To the Editor: Since 1987, morbillivirus  outbreaks among pinnipeds and cetaceans in the Northern Hemisphere have caused high rates of death (Phocine distemper virus (PDV) and Cetacean morbillivirus (CeMV). PDV has been isolated from pinnipeds, and 3 strains of CeMV  have been isolated from dolphins and whales (Tursiops truncatus) has been confirmed in the Southern Hemisphere (in the southwestern Pacific Ocean) (Lagenodelphis hosei) stranded off Brazil and Argentina in 1999 indicate the exposure of South Atlantic cetaceans to morbillivirus , a coastal marine and estuarine species that occurs off the Atlantic Coast of South and Central America. Serologic surveys indicate that morbilliviruses infect marine mammals worldwide  , a monoclonal antibody against the nucleoprotein antigen of canine distemper virus that cross-reacts with cetacean morbilliviruses (Morbillivirus spp.–specific primers were used for PCR: 5′-ATGTTTATGATCACAGCGGT-3′ (forward) and 5′-ATTGGGTTGCACCACTTGTC-3′ (reverse) (http://megasoftware.net/) was used to construct a neighbor-joining phylogenic tree based on the sequenced amplicon from this study (GenBank accession no. KF711855) and 12 other GenBank sequences that represent the 6 morbillivirus species already described in the literature. The analysis placed the Guiana dolphin strain at the CeMV clade, but segregated it from the already described dolphin morbillivirus strains PMV, DMV, and PWMV . We extracted RNA from frozen lung samples by using TRIzol Reagent  according to the manufacturer’s instructions and amplified a 374-bp conserved fragment of the phosphoprotein (P) gene by reverse transcription PCR. The following and PWMV . The samIn summary, sequence analysis of the morbillivirus from the dead Guiana dolphin suggests that the virus is a novel strain of the CeMV species; this conclusion is supported by phylogenic analysis and geographic distribution of the virus and by its distinct host. Emaciation, marked lymphoid depletion, interstitial pneumonia, and meningoencephalitis are common findings in morbillivirus-infected animals (Morbillivirus outbreaks have caused a high number of deaths among pinnipeds and cetaceans and are a major risk to previously unexposed nonimmune populations of aquatic mammals (


Anatids may have spread the virus along their autumn migration routes. During the second half of 2005, highly pathogenic avian influenza (HPAI) H5N1 virus spread rapidly from central Asia to eastern Europe. The relative roles of wild migratory birds and the poultry trade are still unclear, given that little is yet known about the range of virus hosts, precise movements of migratory birds, or routes of illegal poultry trade. We document and discuss the spread of the HPAI H5N1 virus in relation to species-specific flyways of Anatidae species  and climate. We conclude that the spread of HPAI H5N1 virus from Russia and Kazakhstan to the Black Sea basin is consistent in space and time with the hypothesis that birds in the Anatidae family have seeded the virus along their autumn migration routes. Until recent events, HPAI outbreaks or epizootics were assumed to first require transmission of a low pathogenic avian influenza (LPAI) virus from wild birds to domestic poultry  have been recorded in most bird families , represent the highest risk for transmission to domestic poultry , which has an area of 2,745,000 kmhe world . WSL is he world . In westhe world B, the Nohe world .During July and August 2005, several HPAI H5N1 virus outbreaks were reported in Russia and Kazakhstan . These oA mission of the World Organization for Animal Health (OIE) to Kazakhstan established that the first outbreaks were located near important molting sites for migratory waterfowl. This finding is further illustrated in The initial outbreaks of HPAI H5N1 virus in Romania, Turkey, and Ukraine occurred close to wetlands frequented by overwintering migratory waterfowl. These locations were clearly far from any known location where HPAI H5N1 virus had been recorded, while the timing and location match the autumn wildfowl migration ahead of the approaching wave of frost .If these flyways are assumed to be used by infected birds, their geographic extent may be used to estimate an index of risk for virus transmission from wildfowl to domestic poultry. The population-weighted overlay of individual waterfowl species' ranges intersecting with HPAI H5N1 virus locations B can, inOur results indicate that the broad-scale pattern of spread of HPAI H5N1 virus from Russia to the Black Sea basin is consistent with the spatial and temporal pattern of Anatidae migration from Siberia. Given that the first recorded signs of HPAI H5N1 virus in Turkey, Romania, and Ukraine took place in the direct vicinity of important waterfowl overwintering sites, Anatidae could have been implicated in the spread of HPAI H5N1 virus to the Black Sea basin. The search for wild bird species carrying HPAI H5N1 virus is in progress and awaits further classification. Several species demonstrably carry the virus without showing clinical signs, as has been recently reported from studies in Russia . ConverThe broad approach adopted in this study has clear limitations, given the uncertainties regarding the host range of HPAI H5N1 virus within the Anatidae family, the sizes and distribution of the bird populations, their precise migratory patterns, and the demarcation of the summer and winter habitat. A comprehensive retrospective analysis of HPAI H5N1 virus spread in the western Palearctic would require a better description of the dynamic distribution of wild birds  as well as more detailed domestic poultry data  to map the contact points between wild and domestic birds. In addition, local studies could focus on possible introduction points and characterize and detail the specific ecologic conditions in the wild birds–domestic poultry interface that support establishment of the virus, including the local landscape structure (wild bird habitat and farming), climate , and other agro-ecologic conditions.We used the land surface temperature (LST) data products derived from the Moderate Resolution Imaging Spectroradiometer (MODIS) sensor on board the National Aeronautics and Space Administration's Aqua satellite (Daily LST products were aggregated (averaged) to generate 8-day composite LST product (MYD11A2), and 46 of these 8-day composite LST products are generated per year. The LST product has a spatial resolution of 1 km. We downloaded the 8-day composite LST data (MYD11A2) in 2005 from the US Geological Survey Earth Resources Observation and Science (EROS) Data Center. For each individual 1-km pixel, we analyzed time series data of nighttime LST in 2005 and identified the first 8-day period that experienced frost (LST <0°C) in the fall/winter seasons. We assume that the date of early frost events in fall/winter seasons is one of many factors that affect the starting date of wild bird migration from north to south.Distribution data regarding the winter feeding areas and summer breeding areas were extracted from the Global Registry of Migratory Species CD-ROM (


Closteroviridae are associated with GLD. However, Grapevine leafroll-associated virus 3 (GLRaV-3), the type species for the genus Ampelovirus, is regarded as the most important causative agent. Here we provide a general overview on various aspects of GLRaV-3, with an emphasis on the latest advances in the characterization of the genome. The full genome of several isolates have recently been sequenced and annotated, revealing the existence of several genetic variants. The classification of these variants, based on their genome sequence, will be discussed and a guideline is presented to facilitate future comparative studies. The characterization of sgRNAs produced during the infection cycle of GLRaV-3 has given some insight into the replication strategy and the putative functionality of the ORFs. The latest nucleotide sequence based molecular diagnostic techniques were shown to be more sensitive than conventional serological assays and although ELISA is not as sensitive it remains valuable for high-throughput screening and complementary to molecular diagnostics. The application of next-generation sequencing is proving to be a valuable tool to study the complexity of viral infection as well as plant pathogen interaction. Next-generation sequencing data can provide information regarding disease complexes, variants of viral species, and abundance of particular viruses. This information can be used to develop more accurate diagnostic assays. Reliable virus screening in support of robust grapevine certification programs remains the cornerstone of GLD management.Grapevine leafroll disease (GLD) is one of the most important grapevine viral diseases affecting grapevines worldwide. The impact on vine health, crop yield, and quality is difficult to assess due to a high number of variables, but significant economic losses are consistently reported over the lifespan of a vineyard if intervention strategies are not implemented. Several viruses from the family  Grapevine leafroll-associated virus 3 (GLRaV-3) in the genus Ampelovirus that carries the mantle as the “main etiological agent” contributing to the disease.Grapevine leafroll disease (GLD) is one of the most important viral diseases affecting wine, juice, and table grape cultivars, as well as rootstocks. It currently ranks as one of the most important diseases affecting wine grape cultivars, comparable with several fungal diseases , which are based on the comparative sequence analysis, the expression of the encoded proteins and the replication of the genome are discussed. The genetic variability between GLRaV-3 isolates is addressed in detail, and a proposal is made to standardize the naming of the genetic variant groups identified to date. A summary of diagnostic assays employed to detect the virus is also presented, with a special emphasis on the application of next-generation sequencing technologies and its potential to revolutionize our understanding of the metagenomic nature of virus infections in grapevine varieties. The review is concluded with a discussion of the various levels of host-pathogen interactions, highlighting a very intriguing potential role of small RNAs (sRNAs) in this complex plant virus interplay.Vitis vinifera L.) that may have originated in the “Old World,” from where it spread, primarily through commercial trading of propagation material, to attain its current worldwide geographical distribution. Evidence that leafroll occurred in Europe and in other regions of the Mediterranean basin and Near East before the introduction of phylloxera (Dactulosphaira vitifoliae) from the eastern United States in the mid nineteenth century . (iii) Presence of GLD-infected vines in an abandoned vineyard established between 1889 and 1891 by the University of California as a varietal test block in a secluded locality at the foothills of the Sierra Nevada Mountains in Amador County , prior to the wide use of rootstocks made necessary by the spread of phylloxera around 1900  and Grapevine leafroll-associated virus 3 (GLRaV-3) in own-rooted vines from countries where grapes have been grown for centuries, e.g., Cyprus  Prry Gale, . The leaIoannou, , Yemen  1–9 and a group of more recently described viruses . All these viruses belong to the genus Ampelovirus except for GLRaV-2, which is in the genus Closterovirus, and GLRaV-7, which is in the tentative genus Velarivirus  and Little cherry virus 2 (LChV-2). Subgroup II includes GLRaV-4 plus PMWaV-1, PMWaV-3, and Plum bark necrosis stem pitting-associated virus (PBNSPaV). Further biological, serological and molecular data showed that GLRaV-4, -5, -6, -9, -Pr,-De, and -Car are closely related and all could be considered as different strains of GLRaV-4 . The 5′ end of the viral genome is likely to be encapsidated by the virion tail structure, similar to that of other members of the family Closteroviridae, which comprises proteins coded for by ORF4 (HSP70h), ORF5 (p55), and ORF7 (CPm) of the viral genome, and might be instrumental in determining cell-to-cell and systemic transport   functions in cell-to-cell movement . Among these, the sgRNA corresponding to ORF10 (p20B) accumulated to the highest level, followed by sgRNAs encoding products of the ORF8 (p21), ORF9 (p20A), and ORF6 (CP). These results suggest that temporal and quantitative regulation of GLRaV-3 sgRNA transcription occurs during the virus infection cycle, leading to differential expression, and/or accumulation of sgRNAs in a distinct regulation pattern.Similar to other characterized members of the family Ampelovirus is likely distinct from that of the genus Closterovirus  for several GLRaV-3 sgRNAs were determined for isolates GP18 and WA-MR that belong to two different genetic variant groups  combined with sequence analysis of different genomic regions   showed an average of 34.5–38.7% inter-group variation