regulatory pathways
第二章-天然产物有效成分的毒理学安全性与功能性评价PPT课件
体内试验和体外试验各有其优点和局限性，应主要根据实 验研究的目的要求，采用最适当的方法，并且互相验证。
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3. 人体观察
通过中毒事故的处理或治疗，可以直接获得关 于人体的毒理学资料，这是临床毒理学的主要 研究内容。有时可设计一些不损害人体健康的 受控的实验，但仅限于低浓度、短时间的接触， 并且毒作用应有可逆性。
是在人类生活的外界环境中存在、可能与机体接 触并进入机体，在体内呈现一定的生物学作用的一些化 学物质，又称为“外源生物活性物质”。
内源化学物
是指机体内原已存在的和代谢过程中所形成的产 物或中间产物。毒理学研究外源化学物对机体的有害作 用，而不是有益作用(如营养作用、治疗作用等)。
3
毒理学的目的 任何一种化学物质在一定条件下都可能是
❖ 毒理学实验可采用整体动物、游离的动物脏器、组织、 细胞进行。根据所采用的方法不同，可分为体内试验 (in vivo test)和体外试验(in vitro test)。毒理学还 利用限定人体试验和流行病学调查直接研究外源化学 物对人体和人群健康的影响。
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表1-1
化学物 乙醇 氯化钠 硫酸亚铁 硫酸吗啡
第二章 天然产物有效成分的 毒理学安全性和功能性评价
✓ 毒理学评价
✓ 安全性评价 ✓ 功能性评价
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一、毒理学安全性评价
❖ 毒理学(toxicology)
是研究化学、物理和生物因素对机体的损害作用、 生物学机制（biologic mechanisms）、危险度评价 （risk assessment）和危险度管理（risk management）的科学。毒理学主要分为三个研究领域， 即描述毒理学、机制毒理学和管理毒理学。
调控巨噬细胞极化的相关信号通路及其调节机制研究进展
调控巨噬细胞极化的相关信号通路及其调节机制研究进展①刘利萍张焱皓李茂秦欢罗军敏（遵义医科大学免疫学教研室，贵州省免疫分子应用研究工程中心，遵义563000）中图分类号R392.9文献标志码A文章编号1000-484X（2021）06-0747-07［摘要］巨噬细胞是一群具有高度可塑性和异质性的免疫细胞，在维持免疫系统的稳定状态中扮演重要角色。
不同刺激因子作用下，巨噬细胞可极化为M1型和M2型，其极化过程受多种信号通路共同影响。
本文综述巨噬细胞极化过程涉及的主要信号通路及其调节机制的新进展。
［关键词］巨噬细胞；极化；信号通路；调节机制Advances in related signaling pathways and their regulatory mechanisms of macrophage polarizationLIU Li-Ping，ZHANG Yan-Hao，LI Mao，QIN Huan，LUO Jun-Min.Department of Immunology，Zunyi Medical Uni⁃versity，Immune Molecules Application Research Center in Guizhou Province，Guizhou563000，China ［Abstract］Macrophages are a group of highly adaptive and heterogeneous immune cells that play an important role in maintain⁃ing stable state of immune system.Macrophages can be polarized into M1and M2types under stimulation of different cytokines.Pro⁃cess of macrophage polarization is affected by a variety of signal pathways.This review will summarize major signaling pathways of mac⁃rophage polarization as well as their regulatory mechanisms.［Key words］Macrophage；Polarization；Signaling pathway；Regulatory mechanisms作为机体固有免疫系统的重要组成部分，巨噬细胞具有吞噬并杀伤病原微生物、免疫信息传递等功能，在炎症防御、组织发育和维持机体动态平衡等过程发挥重要作用。
Biosynthesis and Biosynthetic Pathways
Biosynthesis and Biosynthetic PathwaysBiosynthesis is the process by which living organisms, both plants and animals, produce complex and essential organic molecules from simpler ones. These organic molecules include amino acids, nucleotides, carbohydrates, lipids, and many other compounds required for the growth, development, and functioning of cells and tissues. Biosynthesis occurs through a series of chemical reactions, which are catalyzed by specific enzymes. These enzymatic reactions are organized into biosynthetic pathways, which are highly regulated and controlled by various mechanisms to ensure a proper balance of substrate utilization, product formation, and energy conservation.Amino Acid BiosynthesisAmino acids are the building blocks of proteins, which are essential macromolecules that perform a wide range of biological functions. There are 20 amino acids that are commonly found in proteins, and each of them can be synthesized de novo by living organisms, starting from simpler precur sors such as glucose, pyruvate, or α-ketoglutarate. The biosynthetic pathways for amino acids are diverse and complex, involving multiple steps and intermediates, as well as various cofactors and regulatory molecules. For example, the biosynthesis of alanine, a non-essential amino acid, involves the conversion of pyruvate to alanine via transamination and reductive amination reactions, which are catalyzed by the enzymes alanine transaminase and alanine dehydrogenase, respectively. Other amino acids, such as lysine and tryptophan, require complex pathways involving multiple enzymes and feedback inhibition mechanisms.Nucleotide BiosynthesisNucleotides are the basic units of DNA and RNA, which are the genetic materials that encode and transmit the genetic information of living organisms. Nucleotides also serve as energy carriers (ATP), signaling molecules (cyclic nucleotides), and cofactors (NAD+, FAD). The biosynthesis of nucleotides is a complex process that involves the synthesis of the purine or pyrimidine bases, the addition of sugar and phosphate groups, and the assembly of the nucleotide triphosphates. The biosynthetic pathways for purinesand pyrimidines are distinct and involve multiple enzymatic reactions and regulatory steps. For example, the biosynthesis of purines, such as adenine and guanine, starts with the synthesis of inosine monophosphate (IMP) from ribose-5-phosphate and amino acids such as glycine and glutamine, via a series of reactions catalyzed by enzymes such as PRPP synthase, adenylosuccinate synthase, and adenylosuccinate lyase.Carbohydrate BiosynthesisCarbohydrates are the primary source of energy for many organisms, including humans. Carbohydrates also serve as structural components of cell walls, extracellular matrix, and glycoproteins. The biosynthesis of carbohydrates involves the conversion of simple sugars such as glucose, fructose, and galactose into more complex polysaccharides through a series of condensation and glycosylation reactions. The biosynthetic pathways for carbohydrates are highly regulated and coordinated, involving enzymes such as hexokinase, phosphofructokinase, and glycogen synthase.Lipid BiosynthesisLipids are a diverse group of molecules that serve many functions in living organisms, including energy storage, membrane structure, signaling, and insulation. The biosynthesis of lipids involves the synthesis of fatty acids, which are then assembled into triglycerides, phospholipids, and other lipid classes. The biosynthetic pathway for fatty acids involves the conversion of acetyl-CoA into malonyl-CoA, which is then used as the building block for fatty acid synthesis. The synthesis of fatty acids is catalyzed by the enzyme fatty acid synthase, which is composed of several subunits that work together to synthesize the fatty acid chain.ConclusionBiosynthesis and biosynthetic pathways are essential processes that contribute to the diversity and complexity of living organisms. These processes involve the utilization of simple precursors such as glucose, amino acids, and nucleotides to synthesize complex organic molecules such as proteins, DNA, and lipids. The biosynthetic pathways for these molecules are highly regulated and controlled, involving multiple enzymatic reactions,intermediates, and regulatory molecules. Understanding the biosynthesis of these molecules is critical for developing new drugs, designing enzymes, and engineering biological systems.。
共享经济英语六级作文
The sharing economy,a burgeoning phenomenon in recent years,has revolutionized the way we perceive ownership and access to resources.It is based on the principle of sharing access to goods and services,facilitated by technology and communitybased online platforms.Here are some key points that can be discussed in an English composition about the sharing economy,suitable for a level akin to the English proficiency required for the CET6College English Test Band6in China.1.Definition and Concept:The sharing economy can be defined as an economic system where idle assets or services are shared among individuals for monetary or nonmonetary benefits.It is driven by technology,allowing people to share resources such as cars, homes,and even skills.2.Rise and Popularity:The sharing economy has gained popularity due to several factors including economic downturns,environmental concerns,and the desire for more flexible and costeffective options.The rise of smartphones and apps has made it easier for people to access these services.3.Examples of Sharing Economy Platforms:There are numerous platforms that embody the sharing economy.For instance,Uber and Lyft for ridesharing,Airbnb for accommodation,and TaskRabbit for various tasks and errands.4.Advantages:CostEffectiveness:Users can save money by sharing resources instead of owning them. Accessibility:Services are often more accessible to a wider audience,including those who cannot afford to own similar assets.Environmental Benefits:Sharing resources can reduce waste and carbon footprint by optimizing the use of existing assets.5.Challenges and Criticisms:Regulatory Issues:The sharing economy often operates in a legal gray area,leading to conflicts with traditional businesses and regulatory bodies.Job Displacement:Traditional jobs can be threatened by the sharing economy,as it disrupts established industries.Security and Privacy Concerns:There are concerns about the safety and privacy of users,especially in accommodations and transportation.6.Impact on Society:The sharing economy can lead to a more collaborative and communityoriented society.It encourages people to think creatively about how resources can be used more efficiently.7.Future Prospects:As technology continues to advance,the sharing economy is likely to expand into new areas.It may also lead to new business models and economic theories that better accommodate this new way of accessing goods and services.8.Personal Experience:If you have used a sharing economy service,you can share your personal experience to illustrate the practical benefits or challenges you faced.9.Conclusion:Conclude by summarizing the main points and expressing your opinion on whether the sharing economy is a positive development for society and the economy.Remember to use a variety of sentence structures and vocabulary to demonstrate your language proficiency.Additionally,ensure that your essay is wellorganized with a clear introduction,body paragraphs that explore each point in detail,and a conclusion that wraps up your thoughts.。
激光植物miRNA讲解
