Neobacillus paridis sp. nov., an endophyte of Paris polyphylla Smith var. yunnanensis.

A novel endophytic strain, designated YIM B02564T, was isolated from the root of Paris polyphylla Smith var. yunnanensis obtained from Yunnan Province, southwest China. By using a polyphasic approach, cells of the strain were characterized as facultative anaerobic, Gram-positive and rod-shaped. The growth conditions of the strain were found to occur at 20-55 °C (optimum, 30 °C), pH 6.0-9.0 (optimum, pH 7.0). Strain YIM B02564T can tolerate 2% NaCl concentration. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain YIM B02564T belonged to the genus Neobacillus and the 16S rRNA gene sequence similarity values of strain YIM B02564T to the type strains of members of this genus ranged from 95.6 to 97.8%. The DNA G+C content of strain YIM B02564T calculated from the whole genome sequence was 41.6 mol%. Values of the ANI and the dDDH between strain YIM B02564T and its closely related Neobacillus species were below 77.9% and 21.5%. Strain YIM B02564T contained MK-7 as the major menaquinone, iso-C15:0 and anteiso-C15:0 as the major fatty acids. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, an unidentified aminophospholipid and four unidentified lipids. It contained meso-diaminopimelic acid in the cell-wall peptidoglycan. On the basis of polyphasic analysis, strain YIM B02564T could be differentiated genotypically and phenotypically from recognized species of the genus Neobacillus. The isolate therefore represents a novel species, for which the name Neobacillus paridis is proposed. The type strain is YIM B02564T (= JCM 34668T = CGMCC 1.18655T).

Janibacter endophyticus sp. nov., an Endophytic Actinobacterium Isolated from the Root of Paris polyphylla Smith var. Yunnanensis.

A novel endophytic actinobacterium, designated as strain YIM B02568T, was isolated from the root of Paris polyphylla Smith var. Yunnanensis obtained from Yunnan Province, southwest China. Strain YIM B02568T was characterized using a polyphasic approach. Phylogenetic analysis indicated that this isolate belonged to the genus Janibacter. The 16S rRNA gene sequence similarity values of strain YIM B02568T to the type strains of members of this genus ranged from 95.8 to 98.6%. However, overall genome relatedness indices were significantly lower than the widely accepted species-defined threshold. The cell wall of strain YIM B02568T contained meso-diaminopimelic acid. The major menaquinone was MK-8(H4). The main polar lipids were phosphatidylglycerol, diphosphatidylglycerol, and phosphatidylinositol. The major cellular fatty acids were comprised of iso-C16:0 and C18:1 ω9c. The DNA G + C content was 71.6 mol%. Based on the data from the polyphasic studies, we propose that strain YIM B02568T represents a novel species within the genus Janibacter, Janibacter endophyticus sp. nov. The type strain is YIM B02568T (= JCM 34639T = CGMCC 1.18658T).

Chryseobacterium paridis sp. nov., an endophytic bacterial species isolated from the root of Paris polyphylla Smith var. yunnanensis.

A Gram-negative, yellow-pigmented, rod-shaped bacterial strain YIM B02567T was isolated from the root of Paris polyphylla Smith var. yunnanensis in China. Strain YIM B02567T grew optimally at 25-30 °C and at pH 7.0 in the absence of NaCl on nutrient agar. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain YIM B02567T belong to the genus Chryseobacterium, and was closely related to Chryseobacterium piperi CTMT and Chryseobacterium soli DSM 19298T. Whole genome sequencing indicated that the genome size was 4,774,612 bp and with a G + C content of 34.5 mol%. Values of the ANI and the dDDH between strain YIM B02567T and its closely related Chryseobacterium species were below 81.72% and 24.7%. Strain YIM B02567T contained menaquinone-6 as the sole isoprenoid quinone, anteiso-C15:0, iso-C17:1 ω9c and iso-C17:0 3-OH as major fatty acids and phosphatidylethanolamine as major polar lipid. Based on the polyphasic analyses, strain YIM B02567T could be differentiated genotypically and phenotypically from recognized species of the genus Chryseobacterium. The isolate, therefore, represents a novel species, for which the name Chryseobacterium paridis sp. nov. is proposed. The type strain is YIM B02567T (= CGMCC 1.18657T).

Paenibacillus paridis sp. nov., an endophytic bacterial species isolated from the root of Paris polyphylla Smith var. yunnanensis.

