BIONIC: biological network integration using convolutions.

Biological networks constructed from varied data can be used to map cellular function, but each data type has limitations. Network integration promises to address these limitations by combining and automatically weighting input information to obtain a more accurate and comprehensive representation of the underlying biology. We developed a deep learning-based network integration algorithm that incorporates a graph convolutional network framework. Our method, BIONIC (Biological Network Integration using Convolutions), learns features that contain substantially more functional information compared to existing approaches. BIONIC has unsupervised and semisupervised learning modes, making use of available gene function annotations. BIONIC is scalable in both size and quantity of the input networks, making it feasible to integrate numerous networks on the scale of the human genome. To demonstrate the use of BIONIC in identifying new biology, we predicted and experimentally validated essential gene chemical-genetic interactions from nonessential gene profiles in yeast.

Dietary lysine restriction induces lipid accumulation in skeletal muscle through an increase in serum threonine levels in rats.

We previously reported that dietary amino acid restriction induces the accumulation of triglycerides (TAG) in the liver of growing rats. However, differences in TAG accumulation in individual cell types or other tissues were not examined. In this study, we show that TAG also accumulates in the muscle and adipose tissues of rats fed a low amino acid (low-AA) diet. In addition, dietary lysine restriction (low-Lys) induces lipid accumulation in muscle and adipose tissues. In adjusting the nitrogen content to that of the control diet, we found that glutamic acid supplementation to the low-AA diet blocked lipid accumulation, but supplementation with the low-Lys diet did not, suggesting that a shortage of nitrogen caused lipids to accumulate in the skeletal muscle in the rats fed a low-AA diet. Serum amino acid measurement revealed that, in rats fed a low-Lys diet, serum lysine levels were decreased, while serum threonine levels were significantly increased compared with the control rats. When the threonine content was restricted in the low-Lys diet, TAG accumulation induced by the low-Lys diet was completely abolished in skeletal muscle. Moreover, in L6 myotubes cultured in medium containing high threonine and low lysine, fatty acid uptake was enhanced compared with that in cells cultured in control medium. These findings suggest that the increased serum threonine in rats fed a low-Lys diet resulted in lipid incorporation into skeletal muscle, leading to the formation of fatty muscle tissue. Collectively, we propose conceptual hypothesis that "amino-acid signal" based on lysine and threonine regulates lipid metabolism.

CARD9 Expression Pattern, Gene Dosage, and Immunodeficiency Phenotype Revisited.

BACKGROUND: CARD9 deficiency is an autosomal recessive primary immunodeficiency underlying increased susceptibility to fungal infection primarily presenting as invasive CNS Candida and/or cutaneous/invasive dermatophyte infections. More recently, a rare heterozygous dominant negative CARD9 variant c.1434 + 1G > C was reported to be protective from inflammatory bowel disease. OBJECTIVE: We studied two siblings carrying homozygous CARD9 variants (c.1434 + 1G > C) and born to heterozygous asymptomatic parents. One sibling was asymptomatic and the other presented with candida esophagitis, upper respiratory infections, hypogammaglobulinemia, and low class-switched memory B cells. METHODS AND RESULTS: The CARD9 c.1434 + 1G > C variant generated two mutant transcripts confirmed by mRNA and protein expression: an out-of-frame c.1358-1434 deletion/ ~ 55 kDa protein (CARD9Δex.11) and an in-frame c.1417-1434 deletion/ ~ 61 kDa protein (CARD9Δ18 nt.). Neither transcript was able to form a complete/functional CBM complex, which includes TRIM62. Based on the index patient's CVID-like phenotype, CARD9 expression was tested and detected in lymphocytes and monocytes from humans and mice. The functional impact of different CARD9 mutations and gene dosage conditions was evaluated in heterozygous and homozygous c.1434 + 1 G > C members of the index family, and in WT (two WT alleles), haploinsufficiency (one WT, one null allele), and null (two null alleles) individuals. CARD9 gene dosage impacted lymphocyte and monocyte functions including cytokine generation, MAPK activation, T-helper commitment, transcription, plasmablast differentiation, and immunoglobulin production in a differential manner. CONCLUSIONS: CARD9 exon 11 integrity is critical to CBM complex function. CARD9 is expressed and affects particular T and B cell functions in a gene dosage-dependent manner, which in turn may contribute to the phenotype of CARD9 deficiency.

Development of a novel ß-1,6-glucan-specific detection system using functionally-modified recombinant endo-ß-1,6-glucanase.

