Carboxypeptidase M modulates BMSCs osteogenesis-adipogenesis via the MAPK/ERK pathway: An integrated single-cell and bulk transcriptomic study.

The pathogenesis of osteoporosis (OP) is closely associated with the disrupted balance between osteogenesis and adipogenesis in bone marrow-derived mesenchymal stem cells (BMSCs). We analyzed published single-cell RNA sequencing (scRNA-seq) data to dissect the transcriptomic profiles of bone marrow-derived cells in OP, reviewing 56 377 cells across eight scRNA-seq datasets from femoral heads (osteoporosis or osteopenia n = 5, osteoarthritis n = 3). Seventeen genes, including carboxypeptidase M (CPM), were identified as key osteogenesis-adipogenesis regulators through comprehensive gene set enrichment, differential expression, regulon activity, and pseudotime analyses. In vitro, CPM knockdown reduced osteogenesis and promoted adipogenesis in BMSCs, while adenovirus-mediated CPM overexpression had the reverse effects. In vivo, intraosseous injection of CPM-overexpressing BMSCs mitigated bone loss in ovariectomized mice. Integrated scRNA-seq and bulk RNA sequencing analyses provided insight into the MAPK/ERK pathway's role in the CPM-mediated regulation of BMSC osteogenesis and adipogenesis; specifically, CPM overexpression enhanced MAPK/ERK signaling and osteogenesis. In contrast, the ERK1/2 inhibitor binimetinib negated the effects of CPM overexpression. Overall, our findings identify CPM as a pivotal regulator of BMSC differentiation, which provides new clues for the mechanistic study of OP.

EEF1B2 regulates bone marrow-derived mesenchymal stem cells bone-fat balance via Wnt/ß-catenin signaling.

The pathological advancement of osteoporosis is caused by the uneven development of bone marrow-derived mesenchymal stem cells (BMSCs) in terms of osteogenesis and adipogenesis. While the role of EEF1B2 in intellectual disability and tumorigenesis is well established, its function in the bone-fat switch of BMSCs is still largely unexplored. During the process of osteogenic differentiation, we observed an increase in the expression of EEF1B2, while a decrease in its expression was noted during adipogenesis. Suppression of EEF1B2 hindered the process of osteogenic differentiation and mineralization while promoting adipogenic differentiation. On the contrary, overexpression of EEF1B2 enhanced osteogenesis and strongly inhibited adipogenesis. Furthermore, the excessive expression of EEF1B2 in the tibias has the potential to mitigate bone loss and decrease marrow adiposity in mice with osteoporosis. In terms of mechanism, the suppression of ß-catenin activity occurred when EEF1B2 function was suppressed during osteogenesis. Our collective findings indicate that EEF1B2 functions as a regulator, influencing the differentiation of BMSCs and maintaining a balance between bone and fat. Our finding highlights its potential as a therapeutic target for diseases related to bone metabolism.

[Genetic analysis of two Chinese pedigrees affected with Hereditary hypofibrinemia due to missense variants].

OBJECTIVE: To retrospectively analyze the clinical phenotypes and genetic variants in two Chinese pedigrees affected with Hereditary hypofibrinemia (IFD) and explore their molecular pathogenesis. METHODS: Two probands and their pedigree members were admitted to the First Affiliated Hospital of Wenzhou Medical University on March 30, 2021 and May 27, 2021, respectively. Clinical phenotypes of the probands were collected, and blood clotting indexes of the probands and their pedigree members were determined. Variants of the FGA, FGB and FGG genes were analyzed by Sanger sequencing, and candidate variants were verified by sequence comparison. Bioinformatic software was used to analyze the conservation of the amino acids and pathogenicity of the proteins. Alteration in protein structure and intermolecular force before and after the variant was analyzed by simulating the protein model. RESULTS: Proband 1, a 18-year-old male, had significantly low plasma fibrinogen activity (Fg:C) and plasma fibrinogen antigen (Fg:Ag), respectively at 0.80 g/L and 1.00 g/L. Proband 2, a 43-year-old male, had slightly low Fg:C and Fg:Ag at 1.35 g/L and 1.30 g/L, respectively. The Fg:C and Fg:Ag of proband 1's father, proband 2's father and son were also below the normal level. Genetic testing showed that proband 1 had harbored a heterozygous missense variant of c.688T>G (p.Phe230Val) in exon 7 of the FGG gene, which was inherited from his father. Proband 2, his father and son all had harbored a heterozygous variant of c.2516A>C (p.Asn839Thr) in exon 6 of the FGA gene. Homology analysis showed that the Phe230 and Asn839 residues were highly conserved among homologous species. Bioinformatic analysis predicted that both p.Phe230Val and p.Asn839Thr were pathogenic variants. CONCLUSION: Analysis of protein simulation model showed that the p.Asn839Thr variant has changed the hydrogen bo`nd between the amino acids, thus affecting the stability of the protein structure. The heterozygous missense variants of p.Phe230Val and p.Asn839Thr probably underlay the IFD in the two pedigrees.

