Identification of common mechanisms and biomarkers for dermatomyositis and atherosclerosis based on bioinformatics analysis.

BACKGROUND: Dermatomyositis (DM) manifests as an autoimmune and inflammatory condition, clinically characterized by subacute progressive proximal muscle weakness, rashes or both along with extramuscular manifestations. Literature indicates that DM shares common risk factors with atherosclerosis (AS), and they often co-occur, yet the etiology and pathogenesis remain to be fully elucidated. This investigation aims to utilize bioinformatics methods to clarify the crucial genes and pathways that influence the pathophysiology of both DM and AS. METHOD: Microarray datasets for DM (GSE128470, GSE1551, GSE143323) and AS (GSE100927, GSE28829, GSE43292) were retrieved from the Gene Expression Omnibus (GEO) database. The weighted gene co-expression network analysis (WGCNA) was used to reveal their co-expressed modules. Differentially expression genes (DEGs) were identified using the "limma" package in R software, and the functions of common DEGs were determined by functional enrichment analysis. A protein-protein interaction (PPI) network was established using the STRING database, with central genes evaluated by the cytoHubba plugin, and validated through external datasets. Immune infiltration analysis of the hub genes was conducted using the CIBERSORT method, along with Gene Set Enrichment Analysis (GSEA). Finally, the NetworkAnalyst platform was employed to examine the transcription factors (TFs) responsible for regulating pivotal crosstalk genes. RESULTS: Utilizing WGCNA analysis, a total of 271 overlapping genes were pinpointed. Subsequent DEG analysis revealed 34 genes that are commonly found in both DM and AS, including 31 upregulated genes and 3 downregulated genes. The Degree Centrality algorithm was applied separately to the WGCNA and DEG collections to select the 15 genes with the highest connectivity, and crossing the two gene sets yielded 3 hub genes (PTPRC, TYROBP, CXCR4). Validation with external datasets showed their diagnostic value for DM and AS. Analysis of immune infiltration indicates that lymphocytes and macrophages are significantly associated with the pathogenesis of DM and AS. Moreover, GSEA analysis suggested that the shared genes are enriched in various receptor interactions and multiple cytokines and receptor signaling pathways. We coupled the 3 hub genes with their respective predicted genes, identifying a potential key TF, CBFB, which interacts with all 3 hub genes. CONCLUSION: This research utilized comprehensive bioinformatics techniques to explore the shared pathogenesis of DM and AS. The three key genes, including PTPRC, TYROBP, and CXCR4, are related to the pathogenesis of DM and AS. The central genes and their correlations with immune cells may serve as potential diagnostic and therapeutic targets.

Chronological deposition record of trace metals in sediment cores from Chaohu Lake, Anhui Province, China.

Lakes located in the mid-low reaches of the Yangtze River watershed have been subjected to various degrees of human perturbation that would have resulted in toxic metal concentrations and would pose potential risk to the natural habitats. Therefore, in the present study, two sediment cores from Chaohu Lake were collected to determine any such concentration, expressed as the enrichment characteristics of major and trace metals (Al, Fe, Mn, Cu, Pb, Zn, Cr, Cd, As, Hg, and Ni) in response to natural and anthropogenic changes. An approximate 180-year (1840-2021) deposition record of trace metals in sediment cores was obtained on the basis of 137Cs and 210Pb dating. Enrichment factors (EFs) and the geo-accumulation index (Igeo) were adopted to evaluate the enrichment state and pollution status of trace metals. The results showed that Cu and Zn were persistently more enriched in sediment, whereas Mn, Pb, Cd, and Hg has shown remarkable increasing trends in the west lake since the 1970s. Evaluation of the Igeo confirmed that sediment was moderately polluted with Cu and Zn within the whole lake and with Cd in the west lake area, whereas it was uncontaminated with Cr and slightly contaminated with other metals. Furthermore, source identification based on multivariate statistical analysis including correlation analysis, principal component analysis, and cluster analysis suggested similar pollution sources for the studied metals. Combined with the natural and anthropogenic changes within the watershed, enhanced soil erosion due to population expansion and agricultural intensification was a major contributor to sedimentary metals before the 1970s while industrial wastewater, urban runoff, and domestic sewage were predominant inputs of trace metals after the 1970s.

Epidemiological studies of accessory cardiac bronchus and a new variant.

