A non-pheromone GPCR is essential for meiosis and ascosporogenesis in the wheat scab fungus.

Meiosis is essential for generating genetic diversity and sexual spores, but the regulation of meiosis and ascosporogenesis is not clear in filamentous fungi, in which dikaryotic and diploid cells formed inside fruiting bodies are not free living and independent of pheromones or pheromone receptors. In this study, Gia1, a non-pheromone GPCR (G protein-coupled receptor) with sexual-specific expression in Fusarium graminearum, is found to be essential for ascosporogenesis. The gia1 mutant was normal in perithecium development, crozier formation, and karyogamy but failed to undergo meiosis, which could be partially rescued by a dominant active mutation in GPA1 and activation of the Gpmk1 pathway. GIA1 orthologs have conserved functions in regulating meiosis and ascosporogenesis in Sordariomycetes. GIA1 has a paralog, GIP1, in F. graminearum and other Hypocreales species which is essential for perithecium formation. GIP1 differed from GIA1 in expression profiles and downstream signaling during sexual reproduction. Whereas the C-terminal tail and IR3 were important for intracellular signaling, the N-terminal region and EL3 of Gia1 were responsible for recognizing its ligand, which is likely a protein enriched in developing perithecia, particularly in the gia1 mutant. Taken together, these results showed that GIA1 encodes a non-pheromone GPCR that regulates the entry into meiosis and ascosporogenesis via the downstream Gpmk1 MAP kinase pathway in F. graminearum and other filamentous ascomycetes.

Dual-Functional Capping Agent-Mediated Transformation of Silver Nanotriangles to Silver Nanoclusters for Dual-Mode Biosensing.

The localized surface plasmon resonance (LSPR) property, depending on the structure (morphology and assembly) of nanoparticles, is very sensitive to the environmental fluctuation. Retaining the colorimetric effect derived from the LSPR property while introducing new optical properties (such as fluorescence) that provide supplementary information is an effective means to improve the controllability in structures and reproducibility in optical properties. DNA as a green and low-cost etching agent has been demonstrated to effectively control the morphology and optical properties (the blue shift of the LSPR peak) of the plasmonic nanoparticles. Herein, taking silver nanotriangles (AgNTs) as a proof of concept, we report a novel strategy to induce precisely tunable LSPR and fluorescence-composited dual-mode signals by using mono-DNA first as an etching agent for etching the morphology of AgNTs and later as a template for synthesizing fluorescent silver nanoclusters (AgNCs). In addition, common templates for synthesizing AgNCs, such as l-glutathione and bovine serum albumin, were demonstrated to have the capability to serve as etching agents. More importantly, these biomolecules as dual-functional capping agents (etching agents and templates) follow the size-dependent rule: as the size of the thiolated biomolecule increases, the blue shift of the LSPR peak increases; at the same time, the fluorescence intensity increases. The enzyme that can change the molecular weight (size) of the biomolecular substrates (DNA, peptides, and proteins) through an enzymatic cleavage reaction was explored to regulate the LSPR and fluorescent properties of the resulting nanoparticles (by etching of AgNTs and synthesis of AgNCs), achieving excellent performance in detection of cancer-related proteases. This study can be expanded to other biopolymers to impact both fundamental nanoscience and applications and provide powerful new tools for bioanalytical biosensors and nanomedicine.

Single Nanovesicles Tracking Reveals Their Heterogeneous Extracellular Adsorptions.

The vigorous nanomedicine offers significant possibilities for effective therapeutics of various diseases, and nanovesicles (NVs) represented by artificial liposomes and natural exosomes and cytomembranes especially show great potential. However, their complex interactions with cells, particularly the heterogeneous extracellular adsorptions, are difficult to analyze spatiotemporally due to the transient dynamics. In this study, by single NVs tracking, the extracellular NVs adsorptions are directly observed and their heterogeneous characteristics are revealed. Briefly, plenty of NVs adsorbed on HCT116 cells are tracked and classified, and it is discovered that they exhibit various diffusion properties from different extracellular regions: stable adsorptions on the rear surface and restricted adsorptions on the front protrusion. After the hydrolysis of hyaluronic acid in the extracellular matrix by hyaluronidase, the restricted adsorptions are further weakened and manifested as dissociative adsorptions, which demonstrated reduced total NVs adsorptions from a single-cell and single-particle perspective. Compared with traditional static analysis, the spatiotemporal tracking and heterogeneous results not only reveal the extracellular NVs-cell interactions but also inspire a wide variety of nanomedicine and their nano-investigations.

