From: bassam al-kindy Date: Tue, 14 Jan 2014 14:58:40 +0000 (+0100) Subject: Update version of the corrections of JF & Arnuld. X-Git-Url: https://bilbo.iut-bm.univ-fcomte.fr/and/gitweb/chloroplast13.git/commitdiff_plain/45f51751b6853ef1ef0687f53f9bd8ef4aca3fda?ds=inline Update version of the corrections of JF & Arnuld. --- diff --git a/annotated.tex b/annotated.tex index b43666b..359b062 100644 --- a/annotated.tex +++ b/annotated.tex @@ -57,33 +57,15 @@ summarizes their distribution in our dataset. Annotation, which is the first stage, is an important task for extracting gene features. Indeed, to extract good gene feature, a good -annotation tool is obviously required. To obtain relevant annotated -genomes, two annotation techniques from NCBI and Dogma are used. The -extraction of gene feature, the next stage, can be anything like gene -names, gene sequences, protein sequences, and so on. Our method -considers gene names, gene counts, and gene sequence for extracting -core genes and producing chloroplast evolutionary tree. The final -stage allows to visualize genomes and/or gene evolution in -chloroplast. Therefore we use representations like tables, -phylogenetic trees, graphs, etc. to organize and show genomes -relationships, and thus achieve the goal of representing gene -evolution. In addition, comparing these representations with ones -issued from another annotation tool dedicated to large population of -chloroplast genomes give us biological perspectives to the nature of -chloroplasts evolution. Notice that a local database linked with each -pipe stage is used to store all the informations produced during the -process. +annotation tool is obviously required. The extraction of gene feature, the next stage, can be anything like gene names, gene sequences, protein sequences, and so on. Our method considers gene names, gene counts, and gene sequence for extracting core genes and producing chloroplast evolutionary tree. The final stage allows to visualize genomes and/or gene evolution in chloroplast. Therefore we use representations like tables, phylogenetic trees, graphs, etc. to organize and show genomes relationships, and thus achieve the goal of representing gene +evolution. In addition, comparing these representations with ones issued from another annotation tool dedicated to large population of chloroplast genomes give us biological perspectives to the nature of chloroplasts evolution. Notice that a local database linked with each pipe stage is used to store all the informations produced during the process. \input{population_Table} \subsection{Genome annotation techniques} -For the first stage, genome annotation, many techniques have been -developed to annotate chloroplast genomes. These techniques differ -from each others in the number and type of predicted genes (for -example: \textit{Transfer RNA (tRNA)} and \textit{Ribosomal RNA -(rRNA)} genes). Two annotation techniques from NCBI and Dogma are -considered to analyze chloroplast genomes. +To obtain relevant annotated genomes, two annotation techniques from NCBI and Dogma are used. For the first stage, genome annotation, many techniques have been developed to annotate chloroplast genomes. These techniques differ +from each others in the number and type of predicted genes (for example: \textit{Transfer RNA (tRNA)} and \textit{Ribosomal RNA (rRNA)} genes). Two annotation techniques from NCBI and Dogma are considered to analyze chloroplast genomes. \subsubsection{Genome annotation from NCBI} @@ -117,16 +99,13 @@ parameters. Protein coding genes are identified in an input genome using sequence similarity of genes in Dogma database. In addition in comparison with NCBI annotation tool, Dogma can produce both \textit{Transfer RNAs (tRNA)} and \textit{Ribosomal RNAs (rRNA)}, -verify their start and end positions. Another difference is also that -there is no gene duplication with Dogma after solving gene -fragmentation. In fact, genome annotation with Dogma can be the key -difference when extracting core genes. +verify their start and end positions. further more, there is no gene duplication with gene annotations from Dogma after applying gene de-fragmentation process. In fact, genome annotation with Dogma can be the key difference when extracting core genes. The Dogma annotation process is divided into two tasks. First, we manually annotate chloroplast genomes using Dogma web tool. The output of this step is supposed to be a collection of coding genes files for each genome, organized in GeneVision file. The second task is to solve -the gene duplication problem and therefore we have use two +the gene duplication problem and therefore we have used two