TA allele of rs2070673 in the CYP2E1 gene is associated with lobular inflammation and nonalcoholic steatohepatitis in patients with biopsy-proven nonalcoholic fatty liver disease.

BACKGROUND AND AIM: Cytochrome P450 2E1 (CYP2E1) plays a role in lipid metabolism, and by increasing hepatic oxidative stress and inflammation, the upregulation of CYP2E1 is involved in development of nonalcoholic steatohepatitis (NASH). We aimed to explore the relationship between CYP2E1-333A>T (rs2070673) and the histological severity of nonalcoholic fatty liver disease (NAFLD). METHODS: We studied 438 patients with biopsy-proven NAFLD. NASH was defined as NAFLD Activity Score ≥ 5 with existence of steatosis, ballooning, and lobular inflammation. CYP2E1-333A>T (rs2070673) was genotyped by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Serum cytokines related to inflammation were measured by the Bio-plex 200 system to investigate possible mediating factors involved in the process. RESULTS: The TA genotype of rs2070673 had a higher prevalence of moderate/severe lobular inflammation (27.6% vs 20.3% vs 13.3%, P < 0.01) and NASH (55.7% vs 42.4% vs 40.5%, P < 0.01) compared with the AA and TT genotypes, respectively. In multivariable regression modeling, the heterozygote state TA was associated with moderate/severe lobular inflammation (adjusted odds ratio: 2.31, 95% confidence interval 1.41-3.78, P < 0.01) or NASH (adjusted odds ratio: 1.82, 95% confidence interval 1.22-2.69, P < 0.01), independently of age, sex, common metabolic risk factors, and presence of liver fibrosis. Compared with no-NASH, NASH patients had significantly higher levels of serum interleukin-1 receptor antagonist, interleukin-18, and interferon-inducible protein-10 (IP-10), whereas only IP-10 was increased with the rs2070673 TA variant (P = 0.01). Mediation analysis showed that IP-10 was responsible for ~60% of the association between the rs2070672 and NASH. CONCLUSIONS: The TA allele of rs2070673 is strongly associated with lobular inflammation and NASH, and this effect appears to be largely mediated by serum IP-10 levels.

Identification of novel biomarkers affecting the metastasis of colorectal cancer through bioinformatics analysis and validation through qRT-PCR.

BACKGROUND: Tumor progression and distant metastasis are the main causes of deaths in colorectal cancer (CRC) patients, and the molecular mechanisms in CRC metastasis have not been completely discovered. METHODS: We identified differentially expressed genes (DEGs) and lncRNAs (DELs) of CRC from The Cancer Genome Atlas (TCGA) database. Then we conducted the weighted gene co-expression network analysis (WGCNA) to investigate co-expression modules related with CRC metastasis. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, DEG-DEL co-expression network and survival analyses of significant modules were also conducted. Finally, the expressions of selected biomarkers were validated in cell lines by quantitative real-time PCR (qRT-PCR). RESULTS: 2032 DEGs and 487 DELs were involved the construction of WGCNA network, and greenyellow, turquoise and brown module were identified to have more significant correlation with CRC metastasis. GO and KEGG pathway analysis of these three modules have proven that the functions of DEGs were closely involved in many important processes in cancer pathogenesis. Through the DEG-DEL co-expression network, 12 DEGs and 2 DELs were considered as hub nodes. Besides, survival analysis showed that 30 DEGs were associated with the overall survival of CRC. Then 10 candidate biomarkers were chosen for validation and the expression of CA2, CHP2, SULT1B1, MOGAT2 and C1orf115 were significantly decreased in CRC cell lines when compared to normal human colonic epithelial cells, which were consistent with the results of differential expression analysis. Especially, low expression of SULT1B1, MOGAT2 and C1orf115 were closely correlated with poorer survival of CRC. CONCLUSION: This study identified 5 genes as new biomarkers affecting the metastasis of CRC. Besides, SULT1B1, MOGAT2 and C1orf115 might be implicated in the prognosis of CRC patients.

