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Nontuberculous mycobacteria (NTM) infections, especially those caused by Mycobacterium abscessus, are extremely difficult to treat due to the organisms' intrinsic resistance to many antimicrobials. Myxin is a naturally occurring phenazine-5,10-dioxide antibiotic from Sorangium sp., notable for its broad-spectrum antimicrobial activity. In this study, we determined the susceptibilities of Myxin against 11 NTM reference strains and 194 clinical M. abscessus isolates using microdilution assays. Additionally, time-kill kinetic experiments were performed to distinguish the clearing efficiency of Myxin in broth. We also evaluated the efficacy of Myxin against intracellular M. abscessus in THP-1 macrophages. Checkerboard assays assessed the interactions between Myxin and eight clinically important anti-NTM antibiotics. Myxin demonstrated potent activity against M. abscessus with low MIC values (MIC50 = 0.125 mg/L and MIC90 = 0.5 mg/L). Treatment with 20 × MIC of Myxin in 7H9 broth resulted in up to 10 log10 CFU/mL reduction after 4 days of incubation. An intracellular concentration-dependent bactericidal effect of Myxin was observed in THP-1 macrophages. Myxin exhibited no antagonism in combination with standard anti-NTM drugs. As such, Myxin represents a promising candidate for the treatment of M. abscessus and other NTM infections.

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The increasing emergence of antibiotic resistance highlights the urgent need for the discovery of new antimicrobial agents. Herein, we report the synthesis and antimicrobial evaluation of a series of phenazine sulfonamides (PSAs) based on 9-chloro phenazine carboxylic acid (1a) and saphenic acid (2). Our initial antimicrobial screening identified compounds with promising activity against methicillin-resistant Staphylococcus aureus (MRSA), S. epidermidis, and Neisseria gonorrhoeae. Notably, compound 7i showed activities in the low micromolar range against MRSA and S. epidermidis. In addition, compounds 7k and 7p exhibited antimicrobial effect against N. gonorrhoeae isolates. In subsequent investigations, 7i demonstrated an additive antibacterial effect against S. aureus in combination with linezolid and vancomycin. Compound 7i also improved the antimicrobial efficacy of vancomycin against vancomycin-resistant S. aureus isolates and demonstrated a powerful biofilm formation inhibition against S. aureus JE2 by suppressing key regulatory genes (agrA and saeR) and impairing virulence-associated exoprotein secretion. In addition, 7i possesses a very promising safety profile: no cytotoxicity towards mammalian cells, and lack of hemolytic and mutagenic action. The lead molecules showed promising activity in a simple in vivo model. Altogether, data from this study suggest PSA are a promising scaffold for developing antimicrobial agents.

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<b>Background and Objective:</b> Phenazines from <i>Streptomyces</i> species are known for their diverse biological activities, including antimicrobial and anticancer properties. However, purified compounds from <i>Streptomyces murinus</i> had not been fully characterized. This study aimed to isolate and identify phenazine-producing <i>Streptomyces murinus</i> ZMA01 from maize rhizosphere soil, evaluate its antimicrobial and anticancer activities, and investigate its molecular mechanisms and ADMET profiles. <b>Materials and Methods:</b> <i>Streptomyces murinus</i> ZMA01 was isolated and identified. Phenazines were extracted, purified, and tested for antimicrobial activity (MIC/MMC) against bacterial and fungal strains. Cytotoxicity IC₅₀ was assessed on Vero, HeLa, HepG2, and MDA-MB-231 cell lines. Apoptotic induction was investigated via morphological changes and Annexin V/PI staining in MDA-MB-231 cells. Molecular docking evaluated interactions with Bcl-2 family proteins. The ADMET parameters were predicted using SwissADME, PreADMET and pkCSM. Statistical analysis was conducted using SPSS (version 11.01). One-way ANOVA and Tukey's <i>post hoc</i> test were employed, with significance defined as p<0.05. <b>Results:</b> <i> Streptomyces murinus</i> ZMA01 yielded 1.25 to 1.39 mg/L of phenazines. Two compounds, methyl-7-carbamoyl-phenazine-1-carboxamide and 7-hydroxyphenazine-1-carboxamide, showed selective antimicrobial activity (MICs: 32 to 256 µg/mL) and potent anticancer effects (IC₅₀: 48.63 to 148.13 µg/mL), particularly inducing apoptosis in MDA-MB-231 cells. Low toxicity to Vero cells (IC₅₀ >400 µg/mL) was noted. Molecular docking confirmed favorable binding to Bcl-2 family proteins, suggesting an apoptotic mechanism. The ADMET predictions indicated good solubility, permeability and low neurotoxicity, with potential for CYP1A2 inhibition. <b>Conclusion:</b> Phenazines isolated from <i>Streptomyces murinus </i>ZMA01 exhibit promising antimicrobial and anticancer activities with favorable ADMET profiles. These findings underscore their therapeutic potential and warrant further <i>in vivo</i> studies and targeted delivery optimization for treating microbial infections and cancers.

