Birefringence and Orientation Direction Control of Photoalignable Liquid Crystalline Copolymer Films Based on In Situ Modification of Mesogenic Groups.

Controlling the birefringence of optical films is imperative to fabricate thin birefringent optical devices. Here, new photoalignable liquid crystalline copolymers (PLCPs) with 4-formylphneyl benzoate (PA) and cinnamic acid (CA) side groups are synthesized, which attain high photoreactivity and controllability of the orientation direction. Additionally, the exposed films are treated with 2-aminofluorene (AF) for an in situ condensation of PA with AF to form a Schiff base with a high inherent birefringence. Birefringence of the photoaligned film can be controlled up to 0.45 without disturbing the photoinduced orientation structure.

Corrigendum: ACE2 knockout hinders SARS-CoV-2 propagation in iPS cell-derived airway and alveolar epithelial cells.

[This corrects the article DOI: 10.3389/fcell.2023.1290876.].

The Uptake of Heparanase into Mast Cells Is Regulated by Its Enzymatic Activity to Degrade Heparan Sulfate.

Mast cells take up extracellular latent heparanase and store it in secretory granules. The present study examined whether the enzymatic activity of heparanase regulates its uptake efficiency. Recombinant mouse heparanase mimicking both the latent and mature forms (L-Hpse and M-Hpse, respectively) was internalized into mastocytoma MST cells, peritoneal cell-derived mast cells, and bone marrow-derived mast cells. The internalized amount of L-Hpse was significantly higher than that of M-Hpse. In MST cells, L-Hpse was continuously internalized for up to 8 h, while the uptake of M-Hpse was saturated after 2 h of incubation. L-Hpse and M-Hpse are similarly bound to the MST cell surface. The expression level of cell surface heparan sulfate was reduced in MST cells incubated with M-Hpse. The internalized amount of M-Hpse into mast cells was significantly increased in the presence of heparastatin (SF4), a small molecule heparanase inhibitor that does not affect the binding of heparanase to immobilized heparin. Enzymatically quiescent M-Hpse was prepared with a point mutation at Glu335. The internalized amount of mutated M-Hpse was significantly higher than that of wild-type M-Hpse but similar to that of wild-type and mutated L-Hpse. These results suggest that the enzymatic activity of heparanase negatively regulates the mast cell-mediated uptake of heparanase, possibly via the downregulation of cell surface heparan sulfate expression.

Correction: A novel cytoskeletal action of xylosides.

[This corrects the article DOI: 10.1371/journal.pone.0269972.].

Low-frequency transmission and persistence of antimicrobial-resistant bacteria and genes from livestock to agricultural soil and crops through compost application.

Livestock excrement is composted and applied to agricultural soils. If composts contain antimicrobial-resistant bacteria (ARB), they may spread to the soil and contaminate cultivated crops. Therefore, we investigated the degree of transmission of ARB and related antimicrobial resistance genes (ARGs) and, as well as clonal transmission of ARB from livestock to soil and crops through composting. This study was conducted at Rakuno Gakuen University farm in Hokkaido, Japan. Samples of cattle feces, solid and liquid composts, agricultural soil, and crops were collected. The abundance of Escherichia coli, coliforms, ß-lactam-resistant E. coli, and ß-lactam-resistant coliforms, as well as the copy numbers of ARG (specifically the bla gene related to ß-lactam-resistant bacteria), were assessed using qPCR through colony counts on CHROMagar ECC with or without ampicillin, respectively, 160 days after compost application. After the application of the compost to the soil, there was an initial increase in E. coli and coliform numbers, followed by a subsequent decrease over time. This trend was also observed in the copy numbers of the bla gene. In the soil, 5.0 CFU g-1 E. coli was detected on day 0 (the day post-compost application), and then, E. coli was not quantified on 60 days post-application. Through phylogenetic analysis involving single nucleotide polymorphisms (SNPs) and using whole-genome sequencing, it was discovered that clonal blaCTX-M-positive E. coli and blaTEM-positive Escherichia fergusonii were present in cattle feces, liquid compost, and soil on day 0 as well as 7 days post-application. This showed that livestock-derived ARB were transmitted from compost to soil and persisted for at least 7 days in soil. These findings indicate a potential low-level transmission of livestock-associated bacteria to agricultural soil through composts was observed at low frequency, dissemination was detected. Therefore, decreasing ARB abundance during composting is important for public health.

