Safety and Feasibility of Intradiscal Administration of Matrilin-3-Primed Adipose-Derived Mesenchymal Stromal Cell Spheroids for Chronic Discogenic Low Back Pain: Phase 1 Clinical Trial.

Functionally enhanced mesenchymal stromal cells participate in the repair of intervertebral disc. This study aimed to assess the safety and tolerability of intradiscal administration of matrilin-3-primed adipose-derived stromal cell (ASC) spheroids with hyaluronic acid (HA) in patients with chronic discogenic low back pain (LBP). In this single-arm, open-label phase I clinical trial, eight patients with chronic discogenic LBP were observed over 6 months. Each patient underwent a one-time intradiscal injection of 1 mL of 6.0 × 106 cells/disc combined with HA under real-time fluoroscopic guidance. Safety and feasibility were gauged using Visual Analogue Scale (VAS) pain and Oswestry Disability Index (ODI) scores and magnetic resonance imaging. All participants remained in the trial, with no reported adverse events linked to the procedure or stem cells. A successful outcome-marked by a minimum 2-point improvement in the VAS pain score and a 10-point improvement in ODI score from the start were observed in six participants. Although the modified Pfirrmann grade remained consistent across all participants, radiological improvements were evident in four patients. Specifically, two patients exhibited reduced high-intensity zones while another two demonstrated decreased disc protrusion. In conclusion, the intradiscal application of matrilin-3-primed ASC spheroids with HA is a safe and feasible treatment option for chronic discogenic LBP.

Multimodal therapy strategy based on a bioactive hydrogel for repair of spinal cord injury.

Traumatic spinal cord injury results in permanent and serious neurological impairment, but there is no effective treatment yet. Tissue engineering approaches offer great potential for the treatment of SCI, but spinal cord complexity poses great challenges. In this study, the composite scaffold consists of a hyaluronic acid-based hydrogel, decellularized brain matrix (DBM), and bioactive compounds such as polydeoxyribonucleotide (PDRN), tumor necrosis factor-α/interferon-γ primed mesenchymal stem cell-derived extracellular vesicles (TI-EVs), and human embryonic stem cell-derived neural progenitor cells (NPC). The composite scaffold showed significant effects on regenerative prosses including angiogenesis, anti-inflammation, anti-apoptosis, and neural differentiation. In addition, the composite scaffold (DBM/PDRN/TI-EV/NPC@Gel) induced an effective spinal cord regeneration in a rat spinal cord transection model. Therefore, this multimodal approach using an integrated bioactive scaffold coupled with biochemical cues from PDRN and TI-EVs could be used as an advanced tissue engineering platform for spinal cord regeneration.

Development of a COX-2-Selective Fluorescent Probe for the Observation of Early Intervertebral Disc Degeneration.

Cyclooxygenase-2 (COX-2) is a biomolecule known to be overexpressed in inflammation. Therefore, it has been considered a diagnostically useful marker in numerous studies. In this study, we attempted to assess the correlation between COX-2 expression and the severity of intervertebral disc (IVD) degeneration using a COX-2-targeting fluorescent molecular compound that had not been extensively studied. This compound, indomethacin-adopted benzothiazole-pyranocarbazole (IBPC1), was synthesized by introducing indomethacin-a compound with known selectivity for COX-2-into a phosphor with a benzothiazole-pyranocarbazole structure. IBPC1 exhibited relatively high fluorescence intensity in cells pretreated with lipopolysaccharide, which induces inflammation. Furthermore, we observed significantly higher fluorescence in tissues with artificially damaged discs (modeling IVD degeneration) compared to normal disc tissues. These findings indicate that IBPC1 can meaningfully contribute to the study of the mechanism of IVD degeneration in living cells and tissues and to the development of therapeutic agents.

Deubiquitinase USP2 stabilizes the MRE11-RAD50-NBS1 complex at DNA double-strand break sites by counteracting the ubiquitination of NBS1.

