Efflux Protein Research Articles

DDDR-33. TNG908, A BRAIN-PENETRANT MTA-COOPERATIVE PRMT5 INHIBITOR, IS EFFICACIOUS IN PRECLINICAL MTAP-DELETED MODELS INCLUDING GLIOBLASTOMA

Abstract Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor in adults. The median overall survival (OS) of GBM patients is poor (1-2 years) with standard of care therapies, demonstrating the significant need for the development of novel therapeutics. TNG908 is a clinical stage MTA-cooperative PRMT5 inhibitor that is selectively active in MTAP-deleted cells by leveraging a synthetic lethal interaction. Approximately 40% of GBM tumors have MTAP loss due to a co-deletion event with the proximal tumor suppressor gene, CDKN2A. In preclinical in vitro studies, TNG908 was 15-fold more potent in MTAP-deleted cancer cell lines than in MTAP WT cells. TNG908 has high passive permeability and is neither a substrate for P-glycoprotein nor Breast Cancer Resistant Protein (BCRP) efflux transporters. Consistent with these favorable attributes, TNG908 demonstrated in vivo brain penetration in multiple preclinical models, including non-human primates and mice. TNG908 on-target pharmacodynamic activity was determined by dose-dependent decreases in SDMA-modified protein levels in a GBM subcutaneous xenograft model. TNG908 demonstrated dose-dependent antitumor activity in multiple hyper- and hypomethylated GBM subcutaneous models, including cell lines and patient-derived xenograft models. Despite 6-fold reduced Kpuu in rodents (Kpuu ~0.15) relative to non-human primates (Kpuu 0.9), oral administration of TNG908 drove near tumor stasis and increased median survival by 3-fold in a highly aggressive murine GBM orthotopic model. In summary, TNG908 is a potent, brain-penetrant, MTA-cooperative PRMT5 inhibitor that drives strong antitumor activity in preclinical models of MTAP-deleted GBM. TNG908 is currently enrolling patients with MTAP-deleted tumors including glioblastoma in a Phase I/II clinical trial (NCT05275478).

Empowering the Battle: Bioenhancers as Allies Against Cancer Drug Resistance.

Drug resistance has been a great hindrance in the path of counteracting diseases like cancer and is driven by drugs misuse and overuse. In terms of cancer, resistance has been developed due to cellular changes, altered growth activation pathways, increased expression of efflux proteins, and changes in the local physiology of cancer (blood supply, tissue hydrodynamics, increased mutation rate/epigenetics, tumor cell heterogeneity). One of the approaches to address these challenges is the use of bioenhancers, which can overcome drug resistance, thereby improving bioavailability (BA). Bioenhancers when combined with drugs can elicit pharmacological activity. They are generally combined with therapeutic agents at low doses, which increase the BA or therapeutic activity of active pharmaceutical ingredient (API). This review sheds light on the synthesis and classification of bio-enhancers. It also discusses different applications of bio-enhancers like piperine, ginger, quercetin, curcumin, etc. in the treatment of cancer. The review also presents some of the recent advancements in terms of nanocarriers for delivering API combined with bioenhancers.

Identification and functional analysis of circulating small extracellular vesicle lncRNA signatures in children with fulminant myocarditis.

Fulminant myocarditis (FM) is the most serious type of myocarditis. However, the molecular mechanism underlying the pathogenesis of FM has not been fully elucidated. Small extracellular vesicles (sEVs) play important roles in many diseases, but any potential role in paediatric FM has not been reported. Here, the differential signatures of lncRNAs in plasma sEVs were studied in FM children and healthy children using transcriptome sequencing followed by functional analysis. Then immune-related lncRNAs were screened to study their role in immune mechanisms, the levels and clinical relevance of core immune-related lncRNAs were verified by qRT-PCR in a large sample size. Sixty-eight lncRNAs had increased levels of plasma sEVs in children with FM and 11 had decreased levels. Functional analysis showed that the sEVs-lncRNAs with different levels were mainly related to immunity, apoptosis and protein efflux. Seventeen core immune-related sEVs-lncRNAs were screened, functional enrichment analysis showed that these lncRNAs were closely related to immune activation, immune cell migration and cytokine pathway signal transduction. The results of the study show that sEVs-lncRNAs may play an important role in the pathogenesis of fulminant myocarditis in children, especially in the mechanism of immune regulation.

