Purpose: This proof-of-concept study investigates gene expression in core needle biopsies (CNB) to predict whether individuals diagnosed with ductal carcinoma in situ (DCIS) on CNB were affected by invasion at the time of diagnosis.

Methods: Using a QuantiGene Plex 2.0 assay, 14 gene expression profiling was performed in 303 breast tissue samples. Preoperative diagnostic performance of a gene was measured by area under receiver-operating characteristic curve (AUC) with 95% confidence interval (CI). The gene mRNA positivity cutoff was computed using Gaussian mixture model (GMM); protein expression was measured by immunohistochemistry; DNA methylation was evaluated by targeted bisulfite sequencing.

Results: mRNA from 69% (34/49) mammoplasties, 72% (75/104) CNB DCIS, and 89% (133/150) invasive breast cancers (IBC) were analyzed. Based on pre-and post-surgery DCIS chart reviews, 21 cases were categorized as DCIS synchronous with invasion and 54 DCIS were pure DCIS without pathologic evidence of invasive disease. The ectopic expression of neuronal cadherin CDH2 was probable in 0% mammoplasties, 6% pure DCIS, 29% synchronous DCIS, and 26% IBC. The CDH2 mRNA positivity in preoperative biopsies showing pure DCIS was predictive of a final diagnosis of invasion (AUC = 0.67; 95% CI 0.53-0.80; P = 0.029). Site-specific methylation of the CDH2 promoter (AUC = 0.76; 95% CI 0.54-0.97; P = 0.04) and measurements of N-cadherin, a pro-invasive cell-cell adhesion receptor encoded by CDH2 (AUC = 0.8; 95% CI 0.66-0.99; P < 0.005) had a discriminating power allowing for discernment of CDH2-positive biopsy.

Conclusions: Evidence of CDH2/N-cadherin expression, predictive of invasion synchronous with DCIS, may help to clarify a diagnosis and direct the course of therapy earlier in a patient's care.

Keywords: CDH2N-cadherin; Diagnostic biopsy, ductal carcinoma in situ (DCIS) of the breast; epigenetics of DCIS; probabilistic modeling.

Objective: To explore the function of miR-543 in endometrial cells and the possible mechanism of regulating the occurrence and development of intrauterine adhesion.

Method: Endometrial epithelial cells and endometrial adenocarcinoma cells were transfected with miR-543 mimics and miR-543 inhibitor as the experimental group and were tested with the control group, using the CCK-8 method, scratch test, and Transwell assay, and flow cytometry was used to detect the proliferation, migration, invasion, and apoptosis of cells. RT-qPCR and Western blot were used to detect the expression of corresponding mRNA and protein.

Results: After the overexpression of miR-543, endometrial epithelial cells and endometrial adenocarcinoma cells have reduced migratory, proliferative, and invasive capabilities, while the apoptosis rate has increased significantly. The mRNA expression of CDH2, COL16A1, vimentin, α-SMA and fibronectin decreased, and the protein expression of CDH2, vimentin, and α-SMA also decreased, while the mRNA and protein expression of CDH1 increased. The result after interfering with miR-543 is opposite, and luciferase reporter gene confirms that CDH2 is the target gene of miR-543.

Conclusion: During the formation of intrauterine adhesions, the expression of CDH2, COL16A1, vimentin, and α-SMA may be inhibited by the high expression of miR-543, which may affect the degree of fibrosis and collagen content in the intrauterine adhesions, thereby inhibiting the occurrence and development of intrauterine adhesions.

To speed up the progress of marker-assisted selection (MAS) in cattle breeding, the dual-specificity tyrosine phosphorylation-regulated kinase 2 (DYRK2), cadherin 2 (CDH2), and kinesin family member 1A (KIF1A) genes were chosen based on our pervious genome-wide association study (GWAS) analysis results. DYRK2 is a kinase that may participate in cell growth and/or development; it shows phosphorylation activity toward serine, threonine, and tyrosine fragments of proteins, and it is different from other protein kinases. The CDH2 gene encodes a classic cadherin, which is a member of the cadherin superfamily. The protein encoded by KIF1A is a member of the kinesin family and plays a role in the transportation of membrane organelles along axon microtubules. We detected insertion/deletion (InDel) variation in these three candidate genes in 438 individual cattle (Xinjiang Brown cattle and Wagyu $\times$ Luxi crossbreed cattle). Only DYRK2-P3-11 bp was polymorphic and genotyped. The polymorphism information content of DYRK2-P3-11 bp was 0.336. Correlation analyses showed that InDel polymorphism was significantly associated with six different milk traits. These findings may aid future analyses of InDel genotypes in cattle breeds, and speed up the progress of MAS in cattle breeding.

