The subjective pain rating was assessed prior to MVIC, except during the POST learn more assessments at visits 2 and 7 (Figure 1) when the subjective DNA Damage inhibitor pain rating was assessed after the MVIC. Resting blood pressure and resting heart rate The resting blood pressure and resting heart rate were measured after the participant had been sitting quietly for a period of at least 5 minutes prior to any other testing. Systolic and

diastolic resting blood pressure were measured in mmHg with an aneroid sphygmomanometer(MDF Instruments, Agoura Hills, CA) and a stethoscope (Marshall Nurse Stethoscope, Riverside, IL) according to the procedures described by Housh et al. [18]. Resting heart rate was measured by palpating the radial artery at the anterior-lateral surface of the wrist in line with the base of the thumb, just medial to the styloid process of the radius. Once the pulse was located, the number of beats that occurred in 30 s was measured and multiplied by two to find more calculate the resting heart rate (bpm). Statistical analyses Four separate two-way repeated measures analyses of variance (ANOVAs) (condition [ANA vs. PLA] x time [PRE vs. POST vs. 24 h vs. 48 h vs. 72 h]) were used to analyze PT, hanging joint angle, relaxed arm circumference, and subjective pain rating. Three separate two-way repeated measures ANOVAs (condition [ANA vs. PLA] × time [PRE vs. 72 h]) were used to analyze systolic blood pressure, diastolic

blood pressure, and resting heart rate. When appropriate, follow-up analyses included one-way repeated measures ANOVAs and Bonferonni-corrected dependent samples t-tests. All statistical analyses were performed using IBM SPSS v. 21 (Chicago, IL), and a type I error rate of 5% was considered statistically significant for all comparisons. Results There were no condition x time (p > 0.05) interactions, there were no main effects for condition (p > 0.05), Vildagliptin but there were main effects for time for PT (p < 0.001), hanging arm joint angle (p < 0.001),

relaxed arm circumference (p < 0.001), and subjective pain rating (p < 0.001). The marginal means for PT (collapsed across condition) decreased (p < 0.001) from PRE to POST, increased (p = 0.001) from POST to 24 h, and then plateaued (p > 0.05) from 48 h to 72 h (Figure 3a). The marginal means for hanging joint angle (collapsed across condition) decreased (p < 0.001) from PRE to POST and then did not change (p > 0.05) from POST to 72 h (Figure 3b). The marginal means for relaxed arm circumference (collapsed across condition) increased from PRE to POST (p < 0.001) and then plateaued (p > 0.05) from POST to 72 h (Figure 3c). The marginal means for subjective pain ratings (collapsed across condition) increased (p < 0.001) from PRE to POST, but did not change (p > 0.05) from POST to 72 h (Figure 3d). Figure 3 Recovery of the non-invasive measures of muscle function.

In addition, biofilm formation is not affected by NO produced by other NO-producing pathways, as neither the NO scavenger nor the addition of exogenous NO had an effect on mature biofilm structures. Previous studies have shown that cellular differentiation and biofilm formation in B. subtilis are controlled by intracellular concentrations of the phosphorylated master regulator Spo0A [14]. Two sensor kinases (KinA and KinC) that control the level of Spo0A phospohrylation possess cytoplasmic PAS sensor domains, which have been implicated to GDC-0449 research buy sense redox potential and O2. In turn, a mutational study of the cytoplasmic PAS domain of B. subtilis’ sensor kinase ResE suggested that it senses NO under anaerobic

conditions [28]. Thus, it is conceivable that KinA and KinC are affected by NO signalling. However, our study indicates that NOS-derived NO and exogenously supplied NO do not affect the PAS domains of KinA and KinC such that biofilm formation and differentiation is significantly altered. This

supports the notion that biofilm formation and differentiation in B. subtilis are rather controlled by specific extracellular molecules, such as signalling peptides [14], as opposed to more broad range redox-based signals like NO. NO is not involved in coordinating swarming of B. subtilis 3610 We tested the influence of NO and NOS activity on the swarming BTK inhibitor motility of B. subtilis 3610 on LB-based swarm agar (Figure 4). Swarm expansion of wild-type B. subtilis on 0.7% LB agar was 9 mm h-1 (± 0.8 mm) and agrees well with swarm expansion of 10 – 14 mm h-1 reported selleck chemicals by Kearns and Losick [13]. Swarm expansion was not significantly affected by the presence of NOS inhibitors, NO scavenger, NO donor and for the nos mutant. This shows that neither NOS-derived NO nor

