Search

Abstract TATA-binding protein associated factor 15 (TAF15) is involved in the pathology of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Although TAF15 accumulates as cytoplasmic aggregates in neurons and the clearance of aggregates may be a therapeutic strategy for ALS, the underlying mechanisms of TAF15 in ALS remain poorly understood. Recently, we found that glutathione transferase omega 2 (GstO2) expression level is significantly reduced in the brain tissue of TAF15-expressing flies. In this study, we demonstrated that GstO2 overexpression in TAF15-induced flies rescues the locomotive activity and neuromuscular junctional defects. Furthermore, TAF15 levels in both cytoplasm and nuclear fractions significantly decreased in the heads of GstO2 co-expressing flies. GstO2-co-overexpression in neurons caused a marked decrease in intracellular reactive oxygen species generation in TAF15-induced flies. Our findings demonstrated that GstO2 was a pathogenic regulator of TAF15-associated proteinopathies. They help expand our understanding of TAF15-associated ALS pathogenesis.

Background: Delayed encephalopathy after carbon monoxide (CO) poisoning is a severe neurologic impairment that occurs after apparent recovery from the acute event. These patients rarely remain neurologically independent, and no effective treatment has yet been validated. Case Report: Two patients had survived acute CO intoxication without neurologic sequelae. Three weeks after each of the two patients recovered, the patients gradually developed cognitive impairment, urinary incontinence, akinetic mutism and parkinsonism. After the administration of high dose prednisolone, the treatment response was prompt within two weeks, and the patients have recovered with mild cognitive impairment within one year. Conclusion: High dose prednisolone can be used for treatment of delayed encephalopathy after CO poisoning.

Transactive response DNA-binding protein-43 (TDP-43) is involved in the pathology of familial and sporadic amyotrophic lateral sclerosis (ALS). TDP-43-mediated ALS models in mice, Drosophila melanogaster, and zebrafish exhibit dysfunction of locomotor function, defective neuromuscular junctions, and motor neuron defects. There is currently no effective cure for ALS, and the underlying mechanisms of TDP-43 in ALS remain poorly understood. In this study, a genetic screen was performed to identify modifiers of human TDP-43 (hTDP-43) in a Drosophila model, and glutathione S-transferase omega 2 (GstO2) was found to be involved in hTDP-43 neurotoxicity. GstO2 overexpressed on recovered defective phenotypes resulting from hTDP-43, including defective neuromuscular junction (NMJ) boutons, degenerated motor neuronal axons, and reduced larvae and adult fly locomotive activity, without modulating the levels of hTDP-43 protein expression. GstO2 modulated neurotoxicity by regulating reactive oxygen species (ROS) produced by hTDP-43 in the Drosophila model of ALS. Our results demonstrated that GstO2 was a key regulator in hTDP-43-related ALS pathogenesis and indicated its potential as a therapeutic target for ALS.

Background:Rhabdomyolysis is a syndrome of skeletal muscle breakdown with leakage of muscle contents into blood stream. Variable disorders affecting muscle membrane or its energy supply can cause rhabdomyolysis. Case Report: We report a patient with rapidly progressive dementia who developed severe parkinsonism after rhabdomyolysis in the course of the illness. Functional neuroimaging studies revealed diffuse cortical hypometabolism and severe nigrostriatal dopaminergic dysfunction. Conclusion:This case suggests that the special attentions should be paid on evaluation for rhabdomyolysis in some demented patients.

Butterfly wings possess distinct micro/nanostructures that contribute to their vibrant coloration, light-trapping capabilities, and sensitivity to various stimuli. These complex features have inspired the creation of diverse devices and systems, such as sensors, photovoltaics, photocatalysis, and robotics. Specifically, the wing scales of the Emerald Swallowtail (Papilio palinurus) display iridescent, polarization-sensitive, and retroreflective colors due to their hierarchical structures. However, current technologies fail to mimic these natural designs fully, limiting their practical application in everyday life. In this study, we introduce a groundbreaking method for fabricating artificial wing scales that emulate the biological structure's functionality with a much simpler geometry. By integrating self-graded lossy media into metallic micro-concavity arrays, we achieve pronounced iridescent effects in both coaxial and non-coaxial arrangements, while preserving retroreflective properties. In particular, the simplified design allows for switchable color patterns based on the viewing angle. Demonstrating the concept, we successfully employ these conspicuous retroreflectors in hydrogen gas detection and the bi-directional/switchable recognition of patterned signals., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

XPA is a central scaffold protein that coordinates the assembly of repair complexes in the global genome (GG-NER) and transcription-coupled nucleotide excision repair (TC-NER) subpathways. Inactivating mutations in XPA cause xeroderma pigmentosum (XP), which is characterized by extreme UV sensitivity and a highly elevated skin cancer risk. Here, we describe two Dutch siblings in their late forties carrying a homozygous H244R substitution in the C-terminus of XPA. They present with mild cutaneous manifestations of XP without skin cancer but suffer from marked neurological features, including cerebellar ataxia. We show that the mutant XPA protein has a severely weakened interaction with the transcription factor IIH (TFIIH) complex leading to an impaired association of the mutant XPA and the downstream endonuclease ERCC1-XPF with NER complexes. Despite these defects, the patient-derived fibroblasts and reconstituted knockout cells carrying the XPA-H244R substitution show intermediate UV sensitivity and considerable levels of residual GG-NER (~50%), in line with the intrinsic properties and activities of the purified protein. By contrast, XPA-H244R cells are exquisitely sensitive to transcription-blocking DNA damage, show no detectable recovery of transcription after UV irradiation, and display a severe deficiency in TC-NER-associated unscheduled DNA synthesis. Our characterization of a new case of XPA deficiency that interferes with TFIIH binding and primarily affects the transcription-coupled subpathway of nucleotide excision repair, provides an explanation of the dominant neurological features in these patients, and reveals a specific role for the C-terminus of XPA in TC-NER.

