Reduction in Activity/Gene Expression of Anthocyanin Degradation Enzymes in Lychee Pericarp is Responsible for the Color Protection of the Fruit by Heat and Acid Treatment

Heat and acid treatments were reported to be a promising substitute for SO2 fumigation in color protection of postharvest lychee （Litchi chinensis） fruits, but the mechanism was not clear. In the present study, hot water （70℃） dipping followed by immersion in 2% HC1 （heat-acid） substantially protected the red color of the fruit during storage at 25℃ and inhibited anthocyanin degradation while hot water dipping alone （heat） led to rapidly browning and about 90% loss in anthocyanin content. The pH values in the pericarp of the heat-acid treated fruit dropped to 3.2, while the values maintained around 5.0 in the heat-treated and control fruit. No significantly different pH values were detected among the arils of heat-acid, heat treated and control fruit. Heat-acid treatment dramatically reduced the activities of anthocyanin degradation enzyme （ADE）, peroxidase （POD） and polyphenol oxidase in the pericarp. A marked reduction in LcPOD gene expression was also detected in heat-acid treated fruit, in contrast, induction was found in heat treated fruit. The pericarp of heat-acid treated fruit exhibited significantly lower respiration rate but faster water loss than that of the untreated or heat treated fruit. Taken together, heat treatment triggered quick browning and anthocyanin loss in lychee fruit, while heat-acid treatment protected the fruit color by a great reduction in the activities/gene expression of anthocyanin degradation enzymes and acidification of lychee pericarp.

Key changes in denervated muscles and their impact on regeneration and reinnervation

The neuromuscular junction becomes progressively less receptive to regenerating axons if nerve repair is delayed for a long period of time. It is difficult to ascertain the denervated muscle＇s residual receptivity by time alone. Other sensitive markers that closely correlate with the extent of denervation should be found. After a denervated muscle develops a fibrillation potential, muscle fiber conduction velocity, muscle fiber diameter, muscle wet weight, and maximal isometric force all decrease; remodeling increases neuromuscular junction fragmentation and plantar area, and expression of myogenesis-related genes is initially up-regulated and then down-regulated. All these changes correlate with both the time course and degree of denervation. The nature and time course of these denervation changes in muscle are reviewed from the literature to explore their roles in assessing both the degree of detrimental changes and the potential success of a nerve repair. Fibrillation potential amplitude, muscle fiber conduction velocity, muscle fiber diameter, mRNA expression levels of myogenic regulatory factors and nicotinic acetylcholine receptor could all reflect the severity and length of denervation and the receptiveness of denervated muscle to regenerating axons, which could possibly offer an important clue for surgical choices and predict the outcomes of delayed nerve repair.

Chronic neuroprotective effects of low concentration lithium on SH-SY5Y cells:possible involvement of stress proteins and gene expression

To investigate the molecular mechanism underlying the neuroprotective effect of lithium on cells, in this study, we exposed SH-SY5Y cells to 0.5 mmol/L lithium carbonate （Li2CO2） for 25-50 weeks and then detected the expression levels of some neurobiology related genes and post-translational modifications of stress proteins in SH-SYSY cells, cDNA arrays showed that pyruvate kinase 2 （PKM2） and calmodulin 3 （CaM 3） expression levels were significantly down-regulated, phosphatase protein PP2A expression was lightly down-regulated, and casein kinase II （CK2）, threonine/tyrosine phosphatase 7 （PYST2）, and dopamine beta-hydroxylase （DBH） expression levels were significantly up-regulated. Besides, western blot analysis of stress proteins （HSP27, HSP70, GRP78 and GRP94） showed an over-expression of two proteins： a 105 kDa protein which is a hyper-phosphorylated isoform of GRP94, and a 108 kDa protein which is a phosphorylated tetramer of HSP27. These results suggest that the neuroprotective effects of lithium are likely related to gene expressions and post-translational modifications of proteins cited above.

Expression analysis of eight amphioxus genes involved in the Wnt/β-catenin signaling pathway

The Wnt/β-catenin signaling pathway plays a crucial role in the embryonic development of metazoans. Although the pathway has been studied extensively in many model animals, its function in amphioxus, the most primitive chordate, remains largely uncharacterized. To obtain basic data for functional analysis, we identified and isolated seven genes （Lrp5/6, Dvl, APC, Ckla, CklS, Gsk3β, and Gro） of the Wnt/β-catenin signaling pathway from the amphioxus （Branchiostoma floridae） genome. Phylogenetic analysis revealed that amphioxus had fewer members of each gene family than that found in vertebrates. Whole-mount in situ hybridization showed that the genes were maternally expressed and broadly distributed throughout the whole embryo at the cleavage and blastula stages. Among them, Dvl was expressed asymmetrically towards the animal pole, while the others were evenly distributed in all blastomeres. At the mid-gastrula stage, the genes were specifically expressed in the primitive endomesoderm, but displayed different patterns. When the embryo developed into the neurula stage, the gene expressions were mainly detected in either paraxial somites or the tail bud. With the development of the embryo, the expression levels further decreased gradually and remained only in some pharyngeal regions or the tail bud at the larva stage. Our results suggest that the Wnt/β-catenin pathway might be involved in amphioxus somite formation and posterior growth, but not in endomesoderm specification.

