Bacillus cereus sensu lato. Here, for the first time, we reported that psychrotolerant Bacillus weihenstephanensis from Northeastern Poland can produce melanin-like pigment. We assessed physicochemical properties of the pigment and the mechanism of its synthesis in relation to B. weihenstephanensis genotypic and phenotypic characteristics. Electron paramagnetic resonance (EPR) spectroscopy displayed a stable free radical signal of the pigment from environmental isolates which are consistent with the commercial melanin. Fourier transform infrared spectroscopy (FT-IR) and physicochemical tests indicated the phenolic character of the pigment. Several biochemical tests showed that melanin-like pigment synthesis by B. weihenstephanensis was associated with laccase activity. The presence of the gene encoding laccase was confirmed by the next generation whole genome sequencing of one B. weihenstephanensis strain. Biochemical (API 20E and 50CHB tests) and genetic  characterization of the isolates revealed their close relation to the psychrotrophic B. weihenstephanensis DSMZ 11821 reference strain. The ability to synthesize melanin-like pigment by soil B. weihenstephanensis isolates and their psychrotrophic character seemed to be a local adaptation to a specific niche. Detailed genetic and biochemical analyses of melanin-positive environmental B. weihenstephanensis strains shed some light on the evolution and ecological adaptation of these bacteria. Moreover, our study raised new biotechnological possibilities for the use of water-soluble melanin-like pigment naturally produced by B. weihenstephanensis as an alternative to commercial non-soluble pigment.Although melanin is known for protecting living organisms from harmful physical and chemical factors, its synthesis is rarely observed among endospore-forming  Bacillus thuringiensis-based biopesticides for UV protection [Cryptococcus neoformans, the conversion of L-DOPA to DOPA-quinone occurs with the participation of laccase [Aspergillus fumigatus, Legionella pneumophila, Pseudomonas aeruginosa or Vibrio cholerae [Melanin is a heterogenous and polymeric pigment found in many Prokaryote and Eukaryote organisms. Melanin production has been considered to be of a great significance, especially with regard to microorganisms in which it has been often associated with virulence in the host and survival advantage in the environment . For insotection . Moreoveotection , and appotection . In mostotection , 12. In  laccase . Subsequ laccase –13. The cholerae –16. Morecholerae  and bactcholerae , respectBacillus weihenstephanensis is a psychrotrophic Gram-positive aerobic or facultative anaerobic bacterium [Bacillus cereus group (B. cereus sensu lato) which includes seven other species commonly occurring in different environments and being extremely important for medical and economic reasons [Bacillus anthracis, etiologic agent of anthrax [Bacillus cereus, an opportunistic pathogen occasionally involved in foodborne illnesses [B. thuringiensis, entomopathogen universally used as a biopesticide [Bacillus mycoides and Bacillus pseudomycoides, characterized by rhizoidal growth on solid media, antifungal activity and stimulation of plants’ growth [B. cereus s.l. also contains recently described thermotolerant Bacillus cytotoxicus [Bacillus toyonensis, a probiotic organisms used in animal feed [B. cereus group members are versatile producers of secondary metabolites, such as antimicrobial substances [B. thuringiensis subsp. dendrolimus L-7601 [B. thuringiensis subsp. kurstaki CCTCC AB90010 [B. weihenstephanensis have not been reported so far. In several studies blackish-brown pigment production in B. cereus s.l. has been obtained through chemical mutagenesis [acterium , commonlacterium  and soilacterium . This ba reasons , 22. The anthrax , (ii) Ballnesses , and , Biebrza National Park , and agricultural land in Jasienowka , Northeastern Poland. All samples from the parks were collected with consent according to Nature Conservation Act adopted on 16 April, 2004 by Polish Parliament . The owner of the farm also permitted collection of soil samples for our study. The strains were isolated during previous studies [A collection of about 950  studies . The fie2) in order to select producers of a blackish-brown pigment diffusing into the medium.The collection included bacilli identified in previous studies as B. cereus, B. thuringiensis, B. mycoides/B. pseudomycoides, and B. weihenstephanensis. B. cereus ATCC 10987 and ATCC 14579 (American Type Culture Collection), B. thuringiensis HD1, HD73, HD567, and HD867 , and B. weihenstephanensis DSMZ 11821 (German Collection of Microorganisms and Cell Cultures), were used as references during phenotypic and genotypic tests of the melanin-positive isolates tested in this study.The isolates were screened by culturing on different media as Luria-Bertani (LB) agar, LB broth, nutrient agar, nutrient broth, and sporulation medium T3 , according to the manufacturer’s procedure and as recommended by Swiecicka and de Vos . BiochemglpF, gmk, ilvD, pta, pur, pycA, tpi) deposited in the MLST database (http://pubmlst.org/bcereus/) during previous studies [Nucleotide sequences of seven housekeeping genes  at 1:1 ratio and placed into slots of a plug mold . After solidification, plugs were treated according to Gaviria Rivera and Priest [-1) and visualized in ChemiDOC XRS System (Bio-Rad).Overnight cultures of strains were grown in LB and centrifuged at 4°C and 2,500 x g for 15 min and resuspended in SE buffer  to an ODd Priest , DNA wasd Priest . PFG Lam2O2), 1% iron (III) chloride (FeCl3), and 5% sodium hydrosulfite (Na2S2O4), were tested. Synthetic melanin  was used as a reference.The pigment was isolated from the four most productive strains: JAS 39/1, JAS 81/4, JAS 83/3, and JAS 86/1. Isolates were inoculated into 200 ml of nutrient broth and incubated at 30°C on rotary shaker at 180 rpm for 96 h until the liquid medium became dark-brown. When the cell mass was removed, the supernatant was acidified by lowering a pH to 2.0 using 1N HCl and incubated at room temperature for one week, followed by boiling of the suspension for 1 h and centrifuging at 14,000 x g for 15 min. The pigment pellet was washed with ethanol as described by Sajjan et al. . The modThe electron paramagnetic resonance measurement for the tested melanin-like pigments and synthetic melanin were performed with the X-band  EPR spectrometer  and the Rapid Scan Unit . Each sample was placed in a thin-walled glass tube free of paramagnetic impurities. Microwave frequency was obtained by the MCM101 Recorder  at magnetic modulation of 100 kHz. The total microwave power of the klystron was 70 mW. The numerical acquisitions of the first-derivative EPR spectra were done at low microwave power of 11 mW. The spectroscopic programs SWAMP (Jagmar) and LabVIEW 8.5  were used.-1. The available spectra range was 400–4000 cm-1. Synthetic melanin  was used as a standard.To quantify the extracted melanin-like pigment, IR spectra were recorded by Nicolet 6700 infrared spectrometer . A small amount of pigment was placed on a diamond window of the spectrometer, and a measurement was done in reflection mode, at a room temperature, by summary of 32 scans with a resolution of 4 cmB. cereus s.l. isolates and incubated at 30°C on a rotary shaker at 180 rpm for 24 h. After the preincubation, arginine [LB was inoculated with melanin-positive arginine  or kojicarginine  as tyrosarginine . In addiarginine  and tricarginine  were useB. cereus strain JAS 83/3, one of the most melanin-productive isolate, was extracted in the QIAcube automat (Qiagen) using the DNeasy Blood and Tissue Kit (Qiagen) with a protocol for Gram-positive bacteria. The draft genome sequence of the strain was determined using an Ion Torrent PGM sequencer (Applied Biosystems) using the Ion 316 chip with 200-bp shotgun sequencing, according to the manufacturer's instructions. The annotation was performed using best-placed reference protein set GeneMarkS+ provided by the National Center for Biotechnology Information (NCBI).Genomic DNA of B. weihenstephanensis JAS 83/3 obtained by shotgun sequencing is under Accession Number: JNLY00000000 . We also deposited three 16S rRNA homologs of B. weihenstephanensis JAS 83/3 (KP006648), B. thuringiensis HD567 (KP006649), and B. thuringiensis HD867 (KP006650). Remaining sequences of 16S rRNA were available under accession number: NR_024697 (B. weihenstephanensis DSMZ 11821), BTK_r29390 (B. thuringiensis HD1), HD73_r36 (B. thuringiensis HD73), BC0007 (B. cereus ATCC 14579), and BCE5759 (B. cereus ATCC 10987).The whole sequence of B. cereus s.l., a large collection of about 950 strains identified as B. cereus, B. thuringiensis, B. mycoides/B. pseudomycoides, and B. weihenstephanensis, was screened by culturing on different growth media  and observed for the blackish-brown pigment diffused in the medium. Although the origins of the strains were diverse , the pigment-positive bacilli were found only among six B. weihenstephanensis isolates from soil samples obtained from Jasienowka farm  and Bialowieza National Park  in Northeastern Poland. However, the production of pigment by isolates from the park was low and observed only on NA plates. In contrast, the farm isolates synthesized the pigment at high quantity both on NA plates and in LB and nutrient broths  1002TCTAGAGATAGA1013 in the 16S rDNA . Pulsed-field fingerprints of isolates do not overlap with PFGE profiles of reference strains.Comparative analysis of 16S rRNA confirmed close relation between melanin-positive isolates and MZ 11821 . On the MZ 11821 . Soil isJAS 86/1 , while BB. weihenstephanensis from the farm  were similar to the EPR signal of the synthetic melanin and all revealed broad curves around 335 mT , and were decolorized by hydrogen peroxide (30% H2O2), as well as by sodium hydrosulfite (5% Na2S2O4). The only feature which differed natural pigment and synthetic melanin was the solubility in water, observed only for pigments from B. weihenstephanensis.Chemical properties of melanin-like pigment produced by soil  similar . PigmentB. weihenstephanensis strain JAS 83/3 was performed to identify genes associated with melanin-like pigment synthesis. We found genes encoding enzymes potentially involved in the synthesis of melanin such as: laccase, phenylalanine 4-monooxygenase, pterin-4-alpha-carbinolamine dehydratase, aromatic amino acid aminotransferase, and 4-hydroxyphenylpyruvate dioxygenase. It is worth adding that JAS 83/3 genome consisted of 5,902,407 bp and contained 5,362 coding sequences (CDSs), 347 pseudogenes, and 99 RNA operons .Shotgun sequencing of B. weihenstephanensis isolates was observed after supplementation of bacterial culture with arginine and kojic acid (tyrosinase inhibitors), sulcotrione (an inhibitor of 4-hydroxyphenylpyruvate dioxygenase in the HGA pathway) and tricyclazole . On the other hand, the addition of 0.1 mM sodium azide, which inhibits laccase, resulted in the inhibition of melanin-like pigment production, indicating that laccase is a key enzyme in the pigment synthesis among B. weihenstephanensis strains. The addition of higher concentrations of sodium azide resulted in inhibition of bacterial growth. The effects of tested chemicals on melanin-like