With the rise of mobile robotics, including self-driving automobiles and drones, developing artificial vision for high-contrast and high-acuity imaging in vertically uneven illumination conditions has become an important goal. In such situations, balancing uneven illumination, improving image contrast for facile object detection, and achieving high visual acuity in the main visual fields are key requirements. Meanwhile, in nature, cuttlefish (genus Sepia ) have evolved an eye optimized for vertically uneven illumination conditions, which consists of a W-shaped pupil, a single spherical lens, and a curved retina with a high-density photoreceptor arrangement and polarized light sensitivity. Here, inspired by the cuttlefish eye, we report an artificial vision system consisting of a W-shaped pupil, a single ball lens, a surface-integrated flexible polarizer, and a cylindrical silicon photodiode array with a locally densified pixel arrangement. The W-shaped pupil integrated on the ball lens balances vertically uneven illumination, and the cylindrical silicon photodiode array integrated with the flexible polarizer enables high-contrast and high-acuity imaging.

The xeroderma pigmentosum protein A (XPA) and replication protein A (RPA) proteins fulfill essential roles in the assembly of the preincision complex in the nucleotide excision repair (NER) pathway. We have previously characterized the two interaction sites, one between the XPA N-terminal (XPA-N) disordered domain and the RPA32 C-terminal domain (RPA32C), and the other with the XPA DNA binding domain (DBD) and the RPA70AB DBDs. Here, we show that XPA mutations that inhibit the physical interaction in either site reduce NER activity in biochemical and cellular systems. Combining mutations in the two sites leads to an additive inhibition of NER, implying that they fulfill distinct roles. Our data suggest a model in which the interaction between XPA-N and RPA32C is important for the initial association of XPA with NER complexes, while the interaction between XPA DBD and RPA70AB is needed for structural organization of the complex to license the dual incision reaction. Integrative structural models of complexes of XPA and RPA bound to single-stranded/double-stranded DNA (ss/dsDNA) junction substrates that mimic the NER bubble reveal key features of the architecture of XPA and RPA in the preincision complex. Most critical among these is that the shape of the NER bubble is far from colinear as depicted in current models, but rather the two strands of unwound DNA must assume a U-shape with the two ss/dsDNA junctions localized in close proximity. Our data suggest that the interaction between XPA and RPA70 is key for the organization of the NER preincision complex.

Distinct cellular DNA damage repair pathways maintain the structural integrity of DNA and protect it from the mutagenic effects of genotoxic exposures and processes. The occurrence of O 6 -carboxymethylguanine (O 6 -CMG) has been linked to meat consumption and hypothesized to contribute to the development of colorectal cancer. However, the cellular fate of O 6 -CMG is poorly characterized and there is contradictory data in the literature as to how repair pathways may protect cells from O 6 -CMG mutagenicity. To better address how cells detect and remove O 6 -CMG, we evaluated the role of two DNA repair pathways in counteracting the accumulation and toxic effects of O 6 -CMG. We found that cells deficient in either the direct repair protein O 6 -methylguanine-DNA methyltransferase (MGMT), or key components of the nucleotide excision repair (NER) pathway, accumulate higher levels O 6 -CMG DNA adducts than wild type cells. Furthermore, repair-deficient cells were more sensitive to carboxymethylating agents and displayed an increased mutation rate. These findings suggest that a combination of direct repair and NER circumvent the effects O 6 -CMG DNA damage., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

ERCC1-XPF is a multifunctional endonuclease involved in nucleotide excision repair (NER), interstrand cross-link (ICL) repair, and DNA double-strand break (DSB) repair. Only two patients with bi-allelic ERCC1 mutations have been reported, both of whom had features of Cockayne syndrome and died in infancy. Here, we describe two siblings with bi-allelic ERCC1 mutations in their teenage years. Genomic sequencing identified a deletion and a missense variant (R156W) within ERCC1 that disrupts a salt bridge below the XPA-binding pocket. Patient-derived fibroblasts and knock-in epithelial cells carrying the R156W substitution show dramatically reduced protein levels of ERCC1 and XPF. Moreover, mutant ERCC1 weakly interacts with NER and ICL repair proteins, resulting in diminished recruitment to DNA damage. Consequently, patient cells show strongly reduced NER activity and increased chromosome breakage induced by DNA cross-linkers, while DSB repair was relatively normal. We report a new case of ERCC1 deficiency that severely affects NER and considerably impacts ICL repair, which together result in a unique phenotype combining short stature, photosensitivity, and progressive liver and kidney dysfunction., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2020 Crown copyright. The government of Australia, Canada, or the UK ("the Crown") owns the copyright interests of authors who are government employees. The Crown Copyright is not transferable.)