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BACKGROUND: Topoisomerase II (Topo II) remains a validated target for anticancer therapy, with many clinically used agents acting via DNA intercalation and enzyme inhibition. However, their clinical use is limited by severe toxicity and resistance. In this study, we investigate aotaphenazine, a rare hydrophenazine derivative isolated from Streptomyces sp. IFM 11694 as a potential novel Topo II inhibitor with selective anticancer activity. METHODS: Molecular docking and 400 ns molecular dynamics (MD) simulations were employed to evaluate aotaphenazine's binding mode within the DNA-Topo II complex (PDB: 3QX3). MM-GBSA calculations quantified interaction energetics, while ProLIF and PLIP analyses detailed the interaction patterns. Topo II inhibition was assessed via in vitro enzymatic assays. Cytotoxicity (MTT) assays were conducted against a panel of human cancer and normal cell lines. Flow cytometry was used to evaluate apoptosis and cell cycle progression in MDA-MB-231 cells. RESULTS: aotaphenazine demonstrated a docking binding energy of -19.12 kcal/mol and remained stably intercalated within the DNA groove during MD simulations. MM-GBSA analysis showed a total binding free energy of -29.81 kcal/mol, driven primarily by van der Waals forces. Interaction profiling identified consistent π-stacking with Cyt8 and Thy9, and strong binding contributions from Ade12 and Gua13. Enzymatic assays confirmed Topo II inhibition with an IC50 of 45.01 nM, comparable to doxorubicin (30.16 nM). In vitro cytotoxicity analysis revealed moderate activity across cancer cell lines (IC50 = 26.30-54.35 µM) and significantly reduced toxicity in normal WI-38 and WISH cells (IC50 = 69.86 µM and 84.72 µM, respectively). Flow cytometry showed that aotaphenazine induced early (20.98%) and late apoptosis (42.80%), along with S-phase cell cycle arrest (43.99%) and a marked reduction in the G2/M population in MDA-MB-231 cells. CONCLUSION: aotaphenazine exhibits a compelling combination of Topo II inhibition, DNA intercalation, and selective anticancer activity, supported by both computational modeling and biological validation. Its lower cytotoxicity toward normal cells and ability to induce apoptosis and cell cycle arrest suggest strong therapeutic potential. These findings establish aotaphenazine as a promising lead compound for the development of safer and more selective Topo II-targeting anticancer agents.

