5 μm. This distance could effectively rake particles of compatible size, such as bacteria [29, 30]. Our previous stable isotope investigations [30] demonstrated that C. servadeii derives

its nutritional requirements from the moonmilk and learn more from dissolved organic matter in the percolating waters. To our knowledge, there are no molecular studies of the gut microbiota of cave invertebrates. The current project aimed at characterizing the feeding behaviour of C. servadeii from Grotta della Foos and the nature of its gut microbiota. The results provided insights pointing towards the existence of a universal guild of bacteria which appears to be common to many animal digestive systems and that suggests to have shared ancestors established prior to their hosts evolution. Methods Sampling site, specimen observation and collection The Grotta della Foos cave system formed within Monte Ciaurlec located in north-eastern Italy, and is underlain by Cretaceous and Triassic limestone units [44] The cave contains over

2600 m of passages. Ten sampling locations within the cave were used for the investigations of behaviour and insect collection. the sites covered altogether 13.3, square EMD 1214063 purchase meters, which is the whole area which Cansiliella is regularly found in Grotta de la Foos cave. The density monitored varied from 1.4 to 1.8 specimens per square meter. Examined specimen were all adults and included both sexes. Live C. servadeii were collected in sterile falcon tubes and transported to the laboratory. Microscopy, insect dissection, and gut content evaluation Insects external teguments were stained with DAPI (5

μg/ml) and observed in visible light and in epifluorescence using a Leica DM4000 inverted microscope equipped with a DFC300 FX camera. Images were acquired by using the LAS software. Insects were dissected to remove the midgut to analyze the intestinal microflora. Before dissection, specimens were stunned by keeping vials at 4°C for 20 min. To extract the midgut, the insect’s abdomen was opened under a stereomicroscope (Figure 1b) in a laminar flow hood using sterile equipment and sterile water. The midgut was transferred in a sterile Eppendorf tube and used for both bacterial culturability tests and bacterial DNA extraction and amplification, 5-FU ic50 and was stored at −20°C until extraction. A segment of each midgut was observed under microscopy after staining with the LIVE/DEAD® BacLight Bacterial Viability Kit (Molecular Probes, California, USA). Slides were also prepared for Gram staining and morphological characterization, which was performed under an Olympus BX60 microscope. Bacterial cultivation In order to examine external bacteria adhering to the insect exoskeletal tegument, live specimens collected with cave water in falcon tubes were handled with sterile forceps and gently touched over the surface of Plate Count Agar (PCA) (Oxoid) plates.

jejuni and C. coli isolates was 23.9% (188/176 samples) while the prevalence of erythromycin (currently recommended for the treatment of laboratory-confirmed

campylobacteriosis) resistance in C. coli was 13.3% (28/210 samples). These levels of resistance are likely to represent an unacceptable frequency of therapeutic failure of the drugs indicated for the treatment of human campylobacteriosis. The high levels of antimicrobial resistance cannot be accounted for exclusively by high numbers of a particular group of resistant genotypes. Rather, there is evidence for widespread acquisition of resistance among LY294002 cost relatively distantly related lineages from retail poultry. This is consistent with a small-scale study of C. jejuni isolated from chicken meat in Senegal where quinolone resistant phenotypes were present in three out of four tested lineages, and also dispersed throughout singleton STs [24]. It is possible that mutations that confer antimicrobial resistance have occurred in multiple lineages. However, bacteria can acquire genetic material, including antimicrobial resistance genes, from relatively distantly related lineages through horizontal gene Daporinad mouse transfer [25, 26]. Horizontal Gene Transfer (HGT) can involve recombination

between lineages, or acquisition of plasmids, which has been demonstrated to be the main mechanism of tetracycline resistance in Campylobacter[27]. There is also evidence that plasmid acquisition may mediate resistance to chloramphenicol and aminoglycosides [28, 29]. Resistance to macrolides is conferred by a 2 bp change in the putative erythromycin binding site. Resistance to fluoroquinolones is most usually the result of a single mutation in the gyrA region [30]. The widespread antimicrobial resistance in the Campylobacter populations, is likely to be the result of horizontal gene transfer as well as multiple independent mutation events. When conditions are such that antimicrobial resistance confers a strong selective advantage,