Clinical value of combined detection of reactive oxygen species modulator 1 and adenosine deaminase in pleural effusion in the identification of NSCLC associated malignant pleural effusion.

BACKGROUND: Reactive oxygen species modulator 1 (ROMO1) is recognized to be involved in cell proliferation and is elevated in serum of various cancer patients. However, ROMO1 had little research in distinguishing between malignant pleural effusions (MPEs) and benign pleural effusions (BPEs). METHODS: Malignant pleural effusion samples from patients with non-small-cell lung cancer (NSCLC) and benign pleural effusion (BPE) samples containing tuberculous and inflammatory pleural effusions were collected. The samples were tested for ROMO1, pleural effusion adenosine deaminase (pADA), pleural effusion carbohydrate antigen (pCA125, pCA153, pCA199), pleural effusion ferritin (pFER), and pleural effusion lactate dehydrogenase (pLDH) levels, and the other relevant partial clinical data that were gathered were used to conduct statistical analysis. RESULTS: The ROMO1, pCA125, pCA199, pCA153, pADA + ROMO1, pCA153 + ROMO1, pCA125 + ROMO1, and pCA199 + ROMO1 levels in MPE were appreciably higher in comparison with BPE group (all P = .000). The concentration of pADA in MPE was markedly lower than BPE (P = .000). When the cutoff = 0.38, the sensitivity of combined detection of ROMO1 + pADA is 98.67% and the specificity is 70.73%, respectively, and the AUC (0.941) is the highest among other parameters. CONCLUSION: The combined detection of ROMO1 + ADA in pleural effusion is an effective biomarker for identifying MPE caused by NSCLC.

A homozygous MITF mutation leads to familial Waardenburg syndrome type 4.

Waardenburg syndrome (WS) is a genetic disorder characterized by hearing loss and pigmentary abnormalities with variable penetrance. Though heterozygous mutations in MITF are a major cause for Waardenburg syndrome type 2 (WS2), homozygous mutations in this gene and the associated phenotype have been rarely characterized. In this study, we identified a novel p.R223H mutation in MITF in a Chinese Han family with variable WS features. Both parents carried a heterozygous p.R223H mutation. They had normal hearing, and premature greying of the hair is their only pigmentary abnormality. In contrast, their two children both carried a homozygous p.R223H mutation and had classic WS features including profound hearing loss, heterochromia irides and marked pigmentary abnormalities in hair and skin. Interestingly, the two affected children also have persistent chronic constipation since the neonatal period, symptoms suggestive of Waardenburg syndrome type 4 (WS4). Our study revealed a likely association between homozygous mutations in MITF and WS4, which implies a dosage effect for the underlying pathogenesis mechanism.

A Rapid UPLC-MS Method for Quantification of Gomisin D in Rat Plasma and Its Application to a Pharmacokinetic and Bioavailability Study.

Gomisin D, a lignan compound isolated from Fructus Schisandra, is a potential antidiabetic and anti-Alzheimer's agent. Recently, gomisin D was used as a quality marker of some traditional Chinese medicine (TCM) formulas. In this study, a rapid ultra-performance liquid chromatography/tandem mass spectrometry method (UPLC-MS/MS) was developed and validated to quantify gomisin D in rat plasma for a pharmacokinetic and bioavailability study. Acetonitrile was used to precipitate plasma proteins. Separations were performed on a BEH C18 column with a gradient mobile phase comprising of acetonitrile and water (0.1% formic acid). An electrospray ionization source was applied and operated in the positive ion mode. The multiple reaction monitoring mode (MRM) was utilized to quantify gomisin D and nomilin (internal standard, IS) using the transitions of m/z 531.2 → 383.1 and m/z 515.3 → 161.0, respectively. The calibration curve was linear over the working range from 1 to 4000 ng/mL (R² = 0.993). The intra- and interday precision ranged from 1.9% to 12.9%. The extraction recovery of gomisin D was in the range of 79.2-86.3%. The validated UPLC-MS/MS method was then used to obtain the pharmacokinetic characteristics of gomisin D after intravenous (5 mg/kg) and intragastric (50 mg/kg) administration to rats. The bioavailability of gomisin D was 107.6%, indicating that this compound may become a promising intragastrical medication. Our results provided useful information for further preclinical studies on gomisin D.

