Abstract
Staphylococcus aureus is a major human pathogen and one of the more prominent pathogens causing biofilm related infections in clinic. Antibiotic resistance in S. aureus such as methicillin resistance is approaching an epidemic level. Antibiotic resistance is widespread among major human pathogens and poses a serious problem for public health. Conventional antibiotics are either bacteriostatic or bacteriocidal, leading to strong selection for antibiotic resistant pathogens. An alternative approach of inhibiting pathogen virulence without inhibiting bacterial growth may minimize the selection pressure for resistance. In previous studies, we identified a chemical series of low molecular weight compounds capable of inhibiting group A streptococcus virulence following this alternative anti-microbial approach. In the current study, we demonstrated that two analogs of this class of novel anti-virulence compounds also inhibited virulence gene expression of S. aureus and exhibited an inhibitory effect on S. aureus biofilm formation. This class of anti-virulence compounds could be a starting point for development of novel anti-microbial agents against S. aureus.
Citation: Ma Y, Xu Y, Yestrepsky BD, Sorenson RJ, Chen M, et al. (2012) Novel Inhibitors of Staphylococcus aureus Virulence Gene Expression and Biofilm Formation. PLoS ONE 7(10): e47255. doi:10.1371/journal.pone.0047255 ?University Medicine Berlin, Germany Editor: Stefan Bereswill, Charite Received July 2, 2012; Accepted September 10, 2012; Published October 15, 2012 Copyright: ?2012 Ma et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by National Institutes of Health (NIH) Grant P01HL573461 (HS), University of Michigan Life Sciences Institute Innovation Partnership grant (HS and SDL), and NIH Pharmacological Sciences Training Program Grant T32 GM007767 (BDY). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: HS, SDL and BDY are co-inventors on a US patent 61/641,590 entitled: Methods and Compositions for treating bacterial infections, filed May 2, 2012. One of the co-authors, MC, is employed by a commercial company (Nanova, Inc.). This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: [email protected] (HS); [email protected] (SDL)

Introduction
Staphylococcus aureus is a major human pathogen that causes skin, soft tissue, respiratory, bone, joint and endovascular infections, including life-threatening cases of bacteremia, endocarditis, sepsis and toxic shock syndrome [1]. Approximately 30% of humans are Staphylococcus aureus carriers without symptoms [2]. S. aureus is also one of the most common pathogens in biofilm related infections of indwelling medical devices which are responsible for billions in healthcare cost each year in the United States [3?]. Bacteria can attach to the surface of biomaterials or tissues and form a multilayered structure consisting of bacterial cells enclosed in an extracellular polymeric matrix [9]. Bacteria in biofilm are particularly resistant to antibiotic treatment [10]. In addition to the difficulty of effectively inhibiting biofilm with conventional antibiotic therapy, treatment is further complicated by the rise of antibiotic resistance among staphylococci. In recent years, methicillin resistance in S. aureus is approaching an epidemic level [2,11?3]. The emergence of antibiotic resistance poses an urgent medical problem worldwide. Current antibiotics target a small set of proteins essential for bacterial survival. As a result, antibiotic resistant strains are subjected to a strong positive selection pressure. Inappropriate and excessive use of antibiotics have contributed to the emergence of pathogens that are highly resistant to most currently available antibiotics [14?6]. The novelapproach of inhibiting pathogen virulence while minimizing the selection pressure for resistance holds great promise as an alternative to traditional antibiotic treatment [17]. The feasibility of such an approach was demonstrated for Vibrio cholerae infections when a novel small molecule was identified that prevented the production of two critical virulence factors, cholera toxin and the toxin coregulated pilus. Administration of this compound in vivo protected infant mice from V. cholerae [18]. In a similar proof-ofconcept (POC) study, a small molecule inhibitor of the membraneembedded sensor histidine kinase QseC was identified. The inhibitor exhibited in vivo protection of mice against infection by Salmonella typhimurium and Francisella tularensis [19]. In a POC study following the same paradigm, we have identified a chemical series of small molecules from a high throughput screen (HTS) that can inhibit expression of the streptokinase (SK) gene in group A streptococcus (GAS) [20]. We previously demonstrated that SK is a key virulence factor for GAS infection [21]. SK activates human plasminogen into an active serine protease that degrades fibrin, a critical component of blood clots and an important line of defense against bacterial pathogens [22,23] Our novel SK gene expression inhibitor also inhibited gene expression of a number of important virulence factors in GAS. The lead compound demonstrated in vivo efficacy at protecting mice against GAS infection, further supporting the feasibility of this novel anti-virulence approach to antibiotic discovery [20].

