XJB-5-131 protects chondrocytes from ferroptosis to alleviate osteoarthritis progression via restoring Pebp1 expression.

Background: Osteoarthritis (OA) is the most common age-related musculoskeletal disease. However, there is still a lack of therapy that can modify OA progression due to the complex pathogenic mechanisms. The aim of the study was to explore the role and mechanism of XJB-5-131 inhibiting chondrocytes ferroptosis to alleviate OA progression. Methods: We treated tert-butyl hydroperoxide (TBHP)-induced ferroptosis of mouse primary chondrocytes with XJB-5-131 in vitro. The intracellular ferroptotic hallmarks, cartilage anabolic and catabolic markers, ferroptosis regulatory genes and proteins were detected. Then we established a mouse OA model via destabilization of the medial meniscus (DMM) surgery. The OA mice were treated with intra-articular injection of XJB-5-131 regularly (2 µM, 3 times per week). After 4 and 8 weeks, we performed micro-CT and histological examination to evaluate the protection role of XJB-5-131 in mouse OA subjects. RNA sequencing analysis was performed to unveil the key downstream gene of XJB-5-131 exerting the anti-ferroptotic effect in OA. Results: XJB-5-131 significantly suppressed TBHP-induced increases of ferroptotic hallmarks (ROS, lipid peroxidation, and Fe2+ accumulation), ferroptotic drivers (Ptgs2, Pgd, Tfrc, Atf3, Cdo1), while restored the expression of ferroptotic suppressors (Gpx4, Fth1). XJB-5-131 evidently promoted the expression of cartilage anabolic and decreased the expression of cartilage catabolic markers. Moreover, intra-articular injection of XJB-5-131 significantly inhibited the expression of Cox2 and Mmp13, while promoted the expression of Col2a1, Gpx4 and Fth1 in DMM-induced mouse articular cartilage. Further, we identified Pebp1 as a potential target of XJB-5-131 by RNA sequencing analysis. The anti-ferroptosis and chondroprotective effects of XJB-5-131 were significantly diminished by Locostatin, a specific antagonist of Pebp1. Conclusion: XJB-5-131 significantly protects chondrocytes from ferroptosis in TBHP-induced mouse primary chondrocytes and DMM surgery-induced OA mice model via restoring the expression of Pebp1. XJB-5-131 is a potential therapeutic drug in the management of OA progression.

Microtubule stabilization targeting regenerative chondrocyte cluster for cartilage regeneration.

Purpose: Chondrocytes (CHs) in cartilage undergo several detrimental events during the development of osteoarthritis (OA). However, the mechanism underlying CHs regeneration involved in pathogenesis is largely unknown. The aim of this study was to explore the underlying mechanism of regeneration of CHs involved in the pathological condition and the potential therapeutic strategies of cartilage repair. Methods and Materials: CHs were isolated from human cartilage in different OA stages and the high-resolution cellular architecture of human osteoarthritis was examined by applying single-cell RNA sequencing. The analysis of gene differential expression and gene set enrichment was utilized to reveal the relationship of cartilage regeneration and microtubule stabilization. Microtubule destabilizer (nocodazole) and microtubule stabilizer (docetaxel) treated-human primary CHs and rats cartilage defect model were used to investing the effects and downstream signaling pathway of microtubule stabilization on cartilage regeneration. Results: CHs subpopulations were identified on the basis of their gene markers and the data indicated an imbalance caused by an increase in the degeneration and disruption of CHs regeneration in OA samples. Interestingly, the CHs subpopulation namely CHI3L1+ CHs, was characterized by the cell regenerative capacity, stem cell potency and the activated microtubule (MT) process. Furthermore, the data indicated that MT stabilization was effective in promoting cartilage regeneration in rats with cartilage injury model by inhibiting YAP activity. Conclusion: These findings lead to a new understanding of CHs regeneration in the OA pathophysiology context and suggest that MT stabilization is a promising therapeutic target for OA and cartilage injury.