exogenously supplied NO influences swarming motility in B. subtilis. Figure 4 Influence of NO and NO synthase (NOS) on the swarm rate of B. subtilis 3610. Swarm expansion DNA Damage inhibitor assays with strain 3610 wild-type (white bars) and strain 3610Δnos (gray bars) were performed on 0.7% LB agar without supplementation (controls) or supplemented with 100 μM L-NAME (NOS inhibitor), 100 μM c-PTIO (NO scavenger) and 20 μM or 200 μM Noc-18 (NO donor). Error bars indicate standard deviation (N = 6). Differences between individual dataset are not statistically significant (α = 0.01; see Material and Methods section for details). NOS-derived NO inhibits biofilm dispersal of B. subtilis 3610 We tested the influence of NOS-derived NO and exogenously supplied NO on the dispersal of B. subtilis 3610 from spot colony biofilms of wild-type and nos mutant cells (Figure 5A). First, biofilms were grown on MSgg agar or MSgg agar supplemented with NOS inhibitor or NO scavenger. To assay dispersal, we mounted a drop of MSgg medium containing a similar treatment as the underlying agar onto mature colony biofilms.

As shown in Figure 8C, the internalized (MTX + PEG)-CS-NPs were found initially to be localized

in the lysosomes, as evidenced by the yellow spots in the merged image obtained from the images of the (MTX + PEG)-CS-NPs (green) and late endosomes/lysosomes (red). The result indicated that the (MTX + PEG)-CS-NPs were internalized via the endocytosis pathway into the late endosomes/lysosome [47]. Indeed, after incubation for 4 h, some green fluorescent FITC-labeled (MTX + PEG)-CS-NPs were no longer located in the red fluorescent late endosomes/lysosomes, indicating the successful endo/lysosomal escape. In agreement with other reports [37, 48], these results combined with the results of in vitro drug release and cell selleck chemicals llc viability studies further proved that MTX was released from the (MTX + PEG)-CS-NPs inside the cells by the intracellular protease-mediated selective cleavage of peptide bond. These findings were also in agreement with other reports in the literature [49] that CS possessed the activity to some extent to escape the endo/lysosome. Conclusions We presented the versatile, robust, and easy MTX-based PEGylated CS-NPs while validating MTX as a successful targeting ligand coordinated with a simple anticancer drug, and established the (MTX + PEG)-CS-NPs as a cocktail platform of specific targeting cooperated with enhanced anticancer activity.

MTX was not prematurely released at off-target site but was intensively released at target site due to its sustained/protease-mediated Selleckchem Y 27632 Aspartate drug release characteristic. To the best of our knowledge, the work for the first time explored the validation of Janus role of MTX based on the nanoscaled drug delivery system in vitro. Additionally, as MTX (a targeting ligand/a first drug) was introduced into one kind

of drug carriers, one further advantage was that the drug delivery systems allowed the further introduction of a second ligand or a second drug for synergistic co-targeted delivery or synergistic co-delivery of drugs. Nevertheless, more details about in vivo targeting and anticancer investigations are indispensable to obtain a better understanding of the therapeutic effect of the (MTX + PEG)-CS-NPs, and relevant studies are in process. Authors’ information Both authors FL and YL contributed equally and mTOR inhibitor should be considered as co-first authors. Acknowledgements Fanghong Luo acknowledges the financial support by the Natural Science Foundation of Fujian Province of China (Grant No. 2013 J01384) and Science and Technology Foundation of Xiamen of China (Grant No. 3502Z20113012). Dr. Yuan Jiang is acknowledged for useful discussions and editing the manuscript. References 1. Peer D, Karp JM, Hong S, Farokhzad OC, Margalit R, Langer R: Nanocarriers as an emerging platform for cancer therapy. Nat Nanotechnol 2007, 2:751–760.