Detection of sequences of endogenous retroviruses was done with the PCR approach. Genomic DNA of examined people was isolated from peripheral EDTA-anticoagulated blood. DNA was extracted making use of protease K digestion and purification on QIAamp silica-gel columns (QIAampH DNA Blood Mini Package Qiagen, Germany). Then, PCRs for detection of HERV-K113 (according to Moyes et al., 2005) [19] and HERVK115 (based mostly on Burmeister et al., 2004) had been done [26]. PCRs were being performed in 10 ml reaction volume in a T3000 thermocycler (Whatman Biometra, Germany) making use of Taq polymerase (Polgen, Poland). PCR primers utilised in described reactions (Symbios, Straszyn, Poland) are offered in Table 2. 3 independent reactions have been expected for detection of HERVK113 for just about every sample. The first response (A), working with the pair of primers K113-F and K113-R (Desk 2), created a 300 bp fragment, corresponding to the insertion website of the endogenous retrovirus (no insertion). The next one (B), working with the pair of primers K113-F and K113-LTR-R (Desk 2), was utilized to detect the 59LTR sequence and HERV-K113 provirus fragment (1253 bp fragment). The amplification with primers pair LTRK113-F and K113-R (Desk 2) (reaction C) was employed to get a 483 bp fragment corresponding to the 39LTR of HERV-K113. A positive end result of the past two reactions confirmed the presence of HERV-K113 in the genome of analyzed persons. The PCR circumstances for HERV-K113 detection ended up as follows: 3 min at 94uC forty cycles: 1 min at 94uC, one min at 55uC (for reactions A and C) or 1 min at 59uC (for reaction B) 1 min at 72uC and the closing extension stage of five min at 72uC. PCR items ended up divided in two% agarose (AppliChem, Darmstadt, Germany) gel stained with ethidium bromide (.five mg/ml) and visualised in UV light (Fig. 1). 4 individual reactions were being carried out for detection of HERVK115 for every sample. The 1st just one (A), with the pair of primers K115-F and K115-R (Table two), was executed to detect the “wildtype” sequence (without HERV-K115 in the genome, 557 bp). The next a single (B), with K115-F and K115-LTR-R primers (Table 2), allow us locate a fragment of 59LTR (380 bp). The third 1 (C) with K115-F and K115-PROV-R primer set (Desk 2) served in detection of provirus presence in the examined genome (1269 bp band). The fourth reaction (D) necessary amplification of K115LTR-F and K115-R primers and gave PCR fragment for 39LTR (436 bp). A good consequence of the final a few reactions indicated the existence of HERV-K115 in the genome of examined individuals. PCRs ended up done as follows: 3 min at 94uC thirty cycles: thirty s at 94uC, 30 s at 54uC, 30 s at 72uC and a last extension set of ten min at 72uC. Amplification solutions were analysed in two% ethidium bromide stained agarose gel and visualised in UV light (Fig. two).
The distribution of HERV-K113 and HERV-K115 was calculated in control (n = 303), EU (n = 121) and HIV(+) (n = 470) groups. Variances among groups had been analysed employing two-sided Fisher specific test. A p price of a lot less than .05 was taken to be significant. A conceivable relation involving studied endogenous retroviruses and HIV an infection was evaluated making use of logistic regression in the normal linear model’s plan. The comparison of genetic and non-genetic factors in EU (n = 114) and HIV(+) (n = 452) teams was carried out. Beside the impression of HERVK113 and HERV-K115, the role of route of publicity to HIV (homosexual, heterosexual and intravenous drug use), sexual intercourse and age of individuals, and HCV carrying have been also tested. Variables were being provided in the product with p,.05. All statistical analyses ended up executed making use of the platform R-CRAN model 2.eight.1
Prevalence of HERV-K113 and HERV-K115 in the Decrease Silesia inhabitants of Poland was eleven.eight% and 7.ninety two%, respectively (manage team, n = 303 Desk three). No homozygous individual or solo LTR was detected in any of the analyzed teams. Genotype distributions of both equally HERVs in control as properly as in EU and HIV(+) teams have been compatible with the Hardy-Weinberg basic principle. Prevalence of HERV-K113 and HERV-K115 in the EU group (exposed uninfected men and women n = 121) was 8.26% and five.71% respectively. In the HIV(+) team (n = 470) these sequences were detected in twelve.ninety eight% for HERV-K113 and seven.23% for HERV-K115. There were being no statistically major variations amongst talked about groups and the manage team (p..05). To analyse the prospective relationship in between researched endogenous retroviral factors and HIV an infection we in comparison HERVK113 and K115 distributions in the group of HIV-infected people (n = 452) and in the group of sufferers exposed frequently for a very long time to HIV, but seronegative (EU, n = 114). We also took into account other factors: age, sex, type of exposure to HIV and HCV carrying. All stated variables were being analysed by logistic regression. The final results of endogenous retroviruses detection in EU and HIV(+) teams with reference to intercourse, manner of exposure and HCV coinfection are presented in Desk 4. Between examined agents only HCV co-an infection (OR = twelve.90 CI95% four.sixty nine?35.48 p = .00002) and homosexual exposure (OR = seven.69 CI95% one.88?one.48 p = .007017) have been identified to be factors rising susceptibility to HIV infection, as we described beforehand [40]. The homosexual EU team was very confined (n = 2) so it demands inclusion of far more patients to validate that conclusion. We discovered no relation among examined HERVs and HIV infection (p..05).

We attempt to comprehend how enzymes originate and evolve. A greater understanding of the guidelines that govern these adaptive processes would fill a information gap in the Darwinian Paradigm, and information protein engineers toward much more effective design and style algorithms. We have realized much from prior laboratory evolution experiments. Evolutionary principle suggests that new biological methods are products of contingency, when “a attribute progressed very long back for a different use has fortuitously permitted survival for the duration of a unexpected and unpredictable adjust in rules” [1]. At the molecular stage, an enzyme that initially progressed to catalyze the conversion of a unique substrate into a unique product or service may also respond likewise with other substrates (substrate ambiguity) or accelerate a diverse chemical transformation (catalytic promiscuity). These weak secondary capabilities could serve as seeds for subsequent evolutionary innovation. Reports of particular person enzymes in vitro showed that wild-kind enzymes could be multi-useful [two,three]. Groundbreaking directed evolution experiments showed that weak secondary routines can be physiologically pertinent and evolvable [four,five,six]. In standard, however with notable exceptions [seven,eight,nine,10,11] artificially advanced enzymes are not as precise or productive as their respective wild-kind ancestors. Why does directed evolution generally drop limited of the natural method? This query is not very easily dealt with [twelve], even following 20 years of directed protein evolution experiments [4,thirteen]. Protein engineers price pace, so they may be inclined to impose exceedingly stringent choices upon little populations for a fairly modest range of generations. We also suspect that laboratory selection problems are additional uni-dimensional than all those in the wild. The TEM-1 beta-lactamase catalyzes the hydrolysis of betalactam antibiotics. It easily evolves, in vitro and in situ, to understand cephalosporin antibiotics, so it serves as a model technique for scientific tests of protein evolution [four,14,15,16,seventeen]. The aminoglycoside phosphotransferases (APH) also present attractive but underutilized techniques for evolutionary research, as they diverged in nature to understand and inactivate a extensive selection of commercially accessible, clinically pertinent antibiotics [eighteen]. APHs are expressed in the cytoplasm, in contrast to beta-lactamase, which is secreted into the periplasm the place its activity can gain other cells. Previous staff have shown that double mutations in 1 family member, aminoglycoside phosphotransferase (299)-IIa, could boost the resistance of host cells to amikacin and isepamecin eight-fold relative to the wild-variety (as measured by distinctions least inhibitory concentrations) [19]. Adjustments of that magnitude are clinically appropriate, but we believe that quantitatively bigger variations are achievable in the laboratory and that these kinds of experiments would be educational. We chose aminoglycoside phosphotransferase (39)-IIIa, or APH(39)-IIIa, for our reports. It is broader in specificity than is APH(299)-IIa [eighteen], and is consequently probably additional evolvable. APH(39)-IIIa is very well characterised [20,21,22], and was the initial APH to be crystallized [23], so the biochemical mechanisms of mutations that accumulate throughout directed evolution can be inferred. We display right here that the enzyme readily evolves to understand amikacin, which is utilized to take care of multidrug resistant gram negative pathogens, and that it is relatively straightforward to purify, shop and assay in vitro. We also display that some mutations that are beneficial underneath choices are affiliated with important physical fitness costs beneath non-selective situations.
E. coli InvaF’ carrying the aph(39)-IIIa-pQBAV3c plasmid had been unfold on LB agar plates with different concentrations of amikacin following every round of selection. In the 1st spherical, 10,000 colonies have been distribute on LB agar supplemented with eighteen micrograms/mL amikacin (“wild-type MIC”). Colonies that shaped ended up then grown to saturation in liquid LB supplemented with chloramphenicol, diluted 1025-fold 50 microliters had been unfold on 106LB plates that contains 22, 26, thirty, 35, forty, forty five, or fifty micrograms/mL amikacin. In the next round, 10,000 colonies have been distribute on LB supplemented with eighty micrograms/mL amikacin. Colonies that formed have been subsequently restruck as above on LB plates made up of 160, one hundred seventy, a hundred and eighty, 200 micrograms/mL amikacin. In the third round, colonies ended up chosen on LB supplemented with 220 micrograms/mL amikacin, and people that shaped ended up then diluted 561024-fold and restruck on LB plates that contains 250, 280, 310, 340, 370, 400, 425, 450, 500, 550, 600, 650, seven-hundred, 750, 800, 825, 850, 875, 900, 950, one thousand, 1050, 1100, 1150, 1200, 2000 micrograms/mL amikacin. Following the fourth spherical, colonies ended up unfold (at around a thousand CFU/plate on ten plates) on LB with 1200 micrograms/mL amikacin. The reported susceptibility of just about every mutant (Desk two) is the minimum amount focus at which it was not able to variety any colonies.

