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ABSTRACTAtg8 and Atg12 are ubiquitin-like proteins, conjugated to phosphatidylethanolamine (PE) and Atg5, respectively, through enzymatic reactions similar to ubiquitylation. The resultant Atg8–PE and Atg12–Atg5 conjugates play crucial roles in autophagy. Structural studies have been extensively performed on all Atg proteins (Atg3, Atg4, Atg5, Atg7, Atg8, Atg10, Atg12, Atg16) involved in these conjugation systems. This review summarizes structural studies and discusses mechanisms of conjugation and deconjugation reactions, as well as autophagic functions of the Atg8 and Atg12 conjugation systems.

Summary: High-speed atomic force microscopy is a technique that allows real-time observation of biomolecules and biological phenomena reconstituted on a substrate. Here, we present a protocol for immobilizing lipid nanorods onto two-dimensional crystals of biotin-binding protein tamavidin 2. We describe steps for the preparation of tamavidin 2 protein, lipid nanorods, and two-dimensional crystals of tamavidin 2 formed on mica. Immobilized lipid nanorods are one of the useful tools for observation of specific proteins in action.For complete details on the use and execution of this protocol, please refer to Fukuda et al. (2023).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Yeast mitofissin Atg44 is a mitochondrial intermembrane space protein that causes membrane fission required for mitophagy. Here, we present a protocol for observing Atg44-mediated membrane fission. We describe steps for recombinant Atg44 purification, lipid nanotube preparation as model membranes, and Atg44-mediated membrane fission real-time observation. We then detail procedures for tube radius estimation using confocal microscopy. This protocol can also be adapted to the study of membrane fission by other proteins.For complete details on the use and execution of this protocol, please refer to Fukuda et al. (2023).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

The Atg8 family of ubiquitin-like proteins play pivotal roles in autophagy and other processes involving vesicle fusion and transport where the lysosome/vacuole is the end station. Nuclear roles of Atg8 proteins are also emerging. Here, we review the structural and functional features of Atg8 family proteins and their protein-protein interaction modes in model organisms such as yeast, Arabidopsis, C. elegans and Drosophila to humans. Although varying in number of homologs, from one in yeast to seven in humans, and more than ten in some plants, there is a strong evolutionary conservation of structural features and interaction modes. The most prominent interaction mode is between the LC3 interacting region (LIR), also called Atg8 interacting motif (AIM), binding to the LIR docking site (LDS) in Atg8 homologs. There are variants of these motifs like “half-LIRs” and helical LIRs. We discuss details of the binding modes and how selectivity is achieved as well as the role of multivalent LIR-LDS interactions in selective autophagy. A number of LIR-LDS interactions are known to be regulated by phosphorylation. New methods to predict LIR motifs in proteins have emerged that will aid in discovery and analyses. There are also other interaction surfaces than the LDS becoming known where we presently lack detailed structural information, like the N-terminal arm region and the UIM-docking site (UDS). More interaction modes are likely to be discovered in future studies.

The UFM1 system, a ubiquitin-like conjugation system is crucial for endoplasmic reticulum (ER) homeostasis. Here, authors found that CYB5R3 is covalently conjugated with UFM1, which becomes a signal for ER-phagy, a selective autophagy of ER.

MEK1 mutations are found in cancer and RASopathies, but their effects remain unclear. Here, the authors reveal a mutant MEK1 structure and qualitative differences in biological properties between the cancer- and RASopathy-associated mutants, providing insights into the pathophysiology, diagnosis, and treatment of these diseases.

Tanigawa et al. investigate the role of Pib2, a vacuolar protein, in the glutamine-dependent upstream regulation of TORC1 in budding yeast. Their study reveals that Pib2 plays dual roles as a glutamine sensor and a direct TORC1 activator, a finding useful for the community working on TORC1 upstream regulation.

Liquid-liquid phase separation of p62/SQSTM1 has been previously described, although the significance in vivo remains unclear. Here the authors show p62 droplets contain ubiquitin, autophagy-related proteins and Keap1 to serve as platform of not only autophagosome formation but also Nrf2 activation.

The ER is subject to autophagy (ER-phagy) for turnover, with Atg40 acting as a receptor to sequester ER with Atg8 in autophagosomes. Here, the authors show that Atg40 is clustered by interaction with Atg8 to generate local membrane curvature and promote autophagosome packing.

Selective autophagy mediates the degradation of various cargoes, including protein aggregates and organelles, thereby contributing to cellular homeostasis. Cargo receptors ensure selectivity by tethering specific cargo to lipidated Atg8 at the isolation membrane. However, little is known about the structural requirements underlying receptor-mediated cargo recognition. Here, we report structural, biochemical, and cell biological analysis of the major selective cargo protein in budding yeast, aminopeptidase I (Ape1), and its complex with the receptor Atg19. The Ape1 propeptide has a trimeric coiled-coil structure, which tethers dodecameric Ape1 bodies together to form large aggregates. Atg19 disassembles the propeptide trimer and forms a 2:1 heterotrimer, which not only blankets the Ape1 aggregates but also regulates their size. These receptor activities may promote elongation of the isolation membrane along the aggregate surface, enabling sequestration of the cargo with high specificity.

The ubiquitin-like protein Atg8, in its lipidated form, plays central roles in autophagy. Yet, remarkably, Atg8 also carries out lipidation-independent functions in non-autophagic processes. How Atg8 performs its moonlighting roles is unclear. Here we report that in the fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae, the lipidation-independent roles of Atg8 in maintaining normal morphology and functions of the vacuole require its interaction with a vacuole membrane protein Hfl1 (homolog of human TMEM184 proteins). Crystal structures revealed that the Atg8-Hfl1 interaction is not mediated by the typical Atg8-family-interacting motif (AIM) that forms an intermolecular β-sheet with Atg8. Instead, the Atg8-binding regions in Hfl1 proteins adopt a helical conformation, thus representing a new type of AIMs (termed helical AIMs here). These results deepen our understanding of both the functional versatility of Atg8 and the mechanistic diversity of Atg8 binding.

