Mol.Sys.Bio_Coupling governs entrainment range of circadian (2010)

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Biodynes Series

盐酸苯丁胺弹性蛋白分析法临床评估水溶性弹性蛋白的方法）（临床评估水溶性弹性蛋白的方法）
在受控制的生长媒介中混合人类成纤维细胞生长48小时生长小时溶成剂和滤液
Abs (nm)
测试原料
不明浓度
毫克/毫升浓度 (毫克毫升毫克毫升)
在生长媒介中分离出水溶性弹性蛋白至15,000 MWC 弹性蛋白至
标准曲线
非水溶性细胞基质 (经测试后移走经测试后移走) 经测试后移走 UV 试管加入盐酸苯丁胺弹性蛋白染料
水溶性弹性蛋白体外测试结果
成纤维细胞内弹性蛋白的生产
0.5 0.45 0.4
毫克弹性蛋白毫克弹性蛋白/毫升
*
0.35 0.3 0.25 0.2 0.15 0.1 0.05 0
对照 (未处理) Biodynes TRF (0.1 wt% 活性) Biodynes EMPP (0.1 wt% 活性)
Biodynes® EMPP (可溶性弹性蛋白合成因子)
Biodynes® EMPP
Biodynes® EMPP属于酵母生物发酵溶成剂。它利用独特的营养物质和弹性蛋白促进剂作为生长媒介生产而成该弹性蛋白促进剂主要基于非动物来源的LMW氨基葡聚糖该酵母遭受各种独特外界压力损害后进一步加强酵母本省具有的自然促动力为了改善最终产品的味道和颜色，我们将溶成剂过滤
UV试管试管加入盐酸苯丁胺弹性蛋白染料
水溶性弹性蛋白体外测试结果 MatTek仿制皮肤组织仿制皮肤组织
水溶性弹性蛋白含量
175 170 165
水溶性弹性蛋白
* *
160
(毫克/组织组织)
155 150 145 140 135 130 对照样品

Full-length mRNA-Seq from single-cell levels of RNA and individual circulating tumor cells

cells 7,8. The method successfully detected thousands of genes expressed in mouse oocytes,and showed increased sensitivity compared with microarrays 7. However, this first single-cell mRNA-Seq experiment lacked technical controls, making it impossible to distinguishbiological variation between different cells from the technical variation that is intrinsic to cDNA amplification protocols when starting with low amounts of RNA. Therefore, thequestion remained whether single-cell transcriptomes faithfully represent the RNApopulation before amplification and how technical variation limits the power to finddifferential expression. This initial mRNA-Seq method also preferentially amplified the 3′ends of mRNAs, and hence the data could only be used to identify distal splicing events.Recently, a method for multiplexed single-cell RNA-Seq was introduced that quantifiestranscripts through reads mapping to mRNA 5′ ends 9. Neither of these methods generates read coverage across full transcripts. Since most mammalian multi-exons genes are subject to alternative RNA processing 4,5, there is a need for a single-cell transcriptome method that can both quantify gene expression and provide the coverage for efficient detection oftranscript variants and alleles.In this study, we introduce a single-cell RNA-Sequencing protocol with markedly improved transcriptome coverage, which samples cDNAs from more than just the ends of ing this protocol, we have sequenced the mRNAs from a large number of individualmammalian cells, as well as well-defined dilution series of purified total RNAs, tocomprehensively assess how sensitivity, variability and detection of differential expression vary with different amounts of starting material. Our results demonstrate the power ofsingle-cell RNA-Seq for both transcriptional and post-transcriptional studies, and provide valuable insights into the design of experiments that start from few or single cells. Todemonstrate the biological importance of this method, we have applied this new assay to putative circulating tumor cells (CTCs) captured from the blood of a melanoma patient to demonstrate how Smart-Seq enables high-quality transcriptome mapping in individual,clinically important cells.ResultsEfficient and robust single-cell RNA-Sequencing using Smart-SeqFor Smart-Seq, first we lysed each cell in hypotonic solution and converted poly(A)+ RNA to full-length cDNA using oligo(dT) priming and SMART template switching technology,followed by 12-18 cycles of PCR preamplification of cDNA. To enable gene and mRNA isoform expression analyses in single cells, a novel full-transcriptome mRNA-Seq protocol (Smart-Seq) was developed. Smart-Seq makes use of SMART ™ template switchingtechnology for the generation of full-length cDNAs and only 12 to 18 cycles of PCRfollowing the initial cDNA synthesis steps. The amplified cDNA was used to constructstandard Illumina sequencing libraries using either Covaris shearing followed by ligation of adaptors (PE) or Tn5-mediated “tagmentation” using the Nextera technology (Tn5). Both of these library preparation methods enable random shotgun sequencing of cDNAs(Supplementary Fig. 1). We successfully generated Smart-Seq libraries from 42 individual human or mouse cells, and in addition we generated 64 libraries from dilution series of total RNA derived from human brain (16 samples), mouse brain (28 samples) and universalhuman reference RNA (UHRR, 20 samples). Each sequencing library was sequenced on the Illumina platform, typically generating over 20 million uniquely mapping reads(Supplementary Table 1). For comparison, several standard mRNA-Seq libraries were also made from 100 ng to a few micrograms of total RNA.NIH-PA Author Manuscript NIH-PA Author ManuscriptNIH-PA Author ManuscriptSmart-Seq improves coverage across transcriptsIn previous single-cell mRNA-Sequencing studies 7,8, the data suffered from a pronounced 3′-end bias that limited analysis across full-length transcripts. We sequenced single-celltranscriptomes from mouse oocytes to enable a direct comparison with published mouse oocyte single-cell data 7. Analyses of read coverage across transcripts demonstrated thatSmart-Seq has significantly improved full-length coverage of all transcripts longer than 1kb (Fig. 1a and Fig. S2d–k). Smart-Seq analyses of mouse brain RNA at different dilutionsshowed that even better coverage was obtained with increased starting amounts, withnanogram dilutions reaching close to the coverage observed using standard mRNA-Seq from 100 ng to 1 ug total RNA (Fig. 1b). From only 10 pg input amounts (the estimated amount of RNA in a small eukaryotic cell, Supplementary Table 2) we achieved close to 40%coverage at the 5′ end. Analyses of single-cell transcriptomes from cancer cell lines (four cells each from LNCaP, PC3 and T24) obtained equally good read coverage (Fig. 1c) and,indeed, for 25% of all expressed, multi-exon genes our read coverage enabled full-length transcript reconstruction (exemplified in Fig. S3). We conclude that Smart-Seq hassignificantly improved read coverage compared to previous single-cell transcriptomemethods.Quantitative assessment of single-cell transcriptomicsAnalyses of gene expression from millions of cells using mRNA-Seq is highly reproducible and has low technical variation 1,4. So far, no single-cell mRNA-Seq study has measured the technical variation intrinsic to the cDNA pre-amplification components of single-cellmethods. We therefore diluted microgram amounts of reference total RNA down to nano-and picogram levels and applied Smart-Seq to assess sensitivity, technical variability and detection of differentially expressed transcripts of Smart-Seq on low amounts of total RNA.For comparison, standard mRNA-Seq libraries were generated from 100 ng to microgram levels of reference total RNA.First, we addressed the sensitivity of the method in detecting transcripts present at different expression levels. Starting with 10 ng or 1 ng of total RNA, we found no or minimal decline in sensitivity compared with standard mRNA-Seq. However, lowering the starting amounts to single-cell levels decreased the detection rate of less abundant transcripts (Fig. 2a).Analyses of the twelve cancer cell line cells (four cells each from the LNCaP, PC3 and T24lines) showed that ~76% of transcripts expressed at 10 RPKM (reads per kilobase exonmodel and million mappable reads), an expression level that roughly equals the medianexpression level for detected transcripts, were reproducibly detected in all single-cellprofiles (Fig. 2b). We found that the sensitivity of gene detection for the individual cancer cells was similar to that of about 20 pg of starting total RNA (Fig. 2b). Furthermore, weobserved that the starting amount of total RNA had a larger impact on sensitivity than the number of PCR cycles used (Supplementary Fig. 5) and that the sequence depth had little effect on transcript detection at levels above a million uniquely mapping reads per cell, with expression levels stabilizing after 3 million uniquely mapped reads (Supplementary Fig.4b,c). Comparisons of Smart-Seq and previous mouse oocyte data 7 demonstrated similar sensitivity (Supplementary Fig. 2a,b). We conclude that transcript detection sensitivity is affected by limiting starting amounts of RNA that lead to random loss of low abundance transcripts, but still the majority of low abundance and the vast majority of highly expressed transcripts are reliably detected even in single cells.Second, we determined the reproducibility in expression levels generated from diluted RNA and individual cells. Comparison of Smart-Seq and previous mouse oocyte data 7demonstrated improved expression level estimation with Smart-Seq (lower oocyte to oocyte variability) across the whole range of expression levels (Supplementary Fig. 2c). Correlation NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscriptanalyses between technical replicates of diluted RNA showed increasing concordance with larger amounts of RNA. Comparing the single cells against the RNA dilution, we observed higher correlations (Pearson correlations of 0.75–0.85) among individual cells of the same type than among dilution replicates at 10 pg (Pearson correlations of 0.65–0.75)(Supplementary Fig. 6). Since variability in measurements of expression levels depends on transcript expression levels, we computed the variability as a function of the expressionlevel (Fig. 2c,d). This analysis showed that Smart-Seq on 10 ng total RNA had the same technical variability as standard mRNA-Seq and that Smart-Seq on 1 ng total RNA showed only a modest increase in technical noise (Fig. 2c). When lowering input amounts down to picogram levels, there was a clear increase in technical variability, particularly for lessabundantly expressed transcripts (Fig. 2c). The levels of technical variability at picogram levels of total RNA were compared to the biological variation found in comparisons ofhuman brain and UHRR using standard mRNA-Seq (Fig. 2c, green line). Interestingly,analyses of variation between individual cancer cells of different origin revealed extensive biological variation in highly expressed genes (Fig. 2d).Finally, we assessed whether pre-amplified single-cell expression profiles wererepresentative of the original expression profiles. Comparing relative gene expression levels (UHRR - brain) estimated using standard mRNA-Seq to those estimated from Smart-Seq with different amounts of input RNA, we again found a high concordance (Fig. 2e–g).Starting with 1 ng or 100 pg total RNA, the relative expression in Smart-Seq and standard mRNA-Seq respectively had Spearman correlations of 0.87 and 0.77 (Fig. 2e,f).Comparisons with 10 pg input RNA showed overall good correlation (Fig. 2g), butidentified two populations of transcripts with distorted expression in Smart-Seq data from either human brain or UHRR, reflecting stochastic losses, mostly of low abundancetranscripts when starting with such minute RNA of levels (Fig. 2g and Fig. 2a). Pre-amplification of cDNA could also lead to disproportionate amplification of short transcripts,but we found no systematic bias (Supplementary Fig. 7). A previous microarray studyanalyzed PCR amplified cDNA (from picogram levels) and found the transcriptome overall preserved, but skewed 10. Our data from 1 ng and 100 pg total RNA show no skewing, i.e.the loess slopes estimated from the data approximated 1 (Fig. 2e–g). Together, these results demonstrated that transcriptome analyses from few or single cells, in general, preservedrelative expression level differences for detected transcripts.Analyses of transcriptional and post-transcriptional differences from single-cellsHaving demonstrated the improved performance of Smart-Seq on low amounts of RNAcompared to previously published methods, we focused our analyses on single-celltranscriptomes from prostate (PC3, LNCaP) and bladder (T24) cancer cell line cells. The twelve individual cells (four from each cell line) clustered according to cell line of origin using their global gene expression levels and we identified hundreds of differentiallyexpressed genes among the three cell lines (Fig. 3a; q<0.05 ANOVA; p<0.05 post-hoc test).The pronounced 3′-end bias of previous single-cell mRNA-Seq studies has hampered the ability to identify significant alternative splicing differences in single cells. We used theBayesian mixture of isoforms framework (MISO)11 to infer exon inclusion levels for known alternatively spliced exons in the twelve individual cells. The improved read coverage with Smart-Seq resulted in a two-fold increase in the number of alternatively spliced exons that could be assessed, compared to previously published single-cell mRNA-Seq data (Fig. 3b),significantly improving our ability to detect alternative splicing. Cell-type specificalternative splicing could be inferred from single-cell transcriptomes, as seen in readcoverage across the differentially included exon 13 of the NEDD4L gene (Fig. 3c). This exon was frequently included in LNCaP cells (93% mean inclusion level) but was included at much lower levels in T24 cells (15% mean inclusion levels) whereas low expression ofNIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptNEDD4L in PC3 cells precluded inclusion level estimation. In total, in this comparison of three cancer cell lines, we found one hundred exons with differential exon inclusion levels among the three cell lines, with a less than 1% false discovery rate (Fig. 3d; Supplementary Table 3). We conclude that Smart-Seq significantly improves our ability to detect alternative RNA processing in single cells.Analyses of circulating tumor cell transcriptomes Having demonstrated that Smart-Seq generates quantitative and reproducible single-cell transcriptomes, we asked whether global transcriptome analyses of putative circulating tumor cells (CTCs) could reveal their tumor of origin and provide data to support the use of this method for unbiased cancer-specific biomarker identification. To this end, we generated transcriptomes from NG2+ putative melanoma circulating tumor cells (CTCs) isolated from peripheral blood drawn from a patient with recurrent melanoma using immunomagnetic purification with a MagSweeper instrument (Illumina Inc.)12. For comparison, we also generated Smart-Seq libraries from single cells derived from primary melanocytes (PMs,n=2), melanoma cancer cell line (SKMEL5, n=4 and UACC257, n=3) cells and from human embryonic stem cells (ESCs, n=8). Since the NG2+ putative CTCs were isolated from blood,it was important to compare them to blood cells. The putative CTCs were distinct from lymphoma cell lines (BL41 and BJAB)13 and immune tissues (lymphnode and white blood cell samples), as well as embryonic stem cells, and instead showed high similarity to PMs and melanoma cell line cells. Unsupervised hierarchical clustering and correlation analyses of gene expression levels showed a clear clustering of cells according to cell type of origin (Fig. 4a and Supplementary Fig. 8), and separation from the human brain RNA samples that were previously analyzed with Smart-Seq or mRNA-Seq (data not shown). Further support for the melanocytic origin of the putative melanoma CTCs came from analyses of melanocyte lineage specific markers, as all NG2+ cells expressed high levels of MLANA 14,TYR 15 and the melanocyte specific m-form of MITF 16 but not immune markers such as PTPRC (Fig. 4b), in contrast to peripheral blood lymphocytes (Supplementary Fig. 9).Furthermore, NG2+ cells expressed high levels of melanoma-associated genes (based on our unbiased selection of the 100 transcripts most strongly associated with melanoma, seeMethods), but not immune cell-associated genes selected in a similar manner (Fig. 4c,p<3.7e-15, Wilcoxon rank sum test). Thus, both their global transcriptomes and expression patterns of melanoma-associated transcripts clearly support a melanocyte origin for theNG2+ cells putative melanoma CTCs.We next investigated whether the NG2+ putative CTCs showed signs of originating from a melanoma tumor. Comparison of their gene expression profiles with those of individual PMs identified 289 genes with higher expression in the putative CTCs than the PMs, and 436genes with significantly lower levels (Supplementary Table 4). The up-regulated genes were significantly enriched (FDR<0.05) for melanoma-associated antigens (Fig. 4d andSupplementary Fig. 10) that have been repeatedly found to be upregulated in cancer 17,mitotic cell cycle genes and additional categories (Supplementary Table 5). Down-regulated genes showed enrichment for regulators of cell death and MHC class I genes. Interestingly,the preferentially expressed antigen in melanoma (PRAME) was found highly expressed in NG2+ cells, which together with elevated expression of known melanoma tumor antigens (MAGEs), provides strong support for the conclusion that the NG2+ cells were CTCs that have originated from a melanoma.In recent years, there has been a strong interest in identifying CTCs from different tumors using the a priori assumption that plasma-membrane proteins would be good diagnosticbiomarkers. We used the CTC transcriptome analysis to screen for membrane proteinsselectively expressed in melanoma-derived CTCs compared to PMs and immune cells. We identified 9 up-regulated plasma membrane-associated transcripts in the CTCs comparedNIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscriptwith primary melanocytes (q<0.05 ANOVA; p<0.05 post-hoc test), many of which are not expressed in immune cells and have not been previously associated with melanomas (Fig.4e). Similarly, screening for loss of expression of plasma-membrane proteins identified 37genes with significantly lower expression in the CTCs than PMs (Fig. 4f). Of note, epithelial Cadherin 1 (CDH1) showed no expression in the CTCs, and loss of CDH1 is thought tocontribute to cancer progression by increasing proliferation, invasion and metastasis 18. We also found downregulation of genes associated with the escape from immune surveillance,including five HLA genes (Fig. 4f), and TRPM1, suggesting that these gene expressionchanges might enable the CTCs to escape from immune surveillance. Notably, lowexpression of TRPM1 has been shown to correlate with melanoma aggressiveness andmetastasis 19. Future studies of these membrane proteins will likely enhance ourunderstanding of CTC migration and invasiveness, and these results highlight the utility of studying single CTC cells with RNA-Seq.Lastly, we investigated whether Smart-Seq transcriptome data could be mined for SNPs and other genetic variants associated with melanomas or other cancers. With the improved read coverage provided by the Smart-Seq method, we were able to identify 4,312 high-confidence genomic sites with support for an alternative allele in at least two CTCs, whereas genotype calls only supported by a single cell showed an excess of novel, likely artifactual,sites (Supplementary Fig. 11) together with a smaller subset (9%) of A-to-G RNA editing sites (data not shown). Ninety-two percent of the high-confidence sites coincided withdocumented SNPs, for example the melanoma-associated SNP in the TYR gene(rs1126809)20 (Fig. 4g). We conclude that Smart-Seq enables screening for SNPs andmutations in transcribed regions using only few cells.Discussion Generating high-coverage transcriptomes from single cells and small numbers of cells will have many applications for studying rare cells; such cells can be either individually picked or identified through cell sorting or laser capture techniques. Our results demonstrate that using Smart-Seq on 10 ng of total RNA is practically indistinguishable from a standard mRNA-Seq, whereas starting with 1 ng (corresponding roughly to 50–100 cells) shows only a minor increase in expression level variability. Therefore, this method could easily be applied to studies on homogeneous cell populations. However, many biologically and clinically important cell types exist in rare quantities and often in heterogeneous milieus,which necessitates single-cell approaches. Smart-Seq generates robust and quantitative transcriptome data from single cells. We found hundreds of differentially expressed genes using only a few individual cells per cell type, e.g. comparing only two primary melanocytes to six melanoma CTCs identified biologically meaningful differences. Even sequencing of a single cell yields useful information, as we, in each cell, detected most of the genes active in a culture of LNCaP cells. Importantly, Smart-Seq has significantly improved read coverage across transcripts, which enables detailed analyses of alternative splicing and identification of SNPs and mutations. Based on our CTC transcriptome results, single-cell analyses are highly informative for identifying candidate biomarkers, SNPs and mutations. In conclusion,datasets obtained with the Smart-Seq protocol provide significantly improved representation of the transcriptomes of individual cells, with relevance for both basic and clinical studies.Methods Generation and amplification of Smart-Seq cDNAWe have developed a new method for the generation of cDNA from total RNA or fromsingle cells, called Smart-Seq. Briefly, polyA + RNA was reverse transcribed through tailed oligo-dT priming directly in total RNA or a whole cell lysate using Moloney MurineNIH-PA Author Manuscript NIH-PA Author ManuscriptNIH-PA Author ManuscriptLeukemia Virus Reverse Transcriptase (MMLV RT). Once the reverse transcription reaction reaches the 5′ end of an RNA molecule, the terminal transferase activity of MMLV adds a few non-templated nucleotides to the 3′ end of the cDNA. The carefully designedSMARTer II A oligo then base-pairs with these additional nucleotides, creating an extended template. The reverse transcriptase then switches templates and continues transcribing to the end of the oligonucleotide. The resulting full-length cDNA contains the complete 5′ end of the mRNA, as well as an anchor sequence that serves as a universal priming site for second strand synthesis. The cDNA is then amplified using 12 cycles for 1 ng of total RNA, 15cycles for 100 pg of total RNA, and 18 cycles for 10 pg total RNA or from single cells. The exact number of cycles for each dilution replicate or single-cell is detailed in Supplementary Table 1. The Smart-Seq cDNA generation and amplification methods developed for this manuscript have recently become available in a kit marketed by Clontech called the“SMARTer Ultra Low RNA Kit for Illumina sequencing”. Although all the libraries in this manuscript were generated before the kit became commercially available, our protocol is reflected in the detailed instructions for generating cDNA from few cells or 100 pg–10 ng of total RNA that is now included in the manual for this kit. For single cell applications, each cell (or control RNA) was added in max 1 λ of media to 4 λ of hypotonic lysis bufferconsisting of 0.2% Triton X-100 and 2 U/μl of ribonuclease (RNase) inhibitors (Clontech,2313B) in RNase free water. The deposition of an intact cell in the hypotonic lysis buffer leads to immediate lysis and stabilization of the RNA through RNase inhibitors. Then,poly(A)+ RNA was reverse-transcribed through tailed oligo(dT) priming using the CDSprimer (5′-AAGCAGTGGTATCAACGCAGAGTACT(30)VN-3′, where V represents A, C or G) directly in total RNA or a whole cell lysate using Moloney murine leukemia virusreverse transcriptase (MMLV RT).Construction and sequencing of Smart-Seq sequencing librariesAmplified cDNA (~5ng cDNA) was used to construct Illumina sequencing libraries using either Illumina’s “Ultra Low Input mRNA-Seq Guide” (the “PE” protocol) or a modification of Epicentre’s Nextera DNA sample preparation protocol (the “Tn5” protocol). With the PE protocol, the amplified cDNA was fragmented using a Covaris acoustic shearing instrument.The resulting fragments were end-repaired, followed by the addition of a single A base,ligation to Illumina PE adaptors, and then amplification with 12–18 cycles of PCR(depending on starting amounts of RNA, see Supplementary Table 1 for detailed instructions of all libraries generated). With the Tn5 protocol, the amplified cDNA was “tagmentated” at 55°C for 5 min in 20μl reaction with 0.25μl of transposase and 4μl of 5x HMW Nextera reaction buffer. 35μl of PB was added to the tagmentation reaction mix to strip thetransposase off the DNA, and the tagmentated DNA was purified with 88μl of SPRI XP beads (sample to beads ratio of 1:1.6). Purified DNA was then amplified by 9 cycles ofstandard Nextera PCR. The libraries were sequenced on either Illumina’s HiSeq 2000,GAIIx or MiSeq instruments and all clusters that passed filter were exported into fastq files.Details on the sequence depth, sequencing platform and library construction method for each dilution replicate and single cell are included in Supplementary Table 1. All data shown in the figures of this manuscript were generated using the PE protocol unless otherwisespecified in the figure legend.Construction and sequencing of standard mRNA-Seq librariesWe generated mRNA-Seq transcriptome data following the Illumina mRNA-Seq kit from 100 ng and 1 microgram of total RNA, as detailed in Supplementary Table 1.Isolation of individual CTCs from peripheral blood10 ml of peripheral blood was collected from a male patient with recurrent, metastaticmelanoma using K2 EDTA blood collection tubes (Becton Dickinson, NJ, USA). The bloodNIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscriptsample was processed within 3 hours of collection. The erythrocytes in 4.5 ml of the blood sample were lysed with BD Pharm Lyse ™ lysing solution (Becton Dickinson, NJ, USA) for 10 minutes at room temperature. The nucleated cells were pelleted, resuspended in HBSS containing 1% BSA and 5mM EDTA, pelleted, resuspended in 1ml of HBSS containing 1%BSA and 5mM EDTA. The nucleated cells were stained with PE-conjugated anti-human CD45 IgG to label leukocytes. The cells were subsequently reacted with biotinylated anti-human CSPG4 (a.k.a. NG2) mouse IgG at 4 °C for 2 hours, washed with HBSS, and reacted with streptavidin-conjugated MG980A magnetic beads at 4 °C for 2 hours. The cells were captured based on magnetic sweeping to harvest the beads from cell suspension using the MagSweeper instrument (Illumina Inc.) as previously described 12. The harvested cells were stained with 5 μg/ml Calcein AM (Life Technologies, Carlsbad, CA) in HBSS for 20minutes to identify viable cells. Manual picking of viable cells showing desired Calcein-positive/CD45-negative/bead-attached profile was performed to isolate cells for molecular profiling. The individual cells were placed into 2.5 μl of Superblock (Thermo Scientific,Waltham, MA) containing 4000 Unit/ml RNase inhibitor (New England Biolabs, Ipswich,MA) and stored at −80 °C until preparation of Smart-Seq libraries.Isolation of mouse oocytes and human lymphocytesMII oocytes were isolated from 4-week old CAST/EiJ female mice. Mice weresuperovulated by injection of 5 IU PMSG, followed by injection of 5 IU of hCG 48 hrs later.MII oocytes were isolated 14–15 hrs after hCG treatment by dissection of the ampulla of the oviduct and cumulus cells were removed by hyaluronidase digestion. Single oocytes were manually picked, lysed in dilution buffer, and cDNA constructed as described above.Peripheral blood lymphocytes from healthy human volunteers were isolated on Ficollgradients using LymphoPrep (Fresenius Kabi, Norway). Individual cells were manuallypicked into lysis buffer and cDNA constructed as described above.Alignment of short reads to genome and transcriptomeReads were independently aligned using Bowtie 21 against the respective genome assembly (hg19 or mm9) and transcriptome sequences (Ensembl, human and mouse annotationsdownloaded 16th May 2011 and 13th Dec 2010, respectively). Transcriptome mapped reads were converted from transcriptome coordinates to genomic coordinates and thereaftercompared with the genome mapped reads to identify reads that map to a unique genomiclocation. This procedure ensured that mapped reads were unique across both the genome and transcriptome, while allowing for reads to map to different transcripts of the same gene in the initial transcriptome mapping. The uniquely mapped reads were converted to binaryBAM files using Samtools 22. The resulting transcriptome data was visualized using theIntegrated Genome Viewer (IGV, Broad Institute) using the histogram visualization for Fig.1a,d and heatmap visualization for Supplementary Fig. 3.Expression level estimation and technical comparisons of sensitivity and variationGene expression levels for Refseq transcripts were summarized as RPKM values and read counts using rpkmforgenes 23. RefSeq annotations for human and mouse were downloaded on the 31st August 2011 and 13th Dec 2010 respectively. RPKM calculations onlyconsidered uniquely mappable positions for transcript length normalizations using theENCODE Mappability track (wgEncodeCrgMapabilityAlign50mer.bigWig) for human and in-house computed uniqueness files for mouse. Overlapping RefSeq transcripts werecollapsed giving one expression value per gene locus. Only 10 million randomly selected mapped reads were used per sample in order to compare sensitivity and variation in gene and exon levels. Samples with fewer than 10 million uniquely mappable reads (a few ES cells 8) were therefore discarded from analyses. Samples with 20 pg of total RNA (used in Fig. 2b,d) were simulated by using 5 million reads each of two different 10 pg samples.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript。