when compared to groups I–IV isolates. The average variation between isolates of groups VI and VI-like (Isolate NZ2) was 28.3%. It is evident that, like ORF2, genetic diversity studies indicate that functional research needs to be performed to better understand the role, if any, of ORF11 and 12 in GLRaV-3 biology.The GLRaV-3 3′UTR length varies; group I–III isolates are 277 nt, except for isolate 139 which is 250 nt; while the 3′UTR of isolates from group VI are 264 nt, except for CA7246 which is 274 nt. The 3′UTR of isolate NZ2 (group VI-like) is 289 nt in length  as nucleotide identities between isolates from phylogenetic groups I to III, VI and VI-like (Isolate NZ2) decrease to as low as 50%. While two regions from nucleotides 18,320–18,382, and 18,430–18,498 (based on GP18 numbering) have low sequence variation, as the average nucleotide identities between group I–III, VI, and VI-like (Isolate NZ2) isolates increase to 90% or greater. Thus, these areas of high conservation may represent possible cis-acting elements important for controlling GLRaV-3 replication.Potential The diversity in the CP gene was examined based on the assumption that viral CPs evolved more rapidly than proteins involved in replication and expression of virus genomes, providing better phylogenetic resolution  from the Sharma et al.  study weIt is clear from Figure The availability of sequence data is crucial to classify GLRaV-3 variant groups. Analysis of the available data suggests that there are at least six well-supported phylogenetic groups within GLRaV-3 populations worldwide Figure . We propWe suggest that ascending, consecutive Roman numerals be used for maintenance of shared terminology by the community. Based on partial sequences and the number of new variants recently found throughout the world, we expect that more genetic variation will be revealed in the future and that more GLRaV-3 phylogenetic groups will be identified.The identification of disease-associated viruses has proven to be challenging since most diseased grapevines are infected with more than one virus. This is further complicated as unrelated viruses can cause similar disease symptoms, new infections typically have a low virus titer, viruses are often unevenly distributed in infected vines, and symptoms in some white cultivars and rootstocks are less noticeable. To date several techniques have been applied to detect viruses associated with GLD in plant material, including biological indexing, serology, nucleic acid-based methods, and next-generation sequencing.V. vinifera cvs Cabernet Franc, Pinot noir, Cabernet Sauvignon or Barbera may be used as indicator host, depending upon personal preferences and/or climatic conditions under which the indicator is grown. Green-grafting is another biological indexing technique used to screen grapevine material for viruses including GLRaV-3  is grafted to an indicator grapevine cultivar , immunofluorescence (IF), and immuno-strip tests  was developed for pathogens with RNA genomes  is also possible for experimental purposes  in whose organs and tissues it is unevenly distributed -miRNAs involved in organ and plant development were observed in infected grapes compared to virus-free plants. Particularly vvi-miRNA 156 and 167, which in Arabidopsis thaliana target “Squamosa promoter binding protein-like” and “Auxin Response Factor (ARF)” transcription factors, respectively, are both down-regulated, whereas the reverse occurs with vvi-miR166, whose increased levels in Arabidopsis thaliana inhibits the expression of its HD-ZIPIII target, thus causing extensive developmental alterations. Surprisingly, a lower expression level of vvi-miR168, which translationally regulates Argonaute 1 (AGO1) expression in A. thaliana and N. benthamiana, was induced by virus infection. A possible explanation of this unexpected finding may be the condition of the analyzed tissues, which were collected from symptomatic leaves during mid-September, when replicating virus titers are low. Indeed these vines had a low viral RNA concentration, as demonstrated by the small number of vsRNAs detected in the library . In line with a similar high-throughput analysis performed on citrus plants affected by Citrus tristeza virus (CTV) , which have been shown to be involved in plant development and plant response to biotic and abiotic stresses. MicroRNAs are a class of small, 21–24-mer, non-coding sRNAs, which are conserved and play a role as “master regulators” of gene expression. In a recent study, Alabi et al.  profiledVitis species) and being phloem-limited, research on GLRaV-3 has largely focused on epidemiology and the development of reliable detection assays. Phylogenetic studies showed the existence of six genetic variant groups and, with the advances in sequencing technologies, more sequence data will be generated that will indubitably lead to the identification of additional genetic variants. It is therefore necessary to have sensitive and rapid diagnostic methods to test material for GLRaV-3 infection that are able to detect all variants that may influence disease etiology. This implies that newly developed and old diagnostic assays, especially PCR based assays, be verified to be able to detect all genetic variants and continuously be reevaluated to ensure that the assay remains valid as new sequence information becomes available. The role of the different genetic variants of GLRaV-3 in GLD etiology is still largely unknown and elucidating this role is an essential next-step. It is also important to investigate the interactions between the different GLRaV-3 variants, and in combination with the mealybug vectors, to potentially explain the dominant occurrence of some of the genetic variants. The successful construction of an infectious clone of GLRaV-3 provides a platform to study viral replication and gene expression, and determine the function of the GLRaV-3 genes that are currently unknown and also the function of the highly variable extended 5′UTR. GLRaV-3 is one of the most important grapevine viruses and with the use of the latest tools in molecular biology a complete understanding of its role in GLD etiology and host-pathogen interaction is attainable.Grapevine leafroll disease is considered to be one of the most economically destructive virus diseases of grapevine and a major constraint to the production of premium wine grapes. GLRaV-3 has been more closely associated with GLD than any other GLRaV, supporting the view that it is the “main etiological agent.” Even though the genetic variation observed in the GLRaV-3 genome has been studied more intensively ever since the publishing of the first near complete genome sequence, research on GLRaV-3 lags behind that of other economically important plant viruses. Due to its narrow host range (infecting only The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In this review, we discuss the possibility that the glycosylation of West Nile (WN) virus E-protein may be associated with enhanced pathogenicity and higher replication of WN virus. The results indicate that E-protein glycosylation allows the virus to multiply in a heat-stable manner and therefore, has a critical role in enhanced viremic levels and virulence of WN virus in young-chick infection model. The effect of the glycosylation of the E protein on the pathogenicity of WN virus in young chicks was further investigated. The results indicate that glycosylation of the WN virus E protein is important for viral multiplication in peripheral organs and that it is associated with the strong pathogenicity of WN virus in birds. The micro-focus reduction neutralization test (FRNT) in which a large number of serum samples can be handled at once with a small volume (15 μL) of serum was useful for differential diagnosis between Japanese encephalitis and WN virus infections in infected chicks. Serological investigation was performed among wild birds in the Far Eastern region of Russia using the FRNT. Antibodies specific to WN virus were detected in 21 samples of resident and migratory birds out of 145 wild bird samples in the region. The West Nile (WN) virus is a mosquito-borne flavivirus of the Japanese encephalitis (JE) serocomplex group that causes lethal encephalitis in humans and horses. WN virus was first isolated in 1937 from the blood of a febrile patient in the WN district of Uganda ,3,4. OutCulex mosquito vectors. Viremic levels of the avian host directly affect the infection rates of vector mosquitoes; birds with higher viremia generate more infected mosquitoes after blood feeding . Th. ThCulex variant . MoreoveWe previously examined the role of the N-linked glycans of E protein in tick-borne encephalitis (TBE) virus particle secretion using subviral particles . SecretiMutations of NS3 or NS4B of the NY strain of WN virus were reported to be responsible for the increased pathogenicity and viremic level in avian or mammalian hosts ,40. Impo5 PFU/mL) in 2-day-old chicks, and high levels of virus were detected in the hearts, spleens, and kidneys  virus ,60,61, iAlthough it is difficult to distinguish the specific flavivirus neutralizing antibodies in multiple infections with heterologous viruses, micro FRNT is able to differentiate antibodies against WN virus and JE virus in single infection. Therefore, micro FRNT is a quite useful method to conduct serological survey in the area where WN and JE viruses are both prevalent.Anseriformes, Charadriiformes, Columbiformes, and Pelecaniformes. Birds in these orders are known to support WN virus propagation with high levels of viremia, and to serve as efficient amplifying hosts for the transmission of WN virus to mosquitoes [Field surveys were conducted in Far East Russia in 2005 and 2006 to know the seroprevalence of WN virus in wild birds using micro FRNT. Neutralizing antibody to WN virus was identified in 21 serum samples taken from 145 wild birds (14.5%)  62]. Bi. Bi62]. squitoes . The JE squitoes ,31. TherColumba livia ) tested and some eastern turtle doves , which are resident birds, had WN virus antibodies and were probably infected with the virus near Khanka Lake. Because Khanka Lake lies far to the east of where WN was first isolated in Russia, the WN virus appears to have been transmitted among wild birds in Far Eastern Russia. The other WN virus-positive birds identified in this study were spotbills (Anas poecilorhyncha), harlequin ducks (Histrionicus histrionicus), red-breasted mergansers (Mergus serrator), black-headed gulls (Larus ridibundus), and common terns (Sterna hirundo), which are all migratory birds, therefore it is possible that these birds were infected with the WN virus in Far Eastern Russia and carried the virus into other regions of East Asia. In the Asia-Pacific region, migratory water birds typically display north-south flying patterns [Sterna hirundo) is a long-distance migratory species that may migrate between Far East Russia and Australasia. The possibility that the WN virus-positive antibodies in common tern might be the result of the Kunjin virus infection could not be excluded because of the limitation of the neutralization test. Recent studies of migration routes of mallard (Anas platryhynchos) determined by satellite telemetry have shown that besides the northward flyway from Japan to Russia, a northwestward flyway also exists in Far East Russia [All of the rock doves ( patterns . Long-dit Russia . In Japat Russia . In Hokkt Russia .The results of this study suggest that WN virus is distributed throughout Far Eastern Russia and that it may spread to East Asian countries with the migration of wild birds. To prepare for the introduction of WN virus to East Asia, the development of a diagnostic test that can accurately differentiate between WN and JE virus infection is needed. In addition, continued epizootiological evaluation of WN virus infection among birds and humans in Far Eastern Russia and East Asia will be important for monitoring the spread of the disease.WN virus causes serious problems in public health since a large numbers of patients with severe encephalitis are reported in various regions of the World. The distribution of the virus is still expanding  and the The highly pathogenic WN virus appeared to emerge in the late 1990s and the glycosylation in E protein may be one of the factors of increased pathogenicity of WN virus. The virus replication in birds is enhanced by the glycosylation of E protein and it may result in the increased transmissibility among bird population and higher pathogenicity in birds. It is quite important to know that only one amino acid substitution of the virus may influence the distribution and pathogenicity of WN virus.Apparently, there is a strong need for vaccine and specific treatments to WN virus infection. To develop these specific measures to WN virus infection, basic research for understanding the replication and pathogenicity of West Nile virus should be further encouraged.


