Changes in brain white matter structure are associated with urine proteins in urologic chronic pelvic pain syndrome (UCPPS): A MAPP Network study

The Multidisciplinary Approach to the Study of Chronic Pelvic Pain (MAPP) Research Network has yielded neuroimaging and urinary biomarker findings that highlight unique alterations in brain structure and in urinary proteins related to tissue remodeling and vascular structure in patients with Urological Chronic Pelvic Pain Syndrome (UCPPS). We hypothesized that localized changes in diffusion tensor imaging (DTI) measurements might be associated with corresponding changes in urinary protein levels in UCPPS. To test this hypothesis, we created statistical parameter maps depicting the linear correlation between DTI measurements (fractional anisotropy (FA) and apparent diffusion coefficient (ADC)) and urinary protein quantification (MMP2, MMP9, NGAL, MMP9/NGAL complex, and VEGF) in 30 UCPPS patients from the MAPP Research Network, after accounting for clinical covariates. Results identified a brainstem region that showed a strong correlation between both ADC (R2 = 0.49, P<0.0001) and FA (R2 = 0.39, P = 0.0002) with urinary MMP9 levels as well as a correlation between both ADC (R2 = 0.42, P = 0.0001) and FA (R2 = 0.29, P = 0.0020) and urinary MMP9/NGAL complex. Results also identified significant correlations between FA and urinary MMP9 in white matter adjacent to sensorimotor regions (R2 = 0.30, P = 0.002; R2 = 0.36, P = 0.0005, respectively), as well as a correlation in similar sensorimotor regions when examining ADC and urinary MMP2 levels (R2 = 0.42, P<0.0001) as well as FA and urinary MMP9/NGAL complex (R2 = 0.33, P = 0.0008). A large, diffuse cluster of white matter was identified as having a strong correlation between both ADC (R2 = 0.35, P = 0.0006) and FA (R2 = 0.43, P<0.0001) with urinary NGAL levels. In contrast, no significant association between DTI measurements and VEGF was observed. Results suggest that elevated MMP9 or MMP9/NGAL in UCPPS may be related to degenerative neuronal changes in brainstem nuclei through excitotoxicity, while also facilitating synaptic plasticity in sensorimotor regions

Association between urinary biomarkers MMP-7/TIMP-2 and reduced renal function in children with ureteropelvic junction obstruction.

ImportanceExtracellular matrix proteins and enzymes involved in degradation have been found to be associated with tissue fibrosis and ureteropelvic junction obstruction (UPJO). In this study we developed a promising urinary biomarker model which can identify reduced renal function in UPJ obstruction patients. This can potentially serve as a non-invasive way to enhance surgical decision making for patients and urologists.ObjectiveWe sought to develop a predictive model to identify UPJO patients at risk for reduced renal function.DesignProspective cohort study.SettingPre-operative urine samples were collected in a prospectively enrolled UPJO biomarker registry at our institution. Urinary MMP-2, MMP-7, TIMP-2, and NGAL were measured as well as clinical characteristics including hydronephrosis grade, differential renal function, t1/2, and UPJO etiology.ParticipantsChildren who underwent pyeloplasty for UPJO.Main outcome measurementPrimary outcome was reduced renal function defined as MAG3 function ResultsWe included 71 patients with UPJO in the original training cohort and 39 in the validation cohort. Median age was 3.3 years (70% male). By univariate analysis, reduced renal function was associated with higher MMP-2 (p = 0.064), MMP-7 (p = 0.047), NGAL (p = 0.001), and lower TIMP-2 (p = 0.033). Combining MMP-7 with TIMP-2, the multivariable logistic regression model predicted reduced renal function with good performance (AUC = 0.830; 95% CI: 0.722-0.938). The independent testing dataset validated the results with good predictive performance (AUC = 0.738).Conclusions and relevanceCombination of urinary MMP-7 and TIMP-2 can identify reduced renal function in UPJO patients. With the high sensitivity cutoffs, patients can be categorized into high risk (aggressive management) versus lower risk (observation)

Changes in brain white matter structure are associated with urine proteins in urologic chronic pelvic pain syndrome (UCPPS): A MAPP Network study.

