Evaluation of Cytotoxic and Antibacterial Effect of Methanolic Extract of Paeonia lactiflora.

BACKGROUND AND OBJECTIVES: Bacterial antibiotics have had several side effects. Therefore, interest in natural substances with less side effects is increasing these days. Paeonia lactiflora, the root of Paeonia lactiflora, is used as a raw material for medicines. In this study, we investigated the antibacterial effect and the cytotoxicity of Paeonia lactiflora extract. MATERIALS AND METHODS: For cytotoxicity, MTT analysis according to ISO 10993-5 was performed. The antibacterial test of the Paeonia lactiflora was determined from bacterial viability, Inhibition zone test, CFU (colony forming unit) and SEM (scanning electron microscope). To confirm the antibacterial component of Paeonia lactiflora, the content of flavonoids and polyphenols was analyzed. RESULTS: Our results showed that Paeonia lactiflora extract contained flavonoids and polyphenols, which exhibited antimicrobial activity against Streptococcus mutans (S. mutans) and Candida albicans (C. ablicans). Further, the cytotoxicity of Paeonia lactiflora extract was low. CONCLUSIONS: We believe that our study makes a significant contribution to the literature because it demonstrates that Paeonia lactiflora extract can be used as an antibiotic.

Preclinical immunogenicity testing using anti-drug antibody analysis of GX-G3, Fc-fused recombinant human granulocyte colony-stimulating factor, in rat and monkey models.

Human granulocyte colony-stimulating factor (G-CSF, this study used Fc-fused recombinant G-CSF; GX-G3) is an important glycoprotein that stimulates the proliferation of granulocytes and white blood cells. Thus, G-CSF treatment has been considered as a crucial regimen to accelerate recovery from chemotherapy-induced neutropenia in cancer patients suffering from non-myeloid malignancy or acute myeloid leukemia. Despite the therapeutic advantages of G-CSF treatment, an assessment of its immunogenicity must be performed to determine whether the production of anti-G-CSF antibodies causes immune-related disorders. We optimized and validated analytical tools by adopting validation parameters for immunogenicity assessment. Using these validated tools, we analyzed serum samples from rats and monkeys injected subcutaneously with GX-G3 (1, 3 or 10 mg/kg once a week for 4 weeks followed by a 4-week recovery period) to determine immunogenicity response and toxicokinetic parameters with serum concentration of GX-G3. Several rats and monkeys were determined to be positive for anti-GX-G3 antibodies. Moreover, the immunogenicity response of GX-G3 was lower in monkeys than in rats, which was relevant to show less inhibition of toxicokinetic profiles in monkeys, at least 1 mg/kg administrated group, compared to rats. These results suggested the establishment and validation for analyzing anti-GX-G3 antibodies and measurement of serum levels of GX-G3 and anti-GX-G3 antibodies, which was related with toxicokinetic profiles. Taken together, this study provides immunogenicity assessment which is closely implicated with toxicokinetic study of GX-G3 in 4-week repeated administrated toxicological studies.

Antimicrobial Effects against Oral Pathogens and Cytotoxicity of Glycyrrhiza uralensis Extract.

We aimed to evaluate the antimicrobial effects of Glycyrrhiza uralensis extract on Streptococcus mutans and Candida albicans and its biocompatibility for dental applications. The antimicrobial activity of the G. uralensis extracts at concentrations of 50, 100, 150, and 200 µg/mL was assessed using agar disk diffusion tests, counting the total number of colony-forming units (CFUs), spectrophotometric growth inhibitory assays, and microbial morphology observations using scanning electron microscopy (SEM; Merin, Carl Zeiss, Oberkochen, Germany). We measured the polyphenol and flavonoid contents of G. uralensis extracts using ultraviolet-visible spectrometry and the cytotoxicity of these extracts using an MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. We identified that G. uralensis extracts had significant antimicrobial effects against S. mutans and C. albicans. The optical density of the experimental groups significantly decreased compared with that of the control group. SEM images revealed that the G. uralensis extract affected the morphology and density of S. mutans and C. albicans. The extract concentration of flavonoids, but not polyphenols, increased with increasing concentrations of the G. uralensis extract. Furthermore, cell viabilities were more than 70% for G. uralensis extracts with concentrations of 50 and 100 µg/mL. Naturally derived G. uralensis is biocompatible and exhibits an excellent antimicrobial effect against oral pathogens such as S. mutans and C. albicans. Thus, G. uralensis extracts can be used for the development of oral products that treat and prevent oral diseases.