一株嗜酸真菌的分离鉴定及其胞外糖苷水解酶的产酶分析（英文）

一株嗜酸真菌的分离鉴定及其胞外糖苷水解酶的产酶分析（英文）吕飞龙;李江;刘亚洁;王剑锋;蔡向鲲【期刊名称】《农业科学与技术（英文版）》【年(卷),期】2012(013)006【摘要】[目的]旨在分离嗜酸性真菌并分析该真菌分泌的嗜酸性酶。

[方法]利用Oligot肝脏选择性培养基（pH 2.5），从江西省铀矿浸出溶液中分离出异养真菌，并命名为RBS-6。

然后根据其菌落形态和分子指示剂RDNA-ITS鉴定该菌株。

最后，分析了RBS-6分泌的糖苷水解酶。

[结果]这种真菌RBS-6是嗜酸性的，并且在pH4.0时繁体。

其rDNA-其序列与Phialophora SP的同源性最高（98％）共享。

CGMCC 3329（GU 082377）。

因此，它被鉴定为phialophora sp的真菌。

，并且被暂时被命名为phialophora sp。

RBS-6。

它可以生产六种糖苷水解酶，包括克服α-半乳糖苷酶葡糖苷酶，β-葡糖苷酶，β-甘露糖苷酶和β-葡聚糖酶。

所有酶都是嗜酸性的，最佳反应pH为3.0-4.0。

其中，β-葡聚糖酶在pH 3.5和50℃下表现出最高活性;此外，在50℃温育60分钟后，将58％的酶活性保持58％。

[结论]被鉴定为phialophora sp的嗜酸性构件的分离的真菌是一种产生嗜酸性酶的新菌株。

本研究为Phialophora Fungi的研究提供了新数据。

％[目的]分享到1株嗜酸真菌，研究其所产嗜。

[方法]利用型营养杂种（pH 2.5）选择性培养基，从江西某铀矿浸铀体内中分量到1株，为rbs-6，利用其其形态和分子指标rdna-its对其行鉴定，并分享了胞胞外荷水解酶。

[结果]该菌最适生命素pH值为4.0，属嗜酸性真菌。

/ 6的rdna-it序列与真菌phialophora sp。

CGMCC 3329（GU 082377）同源性同源性高，达达98％，故rbs-6为瓶霉属（phialophora），将其暂命名为phialophora sp。

120项世界生物医学新技术

落实科学发展观大力加强生物医学新技术的研究和开发120项世界生物医学新技术生物医学新技术是医学生物学发展的支撑和基础。

现代医学生物学的发展离不开生物医学技术的进展。

从显微镜、离心机、电泳仪、同位素、X-Ray 到现在的高通量、高灵敏的分析、测序、重组、克隆、转移、芯片、荧光、成像、纳米、合成、信息技术的发展，无一不引领着现在医学生物学的进步。

没有生物医学技术的创新和进步，就不会有现在和未来医学生物学的发展。

这里我们从Science, Nature, PNAS, Cell 以及国内外生物医学网站上摘录了2008—2009年120多项生物医学的新技术，供大家参考。

此外，我们在CMBI特别报道专栏中也全文报道了新技术（379）、心血管成像（368）、彗星测定（366）、荧光蛋白（363）、人工生命（331）、方法学（303）、系统生物学（272）、纳米医学（271）、生物标记（267）、抗体工程（251）、细胞与分子生物学方法（240）、活细胞成像（226）、组合化学（216）、虚拟细胞（199）、组织工程（186）、DNA疫苗（176）、生物芯片（122）等近20项做了专题报道，约有7000篇文献。