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Phenazines are compounds of medical and biotechnological interest due to their antimicrobial and antitumoral properties. Pseudomonas aeruginosa, a Gram-negative bacterium, naturally produces phenazines such as pyocyanin and phenazine-1-carboxylic acid (PCA). These phenazines can generate reactive oxygen species, which increase oxidative stress in susceptible cells and ultimately lead to their death. Pyocyanin can inhibit the growth of human bacterial pathogens, while PCA serves as a potent antifungal agent that can prevent and control crop diseases. The environmental strain P. aeruginosa ID4365 overproduces pyocyanin, and its derivative strain IDrsmA, which carries a mutation in the rsmA gene, presents a fivefold increase in pyocyanin production compared to the wild-type strain. This mutant is therefore a promising candidate strain for enhancing phenazine production. In this work, we found that the IDrsmA strain has an inactive type three secretion system and is non-cytotoxic. Unlike the wild-type strain ID4365, the IDrsmA strain was unable to infect and kill Galleria mellonella larvae or mice. Besides pyocyanin, the IDrsmA strain also produced increased levels of PCA and 1-hydroxyphenazine. In addition, we showed that the deficient phosphate medium, PPGAS, is the best phenazine production medium compared to PPM or King A media. By optimizing culture conditions, we increased pyocyanin production up to 298 µg/mL using strain IDrsmA. We further modified the IDrsmA strain to produce only PCA, achieving a production level of 303 µg/mL, which is 10 times higher than that of the wild-type strain. Thus, the IDrsmA strain is non-virulent and suitable for phenazine production.

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Obesity leads to the development of several diseases and chronic death worldwide. Mitochondrial dysfunction is one of the vital causes to develop obesity. Targeting mitochondrial uncoupling protein 1 (UCP1) may well be a potential therapeutic approach against obesity or mitochondrial dysfunction-related illnesses. To assess the significance of mitochondrial adenosine triphosphate (ATP) synthesis, mitochondrial DNA quantity and in vitro pharmacodynamics and pharmacokinetics, we used CX-6258 HCl (pan-Pim kinase inhibitor) in this work. CX-6258 HCl significantly reduces ATP production both in white and brown adipocytes and, therefore, improves thermogenesis, which helps to reduce fat in adipocytes. On the HEK293T cell line, no appreciable cell growth was seen. The in silico analysis identifies a potential interaction between CX-6258 HCl and the UCP1 protein. To treat disorders linked to mitochondrial dysfunction or obesity, CX-6258 HCl may be a promising therapeutic option. The role of pan-Pim kinase inhibitor on obesity and mitochondrial dysfunction-related disorders remains unknown. Further investigation will be leading to the development of the mechanism of action and therapeutic potential of CX-6258 HCl (pan-Pim kinase inhibitor).

Epigenetic drugs increase mitochondrial function in both white and brown adipocytes.In silico analysis uncovered macromolecule­drug interface and potent amino acid interaction with protein.Listed new UCP1 protein up-regulator that could be against mitochondrial-related diseases, eye diseases, cardiovascular diseases, cancer, metabolic diseases and obesity.

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BACKGROUND: Biopsies are commonly obtained as an initial step in the evaluation of infection, particularly in immunocompromised patients presenting with concerning skin lesions. We compared the diagnostic utility of special stains with culture as a gold standard for the detection of infection. METHODS: Retrospective review of the surgical pathology files of the Brigham and Women's Hospital, Boston, MA, was performed to identify skin biopsies from immunocompromised patients over a 15-year period. Stains evaluated included Gram, methenamine silver, periodic acid-Schiff diastase, and acid-fast bacilli (Ziehl-Neelsen and Fite). Comparison was made with concurrent tissue culture results as the gold standard and to molecular testing where available. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of each stain were calculated. RESULTS: Three hundred and nineteen skin biopsy specimens were identified which had both special stains and concurrent tissue cultures. We found that fungal stains (methenamine silver and periodic acid-Schiff diastase have moderate sensitivity but high specificity, whereas Gram staining has a low sensitivity and high specificity. Special stains for mycobacteria (Ziehl-Neelsen and Fite) are moderately to highly sensitive and specific. All special stains evaluated have moderate to high NPV. When comparing clinical suspicion for infection with the results of all special stains, sensitivity and NPV were low; however, specificity and PPV were high. CONCLUSION: The use of special stains for the detection of infectious agents is a valuable ancillary tool for the prompt detection of infection in skin biopsies from immunocompromised patients.

