Mollicutes contamination is recognized to be a critical issue for the cultivation of continuous cell lines. In this work we characterized the effect of Mycoplasma hyorhinis contamination on CD133 expression in human colon cancer cell lines.® and mycoplasma agar culture were used to detect mycoplasma contamination on GEO, SW480 and HT-29 cell lines. Restriction fragment length polymorphism assay was used to determine mycoplasma species. All cellular models were decontaminated by the use of a specific antibiotic panel . The percentage of CD133 positive cells was analyzed by flow cytometry on GEO, SW480 and HT-29 cell lines, before and after Mycoplasma hyorhinis eradication.MycoAlertMycoplasma hyorhinis infected colon cancer cell lines showed an increased percentage of CD133+ cells as compared to the same cell lines rendered mycoplasma-free by effective exposure to antibiotic treatment. The percentage of CD133 positive cells increased again when mycoplasma negative cells were re-infected by Mycoplasma hyorhinis.Mycoplasma hyorhinis infection has an important role on the quality of cultured human colon cancer cell lines giving a false positive increase of cancer stem cells fraction characterized by CD133 expression. Possible explanations are (i) the direct involvement of Mycoplasma on CD133 expression or (ii) the selective pressure on a subpopulation of cells characterized by constitutive CD133 expression.In keeping with United Kingdom Coordinating Committee on Cancer Research (UKCCCR) guidelines, the present data indicate the mandatory prerequisite, for investigators involved in human colon cancer research area, of employing mycoplasma-free cell lines in order to avoid the production of non-reproducible or even false data. It is well-known that mycoplasma can have adverse effects on cell cultures such as altered levels of protein and of RNA/DNA synthesis, induction of chromosomal aberrations, changes in cell membrane composition and modification of cellular morphology ,2. FurthSince human continuous cell lines derived from solid tumors are often used to explore biological and functional alterations of tumor cells, the use of mycoplasma-free cell cultures for research and specifically in isolating cancer stem cells (CSCs) is mandatory .Mycoplasma hyorhinis contamination. To address this issue, we employed the well-established GEO, SW480 and HT-29 human colon cell lines [The aim of this study is to show how and to what extent the expression of CD133 antigen can be altered by ll lines  and multMollicutes contamination [Mycoplasma hyorhinis contamination, after antibiotic decontamination it was possible to evidence a decrease in the percentage of CD133 positive cellular population. On these bases we decided to demonstrate that Mollicutes agents can have remarkable effects on the correct identification and characterization of CD133 positive CSCs subpopulation.As imposed by good tissue culture practice, we screened the colon cancer cell lines both for a putative mination ,8 and fomination ,10. In s2 until complete confluence, they were then detached by trypsin/EDTA . During the study, all cell cultures were periodically tested for the absence of bacteria and fungi contaminations by the BacT/ALERT® 3D  instrument, an automated microbial detection system. As regards mycoplasma detection, the cell lines were cultured in absence of antibiotics for at least 2 weeks after thawing and samples were taken after a culture period of at least 2 or 7 days without medium exchange, as suggested by CC Uphoff and HG Drexler [Human colon cancer continuous cell lines were obtained from a research laboratory at CEINGE, Biotecnologie Avanzate . In particular, GEO, SW480 and HT-29 cell lines were cultured in DMEM, RPMI 1640 and McCoy's 5A Medium Modified  respectively, 10% foetal bovine serum  and 1% Ultraglutamine 1 . All cell lines were grown in T-75 flasks  in humidified atmosphere of air containing 5% COvs FSC-Height dot plot was used to identify single cells and exclude doublets. Putative CSCs and immature cells were gated in a CD133 vs empty channel (FITC) dot plot.After enzymatic detachment from saturated cultures, GEO, SW480 and HT-29 cell lines were studied for CD133 expression . For each sample the respective control was prepared in order to determine the level of background cellular autofluorescence without antibody staining. All samples were incubated for 20 minutes at 4°C, washed twice with PBS  and finally analyzed using a FACSAria flow cytometer and the FACSDiva software . Cells were first gated on physical parameters (FSC and SSC) to exclude the majority of debris and dead cells. Afterwards, an FSC-Area ® mycoplasma detection kit  using the MycoAlert® assay control set , as suggested by the manufacturer's instructions.During the study, supernatants of GEO, SW480 and HT-29 cell lines were collected and tested with the MycoAlertAll the samples were also tested by the microbiological aerobic and anaerobic agar culture.Mycoplasma agar and broth were used as media for the detection of all mycoplasma species and were prepared according to the manufacturer's instructions . In particular, the mycoplasma agar was obtained by dissolving 2.84 grams of mycoplasma agar base  in 80 ml of distilled water and sterilized by autoclaving at 121°C for 15 minutes. After cooling, it was combined with 20 ml of mycoplasma selective supplement-G  and dispensed into Petri plates. In order to prepare the mycoplasma broth, 2.04 grams of mycoplasma broth base  were dissolved in 80 ml of distilled water and sterilized by autoclaving at 121°C for 15 minutes. After cooling, the mycoplasma broth was mixed with 20 ml of mycoplasma selective supplement-G  and, finally, aliquots of mycoplasma broth were imparted into tubes.Therefore, cell culture supernatant test samples (100 μl) were pipetted onto the mycoplasma agar plates and incubated anaerobically for 14 days at 37°C. Aliquots (100 μl) of the same test sample were incubated in mycoplasma broth; after a 14 days aerobic incubation, aliquots of 100 μl were subcultured onto mycoplasma agar for 14 days in anaerobic conditions. Development of typical mycoplasma colonies, characterized by a "fried egg" appearance, was observed under the inverted microscope.2, 1.25 U Jump Start Taq DNA polymerase  and 0,2 μM of each primer. The PCR products were subjected to the restriction fragment length polymorphism assay. In particular, the amplicons were digested for 1 hour at 37°C in a final volume of 20 μl with the restriction endonucleases AspI, XbaI, HpaII, and HaeIII . Restriction fragments were then analyzed on a 1.5% agarose gel and visualized by ethidium bromide staining. The fragments polymorphism pattern identified the mycoplasma contaminant species [Mycoplasma positive GEO, SW480 and HT-29 cell lines were cultured for 7 days without antibiotics. One ml of each cell culture supernatant was centrifuged for 10 minutes at 13000 g at 4°C. The obtained pellets were washed twice with PBS  and incubated for 15 minutes at 95°C. Mycoplasmatic DNA was extracted and purified using the Nucleon BACC2 kit . As recommended by CC Uphoff and HG Drexler , the PCR species .Mycoplasma eradication was accomplished by the antibiotic treatment of the infected GEO, SW480 and HT-29 cell lines. Of note, we utilized the three classes of antibiotics that were shown to be highly effective against mycoplasmas . In factIn particular, infected cells were treated with the quinolones enrofloxacin  and Mycoplasma Removal Agent  at concentration of 25 μg/ml and 0.5 μg/ml, respectively, for 7 days. Cells were also exposed to the quinolone ciprofloxacin  at the concentration of 10 μg/ml for 14 days. Moreover, the anti-micoplasmatic treatment, included the BM Cyclin 1 , containing the macrolide tiamulin, for 3 days at the final concentration of 10 μg/ml, followed by the BM Cyclin 2 , containing the tetracycline minocycline, for 4 days at the concentration of 5 μg/ml.® mycoplasma elimination reagent  was used. The kit includes a combination of antibiotic and antimetabolic agents protected by a patent. MycoZap® reagent 1 was used for 4 days, while reagent 2 was used for 12 days, according to the manufacturer's data sheet.Finally, MycoZapAs for mycoplasma detection, all antibiotic-treated cell lines were cultured for at least 4 weeks without any chemotherapic molecules.Mycoplasma hyorhinis-driven infection. The growth of the Mycoplasma hyorhinis was carried out using the media and culture conditions recommended by the supplier .Approximately 80% confluent monolayer of mycoplasma-eradicated GEO cell line was used for the 2. Every 7 days, supernatants were tested for mycoplasma detection and, in case of positive result the cells were detached with trypsin/EDTA and processed for cytometric analysis.On the day of infection, the old medium was replaced with fresh medium containing the dilution of viable mycoplasma at a concentration of about 10 CFU/ml, previously determined by the microbiological aerobic and anaerobic agar culture. The infected cells were then incubated at 37°C, 5% COMollicutes contamination is recognized to be a critical issue for the cultivation of continuous cell lines and about 30% of all cellular models are reported as mycoplasma-infected with a limited number of human species origin [Mollicutes agents can heavily interfere with cellular activities such as growth rate, DNA, RNA and protein synthesis as well as cause significant cell morphology alterations [Mollicutes agent contamination as indicated by good tissue culture practice.s origin . It is werations -16. Cons® assay [Mollicutes agent contamination, being the B/A score of 62, 53 and 16, respectively (data not shown). Later on, these results were confirmed by aerobic and anaerobic microbiological agar culture (data not shown). In order to determine the contaminant species, we performed the analysis of modified restriction fragment length polymorphisms. The application of this method revealed that GEO, SW480 and HT-29 cell lines were infected by Mycoplasma hyorhinis, one of the most common cell culture contaminants encountered [MycoAlert® assay  showed tountered   the direct influence of mycoplasma on anti-CD133 MoAb binding sites or ii) the ability of CD133 positive cells to survive despite the toxic effects exerted by the mycoplasma.Our results demonstrate that Mollicutes contamination should be included in the basic panel of CSCs research techniques. We are confident that technical advancements in cell lines handling will facilitate CSCs studies, positively influencing oncological research improvement in the near future.In conclusion, methods for the detection, elimination and prevention of The authors declare that they have no competing interests.EM conceived the study and performed the cell culture experiments. MG performed flow cytometry experiments. PM and FDA contributed to data analysis. RDN analyzed data and wrote the manuscript. GF participated in data interpretation and revision of the manuscript. LDV conceived the study, designed the experiments and wrote the manuscript together with RDN. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/10/120/prepub


