Seismic performance of soft soil foundation with a new type of assembled wall tuned mass damper.

The design of tuned mass damper (TMD) parameters is influenced by the soil-structure-TMD coupling system; thus, it is important to consider the soil-structure interaction (SSI) for the vibration control effect of the TMD. Recently, the acquisition of TMD parameters considering soil-structure interactions has only remained at the theoretical stage, lacking relevant experimental verification. Traditional TMD face the problems of occupying a large building space, increasing construction costs, and non-replaceable components. In this study, an assembled wall-type damping TMD was designed. By comparing the dynamic response of the uncontrolled and controlled structures equipped with the newly assembled wall-type damping TMD in the shaking table test on a soft soil foundation, we analyzed whether the SSI effect was considered in the TMD design parameters on the damping effect of the newly assembled wall-type tuned mass damper. The TMD parameters optimized using the artificial intelligence algorithm were verified experimentally. The results indicated that the traditional TMD design parameters were discordant because the SSI effect was not considered. The SSI effect in the soil effectively reduces the dynamic response of the superstructure. By considering the SSI effect and improving the multi-population genetic algorithm, a wall-type damping TMD with optimized parameters can achieve a good damping effect.

Combination of theoretical analysis and experiments: Exploring the role of PLA2G7 in human cancers, including renal cancer.

Background: The pivotal role of phospholipase A2 group VII (PLA2G7) has been identified in specific human cancers, such as prostate cancer, diffuse large B cell lymphoma, and melanoma. Given PLA2G7's significant involvement in established tumors, exploring its role in other cancers is highly relevant. Methods: In this study, we acquired and analyzed data from The Cancer Genome Atlas database, the UCSC XENA website, and other online platforms including Gene Set Cancer Analysis, cBioPortal, Tumor Immune Estimation Resource, and TISIDB to investigate PLA2G7's role in human cancers, including renal cancer. Furthermore, in vitro experiments, including immunofluorescence, western blotting, and CCK-8 assays, were conducted to elucidate PLA2G7's role in renal cancer. Finally, the relationship between PLA2G7 and various drug sensitivity was explored. Results: Our findings demonstrate that PLA2G7 is highly expressed and may serve as a valuable candidate biomarker in pan-cancer. PLA2G7 exhibits distinct alteration frequencies across human cancers and is correlated with tumor mutation burden, tumor microenvironment, DNA stemness score, RNA stemness score, tumorigenesis, tumor immunity, and microsatellite instability in pan-cancer. Immunofluorescence and western blotting revealed a relative high level of PLA2G7 protein in renal cancer cell lines (ACHN and 786-O), predominantly localized in the cytoplasm. Treatment with a PLA2G7 gene inhibitor (darapladib) significantly decreased the viability of ACHN and 786-O cell lines. Additionally, we observed an association between PLA2G7 mRNA levels and various drug sensitivity. Conclusions: Our study suggests that PLA2G7 has the potential to serve as a valuable biomarker and therapeutic target for cancer, particularly in the context of renal cancer.

Transcriptomic Insights into Metabolism-Dependent Biosynthesis of Bacterial Nanocellulose.

Bacterial nanocellulose (BNC) is an attractive green-synthesized biomaterial for biomedical applications and various other applications. However, effective engineering of BNC production has been limited by our poor knowledge of the related metabolic processes. In contrast to the traditional perception that genome critically determines biosynthesis behaviors, here we discover that the glucose metabolism could also drastically affect the BNC synthesis in Gluconacetobacter hansenii. The transcriptomic profiles of two model BNC-producing strains, G. hansenii ATCC 53582 and ATCC 23769, which have highly similar genomes but drastically different BNC yields, were compared. The results show that their BNC synthesis capacities were highly related to metabolic activities such as ATP synthesis, ion transport protein assembly, and carbohydrate metabolic processes, confirming an important role of metabolism-related transcriptomes in governing the BNC yield. Our findings provide insights into the microbial biosynthesis behaviors from a transcriptome perspective, potentially guiding cellular engineering for biomaterial synthesis.

Bilateral Renal Ischemia-Reperfusion Model for Acute Kidney Injury in Mice.