Dear Editor,We read with interest the paper entitled “Prevalence of Hepatitis D Virus Infection among Hepatitis B Virus Infected Patients in Qom Province, Center of Iran” by Ghadir et al.  which waFinally we recommend that while there are user-friendly statistical packages, investigators should consult biostatistician at the time of data analysis to avoid the errors due to small sample size or inappropriate statistical tests.


Bemisia tabaci) collected from two important agricultural regions in Florida, USA. VEM successfully characterized the active and abundant viruses that produce disease symptoms in crops, as well as the less abundant viruses infecting adjacent native vegetation. PCR assays designed from the metagenomic sequences enabled the complete sequencing of four novel begomovirus genome components, as well as the first discovery of plant virus satellites in North America. One of the novel begomoviruses was subsequently identified in symptomatic Chenopodium ambrosiodes from the same field site, validating VEM as an effective method for proactive monitoring of plant viruses without a priori knowledge of the pathogens. This study demonstrates the power of VEM for describing the circulating viral community in a given region, which will enhance our understanding of plant viral diversity, and facilitate emerging plant virus surveillance and management of viral diseases.Current knowledge of plant virus diversity is biased towards agents of visible and economically important diseases. Less is known about viruses that have not caused major diseases in crops, or viruses from native vegetation, which are a reservoir of biodiversity that can contribute to viral emergence. Discovery of these plant viruses is hindered by the traditional approach of sampling individual symptomatic plants. Since many damaging plant viruses are transmitted by insect vectors, we have developed “vector-enabled metagenomics” (VEM) to investigate the diversity of plant viruses. VEM involves sampling of insect vectors  from plants, followed by purification of viral particles and metagenomic sequencing. The VEM approach exploits the natural ability of highly mobile adult whiteflies to integrate viruses from many plants over time and space, and leverages the capability of metagenomics for discovering novel viruses. This study utilized VEM to describe the DNA viral community from whiteflies ( Current knowledge of plant virus diversity is heavily biased towards agents of visible and economically important diseases, with less known about the potentially emergent viruses that have not yet made their presence known on crops. However, these undiscovered viruses can provide a reservoir of biodiversity for recombination and reassortment with existing pathogenic plant viruses, and are an essential component of the complex microbial ecology of plants Bemisia tabaci species complex is an important vector of many plant viruses. Whiteflies often occur in high populations on many crops B. tabaci feeds on a very wide range of plants While viruses and virus-like elements can be readily isolated from ∼60% of plants Once the proper samples are obtained, an effective method of viral discovery is needed. Most viral identification techniques have limited capability for characterizing novel viruses due to their specificity. Common methods such as ELISA with antibodies for a specific virus, PCR or microarrays with primers designed for a specific virus, or PCR with degenerate primers that amplify a closely related group of viruses are effective methods for detecting close relatives of known viruses Viral metagenomics, a molecular technique that involves purifying viral particles from samples and shotgun sequencing the viral nucleic acids, circumvents the biases of classic viral identification methods, and has revolutionized the exploration of viral diversity The vector-enabled metagenomics (VEM) approach presented here takes advantage of the highly polyphagous and mobile nature of the whitefly vector, combined with the capability of metagenomics to discover novel viruses without relying on sequence similarity to known viruses. This study utilized VEM to explore the diversity of DNA viruses in whiteflies collected from different crops in two agriculturally important sites in Florida.Bemisia tabaci. BLASTn analysis revealed that 79% of the sequences in the Citra virome and 93% of the sequences in the Homestead virome showed significant similarity to known plant viruses, with nucleotide identities between 70% and 100% to previously described begomoviruses , a begomovirus known to infect watermelon plants at this sampling site . AlignmeAdditionally, two sequences were identified that shared 96-97% nucleotide identities to the DNA-B of Sida golden mosaic virus (SiGMV). Based on one of these metagenomic sequences, PCR primers were designed to amplify, clone, and sequence the entire DNA-B from DNA extracted from the whiteflies. The resulting sequence had characteristics consistent with the DNA-B of SiGMV , but shaStrikingly, one partial sequence of 243 nt was identified in the virome which had only 84% nucleotide identity to the DNA-A of Tobacco leaf rugose virus . PCR priChenopodium ambrosiodes. A full length DNA-A clone (2626 nt) was obtained and sequenced from this plant, which was 96% identical to the complete DNA-A sequence identified from the whiteflies using VEM. This novel virus identified in C. ambrosiodes is tentatively named Chenopodium leaf curl virus - [USA:Florida:Citra: 2009] (ChLCV-[US:FL:Cit:09]), while the novel virus identified in the whiteflies through VEM is tentatively named Chenopodium leaf curl virus - [USA:Florida:Citra: 2007:VEM] (ChLCV-[US:FL:Cit:07:VEM]). Both viruses appear to be most closely related to Desmodium leaf distortion virus, but group with other New World begomoviruses such as Cotton leaf crumple virus, Sida yellow leaf curl virus and Tomato severe leaf curl virus  nucleotide identities to known begomoviruses . The virNumerous metagenomic sequences shared between 88%–93% nucleotide identities to seven known begomoviruses . FurtherPCR primers were designed to further explore two metagenomic sequences that initially showed low levels of identity to known begomoviruses. The complete DNA-A of one sequenced virus initially showed only 88% identity to its closest begomovirus relative; however, it is 94% identical to a Jamaican Sida virus that was deposited in GenBank in December 2010  were cloned and sequenced. The fact that these genomes were present in virions in the whiteflies, have an adenosine-rich region, and do not have an open reading frame that codes for a replication-associated protein, suggests that these sequences are novel begomovirus satellites, which are tentatively named Whitefly VEM Satellite (WfVEM-Sat). The WfVEM-Sat genomes are similar to the Tomato leaf curl virus satellite  begomoviruses belonging to the family a priori knowledge of what viral types are present. First, each virome was dominated by sequences with a high level of similarity to known viruses infecting the crop plants from which the whiteflies were collected. This demonstrates that VEM is capable of detecting and obtaining high levels of sequence coverage from active and abundant viruses in the primary collection host. Second, both viromes contained sequences similar to viruses that infect Sida spp., which are weeds commonly found near many agricultural fields in Florida. This demonstrates that discovery of viruses through VEM is not constrained by the collection host and that VEM is capable of identifying viruses from neighboring crops or weeds. Characterization of viruses from indigenous plants is especially interesting since this is a largely unexplored reservoir of genetic diversity that may contribute to emergence of novel begomovirus strains by host shift or recombination In this study, VEM led to the identification of multiple viruses from each site, exemplifying the advantages of this approach for describing the viral community circulating amongst plants in a given region, without This proof-of-concept study has helped expand our knowledge of begomoviruses in Florida. Vegetable crops in Florida have experienced significant losses due to whitefly-transmitted viruses over the past 15 years. The dominant viruses identified in this study were Tomato yellow leaf curl virus (TYLCV) and Cucurbit leaf crumple virus (CuLCrV), which are both whitefly-transmitted begomoviruses that have been introduced to Florida since the arrival of the silverleaf whitefly in the late 1980's The small amount of sequencing performed in this study was sufficient to uncover numerous metagenomic sequence fragments related to viruses that had not been previously documented in Florida . Some ofIn addition to the discovery of begomoviruses, this study demonstrated the power of the VEM approach for the discovery of novel virus-associated entities such as satellites. This study is the first to demonstrate the presence of begomovirus satellites in the North America. Satellites can play important roles in plant disease, but often remain unrecognized due to limitations of common virus identification methods, especially when there is a lack of sequence homology with the helper virus. Most begomovirus satellites fall into one of several classes: α-satellites, β-satellites, and defective-interfering DNAs (DI-DNA) By enabling the discovery of a wide range of viruses and satellites, VEM is a powerful technique that will significantly enhance our fundamental scientific understanding of plant viral diversity, biogeography, and emergence. Typically, new plant viruses are not identified until an outbreak causing significant crop loss occurs. This has put agricultural biodefense into a reactive mindset – waiting until a new disease becomes a problem before trying to understand and combat the causative agent. The VEM approach circumvents that traditional process by obtaining a comprehensive sample of the viruses actively circulating in an insect vector population within a region, effectively integrating over individual plants, space, and time. Thus, VEM is a proactive molecular surveillance tool that allows rapid identification of emerging viruses before noticeable crop loss occurs, providing precious time for implementation of preventative measures and improving agricultural biosecurity.Future studies using the VEM approach can incorporate high-throughput sequencing, expand the geographical range and frequency of sampling, and investigate both DNA and RNA viruses. The VEM approach can be extended to other insect vectors, such as aphids and leafhoppers, to understand the diversity of plant viruses that is being transmitted through each of these different vectors. In addition, further refinement of this method for begomovirus discovery should focus on recovering whole-genome components in order to allow for accurate analyses of recombination and evolution. When applied on a larger scale, VEM can describe global diversity of plant viruses, which will help refine existing plant