Damaged mitochondria are selectively eliminated by mitophagy. Parkin and PINK1, gene products mutated in familial Parkinson’s disease, play essential roles in mitophagy through ubiquitination of mitochondria. Cargo ubiquitination by E3 ubiquitin ligase Parkin is important to trigger selective autophagy. Although autophagy receptors recruit LC3-labeled autophagic membranes onto damaged mitochondria, how other essential autophagy units such as ATG9A-integrated vesicles are recruited remains unclear. Here, using mammalian cultured cells, we demonstrate that RABGEF1, the upstream factor of the endosomal Rab GTPase cascade, is recruited to damaged mitochondria via ubiquitin binding downstream of Parkin. RABGEF1 directs the downstream Rab proteins, RAB5 and RAB7A, to damaged mitochondria, whose associations are further regulated by mitochondrial Rab-GAPs. Furthermore, depletion of RAB7A inhibited ATG9A vesicle assembly and subsequent encapsulation of the mitochondria by autophagic membranes. These results strongly suggest that endosomal Rab cycles on damaged mitochondria are a crucial regulator of mitophagy through assembling ATG9A vesicles.

Macroautophagy (autophagy) is a bulk protein-degradation system ubiquitously conserved in eukaryotic cells. During autophagy, cytoplasmic components are enclosed in a membrane compartment, called an autophagosome. The autophagosome fuses with the vacuole/lysosome and is degraded together with its cargo. Because autophagy is important for the maintenance of cellular homeostasis by degrading unwanted proteins and organelles, identification of autophagosome cargo proteins (i.e., the targets of autophagy) will aid in understanding the physiological roles of autophagy. In this study, we developed a method for monitoring intact autophagosomes ex vivo by detecting the fluorescence of GFP-fused aminopeptidase I, the best-characterized selective cargo of autophagosomes in Saccharomyces cerevisiae. This method facilitated optimization of a biochemical procedure to fractionate autophagosomes. A combination of LC-MS/MS with subsequent statistical analyses revealed a list of autophagosome cargo proteins; some of these are selectively enclosed in autophagosomes and delivered to the vacuole in an Atg11-independent manner. The methods we describe will be useful for analyzing the mechanisms and physiological significance of Atg11-independent selective autophagy.

A região do Alto Solimões é muito rica em recursos genéticos agrícolas, especialmente frutícolas, mas estes recursos contribuem principalmente para a subsistência em lugar de ter um papel mais amplo no desenvolvimento regional. Um levantamento qualitativo preliminar das espécies frutícolas nativas e exóticas foi executado em 11 comunidades rurais (8 caboclas, 3 Ticunas) nos ecossistemas principais (várzea, terra firme). Observou-se 37 fruteiras nativas em 20 famílias e 10 fruteiras exóticas em 9 famílias; das nativas, 9 foram raras (observadas em 1 ou 2 comunidades) e 3 foram comuns (observadas em 10 ou 11 comunidades); das exóticas, 2 foram raras e as bananas foram encontradas em todas as comunidades, sendo a fruteira mais importante na subsistência e na comercialização. As comunidades com mais acesso imediato à várzea e à terra firme tiveram mais espécies (22,5±6,6 nativas e 6,5±2,2 exóticas), que comunidades com acesso apenas à terra firme (19,0±2,2 nativas e 5,3±0,5 exóticas), que por sua vez tiveram mais espécies que comunidades com acesso apenas à várzea (5±2 nativas e 1,5±0,9 exóticas).

Summary The control of dynamic deformation of a laminate plate is conducted by applying electrical load to a piezoelectric actuator integrated into the laminate. The dynamic behavior of the laminate is analyzed in the paper taking into account the effect of transverse shear. The analytical model of the laminate is composed of fiber-reinforced laminae and piezoelectric layers constituting a symmetric cross-ply laminate rectangular plate with simply-supported egdes. It is subjected to mechanical and electrical loads acting on the piezoelectric actuator in order to compensate the effect of the mechanical loads. The behavior of the laminate is analyzed based on the first-order shear deformation theory. Closed-form solutions are obtained for the following quantities: (1) natural frequencies of the laminate plate, (2) weight functions for the deflections and rotations and (3) transient deflections due to loads varying arbitrarily with time. Illustrative examples are shown for the control of deflections caused by the mechanical loads by means of electrical voltage applied to the piezoelectric actuators. [ABSTRACT FROM AUTHOR]

The diversity of glycerophospholipid species in cellular membranes is immense and affects various biological functions. Glycerol-3-phosphate acyltransferases (GPATs) and lysophospholipid acyltransferases (LPLATs), in concert with phospholipase A1/2s enzymes, contribute to this diversity via selective esterification of fatty acyl chains at the sn-1 or sn-2 positions of membrane phospholipids. These enzymes are conserved across all kingdoms, and in mammals four GPATs of the 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) family and at least 14 LPLATs, either of the AGPAT or the membrane-bound O-acyltransferase (MBOAT) families, have been identified. Here we provide an overview of the biochemical and biological activities of these mammalian enzymes, including their predicted structures, involvements in human diseases, and essential physiological roles as revealed by gene-deficient mice. Recently, the nomenclature used to refer to these enzymes has generated some confusion due to the use of multiple names to refer to the same enzyme and instances of the same name being used to refer to completely different enzymes. Thus, this review proposes a more uniform LPLAT enzyme nomenclature, as well as providing an update of recent advances made in the study of LPLATs, continuing from our JBC mini review in 2009. [ABSTRACT FROM AUTHOR]

