Monodispersed Bioactive Glass Nanoclusters with Ultralarge Pores and Intrinsic Exceptionally High miRNA Loading for Efficiently Enhancing Bone Regeneration

Bioactive glass nanoparticles (BGNs) have attracted much attention in drug delivery and bone tissue regeneration, due to the advantages including biodegradation, high bone‐bonding bioactivity, and facile large‐scale fabrication. However, the wide biomedical applications of BGNs such as efficient gene delivery are limited due to their poor pore structure and easy aggregation. Herein, for the first time, this study reports novel monodispersed bioactive glass nanoclusters (BGNCs) with ultralarge mesopores (10–30 nm) and excellent miRNA delivery for accelerating critical‐sized bone regeneration. BGNCs with different size (100–500 nm) are fabricated by using a branched polyethylenimine as the structure director and catalyst. BGNCs show an excellent apatite‐forming ability and high biocompatibility. Importantly, BGNCs demonstrate an almost 19 times higher miRNA loading than those of conventional BGNs. Additionally, BGNCs–miRNA nanocomplexes exhibit a significantly high antienzymolysis, enhance cellular uptake and miRNA transfection efficiency, overpassing BGNs and commercial Lipofectamine 3000. BGNCs‐mediated miRNA delivery significantly improves the osteogenic differentiation of bone marrow stromal stem cells in vitro and efficiently enhances bone formation in vivo. BGNCs can be a highly efficient nonviral vector for various gene therapy applications. The study may provide a novel strategy to develop highly gene‐activated bioactive nanomaterials for simultaneous tissue regeneration and disease therapy.Monodispersed bioactive glass nanoclusters (BGNCs) with ultra‐large mesopores (10–30 nm) are developed for miRNA delivery to enhance bone regeneration. BGNCs demonstrated an ultrahigh miRNA loading and transfection efficiency, overpassing commercial lipofectamine. BGNCs‐mediated miRNA delivery significantly improved osteogenic differentiation of bone marrow stromal stem cells in vitro and enhanced bone formation in vivo.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/139128/1/adhm201700630-sup-0001-S1.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/139128/2/adhm201700630.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/139128/3/adhm201700630_am.pd

The Chinese pine genome and methylome unveil key features of conifer evolution

Conifers dominate the world's forest ecosystems and are the most widely planted tree species. Their giant and complex genomes present great challenges for assembling a complete reference genome for evolutionary and genomic studies. We present a 25.4-Gb chromosome-level assembly of Chinese pine (Pinus tabuliformis) and revealed that its genome size is mostly attributable to huge intergenic regions and long introns with high transposable element (TE) content. Large genes with long introns exhibited higher expressions levels. Despite a lack of recent whole-genome duplication, 91.2% of genes were duplicated through dispersed duplication, and expanded gene families are mainly related to stress responses, which may underpin conifers' adaptation, particularly in cold and/or arid conditions. The reproductive regulation network is distinct compared with angiosperms. Slow removal of TEs with high-level methylation may have contributed to genomic expansion. This study provides insights into conifer evolution and resources for advancing research on conifer adaptation and development

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

Connexin Gap Junctions and Hemichannels in Modulating Lens Redox Homeostasis and Oxidative Stress in Cataractogenesis

The lens is continuously exposed to oxidative stress insults, such as ultraviolet radiation and other oxidative factors, during the aging process. The lens possesses powerful oxidative stress defense systems to maintain its redox homeostasis, one of which employs connexin channels. Connexins are a family of proteins that form: (1) Hemichannels that mediate the communication between the intracellular and extracellular environments, and (2) gap junction channels that mediate cell-cell communication between adjacent cells. The avascular lens transports nutrition and metabolites through an extensive network of connexin channels, which allows the passage of small molecules, including antioxidants and oxidized wastes. Oxidative stress-induced post-translational modifications of connexins, in turn, regulates gap junction and hemichannel permeability. Recent evidence suggests that dysfunction of connexins gap junction channels and hemichannels may induce cataract formation through impaired redox homeostasis. Here, we review the recent advances in the knowledge of connexin channels in lens redox homeostasis and their response to cataract-related oxidative stress by discussing two major aspects: (1) The role of lens connexins and channels in oxidative stress and cataractogenesis, and (2) the impact and underlying mechanism of oxidative stress in regulating connexin channels

Monodispersed Bioactive Glass Nanoparticles Enhance the Osteogenic Differentiation of Adipose‐Derived Stem Cells through Activating TGF‐Beta/Smad3 Signaling Pathway

