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Background: Cell-cell interaction factors that facilitate the progression of adenoma to sporadic colorectal cancer (CRC) remain unclear, thereby hindering patient survival., Methods: We performed spatial transcriptomics on five early CRC cases, which included adenoma and carcinoma, and one advanced CRC. To elucidate cell-cell interactions within the tumour microenvironment (TME), we investigated the colocalisation network at single-cell resolution using a deep generative model for colocalisation analysis, combined with a single-cell transcriptome, and assessed the clinical significance in CRC patients., Findings: CRC cells colocalised with regulatory T cells (Tregs) at the adenoma-carcinoma interface. At early-stage carcinogenesis, cell-cell interaction inference between colocalised adenoma and cancer epithelial cells and Tregs based on the spatial distribution of single cells highlighted midkine (MDK) as a prominent signalling molecule sent from tumour epithelial cells to Tregs. Interaction between MDK-high CRC cells and SPP1+ macrophages and stromal cells proved to be the mechanism underlying immunosuppression in the TME. Additionally, we identified syndecan4 (SDC4) as a receptor for MDK associated with Treg colocalisation. Finally, clinical analysis using CRC datasets indicated that increased MDK/SDC4 levels correlated with poor overall survival in CRC patients., Interpretation: MDK is involved in the immune tolerance shown by Tregs to tumour growth. MDK-mediated formation of the TME could be a potential target for early diagnosis and treatment of CRC., Funding: Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Science Research; OITA Cancer Research Foundation; AMED under Grant Number; Japan Science and Technology Agency (JST); Takeda Science Foundation; The Princess Takamatsu Cancer Research Fund., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024. Published by Elsevier B.V.)

Patient-derived tumor organoids have considerable potential as an in vitro diagnostic tool for drug susceptibility testing. In the present study, we investigated whether bile collected for diagnostic purposes could be a potential source for the establishment of biliary cancer organoids. Among 68 cases of biliary cancer, we successfully generated 60 bile-derived organoids (BDOs) from individual patients. Consistent with previous reports that described biliary cancer organoids from surgical tissues, the BDOs showed diverse morphologies such as simple cysts, multiloculated cysts, thick capsulated cysts, and solid masses. They also harbored mutations in KRAS and TP53 at frequencies of 15% and 55%, respectively. To enrich the cancer organoids by removing contaminated noncancerous components of BDOs, we attempted to verify the effectiveness of 3 different procedures, including repeat passage, xenografting, and selection with an MDM2 inhibitor for TP53 mutation-harboring BDOs. By monitoring the sequence and expression of mutated TP53, we found that all these procedures successfully enriched the cancer organoids. Our data suggest that BDOs can be established with minimal invasiveness from almost all patients with biliary cancers, including inoperable cases. Thus, despite some limitations with respect to the characterization of BDOs and methods for the enrichment of cancer cell-derived organoids, our data suggest that BDOs could have potential applications in personalized medicine., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Endoscopic ultrasound-guided biliary drainage (EUS-BD) has become comparable to endoscopic retrograde cholangiopancreatography and is now considered a first-line intervention for certain biliary obstructions. Although analysis of experience-related factors may help achieve better outcomes and contribute to its wider adoption, no concrete evidence exists regarding the required operator or institutional experience levels. This study aimed to analyze experience-related factors at beginner multicenters. Patients who underwent EUS-BD using self-expandable metal stents and/or dedicated plastic stents during the study period (up to the first 25 cases since introducing the technique) were retrospectively enrolled from seven beginner institutions and operators. Overall, 90 successful (technical success without early adverse events) and 22 failed (technical failure and/or early adverse events) cases were compared. EUS-BD-related procedures conducted at the time of applicable EUS-BD by each institution/operator were evaluated. The number of institution-conducted EUS-BD procedures (≥7) and operator-conducted EUS screenings (≥436), EUS-guided fine-needle aspirations (FNA) (≥93), and EUS-guided drainages (≥13) significantly influenced improved EUS-BD outcomes ( p = 0.022, odds ratio [OR], 3.0; p = 0.022, OR, 3.0; p = 0.022, OR, 3.0; and p = 0.028, OR, 2.9, respectively). Our threshold values, which significantly divided successful and failed cases, were assessed using receiver operating characteristic curve analysis and may provide useful approximate indications for successful EUS-BD.

