Macrophage transcription factor TonEBP promotes systemic lupus erythematosus and kidney injury via damage-induced signaling pathways.

Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by autoreactive B cells and dysregulation of many other types of immune cells including myeloid cells. Lupus nephritis (LN) is a common target organ manifestations of SLE. Tonicity-responsive enhancer-binding protein (TonEBP, also known as nuclear factor of activated T-cells 5 (NFAT5)), was initially identified as a central regulator of cellular responses to hypertonic stress and is a pleiotropic stress protein involved in a variety of immunometabolic diseases. To explore the role of TonEBP, we examined kidney biopsy samples from patients with LN. Kidney TonEBP expression was found to be elevated in these patients compared to control patients - in both kidney cells and infiltrating immune cells. Kidney TonEBP mRNA was elevated in LN and correlated with mRNAs encoding inflammatory cytokines and the degree of proteinuria. In a pristane-induced SLE model in mice, myeloid TonEBP deficiency blocked the development of SLE and LN. In macrophages, engagement of various toll-like receptors (TLRs) that respond to damage-associated molecular patterns induced TonEBP expression via stimulation of its promoter. Intracellular signaling downstream of the TLRs was dependent on TonEBP. Therefore, TonEBP can act as a transcriptional cofactor for NF-κB, and activated mTOR-IRF3/7 via protein-protein interactions. Additionally, TonEBP-deficient macrophages displayed elevated efferocytosis and animals with myeloid deficiency of TonEBP showed reduced Th1 and Th17 differentiation, consistent with macrophages defective in TLR signaling. Thus, our data show that myeloid TonEBP may be an attractive therapeutic target for SLE and LN.

TonEBP recognizes R-loops and initiates m6A RNA methylation for R-loop resolution.

R-loops are three-stranded, RNA-DNA hybrid, nucleic acid structures produced due to inappropriate processing of newly transcribed RNA or transcription-replication collision (TRC). Although R-loops are important for many cellular processes, their accumulation causes genomic instability and malignant diseases, so these structures are tightly regulated. It was recently reported that R-loop accumulation is resolved by methyltransferase-like 3 (METTL3)-mediated m6A RNA methylation under physiological conditions. However, it remains unclear how R-loops in the genome are recognized and induce resolution signals. Here, we demonstrate that tonicity-responsive enhancer binding protein (TonEBP) recognizes R-loops generated by DNA damaging agents such as ultraviolet (UV) or camptothecin (CPT). Single-molecule imaging and biochemical assays reveal that TonEBP preferentially binds a R-loop via both 3D collision and 1D diffusion along DNA in vitro. In addition, we find that TonEBP recruits METTL3 to R-loops through the Rel homology domain (RHD) for m6A RNA methylation. We also show that TonEBP recruits RNaseH1 to R-loops through a METTL3 interaction. Consistent with this, TonEBP or METTL3 depletion increases R-loops and reduces cell survival in the presence of UV or CPT. Collectively, our results reveal an R-loop resolution pathway by TonEBP and m6A RNA methylation by METTL3 and provide new insights into R-loop resolution processes.

APE1/Ref-1 Inhibits Adipogenic Transcription Factors during Adipocyte Differentiation in 3T3-L1 Cells.

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a multifunctional protein involved in DNA repair and redox regulation. The redox activity of APE1/Ref-1 is involved in inflammatory responses and regulation of DNA binding of transcription factors related to cell survival pathways. However, the effect of APE1/Ref-1 on adipogenic transcription factor regulation remains unknown. In this study, we investigated the effect of APE1/Ref-1 on the regulation of adipocyte differentiation in 3T3-L1 cells. During adipocyte differentiation, APE1/Ref-1 expression significantly decreased with the increased expression of adipogenic transcription factors such as CCAAT/enhancer binding protein (C/EBP)-α and peroxisome proliferator-activated receptor (PPAR)-γ, and the adipocyte differentiation marker adipocyte protein 2 (aP2) in a time-dependent manner. However, APE1/Ref-1 overexpression inhibited C/EBP-α, PPAR-γ, and aP2 expression, which was upregulated during adipocyte differentiation. In contrast, silencing APE1/Ref-1 or redox inhibition of APE1/Ref-1 using E3330 increased the mRNA and protein levels of C/EBP-α, PPAR-γ, and aP2 during adipocyte differentiation. These results suggest that APE1/Ref-1 inhibits adipocyte differentiation by regulating adipogenic transcription factors, suggesting that APE1/Ref-1 is a potential therapeutic target for regulating adipocyte differentiation.