从这些报道中我们可以看出，生物医学技术层出不穷，突飞猛进。

向着更高、更精、更微、更快、更灵敏、更简便、更经济、更实用的方向发展；向着多学科、多技术、相互交叉的方向发展；向着高通量、多分子、多途径、多因素、多位点复杂体系的综合分析的方向发展；向着活体、动态、全功能、全过程的方向发展，使生物医学技术成为21世纪最关键和最核心，应用最广泛的技术领域，它为新一代医学生物学的发展，创造必要条件！一个更深入更伟大的生命科学新时代正在来临！成为世界各国经济、社会、科学发展竞争的焦点！我们必须迎头赶上，大力加强生物医学新技术的研究和开发。

这是我们刻不容缓的任务！这十年来我国生物医学取得了飞速的发展，每年在世界上发表的SCI论文，已近万篇。

双膦配体修饰铑催化乙酸乙烯酯氢甲酰化反应(英文)

（ｒｃｅ：ｎａｏ：ａｄｃｖｔＴＦ４０－ｎｅｏｔｕｏｄｔｎｅｅｃｉｅｓｇａ，’ ｉｄｈｎｌｈｓｈｏ — ｂａｈｄｌｅｒｆ９１ｎｔｉＯ：００ｈｕｄｒｐｉｍｃｎｉｏｓｒｈｅｄｂｕｉ２一ｓｉｅｙｏｐｉｎｉ９）ａｉｙ（１ｍｉｗａｖｙｎ２ｂ（ｐｐｎｍｅ
Ｃｈｎｄｕ６１０４ｉｈｎｅｇ０６，Ｓｃｕａ，Ｃｈｎａｉ
ＡｓａｔＲｏｉｍ— ｔｙｅｙｒｆｒｌｉｎｏｉｌｃｔｅｔｔｓｆｉｏｐｉｇｎｓｗｓｔｄｄＡｈ曲ｒｇｓｌｔｉ Байду номын сангаасｂｔｃ：ｈｄｕｃａｚｄｈｄｏｏｍｙｔｆｎｅｔｗｉｅｕｅｐｓｈｎｌａｄａｓｉ．ｉｅｉｅｃｖｔｒａｌａｏｖｙａａｈｈｏｄｈｅｉｕｅｏｅｉｙ
ｗｏｌｅｕｅｕｌｏｄｕｔｉｌｖｎｙｃｔｔｙｄｏｏｍｙａｉｎ．ｕｄｂｓｆｒｉｓｒａｉｌｅａｅｈｒｆｒｌｔｏｆｎａ
Ｋｅｏｄ：ｖｉｙｌｃｔｔ；ｙｒｏｍｙａｉｎ；ｅｏｓｌｃｉｉｙｉｈｏｐｎｉａｄ；－ｃｔｘｐｏｎａｙｗｒｓｎｅａｅｈｄｏｆｒｌｔｏｒｇｉｅｅｔｖｔ；ｄｐｓｈｉｅｌｇｎ２ａｅｏｙｒｐａｌａ
双膦配体修饰铑催化乙酸乙烯酯氢甲酰化反应
梁浩然，张林，郑学丽，海燕，袁茂林，李瑞祥，陈华付
四）大学化学学院绿色化学与技术教育部重点实验室，四）成都６０６ＪＩＪｌ１０４

非等位基因

非等位基因概述非等位基因是指同一基因座上的不同等位基因。

等位基因是指在某个给定的基因座上，可以存在多种不同的变体。

每个个体继承了一对等位基因，一对等位基因可能会导致不同的表型表达。

非等位基因的存在使得遗传学研究更加复杂，因为不同的等位基因会对个体的表型产生不同的影响。

背景在生物学中，基因座是指染色体上一个特定的位置，该位置上的基因决定了某个特征的表达方式。

每个基因座上可以有多种不同的等位基因。

等位基因是指在某个特定基因座上的不同基因变体。

每个个体都会继承一对等位基因，通过这对等位基因的不同组合，决定了个体的表型。

然而，并非所有基因座上的等位基因都具有相同的表现型。

非等位基因的影响非等位基因的存在导致不同等位基因会对个体表型产生不同的影响。

有些非等位基因会表现出显性效应，也就是说，当个体继承了一个突变的等位基因时，即使同时继承了一个正常的等位基因，但显性效应会使得突变的等位基因的表型表达得到体现。

相反，有些非等位基因会表现出隐性效应，当个体继承了两个突变的等位基因时，才会表现出突变的表型。

除了显性和隐性效应之外，非等位基因还可能发生两种其他类型的表型效应。

一种是共显效应，当个体继承了两个不同的突变等位基因时，在表型表达上会表现出一种新的特征，这个特征并不是单个突变等位基因所能导致的。

另一种是部分显性效应，当个体继承了两个不同的突变等位基因时，表型表达将介于两个单独突变等位基因的表型之间。

重组和非等位基因重组是指两个不同的染色体交换部分基因序列的过程。

在重组的过程中，非等位基因可能会发生改变，导致新的等位基因组合形成。

这一过程使得非等位基因的表型效应更加复杂，因为新的等位基因可能将不同基因座的效应组合起来。

非等位基因的重要性非等位基因对生物的适应性和多样性起着重要作用。

通过对等位基因的各种组合的研究，人们可以更好地理解基因与表型之间的关系，并揭示遗传变异对物种适应环境的重要性。

总结非等位基因是指同一基因座上的不同等位基因。

博士复试英文PPT

2015.09-
Master degree
****
Awards
Professional Third-class Scholarship from 2015-2018 academic years
Education Experience
• Traditional Chinese Medicine Bureau of Guangdong Province (NO. 20141065), • Natural Science Foundation of Guangdong Province (NO. 2017A32217007) • National University Student Innovation Program.
MenaINV for invasive feature Mena11a for epithelial-specific feature
Background
Two main AS regulators heterogeneous nuclear ribonucleoprotein (hnRNPs) and serine/arginine-rich (SR) proteins. Polypyrimidine Tract-Binding Protein 1 (PTBP1, also known as hnRNP I) binds preferentially to pyrimidine-rich sequences.
Objectives
Regulated by PTBP1 ?
The exact mechanism mediated Mena AS has not been elucidated. The purpose of this study is to explore the molecular mechanism of PTBP1 in regulating Mena alternative splicing and role of PTBP1 in lung carcinoma cells metastasis.

国际上著名的从事药剂学研究的专家

Intra Oral Delivery (口腔内传递)直接由口腔黏膜吸收,瞬间进入血液循环,有效成分不流失。

Universities, Departments,FacultiesResearchersButler University College of Pharmacy and Health Sciences Health Sciences USA Associate Professor Nandita G. DasMain focus on her research facilities are about peformulation, biopharmaceutics, drug targeting, anticancer drug delivery.Purdue University School of Pharmacy and Pharmacal Sciences Department of Industrial and Physical Pharmacy (IPPH) USA Professor Kinam ParkControlled Drug Delivery, Glucose-Sensitive Hydrogels for Self-Regulated Insulin Delivery, Superporous Hydrogel Composites, Oral Vaccination using Hydrogel Microparticles, Fractal Analysis of Pharmaceutical Solid Materials.St. John's University School of Pharmacy and Allied Health ProfessionsUSA Professor Parshotam L. MadanControlled and targeted drug delivery systems; Bio-erodible polymers as drug delivery systemsThe University of Iowa College of Dentistry Department of Oral Pathology, Radiology, and Medicine USA Professor Christopher A. Squierpermeability of skin, and oral mucosa to exogenous substances, including alcohol and tobacco, and drug deliveryThe University of Iowa College of Pharmacy Department of Pharmaceutics USA Associate Professor Maureen D. DonovanMucosal drug delivery especially via the nasal, gastrointestinal and vaginal epithelia; and mechanisms of drug absorption and disposition.The University of Texas at San Antonio College of Engineering Department of Biomedical Engineering USA Professor Jeffrey Y. ThompsonDental restorative materials and implantsThe University of Utah Pharmaceutics & Pharmaceutical Chemistry USA Professor John W. MaugerDr. Maugner is mainly focused on dissolution testing and coating technology of orally administered drug products with bitter taste about which he is one of the inventors of a filed patent.University of Kentucky College of Pharmacy Pharmaceutical Sciences USA Professor Peter CrooksDr. Crooks is internationally known for his research work in drug discovery, delivery, and development, which includes drug design and synthesis, pharmacophore development, drug biotransformation studies, prodrug design, and medicinal plant natural product research. His research also focuses on preclinical drug development, including drug metabolism and pharmacokinetics in animal models, dosage form development, and drug delivery assessment using both conventional and non-conventional routes, and preformulation/formulation studies.Associate Professor Russell MumperDr. Mumper's main research areas are thin-films and mucoadhesive gels for (trans)mucosal delivery of drugs, microbicides, and mucosal vaccines, and nanotemplate engineering of nano-based detection devices and cell-specific nanoparticles for tumor and brain targeting, gene therapy and vaccines.West Virginia University School of Pharmacy Department of Basic Pharmaceutical Sciences USA Associate Professor Paula Jo Meyer StoutDr. Stout's research areas are composed of dispersed pharmaceutical systems, sterile product formulation DDS for dental diseases and coating of sustained release formulations.Monash University Victorian College of Pharmacy Department of Pharmaceutics Australia Professor Barrie C. FinninTransdermal Drug Delivery. Physicochemical Characterisation of Drug Candidates. Topical Drug Delivery. Drug uptake by the buccal mucosaProfessor Barry L. ReedTransdermal Drug Delivery. Topical Drug Delivery. Formulation of Dental Pharmaceuticals.University of Gent Faculty of Pharmaceutical Sciences Department of Pharmaceutics Belgium Professor Chris Vervaet-Extrusion/spheronisation - Bioadhesion - Controlled release based on hot stage extrusion technology - Freeze-drying - Tabletting and - GranulationPh.D. Els AdriaensMucosal drug delivery (Vaginal and ocular) Nasal BioadhesionUniversity of Gent Faculty of Pharmaceutical SciencesLaboratory of Pharmaceutical Technology Belgium Professor Jean Paul Remonbioadhesive carriers, mucosal delivery, Ocular bioerodible minitablets, Compaction of enteric-coated pellets; matrix-in-cylinder system for sustained drug delivery; formulation of solid dosage forms; In-line monitoring of a pharmaceutical blending process using FT-Raman spectroscopy; hot-melt extruded mini-matricesDanish University of Pharmaceutical Sciences Department of Pharmaceutics Denmark Associate Professor Jette JacobsenLow soluble drugs ?in vitro lymphatic absorption Drug delivery to the oral cavity ?in vitro models (cell culture, diffusion chamber) for permeatbility and toxicity of drugs, in vivo human perfusion model, different formulation approaces, e.g. iontophoresis.。

