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Purpose This study aimed to investigate the physicochemical factors contributing to stable co-amorphous formations and to design a co-former selection strategy. Methods Non-steroidal inflammatory drugs were used as main components and/or co-formers. Physical mixtures of the materials were melted. Co-amorphization was characterized by the inhibition effect of the co-former on crystallization of the main component from the undercooled melt. The contribution of physicochemical factors to the co-amorphous formation was analyzed by multivariate analysis. Co-amorphous samples prepared by melting were subjected to thermal and spectroscopic analyses and the isothermal crystallization test. Results Naproxen (NAP) was employed as the main component having a rapid crystallization tendency. Some materials used as the co-former inhibited the crystallization of amorphous NAP; decreasing melting temperatures of the components was an indicator of co-amorphization. The contribution of some physicochemical features (e.g., crystallization tendency, glass transition temperature ( Tg )/melting temperature and molecular flexibility) of the co-formers to a co-amorphous formation was suggested by multivariate analysis. Deviation of the glass transition temperature from the theoretical value and changes in the infrared spectra of the co-amorphous samples were correlated with intermolecular interaction. The crystallization behaviors of the co-amorphous samples depended on their Tg . Conclusions The results showed a relationship between stable co-amorphous formation and the physicochemical features of the components, which should inform efficient co-former selection to design stable co-amorphous formations.

Background Endoscopy is an important form of clinical gastroenterology education because it gives students the opportunity to learn about diagnosis procedures and even treatment. During the COVID-19 pandemic, medical students were observed from outside the endoscopy room due to the risk of airborne infection. In this study, we investigated the efficacy of combining endoscopy education with doctor’s-eye-view videos of the procedure obtained using live-action cameras (GoPro®). Methods From February to May 2021, endoscopists wore GoPro Hero8 cameras on their heads to display a doctor’s-eye view video outside the room. The efficacy of the GoPro videos in combination with endoscopic monitoring was evaluated by 15 participating medical students. The participants rated the efficacy on a 5-point scale and commented on the positive and negative points. Results A total of 78.6% of participants evaluated the GoPro as good; 57.2% answered that it increased their understanding, with 71.4% stating that it increased their understanding of procedures in particular. A total of 85.7% of the students answered that their interest in endoscopy had increased, and 85.7% evaluated the benefit of the GoPro videos as good. In addition, 64.3% answered that the method was effective in preventing COVID-19 infection. Education using GoPro videos enabled students to feel as if they were conducting the endoscopy themselves and enabled them to concentrate on learning. Conclusions Practical endoscopic education using a GoPro is an effective educational tool that not only increases understanding of endoscopic practice but also stimulates students’ interest and awareness of their future as doctors.

CRISPR/Cas9-mediated genome editing is a next-generation strategy for genetic modifications, not only for single gene targeting, but also for multiple targeted mutagenesis. To make the most of the multiplexity of CRISPR/Cas9, we established a system for constructing all-in-one expression vectors containing multiple guide RNA expression cassettes and a Cas9 nuclease/nickase expression cassette. We further demonstrated successful examples of multiple targeting including chromosomal deletions in human cells using the all-in-one CRISPR/Cas9 vectors constructed with our novel system. Our system provides an efficient targeting strategy for multiplex genome/epigenome editing, simultaneous activation/repression of multiple genes and beyond.

