Polyethylenimine-complexed plasmid particles targeting focal adhesion kinase function as melanoma tumor therapeutics.

Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase implicated in cell cycle progression and cell migration. Overexpression of FAK in a variety of tumors has suggested that FAK is a promising target for therapeutic intervention. In this study, we took advantage of a modified polyethylenimine (M-PEI) with high transfection efficiency for tumor cells and tissues, and targeted FAK function through both in vitro and in vivo approaches. The results demonstrated that both plasmid-encoded FAK small interfering RNA (siRNA) and overexpression of FAK-related non-kinase (FRNK, FAK dominant negative) dramatically inhibited in vitro B16F10 cell proliferation and invasion. We used two transplantable mouse tumor models of primary and metastatic melanoma to evaluate the therapeutic potential of PEI-complexed plasmids targeting FAK function. The results revealed that intratumoral delivery of PEI-complexed plasmids targeting FAK significantly suppressed primary tumor growth as well as metastasis of B16F10 cells into lung and lymph nodes. Both approaches prolonged the survival of the tumor-bearing mice. Taken together, these results indicate that intratumoral delivery of plasmid DNA targeting FAK function, using M-PEI as a gene carrier, represents a promising avenue for melanoma therapy.

Variations in oral microbiome profiles in rheumatoid arthritis and osteoarthritis with potential biomarkers for arthritis screening.

The key to arthritis management is early diagnosis and treatment to prevent further joint destruction and maximize functional ability. Osteoarthritis (OA) and rheumatoid arthritis (RA) are two common types of arthritis that the primary care provider must differentiate, in terms of diagnosis and treatment. Effective and non-invasive strategies for early detection and disease identification are sorely needed. Growing evidence suggests that RA has a correlation with oral microbiome and may be affected by its dynamic variations. There is already a study comparing oral microbiome in patients with RA and OA, however, it did not screen for potential biomarkers for arthritis. In this study, we assessed the oral microbiome in saliva samples from 110 RA patients, 67 OA patients and 155 healthy subjects, using 16S rRNA gene amplicon sequencing. The structure and differences in oral microbiome between RA, OA and healthy subjects were analyzed. Eight oral bacterial biomarkers were identified to differentiate RA from OA. This report provides proof of oral microbiota as an informative source for discovering non-invasive biomarkers for arthritis screening.

Intracellular calcium and cyclic nucleotide levels modulate neurite guidance by microtopographical substrate features.

Micro- and nanoscale surface features have emerged as potential tools to direct neurite growth into close proximity with next generation neural prosthesis electrodes. However, the signaling events underlying the ability of growth cones to respond to topographical features remain largely unknown. Accordingly, this study probes the influence of [Ca(2+) ]i and cyclic nucleotide levels on the ability of neurites from spiral ganglion neurons (SGNs) to precisely track topographical micropatterns. Photopolymerization and photomasking were used to generate micropatterned methacrylate polymer substrates. Dissociated SGN cultures were plated on the micropatterned surfaces. Calcium influx and release from internal stores were manipulated by elevating extracellular K(+) , maintenance in calcium-free media, or bath application of various calcium channel blockers. Cyclic nucleotide activity was increased by application of cpt-cAMP or 8-Br-cGMP. Elevation of [Ca(2+) ]i by treatment of cultures with elevated potassium reduced neurite alignment to physical microfeatures. Maintenance of cultures in Ca(2+) -free medium or treatment with the non-selective voltage-gated calcium channel blocker cadmium or L-type Ca(2+) channel blocker nifedipine did not signficantly alter SGN neurite alignment. By contrast, ryanodine or xestospongin C, which block release of internal calcium stores via ryanodine-sensitive channels or inositol-1,4,5-trisphosphate receptors respectively, each significantly decreased neurite alignment. Cpt-cAMP significantly reduced neurite alignment while 8-Br-cGMP significantly enhanced neurite alignment. Manipulation of [Ca(2+) ]i or cAMP levels significantly disrupts neurite guidance while elevation of cGMP levels increases neurite alignment. The results suggest intracellular signaling pathways similar to those recruited by chemotactic cues are involved in neurite guidance by topographical features. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2037-2048, 2016.

Comparison of two-stage acid-alkali and alkali-acid pretreatments on enzymatic saccharification ability of the sweet sorghum fiber and their physicochemical characterizations.

Two-stage acid/alkali pretreatment was used to enhance the saccharification efficiency of sweet sorghum fiber. The physicochemical characterizations of the pretreated fibers were evaluated by SEM, FTIR and XRD. The acid and alkali sequence in the two-stage pretreatment process was compared, and their dosage was optimized. The results indicated that the two-stage pretreatment showed better saccharification performance when compared with conventional single stage pretreatment. And compared with the acid-alkali sequence, the alkali-acid sequence achieved higher glucose yield (0.23g·g-1) under the optimized conditions, which was 1.64 and 1.21 times higher than that of the single stage and the acid-alkali pretreatments, respectively. Overall, the two-stage pretreatment process is a promising approach to achieve high fermentable glucose conversion rate of cellulosic material.