Acute kidney injury (AKI) is defined as a rapid decline in renal function, in which persistent kidney dysfunction gradually progresses to chronic kidney disease (CKD) due to the irreversible loss of nephrons and their maladaptive repair. In recent years, the incidence of AKI has been increasing concerning diverse etiologies, including volume depletion, sepsis, nephrotoxicity, muscle injury, and major trauma, in which ischemia-reperfusion injury (IRI) accounts for most episodes. Development of the IRI model in mice is induced by surgical clamping of the renal pedicles, which provides powerful and controllable tools for preclinical models of AKI. Importantly, the IRI model is deployed at different stages of the AKI development, especially in the processes of AKI to CKD. Despite the IRI model being widely practiced in many laboratories, a series of variables still influence the results of this model. Here, we describe the procedure of IRI model development to provide a repeatable and reliable method for researchers to explore the underlying pathogenesis in the development of AKI and the progression of AKI to CKD.

Extrachromosomal circular MiR-17-92 amplicon promotes HCC.

BACKGROUND AND AIMS: Extrachromosomal circular DNAs (eccDNAs) are prevalent in cancer genomes and emerge as a class of crucial yet less characterized oncogenic drivers. However, the structure, composition, genome-wide frequency, and contribution of eccDNAs in HCC, one of the most fatal and prevalent cancers, remain unexplored. In this study, we provide a comprehensive characterization of eccDNAs in human HCC and demonstrate an oncogenic role of microRNA (miRNA)-17-92-containing eccDNAs in tumor progression. APPROACH AND RESULTS: Using the circle-sequencing method, we identify and characterize more than 230,000 eccDNAs from 4 paired samples of HCC tumor and adjacent nontumor liver tissues. EccDNAs are highly enriched in HCC tumors, preferentially originate from certain chromosomal hotspots, and are correlated with differential gene expression. Particularly, a series of eccDNAs carrying the miRNA-17-92 cluster are validated by outward PCR and Sanger sequencing. Quantitative PCR analyses reveal that miRNA-17-92-containing eccDNAs, along with the expression of their corresponding miRNAs, are elevated in HCC tumors and associated with poor outcomes and the age of HCC patients. More intriguingly, exogenous expression of artificial DNA circles harboring the miR-17-92 cluster, which is synthesized by the ligase-assisted minicircle accumulation method, can significantly accelerate HCC cell proliferation and migration. CONCLUSIONS: These findings delineate the genome-wide eccDNAs profiling of HCC and highlight the functional significance of miRNA-containing eccDNAs in tumorigenesis, providing insight into HCC pathogenesis and cancer therapy, as well as eccDNA and miRNA biology.

Trehalose Alleviates Myocardial Ischemia/Reperfusion Injury by Inhibiting NLRP3-Mediated Pyroptosis.

Myocardial ischemia/reperfusion (I/R) injury is a pathological damage secondary to myocardial ischemia that can further aggravate tissue and organ injuries. Therefore, there is an urgent need to develop an effective approach for alleviating myocardial I/R injury. Trehalose (TRE) is a natural bioactive substance that has been shown to have extensive physiological effects in various animals and plants. However, TRE's protective effects against myocardial I/R injury remain unclear. This study aimed to evaluate the protective effect of TRE pre-treatment in mice with acute myocardial I/R injury and to explore the role of pyroptosis in this process. Mice were pre-treated with trehalose (1 mg/g) or an equivalent amount of saline solution for 7 days. The left anterior descending coronary artery was ligated in mice from the I/R and I/R + TRE groups, followed by 2-h or 24-h reperfusion after 30 min. Transthoracic echocardiography was performed to assess cardiac function in mice. Serum and cardiac tissue samples were obtained to examine the relevant indicators. We established an oxygen-glucose deprivation and re-oxygenation model in neonatal mouse ventricular cardiomyocytes and validated the mechanism by which trehalose affects myocardial necrosis via overexpression or silencing of NLRP3. TRE pre-treatment significantly improved cardiac dysfunction and reduced the infarct size in mice after I/R, accompanied by a decrease in the I/R-induced levels of CK-MB, cTnT, LDH, reactive oxygen species, pro-IL-1ß, pro-IL-18, and TUNEL-positive cells. Furthermore, TRE intervention suppressed the expression of pyroptosis-related proteins following I/R. TRE attenuates myocardial I/R injury in mice by inhibiting NLRP3-mediated caspase-1-dependent pyroptosis in cardiomyocytes.

