Identification of proteins involved in intracellular ubiquinone trafficking in Saccharomyces cerevisiae using artificial ubiquinone probe.

Ubiquinone (UQ) is an essential player in the respiratory electron transfer system. In Saccharomyces cerevisiae strains lacking the ability to synthesize UQ6, exogenously supplied UQs can be taken up and delivered to mitochondria through an unknown mechanism, restoring the growth of UQ6-deficient yeast in non-fermentable medium. Since elucidating the mechanism responsible may markedly contribute to therapeutic strategies for patients with UQ deficiency, many attempts have been made to identify the machinery involved in UQ trafficking in the yeast model. However, definite experimental evidence of the direct interaction of UQ with a specific protein(s) has not yet been demonstrated. To gain insight into intracellular UQ trafficking via a chemistry-based strategy, we synthesized a hydrophobic UQ probe (pUQ5), which has a photoreactive diazirine group attached to a five-unit isoprenyl chain and a terminal alkyne to visualize and/or capture the labeled proteins via click chemistry. pUQ5 successfully restored the growth of UQ6-deficient S. cerevisiae (Δcoq2) on a non-fermentable carbon source, indicating that this UQ was taken up and delivered to mitochondria, and served as a UQ substrate of respiratory enzymes. Through photoaffinity labeling of the mitochondria isolated from Δcoq2 yeast cells cultured in the presence of pUQ5, we identified many labeled proteins, including voltage-dependent anion channel 1 (VDAC1) and cytochrome c oxidase subunit 3 (Cox3). The physiological relevance of UQ binding to these proteins is discussed.

Visualizing the spatial distribution of ustalic acid in the fruiting body of Tricholoma kakishimeji.

Imaging mass spectrometry (IMS) was conducted for the first time using ustalic acid (UA) and the fruiting body of Tricholoma kakishimeji to localize mushroom toxins. The mushroom materials were systematically collected in Japan, and analysis of the cross sections of the materials at a resolution of 120 µm using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-IMS) revealed the localization of UA and its biogenically related metabolites. MALDI-IMS confirmed that UA was predominantly located on the entire surface of the fruiting body and accumulated in higher amounts in younger fruiting bodies than in mature ones. UA is the first toxic secondary metabolite in the genus Tricholoma locally identified using IMS in mushrooms.

Optimizing in vitro expression balance of central dogma-related genes using parallel reaction monitoring.

The creation of a self-replicating synthetic cell is an essential to understand life self-replication. One method to create self-replicating artificial cells is to reconstitute the self-replication system of living organisms in vitro. In a living cell, self-replication is achieved via a system called the autonomous central dogma, a system in which central dogma-related factors are autonomously synthesized and genome replication, transcription, and translation are driven by nascent factors. Various studies to reconstitute some processes of the autonomous central dogma in vitro have been conducted. However, in vitro reconstitution of the entire autonomous central dogma system is difficult as it requires balanced expression of several related genes. Therefore, we developed a method to simultaneously quantify and optimize the in vitro expression balance of multiple genes. First, we developed a quantitative mass spectrometry method targeting genome replication-related proteins as a model of central dogma-related factors and acquired in vitro expression profiles of these genes. Additionally, we demonstrated that the in vitro expression balance of these genes can be easily optimized by adjusting the input gene ratio based on the data obtained by the developed method. This study facilitated the easy optimization of the in vitro expression balance of multiple genes. Therefore, extending the scope of this method to other central dogma-related factors will accelerate attempts of self-replicating synthetic cells creation.

Minimum inhibitory concentrations of azithromycin in clinical isolates of Mycobacterium avium complex in Japan.

