A Method To Prevent SARS-CoV-2 IgM False Positives in Gold Immunochromatography and Enzyme-Linked Immunosorbent Assays.

We set out to investigate the interference factors that led to false-positive novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) IgM detection results using gold immunochromatography assay (GICA) and enzyme-linked immunosorbent assay (ELISA) and the corresponding solutions. GICA and ELISA were used to detect SARS-CoV-2 IgM in 86 serum samples, including 5 influenza A virus (Flu A) IgM-positive sera, 5 influenza B virus (Flu B) IgM-positive sera, 5 Mycoplasma pneumoniae IgM-positive sera, 5 Legionella pneumophila IgM-positive sera, 6 sera of HIV infection patients, 36 rheumatoid factor IgM (RF-IgM)-positive sera, 5 sera from hypertensive patients, 5 sera from diabetes mellitus patients, and 14 sera from novel coronavirus infection disease 19 (COVID-19) patients. The interference factors causing false-positive reactivity with the two methods were analyzed, and the urea dissociation test was employed to dissociate the SARS-CoV-2 IgM-positive serum using the best dissociation concentration. The two methods detected positive SARS-CoV-2 IgM in 22 mid-to-high-level-RF-IgM-positive sera and 14 sera from COVID-19 patients; the other 50 sera were negative. At a urea dissociation concentration of 6 mol/liter, SARS-CoV-2 IgM results were positive in 1 mid-to-high-level-RF-IgM-positive serum and in 14 COVID-19 patient sera detected using GICA. At a urea dissociation concentration of 4 mol/liter and with affinity index (AI) levels lower than 0.371 set to negative, SARS-CoV-2 IgM results were positive in 3 mid-to-high-level-RF-IgM-positive sera and in 14 COVID-19 patient sera detected using ELISA. The presence of RF-IgM at mid-to-high levels could lead to false-positive reactivity of SARS-CoV-2 IgM detected using GICA and ELISA, and urea dissociation tests would be helpful in reducing SARS-CoV-2 IgM false-positive results.

MiR-30a regulates the proliferation, migration, and invasion of human osteosarcoma by targeting Runx2.

Osteosarcoma (OS) is the most common primary malignant bone tumor in young patients. However, treatment paradigms and survival rates have not improved in decades. MicroRNAs have been shown to be critical regulators of physiological homeostasis and pathological process, including bone disease. Nearly half of the microRNA (miRNA) genes are located at genomic regions and fragile sites known to be frequently deleted or amplified in various kinds of cancers. In this study, we investigated the role miR-30a in OS. A negative correlation between miR-30a expression and malignant grade was observed in OS cell lines. The overexpression of miR-30a reduced proliferation, migration, and invasion in 143B cells and the inhibitor of miR-30a increased proliferation, migration, and invasion in Saos2 cells. Further studies revealed that runt-related transcription factors 2 (Runx2) was a regulative target gene of miR-30a. Rescue assay significantly reversed the effects of overexpressing or inhibiting miR-30a. miR-30a also suppressed tumor formation and pulmonary metastasis in vivo. All the results suggest a critical role of miR-30a in suppressing proliferation, migration, and invasion of OS by targeting Runx2.

[Diagnostic Cut-Off Value of RDW for Screening Thalassemia and the Combined Determination of MCV, MCH, HBA 2 and RDW].

OBJECTIVE: To explore the value of red cell distribution width (RDW), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and hemoglobin (Hb) A2 combined determination scheme for screening thalassemia. METHODS: The RDW levels of thalassemia group and healthy control group were detected and compared. The efficiency of RDW for screening thalassemia was evaluated by receiver operating characteristic (ROC) curve. The diagnostic cut-off value of RDW was also acquired by Youden index. Then, 3 groups for thalassemia screening scheme were set, including MCV+MCH+HBA 2, MCV+MCH+RDW(>16.0)+HBA 2 and MCV+MCH+RDW(>15.15)+HBA 2. The performances of the 3 groups were evaluated through screening 621 clinical suspected cases of thalassemia. RESULTS: The RDW level in thalassemia group was significantly higher than that in healthy control group (P<0.05). The diagnostic cut-off value for screening thalassemia was RDW>15.15, when the Youden index was the biggest among all data. The sensitivity, specificity, positive predictive value, negative predictive value, false negative rate and consistency rate of MCV+MCH+RDW(>15.15)+HBA 2 group was 75.46%, 48.83%, 26.50%, 89.06%, 24.54%, and 54.06%, respectively. CONCLUSION: The diagnostic cut-off value of RDW for thalassemia screening has been established. The group of MCV(<82.0 fl)+MCH(<27.0 pg)+HBA 2(<2.5% or ≥3.5%)+RDW(>15.15) has a best efficiency among the 3 groups to screen thalassemia.