A large-scale microRNA transcriptome-wide association study identifies two susceptibility microRNAs, miR-1307-5p and miR-192-3p, for colorectal cancer risk.

Transcriptome-wide association studies (TWAS) have identified many putative susceptibility genes for colorectal cancer (CRC) risk. However, susceptibility miRNAs, critical dysregulators of gene expression, remain unexplored. We genotyped DNA samples from 313 CRC East Asian patients and performed small RNA sequencing in their normal colon tissues distant from tumors to build genetic models for predicting miRNA expression. We applied these models and data from genome-wide association studies (GWAS) including 23 942 cases and 217 267 controls of East Asian ancestry to investigate associations of predicted miRNA expression with CRC risk. Perturbation experiments separately by promoting and inhibiting miRNAs expressions and further in vitro assays in both SW480 and HCT116 cells were conducted. At a Bonferroni-corrected threshold of P < 4.5 × 10-4, we identified two putative susceptibility miRNAs, miR-1307-5p and miR-192-3p, located in regions more than 500 kb away from any GWAS-identified risk variants in CRC. We observed that a high predicted expression of miR-1307-5p was associated with increased CRC risk, while a low predicted expression of miR-192-3p was associated with increased CRC risk. Our experimental results further provide strong evidence of their susceptible roles by showing that miR-1307-5p and miR-192-3p play a regulatory role, respectively, in promoting and inhibiting CRC cell proliferation, migration, and invasion, which was consistently observed in both SW480 and HCT116 cells. Our study provides additional insights into the biological mechanisms underlying CRC development.

Large-scale alternative polyadenylation (APA)-wide association studies to identify putative susceptibility genes in human common cancers.

Alternative polyadenylation (APA) modulates mRNA processing in the 3' untranslated regions (3'UTR), which affect mRNA stability and translation efficiency. Here, we build genetic models to predict APA levels in multiple tissues using sequencing data of 1,337 samples from the Genotype-Tissue Expression, and apply these models to assess associations between genetically predicted APA levels and cancer risk with data from large genome-wide association studies of six common cancers, including breast, ovary, prostate, colorectum, lung, and pancreas among European-ancestry populations. At a Bonferroni-corrected P □<□0.05, we identify 58 risk genes, including seven in newly identified loci. Using luciferase reporter assays, we demonstrate that risk alleles of 3'UTR variants, rs324015 ( STAT6 ), rs2280503 ( DIP2B ), rs1128450 ( FBXO38 ) and rs145220637 ( LDAH ), could significantly increase post-transcriptional activities of their target genes compared to reference alleles. Further gene knockdown experiments confirm their oncogenic roles. Our study provides additional insight into the genetic susceptibility of these common cancers.

Nonsterilized Fermentation of Crude Glycerol for Polyhydroxybutyrate Production by Metabolically Engineered Vibrio natriegens.

Polyhydroxybutyrate (PHB) is an attractive biodegradable polymer that can be produced through the microbial fermentation of organic wastes or wastewater. However, its mass production has been restricted by the poor utilization of organic wastes due to the presence of inhibitory substances, slow microbial growth, and high energy input required for feedstock sterilization. Here, Vibrio natriegens, a fast-growing bacterium with a broad substrate spectrum and high tolerance to salt and toxic substances, was genetically engineered to enable efficient PHB production from nonsterilized fermentation of organic wastes. The key genes encoding the PHB biosynthesis pathway of V. natriegens were identified through base editing and overexpressed. The metabolically engineered strain showed 166-fold higher PHB content (34.95 wt %) than the wide type when using glycerol as a substrate. Enhanced PHB production was also achieved when other sugars were used as feedstock. Importantly, it outperformed the engineered Escherichia coli MG1655 in PHB productivity (0.053 g/L/h) and tolerance to toxic substances in crude glycerol, without obvious activity decline under nonsterilized fermentation conditions. Our work demonstrates the great potential of engineered V. natriegens for low-cost PHB bioproduction and lays a foundation for exploiting this strain as a next-generation model chassis microorganism in synthetic biology.

omicsMIC: a Comprehensive Benchmarking Platform for Robust Comparison of Imputation Methods in Mass Spectrometry-based Omics Data.

