Human amniotic epithelial stem cell is a cell therapy candidate for preventing acute graft-versus-host disease.

Graft-versus-host disease (GVHD), an immunological disorder that arises from donor T cell activation through recognition of host alloantigens, is the major limitation in the application of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Traditional immunosuppressive agents can relieve GVHD, but they induce serious side effects. It is highly required to explore alternative therapeutic strategy. Human amniotic epithelial stem cells (hAESCs) were recently considered as an ideal source for cell therapy with special immune regulatory property. In this study, we evaluated the therapeutic role of hAESCs in the treatment of GVHD, based on our previous developed cGMP-grade hAESCs product. Humanized mouse model of acute GVHD (aGVHD) was established by injection of huPBMCs via the tail vein. For prevention or treatment of aGVHD, hAESCs were injected to the mice on day -1 or on day 7 post-PBMC infusion, respectively. We showed that hAESCs infusion significantly alleviated the disease phenotype, increased the survival rate of aGVHD mice, and ameliorated pathological injuries in aGVHD target organs. We demonstrated that hAESCs directly induced CD4+ T cell polarization, in which Th1 and Th17 subsets were downregulated, and Treg subset was elevated. Correspondingly, the levels of a series of pro-inflammatory cytokines were reduced while the levels of the anti-inflammatory cytokines were upregulated in the presence of hAESCs. We found that hAESCs regulated CD4+ subset polarization in a paracrine mode, in which TGFß and PGE2 were selectively secreted to mediate Treg elevation and Th1/Th17 inhibition, respectively. In addition, transplanted hAESCs preserved the graft-versus-leukemia (GVL) effect by inhibiting leukemia cell growth. More intriguingly, hAESCs infusion in HSCT patients displayed potential anti-GVHD effect with no safety concerns and confirmed the immunoregulatory mechanisms in the preclinical study. We conclude that hAESCs infusion is a promising therapeutic strategy for post-HSCT GVHD without compromising the GVL effect. The clinical trial was registered at www.clinicaltrials.gov as #NCT03764228.

SUMOylation Fine-Tunes Endothelial HEY1 in the Regulation of Angiogenesis.

BACKGROUND: Angiogenesis, which plays a critical role in embryonic development and tissue repair, is controlled by a set of angiogenic signaling pathways. As a TF (transcription factor) belonging to the basic helix-loop-helix family, HEY (hairy/enhancer of split related with YRPW motif)-1 (YRPW motif, abbreviation of 4 highly conserved amino acids in the motif) has been identified as a key player in developmental angiogenesis. However, the precise mechanisms underlying HEY1's actions in angiogenesis remain largely unknown. Our previous studies have suggested a potential role for posttranslational SUMOylation in the dynamic regulation of vascular development and organization. METHODS: Immunoprecipitation, mass spectrometry, and bioinformatics analysis were used to determine the biochemical characteristics of HEY1 SUMOylation. The promoter-binding capability of HEY1 was determined by chromatin immunoprecipitation, dual luciferase, and electrophoretic mobility shift assays. The dimerization pattern of HEY1 was determined by coimmunoprecipitation. The angiogenic capabilities of endothelial cells were assessed by CCK-8 (cell counting kit-8), 5-ethynyl-2-deoxyuridine staining, wound healing, transwell, and sprouting assays. Embryonic and postnatal vascular growth in mouse tissues, matrigel plug assay, cutaneous wound healing model, oxygen-induced retinopathy model, and tumor angiogenesis model were used to investigate the angiogenesis in vivo. RESULTS: We identified intrinsic endothelial HEY1 SUMOylation at conserved lysines by TRIM28 (tripartite motif containing 28) as the unique E3 ligase. Functionally, SUMOylation facilitated HEY1-mediated suppression of angiogenic RTK (receptor tyrosine kinase) signaling and angiogenesis in primary human endothelial cells and mice with endothelial cell-specific expression of wild-type HEY1 or a SUMOylation-deficient HEY1 mutant. Mechanistically, SUMOylation facilitates HEY1 homodimer formation, which in turn preserves HEY1's DNA-binding capability via recognition of E-box promoter elements. Therefore, SUMOylation maintains HEY1's function as a repressive TF controlling numerous angiogenic genes, including RTKs and Notch pathway components. Proangiogenic stimuli induce HEY1 deSUMOylation, leading to heterodimerization of HEY1 with HES (hairy and enhancer of split)-1, which results in ineffective DNA binding and loss of HEY1's angiogenesis-suppressive activity. CONCLUSIONS: Our findings demonstrate that reversible HEY1 SUMOylation is a molecular mechanism that coordinates endothelial angiogenic signaling and angiogenesis, both in physiological and pathological milieus, by fine-tuning the transcriptional activity of HEY1. Specifically, SUMOylation facilitates the formation of the HEY1 transcriptional complex and enhances its DNA-binding capability in endothelial cells.