BACKGROUND This study aimed to investigate the expression profile of the phosphatase and tensin homolog (PTEN) gene, the cadherin genes, CDH1 and CDH2, and the cell membrane glycoprotein, CD133, in the Ishikawa human endometrial adenocarcinoma cell line. MATERIAL AND METHODS The Ishikawa endometrial carcinoma cell groups included cells transfected with the pLVX-puro lentiviral expression vector (the Ishikawa-puro group) and cells transfected with the pLVX-puro-PTEN lentiviral expression vector (the Ishikawa-PTEN group). The mRNA expression of the cadherin genes, CDH1 and CDH2, was detected by quantitative reverse transcription-polymerase chain reaction (RT-qPCR). The expression levels of the transmembrane glycoprotein CD133, a cancer stem cell marker, was detected by flow cytometry. RESULTS The expression of CDH1 and CDH2 mRNA in the Ishikawa-PTEN cells was lower than in the control cells. CD133 expression was lower in the Ishikawa-PTEN cells compared with the control cells. CONCLUSIONS This in vitro study showed that in Ishikawa endometrial carcinoma cells, downregulation of PTEN was associated with the expression of the CDH1 and CDH2 genes and upregulated expression of the cell membrane glycoprotein, CD133, which are associated with epithelial-mesenchymal transition (EMT) in malignancy. These findings support the need for further studies to investigate the potential role of PTEN in invasion and metastasis in endometrial carcinoma.

Heroin dependent patients have a high incidence of HIV infection. In contrast to the gene expression method, we developed a systemic correlation analysis method built upon the results of pharmacogenomics study in a methadone maintenance treatment (MMT) cohort consisting of 344 Taiwanese heroin dependent patients. We identified genetic variants and their encoding proteins that may be involved with HIV infection and MMT treatment outcome. Cadherin 2 (CDH2) genetic determinants were identified through the genome-wide pharmacogenomic study. We found significant correlations among HIV infection status, plasma levels of CDH2, cytokine IL-7, ADAM10, and the treatment responses to methadone. Two single nucleotide polymorphisms located within CDH2 gene showed associations with blood pressure and plasma CDH2 concentration. Plasma concentration of CDH2 showed correlations with the level of cytokine IL-7, status of HIV infection, and urine morphine test result. Plasma level of IL-7 was correlated with corrected QT interval (QTc) and gooseflesh skin withdrawal symptom score, while level of ADAM10 was correlated with plasma concentrations of vitamin D metabolite, nicotine metabolite, and R-methadone. The results suggest a novel network involving HIV infection and methadone treatment outcome.

Conformational entropy changes associated with bond-vector motions in proteins contribute to the free energy of ligand-binding. To derive such contributions, we apply the slowly relaxing local structure (SRLS) approach to NMR relaxation from 15N-H bonds or C-CDH2 moieties of several proteins in free and ligand-bound form. The spatial restraints on probe motion, which determine the extent of local order, are expressed in SRLS by a well-defined potential, u(θ). The latter yields the orientational probability density, Peq = exp(-u(θ)), and hence the related conformational entropy, Ŝ = -∫Peq(θ) ln[Peq(θ)] sin θ dθ (Ŝ is "entropy" in units of kBT, and θ represents the bond-vector orientation in the protein). SRLS is applied to 4-oxalocrotonate tautomerase (4-OT), the acyl-coenzyme A binding protein (ACBP), the C-terminal SH2 domain of phospholipase Cγ1 (PLCγ1C SH2), the construct dihydrofolate reductase-E:folate (DHFR-E:folate), and their complexes with appropriate ligands, to determine ΔŜ. Eglin C and its V18A and V34A mutants are also studied. Finally, SRLS is applied to the structurally homologous proteins TNfn3 and FNfn10 to characterize within its scope the unusual "dynamics" of the TNfn3 core. Upon ligand-binding, the backbones of 4-OT, ACBP, and PLCγ1C SH2 show limited, increased, and decreased order, respectively; the cores of DHFR-E:folate and PLCγ1C SH2 become more ordered. The V18A (V34A) mutation increases (decreases) the order within the eglin C core. The core of TNfn3 is less ordered structurally and more mobile kinetically. Secondary structure versus loops, surface-binding versus core insertion, and ligand size emerged as being important in rationalizing ΔŜ. The consistent and general tool developed herein is expected to provide further insights in future work.