The MRE11-RAD50-NBS1 (MRN) complex plays essential roles in the cellular response to DNA double-strand breaks (DSBs), which are the most cytotoxic DNA lesions, and is a target of various modifications and controls. Recently, lysine 48-linked ubiquitination of NBS1, resulting in premature disassembly of the MRN complex from DSB sites, was observed in cells lacking RECQL4 helicase activity. However, the role and control of this ubiquitination during the DSB response in cells with intact RECQL4 remain unknown. Here, we showed that USP2 counteracts this ubiquitination and stabilizes the MRN complex during the DSB response. By screening deubiquitinases that increase the stability of the MRN complex in RECQL4-deficient cells, USP2 was identified as a new deubiquitinase that acts at DSB sites to counteract NBS1 ubiquitination. We determined that USP2 is recruited to DSB sites in a manner dependent on ATM, a major checkpoint kinase against DSBs, and stably interacts with NBS1 and RECQL4 in immunoprecipitation experiments. Phosphorylation of two critical residues in the N terminus of USP2 by ATM is required for its recruitment to DSBs and its interaction with RECQL4. While inactivation of USP2 alone does not substantially influence the DSB response, we found that inactivation of USP2 and USP28, another deubiquitinase influencing NBS1 ubiquitination, results in premature disassembly of the MRN complex from DSB sites as well as defects in ATM activation and homologous recombination repair abilities. These results suggest that deubiquitinases counteracting NBS1 ubiquitination are essential for the stable maintenance of the MRN complex and proper cellular response to DSBs.

Preclinical Study of Human Bone Marrow-Derived Mesenchymal Stem Cells Using a 3-Dimensional Manufacturing Setting for Enhancing Spinal Fusion.

Spinal fusion surgery is a surgical technique that connects one or more vertebrae at the same time to prevent movement between the vertebrae. Although synthetic bone substitutes or osteogenesis-inducing recombinant proteins were introduced to promote bone union, the rate of revision surgery is still high due to pseudarthrosis. To promote successful fusion after surgery, stem cells with or without biomaterials were introduced; however, conventional 2D-culture environments have resulted in a considerable loss of the innate therapeutic properties of stem cells. Therefore, we conducted a preclinical study applying 3D-spheroids of human bone marrow-dewrived mesenchymal stem cells (MSCs) to a mouse spinal fusion model. First, we built a large-scale manufacturing platform for MSC spheroids, which is applicable to good manufacturing practice (GMP). Comprehensive biomolecular examinations, which include liquid chromatography-mass spectrometry and bioinformatics could suggest a framework of quality control (QC) standards for the MSC spheroid product regarding the identity, purity, viability, and potency. In our animal study, the mass-produced and quality-controlled MSC spheroids, either undifferentiated or osteogenically differentiated were well-integrated into decorticated bone of the lumbar spine, and efficiently improved angiogenesis, bone regeneration, and mechanical stability with statistical significance compared to 2D-cultured MSCs. This study proposes a GMP-applicable bioprocessing platform and QC directions of MSC spheroids aiming for their clinical application in spinal fusion surgery as a new bone graft substitute.

Synaptic Cell Adhesion Molecule 3 (SynCAM3) Deletion Promotes Recovery from Spinal Cord Injury by Limiting Glial Scar Formation.

Synaptic cell adhesion molecules (SynCAMs) play an important role in the formation and maintenance of synapses and the regulation of synaptic plasticity. SynCAM3 is expressed in the synaptic cleft of the central nervous system (CNS) and is involved in the connection between axons and astrocytes. We hypothesized that SynCAM3 may be related to the astrocytic scar (glial scar, the most important factor of CNS injury treatment) through extracellular matrix (ECM) reconstitution. Thus, we investigated the influence of the selective removal of SynCAM3 on the outcomes of spinal cord injury (SCI). SynCAM3 knock-out (KO) mice were subjected to moderate compression injury of the lower thoracic spinal cord using wild-type (WT) (C57BL/6JJc1) mice as controls. Single-cell RNA sequencing analysis over time, quantitative real-time polymerase chain reaction (qRT-PCR) analysis, and immunohistochemistry (IHC) showed reduced scar formation in SynCAM3 KO mice compared to WT mice. SynCAM3 KO mice showed improved functional recovery from SCI by preventing the transformation of reactive astrocytes into scar-forming astrocytes, resulting in improved ECM reconstitution at four weeks after injury. Our findings suggest that SynCAM3 could be a novel therapeutic target for SCI.