The cellular interactions in the tumor microenvironment of colorectal cancer (CRC) are poorly understood, hindering patient treatment. In the current study, we investigate whether events occurring at the invasion front are of particular importance for CRC treatment strategies. To this end, we analyze CRC tissues by combining spatial transcriptomics from patients with a public single-cell transcriptomic atlas to determine cell-cell interactions at the invasion front. We show that CRC cells are localized specifically at the invasion front. These cells induce human leukocyte antigen G (HLA-G) to produce secreted phosphoprotein 1 (SPP1)+ macrophages while conferring CRC cells with anti-tumor immunity, as well as proliferative and invasive properties. Taken together, these findings highlight the signaling between CRC cell populations and stromal cell populations at the cellular level., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

AlphaFold2 (AF2) is a protein structure prediction program which provides accurate models. In addition to predicting structural domains, AF2 assigns intrinsically disordered regions (IDRs) by identifying regions with low prediction reliability (pLDDT). Some regions in IDRs undergo disorder-to-order transition upon binding the interaction partner. Here we assessed model structures of AF2 based on the annotations in IDEAL, in which segments with disorder-to-order transition have been collected as Protean Segments (ProSs). We non-redundantly selected ProSs from IDEAL and classified them based on the root mean square deviation to the corresponding region of AF2 models. Statistical analysis identified 11 structural and sequential features, possibly contributing toward the prediction of ProS structures. These features were categorized into two groups: one that contained pLDDT and the other that contained normalized radius of gyration. The typical ProS structures in the former group comprise a long α helix or a whole or part of the structural domain and those in the latter group comprise a short α helix with terminal loops.

Most prokaryotic proteins consist of a single structural domain (SD) with little intrinsically disordered regions (IDRs) that by themselves do not adopt stable structures, whereas the typical eukaryotic protein comprises multiple SDs and IDRs. How eukaryotic proteins evolved to differ from prokaryotic proteins has not been fully elucidated. Here, we found that the longer the internal exons are, the more frequently they encode IDRs in eight eukaryotes including vertebrates, invertebrates, a fungus, and plants. Based on this observation, we propose the "small bang" model from the proteomic viewpoint: the protoeukaryotic genes had no introns and mostly encoded one SD each, but a majority of them were subsequently divided into multiple exons (step 1). Many exons unconstrained by SDs elongated to encode IDRs (step 2). The elongated exons encoding IDRs frequently facilitated the acquisition of multiple SDs to make the last common ancestor of eukaryotes (step 3). One prediction of the model is that long internal exons are mostly unconstrained exons. Analytical results of the eight eukaryotes are consistent with this prediction. In support of the model, we identified cases of internal exons that elongated after the rat-mouse divergence and discovered that the expanded sections are mostly in unconstrained exons and preferentially encode IDRs. The model also predicts that SDs followed by long internal exons tend to have other SDs downstream. This prediction was also verified in all the eukaryotic species analyzed. Our model accounts for the dichotomy between prokaryotic and eukaryotic proteins and proposes a selective advantage conferred by IDRs., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Kynurenine (Kyn), a metabolite of tryptophan (Trp), is a key regulator of mammal immune responses such as cancer immune tolerance. Indoleamine-2,3-dioxygenase (IDO) and tryptophan-2,3-dioxygenase (TDO) are main enzymes regulating the first and rate-limiting step of the Kyn pathway. To identify new small molecule inhibitors of TDO, we selected A172 glioblastoma cell line constitutively expressed TDO. Characterization of this cell line using kinase inhibitor library resulted in identification of MEK/ERK pathway-dependent TDO expression. After knowing the properties for TDO expression, we further proceeded to screen chemical library for TDO inhibitors. We previously determined that S-benzylisothiourea derivatives are enzymatic inhibitors of indoleamine 2,3-dioxygenase 1 (IDO1) and suggested that the isothiourea moiety could be an important pharmacophore for binding to heme. Based on this premise, we screened an in-house library composed of various isothiourea derivatives and identified a bisisothiourea derivative, PVZB3001, as an inhibitor of TDO. Interestingly, PVZB3001 also inhibited the enzymatic activity of IDO1 in both cell-based and cell-free assays but did not inhibit other heme enzymes. Molecular docking studies suggested the importance of isothiourea moieties at the ortho position of the phenyl ring for the inhibition of catalytic activity. PVZB3001 showed competitive inhibition against TDO, and this was supported by the docking simulation. PVZB3001 recovered natural killer (NK) cell viability and functions by inhibiting Kyn accumulation in conditioned medium of both IDO1- and TDO-expressing cells. Furthermore, oral administration of IDO1-overexpressing tumor-bearing mice with PVZB3001 significantly inhibited tumor growth. Thus, we identified a novel selective dual inhibitor of IDO1 and TDO using the Kyn production assay with a glioblastoma cell line. This inhibitor could be a useful pharmacological tool for modulating the Kyn pathway in a variety of experimental systems., Competing Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2022.)

