Third nationwide surveillance of bacterial pathogens in patients with acute uncomplicated cystitis conducted by the Japanese surveillance committee during 2020 and 2021: Antimicrobial susceptibility of Escherichia coli, Klebsiella pneumoniae, and Staphylococcus saprophyticus.

The Japanese surveillance committee conducted a third nationwide surveillance of antimicrobial susceptibility of acute uncomplicated cystitis at 55 facilities throughout Japan between April 2020 and September 2021. In this surveillance, we investigated the susceptibility of Escherichia coli (E. coli), Klebsiella pneumoniae (K. pneumoniae), and Staphylococcus saprophyticus (S. saprophyticus) for various antimicrobial agents by isolating and culturing bacteria from urine samples. In total, 823 strains were isolated from 848 patients and 569 strains of target bacteria, including E. coli (n = 529, 92.9 %), K. pneumoniae (n = 28, 4.9 %), and S. saprophyticus (n = 12, 2.2 %) were isolated. The minimum inhibitory concentrations of 18 antibacterial agents were determined according to the Clinical and Laboratory Standards Institute manual. In premenopausal patients, there were 31 (10.5 %) and 20 (6.8 %) fluoroquinolone (FQ)-resistant E. coli and extended-spectrum ß-lactamase (ESBL)-producing E. coli, respectively. On the other hand, in postmenopausal patients, there were 75 (32.1 %) and 36 (15.4 %) FQ-resistant E. coli and ESBL-producing E. coli, respectively. The rate of FQ-resistant E. coli and ESBL-producing E. coli in post-menopausal women was higher than that for our previous nationwide surveillance (20.7 % and 32.1 %: p = 0.0004, 10.0 % and 15.4 %; p = 0.0259). For pre-menopausal women, there was no significant difference in the rate of FQ-resistant E. coli and ESBL-producing E. coli between this and previous reports, but the frequency of FQ-resistant E. coli and ESBL-producing E. coli exhibited a gradual increase. For appropriate antimicrobial agent selection and usage, it is essential for clinicians to be aware of the high rate of these antimicrobial-resistant bacteria in acute uncomplicated cystitis in Japan.

Development of a Fully Automated Desktop Analyzer and Ultrahigh Sensitivity Digital Immunoassay for SARS-CoV-2 Nucleocapsid Antigen Detection.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak has had a significant impact on public health and the global economy. Several diagnostic tools are available for the detection of infectious diseases, with reverse transcription-polymerase chain reaction (RT-PCR) testing specifically recommended for viral RNA detection. However, this diagnostic method is costly, complex, and time-consuming. Although it does not have sufficient sensitivity, antigen detection by an immunoassay is an inexpensive and simpler alternative to RT-PCR. Here, we developed an ultrahigh sensitivity digital immunoassay (d-IA) for detecting SARS-CoV-2 nucleocapsid (N) protein as antigens using a fully automated desktop analyzer based on a digital enzyme-linked immunosorbent assay. METHODS: We developed a fully automated d-IA desktop analyzer and measured the viral N protein as an antigen in nasopharyngeal (NP) swabs from patients with coronavirus disease. We studied nasopharyngeal swabs of 159 and 88 patients who were RT-PCR-negative and RT-PCR-positive, respectively. RESULTS: The limit of detection of SARS-CoV-2 d-IA was 0.0043 pg/mL of N protein. The cutoff value was 0.029 pg/mL, with a negative RT-PCR distribution. The sensitivity of RT-PCR-positive specimens was estimated to be 94.3% (83/88). The assay time was 28 min. CONCLUSIONS: Our d-IA system, which includes a novel fully automated desktop analyzer, enabled detection of the SARS-CoV-2 N-protein with a comparable sensitivity to RT-PCR within 30 min. Thus, d-IA shows potential for SARS-CoV-2 detection across multiple diagnostic centers including small clinics, hospitals, airport quarantines, and clinical laboratories.

Autoregulation and dual stepping mode of MYA2, an Arabidopsis myosin XI responsible for cytoplasmic streaming.

Arabidopsis thaliana has 13 genes belonging to the myosin XI family. Myosin XI-2 (MYA2) plays a major role in the generation of cytoplasmic streaming in Arabidopsis cells. In this study, we investigated the molecular properties of MYA2 expressed by the baculovirus transfer system. Actin-activated ATPase activity and in vitro motility assays revealed that activity of MYA2 was regulated by the globular tail domain (GTD). When the GTD is not bound to the cargo, the GTD inhibits ADP dissociation from the motor domain. Optical nanometry of single MYA2 molecules, combining total internal reflection fluorescence microscopy (TIRFM) and the fluorescence imaging with one-nanometer accuracy (FIONA) method, revealed that the MYA2 processively moved on actin with three different step sizes: - 28 nm, 29 nm, and 60 nm, at low ATP concentrations. This result indicates that MYA2 uses two different stepping modes; hand-over-hand and inchworm-like. Force measurement using optical trapping showed the stall force of MYA2 was 0.85 pN, which was less than half that of myosin V (2-3 pN). These results indicated that MYA2 has different transport properties from that of the myosin V responsible for vesicle transport in animal cells. Such properties may enable multiple myosin XIs to transport organelles quickly and smoothly, for the generation of cytoplasmic streaming in plant cells.

