Importance of the stem cell microenvironment for ophthalmological cell-based therapy

Cell therapy is a promising treatment for diseases that are caused by cell degeneration or death. The cells for clinical transplantation are usually obtained by culturing healthy allogeneic or exogenous tissue invitro. However, for diseases of the eye, obtaining the adequate number of cells for clinical transplantation is difficult due to the small size of tissue donors and the frequent needs of long-term amplification of cells in vitro, which results in low cell viability after transplantation. In addition, the transplanted cells often develop fibrosis or degrade and have very low survival. Embryonic stem cells(ESCs) and induced pluripotent stem cells(i PS) are also promising candidates for cell therapy. Unfortunately, the differentiation of ESCs can bring immune rejection, tumorigenicity and undesired differentiated cells, limiting its clinical application. Although i PS cells can avoid the risk of immune rejection caused by ES cell differentiation post-transplantation, the low conversion rate, the risk of tumor formation and the potentially unpredictable biological changes that could occur through genetic manipulation hinder its clinical application. Thus, the desired clinical effect of cell therapy is impaired by these factors. Recent research findings recognize that the reason for low survival of the implanted cells not only depends on the seeded cells, but also on the cell microenvironment, which determines the cell survival, proliferation and even reverse differentiation. When used for cell therapy, the transplanted cells need a specific three-dimensional structure to anchor and specific extra cellular matrix components in addition to relevant cytokine signaling to transfer the required information to support their growth. These structures present in the matrix in which the stem cells reside are known as the stem cell microenvironment. The microenvironment interaction with the stem cells provides the necessary homeostasis for cell maintenance and growth. A large number of studies suggest that to explore how to reconstruct the stem cell microenvironment and strengthen its combination with the transplanted cells are key steps to successful cell therapy. In this review, we will describe the interactions of the stem cell microenvironment with the stem cells, discuss the importance of the stem cell microenvironment for cell-based therapy in ocular diseases, and introduce the progress of stem cell-basedtherapy for ocular diseases.     

利用PCR方法鉴定hiTAIL-PCR扩增产物中的质粒骨架片段

T-DNA突变体是研究基因功能的重要资源。高效热不对称交错PCR (hiTAIL-PCR)是克隆突变体中T-DNA插入位点侧翼序列的常用方法。然而我们发现, 利用hiTAIL-PCR克隆到的一些侧翼序列并不对应于宿主的染色体DNA序列, 而是质粒的骨架DNA片段。通过设置1组RB-S4/AC1或者LB-A4/AC1对照反应, 用PCR方法鉴定了hiTAIL-PCR扩增产物中位于T-DNA侧翼的质粒骨架片段。在后续分析中, 通过排除这些片段, 提高了利用hiTAIL-PCR获得宿主染色体DNA片段的效率。同时, 通过调整反应程序, 使得整个PCR的反应时间也大为缩短。在拟南芥(Arabidopsis thaliana) T-DNA突变体drf1侧翼序列的克隆实例中, 对照反应的引入将hiTAIL-PCR中需鉴定的22条扩增产物降至4条, 效率提高了81.8%。     

N and P resorption as functions of the needle age class in two conifer trees

Aims Given the importance of resorption in nutrient conservations, nutrient resorption should change with leaf age if resorption depends on nutrient content, and if nutrient content changes with leaf age. However, no study has addressed this issue.     

Efficient expression of stable recombinant human insulin-like growth factor-1 fusion with human serum albumin in Chinese hamster ovary cells

Insulin-like growth factor-1 (IGF-1) plays a crucial role in cell development, differentiation, and metabolism, and has been a potential therapeutic agent for many diseases. Chinese hamster ovary (CHO) cells are widely used for production of recombinant therapeutic proteins, but the expression level of IGF-1 in CHO cells is very low (1,500?µg/L) and the half-life of IGF-1 in blood circulation is only 4.5?min according to previous studies. Therefore, IGF-1 was fused to long-circulating serum protein human serum albumin (HSA) and expressed in CHO cells. After 8-day fed-batch culture, the expression level of HSA–IGF-1 reached 100?mg/L. The fusion protein HSA–IGF-1 was purified with a recovery of 35% using a two-step chromatographic procedure. According to bioactivity assay, the purified HSA–IGF-1 could stimulate the proliferation of NIH3T3 cells in a dose-dependent fashion and promote the cell-cycle progression. Besides this, HSA–IGF-1 could bind to IGF-1 receptor on cell membrane and activate the intracellular PI3K/AKT signaling pathway. Our study suggested that HSA fusion technology carried out in CHO cells not only provided bioactivity in HSA–IGF-1 for further research but also offered a beneficial strategy to produce other similar cytokines in CHO cells.     

