| 1. |
- Beal, Jacob, et al.
(författare)
-
Robust estimation of bacterial cell count from optical density
- 2020
-
Ingår i: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 3:1
-
Tidskriftsartikel (refereegranskat)abstract
- Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data.
|
|
| 2. |
- Guo, Xiong, et al.
(författare)
-
Kashin-Beck Disease (KBD)
- 2017
-
Ingår i: Endemic disease in China. - Beijing : People's Medical Publishing House. - 9787117247139 ; , s. 150-211
-
Bokkapitel (refereegranskat)
|
|
| 3. |
|
|
| 4. |
- Liu, Huan, et al.
(författare)
-
The first human induced pluripotent stem cell line of Kashin–Beck disease reveals involvement of heparan sulfate proteoglycan biosynthesis and PPAR pathway
- 2022
-
Ingår i: The FEBS Journal. - : John Wiley & Sons. - 1742-464X .- 1742-4658. ; 289:1, s. 279-293
-
Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE: Kashin-Beck disease (KBD) is an endemic osteochondropathy. Due to a lack of suitable animal or cellular disease models, the research progress on KBD has been limited. Our goal was to establish the first disease-specific human induced pluripotent stem cells (hiPSCs) cellular disease model of KBD, and to explore its etiology and pathogenesis exploiting transcriptome sequencing.METHODS: HiPSCs were reprogrammed from dermal fibroblasts of two KBD and one healthy control donors via integration-free vectors. Subsequently, hiPSCs were differentiated into chondrocytes through three-week culture. Gene expression profiles in KBD, normal primary chondrocytes and hiPSC-derived chondrocytes were defined by RNA sequencing. A Venn diagram was constructed to show the number of shared differentially expressed genes (DEGs) between KBD and normal. Gene oncology and Kyoto Encyclopedia of Genes and Genomes annotations were performed, and six DEGs were further validated in other individuals by real-time quantitative reverse transcription PCR (RT-qPCR).RESULTS: KBD cellular disease models were successfully established by generation of hiPSC lines. Seventeen consistent and significant DEGs present in all compared groups (KBD and normal) were identified. RT-qPCR validation gave consistent results with the sequencing data. Glycosaminoglycan biosynthesis-heparan sulfate/heparin, PPAR signaling pathway and cell adhesion molecules (CAMs) pathways were identified to be significantly altered in KBD.CONCLUSION: Differentiated chondrocytes deriving from KBD-origin hiPSCs provide the first cellular disease model for etiological studies of KBD. This study also provides new sights into the pathogenesis and etiology of KBD and is likely to inform the development of targeted therapeutics for its treatment.
|
|
| 5. |
- Ning, Yujie, et al.
(författare)
-
Changes in the NF-κB signaling pathway in juvenile and adult patients with Kashin-Beck disease
- 2019
-
Ingår i: Experimental Cell Research. - : Elsevier. - 0014-4827 .- 1090-2422. ; 379:2, s. 140-149
-
Tidskriftsartikel (refereegranskat)abstract
- To investigate the pathogenesis of Kashin-Beck disease (KBD), we compared the common signaling pathways in peripheral blood mononuclear cells (PBMCs) obtained from healthy juvenile and adults and KBD patients, and also from osteoarthritis (OA) patients. The PBMCs from 12 KBD and 12 healthy juvenile, and those from 20 adult KBD patients and 12 healthy donors were separately collected among the people living in the KBD endemic area. The patients were distinguished according to the national diagnosis criteria. Total RNAs were extracted for the determination of gene expressions by microarray analysis. Ingenuity Pathways Analysis (IPA) was employed to identify the signaling pathways significantly affected by juveniles' and adults' KBD, and OA. The expressions of NFκB-p65, cIAP2 and RANKL in the articular cartilage from both juvenile and adults were detected by immunohistochemistry. NF-κB signaling, apoptosis signaling, death receptor signaling and IL-6 signaling pathways were revealed to be the common affected signaling pathways in the juvenile and adult KBD and the OA. BIRC3 and EGR1 were identified as two common differentially expressed genes. The percentages of positive staining of NFκB-p65, cIAP2 and RANKL were reduced in adult KBD patients but significantly increased in juvenile KBD patients. NF-κB, one of the common signaling pathways between adult and juvenile KBD, was less prominent in the adult KBD patients.
|
|
| 6. |
- Ning, Yujie, et al.