Background Aggressive lipid lowering by high-dose statin treatment has been established for the secondary prevention of coronary artery disease (CAD). Regarding the low-density lipoprotein cholesterol (LDL-C) level, however, the “The lower is the better” concept has been controversial to date. We hypothesized that there is an optimal LDL-C level, i.e., a “threshold” value, below which the incidence of cardiovascular events is no longer reduced. We undertook a subanalysis of the REAL-CAD study to explore whether such an optimal target LDL-C level exists by a novel analysis procedure to verify the existence of a monotonic relationship. Methods For a total of 11,105 patients with CAD enrolled in the REAL-CAD study, the LDL-C level at 6 months after randomization and 5-year cardiovascular outcomes were assessed. We set the “threshold” value of the LDL-C level under which the hazards were assumed to be constant, by including an artificial covariate max (0, LDL-C − threshold) in the Cox model. The analysis was repeated with different LDL-C thresholds (every 10 mg/dl from 40 to 100 mg/dl) and the model fit was assessed by log-likelihood. Results For primary outcomes such as the composite of cardiovascular death, non-fatal myocardial infarction, non-fatal ischemic stroke, and unstable angina requiring emergency hospitalization, the model fit assessed by log-likelihood was best when a threshold LDL-C value of 70 mg/dl was assumed. And in the model with a threshold LDL-C ≥ 70 mg/dl, the hazard ratio was 1.07 (95% confidence interval 1.01–1.13) as the LDL-C increased by 10 mg/dl. Therefore, the risk of cardiovascular events decreased monotonically until the LDL-C level was lowered to 70 mg/dl, but when the level was further reduced, the risk was independent of LDL-C. Conclusions Our analysis model suggests that a “threshold” value of LDL-C might exist for the secondary prevention of cardiovascular events in Japanese patients with CAD, and this threshold might be 70 mg/dl for primary composite outcomes. Trial registration http://www.clinicaltrials.gov . Unique identifier: NCT01042730.

Genome engineering using programmable nucleases enables homologous recombination (HR)-mediated gene knock-in. However, the labour used to construct targeting vectors containing homology arms and difficulties in inducing HR in some cell type and organisms represent technical hurdles for the application of HR-mediated knock-in technology. Here, we introduce an alternative strategy for gene knock-in using transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) mediated by microhomology-mediated end-joining, termed the PITCh (Precise Integration into Target Chromosome) system. TALEN-mediated PITCh, termed TAL-PITCh, enables efficient integration of exogenous donor DNA in human cells and animals, including silkworms and frogs. We further demonstrate that CRISPR/Cas9-mediated PITCh, termed CRIS-PITCh, can be applied in human cells without carrying the plasmid backbone sequence. Thus, our PITCh-ing strategies will be useful for a variety of applications, not only in cultured cells, but also in various organisms, including invertebrates and vertebrates. One challenge facing the use of programmable nucleases in genome engineering is the requirement for homologous recombination. Here, Nakade et al. harness microhomology-mediated end-joining as a means of inserting exogenous coding sequences into the genome using both TALEN and CRISPR/Cas9 technologies.

The CRISPR/Cas9 system provides a powerful tool for genome editing in various model organisms, including zebrafish. The establishment of targeted gene-disrupted zebrafish (knockouts) is readily achieved by CRISPR/Cas9-mediated genome modification. Recently, exogenous DNA integration into the zebrafish genome via homology-independent DNA repair was reported, but this integration contained various mutations at the junctions of genomic and integrated DNA. Thus, precise genome modification into targeted genomic loci remains to be achieved. Here, we describe efficient, precise CRISPR/Cas9-mediated integration using a donor vector harbouring short homologous sequences (10–40 bp) flanking the genomic target locus. We succeeded in integrating with high efficiency an exogenous mCherry or eGFP gene into targeted genes ( tyrosinase and krtt1c19e ) in frame. We found the precise in-frame integration of exogenous DNA without backbone vector sequences when Cas9 cleavage sites were introduced at both sides of the left homology arm, the eGFP sequence and the right homology arm. Furthermore, we confirmed that this precise genome modification was heritable. This simple method enables precise targeted gene knock-in in zebrafish.

The advent of image-activated cell sorting and imaging-based cell picking has advanced our knowledge and exploitation of biological systems in the last decade. Unfortunately, they generally rely on fluorescent labeling for cellular phenotyping, an indirect measure of the molecular landscape in the cell, which has critical limitations. Here we demonstrate Raman image-activated cell sorting by directly probing chemically specific intracellular molecular vibrations via ultrafast multicolor stimulated Raman scattering (SRS) microscopy for cellular phenotyping. Specifically, the technology enables real-time SRS-image-based sorting of single live cells with a throughput of up to ~100 events per second without the need for fluorescent labeling. To show the broad utility of the technology, we show its applicability to diverse cell types and sizes. The technology is highly versatile and holds promise for numerous applications that are previously difficult or undesirable with fluorescence-based technologies. Most current cell sorting methods are based on fluorescence detection with no imaging capability. Here the authors generate and use Raman image-activated cell sorting with a throughput of around 100 events per second, providing molecular images with no need for labeling.

