Identification of activity-based biomarkers for early-stage pancreatic tumors in blood using single-molecule enzyme activity screening.

Single-molecule enzyme activity-based enzyme profiling (SEAP) is a methodology to globally analyze protein functions in living samples at the single-molecule level. It has been previously applied to detect functional alterations in phosphatases and glycosidases. Here, we expand the potential for activity-based biomarker discovery by developing a semi-automated synthesis platform for fluorogenic probes that can detect various peptidases and protease activities at the single-molecule level. The peptidase/protease probes were prepared on the basis of a 7-amino-4-methylcoumarin fluorophore. The introduction of a phosphonic acid to the core scaffold made the probe suitable for use in a microdevice-based assay, while phosphonic acid served as the handle for the affinity separation of the probe using Phos-tag. Using this semi-automated scheme, 48 fluorogenic probes for the single-molecule peptidase/protease activity analysis were prepared. Activity-based screening using blood samples revealed altered single-molecule activity profiles of CD13 and DPP4 in blood samples of patients with early-stage pancreatic tumors. The study shows the power of single-molecule enzyme activity screening to discover biomarkers on the basis of the functional alterations of proteins.

Negative impact of behavior restriction amidst a clustered COVID-19 infection on immobility syndrome in older patients negative for COVID-19: report from a chronic care hospital in Japan.

Previous studies have reported on associations between immobility syndrome and the COVID-19 pandemic. However, little is known about the aggravation of this syndrome in older patients negative for COVID-19 infection amidst behavior restriction due to a clustered COVID-19 infection. Patients hospitalized one month before a clustered COVID-19 infection occurred in our hospital were recruited. Rehabilitation therapy was suspended for 25 days during behavior restriction. The ability of daily living of the patients was evaluated with the functional independence measure and Barthel index. Chronological changes in the functional independence measure and Barthel index scores were evaluated monthly, beginning one month before the clustered COVID-19 infection to one month after re-initiation of rehabilitation therapy. Patients with minimum scores in the functional independence measure (18) and Barthel index (0) prior to the clustered COVID-19 infection were excluded. Functional independence measure scores of 73 older patients and the Barthel index scores of 48 patients were analyzed. The mean total functional independence measure score amidst the behavior restriction significantly changed from 36.3 to 35.1 (p = 0.019), while statistical significance was not detected in the mean motor subtotal (from 21.6 to 20.9 with p = 0.247) or cognitive subtotal functional independence measure scores (from 14.6 to 14.2 with p = 0.478). During the behavior restriction, the mean Barthel index scores declined from 25.8 to 23.2 without statistical significance (p = 0.059). Behavior restriction due to a clustered COVID-19 infection may aggravate immobility syndrome in older patients who are negative for COVID-19.

Amplification-free RNA detection with CRISPR-Cas13.

CRISPR-based nucleic-acid detection is an emerging technology for molecular diagnostics. However, these methods generally require several hours and could cause amplification errors, due to the pre-amplification of target nucleic acids to enhance the detection sensitivity. Here, we developed a platform that allows "CRISPR-based amplification-free digital RNA detection (SATORI)", by combining CRISPR-Cas13-based RNA detection and microchamber-array technologies. SATORI detected single-stranded RNA targets with maximal sensitivity of ~10 fM in <5 min, with high specificity. Furthermore, the simultaneous use of multiple different guide RNAs enhanced the sensitivity, thereby enabling the detection of the SARS-CoV-2 N-gene RNA at ~5 fM levels. Therefore, we hope SATORI will serve as a powerful class of accurate and rapid diagnostics.

Assessment of Oxaliplatin-induced Chronic Neuropathy and Anticancer Efficacy Through Pharmacokinetic and Toxicodynamic Evaluation of a Rat Model of Colorectal Cancer.