(författare)
-
Comparative analysis of the gut microbiota composition between knee osteoarthritis and Kashin-Beck disease in Northwest China
- 2022
-
Ingår i: Arthritis Research & Therapy. - : BioMed Central. - 1478-6362. ; 24:1
-
Tidskriftsartikel (refereegranskat)abstract
- Background: Osteoarthritis (OA) and Kashin-Beck disease (KBD) both are two severe osteochondral disorders. In this study, we aimed to compare the gut microbiota structure between OA and KBD patients.Methods: Fecal samples collected from OA and KBD patients were used to characterize the gut microbiota using 16S rDNA gene sequencing. To identify whether gut microbial changes at the species level are associated with the genes or functions of the gut bacteria between OA and KBD groups, metagenomic sequencing of fecal samples from OA and KBD subjects was performed.Results: The OA group was characterized by elevated Epsilonbacteraeota and Firmicutes levels. A total of 52 genera were identified to be significantly differentially abundant between the two groups. The genera Raoultella, Citrobacter, Flavonifractor, g__Lachnospiraceae_UCG-004, and Burkholderia-Caballeronia-Paraburkholderia were more abundant in the OA group. The KBD group was characterized by higher Prevotella_9, Lactobacillus, Coprococcus_2, Senegalimassilia, and Holdemanella. The metagenomic sequencing showed that the Subdoligranulum_sp._APC924/74, Streptococcus_parasanguinis, and Streptococcus_salivarius were significantly increased in abundance in the OA group compared to those in the KBD group, and the species Prevotella_copri, Prevotella_sp._CAG:386, and Prevotella_stercorea were significantly decreased in abundance in the OA group compared to those in the KBD group by using metagenomic sequencing.Conclusion: Our study provides a comprehensive landscape of the gut microbiota between OA and KBD patients and provides clues for better understanding the mechanisms underlying the pathogenesis of OA and KBD.
|
|
| 7. |
- Ning, Yujie, et al.
(författare)
-
Genetic Variants and Protein Alterations of Selenium- and T-2 Toxin-Responsive Genes Are Associated With Chondrocytic Damage in Endemic Osteoarthropathy
- 2022
-
Ingår i: Frontiers in Genetics. - : Frontiers Media S.A.. - 1664-8021. ; 12
-
Tidskriftsartikel (refereegranskat)abstract
- The mechanism of environmental factors in Kashin-Beck disease (KBD) remains unknown. We aimed to identify single nucleotide polymorphisms (SNPs) and protein alterations of selenium- and T-2 toxin-responsive genes to provide new evidence of chondrocytic damage in KBD. This study sampled the cubital venous blood of 258 subjects including 129 sex-matched KBD patients and 129 healthy controls for SNP detection. We applied an additive model, a dominant model, and a recessive model to identify significant SNPs. We then used the Comparative Toxicogenomics Database (CTD) to select selenium- and T-2 toxin-responsive genes with the candidate SNP loci. Finally, immunohistochemistry was applied to verify the protein expression of candidate genes in knee cartilage obtained from 15 subjects including 5 KBD, 5 osteoarthritis (OA), and 5 healthy controls. Forty-nine SNPs were genotyped in the current study. The C allele of rs6494629 was less frequent in KBD than in the controls (OR = 0.63, p = 0.011). Based on the CTD database, PPARG, ADAM12, IL6, SMAD3, and TIMP2 were identified to interact with selenium, sodium selenite, and T-2 toxin. KBD was found to be significantly associated with rs12629751 of PPARG (additive model: OR = 0.46, p = 0.012; dominant model: OR = 0.45, p = 0.049; recessive model: OR = 0.18, p = 0.018), rs1871054 of ADAM12 (dominant model: OR = 2.19, p = 0.022), rs1800796 of IL6 (dominant model: OR = 0.30, p = 0.003), rs6494629 of SMAD3 (additive model: OR = 0.65, p = 0.019; dominant model: OR = 0.52, p = 0.012), and rs4789936 of TIMP2 (recessive model: OR = 5.90, p = 0.024). Immunohistochemistry verified significantly upregulated PPARG, ADAM12, SMAD3, and TIMP2 in KBD compared with OA and normal controls (p < 0.05). Genetic polymorphisms of PPARG, ADAM12, SMAD3, and TIMP2 may contribute to the risk of KBD. These genes could promote the pathogenesis of KBD by disturbing ECM homeostasis.
|
|
| 8. |
- Ning, Yujie, et al.
