The mitochondrial carboxylase PCCA interacts with Listeria monocytogenes phospholipase PlcB to modulate bacterial survival.

Listeria monocytogenes, a prominent foodborne pathogen responsible for zoonotic infections, owes a significant portion of its virulence to the presence of the phospholipase PlcB. In this study, we performed an in-depth examination of the intricate relationship between L. monocytogenes PlcB and host cell mitochondria, unveiling a novel participant in bacterial survival: the mitochondrial carboxylase propionyl-coenzyme A carboxylase (PCCA). Our investigation uncovered previously unexplored levels of interaction and colocalization between PCCA and PlcB within host cells, with particular emphasis on the amino acids 504-508 of PCCA, which play a pivotal role in this partnership. To assess the effect of PCCA expression on L. monocytogenes proliferation, PCCA expression levels were manipulated by siRNA-si-PCCA or pCMV-N-HA-PCCA plasmid transfection. Our findings demonstrated a clear inverse correlation between PCCA expression levels and the proliferation of L. monocytogenes. Furthermore, the effect of L. monocytogenes infection on PCCA expression was investigated by assessing PCCA mRNA and protein expression in HeLa cells infected with L. monocytogenes. These results indicate that L. monocytogenes infection did not significantly alter PCCA expression. These findings led us to propose that PCCA represents a novel participant in L. monocytogenes survival, and its abundance has a detrimental impact on bacterial proliferation. This suggests that L. monocytogenes may employ PlcB-PCCA interactions to maintain stable PCCA expression, representing a unique pro-survival strategy distinct from that of other intracellular bacterial pathogens. IMPORTANCE: Mitochondria represent attractive targets for pathogenic bacteria seeking to modulate host cellular processes to promote their survival and replication. Our current study has uncovered mitochondrial carboxylase propionyl-coenzyme A carboxylase (PCCA) as a novel host cell protein that interacts with L. monocytogenes PlcB. The results demonstrate that PCCA plays a negative regulatory role in L. monocytogenes infection, as heightened PCCA levels are associated with reduced bacterial survival and persistence. However, L. monocytogenes may exploit the PlcB-PCCA interaction to maintain stable PCCA expression and establish a favorable intracellular milieu for bacterial infection. Our findings shed new light on the intricate interplay between bacterial pathogens and host cell mitochondria, while also highlighting the potential of mitochondrial metabolic enzymes as antimicrobial agents.

Hypoxia-induced HIF-1α promotes Listeria monocytogenes invasion into tilapia.

HIF-1α is a nuclear transcription factor, and its activity is tightly regulated by the level of available oxygen in cells. Here, we investigated the roles of HIF-1α in the invasion of Listeria monocytogenes into tilapia under hypoxic environments. We found that the expression levels of HIF-1α in examined tissues of hypoxic tilapia were significantly upregulated, indicating that the tissue cells have been in hypoxic conditions. After 24-h infection with L. monocytogenes, we found that bacterial burden counts increased significantly in all examined tissues of hypoxic fish. To explore why the bacterial count increased significantly in the tissues of hypoxic fish, we modulated HIF-1α expression through RNAi technology. The results indicated that c-Met expression levels were positively related to HIF-1α expression. Since c-Met is the receptor of InlB that plays critical roles in the adhesion and invasion of L. monocytogenes, the ∆InlB strain was used to further explore the reason for the significant increase in bacterial counts in hypoxic fish. As expected, the decrease in the adhesion ability of ∆InlB suggested that InlB mediates L. monocytogenes infection in tilapia. After being infected with ∆InlB strain, we found that the bacterial counts in hypoxic fish were not affected by hypoxic conditions or HIF-1α expression levels. These findings indicate that HIF-1α may promote the internalization of InlB by upregulating c-Met expression and therefore contributes to the invasion of L. monocytogenes into hypoxic tilapia. IMPORTANCE Listeria monocytogenes is a zoonotic food-borne bacterial pathogen with a solid pathogenicity for humans. After ingestion of highly contaminated food, L. monocytogenes is able to cross the intestine invading phagocytic and nonphagocytic cells and causes listeriosis. China is the world's largest supplier of tilapia. The contamination rate of L. monocytogenes to tilapia products was as high as 2.81%, causing a severe threat to public health. This study revealed the underlying regulatory mechanisms of HIF-1α in the invasion of L. monocytogenes into tilapia under hypoxic environments. This study will be helpful for better understanding the molecular mechanisms of hypoxic environments in L. monocytogenes infection to tilapia. More importantly, our data will provide novel insights into the prevention and control of this pathogen in aquaculture.

