A novel method for high-sensitivity detection of SARS-CoV-2 using dual double-quenched fluorescence probes.

SARS-CoV-2 has infected many people around the world, fast and accurate detection of the virus can help control the spread of the virus. RT-PCR is the gold standard method for SARS-CoV-2 detection. In this study, we improved the RT-PCR by proposing a novel method using dual double-quenched fluorescence probes. We used the improved probes to detect the plasmid DNA and RNA reference materials of SARS-CoV-2 respectively. When the improved probes were used, the background fluorescence intensity has reduced by 50%, the fluorescence increment has increased to 2.8 folds, and the Ct value has significantly reduced by 3 or more, indicating that the detection sensitivity has increased at least eight times. In addition, we also demonstrated that the improved probes have well performance in detecting SARS-CoV-2 with the minimum concentration of 6.2 copies/µL. This study will help biological companies develop better products for SARS-CoV-2 and other clinical pathogen infection.

Missense mutation of c.635 T > C in CAPN3 impairs muscle injury repair in a Limb-Girdel Muscular Dystropy Model.

Limb-girdle muscular dystrophy recessive 1 (LGMDR1), previously known as LGMD2A, is a specific LGMD caused by a gene mutation encoding the calcium-dependent neutral cysteine protease calpain-3 (CAPN3). In our study, the compound heterozygosity with two missense variants c.635 T > C (p.Leu212Pro) and c.2120A > G (p.Asp707Gly) was identified in patients with LGMDR1. However, the pathogenicity of c.635 T > C has not been investigated. To evaluate the effects of this novel likely pathogenic variant to the motor system, the mouse model with c.635 T > C variant was prepared by CRISPR/Cas9 gene editing technique. The pathological results revealed that a limited number of inflammatory cells infiltrated the endomyocytes of certain c.635 T > C homozygous mice at 10 months of age. Compared with wild-type mice, motor function was not significantly impaired in Capn3 c. 635 T > C homozygous mice. Western blot and immunofluorescence assays further indicated that the expression levels of the Capn3 protein in muscle tissues of homozygous mice were similar to those of wild-type mice. However, the arrangement and ultrastructural alterations of the mitochondria in the muscular tissues of homozygous mice were confirmed by electron microscopy. Subsequently, muscle regeneration of LGMDR1 was simulated using cardiotoxin (CTX) to induce muscle necrosis and regeneration to trigger the injury modification process. The repair of the homozygous mice was significantly worse than that of the control mice at day 15 and day 21 following treatment, the c.635 T > C variant of Capn3 exhibited a significant effect on muscle regeneration of homozygous mice and induced mitochondrial damage. RNA-sequencing results demonstrated that the expression levels of the mitochondrial-related functional genes were significantly downregulated in the mutant mice. Taken together, the results of the present study strongly suggested that the LGMDR1 mouse model with a novel c.635 T > C variant in the Capn3 gene was significantly dysfunctional in muscle injury repair via impairment of the mitochondrial function.

Overexpression of DNA (Cytosine-5)-Methyltransferase 1 (DNMT1) And DNA (Cytosine-5)-Methyltransferase 3A (DNMT3A) Is Associated with Aggressive Behavior and Hypermethylation of Tumor Suppressor Genes in Human Pituitary Adenomas.

BACKGROUND Alteration of DNA methylation of tumor suppressor genes (TSGs) is one of the most consistent epigenetic changes in human cancers. DNMTs play several important roles in DNA methylation and development of cancers. Regarding DNMTs protein expressions, little is known about the clinical significance and correlation with promoter methylation status of TSGs in human pituitary adenomas. MATERIAL AND METHODS We analyzed the protein expression of 3 DNMTs using immunohistochemistry and assessed DNA hypermethylation of RASSF1A, CDH13, CDH1, and CDKN2A (p16) in 63 pituitary adenomas. We examined associations between DNMTs expression and clinicopathological features or promoter methylation status of TSGs. RESULTS Overexpression of DNMTs was detected in pituitary adenomas. Frequencies of DNMT1 overexpression were significantly higher in macroadenomas, invasive tumors, and grade III and IV tumors. DNMT3A was frequently detected in invasive tumors and grade IV tumors. In addition, DNMT1 and DNMT3A were frequently detected in high-methylation tumors. Furthermore, in multivariate logistic regression, the significant association between DNMT1 or DNMT3A and high-methylation status persisted after adjusting for clinicopathological features. CONCLUSIONS Our findings suggested that tumor overexpression of DNMT1 and DNMT3A is associated with tumor aggressive behavior and high-methylation status in pituitary adenomas. Our data support a possible role of DNMT1 and DNMT3A in TSG promoter methylation leading to pituitary adenoma invasion and suggest that inhibition of DNMTs has the potential to become a new therapeutic approach for invasive pituitary adenoma.

Synthesis of uniform Pickering microspheres doped with quantum dot by microfluidic technology and its application in tumor marker.

