A comparison study between electrospun polycaprolactone and piezoelectric poly(3-hydroxybutyrate-co-3-hydroxyvalerate) scaffolds for bone tissue engineering.

In this study, bone scaffolds composed of polycaprolactone (PCL), piezoelectric poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and a combination of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and silicate containing hydroxyapatite (PHBV-SiHA) were successfully fabricated by a conventional electrospinning process. The morphological, chemical, wetting and biological properties of the scaffolds were examined. All fabricated scaffolds are composed of randomly oriented fibres with diameters from 800nm to 12µm. Fibre size increased with the addition of SiHA to PHBV scaffolds. Moreover, fibre surface roughness in the case of hybrid scaffolds was also increased. XRD, FTIR and Raman spectroscopy were used to analyse the chemical composition of the scaffolds, and contact angle measurements were performed to reveal the wetting behaviour of the synthesized materials. To determine the influence of the piezoelectric nature of PHBV in combination with SiHA nanoparticles on cell attachment and proliferation, PCL (non-piezoelectric), pure PHBV, and PHBV-SiHA scaffolds were seeded with human mesenchymal stem cells (hMSCs). In vitro study on hMSC adhesion, viability, spreading and osteogenic differentiation showed that the PHBV-SiHA scaffolds had the largest adhesion and differentiation abilities compared with other scaffolds. Moreover, the piezoelectric PHBV scaffolds have demonstrated better calcium deposition potential compared with non-piezoelectric PCL. The results of the study revealed pronounced advantages of hybrid PHBV-SiHA scaffolds to be used in bone tissue engineering.

Hybrid inorganic-organic capsules for efficient intracellular delivery of novel siRNAs against influenza A (H1N1) virus infection.

The implementation of RNAi technology into the clinical practice has been significantly postponing due to the issues regarding to the delivery of naked siRNA predominantly to target cells. Here we report the approach to enhance the efficiency of siRNA delivery by encapsulating the siRNA into new carrier systems which are obtained via the combination of widely used layer-by-layer technique and in situ modification by sol-gel chemistry. We used three types of siRNAs (NP-717, NP-1155 and NP-1496) in encapsulated form as new therapeutic agents against H1N1 influenza virus infection. By employing the hybrid microcontainers for the siRNA encapsulation we demonstrate the reduction of viral nucleoprotein (NP) level and inhibition of influenza virus production in infected cell lines (MDCK and A549). The obtained hybrid carriers based on assembled biodegradable polyelectrolytes and sol-gel coating possess several advantages such as a high cell uptake efficiency, low toxicity, efficient intracellular delivery of siRNAs and the protection of siRNAs from premature degradation before reaching the target cells. These findings underpin a great potential of versatile microencapsulation technology for the development of anti-viral RNAi delivery systems against influenza virus infection.

Mesenchymal Stem Cells Engineering: Microcapsules-Assisted Gene Transfection and Magnetic Cell Separation.

Stem cell engineering-the manipulation and functionalization of stem cells involving genetic modification-can significantly expand their applicability for cell therapy in humans. Toward this aim, reliable, standardized, and cost-effective methods for cell manipulation are required. Here we explore the potential of magnetic multilayer capsules to serve as a universal platform for nonviral gene transfer, stem cell magnetization, and magnetic cell separation to improve gene transfer efficiency. In particular, the following experiments were performed: (i) a study of the process of internalization of magnetic capsules into stem cells, including capsule co-localization with established markers of endo-lysosomal pathway; (ii) characterization and quantification of capsule uptake with confocal microscopy, electron microscopy, and flow cytometry; (iii) intracellular delivery of messenger RNA and separation of gene-modified cells by magnetic cell sorting (MACS); and (iv) analysis of the influence of capsules on cell proliferation potential. Importantly, based on the internalization of magnetic capsules, transfected cells became susceptible to external magnetic fields, which made it easy to enrich gene-modified cells using MACS (purity ∼95%), and also to influence their migration behavior. In summary, our results underline the high potential of magnetic capsules in stem cell functionalization, namely (i) to increase gene-transfer efficiency and (ii) to facilitate enrichment and targeting of transfected cells. Finally, we did not observe a negative impact of the capsules used on the proliferative capacity of stem cells, proving their high biocompatibility.

A gemcitabine sensitivity screen identifies a role for NEK9 in the replication stress response.

