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Proteome is extremely complex as many proteins with a large dynamic range are involved. Nano-liquid chromatography/mass spectrometry-based proteomics has made it possible to separate and identify thousands of proteins in one shot. Although the number of identified proteins in proteomics has significantly improved, it is necessary to increase detection sensitivity to clearly identify low-abundant proteins. In this study, we developed meter-long monolithic columns with a small inner diameter and applied them to selected reaction monitoring-based proteomics for improving proteomic detection sensitivity. Bovine serum albumin tryptic digests were analyzed with optimized selected reaction monitoring methods, and separation efficiency and detection sensitivity in each monolithic column were evaluated. As a result, peak capacity increased by about 1.8-fold and peak area of peptide levels increased by about 2.3-fold. Although flow rate was reduced during analysis with columns of a smaller inner diameter, the peak area reproducibility was maintained. These data displayed the value of meter-long monolithic columns with small inner diameter for selected reaction monitoring-based proteomics. [ABSTRACT FROM AUTHOR]

The demand for ammonia is expected to increase in the future because of its importance in agriculture, industry, and hydrogen transportation. Although the Haber-Bosch process is known as an effective way to produce ammonia, the process is energy-intensive. Thus, an environmentally friendly ammonia production process is desired. In this study, we aimed to produce ammonia from amino acids and amino acid-based biomass-like resources by modifying the metabolism of Escherichia coli. By engineering metabolic flux to promote ammonia production using the overexpression of the ketoisovalerate decarboxylase gene ( kivd), derived from Lactococcus lactis, ammonia production from amino acids was 351 mg/L (36.6% yield). Furthermore, we deleted the glnA gene, responsible for ammonia assimilation. Using yeast extract as the sole source of carbon and nitrogen, the resultant strain produced 458 mg/L of ammonia (47.8% yield) from an amino acid-based biomass-like material. The ammonia production yields obtained are the highest reported to date. This study suggests that it will be possible to produce ammonia from waste biomass in an environmentally friendly process. [ABSTRACT FROM AUTHOR]

Ten secreted aspartic proteases (Saps) of Candida albicans cleave numerous peptides and proteins in the host organism and deregulate its homeostasis. Human kininogens contain two internal antimicrobial peptide sequences, designated NAT26 and HKH20. In our current study, we characterized a Sap-catalyzed cleavage of kininogen-derived antimicrobial peptides that results in the loss of the anticandidal activity of these peptides. The NAT26 peptide was effectively inactivated by all Saps, except Sap10, whereas HKH20 was completely degraded only by Sap9. Proteolytic deactivation of the antifungal potential of human kininogens can help the pathogens to modulate or evade the innate immunity of the host. [ABSTRACT FROM AUTHOR]

• We performed large-scale universal PCR screening for SARS-CoV-2. • Patients who were asymptomatic before admission and their attendants were screened. • Universal PCR screening may be useful for detecting SARS-CoV-2 infected patients. Universal severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; i.e., the causative agent of coronavirus disease 2019 [COVID-19]) polymerase chain reaction (PCR) screening before admission has been adopted by several hospitals to prevent nosocomial SARS-CoV-2 transmission from asymptomatic and pre-symptomatic patients. However, screening usefulness remains unclear because it depends on the regional COVID-19 prevalence, and only a few large-scale studies have been reported. Here we describe the universal PCR screening performed in our hospital before admission of more than 12,000 patients and their attendants to evaluate the usefulness of the screening. We retrospectively described the universal PCR screening results for asymptomatic patients and their attendants before planned admissions at a hospital in Tokyo, Japan, from August 3, 2020, through March 31, 2021. Nasopharyngeal swab samples were collected at an in-hospital PCR center. In total, 12,133 persons (11,859 asymptomatic patients and 274 attendants) underwent PCR screening; nine (0.07%) tested positive for SARS-CoV-2 RNA. Universal PCR screening may be useful for the advanced detection of SARS-CoV-2 infected patients with or without symptoms, which can be a potential source of nosocomial SARS-CoV-2 transmission. [ABSTRACT FROM AUTHOR]

