Ankylosing Spondylitis (AS) is a chronic inflammatory disorder of the spine and sacroiliac joints with unidentified etiology closely associated with metabolic syndrome (MetS). Recent studies have reported that immunological and oxidative stress factors are implicated in AS pathogenesis. The aim of this study was to investigate the oxidative and immunological factors in AS patients with or without MetS compare to control group. Real-Time PCR measured expression level of cytokines, transcription factors and related miRNAs. In addition, Th17 and Treg frequencies and cytokines secretion were evaluated by flowcytometry and ELISA methods, respectively. The oxidative stress biomarkers were also assessed with biochemical methods. In AS patients with MetS, higher Th17 and lower Treg frequency were observed. Increased levels of NF-kB and AP-1 mRNA expression were seen in AS patients with MetS (p = 0.0263 and p = 0.0104, respectively). MiR-146a and miR-223 were significantly decreased (p = 0.0005, p = 0.0161, respectively) and increase in miR-21 (p = 0.0002) was observed in AS patients with MetS compared to AS patients without MetS. Additionally, the secretion of TNF-α (p = 0.0167), IL-1β (p = 0.303), CCL2 (p = 0.0254), CCL3 (p = 0.0119), CXCL8 (p = 0.0364), adiponectin (p = 0.0183) and the levels of SOD (p = 0.0421), NO (p = 0.0451) and CAT (p = 0.0128) were increased in AS patients with MetS. We were not observed significant differences in TOS and GPX levels between studied groups. The higher levels of oxidative stress and immunological inflammatory markers in AS patients with MetS provide further evidences on the oxidative stress and immunological relationship in these patients.

Bot flies (Oestridae) are obligate endoparasites of mammals, and their extraordinary diversification is of great importance in understanding the evolution of parasitism. However, evolutionary analysis of Oestridae has long been impeded by lack of information. Here, the first three mitochondrial genomes of nasal bot flies (Cephalopina titillator, Cephenemyia trompe and Rhinoestrus usbekistanicus) and a comparative mitochondrial genomic analysis between subfamilies of Oestridae are presented. Contrasting to many other parasites, mitochondrial genomes of oestrids are conserved in structure and genes retain the same order and direction as the ancestral insect mitochondrial genome. Nucleotide composition is highly heterogenous, with Gasterophilinae possessing highest GC content and smallest genomic size. Mitochondrial evolutionary rates vary considerably, with Hypodermatinae and Oestrinae exhibiting a faster average rate than Cuterebrinae and Gasterophilinae. In addition, the first phylogenomic analysis covering all four bot fly subfamilies was conducted, supporting monophyly of Oestridae and a sister-group relationship of Hypodermatinae and Oestrinae. The only topological ambiguity is Cuterebrinae being a sister-group of either (Hypodermatinae + Oestridae) or Gasterophilinae. Thus, we suggest that mitochondrial genomes carry a great potential for phylogenetic analysis of Oestridae, and more information of Cuterebrinae is needed to illuminate the early evolutionary radiation and parasite-host coevolution of bot flies.

The gut microbe Akkermansia (A.) muciniphila becomes increasingly important as its prevalence is inversely correlated with different human metabolic disorders and diseases. This organism is a highly potent degrader of intestinal mucins and the hydrolyzed glycan compounds can then serve as carbon sources for the organism itself or other members of the gut microbiota via cross-feeding. Despite its importance for the hosts' health and microbiota composition, exact mucin degrading mechanisms are still mostly unclear. In this study, we identified and characterized three extracellular β-galactosidases (Amuc_0771, Amuc_0824, and Amuc_1666) from A. muciniphila ATCC BAA-835. The substrate spectrum of all three enzymes was analyzed and the results indicated a preference for different galactosidic linkages for each hydrolase. All preferred target structures are prevalent within mucins of the colonic habitat of A. muciniphila. To check a potential function of the enzymes for the degradation of mucosal glycan structures, porcine stomach mucin was applied as a model substrate. In summary, we could confirm the involvement of all three β-galactosidases from A. muciniphila in the complex mucin degradation machinery of this important gut microbe. These findings could contribute to the understanding of the molecular interactions between A. muciniphila and its host on a molecular level.

The metal-assisted nitrone-nitrile cycloaddition reaction is apply to empower chitosan chemistry. The ultrasonic irradiation has proven to efficiently accelerate the cycloaddition affording new heterocyclic (1,2,4-oxadiazoline) chitosan derivatives and avoiding ultrasonic degradation of the chitosan macromolecules. By varying the nitrone nature, both water- and toluene-soluble chitosan derivatives were successfully synthesized. Relying on the ionic gelation approach nanoparticles of heterocyclic chitosan derivatives were prepared. Water-soluble chitosan derivative demonstrated a high antibacterial activity coupled with low toxicity. The toxicity of the synthesized heterocyclic chitosan derivatives and their based nanoparticles are comparable with those of the starting chitosan, while their antibacterial activity is superior. Toluene-soluble derivatives are shown to be efficient homogeneous catalysts towards monoglyceride synthesis via the epoxide ring opening. They efficiently catalyze selective conversion of fatty acids and glycidol into corresponding monoglycerides allowing one to simplify significantly the procedure for separating the reaction product from the catalyst for its recovery and reusage.

In bacteria, protein lysine acetylation circuits can control core processes such as carbon metabolism. In E. coli, cyclic adenosine monophosphate (cAMP) controls the transcription level and activity of protein lysine acetyltransferase (PAT). The M. tuberculosis PatA (Mt-PatA) resides in two different conformations; the activated state and autoinhibited state. However, the mechanism of cAMP allosteric regulation of Mt-PatA remains mysterious. Here, we performed extensive all-atom molecular dynamics (MD) simulations (three independent run for each system) and built a residue-residue dynamic correlation network to show how cAMP mediates allosteric activation. cAMP binds at the regulatory site in the regulatory domain, which is 32 Å away from the catalytic site. An extensive conformational restructuring relieves autoinhibition caused by a molecular Lid (residues 161–203) that shelters the substrate-binding surface. In the activated state, the regulatory domain rotates (~40°) around Ser144, which links both domains. Rotation removes the C-terminus from the cAMP site and relieves the autoinhibited state. Also, the molecular Lid refolds and creates an activator binding site. A conserved residue, His173, was mutated into Lys in the Lid, and during an MD trajectory of the activated state, positioned itself near an acetyl donor molecule in the catalytic domain, suggesting a direct mechanism for acetylation. This study describes the allosteric framework for Mt-PatA and prerequisite intermediate states that permit long-distance signal transmission.