Accessory cardiac bronchus (ACB) is a rare tracheobronchial branching abnormality which originates from the medial wall of the intermediate or main bronchus and is directed to the heart. Three types of ACB have been recognized: type (a) is similar to a short diverticulum, type (b) is a long bronchus ventilating a small undeveloped lobule, and type (c) is an intermediate type with a long diverticulum but no bronchial or alveolar arborization. Herein, we report 40 consecutive cases of ACB detected in 10,287 routine spiral computed tomography (CT) examinations of the chest. The frequency of the anomaly was 0.39%. The study included 17 females and 23 males (female to male ratio 1:1.35). A total of 24 cases belonged to type (a), 14 cases were type (b), and 2 cases were type (c). The mean largest diameter of ACB was 7.9 (range, 4.0 to 12.0) mm and the mean length was 7.5 (range, 3.0 to 18.0) mm. The mean ratio of the largest diameter to length was 1.1, the ratio for (a) was often greater than 1, and the ratios of (b) and (c) were often less than 1. The ACB originated from the intermediate bronchus in 29 cases, which accounted for the largest proportion. The ACB originated from the basal bronchus of the lower lobe in 11 cases (6 cases from the right lower lobe and 5 from the left), which has never been reported before and may be a new variant.

The "2.8 ka BP Cold Event" Indirectly Influenced the Agricultural Exploitation During the Late Zhou Dynasty in the Coastal Areas of the Jianghuai Region.

As a global cooling event, many of the climatic and socio-cultural mechanisms that resulted in changes after the 2. 8 ka BP event remain unclear. In China, this period roughly corresponds with the Zhou Dynasty (1046-212 BC), a critical period when ancient Chinese civilization was experiencing significant cultural and technological changes, including the movement of people to modern-day Jiangsu Province, where they intensively used the natural resources found in this the coastal area. Recent archaeobotanical evidence, and two radiocarbon dates on wheat and foxtail millet, indicate that the Datongpu site, which dates around 2,600 cal a BP, was occupied during this period of transition around the 2.8 ka BP climate event. In total, our investigations recovered 3,399 carbonized seeds from seventy-four flotation samples, of which rice, foxtail millet, broomcorn millet, and wheat seeds where predominant along with 2,296 weed seeds. Additionally, we identified several rice spikelets and wheat rachises. The high number of carbonized rice grains indicates that rice farming was the primary crop in an otherwise mixed rice-dry farming system at Datongpu. In addition, we argue that the "2.8 ka BP cold event" probably influenced population growth and caused food shortages throughout Central China, leading people to migrate southeastward along the Huai River to the coastal areas of Jianghuai Region. We argue that this abrupt shift in the climate indirectly facilitated the exploitation and emergence of large-scale agriculture in this area. Our study provides an example for the indirect impact of climate change in areas with relatively favorable climate conditions.

A CRISPR/Cas9 strategy for the generation of a FLNC knockout hESC line (WAe009-A-70) to model dilated cardiomyopathy and arrhythmogenic right ventricular cardiomyopathy.

The FLNC gene encodes the sarcomeric protein filamin C which plays a central role in cardiomyocytes. Truncating FLNC mutations (stop or frameshift etc.) also cause dilated cardiomyopathy (DCM) and arrhythmogenic right ventricular cardiomyopathy (ARVC). To further understand the exact role of FLNC in DCM, we have generated a human FLNC knockout cell line from the original embryonic stem cell line H9 by CRISPR/Cas9 gene editing technology in this study. The establishment cell line WAe009-A-70 have a compound heterozygous 4 bp deletion/13 bp deletion in the exon 1 of FLNC which resulted in a frameshift in the translation of FLNC. No filamin C protein was detected in cardiomyocytes differentiated from this cell line. Moreover, WAe009-A-70 also expressed pluripotency markers, maintained the ability to differentiate into the three germ layers in vitro and had a normal karyotype.

Preconditioning of mesenchymal stem cells with ghrelin exerts superior cardioprotection in aged heart through boosting mitochondrial function and autophagy flux.