Mass spectrometry is a powerful and widely used tool for generating proteomics, lipidomics, and metabolomics profiles, which is pivotal for elucidating biological processes and identifying biomarkers. However, missing values in spectrometry-based omics data may pose a critical challenge for the comprehensive identification of biomarkers and elucidation of the biological processes underlying human complex disorders. To alleviate this issue, various imputation methods for mass spectrometry-based omics data have been developed. However, a comprehensive and systematic comparison of these imputation methods is still lacking, and researchers are frequently confronted with a multitude of options without a clear rationale for method selection. To address this pressing need, we developed omicsMIC (mass spectrometry-based omics with Missing values Imputation methods Comparison platform), an interactive platform that provides researchers with a versatile framework to simulate and evaluate the performance of 28 diverse imputation methods. omicsMIC offers a nuanced perspective, acknowledging the inherent heterogeneity in biological data and the unique attributes of each dataset. Our platform empowers researchers to make data-driven decisions in imputation method selection based on real-time visualizations of the outcomes associated with different imputation strategies. The comprehensive benchmarking and versatility of omicsMIC make it a valuable tool for the scientific community engaged in mass spectrometry-based omics research. OmicsMIC is freely available at https://github.com/WQLin8/omicsMIC.

STING promotes ferroptosis through NCOA4-dependent ferritinophagy in acute kidney injury.

Ferroptosis in tubules has been implicated in the pathogenesis of acute kidney injury (AKI), whereas the regulatory mechanism remains unclear. The stimulator of interferon genes (STING) is previously recognized as a critical mediator of innate immunity via a DNA-sensing pathway and has been increasingly linked to lipid peroxidation, a hallmark of ferroptosis. Herein we investigated the role and the underlying mechanism of STING in AKI models established by ischemia/reperfusion (IR) in C57BL mice. The expression level of STING was predominantly increased in tubules of kidney after IR treatment. Besides, STING deficiency markedly alleviated IR-induced lipid peroxidation, tissue damage and renal dysfunction. Consistently, in vitro experiments demonstrated that the increase in ferroptotic cell death, lipid ROS production and the decrease in GSH peroxidase 4 (GPX4) expression in renal tubular cells subjected to ferroptosis agonist or hypoxia/reoxygenation intervention were all mitigated by genetic deficiency or pharmacological inhibition of STING, while all exacerbated by STING overexpression. Further, these detrimental effects of STING overexpression relied on the induction of ferritinophagy, i.e. autophagic degradation of ferritin, leading to iron overload. Mechanistically, STING mediated the initiation of ferritinophagy through interacting with nuclear receptor coactivator 4 (NCOA4), a fundamental receptor for the transfer of ferritin into lysosome. Collectively, STING contributes to ferroptosis during ischemic AKI through facilitating NCOA4-mediated ferritinophagy and shows the potential as a promising therapeutic choice for AKI.

TRPM2 protects against cisplatin-induced acute kidney injury and mitochondrial dysfunction via modulating autophagy.

Background: Cisplatin is a widely used anti-tumor agent but its use is frequently limited by nephrotoxicity. Transient receptor potential melastatin 2 (TRPM2) is a non-selective cation channel which is generally viewed as a sensor of oxidative stress, and increasing evidence supports its link with autophagy, a critical process for organelle homeostasis. Methods: Cisplatin-induced cell injury and mitochondrial damage were both assessed in WT and Trpm2-knockout mice and primary cells. RNA sequencing, immunofluorescence staining, immunoblotting and flowcytometry were applied to interpret the mechanism of TRPM2 in cisplatin nephrotoxicity. Results: Knockout of TRPM2 exacerbates renal dysfunction, tubular injury and cell apoptosis in a model of acute kidney injury (AKI) induced by treatment with cisplatin. Cisplatin-caused tubular mitochondrial damage is aggravated in TRPM2-deficient mice and cells and, conversely, alleviated by treatment with Mito-TEMPO, a mitochondrial ROS scavenger. TRPM2 deficiency hinders cisplatin-induced autophagy via blockage of Ca2+ influx and subsequent up-regulation of AKT-mTOR signaling. Consistently, cisplatin-induced tubular mitochondrial damage, cell apoptosis and renal dysfunction in TRPM2-deficient mice are mitigated by treatment with a mTOR inhibitor. Conclusion: Our results suggest that the TRPM2 channel plays a protective role in cisplatin-induced AKI via modulating the Ca2+-AKT-mTOR signaling pathway and autophagy, providing novel insights into the pathogenesis of kidney injury.