Comparison of efficacy between internal fixation of minimally invasive elastic stable intramedullary nail and plate in the treatment of pediatric femoral shaft fracture.

OBJECTIVE: To compare and analyze the clinical effects of internal fixation of minimally invasive elastic stable intramedullary nail and plate in the treatment of pediatric femoral shaft fracture. METHODS: A total of 120 children with femoral shaft fractures who were admitted to our hospital from December 2016 to April 2018 were enrolled. The children were divided into an observation group and a control group by random number table, with 60 children in each group. The children in the observation group underwent internal fixation of minimally invasive elastic stable intramedullary nail, while those in the control group underwent open reduction based on internal fixation of plate. The surgical status and postoperative complications of the two groups were observed and compared, and Kolmert knee function scoring criteria were used for assessing the surgical effects of children. RESULTS: The operation duration, intraoperative blood loss, hospitalization duration, fracture healing time and time of off-bed loaded activity of the observation group were significantly shorter than those of the control group, and the differences were statistically significant (P<0.05). The excellent and good rate of fracture healing in the observation group was 100%, which was higher than that of the control group, 83.33%, and the difference was statistically significant (P<0.05). The total incidence rate of complications in the observation group was 8.33%, which was lower than that of the control group, 10.00%, but the difference was not statistically significant (P>0.05). CONCLUSION: Pediatric femoral shaft fractures can be treated with internal fixation of minimally invasive elastic intramedullary nail, and it has advantages of significant curative effect, small trauma and fast postoperative recovery, which is conducive to fracture healing and worth promoting.

Clinical value of jointly detection pleural fluid Midkine, pleural fluid adenosine deaminase, and pleural fluid carbohydrate antigen 125 in the identification of nonsmall cell lung cancer-associated malignant pleural effusion.

BACKGROUND: Midkine (MK) level has been shown to be elevated in serum of patients with nonsmall cell lung cancer (NSCLC). However, the diagnostic value of MK in pleural effusion in NSCLC has not been well validated and established. METHODS: Samples of NSCLC-associated malignant pleural effusions (MPE) and benign effusions (BPE) were collected. The pleural fluid MK (pMK), pleural fluid adenosine deaminase (pADA), pleural fluid lactate dehydrogenase (pLDH), pleural fluid glucose (pGLU), pleural fluid ferritin (pFER), pleural fluid CA199 (pCA199), pleural fluid CA125 (pCA125), pleural effusion white cell count (pWBC), and pleural effusion red cell count (pRBC) were analyzed, and the clinical data of each group were collected for statistical analysis. RESULT: The level of pMK, pCA125, pMK + pCA125, and pMK + pCA125 + pADA in the MPE was significantly higher than the BPE group (P = .003, .000, .000, .000). The pADA level in the BPE was significantly higher than the MPE group (P = .003). It showed that the area under the ROC curve (AUC) (0.816) of jointly detection pMK, pCA125, and pADA was significantly higher than other markers for the diagnosis of MPE. Therefore, joint detection of pMK + pCA125 + pADA suggested that the sensitivity, specificity, and AUC was 82.54%, 74.19% at the cutoff 0.47 and diagnostic performance was higher than others. CONCLUSION: Joint detection of pMK + pCA125 + pADA can be used as a good indicator for the identification of MPE of NSCLC.

Development of a liquid chromatography with mass spectrometry method for the determination of gelsemine in rat plasma and tissue: Application to a pharmacokinetic and tissue distribution study.

Gelsemine from Gelsemium elegans Benth is a potential anesthetic and analgesic agent with no physical dependence and opiate addiction. This study was aimed at developing an ultrafast liquid chromatography coupled to tandem mass spectrometry method to quantify gelsemine in rat plasma and tissues. Plasma and tissues were processed with acetonitrile precipitation, and dendrobine was chosen as the internal standard. Sample separation was performed on an ACQUITY HSS T3 column. The mobile phase consisted of acetonitrile and 0.1% formic acid aqueous solution. Multiple reactions monitoring mode was utilized to detect the compounds of interest. The mass spectrometer was operated in the positive ion mode for detection. The MS/MS ion transitions monitored were m/z 323.2→70.5 for gelsemine and 264.2→108.05 for dendrobine, respectively. The calibration curves were linear over the range of 1-500 ng/mL in all biological matrices. The lower limit of quantification for rats plasma and tissues was 1.0 ng/mL. The values for inter- and intraday precision and accuracy were well within the ranges acceptable (< 15%). It was successfully applied to the pharmacokinetic and tissue distribution studies of gelsemine after intravenous doses of 5, 2, and 0.5 mg/kg in rats. These data of gelsemine would be useful for clinical application and further development.