Protecting replication fork integrity during DNA replication is essential for maintaining genome stability. Here, we report that SDE2, a PCNA-associated protein, plays a key role in maintaining active replication and counteracting replication stress by regulating the replication fork protection complex (FPC). SDE2 directly interacts with the FPC component TIMELESS (TIM) and enhances its stability, thereby aiding TIM localization to replication forks and the coordination of replisome progression. Like TIM deficiency, knockdown of SDE2 leads to impaired fork progression and stalled fork recovery, along with a failure to activate CHK1 phosphorylation. Moreover, loss of SDE2 or TIM results in an excessive MRE11-dependent degradation of reversed forks. Together, our study uncovers an essential role for SDE2 in maintaining genomic integrity by stabilizing the FPC and describes a new role for TIM in protecting stalled replication forks. We propose that TIM-mediated fork protection may represent a way to cooperate with BRCA-dependent fork stabilization.

Fanconi anemia (FA) is a chromosome instability syndrome characterized by increased cancer predisposition. Specifically, the FA pathway functions to protect genome stability during DNA replication. The central FA pathway protein, FANCD2, locates to stalled replication forks and recruits homologous recombination (HR) factors such as CtBP interacting protein (CtIP) to promote replication fork restart while suppressing new origin firing. Here, we identify alpha-thalassemia retardation syndrome X-linked (ATRX) as a novel physical and functional interaction partner of FANCD2. ATRX is a chromatin remodeler that forms a complex with Death domain-associated protein 6 (DAXX) to deposit the histone variant H3.3 into specific genomic regions. Intriguingly, ATRX was recently implicated in replication fork recovery; however, the underlying mechanism(s) remained incompletely understood. Our findings demonstrate that ATRX forms a constitutive protein complex with FANCD2 and protects FANCD2 from proteasomal degradation. ATRX and FANCD2 localize to stalled replication forks where they cooperate to recruit CtIP and promote MRE11 exonuclease-dependent fork restart while suppressing the firing of new replication origins. Remarkably, replication restart requires the concerted histone H3 chaperone activities of ATRX/DAXX and FANCD2, demonstrating that coordinated histone H3 variant deposition is a crucial event during the reinitiation of replicative DNA synthesis. Lastly, ATRX also cooperates with FANCD2 to promote the HR-dependent repair of directly induced DNA double-stranded breaks. We propose that ATRX is a novel functional partner of FANCD2 to promote histone deposition-dependent HR mechanisms in S-phase., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Monoubiquitination of the Fanconi anemia complementation group D2 (FANCD2) protein by the FA core ubiquitin ligase complex is the central event in the FA pathway. FANCA and FANCG play major roles in the nuclear localization of the FA core complex. Mutations of these two genes are the most frequently observed genetic alterations in FA patients, and most point mutations in FANCA are clustered in the C-terminal domain (CTD). To understand the basis of the FA-associated FANCA mutations, we determined the cryo-electron microscopy (EM) structures of Xenopus laevis FANCA alone at 3.35 Å and 3.46 Å resolution and two distinct FANCA-FANCG complexes at 4.59 and 4.84 Å resolution, respectively. The FANCA CTD adopts an arc-shaped solenoid structure that forms a pseudo-symmetric dimer through its outer surface. FA- and cancer-associated point mutations are widely distributed over the CTD. The two different complex structures capture independent interactions of FANCG with either FANCA C-terminal HEAT repeats, or the N-terminal region. We show that mutations that disturb either of these two interactions prevent the nuclear localization of FANCA, thereby leading to an FA pathway defect. The structure provides insights into the function of FANCA CTD, and provides a framework for understanding FA- and cancer-associated mutations., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The XPA protein functions together with the single-stranded DNA (ssDNA) binding protein RPA as the central scaffold to ensure proper positioning of repair factors in multi-protein nucleotide excision repair (NER) machinery. We previously determined the structure of a short motif in the disordered XPA N-terminus bound to the RPA32C domain. However, a second contact between the XPA DNA-binding domain (XPA DBD) and the RPA70AB tandem ssDNA-binding domains, which is likely to influence the orientation of XPA and RPA on the damaged DNA substrate, remains poorly characterized. NMR was used to map the binding interfaces of XPA DBD and RPA70AB. Combining NMR and X-ray scattering data with comprehensive docking and refinement revealed how XPA DBD and RPA70AB orient on model NER DNA substrates. The structural model enabled design of XPA mutations that inhibit the interaction with RPA70AB. These mutations decreased activity in cell-based NER assays, demonstrating the functional importance of XPA DBD-RPA70AB interaction. Our results inform ongoing controversy about where XPA is bound within the NER bubble, provide structural insights into the molecular basis for malfunction of disease-associated XPA missense mutations, and contribute to understanding of the structure and mechanical action of the NER machinery., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

