Inhibition of osteoblastic Smurf1 promotes bone formation in mouse models of distinctive age-related osteoporosis.

Bone morphogenetic protein (BMP) signaling is essential for osteogenesis. However, recombinant human BMPs (rhBMPs) exhibit large inter-individual variations in local bone formation during clinical spinal fusion. Smurf1 ubiquitinates BMP downstream molecules for degradation. Here, we classify age-related osteoporosis based on distinct intraosseous BMP-2 levels and Smurf1 activity. One major subgroup with a normal BMP-2 level and elevated Smurf1 activity (BMP-2n/Smurf1e) shows poor response to rhBMP-2 during spinal fusion, when compared to another major subgroup with a decreased BMP-2 level and normal Smurf1 activity (BMP-2d/Smurf1n). We screen a chalcone derivative, i.e., 2-(4-cinnamoylphenoxy)acetic acid, which effectively inhibits Smurf1 activity and increases BMP signaling. For BMP-2n/Smurf1e mice, the chalcone derivative enhances local bone formation during spinal fusion. After conjugating to an osteoblast-targeting and penetrating oligopeptide (DSS)6, the chalcone derivative promotes systemic bone formation in BMP-2n/Smurf1e mice. This study demonstrates a precision medicine-based bone anabolic strategy for age-related osteoporosis.

Tumor cell-targeted delivery of CRISPR/Cas9 by aptamer-functionalized lipopolymer for therapeutic genome editing of VEGFA in osteosarcoma.

Osteosarcoma (OS) is a highly aggressive pediatric cancer, characterized by frequent lung metastasis and pathologic bone destruction. Vascular endothelial growth factor A (VEGFA), highly expressed in OS, not only contributes to angiogenesis within the tumor microenvironment via paracrine stimulation of vascular endothelial cells, but also acts as an autocrine survival factor for tumor cell themselves, thus making it a promising therapeutic target for OS. CRISPR/Cas9 is a versatile genome editing technology and holds tremendous promise for cancer treatment. However, a major bottleneck to achieve the therapeutic potential of the CRISPR/Cas9 is the lack of in vivo tumor-targeted delivery systems. Here, we screened an OS cell-specific aptamer (LC09) and developed a LC09-functionalized PEG-PEI-Cholesterol (PPC) lipopolymer encapsulating CRISPR/Cas9 plasmids encoding VEGFA gRNA and Cas9. Our results demonstrated that LC09 facilitated selective distribution of CRISPR/Cas9 in both orthotopic OS and lung metastasis, leading to effective VEGFA genome editing in tumor, decreased VEGFA expression and secretion, inhibited orthotopic OS malignancy and lung metastasis, as well as reduced angiogenesis and bone lesion with no detectable toxicity. The delivery system simultaneously restrained autocrine and paracrine VEGFA signaling in tumor cells and could facilitate translating CRISPR-Cas9 into clinical cancer treatment.

Telomerase Expression in Medaka ( Oryzias melastigma) Pharyngeal Teeth.

Nonmammalian vertebrates have the capacity of lifelong tooth replacement. In all vertebrates, tooth formation requires contact and interaction between the oral or pharyngeal epithelium and the underlying mesenchyme. To secure lifelong replacement, the presence of odontogenic stem cells has been postulated, particularly in the epithelial compartment. This study uses an advanced teleost fish species, the marine medaka Oryzias melastigma, a close relative to Oryzias latipes, to examine the expression and distribution of telomerase reverse transcriptase (Tert), the catalytic unit of telomerase, in developing pharyngeal teeth and to relate these data to the proliferative activity of the cells. The data are complemented by expression analysis of the pluripotency marker oct4 and bona fide stem cell marker lgr5. Tert distribution and tert expression in developing tooth germs show a dynamic spatiotemporal pattern. Tert is present first in the mesenchyme but is downregulated as the odontoblasts differentiate. In contrast, in the epithelial enamel organ, Tert is absent during early stages of tooth formation and upregulated first in ameloblasts. Later, Tert is expressed and immunolocalized throughout the entire inner enamel epithelium. The pattern of Tert distribution is largely mutually exclusive with that of proliferating cell nuclear antigen (PCNA) immunoreactivity: highly proliferative cells, as revealed by PCNA staining, are negative for Tert; conversely, PCNA-negative cells are Tert-positive. Only the early condensed mesenchyme is both Tert- and PCNA-positive. The absence of tert-positive cells in the epithelial compartment of early tooth germs is underscored by the absence of oct4- and lgr5-positive cells, suggesting ways other than stem cell involvement to secure continuous renewal.