SU5416 is a Ligand of the AHR
To confirm that this molecule is a direct ligand of the AHR and not working through some other agonist, we performed competitive binding assays of the AHR using a radioligand. Photoaffinity experiments incubating 125IBr2N3DpD with the hepatic cytosolic fraction from C57BL/6J mice (AHRb isoform) were conducted as described in the Methods [21]. Increasing concentrations of SU5416, TCDD, BNF, and 1,2-Benzanthracene (a ligand of low receptor affinity) were added. As shown in figure 2C, SU5416 competitively displaced the radiolabel with efficacy similar to TCDD.
Figure 2. Induction of DRE-mediated transcription by SU5416 is AHR dependent. A. The AHR-mutant C35 cell line was transfected with the AHRb, lacZ gene and a 36DRE-Luc construct. Controls were transfected with the empty pSPORT vector plus the reporter constructs. After 24 h, the cells were treated with 3 mM SU5416 or 0.3% (v/v) DMSO, then incubated for 18 more h. Induction of AHR activity was determined by normalizing the luciferase activity to b-galactosidase activity. White bars: Empty vector. Grey bars: AHR. Error bars: SD; (n = 3). B. Induction of DRE-mediated transcription by SU5416 is ARNT dependent. The ARNT-deficient C4 cell line was transfected with the human ARNT or the pSPORT parent vector. These cells were also cotransfected, treated and assayed as in A. White bars: Empty vector. Grey Bars: ARNT. Error bars: SD; (n = 3). C. SU5416 is a ligand of the AHR. The hepatic cytosolic fraction from C57BL/6J mice was incubated with 1 nM of the radioligand 125BR2N3DpD, in the presence of increasing concentrations of competitor, SU5416, TCDD, BNF or 1,2-Benzanthracene. Ordinate: Specifically bound radioligand in the presence of competitor divided by specifically bound radioligand in the absence of competitor. Abscissa: The concentration of competing ligand, represented as log of molar concentration. Each data point represents the average of two determinations. Competitive binding to the C57BL/6J
cytosol produced the IC50 values of SU5416 = 2.1 nM, TCDD = 1.5 nM, BNF = 2.8 nM, and 1,2-Benzanthracene = 13.7 nM.

In utero Exposure to SU5416 Stimulates Closure of DV
We have previously shown that genetically altered mice that express only 10% of the AHR display a patent ductus venosus (DV) in the liver in nearly all cases [22]. We additionally identified that in utero activation of the receptor in the hypomorphs with TCDD successfully closed the DV [5]. To test the role of SU5416 as an in vivo ligand and its potential effect on embryology and vascular development, we performed timed matings of female AHRfxneo/+ mice to male AHRfxneo/fxneo mice. The pregnant dams were treated at embryonic day E18.5 with a single dose of SU5416 at 110 mg/kg, or an equivalent volume of the vehicle, corn oil. At 4 weeks of age, the pups were sacrificed, and DV status was examined by hepatic perfusion with trypan blue. As seen in Table 1, only 1 of 25 AHRfxneo/fxneo pups treated with corn oil possessed a closed DV. In the experimental group, 13 of 22 animals of this phenotype exposed to SU5416 had a closed DV.activity indicating loss of binding to the DRE, which is clearly in contrast to the long duration DRE-binding seen with TCDD. Of note, when we did titrate SU5416 doses as high as 10 mM, we did observe as much as 20% of TCDD response (1 nM) as far out as 96 hours (data not shown). This SU5416 data is similar to the known plasma half-life of 30 minutes, although VEGF-receptor inhibitor effects have been shown to last as much as 72 hours in culture [23]. We further analyzed whether the AHR antagonist CH223191 could inhibit the ability of SU5416 to activate the DRE in 101L-hepatoma cells. It has previously been shown that this antagonist inhibits TCDD but not some of the other ligands of the AHR including some polycyclic aromatic hydrocarbons. We first performed a titration of the AHR antagonist in culture with either 1 nM TCDD or 100 nM SU5416. As can be seen in figure S2B, the effects of TCDD are inhibited whereas minimal inhibition is shown for SU5416. In figure S2C, we show a titration of SU5416 with only a small amount of inhibition of activity by the antagonist (10 mM).