Photopolymerized microfeatures for directed spiral ganglion neurite and Schwann cell growth.

Cochlear implants (CIs) provide auditory perception to individuals with severe hearing impairment. However, their ability to encode complex auditory stimuli is limited due, in part, to poor spatial resolution caused by electrical current spread in the inner ear. Directing nerve cell processes towards target electrodes may reduce the problematic current spread and improve stimulatory specificity. In this work, photopolymerization was used to fabricate micro- and nano-patterned methacrylate polymers to probe the extent of spiral ganglion neuron (SGN) neurite and Schwann cell (SGSC) contact guidance based on variations in substrate topographical cues. Micropatterned substrates are formed in a rapid, single-step reaction by selectively blocking light with photomasks which have parallel line-space gratings with periodicities of 10-100 µm. Channel amplitudes of 250 nm-10 µm are generated by modulating UV exposure time, light intensity, and photoinitiator concentration. Gradual transitions are observed between ridges and grooves using scanning electron and atomic force microscopy. The transitions stand in contrast to vertical features generated via etching lithographic techniques. Alignment of neural elements increases significantly with increasing feature amplitude and constant periodicity, as well as with decreasing periodicity and constant amplitude. SGN neurite alignment strongly correlates (r = 0.93) with maximum feature slope. Multiple neuronal and glial types orient to the patterns with varying degrees of alignment. This work presents a method to fabricate gradually-sloping micropatterns for cellular contact guidance studies and demonstrates spatial control of inner ear neural elements in response to micro- and nano-scale surface topography.

Non-syndromic tooth agenesis in two Chinese families associated with novel missense mutations in the TNF domain of EDA (ectodysplasin A).

Here we report two unrelated Chinese families with congenital missing teeth inherited in an X-linked manner. We mapped the affected locus to chromosome Xp11-Xq21 in one family. In the defined region, both families were found to have novel missense mutations in the ectodysplasin-A (EDA) gene. The mutation of c.947A>G caused the D316G substitution of the EDA protein. The mutation of c.1013C>T found in the other family resulted in the Thr to Met mutation at position 338 of EDA. The EDA gene has been reported responsible for X-linked hypohidrotic ectodermal dysplasia (XLHED) in humans characterized by impaired development of hair, eccrine sweat glands, and teeth. In contrast, all the affected individuals in the two families that we studied here had normal hair and skin. Structural analysis suggests that these two novel mutants may account for the milder phenotype by affecting the stability of EDA trimers. Our results indicate that these novel missense mutations in EDA are associated with the isolated tooth agenesis and provide preliminary explanation for the abnormal clinical phenotype at a molecular structural level.

An approach for precise three-dimensional modeling of the human inner ear.

For further morphological and physiological research, it is vital to establish precise three-dimensional models of the whole inner ear including the details of the membranous components. With the system of a projector and a high-resolution digital camera, 2 complete serial unstained celloidin sections of fresh human temporal bones were digitized as high-resolution images which were then sorted, calibrated, aligned and segmented using the 3D-Doctor software. Finally, 2 precise three-dimensional models of the inner ear were generated by simple surface rendering. The contours of tiny structures such as the crista ampullaris, the macula utriculi and the macula sacculi could be observed clearly. Our study suggests that it is technically feasible to employ complete serial unstained celloidin sections for precise three-dimensional reconstruction and that this helps reduce errors and laboratory workload. Moreover, the use of a high-resolution digital camera and the autoalignment function of 3D-Doctor further increase the accuracy of the models.

Cross-linked polyethylenimine as potential DNA vector for gene delivery with high efficiency and low cytotoxicity.

Polyethylenimine (PEI) has been known as an efficient gene carrier with the highest cationic charge potential. High transfection efficiency of PEI, along with its cytotoxicity, strongly depends on its molecular weight. To enhance its gene delivery efficiency and minimize cytotoxicity, we have synthesized small cross-linked PEI with biodegradable linkages and evaluated their transfection efficiencies in vitro. In this study, branched PEI with a molecular weight of 800 Da was cross-linked by small diacrylate [1,4-butanediol diacrylate or ethyleneglycol dimethacrylate (EGDMA)] for 2-6 h. The efficiencies of the cross-linked PEI in in vitro transfection of plasmid DNA containing enhanced green fluorescent protein (EGFP) reporter gene were assessed in melanoma B16F10 cell line and other cell lines. Flow cytometry was used to quantify the cellular entry efficiency of plasmid and the transgene expression level. The cytotoxicities of the cross-linked PEI in these cells were evaluated by MTT assay. EGDMA-PEI 800-4h, a typical cross-linked PEI reported here, mediated a more efficient expression of reporter gene than the commercially available 25-kDa branched PEI control, and resulted in a 9-fold increase in gene delivery in B16F10 cells and a 16-fold increase in 293T cells, while no cytotoxicity was found at the optimized condition for gene delivery. Furthermore, the transfection activity of polyplexes was preserved in the presence of serum proteins.