The Na+-pumping NADH-ubiquinone oxidoreductase (Na+-NQR) is a main ion transporter in many pathogenic bacteria. We previously proposed that N -terminal stretch of the NqrB subunit plays an important role in regulating the ubiquinone reaction at the adjacent NqrA subunit in Vibrio cholerae Na+-NQR. However, since approximately three quarters of the stretch (NqrB-Met1–Pro37) was not modeled in an earlier crystallographic study, its structure and function remain unknown. If we can develop a method that enables pinpoint modification of this stretch by functional chemicals (such as spin probes), it could lead to new ways to investigate the unsettled issues. As the first step to this end, we undertook to specifically attach an alkyne group to a lysine located in the stretch via protein-ligand affinity-driven substitution using synthetic ligands NAS-K1 and NAS-K2. The alkyne, once attached, can serve as an "anchor" for connecting functional chemicals via convenient click chemistry. After a short incubation of isolated Na+-NQR with these ligands, alkyne was predominantly incorporated into NqrB. Proteomic analyses in combination with mutagenesis of predicted target lysines revealed that alkyne attaches to NqrB-Lys22 located at the nonmodeled region of the stretch. This study not only achieved the specific modification initially aimed for but also provided valuable information about positioning of the nonmodeled region. For example, the fact that hydrophobic NAS-Ks come into contact with NqrB-Lys22 suggests that the nonmodeled region may orient toward the membrane phase rather than protruding into cytoplasmic medium. This conformation may be essential for regulating the ubiquinone reaction in the adjacent NqrA. • Korormicin is a highly selective inhibitor of Na+-NQR from Vibrio cholerae. • Korormicin molecular probes that specifically react with lysine were synthesized. • Lys22 located in the N -terminal stretch of NqrB subunit was modified by the probes. • The specific modification explores the dynamic structure of the N -terminal stretch. [ABSTRACT FROM AUTHOR]

R1974 --> Background: Neural networks generate complex behaviors. The relationships between neural networks and behaviors aren't understood well. Optogenetics is a powerful technique that enables on‐demand photo‐regulation of neural activity and exploration of the mechanism of neural computation. Conventional optogenetics presents three difficult challenges: 1. a hypothesis is necessary to conduct experiments; 2. optogenetics is low‐throughput because different transformants are required to test each hypothesis: and 3. single‐cell analysis is difficult due to the lack of single‐cell‐specific promoters. To overcome these difficulties, we have developed a new methodology named "functional cellomics," which can annotate the function of neural networks in a hypothesis‐free, high‐throughput, and single‐cell resolution manner. The key factor is the stochastic labeling of opsin by applying the Cre‐lox system. We designed a genetic circuit in which two sets of lox variants, lox2272 and loxP sequences, are inserted alternately. A transcription factor, QF2w, is interposed between loxP sequences. Induced Cre excises DNA exclusively either between loxP sequences or between lox2272 sequences in every single cell. When Cre is allowed to act on lox2272 sequences, QF2w is expressed. Then, QF2w induces expression of opsin. We succeeded in the stochastic labeling of opsin dependent on the induction of Cre. However, the labeling rate of opsin was about 30% [1]. If opsin is labeled in many neurons, it becomes difficult to distinguish the function of individual neurons. In this study, we developed a new methodology to enable precise control of the labeling rate of opsin to conduct an in‐depth analysis of the function of the nervous system. Methods: In our genetic circuit, the opsin is expressed in neurons in which the Cre‐lox recombination event occurs between the lox2272 sequences. Thus, we hypothesized that the labeling rate of opsin reduces by using lox2272 variants which is difficult to be excised by Cre. We generated a library of randomized lox sequences by PCR and introduced them in S. cerevisiae. Then, we induced Cre production in the S. cerevisiae library and evaluated the excision rates of lox2272 variants by using NGS. To develop a machine learning model to predict the cleavage rate of lox2272 variants, we trained a Gaussian process (GP) model using data containing lox2272 variant sequences and their cleavage rates by Cre. We randomly chose 1000 lox2272 variants as training data and 100 variants to test prediction accuracy. Results and Discussion: We quantified the excision rates of more than 2000 lox2272 variants by NGS and successfully found variants that have excision efficiencies ranging from 0.05% to 100%. We confirmed the results of NGS analysis by an in vivo assay using S. cerevisiae and qPCR. Then, we build a GP model and observed a high correlation between the actual cleavage rates and predicted cleavage rates of the test data (R = 0.93). We used the GP model to predict the efficiency of unevaluated lox2272 sequences and succeeded in the identification of lox2272 variants that had various cleavage rates. These results indicate the feasibility of precise control of the labeling rate of opsin in functional cellomics [2]. 1. Aoki et al, Sci Rep, 8, 10380, 2018. 2. Yamauchi et al, submitted. [ABSTRACT FROM AUTHOR]

R1555 --> 【Research objective】: Due to the constraints regarding cell growth, modification of ribosomal genes in vivo is challenging. The reconstruction of E. coli ribosomes in vitro will be an important step to engineer ribosomes. We are trying to reconstruct the ribosome biogenesis process in E. coli lysates by expressing all of the ribosomal components so that the transcription, translation, and assembly of ribosomal building blocks proceed cooperatively. For the improvement and the validation of this strategy, analytical tools for ribosomal proteins (r‐proteins) are necessary. From this point of view, we tried to develop a highly‐specific method for the quantification of all of the ribosomal proteins (r‐proteins) by using selected reaction monitoring (SRM). The SRM methods will enable highly specific quantification of r‐proteins even if their concentrations are very low in samples. 【Methods】: In this study, we analyzed r‐proteins derived from purified E. coli ribosome, E. coli lysate, and r‐protein‐overexpressed E. coli lysate using nanoLC–MS/MS (LCMS‐8060) and developed optimized SRM methods for each r‐protein. 【Results & Discussion】: Using the SRM methods, we successfully quantified 41 of 54 nascent r‐proteins synthesized in E. coli lysate. To improve production efficiencies of r‐proteins, we optimized translation enhancer region of the plasmids encoding r‐proteins. In conclusion, we successfully developed a highly‐specific and comprehensive analytical method for r‐proteins, which will promote the study of ribosome reconstruction as well as the understanding of ribosome biogenesis. [ABSTRACT FROM AUTHOR]