The latest guidelines include azithromycin as a preferred regimen for treating Mycobacterium avium complex (MAC) pulmonary disease. However, serially collected susceptibility data on clinical MAC isolates are limited, and no breakpoints have been determined. We investigated the minimum inhibitory concentrations (MICs) of azithromycin and clarithromycin for all MAC strains isolated in 2021 from a single center in Japan, excluding duplicates. The MICs were determined using a panel based on the microbroth dilution method, according to the latest Clinical and Laboratory Standards Institute recommendations. The MICs were determined for 318 MAC strains. Although there was a significant positive correlation between the MICs of azithromycin and clarithromycin, the MICs of azithromycin tended to be higher than those of clarithromycin. Among the cases in which the strains were isolated, 18 patients initiated treatment, including azithromycin treatment, after sample collection. Some patients infected with stains with relatively high azithromycin MICs achieved a microbiological cure with azithromycin-containing regimens. This study revealed a higher MIC distribution for azithromycin than clarithromycin, raising questions about the current practice of estimating azithromycin susceptibility based on the clarithromycin susceptibility test result. However, this was a single-center study that included only a limited number of cases treated with azithromycin. Therefore, further multicenter studies that include a greater number of cases treated with azithromycin are warranted to verify the distribution of azithromycin MICs and examine the correlation between azithromycin MICs and treatment effectiveness.IMPORTANCEThe macrolides serve as key drugs in the treatment of pulmonary Mycobacterium avium complex infection, and the administration of macrolide should be guided by susceptibility test results. Azithromycin is recommended as a preferred choice among macrolides, surpassing clarithromycin; however, drug susceptibility testing is often not conducted, and clarithromycin susceptibility is used as a surrogate. This study represents the first investigation into the minimum inhibitory concentration of azithromycin on a scale of several hundred clinical isolates, revealing an overall tendency for higher minimum inhibitory concentrations compared with clarithromycin. The results raise questions about the appropriateness of using clarithromycin susceptibility test outcomes for determining the administration of azithromycin. This study highlights the need for future discussions on the clinical breakpoints of azithromycin, based on large-scale clinical research correlating azithromycin susceptibility with treatment outcomes.

Secretion of mitochondrial DNA via exosomes promotes inflammation in Behçet's syndrome.

Mitochondrial DNA (mtDNA) leakage into the cytoplasm can occur when cells are exposed to noxious stimuli. Specific sensors recognize cytoplasmic mtDNA to promote cytokine production. Cytoplasmic mtDNA can also be secreted extracellularly, leading to sterile inflammation. However, the mode of secretion of mtDNA out of cells upon noxious stimuli and its relevance to human disease remain unclear. Here, we show that pyroptotic cells secrete mtDNA encapsulated within exosomes. Activation of caspase-1 leads to mtDNA leakage from the mitochondria into the cytoplasm via gasdermin-D. Caspase-1 also induces intraluminal membrane vesicle formation, allowing for cellular mtDNA to be taken up and secreted as exosomes. Encapsulation of mtDNA within exosomes promotes a strong inflammatory response that is ameliorated upon exosome biosynthesis inhibition in vivo. We further show that monocytes derived from patients with Behçet's syndrome (BS), a chronic systemic inflammatory disorder, show enhanced caspase-1 activation, leading to exosome-mediated mtDNA secretion and similar inflammation pathology as seen in BS patients. Collectively, our findings support that mtDNA-containing exosomes promote inflammation, providing new insights into the propagation and exacerbation of inflammation in human inflammatory diseases.

Optimizing conditions to construct artificial cells using commercial in vitro transcription-translation system (PUREfrex2.0).

Artificial cells containing in vitro transcription and translation (IVTT) systems inside liposomes are important for the reconstruction and analysis of various biological systems. To improve the accessibility of artificial cell research, it is important that artificial cells can be constructed using only commercially available components. Here, we optimized the construction of artificial cells containing PUREfrex2.0, a commercially available IVTT with high transcriptional and translational activity. Specifically, the composition of the inner and outer s olutions of the liposomes and the concentrations of lipids, glucose/sucrose, potassium glutamate, and magnesium acetate were systematically optimized, and finally we found a protocol for the stable construction of artificial cells containing PUREfre×2.0. These findings are expected to be important in expanding the artificial cell research community.

Application of peptide barcoding to obtain high-affinity anti-PD-1 nanobodies.

Cancer treatment has been revolutionized by immune checkpoint inhibitors, which regulate immune cell function by blocking the interactions between immune checkpoint molecules and their ligands. The interaction between programmed cell death-1 (PD-1) and programmed cell death-ligand 1 (PD-L1) is a target for immune checkpoint inhibitors. Nanobodies, which are recombinant variable domains of heavy-chain-only antibodies, can replace existing immune checkpoint inhibitors, such as anti-PD-1 or anti-PD-L1 conventional antibodies. However, the screening process for high-affinity nanobodies is laborious and time-consuming. Here, we identified high-affinity anti-PD-1 nanobodies using peptide barcoding, which enabled reliable and efficient screening by distinguishing each nanobody with a peptide barcode that was genetically appended to each nanobody. We prepared a peptide-barcoded nanobody (PBNb) library with thousands of variants. Three high-affinity PBNbs were identified from the PBNb library by quantifying the peptide barcodes derived from high-affinity PBNbs. Furthermore, these three PBNbs neutralized the interaction between PD-1 and PD-L1. Our results demonstrate the utility of peptide barcoding and the resulting nanobodies can be used as experimental tools and antitumor agents.