In-Depth Proteomic Analysis of Human Bronchoalveolar Lavage Fluid toward the Biomarker Discovery for Lung Cancers.

PURPOSE: Lung cancer is among the most common cancers. Bronchoalveolar lavage fluid (BALF) can be easily obtained from patients with lung cancers. The aim is to develop a novel proteomic method of the molecule-based sensitive detection of biomarkers from BALF. EXPERIMENTAL DESIGN: BALF samples are collected from segmental bronchus of 14 patients with lung cancers from Kyung Hee University Hospital. First, BALF proteome is depleted using a depletion column, and then peptides are prepared from the enriched low abundant proteins and fractionated by high pH reverse phase liquid chromatography prior to LC-MS/MS. Data are available via ProteomeXchange with identifier PXD012645. RESULTS: A novel method is developed for in-depth proteomic analysis of BALF by combining antibody-based depletion of high abundant proteins from BALF with high pH peptide fractionation. Peptides are analyzed on a high resolution Orbitrap Fusion mass spectrometer. MaxQuant search result in the identification of 4615 protein groups mapped to 4534 genes. CONCLUSIONS AND CLINICAL RELEVANCE: It is found that the method outperformed conventional BALF proteomic methods and it is believed that this method will facilitate the biomarker research for lung cancer. In addition, it is shown that BALF will be a great source of biomarkers of lung diseases.

GDNF family receptor alpha 1 is a reliable marker of undifferentiated germ cells in bulls.

Undifferentiated germ cells, including spermatogonial stem cells (SSCs), make up only a very small proportion of germ cells within the testis; for example, 0.03% of germ cells in the mouse testis are SSCs. In this study, we investigated the characteristics of bovine undifferentiated germ cells and developed an enrichment procedure for these cells on the basis of fluorescence-activated cell sorting (FACS), using the specific cell surface marker glial cell line-derived neurotrophic factor family receptor alpha 1 (GFRα1). FACS analysis showed that only 0.6% of the total testicular cells were GFRα1-positive. These GFRα1-positive cells had a significantly higher expression of UCHL1, ZBTB16, and DDX4 (all markers of undifferentiated spermatogonial and germ cells) than that of fresh testicular cells. Quantitative reverse-transcription PCR analyses also indicated that the gene expression of BCL6B and NANOS2 was significantly higher in GFRα1-positive cells. Furthermore, xenogeneic transplantation of bovine testicular cells into immunodeficient mice resulted in 4.4-fold more colonies of GFRα1-positive cells than those of fresh testicular cells, indicating that FACS with antibodies to GFRα1 had efficiently enriched putative SSCs from total testicular cells. Collectively, these results demonstrate that GFRα1 could be used as a marker of bovine undifferentiated germ cells, including putative SSCs, and that its expression on SSCs has important implications for the further development of techniques for enriching stem cells from other species.

Fabrication of dihydroxyflavone-conjugated hyaluronic acid nanogels for targeted antitumoral effect.

We prepared hyaluronic acid (HA)-based nanogels conjugated with dihydroxyflavone (DHF) and evaluated their cellular uptake and antitumoral efficiency. 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) was used as a conjugation agent for esterification between DHF and HA as well as crosslinking among HA. The conjugations were confirmed by nuclear magnetic resonance spectroscopy, UV/vis spectroscopy, and high-performance liquid chromatography. The size and Zeta-potential of the DHF/HA nanogels were reduced with an increase in the concentration of DMTMM due to the involvement of more HA molecules for the conjugation reactions. The DHF/HA nanogel with a smaller size was greatly taken up by two kinds of tumor cells (HeLa and HepG2), compared to NIH3T3. The cell viabilities were reduced to approximately 60% for HeLa and HepG2 cells after 48 h post treatment with DHF/HA nanogels.

Characterization of the changes in eicosanoid profiles of activated macrophages treated with 20(S)-ginsenoside Rg3.

