An enhancer peptide for membrane-disrupting antimicrobial peptides

<p>Abstract</p> <p>Background</p> <p>NP4P is a synthetic peptide derived from a natural, non-antimicrobial peptide fragment (pro-region of nematode cecropin P4) by substitution of all acidic amino acid residues with amides (i.e., Glu → Gln, and Asp → Asn).</p> <p>Results</p> <p>In the presence of NP4P, some membrane-disrupting antimicrobial peptides (ASABF-α, polymyxin B, and nisin) killed microbes at lower concentration (e.g., 10 times lower minimum bactericidal concentration for ASABF-α against <it>Staphylococcus aureus</it>), whereas NP4P itself was not bactericidal and did not interfere with bacterial growth at ≤ 300 μg/mL. In contrast, the activities of antimicrobial agents with a distinct mode of action (indolicidin, ampicillin, kanamycin, and enrofloxacin) were unaffected. Although the membrane-disrupting activity of NP4P was slight or undetectable, ASABF-α permeabilized <it>S. aureus </it>membranes with enhanced efficacy in the presence of NP4P.</p> <p>Conclusions</p> <p>NP4P selectively enhanced the bactericidal activities of membrane-disrupting antimicrobial peptides by increasing the efficacy of membrane disruption against the cytoplasmic membrane.</p

Generation of novel cationic antimicrobial peptides from natural non-antimicrobial sequences by acid-amide substitution

<p>Abstract</p> <p>Background</p> <p>Cationic antimicrobial peptides (CAMPs) are well recognized to be promising as novel antimicrobial and antitumor agents. To obtain novel skeletons of CAMPs, we propose a simple strategy using acid-amide substitution (i.e. Glu→Gln, Asp→Asn) to confer net positive charge to natural non-antimicrobial sequences that have structures distinct from known CAMPs. The potential of this strategy was verified by a trial study.</p> <p>Methods</p> <p>The pro-regions of nematode cecropin P1-P3 (P1P-P3P) were selected as parent sequences. P1P-P3P and their acid-amide-substituted mutants (NP1P-NP3P) were chemically synthesized. Bactericidal and membrane-disruptive activities of these peptides were evaluated. Conformational changes were estimated from far-ultraviolet circular dichroism (CD) spectra.</p> <p>Results</p> <p>NP1P-NP3P acquired potent bactericidal activities via membrane-disruption although P1P-P3P were not antimicrobial. Far-ultraviolet CD spectra of NP1P-NP3P were similar to those of their parent peptides P1P-P3P, suggesting that NP1P-NP3P acquire microbicidal activity without remarkable conformational changes. NP1P-NP3P killed bacteria in almost parallel fashion with their membrane-disruptive activities, suggesting that the mode of action of those peptides was membrane-disruption. Interestingly, membrane-disruptive activity of NP1P-NP3P were highly diversified against acidic liposomes, indicating that the acid-amide-substituted nematode cecropin pro-region was expected to be a unique and promising skeleton for novel synthetic CAMPs with diversified membrane-discriminative properties.</p> <p>Conclusions</p> <p>The acid-amide substitution successfully generated some novel CAMPs in our trial study. These novel CAMPs were derived from natural non-antimicrobial sequences, and their sequences were completely distinct from any categories of known CAMPs, suggesting that such mutated natural sequences could be a promising source of novel skeletons of CAMPs.</p

Preparation of silk resins by hot pressing Bombyx mori and Eri silk powders

We investigate the mechanical properties and structure of silk resins as potential alternatives to tortoiseshell for producing eyeglass frames and various ornaments. Silk powders are obtained from Bombyx mori and Eri silk waste fibers before the degumming process. The powders are fabricated into resins via simple hot pressing under a pressure of 31.2 MPa at temperatures in the range 150–180 °C. The results indicate that the B. mori resins have higher micro-Vickers hardness, three-point bending strength, and elastic modulus (66 Hv, 122 MPa, and 8.7 GPa, respectively) compared to the Eri silk resins (58 Hv, 95 MPa, and 8.2 GPa, respectively). The better mechanical properties of the fibroin resins are related directly to longer drying times. The optimum drying conditions are found to be at a temperature of 100 °C under a–vacuum of −0.1 MPa for a time of 7 d. ATR-FTIR and XRD results show how the fibroin structure changes after resinification and drying. The morphology and the distribution size of particle of the silk powders and the fractured surfaces of the resins are analyzed from SEM micrographs. The present findings demonstrate that silk resins are suitable materials for developing useful applications because of their favorable mechanical properties