Aptamer-functionalized lipid nanoparticles targeting osteoblasts as a novel RNA interference-based bone anabolic strategy.

Currently, major concerns about the safety and efficacy of RNA interference (RNAi)-based bone anabolic strategies still exist because of the lack of direct osteoblast-specific delivery systems for osteogenic siRNAs. Here we screened the aptamer CH6 by cell-SELEX, specifically targeting both rat and human osteoblasts, and then we developed CH6 aptamer-functionalized lipid nanoparticles (LNPs) encapsulating osteogenic pleckstrin homology domain-containing family O member 1 (Plekho1) siRNA (CH6-LNPs-siRNA). Our results showed that CH6 facilitated in vitro osteoblast-selective uptake of Plekho1 siRNA, mainly via macropinocytosis, and boosted in vivo osteoblast-specific Plekho1 gene silencing, which promoted bone formation, improved bone microarchitecture, increased bone mass and enhanced mechanical properties in both osteopenic and healthy rodents. These results indicate that osteoblast-specific aptamer-functionalized LNPs could act as a new RNAi-based bone anabolic strategy, advancing the targeted delivery selectivity of osteogenic siRNAs from the tissue level to the cellular level.

Expression profile of oestrogen receptors and oestrogen-related receptors is organ specific and sex dependent: the Japanese medaka Oryzias latipes model.

Gene expression of all known subtypes of oestrogen receptor (ER) and oestrogen-related receptor (ERR) in multiple organs and both sexes of the Japanese medaka Oryzias latipes was profiled and systematically analysed. As revealed by statistical analyses and low-dimensional projections, the expressions of ERRs proved to be organ and sex dependent, which is in contrast with the ubiquitous nature of ERs. Moreover, expressions of specific ERR isoforms (ERRγ1, ERRγ2) were strongly correlated with that of all ERs (ERα, ERß1 and ERß2), suggesting the existence of potential interactions. Findings of this study shed light on the co-regulatory role of particular ERRs in oestrogen-ERs signalling and highlight the potential importance of ERRs in determining organ and sex-specific oestrogen responses. Using O. latipes as an alternative vertebrate model, this study provides new directions that call for collective efforts from the scientific community to unravel the mechanistic action of ER-ERR cross-talks, and their intertwining functions, in a cell and sex-specific manner in vivo.

Mechanisms of photosynthetic inactivation on growth suppression of Microcystis aeruginosa under UV-C stress.

This study aims to investigate the effects of UV-C irradiation on photosynthetic processes of Microcystis aeruginosa to unravel the mechanism(s) involved in how and in what ways UV-C mediates growth suppression and cellular recovery. Changes in the concentration of photosynthetic pigments, photochemical efficiency, PS II core protein (D1) content, and the coding genes expressions were measured. The results indicate that UV-C doses at 20-200 mJ cm(-2) lead to rapid reduction in gene expression of both psbA (for D1) and cpc (for phycocyanin), but the suppression was short term and recoverable within 3 d of post-UV incubation. Conversely, UV-C doses at ≥50 mJ cm(-2) could induce marked decline in photochemical efficiency (represented by the optimal PS II quantum yield, FV/FM, and the effective PS II quantum yield, Y) as well as decreases in D1 content and water soluble pigments (phycoerythrins, phycocyanins, allophycocyanins) in M. aeruginosa during the post UV-C incubation period. The results suggest that interruption of both the light energy harvesting apparatus (especially the water soluble pigments) and the photochemical process mainly accounted for the growth suppression effect in UV-C irradiated M. aeruginosa.