Nanosized Ti-Based Perovskite Oxides as Acid-Base Bifunctional Catalysts for Cyanosilylation of Carbonyl Compounds.

The development of effective solid acid-base bifunctional catalysts remains a challenge because of the difficulty associated with designing and controlling their active sites. In the present study, highly pure perovskite oxide nanoparticles with d0-transition-metal cations such as Ti4+, Zr4+, and Nb5+ as B-site elements were successfully synthesized by a sol-gel method using dicarboxylic acids. Moreover, the specific surface area of SrTiO3 was increased to 46 m2 g-1 by a simple procedure of changing the atmosphere from N2 to air during calcination of an amorphous precursor. The resultant SrTiO3 nanoparticles showed the highest catalytic activity for the cyanosilylation of acetophenone with trimethylsilyl cyanide (TMSCN) among the tested catalysts not subjected to a thermal pretreatment. Various aromatic and aliphatic carbonyl compounds were efficiently converted to the corresponding cyanohydrin silyl ethers in good-to-excellent yields. The present system was applicable to a larger-scale reaction of acetophenone with TMSCN (10 mmol scale), in which 2.06 g of the analytically pure corresponding product was isolated. In this case, the reaction rate was 8.4 mmol g-1 min-1, which is the highest rate among those reported for heterogeneous catalyst systems that do not involve a pretreatment. Mechanistic studies, including studies of the catalyst effect, Fourier transform infrared spectroscopy, and temperature-programmed desorption measurements using probe molecules such as pyridine, acetophenone, CO2, and CHCl3, and the poisoning effect of pyridine and acetic acid toward the cyanosilylation, revealed that moderate-strength acid and base sites present in moderate amounts on SrTiO3 most likely enable SrTiO3 to act as a bifunctional acid-base solid catalyst through cooperative activation of carbonyl compounds and TMSCN. This bifunctional catalysis through SrTiO3 resulted in high catalytic performance even without a heat pretreatment, in sharp contrast to the performance of basic MgO and acidic TiO2 catalysts.

Ammonia Production Using Bacteria and Yeast toward a Sustainable Society.

Ammonia is an important chemical that is widely used in fertilizer applications as well as in the steel, chemical, textile, and pharmaceutical industries, which has attracted attention as a potential fuel. Thus, approaches to achieve sustainable ammonia production have attracted considerable attention. In particular, biological approaches are important for achieving a sustainable society because they can produce ammonia under mild conditions with minimal environmental impact compared with chemical methods. For example, nitrogen fixation by nitrogenase in heterogeneous hosts and ammonia production from food waste using microorganisms have been developed. In addition, crop production using nitrogen-fixing bacteria has been considered as a potential approach to achieving a sustainable ammonia economy. This review describes previous research on biological ammonia production and provides insights into achieving a sustainable society.

The lysosomal Ragulator complex activates NLRP3 inflammasome in vivo via HDAC6.

The cellular activation of the NLRP3 inflammasome is spatiotemporally orchestrated by various organelles, but whether lysosomes contribute to this process remains unclear. Here, we show the vital role of the lysosomal membrane-tethered Ragulator complex in NLRP3 inflammasome activation. Deficiency of Lamtor1, an essential component of the Ragulator complex, abrogated NLRP3 inflammasome activation in murine macrophages and human monocytic cells. Myeloid-specific Lamtor1-deficient mice showed marked attenuation of NLRP3-associated inflammatory disease severity, including LPS-induced sepsis, alum-induced peritonitis, and monosodium urate (MSU)-induced arthritis. Mechanistically, Lamtor1 interacted with both NLRP3 and histone deacetylase 6 (HDAC6). HDAC6 enhances the interaction between Lamtor1 and NLRP3, resulting in NLRP3 inflammasome activation. DL-all-rac-α-tocopherol, a synthetic form of vitamin E, inhibited the Lamtor1-HDAC6 interaction, resulting in diminished NLRP3 inflammasome activation. Further, DL-all-rac-α-tocopherol alleviated acute gouty arthritis and MSU-induced peritonitis. These results provide novel insights into the role of lysosomes in the activation of NLRP3 inflammasomes by the Ragulator complex.