Modified Industrial Three-Dimensional Polylactic Acid Scaffold Cell Chip Promotes the Proliferation and Differentiation of Human Neural Stem Cells.

In this study, we fabricated a three-dimensional (3D) scaffold using industrial polylactic acid (PLA), which promoted the proliferation and differentiation of human neural stem cells. An industrial PLA 3D scaffold (IPTS) cell chip with a square-shaped pattern was fabricated via computer-aided design and printed using a fused deposition modeling technique. To improve cell adhesion and cell differentiation, we coated the IPTS cell chip with gold nanoparticles (Au-NPs), nerve growth factor (NGF) protein, an NGF peptide fragment, and sonic hedgehog (SHH) protein. The proliferation of F3.Olig2 neural stem cells was increased in the IPTS cell chips coated with Au-NPs and NGF peptide fragments when compared with that of the cells cultured on non-coated IPTS cell chips. Cells cultured on the IPTS-SHH cell chip also showed high expression of motor neuron cell-specific markers, such as HB9 and TUJ-1. Therefore, we suggest that the newly engineered industrial PLA scaffold is an innovative tool for cell proliferation and motor neuron differentiation.

Diffraction efficiency enhancement and optimization in full-color HOE using the inhibition characteristics of the photopolymer.

A novel and effective simultaneous recording method, to the best of our knowledge, is proposed for improving the diffraction efficiency and uniformity of full-color holographic optical elements (HOE) using the Bayfol HX102 photopolymer. To improve the diffraction efficiency of a full-color HOE, it is important to find the optimal recording beam intensity taking into account the initial and late responses of the medium. The range of optimal beam intensity for recording full-color HOE can be found experimentally by analyzing the inhibition period and response characteristics of the recording medium for three wavelengths. Through this method, a full-color HOE with an average diffraction efficiency of about 56.81% and a standard deviation of about 1.7% was implemented in a single layer photopolymer.

Three-dimensional see-through augmented-reality display system using a holographic micromirror array.

It is difficult to find the micromirror array with desired specifications for augmented-reality displays, and the custom fabricating methods are complicated and unstable. We propose a novel, to our knowledge, three-dimensional see-through augmented-reality display system using the holographic micromirror array. Unlike the conventional holographic waveguide-type augmented-reality displays, the proposed system utilizes the holographic micromirror array as an in-coupler, without any additional elements. The holographic micromirror array is fabricated through the simple, effective, and stable method of applying the total internal reflection-based hologram recording using a dual-prism. The optical mirror and microlens array are set as references, and the specifications can be customized. It reconstructs a three-dimensional image from a displayed elemental image set without using any additional device, and the user can observe a three-dimensional virtual image while viewing the real-world objects. Thus, the principal advantages of the existing holographic waveguide-type augmented-reality system are retained. An optical experiment confirmed that the proposed system displays three-dimensional images exploiting the augmented-reality system simply and effectively.

Drug Evaluation Based on a Multi-Channel Cell Chip with a Horizontal Co-Culture.