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Antimicrobial resistance (AMR) is a mounting global health challenge projected to cause up to 10 million deaths annually by 2050. Despite advances in antibiotic discovery, the rapid emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) pathogens undermines modern medicine, threatening procedures such as surgery, chemotherapy, and organ transplantation. Conventional antibiotics face increasing limitations due to target-site mutations, efflux mechanisms, enzymatic degradation, and biofilm-associated tolerance, underscoring the urgent need for novel antimicrobial strategies. Phenazines, particularly 1-hydroxyphenazine (1-HP), represent promising alternatives owing to their redox activity, broad-spectrum antimicrobial properties, and ecological roles in microbial competition. Recent advances highlight the potential of 1-HP as both a virulence factor and a therapeutic scaffold, with applications spanning agriculture, biotechnology, and medicine. Synthetic biology, metabolic engineering, and nanocarrier-based delivery systems have enabled scalable production and reduced toxicity, while structural modifications such as halogenation have expanded therapeutic potential. This review consolidates historical, mechanistic, and translational insights into 1-HP, emphasizing its dual role as a pathogenic metabolite and a lead compound for future antimicrobial and anticancer development.

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The pleiotropic kinase CK2 plays a crucial role in numerous cellular processes and is frequently deregulated in human diseases. Specifically, elevated CK2 expression and/or activity have been observed in human cancers, thus rendering its inhibition a promising pharmacological strategy for treating malignancies. The most widely used CK2 inhibitor, CX-4945 (Silmitarsetib), was developed by Cylene Pharmaceuticals in 2010. It has been tested in clinical trials for various cancers and, more recently, as a potential therapy for COVID-19 patients. However, it has been demonstrated that CX-4945's specificity is limited, as CX-4945 also inhibits other kinases beyond CK2. A recently developed derivative of CX-4945, SGC-CK2-2, has demonstrated enhanced specificity compared with CX-4945, albeit with reduced potency. In this study, we conducted a detailed analysis of the effects of SGC-CK2-2 in two cancer cell lines, comparing its efficacy with CX-4945 in inhibiting CK2 signaling and in cell death induction. The findings of this study demonstrate the differential sensitivity of CK2 phospho-substrates to these inhibitors, thus indicating that complete inhibition of a single phosphosite, such as S129 Akt, is insufficient to fully suppress CK2 signaling. Furthermore, the results suggest that partial CK2 inhibition with the suppression of the most sensitive phosphosites does not significantly impact cell viability, while a near-complete suppression of CK2 signaling affects cell viability and leads to cell death induction.

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Bacterial interactions can affect the production of secondary metabolites and, therefore, provide a promising approach to exploring new microbial compounds. In this study, we screened actinomycetes isolated from Hegura Island, Ishikawa Prefecture, Japan, to discover new antibiotics through combined-culture with Tsukamurella pulmonis TP-B0596. Three new phenazine-class antibiotics, griseoluteins T (1), C (2), and D (3), along with two known related metabolites, griseoluteic acid (4) and griseolutein A (5), were detected in both mono- and combined-cultures of Streptomyces seoulensis HEK131 with T. pulmonis at different production levels. Detailed spectroscopic analysis revealed that 1 contained a dihydrophenazine core, and was converted to 5 by accepting oxidation spontaneously. 1, containing a dihydrophenazine group, was relatively unstable under oxidative conditions, and the addition of ascorbate was required during the isolation of the compound. 2 and 3 were found to be cysteine-adducts analogous to 4, and their productivity was increased in the combined-culture. We further assessed the antibacterial activities of 1 against clinically significant Gram-positive pathogenic bacteria, including 30 methicillin-resistant Staphylococcus aureus (MRSA), 27 vancomycin-resistant Enterococci (VRE), and 17 Clostridioides difficile. Notably, 1 was found to possess higher antibacterial activity against these microorganisms than several clinically important antibiotics, while displaying lower cytotoxicity against HeLa-S3 cells.