Application of mesenchymal stem cells (MSCs) is considered as a promising cell-based therapy to induce cardioprotection against ischemia-reperfusion (IR) injury. Preconditioning of MSCs is the key strategy to improve MSCs functions in vitro and their efficacy in vivo, especially in elderly subjects in whom cardioprotection is lost. This study investigated the effects of preconditioning of human umbilical cord-derived MSCs with ghrelin and their combination with nicotinamide-mononucleotide (NMN) on cardioprotection, and the role of autophagy flux and mitochondrial function in aged hearts subjected to IR injury. Aged Sprague Dawley rats (20-22 months old) were subjected to LAD occlusion-induced myocardial IR injury and treated with ghrelin-preconditioned or unconditioned-MSCs at early reperfusion. NMN (500 mg/kg, i.p) was also administered at early reperfusion and repeated 12 h later. Intra-myocardial injection of ghrelin-preconditioned MSCs reduced infarct size and cardiotroponin release of aged myocardium, and improved cardiac function following IR injury. MSCs preconditioning with ghrelin restored IR-induced mitochondrial reactive oxygen species and membrane potential depolarization and enhanced ATP production. To reveal possible mechanism, preconditioned-MSCs increased autophagy flux by downregulating the overexpression of Beclin-1 and P62 proteins and increasing the LC3-II expression and LC3-II/LC3-I ratio. Moreover, combining NMN to ghrelin-preconditioned MSCs synergistically augmented its protective effects on infarct size and mitochondrial function. All above effects were abolished by autophagy flux inhibitor, chloroquine. Thus, ghrelin may serve as a promising candidate to improve the cardioprotective efficacy of MSC-based therapy via autophagy/mitochondrial pathway and that NMN serves as a good booster in combination therapy in aged hearts.

Reprogramming of a human induced pluripotent stem cell line from a Marfan syndrome patient harboring a heterozygous mutation of c.2939G > A in FBN1 gene.

Marfan syndrome (MFS) is a connective-tissue disorder caused mainly by heterozygous mutations in the FBN1 gene that encodes fibrillin-1. In this study, human induced pluripotent stem cell (iPSC) line ZZUSAHi003-A was generated from peripheral blood mononuclear cells (PBMCs) isolated from a female patient with MFS using non-integrative Sendai virus. The iPSC line carried the FBN1 gene mutation, showed the normal karyotype, expressed pluripotency markers and had the capacity to differentiate into three germ layers in vivo. This iPS line, ZZUSAHi003-A, could serve as a useful tool for studying pathogenic mechanisms of MFS.

Discovery of BMS-986144, a Third-Generation, Pan-Genotype NS3/4A Protease Inhibitor for the Treatment of Hepatitis C Virus Infection.

The discovery of a pan-genotypic hepatitis C virus (HCV) NS3/4A protease inhibitor based on a P1-P3 macrocyclic tripeptide motif is described. The all-carbon tether linking the P1-P3 subsites of 21 is functionalized with alkyl substituents, which are shown to effectively modulate both potency and absorption, distribution, metabolism, and excretion (ADME) properties. The CF3Boc-group that caps the P3 amino moiety was discovered to be an essential contributor to metabolic stability, while positioning a methyl group at the C1 position of the P1' cyclopropyl ring enhanced plasma trough values following oral administration to rats. The C7-fluoro, C6-CD3O substitution pattern of the P2* isoquinoline heterocycle of 21 was essential to securing the targeted potency, pharmacokinetic (PK), and toxicological profiles. The C6-CD3O redirected metabolism away from a problematic pathway, thereby circumventing the time-dependent cytochrome P (CYP) 450 inhibition observed with the C6-CH3O prototype.

CRISPR/Cas9 mediated establishment of a human CSRP3 compound heterozygous knockout hESC line to model cardiomyopathy and heart failure.

The role of muscle LIM protein (MLP), encoded by CSRP3, is not well understood in humans. CSRP3 knockout mice developed dilated cardiomyopathy with hypertrophy and heart failure after birth. Using CRISPR/Cas9, we generated an MLP deficient human ESC line WAe009-A-41 carrying a compound heterozygous 13 bp deletion/1 bp insertion in the CSRP3 gene. The WAe009-A-41 line exhibited a normal female karyotype (46, XX), expressed pluripotency markers and exhibited capability to differentiate into the three germ layers in vitro. MLP expression was not detectable in WAe009-A-41 line. This cell line can be a useful tool for studying the role of CSRP3 in cardiomyopathy and heart failure.