UV-B Responsive MicroRNA Genes in ArabidopsisthalianaXuefeng Zhou ∗, Guandong Wang ∗and Weixiong Zhang 1, ∗, †Department of Computer Science and Engineering1Department of GeneticsWashington University in Saint Louis Saint Louis, MO 63130-4899, USAAbstractMicroRNAs are small, non-coding RNAs that play critical roles in post-transcriptional gene regulation. In plants, mature microRNAs pair with complementary sites on mRNAs and subse-quently lead to cleavage and degradation of the mRNAs. Many microRNAs target mRNAs that encode transcription factors, therefore, they reg-ulate the expression of many down-stream genes. In this study, we carry out a survey of Arabidop-sis microRNA genes in response to UV-B radia-tion, an important adverse abiotic stress. We de-velop a novel computational approach to identify microRNA genes induced by UV-B radiation and characterize their functions in regulating gene ex-pression. We report that in A. thaliana 21mi-croRNA genes in 11microRNA families are up-regulated under UVB stress condition. We also discuss putative transcriptional down-regulation pathways triggered by the induction of these mi-croRNA genes. Moreover, our approach can be directly applied to miRNAs responding to other abiotic and biotic stresses and extended to miR-NAs in other plants and metazoans.1IntroductionMicroRNAs are approximately 22-nucleotide long, non-coding RNAs that play critical roles in regulating gene expression at the post-transcriptional level (Bartel,2004; He and Han-non, 2004. The discovery of miRNAs (mi-croRNAs has broadened our perspectives on the mechanisms of repression of gene expression, which is an important regulatory mechanism me-diating many biological processes such as de-velopment, cell proliferation and differentiation. In plants, mature miRNAs base-pair with com-plementary sites on target mRNAs and subse-quently direct the mRNAs to be cleaved or de-graded. Plant miRNAs regulate many genes that are involved in developmental control, for example, auxin signaling (Bonnetet al., 2004; Jones-Rhoades and Bartel, 2004, organ polar-ity (Eshedet al., 2001; Kidner and Martienssen, 2004; McConnell et al., 2001, development tran-sitions (Aukermanand Sakai, 2003; Chen, 2004, leaf growth (Palatniket al., 2003 and RNA metabolism (Vaucheretet al., 2004; Xie et al., 2003. Several recent studies showed important functions of miRNAs in response to adverse abi-otic stresses (Bariet al., 2006; Jones-Rhoades and Bartel, 2004; Lu et al., 2005; Sunkar and Zhu, 2004. In Arabidopsis, miR399was identifiedt o be highly expressed under phosphate starva-∗These authors contributed equally to this research. †Corresponding author:zhang@,phone:tion (Bariet al., 2006; Chiou et al., 2006; Fu-(314935-8788,fax:(314935-7302.jii et al., 2005 and miR395was identifiedto be1induced under sulfate starvation (Jones-Rhoadesand Bartel, 2004. Furthermore, quantitative experimental analysis proved that miR393was strongly induced under cold stress (Sunkarand Zhu, 2004. In Populus, moreover, some miR-NAs can be induced by mechanical stress and may function in critical defense systems for structural and mechanical fitness(Luet al., 2005.Many targets genes of miRNAs encode tran-scription factors as well (Bartel,2004, each of which further regulates a set of downstream genes. Thus the activation of miRNA genes un-der abiotic stresses will lead to the repression of many downstream protein-coding genes and affect physiological responses. Among various environmental factors, light plays a particularly important role. Sunlight is not only the energy source for plant photosynthesis, but also regulates several plant developmental processes and some physiological processes, such as photosynthesis, seasonal and diurnal time sensing (Baroliet al., 2004; Chattopadhyay et al., 1998; Chory et al., 1996; Jiao et al., 2005. Similar to adverse en-vironmental factors, such as drought and salinity, light can also have stress effect on plants. It inter-acts with endogenous developmental programs, hence affect plant growth and development. In order to acclimate under such conditions, spe-cificphotoreceptor systems have been developed and evolved to monitor changes of light com-position (Dunaevaand Adamska, 2001; Harvaux and Kloppstech, 2001; Shao et al., 2006. With complex photoreceptors, plant can register UV-B radiation and transduce the information to nu-cleus, hence affect gene expression (Chattopad-hyay et al., 1998; Jiao et al., 2005; Kimura et al., 2003b. Changes in gene expression in response to UV-B radiation include reduction in expression and synthesis of key photosynthetic proteins as well as perturbation of expressions of the genes involved in defense mechanisms (Chattopadhyayet al., 1998; Jiao et al., 2005; Kimura et al., 2003b.Regulation of gene expression plays an impor-tant role in a variety of biological processes, such as development and responses to environmental stimuli. In plants, transcriptional regulation is2mediated by a large number of transcription fac-tors (TFscontrolling the expression of tens or hundreds of target genes in various, sometimes intertwined, signal transduction cascades (Ven-ter and Botha, 2004; Wellmer and Riechmann, 2005. Transcription factor binding sites (TF-BSs are the functional short DNA sequences (cis -elements thatdetermine the timing and lo-cation of transcriptional activities. Many com-putational methods have been developed to re-veal relationships between gene expression pat-terns and TFBSs in the proximal upstream regu-latory regions of the genes of interest. In yeast, motifs with known functions have been related to transcriptional pathways by statistical analysis of the occurrence of known motifs in the promoters of coregulated genes (Bussemakeret al., 2001. However, the presence of individual motifs is only marginally indicative of a gene’sexpression pat-tern. Extended strategies pursue to optimally pre-dict gene expression patterns with promoter cis -elements and their combinations. With a system-atic strategy, the expression of a large proportion of genes in S. cerevisiae was accurately predicted based on promoter sequences (Beerand Tavazoie, 2004. Although distal regulatory elements other than those in proximal upstream promoter regions can modulate gene expression, a recent study em-phasized that the sequences in the 5’-upstreamregions of genes were of primary importance in Arabidopsis gene regulation (Leeet al., 2006. Specifically,promoter sequences were sufficientto recapture the mRNA expression levels for 80%of the TFs studied. This study confirmedthe im-portant role of promoter regions in Arabidopsis gene expression.In light of this transcriptome-based perspec-tive and by taking advantage of the vast available data of genome-scale microarray expression pro-fileof protein-coding genes, we develop an inno-vative computational approach to explore the ex-pression activity of miRNA genes under certain conditions. We focus on identifying and anno-tating miRNAs in A. thaliana which are respon-sive to UV-B radiation, and further consider the regulatory pathways that are probably affected by the putative UV-B inducible miRNA genes. Ourapproach is based on the following two observa-tions. First, plant miRNAs generally direct en-donucleolytic cleavage of target mRNAs (Llaveet al., 2002; Schwab et al., 2005, hence enable rapid clearance of target mRNAs when they are expressed (Axtelland Bartel, 2005; Bartel, 2004. Under a particular condition, if an miRNA is up-regulated, its targetsare most likely to be co-herently down-regulated. Second, miRNA genes are transcribed by RNA polymerase II (Houbaviyet al., 2005; Lee et al., 2004; Xie et al., 2005; Zhou et al., 2007. Hence the 5’proximal pro-moters of miRNA genes are the most important regulatory regions, and significantcis -elements in these regions are important in determining the spatial and temporal expression patterns of the miRNA genes. Therefore, miRNA and protein-coding genes carrying the same or similar cis -elements in their promoters are very likely to be co-regulated under the same condition and conse-quently very likely to be co-expressed.Although we focus on Arabidopsis UV-B re-sponding miRNA genes in this study, our ap-proach can be directly applied to plant miRNA genes functioning under other abiotic or biotic stress conditions.Table 1:Putative UV-B responsive miRNAs. (a:standard deviations of p valuesgene idmiR159/319miR160miR165/166miR167miR169miR170/171miR172miR393miR39 8miR401#targets 12352735632cosine similarity0.270.630.460.640.660.610.660.280.840.73p value 7.11E-031.42E-021.17E-026.10E-023.60E-051.69E-024.25E-044.33E-022.44E-042.96E-02stdv 4.30E-051.94E-041.11E-043.47E-047.00E-061.61E-041.70E-052.67E-046.00E-052.71E-0422.1Results and discussionsUV-B responsive miRNAsOne of the bases of our method for findingstress-responsive miRNA genes is that protein-coding genes targeted by the same miRNA are likely to have coherently down-regulated expression pat-terns. We consider an miRNA to be putatively stress-inducible if the expressions of its target genes are coherently repressed and the coherence is statistically significantabove a threshold. In this study, we only considered bona fidetarget genes reported in the literature. For each miRNA, pairwise cosine similarities of the expressions of its target genes were computed. We measured the coherence of the expressions of its target genes by the average pairwise similarity. The statisticalsignificanceof the coherence was assessed with a3p value from a Monte Carlo simulation. Briefly,for each miRNA with n target genes, we firstcal-culated the average pairwise cosine similarity of the expressions of the target genes. We then ran-domly sampled n genes from the whole set of genes that wereprofiled,and calculated their av-erage pairwise cosine similarity. We repeated the sampling a large number of times, for instance one million times in our study, and took as an em-pirical p value the frequency of observing a sim-ilarity value larger than that of the target genes. For each miRNA, we repeated this simulation 100times, and calculated the average p value and the standard deviation.Table 1shows putative UV-B responsive miR-NAs. For each of these miRNAs, its target genes are coherently down-regulated, and the coherence of their expression patterns is statistically signifi-cant. Except miR167whose p value is less than 0.07, all candidates have p values smaller than 0.05.For miR158, miR162, miR163, miR168, miR395, miR402, miR403, miR404,miR405and miR406, we only found one bona fidetarget gene in the microarray data set, and could not test their coherence, thus excluded them from our study.2.2UV-B responsive miRNA genesWe applied our computational approach, dis-cussed in Section 3.2, to the microarray gene expression data under UV-B radiation treat-ment from the Arabidopsis AtGenExpressproject (/info/expression/ATGenExpress.jsp. We predicted21miRNA genes in 11miRNA families to be up-regulated under UV-B radiation. Table 2lists these UV-B inducible miRNA genes. A pu-tative UV-B responsive miRNA gene must satisfy two criteria:First, the set of protein-coding genes with the same array of motifs in their proximal promoter regions is enriched with UV-B up-regulated genes. Second, its inferred expression (discussedbelow should be anti-correlated with the expressions of its target genes.For each miRNA gene, we analyzed whether the combina tion of significantmotifs in its pro-moter was statistically relevant to the UV-B stress. First, we examined all protein-coding genes in the whole set of gene profiledin the microarray experiments, and found those genes that contain the same or very similar motifs in their proxi-mal promoter regions. We then tested whether these protein-coding genes were enriched with up-regulated genes (seeSections 3.2and 3.5. We further imposed on miRNA genes a crite-rion of anti-correlation between the inferred ex-pression of an miRNA gene and the expressions of its mRNA targets, in order to filterout pos-sible false predictions. Since we did not rely on any direct information of miRNA expression, we used the inferred expression of an miRNA gene and the expressions of its targets to com-pute their anti-correlation (seeSection 3.4. In our study, we chose the fivebest protein-coding genes whose 5’proximal promoters contain ar-rays of cis -elements that most resemble that ofthe corresponding miRNA genes. These fivegenes are most likely to be co-regulated with the corre-sponding miRNA gene, thus their expression pat-terns are most likely to be similar to the expres-sion pattern of the miRNA gene. In the rest of our discussion, we refer to the average expression pattern of the top fiveco-regulated protein-coding genes of an miRNA as its inferred expression pat-tern or expression pattern for short.Before we inferred expression patterns of miRNA genes, we applied the inferring proce-dure to 100randomly selected protein-coding genes with known