virus phylogenies, aid in the development of more inclusive assays for monitoring introduced and emerging viruses, and increase our understanding of plant virus biogeography and evolution.Bemisia tabaci) were collected using battery-operated vacuums Adult whiteflies  and host cells were removed through centrifugation at 10,000×g for 10 minutes, followed by filtration of the supernatant through a 0.22 µm Sterivex filter . The filtrate was treated with 0.2 volumes of chloroform for 10 minutes, then incubated with 2.5 U DNase I per µl sample for 3 hours at 37°C. DNA was extracted from the purified viral particles using the QIAamp MinElute Virus Spin Kit  and amplified with the GenomiPhi V2 DNA Amplification Kit  according to the manufacturer's instructions. The GenomePlex Whole Genome Amplification Kit  was then used to fragment and amplify the viral DNA, which was subsequently cloned into the pCR4 vector using TOPO TA cloning  and sequenced with the M13F forward primer by Beckman Coulter Genomics .Viruses were purified from the whiteflies using a modification of previously described methods The resulting sequences were trimmed for vector and read quality using Sequencher 4.7 . A total of 58 and 158 sequences with >100 nt of good read quality after trimming were obtained from the Citra and Homestead viral metagenomes (viromes), respectively. All metagenomic data was deposited in Genbank under Accession numbers HN153414-HN153629. Metagenomic sequences were analyzed using BLASTn against the Genbank non-redundant database with an E-value cut-off of 1e-5 To further characterize selected metagenomic sequences that likely represented novel virus species or strains, PCR primers were designed to amplify the entire DNA-A, DNA-B, and/or satellite DNA sequences . The resTo characterize the potential host of one of the novel virus sequences identified through the VEM approach, a survey of wild and cultivated plants was conducted at the Citra site in September and November 2009. Young leaves were collected from 203 plants belonging to 11 families and 15 genera and DNA was extracted using the Gentra Purgene Tissue Kit (Qiagen). Degenerate primers were used to screen for the presence of begomoviruses http://tree.bio.ed.ac.uk/software/seal/). Maximum likelihood phylogenetic trees were estimated with PAUP* 4.0 The completely sequenced genome components were aligned with their closest relatives from the GenBank non-redundant database using MUSCLE Table S1Pairwise comparison between the different Whitefly VEM Satellite DNA (WfVEM-Sat).(DOC)Click here for additional data file.Table S2Genes with similarity to the metagenomic sequences with <88% nucleotide identities to known begomoviruses. Sequences range from 100 to 700 nt in length, representing partial genome fragments.(DOC)Click here for additional data file.Table S3Primers used in this study.(DOC)Click here for additional data file.


Shuanglong formula (SLF), a Chinese medicine composed of panax ginseng and salvia miltiorrhiza exhibited significant effect in the treatment of myocardial infarction (MI) in clinical. Because of the complex nature and lack of stringent quality control, it's difficult to explain the action mechanism of SLF.In this study, we present a "system to system" (S2S) mode. Based on this mode, SLF was simplified successively through bioactivity-guided screening to achieve an optimized minimal phytochemical composition (new formula NSLF6) while maintaining its curative effect for MI.Pharmacological test combining with the study of systems biology show that NSLF6 has activity for treatment MI through synergistic therapeutic efficacies between total ginsenosides and total salvianolic acids via promoting cardiac cell regeneration and myocardial angiogenesis, antagonistic myocardial cell oxidative damage.The present S2S mode may be an effective way for the discovery of new composite drugs from traditional medicines. Dr. Zerhouni pointed out in NIH's Roadmap that translational medicine may evoke a great evolution of medicine in 21st century . Since TTCM has not been fully accepted by mainstream medicine whereas it has a long history of clinical practice in China and beyond China. Besides of the complex nature of the formulae, as well as a lack of stringent quality control, the main obstacles of understand TCM may be attributed to its holistic treatment concept representing the interaction of drug system and human system which is quite different with the "P2P" mode of western medicine .During its thousands of years' clinical practice, TCM formulas have being developed according to the routine of "Beside-Bench-Beside" which is also similar with the proposal of Translational Medicine. Many of the TCM formulas have a proven efficacy in clinical application. The pioneering work of Prof. Cheng's group from Yale University proved the efficacy of a TCM formula (PHY906) and interpreted its mechanism by modern pharmacological study , demonstIn the past study we have introduced approaches of Chemomics and systems biology to study the composition of a chemome (e.g. a TCM formula) and the correlation between its change and biological effect ,7. Prof.shuanglong formula (SLF) was presented here. The Chinese medicine SLF, a combination of panax ginseng (PG) and salvia miltiorrhiza (SM) at a ratio of 7:3, has been used for the clinical treatment of cardiovascular diseases such as myocardial infarction (MI) and angina pectoris over ten years by Professor Lianda Li, Xiyuan Hospital of China Academy of Chinese Medical Sciences. Former studies based on MI models of rats, pigs, and dogs showed that SLF alone or combined with mesenchymal cell transplantation could reduce myocardial infarct area and the degree of myocardial injuries, improve cardiovascular function, and increase myocardial blood and myocardial capillary density [Here we present a mode of "system to system" (S2S) by integrating Chemomics and systems biology which is so called Integrative System Biology approach to study the interaction of drug system and biological system . Methods of isoproterenol (ISO)-induced MI rat model and coronary artery ligation-induced MI rat model were the same as before ,13.Method parameters of Metabolomic study of ISO-induced MI rat serum and coronary artery ligation-induced MI rat urine were the same as before ,13.2 at 37°C and the media were replaced at 2-day intervals.Human Umbilical Vein Endothelial Cells (HUVECs) were routinely cultured in Medium RPMI1640 supplemented with 10% fetal bovine serum (FBS), 100 μg/ml penicillin/streptomycin. These cells were cultured in 5% COHUVECs were grown to 70% confluence in 96-well plates and cultured with indicated concentration of GS, TSA and NSLF6. Cell proliferation was both measured using MTT assay and direct counting. Each experiment was carried out with five replicates per treatment and was independently repeated more than three times.4) were placed on the filter in RPMI1640 0.1% bovine serum albumin (BSA) containing indicated concentrations of NSLF. In the lower chamber RPMI1640 supplemented with 5% FBS was added. After incubated 3 h at 37°C, the filter was removed, and the upper side of the filter containing the nonmigrated cells was wiped and rinsed. The filters were fixed with 4% paraformaldehyde and stained with 4',6-diamidino-2-phenylindole (DAPI). Migration was quantified by counting cells. All groups were studied in triplicate.Effect of NSLF on HUVECs migration was studied using BD Chambers with Polycarbonate filters (8.0 μm pore). Suspended cells  and further purified with RNeasy affinity column (MN). First-strand cDNA was synthesized using First strand cDNA synthesis Kit (Fermentas). Primers of Arnt1, Nppa and GAPDH were designed by primer 3.0  were cultured on growth factor reduced-Matrigel (BD Biosciences) coated 24 plates in RPMI1640 0.1% BSA containing 10-40 μg/ml NSLF6 or vehicle. When cultured on matrigel, cells assemble into capillary-like structures. After incubated 18 h, the cord-like structures were observed by an inverted microscope.HUVECs  exhibited appealing therapeutic efficacies on MI .A serum metabolomic study combined with pharmacological tests based on ISO-induced MI rat model was also conducted here to validate the efficacy and optimize the ratio of herbal combination of SLF. As shown in Figure P < 0.05) in CK, LDH, and NEFA levels was observed in rats treated with the TGS + TSA, TGS + TSA + RPG, TGS + TSA + RSM, and TGS + TSA + RPG + RSM  compared with the model group. There were not statistically significant differences (P > 0.05) in the TSA + RPG + RSM, TGS + RPG + RSM, and RPG + RSM groups compared with the model group. The activities of TGS + TSA were almost the same as the TGS + TSA + RPG + RSM. Besides, the results were confirmed by the ECG changes and metabolomic studies  had no significant effect on activities. On the other hand, absence of either/both of ginsenosides and salvianolic acids led to losing of anti-ischemic efficacy. Therefore, we took the components of ginsenosides and salvianolic acids as the indispensable effective components and equivalence contributed to the anti-MI efficacy of SLF. As a result, we obtained a new prescription with simplified compositions and maintaining efficacy, combination of TGS and TSA as the equal ratio of which present in the original formula SLF, named as "NSLF6".1, Rg1, Ro, salvianolic acid B, and lithospermic acid accounted for 74.4% of total solids. Thus, most of the constituents in NSLF6 have been identified and quantifiable so that we have established a stringent quality control, which provided the chemical basis for unveiling the pharmacological mechanism. Additionally, the comparative study of components in plasma of rats before and after administration of NSLF6 as well as NSLF6 was conducted by UPLC/TOF-MS. The results showed that 5 salvianolic acids including salvianolic acid B, rosmarinic acid, propanoid acid, salvianolic E, and lithospermic acid, and 7 ginsenosides concluding ginsenoside Rb1, Rb2, Re, Rd, Rf, M-Rb1, and M-Rb1 from NSLF6 were detected in plasma.The global chromatographic fingerprint of the NSLF6 was established by UPLC/TOF-MS. A total of 20 compounds in NSLF6 were separated and identified, among of which there were 12 ginsenosides and 8 salvianolic acids. The quantitative fingerprints of total ginsenosides and total salvianolic acids were established by HPLC-DAD. As shown in Additional file The activities of NSLF6 were evaluated by metabolomics and pharmacological tests using MI rats induced by coronary artery ligation. As shown in Figure NSLF6 could significantly reduce myocardial infarction area, and one of the mechanisms might be related to promote angiogenesis and antagonize cardiomyocytes oxidative damage.Among NSLF6, TSA and TGS, NSLF6 (20 μg/ml) exhibited the best activity in promoting cells proliferation Figure . The det2O2 was used to study the protective effect of NSLF6 and its effective ingredients. After cardiomyocytes were co-cultured with these drugs for 48 h, 10% H2O2--PBS solution was added to the medium, cultured