DNA repair is critical for maintaining genomic integrity. Finding DNA lesions initiates the entire repair process. In human nucleotide excision repair (NER), XPC-RAD23B recognizes DNA lesions and recruits downstream factors. Although previous studies revealed the molecular features of damage identification by the yeast orthologs Rad4-Rad23, the dynamic mechanisms by which human XPC-RAD23B recognizes DNA defects have remained elusive. Here, we directly visualized the motion of XPC-RAD23B on undamaged and lesion-containing DNA using high-throughput single-molecule imaging. We observed three types of one-dimensional motion of XPC-RAD23B along DNA: diffusive, immobile and constrained. We found that consecutive AT-tracks led to increase in proteins with constrained motion. The diffusion coefficient dramatically increased according to ionic strength, suggesting that XPC-RAD23B diffuses along DNA via hopping, allowing XPC-RAD23B to bypass protein obstacles during the search for DNA damage. We also examined how XPC-RAD23B identifies cyclobutane pyrimidine dimers (CPDs) during diffusion. XPC-RAD23B makes futile attempts to bind to CPDs, consistent with low CPD recognition efficiency. Moreover, XPC-RAD23B binds CPDs in biphasic states, stable for lesion recognition and transient for lesion interrogation. Taken together, our results provide new insight into how XPC-RAD23B searches for DNA lesions in billions of base pairs in human genome., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Global genome nucleotide excision repair (GG-NER) is the main pathway for the removal of bulky lesions from DNA and is characterized by an extraordinarily wide substrate specificity. Remarkably, the efficiency of lesion removal varies dramatically and certain lesions escape repair altogether and are therefore associated with high levels of mutagenicity. Central to the multistep mechanism of damage recognition in NER is the sensing of lesion-induced thermodynamic and structural alterations of DNA by the XPC-RAD23B protein and the verification of the damage by the transcription/repair factor TFIIH. Additional factors contribute to the process: UV-DDB, for the recognition of certain UV-induced lesions in particular in the context of chromatin, while the XPA protein is believed to have a role in damage verification and NER complex assembly. Here we consider the molecular mechanisms that determine repair efficiency in GG-NER based on recent structural, computational, biochemical, cellular and single molecule studies of XPC-RAD23B and its yeast ortholog Rad4. We discuss how the actions of XPC-RAD23B are integrated with those of other NER proteins and, based on recent high-resolution structures of TFIIH, present a structural model of how XPC-RAD23B and TFIIH cooperate in damage recognition and verification., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Fanconi anemia (FA) is an inherited cancer predisposition syndrome characterized by cellular hypersensitivity to DNA interstrand crosslinks (ICLs). To repair these lesions, the FA proteins act in a linear hierarchy: following ICL detection on chromatin, the FA core complex monoubiquitinates and recruits the central FANCI and FANCD2 proteins that subsequently coordinate ICL removal and repair of the ensuing DNA double-stranded break by homology-dependent repair (HDR). FANCD2 also functions during the replication stress response by mediating the restart of temporarily stalled replication forks thereby suppressing the firing of new replication origins. To address if FANCI is also involved in these FANCD2-dependent mechanisms, we generated isogenic FANCI-, FANCD2- and FANCI:FANCD2 double-null cells. We show that FANCI and FANCD2 are partially independent regarding their protein stability, nuclear localization and chromatin recruitment and contribute independently to cellular proliferation. Simultaneously, FANCD2-but not FANCI-plays a major role in HDR-mediated replication restart and in suppressing new origin firing. Consistent with this observation, deficiencies in HDR-mediated DNA DSB repair can be overcome by stabilizing RAD51 filament formation in cells lacking functional FANCD2. We propose that FANCI and FANCD2 have partially non-overlapping and possibly even opposing roles during the replication stress response., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The Fanconi anemia (FA)-BRCA pathway mediates repair of DNA interstrand crosslinks. The FA core complex, a multi-subunit ubiquitin ligase, participates in the detection of DNA lesions and monoubiquitinates two downstream FA proteins, FANCD2 and FANCI (or the ID complex). However, the regulation of the FA core complex itself is poorly understood. Here we show that the FA core complex proteins are recruited to sites of DNA damage and form nuclear foci in S and G2 phases of the cell cycle. ATR kinase activity, an intact FA core complex and FANCM-FAAP24 were crucial for this recruitment. Surprisingly, FANCI, but not its partner FANCD2, was needed for efficient FA core complex foci formation. Monoubiquitination or ATR-dependent phosphorylation of FANCI were not required for the FA core complex recruitment, but FANCI deubiquitination by USP1 was. Additionally, BRCA1 was required for efficient FA core complex foci formation. These findings indicate that FANCI functions upstream of FA core complex recruitment independently of FANCD2, and alter the current view of the FA-BRCA pathway.

Purpose: In the present study, the clinical outcomes and second-look arthroscopic findings of intra-articular injection of stem cells with arthroscopic lavage for treatment of elderly patients with knee osteoarthritis (OA) were evaluated., Methods: Stem cell injections combined with arthroscopic lavage were administered to 30 elderly patients (≥65 years) with knee OA. Subcutaneous adipose tissue was harvested from both buttocks by liposuction. After stromal vascular fractions were isolated, a mean of 4.04 × 10(6) stem cells (9.7 % of 4.16 × 10(7) stromal vascular fraction cells) were prepared and injected in the selected knees of patients after arthroscopic lavage. Outcome measures included the Knee Injury and Osteoarthritis Outcome Scores, visual analog scale, and Lysholm score at preoperative and 3-, 12-, and 2-year follow-up visits. Sixteen patients underwent second-look arthroscopy., Results: Almost all patients showed significant improvement in all clinical outcomes at the final follow-up examination. All clinical results significantly improved at 2-year follow-up compared to 12-month follow-up (P < 0.05). Among elderly patients aged >65 years, only five patients demonstrated worsening of Kellgren-Lawrence grade. On second-look arthroscopy, 87.5 % of elderly patients (14/16) improved or maintained cartilage status at least 2 years postoperatively. Moreover, none of the patients underwent total knee arthroplasty during this 2-year period., Conclusion: Adipose-derived stem cell therapy for elderly patients with knee OA was effective in cartilage healing, reducing pain, and improving function. Therefore, adipose-derived stem cell treatment appears to be a good option for OA treatment in elderly patients., Level of Evidence: Therapeutic case series study, Level IV.