R2247 --> Background: Binding proteins such as antibodies are useful for basic science, diagnosis, and therapy. Nanobodies are also promising binding proteins that achieve comparable binding affinities and specificities to conventional antibodies, despite comprising only a single 15 kDa variable domain. The generation of functional nanobodies typically involves phage, yeast, or ribosome display, which allows screening billions of nanobodies simultaneously. However, display technologies have some drawbacks, one of which is the immobilization effect. The immobilization of nanobodies on the cell surface is necessary to establish genotype–phenotype linkages, but this can affect nanobody functions. Therefore, we have developed peptide barcoding, a technology with which we can evaluate free nanobodies with low bias [1]. Here, we applied the technology to the alanine scanning of anti‐GFP nanobodies and enabled multiplex evaluation of affinities of free nanobodies. Method: We used GFP and anti‐GFP nanobody as model antigen and antibody. GFP was produced by E. coli BL21 (DE3) strain. Anti‐GFP nanobodies were produced by P. pastoris GS115 strain. To construct the alanine‐scanning nanobody library, we substituted each non‐alanine residue of the anti‐GFP nanobody for alanine and prepared 106 kinds of nanobodies. Each nanobody was attached to a genetically encoded unique peptide barcode. The 106 nanobody–peptide barcode fusion proteins (barcodebodies) were produced in one pot by P. pastoris, digested with trypsin, and applied to LC‐MS/MS to confirm the detectability of the peptide barcodes. The peptide barcodes, in this study, were derived from yeast SRM Atlas where tens of thousands of proteotypic peptides are registered [2]. The barcodebody library was mixed with GFP and separated with size‐exclusion chromatography depending on the affinity. The fractions containing high‐affinity barcodebodies or low‐affinity barcodebodies were digested with trypsin and applied to LC‐MS/MS. Results & Discussion: We detected more than 95% of the peptide barcode by LC‐MS/MS. In the fraction containing low‐affinity barcodebodies, we simultaneously identified five peptide barcodes, and in the fraction containing high‐affinity barcodebodies, we identified the other barcodes. We measured the affinity of these five nanobodies by surface plasmon resonance (SPR) and confirmed their decrease of affinities. These results demonstrated the practicality and utility of the peptide barcoding for one‐pot multiplex evaluation of non‐immobilized nanobodies and suggested that this technology could be applied to more high‐throughput screening. [1] Miyamoto et al., PLOS ONE 14(4): e0215993, 2019 [2] Picotti et al., Nature 494(7436), 266‐270, 2013 [ABSTRACT FROM AUTHOR]

Easy preparation of chimeric nanobodies with various scaffolds is important for customizing abilities of nanobodies toward practical utilization. To accomplish high-throughput production of various nanobodies, utilization of microbes is an attractive option. In the present study, various chimeric nanobodies were prepared using the methylotrophic yeast Pichia pastoris. We designed chimeric nanobodies with complementarity-determining regions (CDRs) against green fluorescent protein (GFP) or cluster of differentiation 4 (CD4) based on the scaffold of GFP-nanobody. FLAG-tagged chimeric nanobodies were prepared by one-step cloning and produced using P. pastoris. Secreted chimeric nanobodies were purified from the culture media of P. pastoris transformants. Relative binding abilities of purified chimeric nanobodies to GFP and CD4 was tested using a BIACORE T-200. P. pastoris successfully produced a high yield of FLAG-tagged chimeric nanobodies. FLAG-tagged GFP- and CD4-nanobodies were shown to specifically bind to GFP and CD4, respectively. Chimeric nanobodies, in which the CDR2 or 3 of GFP-nanobody was replaced with CDRs of CD4-nanobody, acquired the ability to bind to CD4 without binding to GFP. These results demonstrate successful production of functional chimeric nanobodies using P. pastoris. These results also suggest that swapping of CDRs, especially CDRs 2 or 3, potentially enables a novel method of creating nanobodies. [ABSTRACT FROM AUTHOR]

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., (© 2024. The Author(s) under exclusive licence to The Japanese Society of Pharmacognosy.)

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., (Copyright © 2024 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: This study was supported by Kyokuto Pharmaceutical Industrial., co. Ltd. (Tokyo, Japan). Kyokuto Pharmaceutical Industrial., co. Ltd. manufactures and distributes BrothMIC NTM panels and BrothMIC SGM panels.