Trimethylamine N-oxide promotes oxidative stress and lipid accumulation in macrophage foam cells via the Nrf2/ABCA1 pathway.

Recently, trimethylamine N-oxide (TMAO) has been considered a risk factor for cardiovascular disease and has a proatherogenic effect. Many studies have found that TMAO is involved in plaque oxidative stress and lipid metabolism, but the specific mechanism is still unclear. In our study, meta-analysis and bioinformatic analysis were firstly conducted in the database, and found that the effect of high plasma TMAO levels on promoting atherosclerotic plaque may be related to the expression of key antioxidant genes nuclear factor erytheroid-derived-2-like 2 (NFE2L2/Nrf2) decreased. Next, we assessed the role of Nrf2-mediated signaling pathway in TMAO-treated foam cells. Our results showed that TMAO can inhibit the expression of Nrf2 and its downstream antioxidant response element such as heme oxygenase-1 (HO-1) and glutathione peroxidase4 (GPX4), resulting in increased production of reactive oxygen species and decreased activity of superoxide dismutase, promoting oxidative stress. And TMAO can also promote lipid accumulation in foam cells by inhibiting cholesterol efflux protein expression. In addition, upregulation of Nrf2 expression partially rescues TMAO-induced oxidative stress and reduces ATP-binding cassette A1 (ABCA1)-mediated lipid accumulation. Therefore, TMAO promotes oxidative stress and lipid accumulation in macrophage foam cells through the Nrf2/ABCA1 pathway, which may provide a potential mechanism for the proatherogenic effect of TMAO.

Role of mitochondrial farnesyltransferase gene in the prevention of the food spoilage fungi Aspergillus flavus by the antimicrobial natural preservative perillaldehyde

Aspergillus flavus, one of the most widely distributed and abundant genus of Aspergillus worldwide, poses an evident threat as a source of food contamination in grains and cereals. Perillaldehyde (PAE), a volatile essential oil extracted from the stem and leaves of Perilla frutescens L., exhibits potent antifungal activity. In our study, we investigated the role of Cox10, a key enzyme in the heme A synthesis pathway crucial for maintaining mitochondrial function. We found that cox10 is a sensitive gene of A. flavus in response to PAE by gene expression assay and GFP fluorescent localization tagging, and then we found that the deletion of this gene affects the growth and development of A. flavus, but the drug resistance is elevated. Through transcriptome sequencing and its experimental validation, the molecular mechanisms of stress triggered by the deletion of cox10 were further clarified, such as the decrease in intracellular drug content due to the increase in the expression of drug efflux proteins, and the increase in the thickness of cell wall due to the increase in the content of cell wall chitin. Clearly, cox10 plays a critical role in regulating various cellular processes of A. flavus, including growth, reproduction, development, as well as pathogenicity and drug resistance. These significant findings establish a solid theoretical foundation for the development of environmentally friendly, safe, and effective antifungal agents to combat A. flavus contamination.

First Report of Plasmid-Mediated Macrolide-Clindamycin-Tetracycline Resistance in a High Virulent Isolate of Cutibacterium acnes ST115.

Cutibacterium acnes, a prevalent skin commensal, has emerged as a significant global challenge due to its widespread antibiotic resistance. To investigate the antibiotic resistance mechanisms and clinical characterization of C. acnes in Korea, we collected 22 clinical isolates from diverse patient specimens obtained from the National Culture Collection for Pathogens across Korea. Among the isolates, KB112 isolate was subjected to whole genome sequencing due to high resistance against clindamycin, erythromycin, tetracycline, doxycycline, and minocycline. The whole genome analysis of KB112 isolate revealed a circular chromosome of 2,534,481 base pair with an average G + C content of 60.2% with sequence type (ST) 115, harboring the potential virulent CAMP factor pore-forming toxin 2 (CAMP2), the multidrug resistance ABC transporter ATP-binding protein YknY, and the multidrug efflux protein YfmO. The genomic sequence also showed the existence of a plasmid (30,947 bp) containing the erm(50) and tet(W) gene, which confer resistance to macrolide-clindamycin and tetracycline, respectively. This study reports plasmid-mediated multi-drug resistance of C. acnes for the first time in Korea.