Protective role of poly(lactic-co-glycolic) acid nanoparticle loaded with resveratrol against isoproterenol-induced myocardial infarction.

Our study is aimed at evaluating the effects of pretreatment with Poly(lactic-co-glycolic) acid nanoparticle loaded with resveratrol (RSV PLGA NPs) compared to conventional resveratrol (RSV) on isoproterenol (ISO) induced myocardial infarction (MI) in rats. Sixty rats were randomly divided into six groups of 10 rats each. RSV and RSV PLGA NPs were given by gavage in two different doses (50 mg/kg body weight [BW] and 100 mg/kg BW) for 3 weeks. RSV and RSV PLGA NPs were given for 2 weeks starting 1 week before ISO administration. The blood samples were taken 24 hr after the last dose of ISO. The antioxidant, anti-inflammatory, and cardioprotective effects were evaluated in all groups. Only 100 mg/kg dose of RSV and both doses of RSV PLGA NPs offered a cardioprotective effect by preventing cardiac troponin T (cTnT) levels, lactate dehydrogenase (LDH), and aspartate aminotransferase (AST) activities leakage from cardiomyocytes, with the best result for RSV PLGA NPs. All the oxidative stress parameters were significantly improved after RSV PLGA NPs compared to RSV pretreatment. RSV PLGA NPs were more efficient than RSV in limiting the increase in inflammatory cytokine expressions such as tumor necrosis factor-alpha (TNF-α), interleukin 1 beta (IL-1ß), and NF-kappaB (NF-kB) expression. In addition, RSV PLGA NPs significantly upregulated eNOS expression and downregulated iNOS expression. RSV PLGA NPs better prevented myocardial necrosis and reduced interstitial edema and neutrophil infiltration than RSV, on histopathological examination. Therefore, improving the bioactivity of RSV by nanotechnology may help limit cardiac injury after myocardial infarction.

P3-P4 ureas and reverse carbamates as potent HCV NS3 protease inhibitors: Effective transposition of the P4 hydrogen bond donor.

A series of tripeptidic acylsulfonamide inhibitors of HCV NS3 protease were prepared that explored structure-activity relationships (SARs) at the P4 position, and their in vitro and in vivo properties were evaluated. Enhanced potency was observed in a series of P4 ureas; however, the PK profiles of these analogues were less than optimal. In an effort to overcome the PK shortcomings, modifications to the P3-P4 junction were made. This included a strategy in which one of the two urea N-H groups was either N-methylated or replaced with an oxygen atom. The former approach provided a series of regioisomeric N-methylated ureas while the latter gave rise to P4 reverse carbamates, both of which retained potent NS3 inhibitory properties while relying upon an alternative H-bond donor topology. Details of the SARs and PK profiles of these analogues are provided.

Potent Inhibitors of Hepatitis C Virus NS3 Protease: Employment of a Difluoromethyl Group as a Hydrogen-Bond Donor.

The design and synthesis of potent, tripeptidic acylsulfonamide inhibitors of HCV NS3 protease that contain a difluoromethyl cyclopropyl amino acid at P1 are described. A cocrystal structure of 18 with a NS3/4A protease complex suggests the presence of a H-bond between the polarized C-H of the CHF2 moiety and the backbone carbonyl of Leu135 of the enzyme. Structure-activity relationship studies indicate that this H-bond enhances enzyme inhibitory potency by 13- and 17-fold compared to the CH3 and CF3 analogues, respectively, providing insight into the deployment of this unique amino acid.

Functionalized triazines as potent HCV entry inhibitors.

A series of potent and novel acylsulfonamide-bearing triazines were synthesized and the structure-activity relationships (SARs) as HCV entry inhibitors were evaluated. This acylsulfonamide series was derived from an early lead, 4-(4-(1-(4-chlorophenyl)cyclopropylamino)-6-(2,2,2-trifluoroethoxy)-1,3,5-triazin-2-ylamino)benzoic acid wherein the carboxylic acid was replaced with an acylsulfonamide moiety. This structural modification provided a class of compounds which projected an additional vector off the terminus of the acylsulfonamide functionality as a means to drive activity. This effort led to the discovery of potent analogues within this series that demonstrated sub-nanomolar EC50 values in the HCV pseudotype particle (HCVpp) assay.