A Gram-reaction-positive, endospore-forming and rod-shaped bacterial strain, designated py1325T, was isolated from the root of Paris polyphylla Smith var. yunnanensis collected from Yunnan Province, PR China, and subjected to a polyphasic taxonomic characterization. It grew optimally with 0-1 % NaCl (w/v), at pH 7 and at 30 °C. The major respiratory quinone was MK-7 and the diagnostic diamino acid in the cell-wall peptidoglycan was meso-diaminopimelic acid. The major cellular fatty acid was anteiso-C15 : 0. The polar lipids consisted of phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, two unidentified aminophospholipids, two unidentified phospholipids and two unidentified lipids. The results of 16S rRNA gene sequence analysis revealed the highest levels of sequence similarity with respect to Paenibacillus luteus R-3T (99.0 %), Paenibacillus sinopodophylli CCTCC AB 2016047T (97.9 %), Paenibacillus castaneae DSM 19417T (97.5 %) and Paenibacillus endophyticus LMG 27297T (97.2 %). The digital DNA-DNA hybridization and average nucleotide identity values between py1325T and these species ranged 20.6-53.3 % and 79.9-93.6 %. The G+C content of the genomic DNA was 47.7 mol%. According to the phylogenetic, phenotypic and chemotaxonomic evidence, strain py1325T clearly represents a novel species of the genus Paenibacillus, for which the name Paenibacillus paridis sp. nov. is proposed. The type strain is py1325T (=CCTCC AB 2015220T=LMG 29068T).

Expression and Clinical Significance of ILF2 in Gastric Cancer.

The aim of this study is to investigate the expression levels and clinical significance of ILF2 in gastric cancer. The mRNA and protein expression levels of ILF2 were, respectively, examined by quantitative real-time PCR (qRT-PCR) and Western blot from 21 paired fresh frozen GC tissues and corresponding normal gastric tissues. In order to analyze the expression pattern of ILF2 in GC, 60 paired paraffin-embedded GC slides and corresponding normal gastric slides were detected by immunohistochemistry (IHC) assay. The correlation between ILF2 protein expression levels and clinicopathological parameters, overall survival (OS), disease-free survival (DFS), and clinical prognosis were analyzed by statistical methods. Significantly higher levels of ILF2 were detected in GC tissues compared with normal controls at both mRNA and protein level. High expression of ILF2 was tightly correlated with depth of invasion, lymph node metastasis, pathological stage, and histological differentiation. Log-rank test showed that high expression of ILF2 was positively associated with poor clinical prognosis. Multivariate analysis identified that ILF2 was an independent prognostic factor for OS and DFS. Our findings suggest that ILF2 may be a valuable biomarker and a novel potential prognosis predictor for GC patients.

Changes of ribbon synapses number of cochlear hair cells in C57BL/6J mice with age(Δ).

To investigate the changes of ribbon synapses (RS) number in cochlear hair cells in C57BL/6J mice with age. Basilar membranes within the cochlea of C57BL/6J mice aged 2, 6, 10 and 12 months were harvested (5 mice in each age group). The presynaptic and postsynaptic membranes were subject to double immunohistochemical staining and observed with a laser confocal microscope. The number of RS in each segment of basilar membrane was counted by using 3D reconstruction technique. Compared with 2-month-old mice, reduction of RS number in basilar membrane inside cochlea mainly occurred to the basal turn among C57BL/6J mice aged 6 months. The number of RS in each turn among 10-month-old mice decreased considerably, and such decrease continued in the top turn and middle turn in mice aged 12 months. In contrast, the number of RS in the basal turn increased slightly. Reduction of RS probably takes place in the early stage of C57BL/6J mice presbycusis. Early prevention of presbycusis can be achieved through measures to mitigate the reduction of RS.

Wnt1 from cochlear schwann cells enhances neuronal differentiation of transplanted neural stem cells in a rat spiral ganglion neuron degeneration model.

Although neural stem cell (NSC) transplantation is widely expected to become a therapy for nervous system degenerative diseases and injuries, the low neuronal differentiation rate of NSCs transplanted into the inner ear is a major obstacle for the successful treatment of spiral ganglion neuron (SGN) degeneration. In this study, we validated whether the local microenvironment influences the neuronal differentiation of transplanted NSCs in the inner ear. Using a rat SGN degeneration model, we demonstrated that transplanted NSCs were more likely to differentiate into microtubule-associated protein 2 (MAP2)-positive neurons in SGN-degenerated cochleae than in control cochleae. Using real-time quantitative PCR and an immunofluorescence assay, we also proved that the expression of Wnt1 (a ligand of Wnt signaling) increases significantly in Schwann cells in the SGN-degenerated cochlea. We further verified that NSC cultures express receptors and signaling components for Wnts. Based on these expression patterns, we hypothesized that Schwann cell-derived Wnt1 and Wnt signaling might be involved in the regulation of the neuronal differentiation of transplanted NSCs. We verified our hypothesis in vitro using a coculture system. We transduced a lentiviral vector expressing Wnt1 into cochlear Schwann cell cultures and cocultured them with NSC cultures. The coculture with Wnt1-expressing Schwann cells resulted in a significant increase in the percentage of NSCs that differentiated into MAP2-positive neurons, whereas this differentiation-enhancing effect was prevented by Dkk1 (an inhibitor of the Wnt signaling pathway). These results suggested that Wnt1 derived from cochlear Schwann cells enhanced the neuronal differentiation of transplanted NSCs through Wnt signaling pathway activation. Alterations of the microenvironment deserve detailed investigation because they may help us to conceive effective strategies to overcome the barrier of the low differentiation rate of transplanted NSCs.