Glycine insertion modulates the fluorescence properties of Aequorea victoria green fluorescent protein and its variants in their ambient environment.

The green fluorescent protein (GFP) derived from Pacific Ocean jellyfish is an essential tool in biology. GFP-solvent interactions can modulate the fluorescent property of GFP. We previously reported that glycine insertion is an effective mutation in the yellow variant of GFP, yellow fluorescent protein (YFP). Glycine insertion into one of the ß-strands comprising the barrel structure distorts its structure, allowing water molecules to invade near the chromophore, enhancing hydrostatic pressure or solution hydrophobicity sensitivity. However, the underlying mechanism of how glycine insertion imparts environmental sensitivity to YFP has not been elucidated yet. To unveil the relationship between fluorescence and ß-strand distortion, we investigated the effects of glycine insertion on the dependence of the optical properties of GFP variants named enhanced-GFP (eGFP) and its yellow (eYFP) and cyan (eCFP) variants with respect to pH, temperature, pressure, and hydrophobicity. Our results showed that the quantum yield decreased depending on the number of inserted glycines in all variants, and the dependence on pH, temperature, pressure, and hydrophobicity was altered, indicating the invasion of water molecules into the ß-barrel. Peak shifts in the emission spectrum were observed in glycine-inserted eGFP, suggesting a change of the electric state in the excited chromophore. A comparative investigation of the spectral shift among variants under different conditions demonstrated that glycine insertion rearranged the hydrogen bond network between His148 and the chromophore. The present results provide important insights for further understanding the fluorescence mechanism in GFPs and suggest that glycine insertion could be a potent approach for investigating the relationship between water molecules and the intra-protein chromophore.

Measurement of microRNA with isothermal DNA amplification on fully automated immunoassay analyzers.

MicroRNAs (miRNAs) in a blood sample are usually measured by quantitative reverse transcription PCR (qRT-PCR), microarray, and next-generation sequencing (NGS) which requires time-consuming pre-treatment, manual operation, and a stand-alone instrument. To overcome these disadvantages, miRNA testing has been developed using the automated analyzers routinely used in clinical laboratories. An isothermal DNA amplification reaction was adapted to a fully automated immunoassay analyzer that conducts extraction, amplification, and detection processes at 37 °C in 44 min. In a reaction vessel, a pre-designed single-stranded signal DNA was amplified in the presence of miRNA, using DNA templates, DNA polymerase, and nicking endonuclease. Then, the amplified signal DNA was hybridized by one DNA probe attached to a magnetic particle and another DNA probe labeled with acridinium ester. After the chemiluminescence reaction, luminescence intensity was automatically measured. The automated assays of cancer-related miRNAs were implemented on the analyzer with throughput of 66 tests per hour. In the assays with one-step amplification, three miRNAs (miR-21-5p, miR-18a-5p, and miR-500a-3p) at concentrations lower than 100 fM were automatically detected and the cross reactivity for miR-21-5p with fifteen similar miRNAs was not higher than 0.02%. In the assay with two-step amplification, detection sensitivity and amplification rate for miR-21-5p were 3 fM and 103-fold, respectively. The coefficient of variations (CVs) in the measurement at the target concentrations from 5 fM to 1000 pM were less than 8%. Furthermore, we also achieved automated nucleic acid detection in human serum. The proposed fully automated miRNA assays showed high sensitivity, low cross reactivity, and reproducibility suitable for clinical use. Graphical abstract.

The use of a genetically encoded molecular crowding sensor in various biological phenomena.

We evaluated usability of a previously developed genetically encoded molecular crowding sensor in various biological phenomena. Molecular crowding refers to intracellular regions that are occupied more by proteins and nucleotides than by water molecules and is thought to have a strong effect on protein function. To evaluate intracellular molecular crowding, usually the diffusion coefficient of a probe is used because it is related to mobility of the surrounding molecular crowding agents. Recently, genetically encoded molecular crowding sensors based on Förster resonance energy transfer were reported. In the present study, to evaluate the usability of a genetically encoded molecular crowding sensor, molecular crowding was monitored during several biological events. Changes in molecular crowding during stem cell differentiation, cell division, and focal adhesion development and difference in molecular crowding in filopodia locations were examined. The results show usefulness of the genetically encoded molecular crowding sensor for understanding the biological phenomena relating to molecular crowding.

Dependence of fluorescent protein brightness on protein concentration in solution and enhancement of it.