Expression analysis of reactive oxygen species detoxifying enzyme genes in Anopheles stephensi during Plasmodium berghei midgut invasion

Objective:To investigate the involvement of reactive oxygen species detoxification system in Anopheles stephensi during Plasmodium berghei midgut invasion.Methods:Eight key reactive oxygen species metabolizing enzymes were cloned and characterized,and their expression was monitored in parasite-infected mosquitoes.Results:Superoxide anion detoxifying superoxide dismutases(Fe/Mn SOD,Cu/Zn SOD 2,Cu/Zn SOD 3A,and Cu/Zn SOD 3B)depicted varied expression patterns.Fe/Mn SOD expression declined,whereas Cu/Zn SOD expression was elevated in the infected mosquitoes.Peroxidases,catalase and glutathione peroxidase showed lack of induction in expression during the Plasmodium berghei infection.Further,expression of thioredoxin reductase increased in the infected mosquitoes,whereas gluthathione S-transferase levels decreased markedly.Conclusions:Detoxification enzymes may play a role in modulating host immunity and parasite transmission.

DIFFERENTIAL EXPRESSION OF GENES INVOLVED IN METABOLISM BETWEEN TUMORIGENITIC HUMAN LEUKEMIA CELL LINES K562 AND K562-n

Objective: To study the molecular mechanism ofdifferent tumorigenicity in nude mice of human leukemiacell lines K562-n and K562. Methods: To analyze the genes differently expressed between K562 and K562-n cells byusing cDNA microarray technique. Results: Among the12800 genes detected, some genes involved in materialmetabolism and material transport were differentlyexpressed between K562-n and K562 cells. These genesinclude homo sapiens placenta-specific ATP-binding cassette transporter gene, dihydrodiol dehydrogenase gene, hepatic dihydrodiol dehydrogenase gene, NAD-dependent methylene tetrahydrofolate dehydrogenase cyclohydrolase,lysophosphatidic acid acyltransferase, alpha gene,argininosuccinate lyase gene, mitochondrial isocitrtatedehydrogenase, adhesion protein SQM1 gene, dimethylarginine dimethylamino-hydrolase gene, M1subunit of ribonucleotide reductase and farnesylpyrophosphate synthetase gene. Conclusion: The hightumorigenicity of K562-n cells is related to the differentexpression of some genes concerned with cell metabolismand material transpoert.

CONSTRUCTION OF THE DICISTRONIC ADENOVIRUS VECTOR EXPRESSING BIOACTIVE HUMAN INTERLEUKIN-12

The full-length cDNA encoding the subunits p40 and p35 of human interleukin12(hIL12) were cloned separately by RTPCR, linked together by internal ribosomal entry site (IRES) of encephalomyocarditis virus which initiates capindependent translation to form a dicistronic gene fragment. The dicistronic fragment was placed between the cytomegalovirus (CMV) promoter and SV40 polyA signal to form a dicistronic expression cassette. Subsequently, the dicistronic expression cassette was inserted into E1 region of Ad5 genome in cosmid vector pAx1cw of E1substitution type. By homologous recombination with EcoT22Idigested Ad5 DNATPC in 293 cells, the replicationdeficient recombinant adenoviruses of hIL12 were generated efficiently. After infected with hIL12 recombinant adenoviruses in vitro, 293 cells, human hepatocellular carcinoma cells HepG2, and primary human skin fibroblasts expressed and secreted hIL12 at comparable levels (30～60ng/ 106cells/24hr), which could stimulate the proliferation and IFNγ production of human lymphoblasts. These suggest that the dicistronic adenovirus vector of hIL12 could effectively mediate the expression of bioactive hIL12 and might be used in cancer gene therapy.

High Efficiency Gene Transfer to Autologous Rabbit Jugular Vein Grafts Using Recombinant Adenovirus─Transferrin／Polylysine─D

Thesedatashowthathigh-efficientgenetransductioncanbeachievedinautologousveingraftsusingadenovirus-transferin/polylysine-DNAcomplexes.Thismethodoferssomeadvantagesoverrecombinantretrovirusofadenovirusvectors.Exvivogenetherapyofveingraftsmayreducegraftocclusionbytheintroductionandhighlevelexpresionofantithromboticand/orantiproliferativegenes.

