Intracellular ATP Levels Research Articles

Intracellular ATP concentration is a key regulator of bacterial cell fate.

ATP, most widely known as the primary energy source for numerous cellular processes, also exhibits the characteristics of a biological hydrotrope. The viable but nonculturable (VBNC) and persister states are two prevalent dormant phenotypes employed by bacteria to survive challenging environments, both of which are associated with low metabolic activity. Here, we investigate the intracellular ATP concentration of individual VBNC and persister cells using a sensitive ATP biosensor QUEEN-7μ and reveal that both types of cells possess a lower intracellular ATP concentration than culturable and sensitive cells, although there is a certain overlap in the intracellular ATP concentrations between antibiotic-sensitive cells and persisters. Moreover, we successfully separated VBNC cells from culturable cells using fluorescence-activated cell sorting based on the intracellular ATP concentration threshold of 12.5 µM. Using an enriched VBNC cell population, we confirm that the precipitation of proteins involved in key biological processes promotes VBNC cell formation. Notably, using green light-illuminated proteorhodopsin (PR), we demonstrate that VBNC cells can be effectively resuscitated by elevating their intracellular ATP concentration. These findings highlight the crucial role of intracellular ATP concentration in the regulation of bacterial cell fate and provide new insights into the formation of VBNC and persister cells.IMPORTANCEThe viable but nonculturable (VBNC) and persister states are two dormant phenotypes employed by bacteria to counter stressful conditions and play a crucial role in chronic and recurrent bacterial infections. However, the lack of precise detection methods poses significant threats to public health. Our study reveals lower intracellular ATP concentrations in these states and establishes an ATP threshold for distinguishing VBNC from culturable cells. Remarkably, we revive VBNC cells by elevating their intracellular ATP levels. This echoes recent eukaryotic studies where modulating metabolism impacts outcomes like osteoarthritis treatment and lifespan extension in Caenorhabditis elegans. Our findings underscore the crucial role of intracellular ATP levels in governing bacterial fate, emphasizing ATP manipulation as a potential strategy to steer bacterial behavior.

USP2 Mitigates Reactive Oxygen Species-Induced Mitochondrial Damage via UCP2 Expression in Myoblasts

Ubiquitin-specific protease 2 (USP2) maintains mitochondrial integrity in culture myoblasts. In this study, we investigated the molecular mechanisms underlying the protective role of USP2 in mitochondria. The knockout (KO) of the Usp2 gene or the chemical inhibition of USP2 induced a robust accumulation of mitochondrial reactive oxygen species (ROS), accompanied by defects in mitochondrial membrane potential, in C2C12 myoblasts. ROS removal by N-acetyl-L-cysteine restored the mitochondrial dysfunction induced by USP2 deficiency. Comprehensive RT-qPCR screening and following protein analysis indicated that both the genetic and chemical inhibition of USP2 elicited a decrease in uncoupling protein 2 (UCP2) at mRNA and protein levels. Accordingly, the introduction of a Ucp2-expressing construct effectively recovered the mitochondrial membrane potential, entailing an increment in the intracellular ATP level in Usp2KO C2C12 cells. In contrast, USP2 deficiency also decreased peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α) protein in C2C12 cells, while it upregulated Ppargc1a mRNA. Overexpression studies indicated that USP2 potentially stabilizes PGC1α in an isopeptidase-dependent manner. Given that PGC1α is an inducer of UCP2 in C2C12 cells, USP2 might ameliorate mitochondrial ROS by maintaining the PGC1α–UCP2 axis in myoblasts.

The anthraquinone derivative KA-4s reduces energy metabolism and enhances the sensitivity of ovarian cancer cells to cisplatin.