Li-metal rechargeable batteries are an attractive option for devices that require an extremely high specific energy density, high robustness, and long-term durability, such as high-altitude platform stations. However, Li dendrite growth during charge-discharge cycling causes short-circuit problems. One technical solution is to form an intermediate layer between the Li metal and electrolyte. This interfacial layer should possess mechanical strength, electrochemical stability in the presence of Li, and Li-ion conductivity. In this study, the Li-ion conductivity of spinel-type LiAl 5 O 8 was investigated using first-principles density functional theory and force field molecular dynamics calculations. The calculation results confirmed that stoichiometric LiAl 5 O 8 compounds do not exhibit Li-ion conductivity, whereas off-stoichiometric compounds with excess Li show long-range Li-ion diffusion. The evaluated activation energy was 0.28 eV, which is as low as that of well-known fast Li-ion conductors, such as garnet-type Li 7 La 3 Zr 2 O 12 . However, the extrapolated Li-ion conductivity at 298 K was relatively low (~ 10 -6 S/cm) owing to the limited formation of migration pathways., (© 2022. The Author(s).)

Non-destructive testing using a magneto-optical effect is a high-resolution non-destructive inspection technique for a metallic structure. It is able to provide high-spatial resolution images of defects. Previously, it has been difficult to fabricate flexible magneto-optical sensors because thermal treatment is necessary to crystallize the magnetic garnet. Therefore, it was not possible to apply magneto-optical imaging to complicated shapes in a test subject, such as a curved surface. In this study, we developed a new process for deposition of the magnetic garnet on the flexible substrate by applying the magnetic garnet powders that have already undergone crystallization. In this new process, as it does not require thermal treatment after deposition, flexible substrates with low heat resistance can be used. In this paper, we report our observations of the optical properties, magnetic hysteresis loop, crystallizability and density of the particles on the flexible substrate deposited by the spin-coating method.

Early detection of pancreatic ductal adenocarcinoma (PDAC) in the general population is difficult due to unknown clinical characteristics. This study was conducted to clarify the factors associated with early stage PDAC. Well-known symptoms and factors associated with PDAC were classified into clinical indicators, risk factors, and imaging findings concomitant with early stage PDAC. To analyze these factors for the detection of patients with early stage PDAC compared to patients without PDAC, we constructed new diagnostic strategies. The factors of 35 patients with early stage PDAC (stage 0 and IA) and 801 patients without PDAC were compared retrospectively. Clinical indicators; presence and number of indicators, elevated pancreatic enzyme level, tumor biomarker level, acute pancreatitis history, risk factors; familial pancreatic cancer, diabetes mellitus, smoking history, imaging findings; presence and number of findings, and main pancreatic duct dilation were significant factors for early stage PDAC detection. A new screening strategy to select patients who should be examined by imaging modalities from evaluating clinical indicators and risk factors and approaching a definitive diagnosis by evaluating imaging findings had a relatively high sensitivity, specificity, and areas under the curve of 80.0%, 80.8%, and 0.80, respectively. Diagnosis based on the new category and strategy may be reasonable for early stage PDAC detection.

Human ATP-dependent Lon protease (LONP1) forms homohexameric, ring-shaped complexes. Depletion of LONP1 causes aggregation of a broad range of proteins in the mitochondrial matrix and decreases the levels of their soluble forms. The ATP hydrolysis activity, but not protease activity, of LONP1 is critical for its chaperone-like anti-aggregation activity. LONP1 forms a complex with the import machinery and an incoming protein, and protein aggregation is linked with matrix protein import. LONP1 also contributes to the degradation of imported, aberrant, unprocessed proteins using its protease activity. Taken together, our results show that LONP1 functions as a gatekeeper for specific proteins imported into the mitochondrial matrix., (© 2021. The Author(s).)

Intrinsically disordered proteins are those proteins with intrinsically disordered regions. One of the unique characteristics of intrinsically disordered proteins is the existence of functional segments in intrinsically dis-ordered regions. These segments are involved in binding to partner molecules, such as protein and DNA, and play important roles in signaling pathways and/or transcriptional regulation. Although there are databases that gather information on such disordered binding regions, data remain limited. Therefore, it is desirable to develop programs to predict the disordered binding regions without using data for the binding regions. We developed a program, NeProc, to predict the disordered binding regions, which can be regarded as intrinsically disordered regions with a structural propensity. We only used data for the structural domains and intrinsically disordered regions to detect such regions. NeProc accepts a query amino acid sequence converted into a position specific score matrix, and uses two neural networks that employ different window sizes, a neural network of short windows, and a neural network of long windows. The performance of NeProc was comparable to that of existing programs of the disordered binding region prediction. This result presents the possibility to overcome the shortage of the disordered binding region data in the development of the prediction programs for these binding regions. NeProc is available at http://flab.neproc.org/neproc/index.html., (2020 THE BIOPHYSICAL SOCIETY OF JAPAN.)

