Synthetic Random Copolymers as a Molecular Platform To Mimic Host-Defense Antimicrobial Peptides.

Synthetic polymers have been used as a molecular platform to develop host-defense antimicrobial peptide (AMP) mimetics which are effective in killing drug-resistant bacteria. In this topical review, we will discuss the AMP-mimetic design and chemical optimization strategies as well as the biological and biophysical implications of AMP mimicry by synthetic polymers. Traditionally, synthetic polymers have been used as a chemical means to replicate the chemical functionalities and physicochemical properties of AMPs (e.g., cationic charge, hydrophobicity) to recapitulate their mode of action. However, we propose a new perception that AMP-mimetic polymers are an inherently bioactive platform as whole molecules, which mimic more than the side chain functionalities of AMPs. The tunable nature and chemical simplicity of synthetic random polymers facilitate the development of potent, cost-effective, broad-spectrum antimicrobials. The polymer-based approach offers the potential for many antimicrobial applications to be used directly in solution or attached to surfaces to fight against drug-resistant bacteria.

Cationic Amphiphilic Polymers with Antimicrobial Activity for Oral Care Applications: Eradication of S. mutans Biofilm.

The antibacterial and antibiofilm activities of cationic amphiphilic methacrylate polymers against cariogenic bacterium S. mutans were investigated. Cationic homopolymer PE0 and copolymer PE31 containing 31 mol % of ethyl methacrylate were synthesized by reversible addition-fragmentation chain transfer polymerization. These polymers displayed bactericidal activity toward S. mutans and prevented biofilm formation by killing the planktonic bacteria. At a concentration of 1000 µg/mL when incubated for 2 h the polymers reduced >80% of biofilm biomass. When the polymer assay solution with the biofilm was vigorously mixed using a pipet for 30 s, >50% of biofilm mass was removed at a polymer concentration of 250 µg/mL. Chlorhexidine and a cationic surfactant failed to reduce the biofilm mass at the same concentration. PE0 was the most effective in removing biofilm and did not show any significant cytotoxicity to human gingival fibroblast and periodontal ligament stem cells when incubated for 10 min.

Improving the Photodynamic Activity of Water-Soluble Porphyrin-Polysaccharide Complexes by Folic Acid Modification.

Studies have shown that folate receptors are highly expressed in various cancer cells. Here, we synthesized folic acid-conjugated pullulan (FAPL) as a solubilizing agent to improve the photodynamic activity of porphyrin derivative-polysaccharide complexes. The porphyrin derivative-FAPL complex exhibited long-term stability in an aqueous solution, attributed to the folic acid modification. Furthermore, in vitro and in vivo experiments highlighted the enhanced photodynamic activity of the porphyrin derivative-FAPL complex toward 4T1 breast-cancer cells, compared with the activities of the porphyrin derivative-pullulan complex and Photofrin. This enhanced activity is attributed to the improvement of intracellular uptake by the folate receptor.

Nanogel-based PspA intranasal vaccine prevents invasive disease and nasal colonization by Streptococcus pneumoniae.

To establish a safer and more effective vaccine against pneumococcal respiratory infections, current knowledge regarding the antigens common among pneumococcal strains and improvements to the system for delivering these antigens across the mucosal barrier must be integrated. We developed a pneumococcal vaccine that combines the advantages of pneumococcal surface protein A (PspA) with a nontoxic intranasal vaccine delivery system based on a nanometer-sized hydrogel (nanogel) consisting of a cationic cholesteryl group-bearing pullulan (cCHP). The efficacy of the nanogel-based PspA nasal vaccine (cCHP-PspA) was tested in murine pneumococcal airway infection models. Intranasal vaccination with cCHP-PspA provided protective immunity against lethal challenge with Streptococcus pneumoniae Xen10, reduced colonization and invasion by bacteria in the upper and lower respiratory tracts, and induced systemic and nasal mucosal Th17 responses, high levels of PspA-specific serum immunoglobulin G (IgG), and nasal and bronchial IgA antibody responses. Moreover, there was no sign of PspA delivery by nanogel to either the olfactory bulbs or the central nervous system after intranasal administration. These results demonstrate the effectiveness and safety of the nanogel-based PspA nasal vaccine system as a universal mucosal vaccine against pneumococcal respiratory infection.