A 3D-Printed Dual Driving Forces Scaffold with Self-Promoted Cell Absorption for Spinal Cord Injury Repair.

Stem cells play critical roles in cell therapies and tissue engineering for nerve repair. However, achieving effective delivery of high cell density remains a challenge. Here, a novel cell delivery platform termed the hyper expansion scaffold (HES) is developed to enable high cell loading. HES facilitated self-promoted and efficient cell absorption via a dual driving force model. In vitro tests revealed that the HES rapidly expanded 80-fold in size upon absorbing 2.6 million human amniotic epithelial stem cells (hAESCs) within 2 min, representing over a 400% increase in loading capacity versus controls. This enhanced uptake benefited from macroscopic swelling forces as well as microscale capillary action. In spinal cord injury (SCI) rats, HES-hAESCs promoted functional recovery and axonal projection by reducing neuroinflammation and improving the neurotrophic microenvironment surrounding the lesions. In summary, the dual driving forces model provides a new rationale for engineering hydrogel scaffolds to facilitate self-promoted cell absorption. The HES platform demonstrates great potential as a powerful and efficient vehicle for delivering high densities of hAESCs to promote clinical treatment and repair of SCI.

Endothelial FIS1 DeSUMOylation Protects Against Hypoxic Pulmonary Hypertension.

BACKGROUND: Hypoxia is a major cause and promoter of pulmonary hypertension (PH), a representative vascular remodeling disease with poor prognosis and high mortality. However, the mechanism underlying how pulmonary arterial system responds to hypoxic stress during PH remains unclear. Endothelial mitochondria are considered signaling organelles on oxygen tension. Results from previous clinical research and our studies suggested a potential role of posttranslational SUMOylation (small ubiquitin-like modifier modification) in endothelial mitochondria in hypoxia-related vasculopathy. METHODS: Chronic hypoxia mouse model and Sugen/hypoxia rat model were employed as PH animal models. Mitochondrial morphology and subcellular structure were determined by transmission electron and immunofluorescent microscopies. Mitochondrial metabolism was determined by mitochondrial oxygen consumption rate and extracellular acidification rate. SUMOylation and protein interaction were determined by immunoprecipitation. RESULTS: The involvement of SENP1 (sentrin-specific protease 1)-mediated SUMOylation in mitochondrial remodeling in the pulmonary endothelium was identified in clinical specimens of hypoxia-related PH and was verified in human pulmonary artery endothelial cells under hypoxia. Further analyses in clinical specimens, hypoxic rat and mouse PH models, and human pulmonary artery endothelial cells and human embryonic stem cell-derived endothelial cells revealed that short-term hypoxia-induced SENP1 translocation to endothelial mitochondria to regulate deSUMOylation (the reversible process of SUMOylation) of mitochondrial fission protein FIS1 (mitochondrial fission 1), which facilitated FIS1 assembling with fusion protein MFN2 (mitofusin 2) and mitochondrial gatekeeper VDAC1 (voltage-dependent anion channel 1), and the membrane tethering activity of MFN2 by enhancing its oligomerization. Consequently, FIS1 deSUMOylation maintained the mitochondrial integrity and endoplasmic reticulum-mitochondria calcium communication across mitochondrial-associated membranes, subsequently preserving pulmonary endothelial function and vascular homeostasis. In contrast, prolonged hypoxia disabled the FIS1 deSUMOylation by diminishing the availability of SENP1 in mitochondria via inducing miR (micro RNA)-138 and consequently resulted in mitochondrial dysfunction and metabolic reprogramming in pulmonary endothelium. Functionally, introduction of viral-packaged deSUMOylated FIS1 within pulmonary endothelium in mice improved pulmonary endothelial dysfunction and hypoxic PH development, while knock-in of SUMO (small ubiquitin-like modifier)-conjugated FIS1 in mice exaggerated the diseased cellular and tissue phenotypes. CONCLUSIONS: By maintaining endothelial mitochondrial homeostasis, deSUMOylation of FIS1 adaptively preserves pulmonary endothelial function against hypoxic stress and consequently protects against PH. The FIS1 deSUMOylation-SUMOylation transition in pulmonary endothelium is an intrinsic pathogenesis of hypoxic PH.