Epithelial-to-mesenchymal transition (EMT) is important in tumor invasiveness and metastasis. We aimed to determine prognostic value of six key EMT markers (CDH1, CDH2, SNAI1, SNAI2, VIM, TWIST1) in clear cell renal cell carcinoma (ccRCC). A total of 533 ccRCC patients with RNASeq data from The Cancer Genome Atlas (TCGA) cohort were included for analysis. Gene expression of these EMT markers was compared between tumor and normal tissues based on Oncomine database and TCGA cohort. Their correlations with progression-free survival (PFS) and overall survival (OS) were also examined in both TCGA cohort and FUSCC (Fudan University Shanghai Cancer Center) cohort. Cox proportional hazards regression model and Kaplan-Meier plot were used to assess the relative factors. Functional enrichment analyses were utilized to describe biologic function annotations and significantly involved hallmarks pathways of each gene. We found that Epithelial marker, CDH1 expression was lower, while mesenchymal markers (CDH2, SNAI1, VIM, TWIST1) expression was higher in ccRCC primary tumors. In the TCGA cohort, we found that patients with higher expression of VIM, TWIST1 or lower expression of CDH1 had worse prognosis. Further, in the FUSCC cohort, we confirmed the predictive ability of mesenchymal markers and epithelial marker expression in PFS and OS of ccRCC patients. After generating Cox regression models, EMT markers (CDH1, SNAI1, VIM, and TWIST1) were independent prognostic factors of both PFS and OS in ccRCC patients. Our preliminary EMT prediction model can facilitate further screening of EMT biomarkers and cast a better understanding of EMT gene function in ccRCC.

Procymidone (PCM), a dicarboximide fungicide, is widely used in agriculture to control plant diseases. In the present study, zebrafish embryos were exposed to PCM at 0, 10, 100 and 1000 ng/L for 72 h, the development and cardiac functioning of larvae were observed and determined. The results showed that hatching rate was significantly decreased in the 1000 ng/L treatment, and pericardial edema rate and spine curvature rate were significantly increased in the 100 and 1000 ng/L groups. The PCM-treated larvae exhibited an increased heart rate as well as arrhythmia, and shortened low jaw. The transcription levels of cardiac development-related genes tbx5, nkx2.5, tnnt2, gata4, myh6, myl7, cdh2, ryr2 were altered, which might be responsible for the cardiac developmental and functioning defects in the larvae. The deformation in bone development might be related with the impaired transcription levels of ihh, shh, bmp2b, bmp4, gh, igf1, sox9, gli2. The activities of Na+/K+-ATPase and Ca2+-ATPase were significantly inhibited by 100 ng/L and 1000 ng/L PCM exposure, which might be a cause for the occurrence of pericardial edema and skeletal deformation. The results of this study will be helpful in evaluating the potential threat of PCM to fish population in the aquatic environment.

Autosomal dominant polycystic kidney disease (ADPKD) affects more than 12 million people worldwide. Mutations in PKD1 and PKD2 cause cyst formation through unknown mechanisms. To unravel the pathogenic mechanisms in ADPKD, multiple studies have investigated transcriptional mis-regulation in cystic kidneys from patients and mouse models, and numerous dysregulated genes and pathways have been described. Yet, the concordance between studies has been rather limited. Furthermore, the cellular and genetic diversity in cystic kidneys has hampered the identification of mis-expressed genes in kidney epithelial cells with homozygous PKD mutations, which are critical to identify polycystin-dependent pathways. Here we performed transcriptomic analyses of Pkd1- and Pkd2-deficient mIMCD3 kidney epithelial cells followed by a meta-analysis to integrate all published ADPKD transcriptomic data sets. Based on the hypothesis that Pkd1 and Pkd2 operate in a common pathway, we first determined transcripts that are differentially regulated by both genes. RNA sequencing of genome-edited ADPKD kidney epithelial cells identified 178 genes that are concordantly regulated by Pkd1 and Pkd2. Subsequent integration of existing transcriptomic studies confirmed 31 previously described genes and identified 61 novel genes regulated by Pkd1 and Pkd2. Cluster analyses then linked Pkd1 and Pkd2 to mRNA splicing, specific factors of epithelial mesenchymal transition, post-translational protein modification and epithelial cell differentiation, including CD34, CDH2, CSF2RA, DLX5, HOXC9, PIK3R1, PLCB1 and TLR6. Taken together, this model-based integrative analysis of transcriptomic alterations in ADPKD annotated a conserved core transcriptomic profile and identified novel candidate genes for further experimental studies.