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Under oxygen-limited conditions, the adaptability and underlying mechanisms of bacterial biofilms have become key areas of interest in microbiology and clinical infection research. Within biofilms-composed of bacterial communities and extracellular matrix-an oxygen gradient commonly forms, resulting in hypoxic or even anoxic microenvironments. Such conditions substantially increase biofilm antibiotic resistance and facilitate the persistence of chronic infections. This review systematically summarizes the adaptive strategies employed by biofilms in hypoxic environments, including anaerobic metabolism, phenazine-mediated electron shuttling, and virulence factor regulation. These adaptive responses are governed by genes involved in anaerobic metabolism, quorum sensing systems, and the secondary messenger 3,5-cyclic diguanylic acid (c-di-GMP), which collectively influence biofilm formation. Key transcriptional regulators such as Anr and Dnr, the two-component system NarXL, along with specific functional genes, form an intricate regulatory network. This article aims to provide a comprehensive overview of the adaptive mechanisms of Pseudomonas aeruginosa biofilms under oxygen-limited conditions, providing a theoretical foundation for the development of novel anti-infective therapies, targeting the biofilm infection microenvironment in cystic fibrosis and chronic wounds.

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Hodgkin lymphoma (HL) develops in the part of the immune system that is centrally aggravated by the NF-κB signaling. Selectively targeting IKKß to downregulate the NF-κB-mediated disease progression helps control this dreadful malignancy. This study evaluated novel and selective IKKß inhibitors to downregulate aberrant NF-κB signaling in HL. GROMACS, GMX_MMPBSA, and PLIP were used after high-throughput virtual screening against the ChemBridge library to identify leads. The in vitro effectiveness was evaluated using flow cytometry, luminometry, and spectrometry on RPMI 666 and Hs 445 cells. HTVS identified SIKB-7543 with favorable binding affinities of -14.2 kcal/mol toward IKKß. MD simulations established stable bonding for SIKB-7543 and IKKß with RSMD values around 0.07 nm. The ΔG binding calculation was -50.46 kcal/mol, favoring sturdy binding. ADME analysis favored small-molecule characteristics. SIKB-7543 inhibited IKKß activity with an IC50 value of 118 nM. The compound effectively controlled the proliferation of RPMI 666 and Hs 445 cells with GI50 values of 345.6 nM and 320.5 nM, respectively. SIKB-7543 prompted dose-responsive apoptosis in the HL cells. Cell-cycle analysis demonstrated a concentration-dependent increase in the sub-G0 population in both cell lines following SIKB-7543 treatment, while decreasing the NF-κB-p65 (Rel A) positive populations in TNFα-stimulated RPMI 666 and Hs 445 cells dose-dependently. Results suggest SIKB-7543 is a selective IKKß inhibitor that downregulates aberrant NF-κB signaling, controls proliferation, and induces apoptosis, warranting further preclinical developments to counter HL malignancy.

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Phenazines are bioactive secondary metabolites with antifungal, anticancer, and insecticidal properties, while hydroxylated derivatives often exhibit enhanced bioactivity. 2-hydroxyphenazine (2-OH-PHZ), which is synthesised by the flavin-dependent monooxygenase PhzO from phenazine-1-carboxylic acid (PCA), shows better bioactivity against the pathogenic fungus Gaeumannomyces graminis vars. tritici. However, the low catalytic efficiency (10%-20% conversion) of PhzO limited 2-OH-PHZ production. To boost PhzO activity, engineering flavin reductase (Fre)-mediated FADH2 regeneration was applied to Pseudomonas chlororaphis LX24AE. Remarkably, this approach improved catalytic efficiency from 25% to 40% and increased the production of a novel dihydroxylated derivative. Then, it was first characterised by UPLC-MS and NMR, and identified as 3,4-dihydroxyphenazine-1-carboxylic acid (3,4-OH-PCA). Next, the Fre-PhzO module through heterologous co-expression in P. putida KT2440 demonstrated a 4.5-fold enhancement in hydroxylation efficiency relative to the PhzO mono-component system, which also confirmed that PhzO and flavin reductase are essential for 3,4-OH-PCA biosynthesis. Moreover, in vitro assays further verified that PhzO exhibits FAD-dependent catalytic promiscuity, simultaneously generating 2-OH-PCA and 3,4-OH-PCA. Furthermore, in vitro and in vivo assays demonstrated that substrate concentration affected the distribution of products. Finally, cytotoxicity evaluation of the isolated 3,4-OH-PCA was performed, and it showed substantial inhibition against oesophageal cancer TE-1 cells and human cervical cancer HeLa cells with an IC50 value of 8.55 µM and 17.69 µM, respectively. This work redefines PhzO as a promiscuous biocatalyst capable of dual hydroxylation, offering a modular platform for engineering bioactive phenazine derivatives.