Several amyotrophic lateral sclerosis (ALS)-related proteins such as FUS, TDP-43, and hnRNPA1 demonstrate liquid-liquid phase separation, and their disease-related mutations correlate with a transition of their liquid droplet form into aggregates. Missense mutations in SQSTM1/p62, which have been identified throughout the gene, are associated with ALS, frontotemporal degeneration (FTD), and Paget's disease of bone. SQSTM1/p62 protein forms liquid droplets through interaction with ubiquitinated proteins, and these droplets serve as a platform for autophagosome formation and the antioxidative stress response via the LC3-interacting region (LIR) and KEAP1-interacting region (KIR) of p62, respectively. However, it remains unclear whether ALS/FTD-related p62 mutations in the LIR and KIR disrupt liquid droplet formation leading to defects in autophagy, the stress response, or both. To evaluate the effects of ALS/FTD-related p62 mutations in the LIR and KIR on a major oxidative stress system, the Keap1-Nrf2 pathway, as well as on autophagic turnover, we developed systems to monitor each of these with high sensitivity. These methods such as intracellular protein-protein interaction assay, doxycycline-inducible gene expression system, and gene expression into primary cultured cells with recombinant adenovirus revealed that some mutants, but not all, caused reduced NRF2 activation and delayed autophagic cargo turnover. In contrast, while all p62 mutants demonstrated sufficient ability to form liquid droplets, all of these droplets also exhibited reduced inner fluidity. These results indicate that like other ALS-related mutant proteins, p62 missense mutations result in a primary defect in ALS/FTD via a qualitative change in p62 liquid droplet fluidity. [ABSTRACT FROM AUTHOR]

The PROPPIN family member Atg18 is a phosphoinositide-binding protein that is composed of a seven β-propeller motif and is part of the conserved autophagy machinery. Here, we report that the Atg18 phosphorylation in the loops in the propellar structure of blade 6 and blade 7 decreases its binding affinity to phosphatidylinositol 3,5-bisphosphate in the yeast Pichia pastoris. Dephosphorylation of Atg18 was necessary for its association with the vacuolar membrane and caused septation of the vacuole. Upon or after dissociation from the vacuolar membrane, Atg18 was rephosphorylated, and the vacuoles fused and formed a single rounded structure. Vacuolar dynamics were regulated according to osmotic changes, oxidative stresses, and nutrient conditions inducing micropexophagy via modulation of Atg18 phosphorylation. This study reveals how the phosphoinositide-binding activity of the PROPPIN family protein Atg18 is regulated at the membrane association domain and highlights the importance of such phosphoregulation in coordinated intracellular reorganization. [ABSTRACT FROM AUTHOR]

The presence of multiple homologues of the same yeast Atg protein endows an additional layer of complexity on the autophagy pathway in higher eukaryotes. The physiological function of the individual genes, however, remains largely unknown. Here we investigated the role of the two Caenorhabditis elegans homologues of the cysteine protease Atg4 in the pathway responsible for degradation of protein aggregates. Loss of atg-4.1 activity causes defective degradation of a variety of protein aggregates, whereas atg-4.2 mutants remove these substrates normally. LGG-1 precursors accumulate in atg-4.1 mutants, but not atg-4.2 mutants. LGG-1 puncta, formation of which depends on lipidation of LGG-1, are present in atg-4.1 and atg-4.2 single mutants, but are completely absent in atg-4.1; atg-4.2 double mutants. In vitro enzymatic analysis revealed that ATG-4.1 processes LGG-1 precursors about 100-fold more efficiently than ATG-4.2. Expression of a mutant form LGG-1, which mimics the processed precursor, rescues the defective autophagic degradation of protein aggregates in atg-4.1 mutants and, to a lesser extent, in atg-4.1; atg-4.2 double mutants. Our study reveals that ATG-4.1 and ATG-4.2 are functionally redundant yet display differential LGG-1 processing and deconjugating activity in the aggrephagy pathway in C. elegans. [ABSTRACT FROM AUTHOR]

Droplet microfluidic-based technology is a powerful tool for biotechnology, and it is also expected that it will be applied to culturing and screening methods. Using this technology, a new high-throughput screening method for lactic acid bacteria was developed. In this study, the conventional culture of lactic acid bacteria that form clear zones on an agar medium was reproduced in water-in-oil droplets, and only the droplets in which lactic acid bacteria grew were collected one by one. Using this method, the specific recovery of Lactiplantibacillus plantarum from a mixture of L. plantarum and Escherichia coli and the acquirement of lactic acid bacteria from an environmental sample were successful. This method could be applied to various conventional screening methods using the clear zone as a microbial growth indicator. This has expanded the possibilities of applying droplet microfluidic-based technology to microbial cultivations., Competing Interests: YO and NH were employed by On-chip Biotechnologies Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Mori, Watanabe, Nanri, Matsukura, Ota, Homma and Noda.)

Background: The serum markers Krebs von den Lungen-6 (KL-6), surfactant protein A (SP-A), and surfactant protein D (SP-D) have been used for the diagnosis, differential diagnosis, and prognosis prediction of interstitial pneumonia. However, the significance of measuring the serum and bronchoalveolar lavage fluid (BALF) KL-6, SP-D, and SP-A levels in predicting the prognosis of chronic fibrosing interstitial pneumonia (CFIP), idiopathic pulmonary fibrosis, and idiopathic nonspecific interstitial pneumonia remains unclear. We aimed to clarify the significance of measuring the serum and BALF KL-6, SP-A, and SP-D levels in predicting the prognosis of patients with CFIP., Methods: Among 173 patients who were diagnosed with CFIP between September 2008 and February 2021, 39 who underwent bronchoalveolar lavage were included in this study. Among these, patients experiencing an annual decrease in forced vital capacity (FVC) of ≥10% or those facing challenges in undergoing follow-up pulmonary function tests owing to significant deterioration in pulmonary function were categorized as the rapidly progress group. Conversely, individuals with an annual decrease in the FVC of <10% were classified into the slowly progress group. The serum and BALF KL-6, SP-D, and SP-A levels, as well as BALF/serum SP-D and SP-A ratios were compared between the two groups., Results: Among the patients with CFIP, the BALF SP-D level (p=0.0111), BALF SP-A level (p<0.0010), BALF/serum SP-D ratio (p=0.0051), and BALF/serum SP-A ratio (p<0.0010) were significantly lower in the rapidly than in the slowly progress group (p<0.0010). The receiver operating characteristics analysis results demonstrated excellent performance for diagnosing patients with CFIP, with the BALF SP-D level (area under the curve [AUC], 0.7424), BALF SP-A level (AUC, 0.8842), BALF/serum SP-D ratio (AUC, 0.7673), and BALF/serum SP-A ratio (AUC, 0.8556). Moreover, the BALF SP-A level showed a notably superior CFIP diagnostic capability. Survival analysis using the Kaplan-Meier method revealed that patients with a BALF SP-A level of <1500 ng/mL and BALF/serum SP-A ratio of <15.0 had poor prognoses., Conclusions: Our results suggest that BALF SP-A measurement may be useful for predicting the prognosis in patients with CFIP., (© 2024. The Author(s).)