methods. The first method, based on gene name, translates each genome into a set of genes without duplicates. The second method avoid gene duplication through a defragment process. In each iteration, this @@ -161,12 +140,9 @@ method can be stated as follows: how can we ensure that the gene which is predicted in core genes is the same gene in leaf genomes? The answer to this problem is that if the sequences of any gene in a genome annotated from Dogma and NCBI are similar with respect to a -given threshold, then we do not have any problem with this -method. When the sequences are not similar we have a problem, because -we cannot decide which sequence belongs to a gene in core genes. +given threshold, the method is operational when the sequences are not similar. The problem of attribution of a sequence to a gene in the core genome come to light. -The second method is based on the underlying idea: we can predict the -the best annotated genome by merging the annotated genomes from NCBI +The second method is based on the underlying idea that it is possible to predict the the best annotated genome by merging the annotated genomes from NCBI and Dogma according to a quality test on genes names and sequences. To obtain all quality genes of each genome, we consider the following hypothesis: any gene will appear in the predicted genome if and only @@ -286,7 +262,7 @@ core genes with its two genomes parents. \subsection{Features visualization} -The goal is to visualize results by building a tree of evolution. All +The goal is to visualize results by building an evolutionary tree. All core genes generated represent an important information in the tree, because they provide ancestor information of two or more genomes. Each node in the tree represents one chloroplast genome or @@ -294,8 +270,8 @@ one predicted core and labelled as \textit{(Genes count:Family name\_Scientific names\_Accession number)}. While an edge is labelled with the number of lost genes from a leaf genome or an intermediate core gene. Such numbers are very interesting because they give an information about -the evolution: how many genes were lost between two species whether -they belong to the same lineage or not. Phylogenetic relationships are mainly built by comparison of sets of coding and non-coding sequences. Phylogenies of photosynthetic plants are important to assess the origin of chloroplasts (REF) and the modalities of gene loss among lineages. These phylogenies are usually done using less than ten chloroplastic genes (REF), and some of them may not be conserved by evolution process for every taxa. As phylogenetic relationships inferred from data matrices complete for each species included and with the same evolution history are better assumptions, we selected core genomes for a new investivation of photosynthetic plants phylogeny. To depict the links between +evolution: how many genes were lost between two species whether +they belong to the same lineage or not. To depict the links between species clearly, we built a phylogenetic tree showing the relationships based on the distances among genes sequences. Many tools are available to obtain a such tree, for example: @@ -323,18 +299,14 @@ the distances and finally draw the phylogenetic tree. \section{Implementation} -The different algorithms have been implemented using Python version -2.7, on a laptop running Ubuntu~12.04~LTS. More precisely, the -computer is a Dell Latitude laptop - model E6430 with 6~GiB memory and +All the different algorithms have been implemented using Python on a personal computer running Ubuntu~12.04 with 6~GiB memory and a quad-core Intel core~i5~processor with an operating frequency of -2.5~GHz. Many python packages such as os, Biopython, memory\_profile, -re, numpy, time, shutil, and xlsxwriter were used to extract core +2.5~GHz. All the programs can be downloaded at \url{http://......} . genes from large amount of chloroplast genomes. \begin{center} -\begin{table}[b] -\caption{Type of annotation, execution time, and core genes -for each method}\label{Etime} +\begin{table}[H] +\caption{Type of annotation, execution time, and core genes.