Subjects were asked to step up (concentric muscle action) onto a 40 cm box then step down (eccentric muscular contraction) and the soreness in doing so was rated. The three scales (for the three mornings) were all contained on one sheet see more of paper, but marked soreness values from preceding mornings were covered on the second and third mornings to avoid comparison by the subject. Biochemical analyses Creatine kinase. Analysis of the muscle damage marker creatine kinase (CK), in serum collected before and 12, 36 and 60 hours

post damage, was carried out at a commercial blood testing laboratory (MedLab Central, Palmerston North, New Zealand). An enzymatic ‘reverse reaction’ method was employed, which photometrically measures the rate of NADPH formation as a final product of the last of three reactions, to quantify CK activity. Results are expressed as % change from pre-damage levels. Plasma protein carbonyls. Plasma protein carbonyls were measured using the method previous described by Levine et al.[24]. Briefly, 50 μL of plasma was added to an equal GS-9973 concentration volume of 2,4-dinitro-phenylhydrazine (DNPH, Sigma-Aldrich, Auckland, New Zealand) in 2 M HCl (control = DNPH/HCl in the absence

of plasma) and incubated in the dark for 1 hour. Protein was precipitated with 50% trichloroacetate (TCA, Sigma-Aldrich, Auckland, New Zealand) and the pellet washed GF120918 datasheet three times with ethanol:ethylacetate (1:1). The pellet was then re-suspended in 1 mL 6 M guanidine hydrochloride (Merck NZ Ltd., Palmerston North, New Zealand) at 37°C for approximately 15 min, followed by the absorbance being measured at 360 nm in a UV-visible 1601 spectrophotometer (Shimadza Corporation, Kyoto, Japan). Protein carbonyl levels were then calculated from the absorbance difference

between test and control many using the molar absorption coefficient (ϵ): 22,000 M-1 cm-1. Plasma protein levels were measured using the Bradford method [25] using commercial Bradford reagent (BioRad Laboratories). Results are calculated as nmol of protein carbonyls/mg total protein and expressed as % change from pre-damage levels. Plasma radical oxygen species (ROS)-generating potential. Hydrolysed carboxy-dihydro-2′,7′-dichlorohydrofluorescein diacetate (carboxy-H2DCFDA, Merck, Ltd., Palmerston North, New Zealand) was used to assess the ROS-generating capacity of plasma, using a method previously described by Hurst et al.[26]. Briefly, dihydro-2′,7′-dichlorohydrofluorescein (DCF), which is fluorescent when oxidised was added to diluted (1:4) plasma collected pre and post damage at 12, 36 and 60 hours in phosphate buffered saline [PBS], pH 7.4, Invitrogen NZ Ltd., Auckland, New Zealand), or PBS control, then 0.

It should be noted that most of the wounds we have evaluated in the past have relatively high overall numbers of bacteria (>105 per mg debridement, based upon quantitative buy AZD2281 molecular methods) so even relatively low percentages of individual species such as 2% Anaerococcus spp. may potentially represents a large number of individual bacteria propagating within wound biofilms. Conclusion Dowd et al [15] first used pyrosequencing to survey pooled samples of VLU, diabetic foot ulcers and decubitous ulcers and later did a more comprehensive survey of diabetic foot ulcers [9]. This study takes a similar

but more comprehensive approach with VLU in order to better elucidate the individual ecologies in a large population of such chronic wounds. Here we show that individual wounds CHIR-99021 in vitro have distinct ecological footprints. We also show that within individual wounds there can be both significant site specific differences and relative uniformity in the bacterial ecology. The bottom line appears to be that each wound must be carefully evaluated and that no single pathogen is likely to be the causative agent of such infections. The wound care scientific and clinical opinion leaders have come to accepted the abundance of data showing that these polymicrobial biofilms represent

a primary impediment to wound www.selleckchem.com/products/AZD8931.html healing [9, 14, 20–22, 22–25, 25–30]. Based upon the current Gemcitabine datasheet work and previous efforts we can deduce that the unique profiles of each individual wound indicate that a personalized approach to therapeutics combined with the multiple concurrent strategies of biofilm-based wound care [26] will revolutionize wound care. As Tom Pollard indicated in a commentary recently, biofilm-based wound care is “” a significant shift in our whole approach

to wound healing.”" [31]. Biofilm-based wound care combined with individualized therapeutic approaches and accurate rapid molecular diagnostics provides renewed found hope for those suffering with chronic wounds. Methods General sample collection methods Patients were identified with VLU that have been persistent for over 6 months. These patients were enrolled in the study protocol after being educated and signing the informed consent protocol in compliance with Western Institutional Review Board approved protocols 56-RW-004 WIRB® Protocol #20062347. Necessary details of the study including the protocols, guidelines and requirements were thoroughly explained to all the patients. Following these explanations, written consents was obtained in the presence of a third party witness. A copy of the consent form has been provided to journal editors. The patients were well informed that they have the right to opt out of the study at any time in spite of their written consent. VLU wound beds were debrided to remove superficial debris and cleansed with sterile saline.