Design and fabrication of a liquid crystal polarization grating for mid- and far-infrared wavelengths.

A lot of research on liquid crystal polarization gratings (LCPGs) that can separate circularly polarized light with 100% diffraction efficiency has been conducted in the visible and near-infrared wavelength regions. In this paper, we tried to design and fabricate the LCPGs that are available for use in the mid- and far-infrared (MIR and FIR) wavelength regions. The materials for making LCPGs were selected in view of low absorption characteristics measured by the use of a Fourier-transform infrared (FT-IR) spectrometer. LCPGs designed for 3.88 µm and 9.5-10.6 µm were fabricated, and we evaluated their diffraction properties experimentally. The MIR and FIR LCPGs should open new application fields of LC technologies including polarimetry, spectroscopy, and beam steering.

Comparative evaluation of analytical pipelines for illumina short- and nanopore long-read 16S rRNA gene amplicon sequencing with mock microbial communities.

Utility of a recently developed long-read pipeline, Emu, was assessed using an expectation-maximization algorithm for accurate read classification. We compared it to conventional short- and long-read pipelines, using well-characterized mock bacterial samples. Our findings highlight the necessity of appropriate data-processing for taxonomic descriptions, expanding our understanding of the precise microbiome.

Harnessing a T1 Phage-Derived Spanin for Developing Phage-Based Antimicrobial Development.

The global increase in the prevalence of drug-resistant bacteria has necessitated the development of alternative treatments that do not rely on conventional antimicrobial agents. Using bacteriophage-derived lytic enzymes in antibacterial therapy shows promise; however, a thorough comparison and evaluation of their bactericidal efficacy are lacking. This study aimed to compare and investigate the bactericidal activity and spectrum of such lytic enzymes, with the goal of harnessing them for antibacterial therapy. First, we examined the bactericidal activity of spanins, endolysins, and holins derived from 2 Escherichia coli model phages, T1 and T7. Among these, T1-spanin exhibited the highest bactericidal activity against E. coli. Subsequently, we expressed T1-spanin within bacterial cells and assessed its bactericidal activity. T1-spanin showed potent bactericidal activity against all clinical isolates tested, including bacterial strains of 111 E. coli, 2 Acinetobacter spp., 3 Klebsiella spp., and 3 Pseudomonas aeruginosa. In contrast, T1 phage-derived endolysin showed bactericidal activity against E. coli and P. aeruginosa, yet its efficacy against other bacteria was inferior to that of T1-spanin. Finally, we developed a phage-based technology to introduce the T1-spanin gene into target bacteria. The synthesized non-proliferative phage exhibited strong antibacterial activity against the targeted bacteria. The potent bactericidal activity exhibited by spanins, combined with the novel phage synthetic technology, holds promise for the development of innovative antimicrobial agents.

Near-infrared hyperspectral circular polarization imaging and object classification with machine learning.

We constructed a hyperspectral circular polarization (S3) imaging system in the near-infrared (NIR) region comprising a circularly polarized broadband light source, a polarization grating, and a commercial hyperspectral camera. With this system, we captured hyperspectral S3 images of plastic samples. We then demonstrated the classification with machine learning and found that the hyperspectral S3 images showed higher classification precision than the conventional NIR hyperspectral images. This result indicates that the hyperspectral S3 imaging has potential for object classification even for samples with similar absorption spectra. This hyperspectral S3 imaging system can be applied in garbage classification in recycling plants.

Direct visualization of ribosomes in the cell-free system revealed the functional evolution of aminoglycoside.