Molecular crosstalk between articular cartilage, meniscus, synovium, and subchondral bone in osteoarthritis.

AIMS: Osteoarthritis (OA) is a common degenerative joint disease worldwide, which is characterized by articular cartilage lesions. With more understanding of the disease, OA is considered to be a disorder of the whole joint. However, molecular communication within and between tissues during the disease process is still unclear. In this study, we used transcriptome data to reveal crosstalk between different tissues in OA. METHODS: We used four groups of transcription profiles acquired from the Gene Expression Omnibus database, including articular cartilage, meniscus, synovium, and subchondral bone, to screen differentially expressed genes during OA. Potential crosstalk between tissues was depicted by ligand-receptor pairs. RESULTS: During OA, there were 626, 97, 1,060, and 2,330 differentially expressed genes in articular cartilage, meniscus, synovium, and subchondral bone, respectively. Gene Ontology enrichment revealed that these genes were enriched in extracellular matrix and structure organization, ossification, neutrophil degranulation, and activation at different degrees. Through ligand-receptor pairing and proteome of OA synovial fluid, we predicted ligand-receptor interactions and constructed a crosstalk atlas of the whole joint. Several interactions were reproduced by transwell experiment in chondrocytes and synovial cells, including TNC-NT5E, TNC-SDC4, FN1-ITGA5, and FN1-NT5E. After lipopolysaccharide (LPS) or interleukin (IL)-1ß stimulation, the ligand expression of chondrocytes and synovial cells was upregulated, and corresponding receptors of co-culture cells were also upregulated. CONCLUSION: Each tissue displayed a different expression pattern in transcriptome, demonstrating their specific roles in OA. We highlighted tissue molecular crosstalk through ligand-receptor pairs in OA pathophysiology, and generated a crosstalk atlas. Strategies to interfere with these candidate ligands and receptors may help to discover molecular targets for future OA therapy.Cite this article: Bone Joint Res 2022;11(12):862-872.

Articular fibrocartilage-targeted therapy by microtubule stabilization.

The fibrocartilage presented on the joint surface was caused by cartilage injury or degeneration. There is still a lack of effective strategies for fibrocartilage. Here, we hypothesized that the fibrocartilage could be viewed as a raw material for the renewal of hyaline cartilage and proposed a previously unidentified strategy of cartilage regeneration, namely, "fibrocartilage hyalinization." Cytoskeleton remodeling plays a vital role in modifying the cellular phenotype. We identified that microtubule stabilization by docetaxel repressed cartilage fibrosis and increased the hyaline cartilage extracellular matrix. We further designed a fibrocartilage-targeted negatively charged thermosensitive hydrogel for the sustained delivery of docetaxel, which promoted fibrocartilage hyalinization in the cartilage defect model. Moreover, the mechanism of fibrocartilage hyalinization by microtubule stabilization was verified as the inhibition of Sparc (secreted protein acidic and rich in cysteine). Together, our study suggested that articular fibrocartilage-targeted therapy in situ was a promising strategy for hyaline cartilage repair.

Single cell RNA-seq analysis identifies ferroptotic chondrocyte cluster and reveals TRPV1 as an anti-ferroptotic target in osteoarthritis.

