Akihiro Sanda

Identification of a novel serum biomarker for pancreatic cancer, C4b-binding protein α -chain (C4BPA) by quantitative proteomic analysis using tandem mass tags

Background:Pancreatic ductal adenocarcinoma (PDAC) remains a devastating disease due to the lack of specific early diagnostic markers. To improve the outcomes, proteomic approaches are being developed for the discovery of novel biomarkers of PDAC.Methods:Using tandem mass tag labelling and LC-MS/MS, we performed comparative analyses of pre- and postoperative sera from PDAC patients to identify specific serum biomarkers for PDAC. In validation studies, we evaluated the discriminatory power of candidate proteins.Results:Among the 302 proteins analysed, 20 were identified as potential biomarkers, with C4b-binding protein α-chain (C4BPA) and polymeric immunoglobulin receptor (PIGR) being selected for further analysis. The sera levels of C4BPA and PIGR were significantly higher in the preoperative PDAC patients than in the postoperative ones (P<0.008, P<0.036, respectively). In addition, serum C4BPA levels, but not PIGR, in patients with PDAC were significantly higher than those in healthy controls as well as in patients with pancreatitis and other malignancies including biliary tract cancers (BTC) (P<0.001). The respective area under the receiver operator characteristics (ROC) curve (AUC) was 0.860 for C4BPA, 0.846 for CA19-9 and 0.930 for the combination of C4BPA and CA19-9 in PDAC vs non-cancer individuals. The respective AUC was 0.912 for C4BPA, 0.737 for CA19-9 in Stages I and II of PDAC, 0.854 for C4BPA and 0.264 for CA19-9 in PDAC vs BTC.Conclusions:We have demonstrated that C4BPA is a novel serum biomarker for detecting early stage PDAC, as well as for distinguishing PDAC from other gastroenterological cancers. Further analysis in large cohort studies will warrant C4BPA as a promising biomarker of PDAC in clinical use.

Urinary albumin and transferrin as early diagnostic markers of chronic kidney disease

Feline renal diseases are increasingly noted in veterinary practice. It is important to diagnose and identify the pathological basis of renal dysfunction accurately at an early stage, but there are only a few reports on this area in clinical veterinary medicine. We investigated the efficacy of measurement of urinary albumin (u-Alb) and urinary transferrin (u-Tf) for early diagnosis using 5-µl urine samples collected noninvasively by catheterization from normal (IRIS stage I) cats and cats with stage I chronic kidney disease (CKD). The u-Alb levels in normal and stage I CKD cats were 6.0 ± 4.5 and 11.2 ± 8.4 mg/dl, respectively, and the u-Tf levels were 0.09 ± 0.42 and 0.52 ± 0.79 mg/dl, respectively. Based on ROC curve analysis, the sensitivity and specificity of u-Alb and u-Tf were higher than those of the currently used biomarker, the plasma creatinine level. The sensitivity of u-Alb was higher than that of u-Tf, whereas the specificity of u-Tf was higher than that of u-Alb. The validity of the threshold albumin level (20 mg/dl) was confirmed by measurements using SDS-PAGE. Since leakage of u-Tf in urine precedes leakage of u-Alb, inclusion of u-Tf in biochemistry tests may be appropriate for IRIS staging as a diagnostic marker of early diagnosis of renal disorder in cats.

Studies on the Specificity of Ribonuclease from Rhizopus sp.

In order to investigate the base specificity of the RNase from Rhizopus sp. (RNase Rh), its kinetic parameters were measured with 16 dinucleoside phosphates (XpY, where X or Y represents one of adenosine, guanosine, uridine and cytidine) as substrates at pH 5.5 and 25°. The average Km values of ApY, GpY, CpY and UpY increased in the order A, G, C and U. The average Km values of ApY, GpY, CpY and UpY increased in the order A, G, U and C. The average Vmax values of ApY, GpY, CpY and UpY were larger for dinucleoside phosphates having A and G at X. However, the average Vmax values of XpA, XpG, XpC and XpU were relatively constant. It was concluded that the X base in XpY contributes mainly to the specificity of the enzyme and the Y base may modify this somewhat. The K1 values of various nucleotides towards RNase Rh were measured at pH 5.5. These compounds inhibited RNase Rh competitively. Although the inhibitory effect depends on the base, sugar and location of the phosphate moiety, for a given location of phosphate on the sugar, the K1 values of ribonucleotides decreased in the order U, C, G and A and those of deoxyribonucleotides decreased in the order T, C, G and A. These data also show that purine bases, especially adenine, have high affinities for RNase Rh.

Rapid Discrimination between Methicillin-Sensitive and Methicillin-Resistant Staphylococcus aureus Using MALDI-TOF Mass Spectrometry

　Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major pathogens responsible for nosocomial infections. The presence of MRSA in a hospital is detrimental to patients and to hospital management. Thus, rapid identification of MRSA is needed. Here, we report on a prospective method to rapidly discriminate of MSSA from MRSA using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and support vector machine (SVM) analysis in 160 clinical isolates of S. aureus. The predictive model was tested using 100 S. aureus isolates (50 MSSA and 50 MRSA). The identification rates were 90.0% for MSSA and 87.5% for MRSA in a 10-fold cross-validation SVM. In blind test sets, 60 S. aureus isolates (30 MSSA and 30 MRSA) were correctly classified, with identification rates of 93.3% for MSSA and 86.7% for MRSA. The method proposed in this study using the predictive model enables detection of one colony in 5 minutes, and thus is useful at clinical sites at which rapid discrimination of MRSA from MSSA is required.