Unmodificated stepless regulation of CRISPR/Cas12a multi-performance.

As CRISPR technology is promoted to more fine-divided molecular biology applications, its inherent performance finds it increasingly difficult to cope with diverse needs in these different fields, and how to more accurately control the performance has become a key issue to develop CRISPR technology to a new stage. Herein, we propose a CRISPR/Cas12a regulation strategy based on the powerful programmability of nucleic acid nanotechnology. Unlike previous difficult and rigid regulation of core components Cas nuclease and crRNA, only a simple switch of different external RNA accessories is required to change the reaction kinetics or thermodynamics, thereby finely and almost steplessly regulating multi-performance of CRISPR/Cas12a including activity, speed, specificity, compatibility, programmability and sensitivity. In particular, the significantly improved specificity is expected to mark advance the accuracy of molecular detection and the safety of gene editing. In addition, this strategy was applied to regulate the delayed activation of Cas12a, overcoming the compatibility problem of the one-pot assay without any physical separation or external stimulation, and demonstrating great potential for fine-grained control of CRISPR. This simple but powerful CRISPR regulation strategy without any component modification has pioneering flexibility and versatility, and will unlock the potential for deeper applications of CRISPR technology in many finely divided fields.

PGRN inhibits CD8+T cell recruitment and promotes breast cancer progression by up-regulating ICAM-1 on TAM.

BACKGROUND: Tumor microenvironment actually reduces antitumor effect against the immune attack by exclusion of CD8+T cells. Progranulin (PGRN) is a multifunctional growth factor with significant pathological effects in multiple tumors; however, its role in immunity evasion of breast cancer (BCa) is not completely understood. METHODS: We depleted GRN (PGRN gene) genetically in mice or specifically in PY8119 murine BCa cell line, and mouse models of orthotopic or subcutaneous transplantation were used. Chimeric mice-deficient of PGRN (Grn-/-) in bone marrow (BM) compartment was also generated. Association of PGRN expression with chemokine production or BCa development was investigated by histological and immunological assays. RESULTS: We found PGRN was involved in exhaustion of cytotoxic CD8+T cell in BCa with the increasing expressions of M2 markers and intercellular cell adhesion molecule-1 (ICAM-1) on macrophages. Specifically, ablation of PGRN in PY8119 cells reduced tumor burden, accompanied by the infiltrating of cytotoxic CD8+T cells into tumor nests. Moreover, our result revealed that blockade of PD-1 in PGRN-depleted tumors exhibited better antitumor effect in vivo and significantly decreased tumor burden. CONCLUSION: These findings suggest that inhibition of PGRN may act as a potential immune-therapeutic strategy by recovering infiltration of CD8+T cell in BCa tissue and thereby enhancing the response to anti-PD-1 therapy.

miR-486-5p Attenuates Steroid-Induced Adipogenesis and Osteonecrosis of the Femoral Head Via TBX2/P21 Axis.

Enhanced adipogenic differentiation of mesenchymal stem cells (MSCs) is considered as a major risk factor for steroid-induced osteonecrosis of the femoral head (SOFNH). The role of microRNAs during this process has sparked interest. miR-486-5p expression was down-regulated significantly in femoral head bone tissues of both SONFH patients and rat models. The purpose of this study was to reveal the role of miR-486-5p on MSCs adipogenesis and SONFH progression. The present study showed that miR-486-5p could significantly inhibit adipogenesis of 3T3-L1 cells by suppressing mitotic clonal expansion (MCE). And upregulated expression of P21, which was caused by miR-486-5p mediated TBX2 decrease, was responsible for inhibited MCE. Further, miR-486-5p was demonstrated to effectively inhibit steroid-induced fat formation in the femoral head and prevented SONFH progression in a rat model. Considering the potent effects of miR-486-5p on attenuating adipogenesis, it seems to be a promising target for the treatment of SONFH.

MiR-601-induced BMSCs senescence accelerates steroid-induced osteonecrosis of the femoral head progression by targeting SIRT1.