Gypenoside inhibits interleukin‐1β‐induced inflammatory response in human osteoarthritis chondrocytes

Gypenoside (GP), the main active ingredient of Gynostemma pentaphyllum, possesses a variety of pharmacological capacities including anti‐inflammation, anti‐oxidation, and anti‐tumor. However, the effects of GP on IL‐1β‐stimulated human osteoarthritis (OA) chondrocytes are still unknown. Therefore, this study aimed to investigate the anti‐inflammatory effects of GP on IL‐1β‐stimulated human OA chondrocytes and explore the possible mechanism. Our results showed that GP dose‐dependently inhibited IL‐1β‐induced NO and PGE2 production in human OA chondrocytes. In addition, treatment of GP inhibited the expression of MMP3 and MMP13, which was increased by IL‐1β. Finally, we found that pretreatment of GP obviously suppressed NF‐κB activation in IL‐1β‐stimulated human OA chondrocytes. Taken together, the results demonstrated that GP has chondro‐protective effects, at least in part, through inhibiting the activation of NF‐κB signaling pathway in human OA chondrocytes. Thus, these findings suggest that GP may be considered as an alternative therapeutic agent for the management of OA patients.     

<Emphasis Type="Italic">FLOURY ENDOSPERM8</Emphasis>, encoding the UDP-glucose pyrophosphorylase 1, affects the synthesis and structure of starch in rice endosperm

Cereal opaque-kernel mutants are ideal genetic materials for studying the mechanism of starch biosynthesis and amyloplast development. Here we isolated and identified two allelic floury endosperm 8 (flo8) mutants of rice, named flo8-1 and flo8-2. In the flo8 mutant, the starch content was decreased and the normal physicochemical features of starch were altered. Map-based cloning and subsequent DNA sequencing analysis revealed a single nucleotide substitution and an 8-bp insertion occurred in UDP-glucose pyrophosphorylase 1 (Ugp1) gene in flo8-1 and flo8-2, respectively. Complementation of the flo8-1 mutant restored normal seed appearance by expressing full length coding sequence of Ugp1. RT-qPCR analysis revealed that Ugp1 was ubiquitously expressed. Mutation caused the decreased UGPase activity and affected the expression of most of genes associated with starch biosynthesis. Meanwhile, western blot and enzyme activity analyses showed the comparability of protein levels and enzyme activity of most tested starch biosynthesis related genes. Our results demonstrate that Ugp1 plays an important role for starch biosynthesis in rice endosperm.     

Associations of the Polymorphisms in DHRS4, SERPING1, and APOR Genes with Postmortem pH in Berkshire Pigs

Postmortem pH is a main factor influencing the meat quality in pigs. This study investigated the association of postmortem pH with single-nucleotide polymorphisms (SNPs) in the fourth member of the short-chain dehydrogenase/reductase family (DHRS4), the first member of serpin peptidase inhibitor, clade G (complement inhibitor) (SERPING1), and the apolipoprotein R precursor (APOR) genes in Berkshire pigs. The study included 437 pigs, and genotyping was conducted using the GoldenGate Assay (Illumina, San Diego, CA, USA). DHRS4, SERPING1, and APOR polymorphisms were significantly associated with pH₄₅ or pH₂₄ (p?SERPING1 was also statistically significantly associated with water holding capacity (p?DHRS4, SERPING1, and APOR genes have potential for use as genetic markers for the meat quality in pigs.     

