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Being an active scaffold in bone tissue engineering, hydrogel self-assembled from biomimetic peptide amphiphile (PA) has excellent ability to induce osteogenic differentiation and osteogenesis. Here, a multifunctional scaffold based on bone morphogenetic protein-2 (BMP-2) mimicking peptide, RGDS, and phosphoserine has been developed to enhance osteogenesis. Cell experiments in vitro displayed that the hydrogel could effectively promote rat messenchymal stem cells (rMSCs) proliferation and induce them differentiation into oesteblasts. The up-regulated RNA expression of osteogenic marker genes, like BMP-2, osteopontin (OPN), osteocalcin (OCN) and runt-related transcription factor 2 (Runx2) revealed that the scaffold could accelerate rMSCs differentiation at RNA level. Further studies on rat skull defect model demonstrated that the multifunctional scaffold exhibited excellent repair ability due to a potential synergistic effect of biomimetic peptide and phosphoserine. Histochemical/immunohistochemical staining results showed that expressions of alkaline phosphatase (ALP) and OCN was significantly up-regulated, indicating that the hydrogel could accelerate maturation of osteoblast precursors during the whole repairing process and be a promising bioactive scaffold for bone repairing.

The aim of the present study was to identify key genes and signaling pathways associated with the pathogenesis of juvenile spondyloarthritis (JSA). The gene expression profile dataset {"type":"entrez-geo","attrs":{"text":"GSE58667","term_id":"58667"}}GSE58667, including data from 15 human whole blood samples collected from 11 patients with JSA and four healthy controls, was analyzed to identify differentially expressed genes (DEGs) associated with disease characteristics. Additionally, Gene Ontology term and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses of the DEGs were performed. Protein-protein, microRNA-transcription factor and chemical-gene interaction networks were constructed. A total of 326 DEGs, 196 upregulated and 130 downregulated, were identified. DEGs, including C-X-C motif chemokine ligand 5 (CXCL5), BCL2 interacting protein 3 like (BNIP3L), dual specificity phosphatase 5 (DUSP5) and tumor protein p53 (TP53) were enriched in functions associated with apoptosis, the cell cycle and immune responses. KEGG pathway enrichment analysis revealed that pathways associated with inflammation and the mitogen-activated protein kinase 1 (MAPK) signaling pathway were the most enriched by DEGs. The results of the present study indicated that the MAPK signaling pathway and four genes, including CXCL5, BNIP3L, DUSP5 and TP53, may be implicated in the pathogenesis of JSA.

Bone morphogenetic protein-2 biomimetic peptide (BMPBP) is a potent osteoinductive cytokine and plays a critical role during bone regeneration. Efforts to prepare hydrogels with surface modification or physical absorption of bioactive molecules do not provide sufficient bioactivity to meet the requirements of clinical application. The goal of this study was to form a three-dimensional hydrogel comprised of BMP-2 core sequence oligopeptide, phosphoserine, a synthetic cell adhesion peptide (RGDS), and polyaspartic acid to synergistically promote osteogenesis. Experiments performed in vitro revealed that the peptide gel was conducive to adhesion and proliferation of rat marrow mesenchymal stem cells (rMSCs). In addition, RT-PCR analysis indicated that rMSCs allowed better expression of osteogenesis-related genes such as BMP-2, runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN). Use of the rat cranial bone defects model with micro-CT 3D reconstruction showed that bone regeneration patterns occurred from one side edge toward the center of the area implanted with the prepared biomimetic peptide hydrogels, demonstrating significantly accelerated bone regeneration. This work will provide a basis to explore the further application potential of this bioactive scaffold.

