DPSC-Derived Extracellular Vesicles Promote Rat Jawbone Regeneration.

Repair and functional reconstruction of large jawbone defects remain one of the challenges in the field of head and neck surgery. The recent progress in tissue engineering technologies and stem cell biology has significantly promoted the development of regenerative reconstruction of jawbone defects. The multiple trophic activities of extracellular vesicles (EVs) produced by mesenchymal stem cells (MSCs) may play a critical role in their therapeutic effects. Accumulating evidence has shown the promise of dental pulp stem cells (DPSCs) in bone regeneration, but less is known about the regenerative effects of DPSC-EVs on jawbone defects. The purpose of this study is to explore the osteogenic effects of DPSC-EVs on jawbone marrow-derived MSCs (JB-MSCs) in vitro and their osteoinductive effects in a mandibular bone defect model in rats. Our results showed that JB-MSCs could efficiently uptake DPSC-EVs, which in turn significantly promoted the expression of osteogenic genes, such as runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), and osteocalcin (OCN), as well as the osteogenic differentiation capability of JB-MSCs. Meanwhile, we found that the pro-osteogenic effect in vitro induced by DPSC-EVs was comparable to that induced by BMP-2 (bone morphogenetic protein 2), currently the only Food and Drug Administration-approved osteoinductive growth factor. In vivo, animals that were locally treated with DPSC-EVs laden with a commercially available collagen membrane exhibited a relatively fast wound closure and increased new bone density at the mandible defects. Our results provide evidence for the osteogenic and osteoinductive effects of DPSC-EVs on jawbone regeneration. Due to the accessibility, rapid proliferation, and osteogenic propensity of DPSCs, DPSC-EVs may represent a safe cell-free therapeutic approach for craniofacial bone regeneration.

Extracellular Vesicles of GMSCs Alleviate Aging-Related Cell Senescence.

Healthy aging is a complex biological process with progressive accumulation of senescent cells characterized by stable cell cycle arrest, resulting in impaired homeostasis, regenerative potential, and gradual functional decline in multiple tissues and organs, whereby the aberrant activation of mammalian target of rapamycin (mTOR) signaling networks plays a central role. Herein, we explored the effects of extracellular vesicles (EVs) released by gingiva-derived mesenchymal stem cells (GMSC-EVs) on oxidative stress-induced cellular senescence in human endothelial cells and skin fibroblasts and their antiaging potentials. Our results showed that GMSC-EVs robustly abrogated oxidative stress-induced upregulation in the expression of cellular senescence-related genes, such as ß-galactosidase, p21, p53, and γH2AX, and mTOR/pS6 signaling pathway, in human umbilical vein endothelial cells (HUVECs) and skin fibroblasts. Meanwhile, GMSC-EVs restored oxidative stress-induced impairment in proliferation and tube formation by HUVECs. Systemic administration of GMSC-EVs attenuated aging-associated elevation in the expression levels of p21, mTOR/pS6, interleukin 6, and tumor necrosis factor α in skin and heart tissues of aged mice. These findings suggest that GMSC-EVs could be a potential alternative source of cell-free product for attenuation of aging-related skin and vascular dysfunctions due to their potent inhibitory effects on oxidative stress-induced cellular senescence in endothelial cells and skin fibroblasts.

Global transcriptome analysis provides new insights in Thellungiella salsuginea stress response.

