Hybridomas reacting specifically with Cronobacter were expanded and cloned at least three times by limiting dilution. Positive clones were frozen in recovery cell culture freezing media® or FCS HDAC inhibitor supplemented with 4% (v/v) DMSO and stored at -80°C overnight before being transferred to liquid nitrogen. The positive clones were propagated either in tissue culture or by ascitic fluid using the procedure of Harlow and Lane [28]. Isotypes of purified monoclonal antibodies from ascites or spent medium were determined using the mouse type subisotyping

kit according to the manufacturer’s instructions. Immunochemical Methods Elisa Screening of antisera spent medium and ascites for the presence Semaxanib research buy of antibodies against Cronobacter selleck inhibitor was performed by an indirect non-competitive ELISA. Flat-bottom 96 well plates were coated with 0.1 ml of (108 heat-killed cells ml-1) of whole cell antigen diluted in 0.05 M carbonate buffer (pH 9.6) overnight at 4°C. Alkaline phosphatase-conjugate goat anti-mouse immunoglobulin and p-nitrophenyl phosphate were used as secondary antibodies and substrate, respectively. Additive index elisa Additive index

ELISA was performed on paired MAbs as described by Friguet et al., [29]. An additive index for each pair of MAbs was calculated according to the formula [2A 1+2/(A 1 + A 2)] – 1 × 100, where A 1, A 2, and A 1+2 are absorbance values with antibody 1 alone, antibody 2 alone, and the two antibodies together, respectively. Gel electrophoresis Profiles of Cronobacter OMPs were examined using SDS-PAGE following the method described by Laemmli [30]. The runs were performed in 4% stacking and 12.5% separating gels. Equal concentrations of Cronobacter OMPs (20 μg well-1) were mixed with sample buffer at a ratio of 1:5, boiled for 5 min and loaded (approx. 20 μl/lane). Gels were either stained Edoxaban with 1% (w/v) Coommassie Brilliant Blue G-250 or used for immunoblotting. Likewise,

LPS preparations from Cronobacter were examined using Deoxycholate-polyacrylamide gel electrophoresis (DOC-PAGE) following the method described by Reuhs et al., [31]. Briefly, the runs were performed using 4% (v/v) stacking and 12.5% (v/v) separating gels. Equal concentrations of Cronobacter LPS (5 μg well-1) were mixed with sample buffer [2 ml of 22.7% (w/v) Tris-base solution; 1 ml of 50% (v/v) glycerol; 1 ml of 1% (w/v) bromophenol blue and 6 ml distilled water] at a ratio of 1:5. The gels were pre-run in DOC-electrophoresis buffer (Tris-base, 4.5 g; glycine, 21.7 g; 2.5 g sodium deoxycholate, pH adjusted to 8.3 and volume adjusted to 1 liter) for 10 min at 80 volts before loading the samples. Samples were run in the same buffer at 80 and 120 volts for the stacking and separating gels, respectively. Upon completion, gels were either stained using the PageSilver™ silver staining kit (Fermentas) or were used for immunoblotting.

Furthermore, PilA of ssp. novicida was recently shown to be involved in protein secretion that was coupled to Tfp [20, 25]. Interestingly,

mutation of pilA and loss of protein secretion resulted in increased virulence in a mouse infection model [25]. As the human pathogenic type A and type B strains do not secrete detectable selleck kinase inhibitor levels of proteins in vitro, it is possible that one step in the evolution of human pathogenic variants of F. tularensis from ssp. novicida has involved loss of protein secretion PLK inhibitor as a consequence of changes in PilA structure and function. In this work we wanted to address the question if PilA is involved in virulence of the highly pathogenic type A strain SCHU S4, similarly to