The aim of this study was to analyzing the impact of germination time on the morphology, crystallinity, gelatinization and viscosity properties on the starch of Esmeralda and Perla barley variety. The two barley were germinated for 1 to 8 days, at 26 °C and 65% relative humidity. Micrographs showed the presence of pinholes and eroded surfaces. Starch in Esmeralda was hydrolyzed completely at 8 days of germination. Birefringence was reduced from day 4, losing molecular structuring of the crystalline area. Morphometric data: fractal dimension, area, perimeter, circularity, and roundness decreased significantly along germination time in both varieties. The entropy increased significantly, from 0.79 to 10.09 in Esmeralda and from 0.46 to 7.57 in Perla. Relative crystallinity decreased significantly in the Perla from 24.7% to 23.6%. Viscosity peaks were also significantly reduced, pasting temperature was constant in Esmeralda but in Perla was significantly reduced from 95.43 to 95.19 °C with germination, the gelatinization temperature increased significantly in the Esmeralda while in Perla it remained constant. Enthalpy decreased significantly to 75.8% and 37% in Esmeralda and Perla respectively. The study of germination impact on structural and physicochemical properties is important to identify the use of hydrolyzed starches in the food industry or others.

Previous studies have shown that crude polysaccharides from the Lycium barbarum fruit could inhibit cancer cell growth, but the major effective constituents are yet to be identified. In this study, we compared the effects of L. barbarum fruit polysaccharide fractions on the growth of hepatoma cells (SMMC-7721 and HepG2), cervical cancer cells (HeLa), gastric carcinoma cells (SGC-7901), and human breast cancer cells (MCF-7). LBGP-I-3 showed stronger inhibitory effects on MCF-7 cells (cell viability of 48.96%) than SMMC-7721 (cell viability of 78.91%) and HeLa cells (cell viability of 55.94%), and had no effect on HepG2 and SGC-7901 cells. In addition, LBGP-I-3 had no inhibitory effect on normal liver cells (L02, cell viability of 115.58%). Investigation of the underlying mechanism suggested that LBGP-I-3 inhibited the growth of cancer cells by cell cycle arrest and apoptosis. LBGP-I-3 arrested the cell cycle at the G0/G1 phase, altered mitochondrial function, activated oxidative stress, and regulated the MAPK signaling pathway to induce apoptosis. Thus, LBGP-I-3 may be a potential functional food ingredient for the prevention of cancer without toxicity to normal cells in vitro. These results could help further elucidate the structure–activity relationship of L. barbarum fruit polysaccharides and functional food development.

The present study investigated effects of low-level laser therapy with cellulose nanocrystals/cellulose nanofibrils loaded in nanoemulsion (NE) against skin cancer cells on apoptosis. The nanoemulsion was fabricated and characterized by the standard methods. The toxicity level by cytotoxicity assays, generation of reactive singlet oxygen (ROS) and antioxidant potential, cell proliferation and migration were confirmed by using standard assays. The cellular uptake efficacy was evaluated by differential staining. The protein levels of EGFR, PI3K, AKT, ERK, GAPDH, and β-actin were detected by western blot. The samples showed a spherical shaped structure with the average size confirmed strong and stable hydrogen bonding forces with high degradation temperature and endothermic transition peaks. The fabricated samples showed no toxicity and high cell proliferation by generating more singlet oxygen levels and antioxidants. The intracellular signaling pathways was regulated with high protein expression levels, which was stimulated by specific molecules for cell proliferation, migration, and differentiation in cancer cells. The results proved that combined treatment regulated the intracellular signaling pathways in cancer cells. The current study showed a novel strategy for improving therapeutic efficacy of nanoemulsion by using low-level laser therapy. Further, the current favorable outcomes will be evaluated in in vivo animal models.

Despite its long half-life and physiological role, elastin undergoes irreversible changes (i.e elastolysis and/or calcification) impairing resilience of soft connective tissues. At present, it is still undefined: 1) to which extent elastin fibers have to be fragmented in order to increase their susceptibility to calcify; 2) which is the contribution of ionic environment on elastin mineralization; 3) why, in the same tissue area, mineralized coexist with non-mineralized fibers. The in vitro mineralization process was investigated on insoluble elastin, hydrolyzed or not-hydrolyzed, and incubated in different cell-free ionic environments. Mineral deposition is favored on hydrolyzed fibrillar structures due to exposure of multiple charged sites increasing the adsorption of Ca2+ that can attract phosphate and increase the local ion concentration over the point of supersaturation, representing the minimum requirement for hydroxyapatite nucleation sites. At physiological pH, the degree of elastin mineralization is influenced by hydrolysis and complexity of medium composition, since ionic species, as sodium, potassium, magnesium, in addition to calcium and phosphorus, interfere with the calcification process. These findings broaden the knowledge on the factors controlling hydroxyapatite deposition on insoluble elastin and can also explain why, in vivo, calcified and non-calcified fibers can be observed within the same tissue.

A high-efficiency graphene oxide-terminated hyperbranched amino polymer-carboxymethyl cellulose ternary nanocomposite (GO-HBP-NH2-CMC) was fabricated for adsorbing heavy metals from aqueous solutions. The adsorbent was characterized by SEM, FT-IR, Raman, and XPS analyses showing its porous architecture, rough surface, abundant N- and O-containing functional groups providing enhanced binding ability towards Pb2+ and Cu2+. Experimental adsorption data fitted well to the pseudo-second-order kinetics and Langmuir isotherm models, indicating the adsorption of GO-HBP-NH2-CMC towards Pb2+ and Cu2+ being a chemical and monolayer process. The maximum adsorption capacities of GO-HBP-NH2-CMC for Pb2+ and Cu2+ at 25 °C comprised 152.86 and 137.48 mg/g, respectively. The laboratory-scale experimental study into the Pb2+ and Cu2+ adsorption in a fixed-bed column was undertaken. Effects of flow rate, bed depth and influent metals concentration on the adsorption performance were assessed. Experimental data successfully correlated with the Adams-Bohart, Thomas and Yoon-Nelson models with the R2 exceeding 0.79. Density functional theory calculation was adopted to study interactions between functional groups at GO-HBP-NH2-CMC and heavy metals showing OH, NH2 and COOH moieties in GO-HBP-NH2-CMC being more likely to bind Pb2+ rather than Cu2+, while the binding abilities of -CONH- towards Pb2+ and Cu2+ were similar.