BACKGROUND: Osteoarthritis (OA) is the most common degenerative joint disease primarily characterized by cartilage destruction. The aim of this study was to investigate the role, molecular characteristics and potential therapeutic target of chondrocyte ferroptosis in the pathogenesis of OA. METHODS: The expression of ferroptotic hallmarks (iron and lipid peroxidation accumulation, glutathione deletion) were analyzed in paired intact and damaged cartilages from OA patients. Single cell RNA sequencing (scRNA-seq) analysis was performed on 17,638 chondrocytes to verify the presence, investigate the molecular signatures and unveil the potential therapeutic target of ferroptotic chondrocyte cluster in human OA cartilages. Destabilization of medial meniscus (DMM)-induced OA model and tert-butyl hydroperoxide (TBHP)-treated primary mouse chondrocytes and human cartilage explants were used to evaluate the protective effect of pharmacologically activated transient receptor potential vanilloid 1 (TRPV1). The downstream molecular mechanisms of TRPV1 was further investigated in glutathione peroxidase 4 (Gpx4) heterozygous genetic deletion mice (Gpx4+/-). FINDINGS: The concentrations of iron and lipid peroxidation and the expression of ferroptotic drivers in the damaged areas of human OA cartilages were significantly higher than those in the intact cartilage. scRNA-seq analysis revealed a chondrocyte cluster characterized by preferentially expressed ferroptotic hallmarks and genes, namely ferroptotic chondrocyte cluster. Comprehensive gene set variation analysis revealed TRPV1 as an anti-ferroptotic target in human OA cartilage. Pharmacological activation of TRPV1 significantly abrogated cartilage degeneration by protecting chondrocytes from ferroptosis. Mechanistically, TRPV1 promoted the expression of GPX4, and its anti-ferroptotic role was largely mitigated in the OA model of Gpx4+/- mice. INTERPRETATION: TRPV1 activation protects chondrocytes from ferroptosis and ameliorates OA progression by upregulating GPX4. FUNDING: National Key R&D Program of China (2018YFC1105904), Key Program of NSFC (81730067), National Science Foundation of China (81772335, 81941009, 81802196), Natural Science Foundation of Jiangsu Province, China (BK20180127), Jiangsu Provincial Key Medical Talent Foundation, Six Talent Peaks Project of Jiangsu Province (WSW-079).

Targeting macrophagic SHP2 for ameliorating osteoarthritis via TLR signaling.

Osteoarthritis (OA), in which M1 macrophage polarization in the synovium exacerbates disease progression, is a major cause of cartilage degeneration and functional disabilities. Therapeutic strategies of OA designed to interfere with the polarization of macrophages have rarely been reported. Here, we report that SHP099, as an allosteric inhibitor of src-homology 2-containing protein tyrosine phosphatase 2 (SHP2), attenuated osteoarthritis progression by inhibiting M1 macrophage polarization. We demonstrated that M1 macrophage polarization was accompanied by the overexpression of SHP2 in the synovial tissues of OA patients and OA model mice. Compared to wild-type (WT) mice, myeloid lineage conditional Shp2 knockout (cKO) mice showed decreased M1 macrophage polarization and attenuated severity of synovitis, an elevated expression of cartilage phenotype protein collagen II (COL2), and a decreased expression of cartilage degradation markers collagen X (COL10) and matrix metalloproteinase 3 (MMP3) in OA cartilage. Further mechanistic analysis showed thatSHP099 inhibited lipopolysaccharide (LPS)-induced Toll-like receptor (TLR) signaling mediated by nuclear factor kappa B (NF-κB) and PI3K-AKT signaling. Moreover, intra-articular injection of SHP099 also significantly attenuated OA progression, including joint synovitis and cartilage damage. These results indicated that allosteric inhibition of SHP2 might be a promising therapeutic strategy for the treatment of OA.

Identification of Transcription Factor Networks during Mouse Hindlimb Development.

Mammalian hindlimb development involves a variety of cells and the regulation of spatiotemporal molecular events, but regulatory networks of transcription factors contributing to hindlimb morphogenesis are not well understood. Here, we identified transcription factor networks during mouse hindlimb morphology establishment through transcriptome analysis. We used four stages of embryonic hindlimb transcription profiles acquired from the Gene Expression Omnibus database (GSE30138), including E10.5, E11.5, E12.5 and E13.5, to construct a gene network using Weighted Gene Co-expression Network Analysis (WGCNA), and defined seven stage-associated modules. After filtering 7625 hub genes, we further prioritized 555 transcription factors with AnimalTFDB3.0. Gene ontology enrichment showed that transcription factors of different modules were enriched in muscle tissue development, connective tissue development, embryonic organ development, skeletal system morphogenesis, pattern specification process and urogenital system development separately. Six regulatory networks were constructed with key transcription factors, which contribute to the development of different tissues. Knockdown of four transcription factors from regulatory networks, including Sox9, Twist1, Snai2 and Klf4, showed that the expression of limb-development-related genes was also inhibited, which indicated the crucial role of transcription factor networks in hindlimb development.