Comprehensive Analysis of the Oncogenic, Genomic Alteration, and Immunological Landscape of Cation-Chloride Cotransporters in Pan-Cancer.

Background: Assessing the phenotypic diversity underlying tumor progression requires the identification of variations in the respective molecular interaction in the tumor microenvironment (TME). Despite emerging studies focusing on the association between cation-chloride cotransporters (CCCs) and carcinogenesis, direct evidence that CCCs (KCC2 and NKCC1) mediate tumor progression in pan-cancer remains unclear. Methods: We conducted a comprehensive assessment of the expression, DNA variation profiles, and prognostic and immunologic implications of CCCs based on a large-scale pan-cancer population, including 10,967 cancer patients from the Cancer Genome Atlas, 9,162 cancer patients from Genomics Expression Omnibus, 48,834 cancer patients from 188 independent studies, and 356 cancer patients from three real-world cohorts. Results: In this study, we first found that CCCs were highly expressed in most tumors, and prominently associated with prognosis. Kaplan-Meier analysis and Cox regression analysis revealed that KCC2 and NKCC1 significantly predicted survival for patients with pan-cancer, suggesting that CCCs have inconsistent tumorigenesis regulatory mechanisms in cancers. Next, we examined the DNA variation landscape of KCC2 and NKCC1 and their prognostic implications in pan-cancer. The results demonstrated that UCEC patients with somatic copy number variation (CNV) of NKCC1 received significantly better outcomes (p < 0.05). Besides emphasizing the clinical implications of CNV of CCCs for cancer patients, we found that NKCC1MUT could prominently prolong progression-free survival (p = 2.59e-04), disease-specific survival (p = 0.019), and overall survival (p = 0.034) compared with NKCC1WT cancer patients possibly via regulation of cell proliferation and oncogenic stress pathways. Additionally, KCC2 positively correlated with the levels of tumor-infiltrating macrophages and CD4+ T cells, but NKCC1 showed a significantly widely negative association with tumor-infiltrated lymphocytes, suggesting an immune-excluded TME in cancers. Similarly, expression of KCC2, rather than NKCC1, was positively correlated with the immune checkpoint molecules, indicating its role as an immune regulator in a wide variety of cancers. Finally, to verify our hypothesis and altered expression of CCCs, we performed IHC analysis and revealed the staining distribution in tumor and adjacent normal tissues of glioma, clear cell renal cell carcinoma, papillary cell renal cell carcinoma, and hepatocellular and breast cancer from three real-world cohorts, and validated prominently prognostic implications of CCCs in patients with clear cell renal cell carcinoma. Conclusion: This study first comprehensively investigated the molecular and clinical role of CCCs, and illustrated the significant association among KCC2/NKCC1 expression, DNA variation profiles prognosis, and TME of pan-cancer. The pan-cancer findings provided an in-depth understanding of potential oncogenic and immunologic of differential expression and DNA alteration of KCC2/NKCC1 cancers.

Molecular-scale cage-confinement pyrolysis route to size-controlled molybdenum carbide nanoparticles for electrochemical sensor.