BACKGROUND: The imbalance between osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is not only the primary pathological feature but also a major contributor to the pathogenesis of steroid-induced osteonecrosis of the femoral head (SONFH). Cellular senescence is one of the main causes of imbalanced BMSCs differentiation. The purpose of this study was to reveal whether cellular senescence could participate in the progression of SONFH and the related mechanisms. METHODS: The rat SONFH model was constructed, and rat BMSCs were extracted. Aging-related indicators were detected by SA-ß-Gal staining, qRT-PCR and Western Blot experiments. Using H2O2 to construct a senescent cell model, and overexpressing and knocking down miR-601 and SIRT1 in hBMSCs, the effect on BMSCs differentiation was explored by qRT-PCR, Western Blot experiment, oil red O staining (ORO), alizarin red staining (ARS), and luciferase reporter gene experiment. A rat SONFH model was established to test the effects of miR-601 and metformin in vivo. RESULTS: The current study showed that glucocorticoids (GCs)-induced BMSCs senescence, which caused imbalanced osteogenesis and adipogenesis of BMSCs, was responsible for the SONFH progression. Further, elevated miR-601 caused by GCs was demonstrated to contribute to BMSCs senescence through targeting SIRT1. In addition, the anti-aging drug metformin was shown to be able to alleviate GCs-induced BMSCs senescence and SONFH progression. CONCLUSIONS: Considering the role of BMSCs aging in the progression of SONFH, this provides a new idea for the prevention and treatment of SONFH.

PDK1-stabilized LncRNA SPRY4-IT1 promotes breast cancer progression via activating NF-κB signaling pathway.

Pyruvate dehydrogenase kinase 1 (PDK1) is a widely known glycolytic enzyme, and some evidence showed that PDK1 promoted breast cancer by multiple approaches. However, very few lncRNAs have been identified to be associated with PDK1 in breast cancer in previous research. In this study, we found that lncRNA sprouty4-intron transcript 1 (SPRY4-IT1) was regulated by PDK1 with correlation analysis, and PDK1 upregulated SPRY4-IT1 remarkably in breast cancer cells, as PDK1 interacted with SPRY4-IT1 in the nucleus and significantly enhanced the stability of SRPY4-IT1. Furthermore, SPRY4-IT1 was highly expressed in breast cancer, significantly promoted the proliferation and inhibited apoptosis of breast cancer cells. In terms of mechanism, SPRY4-IT1 inhibited the transcription of NFKBIA and the expression of IκBα, thus promoting the formation of p50/p65 complex and activating NF-κB signaling pathway, which facilitated survival of breast cancer cells. Therefore, our finding reveals that PDK1/SPRY4-IT1/NFKBIA axis plays a crucial role that promoting tumor progression, and SPRY4-IT1 knockdown incombined with PDK1 inhibitor is promising to be a new therapeutic strategy in breast cancer.

Dual-aptamer-engineered M1 macrophage with enhanced specific targeting and checkpoint blocking for solid-tumor immunotherapy.

Chimeric antigen receptor T (CAR-T) cell therapy has faced a series of challenges and has shown very little efficacy in solid tumors to date. Although genetically engineered macrophages have achieved definite therapeutic effect in solid tumors, heterogeneous expression of engineered proteins and the potential for toxicity limit further applications. Herein, we propose a nongenetic and simple macrophage cell engineering strategy through glycan metabolic labeling and click reaction for the treatment of solid tumors. The aptamer-engineered M1 macrophage (ApEn-M1) showed enhanced active targeting ability for tumor cells in vitro and in vivo, resulting in significant cytotoxicity effects. Moreover, ApEn-M1 exhibited superior antitumor efficacy in a breast cancer xenograft mouse model and a lung metastasis mouse model of breast cancer. Interestingly, the ApEn-M1 could reprogram the immunity microenvironment by increasing T cell infiltration and enhancing T cell activity in the tumor region. Additionally, the administration of ApEn-M1 showed no obvious systemic side effects. With glycan metabolic labeling, the macrophages could be efficiently labeled with aptamers on the cell surface via click reaction without genetic alteration or cell damage. Hence, this study serves as a proof of concept for cell-surface anchor engineering and expands the range of nongenetic macrophage cell engineering strategies.

The miR-345-3p/PPP2CA signaling axis promotes proliferation and invasion of breast cancer cells.