In this study, we used ultra-performance liquid chromatography coupled with tandem mass spectrometry to assess the levels of eicosanoids from RAW264.7 macrophages treated with lipopolysaccharides (LPS) and 20(S)-ginsenoside Rg3 (Rg3). The production of nitric oxide (NO) and the secretion of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were increased in inflammatory macrophages treated with LPS. Rg3 treatment, however, decreased the levels of NO, TNF-α, and IL-6 in activated macrophages. Eicosanoids, known as major metabolites correlated with inflammation, have pro- or anti-inflammatory activities. For a detailed characterization of the eicosanoids altered by treatment with LPS and Rg3, the eicosanoids were profiled by multiple reaction monitoring. A total of 69 macrophage eicosanoids were analyzed and the profiling dataset was statistically analyzed. Principal component and hierarchical cluster analyses differentiated control cells from cells treated with LPS, Rg3, or LPS+Rg3 for 12 or 24h. Furthermore, 18 differentially regulated eicosanoids were found between macrophages treated with LPS for 24h and those treated with LPS+Rg3 for 24h (fold change>2, p value<0.05). These results indicate that Rg3 alters eicosanoid metabolism in activated macrophages treated with LPS. Furthermore, we also identified several eicosanoids correlated with the anti-inflammatory activity of Rg3.

PSMB8 and PBK as potential gastric cancer subtype-specific biomarkers associated with prognosis.

Gastric adenocarcinoma is a common form of cancer associated with a poor prognosis. We analyzed microarray profiling data from 48 patients with gastric adenocarcinoma to characterize gastric cancer subtypes and identify biomarkers associated with prognosis. We identified two major subtypes of gastric adenocarcinoma differentially associated with overall survival (P = 0.025). Genes that were differentially expressed were identified using specific criteria (P < 0.001 and >1.5-fold); expression of 294 and 116 genes was enriched in good and poor prognosis subtypes, respectively. Genes related to translational elongation and cell cycle were upregulated in the poor prognosis group. Of these genes, upregulation of proteasome subunit beta type 8 PSMB8 and PDZ binding kinase PBK was confirmed by real-time reverse transcription-PCR analysis. PSMB8 or PBK knockdown had no effect on gastric cancer cell proliferation but suppressed cell migration and invasion, respectively. Furthermore, immunohistochemistry analysis of 385 gastric cancer patients revealed that increased nuclear expression of PSMB8 and PBK was correlated with depth of invasion, lymph node metastasis, and lower survival rates. Taken together, two gastric adenocarcinoma subtypes were predictive of prognosis. PSMB8 and PBK were predictive of gastric cancer prognosis and could be potential gastric cancer subtype-specific biomarkers.

A Mutation in PMP2 Causes Dominant Demyelinating Charcot-Marie-Tooth Neuropathy.

Charcot-Marie-Tooth disease (CMT) is a heterogeneous group of peripheral neuropathies with diverse genetic causes. In this study, we identified p.I43N mutation in PMP2 from a family exhibiting autosomal dominant demyelinating CMT neuropathy by whole exome sequencing and characterized the clinical features. The age at onset was the first to second decades and muscle atrophy started in the distal portion of the leg. Predominant fatty replacement in the anterior and lateral compartment was similar to that in CMT1A caused by PMP22 duplication. Sural nerve biopsy showed onion bulbs and degenerating fibers with various myelin abnormalities. The relevance of PMP2 mutation as a genetic cause of dominant CMT1 was assessed using transgenic mouse models. Transgenic mice expressing wild type or mutant (p.I43N) PMP2 exhibited abnormal motor function. Electrophysiological data revealed that both mice had reduced motor nerve conduction velocities (MNCV). Electron microscopy revealed that demyelinating fibers and internodal lengths were shortened in both transgenic mice. These data imply that overexpression of wild type as well as mutant PMP2 also causes the CMT1 phenotype, which has been documented in the PMP22. This report might expand the genetic and clinical features of CMT and a further mechanism study will enhance our understanding of PMP2-associated peripheral neuropathy.

A novel homozygous MPV17 mutation in two families with axonal sensorimotor polyneuropathy.