The rapid emergence of multi-drug-resistant bacteria has raised a serious public health concern. Therefore, new antibiotic developments have been highly desired. Here, we propose a new method to visualize antibiotic actions on translating ribosomes in the cell-free system under macromolecular crowding conditions by cryo-electron microscopy, designated as the DARC method: the Direct visualization of Antibiotic binding on Ribosomes in the Cell-free translation system. This new method allows for acquiring a more comprehensive understanding of the mode of action of antibiotics on the translation inhibition without ribosome purification. Furthermore, with the direct link to biochemical analysis at the same condition as cryo-EM observation, we revealed the evolution of 2-DOS aminoglycosides from dibekacin (DBK) to arbekacin (ABK) by acquiring the synthetic tailored anchoring motif to lead to stronger binding affinity to ribosomes. Our cryo-EM structures of DBK and ABK bound ribosomes in the cell-free environment clearly depicted a synthetic tailored γ-amino-α-hydroxybutyryl (HABA) motif formed additional interactions with the ribosome enhancing antibiotic bindings. This new approach would be valuable for understanding the function of antibiotics for more efficient drug development.

Design of two-dimensional multilevel optical anisotropic diffraction gratings with a generative adversarial network.

This study uses a generative adversarial network to design multilevel optical anisotropic diffraction gratings with specific customizable characteristics. As input, this method uses the far electric field of polarization and intensity in each diffracted light through the gratings to design. Using the finite-difference time-domain method, the designed structures are numerically evaluated, confirming that they can be created with the intended parameters. Multilevel optical anisotropic diffraction gratings created this way can be used in various fields to develop improved optical elements.

Mobile class A ß-lactamase gene bla GMA-1.

IMPORTANCE: Despite increasing reports, class A ß-lactamases of environmental bacteria remain very poorly characterized, with limited understanding of their transmission patterns. To address this knowledge gap, we focused on a recently designated GMA family ß-lactamase gene, bla GMA-1, found in marine bacterial genera such as Vibrio. This study shows that gammaproteobacterial mobile class A ß-lactamase is specialized for penicillin degradation, and bla GMA-1 is frequently linked to strand-biased circularizing integrative elements (SEs) in sequences in the RefSeq/GenBank database. Evidence for the implication of SEs in ß-lactamase environmental transmission provides insights for future surveillance studies of antimicrobial resistance genes in human clinical settings.

Target degradation specificity of phytoplasma effector phyllogen is regulated by the recruitment of host proteasome shuttle protein.

Phytoplasmas infect a wide variety of plants and can cause distinctive symptoms including the conversion of floral organs into leaf-like organs, known as phyllody. Phyllody is induced by an effector protein family called phyllogens, which interact with floral MADS-box transcription factors (MTFs) responsible for determining the identity of floral organs. The MTF/phyllogen complex then interacts with the proteasomal shuttle protein RADIATION SENSITIVE23 (RAD23), which facilitates delivery of the MTF/phyllogen complex to the host proteasome for MTF degradation. Previous studies have indicated that the MTF degradation specificity of phyllogens is determined by their ability to bind to MTFs. However, in the present study, we discovered a novel mechanism determining the degradation specificity through detailed functional analyses of a phyllogen homologue of rice yellow dwarf phytoplasma (PHYLRYD ). PHYLRYD degraded a narrower range of floral MTFs than other phyllody-inducing phyllogens, resulting in compromised phyllody phenotypes in plants. Interestingly, PHYLRYD was able to bind to some floral MTFs that PHYLRYD was unable to efficiently degrade. However, the complex of PHYLRYD and the non-degradable MTF could not interact with RAD23. These results indicate that the MTF degradation specificity of PHYLRYD is correlated with the ability to form the MTF/PHYLRYD /RAD23 ternary complex, rather than the ability to bind to MTF. This study elucidated that phyllogen target specificity is regulated by both the MTF-binding ability and RAD23 recruitment ability of the MTF/phyllogen complex.