The synthesis and functionalization of metal organic frameworks and their applications for the selective separation of proteins/peptides.

Recently, proteins separation has drawn great interest for the full investigation of a proteome because the proteins separation is the precondition when conducting clinical research or proteomics research. Metal organic frameworks (MOFs) are fabricated via covalent connection between organic ligands and metal ions/clusters units. MOFs have attracted much attention due to the ultra-high specific surface area, tunable structure, more metal site or unsaturated site, and chemical stability. Over the past decade, different functionalization types of MOFs have been reported in combination with amino acids, nucleic acids, proteins, polymers, and nanoparticles for various applications. In this review, the synthesis and functionalization of MOFs have been thoroughly discussed, and we introduced the existing problems and development trends in these fields. Furthermore, MOFs as advanced adsorbents for selective separation of proteins/peptides are summarized. Additionally, we present a comprehensive prospects and challenges in the preparation of robust functional MOFs-based adsorbents and make a final outlook on their future development prospects in selective separation of proteins/peptides.

Genome-Wide Identification and Expression Analysis of the Stearoyl-Acyl Carrier Protein Δ9 Desaturase Gene Family under Abiotic Stress in Barley.

Stearoyl-acyl carrier protein (ACP) Δ9 desaturase (SAD) is a critical fatty acid dehydrogenase in plants, playing a prominent role in regulating the synthesis of unsaturated fatty acids (UFAs) and having a significant impact on plant growth and development. In this study, we conducted a comprehensive genomic analysis of the SAD family in barley (Hordeum vulgare L.), identifying 14 HvSADs with the FA_desaturase_2 domain, which were divided into four subgroups based on sequence composition and phylogenetic analysis, with members of the same subgroup possessing similar genes and motif structures. Gene replication analysis suggested that tandem and segmental duplication may be the major reasons for the expansion of the SAD family in barley. The promoters of HvSADs contained various cis-regulatory elements (CREs) related to light, abscisic acid (ABA), and methyl jasmonate (MeJA). In addition, expression analysis indicated that HvSADs exhibit multiple tissue expression patterns in barley as well as different response characteristics under three abiotic stresses: salt, drought, and cold. Briefly, this evolutionary and expression analysis of HvSADs provides insight into the biological functions of barley, supporting a comprehensive analysis of the regulatory mechanisms of oil biosynthesis and metabolism in plants under abiotic stress.

Bioactive Constituents of Verbena officinalis Alleviate Inflammation and Enhance Killing Efficiency of Natural Killer Cells.

Natural killer (NK) cells play key roles in eliminating pathogen-infected cells. Verbena officinalis (V. officinalis) has been used as a medical plant in traditional and modern medicine for its anti-tumor and anti-inflammatory activities, but its effects on immune responses remain largely elusive. This study aimed to investigate the potential of V. officinalis extract (VO extract) to regulate inflammation and NK cell functions. We examined the effects of VO extract on lung injury in a mouse model of influenza virus infection. We also investigated the impact of five bioactive components of VO extract on NK killing functions using primary human NK cells. Our results showed that oral administration of VO extract reduced lung injury, promoted the maturation and activation of NK cells in the lung, and decreased the levels of inflammatory cytokines (IL-6, TNF-α and IL-1ß) in the serum. Among five bioactive components of VO extract, Verbenalin significantly enhanced NK killing efficiency in vitro, as determined by real-time killing assays based on plate-reader or high-content live-cell imaging in 3D using primary human NK cells. Further investigation showed that treatment of Verbenalin accelerated the killing process by reducing the contact time of NK cells with their target cells without affecting NK cell proliferation, expression of cytotoxic proteins, or lytic granule degranulation. Together, our findings suggest that VO extract has a satisfactory anti-inflammatory effect against viral infection in vivo, and regulates the activation, maturation, and killing functions of NK cells. Verbenalin from V. officinalis enhances NK killing efficiency, suggesting its potential as a promising therapeutic to fight viral infection.