Modulation of ion transporter expression in gill mitochondrion-rich cells of eels acclimated to low-Na(+) or-Cl(-) freshwater.

In this study, we aimed to establish an experimental model to study the role of the gill mitochondrion-rich cells (MRCs) of freshwater fish in Na(+) uptake and to examine the effect of adjusting external Na(+) and Cl(-) ions on selected ion transporters in gill MRCs. Japanese eels (Anguilla japonica) acclimated to deionized (DI) water for 2 weeks were transferred directly to (a) ion-supplemented artificial freshwater (AF), (b) Na(+) -deficient AF, or (c) Cl(-) -deficient AF for 2 days. The effects of the transfer on the expression levels of ion transporters in isolated gill cells were investigated. Our data demonstrated that the 2-day acclimation in ion-supplemented AF, Na(+) -deficient AF, or Cl(-) -deficient AF led to a significant increase in serum osmolarity attributed mainly to an increase in serum Na(+) and/or Cl(-) levels when compared with DI-acclimated eel. Significant inductions of V-type H(+) -ATPase (V-H(+) -ATPase) and cotransporter (NBC1) mRNA expression in gill MRCs were detected in AF-acclimated fish. In fish acclimated to Na(+) -deficient AF, mRNA expression levels of V-H(+) -ATPase, NBC1, and Na(+) /H(+) -exchanger-3 (NHE3) were significantly increased in MRCs. Fish acclimated to Cl(-) -deficient AF showed no observable change in expression levels of ion transporters in gill MRCs. In addition, expression levels of ion transporters in pavement cells were stable throughout the 2-day experiments. These data indicate that the level of Na(+) in freshwater is important for altering the mRNA expression of ion transporters in gill MRCs, which supports the notion that gill MRCs play important roles in freshwater Na(+) uptake.

In situ hybridization to detect spatial gene expression in medaka.

A whole-animal tissue section in situ hybridization (ISH) system with radio-labeled probes was developed to detect differential gene expression among tissues of the small, oviparous teleost fish, Japanese medaka (Oryzias latipes). Because of its tissue- and gender-specific expression, gonadal aromatase (CYP19a) was selected as a model gene to demonstrate the potential of the system. The ISH system was validated with a 7d exposure to the model aromatase inhibitor, fadrozole. Fadrozole did not affect the magnitude of gene expression in testes, but significantly up-regulated CYP19a gene expression in ovaries. These results were confirmed with quantitative real-time-polymerase chain reaction (RT-PCR). Histological evaluation revealed that females exposed to 100microg/L fadrozole lacked mature oocytes. Male gonadal morphology was normal in all treatments. The ISH method developed in this study allowed tissue-specific resolution of gene expression in a whole animal model, as well as the ability to analyze cellular morphological detail in the same organism.

Development of a marine fish model for studying in vivo molecular responses in ecotoxicology.

A protocol for fixation and processing of whole adult marine medaka (Oryzias melastigma) was developed in parallel with in situ hybridization (ISH) and immunohistochemistry (IHC) for molecular analysis of in vivo gene and protein responses in fish. Over 200 serial sagittal sections (5microm) can be produced from a single adult medaka to facilitate simultaneous localization and quantification of gene-specific mRNAs and proteins in different tissues and subcellular compartments of a single fish. Stereological analysis (as measured by volume density, V(v)) was used to quantify ISH and IHC signals on tissue sections. Using the telomerase reverse transcriptase (omTERT) gene, omTERT and proliferating cell nuclear antigen (PCNA) proteins as examples, we demonstrated that it is possible to localize, quantify and correlate their tissue expression profiles in a whole fish system. Using chronic hypoxia (1.8+/-0.2 mgO(2)L(-1) for 3 months) as an environmental stressor, we were able to identify significant alterations in levels of omTERT mRNA, omTERT protein, PCNA (cell proliferation marker) and TUNEL (apoptosis) in livers of hypoxic O. melastigma (p<0.05). Overall, the results suggest that O. melastigma can serve as a model marine fish for assessing multiple in vivo molecular responses to stresses in the marine environment.