Self-assembled pH-sensitive cholesteryl pullulan nanogel as a protein delivery vehicle.

A self-assembled nanogel, derived from an acid-labile cholesteryl-modified pullulan (acL-CHP), was prepared by grafting vinyl ether-cholesterol substituents onto a 100 kD pullulan main chain polymer backbone. Stable nanogels are formed by acL-CHP self-assemblies at neutral pH. The hydrodynamic radius of the nanogels, observed to be 26.5 ± 5.1 nm at pH 7.0, increased by ~135% upon acidification of the solution to pH 4.0. SEC analysis of the acL-CHP nanogel at pH 4.0 showed that the grafts were nearly 80% degraded after 24 h, whereas little or no degradation was observed over the same time period for a pH stable analog (acS-CHP) at pH 4.0 or the acL-CHP at pH 7.0. Complexation of BSA with the acL-CHP nanogel was observed at pH 7.0 with subsequent release of the protein upon acidification. These findings suggest that stimuli-responsive, self-assembled nanogels can release protein cargo in a manner that is controlled by the degradation rate of the cholesterol-pullulan grafting moiety.

Activation of platelets upon contact with a vitamin E-coated/non-coated surface.

The purpose of this study was to determine the effects of a vitamin E-coated surface on platelet activation, focusing on the interactions among the vitamin E-coated surface, platelets and leukocytes. Platelet-rich plasma (PRP) or PRP containing leukocytes (LPRP) was used. No difference was observed in platelet activation between PRP and LPRP for a vitamin E-coated membrane, meaning that platelet activation triggered by leukocytes was suppressed in plasma coming in contact with a vitamin E-coated membrane, while the membrane itself directly induced platelet activation. The antioxidant capacity of the vitamin E-coated membrane in contact with PRP or LPRP was partially reduced, but sufficient residual capacity remained. The in vitro experiments using an oxidized vitamin E-coated surface revealed that P-selectin expression and superoxide anion production in the platelets and platelet adhesion were induced by contact with the oxidized vitamin E-coated surface. We conclude that contact with a vitamin E-coated surface reduces platelet activation mediated by superoxide anions, probably by reducing superoxide anions, but during the process of the reduction, the vitamin E-coated surface itself becomes oxidized, which again causes platelet activation. The beneficial effects of a vitamin E-coated dialyzer in respect of platelet activation were counteracted by the formation of oxidized vitamin E.

Nanogel-based antigen-delivery system for nasal vaccines.

Nasal vaccination is considered a potent and practical immunization route for the induction of effective immunity to infectious diseases. Successful nasal vaccines require efficient delivery to, and retention of antigens within, nasal mucosa, including both the inductive (e.g., nasopharynx-associated lymphoid tissues) and effector (e.g., turbinate covered with single-layer epithelium) tissues, where antigen-specific immune responses are initiated and executed, respectively. We developed an approach towards successful nasal vaccination by using self-assembled nano-sized hydrogel particles, known as nanogels, which are composed of a cationic type of cholesteryl group-bearing pullulan. Here, we review the merging of nanotechnological and immunological concepts leading to the development of next-generation nasal vaccines, and demonstrate the applicability of novel nanogel-based vaccine for the prevention of infectious diseases.

CHRNA1 and its correlated-myogenesis/cell cycle genes are prognosis-related markers of metastatic melanoma.