Structure Elucidation and Interaction Dynamics of MefA-MsrD Efflux Proteins in Streptococcus pneumoniae: Impact on Macrolide Susceptibility.

Macrolides are empirically used to treat bacterial community-acquired pneumonia (CAP). Streptococcus pneumoniae, being the major pathogen responsible for bacterial CAP with high mortality rates, express MefA-MsrD efflux pumps to hinder macrolide susceptibility. Despite its importance, the structural features of the efflux-protein complex and its impact on macrolide susceptibility have not yet been elucidated explicitly. Therefore, in the present study, combining homology, threading, and dynamics approaches, MefA and MsrD proteins in pathogenic S. pneumoniae were modeled. Both membrane (lipid-bilayer) and cytoplasmic (aqueous) environments were considered to simulate the MefA and MsrD proteins in their ideal cellular conditions followed by dynamics analyses. The simulated MefA structure represented a typical major facilitator superfamily protein structure with 13 transmembrane helices. MefA-MsrD interaction via clustering-based docking revealed low-energy conformers with stable intermolecular interactions. The higher clinical MIC value of azithromycin over erythromycin was reflected upon erythromycin eliciting stronger interactions (dissociation constant or ki = ∼52 μM) with the cytoplasmic ATP-binding MsrD than azithromycin (ki = ∼112 μM). The strong (binding energy = -132.1 ± 9.5 kcal/mol) and highly stable (root-mean-square fluctuation < 1.0 Å) physical association between MefA with MsrD was validated and was found to be unaffected by the antibiotic binding. Higher propensity of the macrolides to interact with MsrD than MefA established the importance of the former in macrolide susceptibility. Ours is probably the first report on the structural arrangements in the MefA-MsrD efflux complex and the macrolide susceptibility in S. pneumoniae. This study provides a novel lead for experimental explorations and efflux-pump inhibitor designs.

THU490 Role Of Cholesterol Efflux Pump ABCA1 In Macrophages And Subsequent Modulation Of The Tumor Microenvironment

Abstract Disclosure: S.V. Bendre: None. N. Krawczynska: None. S. Bhogale: None. A. Das Gupta: None. A. Nelczyk: None. H. Vidana Gamage: None. S. Han: None. E. Tajkhorshid: None. S. Sinha: None. W. Cho: None. E.R. Nelson: None. Breast cancer constitutes about 30% of all new female cancers each year. Although there has been an overall decrease in death rates, mortalities associated with recurrent metastatic cancer remain high. Hence, development of targeted therapeutics against metastatic breast cancer is of utmost importance. Although breast cancer is generally considered immunologically “cold”, clinical studies have shown that immunotherapy has potential to improve patient outcomes. One of the challenges of immunotherapy is overcoming the highly immune suppressive tumor microenvironment. Myeloid cells, such as macrophages, which are abundant within the tumor micro-environment, play a key role in cancer cell survival, progression, and metastasis. Several aspects of cholesterol metabolism and regulation have been implicated in breast cancer progression. ABCA1 (ATP Binding Cassette subfamily A member 1) is a membrane bound cholesterol efflux protein, which facilitates cholesterol transport across, as well as between, membrane leaflets. Although implicated in macrophage biology, little is known about how ABCA1 impacts macrophage function in terms of tumor biology. Therefore, the goal of the current work is to determine the effect of altering ABCA1 within macrophages and its subsequent effect on the tumor microenvironment. Intriguingly, our analysis of publicly available transcriptomes indicates that ABCA1 expression within breast tumors is associated with good prognosis, supporting a role for ABCA1 in tumor progression. Important aspects of macrophage function are efferocytosis, antigen presentation, interaction with and activation of T cells, and promotion of angiogenesis. Therefore, we investigated each of these aspects under conditions where ABCA1 expression was manipulated. Our results indicate that knocking down ABCA1 in macrophages decreased the activation and expansion of T cells. Those T cells that were activated, had a decreased anti-cancer cytotoxic response. Over expression of ABCA1 had the opposite effects, supporting our conclusion that ABCA1 in macrophages is critical for normal T cell activation and function. Furthermore, we are finding that ABCA1 is involved in several other aspects of macrophage biology with relevance to tumor progression. Specifically, knockdown of ABCA1 increased the efficiency of efferocytosis, increased angiogenesis, and decreased macrophage infiltration into breast cancer organoids. All these activities suggest that the loss of ABCA1 shifts macrophages into a functional state that is immune-suppressive and pro-tumorigenic. In summary, our results to date indicate that ABCA1 expression within macrophages reduces their pro-tumor functions. Therefore, it will be important to explore this biology in the hopes of developing ABCA1 as a therapeutic target to shape the tumor microenvironment into being less permissive to tumor growth. Presentation: Thursday, June 15, 2023

