Novel Sarcoscypha Species from National Parks in Korea: Sarcoscypha humida sp. nov.

Sarcoscypha (Sarcoscyphaceae, Pezizales) is a saprobic fungus characterized by the cup or disc-shaped blight red apothecium and oblong to ellipsoid ascospores. The 18 species of Sarcoscypha were known to occur in Europe, North America, and tropical Asia. However, up to date, only two Sarcoscypha species have been reported in Korea. In this study, novel Sarcoscypha specimens were collected from Juwangsan, Odaesan, and Taebaeksan National Parks from September to October in Korea. This species is well distinguished from other Sarcoscypha species according to the molecular and phylogenetic analysis based on internal transcribed spacer (ITS) region. Here, we provided detailed descriptions with illustrations and a phylogenetic tree to report our specimens as novel Sarcoscypha species.

Cloning and Expression Analysis of Bioluminescence Genes in Omphalotus guepiniiformis Reveal Stress-Dependent Regulation of Bioluminescence.

Bioluminescence is a type of chemiluminescence that arises from a luciferase-catalyzed oxidation reaction of luciferin. Molecular biology and comparative genomics have recently elucidated the genes involved in fungal bioluminescence and the evolutionary history of their clusters. However, most studies on fungal bioluminescence have been limited to observing the changes in light intensity under various conditions. To understand the molecular basis of bioluminescent responses in Omphalotus guepiniiformis under different environmental conditions, we cloned and sequenced the genes of hispidin synthase, hispidin-3-hydroxylase, and luciferase enzymes, which are pivotal in the fungal bioluminescence pathway. Each gene showed high sequence similarity to that of other luminous fungal species. Furthermore, we investigated their transcriptional changes in response to abiotic stresses. Wound stress enhanced the bioluminescence intensity by increasing the expression of bioluminescence pathway genes, while temperature stress suppressed the bioluminescence intensity via the non-transcriptional pathway. Our data suggested that O. guepiniiformis regulates bioluminescence to respond differentially to specific environmental stresses. To our knowledge, this is the first study on fungal bioluminescence at the gene expression level. Further studies are required to address the biological and ecological meaning of different bioluminescence responses in changing environments, and O. quepiniiformis could be a potential model species.

Advancing Biomimetic Functions of Synthetic Cells through Compartmentalized Cell-Free Protein Synthesis.

Synthetic cells are artificial constructs that mimic the structures and functions of living cells. They are attractive for studying diverse biochemical processes and elucidating the origins of life. While creating a living synthetic cell remains a grand challenge, researchers have successfully synthesized hundreds of unique synthetic cell platforms. One promising approach to developing more sophisticated synthetic cells is to integrate cell-free protein synthesis (CFPS) mechanisms into vesicle platforms. This makes it possible to create synthetic cells with complex biomimetic functions such as genetic circuits, autonomous membrane modifications, sensing and communication, and artificial organelles. This Review explores recent advances in the use of CFPS to impart advanced biomimetic structures and functions to bottom-up synthetic cell platforms. We also discuss the potential applications of synthetic cells in biomedicine as well as the future directions of synthetic cell research.

Rational design and engineering of polypeptide/protein vesicles for advanced biological applications.

Synthetic vesicles have gained considerable popularity in recent years for numerous biological and medical applications. Among the various types of synthetic vesicles, the utilization of polypeptides and/or proteins as fundamental constituents has garnered significant interest for vesicle construction owing to the unique bio-functionalities inherent in rationally designed amino acid sequences. Especially the incorporation of functional proteins onto the vesicle surface facilitates a wide range of advanced biological applications that are not easily attainable with traditional building blocks, such as lipids and polymers. The main goal of this review is to provide a comprehensive overview of the latest advancements in polypeptide/protein vesicles. Moreover, this review encompasses the rational design and engineering strategies employed in the creation of polypeptide/protein vesicles, including the synthesis of building blocks, the modulation of their self-assembly, as well as their diverse applications. Furthermore, this work includes an in-depth discussion of the key challenges and opportunities associated with polypeptide/protein vesicles, providing valuable insights for future research. By offering an up-to-date review of this burgeoning field of polypeptide/protein vesicle research, this review will shed light on the potential applications of these biomaterials.

Temperature-responsive membrane permeability of recombinant fusion protein vesicles.