Structural and functional characterization of a monoclonal antibody blocking TIGIT.

TIGIT is an immune checkpoint receptor that is expressed on subsets of activated T cells and natural killer (NK) cells. Several ligands for TIGIT, including poliovirus receptor (PVR), are expressed on cancer cells and mediate inhibitory signaling to suppress antitumor activities of the immune cells. Many studies support that the TIGIT signaling is a potential target for cancer immunotherapy. We developed an IgG4-type monoclonal antibody against human TIGIT, designated as MG1131, using a phage display library of single-chain variable fragments (scFvs). MG1131 interacts with TIGIT much more tightly than PVR does. The crystal structure of a scFv version of MG1131 bound to TIGIT was determined, showing that MG1131 could block the PVR-TIGIT interaction and thus the immunosuppressive signaling of TIGIT. Consistently, MG1131 is bound to TIGIT-expressing cells and interferes with PVR binding to these cells. Moreover, MG1131 increased NK cell-mediated tumor killing activities, inhibited immunosuppressive activity of regulatory T (Treg) cells from healthy donors, and restored interferon-γ secretion from peripheral blood mononuclear cells derived from multiple myeloma patients. MG1131 also increased T cell infiltration to the tumor site and inhibited tumor growth in mice. Collectively, these data indicate that MG1131 modulates the effector functions of T cells and NK cells positively and Treg cells negatively.

Bazedoxifene, a Selective Estrogen Receptor Modulator, Promotes Functional Recovery in a Spinal Cord Injury Rat Model.

In research on various central nervous system injuries, bazedoxifene acetate (BZA) has shown two main effects: neuroprotection by suppressing the inflammatory response and remyelination by enhancing oligodendrocyte precursor cell differentiation and oligodendrocyte proliferation. We examined the effects of BZA in a rat spinal cord injury (SCI) model. Anti-inflammatory and anti-apoptotic effects were investigated in RAW 264.7 cells, and blood-spinal cord barrier (BSCB) permeability and angiogenesis were evaluated in a human brain endothelial cell line (hCMEC/D3). In vivo experiments were carried out on female Sprague Dawley rats subjected to moderate static compression SCI. The rats were intraperitoneally injected with either vehicle or BZA (1mg/kg pre-SCI and 3 mg/kg for 7 days post-SCI) daily. BZA decreased the lipopolysaccharide-induced production of proinflammatory cytokines and nitric oxide in RAW 264.7 cells and preserved BSCB disruption in hCMEC/D3 cells. In the rats, BZA reduced caspase-3 activity at 1 day post-injury (dpi) and suppressed phosphorylation of MAPK (p38 and ERK) at dpi 2, hence reducing the expression of IL-6, a proinflammatory cytokine. BZA also led to remyelination at dpi 20. BZA contributed to improvements in locomotor recovery after compressive SCI. This evidence suggests that BZA may have therapeutic potential to promote neuroprotection, remyelination, and functional outcomes following SCI.

Stable maintenance of the Mre11-Rad50-Nbs1 complex is sufficient to restore the DNA double-strand break response in cells lacking RecQL4 helicase activity.