Nicotinic acetylcholine receptors (CHRNs) expression and their critical role in various types of cancer have been reported. However, it is still unclear which CHRNs and their associated genes play essential roles in metastasis in melanoma patients. Here, we performed bioinformatics analyses on publicly available bulk RNA sequencing (RNA-seq) data of patients with melanoma to identify the CHRNs highly expressed in metastatic melanoma. We found that CHRNA1 was highly expressed in metastatic melanoma samples compared to primary melanoma samples and was strongly associated with CHRNB1 and CHRNG. These muscle-type CHRNs (CHRNA1, CHRNB1, and CHRNG) were correlated with the ZEB1 and Rho/ROCK pathway-related genes in metastatic melanoma samples. Pairwise correlations and enrichment analyses revealed that CHRNA1 was significantly associated with myogenesis/muscle contraction and cell cycle genes. Kaplan-Meier curves illustrated the involvement of CHRNA1, four of its correlated genes (DES, FLNC, CDK1, and CDC20), and the myogenesis gene signature in the prognosis of melanoma patients. Following the bulk RNA-seq analysis, single-cell RNA-seq (scRNA-seq) analysis showed that the CHRNA1-expressing melanoma cells are primarily metastatic and had high expression levels of CHRNB1, CHRNG, and myogenesis/cell cycle-related genes. Our bioinformatics analyses of the bulk RNA-seq and scRNA-seq data of patients with melanoma revealed that CHRNA1 and its correlated myogenesis/cell-related cycle genes are critical prognosis-related markers of metastatic melanoma.

Microarray transcriptome datasets of maternal-zygotic DNA methyltransferase 3aa -/- zebrafish during early developmental stages.

DNA methylation is an epigenetic regulator mediated by DNA methyltransferases (Dnmts). The methylation is involved in control of gene expression in vertebrates. It has been reported that there are mainly two types of de novo Dnmts, Dnmt3a and Dnmt3b, in mammals. These two Dnmts function in DNA methylation in the distinct or overlapping genomic regions. The zebrafish homologs of mammalian Dnmt3a are Dnmt3aa and Dnmt3ab. We generated a maternal-zygotic dnmt3aa deficient mutant (MZdnmt3aa) to identify the specific target regions for DNA methylation in the zebrafish genome and their function in the developmental process. Microarray analysis revealed alterations in gene expression by knock-out of dnmt3aa in early zebrafish development. Microarray datasets were produced from samples at five different developmental stages: 1-2 cell, shield, 5-somite, 1-day post fertilization (dpf), and 2 dpf. Herein, we present novel raw and processed transcriptome datasets generated by analysis of the MZdnmt3aa -/- mutant. The raw microarray data are available through the Gene Expression Omnibus (GEO), accession number GSE202646. These transcriptome data may be useful for comparing differences in gene expression among species of Dnmt3a mutants and for analyzing human diseases caused by DNMT3A such as acute myelogenous leukemia (AML).

Biomimetic antimicrobial polymers-Design, characterization, antimicrobial, and novel applications.

Biomimetic antimicrobial polymers have been an area of great interest as the need for novel antimicrobial compounds grows due to the development of resistance. These polymers were designed and developed to mimic naturally occurring antimicrobial peptides in both physicochemical composition and mechanism of action. These antimicrobial peptide mimetic polymers have been extensively investigated using chemical, biophysical, microbiological, and computational approaches to gain a deeper understanding of the molecular interactions that drive function. These studies have helped inform SARs, mechanism of action, and general physicochemical factors that influence the activity and properties of antimicrobial polymers. However, there are still lingering questions in this field regarding 3D structural patterning, bioavailability, and applicability to alternative targets. In this review, we present a perspective on the development and characterization of several antimicrobial polymers and discuss novel applications of these molecules emerging in the field. This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Therapeutic Approaches and Drug Discovery > Nanomedicine for Infectious Disease.

Unified approach to catechin hetero-oligomers: first total synthesis of trimer EZ-EG-CA isolated from Ziziphus jujuba.

A catechin hetero-trimer isolated from Ziziphus jujuba has been synthesized. Among three constituent monomers, (-)-epiafzelechin and (-)-epigallocatechin were prepared by de novo synthesis. Trimer formation relied on the unified approach to oligomers based on the bromo-capping and the orthogonal activation, reaching the reported structure of the natural product.