expression patterns, and then4Table 2:Putative UV-B responsive miRNA genes. (a:p values for assessing the enrichment of UVB up-regulated genes in the set of coding genes that contain the same arrays of motifs as the miRNA genes. (bp values for as sessing the significanceof the cosine similaries. (c:Standard deviations of the p values (b .gene id miR156e miR156f miR156h miR157c miR159a miR159b miR160cmiR165a miR166c miR166f miR167d miR169d miR169j miR170miR171a miR172c miR172e miR393a miR398a miR401miR395c miR395ep value 2.48E-059.39E-021.67E-068.93E-028.51E-029.94E-043.48E-042.11E-114.55E-022.50E-072.52E-061.79E-025.36E-101.27E-024.66E-021.16E-028.21E-051.08E-054.89E-021.02E-128.09E-028.63E-03cosine similarity-0.42-0.42-0.41-0.32-0.41-0.48-0.53-0.52-0.47-0.47-0.72-0.41-0.41-0.69-0.75-0.75-0.77-0.60-0.78-0.71p value 5.81E-022.69E-025.76E-028.32E-023.09E-021.25E-027.14E-025.24E-027.50E-029.45E-027.81E-029.10E-029.37E-022.82E-028.32E-031.15E-032.60E-044.52E-025.94E-028.51E-02stdv 1.76E-041.05E-042.73E-042.76E-041.95E-041.11E-043.51E-042.10E-041.60E-043.64E-041.77E-043.21E-041.93E-041.83E-049.10E-054.20E-051.80E-051.66E-042.13E-041.97E-04assessed the similarities between their inferred and actual expression patterns. For all these 100genes, the cosine similarity values of their in-ferred and actual expression patterns are between 0.3and 0.89, and the average of these values is 0.51. Figure 1shows the inferred expression pat-tern and the actual expression pattern of protein coding gene, At1g19770. Th is figuregives a pic-torial view of the similarity of the inferred and original expression patterns. The cosine simi-larity value of these two expression patterns ofAt1g19770is 0.76.For each putative UV-B responsive miRNA gene, we calculated the average cosine similar-ity between its inferred expression and the ex-pressions of its targets. We assessed the statisti-cal significanceof the similarity with a p value. Similar to the analysis of expression coherence of target genes in Section 2.1, the p value was alsoExpression pattern of AT1G19770 and its top 5 correlated genesFigure 1:The expressions of At1g19770in root and shoot, respectively, the expressions of thefiveprotein-coding genes that are most correlated to it, and their mean expression. obtained by a Monte Carlo simulation. We took as an empirical p value the frequency to observe a cosine similarity value smaller than that in the real data. For each miRNA, the simulation was also repeated 100times to obtain an average p value and a standard deviation.Forty miRNA genes satisfy the firstcriterion. However, as shown in Table 2, inferred expres-sions of 21miRNA genes are anti-correlated to the expressions of their target genes (cosinesim-ilarity less than 0, and the anti-correlations re-flectedby the average cosine similarities of in-ferred expressions and expressions of target genes are statistically significant.These 21genes are our predicted UV-B responsive miRNA genes. In all putative UV-B responsive miRNA fami-lies shown in Table 1, at least one member gene from each family was predicted to be up-regulated under UV-B radiation. However,none of the members in other miRNA families was predicted to be UV-B responsive. Three miRNA genes, miR168a , miR395c , and miR395e , might also be5UV-B responsive. The arrays of motifs in their proximal promoter regions are statistically signif-icantly relevant to UV-B stress, shown by small p values obtained from an accumulative hyper-geometric test. Protein-coding genes sharing the same array of motifs with them have enriched GO (GeneOntology terms that are related to stress response (seediscussion in Section 2.3. How-ever, since these miRNAs have fewer than two experimental validated target genes, the coher-ence of their target gene expressions and the anti-correlations between their expressions and ex-pressions of their targets could not be analyzed. Hence these genes will not be discussed further.Table 3:Stress-related GO terms are enriched in the annotations of protein-coding genes that contain all the motifs present in corresponding putative UV-B responsive miRNA genes.2.2E-52.3E-52.4E-53.1E-53.9E-51.4E-56.8E-56.5E-104.2E-87.8E-71.2E-61.3E-67.9E-62.5E-55.0E-51.6E-47.0E-64.8E-57.3E-59.4E-53.2E-83.8E-89.5E-71.2E-61.4E-63.3E-64.2E-63.4E-53.4E-58.2E-55.0E-51.0E-41.5E-41.7E-55.5E-51.3E-51.6E-46.8E-51.1E-181.1E-186.9E-131.1E-121.3E-124.8E-124.9E-111.3E-051.5E-41.5E-41.8E-4binding/ligandtranscription factor activity transcription regulator activityhydrolase activity, acting on glycosyl bonds/N-glycosylase/glycosylasecatalytic activity/enzymeactivityhydrolase activity, hydrolyzing O-glycosyl compounds/O-glucosylhydrolase copper, zinc superoxide dismutase activity/zincsuperoxide oxidoreductase oxidoreductaseactivity/redoxactivity indole derivative metabolism response to wounding response to stressresponse to external stimulus oxidoreductase activity, acting on diphenols and related substances as donors, oxygen as acceptor oxidoreductase activity, acting on diphenols and related substances as donors oxidoreductase activity, acting on diphenols and related substances as donors, oxygen as acceptor oxidoreductase activity, acting on diphenols and related substances as donors transcription regulator activityflavonoid3’-monooxygenaseactivity/flavonoid3’-hydroxylaseDNA bindingresponse to stimulus response to pest, pathogene or parasite response to external biotic stimulus response to biotic stimulus anthranilate synthase activity response to stressresponse to external stimulus oxo-acid-lyase activityethylene biosynthesis/ethenebiosynthesis ethylene metabolism/ethenemetabolism amino acid derivative biosynthesistranscription factor activity transcription regulator activityhydrolase activity, acting on glycosyl bonds/N-glycosylase/glycosylaseoxidoreductase activity, acting on diphenols and related substances as donors, oxygen as acceptor oxidoreductase activity, acting on diphenols and related substances as donors transcription factor activity DNA bindingcarotene metabolismresponse to stress response to stimulusresponse to abiotic stimulus response to biotic stimulus response to woundingcatalytic activity/enzymeactivity defense response/defenceresponsecarotene metabolismtetraterpenoid metabolism carotenoid metabolism fucosyltransferase activity2.3Functions of protein-coding genes that share the same ar-rays of motifs as putative UV-B responsive miRNA genesdue to lack of reported target genes. However, protein-coding genes containing the same arrays of motifs as these three miRNA genes, especially miR168a and miR395c , are enriched with stress-related GO terms.For each putative UV-B responsive miRNA gene shown in Table 2, there are some protein-coding genes containing in their proximal promoter re-gions the same array of motifs. These protein-coding genes are very likely to share the same regulatory program, hence co-express with the miRNA gene. In order to further interpret their relevance to UV-B stress, hence to confirmthe relevance of the miRNA gene to UV-B stress, we calculated the enrichment of GO functional terms in the annotations of these protein-coding genes. As shown in Table 3, for 13out of 21putative UV-B responsive miRNA genes, we identifiedsignif-icantly enriched stress-related GO terms, by us-ing the webserver for gene annotation analysis, FuncAssociate(/cgi/func/funcassociate. In the table, the p values represent the statistical significanceof the GO terms.These enriched GO terms can be grouped into three major categories. Thefirstcategory is re-lated to transcription regulation. Protein-coding genes sharing the same regulatory regions as four miRNA genes, miR156b , miR165a , miR169j and miR172c , fall into this category. The second cate-gory is related to direct response to stress or exter-nal stimuli. Protein-coding genes corresponding to miR156h and miR166f are in thiscategory. The last category includes hydrolase activity and ox-idoreductase activity. Protein-coding genes that are likely regulated by the same regulatory pro-grams as the rest seven miRNA genes are in this category. It has been well studied that hydro-lase and oxidoreductase are involved in response to many stresses, including light stress (Apeland Hirt, 2004; Kimura et al., 2001, 2003a. The anal-ysis of GO term enrichment provides additional evidence that these 13miRNA genes are very likely to be involved in the responses to the UVB stress. Three miRNA genes, miR168a , miR395c and miR395e , were excluded from our prediction2.4Presence of known light-relevantcis -elements in the promoters of miRNA genesUsing the WordSpy genome-wide motif-findingalgorithm (Wanget al., 2005; Wang and Zhang, 2006, we identifiedmany significantcis -elements that are characteristic of UV-B respon-siveness of the 21miRNA genes listed in Ta-ble 2. Some of them are well characterized in plant motif databases such as PLACE (Higoet al., 1999 and discussed in the litera-ture (Terzaghiand Cashmore, 1995; Narusaka et al., 2004; Yamaguchi-Shinozaki and Shinozaki, 2005; Zhang et al., 2004. The cis -elements, which have been experimentally characterized in light-regulated genes, include the G-box (CACGTG,the GT-1site (GGTTAA,I-boxes (GATAAGA,TGA-box (TGACGT,GATA-box (GATATTT,H-box (CCTACCand CCAAT (CCAAT(Terzaghiand Cashmore, 1995. As shown in Table 4, these motifs appear in the pro-moters of some of miRNA genes that are up-regulated by UVB stimuli.The presence of the well studied light-related motifs shed light on the possible mechanisms ac-tivating the miRNA genes. For these 21miR-NAs, the most prevalent cis -elements are GT-1site, I-box core and CCAAT-box. These miRNA genes all have GT-1site in their promoters, all ex-cept one contain I-box core, and 17of them con-tain CCAAT-box. The involvement of GT-1site, I-box and CCAAT-box in abiotic stressregula-tion has been well studied (Arguello-Astorgaand Herrera-Estrella, 1998; Shinozaki et al., 2003; Teakle and Kay, 1995; Yamaguchi-Shinozaki and Shinozaki, 2005; Zhang et al., 2004, therefore, it is not surprising to findthem in almost all of these UV-B induced miRNA genes. Among the 21miRNA genes, 7contain GATA-box in their pro-Table 5:Known stress-related motifs shared byUVB up-regulated miRNA gene, miR167d , and its co-regulated genes. GATA-BOX:GATATTT, GT-1site:GGTTAA, I-box:GATAA, TGA-box:TGACG, CCAAT-box:CCAATAT1G20823AT4G16630AT2G27830AT3G03270AT3G12510GATA-box GATA-box GATA-box GATA-boxGT-1I-box I-box I-box I-boxTGA-box TGA-boxCCAAT CCAAT CCAAT CCAAT CCAATGT-1TGA-boxTable 4:Known light-related motifs in the upstream moters, which has been shownto regulate light-regions of miRNA genes that are predicted to be responsive genes (Arguello-Astorgaand Herrera-up-regulated by UV-B. 1:GT-1site, 2:I-box core Estrella, 1998; Teakle and Kay, 1995. (GATAA,3:CCAAT-box (CCAAT,4:TGA-boxThese motifs were previously analyzed on(TGACG,5:TGACGT, 6:GATA-box (GATATTT,protein-coding genes. Their presence suggests7:I-box (GATAAGA,8:G-box (CACGTG,9:H-that these miRNA genes are regulated similarly box (CCTACC.as light-responsive protein-coding genes. To be specific,we list in Table 5the known motifs that miR167d shares with its possible co-regulated miR156e X X X Xprotein-coding genes. These shared motifs pro-miR156f X X X X miR156h X X vide additional evidence that