what we have previously shown for type B strains, and if Tfp secretion and assembly genes are required for virulence. Results Construction of non-polar pilin gene mutants In a recent study, we were able to demonstrate that the pilA gene can be lost by a deletion event mediated by direct repeats flanking the gene [22]. Type B strains lacking pilA were found to be attenuated for virulence in a mouse infection model. In this study we wanted to extend this work to the highly pathogenic type A strain SCHU S4, and therefore we constructed a specific pilA deletion mutant using our previously described allelic exchange technique [7]. In addition, to address the significance of secretion and CBL-0137 datasheet assembly of PilA, we also engineered in-frame deletions in pilC and pilQ, encoding a transmembrane protein and a secretin, respectively. For some pathogens, Tfp expression is associated with a unique ability to retract the pili, a phenotype depending on the ATPase PilT. Interestingly, pilT appears to be functional in type A

strains, while it is a pseudogene in the less pathogenic type B strains. In order to elucidate if the expression of PilT could be correlated to the higher virulence of type A strains, we also constructed an in-frame deletion in the pilT Cyclooxygenase (COX) gene. In order to verify that the mutations did not have a major impact on neighboring gene transcription, each region was analysed by RT-PCR on mRNA extracted from the mutant strains and compared to the isogenic wild-type strain (Fig. 1). Thereby we could confirm that none of the deletion events caused any polar effects on transcription. Both pilC and pilT are flanked by pseudogenes situated directly downstream of each gene that were found not to be transcribed neither in the wild-type nor in the pilC or pilT mutant strains. The upstream genes of pilC and pilT were readily transcribed at similar levels in the wild-type and mutant strains. In the case of the pilQ mutant, we could verify non-polarity on the downstream aroK gene. Figure 1 A-D. Analysis of gene transcription in wild-type and mutant strains of F. tularensis using RT-PCR of mRNA.

Methods Materials and coating preparation Bionic lotus polymer surfaces were fabricated through engineering materials, such as stainless steel or other metal substrates (Al/Cu), by using a certain volume of water-soluble PTFE emulsion and polyphenylene sulfide

dispersion in mixed solvent (distilled water/ethanol/isobutyl alcohol in a volume fraction of 2:5:1), non-ionic surfactant (octylphenol polyoxyethylene ether: (C8H17-Ph-O(C2H4O)nH, n ~ 10), and industrial raw material ammonium carbonate ((NH4)2CO3) [18, 20]. The steel/alumina/copper block was polished with 500# and 900# sand papers in turn, and then cleaned with acetone in an ultrasonic bath for 5 min. The wet coatings on stainless steel or various metal substrate blocks were prepared #see more randurls[1|1|,|CHEM1|]# by spraying the coating precursors with 0.2 MPa nitrogen gas and curing at temperature 150°C for 1 h and 390°C for 1.5 h. External macroscopic force interference In order to investigate the impact of external macroscopic force interference on polymer nano-fibers, pure PTFE coating (P1 coating) https://www.selleckchem.com/products/Trichostatin-A.html sample was naturally cooled to 20°C in the sintering furnace after curing at 390°C for 1.5 h. In contrast to P1 coating, H2 gas flow was passed into the sintering furnace during the same curing and cooling

process as P1 coating for PTFE/PPS superhydrophobic coating (P2 coating) sample. Internal Cyclic nucleotide phosphodiesterase microscopic force interference Internal microscopic force interference was introduced to further investigate controllable polymer nano-papules or nano-wires.

After curing at 390°C for 1.5 h in the sintering furnace, the PTFE/PPS superhydrophobic coating samples were cooled at four different conditions, respectively, as shown in Table  1. There are three coating samples cooled in the uniform cooling mediums: the Q1 and Q2 coating were quenched in the air at room temperature (20°C) and the cryogenic liquid medium (ethanol + dry ice) at -60°C, respectively. In addition, the Q3 coating was quenched in the non-uniform cooling medium (pure dry ice cooling environment at -78.5°C). Table 1 Various cooling conditions for superhydrophobic polymer coatings after curing Samples Crystallization interference methods Thermal conductivity of the mediums [23] Q1 coating Quenched in the air at 20°C K ≈ 0.026 [W/(m K)] Q2 coating Quenched in the mixture of dry ice and ethanol at -60°C K ≈ 0.24 [W/(m K)] Q3 coating Quenched in the pure dry ice at -78.5°C K ≈ 0.099 [W/(m K)] Characterization Microstructures of the bionic lotus polymer coating surfaces were observed by a scanning electron microscopy (JSM-5600LV and field emission scanning electron microscopy (FE-SEM), JEOL, Akishima, Japan).