Ovarian cancer is the leading cause of death from female gynecological cancers. Cisplatin (DDP)is a first-line drug for ovarian cancer treatment. Due to DDP resistance, there is an urgent need for novel therapeutic drugs with improved antitumor activity. AMPK-mediated metabolic regulatory pathways are related to tumor drug resistance. Our study aimed to determine the relationship between reversing DDP resistance with the anthraquinone derivative KA-4s and regulating AMPK energy metabolism in ovarian cancer. The results showed that KA-4s inhibited the proliferation of ovarian cancer cells. The combination of KA-4s with DDP effectively promoted drug-resistant ovarian cancer cell apoptosis and inhibited cell migration and invasion. Moreover, KA-4s decreased the intracellular ATP level and increased the calcium ion level, leading to AMPK phosphorylation. Further studies suggested that the AMPK signaling pathway may be involved in the mechanism through which KA-4s reduce drug resistance. KA-4s inhibited mitochondrial respiration and glycolysis; downregulated the glucose metabolism-related proteins GLUT1 and GLUT4; the lipid metabolism-related proteins SREBP1 and SCD1; and the drug resistance-related proteins P-gp, MRP1, and LRP. The inhibitory effect of KA-4s on GLUT1 was confirmed by the application of the GLUT1 inhibitor BAY-876. KA-4s combined with DDP significantly increased the expression of p-AMPKand reduced the expression of P-gp. In a xenograft model of ovarian cancer, treatment with KA-4s combined with DDP reduced energy metabolism and drug resistance, inducing tumor apoptosis. Consequently, KA-4s might be evaluated as a new agent for enhancing the chemotherapeutic efficacy of treatment for ovarian cancer.

The effect of biocide chloromethylisothiazolinone/methylisothiazolinone (CMIT/MIT) mixture on C2C12 muscle cell damage attributed to mitochondrial reactive oxygen species overproduction and autophagy activation

ABSTRACT The mixture of 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one (CMIT/MIT) is a biocide widely used as a preservative in various commercial products. This biocide has also been used as an active ingredient in humidifier disinfectants in South Korea, resulting in serious health effects among users. Recent evidence suggests that the underlying mechanism of CMIT/MIT-initiated toxicity might be associated with defects in mitochondrial functions. The aim of this study was to utilize the C2C12 skeletal muscle model to investigate the effects of CMIT/MIT on mitochondrial function and relevant molecular pathways associated with skeletal muscle dysfunction. Data demonstrated that exposure to CMIT/MIT during myogenic differentiation induced significant mitochondrial excess production of reactive oxygen species (ROS) and a decrease in intracellular ATP levels. Notably, CMIT/MIT significantly inhibited mitochondrial oxidative phosphorylation (Oxphos) and reduced mitochondrial mass at a lower concentration than the biocide amount, which diminished the viability of myotubes. CMIT/MIT induced activation of autophagy flux and decreased protein expression levels of myosin heavy chain (MHC). Taken together, CMIT/MIT exposure produced damage in C2C12 myotubes by impairing mitochondrial bioenergetics and activating autophagy. Our findings contribute to an increased understanding of the underlying mechanisms associated with CMIT/MIT-induced adverse skeletal muscle health effects.

A novel VCP modulator, KUS121, attenuates atherosclerosis progression by maintaining intracellular ATP and mitigating ER stress in endothelial cells

Abstract Back ground: Endoplasmic reticulum (ER) stress signaling pathways have pivotal roles in atherosclerosis progression. Recently, we have developed Kyoto University Substance (KUS) 121, which selectively inhibits ATPase activities of valosin-containing protein (VCP) and consequently saves intracellular ATP consumption and mitigates ER stress. KUS121 are known to have protective effects on several disease models including ischemic heart disease and heart failure. However, the efficacy of KUS121 against atherosclerosis was still unclear. Purpose To elucidate the effect of KUS121 on atherosclerosis and its mechanisms. Methods and Results The daily treatment of KUS121 reduced atherosclerosis progression by approximately 40% and macrophage burden in the plaques were also significantly decreased in ApoE knockout mice on high fat diet. Interestingly, we found that C/EBP homologous protein (CHOP), an established ER stress marker, was mainly expressed in plaque endothelium. Therefore, we assessed the action of KUS 121 in endothelial cells using the EA.hy926 cell line. Consequently, it was demonstrated that KUS121 attenuated ER stress-induced apoptosis and downregulated the IRE1α-associated inflammatory pathways. Consistent with these in vitro findings, KUS121 treatment also significantly reduced endothelial apoptosis assessed by TUNEL staining and inflammation examined by immunostaining of NF-κB and ICAM1 in atherosclerotic plaques. Moreover, KUS121 treatment attenuated the recruitment of inflammatory Ly6C high monocytes assessed by bead-labeling technique, which could be due to the reduction of endothelial ICAM1 expression. Furthermore, we also demonstrated that maintenance of intracellular ATP levels mitigates ER stress and prevents pathological states in endothelial cells. Conclusion KUS121 can be a new therapeutic option for atherosclerotic diseases by maintaining intracellular ATP levels and attenuating ER stress-induced apoptosis and inflammation in plaque endothelium.