Effective Protein A column regeneration is important in therapeutic monoclonal antibody (mAb) production for product quality and process economics. To design a robust and effective regeneration strategy, resin compatibility, microbial inactivation efficiency and cleaning effectiveness are three major considerations. Regenerating Protein A column using acidic (e.g. acetic acid) and/or caustic (e.g. NaOH) solutions is a common approach. However, it is challenging to balance between resin performance decline and adequate microbial control in choosing the right caustic solution concentration, even for resin with enhanced caustic tolerance such as MabSelect SuRe. This report introduces an enhanced regeneration strategy for MabSelect SuRe resin. The approach applies benzyl alcohol in low concentration acetic acid and NaOH (100 mM) solutions in sequential steps to regenerate MabSelect SuRe resin for reuse. Such solutions demonstrated good resin compatibility in resin functional test after extended solution incubation time, and demonstrated superior microbial control using eight test microorganisms. The cleaning effectiveness of the proposed strategy is demonstrated by carryover study, where product and impurity carryover was below detection limit. Finally, during a resin lifetime study, consistent step yields and product quality attributes were demonstrated, and no carryover was observed, up to 150 purification cycles. A viral clearance study further demonstrated that aged resin at the end of the resin lifetime had comparable viral clearance performance compared to new resin. No active virus was detected in the elution pool of the subsequent blank runs. These studies further verified the robustness of the proposed regeneration strategy for MabSelect SuRe resin.

Rad51 and Rad54 play crucial roles during homologous recombination. The biochemical activities of human Rad51 (hRad51) and human Rad54 (hRad54) and their interactions with each other are well documented. However, it is not known how these two proteins work together to bypass heterologous sequences; i.e. mismatched base pairs, during homologous recombination. In this study, we used a fluorescence resonance energy transfer assay to monitor homologous recombination processes in real time so that the interactions between hRad54 and hRad51 during DNA strand exchange and branch migration, which are two core steps of homologous recombination, could be characterized. Our results indicate that hRad54 can facilitate hRad51-promoted strand exchange through various degrees of mismatching. We propose that the main roles of hRad51 in homologous recombination is to initiate the homology recognition and strand-exchange steps and those of hRad54 are to promote efficient branch migration, bypass potential mismatches and facilitate long-range strand exchanges through branch migration of Holliday junctions.

Human RAD51 and RAD54 are key players in homologous recombination, a process that requires homology recognition and strand invasion by a RAD51-single-stranded DNA (ssDNA) nucleoprotein filament and chromatin remodeling by RAD54. Here we use in vitro chromatin reconstitution systems to show that RAD51-ssDNA stimulates RAD54-dependent chromatin remodeling in a homology-dependent, polarity-independent manner. This stimulation was not seen with RAD54B or other remodelers. Chromatin remodeling by RAD54 enabled strand invasion by RAD51-ssDNA on nucleosomal templates, which was homology- and polarity-dependent. Three natural RAD54 mutants found in primary cancer cells showed specific defects in remodeling or in the RAD54-RAD51 interaction. We propose that RAD54 is recruited by RAD51-ssDNA filament to the chromatin of the intact chromosome and that it remodels that chromatin to facilitate accessibility for strand exchange.

Purpose: To investigate whether secreted phosphoprotein-1iSPP1j promoter variants are associated with susceptibility to ankylosing spondylitis (AS) in the western China.Methods: The rs5860110 variant of SPP1 promoter was genotyped using direct sequencing in 120 unrelated AS patients and 106 ethnically matched healthy controls. All the patients were human leukocyte antigen iHLAj-B27 positive. Frequencies of different genotypes and alleles were analyzedamong the AS patients and the controls.Results: In AS patient group, frequency of +254 SPP1 promoter genotype was 0.483 in base TG/TG homozygotes and 0.517 in TG-(deletion)/TG-(deletion) homozygotes, while the values for the control group were 0.311 and 0.689, respectively. However, TG-(deletion)/TG heterozygote was not detected in this study. The rs5860110 single nucleotide polymorphism (SNP) in SPP1 was significantly different when all AS patients were compared to controls (p < 0.01).Conclusion: The rs5860110 SNP in SPP1 has a protective role against AS in the selected Chinese population.Keywords: Polymorphism, Ankylosing spondylitis, Secreted phosphoprotein-1, Sequencing, Genotype, Alleles

To initiate homologous recombination, sequence similarity between two DNA molecules must be searched for and homology recognized. How the search for and recognition of homology occurs remains unproven. We have examined the influences of DNA topology and the polarity of RecA–single-stranded (ss)DNA filaments on the formation of synaptic complexes promoted by RecA. Using two complementary methods and various ssDNA and duplex DNA molecules as substrates, we demonstrate that topological constraints on a small circular RecA–ssDNA filament prevent it from interwinding with its duplex DNA target at the homologous region. We were unable to detect homologous pairing between a circular RecA–ssDNA filament and its relaxed or supercoiled circular duplex DNA targets. However, the formation of synaptic complexes between an invading linear RecA–ssDNA filament and covalently closed circular duplex DNAs is promoted by supercoiling of the duplex DNA. The results imply that a triplex structure formed by non-Watson–Crick hydrogen bonding is unlikely to be an intermediate in homology searching promoted by RecA. Rather, a model in which RecA-mediated homology searching requires unwinding of the duplex DNA coupled with local strand exchange is the likely mechanism. Furthermore, we show that polarity of the invading RecA–ssDNA does not affect its ability to pair and interwind with its circular target duplex DNA.