Valencene, Nootkatone and Their Liposomal Nanoformulations as Potential Inhibitors of NorA, Tet(K), MsrA, and MepA Efflux Pumps in Staphylococcus aureus Strains.

Valencene and nootkatone are aromatic sesquiterpenes with known biological activities, such as antimicrobial, antioxidant, anti-inflammatory, and antitumor. Given the evidence that encapsulation into nanosystems, such as liposomes, could improve the properties of several compounds, the present study aimed to evaluate the activity of these sesquiterpenes in their isolated state or in liposomal formulations against strains of Staphylococcus aureus carrying efflux pumps. The broth microdilution method evaluated the antibiotic-enhancing activity associated with antibiotics and ethidium bromide (EtBr). The minimum inhibitory concentration was assessed in strains of S. aureus 1199B, IS-58, and RN4220, which carry the efflux proteins NorA, Tet(K), and MsrA. In tests with strain 1199B, valencene reduced the MIC of norfloxacin and EtBr by 50%, while the liposomal formulation of this compound did not show a significant effect. Regarding the strain IS-58, valencene, and its nanoformulation reduced norfloxacin MIC by 60.3% and 50%, respectively. In the non-liposomal form, the sesquiterpene reduced the MIC of EtBr by 90%. Against the RN4220 strain, valencene reduced the MIC of the antibiotic and EtBr by 99% and 93.7%, respectively. Nootkatone and its nanoformulation showed significant activity against the 1199B strain, reducing the EtBr MIC by 21.9%. Against the IS-58 strain, isolated nootkatone reduced the EtBr MIC by 20%. The results indicate that valencene and nootkatone potentiate the action of antibiotics and efflux inhibitors in strains carrying NorA, Tet(K), and MsrA proteins, which suggests that these sesquiterpenes act as efflux pump inhibitors in S. aureus. Therefore, further studies are needed to assess the impact of incorporation into liposomes on the activity of these compounds in vivo.

Development of Novel 11C-Labeled Selective Orexin-2 Receptor Radioligands for Positron Emission Tomography Imaging.

Orexin 2 receptors (OX2R) represent a vital subtype of orexin receptors intricately involved in the regulation of wakefulness, arousal, and sleep-wake cycles. Despite their importance, there are currently no positron emission tomography (PET) tracers available for imaging the OX2R in vivo. Herein, we report [11C]1 ([11C]OX2-2201) and [11C]2 ([11C]OX2-2202) as novel PET ligands. Both compounds 1 (Ki = 3.6 nM) and 2 (Ki = 2.2 nM) have excellent binding affinity activities toward OX2R and target selectivity (OX2/OX1 > 600 folds). In vitro autoradiography in the rat brain suggested good to excellent in vitro binding specificity for [11C]1 and [11C]2. PET imaging in rat brains indicated that the low brain uptake of [11C]2 may be due to P-glycoprotein and/or breast cancer resistance protein efflux interaction and/or low passive permeability. Continuous effort in medicinal chemistry optimization is necessary to improve the brain permeability of this scaffold.