SU5416-induced Upregulation of CYP1A1 is Similar in Murine AHRb and AHRd Splenocytes
As the above in vitro experiments were performed in cell lines, we next utilized AHRb (C57BL/6J) and AHRd congenic mice (on a C57BL/6J background). Spleens from these mice were harvested and suspended in culture media, and exposed to titrating doses of TCDD and SU5416. These data are presented in figure 4, where the graphs show normalized data from 0 to 100% response. Normalized data was chosen to allow comparison of CYP1A1 upregulation to its maximum in AHRb versus AHRd mice. After 4 hours of culture, TCDD induced CYP1A1 more rapidly and to a higher degree in wild-type than AHRd splenocytes, with an EC50 of 0.461 nM in wild-type and 1.894 nM in AHRd animals. Figure 4B shows that SU5416 induced CYP1a1 similarly in AHRb and AHRd mice, with an EC50 of 0.682 nM in wild-type and 0.730 nM in AHRd mice. Figures S3A and B show the total fold change seen by qPCR analysis of splenocytes after exposure to TCDD and SU5416, to allow an assessment of the potency of AHR activation of these two ligands with CYP1A1 induction as the readout. As can be seen in the figure, TCDD elicits more CYP1A1 in AHRb compared to AHRd mice, whereas SU5416 leads to the same or more CYP1A1 in AHRd mice. By this readout, TCDD and SU5416 have similar potency in AHRd cells, and TCDD is a stronger ligand in AHRb cells.

SU5416 Upregulates CYP1A1 and CYP1B1
The above data clearly shows that SU5416 is a ligand of the AHR. We now focused our attention on the strong response of SU5416 to the AHRd polymorphism in the screening assay, and compared the activity of this ligand in the high and low affinity polymorphisms. We utilized the wild type rat hepatoma cell line, 5L, which harbors the high affinity AHR isoform, and our newly created AHRd-15 cell line. As seen in figure 3A, we first performed a titration with TCDD and measured EROD activity. As expected, the activity of TCDD was shifted by 1.5 orders of magnitude to the left for the AHRb isoform. In contrast, when SU5416 was tested in vitro, the two curves virtually overlapped (figure 3B), showing equal potency for cytochrome P450 induction using the two cell lines. We also tested BNF, which as expected, showed a strong response with the 5L cell line and no response with the AHRd-15 cell line (figure 3C). As these experiments were done in cell lines, and in addition the AHRd-15 line combines a rat cell line with a transfected murine AHR, we further tested the ability of SU5416 to activate the AHR in vivo. Six-week old C57BL/6J mice (AHRb) and DBA/2J (AHRd) were orally administered 30, 80, or 120 mg of SU5416 per kg of body weight.

Phosphorylations were also attempted with di-tert-butyl or dicyanoethyl phosphoramidites to produce di-tert-butyl or dicyanoethyl instead of dibenzyl phosphate. Neither of these phosphates was stable on silica gel, and b-elimination products were obtained after chromatography. TFA deprotection of crude di-tert-butyl phosphate, and NH4OH deprotection of crude dicyanoethyl phosphate both gave b-elimination products as well. Thus, the dibenzylphosphate was chosen to carry through to the final products 1 and rac-2. Hydrogenation of the crude dibenzyl phosphate (1S,3R,4R)13 went very slowly, giving a complex crude mixture. Thus, (1S,3R,4R)-13 was purified by reverse-phase semi-preparative high performance liquid chromatography (HPLC). With pure dibenzyl phosphate, hydrogenation at atmospheric pressure worked very well, and gave a very clean final product 1, similar to our experience with a-ketoamides [14].X-ray crystallography
During the synthesis of the inhibitors, Michael addition of tristhiomethyl methide to an a,b-unsaturated ketone 8 produced three stereoisomers of 9, which could not be readily separated (Figure 3). Two diastereomers of a subsequent synthetic intermediate, (1S,3R,4R)-11 and rac-11, were separated by chromatography. Each diastereomer was crystallized, and the relative stereochemistry was determined.

Figure 4. X-ray crystal structures of intermediates (1S,3R,4R)-11 and rac-11 are shown above as displacement ellipsoid drawings (50%). The positional disorder of the benzyl group in rac11 is shown as lighter lines. Hydrogen atoms are omitted for clarity. Structural depiction of the stereochemistries of (1S,3R,4R)-11 and rac11 are shown below each crystal structure. ?after geometry optimization was 3.16 A; with the trans-pyrrolidine torsion angle fixed during geometry optimization, the distance was ?3.67 A (Figure 6).Discussion Stereochemical results of inhibitor synthesisFigure 3. Cyclohexyl ketone inhibitor 1 was synthesized by the method shown. Thermodynamic control in the Michael addition resulted in the anti-Ser-trans-cyclohexyl stereoisomer of 9 as the major product (Figure 4). The chiral center adjacent to the Ser carbonyl was easily epimerized due to the electron-withdrawing effects of both the a-amide and a-ketone, resulting in an enantiomeric mixture of a second diastereomer, rac-9. Because the unnatural D-Thr-the original Ser configuration intact (Figure 4). The minor isomer, rac-11, proved to be a racemic mixture. The absolute configurations were assigned as (1R,3R,4R)-11 and (1S,3S,4S)-11, in which the stereocenter of the Ser analogue was partially epimerized to the syn-Ser-trans-cyclohexyl configuration (Figure 4).Pin1 PPIase Enzyme Assays
The a-chymotrypsin protease-coupled assay was used to evaluate inhibition of Pin1 by compounds 1 and rac-2 with the same substrate concentration as described previously [10,14]. The IC50 values of the two diastereomers were determined to be 260630 mM for 1, and 6168 mM for rac-2. Preincubation with Pin1 for 15 minutes did not result in improved inhibition.