lineages that trace ancestry to resistant isolates will increase as a proportion of the population [31]. Under these circumstances a phylogenetic tree will show clusters of resistant lineages that have expanded clonally. Consistent with this, statistical analyses of the ClonalFrame tree Ketotifen of retail poultry isolates indicated that resistant phenotypes were not randomly distributed but showed some clustering within lineages. At the highest level there was a species-specific association with erythromycin resistance correlated with C. coli, as in previous studies of Campylobacter in pigs, turkeys and chickens [32–35]. Resistance to tetracycline, quinolones and chloramphenicol showed no association with either Campylobacter species, but all were non-randomly distributed among C. jejuni lineages. This could indicate that antimicrobial resistance, having arisen in an ancestral lineage, is propagated clonally by vertical transmission.

CA Cancer J Clin 2014, 64(1):9–29.PubMedCrossRef 3. Wang X, Lin YW, Wang S, Wu J, Mao QQ, Zheng XY, Xie LP: A meta-analysis of tea consumption and the risk of bladder cancer. Urol

Int 2013, 90(1):10–16.PubMedCrossRef 4. Chiong E, Kesavan A, Mahendran R, Chan YH, Sng JH, Lim YK, Kamaraj R, Tan TM, Esuvaranathan K: NRAMP1 and hGPX1 gene polymorphism and response to bacillus Calmette-Guerin therapy for bladder cancer. Eur Urol 2011, 59(3):430–437.PubMedCrossRef 5. Casadio V, Molinari C, Calistri D, Tebaldi M, Gunelli R, Serra learn more L, Falcini F, Zingaretti C, Silvestrini R, Amadori D, Zoli W: DNA Methylation profiles as predictors of recurrence in non muscle invasive bladder cancer: an MS-MLPA approach. J Exp Clin Cancer Res 2013, 32:94.PubMedCrossRef 6. Bartel DP: MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004, 116(2):281–297.PubMedCrossRef 7. Bartel DP: MicroRNAs: target recognition and regulatory functions. Cell 2009, 136(2):215–233.PubMedCentralPubMedCrossRef 8. Calin GA, Liu CG, Sevignani C, Ferracin M, Felli N, Dumitru CD, Shimizu M, Cimmino A, Zupo S, Dono M, Dell’Aquila ML, Alder H, Rassenti

L, Kipps TJ, Bullrich F, Negrini M, Croce CM: MicroRNA profiling reveals distinct signatures in B cell chronic lymphocytic leukemias. Proc Natl Acad Sci U S A 2004, 101(32):11755–11760.PubMedCentralPubMedCrossRef 9. Zhou B, Chen H, Wei D, Kuang Y, Zhao X, Li G, Xie J, Chen P: A novel miR-219-SMC4-JAK2/Stat3 regulatory pathway in human BYL719 ic50 hepatocellular carcinoma. J Exp Clin Cancer Res 2014, 33(1):55.PubMedCentralPubMedCrossRef 10. Ma C, Nong K, Wu B, Dong B,

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The filter was then mounted on an aluminium stub, sputter coated

with gold/palladium using a Cressington 208 HR High Resolution Sputter Coater, and observed with a Hitachi S-4700 field emission scanning electron microscope. Cells isolated from the surrounding sediment were pre-fixed for transmission electron microscopy (TEM) using 4% (v/v) glutaraldehyde in 0.2 M sodium cacodylate buffer (SCB) (pH 7.2) with the addition of 0.3 M sorbitol. Roxadustat purchase The pre-fixed cells were washed in 0.2 M SCB (pH 7.2) three times and embedded in 2% of low melting temperature agarose and post-fixed in 1% (w/v) osmium tetroxide in 0.2 M SCB (pH 7.2) at room temperature for 1 hr, before being dehydrated through a graded series of ethanol and 100% acetone. The dehydrated cells were then infiltrated with acetone-Epon 812 resin mixtures and 100% Epon 812 resin. Ultra-thin serial sections were collected on copper Formvar-coated slot grids, stained with 2% (w/v) uranyl acetate and lead citrate, Hydroxychloroquine nmr and observed using a Hitachi H7600 electron microscope. DNA extraction, PCR amplification, alignment and phylogenetic analysis Genomic DNA was extracted using the MasterPure Complete DNA and RNA purification Kit (Epicentre, WI, USA) from 30 cells that were individually isolated and washed three times in sterile seawater