Telomerase reverse transcriptase promoter mutations in glandular lesions of the urinary bladder.

Glandular lesions of the urinary bladder include a broad spectrum of entities ranging from completely benign to primary and secondary malignancies. The accurate diagnosis of these lesions is both important and challenging. Recently, studies suggest that telomerase reverse transcriptase (TERT) promoter mutations could be a biomarker for urothelial carcinoma (UC). We hypothesized that these mutations can distinguish UC with glandular differentiation from nephrogenic adenoma, primary adenocarcinoma of the urinary bladder (PAUB), or secondary malignancies. Twenty-five cases of benign glandular lesions (including nephrogenic adenoma); 29 cases of UC with glandular differentiation; 10 cases of PAUB; and 10 cases each of metastatic colon cancer, prostatic carcinoma, and carcinoma from Mullerian origin were collected. Slides were reviewed and selected to make sure the lesion was at least 10% to 20% of all tissue. Macrodissection was performed in some of cases, and genomic DNA was extracted from the tissue. Telomerase reverse transcriptase promoter mutations were determined by standard polymerase chain reaction sequencing. Twenty-one cases (72%) of UC with glandular differentiation were positive for TERT promoter mutations. However, none of the remaining cases (total 65 cases of benign lesions, PAUB, and metastatic carcinomas) was positive for TERT promoter mutation. Telomerase reverse transcriptase promoter mutations were highly associated with UC including UC with glandular differentiation but not other glandular lesions of bladder. Therefore, in conjunction with morphologic features, Immunohistochemistry stain profile, and clinical information, TERT promoter mutations could distinguish UC with glandular differentiation from other bladder glandular lesions. In addition, lack of TERT promoter mutations in primary adenocarcinoma of bladder suggests that this entity may have different origin or carcinogenesis from those of UC.

Application of a liquid chromatography-tandem mass spectrometry method to the pharmacokinetics, bioavailability and tissue distribution of neohesperidin dihydrochalcone in rats.

1. This study was aimed at developing a high sensitive and selective liquid chromatography-tandem mass spectrometry method to quantify neohesperidin dihydrochalcone (NHDC) in rat plasma and tissues for pharmacokinetic, bioavailability and tissue distribution studies. 2. Biological samples were processed with one-step protein precipitation. Rutin was chosen as the internal standard (IS). Chromatographical separation was achieved on an SB-C18 (2.1 mm× 150 mm, 5 µm) column with acetonitrile--0.1% formic acid in water as the mobile phase with gradient elution. Electrospray ionization (ESI) source was applied and operated in negative ion mode; selected ion monitoring mode was used for quantification using target fragment ions m/z 611.4 for NHDC and m/z 609.1 for IS. 3. Calibration plots were linear over the range of 10-3000 ng/mL for NHDC. Lower limit of quantification (LLOQ) for NHDC was 10 ng/mL. Mean recovery of NHDC from plasma and tissues was better than 80.3%. Coefficient of variation of intra-day and inter-day precision were both less than 15%. The bioavailability of NHDC was 21.8%. 4. In conclusion, a sensitive, simple and specific LC-ESI-MS method for the determination of NHDC in rat biological samples was developed. This developed method is successfully used in the pharmacokinetic and tissue distribution study of NHDC in rats.

A Novel Homozygous Missense Mutation (Ile583Asn) in a Consanguineous Marriage Family with Hereditary Factor XII Deficiency: A Case Report.

BACKGROUND: Hereditary coagulation factor XII (FXII) deficiency is an autosomal recessive disorder. At present, the contribution of severe FXII deficiency to the development of thromboembolism is still undetermined. There are limited reports on the relationship between the FXII defect and thromboembolism. CASE PRESENTATION: A 27-year-old woman came to our hospital for the treatment of shoulder trauma and cervical disc herniation caused by a car accident. The shoulder trauma was treated with five stitches. After physical examination, imaging examination, and routine coagulation examination, cervical disc herniation was treated conservatively. Combined with the examination results, the patient was diagnosed with FXII deficiency. Unfortunately, the patient was readmitted 10 days after the trauma with edema in the lower limbs and secondary varicose veins. The D-dimer increased to 6.22 mg/L. Thrombus in the inferior vena cava and right common iliac was shown by lower limb venography. According to the patient's medical history, the F12 gene was analyzed by direct sequencing. The patient was also screened for other thrombotic risk factors. Genetic analysis showed that the patient had a c.1748T > A (p.Ile583Asn) homozygous missense mutation in exon 14 of the F12 gene. No other hereditary thrombophilia risk factors screened were positive in the patient. CONCLUSION: The p.Ile583Asn missense mutation in exon 14 of the F12 gene might be responsible for the reduction of the FXII level in the patient.