The δ isozyme of diacylglycerol kinase (DGKδ) plays critical roles in lipid signaling by converting diacylglycerol (DG) to phosphatidic acid (PA). We previously demonstrated that DGKδ preferably phosphorylates palmitic acid (16:0)- and/or palmitoleic acid (16:1)-containing DG molecular species, but not arachidonic acid (20:4)-containing DG species, which are recognized as DGK substrates derived from phosphatidylinositol turnover, in high glucose-stimulated myoblasts. However, little is known about where these DG molecular species come from. DGKδ and two DG-generating enzymes, sphingomyelin synthase (SMS) 1 and SMS-related protein (SMSr), contain a sterile α motif domain (SAMD). In the present study, we found that SMSr-SAMD, but not SMS1-SAMD, co-immunoprecipitates with DGKδ-SAMD. Full-length DGKδ co-precipitated with full-length SMSr more strongly than with SMS1. However, SAMD-deleted variants of SMSr and DGKδ interacted only weakly with full-length DGKδ and SMSr, respectively. These results strongly suggested that DGKδ interacts with SMSr through their respective SAMDs. To determine the functional outcomes of the relationship between DGKδ and SMSr, we used LC-MS/MS to investigate whether overexpression of DGKδ and/or SMSr in COS-7 cells alters the levels of PA species. We found that SMSr overexpression significantly enhances the production of 16:0- or 16:1-containing PA species such as 14:0/16:0-, 16:0/16:0-, 16:0/18:1-, and/or 16:1/18:1-PA in DGKδ-overexpressing COS-7 cells. Moreover, SMSr enhanced DGKδ activity via their SAMDs in vitro. Taken together, these results strongly suggest that SMSr is a candidate DG-providing enzyme upstream of DGKδ and that the two enzymes represent a new pathway independent of phosphatidylinositol turnover.

Herpesviruses uniquely express two essential nuclear egress–regulating proteins forming a heterodimeric basic structure of the nuclear-egress complex (core NEC). These core NECs serve as a hexameric lattice-structured platform for capsid docking and recruit viral and cellular NEC-associated factors that jointly exert nuclear lamina- and membrane-rearranging functions (multicomponent NEC). Here, we report the X-ray structures of β- and γ-herpesvirus core NECs obtained through an innovative recombinant expression strategy based on NEC-hook::NEC-groove protein fusion constructs. This approach yielded the first structure of γ-herpesviral core NEC, namely the 1.56 Å structure of Epstein–Barr virus (EBV) BFRF1-BFLF2, as well as an increased resolution 1.48 Å structure of human cytomegalovirus (HCMV) pUL50-pUL53. Detailed analysis of these structures revealed that the prominent hook segment is absolutely required for core NEC formation and contributes approx. 80% of the interaction surface of the globular domains of NEC proteins. Moreover, using HCMV::EBV hook domain swap constructs, computational prediction of the roles of individual hook residues for binding, and quantitative binding assays with synthetic peptides presenting the HCMV- and EBV-specific NEC hook sequences, we characterized the unique hook-into-groove NEC interaction at various levels. Although the overall physicochemical characteristics of the protein interfaces differ considerably in these β- and γ-herpesvirus NECs, the binding free energy contributions of residues displayed from identical positions are similar. In summary, the results of our study reveal critical details of the molecular mechanism of herpesviral NEC interactions and highlight their potential as an antiviral drug target.

Understanding the mechanisms by which viruses evade host-cell immune defenses is important for developing improved antiviral therapies. In an unusual twist, human cytomegalovirus (HCMV) co-opts the antiviral radical SAM enzyme, viperin (Virus inhibitory protein, endoplasmic reticulum-associated, interferon-inducible), to enhance viral infectivity. This process involves translocation of viperin to the mitochondrion where it binds the β-subunit (HADHB) of the mitochondrial trifunctional enzyme complex that catalyzes the thiolysis of β-ketoacyl-CoA esters as part of fatty acid β-oxidation. Here, we investigated how the interaction between these two enzymes alters their activities and affects cellular ATP levels. Experiments with purified enzymes indicated that viperin inhibits the thiolase activity of HADHB, but, unexpectedly, HADHB activates viperin leading to the synthesis of the antiviral nucleotide 3ʹ-deoxy-3′,4ʹ-didehydro-CTP. Measurements of enzyme activities in lysates prepared from transfected HEK 293T cells expressing these enzymes mirrored the findings obtained with purified enzymes. Thus, localizing viperin to the mitochondria decreased thiolase activity and co-expression of HADHB significantly increased viperin activity. Furthermore, targeting viperin to mitochondria also increased the rate at which HADHB is retro-translocated out of mitochondria and degraded, providing an additional mechanism by which viperin reduces HADHB activity. Targeting viperin to the mitochondria decreased cellular ATP levels by > 50 %, consistent with the enzyme disrupting fatty acid catabolism. These results provide biochemical insight into the mechanism by which HCMV subverts viperin; they also provide a biochemical rationale for viperin’s recently discovered role in regulating thermogenesis in adipose tissues.

MS-based metabolomics methods are powerful techniques to map the complex and interconnected metabolic pathways of the heart; however, normalization of metabolite abundance to sample input in heart tissues remains a technical challenge. Herein, we describe an improved GCMS-based metabolomics workflow that uses insoluble protein–derived glutamate for the normalization of metabolites within each sample and includes normalization to protein-derived amino acids to reduce biological variation and detect small metabolic changes. Moreover, glycogen is measured within the metabolomics workflow. We applied this workflow to study heart metabolism by first comparing two different methods of heart removal: the Langendorff heart method (reverse aortic perfusion), and in situ freezing of mouse heart with a modified tissue freeze-clamp approach. We then used the in situ freezing method to study the effects of acute β-adrenergic receptor stimulation (through isoproterenol treatment (ISO)) on heart metabolism. Using our workflow and within minutes, ISO reduced the levels of metabolites involved in glycogen metabolism, glycolysis, and the Krebs cycle, but the levels of pentose phosphate pathway metabolites and of many free amino acids remained unchanged. This observation was coupled to a 6-fold increase in phosphorylated adenosine nucleotide abundance. These results support the notion that ISO acutely accelerates oxidative metabolism of glucose to meet the ATP demand required to support increased heart rate and cardiac output. In summary, our MS-based metabolomics workflow enables improved quantification of cardiac metabolites and may also be compatible with other methods such as liquid chromatography or capillary electrophoresis.