Metabolic energetic adaptation of Atlantic salmon phagocytes to changes in carbon sources and exposure to PAMPs

Phagocytic cells are pivotal for host homeostasis and infection defense, necessitating metabolic adaptations in glycolysis, the tricarboxylic acid (TCA) cycle, and oxidative phosphorylation (OXPHOS). While mammalian phagocytes shift towards glycolysis and glutaminolysis during polarization, research on fish phagocyte metabolic reprogramming is limited. To address this, the Atlantic salmon phagocytic cell line, SHK-1, serves as a valuable model. Using the Seahorse XFe96 Flux Analyzer, this study compares SHK-1 bioenergetics under glucose-restricted (L-15 medium) and glucose-supplemented (PM) conditions, providing insights into metabolic characteristics and responses to Piscirickettsia salmonis bacterium Pathogen-associated molecular patterns (PAMPs). A standardized protocol for the study of real-time changes in the metabolism study of SHK-1 in PM and L-15 media, determining oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) is shown. Exhibiting metabolic adaptations, SHK-1 cells in the PM medium have higher basal and maximal OCR and spare capacity (SRC), while those grown in the L-15 medium favor OXPHOS, showing minimal glycolytic function. Despite metabolic differences, intracellular ATP levels are comparable, highlighting the metabolic plasticity and adaptability of SHK-1 cells to various carbon sources. Exposure to PAMPs from Piscirickettsia salmonis induces a metabolic shift, increasing glycolysis and OXPHOS, influencing ATP, lactate, glutamine, and glutamate levels. These findings highlight the role of mitochondrial bioenergetics and metabolic plasticity in salmon phagocytes, offering novel nutritional strategies for host-pathogen interventions based on energy metabolism.

SLC45A4 is involved in malignant progression of ovarian cancer through glycolytic metabolic reprogramming

Tumor cells promote malignant behaviors such as proliferation, invasion, and metastasis of cancer cells through glucose metabolic reprogramming, but the role of the H-dependent sugar cotransporter SLC45A4 in regulating metabolic reprogramming in ovarian cancer (OC) remains largely unknown. This study aimed to investigate the effects of SLC45A4 silencing on the transcriptome spectrum of ovarian cancer cells (OCC), glucose uptake, lactic acid production, intracellular ATP levels, and the expression and activity of HIF-α glycolysis signaling pathway. The results showed that SLC45A4 is overexpressed in OC and its elevated expression correlates with adverse clinical outcomes in OC patients. Silencing of SLC45A4 significantly inhibited the proliferation, invasion, and metastasis of OCC by suppressing glucose uptake and glycolysis, and it also reduced the expression of HIF-α glycolysis signaling pathway in OC tissues. In vivo experiments using shRNA to knock down SLC45A4 in xenograft models in nude mice demonstrated a significant inhibition of tumor growth. These findings suggest that SLC45A4 silencing can restrain the malignant progression of OC by inhibiting glucose uptake in OCC and affecting the reprogramming of glycolytic energy metabolism, indicating that SLC45A4 may serve as a potential therapeutic target for OC intervention.

Biomimetic Single-Atom Nanozyme for Dual Starvation-Enhanced Breast Cancer Immunotherapy.

Cold exposure (CE) therapy is an innovative and cost-efficient cancer treatment that activates brown adipose tissue to compete for glucose uptake, leading to metabolic starvation in tumors. Exploring the combined antitumor mechanisms of CE and traditional therapies (such as nanocatalysis) is exciting and promising. In this study, a platelet membrane biomimetic single-atom nanozyme (SAEs) nanodrug (PFB) carrying bis-2-(5-phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethyl sulfide (BPTES) is developed for use in cancer CE therapy. Owing to the platelet membrane modification, PFB can effectively target tumors. Upon entering cancer cells, the dual starvation effect induced by CE treatment and BPTES can significantly diminish intracellular glucose and ATP levels, resulting in a substantial reduction in cellular (glutathione) GSH, which can enhance the cytotoxic efficacy of reactive oxygen species generated by SAEs. This strategy not only boosts ROS production in tumors, but also strengthens immune responses, particularly by increasing memory T-cell abundance and suppressing distant tumor growth and tumor metastasis. Compared with SAEs therapy alone, this combined approach offers superior benefits for tumor immunotherapy. This study achieves a combination of CE and nanomedicines for the first time, providing new ideas for future nanomedicine combination therapy modalities.