EMBO J 1997, 16:2161–2169.PubMedCrossRef 39. Fernandez S, Sorokin A, Alonso JC: Genetic recombination in Bacillus R428 clinical trial subtilis 168: effects of recU and recS mutations on DNA repair and homologous recombination. J Bacteriol 1998, 180:3405–3409.PubMed 40. Kelly SJ, Li J, Setlow P, Jedrzejas MJ: Structure, flexibility, and mechanism of the Bacillus stearothermophilus RecU holliday junction resolvase. Proteins 2007, 68:961–971.PubMedCrossRef

41. Sluijter M, Aslam M, Hartwig NG, van Rossum AM, Vink C: Identification of amino acid residues critical for catalysis of Holliday junction resolution by Mycoplasma genitalium RecU. J Bacteriol 2011, 193:3941–3948.PubMedCrossRef 42. Ayora S, Carrasco B, Cardenas PP, Cesar CE, Canas C, Yadav T, Marchisone C, Alonso JC: Double-strand break repair in bacteria: Adriamycin research buy a view from Bacillus subtilis. FEMS Microbiol Rev 2011, 35:1055–1081.PubMedCrossRef 43. Smith BT, Grossman AD, Walker GC: Localization of UvrA and effect of DNA damage on the chromosome of Bacillus subtilis. J Bacteriol 2002, 184:488–493.PubMedCrossRef

44. Levin-Zaidman S, Frenkiel-Krispin D, Shimoni E, Sabanay I, Wolf SG, Minsky A: Ordered intracellular RecA-DNA assemblies: a potential site of in vivo RecA-mediated activities. Proc Natl Acad Sci U S A 2000, 97:6791–6796.PubMedCrossRef 45. Odsbu I, Morigen , Skarstad K: A reduction in ribonucleotide reductase activity slows down the chromosome replication fork but does PI3K Inhibitor Library not change its localization. PLoS One 2009, 4:e7617.PubMedCrossRef 46. Barre FX, Tolmetin Soballe B, Michel B, Aroyo M, Robertson M, Sherratt D: Circles: the replication-recombination-chromosome segregation connection. Proc Natl Acad Sci U S A 2001, 98:8189–8195.PubMedCrossRef 47. Michel B, Recchia GD, Penel-Colin M, Ehrlich SD, Sherratt DJ: Resolution of Holliday junctions by RuvABC prevents dimer formation in rep mutants and UV-irradiated cells. Mol Microbiol 2000, 37:180–191.PubMedCrossRef 48. Hendricks EC, Szerlong H, Hill T, Kuempel P: Cell division, guillotining of dimer chromosomes and SOS induction

in resolution mutants (dif, xerC and xerD) of Escherichia coli. Mol Microbiol 2000, 36:973–981.PubMedCrossRef 49. Kaimer C, Schenk K, Graumann PL: Two DNA translocases synergistically affect chromosome dimer resolution in Bacillus subtilis. J Bacteriol 2011, 193:1334–1340.PubMedCrossRef 50. Boyle-Vavra S, Yin S, Challapalli M, Daum RS: Transcriptional induction of the penicillin-binding protein 2 gene in Staphylococcus aureus by cell wall-active antibiotics oxacillin and vancomycin. Antimicrob Agents Chemother 2003, 47:1028–1036.PubMedCrossRef Competing interests The authors declare that they have no competing interests. Authors’ contributions ARP, PR and MGP designed research, analyzed data and wrote the paper, HV contributed with new genetic constructs, ARP performed research. All authors read and approved the final manuscript.