全部床義歯装着が舌骨の位置と咽頭の幅径に与える影響

高齢者の摂食・嚥下機能を考える上では,加齢変化による摂食・嚥下機能の予備力低下を考慮する必要がある.特に,加齢による中咽頭拡大という器質的な変化は,咽頭残留の増加などの機能的変化の一因となると考えられている.一方,高齢者の口腔には,上下顎に全部床義歯が装着されていることが多く,義歯の装着と撤去は下顎位を偏位させて,中咽頭の形態に影響を及ぼすと推察されるが,その詳細は明らかになっていない.そこで本研究では,全部床義歯の装着状況が中咽頭の三次元的形態に与える影響を明らかにすることを目的とした.対象は,全部床義歯装着者17名(平均年齢72.9±9.2歳)とした.上下顎全部床義歯装着(上下顎あり),上顎全部床義歯装着(上顎のみ),上下顎全部床義歯非装着(上下顎なし)の3条件において,CBCTによる撮影を行い,三次元解析を用いて1)下顎の位置(FMA),2)舌骨の位置,3)中咽頭の幅径を測定し,各条件間で比較検討を行った.FMAは,上下顎ありに比べて,他の2条件では有意に減少した.舌骨の位置は,上下顎なしでは,上下顎ありに比べ有意に上方また前方に移動した.中咽頭の前後径は,喉頭蓋谷最深点の高さにおいて,上下顎なしでは,他の2条件に比べ有意に増加した.左右径は,上下顎なしでは,上下顎ありに比べ舌背と舌根の境界の高さ,および喉頭蓋谷最深点での高さにおいて有意に増加した.全部床義歯の撤去は,下顎を前上方に回転させ,舌骨を前上方に牽引する.その結果,舌根部が前方移動し,中咽頭の下部が前後左右方向に拡大したものと考えられた.義歯の撤去は,加齢によって生じた中咽頭の拡大という予備力低下のさらなる増悪に通じ,嚥下の際により大きな筋の収縮を必要とさせると考えられた.摂食・嚥下障害を有する高齢者は義歯が必要にも関わらず装着していないことも多く,本研究の結果より,義歯装着の新たな重要性が示された.In rehabilitation for dysphagia in the elderly, it is necessary to take into account both age-related changes and reduced reserve capacity for swallowing. Especially, since it was thought that the oropharyngeal expansion as morphological age-related change causes functional changes such as pharyngeal residue. Wearing complete dentures is common in elderly people, and although wearing and removing dentures is thought to affect the morphology of the oropharynx, the details of this effect remain unclear. The present study aimed to elucidate the relationship between wearing dentures and changes in the hyoid bone position and pharyngeal diameter. The subjects were 17 elderly people fitted with complete dentures who underwent cone-beam computed tomography imaging while wearing maxillary and mandibular dentures (MM), maxillary denture only (OM), and with neither maxillary nor mandibular dentures (ED). Computed tomography images were subjected to analysis of mandibular position (FMA), hyoid bone position, and antero-posterior and left-right oropharyngeal diameters. FMA decreased significantly in the order MM, OM, ED. In the ED condition, the hyoid bone shifted significantly in the antero-superior direction as compared with the MM and OM conditions. Antero-posterior and left-right oropharyngeal diameters in the ED condition were significantly larger than those in the MM condition at the height of the base of the epiglottis. Removal of complete dentures causes an antero-superior shift of the mandible, resulting in an anterior and superior shift of the hyoid bone and the expansion of the inferior part of the oropharynx. These findings suggest that removal of dentures exacerbate reduced reserve capacity for swallowing by age-related changes in the elderly

Production of scFv-Conjugated Affinity Silk Powder by Transgenic Silkworm Technology

Bombyx mori (silkworm) silk proteins are being utilized as unique biomaterials for medical applications. Chemical modification or post-conjugation of bioactive ligands expand the applicability of silk proteins; however, the processes are elaborate and costly. In this study, we used transgenic silkworm technology to develop single-chain variable fragment (scFv)-conjugated silk fibroin. The cocoons of the transgenic silkworm contain fibroin L-chain linked with scFv as a fusion protein. After dissolving the cocoons in lithium bromide, the silk solution was dialyzed, concentrated, freeze-dried, and crushed into powder. Immunoprecipitation analyses demonstrate that the scFv domain retains its specific binding activity to the target molecule after multiple processing steps. These results strongly suggest the promise of scFv-conjugated silk fibroin as an alternative affinity reagent, which can be manufactured using transgenic silkworm technology at lower cost than traditional affinity carriers

