Myristica fragrans Extract Inhibits Platelet Desialylation and Activation to Ameliorate Sepsis-Associated Thrombocytopenia in a Murine CLP-Induced Sepsis Model.

Sepsis, characterized by an uncontrolled host inflammatory response to infections, remains a leading cause of death in critically ill patients worldwide. Sepsis-associated thrombocytopenia (SAT), a common disease in patients with sepsis, is an indicator of disease severity. Therefore, alleviating SAT is an important aspect of sepsis treatment; however, platelet transfusion is the only available treatment strategy for SAT. The pathogenesis of SAT involves increased platelet desialylation and activation. In this study, we investigated the effects of Myristica fragrans ethanol extract (MF) on sepsis and SAT. Desialylation and activation of platelets treated with sialidase and adenosine diphosphate (platelet agonist) were assessed using flow cytometry. The extract inhibited platelet desialylation and activation via inhibiting bacterial sialidase activity in washed platelets. Moreover, MF improved survival and reduced organ damage and inflammation in a mouse model of cecal ligation and puncture (CLP)-induced sepsis. It also prevented platelet desialylation and activation via inhibiting circulating sialidase activity, while maintaining platelet count. Inhibition of platelet desialylation reduces hepatic Ashwell-Morell receptor-mediated platelet clearance, thereby reducing hepatic JAK2/STAT3 phosphorylation and thrombopoietin mRNA expression. This study lays a foundation for the development of plant-derived therapeutics for sepsis and SAT and provides insights into sialidase-inhibition-based sepsis treatment strategies.

Mutations in OsDET1, OsCOP10, and OsDDB1 confer embryonic lethality and alter flavonoid accumulation in Rice (Oryza sativa L.) seed.

Morphological and biochemical changes accompanying embryogenesis and seed development are crucial for plant survival and crop productivity. Here, we identified a novel yellowish-pericarp embryo lethal (yel) mutant of the japonica rice cultivar Sindongjin (Oryza sativa L.), namely, yel-sdj. Seeds of the yel-sdj mutant showed a yellowish pericarp and black embryo, and were embryonic lethal. Compared with wild-type seeds, the yel-sdj mutant seeds exhibited significantly reduced grain size, grain weight, and embryo weight, and a remarkably lower rate of embryo retention in kernels subjected to milling. However, the volume of air space between embryo and endosperm, density of embryo, and total phenolic content (TPC) and antioxidant activity of mature grains were significantly higher in the yel-sdj mutant than in the wild type. Genetic analysis and mapping revealed that the yel-sdj mutant was non-allelic to the oscop1 null mutants yel-hc, yel-cc, and yel-sk, and its phenotype was controlled by a single recessive gene, LOC_Os01g01484, an ortholog of Arabidopsis thaliana DE-ETIOLATED 1 (DET1). The yel-sdj mutant carried a 7 bp deletion in the second exon of OsDET1. Seeds of the osdet1 knockout mutant, generated via CRISPR/Cas9-based gene editing, displayed the yel mutant phenotype. Consistent with the fact that OsDET1 interacts with CONSTITUTIVE PHOTOMORPHOGENIC 10 (OsCOP10) and UV-DAMAGED DNA BINDING PROTEIN 1 (OsDDB1) to form the COP10-DET1-DDB1 (CDD), seeds of oscop10 and osddb1 knockout mutants also showed the yel phenotype. These findings will enhance our understanding of the functional roles of OsDET1 and the CDD complex in embryogenesis and flavonoid biosynthesis in rice seeds.

Complete genome sequence of Bacillus velezensis NST6 and comparison with the species belonging to operational group B. amyloliquefaciens.

Bacillus spp. play important roles in production of bioactive natural products with potential agricultural and medical applications. The three families of lipopeptides produced by Bacillus spp. have been most recognized for their antagonistic activity against other microbes, i.e. fengycin, iturin, and surfactin. A novel strain NST6 was isolated from soil and identified as B. velezensis based on phylogenomic analysis. Genome analysis revealed 21 putative biosynthetic gene clusters including the ones responsible for producing bacillomycin and surfactin. However, fengycin cluster was compromised with absence or partial disruption of three non-ribosomal peptide synthetases. Distribution of biosynthetic gene clusters showed that clusters for iturin families were well conserved in 327 genomes of the species belonging to the operational group B. amyloliquefaciens. However, clusters for fengycin and surfactin showed dynamic distribution at gene level. Comparative analysis of closely related species would provide new insights to the diversity in genetic elements for secondary metabolites.