Functional Characterization and Screening of Promiscuous Kinases and Isopentenyl Phosphate Kinases for the Synthesis of DMAPP via a One-Pot Enzymatic Cascade.

Dimethylallyl diphosphate (DMAPP) is a key intermediate metabolite in the synthesis of isoprenoids and is also the prenyl donor for biosynthesizing prenylated flavonoids. However, it is difficult to prepare DMAPP via chemical and enzymatic methods. In this study, three promiscuous kinases from Shigella flexneri (SfPK), Escherichia coli (EcPK), and Saccharomyces cerevisiae (ScPK) and three isopentenyl phosphate kinases from Methanolobus tindarius (MtIPK), Methanothermobacter thermautotrophicus str. Delta H (MthIPK), and Arabidopsis thaliana (AtIPK) were cloned and expressed in Escherichia coli. The enzymatic properties of recombinant enzymes were determined. The Kcat/Km value of SfPK for DMA was 6875 s-1 M-1, which was significantly higher than those of EcPK and ScPK. The Kcat/Km value of MtIPK for DMAP was 402.9 s-1 M-1, which was ~400% of that of MthIPK. SfPK was stable at pH 7.0-9.5 and had a 1 h half-life at 65 °C. MtIPK was stable at pH 6.0-8.5 and had a 1 h half-life at 50 °C. The stability of SfPK and MtIPK was better than that of the other enzymes. Thus, SfPK and MtIPK were chosen to develop a one-pot enzymatic cascade for producing DMAPP from DMA because of their catalytic efficiency and stability. The optimal ratio between SfPK and MtIPK was 1:8. The optimal pH and temperature for the one-pot enzymatic cascade were 7.0 and 35 °C, respectively. The optimal concentrations of ATP and DMA were 10 and 80 mM, respectively. Finally, maximum DMAPP production reached 1.23 mM at 1 h under optimal conditions. Therefore, the enzymatic method described herein for the biosynthesis of DMAPP from DMA can be widely used for the synthesis of isoprenoids and prenylated flavonoids.

Tribological Properties of Laser Cladded Alloys for Repair of Rail Components.

Tram or light rail systems are heavily relied upon for passenger transit; however, low-carbon steel grades commonly used in special trackwork, such as in switches, are prone to wear, rolling contact fatigue (RCF), and deformation under cyclic wheel-rail contact. To address this, laser cladding can be used to apply a metal coating to protect the underlying substrate and rebuild the worn rail profiles. Laser cladding may also be applied to remove cracking by rebuilding the rail head. The tribological characteristics of light rail components after laser cladding with Stellite 6 and a newly developed martensitic stainless steel were investigated, using roller-on-disc wear testing. Analysis of the microstructure, mechanical properties, and wear performance was undertaken to develop a comprehensive understanding of the influence of the laser cladding type on the wear and surface fatigue performance. Both cladding alloys significantly improved the tribological performance. These findings were compared to those for a laser cladded hypereutectoid rail type (reported in our previous study). It was found that laser cladding with a suitable alloy was an effective technique for improving the tribological properties, increasing the wear resistance, and increasing the retardation of cracking on both substrates. These findings suggest laser cladding may be used to repair light rail components, and this technique can be optimized to suit different rail grades. This makes laser cladding a flexible and versatile maintenance strategy, in both coating and repair applications, to prolong the operational lifetime of critical components for the railway industry.

Therapeutic Effect of Biomimetic Scaffold Loaded with Human Amniotic Epithelial Cell-Derived Neural-like Cells for Spinal Cord Injury.

