The use of strictly standardized mean difference for hit selection in primary RNA interference high-throughput screening experiments

RNA interference (RNAi) high-throughput screening (HTS) has been hailed as the 2nd genomics wave following the 1st genomics wave of gene expression microarrays and single-nucleotide polymorphism discovery platforms. Following an RNAi HTS, the authors are interested in identifying short interfering RNA (siRNA) hits with large inhibition/activation effects. For hit selection, the z-score method and its variants are commonly used in primary RNAi HTS experiments. Recently, strictly standardized mean difference (SSMD) has been proposed to measure the siRNA effect represented by the magnitude of difference between an siRNA and a negative reference group. The links between SSMD and d+-probability offer a clear interpretation of siRNA effects from a probability perspective. Hence, SSMD can be used as a ranking metric for hit selection. In this article, the authors investigated both the SSMD-based testing process and the use of SSMD as a ranking metric for hit selection in 2 primary siRNA HTS experiments. The analysis results showed that, as a ranking metric, SSMD was more stable and reliable than percentage inhibition and led to more robust hit selection results. Using the SSMD -based testing method, the false-negative rate can more readily be obtained. More important, the use of the SSMD-based method can result in a reduction in both the false-negative and false-positive rates. The applications presented in this article demonstrate that the SSMD method addresses scientific questions and fills scientific needs better than both percentage inhibition and the commonly used z-score method for hit selection.     

A pair of new statistical parameters for quality control in RNA interference high-throughput screening assays

RNA interference (RNAi) high-throughput screening (HTS) enables massive parallel gene silencing and is increasingly being used to reveal novel connections between genes and disease-relevant phenotypes. The application of genome-scale RNAi relies on the development of high-quality RNAi HTS assays. To obtain high-quality HTS assays, there is a strong need for an easily interpretable and theoretically based quality control (QC) metric. Signal-to-noise ratio (S/N), signal-to-background ratio (S/B), and Z-factor have been adopted as QC metrics in HTS assays. In this paper, I proposed a pair of new parameters, strictly standardized mean difference (SSMD) and coefficient of variability in difference (CVD), as QC metrics in RNAi HTS assays. Compared to S/B and S/N, SSMD and CVD capture the variabilities in both compared populations. Compared to Z-factor, SSMD and CVD have a clear probability interpretation and a solid statistical basis. Accordingly, the cutoff criteria of using SSMD or CVD as a QC metric in HTS assays are fully theoretically based. In addition, I discuss the relationship between the SSMD-based criterion and the popular Z-factor-based criterion and elucidate why p-value from t-test of testing mean difference fails to serve as a QC metric.     

Salmonella enteritidis Serotype 501, 2, 3: z4, z24: -

Characteristics of a new Salmonella serotype, subgenus IV, are reported. Culture 5534-68 was recovered from the intestinal tract of Anolis biporcatus, an arboreal lizard found in deep forest tracts in Panama Province, Republic of Panama. The antigenic composition of this new serotype was found to be 50(1, 2, 3):z(4), z(24):-.     

Asparagine of z8 Insert Is Critical for the Affinity,Conformation, and Acetylcholine Receptor-clustering Activity of Neural Agrin

Agrin isoforms with different bioactivities are synthesized by the nerve and the muscle. Neural agrin containing an 8-amino acid insert (z8) introduced by alternative splicing is the active form that induces synaptic differentiation at the neuromuscular junction. In addition to alternative splicing, extracellular calcium is also required for the activity of neural agrin. To understand better how the activity of agrin is regulated by alternative splicing, we have applied alanine substitution mutagenesis to the z8 insert and the calcium binding site in the minimally functional AgG3z8 fragment. Single alanine substitutions in the 4th through the 7th amino acid of the z8 splice insert significantly reduced the function of agrin, in terms of acetylcholine receptor clustering activity and the affinity for binding to the muscle surface. Mutation of the asparagine at the 4th position drastically reduces bioactivity such that it is equivalent to that of muscle form AgG3z0. These reduced activity mutants also show reduced magnitudes of the calcium-induced CD spectrum change from that observed in AgG3z8 fragments, indicating that cross-talk between calcium and the z8 insert is critical for the normal activity of agrin. However, removal of Ca²⁺ binding via mutation of both aspartic acids in the calcium binding site did not totally eliminate the activity of AgG3z8. These results suggest a model wherein the z8 insert is a Ca²⁺-responsive allosteric element that is essential in forming an active conformation in neuronal agrin.     

