MicroRNA Profiling Implies New Markers of Gemcitabine Chemoresistance in Mutant p53 Pancreatic Ductal Adenocarcinoma.

No reliable predictors of susceptibility to gemcitabine chemotherapy exist in pancreatic ductal adenocarcinoma (PDAC). MicroRNAs (miR) are epigenetic gene regulators with tumorsuppressive or oncogenic roles in various carcinomas. This study assesses chemoresistant PDAC for its specific miR expression pattern.Gemcitabine-resistant variants of two mutant p53 human PDAC cell lines were established. Survival rates were analyzed by cytotoxicity and apoptosis assays. Expression of 1733 human miRs was investigated by microarray and validated by qRT-PCR. After in-silico analysis of specific target genes and proteins of dysregulated miRs, expression of MRP-1, Bcl-2, mutant p53, and CDK1 was quantified by Western blot.Both established PDAC clones showed a significant resistance to gemcitabine (p<0.02) with low apoptosis rate (p<0.001) vs. parental cells. MiR-screening revealed significantly upregulated (miR-21, miR-99a, miR-100, miR-125b, miR-138, miR-210) and downregulated miRs (miR-31*, miR-330, miR-378) in chemoresistant PDAC (p<0.05). Bioinformatic analysis suggested involvement of these miRs in pathways controlling cell death and cycle. MRP-1 (p<0.02) and Bcl-2 (p<0.003) were significantly overexpressed in both resistant cell clones and mutant p53 (p = 0.023) in one clone.Consistent miR expression profiles, in part regulated by mutant TP53 gene, were identified in gemcitabine-resistant PDAC with significant MRP-1 and Bcl-2 overexpression. These results provide a basis for further elucidation of chemoresistance mechanisms and therapeutic approaches to overcome chemoresistance in PDAC

Motion preservation surgery for scoliosis with a vertebral body tethering system: a biomechanical study

Purpose!#!There is a paucity of studies on new vertebral body tethering (VBT) surgical constructs especially regarding their potentially motion-preserving ability. This study analyses their effects on the ROM of the spine.!##!Methods!#!Human spines (T10-L3) were tested under pure moment in four different conditions: (1) native, (2) instrumented with one tether continuously connected in all vertebrae from T10 to L3, (3) additional instrumented with a second tether continuously connected in all vertebrae from T11 to L3, and (4) instrumented with one tether and one titanium rod (hybrid) attached to T12, L1 and L2. The instrumentation was inserted in the left lateral side. The intersegmental ROM was evaluated using a magnetic tracking system, and the medians were analysed. Please check and confirm the author names and initials are correct. Also, kindly confirm the details in the metadata are correct. The mentioned information is correct RESULTS: Compared to the native spine, the instrumented spine presented a reduction of less than 13% in global ROM considering flexion-extension and axial rotation. For left lateral bending, the median global ROM of the native spine (100%) significantly reduced to 74.6%, 66.4%, and 68.1% after testing one tether, two tethers and the hybrid construction, respectively. In these cases, the L1-L2 ROM was reduced to 68.3%, 58.5%, and 38.3%, respectively. In right lateral bending, the normalized global ROM of the spine with one tether, two tethers and the hybrid construction was 58.9%, 54.0%, and 56.6%, respectively. Considering the same order, the normalized L1-L2 ROM was 64.3%, 49.9%, and 35.3%, respectively.!##!Conclusion!#!The investigated VBT techniques preserved global ROM of the spine in flexion-extension and axial rotation while reduced the ROM in lateral bending

Protein target expression.

<p>Protein expression of MRP-1, mutant p53 (mt p53 R273H and mt p53 R248W), CDK1, Bcl-2, and actin (loading control) in parental (PANC-1, MIA-PaCa-2) and gemcitabine resistant PDAC cell clones (PANC-1-GR, MIA-PaCa-2-GR) by Western blot.</p

MicroRNA microarray validation.

<p>MiR microarray validation with qRT-PCR for significantly dysregulated miRs in PANC-1-GR (A) and MIA-PaCa-2-GR cell clones (B). Fold change from miR microarray is demostrated by log2 values (left y-axis, dark grey bars). Fold change from qRT-PCR was determined using the 2^-ΔΔCt method (right y-axis, light grey bars). Error bars represent the standard deviation of mean.</p

Establishment of chemoresistant PDAC cell clones.

<p>Generation of chemoresistant PDAC cell clones by repeated pulsatile treatment over 3 days with constant sublethal concentrations of 0.4μM (A) or 0.06μM (D) gemcitabine followed by recovery-periods and quantification of cell viability by MTT assay as well as apoptosis assay in parental vs. chemoresistant PANC-1 (A, B, C) and MIA-PaCa-2 (D, E, F) cell clones.</p

MicroRNA profiling.

<p>Procedure and results of miR expression profiling by GeneChip microarray and qRT-PCR validation in parental and gemcitabine resistant PANC-1 and MIA-PaCa-2 cell clones.</p

Morphologic changes in parental and chemoresistant PANC-1 and MIA-PaCa-2.

<p>Representative attached epithelial cells with spindle-shaped cells in untreated MIA-PaCa-2 (A) and PANC-1 cells (B) compared to more plump rounded morphology and enhanced formation of pseudopodia in their chemoresistant cell clones (C, D).</p

Intrinsic drug sensitivity of parental PDAC cell lines.

<p>Relative cell viability of five human PDAC cell lines following 72 h exposure to ascending concentrations of gemcitabine by MTT cytotoxicity assay.</p

Protein densitometry.

<p>Adjusted relative density of MRP-1, mutant p53 (mt p53 R273H and mt p53 R248W), CDK1, Bcl-2, and their loading controls measured by ImageJ densitometry software. Asterisks indicates to a significant difference of p<0.05, respectively.</p