One of the major complications in diabetes is impaired wound healing. Unfortunately, effective therapies are currently lacking. Epithelial to mesenchymal transition (EMT) is a critical process involved in cutaneous wound healing. In response to injury, EMT is required to activate and mobilize stationary keratinocytes in the skin toward the wound bed, which allows for re-epithelialization. This process is stalled in diabetic wounds. In this study, we investigate the role of long non-coding RNA (lncRNA), MALAT1, in transforming growth factor beta 1(TGF-β1)-induced EMT of human keratinocyte (HaCaT) cells. Initially, we detected MALAT1 and TGF-β1 expression in non-diabetic and diabetic wounds and found that these expression are significantly up-regulated in diabetic wounds. Then, HaCaT cells were cultured and exposed to TGF-β1. The EMT of HaCaT cells were confirmed by the increased expression of CDH2, KRT10, and ACTA2, in addition to the down-regulation of CDH1. Knockdown of MALAT1 was achieved by transfecting a small interfering RNA (SiRNA). MALAT1 silencing attenuates TGFβ1-induced EMT. Mechanistically, MALAT1 is involved in TGF-β1 mediated EMT through significantly induced ZEB1 expression, a critical transcription factor for EMT. In summary, lncRNA MALAT1 is involved in TGFβ1-induced EMT of human HaCaT cells and provides new understanding for the pathogenesis of diabetic wounds.

Keywords: MALAT1; diabetic wounds; epithelial mesenchymal transition; long non-coding RNA.

Direct neural reprogramming involves a rapid conversion of somatic cells into neural cells without passing through the intermediate pluripotent stage. This phenomenon can be mediated in the starting somatic cells by the introduction of lineage-specific master transcription factors or by pluripotency factors routinely used in iPS cell generation. In the latter process known as Pluripotency factor-mediated Direct Reprogramming (PDR), the pluripotency factors are used to elicit epigenetic changes producing a permissive state in the starting cells which are then driven to the neural lineages by simple manipulations of the culture conditions. When genes are exogenously introduced to achieve such conversion, their persistent expression after completion of the reprogramming can affect the properties of the resulting cells. Here, we describe a robust method for direct neural reprogramming using the episomal vectors that incorporate a suicide gene scFCY1 (encoding cytosine deaminase) that allows rapid and efficient generation of a homogenous population of transgene-free human-induced neural progenitor cells (hiNPCs). The resulting NESTIN⁺/PAX6⁺/CDH2⁺ hiNPCs can be expanded and cryopreserved and can be further differentiated into neurons and glia.

Keywords: Cell fate change; Cytosine deaminase; Direct conversion; Direct reprogramming; Episomal vector; Induced neural progenitor; Induced neural stem cell; Suicide gene.

Mechanicals forces are known to influence cell behavior. In vivo, the corneal endothelium is under the influence of various mechanical forces, such as intraocular pressure (IOP) and fluid flow. In this study, we used a corneal bioreactor to understand the effect of these hydrodynamic forces on the transcription of intercellular junctions associated genes in the corneal endothelium. Native and tissue-engineered (TE) corneal endothelium were cultured in a corneal bioreactor for 7 days with 16 mmHg IOP and 5 μl/ml of medium flow. RNA was harvested, and gene expression was quantified. Cells that were used to reconstruct the TE corneal endothelium were also seeded on plastic to characterize their cell morphology by calculating their circularity index. For native endothelia, hydrodynamic forces increased gene expression of GJA1 (connexin 43), CDH2 (N-cadherin), TJP1 (ZO-1), ITGAV (integrin subunit αv), ITGB5 (integrin subunit β5) and CTNND1 (p120-ctn) by 1.68 ± 0.40, 1.10 ± 0.27, 3.80 ± 0.56, 1.82 ± 0.33, 1.32 ± 0.21 and 3.04 ± 0.63, respectively. For TE corneal endothelium, this fold change was 1.72 ± 0.31, 1.58 ± 0.41, 6.18 ± 1.03, 1.80 ± 0.71, 1.77 ± 0.55, 2.42 ± 0.71. Furthermore, gene transcription fold changes (hydrodynamic/control) increased linearly with TE corneal endothelium cells population morphology with r = 0.83 for TJP1 (ZO-1) and r = 0.58 for CTNND1 (p120-ctn). In fact, the more elongated the cells populations were, the greater hydrodynamic conditions increased the transcription of TJP1 (ZO-1) and CTNND1 (p120-ctn). These results suggest that hydrodynamic forces contribute to the maintenance of tight and adherens junctions of native corneal endothelial cells, as well as to the formation of tight and adherens junctions of corneal endothelial cells that are in the process of forming a functional endothelial barrier.

Keywords: CTNND1; Circularity; Corneal bioreactor; Corneal endothelium; Pressure; TJP1; Tight junctions; ZO-1; p120-ctn.