Evaluation of Cell-Penetrating Peptides Using Microfluidic In Vitro 3D Brain Endothelial Barrier.

In drug delivery to the human brain, blood vessels are a significant hurdle because they restrict the entry of most solutes to protect brain. To overcome this hurdle, an in vitro 3D model for brain endothelial barrier is developed using a microfluidic device with hydrogel providing a 3D extracellular matrix scaffold. Using the model, peptides known to utilize receptor-mediated transcytosis are verified, which has been one of the most promising mechanisms for brain-specific penetration. The cytotoxicity and cellular damage to the peptide are investigated and the receptor-mediated transcytosis and brain endothelial specific penetrating abilities of the peptides in a quantitative manner are demonstrated. As a preclinical test, applying the quantification assays conducted in this study are suggested, including the penetrating ability, cytotoxicity, endothelial damage, and receptor specificity. Using this microfluidic device as an in vitro platform for evaluating various brain targeting drugs and drug carrier candidates is also proposed.

Supramolecular Peptide Hydrogel-Based Soft Neural Interface Augments Brain Signals through a Three-Dimensional Electrical Network.

Recording neural activity from the living brain is of great interest in neuroscience for interpreting cognitive processing or neurological disorders. Despite recent advances in neural technologies, development of a soft neural interface that integrates with neural tissues, increases recording sensitivity, and prevents signal dissipation still remains a major challenge. Here, we introduce a biocompatible, conductive, and biostable neural interface, a supramolecular ß-peptide-based hydrogel that allows signal amplification via tight neural/hydrogel contact without neuroinflammation. The non-biodegradable ß-peptide forms a multihierarchical structure with conductive nanomaterial, creating a three-dimensional electrical network, which can augment brain signal efficiently. By achieving seamless integration in brain tissue with increased contact area and tight neural tissue coupling, the epidural and intracortical neural signals recorded with the hydrogel were augmented, especially in the high frequency range. Overall, our tissuelike chronic neural interface will facilitate a deeper understanding of brain oscillation in broad brain states and further lead to more efficient brain-computer interfaces.

Lipopeptide-Based Nanosome-Mediated Delivery of Hyperaccurate CRISPR/Cas9 Ribonucleoprotein for Gene Editing.

A transient cytosolic delivery system for accurate Cas9 ribonucleoprotein is a key factor for target specificity of the CRIPSR/Cas9 toolkit. Owing to the large size of the Cas9 protein and a long negative strand RNA, the development of the delivery system is still a major challenge. Here, a size-controlled lipopeptide-based nanosome system is reported, derived from the blood-brain barrier-permeable dNP2 peptide which is capable of delivering a hyperaccurate Cas9 ribonucleoprotein complex (HypaRNP) into human cells for gene editing. Each nanosome is capable of encapsulating and delivering ≈2 HypaRNP molecules into the cytoplasm, followed by nuclear localization at 4 h post-treatment without significant cytotoxicity. The HypaRNP thus efficiently enacts endogenous eGFP silencing and editing in human embryonic kidney cells (up to 27.6%) and glioblastoma (up to 19.7% frequency of modification). The lipopeptide-based nanosome system shows superior delivery efficiency, high controllability, and simplicity, thus providing biocompatibility and versatile platform approach for CRISPR-mediated transient gene editing applications.

Effects of treatment with aqueous and acid-based coloring liquid on the color of zirconia.

STATEMENT OF PROBLEM: To preserve the mechanical property of color-treated zirconia for optimal restoration longevity, aqueous colorants have been developed as an alternative to acid-based coloring agents. However, little is known regarding the effects of aqueous colorants on the color of zirconia. PURPOSE: The purpose of this in vitro study was to compare the effect of aqueous coloring liquids with acid-based coloring liquids on the color of zirconia. MATERIAL AND METHODS: Eighty monolithic zirconia specimens (10×10×2 mm) were fabricated and divided into 4 groups according to their color treatment: unshaded zirconia (control), precolored zirconia, aqueous coloring liquid on zirconia, and acid-based coloring liquid on zirconia. The shaded zirconia specimens were further divided into 3 subgroups based on the number of coloring applications used (1, 3, or 6). The International Commission on Illumination (CIELab) color coordinates were measured by spectrophotometry. RESULTS: Significant differences in the CIE a∗ and b∗ values were observed between the specimen groups treated with the aqueous coloring liquid and the acid-based coloring liquid (P<.001). Increasing the number of colorings resulted in an increase in the CIE a∗ and b∗ values and a decrease in the CIE L∗ values in all the groups (P<.001). CONCLUSIONS: Treatment with aqueous coloring liquid on zirconia produced a greater redness or yellowness compared with treatment with acid-based coloring liquid. The coloring of zirconia lowered its brightness and imparted a red/yellow hue.