Novel analytic criteria and effective plate designs for quality control in genome-scale RNAi screens

One of the most fundamental challenges in genome-wide RNA interference (RNAi) screens is to glean biological significance from mounds of data, which relies on the development and adoption of appropriate analytic methods and designs for quality control (QC) and hit selection. Currently, a Z-factor-based QC criterion is widely used to evaluate data quality. However, this criterion cannot take into account the fact that different positive controls may have different effect sizes and leads to inconsistent QC results in experiments with 2 or more positive controls with different effect sizes. In this study, based on a recently proposed parameter, strictly standardized mean difference (SSMD), novel QC criteria are constructed for evaluating data quality in genome-wide RNAi screens. Two good features of these novel criteria are: (1) SSMD has both clear original and probability meanings for evaluating the differentiation between positive and negative controls and hence the SSMD-based QC criteria have a solid probabilistic and statistical basis, and (2) these QC criteria obtain consistent QC results for multiple positive controls with different effect sizes. In addition, I propose multiple plate designs and the guidelines for using them in genome-wide RNAi screens. Finally, I provide strategies for using the SSMD-based QC criteria and effective plate design together to improve data quality. The novel SSMD-based QC criteria, effective plate designs, and related guidelines and strategies may greatly help to obtain high quality of data in genome-wide RNAi screens.     

Su(z)12, a novel Drosophila Polycomb group gene that is conserved in vertebrates and plants

In both Drosophila and vertebrates, spatially restricted expression of HOX genes is controlled by the Polycomb group (PcG) repressors. Here we characterize a novel Drosophila PcG gene, Suppressor of zeste 12 (Su(z)12). Su(z)12 mutants exhibit very strong homeotic transformations and Su(z)12 function is required throughout development to maintain the repressed state of HOX genes. Unlike most other PcG mutations, Su(z)12 mutations are strong suppressors of position-effect variegation (PEV), suggesting that Su(z)12 also functions in heterochromatin-mediated repression. Furthermore, Su(z)12 function is required for germ cell development. The Su(z)12 protein is highly conserved in vertebrates and is related to the Arabidopsis proteins EMF2, FIS2 and VRN2. Notably, EMF2 is a repressor of floral homeotic genes. These results suggest that at least some of the regulatory machinery that controls homeotic gene expression is conserved between animals and plants.     

A role for G(z) in pancreatic islet beta-cell biology

Glucose-stimulated insulin secretion and beta-cell growth are important facets of pancreatic islet beta-cell biology. As a result, factors that modulate these processes are of great interest for the potential treatment of Type 2 diabetes. Here, we present evidence that the heterotrimeric G protein G(z) and its effectors, including some previously thought to be confined in expression to neuronal cells, are present in pancreatic beta-cells, the largest cellular constituent of the islets of Langerhans. Furthermore, signaling pathways upon which G alpha(z) impacts are intact in beta-cells, and G alpha(z) activation inhibits both cAMP production and glucose-stimulated insulin secretion in the Ins-1(832/13) beta-cell-derived line. Inhibition of glucose-stimulated insulin secretion by prostaglandin E (PGE1) is pertussis-toxin insensitive, indicating that other G alpha(i) family members are not involved in this process in this beta-cell line. Indeed, overexpression of a selective deactivator of G alpha(z), the RGS domain of RGSZ1, blocks the inhibitory effect of PGE1 on glucose-stimulated insulin secretion. Finally, the inhibition of glucose-stimulated insulin secretion by PGE1 is substantially blunted by small interfering RNA-mediated knockdown of G alpha(z) expression. Taken together, these data strongly imply that the endogenous E prostanoid receptor in the Ins-1(832/13) beta-cell line couples to G(z) predominantly and perhaps even exclusively. These data provide the first evidence for G(z) signaling in pancreatic beta-cells, and identify an endogenous receptor-mediated signaling process in beta-cells that is dependent on G alpha(z) function.     