One anti-tumoral compound isolated from several plant-derived products is cinnamic acid. Cinnamic acid and its associated compounds can be found in coffee, apples, citric HSP inhibitor fruits, vegetable oils, propolis and wine. Cinnamic acid has a long history of human use as a component of plant-derived scents and flavoring agent [13]. Liu et al. [5] found that this compound induced tumor cell differentiation by modulating the expression of genes implicated in tumor metastasis and immunogenicity in cultured human melanoma cells. Several researchers have also demonstrated the antioxidant activity of caffeic acid and its derivatives

[14, 15], which may be associated with cell death. Lee et al. [8] demonstrated that natural antioxidant compounds in diet, such as polyphenols in green tea, activate the MAPK pathway. Moreover, at high concentrations, these substances activate the caspase signaling

cascade, which induces apoptosis in normal cells [8]. Lamartiniere et al. [16] showed that soy isoflavones such as genistein (another polyphenolic compound) act as chemopreventive agents against prostate and mammary cancers. One of the chemopreventive mechanisms against cancer is the induction of irreversible DNA damage, which results in cell death via apoptosis [17]. Impaired function of p53 increases the probability of proliferating cells with genetic abnormalities in some conditions [18, 19]. This is due to the activation of p53 in response to learn more unfavorable treatments, which results in genetic abnormalities such as DNA breakages this website [20, 21], disruption Selleckchem GSI-IX of microtubules [22], lack of chromosome

segregation at mitosis [23] or the incorrect termination of cell division, which can result in micronuclei formation [22]. The micronucleus test is widely used to detect chromosomal aberrations because micronuclei can originate from chromosomal fragments or disruptions in the mitotic spindle [24, 25]. This assay has been used to evaluate the exposure levels of the human population to mutagenic or genotoxic agents [26–30] as well as in cell cultures to determine the mutagenic potential of drugs and/or natural compounds [31–33]. The screening of new compounds with anti-microbial and anti-inflammatory activities has resulted in the discovery of anti-tumor and chemopreventive properties of cinnamic acid and its derivatives [5, 34–36]. Selective cytotoxicity in tumor cells is an important role to be analyzed to compare drug effects in cultured cells [37, 38]. This study aimed to compare the cytotoxic and genotoxic potential of cinnamic acid in both a human melanocyte cell line of blue nevus and in cultured melanoma human cells. Materials and methods Cell cultures HT-144 cell line, derived from malignant cutaneous melanoma, was obtained from American Type Culture Collection (ATCC). NGM cell line, derived from melanocytes of blue nevus, was obtained from Cell Bank of Rio de Janeiro (Brazil).

After determinations of the OD600 and centrifugation of the sample (13,000 g, 5 min) aliquots of the supernatant were used to determine concentrations of glucose and D/L-lactate by reverse-phase high-pressure liquid chromatography (HPLC) as described by Engels et al. 2008. To discriminate between the D- and L- isomers of lactate enzymatic determinations were performed as described by the manufacturer (R-Biopharm, Darmstadt, Germany). D-lactate dehydrogenase

assay For determination of enzyme activities, exponentially growing cells were harvested by MX69 mouse centrifugation (4,500 g, 5 min, 4°C) and washed twice with 50 mM ice-cold KH2PO4, pH 7.0. Cell pellets were resuspended in 1 ml of 50 mM KH2PO4, pH 7.0, directly or after storage at -70°C. After disruption by ultrasonic treatment at 4°C (UP 200S; Dr. Hielscher GmbH, Teltow, Germany) at an amplitude of 55% and a duty cycle of 0.5 for 6 min and centrifugation at 4°C for 60 min at 13,000 g, enzyme activity was determined immediately in the cell-free supernatant. D-Lactate dehydrogenase activity was determined by a modified assay according to [31]. Reaction mixtures of 1 ml contained 100 mM KH2PO4 (pH 7.5), 50 μM 2,6-dichloroindophenol (DCPIP) and 20 μl crude extract. The reaction was started

by addition of 10 mM D-lactate and quinone-dependent D-lactate dehydrogenase was assayed spectrophotometrically at 30°C by determining the decrease in absorbance of DCPIP (ε600 = 20 mM-1 cm-1). Construction of plasmids and strains The oligonucleotides listed in Table 4SC-202 molecular weight 1 were obtained from Operon (Cologne, Germany). Standard methods such as PCR, restriction, and ligation were carried out as described previously [29]. Plasmids were constructed in Escherichia coli DH5α from PCR-generated fragments (KOD, Novagen) and isolated with the QIAprep spin miniprep kit (QIAGEN, Hilden, Germany).