pigment production in isolates are presented in In a set of biochemical tests, no inhibitory effect on the melanin-like pigment synthesis by environmental B. cereus s.l. species able to produce this pigment are extremely rare in nature [B. weihenstephanensis melanin-positive isolates within a B. cereus s.l. collection of about 950 strains of different origin. However, only four strains originated from Jasienowka farm were able to produce melanin-like pigment in/on different media steadily when the experiments were conducted. Chemicals used in agriculture, such as mineral fertilizers or plant protection products, change bacterial communities [B. weihenstephanensis DSMZ 11821 reference strain [B. cereus group, the adaptation to low temperatures was regarded to be a characteristic of B. weihenstephanensis [B. cereus group isolated from Northeastern Poland, one of the coldest region in Poland , and revealed the existence of thermal ecotype among soil isolates [B. weihenstephanensis strains able to grow at low temperatures seem to be perfectly adapted to the habitat they occupy in Northeastern Poland.Although many microorganisms have been known to synthesize melanin –15, B. cn nature , 33. In munities  and may munities , 53. Mele strain . Within hanensis . Howeverisolates , 41. B. B. cereus s.l. strains from Northeastern Poland was genetically diverse, and this diversity mostly resulted from mutation events [B. weihenstephanensis might be a local adaptation to specific environmental niches which protects these bacteria from adverse environmental factors, such as UV light [Vibrio cholerae strains and suggested an environmental protective function of melanin in the V. cholerae community. This raised a question, why bacteria able to produce melanin are very rare in natural environments? We assumed that melanin-like pigment synthesis among B. weihenstephanensis entails a fitness cost due to the energy expensive pathway involving laccase and/or polymerization of the pigment. Yet, the benefit of extracellular melanin-like pigment production could be shared with co-occurring bacteria which do not synthesize the pigment. Such a cooperation of bacteria to reduce production costs were observed for entomopathogenic toxin synthesis by B. thuringiensis [B. weihenstephanensis [The cluster analysis based on biochemical properties, 16S rRNA and housekeeping genes sequences indicated a close relationship among melanin-like pigment producers. A previous study showed that the community of soil n events . Belotten events  noted thUV light  extreme UV light  or heavyUV light . SimilarUV light  also obsngiensis , a closehanensis , 21.B. weihenstephanensis isolates. It was shown that o-Semiquinone free radicals (S = 1/2) with unpaired electrons localized on oxygen atoms were responsible for these melanin spectra [B. weihenstephanensis isolates and synthetic melanin, indicating their phenolic character. Some differences in FT-IR spectra of environmental pigment and commercial melanin could result from the procedure of pigment extraction and purification, e.g. the usage of ethanol. According to the actual definition, melanin is a dark in color substance, insoluble in aqueous or organic solvents, resistant to concentrated acid and susceptible to bleaching by oxidizing agents [B. weihenstephanensis melanin-like pigment and the synthetic one are comparable. It is worth emphasizing that the precipitation with ferric chloride, decolorization by hydrogen peroxide and infrared spectra, indicated the phenolic nature of melanin-like pigments synthesized by environmental B. weihenstephanensis. The solubility in water of “environmental” melanin was the only difference. Similar results, including also melanin-like pigment water-solubility, were observed by Aghajanyan et al. [B. thuringiensis subsp. galleriae strain K1 obtained by chemical mutagenesis from industrial bioinsecticide B. thuringiensis 69–6. However, in general melanin produced by bacteria was water-insoluble [B. weihenstephanensis isolates might have broader biotechnological applications in comparison to the insoluble pigment.The presence of stable free radicals in all melanin pigments allowed the identification of the pigment as melanin using EPR spectroscopy. The similar shapes of EPR spectra of natural dark particles and the synthetic melanin confirmed the production of melanin pigment by environmental  spectra . Moreove spectra . FT-IR scals S = /2 with un et al.  for B. tnsoluble , 59. CerB. thuringiensis CCTCC AB 90010; however, we did not identify the gene encoding tyrosinase in B. weihenstephanensis JAS 83/3 genome. Nevertheless, there are several other enzymes potentially involved in melanin production, such as laccase or polyketide synthases [B. cereus s.l. Here, for the first time, we pointed out that laccase might be involved in the production of melanin-like pigment among strains belonging to the B. cereus group. While laccase is widely distributed in fungi and plants, in which it can be involved in melanin formation, lignolysis or detoxification, the activity of this enzyme in bacteria has been rarely documented [In microbial melanogenesis the most important enzyme seems to be tyrosinase, a monooxygenase which binds two copper ions within the active site by three conserved histidines residues . Indeed,ynthases , 13, 17.ynthases , 53, yetcumented , 61. Laccumented . In factB. weihenstephanensis isolates naturally produce melanin-like pigment. This process is probably associated with laccase activity. B. weihenstephanensis pigment producers from Northeastern Poland demonstrated a high level of phenotypic and genotypic similarity. Our study shed new light on the evolution and adaptation of B. weihenstephanensis to specific habitats. Because of possible applications, water-soluble melanin-like pigment produced by B. weihenstephanensis might be an alternative to commercial pigment, thus, it is worth further investigation.Only a limited number of S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Lonicera japonica is an important herbal medicine with great economic value. However, gene expression information for flowers and leaves of L. japonica remains elusive, which greatly impedes functional genomics research on this species. In this study, transcriptome profiles from leaves and flowers of L. japonica were examined using next-generation sequencing technology. A total of 239.41 million clean reads were used for de novo assembly with Trinity software, which generated 150,523 unigenes with N50 containing 947 bp. All the unigenes were annotated using Nr, SwissProt, COGs (Clusters of Orthologous Groups), GO (Gene Ontology), and KEGG (Kyoto Encyclopedia of Genes and Genomes) databases. A total of 35,327 differentially expressed genes  between leaves and flowers were detected. Among them, a total of 6602 DEGs were assigned with important biological processes including “Metabolic process,” “Response to stimulus,” “Cellular process,” and etc. KEGG analysis showed that three possible enzymes involved in the biosynthesis of chlorogenic acid were up-regulated in flowers. Furthermore, the TF-based regulation network in L. japonica identified three differentially expressed transcription factors between leaves and flowers, suggesting distinct regulatory roles in L. japonica. Taken together, this study has provided a global picture of differential gene expression patterns between leaves and flowers in L japonica, providing a useful genomic resource that can also be used for functional genomics research on L. japonica in the future.The perennial and evergreen twining vine,  Lonicera japonica Thunb is widely cultivated in Asian countries such as China, Japan, and Korea  in herbal medicine trading markets  between leaves and flowers were identified and the three most important pathways were revealed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Finally, multiple transcription factors (TFs) were detected in unigene libraries of leaves and flowers. Taken together, the transcriptome assembly and annotation of L. japonica tissues provide important genome information that will facilitate functional genomic research and metabolism regulation of medicinal composition in L. japonica in the future.In this study, the transcriptome profiles of leaves and flowers in L. japonica were harvested and stored at −80°C. TRIzol (Invitrogen) was used to isolate total RNAs of the flowers and leaves according to the manufacturer's instructions. An Agilent 2100 Bioanalyzer (Agilent Technologies Inc.) was used to assess the integrity of the isolated total RNAs. DNase I (Promega) was then used to remove genomic DNA contamination. The isolated total RNAs were further quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific Inc.), and the purity of the total RNA was measured by calculating the A260/280 and A260/230 ratios. Finally, the purified RNA was dissolved in RNase-free water and stored in a −80°C freezer.The flowers and leaves of cDNA libraries were prepared using a TruSeqTM RNA sample preparation kit (Illumina) according to the manufacturer's instructions. In short, poly-A mRNA from total RNA of the flowers and leaves was purified using oligo (dT) magnetic beads (NEB). The collected mRNAs were first fragmented and further used to synthesize first-strand cDNAs with hexamer and reverse transcriptase (Promega). Subsequently, the second-strand cDNAs were synthesized with DNA polymerase I and RNase H. The obtained cDNA fragments were then purified, end-repaired, A-tailed and ligated to index adapters (Illumina). The ligation products were amplified by PCR and sequenced using the Illumina HiSeq2000 platform and a 100-bp pair-end sequencing protocol was employed.De novo assembly of the unigenes in L. japonica was performed as described by Zhang et al. (SRP052594). The Trinity program was used to assemble the clean reads and obtain non-redundant unigenes  was used to annotate the pathway of these unigenes.Annotation of the assembled unigenes was performed by searching transcripts against the NCBI non-redundant protein (Nr), COG (Cluster of Orthologous Groups), Swiss-Prot, and Trembl databases. The search was conducted using BLASTx with an P < 0.005).The RPKM (Reads Per kb per Million reads) of each unigene in leaf and flower tissues was calculated and used to measure gene expression level as described by Mortazavi et al. . The edgL. japonica were determined by quantitative reverse transcription-PCR (qRT-PCR) assay. Of total RNA, 1 μg was reverse-transcribed using SuperScript III Reverse Transcriptase (Invitrogen) and oligo (dT) 18 according to the manufacturer's protocol. The qPCR experiment was carried out using an ABI 7300 Real-Time PCR System (ABI) and each reaction was performed in triplicate. For mRNA expression detection, U6 RNA was set as an internal reference gene. The primers for qPCR are listed in Supplementary Dataset mRNA expression levels in the leaf and flower tissues of The GO functional enrichment analysis of down- and up-regulated DEGs in the network was performed by Bingo plugin in Cytoscape software  to identify TFs using BLASTx  were obtained after removing adaptor sequences and low-quality reads. The Trinity program was then used for de novo assembly of all clean reads, which generated a total of 150,523 unigenes