Mussel Inspired Trigger-Detachable Adhesive Hydrogel.

Adhesion to many kinds of surfaces, including biological tissues, is important in many fields but has been proved to be extremely challenging. Furthermore, peeling from strong adhesion is needed in many conditions, but is sometimes painful. Herein, a mussel inspired hydrogel is developed to achieve both strong adhesion and trigger-detachment. The former is actualized by electrostatic interactions, covalent bonds, and physical interpenetration, while the latter is triggered, on-demand, through combining a thixotropic supramolecular network and polymer double network. The results of the experiments show that the hydrogel can adhere to various material surfaces and tissues. Moreover, triggered by shear force, non-covalent interactions of the supramolecular network are destroyed. This adhesion can be peeled easily. The possible mechanism involved is discussed and proved. This work will bring new insight into electronic engineering and tissue repair like skin care for premature infants and burn victims.

Affimer-Based Europium Chelates Allow Sensitive Optical Biosensing in a Range of Human Disease Biomarkers.

The protein biomarker measurement has been well-established using ELISA (enzyme-linked immunosorbent assay), which offers good sensitivity and specificity, but remains slow and expensive. Certain clinical conditions, where rapid measurement or immediate confirmation of a biomarker is paramount for treatment, necessitate more rapid analysis. Biosensors offer the prospect of reagent-less, processing-free measurements at the patient's bedside. Here, we report a platform for biosensing based on chelated Eu3+ against a range of proteins including biomarkers of cardiac injury (human myoglobin), stroke (glial fibrillary acidic protein (GFAP)), inflammation (C-reactive protein (CRP)) and colorectal cancer (carcinoembryonic antigen (CEA)). The Eu3+ ions are chelated by modified synthetic binding proteins (Affimers), which offer an alternative targeting strategy to existing antibodies. The fluorescence characteristics of the Eu3+ complex with modified Affimers against human myoglobin, GFAP, CRP and CEA were measured in human serum using λex = 395 nm, λem = 590 and 615 nm. The Eu3+-Affimer based complex allowed sensitive detection of human myoglobin, GFAP, CRP and CEA proteins as low as 100 fM in (100-fold) diluted human serum samples. The unique dependence on Eu3+ fluorescence in the visible region (590 and 615 nm) was exploited in this study to allow rapid measurement of the analyte concentration, with measurements in 2 to 3 min. These data demonstrate that the Affimer based Eu3+ complexes can function as nanobiosensors with potential analytical and diagnostic applications.

Simultaneous Assay of Oxygen-Dependent Cytotoxicity and Genotoxicity of Anticancer Drugs on an Integrated Microchip.

Oxygen deprivation is a common feature in a variety of cancer tissues and associated with tumor progression, acquisition of antiapoptotic potential, and clinical therapeutic resistance. Thus, great interest has been aroused to develop new platforms or approaches of activity assays to impact on the hypoxic microenvironment and oxygen-dependent drug responses to improve the productivity of new drug discovery. In this study, an integrated microsystem is established to combine the cytotoxic and genotoxic tests together for continuous multiple measurements under mimicking hypoxic tumor microenvironment. We fabricated a double-layer chip device by combining a single-cell-arrayed agarose layer with a microfluidics-based oxygen gradient-generating layer using a PDMS membrane. Using tirapazamine (TPZ) and blemycin (BLM) as model anticancer drugs, we demonstrated its application and performance in single cell loading, cell cultivation, and subsequent drug treatment as well as in situ analysis of oxygen-dependent cytotoxicity and genotoxicity of anticancer drugs. The results demonstrated the opposite oxygen-dependent toxicity of TPZ and BLM, which also indicated that the formation of DNA breaks is related with cell apoptosis. Compared with the traditional assays, this device takes advantage of microfluidic phenomena to generate various oxygen concentrations while exhibiting the combinatorial diversities achieved by the single cell microarray, offering a powerful tool to study single cell behaviors and responses under different oxygen conditions with desired high-content and high-throughput capabilities.

Aquaporin1 regulates development, secondary metabolism and stress responses in Fusarium graminearum.