Acoustical stimulus changes the expression of stromal cell-derived factor-1 in the spiral ganglion neurons of the rat cochlea.

Neural stem cell (NSC) transplantation into the cochlea has been tested as a treatment for spiral ganglion neuron (SGN) degenerative disease and injury in various animal models. A recent study has shown evidence of functional recovery after transplantation of the stem cells into a degenerated-SGN model. Chemokine stromal cell-derived factor-1 (SDF-1, or known as CXC chemokine ligand-12, CXCL-12) signaling through CXCR4 has previously been identified as a key step in the homing of the stem cells within the injury areas; meanwhile, studies have revealed that the SDF-1/CXCR4 axis is also involved in axon guidance and pathfinding. A study found that transplanted neural precursor cells can migrate to the root of the auditory nerve when animals are subjected to an augmented acoustic environment (AAE). In accordance with these studies, we hypothesize that AAE will up-regulate the expression of SDF-1 in acoustic nerves. We tested our hypothesis by examining the expression of SDF-1 in different acoustic environments, and the results were confirmed by the auditory brainstem response (ABR), immunohistochemical and RT-PCR analyses. The results showed that SDF-1 was expressed at a relatively low level in the SGNs under normal animal unit acoustic conditions (40-50 dB). Moreover, it was significantly up-regulated in the SGNs under the 75 dB (augmented physiological process without hearing loss) and 90 dB AAE (pathological process with light hearing loss) conditions; however, under the 115 dB AAE (pathological process with severe hearing loss) condition, the expression of SDF-1 was not up-regulated. The results confirmed that appropriately augmented acoustical stimuli lead to the up-regulation of SDF-1, which may assist in the migration of the transplanted cells and the subsequent establishment of essential synaptic contacts between the exogenous cells and the host auditory pathway.

Stem cell transplantation via the cochlear lateral wall for replacement of degenerated spiral ganglion neurons.

Spiral ganglion neurons (SGNs) are poorly regenerated in the mammalian inner ear. Because of this, stem cell transplantation has been used to replace injured SGNs, and several studies have addressed this approach. However, the difficulty of delivering stem cells into the cochlea and encouraging their migration to Rosenthal's canal (RC), where the SGNs are located, severely restricts this therapeutic strategy. In this study, we attempted to establish a new stem cell transplantation route into the cochlea via the cochlear lateral wall (CLW). First, we tested the precision of this route by injecting Fluorogold into the CLW and next assessed its safety by mock surgeries. Then, using a degenerated SGN animal model, we transplanted neural stem cells (NSCs), derived from the olfactory bulb of C57BL/6-green fluorescent protein (GFP) mice, via the CLW route and examined the cells' distribution in the cochlea. We found the CLW transplantation route is precise and safe. In addition, NSCs migrated into RC with a high efficiency and differentiated into neurons in a degenerated SGN rat model after the CLW transplantation. This result revealed that the basilar membrane (BM) may have crevices permitting the migration of NSCs. The result of this study demonstrates a novel route for cell transplantation to the inner ear, which is important for the replacement of degenerated SGNs and may contribute to the treatment of sensorineural hearing loss.

Up-regulation of stromal cell-derived factor-1 enhances migration of transplanted neural stem cells to injury region following degeneration of spiral ganglion neurons in the adult rat inner ear.

Neural stem cell (NSC) transplantation into the cochlea is widely used for the treatment of spiral ganglion neuron (SGN) degenerative disease and injury in the animal models, but the migration of the transplanted NSCs to the injury region is difficult and the mechanism is still unclear. In this study, we aimed to validate whether the SGN-degenerated cochlear microenvironment plays a role in the NSC migration and investigated whether stromal cell-derived factor-1 (SDF-1) was involved in the NSCs migration. Using a rat SGN degeneration model, we demonstrated that the transplanted NSCs are more likely to migrate to the injury region during the early post-injury (EPI) than the late post-injury (LPI) stage and the control cochlea. We found that the expressions of SDF-1 increased transiently after SGN degeneration. Additionally, we showed that the NSCs express CXCR4, a receptor for SDF-1. We observed that the region to which the transplanted NSC localized coincides with the region where the SDF-1 is highly expressed following the degeneration of SGNs. Finally, we observed that the increased SDF-1 is derived from the Schwann cells in the SGN-degenerated model. These results suggest that SDF-1, which is derived from cochlear Schwann cells and up-regulated in the early injury microenvironment, plays a beneficial role in the NSC migration to the injury region. Optimizing SDF-1 expression in the host microenvironment or increasing the CXCR4 expression of the donor stem cells may improve the migration efficiency of transplanted cells toward the injury region in the cochlea.