MiR-539-3p inhibited chondrogenic differentiation in human adipose stem cells by targeting Sox9.

BACKGROUND: Mesenchymal stem cells (MSCs) have emerged as the attractive candidates for cell therapy for cartilage repair in clinical therapy of osteoarthritis (OA). MiR-539-3p was reported to differentially express during chondrogenic differentiation of adipose stem cells (ASCs) by miRNA microarrays. The aim of the study was to investigate the effects and underlying mechanisms of miR-539-3p on chondrogenic differentiation of ASCs. METHODS: Human ASCs (hASCs) were obtained from liposuction and transfected with miR-539-3p mimic or inhibitor. Then, the cells were cultured in chondrogenic differentiation medium including transforming growth factor-ß1 (TGF-ß1). RESULTS: Our results found that miR-539-3p was gradually down-regulated during chondrogenic differentiation of hASCs. MiR-539-3p overexpression inhibited TGF-ß1-induced chondrogenic differentiation of hASCs, as supported by reducing the gene and protein expression of chondrogenic differentiation markers type II collagen alpha 1 (COL2A1), aggrecan (ACAN), and type II collagen. In contrast, miR-539-3p inhibitor significantly promoted the chondrogenic differentiation of hASCs. Dual luciferase reporter assay demonstrated that Sox9 was a direct target gene of miR-539-3p. The expression of SRY-box transcription factor 9 (Sox9) was up-regulated progressively over time during chondrogenic differentiation of hASCs. Additionally, Sox9 overexpression notably reversed chondrogenic differentiation of hASCs inhibited by miR-539-3p mimic, as demonstrated by the decreased expression of COL2A1, ACAN, and type II collagen. CONCLUSIONS: Altogether, miR-539-3p inhibited chondrogenic differentiation of hASCs by targeting Sox9. MiR-539-3p may have significant clinical applications for use as a targeted therapy of OA.

A prospective evaluation of AI-augmented epidemiology to forecast COVID-19 in the USA and Japan.

The COVID-19 pandemic has highlighted the global need for reliable models of disease spread. We propose an AI-augmented forecast modeling framework that provides daily predictions of the expected number of confirmed COVID-19 deaths, cases, and hospitalizations during the following 4 weeks. We present an international, prospective evaluation of our models' performance across all states and counties in the USA and prefectures in Japan. Nationally, incident mean absolute percentage error (MAPE) for predicting COVID-19 associated deaths during prospective deployment remained consistently <8% (US) and <29% (Japan), while cumulative MAPE remained <2% (US) and <10% (Japan). We show that our models perform well even during periods of considerable change in population behavior, and are robust to demographic differences across different geographic locations. We further demonstrate that our framework provides meaningful explanatory insights with the models accurately adapting to local and national policy interventions. Our framework enables counterfactual simulations, which indicate continuing Non-Pharmaceutical Interventions alongside vaccinations is essential for faster recovery from the pandemic, delaying the application of interventions has a detrimental effect, and allow exploration of the consequences of different vaccination strategies. The COVID-19 pandemic remains a global emergency. In the face of substantial challenges ahead, the approach presented here has the potential to inform critical decisions.

MiR-105 enhances osteogenic differentiation of hADSCs via the targeted regulation of SOX9.

OBJECTIVE: To investigate whether miR-105 can regulate the osteogenic differentiation of human adipose-derived mesenchymal stem cells (hADSCs) by targeting SOX9. METHODS: The hADSCs were grouped for subsequent transfection and induction of osteogenic differentiation as follows: control, miR-NC, miR-105 mimics, miR-105 inhibitors, SOX9, SOX9 siRNA, miR-105 mimics + SOX9 and miR-105 inhibitors + SOX9 siRNA groups. Next, hADSCs were stained for alkaline phosphatase (ALP), and Alizarin Red S staining (ARS) was performed. Osteogenic differentiation-related genes and miR-105 expression were assessed by qRT-PCR, while SOX9 protein expression was determined by Western blotting. RESULTS: MiR-105 expression was increased and SOX9 protein expression was decreased during the osteogenic differentiation of hADSCs. A dual-luciferase reporter assay confirmed SOX9 to be a target gene of miR-105. Compared with the control group, the miR-105 mimics and SOX9 siRNA groups had elevated BMP2, OPN, OCN, BSP, Osx and Runx2 mRNA expression with reduced SOX9 expression, as well as increased ARS intensity and ALP activity. After transfection of miR-105 inhibitors/SOX9 into hADSCs, the results were the opposite. Overexpressing SOX9 reversed the effect of miR-105 in promoting the osteogenic differentiation of hADSCs. CONCLUSION: MiR-105 could target SOX9 to improve the expression of osteogenic differentiation genes and thus enhance the osteogenic differentiation of hADSCs.

Efficacy of One-Stage Posterior Debridement and Bone Grafting with Internal Fixation in the Treatment of Monosegmental Thoracolumbar Tuberculosis.