Combining the strength of flow cytometry with fluorescence imaging and digital image analysis, imaging flow cytometry is a powerful tool in diverse fields including cancer biology, immunology, drug discovery, microbiology, and metabolic engineering. It enables measurements and statistical analyses of chemical, structural, and morphological phenotypes of numerous living cells to provide systematic insights into biological processes. However, its utility is constrained by its requirement of fluorescent labeling for phenotyping. Here we present label-free chemical imaging flow cytometry to overcome the issue. It builds on a pulse pair-resolved wavelength-switchable Stokes laser for the fastest-to-date multicolor stimulated Raman scattering (SRS) microscopy of fast-flowing cells on a 3D acoustic focusing microfluidic chip, enabling an unprecedented throughput of up to ∼140 cells/s. To show its broad utility, we use the SRS imaging flow cytometry with the aid of deep learning to study the metabolic heterogeneity of microalgal cells and perform marker-free cancer detection in blood.

Catheter ablation is an important non-pharmacological intervention for atrial fibrillation (AF), but its effect on the incidence of asymptomatic cerebral emboli and long-term effects on cognitive function remain unknown. We prospectively enrolled 101 patients who underwent AF ablation. Brain magnetic resonance imaging (MRI) (72 patients) and neuropsychological assessments (66 patients) were performed 1–3 days (baseline) and 6 months after ablation. Immediately after ablation, diffusion-weighted MRI and 3-dimensional double inversion recovery (3D-DIR) detected embolic microinfarctions in 63 patients (87.5%) and 62 patients (86.1%), respectively. After 6 months, DIR lesions disappeared in 41 patients. Microbleeds (MBs) increased by 17%, and 65% of the de novo MBs were exactly at the same location as the microinfarctions. Average Mini-Mental State Examination scores improved from 27.9 ± 2.4 to 28.5 ± 1.7 ( p  = 0.037), and detailed neuropsychological assessment scores showed improvement in memory, constructional, and frontal lobe functions. Ejection fraction, left atrial volume index and brain natriuretic peptide level improved from baseline to 3–6 months after ablation. Despite incidental microemboli, cognitive function was preserved 6 months after ablation.

ATF6α is an endoplasmic reticulum (ER)-embedded transcription factor which is rapidly activated by ER stress, and a major regulator of ER chaperone levels in vertebrates. We previously suggested that ATF6α occurs as a monomer, dimer and oligomer in the unstressed ER of Chinese hamster ovary cells due to the presence of two evolutionarily conserved cysteine residues in its luminal region (C467 and C618), and showed that ATF6α is reduced upon ER stress, such that only reduced monomer ATF6α is translocated to the Golgi apparatus for activation by proteolysis. However, mutagenesis analysis (C467A and C618A) revealed that the C618A mutant behaves in an unexpected manner (monomer and oligomer) during non-reducing SDS-PAGE, for reasons which remained unclear. Here, we used human colorectal carcinoma-derived HCT116 cells deficient in ATF6α and its relevant ATF6β, and found that ATF6α dimer and oligomer are both dimers, which we designated C618-dimer and C467-dimer, respectively. We demonstrated that C467-dimer (previously considered an oligomer) behaved bigger than C618-dimer (previously considered a dimer) during non-reducing SDS-PAGE, based on their disulfide-bonded structures. Furthermore, ATF6α monomer physically associates with another ATF6α monomer in the absence of disulfide bonding, which renders two C467 residues in close proximity so that formation of C467-dimer is much easier than that of C618-dimer. In contrast, C618-dimer is more easily reduced upon ER stress. Thus, our analysis revealed that all forms of ATF6α, namely monomer, C618-dimer and C467-dimer, are activated by single reduction of a disulfide bond in response to ER stress, ensuring the rapidity of ATF6α activation.Key words: disulfide-bonded structure, endoplasmic reticulum, membrane-bound transcription factor, non-reducing SDS-PAGE, unfolded protein response