When ‘Open’ and ‘regrown’ were pooled to ‘non-Park’ and compared to ‘Park’ nine species showed significant association and 155 no association (Table 5). Among the significantly associated species, three were living in hollows (Table 5) and all these three were mainly found in ‘Park’. Table 5 The species with significant association to one of the

(site-) ‘types’ according to IndVal analyses, either as compared between all three site types (Park/Open/Regrown) or compared between ‘Park’ or A-1155463 clinical trial ‘non-Park’. Also the percentage of sites in which they occurred within ‘Park’ or ‘non-Park’ are shown. Wood types are defined as: w wood and bark, h hollows. For ‘Park’ n = 8, ‘Open’ n = 8 and ‘regrown’ n = 11 Species Wood type Test with three types Test with two types % sites w. occurrence Maxgrp IndVal P Maxgrp IndVa P Park non-Park Euglenes oculatus h Open 66.0 0.001 Non-park 47.4 0.048 0 47.4 Trichoceble memnonia w Park 56.8 0.004 Park 60 0.002 62.5 5.3

Stenichnus godarti w Open 55.0 0.004 Non-park 47.4 0.049 0 47.4 Rhizophagus parvulus w Regrown 54.5 0.005 – – n.s 0 31.6 Gabrius splendidulus w Regrown 55.2 0.007 – – n.s. 0 42.1 Prionocyphon serricornis h Park 49.5 0.012 Park 55.6 0.007 62.5 21.1 Trichoceble floralis w Open 45.6 0.024 – – n.s. 37.5 36.8 Cryptophagus confusus h Park 43.0 0.027 Park 51.6 0.012 62.5 10.5 Schizotus pectinicornis w Regrown 36.4 0.027 – – n.s. 0 21.0 this website Orthocis festivus w Regrown 36.4 0.028 – – n.s. 0 21.0 Synchita humeralis w Regrown 45.7 0.031 Non-park 52.6 0.027 0 52.6 Phloeopara corticalis w Open 37.5 0.038 – – n.s. 0 15.8 Calambus bipustulatus w Open 40.0 0.040 – – n.s. Epigenetics inhibitor 12.5 21.0 Hylesinus fraxini w Park 34.0 0.045 Park 35.4 0.019 37.5 5.3 Cryptophagus populi w Open 37.3 0.045 – – n.s. 25.0 26.3 Scolytus

laevis w Regrown 40.6 0.049 – – n.s. 0 42.1 Hapalaraea melanocep. w – – n.s. Park 38 0.042 50.0 10.5 Mycetophagus Tangeritin multipun. w – – n.s. Park 35 0.049 37.5 5.3 Discussion For saproxylic beetle species living in tree hollows and for red-listed saproxylic beetles species, species numbers did not differ between parks and the more natural sites. Also for species associated with wood and bark rather high numbers were found in the ‘Park’ sites, but their numbers were significantly lower than in the ‘Open’ sites. This shows that the old trees in parks harbour a rich fauna in spite of the more intensive management. The removal of wood from parks probably explains the significantly lower number of species associated with wood and bark. However, even among them, the red-listed species showed no such pattern, indicating that they could be living within the dead wood still attached to the living parts of old park trees.

The operon iniBAC was previously found to confer multidrug tolerance to M. bovis BCG through an associated pump-like activity, and was induced by isoniazid and ethambutol [19, 20]. These findings suggest that the mtrA gene might be involved in drug resistance. In the current study, we have confirmed that MtrA could bind the iniB promoter region. The recombinant M. smegmatis strain was found

to become sensitive to the anti-TB drugs, isoniazid and streptomycin, when mtrA gene expression was inhibited by an antisense mRNA technique (Fig. 5A). In M. avium, mtrAB was shown to play a role in regulating the composition and permeability of mycobacterial cell walls and was required for morphotypic multidrug A-1210477 cell line resistance [14]. In the current study, the recombinant M. smegmatis cells were