Microtubule Stabilization Enhances the Chondrogenesis of Synovial Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are well known for their multi-directional differentiation potential and are widely applied in cartilage and bone disease. Synovial mesenchymal stem cells (SMSCs) exhibit a high proliferation rate, low immunogenicity, and greater chondrogenic differentiation potential. Microtubule (MT) plays a key role in various cellular processes. Perturbation of MT stability and their associated proteins is an underlying cause for diseases. Little is known about the role of MT stabilization in the differentiation and homeostasis of SMSCs. In this study, we demonstrated that MT stabilization via docetaxel treatment had a significant effect on enhancing the chondrogenic differentiation of SMSCs. MT stabilization inhibited the expression of Yes-associated proteins (YAP) and the formation of primary cilia in SMSCs to drive chondrogenesis. This finding suggested that MT stabilization might be a promising therapeutic target of cartilage regeneration.

Nitric Oxide Nanomotor Driving Exosomes-Loaded Microneedles for Achilles Tendinopathy Healing.

The microneedle (MN) provides a promising strategy for transdermal delivery of exosomes (EXO), in which the therapeutic effects and clinical applications are greatly reduced by the fact that EXO can only partially reach the injury site by passive diffusion. Here, we designed a detachable MN array to deliver EXO modified by a nitric oxide nanomotor (EXO/MBA) for Achilles tendinopathy (AT) healing. With the releasing of EXO/MBA, l-arginine was converted to nitric oxide by NOS or ROS as the driving force. Benefiting from the motion ability and the property of MPC tending to lower pH, EXO could accumulate at the injury site more efficiently. This work demonstrated that EXO/MBA-loaded MN notably suppressed the inflammation of AT, facilitated the proliferation of tendon cells, increased the expression of Col1a, and prevented extracellular matrix degradation, indicating its potential value in enthesiopathy healing and other related biomedical fields.

Genetic Dissection of Hypertrophic Cardiomyopathy with Myocardial RNA-Seq.

Hypertrophic cardiomyopathy (HCM) is an inherited disorder of the myocardium, and pathogenic mutations in the sarcomere genes myosin heavy chain 7 (MYH7) and myosin-binding protein C (MYBPC3) explain 60%-70% of observed clinical cases. The heterogeneity of phenotypes observed in HCM patients, however, suggests that novel causative genes or genetic modifiers likely exist. Here, we systemically evaluated RNA-seq data from 28 HCM patients and nine healthy controls with pathogenic variant identification, differential expression analysis, and gene co-expression and protein-protein interaction network analyses. We identified 43 potential pathogenic variants in 19 genes in 24 HCM patients. Genes with more than one variant included the following: MYBPC3, TTN, MYH7, PSEN2, and LDB3. A total of 2538 protein-coding genes, six microRNAs (miRNAs), and 1617 long noncoding RNAs (lncRNAs) were identified differentially expressed between the groups, including several well-characterized cardiomyopathy-related genes (ANKRD1, FHL2, TGFB3, miR-30d, and miR-154). Gene enrichment analysis revealed that those genes are significantly involved in heart development and physiology. Furthermore, we highlighted four subnetworks: mtDNA-subnetwork, DSP-subnetwork, MYH7-subnetwork, and MYBPC3-subnetwork, which could play significant roles in the progression of HCM. Our findings further illustrate that HCM is a complex disease, which results from mutations in multiple protein-coding genes, modulation by non-coding RNAs and perturbations in gene networks.

Genome-Wide Analysis of MicroRNA-related Single Nucleotide Polymorphisms (SNPs) in Mouse Genome.