It is important to understand the interaction mechanisms between nanomaterials and adipose‐derived stem cells for biomedical application. Nanoscale bioactive glass has positive effects on guiding osteoblasts differentiation and bone regeneration. However, the effects and molecular mechanism of monodispersed bioactive glass nanoparticles on the osteogenic differentiation of adipose‐derived stem cells are still not clear up to now. In this study, the effects and underlying molecular mechanism of monodispersed bioactive glass nanoparticles on the osteogenic differentiation of adipose‐derived stem cells are investigated in minute detail. The results show that nanoparticles (100–200 nm) can be absorbed by stem cells and is distributed in cytoplasm and nucleus. In both culture conditions (normal and osteoinductive), nanoparticles (80 µg mL−1) can significantly enhance the osteogenic differentiation of stem cells through upregulating the alkaline phosphatase activity, osteogenic genes and protein expressions, as well as calcium deposition. Further study suggests that the activation of transforming growth factor‐beta/Smad3 signaling pathway plays an important role in the osteogenic differentiation of adipose‐derived stem cells enhanced by monodispersed nanoparticles. This study may have important implications for better understanding of stem cells fate induced by monodispersed nanoparticles and provide a promising approach toward stem cells‐based bone regeneration.Monodispersed bioactive glass nanoparticles (BGNs) are used to regulate the osteogenic differentiation of adipose‐derived stem cells (ASCs). BGNs (100–200 nm) with 80 µg mL−1 can significantly enhance the osteogenic differentiation of ASCs through upregulating the alkaline phosphatase activity, osteogenic genes and protein expressions (Runx2 and Opn), as well as calcium deposition, through the activation of transforming growth factor‐beta/Smad3 signaling pathway.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/145388/1/ppsc201800087.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/145388/2/ppsc201800087_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/145388/3/ppsc201800087-sup-0001-S1.pd

Protocol for altering connexin hemichannel function in primary chicken lens fiber cells using high-titer retroviral RCAS(A) infection

Summary: Connexins (Cxs) play a crucial role in maintaining lens transparency. Here, we present a protocol for altering Cx hemichannel (HC) function in primary chicken lens fiber cells using high-titer retroviral replication competent avian sarcoma-leukosis virus long terminal repeat with splice acceptor (A) infection. We describe steps for incubating eggs, isolating lenses, culturing cells, preparing reagents, and infecting cells. We then detail cell treatment and detection of apoptosis and death. This protocol can assess protein kinase A, HC activity, and increased glutathione transport for protecting lens fiber cells against oxidative stress.For complete details on the use and execution of this protocol, please refer to Liu et al.,1 Riquelme et al.,2 Shi et al.,3 Jiang,4 and Rath et al.5 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

Osteocytic Connexin Hemichannels Modulate Oxidative Bone Microenvironment and Breast Cancer Growth

Osteocytes, the most abundant bone cell types embedded in the mineral matrix, express connexin 43 (Cx43) hemichannels that play important roles in bone remodeling and osteocyte survival. Estrogen deficiency decreases osteocytic Cx43 hemichannel activity and causes a loss in osteocytes&rsquo; resistance to oxidative stress (OS). In this study, we showed that OS reduced the growth of both human (MDA-MB-231) and murine (Py8119) breast cancer cells. However, co-culturing these cells with osteocytes reduced the inhibitory effect of OS on breast cancer cells, and this effect was ablated by the inhibition of Cx43 hemichannels. Py8119 cells were intratibially implanted in the bone marrow of ovariectomized (OVX) mice to determine the role of osteocytic Cx43 hemichannels in breast cancer bone metastasis in response to OS. Two transgenic mice overexpressing dominant-negative Cx43 mutants, R76W and &Delta;130-136, were adopted for this study; the former inhibits gap junctions while the latter inhibits gap junctions and hemichannels. Under normal conditions, &Delta;130-136 mice had significantly more tumor growth in bone than that in WT and R76W mice. OVX increased tumor growth in R76W but had no significant effect on WT mice. In contrast, OVX reduced tumor growth in &Delta;130-136 mice. To confirm the role of OS, WT and &Delta;130-136 mice were administered the antioxidant N-acetyl cysteine (NAC). NAC increased tumor burden and growth in &Delta;130-136 mice but not in WT mice. Together, the data suggest that osteocytes and Cx43 hemichannels play pivotal roles in modulating the oxidative microenvironment and breast cancer growth in the bone

Intrinsic Ultrahigh Drug/miRNA Loading Capacity of Biodegradable Bioactive Glass Nanoparticles toward Highly Efficient Pharmaceutical Delivery

The lack of safe and efficient drug and gene delivery vectors has become a major obstacle for the clinical applications of drug and nonviral gene therapy. To date, for nonviral gene vectors, most studies are focused on cationic polymers, liposomes, and modified inorganic nanoparticles which have shown high cellular toxicity, low transfection efficiency, or nondegradation. Additionally, few biodegradable biomaterials demonstrate intrinsic high binding abilities to both drug and gene. Bioactive glasses (BGs) have achieved successful applications in bone regeneration due to their high biocompatibility and biodegradation. Here, for the first time, we demonstrate the intrinsic ultrahigh drug and miRNA binding ability of bioactive glass nanoparticles (BGNs) without any cationic polymer modification. BGNs demonstrate an over 45-fold improvement in hydrophilic drug loading (diclofenac sodium) and 7-fold enhancement in miRNA binding over their corresponding silica nanoparticles. The hydrophilic drug loading ability of BGNs (>45 wt % loading) is also higher than that of most other reported inorganic nanoparticles, including mesoporous silica nanoparticles. BGNs show significantly lower cytotoxicity and higher cellular uptake and miRNA transfection efficiency compared to those of commercial transfection reagents polyethylenimine and lipofectamine 3000. Our results demonstrate that BGNs may become a new competitive vehicle for drug and gene delivery applications. This study may also provide a new strategy to develop novel biomaterials with intrinsic drug and gene binding ability for disease therapy