observed to increase in length. This is most likely due to the changes of the mycobacterial cell wall, which would contribute to mycobacterial sensitivity to anti-TB drugs. All evidence makes MtrA a good target candidate for drug design. Conclusions The two-component systems of M. tuberculosis are apparently required for its growth and resistance in hostile Captisol cost host environments, in which MtrAB has been reported to regulate the expression of the M. tuberculosis replication initiator gene, dnaA. In the current study, we have identified the conserved sites for the recognition of MtrA in the dnaA promoter as well as approximately 420 potential target genes. Further in vivo studies about a related organism, M. smegmatis, reveal changes in both cell morphology and drug resistance when MtrA gene expression is inhibited. The data presented here significantly enhance our understanding of the regulatory mechanisms of the essential two-component MtrAB system and its role in the drug resistance

of M. smegmatis. Methods Cloning, expression and purification of recombinant proteins All DNA primers (Additional file 7) and oligonucleotides (Additional file 8) were synthesized by Invitrogen. M. tuberculosis mtrA was amplified using primers from genomic DNA. The MtrA genes were cloned into the overexpression vectors Oxalosuccinic acid pET28a or pGEX-4T-1 to produce recombinant plasmids (Additional file 1). E. coli BL21(DE3) cells that were transformed with the recombinant plasmid were grown at 37°C in 1 L of LB medium containing 30 μg/mL kanamycin or 100 μg/mL ampicillin, respectively. Protein purification was carried out as described in earlier reports [21–24]. Bacterial one-hybrid analysis The interaction between the regulatory Selleckchem RepSox region of the M. tuberculosis dnaA gene and MtrA was assayed using the bacterial one-hybrid technique [24]. The reporter vector pBXcmT and pTRG vectors containing MtrA were generated (Additional file 1). The bacterial one-hybrid assays were carried out as described in a previous study [24].

These results are of extreme importance

as this route of phage administration can provide a viable strategy for delivery of phage in a commercial context. Phages could also be given in SRT1720 order the drinking water, however preliminary experiments showed that phage needed to be administrated with antacid and this could prove more difficult to deliver with the water than as an inclusion in the feed. Moreover, in our study the phage cocktail was administered as a single dose to Campylobacter-infected chicks 7dpi. A single dose of phage is, in comparison to multiple doses [41], an easier and more this website feasible strategy in a farm situation. It must be noted that the present model does not comprise all the variables that can play a role in the use of phages to control Campylobacter in poultry. Firstly, this model considers the use of phages as a therapy and not as a prophylactic measure. Secondly, in the

present work birds were challenged with Campylobacter at one-year-old, but in a real commercial context birds just get colonized with Campylobacter AZD1480 mouse after two weeks of age. However, these conditions were not tested in our experiments as it is very difficult to maintain chicks free of pathogens. An additional limitation of the model was the limited time course of the experiments (seven days). Nevertheless, the model described herein is a proof of principle that Campylobacter phages given orally or administered in feed can effectively reduce the Campylobacter colonization levels. Further studies need to be undertaken in order to test phage Carnitine dehydrogenase effectiveness in older chickens, their use as prophylactic agents and longer time course trials in order to reflect the production cycle. Conclusions The phage cocktail was able to reduce C. coli and C. jejuni in infected poultry by approximately 2 log10cfu/g, which is of great importance as they are the most prevalent Campylobacter species found in positive

Campylobacter flocks. Moreover mathematical models indicate that a 2 log10cfu/g reduction of Campylobacter on the chicken carcasses could lead to a 30-fold reduction in the incidence of campylobacteriosis associated with consumption of chicken meals [48]. The phage cocktail administered in feed led to an earlier reduction in Campylobacter titre than when given by oral gavage and thus this method can be easily and successfully used under commercial condition in a poultry unit. Another important aspect of the present study is that as the phages that composed the cocktail were isolated from poultry carcasses, their use to reduce Campylobacter colonisation in the live birds would not introduce any new biological entity into the food chain. Methods Bacterial strains For the single-step growth experiments, two wild type strains of C. coli, isolated from poultry and poultry products, were used as the hosts of the three phages that composed the cocktail (C.