National genomic surveillance integrating standardized quantitative susceptibility testing clarifies antimicrobial resistance in Enterobacterales.

Antimicrobial resistance is a global health concern; Enterobacterales resistant to third-generation cephalosporins (3GCs) and carbapenems are of the highest priority. Here, we conducted genome sequencing and standardized quantitative antimicrobial susceptibility testing of 4,195 isolates of Escherichia coli and Klebsiella pneumoniae resistant to 3GCs and Enterobacterales with reduced meropenem susceptibility collected across Japan. Our analyses provided a complete classification of 3GC resistance mechanisms. Analyses with complete reference plasmids revealed that among the blaCTX-M extended-spectrum ß-lactamase genes, blaCTX-M-8 was typically encoded in highly similar plasmids. The two major AmpC ß-lactamase genes were blaCMY-2 and blaDHA-1. Long-read sequencing of representative plasmids revealed that approximately 60% and 40% of blaCMY-2 and blaDHA-1 were encoded by such plasmids, respectively. Our analyses identified strains positive for carbapenemase genes but phenotypically susceptible to carbapenems and undetectable by standard antimicrobial susceptibility testing. Systematic long-read sequencing enabled reconstruction of 183 complete plasmid sequences encoding three major carbapenemase genes and elucidation of their geographical distribution stratified by replicon types and species carrying the plasmids and potential plasmid transfer events. Overall, we provide a blueprint for a national genomic surveillance study that integrates standardized quantitative antimicrobial susceptibility testing and characterizes resistance determinants.

ACE2 knockout hinders SARS-CoV-2 propagation in iPS cell-derived airway and alveolar epithelial cells.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, continues to spread around the world with serious cases and deaths. It has also been suggested that different genetic variants in the human genome affect both the susceptibility to infection and severity of disease in COVID-19 patients. Angiotensin-converting enzyme 2 (ACE2) has been identified as a cell surface receptor for SARS-CoV and SARS-CoV-2 entry into cells. The construction of an experimental model system using human iPS cells would enable further studies of the association between viral characteristics and genetic variants. Airway and alveolar epithelial cells are cell types of the lung that express high levels of ACE2 and are suitable for in vitro infection experiments. Here, we show that human iPS cell-derived airway and alveolar epithelial cells are highly susceptible to viral infection of SARS-CoV-2. Using gene knockout with CRISPR-Cas9 in human iPS cells we demonstrate that ACE2 plays an essential role in the airway and alveolar epithelial cell entry of SARS-CoV-2 in vitro. Replication of SARS-CoV-2 was strongly suppressed in ACE2 knockout (KO) lung cells. Our model system based on human iPS cell-derived lung cells may be applied to understand the molecular biology regulating viral respiratory infection leading to potential therapeutic developments for COVID-19 and the prevention of future pandemics.

Intracellular polyamine depletion induces N-linked galactosylation of the monoclonal antibody produced by CHO DP-12 cells.

The heterogeneity of the N-linked glycan profile of therapeutic monoclonal antibodies (mAbs) derived from animal cells affects therapeutic efficacy and, therefore, needs to be appropriately controlled during the manufacturing process. In this study, we examined the effects of polyamines on the N-linked glycan profiles of mAbs produced by CHO DP-12 cells. Normal cell growth of CHO DP-12 cells and their growth arrest by α-difluoromethylornithine (DFMO), an inhibitor of the polyamine biosynthetic pathway, was observed when 0.5% fetal bovine serum was added to serum-free medium, despite the presence of cadaverine and aminopropylcadaverine, instead of putrescine and spermidine in cells. Polyamine depletion by DFMO increased IgG galactosylation, accompanied by ß1,4-galactosyl transferase 1 (B4GAT1) mRNA elevation. Additionally, IgG production in polyamine-depleted cells was reduced by 30% compared to that in control cells. Therefore, we examined whether polyamine depletion induces an ER stress response. The results indicated increased expression levels of chaperones for glycoprotein folding in polyamine-depleted cells, suggesting that polyamine depletion causes ER stress related to glycoprotein folding. The effect of tunicamycin, an ER stress inducer that inhibits N-glycosylation, on the expression of B4GALT1 mRNA was examined. Tunicamycin treatment increased B4GALT1 mRNA expression. These results suggest that ER stress caused by polyamine depletion induces B4GALT1 mRNA expression, resulting in increased IgG galactosylation in CHO cells. Thus, introducing polyamines, particularly SPD, to serum-free CHO culture medium for CHO cells may contribute to consistent manufacturing and quality control of antibody production.