Type I and type II classical cadherins comprise a family of cell adhesion molecules that regulate cell sorting and tissue separation by forming specific homo and heterophilic bonds. Factors that affect cadherin-mediated cell-cell adhesion include cadherin binding affinity and expression level. This study examines the expression pattern of type I cadherins (Cdh1, Cdh2, Cdh3, and Cdh4), type II cadherins (Cdh6, Cdh7, Cdh8, Cdh9, Cdh10, Cdh11, Cdh12, Cdh18, Cdh2, and Cdh2), and the atypical cadherin 13 (Cdh13) during distinct morphogenetic events in the developing mouse central nervous system from embryonic day 11.5 to postnatal day 56. Cadherin mRNA expression levels obtained from in situ hybridization experiments carried out at the Allen Institute for Brain Science (https://alleninstitute.org/) were retrieved from the Allen Developing Mouse Brain Atlas. Cdh2 is the most abundantly expressed type I cadherin throughout development, while Cdh1, Cdh3, and Cdh4 are expressed at low levels. Type II cadherins show a dynamic pattern of expression that varies between neuroanatomical structures and developmental ages. Atypical Cdh13 expression pattern correlates with Cdh2 in abundancy and localization. Analyses of cadherin-mediated relative adhesion estimated from their expression level and binding affinity show substantial differences in adhesive properties between regions of the neural tube associated with the segmentation along the anterior-posterior axis. Differences in relative adhesion were also observed between brain nuclei in the developing subpallium (basal ganglia), suggesting that differential cell adhesion contributes to the segregation of neuronal pools. In the adult cerebral cortex, type II cadherins Cdh6, Cdh8, Cdh10, and Cdh12 are abundant in intermediate layers, while Cdh11 shows a gradated expression from the deeper layer 6 to the superficial layer 1, and Cdh9, Cdh18, and Cdh2 are more abundant in the deeper layers. Person's correlation analyses of cadherins mRNA expression patterns between areas and layers of the cerebral cortex and the nuclei of the subpallium show significant correlations between certain cortical areas and the basal ganglia. The study shows that differential cadherin expression and cadherin-mediated adhesion are associated with a wide range of morphogenetic events in the developing central nervous system including the organization of neurons into layers, the segregation of neurons into nuclei, and the formation of neuronal circuits.

Keywords: basal ganglia; cell adhesion molecules; cerebral cortex; classical cadherins; differential cell adhesion; neural circuit formation; neural development; tissue morphogenesis.

Background: We aimed to explore the biomarkers associated with atrial fibrillation (AF) with mitral regurgitation (MR).

Methods: The gene expression profile data GSE115574 were downloaded from Gene Expression Omnibus database, which were obtained from patients with degenerative MR with AF and sinus rhythm (SR). The differentially expressed genes (DEGs) in samples of AF with MR compared with those of SR with MR were selected, followed by functional enrichment analysis, protein-protein interaction (PPI) network analysis, transcription factor (TF) prediction, and drug-gene interaction prediction.

Results: By comparing the genes' expression profiles between AF with MR and SR with MR, 379 DEGs were obtained. The upregulated genes, such as NMNAT2, LDHB, and hexosaminidase subunit beta (HEXB), were significantly enriched in metabolic pathways. Hub genes, such as amyloid beta precursor protein (APP), CDH2, SPP1, and STC2, were significantly associated with functions related to extracellular matrix organization and vitamin D response. Additionally, two TFs, PRDM3 and LSM6, were predicted for the key module genes. APP predicted the most drug molecules, that is, 22 molecules, and SPP1 predicted 10 drug molecules.

Conclusions: Dysregulation of the metabolic pathway may play a critical role in AF with MR. Changes in functions related to the extracellular matrix and vitamin D response may also be associated with AF progression in patients with MR. Furthermore, APP, STC2, and SPP1 may serve as potential therapeutic targets of AF.

During the development of natural cartilage, mesenchymal condensation is the starting event of chondrogenesis, and mesenchymal stem cells (MSCs) experienced a microenvironment transition from primarily cell-cell interactions to a later stage, where cell-extracellular matrix (ECM) interactions dominate. Although micromass pellet culture has been developed to mimic mesenchymal condensation in vitro, the molecular mechanism remains elusive, and the transition from cell-cell to cell-ECM interactions has been poorly recapitulated. In this study, we first constructed MSC microspheres (MMs) and investigated their chondrogenic differentiation with functional blocking of N-cadherin. The results showed that early cartilage differentiation and cartilage-specific matrix deposition of MSCs in the group with the N-cadherin antibody were significantly postponed. Next, poly(l-lysine) treatment was transiently applied to promote the expression of N-cadherin gene, CDH2, and the treatment-promoted MSC chondrogenesis. Upon one-day culture in MMs with established cell-cell adhesions, collagen hydrogel-encapsulated MMs (CMMs) were constructed to simulate the cell-ECM interactions, and the collagen microenvironment compensated the inhibitory effects from N-cadherin blocking. Surprisingly, chondrogenic-differentiated cell migration, which has important implications in cartilage repair and integration, was found in the CMMs without N-cadherin blocking. In conclusion, our study demonstrated that N-cadherin plays the critical role in early mesenchymal condensation, and the collagen hydrogel provides a supportive microenvironment for late chondrogenic differentiation. Therefore, sequential presentations of cell-cell adhesion and cell-ECM interaction in an engineered microenvironment seem to be a promising strategy to facilitate MSC chondrogenic differentiation.