for 2 h, thereafter high content screening (HCS) system was used to analyze the health degree of cardiomyocytes [2O2  also confirmed that NSLF6 had the protective effect on damaged cardiomyocytes, and such a pharmacological action was mainly contributed by TGS   were confirmed by Real-Time PCR, and obtained the functional relationship  is known as multi-component drug capable of targeting multiple sites through multiple mechanism of action at multiple pathological steps. But these actions represent integral regulation other than the sum of individual target. Therefore, chemomics-integrated systems biology , a holistic methodology, was developed for the study of interaction between external intervention system (TCM) and biological response system (human system) based on S2S mode. The Traditional Chinese Formula is a compatible combination of raw herbs, in which indispensable components co-exist with ineffective components. For further drug development, it is necessary to remove the ineffective components while maintaining its curative effect for a specific disease. Consequently, we developed a S2S mode by which a complex formula can be simplified successively through bioactivity-guided screening to achieve a minimal effective composition with definite constituents and controllable quality, so that its mechanism can also be interpreted. In the present study, TGS and TSA were screened from SLF according to the studies of chemomics, serum pharmacochemistry, metabolomics and activity evaluation. NSLF6, the simplified prescription with components combination of TSA and TGS was obtained, and the systems study on the action mechanism of NSLF6 was conducted.According pharmacological studies based on the MI rat model induced by ISO or the coronary artery ligation, NSLF6 can treat MI via several mechanisms such as promoting cardiomyocytes renewal, promoting angiogenesis and antagonizing cardiomyocytes oxidative damage.in vivo experiments showed that SLF with bone marrow mononuclear cells (BMMCs) auto-transplanted into myocardial infarction model in swines could significantly improve the survival, differentiation and amplification of the transplanted cells in myocardial tissue, generate a raft of new cardiomyocytes and microvascular, and eventually promote lesions recovery, compared with BMMCs group [in vitro study proved that SAB, the effective ingredient of NSLF6, effectively induced the differentiation of mESCs into cardiomyocytes with physiological beating frequency. Except Vc and retinoic acid, there has been few drug reported to have the inducing effect on ESC differentiating into cardiomyocytes. Thus, NSLF6 has a good potential and specific advantage in the treatment of MI. The study of genomic and metabolomic above displayed the NSLF6 activity of regulating energy metabolism, transcription factors and oxide reductase activity, which was reported to be involved in the differentiation of mESCs into cardiomyocytes [It has been reported that there are stem cells existing in the heart of adult . WhetherCs group . The abomyocytes .1, one of ingredients in NSLF6, could stimulate the expression of VEGF to enhance the angiogenesis in previous report [Partial angiogenesis and remodeling after MI is an important part of Cardiac Tissue Repair. Therapeutic angiogenesis is the clinical use of methods to enhance or promote the development of collateral blood vessels in ischemic tissue, and vividly described as "self-medication for heart bypass". The study of genomic and metabolomic above displayed the NSLF6 activity of regulating the energy metabolism, ion binding, MAPK and VEGF pathway which was reported to be the most Specific and powerful regulators to promote angiogenesis . Therefos report .2O2 oxidative damage model of myocardial cells was widely used in the study of ischemia/reperfusion injury and ischemic preconditioning. It presents as the destruction of nuclear integrity and membrane permeability, increased mitochondrial membrane potential, oxygen free radicals, myocardial damage induced increase of enzyme activity, cell apoptosis, calcium overload and so on [The Hnd so on . The actnd so on , SOD [27nd so on  were cloBased on the integrative system biology mode, the traditional Chinese medicine SLF, as a clinical effective medicine, was developed to a new formula--NSLF6 with comparatively clear phytochemical composition, comparatively clear mechanism, and controllable quality. The stringent quality control system has been established using combination of fingerprint and multi-component determination. NSLF6 was proved to be effective on MI by pharmacological test  combining with the study of gene, protein, and metabolite networks. The results revealed that SLF produced efficacy against MI through synergistic therapeutic efficacies between TGS and TSA. The present S2S mode may be an effective way for the discovery of new compound medicines.Shuanglong formula; MI: myocardial infarction; P2P: point to point; P2S: point to system; S2S: system to system; TCM: Traditional Chinese Medicine; PG: panax ginseng; SM: salvia miltiorrhiza; ECG: electrocardiogram; TGS: total ginsenosides; RPG: the remainder of PG; RSM: the remainder of SM; HUVECs; Human Umbilical Vein Endothelial cells; HCS: high content screening; H/R: Hypoxia/Reoxygenation; TCCM: Traditional Chinese Compound Medicine; BMMCs: bone marrow mononuclear cells; ISO: isoproterenol; LDH: lactate dehydrogenase; CK: creatines kinases; NEFA: nonesterified fatty acid; MDA: malondialdehyde.SLF: The authors declare that they have no competing interests.Q-LL, X-PL, and H-YZ performed the experiments (metabonomics). Y-YX, Y-JC and JW performed the experiments (TCM research). Q-BX, Q-ZX, XL, S-FL and X-MF performed the experiments (genomics & proteomics). XC, RG, and R-LZ performed the experiments . Q-LL, X-PL, Y-YX, S-FL, X-MF and Q-LZ participated in writing. G-AL designed and guided the whole experiments and wrote the paper. All authors read and approved the final manuscript.Supplementary materials.Click here for file


The objective of this study was to assess whether carboxymethyl cellulose- (CMC-) based hydrogel containing BioC ; tricalcium phosphate (TCP) : hydroxyapatite (Hap) = 70 : 30) and bone morphogenic protein-2 (BMP-2) led to greater bone formation than CMC-based hydrogel containing BioC without BMP-2. In order to demonstrate bone formation at 4 and 8 weeks, plain radiographs, microcomputed tomography (micro-CT) evaluation, and histological studies were performed after implantation of all hybrid materials on an 8 mm defect of the right tibia in rats. The plain radiographs and micro-CT analyses revealed that CMC/BioC/BMP-2 (0.5 mg) led to much greater mineralization at 4 and 8 weeks than did CMC/BioC or CMC/Bio/BMP-2 (0.1 mg). Likewise, bone formation and bone remodeling studies revealed that CMC/BioC/BMP-2 (0.5 mg) led to a significantly greater amount of bone formation and bone remodeling at 4 and 8 weeks than did CMC/BioC or CMC/BioC/BMP-2 (0.1 mg). Histological studies revealed that mineralized bone tissue was present around the whole circumference of the defect site with CMC/BioC/BMP-2 (0.5 mg) but not with CMC/BioC or CMC/BioC/BMP-2 (0.1 mg) at 4 and 8 weeks. These results suggest that CMC/BioC/BMP-2 hybrid materials induced greater bone formation than CMC/BioC hybrid materials. Thus, CMC/BioC/BMP-2 hybrid materials may be used as an injectable substrate to regenerate bone defects. More than a million people in the United States and other countries require bone graft to regenerate bone defects caused by fracture, trauma, or tumor resection , 2. AutoHydroxyapatite (HAp) and calcium phosphate (CaP) have been successfully used as bone graft materials for the regeneration of bone defects due to their physical and chemical properties and their structural similarity to natural bone. CaP-based materials have been approved by the Food and Drug Administration (FDA) for clinical applications in the fields of dental and orthopedic surgery. CaP-based materials possess bioactive, biocompatible, and osteoconductive properties . Althoug in vitro [BMPs have osteoinductive properties. Among BMP family members, BMP-2 is one of the most osteoinductive growth factors. Previous studies have demonstrated that BMP-2 can induce mesenchymal stem cells (MSCs) to differentiate into osteoblast lineages and regenerate bone –13. In ain vitro , 15. Recin vitro –21. in vitro study, Leone et al. demonstrated that amidated carboxymethyl cellulose (CMCA) hydrogel is a potential filler for cartilage defects [Carboxymethyl cellulose (CMC) has natural biodegradable and biocompatible properties , 23 and  defects . Moreove defects .On the basis of these results, we hypothesized that a CMC-based hydrogel containing BioC ; tricalcium phosphate (TCP): hydroxyapatite (Hap) = 70 : 30) and BMP-2 would promote greater bone formation in a rat tibial defect model than would a CMC-based hydrogel containing BioC without BMP-2.The cellulose material (CMC-based hydrogel containing BioC) has been integrated with two different BMP-2 solutions  with a simple soaking manner. Released BMP-2 from CMC/BioC/BMP-2 (0.1 mg) and CMC/Bio-C/BMP-2 (0.5 mg) hybrid materials was evaluated with an enzyme-linked immunosorbent assay (ELISA) in accordance with the manufacturer's instructions by using a microplate reader  at a wavelength of 450 nm. In brief, CMC/BioC/BMP-2 (0.1 mg) and CMC/BioC/BMP-2 (0.5 mg) blended materials were, respectively, soaked in a membrane bag , 15 mL tube with 1 mL of phosphate buffer saline (PBS) . The tube was incubated at 37°C with gentle shaking at 100 rpm. At predetermined time intervals of 1, 3, 5, and 10 hr and 1, 3, 5, 7, 14, 21, and 28 days, supernatants were collected and replaced with fresh PBS for 28 days. The absorbance of the collected samples was determined with a microplate reader. in vivo evaluation of CMC/BioC, CMC/BioC/BMP-2 (0.1 mg), and CMC/BioC/BMP-2 (0.5 mg) groups. The experimental protocol was approved by the Institutional Animal Care and Use Committee of the Korea University Medical Center (KUIACUC-2012-128). Experimental animals were divided into three groups: group I (n = 4) was implanted with CMC/BioC, group II (n = 4) was implanted with CMC/BioC/BMP-2 (0.1 mg), and group III (n = 4) was implanted with CMC/BioC/BMP-2 (0.5 mg). The rats were anesthetized with tiletamine/zolazepam  and xylazine . After shaving the right tibia, the periosteum and soft tissue were carefully retracted and two 0.9 mm K-wires  were fixed to the right tibia. K-wires were clamped bilaterally with the author's own-designed external fixator . An 8 mm defect of the right tibia was created with a cutting burr, and 150 μL of CMC/BioC, CMC/BioC/BMP-2 (0.1 mg), or CMC/BioC/BMP-2 (0.5 mg) was injected into the right tibia defect, as appropriate, respectively. The subcutaneous tissue and skin were sutured with absorbable 4-0 vicryl   were used for theNJ, USA) . The ratPlain Radiographs. At 4 and 8 weeks after injection, samples were fixed in 3.7% paraformaldehyde solution. Radiographs of