Thellungiella salsuginea is highly tolerant to abiotic stress, while its a close relative Arabidopsis thaliana is sensitive to stress. This characteristic makes T. salsuginea an excellent model for uncovering the mechanisms of abiotic stress tolerance. Abscisic acid (ABA) plays essential roles in plant abiotic and biotic stress tolerance. To test the changes in gene expression of T. salsuginea under ABA treatment, in this study, the transcriptomes of T. salsuginea roots and leaves were compared in response to exogenously application of ABA. The results showed that ABA treatment caused different expression of 2,200 and 3,305 genes in leaves and roots, respectively, compared with the untreated control. In particular, genes encoding transcription factors such as WRKY, MYB, NAC, GATA, ethylene-responsive factors (ERFs), heat stress transcription factors, basic helix-loop-helix, PLATZ and B3 domain-containing family members were enriched. In addition, 49 and 114 differentially expressed genes were identified as ABA-regulated genes, separately in leaves and roots, respectively, which were related to biotic and abiotic stresses. The expression levels of some genes were validated by qRT-PCR. Different responses of genes to ABA treatment were discovered in T. salsuginea and A. thaliana. This transcriptome analysis expands our understanding of the role of ABA in stress tolerance in T. salsuginea. Our study provides a wealth of information for improving stress tolerance in crop plants.

Visible red and infrared light alters gene expression in human marrow stromal fibroblast cells.

OBJECTIVES: This study tested whether or not gene expression in human marrow stromal fibroblast (MSF) cells depends on light wavelength and energy density. MATERIALS AND METHODS: Primary cultures of isolated human bone marrow stem cells (hBMSC) were exposed to visible red (VR, 633 nm) and infrared (IR, 830 nm) radiation wavelengths from a light emitting diode (LED) over a range of energy densities (0.5, 1.0, 1.5, and 2.0 Joules/cm2) Cultured cells were assayed for cell proliferation, osteogenic potential, adipogenesis, mRNA and protein content. mRNA was analyzed by microarray and compared among different wavelengths and energy densities. Mesenchymal and epithelial cell responses were compared to determine whether responses were cell type specific. Protein array analysis was used to further analyze key pathways identified by microarrays. RESULT: Different wavelengths and energy densities produced unique sets of genes identified by microarray analysis. Pathway analysis pointed to TGF-beta 1 in the visible red and Akt 1 in the infrared wavelengths as key pathways to study. TGF-beta protein arrays suggested switching from canonical to non-canonical TGF-beta pathways with increases to longer IR wavelengths. Microarrays suggest RANKL and MMP 10 followed IR energy density dose-response curves. Epithelial and mesenchymal cells respond differently to stimulation by light suggesting cell type-specific response is possible. CONCLUSIONS: These studies demonstrate differential gene expression with different wavelengths, energy densities and cell types. These differences in gene expression have the potential to be exploited for therapeutic purposes and can help explain contradictory results in the literature when wavelengths, energy densities and cell types differ.

Linker length affects expression and bioactivity of the onconase fusion protein in Pichia pastoris.

The aim of this study was to analyze the effect of linker length on the expression and biological activity of recombinant protein onconase (ONC) in fusion with human serum albumin (HSA) in Pichia pastoris. Four flexible linkers with different lengths namely Linker L0, L1: (GGGGS)1, L2: (GGGGS)2, and L3:(GGGGS)3 were inserted into the fusion gene and referred to as HSA-n-ONC, where N = 0, 5, 10, or 15. The sequence of the fusion gene HSA-ONC was designed based on the GC content and codon bias in P. pastoris; the signal peptide of albumin was used as the secretion signal. Gene sequences coding for the fusion protein with different linkers were inserted into pPICZα-A to form recombinant plasmids pPICZα-A/HSA-n-ONC, which were then transformed into P. pastoris X-33 for protein expression. Ideal conditions for expression of the fusion proteins were optimized at a small scale, using shake flasks before proceeding to mass production in 10-L fermenters. The recombinant fusion proteins were purified by aqueous two-phase extraction coupled with DEAE anion exchange chromatography, and their cytotoxic effect on the tumor cell was evaluated by the sulforhodamine B assay. The results showed that the expressed amount of fusion proteins had no significant relationship with the length of different linkers and rHSA-0-ONC had no cytotoxic effect on the tumor cells. While rHSA-5-ONC and rHSA-10-ONC had a weak cytotoxic effect, rHSA-15-ONC could kill various tumor cells in vitro. In summary, the biological activity of the fusion protein gradually improved with increasing length of the linker.