Keywords: N-cadherin; chondrogenesis; collagen hydrogel; mesenchymal stem cell; microsphere.

The limited ability of articular cartilage to self-repair has motivated the development of tissue engineering strategies that aim to harness the regenerative potential of mesenchymal stem/marrow stromal cells (MSCs). Understanding how environmental factors regulate the phenotype of MSCs will be central to unlocking their regenerative potential. The biophysical environment is known to regulate the phenotype of stem cells, with factors such as substrate stiffness and externally applied mechanical loads known to regulate chondrogenesis of MSCs. In particular, hydrostatic pressure (HP) has been shown to play a key role in the development and maintenance of articular cartilage. Using a collagen-alginate interpenetrating network (IPN) hydrogel as a model system to tune matrix stiffness, this study sought to investigate how HP and substrate stiffness interact to regulate chondrogenesis of MSCs. If applied during early chondrogenesis in soft IPN hydrogels, HP was found to downregulate the expression of ACAN, COL2, CDH2 and COLX, but to increase the expression of the osteogenic factors RUNX2 and COL1. This correlated with a reduction in SMAD 2/3, HDAC4 nuclear localization and the expression of NCAD. It was also associated with a reduction in cell volume, an increase in the average distance between MSCs in the hydrogels and a decrease in their tendency to form aggregates. In contrast, the delayed application of HP to MSCs grown in soft hydrogels was associated with increased cellular volume and aggregation and the maintenance of a chondrogenic phenotype. Together these findings demonstrate how tailoring the stiffness and the timing of HP exposure can be leveraged to regulate chondrogenesis of MSCs and opens alternative avenues for developmentally inspired strategies for cartilage tissue regeneration.

Keywords: HDAC4; bioreactor 3D cell culture; interpenetrating polymer network; mechanobiolgy; tissue engineering.

The combination of macro- and microporosity is a potent manner of enhancing osteogenic potential, but the biological events leading to this increase in osteogenesis are not well understood. In this study, we investigated the effect of a dual pore size scaffold on the physical and biological properties, with the hypothesis that cell condensation is the determining factor for enhanced osteogenic differentiation. To this end, a hierarchical scaffold possessing a dual (large and small) pore size was fabricated by combining two additive manufacturing techniques: melt electrospinning writing (MEW) and fused deposition modeling (FDM). The scaffolds showed a mechanical stiffness of 23.2 ± 1.5 MPa similar to the FDM control scaffold, while the hybrid revealed an increased specific surface area of 1.4 ± 0.1 m²/g. The scaffold was cultured with primary human osteoblasts for 28 days, which showed enhanced cell adhesion and proliferation. The hierarchical structure was also beneficial for in vitro alkaline phosphate activity and mineralization and showed an increased expression of osteogenic protein and genes. Mesenchymal condensation markers related to osteoblastic differentiation (CDH2, RhoA, Rac1, and Cdc42) were upregulated in the hybrid construct, demonstrating that the MEW membrane provided an environment more suitable for the recapitulation of cell condensation, which in turn leads to higher osteogenic differentiation. In summary, this study demonstrated that the hierarchical scaffold developed in this paper leads to a significant improvement in the scaffold properties such as increased specific surface area, initial cell adhesion, cell proliferation, and in vitro osteogenesis.

Keywords: dual-scale scaffold; fused deposition modeling; hierarchical scaffold; melt electrospinning writing; osteogenesis.

Background: Given the increased use and diversity of diagnostic procedures, it is important to understand genetic susceptibility to radiation-induced thyroid cancer.

Methods: Based on self-declared diagnostic radiology examination records in addition to existing literature, we estimated the radiation dose delivered to the thyroid gland from diagnostic procedures during childhood and adulthood in two case-control studies conducted in France. A total of 1071 differentiated thyroid cancer (DTC) cases and 1188 controls from the combined studies were genotyped using a custom-made Illumina OncoArray DNA chip. We focused our analysis on variants in genes involved in DNA damage response and repair pathways, representing a total of 5817 single-nucleotide polymorphisms in 571 genes. We estimated the odds ratio per milli-Gray (OR/mGy) of the radiation dose delivered to the thyroid gland using conditional logistic regression. We then used an unconditional logistic regression model to assess the association between DNA repair gene variants and DTC risk. We performed a meta-analysis of the two studies.