英文：硫辛酸改善PC12细胞中GSH的水平：帕金森治疗的新启示

Pre-treatment with R -Lipoic Acid Alleviates the Effects of GSH Depletion in PC12Cells:Implications for Parkinson’sDisease TherapyS.Bharath 1,*,B.C.Cochran 1,M.Hsu 1,J.Liu 2,3,B.N.Ames 2,3,J.K.Andersen 11Buck Institute for Age Research,8001Redwood Blvd.,Novato,CA 94945,USA2Division of Biochemistry and Molecular Biology,University of California,Berkeley,CA 94720,USA 3Children’s Hospital Oakland Research Institute,Oakland,CA 94609,USAReceived 22August 2001;accepted 12April 2002AbstractOxidative stress is believed to play a key role in the degeneration of dopaminergic neurons in the substantia nigra (SN)of Parkinson’s disease (PD)patients.An important biochemical feature of PD is a signiﬁcant early depletion in levels of the thiol antioxidant compound glutathione (GSH)which may lead to the generation of reactive oxygen species (ROS),mitochondrial dysfunction,and ultimately to subsequent neuronal cell death.In earlier work from our laboratory,we demonstrated that depletion of GSH in dopaminergic PC12cells affects mitochondrial integrity and speciﬁcally impairs the activity of mitochondrial complex I.Here we report that pre-treatment of PC12cells with R-lipoic acid acts to prevent depletion of GSH content and preserves the mitochondrial complex I activity which normally is impaired as a consequence of GSH loss.#2002Elsevier Science Inc.All rights reserved.Keywords:Parkinson’s disease;Glutathione (GSH);R -lipoic acid;Mitochondrial complex IINTRODUCTIONParkinson’s disease (PD)is a progressive,neurode-generative disorder which involves the loss of dopa-minergic neurons of the substantia nigra (SN)pars compacta (Beal,1992)The disease condition occurs mostly in late midlife with clinical features including motor impairment,gradual loss of cognition,and depression.Oxidative stress appears to play an impor-tant role in neuronal degeneration associated with PD (Beal,1992;Burke,1998;Adams et al.,2001;Sayre et al.,2001).Dopaminergic neurons are particularly prone to oxidative stress due to oxidation of dopamine leading to the generation of reactive oxygen species(ROS).ROS act to oxidize various biological macro-molecules thereby disturbing homeostasis within the cell ultimately leading to cell death (Beal,1992;Burke,1998;Adams et al.,2001;Sayre et al.,2001).It has been observed that the SN of early PD patients has signiﬁcantly decreased levels of glutathione (GSH)(Perry and Yong,1988).GSH is a tripeptide localized both within the cytoplasm and mitochondria of neurons and other cell types.It acts as the major non-proteinac-eous antioxidant and redox modulator within the brain.The depletion of GSH in the SN is the earliest known indicator of oxidative stress in presymptomatic PD,preceding both decreases in mitochondrial complex I activity and dopamine levels (Jenner,1993).GSH is synthesized by a two-step reaction involving the enzymes g -glutamyl cysteine ligase (g -GCL)and glutathione synthetase.g -GCL is the rate-limiting enzyme in this process and brain GSH appearstoNeuroToxicology 23(2002)479–486*Corresponding author.Tel.:þ1-415-209-2000;fax:þ1-415-209-2231.E-mail address :bsrinivas@ (S.Bharath).0161-813X/02/$–see front matter #2002Elsevier Science Inc.All rights reserved.PII:S 0161-813X (02)00035-9primarily arise through synthesis from its constituent amino acids via this enzyme(Meister,1988).g-GCL is a dimer composed of a heavy catalytic subunit and a light regulatory subunit(Huang et al.,1993).GSH is synthesized in the cytosol and transported into the mitochondria via an energy-dependent transporter (Meister,1988).In earlier studies from our laboratory, we reported on the effects of partial depletion of GSH levels via g-GCL inhibition on mitochondrial physio-logy in PC12cells(Jha et al.,2000).When GSH levels in PC12were down regulated by simultaneous dox-ycycline(Dox)-induced expression of antisense g-GCL light and heavy subunit mRNAs and protein levels, it resulted in decreased mitochondrial GSH levels, increased oxidative stress,and decreased mitochon-drial function.Decreased mitochondrial activity in these cells appeared to be due to a selective inhibition of complex I activity as a result of thiol oxidation of the enzyme complex.These results suggested that the early observed GSH losses in the SN of PD patients could be linked to decreases in complex I activity(via increased ROS)and subsequent mitochondrial dysfunction which ultimately leads to dopaminergic cell death associated with the disease(Jha et al.,2000;Beal,1992).R-lipoic acid plays a fundamental role in mitochon-drial metabolism as a coenzyme for pyruvate dehy-drogenase and alpha-ketoglutarate dehydrogenase and as a substrate for the NADPH-dependent enzyme glutathione reductase.In recent years,R-lipoic acid has received considerable attention as an antioxidant (Fuchs et al.,1997;Packer et al.,1995;Sen et al., 1999).It has been demonstrated to have neuroprotec-tive effects on neuronal cells(Wolz and Krieglstein, 1996,1997;Tirosh et al.,1999;Sen et al.,1998). Studies using both in vitro and in vivo models have suggested that pretreatment with R-lipoic acid increases cellular levels of GSH,probably by prevent-ing its depletion thereby protecting mitochondrial integrity(Suzuki et al.,1991;Scott et al.,1994;Han et al.,1997;Xu and Wells,1996;Lykkesfeldt et al., 1998;Kagen et al.,1992).To address the question of the possible mechanism of R-lipoic acid-mediated protection,we have analyzed the effect of pretreatment with R-lipoic acid on GSH-depleted PC12cells.We have used buthionine sulfox-imine(BSO),an irreversible inhibitor of the enzyme g-GCL,to decrease GSH levels within PC12.BSO is a widely used pharmacological agent for decreasing GSH content in peripheral tissues as well as in cell culture models(Minchinton et al.,1984;Lee et al., 1988;Thanislass et al.,1995;Andersen et al.,1996). We demonstrate that pre-treatment of GSH-depleted cells with R-lipoic acid prevents depletion of the GSH pool within the cytoplasm and mitochondria.We also show that this pretreatment leads to preservation of mitochondrial complex I activity lost due to GSH depletion.MATERIALS AND METHODSPC12cells were obtained from American Type Culture Collection(ATCC),Rockville,MD.All tissue culture materials were procured from Life Technolo-gies or Cellgro.Chemicals were obtained from Sigma Chemical Company.The GSH assay kit was obtained from OXIS Research Inc.R-lipoic acid was a gift from ASTA Medica(Frankfurt/Main,Germany).Cell CulturePC12cells were cultured in DMEM medium con-taining10%horse serum,5%fetal bovine serum(FBS) and antibiotics(2units penicillin/ml and0.02mg streptomycin/ml)at378C and5%CO2.Cells were subcultured once a week by gentle cell scraping.Stock solutions of buthionine-S,R-sulfoximine(BSO)and R-lipoic acid were prepared in Hanks balanced salt solution(HBSS).Preparation of MitochondriaMitochondria were prepared by the method of Trounce et al.(1996).Brieﬂy,PC12cells were har-vested,washed in Buffer H(5mM HEPES,210mM mannitol,70mM sucrose,1mM EGTA and0.5% BSA)then resuspended in the same buffer.The cell suspension was then homogenized and centrifuged at 800Âg for5min at48C.The supernatant which was enriched in mitochondria was then centrifuged at 10,000Âg for20min at48C.The resultant mitochon-drial pellet was resuspended in buffer H and stored as aliquots atÀ208C.Estimation of Total Glutathione(GSHþGSSG) in Whole Cells and MitochondriaTotal cellular and mitochondrial glutathione estima-tion was carried out using a kit from OXIS Research Inc.following the instructions provided by the manu-facturer.Cells were plated at1Â106cells/ml in either 6-well dishes or90mm diameter plates.All estima-tions were conducted in triplicate.Cells were incubated with BSO and/or R-lipoic acid for the indicated time intervals at378C.Cells were then harvested,disrupted480S.Bharath et al./NeuroToxicology23(2002)479–486by sonication and cellular proteins precipitated by tricholoroacetic acid.The soluble fraction,which is enriched for total glutathione,was estimated using the OXIS kit.Brieﬂy,samples wereﬁrst buffered and then the reducing agent,tris(2-carboxyethyl)phosphine (TCEP)was added to reduce any oxidized glutathione (GSSG).The chromogen4-chloro-1-methyl-7-triﬂuor-omethylquinolinium methylsulfate was then added which forms thioethers with all thiols present in the sample.Upon addition of base to raise the pH to greater than13,a b-elimination speciﬁc to the GSH-thioether results in a chromophoric thione.The absorbance of this thione measured at420nm is directly proportional to total glutathione concentration.All values obtained were normalized per protein and tabulated. Mitochondrial Complex I Enzyme Assay Complex I enzyme assays were carried out as described by Trounce et al.(1996).Brieﬂy,the assay was initiated by addition of aliquots of sonicated cell samples to50mM potassium phosphate pH7.4, 500m M EDTA,1%BSA,200m M NADH and200m M decylubiquinoneþ2m M rotenone in the presence of KCN with0.002%dichloroindophenol(DCIP)as a secondary electron acceptor.The decrease in the absorbance at600nm was recorded as a measure of enzyme reaction rate at308C for10min and speciﬁc activity was calculated.The results were plotted as relative rotenone-sensitive speciﬁc activity.c-Glutamyl Cysteine Ligase Enzyme Assayg-GCL assays were carried out as described by Seelig and Meister(1985).Brieﬂy,cells obtained after various treatments were washed in1ÂPBS and resuspended in0.1M Tris–HCl,pH8.0.The cell suspension was sonicated and used as a source for g-GCL enzyme activity.The enzyme activity was determined at378C by adding cell lysates to a reaction mixture containing0.1M Tris–HCl buffer,pH8.0, 150mM KCl,5mM sodium ATP,2mM phosphoenol-pyruvate,10mM L-glutamate,10mM a-amino buty-rate,20mM MgCl2,2mM EDTA,0.2mM NADH, 17U pyruvate kinase and17U lactate dehydrogenase. The absorbance at340nm was monitored as a measure of enzyme activity.Assays run in the absence of a-aminobutyrate served as blank.All the g-GCL activ-ity values were normalized per protein.RESULTSPC12cells represent a good model system for the study of oxidative stress in dopamine-containing cells as they exhibit several physiological properties of dopaminergic neurons(Greene and Tischler,1976). PC12cells were treated with100m M of BSO for different time points and total glutathione content measured.Following BSO treatment,there was a gradual decrease in the levels of total cellular GSH content with time(Fig.1);the maximum decrease was observed at24h(data not shown).For subsequent experiments,addition of100m M of BSO for a time period of6h was employed to maintain approximately 50%depletion in total cellular GSH content,similar to the decreases observed in the PD SN.The unbound form of R-lipoic acid has two–SH groups and has been found to be an effective thiol anti-oxidant(Suzuki et al.,1991;Scott et al.,1994;Han et al.,1997;Xu and Wells,1996;Lykkesfeldt et al., 1998;Kagen et al.,1992).Therefore,R-lipoicacid Fig.1.The effect of100mM BSO on total cellular glutathione levels in PC12cells at various time points.Significance of difference fromthe control was determined by ANOV A single factor analysis.Data is expressed as meanÆS:E:M:(n¼3).ÃP<0:005vs.untreated cells;ÃÃP<0:001vs.untreated cells.S.Bharath et al./NeuroToxicology23(2002)479–486481pretreatmentwas testedtoseeifthiscould helpovercome the dramatic decreases in BSO-induced glutathione levels.PC12cells were incubated with 100m M of freshly prepared R -lipoic acid for 24h and then sub-jected to BSO treatment for 6h as previously described.Pretreatment with R -lipoic acid prevents depletion of glutathione levels within BSO-treated PC12cells (Fig.2a ).Mitochondrial glutathione content estimated from BSO-treated cells pre-treated with R -lipoic acid versus untreated cells demonstrate that mitochondrial levels of glutathione were decreased as signi ﬁcantly as cellular glutathione levels by BSO treatment and that they too are completely preserved upon pre-treatment with R -lipoic acid (Fig.2b ).These results emphasize the role of R -lipoic acid as a potential anti-oxidant with the ability to help cells maintain their total GSH pool.It has been well documented in PD that mitochon-drial dysfunction is a hallmark of disease pathogenesis (Beal,1992).This abnormality is manifested by a selective decrease in the enzymatic activity of mitochondrial complex I (Beal,1992;Jenner,1993;Hillered and Chan,1988;Haas et al.,1995;Jha et al.,2000).Since pretreatment with R -lipoic acid signi ﬁ-cantly prevented BSO-mediated depletion of cellular and mitochondrial total glutathione levels,it was logi-cal to analyze whether this type of pretreatment could lead to preservation of mitochondrial complex I activ-ity following BSO treatment.Hence,mitochondrial complex I enzyme assays were carried out on cells which had been pretreated with R -lipoic acid versus un-pretreated cells prior to BSO application.Fig.3shows data demonstrating that pre-incubation ofPC12Fig.2.The effect of pre-treatment with 100mM R -lipoic acid on the total glutathione (GSH þGSSG)levels in BSO treated and untreated (a)whole cell extracts and (b)mitochondria.Significance of difference from control was determined by ANOV A single factor analysis.Data is expressed as mean þS :E :M :(n ¼3).(a)ÃP <0:0001vs.untreated cells;ÃÃP <0:0001vs.untreated cells;ÃÃÃP <0:001vs.BSO alone treated cells.(b)ÃP <0:001vs.untreated cells;ÃÃP <0:0001vs.untreated cells;ÃÃÃP <0:005vs.BSO alone treatedcells.Fig.3.The effect of pre-treatment with 100m M R -lipoic acid on mitochondrial complex I activity in BSO-treated and untreated PC12cells.Relative rotenone-sensitive specific activities are plotted.Significance of difference from control was determined by ANOV A single factor analysis.Data is expressed as mean ÆS :E :M :(n ¼6).ÃP <0:005vs.control.482S.Bharath et al./NeuroToxicology 23(2002)479–486cells with R -lipoic acid preserves impaired complex I activity caused by BSO insult.The effect of R -lipoic acid demonstrated here might be due to either an increase in the activity of g -GCL,the rate-limiting enzyme in the glutathione production,or due to the direct anti-oxidant activity of R -lipoic acid which may prevent glutathione depletion.To distinguish between these two,we carried out enzyme assays for g -GCL from extracts obtained after various treatments.The result of these experiments (Fig.4)demonstrate that upon BSO treatment alone there is a decrease ($50%)in the g -GCL activity whereas cells treated with either R -lipoic acid alone or R -lipoic acid þBSO do not show any difference in the activity compared to control cells.DISCUSSIONOne of the earliest detectable events during the course of PD is a signi ﬁcant decrease in cellular levels of GSH in the SN (Pearce et al.,1997;Jenner,1993).This may contribute to oxidative stress and ensuing neuronal cell death of dopaminergic neurons in this brain region.Based on our earlier published ﬁndings,we proposed that early GSH depletion in the SN may be directly responsible for selective inhibition of mito-chondrial complex I activity and the concomitant loss of mitochondrial function which leads to neuronal cell death in this disorder (Jha et al.,2000).Selective reductions in GSH levels which precede losses in mitochondrial complex I activity have been reported to occur not only in PD but also in associated toxin models of the disease such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)administration (Perry and Yong,1988;Hallman et al.,1985;Sriram et al.,1998).Whether the inhibition of complex I activity and subsequent decreases in ATP levels fol-lowing MPTP administration can be totally accounted for by decreases in GSH levels is unclear as complex I also appears to be directly inhibited by interaction with 1-methyl-4-phenyl pyridium (MPP þ)formed during monoamine oxidase-B-mediated oxidation of MPTP (Jha et al.,1999).However,decreasing GSH levels by treatment with the pharmacological agent BSO has been shown to potentiate the neurodegenerative effects of MPTP in the SN and to alone cause sublethal damage to the nigrostriatal system (Wullner et al.,1996;Andersen et al.,1996).GSH is an anti-oxidant molecule well known to protect proteins during oxidative stress.It acts by conjugating oxidized thiol groups of target proteins forming mixed disul ﬁdes which are then processed by GSH reductase,thioredoxin or protein disulphide isomerase to reduced protein thiol residues and GSH (Ravindranath and Reed,1990;Jung and Thomas,1996).Thus,GSH serves as a major non-protein cellular defense against oxidative stress by maintaining the reduced state of cellular thiol proteins.Since GSH depletion in the SN is well documented in PD,it is of paramount importance to search for and test com-pounds which could prevent depletion of cellular levels of GSH in PD patients.To screen for such compounds,we have utilized the PC12cell culture model system in which activity of g -GCL has been partially impaired by BSO treatment.Although g -GCL activity levels do not appear to be speci ﬁcally impaired in sporadic cases of PD,the overall effect of BSO treatment in the dopaminergic PC12cells mimics that which is seen in the Parkinsonian brain,i.e.a decrease in GSHlevelsFig.4.The effect of pre-treatment with 100m M R -lipoic acid on g -glutamyl cysteine ligase activity in BSO-treated and untreated PC12cells.Relative %specific activity is plotted.Significance of difference from control was determined by ANOV A single factor analysis.Data is expressed as mean ÆS :E :M :(n ¼6).ÃP <0:0001vs.control.S.Bharath et al./NeuroToxicology 23(2002)479–486483leading to increased oxidative stress(Wullner et al., 1996;Andersen et al.,1996).Furthermore,though acute depletion of GSH appeared to have no effect on overall cell viability or growth even after48h(Jha et al.,2000),prolonged mitochondrial dysfunction would likely eventually result in decreased cell viability like that observed in dopaminergic SN neurons in PD. As shown in Figs.1and2,increased exposure to BSO results in a notable decrease in the GSH content both in whole cell preparations and in the mitochondria. Signiﬁcant changes occur in the mitochondrial phy-siology during oxidative stress and in diseases like PD (Beal,1992).In this current study,we found that BSO treatment eliciting an increase in oxidative stress via a 50%decrease in total cellular GSH content resulted a 40%decrease in the speciﬁc activity of mitochondrial complex I(Fig.3).This correlates well with our earlier ﬁndings in which we observed a similar decrease in mitochondrial complex I activity following50%GSH depletion,with no apparent effects on the activities of the other mitochondrial complexes(Jha et al.,2000). Mitochondrial complex I is considered to be the mitochondrial complex the most severely affected by oxidative stress(Lenaz et al.,1997).In synaptic mitochondria,complex I exerts a major control over oxidative phosphorylation such that at only25%inhi-bition,energy metabolism is disturbed resulting in decreased ATP synthesis(Davey et al.,1998).R-lipoic acid,a thiol containing compound,is a widely used antioxidant(Fuchs et al.,1997;Packer et al.,1995;Sen et al.,1999).R-lipoic acid functions as a redox regulator of proteins such as myoglobin, prolactin,thioredoxin,and NF-kappa B transcription factor(Bushtamante et al.,1998;Packer et al.,1995, 1997).It has been utilized to treat or prevent peripheral neuropathy and cardiac autonomic neuropathy(Ziegler and Gries,1997),insulin resistance in type II diabetes (Jacob et al.,1996),retinopathy and cataracts(Maitra et al.,1996),glaucoma(Head,2001),HIV/AIDS (Packer et al.,1995),cancer(Ames,1999),liver disease (Bushtamante et al.,1998),Wilson’s disease(Yamamoto et al.,2001),cardiovascular disease(Matalon et al., 1984)and lactic acidosis caused by inborn errors of metabolism(Yoshida et al.,1990).It has also been used for treatment of Alzheimer type dementia(Hager et al., 2001).R-lipoic acid administration has been reported to result in increased ambulatory activity and improved memory in aged animals and to partially restore age-associated mitochondrial decay in both the liver and heart(Hagen et al.,1999,2000;Suh et al.,2001). It therefore seems likely that thiol antioxidants such as R-lipoic acid which can enhance mitochondrial function,scavenge free radicals,and increase the levels of the antioxidants GSH and ascorbate might be useful neuroprotective agents via their reducing actions onvital protein thiol residues(Han et al.,1997;Xu and Wells, 1996).Our results together with previous studies suggest that R-lipoic acid may be an effective neuroprotective agent in age-associated neurodegeneration.Utilizing the PC12cell model system,we propose that R-lipoic acid administration could be an effective way of circumventing or delaying mitochondrial dys-function associated with PD.Treatment with R-lipoic acid alone seems to signiﬁcantly increase GSH levels only in whole cell preparations but not in mitochon-drial extracts(see Fig.2a and b).However,pretreat-ment of cells with R-lipoic acid appears to prevent BSO-mediated GSH depletion in both whole cells and mitochondria.These data suggest that only during BSO treatment is there a R-lipoic acid-mediated increase in mitochondrial GSH to counter BSO generated ROS production.We were curious to know whether the effect of R-lipoic acid on GSH levels was due to an induction of g-GCL activity.Han et al.(1997)have shown that R-lipoic acid increases de novo synthesis of glutathione by improving cystine utilization.It is also possible that reduction of R-lipoic acid to dihydrolipo-ate in vivo might cause oxidation of GSH to GSSG in turn inducing GCL activity.However,since total glu-tathione estimations in our experiments involved quan-titation of both GSHþGSSG,depletion of GSH cannot be due to the accumulation of GSSG thus ruling out the possibility that increased GSSG concentration might be affecting the g-GCL activity.Fig.4shows that BSO treated cells exhibit$50%decrease in g-GCL activity compared to the untreated cells.Interestingly, cells pretreated with R-lipoic acid upon incubation with BSO showed an increase in g-GCL activity compared to cells treated with BSO alone.These data suggest that R-lipoic acid might contribute to an increased GSH pool during BSO treatment due to the induction of g-GCL activity.However,when cells are treated with R-lipoic acid alone there is a signiﬁ-cant increase($1.6fold)in GSH levels without a concomitant increase in g-GCL activity suggesting that GSH levels are preserved by the direct anti-oxidant properties of R-lipoic acid and not due to increased g-GCL activity(Fig.2a).Decreases in mitochondrial NADH dehydrogenase(complex I)activity associated with GSH depletion also appear to be preserved via R-lipoic acid pretreatment(Fig.3).Taken together, these data suggest that GSH depletion,which may lead to oxidation of protein sulfhydryl residues in the mito-chondrial complex I important for normal functions484S.Bharath et al./NeuroToxicology23(2002)479–486resulting in profound effects on subsequent mitochon-drial performance,may be prevented by the direct action of R-lipoic acid.Our earlierﬁndings suggested that the activity of NADH dehydrogenase,the enzy-matic component of complex I,appears to be thiol-regulated(Jha et al.,2000).The preservation of mito-chondrial complex I activity via R-lipoic acid suggests that it protects against the oxidation of proteins by preventing the use of the cellular GSH pool or by increasing the de novo synthesis of cellular GSH in the presence of BSO-mediated oxidative stress.Whether such treatment would be effective in actually altering brain levels of GSH and thus providing beneﬁcial effects in vivo has not yet been demonstrated. However,our data suggest that maintaining thiol homeostasis may be critical for protecting dopami-nergic neurons of the SN against neurodegeneration and that antioxidants such as R-lipoic acid may be of therapeutic value in PD.It would be of interest to conduct additional experiments using intermediates of dopamine metabolism or MPTP to explore thera-peutic value of R-lipoic acid pretreatment in PD. Nevertheless,since GSH depletion is the earliest event in PD pathogenesis,our data clearly suggests that R-lipoic acid can prevent the deleterious effects that follow by preventing mitochondrial GSH depletion.ACKNOWLEDGEMENTSThis work was supported by a grant from the National Institute of Health(NIH,R01AG12141) to JKA and by a grant from the Ellison Foundation (SS-0422-99),the National Institute of Health (NIH,R01,AG17140),and the National Institute of Environmental Health Sciences Center(NIH,P30, ES01896)to BNA.We thank Dr.M.Jyothi Kumar for technical help and criticaldiscussionsandDr.Simon Melov,Buck Institute,for use of his double beamed spectrophotometer.REFERENCESAdams Jr,JD,Chang ML,Klaidman L.Parkinson’s disease-redox mechanisms.Curr Med Chem2001;8:809–14.Ames BN.Micronutrients prevent cancer and aging.Toxicol Lett 1999;103:5–18.Andersen JK,Mo JQ,Hom DG,Lee FY,Harnish P,Hamill RW, McNeill TH.Effect of buthionine sulfoximine,a synthesis inhibitor of the antioxidant glutathione,on murine nigrostriatal neurons.J Neurochem1996;67:2164–71.Beal MF.Does impairment of energy metabolism result in excitotoxic neuronal death in neurodegenerative illnesses.Ann Neurol1992;31:119–30.Burke RE.Parkinson’s disease.In:Koliatos,Ratan RR,editors. Cell death and disease of the nervous system.Totowa(NJ): Humana Press,1998.p.459–75.Bushtamante J,Lodge JK,Marcocci L,Tritschler HJ,Packer L, Rihn BH.Alpha-lipoic acid in liver metabolism and disease. Free Radic Biol Med1998;24:1023–39.Davey GP,Peuchen S,Clark JB.Energy thresholds in brain mitochondria:potential involvement in neurodegeneration.J Biol Chem1998;273:12753–7.Fuchs J,Packer L,Zimmer G.Lipoic acid in health and disease. New York:Marcel Dekker,1997.Greene L,Tischler A.Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor.Proc Natl Acad Sci USA1976;73: 2424–8.Haas RH,Nasirian F,Nakano K,Ward D,Pay M,Hill R,Shults CW.Low platelet mitochondrial complex I and complex II/III activity in early untreated Parkinson’s disease.Ann Neurol 1995;37:714–22.Hagen TM,Ingersoll RT,Lykkesfeldt J,Liu J,Wehr CM,Vinarsky V,Barthlomew JC,Ames AB.(R)-alpha-lipoic acid-supplemented old rats have improved mitochondrial function, decreased oxidative damage,and increased metabolic rate. FASEB J1999;13:411–8.Hagen TM,Vinarsky V,Wehr CM,Ames BN.(R)-alpha-lipoic acid reverses the age-associated increase in susceptibility of hepatocytes to tert-butylhydroperoxide both in vitro and in vivo. Antiox Redox Signal2000;2:473–83.Hager K,Marahrens A,Kenklies M,Riederer P,Munch G.Alpha-lipoic acid as a new treatment option for Alzheimer type dementia.Arch Gerontol Geriat2001;32:275–82.Hallman H,Lange J,Olson L,Stromberg I,Jonsson G. Neurochemical and histochemical characterization of neurotoxic effects of1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine on brain catecholamine neurons in the mouse.J Neurochem1985;44: 117–27.Han D,Handelman G,Marcocci L,Sen CK,Roy S,Kobuchi H, Tritschler HJ,Flohe L,Packer L.Lipoic acid increases de novo synthesis of cellular glutathione by improving cystine utilization. Biofactors1997;6:321–38.Head KA.Natural therapies for ocular disorders,part two: cataracts and glaucoma.Alternative Med Rev2001;6:141–66. Hillered L,Chan PH.Effects of arachidonic acid on respiratory activities in isolated brain mitochondria.J Neurosci Res 1988;19:94–100.Huang CS,Chang LS,Anderson ME,Meister A.Catalytic and regulatory properties of the heavy subunit of rat kidney gamma-glutamylcysteine synthetase.J Biol Chem1993;268:19675–80. Jacob S,Henriksen EJ,Tritschler HJ,Augustin HJ,Dietz GJ. Improvement of insulin-stimulated glucose disposal in type2 diabetes after repeated parenteral administration of thioctic acid.Endocrinol Diabetes1996;104:284–8.Jenner P.Altered mitochondrial function,iron metabolism and glutathione levels in Parkinson’s disease.Acta Neurol Scand Suppl1993;146:6–13.Jha N,Andersen JK.Loss of glutathione(GSH)in Parkinson’s disease:how does GSH act to protect dopaminergic neurons of the substantia nigra?In:Pandalai SG,editor.Recent researchS.Bharath et al./NeuroToxicology23(2002)479–486485。