Spinal cord injury (SCI) results in devastating consequences for the motor and sensory function of patients due to neuronal loss and disrupted neural circuits, confronting poor prognosis and lack of effective therapies. A new therapeutic strategy is urgently required. Here, human amniotic epithelial cells (hAEC), featured with immunocompatibility, non-tumorgenicity and no ethical issues, were induced into neural-like cells by a compound cocktail, as evidenced with morphological change and the expression of neural cell markers. Interestingly, the hAEC-neural-like cells maintain the characteristic of low immunogenicity as hAEC. Aiming at SCI treatment in vivo, we constructed a 3D-printed GelMA hydrogel biomimetic spinal cord scaffold with micro-channels, in which hAEC-neural-like cells were well-induced and grown. In a rat full transection SCI model, hAEC-neural-like cell scaffolds that were implanted in the lesion demonstrated significant therapeutic effects; the neural circuit and hindlimb locomotion were partly recovered compared to little affection in the SCI rats receiving an empty scaffold or a sham implantation operation. Thus, the establishment of hAEC-neural-like cell biomimetic scaffolds may provide a safe and effective treatment strategy for SCI.

Atmospheric turbulence resistant heterodyne coherent receiver of few-mode fiber.

We propose and implement a free-space optical (FSO) communication system based on few-mode heterodyne detection that can effectively suppress atmospheric turbulence effects. The experimental results show that the received power gain of the FSO communication system using six-mode fibres is about 6 dB over that using SMF under moderate to strong turbulence conditions.In addition, we have built a coherent detection system for space laser communications with few-mode heterodyne detection and reception, and verified the compensation of atmospheric turbulence effects by the few-mode heterodyne detection and reception technique. Experimental results show that the proposed scheme improves the power budget by 4∼5dB over the single-mode heterodyne coherent reception scheme at BER = 3.8×10-3 under moderate to strong turbulence conditions.

Application of a New Alloy and Post Processing Procedures for Laser Cladding Repairs on Hypereutectoid Rail Components.

The development of a laser cladding repair strategy is critical for the continued growth of heavy-haul railway networks. Premium hypereutectoid rails have undergone laser cladding using a new martensitic stainless-steel alloy, 415SS, developed for high carbon rails after standard cladding metals were found to be incompatible. Non-destructive neutron diffraction techniques were used to measure the residual stress in different layers generated across a dissimilar metal joint during laser cladding. The internal stress distribution across the cladding, heat-affected zone (HAZ), and substrate was measured in the untempered rail, after 350 °C and 540 °C heat treatment procedures and two surface grinding operations. The martensitic 415SS depositions produce compressive stress in the cladding, regardless of tempering procedures, which may inhibit fatigue crack propagation whilst grinding operations locally relive surface stress. Balancing tensile stresses were recorded below the fusion boundary in the HAZ due to thermal gradients altering the microstructure. The combination of 540 °C tempering and 0.5 mm surface layer removal produced a desirable combination of compression in the cladding deposition with significantly reduced tensile stresses in the HAZ. A comparison with the current literature shows that this alloy achieves a unique combination of desirable hardness, low tensile stress, and compression in the cladding layer. Data obtained during strain scanning has been used to determine the location of microstructural changes at the fusion boundary and HAZ through correlation of the stress, strain, full width at half maximum (FWHM), and intensity profiles. Therefore, neutron diffraction can be used for both the accurate measurement of internal residual stress and to obtain microstructural information of a metallurgical join non-destructively.

Repair of Retinal Degeneration by Human Amniotic Epithelial Stem Cell-Derived Photoreceptor-like Cells.

The loss of photoreceptors is a major event of retinal degeneration that accounts for most cases of untreatable blindness globally. To date, there are no efficient therapeutic approaches to treat this condition. In the present study, we aimed to investigate whether human amniotic epithelial stem cells (hAESCs) could serve as a novel seed cell source of photoreceptors for therapy. Here, a two-step treatment with combined Wnt, Nodal, and BMP inhibitors, followed by another cocktail of retinoic acid, taurine, and noggin induced photoreceptor-like cell differentiation of hAESCs. The differentiated cells demonstrated the morphology and signature marker expression of native photoreceptor cells and, intriguingly, bore very low levels of major histocompatibility complex (MHC) class II molecules and a high level of non-classical MHC class I molecule HLA-G. Importantly, subretinal transplantation of the hAESCs-derived PR-like cells leads to partial restoration of visual function and retinal structure in Royal College of Surgeon (RCS) rats, the classic preclinical model of retinal degeneration. Together, our results reveal hAESCs as a potential source of functional photoreceptor cells; the hAESCs-derived photoreceptor-like cells could be a promising cell-replacement candidate for therapy of retinal degeneration diseases.