Data‐Independent Acquisition (DIA) generates comprehensive yet complex mass spectrometric data, which imposes the use of data‐dependent acquisition (DDA) libraries for deep peptide‐centric detection. We here show that DIA can be redeemed from this dependency by combining predicted fragment intensities and retention times with narrow window DIA. This eliminates variation in library building and omits stochastic sampling, finally making the DIA workflow fully deterministic. Especially for clinical proteomics, this has the potential to facilitate inter‐laboratory comparison.

Progesterone receptor (PR) is expressed in a wide variety of human tissues, including both reproductive and non-reproductive tissues. Upon binding to the PR, progesterone can display several non-reproductive functions, including neurosteroid activity in the central nervous system, inhibition of smooth muscle contractile activity in the gastrointestinal tract, and regulating the development and maturation of the lung. PR exists as two major isoforms, PRA and PRB. Differential expression of these PR isoforms reportedly contributes to different biological activities of the hormone. However, the distribution of the PR isoforms in human tissues has remained virtually unexplored. In this study, we immunolocalized PR expression in various human tissues using PR (1294) specific antibody, which is capable of detecting both PRA and PRB, and PRB (250H11) specific antibody. Tissues from the uterus, ovary, breast, placenta, prostate, testis, cerebrum, cerebellum, pituitary, spinal cord, esophagus, stomach, small intestine, colon, pancreas, liver, kidney, urinary bladder, lung, heart, aorta, thymus, adrenal gland, thyroid, spleen, skin, and bone were examined in four different age groups (fetal, pediatric, young, and old) in male and female subjects. PR and PRB were detected in the nuclei of cells in the female reproductive system, in both the nuclei and cytoplasm of pituitary gland and pancreatic acinar cells, and only in the cytoplasm of cells in the testis, stomach, small intestine, colon, liver, kidney, urinary bladder, lung, adrenal gland, and skin. Of particular interest, total PRB expression overlapped with that of total PR expression in most tissues but was negative in the female fetal reproductive system. The findings indicate that progesterone could affect diverse human organs differently than from reproductive organs. These findings provide new insights into the novel biological roles of progesterone in non-reproductive organs.

Dynamin‐superfamily proteins (DSPs) are large self‐assembling mechanochemical GTPases that harness GTP hydrolysis to drive membrane remodeling events needed for many cellular processes. Mutation to alanine of a fully conserved lysine within the P‐loop of DSP GTPase domains results in abrogation of GTPase activity. This mutant has been widely used in the context of several DSPs as a dominant‐negative to impair DSP‐dependent processes. However, the precise deficit within the GTPase domain of the P‐loop K to A mutation remains an open question. Here, we use biophysical, biochemical and structural approaches to characterize this mutant in the context of the endosomal DSP Vps1. We show that the Vps1 P‐loop K to A mutant binds nucleotide with an affinity similar to wild type but exhibits defects in the organization of the GTPase active site that explain the lack of hydrolysis. In cells, Vps1 and Dnm1 bearing the P‐loop K to A mutation are defective in disassembly. These mutants become trapped in assemblies at the typical site of action of the DSP. This work provides mechanistic insight into the widely‐used DSP P‐loop K to A mutation and the basis of its dominant‐negative effects in the cell.

A distinguishing feature of camel (Camelus dromedarius) VHH domains are non‐canonical disulfide bonds between CDR1 and CDR3. The disulfide bond may provide an evolutionary advantage, as one of the cysteines in the bond is germline encoded. It has been hypothesized that this additional disulfide bond may play a role in binding affinity by reducing the entropic penalty associated with immobilization of a long CDR3 loop upon antigen binding. To examine the role of a non‐canonical disulfide bond on antigen binding and the biophysical properties of a VHH domain, we have used the VHH R303, which binds the Listeria virulence factor InlB as a model. Using site directed mutagenesis, we produced a double mutant of R303 (C33A/C102A) to remove the extra disulfide bond of the VHH R303. Antigen binding was not affected by loss of the disulfide bond, however the mutant VHH displayed reduced thermal stability (Tm = 12 °C lower than wild‐type), and a loss of the ability to fold reversibly due to heat induced aggregation. X‐ray structures of the mutant alone and in complex with InlB showed no major changes in the structure. B‐factor analysis of the structures suggested that the loss of the disulfide bond had no major change on the flexibility of the CDR loops, and revealed no evidence of loop immobilization upon antigen binding. These results suggest that the non‐canonical disulfide bond found in camel VHH may have evolved to stabilize the biophysical properties of the domain, rather than playing a significant role in antigen binding.

Surface hydrophobization of cellulose nanomaterials has been used in the development of nanofiller reinforced polymer composites and formulations based on Pickering emulsions. Despite well known effect of hydrophobic domains on self assembly or association of water soluble polymer amphiphiles, very few studies have addressed the behavior of hydrophobized cellulose nanomaterials in aqueous media. In this study, we investigate the properties of hydrophobized cellulose nanocrystals (CNCs) and their self assembly and amphiphilic properties in suspensions and gels. CNCs of different hydrophobicity were synthesized from sulfated CNCs by coupling primary alkylamines of different alkyl chain lengths (6, 8 and 12 carbon atoms). The synthetic route permitted the retention of surface charge, ensuring good colloidal stability of hydrohobized CNCs in aqueous suspensions. We compare surface properties (surface charge, Zeta potential), hydrophobicity (water contact angle, microenvironment probing using pyrene fluorescence emission) and surface activity (tensiometry) of different hydrophobized CNCs and hydrophilic CNCs. Association of hydrophobized CNCs driven by hydrophobic effects is confirmed by X ray scattering (SAXS) and autofluorescent spectroscopy experiments. As a result of CNC association, CNCs suspensions/gels can be produced with a wide range of rheological properties depending on the hydrophobic/hydrophilic balance. In particular, sol gel transitions for hydrophobized CNCs occur at lower concentrations then hydrophilic CNCs and more robust gels are formed by hydrophobized CNCs. Our work illustrates that amphiphilic CNCs can complement associative polymers as modifiers of rheological properties of water based systems.