(i.e., “”isolate 1″”). This procedure was repeated three months later on a different sample of 30 individually isolated cells (i.e., Immune system “”isolate 2″”). Polymerase chain reactions (PCR) were performed using PuRe Taq Ready-To-Go PCR beads kit (GE Healthcare, Buckinghamshire,

UK). Nearly the entire eukaryotic SSU rDNA gene was amplified from each isolate using the eukaryotic universal primers 5′- TGATCCTTCTGCAGGTTCACCTAC-3′ [49] and 5′-GCGCTACCTGGTTGATCCTGCCAGT-3′ [50]. PCR amplifications consisted of an initial denaturing period (95°C for 3 min), 35 cycles of denaturing (93°C for 45 s), annealing (5 cycles at 45°C and 30 cycles at 55°C, for 45 s), extension (72°C for 2 min), and a final extension period (72°C for 5 min). The amplified DNA fragments were purified from agarose gels using UltraClean 15 DNA Purification Kit (MO Bio, CA, USA), and subsequently cloned into the TOPO TA Cloning Kit (Invitrogen, CA, USA). Two clones of the eukaryotic SSU rRNA gene, from each of the two isolates (i.e., four clones in total), were sequenced with the ABI Big-Dye reaction mix using the vector primers and internal primers oriented in both directions. The new sequences were screened with BLAST, identified by molecular phylogenetic analysis, and deposited into GenBank: HM004353, HM004354. The SSU rDNA sequences from B.

prev. year – -6.05% -0.02% -7.88% -1.17% +1.10% > 75 4 497 4 464 4 604 4 607 4 326 4 249 % increase vs.

prev. year – -0.73% +3.13% +0.06% -6.09% -1.77% Total 17 283 17 281 17 453 16 666 16 205 15 857 % increase vs. prev. year – -0.01% +0.99% -4.50% -2.76% -2.14% Table 3 Quadrantectomies performed in Italy between 2000 and 2005 (SDO Italian hospitalizations database) Age group 2000 2001 see more 2002 2003 2004 2005 25–44 3 438 3 714 3 940 4 032 4 610 4 808 % increase vs. prev. year – +8.02% +6.08% +2.33% +14.33% +4.29% 45–64 12 780 13 761 14 354 14 551 15 113 15 518 % increase vs. prev. year – +7.67% +4.30% +1.37% +3.86% +2.67% 65–74 5 443 5 806 6 197 6 314 7 423 6 980 % increase vs. prev. year – +6.66% +6.73% +1.88% +17.56% -5.96% > 75 2 664 2 881 2 547 3 502 3 734 4 037 % increase vs. prev. year – +8.14% -11.59% +37.49% +6.62% +8.11% Total 24 325 26 162 27 038 28 399 30 880 31 343 % increase vs. prev. year – +7.55% +3.34% +5.03% +8.73% +1.49% The total number of mastectomies went from 17,283 in the year 2000 to 15,857 in 2005 (with a reduction of about -8.2% across the six examined years). We observed in most age groups (45–64, 65–74 and ≥ 75 years) a reduction in the number of mastectomies between year 2005 vs. year 2000, with the only Ku-0059436 nmr exception of women aged <45 years old (an age group excluded from national screening campaigns), where an increase of 7.9% in the number of mastectomies was found (Table 2).