This study aimed to reveal the preferred initial treatment for Koos grade 3 vestibular schwannomas (VS). We performed a two-institutional retrospective study on 21 patients with Koos grade 3 VS undergoing resection at Yokohama Medical Center and 37 patients undergoing radiosurgery at Yokohama Rosai Hospital from 2010 to 2021. Tumor control, complications, and functional preservation were compared. The median pre-treatment volume and follow-up duration were 2845 mm 3 and 57.0 months, respectively, in the resection group and 2127 mm 3 and 81.7 months, respectively, in the radiosurgery group. In the resection group, 16 (76.2%) underwent gross total resection, and three patients (14.3%) experienced regrowth; however, no one required additional treatment. In the radiosurgery group, the tumor control rate was 86.5%, and three cases (8.1%) required surgical resection because of symptomatic brainstem compression. Kaplan-Meier analyses revealed that tumors with delayed continuous enlargement and large thin-walled cysts were significantly associated with poor prognostic factors (p = 0.0027, p < 0.001). The pre-radiosurgery growth rate was also associated with the volume increase (p = 0.013). Two cases (9.5%) required additional operation due to complications such as post-operative hematoma and cerebrospinal fluid leaks in the resection group, whereas temporary cranial neuropathies were observed in the radiosurgery group. Two patients (9.5%) had poor facial nerve function (House-Brackmann grading grade 3) in the resection group, while no one developed facial paresis in the radiosurgery group. Trigeminal neuropathy improved only in the resection group.Radiosurgery can be considered for the treatment of Koos grade 3 VS for functional preservation. However, resection may also be considered for patients with severe trigeminal neuropathy or a high risk of volume increments, such as large thin-walled cysts and rapid pre-treatment growth., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Congenital antithrombin (AT) or serpin C1 deficiency, caused by a SERPINC1 abnormality, is a high-risk factor for venous thrombosis. SERPINC1 is prone to genetic rearrangement, because it contains numerous Alu elements. In this study, a Japanese patient who developed deep vein thrombosis during pregnancy and exhibited low AT activity underwent SERPINC1 gene analysis using routine methods: long-range polymerase chain reaction (PCR) and real-time PCR. Sequencing using long-range PCR products revealed no pathological variants in SERPINC1 exons or exon-intron junctions, and all the identified variants were homozygous, suggesting a deletion in one SERPINC1 allele. Copy number quantification for each SERPINC1 exon using real-time PCR revealed half the number of exon 1 and 2 copies compared with controls. Moreover, a deletion region was deduced by quantifying the 5'-upstream region copy number of SERPINC1 for each constant region. Direct long-range PCR sequencing with primers for the 5'-end of each presumed deletion region revealed a large Alu-mediated deletion (∼13 kb) involving SERPINC1 exons 1 and 2. Thus, a large deletion was identified in SERPINC1 using conventional PCR methods., (© 2024. Japanese Society of Hematology.)

Nickel-catalyzed syntheses of oligothiophene and polythiophene were carried out with Ni(cod)(dq) (COD: 1,5-cycloctadiene; DQ: duroquinone) as a catalyst precursor. Studies on the ligand exchange of Ni(cod)(dq) revealed that a high temperature was necessary to replace COD and DQ with PPh 3 and N-heterocyclic carbene IPr. A coupling reaction of a metalated 3-hexylthiophene with 2-chloro-3-hexylthiophene employing Ni(cod)(dq) with IPr proceeded with a remarkably reduced amount of homocoupling byproduct. Polymerization of 2-chloro-3-hexylthiophene with Ni(cod)(dq)/DPPP also resulted in the reduction of the regioregularity defect.

Objective: Different mitochondrial DNA genotypes can coexist in a cell population as well as in a single cell, a condition known as heteroplasmy. Here, we accurately determined the heteroplasmy levels of the m.3243A>G mutation, which is the most frequently identified mutation in patients with mitochondrial diseases, using droplet digital polymerase chain reaction (ddPCR)., Methods: The m.3243A>G heteroplasmy levels in artificial heteroplasmy controls mixed with various proportions of wild-type and mutant plasmids were measured using ddPCR, PCR-restriction fragment length polymorphism, and Sanger sequencing. The m.3243A>G heteroplasmy levels in DNA, extracted from the peripheral blood of patients with suspected mitochondrial disease and healthy subjects, were determined using ddPCR., Results: The accuracy of the ddPCR method was high. The lower limit of detection was 0.1%, which indicated its higher sensitivity compared with other methods. The m.3243A>G heteroplasmy levels in peripheral blood, measured using ddPCR, correlated inversely with age at the time of analysis. The m.3243A>G mutation may be overlooked in the peripheral blood-derived DNA of elderly people, as patients >60 years of age have heteroplasmy levels <10%, which is difficult to detect using methods other than the highly sensitive ddPCR., Conclusion: ddPCR may be considered an accurate and sensitive method for measuring m.3243 A>G heteroplasmy levels of mitochondrial DNA., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Society for Clinical Pathology.)