}\label{Etime} {\scriptsize \begin{tabular}{p{2cm}p{0.5cm}p{0.25cm}p{0.5cm}p{0.25cm}p{0.5cm}p{0.25cm}p{0.5cm}p{0.25cm}p{0.5cm}p{0.2cm}} \hline\hline @@ -355,15 +327,15 @@ Gene Quality & $\surd$ & $\surd$ & $\surd$ & $\surd$ & \multicolumn{2}{c}{$\sime Table~\ref{Etime} presents for each method the annotation type, execution time, and the number of core genes. We use the following notations: \textbf{N} denotes NCBI, while \textbf{D} means DOGMA, -and \textbf{Seq} is for sequence. The first {\it Annotation} columns -represent the algorithm used to annotate chloroplast genomes, the {\it +and \textbf{Seq} is for sequence. The first two {\it Annotation} columns +represent the algorithm used to annotate chloroplast genomes. The next two ones {\it Features} columns mean the kind of gene feature used to extract core genes: gene name, gene sequence, or both of them. It can be seen that -almost all methods need low {\it Execution time} to extract core genes -from large chloroplast genome. Only the gene quality method requires -several days of computation (about 3-4 days) for sequence comparisons, -once the quality genomes are construced it takes just 1.29~minutes to -extract core gene. Thanks to this low execution times we can use these +almost all methods need low {\it Execution time} expended in minutes to extract core genes +from the large set of chloroplast genomes. Only the gene quality method requires +several days of computation (about 3-4 days) for sequence comparisons. However, +once the quality genomes are well constructed, it only takes 1.29~minutes to +extract core gene. Thanks to this low execution times that gave us a privilege to use these methods to extract core genes on a personal computer rather than main frames or parallel computers. The lowest execution time: 1.52~minutes, is obtained with the second method using Dogma annotations. The number @@ -373,18 +345,13 @@ biological background of chloroplasts. With NCBI we have 28 genes for 96 genomes, instead of 10 genes for 97 genomes with Dogma. Unfortunately, the biological distribution of genomes with NCBI in core tree do not reflect good biological perspective, whereas with -DOGMA the distribution of genomes is biologically relevant. {\it Bad -genomes} gives the number of genomes that destroy core genes due to -low number of gene intersection. \textit{NC\_012568.1 Micromonas -pusilla} is the only genome which destroyed the core genome with NCBI +DOGMA the distribution of genomes is biologically relevant. Some a few genomes maybe destroying core genes due to +low number of gene intersection. More precisely, \textit{NC\_012568.1 Micromonas pusilla} is the only genome who destroyes the core genome with NCBI annotations for both gene features and gene quality methods. -The second important factor is the amount of memory being used by each +The second important factor is the amount of memory nessecary in each methodology. Table \ref{mem} shows the memory usage of each -method. We used a package from PyPI~(\textit{the Python Package -Index}) named \textit{Memory\_profile} (located at~{\tt -https://pypi.python.org/pypi}) to extract all the values in -table~\ref{mem}. In this table, the values are presented in megabyte +method. In this table, the values are presented in megabyte unit and \textit{gV} means genevision~file~format. We can notice that the level of memory which is used is relatively low for all methods and is available on any personal computer. The different values also @@ -392,14 +359,16 @@ show that the gene features method based on Dogma annotations has the more reasonable memory usage, except when extracting core sequences. The third method gives the lowest values if we already have the quality genomes, otherwise it will consume far more -memory. Moreover, the amount of memory used by the third method also +memory. Moreover, the amount of memory, which is used by the third method also depends on the size of each genome. -\begin{center} + \begin{table}[H] +\centering \caption{Memory usages in (MB) for each methodology}\label{mem} +\tabcolsep=0.11cm {\scriptsize -\begin{tabular}{p{2.5cm}p{1.5cm}p{1cm}p{1cm}p{1cm}p{1cm}p{1cm}p{1cm}} +\begin{tabular}{p{2.5cm}@{\hskip 0.1mm}p{1.5cm}@{\hskip 0.1mm}p{1cm}@{\hskip 0.1mm}p{1cm}@{\hskip 0.1mm}p{1cm}@{\hskip 0.1mm}p{1cm}@{\hskip 0.1mm}p{1cm}@{\hskip 0.1mm}p{1cm}} \hline\hline Method& & Load Gen. & Conv. gV & Read gV & ICM & Core tree & Core Seq. \\ \hline @@ -411,7 +380,7 @@ Gene Quality & ~ & 15.3 & $\le$3G & 16.1 & 17 & 17.1 & 24.4\\ \end{tabular} } \end{table} -\end{center} + diff --git a/biblio.bib b/biblio.bib index 510d794..22ebe6d 100644 --- a/biblio.bib +++ b/biblio.bib @@ -1,5 +1,5 @@ @article{Sayers01012011, -author = {Sayers, Eric W. and Barrett, Tanya and Benson, Dennis A. and Bolton, Evan and Bryant, Stephen H. and Canese, Kathi and Chetvernin, Vyacheslav and Church, Deanna M. and DiCuccio, Michael and Federhen, Scott and Feolo, Michael and Fingerman, Ian M. and Geer, Lewis Y. and Helmberg, Wolfgang and Kapustin, Yuri and Landsman, David and Lipman, David J. and Lu, Zhiyong and Madden, Thomas L. and Madej, Tom and Maglott, Donna R. and Marchler-Bauer, Aron and Miller, Vadim and Mizrachi, Ilene and Ostell, James and Panchenko, Anna