We evaluated the inclusion of a cryptic species in a Japanese Amanita caesareoides population. We sampled A. caesareoides specimens under various vegetation and climate conditions, and then conducted phylogenetic analyses on sequences from seven loci. The A. caesareoides specimens showed two distinct groups, except when the ITS phylogeny was considered. These two phylogroups showed different distributions: subalpine-cool temperate and temperate-subtropical areas. Although these two phylogroups overlapped in terms of basidiospore size, the latter tended to exhibit smaller basidiospores. In addition, only the former showed mycelial growth on nutrient agar. Based on these phylo-morpho-ecophysiological characteristics, we separated the specimens labeled with the name A. caesareoides into two species. As the lectotype of A. caesareoides showed similarity to the former by DNA analysis, the latter was described as a new species, namely A. satotamagotake . Based on the geographic patterns of the two species, A. satotamagotake may have invaded the natural habit of A. caesareoides because of global warming., Competing Interests: The authors of this study have no conflicts of interest to declare., (2024, by The Mycological Society of Japan.)

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., (Copyright © 2023 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2023 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

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 d 0 -transition-metal cations such as Ti 4+ , Zr 4+ , and Nb 5+ as B -site elements were successfully synthesized by a sol-gel method using dicarboxylic acids. Moreover, the specific surface area of SrTiO 3 was increased to 46 m 2 g -1 by a simple procedure of changing the atmosphere from N 2 to air during calcination of an amorphous precursor. The resultant SrTiO 3 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, CO 2 , and CHCl 3 , 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 SrTiO 3 most likely enable SrTiO 3 to act as a bifunctional acid-base solid catalyst through cooperative activation of carbonyl compounds and TMSCN. This bifunctional catalysis through SrTiO 3 resulted in high catalytic performance even without a heat pretreatment, in sharp contrast to the performance of basic MgO and acidic TiO 2 catalysts.

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., (© 2022 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

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.

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., Competing Interests: Kyoto Agriculture Research Institute KARI provided support in the form of salaries for KM, SH, and TM. MH and HT are shareholders in bitBiome, Inc., which provides single-cell genomics services using the SAG-gel workflow as bit-MAP. MK is employed at bitBiome, Inc. SM is employed at WORLD INTEC 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 © 2022 Aoki, Kogawa, Matsuda, Matsubara, Hirata, Nishikawa, Hosokawa, Takeyama, Matoh and Ueda.)

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., Competing Interests: The authors have declared that no competing interests exist.

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 β2 tubulin gene, with a moderate bootstrap value. In contrast, based on analyses of three loci, i.e., rpb 2, tef 1, and the β2 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., (2022, by The Mycological Society of Japan.)

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

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., (© 2022. The Author(s).)

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 .

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., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Optimisation of protein binders relies on laborious screening processes. Investigation of sequence-function relationships of protein binders is particularly slow, since mutants are purified and evaluated individually. Here we developed peptide barcoding, a high-throughput approach for accurate investigation of sequence-function relationships of hundreds of protein binders at once. Our approach is based on combining the generation of a mutagenised nanobody library fused with unique peptide barcodes, the formation of nanobody-antigen complexes at different ratios, their fine fractionation by size-exclusion chromatography and quantification of peptide barcodes by targeted proteomics. Applying peptide barcoding to an anti-GFP nanobody as a model, we successfully identified residues important for the binding affinity of anti-GFP nanobody at once. Peptide barcoding discriminated subtle changes in K D at the order of nM to sub-nM. Therefore, peptide barcoding is a powerful tool for engineering protein binders, enabling reliable one-pot evaluation of sequence-function relationships., (© 2021. The Author(s).)

" Kakishimeji " identified as Tricholoma ustale and belonging to Tricholoma sect. Genuina is a common poisonous mushroom in Japan. Kakishimeji contains the toxic compound ustalic acid and causes digestive trouble. However, this fungus is consumed in some regions of Japan without any digestive issues. We clarified the probable species complex of Kakishimeji based on a phylogenetic analysis. We collected 89 basidioma specimens of Kakishimeji and related species from various forest sites in Japan and conducted phylogenetic analyses using 7 nuclear and mitochondrial gene sequences. Kakishimeji was found to consist of four distinct phylogenetic clades based on all DNA regions tested. Of these, two clades included European T. stans and T. albobrunneum type specimens. Another two clades consisted of sister clades to T. pessundatum and T. ustaloides . In addition, all four phylogenetic clades of Kakishimeji had different spore and basidium sizes. Therefore, we regarded the latter two clades as two new Tricholoma species: T. kakishimeji and T. kakishimejioides ., (2021, by The Mycological Society of Japan.)