Investigation of the Effect of Lasmiditan on the Pharmacokinetics of P-Glycoprotein and Breast Cancer Resistance Protein Substrates.

Lasmiditan is an invitro inhibitor of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) efflux transporters. We aimed to confirm predictions from physiologically based pharmacokinetic models of lasmiditan, and assess the safety and tolerability of rosuvastatin and dabigatran co-administered with lasmiditan. In this open-label, post-marketing drug-drug interaction, phase1 clinical trial, eligible participants were adults aged 21-70years with a body mass index of 18.5-35.0kg/m2 . Part1 (P-gp, 150mg dabigatran etexilate with 200mg lasmiditan) and part2 (BCRP, 10mg rosuvastatin with 200mg lasmiditan) employed similar designs: a single dose of probe substrate administered on day-2 with pharmacokinetic evaluation; 1-week washout; lasmiditan administered on days8 and 9 alone; lasmiditan co-administered with a single dose of probe substrate on day10, with pharmacokinetic evaluation of probe substrate and lasmiditan. Sixty-six participants were included in part1 and 30 participants were included in part2. Following dabigatran co-administration with lasmiditan, versus dabigatran alone, 90% confidence intervals for geometric least-squares (LS) mean ratios of area under the plasma concentration-time curve from time0 extrapolated to infinity (AUC0-∞ ) and maximum observed drug concentration (Cmax ) were not contained within the non-effect boundaries (0.80 to 1.25). Dabigatran AUC0-∞ increased by 25% and Cmax increased by 22%. The median time of maximum observed drug concentration (tmax ) for dabigatran was 2.0 to 3.0hours. Following rosuvastatin co-administration with lasmiditan, versus rosuvastatin alone, 90%CIs for geometric LS mean ratios of AUC0-∞ and Cmax were contained within non-effect boundaries (0.80-1.25). Rosuvastatin AUC0-∞ increased by 15% and Cmax increased by 7%. The median tmax for rosuvastatin was 4.0hours. Results suggest that lasmiditan has a weak effect on P-gp substrates and no clinically relevant effect on BCRP substrates.

Cryo-Electron Microscopy Structures of a Campylobacter Multidrug Efflux Pump Reveal a Novel Mechanism of Drug Recognition and Resistance.

Campylobacter jejuni is a bacterium that is commonly present in the intestinal tracts of animals. It is also a major foodborne pathogen that causes gastroenteritis in humans. The most predominant and clinically important multidrug efflux system in C. jejuni is the CmeABC (Campylobacter multidrug efflux) pump, a tripartite system that includes an inner membrane transporter (CmeB), a periplasmic fusion protein (CmeA), and an outer membrane channel protein (CmeC). This efflux protein machinery mediates resistance to a number of structurally diverse antimicrobial agents. A recently identified CmeB variant, termed resistance enhancing CmeB (RE-CmeB), can increase its multidrug efflux pump activity, likely by influencing antimicrobial recognition and extrusion. Here, we report structures of RE-CmeB in its apo form as well as in the presence of four different drugs by using single-particle cryo-electron microscopy (cryo-EM). Coupled with mutagenesis and functional studies, this structural information allows us to identify critical amino acids that are important for drug resistance. We also report that RE-CmeB utilizes a somewhat unique subset of residues to bind different drugs, thereby optimizing its ability to accommodate different compounds with distinct scaffolds. These findings provide insights into the structure-function relationship of this newly emerged antibiotic efflux transporter variant in Campylobacter. IMPORTANCE Campylobacter jejuni has emerged as one of the most problematic and highly antibiotic-resistant pathogens, worldwide. The Centers for Disease Control and Prevention have designated antibiotic-resistant C. jejuni as a serious antibiotic resistance threat in the United States. We recently identified a C. jejuni resistance enhancing CmeB (RE-CmeB) variant that can increase its multidrug efflux pump activity and confers an exceedingly high-level of resistance to fluoroquinolones. Here, we report the cryo-EM structures of this prevalent and clinically important C. jejuni RE-CmeB multidrug efflux pump in both the absence and presence of four antibiotics. These structures allow us to understand the action mechanism for multidrug recognition in this pump. Our studies will ultimately inform an era in structure-guided drug design to combat multidrug resistance in these Gram-negative pathogens.

