Selective-plane-activation structured illumination microscopy.

The background light from out-of-focus planes hinders resolution enhancement in structured illumination microscopy when observing volumetric samples. Here we used selective plane illumination and reversibly photoswitchable fluorescent proteins to realize structured illumination within the focal plane and eliminate the out-of-focus background. Theoretical investigation of the imaging properties and experimental demonstrations show that selective plane activation is beneficial for imaging dense microstructures in cells and cell spheroids.

On-the-fly Raman microscopy guaranteeing the accuracy of discrimination.

Accelerating the measurement for discrimination of samples, such as classification of cell phenotype, is crucial when faced with significant time and cost constraints. Spontaneous Raman microscopy offers label-free, rich chemical information but suffers from long acquisition time due to extremely small scattering cross-sections. One possible approach to accelerate the measurement is by measuring necessary parts with a suitable number of illumination points. However, how to design these points during measurement remains a challenge. To address this, we developed an imaging technique based on a reinforcement learning in machine learning (ML). This ML approach adaptively feeds back "optimal" illumination pattern during the measurement to detect the existence of specific characteristics of interest, allowing faster measurements while guaranteeing discrimination accuracy. Using a set of Raman images of human follicular thyroid and follicular thyroid carcinoma cells, we showed that our technique requires 3,333 to 31,683 times smaller number of illuminations for discriminating the phenotypes than raster scanning. To quantitatively evaluate the number of illuminations depending on the requisite discrimination accuracy, we prepared a set of polymer bead mixture samples to model anomalous and normal tissues. We then applied a home-built programmable-illumination microscope equipped with our algorithm, and confirmed that the system can discriminate the sample conditions with 104 to 4,350 times smaller number of illuminations compared to standard point illumination Raman microscopy. The proposed algorithm can be applied to other types of microscopy that can control measurement condition on the fly, offering an approach for the acceleration of accurate measurements in various applications including medical diagnosis.

Monitoring of blood levels in patients administered CYP3A4 inhibitor during the maintenance phase of venetoclax administration
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OBJECTIVE: Venetoclax, an oral B-cell lymphoma-2 inhibitor, necessitates dose adjustment when combined with a CYP3A4 inhibitor; however, the dosing regimen remains unclear on adding a CYP3A4 inhibitor after venetoclax administration. CASE SUMMARY: We present a case report of a patient who was simultaneously treated with a CYP3A4 inhibitor and a steady daily dose of venetoclax. A 30-year-old male diagnosed with acute myeloid leukemia received a combination of venetoclax and azacitidine as remission induction therapy. He was prescribed 400 mg/day venetoclax at a steady daily dose, with fosfluconazole initiated on day 18. Given that fosfluconazole can induce moderate CYP3A4 inhibitory effects, the venetoclax dosage was reduced to 200 mg/day on the same day. Despite dose reduction, plasma trough levels of venetoclax continued rising gradually. Nearly 10 days were required to decrease blood levels to a steady state. CONCLUSION: The risk of elevated venetoclax blood levels needs to be considered when initiating CYP3A4 inhibitors and reducing venetoclax dosage on the same day.

Risk of delirium with antiepileptic drug use: a study based on the Japanese Adverse Drug Event Report database.

BACKGROUND: Antiepileptic drugs may cause delirium, and the risk may vary with each drug. However, related studies have provided inconsistent results. AIM: The aim of this study was to investigate whether the use of antiepileptic drugs is a risk factor for delirium development. METHOD: Using the Japanese Adverse Drug Event Report database, we analysed 573,316 reports pertaining to the period from 2004 to 2020. Reporting odds ratios and 95% confidence intervals of delirium associated with use of antiepileptic drugs were calculated after adjusting for potential confounders. Furthermore, for each antiepileptic drug, we performed an analysis stratified based on older age and benzodiazepine receptor agonist usage. RESULTS: There were 27,439 reports of antiepileptic drug-related adverse events. Of these, 191 reports were associated with antiepileptic drugs and delirium (crude reporting odds ratio [cROR], 1.66; 95% confidence interval [CI], 1.43-1.93). The use of lacosamide (adjusted reporting odds ratio [aROR], 2.44; 95% CI, 1.24-4.80), lamotrigine (aROR, 1.54; 95% CI, 1.05-2.26), levetiracetam (aROR, 1.91; 95% CI, 1.35-2.71), and valproic acid (aROR, 1.49; 95% CI, 1.16-1.91) was related to a significantly higher reporting odds ratio for delirium, even after adjustment for possible confounding factors. However, when used in combination with benzodiazepine receptor agonists, none of the antiepileptic drugs were found to be associated with delirium. CONCLUSION: Our study's findings suggest that antiepileptic drug usage may be associated with delirium development.

Bessel-beam illumination Raman microscopy.

We demonstrate the use of Bessel beams for side illumination slit-scanning Raman imaging for label-free and hyperspectral analysis of cell spheroids. The background elimination by the side illumination and the aberration-resistant Bessel beam drastically improves the image contrast in Raman observation, allowing label-free investigation of intracellular molecules in thick biological samples. Live cell spheroids were observed to confirm the improvement in image contrast and background reduction with Bessel illumination compared to conventional epi-line illumination.

Saturated-excitation image scanning microscopy.

Image scanning microscopy (ISM) overcomes the trade-off between spatial resolution and signal volume in confocal microscopy by rearranging the signal distribution on a two-dimensional detector array to achieve a spatial resolution close to the theoretical limit achievable by infinitesimal pinhole detection without sacrificing the detected signal intensity. In this paper, we improved the spatial resolution of ISM in three dimensions by exploiting saturated excitation (SAX) of fluorescence. We theoretically investigated the imaging properties of ISM, when the fluorescence signals are nonlinearly induced by SAX, and show combined SAX-ISM fluorescence imaging to demonstrate the improvement of the spatial resolution in three dimensions. In addition, we confirmed that the SNR of SAX-ISM imaging of fluorescent beads and biological samples, which is one of the challenges in conventional SAX microscopy, was improved.