Introduction: Rapid antigen detection (RAD) tests are convenient tools for detecting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in clinics, and testing using saliva samples could decrease the risk of infection during sample collection. This study aimed to assess the accuracy of the SARS-CoV-2 RAD for testing of nasopharyngeal swab specimens and saliva samples in comparison with the RT-PCR tests and viral culture for detecting viable virus., Methods: One hundred seventeen nasopharyngeal swab specimens and 73 saliva samples with positive results on RT-PCR were used. Residual samples were assayed using a commercially available RAD test immediately, and its positivity was determined at various time points during the clinical course. The concordance between 54 nasopharyngeal swab samples and saliva samples that were collected simultaneously was determined. Viral culture was performed on 117 samples and compared with the results of the RAD test., Results: The positive rate of RAD test using saliva samples was low throughout the clinical course. Poor concordance was observed between nasopharyngeal swab specimens and saliva samples (75.9%, kappa coefficient 0.310). However, a substantially high concordance between the RAD test and viral culture was observed in both nasopharyngeal swab specimens (86.8%, kappa coefficient 0.680) and saliva samples (95.1%, kappa coefficient 0.643)., Conclusions: The sensitivity of the SARS-CoV-2 RAD test was insufficient, particularly for saliva samples. However, a substantially high concordance with viral culture suggests its potential utility as an auxiliary test for estimating SARS-CoV-2 viability., (Copyright © 2021 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

The development of methods for the detection and qualification of toxic substances in mushrooms is a rapidly growing research area in forensic toxicology. This study aimed to determine liquid chromatographic and mass spectrometric conditions applicable to the analysis of ustalic acid (UA) in Tricholoma ustale. We used HPLC coupled with single quadrupole mass spectrometry (Q-MS) with electrospray ionization in its negative ion mode to analyze UA. We performed HPLC separations on a C18 reversed-phase column with gradient elution using mobile phases containing water, acetonitrile, and formic acid. The MS showed that UA formed the deprotonated molecular ion [M-H] - at m/z 337, which was sufficient for the quantitative analysis of the compound. The average recovery rates of UA from four edible mushrooms (Flammulina velutipes, Pleurotus eryngii, Lentinula edodes, and Grifola frondosa) to which 10.0 μg/g of UA was added were 108%, 104%, 108%, and 107%, respectively, and the relative standard deviation values ranged from 4.1 to 6.4%. Quantitative analysis of UA in three systematically collected individual mushrooms of T. ustale revealed 41.9-155.7 ppm in each dry material. We also explored the fragmentation behaviors of UA in triple quadrupole mass spectrometry and the proposed structures for the product ions. The data suggest that conventional Q-MS with authenticated UA would be able to identify this compound in T. ustale when used for the immediate inspection of incidences of poisoning. Confirmation of the presence of UA in T. ustale would ultimately allow for the chemotaxonomic discrimination of Tricholoma species.

Yeast cell surface display (YSD) has been used to engineer various proteins, including antibodies. Directed evolution, which subjects a gene to iterative rounds of mutagenesis, selection and amplification, is useful for protein engineering. In vivo continuous mutagenesis, which continuously diversifies target genes in the host cell, is a promising tool for accelerating directed evolution. However, combining in vivo continuous evolution and YSD is difficult because mutations in the gene encoding the anchor proteins may inhibit the display of target proteins on the cell surface. In this study, we have developed a modified YSD method that utilises SpyTag/SpyCatcher-based in vivo protein ligation. A nanobody fused with a SpyTag of 16 amino acids and an anchor protein fused with a SpyCatcher of 113 amino acids are encoded by separate gene cassettes and then assembled via isopeptide bond formation. This system achieved a high display efficiency of more than 90%, no intercellular protein ligation events, and the enrichment of target cells by cell sorting. These results suggested that our system demonstrates comparable performance with conventional YSD methods; therefore, it can be an appropriate platform to be integrated with in vivo continuous evolution.

Unlike other snakes, most species of Rhabdophis possess glands in their dorsal skin, sometimes limited to the neck, known as nucho-dorsal and nuchal glands, respectively. Those glands contain powerful cardiotonic steroids known as bufadienolides, which can be deployed as a defense against predators. Bufadienolides otherwise occur only in toads (Bufonidae) and some fireflies (Lampyrinae), which are known or believed to synthesize the toxins. The ancestral diet of Rhabdophis consists of anuran amphibians, and we have shown previously that the bufadienolide toxins of frog-eating species are sequestered from toads consumed as prey. However, one derived clade, the Rhabdophis nuchalis Group, has shifted its primary diet from frogs to earthworms. Here we confirm that the worm-eating snakes possess bufadienolides in their nucho-dorsal glands, although the worms themselves lack such toxins. In addition, we show that the bufadienolides of R. nuchalis Group species are obtained primarily from fireflies. Although few snakes feed on insects, we document through feeding experiments, chemosensory preference tests, and gut contents that lampyrine firefly larvae are regularly consumed by these snakes. Furthermore, members of the R. nuchalis Group contain compounds that resemble the distinctive bufadienolides of fireflies, but not those of toads, in stereochemistry, glycosylation, acetylation, and molecular weight. Thus, the evolutionary shift in primary prey among members of the R. nuchalis Group has been accompanied by a dramatic shift in the source of the species' sequestered defensive toxins., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Yeast surface display is a powerful technology used to isolate and engineer proteins to improve their activity, specificity, and stability. In this method, gene expression is regulated by promoters, and secretion efficiency is affected by secretion signals. Furthermore, both the accessibility and activity of the displayed proteins are affected by the length of anchor proteins. The ideal promoter, secretion signal, and anchor protein combination depend on the proteins of interest. In this study, we optimized a yeast surface display suitable for nanobody evaluation. We designed five display systems that used different combinations of promoters, secretion signals, and anchor proteins. Anti-hen egg-white lysozyme nanobody was used as the model nanobody. The amount of nanobodies displayed on yeast cells, the number of antigens bound to the displayed nanobodies, and the display efficiency were quantified. Overall, we improved the yeast display system for nanobody engineering and proposed its optimization.

