Spatial and single-cell colocalisation analysis reveals MDK-mediated immunosuppressive environment with regulatory T cells in colorectal carcinogenesis.

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.

[Successful treatment of the diverticular lesion of the colon via EMR with over-the-scope clip (OTSC):a case report].

A 65-year-old woman presented with a flat elevated lesion of about 1cm in the cecal diverticulum during a lower gastrointestinal endoscopy that was performed previously by another physician during a medical checkup. The patient was referred to our department for resection. Considering the risk of perforation owing to the diverticular lesion, positive nonlifting sign, and Group 5 diagnosis on the previous biopsy, EMR with over-the-scope clip (OTSC) (EMRO) was selected, and complete resection was achieved without complications.

Experience-Related Factors in the Success of Beginner Endoscopic Ultrasound-Guided Biliary Drainage: A Multicenter Study.

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.

Computational Methods to Predict Intrinsically Disordered Regions and Functional Regions in Them.

Intrinsically disordered regions (IDRs) are protein regions that do not adopt fixed tertiary structures. Since these regions lack ordered three-dimensional structures, they should be excluded from the target portions of homology modeling. IDRs can be predicted from the amino acid sequences, because their amino acid compositions are different from that of the structured domains. This chapter provides a review of the prediction methods of IDRs and a case study of IDR prediction.

Efficient Establishment of Bile-Derived Organoids From Biliary Cancer Patients.

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.

How AlphaFold2 Predicts Conditionally Folding Regions Annotated in an Intrinsically Disordered Protein Database, IDEAL.

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.

Spatial and single-cell transcriptomics decipher the cellular environment containing HLA-G+ cancer cells and SPP1+ macrophages in colorectal cancer.

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.

Exon Elongation Added Intrinsically Disordered Regions to the Encoded Proteins and Facilitated the Emergence of the Last Eukaryotic Common Ancestor.

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.

Classification of proteins inducing liquid-liquid phase separation: sequential, structural and functional characterization.

Liquid-liquid phase separation (LLPS) within the cell can form biological condensates, which are increasingly recognized to play important roles in various biological processes. Most proteins involved in LLPS are known to be intrinsically disordered proteins containing intrinsically disordered regions (IDRs) with low complexity regions (LCRs). The proteins driving LLPS were selected from databases of LLPS-related proteins and then classified into three classes according to the components in the condensates. Through in silico analyses, we found that proteins in the homo class, those that induce LLPS without partner molecules, have different IDRs and LCRs compared with the reference proteome. In contrast, proteins in the other classes, those that induce LLPS with partner proteins (the hetero class) or nucleic acids (the mixed class), did not show a clear difference to the reference proteome in IDRs and LCRs. The hetero-class proteins contained structural domains to serve protein-protein interactions, and the mixed-class ones had the structural domains associated with nucleic acids. These results suggest that IDRs in the homo-class proteins have unique IDRs, which provide multivalent interaction sites required for LLPS, whereas the hetero- and mixed-class proteins can induce LLPS through the combination of the interaction among LCRs, structural domains and nucleic acids.

[A case report of intussusception in adult patient with long-term percutaneous endoscopic gastrojejunostomy].

An 87-year-old man had a self-extracted percutaneous endoscopic gastrojejunostomy tube (PEG-J tube) and underwent PEG-J replacement. He vomited 2 days after the replacement, and bowel bleeding occurred 5 days after the replacement. Therefore, he presented to our hospital. A simple abdominal computed tomography scan showed a target sign in the jejunum on the anus side rather than on the PEG-J tube tip portion, resembling intussusception. We confirmed mucosa necrosis in the intussusception by small-diameter endoscopy and attempted internal treatment. However, resistance was so strong that the internal treatment was difficult;therefore, he underwent a jejunal resection. Although diarrhea and dumping syndrome are common PEG-J complications, there have been few reports of intussusception caused by PEG-J. We report a case of adult-onset intussusception, which was thought to be caused by long-term PEG-J implantation.

Diagnostic Strategy of Early Stage Pancreatic Cancer via Clinical Predictor Assessment: Clinical Indicators, Risk Factors and Imaging Findings.

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.

Mitochondrial Lon protease is a gatekeeper for proteins newly imported into the matrix.

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.

A case of dengue fever that should be considered as imported infectious disease with digestive symptoms.

Patients with dengue fever usually present with fever and rash, but non-specific symptoms such as headache, myalgia, arthralgia, and digestive symptoms are sometimes seen. We report a case of dengue fever with digestive symptoms in a patient who traveled to Indonesia. A 35-year-old man presented with fever, diarrhea, headache, and arthralgia. He later developed generalized rash. Dengue fever was clinically suspected from the travel history and confirmed by laboratory tests. He tested positive for anti-dengue virus antibodies, so dengue fever was diagnosed. Dengue fever should be included in the differential diagnosis of patients with digestive symptoms after returning to Japan.

NeProc predicts binding segments in intrinsically disordered regions without learning binding region sequences.

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.

[A case report of adult-onset IgA vasculitis with repeated migrating abdominal lesions and manifestations].

A 68-year-old woman was referred to our hospital due to widespread purpura on her legs. A diagnosis of IgA vasculitis was made based on the findings of a skin biopsy. However, after being admitted to our hospital, abdominal pain and lower gastrointestinal hemorrhaging developed. The purpura disappeared gradually, whereas the abdominal pain migrated and persisted. Treatment with prednisolone was initiated, and the clinical course improved temporarily. However, her severe abdominal symptoms recurred while, in addition, the intestinal tract lesions migrated after the prednisolone dosage was tapered. Therefore, intravenous high-dose methylprednisolone was administered followed by oral steroids. The dose was thereafter carefully tapered, and the steroid dose reduction was successful with this treatment. We herein report the clinical course of the case along with a review of the relevant literature.

Genomic Analyses of Bifidobacterium moukalabense Reveal Adaptations to Frugivore/Folivore Feeding Behavior.

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.

Functional Segments on Intrinsically Disordered Regions in Disease-Related Proteins.

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.

Both Intrinsically Disordered Regions and Structural Domains Evolve Rapidly in Immune-Related Mammalian Proteins.

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.

Discovery of Cryoprotective Activity in Human Genome-Derived Intrinsically Disordered 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.

Large-scale aggregation analysis of eukaryotic proteins reveals an involvement of intrinsically disordered regions in protein folding.

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.