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Protein kinase CK2 has emerged as a pivotal regulator of cellular processes involved in skin homeostasis, including cell proliferation, differentiation and inflammatory response regulation. In fact, CK2 activity dysregulation is implicated in the pathogenesis of different skin diseases, such as psoriasis, cancer and inflammatory dermatoses. CK2 overactivation fosters keratinocyte proliferation and pro-inflammatory cytokine production through the STAT3 and Akt pathways in psoriasis, thus contributing to epidermal hyperplasia and inflammation. In the realm of oncology, CK2 overexpression correlates with tumor progression, facilitating cell survival and metastasis in melanoma and non-melanoma skin cancers. Pharmacological inhibition of CK2 has demonstrated therapeutic potential, with CX-4945 (Silmitasertib) as the most studied adenosine triphosphate-competitive inhibitor (ATP-competitive inhibitor). Preclinical models reveal that CK2 inhibitors effectively mitigate pathological features of psoriasis, regulate keratinocyte differentiation, and suppress tumor growth in skin cancers. These inhibitors also potentiate the efficacy of conventional chemotherapeutics and exhibit anti-inflammatory effects in dermatological conditions. Future research will aim to enhance the specificity and delivery of CK2-targeting therapies, including topical formulations, to minimize systemic side effects. Combination therapies integrating CK2 inhibitors with other agents might offer synergistic benefits in managing skin diseases. This review underscores CK2's critical role in skin and its therapeutic potential as a pharmacological target, advocating for innovative approaches to harness CK2 inhibition in dermatology.

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Cuproptosis, a recently recognized regulated cell death, distinct from established death mechanisms, offers promising cancer therapy. However, its efficacy relies on intracellular copper availability and homeostasis. Herein, a novel Copper(II) dipyridohenazine complex, Cu(L1)2Cl acts as an oxidative stress amplifier and glutathione (GSH) disrupter for synergistic cuproptosis/chemodynamic anticancer therapy for the treatment of challenging triple negative breast cancer. Cu(L1)2Cl followed the endocytosis pathway to enter tumor cells and depleted GSH to release Cu+ ions which result in the production of.OH radicals generated from H2O2, leading to chemodynamic therapy. The spike in ROS generation disrupts cellular redox homeostasis, causing impaired mitochondrial function, ATP depletion, and endoplasmic reticulum stress generation. ATP depletion directly affects the function of copper-transporting ATPase 1 (ATP7A), resulting in a large amount of Cu+ trapped inside cancer cells, causing oligomerization of dihydrolipoamide S-acetyltransferase (DLAT), and depletion of Lipoyl synthase (LIAS), and leading to cellular cuproptosis. Subsequently, Cu(L1)2Cl interrupts tumor metastasis and evokes immunogenic cell death (ICD) by promoting high mobility group protein (HMGB1), ATP and lactate dehydrogenase (LDH) release, calreticulin (CRT) exposure, and inhibiting programmed death ligand 1 (PD-L1). The in vivo studies on 4T1 tumor bearing Balb/c mice validate its potent antitumor efficacy, thereby providing a new therapeutic paradigm to augment cuproptosis-related therapies.