This finding could be related to a late diagnosis of breast tumors in women aged 25–44, thus requiring disruptive surgery. On the other hand, there was an increase of 28.8% in the overall number of quadrantectomies, passing from 24,325 (year 2000) to 31,343 in 2005. The

increase of quadrantectomies was shown in all the four age groups (Table 3). Even in the youngest age group, quadrantectomies increased more than mastectomies, as Vorinostat molecular weight a 28.6% increase (+1517 cases) in the overall number of procedures (mainly quadrantectomies) was found in women <45 years of age, and accounted for about 15% of the overall increase observed across the six examined years in the total number of surgeries. A total of 38,164 mastectomies and 86,077 quadrantectomies were performed in patients aged between 45 and 64 years across the six years examined, with quadrantectomies increasing by a rate of about 21.0%. Similarly, in patients aged 65–74 and ≥ 75 years old, we observed an increase of 28.3% (+1537 cases) and 51.5% (+1373 cases) respectively, concerning the number of quadrantectomies performed between 2000 and 2005. In table 2 and table 3 we have also shown the percentage of average yearly increase, and the % increase vs. previous year per each age group. According to our data concerning major breast surgeries, the overall incidence of breast cancer per 100.000 women aged 0–84 years old was 141.80 in year 2000 and 160.85 in 2005, with a 13.4% increase (Table 4).

9/4.15 68.0/5.5

1/0% +4.1 42 Biogenesis of cellular components 42.27 Extracellular/secretion protein 432 OmpW family outer memb. prot. precursor 151 Q3BP00_XANC5 X. c. pv. vesicatoria XAC3664 23.8/4.97 17.0/6.1 5/13% +2.2 a Gene accession number in X. axonopodis pv. citri genome of the identified protein. b Fold change in biofilm compared to planktonic cultures. * Protein spots 55 and 38 were previously identified https://www.selleckchem.com/products/azd9291.html as “outer membrane active sucrose transporter” and “ferric enterobactin receptor” are now classified as TonB-dependent receptor, while protein spots 526 and 555 were previously identified as “carbohydrate selective porin” and is now classified as Regulator of pathogenecity factors. Functional characterization of differentially regulated X. a. pv. citri biofilm

proteins The identified differentially expressed proteins were used to determine enriched GO categories in biological processes, molecular function and cellular localization. The main enriched categories for the up- and down-regulated proteins with an average fold change of minimum ±1.5 are represented graphically (Figure 3). The major biological processes and cellular localization categories that changed learn more in the X. a. pv. citri biofilms are ‘transporter activity’ and ‘external encapsulating structure’, respectively. The categories that showed enrichment in the up-regulated proteins include ‘catabolic process’, ‘external encapsulating structure’, ‘receptor activity’ and ‘transporter activity’; while most of the down-regulated proteins were in the categories of ‘biosynthetic process’, ‘nucleobase, nucleoside, nucleotide and nucleic acid metabolic process’, ‘metabolic process’, ‘catabolic process’ and ‘generation of precursor metabolites and energy’. Figure 3 Gene ontology (GO) terms enriched in the identified up-and down-regulated proteins in X . a . pv . citri biofilms compared to planktonic cultures. Proteins were considered differentially

expressed in X. a. pv. citri Avelestat (AZD9668) biofilms when variation was a minimum of 1.5-fold (p < 0.05). The GO enrichment analysis was performed using Blast2GO. It is noteworthy that among the identified proteins, some have previously been shown to be involved in biofilm formation or regulation in other pathogenic bacteria. These include a the non-fimbrial adhesin, YapH [26], the FadL porin [27], citrate synthase [28], UDP-glucose dehydrogenase [19], the molecular chaperone DnaK [29–31], the elongation factor Ef-Tu [29, 32], the polynucleotide phosphorylase [33] and a TonB-dependent receptor protein [19] (Table 2). These findings further validate our experimental results. Table 2 Differentially expressed proteins detected previously in biofilms Protein Species Reference Non-fimbrial adhesion, YapH X. axonopodis pv. phaseoli 26 Outer membrane protein, FadL P. fluorescens 27 Citrate synthase B. cenocepacia 28 UDP-glucose dehydrogenase X. axonopodis pv. citri 19 Molecular chaperone DnaK S. pneumoniae, S. mutants, P.

