Collagen fiber alignment and maximum principal strain in the glenohumeral capsule predict location of failure during uniaxial extension

The glenohumeral joint is frequently dislocated resulting in injury to the glenohumeral capsule. Repair techniques that focus on restoring the capsule after dislocation to re-establish its stabilizing function could benefit from predictions of the location of failure in this continuous sheet of tissue with a random collagen fiber alignment in the unloaded state. Therefore, the objective of this study was to determine the collagen fiber alignment and maximum principal strain in all regions of the capsule during uniaxial extension to failure and to determine whether these parameters could predict the location of tissue failure. Collagen fiber alignment, quantified using a small-angle light-scattering device, and maximum principal strain in the capsule were determined at 5 % increments of elongation until tissue failure. A contingency table analyzed with Fischer’s exact test demonstrated that peak collagen fiber alignment, represented by the normalized orientation index ( $p < 0.001$ ) and maximum principal strain ( $p < 0.001$ ), is significant in predicting location of failure. The direct correlation between the maximum principal strain and collagen fiber alignment measured prior to failure to the location of tissue failure suggests these parameters can be used as a predictive tool to help locate the areas of the glenohumeral capsule that are susceptible to failure. In the future, changes in collagen fiber alignment following injury could be used to develop a constitutive model for injured capsular tissue.     

Adenovirus-Mediated Efficient Gene Transfer into Cultured Three-Dimensional Organoids

Three-dimensional organoids have been recently established from various tissue-specific progenitors (such as intestinal stem cells), induced pluripotent stem cells, or embryonic stem cells. These cultured self-sustaining stem cell–based organoids may become valuable systems to study the roles of tissue-specific stem cells in tissue genesis and disease development. It is thus conceivable that effective genetic manipulations in such organoids may allow us to reconstruct disease processes and/or develop novel therapeutics. Recombinant adenoviruses are one of the most commonly used viral vectors for in vitro and in vivo gene deliveries. In this study, we investigate if adenoviruses can be used to effectively deliver transgenes into the cultured “mini-gut” organoids derived from intestinal stem cells. Using adenoviral vectors that express fluorescent proteins, we demonstrate that adenoviruses can effectively deliver transgenes into the cultured 3-D “mini-gut” organoids. The transgene expression can last at least 10 days in the cultured organoids. As a proof-of-principle experiment, we demonstrate that adenovirus-mediated noggin expression effectively support the survival and self-renewal of mini-gut organoids, while adenovirus-mediated expression of BMP4 inhibits the self-sustainability and proliferation of the organoids. Thus, our results strongly suggest that adenovirus vectors can be explored as effective gene delivery vehicles to introduce genetic manipulations in 3-D organoids.     

Chronic Electrical Stimulation with a Suprachoroidal Retinal Prosthesis: A Preclinical Safety and Efficacy Study

Purpose

To assess the safety and efficacy of chronic electrical stimulation of the retina with a suprachoroidal visual prosthesis.

Methods

Seven normally-sighted feline subjects were implanted for 96–143 days with a suprachoroidal electrode array and six were chronically stimulated for 70–105 days at levels that activated the visual cortex. Charge balanced, biphasic, current pulses were delivered to platinum electrodes in a monopolar stimulation mode. Retinal integrity/function and the mechanical stability of the implant were assessed monthly using electroretinography (ERG), optical coherence tomography (OCT) and fundus photography. Electrode impedances were measured weekly and electrically-evoked visual cortex potentials (eEVCPs) were measured monthly to verify that chronic stimuli were suprathreshold. At the end of the chronic stimulation period, thresholds were confirmed with multi-unit recordings from the visual cortex. Randomized, blinded histological assessments were performed by two pathologists to compare the stimulated and non-stimulated retina and adjacent tissue.

Results

All subjects tolerated the surgical and stimulation procedure with no evidence of discomfort or unexpected adverse outcomes. After an initial post-operative settling period, electrode arrays were mechanically stable. Mean electrode impedances were stable between 11–15 kΩ during the implantation period. Visually-evoked ERGs & OCT were normal, and mean eEVCP thresholds did not substantially differ over time. In 81 of 84 electrode-adjacent tissue samples examined, there were no discernible histopathological differences between stimulated and unstimulated tissue. In the remaining three tissue samples there were minor focal fibroblastic and acute inflammatory responses.

Conclusions

Chronic suprathreshold electrical stimulation of the retina using a suprachoroidal electrode array evoked a minimal tissue response and no adverse clinical or histological findings. Moreover, thresholds and electrode impedance remained stable for stimulation durations of up to 15 weeks. This study has demonstrated the safety and efficacy of suprachoroidal stimulation with charge balanced stimulus currents.     