MicroRNAs are widely referred to as gene expression regulators for different diseases. The integration between single nucleotide polymorphisms (SNPs) and miRNAs has been associated with both human and animal diseases. In order to gain new insights on the effects of SNPs on miRNA and their related sequences, we steadily characterized a whole mouse genome miRNA related SNPs, analyzed their effects on the miRNA structural stability and target alteration. In this study, we collected 73643859 SNPs across the mouse genome, analyzed 1187 pre-miRNAs and 2027 mature miRNAs. Upon mapping the SNPs, 1700 of them were identified in 702 pre-miRNAs and 609 SNPs in mature miRNAs. We also discovered that SNP densities of the pre-miRNA and mature miRNAs are lower than the adjacent flanking regions. Also the flanking regions far away from miRNAs appeared to have higher SNP density. In addition, we also found that transitions were more frequent than transversions in miRNAs. Notably, 841 SNPs could change their corresponding miRNA's secondary structure from stable to unstable. We also performed target gain and loss analysis of 163 miRNAs and our results showed that few miRNAs remained unchanged and many miRNAs from wild mice gained target site. These results outline the first case of SNP variations in the mouse whole genome scale. Those miRNAs with changes in structure or target could be of interest for further studies.

A multiplex SNP genotyping by allele-specificspecific PCR based on stem-loop and universal fluorescent primers of Chr1daxin mice.

Single nucleotide polymorphisms (SNPs) are one of the most common markers in mammals. Rapid, accurate, and multiplex typing of SNPs is critical for subsequent biological and genetic research. In this study, we have developed a novel method for multiplex genotyping SNPs in mice. The method involves allele-specific PCR amplification of genomic DNA with two stem-loop primers accompanied by two different universal fluorescent primers. Blue and green fluorescent signals were conveniently detected on a DNA sequencer. We verified four SNPs of 65 mice based on the novel method, and it is well suited for multiplex genotyping as it requires only one reaction per sample in a single tube with multiplex PCR. The use of universal fluorescent primers greatly reduces the cost of designing different fluorescent probes for each SNP. Therefore, this method can be applied to many biological and genetic studies, such as multiple candidate gene testing, genome-wide association study, pharmacogenetics, and medical diagnostics.

Candidate Regulators of Dyslipidemia in Chromosome 1 Substitution Lines Using Liver Co-Expression Profiling Analysis.

Dyslipidemia is a major risk factor for cardiovascular disease. Although many genetic factors have been unveiled, a large fraction of the phenotypic variance still needs further investigation. Chromosome 1 (Chr 1) harbors multiple gene loci that regulate blood lipid levels, and identifying functional genes in these loci has proved challenging. We constructed a mouse population, Chr 1 substitution lines (C1SLs), where only Chr 1 differs from the recipient strain C57BL/6J (B6), while the remaining chromosomes are unchanged. Therefore, any phenotypic variance between C1SLs and B6 can be attributed to the differences in Chr 1. In this study, we assayed plasma lipid and glucose levels in 13 C1SLs and their recipient strain B6. Through weighted gene co-expression network analysis of liver transcriptome and "guilty-by-association" study, eight associated modules of plasma lipid and glucose were identified. Further joint analysis of human genome wide association studies revealed 48 candidate genes. In addition, 38 genes located on Chr 1 were also uncovered, and 13 of which have been functionally validated in mouse models. These results suggest that C1SLs are ideal mouse models to identify functional genes on Chr 1 associated with complex traits, like dyslipidemia, by using gene co-expression network analysis.

miR-505-3p is a repressor of the puberty onset in female mice.