The Multidisciplinary Approach to the Study of Chronic Pelvic Pain (MAPP) Research Network has yielded neuroimaging and urinary biomarker findings that highlight unique alterations in brain structure and in urinary proteins related to tissue remodeling and vascular structure in patients with Urological Chronic Pelvic Pain Syndrome (UCPPS). We hypothesized that localized changes in diffusion tensor imaging (DTI) measurements might be associated with corresponding changes in urinary protein levels in UCPPS. To test this hypothesis, we created statistical parameter maps depicting the linear correlation between DTI measurements (fractional anisotropy (FA) and apparent diffusion coefficient (ADC)) and urinary protein quantification (MMP2, MMP9, NGAL, MMP9/NGAL complex, and VEGF) in 30 UCPPS patients from the MAPP Research Network, after accounting for clinical covariates. Results identified a brainstem region that showed a strong correlation between both ADC (R2 = 0.49, P&lt;0.0001) and FA (R2 = 0.39, P = 0.0002) with urinary MMP9 levels as well as a correlation between both ADC (R2 = 0.42, P = 0.0001) and FA (R2 = 0.29, P = 0.0020) and urinary MMP9/NGAL complex. Results also identified significant correlations between FA and urinary MMP9 in white matter adjacent to sensorimotor regions (R2 = 0.30, P = 0.002; R2 = 0.36, P = 0.0005, respectively), as well as a correlation in similar sensorimotor regions when examining ADC and urinary MMP2 levels (R2 = 0.42, P&lt;0.0001) as well as FA and urinary MMP9/NGAL complex (R2 = 0.33, P = 0.0008). A large, diffuse cluster of white matter was identified as having a strong correlation between both ADC (R2 = 0.35, P = 0.0006) and FA (R2 = 0.43, P&lt;0.0001) with urinary NGAL levels. In contrast, no significant association between DTI measurements and VEGF was observed. Results suggest that elevated MMP9 or MMP9/NGAL in UCPPS may be related to degenerative neuronal changes in brainstem nuclei through excitotoxicity, while also facilitating synaptic plasticity in sensorimotor regions

Changes in brain white matter structure are associated with urine proteins in urologic chronic pelvic pain syndrome (UCPPS): A MAPP Network study.

The Multidisciplinary Approach to the Study of Chronic Pelvic Pain (MAPP) Research Network has yielded neuroimaging and urinary biomarker findings that highlight unique alterations in brain structure and in urinary proteins related to tissue remodeling and vascular structure in patients with Urological Chronic Pelvic Pain Syndrome (UCPPS). We hypothesized that localized changes in diffusion tensor imaging (DTI) measurements might be associated with corresponding changes in urinary protein levels in UCPPS. To test this hypothesis, we created statistical parameter maps depicting the linear correlation between DTI measurements (fractional anisotropy (FA) and apparent diffusion coefficient (ADC)) and urinary protein quantification (MMP2, MMP9, NGAL, MMP9/NGAL complex, and VEGF) in 30 UCPPS patients from the MAPP Research Network, after accounting for clinical covariates. Results identified a brainstem region that showed a strong correlation between both ADC (R2 = 0.49, P<0.0001) and FA (R2 = 0.39, P = 0.0002) with urinary MMP9 levels as well as a correlation between both ADC (R2 = 0.42, P = 0.0001) and FA (R2 = 0.29, P = 0.0020) and urinary MMP9/NGAL complex. Results also identified significant correlations between FA and urinary MMP9 in white matter adjacent to sensorimotor regions (R2 = 0.30, P = 0.002; R2 = 0.36, P = 0.0005, respectively), as well as a correlation in similar sensorimotor regions when examining ADC and urinary MMP2 levels (R2 = 0.42, P<0.0001) as well as FA and urinary MMP9/NGAL complex (R2 = 0.33, P = 0.0008). A large, diffuse cluster of white matter was identified as having a strong correlation between both ADC (R2 = 0.35, P = 0.0006) and FA (R2 = 0.43, P<0.0001) with urinary NGAL levels. In contrast, no significant association between DTI measurements and VEGF was observed. Results suggest that elevated MMP9 or MMP9/NGAL in UCPPS may be related to degenerative neuronal changes in brainstem nuclei through excitotoxicity, while also facilitating synaptic plasticity in sensorimotor regions