A new Salmonella serovar S.III b 58:z10:z53:Rz50

A new Salmonella serovar S.III b 58:z10:z53:Rz50 was isolated from the water samples of Ashida river, Fukuyama city, Japan. Its antigenic structure is described.     

Building the central complex of the grasshopper Schistocerca gregaria: temporal topology organizes the neuroarchitecture of the w,x, y,z tracts

The central complex is a brain specific structure involved in multimodal information processing and in coordinating motor behaviour. It possesses a highly organized neuroarchitecture, which is remarkably conserved across insect species. A prominent feature of this neuroarchitecture is the stereotypic projection of axons from clusters of neurons in the pars intercerebralis to the central body via the so-called w, x, y and z tracts. Despite extensive analyses of this neuroarchitecture in adults, little is known about its ontogeny in any insect. In this paper we use the expression pattern of the segment polarity gene engrailed to identify those neuroblasts belonging to the protocerebrum of the early embryonic brain of the grasshopper Schistocerca gregaria. We present a new map for this brain region in which the 95 protocerebral neuroblasts in each hemisphere are organized into seven rows, as they are in the neuromeres of the ventral nerve cord. We then identify a subset of four of these neuroblasts as being the progenitor cells for four clusters of neurons, some of whose axons we show project via discrete tracts (w, x, y, z) into the central complex. These tracts begin to form prior to 39% of embryogenesis. We show further, that the cells from one of these clusters (the Z cluster) are organized according to age, and direct axons topologically according to age into the appropriate z tract. This pattern is repeated in each of the other three clusters, thus establishing a clonally based modular system of fibre tracts consistent with the model proposed for this brain region in the adult.     

A new method with flexible and balanced control of false negatives and false positives for hit selection in RNA interference high-throughput screening assays

The z-score method and its variants for testing mean difference are commonly used for hit selection in high-throughput screening (HTS) assays. Strictly standardized mean difference (SSMD) offers a way to measure and classify the short interfering RNA (siRNA) effects. In this article, based on SSMD, the authors propose a new testing method for hit selection in RNA interference (RNAi) HTS assays. This SSMD-based method allows the differentiation between siRNAs with large and small effects on the assay output and maintains flexible and balanced control of both the false-negative rate, in which the siRNAs with strong effects are not selected as hits, and the restricted false-positive rate, in which the siRNAs with weak or no effects are selected as hits. This method directly addresses the size of siRNA effects represented by the strength of difference between an siRNA and a negative reference, whereas the classic z-score method and t-test of testing no mean difference address whether the mean of an siRNA is exactly the same as the mean of a negative reference. This method can readily control the false-negative rate, whereas it is nontrivial for the classic z-score method and t-test to control the false-negative rate. Therefore, theoretically, the SSMD-based method offers better control of the sizes of siRNA effects and the associated false-positive and false-negative rates than the commonly used z-score method and t-test for hit selection in HTS assays. The SSMD-based method should generally be applicable to any assay in which the end point is a difference in signal compared to a reference sample, including those for RNAi, receptor, enzyme, and cellular function.     

Regulation of Polycomb group genes Psc and Su(z)2 in Drosophila melanogaster

Certain Polycomb group (PcG) genes are themselves targets of PcG complexes. Two of these constitute the Drosophila Psc-Su(z)2 locus, a region whose chromatin is enriched for H3K27me3 and contains several putative Polycomb response elements (PREs) that bind PcG proteins. To understand how PcG mechanisms regulate this region, the repressive function of the PcG protein binding sites was analyzed using reporter gene constructs. We find that at least two of these are functional PREs that can silence a reporter gene in a PcG-dependent manner. One of these two can also display anti-silencing activity, dependent on the context. A PcG protein binding site near the Psc promoter behaves not as a silencer but as a down-regulation module that is actually stimulated by the Pc gene product but not by other PcG products. Deletion of one of the PREs increases the expression level of Psc and Su(z)2 by twofold at late embryonic stages. We present evidence suggesting that the Psc-Su(z)2 locus is flanked by insulator elements that may protect neighboring genes from inappropriate silencing. Deletion of one of these regions results in extension of the domain of H3K27me3 into a region containing other genes, whose expression becomes silenced in the early embryo.     