We developed a multi-channel cell chip containing a three-dimensional (3D) scaffold for horizontal co-culture and drug toxicity screening in multi-organ culture (human glioblastoma, cervical cancer, normal liver cells, and normal lung cells). The polydimethylsiloxane (PDMS) multi-channel cell chip (PMCCC) was based on fused deposition modeling (FDM) technology. The architecture of the PMCCC was an open-type cell chip and did not require a pump or syringe. We investigated cell proliferation and cytotoxicity by conducting 3-(4,5-dimethylthiazol-2-yl)-2,5-dphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays and analysis of oleanolic acid (OA)-treated multi-channel cell chips. The results of the MTT and LDH assays showed that OA treatment in the multi-channel cell chip of four cell lines enhanced chemoresistance of cells compared with that in the 2D culture. Furthermore, we demonstrated the feasibility of the application of our multi-channel cell chip in various analysis methods through Annexin V-fluorescein isothiocyanate/propidium iodide staining, which is not used for conventional cell chips. Taken together, the results demonstrated that the PMCCC may be used as a new 3D platform because it enables simultaneous drug screening in multiple cells by single point injection and allows analysis of various biological processes.

Integration of air quality model with GIS for the monitoring of PM2.5 from local primary emission at a rural site.

Local primary emissions of air pollutants are responsible for public health, decreasing productivity, and cultural activities in local residential areas. In this study, an integrated air quality observation and modeling system with a geographical information system (GIS) was developed to characterize the air pollution caused by local primary emission sources. This integrated system could provide air quality monitoring, data analysis, and visualization results that reflect air pollutant concentration data in a study area containing a local rural village (LRV) and an asphalt manufacturing facility (AMF). Additionally, the model was used to estimate the contributions of air quality from an emission source at the receptor and determine the control factor for the emission rate or meteorological changes. From the forward and backward modeling results, we found that the concentrations of particulate matter smaller than 2.5 µm (PM2.5) concentrations in the village were affected by the unique meteorological and emission conditions. The PM2.5 concentration was significantly increased for the cases with a slow wind speed of 1 m/s or high wind speed of 3 m/s, with an emission rate of 10 g/s. The contribution of AMF emissions was explained by contribution factor analysis. During the study period of December 2014-December 2015, the incoming contribution of PM2.5 at the LRV measurement station was approximately 47.6%. These results suggest that the proposed method can be useful for understanding adverse air quality conditions and estimating the emissions of air pollutants from primary sources for local environmental and public health authorities.

Microglial TonEBP mediates LPS-induced inflammation and memory loss as transcriptional cofactor for NF-κB and AP-1.

BACKGROUND: Microglia are brain-resident myeloid cells involved in the innate immune response and a variety of neurodegenerative diseases. In macrophages, TonEBP is a transcriptional cofactor of NF-κB which stimulates the transcription of pro-inflammatory genes in response to LPS. Here, we examined the role of microglial TonEBP. METHODS: We used microglial cell line, BV2 cells. TonEBP was knocked down using lentiviral transduction of shRNA. In animals, TonEBP was deleted from myeloid cells using a line of mouse with floxed TonEBP. Cerulenin was used to block the NF-κB cofactor function of TonEBP. RESULTS: TonEBP deficiency blocked the LPS-induced expression of pro-inflammatory cytokines and enzymes in association with decreased activity of NF-κB in BV2 cells. We found that there was also a decreased activity of AP-1 and that TonEBP was a transcriptional cofactor of AP-1 as well as NF-κB. Interestingly, we found that myeloid-specific TonEBP deletion blocked the LPS-induced microglia activation and subsequent neuronal cell death and memory loss. Cerulenin disrupted the assembly of the TonEBP/NF-κB/AP-1/p300 complex and suppressed the LPS-induced microglial activation and the neuronal damages in animals. CONCLUSIONS: TonEBP is a key mediator of microglial activation and neuroinflammation relevant to neuronal damage. Cerulenin is an effective blocker of the TonEBP actions.

Identification of pre-leukaemic haematopoietic stem cells in acute leukaemia.