Despite the promising clinical efficacy of the second-generation anaplastic lymphoma kinase (ALK) inhibitor alectinib in patients with ALK-rearranged lung cancer, some tumor cells survive and eventually relapse, which may be an obstacle to achieving a cure. Limited information is currently available on the mechanisms underlying the initial survival of tumor cells against alectinib. Using patient-derived cell line models, we herein demonstrate that cancer cells survive a treatment with alectinib by activating Yes-associated protein 1 (YAP1), which mediates the expression of the anti-apoptosis factors Mcl-1 and Bcl-xL, and combinatorial inhibition against both YAP1 and ALK provides a longer tumor remission in ALK-rearranged xenografts when compared with alectinib monotherapy. These results suggest that the inhibition of YAP1 is a candidate for combinatorial therapy with ALK inhibitors to achieve complete remission in patients with ALK-rearranged lung cancer.

Since G-protein coupled receptors (GPCRs) are linked to various diseases, screening of functional ligands against GPCRs is vital for drug discovery. In the present study, we developed a high-throughput functional cell-based assay by combining human culture cells producing a GPCR, yeast cells secreting randomized peptide ligands, and a droplet microfluidic device. We constructed a reporter human cell line that emits fluorescence in response to the activation of human glucagon-like peptide-1 receptor (hGLP1R). We then constructed a yeast library secreting an agonist of hGLP1R or randomized peptide ligands. We demonstrated that high-throughput identification of functional ligands against hGLP1R could be performed by co-culturing the reporter cells and the yeast cells in droplets. We identified functional ligands, one of which had higher activity than that of an original sequence. The result suggests that our system could facilitate the discovery of functional peptide ligands of GPCRs.

The coral microbiome has attracted increased attention because of its potential roles in host protection against deadly diseases. However, little is known about the role of coral-associated bacteria against the temperature-dependent opportunistic pathogen Vibrio coralliilyticus. In this study, we tested whether bacteria associated with the reef-building coral Galaxea fascicularis could inhibit the growth of V. coralliilyticus. Twenty-nine cultivable bacteria were successfully isolated from a healthy colony of G. fascicularis kept in an aquarium. Among the bacterial isolates, three Ruegeria sp. strains inhibited the growth of V. coralliilyticus P1 as a reference strain and Vibrio sp. isolated in this study. Ruegeria sp. strains were also detected from other G. fascicularis colonies in the aquarium and in previous field studies by 16S rRNA amplicon sequencing, suggesting that Ruegeria sp. strains are common among G. fascicularis colonies. These results illuminate the potential role of Ruegeria sp. in protecting corals against pathogenic Vibrio species.

Coral reefs are one of the most biologically diverse and economically important ecosystems on earth. However, the destruction of coral reefs has been reported worldwide owing to rising seawater temperature associated with global warming. In this study, we investigated the potential of a redox nanoparticle (RNP O ) to scavenge reactive oxygen species (ROS), which are overproduced under heat stress and play a crucial role in causing coral mortality. When reef-building coral (Acropora tenuis) larvae, without algal symbionts, were exposed to thermal stress at 33 °C, RNP O treatment significantly increased the survival rate. Proteome analysis of coral larvae was performed using nano-liquid chromatography-tandem mass spectrometry for the first time. The results revealed that several proteins related to ROS-induced oxidative stress were specifically identified in A. tenuis larvae without RNP O treatment, whereas these proteins were absent in RNP O -treated larvae, which suggested that RNP O effectively scavenged ROS from A. tenuis larvae. Results from this study indicate that RNP O treatment can reduce ROS in aposymbiotic coral larvae and would be a promising approach for protecting corals from thermal stress.