In this study, we investigate the changes in the permeability of the recombinant fusion protein vesicles with different membrane structures as a function of solution temperature. The protein vesicles are self-assembled from recombinant fusion protein complexes composed of an mCherry fused with a glutamic acid-rich leucine zipper and a counter arginine-rich leucine zipper fused with an elastin-like polypeptide (ELP). We have found that the molecular weight cut-off (MWCO) of the protein vesicle membranes varies inversely with solution temperature by monitoring the transport of fluorescent-tagged dextran dyes with different molecular weights. The temperature-responsiveness of the protein vesicle membranes is obtained from the lower critical solution temperature behavior of ELP in the protein building blocks. Consequently, the unique vesicle membrane structures with different single-layered and double-layered ELP organizations impact the sensitivity of the permeability responses of the protein vesicles. Single-layered protein vesicles with the ELP domains facing the interior show more drastic permeability changes as a function of temperature than double-layered protein vesicles in which ELP blocks are buried inside the membranes. This work about the temperature-responsive membrane permeability of unique protein vesicles will provide design guidelines for new biomaterials and their applications, such as drug delivery and synthetic protocell development.

Determination and Analysis of Hyper-Variable A Mating Types in Wild Strains of Lentinula edodes in Korea.

The diversity of A mating type in wild strains of Lentinula edodes was extensively analyzed to characterize and utilize them for developing new cultivars. One hundred twenty-three A mating type alleles, including 67 newly discovered alleles, were identified from 106 wild strains collected for the past four decades in Korea. Based on previous studies and current findings, a total of 130 A mating type alleles have been found, 124 of which were discovered from wild strains, indicating the hyper-variability of A mating type alleles of L. edodes. About half of the A mating type alleles in wild strains were found in more than two strains, whereas the other half of the alleles were found in only one strain. About 90% of A mating type combinations in dikaryotic wild strains showed a single occurrence. Geographically, diverse A mating type alleles were intensively located in the central region of the Korean peninsula, whereas only allele A17 was observed throughout Korea. We also found the conservation of the TCCCAC motif in addition to the previously reported motifs, including ATTGT, ACAAT, and GCGGAG, in the intergenic regions of A mating loci. Sequence comparison among some alleles indicated that accumulated mutation and recombination would contribute to the diversification of A mating type alleles in L. edodes. Our data support the rapid evolution of A mating locus in L. edodes, and would help to understand the characteristics of A mating loci of wild strains in Korea and help to utilize them for developing new cultivars.

Advances in cell coculture membranes recapitulating in vivo microenvironments.

Porous membranes play a critical role in in vitro heterogeneous cell coculture systems because they recapitulate the in vivo microenvironment to mediate physical and biochemical crosstalk between cells. While the conventionally available Transwell® system has been widely used for heterogeneous cell coculture, there are drawbacks to precise control over cell-cell interactions and separation for implantation. The size and numbers of the pores and the thickness of the porous membranes are crucial in determining the efficiency of paracrine signaling and direct junctions between cocultured cells, and significantly impact on the performance of heterogeneous cell cultures. These opportunities and challenges have motivated the design of advanced coculture platforms through improvement of the structural and functional properties of porous membranes.

Heterogeneous Synthetic Vesicles toward Artificial Cells: Engineering Structure and Composition of Membranes for Multimodal Functionalities.

The desire to develop artificial cells to imitate living cells in synthetic vesicle platforms has continuously increased over the past few decades. In particular, heterogeneous synthetic vesicles made from two or more building blocks have attracted attention for artificial cell applications based on their multifunctional modules with asymmetric structures. In addition to the traditional liposomes or polymersomes, polypeptides and proteins have recently been highlighted as potential building blocks to construct artificial cells owing to their specific biological functionalities. Incorporating one or more functionally folded, globular protein into synthetic vesicles enables more cell-like functions mediated by proteins. This Review highlights the recent research about synthetic vesicles toward artificial cell models, from traditional synthetic vesicles to protein-assembled vesicles with asymmetric structures. We aim to provide fundamental and practical insights into applying knowledge on molecular self-assembly to the bottom-up construction of artificial cell platforms with heterogeneous building blocks.

A novel combination therapy for multidrug resistant pathogens using chitosan nanoparticles loaded with ß-lactam antibiotics and ß-lactamase inhibitors.

Antimicrobial resistance is one of the greatest global threats. Particularly, multidrug resistant extended-spectrum ß-lactamase (ESBL)-producing pathogens confer resistance to many commonly used medically important antibiotics, especially beta-lactam antibiotics. Here, we developed an innovative combination approach to therapy for multidrug resistant pathogens by encapsulating cephalosporin antibiotics and ß-lactamase inhibitors with chitosan nanoparticles (CNAIs). The four combinations of CNAIs including two cephalosporin antibiotics (cefotaxime and ceftiofur) with two ß-lactamase inhibitors (tazobactam and clavulanate) were engineered as water-oil-water emulsions. Four combinations of CNAIs showed efficient antimicrobial activity against multidrug resistant ESBL-producing Enterobacteriaceae. The CNAIs showed enhanced antimicrobial activity compared to naïve chitosan nanoparticles and to the combination of cephalosporin antibiotics and ß-lactamase inhibitors. Furthermore, CNAIs attached on the bacterial surface changed the permeability to the outer membrane, resulting in cell damage that leads to cell death. Taken together, CNAIs have provided promising potential for treatment of diseases caused by critically important ESBL-producing multidrug resistant pathogens.