Results: The OR/mGy was 1.02 (95% CI: 1.00, 1.03). We found significant associations between DTC and rs7164173 in CHD2 (p = 5.79 10-5), rs6067822 in NFATc2 (p = 9.26 10-5), rs1059394 and rs699517 both in ENOSF1/THYS, rs12702628 in RPA3, and an interaction between rs7068306 in MGMT and thyroid radiation doses (p= 3.40 10-4).

Conclusions: Our results suggest a role for variants in CDH2, NFATc2, ENOSF1/THYS, RPA3 and MGMT in DTC risk.

Impact: CDH2, NFATc2, ENOSF1/THYS and RPA3 have not previously been shown to be associated with DTC risk.

The main problem in the treatment of non-small cell lung cancer (NSCLC) is metastasis. Epithelial-mesenchymal transition (EMT) is known as the critical signaling in tumor progression, metastasis, and also the drug resistance. In this study, we reported a novel gene Polymerase delta-interacting protein 2 (POLDIP2) was downregulated in NSCLC tissues and first demonstrated that overexpression of POLDIP2 increased the anchorage-independent growth (AIG) and invasiveness of H1299 cells. In addition, we examined that knockdown of POLDIP2 in H1299 and A549 cells reduced tumorigenicity and metastatic capacity in vitro and also in vivo. Moreover, downregulation of the cell proliferation marker cyclin D1 and EMT markers CDH2, Slug, and Twist was showed in H1299 cells by POLDIP2 knockdown, suggesting that the inhibition of malignancy was affected by modulating key genes for tumor growth and invasiveness. Taken together, our study is the first study that demonstrated that POLDIP2 gene was function as an oncogene in NSCLC and implied the oncogenic ability might be through promoting cell proliferation or EMT.

In our previous study, we identified differences in the levels of CDH2 and vascular endothelial growth factor (VEGF) between effective and ineffective clones of human umbilical cord blood (hUCB) mesenchymal stem cells (MSCs), with regard to the infarcted rat myocardium. In this study, we compared gene expression profiles between the effective and ineffective clones and identified that endothelin-1 (EDN1) is enriched in the effective clone. In the mechanistic analyses, EDN1 significantly increased expression of CDH2 and VEGF through endothelin receptor A (EDNRA), which was prevented by EDNRA blocker, BQ123. To decipher how EDN1 induced gene expression of CDH2, we performed a promoter activity assay and identified GATA2 and MZF1 as inducers of CDH2. EDN1 significantly enhanced the promoter activity of the CDH2 gene, which was obliterated by the deletion or point mutation at GATA2 or MZF1 binding sequence. Next, therapeutic efficacy of EDN1-priming of hUCB-MSCs was tested in a rat myocardial infarction (MI) model. EDN1-primed MSCs were superior to naive MSCs at 8 weeks after MI in improving myocardial contractility (p < 0.05), reducing fibrosis area (p < 0.05), increasing engraftment efficiency (p < 0.05), and improving capillary density (p < 0.05). In conclusion, EDN1 induces CDH2 and VEGF expression in hUCB-MSCs, leading to the improved therapeutic efficacy in rat MI, suggesting that EDN1 is a potential priming agent for MSCs in regenerative medicine.

A recent study from our lab revealed that the inhibition of cyclooxygenase-2 (COX-2) exclusively reduces the level of PGE₂ (Prostaglandin E₂) among prostanoids and hampers the normal development of several structures, strikingly the cranial vault, in chick embryos. In order to unearth the mechanism behind the deviant development of cranial features, the expression pattern of various factors that are known to influence cranial neural crest cell (CNCC) migration was checked in chick embryos after inhibiting COX-2 activity using etoricoxib. The compromised level of cell adhesion molecules and their upstream regulators, namely CDH1 (E-cadherin), CDH2 (N-cadherin), MSX1 (Msh homeobox 1), and TGF-β (Transforming growth factor beta), observed in the etoricoxib-treated embryos indicate that COX-2, through its downstream effector PGE₂, regulates the expression of these factors perhaps to aid the migration of CNCCs. The histological features and levels of FoxD3 (Forkhead box D3), as well as PCNA (Proliferating cell nuclear antigen), further consolidate the role of COX-2 in the migration and survival of CNCCs in developing embryos. The results of the current study indicate that COX-2 plays a pivotal role in orchestrating craniofacial structures perhaps by modulating CNCC proliferation and migration during the embryonic development of chicks.