In acute myeloid leukaemia (AML), the cell of origin, nature and biological consequences of initiating lesions, and order of subsequent mutations remain poorly understood, as AML is typically diagnosed without observation of a pre-leukaemic phase. Here, highly purified haematopoietic stem cells (HSCs), progenitor and mature cell fractions from the blood of AML patients were found to contain recurrent DNMT3A mutations (DNMT3A(mut)) at high allele frequency, but without coincident NPM1 mutations (NPM1c) present in AML blasts. DNMT3A(mut)-bearing HSCs showed a multilineage repopulation advantage over non-mutated HSCs in xenografts, establishing their identity as pre-leukaemic HSCs. Pre-leukaemic HSCs were found in remission samples, indicating that they survive chemotherapy. Therefore DNMT3A(mut) arises early in AML evolution, probably in HSCs, leading to a clonally expanded pool of pre-leukaemic HSCs from which AML evolves. Our findings provide a paradigm for the detection and treatment of pre-leukaemic clones before the acquisition of additional genetic lesions engenders greater therapeutic resistance.

Tonicity-responsive enhancer-binding protein promotes hepatocellular carcinogenesis, recurrence and metastasis.

OBJECTIVES: Hepatocellular carcinoma (HCC) is a common cancer with high rate of recurrence and mortality. Diverse aetiological agents and wide heterogeneity in individual tumours impede effective and personalised treatment. Tonicity-responsive enhancer-binding protein (TonEBP) is a transcriptional cofactor for the expression of proinflammatory genes. Although inflammation is intimately associated with the pathogenesis of HCC, the role of TonEBP is unknown. We aimed to identify function of TonEBP in HCC. DESIGN: Tumours with surrounding hepatic tissues were obtained from 296 patients with HCC who received completion resection. TonEBP expression was analysed by quantitative reverse transcription-quantitative real-time PCR (RT-PCR) and immunohfistochemical analyses of tissue microarrays. Mice with TonEBP haplodeficiency, and hepatocyte-specific and myeloid-specific TonEBP deletion were used along with HCC and hepatocyte cell lines. RESULTS: TonEBP expression is higher in tumours than in adjacent non-tumour tissues in 92.6% of patients with HCC regardless of aetiology associated. The TonEBP expression in tumours and adjacent non-tumour tissues predicts recurrence, metastasis and death in multivariate analyses. TonEBP drives the expression of cyclo-oxygenase-2 (COX-2) by stimulating the promoter. In mouse models of HCC, three common sites of TonEBP action in response to diverse aetiological agents leading to tumourigenesis and tumour growth were found: cell injury and inflammation, induction by oxidative stress and stimulation of the COX-2 promoter. CONCLUSIONS: TonEBP is a key component of the common pathway in tumourigenesis and tumour progression of HCC in response to diverse aetiological insults. TonEBP is involved in multiple steps along the pathway, rendering it an attractive therapeutic target as well as a prognostic biomarker.

Tonicity-Responsive Enhancer-Binding Protein Mediates Hyperglycemia-Induced Inflammation and Vascular and Renal Injury.

Diabetic nephropathy (DN) has become the single leading cause of ESRD in developed nations. Bearing in mind the paucity of effective treatment for DN and progressive CKD, novel targets for treatment are sorely needed. We previously reported that increased activity of tonicity-responsive enhancer-binding protein (TonEBP) in monocytes was associated with early DN in humans. We now extend these findings by testing the hypotheses that TonEBP in macrophages promotes hyperglycemia-mediated proinflammatory activation and chronic renal inflammation leading to DN and CKD, and TonEBP genetic variability in humans is associated with inflammatory, renal, and vascular function-related phenotypes. In a mouse model of DN, compared with the wild-type phenotype, TonEBP haplodeficiency associated with reduced activation of macrophages by hyperglycemia, fewer macrophages in the kidney, lower renal expression of proinflammatory genes, and attenuated DN. Furthermore, in a cohort of healthy humans, genetic variants within TonEBP associated with renal function, BP, and systemic inflammation. One of the genetic variants associated with renal function was replicated in a large population-based cohort. These findings suggest that TonEBP is a promising target for minimizing diabetes- and stress-induced inflammation and renovascular injury.