The proper cellular response to DNA double-strand breaks (DSBs) is critical for maintaining the integrity of the genome. RecQL4, a DNA helicase of which mutations are associated with Rothmund-Thomson syndrome (RTS), is required for the DNA DSB response. However, the mechanism by which RecQL4 performs these essential roles in the DSB response remains unknown. Here, we show that RecQL4 and its helicase activity are required for maintaining the stability of the Mre11-Rad50-Nbs1 (MRN) complex on DSB sites during a DSB response. We found using immunocytochemistry and live-cell imaging that the MRN complex is prematurely disassembled from DSB sites in a manner dependent upon Skp2-mediated ubiquitination of Nbs1 in RecQL4-defective cells. This early disassembly of the MRN complex could be prevented by altering the ubiquitination site of Nbs1 or by expressing a deubiquitinase, Usp28, which sufficiently restored homologous recombination repair and ATM, a major checkpoint kinase against DNA DSBs, activation abilities in RTS, and RecQL4-depleted cells. These results suggest that the essential role of RecQL4 in the DSB response is to maintain the stability of the MRN complex on DSB sites and that defects in the DSB response in cells of patients with RTS can be recovered by controlling the stability of the MRN complex.

Genetic Therapy for Intervertebral Disc Degeneration.

Intervertebral disc (IVD) degeneration can cause chronic lower back pain (LBP), leading to disability. Despite significant advances in the treatment of discogenic LBP, the limitations of current treatments have sparked interest in biological approaches, including growth factor and stem cell injection, as new treatment options for patients with chronic LBP due to IVD degeneration (IVDD). Gene therapy represents exciting new possibilities for IVDD treatment, but treatment is still in its infancy. Literature searches were conducted using PubMed and Google Scholar to provide an overview of the principles and current state of gene therapy for IVDD. Gene transfer to degenerated disc cells in vitro and in animal models is reviewed. In addition, this review describes the use of gene silencing by RNA interference (RNAi) and gene editing by the clustered regularly interspaced short palindromic repeats (CRISPR) system, as well as the mammalian target of rapamycin (mTOR) signaling in vitro and in animal models. Significant technological advances in recent years have opened the door to a new generation of intradiscal gene therapy for the treatment of chronic discogenic LBP.

Safety and efficacy of Wharton's jelly-derived mesenchymal stem cells with teriparatide for osteoporotic vertebral fractures: A phase I/IIa study.

Osteoporotic vertebral compression fractures (OVCFs) are serious health problems. We conducted a randomized, open-label, phase I/IIa study to determine the feasibility, safety, and effectiveness of Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) and teriparatide (parathyroid hormone 1-34) in OVCFs. Twenty subjects with recent OVCFs were randomized to teriparatide (20 µg/day, daily subcutaneous injection for 6 months) treatment alone or combined treatment of WJ-MSCs (intramedullary [4 × 107 cells] injection and intravenous [2 × 108 cells] injection after 1 week) and teriparatide (20 µg/day, daily subcutaneous injection for 6 months). Fourteen subjects (teriparatide alone, n = 7; combined treatment, n = 7) completed follow-up assessment (visual analog scale [VAS], Oswestry Disability Index [ODI], Short Form-36 [SF-36], bone mineral density [BMD], bone turnover measured by osteocalcin and C-terminal telopeptide of type 1 collagen, dual-energy x-ray absorptiometry [DXA], computed tomography [CT]). Our results show that (a) combined treatment with WJ-MSCs and teriparatide is feasible and tolerable for the patients with OVCFs; (b) the mean VAS, ODI, and SF-36 scores significantly improved in the combined treatment group; (c) the level of bone turnover markers were not significantly different between the two groups; (d) BMD T-scores of spine and hip by DXA increased in both control and experimental groups without a statistical difference; and (e) baseline spine CT images and follow-up CT images at 6 and 12 months showed better microarchitecture in the combined treatment group. Our results indicate that combined treatment of WJ-MSCs and teriparatide is feasible and tolerable and has a clinical benefit for fracture healing by promoting bone architecture. Clinical trial registration: https://nedrug.mfds.go.kr/, MFDS: 201600282-30937.