Once in a while, a conceptual or technical breakthrough changes the way we approach science. In microbiology, Leeuwenhoek’s lenses, Koch’s solid growth medium, Fleming’s penicillin, and Woese’s recognition of the 16S rRNA sequence as a universal chronometer all changed the way discoveries were made, each providing a new way to study or understand microbial life. Following Woese’s groundbreaking insight, Pace applied the polymerase chain reaction to the 16S rRNA gene, making it possible to characterize the molecular diversity of microorganisms in natural environments without culturing bias.(1) The cascade of publications that followed made it clear that microbiologists were ignorant about the staggering diversity that had eluded detection by the culture-based methods that had dominated microbiology research since Koch’s 19th century innovation. Also made clear was the fact that many of the as-yet unculturable organisms were deeply divergent from those known from culture, presenting an opportunity to redefine the limits of microbial life. Pace then proposed that it was time to extract and clone DNA directly from environmental samples to conduct genomic analysis on the cornucopia of organisms that could not be cultured. This approach eventually became known as metagenomics—the analysis of collective genomes extracted from an environment. For the past 20 years, metagenomics research has primarily focused on sequence-based information and only a small proportion has been dedicated to functional metagenomics, which involves expressing DNA extracted from an environmental sample in a surrogate host to discover new metabolites and proteins.(2−4) Functional metagenomics has been plagued by the hurdle of heterologous gene expression, and consequently, progress has been slow. Recently, Sugimoto et al.(5) presented a game-changing approach to functional metagenomics that has the potential to revolutionize discovery. Sugimoto et al. offer two innovations in the search for gene function and new secondary metabolites encoded in microbial genomes. First, they present a major step forward in the characterization of biosynthetic pathways directly from sequencing reads, rather than from metagenomic assemblies, allowing them to avoid the fragmentation that often arises from variable coverage of shotgun community sequencing. Working directly with sequencing reads also allowed them to explore new taxonomic and functional genomic space and mitigated the uneven sampling of previously sequenced or culturable organisms (biased toward the Western, heathy human gut). Their new algorithm, designated MetaBGC, identifies biosynthetic gene cluster (BGC) reads from shotgun metagenomes with systematically tested and tuned sequence-scoring models that work in read-length sized chunks. Reads are binned for targeted assembly enabling reconstitution of entire pathways. The authors first focus on pathways containing type II polyketide synthases (T2PKSs) from a variety of human microbiome databases and show impressive results, even at very low coverage and abundance. Importantly, their method facilitates easy separation of true-positive and false-positive BGC bins. Looking across 3203 human metagenome samples from all major human body sites, the authors identify many new T2PKS BGCs, many from organisms not found in reference databases. BGC abundance was then correlated to body site, taxonomy of the organism of origin, and metatranscriptome information to provide further insight into the diversity and distribution of chemistry in the human microbiome. Sugimoto et al. focus on two novel BGC pathways for expression and chemical structure elucidation. The first, BGC3 from the oral microbiome, was found in a strain that had not yet been cultured. Another innovation in the work is that the authors opted to synthesize the entire BGC, inserting strong promoters and optimizing codon usage for expression in Streptomyces, and expressed the pathway in the heterologous host Staphylococcus albus. The resulting suite of molecules, dubbed metamycins, show strong inhibitory activity against Gram-positive isolates, especially those isolated from the human oral cavity (Figure 1). Transcriptomics analysis showed that the metamycin BGC is expressed under physiological conditions in human plaque during early biofilm formation. A second novel pathway, BGC6 from the gut microbiome, was also found in a cultured isolate. The authors amplified the BGC from Blautia wexlerae and expressed it in Bacillus subtilis to discover wexrubicin, a tetracyclic anthracycline. Unlike other anthracyclines (e.g., doxyrubicin), wexrubicin displays no toxicity in human cell assays. Figure 1. Innovation in metagenomics from Donia and colleagues uncovers new chemical diversity in the microbiome. New molecules, metamycin C and D, are active against bacteria sourced from the human microbiome at levels similar to that of the antibiotic tetracycline. MIC-A is the minimum inhibitory concentration on solid media. The discoveries reported by Sugimoto et al. advance natural products chemistry and drug discovery from uncultured organisms to a new level by making it more routine to access the biosynthetic machinery of the microbiome. But the work is equally impactful on microbial ecology. By returning to the source of the metagenome and examining expression of the pathways discovered in vitro, Sugimoto et al. have been able to propose ecological roles for the newly discovered molecules in the microbiome of origin. The metamycin biosynthetic pathway is expressed in the oral microbiome during initial biofilm formation, and the metamycins target Gram-positive bacteria in the human oral microbiome. The contributions to both chemical discovery and ecology make this research a turning point in understanding the diversity and roles of microbial metabolites in the microbial communities in which they are produced. Despite advances in molecular biology and sequencing over the past four decades, our understanding of microbiomes and the genes and molecules underlying microbial interaction networks remains in its infancy. The astounding diversity of microbial life begets exponential chemical complexity in the communities that comprise it, inviting microbiologists and chemists to unravel the structures, activities, and ecological roles of the cornucopia of compounds encoded in these communities’ metagenomes. We have learned that the intricate networks underpinning microbial community interactions form miniature versions of Darwin’s tangled bank, but work has been stymied by the inaccessibility of the tangible products of many functions encoded in metagenomes. Just as microscopy, culturing, antibiotics, and culture-independent analyses of microbes have all revolutionized microbiology, Sugimoto et al.’s advance will accelerate understanding microbiome functions in many biological processes, including global nutrient cycling, agriculture, and human health. The authors are thankful for Army Research Office MURI Grant W911NF1910269. The authors declare no competing financial interest. This article references 5 other publications.

Colibactin is a genotoxic gut microbiome metabolite long suspected of playing an etiological role in colorectal cancer. Evidence suggests colibactin forms DNA interstrand cross-links (ICLs) in eukaryotic cells and activates ICL repair pathways, leading to the production of ICL-dependent DNA double-strand breaks (DSBs). Here we show that colibactin ICLs can evolve directly to DNA DSBs. Using the topology of supercoiled plasmid DNA as a proxy for alkylation adduct stability, we find that colibactin-derived ICLs are unstable toward depurination and elimination of the 3¢ phosphate. This ICL degradation pathway leads progressively to single strand breaks (SSBs) and subsequently DSBs. The spontaneous conversion of ICLs to DSBs is consistent with the finding that non-homologous end joining repair-deficient cells are sensitized to colibactin-producing bacteria. The results herein further our understanding of colibactin-derived DNA damage and underscore the complexities underlying the DSB phenotype.