with an average length of 632 bp and an N50 of 947 bp  of common protein databases including the NCBI COG , Thellungiella salsuginea (54.83%), Brassica rapa (54.39%), and B. oleracea (50%). As L. japonica is a type of vine, we performed a distribution analysis among L. japonica, Vitis vinifera, Populus euphratica, and P. trichocarpa Torr. & Gray. The result showed that L. japonica had 25,353 homologous genes with V. vinifera, P. euphratica and P. trichocarpa; and L. japonica had more hits when compared with P. euphratica (62.3%), P. trichocarpa (62.1%), and V. vinifera . There were 6602 DEGs assigned with Biological Process terms . The top three pathways included “Ribosome” , “Oxidative phosphorylation” , and “Glycolysis/gluconeogenesis” . This result suggested that the DEGs were associated with the growth and metabolism activities of flower and leaf tissues in L. japonica. Among them, ribosomes are found in all cellular organisms and are responsible for synthesis of proteins in cells, while oxidative phosphorylation is the metabolic pathway in which mitochondria in cells use the enzymes and energy released by the oxidation of nutrients to synthesize ATP. For example, many other DEGs were involved in the process of “Glycolysis/gluconeogenesis”  against the PlnTFDB, and 144 potential TFs were identified , followed by 300–400 bp (14.91%), >1000 bp (20.21%), 400–500 bp (8.69%), 500–600 bp (6.83%), 700–800 bp (6.21%), 800–900 bp (2.83%), 600–700 bp (5.59%), and 900–1000 bp (3.08%; Figure The assembled unigenes in flowers and leaves were subjected to BLASTx similarity analysis (d Figure . The lenL. japonica. A total of 10 TFs, such as the MYB family, ein3, eil1, gbf1, and nap, were involved in the complicated regulation network (Figure nap was down-regulated in leaves. Ein3 and its closest homolog eil1 are two primary TFs downstream of ein2 and are very important for the induction of multiple ethylene response genes (Alonso et al., Arabidopsis. In the present study, network analysis showed that ein3 interacted with two targets including eil1 and ap2. Both eil1 and ap2 were linked with the ethylene-mediated signaling pathway. Therefore, the results indicate that ein3, eil1, and ap2 may form a complex gene network to regulate the process of leaf senescence in L. japonica. Furthermore, we observed that ein3 was down-regulated in flowers, suggesting that there was a different signal pathway in the regulatory process of flower senescence in L. japonica. Meanwhile, we observed that ap3 interacted with nap in the network; and ap3 was also down-regulated in leaf tissue (Figures Cytoscape software was used to construct a TF-based regulation network between TFs and their gene targets. There were 84 nodes and 171 edges obtained in k Figure . Among t Figures , suggest Figures .L. japonica is a woody perennial, evergreen to semi-evergreen vine. The dried leaves and flowers are employed in traditional Chinese medicine to treat fever, headache, cough, thirst and sore throat. Components isolated from L. japonica include phenolic acids and flavonoids, which have been reported to have pharmacological functions (Tang, L. japonica, functional genomic studies and gene discovery are greatly limited in L. japonica. NGS technologies have provided powerful tools for high-throughput sequencing, allowing easy discovery of novel genes by obtaining massive sequence information with enormous depth and coverage. For example, Li et al. (Sophora moorcroftiana by use of the Trinity program. They identified a large number of important genes involved in drought tolerance.ns Tang, . Howeveri et al.  obtainedL. japonica leaves and flowers using the Illumina Hiseq 2000 platform and obtained 150,523 unigenes. Notably, more unigenes were identified in this study than identified in previous work; for instance, Yuan et al. (L. japonica buds. Also, He et al. (L. japonica buds and leaves using the Roche/454 GS FLX platform. Nevertheless, we observed that < 50% of the unigenes were successfully annotated by BLAST against public databases including Nr, Swiss-Prot and COG. The possible cause of this result is the absence of genome information of L. japonica and technical limitations such as sequencing depth and read length. Gene expression profiles in different tissue types have been extensively studied to reveal tissue-specific gene functions. For example, based on microarray technology, Kram et al. (SK1, C4H, and HCT, were up-regulated in flowers (Figure L. japonica. This result suggested the content of chlorogenic acid was higher in L. japonica flowers than leaves. Previous study reported that L. japonica flower buds contained a higher proportion of chlorogenic acid than flowers (Geng et al., L. japonica tissues.In the present study, we sequenced two cDNA libraries from n et al.  obtainede et al.  obtainedm et al.  identifim et al.  detectedL. japonica unigenes with the PlnTFDB. Among them, the main TF families include MYB, C2H2, and ERF. For instance, the MYB family was reported to be the largest TF family in Arabidopsis (Yanhui et al., L. japonica was thus constructed, in which ein3 and gbf1 were up-regulated, while nap was down-regulated in leaves compared with flowers. This result indicated that some TFs have special distributions in plant tissues, which may be linked with their regulatory roles in metabolic activity in L. japonica.TFs are important DNA-binding proteins. TFs affect the access of RNA polymerase to the gene promoter (Udvardi et al., L. japonica using NGS technology. A total of 35,327 DEGs were identified between leaves and flowers. Among them, 6602 DEGs were assigned within some important biological processes including “Metabolic process,” “Response to stimulus,” and “Cellular process.” KEGG analysis showed that three possible enzymes involved in the biosynthesis of chlorogenic acid were up-regulated in flowers, which revealed new insight into the molecular regulation of chlorogenic acid metabolism in the flower tissues. Meanwhile, the TF-based regulation network in L. japonica showed three DEGs for TFs between leaves and flowers, suggesting their regulatory roles in metabolic activity in L. japonica. Overall, this study provided a global picture of different gene expression patterns between leaves and flowers in L. japonica. These results not only provide a useful genomic resource of L. japonica but will also shed light on functional genomics research on L. japonica in the future.This study characterized the transcriptome profiles in the leaves and flowers of ML initiated and designed the research. LZ, YL, CF performed and analyzed the experiments. JX, JG, GW, and HJ Contributed reagents/materials/analysis tools. LZ wrote the paper. YL and ML revised the paper.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.


Escherichia coli infection in vivo and uncovers differences between the consequences of E. coli vs. Staphylococcus aureus infection of primary mammary epithelial cells (PMECs). In udders, live E. coli elicits inflammatory and immune defenses through numerous cytokines and chemokines. Importantly, E. coli infection causes downregulation of genes encoding lipid biosynthesis enzymes that are involved in milk production. Additionally, host metabolism is generally suppressed. Finally, defensins and bacteria-recognition genes are upregulated, while the expression of the extracellular matrix protein transcripts is silenced. In PMECs, heat-inactivated E. coli elicits expression of ribosomal, cytoskeletal and angiogenic signaling genes, and causes suppression of the cell cycle and energy production genes. We hypothesize that heat-inactivated E. coli may have prophylactic effects against mastitis. Heat-inactivated S. aureus promotes stronger inflammatory and immune defenses than E. coli. Lipopolysaccharide by itself induces MHC antigen presentation components, an effect not seen in response to E. coli bacteria. These results provide the basis for strategies to prevent and treat mastitis and may lead to the reduction in the use of antibiotics.Bovine mastitis is a widespread disease in dairy cows, and is often caused by bacterial mammary gland infection. Mastitis causes reduced milk production and leads to excessive use of antibiotics. We present meta-analysis of transcriptional profiles of bovine mastitis from 10 studies and 307 microarrays, allowing identification of much larger sets of affected genes than any individual study. Combining multiple studies provides insight into the molecular effects of  E. coli, Streptococcus uberis and Staphylococcus aureus [Mastitis is, arguably, the most important disease of dairy cattle , 2. It is aureus –6. Mastis aureus .Escherichia coli and S. aureus infections result in different symptoms and cellular responses. Escherichia coli infection is typically associated with an acute and severe form of mastitis, while S. aureus causes often a chronic but sub-clinical disease. In bovine primary mammary epithelial cells (PMECs), E. coli infection induces the expression of Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4), and cytokines Tumor Necrosis Factor-α, Interleukin-1α, Interleukin-6 and Interleukin-8, and activation of the NFκB pathway; on the other hand, while S. aureus infection induces TLR2 expression, other molecular responses are delayed if present at all [t at all .E. coli. [There have been significant attempts to prevent or ameliorate the consequences of bovine mastitis. For example, lipopolysaccharide (LPS) can be used to stimulate the inflammatory reactions in udders; such treatments may reduce the severity of subsequent infections , 13. LipE. coli. . MastitiE. coli. , which hE. coli. .in vivo, as well as by treating PMECs with heat-inactivated bacteria in vitro [E. coli infection [The responses to mastitis infection have been studied using transcriptional profiling, both in infected udders in vitro –23. Drawin vitro . Recentlin vitro –27. Whilin vitro , differenfection –31. Combnfection , 33.E. coli and S. aureus. Combining multiple studies, we were able to identify large sets of differentially regulated genes, which allowed us insights into the molecular effects of E. coli infection in vivo. Additionally, we found differences between E. coli and S. aureus infections of PMECs. We found that lipid biosynthesis enzymes involved in milk production are repressed under E. coli infection, which provides molecular insight into reduced milk production in infected animals. We defined the specific effects of heat-treated E. coli in vitro, which, we propose, may have prophylactic effects against mastitis. We also identify responses to bacterial LPS that are not elicited by live bacteria. The results provide insight for developing strategies to prevent and treat mastitis and may lead to the reduction in the use of antibiotics in its treatment.Here we assemble and present a meta-analysis comprising 307 microarrays from 10 individual studies of mastitis-related transcriptional profiling of responses to mastitis” and selecting “Bos taurus” as the organism yielded twenty nine data sets as output. From these, we selected studies focused on responses of the epithelial cells to a mastitis-causing bacterium, E. coli or S. aureus, either conducted in vivo (udder tissue) or in vitro . We did not analyze systemic responses in blood cells. The selected studies used the “Affymetrix Bovine Genome Array” platform containing 24128 genes. Additional studies were found using non-Affymetrix microarrays, but we decided not to include these for the following reasons: 1. such studies mostly used in-house microarrays, which incompletely overlap the Affymetrix arrays, and therefore would significantly reduce the total number of genes studied; 2. Each of the in-house array is used in just a few datasets ; 3. They used two-color RNA labeling approach, which yields relative expression values, which are not easily integrated with the Affymetrix studies; 4. The Affymetrix studies can analyze a high number of samples, and employ standardized quality controls and analysis algorithms, which can be used across different studies. The.CEL or.TXT files deposited from these studies were downloaded and unzipped, then log2 transformed. Datasets obtained were combined and analyzed using RMAExpress for quality control [Searching GEO Datasets for the key term “ control , 34. ForE. coli-treated and control samples, we combined microarray data containing the 177 microarrays from the E. coli experiments into a single spreadsheet, using data-loader. We performed four separate analyses: 1) 4 studies comprising 89 microarrays for control and E. coli-infected udder biopsies. Differentially expressed genes in each of the class were recorded [E. coli-treated PMEC containing three data sets with 49 treated samples and 39 controls [S. aureus [E. coli and S. aureus were used in these studies, specifically, E. coli 1303, E. coli k2bh2, E. coli ECC-Z, S. aureus M60 and S. aureus 1027  Dcontrols –20. 