Resolvin D3 Promotes Inflammatory Resolution, Neuroprotection, and Functional Recovery After Spinal Cord Injury.

Resolvins, a new family from the endogenous specialized pro-resolving mediators (SPMs), promote the resolution of the inflammatory response. Resolvin D3 (RvD3), a docosahexaenoic acid (DHA) product, has been known to suppress the inflammatory response. However, the anti-inflammatory and neuroprotective effects of RvD3 are not known in a model of spinal cord injury (SCI). Here, we investigated the anti-inflammatory and neuroprotective effect of RvD3 in a mouse model of SCI. Processes associated with anti-inflammation and angiogenesis were studied in RAW 264.7 cells and the human brain endothelial cell line hCMEC/D3, respectively. Additionally, female C57BL/6 mice were subjected to moderate compression SCI (20-g weight compression for 1 min) followed by intrathecal injection of vehicle or RvD3 (1 µg/20 µL) at 1 h post-SCI. RvD3 decreased the lipopolysaccharide (LPS)-induced production of inflammatory mediators and nitric oxide (NO) in RAW 264.7 cells and promoted an angiogenic effect in the hCMEC/D3 cell line. Treatment with RvD3 improved locomotor recovery and reduced thermal hyperalgesia in SCI mice compared with vehicle treatment at 14 days post-SCI. Remarkably, RvD3-treated mice exhibited reduced expression of inflammatory cytokines (TNF-α, IL6, IL1ß) and chemokines (CCL2, CCL3). Also, RvD3-treated mice exhibited increased expression of tight junction proteins such as zonula occludens (ZO)-1 and occludin. Furthermore, immunohistochemistry showed a decreased level of gliosis (GFAP, Iba-1) and neuroinflammation (CD68, TGF-ß) and enhanced neuroprotection. These data provide evidence that intrathecal injection of RvD3 represents a promising therapeutic strategy to promote inflammatory resolution, neuroprotection, and neurological functional recovery following SCI.

Organizing an in-class hackathon to correct PDF-to-text conversion errors of Genomics & Informatics 1.0.

This paper describes a community effort to improve earlier versions of the full-text corpus of Genomics & Informatics by semi-automatically detecting and correcting PDF-to-text conversion errors and optical character recognition errors during the first hackathon of Genomics & Informatics Annotation Hackathon (GIAH) event. Extracting text from multi-column biomedical documents such as Genomics & Informatics is known to be notoriously difficult. The hackathon was piloted as part of a coding competition of the ELTEC College of Engineering at Ewha Womans University in order to enable researchers and students to create or annotate their own versions of the Genomics & Informatics corpus, to gain and create knowledge about corpus linguistics, and simultaneously to acquire tangible and transferable skills. The proposed projects during the hackathon harness an internal database containing different versions of the corpus and annotations.

An Injectable Hyaluronan-Methylcellulose (HAMC) Hydrogel Combined with Wharton's Jelly-Derived Mesenchymal Stromal Cells (WJ-MSCs) Promotes Degenerative Disc Repair.

Intervertebral disc (IVD) degeneration is one of the predominant causes of chronic low back pain (LBP), which is a leading cause of disability worldwide. Despite substantial progress in cell therapy for the treatment of IVD degeneration, significant challenges remain for clinical application. Here, we investigated the effectiveness of hyaluronan-methylcellulose (HAMC) hydrogels loaded with Wharton's Jelly-derived mesenchymal stromal cell (WJ-MSCs) in vitro and in a rat coccygeal IVD degeneration model. Following induction of injury-induced IVD degeneration, female Sprague-Dawley rats were randomized into four groups to undergo a single intradiscal injection of the following: (1) phosphate buffered saline (PBS) vehicle, (2) HAMC, (3) WJ-MSCs (2 × 104 cells), and (4) WJ-MSCs-loaded HAMC (WJ-MSCs/HAMC) (n = 10/each group). Coccygeal discs were removed following sacrifice 6 weeks after implantation for radiologic and histologic analysis. We confirmed previous findings that encapsulation in HAMC increases the viability of WJ-MSCs for disc repair. The HAMC gel maintained significant cell viability in vitro. In addition, combined implantation of WJ-MSCs and HAMC significantly promoted degenerative disc repair compared to WJ-MSCs alone, presumably by improving nucleus pulposus cells viability and decreasing extracellular matrix degradation. Our results suggest that WJ-MSCs-loaded HAMC promotes IVD repair more effectively than cell injection alone and supports the potential clinical use of HAMC for cell delivery to arrest IVD degeneration or to promote IVD regeneration.