ß-1,3-d-Glucan is a ubiquitous glucose polymer produced by plants, bacteria, and most fungi. It has been used as a diagnostic tool in patients with invasive mycoses via a highly-sensitive reagent consisting of the blood coagulation system of horseshoe crab. However, no method is currently available for measuring ß-1,6-glucan, another primary ß-glucan structure of fungal polysaccharides. Herein, we describe the development of an economical and highly-sensitive and specific assay for ß-1,6-glucan using a modified recombinant endo-ß-1,6-glucanase having diminished glucan hydrolase activity. The purified ß-1,6-glucanase derivative bound to the ß-1,6-glucan pustulan with a KD of 16.4 nm We validated the specificity of this ß-1,6-glucan probe by demonstrating its ability to detect cell wall ß-1,6-glucan from both yeast and hyphal forms of the opportunistic fungal pathogen Candida albicans, without any detectable binding to glucan lacking the long ß-1,6-glucan branch. We developed a sandwich ELISA-like assay with a low limit of quantification for pustulan (1.5 pg/ml), and we successfully employed this assay in the quantification of extracellular ß-1,6-glucan released by >250 patient-derived strains of different Candida species (including Candida auris) in culture supernatant in vitro We also used this assay to measure ß-1,6-glucan in vivo in the serum and in several organs in a mouse model of systemic candidiasis. Our work describes a reliable method for ß-1,6-glucan detection, which may prove useful for the diagnosis of invasive fungal infections.

Split Enzyme-Based Biosensors for Structural Characterization of Soluble and Insoluble ß-Glucans.

ß-Glucan is widely distributed in various plants and microorganisms and is composed of ß-1,3-linked d-glucose units. It may have a branched short or long side chain of glucose units with ß-1,6- or ß-1,4-linkage. Numerous studies have investigated different ß-glucans and revealed their bioactivities. To understand the structure-function relationship of ß-glucan, we constructed a split-luciferase complementation assay for the structural analysis of long-chain ß-1,6-branched ß-1,3-glucan. The N- and C-terminal fragments of luciferase from deep-sea shrimp were fused to insect-derived ß-1,3-glucan recognition protein and fungal endo-ß-1,6-glucanase (Neg1)-derived ß-1,6-glucan recognition protein, respectively. In this approach, two ß-glucan recognition proteins bound to ß-glucan molecules come into close proximity, resulting in the assembly of the full-length reporter enzyme and induction of transient luciferase activity, indicative of the structure of ß-glucan. To test the applicability of this assay, ß-glucan and two ß-glucan recognition proteins were mixed, resulting in an increase in the luminescence intensity in a ß-1,3-glucan with a long polymer of ß-1,6-glucan in a dose-dependent manner. This simple test also allows the monitoring of real-time changes in the side chain structure and serves as a convenient method to distinguish between ß-1,3-glucan and long-chain ß-1,6-branched ß-1,3-glucan in various soluble and insoluble ß-glucans.

Development of a Highly Sensitive ß-Glucan Detection System Using Scanning Single-Molecule Counting Method.

To overcome the limitations of the Limulus amebocyte lysate (LAL) assay method for the diagnosis of invasive fungal infection, we applied a reaction system combining recombinant ß-glucan binding proteins and a scanning single-molecule counting (SSMC) method. A novel (1→3)-ß-D-glucan recognition protein (S-BGRP) and a (1→6)-ß-glucanase mutant protein were prepared and tested for the binding of (1→6)-branched (1→3)-ß-D-glucan from fungi. S-BGRP and (1→6)-ß-glucanase mutant proteins reacted with ß-glucan from Candida and Aspergillus spp. Although LAL cross-reacted with plant-derived ß-glucans, the new detection system using the SSMC method showed low sensitivity to plant (1→3)-ß-D-glucan, which significantly improved the appearance of false positives, a recognized problem with the LAL method. Measurement of ß-glucan levels by the SSMC method using recombinant ß-glucan-binding proteins may be useful for the diagnosis of fungal infections. This study shows that this detection system could be a new alternative diagnostic method to the LAL method.

Possibility of Japanese Cedar Pollen Causing False Positives in the Deep Mycosis Test.

Because Japanese cedar pollen (JCP) contains beta-1,3-d-glucan (BG), there is concern that its lingering presence in the atmosphere, especially during its scattering period, may cause false positives in the factor-G-based Limulus amebocyte lysate (LAL) assay used to test for deep mycosis (i.e., G-test). Hence, we examined whether the LAL assay would react positively with substances contained in JCP by using the G-test to measure JCP particles and extracts. BG was purified from the JCP extract on a BG-specific affinity column, and the percentage extractability was measured using three different BG-specific quantitative methods. The G-test detected 0.4 pg BG in a single JCP particle and 10 fg from a single particle in the extract. The percentage extractability of JCP-derived BG was not significantly different among the three quantitative methods. As the JCP particles should technically have been removed during serum separation, they should be less likely to be a direct false-positive factor. However, given that the LAL-assay-positive substances in the JCP extract were not distinguishable by the three BG-specific quantitative methods, we conclude that they may cause the background to rise. Therefore, in Japan false positives arising from JCP contamination should be considered when testing patients for deep mycosis.

