Oleanolic acid regulates the proliferation and extracellular matrix of keloid fibroblasts by mediating the TGF-β1/SMAD signaling pathway

Background

Keloid (KD) is a unique pathological fibroproliferative disease that seriously affects the appearance of patients. This study investigated the effect of oleanolic acid (OA) on the proliferation of keloid fibroblasts (KFs) and the expression of extracellular matrix (ECM)-related proteins.

Methods

The proliferation of KFs was evaluated using an MTT assay. The effects of OA on intra- and extracellular levels of fibronectin (FN), procollagen I, matrix metalloproteinase-1 (MMP-1), and α-smooth muscle actin (α-SMA) were evaluated using Western blotting. To simulate the KD microenvironment, TGF-β1 was added to the serum-free culture medium, and KFs were incubated with TGF-β1 and OA for 24 h. The intra- and extracellular levels of the ECM-related proteins and the effect of OA on TGF-β1-induced phosphorylation of the SMAD2 and SMAD3 proteins were evaluated using Western blotting.

Results

OA inhibited the proliferation of KFs in a concentration- and time-dependent manner. Furthermore, OA treatment of KFs reduced the intra- and extracellular levels of FN, procollagen I, and α-SMA and increased those of MMP-1. OA also reduced TGF-β1-induced increases in the intra- and extracellular levels of FN, procollagen I, and α-SMA and increased the levels of the MMP-1 protein. Additionally, OA significantly reduced TGF-β1-induced phosphorylation of SMAD2 and SMAD3 in KFs.

Conclusions

OA inhibited KF proliferation and reduced ECM deposition through the TGF-β1/SMAD pathway, which suggests that OA may be an effective drug for the prevention and treatment of KD.     

Assessment of the ForenSeq mtDNA control region kit and comparison of orthogonal technologies

The ForenSeq® mtDNA Control Region Kit, MiSeq FGx®, and Universal Analysis Software (UAS) were assessed to better define the performance and limitations of the system with forensically relevant samples to provide data for its transition into practice. A total of six MiSeq FGx sequencing runs of ForenSeq mtDNA Control Region kit, three runs of additional orthogonal sequencing chemistries, and Sanger sequencing results for 14 samples were used to test for concordance. Sensitivity, reproducibility, mixture detection studies, as well as studies to measure the performance of amplification and sequencing controls were performed. The use and reliability of the UAS for data analysis was also examined. With a variety of sample types and controls representing many mitochondrial haplotypes, the recently developed mtDNA Control Region Kit, with the MiSeq FGx and UAS, was found to be fit for purpose as reliable, reproducible, and robust. Sensitivity down to 1 pg of input genomic DNA was demonstrated, which allows the system to offer low limits of detection for better interrogation of potential heteroplasmy in samples. Concerns for implementing next generation sequencing (NGS) for mtDNA in laboratories were addressed in this research, including initial template quantification and confirmation of haplotypes generated by UAS software regarding length-based polymorphisms. To improve performance with forensic samples, laboratories could implement mitochondrial-specific qPCR assays for quantification and perform the optional manual normalization protocol. Additional optimization on sample multiplexing can provide methods that either increase sensitivity or cost efficiency of the assay.     

Development and validation of a fast and automated DNA identification line

The importance of DNA evidence for gaining investigative leads demands a fast workflow for forensic DNA profiling performed in large volumes. Therefore, we developed software solutions for automated DNA profile analysis, contamination check, major donor inference, DNA database (DDB) comparison and reporting of the conclusions. This represents the Fast DNA IDentification Line (FIDL) and this study describes its development, validation and implementation in criminal casework at the authors’ institute. This first implementation regards single donor profiles and major contributors to mixtures. The validation included testing of the software components on their own and examination of the performance of different DDB search strategies. Furthermore, end-to-end testing was performed under three conditions: (1) testing of scenarios that can occur in DNA casework practice, (2) tests using three months of previous casework data, and (3) testing in a casework production environment in parallel to standard casework practices. The same DNA database candidates were retrieved by this automated line as by the manual workflow. The data flow was correct, results were reproducible and robust, results requiring manual analysis were correctly flagged, and reported results were as expected. Overall, we found FIDL valid for use in casework practice in our institute. The results from FIDL are automatically reported within three working days from receiving the trace sample. This includes the time needed for registration of the case, DNA extraction, quantification, polymerase chain reaction and capillary electrophoresis. FIDL itself takes less than two hours from intake of the raw CE data to reporting. Reported conclusions are one of five options: (1) candidate retrieved from DDB, (2) no candidate retrieved from DDB, (3) high evidential value with regards to reference within the case, (4) results require examination of expert, or (5) insufficient amount of DNA obtained to generate a DNA profile. In our current process, the automated report is sent within three working days and a complete report, with confirmation of the FIDL results, and signed by a reporting officer is sent at a later time. The signed report may include additional analyses regarding e.g. minor contributors. The automated report with first case results is quickly available to the police enabling them to act upon the DNA results prior to receiving the full DNA report. This line enables a uniform and efficient manner of handling large numbers of traces and cases and provides high value investigative leads in the early stages of the investigation.