5 (3–25)   · ISS 25 (9–50)   · NISS 33 (13–66) IAP (# patients)     · <12 mmHg 10   · >12 mmHg (IAH) 10 IAP = intra-abdominal pressure; IAH = intra-abdominal hypertension as defined by selleck inhibitor Cheatham et al. 2007 [9]. Primary objective – fascial closure rate Fascial closure was achieved in 13 out of 20 patients (65% of patients on an intent-to-treat basis) (see Table 3; see supplemental data for Kaplan-Meier estimate data). Fascial closure rate expressed as the percentage of survivors was 75% (12/16 patients) (data not shown). One patient died following fascial closure but the remaining 12

closed abdomens were stable at a follow up 8 days after closure although a superficial wound sepsis was present in one. The median time to achieve primary fascial closure was 3 days (CI) (n=20). Two patients were withdrawn from the study after 19 and 24 days of NPWT therapy because they developed a Grade 4 (fixed) abdomen and fascial closure was no longer an option (i.e. see more they could no longer contribute to the primary objective). Each open abdomen was graded according to the WSACS classification [7] (Table 1) at the initial application of NPWT and at each subsequent dressing

change, including the final Barasertib manufacturer removal of the dressing. The grade of open abdomen for the majority of patients improved during the course of therapy. Table 3 Progression of open abdominal wounds from initial presentation to end Montelukast Sodium of therapy Grade Baseline End of therapy Closed 0 13 (65%) 1a 14 (70.0%) 2 (10%) 1b 5 (25.0%) 1 (5%) 2 1 (5.0%) 2 (10%) 2c 0 0 3 0 0 4 0 2 (10%) N 20 (100%) 20 (100%)* Progress of the wounds during therapy was assessed using the Bjorck et al. classification system. *one patient died less than 24 hours after having a baseline assessment. As no other data was available, it was assumed that the wound grade at death was the same as the baseline assessment (Grade 1A). Secondary objectives SOFA and APACHE11 scores decreased from medians of 11 and 14.5 at baseline to 9 and 12 respectively at the end of

therapy. There was no apparent relationship between IAP at baseline and achievement of fascial closure. Median time in ICU was 8 days (range 1–28 days, n=20). In the remaining patients, reasons for discontinuation of NPWT were death, (3/20; 15%), poor compliance (1/20; 5%), withdrawal for other reasons (1/20; 5% – persistent bowel hematic as a consequence of an extremely large viscera). Fluid contained in the waste canister was approximately measured and this formed part of the daily fluid management of the patient. A mean volume of 871 ml (median 700 ml) was present in the canister at dressing change. Blood loss into the canister was also an early sign of internal bleeding and allowed rapid intervention (data not shown). A range of complications were assessed and results are shown in Table 4. One fistula (5%) was observed during the study in a single patient who had received penetrating trauma.

With this approach a total of 84 putative ORFs were identified. In a second approach we used the NCBI ORF Finder program coupled with the program blastp and

compared the translated proteins with the proteins of the PB1-like phages [26, 32]. Combination of the results of both approaches revealed a total of 94 predicted ORFs as well as one unique ORF in phage JG024. No RNA polymerase was detected suggesting that this phage uses the host transcriptional GS-4997 supplier machinery, as it was also suggested for the PB1-like family of phages. We detected a putative structural gene cluster which contains genes encoding for putative head structure proteins (ORF 18 and 19) as well as for tail and baseplate proteins (ORF 22-47). Moreover, ORF 40 was designated as a lytic tail protein. It

was shown for the phages 14-1 and LBL3 that this protein has a transglycosylase domain with a N-acetyl-D-glucosamine binding site, which shows a specific degradation of peptidoglycan [15]. ORF 48 encodes a putative endolysin with a high similarity to the endolysin of phage LMA2 (98.6%) and belongs to a lysozyme-like superfamily. A MI-503 putative holin may be encoded by ORF 52, which shares a 100% identity to ORF 50 of phage F8 and to ORF 51 of phage 14-1. It was suggested that these ORFs encode probable holins since they are located near the endolysin gene and they encode a small protein (201 aa) containing three transmembrane domains [15]. Additionally, a complete DNA replication machinery was detected suggesting that the DNA replication is host independent as described for the PB1-like phages. The respective gene cluster contains a DNA ligase (ORF 50), a helicase (ORF 55 and 56), a DNA polymerase III (ORF 57 and 58), as well as a thymidylate synthase (ORF 61). A putative primase was also found but is not included in this gene cluster (ORF 77), as shown for the other PB1-like phages [15]. Also, differences between the PB1-like phages