The Ascomycete fungus Fusarium graminearum, the causal agent of Fusarium head blight of wheat and barley, has become a predominant model organism for the study of fungal phytopathogens. Aquaporins (AQPs) have been implicated in the transport of water, glycerol, and a variety of other small molecules in yeast, plants and animals. However, the role of these proteins in phytopathogenic fungi is not well understood. Here, we identified and attempted to elucidate the function of the five aquaporin genes in F. graminearum. The phylogenetic analysis revealed that FgAQPs are divided into two clades, with FgAQP1 in the first clade. The ∆AQP1 mutant formed whitish colonies with longer aerial hyphae and reduced conidiation and perithecium formation. The ∆AQP1 mutant conidia were morphologically abnormal and appeared to undergo abnormal germination. The ∆AQP1 mutant and the wild type strain were equally pathogenic, while the mutant produced significantly higher quantities of deoxynivalenol (DON). The ∆AQP1 mutant also exhibited increased resistance to osmotic and oxidative stress as well as cell-wall perturbing agents. Using FgAQP1-GFP and DAPI staining, we found that FgAQP1 is localized to the nuclear membrane in conidia. Importantly, deletion of FgAQP1 increased the severity of conidium autophagy. Taken together, these results suggest that FgAQP1 is involved in hyphal development, stress responses, secondary metabolism, and sexual and asexual reproduction in F. graminearum. Unlike the ∆AQP1 mutant, the ∆AQP2, ∆AQP3, ∆AQP4 and ∆AQP5 mutants had no variable phenotypes.

Metal-organic framework based in-syringe solid-phase extraction for the on-site sampling of polycyclic aromatic hydrocarbons from environmental water samples.

In-syringe solid-phase extraction is a promising sample pretreatment method for the on-site sampling of water samples because of its outstanding advantages of portability, simple operation, short extraction time, and low cost. In this work, a novel in-syringe solid-phase extraction device using metal-organic frameworks as the adsorbent was fabricated for the on-site sampling of polycyclic aromatic hydrocarbons from environmental waters. Trace polycyclic aromatic hydrocarbons were effectively extracted through the self-made device followed by gas chromatography with mass spectrometry analysis. Owing to the excellent adsorption performance of metal-organic frameworks, the analytes could be completely adsorbed during one adsorption cycle, thus effectively shortening the extraction time. Moreover, the adsorbed analytes could remain stable on the device for at least 7 days, revealing the potential of the self-made device for on-site sampling of degradable compounds in remote regions. The limit of detection ranged from 0.20 to 1.9 ng/L under the optimum conditions. Satisfactory recoveries varying from 84.4 to 104.5% and relative standard deviations below 9.7% were obtained in real samples analysis. The results of this study promote the application of metal-organic frameworks in sample preparation and demonstrate the great potential of in-syringe solid-phase extraction for the on-site sampling of trace contaminants in environmental waters.

A porous graphene sorbent coated with titanium(IV)-functionalized polydopamine for selective lab-in-syringe extraction of phosphoproteins and phosphopeptides.

A novel polydopamine coated three-dimensional porous graphene aerogel sorbent carrying immobilized titanium(IV) ions (denoted as Ti4+@PDA@GA) was fabricated without using an organic solvent. The material is shown to be a viable carbon foam type of monolithic sorbent for selective lab-in-syringe enrichment of phosphoproteins and phosphopeptides. The phosphoproteins can be separated from a sample by aspiration and then bind to the sorbent. The analytes then can be dispensed within 5 min. The weight percent of titanium in the monolith typically is 14%, and the absorption capacities for the model proteins ß-casein and κ-casein are 1300 and 1345 mg g-1, respectively. The absorption capacities for nonphosphoproteins are much smaller, typically 160 mg g-1 for ß-lactoglobulin, 125 mg g-1 for bovine serum, and 4.8 mg g-1 for lysozyme. The results demonstrate that the selectivity for phosphoproteins was excellent on multiple biological samples including standard protein mixtures, spiked human blood serum, and drinking milk. The selective enrichment of phosphopeptides also makes the method a promising tool in phosphoproteomics. Graphical abstract Schematic of a polydopamine coated three-dimensional porous graphene aerogel for immobilization of titanium(IV) ions. The material served as a monolithic sorbent for selective enrichment of phosphopeptides and phosphoproteins from biological samples. The enrichment process can be carried out conveniently using a lab-in-syringe way.