3) Mcontrols . 4) Two . aureus , 20. Seveus 1027 . The ani−4, when compared with respective controls in the following data sets: global, live E. coli-, heat-inactivated E. coli- and heat-inactivated S. aureus-treated samples. For each analysis, the number of genes induced or suppressed in the respective comparison is recorded in We used the RankProd Software to identify the differentially expressed genes with p-values better than 10−3. We also generated clusters of ontological categories containing extensively overlapping sets of genes, which condensed some redundancies in the regulated ontological categories. We separately identified ontological data for the induced and suppressed ontological clusters and genes in each comparison.We chose genes with p-values better than our threshold from RankProd output and used online Database for Annotation, Visualization and Integrated Discovery (DAVID) software for further analysis as described before , 35. ForThe PRISMA Checklist is included as mastitis” and “Bos taurus” and selected studies using Affymetrix bovine microarrays platform only. We found that studies describing transcriptional responses to live E. coli strains were conducted in vivo in udder tissues, while the responses to heat-inactivated E. coli, S. aureus or LPS were studied in primary cultures of mammary epithelial cells. We analyzed the gene ontologies upregulated and downregulated in these data sets separately  receptor signaling pathways, are upregulated but less prominent in heat-inactivated ed PMECs , where pE. coli regulates a different set of genes from the one regulated by live E. coli: specifically 1) the metabolic enzymes of lipid biosynthesis and sugar transport are not suppressed and 2) inflammation- and defense-related genes are much attenuated in response to heat-inactivated E. coli.Overall, the heat-inactivated S. aureus tend to be milder and cause less significant mastitis morbidity than those with E. coli [S. aureus treatment of PMECs [E. coli-treated PMECs shown above. In the S. aureus treated PMECs, the most prominently induced cluster comprises inflammatory, immune and defense responses Click here for additional data file.


Theileria spp. are tick transmitted protozoa that can infect large and small ruminants causing disease and economic losses. Diagnosis of infections is often challenging, as parasites can be difficult to detect and identify microscopically and serology is unreliable. While there are PCR assays which can identify certain Theileria spp., there is no one PCR that has been designed to identify all recognized species that occur in ruminants and which will greatly simplify the laboratory diagnoses of infections.Theileria FRET-qPCR were selected by comparing sequences of recognized Theileria spp. in GenBank and the test validated using reference organisms. The assay was also tested on whole blood samples from large and small ruminants from nine provinces in China.Primers and probes for a genus-specific pan-Theileria FRET-qPCR detected all recognized species but none of the closely related protozoa. In whole blood samples from animals in China, Theileria spp. DNA was detected in 53.2% of the sheep tested (59/111), 44.4% of the goats (120/270) and 30.8% of the cattle . Water buffaloes (n = 29) were negative. Sequencing of some of the PCR products showed cattle in China were infected with T. orientalis/T. sergenti/T. buffeli group while T. ovis and T. luwenshuni were found in sheep and T. luwenshuni in goats. The prevalence of Theileria DNA was significantly higher in Bos p. indicus than in Bos p. taurus (77.7% vs. 18.3%) and copy numbers were also significantly higher (104.88 vs. 103.00Theileria 18S rRNA gene copies/per ml whole blood).The pan-Theileria FRET-qPCR can detect all recognized Theileria spp. of ruminants in a single reaction. Large and small ruminants in China are commonly infected with a variety of Theileria spp.The pan- Theileria spp. are tick-transmitted, intracellular protozoan parasites infecting leukocytes and erythrocytes of wild and domestic large and small ruminants. Several Theileria spp., transmitted by ixodid ticks of the genera Rhipicephalus, Hyalomma, Amblyomma and Haemaphysalis, have been described in cattle, water buffaloes, sheep and goats in different geographical zones of the world [Theileria spp. can cause fever, anemia and hemoglobinuria and, in severe cases, death although many species are benign. Animals recovered from acute or primary infections usually remain persistently infected and may act as reservoirs of infecting ticks [he world –5. Theilhe world –11. Infeng ticks , 13.Theileria spp. Although molecular studies have been performed, these have been to detect Theileria of specific domestic animal species, for example sheep and goats [Theileria spp. might occur. To address this problem, we developed and validated a highly sensitive genus-specific Theileria FRET-qPCR that detects the recognized Theileria spp. of domestic animals and investigated the molecular prevalence of Theileria in cattle, water buffaloes, goats and sheep from nine provinces in China.While there have been numerous reports of theileriosis in various animal species in China since 1958 –27, manyBos primigenius (p.) taurus studied (n = 975) were Holsteins, Simmentals, Bohai blacks, Luxis and Wannans while the Bos. p. indicus (n = 260) were the Yunlings, Minnans, and Leiqiongs  were collected in EDTA from apparently healthy cattle , water buffaloes (29), goats (270) and sheep (111) from 9 provinces/municipality of China , T. buffeli (HQ840967), T. annulata (KF429799), T. sergenti (EU083804), T. luwenshuni (JX469527), T. velifera (AF097993), T. ovis (AY508458), T. parva (L02366), T. uilenbergi (JF719835), T. equi (AB515310), T. lestoquardi (JQ917458), T. separata (AY260175), T. capreoli (AY726011), T. cervi (AY735119), T. bicornis (AF499604), T. taurotragi (L19082), T. mutans (FJ213585); Babesia bovis (KF928529), B. divergens (LK935835), B. bigemia (LK391709), Hepatozoon americanum (AF176836), Cytauxzoon felis (AY679105) and Toxoplasma gondii (L37415)  were compared with the existing Theileria sequences in the GenBank using BLAST. The specificity of the PCR was further verified with the amplification of T. orientalis rRNA-containing pIDTSMART cloning Vector  and 100 DNA copies of the rRNA gene of B. canis, H. americanum, C. felis and T. gondii .PCR products were verified using electrophoresis (1.5% MetaPhor agarose gels), followed by purification with a QIAquick PCR Purification Kit  and genomic sequencing . The sequencing data from randomly selected positive Theileria species  were gel purified using a QIAquick Gel Extraction Kit . After using the estimated molecular mass of the rRNA gene and the Quanti-iT TM PicoGreen ® dsDNA Assay Kit  to calculate the molarity of the solution, dilutions were made to give solutions containing 10,000, 1,000, 100, 10, 1 gene copies per PCR reaction system. These dilutions, and further dilutions providing 2, 4, 6 and 8 gene copies per PCR reaction, were used to determine the minimal detection limit. The 10-fold dilutions were used as quantitative standards in the FRET-PCR surveys to enable standard curves to be developed for the calculation of the gene copy numbers in positive samples.For use as quantitative standards, the PCR products of DNAs of 5 Theileria FRET-qPCR we established has a sequence which is highly conserved among the different Theileria species. To differentiate Theileria spp. in a positive reaction, we used a standard PCR to amplify a highly polymorphic region of the 18S rRNA gene (591–594 nucleotides for different Theileria spp.) and sequenced the products . For the PCR we designed a forward primer (5′-CCTGAGAAACGGCTACCACATCT-3′) that amplified all Theileria species and used a previously described reverse primer (5′-GGACTACGACGGTATCTGATCG-3′) that also amplified all species [The amplicon of the pan- species .Theileria spp. were analyzed by Chi-squared Test while numbers of copies of the Theileria 18S rRNA gene determined in the Theileria FRET-qPCR were log10-transformed and analyzed using the Student’s T-test. Differences of P < 0.05 were considered statistically significant.Differences in positivity of Theileria spp. we used showed the region is highly conserved, but is substantially different from those in closely related protozoan species  with Theileria spp. DNA. Using the gel-purified PCR products as quantitative standards, we determined the detection limit of the pan-Theileria FRET-qPCR was 2 copies of the Theileria 18S rRNA gene per reaction for T. orientalis, T. sergenti and T. luwenshuni, T. buffeli and T. ovis.Comparison of the sequences in the highly conserved region of the s Figure . The twoTheileria were found in each of the nine provinces sampled with several animals of each species being positive at each location, except in the case of water buffaloes which were all negative in the one site they were studied. The overall prevalences of Theileria spp. DNA in sheep  and goats  were significantly higher than in cattle  . The pan-Theileria FRET-PCR showed that sheep had an average of 102.4 copies of Theileria 18S rRNA/ml whole blood which was significantly lower than the 104.3 copies in cattle and 105.8 copies in goats . While the prevalence of Theileria spp. DNA varied greatly from 3.5% (9/255) in Holsteins from Shanghai to 100% in Luxi cattle from Shandong (40/40) and Leiqiong cattle from Hainan (74/74), the prevalence did not differ significantly in sheep from Inner Mongolia and Shandong  from li group  were preTheileria spp. DNA in cattle we found that Bos p. indicus animals had significantly higher