Keywords: cell migration; cranial neural crest cells; development; embryogenesis.

Tumor growth and metastases are dependent on interactions between cancer cells and the local environment. Expression of the cell-cell adhesion molecule N-cadherin (Ncad) is associated with highly aggressive cancers, and its expression by osteogenic cells has been proposed to provide a molecular "dock" for disseminated tumor cells to establish in pre-metastatic niches within the bone. To test this biologic model, we conditionally deleted the Ncad gene (Cdh2) in osteolineage cells using Osx-cre (cKO). Contrary to expectations, the metastatic breast cancer cell line PyMT-BO1 was able to form tumors in bone and to induce osteolysis in cKO as well as in control mice. Despite absence of Ncad, bone marrow stromal cells isolated from cKO mice were able to engage in direct cell-cell interactions with tumor cells expressing either N- or E-cadherin. However, subcutaneous PyMT-BO1 and B16F10 tumors grew larger in cKO relative to control littermates. Cell tracking experiments using the Ai9 reporter revealed the presence of Osx+ and Ncad+ cells in the stroma of extra-skeletal tumors and in a small population of lung cells. Gene expression analysis by RNAseq of Osx+ cells isolated from extra-skeletal tumors revealed alterations of pro-tumorigenic signaling pathways in cKO cells relative to control Osx+ cells. Thus, Ncad in Osx+ cells is not necessary for the establishment of bone metastases, but in extra-skeletal tumors it regulates pro-tumorigenic support by the microenvironment.

Keywords: Bone-tumor cell interactions; N-cadherin; Transcriptomics; Tumor microenvironment.

Background: Arrhythmogenic cardiomyopathy (ACM) is a genetic disorder with an estimated prevalence between 1:2,000 and 1:5,000 and is characterized by the fibrofatty replacement of cardiomyocytes that predisposes to malignant arrhythmias, heart failure, and sudden cardiac death. The diagnosis is based on the 2010 Task Force Criteria including family history, electrocardiographic traits and arrhythmogenic pattern, specific gene mutations, and structural and/or histological abnormalities. Most ACMs display an autosomal dominant mode of inheritance often with incomplete penetrance and variable expressivity. Genetic screening of patients with ACM identifies pathogenic or likely pathogenic variants, prevalently in genes encoding the cardiac desmosome (PKP2, DSP, DSC2, DSG2, and JUP) or less frequently in non-desmosomal genes (CTNNA3, PLN, TMEM43, RYR2, SCN5A, CDH2, and DES). Methods: In the present study, we performed molecular autopsy in a boy who died suddenly during physical exertion. In addition to post-mortem examination, a DNA sample was analyzed with next-generation sequencing (NGS). Results: The genetic analysis revealed the presence of pathogenic heterozygous c.314del (p.Pro105Leufs^*Conclusions: Molecular autopsy can establish cardiogenetic diagnosis and allow appropriate preventative measures in high-risk relatives.

Keywords: arrhythmogenic cardiomyopathy; juvenile sudden death; molecular autopsy; novel PKP2 mutation; sport restriction.

Cancer stem cells (CSCs) have garnered attention with their potential for early diagnosis and prognosis of oral squamous cell carcinoma (OSCC). It is still indistinct whether CSCs are recognized with a specific set of characteristics. The present study aimed to assess the association of CD44 with stemness-related, Epithelial Mesenchymal Transition EMT-related genes and the secretome of the CSCs. The single-cell suspension from primary OSCC tumors was prepared by enzymatic digestion and the cells were cultured in-vitro. The cancer stem cells were isolated by CD44+ selection using magnetic cell-sorting. The expression of CD44, proliferation rate, gene expression of EMT-related transcription factors, stemness markers, cytokine levels and angiogenic factors in both cell population was assessed. The sorted CD44+ cells showed significantly higher proliferation rate than heterogenous population. The CD44 expression was >90% in the sorted cells which was higher than the heterogenous cells. The CD44+ CSCs cells demonstrated significant increased levels of EMT-related genes TWIST1 and CDH2 (N-cadherin), CSC-related genes CD44 and CD133 (PROM1), stemness-related genes OCT4, SOX2, inflammatory cytokines IL-1ß, IL-12, IL-18 and TNF-α and angiogenic factors Angiopoietin-1, Angiopoietin-2, bFGF and VEGF while levels of epithelial gene CDH1 (E-cadherin) decreased in comparison to mixed cell population. The genetic and secretome profiling of the CD44+ CSCs could serve as diagnostic and prognostic tools in the treatment of oral cancers.

Keywords: CD44; cancer stem cells; oral squamous cell carcinoma; secretome.