Image of the Micro-Computed Tomography and Atomic-Force Microscopy of Bone in Osteoporosis Animal Model.

Osteoporosis, a major public health problem, is characterised by a reduction in and deterioration of bone tissue microarchitecture, with a consequent increase in bone frailty and susceptibility to fractures. We performed ex vivo micro-computed tomography (micro-CT) and atomic-force microscopy (AFM) between normal and surgically ovariectomized animals (OVX). The tibial architecture was investigated using maximum intensity projection images. Micro-CT analysis of the tibia metaphysis was used to calculate the trabecular spacing (Tb.Sp). Following OVX surgery, the rats exhibited increased Tb.Sp. Between the bundles, there are holes in the fabric with a diameter in the range of 3 to 5 µm in normal tibia. The diameter of hole in the osteoporosis tibia is the range of 5 to 10 µm, and its area increased than normal tibia. AFM can be used for the inspection of bone surfaces and offers the possibility of quantification of local geometries, such as pit size, collagen fibril diameters, or collagen ultrastructure. Our results suggest that AFM will surely become one of the most important tools in the realm of medical research for imaging such as bone and tissues.

Removal of anionic dyes (Reactive Black 5 and Congo Red) from aqueous solutions using Banana Peel Powder as an adsorbent.

The adsorption characteristics of Reactive Black 5 (RB5) and Cong Red (CR) onto Banana Peel Powder (BPP) from aqueous solution were investigated as a function of pH, contact time, initial dye concentration and temperature. The BPP was characterized by Fourier Transform Infrared Spectroscopy (FTIR) and Scanning Electron Microscopy (SEM) analysis. FTIR results revealed that hydroxyl (-OH), amine (-NH) and carboxyl (-CË­O) functional groups present on the surface of BPP. The SEM results show that BPP has an irregular and porous surface morphology which is adequate for dye adsorption. The equilibrium data were analyzed using Langmuir and Freundlich isotherm models. Experimental results were best represented by the Langmuir isotherm model. The adjustments of models were confirmed by the Chi-square (χ2) test and the correlation coefficients (R2). The maximum monolayer adsorption capacities of RB5 and CR on BPP calculated from Langmuir isotherm model were 49.2 and 164.6mg/g at pH 3.0 and 298K. Experimental data were also tested in terms of adsorption kinetics using pseudo-first-order and pseudo-second-order kinetic models. The results showed that the adsorption processes of both RB5 and CR followed well pseudo-second-order kinetic models. The calculated thermodynamic parameters ΔG°, ΔH° and ΔS° showed that the adsorption of RB5 and CR onto BPP was feasible, spontaneous and endothermic in the temperature range 298-318K. The RB5 and CR were desorbed from BPP using 0.1M NaOH. The recovery for both anionic dyes was found to be higher than 90%. Based on these it can be concluded that BPP can be used as an effective, low cost, and eco-friendly adsorbent for CR removal than RB5 from aqueous solution.

Mitigation Effects of a Novel Herbal Medicine, Hepad, on Neuroinflammation, Neuroapoptosis, and Neuro-Oxidation.

Parkinson's disease (PD), a common adult-onset neurodegenerative disorder with complex pathological mechanisms, is characterized by the degeneration of dopaminergic nigrostriatal neurons. The present study demonstrated that the herbal medicines Hepad 1 and 2 protected against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neurotoxicity in C57BL/6 mice and SH-SY5Y cells. Hepad 1 and 2 remarkably alleviated the enhanced expression of pro-inflammatory cytokines (tumor necrosis factor-α, interleukin-6, inducible nitric oxide synthase, cyclooxygenase-2, macrophage-1, and phosphorylated iκB-α) and apoptotic signals (Bcl-2-associated X protein, caspase-3, and poly [ADP-ribose] polymerase-1). Additionally, Hepad reduced MPTP-induced oxidative damage by increasing the expression of anti-oxidant defense enzymes (superoxide dismutase and glutathione S-transferase) and downregulating the levels of nicotinamide adenine dinucleotide phosphate oxidase 4. This study also showed that the neuroprotective effects of Hepad include anti-inflammatory, anti-apoptotic, and anti-oxidative properties, in addition to activation of the protein kinase B, extracellular-signal-regulated kinase, and c-Jun N-terminal kinase signaling pathways. Furthermore, oral administration of Hepad 1 and 2 attenuated the death of tyrosine hydroxylase-positive substantia nigra neurons that was induced by 20 mg/kg MPTP. Therefore, our results suggest that Hepad 1 and 2 are useful for treating PD and other disorders associated with neuro-inflammatory, neuro-apoptotic, and neuro-oxidative damage.