Combined Treatment with Fasudil and Menthol Improves Functional Recovery in Rat Spinal Cord Injury Model.

Neuroprotective measures by preventing secondary spinal cord injury (SCI) are one of the main strategies for repairing an injured spinal cord. Fasudil and menthol may be potent neuroprotective agents, which act by inhibiting a rho-associated protein kinase (ROCK) and suppressing the inflammatory response, respectively. We hypothesized that combined treatment of fasudil and menthol could improve functional recovery by decreasing inflammation, apoptosis, and glial scar formation. We tested our hypothesis by administering fasudil and menthol intraperitoneally (i.p.) to female Sprague Dawley rats after moderate static compression (35 g of impounder for 5 min) of T10 spinal cord. The rats were randomly divided into five experimental groups: (i) sham animals received laminectomy alone, (ii) injured (SCI) and untreated (saline 0.2 mL/day, i.p.) rats, (iii) injured (SCI) rats treated with fasudil (10 mg/kg/day, i.p.) for two weeks, (iv) injured (SCI) rats treated with menthol (10 mg/kg/day, i.p.) for twoweeks, (v) injured (SCI) rats treated with fasudil (5 mg/kg/day, i.p.) and menthol (10 mg/kg/day, i.p.) for two weeks. Compared to single treatment groups, combined treatment of fasudil and menthol demonstrated significant functional recovery and pain amelioration, which, thereby, significantly reduced inflammation, apoptosis, and glial/fibrotic scar formation. Therefore, combined treatment of fasudil and menthol may provide effective amelioration of spinal cord dysfunction by a synergistic effect of fasudil and menthol.

CORM-2-Solid Lipid Nanoparticles Maintain Integrity of Blood-Spinal Cord Barrier After Spinal Cord Injury in Rats.

Spinal cord injury (SCI) is a devastating condition of the central nervous system that can lead to permanent motor and sensory deficits. Carbon monoxide-releasing molecule-2 (CORM-2) has been shown to have anti-inflammatory, anti-apoptotic, and angiogenic properties that may be useful for the treatment of SCI. However, it has a short carbon monoxide (CO) release half-life (approximately 1 min). To address this challenge, we developed a CORM-2-incorporated solid lipid nanoparticle (CORM-2-SLN) and evaluated its ameliorating effects for preventing blood-spinal cord barrier (BSCB) disruption and endothelial cell death following SCI. After a moderate compression injury of the spinal cord (compression with a 35-g impounder for 5 min), groups of rats were treated with a CORM-2-solution and CORM-2-SLNs at an equal dose of 10 mg/kg each via an intraperitoneal injection for 8 consecutive days. Behavior analysis was performed and animals were later sacrificed at different time points and evaluated for whether the CORM-2-SLNs prevented BSCB disruption and rescued endothelial cell damage following SCI. The CORM-2-SLN-treated group showed significantly diminished extravasation of Evans Blue dye with enhanced expression of tight junction proteins following SCI. Likewise, significantly diminished endothelial cell markers after SCI were optimally stabilized at 21 days. Additionally, lipopolysaccharide (LPS)-induced loss of tight junction integrity was significantly preserved after CORM-2-SLN treatment in human cerebral microvascular endothelial cell line (hCMEC/D3). Clinically, CORM-2-SLNs were associated with a significantly improved functional recovery, as compared with the CORM-2-solution. CORM-2-SLNs may help potentially to maintain BSCB integrity following SCI.