Puberty onset is a complex trait regulated by multiple genetic and environmental factors. In this study, we narrowed a puberty related QTL region down to a 1.7 Mb region on chromosome X in female mice and inferred miR-505-3p as the functional gene. We conducted ectopic expression of miR-505-3p in the hypothalamus of prepubertal female mice through lentivirus-mediated orthotopic injection. The impact of miR-505-3p on female puberty was evaluated by the measurement of pubertal/reproduction events and histological analysis. The results showed that female mice with overexpression of miR-505-3p in the hypothalamus manifested later puberty onset timing both in vaginal opening and ovary maturation, followed by weaker fertility lying in the longer interval time between mating and delivery, higher abortion rate and smaller litter size. We also constructed miR-505-3p knockout mice by CRISPR/Cas9 technology. MiR-505-3p knockout female mice showed earlier vaginal opening timing, higher serum gonadotrophin and higher expression of puberty-related gene in the hypothalamus than their wild type littermates. Srsf1 was proved to be the target gene of miR-505-3p that played the major role in this process. The results of RNA Immunoprecipitation-sequencing showed that SRSF1 (or SF2), the protein product of Srsf1 gene, mainly bound to ribosome protein (RP) mRNAs in GT1-7 cells. The collective evidence implied that miR-505-3p/SRSF1/RP could play a role in the sexual maturation regulation of mammals.

Characterization of the dynamic change of microRNA expression in mice hypothalamus during the time of female puberty.

Puberty onset is a milestone in sexual development. A tumor suppress gene (TSG) network had been reported to be involved in the regulation of female puberty onset. The observations in rodents and primates showed a potential link between microRNAs and puberty onset. To figure out what miRNAs play roles in this important biological process, profilings of microRNAs in the hypothalamus of female mice from three different pubertal stages, juvenile [postnatal day (P10)], early pubertal (P25) and pubertal (P30) were performed on the Affymetrix GeneChip miRNA 3.0 Arrays, the cerebral cortex (CTX) was used as a control tissue. 20 miRNAs were shown to be differentially expressed in hypothalamus (fold change > 1.5, P < 0.05), but not in CTX during the transition from juvenile to pubertal. Four of them were validated by real-time quantitative RT-PCR (qRT-PCR) method. 1018 genes were predicted as the targets of these miRNAs. Further bioinformatics analysis suggested that these target genes were involved in many important signaling pathways, especially in the cancer related pathways. We also found that about 90% of these target genes were expressed in the hypothalamus, as well as in the immortalized GnRH-producing GT1-7 cells, which provided additional evidence that these miRNAs could be female puberty onset related. Here we present a novel comprehensive data set of miRNA gene expression during the puberty onset; and it provides an important recourse for the future functional characterization of individual miRNAs and their targets in mouse hypothalamus and in GT1-7 cells.

Sequence analysis of chromosome 1 revealed different selection patterns between Chinese wild mice and laboratory strains.

Both natural and artificial selection play a critical role in animals' adaptation to the environment. Detection of the signature of selection in genomic regions can provide insights for understanding the function of specific phenotypes. It is generally assumed that laboratory mice may experience intense artificial selection while wild mice more natural selection. However, the differences of selection signature in the mouse genome and underlying genes between wild and laboratory mice remain unclear. In this study, we used two mouse populations: chromosome 1 (Chr 1) substitution lines (C1SLs) derived from Chinese wild mice and mouse genome project (MGP) sequenced inbred strains and two selection detection statistics: Fst and Tajima's D to identify the signature of selection footprint on Chr 1. For the differentiation between the C1SLs and MGP, 110 candidate selection regions containing 47 protein coding genes were detected. A total of 149 selection regions which encompass 7.215 Mb were identified in the C1SLs by Tajima's D approach. While for the MGP, we identified nearly twice selection regions (243) compared with the C1SLs which accounted for 13.27 Mb Chr 1 sequence. Through functional annotation, we identified several biological processes with significant enrichment including seven genes in the olfactory transduction pathway. In addition, we searched the phenotypes associated with the 47 candidate selection genes identified by Fst. These genes were involved in behavior, growth or body weight, mortality or aging, and immune systems which align well with the phenotypic differences between wild and laboratory mice. Therefore, the findings would be helpful for our understanding of the phenotypic differences between wild and laboratory mice and applications for using this new mouse resource (C1SLs) for further genetics studies.

Genome Sequencing of Chromosome 1 Substitution Lines Derived from Chinese Wild Mice Revealed a Unique Resource for Genetic Studies of Complex Traits.