Toxin-antitoxin systems are preserved by nearly every prokaryote. The type II toxin MazF acts as a sequence-specific endoribonuclease, cleaving ribonucleotides at specific sequences that vary from three to seven bases, as has been reported in different host organisms to date. The present study characterized the MazEF module (MazEF-sth) conserved in the Symbiobacterium thermophilum IAM14863 strain, a Gram-negative syntrophic bacterium that can be supported by co-culture with multiple bacteria, including Bacillus subtilis . Based on a method combining massive parallel sequencing and the fluorometric assay, MazF-sth was determined to cleave ribonucleotides at the UACAUA motif, which is markedly similar to the motifs recognized by MazF from B. subtilis (MazF-bs), and by several MazFs from Gram-positive bacteria. MazF-sth, with mutations at conserved amino acid residues Arg29 and Thr52, lost most ribonuclease activity, indicating that these residues that are crucial for MazF-bs also play significant roles in MazF-sth catalysis. Further, cross-neutralization between MazF-sth and the non-cognate MazE-bs was discovered, and herein, the neutralization mechanism is discussed based on a protein-structure simulation via AlphaFold2 and multiple sequence alignment. The conflict between the high homology shared by these MazF amino acid sequences and the few genetic correlations among their host organisms may provide evidence of horizontal gene transfer.

The sequence-specific endoribonuclease MazF is widely conserved among prokaryotes. Approximately 20 different MazF cleavage sequences have been discovered, varying from three to seven nucleotides in length. Although MazFs from various prokaryotes were found, the cleavage sequences of most MazFs are unknown. Here, we characterized the conserved MazF of Salmonella enterica subsp. arizonae (MazF-SEA). Using massive parallel sequencing and fluorometric assays, we revealed that MazF-SEA preferentially cleaves the sequences U ∧ ACG and U ∧ ACU ( ∧ represents cleavage sites). In addition, we predicted the 3D structure of MazF-SEA using AlphaFold2 and aligned it with the crystal structure of RNA-bound Bacillus subtilis MazF to evaluate RNA interactions. We found Arg-73 of MazF-SEA interacts with RNAs containing G and U at the third position from the cleavage sites (U ∧ ACG and U ∧ ACU). We then obtained the mutated MazF-SEA R73L protein to evaluate the significance of Arg-73 interaction with RNAs containing G and U at this position. We also used fluorometric and kinetic assays and showed the enzymatic activity of MazF-SEA R73L for the sequence UACG and UACU was significantly decreased. These results suggest Arg-73 is essential for recognizing G and U at the third position from the cleavage sites. This is the first study to our knowledge to identify a single residue responsible for RNA recognition by MazF. Owing to its high specificity and ribosome-independence, MazF is useful for RNA cleavage in vitro. These results will likely contribute to increasing the diversity of MazF specificity and to furthering the application of MazF in RNA engineering., Competing Interests: Conflict of interest The authors declare no conflicts of interest regarding the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Bacteriophages are the most abundant entities on Earth. In contrast with the number of phages considered to be in existence, current phage isolation and screening methods lack throughput. Droplet microfluidic technology has been established as a platform for high-throughput screening of biological and biochemical components. In this study, we developed a proof-of-concept method for isolating phages using water-in-oil droplets (droplets) as individual chambers for phage propagation and co-cultivating T2 phage and their host cell Escherichia coli within droplets. Liquid cultivation of microbes will facilitate the use of microbes that cannot grow on or degrade agar as host cells, ultimately resulting in the acquisition of phages that infect less known bacterial cells. The compartmentalizing characteristic of droplets and the use of a fluorescent dye to stain phages simultaneously enabled the enumeration and isolation of viable phage particles. We successfully recultivated the phages after simultaneously segregating single phage particles into droplets and inoculating them with their host cells within droplets. By recovering individual droplets into 96-well plates, we were able to isolate phage clones derived from single phage particles. The success rate for phage recovery was 35.7%. This study lays the building foundations for techniques yet to be developed that will involve the isolation and rupturing of droplets and provides a robust method for phage enumeration and isolation., Competing Interests: YO and YM were employed by On-chip Biotechnologies Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Hoshino, Ota, Suyama, Morishita, Tsuneda and Noda.)

Flow imaging microscopy (FIM) is widely used to characterize biopharmaceutical subvisible particles (SVPs). The segmentation threshold, which defines the boundary between the particle and the background based on pixel intensity, should be properly set for accurate SVP quantification. However, segmentation thresholds are often subjectively and empirically set, potentially leading to variations in measurements across instruments and operators. In the present study, we developed an objective method to optimize the FIM segmentation threshold using poly(methyl methacrylate) (PMMA) beads with a refractive index similar to that of biomolecules. Among several candidate particles that were evaluated, 2.5-µm PMMA beads were the most reliable in size and number, suggesting that the PMMA bead size analyzed by FIM could objectively be used to determine the segmentation threshold for SVP measurements. The PMMA bead concentrations measured by FIM were highly consistent with the indicative concentrations, whereas the PMMA bead size analyzed by FIM decreased with increasing segmentation threshold. The optimal segmentation threshold where the analyzed size was closest to the indicative size differed between an instrument with a black-and-white camera and that with a color camera. Inter-instrument differences in SVP concentrations in acid-stressed recombinant adeno-associated virus (AAV) and protein aggregates were successfully minimized by setting an optimized segmentation threshold specific to the instrument. These results reveal that PMMA beads can aid in determining a more appropriate segmentation threshold to evaluate biopharmaceutical SVPs using FIM., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Abscisic acid (ABA)-mediated abiotic stress tolerance causes plant growth inhibition. Under such stress conditions, some mosses generate de novo stress-resistant stem cells, also called brood cells or brachycytes, that do not exist under normal conditions. However, the cell physiological basis of the growth inhibition and the stem cell formation is not well understood. Here, we show that the ABA-induced growth inhibition of the moss Physcomitrium patens apical protonemal cells (protonemal stem cells) is mediated through a shift from asymmetric to symmetric cell division. This change of the cell division mode, and consequently change of stem cell activity, is substantiated by dampening cell polarity and cell proliferative activity through the altered distribution of cytoskeletal elements, the mitotic spindle and the vacuole, which results in the production of stress-resistant stem cells. Alteration of the cell physiological data is supported by the results of RNAseq analysis indicating rapid changes in both cell polarity and cell cycle regulation, while long-term treatments with ABA for 5 to 10 days impact mainly the transcriptional and translational regulation. The regulation of cell polarity and cell cycle genes suggests growth arrest mediated by small GTPases (ROPs) and their guanine exchange factors (ROPGEFs) and by cyclin and cyclin-dependent-kinase complex, respectively. Our data suggest that a tradeoff relationship between growth ability and abiotic stress response in the moss is substantiated by ABA signaling to suppress cell polarity and asymmetric cell growth and may play a pivotal role in stem cell fate conversion to newly produced stress-resistant stem cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Beier, Jinno, Noda, Nakamura, Sugano, Suzuki and Fujita.)