Breast cancer is the most common malignancy among women worldwide. Functional studies have demonstrated that miRNA dysregulation in many cases of cancer, in which miRNAs act as either oncogenes or tumor suppressor. Here we report that miR-345-3p is generally upregulated in breast cancer tissues and breast cancer cell lines. Overexpression and inhibition of miR-345-3p revealed its capacity in regulating proliferation and invasion of breast cancer cells. Further research identified protein phosphatase 2 catalytic subunit alpha (PPP2CA), a suppressor of AKT phosphorylation, as a candidate target of miR-345-3p. In vitro, miR-345-3p mimics promoted AKT phosphorylation by targeting its negative regulator, PPP2CA. Blocking miR-345-3p relieved its inhibition of PPP2CA, which attenuated PI3K-AKT signaling pathway. In vivo, inhibiting miR-345-3p by miR-345-3p-inhibition lentivirus suppressed tumor growth and invasiveness in mice. Together, the miR-345-3p/PPP2CA signaling axis exhibits tumor-promoting functions by regulating proliferation and invasion of breast cancer cells. These data provide a clue to novel therapeutic approaches for breast cancer.

Increased Protection of Earlier Use of Immunoprophylaxis in Preventing Perinatal Transmission of Hepatitis B Virus.

BACKGROUND: Passive-active immunoprophylaxis against mother-to-child transmission (MTCT) of hepatitis B virus (HBV) recommends administering hepatitis B immunoglobulin (HBIG) and birth-dose hepatitis B vaccine in infants within 12 or 24 hours after birth. With this protocol, MTCT of HBV still occurs in 5-10% infants of HBV-infected mothers with positive hepatitis B e antigen (HBeAg). The present study aimed to investigate whether earlier administration of HBIG and hepatitis B vaccine after birth can further increase protection efficacy. METHODS: We conducted a prospective, multi-center observational study in infants born to mothers with HBV infection, in whom neonatal HBIG and birth dose hepatitis B vaccine were administered within one hour after birth. The infants were followed up for HBV markers at 7-14 months of age. RESULTS: A total of 1140 pregnant women with HBV were enrolled, and 982 infants (9 twins) of 973 mothers were followed up at 9.6 ± 1.9 months of age. HBIG and birth-dose vaccine were administered in newborn infants within a median of 0.17 (0.02-1.0) hours after birth. The overall rate of MTCT was 0.9% (9/982), with none (0%) of the 607 infants of HBeAg-negative mothers and 9 (2.4%) of 375 infants of HBeAg-positive mothers acquiring HBV. All 9 HBV-infected infants were born to mothers with HBV DNA >2.75 × 106 IU/mL. Maternal HBV DNA levels >2 × 106 IU/mL were an independent risk factor (odds ratio, 10.627; 95% confidence interval, 2.135-∞) for immunoprophylaxis failure. CONCLUSIONS: Earlier use (within 1 hour after birth) of HBIG and hepatitis B vaccine can provide better protection efficacy against MTCT of HBV.

A genome-wide association study reveals a substantial genetic basis underlying the Ebbinghaus illusion.

The Ebbinghaus illusion (EI) is an optical illusion of relative size perception that reflects the contextual integration ability in the visual modality. The current study investigated the genetic basis of two subtypes of EI, EI overestimation, and EI underestimation in humans, using quantitative genomic analyses. A total of 2825 Chinese adults were tested on their magnitudes of EI overestimation and underestimation using the method of adjustment, a standard psychophysical protocol. Heritability estimation based on common single nucleotide polymorphisms (SNPs) revealed a moderate heritability (34.3%) of EI overestimation but a nonsignificant heritability of EI underestimation. A meta-analysis of two phases (phase 1: n = 1986, phase 2: n = 839) of genome-wide association study (GWAS) discovered 1969 and 58 SNPs reaching genome-wide significance for EI overestimation and EI underestimation, respectively. Among these SNPs, 55 linkage-disequilibrium-independent SNPs were associated with EI overestimation in phase 1 with genome-wide significance and their associations could be confirmed in phase 2 cohort. Gene-based analyses found seven genes to be associated with EI overestimation at the genome-wide level, two from meta-analysis, and five from classical two-stage analysis. Overall, this study provided consistent evidence for a substantial genetic basis of the Ebbinghaus illusion.

Genomic Analyses of Visual Cognition: Perceptual Rivalry and Top-Down Control.