and Phan, Lon and Pruitt, Kim D. and Schuler, Gregory D. and Sequeira, Edwin and Sherry, Stephen T. and Shumway, Martin and Sirotkin, Karl and Slotta, Douglas and Souvorov, Alexandre and Starchenko, Grigory and Tatusova, Tatiana A. and Wagner, Lukas and Wang, Yanli and Wilbur, W. John and Yaschenko, Eugene and Ye, Jian}, +author = {Sayers \emph{et al}}, title = {Database resources of the National Center for Biotechnology Information}, volume = {39}, number = {suppl 1}, @@ -283,4 +283,26 @@ ISSN = {1088-9051} title={DNASTAR- GenVision Software for Genomic Visualizations}, author={DNASTAR}, url = {http://www.dnastar.com/products/genvision.php} -} \ No newline at end of file +} + +@article{mcfadden2001primary, + title={Primary and secondary endosymbiosis and the origin of plastids}, + author={McFadden, Geoffrey Ian}, + journal={Journal of Phycology}, + volume={37}, + number={6}, + pages={951--959}, + year={2001}, + publisher={Wiley Online Library} +} + +@article{li2013complete, + title={Complete Chloroplast Genome Sequence of Holoparasite Cistanche deserticola (Orobanchaceae) Reveals Gene Loss and Horizontal Gene Transfer from Its Host Haloxylon ammodendron (Chenopodiaceae)}, + author={Li, Xi and Zhang, Ti-Cao and Qiao, Qin and Ren, Zhumei and Zhao, Jiayuan and Yonezawa, Takahiro and Hasegawa, Masami and Crabbe, M James C and Li, Jianqiang and Zhong, Yang}, + journal={PloS one}, + volume={8}, + number={3}, + pages={e58747}, + year={2013}, + publisher={Public Library of Science} +} diff --git a/conclusion.tex b/conclusion.tex index 25c5c57..6f2c431 100644 --- a/conclusion.tex +++ b/conclusion.tex @@ -1,2 +1,2 @@ -In this paper, we applied three methodologies for extracting core genes from large chloroplastes genomes. Extracted core genes depends on gene features and sequences. We developed a program using python to extract the core genes based on three methodologies. We considered first to extract core genes by sequence comparisons based on NCBI annotation. But the method failed to produce a core gene with different similarity thresholds because of NCBI annotation problems. We considered then to use DOGMA annotation tool to enhance core genes. Second and third methods used the annotation from NCBI and DOGMA. Second method is to extract gene names from gene features. An Intersection core metrix built where each position stores the intersection score by intersect two genomes (\emph{i.e. set of genes}) at a time. Core genes then constructed by selecting the maximum IS from ICM, remove the two intersected genomes with maximum IS, and add the corresponding core genes to ICM. In third method, a gene quality test is considered to ensure that the gene produced from NCBI annotation is the same gene (\emph{i.e.} gene name and sequence) produced by DOGMA. A gene quality test take place to construct new genomes according to the genes that pass a specific similarity threshold of 65\%, ICM then will take place to extract the core genes.\\ -Core tree are generated from each method to display the distribution of chloroplastes and core genes. The core tree from second method based on DOGMA annotation shows that the distribution of chloroplastes (\emph{i.e. Green Algae, Red Algae, and Land plants}) are match chloroplastes evolution history where each endosymbiosis version is branched well in the tree. \ No newline at end of file +In this paper, we applied three methodologies for extracting core genes from large chloroplasts genomes. Extracted core genes depend on gene features and sequences. We developed a program using python to extract the core genes based on three methodologies. We considered first to extract core genes by sequence comparisons based on NCBI annotation. But the method failed to produce a core gene with different similarity thresholds because of NCBI annotation problems. We considered then to use DOGMA annotation tool to enhance core genes. Second and third methods used the annotation from NCBI and DOGMA. Second method is to extract gene names from gene features. An Intersection core metrix built where each position stores the intersection score by intersect two genomes (\emph{i.e. set of genes}) at a time. Core genes then constructed by selecting the maximum IS from ICM, remove the two intersected genomes with maximum IS, and add the corresponding core genes to ICM. In third method, a gene quality test is considered to ensure that the gene produced from NCBI annotation is the same gene (\emph{i.e.} gene name and sequence) produced by DOGMA. A gene quality test take place to construct new genomes according to the genes that pass a specific similarity threshold of 65\%, ICM then will take place to extract the core genes.