Despite the well-established role of oxidative stress in the pathogenesis of age-related macular degeneration (AMD), the mechanism underlying phototoxicity remains unclear. Herein, we used a drug repurposing approach to isolate an FDA-approved drug that blocks the aggregation of the photoinducible major fluorophore of lipofuscin, the bis-retinoid N -retinylidene- N -retinylethanolamine (A2E). Our fluorescence-based screening combined with dynamic light scattering (DLS) analysis led to the identification of entacapone as a potent inhibitor of A2E fluorescence and aggregation. The entacapone-mediated inhibition of A2E aggregation blocks its photodegradation and offers photoprotection in A2E-loaded retinal pigment epithelial (RPE) cells exposed to blue light. In-depth mechanistic analysis suggests that entacapone prevents the conversion of toxic aggregates by redirecting A2E into off-pathway oligomers. These findings provide evidence that aggregation contributes to the phototoxicity of A2E.

Protein or peptide cancer vaccines usually include immune potentiators, so-called adjuvants. However, it remains challenging to identify structurally simple, chemically accessible synthetic molecules that are effective and safe as vaccine adjuvant. Here, we present cholicamideβ ( 6 ), a self-assembling small-molecule vaccine adjuvant with an improved toxicity profile and proven efficacy in vivo . We demonstrate that cholicamideβ ( 6 ), which is less cytotoxic than its parent compound, forms virus-like particles to potently activate dendritic cells with the concomitant secretion of cytokines. When combined with a peptide antigen, cholicamideβ ( 6 ) potentiated the antigen presentation on dendritic cells to induce antigen-specific T cells. As a therapeutic cancer vaccine adjuvant in mice, a mixture of cholicamideβ ( 6 ) and a peptide antigen protected mice from the challenges of malignant cancer cells without overt toxicity. Cholicamideβ ( 6 ) may offer a translational opportunity as an unprecedented class of small-molecule cancer vaccine adjuvants.

Analyzing the way of deposition of saliva stains contributes to appropriate interpretation of saliva as evidence in court, particularly in sexual assault cases. In this proof-of-concept study, we aimed to confirm the difference between drooling-derived (non-contact) saliva and licking-derived (contact) saliva and clarify whether objectively distinguishing between the two saliva is possible. To allow discrimination between these two samples, an indicator was devised where the relative Streptococcus salivarius DNA quantity was calculated by dividing the S. salivarius DNA copies by the amount of stained saliva from the same saliva sample using quantitative polymerase chain reaction and salivary α-amylase activity assays. The study findings reveal that the value of the proposed indicator of licking-derived saliva was 100-fold significantly greater than that of drooling-derived saliva (P < 0.05, Welch's t-test). However, theoretical and technical challenges preclude the application of this indicator as a practical method. We believe that this saliva-specific bacterial DNA-based approach could allow estimation of the saliva stain deposition method., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Purpose: To compare the diagnostic capabilities of orbital synchronized helical scanning at lower extremity computed tomography angiography between the Add/Sub software and the deformable image registration., Methods: From March 2015 to December 2016, 100 dialysis patients underwent orbital synchronized lower limb CT subtraction angiography and lower limb endovascular treatment within 4 months. For the visual evaluation of blood vessels in the lower extremities, a stenosis rate of 50% or more was considered to be stenosis. It was classified into two areas: above-knee (AK) region (superficial femoral artery and popliteal artery) and below-knee (BK) region (anterior tibial artery, posterior tibial artery, and fibula artery). Considering angiography for the lower limb endovascular treatment as the golden standard, we calculated the sensitivity, specificity, positive-predictive value, negative-predictive value, and diagnostic capabilities. Receiver operating characteristic curve (ROC) analysis was performed to calculate the area under curve (AUC)., Results: Calcification subtraction failure was observed to be 11% in the AK region and 2% in the BK region using the Add/Sub software. The specificity, positive-predictive value, diagnostic capabilities, and AUC of the deformable image registration were lower than those of the Add/Sub software., Conclusions: Add/Sub software and deformable image registration have high diagnostic capability to remove calcification. On the other hand, the specificity and AUC of the deformable image registration were lower than those of the Add/Sub software. Also, even if the same deformable image registration is used, caution is required because the diagnostic performance varies depending on the site.

The [GADV]-protein, consisting only of glycine (G), alanine (A), aspartic acid (D), and valine (V), is frequently studied as a candidate for a primitive protein that existed at the beginning of life on Earth. The number of proteogenic amino acids increased during evolution, and glutamic acid may have been added as the fifth amino acid. In this study, we used molecular dynamics simulations to estimate the conformation of random peptides when glutamate is added to G, A, D, and V ([GADVE]), when leucine is added ([GADVL]), and when the frequency of alanine is doubled ([GADVA]). The results showed that the secondary structure contents of the [GADVE]-peptide and [GADVL]-peptide were higher than that of the [GADVA]-peptide. Although the [GADVL]-peptide had a higher secondary structure formation ability than the [GADVE]-peptide, it was less water soluble, suggesting that it may not be a primitive protein. The [GA(D/E)V]-peptide with G:A:D:V:E = 2:2:1:2:1 according to the occurrence ratio in the codon table also increased the secondary structure contents compared to the [GADV]-peptide, indicating that the addition of glutamic acid increased the structure formation ability of the primitive protein candidates.