Various mutations compensate for a deleterious lacZα insert in the replication enhancer of M13 bacteriophage

<div><p>M13 and other members of the Ff class of filamentous bacteriophages have been extensively employed in myriad applications. The Ph.D. series of phage-displayed peptide libraries were constructed from the M13-based vector M13KE. As a direct descendent of M13mp19, M13KE contains the lacZα insert in the intergenic region between genes IV and II, where it interrupts the replication enhancer of the (+) strand origin. Phage carrying this 816-nucleotide insert are viable, but propagate in <i>E</i>. <i>coli</i> at a reduced rate compared to wild-type M13 phage, presumably due to a replication defect caused by the insert. We have previously reported thirteen compensatory mutations in the 5’-untranslated region of gene II, which encodes the replication initiator protein gIIp. Here we report several additional mutations in M13KE that restore a wild-type propagation rate. Several clones from constrained-loop variable peptide libraries were found to have ejected the majority of lacZα gene in order to reconstruct the replication enhancer, albeit with a small scar. In addition, new point mutations in the gene II 5’-untranslated region or the gene IV coding sequence have been spontaneously observed or synthetically engineered. Through phage propagation assays, we demonstrate that all these genetic modifications compensate for the replication defect in M13KE and restore the wild-type propagation rate. We discuss the mechanisms by which the insertion and ejection of the lacZα gene, as well as the mutations in the regulatory region of gene II, influence the efficiency of replication initiation at the (+) strand origin. We also examine the presence and relevance of fast-propagating mutants in phage-displayed peptide libraries.</p></div

Comparison of propagation rates of wild-type M13 mutants.

<p>(A) Time course for amplification of WT-M13, WT-G6792T, and M13KE. Each phage clone was amplified in three separate early log cultures of <i>E</i>. <i>coli</i> ER2738. From each culture, one aliquot was diluted and plated at the indicated incubation times, and the concentration of phage (pfu/μL) in each growing culture was determined based on plaque counts. Data points represent the mean log(pfu/μL) of the three separate cultures for each type of phage, and the error bars show the 95% confidence interval. Statistical analysis indicated significant differences among the phage concentrations for the three sets of data at 135 minutes (ANOVA; F_<b>2,8</b> = 318.2, P < 0.0001). Post-hoc analysis showed that WT-M13 and WT-G6792T were not significantly different from each other (Tukey’s HSD; α = 0.05, P = 0.0981). Both WT-M13 and WT-G6792T were significantly different from M13KE (P < 0.0001). (B) Phage concentrations of all WT-M13 mutant clones at 135 minutes of incubation. Each phage clone was amplified separately in an ER2738 culture. At 135 minutes, three aliquots from each flask of growing culture were diluted and plated, and the concentration of phage (pfu/μL) was determined based on plaque counts. The M13KE control was run 12 times for a total of n = 36 platings. WT-M13 was run 6 times (n = 18 platings) and all other phage clones were run twice each (n = 6 platings). Each bar represents the mean log(pfu/μL) of all platings for a given clone, and the error bars show the 95% confidence interval. Statistical analysis indicated significant differences among the phage concentrations for all the data sets (ANOVA; F_<b>5,77</b> = 239.3, P < 0.0001). Post-hoc analysis showed that M13KE is significantly different from all the other clones (Tukey’s HSD; α = 0.05, P < 0.0001) and that there is no significant difference between any pair among WT-M13 and the WT-mutant clones (range in P = 0.34–1.00).</p

Schematics of the M13KE genome.