and JG024 were found. Phage 14-1 (ORF 71) and phage LBL3 (ORF 68) encode a hypothetical protein with a size of 434 aa. Interestingly, this protein is encoded by two ORFs in phage JG024 designated ORF 72 (362 aa) and 73 (60 aa). The two ORFs are separated by only 116 bp. Moreover, ORF 79 is a small predicted HAS1 gene with a size of 132 bp and encodes for a unique protein in phage JG024. This ORF was identified by two programs, GeneMark and ORF Finder, independently. No functional indication could be pointed out since there are no similarities to other proteins in the databases and no conserved domains have been detected in ORF 79. We also searched the genome of phage JG024 for CHIR-99021 supplier promoters, terminators and regulatory elements, see Methods. The PB1 phages do not contain a phage RNA-polymerase and depend on the transcriptional machinery of the host bacterium. Putative sigma 70-promoter regions have been predicted in PB1 phages [15].

Proc Natl Acad Sci USA 2003,100(14):8176–8181.PubMedCrossRef 71. Summer E, Berry J, Tran T, Niu

L, Struck Go6983 chemical structure D, Young R: Rz/Rz1 lysis gene equivalents in pahges of Gram-negative hosts. J Mol Biol 2007, in press. 72. Savva CG, Dewey JS, Deaton J, White RL, Struck DK, Holzenburg A, Young R: The holin of bacteriophage lambda forms rings with large diameter. Mol Microbiol 2008,69(4):784–793.PubMedCrossRef 73. Grundling A, Smith DL, Blasi U, Young R: Dimerization between the holin and holin inhibitor of phage lambda. Journal of bacteriology 2000,182(21):6075–6081.PubMedCrossRef 74. Ranade K, Poteete AR: Superinfection exclusion (sieB) genes of bacteriophages P22 and lambda. J Bacteriol 1993,175(15):4712–4718.PubMed 75. Ranade K, Poteete AR: A switch in translation mediated by an antisense RNA. Genes Dev 1993,7(8):1498–1507.PubMedCrossRef 76. Sergueev K, Court D, Reaves L, Austin S: E.coli cell-cycle regulation by bacteriophage lambda. J Mol Biol 2002,324(2):297–307.PubMedCrossRef 77. Stayrook S, Jaru-Ampornpan P, Ni J, Hochschild A,

Lewis M: Crystal structure of the lambda repressor and a model for pairwise cooperative operator binding. Nature 2008,452(7190):1022–1025.PubMedCrossRef 78. Jain D, click here Kim Y, Maxwell KL, Beasley S, Zhang R, Gussin GN, Edwards AM, Darst SA: Crystal structure of bacteriophage lambda cII and its DNA complex. Mol Cell 2005,19(2):259–269.PubMedCrossRef 79. Datta AB, Roy S, Parrack P: Role of C-terminal residues in oligomerization and stability of lambda CII: implications for lysis-lysogeny decision of the phage. J Mol Biol 2005,345(2):315–324.PubMedCrossRef 80. Hall BM, Roberts SA, Heroux A, Cordes MH: Two structures of a lambda Cro variant highlight dimer flexibility but disfavor major dimer distortions upon PAK5 specific binding of cognate

DNA. J Mol Biol 2008,375(3):802–811.PubMedCrossRef 81. Newlove T, Atkinson KR, Van Dorn LO, Cordes MH: A trade between similar but nonequivalent intrasubunit and intersubunit contacts in Cro dimer evolution. Biochemistry 2006,45(20):6379–6391.PubMedCrossRef 82. Iwai H, Forrer P, Pluckthun A, Guntert P: NMR solution structure of the monomeric form of the bacteriophage lambda capsid stabilizing protein gpD. J Biomol NMR 2005,31(4):351–356.PubMedCrossRef 83. Chang C, Pluckthun A, Wlodawer A: Crystal structure of a truncated version of the phage lambda protein gpD. Proteins 2004,57(4):866–868.PubMedCrossRef 84. Kovall R, Matthews BW: Toroidal structure of lambda-exonuclease. AZD8931 Science 1997,277(5333):1824–1827.PubMedCrossRef 85. Maxwell KL, Yee AA, Arrowsmith CH, Gold M, Davidson AR: The solution structure of the bacteriophage lambda head-tail joining protein, gpFII. J Mol Biol 2002,318(5):1395–1404.PubMedCrossRef 86.