these miRNA genes miR157c X X X Xand the corresponding protein-coding genes are miR159a X X XmiR159b X X X X X Xregulated by similar mechanisms under UV-B miR160c X X Xstimuli. miR165a X X X X X XmiR166cmiR166f miR167d miR169d miR169j miR170miR171a miR172c miR172emiR393a miR398a miR401X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X XX X XXXXXX X XX2.5Expression-repression pathwaysthat UV-B responsive miRNAs may be involved inLight can trigger the transcription of a set of miRNA genes, which direct their target protein-coding mRNAs to be degraded quickly. Remark-ably, 8of the 11putative light-inducible miRNAs (exceptmiR393, miR398and miR401 have tar-gets that encode transcription factors. These tar-geted transcription factors can subsequently af-fect the expressions of their downstream genes. Hence, as in developmental stages, under light stress conditions there are down-regulation path-ways that are initiated by miRNAs, which cascade to the targets of these miRNAs, the targets of the targets of miRNAs, and so on. Table 6shows the targets of the putative UV-B responsive miRNAs. A striking observation is that auxin signalingTable 6:Target genes of UV-B responsive miRNAs.At1g53160(SPL4,At1g69170(SPL4At2g33810(SPL3,At5g43270(SPL2At3g15270( SPL5At2g35320, At2g45990, At3g28690At4g12080, At4g28660, At4g36860At5g18590, At5g08620, At5g38610At3g06450, At3g61740, At3g15030At4g37770, At5g09410,At4g26930At5g17580, At5g56790, At5g55020At2g26950, At5g67090,At2g32460At1g30210, At1g53230, At2g26960At2g31070At1g77850(ARF17At4g32880(ATHB-8,At5g60690(REVAt1g30490At1g02800, At3g61310At3g20910, At5g12840At1g70700, At1g80770At2g45160, At3g60630At5g60120(TOE2,At5g67180(TOE3At2g39250At2g42380At5g65790(MYB68At3g14770At5g36870At3g44690, At5g12900, At5g19560At3g62980(TIR1,At4g03190At3g23690, At3g26830binding protein-like transcription factorHAP2-like factorsAPETALA2-like factor APETALA2-like factor bZIP family (G-boxMYBnodulin MtN3family protein glycosyl transferaseF-box protein bHLHCCACGTGGFigure 2:Auxin signaling pathways that putative UV-B responsive miRNAs may be involved in. pathways can be affected by several light-induced miRNA genes. Auxin (principallyindole-3-acetic acid is an important hormone in plants. It affects many aspects of plant growth and development by influencingAUXIN RESPONSE FACTORS (ARF,a plant-spec ificfamily of DNA binding proteins. ARFs regulate the expression of auxin-inducible genes, such as GH3and auxin/indole-3-acetic acid (Aux/IAAby binding auxin re-sponse elements (AREs.Figure 2illustrates pos-sible auxin signaling pathways that four miRNAs, miR160, miR165/166, miR167and miR393may be involved in. As shown, these miRNAs will af-fect auxin signaling pathways by regulating dif-ferent transcription factors under UV-B stimuli.miR420which were not reported to be bona fidemiRNAs (Xieet al., 2005. Thre e pairs of polycistronic miRNA genes, miR169i and miR169j , miR169k and miR169l , and miR169m and miR169n are referred to as miR169j , miR169l , and miR169n , respectively, in this paper. Differ-ent from many animal miRNA genes, all these A.。
Plant tolerance to drought and salinity stress regulating
REVIEWPlant tolerance to drought and salinity:stress regulating transcription factors and their functional significance in the cellular transcriptional networkDortje Golldack •Ines Lu¨king •Oksoon Yang Received:18February 2011/Revised:25March 2011/Accepted:25March 2011/Published online:8April 2011ÓSpringer-Verlag 2011Abstract Understanding the responses of plants to the major environmental stressors drought and salt is an important topic for the biotechnological application of functional mechanisms of stress adaptation.Here,we review recent discoveries on regulatory systems that link sensing and signaling of these environmental cues focusing on the integrative function of transcription activators.Key components that control and modulate stress adaptive pathways include transcription factors (TFs)ranging from bZIP,AP2/ERF,and MYB proteins to general TFs.Recent studies indicate that molecular dynamics as specific homodimerizations and heterodimerizations as well as modular flexibility and posttranslational modifications determine the functional specificity of TFs in environ-mental adaptation.Function of central regulators as NAC,WRKY,and zinc finger proteins may be modulated by mechanisms as small RNA (miRNA)-mediated posttran-scriptional silencing and reactive oxygen species signaling.In addition to the key function of hub factors of stress tolerance within hierarchical regulatory networks,epige-netic processes as DNA methylation and posttranslational modifications of histones highly influence the efficiency of stress-induced gene prehensive elucidation of dynamic coordination of drought and salt responsive TFs in interacting pathways and their specific integration in the cellular network of stress adaptation will provide newopportunities for the engineering of plant tolerance to these environmental stressors.Keywords Drought ÁEpigenetics ÁTranscription factor ÁRNAi ÁSalt tolerance ÁArabidopsisIntroductionA major challenge for current agricultural biotechnology is to satisfy an ever increasing demand in food production facing a constantly increasing world population that will reach more than 9billion in 2050(Godfray et al.2010;Tester and Langridge 2010).This growing demand for food is paralleled by dramatic losses of arable land due to increasing severity of soil destruction by abiotic environ-mental conditions.Thus,drought and salinity are the two major environmental factors that adversely affect plant growth and development and have a crucial impact on agricultural productivity and yields.Drought due to short-age of water is critical for crop production in large agro-nomic areas worldwide and it is usually coped with extensive irrigations.Although earth is rich in water,most water resources are highly salinized whereas high quality fresh water that is suitable for irrigation is extremely lim-ited.Accordingly,not only drought but also soil salinity becomes increasingly an agricultural problem due to extensive spreading of agricultural practices as irrigation (Flowers 2004)and it urgently requires the breeding of crops with increased water use efficiency and salt tolerance.Exposure of plants to excess salt causes ion imbalance and ion toxicity-induced imbalances in metabolism.Another component of salinity is hyperosmotic stress that results in water deficit in a comparable way to drought-induced water deficit.Plants basically counteract theCommunicated by R.Reski.D.Golldack (&)ÁI.Lu¨king ÁO.Yang Department of Biochemistry and Physiology of Plants,Faculty of Biology,Bielefeld University,33615Bielefeld,Germanye-mail:dortje.golldack@uni-bielefeld.dePlant Cell Rep (2011)30:1383–1391DOI 10.1007/s00299-011-1068-0negative effects of salinity and drought by activation of biochemical responses that include(1)the synthesis and accumulation of osmolytes,(2)maintaining the intracel-lular ion homeostasis,and(3)scavenging of reactive oxygen species(ROS)generated as a secondary effect of drought(Flowers2004;Ashraf and Akram2009).Plant engineering strategies for cellular and metabolic reprogramming to increase the efficiency of plant adaptive processes may either focus on(1)conferring stress toler-ance by directly re-programming ion transport processes and primary metabolism or(2)by modulating signaling and regulatory pathways of the adaptive mechanisms.The second approach seems to be more perspective because it is likely that signaling and regulatory factors orchestrate as key signaling components the transcriptional and transla-tional control of group(1)adaptive mechanisms(Die´dhiou et al.2008;Popova et al.2008).Accordingly,molecular re-programming to enhance stress tolerance of plants would probably require the genetic engineering of a single or a few master regulators of adaptation instead of modulating numerous metabolic and cellular adaptive mechanisms.However,although several plant stress signaling com-ponents have been dissected in detail the knowledge on integration of regulatory mechanisms in stress signaling cascades and on key regulators is still limited,although knowledge on the regulating key factors of stress adap-tation is highly necessary for biotechnological engineering of stress tolerance.In this review,we focus on recent advances in transcription factor(TF)-based engineering of increased drought and salt adaptation.Putative integra-tions and links of TFs in stress adaptive signaling net-works coordinating the endogenous programs of environmental adaptation will be highlighted.Accord-ingly,for this review TFs out of the wider range of all stress inducible TFs were selected so that we do not comprehensively cover all stress-related factors.Major criterion for this selection was a putative potential of the TFs in controlling sub-regulons of stress-adaptive cellular mechanisms within the hierarchical transcriptional net-work that will be discussed in the review.Arabidopsis and related model species:learningfrom species with different natural stress tolerance Traditional breeding attempts for sustainable agricultural use of dry and salinized soils have been clearly facilitated and stimulated by the wealth of knowledge of genomics and transcriptomics data available from the model species Arabidopsis(Arabidopsis thaliana)and rice(Oryza sativa). Linear general frameworks of plant drought and salt adaptation have been established that were mainly based on systematic and comprehensive mutant analyses.Thus,it is now accepted that changes in membrane integrity and modulation of lipid synthesis are key factors in the primary sensing of drought and salt(Kader and Lindberg2010).Secondary,osmotic stress-induced sig-naling involves changes in plasma membrane H?-ATPase and Ca2?