Despite the essential role of Bifidobacterium in health-promoting gut bacteria in humans, little is known about their functions in wild animals, especially non-human primates. It is difficult to determine in vivo the function of Bifidobacterium in wild animals due to the limited accessibility of studying target animals in natural conditions. However, the genomic characteristics of Bifidobacterium obtained from the feces of wild animals can provide insight into their functionality in the gut. Here, we analyzed the whole genomes of 12 B. moukalabense strains isolated from seven feces samples of wild western lowland gorillas ( Gorilla gorilla gorilla ), three samples of wild central chimpanzees ( Pan troglodytes troglodytes ) and two samples of wild forest elephants ( Loxodonta cyclotis ) in Moukalaba-Doudou National Park, Gabon. In addition, we analyzed the fecal bacterial communities of six wild western lowland gorillas by meta 16S rRNA gene analyses with next generation sequencing. Although the abundance of the genus Bifidobacterium was as low as 0.2% in the total reads, a whole genome analysis of B. moukalabense suggested its contribution digestion of food and nutrition of frugivore/folivore animals. Specifically, the whole genome analysis indicated the involvement of B. moukalabense in hemicellulose degradation for short chain fatty acid production and nucleic acid utilization as nitrogen resources. In comparison with human-associated Bifidobacterium spp., genes for carbohydrate transport and metabolism are not conserved in these wild species. In particular the glycosidases, which are found in all 12 strains of B. moukalabense , were variably detected, or not detected, in human-associated species.

One of the unique characteristics of intrinsically disordered proteins (IPDs) is the existence of functional segments in intrinsically disordered regions (IDRs). A typical function of these segments is binding to partner molecules, such as proteins and DNAs. These segments play important roles in signaling pathways and transcriptional regulation. We conducted bioinformatics analysis to search these functional segments based on IDR predictions and database annotations. We found more than a thousand potential functional IDR segments in disease-related proteins. Large fractions of proteins related to cancers, congenital disorders, digestive system diseases, and reproductive system diseases have these functional IDRs. Some proteins in nervous system diseases have long functional segments in IDRs. The detailed analysis of some of these regions showed that the functional segments are located on experimentally verified IDRs. The proteins with functional IDR segments generally tend to come and go between the cytoplasm and the nucleus. Proteins involved in multiple diseases tend to have more protein-protein interactors, suggesting that hub proteins in the protein-protein interaction networks can have multiple impacts on human diseases., Competing Interests: The authors declare no conflict of interest.

Eukaryotic proteins consist of structural domains (SDs) and intrinsically disordered regions (IDRs), i.e., regions that by themselves do not assume unique three-dimensional structures. IDRs are generally subject to less constraint and evolve more rapidly than SDs. Proteins with a lower number of protein-to-protein interactions (PPIs) are also less constrained and tend to evolve fast. Extracellular proteins of mammals, especially immune-related extracellular proteins, on average have relatively high evolution rates. This article aims to examine if a high evolution rate in IDRs or that in SDs accounts for the rapid evolution of extracellular proteins. To this end, we classified eukaryotic proteins based on their cellular localizations and analyzed them. Moreover, we divided proteins into SDs and IDRs and calculated the respective evolution rate. Fractional IDR content is positively correlated with evolution rate. For their fractional IDR content, immune-related extracellular proteins show an aberrantly high evolution rate. IDRs evolve more rapidly than SDs in most subcellular localizations. In extracellular proteins, however, the difference is diminished. For immune-related proteins in mammals in particular, the evolution rates in SDs come close to those in IDRs. Thus high evolution rates in both IDRs and SDs account for the rapid evolution of immune-related proteins.

Intrinsically disordered proteins (IDPs) are an emerging phenomenon. They may have a high degree of flexibility in their polypeptide chains, which lack a stable 3D structure. Although several biological functions of IDPs have been proposed, their general function is not known. The only finding related to their function is the genetically conserved YSK₂ motif present in plant dehydrins. These proteins were shown to be IDPs with the YSK₂ motif serving as a core region for the dehydrins' cryoprotective activity. Here we examined the cryoprotective activity of randomly selected IDPs toward the model enzyme lactate dehydrogenase (LDH). All five IDPs that were examined were in the range of 35-45 amino acid residues in length and were equally potent at a concentration of 50 μg/mL, whereas folded proteins, the PSD-95/Dlg/ZO-1 (PDZ) domain, and lysozymes had no potency. We further examined their cryoprotective activity toward glutathione S -transferase as an example of the other enzyme, and toward enhanced green fluorescent protein as a non-enzyme protein example. We further examined the lyophilization protective activity of the peptides toward LDH, which revealed that some IDPs showed a higher activity than that of bovine serum albumin (BSA). Based on these observations, we propose that cryoprotection is a general feature of IDPs. Our findings may become a clue to various industrial applications of IDPs in the future., Competing Interests: The authors declare no conflict of interest.