Visual cognition in humans has traditionally been studied with cognitive behavioral methods and brain imaging, but much less with genetic methods. Perceptual rivalry, an important phenomenon in visual cognition, is the spontaneous perceptual alternation that occurs between two distinct interpretations of a physically constant visual stimulus (e.g., binocular rivalry stimuli) or a perceptually ambiguous stimulus (e.g., the Necker cube). The switching rate varies dramatically across individuals and can be voluntarily modulated by observers. Here, we adopted a genomic approach to systematically investigate the genetics underlying binocular rivalry, Necker cube rivalry and voluntary modulation of Necker cube rivalry in young Chinese adults (Homo sapiens, 81% female, 20 ± 1 years old) at multiple levels, including common single nucleotide polymorphism (SNP)-based heritability estimation, SNP-based genome-wide association study (GWAS), gene-based analysis, and pathway analysis. We performed a pilot GWAS in 2441 individuals and replicated it in an independent cohort of 943 individuals. Common SNP-based heritability was estimated to be 25% for spontaneous perceptual rivalry. SNPs rs184765639 and rs75595941 were associated with voluntary modulation, and imaging data suggested genotypic difference of rs184765639 in the surface area of the left caudal-middle frontal cortex. Additionally, converging evidence from multilevel analyses associated genes such as PRMT1 with perceptual switching rate, and MIR1178 with voluntary modulation strength. In summary, this study discovered specific genetic contributions to perceptual rivalry and its voluntary modulation in human beings. These findings may promote our understanding of psychiatric disorders, as perceptual rivalry is a potential psychiatric biomarker.SIGNIFICANCE STATEMENT Perceptual rivalry is an important visual phenomenon in which our perception of a physically constant visual input spontaneously switches between two different states. There are individual variations in perceptual switching rate and voluntary modulation strength. Our genomic analyses reveal several loci associated with these two kinds of variation. Because perceptual rivalry is thought to be relevant to and potentially an endophenotype for psychiatric disorders, these results may help understand not only visual cognition, but also psychiatric disorders.

Global urinary metabolic profiling of the osteonecrosis of the femoral head based on UPLC-QTOF/MS.

INTRODUCTION: Osteonecrosis of the femoral head (ONFH), one of the widespread orthopedic diseases with a decrease in bloodstream to the femoral head, is frequently accompanied by cellular death, trabecula fracture, and collapse of the articular surface. The exactly pathological mechanism of ONFH remains to explore and further identify. OBJECTIVES: The aim was to identify the global urinary metabolic profiling of ONFH and to detect biomarkers of ONFH. METHODS: Urine samples were collected from 26 ONFH patients and 26 healthy people. Ultra-performance liquid chromatography-quadrupole time of flight tandem mass spectrometry (UPLC-QTOF/MS) in combination with multivariate statistical analysis was developed and performed to identify the global urinary metabolic profiling of ONFH. RESULTS: The urinary metabolic profiling of ONFH group was significantly separated from the control group by multivariate statistical analysis. 33 distinctly differential metabolites were detected between the ONFH patients and healthy people. Sulfate, urea, Deoxycholic acid and PE(14:0/14:1(9Z)) were screened as the potential biomarkers of ONFH. In addition, the up/down-regulation of sulfur metabolism, cysteine and methionine metabolism, glycerophospholipid metabolism, and histidine metabolism were clearly be associated with the ONFH pathogenic progress. CONCLUSION: Our results suggested that metabolomics could serve as a promising approach for identifying the diagnostic biomarkers and elucidating the pathological mechanism of ONFH.

Nature vs. nurture in human sociality: multi-level genomic analyses of social conformity.

Social conformity is fundamental to human societies and has been studied for more than six decades, but our understanding of its mechanisms remains limited. Individual differences in conformity have been attributed to social and cultural environmental influences, but not to genes. Here we demonstrate a genetic contribution to conformity after analyzing 1,140 twins and single-nucleotide polymorphism (SNP)-based studies of 2,130 young adults. A two-step genome-wide association study (GWAS) revealed replicable associations in 9 genomic loci, and a meta-analysis of three GWAS with a sample size of ~2,600 further confirmed one locus, corresponding to the NAV3 (Neuron Navigator 3) gene which encodes a protein important for axon outgrowth and guidance. Further multi-level (haplotype, gene, pathway) GWAS strongly associated genes including NAV3, PTPRD (protein tyrosine phosphatase receptor type D), ARL10 (ADP ribosylation factor-like GTPase 10), and CTNND2 (catenin delta 2), with conformity. Magnetic resonance imaging of 64 subjects shows correlation of activation or structural features of brain regions with the SNPs of these genes, supporting their functional significance. Our results suggest potential moderate genetic influence on conformity, implicate several specific genetic elements in conformity and will facilitate further research on cellular and molecular mechanisms underlying human conformity.