Analysis of Melanoma Gene Expression Signatures at the Single-Cell Level Uncovers 45-Gene Signature Related to Prognosis.

Since the current melanoma clinicopathological staging system remains restricted to predicting survival outcomes, establishing precise prognostic targets is needed. Here, we used gene expression signature (GES) classification and Cox regression analyses to biologically characterize melanoma cells at the single-cell level and construct a prognosis-related gene signature for melanoma. By analyzing publicly available scRNA-seq data, we identified six distinct GESs (named: "Anti-apoptosis", "Immune cell interactions", "Melanogenesis", "Ribosomal biogenesis", "Extracellular structure organization", and "Epithelial-Mesenchymal Transition (EMT)"). We verified these GESs in the bulk RNA-seq data of patients with skin cutaneous melanoma (SKCM) from The Cancer Genome Atlas (TCGA). Four GESs ("Immune cell interactions", "Melanogenesis", "Ribosomal biogenesis", and "Extracellular structure organization") were significantly correlated with prognosis (p = 1.08 × 10-5, p = 0.042, p = 0.001, and p = 0.031, respectively). We identified a prognostic signature of melanoma composed of 45 genes (MPS_45). MPS_45 was validated in TCGA-SKCM (HR = 1.82, p = 9.08 × 10-6) and three other melanoma datasets (GSE65904: HR = 1.73, p = 0.006; GSE19234: HR = 3.83, p = 0.002; and GSE53118: HR = 1.85, p = 0.037). MPS_45 was independently associated with survival (p = 0.002) and was proved to have a high potential for predicting prognosis in melanoma patients.

Understanding Breast Cancers through Spatial and High-Resolution Visualization Using Imaging Technologies.

Breast cancer is the most common cancer affecting women worldwide. Although many analyses and treatments have traditionally targeted the breast cancer cells themselves, recent studies have focused on investigating entire cancer tissues, including breast cancer cells. To understand the structure of breast cancer tissues, including breast cancer cells, it is necessary to investigate the three-dimensional location of the cells and/or proteins comprising the tissues and to clarify the relationship between the three-dimensional structure and malignant transformation or metastasis of breast cancers. In this review, we aim to summarize the methods for analyzing the three-dimensional structure of breast cancer tissue, paying particular attention to the recent technological advances in the combination of the tissue-clearing method and optical three-dimensional imaging. We also aimed to identify the latest methods for exploring the relationship between the three-dimensional cell arrangement in breast cancer tissues and the gene expression of each cell. Finally, we aimed to describe the three-dimensional imaging features of breast cancer tissues using noninvasive photoacoustic imaging methods.

Methylome data derived from maternal-zygotic DNA methyltransferase 3aa-/- zebrafish.

Genomic DNA methylation is an epigenetic marker mediated by DNA methyltransferases (Dnmts); in vertebrates, it comprises of a maintenance DNA methyltransferase, Dnmt1, and two de novo DNA methyltransferases (Dnmt3a and Dnmt3b). In zebrafish, there are two homologs of the mammalian Dnmt3a: Dnmt3aa and Dnmt3ab. A knockout (KO) mutant of zebrafish dnmt3aa was generated using the CRISPR/Cas9 genome-editing system as a new model for DNA methylation research. Since zebrafish dnmt3aa KO mutants were viable and fertile, a maternal-zygotic dnmt3aa deficient mutant (MZdnmt3aa) was generated. We performed whole-genome bisulfite sequencing (WGBS) to reveal the DNA methylation profile using this mutant and identified genomic regions with altered CpG methylation as differentially methylated regions (DMRs) in this mutant compared to those in the wild-type fish. We provided novel raw and processed datasets using the MZdnmt3aa KO mutant, and the raw data of WGBS are available through the Gene Expression Omnibus (GEO), accession number GSE178690.

Radiomic Analysis for Pretreatment Prediction of Recurrence Post-Radiotherapy in Cervical Squamous Cell Carcinoma Cancer.