Oleanolic acid promotes porcine oocyte maturation by activating the Nrf2/HO-1 signalling pathway

This study investigated the potential role and underlying mechanisms of oleanolic acid (OA), a pentacyclic triterpene with antioxidant and anti-inflammatory properties, in porcine oocytes during in vitro maturation (IVM). The results showed that supplementation with 5 μM OA during IVM resulted in a greater percentage of mature oocytes, parthenogenetically activated embryos and somatic cell nuclear-transferred embryos. This was evidenced by significant increases in the rate of first polar body expulsion, the expansion of cumulus granulosa cells and the total cell number in blastocysts. Further analysis revealed that OA promoted fatty acid accumulation and upregulated the mRNA expression of genes involved in fatty acid β-oxidation. OA significantly increased the intracellular mitochondrial membrane potential and ATP levels and effectively inhibited BAX/BCL2 and Cleaved Caspase3 protein expression. Notably, OA increased the protein levels of intracellular Nrf2 and HO-1, and the GSH levels and the activities of the antioxidant enzymes SOD and catalase (CAT), while reducing ROS levels. Mechanistically, OA activated the Nrf2/HO-1 signalling pathway, which is crucial for regulating the expression of antioxidant-related targets in IVM porcine oocytes. Our findings indicated that OA improved antioxidant capacity by activating the Nrf2/HO-1 signalling pathway, thereby promoting porcine oocyte maturation.

Valnemulin restores colistin sensitivity against multidrug-resistant gram-negative pathogens

Colistin is one of the last-resort antibiotics in treating infections caused by multidrug-resistant (MDR) pathogens. Unfortunately, the emergence of colistin-resistant gram-negative strains limit its clinical application. Here, we identify an FDA-approved drug, valnemulin (Val), exhibit a synergistic effect with colistin in eradicating both colistin-resistant and colistin-susceptible gram-negative pathogens both in vitro and in the mouse infection model. Furthermore, Val acts synergistically with colistin in eliminating intracellular bacteria in vitro. Functional studies and transcriptional analysis confirm that the combinational use of Val and colistin could cause membrane permeabilization, proton motive force dissipation, reduction in intracellular ATP level, and suppression in bacterial motility, which result in bacterial membrane disruption and finally cell death. Our findings reveal the potential of Val as a colistin adjuvant to combat MDR bacterial pathogens and treat recalcitrant infections.

Miltefosine induces reproductive toxicity during sperm capacitation by altering PI3K/AKT signaling pathway

Miltefosine is the first and only drug approved for the treatment of leishmaniasis. It is also known as a PI3K/AKT signaling pathway inhibitor utilized in anti-cancer or anti-viral therapies. However, the impact of miltefosine on male fertility has not been fully understood. Therefore, this study was performed to investigate the effects of miltefosine on sperm function during capacitation. Duroc spermatozoa were exposed to 0, 2.5, 5, 10, 20, 40, and 80 μM miltefosine and induced for capacitation. Our results showed that miltefosine dramatically increased the expression of PI3K/AKT signaling pathway-associated proteins. Sperm motility, motion kinetics, capacitation, and tyrosine phosphorylation were significantly suppressed by miltefosine. However, intracellular ATP levels and cell viability were not significantly affected. Our findings suggest that miltefosine may disrupt sperm function by abnormally increasing the levels of PI3K/AKT signaling pathway-associated proteins. Therefore, the harmful effects of miltefosine on male reproduction should be considered when using this drug.

Class IIb Microcin MccM Interferes with Oxidative Phosphorylation in Escherichia coli.