The radical SAM enzyme, viperin, exerts a wide range of antiviral effects through both the synthesis of the antiviral nucleotide 3’-deoxy-3’, 4’-didehydro-CTP (ddhCTP) and through its interactions with various cellular and viral proteins. Here we investigate the interaction of viperin with hepatitis C virus non-structural protein 5A (NS5A) and the host sterol regulatory protein, vesicle-associated membrane protein A (VAP-33). NS5A and VAP-33 form part of the viral replication complex that is essential for replicating the RNA genome of hepatitis C virus. Using transfected enzymes in HEK293T cells, we show that viperin binds to both NS5A and the C-terminal domain of VAP-33 (VAP-33C) independently and that this interaction is dependent on the proteins being co-localized to the ER membrane. Co-expression of VAP-33C and NS5A resulted in changes to the catalytic activity of viperin that depended upon viperin being co-localized to the ER membrane. The viperin-NS5A-VAP-33 complex exhibited the lowest activity, indicating that NS5A may inhibit viperin’s ability to synthesize ddhCTP. Co-expression of viperin with NS5A was also found to significantly reduce cellular NS5A levels, most likely by increasing the rate of proteasomal degradation. An inactive mutant of viperin, unable to bind the iron-sulfur cluster, was similarly effective at reducing cellular NS5A levels.

Hepatocellular carcinoma (HCC), which is one of the three major cancers, has attracted growing attention due to its high mortality, health-care cost and circumscribed therapeutic methods. Hence, the development of a fast, accurate and flexible method to detect alpha fetoprotein (AFP), the specific marker of HCC, is significant for diagnosis and treatment of cancer. Here, we constructed a novel SERS biosensing platform combining the target-responsive DNA hydrogel for the sensitive detection of AFP. The linker strand in DNA hydrogel is an aptamer that can specifically recognize AFP and accurately control the release of immunoglobulin G (IgG) encapsulated in hydrogel. In the presence of AFP, the hydrogels were disentangled and the IgG were released. Thereafter, the released IgG was captured by SERS probes and bio-functional magnetic beads through forming sandwich-like structures, resulting in the signal decreasing of Raman tags in supernatant after magnetic separation. Due to the ultra-high sensitivity of the SERS biosensor, the proposed method has the wide detection linear range (50 pg/mL～0.5 μg/mL) and the detection limit down to 50 pg/mL. Moreover, the sequence of linker strand in DNA hydrogel can be specifically encoded into a new aptamer that responds to other cancer markers. This convenient and inexpensive detection method provides a new strategy for the detection of tumor markers.

Identification of the enzyme(s) involved in complex biosynthetic pathways can be challenging. An alternative approach might be to deliberately diverge from the original natural enzyme source and use promiscuous enzymes from other organisms. In this paper, we have tested the ability of a series of human and animal cytochromes P450 involved in xenobiotic detoxification to generate derivatives of (+)-epi-α-bisabolol and attempt to produce the direct precursor of hernandulcin, a sweetener from Lippia dulcis for which the last enzymatic steps are unknown. Screening steps were implemented in vivo in S. cerevisiae optimized for the biosynthesis of oxidized derivatives of (+)-epi-α-bisabolol by coexpressing two key enzymes: the (+)-epi-α-bisabolol synthase and the NADPH cytochrome P450 reductase. Five out of 25 cytochromes P450 were capable of producing new hydroxylated regioisomers of (+)-epi-α-bisabolol. Of the new oxidized bisabolol products, the structure of one compound, 14-hydroxy-(+)-epi-α-bisabolol, was fully elucidated by NMR while the probable structure of the second product was determined. In parallel, the production of (+)-epi-α-bisabolol derivatives was enhanced through addition of a supplementary genomic copy of (+)-epi-α-bisabolol synthase that augmented the final titer of hydroxylated product to 64 mg/L. We thus demonstrate that promiscuous drug metabolism cytochromes P450 can be used to produce novel compounds from a terpene scaffold.

Cell-free systems provide a versatile platform for the development of low-cost, easy-to-use sensors for diverse analytes. However, sensor affinity dictates response sensitivity, and improving binding affinity can be challenging. Here we describe efforts to address this problem while developing a biosensor for Vitamin B12, a critical micronutrient. We first use a B12-responsive transcription factor to enable B12-dependent output in a cell-free reaction, but the resulting sensor responds to B12 far above clinically relevant concentrations. Surprisingly, when expressed in cells, the same sensor mediates a much more sensitive response to B12. The sensitivity difference is partly due to regulated import that accumulates cytoplasmic B12. Overexpression of importers further improves sensitivity, demonstrating an inherent advantage of whole-cell sensors. The resulting cells can respond to B12 in serum, can be lyophilized, and are functional in a minimal-equipment environment, showing the potential utility of whole-cell sensors as sensitive, field-deployable diagnostics.

Protein-protein interactions control a wide variety of natural biological processes. α-Helical coiled coils frequently mediate such protein-protein interactions. Due to the relative simplicity of their sequences and structures and the ease with which properties such as strength and specificity of interaction can be controlled, coiled coils can be designed de novo to deliver a variety of non-natural protein-protein-interaction domains. Herein, several de novo designed coiled coils are tested for their ability to mediate protein-protein interactions in Escherichia coli cells. The set includes a parallel homodimer, a parallel homotetramer, an antiparallel homotetramer and a newly designed heterotetramer, all of which have been characterized in vitro by biophysical and structural methods. Using a transcription repression assay based on reconstituting the Lac repressor, we find that the modules behave as designed in the cellular environment. Each design imparts a different property to the resulting Lac repressor-coiled coil complexes, resulting in the benefit of being able to reconfigure the system in multiple ways. Modification of the system also allows the interactions to be controlled: assembly can be tuned by controlling the expression of the constituent components; and complexes can be disrupted through helix sequestration. The small and straightforward de novo designed components that we deliver are highly versatile and have considerable potential as protein-protein-interaction domains in synthetic biology where proteins must be assembled in highly specific ways. The relative simplicity of the designs makes them amenable to future modifications to introduce finer control over their assembly and to adapt them for different contexts.