Mycobacterium tuberculosis in human remains. We screened long bones from an 18th century cemetery and skulls from the anatomical "Weisbach collection" (19th century). In addition, besides the study of abundance of tuberculosis in inmates of the poorhouse itself, we were interested to test whether in this particular instance tuberculosis can be identified from cortical bones, which are rarely affected by tuberculosis, but mostly better preserved than the vertebral bodies or epiphyses.In the present pilot study we applied recently published protocols for detecting The DNA extractions from the bone samples were obtained following established ancient DNA protocols. Subsequently extracts were subjected to a series of PCR amplifications using primer pairs published previously ,2. PCR p6110 insertion sequence yielded PCR products of appropriate size. In one sample only (skull sample WB354 of the "Weisbach collection") sequence analysis revealed an authentic M. tuberculosis sequence that matched to a reference sequence from GenBank.Only primers targeting the repetitive ISM. tuberculosis DNA in historic human femurs from an 18th century cemetery relating to a poor house in Kaiserebersdorf, Austria. Our data may indicate that in this particular case, thoracic or lumbar vertebrae, i.e. bones that are severely affected by the disease, would be more suitable for molecular diagnostics than long bones. However, the unpredictable state of DNA preservation in bones from museum collections does not allow any general recommendation of any type of bone.With a variety of established PCR approaches we failed to detect  Spondylitis tuberculosa, "Pott's disease") and joint tuberculosis are caused by Mycobacterium tuberculosis and appear among others as late manifestations of a tuberculosis infection. Because of the inflammatory bony changes, e.g., partial or total destruction of the vertebral bodies or joint elements, manifestations of tuberculosis infections of (pre)historic human skeletal remains are very often identifiable by means of macroscopic inspections. Since the spread of M. tuberculosis depends on population density, its verification is also of high interest for the reconstruction of population dynamic processes in archaeology and anthropology [Bone tuberculosis . Such bones are rarely affected by tuberculosis but are frequently much better preserved than the vertebral bodies or epiphyses, and preservation of authentic ancient DNA in the bones' middle regions has been shown likely [In the present pilot study, we screened 10 long bones from the 18n likely . Accordin likely .All samples were taken from the collections housed at the Department of Anthropology, Natural History Museum Vienna, Austria. Ten samples were taken from the midshaft femur of ten different individuals recovered from the burial site at the castle of Kaiserebersdorf, south-east of Vienna: KE1, KE8, KE18, KE20, KE22b, KE26b, KE22, KE23, KE26c, KE29. . Tubercuth century were used. Individual data such as sex, age-of-death, and cause-of-death were recorded in handwritten protocols [As positive controls, three bone samples (core samples obtained from skull vaults) from the anatomical "Weisbach collection", dated to the end of the 19rotocols : WB 565,investigated = 2000). Therefore, we considered them as negative controls.Finally, two bone samples  were taken from the site Hainburg (Lower Austria) that dates back to the early Bronze Age : HB284 , and HB310 . From this epoch, bones with morphological alterations due to tuberculosis have never been reported from Austrian localities  or the DyNAzyme  DNA polymerase in 1× Reaction Buffer, 0.2 mmol/L of each dNTP, 0.04% bovine serum albumin (BSA), 1 μmol/L of each primer, and 2 μL of DNA extract (DNA concentration not quantified). Thermal cycling conditions were: 94°C for 2 min; 32 cycles of 94°C for 50 sec, 48° – 60°C for 50 sec, and 72°C for 1 min; and a final extension at 72°C for 10 min. PCR products were purified for direct sequencing using 8 μL 10× diluted exoSAP-IT solution  per reaction. Cycle sequencing, using the same primers as in the PCR reaction, was performed in 10 μL reactions using 2 μL BigDye Terminator Cycle Sequencing Ready Reaction Kit , 2 μL 5× Sequencing Buffer, 10 pmol primer, and 3 μL purified PCR product. Sequencing products were purified with ethanol precipitation and analyzed using an ABI 3100 Genetic Analyzer .PCR amplifications were performed using the PCR products that could not be sequenced directly were cloned in the pCR4Blunt-TOPO plasmid vector by means of the TOPO TA Cloning Kit (Invitrogen) and individual clones were subsequently sequenced.Taq DNA polymerase (Roche) in 1× Reaction Buffer, 0.2 mmol/L of each dNTP, 1 μmol/L of each primer, 50 ng genomic DNA of Microtus maximowiczii and 2 μL of DNA extract (DNA concentration not quantified). Thermal cycling conditions were: 94°C for 2 min; 35 cycles of 94°C for 20 sec, 56°/57°C for 30 sec, and 72°C for 60/40 sec; and a final extension at 72°C for 7 min. The primers were Pro+ (5'-ACC ATC AGC ACC CAA AGC TG-3') and Phe- (5'-AAG CAT TTT CAG TGC TTT GCT T-3') for the ~1000 bp fragment, and Pro+ and mico3- (5'-GTA AAA GAA GCA TTA ATT AAA-3') for the ~300 bp fragment [Ancient DNA extracts may contain PCR inhibiting components that can cause misleading results. In such instances failure to obtain PCR products cannot be assigned to the absence of the target DNA but simply to inhibition of the reaction. In order to test for co-extracted PCR inhibitors in the samples used in the current study, established test PCRs targeting a ~1000 bp and a ~300 bp fragment of the mitochondrial genome of microtine rodents were spiked with ancient DNA extracts. Spiking PCR amplifications  were perfragment . None ofThe bone samples were processed independently in the DNA labs of the Natural History Museum Vienna and the Natural History Museum Oslo following standards to avoid contamination of samples: These include in particular, usage of DNA, DNAse, and RNAse-free consumables and reagents, as well as pipette tips with aerosol protection filters, and UV-treatment of tubes prior to extraction and PCR amplifications. Negative controls for extractions (without any sample) as well as PCR reactions (without any DNA) were included in the experimental setup. The samples from Hainburg  were considered as additional negative controls. Samples from presumably TB-containing cases were never processed before the experiments on the negative controls and samples of unknown infection status had been finished. In both labs no studies of modern mycobacterial or human DNA had been performed before, and especially no modern mycobacterial DNA was used as positive PCR controls.Mycobacterium tuberculosis, several PCR products were obtained. OxyR amplified for the skull WB594 using the primer pair R1/F4. The PCR targeting the IS6110 insertion sequences yielded PCR products of appropriate size for the skulls WB354, WB565, and WB594 using primer pair TbA/TbB, and for the long bones KE23 and HB284, and the skulls WB354 and WB594 using primer pair TbC/TbD.When subjecting the long bone samples from Kaiserebersdorf, Austria, as well as the positive and negative controls to PCR amplification targeting various regions in the genome of 6110 insertion sequences of M. tuberculosis (reference sequence: AF 181860), and was therefore considered positive. The sequence has been deposited in GenBank under the accession number FJ177511. None of the other cloned PCR products yielded sequences with similarity to M. tuberculosis but proved unspecific PCR amplification .When sequencing the obtained PCR products only WB354 amplified with the primer pair TbC/TbD yielded a sequence identical with the targeted ISWith the exception of the unspecific amplification of non-target DNA of samples KE23 and HB284 when using the TbC/TbD primer pair, DNA extracts from long bones never yielded PCR products that were visible as distinct band of appropriate size on ethidium-bromide stained agarose gels. Even reamplification attempts were negative for the various primer pairs.th century burials in Kaiserebersdorf, Austria. A positive result, i.e., PCR amplification of a DNA fragment with a nucleotide sequence matching the IS6110 insertion sequences of M. tuberculosis deposited in GenBank, was only obtained for the skull WB354. This was not unexpected since this sample served as positive control and tuberculosis had been diagnosed earlier in this individual by means of the spoligotyping approach [6110 insertion sequence proofs the suitability of the applied PCR approach. However, also for this sample positive PCR amplification was only obtained with the primer pair TbC/TbD that targets the repetitive insertion sequences IS6110, but not for any of the other molecular markers tested. This may indicate that only very limited amount of template DNA could be extracted from this particular sample. The failure to amplify any tuberculosis specific target DNA from the other positive skull samples may further support this interpretation. It should be considered that a M. tuberculosis strain which did not contain IS6110 has been reported earlier [6110 as a single marker for PCR-facilitated detection M. tuberculosis might be insufficient. Nevertheless, the IS6110 insertion sequence is very likely the most sensitive marker [M. tuberculosis genome and yielded positive results while other markers such as, e.g., oxyR failed [In the present study we aimed at diagnosing tuberculosis in historic human long bones from 18approach . The amp earlier . Thus, te marker  in the MR failed .6110 insertion sequence could not de determined in the long bones of the samples KE20 and KE23 from the poor house in Kaiserebersdorf, Austria. Given the difficulties in amplifying target DNA of M. tuberculosis in the positive skull samples, it may not be surprising that none of the test bones from the burials in Kaiserebersdorf yielded any specific PCR product, although it seems likely from other anthropological evidence that at least samples KE20 and KE23 relate to individuals that suffered from tuberculosis.However, even the ISM. tuberculosis DNA from the samples from Kaiserebersdorf may relate to the bone samples themselves. Long bones such as, e.g., the femurs that are rarely affected by tuberculosis may represent material that is hardly suitable for the molecular detection of M. tuberculosis. This interpretation may hold for the particular samples under study, but it has been shown earlier that M. tuberculosis can be detected in historic femur samples [Several reasons can account for the negative results on the long bone samples from Kaiserebersdorf: First, some individuals may not have suffered from tuberculosis. This can almost certainly be excluded for samples KE20 and KE23. Second, no DNA was preserved in the samples from Kaiserebersdorf. With the data at hand, we can currently not reject such interpretation. We refrained from amplifying other human target DNA in order to test for the presence of amplifiable DNA. The human actin gene has been suggested for this purpose . However samples  althoughIn the current study, we failed to positively diagnose tuberculosis in DNA extracts from historic human long bones from inmates of a poor house in Kaiserebersdorf, Austria. Using thoracic or lumbar vertebrae for diagnosing tuberculosis, i.e., bones that are severely affected by the disease might have been more successful than long bones. This issue will be further explored in a future study. We conclude that the unpredictable state of DNA preservation in bones from museum collections does not allow any general recommendation of any type of bone.LB planned the project, conducted the labwork at the NHM, Vienna, and participated in writing and editing the manuscript. BD and LK conducted the labwork at the NHM, Vienna. CL conducted the labwork at the NHM, Oslo. MTN planned the project, contributed the samples, and edited the manuscript. EH planned the project and participated in writing and editing the manuscript. All authors approved the final version of the manuscript.