Background: Recently, mesenchymal stem cells (MSCs) have been suggested as a source for cell-based treatment of cartilage lesions based on the ability of these cells to differentiate into chondrocytes., Purpose: To characterize MSCs derived from the synovial fluid in ankle joints with osteochondral lesion of the talus (OLT)., Study Design: Controlled laboratory study., Methods: Synovial fluid was collected from the ankle joints of 28 patients with OLT who underwent arthroscopic marrow stimulation between September 2011 and April 2012. Epitope profiles and multilineage differentiation were assessed to characterize the synovial fluid MSCs. To clarify the origin of synovial fluid MSCs, we assessed gene profiles of MSCs derived from various mesenchymal tissues by reverse transcription-polymerase chain reaction (RT-PCR) analysis., Results: Synovial fluid MSCs expressed CD90 and CD105, showed low expression of CD14 and CD34, and underwent multilineage differentiation in vitro. The RT-PCR revealed strong expression of CD90, CD44, and CD73, whereas CD45 and CD133 were not detected. The colony number of synovial fluid MSCs from OLT significantly increased in stages C and D, as defined by arthroscopic classification. Gene expression profiles indicated that synovial fluid MSCs derived from the patients with OLT were more similar to MSCs from synovium than to MSCs from bone marrow and adipose tissue., Conclusion: This study confirmed that human synovial fluid is a good source of MSCs, with the capacity to differentiate toward several cell lineages. Further study with matched controls of synovial fluid MSCs derived from ankle joints without OLT is required for a more accurate evaluation of synovial fluid MSCs., Clinical Relevance: The findings of this study provide a platform for exploring the potential role of synovial fluid MSCs in OLT and their therapeutic potential in novel joint regeneration strategies., (© 2014 The Author(s).)

DNA-protein cross-links (DPCs) are bulky, helix-distorting DNA lesions that form in the genome upon exposure to common antitumor drugs, environmental/occupational toxins, ionizing radiation, and endogenous free-radical-generating systems. As a result of their considerable size and their pronounced effects on DNA-protein interactions, DPCs can interfere with DNA replication, transcription, and repair, potentially leading to mutagenesis, genotoxicity, and cytotoxicity. However, the biological consequences of these ubiquitous lesions are not fully understood due to the difficulty of generating DNA substrates containing structurally defined, site-specific DPCs. In the present study, site-specific cross-links between the two biomolecules were generated by copper-catalyzed [3 + 2] Huisgen cycloaddition (click reaction) between an alkyne group from 5-(octa-1,7-diynyl)-uracil in DNA and an azide group within engineered proteins/polypeptides. The resulting DPC substrates were subjected to in vitro primer extension in the presence of human lesion bypass DNA polymerases η, κ, ν, and ι. We found that DPC lesions to the green fluorescent protein and a 23-mer peptide completely blocked DNA replication, while the cross-link to a 10-mer peptide was bypassed. These results indicate that the polymerases cannot read through the larger DPC lesions and further suggest that proteolytic degradation may be required to remove the replication block imposed by bulky DPC adducts.

Transforming growth factor-beta (TGF-β) superfamily proteins play a critical role in proliferation, differentiation, and other functions of mesenchymal stem cells (MSCs). During chondrogenic differentiation of MSCs, TGF-β up-regulates chondrogenic gene expression by enhancing the expression of the transcription factor SRY (sex-determining region Y)-box9 (Sox9). In this study, we investigated the effect of continuous TGF-β1 overexpression in human synovium-derived MSCs (hSD-MSCs) on immunophenotype, differentiation potential, and proliferation rate. hSD-MSCs were transduced with recombinant retroviruses (rRV) encoding TGF-β1. The results revealed that continuous overexpression of TGF-β1 did not affect their phenotype as evidenced by flow cytometry and reverse transcriptase PCR (RT-PCR). In addition, continuous TGF-β1 overexpression strongly enhanced cell proliferation of hSD-MSCs compared to the control groups. Also, induction of chondrogenesis was more effective in rRV-TGFB-transduced hSD-MSCs as shown by RT-PCR for chondrogenic markers, toluidine blue staining and glycosaminoglycan (GAG)/DNA ratio. Our data suggest that overexpression of TGF-β1 positively enhances the proliferation and chondrogenic potential of hSD-MSCs., (Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.)