Dielectric and dynamic antibacterial investigations of organic–inorganic conductive membranes based on oxidized cellulose with BNKT nanoceramics

The development of eco-friendly materials for advanced applications is highly demanded. The current study focuses on the preparation of conductive membranes based on tricarboxylic cellulose (TCC) loaded with cubic bismuth sodium titanate (BNKT) nanoceramics. FTIR, SEM, and EDX analyses confirm the presence of loaded BNKT on the membranes. The electrical response of the cellulose/xBNKT (x = 5, 10, 15, and 20% wt/wt) membrane is investigated using impedance spectroscopy. The real part (Z′) and the imaginary part (Z″) of the complex impedance are studied as a function of frequency (4Hz ~ 8MHz) and temperature (20 ~ 160 °C) for the different compositions. Impedance and modulus studies reveal a Debye-type relaxation phenomenon. The dielectric studies manifest promising dielectric properties. The bactericidal performance of all nanomembranes is evaluated. The nanomembrane with 20% BNKT (C20) exhibits bactericidal activity against Gram-negative and Gram-positive bacteria, with 6 log CFU reductions observed after an exposure time of 180 min. Treatment with the C20 nanomembrane shows the highest amounts of protein efflux. The results indicate that the C20 nanomembrane layer eradicated all bacterial cells. The findings suggest that the C20 nanomembrane is recommended as an intelligent and innovative antibacterial nano-system for bio-applications.

Impact of dietary plant flavonoids on 7,8-dihydroxyflavone transepithelial transport in human intestinal Caco-2 cells.

7,8-dihydroxyflavone (7,8-DHF) is a biologically active flavone with various physiological activities, including neuroprotection, anti-inflammation, and weight loss. Previous studies have found that the efflux protein P-glycoprotein (P-gp) significantly affects the transepithelial transport of 7,8-DHF in the intestine, resulting in its low oral bioavailability. Based on this, in this study, a Caco-2 monolayer cell model was used to investigate 14 dietary plant flavonoids as potential P-gp inhibitors, and their effects on the transepithelial transport and in vitro digestion of 7,8-DHF were explored. The results showed that among the 14 plant flavonoids, hesperetin, epigallocatechin gallate, fisetin, kaempferol, quercetin, and isoorientin increased and the apparent permeability coefficients (P app) of 7,8-DHF at AP → BL direction and lowered P app value at BL → AP direction to varying degrees, reducing the efflux ratio of 7,8-DHF less than 1.5. In particular, kaempferol and quercetin exhibited the best effect on promoting the transepithelial transport of 7,8-DHF, especially when used at molar concentration ratios of 1:1 and 1:2 with 7,8-DHF. This is beneficial for improving the oral bioavailability of 7,8-DHF. Meanwhile, 7,8-DHF was found to maintain structural stability in simulated saliva, gastric juice, and intestinal juice, and its stability was not affected by the coexistence of quercetin and kaempferol. Overall, this study provided a theoretical basis for seeking natural and safe P-gp inhibitors to improve the oral absorption of natural products.