The genus Arthrinium (Ascomycota, Sordariomycetes, Apiosporaceae) from marine habitats from Korea, with eight new species.

Species of Arthrinium are well-known plant pathogens, endophytes, or saprobes found in various terrestrial habitats. Although several species have been isolated from marine environments and their remarkable biological activities have been reported, marine Arthrinium species remain poorly understood. In this study, the diversity of this group was evaluated based on material from Korea, using morphological characterization and molecular analyses with the internal transcribed spacer (ITS) region, ß-tubulin (TUB), and translation elongation factor 1-alpha (TEF). A total of 41 Arthrinium strains were isolated from eight coastal sites which represented 14 species. Eight of these are described as new to science with detailed descriptions.

Tuning the Structural Integrity and Mechanical Properties of Globular Protein Vesicles by Blending Crosslinkable and NonCrosslinkable Building Blocks.

Vesicles made from functionally folded, globular proteins that perform specific biological activities, such as catalysis, sensing, or therapeutics, show potential applications as artificial cells, microbioreactors, or protein drug delivery vehicles. The mechanical properties of vesicle membranes, including the elastic modulus and hardness, play a critical role in dictating the stability and shape transformation of the vesicles under external stimuli triggers. Herein, we have developed a strategy to tune the mechanical properties and integrity of globular protein vesicle (GPV) membranes of which building molecules are recombinant fusion protein complexes: a mCherry fused with an acidic leucine zipper (mCherry-ZE) and a basic leucine zipper fused with an elastin-like polypeptide (ZR-ELP). To control the mechanical properties of GPVs, we introduced a nonstandard amino acid (para-azidophenylalanine (pAzF)) into the ELP domains (ELP-X), which enabled the creation of crosslinked vesicles under ultraviolet (UV) irradiation. Crosslinked GPVs made from mCherry-ZE/ZR-ELP-X complexes presented higher stability than noncrosslinked GPVs under hypotonic osmotic stress. The degree of swelling of GPVs increased as less crosslinking was achieved in the vesicle membranes, which resulted in the disassembly of GPVs into membraneless coacervates. Nanoindentation by atomic force microscopy (AFM) confirmed that the stiffness and Young's elastic modulus of GPVs increase as the blending molar ratio of ZR-ELP-X to ZR-ELP increases to make vesicles. The results obtained in this study suggest a rational design to make GPVs with tunable mechanical properties for target applications by simply varying the blending ratio of ZR-ELP and ZR-ELP-X in the vesicle self-assembly.

Selective Killing of Shiga Toxin-Producing Escherichia coli with Antibody-Conjugated Chitosan Nanoparticles in the Gastrointestinal Tract.

Shiga toxin-producing Escherichia coli (STEC) are critical foodborne pathogens, which cause serious human health issues, including hemolytic uremic syndrome. Illnesses caused by STEC lack effective treatments that target the elimination of these bacteria from the gastrointestinal tract without causing an adverse effect. Reducing this pathogen from a reservoir of STEC is an effective strategy, but the challenges remain due to the lack of efficient, selective antimicrobial agents. We developed specific antibody-conjugated chitosan nanoparticles (CNs) to selectively target and treat STEC in the gastrointestinal tract. Given the great broad-spectrum antimicrobial activity of CN, we conjugated antibodies to CN. Antibodies were raised and purified from egg yolks after immunization of hens with seven different O-side-chain antigens isolated from STEC (O26, O45, O103, O111, O121, O145, and O157). We prepared CN-immunoglobulin Y (IgY) conjugates by forming amide bonds at different ratios of CN:IgY (10:1, 10:2, and 10:4). The CN-IgY conjugated at a 10:2 ratio demonstrated significantly enhanced antimicrobial activity against E. coli O157:H7. Conjugates of CN and anti-STEC IgY antibodies killed corresponding STEC serotypes specifically and selectively, while showing no significant impact on nontargeted bacteria, including Salmonella enterica and Lactobacillus plantarum. The enhanced antimicrobial activity of CN-IgY against STEC was also confirmed in synthetic intestinal fluid, as well as an in vivo animal model of Caenorhabditis elegans. These results suggest that the CN-IgY conjugates have strong and specific antimicrobial activity and that they are also great candidates to eliminate pathogens selectively in the gastrointestinal tract without inhibiting beneficial bacteria.