Improved in situ sequencing for high-resolution targeted spatial transcriptomic analysis in tissue sections

Spatial transcriptomics enables the study of localization-indexed gene expression activity in tissues,providing the transcriptional landscape that in turn indicates the potential regulatory networks of gene expression.In situ sequencing(ISS)is a targeted spatial transcriptomic technique,based on padlock probe and rolling circle amplification combined with next-generation sequencing chemistry,for highly multiplexed in situ gene expression profiling.Here,we present improved in situ sequencing(IISS)that exploits a new probing and barcoding approach,combined with advanced image analysis pipelines for high-resolution targeted spatial gene expression profiling.We develop an improved combinatorial probe anchor ligation chemistry using a 2-base encoding strategy for barcode interrogation.The new encoding strategy results in higher signal intensity as well as improved specificity for in situ sequencing,while maintaining a streamlined analysis pipeline for targeted spatial transcriptomics.We show that IISS can be applied to both fresh frozen tissue and formalin-fixed paraffin-embedded tissue sections for single-cell level spatial gene expression analysis,based on which the developmental trajectory and cell-cell communication networks can also be constructed.

TIGER:A Web Portal of Tumor Immunotherapy Gene Expression Resource

Immunotherapy is a promising cancer treatment method;however,only a few patients benefit from it.The development of new immunotherapy strategies and effective biomarkers of response and resistance is urgently needed.Recently,high-throughput bulk and single-cell gene expression profling technologies have generated valuable resources.However,these resources are not well organized and systematic analysis is difficult.Here,we present TIGER,a tumor immunotherapy gene expression resource,which contains bulk transcriptome data of 1508 tumor samples with clinical immunotherapy outcomes and 11,057 tumor/normal samples without clinical immunotherapy outcomes,as well as single-cell transcriptome data of 2,116,945 immune cells from 655 samples.TIGER provides many useful modules for analyzing collected and user-provided data.Using the resource in TIGER,we identified a tumor-enriched subset of CD4^(+)T cells.Patients with melanoma with a higher signature score of this subset have a significantly better response and survival under immunotherapy.We believe that TIGER will be helpful in understanding anti-tumor immunity mechanisms and discovering effective biomarkers.

Survivin的肿瘤临床研究

Survivin是凋亡抑制蛋白家族的新成员,其具有肿瘤特异性,只表达于肿瘤和胚胎组织,且与肿瘤细胞的分化增殖及浸润转移密切相关。本文综述了survivin的生物学特性,并着重阐述了survivin与bcl-2和bax基因的关系及其在肿瘤诊断和治疗中的新进展。

Rutaecarpine Inhibits Angiotensin Ⅱ-Induced Proliferation in Rat Vascular Smooth Muscle Cells

Objective： To evaluate the effects and possible mechanisms of rutaecarpine on angiotensin Ⅱ （Ang Ⅱ ）-induced proliferation in cultured rat vascular smooth muscle cells （VSMCs）. Methods： VSMCs were isolated from Male Sprague-Dawley rat aorta, and cultured by enzymic dispersion method. Experiments were performed with cells from passages 3-8. The cultured VSMCs were randomly divided into control, model （Ang Ⅱ 0.1 μ moVL）, and rutaecarpine （0.3-3.0μmol/L） groups. VMSC proliferation was induced by Ang Ⅱ, and was evaluated by the 3-（4, 5-dimethylthiazol-2-yl）-2, 5-diphenyltetrazolium bromide assay and cell counting. To examine the mechanisms involved in anti-proliferative effects of rutaecarpine, nitric oxide （NO） levels and NO synthetase （NOS） activity were determined. Expressions of VSMC proliferation-related genes including endothelial nitric oxide synthase （eNOS）, and c-myc hypertension related gene-1 （HRG-1） were determined by real-time reverse chain reaction （RT-PCR）. Results： Rutaecarpine （0.3-3.0μmol/l_） inhibited Ang R-induced VSMC proliferation and the best effects were achieved at 3.0 μmol/L. The Ang Ⅱ-induced decreases in cellular NO contents and NOS activities were antagonized by rutaecarpine （P〈0.05）. Ang Ⅱ administration suppressed the expressions of eNOS and HRG-1, while increased c-myc expression （P〈0.05）. All these effects were attenuated by 3.0μmol/L rutaecarpine （P〈0.05）. Conclusion： Rutaecarpine is effective against Ang Ⅱ-induced rat VSMC proliferation, and this effect is due, at least in part, to NO production and the modulation of VMSC proliferation-related gene expressions.