Synergetic analysis of springtime air pollution episodes over Gwangju, Korea.

The characteristics of springtime aerosols, including their optical and microphysical properties, were analyzed for the months of March to May of 2009 in Gwangju (35.23°N, 126.84°E), Korea. A high Light Detection and Ranging (LIDAR)-derived aerosol depolarization ratio (δ) of 0.25±0.04 was determined on dust particles during the observation period. The Ångström exponent values of the 440-870nm wavelength pair (Å440-870) and single-scattering albedo at 675nm (Ω675) measured by a CIMEL sun/sky radiometer were 0.77±0.19 and 0.95±0.01, respectively. The elevated dust layers reached a maximum elevation of 4km above sea level. Anthropogenic/smoke particles that originated from highly populated/industrialized regions could be distinguished by their relatively smaller particle size (Å440-870 ranged between 1.33 and 1.36) and higher light-absorbing (Ω675 of 0.92±0.01) characteristics. These aerosols are mostly distributed at altitudes <1.2km. The root-mean-square deviation (RMSD) between the aerosol optical depth (AOD, τ) derived from LIDAR (τLIDAR) and from the CIMEL sun/sky radiometer (τCIMEL) varied with respect to the surface PM10 concentration. The RMSD between τLIDAR and τCIMEL was as low as 13% under lower PM10 concentration levels (<100µg/m3). In contrast, the RMSD between τLIDAR and τCIMEL increased three times (~31%) under high surface PM10 concentration levels (>100µg/m3). These results suggest that the accuracy of τLIDAR is influenced by specific atmospheric conditions, regardless of its uncertainty.

SP, CGRP changes in pyridoxine induced neuropathic dogs with nerve growth factor gene therapy.

BACKGROUND: Nerve growth factor (NGF) is known not only as a major factor for neuronal plasticity but also as a pain stimulator. Although there have been several trials with NGF for its application in the regeneration or protection of the nervous system, the pain induced by NGF remains a challenge to be overcome. In this study, the pain induced by NGF gene therapy was evaluated. RESULTS: Vehicle or recombinant dog NGF plasmid was administered into the intrathecal space of dogs. Twenty-four hours after the vehicle or NGF plasmid inoculation, dogs were subcutaneously treated with 150 mg/kg pyridoxine every day for 7 days. For pain assessment, physical examination and electrophysiological recording were performed. Only in the vehicle-treated group, weight loss occurred, while NGF plasmid inoculation significantly improved this physical abnormalities. In the vehicle-treated group, electrophysiological recordings showed that H-reflex disappeared at 24 h after the last pyridoxine treatment. However, in the NGF plasmid inoculated group, the H-reflex were normal. In the results of immunohistochemistry, the NGF plasmid administration efficiently expressed in the dorsal root ganglia and significantly increased the pyridoxine-induced reduction of calcitonin gene-related peptide (CGRP) immunoreactive neurons, but not in substance P immunoreactive neurons, in the dorsal root ganglia. CONCLUSIONS: Given these results, we reason that NGF gene therapy in pyridoxine induced neuropathic dogs does not induce neuropathic pain with this dosage, even with increasing the expression of CGRP.