BACKGROUND: Mutations in MPV17 cause the autosomal recessive disorder mitochondrial DNA depletion syndrome 6 (MTDPS6), also called Navajo neurohepatopathy (NNH). Clinical features of MTDPS6 is infantile onset of progressive liver failure with seldom development of progressive neurologic involvement. METHODS: Whole exome sequencing (WES) was performed to isolate the causative gene of two unrelated neuropathy patients (9 and 13 years of age) with onset of the syndrome. Clinical assessments and biochemical analysis were performed. RESULTS: A novel homozygous mutation (p.R41Q) in MPV17 was found by WES in both patients. Both showed axonal sensorimotor polyneuropathy without liver and brain involvement, which is neurophysiologically similar to axonal Charcot-Marie-Tooth disease (CMT). A distal sural nerve biopsy showed an almost complete loss of the large and medium-sized myelinated fibers compatible with axonal neuropathy. An in vitro assay using mouse motor neuronal cells demonstrated that the abrogation of MPV17 significantly affected cell integrity. In addition, the expression of the mutant protein affected cell proliferation. These results imply that both the loss of normal function of MPV17 and the gain of detrimental effects of the mutant protein might affect neuronal function. CONCLUSION: We report a novel homozygous mutation in MPV17 from two unrelated patients harboring axonal sensorimotor polyneuropathy without hepatoencephalopathy. This report expands the clinical spectrum of diseases caused by mutations of MPV17, and we recommend MPV17 gene screening for axonal peripheral neuropathies.

Overexpression of mutant HSP27 causes axonal neuropathy in mice.

BACKGROUND: Mutations in heat shock 27 kDa protein 1 (HSP27 or HSPB1) cause distal hereditary motor neuropathy (dHMN) or Charcot-Marie-Tooth disease type 2 F (CMT2F) according to unknown factors. Mutant HSP27 proteins affect axonal transport by reducing acetylated tubulin. RESULTS: We generated a transgenic mouse model overexpressing HSP27-S135F mutant protein driven by Cytomegalovirus (CMV) immediate early promoter. The mouse phenotype was similar to dHMN patients in that they exhibit motor neuropathy. To determine the phenotypic aberration of transgenic mice, behavior test, magnetic resonance imaging (MRI), electrophysiological study, and pathology were performed. Rotarod test showed that founder mice exhibited lowered motor performance. MRI also revealed marked fatty infiltration in the anterior and posterior compartments at calf level. Electrophysiologically, compound muscle action potential (CMAP) but not motor nerve conduction velocity (MNCV) was reduced in the transgenic mice. Toluidine staining with semi-thin section of sciatic nerve showed the ratio of large myelinated axon fiber was reduced, which might cause reduced locomotion in the transgenic mice. Electron microscopy also revealed abundant aberrant myelination. Immunohistochemically, neuronal dysfunctions included elevated level of phosphorylated neurofilament and reduced level of acetylated tubulin in the sural nerve of transgenic mice. There was no additional phenotype besides motor neuronal defects. CONCLUSIONS: Overexpression of HSP27-S135F protein causes peripheral neuropathy. The mouse model can be applied to future development of therapeutic strategies for dHMN or CMT2F.

Effects of paracrine factors on CD24 expression and neural differentiation of male germline stem cells.

Spermatogonial stem cells (SSCs) are adult male germ cells that develop after birth. Throughout the lifetime of an organism, SSCs sustain spermatogenesis through self-renewal and produce daughter cells that differentiate into spermatozoa. Several studies have demonstrated that SSCs can acquire pluripotency under appropriate culture conditions, thus becoming multipotent germline stem cells (mGSCs) that express markers of pluripotency in culture and form teratomas following transplantation into immunodeficient mice. In the present study, we generated neural precursor cells expressing CD24, a neural precursor marker, from pluripotent stem cell lines and demonstrated that these cells effectively differentiated along a neural lineage in vitro. In addition, we found that paracrine factors promoted CD24 expression during the neural differentiation of mGSCs. Our results indicated that the expression of CD24, enhanced by a combination of retinoic acid (RA), noggin and fibroblast growth factor 8 (FGF8) under serum-free conditions promoted neural precursor differentiation. Using a simple cell sorting method, we were able to collect neural precursor cells with the potential to differentiate from mGSCs into mature neurons and astrocytes in vitro.

A family with axonal sensorimotor polyneuropathy with TUBB3 mutation.