Fanconi anemia (FA) is a chromosome instability syndrome characterized by increased cancer predisposition. Within the FA pathway, an upstream FA core complex mediates monoubiquitination and recruitment of the central FANCD2 protein to sites of stalled replication forks. Once recruited, FANCD2 fulfills a dual role towards replication fork recovery: (i) it cooperates with BRCA2 and RAD51 to protect forks from nucleolytic degradation and (ii) it recruits the BLM helicase to promote replication fork restart while suppressing new origin firing. Intriguingly, FANCD2 and its interaction partners are also involved in homologous recombination (HR) repair of DNA double-strand breaks, hinting that FANCD2 utilizes HR proteins to mediate replication fork recovery. One such candidate is CtIP (CtBP-interacting protein), a key HR repair factor that functions in complex with BRCA1 and MRE11, but has not been investigated as putative player in the replication stress response. Here, we identify CtIP as a novel interaction partner of FANCD2. CtIP binds and stabilizes FANCD2 in a DNA damage- and FA core complex-independent manner, suggesting that FANCD2 monoubiquitination is dispensable for its interaction with CtIP. Following cellular treatment with a replication inhibitor, aphidicolin, FANCD2 recruits CtIP to transiently stalled, as well as collapsed, replication forks on chromatin. At stalled forks, CtIP cooperates with FANCD2 to promote fork restart and the suppression of new origin firing. Both functions are dependent on BRCA1 that controls the step-wise recruitment of MRE11, FANCD2 and finally CtIP to stalled replication forks, followed by their concerted actions to promote fork recovery., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Rheumatoid arthritis (RA) and osteoarthritis (OA) are primarily chronic inflammatory diseases. Mesenchymal stem cells (MSCs) have the ability to differentiate into cells of the mesodermal lineage, and to regulate immunomodulatory activity. Specifically, MSCs have been shown to secrete insulin-like growth factor 1 (IGF-1). The purpose of the present study was to examine the inhibitory effects on inflammatory activity from a co-culture of human synovium-derived mesenchymal stem cells (hSDMSCs) and sodium nitroprusside (SNP)-stimulated chondrocytes. First, chondrocytes were treated with SNP to generate an in vitro model of RA or OA. Next, the co-culture of hSDMSCs with SNP-stimulated chondrocytes reduced inflammatory cytokine secretion, inhibited expression of inflammation activity-related genes, generated IGF-1 secretion, and increased the chondrocyte proliferation rate. To evaluate the effect of IGF-1 on inhibition of inflammation, chondrocytes pre-treated with IGF-1 were treated with SNP, and then the production of inflammatory cytokines was analyzed. Treatment with IGF-1 was shown to significantly reduce inflammatory cytokine secretion in SNP-stimulated chondrocytes. Our results suggest that hSDMSCs offer a new strategy to promote cell-based cartilage regeneration in RA or OA., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The purpose of this study was to investigate the anti-inflammatory effect of platelet-rich plasma (PRP) with collagen matrix on human nucleus pulposus (NP) cell in response to pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) and interleukin-1 (IL-1). NP cells from human disks were cultured in a monolayer and maintained in the collagen matrix prior to the addition of recombinant human IL-1 and TNF-α. After applying IL-1 and TNF-α, PRP prepared by using a commercially available platelet concentration system was added. The response was investigated using real-time PCR for mRNA expression of type II collagen, aggrecan, matrix metalloproteinase-3 (MMP-3), and cyclooxygenase-2 (COX-2). The combination of IL-1β and TNF-α led to decrease of matrix synthesis gene expression such as collagen type II and aggrecan and increase of the degradation gene expression of COX-2 and MMP-3, compared to the control. Consecutive PRP exposure significantly recovered the down-regulated gene expression of collagen type II and aggrecan and significantly reduced the increased MMP-3 and COX-2 gene expression, compared to that of control groups with pro-inflammatory cytokines. The administration of PRP with collagen matrix markedly suppressed cytokine-induced pro-inflammatory degrading enzymes and mediators in the NP cell. It also rescued gene expression concerning matrix synthesis, thereby stabilizing NP cell differentiation., (© 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Investigators conducting diabetes-related research have focused on islet transplantation as a radical therapy for type 1 diabetes mellitus. Pancreatic islet isolation, an essential process, is a very demanding work because of the proteolytic enzymes, species, treatment time, and individual difference. Replacement of primary isolated pancreatic islets must be carried out continuously for various in vitro tests, making primary isolated islets a useful tool for cell transplantation research. Hence, we sought to develop pseudoislets from commercial pancreas-derived cell lines. In this study, we used RIN-5F and RIN-m cells, which secrete insulin, somatostatin, or glucagon. To manufacture hybrid cellular spheroids, the cells were cultured under hanging drop plate and nonadhesive plate methods. We observed that hybrid cellular pseudoislets exhibited an oval shape, with sizes ranging from 590 to 1200 μm. Their morphology was similar to naïve islets. Cell line pseudoislets secreted and expressed insulin, glucagon, and somatostatin, as confirmed by reverse transcriptase polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistochemistry analyses. Thus, the current artificially manufactured biomimetic pseudoislets resembled pancreatic islets of the endocrine system, appearing as cellular aggregates that secreted insulin, glucagon, and somatostatin. Enhanced immunoisolation techniques may lead to the development of new islet sources for pancreatic transplantation through this pseudoislet strategy., (Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.)

Background: The importance of drug repositioning has been gaining attention in the last few years, allowing existing pharmaceutical products to be reevaluated for potential alternative therapeutic applications. The purpose of this study was to evaluate the effects of fragmin/protamine microparticles (F/P MPs) on cell aggregates under the concept of drug repositioning., Methods: Mesenchymal stem cells (MSCs) and embryonic rat heart-derived cardiac H9C2 cells were mixed with D-PBS, basal medium, fragmin, protamine, and F/P MPs to manufacture aggregates intended for cell transplantation. To evaluate their adhesive properties as cell carriers, we injected combinations of MSC aggregates into cartilage tissue, observing their leakage from the implantation site., Results: Our data demonstrated that MSCs and H9C2 cells mixed with F/P MPs rapidly produced large, viscous cellular aggregates. F/P MPs were bound to the surface of MSCs and H9C2 cells; thus, F/P MPs induced the formation of F/P MP-cell aggregates. Cell aggregates were prevented from leaking from the transplanted site., Conclusion: Aggregation induced by F/P MPs may improve the efficiency of cell therapy, a novel method for transplantation., (Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.)