Dysbiosis of the human gut microbiota is linked to numerous diseases. Understanding the molecular mechanisms by which microbes interact and compete with one another is required for developing successful strategies to modulate the microbiome. The natural product Microcin M (MccM) consists of a 77-residue bioactive peptide conjugated to a siderophore and is a class II microcin involved in microbial competition with an enigmatic mode-of-action. In this work, we investigated the basis for MccM activity and leveraged bioinformatics to expand the known chemical diversity of class II microcins. We applied automated fast-flow solid phase peptide synthesis coupled with chemoenzymatic chemistry to acquire MccM and demonstrated that its activity was bacteriostatic. We then used our synthetic molecule to ascertain that catecholate siderophore transporters in Escherichia coli K-12 are necessary for MccM import. Once inside the cell, we found that MccM treatment decreased the levels of intracellular ATP and interfered with gene expression. These effects were ameliorated in genetic mutants lacking ATP synthase or in conditions that support substrate-level phosphorylation. Further, we showed that MccM elevated the levels of reactive oxygen species within the target cell. We propose that MccM effects its bacteriostatic activity by decreasing the total energy level of the cell through inhibition of oxidative phosphorylation. Lastly, using genome mining, we bioinformatically identified 171 novel putative class II microcins. Our investigation sheds light on the natural processes involved in microbial competition and provides inspiration, in the form of new molecules, for future therapeutic endeavors.

Lonidamine Increases the Cytotoxic Effect of 1-[(4-Amino-2-methyl-5-pyrimidinyl)methyl]-3-(2-chloroethyl)-3-nitrosourea via Energy Inhibition, Disrupting Redox Homeostasis, and Downregulating MGMT Expression in Human Lung Cancer Cell Line.

Lung cancer ranks as the second most diagnosed cancer and the leading cause of cancer-related deaths worldwide. Novel chemotherapeutic strategies are crucial to efficiently target tumor cells while minimizing toxicity to normal cells. In this study, we proposed a combination strategy using energy blocker lonidamine (LND) and cytotoxic drug nimustine (ACNU, 1-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-3-(2-chloroethyl)-3-nitrosourea) to enhance the killing of a human lung cancer cell line and investigated the potential chemo-sensitizing mechanism of LND. LND was found to remarkably increase the cytotoxicity of ACNU to A549 and H1299 cells without significantly affecting normal lung BEAS2B cells. The combination of LND and ACNU also produced significant effects on cell apoptosis, colony formation, cell migration, and invasion assays compared to single drug treatment. Mechanistically, LND decreased intracellular ATP levels by inhibiting glycolysis and inducing mitochondrial dysfunction. Furthermore, the combination of LND and ACNU could intensify cellular oxidative stress, decrease cellular GSH contents, and increase reactive oxygen species (ROS) production. Notably, LND alone dramatically downregulated the expression of DNA repair protein MGMT (O6-methylguanine-DNA methyltransferase), enhancing DNA interstrand cross-link formation induced by ACNU. Overall, LND represents a potential chemo-sensitizer to enhance ACNU therapy through energy inhibition, disrupting redox homeostasis and downregulating MGMT expression in human lung cancer cell line under preclinical and clinical background.

Three-dimensional culture of human proximal tubular epithelial cells for an in vitro evaluation of drug-induced kidney injury

Drug-induced kidney injury (DIKI) is the major cause of acute kidney injury (AKI). Renal proximal tubular epithelial cells (RPTECs) are the primary target sites of DIKI and express transporters involved in renal drug disposition. In the present study, we focused on three-dimensionally cultured human RPTECs (3D-RPTECs) with elevated expression of drug transporters to investigate their utility in DIKI evaluation. Intracellular ATP levels in 3D-RPTECs are reduced by tenofovir and cisplatin that are substrates of an organic anion transporter 1 and an organic cation transporter 2, respectively. In addition, 3D-RPTECs were exposed to 17 and 15 drugs that are positive and negative to RPTEC toxicity, respectively, for up to 28 d. The 20 % decreasing concentration of drugs for ATP amount (EC20) was obtained, and the ratio of EC20 values and clinical maximum concentration (Cmax) ≤100 were used as cut-off value to evaluate potential of DIKI. The sensitivities of 3D-RPTECs were 82.4 % and 88.2 % after 7 d and 28 d of drug exposure, respectively, and the specificities were 100 % and 93.3 %, respectively. The predictive performance of 3D-RPTECs was higher than that of two-dimensional cultured RPTECs and the kidney cell line HK-2. In conclusion, 3D-RPTECs are useful for in vitro evaluation of RPTEC injury by measuring intracellular ATP levels.