(författare)
-
Imbalance of dietary nutrients and the associated differentially expressed genes and pathways may play important roles in juvenile Kashin-Beck disease
- 2018
-
Ingår i: Journal of Trace Elements in Medicine and Biology. - : Elsevier. - 0946-672X .- 1878-3252. ; 50, s. 441-460
-
Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND:Kashin-Beck disease (KBD) is a childhood-onset endemic osteoarthropathy in China. Nutrients including trace elements may play active roles in the development of KBD.OBJECTIVE:This study aimed to estimate the nutrient intakes of children in endemic areas and to identify the imbalanced nutrients associated differentially expressed genes in the juvenile patients with KBD.METHODS:In this cross-sectional study, a consecutive 3 day 24 h semi-quantitative dietary retrospect questionnaire was conducted to estimate the daily nutrient intakes of children using CDGSS 3.0 software. Gene profile analysis was employed to identify differentially expressed genes in peripheral blood mononuclear cells of children with KBD. GOC, CTD, KEGG, and REACTOME databases were used to establish the relationship between nutrients and nutrients-associated differentially expressed genes and pathways. Statistical analyses were accomplished by SPSS 18.0 software.RESULTS:Daily Se intakes without supplementation of children were significantly lower in Se-supplemented (Se + ) KBD areas (29.3 ∼ 29.6 mg/d) and non-endemic area (27.8 ± 7.9 mg/d) compared to non-Se-supplemented (Se-) KBD area (32.9 ± 7.9 mg/d, c2 = 20.24, P < .01). Children in Se+ KBD areas were suffering more serious insufficient intake of multiple nutrients, including vitamins-B2/-C/-E, Ca, Fe, Zn and I. Gene profile analysis combined with bioinformatics technique identified 34 nutrients associated differentially expressed genes and 10 significant pathways which are related to the pathological changes in juvenile KBD.CONCLUSIONS:Imbalance of dietary nutrients and nutrients-associated differentially expressed genes and pathways may play important roles in the development of juvenile KBD.
|
|
| 9. |
- Ning, Yujie, et al.
(författare)
-
Nutrients other than selenium are important for promoting children's health in Kashin-Beck disease areas
- 2018
-
Ingår i: Biological Trace Element Research. - : Springer. - 0163-4984 .- 1559-0720. ; 183:2, s. 233-244
-
Tidskriftsartikel (refereegranskat)abstract
- Overall nutritional status has been proved associated with people's health. The overall nutritional status of children in Kashin-Beck disease (KBD) areas has been overlooked for decades. Therefore, it is worth investigating in the current generation to gather evidence and make suggestions for improvement. A cross-sectional study with three 24-h dietary recalls was conducted to collect raw data on the daily food intake of children. Recorded food was converted into daily nutrient intakes using CDGSS 3.0 software. WHO AnthroPlus software was used to analyse the BMI-for-age z-score (BAZ) for estimating the overall nutrition status of children. All the comparisons and regression analyses were conducted with SPSS 18.0 software. Multiple nutrient intakes among children from the Se-supplemented KBD-endemic were under the estimated average requirement. The protein-to-carbohydrate ratio (P/C ratio) was significantly higher in children from the non-Se-supplemented KBD-endemic area than the other areas (P < 0.001). The children's BAZ was negatively associated with age (B = -0.095, P < 0.001) and the number of KBD relatives (B = -0.277, P = 0.04), and it was positively associated with better housing conditions, receiving colostrum, and daily intakes of niacin and zinc by multivariate regression analysis (F = 10.337, R = 0.609, P < 0.001).Compared to non-Se-supplemented KBD-endemic area and non-endemic areas, children in Se-supplemented KBD-endemic areas have an insufficient intake of multiple nutrients. School breakfast and lunch programmes are recommended, and strict implementation is the key to ensuring a positive effect.
|
|
| 10. |
- Wang, Xi, et al.