Genome Sequence of a Heavy Metal-Detoxifying Actinomycete, Microbacterium proteolyticum ustc.

A complete genome is presented for Microbacterium proteolyticum ustc, a member of the Gram-positive order Micrococcales of the phylum Actinomycetota that is resistant to high concentrations of heavy metals and participates in metal detoxification. The genome consists of one plasmid and one chromosome.

A tunable fluorescent probe for superoxide anion detection during inflammation caused by Treponema pallidum.

Syphilis, caused by Treponema pallidum (T. pallidum), is associated with the oxidative stress due to its inflammation-like symptom, and detecting the reactive oxygen species (ROS) is crucial for monitoring the infectious process. Herein, we design and synthesize a perylene-based tunable fluorescent probe, PerqdOH, which can detect endogenous O2˙- during T. pallidum infection. The fluorescence peak shifted from 540 nm to 750 nm with increasing O2˙- levels. Besides, both decreased green fluorescence and enhanced red fluorescence could be observed simultaneously during the in vitro infection, providing the real-time monitoring of intracellular O2˙- caused by T. pallidum. Furthermore, the probe exhibited a remarkable signal in the treponemal lesions on the back of a rabbit model. Taken together, our synthesized PerqdOH holds great potential for application in clarifying the infectious process caused by T. pallidum in real time.

Virulence and Adhesion of the Treponema pallidum Nichols Strain Simultaneously Decrease in a Continuous-Infection New Zealand White Rabbit Model.

Syphilis is a sexually transmitted disease caused by T. pallidum, and the T. pallidum Nichols strain is widely used with the New Zealand white rabbit model for evaluating drug and vaccine protection. However, changes in the virulence of T. pallidum during transmission are still unknown. Herein, we explored the virulence of T. pallidum in the rabbit model of continuous infection through phenotype observation and further investigated the relationship between virulence and adhesion. During the construction of the syphilis rabbit model, the optimal dose of 104/site of T. pallidum was determined to effectively observe the depiction of syphilis lesions and immune responses for further virulence evaluation. Its virulence was gradually weakened during the interaction with host cells or the testicular passage, which was also proven using the pathological phenotype of the syphilis rabbit model. In addition, the adhesive ability of T. pallidum was reduced with increasing generation, which was verified via the co-incubation of the pathogen with Sf1Ep cells. This study provides insight into the relationship by which the virulence and adhesion of T. pallidum were decreased in a New Zealand white rabbit model of continuous infection and contributes to our knowledge regarding the development of syphilis.

Emergency Treatment and Photoacoustic Assessment of Spinal Cord Injury Using Reversible Dual-Signal Transform-Based Selenium Antioxidant.

Spinal cord injury (SCI), following explosive oxidative stress, causes an abrupt and irreversible pathological deterioration of the central nervous system. Thus, preventing secondary injuries caused by reactive oxygen species (ROS), as well as monitoring and assessing the recovery from SCI are critical for the emergency treatment of SCI. Herein, an emergency treatment strategy is developed for SCI based on the selenium (Se) matrix antioxidant system to effectively inhibit oxidative stress-induced damage and simultaneously real-time evaluate the severity of SCI using a reversible dual-photoacoustic signal (680 and 750 nm). Within the emergency treatment and photoacoustic severity assessment (ETPSA) strategy, the designed Se loaded boron dipyrromethene dye with a double hydroxyl group (Se@BDP-DOH) is simultaneously used as a sensitive reporter group and an excellent antioxidant for effectively eliminating explosive oxidative stress. Se@BDP-DOH is found to promote the recovery of both spinal cord tissue and locomotor function in mice with SCI. Furthermore, ETPSA strategy synergistically enhanced ROS consumption via the caveolin 1 (Cav 1)-related pathways, as confirmed upon treatment with Cav 1 siRNA. Therefore, the ETPSA strategy is a potential tool for improving emergency treatment and photoacoustic assessment of SCI.

Enhanced nuclear translation is associated with proliferation and progression across multiple cancers.