A Novel Fibrinogen Mutation p.BßAla68Asp Causes an Inherited Dysfibrinogenemia.

OBJECTIVE: Our study aimed to analyze the phenotype and genotype of a pedigree with inherited dysfibrinogenemia, and preliminarily elucidate the probable pathogenesis. METHODS: The one-stage clotting method was used to test the fibrinogen activity (FIB:C), whereas immunoturbidimetry was performed to quantify the fibrinogen antigen (FIB:Ag). Furthermore, DNA sequence analysis was conducted to confirm the site of mutation. Conservation analysis and protein model analysis were performed using online bioinformatics software. RESULTS: The FIB:C and FIB:Ag of the proband were 1.28 and 2.20 g/L, respectively. Gene analysis revealed a heterozygous c.293C > A (p.BßAla68Asp) mutation in FGB. Bioinformatics and modeling analysis suggested that the missense mutation could potentially have a deleterious effect on fibrinogen. CONCLUSION: The BßAla68Asp mutation in exon 2 of FGB may account for the reduced FIB:C levels observed in the pedigree. To our knowledge, this point mutation is the first report in the world.

Temporal transcriptome features identify early skeletal commitment during human epiphysis development at single-cell resolution.

Human epiphyseal development has been mainly investigated through radiological and histological approaches, uncovering few details of cellular temporal genetic alternations. Using single-cell RNA sequencing, we investigated the dynamic transcriptome changes during post-conception weeks (PCWs) 15-25 of human distal femoral epiphysis cells. We find epiphyseal cells contain multiple subtypes distinguished by specific markers, gene signatures, Gene Ontology (GO) enrichment analysis, and gene set variation analysis (GSVA). We identify the populations committed to cartilage or ossification at this time, although the secondary ossification centers (SOCs) have not formed. We describe the temporal alternation in transcriptional expression utilizing trajectories, transcriptional regulatory networks, and intercellular communication analyses. Moreover, we find the emergence of the ossification-committed population is correlated with the COL2A1-(ITGA2/11+ITGB1) signaling. NOTCH signaling may contribute to the formation of cartilage canals and ossification via NOTCH signaling. Our findings will advance the understanding of single-cell genetic changes underlying fetal epiphysis development.

Single-cell transcriptome analysis reveals aberrant stromal cells and heterogeneous endothelial cells in alcohol-induced osteonecrosis of the femoral head.

Alcohol-induced osteonecrosis of the femoral head (ONFH) is a disabling disease with a high incidence and elusive pathogenesis. Here, we used single-cell RNA sequencing to explore the transcriptomic landscape of mid- and advanced-stage alcohol-induced ONFH. Cells derived from age-matched hip osteoarthritis and femoral neck fracture samples were used as control. Our bioinformatics analysis revealed the disorder of osteogenic-adipogenic differentiation of stromal cells in ONFH and altered regulons such as MEF2C and JUND. In addition, we reported that one of the endothelial cell clusters with ACKR1 expression exhibited strong chemotaxis and a weak angiogenic ability and expanded with disease progression. Furthermore, ligand-receptor-based cell-cell interaction analysis indicated that ACKR1+ endothelial cells might specifically communicate with stromal cells through the VISFATIN and SELE pathways, thus influencing stromal cell differentiation in ONFH. Overall, our data revealed single cell transcriptome characteristics in alcohol-induced ONFH, which may contribute to the further investigation of ONFH pathogenesis.

Identification and validation of immunotherapy for four novel clusters of colorectal cancer based on the tumor microenvironment.