BACKGROUND: We explored the clinical effect of one-stage posterior debridement and bone grafting with internal fixation for the treatment of monosegmental thoracolumbar tuberculosis (TB). METHODS: The data from 90 patients with thoracolumbar TB, who had undergone one-stage posterior debridement and bone grafting with internal fixation, were retrospectively reviewed. Data on the operative time, blood loss, length of hospital stay, erythrocyte sedimentation rate, C-reactive protein, improvement of neurological function, visual analog scale score, vertebral Cobb angle, bone healing, and complications were collected. RESULTS: A total of 88 patients were finally included in the present retrospective study, included 42 men and 46 women. The mean patient age was 45.4 ± 12.3 years (range, 27-70), and the mean duration of disease until treatment was 11 ± 4.5 months (range, 3-19). The mean operative time was 167.0 minutes (range, 130-210), and the mean blood loss was 767.4 mL (range, 500-1150). At the final follow-up examination, the correction in the Cobb angle was 19°, the visual analog scale score had decreased to 3 ± 1.72, the neurologic deficits using the Frankel grade had improved, and the erythrocyte sedimentation rate and C-reactive protein level had returned to normal levels. CONCLUSION: One-stage posterior debridement and bone grafting with internal fixation might be a better choice for treating patients with monosegment thoracolumbar TB.

Active Learning Using Hint Information.

The abundance of real-world data and limited labeling budget calls for active learning, an important learning paradigm for reducing human labeling efforts. Many recently developed active learning algorithms consider both uncertainty and representativeness when making querying decisions. However, exploiting representativeness with uncertainty concurrently usually requires tackling sophisticated and challenging learning tasks, such as clustering. In this letter, we propose a new active learning framework, called hinted sampling, which takes both uncertainty and representativeness into account in a simpler way. We design a novel active learning algorithm within the hinted sampling framework with an extended support vector machine. Experimental results validate that the novel active learning algorithm can result in a better and more stable performance than that achieved by state-of-the-art algorithms. We also show that the hinted sampling framework allows improving another active learning algorithm designed from the transductive support vector machine.

Target deletion of the cytoskeleton-associated protein palladin does not impair neurite outgrowth in mice.

Palladin is an actin cytoskeleton-associated protein which is crucial for cell morphogenesis and motility. Previous studies have shown that palladin is localized to the axonal growth cone in neurons and may play an important role in axonal extension. Previously, we have generated palladin knockout mice which display cranial neural tube closure defect and embryonic lethality before embryonic day 15.5 (E15.5). To further study the role of palladin in the developing nervous system, we examined the innervation of palladin-deficient mouse embryos since the 200 kd, 140 kd, 90-92 kd and 50 kd palladin isoforms were undetectable in the mutant mouse embryo brain. Contrary to the results of previous studies, we found no inhibition of the axonal extension in palladin-deficient mouse embryos. The cortical neurons derived from palladin-deficient mice also showed no significant difference in neurite outgrowth as compared with those from wild-type mice. Moreover, no difference was found in neurite outgrowth of neural stem cell derived-neurons between palladin-deficient mice and wild-type mice. In conclusion, these results suggest that palladin is dispensable for normal neurite outgrowth in mice.

Ultra-sensitive detection of mutated papillary thyroid carcinoma DNA using square wave stripping voltammetry method and amplified gold nanoparticle biomarkers.

This study presents an ultra-sensitive technique for the electrochemical detection of the mutated BRAF gene associated with papillary thyroid carcinomas (PTC). In the proposed approach, a biotinylated 30-nucleotides probe DNA was immobilized in a streptavidin-modified 96-well microtiter plate and the free active sites of the streptavidin were blocked using biotinylated bovine serum albumin (BSA). The biotinylated target DNA was then added and allowed to hybridize with the immobilized probe DNA for 30min. Subsequently, streptavidin-labeled gold nanoparticles were added, and a nanoparticle enlargement process was performed using gold ion solution and formaldehyde reductant. The gold particles were then dissolved in bromide and DNA hybridization detection process was performed using a square wave stripping voltammetry (SWSV) technique. The results indicated a stable SWSV response in differential detection between blank solution and target DNA solution with a concentration of 130aM. Moreover, the coefficient of determination (R(2)) of the semi-log plot of the SWSV response current against the target DNA concentration (0.52-1300aM) was found to be 0.9982. The detection limit was estimated to be 0.35aM (based on a signal-to-noise ratio of 3:1). This value was approximately three orders of magnitude lower than that obtained using the same method but without gold amplification process. Finally, the proposed approach is successful in differentiating between the mutant and wildtype BRAF sequences that are present in genuine 224-nucleotides DNA.

Regulation of DNA methylation activity through Dnmt3L promoter methylation by Dnmt3 enzymes in embryonic development.