\\ +Core tree are generated from each method to display the distribution of chloroplasts and core genomes. The tree from second method based on DOGMA annotation shows that the distribution of chloroplasts (\emph{i.e. Green Algae, Red Algae, and Land plants}) match chloroplasts evolution history where each endosymbiosis event is branched well in the tree. \ No newline at end of file diff --git a/discussion.tex b/discussion.tex index 97fcb3f..9491806 100644 --- a/discussion.tex +++ b/discussion.tex @@ -3,41 +3,40 @@ a lineage comprising \textit{Red Algae, Green Algae} and \textit{Land Plants} (t Several Second Enbiosymbioses occurred then: two involving a Red Algae and other heterotrophic eucaryotes and giving birth to both Brown Algae and Dinoflagellates lineages; another involving a Green Algae and -a heterotrophic eucaryot and giving birth to Euglens.\\ -The interesting with the tree produced (especially from DOGMA) is +a heterotrophic eucaryot and giving birth to Euglens\cite{mcfadden2001primary}.\\ +The interesting point with the tree produced (especially from DOGMA) is that organisms resulting from the first endosymbiosis are distributed in every of the lineage found in the chloroplast genome structure evolution: with Red Algae chloroplasts together in one lineage, and -Green Algae and Land Plants chloroplasts together in antoher lineage; -while oranisms resulting from secondary endosymbioses are localized in +Green Algae and Land Plants chloroplasts together in another lineage; +while organisms resulting from secondary endosymbioses are more localized in the tree: both the chloroplasts of Brown Algae and Dinoflagellates representatives are found exclusively in the lineage also comprising the Red Algae chloroplasts from which they evolved, while the Euglens chloroplasts are related to the Green Algae chloroplasts from which they -evolved. This make sense in term of biology and history of lineages and +evolved. This makes sense in terms of biology, history of lineages, and theories of chloroplasts (and so photosynthetic ability) origins in -different Eucaryotic lineages. +different Eucaryotic lineages\cite{mcfadden2001primary}. Interestingly, The sole organisms included that possesses a chloroplast (and so a chloroplastic genome) but that have lost the photosynthetic ability (being parasitic plants) are found at the base of -the tree, and not together with its related species phylogenetically, -meaning that functional chloroplast genes are evolutionnary constrained +the tree, and not together with their phylogenetically related species. This means that functional chloroplast genes are evolutionnary constrained when used in photosynthetic process, but loose rapidly their efficiency -when not used. They are Cuscuta-grovonii an Angiosperm (flowering plant) +when not used, as recently observed for a species of Angiosperms\cite{li2013complete}. These species are \textit{Cuscuta-grovonii} an Angiosperm (flowering plant) at the base of the DOGMA Angiosperm-Conifers branch, and -Epipactis-virginiana also an Angiosperm at the complete base of the tree. +\textit{Epipactis-virginiana} also an Angiosperm at the complete base of the tree. Another interesting result is that land plants that -represent single sublineage originating from the large and diverse +represent a single sublineage originating from the large and diverse lineage of green algae in Eucaryots history are present in two different branches of the DOGMA tree, associated with Green Algae, one branch comprising the basal grade of land plants (mosses and ferns) and the second -comprising the most internal lineage of land plants (Conifers and flowering plants). +comprising the most internal lineages of land plants (Conifers and flowering plants). But independently of their split in two distinct branches of the DOGMA tree, the Land Plants always show a higher number of functional genes in -their chloroplasts than the green algae from which they emerged, probably meaning that +their chloroplasts than the green algae from which they emerged, probably meaning that the terrestrial way of life necessitates more functional genes for an -optimal photosynthesis than marine way of life. But a more detailed -analysis of selected genes is necessary to better understad the reasons why? +optimal photosynthesis than the marine way of life. However, a more detailed +analysis of selected genes is necessary to better understand the reasons why? diff --git a/intro.tex b/intro.tex index 9a70444..2a6cac9 100644 --- a/intro.tex +++ b/intro.tex @@ -6,21 +6,22 @@ annotated from NCBI \cite{Sayers01012011} and Dogma \cite{RDogma}: how can we identify the best core genome and what is the evolutionary scenario of these chloroplasts.