Fluorescent proteins have been widely used in biology because of their compatibility and varied applications in living specimens. Fluorescent proteins are often undesirably sensitive to intracellular conditions such as pH and ion concentration, generating considerable issues at times. However, harnessing these intrinsic sensitivities can help develop functional probes. In this study, we found that the fluorescence of yellow fluorescent protein (YFP) depends on the protein concentration in the solution and that this dependence can be enhanced by adding a glycine residue in to the YFP; we applied this finding to construct an intracellular protein-crowding sensor. A Förster resonance energy transfer (FRET) pair, involving a cyan fluorescent protein (CFP) insensitive to protein concentration and a glycine-inserted YFP, works as a genetically encoded probe to evaluate intracellular crowding. By measuring the fluorescence of the present FRET probe, we were able to detect dynamic changes in protein crowding in living cells.

Tyrosine kinase inhibitor-induced vasculopathy in clear cell renal cell carcinoma: an unrecognized antitumour mechanism.

AIMS: To evaluate the pathological features of clear cell renal cell carcinoma (CCRCC) treated with tyrosine kinase inhibitors (TKIs), and to elucidate the mechanism of action of TKIs. METHODS AND RESULTS: Twenty cases of CCRCC treated with TKIs (sorafenib or sunitinib) were retrospectively analysed: 16 were patients who had undergone radical nephrectomy after neoadjuvant TKI therapy, and four were autopsy cases of patients who received TKI treatment. All tumours had two distinct regions: one characterized by necrosis and/or degeneration, indicating antitumour activity; and the other characterized by no or few pathological changes, indicating the absence of antitumour activity. Vasculopathy of tumour vessels was observed in or adjacent to the necrotic or degenerative areas; a decreased density of endothelial cells was noted in the tumour vessels. Few or no changes of vasculopathy were observed in tumour vessels in the other CCRCC areas, indicating the absence or low levels of antitumour activity. CONCLUSIONS: This is the first pathological report of vasculopathy in TKI-treated CCRCC cases. Our data suggest that TKIs initially induce vasculopathy in tumour vessels, and consequently cause reduction or diminution of blood supply to the CCRCCs, resulting in antitumour activity characterized by necrosis and hyalinization.

Glycine insertion makes yellow fluorescent protein sensitive to hydrostatic pressure.

Fluorescent protein-based indicators for intracellular environment conditions such as pH and ion concentrations are commonly used to study the status and dynamics of living cells. Despite being an important factor in many biological processes, the development of an indicator for the physicochemical state of water, such as pressure, viscosity and temperature, however, has been neglected. We here found a novel mutation that dramatically enhances the pressure dependency of the yellow fluorescent protein (YFP) by inserting several glycines into it. The crystal structure of the mutant showed that the tyrosine near the chromophore flipped toward the outside of the ß-can structure, resulting in the entry of a few water molecules near the chromophore. In response to changes in hydrostatic pressure, a spectrum shift and an intensity change of the fluorescence were observed. By measuring the fluorescence of the YFP mutant, we succeeded in measuring the intracellular pressure change in living cell. This study shows a new strategy of design to engineer fluorescent protein indicators to sense hydrostatic pressure.

[Robot-assisted laparoscopic partial nephrectomy using daVinci S-surgical system for localized renal tumor: report of initial five cases].

OBJECTIVES: For the management of patients with small renal tumor, laparoscopic partial nephrectomy (LPN) provides similar oncological control as radical nephrectomy (RN) and is superior to RN with respect to preserving renal function and preventing chronic kidney disease (CKD). The challenge of LPN is to resect a tumor in a bloodless field within a limited warm ischemia time (WIT) , followed by hemostatic renorrhaphy under restricted movement of laparoscopic forceps. Therefore, LPN still remains challenging to even experienced laparoscopic surgeon. DaVinci device improved the movability of forceps in LPN and provided three-dimensional visualization. We evaluated outcome and safety of our first series of robot-assisted laparoscopic partial nephrectomy (RALPN) for localized kidney tumor. There was no previous report of RALPN undertaken in our country. PATIENTS AND METHODS: Since August 2010, our team carried out RALPN for a total of five cases of renal tumor. There were four males and one female with an age range of 41 to 65 years-old. Size of tumor ranged from 15 to 28 mm, located in exophytic region, and four cases in right side and one in left. RALPN was undertaken by single surgeon through transperitoneal approach in two cases and retroperitoneal in tree. RESULTS: RALPN was completed in all patients without conversion to open or hand-assisted surgery. The median operative time and the estimated blood loss were 189 minutes, ranged from 150 to 264, and 29 ml, from 10 to 50, respectively. The median volume of removed tumor and the length of WIT were 7 g, ranged from 4 to 13 g, and 18 minutes, from 13 to 26 minutes, respectively. No complications or reoperations were associated during or post our RALPN cases. Pathological examination of removed tumor showed renal cell carcinoma with negative surgical margin in all cases. CONCLUSIONS: Introduction of daVinci device to LPN made this procedure, RALPN, a secured and promising one, which leading to shorten the WIT and to achieve satisfied renorrhaphy. Even for the complex and technically challenging renal tumors, robotic assistance is expected to provide patients the benefit of minimally invasive surgery with safety and satisfactory renal function.