-ATPase activities that trigger concerted changes of Ca2?influx,cytoplasmic pH,and apoplastic production of ROS(Beffagna et al.2005).In addition,osmotic stress-induced Ca2?fluxes are linked to abscisic acid(ABA),and calcium-responsive protein kinases act as key regulators in drought and salinity-induced signaling cascades(Die´dhiou et al.2008).As convergent down-stream elements of transcriptional activation,many genes that are responsive to drought and to salinity belong to the ABA-responsive element(ABRE)and dehydration-responsive element/C-repeat element(DRE/CRT)regulons(Yamaguchi-Shino-zaki and Shinozaki2005).Despite this knowledge derived from the model plants Arabidopsis and rice,the applicability of these data for biotechnological engineering of increased drought and salt tolerance is clearly prehensive comparisons of the salt inducible transcriptomes of the salt-sensitive spe-cies Arabidopsis and rice and,for example,transcriptional data of the closely related salt-tolerant model species Lobularia maritima(Brassicaceae)and Festuca rubra ssp. litoralis(Poaceae)show extensive differences in salt responsive expressional regulations(Popova et al.2008; Die´dhiou et al.2009b;Fig.1).In contrast to salt excluding and avoiding halophyte models as Thellungiella halophila with a very limited number of salt responsive transcripts, the salt-accumulating and-detoxifying halophytes L. maritima and F.rubra ssp.litoralis allowed identification of a wide range of transcripts with different salt responsive regulation in the salt-sensitive and salt-tolerant species (Volkov et al.2003;Taji et al.2004;Popova et al.2008; Die´dhiou et al.2009b).In addition,transgenic modulation of regulatory and signaling elements in Arabidopsis and rice according to the pattern in the halophytes L.maritima and F.rubra ssp.litoralis successfully activated stress adaptation in the sensitive model species(Die´dhiou et al. 2008;Yang et al.2009).Accordingly,understanding of stress-induced signaling complexity in stress-sensitive model species has to be complemented by comparisons with naturally tolerant species for a systematic identifica-tion of key regulators of stress tolerance with the potential of biotechnological application.bZIP TFs and their role in conferring stress tolerance to plantsResearch on salt and drought regulatory TFs has mainly focused on single factors and linear pathways.Emergingfindings increasingly suggest,however,integration of the TFs in dynamic network hubs as well as interaction and competition of pathways manifesting complexity of molecular links in stress adaptation.The emerging view of the salt-and drought-signaling network unequivocally supports a key and integrative function of members of the bZIP TFs in these regulatory networks(Fig.2)and the potential of these factors to confer enhanced stress tolerance has been demonstrated repeatedly.A key regulator of salt stress adaptation,the group F bZIP TF bZIP24,was identified by differential screening of salt-inducible transcripts in A.thaliana and a halophytic Arabidopsis-relative model species(Yang et al. 2009).Expressional regulation of bZIP24was different with induced transcription in the salt-sensitive and tran-scriptional repression in the halotolerant species,and RNAi-mediated repression of the factor conferred increased salt tolerance to Arabidopsis.The improved tolerance was mediated by stimulated transcription of a wide range of stress-inducible genes that are e.g.involved in cytoplasmic ion homeostasis,osmotic adjustment,as well as in plant growth and development demonstrating a central function of bZIP24in salt tolerance by regulatingmultiple mechanisms that are essential for stress adaptation (Yang et al.2009).Next to bZIP24and its function in salt adaptation,group A bZIP factors AREB1,AREB2,and ABF3have a key regulatory role in ABA signaling under drought stress.Thus,A.thaliana areb1areb2abf3triple knock out mutants had increased tolerance to ABA and reduced drought tolerance(Yoshida et al.2010).In addi-tion,in other species as rice and tomato transgenic modi-fication of group A bZIP TFs modified the tolerance of plants to water deficit and to salt stress(Amir Hossain et al. 2009;Hsieh et al.2010)strongly suggesting trans-species potential of these factors for increasing stress tolerance.From animal systems dynamic coordinations of numer-ous bZIP controlled signal transduction pathways by molecular re-organization and by posttranslational mech-anisms are well-known(Jindra et al.2004;Miller2009). Thus,specific homodimerizations and heterodimerizations within the class of bZIP TFs as well as modularflexibility of the interacting proteins and posttranslational modifica-tions might determine the functional specificity of bZIP factors in cellular transcription networks(Miller2009). Excitingly,evidences for involvement of homologous mechanisms in signaling hubs in plant systems are just now 20304050At Lm Os Fremerging.As an example,the three factors AREB1,AREB2,and ABF3can form homodimers and heterodi-mers as well as interact with a SnRK2protein kinase suggesting ABA-dependent phosphorylation of the proteins (Yoshida et al.2010).As another example for the function of bZIP factors in salt adaptation in A.thaliana ,salt stress induced proteolytic processing and translocation of the group B factor AtbZIP17to the nucleus followed by transcriptional up-regulation of salt-responsive transcripts (Liu et al.2007).The group F factor AtbZIP24shows salt-inducible subcellular re-targeting to the nucleus and for-mation of homodimers suggesting that molecular dynamics of bZIP factors could mediate new signaling connections within the complex cellular signaling network (Yang et al.2009).In contrast to the homodimerization of bZIP24,specific heterodimerization was shown for the salt-responsive group S AtbZIP1with group C bZIP TFs (Weltmeier et al.2009).In conclusion,it might be hypothesized that specific homodimerizations and hetero-dimerizations as well as posttranslational modifications (e.g.phosphorylations)might determine the functional specificity of bZIP factors in the cellular transcription networks of drought and salt adaptation.Interestingly,transgenic over-expression of rice SnRK2-type SAPK4in rice regulated ion and ROS homeostasis under salt stresssupporting the hypothesis of key functions of SnRK kina-ses in the intracellular signaling cascades of osmotic adaptation thus further supporting key modulatory function of posttranslational phosphorylations in diverse plant sys-tems that might,e.g.target bZIP factors (Die´dhiou et al.2008;Fig.2).Recently,it was recognized that general TFs might also have an important role in stress-responsive transcription.Thus,the TBP-associated factor AtTAF10has a specific and key function in plant salt and osmotic stress adaptation by regulating accumulation of Na ?and proline (Gao et al.2006).This functional overlap to bZIP24(Yang et al.2009)strongly suggests linked regulation and cofunctions of bZIP proteins and TAFs within the complex drought and salt signaling network—a hypothesis that awaits further clarification (Fig.2).The role of WRKY TFs and Cys2/His2zinc finger proteins in the regulation of adaptation to osmotic stressOur understanding of plant stress-inducible signaling has been greatly facilitated by research on TFs that regulate and control subsets of stress-responsive geneexpression.Fig.2Model of signaling pathways and regulatorytranscription factors involved in plant adaptation to drought and saltThus,WRKY proteins regulate diverse plant processes ranging from development to various biotic and abiotic stresses as well as hormone-mediated pathways(Rama-moorthy et al.2008).Involvement of WRKY factors in plant salt adaptation were shown for WRKY25and WRKY33that increased salt tolerance and ABA sensitivity independent of the SOS-pathway when over-expressed in A.thaliana(Jiang and Deyholos2009).In A.thaliana, wrky63knock out mutants showed decreased sensitivity to ABA and drought(Ren et al.2010).In these plants,the stomatal closure and the expression of the AREB1/ABF2 TF were affected indicating involvement of WRKY fac-tors in the ABA-dependent pathway of drought and salt adaptation(Ren et al.2010).Potential of WRKY-type TFs to confer increased salt tolerance by transgenic expression is further supported by the different salt-induced regula-tion of a WRKY protein in salt-sensitive rice and a hal-ophytic rice-relative model species(Die´dhiou et al.2009a, b).Interestingly,A.thaliana WRKY25and WRKY33are not only responsive to osmotic stresses but they are also regulated by oxidative stress(Miller et al.2008).In addition,down-stream regulated target genes of WRKY33 include transcripts with function in ROS detoxification as peroxidases and glutathione-S-transferases(Jiang and Deyholos2009)suggesting function of WRKY factors as key regulators in both osmotic and oxidative stress adaptation.Alternatively,it is tempting to hypothesize involvement of WRKY factors in the osmotic stress sig-naling via control of the intracellular stress-induced ROS levels(Fig.2).Interestingly,Zat proteins(TFIIIA-type Cys2/His2zinc finger proteins)have been suggested to control and regulate WRKY functions(Miller et al.2008).Thus,in soybean overexpression of GmWRKY54conferred increased salt and drought tolerance and regulation of the GmWRKY54 by Zat10/STZ was hypothesized(Zhou et al.2008).In addition,in rice stomatal closure is regulated by the Cys2/ His2zincfinger protein DST(drought and salt tolerance) via ABA-independent targeting of genes that are involved in ROS homeostasis(Huang et al.2009).Thesefindings further support involvement of zincfinger proteins and probably WRKY TFs in osmotic adaptation via ROS sig-naling(Fig.2).Interestingly,although both drought and salt stress might result in intracellular accumulation of toxic amounts of ROS,hydrogen peroxide(H2O2)and nitric oxide(NO)also function as signaling molecules in ABA-mediated stomatal responses(Miller et al.2010; Wilkinson and Davies2010).Mutation of a cellulose synthase-like protein induced accumulation of ROS, changed sensitivity to salt stress and to water deficit,and regulation of plant osmotic stress tolerance via control of intracellular stress-induced ROS levels has been suggested (Zhu et al.2010a).Stress adaptation and multi-transcriptional regulation: AP2/ERF,MYB,and bHLH TFsNext to TFs with possible upstream position in the hier-archical network of stress adaptation as the bZIP factors described above,integrative stress-adaptive functional roles of regulatory proteins from other diverse groups have been reported.These factors might be either integrated in the main pathways of environmental adaptation,likely under control of the key regulatory TFs,or they might have functions in regulating sub-networks of adaptation to drought and salt stress and in linking these stress adapta-tions to other stresses,developmental and hormonal responses.Thus,dual roles in both biotic and abiotic stress responses have been demonstrated for AP2/ERF proteins as soybean GmERF3and the ABA-responsive RAP2.6from A.thaliana(Zhang et al.2009;Zhu et al.2010b).Over-expression of Arabidopsis light and drought responsive RAP2.4led to defects in multiple developmental processes regulated by light and ethylene as well as drought tolerance (Lin et al.2008).Complementary to these observations, overexpression of AP2/ERF GmERF3in tobacco induced the expression of PR genes and of osmotin accompanied by enhanced accumulation of free proline and soluble carbo-hydrates(Zhang et al.2009).Members of the DREB/CBF subfamily of the AP2/ERF TFs have been recognized for a decade for their roles in stress tolerance via ABA-depen-dent and-independent pathways and for their regulation of a stress-response sub-transcriptome with more than hun-dred target genes inclusive regulatory factors as ZAT12 and RAP2.1(Shinozaki and Yamaguchi-Shinozaki2000). However,constitutive overexpression of the DREB/CBF pathway led to serious developmental defects of transgenic plants although accompanied by increased tolerance to drought,salt,and cold(Kasuga et al.1999).These data clearly demonstrate complexity of the stress adaptive net-work that requires major control points of the multiple transcriptional sub-regulons as well as cooperative and integrative function of the different stress sub-clusters to prevent impairing side effects.Nevertheless,members of the AP2/ERF TF family are integrated as a hub in signaling interconnections of complex biotic and abiotic environ-mental cues.Supporting the undeniable key function of AP2/ERF in terms of drought and salt tolerance the picture of integrative function of these factors in plant develop-mental processes as well as biotic and/or abiotic stress signaling in an interconnecting and