Elevated TRPV4 Levels Contribute to Endothelial Damage and Scarring in Experimental Spinal Cord Injury.

Currently, the role of transient receptor potential vanilloid type 4 (TRPV4), a nonselective cation channel in the pathology of spinal cord injury (SCI), is not recognized. Herein, we report the expression and contribution of TRPV4 in the pathology of scarring and endothelial and secondary damage after SCI. TRPV4 expression increased during the inflammatory phase in female rats after SCI and was expressed primarily by cells at endothelial-microglial junctions. Two-photon microscopy of intracellular-free Ca2+ levels revealed a biphasic increase at similar time points after SCI. Expression of TRPV4 at the injury epicenter, but not intracellular-free Ca2+, progressively increases with the severity of the injury. Activation of TRPV4 with specific agonist altered the organization of endothelial cells, affected tight junctions in the hCMEC/D3 BBB cell line in vitro, and increases the scarring in rat spinal cord as well as induced endothelial damage. By contrast, suppression of TRPV4 with a specific antagonist or in female Trpv4 KO mouse attenuated inflammatory cytokines and chemokines, prevented the degradation of tight junction proteins, and preserve blood-spinal cord barrier integrity, thereby attenuate the scarring after SCI. Likewise, secondary damage was reduced, and behavioral outcomes were improved in Trpv4 KO mice after SCI. These results suggest that increased TRPV4 expression disrupts endothelial cell organization during the early inflammatory phase of SCI, resulting in tissue damage, vascular destabilization, blood-spinal cord barrier breakdown, and scarring. Thus, TRPV4 inhibition/knockdown represents a promising therapeutic strategy to stabilize/protect endothelial cells, attenuate nociception and secondary damage, and reduce scarring after SCI.SIGNIFICANCE STATEMENT TRPV4, a calcium-permeable nonselective cation channel, is widely expressed in both excitable and nonexcitable cells. Spinal cord injury (SCI) majorly caused by trauma/accidents is associated with changes in osmolarity, mechanical injury, and shear stress. After SCI, TRPV4 was increased and were found to be linked with the severity of injury at the epicenter at the time points that were reported to be critical for repair/treatment. Activation of TRPV4 was damaging to endothelial cells that form the blood-spinal cord barrier and thus contributes to scarring (glial and fibrotic). Importantly, inhibition/knockdown of TRPV4 prevented these effects. Thus, the manipulation of TRPV4 signaling might lead to new therapeutic strategies or combinatorial therapies to protect endothelial cells and enhance repair after SCI.

Changes in FGF-23, Neutrophil/Platelet Activation Markers, and Angiogenin in Advanced Chronic Kidney Disease and Their Effect on Arterial Stiffness.