(författare)
-
Alterations in the gut microbiota and metabolite profiles of patients with Kashin-Beck disease, an endemic osteoarthritis in China
- 2021
-
Ingår i: Cell Death and Disease. - : Nature Publishing Group. - 2041-4889. ; 12:11
-
Tidskriftsartikel (refereegranskat)abstract
- Kashin-Beck disease (KBD) is a severe osteochondral disorder that may be driven by the interaction between genetic and environmental factors. We aimed to improve our understanding of the gut microbiota structure in KBD patients of different grades and the relationship between the gut microbiota and serum metabolites. Fecal and serum samples collected from KBD patients and normal controls (NCs) were used to characterize the gut microbiota using 16S rDNA gene and metabolomic sequencing via liquid chromatography-mass spectrometry (LC/MS). To identify whether gut microbial changes at the species level are associated with the genes or functions of the gut bacteria in the KBD patients, metagenomic sequencing of fecal samples from grade I KBD, grade II KBD and NC subjects was performed. The KBD group was characterized by elevated levels of Fusobacteria and Bacteroidetes. A total of 56 genera were identified to be significantly differentially abundant between the two groups. The genera Alloprevotella, Robinsoniella, Megamonas, and Escherichia_Shigella were more abundant in the KBD group. Consistent with the 16S rDNA analysis at the genus level, most of the differentially abundant species in KBD subjects belonged to the genus Prevotella according to metagenomic sequencing. Serum metabolomic analysis identified some differentially abundant metabolites among the grade I and II KBD and NC groups that were involved in lipid metabolism metabolic networks, such as that for unsaturated fatty acids and glycerophospholipids. Furthermore, we found that these differences in metabolite levels were associated with altered abundances of specific species. Our study provides a comprehensive landscape of the gut microbiota and metabolites in KBD patients and provides substantial evidence of a novel interplay between the gut microbiome and metabolome in KBD pathogenesis.
|
|
| 11. |
- Wang, Xi, et al.
(författare)
-
Comparison of the major cell populations among osteoarthritis, Kashin-Beck disease and healthy chondrocytes by single-cell RNA-seq analysis
- 2021
-
Ingår i: Cell Death and Disease. - : Springer Nature. - 2041-4889. ; 12:6
-
Tidskriftsartikel (refereegranskat)abstract
- Chondrocytes are the key target cells of the cartilage degeneration that occurs in Kashin-Beck disease (KBD) and osteoarthritis (OA). However, the heterogeneity of articular cartilage cell types present in KBD and OA patients and healthy controls is still unknown, which has prevented the study of the pathophysiology of the mechanisms underlying the roles of different populations of chondrocytes in the processes leading to KBD and OA. Here, we aimed to identify the transcriptional programmes and all major cell populations in patients with KBD, patients with OA and healthy controls to identify the markers that discriminate among chondrocytes in these three groups. Single-cell RNA sequencing was performed to identify chondrocyte populations and their gene signatures in KBD, OA and healthy cells to investigate their differences as related to the pathogenetic mechanisms of these two osteochondral diseases. We performed immunohistochemistry and quantitative reverse-transcription PCR (qRT-PCR) assays to validate the markers for chondrocyte population. Ten clusters were labelled by cell type according to the expression of previously described markers, and one novel population was identified according to the expression of a new set of markers. The homeostatic and mitochondrial chondrocyte populations, which were identified by the expression of the unknown markers MT1X and MT2A and MT-ND1 and MT-ATP6, were markedly expanded in KBD. The regulatory chondrocyte population, identified by the expression of CHI3L1, was markedly expanded in OA. Our study allows us to better understand the heterogeneity of chondrocytes in KBD and OA and provides new evidence of differences in the pathogenetic mechanisms between these two diseases.
|
|
| 12. |
- Wang, Xi, et al.