Structure-activity relationships of 4-hydroxy-4-biaryl-proline acylsulfonamide tripeptides: A series of potent NS3 protease inhibitors for the treatment of hepatitis C virus.

The design and synthesis of a series of tripeptide acylsulfonamides as potent inhibitors of the HCV NS3/4A serine protease is described. These analogues house a C4 aryl, C4 hydroxy-proline at the S2 position of the tripeptide scaffold. Information relating to structure-activity relationships as well as the pharmacokinetic and cardiovascular profiles of these analogues is provided.

Discovery of a Potent Acyclic, Tripeptidic, Acyl Sulfonamide Inhibitor of Hepatitis C Virus NS3 Protease as a Back-up to Asunaprevir with the Potential for Once-Daily Dosing.

The discovery of a back-up to the hepatitis C virus NS3 protease inhibitor asunaprevir (2) is described. The objective of this work was the identification of a drug with antiviral properties and toxicology parameters similar to 2, but with a preclinical pharmacokinetic (PK) profile that was predictive of once-daily dosing. Critical to this discovery process was the employment of an ex vivo cardiovascular (CV) model which served to identify compounds that, like 2, were free of the CV liabilities that resulted in the discontinuation of BMS-605339 (1) from clinical trials. Structure-activity relationships (SARs) at each of the structural subsites in 2 were explored with substantial improvement in PK through modifications at the P1 site, while potency gains were found with small, but rationally designed structural changes to P4. Additional modifications at P3 were required to optimize the CV profile, and these combined SARs led to the discovery of BMS-890068 (29).

Knockdown of SCARA5 inhibits PDGF-BB-induced vascular smooth muscle cell proliferation and migration through suppression of the PDGF signaling pathway.

Vascular smooth muscle cell (VSMC) proliferation and migration are critical in the progression of atherosclerosis and can be induced by platelet-derived growth factor (PDGF). Several studies have demonstrated that scavenger receptor class A, member 5 (SCARA5) is important in cancer cell migration and invasion. However, the role of SCARA5 in VSMCs remains to be elucidated in the development of atherosclerosis. Therefore, the role of SCARA5 was investigated in PDGF­BB­stimulated VSMC proliferation and migration. In the present study, it was shown that SCARA5 expression was enhanced by PDGF­BB in human aortic smooth muscle cells (HASMCs). Knockdown of SCARA5 by small interfering (si)RNA significantly inhibited PDGF­BB­induced HASMC proliferation and migration. Furthermore, siRNA­SCARA5 significantly inhibited the phosphorylation of PDGF receptor (PDGFR) ß, AKT and extracellular signal­regulated kinase 1/2 in PDGF­BB­stimulated HASMCs. In conclusion, this study demonstrated that knockdown of SCARA5 inhibits PDGF­BB­induced HASMC proliferation and migration through suppression of the PDGF signaling pathway. Thus, SCARA5 may be a novel therapeutic target for preventing or treating vascular diseases involving VSMC proliferation and migration.

Quercetin increases macrophage cholesterol efflux to inhibit foam cell formation through activating PPARγ-ABCA1 pathway.

The accumulation of cholesterol in macrophages could induce the formation of foam cells and increase the risk of developing atherosclerosis. We wonder if quercetin, one of flavonoids with anti-inflammation functions in different cell types, could elevate the development of foam cells formation in atherosclerosis. We treated foam cells derived from oxLDL induced THP-1 cells with quercetin, and evaluated the foam cells formation, cholesterol content and apoptosis of the cells. We found that quercetin induced the expression of ABCA1 in differentiated THP-1 cells, and increased the cholesterol efflux from THP-1 cell derived foam cells. Eventually, cholesterol level and the formation of foam cell derived from THP-1 cells decreased after quercetin treatment. In addition, quercetin activated PPARγ-LXRα pathway to upregulate ABCA1 expression through increasing protein level of PPARγ and its transcriptional activity. Inhibition of PPARγ activity by siRNA knockdown or the addition of chemical inhibitor, GW9662, abolished quercetin induced ABCA1 expression and cholesterol efflux in THP-1 derived macrophages. Our data demonstrated that quercetin increased cholesterol efflux from macrophages through upregulating the expressions of PPARγ and ABCA1. Taken together, increasing uptake of quercetin or quercetin-rich foods would be an effective way to lower the risk of atherosclerosis.