ATP-dependent chromatin-remodeling enzymes are linked to changes in gene expression; however, it is not clear how the multiple remodeling enzymes found in eukaryotes differ in function and work together. In this report, we demonstrate that the ATP-dependent remodeling enzymes ACF and Mi2β can direct consecutive, opposing chromatin-remodeling events, when recruited to chromatin by different transcription factors. In a cell-free system based on the immunoglobulin heavy chain gene enhancer, we show that TFE3 induces a DNase I-hypersensitive site in an ATP-dependent reaction that requires ACF following transcription factor binding to chromatin. In a second step, PU.1 directs Mi2β to erase an established DNase I-hypersensitive site, in an ATP-dependent reaction subsequent to PU.1 binding to chromatin, whereas ACF will not support erasure. Erasure occurred without displacing the transcription factor that initiated the site. Other tested enzymes were unable to erase the DNase I-hypersensitive site. Establishing and erasing the DNase I-hypersensitive site required transcriptional activation domains from TFE3 and PU.1, respectively. Together, these results provide important new mechanistic insight into the combinatorial control of chromatin structure.

The DNA-dependent ATPase activity of the Escherichia coli RecA protein has been recognized for more than two decades. Yet, the role of ATP hydrolysis in the RecA-promoted strand exchange reaction remains unclear. Here, we demonstrate that ATP hydrolysis is required as part of a proofreading process during homology recognition. It enables the RecA-ssDNA complex, after determining that the strand-exchanged duplex is mismatched, to dissociate from the synaptic complex, which allows it to re-initiate the search for a "true" homologous region. Furthermore, the results suggest that when non-homologous sequences are present at the proximal end, ATP hydrolysis is required to allow ssDNA-RecA to reinitiate the strand exchange from an internal homologous region.

It has been shown in vitro thatSaccharomyces cerevisiae strand exchange protein 1 (Sep1) promotes the transfer of one strand of a linear duplex DNA to a homologous single-stranded DNA circle. Sep1 also has an exonuclease active on DNA and RNA. By using exonuclease III-treated linear duplex DNA with various lengths of single-stranded tail as well as Ca2+ to inhibit the exonuclease activity of Sep1, we show that the processivity of exonuclease activity of Sep1 is greater than previously reported. The results in this work also demonstrate that the joint molecule between the linear duplex and single-stranded circle observed from the Sep1-promoted strand transfer reaction is just the pairing between the long single-stranded tail of the linear duplex DNA (generated by the exonuclease activity of Sep1) and the single-stranded circular DNA. When a synthetic Holliday junction was used as substrate, branch migration facilitated by Sep1 could not be detected. Finally, using electron microscopy no α-structure, a joint molecule with displaced single-stranded DNA tail that indicates branch migration could be observed. The results imply that Sep1 cannot promote branch migration in vitro. Further investigation is needed to determine the role of Sep1 in recombination in vivo.

SummaryAlthough human induced pluripotent stem cells (hiPSCs) have enormous potential in regenerative medicine, their epigenetic variability suggests that some lines may not be suitable for human therapy. There are currently few benchmarks for assessing quality. Here we show that X-inactivation markers can be used to separate hiPSC lines into distinct epigenetic classes and that the classes are phenotypically distinct. Loss of XIST expression is strongly correlated with upregulation of X-linked oncogenes, accelerated growth rate in vitro, and poorer differentiation in vivo. Whereas differences in X-inactivation potential result in epigenetic variability of female hiPSC lines, male hiPSC lines generally resemble each other and do not overexpress the oncogenes. Neither physiological oxygen levels nor HDAC inhibitors offer advantages to culturing female hiPSC lines. We conclude that female hiPSCs may be epigenetically less stable in culture and caution that loss of XIST may result in qualitatively less desirable stem cell lines.