E. coli was transformed by the RbCl2 method [32], while C. glutamicum was transformed via electroporation [33]. All cloned DNA fragments were shown to be correct by sequencing (BigDye Terminator Inositol monophosphatase 1 v3.1 Cycle Sequencing Kit and ABI Prism Capillary Sequencer Model 3730, Applied Biosystems, Forster-City, USA). Disruption of dld To construct a C. glutamicum dld inactivation mutant, an internal 1224-bp fragment of dld was amplified by using primer pair Cg-dld-SalI-N498 and Cg-dld-C1716-SalI which was subsequently cloned into pT7-blue T-vector (Novagen). The SalI restricted PCR fragment was ligated into the SalI site of pK18mob. Gene inactivation with pk18mobN498dld was carried out as described previously [24]. The correct genotype of the GANT61 insertion mutant was verified by PCR analysis and determination of enzyme activity.

On the other hand, suspensions with methyl orange and the different

TiO2 powders were prepared as described before but they were not subjected to irradiation: in such dark conditions, no changes in the methyl orange concentration were observed for these suspensions all along the test, so absorption to the TiO2 surface was discarded in all cases. Results and discussion FESEM and TEM micrographs in Figure  1 shows the TiO2 Proteasome inhibitor powder as synthesized following the methodology described. It is constituted by spherical particles with a mean size around 1 to 2 μm and formed in turn by the agglomeration of a myriad of smaller nanoparticles. Furthermore, this hierarchical configuration, from now labelled as Tisph powder, displays an outstanding specific surface area, as large as S s = 322 m2 · g-1, indicating the presence of interparticle Selleck ITF2357 porosity (meso- and microporosity). Figure 1 FESEM (a) and TEM (b) micrographs of the Ti sph as-prepared powder. Certainly, such a high specific surface on a micron-sized powder may have tremendous potential for photocatalytic applications, but when going to XRD measurements, no trace of crystalline order was ever observed, see Figure  2a.

This represents a serious problem since as mentioned, a high degree of crystallinity is essential for an efficient photocatalytic performance. In fact, photoreactivity demands a compromise between crystallinity, specific surface and porosity, so here is where we took our amorphous Tisph powder selleck kinase inhibitor to fast microwave crystallization, trying to improve the crystallinity of the TiO2 spheres with the minor loss in specific surface area and porosity (i.e. keeping the hierarchical microstructure).

In this sense, Celecoxib Figure  2 evinces that after 7 min of microwave (MW) radiation, XRD peaks of the TiO2 anatase phase can be already detected in the powder sample. As the exposure time is increased, an increase in the structural order is also observed (narrower peaks) and after 15 min, no further improvement in the crystallinity seems to be attained with the MW treatment. Moreover, the XRD analyses also showed that 10 min under MW radiation produced a crystallinity comparable to that obtained after 1 h at 400°C in a conventional electric furnace (similar width of XRD peaks in diffractograms of Figure  2c and 2f). Figure 2 X-ray diffractograms. Of as-synthesized Tisph powder (curve a) and after 7 min (curve b), 10 min (curve c), 15 min (curve d) and 30 min (curve e) of MW treatment. XRD of the same powder treated at 400°C/1 h in a conventional electric furnace (f). All peaks corresponding to TiO2 anatase (JCPDS file no. 21-1272). When going to the microscope, Figure  3 shows that the spherical morphology is retained after all the heating treatments.