BACKGROUND: The current study aims to predict the recurrence of cervical cancer patients treated with radiotherapy from radiomics features on pretreatment T1- and T2-weighted MR images. METHODS: A total of 89 patients were split into model training (63 patients) and model testing (26 patients). The predictors of recurrence were selected using the least absolute shrinkage and selection operator (LASSO) regression. The machine learning used neural network classifiers. RESULTS: Using LASSO analysis of radiomics, we found 25 features from the T1-weighted and 4 features from T2-weighted MR images, respectively. The accuracy was highest with the combination of T1- and T2-weighted MR images. The model performances with T1- or T2-weighted MR images were 86.4% or 89.4% accuracy, 74.9% or 38.1% sensitivity, 81.8% or 72.2% specificity, and 0.89 or 0.69 of the area under the curve (AUC). The model performance with the combination of T1- and T2-weighted MR images was 93.1% accuracy, 81.6% sensitivity, 88.7% specificity, and 0.94 of AUC. CONCLUSIONS: The radiomics analysis with T1- and T2-weighted MR images could highly predict the recurrence of cervix cancer after radiotherapy. The variation of the distribution and the difference in the pixel number at the peripheral and the center were important predictors.

Identification of aberrant transcription termination at specific gene loci with DNA hypomethylated transcription termination sites caused by DNA methyltransferase deficiency.

CpG methylation of genomic DNA is a well-known repressive epigenetic marker in eukaryotic transcription, and DNA methylation of promoter regions is correlated with gene silencing. In contrast to the promoter regions, the function of DNA methylation during transcription termination remains to be elucidated. A recent study revealed that mouse DNA methyltransferase 3a (Dnmt3a) mainly functions in de novo methylation in the promoter and gene body regions, including transcription termination sites (TTSs), during development. To investigate the relationship between DNA methylation overlapping the TTSs and transcription termination, we performed bioinformatics analysis using six pre-existing Dnmt-/- mouse cell datasets: four types of neurons (three Dnmt3a-/- and one Dnmt1-/- mutants) and two types of embryonic fibroblasts (MEFs) (Dnmt3a-/- and Dnmt3b-/- mutants). Combined analyses using methylome and transcriptome data revealed that read counts downstream of hypomethylated TTSs were increased in three types of neurons (two Dnmt3a-/- and one Dnmt1-/- mutants). Among these, an increase in chimeric transcripts downstream of the TTSs was observed in Dnmt3a-/- mature olfactory sensory neurons and Dnmt3a-/- agouti-related peptide (protein)-producing neurons, thereby indicating that read-through occurs in hypomethylated TTSs at specific gene loci in these two mutants. Conversely, in Dnmt3a-/- MEFs, we detected reductions in read counts downstream of hypomethylated TTSs. These results indicate that the hypomethylation of TTSs can both positively and negatively regulate transcription termination, dependent on Dnmt and cell types. This study is the first to identify the aberrant termination of transcription at specific gene loci with DNA hypomethylated TTSs attributable to Dnmt deficiency.

Nanogel antigenic protein-delivery system for adjuvant-free intranasal vaccines.

Nanotechnology is an innovative method of freely controlling nanometre-sized materials. Recent outbreaks of mucosal infectious diseases have increased the demands for development of mucosal vaccines because they induce both systemic and mucosal antigen-specific immune responses. Here we developed an intranasal vaccine-delivery system with a nanometre-sized hydrogel ('nanogel') consisting of a cationic type of cholesteryl-group-bearing pullulan (cCHP). A non-toxic subunit fragment of Clostridium botulinum type-A neurotoxin BoHc/A administered intranasally with cCHP nanogel (cCHP-BoHc/A) continuously adhered to the nasal epithelium and was effectively taken up by mucosal dendritic cells after its release from the cCHP nanogel. Vigorous botulinum-neurotoxin-A-neutralizing serum IgG and secretory IgA antibody responses were induced without co-administration of mucosal adjuvant. Importantly, intranasally administered cCHP-BoHc/A did not accumulate in the olfactory bulbs or brain. Moreover, intranasally immunized tetanus toxoid with cCHP nanogel induced strong tetanus-toxoid-specific systemic and mucosal immune responses. These results indicate that cCHP nanogel can be used as a universal protein-based antigen-delivery vehicle for adjuvant-free intranasal vaccination.