FGFR1 SUMOylation coordinates endothelial angiogenic signaling in angiogenesis.

Angiogenesis contributes fundamentally to embryonic development, tissue homeostasis, and wound healing. Basic fibroblast growth factor (FGF2) is recognized as the first proangiogenic molecule discovered, and it facilitates angiogenesis by activating FGF receptor 1 (FGFR1) signaling in endothelial cells. However, the precise roles of FGFR and the FGF/FGFR signaling axis in angiogenesis remain unclear, especially because of the contradictory phenotypes of in vivo FGF and FGFR gene deficiency models. Our previous study results suggested a potential role of posttranslational small ubiquitin-like modifier modification (SUMOylation), with highly dynamic regulatory features, in vascular development and disorder. Here, we identified SENP1-regulated endothelial FGFR1 SUMOylation at conserved lysines responding to proangiogenic stimuli, while SENP1 functioned as the deSUMOylase. Hypoxia-enhanced FGFR1 SUMOylation restricted the tyrosine kinase activation of FGFR1 by modulating the dimerization of FGFR1 and FGFR1 binding with its phosphatase PTPRG. Consequently, it facilitated the recruitment of FRS2α to VEGFR2 but limited additional recruitment of FRS2α to FGFR1, supporting the activation of VEGFA/VEGFR2 signaling in endothelial cells. Furthermore, SUMOylation-defective mutation of FGFR1 resulted in exaggerated FGF2/FGFR1 signaling but suppressed VEGFA/VEGFR2 signaling and the angiogenic capabilities of endothelial cells, which were rescued by FRS2α overexpression. Reduced angiogenesis and endothelial sprouting in mice bearing an endothelial-specific, FGFR1 SUMOylation-defective mutant confirmed the functional significance of endothelial FGFR1 SUMOylation in vivo. Our findings identify the reversible SUMOylation of FGFR1 as an intrinsic fine-tuned mechanism in coordinating endothelial angiogenic signaling during neovascularization; SENP1-regulated FGFR1 SUMOylation and deSUMOylation controls the competitive recruitment of FRS2α by FGFR1 and VEGFR2 to switch receptor-complex formation responding to hypoxia and normoxia angiogenic environments.

Residual Stress Measurement Techniques for Metal Joints, Metallic Coatings and Components in the Railway Industry: A Review.

Manufacturing and maintenance procedures in the railway industry regularly implement welding and metal deposition operations to produce joints, coatings and repair structures. During these processes, residual stresses arise through the generation of heat affected zones and plastic deformation. This makes accurate measurements of the internal stresses a critical aspect of manufacturing, monitoring, repair and model validation in the develop new metallic coating and joining technologies. Selection of an appropriate residual stress measurement method has many important factors including component size, resolution and the magnitude and location of internal stresses, often resulting in a combination of techniques required to obtain complete assessment of the stress state. This paper offers a review of residual stress measurement techniques for railway components including rail joints and coatings through comparison of destructive and non-destructive approaches, their measurement capabilities, benefits and limitations. A comprehensive discussion of different applications is provided with a summary of facilities available to both research and industry.

Analysis of factors influencing high frequency hearing loss among noise-exposed workers in an air conditioner manufacturing industry / 中国职业医学