Molecular modeling
Each of the three cyclohexyl ketone inhibitors was docked flexibly, with geometry minimization, into the Pin1 active site. The resulting docked stereoisomers, (1S,3R,4R)-1, (1R,3R,4R)-2, and (1S,3S,4S)-2, are shown in Figure 5. The total energies, Cys113?S–C = O ketone distances, and angles are reported in Table 1. Figure 5. Models of cyclohexyl ketone inhibitors were docked with dynamic minimization. (A) (1S,3R,4R)-1 in orange, (B) (1R,3R,4R)-2 in blue, (C) (1S,3S,4S)-2 in green, and (D) superposition of all atoms of 1 and rac-2. Models were based on PDB 2Q5A [32], and minimized using Sybyl 8.1.1 [42]. Images were prepared using MacPyMol [44]. Figure 6. Pin1 is proposed to stretch the prolyl ring by binding phosphate and C-terminal residues tightly, creating a transpyrrolidine conformation of the substrate and forcing pyramidalization of the prolyl nitrogen in the twisted-amide mechanism. Distance measurements are from calculated structures of AcroH in the ground state and the trans-pyrrolidine transition state.
containing inhibitors were more potent than the L-Thr in work by Zhang et al [32], both diastereomers 1 and rac-2 were tested for Pin1 inhibition. Inhibitor 1, corresponding to the native L-Ser-LPro stereochemistry of Pin1 substrates, had an IC50 value of 260 mM, while rac-2, an enantiomeric mixture of D-Ser-L-Pro and L-Ser-D-Pro analogues, had an IC50 value of 61 mM. Preincubation did not result in improved inhibition, suggesting that they are not slow-binding inhibitors. We obtained a crystal structure of the similarly substituted, reduced amide inhibitor 4 bound in the Pin1 active site, suggesting that the ketones also bind in the active site [27].

conformation, and the trans-pyrrolidine AcroH conformation ?was 0.51 A (Figure 6). This effect of stretching the ring conformation may provide insight into the mechanism of Pin1. In either of the proposed mechanisms: (1) nucleophilic-addition [26], or (2) twisted-amide [25], the nitrogen of the prolyl ring must become pyramidalized and deconjugated from the carbonyl in the transition state [22,24,25]. If binding of substrate to the catalytic site forces the Pro ring into a trans-pyrrolidine conformation, the nitrogen lone pair and the carbonyl p-bond would no longer be conjugated (Figure 6).

Tracheas were fastened with 4% paraformaldehyde in PBS and possibly embedded in paraffin or OCT just before serial sectioning at 7 mm. Antigen retrieval was executed utilizing ten mM sodium citrate in a 2100 Antigen Retriever (Aptum Biologics Ltd.). Sections were subsequently stained making use of the following antibodies: mouse anti acetylated tubulin, 1:1000 (Sigma) rooster anti GFP, 1:five hundred (Aves Labs) rabbit anti Keratin5, 1:five hundred (Convance) mouse anti Keratin14, one:one hundred (Thermo Scientific Lab Eyesight) rabbit anti Keratin14, 1:500 (Covance) rabbit anti Lactotransferrin, one:5000 (Millipore), mouse anti alpha Clean muscle mass actin, 1:200 (Sigma), rat anti Keratin8/Troma 1c, one:a hundred (Developmental Research Hybridoma Lender). Alexa488, Alexa555 and Alexa647 secondary antibodies ended up from Molecular Probes. Sections were analyzed employing a Zeiss 710 inverted confocal microscope method. For quantification of Krt5+ basal cells midline sections have been chosen from three younger (3 month) and 3 more mature (22 month) male and female mice, stained with antibodies to Krt5, Krt8, acetylated tubulin and DAPI and photographed in between cartilages four and ten utilizing a Zeiss Axio Imager D2 microscope. Overall Krt5+ cells were counted along equally lateral surfaces and calculated as a proportion of complete DAPI+ cells. ARGLS and epithelial cells underneath the surface area epithelium have been not provided. In situ hybridization was done as described by Wansleeben et al. 2010 [forty one]. For analysis of immune cells tracheas were harvested, cleaned of connected connective tissue, and digested with one.5 mg/ml Collagenase A (Roche, Indianapolis, IN), .four mg/ml DNase I (Roche, Indianapolis, IN), and two U/ml Dispase II (Sigma-Aldrich, St. Louis, MO) in Hanks well balanced salt solution (Gibco) with 3% FBS and ten mM HEPES pH 7.two?.5. One mobile suspensions ended up washed and around, 56105 cells for every trachea utilised for eleven color movement cytometry. Antibodies employed provided the pursuing: CD45, CD11c, and IA/IE (eBioscience, San Diego, CA), CD11b and Ly6G (BD Biosciences, San Jose, CA), and F4/80, CD64, CD24, and CD31 (Biolegend, San Diego, CA). At minimum 1 channel was utilized for detecting autofluorescence. In addition, Invitrogen Aqua Live/Useless (Grand Island, NY) was utilised to exclude dead cells. Information were gathered with a BD LSRII circulation cytometer and analyzed with Flowjo computer software.
Determine S1 Fix of tracheal epithelium following reduction of luminal cells. Sections of tracheas of youthful (3 thirty day period) and outdated (22month) male mice seven times right after publicity to sulfur dioxide. Sections ended up examined by immunohistochemistry (A, B) and haematoxylin and eosin staining (A9, B9). Equivalent results were witnessed in two other mice in each and every team. Staining of sections 24 hrs right after publicity verified that the extent of damage to luminal cells was equivalent in youthful and previous mice Scale bars 50 um. (TIF) Desk S1 Genes differentially expressed at more than 2 fold increased ranges in tracheas of aged (14 thirty day period) vs . youthful (two thirty day period) mice (p,.05). (DOCX) Desk S2 Genes differentially expressed at far more than 2 fold decrease levels in tracheas of previous versus younger mice. (DOCX) Desk S3 Primers for qPCR investigation.Total RNA from each of the four organic replicates utilized for the microarray was used to synthesize cDNA utilizing iScript cDNA Synthesis Package (Bio-Rad). Gene expression levels had been quantified by qRT-PCR on the StepOnePlus Actual-Time PCR Method (Applied Biosystems). Threshold cycle values (Ct) for samples ended up normalized to GAPDH (DCt), and these values throughout samples ended up compared (DDCt) to quantify relative expression. Bars symbolize common relative expression, with expression amounts in 2 thirty day period outdated mice established to one. Mistake bars depict 95% self confidence interval. Primers are outlined in Table S3.Total RNA from distal tracheas and carinas of 4 young (2 thirty day period) and four older (14 month) C57Bl/six feminine mice was extracted using QIAshredder and RNeasy Micro Kits (QIAGEN). The high quality was checked with a 2100 Bioanalyzer (Agilent Systems). Overall RNA was processed making use of Ambion MessageAmp Leading by the Duke Microarray Facility. Normal Affymetrix protocols and Affymetrix GeneChip Mouse Genome 430 two. Array chips had been used to make cel data files. Data analysis was done using Genomics Suite 6.five (Partek) computer software and strong multichip investigation normalization was performed on each and every dataset. Two-way ANOVA and fold-modify analyses have been executed to decide on genes that had been differentially expressed among 2 month and fourteen thirty day period previous C57Bl/6 datasets.