<p>(A) The map of M13KE is shown. The (+) strand origin is divided into Domains A and B [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176421#pone.0176421.ref020" target="_blank">20</a>]. Domain A (nucleotides 5769–5819 in both WT-M13 and M13KE) is the “core origin” and is required for both (+) strand initiation and termination. Domain A is extremely sensitive to deletions and insertions, which reduce biological activity to ≤ 0.01% [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176421#pone.0176421.ref020" target="_blank">20</a>]. Domain B stretches from position 5820 to about 5910 in wild-type M13 (WT-M13), but it is interrupted in M13KE by the lacZα insert (the separated segments are indicated as B1 and B2). Dubbed the “replication enhancer,” Domain B is required for (+) strand initiation and is moderately sensitive to inserts and deletions, which reduce biological activity to ≥ 1% [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176421#pone.0176421.ref020" target="_blank">20</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176421#pone.0176421.ref031" target="_blank">31</a>]. The locations of spontaneous mutations and ejections in M13KE are labeled as gene II 5’-UTR, ΔlacZα, and T5091C. The T5091C mutation is a reversion back to the WT-M13 nucleotide at position 5092 (the number is lower by 1 nt due a missing 1565T in M13mp18,19 and M13KE). The map of WT-M13 would be identical to M13KE with the exception of the lacZα insert (all downstream numbering is 815 nt lower in WT-M13). The (-) strand origin (not labeled) is upstream of the (+) strand origin in the intergenic region. The map was constructed using SnapGene^®. (B) The exact locations of the lacZα insert and ejections are indicated. Nucleotide numbering corresponds to WT-M13. Domain A is not shown except for last downstream base, 5819C. In M13mp-based phage, an 816-nt insert containing the lacZα gene is placed in Domain B between nucleotides 5868 and 5869. Two different spontaneous ejections have arisen in M13KE, ΔlacZα-827 and ΔlacZα-838, both of which left behind eleven nucleotides of the lacZα insert. The vast majority of the lacZα insert was removed, in addition to a small section of Domain B: 22 nucleotides in the smaller (827-nt) ejection and 33 nt in the larger (838-nt) ejection.</p

Comparison of propagation rates for various M13-based phage.

<p>(A) Time course for amplification of WT-M13, M13KE, and M13mp18. Each phage clone was amplified in three separate early log cultures of <i>E</i>. <i>coli</i> ER2738. From each culture, one aliquot was diluted and plated at the indicated incubation times, and the concentration of phage (pfu/μL) in each growing culture was determined based on plaque counts. Data points represent the mean log(pfu/μL) of the three separate cultures for each type of phage, and the error bars show the 95% confidence interval. Statistical analysis indicated significant differences among the phage concentrations for the three sets of data at 135 minutes (ANOVA; F_<b>2,8</b> = 89.2, P < 0.0001). Post-hoc analysis showed that M13KE and M13mp18 are not significantly different from each other (Tukey’s HSD; α = 0.05, P = 0.4471). Both M13KE and M13mp18 are significantly different from WT-M13 (P < 0.0001). (B) Phage concentrations of various M13-based clones at 135 minutes of incubation. Each phage clone was amplified separately in an ER2738 culture. At 135 minutes, three aliquots from each flask of growing culture were diluted and plated, and the concentration of phage (pfu/μL) was determined based on plaque counts. The M13KE control was run 21 times for a total of n = 63 platings. WT-M13 was run 6 times (n = 18 platings), M13mp18 and the Ph.D.-7 library were run 4 times each (n = 12 platings), and all other phage clones were run twice each (n = 6 platings). Each bar represents the mean log(pfu/μL) of all platings for a given clone, and the error bars show the 95% confidence interval. Statistical analysis indicated significant differences among the phage concentrations for all the data sets (ANOVA; F_{<b>12,158</b>} = 139.1, P < 0.0001). Post-hoc analysis showed that the 135-min concentrations of some clones are significantly different from one another, while others are not (Tukey’s HSD; α = 0.05, see the connecting letters at the bottom of the bars).</p