Bone 31:276–281PubMedCrossRef 24. Fujiwara S, Nakamura T, Orimo H, Hosoi T, Gorai I, Oden A (2008) Development and application of a Japanese model of the WHO fracture risk assessment tool (FRAX™). Osteoporos Int 19:429–435PubMedCrossRef 25. Hagino H, Yamamoto K, Ohshiro H, Nakamura T, Kishimoto H, Nose T (1999) Changing incidence of hip, distal radius, and proximal humerus fractures in Tottori Prefecture, Japan. Bone 4(3):265–270CrossRef 26. Fujiwara S, Kasagi F, Masunari N, Naito K, Suzuki G, Fukunaga M (2003) Fracture prediction from bone mineral density in Japanese men and women. J Bone Miner Res 18(8):1547–1553PubMedCrossRef 27. Kanis JA, Oden A, Johnell O, MK-2206 manufacturer de Jonsson B, Laet

C, Dawson A Small molecule library concentration (2001) The burden of osteoporotic fractures: a method for setting intervention thresholds. Osteoporos Int 12:417–427PubMedCrossRef 28. Kanis JA, Johnell O, Oden A, Sernbo I, Redlund-Johnell I, Dawson A, De Laet C, Jonsson B (2000) Long-term

risk of osteoporotic fracture in Malmo. Osteoporos Int 11:669–674PubMedCrossRef 29. Kung AWC, Lee KK, Ho AYY, Tang G, Luk KDK (2007) Ten-year risk of osteoporotic fractures in postmenopausal Chinese women according to clinical risk factors and BMD T-scores: a prospective study. J Bone Miner Res 22(7):1080–1087PubMedCrossRef 30. Melton LJ 3rd (1995) How many women have osteoporosis now? J Bone Miner Res 10(2):175–177PubMedCrossRef 31. Bow CH, Tsang SWY, Loong CH, Soong SS, Yeung SC, Kung AW (2010) Bone mineral density enhances use of clinical risk factors in predicting ten-year risk of osteoporosis fractures in Chinese men: the Hong Kong osteoporosis study. Osteoporos Int. doi:10.​1007/​s00198-010-1490-0 PubMed”
“Introduction Osteoporosis is a chronic disease requiring chronic treatment. It is therefore Isotretinoin essential to evaluate the efficacy and safety of osteoporosis treatments for the longest time possible, i.e. well beyond the 3 to 5 years recommended by the regulatory authorities. Thus, clinical studies for the bisphosphonates zoledronic acid, risedronate, and alendronate have been extended to 6, 7, and 10 years,

respectively [1–3]; the selective estrogen receptor modulator raloxifene has been evaluated up to 8 years [4, 5]; and results at 5 to 6 years are available for the human monoclonal antibody denosumab [6, 7]. These studies generally indicate sustained increases in the surrogate marker of antifracture efficacy, bone mineral density (BMD). The study designs, notably excluding a placebo group for ethical reasons, preclude direct measurement of long-term reductions in fracture incidence. The orally active agent strontium ranelate is indicated for the management of postmenopausal osteoporosis. Its mode of action in osteoporotic bone includes opposite effects on resorption and formation, which is associated with an improvement in the material or structural properties of bone [8].

Funding for the collection of sediments and participation of VPE and JMB

in this research was provided by the US National Science Foundation grant MCB-060484. We also acknowledge the constructive Ponatinib cost feedback from four anonymous reviewers. Electronic supplementary material Additional file 1: Maximum likelihood (ML) analysis of 29 taxa focusing on the position of Calkinsia aureus within the Euglenozoa clade. Two jakobids, Andalucia incarcerata and A. godoyi, are used as outgroups in this analysis. The short environmental sequences are excluded from the dataset used in Figure 11 and fast-evolve euglenids sequences, Ploeotia, Menoidium and Astasia, are included. ML bootstrap values greater than 50% are shown. Thick branches indicate Bayesian posterior probabilities over 0.95. Ba, bacteriotroph; click here Eu, eukaryotroph; Os, osmotroph; Ph, phototroph. GenBank accession numbers of the sequences analyzed are shown in parentheses. (EPS 405 KB) Additional file 2: Maximum likelihood (ML) analysis of 25 taxa focusing on the position of Calkinsia aureus within the Euglenozoa clade. Two jakobids, Andalucia incarcerata and A. godoyi, are used as outgroups in this analysis. The short environmental sequences are removed from the dataset used in Figure 11 and fast-evolve euglenids sequences, Dinema, Ploeotia, Menoidium and Astasia, are excluded. ML bootstrap values greater than 50% are shown. Thick branches

indicate Bayesian posterior probabilities over 0.95. PIK3C2G Ba, bacteriotroph; Eu, eukaryotroph; Ph, phototroph. GenBank accession numbers of the sequences analyzed are shown in parentheses. (EPS 400 KB) References 1. Keeling PJ, Burger G, Durnford DG, Lang BF, Lee RW, Pearlman RE, Roger AJ, Gray MW: The tree of eukaryotes. Trends Ecol