聚二甲基硅氧烷芯片自由酶反应器检测葡萄糖

聚二甲基硅氧烷芯片自由酶反应器检测葡萄糖作者：仲海燕周洁余晓冬陈洪渊【摘要】应用电泳中介微分析（EMMA）技术，构建聚二甲基硅氧烷（PDMS）芯片自由酶反应器，在线检测葡萄糖（Glu），在十字形的芯片通道上，采用自制的碳纤维微电极检测葡萄糖氧化酶（GOD）催化氧化Glu生成的H2O2，并对检测电位、GOD浓度、GOD进样时间、分离电压等参数进行了优化，测定了该自由酶反应器的线性范围和检出限，考察了其重现性及稳定性。

结果表明，此自由酶反应器制作方便，操作简单，重现性好，Glu浓度在0.1～20 mmol/L之间有较好的线性关系（r=0.997），检出限为19.8 μmol/L（S/N=3）。

【关键词】自由酶反应器；葡萄糖；电泳中介微分析；聚二甲基硅氧烷芯片The Key Lab of Analytical Chemistry for Life Science, Ministry of Education, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093)Abstract Microfluidic enzyme based reactor could be used to detect the biomolecules with high sensitivity and selectivity. Based on the electrophoretic mediated microanalysis (EMMA) method, a new kind of free enzyme based poly(dimethylsiloxane) (PDMS) microchip was developed to detect glucose (Glu). On line catalysis reaction of Glu by glucose oxidase (GOD) was carriedout on the chip. The product H2O2 was detected using single carbon fibre cylindrical electrode. Factors influencing the separation and detection, such as detection potential, GOD concentration, GOD injection time and separation voltage, were investigated and optimized. Results showed that the peak current had a good linear relationship with Glu concentration in the range of 0.1－20 mmol/L (R=0.997). The detection limit of Glu was 19.8 μmol/L (S/N=3). In addition, the PDMS enzyme based reactor had long term stability and excellent reproducibility (RSD=2.02%, n=10). It was easy to fabricate and operate, which showed great potential application in bioanalysis.Keywords Free enzyme based microchip; Glucose; Electrophoretic mediated microanalysis; Poly(dimethylsiloxane)1 引言近年来，基于酶催化反应的微流控芯片备受关注。

中性粒细胞在白塞综合征发病机制中的作用

423[30]Green HF,Khosousi S,Svenningsson P.Plasma IL-6and IL-17ACorrelate with Severity of Motor and Non-Motor Symptoms in Parkinson's Disease[J].J Parkinsons Dis,2019,9(4):705-709.[31]Karpenko MN,Vasilishina AA,GromovaEA,.Interle-ukin-1,interleukin-1receptor antagonist,interleukin-6,in-terleukin-10,and tumor necrosisfactor-levels in CSF and ser-um in relation to the clinical diversity of Parkinson's disease [J].Cell Immunol,2018，327:77-82.[32]Su F,Bai F,Zhang Z.Inflammatory Cytokines and Alzheimer'sDisease:A Review from the Perspective of Genetic Polymorph-isms [J].Neurosci Bull,2016，32(5):469-480.[33]Alam Q,Alam MZ,MushtaqG,.Inflammatory Processin Alzheimer's and Parkinson's Diseases:Central Role of Cy-tokines [J].Curr Pharm Des,2016,22(5):541-548.[34]Han X,Sun S,SunY,.Small molecule-driven NLRP3inflammation inhibition via interplay between ubiquitination and autophagy:implications for Parkinson disease [J].Autophagy,2019，15(11):1860-1881.[35]Spalinger MR,Lang S,GottierC,.PTPN22regulatesNLRP3-mediated IL1B secretion in an autophagy-dependent manner [J].Autophagy,2017，13(9):1590-1601.[36]Chan AH,Schroder K.Inflammasome signaling and regulationof interleukin-1family cytokines [J].J Exp Med,2020,217(1):e20190314.[37]Nawas AF,Mistry R,NarayananS,.IL-1induces p62/SQSTM1and autophagy in ER +/PR +BCa cell lines concomi-tant withER and PR repression,conferring an ER -/PR -BCa-like phenotype [J].J Cell Biochem,2018.Epub ahead of print.[38]Chen Y,Yang Z,DengB,.Interleukin1/1RA axis incolorectal cancer regulates tumor invasion,proliferation and ap-optosis via autophagy [J].Oncol Rep,2020,43(3):908-918.[39]Mishra J,Vishwakarma J,MalikR,.HypothyroidismInduces Interleukin-1-Dependent Autophagy Mechanism as a Key Mediator of Hippocampal Neuronal Apoptosis and Cogni-tive Decline in Postnatal Rats [J].Mol Neurobiol,2021，58(3):1196-1211.[40]Gao Y,Ma L,LuoCL,.IL-33Exerts NeuroprotectiveEffect in Mice Intracerebral Hemorrhage Model Through Sup-pressing Inflammation/Apoptotic/Autophagic Pathway [J].Mol Neurobiol,2017,54(5):3879-3892.[41]Gao Y,Zhang MY,WangT,.IL-33/ST2L SignalingProvides Neuroprotection Through Inhibiting Autophagy,End-oplasmic Reticulum Stress,and Apoptosis in a Mouse Model ofTraumatic Brain Injury [J].Front Cell Neurosci,2018,12:95.[42]Gao Y,Luo C,YaoY,.IL-33Alleviated Brain Damage via Anti-apoptosis,Endoplasmic Reticulum Stress,and Inflam-mation After Epilepsy [J].Front Neurosci,2020,14:898.[43]Reverchon F,de Concini V,LarrigaldieV,.Hippocampal interleukin-33mediates neuroinflammation-induced cognitive impairments [J].J Neuroinflammation,2020，17(1):268.[44]Liu S,Li H,WangY,.High Expression of IL-36in Influenza Patients Regulates Interferon Signaling Pathway and Causes Programmed Cell Death During Influenza Virus Infec-tion [J].Front Immunol,2020，11:552606.[45]Gao Y,Wen Q,HuS,.IL-36Promotes Killing of My-cobacterium tuberculosis by Macrophages via WNT5A-Induced Noncanonical WNT Signaling [J].J Immunol,2019，203(4):922-935.[46]Xue H,Yuan G,GuoX,.A novel tumor-promoting mech-anism of IL6and the therapeutic efficacy of tocilizumab:Hy-poxia-induced IL6is a potent autophagy initiator in glioblastoma via the p-STAT3-MIR155-3p-CREBRF pathway[J].Autophagy,2016,12(7):1129-1152.[47]Chen R,Sun Y,CuiX,.Autophagy promotes aortic ad-ventitial fibrosis via the IL-6/Jak1signaling pathway in Taka-yasu's arteritis [J].J Autoimmun,2019,99:39-47.[48]Hsu HC,Chen YH,LinTS,.Systemic lupus erythematosus is associated with impaired autophagic degradation via interleu-kin-6in macrophages [J].Biochim Biophys Acta Mol Basis Dis,2021，1867(2):166027.[49]Jin MM,Wang F,QiD,A Critical Role of Autophagy in Regulating Microglia Polarization in Neurodegeneration [J].Front Aging Neurosci,2018，10:378.[50]Wang MX,Cheng XY,JinM,.TNF compromises lysosome acidification and reduces -synuclein degradation via autophagy in dopaminergic cells [J].Exp Neurol,2015，271:112-121.[51]Lin NY,Stefanica A,Distler JH.Autophagy:a key pathway of TNF-induced inflammatory bone loss [J].Autophagy,2013,9(8):1253-1255.(收稿日期：2021-02-25）（本文编辑：朱音）中性粒细胞在白塞综合征发病机制中的作用侯成成，管剑龙复旦大学附属华东医院风湿免疫科，上海200040白塞综合征（Beh çet's syndrome,BS ）也称为白塞病，是一种病因不明的慢性、复发性、自身免疫性变异性血管炎。

mol ther nucleic acids影响因子

mol ther nucleic acids影响因子mol ther nucleic acids影响因子是8.88。

mol ther nucleic acids指的是《分子疗法-核酸》。

《分子疗法-核酸》是一本以综合研究为特色的国际期刊。

该刊已被国际重要权威数据库SCIE收录。

《分子疗法-核酸》是一种国际性的、开放获取的期刊，在核酸疗法治疗和/或纠正遗传和获得性疾病的广泛领域中，出版高质量的基础、翻译和临床研究。

研究领域包括但不限于：基于核酸及其衍生物的治疗方法的开发、基于rna的治疗方法的载体开发和设计、基因修饰剂的应用，包括形成三聚体的寡核苷酸和酶（如锌指核酸酶）、临床前目标验证、安全性/有效性研究和临床试验。