linking way is,how-ever,only emerging.As another example for multi-functional regulations,the R2R3-MYB TF AtMYB41is transcriptionally induced in response to ABA,drought,salinity,and cold(Lippold et al. 2009).In addition,the factor influences cell expansion and cuticle deposition suggesting a linking function in abioticstress response and cell wall modifications(Cominelli et al. 2008).Interaction and competition of complex signaling pathways infine-tuning cellular responses is further illustrated by the A.thaliana basic-helix-loop helix TF bHLH92.The factor regulates only the expression of a subset of salt-and drought-responsive genes(Jiang et al. 2009).However,different peroxidases are down-stream targets of the factor and bHLH92might have a function in the control of ROS-mediated signaling thus linking salt and drought adaptation to ROS signaling(Fig.2). Here,more detailed work will be necessary to elucidate the precise integration of the diverse TFs in the cellular network of stress adaptation and to understand their potential in genetic engineering of improved stress tol-erance,probably via targeted engineering of defined subsets of stress adaptive mechanisms or sub-pathways of signaling to customize specific features of stress adaptation.NAC-triggered gene expression and miRNANAC type proteins are not only involved in diverse pro-cesses as developmental programs,defense,and biotic stress responses(Olsen et al.2005)but they also have a key function in abiotic stress tolerance inclusive drought and salinity.Thus,in rice ONAC5and ONAC6are transcrip-tionally induced by ABA,drought,and salt stress(Rabbani et al.2003;Takasaki et al.2010).ONAC5and ONAC6 transcriptionally activate stress-inducible genes as OsLEA3 by direct binding to the promoter and they interact in vitro suggesting functional dimerization of these TFs(Takasaki et al.2010).Interestingly,overexpression of the Arabid-opsis factors ANAC019,ANAC055,and ANAC072caused increased drought tolerance of transgenic plants but they only changed transcription of a limited number of non-particularly salt-and drought-responsive genes(Tran et al. 2004).These important results strongly suggest interaction or co-regulation of NAC factors with other regulatory pathways or subsets of stress-inducible molecular mecha-nisms for achieving the significant increased stress toler-ance that was observed(Tran et al.2004).Improved drought and salt tolerance could also be achieved by transgenic overexpression of diverse NAC factors in spe-cies ranging from A.thaliana and rice to chickpea,wheat, and tomato(Peng et al.2009;Yokotani et al.2009;Xia et al.2010;Yang et al.2011).Interestingly,in tomato two NAC TFs were salt-inducible in a salt-tolerant cultivar but showed different expression in salt-sensitive tomato plants (Yang et al.2011).These data indicate that differences in plant salt tolerance might be due to different and specific transcriptional activation of NAC-dependent regulatory pathways.As important examples for conferring increased stress tolerance underfield conditions,in rice transgenic over-expression of SNAC1enhanced salt and drought tolerance and OsNAC10improved drought tolerance and grain yield (Hu et al.2006;Jeong et al.2010).OsNAC10-regulated target genes mainly included protein kinases and TFs of AP2,WRKY,LRR,NAC,and Zn-finger types as well as the stress-responsive genes cytochrome P450and the potassium transporter HAK5(Jeong et al.2010).These results support the view that NAC type TFs might be part of the general frameworks of drought and salt adaptation by connecting or regulating subsets of linear adaptive pathways but the NAC factors themselves are likely to be controlled by global regulatory factors of the network of stress adaptive transcription and metabolism.Thus, important evidence for cooperative regulation of stress responses by members of different TF families was pro-vided by the study of Tran et al.(2007)that showed interaction and co-function of the drought,salt,and ABA inducible zincfinger protein ZFHD1and a NAC factor.As it was recognized recently,members of the CCAAT-HAP TF family also have a potential key function in conferring stress tolerance to crops.Transgenic maize plants with increased expression of the CCAAT-HAP-type factor ZmNF-YB2showed improved drought tolerance underfield conditions(Nelson et al.2007).This effect was achieved by mechanisms independent of ABA and DREB/ CBF pathways supporting the hypothesis of concerted action of different TF families within subsets of regulatory modules in the cellular stress-response network.Interestingly,members of the NAC TF family are potential regulatory targets of the small RNA(miRNA) posttranscriptional silencing machinery(Rhoades et al. 2002;Guo et al.2005).As an example,recently a NAC domain containing TF was identified as a target of miR164 in switchgrass(Matts et al.2010).Thus,regulation of NAC TFs by miRNA-mediated cleavage of mRNAs together with data showing differential regulation of NAC factors in response to drought and salt stress indicate that these TFs might participate in the regulation of environmental adaptation through miRNA pathways.Next to NAC pro-teins,TFs e.g.of SCL,MYB,and TCP types were iden-tified as targets of drought and salt inducible miRNAs as miR159,miR168,miR171,and miR396(Liu et al.2008). Accordingly,it might be hypothesized that the cellular networks of drought-and salt-stress tolerance are regulated by miRNA-mediated targeting of convergent and divergent adaptive pathways under control of different stress-specific TFs.Accordingly,relevance of modification of drought and salt stress-specific signaling pathways via the miRNA machinery in a biotechnological context might be a pow-erful approach for genetic engineering of improved toler-ance but remains to be discovered.Epigenetics:what is next in terms of biotechnological application?Next to transcriptional regulations of abiotic stress responses,epigenetic processes are becoming a new and current chapter in plant environmental adaptation.Effi-ciency of gene expression is highly influenced by chro-matin structure that might be modulated epigenetically by processes as DNA methylation and posttranslational mod-ifications of histones.The histone-mediated structure of nucleosomes in the chromatin might be posttranslationally modified at the N-terminal tails of the core histone com-plexes(H2A,H2B,H3,H4)and thus influence nucleosome density,binding efficiency of TFs,and transcriptional activity(Chinnusamy and Zhu2009;Kim et al.2010).In addition to methylations of histones,also acetylations and phosphorylations as well as other posttranslational modi-fications of histones as ubiquitination,biotinylation,and sumoylation might have a modulating impact on the reg-ulation of stress-specific gene expression(Chinnusamy et al.2008).Meanwhile,it is accepted knowledge that phenotypes within one species may transmit different epigenetic information based on covalent modifications of DNA or histones(Fazzari and Greally2004).Thus,plant popula-tions from stress exposed habitats may carry inherited memories of stress adaptation and transfer this epigeneti-cally to next generations.As an example,the desert shrub Zygophyllum dumosum was posttranslationally methylated at histone H3under wet but less under dry growth condi-tions indicating posttranslational regulation of gene expression activity(Granot et al.2009).As it was also reported recently,natural populations of mangroves were DNA hypomethylated when grown under saline conditions in contrast to populations from non-saline sites(Lira-Medeiros et al.2010).Based on these results,it seems obvious to think on simulation of inherited memories of stress adaptation in biotechnological applications to confer increased drought and salt tolerance to naturally sensitive species.However,in contrast to the detailed knowledge on influences of epigenetic mechanisms on developmental processes,information on epigenetic regulation of abiotic stress resistance is still rare.As a few examples,salinity-induced phosphorylation of histone H3and acetylation of histone H4in A.thaliana and tobacco have been reported(Sokol et al.2007).In addition, altered acetylation as well as trimethylation of histone H3 under drought stress in drought-responsive genes of A. thaliana have been observed(Kim et al.2008).In rice, expression of cytosine DNA methyltransferases was mod-ified by salt stress indicating functional importance of epigenetic modulation of genome activity also in monocot species(Sharma et al.2009).Detailed knowledge on the specific mechanisms that underlay epigenetic regulation under environmental expo-sure is,however,only slowly emerging.Thus,trans-gen-erational modifications of stress adaptations as salt stress include altered genomic DNA methylation as well as function of Dicer-like proteins suggesting involvement of small RNA pathways in epigenetic regulations(Boyko et al.2010).Interestingly,in barley expression of Poly-comb proteins with function in histone methylation was influenced by abscisic acid(ABA)suggesting involvement of ABA-mediated pathways in epigenetic modifications (Kapazoglou et al.2010).Thus,according to the current knowledge,an applica-tion of epigenetic processes to improve the stress-regulat-ing function of TFs will be a challenging and novel biotechnological approach for the engineering of plant tolerance to drought and salinity,however,many detailed information are still missing.Particularly,despite the importance of elucidating epigenetic mechanisms in model plants,it will be obligatory to extend investigations to systematic and comprehensive comparisons of stress rele-vant epigenetics in sensitive-and naturally tolerant species. Linking epigenetic processes to the key regulatory com-ponents of the general stress adaptive frameworks will be essential to further support the feasibility of epigenetics in the customized engineering of stress adaptation. Conclusion and perspectivesCellular effects of environmental stresses as drought and salinity are not only imbalances of ionic and osmotic homeostasis but also impaired photosynthesis,cellular energy depletion,and redox imbalances.Regulatory sys-tems inclusive TFs that link sensing and signaling of the environmental conditions and the cellular adaptive responses are emerging but are not well understood yet.As a next step,it will be important to identify master regula-tors and master pathways of stress adaptation in naturally stress-tolerant species as well as integration of the diverse regulatory factors in the network of intracellular stress adaptation pathways(Fig.2).Within this hierarchical net-work,cellular stress responses might befine tuned by interaction and competition of TFs that regulate sub-clus-ters of the stress transcriptome.Here,systematic and comprehensive data on the timing of all stress responsive TFs upon stress will be indispensable for detailed hierar-chical linking of all regulatory factors.In addition,more detailed understanding of shared and competing transcrip-tional regulation as well as modulated intramolecular interactions of different factors and epigenetic processes will be essential for targeted and efficient genetic engi-neering of improved drought and salt tolerance in plants.。
A case for regulatory B cells.