AIMS: The aims of this study were to measure changes in fibroblast growth factor 23 (FGF-23), neutrophil (elastase, lactoferrin)/platelet activation marker (mean platelet volume-to-platelet count ratio [MPR]), and angiogenin according to the stage of chronic kidney disease (CKD), and to evaluate the association of FGF-23, elastase, lactoferrin, MPR, and angiogenin with arterial stiffness using brachial-ankle pulse wave velocity (ba-PWV) in CKD patients. METHODS: According to the estimated glomerular filtration rate (eGFR) calculated using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation, the patients were allocated to five groups: (1) normal controls (eGFR ≥90 mL/min/1.73 m2 without pathologic, urine [proteinuria], blood [electrolyte], and imaging abnormalities; n = 22); (2) CKD stage 2 (eGFR 60-89 mL/min/1.73 m2; n = 17); (3) CKD stage 3 (eGFR 30-59 mL/min/1.73 m2; n = 22); (4) CKD stage 4 (eGFR 15-30 mL/min/1.73 m2; n = 17); and (5) CKD stage 5-hemodialysis (HD) (n = 30). All the patients were free of clinically apparent cardiovascular disease. Serum FGF-23, elastase, lactoferrin, and angiogenin concentrations and the MPR were measured to study the association of the above parameters with the clinical (age, sex, presence of diabetes mellitus, and blood pressure), biochemical (calcium, phosphorus, uric acid, intact parathyroid hormone [PTH], low-density lipoprotein cholesterol, and high-sensitivity C-reactive protein), and ba-PWV values of the CKD patients. RESULTS: (1) The mean ba-PWV values were 1,497.2 ± 206.4 cm/s in the controls, 1,649.0 ± 247.9 cm/s in the CKD stage 2 group (p < 0.05 vs. controls), 1,655.8 ± 260.3 cm/s in the CKD stage 3 group (p < 0.05 vs. controls), 1,823.0 ± 402.4 cm/s in the CKD stage 4 group (p < 0.05 vs. controls and CKD stages 2 and 3), and 1,905.2 ± 374.1 cm/s in the CKD stage 5-HD group (p < 0.05 vs. controls and CKD stage 2). (2) The mean log10(FGF-23) concentration values were 0.77 ± 0.27, 0.97 ± 0.48, 1.10 ± 0.35 (p < 0.05 vs. controls and CKD stage 2), 1.35 ± 0.48 (p < 0.05 vs. controls and CKD stages 2 and 3), and 2.12 ± 0.82 (p < 0.05 vs. controls and CKD stages 2-4); the mean angiogenin levels were 230.6 ± 70.5 pg/mL, 283.0 ± 53.5 pg/mL (p < 0.05 vs. controls), 347.3 ± 76.9 pg/mL (p < 0.05 vs. controls and CKD stage 2), 445.9 ± 90.6 pg/mL (p < 0.05 vs. controls and CKD stages 2 and 3), and 370.9 ± 142.4 pg/mL (p < 0.05 vs. controls and CKD stages 2 and 3). (3) In the stage 3-4 CKD/HD patients, the mean elastase-to-neutrophil and lactoferrin-to-neutrophil ratios were significantly lower than in the controls and the stage 2 CKD patients. (4) Our multivariate linear regression analyses showed that age, pulse pressure, mean arterial pressure, PTH, and FGF-23 were independently associated with ba-PWV values. CONCLUSIONS: Circulating FGF-23 and angiogenin concentrations gradually increased as CKD advanced, whereas neutrophil activation markers were significantly lower in the stage 3-4 CKD/HD patients than in the controls and stage 2 CKD patients. FGF-23 was weakly associated with ba-PWV values in patients with CKD/HD and no previous cardiovascular disease.

Effects of Matrices and Additives on Multiple Charge Formation of Proteins in MALDI-MS Analysis.

The sinapinic acid (SA) matrix has frequently been used for protein analysis in matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). However, the SA matrix does not result in the formation of distinctive multiple protein charge states, whereas the 2-nitrophloroglucinol (2-NPG) matrix is capable of this. The formation of multiple charge states in the MALDI-MS analysis of proteins is advantageous in that it results in higher accuracy. In this study, the mass spectra of several common standard proteins, namely cytochrome c, myoglobin, bovine serum albumin (BSA), and immunoglobulin G (IgG), were compared using various matrices (2,5-dihydroxybenzoic acid, α-cyano-hydroxycinnamic acid, SA, and 2-NPG). Furthermore, the mass spectra of two large standard proteins (BSA and IgG) using various acid additives (H3PO4, HNO3, H2SO4, HCl, and trifluoroacetic acid) with the 2-NPG matrix were also compared. Among the different matrices, 2-NPG provided the broadest range of multiple protein charge states, while, among the different additives, the 2-NPG matrix in combination with HCl generated the broadest multiple charge states as well as the most intense protein peaks.