Wavefront-sensorless adaptive optics with a laser-free spinning disk confocal microscope.

Adaptive optics is being applied widely to a range of microscopies in order to improve imaging quality in the presence of specimen-induced aberrations. We present here the first implementation of wavefront-sensorless adaptive optics for a laser-free, aperture correlation, spinning disk microscope. This widefield method provides confocal-like optical sectioning through use of a patterned disk in the illumination and detection paths. Like other high-resolution microscopes, its operation is compromised by aberrations due to refractive index mismatch and variations within the specimen. Correction of such aberrations shows improved signal level, contrast and resolution.

Delirium risk of histamine-2 receptor antagonists and proton pump inhibitors: A study based on the adverse drug event reporting database in Japan.

OBJECTIVE: Although histamine-2 receptor antagonists (H2RAs) have been shown to be more likely to cause delirium than proton pump inhibitors (PPIs), these results were not adjusted for potential confounding factors. Accordingly, we investigated whether H2RAs and PPIs are risk factors for delirium, even when adjusting for other risk factors by analyzing adverse drug event reports compiled in the post-marketing stages of drugs provided by the Japanese regulatory authorities. METHOD: We analyzed 577,431 reports in the Japanese Adverse Drug Event Report database from April 2004 to July 2020. RESULTS: Of all reports analyzed, 2532 described delirium, and 574,899 described other adverse events. Delirium was associated with H2RAs (crude reporting odds ratio, ROR, 4.17; 95% CI, 3.34-5.22) but not PPIs (crude ROR 0.62; 95% CI 0.43-0.90). Even with adjustment for age, sex, history of dementia or depression, and concomitant drugs reported as risk factors for delirium, the use of H2RAs showed a significantly higher adjusted ROR than that of PPIs (H2RAs: adjusted ROR 3.99; 95% CI 3.18-5.01 and PPIs: adjusted ROR 0.58; 95%CI 0.40-0.84). CONCLUSIONS: These results suggest that, from a cognitive perspective, PPIs may be preferable to H2RAs for patients with or at risk for delirium.

Hyperspectral two-photon excitation microscopy using visible wavelength.

We demonstrate hyperspectral imaging by visible-wavelength two-photon excitation microscopy using line illumination and slit-confocal detection. A femtosecond pulsed laser light at 530 nm was used for the simultaneous excitation of fluorescent proteins with different emission wavelengths. The use of line illumination enabled efficient detection of hyperspectral images and achieved simultaneous detection of three fluorescence spectra in the observation of living HeLa cells with an exposure time of 1 ms per line, which is equivalent to about 2 µs per pixel in point scanning, with 160 data points per spectrum. On combining linear spectral unmixing techniques, localization of fluorescent probes in the cells was achieved. A theoretical investigation of the imaging property revealed high-depth discrimination property attained through the combination of nonlinear excitation and slit detection.

(-)-Epigallocatechin-3-gallate inhibits RANKL-induced osteoclastogenesis via downregulation of NFATc1 and suppression of HO-1-HMGB1-RAGE pathway.

Osteoporosis disturbs the balance of bone metabolism, and excessive bone resorption causes a decrease in bone density, thus increasing the risk of fracture. (-)-Epigallocatechin-3-gallate (EGCG) is the most abundant catechin contained in green tea. EGCG has a variety of pharmacological activities. Recently, it was reported that EGCG inhibits osteoclast differentiation, but the details of the mechanism underlying the EGCG-mediated suppression of osteoclastogenesis are unknown. In this study, we investigated the effects of EGCG on several signaling pathways in osteoclastogenesis. EGCG suppressed the expression of the nuclear factor of activated T cells cytoplasmic-1 (NFATc1), the master regulator of osteoclastogenesis. EGCG decreased the expression of cathepsin K, c-Src, and ATP6V0d2 and suppressed bone resorption. We also found that EGCG upregulated heme oxygenase-1 (HO-1) and suppressed the extracellular release of high-mobility group box 1 (HMGB1). In addition, EGCG decreased the expression of the receptor for advanced glycation end products (RAGE), which is the receptor of HMGB1, in osteoclastogenesis. In summary, our study showed that EGCG could inhibit osteoclast differentiation through the downregulation of NFATc1 and the suppression of the HO-1-HMGB1-RAGE pathway. EGCG might have the potential to be a lead compound that suppresses bone resorption in the treatment of osteoporosis.

Visible-wavelength two-photon excitation microscopy with multifocus scanning for volumetric live-cell imaging.

Two-photon excitation microscopy is one of the key techniques used to observe three-dimensional (3-D) structures in biological samples. We utilized a visible-wavelength laser beam for two-photon excitation in a multifocus confocal scanning system to improve the spatial resolution and image contrast in 3-D live-cell imaging. Experimental and numerical analyses revealed that the axial resolution has improved for a wide range of pinhole sizes used for confocal detection. We observed the 3-D movements of the Golgi bodies in living HeLa cells with an imaging speed of 2 s per volume. We also confirmed that the time-lapse observation up to 8 min did not cause significant cell damage in two-photon excitation experiments using wavelengths in the visible light range. These results demonstrate that multifocus, two-photon excitation microscopy with the use of a visible wavelength can constitute a simple technique for 3-D visualization of living cells with high spatial resolution and image contrast.