the specimens were obtained with a plain radiograph apparatus  at 43 KVP, 2 mA, and 44 cm film-radiation beam distance for a 1.5 s exposure time.Microcomputed Tomography (Micro-CT) Evaluation. At predetermined time intervals of 4 and 8 weeks, bone volume was obtained with a micro-CT system . The CT system was operated at a voltage of 40 kV, and a current of 250 μA was used with a nominal resolution of 9 μm/pixel. Image analysis was performed with a bone analyzer .Histological Study. The specimens were retrieved at 4 and 8 weeks. After decalcification, the samples were embedded in paraffin. The tissues were cross-sectioned at an 8 μm thickness in the longitudinal parallel direction and stained with hematoxylin and eosin (H&E) and Masson's trichrome staining. The cytoplasm of osteoblasts and bone formation were assessed with H&E staining. Mineralized bone matrix and osteoid were evaluated by Masson's trichrome staining.P < 0.05 and **P < 0.001.Data are presented as mean ± standard deviation. Statistical comparisons were carried out via one-way analysis of variance using Systat software . Differences were considered statistically significant at *On the first day, the released amounts of BMP-2 were 71.55 ± 2.24 ng with CMC/BioC/BMP-2 (0.1 mg) and 83.75 ± 1.12 ng with CMC/Bio-C/BMP-2 (0.5 mg) . At 28 dP < 0.001). A significant difference in bone formation was observed between the CMC/BioC/BMP-2 group (0.5 mg) and CMC/BioC group (**P < 0.001) and between the CMC/BioC/BMP-2 group (0.5 mg) and the CMC/BioC/BMP-2 (0.1 mg) group (*P < 0.05) at 8 weeks. At 4 weeks, there was a statistically significant difference in bone remodeling of the defect area between the CMC/BioC/BMP-2 group (0.5 mg) and the CMC/BioC group (*P < 0.05), as well as between the CMC/BioC/BMP-2 group (0.5 mg) and the CMC/BioC/BMP-2 (0.1 mg) group (*P < 0.05) (P < 0.001) or the CMC/BioC/BMP-2 (0.1 mg) group at 8 weeks (*P < 0.05).At 4 weeks after surgery, the plain radiographs revealed no mineralization at the defect area for the CMC/BioC group but slight mineralization for the CMC/BioC/BMP-2 (0.1 mg) and CMC/BioC/BMP-2 (0.5 mg) groups . At 8 we < 0.05) . In addiThe H&E staining analysis revealed that the tissue of the defect area showed negligible mineralized bone tissue for the CMC/BioC group and partially mineralized bone tissue for the CMC/BioC/BMP-2 (0.1 mg) group. However, the tissue of the defect area showed a much greater amount of mineralized bone tissue for the CMC/BioC/BMP-2 (0.5 mg) group than for the CMC/BioC or CMC/BioC/BMP-2 (0.1 mg) group at 4 weeks Figures . In all Osteoconductive materials, such as CaP-based materials, allow a framework for vascular invasion and cellular infiltration but do not induce mesenchymal cells to differentiate into mature bone cells. Osteoinductive materials, including growth factors such as BMPs, basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), and vascular endothelial growth factor (VEGF), induce new bone formation but they do not provide a framework for vascular invasion and cellular infiltration. Thus, osteoconductive materials combined with osteoinductive materials may be ideal for bone regeneration.The objective of this study was to assess whether a CMC-based hydrogel containing BioC and BMP-2 induced a greater amount of new bone formation than did a CMC-based hydrogel containing BioC without BMP-2. The results from the plain radiographs and micro-CT demonstrated that bone formation was significantly greater in the CMC/BioC/BMP-2 (0.5 mg) group than in the CMC/BioC or CMC/BioC/BMP-2 (0.1 mg) group at 4 and 8 weeks. Thus, these findings demonstrate that CMC/BioC/BMP-2 (0.5 mg) induces rapid bone formation at an earlier stage than does CMC/BioC or CMC/BioC/BMP-2 (0.1 mg). As expected from the plain radiographs and micro-CT data, the histological study data revealed that the CMC/BioC/BMP-2 (0.5 mg) group had more mineralized bone tissue at the defect site than did the CMC/BioC and CMC/BioC/BMP-2 (0.1 mg) groups at 4 weeks. In all groups, a greater amount of mineralized bone tissue was observed at the defect site at 8 weeks than at 4 weeks. The entire defect area was covered with mineralized bone tissue at 8 weeks in the CMC/BioC/BMP-2 (0.5 mg) group but not in the CMC/BioC or CMC/BioC/BMP-2 (0.1 mg) group. in vitro study and greater ectopic bone formation in an in vivo study than the control group. In a previous study, we found that woven bone covered the whole circumference more often in a BMP-2-coated tricalcium phosphate/hydroxyapatite group than in a tricalcium phosphate/hydroxyapatite group in a rat model of femoral distraction osteogenesis [These results are consistent with those of previous studies. Lin et al.  reportedogenesis , 31. Kimogenesis  and Parkogenesis  reportedogenesis  showed togenesis . in vivo study has some limitations. For example, long-term evaluation greater than 8 weeks is needed to compare the quality and architecture of new bone formation between control and implanted groups. More comprehensive analyses that include the histology and angiogenesis of new bone and biomechanical testing are needed. Finally, the effects of long-term release of BMP-2 require further investigation.Our results suggest that CMC/BioC/BMP-2 hybrid materials induce greater bone formation at an earlier stage through release of BMP-2 than CMC/BioC hybrid materials. However, ourIn conclusion, CMC/BioC/BMP-2 (0.5 mg) hybrid materials implanted in a rat tibial defect model led to greater bone formation than did CMC/BioC and CMC/BioC/BMP-2 (0.1 mg) hybrid materials. Thus, CMC/BioC/BMP-2 hybrid materials may be useful in an injectable substrate for the clinical application of the regeneration of bone defects in the orthopedic field.


The scientific literature contains millions of microbial gene identifiers within the full text and tables, but these annotations rarely get incorporated into public sequence databases. We propose to utilize the Open Access (OA) subset of PubMed Central (PMC) as a gene annotation database and have developed an R package called pmcXML to automatically mine and extract locus tags from full text, tables and supplements.Burkholderia pseudomallei K96243 for comparison. There were 168 publications containing 48,470 locus tags and 83% of mentions were from supplementary materials and 9% from publications outside the OA subset.We mined locus tags from 1835 OA publications in ten microbial genomes and extracted tags mentioned in 30,891 sentences in main text and 20,489 rows in tables. We identified locus tag pairs marking the start and end of a region such as an operon or genomic island and expanded these ranges to add another 13,043 tags. We also searched for locus tags in supplementary tables and publications outside the OA subset in B. pseudomallei locus tags within the full text and tables of OA publications represent only a small fraction of the total mentions in the literature. For microbial genomes with very few functionally characterized proteins, the locus tags mentioned in supplementary tables and within ranges like genomic islands contain the majority of locus tags. Significantly, the functions in the R package provide access to additional resources in the OA subset that are not currently indexed or returned by searching PMC. The rapid growth of next generation sequencing and transcriptomic studies, particularly on the causative agents of infectious diseases, requires accurate genome annotations to confidently analyze the sequencing data and identify and compare functions, pathways and networks. There are many resources available for genome annotation and most rely on transferring annotations from model organism or protein family databases that vary greatly in content and quality . For michttp://text.soe.ucsc.edu). A related project called pubmed2ensembl links millions of articles to thousands of genes from 50 eukaryotic species using six data sources containing gene to literature links  returns 47 results).Searching for all locus tags in PMC requires other steps that are outlined in the flowchart in Figure Yersinia pestis CO92: (YPO0* OR YPO1* OR YPO2* OR YPO3* OR YPO4*) AND (Yersinia [ABSTRACT] OR Yersinia[TITLE]) AND open access[FILTER]. In some cases, the results returned a warning that the wildcard search used only the first 600 variations and therefore we lengthened the root word to include two digits.We used the locus tag prefix and first digit from the GFF3 file to build wildcard searches and find PMC articles with a matching locus tag  and optional suffixes. We then expanded locus tag pairs marking the start and end of a region such as an operon or genomic island using the ordered list of tags in the GFF3 file. We saved the PMC id, locus tag, section title or table caption, full sentence or table row, and a flag indicating if the tag was mentioned indirectly within a range. We manually checked all ranges with ten or more locus tags to ensure valid range expansions. Finally, we searched for additional locus tags in B. pseudomallei from supplementary tables and from full text articles outside the OA subset.For each publication, we passed the PMC id to the Open Archives Initiative service and downloaded the XML version of the full text article. We parsed the XML into text by splitting the document into main sections, and each section was further divided into complete sentences. We parsed XML tables using rowspan and colspan attributes to correctly position and repeat cell values and then joined column names and cell values into a single delimited list to preserve the table structure in a single row. We extracted tags from both main text and tables by matching a prefix followed by four digits (or three digits in https://github.com/cstubben/pmcXML) for further community development.A complete description of the R functions listed in Figure There are over 2.88 million articles in PMC and 681,814 articles (23.6%) are included in the Open Access subset . The number of OA publications is increasing rapidly each year , while the number of tags mentioned indirectly within other genomes included 5% from Francisella, 8% from Helicobacter, 14% from Chlamydia and Listeria, and 20-23% from the other five genomes.We identified 30,891 locus tags in main text and 20,489 tags in tables  and over half the tags were from two tables listing type VI secretion system homologs in B. pseudomallei. We also identified seven PMC articles not found in the search results, including five with tags in supplementary tables only. For example, the study by Schell et al. [In order to better estimate the fraction of locus tags indexed by OA publications, we also checked supplementary tables and other publications in l et al.  lists 65The 63 supplementary tables contained the majority of all locus tags (83%) and included 40,122 total mentions from 30 publications. The supplements included 21 Word tables, 19 PDF tables, 14 Excel files, four HTML tables, three zipped files and two GenBank files  and includes a known structure in the Protein Data Bank (PDB) [Burkholderia Oca-like adhesin