Cocos nucifera L., the coconut, the origins of which have been one of the “abominable mysteries” of palm systematics for decades. Previous studies with predominantly plastid genes weakly supported American ancestry for the coconut but ambiguous sister relationships. In this paper, we use multiple single copy nuclear loci to address the phylogeny of the Cocoseae subtribe Attaleinae, and resolve the closest extant relative of the coconut.The Cocoseae is one of 13 tribes of Arecaceae subfam. Arecoideae, and contains a number of palms with significant economic importance, including the monotypic and pantropical Syagrus as sister to Cocos and resolve novel and well-supported relationships among the other genera of the Attaleinae. We also address incongruence among the gene trees with gene tree reconciliation analysis, and assign estimated ages to the nodes of our tree.We present the results of combined analysis of DNA sequences of seven WRKY transcription factor loci across 72 samples of Arecaceae tribe Cocoseae subtribe Attaleinae, representing all genera classified within the subtribe, and three outgroup taxa with maximum parsimony, maximum likelihood, and Bayesian approaches, producing highly congruent and well-resolved trees that robustly identify the genus Cocos and Syagrus. This is not only of biogeographic interest, but will also open fruitful avenues of inquiry regarding evolution of functional genes useful for crop improvement. Establishment of two major clades of American Attaleinae occurred in the Oligocene (ca. 37 MYBP) in Eastern Brazil. The divergence of Cocos from Syagrus is estimated at 35 MYBP. The biogeographic and morphological congruence that we see for clades resolved in the Attaleinae suggests that WRKY loci are informative markers for investigating the phylogenetic relationships of the palm family.This study represents the as yet most extensive phylogenetic analyses of Cocoseae subtribe Attaleinae. We present a well-resolved and supported phylogeny of the subtribe that robustly indicates a sister relationship between  Cocos nucifera L., the coconut, is a charismatic monotypic genus forming a dominant part of littoral vegetation across the tropics. Besides its paradisiacal connotation, the coconut plays a vital role at many different economic levels Cocos nucifera is pantropically distributed, a present day range significantly influenced both by a seed well-adapted to oceanic dispersal and the species' importance to humans Cocos was first proposed by de Candolle Cocos nucifera belongs to the monophyletic Cocoseae Elaeis guineensis Jacq. , Bactris gasipaes Kunth , and many other species of value in local economies Bactris Jacq., Cocos L., and Elaeis Jacq. alliances. Uhl and Dransfield Elaeis Jacq., has both a species endemic to tropical America and another in Africa.Cocos, two endemics in Madagascar , and Jubaeopsis Becc. , the majority of the genera are Neotropical endemics Within the Cocoseae, the coconut is part of the moderate-sized subtribe Attaleinae Despite the importance of the Cocoseae, a well-resolved phylogeny for this tribe has been elusive Theobroma cacao L. T. cacaoC. nucifera germplasm collections The utility of WRKY loci for determining infraspecific relationships has been demonstrated by genetic mapping in Cocos nucifera in order to resolve the closest extant relative of the coconut, the evolutionary relationships of the other genera in the subtribe, determine how paleohistorical events shaped the evolution and biogeography of the Attaleinae, and demonstrate the utility of WRKY loci for phylogenetic inference within the Arecaceae.Our study focuses on reconstructing the phylogenetic relationships within the subtribe Attaleinae, and represents the most intensive sampling of the group so far in a molecular analysis. We use sequences of seven putatively independent, single copy WRKY loci originally isolated from Excluding gaps, the number of phylogenetically informative characters  ranged fExamination of the individual consensus trees and partitioned Bremer indices , 2 indicThe combined sequence matrix consisted of 5831 total characters of which 974 were parsimony informative (17%). Heuristic search with parsimony found 446 equally most parsimonious trees of length = 2614, CI = 0.73 and RI = 0.87. Combining all seven WRKY loci yielded the most fully resolved trees and the highest bootstrap support , both wiBactris (Bactridinae). Most clades had posterior probability (PP) scores = 100%. The only clades with <90% were at several terminal nodes in Syagrus.The ML  tree wasSyagrus, which is paraphyletic with Lytocaryum. The very well-supported subtribe  consists of three clades. The first represents the African genera (91/95% BP), within which Becarriophoenix is sister to a Jubaeopsis/Voanioala clade (84/84% BP). Partitioned Bremer support for this clade (90% BP) consists of Cocos strongly supported as sister to Syagrus inclusive of Lytocaryum . Only WRKY2 is incongruent with this resolution (Lytocaryum as nested within Syagrus (96% BP), clade of American genera unites Butia and Jubaea in a clade (100/100% BP) that is sister to a fairly well-supported clade (88/96% BP) of Allagoptera (100/100% BP), Parajubaea (100/100% BP) and Polyandrococos. Allagopetra and Polyandrococos form a clade with moderate to high support (84/97%). A monophyletic Attalea (100/100% BP) is sister to the rest of this clade. Within Attalea, monophyletic subg. Attalea (100/100% BP) and Scheelea (75/81% BP) subclades are resolved, while the Orbignya group appears paraphyletic to Scheelea (Syagrus clade includes a “rain forest” group (86/98% BP), uniting species from Amazonas, the Andean foothills, the Caribbean and the Atlantic rain forest of Brazil  unites three clustering (multiple stems) species with the solitary-stemmed S. macrocarpa.The monophyly of all genera of the Attaleinae is supported by the combined WRKY data matrix , with this clade  is positsolution . Four of Syagrus –4. The sScheelea . Substruf Brazil , to whicReconciling individual gene trees to the species trees  necessitated significant costs in terms of deep coalescence events . Locus WSyagrus in which Lytocaryum is embedded (some of the gene trees used as input resolved Lytocaryum as sister to Syagrus). The American and African clades are resolved. In the African clade, Jubaeopsis and Voanioala are sisters, with Beccariophoenix subtending, as with MP, ML and BA  would appear to be the oldest genus in the American Attaleinae, with a crown age of 27 MYBP (15.4–30.5 95% HPD), while the equally speciose Attalea is relatively young at 13 MYBP (8.6–20.5 95% HPD). The three main clades of Syagrus s. s. share crown node ages of ca. 16.6–17.5 MYBP.The maximum-clade-credibility tree  producedJubaeopsis). Both major clades of the American Attaleinae remained restricted to eastern Brazil until an Eocene/early Oligocene range extension to coastal Chile or Southern Brazil by the MCRA of Allagoptera, Butia, Jubaea, Polyandrococos and Parajubaea, and Miocene dispersals to Amazonas and the West Indies . Range extensions of Syagrus, first into contiguous Southern Brazil and Argentina-Paraguay, did not occur until the mid-Miocene  and Eastern Brazil (E) as the ancestral distribution for the Attaleinae, with subsequent vicariance between the two hemispheres , and one-Miocene , 3, withAllagoptera, Parajubaea and Polyandrococos all have have nonvascular bundles attached to their hypodermis layers  lent support to one of the clades resolved by the WRKY genes. s layers , but onldrococos . Howevergoptera. Giardia lamblia Kofoid & Christiansen and the mycetozoan Dictylostelium discoideum Raper, but are absent from the genomes of fungi and animals 2H2- or C2HC-type zinc finger motif. Eulgem et al. Arabidopsis (DC.) Heynh. Group 1 WRKY genes were defined by the presence of two WRKY domains, each of the C2H2-type zinc finger motif, with only the C-terminal WRKY domain actively binding DNA. Group 2 WRKY genes contained only a single WRKY domain, and were classified into subgroups a-e based upon additional amino acid motifs found outside the WRKY domain. Group 3 WRKY genes were defined by the presence of the C2HC-type zinc finger motif in the DNA-binding WRKY domain. Further annotation of Arabidopsis WRKY genes Oryza L. and Arabidopsis WRKY gene families have resulted in several minor modifications to the original classification scheme WRKY transcription factors are predominantly plant-specific proteins, broadly distributed across the genome D. discoideum, G. lamblia, and Chlamydamonas reinhardtii Dangeard, several from the moss Physcomitrella patens (Hedw.) B.S.G. and the fern Ceratopteris richardii Brongn. Oryza sativa L. A feature common to WRKY genes is interruption of the coding region of the highly conserved, DNA-binding functional WRKY domain  with an intron. The size and sequence of the intron varies in each gene, but its position is highly conserved within each group/subgroup Oryza sativa, of the 102 WRKY loci described from subsp. indica, 99 are unigenes; of the 98 copies characterized in subsp. japonica, 97 are unique Theobroma, Persea and Oryza than with each other. One locus (WRKY21) belongs to group 2b. Four of the loci belong to Group 1 (two WRKY domains). Two of these (WRKY6 and WRKY7) resolve in two distant clusters based on either the C- or N-terminus domains. Two loci , tended to cluster close together based on their conserved domains, thus raising the possibility that they could represent recent paralogs. Nonetheless, primers designed for each of these three amplifies a single product in the vast majority of species that we sampled, suggesting that their divergence occurred before the diversification of the Cocoseae. Further evidence of the independence of these loci is the fact that the sequences of all seven amplified from a single species could not be aligned except over portions of their highly conserved WRKY domains.Paralogy is the leading concern when using nuclear genes, especially members of multigene families. Paralogous sequences from gene duplication events due to unequal crossing over, replicative transpostition or ancient polyploidization events, when unrecognized, may lead to erroneous phylogenetic inferences prk from across all 20 genera of Cocoseae supported recognition of a “spiny” clade , and a “non-spiny” clade, i.e., the Attaleinae. Asmussen et al.'s Voanioala and Jubaeopsis as sister genera. Gunn's n = 16 chromosome number that is characteristic of the American clade Beccariophoenix madagascariensis has n = 18, while Jubaeopsis (n = 80–100) and Voanioala (n = 298) have been suggested as autopolyploids Jubaeopsis and Voanioala are polyploids, while Beccariophoenix has a chromosome number much closer to the American Attaleinae.Gunn's Cocos alliance” resolved by WRKY genes consist of only Cocos nucifera, Syagrus and Lytocaryum (embedded in Syagrus), positioned as sister to the “Attalea alliance” of all other American genera. As in