Comparison of hepatitis B viral loads and viral antigen levels in child-bearing age women with and without pregnancy.

BACKGROUND: Pregnancy is a unique physiological condition with the cellular immune functions compromised at some extents to allow the mature of growing fetus. Whether pregnancy may influence the replication of hepatitis B virus (HBV) is less studied. The present study aimed to investigate the influence of pregnancy on the replication of HBV and expression of viral antigens by comparing the levels of HBV DNA and viral antigens in pregnant and non-pregnant women. METHODS: A total of 727 HBsAg-positive serum samples, collected from 214 pregnant women and 513 non-pregnant women of childbearing age, were included. Based on the pregnancy status, subjects were divided into four groups: nulliparous (n = 158), pregnant (n = 214), 7-12 months postpartum (n = 170), and 2-5 years postpartum (n = 185). The levels of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) were quantitatively measured with microparticle enzyme immunoassay. HBV DNA levels were detected by fluorescent real-time PCR. RESULTS: The median ages of four groups were 25.0, 25.3, 26.2 and 29.3 years, respectively (p < 0.01). HBeAg-positive proportions were 34.2, 33.6, 35.3 and 29.2%, respectively (p = 0.624). HBV DNA levels in HBeAg-positive women were higher than those in HBeAg-negative women (7.88 vs 2.62 log IU/ml, p < 0.001). HBV DNA levels in the four groups with positive HBeAg were 7.8, 7.7, 8.0 and 8.0 log IU/ml, respectively (p = 0.057), while HBsAg titers were 4.4, 4.5, 4.6 and 4.8 log IU/ml (p = 0.086) and HBeAg titers were 3.1, 3.0, 3.1 and 3.0 log S/CO (p = 0.198). In the four groups with negative HBeAg, HBV DNA levels were 2.3, 2.6, 2.5 and 2.8 log IU/ml, respectively (p = 0.085), while HBsAg titers were 3.1, 3.3, 3.3 and 3.0 log IU/ml (p = 0.06). CONCLUSIONS: Serum levels of HBV DNA and viral antigens showed no significant changes in nulliparous, pregnant, and postpartum women, regardless of the HBeAg status. The results indicate that pregnancy has little influence on the replication of HBV and the expression of viral antigens.

Cartilaginous Metabolomic Study Reveals Potential Mechanisms of Osteophyte Formation in Osteoarthritis.

Osteophyte is one of the inevitable consequences of progressive osteoarthritis with the main characteristics of cartilage degeneration and endochondral ossification. The pathogenesis of osteophyte formation is not fully understood to date. In this work, metabolomic approaches were employed to explore potential mechanisms of osteophyte formation by detecting metabolic variations between extracts of osteophyte cartilage tissues (n = 32) and uninvolved control cartilage tissues (n = 34), based on the platform of ultraperformance liquid chromatography tandem quadrupole time-of-flight mass spectrometry, as well as the use of multivariate statistic analysis and univariate statistic analysis. The osteophyte group was significantly separated from the control group by the orthogonal partial least-squares discriminant analysis models, indicating that metabolic state of osteophyte cartilage had been changed. In total, 28 metabolic variations further validated by mass spectrum (MS) match, tandom mass spectrum (MS/MS) match, and standards match mainly included amino acids, sulfonic acids, glycerophospholipids, and fatty acyls. These metabolites were related to some specific physiological or pathological processes (collagen dissolution, boundary layers destroyed, self-restoration triggered, etc.) which might be associated with the procedure of osteophyte formation. Pathway analysis showed phenylalanine metabolism (PI = 0.168, p = 0.004) was highly correlative to this degenerative process. Our findings provided a direction for targeted metabolomic study and an insight into further reveal the molecular mechanisms of ostophyte formation.

Invalidation of mitophagy by FBP1-mediated repression promotes apoptosis in breast cancer.