Herein, size-controllable molybdenum carbide nanoparticles (Mo2C NPs) encapsulated by N, P-codoped carbon shells which simultaneously wrapping on the surface of carbon nanotube (Mo2C@NPC/CNT) is synthesized through a molecular-scale cage-confinement pyrolysis route. Such confinement achieves a good coating and protection of Mo2C and the effective control over the size of Mo2C NPs ranging from 2.5 to 10 nm facilitates a rational investigation into their electrochemical sensor behavior at nanometer scales. The optimized structure consisting of Mo2C nanoparticles with size of ~5 nm showed an outstanding electrochemical response toward dopamine (DA) and acetaminophen (AC) with detection limits (S/N = 3) of 0.008 µM for AC and 0.01 µM for DA.

Protective effect of Ganoderic acid A on adjuvant-induced arthritis.

The purpose of the experiment was to explore the effect of Ganoderic acid A (GAA) on adjuvant-induced arthritis in rats. In this study, the rat model of collagen-induced rheumatoid arthritis (CIA) was established with type II collagen plus Freund's complete adjuvant. Arthritis index, joint pathology, toe swelling, hemorheology, synovial cell apoptosis, related cytokines and JAK3/STAT3 and nuclear factor-κB (NF-κB) signaling pathway were measured in rats. We found that GAA can significantly inhibit the arthritis index, improve joint pathology, reduce toe swelling, improve blood rheology, improve synovial cell apoptosis, and restore related cytokine negative regulation JAK3/STAT3 and NF-κB signaling pathways. In conclusion, GAA has an obvious therapeutic effect on joint inflammation of toes in CIA model rats, which may be due to the regulation of JAK3/STAT3 and NF-κB signaling pathway.

Confinement of single polyoxometalate clusters in molecular-scale cages for improved flexible solid-state supercapacitors.

Herein, we realized the supramolecular confinement of a single polyoxometalate (POM) cluster precisely in a polypyrrole (PPy) hydrogel-wrapped CNT framework with molecular-scale cages. This hybrid hydrogel framework demonstrated an ultra-high loading (67.5 wt%) and extremely uniform dispersion of individual of H3[P(Mo3O10)4] (PMo12) molecules, as demonstrated by sub-ångström-resolution HAADF-STEM. Consequently, it exhibited a better supercapacitor performance than that of the conventional composite system. The flexible solid-state supercapacitor exhibited a high energy density of 67.5 µW h cm-2 at a power density of 700 µW cm-2 and delivered a high capacitance retention of 85.7% after 3000 cycles. Moreover, the flexible device exhibited excellent mechanical stability. Density functional theory calculations revealed that the wrapped "fishnet-like" hydrogel creates a cage structure with a size of 1.8 nm for the precise storage of the PMo12 molecule (diameter = 1.05 nm), leading to the mono-dispersion of single PMo12 molecules on the hybrid hydrogel. The "caging" effect also activates the PMo12 molecule to enhance its charging/discharging performance by introducing new reactive sites for proton transfer. We believe that this design for suitable cage structures can be used for the construction of other POM-based hybrid hydrogels, thereby achieving mono-dispersity and performance enhancement.

From Genotype to Phenotype: Cytochrome P450 2D6-Mediated Drug Clearance in Humans.

How genotypic variation results in phenotypic differences is still a challenge for biology. In the field of drug metabolism, the means by which specific cytochrome P4502D6 (CYP2D6) genotypes yield different phenotypes at various levels (molecular, cellular, and organismal) is an important question, as differences in CYP2D6 activity can contribute to adverse drug reactions. Herein, the genotype of CYP2D6 was determined along with the absolute content of CYP2D6 and microsomal protein per gram of liver in human liver microsomes, the molecular, cellular (microsomal, tissue, organ), and organismal phenotype of CYP2D6 determined; the effect of genotype on each phenotype of CYP2D6-mediated dextromethorphan clearance (CL) was delineated, and the overall genotype-phenotype relationship for CYP2D6 was charted. We demonstrate that changes in the cellular and organismal CL phenotypes are markedly greater than changes seen at the molecular level. With individuals carrying the 1661CC polymorphism, for example, the most noticeable change took place in organ CL phenotype (4.17-fold), followed by tissue (3.75-fold), organism (3.69-fold), microsomal (3.09-fold), and molecular (1.66-fold) phenotypes. In addition, the biggest intragenotype individual coefficient of variation in organismal phenotype was observed in the 1661GG individuals, which reached 104.5%, followed by that of 100TT, 100CT, 1661GC, 100CC, and 1661CC polymorphisms (102.7%, 62.4%, 53.5%, 49.7%, and 44.8%, respectively). Our study has allowed us to chart the genotype-phenotype relationship for CYP2D6 from the molecular to the organismal level as well as allowed us to determine intragenotype individual variation in phenotype with each genotype.