Gunn's Jubaea and Butia is very well supported, congruent with their similar leaf Attalea as sister to Butia/Jubaea has high support only with ML and BA , plus both morphological and RFLP datasets, did not help resolve the exact position of Cocos within the Attaleinae Parajubaea in a clade with only 52% BP. The WRKY consensus positions C. nucifera as sister to the South American genus Syagrus, and the clade has very strong support  when outgroups were removed. When Syagrus and Lytocaryum were removed from the alignment, Cocos resolved as the first branch after the African clade and thus sister to the remaining American Attaleinae. With Syagrus excluded, Cocos resolved as sister to Lytocaryum. With even just a single species of Syagrus included in the analysis (we successively included just one species from each of the three main Syagrus subclades), Cocos resolved as its sister. We believe that the relationship between Cocos and Syagrus resolved by seven concatenated WRKY gene alignments is real, considering our substantial depth of sampling of the Attaleinae, and that the number of steps as well as the appreciable time duration (>20 MY) between the stem and crown nodes of Cocos reflects intervening extinction events.With three plastid genes  support –2, last  support , Table 5s WRKY7: , S4, onlSyagrus is concentrated in Eastern Brazil  used in this paper were introgressed with other cultivars Parajubaea with Allagoptera and Polyandrococos . Gunn's prk sequences embedded Polyandrococos within Allagoptera, and Dransfield et al. Allagoptera. Polyandrococos is sister to Allagoptera, rather than embedded within it as Gunn prk, thereby rendering Dransfield et al.'s Polyandrococos to Allagoptera equivocal. Both Allagoptera and Polyandrococos have spicate inflorescences and share similar pinnae phylotaxy Parajubaea, still uncertainly consisting of 1–3 species JubaeaAllagopteraPolyandrococosParajubaea was aligned with Butia, Jubaea and Syagrus by Uhl and Dranfield Allagoptera, Parajubaea and Polyandrococos.The most surprising clade is that which unites Jubaeopsis), and two Madagascar endemic genera  that are both high polyploids, Voanioala with the highest chromosome number known in the monocotyledons Plant species exchange between Africa and South America continued after direct connections were severed in the late Cretaceous, at least through the early Paleocene, possibly via the Walvis Ridge/Rio-Grande Rise and Sierra Leone Ridges Allagoptera (including Polyandrococos), Butia, Jubaea and Parajubaea had dispersed to southern Brazil, the Andes and coastal Chile by the early Oligocene  and in the wild from the Andes of southern Bolivia  Jubaea/Butia and Parajubaea/Allagoptera (including Polyandrococos) in the proto-Andean region at this time is congruent with a Pacific marine incursion known as the Western Andean Portal (WAP) or Guayaquil gap, which a number of studies have proposed took place from the Eocene to the mid Miocene Parajubaea and the eastern Brazilian Allagoptera (including Polyandrococos) corresponds to the flooding of western Amazonia caused by the uplift of the Eastern Cordillera of the Central Andes in the early Miocene and subsequent Caribbean marine incursion to the north Jubaea and Butia, however, appears to have occurred considerably later (14.5 MYBP), perhaps as the central Andes rose to sufficient elevation to obstruct dispersal or fragment a broad ancestral range of their MRCA.Establishment of the two major clades of American Attaleinae on the other hand does appear congruent with the terminal Eocene cooling event .Based on the estimated dates, much of the subsequent diversification in the Attaleinae can be attributed to the Andean uplift from late Miocene through the Pliocene Cocos nucifera, the only two species of Attaleinae found in the Caribbean are both endemics: Attalea crassispatha (9 MYBP) in Haiti and Syagrus amara (8.4 MYBP) in the Lesser Antilles with essentially contemporaneous late Miocene divergence dates from their respective congenors.Other than the pantropical Cocos and Syagrus. Branch age estimates of our phylogeny are, in many cases, congruent with known paleohistorical events in South America. The biogeographic and morphological congruence that we see in the resolution of the larger genera such as Attalea and Syagrus  and Elaeis oleifera (Elaeidinae). The strategy for larger genera was to sample evenly from all recognized geographic or morphological groups. Multiple individuals were sampled for several species in several genera as a consistency check.DNA was isolated from living accessions of 75 taxa , mostly DNA was extracted from silica gel dried leaf samples using the BIO101 kit as described in Mauro-Herrera et al. Cocos nuciferaC. nucifera were subjected to BLAST analyses against the non-redundant database at GenBank Arabidopsis thaliana, Theobroma cacao, Oryza sativa, and Persea americana WRKY proteins using ClustalX Cocos nucifera.By using a degenerate primer pair 4, 0.025 U/µl reaction volume Taq DNA Polymerase  and 10 ng of template DNA brought to a total volume of 15 µls with nuclease-free H20. Amplifications were conducted using PTC-225 thermalcyclers . Conditions were: 95°C, 2 min;  x 35 cycles; 72°C, 10 min; 4 C, hold. Amplification success was determined by agarose gel electrophoresis in 1.2% agarose, 0.5 x TBE buffer, ethidium bromide-stained, and visualized with UV light. Amplifications were treated with Exonuclease I and Shrimp Alkaline Phophatase to remove any unincorporated PCR primers and dNTP's, ethanol precipitated, and resuspended in sterile H2O. Direct sequencing was done in both directions on 1–2 µl of the treated amplification product with either the forward or reverse primer used for the initial amplification. All sequencing was done by capillary electrophoresis on an ABI 3100 or 3730 Genetic Analyzer using the BigDye Terminator Cycle Sequencing Ready Reaction Kit v3.1 .Amplifications contained: 0.200 nM forward and reverse primer, 200 µM dNTPs, 1 mg/ml bovine serum albumin, 1 x amplification buffer with 2 mM MgSOIn the preliminary screening, seven WRKY loci produced a single band migrating at the expected mobility upon gel electrophoresis for each of three taxa amplified. Direct sequencing of the amplification products of most samples  gave cleAll sequences have been deposited in GenBank  and assiCloning was necessary for several taxa for six of the seven loci . For preAttalea, Butia and particularly Syagrus exhibited small indel polymorphisms among the clones, often evidence of interspecific hybridization, which has been reported in these three genera In most of the cases where cloning was necessary, clones showed allelic variation at SNPs or microsatellite repeats within introns, but consistently resolved as sisters with 100% BP when incorporated into the aligned matrix. For these, which constituted the majority, we used consensus sequences in our final alignments. Several species of Sequences were aligned using MAFFT Aligned sequences for the seven WRKY loci were analyzed separately and in combination using MP with PAUP* v. 4.0b10 MP tree searches were heuristic, conducted under the Fitch  weights Treefinder Elaeis oleifera was designated as outgroup. Two and one half million Markov chain Monte Carlo (MCMC) generations were run, with convergence diagnostics calculated every 1000th generation for monitoring the stabilization of log likelihood scores. Convergence was also evaluated for each of the two parallel runs using Tracer v.1.4 Two parallel runs were performed in MrBayes, each consisting of four chains, one “cold” and three incrementally heated. de novo species trees by conducting 25 heuristic searches in GeneTree with random tree starting points, no constraints, and gene tree bootstrapping . A strict consensus tree was generated in PAUP* from the species trees found by GeneTree.Different loci used to infer phylogenetic relationships of the same taxa often result in incongruent topologies Attaleineae and dated to ∼60 MYBP Cocos” Cocos nucifera would be rash . Although normal prior distributions are used when dating trees under an indirect approach http://cbsuapps.tc.cornell.edu/beast.aspx). This cluster imposes a time limit of three days (72 hours) per analysis but allows several runs of the same analysis simultaneously. Analyses were undertaken by sampling every 1000th generation. Tracer v.1.4 A total of 10 different runs of 10 million generations each were undertaken on the online cluster of the Computational Biology Service Unit from Cornell University , and all parameter estimates were consistent between runs. Runs were thus combined, after removing a burnin of 100 trees each , into a single run of ca. 100 million generations. All parameters, including age estimates, reached acceptable ESS values and were thus deemed reliable (ESS>100). The tree files were combined after a burnin of 100 trees for each run. As the resulting combined tree file was too large to analyze in TreeAnnotator, combining of the different runs was redone. It was thus re-sampled at a lower frequency of every 5,000nnotator .The results presented in this paper are based on a non-partitioned analysis. A partitioned analysis , and O = western Amazonas. For Cocos nucifera, which is pantropical, we assigned only those areas of these fifteen where the species is currently found.The biogeographic patterns inferred from our gene trees were assessed using the dispersal-vicariance method of analysis Allagopetra, Parajubaea and Polyandrococos were hand sectioned with a double edged razor blade on a cutting board after folding the leaflet back and forth on itself various times to facilitate sectioning. Sections were not stained. Dried material was boiled for 5–10 minutes in water with Aerosol Detergent .Leaflets of Figure S1Strict consensus trees from parsimony analysis for loci WRKY2-12 (four loci).(1.31 MB TIF)Click here for additional data file.Figure S2Strict consensus trees from parsimony analysis for loci WRKY16, 19, and 21 (three loci).(1.00 MB TIF)Click here for additional data file.Figure S3Maximum likelihood bootstrap consensus trees for each of four WRKY loci: WRKY2, 6, 7, and 12.(0.87 MB TIF)Click here for additional data file.Figure S4Maximum likelihood bootstrap consensus trees for each of three WRKY loci: WRKY16, 19, and 21, as well as the combined analysis .(0.94 MB TIF)Click here for additional data file.Figure S5Strict consensus tree of 190 lowest “cost” reconciled species trees found by 25 heuristic searches by the program GeneTree using gene tree bootstrapping.(0.08 MB TIF)Click here for additional data file.Table S1All 75 taxa used in the study are listed with voucher specimens and GenBank accession numbers for the WRKY sequences.(0.12 MB DOC)Click here for additional data file.Table S2This table lists the specific primers used to amplify and sequence the seven WRKY loci from members of Arecaceaea tribe Cocoseae.(0.03 MB DOC)Click here for additional data file.