Environmental concentrations of antibiotics, biocides, and heavy metals fail to induce phenotypic antimicrobial resistance in Escherichia coli

Most anthropogenically affected environments contain mixtures of pollutants from different sources. The impact of these pollutants is usually the combined effect of the individual polluting constituents. However, how these stressors contribute to the development of antimicrobial resistance in environmental microorganisms is poorly understood. Thus, a 30-day exposure experiment to environmental and sub-inhibitory concentrations of oxytetracycline, amoxicillin, zinc, copper, BAC (benzalkonium chloride) 10 and DADMAC (diallyldimethylammonium chloride) 12, was conducted using fully susceptible E. coli ATCC 25922 to ascertain any development of phenotypic or genotypic resistance. Furthermore, wild-type isolates were collected from the same aquatic environment as the stressors, analysed for phenotypic resistance using the disk diffusion method and genotypically through whole genome sequencing. Exposure to the various concentrations and combinations of the stressors did not trigger phenotypic resistance in the experimental bacteria. Furthermore, genotypic analysis of the WGS on the exposed isolates only found the macrolide resistance mdf(A) gene (also present in the control strain) and the disinfectant resistance gene sitABCD. With further analysis for single nucleotide variants (SNV), mutations were detected for 19 genes that encoded for oxidative stress, DNA repair, membrane proteins efflux systems, growth and persister formations except for the robA, a transcription protein subset of the ArcC/XylS family of proteins, which confer multidrug resistance in E. coli. This indicates that exposure to sub-inhibitory concentrations of antibiotics, heavy metals and biocide residues in the aquatic environmental concentrations of the stressors identified in the current study could not induce phenotypic or genotypic resistance but encoded for genes responsible for the development of persistence and tolerance in bacteria, which could be a precursor to the development of resistance in environmental bacteria.

Orchestrated copper-based nanoreactor for remodeling tumor microenvironment to amplify cuproptosis-mediated anti-tumor immunity in colorectal cancer

Owing to the deficiency of infiltrating immune cells and the accumulation of immunosuppressive cells in tumor microenvironment (TME), immunotherapy remains enormous challenges in colorectal cancer (CRC). Here, CRC was found to be sensitive to cuproptosis which could further evoke immune responses and mediate immune resistance. Thus, a cuproptosis-mediated immunotherapy nanoreactor (CCJD-FA) was constructed by encapsulating copper-based shell-coated CaO2 and bromodomain-containing protein 4 inhibitor JQ-1 with DSPE-PEG-FA. Within tumor cells, CCJD-FA was disassembled under GSH environment, and the exposed CaO2 could generate H2O2 under the acidic condition, which reacted with the released Cu2+ via Fenton-like reaction to produce O2 for relieving hypoxia and Cu+ for inducing cuproptosis. Furthermore, CCJD-FA could also inhibit intracellular glycolysis and ATP generation, then blocking the Cu+ efflux protein ATP7B. Meanwhile, the IFN-γ-induced PD-L1 expression could be exactly reduced by the BRD4 suppression and oxygen supply. These effects sensitized cancer cells to cuproptosis, further evoking systemic immune responses and reshaping immunosuppressive TME to inhibit tumor growth.

A bioinformatic analysis of zinc transporters in intestinal Lactobacillaceae.

As the second most abundant transition element and a crucial cofactor for many proteins, zinc is essential for the survival of all living organisms. To maintain required zinc levels and prevent toxic overload, cells and organisms have a collection of metal transport proteins for uptake and efflux of zinc. In bacteria, metal transport proteins are well defined for model organisms and many pathogens, but fewer studies have explored metal transport proteins, including those for zinc, in commensal bacteria from the gut microbiota. The healthy human gut microbiota comprises hundreds of species and among these, bacteria from the Lactobacillaceae family are well documented to have various beneficial effects on health. Furthermore, changes in dietary metal intake, such as for zinc and iron, are frequently correlated with changes in abundance of Lactobacillaceae. Few studies have explored zinc requirements and zinc homeostasis mechanisms in Lactobacillaceae, however. Here we applied a bioinformatics approach to identify and compare predicted zinc uptake and efflux proteins in several Lactobacillaceae genera of intestinal relevance. Few Lactobacillaceae had zinc transporters currently annotated in proteomes retrieved from the UniProt database, but protein sequence-based homology searches revealed that high-affinity ABC transporter genes are likely common, albeit with genus-specific domain features. P-type ATPase transporters are probably also common and some Lactobacillaceae genera code for predicted zinc efflux cation diffusion facilitators. This analysis confirms that Lactobacillaceae harbor genes for various zinc transporter homologs, and provides a foundation for systematic experimental studies to elucidate zinc homeostasis mechanisms in these bacteria.

