Differentiation of Prostate Cancer from Normal Tissue in Radical Prostatectomy Specimens by Desorption Electrospray Ionization and Touch Spray Ionization Mass Spectrometry.

Radical prostatectomy is a common treatment option for prostate cancer before it has spread beyond the prostate. Examination for surgical margins is performed post-operatively with positive margins reported to occur in 6.5 – 32% of cases. Rapid identification of cancerous tissue during surgery could improve surgical resection. Desorption electrospray ionization (DESI) is an ambient ionization method which produces mass spectra dominated by lipid signals directly from prostate tissue. With the use of multivariate statistics, these mass spectra can be used to differentiate cancerous and normal tissue. The method was applied to 100 samples from 12 human patients to create a training set of MS data. The quality of the discrimination achieved was evaluated using principal component analysis - linear discriminant analysis (PCA-LDA) and confirmed by histopathology. Cross validation (PCA-LDA) showed >95% accuracy. An even faster and more convenient method, touch spray (TS) mass spectrometry, not previously tested to differentiate diseased tissue, was also evaluated by building a similar MS data base characteristic of tumor and normal tissue. An independent set of 70 non-targeted biopsies from six patients was then used to record lipid profile data resulting in 110 data points for an evaluation dataset for TS-MS. This method gave prediction success rates measured against histopathology of 93%. These results suggest that DESI and TS could be useful in differentiating tumor and normal prostate tissue at surgical margins and that these methods should be evaluated intra-operatively

Ambient ionization mass spectrometric analysis of human surgical specimens to distinguish renal cell carcinoma from healthy renal tissue

Touch spray - mass spectrometry (TS-MS) is an ambient ionization technique (ionization of unprocessed samples in the open air) that may find intraoperative applications in quickly identifying the disease state of cancerous tissues and in defining surgical margins. In this study, TS-MS was performed on fresh kidney tissue (~1–5 cm3), within one hour of resection, from 21 human subjects afflicted by renal cell carcinoma (RCC). The preliminary diagnostic value of TS-MS data taken from freshly resected tissue was evaluated. Principal component analysis (PCA) of the negative ion mode (m/z 700–1000) data provided separation between RCC (16 samples) and healthy renal tissue (13 samples). Linear discriminant analysis (LDA) on the PCA compressed data estimated sensitivity (true positive rate) and specificity (true negative rate) of 98% and 95%, respectively, based on histopathological evaluation. The results indicate that TS-MS might provide rapid diagnostic information in spite of the complexity of unprocessed kidney tissue and the presence of interferences such as urine and blood. Desorption electrospray ionization imaging (DESI-MSI) in the negative ionization mode was performed on the tissue specimens after TS-MS analysis as a reference method. The DESI imaging experiments provided phospholipid profiles (m/z 700–1000) that also separated RCC and healthy tissue in the PCA space, with PCA-LDA sensitivity and specificity of 100% and 89%, respectively. The TS and DESI loading plots indicated that different ions contributed most to the separation of RCC from healthy renal tissue (m/z 794 [PC 34:1+Cl]− and 844 [PC 38:4+Cl]− for TS vs. m/z 788 [PS 36:1-H]− and 810 [PS 38:4-H]− for DESI), while m/z 885 ([PI 38:4-H]−) was important in both TS and DESI. The prospect, remaining hurdles, and future work required for translating TS-MS into a method of intraoperative tissue diagnosis is discussed.

Cholesterol Sulfonation Enzyme, SULT2B1b, Modulates AR and Cell Growth Properties in Prostate Cancer

Cholesterol accumulates in prostate lesions and has been linked to prostate cancer (PCa) incidence and progression. However, how accumulated cholesterol contributes to PCa development and progression is not completely understood. Cholesterol sulfate (CS), the primary sulfonation product of cholesterol sulfotransferase (SULT2B1b), accumulates in human prostate adenocarcinoma and precancerous prostatic intraepithelial neoplasia (PIN) lesions compared to normal regions of the same tissue sample. Given the enhanced accumulation of CS in these lesions, it was hypothesized that SULT2B1b-mediated production of CS provides a growth advantage to these cells. To address this, PCa cells with RNAi-mediated knockdown (KD) of SULT2B1b were used to assess the impact on cell growth and survival. SULT2B1b is expressed and functional in a variety of prostate cells and the data demonstrate that SULT2B1b KD, in LNCaP and other androgen-responsive (VCaP and C4-2) cells, results in decreased cell growth/viability and induces cell death. SULT2B1b KD also decreases androgen receptor (AR) activity and expression at mRNA and protein levels. While AR overexpression has no impact on SULT2B1b KD-mediated cell death, addition of exogenous androgen is able to partially rescue the growth inhibition induced by SULT2B1b KD in LNCaP cells. These results suggest that SULT2B1b positively regulates the AR either through alterations in ligand availability or by interaction with critical co-regulators that influence AR activity

Identification of Critical Residues in the Propeptide of LasA Protease of Pseudomonas aeruginosa Involved in the Formation of a Stable Mature Protease▿