Genomic sequence of an avian paramyxovirus type 1 strain isolated from Muscovy duck (Cairina moschata) in China.

The complete sequence of an avian paramyxovirus type 1 (APMV-1) strain, FP1/02, isolated from Muscovy duck in China, was determined. Sequence analysis indicated that the complete genome of strain FP1/02 contained 15,192 nucleotides (nt), following the rule of six. The genome contained an extra 6-nt insertion in the non-coding region of the NP gene when compared with other APMV-1 strains, such as strains La Sota and Beaudette C. The cleavage site of the F protein was (112)R-R-Q-K-R↓F(117), indicating that the FP1/02 strain was virulent, but the morbidity and mortality varied with the species of duck. Genotypic analysis based on the F gene revealed that APMV-1 FP1/02 was a member of genotype VII. Phylogenetic analysis showed that the FP1/02 strain shared high identity with other APMV-1 strains such as ZJ1, SF02 and NA-1 isolated from geese.

Multidrug resistant gram-negative bacilli as predominant bacteremic pathogens in liver transplant recipients.

BACKGROUND: Bacteremias, which are often caused by gram-negative bacteria, are the most frequently occurring infectious complications after liver transplantation (LT). The aim of this study was to investigate bacteremic incidence, pathogenic spectrum, risk factors for bacteremia due to multidrug resistant (MDR) gram-negative bacilli, and its impact on mortality after LT. METHODS: A cohort analysis of prospectively recorded data was done in 475 LT recipients, who were divided into 3 categories: cases with gram-negative bacteremia, cases with MDR gram-negative bacteremia, and cases without bacteremia as controls. RESULTS: In 475 LT recipients, there were 152 (32.0%) patients with gram-negative bacillus bacteremia in the first 6 months after LT. Out of 152 patients, there were 225 bacteremic episodes, which accounted for 69.7% in a total 323 bacteremic episodes. A total of 190 bacteremic episodes were caused by Stenotrophomonas maltophilia, Enterobacteriaceae, Ochrobactrum anthropi, Pseudomonas, and Acinetobacter baumanii, all of which were the most frequent gram-negative isolates in this study, and MDR bacilli constituted 56.3%. The most frequent source was intravascular catheters. There were 70 patients with MDR gram-negative bacillus bacteremia. Independent risk factors for bacteremia due to MDR gram-negative bacillus were as follows: post-LT abdominal infection (P<0.0001, odds ratio [OR] 0.066, 95% confidence interval [CI] 0.019-0.226), post-LT reoperative episodes (P<0.0001, OR 10.505, 95% CI 3.055-36.121), or one or more episodes of acute rejection (P=0.042, OR 4.457, 95% CI 0.988-20.103). In the first 6 months after LT, MDR gram-negative bacillus bacteremia-related mortality was significantly higher than that due to antibiotic-susceptible bacillus (38.6% vs. 14.6%, P<0.001). CONCLUSION: Post-LT bacteremias caused by MDR gram-negative bacilli are common, and associated with allograft acute rejection, post-LT reoperation, and abdominal infection. The increasing isolates of MDR gram-negative bacilli pose a great challenge for clinical treatment.

ITS regions in diploids of Aegilops (Poaceae) and their phylogenetic implications.

The nucleotide sequences of the internal transcribed spacer (ITS) of nuclear ribosomal DNA in nine diploid species representing six sections of Aegilops were determined by direct sequencing of PCR-amplified DNA fragments. These sequences were aligned with two ITS sequences of additional species from Genbank. Sequence divergences were estimated using Kimura two-parameter model, and the phylogenetic analyses were performed using the maximum parsimony (MP) and the neighbor-joining (NJ) methods with PAUP and PHYLIP, respectively. The sequence divergences between the diploid species varied from 0.5% to 4.68%. The resulting MP tree and NJ tree showed relatively congruent phylogenetic relationships among these species, except Ae. caudata. Particularly, Ae. speltoides was basal within the two trees. The paraphyletic relationships between Ae. speltoides and two species of Sect. Sitopsis, and between Ae. uniaristata and two species of Sect. Comopyrum were supported strongly. The ITS data suggest that currently recognized sections within Aegilops should be reconsidered.