@#Objective （ ） To explore the feasibility of using generalized estimating equation GEE to analyze the influencing - （ ） factors of high frequency hearing loss HFHL among noise exposed workers in an air conditioner manufacturing enterprise. Methods - The noise exposed workers in an air conditioner manufacturing industry who had been tested for pure tone hearing threshold twice or more from 2015 to 2019 were selected as the research subjects using the judgment sampling method. Data ， ， ， ， ， （ ） such as age length of service gender smoking alcohol consumption body mass index BMI and HFHL were collected. The Results influencing factors of HFHL were analyzed using the GEE. The detection rates of HFHL from 2015 to 2019 were ， ， ， ， ， 22.2% 23.8% 24.2% 24.1% and 20.9% respectively. Among them the detection rate of HFHL in 2019 was lower than that （ P ） ， ， in 2017 and 2018 all <0.001 . The GEE analysis results showed that the risks of HFHL in 2015 2016 2017 and 2018 were （ P ）， higher than that in 2019 all <0.01 regardless of interaction effects and after adjusting for confounding factors such as ， ［OR（ CI）］ （ - duration of noise exposure smoking and BMI. The odds ratios and 95% confidence intervals 95% were 1.19 1.07 ）， （ - ）， （ - ） （ - ）， 1.33 1.26 1.13 1.39 1.30 1.18 1.43 and 1.27 1.15 1.39 respectively. The risk of HFHL was higher in males than in （P ）， OR（ CI） （ - ） ， （P ）， OR females <0.01 and 95% was 3.78 3.00 4.77 . The older the age the higher the risk of HFHL <0.01 and （ CI） （ - ） Conclusion - 95% was 1.07 1.05 1.09 . The influencing factors of HFHL among noise exposed workers in the air conditioner industry are age and gender. GEE can be used to analyze the factors influencing the longitudinal data of HFHL in workers with noise exposure.

AGEs-Induced Calcification and Apoptosis in Human Vascular Smooth Muscle Cells Is Reversed by Inhibition of Autophagy.

Vascular calcification (VC) in macrovascular and peripheral blood vessels is one of the main factors leading to diabetes mellitus (DM) and death. Apart from the induction of vascular calcification, advanced glycation end products (AGEs) have also been reported to modulate autophagy and apoptosis in DM. Autophagy plays a role in maintaining the stabilization of the external and internal microenvironment. This process is vital for regulating arteriosclerosis. However, the internal mechanisms of this pathogenic process are still unclear. Besides, the relationship among autophagy, apoptosis, and calcification in HASMCs upon AGEs exposure has not been reported in detail. In this study, we established a calcification model of SMC through the intervention of AGEs. It was found that the calcification was upregulated in AGEs treated HASMCs when autophagy and apoptosis were activated. In the country, AGEs-activated calcification and apoptosis were suppressed in Atg7 knockout cells or pretreated with wortmannin (WM), an autophagy inhibitor. These results provide new insights to conduct further investigations on the potential clinical applications for autophagy inhibitors in the treatment of diabetes-related vascular calcification.

Biochemical Characterization of a Novel Prenyltransferase from Streptomyces sp. NT11 and Development of a Recombinant Strain for the Production of 6-Prenylnaringenin.

Prenyl groups increase the lipophilicity of flavonoids, endowing them with a special activity, selectivity, and pharmacological properties by prenylation. Herein, a novel prenyltransferase (ShFPT) gene from Streptomyces sp. NT11 was expressed in Escherichia coli, and its biochemical characteristics were determined. ShFPT exhibited high selectivity to prenylate naringenin at C-6 to generate 6-prenylnaringenin. The optimal activity was observed at pH 6.0 and 55 °C. The Kcat and Km for naringenin were 0.0095 s-1 and 0.20 mM, respectively. Several promiscuous kinase and isopentenyl phosphate kinase genes were screened to develop the most efficient dimethylallyl diphosphate (DMAPP) synthesis pathway for 6-prenylnaringenin synthesis in E. coli. The 6-prenylnaringenin production was improved by changing the induction strategies and optimizing the bioconversion conditions. Finally, 6-prenylnaringenin production reached the highest yield of 69.9 mg/L with average productivity of 4.0 mg/L/h after 16 h incubation, which is the highest yield for any prenylated flavonoid reported to date in E. coli. Therefore, this study provides an efficient method for 6-prenylnaringenin production and reveals the DMAPP synthesis pathway.

Human Amniotic Epithelial Stem Cell-Derived Retinal Pigment Epithelium Cells Repair Retinal Degeneration.