In Caenorhabditis elegans, which has only 302 neurons, relationships between behaviors and neural networks are not easily elucidated. In this study, we proposed a novel cellomics approach enabling high-throughput and comprehensive exploration of the functions of a single neuron or a subset of neurons in a complex neural network on a particular behavior. To realize this, we combined optogenetics and Brainbow technologies. Using these technologies, we established a C. elegans library where opsin is labeled in a randomized pattern. Behavioral analysis on this library under light illumination enabled high-throughput annotation of neurons affecting target behaviors. We applied this approach to the egg-laying behavior of C. elegans and succeeded in high-throughput confirmation that hermaphrodite-specific neurons play an important role in the egg-laying behavior. This cellomics approach will lead to the accumulation of neurophysiological and behavioral data of the C. elegans neural network, which is necessary for constructing neuroanatomically grounded models of behavior.

Main Conclusion: Latexes in immature fruit, young petioles and lignified trunks of fig trees protect the plant using toxic proteins and metabolites in various organ-dependent ways. Latexes from plants contain high amounts of toxic proteins and metabolites, which attack microbes and herbivores after exudation at pest-induced wound sites. The protein and metabolite constituents of latexes are highly variable, depending on the plant species and organ. To determine the diversity of latex-based defense strategies in fig tree (Ficus carica) organs, we conducted comparative proteomic, transcriptomic and metabolomic analyses on latexes isolated from immature fruit, young petioles and lignified trunks of F. carica after constructing a unigene sequence library using RNA-seq data. Trypsin inhibitors were the most abundant proteins in petiole latex, while cysteine proteases ("ficins") were the most abundant in immature fruit and trunk latexes. Galloylglycerol, a possible defense-related metabolite, appeared to be highly accumulated in all three latexes. The expression levels of pathogenesis-related proteins were highest in the latex of trunk, suggesting that this latex had adapted a defensive role against microbe attacks. Although young petioles and immature fruit are both unlignified soft organs, and potential food for herbivorous insects, unigenes for the sesquiterpenoid pathway, which likely produces defense-associated volatiles, and the phenylpropanoid pathway, which produces toxic furanocoumarins, were expressed less in immature fruit latex. This difference may indicate that while petioles and fruit protect the plant from attack by herbivores, the fruit must also attract insect pollinators at younger stages and animals after ripening. We also suggest possible candidate transcription factors and signal transduction proteins that are involved in the differential expression of the unigenes.

During 2006 through 2012, spontaneous group B Streptococcus infections were reported in 22 female KK-Ay mice, an animal model of type 2 diabetes. The affected mice were 5 to 27 wk old, and the cases involved various body sites, including cases of submandibular, caudal, and lumbar abscesses (n =18) or led to torticollis (n = 2), hydrocephalus (n = 1), or moribund clinical signs (n = 1). At necropsy, the mouse with hydrocephalus also demonstrated retained exudate in the uterus, and the moribund mouse showed renal inflammation. Streptococcus agalactiae was isolated in pure culture from all except 2 cases: the facial abscess also yielded Klebsiella pneumoniae, and the uterine exudate was coinfected with Staphylococcus aureus. In addition, S. agalactiae was isolated from the oral cavity and feces of normal KK-Ay mice. S. agalactiae potentially can cause a clinically significant spontaneous infection in a mouse model of diabetes.

Candida albicans, a causative agent of opportunistic fungal infections in immunocompromised patients, uses ten secreted aspartic proteases (SAPs) to deregulate the homeostasis of the host organism on many levels. One of these deregulation mechanisms involves a SAP-dependent disturbance of the control over proteolytic enzymes of the host by a system of dedicated proteinase inhibitors, with one important example being the neutrophil elastase and alpha1-proteinase inhibitor (A1PI). In this study, we found that soluble SAPs 1-4 and the cell membrane-anchored SAP9 efficiently cleaved A1PI, with the major cleavage points located at the C-terminal part of A1PI in a close vicinity to the reactive-site loop that plays a critical role in the inhibition mechanism. Elastase is released by neutrophils to the environment during fungal infection through two major processes, a degranulation or formation of neutrophil extracellular traps (NET). Both, free and NET-embedded elastase forms, were found to be controlled by A1PI. A local acidosis, resulting from the neutrophil activity at the infection sites, favors A1PI degradation by SAPs. The deregulation of NET-connected elastase affected a NET-dependent damage of epithelial and endothelial cells, resulting in the increased susceptibility of these host cells to candidal colonization. Moreover, the SAP-catalyzed cleavage of A1PI was found to decrease its binding affinity to a proinflammatory cytokine, interleukin-8. The findings presented here suggest a novel strategy used by C. albicans for the colonization of host tissues and overcoming the host defense.