Background: Before cell or tissue transplantation, cells or tissues have to be maintained for a certain period in vitro using culture medium and methods. Most culture media contain substances such as pH indicators and buffers. It is not known whether some of these substances are safe for subsequent application in the transplantation of cells or tissues into the human body. We investigated culture media and methods with respect to the safety of the components in future transplantation applications., Methods: A modified culture medium--medical fluid-based culture medium (FCM)--was designed by using various fluids and injectable drugs that are already currently permitted for use in clinical medicine. Medium components necessary for optimal cell growth were obtained from approved drugs. FCM was manufactured with adjusted final concentrations of the medium components similar to those in commercial Dulbecco's modified Eagle's medium (DMEM). In particular, 1029.40 mg/L amino acids, approximately 88.85 mg/L vitamins, 13,525.77 mg/L inorganic salts, and 4500 mg/L D-glucose comprise the high-glucose FCM. Next, human fat synovium-derived mesenchymal stem cells and rat H9c2 (2-1) cells were cultured under 2 conditions: (1) DMEM-high glucose (HG), an original commercial medium, and (2) optimized FCM-HG. We assessed the morphologies and proliferation rates of these cells., Results: We observed that FCM-HG was able to induce the growth of FS-MSC and commercially available H9c2 cell. The morphologies and proliferation patterns of these cells cultured under FCM-HG showed no differences compared with cells grown in DMEM-HG., Conclusion: Our data suggest that FCM, which we developed for the first time according to the concept of drug repositioning, was a useful culture medium, especially in cultured cells intended for human cell transplantation., (Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.)

Nucleotide excision repair (NER) removes lesions caused by environmental mutagens or UV light from DNA. A hallmark of NER is the extraordinarily wide substrate specificity, raising the question of how one set of proteins is able to recognize structurally diverse lesions. Two key features of good NER substrates are that they are bulky and thermodynamically destabilize DNA duplexes. To understand what the limiting step in damage recognition in NER is, we set out to test the hypothesis that there is a correlation of the degree of thermodynamic destabilization induced by a lesion, binding affinity to the damage recognition protein XPC-RAD23B, and overall NER efficiency. We chose to use acetylaminofluorene (AAF) and aminofluorene (AF) adducts at the C8 position of guanine in different positions within the NarI (GGCGCC) sequence, as it is known that the structures of the duplexes depend on the position of the lesion in this context. We found that the efficiency of NER and the binding affinity of the damage recognition factor XPC-RAD23B correlated with the thermodynamic destabilization induced by the lesion. Our study is the first systematic analysis correlating these three parameters and supports the idea that initial damage recognition by XPC-RAD23B is a key rate-limiting step in NER.

Exposure to aristolochic acid (AA), a component of Aristolochia plants used in herbal remedies, is associated with chronic kidney disease and urothelial carcinomas of the upper urinary tract. Following metabolic activation, AA reacts with dA and dG residues in DNA to form aristolactam (AL)-DNA adducts. These mutagenic lesions generate a unique TP53 mutation spectrum, dominated by A:T to T:A transversions with mutations at dA residues located almost exclusively on the non-transcribed strand. We determined the level of AL-dA adducts in human fibroblasts treated with AA to determine if this marked strand bias could be accounted for by selective resistance to global-genome nucleotide excision repair (GG-NER). AL-dA adduct levels were elevated in cells deficient in GG-NER and transcription-coupled NER, but not in XPC cell lines lacking GG-NER only. In vitro, plasmids containing a single AL-dA adduct were resistant to the early recognition and incision steps of NER. Additionally, the NER damage sensor, XPC-RAD23B, failed to specifically bind to AL-DNA adducts. However, placing AL-dA in mismatched sequences promotes XPC-RAD23B binding and renders this adduct susceptible to NER, suggesting that specific structural features of this adduct prevent processing by NER. We conclude that AL-dA adducts are not recognized by GG-NER, explaining their high mutagenicity and persistence in target tissues.

Over the years, cardiovascular diseases continue to increase and affect not only human health but also the economic stability worldwide. The advancement in tissue engineering is contributing a lot in dealing with this immediate need of alleviating human health. Blood vessel diseases are considered as major cardiovascular health problems. Although blood vessel transplantation is the most convenient treatment, it has been delimited due to scarcity of donors and the patient's conditions. However, tissue-engineered blood vessels are promising alternatives as mode of treatment for blood vessel defects. The purpose of this paper is to show the importance of the advancement on biofabrication technology for treatment of soft tissue defects particularly for vascular tissues. This will also provide an overview and update on the current status of tissue reconstruction especially from autologous stem cells, scaffolds, and scaffold-free cellular transplantable constructs. The discussion of this paper will be focused on the historical view of cardiovascular tissue engineering and stem cell biology. The representative studies featured in this paper are limited within the last decade in order to trace the trend and evolution of techniques for blood vessel tissue engineering.