BACKGROUND/AIM: Oxaliplatin-induced chronic neuropathy is a prominent factor for dose reduction and not completing all cycles of chemotherapy for patients with colorectal cancer (CRC). The aim of the study was to investigate the pharmacokinetics and toxicodynamics of oxaliplatin-induced chronic neuropathy in CRC rats to ensure effective management. MATERIALS AND METHODS: A rat model of CRC was developed using 1,2-Dimethylhydrazine and dextran sulfate. Oxaliplatin (L-OHP) was administered intravenously to CRC rats every week. The pharmacokinetic profiles and tumor distribution of L-OHP and chronic neuropathies were investigated for over four weeks. RESULTS: The mean values of the area under the concentration-time curve for L-OHP showed a dose-dependent increase. Chronic neuropathy occurred from Day 14 in the 8 mg/kg group and Day 19 in the 3 and 5 mg/kg groups. CONCLUSION: These results provide preliminary information for the development of a pharmacokinetic and toxicodynamic model of L-OHP for CRC therapy cycles.

Vehicle-dependent Effects of Sphingosine 1-phosphate on Plasminogen Activator Inhibitor-1 Expression.

AIM: Sphingosine 1-phosphate (S1P) has been suggested to be a positive regulator of plasminogen activator inhibitor 1 (PAI-1) in adipocytes, while some studies are not consistent with this prothrombotic property of S1P. Since S1P is bound to apolipoprotein M (apoM) on HDL or to albumin in plasma, we compared the properties of these two forms on the PAI-1 induction. METHODS: We investigated the associations of S1P, apoM, and PAI-1 concentrations in the plasma of normal coronary artery (NCA), stable angina pectoris (SAP), and acute coronary syndrome (ACS) subjects (n=32, 71, and 38, respectively). Then, we compared the effects of S1P with various vehicles on the PAI-1 expression in 3T3L1 adipocytes. We also investigated the modulation of the PAI-1 levels in mice infected with adenovirus coding apoM. RESULTS: Among ACS subjects, the PAI-1 level was positively correlated with the S1P level, but not the apoM level. In adipocytes, S1P bound to an apoM-rich vehicle induced PAI-1 expression to a lesser extent than the control vehicle, while S1P bound to an apoM-depleted vehicle induced PAI-1 expression to a greater extent than the control vehicle in 3T3L1 adipocytes. Additionally, apoM overexpression in mice failed to modulate the plasma PAI-1 level and the adipose PAI-1 expression level. S1P bound to albumin increased PAI-1 expression through the S1P receptor 2-Rho/ROCK-NFκB pathway. CONCLUSION: S1P bound to albumin, but not to apoM, induces PAI-1 expression in adipocytes, indicating that S1P can exert different properties on the pathogenesis of vascular diseases, depending on its vehicle.

[Measurement of the Dose Rate Using Dosimeters in Interventional Radiology and Its Difficulty].

In equipment used for interventional radiology (IVR), automatic exposure control (AEC) is incorporated to obtain the X-ray output suitable for the treatment of targeted lesions. For the AEC, users select a region as the signal sensing region (measuring field, MF) in the flat panel detector; MFs with various sizes and shapes were pre-defined and prepared in the system. The aim of this study was to examine the change of measured dose rate with the selection of MFs, the type of dosimeters (the ionization chamber dosimeter and the semiconductor dosimeter), and the dosimeter placement relative to the direction of X-ray tube (from cathode to anode). The IVR equipment was Allura Xper FD20/10 (Philips Medical Systems), and six kinds of built-in MFs were used. It was found that dose rate measured by the ionization chamber dosimeter showed a variation of -2 mGy/min with the MFs and the ionization chamber dosimeter placement. The dose rate measured by the semiconductor dosimeter showed more variation than the ionization chamber dosimeter. The change of dose rate with the dosimeter placement would be caused by the MF overlapping the dosimeter which would affect the AEC (the X-ray output). Also, the change of dose rate with the dosimeter placement was considered to be related to the heel effect of the X-ray beam. When performing dose rate measurements, we should notice that the selection of MFs, the type of dosimeters, and the dosimeter placement would affect the measured values.