A subset of the proteome is prone to aggregate formation, which is prevented by chaperones in the cell. To investigate whether the basic principle underlying the aggregation process is common in prokaryotes and eukaryotes, we conducted a large-scale aggregation analysis of ~500 cytosolic budding yeast proteins using a chaperone-free reconstituted translation system, and compared the obtained data with that of ~3,000 Escherichia coli proteins reported previously. Although the physicochemical properties affecting the aggregation propensity were generally similar in yeast and E. coli proteins, the susceptibility of aggregation in yeast proteins were positively correlated with the presence of intrinsically disordered regions (IDRs). Notably, the aggregation propensity was not significantly changed by a removal of IDRs in model IDR-containing proteins, suggesting that the properties of ordered regions in these proteins are the dominant factors for aggregate formation. We also found that the proteins with longer IDRs were disfavored by E. coli chaperonin GroEL/ES, whereas both bacterial and yeast Hsp70/40 chaperones have a strong aggregation-prevention effect even for proteins possessing IDRs. These results imply that a key determinant to discriminate the eukaryotic proteomes from the prokaryotic proteomes in terms of protein folding would be the attachment of IDRs.

Codon usage tends to be optimized in highly expressed genes. A plausible explanation for this phenomenon is that translational accuracy is increased in highly expressed genes with infrequent use of rare codons. Besides structural domains (SDs), eukaryotic proteins generally have intrinsically disordered regions (IDRs) that by themselves do not assume unique three-dimensional structures. As IDRs are free from structural constraint, they can probably accommodate more translational errors than SDs can. Thus, codon usage in IDRs is likely to be less optimized than that in SDs. Codon usage in all the genes of seven eukaryotes was examined in terms of both tRNA adaptation index and codon adaptation index. Different amino acid compositions in different protein regions were taken into account in calculating expected adaptation indices, to which observed indices were compared. Codon usage is less optimized in gene regions encoding IDRs than in those corresponding to SDs. The finding does not depend on whether IDRs are located at the N-terminus, in the middle, or at the C-terminus of proteins. Furthermore, the observation remains unchanged in two different algorithms used to predict IDRs in proteins. The result is consistent with the idea that IDRs tolerate more translational errors than SDs., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Protein-protein interactions are fundamental for all biological phenomena, and protein-protein interaction networks provide a global view of the interactions. The hub proteins, with many interaction partners, play vital roles in the networks. We investigated the subcellular localizations of proteins in the human network, and found that the ones localized in multiple subcellular compartments, especially the nucleus/cytoplasm proteins (NCP), the cytoplasm/cell membrane proteins (CMP), and the nucleus/cytoplasm/cell membrane proteins (NCMP), tend to be hubs. Examinations of keywords suggested that among NCP, those related to post-translational modifications and transcription functions are the major contributors to the large number of interactions. These types of proteins are characterized by a multi-domain architecture and intrinsic disorder. A survey of the typical hub proteins with prominent numbers of interaction partners in the type revealed that most are either transcription factors or co-regulators involved in signaling pathways. They translocate from the cytoplasm to the nucleus, triggered by the phosphorylation and/or ubiquitination of intrinsically disordered regions. Among CMP and NCMP, the contributors to the numerous interactions are related to either kinase or ubiquitin ligase activity. Many of them reside on the cytoplasmic side of the cell membrane, and act as the upstream regulators of signaling pathways. Overall, these hub proteins function to transfer external signals to the nucleus, through the cell membrane and the cytoplasm. Our analysis suggests that multiple-localization is a crucial concept to characterize groups of hub proteins and their biological functions in cellular information processing.

Aim: To identify factors that influence long-term liver function following radiofrequency ablation (RFA) in patients with viral hepatitis-related hepatocellular carcinoma., Methods: A total of 123 patients with hepatitis B virus- or hepatitis C virus-related hepatocellular car-cinoma (HCC) (n = 12 and n = 111, respectively) were enrolled. Cumulative rates of worsening Child-Pugh (CP) scores (defined as a 2-point increase) were examined., Results: CP score worsening was confirmed in 22 patients over a mean follow-up period of 43.8 ± 26.3 mo. Multivariate analysis identified CP class, platelet count, and aspartate aminotransferase levels as signi-ficant predictors of a worsening CP score (P = 0.000, P = 0.011 and P = 0.024, respectively). In contrast, repeated RFA was not identified as a risk factor for liver function deterioration., Conclusion: Long-term liver function following RFA was dependent on liver functional reserve, the degree of fibrosis present, and the activity of the hepatitis condition for this cohort. Therefore, in order to maintain liver function for an extended period following RFA, suppression of viral hepatitis activity is important even after the treatment of HCC.