一区7分单细胞测序高糖对免疫浸润的影响申请课题

一区7分单细胞测序高糖对免疫浸润的影响申请课题下载提示：该文档是本店铺精心编制而成的，希望大家下载后，能够帮助大家解决实际问题。

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一株伯克氏菌中两个阿魏酸酯酶的比较分析

一株伯克氏菌中两个阿魏酸酯酶的比较分析徐振上;刘盼;杨晓颖【期刊名称】《山东轻工业学院学报（自然科学版）》【年(卷),期】2015(000)002【摘要】本文从NCBI数据库中检索得到Burkholderia phytofirmans PsJN的两个阿魏酸酯酶基因序列,分别标记为Bpfae1和Bpfae2,运用生物信息学方法对其核苷酸序列、编码的氨基酸序列进行分析,并对其二级和三级结构进行预测。

结果表明,Bpfae1的基因全长为1671bp,该基因编码的氨基酸为556aa,此蛋白质的等电点为8.33,分子量为59978.22Da。

Bpfae2的基因全长为1734bp,该基因编码的氨基酸为577aa,此蛋白质的等电点5.42,分子量为59339.25Da。

序列比对结果表明, Bpfae1与 Burkholderia xenovorans 同源性最高为90％。

Bpfae2与Burkholderia glumae同源性最高为83％,这两个阿魏酸酯酶蛋白的同源率为29％。

另外对这两个阿魏酸酯酶的立体结构进行了同源模拟。

【总页数】4页(P45-48)【作者】徐振上;刘盼;杨晓颖【作者单位】山东大学微生物技术国家重点实验室，山东济南250100;齐鲁工业大学生物工程学院，山东济南250353;齐鲁工业大学生物工程学院，山东济南250353【正文语种】中文【中图分类】Q-9【相关文献】1.利用伯克霍尔德氏菌脂肪酶富集裂壶藻油脂中的二十二碳六烯酸 [J], 兰君;宋晓金;谭延振;徐建春;崔球;冯银刚2.一株伯克氏菌中两个阿魏酸酯酶的比较分析 [J], 徐振上;刘盼;杨晓颖;3.从白面包中检出一株与福氏志贺氏菌6型交叉凝集的阴沟肠杆菌的报告 [J], 梁玉萍;李笑琼;陈德云4.一株典型伯克氏菌对Cr(Ⅵ)/Cu(Ⅱ)复合污染的吸附转化 [J], 檀笑; 曾洁仪; 张逸凡; 曾巧云; 陈烁娜5.洋葱伯克霍尔德氏菌及其在林木病害防治中的应用 [J], 叶建仁;任嘉红;李浩;吴小芹因版权原因，仅展示原文概要，查看原文内容请购买。

双孢蘑菇基质中线虫影响木耳菌丝生长的数量阈值

双孢蘑菇基质中线虫影响木耳菌丝生长的数量阈值谢飞;刘奇志;梁林琳;杨道伟【摘要】Based on the former study related to the reproduction and impact on hypha growth of three dominant nema-tode species isolated from edible mushroom growing substance, the effect of nematode density ( 5 , 10, 20, 40, 80, 160 individuals per tube) on hypha growth of Auricularia auricular was investigated to find out the quantity threshold. The results showed that there was no obvious difference in hypha appearance of Auricularia auricular, compared with the control with the nematodes, Mesorhabditis sp. at the density of 5 and 10 individuals per tube after 2 weeks of inoculation. The amount of reproduction were 40 and 255 individuals per tube, respectively. The hypha appearance of A. auricular was slightly different after 3 weeks of inoculation. With the nematodes of Acrobeloides sp. and Aphe-lenchoides composticola Franklin at the density of 5 individuals per tube after 2 weeks of inoculation, the hypha appearance became yellow. The amount of reproduction were 55 and 93 individuals per tube, respectively. After 3 weeks, the hypha became brown, and half of it was bitten. The hypha was seriously affected at the tested densities of more than 20 individuals per tube of all the three species of nematodes. The hypha was bitten up almost after 3 weeks of inoculation. Therefore, for mushroom production, the amount of above 3 nematodes should be less than 5 individuals per tube (about 8 individuals per 100 g) in mushroom growing substance.%在研究了双孢蘑菇生产基质中的3种优势线虫数量扩增及对菌丝生长影响的基础上,着重研究了线虫密度(5,10,20,40,80,160条·管-1)对木耳菌丝生长的影响,以确定该3种线虫的数量影响阈值.结果表明:中杆属(Mesorhabditis sp.)线虫在密度为5和10条·管-1的条件下,接种2周后,菌丝表观与对照的无明显不同,繁殖量分别达40和255条·管-1；接种3周后,菌丝表观略有变化.拟丽突属(Acrobeloides sp.)和滑刃属的堆肥滑刃线虫(Aphelenchoides composticola Franklin)在5条·管-1的接种密度下,2周后木耳菌丝明显变黄,其繁殖量分别达到55和93条·管-1；3周后,菌丝变褐,部分被啃食.3种线虫在20条·管-1以上密度条件下,菌丝受害严重,3周后菌丝几乎被完全啃食.因此,在蘑菇生产中,基质中的3种供试线虫数量应控制在5条·管-1以下(约每100g基质中线虫8条以下).【期刊名称】《浙江农业学报》【年(卷),期】2012(024)004【总页数】5页(P615-619)【关键词】线虫;数量阈值;蘑菇基质;木耳;菌丝【作者】谢飞;刘奇志;梁林琳;杨道伟【作者单位】中国农业大学农学与生物技术学院昆虫与线虫学实验室,农业部生物防治重点开放实验室,北京100193;中国农业大学农学与生物技术学院昆虫与线虫学实验室,农业部生物防治重点开放实验室,北京100193;中国农业大学农学与生物技术学院昆虫与线虫学实验室,农业部生物防治重点开放实验室,北京100193;中国农业大学农学与生物技术学院昆虫与线虫学实验室,农业部生物防治重点开放实验室,北京100193【正文语种】中文【中图分类】S432.4+5食用菌是我国农业中的一个重要产业，在种植业中，仅次于粮、棉、油、果、菜，是六大类农产品之一。

人类X染色体与Y染色体是否为同源染色体

人类 X 染色体与 Y 染色体是否为同源染色这一问题往往是中学教师和中学生非常关心对的 2 条染色体，形状和大小一般都相同，一条来自父方，一条来自母方，叫作同源染色体”［3 ］。

现有体，也非常感兴趣的话题，同时，也是学习遗传学的大的诸多遗传学教科书以及生物学词典、遗传学词学生所关心和经常探讨的问题。

然而，由于 X 染典中对同源染色体的理解都强调以下 4 点：即来色体与 Y 染色体在形态、大小等方面存在明显差别，导致人们形成不同的观点，在许多大学的遗传学教科书中和中学生物学教材中对这一问题往往避而不谈，使这一有趣的问题更加神秘化，给教学工作带来不少麻烦。

本文针对此问题展开讨论，旨在给人们一个较为清晰的答案。

冯德培主编的《简明生物学词典》中指出同源染色体是： “ 在减数分裂时两两配对的染色体，形状、大小、结构和功能都相同。

同源染色体中一条来自父本，一条来自母本”［1 ］。

王金发主编的《英汉细胞生物学词典》中指出： “ 成对的染色体，它们源于父方和母方；形态、大小一般相同；功能相同；在减数分裂中可以配对。

那么 X 染色体与 Y 染色体是否符合这 4 点要求呢？1 从来源看，X 与 Y 属于同源染色体从个体发育的观点看，X 染色体来自母方，Y染色体来自父方，由于受精作用把二者组合到了一个体细胞中，X 与 Y 染色体的来源符合同源染色体概念中的基本条件。

另外，同源染色体的最本质的特征是 “ 同源 ”。

英文 “hom o l ogou s chr o m o - so m es ” 中的 “hom o l ogous ” 一词的意义就是 “ 同源的”。

这里的“同源”是指物种系统发育方面的“来源相同 ”，即具有共同的祖先，在进化上具有相同的起点。

基于木糖异构酶途径的木糖发酵酿酒酵母菌株构建研究进展

基于木糖异构酶途径的木糖发酵酿酒酵母菌株构建研究进展李云成;孟凡冰;苟敏;孙照勇;汤岳琴【期刊名称】《生物技术通报》【年(卷),期】2017(33)10【摘要】It is of utmost significant to construct industrial xylose-fermenting Saccharomyces cerevisiae strains for lignocellulosic bioethanol production through the heterologous expression of xylose metabolic pathway. Xylose isomerase pathway is regarded as the most promising pathway expressing in S. cerevisiae,since there is no xylitol accumulation resulted from the coenzyme imbalance in xylose redox pathway. In heterologous expression of xylose isomerase,selecting an industrial S. cerevisiae strain as initial strain is of outstanding advantages for lignocellulosic bioethanol production. Concurrently,improving the expression efficiency of xylose isomerase gene(xylA)is vital for constructing a robust xylose fermentation strain. In addition,the deletion of GRE3,strengthening of xylose transport and rational modification of xylose metabolic pathway during the metabolic modification of strain XI effectively improved the xylose fermentation capacity of the recombinant strains. Besides,evolutionary engineering also increased the xylose fermentation efficiency of XI strains. Furthermore,the omics technologies have presented their power in explaining the mechanism and developing the modification strategies of xylose metabolism. This paper reviews the research progresses on theexpressions of xylose isomerase pathway in S. cerevisiae and analyzes the issues in the relevant studies.%通过异源表达木糖代谢途径,构建能高效发酵木糖产乙醇的工业酿酒酵母菌株,对木质纤维素燃料乙醇的开发具有重要意义.与氧化还原途径相比,木糖异构化途径的表达不会因辅酶不平衡而造成中间产物木糖醇的累积,因此被视为是理想的木糖代谢途径.在木糖异构途径的表达过程中,选择工业酿酒酵母作为出发菌株进行木糖异构酶途径的表达具有突出优势.同时,提高木糖异构酶基因xylA的表达效率对木糖异构菌株的构建至关重要.另外,在对XI菌株进行代谢工程改造时,GRE3的敲除、木糖转运的提升、木糖代谢途径的定向改造等均能有效改善菌株发酵木糖产乙醇的能力.除此之外,进化工程也是提升XI菌株木糖发酵效率的重要方法之一.而在相关机理阐释和改造策略的制定过程中,组学技术已显示出强大的功能.综述了近年来木糖异构酶途径在酿酒酵母中的表达研究进展,同时还对相关研究存在的问题进行了分析.【总页数】9页(P88-96)【作者】李云成;孟凡冰;苟敏;孙照勇;汤岳琴【作者单位】成都大学药学与生物工程学院,成都 610106;成都大学药学与生物工程学院,成都 610106;四川大学建筑与环境学院,成都 610207;四川大学建筑与环境学院,成都 610207;四川大学建筑与环境学院,成都 610207【正文语种】中文【相关文献】1.工业酿酒酵母木糖代谢途径的建立及酒精发酵初步研究 [J], 郭亭;梁达奉;鲍晓明2.木糖异构酶酿酒酵母孢子“微胶囊”的构建及酶学性质分析 [J], 李毅;李子杰;中西秀树;高晓冬3.稳定高效表达木糖还原酶基因工业酿酒酵母的构建及木糖醇发酵的初步研究 [J], 李敏;马涛;生举正;郭亭;鲍晓明4.酿酒酵母木糖发酵酒精途径工程的研究进展 [J], 沈煜;王颖;鲍晓明;曲音波5.宿主遗传背景对重组酿酒酵母木糖共发酵影响的研究进展 [J], 程诚;熊亮;于欣水;赵心清;徐友海;胡世洋;白凤武因版权原因，仅展示原文概要，查看原文内容请购买。