Essential oils from two Allium species exert effects on cell proliferation and neuroblast differentiation in the mouse dentate gyrus by modulating brain-derived neurotrophic factor and acetylcholinesterase.

BACKGROUND: In the present study, we investigated the effects of oil products from two Allium species: Allium sativum (garlic) and Allium hookeri (Chinese chives) on cell proliferation and neuroblast differentiation in the mouse dentate gyrus. METHODS: Using corn oil as a vehicle, the essential oil from garlic (10 ml/kg), or Chinese chives (10 ml/kg) was administered orally to 9-week-old mice once a day for 3 weeks. One hour following the last treatment, a novel object recognition test was conducted and the animals were killed 2 h after the test. RESULTS: In comparison to the vehicle-treated group, garlic essential oil (GO) treatment resulted in significantly increased exploration time and discrimination index during the novel object recognition test, while Chinese chives essential oil (CO) reduced the exploration time and discrimination index in the same test. In addition, the number of Ki67-immunoreactive proliferating cells and doublecortin-immunoreactive neuroblasts significantly increased in the dentate gyrus of GO-treated animals. However, administration of CO significantly decreased cell proliferation and neuroblast differentiation. Administration of GO significantly increased brain-derived neurotrophic factor (BDNF) levels and decreased acetylcholinesterase (AChE) activity in the hippocampal homogenates. In contrast, administration of CO decreased BDNF protein levels and had no significant effect on AChE activity, compared to that in the vehicle-treated group. CONCLUSIONS: These results suggest that GO significantly improves novel object recognition as well as increases cell proliferation and neuroblast differentiation, by modulating hippocampal BDNF protein levels and AChE activity, while CO impairs novel object recognition and decreases cell proliferation and neuroblast differentiation, by reducing BDNF protein levels in the hippocampus.

Effects of Extracellular Matrix Protein-derived Signaling on the Maintenance of the Undifferentiated State of Spermatogonial Stem Cells from Porcine Neonatal Testis.

In general, the seminiferous tubule basement membrane (STBM), comprising laminin, collagen IV, perlecan, and entactin, plays an important role in self-renewal and spermatogenesis of spermatogonial stem cells (SSCs) in the testis. However, among the diverse extracellular matrix (ECM) proteins constituting the STBM, the mechanism by which each regulates SSC fate has yet to be revealed. Accordingly, we investigated the effects of various ECM proteins on the maintenance of the undifferentiated state of SSCs in pigs. First, an extracellular signaling-free culture system was optimized, and alkaline phosphatase (AP) activity and transcriptional regulation of SSC-specific genes were analyzed in porcine SSCs (pSSCs) cultured for 1, 3, and 5 days on non-, laminin- and collagen IV-coated Petri dishes in the optimized culture system. The microenvironment consisting of glial cell-derived neurotrophic factor (GDNF)-supplemented mouse embryonic stem cell culture medium (mESCCM) (GDNF-mESCCM) demonstrated the highest efficiency in the maintenance of AP activity. Moreover, under the established extracellular signaling-free microenvironment, effective maintenance of AP activity and SSC-specific gene expression was detected in pSSCs experiencing laminin-derived signaling. From these results, we believe that laminin can serve as an extracellular niche factor required for the in vitro maintenance of undifferentiated pSSCs in the establishment of the pSSC culture system.

Full-color holographic diffuser using time-scheduled iterative exposure.

A compact wavelength multiplexing technique is proposed and experimentally investigated to improve the efficiency of a full-color holographic diffuser using photopolymer. The exposure responses of a monochromatic hologram and a three wavelength multiplexed hologram recorded in photopolymer film are presented. The time-scheduled exposure energies at wavelengths of 633, 532, and 473 nm were chosen to optimize the uniform diffraction efficiency of the wavelength multiplexed hologram. These three wavelength iterative sequences of exposures are applied to achieve a specific color balance for a multicolor holographic diffuser. The experimental results confirm that the fabrication method is well suited to the manufacture of holographic diffusers for full-color display applications.