proteins or BoaB and BoaA [Burkholderia pseudomallei adhesion A protein or BpaA [B. pseudomallei[There were 22 ns Table . Seven onk (PDB) . BPSL170 or BpaA . It was udomallei. BPSS138udomallei. BPSS149udomallei,22 with Since the number of OA publications is increasing rapidly Figure , tools tB. pseudomallei, over half of RefSeq genes have alternate starts in the Genemark, Glimmer or Prodigal predictions available in the same genomes FTP directory at NCBI. Therefore, finding verified start coordinates in the primary literature based on either experimental or computational evidence would be very useful. Prodigal has been recommended for Burkholderia genomes due to their high GC content, and Dunbar et al. [B. pseudomallei are from strain 1710b. Since many of these protein sequences have 100% similarity to the corresponding protein sequences of K96243, locus tags in closely related strains would be another valuable resource to improve annotations. In addition, the B. pseudomallei genome reannotation by Nandi et al. [Structural annotations include revised gene starts, novel genes, doubtful coding regions and mobile regions. In r et al.  includesi et al.  includedOther useful sources of structural annotations in the primary literature include novel genes and doubtful coding regions. The reannotation by Nandi et al. identified 283 novel genes and 120 doubtful CDSs in the supplementary tables . One of Burkholderia intracellular motility A protein (BimA) from Stevens et al. in 2005 [Burkholderia; however, the gene name bimA is not included in any public sequence database for strain K96243 including NCBI, UniProt and Ensembl. This gene name is also missing from annotations provided by IMG [Burkholderia annotations were available from the now defunct Pathema database at JCVI [Functional annotations include gene names, definitions and less often terms from controlled vocabularies describing functions and other characteristics. Protein definitions and gene names are critical for comparative analyses since they are the most commonly used source of information transfer . Clearly in 2005 . There ad by IMG , and sped by IMG  or Burkhd by IMG  as well d by IMG . In 2004d by IMG . As part at JCVI  and BimAfliC, bsaQ, and rpoS correspond to BPSL3319, BPSS154, and BPSL1505 respectively in the RefSeq GFF3 file; therefore, extracting gene names from OA publications will definitely improve microbial genome annotations. In an effort to obtain the most accurate annotations for B. pseudomallei genomes, we are continuing to develop R scripts to extract these gene names.In this study, we focused only on locus tags. However, there are many other gene identifiers that should be extracted. In fact, many tables and text sources list gene names by default, and only use a locus tag if a gene name was not assigned by RefSeq or other annotation source. For example, this sentence in Bartpho et al.  is typicB. pseudomallei in particular, we believe that freely available supplementary materials and locus tags mentioned indirectly within ranges are important sources for acquiring gene annotations. Other sources including gene names, accession numbers and coordinates should also be collected before proceeding with future efforts to summarize functions, interactions and pathways.There are many challenges in extracting gene identifiers from the literature, and some groups like the UCSC Genocoding project are actively trying to mine articles outside the OA subset to expand access to human gene and sequence mentions . At leasB. pseudomallei locus tags are mentioned directly in the main text and tables of the OA subset and are indexed and available for searching in PMC. This represents 3% of the total number of B. pseudomallei locus tags mentioned in the literature, since most locus tags are available in supplementary tables or within ranges. Both of these are valuable annotation sources and we developed queries and tools in the pmcXML package to improve access to these data sources.Only 1514 Due to the rapid growth of OA submissions, extracting gene and locus tags from the literature would clearly benefit efforts to improve microbial genome annotation. The next challenge will involve developing the data mining algorithms needed to automatically summarize the gene mentions to identify names and functions of experimentally characterized proteins such as virulence factors and antibiotic resistance genes directly from the literature database. If successful, this would help to convert a full text database into a functional gene annotation database first envisioned by Bourne , and wouProject name: pmcXMLProject home page:https://github.com/cstubben/pmcXMLOperating system(s): Platform independent, however loading supplementary tables requires a number of Unix dependencies  to read zip, Word tables and pdf filesProgramming language: ROther requirements: Package dependencies include stringr and gdata from CRAN, genomes from Bioconductor and genomes2 from GitHubLicense: GPLAny restrictions to use by non-academics: noneThe authors declare that no competing interests exist.B. pseudomallei genomes and edited the manuscript. Both authors read and approved the final manuscript.CJS wrote the R code to mine locus tags and wrote the manuscript. JFC defined the data mining task applied to Summary of NCBI searches in PubMed and PMC using locus tag prefixes and a summary of Gene-PubMed links in the Entrez Gene database.Click here for fileA description of R functions in the pmcXML package and R code used to parse locus tags.Click here for fileA list of 1446 publications from ten reference genomes and 64,423 locus tag mentions in the PMC Open Access subset.Click here for fileLocus tag pairs detected as ranges that should not be expanded.Click here for fileBurkholderia pseudomallei K96243 and 48,470 locus tags mentions in the scientific literature. The publications include counts of locus tags in full text, tables or supplements.A list of 168 publications from Click here for fileBurkholderia pseudomallei with counts of locus tags mentions, file types and formatting notes.A list of 63 supplementary tables from Click here for file


Scylla paramamosain). We then tested the association between these microsatellite markers and the growth performance of S. paramamosain. A total of 129 polymorphic microsatellite markers were identified, with an observed heterozygosity ranging from 0.19 to 1.00 per locus, an expected heterozygosity ranging from 0.23 to 0.96 per locus, and a polymorphism information content (PIC) ranging from 0.21 to 0.95 per locus. Of these microsatellite markers, 30 showed polymorphism in 96 full-sib individuals of a first generation family. Statistical analysis indicated that three microsatellite markers were significantly associated with 12 growth traits of S. paramamosain. Of these three markers, locus Scpa36 was significantly associated with eight growth traits, namely, carapace length, abdomen width (AW), body height (BH), fixed finger length of the claw, fixed finger width of the claw, fixed finger height of the claw, meropodite length of pereiopod 2, and meropodite length of pereiopod 3 (MLP3) (P<0.05). Locus Scpa75 was significantly associated with five growth traits, namely, internal carapace width, AW, carapace width at spine 8, distance between lateral spine 2 (DLS2), and MLP3 (P<0.05). Locus Spm30 was significantly associated with BH, DLS2, and body weight (P<0.05). Further analysis suggested a set of genotypes  that have great potential in the selection of S. paramamosain for growth traits. These findings will facilitate the development of population conservation genetics and molecular marker-assisted selective breeding of S. paramamosain and other closely related species.Microsatellite markers from a transcriptome sequence library were initially isolated, and their genetic variation was characterized in a wild population of the mud crab ( Scylla paramamosain) is a highly commercially valuable species, mainly distributed along the southeastern coasts of China and other Asian countries such as Japan, Vietnam, and the Philippines. S. paramamosain is an important aquaculture and capture marine species in China. Records of S. paramamosain aquaculture date back more than 100 years in China S. paramamosain mate inshore and the gravid females generally migrate offshore to spawn eggs S. paramamosain has received increasing attention over the years and has been cultured by fishermen because of its wonderful flavor and fast growth rate. The aquaculture production in China reached 110,000 tons in 2011 The mud crab  Macrobrachium rosenbergii) are significantly associated with three growth traits  Paralichthys olivaceus) has been developed using microsatellite-assisted selection Microsatellites are nuclear genetic markers that are considered an ideal molecular marker system for investigating genetic diversity S. paramamosain and investigated the correlation of different growth traits S. paramamosain and other closely related species.We recently constructed first generation families of All animal experiments in this study were conducted according to relevant national and international guidelines. This project was approved by East China Sea Fisheries Research Institute. In China, catching wild mud crab from seawater does not require specific permits. This study does not involve endangered or protected species.S. paramamosain was collected from the coastal waters along Wenchang City, China in September 2011. This population was used to evaluate the polymorphism of transcriptome-derived microsatellite markers. A first generation (G1) family of S. paramamosain was bred in June 2012 and cultured on Hainan Island, China. The G1 crabs were all cultured in the same pond to maintain them under the same environmental condition. A total of 96 full-sib individuals approximately three months of age were randomly collected from the G1 family in September 2012. The average weight of these individuals is 82.47 g.A wild population of 32 Sixteen growth traits of the 96 full-sib individuals were measured according to Keenan et al. Genomic DNA was extracted from the muscle tissues of 32 wild and 96 full-sib individuals following the traditional proteinase K and phenol–chloroform extraction method as described by Ma et al. S. paramamosain from four tissues  of 12 individuals (the manuscript is being prepared). A total of 540 Mbp of data was produced, and 78,268 unigenes were obtained through sequence similarity with known proteins (E ≤0.00001) in the UniProt and non-redundant (NR) protein databases. A total of 19,011 microsatellites were identified from unigenes using the MISA software under default settings. In the present study, we designed primers for the microsatellite sequences based on the following criteria: a minimum of eight repeats for dinucleotide, trinucleotide, and tetranucleotide repeats and sufficient flanking regions using the software Primer Premier 5.0.In our previous study, we carried out 454 high-throughput pyrosequencing on a mixed cDNA library of 