Fructose-1,6-bisphosphatase 1, a rate-limiting enzyme in gluconeogenesis, was recently shown to be a tumor suppressor. However, the functions of fructose-1,6-bisphosphatase 1 in the regulation of mitophagy and apoptosis remain unknown. Here, we investigated the effects of fructose-1,6-bisphosphatase 1 on mitophagy and apoptosis as well as their underlying mechanisms in breast cancer cells. In this work, the messenger RNA and protein expression of various molecules were determined by quantitative realtime polymerase chain reaction and western blot, respectively. Gene-expression correlations were obtained from The Cancer Genome Atlas Breast Cancer database and analyzed using cBioPortal. The levels of cellular reactive oxygen species and apoptotic index were detected by flow cytometry. The mitochondrial membrane potentials were assessed with a JC-1 fluorescent sensor. Subcellular structures were observed under a transmission electron microscope. The intracellular distribution of translocase of outer membrane 20 was detected by immunofluorescence staining. Protein-protein interactions were analyzed by immunoprecipitation. Our results indicated that fructose-1,6-bisphosphatase 1 expression was negatively correlated with autophagy level in breast cancer. Fructose-1,6-bisphosphatase 1 restrained autophagy activity by increasing the level of p62 and decreasing the levels of LC3 and Beclin 1. Additionally, fructose-1,6-bisphosphatase 1 promoted cell apoptosis by upregulating the levels of intracellular ROS and expression of pro-apoptotic proteins such as cleaved PARP, cleaved Caspase 3, and Bax and downregulating the levels of anti-apoptotic proteins such as PARP, Caspase 3, and Bcl-2. Finally, fructose-1,6-bisphosphatase 1 limited the efficient removal of diseased mitochondria and reduced the messenger RNA and protein expressions of HIF-1α, BNIP3L/NIX, and BNIP3. More importantly, fructose-1,6-bisphosphatase 1 facilitated co-action between Bcl-2 and Beclin 1, which may be important in the mechanism of fructose-1,6-bisphosphatase 1-mediated mitophagy inhibition. In summary, loss of mitophagy by fructose-1,6-bisphosphatase 1-mediated repression promotes apoptosis in breast cancer.

Pyruvate kinase M2 interacts with mammalian sterile 20-like kinase 1 and inhibits tamoxifen-induced apoptosis in human breast cancer cells.

Tamoxifen has been reported to be associated with antagonism of estrogen-mediated cell growth signaling and activation of estrogen receptor-independent apoptosis events. It has been demonstrated that mammalian sterile 20-like kinase 1 is a direct target of Caspases to amplify the apoptotic signaling pathway. Here, we presented that breast cancer MCF-7 and SKBR3 cells under treatment with 4-hydroxytamoxifen displayed decreased level of pyruvate kinase M2. Western blot results also showed that 4-hydroxytamoxifen induced the activity of pro-apoptotic protein Caspase-3 in MCF-7 and SKBR3 cells, as evidenced by the cleavage of mammalian sterile 20-like kinase 1 substrate in a dose-dependent manner. Co-immunoprecipitation and immunofluorescence experiments were performed to clarify the relationship between pyruvate kinase M2 and mammalian sterile 20-like kinase 1. The results indicated that mammalian sterile 20-like kinase 1 was associated with pyruvate kinase M2 in cultured mammalian cells, and the interaction between mammalian sterile 20-like kinase 1 and pyruvate kinase M2 was decreased in response to 4-hydroxytamoxifen treatment. In addition, knockdown of pyruvate kinase M2 upregulated the level of cleaved Caspase-3 and subsequently facilitated the nuclear translocation of mammalian sterile 20-like kinase 1. Our data further supplemented the extensive functions of pyruvate kinase M2 in mediating breast cancer cell viability by substantially abating the mammalian sterile 20-like kinase 1-mediated apoptosis. In summary, our results identified that mammalian sterile 20-like kinase 1 is a novel downstream target of pyruvate kinase M2, and knockdown of pyruvate kinase M2 contributes apoptosis via promoting nuclear translocation of mammalian sterile 20-like kinase 1 by enhancing Caspase-3-dependent cleavage.

Metabolic analysis of osteoarthritis subchondral bone based on UPLC/Q-TOF-MS.