Latent 1,3-ß-D-glucan acts as an adjuvant for allergen-specific IgE production induced by Japanese cedar pollen exposure.

BACKGROUND: The pollen grains of several plant species contain 1,3-ß-D-glucan (BG). BG activates dendritic cells (DCs) and subsequently regulates the innate immune responses. Within Japan, the most common disease associated with type-I hypersensitivity is Japanese cedar pollinosis. However, the role of BG in Japanese cedar pollen (JCP) remains unclear. This study examined the localization and immunological effects of BG in JCP. METHODS: The localization of BG in JCP grain was determined by immunohistochemical staining using a soluble dectin-1 protein probe and a BG recognition protein (BGRP). The content of BG extracted from JCP was measured by a BGRP-based ELISA-like assay. The cytokine production by bone marrow-derived DCs (BMDCs) obtained from wild-type and BG receptor (dectin-1) knock-out mice was examined in vitro. The mice were intranasally administered JCP grains and the specific serum Ig levels were then quantified. RESULTS: BG was detected in the exine and cell wall of the generative cell and tube cell of the JCP grain. Moreover, BG in the exine stimulated production of TNF-α and IL-6 in the BMDCs via a dectin-1-dependent mechanism. Meanwhile, JCP-specific IgE and IgG were detected in the serum of wild-type mice that had been intranasally administered with JCP grains. These mice also exhibited significantly enhanced sneezing behavior. However, dectin-1 knock-out mice exhibited significantly lower JCP-specific IgE and IgG levels compared to wild-type mice. CONCLUSIONS: Latent BG in JCP can act as an adjuvant to induce JCP-specific antibody production via dectin-1.

Coronary Vasculitis Induced in Mice by the Cell Wall Mannoprotein of Candida krusei.

Kawasaki disease (KD) is an inflammatory disease that was identified by Professor Tomisaku Kawasaki in 1961. Candida albicans-derived substances, such as the hot water extract of C. albicans (CADS) and Candida water-soluble fraction (CAWS), induced coronary vasculitis similar to KD in mice. An increasing proportion of deep-seated candidiasis cases are caused by non-albicans Candida and are often resistant to antifungal drugs. We herein investigated whether the hot water extract of C. krusei, inherently resistant to fluconazole, induces vasculitis in mice. Three strains of C. krusei, NBRC1395, NBRC1162, and NBRC10737, were cultured in natural (Y) and chemically defined (C) media and cell wall mannoprotein (MN) fractions were prepared by autoclaving cells (CKY1395MN, CKC1395MN, CKY1162MN, CKC1162MN, CKY10737MN, and CKC10737MN). All MN fractions reacted strongly with Concanavalin A (Con A) and dectin-2 and induced anaphylactoid shock in ICR mice. MNs induced severe coronary vasculitis in DBA/2 mice, resulting in cardiac hypertrophy. MNs also induced coronary vasculitis in C57Bl/6 mice. These results suggest that the MNs of non-albicans Candida, such as C. krusei, induce similar toxicity to those of C. albicans.

N-Terminal (1→3)-ß-d-Glucan Recognition Proteins from Insects Recognize the Difference in Ultra-Structures of (1→3)-ß-d-Glucan.

Recognition of (1→3)-ß-d-glucans (BGs) by invertebrate ß-1,3-d-glucan recognition protein (BGRP) plays a significant role in the activation of Toll pathway and prophenoloxidase systems in insect host defense against fungal invasion. To examine the structure diversity of BGRPs for the recognition of physiochemically different BGs, the binding specificity of BGRPs cloned from four different insects to structure different BGs was characterized using ELISA. Recombinant BGRPs expressed as Fc-fusion proteins of human IgG1 bound to the solid phase of BGs. Based on the binding specificities, the BGRPs were categorized into two groups with different ultrastructures and binding characters; one group specifically binds BGs with triple-helical conformation, while the other group recognizes BGs with disordered conformations like single-helical or partially opened triple helix. The BGRPs from the silkworm and the Indian meal moth bound to the BGs with a triple-helical structure, whereas BGRPs from the red flour beetle and yellow mealworm beetle showed no binding to triple-helical BGs, but bound to alkaline-treated BGs that have a partially opened triple-helical conformation. This evidence suggests that the insect BGRPs can distinguish between different conformations of BGs and are equipped for determining the diversity of BG structures.