基于生物信息学的假丝酵母尿酸氧化酶聚集特性改造及去免

基于生物信息学的假丝酵母尿酸氧化酶聚集特性改造及去免摘要尿酸氧化酶是一种重要的生物催化剂，可广泛应用于药物制剂、生物传感器等领域。

然而，尿酸氧化酶在应用过程中存在固有的不稳定性和催化活性低的缺点，限制了其应用范围。

因此，寻找一种改造策略来提高尿酸氧化酶的稳定性和催化活性，已成为研究的热点问题。

本研究借助生物信息学分析，设计了一种基于假丝酵母尿酸氧化酶聚集特性改造及去免策略，旨在提高尿酸氧化酶的稳定性和催化活性。

首先，对假丝酵母尿酸氧化酶的序列进行分析，发现其具有聚集倾向。

然后，结合仿生学原理，设计了一种新型的尿酸氧化酶融合结构。

该结构由一个具有聚集抑制作用的多肽序列和一个尿酸氧化酶结构域组成。

为了实现该融合结构的生物合成，采用了基因重组技术和原核表达系统，成功表达出融合蛋白。

接着，对融合蛋白的生化性质进行了表征。

结果显示，与野生型尿酸氧化酶相比，融合蛋白具有更高的催化活性和稳定性。

通过热力学和动力学实验，发现多肽序列通过抑制聚集反应，提高了融合蛋白的稳定性，同时尿酸氧化酶结构域保持其催化活性。

在适宜条件下，融合蛋白的最高催化活性提高了50%以上。

最终，采用酵母表达系统，成功实现了大规模生产融合蛋白的目标。

该研究为基于生物信息学的尿酸氧化酶改造提供了一种新的思路和方法，有望为尿酸氧化酶的应用提供更广泛的应用范围和更高的经济效益。

关键词：尿酸氧化酶；生物信息学；聚集特性；改造；催化活性AbstractUric acid oxidase is an important biocatalyst, which can be widely used in pharmaceutical formulations, biosensors and other fields. However, uric acid oxidase has inherent instability and low catalytic activity in the application process, which limits its application scope. Therefore, it has become a hot issue to find a modification strategy to improve the stability and catalytic activity of uric acid oxidase. In this study, based on bioinformatics analysis, a strategy of modifying and removing immunity based on the aggregation characteristics of Pichia pastoris uric acid oxidase was designed to improve the stability and catalytic activity of uric acid oxidase.Firstly, the sequence of Pichia pastoris uric acid oxidase was analyzed, and it was found that it had aggregation tendency. Then, based on the bionic principle, a novel uric acid oxidase fusion structure was designed. The structure consists of a polymer sequence with anti-aggregation activity and a uric acid oxidase structure domain. In order to realize the biosynthesis of the fusion structure, genetic recombination technology and bacterial expression system were used to successfully express the fusion protein.Then, the biochemical properties of the fusion protein were characterized. The results showed that compared with the wild type uric acid oxidase, the fusion protein had higher catalytic activity and stability. Through thermodynamic and kinetic experiments, it was found that the polymer sequence improved the stability of the fusion protein by inhibiting the aggregation reaction, while the uric acid oxidase structure domain maintained its catalytic activity. In suitable conditions, the maximum catalytic activity of the fusion protein increased by more than 50%.Finally, a yeast expression system was used to successfully achieve the goal of large-scale production of the fusion protein. This study provides a new idea and method for the modification of uric acid oxidase based on bioinformatics, and is expected toprovide a wider range of application and higher economic benefits for the application of uric acid oxidase.Keywords: uric acid oxidase; bioinformatics; aggregation characteristics; modification; catalytic activity.。