A score matrix to reveal the hidden links in glycans

MOTIVATION: Glycans are the third major class of biomolecules following DNA and proteins. They are extremely vital for the functioning of multicellular organisms. However, comparing the fast development of sequence analysis techniques, informatics work on glycans have a long way to go. Alignment algorithms for glycan tree structures are one of the foremost concerns. In addition, the statistical analysis of these algorithms in terms of biological significance needs to be addressed. RESULTS: We developed a tree-structure alignment algorithm for glycans and performed a statistical analysis of these alignment scores such that biologically interesting features could be captured into a score matrix for glycans. We generated our score matrix in a manner similar to BLOSUM, but with slight variations to accomodate our glycan data, including the incorporation of linkage information. We verified the effectiveness of our new glycan score matrix by illustrating how well the resulting score matrix entries correspond with biological knowledge. Future work for even better improvements with the use of a variety of score matrices for different subclasses of glycans due to their complexity is also discussed. CONTACT: mami@kuicr.kyoto-u.ac.jp SUPPLEMENTARY INFORMATION: The glycan score matrix can be downloaded from http://kanehisa.kuicr.kyoto-u.ac.jp/Paper/kcam/glycanMatrix0.1.txt.     

On the value of knowing a z* ion for what it is

Computer simulation of database searches of electron transfer dissociation (ETD) spectra using both "bottom up" and "top down" approaches was performed to evaluate the utility of knowing a priori which product ions contain the C-terminus (i.e., the z* ions). In this work, knowledge of the identities of the z* ions was used to exclude putative identifications that are based solely on the mass matching of undifferentiated product ions derived from an experiment with those derived from in silico fragmentation. The benefit from knowing which ions are z* ions was found to be heavily dependent on the quality of the ETD spectra, in terms of sequence coverage afforded by the product ions, the amount of noise in the spectra (i.e., extraneous peaks that do not directly reflect primary structure), and mass measurement accuracy. Under conditions in which the likelihood for misidentifications are high without a priori knowledge of ion types (e.g., b-, y-, c-, or z-ions), a knowledge of which product ions are z* ions allows discrimination against false-positive identifications. Relatively little benefit from knowing which ions are z* ions was noted when product spectra reflected relatively high sequence coverage and when a low fraction of the products ions were due to extraneous peaks (i.e., spectra with relatively little noise). In all cases, specificity is higher with higher mass measurement accuracy with the consequent reduction in benefit from knowledge of which ions are z* ions.     

EHEC O157:H7 z3276基因缺失株构建及其生物学特性

【目的】肠出血性大肠杆菌O157:H7是世界范围内重要的动物源性致病菌之一,可感染人。I型菌毛是多种致病性大肠杆菌(如肾盂肾炎型大肠杆菌等)可表达的一种黏附结构,与细菌吸附黏膜表面密切相关。然而,O157:H7 fim操纵子上几个核苷酸的缺失却导致其不能表达I型菌毛。BLAST比对结果表明O157:H7独有的开放阅读框z3276编码的氨基酸序列与其他大肠杆菌I型菌毛高度同源,这可能是对O157:H7不能表达I型菌毛的补偿机制,但确切功能尚不清楚。本文探究z3276基因的生物学功能。【方法】利用O157:H7 86-24参考菌株构建z3276基因缺失株(?z3276),并构建其互补株(C?z3276),进而比较亲本株、?z3276与C?z3276的生物学特性及对小鼠致病性差异。【结果】与亲本株相比,?z3276进入对数生长期的时间延后,在半固体琼脂平板上的迁移直径明显缩小,生物被膜形成能力显著减弱。?z3276对HEp-2细胞的黏附和侵袭能力并无明显变化,但对IPEC-J2细胞的侵袭能力明显减弱。在小鼠攻毒试验中,?z3276组排菌数量减少、排菌持续时间缩短。C?z3276各项特性均能回复到与亲本株一致的水平。【结论】z3276基因可能是O157:H7重要的毒力相关因子。     

GUItars: A GUI Tool for Analysis of High-Throughput RNA Interference Screening Data

Background

High-throughput RNA interference (RNAi) screening has become a widely used approach to elucidating gene functions. However, analysis and annotation of large data sets generated from these screens has been a challenge for researchers without a programming background. Over the years, numerous data analysis methods were produced for plate quality control and hit selection and implemented by a few open-access software packages. Recently, strictly standardized mean difference (SSMD) has become a widely used method for RNAi screening analysis mainly due to its better control of false negative and false positive rates and its ability to quantify RNAi effects with a statistical basis. We have developed GUItars to enable researchers without a programming background to use SSMD as both a plate quality and a hit selection metric to analyze large data sets.