Yes-associated protein 1 mediates initial cell survival during lorlatinib treatment through AKT signaling in ROS1-rearranged lung cancer.

Tyrosine kinase inhibitors (TKIs) that target the ROS proto-oncogene 1, receptor tyrosine kinase (ROS1) gene have shown dramatic therapeutic effects in patients with ROS1-rearranged non-small-cell lung cancer (NSCLC). Nevertheless, advanced ROS1-rearranged NSCLC is rarely cured as a portion of the tumor cells can survive the initial stages of ROS1-TKI treatment, even after maximum tumor shrinkage. Therefore, understanding the mechanisms underlying initial cell survival during ROS1-TKI treatment is necessary to prevent cell survival and achieve a cure for ROS1-rearranged NSCLC. In this study, we clarified the initial survival mechanisms during treatment with lorlatinib, a ROS1 TKI. First, we established a patient-derived ezrin gene-ROS1-rearranged NSCLC cell line (KTOR71). Then, following proteomic analysis, we focused on yes-associated protein 1 (YAP1), which is a major mediator of the Hippo pathway, as a candidate factor involved in cell survival during early lorlatinib treatment. Yes-associated protein 1 was activated by short-term lorlatinib treatment both in vitro and in vivo. Genetic inhibition of YAP1 using siRNA, or pharmacological inhibition of YAP1 function by the YAP1-inhibitor verteporfin, enhanced the sensitivity of KTOR71 cells to lorlatinib. In addition, the prosurvival effect of YAP1 was exerted through the reactivation of AKT. Finally, combined therapy with verteporfin and lorlatinib was found to achieve significantly sustained tumor remission compared with lorlatinib monotherapy in vivo. These results suggest that YAP1 could mediate initial cell resistance to lorlatinib in KTOR71 cells. Thus, combined therapy targeting both YAP1 and ROS1 could potentially improve the outcome of ROS1-rearranged NSCLC.

Massively parallel single-cell genomics of microbiomes in rice paddies.

Plant growth-promoting microbes (PGPMs) have attracted increasing attention because they may be useful in increasing crop yield in a low-input and sustainable manner to ensure food security. Previous studies have attempted to understand the principles underlying the rhizosphere ecology and interactions between plants and PGPMs using ribosomal RNA sequencing, metagenomic sequencing, and genome-resolved metagenomics; however, these approaches do not provide comprehensive genomic information for individual species and do not facilitate detailed analyses of plant-microbe interactions. In the present study, we developed a pipeline to analyze the genomic diversity of the rice rhizosphere microbiome at single-cell resolution. We isolated microbial cells from paddy soil and determined their genomic sequences by using massively parallel whole-genome amplification in microfluidic-generated gel capsules. We successfully obtained 3,237 single-amplified genomes in a single experiment, and these genomic sequences provided insights into microbial functions in the paddy ecosystem. Our approach offers a promising platform for gaining novel insights into the roles of microbes in the rice rhizomicrobiome and to develop microbial technologies for improved and sustainable rice production.

Evaluation of a library of loxP variants with a wide range of recombination efficiencies by Cre.

Sparse labeling of individual cells is an important approach in neuroscience and many other fields of research. Various methods have been developed to sparsely label only a small population of cells; however, there is no simple and reproducible strategy for managing the probability of sparse labeling at desired levels. Here, we aimed to develop a novel methodology based on the Cre-lox system to regulate sparseness at desired levels, and we purely analyzed cleavage efficiencies of loxP mutants by Cre. We hypothesized that mutations in the loxP sequence reduce the recognition efficiency by Cre, which enables the regulation of the sparseness level of gene expression. In this research, we mutagenized the loxP sequence and analyzed a library of loxP variants. We evaluated more than 1000 mutant loxP sequences, including mutants with reduced excision efficiencies by Cre ranging from 0.51% to 59%. This result suggests that these mutant loxP sequences can be useful in regulating the sparseness of genetic labeling at desired levels.

Enhancement of PET degradation by PET depolymerase with the microbe addition.

The degradation of polyethylene terephthalate (PET) by modified PET depolymerase has recently attracted much attention. We found that mixing a PET depolymerase with non-genetically modified Thermus sp. can enhance its PET-degrading activity by 7.7-fold. This approach is attractive for constructing a sustainable PET recycling system.