Recent technological advances have re-invigorated the interest in nuclear translation (NT), but the underlying mechanisms and functional implications of NT remain unknown. Here we show that NT is enhanced in malignant cancer cells and is associated with rapid cell growth. Nuclear ribopuromycylation analyses in a panel of diverse cell lines revealed that NT is scarce in normal immortalized cells, but is ubiquitous and robust in malignant cancer cells. Moreover, NT occurs in the nucleolus and requires normal nucleolar function. Intriguingly, NT is reduced by cellular stresses and anti-tumor agents and positively correlates with cancer cell proliferation and growth. By using a modified puromycin-associated nascent chain proteomics, we further identified numerous oncoproteins that are preferentially translated in the nucleus, such as transforming growth factor-beta 2 (TGFB2) and nucleophosmin 1 (NMP1). Specific overexpression of TGFB2 and NMP1 messenger RNAs in the nucleus can increase their protein levels and promote tumorigenesis. These findings establish a previously unknown link between NT and malignancy and suggest that cancer cells might have adapted a mechanism of NT to support their need for rapid growth, which highlight the potential of NT in tumorigenesis and might also open up new possibilities for therapeutic targeting of cancer-specific cellular functions.

Transcriptome analysis of the muscle of fast- and slow-growing phoenix barb (Spinibarbus denticulatus denticulatus).

Growth rate is a commercial trait in aquaculture that is influenced by multiple factors, among which genetic composition plays a fundamental role in the growth rate of species. The phoenix barb (Spinibarbus denticulatus denticulatus) is a widely distributed freshwater fish species in South China. Although S. d. denticulatus is reared in South China, the molecular mechanisms underlying the growth rate of the species remain unclear. Here, the authors performed transcriptome analysis of muscle tissues from fast-growing (FG) and slow-growing (SG) S. d. denticulatus at 90, 150, and 300 days after hatch (DAH) to elucidate its growth mechanism. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that differentially expressed genes (DEGs) between the two groups were enriched in pathways related to muscle growth, glycolysis, and energy and lipid metabolism. Nonetheless, a higher number of DEGs were identified in the FG vs. SG groups at 90 and 300 DAH compared with 150 DAH. DEGs identified at 90 DAH were mainly enriched in the GH/IGF axis, PI3K-Akt signalling pathway, AMPK signalling pathway and lipid metabolism highly expressed in FG individuals. DEGs identified at 300 DAH were mainly enriched in PI3K-Akt signalling pathway, glycolysis/gluconeogenesis, gene translation and lipid metabolism. In addition, some genes were expressed during the early growth stage in FG individuals but expressed during the late stage in SG individuals, indicating considerable variations in the expression profiles of growth-related genes at different developmental stages. Overall, these findings contribute to the understanding of the growth mechanism of S. d. denticulatus, which would be useful for the propagation of fast-growing breeds.

Serum IL-12p40: A novel biomarker for early prediction of minimal change disease relapse following glucocorticoids therapy.

Background: Minimal change disease (MCD) has a high recurrence rate, but currently, no biomarker can predict its recurrence. To this end, this study aimed at identifying potential serum cytokines as valuable biomarkers for predicting the risk of MCD recurrence. Materials and methods: Raybiotech 440 cytokine antibody microarray was used to detect the serum samples of eight relapsed, eight non-relapsed MCD patients after glucocorticoid treatment, and eight healthy controls. The differentially expressed cytokines were confirmed by enzyme-linked immunosorbent assay (ELISA) with serum samples from 29 non-relapsed and 35 relapsed MCD patients. The study used the receiver operating characteristic (ROC) curve analysis to investigate the sensitivity and specificity of a serum biomarker for predicting the MCD relapse. Results: Serum IL-12p40 levels increased significantly in the relapsed group. The Area Under the ROC Curve (AUC) of IL-12p40 was 0.727 (95%CI: 0.597-0.856; P < 0.01). The RNA-sequencing analysis and qPCR assay performed on the IL-12 treated mouse podocytes and the control group showed increased expression of podocyte damage genes, such as connective tissue growth factor (CTGF), matrix metallopeptidase 9 (MMP9), secreted phosphoprotein 1 (SPP1), and cyclooxygenase-2 (COX-2) in the former group. Conclusion: IL-12p40 may serve as a new biomarker for predicting the risk of MCD recurrence after glucocorticoid treatment, and it may be involved in the pathogenesis and recurrence of MCD.