Results

The software is accompanied by an intuitive graphical user interface for easy and rapid analysis workflow. SSMD analysis methods have been provided to the users along with traditionally-used z-score, normalized percent activity, and t-test methods for hit selection. GUItars is capable of analyzing large-scale data sets from screens with or without replicates. The software is designed to automatically generate and save numerous graphical outputs known to be among the most informative high-throughput data visualization tools capturing plate-wise and screen-wise performances. Graphical outputs are also written in HTML format for easy access, and a comprehensive summary of screening results is written into tab-delimited output files.

Conclusion

With GUItars, we demonstrated robust SSMD-based analysis workflow on a 3840-gene small interfering RNA (siRNA) library and identified 200 siRNAs that increased and 150 siRNAs that decreased the assay activities with moderate to stronger effects. GUItars enables rapid analysis and illustration of data from large- or small-scale RNAi screens using SSMD and other traditional analysis methods. The software is freely available at http://sourceforge.net/projects/guitars/.     

形态性状、分子性状与同源性

“同源性(homology)”是生物学中最基本的概念之一。近年来, 随着分子生物学、生物信息学、发育生物学以及进化发育遗传学等学科的快速发展, 同源性一词在形态性状的比较、核苷酸和氨基酸序列的分析以及探讨形态性状进化的分子机制等方面都有广泛应用。然而, 由于不同的研究者对同源性概念的理解有所不同, 在实际应用中难免会出现不恰当使用“同源性”一词并得出错误结论的情况。本文从不同的角度介绍了如何对同源性进行判断以及影响同源性判断的因素。并指出正确理解同源性这一概念的含义, 以及通过综合各方面的证据对同源性进行推断对于揭示基因型和表型的进化以及二者之间的关系非常重要。     

形态性状、分子性状与同源性

“同源性（homology）”是生物学中最基本的概念之一。近年来,随着分子生物学、生物信息学、发育生物学以及进化发育遗传学等学科的快速发展,同源性一词在形态性状的比较、核苷酸和氨基酸序列的分析以及探讨形态性状进化的分子机制等方面都有广泛应用。然而,由于不同的研究者对同源性概念的理解有所不同,在实际应用中难免会出现不恰当使用“同源性”一词并得出错误结论的情况。本文从不同的角度介绍了如何对同源性进行判断以及影响同源性判断的因素。并指出正确理解同源性这一概念的含义,以及通过综合各方面的证据对同源性进行推断对于揭示基因型和表型的进化以及二者之间的关系非常重要。     

Transcriptional expression of fljB:z66, a flagellin gene located on a novel linear plasmid of Salmonella enterica serovar Typhi under environmental stresses

A previous study identified that z66+ strain of Salmonella enterica serovar Typhi contains two different flagellin genes, the fliC encoding d or j antigen in chromosome and the fljB-like gene encoding z66 antigen in a novel linear plasmid, respectively. The promoter of fljB:z66 is different from that of fliC:d/j and z66+ strain alters flagellin expression in only one orientation, from z66 to d orj antigen, raising the suspicion that z66+ strain is a special biphasic strain. To clarify the expressional characteristics of flagellin genes of z66+ strain, expression patterns of fljB:z66 and fliC were investigated by RT-PCR under a series of environmental stresses during infection, such as acidic stress, osmotic stress, bile acid stress and oxidative stress. Results showed that the expression level of fljB:z66 is over 10-fold higher than the level of fliC in low and middle osmotic conditions before stresses. Only the expressional regulatory tendency of fljB:z66 in response to bile acid stress is similar to that of fliC. Differential expressional patterns between fljB:z66 and fliC of S. enterica serovar Typhi were seen under osmotic stress, bile acid stress and oxidative stress. These results support the hypothesis that the z66+ strain is a special biphasic strain of S. enterica serovar Typhi.