Arabidopsis thaliana (L.) Heynh and Oryza sativa L., a large number of genes encode proteins of unknown functions, whose characterization still remains one of the major challenges. With an aim to characterize these unknown proteins having defined features (PDFs) in plants, we have chosen to work on proteins having a cystathionine β-synthase (CBS) domain. CBS domain as such has no defined function(s) but plays a regulatory role for many enzymes and thus helps in maintaining the intracellular redox balance. Its function as sensor of cellular energy has also been widely suggested.In Arabidopsis and 59 in Oryza. In most of these proteins, CBS domain coexists with other functional domain(s), which may indicate towards their probable functions. In order to investigate the role(s) of these CDCPs, we have carried out their detailed analysis in whole genomes of Arabidopsis and Oryza, including their classification, nomenclature, sequence analysis, domain analysis, chromosomal locations, phylogenetic relationships and their expression patterns using public databases (MPSS database and microarray data). We have found that the transcript levels of some members of this family are altered in response to various stresses such as salinity, drought, cold, high temperature, UV, wounding and genotoxic stress, in both root and shoot tissues. This data would be helpful in exploring the so far obscure functions of CBS domain and CBS domain-containing proteins in plant stress responses.Our analysis has identified 34 CBS domain containing proteins (CDCPs) in Arabidopsis and Oryza. A comprehensive analysis of expression patterns for CDCPs using the already existing transcriptome profiles and MPSS database reveals that a few CDCPs may have an important role in stress response/tolerance and development in plants, which needs to be validated further through functional genomics.We have identified, classified and suggested the nomenclature of CDCPs in  It has been reported that some of these proteins of unknown function(s) can indeed improve tolerance of transgenic plants to oxidative stress [All eukaryotic genomes sequenced so far, contain a number of genes that encode for proteins whose functions are still unknown. These proteins have been documented to be induced under specific set of conditions and participate in protein-protein interactions and/or sometimes are also associated with mutant phenotypes . These pin/motif ,2. A proin/motif . Recentle stress .Oryza sativa, we have made an attempt to characterize several unknown members of the stress responsive machinery [To understand the probable mechanism of abiotic stress tolerance in achinery . A groupinosine-5'-monophosphate dehydrogenase (IMPDH), AMP-activated protein kinase (AMPK), chloride channels (CLC) and cystathionine-β-synthase (CBS). The importance of CBS domain was realized by the observation that point mutations in the CBS domain cause several hereditary diseases in humans [Methanococcus jannaschii as a conserved domain in a group of proteins. CBS domain exists not only in archaebacterial proteins, but also in eubacterial and eukaryotic proteins [CBS domains are found to be associated with several proteins of unrelated functions, such as n humans . CBS domn humans  in the gproteins . The namproteins .et al. [CLC-Nt1) was identified from tobacco [Arabidopsis [In CBS protein, C-terminal CBS domain exerts an autoinhibitory effect on the CBS activity, while binding of SAM (S-adenosylmethionine) with CBS domain induces a conformational change which relieves the autoinhibitory effect. Mutation in CBS domains abolish or strongly reduce activation by SAM and cause homocystinuria. CBS domain of γ-subunit of AMPK acts as sensor of cellular energy status and mutations cause a glycogen storage disease, which is clinically expressed as a familial hypertrophic cardiomyopathy (Wolff-Parkinson-White syndrome) -12. Scotet al.  reportedet al. . Earlieret al. . In CLCset al. -21. Howe tobacco , thereafbidopsis -25 and rbidopsis , but funArabidopsis an ideal plant for study of newly identified protein family [Arabidopsis thaliana and Oryza sativa where we have identified and classified the CDCPs based on their conserved features. To further establish their possible involvement in development and abiotic stresses, we have analyzed the expression of genes encoding CDCPs using MPSS database and already existing Arabidopsis microarray database .The availability of complete genome sequence along with the microarray expression and Massively Parallel Signature Sequencing (MPSS) data makes n family . In the Arabidopsis thaliana and Oryza sativa. For this, the CDCPs were searched against the protein sequence database for Arabidopsis and Oryza, using the HMM profile of CBS domain obtained from Pfam database as described earlier [In the present study, we provide information on the CDCPs in  earlier . We propArabidopsis and Oryza, employing the standard bioinformatics tools (described in methods), identified a total of 34 proteins (encoded by 33 genes) in Arabidopsis and 59 proteins (encoded by 37 genes) in Oryza with a distinct CBS domain  [pentatricopeptide repeats (PPR) [Phox and Bem1p (PB1) [inosine monophosphate dehydrogenase (IMPDH) [domain of unknown function (DUF21), while few other proteins only possess the CBS domain(s) in their sequences. In Arabidopsis, 25 proteins containing a single CBS domain were found which are encoded by 24 genes since one gene encoded for two alternate splice variants, whereas in Oryza 46 proteins containing a single CBS domain were found to be encoded by 37 genes. Interestingly, in Oryza an additional class of CDCPs having IMPDH domain was observed, which was not found in Arabidopsis. The existence of more number of CDCPs in Oryza is due to alternative splice events which correspond to the earlier report where it has been shown that in Oryza 36,650 alternate splicing events affected 8,772 genes, while in Arabidopsis 16,252 alternate splicing events affected 5,313 genes [Arabidopsis, as no case of alternative splicing was observed, whereas in Oryza 8 genes encode for 13 proteins. The nomenclature has been assigned according to the domain(s) present in the given sequence such as CBSX for proteins containing only a single CBS domain, CBSDUFCH1 for protein containing one CBS domain along with DUF and CorC_HlyC domains, CBSCLC for proteins containing one CBS domain and a CLC domain, CBSDUF for proteins containing one CBS domain and a DUF domain, CBSSIS for proteins containing one CBS domain and a SIS domain and CBSPPR for proteins containing one CBS and a PPR domain CBSIMPDH for proteins containing one CBS domain along with an IMPDH domain. Whereas, for two CBS domain containing proteins, CBSCBS nomenclature was given to the proteins containing only two CBS domains, and CBSCBSPB for proteins containing two CBS domains and one PB1 domain  , pentatrts (PPR) -38, Phox1p (PB1) -41 and i (IMPDH) . Some of13 genes . In caseArabidopsis reveals the presence of 24 genes which code for 25 proteins containing only single CBS domain, while in Oryza 29 genes coding for 46 proteins containing only single CBS domain were found. In case of two CBS domain proteins, 9 genes code for 9 proteins in Arabidopsis, while in Oryza 8 genes code for 13 proteins [Table Whole genome analysis of CDCPs in , however the biological significance of these domains remains to be elucidated. These CDCPs can act as binding domains for adenosine derivatives and may regulate the activity of attached enzymatic or other domains [Arabidopsis CBSX proteins (CDCP2) has been purified and crystallized [Arabidopsis, 6 genes were found to encode for 6 CBSX proteins, while in Oryza, a total of 22 CBSX proteins were categorized in this subgroup, which were encoded by 12 genes. In Oryza, all the CBSX genes code for only one protein, except for OsCBSX3, OsCBSX4 and OsCBSX7, which code for 5, 6 and 2 proteins, respectively through the alternate splicing mechanism.A fraction of predicted CBS domain-containing proteins harbor only single CBS domain (PF00571). CDCPs have been reported to have the regulatory functions  domains . In some domains . Recentltallized . In caseArabidopsis, this subgroup contains two proteins which are encoded by two genes, while in case of Oryza, two proteins are encoded by a single gene. These proteins contain a domain of unknown function (DUF21) (PF01595) at the N-terminus adjacent to a CBS domain and a CorC_HlyC domain (PF03471) at the C-terminus. DUF21 domain has no known function and is usually present at the N-terminus of the proteins adjacent to a CBS domain. The CorC_HlyC is a transport associated domain and is found at the C-terminus of the proteins. CorC_HlyC domain is also found in magnesium and cobalt efflux protein CorC and some of the Na+/H+ antiporters. The function of this domain is unknown but it might be involved in modulating the transport of ion substrates .In case of Oryza, this subgroup contains four proteins, which are encoded by three genes, as OsCBSDUF1 gene codes for two proteins whereas, in case of Arabidopsis, this subgroup contains seven proteins, which are encoded by seven genes.Proteins classified in this subgroup contain a DUF21 domain at the N-terminus, along with a CBS domain. In case of Oryza and Arabidopsis [Oryza. Whereas in case of Arabidopsis, our results are in accordance with the previous report as we found 8 CLC proteins encoded by 7 genes.These proteins belong to chloride channel protein (CLC) family which sustains a wide variety of cellular functions, including membrane excitability, synaptic communication, transepithelial transport, cell volume recognition, cell proliferation, and acidification of endosomes and lysosomes. Earlier chimeric and deletion approaches had suggested that CBS domain may influence gating of CLCs. Past studies have suggested that mutation in the CBS domain affects protein-protein interaction within CLC protein subunits as well as between two subunits of the dimer and that they influence the voltage dependence of gating through the common gate . Role ofbidopsis . HoweverArabidopsis and Oryza. The SIS domain is widespread and found in all species including, prokaryotic, archaebacterial and eukaryotic proteins. In general SIS domain is found in proteins that have a common role in phosphosugar isomerization as SIS domain functions by binding to the phosphosugars. The SIS domains are also found in a family of bacterial transcriptional regulators [Escherichia coli iron transporters [Proteins containing sugar isomerase (SIS) domain along with a CBS domain have been classified in this subgroup. We have identified one CBSSIS gene encoding only one protein in cases of both, gulators  as well sporters .Arabidopsis and Oryza, this subgroup is composed of a single gene encoding for a single protein, containing a pentatricopeptide repeat (PPR) motif and a CBS domain. The PPR motif, first described by Small et al. [Arabidopsis PPR family proteins has identified 441 members and their further analysis revealed that PPR proteins play constitutive, often essential roles in mitochondria and chloroplast, probably via binding to organeller transcripts [Oryza and Arabidopsis, contains a CBS domain in the same reading frame. Occurrence of CBS domain along with PPR repeat in a protein suggests that this protein might be involved in the various cellular processes by sensing the cell energy status [In both l et al.  is a degl et al. . A genomnscripts . Many plnscripts . Interesy status . StructuOryza, only one CBSIMPDH gene has been identified, which