Ethylene oxide suppresses boar sperm function during capacitation

Ethylene oxide (E.O) is an epoxide compound, and it has been utilized as a sterilizer or production of ether compounds in several industries. Although the toxic effects of E.O on bacteria and mammals have been reported, its effects on male reproductive toxicity during sperm capacitation are not fully understood. Therefore, this study was designed to evaluate the effects of E.O exposure during sperm capacitation. Boar spermatozoa were treated with various E.O concentrations (0, 0.1, 1, 10, and 100 μМ). After exposure, sperm motility, motion kinematics, capacitation status, intracellular ATP levels, cell viability, expression levels of protein kinase A (PKA) activation, and tyrosine phosphorylation were evaluated. Results revealed that E.O exposure significantly decreased sperm motility, motion kinematics, and intracellular ATP levels but significantly increased the capacitated spermatozoa. In addition, the PKA activation and tyrosine phosphorylation were abnormally changed. According to our results, E.O may cause toxic effects on sperm function during capacitation, which induces male reproductive toxicity. Consequently, we suggest that male reproductive toxicity should be considered when using E.O.

A promising metabolite, 9-aminominocycline, restores the sensitivity of tigecycline against tet(X4)-positive Escherichia coli.

The emergence and widespread of tigecycline resistance undoubtedly poses a serious threat to public health globally. The exploration of combination therapies has become preferred antibacterial strategies to alleviate this global burden. In this study, tigecycline-resistant tet(X4)-positive Escherichia coli were selected for adjuvant screening. Interestingly, 9-aminominocycline (9-AMC), one of the tigecycline metabolites, exhibits synergistic antibacterial activity with tigecycline using checkerboard assay. The efficacy in vitro and in vivo was evaluated, and the synergistic mechanism was further explored. The results suggested that 9-AMC combined with tigecycline could inhibit the growth of antibiotic resistant bacteria, efficiently retard the evolution of tet(X4) gene and narrow the drug mutant selection window. In addition, the combination of tigecycline and 9-AMC could destroy the normal membrane structure of bacteria, inhibit the formation of biofilm, remarkably reduce the level of intracellular ATP level, and accelerate the oxidative damage of bacteria. Furthermore, 9-AMC is more stable in the bind of Tet(X4) inactivating enzyme. The transcriptomics analysis revealed that the genes related to the 9-AMC and tigecycline were mainly enriched in ABC transporters. Collectively, the results reveal the potentiation effects on tigecycline and the probability of 9-AMC as a novel tigecycline adjuvant against tet(X4)-positive Escherichia coli, which provides new insights for adjuvant screening.

N-acetylglucosamine kinase (BbHxk1) has pleiotropic effects on vegetative growth, cell wall integrity, morphological transition, cuticle infection, and metabolic modulation in the biological pesticide Beauveria bassiana

Beauveria bassiana is a popular and eco-friendly biopesticide. During its pathogen-pest interaction, both N-acetylglucosamine (GlcNAc) catabolism and anabolism are crucial for nutrient supply and cell-wall construction. The initiation of GlcNAc metabolism relies on the catalysis of GlcNAc kinase, which has been extensively studied in the human pathogen Candida albicans. However, the physiological function of GlcNAc kinase remains poorly understood in entomopathogenic fungi. In the present study, a GlcNAc kinase homolog was identified and designated as BbHxk1 in B. bassiana. Deletion of BbHxk1 resulted in viable but reduced vegetative growth on various carbon sources. ΔBbHxk1 mutants displayed severe defects in cell wall integrity, making them more susceptible to cell wall stress cues. Furthermore, the absence of BbHxk1 resulted in an increase in conidial yield and blastospore production, and a faster rate of germination and filamentation, potentially attributed to higher intracellular ATP levels. BbHxk1 deficiency led to a reduction in the activities of cuticle-degrading enzymes, which might contribute to the attenuated pathogenicity specifically through cuticle penetration rather than hemocoel infection towards Galleria mellonella larvae. Being different from C. albicans Hxk1, which facultatively acts as a catalyzing enzyme and transcriptional regulator, BbHxk1 primarily acts as a catalyzing enzyme and metabolic regulator. The altered metabolomic profiling correlated with the phenotypic defects in ΔBbHxk1 mutants, further implicating a potential metabolism-dependent mechanism of BbHxk1 in mediating physiologies of B. bassiana. These findings not only unveil a novel role for GlcNAc kinase in B. bassiana, but also provide a solid theoretical basis to guide metabolic reprogramming in order to maintain or even enhance the efficiency of fungi for practical applications.