The Replication Stress Response (RSR) is a signaling network that recognizes challenges to DNA replication and coordinates diverse DNA repair and cell-cycle checkpoint pathways. Gemcitabine is a nucleoside analogue that causes cytotoxicity by inducing DNA replication blocks. Using a synthetic lethal screen of a RNAi library of nuclear enzymes to identify genes that when silenced cause gemcitabine sensitization or resistance in human triple-negative breast cancer cells, we identified NIMA (never in mitosis gene A)-related kinase 9 (NEK9) as a key component of the RSR. NEK9 depletion in cells leads to replication stress hypersensitivity, spontaneous accumulation of DNA damage and RPA70 foci, and an impairment in recovery from replication arrest. NEK9 protein levels also increase in response to replication stress. NEK9 complexes with CHK1, and moreover, NEK9 depletion impairs CHK1 autophosphorylation and kinase activity in response to replication stress. Thus, NEK9 is a critical component of the RSR that promotes CHK1 activity, maintaining genome integrity following challenges to DNA replication.

CHD7 expression predicts survival outcomes in patients with resected pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with poor outcomes with current therapies. Gemcitabine is the primary adjuvant drug used clinically, but its effectiveness is limited. In this study, our objective was to use a rationale-driven approach to identify novel biomarkers for outcome in patients with early-stage resected PDAC treated with adjuvant gemcitabine. Using a synthetic lethal screen in human PDAC cells, we identified 93 genes, including 55 genes linked to DNA damage responses (DDR), that demonstrated gemcitabine sensitization when silenced, including CHD7, which functions in chromatin remodeling. CHD7 depletion sensitized PDAC cells to gemcitabine and delayed their growth in tumor xenografts. Moreover, CHD7 silencing impaired ATR-dependent phosphorylation of CHK1 and increased DNA damage induced by gemcitabine. CHD7 was dysregulated, ranking above the 90th percentile in differential expression in a panel of PDAC clinical specimens, highlighting its potential as a biomarker. Immunohistochemical analysis of specimens from 59 patients with resected PDAC receiving adjuvant gemcitabine revealed that low CHD7 expression was associated with increased recurrence-free survival (RFS) and overall survival (OS), in univariate and multivariate analyses. Notably, CHD7 expression was not associated with RFS or OS for patients not receiving gemcitabine. Thus, low CHD7 expression was correlated selectively with gemcitabine sensitivity in this patient population. These results supported our rationale-driven strategy to exploit dysregulated DDR pathways in PDAC to identify genetic determinants of gemcitabine sensitivity, identifying CHD7 as a novel biomarker candidate to evaluate further for individualizing PDAC treatment.

Pronecrotic mixed lineage kinase domain-like protein expression is a prognostic biomarker in patients with early-stage resected pancreatic adenocarcinoma.

BACKGROUND: Mixed lineage kinase domain-like protein (MLKL) is a necrosome component mediating programmed necrosis that may be an important determinant of cancer cell death. The goal of the current study was to evaluate the prognostic value of MLKL expression in patients with pancreatic adenocarcinoma (PAC). METHODS: Tissue from 80 patients was collected from a prospectively maintained database of patients with PAC who underwent pancreaticoduodenectomy between January 2000 and October 2008. Immunohistochemistry analysis was performed and scored using an established scoring system. Kaplan-Meier survival curves were generated for recurrence-free survival (RFS) and overall survival (OS) for all patients and for patients receiving adjuvant chemotherapy. MLKL scores were correlated with RFS and OS using univariate and multivariate Cox regression analyses incorporating clinically relevant covariates. RESULTS: The median age of the patients was 63 years and 53% were men. Low MLKL expression was associated with decreased OS (6 months vs 17 months; P = .006). In the subset of 59 patients who received adjuvant chemotherapy, low MLKL expression was associated with decreased RFS (5 months vs 15 months; P = .006) and decreased OS (6 months vs 19 months; P < .0001). On multivariate analysis, low MLKL expression was associated with poor OS in all patients (hazards ratio, 4.6 [95% confidence interval, 1.6-13.8]; P = .006) and in patients receiving adjuvant chemotherapy (hazards ratio, 8.1 [95% confidence interval, 2.2-29.2]; P = .002). CONCLUSIONS: Low expression of MLKL is associated with decreased OS in patients with resected PAC and decreased RFS and OS in the subset of patients with resected PAC who receive adjuvant chemotherapy. The use of this biomarker in patients with PAC may provide important prognostic information.