Optimization of the proliferation and persistency of CAR T cells derived from human induced pluripotent stem cells

CARシグナルを補完する遺伝子改変により *iCAR-T細胞の固形がん治療効果が改善される. 京都大学プレスリリース. 2022-12-13.Genetic modifications boosting CAR signaling improve the therapeutic efficacy of iPSC-derived CAR-T cells against solid tumors. 京都大学プレスリリース. 2022-12-13.The effectiveness of chimaeric antigen receptor (CAR) T-cell immunotherapies against solid tumours relies on the accumulation, proliferation and persistency of T cells at the tumour site. Here we show that the proliferation of CD8αβ cytotoxic CAR T cells in solid tumours can be enhanced by deriving and expanding them from a single human induced-pluripotent-stem-cell clone bearing a CAR selected for efficient differentiation. We also show that the proliferation and persistency of the effector cells in the tumours can be further enhanced by genetically knocking out diacylglycerol kinase, which inhibits antigen-receptor signalling, and by transducing the cells with genes encoding for membrane-bound interleukin-15 (IL-15) and its receptor subunit IL-15Rα. In multiple tumour-bearing animal models, the engineered hiPSC-derived CAR T cells led to therapeutic outcomes similar to those of primary CD8 T cells bearing the same CAR. The optimization of effector CAR T cells derived from pluripotent stem cells may aid the development of long-lasting antigen-specific T-cell immunotherapies for the treatment of solid tumours

Effects of RGDS sequence genetically interfused in the silk fibroin light chain protein on chondrocyte adhesion and cartilage synthesis.

Initial chondrocyte-silk fibroin interactions are implicated in chondrogenesis when using fibroin as a scaffold for chondrocytes. Here, we focused on integrin-mediated cell-scaffold adhesion and prepared cell adhesive fibroin in which a tandem repeat of the Arg-Gly-Asp-Ser (RGDS) sequence was genetically interfused in the fibroin light chain (L-chain) (L-RGDSx2 fibroin). We investigated the effects of the sequence on chondrocyte adhesion and cartilage synthesis, in comparison to the effects of fibronectin. As the physicochemical surface properties (e.g., wettability and zeta potential) of the fibroin substrate were not affected by the modification, specific cell adhesion to the RGDS predominately changed the chondrocyte adhesive state. This suggestion was also supported by the competitive inhibition of chondrocyte attachment to the L-RGDSx2 fibroin substrate with soluble RGD peptides in the medium. Unlike fibronectin, the expression of RGDS in the fibroin L-chain had no effect on chondrocyte spreading area but enhanced mRNA expression levels of integrins alpha5 and beta1, and aggrecan at 12 h after seeding. Although both the sequence and fibronectin increased cell adhesive force, chondrocytes grown on the fibroin substrate exhibited a peak in the force with time in culture. These results suggested that moderate chondrocyte adhesion to fibroin induced by the RGDS sequence was able to maintain the chondrogenic phenotype and, from the histology findings, the sequence could facilitate chondrogenesis

System-Based Differential Gene Network Analysis for Characterizing a Sample-Specific Subnetwork

Gene network estimation is a method key to understanding a fundamental cellular system from high throughput omics data. However, the existing gene network analysis relies on having a sufficient number of samples and is required to handle a huge number of nodes and estimated edges, which remain difficult to interpret, especially in discovering the clinically relevant portions of the network. Here, we propose a novel method to extract a biomedically significant subnetwork using a Bayesian network, a type of unsupervised machine learning method that can be used as an explainable and interpretable artificial intelligence algorithm. Our method quantifies sample specific networks using our proposed Edge Contribution value (ECv) based on the estimated system, which realizes condition-specific subnetwork extraction using a limited number of samples. We applied this method to the Epithelial-Mesenchymal Transition (EMT) data set that is related to the process of metastasis and thus prognosis in cancer biology. We established our method-driven EMT network representing putative gene interactions. Furthermore, we found that the sample-specific ECv patterns of this EMT network can characterize the survival of lung cancer patients. These results show that our method unveils the explainable network differences in biological and clinical features through artificial intelligence technology