Age-related macular degeneration (AMD), featured with dysfunction and loss of retinal pigment epithelium (RPE), is lacking efficient therapeutic approaches. According to our previous studies, human amniotic epithelial stem cells (hAESCs) may serve as a potential seed cell source of RPE cells for therapy because they have no ethical concerns, no tumorigenicity, and little immunogenicity. Herein, trichostatin A and nicotinamide can direct hAESCs differentiation into RPE like cells. The differentiated cells display the morphology, marker expression and cellular function of the native RPE cells, and noticeably express little MHC class II antigens and high level of HLA-G. Moreover, visual function and retinal structure of Royal College of Surgeon (RCS) rats, a classical animal model of retinal degeneration, were rescued after subretinal transplantation with the hAESCs-derived RPE like cells. Our study possibly makes some contribution to the resource of functional RPE cells for cell therapy. Subretinal transplantation of hAESCs-RPE could be an optional therapeutic strategy for retinal degeneration diseases.

Biosynthesis of 3'-O-methylisoorientin from luteolin by selecting O-methylation/C-glycosylation motif.

Glycosylation and methylation of flavonoids are the main types of structural modifications and can endow flavonoids with greater stability, bioactivity, and bioavailability. In this study, five types of O-methyltransferases were screened for producing O-methylated luteolin, and the biosynthesis strategy of 3'-O-methylisoorientin from luteolin was determined. To improve the production of 3'-O-methylluteolin, the S-adenosyl-l-methionine synthesis pathway was reconstructed in the recombinant strain by introducing S-adenosyl-l-methionine synthetase genes. After optimizing the conversion conditions, maximal 3'-O-methylluteolin production reached 641 ± 25 mg/L with a corresponding molar conversion of 76.5 %, which was the highest titer of methylated flavonoids reported to date in Escherichia coli. 3'-O-Methylluteolin (127 mg) was prepared from 250 mL of the broth by silica gel column chromatography and preparative HPLC with a yield of 79.4 %. Subsequently, we used the biocatalytic cascade of Gentiana triflora C-glycosyltransferase (Gt6CGT) and Glycine max sucrose synthase (GmSUS) to biosynthesize 3'-O-methylisoorientin from 3'-O-methylluteolin in vitro. By optimizing the coupled reaction conditions and using the fed-batch operation, maximal 3'-O-methylisoorientin production reached 226 ± 8 mg/L with a corresponding molar conversion of 98 %. Therefore, this study provides an efficient method for the production of novel 3'-O-methylisoorientin and the biosynthesis strategy for methylated C-glycosylation flavonoids by selective O-methylation/C-glycosylation motif on flavonoids.

Orientin and vitexin production by a one-pot enzymatic cascade of a glycosyltransferase and sucrose synthase.

Orientin and vitexin, important components of bamboo-leaf extracts, are C-glycosylflavones which exhibit a number of interesting biological properties. In this work, we developed an efficient biocatalytic cascade for orientin and vitexin production consisting of Trollius chinensis C-glycosyltransferase (TcCGT) and Glycine max sucrose synthase (GmSUS). In order to relieve the bottleneck of the biocatalytic cascade, the biocatalytic efficiency, reaction condition compatibilities and the ratio of the enzymes were determined. We found that the specific activity of TcCGT was significantly influenced by enzyme dose and Triton X-100 or Tween 20 (0.2%). Co-culture of BL21-TcCGT-Co and BL21-GmSUS-Co affected the catalytic efficiency of TcCGT and GmSUS, and the maximum orientin production rate reached 47 µM/min at the inoculation ratio of 9:1. The optimal pH and temperature for the biocatalytic cascade were pH 7.5 and 30 °C, respectively. Moreover, the high dose of the enzymes can improve the tolerance of biocatalytic cascade to substrate inhibition in the one-pot reaction. By using a fed-batch strategy, maximal titers of orientin and vitexin reached 7090 mg/L with a corresponding molar conversion of 98.7% and 5050 mg/L with a corresponding molar conversion of 97.3%, respectively, which is the highest titer reported to date. Therefore, the method described herein for efficient production of orientin and vitexin by modulating catalytic efficiencies of enzymes can be widely used for the C-glycosylation of flavonoids.

Study on the transcriptome for breast muscle of chickens and the function of key gene RAC2 on fibroblasts proliferation.