Correlation of Cytochrome P450 Oxidoreductase Expression with the Expression of 10 Isoforms of Cytochrome P450 in Human Liver.

Human cytochrome P450 oxidoreductase (POR) provides electrons for all microsomal cytochromes P450 (P450s) and plays an indispensable role in drug metabolism catalyzed by this family of enzymes. We evaluated 100 human liver samples and found that POR protein content varied 12.8-fold, from 12.59 to 160.97 pmol/mg, with a median value of 67.99 pmol/mg; POR mRNA expression varied by 26.4-fold. POR activity was less variable with a median value of 56.05 nmol/min per milligram. Cigarette smoking and alcohol consumption clearly influenced POR activity. Liver samples with a 2286822 TT genotype had significantly higher POR mRNA expression than samples with CT genotype. Homozygous carriers of POR2286822C>T, 2286823G>A, and 3823884A>C had significantly lower POR protein levels compared with the corresponding heterozygous carriers. Liver samples from individuals homozygous at 286823G>A, 1135612A>G, and 10954732G>A generally had lower POR activity levels than those from heterozygous or wild-type samples, whereas the common variant POR*28 significantly increased POR activity. There was a strong association between POR and the expression of P450 isoforms at the mRNA and protein level, whereas the relationship at the activity level, as well as the effect of POR protein content on P450 activity, was less pronounced. POR transcription was strongly correlated with both hepatocyte nuclear factor 4 alpha and pregnane X receptor mRNA levels. In conclusion, we have elucidated some potentially important correlations between POR single-nucleotide polymorphisms and POR expression in the Chinese population and have developed a database that correlates POR expression with the expression and activity of 10 P450s important in drug metabolism.

Gene polymorphisms and contents of cytochrome P450s have only limited effects on metabolic activities in human liver microsomes.

Extensive inter-individual variations in pharmacokinetics are considered as a major reason for unpredictable drug responses. As the most important drug metabolic enzymes, inter-individual variations of cytochrome P450 (CYP) activities are not clear in human liver. In this paper, metabolic activities, gene polymorphisms and protein contents of 10 CYPs were determined in 105 human normal liver microsomes. The results indicated substantial inter-individual variations in CYP activities, with the greatest being CYP2C19 activity (>600-fold). Only half of 10 CYP isoforms and 26 gene polymorphism sites had limited effects on metabolic activities, such as CYP2A6, CYP2B6, CYP2C9, CYP2D6 and CYP3A4/5, others had almost no effects. Compared with their respective wild type, Km, Vmax, and CLint decreased by 51.6%, 88.7% and 70.7% in CYP2A6*1/*4 genotype, Vmax and CLint decreased by 32.8% and 60.2% in CYP2C9*1/*3 genotype, Km increased by 118.4% and CLint decreased by 65.2% in CYP2D6 100TT genotype, respectively. Moreover, there were only 4 CYP isoforms, CYP1A2, CYP2A6, CYP2E1 and CYP3A5, which had moderate or weak correlations between Vmax values and corresponding contents. In conclusions, the genotypes and contents of some CYPs have only limited effects on metabolic activities, which imply that there are other more important factors to influence inter-individual variations.

Significant change of cytochrome P450s activities in patients with hepatocellular carcinoma.