Osteoarthritis (OA), one of the most widespread musculoskeletal joint diseases among the aged, is characterized by the progressive loss of articular cartilage and continuous changes in subchondral bone. The exact pathogenesis of osteoarthritis is not completely clear. In this work, ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC/Q-TOF-MS) in combination with multivariate statistical analysis was applied to analyze the metabolic profiling of subchondral bone from 42 primary osteoarthritis patients. This paper described a modified two-step method for extracting the metabolites of subchondral bone from primary osteoarthritis patients. Finally, 68 metabolites were identified to be significantly changed in the sclerotic subchondral bone compared with the non-sclerotic subchondral bone. Taurine and hypotaurine metabolism and beta-alanine metabolism were probably relevant to the sclerosis of subchondral bone. Taurine, L-carnitine, and glycerophospholipids played a vital regulation role in the pathological process of sclerotic subchondral bone. In the sclerotic process, beta-alanine and L-carnitine might be related to the increase of energy consumption. In addition, our findings suggested that the intra-cellular environment of sclerotic subchondral bone might be more acidotic and hypoxic compared with the non-sclerotic subchondral bone. In conclusion, this study provided a new insight into the pathogenesis of subchondral bone sclerosis. Our results indicated that metabolomics could serve as a promising approach for elucidating the pathogenesis of subchondral bone sclerosis in primary osteoarthritis. Graphical Abstract Metabolic analysis of osteoarthritis subchondral bone.

[Establishment and preliminary application of detection of Mycobacterium tuberculosis in sputum based on variable number tandem repeat].

OBJECTIVE: To establish a laboratory method for detection of Mycobacterium tuberculosis in sputum based on variable number tandem repeat (VNTR). METHODS: Mycobacterium tuberculosis was tested by VNTR and fluorescent quantitative reverse transcription polymerase chain reaction (FQ-PCR) in 130 sputum samples from patients with pulmonary tuberculosis and 200 specimens from patients with other lung diseases. According to the amplification conditions and clinical detection needs, MTUB21, MUTB04, QUB18, QUB26, QUB11b, MIRU31, MIRU10 and MIRU26 were selected as test targets. The results of VNTR and FQ-PCR were compared with Lowenstein-Jensen culture and clinical diagnosis, and analyzed by chi-square test. RESULTS: With the results of L-J culture as the standard, the sensitivity and specificity of VNTR were 93.1% (108/116) and 97.7% (209/214), and those of FQ-PCR were 94.0% (109/116) and 96.7% (207/214), respectively; no significant difference was observed between two groups (χ2=0.352, P=0.569). Using the clinical diagnosis as the standard, the sensitivity and specificity of VNTR were 86.9% (113/130) and 100% (200/200), and those of FQ-PCR were 87.7% (114/130) and 99.0% (198/200), respectively; the difference was not statistically significant (χ2=0.030, P=0.862). In 113 VNTR positive samples, the molecular codes differed from each other in 98.2% samples (111/113); only 2 samples had identical code (5-4-6-8-5-5-3-8). CONCLUSION: The study suggests that VNTR provides a promising method for diagnosis of clinical tuberculosis.

TROP2 is highly expressed in triple-negative breast cancer CTCs and is a potential marker for epithelial mesenchymal CTCs.

Circulating tumor cells (CTCs) are the seeds of distant metastases of malignant tumors and are associated with malignancy and risk of metastasis. However, tumor cells undergo epithelial-mesenchymal transition (EMT) during metastasis, leading to the emergence of different types of CTCs. Real-time dynamic molecular and functional typing of CTCs is necessary to precisely guide personalized treatment. Most CTC detection systems are based on epithelial markers that may fail to detect EMT CTCs. Therefore, it is clinically important to identify new markers of different CTC types. In this study, bioinformatics analysis and experimental assays showed that trophoblast cell surface antigen 2 (TROP2), a target molecule for advanced palliative treatment of triple-negative breast cancer (TNBC), was highly expressed in TNBC tissues and tumor cells. Furthermore, TROP2 can promote the migration and invasion of TNBC cells by upregulating EMT markers. The specificity and potential of TROP2 as an EMT-associated marker of TNBC CTCs were evaluated by flow cytometry, immunofluorescence, spiking experiments, and a well-established CTC assay. The results indicated that TROP2 is a potential novel CTC marker associated with EMT, providing a basis for more efficacious markers that encompass CTC heterogeneity in patients with TNBC.