The incidence and mortality of colorectal cancer (CRC) are increasing year by year. The accurate classification of CRC can realize the purpose of personalized and precise treatment for patients. The tumor microenvironment (TME) plays an important role in the malignant progression and immunotherapy of CRC. An in-depth understanding of the clusters based on the TME is of great significance for the discovery of new therapeutic targets for CRC. We extracted data on CRC, including gene expression profile, DNA methylation array, somatic mutations, clinicopathological information, and copy number variation (CNV), from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO) (four datasets-GSE14333, GSE17538, GSE38832, and GSE39582), cBioPortal, and FireBrowse. The MCPcounter was utilized to quantify the abundance of 10 TME cells for CRC samples. Cluster repetitive analysis was based on the Hcluster function of the Pheatmap package in R. The ESTIMATE package was applied to compute immune and stromal scores for CRC patients. PCA analysis was used to remove batch effects among different datasets and transform genome-wide DNA methylation profiling into methylation of tumor-infiltrating lymphocyte (MeTIL). We evaluated the mutation differences of the clusters using MOVICS, DeconstructSigs, and GISTIC packages. As for therapy, TIDE and SubMap analyses were carried out to forecast the immunotherapy response of the clusters, and chemotherapeutic sensibility was estimated based on the pRRophetic package. All results were verified in the TCGA and GEO data. Four immune clusters (ImmClust-CS1, ImmClust-CS2, ImmClust-CS3, and ImmClust-CS4) were identified for CRC. The four ImmClusts exhibited distinct TME compositions, cancer-associated fibroblasts (CAFs), functional orientation, and immune checkpoints. The highest immune, stromal, and MeTIL scores were observed in CS2, in contrast to the lowest scores in CS4. CS1 may respond to immunotherapy, while CS2 may respond to immunotherapy after anti-CAFs. Among the four ImmClusts, the top 15 markers with the highest mutation frequency were acquired, and CS1 had significantly lower CNA on the focal level than other subtypes. In addition, CS1 and CS2 patients had more stable chromosomes than CS3 and CS4. The most sensitive chemotherapeutic agents in these four ImmClusts were also found. IHC results revealed that CD29 stained significantly darker in the cancer samples, indicating that their CD29 was highly expressed in colon cancer. This work revealed the novel clusters based on TME for CRC, which would guide in predicting the prognosis, biological features, and appropriate treatment for patients with CRC.

catena-Poly[[diaqua-bis-[2-(4-tolyl-sulfanyl)acetato-κO]manganese(II)]-µ-4,4'-bipyridine-κN:N'].

In the polymeric title complex, [Mn(C(9)H(9)O(2)S)(2)(C(10)H(8)N(2))(H(2)O)(2)](n), the Mn(2+) cation and the 4,4'-bipyridine ligand lie on a twofold rotation axis. The cation has an MnN(2)O(4) octa-hedral environment, being coordinated by the O atoms of two water mol-ecules and two monodentate (4-tolyl-sulfan-yl)acetate anions, and by two N atoms of two 4,4'-bipyridine ligands. The latter bridge adjacent cations into linear chains parallel to [010]. The chains are further linked with each other into a two-dimensional network parallel to (100) via inter-molecular O-H⋯O hydrogen bonds.

Hyperactivation of Ha-ras oncogene, but not Ink4a/Arf deficiency, triggers bladder tumorigenesis.

Although ras is a potent mitogenic oncogene, its tumorigenicity depends on cellular context and cooperative events. Here we show that low-level expression of a constitutively active Ha-ras in mouse urothelium induces simple urothelial hyperplasia that is resistant to progression to full-fledged bladder tumors even in the absence of Ink4a/Arf. In stark contrast, doubling of the gene dosage of the activated Ha-ras triggered early-onset, rapidly growing, and 100% penetrant tumors throughout the urinary tract. Tumor initiation required superseding a rate-limiting step between simple and nodular hyperplasia, the latter of which is marked by the emergence of mesenchymal components and the coactivation of AKT and STAT pathways as well as PTEN inactivation. These results indicate that overactivation of Ha-ras is both necessary and sufficient to induce bladder tumors along a low-grade, noninvasive papillary pathway, and they shed light on the recent findings that ras activation, via point mutation, overexpression, or intensified signaling from FGF receptor 3, occurs in 70%-90% of these tumors in humans. Our results highlight the critical importance of the dosage/strength of Ha-ras activation in dictating its tumorigenicity--a mechanism of oncogene activation not fully appreciated to date. Finally, our results have clinical implications, as inhibiting ras and/or its downstream effectors, such as AKT and STAT3/5, could provide alternative means to treat low-grade, superficial papillary bladder tumors, the most common tumor in the urinary system.