MicroRNA-155 inhibits the osteogenic differentiation of mesenchymal stem cells induced by BMP9 via downregulation of BMP signaling pathway.

Previous studies have indicated that bone morphogenetic protein 9 (BMP9) can promote the osteogenic differentiation of mesenchymal stem cells (MSCs) and increase bone formation in bone diseases. However, the mechanisms involved remained poorly understood. It is necessary to investigate the specific regulatory mechanisms of osteogenic differentiation that were induced by BMP9. During the process of osteogenic differentiation induced by BMP9, the expression of microRNA-155 (miR-155) exhibited a tendency of increasing at first and then decreasing, which made us consider that miR-155 may have a modulatory role in this process, but the roles of this process have not been elucidated. This study aimed to uncover miR-155 capable of concomitant regulation of this process. mmu-miR-155 mimic (miR-155) was transfected into MSCs and osteogenesis was induction by using recombinant adenovirus expressing BMP9. Overexpressed miR-155 in MSCs led to a decrease in alkaline phosphatase (ALP) staining and Alizarin red S staining during osteogenic differentiation, and reduced the expression of osteogenesis-related genes, such as runt-related transcription factor 2 (Runx2), osterix (OSX), osteocalcin (OCN) and osteopontin (OPN). On protein levels, overexpressed miR-155 markedly decreased the expression of phosphorylated Smad1/5/8 (p-Smad1/5/8), Runx2, OCN and OPN. Luciferase reporter assay revealed Runx2 and bone morphogenetic protein receptor 9 (BMPR2) are two direct target genes of miR-155. Downregulation of the expression of Runx2 and BMPR2, respectively could offset the inhibitory effect of miR-155 in the osteogenesis of MSCs. In vivo, subcutaneous ectopic osteogenesis of MSCs in nude mice showed miR-155 inhibited osteogenic differentiation. In conclusion, our results demonstrated that miR-155 can inhibit the osteogenic differentiation induced by BMP9 in MSCs.

[Overexpression of IL-8 promotes migration of BT549 breast cancer cells].

OBJECTIVE: To construct a recombinant adenovirus vector containing IL-8 gene and observe its effect on the proliferation, cell cycle and migration of BT549 breast cancer cells. METHODS: IL-8 gene was amplified by PCR using the cDNA from 143B bone sarcoma cells and inserted into shuttle plasmid pAdTrack-TO4. The recombinant shuttle plasmid pAdTrack-TO4-IL-8 was digested by PmeI and then transformed to AdEasier competent cells. The obtained recombinant adenovirus plasmid pAdIL-8 was digested by PacI, and then transfected to HEK293 cells for package and amplification by Lipofectamine(TM) 2000. The titer was tested by dilution assay. The expression of IL-8 mRNA and protein in BT549 cells was detected by reverse transcription PCR and ELISA, respectively. Effect of IL-8 overexpression on proliferation, cell cycle and migration in BT549 cells was respectively investigated by MTT assay, flow cytometry and wound-healing test. RESULTS: PCR and DNA sequence analysis verified the recombinant shuttle plasmid pAdTrack-TO4-IL-8. Restriction enzymes PacI confirmed the recombinant adenovirus plasmid pAdIL-8. IL-8 was overexpressed in BT549 cells after AdIL-8 infection. Overexpression of IL-8 promoted BT549 cell migration and arrested the cell cycle in the S phase, but it made no significant difference in the proliferation of BT549 cells. CONCLUSION: IL-8 overexpression can promote migration of BT549 breast cancer cells.

BMP9 inhibits the growth and migration of lung adenocarcinoma A549 cells in a bone marrow stromal cell­derived microenvironment through the MAPK/ERK and NF-κB pathways.

Bone is the most common distant metastatic site of lung cancer, and is particularly prone to osteolytic damage. Soluble factors secreted from bone marrow-derived cells and tumor cells contribute to the growth and metastasis of cancer cells, and enhance osteolytic damage. BMP9, as the most powerful osteogenetic factor of the bone morphogenetic protein (BMP) family, can regulate the development of various tumors. However, the effects and underlying mechanisms of BMP9 in regards to lung cancer and the bone metastatic microenvironment are poorly understood. Here, we determined the inhibitory effects of BMP9 on the proliferation and migration of lung adenocarcinoma A549 cells. When a co-culture system of A549 cells and bone marrow-derived cells (HS-5) was established, it was shown that HS-5 cells promoted the proliferation and migration of A549 cells, and metastasis and osteoclast-related factors IL-6 and IL-8 were increased in the A549 and HS-5 cells. However, BMP9 inhibited the proliferation and migration of the A549 cells in the bone microenvironment, and decreased the levels of IL-6 and IL-8. In addition, mitogen-activated protein kinase (MAPK/ERK) and nuclear factor-κB (NF-κB) signaling pathway may be involved in these effects.