0 (Applied Biosystems, Foster city, CA, USA) according to the recommendations of the manufacturer (Table 5). This software was used to choose the best combinations of each primers-probe set www.selleckchem.com/products/ldn193189.html values. Finally, the selected primers and probes were checked for homology to non-target sequences by a search with the BLAST program of the National Center for Biotechnology Information (NCBI). Primers and MgB probes were synthesized by Applied Biosystems and stored at -20°C prior to use. Real-time PCR amplification Reactions were done in 20 μL PCR mixtures containing 10 μL of 1X Taqman Universal PCR

Mastermix (AmpliTaq Gold™ DNA polymerase, dNTPs, Passive reference (ROX), and optimised buffer components including 5 mM MgCl2), 400 nM of each primer (glyA-R Ilomastat molecular weight and glyA-F for C. coli real-time PCR assay, hipO-R and hipO-F for C. jejuni real-time PCR assay), 200 nM of the probe (glyA-P

and hipO-P respectively), and 5 μL of template DNA. The thermal cycle protocol used was the following: activation of the Taq DNA polymerase at 95°C for 10 min, then 45 or 48 cycles of 15 s at 95°C and 60 s at 60°C. Thermal cycling, fluorescent data collection, and data analysis were carried out with the ABI PRISM® 7300 Sequence Detection System (Applied Biosystems) according to the manufacturer’s instructions. Fluorescence of FAM and VIC was measured at their respective wavelengths during the annealing/elongation step of each cycle. After real-time data acquisition, the baseline cycles for the FAM and VIC signals were set from cycle three to three cycles below the cycle at which the first signal

appeared and the threshold value at the point at which the fluorescence exceeded 10 times the standard deviation of the mean baseline emission. The threshold cycle (Ct) is the first PCR cycle at which a statistically Vitamin B12 significant increase in fluorescent signal is detected. All reactions were carried out alongside a non template control containing all reagents except DNA, positive controls containing DNA from reference strains (C. jejuni NCTC 11168 and/or C. coli CIP 70.81), and negative controls containing DNA from Listeria monocytogenes ATCC 19115 and from Escherichia coli CIP V517. All the DNA extractions were done as described before. Each control was run in triplicate and each sample in duplicate. Evaluation of performance of the real-time PCR assays Specificity and sensitivity The specificity of each real-time PCR assay was first assessed with purified genomic DNA preparations (about 106 genome copies per PCR reaction) of different bacterial strains (Table 1) and then with DNA www.selleckchem.com/products/bmn-673.html extracted from 30 Campylobacter-negative faecal, feed, and environmental samples as defined above. This screening strategy, described previously by Lagier et al. (2004) [33], ensure the specificity of the primers and probes for C. jejuni and C. coli only in field samples.

These include restrictions on animal movement and trade for affected countries, with disease and THZ1 purchase infection https://www.selleckchem.com/products/MGCD0103(Mocetinostat).html control measures increasing production costs owing to antibody testing, vaccination programs and extra

labor. Although PRV has been widely studied (especially its agricultural impact, its viral pathogenesis, its molecular biology, its use as a neuronal tracer, and in DNA vaccine exploration [1]) how the native host responds globally after infection with wild type PRV is still poorly understood. Clinically, infection in older pigs ranges from asymptomatic to severe respiratory disease but with limited mortality. Young piglets exhibit more serious clinical signs and often succumb to fatal encephalitis

preceded by typical behaviors consistent with infection of the central nervous system. In recent years, microarray technology has proven useful to assess the cellular selleck products transcriptional responses to herpesvirus infections in human and mouse cell lines [3–5]. It has been used to study host gene expression after PRV infection of rat embryo fibroblasts [5], and the central nervous system (CNS) in rodent brain at various times post infection in vivo [6]. However few porcine genome-wide expression studies have been published. Most experiments have used ‘in-house’ cDNA arrays to study transcriptional events in pig tissues, such as the stress-genes related to early weaning of piglets [7]. The down side of these cDNA-based clone libraries is that the genes represented on the