Glycans play important roles in such cell-cell interactions as signaling and adhesion, including processes involved in pathogenic infections, cancers, and neurological diseases. Glycans are biosynthesized by multiple glycosyltransferases (GTs), which function sequentially. Excluding mucin-type O-glycosylation, the non-reducing terminus of glycans is biosynthesized in the Golgi apparatus after the reducing terminus is biosynthesized in the ER. In the present study, we performed genome-wide analyses of human GTs by investigating the degree of conservation of homologues in other organisms, as well as by elucidating the phylogenetic relationship between cephalochordates and urochordates, which has long been controversial in deuterostome phylogeny. We analyzed 173 human GTs and functionally linked glycan synthesis enzymes by phylogenetic profiling and clustering, compiled orthologous genes from the genomes of other organisms, and converted them into a binary sequence based on the presence (1) or absence (0) of orthologous genes in the genomes. Our results suggest that the non-reducing terminus of glycans is biosynthesized by newly evolved GTs. According to our analysis, the phylogenetic profiles of GTs resemble the phylogenetic tree of life, where deuterostomes, metazoans, and eukaryotes are resolved into separate branches. Lineage-specific GTs appear to play essential roles in the divergence of these particular lineages. We suggest that urochordates lose several genes that are conserved among metazoans, such as those expressing sialyltransferases, and that the Golgi apparatus acquires the ability to synthesize glycans after the ER acquires this function.

The solute carrier 27A (SLC27A) gene family encodes fatty acid transport proteins (FATPs) and includes 6 members. During fetal and postnatal periods of development, the growing brain requires a reliable supply of fatty acids. Because autism spectrum disorders (ASD) are now recognized as disorders caused by impaired early brain development, it is possible that functional abnormalities of SLC27A genes may contribute to the pathogenesis of ASD. Here, we confirmed the expression of SLC27A3 and SLC27A4 in human neural stem cells derived from human induced pluripotent stem cells, which suggested their involvement in the developmental stage of the central nervous system. Additionally, we resequenced the SLC27A3 and SLC27A4 genes using 267 ASD patient and 1140 control samples and detected 47 (44 novel and 29 nonsynonymous) and 30 (17 novel and 14 nonsynonymous) variants for the SLC27A3 and SLC27A4, respectively, revealing that they are highly polymorphic with multiple rare variants. The SLC27A4 Ser209 allele was more frequently represented in ASD samples. Furthermore, we showed that a SLC27A4 Ser209 mutant resulted in significantly higher fluorescently-labeled fatty acid uptake into bEnd3 cells, a mouse brain capillary-derived endothelial cell line, compared with SLC27A4 Gly209, suggesting that the functional change may contribute to ASD pathophysiology.

Intrinsically disordered proteins (IDPs) are an emerging concept. IDPs have high flexibility in their polypeptide chains, lacking a stable 3-dimensional structure. Because of the difficulty in performing X-ray crystallography for IDPs, nuclear magnetic resonance (NMR) spectroscopy is the first choice for atomic-level investigation of their nature. Given that isotopically labeled IDP samples are necessary for NMR study, a robust and cost-effective protocol for bacterial expression and purification of IDP is also needed. We employed the N pro (EDDIE)-autoprotease fusion protein system. Although IDPs are believed to be readily degraded by endogenous proteases when expressed in Escherichia coli , N pro -fused IDPs showed excellent resistance to degradation. Seven IDPs of uncharacterized function sampled from the human genome as well as 3 constructs from IDP regions derived from human FancM and Thermococcus kodakarensis Hef were prepared. We improved the protocol of refolding of N pro (EDDIE) to use dialysis, which is convenient for subsequent purification using reversed-phase (RP) HPLC. The method is robust and widely applicable to any IDP sample, promoting the acquisition of experimental data for IDPs in a high-throughput manner.

Background: Histone H3 methylation at lysine 9 (H3K9) is a conserved epigenetic signal, mediating heterochromatin formation by trimethylation, and transcriptional silencing by dimethylation. Defective GLP (Ehmt1) and G9a (Ehmt2) histone lysine methyltransferases, involved in mono and dimethylation of H3K9, confer autistic phenotypes and behavioral abnormalities in animal models. Moreover, EHMT1 loss of function results in Kleefstra syndrome, characterized by severe intellectual disability, developmental delays and psychiatric disorders. We examined the possible role of histone methyltransferases in the etiology of autism spectrum disorders (ASD) and suggest that rare functional variants in these genes that regulate H3K9 methylation may be associated with ASD., Methods: Since G9a-GLP-Wiz forms a heteromeric methyltransferase complex, all the protein-coding regions and exon/intron boundaries of EHMT1, EHMT2 and WIZ were sequenced in Japanese ASD subjects. The detected variants were prioritized based on novelty and functionality. The expression levels of these genes were tested in blood cells and postmortem brain samples from ASD and control subjects. Expression of EHMT1 and EHMT2 isoforms were determined by digital PCR., Results: We identified six nonsynonymous variants: three in EHMT1, two in EHMT2 and one in WIZ. Two variants, the EHMT1 ankyrin repeat domain (Lys968Arg) and EHMT2 SET domain (Thr961Ile) variants were present exclusively in cases, but showed no statistically significant association with ASD. The EHMT2 transcript expression was significantly elevated in the peripheral blood cells of ASD when compared with control samples; but not for EHMT1 and WIZ. Gene expression levels of EHMT1, EHMT2 and WIZ in Brodmann area (BA) 9, BA21, BA40 and the dorsal raphe nucleus (DoRN) regions from postmortem brain samples showed no significant changes between ASD and control subjects. Nor did expression levels of EHMT1 and EHMT2 isoforms in the prefrontal cortex differ significantly between ASD and control groups., Conclusions: We identified two novel rare missense variants in the EHMT1 and EHMT2 genes of ASD patients. We surmise that these variants alone may not be sufficient to exert a significant effect on ASD pathogenesis. The elevated expression of EHMT2 in the peripheral blood cells may support the notion of a restrictive chromatin state in ASD, similar to schizophrenia.