Mupirocin, a commercially available antibiotic produced by Pseudomonas fluorescens and thiomarinol, isolated from the marine bacterium Pseudoalteromonas sp. SANK 73390, both consist of a polyketide-derived monic acid homologue esterified with ei-ther 9-hydroxynonanoic acid (mupirocin, 9HN) or 8-hydroxyoctanoic acid (thiomarinol, 8HO). The mechanisms of formation of these deceptively simple 9HN and 8HO fatty acid moieties in mup and tml respectively remain unresolved. To define starter unit generation the purified mupirocin proteins MupQ, MupS and MacpD and their thiomarinol equivalents (TmlQ, TmlS and TacpD) have been expressed and shown to convert malonyl-coenzyme A (CoA) and succinyl-CoA to 3-hydroxypropionoyl (3-HP) or 4-hydroxybutyryl (4-HB) fatty acid starter units respectively via the MupQ/TmlQ catalysed generation of an unusual bis-CoA/acyl carrier protein (ACP) thioester, followed by MupS/TmlS catalysed reduction. Mix and match experiments show MupQ/TmlQ to be highly selective for the correct CoA, MacpD/TacpD were interchangeable but an alternate trans-acting ACPs from the mupirocin pathway (MacpA/TacpA) or a heterologous ACP (BatA) were non-functional. MupS and TmlS selectivity was more varied and these reductases differed in their substrate and ACP selectivity. The solution structure of MacpD determined by NMR revealed a C-terminal extension with partial helical character that has been shown to be important for maintaining high titres of mupirocin. We generated a truncated MacpD construct, MacpD_T, which lacks this C-terminal extension but retains an ability to generate 3-HP with MupS and MupQ, suggesting further downstream roles in protein-protein interactions for this re-gion of the ACP.

Two thermostable isoforms of a hexosaminidase were purified to homogeneity from the soluble extract of freshwater mussel Lamellidens corrianus, employing a variety of chromatographic techniques. Hexosaminidase A (HexA) is a heterodimer with subunit masses of ~80 and 55 kDa. Hexosaminidase B (HexB) is a homodimer with a subunit mass of 55–60 kDa. Circular dichroism spectroscopic studies indicated that both HexA and HexB contain β-sheet as the major secondary structural component with considerably lower content of α-helix. The temperature and pH optima of both the isoforms were found to be 60 °C and 4.0, respectively. The IC50 values for HexA with N-acetyl-d-galactosamine, N-acetyl-d-glucosamine, d-galactosamine, d-glucosamine, methyl α-d-mannopyranoside and d-mannose are 3.7, 72.8, 307, 216, 244 and 128 mM, respectively, whereas the corresponding IC50 values for HexB were estimated as 5.1, 61, 68, 190, 92 and 133 mM, respectively. Kinetic parameters KM and Vmax for HexA and B with p-nitrophenyl N-acetyl-β-d-glucosaminide are 4 mM, 0.23 μmol·min−1·mL−1 and 2.86 mM, 0.29 μmol·min−1·mL−1, respectively, and with p-nitrophenyl N-acetyl-β-d-galactosaminide are 4.5 mM, 0.054 μmol·min−1·mL−1 and 1.4 mM, 0.14 μmol·min−1·mL−1, respectively. GalNAc inhibited both isoforms in a non-competitive manner, whereas a mixed mode of inhibition was observed with GlcNAc with both forms.

In the present study, we investigated the neuroprotective effects of a purified Corydalis yanhusuo polysaccharide (CYP) on Aβ (25–35)-induced neurotoxicity and explored its underlying molecular mechanisms in PC12 cells. The results showed pretreatment with CYP (25, 50, and 100 μg/ml) prior to Aβ (25–35) exposure significantly protected PC12 cells from Aβ (25–35)-induced cell death, lactate dehydrogenase (LDH) release, DNA fragmentation, mitochondrial dysfunction and mitochondrial cytochrome c release. Moreover, Aβ (25–35)-induced increase of ratio between Bax and Bcl-2 protein expression was dramatically reversed by CYP pretreatment. Furthermore, the addition of CYP led to a significant repressing effect on the elevated protein expression of cleaved caspase-8, caspase-9, and caspase-3 in Aβ (25–35)-treated PC12 cells. Taken together, these findings indicated that protective effect of CYP against Aβ (25–35)-induced toxicity in PC12 cells was probably mediated by inhibition of apoptosis via both mitochondrial apoptotic pathway and death receptor pathway.

In this paper, the influence of a variety of salts (NaCl, CaCl2, and KCl) at different concentrations (0, 0.1, 0.5 and 1% w/w) on rheological and functional properties of basil seed gum (BSG) were investigated. BSG produced a high viscosity solution with yield stress, which was a function of salt type and concentration. In all samples, viscosity decreased as the electrostatic interactions between the BSG chains altered by salts. Flow behavior index increased by salt addition, which shows BSG had weaker shear-thinning behavior and worse mouthfeel in the presence of salts. The viscoelasticity of BSG strongly influenced by the addition of salt type as well as concentration. Larger cations (Ca+2) shield the electrostatic interaction between BSG chains more strongly compared to smaller cations as they have larger hydrated radius. As a result divalent salts decreased the viscosity and viscoelasticity more significantly. Emulsion capacity improved by salts addition, especially at high concentrations of salts. The foam capacity increased in the presence of CaCl2 and KCl increased foaming capacity of BSG. The results suggest that the addition of the different types of salt can alter or modify the rheological and functional properties of BSG, depending on the salt concentration.

Zedoary turmeric oil (ZTO) has a strong antitumor activity. However, its volatility, insolubility, low bioavailability, and difficulty of medication owing to oily liquid limit its clinical applications. Solid lipid nanoparticles can provide hydrophobic environment to dissolve hydrophobic drug and solidify the oily active composition to decrease the volatility and facilitate the medication. Chitosan has been widely used in pharmaceutics in recent years and coating with chitosan further enhances the internalization of particles by cells due to charge attract. Here, Chitosan (CS)-coated solid lipid nanoparticles (SLN) loaded with ZTO was prepared and characterized using dynamic laser scanner (DLS) and transmission electron microscope (TEM). The uptake and distribution of drug were evaluated in vitro and in vivo. The average sizes of ZTO-SLN and CS-ZTO-SLN were 134.3 ± 3.42 nm and 210.7 ± 4.59 nm, respectively. CS coating inverted the surface charge of particles from −8.93 ± 1.92 mV to +9.12 ± 2.03 mV. The liver accumulation of CS-ZTO-SLN was higher than ZTO-SLN (chitosan-uncoated particles) by analysis of tissue homogenate using HPLC, and the bioavailability of ZTO was also obviously improved. The results suggested that SLN coated with CS improved the features of ZTO formulation and efficiently deliver drug to the liver.