array are often very focused on a given biological system or process and lack a whole genome overview. In this study, piglet samples were hybridized onto an Illumina Human Refset Chip (Illumina Inc. San Diego), corresponding to 23,000 transcript probes. This cross-species comparison potentially allows the study of the whole transcriptome. Branched chain aminotransferase There are now porcine arrays available from commercial suppliers (e.g. Affymetrix and Qiagen), but these are not all representative of the entire pig genome and were not widely available at the time of this study. In the absence of a comprehensive species-specific array deeper interrogation of the pig gene complement was afforded by the use of the better annotated human geneset. Although the use of this approach can only be partially informative when there are no confirmed pig orthologues in the public databases, we have identified host cellular genes whose mRNA levels change during natural PRV infection of piglet brain and lung. The resulting data define key pathways of host-gene expression that characterize the host response to an acute central nervous system (CNS) and respiratory infection. Methods Experimental pigs and housing The experimental animals were sourced from an outbreak of PRV that occurred in the farrowing house of a local commercial farmer due to a reduced level of protection via maternal antibody.

For example, though Andrade-Linares Protein Tyrosine Kinase inhibitor et al. (2011) did not measure antioxidant or reactive oxygen species production they reported a potential negative, life stage response of the host to FK228 ic50 endophyte colonization. In their study three dark septate endophyte species colonizing tomato (Lycopersicum esculentum) successfully decreased the negative effects of the fungal pathogen Verticillium dahlia but only when the pathogen was presented in low doses. At higher pathogen doses the endophyte effect on host biomass loss was not significantly different from controls. The same study found no significant difference

in terms of reproductive output between E + and E- plants except at the earliest harvest date. Fruit number and biomass at first harvest were significantly higher in E + versus E- hosts. Thus positive impacts on host vegetative growth and reproductive output appear to be life stage dependent, but whether they extend to increased host lifetime fitness has not been determined. Shoot and whole plant endophytes

Several studies on various host species and their shoot associated fungal endophytes support increased host stress tolerance due to increased antioxidant production in E + hosts (Table 1) compared to E- hosts. A comparison of cellular level reactive oxygen species scavenging activity in Phyllosticta colonized versus E- Guazuma tomentosa revealed significantly higher scavenging activity in the former (Srinivasan E7080 in vivo et al. 2010). Neotyphodium–endophyte colonized grasses showed significantly higher glutamine synthetase and total amino acid activity (Lyons et al. 1990) in response to nutrient treatments which positively correlated with host biomass. In response to temperature, ID-8 drought, and salt stress, E + hosts produced significantly more biomass than their E- counterparts (Redman et al. 2001 and 2002; Márquez et al. 2007; Rodriguez et al. 2008; Redman et al. 2011). Regardless of plant host or fungal endophyte genera, symbiosis resulted in increased plant biomass production

and/or survival in response to all three stress treatments and the mechanism appeared to be increased antioxidant activity leading to higher reactive oxygen species scavenging rates and lower reactive oxygen species accumulation in E + host tissues (Rodriguez et al. 2008). This leads to the general conclusion that habitat-specific stress tolerance can be effectively conferred via symbiotic interactions with fungal endophytes from diverse genera (Rodriguez et al. 2008). Additional studies reported a virus present in the endophyte Curvularia protuberata was needed for the endophyte to confer heat tolerance (Márquez et al. 2007). Both a monocot and dicot colonized by the virus-endophyte combination were able to successfully tolerate root zone temperatures of up to 65°C.

*P < 0.05 and # P < 0.01 vs CS; ★ P < 0.05 and ※< 0.01 vs SE; △ P < 0.01 vs ES. Exhaustive exercise induces the generation of free radicals which may cause an increase in lipid peroxidation [21]. Measuring MDA is one of the most widely used approaches for evaluating oxidative damage to lipids. Figure 3b illustrates that the plasmic MDA levels of SE or ES-LBP rats significantly decreased compared with that of ES rats (P<0.05 and P< 0.01 respectively). This result indicates that LBPs can attenuate lipid peroxidation. NO is an important vasodiator factor produced by vascular endothelial cells. We found that there was a significant increase in the SE

group. As expected, the NO level was significantly reduced by exhaustive exercise. Further, XAV-939 cost we found this reduction induced by exhaustive exercise could be reversed by LBPs treatment (Figure 3c). The expression of heat shock proteins (HSPs) is induced by hyperthermia check details ischemia, oxidative cytokine, muscular stress, glucose deprivation, alterations in calcium and pH [22]. HSP70 is a group of binding proteins with molecular weight of 70 KD, which is significantly https://www.selleckchem.com/products/ly2835219.html increased by high-intensity exercise [23]. To determine the expression of HSP70 after exercise and supplement with LBPs, the plasmic level of HSP70, analyzed by ELISA, showed

