1 is essential for viability on LB medium, and this essentiality was suppressed by EP overexpression, revealing that EP toxicity both mitigates, and is mitigated by, a defect in dacA1. A subsequent suppressor screen to restore viability of ΔdacA1 in LB medium identified hypomorphic mutants in the PG synthesis pathway, as well as mutations that promote EP activation. Our data thus reveal a more complex role of DacA1 in maintaining PG homeostasis than previously assumed.