(författare)
-
Gene expression signature in endemic osteoarthritis by microarray analysis
- 2015
-
Ingår i: International Journal of Molecular Sciences. - Basel, Switzerland : MDPI. - 1661-6596 .- 1422-0067. ; 16:5, s. 11465-11481
-
Tidskriftsartikel (refereegranskat)abstract
- Kashin-Beck Disease (KBD) is an endemic osteochondropathy with an unknown pathogenesis. Diagnosis of KBD is effective only in advanced cases, which eliminates the possibility of early treatment and leads to an inevitable exacerbation of symptoms. Therefore, we aim to identify an accurate blood-based gene signature for the detection of KBD. Previously published gene expression profile data on cartilage and peripheral blood mononuclear cells (PBMCs) from adults with KBD were compared to select potential target genes. Microarray analysis was conducted to evaluate the expression of the target genes in a cohort of 100 KBD patients and 100 healthy controls. A gene expression signature was identified using a training set, which was subsequently validated using an independent test set with a minimum redundancy maximum relevance (mRMR) algorithm and support vector machine (SVM) algorithm. Fifty unique genes were differentially expressed between KBD patients and healthy controls. A 20-gene signature was identified that distinguished between KBD patients and controls with 90% accuracy, 85% sensitivity, and 95% specificity. This study identified a 20-gene signature that accurately distinguishes between patients with KBD and controls using peripheral blood samples. These results promote the further development of blood-based genetic biomarkers for detection of KBD.
|
|
| 13. |
- Wu, Cuiyan, et al.
(författare)
-
Long noncoding RNA expression profile reveals lncRNAs signature associated with extracellular matrix degradation in kashin-beck disease
- 2017
-
Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 7
-
Tidskriftsartikel (refereegranskat)abstract
- Kashin-Beck disease (KBD) is a deformative, endemic osteochondropathy involving degeneration and necrosis of growth plates and articular cartilage. The pathogenesis of KBD is related to gene expression and regulation mechanisms, but long noncoding RNAs (lncRNAs) in KBD have not been investigated. In this study, we identified 316 up-regulated and 631 down-regulated lncRNAs (≥ 2-fold change) in KBD chondrocytes using microarray analysis, of which more than three-quarters were intergenic lncRNAs and antisense lncRNAs. We also identified 232 up-regulated and 427 down-regulated mRNAs (≥ 2-fold change). A lncRNA-mRNA correlation analysis combined 343 lncRNAs and 292 mRNAs to form 509 coding-noncoding gene co-expression networks (CNC networks). Eleven lncRNAs were predicted to have cis-regulated target genes, including NAV2 (neuron navigator 2), TOX (thymocyte selection-associated high mobility group box), LAMA4 (laminin, alpha 4), and DEPTOR (DEP domain containing mTOR-interacting protein). The differentially expressed mRNAs in KBD significantly contribute to biological events associated with the extracellular matrix. Meanwhile, 34 mRNAs and 55 co-expressed lncRNAs constituted a network that influences the extracellular matrix. In the network, FBLN1 and LAMA 4 were the core genes with the highest significance. These novel findings indicate that lncRNAs may play a role in extracellular matrix destruction in KBD.
|
|
| 14. |
- Yang, Lei, et al.
(författare)
-
Gene expression profiles and molecular mechanism of cultured human chondrocytes' exposure to T-2 toxin and deoxynivalenol
- 2017
-
Ingår i: Toxicon. - Amsterdam : Elsevier. - 0041-0101 .- 1879-3150. ; 140, s. 38-44
-
Tidskriftsartikel (refereegranskat)abstract
- T-2 toxin and deoxynivalenol (DON) are secondary metabolites produced by Fusarium fungi and are commonly found on food and feed. Although T-2 toxin and DON have been suggested as the etiology of Kashin-Beck disease (KBD), an endemic osteochondropathy, little is known about the mechanism when human chondrocytes are exposed to T-2 toxin and DON. The purpose of this study is to identify the gene expression differences and underlying molecular changes modulated by T-2 toxin and DON in vitro in human chondrocytes. After the experiments of cell viability, the gene expression profiles were analyzed in cells that were treated with 0.01 μg/ml T-2 toxin and 1.0 μg/ml DON for 72 h by Affymetrix Human Gene Chip. The array results showed that 882 and 2118 genes were differentially expressed for T-2 toxin and DON exposure, respectively. Enrichment analysis revealed that diverse cellular processes including DNA damage, cell cycle regulation and metabolism of extracellular matrix were affected when human chondrocytes were exposed to T-2 toxin and DON. These results demonstrate the gene expression differences and molecular mechanism of cultured human chondrocytes exposure to T-2 toxin and DON, and provide a new insight into future research in the etiology of KBD.
|
|