Evol 2005, 20:670–676.CrossRefPubMed 2. Yoon HS, Grant J, Tekle YI, Wu M, Chaon BC, Cole JC, Logsdon JM Jr, Patterson DJ, Bhattacharya D, Katz LA: Broadly sampled multigene trees of eukaryotes. BMC Evol Biol 2008, 8:14.CrossRefPubMed 3. Adl SM, Simpson AGB, Farmer MA, Andersen RA, Anderson OR, Barta JR, Bowser SS, Brugerolle G, Fensome RA, Fredericq S, James TY, Karpov S, Kugrens P, Krug J, Lane CE, Lewis LA, Lodge J, Lynn DH, Mann DG, McCourt RM, Mendoza L, Moestrup Ø, Mozley-Standridge SE, Nerad TA, Shearer CA, Smirnov AV, Spiegel FW, Taylor MF: The new higher level classification of eukaryotes with emphasis on the taxonomy of protists. J Eukaryot Microbiol 2005, 52:399–451.CrossRefPubMed 4. Adl SM, Leander BS, Simpson AGB, Archibald JM, Anderson OR, Bass D, Bowser SS, Brugerolle G, Farmer MA, Karpov S, Kolisko M, Lane CE, Lodge DJ, Mann DG, Meisterfeld R, Mendoza L, Moestrup Ø, Mozley-Standridge SE, Smirnov AV, Spiegel F: Diversity, nomenclature, and taxonomy of protists. Syst Biol 2007, 56:684–689.CrossRefPubMed 5. Cavalier-Smith T: Kingdom protozoa and its 18 phyla. Microbiol Rev 1993, 57:953–994.PubMed 6.

(2009). For densitometry

gels were analysed by Image Studio Lite (LI-COR, Inc). Results and discussion CyanoQ associates with PSII complexes isolated from T. elongatus The CyanoP and CyanoQ orthologues in T. elongatus click here are encoded by tlr2075 (Michoux et al. 2010) and tll2057, respectively. Despite detailed analysis of the subunit composition of His-tagged PSII complexes isolated from T. elongatus by mass spectrometry (Sugiura et al. 2010), neither CyanoQ nor CyanoP has been detected. To investigate whether CyanoQ or CyanoP are able to associate with PSII isolated from T. elongatus, we first performed pull-down experiments by binding solubilised membrane extracts obtained from a His-tagged CP43 strain of T. elongatus (CP43-His) to a cobalt resin and analysing bound proteins released by 100-mM imidazole. Immunoblotting experiments revealed that a significant proportion of CyanoQ co-purified with CP43-His (Fig. 1). By contrast, no detectable CyanoQ bound to the cobalt resin when a non-tagged WT sample was tested. As expected, the D1 and PsbO subunits of PSII co-purified with His-tagged CP43, as did significant amounts of Psb27, which is known to be a component of non-oxygen-evolving PSII Selumetinib cell line complexes (Nowaczyk et al. 2006; Grasse et al. 2011). In contrast only trace amounts of CyanoP co-purified with CP47-His under the experimental conditions used. Fig. 1 Association of CyanoQ

with His-tagged CP43. Detergent solubilised membrane extracts from either WT or His-tagged CP43 strains of T. elongatus (CP43-His)