Endogenous Serotonin Accumulation Coincides with Reorganization of Auxin Efflux Protein (PIN1) and Actin (ACT8) Accompanying Primary Root Growth Inhibition in NaCl-Stress-Induced Etiolated Sunflower (Helianthus annuus; cv. KBSH 44) Seedlings

Endogenous Serotonin Accumulation Coincides with Reorganization of Auxin Efflux Protein (PIN1) and Actin (ACT8) Accompanying Primary Root Growth Inhibition in NaCl-Stress-Induced Etiolated Sunflower (Helianthus annuus; cv. KBSH 44) Seedlings

PNPLA3-I148M Variant Promotes the Progression of Liver Fibrosis by Inducing Mitochondrial Dysfunction.

Patatin-like phospholipase domain-containing 3 (PNPLA3) rs738409 polymorphism (I148M) is strongly associated with non-alcoholic steatohepatitis and advanced fibrosis; however, the underlying mechanisms remain largely unknown. In this study, we investigated the effect of PNPLA3-I148M on the activation of hepatic stellate cell line LX-2 and the progression of liver fibrosis. Immunofluorescence staining and enzyme-linked immunosorbent assay were used to detect lipid accumulation. The expression levels of fibrosis, cholesterol metabolism, and mitochondria-related markers were measured via real-time PCR or western blotting. Electron microscopy was applied to analyze the ultrastructure of the mitochondria. Mitochondrial respiration was measured by a Seahorse XFe96 analyzer. PNPLA3-I148M significantly promoted intracellular free cholesterol aggregation in LX-2 cells by decreasing cholesterol efflux protein (ABCG1) expression; it subsequently induced mitochondrial dysfunction characterized by attenuated ATP production and mitochondrial membrane potential, elevated ROS levels, caused mitochondrial structural damage, altered the oxygen consumption rate, and decreased the expression of mitochondrial-function-related proteins. Our results demonstrated for the first time that PNPLA3-I148M causes mitochondrial dysfunction of LX-2 cells through the accumulation of free cholesterol, thereby promoting the activation of LX-2 cells and the development of liver fibrosis.

Variable resistance to zinc intoxication among Streptococcus agalactiae reveals a novel IS1381 insertion element within the zinc efflux transporter gene czcD.

Streptococcus agalactiae, also known as group B Streptococcus, is an important human and animal pathogen. Zinc (Zn) is an essential trace element for normal bacterial physiology but intoxicates bacteria at high concentrations. Molecular systems for Zn detoxification exist in S. agalactiae, however the degree to which Zn detoxification may vary among different S. agalactiae isolates is not clear. We measured resistance to Zn intoxication in a diverse collection of clinical isolates of S. agalactiae by comparing the growth of the bacteria in defined conditions of Zn stress. We found significant differences in the ability of different S. agalactiae isolates to resist Zn intoxication; some strains such as S. agalactiae 18RS21 were able to survive and grow at 3.8-fold higher levels of Zn stress compared to other reference strains such as BM110 (6.4mM vs 1.68mM Zn as inhibitory, respectively). We performed in silico analysis of the available genomes of the S. agalactiae isolates used in this study to examine the sequence of czcD, which encodes an efflux protein for Zn that supports resistance in S. agalactiae. Interestingly, this revealed the presence of a mobile insertion sequence (IS) element, termed IS1381, in the 5' region of czcD in S. agalactiae strain 834, which was hyper-resistant to Zn intoxication. Interrogating a wider collection of S. agalactiae genomes revealed identical placement of IS1381 in czcD in other isolates from the clonal-complex-19 (CC19) 19 lineage. Collectively, these results show a resistance spectrum among S. agalactiae isolates enables survival in varying degrees of Zn stress, and this phenotypic variability has implications for understanding bacterial survival in metal stress.