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Coupling governs entrainment range of circadianclocksUte Abraham1,3,Adria´n E Granada2,3,Pa˚l O Westermark2,3,Markus Heine1,3,4,Achim Kramer1,*and Hanspeter Herzel21Laboratory of Chronobiology,Charite´Universita¨tsmedizin Berlin,Berlin,Germany and2Institute for Theoretical Biology,Humboldt University Berlin,Berlin,Germany 3These authors contributed equally to this work4Present address:Department of Anatomy II,University Medical Center Hamburg-Eppendorf,Hamburg20246,Germany*Corresponding boratory of Chronobiology,Charite´Universita¨tsmedizin Berlin,Hessische Str.3-4,Berlin10115,Germany.Tel.:þ4930450524263; Fax:þ4930450524942;E-mail:achim.kramer@charite.deReceived31.3.10;accepted7.10.10Circadian clocks are endogenous oscillators driving daily rhythms in physiology and behavior.Synchronization of these timers to environmental light–dark cycles(‘entrainment’)is crucial for anorganism’sﬁtness.Little is known about which oscillator qualities determine entrainment,i.e.,entrainment range,phase and amplitude.In a systematic theoretical and experimental study,weuncovered these qualities for circadian oscillators in the suprachiasmatic nucleus(SCN—the masterclock in mammals)and the lung(a peripheral clock):(i)the ratio between stimulus(zeitgeber)strength and oscillator amplitude and(ii)the rigidity of the oscillatory system(relaxation rate uponperturbation)determine entrainment properties.Coupling among oscillators affects both qualitiesresulting in increased amplitude and rigidity.These principles explain our experimentalﬁndingsthat lung clocks entrain to extreme zeitgeber cycles,whereas SCN clocks do not.We conﬁrmed ourtheoretical predictions by showing that pharmacological inhibition of coupling in the SCN leads tolarger ranges of entrainment.These differences between master and the peripheral clocks suggestthat coupling-induced rigidity in the SCNﬁlters environmental noise to create a robust circadiansystem.Molecular Systems Biology6:438;published online30November2010;doi:10.1038/msb.2010.92Subject Categories:computational methods;metabolic&regulatory networksKeywords:circadian clock;coupling;entrainment;mathematical modeling;oscillatorThis is an open-access article distributed under the terms of the Creative Commons AttributionNoncommercial No Derivative Works3.0Unported License,which permits distribution and reproductioninanymedium,providedtheoriginalauthorandsourceare credited.Thislicense does notpermitcommercialexploitation or the creation of derivative works without speciﬁc permission.IntroductionDaily rhythms in physiology,metabolism and behavior are controlled by an endogenous circadian timing system,which has evolved to synchronize an organism to periodically recurring environmental conditions,such as light–dark or temperature cycles.In mammals,the circadian system relies on cell-autonomous oscillators residing in almost every cell of the body.Essentially the same molecular components are arranged as interlocked transcriptional–translational feedback loops generating B24-h rhythms at the molecular level(for a review see Reppert and Weaver,2002).Precise synchroniza-tion of rhythms is an essential part of circadian organization, which usually follows hierarchically organized steps:(i) Periodic light detected by the eyes entrains(i.e.,synchronizes) B20000neurons in the bilateral suprachiasmatic nucleus (SCN)of the hypothalamus via neuronal signals traveling along the retinohypothalamic tract(for a review see Maywood et al,2007).(ii)Within SCN tissue,individual neurons synchronize each other via neuropeptide coupling/synaptic signaling and/or gap junctions(Shirakawa et al,2001;Aton and Herzog,2005).This generates precise and self-sustained B24-h oscillations,e.g.,of electrical activity in the SCN or of locomotor behavior,even in the absence of external signals(Pittendrigh and Daan,1976;Reppert and Weaver, 2002;Herzog et al,2004).Inhibition of this intra-SCN synchronization shows that the periods of the individual SCN neurons are quite variable ranging from20to28h(Welsh et al,1995;Honma et al,2004).(iii)Subsequently,the SCN synchronizes other peripheral tissues orchestrating the rhythmicity and phasing of their circadian clocks.Little is known about the mechanisms of this communication; however,hormonal signals,sympathetic enervation and/or indirect cues,such as body temperature,feeding time and activity rhythms,have been discussed(Levi and Schibler, 2007).In contrast to the SCN,cellular clocks within a peripheral tissue seem to be much less coupled,leading to rapid desynchronization of individual oscillator cells when a rhythmic environment is removed(Nagoshi et al,2004; Liu et al,2007).Molecular Systems Biology6;Article number438;doi:10.1038/msb.2010.92 Citation:Molecular Systems Biology6:438&2010EMBO and Macmillan Publishers Limited All rights reserved1744-4292/10 Many theoretical studies underline that coupled rhythms of similar periods synchronize (Huygens,1673;von Holst,1939;Winfree,1980;Kuramoto,1984;Balanov et al ,2009).They discuss detailed bifurcation diagrams,including toroidal oscillations and deterministic chaos (Glass et al ,1987;Pikovsky et al ,2001;Anishchenko et al ,2007).Even though in circadian rhythms complex nonlinear phenomena are found under certain experimental circumstances (Helfrich-Fo ¨rster,2004;de la Iglesia et al ,2000,2004),a stable entrainment is observed in most physiological situations.Nevertheless,the robust and relatively fast synchronization of circadian rhythms in mammals is nontrivial due to the quite limited synchronization strengths (i.e.,entrainment signals,intra-SCN signals and signals from the SCN to the periphery).In general,immediate synchronization is not surprising when the stimuli are very strong (e.g.,deﬁbrillation of the heart).Y et a robust and fast synchronization in systems with weak synchroniza-tion signals probably requires a speciﬁc evolutionary design of oscillator properties and coupling schemes.Little is known about which oscillator properties govern synchronization characteristics of circadian oscillators,such as the range of entrainment (i.e.,the range of zeitgeber periods to which a cell,a tissue or an organism is able to entrain (Pittendrigh and Daan,1976;Chiesa et al ,2007)).In addition,the oscillator properties of SCN and peripheral tissues that govern entrain-ment behavior have not been systematically studied,although examples indicate that entrainment properties are substan-tially different:The circadian oscillator in mouse liver entrains to an inverted environmental temperature cycle,while the SCN does not (Brown et al ,2002).Moreover,SCN oscillations usually exhibit moderate phase shifts in response to a zeitgeber stimulus (Prosser and Gillette,1989;Piggins et al ,1995;Wisor and T akahashi,1997;Gribkoff et al ,1998;Best et al ,1999),while organotypic lung slices were recently shown to exhibit remarkably large phase responses (Gibbs et al ,2009).Here,we report a systematic theoretical and experimental study of the entrainment range of the central circadian clock (SCN)on one hand and a peripheral clock (lung)on the other hand.We subjected lung and SCN tissue slices to identical temperature cycles to compare their entrainment properties.We show that—in contrast to lung tissue—the SCN constitutesa rigid oscillator that cannot easily entrain to temperature cycles with experimental periods of 20or 28h.We further set out to understand theoretically which properties of a tissue determine the entrainment range.This approach allowed us to infer features of the tissue,particularly about the involved biological oscillators and their ing generic oscillator models we show how the period,the relaxation rate,the coupling strength,as well as the ratio between zeitgeber strength and amplitude govern the entrainment range.In particular,simulations predict that reduced coupling strength leads to a weak oscillatory state and consequently a larger entrainment range.We tested and conﬁrmed these predictions experimentally by studying the entrainment of SCN slices treated with drugs that attenuate coupling.ResultsAmplitude and zeitgeber strength determine the entrainment rangeT o set the stage for a systematic theoretical investigation of entrainment,we ﬁrst summarize established concepts of entrainment and state our assumptions and deﬁnitions.Figure 1A schematically describes the concept of oscillator entrainment to external signals (zeitgebers, e.g.,light,temperature or food).Zeitgeber cycles interact with the endogenous oscillator in such a way that the frequencies are locked in a 1:1entrainment ratio,leading to a stable phase relation between the external entraining signal and the entrained oscillator.Critically,upon release into constant conditions,entrained oscillations persist with a phase predicted from the previous zeitgeber cycle.Note that we do not yet assume anything about the physical nature of the oscillator:it could be a single-cell biochemical oscillator (Leloup and Goldbeter,2003;Forger and Peskin,2003;Becker-Weimann et al ,2004)or a synchronized population of cells,such as the SCN.However,many details of the kinetic mechanisms are not known,and in particular,the chosen nonlinearities in detailed models,which shape the dynamics of the self-sustained oscillations,are not based on experi-mental data.Consequently,we follow in this section the tradition of Winfree (1980),Kronauer et al (1982)andGlassABFigure 1Basic concepts of entrainment.(A )Schematic representation of a circadian rhythm entrained by 24-h temperature cycles.The quasi-square-wave temperature cycle represents the zeitgeber cycle used in the experiments of this study (see Figures 4and 6).Note that upon complete entrainment the phase angle between rhythmic variable and zeitgeber cycle is constant.(B )Schematic representation of the entrainment region.The entrainment region is dependent on zeitgeber period (T )and zeitgeber strength and is also known as 1:1Arnold tongue.Small-amplitude oscillators exhibit a broader range of entrainment than large-amplitude oscillators.For a constant zeitgeber strength,the entrainment region is conﬁned between its lower (T Low )and upper (T High )limit.Coupling governs circadian entrainment U Abraham et aland Mackey(1988)and study generic amplitude-phase oscillators.Figure1B illustrates that the range of entrainment depends on zeitgeber strength and oscillator amplitude.Entrainment is most easily achieved if the endogenous and zeitgeber periods are similar.For example,the circadian behavior in laboratory rodents can be entrained to zeitgeber cycles with deviations of up to2h from the endogenous B24-h period.The entrainment range can be enlarged if the zeitgeber strength is increased. Thus,the entrainment region typically shows a tongue-shaped form(Berge et al,1984).Alternatively,small-amplitude oscillators can be entrained more easily as the effect of the zeitgeber is stronger(Pittendrigh et al,1991;Vitaterna et al, 2006;Brown et al,2008).Proceeding in general terms,we assume the oscillator can be characterized by its amplitude,its intrinsic period and its stability with respect to amplitude perturbations(amplitude relaxation rate or Floquet exponent(Guckenheimer and Holmes,1983)).These general oscillator properties are conveniently parametrized by the Poincare´oscillator(Glass and Mackey,1988),which is given by the following equations in polar coordinates with the radial coordinate r and phase f:d rd t¼l rðA0ÀrÞd f¼2pð1ÞA0and t denote amplitude and intrinsic period of the oscillator. The parameter l quantiﬁes the relaxation of amplitudes to the stable oscillation(limit cycle)characterized by r¼A0.We term oscillators with small radial relaxation rates as l weak oscillators.Conversely,we term oscillators with large relaxa-tion rates as rigid oscillators.In terms of timescales,we deﬁne an oscillator as rigid if the radial relaxation time1/l is faster than the intrinsic period,i.e.,1/l o t.From dynamical systems theory,it is known that a set of synchronized coupled oscillators can be dynamically described as a one-oscillator model,such as the Poincare´oscillator,where the parameters and variables represent the whole synchronized population. Thus,we demonstrate(see below and Supplementary Information)that the results obtained by studying this generic oscillator are general enough to apply to either single cells,or asynchronized rhythm generated by coupled oscillators. Relaxation rate affects range of entrainment,entrained amplitude and phase shifting properties Which properties of an oscillator are crucial for determining its range of entrainment?T o test how the amplitude relaxation rate affects the response to zeitgeber cycles,i.e.,the entrainment range,we calculated the entrainment range for different relaxation rates l for the Poincare´oscillator (Figure2A).Such an illustration of the entrainment region is also termed1:1‘Arnold tongue’(Berge et al,1984).Even for a relative small zeitgeber strength,entrainment to cycles with deviations of up to2h from the endogenous B24-h period can be achieved(with A0¼1and a zeitgeber strength of0.05hÀ1in these simulations).The Arnold tongue is somewhat skewed, i.e.,an external signal with long periods can entrain the oscillator more easily.The comparison of the inner curves(fast amplitude relaxation l¼1hÀ1)and the outer curves(slower relaxation l¼0.03hÀ1)reveals that weak oscillators are more easily entrained.These particular values of the amplitude relaxation rates l are comparable with experimental measure-ments(Brown et al,2008;Westermark et al,2009).Our results for the generic Poincare´oscillator also hold for other oscillator models,such as the standard Hopf oscillator(Guckenheimer and Holmes,1983)or the more realistic biophysical circadian clock Becker-Weimann–Bernard model(Becker-Weimann et al,2004;Bernard et al,2007)(see Supplementary Informa-tion and Supplementary Figures S1and S2). Interestingly,also the oscillator amplitude during entrain-ment depends on the amplitude relaxation rate l(Figure2B). Weak oscillators exhibit a considerable amplitude expansion (A ent4A0)for zeitgeber periods close to the oscillator’s intrinsic period,and an amplitude reduction(A ent o A0)for zeitgeber periods close to both upper and lower limit of entrainment(LLE).Rigid oscillators exhibit almost no amplitude variations(A ent E A0)within the range of entrain-ment.Some of these effects were described earlier inexperimental and theoretical studies(Wever,1972;Aschoff and Pohl,1978;Roenneberg et al,2005;KurosawaandFigure2Entrainment range and amplitude depend on the oscillator relaxation rate.(A)Numerically calculated entrainment region for a Poincare´oscillator with radius 1plotted as a function of zeitgeber period and zeitgeber strength.The entrainment range is broader for weak oscillators with low relaxation rates l.(B)Entrained amplitude of weak and rigid oscillators within the entrainment range.Weak oscillators exhibit a strong amplitude expansion for zeitgeber periods close to the oscillator’s intrinsic period(24h),and an amplitude reduction for zeitgeber periods close to both upper and lower limit of entrainment.Rigid oscillators remain almost unperturbed along the entrainment range.Numerical simulations results(dots)and analytically derived curves(lines)are in good agreement.Coupling governs circadian entrainmentU Abraham et alGoldbeter,2006).Here,we also ﬁnd amplitude expansion upon entrainment of lung tissue (Figure 4A and C,for details see below).The effect of the amplitude relaxation rate l on entrainment is also reﬂected in its effect on the shape of the phase–response curves (describing the response of the oscillator to single zeitgeber pulses)of weak and rigid oscillators.Already Pittendrigh (1965)and later Honma et al (1985)argued that entrainment range and phase–response curve amplitude are closely related.We also ﬁnd in our simulations that weak oscillators—while having a larger range of entrainment—also react with overall larger phase shifts to a zeitgeber stimulus than rigid oscillators (Supplementary Figure S3A).Experi-mentally,several studies have shown that the SCN reacts with small-to-moderate phase shifts upon zeitgeber stimuli (Wisor and T akahashi,1997),whereas peripheral clocks such as the lung elicit large phase shifts upon zeitgeber stimuli (Gibbs et al ,2009),suggesting that SCN might constitute a rigid and lung a weak oscillator.We conﬁrmed these ﬁndings with forskolin as a zeitgeber—forskolin activates cAMP-mediated signaling to the clock (Y agita and Okamura,2000;Obrietan et al ,1999;Brown et al ,2008)—in direct comparison of these two tissues (Supplementary Figure S3B).The ratio of zeitgeber strength to oscillator amplitude determines the range of entrainmentIt has been discussed earlier (Pittendrigh et al ,1991;Vitaterna et al ,2006;Brown et al ,2008;van der Leest et al ,2009)that zeitgeber stimuli can have a strong effect on small-amplitude oscillators.We hypothesized that a single quantity,the ratio between zeitgeber strength and oscillator amplitude,can predict the entrainment range if the amplitude relaxation and the oscillator period are known.T o test this theoretically,we numerically calculated the LLE T low as a function of the zeitgeber strength to oscillator–amplitude ratio (Figure 3and Supplementary Figure S4).The dots represent values for thePoincare´oscillator for a large number of combinations of zeitgeber strengths and amplitudes.In addition to thesenumerical results for the Poincare´oscillator,we derived analytically the LLE under very general assumptions.The analytically derived relationship (solid curve)is in good agreement with the numerical results.We conclude that the ratio between the zeitgeber strength and oscillator amplitude indeed determines the entrainment range for a given oscillator period and relaxation rate l .In the Supplementary Informa-tion section,we apply an analytical theory of entrainment from which the phase of entrainment C ent and entrained amplitude A ent are obtained.In Equation 2,these analytical results are given as functions of the oscillator period t ,amplitude A 0,zeitgeber strength B ,zeitgeber period T ,and amplitude relaxation rate l and detuning deﬁned as D ¼2p /t À2p /T .T low ¼t1þðB t =4p A 0ÞC ent ¼arcsin 2A 0DA ent ¼l 2A 0l 2þD 2þﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃð2l 2A 0Þ2Àðl 2þD 2Þð4l 2A 20ÀB 2Þ4ðl 2þD 2Þ2s ð2ÞT ogether,the results shown above (Figures 2and 3)reﬂectvery general entrainment properties of simple,generic oscillator models:the entrainment behavior of an oscillator is essentially determined by its intrinsic period,its amplitude relaxation rate and by the ratio between zeitgeber strength and amplitude.We justify these claims by analytical calculations,as well as by numerical simulations of a generic Hopf oscillator and the more realistic biophysical circadian clock Becker-Weimann–Bernard model (see Supplementary Information and Supplementary Figures S1,S2and S3A).Lung tissue behaves as a weak oscillator and SCN as a rigid oscillatorBased on our theoretical considerations elaborated above,we predict that weak,small-amplitude oscillators exhibit a larger range of entrainment compared with more rigid,high-amplitude oscillators.T o test these predictions experimentally,we investigated the entrainment of circadian oscillators using temperature as a zeitgeber.T emperature is a key environ-mental factor for the regulation of circadian rhythms of many species (Sweeney and Hastings,1960;Rensing and Ruoff,2002).In addition to being an environmental input,tempera-ture cycles have also been shown to act as a synchronizing factor in vivo (Brown et al ,2002).Mice usually experiencedaily temperature variations of up to 21C (Kapa´s and Krueger,1996),which are considered to contribute to tissue clock synchronization in vivo .Thus,we used a quasi-square-wave temperature proﬁle ranging from 35.51C (cold)to 371C (warm)with gradual transitions between these two conditions (see Materials and methods).We tested two different temperature cycles consisting of either 10h of cold and 10h of warm,or 14h of cold and 14h of warm.Hence,these cycles represent zeitgeber periods T of 20or 28h,which both are presumably close to the borderline of the entrainment range for circadian oscillators.As readout for clock dynamics,we used a bioluminescence-based mouse model,where therhythmicFigure 3The ratio of zeitgeber strength to oscillator amplitude determines entrainment range.The lower limit of entrainment is plotted as a function of the ratio of zeitgeber strength to oscillator amplitude.The area under the analytically derived curve represents the entrainment region.The dots are numericallysimulated lower limits of entrainment for a Poincare´oscillator with l ¼1h Àputational details are given in Material and methods,and theoretical details are provided in Supplementary Information.Coupling governs circadian entrainment U Abraham et alclock protein PER2(a state variable for the circadian clock)is fused to luciferase as a reporter(Y oo et al,2004).Tissue explants from PER2::LUC mice were subjected to temperature cycles and simultaneously monitored for biolu-minescence rhythms.T o test for predicted differences in the entrainment behavior of rigid and weak oscillators,we used SCN tissue as an example of a putatively rigid oscillator(Wisor and T akahashi,1997)and lung tissue as an example of a putatively weak,peripheral oscillator(Gibbs et al,2009;see also Supplementary Figure S3).Whereas explanted lung tissue entrained to both the20-h and the28-h temperature cycles, SCN tissue did not entrain to either(Figure4).It took the lung tissue about three so-called‘transient cycles’(days4–6, Figure4B and D)to adopt a stable phase relationship to the zeitgeber cycles.This can be,for example,nicely seen in Figure 4A,as initially two daily peaks of PER2abundance:one decreasing peak during the cold phase corresponding to the phase before entrainment,and a second,increasing peak during the warm phase representing the new,entrained phase (Figure4A).This increased amplitude of the second peak corresponds nicely to our theoretical prediction,concerning amplitude expansion upon entrainment(Figure2B).The phase of the entrained PER2abundance persisted upon release into constant temperature,strongly suggesting true entrain-ment of the lung clock to the temperature cycle rather than direct temperature effects(so-called‘masking’).In contrast to lung,SCN oscillation appeared unperturbed by temperature (e.g.,we did not observe any transient effects on SCN oscillation,such as an increasing secondary peak of PER2 abundance).This becomes even clearer in Figure4B and D, where peak expression times in the lung moved along with the daily progression of cold phases,while SCN tissue peaked unaltered at similar times each day,i.e.,is presumably free-running.The successful entrainment of lung tissue,but not SCN tissue,to such extreme zeitgeber periods with relatively small temperature differences conﬁrms the predictions of our model.It further indicates that lung tissue is probably indeed a weak oscillator,whereas the SCN is a rigid oscillator.Our theoretical considerations also predict that increasing the zeitgeber strength enlarges the range of entrainment (Figure2A),and at the same time affects the phase of entrainment(Supplementary Figure S5A)as already noted by24681012DaysTime (h)BA0246810120.751.001.2520hRel.bioluminescenceTime (days)024********1.002.00Rel.bioluminescenceC24681012DaysD Time (h)Figure4Lung tissue behaves like a weak and the SCN like a rigid circadian oscillator.(A,C)Temperature entrainment experiment with SCN and lung slices from PER2::LUC knockin mice.Tissues were cultured in luciferin-containing medium at371C,and bioluminescence derived from rhythmic PER2-LUC abundance was continuously monitored.Between days4and8or9,tissues were subjected to20or28-h temperature cycles with10or14h35.51C(blue boxes)and10h or14h371C before releasing them in constant371C conditions.Periods before,during and after entrainment are given in Supplementary Table S1.(B,D)Double plots of the peak times(±s.d.;n¼4)of PER2-LUC bioluminescence.The circadian clock of lung tissue entrains to the D T¼1.51C temperature cycles(blue:low temperature),while the SCN clock does not:The bioluminescence peaks of the lung slices adopt a stable phase relation to the temperature cycle,while the SCN-derived peaks‘run through’. After release in constant371C(from day9on),lung-derived bioluminescence rhythms continue with a phase that is predicted from the previous temperature cycles,thus excluding temperature-driven,so-called,masking effects.Source data is available for thisﬁgure at /msb.Coupling governs circadian entrainmentU Abraham et alothers(Roenneberg et al,2003).Thus,we speculated that—although having a smaller range of entrainment than lung clocks—SCN clocks might entrain to extreme zeitgeber cycles, if we would use a stronger zeitgeber.Therefore,we again applied an extreme temperature cycle(T¼20h)to lung and SCN explant cultures,but this time with increasing zeitgeber strength(0.75,1.5,3and61C temperature variation).We found that(i)the SCN clock still did not entrain to these stronger zeitgebers and(ii)the lung clocks entrained in every case,but with a zeitgeber dose dependence of the phase of entrainment,as predicted by theory(Supplementary Figure S5). Thus,the entrainment range of SCN clocks seems to be even smaller than anticipated.T o verify that SCN clocks are able to entrain to temperature cycles at all(as shown by others,e.g., Herzog and Huckfeldt,2003),we then applied a less extreme zeitgeber cycle(T¼22h)again with increasing zeitgeber strength (4,6and81C temperature variation).Now,we observe entrainment for the two stronger zeitgebers(with,again, different phases of entrainment),but not for the weaker zeitgeber (Supplementary Figure S6).T ogether,these experiments nicely recapitulate the theoretical entrainment concepts illustrated by the so-called Arnold tongue:whether an oscillator entrains depends on how close the zeitgeber cycle is to the intrinsic period of the oscillator and how strong the zeitgeber is.In addition, zeitgeber strength also determines the phase of entrainment. Coupling makes oscillators more rigidWhat is the fundamental difference between lung and SCN tissue that so profoundly affects entrainment behavior?As suggested earlier(Bernard et al,2007;van der Leest et al,2009), coupling of circadian rhythms can have a huge effect on the properties of oscillators.So far,we have considered oscillators in a general sense:they could be individual cells or a synchronized, orchestrated population of a large number of coupled cells.T o investigate the inﬂuence of coupling strength on the entrainment behavior,we focus on coupled oscillators(e.g.,SCN cells coupled via neuropeptides,Aton and Herzog,2005)and analyze period,amplitudes and relaxation rate of the synchronized system.As an elucidating example,we study two coupled Poincare´oscillators described by the following equations:d x i d t ¼Àg x iðr iÀA0ÞÀ2pty iþMþFd y i¼Àg y iðr iÀA0Þþ2px ið3ÞThe single-uncoupled oscillator i has an intrinsic period of t¼24h.Here,we consider two oscillators:i¼1or2with r i¼(x iþy i)1/2being equivalent to the radial coordinate in the polar representation of the oscillator(Equation1).A0is the amplitude of the stable limit cycle oscillation,and g is the amplitude relaxation rate of the single-non-coupled oscillator (note,in Equation1we use l for the relaxation rate to emphasize that it characterizes either a single oscillator or an oscillatory system).Coupling is modeled as an average of the states of the two oscillators(a meanﬁeld M,Kuramoto,1984):M¼K2ðx1þx2ÞThis type of coupling has been previously suggested for coupled SCN neurons assuming fast(relative to the24-h oscillation period)diffusion of coupling neuropeptides(Gonze et al,2005; Locke et al,2008).Finally,we allow the system to be entrained by a square-wave forcing term F that represents,for example, light or temperature entrainment.Our conclusions derived from this simple system of two coupled oscillators(see below)are also valid for more realistic biochemical circadian clock models (see Supplementary Information and Supplementary Figure S2). T o understand how coupling governs the dynamical proper-ties of a system of coupled oscillators,weﬁrst analyzed the model(Equation3)without entrainment(i.e.,F¼0).In this case,the resulting limit cycle of the coupled system can be again characterized by its period,amplitude and relaxation rate.Weﬁnd that increasing coupling strength leads to a drastic increase of the amplitude most pronounced for small values of g,the relaxation rate of the individual oscillators (Figure5A)—a well-known resonance phenomenon(Guck-enheimer and Holmes,1983).It implies that coupling of resonating clock cells,i.e.,weak oscillators,may lead to increased amplitudes of the oscillating system and,as a consequence(Figure3),to a smaller range of entrainment. Interestingly,the amplitude relaxation rate of the coupled system l is primarily dominated by the coupling strength,and, surprisingly,this rate is independent of the radial relaxation rates g of the individual oscillators(Figure5B).The coupling rather than the single-cell oscillators determines the amplitude relaxation rate of the synchronized system—with the described implications for entrainment(Figure2).This intriguingﬁnding reﬂects the fact that deviations from the synchronized state are damped via the coupling term (Pikovsky,1984).In other words,in a network of oscillators almost all perturbations affect synchrony.This implies that the relaxation to the equilibrium is strongly inﬂuenced by the coupling that brings the network back to synchrony. For sufﬁciently small coupling,the dominant amplitude relaxation rate of the coupled system is proportional to the coupling strength as shown in Figure5B,and determines the relaxation to synchrony rather than the individual oscillator properties.In summary,coupling makes the synchronized oscillatory state more rigid in two aspects:(i)resonance increases the amplitude and(ii)coupling leads to faster relaxation.These theoretical considerations now predict that decreas-ing the coupling should enlarge the range of entrainment,as (i)amplitude should decrease,and(ii)the coupled oscillatory state should become weaker(i.e.,the relaxation rate should decrease).This is indeed the case for coupled Poincare´oscillators(Equation3):When we calculate the LLE as a function of coupling strength(for different single-oscillator relaxation rates),we see that the LLE increases as the coupling strength increases(Figure5C),i.e.,coupling makes the system harder to entrain.Furthermore,the LLE also generally decreases with increasing relaxation rates of the single oscillators.In other words,the weaker the individual oscillators within a coupled system are,the harder is it to entrain this system.This atﬁrst glance seems counterintuitive (because for individual oscillators the opposite is true, Figure2A),but is due to the amplitude expansion that occurs when the single-oscillator relaxation rates are low(Figure5A). This increase in amplitude of the coupled system leads to smaller ranges of entrainment(Figure3).Coupling governs circadian entrainment U Abraham et al。