Xylodon flaviporus-Derived Drimane Sesquiterpenoids That Inhibit Osteoclast Differentiation.

The presence of excessive osteoclasts is a major factor in skeletal diseases. The present study aimed to discover osteoclast differentiation inhibitors from the basidiomycete Xylodon flaviporus. Seven new drimane sesquiterpenoids (1-7) and 7-ketoisodrimenin-5-ene (8) were obtained and characterized by various spectroscopic methods. The isolated compounds were evaluated for their inhibitory effects against receptor activator of nuclear factor-kappa-B ligand-induced osteoclastogenesis in mouse bone marrow macrophages. Compounds 1, 3, and 6 showed potent activities with IC50 values of 1.6, 0.9, and 2.1 µM, respectively, while 4, 5, and 7 exhibited relatively weak activities with IC50 values of 10.7, 10.1, and 8.5 µM, respectively.

Comprehensive genomic and transcriptomic analysis of polycyclic aromatic hydrocarbon degradation by a mycoremediation fungus, Dentipellis sp. KUC8613.

The environmental accumulation of polycyclic aromatic hydrocarbons (PAHs) is of great concern due to potential carcinogenic and mutagenic risks, as well as their resistance to remediation. While many fungi have been reported to break down PAHs in environments, the details of gene-based metabolic pathways are not yet comprehensively understood. Specifically, the genome-scale transcriptional responses of fungal PAH degradation have rarely been reported. In this study, we report the genomic and transcriptomic basis of PAH bioremediation by a potent fungal degrader, Dentipellis sp. KUC8613. The genome size of this fungus was 36.71 Mbp long encoding 14,320 putative protein-coding genes. The strain efficiently removed more than 90% of 100 mg/l concentration of PAHs within 10 days. The genomic and transcriptomic analysis of this white rot fungus highlights that the strain primarily utilized non-ligninolytic enzymes to remove various PAHs, rather than typical ligninolytic enzymes known for playing important roles in PAH degradation. PAH removal by non-ligninolytic enzymes was initiated by both different PAH-specific and common upregulation of P450s, followed by downstream PAH-transforming enzymes such as epoxide hydrolases, dehydrogenases, FAD-dependent monooxygenases, dioxygenases, and glycosyl- or glutathione transferases. Among the various PAHs, phenanthrene induced a more dynamic transcriptomic response possibly due to its greater cytotoxicity, leading to highly upregulated genes involved in the translocation of PAHs, a defense system against reactive oxygen species, and ATP synthesis. Our genomic and transcriptomic data provide a foundation of understanding regarding the mycoremediation of PAHs and the application of this strain for polluted environments.

Understanding the Coacervate-to-Vesicle Transition of Globular Fusion Proteins to Engineer Protein Vesicle Size and Membrane Heterogeneity.

Protein-rich coacervates are liquid phases separate from the aqueous bulk phase that are used by nature for compartmentalization and more recently have been exploited by engineers for delivery and formulation applications. They also serve as an intermediate phase in an assembly path to more complex structures, such as vesicles. Recombinant fusion protein complexes made from a globular protein fused with a glutamic acid-rich leucine zipper (globule-ZE) and an arginine-rich leucine zipper fused with an elastin-like polypeptide (ZR-ELP) show different phases from soluble, through an intermediate coacervate phase, and finally to vesicles with increasing temperature of the aqueous solution. We investigated the phase transition kinetics of the fusion protein complexes at different temperatures using dynamic light scattering and microscopy, along with mathematical modeling. We controlled coacervate growth by aging the solution at an intermediate temperature that supports coacervation and confirmed that the size of the coacervate droplets dictates the size of vesicles formed upon further heating. With this understanding of the phase transition, we developed strategies to induce heterogeneity in the organization of globular proteins in the vesicle membrane through simple mixing of coacervates containing two different globular fusion proteins prior to the vesicle transition. This study gives fundamental insights and practical strategies for development of globular protein-rich coacervates and vesicles for drug delivery, microreactors, and protocell applications.

Development of Multimodal Antibacterial Surfaces Using Porous Amine-Reactive Films Incorporating Lubricant and Silver Nanoparticles.