BACKGROUND: Growth performance is significant in broiler production. In the growth process of broilers, gene expression varies at different growth stages. However, limited research has been conducted on the molecular mechanisms of muscle growth and development in yellow-feathered male chickens. RESULTS: In the study, we used RNA-seq to study the transcriptome of the breast muscle of male Jinghai yellow chickens at 4 (M4F), 8 (M8F) and 12 weeks (M12F) of age. The results showed that 4608 differentially expressed genes (DEGs) were obtained by comparison in pairs of the three groups with Fold Change (FC) ≥ 2 and False Discovery Rate (FDR) ≤ 0.05, and 83, 3445 and 3903 DEGs were obtained separately from M4FvsM8F, M4FvsM12F and M8FvsM12F. Six genes were found as co-differentially expressed in the three age groups, namely SNCG, MYH1A, ARHGDIB, ENSGALG00000031598, ENSGALG00000035660 and ENSGALG00000030559. The GO analysis showed that 0, 304 and 408 biological process (BP) were significantly enriched in M4FvsM8F, M4FvsM12F and M8FvsM12F groups, respectively. KEGG pathway enrichment showed that 1, 2, 4 and 4 pathways were significantly enriched in M4FvsM8F, M4FvsM12F, M8FvsM12F and all DEGs, respectively. They were steroid biosynthesis, carbon metabolism, focal adhesion, cytokine-cytokine receptor interaction, biosynthesis of amino acids and salmonella infection. We constructed short hairpin RNA (shRNA) to interfere the differentially expressed gene RAC2 in DF-1 cells and detected mRNA and protein expression of the downstream genes PAK1 and MAPK8. Results of qPCR showed that RAC2, PAK1 and MAPK8 mRNA expression significantly decreased in the shRAC2-2 group compared with the negative control (NC) group. Western Blot (WB) results showed that the proteins of RAC2, PAK1 and MAPK8 also decreased in the shRAC2-2 group. Cell Counting Kit-8 (CCK-8) and 5-Ethynyl-2'-deoxyuridine (EdU) assay both showed that the proliferation of DF-1 cells was significantly inhibited after transfection of shRAC2-2. CONCLUSIONS: The results of RNA-seq revealed genes, BP terms and KEGG pathways related to growth and development of male Jinghai yellow chickens, and they would have important guiding significance to our production practice. Further research suggested that RAC2 might regulate cell proliferation by regulating PAKs/MAPK8 pathway and affect growth of chickens.

A novel TSC1 frameshift mutation c.1550_1551del causes tuberous sclerosis complex by aberrant splicing and nonsense-mediated mRNA degradation (NMD) simultaneously in a Chinese family.

BACKGROUND: Tuberous sclerosis complex (TSC), belongs to autosomal dominant genetic disorder, which affects multiple organ systems in the body, including the skin, brain, lungs, kidneys, liver, and eyes. Mutations in TSC1 or TSC2 was proved to be associated with these conditions. METHODS: Gene-panel Sequence of NGS was used to detect the mutation in a Chinese family. The research further investigates whether aberrant splicing and nonsense-mediated mRNA degradation (NMD) could serve as a mechanism cause by TSC1 mutation. MINI-Gene assay apply by pcMINI-TSC1wt/mut plasmids delivered in HeLa and 293T cell lines. Recombinant plasmids expressing wild-type and mutant-type EGFP-TSC1 were constructed and transiently transfected into human embryonic kidney cells 293T by lipofectamine. Real-time PCR and Western Blot were performed to analyze the expression of mRNAs and proteins of EGFP-TSC1 and NMD factor UPF1. RESULTS: The gene test verified a novel heterozygous TSC1 frameshift mutation (TSC1 c.1550_1551del) in the proband and her mother. From MINI-Gene assay, the agarose gel showed that both the mutant and wild-type mRNA possess two main bands, indicating two splicing modes, named band A and B, respectively. The mutation c.1550_1551del has not produced new splicing site, but there is a selective splicing in varying degree significantly after mutation. On the contrary, function validation assay showed that cells transfected with the mutant TSC1 plasmids expressed significantly lower TSC1 in mRNAs and proteins levels, compared with the wild-type TSC1 plasmid transfection. A translation inhibitor cycloheximide and small interfering RNA of UPF1 (siRNA-UPF1) increased mRNA or protein expression of TSC1 significantly in cells transfected with the mutant plasmids. CONCLUSION: Our study demonstrated that the novel TSC1 frameshift mutation (TSC1 c.1550_1551del) trigger aberrant splicing and NMD simultaneously, causing decrease of hamartin, then, leading to tuberous sclerosis complex formation.