\\ Chloroplast (such as mitochondria) are fondamental key elements in -living organisms history. Indeed, chlorplast in Eucaryotes are organites responsible for +living organisms history. Indeed, chloroplast in Eucaryotes are organites responsible for photosynthesis. Photosynthesis is the main way to produce organic matter from mineral matter, using solar energy. Consequently photosynthetic organisms are at the base of most ecosystems trophic chains and -photosynthesis in eucaryotes allowed a great speciation in the lineage +photosynthesis in Eucaryotes allowed a great speciation in the lineage (to a great biodiversity). From an ecological point of view, photosynthetic organisms are at the origin of the presence of dioxygen in the atmosphere (allowing extant life) and are the main source of mid- -to long term carbon stockage (using atmospheric CO2, important in the -context of climate change). Chloroplast found in Eucaryots have an endosymbiotic origin, meaning -that they are a fusion of a photosynthetic bacteria (Cyanobacteria) and -a eucaryotic cell (enable to produce organic matter from solar energy = heterotrophic). \\ +to long term carbon stockage (using atmospheric CO2) an important feature in the +context of climate change. Chloroplasts found in Eucaryotes have an endosymbiotic origin, meaning +that they from the incorporation of a photosynthetic bacteria (Cyanobacteria) within an eucaryotic cell. \\ -By the principle of -classification, a small number of genes lost among species indicates -that these species are close to each other and belong to same family, -while a large lost means that we have an evolutionary relationship -between species from different families. +By the principle of phylogenetic classification, a mutation in the DNA shared by two to several taxa has a higher probability to be inherited from common ancestor than to have evolved independently. In such a process, shared changes in the genomes allow to build relationships between species. In the case of chloroplasts, an important category of changes in the genome is the loss of functional genes, when inoperant or when transferred to the nucleus. Thereby, we hypothesize that small number of gene losses among species indicates +that these species are close to each other and belong to same lineage, +while a large loss means that we have an evolutionary relationship +between species from much more distant lineages. Phylogenetic relationships are mainly built by comparison of sets of coding and non-coding sequences. Phylogenies of photosynthetic plants are important to assess the origin of chloroplasts (REF) and the modalities of gene loss among lineages. These phylogenies are usually done using less than ten chloroplastic genes (REF), and some of them may not be conserved by evolution process for every taxa. As phylogenetic relationships inferred from data matrices complete for each species included and with the same evolution history are better assumptions, we selected core genomes for a new investigation of photosynthetic plants phylogeny. To depict the links between species clearly, we here intend to built a phylogenetic tree showing the relationships based on the distances among gene sequences of a core genome. The circumscription of the core chloroplast genomes for a given set of photosynthetic organisms needs bioinformatic tools for sequence annotation and comparison that we describe here. + +Other possible scientific questions to consider for introduction improvement: +Which bioinformatic tools are necessary for genes comparison in selected complete chloroplast genomes? Which bioinformatic tools are necessary to build a phylogeny of numerous genes and species, etc? \ No newline at end of file diff --git a/main.tex b/main.tex index bebdb14..fd5f236 100755 --- a/main.tex +++ b/main.tex @@ -23,14 +23,14 @@ \title{Finding the Core-Genes of Plant Species Chloroplast} -\author[1]{Bassam AlKindy} %\footnote{email: bassam.al-kindy@univ-fcomt\'{e}.fr} -\author[1]{Jacques Bahi} +\author[1]{Bassam AlKindy} %\footnote{email: bassam.al-kindy@univ-fcomte.fr} \author[1]{Jean-Fran\c{c}ois Couchot} \author[1]{Christophe Guyeux} \author[2]{Arnaud Mouly} \author[1]{Michel Salomon} +\author[1]{Jacques Bahi} \affil[1]{FEMTO-ST Institute, UMR 6174 CNRS, Computer Science Department DISC, Universit\'{e} de Franche-Comt\'{e}, France} -\affil[2]{Lab. Chrono-Environnement, UMR 6174 CNRS, Universit\'{e} de Franche-Comt\'{e}, France} +\affil[2]{Lab. Chrono-Environnement, UMR 6249 CNRS, Universit\'{e} de Franche-Comt\'{e}, France} %{\small \it Authors in alphabetic order} \renewcommand\Authands{ and }