The genomic DNA is methylated by de novo methyltransferases Dnmt3a and Dnmt3b during early embryonic development. The establishment of appropriate methylation patterns depends on a fine regulation of the methyltransferase activity. The activity of both enzymes increases in the presence of Dnmt3L, a Dnmt3a/3b-like protein. However, it is unclear how the function of Dnmt3L is regulated. We found here that the expression of Dnmt3L is controlled via its promoter methylation during embryonic development. Genetic studies showed that Dnmt3a, Dnmt3b and Dnmt3L are all involved in the methylation of the Dnmt3L promoter. Disruption of both Dnmt3a and Dnmt3b genes in mouse rendered the Dnmt3L promoter devoid of methylation, causing incomplete repression of the Dnmt3L transcription in embryonic stem cells and embryos. Disruption of either Dnmt3a or Dnmt3b led to reduced methylation and increased transcription of Dnmt3L, but severe hypomethylation occurred only when Dnmt3b was deficient. Consistent with the major contribution of Dnmt3b in the Dnmt3L promoter methylation, methylation of Dnmt3L was significantly reduced in mouse models of the human ICF syndrome carrying point mutations in Dnmt3b. Interestingly, Dnmt3L also contributes to the methylation of its own promoter in embryonic development. We thus propose an auto-regulatory mechanism for the control of DNA methylation activity whereby the activity of the Dnmt3L promoter is epigenetically modulated by the methylation machinery including Dnmt3L itself. Insufficient methylation of the DNMT3L promoter during embryonic development due to deficiency in DNMT3B might be implicated in the pathogenesis of the ICF syndrome.

Temporal and parental-specific expression of imprinted genes in a newly derived Chinese human embryonic stem cell line and embryoid bodies.

Although the study of imprinted genes in human development is very important, little is known about their expression and regulation in the early differentiation of human tissues due to lack of an appropriate model. In this study, a Chinese human embryonic stem (hES) cell line, SHhES1, was derived and fully characterized. Expression profiles of human imprinted genes were determined by Affymetrix Oligo micro-array in undifferentiated SHhES1 cells and SHhES1-derived embryoid bodies (EBs) at day 3, 8, 13 and 18. Thirty-two known human imprinted genes were detected in undifferentiated ES cells. Significantly, differential expression was found in nine genes at different stages of EB formation. Expression profile changes were confirmed by quantitative real-time reverse transcriptase-polymerase chain reaction in SHhES1 cells as well as in another independently derived hES cell line, HUES-7. In addition, the monoallelic expressions of four imprinted genes were examined in three different passages of undifferentiated ES cells and EBs of both hES cell lines. The monoallelic expressions of imprinted genes, H19, PEG10, NDNL1 and KCNQ1 were maintained in both undifferentiated hES cells and derived EBs. More importantly, with the availability of maternal peripheral blood lymphocyte sample, we demonstrated that the maternal expression of KCNQ1 and the paternal expression of NDNL1 and PEG10 were maintained in SHhES1 cells. These data provide the first demonstration that the parental-specific expression of imprinted genes is stable in EBs after extensive differentiation, also indicating that in vitro fertilization protocol does not disrupt the parental monoallelic expression of the imprinted genes examined.

No correlation between BRAFV600E mutation and clinicopathological features of papillary thyroid carcinomas in Taiwan.

OBJECTIVE: Genetic alterations in four oncogenes, namely RAS point mutations, RET rearrangements (RET/PTC), NTRK1 rearrangements (TRK) and BRAF point mutations have been identified in human papillary thyroid carcinomas (PTCs). These oncogenes act along the RET/PTC(TRK)-RAS-BRAF-MEK-MAPK kinase pathway, mediating a number of cellular fates including growth, proliferation and survival in thyroid cells. In this study, we analysed mutations of BRAF in a cohort of PTCs. METHODS: To screen for BRAF mutations, the genomic DNA of 105 PTCs were amplified by polymerase chain reaction (PCR) with primers flanking exon 15 and PCR products were directly sequenced with an automatic sequencer. These results, together with data from our previous studies on RAS, RET rearrangements and NTRK1 rearrangements in the same tumours, were compared to determine their individual significance in the pathogenesis of PTCs in Taiwan. RESULTS: BRAF mutations were detected in 49 of 105 (47%) tumour samples. All mutations involved a thymine-to-adenine transversion at nucleotide 1799 and were heterozygous. There was no overlap between papillary carcinomas harbouring RET rearrangements, NTRK1 rearrangements and BRAF mutations. In this cohort, correlation between BRAF mutations and various clinicopathological parameters in 101 papillary carcinomas did not reveal any association with age at diagnosis, sex, tumour size, histological variants of PTC, multicentricity, cervical lymph node metastases, extrathyroidal invasion, distant metastases and clinical stage. CONCLUSIONS: BRAFV600E mutation is the most prevalent oncogene in PTCs in Taiwan. Our data did not suggest that BRAFV600E mutation could be a potentially useful marker of prognosis in patients with papillary carcinomas in the population studied.