Protocol for detecting Helicobacter suis infection in gastric biopsies and serum by PCR and ELISA.

Infection with Helicobacter suis, which causes many cases of gastric disease, is not reliably diagnosed. Here, we present a protocol for detecting H. suis infection. We describe steps for collecting gastric biopsies and sera from patients, preparing DNA for PCR, and targeting the H. suis-specific gene. We then define procedures for inoculating biopsies onto primary agar plates and transferring colonies to secondary agar plates. Finally, we detail whole-genome sequencing of bacteria and assess H. suis infection in sera with ELISA. For complete details on the use and execution of these protocols, please refer to Matsui et al.1.

Dielectrophoretic trapping of nanosized biomolecules on plasmonic nanohole arrays for biosensor applications: simple fabrication and visible-region detection.

Surface plasmon resonance is an optical phenomenon that can be applied for label-free, real-time sensing to directly measure biomolecular interactions and detect biomarkers in solutions. Previous studies using plasmonic nanohole arrays have monitored and detected various biomolecules owing to the propagating surface plasmon polaritons (SPPs). Extraordinary optical transmission (EOT) that occurs in the near-infrared (NIR) and infrared (IR) regions is usually used for detection. Although these plasmonic nanohole arrays improve the sensitivity and throughput for biomolecular detection, these arrays have the following disadvantages: (1) molecular diffusion in the solution (making the detection of biomolecules difficult), (2) the device fabrication's complexities, and (3) expensive equipments for detection in the NIR or IR regions. Therefore, there is a need to fabricate plasmonic nanohole arrays as biomolecular detection platforms using a simple and highly reproducible procedure based on other SPP modes in the visible region instead of the EOT in the NIR or IR regions while suppressing molecular diffusion in the solution. In this paper, we propose the combination of a polymer-based gold nanohole array (Au NHA) obtained through an easy process as a simple platform and dielectrophoresis (DEP) as a biomolecule manipulation method. This approach was experimentally demonstrated using SPP and LSPR modes (not EOT) in the visible region and simple, label-free, rapid, cost-effective trapping and enrichment of nanoparticles (trapping time: <50 s) and bovine serum albumin (trapping time: <1000 s) was realized. These results prove that the Au NHA-based DEP devices have great potential for real-time digital and Raman bioimaging, in addition to biomarker detection.

Complete genome constellations of two bovine rotavirus A strains isolated in Japan reveal a unique T9 NSP3 genotype.

Full genome sequencing of two bovine rotavirus A (RVA) strains isolated in Japan in 2019 revealed two genotype constellations; one had a constellation of G8-P[1]-I2-R2-C2-M2-A3-N2-T9-E2-H3. Thereupon, genotype T9 carried by RVA/Bovine-tc/JPN/AH1041/2022/G8P[1], constitutes a rare NSP3 genotype, and only two unusual Japanese bovine RVA strains have thus far been reported to carry this genotype. The other RVA/Bovine-tc/JPN/AH1207/2022/G6P[5] strain possessed a constellation of G6-P[5]-I2-R2-C2-M2-A3-N2-T6-E2-H3. Phylogenetic analyses indicate that the majority of gene segments were most closely related to Japanese bovine RVAs, suggesting that both strains might have derived through multiple reassortment events from RVA strains circulating within Japanese cattle. The emergence of RVA strains in Japan and their reassortment with locally circulating atypical RVAs could have implications for current vaccination strategies.