Tumor markers play a significant role in early cancer diagnosis, evaluation of the extent of the disease, and monitoring of therapy response. In this study, we described the Pickering emulsion polymerization method to synthesize uniform magnetic/fluorescent microspheres. A Pickering-structure composed of a lot silica nanoparticle closely covered onto the quantum dot-encoded magnetic microbeads is designed and synthesized. The uniform magnetic/fluorescent microspheres were prepared using a microfluidic device and the performance of the microspheres synthesized by the instruments was evaluated by flow cytometry. To avoid fluorescence quenching and intrinsic toxicity, CdSe/ZnS core-shell quantum dot and Fe3O4 nanoparticle were successfully encapsulated into MFM microspheres using the microfluidic technology. Using this structure enables the facile realization of a theoretical 4 × 4 barcoding matrix combining two colors and four fluorescence intensity levels. Then, different optical codes were prepared by simple changing the emission wavelength and the intensity of the quantum dots. The resulting microsphere are combined with flow cytometer using two lasers for decoding of multiplex tumor markers. Moreover, the stability testing of microspheres demonstrated good performance for further application in detection of tumor markers as well. When applied for the high-throughput ultrasensitive detection of three tumor markers (CEA, CA125 and CA199) in a single sample, the detection limits of 0.027 ng/mL for CEA, 1.48 KU/L for CA125 and 1.09 KU/L for CA199 are achieved, which exhibit superior detection performance. Thus, Pickering-structure magnetic/fluorescent microspheres are promising for application in tumor markers.

Effectiveness and safety of leukapheresis in hyperleukocytic leukemias: a retrospective multicenter study.

Leukapheresis is an effective adjuvant therapy for leukemia patients with hyperleukocytosis, but few studies have reported recent data with modern modalities and comparisons among different leukemia types. We conducted a retrospective study on leukapheresis among 420 patients with AML, ALL and CML in four local centers. WBC counts decreased significantly (p < 0.001) postleukapheresis in all three cohorts. Clearance efficiency was higher in acute leukemia patients than CML patients (p < 0.01). Concomitant leukocytoreduction drugs improved WBC reduction only in AML patients (p < 0.05). Leukocyte, hemoglobin and platelet levels preleukapheresis might affect the clearance efficiency in AML and/or ALL patients. Hematological toxicities were the major concerns, but most of them were mild, and only 11 patients died of all causes within one week postleukapheresis. In conclusion, leukapheresis can safely reduce the leukemic burden, especially for patients with acute leukemias.

Cis-acting: A pattern of lncRNAs for gene regulation in induced pluripotent stem cells from patients with Down syndrome determined by integrative analysis of lncRNA and mRNA profiling data.

Down syndrome (DS), caused by the trisomy of chromosome 21, is one of the common chromosomal disorders, the main clinical manifestations of which are delayed nervous development and intellectual disability. Long non-coding RNAs (lncRNAs) have critical roles in various biological processes, including cell growth, cell cycle regulation and differentiation. The roles of abnormally expressed lncRNAs have been previously reported; however, the biological functions and regulatory patterns of lncRNAs in DS have remained largely elusive. The aim of the present study was to perform a whole-genome-wide identification of lncRNAs and mRNAs associated with DS. In addition, global expression profiling analysis of DS-induced pluripotent stem cells was performed and differentially expressed (DE) lncRNAs and mRNAs were screened. Furthermore, the target genes and functions of the DE lncRNAs were predicted using Gene Ontology annotation and Kyoto Encyclopedia of Genes and Genomes signaling pathway enrichment analysis. The results revealed that the majority of the lncRNAs exerted functions in DS via cis-acting target genes. In addition, the results of the enrichment analysis indicated that these target genes were mainly involved in nervous and muscle development in DS. In conclusion, this integrative analysis using lncRNA and mRNA profiling provided novel insight into the pathogenesis of DS and it may promote the diagnosis and development of novel therapeutics for this disease.

Co-targeting CK2α and YBX1 suppresses tumor progression by coordinated inhibition of the PI3K/AKT signaling pathway.

Protein kinase CK2 alpha (CK2α) is involved in the development of multiple malignancies. Overexpression of Y-box binding protein 1 (YBX1) is related to tumor proliferation, drug resistance, and poor prognosis. Studies have demonstrated that both CK2 and YBX1 could regulate the PI3K/AKT pathway. In addition, we predicted that CK2 might be the upstream kinase of YBX1 through the Human Protein Reference Database (HPRD). Herein, we hypothesize that CK2 may interact with YBX1 and they regulate the PI3K/AKT signaling pathway together. Expressions of CK2α and YBX1 in cancer cell lines were evaluated by immunoblotting. The results showed that CK2α could regulate the expression of YBX1 at the transcriptional level, which is dependent on its enzymatic activity. Synergistic effects of PI3K/AKT pathway inactivation could be observed through combined inhibition of CK2α and YBX1, and YBX1 was required for CK2α-induced PI3K/AKT pathway activation. Further results demonstrated that CK2α could interact with YBX1 and PI3K/AKT antagonist decreased cell resistance to doxorubicin induced by co-activation of CK2α and YBX1. These results indicated that combined inhibition of CK2α and YBX1 showed synergistic effects in inactivating the PI3K/AKT signaling pathway and may be one of the mechanisms involved in tumor growth and migration.