Candida albicans is an opportunistic pathogen that causes fatal diseases in immunocompromised hosts. Host resistance against C. albicans relies on ingestion of the pathogen by macrophages. Analysis of the escaping behavior of C. albicans from macrophages is required to understand the onset of systemic candidiasis. In this study, native interactions of C. albicans with macrophages were investigated by proteome analysis using high efficiency of long monolithic silica capillary column. Using this system, we developed a method of "mixed and quantitative proteome analysis" in which C. albicans and macrophages were simultaneously analyzed by nanoLC-MS/MS without the need to isolate the two individual living cells. Two hundred twenty-seven proteins from C. albicans and five proteins from macrophages were identified as candidate interaction-specific molecules. C. albicans seemed to produce glucose through a β-oxidation pathway, a glyoxylate cycle, and gluconeogenesis for escape from macrophages. Up-regulation of stress-related and candidate pathogenic proteins in C. albicans indicated how C. albicans endured the harsh environment inside the macrophages. Down-regulation of apoptosis-associated protein NOA1- and chaperone HSPA1A-syntheses in macrophage indicated that C. albicans was able to escape from macrophages in part by suppressing the production of these macrophage proteins.

Candidiasis is a common fungal infection that is prevalent in immunocompromised individuals. In this study, an oral vaccine against Candida albicans was developed by using the molecular display approach. Enolase 1 protein (Eno1p) of C. albicans was expressed on the Lactobacillus casei cell surface by using poly-gamma-glutamic acid synthetase complex A from Bacillus subtilis as an anchoring protein. The Eno1p-displaying L. casei cells were used to immunize mice, which were later challenged with a lethal dose of C. albicans. The data indicated that the vaccine elicited a strong IgG response and increased the survival rate of the vaccinated mice. Furthermore, L. casei acted as a potent adjuvant and induced high antibody titers that were comparable to those induced by strong adjuvants such as the cholera toxin. Overall, the molecular display method can be used to rapidly develop vaccines that can be conveniently administered and require minimal processing.

Candida albicans is an opportunistic pathogen that causes fatal disease if the host immunity is compromised. The mortality rate of systemic candidiasis is very high; hence, there is a ceaseless demand for novel pharmaceuticals. In this study, quantitative time-course proteomics of C. albicans during adaptation to fetal bovine serum (FBS) is described. Survival in blood is essential for virulence of C. albicans, and a detailed analysis is required. We cultivated C. albicans in FBS for 0-180min, and determined quantitative time-course variations of 1024 proteins in the cultured cells by using a LC-MS/MS system with a long monolithic silica capillary column. Clustering analysis identified FBS-induced proteins associated with detoxification of oxidative species, high-affinity glucose transport, citrate cycle, oxidative phosphorylation, and iron acquisition. Furthermore, we identified possible virulence factors such as orf19.4914.1 (named Blood-induced peptide 1, Blp1). Heterologous expression of BLP1 in Saccharomyces cerevisiae shortened the lag phase and resulted in a pleiotropic stress-tolerance phenotype, indicating a possible role for quick adaptation to a stressful environment. While further experiments are necessary to prove virulence of the identified factors, systematic identification of candidate virulence proteins in this study will lead to profound understanding of virulence of C. albicans., Biological Significance: This paper describes time-course proteomics of C. albicans during adaptation to serum, which is an essential process for fatal systemic candidiasis. Using a LC-MS/MS system with a monolithic silica capillary column, we have successfully characterized time-course variations of 1024 proteins. Among them, orf19.4914.1 (Blp1) was identified as a novel pleiotropic stress-tolerance peptide, which could have an important role for virulence of C. albicans., (© 2013.)

We developed a novel method to coat living non-genetically modified (GM) cells with functional recombinant proteins. First, we prepared GM yeast to secrete constructed proteins that have two domains: a functional domain and a binding domain that recognizes other cells. Second, we cocultivated GM and non-GM yeasts that share and coutilize the medium containing recombinant proteins produced by GM yeasts using a filter-membrane-separated cultivation reactor. We confirmed that GM yeast secreted enhanced green fluorescent protein (EGFP) fusion proteins to culture medium. After cocultivation, EGFP fusion proteins produced by GM yeast were targeted to non-GM yeast (Saccharomyces cerevisiae BY4741DeltaCYC8 strain) cell surface. Yeast cell-surface engineering is a useful method that enables the coating of GM yeast cell surface with recombinant proteins to produce highly stable and accumulated protein particles. The results of this study suggest that development of cell-surface engineering from GM organisms (GMOs) to living non-GMOs by our novel cocultivation method is possible.

Poor detection of muscle motor evoked potential (m-MEP) by electrical cortical stimulation has often been encountered during neurosurgical operations, although widely known optimal factors for reliable detection have been put into practice. We hypothesized that the depth of anesthesia influenced the detection of m-MEP. Bispectral Index (BIS) monitoring was used to assess the depth of anesthesia for 16 patients during brain tumor operations. The doses of intravenous anesthetics, propofol, were titrated according to BIS indices during operations. When BIS indices were 40 or below, detection was difficult and higher intensities of electrical stimulation were necessary to evoke m-MEPs. Furthermore, risk of seizure was relatively high due to stronger electrical stimulation. Our results suggested that a BIS index above 50 and below 70 was optimal range. BIS monitoring is a useful method for setting the optimal dose of anesthetics for reliable intraoperative cortical mapping.