Serial paraffin embedded tissue sections (four mm thickness) had been deparaffinized in xylene, hydrated by way of graded alcoholic beverages series, pre-addressed in a microwave oven for fifteen min in TrisEDTA (.one M, pH = 9.) made up of Tween 20 (.05% v/v) for antigen retrieval [31]. Slides had been washed with Tris-buffered saline (TBS, .1 M, pH = seven.2) containing Triton X-a hundred (.1%) followed by cure with .three% H2O2 at place temperature for ten minutes to block the endogenous peroxidase activity. Thereafter, sections had been incubated with standard horse serum (10%) well prepared in 5% bovine serum albumin (BSA) to preclude any non-distinct binding. The sections were being incubated with either TG2 antibody (mouse mAb cat # MS-three hundred-PABX, one:four,000 dilution, Lab Vision Corporation, Fremont, CA)/Nepsilon gamma-glutamyl lysine amino residues antibody (mouse mAb cat # ab424, Abcam, Cambridge) for sixty minutes/antiFAK (phospho Y397) antibody (rabbit pAb cat # ab4803, Abcam, Cambridge)/anti-ERK1+ERK2 (phospho T202+ T185+ Y187) antibody (rabbit mAb cat # ab32538, Abcam, Cambridge). Slides had been washed with Tris-buffered saline (TBS, .1 M, pH = seven.2) made up of Triton X-100 (.1%) followed by incubation with biotinylated secondary antibodies for 20 minutes. The sections ended up finally incubated with VECTASTAIN Elite ABC Reagent (Vector labs, Burlingame, CA) and diaminobenzidine was utilised as the chromogen. All methods ended up carried out at space temperature unless of course normally specified. Slides were being washed with Tris-buffered saline (TBS, .one M, pH = 7.4), 3? times following just about every move. Eventually, the sections have been counterstained with Mayer’s hematoxylin and mounted with D.P.X mountant. In negative regulate tissue sections, the primary antibody was replaced by isotype-particular non-immune mouse IgG. The sections were evaluated by gentle microscopic examination.
To establish the medical significance of TG2 overexpression in cytoplasm/stroma, immunohistochemistry was performed in breast typical (n = 40) and cancer tissues (n = 253). Scatter plot examination demonstrated in Figure 1(A) depicts the distribution of IHC scores for TG2 immunostaining in breast normal and most cancers tissues. Of the 40 breast standard tissues, fourteen instances (35%) confirmed weak to moderate immunostaining for TG2 in cytoplasm of epithelial cells (Determine 2(i), a, Desk 1). Nevertheless, no TG2 immunostaining was observed in stroma of the breast typical tissues employed in this analyze (Figure two(i), b, Desk one). Immunohistochemical analysis of 253 breast cancers revealed 199 instances (seventy eight.six%) demonstrating strong TG2 immunostaining either in cytoplasm (33.six%) or stroma (forty five.%, Table one). Among DCIS, 22 of sixty (36.seven%) showed cytoplasmic TG2, although bulk of the circumstances (fifty/60 83.3%) confirmed no detectable TG2 expression in stroma (Figure 2(ii), a and b, Table one). Fifty 4 of 168 (32.1%) IDCs confirmed cytoplasmic TG2, while 97 situations (57.7%) confirmed TG2 expression in stroma (Figure 2(iii), a and b, Desk one). Of 16 invasive lobular carcinomas, six (37.5%) showed cytoplasmic TG2, even though only 4 cases (twenty five.%) showed TG2 overexpression in stroma (Determine 2(iv), a and b, Table one). Amongst IMCs analyzed in this examine, 3 of nine (33.three%) showed cytoplasmic TG2 although only 3 scenarios (33.three%) showed TG2 expression in stroma (Figure two(v), a and b.
IHC scoring was performed less than supervision of the pathologist (MC). Immunopositive staining was evaluated in five pathological places of the tissue sections as described before [31]. Immunostaining for all the proteins in this analyze was evaluated independently in tumor mobile cytoplasm, nucleus and stroma by the intensity and percentage of good staining. Sections were scored as constructive if TG2/N-epsilon gamma-glutamyl lysine amino residues/anti-FAK (phospho Y397)/anti-ERK1+ERK2 (phospho Desk one). Detrimental regulate sections, wherein major antibody was changed by isotype IgG, no immunostaining was observed in cytoplasm/stroma of breast cancer tissue sections (info not revealed). Box plot assessment discovered important improve in stromal TG2 with advancing stage (p = .020), tumor dimension (p,.001), lymph node metastasis (p,.001) and recurrence (loco-regional recurrence/distant metastasis) (p,.001) (Figure 3A?D respectively Desk two).Scatter plot analysis. All breast tissue sections utilised for TG2 immunostaining ended up scored on the basis of % positivity and depth. The overall rating was calculated as sum complete of scores for % positivity and intensity as explained in Materials and Techniques. Panel A reveals the score distribution of TG2 (cytoplasm/stroma) in breast usual and cancer tissues. Panel B and C demonstrates the score distribution of TG2 between unique histological sorts of breast cancer in cytoplasm and stroma respectively.