positivity  and copy number of the Theileria 18S rRNA gene  than Bos p. taurus animals. Similarly, Bos p. indicus cattle were more likely to be positive  and have higher copy numbers of the Theileria 18S rRNA gene  than Bos p. taurus animals. The cattle from southern China had significantly higher Theileria 18S rRNA gene copy numbers  than those from northern China but this difference in the prevalence was not significant (31.8% vs. 26.5%). In cattle from Yunnan province where gender information was available, female animals were more commonly positive  and copy numbers  than males.When we analyzed factors that might be associated with the prevalence of Theileria rRNA nucleotide sequences obtained in this study that were not identical to existing entries in GenBank were deposited with the following gene accession numbers: KJ850933 and KJ850938 (T. sergenti); KJ850936 and KJ850940 (T. buffeli); KJ850934, KJ850937, KJ850943, KJ850939 and KJ850941 ; KJ850942 (T. ovis); KJ850935 and KM016463 (T. luwenshuni). The sequences obtained were very similar (0–4 nucleotide mismatches) to Theileria spp. sequences deposited by other laboratories in China, USA, France, Australia and Iran . Such variability is probably due to tick prevalence rates, geoclimatic factors and livestock management systems. Further research using sensitive detection methods will be important in determining the best mechanisms to control infections.We found both goats and sheep were infected with ni Table  using thlenbergi, 38, then et al. found thT. ovis was first identified in China in 2011 by PCR and sequencing and an infection rate of 78% was found in Xinjiang but no positive animals were found in twelve other provinces [T. ovis is considered benign and economically unimportant as it might only cause signs in some animals which are stressed [rovinces , 27, 39.stressed .Theileria spp. than local stocks. Recent studies, however, have indicated that different breeds of animals might have similar susceptibilities to infections with Theileria spp. although some breeds are more capable of controlling the pathogenic effects of the organism [It is well recognized that exotic breeds are more susceptible to disease following infection with organism .B. p. taurus breeds are more susceptible to theileriosis than B. p. indicus breeds which might be associated with innate or immune mechanisms and/or general resistance to ticks. These factors have been mainly investigated with T. parva infections and thus it is interesting that our pan-Theileria FRET-PCR testing showed that in China it is the B. p. indicus breeds that are more likely to be positive than the B. p. taurus breeds and that the former generally had higher copy numbers indicating heavier infections. While our sample sizes were small and we cannot exclude the possibility of sample bias, our findings might be due to host genetic factors relating to infections with less pathogenic Theileria spp. or other factors such as differences in tick control and husbandry practices on different farms. Further studies taking these factors into account are needed to more precisely investigate the relationships between infections with benign Theileria spp. and the genetic background of the host.Generally, Theileria spp. The pan-Theileria FRET-PCR should be a useful diagnostic tool as it will enable diagnostic laboratories to detect infections in all domestic species with a single test.In summary, our study has described the development and testing of a FRET-PCR which can detect recognized


Brassica oleracea and Brassica rapa provides an important opportunity for researchers to identify and characterize NBS-encoding R genes in Brassica species and to compare with analogues in Arabidopsis thaliana based on a comparative genomics approach. However, little is known about the evolutionary fate of NBS-encoding genes in the Brassica lineage after split from A. thaliana.Plant disease resistance (R) genes with the nucleotide binding site (NBS) play an important role in offering resistance to pathogens. The availability of complete genome sequences of B. oleracea, B. rapa and A. thaliana. Through the employment of HMM search and manual curation, we identified 157, 206 and 167 NBS-encoding genes in B. oleracea, B. rapa and A. thaliana genomes, respectively. Phylogenetic analysis among 3 species classified NBS-encoding genes into 6 subgroups. Tandem duplication and whole genome triplication (WGT) analyses revealed that after WGT of the Brassica ancestor, NBS-encoding homologous gene pairs on triplicated regions in Brassica ancestor were deleted or lost quickly, but NBS-encoding genes in Brassica species experienced species-specific gene amplification by tandem duplication after divergence of B. rapa and B. oleracea. Expression profiling of NBS-encoding orthologous gene pairs indicated the differential expression pattern of retained orthologous gene copies in B. oleracea and B. rapa. Furthermore, evolutionary analysis of CNL type NBS-encoding orthologous gene pairs among 3 species suggested that orthologous genes in B. rapa species have undergone stronger negative selection than those in B .oleracea species. But for TNL type, there are no significant differences in the orthologous gene pairs between the two species.Here we present genome-wide analysis of NBS-encoding genes in B. rapa and B. oleracea based on whole genome sequences. Through tandem duplication and whole genome triplication analysis in B. oleracea, B. rapa and A. thaliana genomes, our study provides insight into the evolutionary history of NBS-encoding genes after divergence of A. thaliana and the Brassica lineage. These results together with expression pattern analysis of NBS-encoding orthologous genes provide useful resource for functional characterization of these genes and genetic improvement of relevant crops.This study is first identification and characterization of NBS-encoding genes in The online version of this article (doi:10.1186/1471-2164-15-3) contains supplementary material, which is available to authorized users. The interactions of this gene-for-gene (or genes-for-genes) manner activate the signal transduction cascades that turn on complex defense responses against pathogen attack and this is called incompatible interaction  formed a tetraploidy species  [B. oleracea and B. rapa and provide insights into their evolutionary history and disease resistance.The genera  ~16 MYA –25. In Bn = 34)] . This weArabidopsis thaliana, Brassica rapa and Brassica oleracea genomic and annotation data was downloaded from the TAIR10 (http://www.arabidopsis.org)  and the rest [36 (22.9%) genes in B. oleracea and 9 (4.4%) genes in B. rapa] were located on the unanchored scaffolds  have more genes and the rest chromosomes have fewer genes (e. g. C05 in B. oleracea), and many of these genes reside in a cluster manner. R genes existing in clusters may facilitate the evolutionary process through producing novel resistance genes via genome duplication, tandem duplication and gene recombination [B. oleracea (60.3%) and A. thaliana (61.7%) is higher than that of B. rapa (59.4%). In B. oleracea, 73 NBS genes, representing 60.3% of total genes on chromosomes, were located in 24 clusters and the remaining 48 genes were singletons. Five clusters containing 19 NBS genes were identified on the chromosome C07  NBS genes with TIR domain and CC motif in 43 clusters and remaining 80 genes were found as singletons on chromosomes. Among the 43 clusters, 11 with 31 genes were located on chromosome A09  NBS genes with TIR domain and CC motif were mapped in 37 clusters whereas the remaining 64 genes were found as singletons. The numbers of genes in clusters ranged from two to six in both Brassica species and two to nine in A. thaliana.NBS-encoding genes for the three species were mapped onto pseudo-molecules/ chromosomes [121 (77.1%) genes in bination . Accordibination  and Meye and 167 0% genes B. rapa and A. thaliana than B. oleracea. In B. oleracea among 24 identified clusters, 5 were homogenous and one of them containing four genes  with TN domain configuration was located on chromosome C06. Most of the clusters (18) are heterogenous with distantly related NBS domains. Fifteen clusters in each of B. rapa and A. thaliana were found to be homogenous containing the NBS-encoding genes mostly from TNL domain combination.Further, more numbers of homogenous clusters was observed in B. oleracea, A. thaliana and B. rapa represents two major groups of TNL (348 genes) and CNL (138 genes) containing genes from three species. In composite phylogenetic tree, TNL and CNL groups were further divided into three sub-groups, TNL-I-III and CNL-I-III . This subgroup included the largest part of the full length TNLs and second and third prevalent classes are TN and N type genes respectively. The domain arrangement was found to be highly diverse and NBS-encoding genes from three species with thirteen different complex and unusual domain combinations of TNNL, TCNL, TNTN, TNLT, TNNTNNL, NLTNL, NNL, TNLTNL, CTN, TNN, TTN, TNLN and LTNL were identified in this subgroup. In subgroup TNL-II, more than half of the genes were from B. oleracea and others were from B.rapa and A. thaliana. This subgroup along with various complex domain arrangement containing genes also carried most of the full length TNLs. TNL-III was the smallest subgroup with majority of genes from B. oleracea (5 genes) and a single gene from each of B. rapa and A. thaliana. B. oleracea gene, Bol044437 with unusual domain arrangement TNNL also clustered in this subgroup.Comparative phylogenetic relationship of NBS-encoding genes in A. thaliana. However, CNL group is not much variant and only few complex domain arrangements are evident; NNL, CNNL and CNNN. In CNL-1 subgroup, out of 5 clustered A. thaliana genes, 4 genes  were also grouped in the respective A. thaliana CNL-A subgroup as identified and described by Meyers et al. 2003. Both CNL-II and CNL-III subgroups included most of NBS-encoding genes from B. rapa and A. thaliana and fewer genes from B. oleracea species. NBS-encoding genes with N and CN type truncated domains were observed more in CNL-II subgroup and one B. rapa gene (Bra037453) with unusual domain, CNNN also clustered here. Subgroup CNL-III was represented by 73 genes and most of the members (36) were full length CNL ORFs. Four B. rapa genes  with unusual domains NNL and CNNL were also identified in this subgroup.CNL group was further divided into three distinct subgroups represented by genes from all the three species and we also observed one CNL subgroup which was already recognized in B. oleracea could be classified into two major groups (Bol-A and Bol-B)  expressed relatively higher in root and stalk indicating their tissue-specific role in these tissues. Majority of genes in subgroup Bol-A2 were found to be upregulated in root and callus  but down regulated in stalk, leaf, flower and silique. Up regulation of these genes in callus suggests their induction under wounding. However, eighteen genes in group Bol-B displayed differential expression in different tissues and among all the genes in this subgroup, Bol009890 exhibited highest expression in leaf and Bol036980 showed more transcript level in flower tissue.To investigate the expression pattern of NBS-encoding