This study aimed to analyze circulating tumor cells (CTCs) from patients with colorectal cancer (CRC). We designed a dielectrophoresis-based micropore system and tested its cell capture with HT29 colon cancer cells. Then, blood samples were drawn from 24 patients with stages II-IV CRC. Mononuclear cells were isolated and loaded into the micropore system. Single cells were positioned into small pores with dielectrophoresis. After labeling the cells with the appropriate antibodies, tumor-like cells were collected with an automated micromanipulator. We collected 43 CTCs from 15 out of 24 patient samples. The presence of CTC was significantly associated with ling metastasis. We performed whole genome amplification, followed by PCR and Sanger sequencing, to examine the point mutations in the KRAS , BRAF , and PIK3CA genes. This mutation analysis was successfully performed in 35 cells. Among the 14 cytokeratin (CK)-positive cells, we found PIK3CA mutations in three cells (21%) from two patients. Among the 21 CK-negative cells, we found a KRAS mutation in one cell (5%) from one patient and a PIK3CA mutation in one cell (5%) from one patient. It is noteworthy that these mutations were not detected in the corresponding primary tumors. In conclusion, dielectrophoresis-based capture in a micropore system was useful for detecting both CK-positive and CK-negative CTCs. This simple method could be applied to various tumor types.

Roseateles depolymerans is an obligately aerobic bacterium that produces a photosynthetic apparatus only under the scarcity of carbon substrates. We herein examined changes in the transcriptomes of R. depolymerans cells to clarify the expression of photosynthesis genes and their upstream regulatory factors under carbon starvation. Transcriptomes 0, 1, and 6‍ ‍h after the depletion of a carbon substrate indicated that transcripts showing the greatest variations (a 500-fold increase [6 h/0 h]) were light-harvesting proteins (PufA and PufB). Moreover, loci with more than 50-fold increases (6 h/0‍ ‍h) were fully related to the photosynthetic gene cluster. Among 13 sigma factor genes, the transcripts of a sigma 70 family sigma factor related to RpoH (SP70) increased along photosynthesis genes under starvation; therefore, a knockout experiment of SP70 was performed. ΔSP70 mutants were found to lack photosynthetic pigments (carotenoids and bacteriochlo-rophyll a) regardless of carbon starvation. We also examined the effects of heat stress on ΔSP70 mutants, and found that SP70 was also related to heat stress tolerance, similar to other RpoH sigma factors (while heat stress did not trigger photosystem production). The deficient accumulation of photosynthetic pigments and the heat stress tolerance of ΔSP70 mutants were both complemented by the introduction of an intact SP70 gene. Furthermore, the transcription of photosynthetic gene operons (puf, puh, and bch) was markedly reduced in the ΔSP70 mutant. The RpoH homologue SP70 was concluded to be a sigma factor that is essential for the transcription of photosynthetic gene operons in R. depolymerans.

The organocatalytic enantioselective hydrophosphinylation of various secondary phosphine sulfides with aromatic and aliphatic nitroalkenes is presented in this study. The reaction produced chiral β-nitrophosphine sulfides with excellent yields and enantioselectivities (up to 99% yield and 99% ee). Furthermore, the chiral β-nitrophosphine sulfides can be easily converted into α-substituted β-aminophosphine, which is a family of useful P, N-ligands and phosphine catalysts., (© 2022 Wiley-VCH GmbH.)

Introduction: Acute respiratory distress syndrome (ARDS) is considered a poor prognostic factor for miliary tuberculosis (MTB), but little is known about the effectiveness of steroid pulse therapy for MTB complicated by ARDS., Patients and Methods: Medical records were used to retrospectively investigate the prognosis and clinical information of 13 patients diagnosed with MTB complicated by ARDS among 68 patients diagnosed with MTB at our hospital between January 1994 and October 2016. None of the patients had multidrug resistant tuberculosis (TB). MTB was diagnosed by 1 radiologist and 2 respiratory physicians based on the observation of randomly distributed, uniformly sized diffuse bilateral nodules on chest computed tomography and the detection of mycobacterium TB from clinical specimens. ARDS was diagnosed based on the Berlin definition of ARDS. The effect of steroid pulse therapy on death within 3 months of hospitalization was examined using Cox proportional hazards models. Variables were selected by the stepwise method (variable reduction method)., Results: Six of 8 patients with MTB complicated by ARDS were alive 3 months after hospitalization in the steroid pulse therapy group, whereas only 1 of 5 patients was alive in the non-steroid pulse therapy group. Analysis of factors related to the survival of patients with MTB complicated by ARDS revealed that steroid pulse therapy was the strong prognostic factor (hazard ratio = 0.136 (95 % CI: 0.023-0.815))., Conclusion: Our findings suggest that steroid pulse therapy improves the short-term prognosis of patients with MTB complicated by ARDS., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

The purpose of this research is to install a handrail on sliding doors used in hospitals and nursing facilities to support the elderly and disabled to walk by themselves. Semi-automatic lifting equipment is utilised for the retractable handrail to make sure people in bad health are able to open the door using minimal force. This paper summarises ergonomic considerations to develop the product above. For example, the maximum opening force should be a certain value or less for the elderly and disabled to open the product effortlessly. Additionally, the initial opening force is required to be a certain degree for safe use. When the product is used as a handrail, the handrail is required to be stable and unshakable in opening or closing directions. The results obtained by having the elderly and disabled use the prototype demonstrated that it improves their walking abilities.