An ultrastructural study of phagocytosis and shrinkage in nutritive phagocytes of the sea urchin Anthocidaris crassispina.

The ultrastructural mechanisms of waste-sperm phagocytosis and postspawning shrinkage were studied for accessory cells (nutritive phagocytes; NPs) of the sea urchin Anthocidaris crassispina. Sperm cells were phagocytosed by NPs; they penetrated into the cytoplasm of the NPs inside heterophagosomes formed by an invagination of the cell membrane. Single-sperm-containing heterophagosomes aggregated to form large multisperm heterophagosomes that were accompanied by cytoplasmic vesicles and lipids. Two types of vesicle, viz., Golgi-complex-derived electron-dense vesicles ("zymogen granules") and smooth-endoplasmic-reticulum-derived electron-lucent vesicles, were incorporated within multisperm heterophagosomes. Completed multisperm heterophagosomes were transformed into electron-dense remnant bodies, the content of which underwent destruction, resulting in "empty" vacuoles inside the remnant body. The "empty" vacuoles were then compressed by the surrounding cytoplasm. Shrinkage of NPs occurred upon completion of sperm degeneration in gonad tubules. This process was undertaken by structures termed cell-size-reducing autolysosomes, which performed two types of autolysis, and resulted in the formation of "cheese-hole"-like vacuoles in the cytoplasm of NPs. Subsequent cytoplasmic compression of these vacuoles was required for the reduction in size of NPs, an essential event for remodeling the cell for the next gametogenetic cycle.

The application of histo-cytopathological biomarkers in marine pollution monitoring: a review.

During the past two decades, a variety of histopathological alterations in fish and bivalves have been developed and used as biomarkers in pollution monitoring. Some of these have been successfully adopted in major national monitoring programmes, while others, although show promise, are still in the experimental stage. This paper critically reviews the scientific basis, cause and effect relationship, reliability, advantages and limitations of 14 histo-cytopathological biomarkers. The usefulness and practical application of each biomarker have been evaluated against a number of objective criteria including: ecological relevance, sensitivity, specificity, dose-response relationship, confounding factors, technical difficulties and cost-effectiveness.

Impairment of sea urchin sperm quality by UV-B radiation: predicting fertilization success from sperm motility.

Sperm quality of the sea urchin, Anthocidaris crassispina, after exposure to environmentally realistic UV-B irradiances, was assessed by changes in sperm motility (measured by the computer-assisted sperm analysis (CASA) system), and related to subsequent fertilization success. Percentage motile sperm of A. crassispina declined significantly after exposure to a UV-B dose of 16.2 kJ m(-2), while sperm motion velocity as measured by curvilinear velocity (VCL), straight line velocity (VSL), and average path velocity (VAP) showed significant reduction after exposure to a UV-B dose of 5.4 kJ m(-2). A parallel study showed that fertilization success was significantly reduced after sperm were exposed to UV-B doses > or = 5.4 kJ m(-2). Notably, the four sperm motility parameters were strongly correlated with fertilization success (P < 0.001), followed the increasing order: VSL (r = 0.8) < % motile sperm (r = 0.804) < VCL (r = 0.912) < VAP (r = 0.928). Fertilization success is best predicted by VAP using the exponential model: y = 8.678 + 90.202/[1 + exp(82.83 - x)/10.27)] (r(2) = 0.95). Thus, impairment of sperm motility of sea urchin, as measured by the CASA method, can be used to predict reproductive success and ecological effects.