1512 ± 0.0278 0.4604 ± 0.0331✩ 0.7453 ± 0.0636✩ 0.9071 ± 0.4985✩ Hut 78 0.5282 ± 0.0537⋆ 0.6943 ± 0.0365⋆▵ 0.8477 ± 0.0513⋆▴ 0.8710 ± 0.0485▴ ⋆Compared with the corresponding group of Jurkat cells, P < 0.01; ✩Compared with the other groups of Jurkat cells (including the AZD1080 research buy control group), P < 0.01; ▴Compared with the control

group and S50 group of Hut 78 cells, P < 0.01; ▵Compared with the other groups of Hut 78 cells (including the control group), P < 0.01. Figure 2 The expression of CCR7 mRNA and protein in Jurkat and Hut cells after CCL21 co-culture in vitro. RT-PCR amplication and Western Blot 3-MA mouse analysis of the two cell lines under the different concentration of CCL21,

which was performed as described in Methods. β-actin is positive control in RT-PCR amplication and GAPDH is positive control in Western Blot analysis. The relative grey scale of CCR7 mRNA and protein in Hut cell were both higher than that in Jurkat cell with corresponding concentration of CCL21. In the group with AZD1152 supplier different concentration of CCL21 of each cell lines, there were some differences on the grey scale as described in the result. According to the relative grey scale, the numbers of CCR7 transcripts of the two cell lines in all concentration groups were higher than that in the control group (P < 0.01). The CCR7 transcripts of the Hut 78 cells in control, S50, and S100 groups were higher than that in the corresponding groups of Jurkat cells (P < 0.01). The CCR7 transcripts of the two cell lines in the higher concentration group were higher than that in the lower concentration group, except for S100 and

S200 groups in the Hut 78 cell line (P < 0.01). (2) Expression of CCR7 protein (Table 5, Figure 2) this website Table 5 The relative grey scale of CCR7 protein ( ± s, n = 9)   Control group S50 group S100 group S200 group Jurkat 0.5053 ± 0.0336 0.4870 ± 0.0278 0.6916 ± 0.0238✩ 0.7095 ± 0.0332✩ Hut 78 1.1037 ± 0.1135⋆ 1.0700 ± 0.1121⋆ 1.4792 ± 0.2500⋆▴ 1.4804 ± 0.2524⋆▴ ⋆Compared with the corresponding group of Jurkat cells, P < 0.01; ✩Compared with the control group and the S50 group of Jurkat cells, P < 0.01; ▴Compared with the control group and the S50 group of Hut 78 cells, P < 0.01. In both cell lines, the relative expression of the CCR7 protein in the S100 and S200 groups were higher than that in the control group, whereas the CCR7 expression in the S100 group was higher than that in the S50 group (P < 0.01). The CCR7 expression of the Hut 78 cell line in the control, S50, S100, and S200 groups were higher than those of the Jurkat cell line (P < 0.01).

jejuni 81-176 Wild-type Tet [32] E. coli        MG1655 Wild-type   [33]  TRMG1655 csrA::kan Kan [33]  TOP10 Cloning host Strep Invitrogen Plasmids        pBAD-TOPO Cloning vector containing araBAD promoter Amp Invitrogen  pBADcsrAEC E. coli csrA cloned into pBAD-TOPO Amp This study  pBADcsrACJ C. jejuni csrA cloned into pBAD-TOPO Amp This

study #Tet, tetracycline; Kan, kanamycin; Strep, streptomycin; Alvocidib mw Amp, ampicillin. Phylogenetic analyses Phylogenetic comparison of CsrA orthologs was Idasanutlin mouse performed by neighbor joining using CLUSTALW [34] within the VectorNTI 7.1 program suite (Invitrogen, Carlsbad, CA). Accession numbers for CsrA proteins used in the comparisons are listed in Additional file 1: Table S1. Bootstrapping (500 replicates) was performed to determine

the PI3K inhibitor statistical robustness of the clusters, and the percent of bootstraps that supported the clusters are indicated at each tree node (Figure 1A). Figure 1 C. jejuni CsrA is divergent from the E. coli ortholog, including in the RNA binding domains. A) CsrA orthologs from 20 diverse pathogenic and non-pathogenic bacterial species were aligned using CLUSTALW (neighbor joining). Numbers at tree nodes indicate the percent of bootstrap replicates that support the adjacent branches. Protein lengths (number of amino acids) are indicated to the right of each ortholog. Accession numbers for each protein are listed in Additional file 1: Table S1. B) Alignment of the amino acid sequences of CsrA orthologs. Regions 1 and 2 of E. coli CsrA important for RNA binding [35] are indicated by boxes and other amino acid residues important