Mutations in the ß­tubulin isotype III (TUBB3) gene result in TUBB3 syndrome that includes congenital fibrosis of the extraocular muscle type 3 (CFEOM3), intellectual impairments and/or an axonal sensorimotor neuropathy. In the present study, a TUBB3 D417N mutation was identified in a family with axonal sensorimotor polyneuropathy by whole exome sequencing. The proband exhibited gait disturbance at the age of 12 years and was wheelchair bound at 40 years. However, the proband's cousin exhibited gait disabilities at 45 years of age and was still able to walk when he was 60 years old. Ophthalmoplegia and intellectual impairment were not observed in either patient. A sural nerve biopsy identified an absence of large myelinated fibers without demyelinating degeneration. Based on these clinical features, the two patients exhibited an axonal peripheral neuropathy without CFEOM3. These results therefore suggested that certain TUBB3 mutations may predominantly be associated with axonal peripheral neuropathy. Furthermore, the results also suggested that TUBB3 mutations may be implicated in modulating the inter­ and intra­familial heterogeneity of clinical phenotypes.

Cellular attachment and differentiation on titania nanotubes exposed to air- or nitrogen-based non-thermal atmospheric pressure plasma.

The surface topography and chemistry of titanium implants are important factors for successful osseointegration. However, chemical modification of an implant surface using currently available methods often results in the disruption of topographical features and the loss of beneficial effects during the shelf life of the implant. Therefore, the aim of this study was to apply the recently highlighted portable non-thermal atmospheric pressure plasma jet (NTAPPJ), elicited from one of two different gas sources (nitrogen and air), to TiO2 nanotube surfaces to further improve their osteogenic properties while preserving the topographical morphology. The surface treatment was performed before implantation to avoid age-related decay. The surface chemistry and morphology of the TiO2 nanotube surfaces before and after the NTAPPJ treatment were determined using a field-emission scanning electron microscope, a surface profiler, a contact angle goniometer, and an X-ray photoelectron spectroscope. The MC3T3-E1 cell viability, attachment and morphology were confirmed using calcein AM and ethidium homodimer-1 staining, and analysis of gene expression using rat mesenchymal stem cells was performed using a real-time reverse-transcription polymerase chain reaction. The results indicated that both portable nitrogen- and air-based NTAPPJ could be used on TiO2 nanotube surfaces easily and without topographical disruption. NTAPPJ resulted in a significant increase in the hydrophilicity of the surfaces as well as changes in the surface chemistry, which consequently increased the cell viability, attachment and differentiation compared with the control samples. The nitrogen-based NTAPPJ treatment group exhibited a higher osteogenic gene expression level than the air-based NTAPPJ treatment group due to the lower atomic percentage of carbon on the surface that resulted from treatment. It was concluded that NTAPPJ treatment of TiO2 nanotube surfaces results in an increase in cellular activity. Furthermore, it was demonstrated that this treatment leads to improved osseointegration in vitro.

Mutations in the PLEKHG5 gene is relevant with autosomal recessive intermediate Charcot-Marie-Tooth disease.

BACKGROUND: Mutations in the Pleckstrin homology domain-containing, family G member 5 (PLEKHG5) gene has been reported in a family harboring an autosomal recessive lower motor neuron disease (LMND). However, the PLEKHG5 mutation has not been described to cause Charcot-Marie-Tooth disease (CMT). METHODS: To identify the causative mutation in an autosomal recessive intermediate CMT (RI-CMT) family with childhood onset, whole exome sequencing (WES), histopathology, and lower leg MRIs were performed. Expression and activity of each mutant protein were analyzed. RESULTS: We identified novel compound heterozygous (p.Thr663Met and p.Gly820Arg) mutations in the PLEKHG5 gene in the present family. The patient revealed clinical manifestations of sensory neuropathy. Fatty replacements in the distal lower leg muscles were more severe than in the thigh muscles. Although the symptoms and signs of this patient harboring slow nerve conduction velocities suggested the possibility of demyelinating neuropathy, a distal sural nerve biopsy was compatible with axonal neuropathy. Immunohistochemical analysis revealed that the patient has a low level of PLEKHG5 in the distal sural nerve and an in vitro assay suggested that the mutant proteins have a defect in activating the NF-κB signaling pathway. CONCLUSIONS: This study identifies compound heterozygous PLEKHG5 mutations as the cause of RI-CMT. We suggest that PLEKHG5 might play a role in the peripheral motor and sensory nervous system. This study expands the phenotypic spectrum of PLEKHG5 mutations.