were mixed with cobalt resin and the bound proteins eluted by 100-mM imidazole (100 mM) followed by SDS solubilising buffer (SDS) for analysis by a SDS-PAGE and silver staining and b immunoblotting. Pre solubilised extract added to resin; Post solubilised extract after incubation with cobalt resin; Wash last wash before elution; Ctrl control in which resin lacking Co was used A commonly used method to isolate highly active oxygen-evolving dimeric PSII complexes from T. elongatus for structural studies involves a two-step anion-exchange chromatography protocol (Kern et al. 2005). This type of preparation has been successfully used to generate high-quality PSII crystals yielding diffraction data Doxorubicin in vitro of up to 3 Å resolution (Loll et al. 2005; Murray et al. 2008a, b). The PSII preparation analysed here (which produced 400-µm-long PSII crystals) also contained detectable levels of the alpha subunit of the ATPase (Tlr0435) and, interestingly, a predicted thioredoxin peroxidase/peroxiredoxin (Tll1454), which is homologous to a peroxiredoxin (2-CysPrx) thought to interact with PSII in chloroplasts (Muthuramalingam et al. 2009) (Fig. 2). Immunoblotting of the PSII complex revealed that CyanoQ was indeed present and had been purified to about the same degree as the D1 subunit (approximate 10-fold enrichment on chlorophyll basis compared with thylakoid membranes).

Multiplex PCR and Southern blot analysis further confirmed that all albino transformants tested were true car2 null mutants (Figure 4B). The albino phenotype was directly caused by the deletion of CAR2 because the phenotype was completely restored when re-integrating a wild type gene fragment (Additional file 1). Whereas the targeted deletion frequency for CAR2 was estimated to be 10.5% in WT, it was increased to 75.3% in the ∆ku70e background, a more than 7-fold improvement.

Dramatically increased gene deletion frequencies were also observed at both STE20 and URA3 loci (Table 2), with the deletions verified by Southern blot and phenotypic analyses (Figure 5). Figure 4 Phenotypic and genotypic characterization of pKOCAR2 find more transformants. (A) A transformation plate showing both red and albino transformants, with black arrow heads marking some albino transformants. (B) Southern blot results using the 5′ flanking sequence of CAR2 as a probe. Genomic DNA was digested with PvuI and a band shift from 5.0 kb (WT) to 3.0 kb indicates successful deletion of CAR2 gene. Figure 5 Targeted deletion of STE20 and URA3 genes. (A) and (D) Illustration of gene deletion constructs; (B) and (E): Southern blots

Tipifarnib using probes shown in (A) and (D); (C) Colony phenotype of WT and ∆ste20 strains mated with R. toruloides ATCC 10788; (F) Growth phenotype of WT and ∆ura3 strains derived from 10-fold serially diluted cells. The latter showed resistance to 5-FOA (1 g/L) – a substrate that can be converted to a toxic intermediate by the URA3-encoded enzyme [27]. Effect of homology sequence length on deletion frequency To understand the effects of homology sequence length on gene deletion frequency, pKOCAR2 was modified to have various lengths of homology sequence, ranging from 50 to 1500 bp (Additional file 2). The minimum homology length

necessary for CAR2 deletion in WT was at least 250 bp with a gene deletion frequency of 0.7%, while only 100 bp was sufficient in the ∆ku70e strain, which gave gene deletion frequency of approximately 20%. Homology length of at least 1 kb was required to achieve gene deletion frequency of more than 90% using the ∆ku70e strain Parvulin (Table 3). Table 3 Effects of homologous sequence length on CAR2 deletion frequency Homology length (bp) Gene deletion frequencya Improvement (folds) WT ∆ku70e 50 0 (780) 0 (8) – 100 0 (620) 21.4% (14) – 250 0.7% (1668) 30.3% (33) 43.3 500 11.2% (2124) 67.0% (778) 6 750 10.5% (6152) 75.3% (885) 7.2 1000 30.4% (2280) 91.7% (2196) 3 1500 20.5% (2730) 91.0% (4304) 4.4 Note: aNumber in parenthesis indicate number of transformants screened. Sensitivity of KU70 deficient mutant to DNA damaging agents Deficiency in Ku complex encoding genes have been linked to elevated sensitivity to DNA-damaging agents due to the defects in DNA repair [12]. As expected, the ∆ku70 strain displayed higher susceptibility to DNA damage induced by methyl methane sulfonate (MMS) and exposure to ultraviolet (UV) radiation compared to WT.