FGF21 protects against ox-LDL induced apoptosis through suppressing CHOP expression in THP1 macrophage derived foam cells.

BACKGROUND: FGF21,as a member of the fibroblast growth factor superfamily, is an important endogenous regulator to systemic glucose and lipid metabolism. Elevated serum FGF21 levels have been reported in subjects with coronary heart disease and carotid artery plaques. The formation and apoptosis of foam cell, induced by ox-LDL and oxysterols, are key steps in the development of atherosclerosis. METHODS: In this study, THP1 derived macrophages were induced into foam cells by ox-LDL or sterols. The formation and apoptosis of foam cells treated with or without FGF21 were analyzed. RESULTS: We demonstrated that the accumulation of cholesterol was decreased after FGF21 treatment in THP1 macrophage derived foam cells. Consistently, the apoptosis of macrophage was alleviated dramatically with FGF21 treatment. ERK1/2 knockdown didn't abrogate the effect of FGF21 on THP1 macrophage derived foam cells. However, FGF21 suppressed the induced expression of CHOP and DR5 in THP1 macrophage derived foam cells. CONCLUSION: FGF21 protects against the formation and apoptosis of THP1 macrophages derived foam cells through suppressing the expression of CHOP.

The effects of epidural/spinal opioids in labour analgesia on neonatal outcomes: a meta-analysis of randomized controlled trials.

PURPOSE: Epidural/spinal opioids are increasingly used to relieve parturients' pain in labour. Some studies indicate that opioids can induce side effects in neonates, such as respiratory depression and neurobehavioural changes. This meta-analysis aimed to clarify the effects of opioids in labour analgesia on neonates. SOURCE: PubMed, Cochrane Central Register of Controlled Trials (CENTRAL), and EMBASE™ were searched for relevant randomized controlled trials (RCTs). The neonatal data of Apgar scores, Neurological and Adaptive Capacity Scores (NACS), and umbilical cord pH values were extracted. Statistical analyses were carried out using Review Manager 5.2 and Stata(®) 10. PRINCIPAL FINDINGS: Twenty-one trials with 2,859 participants were included in our meta-analysis. No difference in the incidence of Apgar scores < 7 was shown between the opioid and control groups at one minute (risk difference [RD] 0.0%, 95% confidence interval [CI]: -3.0 to 2.0, P = 0.78; I (2) = 0%, 95% CI: 0 to 50) and at five minutes (RD -1.0%, 95% CI: -2.0 to 1.0, P = 0.31; I(2) = 0%, 95% CI: 0 to 50). No significant differences were found in the NACS at two hours (mean difference [MD] -0.35, 95% CI: -1.70 to 1.01, P = 0.62; I(2) = 0%, 95% CI: 0 to 79) and at 24 hr (MD -0.45, 95% CI: -1.36 to 0.46, P = 0.33; I(2) = 3%, 95% CI: 0 to 26). Also, no significant differences were found in umbilical cord artery pH (MD -0.02, 95% CI: -0.06 to 0.03, P = 0.48; I(2) = 80%, 95% CI: 46 to 92) and vein pH (MD -0.03, 95% CI: -0.07 to 0.00, P = 0.08; I(2) = 77%, 95% CI: 36 to 91). No significant publication bias was found. CONCLUSION: The common doses of fentanyl and sufentanil used with an epidural/spinal technique in labour analgesia are safe for neonates up to 24 hr after delivery. In future studies, more attention should be paid to the long-term side effects in neonates.

The discovery of asunaprevir (BMS-650032), an orally efficacious NS3 protease inhibitor for the treatment of hepatitis C virus infection.

The discovery of asunaprevir (BMS-650032, 24) is described. This tripeptidic acylsulfonamide inhibitor of the NS3/4A enzyme is currently in phase III clinical trials for the treatment of hepatitis C virus infection. The discovery of 24 was enabled by employing an isolated rabbit heart model to screen for the cardiovascular (CV) liabilities (changes to HR and SNRT) that were responsible for the discontinuation of an earlier lead from this chemical series, BMS-605339 (1), from clinical trials. The structure-activity relationships (SARs) developed with respect to CV effects established that small structural changes to the P2* subsite of the molecule had a significant impact on the CV profile of a given compound. The antiviral activity, preclincial PK profile, and toxicology studies in rat and dog supported clinical development of BMS-650032 (24).