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The bifunctions of fluorescence activity and peroxidase mimics were well-integrated by a hemin chloride-modified Zr-based metal-organic framework (Hemin@NH2-UiO-66), which can catalyze the oxidation of o-phenylenediamine (OPD) to generate 2,3-diaminophenazine (DAP), presenting typical peroxidase mimic properties. The influence of the introduction of hemin chloride upon the enhanced peroxidase mimic activity was clarified, and the nanozyme catalytic parameters were optimized. Interestingly, the oxidized product of DAP presents strong fluorescence emission at 564 nm; combining it with the intrinsic fluorescence emission of NH2-UiO-66 at 452 nm, a dual-emission system could be built up by the resulting Hemin@NH2-UiO-66 in the presence of definite OPD and H2O2. Moreover, the fluorescence quenching effect (564 nm) was observed by adding l-cysteine (l-Cys); based on that, a straight-line dependence of the fluorescence intensity ratio (I452/I564) upon the concentrations of l-Cys was established. The detection limit was 0.21 µmol, and the analytical selectivity for l-Cys was also demonstrated. The work highlights the idea of combining the intrinsic fluorescence property and nanozyme catalytic activity in a functional MOF, and its special usability is found in the ratiometric fluorescence sensing analyses.

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Pseudomonas species are recognized for producing a diverse array of microbial metabolites with significant potential across various fields. Pyrrolnitrin (PRN), a halogenated metabolite based on phenylpyrrole, exhibits potent antibiotic properties. This research aimed to examine the influence of pyrrolnitrin on the antagonistic properties of Pseudomonas chlororaphis strains PB-St2, FS2, and RP4. Mutants of P. chlororaphis were generated by inhibiting prnA using two distinct suicide vectors, pEX18Tc and pKC1132. Analysis via high performance liquid chromatography (HPLC) revealed that pyrrolnitrin production was completely eliminated in the pKC1132 mutant and decreased by 82.5% in the pEX18Tc mutant. Both mutants also exhibited reduced phenazine production, with pKC1132 mutants showing a 61.1% reduction in phenazine-1-carboxylic acid (PCA) and pEX18Tc-induced mutants displaying a 39.9% decrease in PCA. To further elucidate the dependence of pyrrolnitrin production on other regulatory elements, the complete prnABCD operon with its native promoter was cloned and expressed in Escherichia coli (BL21). The antimicrobial potential of purified pyrrolnitrin was evaluated against fungal plant pathogens, human bacterial pathogens, and cancer cell lines (HepG-2 and SF767). The most pronounced inhibitory effect on Alternaria alternata was observed with 100 µg of pyrrolnitrin, resulting in an 82% reduction in spore formation followed by its effect on Aspergillus niger, causing a 75% decrease in spore production. Pyrrolnitrin's antibacterial activity produced inhibition zones of 1.8 cm against Salmonella enterica, 3.4 cm against Bacillus cereus, and 1.4 cm against Staphylococcus sp. at a concentration of 75 µg. The antiproliferative effects of pyrrolnitrin on cancer cell lines demonstrated 50% inhibition of both HepG-2 and SF767 cell lines at concentrations of 15 µg and 25 µg, respectively. Pyrrolnitrin exhibited significant antifungal and antibacterial activities, as well as cytotoxic effects on cancer cell lines, indicating its potential as both a biocontrol agent and therapeutic compound.

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Phenazine derivatives, a class of nitrogen-containing heterocyclic compounds, exhibit broad-spectrum antifungal, anticancer, and antimalarial activities. Pseudomonas and Streptomyces are the primary microbial strains responsible for the synthesis of phenazine derivatives. In general, Pseudomonas strains use phenazine-1-carboxylic acid (PCA) as a precursor for enzymatic modification, while Streptomyces strains employ phenazine-1,6-dicarboxylic acid (PDC) as the precursor. Pseudomonas is considered an ideal platform for the efficient biosynthesis of various phenazine derivatives due to its rapid growth rate, ease of genetic manipulation, and well-established fermentation systems. However, the synthesis of phenazine derivatives in Pseudomonas largely relies on previously reported natural biosynthetic pathways from other microbial strains. The biosynthesis of phenazine derivatives through unknown pathways often presents significant challenges for researchers. The concept of combinatorial biosynthesis offers a promising solution to overcome these difficulties. In this study, we designed and constructed a platform Pseudomonas strain producing 15 phenazine derivatives by exchanging and combining the modifying enzymes of PCA and PDC, besides 16 constructed modification pathways. Among these, three derivatives feature novel chemical structures, while 13 represent previously unreported biosynthetic pathways. With the discovery of new phenazine modifying enzymes, they can be quickly incorporated into our platform, enabling the rapid synthesis of a wide variety of phenazine derivatives. This work demonstrates the potential of designing non-natural metabolic pathways to enable the production of diverse phenazine derivatives, thereby enhancing bacterial capacity for the synthesis of high-value phenazine compounds. This combinatorial biosynthetic approach provides a potential alternative for exploring unknown biosynthetic routes and for the development of unexplored natural biosynthetic pathways for phenazine derivatives.