We subsequent wished to decide whether balsamin antiviral exercise also extends to key T cells. For that, we assessed the effect of raising doses of balsamin on HIV-1 replication in these cells. Balsamin inhibited HIV-one replication in a dose-dependent way, as assessed by RT assay executed on the cells supernatant three days right after infection (Determine 3A). Accordingly, intracellular HIV-one p24 degrees showed a comparable dose-dependent decrease when monitored by Western blotting of balsamin-dealt with cells (Determine 3B). Importantly, measuring in parallel the cytotoxicity of balsamin on these cells confirmed an absence of deleterious influence on mobile wellness, each above the range of balsamin doses employed and more than the period of time of the assay (Determine 3C). As in determine two, we estimated the TC50 of balsamin in principal CD4+ T cells to be ,8.seventy five mM by both equally Trypan blue exclusion counting approach (Determine 3D) and Annexin V/seven-AAD staining (Determine 3E). In comparison, TC50 of AZT in key CD4+ T cells was ,31 mM (data not revealed).We conclude that balsamin potently inhibits HIV-one replication in major T cells, with out influencing cellular viability.
How RIP have an effect on viral replication is a subject of debate [24]. To get rid of some mild on how balsamin inhibits HIV-1 replication, we tried using to delineate far more specifically at which action of the viral replication cycle balsamin exerts its antiviral exercise. We initially infected Jurkat T cells with two various doses of HIV-1 and, fortyeight hours later on, viral supernatant was gathered and its information in HIV-1 RT was measured (Figure 4B), which confirmed that balsamin exerted a robust effect on viral replication as anticipated. In parallel, the accumulation of viral DNA in cells was monitored by PCR (Determine 4A). This showed that balsamin experienced no result on the capability of the virus to reverse-transcribe its genome into DNA. Warmth-inactivation of viral particles prior to infection served as negative regulate. This demonstrates that balsamin exerts its antiviral activity at a action later on than reverse transcription but prior to viral particle generation. These outcomes proposed that balsamin may possibly exert its exercise at the translation move of viral proteins, between reverse transcription of incoming viral genome and launch of recently developed viral particles. In buy to assess this risk, we designed use of a singleround HIV-1 process, wherever the virus is only capable for a solitary replication cycle. This process allows to specifically check the translation of viral proteins during a single solitary round of replication, thus avoiding the perhaps confounding outcome of subsequent rounds of replication. For that we utilized a HIV-one construct that is equipped to carry out only a single spherical of viral replication, due to a deletion of the envelope gene. The first infection is carried out owing to the incorporation of the VSVG envelope protein in the viral particles in the course of the output of these viruses. During the first round of an infection, the HIV-one proviral genome is built-in in the host DNA, which drives the typical transcription, translation, assembly and release of viral proteins. However, because of to the absence of any viral glycoprotein, no infectious viruses are made and for that reason subsequent rounds of replications are completely abrogated [25]. Main CD4+ T cells had been infected with two various viral doses of this one-spherical env-deleted HIV-1 virus. Forty-8 several hours afterwards, viral supernatant was collected and its content material in HIV1 RT was measured (Figure 5B), which confirmed that balsamin exerted a solid outcome on viral replication even in this solitary round setting. Curiously, Western blot analysis was carried out in parallel on mobile extracts showed a comparable reduce of viral proteins accumulation in contaminated cells (Determine 5A). This exhibits that balsamin exerts its antiviral action at or in advance of the translation of viral proteins. We conclude from these outcomes merged with outcomes from Determine 4 that that balsamin exerts its action between reverse transcription and the apparition of viral proteins, probably at the translation stage.
In buy to figure out no matter whether the antiviral action of balsamin extends to other course of viruses, we resolved no matter whether it was equipped to inhibit replication of the influenza virus, a RNA virus possessing a segmented negative strand genome. For that, A549 cells have been contaminated with the PR8 pressure of influenza virus in the existence of growing concentration of balsamin. 20-4 hrs later on, a Western blot assessment was performed on mobile extracts (Figure 6A) to keep track of the accumulation of the viral M1 protein. This demonstrated a powerful dose-dependent inhibition of influenza replication by balsamin. Notably, visual inspection of the cells excluded that this decrease would be because of to balsamin-induced cell struggling (information not shown). In parallel, viral supernatants have been collected, and their material of infectious particles had been decided by titration on MDCK cells (Figure 6B). This verified that balsamin cure led to a powerful dose-dependent reduction of manufacturing of new viral particles. Taken jointly, info recommend that balsamin antiviral action is lively in opposition to a broad range of viruses.

The circadian clock is a self-sustained endogenous oscillator that generates everyday rhythms in actions and physiology with a time period of about 24 several hours, even in absence of exterior cues [1]. Synchronizing this clock to the environmental lightdark cycle is thought to offer a survival gain by letting organisms to predict environmental modifications and optimize the relative timing of their habits and interior physiology [2,three]. A variety of physiological procedures are controlled by the circadian clock, such as the snooze-wake cycle, entire body temperature, feeding habits, fat burning capacity, cell cycle progression and gastrointestinal perform. Big digestive activities display a everyday rhythm, which includes motility, servicing and substitute of the protecting epithelial barrier, nutrient absorption and manufacturing of digestive enzymes [4,five]. Of certain value is the actuality that intestinal epithelial cells exhibit rhythmic cell division, differentiation and apoptosis [six?]. The classical watch of circadian clock business in the greater part of animal species was 1 of a central, master pacemaker, both in the suprachiasmatic nucleus (SCN) of mammals, or in the eyes and pineal gland of lower vertebrates. This view has modified significantly above the many years, with substantial proof for impartial circadian oscillators inside of several, if not all, peripheral tissues. In mammals, this incorporates the existence of peripheral clocks in digestive tissues, such as pancreas, liver, belly and intestine [9?two]. Circadian clock corporation in zebrafish is even more decentralized than in mammals, as most zebrafish tissues not only possess an endogenous clock, but also are right light responsive [thirteen,14]. In spite of this truth, the existence and function of peripheral clocks in the zebrafish gastrointestinal tract stays largely unexplored. The renewal of cells within the intestine is a crucial aspect of its physiology. In mammals, new cells are created from a stem mobile inhabitants observed at the foundation of the intestine in crypts, prior to differentiating and migrating alongside the size of the intestinal villi [15]. The timing of this cell division is beneath the management of the circadian clock, and clock genes have been revealed to oscillate in the course of the mouse intestinal tract [twelve]. Nevertheless, comparatively small is known about how the clock regulates mobile cycle timing or which distinct cell cycle genes may possibly be less than direct clock control in this distinct tissue. In addition, entrainment of the intestinal clock in mammals seems to be really difficult, with systemic alerts from the central clock in the SCN playing a function, in coordination with community cellular clocks, as very well as entraining indicators transpiring directly from the ingestion of foodstuff [sixteen]. How these alerts are then integrated to regulate cell cycle timing and gene expression in the intestine is not yet distinct. Such an knowing is of obvious clinical importance given the overpowering proof that disruption of circadian clock purpose can lead to an elevated possibility of cancer [17,eighteen]. To investigate these issues even further, we have examined circadian clock functionality in grownup zebrafish gut. We monitored the day-to-day timing of mobile division and discovered a number of mobile cycle genes that are less than clock control. While the circadian process in zebrafish is remarkably decentralized, the presence of a circadian clock has under no circumstances been proven in adult intestine, nor has the existence of clock-controlled mobile cycle progression. Zebrafish, therefore, characterize a novel model method with which to analyze this part of intestinal purpose and physiology. Assessment of rhythmic cell cycle gene expression in the gut may possibly offer clues to the system by which clock-cell cycle regulation happens. Furthermore, the direct light sensitivity of zebrafish tissues lets us to examine entrainment of the intestinal clock to light, as well as to foods. The influence and integration of the two of these cues on clock-mobile cycle regulation will be decided. Last but not least, we will investigate the consequences of foods deprivation on the two circadian clock purpose, as well as mobile proliferation in the intestine.
Not remarkably, the circadian clock features in the gut in vivo, but to exhibit that this clock is endogenous, we manufactured use of the transgenic period3 (per3)-luciferase fish and in vitro tissue society techniques [23]. Bioluminescent traces of intestinal tissue from per3-luciferase fish reveals higher amplitude rhythms of per3 expression on a LD cycle, with a peak at ZT5 and a period of 24. ?.4 hours (Figure 1C). In DD, per3 expression remains rhythmic with an normal period of 26.one ?.1 several hours (mean ?SEM). When the cultures are returned to a LD regime, per3 rhythms are re-set up with a peak at ZT5 (Figure 1C). To display the direct light-weight sensitivity of the zebrafish intestine, tissues entrained to a LD cycle have been then exposed to a reversed LD cycle, 12 hrs out of stage. The waveform of per3 expression acutely alters during re-entrainment, but inside of one particular circadian cycle, the cultured intestines have now stably re-entrained to the new, reversed LD cycle (Figure 1D).