for CsrA regulation are indicated by an asterisk (*). Red shading indicates amino acids that are identical to those of E. coli fantofarone CsrA; purple shading indicates amino acids that are different from E. coli CsrA but identical within the C. jejuni-containing clade of Figure 1A. Amino acids within RNA binding sequences 1 and 2 of C. jejuni CsrA that are conservative substitutions compared to E. coli CsrA are underlined. DNA and protein techniques Genomic DNA from E. coli and C. jejuni strains for use in PCR amplification was purified using the Generation Capture Column Kit (Qiagen, Chatsworth, CA). The plasmids used in this study were extracted and purified using the QIAprep Spin Miniprep Kit (Qiagen). PCR reactions were carried out using the Expand High Fidelity PCR System (Roche, Mannheim, Germany). Primers for PCR (Table 2) were synthesized by Integrated DNA Technologies (Coralville, IA). All DNA sequencing was performed by the GHSU Genomics Core Facility using an ABI Prism 337 XL DNA sequencer (Applied Biosystems, Foster City, CA). Western blots to validate the expression of CsrAEC and CsrACJ were performed by using standard methods, with anti-his primary antibody (Penta-His Mouse Monoclonal, Qiagen; 1:1000 dilution) and goat, anti-mouse IgG-horseradish peroxidase secondary antibody (Pierce).

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Previous studies and our results indicated that there might be apparent differences between EGFR phosphorylation pattern and function of different tyrosine phosphorylation sites. EGFR phosphorylation is likely to be of biological relevance in NSCLC [5, 38]. see more expression of pTyr1068 in tumor samples evaluated by IHC here exhibits a strong predictive value for EGFR-TKIs therapy, especially in patients without EGFR mutations. In the entire patient population, those with pTyr1068 expression have a significantly improved response rate and prolonged PFS compared with expression negative ones. Moreover, its predictive role is not just for efficacy

in patients with concomitant EGFR mutation. Patients with pTyr1068 expression achieved a superior benefit of PFS (median 4.2 months v 1.2 months; P < 0.001). Especially, sixteen patients with both wild-type EGFR and pTyr1068 who have responded to EGFR-TKIs selleck screening library possessed a median PFS of 15.6 months (95%CI: 7.28-23.9). The results suggested pTyr1068 expression may be a supplementary predictor for EGFR-TKIs in selecting proper patients to EGFR-TKIs among those with wild-type EGFR. Prior studies have demonstrated that the specific phosphorylation sites inside the intracellular tail often serve as docking sites for a range of proteins and initiate cascades of separate and functional distinct downstream signaling pathways [14, 39], pTyr1068 is involved

in MAPK and Akt pathways activation [17, 20, 40] being considered a marker of EGFR Astemizole https://www.selleckchem.com/products/smoothened-agonist-sag-hcl.html activation. Helfrich et al. showed not

only EGFR mutant cell line (H3255) but also EGFR TKIs sensitive wild-type cell lines (H322 and Calu3) had higher pTyr1068 expression and more sensitivity to gefitinib [41]. Amann et al. showed that EGFR was constitutively phosphorylated in gefitinib-sensitive cell lines yet the level of phosphorylation of the EGFR mutant cell line was comparable with that in wild-type cells [42]. These findings suggest that EGFR activation (phosphorylation) can be triggered and then affect subsequent steps of signal transduction regardless of EGFR mutational status. In the present study, the patients with EGFR wild-type might also show high phosphorylated EGFR expression, which may account for why 10–20% of NSCLC patients in absence of EGFR mutation have responded to treatment with gefitinib or erlotinib. Hijiya et al. investigated another autophosphorylation site Tyr1173 and found that no correlation with clinical responsiveness to gefitinib [43]. Emery et al. noted that the higher level of pTyr1173 was associated with longer time to progression (TTP) of EGFR-TKIs [29]. In contrast, there appears a negative correlation between pTyr1173 expression and clinical outcomes in our study. pTyr1173 expression is not only significantly associated with worse PFS in the univariate analysis; it also maintains independently poor prognostic significance in the multivariate analysis.