A cell clone strain from <Emphasis Type="Italic">Mythimna separata</Emphasis> (Lepidoptera: Noctuidae) highly susceptible to <Emphasis Type="Italic">Autographa californica</Emphasis> multiple nucleopolyhedrovirus (AcMNPV) and <Emphasis Type="Italic">M. separata</Emphasis> NPV (MsNPV)

In this study, we describe a cell line, Ms-10C, cloned from the line QAU-Ms-E-10 (simplified Ms-10), an embryonic line from Mythimna separata. The cloned cell line was significantly more sensitive to nucleopolyhedrovirus (NPV). Ms-10C cells were mainly spherical with a diameter of 14.42 ± 2.23 μm. DNA amplification fingerprinting (DAF) confirmed the profile of PCR-amplified bands of the cloned cell line was consistent with those of the parental cell line, Ms-10. The sequencing result of the mitochondrial cytochrome c oxidase I (mtCO I) fragment confirmed that the amplified 636-bps mtCOI fragment was 100% identical to that of M. separata. Its chromosomes exhibited the typical characters of lepidopteran cell lines. Its population doubling time was 42.2 h at 27°C. Ms-10C was more sensitive than Ms-10 to both Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and M. separata nucleopolyhedrovirus (MsNPV). At 4 d post infection, the infection rates of two viruses reached 94.2 and 92.3%, respectively. The availability of this cell clone strain will provide a useful tool for the basic research on nucleopolyhedrovirus and for potential application in expression of recombinant proteins with baculovirus expression vector system.     

Impacts of sediment type on the performance and composition of submerged macrophyte communities

To restore deteriorated lake ecosystems, it is important to identify environmental factors that influence submerged macrophyte communities. While sediment is a critical environmental factor for submerged macrophytes and many studies have examined effects of sediment type on the growth of individual submerged macrophytes, very few have tested how sediment type affects the growth and species composition of submerged macrophyte communities. We constructed submerged macrophyte communities containing four co-occurring submerged macrophytes (Hydrilla verticillata, Myriophyllum spicatum, Ceratophyllum demersum and Chara fragilis) and subjected them to three sediment treatments, i.e., clay, a mixture of clay and quartz sand at a volume ratio of 1:1 and a mixture at a volume ratio of 1:4. Compared to the clay, the 1:1 mixture treatment greatly increased overall biomass, number of shoot nodes and shoot length of the community, but decreased its diversity. This was because it substantially promoted the growth of H. verticillata within the community, making it the most abundant species in the mixture sediment, but decreased that of M. spicatum and C. demersum. The sediment type had no significant effects on the growth of C. fragilis. As a primary nutrient source for plant growth, sediment type can have differential effects on various submerged macrophyte species and 1:1 mixture treatment could enhance the performance of the communities, increasing the overall biomass, number of shoot nodes and shoot length by 39.03%, 150.13% and 9.94%, respectively, compared to the clay treatment. Thus, measures should be taken to mediate the sediment condition to restore submerged macrophyte communities with different dominant species.     

Dynamic metabolic responses of brown planthoppers towards susceptible and resistant rice plants

Brown planthopper (Nilaparvata lugens Stål, BPH) causes huge economic losses in rice‐growing regions, and new strategies for combating BPH are required. To understand how BPHs respond towards BPH‐resistant plants, we systematically analysed the metabolic differences between BPHs feeding on the resistant and susceptible plants using NMR and GC‐FID/MS. We also measured the expression of some related genes involving glycolysis and biosyntheses of trehalose, amino acids, chitin and fatty acids using real‐time PCR. BPH metabonome was dominated by more than 60 metabolites including fatty acids, amino acids, carbohydrates, nucleosides/nucleotides and TCA cycle intermediates. After initial 12 h, BPHs feeding on the resistant plants had lower levels of amino acids, glucose, fatty acids and TCA cycle intermediates than on the susceptible ones. The levels of these metabolites recovered after 24 h feeding. This accompanied with increased level in trehalose, choline metabolites and nucleosides/nucleotides compared with BPH feeding on the susceptible plants. Decreased levels of BPH metabolites at the early feeding probably resulted from less BPH uptakes of sap from resistant plants and recovery of BPH metabolites at the later stage probably resulted from their adaptation to the adverse environment with their increased hopping frequency to ingest more sap together with contributions from yeast‐like symbionts in BPHs. Throughout 96 h, BPH feeding on the resistant plants showed significant up‐regulation of chitin synthase catalysing biosynthesis of chitin for insect exoskeleton, peritrophic membrane lining gut and tracheae. These findings provided useful metabolic information for understanding the BPH–rice interactions and perhaps for developing new BPH‐combating strategies.