an immediate increase after both exercise sessions. As shown in Figure 3d, the HSP70 levels of SE or ES rats were increased. Furthermore, LBPs treatment induced a much higher increase in the ES group (P< 0.01). Expression of eNOS mRNA As the NO level can be up-regulated by LBPs, we therefore examined the effect of LBPs on the expression of eNOS in the aorta after exhaustive exercise. The expression of eNOS mRNA in aorta of four groups was shown C-X-C chemokine receptor type 7 (CXCR-7) in Figure 4. There were significant differences in the eNOS mRNA expression level among different groups. The eNOS expression was increased in both SE and ES-LBP groups (P < 0.01). However, the level of eNOS expression was significantly attenuated in rats after exhaustive exercise (P < 0.01). LBPs treatment significantly

reversed the inhibition of the eNOS expression in rats from ES group (p < 0.01). Figure 4 Effects of LBPs on eNOS mRNA expression in thoracic aorta separated from rats in different groups. Values are expressed as mean ± SD (n = 10). # P<0.01 vs CS; △ P<0.01 vs ES. Discussion The effects of LBPs on vascular vasoreactivity in exhaustive exercise rats were investigated. The major finding of this study was that the contraction induced by NA in thoracic aorta was increased in the presence of exhaustive exercise. Furthermore, supplementation with the LBPs for 4 weeks remarkably improved the vascular reactivity of ES-LBP rats compared to the ES rats (Figure 1). As the arterial compliance is judged by the responsiveness to NA, the results showed that the compliance or distensibility of aorta was increased in LBPs treated animals [24].

Primers (available upon request) were designed using Primer Express (Applied Biosystems). The RT-qPCR assays were done using the ABI 7000 SDS, SYBR green chemistry, and optical plates (Applied

Biosystems), as previously described [52]. At each time point, raw RT-qPCR data for each gene were normalized against the data obtained for the 16S rRNA transcript, as it was previously demonstrated that this is an adequate endogenous control [52]. The final results were based on three independent experiments. Results Selection of C. trachomatis proteins analyzed in this work To search for previously unidentified T3S substrates of C. trachomatis, we first surveyed the INK1197 genome of strain L2/434 for genes encoding mostly uncharacterized proteins, or with a putative biochemical activity compatible with the function of a T3S effector (e.g., proteases). Among these genes, we selected those encoding proteins not predicted to have a signal sequence characteristic of the general secretory pathway (according to Psortb v3.0) and that had not been previously analyzed experimentally for the presence of a T3S signal. This singled out 32 proteins (CT016, CT017, CT031, CT051, CT053, CT080, CT105, CT142,

CT143, CT144, CT153, CT161, CT172, CT273, CT277, CT289, CT309, CT330, CT338, CT386, CT425, CT568, CT583, CT590, CT631, CT635, CT656, CT696, CT702, CT837, CT845, and CT849; we used the nomenclature of the annotated C. trachomatis D/UW3 strain [53]; the names of the corresponding genes as annotated A1155463 for strain L2/434 [54] can be found in Additional file 3: Table S3). Furthermore, for comparison purposes, we considered proteins that had been tested for the presence of a T3S signal using Shigella flexneri as a heterologous bacteria: eight proteins whose first ~40 amino acids of the corresponding C. pneumoniae homologs did not drive secretion of an adenylate cyclase Glutathione peroxidase (Cya) reporter protein by S. flexneri (CT066, CT429, GrgA/CT504, CT538, CT584, CT768, CT779, CT814), and three

proteins whose N-terminal region of the C. pneumoniae homologs drove secretion of a Cya reporter protein by S. flexneri (CT203, CT577, CT863) [21]. Please note that at the time this work was initiated GrgA/CT504 was an uncharacterized protein; however, it was recently described as a transcriptional ICG-001 clinical trial activator [55]. Finally, throughout this study we used as positive controls a C. trachomatis bona-fide T3S effector (CT694) [14] and a C. trachomatis likely T3S substrate (CT082) that we had previously identified [26], and which was recently independently confirmed [27], and as negative control a predicted ribosomal protein (RplJ/CT317). In summary, in experiments that will be described below, we analyzed T3S signals in 46 C. trachomatis proteins (~5% of all proteins encoded by the L2/434 strain): 32 hypothetical proteins previously not analyzed experimentally for T3S signals, 11 proteins whose C. pneumoniae homologs were previously analyzed for T3S signals using S.