genes, we compared the transcript abundance in different tissues using RNA-seq data from GEO database. The expression profile of NBS-encoding genes in B. rapa, genes could be categorized into two main groups, Bra-A and Bra-B  and Bra004192 and Bra035103 highly expressed in stalk and leaf. In subgroup Bra-A2, where a number of genes were expressed more in root and callus. However, Bra018810 displayed highest expression in silique suggesting its silique-specific role. In Subgroup Bra-A3, some genes showed the preferential transcript level in stalk and flower and some genes relatively expressed higher in flower, silique and callus. For example, Bra008055 accumulated more transcripts in leaf, flower and callus, Bra008056 in flower and Bra026094 in stalk and silique. Most of genes in group Bra-B showed high expression in stalk and leaf as compared to other tissues and Bra009882, Bra008053, Bra018834, Bra027866, Bra026368 and Bra030778 highly expressed in leaf tissues. This may specify that genes in this subgroup act as positive regulator in leaf tissues.In B. oleracea and B. rapa genomes. Some NBS-encoding genes showed higher expression in same tissue indicating their functional conservation, but others were more abundant in different tissues which point toward their functional differences. According to expression pattern of NBS-encoding genes in different tissues, it would be interesting to functionally characterize these genes for pathogen defense response, especially race- and species-specific pathogens in Brassica species.Taken together, we suggest that NBS-encoding genes exhibited differential expression pattern in different tissues and several genes are induced by wounding in A. thaliana genome has experienced two recent whole genome duplication (named α and β) within the crucifer (Brassicaceae) lineage and one triplication event (γ) that is probably shared by most dicots (asterids and rosids) [Brassica species and A. thaliana lineages diverged about 20 MYA and subsequently a whole genome triplication (WGT) event occurred in the Brassica ancestor approximately 16 MYA. As WGT of the Brassica ancestor, NBS-encoding genes in the A. thaliana genome might have triplicated orthologous copies in B. rapa and B. oleracea. Since, A. thaliana is considered a model plant system for plant molecular biology research and most of its genes have been functionally characterized. Therefore, we traced these orthologous gene pairs between A. thaliana and Brassica species to detect the NBS-encoding genes in evolutionary history. From analysis of orthologous regions for genome-wide comparative analysis, we obtained 42 orthologous gene pairs between A. thaliana and B. oleracea, 62 between A. thaliana and B. rapa and 24 between B. oleracea and B. rapa, which are shown in Figure  rosids) . The ancA. thaliana and B. oleracea, 26 A. thaliana NBS genes were shown to retain one copy, 5 A. thaliana NBS genes retained two copies and only 2 genes corresponding to AT4G19500.1 and AT4G19510.1 each preserved tripled copies after triplication in B. oleracea. In total, 42 NBS genes in B. oleracea genome have 33 corresponding genes in A. thaliana genome. A. thaliana corresponding genes in B. oleracea were located on different chromosomes and some gene pairs (which retained single copy in B. oleracea) and 3 out of 5 A. thaliana corresponding genes (which retained two copies in B. oleracea) preserved domain structure  preserved tripled copies in B. rapa. At last, we got 50 A. thaliana NBS genes compared to 62 NBS genes in B. rapa genome. Gene pairs in B. rapa corresponding to A. thaliana were located on different chromosomes. Further, some genes (which retained single copy in B. rapa), 5 out of 8 A. thaliana NBS genes (which retained two copies in B. rapa) and 2 genes (which retained tripled copies in B. rapa) preserved domain configuration in B. rapa  and C09 (2–4 genes in arrays) carried two and three tandem arrays respectively. The highest number of tandem arrays (6) with 17 genes was found on chromosome C07 which contains the highest number of R genes in the genome. In A. thaliana genome, out of 167 NBS genes 93 (55.7%) genes were tandemly duplicated and positioned on chromosomes in 37 tandem arrays. The tandem duplicated genes were distributed in tandem arrays of 2–6 genes. In B. rapa genome, 97 genes (47.1%) were tandemly duplicated and 93 genes were located on chromosomes in 38 tandem arrays while two tandem arrays were located on scaffold sequences. The number of duplicated genes range from 2–5 genes in tandem arrays  preserved tripled copies and other two genes (AT4G19520 and AT4G19530) have retained one copy in B. oleracea genome respectively. In B. rapa genome, we found that only AT4G19500 gene preserved two copies and other members of this tandem arrays were missed or deleted  in B. rapa, which were clustered in one phylogenetic sub tree corresponding to syntenic relationship. The second member of tandem array, AT4G19510.1 have three homologous genes  only in B. oleracea and did not retain any copy in B. rapa genome, indicating syntenic relationship between two species. Each of tandem array member, AT4G19520.1 and AT4G19530.1 have one homologous genes (Bol024371 and Bol024372) only in B. oleracea respectively, which appeared in one phylogenetic sub tree  in A. thaliana diseases resistance, so they retained three copies after triplication in B. oleracea genome. AT4G19520 and AT4G19530 might have subjected to less evolutionary pressure leading to other two other two duplicated copies lost in B. oleracea genome. We hypothesize that these homologous genes of B. oleracea might be resistant to species-specific pathogens or diseases in B. oleracea genome. After WGT of Brassica ancestor, genomic components were triplicated and redundance data was generated. From evolutionary pressure or environment selection, critical components were retained and others were deleted or lost.The genes AT4G19500 and AT4G19510 in tandem array might have important role in process of A. thaliana which have their corresponding orthologous and paralogous genes in B. rapa and B. oleracea genomes to investigate expression pattern divergence among 3 species.Differential expression level of orthologous and paralogous gene pairs for NBS-encoding genes can reflect expression pattern divergence of orthologous and paralogous genes after WGT. Through syntenic analysis among 3 species, we focused on transcript expression level of 5 CNL and 16 TNL NBS-encoding genes in different tissues in A. thaliana gene AT1G50180.1), one in B. oleracea (Bol011780) and one in B. rapa (Bra014241), was found to be different across the different tissues. Bol011780 showed reduced expression in stalk, silique and moderately expressed in callus, on the other hand we only observed the reduced expression of Bra014241 in stalk, leaf and flower. Orthologous gene Bol039594 in B. oleracea  expressed only in root, silique and callus while the expression of its corresponding analogue Bra013947 in B. rapa was confined to stalk and callus. Further, Bol042325  expressed in leaf, flower and callus but expression of its orthologous gene in B. rapa (Bra019063) was significantly decreased in all tissues. Another orthologous gene in B. oleracea, Bol005097  was abundantly expressed in leaf, callus and moderately expressed in root, stalk and silique and its orthologous gene, Bra027333 in B. rapa displayed high expression in stalk, leaf, flower, callus and reduced expression in root and silique. Bol005098 in B. oleracea  abundantly expressed in root, stalk, leaf, callus and exhibited reduced expression in flower and silique, whereas its orthologous gene Bra027332 in B. rapa, was ubiquitously expressed in all tissues  are also located in a tandem array and two paralogs in each of B. oleracea (Bol005097 and Bol 005098) and B. rapa (Bra027332 and Bra027333) were generated by their tandem duplication. When we compared the expression profile between these two paralogs (Bol005097 and Bol 005098) in B. oleracea, we found that there was a clear difference in expression level of these two paralogs in different tissues except the root and callus where they transcribe almost at the same level. In B. rapa, the expression of Bra027332 and Bra027333 paralogs was significantly high in stalk, leaf, flower and callus, but Bra027332 exhibited moderate and Bra027333 showed low expression level in root and siliques  corresponding to A. thaliana gene AT1G72890.1 have been identified. In B. oleracea Bol026304 was observed to express in stalk, leaf, flower and callus whereas Bol040038 only expressed in stalk and callus. In B. rapa one of the retained orthologous copy (Bra003864) expressed in vegetative tissues and other orthologous gene (Bra016029) was down regulated in all tissues. Another A. thaliana gene (AT1G72950.1) retained a single ortholog in each of B. oleracea (Bol026303) and B. rapa (Bra016028), where Bol026303 was noticed in all tissues but specifically highly expressed in root and callus, while its orthologous gene Bra016028 transcribe at too low level to be detected. In one more case in TNL type, A. thaliana gene AT4G12010.1 retained corresponding two orthologous genes in each of B. oleracea (Bol008302 and Bol030522) and B. rapa (Bra029431 and Bra000759). One of the ortholog Bol030522 expressed more or less in all tissues while rest of the orthologs transcribed at too low level. Furthermore, a single copy in each of B. oleracea (Bol030521) and B. rapa (Bra000758) was retained corresponding to A. thaliana gene AT4G12020.2. Bol030521 ubiquitously expressed in most of the tissues but the expression level of its ortholog, Bra000758 in B. rapa was very reduced in all tissues  in each of B. oleracea  and B. rapa . Through expression profile comparison it is clear that in B. oleracea, Bol030521 highly expressed in root, leaf, flower, silique and callus, Bol030522 distinctly expressed in leaf, silique and callus while the expression level of Bol008302 was very low across all tissues studied. In B. rapa, the expression of two paralogs (Bra000758 and Bra029431) was significantly reduced in all tissues while Bra000759 was detected at very low level. In addition to that the other two genes in A. thaliana, AT1G72890.1 and AT1G72950.1 have also generated three paralogs (again two generated by tandem duplication and one by genome triplication) in B. oleracea and B. rapa. In B. oleracea, Bol026303 was noticeably expressed in root, flower and callus, Bol026304 showed clear expression only in stalk, leaf and callus, whereas the third paralog Bol040038 was only detected in stalk and callus. In B. rapa, Bra003864 showed significantly wide expression in root, stalk, leaf and callus whereas the other two paralogs Bra016028 and Bra016029 exhibited very low expression level in all tissues  than that (0.235) of B. rapa genome. The Ka/Ks values of orthologous gene pairs of CNL type R genes were found to show significant differences in B. oleracea and B. rapa species  and the number of synonymous substitution per synonymous site (Ks). The ratios of the rates of nonsynonymous to synonymous substitutions (Ka/Ks) of orthologous gene pairs were estimated for each branch of the phylogenetic tree using PAML software . The Ka/s Figure . For CNL greater 2.72% tha greater 2.72% thaB. rapa