3D in vitro co-culture disc for spatiotemporal image analysis of cancer-stromal cell interaction.

Assessing phenotypic changes in both cancer cells and surrounding cells, which construct the tumour microenvironment, is essential for understanding the role of bi-directional communication among cells in the tumorigenic process. Here, a 3D in vitro cancer-stroma co-culture system, a co-culture disc, was reported for the spatiotemporal image analysis of cancer-stromal cell interactions. Due to their centre-open disc structure, the lung cancer A549 spheroids could be co-cultured with a high concentration of fibroblasts, without gel shrinkage in the long term (>1 month). In the co-culture disc, some populations of applied normal human lung fibroblasts showed morphological and phenotypic changes into activated myofibroblasts (AMFs) with high expression of myo-fibrotic α-smooth muscle actin fibre in the cell, which is a well-known feature of cancer-associated fibroblasts. The AMFs appeared heterogeneously at the boundary of cancer spheroids, which could not be detected by standard mass analysis using a quantitative RT-qPCR system, and they led to A549 cancer cell migration. In addition, the effects of oncogenic or medicinal additives were quantitatively assessed by combining co-culture discs with image analysis. This system provides a new potential technique to analyse the complicated crosstalk among cancer tissue-constructing cells during the tumorigenic process and provides insight into applications for the quantitative evaluation of substances inducing tumorigenesis as well as drugs to prevent and inhibit cancer progression.

Amphiphilic polymer therapeutics: an alternative platform in the fight against antibiotic resistant bacteria.

As we are on the cusp of the "post-antibiotic" era due to rapid spread of drug resistant bacteria, there is an urgent need for new antimicrobials that are not susceptible to bacterial resistance mechanisms. In this review, we will discuss the recent development of "polymer therapeutics" with antimicrobial activity. Learning from host-defence peptides, we propose the biomimetic design of synthetic polymers to target bacterial cell membranes, which act by compromising the membrane integrity. The discussion is extended to the future challenges and opportunities of antimicrobial polymers for clinical applications.

Synthetic Biomimetic Polymethacrylates: Promising Platform for the Design of Anti-Cyanobacterial and Anti-Algal Agents.

Extensive, uncontrolled growth of algae and cyanobacteria is an environmental, public health, economic, and technical issue in managing natural and engineered water systems. Synthetic biomimetic polymers have been almost exclusively considered antimicrobial alternatives to conventional antibiotics to treat human bacterial infections. Very little is known about their applicability in an aquatic environment. Here, we introduce synthetic biomimetic polymethacrylates (SBPs) as a cost-effective and chemically facile, flexible platform for designing a new type of agent suitable for controlling and mitigating photosynthetic microorganisms. Since SBPs are cationic and membranolytic in heterotrophic bacteria, we hypothesized they could also interact with negatively charged cyanobacterial or algal cell walls and membranes. We demonstrated that SBPs inhibited the growth of aquatic photosynthetic organisms of concern, i.e., cyanobacteria (Microcystis aeruginosa and Synechococcus elongatus) and green algae (Chlamydomonas reinhardtii and Desmodesmus quadricauda), with 50% effective growth-inhibiting concentrations ranging between 95 nM and 6.5 µM. Additionally, SBPs exhibited algicidal effects on C. reinhardtii and cyanocidal effects on picocyanobacterium S. elongatus and microcystin-producing cyanobacterium M. aeruginosa. SBP copolymers, particularly those with moderate hydrophobic content, induced more potent cyanostatic and cyanocidal effects than homopolymers. Thus, biomimetic polymers are a promising platform for the design of anti-cyanobacterial and anti-algal agents for water treatment.