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KD025(belumosudil), a selective ROCK2 inhibitor, exhibits unique anti-adipogenic activity through inhibition of casein kinase 2 (CK2). This study investigated the dual inhibitory effects of KD025 on metabolism in a diet-induced obese model. C57BL/6 mice on a high fat diet (HFD) were treated with KD025 for 4 weeks, while fasudil (a pan-ROCK inhibitor) and CX-4945 (a CK2-specific inhibitor) served as comparison treatments. KD025 significantly reduced body weight gain without affecting food intake, serum insulin, or fasting blood glucose levels. In contrast, while both CX-4945 and fasudil treatments showed a trend toward weight reduction, these results were not statistically significant. KD025 improved lipid metabolism by significantly lowering LDL cholesterol and triglyceride, although it slightly impaired glucose metabolism, as observed in insulin and glucose tolerance tests. Weight reduction in the KD025- and CX-4945-treated groups was attributed to decreased adipose tissue mass, particularly in inguinal (ingWAT) and epididymal (epiWAT) fat depots. Hematoxylin and eosin (H&E) staining confirmed smaller adipocyte size in these groups. KD025 had no significant effect on serum levels of tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), or monocyte chemoattractant protein-1 (MCP-1) with varied inflammatory responses. Furthermore, KD025 and CX-4945 upregulated adipogenic and browning markers, such as Cebpa, Cidea, and Pparg in the epiWAT, though without significant UCP1 expression. Overall, KD025 effectively reduced weight gain in HFD-fed mice through dual inhibition of CK2 and ROCK2, highlighting its potential as a therapeutic agent for obesity-related conditions.

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Abnormal alkaline phosphatase (ALP) levels in the human body are closely associated with various diseases, particularly hepatobiliary diseases and bone diseases. Herein, we constructed a ratiometric sensor based on Förster resonance energy transfer (FRET) using strongly photoluminescent copper nanoclusters (Cu NCs) for the detection of ALP with high sensitivity and specificity. The cysteine-stabilized Cu NCs (Cys-Cu NCs) were synthesized through a ligand-exchange reaction and core-size etching focusing, which displayed bright photoluminescence (PL) with a quantum yield (QY) of 10.5 %. Multispectral characterization indicated that zwitterionic cysteine ligands without obvious steric hindrance could significantly enhance intra/inter-ligand-involved charge transfer, leading to a significant increase in fluorescence emission (∼14 folds) compared to precursor Cu NCs. A FRET-based ratiometric sensor for ALP detection was constructed by combining Cys-Cu NCs with a Cu2+-assisted oxidation reaction of o-phenylenediamine (OPD) to generate fluorescent 2,3-diaminophenazine (DAP). The strong coordination interaction between the ALP substrate and Cu2+ significantly affected the FRET process between Cys-Cu NCs and DAP, thereby altering the fluorescence ratio. Based on the specific response of ALP to its substrate, the ratiometric sensors showed good linear relationship within the range of 0.1-50 U/L, with a detection limit (LOD) of 0.075 U/L. Furthermore, the FRET-based ratiometric sensor was integrated with a polymer hydrogel to fabricate a portable hydrogel sensor for simple and visual detection of ALP.