species experienced stronger pressure about negative selection than B. oleracea species which specify that B. oleracea species experienced stronger evolutionary constraints than B. rapa species in CNL type NBS-encoding orthologous genes. But for TNL group, we could not find significant differences between the two species about the orthologous gene pairs, although the mean Ka/Ks values of all orthologous gene pairs for TNL group in B. rapa genome was slightly higher than that of B. oleracea genome.Comparing the mean Ka/Ks ratios for CNL group, we observed that orthologous genes of CNL type NBS-encoding genes in A. thaliana through PRGdb (http://prgdb.crg.eu/wiki) database [Brassica species and totally four NBS disease resistance genes were obtained in A. thaliana which retained the orthologous gene pairs in Brassica species. In A. thaliana, RPM1 gene enables dual specificity resistance to two avirulence products (AvrRpm1 or AvrB type III effector protein) of Pseudomonas syringae, a causal agent of bacterial blight disease [B. oleracea (Bol008133) analogous to the RPM1. We also recognized one conserved gene, Bra040356 in B. rapa corresponding to RPM1 but its domain type could not identified. Another disease resistance gene, RPS2 in A. thaliana is known to have NBS, LRR and LZ domains and confers resistance to P. syringae avirulence gene avrRpt2[RPS2 homologs with high nucleotide similarity in Brassica species, like B. montana AF180358 (98% identity), B. oleracea AF180357 (98% identity) and B. rapa AF180359 (95% identity). Later Malvas et al. [B. oleracea RPS2 homolog in disease resistance which is constitutively expressed. In our study, two CNL type NBS genes, one in B. rapa (Bra013947-previous GenBank ID AF180359) and one in B. oleracea (Bol039594- previous GenBank ID AF180357) were found to be conserved parallel to the RPS2 gene with 94.59% nucleotide similarity. In addition to that both B. oleracea and B. rapa have retained one copy of (Bra027599) and (Bol032054) genes parallel to RPS4 which was previously recognized to specify resistance to avirulence gene avrRps4 from P. syringae pv. Pisi in A. thaliana[B. rapa (Bra019754 and Bra016781) were preserved with RPS5 disease resistance gene in A. thaliana that is responsible for recognizing avirulence gene avrPphB from P. syringae pv.Phaseolicola[We retrieved the characterized gene-for-gene type NBS proteins in database  and theidatabase . These p disease . In the e avrRpt2. Earliere avrRpt2 identifis et al.  also des thaliana. Two genseolicola.Brassicaceae family  however, comparisons across plant species demonstrated that in T. cacao, P. trichocarpa, V. vinifera; and M. truncatula the CNL class predominates. In contrast to the NBS-encoding genes, the current analysis revealed a higher number of TIR-X (TX-lacking both NBS and LRRs) and TIR-NBS (TN-lacking LRRs) genes in B. oleracea (82 and 29) than A. thaliana (46 and17), B. rapa (42 and 23), Theobroma cacao (17 and 4), Populus trichocarpa (67 and 10) and Vitis vinifera (10 and 14). Furthermore, in three Brassica species  the number of both TX and TN genes and only TN genes was found to be higher than T. cacao, V. vinifera and P. trichocarpa respectively. B. oleracea despite having a larger genome (630 Mb) contained less NBS genes than A. thaliana (123 Mb) and B. rapa (485 Mb) which is may be because of prevalence of higher number of TX, TN and N type genes. TX, TN and TNL genes are prevalent in both conifers and dicots which point out their presence approximately 300 MYA before the divergence of these taxa [The present study suggests the abundance of TNL class in ese taxa . The fun 82 and 2 than A. B. oleracea, 55.7% in A. thaliana and 47.1% in B. rapa were formed by tandem duplication and distributed in tandem arrays. Tandem duplicated analysis reveal that the rate of tandem duplicated genes was found to be higher in A. thaliana than Brassica species. After tracing the fate of tandem arrays before or after complete genome triplication, 50 NBS genes were tandem duplicated genes after divergence of B. oleracea and B. rapa species, representing 31.8% of total NBS-encoding genes in B. oleracea genome. There are 83 NBS genes generated after divergence of B. oleracea and B. rapa species, which represents 40.3% of all NBS genes in B. rapa genome. Through analysis of whole genome triplication, 42 B. oleracea and 62 B. rapa NBS genes were retained on triplicated regions in genomes, representing 26.75% and 30.1% of total NBS genes in B. oleracea and B. rapa genomes, respectively.Plants have experienced more genome duplication events than any other eukaryotes on the earth. After the duplication, genes can follow one of the three functional outcomes, gene loss, neo-functionalization and sub-functionalization . In duplB. oleracea, B. rapa and A. thaliana, we speculated that after WGT of Brassica ancestor approximately 16 MYA, orthologous gene pairs for NBS-encoding genes on triplicated regions in genome of Brassica ancestor were lost or deleted quickly. The quick loss of paralogs from whole genome duplication might be due to the gene dosage imbalance issue [B. rapa and B. oleracea approximately 3.7 MYA, NBS-encoding genes in Brassica species experienced species-specific gene amplification by mechanism of tandem duplication to adopt environment selection. The increase of gene dosage by tandem paralogs might have some advantage to the plant pathogen defense.Polyploidy event provide rich genomic resources to study retention and loss of multi-copy genes. Through comparative analysis of NBS-encoding genes among ce issue , 59. ButBrassica species made some progress. We found eight conserved genes in Brassica species corresponding to the four A. thaliana R genes  species in the conserved orthologous region. In present study, we observed one (N type) gene in B. oleracea and two (CNL type) genes in B. oleracea and B. rapa corresponding to RPM1 and RPS2 respectively. Therefore, we propose that these conserved genes in the Brassica species may offer gene-to-gene resistance to specific avirulence products from Pseudomonas syringae pathogen. A. thaliana gene RPS4 (AT5G45250) retained one corresponding orthologous gene in B. oleracea (Bol032054) and B. rapa (Bra027599) genomes respectively, so we assume that these two genes in Brassica species might be race-specific resistant to Pisi and Phaseolicola subspecies of Pseudomonas syringae pathogen. RPS5 retained two copies only in B. rapa and these gene copies are species-specific disease resistance genes in B. rapa species.To environmentally friendly control plant diseases, one of the efficient methods is the employment of genetically disease resistant plants. In the last few years, the studies regarding disease resistance inheritance and molecular cloning of R genes in A. thaliana - B. rapa and A. thaliana - B. oleracea lineages. A. thaliana - B. oleracea lineage exhibited higher mean Ka/Ks ratios in their orthologous gene pairs than those of A. thaliana - B. rapa lineage in CNL type NBS-encoding R genes. We conclude that these NBS-encoding genes in B. rapa species have undergone stronger negative selection than those in B. oleracea species. So, the corresponding NBS-encoding genes in B. oleracea species would have experienced stronger evolutionary constraints to adopt changes in the environment. But for TNL type, there are no significant differences between the two species about the orthologous gene pairs. We speculated that these NBS-encoding genes in B. rapa and B .oleracea species may have undergone different selection pressure to offer resistance to same pathogen or some pathogens may be species-specific pathogens for Brassica species.For evolutionary relationship of orthologous gene pairs for race-specific NBS-encoding genes among three species, we compared Ka/Ks values of orthologous gene pairs between A. thaliana, B. rapa and B. oleracea genomes respectively and total number of NBS-encoding genes in these three species is very close in spite of genome size and WGD/WGT events. Genomic organization and composite phylogenetic analysis facilitate the identification and classification of NBS-encoding genes among A. thaliana, B. rapa and B. oleracea. Expression profiling showing the differential expression pattern of orthologous NBS-encoding genes provides a blueprint for further characterization of these genes in B. oleracea and B. rapa. The expression profile of different NBS-coding members can be separated into different groups, indicative of functional divergence. Although, orthologous NBS-encoding genes in B. oleracea and B. rapa are highly divergent but expression pattern divergence among paralogs within a species exceeds the level of divergence among orthologs in each type of NBS-encoding genes. Paralogs might contribute more to functional divergence than orthologs over the evolution of Brassicaceae. Through comparative analysis of tandem duplication and whole genome triplication in NBS-encoding genes among three species, there are fewer paralogous NBS-encoding genes retention after whole genome triplication than those from tandem duplication. So, tandem duplication might play more important influence than whole genome duplication in generation of NBS-encoding genes. We speculated that the quick loss of paralogs from whole genome duplication might be due to the gene dosage imbalance issue. The increase of gene dosage by tandem paralogs might have some advantage to the plant pathogen defense. Our evolutionary studies illustrate that CNL type orthologous genes in B. rapa species compared to A. thaliana have undergone stronger negative selection than those in B. oleracea species compared to A. thaliana and opposite to that orthologous genes in B. oleracea species experienced stronger evolutionary constraints than those in B. rapa species for CNL type R genes. For TNL type NBS-encoding genes, we did not observed significant difference between the two species about the orthologous gene pairs using Mann–Whitney U-test. It is indicated that these orthologous NBS-encoding genes in B. rapa and B .oleracea species maybe undergone different selection pressure to resist the same pathogen or some pathogens may act as species-specific pathogens for different Brassica species. Through comparative analysis of NBS-encoding genes among A. thaliana, B. rapa and B. oleracea, we hope to explore the evolutionary fate of NBS-encoding genes in Brassica lineage after split from Arabidopsis thaliana and advance the understanding of disease resistance between B. oleracea and B. rapa species, which will provide a valuable model for studying functional and evolutionary aspects within the Brassica genus and the crucifer lineage.We have identified 157, 206 and 167 NBS-encoding genes in B. oleracea, B. rapa and A. thaliana. This table contain the type, distribution of NBS domains, protein full length, subfamily, lists of predicted domains, coding sequences and peptide sequences of NBS-encoding genes in B. oleracea, B. rapa and A. thaliana genomes. (XLS 2 MB)Additional file 1: Table S1: Information of NBS-encoding genes in B. oleracea, A. thaliana and B. rapa. The Maximum Likelihood tree was constructed by MEGA 5.0 software with 1000 replications. CNL type of NBS-encoding genes was divided into three sub-groups and TNL type was divided into three sub-groups. Each species was shown by different colors. (JPEG 723 KB)Additional file 2: Figure S1: Phylogenetic relationship of NBS-encoding genes among B. oleracea and B. rapa genomes. I. Heat map representation of NBS-encoding genes in B. oleracea genomes. II. Heat map representation of NBS-encoding genes in B. rapa genomes. The tissues used for expression profiling are indicated at the top of each column. The genes are on right expression bar. Color scale bar at the bottom of each heat map represents log2 transformed FPKM values, thereby values more than 2, 0 and less than -2 represent positive, zero and negative expression, respectively. (JPEG 1 MB)Additional file 3: Figure S2: Heat map representation of NBS-encoding genes in B. oleracea, B. rapa and A. thaliana. This table contain name, location, gene numbers, gene lists of tandem arrays in B. oleracea, B. rapa and A. thaliana genomes. (XLS 53 KB)Additional file 4: Table S2: List of Tandem arrays of NBS-encoding genes among A. thaliana compared to B. oleracea and A. thaliana compared to B. rapa genomes. This table contain co-retained tandem duplicated genes of NBS-encoding genes in A. thaliana compared to B. oleracea and A. thaliana compared to B. rapa genomes. (XLS 24 KB)Additional file 5: Table S3: Co-retained tandem duplicated genes of NBS-encoding genes in