code for two CBSIMPDH proteins while in Arabidopsis, no member of this subgroup has been identified. Interestingly, in Oryza CBSIMPDH proteins, CBS domain lies within the IMPDH domain. IMPDH domain has been identified in the sequence of IMPDH enzyme, which is a key enzyme in the de novo guanosine nucleotide biosynthesis. Scott et al. [in vitro and that the tetrameric IMPDH binds ATP in a positive, cooperative way. They have also observed that IMPDH was activated by ATP, which was never reported earlier. This observation strongly supported the verity that ATP binding to the CBS domain allosterically activates IMPDH and consequently XMP synthesis. If so, this mechanism would couple the GTP/dGTP biosynthesis to the cellular energy status i.e., high ATP levels [Proteins containing inosine-5'-monophosphate dehydrogenase (IMPDH) domain (PF00478) along with CBS domain has been classified in this subgroup. In t et al.  have shoP levels .Oryza, 8 CBSCBS proteins have been identified, which are encoded by 5 different genes. The OsCBSX7 gene, which has been classified in CBSX subgroup, also codes for a protein OsCBSCBS1 containing two CBS domains. The OsCBSCBS4 gene encodes for four proteins through an alternative splicing mechanism whereas, in case of Arabidopsis, AtCBSCBS subgroup contains four genes, which encode four proteins.In this subgroup, proteins containing only two CBS domains have been classified. In case of Oryza, this subgroup possess 4 genes, which encode for 4 proteins, while in Arabidopsis 5 CBSCBSPB genes encode for 5 proteins. The PB1 domains are present in many eukaryotic cytoplasmic signaling proteins. They are dimerization/oligomerization domains present in adaptor and scaffold proteins and kinases that serve to organize platforms that ensure specificity and fidelity during cellular signaling. Recently, a number of studies have provided valuable information on the structural details that govern binding between the different PB1 modules and explain how they direct the formation of different macromolecular signaling complexes [These proteins contain Phox/Bemp1 (PB1) domain along with the CBS domain. In case of omplexes . Proteinomplexes . The funArabidopsis, the family of 33 CDCP genes was found to be distributed randomly on all the 5 chromosomes  CDCP genes, were found to be located on chromosome V, followed by 9 on chromosome I, 7 on chromosome IV, 5 on chromosome III and 2 on chromosome II. While in Oryza maximum (9 in number) CDCP genes were found to be located on chromosome I, followed by 7 on chromosome III, 6 each on chromosome II and IV, 3 on chromosome VIII, 2 each on chromosomes IX and XII; and 1 each on chromosomes V and VII.In s Figure , while is Figure . In caseArabidopsis and 9 chromosome of Oryza, at which they were found to be located, is not uniform. Their chromosomal distribution pattern reveals that some CDCP genes are found in clusters on certain chromosomes at various chromosomal regions. Occurrence of cluster of genes belonging to a family at certain chromosomes and chromosomal regions is common. Jain et al. [OsbZIP) have also been reported to be present in clusters at certain chromosomes and chromosomal regions [Oryza, 4 genes encoding two CBS domain containing proteins were found in close vicinity at chromosome I. Percent identity amongst all the four genes clustered was found to be in the range of 30 to 60%.The distribution of the CDCP genes on the 5 chromosome of n et al.  have rep regions . In OryzArabidopsis [Oryza [Arabidopsis genome, with many losses and rearrangements, leaving a mosaic of "segmental duplications" or "duplication blocks" [Arabidopsis and Oryza. Figure Arabidopsis and Figure Oryza. In Arabidopsis, AtCBSX4 at chromosome I seem to be duplicated segmentally, and a domain duplication event might have occurred, which have resulted in the appearance of AtCBSCBS4 gene on the same chromosome. AtCBSDUFCH2 gene at chromosome I was observed to be duplicated to chromosome III as AtCBSDUFCH1. These duplicated genes show 85% identity at the nucleotide level and 79% identity at the protein level. AtCBSCLC8, present on the chromosome III seems to be duplicated segmentally to chromosome V as AtCBSCLC6 and at the same time the gene observed to have undergone inversion. AtCBSCBSPB2 present at chromosome II seems to be segmentally duplicated at chromosome III as AtCBSCBSPB3. These genes share 86% identity at the nucleotide level and 67% identity at the protein level. Interestingly in Oryza, it was observed that during the course of duplication the structural domains accumulate variations in order to adapt to new function. For example OsCBSDUF2 gene present on chromosome III got duplicated on chromosome I as OsCBSCBS5. In other instances the CDCP genes were duplicated but retained their structural domains, like OsCBSCBSPB2 present on chromosome XI got duplicated as OsCBSCBSPB4 on chromosome XII. OsCBSCLC9 on chromosome II was found to be duplicated as OsCBSCLC8 on chromosome VIII. OsCBSCBS2 located on chromosome I was observed to be duplicated as OsCBSCBS3 on chromosome IV.The sequence information and analysis of the bidopsis -60 and Os [Oryza  whole ge blocks" -65. AnalOryza have been observed to have a percent identity of 55% to 60% within themselves, and 30% with that of Arabidopsis except for the AtCBSX1 which showed identity of more than 80% with OsCBSX1 and OsCBSX2. Similar identity pattern was also observed for the OsCBSCLC proteins, which showed identity ranging from 30% to 80% within their own subgroups and also with AtCBSCLC proteins. The OsCBSDUF proteins were observed to have identity ranging from 50% to 60% with the AtCBSDUF proteins. The OsCBSSIS1 protein shares 80% identity with AtCBSSIS1 and OsCBSPPR1 protein shares 60% identity with AtCBSPPR1 protein. OsCBSCBS proteins share 40% to 75% identity among themselves. The two CBS domain proteins also showed 50% to 70% identity with the other two CBS domain members. Apart from having variations, the CBS domain also accumulates large insertions. The large insertion or deletion might have helped the CBS domain proteins to evolve in order to perform specific functions. Results from the sequence alignment suggested that the sequences might have evolved according to the other functional domains present in the sequences which might have led to their specialized functions.Sequence analysis of all the CDCPs shows an overall homology amongst their own respective groups  provides a sensitive quantitative measure of gene expression for nearly all genes in the genome . To studArabidopsis and Oryza showed that AtCBSX4 has no expression value in any of the tissues under any condition according to the MPSS database while in Oryza OsCBX12 has no expression values in the MPSS database. In Arabidopsis, genes encoding for proteins with CorC_HlyC functional domain, which has a major role in magnesium and cobalt efflux, along with CBS domain seems to be expressed more in siliqua, leaves and inflorescence. In Oryza, OsCBSDUFCH1 gene was observed to show more expression in leaves and other plant tissues, except roots. The gene AtCBSDUFCH1 is expressed more in inflorescence of ap1-10, ap3-6 mutants than in the normal condition of inflorescence. AtCBSDUFCH2, which is duplicated from AtCBSDUFCH1 on chromosome III, is expressed more in leaves and seed. In case of Arabidopsis, CBSCLC genes showed more expression in callus, roots and leaves overall in the MPSS analysis, while in case of Oryza, OsCBSCLC7 seems to be more expressed in root tissues, while other members were found to be expressed more in aerial part of the plant. It was observed that in TIGR ver 6, the data for OsCBSCLC10 was not available in the MPSS database. The CBSSIS1 gene seems to be expressed more in callus, normal inflorescence and leaves, while CBSPPR1 gene showed more expression in leaves, seed, siliqua and roots in Arabidopsis. In Oryza CBSSIS1, CBSPPR1 and CBSIMPDH1 genes were observed to maintain a constant level of expression in all the plant tissues.Analysis of CDCP genes in Oryza, whereas in case of Arabidopsis, AtCBSCBS4 does not show expression in any of the plant part considered for the MPSS analysis. AtCBSCBS2 showed enhanced levels of expression in normal inflorescence, leaves and callus. While, AtCBSCBS3 showed expression in other inflorescence such as ap1-10, ap3-6 and agamous mutant conditions but does not show any expression in the normal inflorescence.Among the groups of genes having two CBS domains, MPSS analysis showed higher expression for all the CBSCBS genes in all the plant parts in Arabidopsis, AtCBSCBSPB1, AtCBSCBSPB2 and AtCBSCBSPB3 showed expression in MPSS analysis. Whereas in Oryza, OsCBSCBSPB3 was observed to show expression in the meristematic tissues and root while rest of the genes of this subclass were found to have been expressed in leaves also. In Arabidopsis, AtCBSCBSPB1 showed more expression in callus, root and normal inflorescence, while AtCBSCBSPB2 showed expression only in normal inflorescence and siliqua. This data indicates that CDCP genes exhibit a strict tissue specific expression which is also developmentally controlled.In Arabidopsis, we took advantage of the available data on transcriptional profiling . Analysis of microarray data indicated that some of the CDCP genes are regulated by various abiotic stress conditions  light stress causes the formation of oxygen radicals in chloroplasts and has the potential to damage them. However, plants are able to respond to this stress and protect chloroplasts by various means, including transcriptional regulation in the nucleus. Although the corresponding signaling pathway is unknown, there have been attempts to study its regulation [Arabidopsis [Arabidopsis [2O2), is a challenge faced by all aerobic organisms [Thomashow  has showgulation . When exbidopsis . Woundinbidopsis . On expobidopsis . Membranbidopsis ,73 and abidopsis -77. Whenbidopsis . Earlierbidopsis . Under trganisms ,81. Althrganisms . OxidatiThis analysis revealed two important facts about the CDCP genes. On the one hand, we could identify some members  whose expression was found to be unaltered under any stress conditions, in either roots or shoots. On the other hand, some genes could be identified  present in them. The chromosomal position of genes encoding CDCPs gives an insight into their distribution in the whole genome of Arabidopsis and Oryza. In addition, MPSS analysis reveals the differential expression of these genes in various tissues and parts under stress conditions in these plant species. Moreover, microarray expression data gives an overview that the CDCPs might play an important role in stress response/tolerance in Arabidopsis under various stress conditions. Thus, it can be concluded that CDCP genes exhibit strict stress and developmentally regulated expression patterns in Arabidopsis and Oryza. Certainly, there is a need to functionally validate the role of these CDCPs which exhibit "induced expression patterns" under multiple stresses. Another major finding of this work is the observed expansion of CDCP gene family in Oryza as compared to Arabidopsis. This expansion has been noticed in Oryza primarily because of presence of relatively more cases of alternative splicing as well as gene duplications, possibly indicating towards the significant involvement of CDCPs in development and stress responses. This study will be helpful in commenting on the structural and functional aspects of these unexplored proteins with respect to their roles under various abiotic stresses. Tools of