A critical role of an oxygen-responsive gene for aerobic nitrogenase activity in Azotobacter vinelandii and its application to Escherichia coli.

Since nitrogenase is irreversibly inactivated within a few minutes after exposure to oxygen, current studies on the heterologous expression of nitrogenase are limited to anaerobic conditions. This study comprehensively identified genes showing oxygen-concentration-dependent expression only under nitrogen-fixing conditions in Azotobacter vinelandii, an aerobic diazotroph. Among the identified genes, nafU, with an unknown function, was greatly upregulated under aerobic nitrogen-fixing conditions. Through replacement and overexpressing experiments, we suggested that nafU is involved in the maintenance of nitrogenase activity under aerobic nitrogenase activity. Furthermore, heterologous expression of nafU in nitrogenase-producing Escherichia coli increased nitrogenase activity under aerobic conditions by 9.7 times. Further analysis of NafU protein strongly suggested its localization in the inner membrane and raised the possibility that this protein may lower the oxygen concentration inside the cells. These findings provide new insights into the mechanisms for maintaining stable nitrogenase activity under aerobic conditions in A. vinelandii and provide a platform to advance the use of nitrogenase under aerobic conditions.

Tricholoma alpinum, sp. nov., under five-needle pines in alpine and subalpine zones in Japan.

Alpine regions in Japan are characterized by the Siberian dwarf pine, Pinus pumila. Although these regions are conserved as national parks due to their unique biome, few reports of the fungal biota are available. We examined mushroom fungi under such vegetation and found a unique yellowish-capped Tricholoma species. Multilocus molecular phylogenetic analyses and morphological observation of specimens showed that the Tricholoma species is very similar to T. fumosoluteum known in North America. The Japanese yellowish-capped T. aff. fumosoluteum had larger basidiospores and basidia and shorter pileipellis hyphae compared with T. fumosoluteum. Therefore, we describe the Japanese entity as a new species, T. alpinum.

Production of Single-Domain Antibodies in Pichia pastoris.

Single-domain antibodies (sdAbs) are binders that consist of a single immunoglobulin domain. SdAbs have gained importance as therapeutics, diagnostic reagents, and research tools. Functional sdAbs are commonly produced in Escherichia coli, which is a simple and widely used host for production of recombinant proteins. However, there are drawbacks of the E. coli expression system, including the potential for misfolded recombinant proteins and pyrogenic contamination with toxic lipopolysaccharides. Pichia pastoris is an alternative host for the production of heterologous proteins because of its high recombinant protein yields and the ability to produce soluble, properly folded proteins without lipopolysaccharide contamination. Here, we describe a method to produce sdAbs in P. pastoris. We present methods for the cloning of sdAb-encoding genes into a P. pastoris expression vector, production and purification of sdAbs, and measurement of sdAb-binding kinetics. Functional sdAbs are easily and routinely obtained using these methods.

New findings on the fungal species Tricholoma matsutake from Ukraine, and revision of its taxonomy and biogeography based on multilocus phylogenetic analyses.

Matsutake mushrooms are among the best-known edible wild mushroom taxa worldwide. The representative Tricholoma matsutake is from East Asia and the northern and central regions of Europe. Here, we report the existence of T. matsutake under fir trees in Eastern Europe (i.e., Ukraine), as confirmed by phylogenetic analysis of nine loci on the nuclear and mitochondrial genomes. All specimens from Japan, Bhutan, China, North Korea, South Korea, Sweden, Finland, and Ukraine formed a T. matsutake clade according to the phylogeny of the internal transcribed spacer region. The European population of T. matsutake was clustered based on the ß2 tubulin gene, with a moderate bootstrap value. In contrast, based on analyses of three loci, i.e., rpb2, tef1, and the ß2 tubulin gene, T. matsutake specimens sampled from Bhutan and China belonged to a clade independent of the other specimens of this species, implying a genetically isolated population. As biologically available type specimens of T. matsutake have not been designated since its description as a new species from Japan in 1925, we established an epitype of this fungus, sampled in a Pinus densiflora forest in Nagano, Japan.

Efficient automated semi-quantitative urine culture analysis via BD Urine Culture App.

We aimed to assess the clinical utility of BD KiestraTM Urine Culture App (UCA). High concordance rates were observed between the urine culture colony counts obtained by medical technologists and those produced using UCA. This application may increase the efficiency of obtaining semi-quantitative urine culture results.