Anti-biofouling has been improved by passive or active ways. Passive antifouling strategies aim to prevent the initial adsorption of foulants, while active strategies aim to eliminate proliferative fouling by destruction of the chemical structure and inactivation of the cells. However, neither passive antifouling strategies nor active antifouling strategies can solely resist biofouling due to their inherent limitations. Herein, we successfully developed multimodal antibacterial surfaces for waterborne and airborne bacteria with the benefit of a combination of antiadhesion (passive) and bactericidal (active) properties of the surfaces. We elaborated multifunctionalizable porous amine-reactive (PAR) polymer films from poly(pentafluorophenyl acrylate) (PPFPA). Pentafluorophenyl ester groups in the PAR films facilitate creation of multiple functionalities through a simple postmodification under mild condition, based on their high reactivity toward various primary amines. We introduced amine-containing poly(dimethylsiloxane) (amine-PDMS) and dopamine into the PAR films, resulting in infusion of antifouling silicone oil lubricants and formation of bactericidal silver nanoparticles (AgNPs), respectively. As a result, the PAR film-based lubricant-infused AgNPs-incorporated surfaces demonstrate outstanding antibacterial effects toward both waterborne and airborne Escherichia coli, suggesting a new door for development of an effective multimodal anti-biofouling surface.

Nature-Inspired and "Water-Skating" Paper and Polyester Substrates Enabled by the Molecular Structure of Poly(γ-stearyl-α,l-glutamate) Homopolypeptide.

We demonstrate that the molecular structure of a synthetic homopolypeptide that resembles the leg architecture of water strider insects is effective to impart flexible polymeric surfaces with superhydrophobic behavior. Filter paper (FP) and polyester (PET) were modified with a coating consisting of low-molecular weight α-helical poly(γ-stearyl-α,l-glutamate) (PSLG, Mw = 4500 Da) homopolypeptide. PSLG-coated substrates displayed near to and superhydrophobic behavior (≥150°) as reflected by the contact angle values. Despite being physically adsorbed, the PSLG coating uniformly covered and was strongly adhered to the substrate surfaces. The thin coating layer displayed remarkable mechanical abrasion resistance and was insensitive to long-time exposure to ambient conditions. PLSG-coated textile fibers exhibited useful and interesting properties. Under an iron-containing load, PSLG-coated PET was able to float and "walk" on water when exposed to a magnet. The PSLG coating was able to reduce the adhesion of Escherichia coli, model Gram-negative bacteria. The results indicated that the molecular geometry of PSLG homopolypeptide, which possesses a α-helical backbone sprouting out of highly hydrophobic stearyl side chains, was the key feature responsible for the observed behaviors. This study is relevant for a broad range of potential applications: from crop and drinking water management in arid geographic areas to biomedical devices and implants.

New Report of Three Unrecorded Species in Trichoderma harzianum Species Complex in Korea.

The genus Trichoderma (Hypocreaceae, Ascomycota) consists of globally distributed fungi. Among them, T. harzianum, one of the most commonly collected Trichoderma species, had been known as a polyphyletic or aggregate species. However, a total of 19 species were determined from the polyphyletic groups of T. harzianum. Thus, we explored Korean "T. harzianum" specimens that were collected in 2013-2014. These specimens were re-examined based on a recent study with translate elongation factor 1-alpha (EF1α) sequences to reveal cryptic Trichoderma species in Korea. As a result, four different species, T. afroharzianum, T. atrobruneum, T. pyramidale, and T. harzianum, were identified. Except T. harzianum, the other three species have not been reported in Korea. In this work, we describe these species and provide figures.

Cooperative Catechol-Functionalized Polypept(o)ide Brushes and Ag Nanoparticles for Combination of Protein Resistance and Antimicrobial Activity on Metal Oxide Surfaces.

Prevention of biofouling and microbial contamination of implanted biomedical devices is essential to maintain their functionality and biocompatibility. For this purpose, polypept(o)ide block copolymers have been developed, in which a protein-resistant polysarcosine (pSar) block is combined with a dopamine-modified poly(glutamic acid) block for surface coating and silver nanoparticles (Ag NPs) formation. In the development of a novel, versatile, and biocompatible antibacterial surface coating, block lengths pSar were varied to derive structure-property relationships. Notably, the catechol moiety performs two important tasks in parallel; primarily it acts as an efficient anchoring group to metal oxide surfaces, while it furthermore induces the formation of Ag NPs. Attributing to the dual function of catechol moieties, antifouling pSar brush and antimicrobial Ag NPs can not only adhere stably on metal oxide surfaces, but also display passive antifouling and active antimicrobial activity, showing good biocompatibility simultaneously. The developed strategy seems to provide a promising platform for functional modification of biomaterials surface to preserve their performance while reducing the risk of bacterial infections.