Expression pattern and tissue localization of the class B scavenger receptor BmSCRBQ4 in Bombyx moil

Class B scavenger receptors （SR-Bs） are cell surface glycoproteins involved in various physiological processes in vivo, including the transport and metabolism of lipids, binding and phagoeytosis of xenobiotics, and signaling. But little information is available about silkworm SR-Bs; it is necessary to study these SR-Bs for revealing their function. In this study, we cloned the full-length coding sequence of BrnSCRBQ4, a SR-B gene from the silkworm Bombyx mori L. We found that the BmSCRBQ4 gene consists of nine exons and eight introns, with an open reading frame of 1371 bp encoding 456 amino acids. Gene expression studies determined that BmSCRBQ4 messenger RNA （mRNA） was expressed in unfertilized eggs, during embryonic development and throughout the majority of the larval period. Expression of mRNA was detected in the mid gut, middle silk gland, posterior silk gland, head, integumentum, fat body, testes and the ovaries of the larval B. mori Dazao strain, as well as in the silkworm cell lines BmN and BmE. Protein expression studies found BmSCRBQ4 protein was expressed only in the testes, fat body and middle silk gland of larvae, as well as in the silkworm cell lines BmN and BmE. The BmSCRBQ4 protein showed variability in banding patterns in different tissues and cells when analyzed by Western blotting. Immunohistochemical staining showed that the BmSCRBQ4 protein localizes to the constitutive membranes or cellular membranes of these tissues. These results indicated that BmSCRBQ4 gene may play some physiologically relevant roles at the cell surface in each tissue.

Gene expression and physiological responses to salinity and water stress of contrasting durum wheat genotypes

Elucidating the relationships between gene expression and the physiological mechanisms remains a bottleneck in breeding for resistance to salinity and drought. This study related the expression of key target genes with the physiological performance of durum wheat under different combinations of salinity and irrigation. The candidate genes assayed included two encoding for the DREB（dehydration responsive element binding） transcription factors Ta DREB1 A and Ta DREB2 B, another two for the cytosolic and plastidic glutamine synthetase（Ta GS1 and Ta GS2）, and one for the specific Na^＋/H^＋ vacuolar antiporter（Ta NHX1）. Expression of these genes was related to growth and different trait indicators of nitrogen metabolism（nitrogen content, stable nitrogen isotope composition, and glutamine synthetase and nitrate reductase activities）, photosynthetic carbon metabolism（stable carbon isotope composition and different gas exchange traits） and ion accumulation. Significant interaction between genotype and growing conditions occurred for growth, nitrogen content, and the expression of most genes.In general terms, higher expression of Ta GS1, Ta GS2,Ta DREB2 B, and to a lesser extent of Ta NHX1 were associated with a better genotypic performance in growth, nitrogen, and carbon photosynthetic metabolism under salinity and water stress. However, Ta DREB1 A was increased in expression under stress compared with control conditions, with tolerant genotypes exhibiting lower expression than susceptible ones.

Arbuscular mycorrhizal fungi inoculation and exogenous indole-3-acetic acid application induce antioxidant defense response to alleviate cadmium toxicity in Broussonetia papyrifera

Cadmium(Cd)contamination in soil poses a huge threat to plants even at low concentrations;Broussonetia papyrifera has great potential in remediation of soil heavy metal contamination.However,whether exogenous indole-3-acetic acid(IAA)application and arbuscular mycorrhizal fungi(AMF)have synergistic effects on Cd tolerance of B.papyrifera remains unclear.To investigate the effects of AMF inoculation and IAA application on the tolerance of B.papyrifera to Cd stress,two experiments were conducted:the first to investigate the effect of AMF(Rhizophagus irregularis)inoculation on the tolerance of B.papyrifera to Cd stress and the second to investigate the combined effects of AMF inoculation and IAA application on the tolerance of B.papyrifera to Cd stress.Parameters including endogenous hormone concentration,antioxidant defense response,malondialdehyde(MDA)content,and gene expression related to antioxidant enzyme system and hormone were measured.The results indicated that AMF alleviated Cd toxicity of B.papyrifera by reducing MDA content and improving antioxidant enzyme activities and Cd absorption capacity.Furthermore,the combination of AMF inoculation and IAA application had a synergetic effect on the tolerance of B.papyrifera to Cd stress through upregulating BpAUX1 and BpAUX2,which might contribute to root growth and root xylem synthesis,and by upregulating BpSOD2 and BpPOD34 to enhance the antioxidant enzyme system.This work provides a new insight into the application of IAA in the remediation of soil Cd pollution by mycorrhizal plants.