Antimicrobial and anti-inflammatory activities of chemokine CXCL14-derived antimicrobial peptide and its analogs.

CXCL14 is a CXC chemokine family that exhibits antimicrobial activity and contains an amphipathic cationic α-helical region in the C-terminus, a characteristic structure of antimicrobial peptides (AMPs). In this study, we designed three analogs of CXCL1459-75 (named CXCL14-C17) corresponding to the C-terminal α-helix of CXCL14, which displayed potential antimicrobial activity against a wide variety of gram-negative and gram-positive bacteria with minimum inhibitory concentrations of 4-16 µM without mammalian cell toxicity. Furthermore, two CXCL14-C17 analogs (CXCL14-C17-a1 and CXCL14-C17-a3) with improved cell selectivity were engineered by introducing Lys, Arg, or Trp in CXCL14-C17. Additionally, CXCL14-C17 analogs showed much greater synergistic effect (FICI: 0.3125-0.375) with chloramphenicol and ciprofloxacin against multidrug-resistant Pseudomonas aeruginosa (MDRPA) than LL-37 did (FICI: 0.75-1.125). CXCL14-C17 analogs were more active against antibiotic-resistant bacteria including methicillin-resistant Staphylococcus aureus (MRSA), MDRPA, and vancomycin-resistant Enterococcus faecium (VREF) than LL-37 and melittin. In particular, CXCL14-C17-a2 and CXCL14-C17-a3 completely inhibited the biofilm formation at sub-MIC and all of the peptides were able to eliminate pre-formed biofilm as well. Membrane depolarization, flow cytometry, sytox green uptake, ONPG hydrolysis and confocal microscopy revealed the possible target of the native peptide (CXCL14-C17) to likely be intracellular, and the amphipathic designed analogs targeted the bacterial membrane. CXCL14-C17 also showed DNA binding characteristic activity similar to buforin-2. Interestingly, CXCL14-C17-a2 and CXCL14-C17-a3 effectively inhibited the production and expression of nitric oxide (NO), tumor necrosis factor (TNF)-α, interleukin (IL)-6, and monocyte chemoattractant protein (MCP)-1 from lipopolysaccharide (LPS)-stimulated RAW264.7 cells, suggesting that these peptides could be promising anti-inflammatory and antimicrobial agents.

Structural and Functional Assessment of mBjAMP1, an Antimicrobial Peptide from Branchiostoma japonicum, Revealed a Novel α-Hairpinin-like Scaffold with Membrane Permeable and DNA Binding Activity.

Here we describe the three-dimensional structure and antimicrobial mechanism of mBjAMP1, an antimicrobial peptide (AMP) isolated from Branchiostoma japonicum. The structure of mBjAMP1 was determined by 2D solution NMR spectroscopy and revealed a novel α-hairpinin-like scaffold stabilized by an intramolecular disulfide bond. mBjAMP1 showed effective growth inhibition and bactericidal activities against pathogenic bacteria but was not cytotoxic to mammalian cells. Antimicrobial mechanism studies using fluorescence-based experiments demonstrated that mBjAMP1 did not disrupt membrane integrity. Laser-scanning confocal microscopy indicated that mBjAMP1 is able to penetrate the bacterial cell membrane without causing membrane disruption. Moreover, gel retardation assay suggested that mBjAMP1 directly binds to bacterial DNA as an intracellular target. Collectively, mBjAMP1 may inhibit biological functions by binding to DNA or RNA after penetrating the bacterial cell membrane, thereby causing cell death. These results suggest that mBjAMP1 may present a promising template for the development of peptide-based antibiotics.

Peptide-Programmable Nanoparticle Superstructures with Tailored Electrocatalytic Activity.