Differential roles of three FgPLD genes in regulating development and pathogenicity in Fusarium graminearum.

Phospholipase D (PLD) is an important phospholipid hydrolase that plays critical roles in various biological processes in eukaryotic cells. However, little is known about its functions in plant pathogenic fungi. In this study, we identified three FgPLD genes in Fusarium graminearum that are homologous to the Saccharomyces cerevisiae Spo14 gene. We constructed deletion mutants of all three FgPLD genes using homologous recombination. Deletion of FgPLD1 (Δpld1), but not FgPLD2 or FgPLD3, affected hyphal growth, conidiation, and perithecium formation. The Δpld1 mutant showed reduced deoxynivalenol (DON) production and virulence in flowering wheat heads and corn silks. Furthermore, three FgPLD proteins have the same subcellular localization and localize to the cytoplasm in F. graminearum. Taken together, these results indicate that FgPLD1, but not FgPLD2 or FgPLD3, is important for hyphal growth, sexual or asexual reproduction, and plant infection.

One-Step Facile Synthesis of Aptamer-Modified Graphene Oxide for Highly Specific Enrichment of Human A-Thrombin in Plasma.

The enrichment of low-abundance proteins in complex biological samples plays an important role in clinical diagnostics and biomedical research. This work reports a novel one-step method for the synthesis of aptamer-modified graphene oxide (GO/Apt) nanocomposites, without introducing the use of gold, for the rapid and specific separation and enrichment of human α-thrombin from buffer solutions with highly concentrated interferences. The obtained GO/Apt nanocomposites had remarkable aptamer immobilization, up to 44.8 nmol/mg. Furthermore, GO/Apt nanocomposites exhibited significant specific enrichment efficiency for human α-thrombin (>90%), even under the presence of 3000-fold interference proteins, which was better than the performance of other nanomaterials. Finally, the GO/Apt nanocomposites were applied in the specific capturing of human α-thrombin in highly concentrated human plasma solutions with negligible nonspecific binding of other proteins, which demonstrated their prospects in rare protein analysis and biosensing applications.

Single-Cell-Arrayed Agarose Chip for in Situ Analysis of Cytotoxicity and Genotoxicity of DNA Cross-Linking Agents.

Development of approach or device to allow continuous multiple measurements, such as integrating cytotoxic and genotoxic analysis, is quite appealing for study of the drug's activity and mechanism of action or resistance. In this study, a single-cell-arrayed agarose chip system was developed to combine cell cultivation with subsequent in situ analysis of cytotoxicity and genotoxicity of the chemotherapeutic agent. The modified alkaline comet assay coupled with the Live/Dead assay was used to monitor the interstrand cross-links (ICLs) formation and the cytotoxic effects in different glioma cell lines. In addition, the ICL-induced double strand breaks (DSBs) was measured on the chip to reflect the level of ICLs indirectly. Compared with the traditional methods, the microarray agarose device offers higher throughput, reproducibility, and robustness, exhibiting good potential for high-content drug screening.

The phospholipase C (FgPLC1) is involved in regulation of development, pathogenicity, and stress responses in Fusarium graminearum.

Phospholipase C (PLC) is an important phospholipid hydrolase that plays critical roles in various biological processes in eukaryotic cells. To elucidate the functions of PLC in morphogenesis and pathogenesis in Fusarium graminearum, deletion mutants were constructed of all six FgPLC genes identified in this study. Deletion of FgPLC1, but not the other five FgPLC genes, affected hyphal growth and conidiation. The FgPLC1 deletion mutant (Δplc1) also was defective in conidium germination and germ tube growth. It was sterile in selfing crosses and had increased sensitivities to hyperosmotic and cell wall stresses. The Δplc1 mutant showed reduced DON production and virulence during infection in flowering wheat heads. Deletion of FgPLC1 decreased the phosphorylation levels of both Gpmk1 and Mgv1 MAP kinases. qRT-PCR analysis showed that several genes related to defective phenotypes were down-regulated in the Δplc1 mutant. Taken together, these results indicated that FgPLC1 is important for hyphal growth, plant infection, and sexual or asexual reproduction, and it may be functionally related to MAP kinases in F. graminearum.