The present research aimed to study the nanofibers from bacterial cellulose (BC) by HCl and explore its new potential application in fresh-cut apples. Bacterial cellulose nanofibers (BCNs) showed low and more homogeneity particle size, as well as higher zeta potential and transparency in comparison with BC, which was confirmed by morphological analysis. Physical properties analysis showed that BCNs was more excellent semi-crystalline polymer with higher thermal stability as compared with BC. Rheological results displayed that BCNs suspensions presented a shear thinning behavior with higher apparent viscosity, storage (G′) and loss (G′′) moduli at the same concentration in comparison with BC. Furthermore, BCNs suspensions were more stable than BC suspensions under storage condition of 4 °C. Additionally, 2% (wt%) of BCNs suspensions were coated on fresh-cut apples. Results showed that the samples coated with BCNs suspension displayed more excellent properties of keeping fresh-cut apples as compared with that coated with BC suspensions, including delaying weight loss, improving firmness and soluble solids content, reducing browning index and titratable acidity. Therefore, the low cost and high biocompatibility of BCNs can be used as new coatings for fresh-cut apples and have great potential to coat fresh-cut fruits and vegetables in food industry.

The polysaccharides and phenylethanoid glycosides from Cistanche deserticola have been demonstrated with various health benefits, however the interactive effect between these two kinds of compounds in vivo are not in detail known. The objective of this study was to investigate the synergistic actions of cistanche polysaccharides with phenylethanoid glycoside and the effects of polysaccharides on gut microbiota. Sprague-Dawley rats were fed with different kinds of cistanche polysaccharides for 20 days, on the last day, all rats were administered the echinacoside at 100 mg/kg. The results were compared mainly on the difference of pharmacokinetic parameters, gut microbiota composition, and short chain fatty acids contents. The results indicated that all the cistanche polysaccharides, including crude polysaccharide, high molecular weight polysaccharide and low molecular weight polysaccharide, could regulate the gut microbiota diversity, increase beneficial bacteria and particularly enhance the growth of Prevotella spp. as well as improve the production of short chain fatty acids and the absorption of echinacoside. By exploring the synergistic actions of polysaccharides with small molecules, these findings suggest that cistanche polysaccharides, particularly low molecular weight polysaccharides, could be used as a gut microbiota manipulator for health promotion.

Hot water extraction and chromatographic purification methods were used to extract and purify two polysaccharides (RAPS-1 and RAPS-2) from the roots of alfalfa. Subsequently, RAPS-2 was modified using the HNO3/Na2SeO3 method to obtain Se-RAPS-2. The structural features, antioxidant and in vitro anti-tumor activities of the three polysaccharides were evaluated. The structural analysis revealed that RAPS-1 (Mw = 10.0 kDa) was composed of rhamnose, xylose, arabinose, galacturonic acid, mannose and glucose, whereas RAPS-2 (Mw = 15.8 kDa) consisted of rhamnose, xylose, galacturonic acid, mannose, glucose and galactose. RAPS-1 contained 1 → 2, 1 → 4, 1 → 3, and 1 → 6 or 1 → glycosidic bonds; however, while RAPS-2 lacked 1 → 4 glycosidic linkages. The molecular weight of Se-RAPS-2 was 11.0 kDa less than that of RAPS-2. The results of activities demonstrated that Se-RAPS-2 displayed superior antioxidant activity and inhibitory effect in HepG2 cells than RAPS-1 and RAPS-2.

The development of ideal organic-inorganic composite scaffold with porous structure and favorable osteoinductive properties that mimics the extracellular matrix composition of bone, is essential for the guidance of new bone formation in orthopaedic practice. Nowadays, numerous efforts have been dedicated to constructing implantable biocomposite scaffolds with appropriate structure and bioactivity for repairing bone defects. In this study, we fabricated chitosan-alginate-gelatin (CAG)-based porous biocomposite scaffolds with calcium phosphate coating on the surface and dexamethasone (DEX)-loaded mesoporous silica nanoparticles within the scaffold, which allows sustained release of DEX for bone tissue engineering application. The inorganic components of calcium phosphate crystals formed on the wall of scaffolds were obtained through electrochemical deposition method. The hybrid mineralized scaffolds demonstrate significantly high mechanical strength and reduced swelling property compared with pristine CAG scaffolds. The in vitro proliferation and osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells (BMSCs) cultured on biocomposite scaffolds were significantly enhanced. Furthermore, in vivo experiments revealed that biocomposite scaffolds with minerals deposition and DEX loading showed better new bone formation ability, as compared to pure CAG scaffold and single mineralized scaffold. Therefore, the developed biocomposite scaffolds may be highly promising as local implantable scaffolds for potential applications in bone tissue engineering.

The venom protein components of Malabar pit viper (Trimeresurus malabaricus) were identified by combining SDS-PAGE and ion-exchange chromatography pre-fractionation techniques with LC-MS/MS incorporating Novor and PEAKS-assisted de novo sequencing strategies. Total 97 proteins that belong to 16 protein families such as L-amino acid oxidase, metalloprotease, serine protease, phospholipase A2, 5′-nucleotidase, C-type lectins/snaclecs and disintegrin were recognized from the venom of a single exemplar species. Of the 97 proteins, eighteen were identified through de novo approaches. Immunological cross-reactivity assessed through ELISA and western blot indicate that the Indian antivenoms binds less effectively to Malabar pit viper venom components compared to that of Russell's viper venom. The in vitro cell viability assays suggest that compared to the normal cells, MPV venom induces concentration dependent cell death in various cancer cells. Moreover, crude venom resulted in chromatin condensation and apoptotic bodies implying the induction of apoptosis. Taken together, the present study enabled in dissecting the venom proteome of Trimeresurus malabaricus and revealed the immuno-cross-reactivity profiles of commercially available Indian polyvalent antivenoms that, in turn, is expected to provide valuable insights on the need in improving antivenom preparations against its bite.

Grifola frondosa is an edible and medicinal mushroom with great nutritional values and bioactivities. In the present study, a soluble homogeneous β-glucan, GFPS, with high molecular mass of 5.42 × 106 Da was purified from the fruit bodies of Grifola frondosa using 5% cold NaOH. The structure of GFPS was determined with FT-IR, NMR, and monosaccharide composition analysis, and was identified to be a β-D-(1-3)-linked glucan backbone with a single β-D-(1-6)-linked glucopyranosyl residue branched at C-6 on every third residue. Our results indicated that GFPS had a triple helical structure and could form complex with polydeoxyadenylic acid (poly[A]). Further studies demonstrated that GFPS could interact with poly[A] moiety of a designed antisense oligonucleotide (ASO) targeting the primary transcript of proinflammatory cytokine TNFα (TNFα-A60). This GFPS-based complex could incorporate TNFα-A60 into the macrophage cells via dectin-1 receptor and attenuate lipopolysaccharide-induced secretion of TNFα. Our results suggested that GFPS could be applied to deliver therapeutic oligonucleotides for the treatment of diseases such as inflammation and cancers.