functional genomics based on transgenic approach can further help in testing the "candidature" of these proteins with defined features towards improving stress tolerance in crop plants. The leads provided here would also pave the way for elucidating the precise role of individual CDCP proteins in plants.The cystathionine β-synthase (CBS) domain containing proteins (CDCPs) comprise of a large superfamily of evolutionarily conserved proteins which are present in all kingdoms of life. In plants, CDCPs were never reported and hence their occurrence and possible function is still a mystery. The present study has identified CDCPs in Arabidopsis thaliana (TIGR version 5 and TAIR 8) and Oryza sativa (TIGR version 6). The sequences obtained from TIGR were also cross checked with TAIR8 sequences for any new instances of CDCP proteins in Arabidopsis. The domain structure of the CBS was used to identify and classify the CDCP proteins using the TIGR A. thaliana genome sequence version 5.0 and O. sativa version 6.0. Profiles unique to the CBS domain were used to screen all predicted proteins using the HMMER software  [Arabidopsis.The CDCP protein sequences were retrieved from whole genome sequences of PF00571) . We havePF00571) . All theArabidopsis thaliana and Os is for Oryza sativa. The chromosomal positions of the CDCP genes were obtained from TIGR version 6 for O. sativa and TIGR version 5, TAIR version 8 for A. thaliana, and plotted using Dia version 0.96.1, an open source gtk+ based diagram creation program.For convenience, we have assigned name to protein sequence according to the domains observed in the respective protein sequence, where At is for Multiple alignment analyses were performed using MUSCLE (version 3.6) program . The unrArabidopsis MPSS project  mapped to Arabidopsis and Oryza gene models. The signature was considered to be significant if it uniquely identifies an individual gene and shows perfect match (100% identity over 100% length of the tag). The normalized abundance  of these signatures for a given gene in a given library represents a quantitative estimate of expression of that gene.Expression evidence from MPSS  tags was determined from the A. thaliana is: CAF – Callus, hardened tissue that forms to protect the exposed areas of cuttings; INF – Inflorescence, part of the plant that consists of flower bearing stalks; LEF – Leaves – 21 day, untreated, classic MPSS; ROF – Root – 21 day, untreated, classic MPSS; SIFSilique (Seedpod) – 24 to 48 hr post-fertilization, classic MPSS; AP1 – ap1-10 inflorescence  – mixed stage, immature buds; AP3 – ap3-6 inflorescence  – mixed stage, immature buds; AGM – agamous inflorescence  – mixed stage, immature buds; INS – Inflorescence – mixed stage, immature buds; ROS – Root – 21 day, untreated; SAP – sup/ap1 inflorescence – mixed stage, immature buds; S04 – Leaves, 4 hr after salicylic acid treatment; S52 – Leaves, 52 hr after salicylic acid treatment; LES – Leaves – 21 day, untreated; GSE – Germinating seedlings; CAS – Callus (hardened tissue that forms to protect the exposed areas of cuttings) – actively growing, signature MPSS; SIS – Silique (Seedpod) – 24 to 48 hr post-fertilization, signature MPSS.The description of MPSS libraries in O. sativa is: NYR -14 days – Young Roots, NRA – 60 days – Mature Roots – Replicate A, NRB – 60 days – Mature Roots – Replicate B, NGD – 10 days – Germinating seedlings grown in dark, NST – 60 days – Stem, NYL – 14 days – Young leaves, NLA – 60 days – Mature Leaves – Replicate A, NLB – 60 days – Mature Leaves – Replicate B, NLC – 60 days – Mature Leaves – Replicate C, NLD – 60 days – Mature Leaves – Replicate D, NME – 60 days – Crown vegetative meristematic tissue, NPO – Mature Pollen, NOS – Ovary and mature stigma, NIP – 90 days – Immature panicle, NGS – 3 days – Germinating seed, NCA – 35 days – Callus, NSR – 14 days – Young roots stressed in 250 mM NaCl for 24 h, NSL – 14 days – Young leaves stressed in 250 mM NaCl for 24 h, NDR – 14 days – Young roots stressed in drought for 5 days, NDL – 14 days – Young leaves stressed in drought for 5 days, NCR– 14 days – Young roots stressed in 4 C cold for 24 h, NCL – 14 days – Young leaves stressed in 4 C cold for 24 h, 9RO – Roots, I9RR – Roots – Replicate, 9LA – Leaves, 9LB – Leaves – Replicate, 9LC – Leaves, 9LD – Leaves – Replicate, 9ME – Meristematic Tissue, FRO – F1 Hybrid 60 days Mature Root, FRR – F1 Hybrid 60 days Mature Root-Repl, FLA – F1 Hybrid 60 days Mature Leaf Replicate A, FLB – F1 Hybrid 60 days Mature Leaf Replicate B, FLC – F1 Hybrid 60 days Mature Leaf Replicate C, FLD – F1 Hybrid 60 days Mature Leaf Replicate D, FME – F1 Hybrid 60 days Meristematic tissue, PSC – rice developing seeds, 6 days old cypress high milling (99–1710), PSI – rice developing seeds,6 days old, Ilpumbyeo – High Taste, PSL – rice developing seeds, 6 days old, LaGrue-Low Milling, PSN – rice developing seed, 6 days old, Nipponbare-Grain quality control, PSY – rice developing seeds, 6 days old, Expression values obtained from MPSS database for respective CDCP genes were used for making the heatmap using gplots package of open source R software.The description of MPSS libraries in Arabidopsis Information Resource [The microarray data of the selected gene expression for various abiotic stress conditions such as cold, UV, wound, genotoxic stress, drought, osmotic, salt and oxidative stress were retrieved from the Resource . The datSKS, SLS-P and AP made contributions to the conception of the study and in the preparation of the final draft of the manuscript. HRK, AKS and AP devised overall strategy, performed analysis, drafted and edited the manuscript. HRK developed relevant programs for sequence and microarray analysis. All authors read and approved the final manuscript.Multiple sequence alignment of amino acid sequence of single CBS domain in Arabidopsis thaliana and Oryza sativa. Multiple sequence alignment of amino acid sequence of single CBS domain in Arabidopsis and Oryza. The alignments of the domain sequences were obtained using MUSCLE software and figures were prepared using multiple sequence alignment editor-Jalview. The consensus sequence is shown below these graphs.Click here for fileMultiple sequence alignment of amino acid sequence of two CBS domains in Arabidopsis thaliana and Oryza sativa. Multiple sequence alignment of amino acid sequence of two CBS domains in Arabidopsis and Oryza. The alignments of the domain sequences were obtained using MUSCLE software and figures were prepared using multiple sequence alignment editor-Jalview. The consensus sequence is shown below these graphs.Click here for fileOryza sativaMPSS analysis of CBS domain containing proteins in . Expression evidence from MPSS tags was determined from the Oryza sativa MPSS project . The normalized abundance  of these signatures for a given gene in a given library represents a quantitative estimate of expression of that gene. The description of these libraries is given in methods.Click here for fileMPSS analysis of CBS domain containing proteins in A. thaliana. Expression evidence from MPSS tags was determined from the Arabidopsis MPSS project http://mpss.udel.edu/at/. The normalized abundance  of these signatures for a given gene in a given library represents a quantitative estimate of expression of that gene. The description of these libraries is given in methods.Click here for file


LOXL1) polymorphisms are associated with primary open-angle glaucoma (POAG) and exfoliation syndrome (XFS).To assess whether lysyl oxidase-like 1 . Demographic and clinical features of POAG patients and control subjects were compared in terms of the TT/GG compound genotype of rs1048661 and rs3825942.Japanese patients with POAG (n=213) or XFS (n=89) and 191 control subjects were analyzed for rs1048661 and the G allele of rs3825942 were significantly higher in XFS patients than in control subjects . Except for one who had the TG/AG compound genotype, all XFS patients had the TT/GG compound genotype. An almost 250 fold increase in the risk of XFS  was found in patients with the TT/GG compound genotype compared to those without the genotype. There were no significant differences in the genotype and allele frequencies between POAG patients and control subjects. Furthermore, no significant differences were noted in the demographic and clinical features of POAG patients as well as control subjects with and without the TT/GG high-risk compound genotype.There was a significant difference in the genotype frequencies between XFS patients and control subjects (p<0.0001). Frequencies of the T allele of LOXL1 polymorphisms were associated with XFS. However, the frequencies of the polymorphisms differed between Japanese and Caucasian XFS patients. These polymorphisms had no influence on the phenotypic features of POAG patients. Exfoliation syndrome (XFS) is characterized by the accumulation of extracellular fibrillar materials in various organs of the body ,2. In thrs1048661 (R141L) and rs3825942 (G153D), in exon 1 of the lysyl oxidase-like 1 (LOXL1) gene confer risk of XFG, possibly through XFS, in patients from Iceland and Sweden. They reported that approximately 25% of the general population was homozygous for the highest risk haplotype and that the risk of XFG for those 25% was more than 700 times that of individuals carrying only low risk haplotypes. They also reported that the two SNPs in LOXL1 were not associated with primary open-angle glaucoma (POAG). However, the LOXL1 high risk genotype for XFS may affect the phenotypic features such as maximum intraocular pressure (IOP) of patients with POAG. In this study, we investigated whether the two SNPs in LOXL1 were associated with XFS and POAG in the Japanese population. Furthermore, we also investigated in detail the association between the two SNPs and the phenotypic features of patients with POAG.Thorleifsson et al. , in 2007Japanese patients with POAG or XFS including XFG were recruited from ophthalmology practices in the University of Yamanashi Hospital, Enzan Municipal Hospital, Uenohara City Hospital, and Oizumi Clinic in Yamanashi or Nagano Prefecture, which are all located in the central portion of Japan (mostly the same population as that previously reported from Japan ). A diagrs1048661  and rs3825942 , both located in exon 1 of LOXL1, were genotyped by restriction fragment length polymorphism assay. The following primers were used for amplification: forward primer: 5′-GCT GAT CCA GTG GGA GAA CAA-3′ and reverse primer: 5′-GGG CTG GTA GGG GTA GAT GA-3′.Genomic DNA was purified with a Flexi Gene® DNA Kit , and two nonsynonymous SNPs, LOXL1 polymorphic sites. Amplification product (5 μl) was digested with 4 units of Apa I  for 2 h at 25 °C to genotype rs1048661 or digested with 4 units of Hinf I  for 2 h at 37 °C to genotype rs3825942. DNA fragments with G at codon 141 (corresponding to an arginine residue) contained the Apa I restriction site, resulting in fragments of 304 bp+264 bp, while DNA fragments with T at codon 141 (corresponding to a leucine residue) lacked the Apa I restriction site, resulting in a fragment of 568 bp. DNA fragments with A at codon 153 (corresponding to an aspartic acid residue) contained the Hinf I restriction site, resulting in fragments of 335 bp+233 bp, while DNA fragments with G at codon 153 (corresponding to a glycine residue) lacked the Hinf I restriction site, resulting in a fragment of 568 bp. After the amplification products were loaded on 2% agarose gels and electrophoresed, the gels were stained with ethidium bromide and genotypes were