LasA protease is a 20-kDa elastolytic and staphylolytic enzyme secreted by Pseudomonas aeruginosa. LasA is synthesized as a preproenzyme that undergoes proteolysis to remove a 22-kDa amino-terminal propeptide. Like the propeptides of other bacterial proteases, the LasA propeptide may act as an intramolecular chaperone that correctly folds the mature domain into an active protease. To locate regions of functional importance within proLasA, linker-scanning insertional mutagenesis was employed using a plasmid containing lasA as the target. Among the 5 missense insertions found in the mature domain of proLasA, all abolished enzymatic activity but not secretion. In general, the propeptide domain was more tolerant to insertions. However, insertions within a 9-amino-acid region in the propeptide caused dramatic reductions in LasA enzymatic activity. All mutant proLasA proteins were still secreted, but extracellular stability was low due to clustered insertions within the propeptide. The codons of 16 residues within and surrounding the identified 9-amino-acid region were subjected to site-directed mutagenesis. Among the alanine substitutions in the propeptide that had a major effect on extracellular LasA activity, two (L92A and W95A) resulted in highly unstable proteins that were susceptible to proteolytic degradation and three (H94A, I101A, and N102A) were moderately unstable and allowed the production of a LasA protein with low enzymatic activity. These data suggest that these clustered residues in the propeptide may play an important role in promoting the correct protein conformation of the mature LasA protease domain

Novel Three-Dimensional Organoid Model for Evaluation of the Interaction of Uropathogenic Escherichia coli with Terminally Differentiated Human Urothelial Cells

Human bladder 5637 cells cultivated under microgravity conditions formed organoids that displayed characteristics of in vivo tissue-specific differentiation. Uropathogenic Escherichia coli (UPEC) strain CP9 colonized and penetrated the organoids and induced α-hemolysin-mediated exfoliation of uroepithelial cells. We propose these uro-organoids as models that simulate the interactions between UPEC and terminally differentiated human urothelium

Hemolysin of Uropathogenic Escherichia coli Evokes Extensive Shedding of the Uroepithelium and Hemorrhage in Bladder Tissue within the First 24 Hours after Intraurethral Inoculation of Mice▿

Many uropathogenic Escherichia coli (UPEC) strains produce both hemolysin (Hly) and cytotoxic necrotizing factor type 1 (CNF1), and the loci for these toxins are often linked. The conclusion that Hly and CNF1 contribute to urovirulence is supported by the results of epidemiological studies associating the severity of urinary tract infections (UTIs) with toxin production by UPEC isolates. Additionally, we previously reported that mouse bladders and rat prostates infected with UPEC strain CP9 exhibit a more profound inflammatory response than the organs from animals challenged with CP9cnf1 and that CNF1 decreases the antimicrobial activities of polymorphonuclear leukocytes. More recently, we created an Hly mutant, CP9ΔhlyA1::cat, and showed that it was less hemolytic and destructive for cultured bladder cells than CP9 was. Here we evaluated the relative effects of mutations in hlyA1 or cnf1 alone or together on the pathogenicity of CP9 in a mouse model of ascending UTI. To do this, we constructed an hlyA1-complemented clone of CP9ΔhlyA1::cat and an hlyA1 cnf1 CP9 double mutant. We found that Hly had no influence on bacterial colonization of the bladder or kidneys in single or mixed infections with the wild type and CP9ΔhlyA1::cat but that it did provoke sloughing of the uroepithelium and bladder hemorrhage within the first 24 h after challenge. Finally, we confirmed that CNF1 expression induces bladder inflammation and, in particular, as shown in this study, submucosal edema. From these data, we speculate that Hly and CNF1 may be largely responsible for the signs and symptoms of cystitis in humans infected with toxigenic UPEC

Separating the pH-Dependent Behavior of Water in the Stern and Diffuse Layers with Varying Salt Concentration

Vibrational sum frequency generation (SFG) spectroscopy was utilized to distinguish different populations of water molecules within the electric double layer (EDL) at the silica/water interface. By systematically varying the electrolyte concentration, surface deprotonation, and SFG polarization combinations, we provide evidence of two regions of water molecules that have distinct pH-dependent behavior when the Stern layer is present (with onset between 10 and 100 mM NaCl). For example, water molecules near the surface in the Stern layer can be probed by the pss polarization combination, while other polarization combinations (ssp and ppp) predominantly probe water molecules further from the surface in the diffuse part of the electrical double layer. For the water molecules adjacent to the surface within the Stern layer, upon increasing the pH from the point-of-zero charge of silica (pH ∼2) to higher values (pH ∼12), we observe an increase in alignment consistent with a more negative surface with increasing pH. In contrast, water molecules further from the surface appear to exhibit a net flip in orientation upon increasing the pH over the same range, which we attribute to the presence of the Stern layer and possible overcharging of the EDL at lower pH. The opposing pH-dependent behavior of water in these two regions sheds new light on our understanding of the water structure within the EDL at high salt concentrations when the Stern layer is present