The GP129, GP131 and GP133 genes of guinea pig cytomegalovirus (GPCMV) are homologues of human cytomegalovirus UL128, UL130 and UL131A, respectively, which are essential for infection of endothelial and epithelial cells, and for viral transmission to leukocytes. Our previous study demonstrated that a GPCMV strain lacking the 1.6 kb locus that contains the GP129, GP131 and GP133 genes had a growth defect in animals. Here, we demonstrated that the WT strain, but not the 1.6 kb-deleted strain, formed capsids in macrophages prepared from the peritoneal fluid. To understand the mechanism, we prepared GPCMV strains defective in each of GP129, GP131 and GP133, and found that they were all essential for the infection of peritoneal, splenic and PBMC-derived macrophages/monocytes, and for expression of immediate-early antigens in the macrophages/monocytes, although they were dispensable for infection of fibroblasts. Monocyte/macrophage tropism could be one of the important determinants for viral dissemination in vivo., (© 2014 The Authors.)

IDEAL (Intrinsically Disordered proteins with Extensive Annotations and Literature, http://www.ideal.force.cs.is.nagoya-u.ac.jp/IDEAL/) is a collection of intrinsically disordered proteins (IDPs) that cannot adopt stable globular structures under physiological conditions. Since its previous publication in 2012, the number of entries in IDEAL has almost tripled (120 to 340). In addition to the increase in quantity, the quality of IDEAL has been significantly improved. The new IDEAL incorporates the interactions of IDPs and their binding partners more explicitly, and illustrates the protein-protein interaction (PPI) networks and the structures of protein complexes. Redundant experimental data are arranged based on the clustering of Protein Data Bank entries, and similar sequences with the same binding mode are grouped. As a result, the new IDEAL presents more concise and informative experimental data. Nuclear magnetic resonance (NMR) disorder is annotated in a systematic manner, by identifying the regions with large deviations among the NMR models. The ordered/disordered and new domain predictions by DICHOT are available, as well as the domain assignments by HMMER. Some examples of the PPI networks and the highly deviated regions derived from NMR models will be described, together with other advances. These enhancements will facilitate deeper understanding of IDPs, in terms of their flexibility, plasticity and promiscuity.

Intrinsically disordered (ID) proteins (IDPs) are abundant in eukaryotes but are scarce in prokaryotes. Mitochondria, cellular organelles that descended from Rickettsia-like α-proteobacteria, are at the intersection between prokaryotes and eukaryotes. Although IDPs are reportedly as rare in mitochondria as in bacteria, these details remained to be clarified. Human mitochondrial proteins (n = 706) were obtained from the UniProt database, and information on ID regions of all human proteins was extracted from the DICHOT database. A BLAST search carried out against all α-proteobacterial proteins identified two types of mitochondrial proteins: those with (B) and without (E) bacterial homologues. The B-type proteins (n = 387) descended from a bacterial ancestor, whereas the E-type proteins (n = 319) were more recently added to the mitochondria via the host cell during the early evolution of eukaryotes. The average ID ratios of B-type/E-type proteins are 10.3% and 21.4%, respectively. The 706 proteins were further classified into four groups based on the mitochondrial subcompartment, namely, the matrix, intermembrane space, inner membrane, or outer membrane. The ID ratios in these different locations suggest that the frequency of IDPs in mitochondria might be due to the evolutionary origin (B-type/E-type) of the protein, rather than differences in its functional environment., (© 2012 The Authors Journal compilation © 2012 by the Molecular Biology Society of Japan/Wiley Publishing Ltd.)

IDEAL, Intrinsically Disordered proteins with Extensive Annotations and Literature (http://www.ideal.force.cs.is.nagoya-u.ac.jp/IDEAL/), is a collection of knowledge on experimentally verified intrinsically disordered proteins. IDEAL contains manual annotations by curators on intrinsically disordered regions, interaction regions to other molecules, post-translational modification sites, references and structural domain assignments. In particular, IDEAL explicitly describes protean segments that can be transformed from a disordered state to an ordered state. Since in most cases they can act as molecular recognition elements upon binding of partner proteins, IDEAL provides a data resource for functional regions of intrinsically disordered proteins. The information in IDEAL is provided on a user-friendly graphical view and in a computer-friendly XML format.