Bacillus Calmette-Guerin (BCG) therapy for bladder cancer. An 82-year-old man presented with low back pain. Past medical history revealed bladder cancer diagnosed and treated 16 months previously by intravesical BCG. Magnetic resonance imaging of the thoracic spine showed destruction of the T5 and T6 vertebrae and an epidural soft tissue mass with anterior dural sac compression. Due to the progression of vertebral destruction, posterior spinal segmental fusion was performed. Mycobacterium bovis (M. bovis) was identified using multiplex polymerase chain reaction of surgical tissue specimens. The patient was started on an antituberculosis treatment regimen including isoniazid, rifampicin, and ethambutol. After surgery, his back pain resolved completely. At the latest examination, the patient was pain-free with no functional limitations or recurrent infection in clinical or imaging findings. Patients undergoing BCG therapy should be monitored for possible hematogenous spread of mycobacteria to the spine for months or even years after treatment.We present a rare case of tuberculous spondylitis following intravesical Bacillus Calmette-Guerin (BCG), an attenuated derivative of the virulent strain of Mycobacterium bovis (M. bovis), is widely used and effective in preventing superficial bladder cancer recurrence [ M. bovis tuberculosis spondylitis occurring 16 months after intravesical BCG therapy in an immunocompetent patient with bladder carcinoma.currence –3. Seriocurrence . The incAn 82-year-old man was admitted with a 2-month history of low back pain and no history of trauma, fever, night sweats, or cough. Past medical history revealed bladder cancer diagnosed 16 months previously and treated with 8 cycles of intravesical BCG.The patient was admitted with back pain and tenderness on palpation and percussion over the spinous processes of the upper thoracic vertebrae. A lateral pelvic compression test and extreme range of motion of the hips and sacroiliac joints were painless. Deep tendon reflexes were normal. There was no motor weakness or paresthesia. Initial routine laboratory examination showed mild normochromic normocytic anemia, normal white blood cell count, an erythrocyte sedimentation rate of 33 mm/first hour, and a C-reactive protein of 0.75 mg/dL. The QuantiFERON-TB test result was negative. Plain radiographs showed T5 and T6 compression fractures Figures . A chest M. bovis was subsequently identified using multiplex polymerase chain reaction (Computed tomography-guided needle biopsy was performed for suspicion of a tuberculous infection. Histological examination of the biopsy specimen revealed a granulomatous infection. Due to progressive vertebral destruction and severe low back pain, T3–8 posterior spinal segmental instrumentation and fusion was performed via a posterior approach Figures . Tissue reaction . The diaAfter surgery, the patient's back pain resolved completely. Antituberculous treatment was completed 6 months after diagnosis and surgical treatment. One year after surgery, the patient was pain-free with no functional limitations or recurrent infection.Intravesical BCG immunotherapy for treatment of superficial bladder cancer and in situ carcinomas has a low risk of adverse effects, most of which are localized and self-limited . Althoug M. bovis bacilli obtained from a distant site strongly suggest dissemination of BCG infection [There is no obvious evidence regarding the mechanism of systemic manifestations that are related to BCG immunotherapy or vaccination. The possibility of both dissemination of BCG infection and hypersensitivity reactions has been debated. However, positive cultures fornfection , 8. Hemanfection , predictnfection . The timnfection . Therefo M. bovis bacilli. These are a bladder epithelium injury, urethral injury during BCG instillation, deep bladder tumor resection, pelvic radiation, severe cystitis and bladder biopsy, or prostate resection [Several risk factors are considered with an incidence of dissemination of viableesection , 16, 17. M. bovis BCG strains from M. tuberculosis to differentiate between BCG reactivation and reinfection with M. tuberculosis. Recently, rapid molecular diagnostic testing has been developed that differentiates M. bovis BCG from its parent M. bovis strain [ M. bovis [It is very important to reliably distinguishs strain . Other mM. bovis , 18.The role of prophylactic antituberculous therapy accompanying intravesical BCG therapy is debated. Rawls et al. recommended 3 days of prophylactic isoniazid therapy beginning the morning before treatment . However M. tuberculosis, all strains of M. bovis are resistant to pyrazinamide. Hence, isoniazid and rifampicin, with or without ethambutol, should be used as first-line agents to treat M. bovis infections [ M. bovis tuberculous spondylitis, surgical intervention is usually necessary for spinal instability [Unlikefections , 20. In fections . For M. tability , 12, 13. M. bovis spondylitis. Predisposing factors to dissemination should be addressed to minimize the risk of hematogenous spread of live attenuated BCG to the spine. Furthermore, the timeframe for BCG spondylitis onset after intravesical BCG therapy is variable. Patients having BCG therapy should be monitored for possible hematogenous spread of mycobacteria, which can arise months or even years after therapy. If BCG spondylitis is suspected, diagnosis and treatment are needed to avoid unfavorable sequelae.In conclusion, we presented a rare case of intravesical BCG therapy complicated by


Borrelia bavariensis was detected by PCR in 2 aspirates obtained from different joints. Complete recovery was achieved after a 4-week course with amoxicillin. Lyme arthritis must be considered in patients from Europe who have persisting joint effusions.A case of Lyme oligoarthritis occurred in an 11-year-old boy in Vienna, Austria. DNA of  Borrelia burgdorferi sensu lato (s.l.) complex. In Europe, several genospecies of B. burgdorferi s.l. cause the disease, whereas in North America, B. burgdorferi sensu stricto is the only agent of Lyme borreliosis. This difference causes variability in clinical manifestations  and was referred to another hospital because of persistent effusions of all 3 joints. Clinical investigation revealed swelling of both knees, which were not warm or red, and a swollen, hot, red left ankle.A routine blood test showed a normal leukocyte count, elevated C-reactive protein levels , and elevated erythrocyte sedimentation rates . Tests for rheumatoid factor and other autoantibodies  showed negative results. The patient underwent needle aspiration of all 3 joints under general anesthetic to obtain synovial fluid: 18 mL from the right knee, 60 mL from the left knee, and 6 mL from the left ankle. The patient was given a preliminary diagnosis of juvenile idiopathic arthritis and treated with nonsteroidal antiinflammatory drugs. A few days later, another aspiration of the left knee was performed, followed by an intraarticular injection of steroids.Chlamydia spp. (IgG and IgA enzyme immunoassays), Mycoplasma pneumoniae (IgG and IgA enzyme immunoassays), Salmonella spp. (agglutination assay), and Yersinia spp. (agglutination assay). Antibodies against B. burgdorferi s.l. were detected by Borrelia ELISA ; IgG levels were highly elevated . Test results obtained by using the Anti-Borrelia Euroline Westernblot  were positive for IgG with strong band intensities for VlsE, p83, p39, p30, p21, p19, and p17 and weak band intensity for p25 (OspC). Cytologic test results for synovial fluid showed an inflammatory infiltrate with lymphocytes and segmented neutrophils. Culture for bacterial pathogens was negative.Infection with pathogens associated with reactive arthritis was ruled out by negative serologic test results for B. burgdorferi s.l. strain was used as a positive control, and PCR-grade water was used as a negative control. To check for PCR inhibition, we used samples spiked with borrelial DNA in an extra well. In 2 of the 3 joint aspirates (left knee and left ankle), borrelial DNA was detected by both assays. For genotype identification, samples were subjected to a previously described nested PCR targeting the 5S-23S intergenic spacer region . Two TaqMan-based real-time PCR assays targeting the 16S rDNA gene (Borrelia strain in both joints. When compared with known sequences by using BLAST (http://www.ncbi.nlm.nih.gov/blast), the sequences showed 100% identity with that of the PBi strain.Amplicons were purified by using the QIAquick PCR Purification Kit  and sent to MWG Eurofins  for bidirectional sequencing by using primers B5S-23S_Fn and B5S-23–S_Rn  (B. burgdorferi s.l. Published studies have most commonly identified B. burgdorferi sensu stricto in joints from Lyme arthritis patients in Europe (B. bavariensis, the pathogen that caused the illness in the patient we describe, was formerly classified as B. garinii genospecies characterized by OspA serotype 4 (B. garinii strains. Furthermore, the 2 genotypes differ in their hosts: B. bavariensis is a rodent-associated strain, whereas other B. garinii serotypes can be found in birds.In Europe, Lyme arthritis can be caused by several genospecies of n Europe . B. bavaMost Lyme arthritis patients respond well to a single course of treatment with antimicrobial drugs, although in a small percentage of cases persistent synovitis can develop months or even years after treatment. For those patients whose synovial fluid PCR result is negative, intraarticular application of corticosteroids can be beneficial (This case illustrates that Lyme arthritis must be taken into account in patients in Europe who have persisting joint effusions. Treatment with antimicrobial drugs is highly effective. We did not find any other report of cases in which the pathogen was detected in multiple joints by using a direct identification method. This case is further evidence for the systemic characteristics of Lyme borreliosis.