A total of 67 secreted proteins of M. oryzae was experimentally demonstrated to be secreted through cloning into an overexpression vector and expressed in M. oryzae transformants (Ebbole and Dean, unpublished data). These 67 secreted proteins were annotated with a biological process term GO:TPCA-1 order 0009306 (“”protein secretion”") and a cellular component term GO:0005576 (extracellular region). An evidence code IDA was assigned to annotations of these 67 proteins since function was determined through direct assay. A total of 128 curated cytochrome P450′s of M. oryzae were validated by comparison and analysis of gene

location and Small molecule library solubility dmso structure, clustering of genes, and phylogenetic reconstruction [28]. Different subsets of these proteins were annotated with different GO terms. For example, 75 of the 128 P450 proteins were annotated with the molecular function term GO:0005506 (“”iron ion binding”"), and 40 P450 proteins with the molecular function term GO:0016491 (“”oxidoreductase activity”"). An evidence code IGC was assigned to annotations of these P450 proteins since annotations were based on genomic context.

A total of 428 putative transcription factors of M. oryzae were validated by integrated computational analysis of whole genome Sapanisertib microarray expression data, and matches to InterPro, pfam, and COG [3]. Again, different subsets of the 428 proteins were annotated with different GO terms. For example, 36 proteins were annotated with GO:0005975 (“”carbohydrate metabolic process”"), and 12 proteins were annotated with GO:0006520

(“”amino acid metabolic process”"). An evidence code RCA was assigned to annotations of the 428 transcription factors since the annotations were based on reviewed computational analysis. A total of 2,548 conserved domains from NCBI CDD were used as evidence for cross-checking putative functions, but no GO annotation was made based solely on identification of these domains. In addition, the evidence code ISS was assigned to annotations of 216 M. oryzae proteins for the following reasons: 1) These proteins have significant similarity to experimentally-characterized homologs over the majority (at least 80%) of the full length sequences. GNA12 2) The pairwise alignments of good matches between the characterized proteins and the proteins of M. oryzae were manually reviewed. 3) Functional domains were conserved between the M. oryzae proteins and their homologs. 4) The GO assignments from the characterized match proteins to the M. oryzae proteins were manually determined to be biologically relevant. The remaining 1,343 proteins with a reciprocal BLASTP best match of e-value > 10-20 and pid < 40% were assigned GO terms from their characterized matches, but the evidence codes were identified as IEA (Inferred from Electronic Annotation). In sum, GO terms were assigned to 6,286 proteins of M. oryzae.

Authors’ contributions SD, VD and MP searched the literature for relevant contributions and helped to draft the manuscript. LS, MDA and MB conceived of the review, designed it and refined the draft version of the manuscript. All authors read and approved the final manuscript.”
“Background Increasing evidence suggests that immune responses play an important role in the control of cancer and manipulation of the immune system to recognize and attack cancer cells may be a valuable form of therapy [1]. Hepatocellular carcinoma (HCC), which is the third most common cause of cancer death world-wide [2], is a potential target for immunotherapy [3] because there are numerous documented

cases of spontaneous regression [4] and the presence of cytotoxic see more tumour infiltrating lymphocytes (TIL) at histological FK228 in vitro examination is associated with a better prognosis after liver resection or transplantation [5]. Infusion of T lymphocytes, activated with SN-38 anti CD3 and interleukin 2 (IL2), improved disease-free survival after HCC resection, suggesting a role for T cell immunotherapy in this setting [6]. However, current methods of isolation and in vitro expansion of T lymphocytes are cumbersome and expensive, and the durability of any anti-tumour immune response

induced by administration of non-antigen specific, in vitro expanded T cells is unknown [7]. Many tumours, including HCC, express tumour-associated antigens (TAA) that might serve as potential targets for antigen-specific T cell immunotherapy. Glypican 3 (GPC-3), a 580 amino acid glycosylphosphatidylinositol-linked heparan sulphate proteoglycan, is