J Immunol. 2006 Jan 15;176(2):705-10.A case for regulatoryB cells. Mizoguchi A, Bhan AK.SourceImmunopathology Unit, Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.AbstractB cells are typically characterized by their ability to produce Abs, including autoantibodies.B细胞能产生抗体，包括自身抗体，为其显著特征。
However, B cells possess additional immune functions, including the production of cytokines and the ability to function as a secondary APC.然而，B细胞表现另一免疫功能，包括细胞因子的功能，以及作为次级抗原提呈细胞的功能。
As with T cells, the B cell population contains functionally distinct subsets capable of performing both pathogenic and regulatory functions.与T细胞一样，B细胞家族包含某些从功能上有区别的因子，这些因子表现出致病及调节功能。
Recent studies indicate that regulatory B cells develop in several murine models of chronic inflammation, including inflammatory bowel disease, rheumatoid arthritis, and experimental autoimmune encephalomyelitis.最近的研究发现调节性B细胞在一些慢性炎症的大鼠模型中形成，包括肠炎症疾病，风湿性关节炎，以及实验性自身免疫性脑脊髓炎。
The 505(b)(2)
The 505(b)(2) Drug Development Pathway:When and How toTake Advantage of aUnique AmericanRegulatory Pathway By Mukesh Kumar, PhD, RAC andHemant Jethwani, MSThe 505(b)(2) regulation offers a less expensive and faster new drug development pathway that may be particularly attractive to a manufacturer with experience in developing generic products. It involves making significant changes to an existing product approved by the US Food and Drug Administration (FDA), called the reference product, to create a new drug with its own indica-tion, formulation, target population and/or other differences that need to be supported with clini-cal studies. A major advantage of this pathway is that it allows a sponsor to rely, at least in part, on FDA’s findings of safety and/or effectiveness for the previously approved drug, thereby reducing the number of clinical trials required for approval. Another incentive is three to five years of market exclusivity for 505(b)(2) products, depending upon the extent of changes to the reference prod-uct and the type of clinical data included in the approved New Drug Application (NDA).However, like all drug development strat-egies, the 505(b)(2) pathway requires careful consideration and planning. Important issues to consider include intellectual property concerns, the amount and quality of supporting informa-tion available from reference products or the literature, the logistics of conducting clinical trials with generic-like products, market compe-tition for approved products and requirements for an international product launch. Here we discuss practical strategies for drug development via the 505(b)(2) regulatory pathway.Drugs Can be Approved via One of Three Regulatory PathwaysNew drug products can belong to one of two broad categories: brand new drugs and identical or close copies of previously approved drugs, also called generics. Globally, separate regulatory pathways for innovator products and generic drugs are well established. US regulations, how-ever, divide these drugs into three categories: (1) new drugs, covered under Section 505(b)(1) of the Food, Drug, and Cosmetics Act (FD&C Act); (2) generic drugs, covered under Section 505(j) of the FD&C Act; and (3) “similar” drugs, covered under Section 505 (b)(2). It is the third category that is discussed here.The generic and 505(b)(2) categories were added by the Drug Price Competition and Patent Term Restoration Act of 1984, usually referred to as the Hatch-Waxman Act. The Hatch-Waxman Act aimed to promote generics while leaving intact a financial incentive for new product research and development. It was an attempt to balance the need for innovation with the desire for lower-cost alternatives within a reasonable length of time. Drug companies were given the opportu-nity to create not only exact copies of previously approved drugs, provided there was no infringe-ment of patents, but also improved versions of previously approved drugs by updating formu-lations or finding new uses. Table 1 describes the three pathways under the FD&C Act.Despite existing for more than 25 years, along with generic drugs, the 505(b)(2) prod-ucts have only recently become popular with drug companies due to increased challenges to discover and develop new chemical entities. As with innovator drugs, products following the 505(b)(2) pathway are subject to the full userfee under the Prescription Drug User Fee Act (PDUFA). They also may require several clinical and nonclinical studies that could involve signifi-cant resources, albeit less than for an innovator product but much higher than for a generic drug. Some key parameters for the three product cat-egories are listed in Table 2.The 505(b)(2) Pathway is Unique to the USThe 505(b)(2) application is intended to encour-age sponsors to develop improved generics, i.e., drugs similar to an approved product with some significant changes that are not permitted under Abbreviated New Drug Application (ANDA) rules. The 505(b)(2) pathway replaced the “Paper NDA” pathway used prior to the Hatch-WaxmanTable 1. Regulatory Pathways for New Drug ProductsAct, whereby FDA could approve NDAs that relied on published studies and lacked any reference to innovator safety and effectiveness data. Paper NDAs were frequently challengedby innovator product manufacturers, citing lack of sufficient safety and efficacy data. Under the 505(b)(2) regulation, FDA has the authority to approve new products based on fewer new stud-ies to demonstrate their safety and efficacy and relying extensively on the agency’s previous findings of safety and efficacy for the reference product. The sponsor of a 505(b)(2) product is not required to obtain a right of reference from the innovator product manufacturer. However, the sponsor needs to include data from bridging stud-ies to support changes from the reference drug.As mentioned, the 505(b)(2) application applies when certain changes are made to the innovator drug to either create a new formu-lation or include new uses/indications. The following are examples of changes to approved drugs that would fall under the 505(b)(2) mechanism:changes in dosage form, strength, for-•mulation, dosing regimen or route ofadministrationnew combination product, including •substitution of an active ingredientmodified active ingredient (e.g., salt, •chelate, ester, complex, etc.)new indications for previously•approved drugsover-the-counter switch of an approved •prescription drugBecause 505(b)(2) products are considered new products, they are subject to the PDUFA user fee requirement. Review by FDA is similar in duration to that of traditional NDAs, and the approved product is eligible for a minimum of three years of market protection from generics if the bridging studies were other than bioavailabil-ity (BA) and bioequivalence (BE) studies. This regulatory process is unique to the US. Products approved under the 505(b)(2) pathway typically are approved either as generics or new products in other countries. The 505(b)(2) Pathway Offers Many Advantages to Manufacturers and PatientsThere are advantages for all stakeholders from developing 505(b)(2) products. This pathway eliminates duplication of experiments and encourages developers to conduct new stud-ies that add value to the final product, suchas a better understanding of mechanisms of action, improved formulation and utilization of the same product for multiple diseases. Also, development of such products creates new intel-lectual property while protecting the rights of the original product, and providing a fair incentive for the investment. Since 505(b)(2) products are derived from reference products for which exten-sive safety and efficacy information is available, they generally carry less risk, cost less and can achieve FDA approval in a much shorter time. Some 505(b)(2) products have been created with less than $30 million in additional investment (in terms of new clinical and nonclinical studies conducted) and in about three years, which is remarkable compared to the cost and timeline for a traditional new drug.Perhaps the biggest incentive to develop 505(b)(2) products is the three to five years of market exclusivity in the US, depending upon the extent of changes to the previously approved drug and the amount of data submitted to FDA. This is an apparent advantage when compared to ANDA approval, where exclusivity can be held for only 180 days and applies only to the first generic product. Table 3 lists the different terms of market exclusivity available by product cat-egory and target indication.Market exclusivity enables manufacturersto take advantage of greater pricing flexibil-ity. During the market exclusivity period they can promote their product over the innovator drug and build their own brand with an attrac-tive price without fear of price erosion due to generic competition. Most importantly, 505(b)(2) products may receive an “AB” substitutability rating in the Orange Book. Thus, from a thera-peutic substitution perspective and under state formulary laws, the 505(b)(2) product is not at a disadvantage relative to a generic drug.Table 2. Comparison Between Conventional NDA, ANDA and 505(b)(2) Drug SubmissionsChallenges for Developing 505(b)(2) Drug ProductThere are some unique challenges facing 505(b) (2) applications. They often require substantial additional innovative work to bring the prod-uct to market. Since similars involve significant changes to the reference product formulation, either by including additional components or making changes to the active pharmaceuti-cal ingredient, the impact of these changeson safety and efficacy must be evaluated via clinical and/or nonclinical studies. Such stud-ies could uncover new issues, leading to further investigations and associated costs. Also, unlike generic drugs, such products involve extensive interactions with FDA to proactively understand regulatory, scientific and technical requirements. Such products are considered new and unique by FDA; hence, the review process is analogous to that for traditional NDAs.Also, since portions of the 505(b)(2) appli-cation could utilize self-generated proprietary data, this information needs to be protected via a patent or trade secret agreement, as applicable. Still, significant portions of the information could be in the public domain in existing patents for the innovator product. Unlike the traditional NDA, wherein the sponsor owns all the data necessary for approval (or has obtained the right to reference), the filing or approval of a 505(b) (2) application may be delayed due to reference drug patent or exclusivity protection. Sponsors filing 505(b)(2) applications must include patent certifications in their applications and must also provide notice of certain patent certifications to reference drug NDA and patent holders.Determining what additional information may be required