Certified health care practitioners who managed sufferers with the above-named neurological problems and had been in excellent standing with their state evaluation boards ended up suitable to take part. Investigators registered to participate in PRISM via a centralized web portal [57] and submitted needed information and files to the central IRB for approval. Web sites could also work by way of a local IRB if required. Websites were granted entry to begin enrolling sufferers upon IRB approval.Investigators were instructed to supply the chance to participate in PRISM to roughly twenty consenting individuals, age 18 or older, with any of the 6 eligible neurological situations. Taking part individuals (or their caregivers, when clients were not able to do so because of cognitive or other disabilities) accomplished a information capture form, which incorporated an knowledgeable consent assertion and demographic info like day of delivery, sex, primary neurological prognosis, approximate day of analysis, and use of antipsychotic or antidepressant medications. In addition, the patients (or their caregivers) completed the CNSLS and a QOL evaluate. The QOL evaluate rated the response to the issue, “How has your neurological condition afflicted your good quality of lifestyle?” on a horizontal eleven-level integer scale ranging from `09 (not at all) on the still left to `109 (strongly influenced) on the correct. No further screening was performed for other neurological/psychiatric ailments. The data were then entered by clinic employees into the PRISM registry through the centralized net portal.
The presence of PBA indicators was defined as a CNS-LS score $thirteen absence of PBA signs and symptoms was outlined as a CNS-LS ,thirteen. A much more restrictive definition was also evaluated utilizing a CNS-LS $21. This rating is regular with imply CNS-LS scores of PBA sufferers who participated in current clinical trials [seven] and has been used in other prevalence surveys to identify a subset of sufferers likely to have a lot more regular and serious PBA indicators [7,eight]. The CNS-LS is the initial self-report evaluate of PBA indicators to be established and validated it is made up of subscales for laughter (4 objects) and for crying (three items), with every product scored on a 5-position scale (1 = applies in no way 5 = applies most of the time) for a overall score ranging from seven (no symptoms) to 35 (maximum) [32,49]. In patients with ALS (n = ninety nine), a CNS-LS rating $thirteen accurately predicted neurologists’ diagnoses of PBA for eighty two% of individuals (sensitivity of .eighty four specificity of .eighty one) the CNS-LS also showed great check-retest trustworthiness (.88) and interior regularity (Cronbach’s a coefficient = .87) [32]. In clients with MS (n = 90), a CNS-LS rating $13 correctly predicted physicians’ diagnoses of PBA for seventy eight% of clients (sensitivity of .96 specificity of .fifty five), and a CNS-LS rating $17 correctly predicted 89% of physicians’
PRISM was created to be a basic affected person registry enabling healthcare pros to capture the prevalence and medical correlates of PBA indicators. PRISM aimed to recruit 500 internet sites nationwide, with every enrolling around 20 sufferers with any of 6 picked neurological conditions acknowledged to be related.diagnoses (sensitivity of .ninety four specificity of .83) [49]. The CNS-LS has not been validated in other neurological conditions. In individuals with PD, a CNS-LS $eleven had 100% sensitivity for physician prognosis of involuntary psychological expression problem (IEED) but specificity was 48% [18] and as a result considered to have bad discriminant validity as a screening device for PBA.
Printed PBA symptom prevalence estimates by primary neurological issue. Shading implies a number of estimates. Advert, Alzheimer’s condition ALS, amyotrophic lateral sclerosis CNS-LS, Centre for Neurologic Review capability Scale MS, a number of sclerosis PBA, pseudobulbar influence PD, Parkinson’s disease PRISM, PBA Registry Collection TBI, traumatic mind harm. aPatient interview bCNS-LS $thirteen (greater estimate), CNS-LS $21, reduce estimate c Poeck criteria: pathological influence could be mood congruent (emotional lability) or incongruent (pathological laughing and crying) d Retrospective overview of hospital or clinic information eMailed questionnaire fEmotional lability questionnaire (ELQ) gAscertainment strategy mysterious h Individual job interview, Poeck requirements iBrief questionnaire (uncontrollable laughing/crying when not pleased/unfortunate) jCNS-LS $13 (maximum estimate), CNS-LS $17 (center estimate), Cummings Involuntary Psychological Expression Disorder criteria (least expensive estimate) kCNS-LS $seventeen (lower estimate), CNS-LS $13 (larger estimate) lPathological Laughing and Crying Scale (PLACS) $10 and score of $two on PLACS products two (frequency), 13 (loss of voluntary manage), and eighteen (distress/humiliation) mPatient interview Property (reduce estimate), and Kim (larger estimate) requirements nPatient interview Property conditions o Individual interview Kim criteria pPatient interview Kim criteria (reduce estimate n = 516) and modified Kim criteria (client report only without corroboration from family higher estimate) qPatient job interview Kim criteria at hospital admission (reduce estimate) and at 3 months (larger estimate) following stroke.