Cross‐talk between EGF and BMP9 signalling pathways regulates the osteogenic differentiation of mesenchymal stem cells

Mesenchymal stem cells (MSCs) are multipotent progenitors, which give rise to several lineages, including bone, cartilage and fat. Epidermal growth factor (EGF) stimulates cell growth, proliferation and differentiation. EGF acts by binding with high affinity to epidermal growth factor receptor (EGFR) on the cell surface and stimulating the intrinsic protein tyrosine kinase activity of its receptor, which initiates a signal transduction cascade causing a variety of biochemical changes within the cell and regulating cell proliferation and differentiation. We have identified BMP9 as one of the most osteogenic BMPs in MSCs. In this study, we investigate if EGF signalling cross‐talks with BMP9 and regulates BMP9‐induced osteogenic differentiation. We find that EGF potentiates BMP9‐induced early and late osteogenic markers of MSCs in vitro, which can be effectively blunted by EGFR inhibitors Gefitinib and Erlotinib or receptor tyrosine kinase inhibitors AG‐1478 and AG‐494 in a dose‐ and time‐dependent manner. Furthermore, EGF significantly augments BMP9‐induced bone formation in the cultured mouse foetal limb explants. In vivo stem cell implantation experiment reveals that exogenous expression of EGF in MSCs can effectively potentiate BMP9‐induced ectopic bone formation, yielding larger and more mature bone masses. Interestingly, we find that, while EGF can induce BMP9 expression in MSCs, EGFR expression is directly up‐regulated by BMP9 through Smad1/5/8 signalling pathway. Thus, the cross‐talk between EGF and BMP9 signalling pathways in MSCs may underline their important roles in regulating osteogenic differentiation. Harnessing the synergy between BMP9 and EGF should be beneficial for enhancing osteogenesis in regenerative medicine.     

Effects of green tea polyphenol on the quality of canine semen after long-term storage at 5 °C

The aim of the present study was to determine the effects of green tea polyphenol on the quality of canine semen after long-term storage at 5 °C. The supplementation of a Tris-egg yolk extender with polyphenol (0.5, 0.75, or 1 mg/mL) increased the motility and viability of sperm preserved for four weeks at 5 °C.     

The effect of relaxin supplementation of in vitro maturation medium on the development of cat oocytes obtained from ovaries stored at 4 °C

Relaxin is a member of the insulin-like family of hormones that promotes growth in a number of reproductive tissues, including the granulosa and theca cells. Cat oocytes collected from cold-stored ovaries remain capable of maturing in vitro, but the developmental ability of the oocytes decreases after 24 h of cold storage. To improve the developmental ability of cat oocytes from cold-stored ovaries, we investigated the effect of relaxin supplementation of maturation medium on their meiotic ability and subsequent development. Cat oocytes were collected from ovaries stored at 4 °C for one day and cultured in maturation medium supplemented with different concentrations (0, 10, 20, and 40 ng/ml) of relaxin for 24 h. They were then fertilized in vitro for 12 h with frozen-thawed spermatozoa. After in vitro fertilization, the zygotes were cultured in synthetic oviduct fluid medium for 8 days. There were no significant differences in the maturation rates and glutathione contents of oocytes among the groups, irrespective of relaxin supplementation. The rate of blastocyst formation from oocytes matured with 10 ng/ml relaxin (16.0%) was higher (p < 0.05) than that from oocytes matured without relaxin (5.9%). Our findings indicate that supplementation of 10 ng/ml relaxin into maturation medium may improve blastocyst formation of cat oocytes after in vitro fertilization.     

Cell-surface expression of Aspergillus saitoi-derived functional α-1,2-mannosidase on Yarrowia lipolytica for glycan remodeling

Expression of proteins on the surface of yeast has a wide range of applications, such as development of live vaccines, screening of antibody libraries, and use as whole-cell biocatalysts. The hemiascomycetes yeast Yarrowia lipolytica has been raised as a potential host for heterologous expression of recombinant proteins. In this study, we report the expression of Aspergillus saitoi α-1,2-mannosidase, encoded by the msdS gene, on the cell surface of Y. lipolytica. As the first step to achieve the secretory expression of msdS protein, four different signal sequences-derived from the endogenous Y. lipolytica Lip2 and Xpr2 prepro regions and the heterologous A. niger α-amylase and rice α-amylase signal sequences-were analyzed for their secretion efficiency. It was shown that the YlLip2 prepro sequence was most efficient in directing the secretory expression of msdS in fully N-glycosylated forms. The surface display of msdS was subsequently directed by fusing GPI anchoring motifs derived from Y. lipolytica cell wall proteins, YlCwp1p and YlYwp1p, respectively, to the C-terminus of the Lip2 prepro-msdS protein. The expression of actively functional msdS protein on the cell surface was confirmed by western blot, flow cytometry analysis, along with the α-1,2-mannosidase activity assay using intact Y. lipolytica cells as the enzyme source. Furthermore, the glycoengineered Y. lipolytica Δoch1Δmpo1 strains displaying α-1,2-mannosidase were able to convert Man₈GlcNAc₂ to Man₅GlcNAc₂ efficiently on their cell-wall mannoproteins, demonstrating its potential used for glycoengineering in vitro or in vivo.     