expressed in foetal liver and plays an important role in foetal development because it facilitates the interaction of growth factors with their cognizant receptors [8]. It is rarely detected in adult liver but is reactivated in 72% of HCC [9], where its expression is correlated with a poor prognosis [10]. Intradermal vaccination of BALB/c mice with a GPC-3 peptide (EYILSLEEL), restricted to the murine MHC-I molecule H-2Kd, mixed with incomplete Freund’s adjuvant induced epitope specific, cytotoxic T lymphocytes (CTL) [11] and immunization using dendritic cells (DC) pulsed with this peptide prevented the growth of GPC-3 positive tumours [12]. Mice vaccinated with DC expressing Avelestat (AZD9668) GPC-3 as a transgene were also found to have protective immunity against subsequent challenge with GPC-3 positive melanoma cells [13]. In a study of 20 HCC patients treated with locoregional therapy, 16 (80%) were found to have TAA-specific CD8+ T cells, including T cells directed against GPC-3 [14]. Furthermore, the magnitude of the TAA-specific CD8+ T-cell response was a significant independent prognostic factor for tumour-free survival. These data suggest that GPC-3 is a novel HCC-associated antigen but further studies are required to investigate the immunogenicity of human GPC-3 and to establish any therapeutic potential.

YitA AZ 628 cell line and YipA persist for several hours following a growth temperature shift to 37°C YitA and YipA levels over time following a temperature shift from 22°C to 37°C was determined by Western blot analysis. YitA and YipA synthesized by KIM6+ (pCR-XL-TOPO::yitR) following growth in BHI at 22°C were still present 7 hours after an upshift to 37°C (Figure 4, lanes 5, 8, 11, 14, and 18). After 9 hours, a slight reduction

in YitA and YipA protein was seen in the 37°C culture compared to the matched culture maintained at 22°C (Figure 4, lanes 21 and 20, respectively). After 24 hours, there was a significant decrease in detectable YitA and YipA (Figure 4, lane 24) in the 37°C culture. After 30 hours at 37°C, only a small quantity of YitA remained and no detectable YipA (Figure 4, lane 27). Figure 4 YitA and YipA proteins persist in Y. pestis for at least 9 hours after transfer to 37 °C . Y. pestis KIM6+ (pCR-XL-TOPO::yitR) (lanes 5, 8, 11, 14, 18, 21, 24, and 27) and KIM6+ΔyitA-yipB (pCR-XL-TOPO::yitR) (lanes 3, 6, 9, 12,

15, 19, 22, 25, and 28) were grown overnight at 22°C and subsequently transferred to 37°C for the indicated amount of time prior to sample collection. A matched KIM6+ (pCR-XL-TOPO::yitR) was maintained at 22°C as a positive reference control (lanes 2, 4, 7, 10, 13, 17, 20, 23, and 26). T0 = initial time point, T(x) = x hours at 37°C. Panels show Western blots probed with anti-YitA, anti-YipA, or anti-Ail (sample loading control) antiserum. Evidence for post-translational

processing of YipA Carnitine palmitoyltransferase II Two forms of YipA were typically detected by Western Belnacasan order blot: the predicted full-length protein at ~106 kDa and a smaller protein of ~73 kDa, with the smaller form often predominating (Figures 2, 3, 4). To determine which of the bands detected using anti-YipA serum correspond to the N-terminal region and the C-terminal region of YipA, Y. pestis strains containing translational fusions of mature β-lactamase (~28.9 kDa) to the C-terminus of YitA or YipA were constructed (Figure 1B). After overnight growth at 22°C, Y. pestis YitA-β-lactamase and YipA-β-lactamase with or without Luminespib concentration plasmid pCR-XL-TOPO::yitR were assayed by Western blot. YitA-β-lactamase was detected by anti-YitA serum as a single band at ~123 kDa (due to the addition of the mature β-lactamase) with a light smear of smaller bands (Figure 5A, lane 2), whereas wild-type YitA was detected around 95 kDa (Figure 5A, lane 4). Anti-β-lactamase antibody also detected full length YitA-β-lactamase at ~123 kDa as a prominent band and a smear of several smaller bands (Figure 5C, lane 2). Figure 5 Characterization of post-translational processing of YipA. KIM6+ (pCR-XL-TOPO::yitR) with the C-terminus of YitA (Lane 2) or YipA (Lane 4) tagged with mature β-lactamase were grown overnight at 22°C in BHI broth.