One-step green synthesis of platinum mesoporous nanoparticles by riboflavin for light activated antitumoral therapy

Photodynamic therapy (PDT) has been established as one of the most promising novel cancer therapies with fewer side-effects and enhanced efficacy compared to the currently available conventional treatments. However, its application has been hindered by the limitations that photosensitizers (PS) have. The combination of PS with metallic nanoparticles like platinum nanoparticles (PtNPs), can help to overcome these intrinsic drawbacks. In this work, the combination of PtNPs and the natural photosensitizer riboflavin (RF) is proposed. PtNPs are synthesized using RF (Pt@RF) as reducing and stabilizing agent in a one-step method, obtaining nanoparticles with mesoporous structure for UV triggered PDT. In view of possible future UV irradiation treatments, the degradation products of RF, ribitol (RB) and lumichrome (LC), this last being a photosensitizing byproduct, are also employed for the synthesis of porous PtNPs, obtaining Pt@LC and Pt@RB. When administered in vitro to lung cancer cells, all the samples elicit a strong decrease of cell viability and a decrease of intracellular ATP levels. The antitumoral effect of both Pt@RF and Pt@LC is triggered by UV-A irradiation. This antitumoral activity is caused by the induction of oxidative stress, shown in our study by the decrease in intracellular glutathione and increased expression of antioxidant enzymes.

DNA-controlled protein fluorescence: Design of aptamer-split peptide hetero-modulator for GFP to respond to intracellular ATP levels.

Enabling the precise control of protein functions with artificially programmed reaction patterns is beneficial for investigating biological processes. Although several strategies have been established that employ the programmability of nucleic acid, they have been limited to DNA hybridization without external stimuli or target binding. Here, we report an approach for the DNA-mediated control of the tripartite split-GFP assembly via aptamers with responsiveness to intracellular small molecules as stimuli. We designed a novel structure-switching aptamer-peptide conjugate as a hetero modulator for splitGFP in response to ATP. By conjugating two peptides (S10/11) derived from the tripartite split-GFP to ATP aptamer, we achieved GFP reassembly using only ATP as a trigger molecule. The response to ATP at≥4 mM concentrations indicated that it can be applied to respond to intracellular ATP in live cells. Furthermore, our hetero-modulator exhibited high and long-term stability, with a half-life of approximately four days in a serum stability assay, demonstrating resistance to nuclease degradation. We validated that our aptamer-modulator splitGFP was successfully reconstituted in the cell in response to intracellular ATP levels. Our aptamer-modulated splitGFP platform can be utilized to monitor a wide range of intracellular metabolites by replacing the aptamer sequence.

Treatment of Rheumatoid Arthritis Based on the Inherent Bioactivity of Black Phosphorus Nanosheets.

Rheumatoid arthritis (RA) is an autoimmune disease that affects the living quality of patients, especially the elderly population. RA-related morbidity and mortality increase significantly with age, while current clinical drugs for RA are far from satisfactory and may have serious side effects. Therefore, the development of new drugs with higher biosafety and efficacy is demanding. Black phosphorus nanosheets (BPNSs) have been widely studied because of their excellent biocompatibility. Here, we focus on the inherent bioactivity of BPNSs, report the potential of BPNSs as a therapeutic drug for RA and elucidate the underlying therapeutic mechanism. We find that BPNSs inhibit autophagy at an early stage via the AMPK-mTOR pathway, switch the energy metabolic pathway to oxidative phosphorylation, increase intracellular ATP levels, suppress apoptosis, reduce inflammation and oxidative stress, and down-regulate senescence-associated secretory phenotype (SASP)-related genes in rheumatoid arthritis synovial fibroblasts (RA-SFs). Further, BPNSs induce the apoptosis of macrophages and promote their transition from the M1 to the M2 phenotype by regulating related cytokines. Significantly, the administration of BPNSs can alleviate key pathological features of RA in mice, revealing great therapeutic potential. This study provides a novel option for treating RA, with BPNSs emerging as a promising therapeutic candidate.