for approval is a critical strategic requirement. Information requirements usually are subject to case-by-case determination by FDA. FDA guidance documents and discussions with regulatory professionals experienced in the 505(b) (2) approval route, as well as with the relevant FDA review division, are critical in understand-ing what data are necessary and adequate. The biggest risk: if the required studies are only BA/ BE studies, the product will receive a 505(b)(2) designation and be subject to associated user fees (which are about $1.4 million (US) in 2010) without being eligible for any market exclusivity and thus subject to generic competition from the beginning of market approval.There are few additional challenges associated with the use of the 505(b)(2) pathway. These products face fierce competition from generics with similar biological properties and since they are more expensive than generics, a robust marketing campaign may be required to attract customer attention. On the other hand, 505(b)(2) products offer certain advantages over innovator and/or generic drugs, enabling the manufacturer to promote these benefits directly to patients. These could be marketing advantages such as a formulation that is easier for patients to take, extended dosage, different strength, etc.Strategies for Developing 505(b)(2) ProductsFor small drug companies, the 505(b)(2) pathway for a new product could prove an attractive busi-ness model for the simple reason that it takes much less time, cost and risk to get the product onto the market compared to innovator drugs, and could yield significantly higher returns on investment compared to generic drugs.A good strategy could mean the difference between a successful, i.e., profitable, and unsuc-cessful product. The following are key strategic considerations for a 505(b)(2) product:extent of innovation/modification made •to the innovator product:these modifi-cations decide whether the product isapplicable for a 505(b)(2) review or not,and help determine the number of yearsof market exclusivity grantedthorough analysis of available data:•before embarking on manufacturing a505(b)(2) product, a company shouldthoroughly analyze the data available,including the scientific basis of approvalof the reference drug, published litera-ture, particularly since the innovatordrug was approved, market competi-tion, etc. (The amount of available datapreviously submitted to FDA deter-mines whether this is a viable project.)development strategy:• careful analysis of data should lead to a list of the addi-tional studies that may be required for agiven 505(b)(2) product; bridging stud-ies are required to show that changesto the innovator product lead to thedesired impact on safety, efficacy andtolerance of the proposed drug productTable 3. Market Exclusivity Available to FDA-Approved ProductsFDA discussions: there is no substitute •for robust discussions with the relevantFDA review division regarding theproposed and executed developmentstrategy; FDA offers significant adviceregarding final requirements for anapproval, and it has been statisticallydemonstrated that companies thatinvolve FDA in discussions early intheir product development plans andimplement the agency’s suggestionsincrease their chances for first cycleapproval almost three-fold, leading toenormous time and cost savings and,hence, higher returns on investmentimplementation of strategy: exhaustive •implementation planning is the path tosuccess; timelines should be diligentlyobserved and any deviations aggres-sively addressedcost control:• cost incurred depends upon the preclinical and clinical stud-ies required, amount of informationavailable regarding the reference drug,advancements in analytical technologyand various other such factors; bridgingstudies should be scientifically justifiedand strategically executed to control costmarketing and branding strategy:• as 505(b)(2) products are generally moreexpensive than generic versions ofthe innovator drug, the manufacturershould have a robust marketing plan ConclusionOver the years, the 505(b)(2) regulatory pathway has become very attractive to companies of all sizes. It is the proverbial “low-hanging fruit” for manufacturers due to the short time of marketing with attractive returns on investment. Every year FDA approves about twice as many 505(b)(2) applications as traditional 505(b)(1) applications. It is projected that due to increased challenges in creating new products, 505(b)(2) products might comprise more than 70% of all FDA approvals within 10 years. This pathway is particularly attractive to manufacturers transitioning from generic drugs to innovator products. Due to the similarities to traditional drug development, these products offer a low-risk market entry point by training the work force in the traditional development processes. However, there are unique scientific, regulatory, logistical and finan-cial challenges to developing such products––all of which could convert a potentially attractive project into a constant headache.AuthorsMukesh Kumar, PhD, RAC is a senior director, Regulatory Affairs, at Amarex Clinical Research, LLC, located in Germantown, MD, which is a full-service CRO offering regula-tory consultancy, strategic planning, trial management, data management and statistical analysis services for global clinical trials. Kumar is a member of the RAPS Board of Editors for Regulatory Focus and can be reached at mukeshk@amarexcro. com. Hemant Jethwani, MS is a regulatory affairs associate at Amarex Clinical Research LLC. He can be reached athemantj@.Do you want to be a part of an organization whose mission it is to protectthe health of the public by ensuring the safety and effectiveness ofmedical devices? Do you have a BA/BS degree in the sciences, and areyou knowledgeable in the fields of Engineering, Interdisciplinary Scienceand/or Nursing? Then, the Office of Compliance, Center for Devicesand Radiological Health, FDA wants you!The Office of Compliance mission is to promote and protect the healthof the public by ensuring the safety and effectiveness of medical devices.We enforce regulations and laws to which regulated industry is subject,without hindering innovation or access to medical devices. We are anorganization that hires scientists interested in protecting the public health,performing regulatory activities and working in a state of thescientific environment.To apply, email/send resume to:Collin Figueroa, Program Management Officer, OC,CDRH10903 New Hampshire Ave., Bldg. 66 Room 3438Silver Spring, Maryland 20993-0002e-mail: collin.figueroa@。
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Other Relevant ODE Programs
• Humanitarian Device Exemptions (HDEs)
Essentially same as PMA in both form and content, but exempt from the effectiveness requirement Approved HDE authorizes marketing of the humanitarian use device (HUD) Available if < 8,000 patients / year have the condition to be treated
Section 3: Regulatory Pathways
1
Device Classification
2
FDA Device Classification: Risk-Based Approach
• Class 1:
Common, low-risk devices General controls Most exempt from pre-market submission
4
FDA Regulatory Pathways
5
Three Steps to Obtaining Marketing Clearance from FDA
Step 1: • Make sure the product that you wish to market is in fact a medical device
9
Pre-market Approval (PMA) Application
• Establish safety and effectiveness
Usual process: bench – animal – clinical (human) testing Investigational device exemption (IDE) needed for human testing Feasibility studies: answer design-related questions not previously answered; preliminary safety data Safety studies (phase II): finalize design and protocol of study; investigate safety in limited number of patients; preliminary effectiveness data; info needed for pivotal study Pivotal Safety and Effectiveness Study: controlled trial, if possible masked, method of use consistent with proposed labeling, statistical validity.
• Class 2:
More complex, higher risk Special controls Pre-market notification [510(k)]
• Class 3:
Most complex, highest risk (Devices which support or sustain human life; devices which pose potential unreasonable risk of illness or injury) Comprehensive data needed Pre-market application [PMA]
6
Pre-market Notification vs. Pre-market Approval
Pre-market Notification 510(k) Requires: Demonstration of Substantial Equivalence to Predicate Device(s)* “As safe and effective as the predicate device(s)” Classes: I, II, some III
Step 2: • • Determine the class of your device (I, II, or III) This classification will identify the marketing process: 510(k) or PMA
Step 3: • • Develop data and/or information that is necessary to submit application For some 510(k)s and most PMAs, clinical performance data is required; In these cases, a trial must be conducted in accordance with FDA’s investigational device exemption (IDE) regulation
11
Clinical Trials and Study Design
• Questions of appropriate endpoints:
Physiological endpoints (e.g., plasma levels, blood pressure measurements) Clinical events (e.g., hospitalizations, symptoms, functional capacity) Mortality Surrogate endpoints use when true endpoint is rare, delayed, confounded, etc. valid if surrogate endpoint is correlated with true clinical endpoint
* Device that is not subject to PMA; predicate devices: legally marketed before May 28, 1976 (preamendments device), or device that has been reclassified from class III to class II or I, or device that has been found SE to one of these devices through the 510(k) process.
510(k)s
Original HDE HDE Supplements
4,458
4,202
4,248
4,320
4,247
3,635
Source: FDA ODE Annual Report FY 2004
Sources: Prentice 1989; FDA, 2003
12
Device and Approval Information on FDA’s Website
e.g., Recent Device Approvals: /cdrh/consumer/mda/
• Product Development Protocols (PDPs)
Second designated pathway for class III devices (introduced 1976) Based upon early consultation between the sponsor and the FDA leading to device development and testing plan acceptable to both parties Seldomly used
• Questions of study design:
Type of control used; inclusion/exclusion criteria, monitoring, statistical methodology, analysis of potential biases and of covariates
13
FDA – Key Performance Indices
14
Types and Numbers of Applications Submitted to FDA
TYPE OF SUBMISSION TO CDRH
FY1999
FY2000
FY2001
FY2002
FY2003
FY2004
Original PMAs PMA Supplements Original IDEs IDE Amendments IDE Supplements
8
Investigational Device Exemption (IDE)
• IDE according to 21 CFR 812: Allows investigational devices to be distributed for purposes of conducting a clinical study Clinical studies conducted to collect safety and effectiveness data in support of a premarket notification (510(k)) or premarket approval (PMA) • Basis for IDE: Proof of reasonable safety and effectiveness profiles in bench and animal testing