2, 0.4 units of Taq polymerase , and approximately 50 ng of DNA. The following conditions were used for the PCR: 1 cycle of denaturation at 94°C for 4 min and 30 cycles of 30 s at 94°C, 50 s at a primer-specific annealing temperature .We used a single population of 32 wild individuals to evaluate the polymorphism of the microsatellite markers derived from the transcriptome. Polymerase chain reaction (PCR) was performed using a 12.5 µl total volume that contained 0.4 µM each primer, 0.2 mM each dNTP, 1× PCR buffer, 1.5 mM MgClperature , and 50 1 family, with 30 loci exhibiting polymorphism. We tested the association between the 30 microsatellite loci and 16 growth traits . The PCRs and electrophoresis were conducted as described above.All 129 polymorphic microsatellite markers developed from the transcriptome were genotyped in 96 individuals of the GNa), effective number of alleles (Ne), observed heterozygosity (HO), expected heterozygosity (HE), polymorphism information content (PIC), Chi-square tests for Hardy–Weinberg equilibrium (HWE), and linkage disequilibrium (LD). Significance values for all multiple tests were corrected through sequential Bonferroni procedure The genetic diversity indices of the microsatellites were calculated using the software POPGENE version 1.31 ijk = μ+Gi+Sj+eijk, where Yijk is the observed value of the ijkth trait; μ is the mean value of the trait; Gi is the effect of the ith genotype; Sj is the effect of the jth sex; and eijk is the random error effect. All G1 individuals used in the association analysis were derived from the same family, cultured under the same conditions, and collected at the same age, so other effects such as batch, generation, family, age, and site were not considered in the statistical model. Significant differences in growth traits among the different genotypes were calculated through multiple comparison analysis using the S-N-K method. Differences with P values of 0.05 were considered statistically significant.The associations between microsatellite markers and growth traits  were tested using the General Linear Model (GLM) procedure in the software SPSS version 19. A linear animal model with the fixed effects was used as follows: YP<0.00039), and all loci exhibited no evidence of stuttering and allelic dropout. Furthermore, no significant LD was found in all pairs of loci.A total of 563 pairs of primers were successfully designed based on the transcriptome-derived microsatellite sequences. Of these primer pairs, 129 showed polymorphism in a wild population of 32 individuals , whereas1 family . At this locus (P<0.05). The individuals with genotype BC showed a significantly faster growth rate than those with genotypes AB and BB in terms of BH (P<0.05). The individuals with genotypes BC and AC grew significantly faster than AB individuals in terms of FFWC (P<0.05). However, the individuals with genotype BC grew significantly faster than those with AB and BB in terms of MLP3 (P<0.05). Meanwhile, the individuals with genotype AC grew significantly faster than those with AB in terms of MLP3 (P<0.05).Of the 30 polymorphic microsatellite loci in the Gis locus , the indP<0.05). At this locus (P<0.05). By contrast, no significant differences in phenotypic values were detected between genotype pairs in terms of ICW, AW, CWS8, and MLP3 (P>0.05) at this locus.Locus Scpa75 was significantly associated with traits ICW, AW, CWS8, DLS2, and MLP3 . At this locus (P<0.05). The individuals with genotypes AC, BD, and AB grew significantly faster than those with genotype CD in terms of BW (P<0.05).Locus Spm30 was significantly associated with BH, DLS2, and BW .No microsatellite marker was significantly associated with CW, CFW, DLS1, and MLP1  of the individuals with genotype BC (N = 21) was much higher than that (24.92 mm) of the individuals with the other three genotypes (N = 63). At locus Scpa75, the average AW (26.85 mm) of the individuals with genotype BD (N = 24) was much higher than that (24.86 mm) of the individuals with the other three genotypes (N = 63). Therefore, genotype BC at locus Scpa36 has greater potential in selecting for AW than BD at locus Scpa75.Trait BH was significantly associated with loci Scpa36 and Spm30. At locus Scpa36, the average BH (32.21 mm) of the individuals with genotype BC (N = 21) was much higher than that (29.43 mm) of the individuals with the other three genotypes (N = 63). At locus Spm30, the average BH (30.96 mm) of the individuals with genotypes AC and BD (N = 54) was much higher than that (28.59 mm) of the individuals with the other two genotypes (N = 42). Therefore, genotype BC at locus Scpa36 would have a more important role in selecting for BH than AC and BD at locus Spm30.Trait DLS2 was significantly associated with loci Scpa75 and Spm30. At locus Scpa75, the average DLS2 (48.69 mm) of the individuals with genotype BC (N = 14) was much higher than that (45.43 mm) of the individuals with the other three genotypes (N = 73). At locus Spm30, the average DLS2 (47.38 mm) of the individuals with genotypes AC and BD (N = 54) was much higher than that (43.64 mm) of the individuals with the other two genotypes (N = 42). This result shows that genotype BC at locus Scpa75 is better in selecting for DLS2 than AC and BD at locus Spm30.MLP3 was significantly associated with loci Scpa36 and Scpa75. At locus Scpa36, the average MLP3 (25.75 mm) of the individuals with genotype BC (N = 21) was much higher than that (23.29 mm) of the individuals with the other three genotypes (N = 63). At locus Scpa75, the average MLP3 (25.51 mm) of the individuals with genotype BC (N = 14) was much higher than that (23.64 mm) of the individuals with the other three genotypes (N = 73). Therefore, genotype BC at locus Scpa36 is more useful in selecting for MLP3 than BC at locus Scpa75.CL, FFLC, FFWC, FFHC, MLP2, ICW, CWS8, and BW were significantly associated with one microsatellite locus. CL, FFLC, FFWC, FFHC, and MLP2 were significantly associated with locus Scpa36. At this locus, the phenotypic values of these five growth traits  of the individuals with genotype BC were much higher than those of the individuals with the other three genotypes . Therefore, genotype BC at microsatellite locus Scpa36 is useful in selecting for these five growth traits. ICW and CWS8 were significantly associated with locus Scpa75. At this locus, the phenotypic values of the two growth traits of the individuals with genotypes BC and BD  were much higher than those of the individuals with the other two genotypes . Therefore, these two genotypes (BC and BD) at locus Scpa75 have great potential in selecting for ICW and CWS8. BW was significantly associated with locus Spm30, and its phenotypic value (92.99 mm) of the individuals with genotype AC was much higher than that (78.74 mm) of the individuals with the other three genotypes. Therefore, genotype AC at locus Spm30 could be useful in selecting for BW in breeding programs.S. paramamosain) have been reported, but most of them are randomly derived from genomic DNA S. paramamosain, which have several advantages compared with DNA-derived microsatellites, such as their correlation with potential known genes, better transferability among different species, and better suitability for comparative mapping and genomic studies Next-generation sequencing has recently been used to discover microsatellite markers and SNPs in aquaculture species Na of 7.00 and HO of 0.68) are nearly equal to those isolated from genes (average Na of 5.90 and HO of 0.67) Na of 6.80 and HO of 0.76) Crassostrea gigas) Cyprinus carpio L.) Type I microsatellite markers are predicted to be relatively less polymorphic than those derived from genomic DNA 1 family, whereas the others were monomorphic. A total of 78 effective alleles were detected at 30 loci, which is nearly equal to the observed number of alleles (85). This finding indicates that the alleles in the G1 family are uniform distributed, and the variation of microsatellite loci is not significantly affected by selection pressure HO of 0.74 and average PIC of 0.49), which is similar to that found in other G1 families in a previous study (HO ranging from 0.46 to 0.85 and PIC ranging from 0.40 to 0.77) HO of 0.76 and PIC of 0.67) Of the 129 microsatellite loci, 30 showed polymorphism in the GS. paramamosain, and tried to identify potential markers for molecular MAS. Among the 30 microsatellite markers used in association analysis, Scpa36, Scpa75, and Spm30 were confirmed to be significantly associated with 12 of the 16 growth traits of S. paramamosain. All three loci showed high polymorphism in the G1 family, with the observed number of alleles ranging from 3 to 4 per locus and the PIC ranging from 0.56 to 0.70 per locus. The loci with low polymorphism  were not associated with any growth traits (P>0.05). This phenomenon indicates that the lowly polymorphic microsatellite loci have less advantage in association analysis than the highly polymorphic loci. A similar trend was found in other aquatic animals such as GIFT , the common carp (Cyprinus carpio L.), and the Japanese scallop (Patinopecten yessoensis) Scientists have identified a set of microsatellite markers that were significantly associated with growth traits and other economically important traits in aquatic animals Cynoglossus semilaevis) through the mapping of high-density genetic maps Scophthalmus maximus) S. paramamosain, even though we did not conduct a linkage analysis. The linkage between these markers and QTLs should be confirmed and their genetic distances should be determined through mapping on genetic linkage maps in subsequent studies.If a microsatellite marker is closely linked to a phenotypic trait, it would be detected in terms of a significant association according to the theory of linkage disequilibrium S. paramamosain. Similar cases have also been found in other animals, such as the largemouth bass  P. trituberculatus) S. paramamosain for growth performance.The growth traits AW, BH, DLS2, and MLP3 were significantly associated with two microsatellite loci, and eight traits  were significantly associated with one microsatellite locus. Meanwhile, one microsatellite marker was significantly associated with several different growth traits, and different markers were simultaneously and significantly associated with one trait. This phenomenon indicates that one locus contributes to multiple growth traits and multiple loci influenced the same growth trait of Scylla paramamosain). These findings are helpful in investigations on the population conservation genetics, construction of genetic maps, and molecular MAS of S. paramamosain and other closely related species.We initially isolated 129 transcriptome-derived polymorphic microsatellite markers. We then identified three markers that were significantly associated with 12 phenotypic growth traits of the mud crab (Table S1S. paramamosain.Characterization of 129 polymorphic microsatellite markers derived from a transcriptome sequence library in (DOCX)Click here for additional data file.