Background: Although structural domains in proteins (SDs) are important, half of the regions in the human proteome are currently left with no SD assignments. These unassigned regions consist not only of novel SDs, but also of intrinsically disordered (ID) regions since proteins, especially those in eukaryotes, generally contain a significant fraction of ID regions. As ID regions can be inferred from amino acid sequences, a method that combines SD and ID region assignments can determine the fractions of SDs and ID regions in any proteome., Results: In contrast to other available ID prediction programs that merely identify likely ID regions, the DICHOT system we previously developed classifies the entire protein sequence into SDs and ID regions. Application of DICHOT to the human proteome revealed that residue-wise ID regions constitute 35%, SDs with similarity to PDB structures comprise 52%, while SDs with no similarity to PDB structures account for the remaining 13%. The last group consists of novel structural domains, termed cryptic domains, which serve as good targets of structural genomics. The DICHOT method applied to the proteomes of other model organisms indicated that eukaryotes generally have high ID contents, while prokaryotes do not. In human proteins, ID contents differ among subcellular localizations: nuclear proteins had the highest residue-wise ID fraction (47%), while mitochondrial proteins exhibited the lowest (13%). Phosphorylation and O-linked glycosylation sites were found to be located preferentially in ID regions. As O-linked glycans are attached to residues in the extracellular regions of proteins, the modification is likely to protect the ID regions from proteolytic cleavage in the extracellular environment. Alternative splicing events tend to occur more frequently in ID regions. We interpret this as evidence that natural selection is operating at the protein level in alternative splicing., Conclusions: We classified entire regions of proteins into the two categories, SDs and ID regions and thereby obtained various kinds of complete genome-wide statistics. The results of the present study are important basic information for understanding protein structural architectures and have been made publicly available at http://spock.genes.nig.ac.jp/~genome/DICHOT.

An allophanic soil (AS) catalyzed the formation of dark-colored polymers via polycondensation reactions between catechol and glycine. The organic carbon content of the AS was increased from 0.16% to 1.3%, indicating that some of the dark-colored polymers had been adsorbed to the AS. The characteristics of the dark-colored polymers adsorbed on the AS were similar to those of a humin that is not extractable with an aqueous alkaline solution. Such a humin-like substance (HuLS) was separated from the AS by treatment with a mixture of HF and HCl. The HuLS and humic acid-like substance (HaLS), comprising the acid-insoluble fraction in the reaction mixture, were characterized by elemental analysis, size exclusion chromatography, pyrolysis-GC/MS and (13)C NMR. However, the structural features of HaLS and HuLS had many points in common. These results suggest that HuLS-AS can be regarded as an organo-clay complex formed by the strong adsorption of HaLS to the AS. The adsorption of pentachlorophenol (PCP) to AS and HuLS-AS was examined at pH 5.5. At this pH, the zeta potential of the HuLS-AS showed a negative value. It would, therefore, be expected that pentachlorophenolate anions would adsorb with difficulty to HuLS-AS because of electrostatic repulsion. Nevertheless, the adsorption coefficient for PCP to HuLS-AS, as estimated by the Freundlich isotherm, was seven times larger than that for AS. These results show that HuLS, when adsorbed on the AS surface, has the capability to enhance the adsorption of PCP., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The molecular mechanism for meiotic entry remains largely elusive in flowering plants. Only Arabidopsis SWI1/DYAD and maize AM1, both of which are the coiled-coil protein, are known to be required for the initiation of plant meiosis. The mechanism underlying the synchrony of male meiosis, characteristic to flowering plants, has also been unclear in the plant kingdom. In other eukaryotes, RNA-recognition-motif (RRM) proteins are known to play essential roles in germ-cell development and meiosis progression. Rice MEL2 protein discovered in this study shows partial similarity with human proline-rich RRM protein, deleted in Azoospermia-Associated Protein1 (DAZAP1), though MEL2 also possesses ankyrin repeats and a RING finger motif. Expression analyses of several cell-cycle markers revealed that, in mel2 mutant anthers, most germ cells failed to enter premeiotic S-phase and meiosis, and a part escaped from the defect and underwent meiosis with a significant delay or continued mitotic cycles. Immunofluorescent detection revealed that T7 peptide-tagged MEL2 localized at cytoplasmic perinuclear region of germ cells during premeiotic interphase in transgenic rice plants. This study is the first report of the plant RRM protein, which is required for regulating the premeiotic G1/S-phase transition of male and female germ cells and also establishing synchrony of male meiosis. This study will contribute to elucidation of similarities and diversities in reproduction system between plants and other species., Competing Interests: The authors have declared that no competing interests exist.