The lack of information concerning individual variation in drug-metabolizing enzymes is one of the most important obstacles for designing personalized medicine approaches for hepatocellular carcinoma (HCC) patients. To assess cytochrome P450 (CYP) in the metabolism of endogenous and exogenous molecules in an HCC setting, the activity changes of 10 major CYPs in microsomes from 105 normal and 102 HCC liver tissue samples were investigated. We found that CYP activity values expressed as intrinsic clearance (CLint) differed between HCC patients and control subjects. HCC patient samples showed increased CLint for CYP2C9, CYP2D6, and CYP2E1 compared to controls. Meanwhile, CYP1A2, CYP2C8, and CYP2C19 CLint values decreased and CYP2A6, CYP2B6, and CYP3A4/5 activity was unchanged relative to controls. For patients with HCC accompanied by fibrosis or cirrhosis, the same activity changes were seen for the CYP isoforms, except for CYP2D6 which had higher values in HCC patients with cirrhosis. Moreover, CYP2D6*10 (100C>T), CYP2C9*3 (42614 A>C), and CYP3A5*3 (6986A>G) polymorphisms had definite effects on enzyme activities. In the HCC group, the CLint of CYP2D6*10 mutant homozygote was decreased by 95% compared to wild-type samples, and the frequency of this homozygote was 2.8-fold lower than the controls.In conclusion, the activities of CYP isoforms were differentially affected in HCC patients. Genetic polymorphisms of some CYP enzymes, especially CYP2D6*10, could affect enzyme activity. CYP2D6*10 allelic frequency was significantly different between HCC patients and control subjects. These findings may be useful for personalizing the clinical treatment of HCC patients as well as predicting the risk of hepatocarcinogenesis.

Safflower yellow inhibits angiotensin II-induced adventitial fibroblast proliferation and migration.

Safflower yellow (SY) has been widely used in Chinese medicine for the treatment of ischemic cardiocerebrovascular disease. Recent studies have indicated that SY has a reverse effect on vascular remodeling (VR). However, its detailed mechanisms require further study to provide more scientific evidence for the clinical treatment of VR. This study aims to investigate the effects of SY on angiotensin II (Ang II)-induced cell proliferation, migration, apoptosis, and extracellular matrix in rat aortic adventitial fibroblasts (AFs). The proliferation and migration rates of AFs treated with Ang II for 24 h were higher than those of untreated AFs; and increases in the expression of p-ERK1/2, AP-1, collagen I, and collagen III were observed. Treatment with SY significantly downregulated cell proliferation, migration, and the expression of p-ERK1/2, AP-1, collagen I, and collagen III. We also found that the cell percentage of apoptosis of AFs treated with Ang II for 24 h was lower than those of untreated AFs. After treatment with SY, the percentage of apoptosis was increased. SY exhibits anti-proliferative, anti-migratory, and pro-apoptotic activities in rat aortic AFs, perhaps through the Ang II/ERK/AP-1 signaling pathway. The present findings may provide new clues regarding the potential function of SY to treat or prevent VR.

Identification and evaluation of apoptotic compounds from Garcinia oligantha.

Four new compounds, oliganthins A-D (1-4), and one known caged xanthone gaudichaudione H (5) were isolated from the stems of Garcinia oligantha. The structures of the new compounds were elucidated by spectroscopic evidences. All of the five compounds were evaluated for their apoptosis-inducing effects using HeLa-C3 cells which have been genetically engineered to produce a fluorescent biosensor capable of detecting caspase-3 activation. All of them induced cell apoptosis at 10 µM or lower concentrations. The apoptotic activity of oliganthins A, B and gaudichaudione H were further confirmed by detecting the cleavage of PARP, which is the substrate of activated caspase-3, in these compounds-treated cells using the method of Western blot. Moreover, the values of IC(50) were measured for all five compounds on HeLa cells using the MTT assay. Among them, gaudichaudione H had the lowest IC(50) value of 0.90 µM, while the other four new compounds had IC(50) values of 1.58, 1.52, 4.15, and 7.82 µM, respectively. These results show that gaudichaudione H has the strongest apoptosis-inducing effect and cell growth inhibition effect among these xanthones and it may have the potential to be developed into a new anticancer agent.