Metal-organic frameworks@graphene hybrid aerogels for solid-phase extraction of non-steroidal anti-inflammatory drugs and selective enrichment of proteins.

Graphene aerogel (GA)-supported metal-organic framework (MOF) particles with a three-dimensional (3D) architecture were fabricated for the first time via a facile template-free "sol-cryo" method. The prepared MOFs@graphene hybrid aerogels exhibit a 3D interconnected macroporous framework of graphene sheets with uniform dispersion of MOF particles. We also report the first attempt at using the hybrid aerogels as adsorbents for the solid-phase extraction (SPE) of non-steroidal anti-inflammatory drugs (NSAIDs) and the selective enrichment of proteins. The macroporous skeletons of GA provide both low backpressure and rapid mass transfer in SPE application, thus overcoming the obstacle of high backpressure caused by directly packing submicron or micron sized MOF particles into SPE cartridges. Excellent performances including satisfactory recoveries, high sensitivity and good reproducibility were achieved in the extraction of five NSAIDs. The hybrid aerogels also showed an interesting ability for selective enrichment of ribonuclease A (RNase A) and simultaneous exclusion of cytochrome C (Cyt C) and lysozyme (Lyz), which could be attributed to the electrostatic interactions between proteins and the positively charged coordinatively unsaturated metal sites (CUS) in MIL-101. We believe that this work will promote the application of MOFs in adsorption and separation, and our synthetic strategy could be further extended to develop other graphene-based hybrid aerogels.

Magnetic metal-organic frameworks for selective enrichment and exclusion of proteins for MALDI-TOF MS analysis.

We firstly report magnetic metal-organic frameworks for selective enrichment and exclusion of proteins for MALDI-TOF MS analysis. Fe3O4@MIL-100(Fe) nanoparticles were achieved by step-by-step assembly on poly(acrylic acid) modified Fe3O4.

Self-made microextraction by packed sorbent device for the cleanup of polychlorinated biphenyls from bovine serum.

Microextraction by packed sorbent, a miniaturized form of the solid-phase extraction, is a new sample pretreatment technology mainly used for bioanalysis. In this work, self-made device was fabricated by packing C18 sorbent into a microinjection needle (50 µL) and then applied for the analysis of polychlorinated biphenyls in bovine serum followed by gas chromatography with mass spectrometry determination. Compared with conventional solid-phase extraction, the developed method bears many intriguing properties such as low consumption of the sample and organic solvent, time-saving and easy operation, which are of great interest and desire for bioanalysis applications. A series of parameters that affect the analytical performance, such as the type of elution, the aspirating/dispensing cycles of sample loading and elution, washing solution, and matrix effects, was investigated in detail. Under the optimized conditions, the proposed method presented a good linearity (R ≥ 0.986) and satisfactory sensitivity and limits of detection (0.06-0.53 ng/mL) and quantification (0.20-1.77 ng/mL), respectively. In addition, satisfactory recoveries (60.0-91.4%) and accuracy (RSD ≤ 5.72%) were achieved after optimizing the conditions when applying the developed method to real sample analysis. The screening of polychlorinated biphenyls residues in bovine serum samples by the developed method demonstrated that the assay is ideally suited as a monitoring method for polychlorinated biphenyls residues in bioanalysis.

Research on the interaction of hydrogen-bond acidic polymer sensitive sensor materials with chemical warfare agents simulants by inverse gas chromatography.

Hydrogen-bond acidic polymers are important high affinity materials sensitive to organophosphates in the chemical warfare agent sensor detection process. Interactions between the sensor sensitive materials and chemical warfare agent simulants were studied by inverse gas chromatography. Hydrogen bonded acidic polymers, i.e., BSP3, were prepared for micro-packed columns to examine the interaction. DMMP (a nerve gas simulant) and 2-CEES (a blister agent simulant) were used as probes. Chemical and physical parameters such as heats of absorption and Henry constants of the polymers to DMMP and 2-CEES were determined by inverse gas chromatography. Details concerning absorption performance are also discussed in this paper.