obtained  and 5′-Texas Red labeling M13-forward or M13-reverse primer. Sequence data were compared with the sequence registered in the NCBI GenBank (NM_005576), and 141Leu-153Gly homozygosity and 141Arg-153Asp homozygosity were identified and used as positive controls. Heterozygosity control was obtained by mixing these two homozygosity plasmids.After amplification, nucleotide sequences were determined with a Thermo Sequenase2 analysis of the Hardy–Weinberg equilibrium for LOXL1 genotypes was performed for patients and control subjects. Genotype and allele frequency differences between POAG or XFS patients and control subjects were estimated by the χ2 test and Fisher’s exact test, respectively. Statistical analyses of the compound genotype of rs1048661 and rs3825942 were conducted using the χ2 test. We also used a logistic regression model to simultaneously study the effects of multiple variables when comparing XFS patients with control subjects. The predictor variables were age, gender, and TT/GG compound genotype of rs1048661 and rs3825942. The odds ratio of age is per year. We also compared demographic and clinical features including age at the time of blood sampling and diagnosis, gender, family history of glaucoma, refractive error, maximum IOP, and history of laser trabeculoplasty and glaucoma surgery of control subjects and POAG patients with and without the TT/GG compound genotype using the Student t-test for continuous variables and Fisher’s exact test for comparison of proportions. p<0.05 was considered to be statistically significant.Data were analyzed using SAS statistical software . χrs1048661 and rs3825942, in POAG patients, XFS patients, and control subjects are shown in rs1048661 and rs3825942 was noted between XFS patients and control subjects . Frequencies of the T allele of rs1048661 and the G allele of rs3825942 were significantly higher in XFS patients than in control subjects . The compound genotype frequencies of these two SNPs in POAG patients, XFS patients, and control subjects are shown in rs1048661 and rs3825942. A significant difference in compound genotype frequencies was noted between XFS patients and control subjects . After adjusting for age and gender, an almost 250 fold increase in risk of XFS  was found in patients with the TT/GG compound genotype compared to those with no such genotype. No significant differences were noted in the demographic and clinical features of POAG patients as well as control subjects with and without the TT/GG compound genotype , and 191 control subjects were enrolled in this study. The mean age at the time of blood sampling was 62.9±14.8 (standard deviation) years for POAG patients (range: 20–95 years), 76.5±6.6 years for XFS patients (range: 60–92 years), and 65.7±11.4 years for control subjects (range: 41–87 years). The mean age at the time of diagnosis was 54.4±15.3 years and 70.4±8.2 years for POAG and XFS patients, respectively. Genotype and allele frequencies of the two SNPs, genotype .LOXL1 is a member of the lysyl oxidase family of proteins that catalyzes oxidative deamination of lysine residues of tropoelastin, which leads to their spontaneous cross-linking with consequent formation of elastin polymer fibers [rs1048661 and rs3825942 for XFS, the  haplotype had the highest risk, and the risk G allele of rs1048661 was associated with low expression of LOXL1 mRNA, which suggests that low LOXL1 expression predisposes to XFS. Similar frequencies of LOXL1 SNP alleles, genotypes, and haplotypes were also reported in Caucasian Australian [rs1048661 and rs3825942, were associated with XFS. However, the allele, genotype, and compound genotype frequencies of these two SNPs differed between our XFS patients and Caucasian XFS patients, as Hayashi et al. [rs1048661 and rs3825942 and that allele T and not G was the risk allele of rs1048661. It is difficult to provide an explanation why allele T of rs1048661 is associated with XFS in the Japanese population alone. However, these data suggest that rs1048661 (R141L) may be little related to the occurrence of XFS, and the reduced LOXL1 expression by allele G of rs1048661 may be a small change and not necessarily biologically meaningful as Thorleifsson et al. [rs1048661 plays an important role as a risk allele. In our control subjects, the frequency of T allele of rs1048661 was higher than that of G allele, contrary to the reported allele frequencies in Caucasian populations [LOXL1 may affect the low prevalence of XFS in the Japanese population. It remains to be determined whether there are subtypes of XFS with different patterns of manifestation. XFS patients in this study presented with either unilateral or bilateral involvement with or without elevated IOP. It seems difficult to explain these phenotype differences on the basis of the genotypes of LOXL1 because most XFS patients had the same compound genotype. Several susceptibility loci [LOXL1 may be associated with the phenotype differences in patients with XFS.The pathology of XFS is characterized by chronic accumulation of abnormal fibrillar material. The current pathogenetic concept describes XFS as an elastic microfibrillopathy  . LOXL1 ir fibers ,10. Thorr fibers  reportedstralian , Americastralian -16, and stralian  populatin et al.  describeulations ,11-16. Tulations , which iulations . The chaity loci -22 and oity loci ,24 for XLOXL1. It was also found that exfoliation materials that form before any clinical signs of XFS may affect phenotypic features including maximum IOP in patients with POAG. However, as shown in Signs of XFS often appear later in eyes initially diagnosed as having POAG. It is not clear whether this reflects an inaccurate clinical diagnosis or a coincidence of two different conditions. By thorough biomicroscopic examination, a diffuse-matte homogeneous film that consists of a layer of microfibrils can be observed on the entire surface of the anterior lens capsule before the formation of typical XFS deposits . A prospLOXL1 polymorphisms were associated with XFS. However, the allele, genotype, and compound genotype frequencies of these two SNPs differed between Japanese XFS patients and Caucasian XFS patients. These polymorphisms had no influence on the phenotypic feature of POAG patients. Further studies in the other ethnic populations should be performed to clarify how changes of LOXL1 lead to XFS.In conclusion,


To review the evidence in literature regarding the occurrence of Chronic Kidney Disease (CKD) after the treatment of small renal tumors with either radical nephrectomy (RN) or partial nephrectomy (PN).Current literature reviewed using Mediline search regarding renal functional outcomes following surgical treatment of small renal tumours.Studies have clearly shown that RN leads to CKD more often than PN and RN remains an independent risk factor for patients developing new onset renal insufficiency. There is independent, graded association between a reduced estimated GFR and the risk of death, cardiovascular events, and hospitalization. PN achieves a better Health Related Quality Of Life due to better preservation of renal function. Radical nephrectomy is significantly associated with death from any cause compared with partial nephrectomy.Removal of entire kidney is definitely an over-treatment for small renal tumors and PN should be the standard of care for these small renal tumors even in the setting of a normal contralateral kidney. Small renal tumors (SRT) that are 4 cm or smaller now account for the largest proportion of newly diagnosed renal masses. Despite et al. from Memorial Sloan-Kettering Cancer Center also showed that when controlled for preoperative risk factors for renal insufficiency i.e., hypertension, diabetes etc., patients undergoing a RN are at a greater risk of CKD than a similar cohort of patients undergoing a PN.[In a retrospective study from the Mayo Clinic with 10-year follow-up data that compared the outcome of RNs n=1492) and PNs (n=189) for unilateral single tumors with normal contralateral kidneys, patients who underwent a RN had a significantly higher risk for proteinuria and CKD i.e., creatinine of more than 2.0 mg/dL . A simila92 and PN2 as Stage 3 CKD.[et al. conducted a retrospective cohort study of 662 patients with a normal concentration of serum creatinine who had elective partial or radical nephrectomy for a solitary, renal cortical tumor (≤4 cm). They reported that a RN was significantly associated with new onset GFR less than 45 ml per minute compared with a PN (HR 11.8). The estimated 5-year probability of a postoperative GFR less than 45 ml per minute was 7% vs. 43% in patients who underwent a PN vs. a RN. A multivariable analysis showed that a RN remained an independent risk factor for patients developing new onset renal insufficiency.[et al. it has been shown that preoperatively Stage 3 CKD was identified in 26.7% of patients undergoing treatment for small renal tumors. Following intervention, the 3-year freedom from a GFR decrease of below 60 ml per minute per 1.73 m2 for radio frequency ablation, PN and RN was 95.2%, 70.7%, and 39.9%, respectively.[Current guidelines define a glomerular filtration rate (GFR) of lower than 60 mL/min per 1·73 mge 3 CKD. Huang, eet al. found an independent, graded association between a reduced estimated GFR and the risk of death, cardiovascular events, and hospitalization. Patinets with Stage 3 CKD, which means a GFR of <60 ml/m2, had a 10% increased risk of hospitalization, a 40% increased risk for cardiovascular events, and a 20% increased risk of death as compared with those who had a GFR of >60 ml/m2.[et al. found that a partial nephrectomy achieves a better Health Related Quality Of Life (HRQOL) due to better preservation of renal function and overall quality of life.[In a large, community-based population study of more than one million patients Go, 60 ml/m2. The asso60 ml/m2. In a comet al. compared overall survival in patients with sporadic, unilateral, solitary, and localized renal masses of 4 cm or less that have been treated by RN or PN. In patients younger than 65 years old, a RN was significantly associated with death from any cause compared with a PN . The increased risk of death persisted after adjusting for year of surgery, preoperative creatinine, Charlson-Romano index, symptoms at presentation, diabetes at presentation, and histology.[In a recent study of 648 patients (RN in 290 patients and PN in 358 patients), Thompson, istology. The findistology. These twThese data indicate that many patients are at risk for CKD after having a RN as treatment for small, renal cortical tumors. Therefore, these findings should have important implications on the contemporary management of these tumors. Preoperative GFR estimates should be an integral part of the decision algorithm during the planning of the surgical strategy for patients with renal cortical tumors, irrespective of tumor size.Based on the increasing number of studies showing long-term deleterious effects of CKD, urologists should be aware of the fact that normal serum creatinine may not necessarily mean normal GFR for the age and blanket treatment of small, renal cortical tumors as a RN could have a substantial effect on the quality and length of the survival of these patients. Collectively, these observations show that the removal of the entire kidney, even if performed laparoscopically, is definitely an overtreatment for small renal tumors and a PN should be the standard of care for these small renal tumors even in the setting of a normal contralateral kidney.