Mechanisms and effects of retention of over-expressed aquaporin AtPIP2;1 in the endoplasmic reticulum

Plasma membrane intrinsic proteins (PIPs) are aquaporins that mediate water transport across the plant plasma membrane (PM). The present work addresses, using Arabidopsis AtPIP2;1 as a model, the mechanisms and significance of trafficking of newly synthesized PIPs from the endoplasmic reticulum (ER) to the Golgi apparatus. A functional diacidic export motif (Asp4-Val5-Glu6) was identified in the N-terminal tail of AtPIP2;1, using expression in transgenic Arabidopsis of site-directed mutants tagged with the green fluorescent protein (GFP). Confocal fluorescence imaging and a novel fluorescence recovery after photobleaching application based on the distinct diffusion of PM and intracellular AtPIP2;1-GFP forms revealed a retention in the ER of diacidic mutated forms, but with quantitative differences. Thus, the individual role of the two acidic Asp4 and Glu6 residues was established. In addition, expression in transgenic Arabidopsis of ER-retained AtPIP2;1-GFP constructs reduced the root hydraulic conductivity. Co-expression of AtPIP2;1-GFP and AtPIP1;4-mCherry constructs suggested that ER-retained AtPIP2;1-GFP may interact with other PIPs to hamper their trafficking to the PM, thereby contributing to inhibition of root cell hydraulic conductivity.     

trnL-F is a powerful marker for DNA identification of field vittarioid gametophytes (Pteridaceae)

Background and Aims

The gametophyte phase of ferns plays an important role in habitat selection, dispersal, adaptation and evolution. However, ecological studies on fern gametophytes have been impeded due to the difficulty of species identification of free-living gametophytes. DNA barcoding provides an alternative approach to identifying fern gametophytes but is rarely applied to field studies. In this study, an example of field vittarioid gametophyte identification using DNA barcoding, which has not been done before, is given.

Methods

A combination of distance-based and tree-based approaches was performed to evaluate the discriminating power of three candidate barcodes (matK, rbcL and trnL-F) on 16 vittarioid sporophytes. Sequences of the trnL-F region were generated from 15 fern gametophyte populations by tissue-direct PCR and were compared against the sporophyte dataset, using BLAST.

Key Results trnL-F

earns highest primer universality and discriminatory ability scores, whereas PCR success rates were very low for matK and rbcL regions (10·8 % and 41·3 %, respectively). BLAST analyses showed that all the sampled field gametophytes could be successfully identified to species level. Three gametophyte populations were also discovered to be living beyond the known occurrence of their sporophyte counterparts.

Conclusions

This study demonstrates that DNA barcoding (i.e. reference databasing, tissue-direct PCR and molecular analysis), especially the trnL-F region, is an efficient tool to identify field gametophytes, and has considerable potential in exploring the ecology of fern gametophytes.     

High-Resolution Optical Molecular Imaging of Changes in Choline Metabolism in Oral Neoplasia

This study was aimed at developing an optical molecular imaging approach to measure differences in uptake and intracellular retention of choline in clinically isolated tissue biopsies from head and neck cancer patients. An optically detectable analogue of choline (propargyl choline) was synthesized and evaluated in 2D and 3D models and clinically isolated paired biopsies (n = 22 biopsies). Fluorescence contrast between clinically abnormal and normal tissues based on uptake and intracellular retention of propargyl choline was measured and correlated with pathologic diagnosis. Results in 2D and 3D models demonstrated a rapid uptake of propargyl choline in cancer cells, uniform permeation in tissue models, and specific detection of intracellular entrapped propargyl choline using the click chemistry reaction with an azide-modified Alexa 488 dye. Fluorescence imaging measurements following topical delivery of propargyl choline in clinically isolated biopsies showed that the mean fluorescence intensity (MFI) of neoplastic tissues was four-fold to five-fold higher than the MFI of clinically and pathologically normal samples. This difference in fluorescence contrast was measured on the basis of comparison of paired biopsy sets isolated from individual patients as well as comparison of clinically abnormal and normal biopsies independent of anatomic locations in the head and neck cavity and across diverse patients. In conclusion, a novel imaging approach based on monoalkyne-modified choline was developed and validated using cell and tissue models. Results in clinically isolated tissue biopsies demonstrate a significant fluorescent contrast between neoplastic and normal tissues and illustrate high specificity of the optical imaging approach.