A ring-expansion protocol that consisted of the 1,2-addition of various enolate nucleophiles to 6-trimethylsiloxy-2-cyclohexene-1-one (1) and the NaIO(4)-promoted oxidative ring opening of the resulting diols 2, followed by an intramolecular Knoevenagel condensation, furnished versatile dihydrotropones 6. Maintaining Z-configuration in the oxidative ring-opening products 3 is crucial for the success of the ring-expansion strategy. Dihydrotropones 6 are ripe for further elaborations such as oxidation to tropones 8 and Diels-Alder reaction with the Danishefsky's diene 10 to afford polycyclic compounds 12.

The primary objective of this study was to determine the possible apoptotic cell death preventive effects of the antioxidant selenium using an experimental rat spinal cord injury (SCI) model and cultured spinal cord-derived neural progenitor cells (NPCs). Sodium selenite treatment exerted a profound preventive effect on apoptotic cell death, including p-P38, p-SAPK/JNK, caspases, and PARP activity, and ameliorated astrogliosis and hypomyelination, which occurs in regions of active cell death in the spinal cords of SCI rats. The foremost protective effect of selenite in SCI would therefore be manifested in the suppression of acute secondary apoptotic cell death. However, selenite does not appear to exert an anti-inflammatory function associated with active microglia and macrophage propagation or infiltration into the lesion site. Selenite-mediated neuroprotection has been linked to selenite's attenuation or inhibition of p38 mitogen-activated protein kinase, pSAPK/JNK, and Bax activation in in vitro and in vivo SCI lesion sites. Selenite also attenuated cell death via the prevention of cytochrome c release, caspase activation, and ROS accumulation in the cytosol. Also, our study showed that selenite administered immediately after SCI significantly diminishes functional deficits. The selenite-treated group recovered hind limb reflexes more rapidly, and a higher percentage of these rats regained responses to a greater degree than was seen in the untreated injured rats. Our data indicate that the therapeutic outcome of selenite is most likely the consequence of its comprehensive apoptotic cell death blocking effects, resulting in the protection of white matter, oligodendrocytes, and neurons, and the inhibition of astrogliosis. The finding that the administration of selenite prevents secondary pathological events in traumatic spinal cord injuries, and promotes the recovery of motor function in an animal model. Its efficacy may facilitate the development of novel drug targets for the treatment of SCI.

This study was designed to investigate possible prevention of apoptotic cell death by selenium, an antioxidant, using cultured brain-derived neural progenitor cells (NPCs) and an experimental mouse brain trauma (BT) model. We tested some of the neuroprotective effects of sodium selenite in NPC cells by monitoring thioredoxin reductase (TR) expression, optimum H(2)O(2) removal, and consequent inhibition of pro-apoptotic events including cytochrome c release and caspase 3 and 9 activation. Analysis of key apoptotic regulators during H(2)O(2)-induced apoptosis of NPCs showed that selenite blocks the activation of c-jun N-terminal protein kinase (JNK)/P38 mitogen-activated protein kinase (MAPK), and Akt survival protein. Moreover, selenite activates p44/42 MAPK and inhibits the downregulation of Bcl2 in selenite-treated NPC cells. For in vivo experiments, the effects of selenite on H(2)O(2) neurotoxicity were tested using several biochemical and morphologic markers. Here we show that selenite potentially inhibits H(2)O(2)-induced apoptosis of NPCs and in traumatic brain injury. This in vivo protective function was also associated with inhibition of H(2)O(2)-induced reactive oxygen species (ROS) generation, cytochrome c release and caspase 3 and 9 activation. Our data show that the protective function of selenite through attenuation of secondary pathological events most likely results from its comprehensive effects that block apoptotic cell death, resulting in the maintenance of functional neurons and in inhibition of astrogliosis. The finding that selenite administration prevents secondary pathological events in an animal model of traumatic brain injury, as well as its efficacy, may provide novel drug targets for treating brain trauma.

Spinal cord injury (SCI) typically results from sustained trauma to the spinal cord, resulting in loss of neurologic function at the level of the injury. However, activation of various physiological mechanisms secondary to the initial trauma including edema, inflammation, excito-toxicity, excessive cytokine release and apoptosis may exacerbate the injury and/or retard natural repair mechanisms. Herein, we demonstrate that cytoplasmic extracts prepared from adipose tissue stromal cells (ATSCs) inhibits H(2)O(2)-mediated apoptosis of cultured spinal cord-derived neural progenitor cells (NPCs) resulting in increased cell survival. The ATSC extracts mediated this effect by decreasing caspase-3 and c-Jun-NH2-terminal kinase (SAPK/JNK) activity, inhibiting cytochrome c release from mitochondria and reducing Bax expression levels in cells. Direct injection of ATSC extracts mixed with Matrigel into the spinal cord immediately after SCI also resulted in reduced apoptotic cell death, astrogliosis and hypo-myelination but did not reduce the extent of microglia infiltration. Moreover, animals injected with the ATSC extract showed significant functional improvement of hind limbs as measured by the BBB (Basso, Beattie and Bresnahan) scale. Collectively, these studies show a prominent therapeutic effect of ATSC cytoplasmic extracts on SCI principally caused by an inhibition of apoptosis-mediated cell death, which spares white matter, oligodendrocytes and neurons at the site of injury. The ability of ATSC extracts to prevent secondary pathological events and improve neurologic function after SCI suggests that extracts prepared from autologous cells harvested from SCI patients may have clinical utility.