Driving AMPA receptors into synapses by LTP and CaMKII: requirement for GluR1 and PDZ domain interaction.

To elucidate mechanisms that control and execute activity-dependent synaptic plasticity, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (AMPA-Rs) with an electrophysiological tag were expressed in rat hippocampal neurons. Long-term potentiation (LTP) or increased activity of the calcium/calmodulin-dependent protein kinase II (CaMKII) induced delivery of tagged AMPA-Rs into synapses. This effect was not diminished by mutating the CaMKII phosphorylation site on the GluR1 AMPA-R subunit, but was blocked by mutating a predicted PDZ domain interaction site. These results show that LTP and CaMKII activity drive AMPA-Rs to synapses by a mechanism that requires the association between GluR1 and a PDZ domain protein.

Evolution of parental ITS regions of nuclear rDNA in allopolyploid Aegilops (Poaceae) species.

The genus Aegilops comprises approximately 25 diploid, tetraploid and hexaploid species, in which the genome types of all allopolyploids involve either U or D genome, or both of them. The internal transcribed spacer (ITS) region of 18S-26S nuclear ribosomal DNA (rDNA) from 11 allopolyploid species and 7 related diploid species in the genus were directly sequenced by pooled PCR products. Phylogenetic analyses for tracing evolutionary patterns of parental rDNA in allopolyploid species were performed using the neighbor-joining method. The D genome involved tree included three clades (CC-DDCC, DDMM-DDMMSS-DDMMUU, and MM-MhMh-DDNN), but did not include Ae. squarrosa (DD). It indicated that the rDNA of ancestral D genome had been somewhat differentiated in allopolyploids. The U genome involved tree showed that the allopolyploids and their common ancestor, Ae. umbellulata, formed a clade, suggesting that rDNA in UUMM and UUSS genomes has been homogenizing toward that of ancestral U genome. The phylogenetic pattern of U genome based on ITS sequences also supported the "pivotal-differential" hypothesis.

Two-photon imaging in living brain slices.

Two-photon excitation laser scanning microscopy (TPLSM) has become the tool of choice for high-resolution fluorescence imaging in intact neural tissues. Compared with other optical techniques, TPLSM allows high-resolution imaging and efficient detection of fluorescence signal with minimal photobleaching and phototoxicity. The advantages of TPLSM are especially pronounced in highly scattering environments such as the brain slice. Here we describe our approaches to imaging various aspects of synaptic function in living brain slices. To combine several imaging modes together with patch-clamp electrophysiological recordings we found it advantageous to custom-build an upright microscope. Our design goals were primarily experimental convenience and efficient collection of fluorescence. We describe our TPLSM imaging system and its performance in detail. We present dynamic measurements of neuronal morphology of neurons expressing green fluorescent protein (GFP) and GFP fusion proteins as well as functional imaging of calcium dynamics in individual dendritic spines. Although our microscope is a custom instrument, its key advantages can be easily implemented as a modification of commercial laser scanning microscopes.

Rapid spine delivery and redistribution of AMPA receptors after synaptic NMDA receptor activation.

To monitor changes in alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor distribution in living neurons, the AMPA receptor subunit GluR1 was tagged with green fluorescent protein (GFP). This protein (GluR1-GFP) was functional and was transiently expressed in hippocampal CA1 neurons. In dendrites visualized with two-photon laser scanning microscopy or electron microscopy, most of the GluR1-GFP was intracellular, mimicking endogenous GluR1 distribution. Tetanic synaptic stimulation induced a rapid delivery of tagged receptors into dendritic spines as well as clusters in dendrites. These postsynaptic trafficking events required synaptic N-methyl-D-aspartate (NMDA) receptor activation and may contribute to the enhanced AMPA receptor-mediatedtransmission observed during long-term potentiation and activity-dependent synaptic maturation.