Chrysanthemum morifolium is one of the most popular ornamental plants globally. Owing to its large and complex genome (around 10 Gb, segmental hexaploid), it has been difficult to obtain comprehensive transcriptome, which will promote to perform new breeding technique, such as genome editing, in C. morifolium. In this study, we used single-molecule real-time (SMRT) sequencing and RNA-seq technologies, combined them with an error-correcting process, and obtained high-coverage ray-floret transcriptome. The SMRT-seq data increased the ratio of long mRNAs containing complete open-reading frames, and the combined dataset provided a more complete transcriptomic data than those produced from either SMRT-seq or RNA-seq-derived transcripts. We finally obtained 'Sei Arabella' transcripts containing 928,645 non-redundant mRNA, which showed 96.6% Benchmarking Universal Single-Copy Orthologs (BUSCO) score. We also validated the reliability of the dataset by analyzing a mapping rate, annotation and transcript expression. Using the dataset, we searched anthocyanin biosynthesis gene orthologs and performed a qRT-PCR experiment to assess the usability of the dataset. The assessment of the dataset and the following analysis indicated that our dataset is reliable and useful for molecular biology. The combination of sequencing methods provided genetic information and a way to analyze the complicated C. morifolium transcriptome., (© 2022. The Author(s).)

EPI-589 attenuates oxidative stress due to the radical scavenging activity of the reduced form and affects mitochondrial energy metabolism as a substrate of quinone-oxidoreductases. Given the effects of EPI-589 on oxidative stress and mitochondrial dysfunction, EPI-589 shows promise as a potential therapy for patients with amyotrophic lateral sclerosis. This phase 1 study evaluated the safety, tolerability, and pharmacokinetic profiles of EPI-589. Sixty-eight healthy participants were randomly assigned to EPI-589 or placebo. All adverse events were mild or moderate in severity, and no severe adverse events were reported. After single-dose administration under fasting conditions, time to maximum plasma concentration (t max ) occurred 0.25 to 1.00 hour after administration. Both peak plasma concentration (C max ) and area under the plasma concentration-time curve (AUC) were approximately linear with increases in single doses over a dose range of 250-1000 mg. Under fed conditions, the C max decreased to 62.6% of the C max under fasting conditions, the AUC was slightly increased, and the t max was delayed by 1 hour. When EPI-589 was administered daily on days 1 and 7 with twice-daily dosing on days 2 through 6, the plasma trough concentration appeared to reach steady state by day 3. At both doses studied (500 mg twice daily and 750 mg twice daily), C max, t max , and AUC were generally comparable between day 1 and day 7 and between the Japanese and White participants. EPI-589 was well tolerated as a single daily dose up to 1000 mg and as twice-daily doses up to 750 mg, with a linear pharmacokinetic profile., (© 2022, The American College of Clinical Pharmacology.)

Monoclonal antibodies have been established as the largest product class of biopharmaceuticals. Since extensive characterization is required for development and quality control of monoclonal antibody, a widely available reference material (RM) is needed. Herein, a humanized IgG1κ monoclonal antibody reference material, RM 6208-a, AIST-MAB, was established by the National Metrology Institute of Japan, National Institute of Advanced Industrial Science and Technology (NMIJ/AIST). The monoclonal antibody solution was produced as a pharmaceutical grade using a Chinese hamster ovary-derived cell line. The assigned indicative value represents the concentration of the antibody with a heterotetrameric structure including oligomeric forms, determined by an amino acid analysis using isotope dilution mass spectrometry, and their homogeneity and stability were assessed. In addition to antibody concentration, various physicochemical properties, including peptide mapping data, charge variants, and aggregates, were examined. This RM is intended for use in validation of analytical procedures and instruments such as a system suitability test for quantification of antibody. It is also intended for comparing and evaluating the results of antibody analyses across analytical methods and analytical laboratories such as inter-laboratory comparison. Both the material and the set of data from our study provide a tool for an accurate and reliable characterization of product quality attributes of monoclonal antibodies in biopharmaceutical and metrology communities., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kinumi, Saikusa, Kato, Kojima, Igarashi, Noda and Honda.)

Chemotherapy is a standard therapy for muscle-invasive bladder cancer (MIBC). However, genomic alterations associated with chemotherapy sensitivity in MIBC have not been fully explored. This study aimed to investigate the genomic landscape of MIBC in association with the response to chemotherapy and to explore the biological role of genomic alterations. Genomic alterations in MIBC were sequenced by targeted exome sequencing of 409 genes. Gene expression in MIBC tissues was analyzed by western blotting, immunohistochemistry, and RNA microarray. Cellular sensitivity to gemcitabine and gemcitabine metabolite was examined in bladder cancer cells after modulation of candidate gene. Targeted exome sequencing in 20 cases with MIBC revealed various genomic alterations including pathogenic missense mutation of DPYD gene encoding dihydropyrimidine dehydrogenase (DPD). Conversely, high DPYD and DPD expression were associated with poor response to gemcitabine-containing chemotherapy among patients with MIBC, as well as gemcitabine resistance in bladder cancer cells. DPD suppression rendered cells sensitive to gemcitabine, while DPD overexpression made cells gemcitabine-resistant through reduced activity of the cytotoxic gemcitabine metabolite difluorodeoxycytidine diphosphate. This study revealed the novel role of DPD in gemcitabine metabolism. It has been suggested that DPYD genomic alterations and DPD expression are potential predictive biomarkers in gemcitabine treatment., (© 2022. The Author(s).)

Phase-separated membraneless organelles or biomolecular condensates play diverse functions in cells, however recapturing their characteristics using small organic molecules has been a challenge. In the present study, cell-lysate-based screening of 843 self-assembling small molecules led to the discovery of a simple organic molecule, named huezole, that forms liquid droplets to selectively sequester tubulin. Remarkably, this small molecule enters cultured human cells and prevents cell mitosis by forming tubulin-concentrating condensates in cells. The present study demonstrates the feasibility of producing a synthetic condensate out of non-peptidic small molecules for exogenous control of cellular processes. The modular structure of huezole provides a framework for designing a class of organelle-emulating small molecules., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)