Mouse resources such as Collaborative Cross, outbred stocks, Hybrid Mouse Diversity Panel, and chromosome substitution strains have been instrumental to many progresses in the studies of complex traits genetics. We have established a population of chromosome 1 (Chr 1) substitution lines (C1SLs) in which donor chromosomes were derived from Chinese wild mice. Genome sequencing of 18 lines of this population showed that Chr 1 had been replaced by the donor chromosome. About 4.5 million unique single nucleotide polymorphisms and indels were discovered on Chr 1, of which 1.3 million were novel. Compared with sequenced classical inbred strains, Chr 1 of each C1SL had fivefold more variants, and more loss of function and potentially regulatory variants. Further haplotype analysis showed that the donor chromosome accumulated more historical recombination events, with the largest haplotype block being only 100 kb, and about 57% of the blocks were <1 kb. Subspecies origin analysis showed that these chromosomes had a mosaic genome structure that dominantly originated from Mus musculus musculus and M. m. castaneus subspecies, except for the C57BL/6J-Chr1KM line from M. m. domesticus In addition, phenotyping four of these lines on blood biochemistry suggested that there were substantial phenotypic variations among our lines, especially line C57BL/6J-Chr1HZ and donor strain C57BL/6J. Further gene ontology enrichment revealed that the differentially expressed genes among liver-expressed genes between C57BL/6J and C57BL/6J-Chr1HZ were enriched in lipid metabolism biological processes. All these characteristics enable C1SLs to be a unique resource for identifying and fine mapping quantitative trait loci on mouse Chr 1, and carrying out systems genetics studies of complex traits.

A novel three-round multiplex PCR for SNP genotyping with next generation sequencing.

Owing to the high throughput and low cost, next generation sequencing has attracted much attention for SNP genotyping application for researchers. Here, we introduce a new method based on three-round multiplex PCR to precisely genotype SNPs with next generation sequencing. This method can as much as possible consume the equivalent amount of each pair of specific primers to largely eliminate the amplification discrepancy between different loci. After the PCR amplification, the products can be directly subjected to next generation sequencing platform. We simultaneously amplified 37 SNP loci of 757 samples and sequenced all amplicons on ion torrent PGM platform; 90.5 % of the target SNP loci were accurately genotyped (at least 15×) and 90.4 % amplicons had uniform coverage with a variation less than 50-fold. Ligase detection reaction (LDR) was performed to genotype the 19 SNP loci (as part of the 37 SNP loci) with 91 samples randomly selected from the 757 samples, and 99.5 % genotyping data were consistent with the next generation sequencing results. Our results demonstrate that three-round PCR coupled with next generation sequencing is an efficient and economical genotyping approach. Graphical Abstract The schematic diagram of three-round PCR.

[The reason and treatment of portal vein thrombosis in patients with portal hypertension postoperation].

OBJECTIVE: To investigate reason and the management of portal vein thrombosis in patients with portal hypertension postoperatively. METHODS: 329 patients with portal hypertension in liver cirrhosis who had splenectomy was reviewed from 1992 to 2001. In whom 43 (13.1%) patients with portal vein thrombosis postoperative were analyzed. RESULTS: In these patients, except 1 died for portal vein phlebitis, all patients were recovered. There are 138 patients who underwent splenectomy or splenectomy and devascularization, 26 (18.8%) of them had thrombosis. 191 patients underwent splenectomy and portacaval or portasplenic shut, 17 (8.9%) of them had thrombosis. The data of these two groups have significant difference (chi(2) = 8.44, P < 0.01). CONCLUSIONS: Thrombocytosis postsplenectomy as well as the changes of portal hemodynamics is the main reason of portal vein thrombosis. Portal vein thrombosis is also in association with the operative ways. Operation standardization, dynamic examining platelet count, routine color ultrasonography examining and early anticoagulation therapy are the effective methods in preventing and managing portal thrombosis postoperation for portal hypertension.