Biomaterials derived via programmable supramolecular protein assembly provide a viable means of constructing precisely defined structures. Here, we present programmed superstructures of AuPt nanoparticles (NPs) on carbon nanotubes (CNTs) that exhibit distinct electrocatalytic activities with respect to the nanoparticle positions via rationally modulated peptide-mediated assembly. De novo designed peptides assemble into six-helix bundles along the CNT axis to form a suprahelical structure. Surface cysteine residues of the peptides create AuPt-specific nucleation site, which allow for precise positioning of NPs onto helical geometries, as confirmed by 3-D reconstruction using electron tomography. The electrocatalytic model system, i.e., AuPt for oxygen reduction, yields electrochemical response signals that reflect the controlled arrangement of NPs in the intended assemblies. Our design approach can be expanded to versatile fields to build sophisticated functional assemblies.

Analysis of two quorum sensing-deficient isolates of Pseudomonas aeruginosa.

Three strains of Pseudomonas aeruginosa were isolated: wild-type (WT, NO4) showed normal quorum sensing (QS), whereas QSD3 and QSD7 were QS-deficient (QSD) containing limited N-butyryl homoserine lactone (C4-HSL). The autoinducer activity produced by NO4 was found to be at least 50-fold higher than those by the QSD3 and the QSD7 strains. The QSDs produced lower levels of phenazine compounds (pyocyanin), siderophores (pyoverdine) and biosurfactants (rhamnolipids) than NO4. Therefore, the swarming motility and the swimming motility of the QSD3 and the QSD7 strains also decreased. Treatment with exogenous C4-HSL completely restored rhamnolipid production in both QSDs, suggesting that the biosynthesis of C4-HSL is defective. However, the biofilm production of the QSDs reached much higher levels than those of wild-types (NO4 and P. aeruginosa PAO1). And both QSD strains were more resistant than wild-type cell (NO4) against kanamycin and tobramycin. The RpoS gene, which function is related with QS, is point-nonsense mutated in QSD3 strain. But eleven QS-related genes in QSD3 were not mutated, compared to those of PAO1, which carries intact QS genes and is used as a positive control. This study is helpful in the development of novel approaches in the treatment of P. aeruginosa infections.

Effects of aqueous and acid-based coloring liquids on the hardness of zirconia restorations.

STATEMENT OF PROBLEM: The effects of the application of aqueous coloring liquids on the mechanical properties of zirconia have not yet been investigated. PURPOSE: The purpose of this in vitro study was to evaluate the effects of 3 different coloring techniques and the number of coloring liquid applications on the hardness of zirconia. MATERIAL AND METHODS: Eighty specimens were divided into 8 groups (n=10); nonshaded zirconia, preshaded zirconia, acid-based coloring liquid zirconia, and aqueous coloring liquid zirconia (1, 3, 6). Vickers hardness was measured. Data were analyzed via 1-way and 2-way ANOVAs. Multiple comparisons were performed using a Scheffé test (α=.05). RESULTS: Statistically significant differences in hardness were found between acid-based coloring liquid zirconia and aqueous coloring liquid zirconia (P<.001). Increasing the number of coloring liquid applications decreased the hardness value of acid-based coloring liquid zirconia (P<.001) but had no effect on the hardness of aqueous coloring liquid zirconia (P>.05). CONCLUSIONS: Within the limitations of this study, the hardness of zirconia was influenced to differing degrees depending on coloring technique. The number of coloring liquid applications affected the hardness of zirconia colored with the acid-based coloring liquid but not the hardness of zirconia colored with the aqueous coloring liquid.

Screening of herbal medicines for the recovery of cisplatin-induced nephrotoxicity.

The goal of this study was to quantitatively determine the recovery effects of herbal medicines (HM) on the cisplatin-induced nephrotoxicity. In the present study, the recovery effects of 239 HM on HEK 293 cells that had been damaged by cisplatin were evaluated by a mitochondrial activity MTS assay. After the first round of screening, candidate HM were selected based on a recovery rate of greater than 20%. The efficacy of the selected herbs was then determined by dose response kinetic analysis. Of the extracts evaluated, 7 HM (Paeonia suffruticosa (PS), Curcuma longa (CL), Centipeda minima (CM), Loranthus parasiticus (LP), Pulsatilla dahurica (PD), Sinapis alba (SA), and Scutellaria barbata (SB)) had a strong recovery effect on cisplatin-induced damage in HEK 293 cells. An LDH assay showed that LP, CM, SB, CL, SA, and PS had the best recovery effect, whereas a comet assay indicated that PS, SB, SA, PD, and CL had the best recovery effect. Taken together, these results suggest that SB, CL, PS, and SA are the best candidate HM for the recovery of cisplatin-induced nephrotoxicity. Therefore, additional studies should be conducted to determine if these HM possess novel therapeutic agents that can be used for the prevention or treatment of renal disorders.