Hepatocellular carcinoma biomarkers screening based on hydrogel photonic barcodes with tyramine deposition amplified ELISA.

Hepatocellular carcinoma (HCC), as one of the most lethal cancers, significantly impacts human health. Attempts in this area tends to develop novel technologies with sensitive and multiplexed detection properties for early diagnosis. Here, we present novel hydrogel photonic crystal (PhC) barcodes with tyramine deposition amplified enzyme-linked immunosorbent assay (ELISA) for highly sensitive and multiplexed HCC biomarker screening. Because of the abundant amino groups of acrylic acid (AA) component, the constructed hydrogel PhC barcodes with inverse opal structure could facilitate the loading of antibody probes for subsequent detection of tumor markers. By integrating tyramine deposition amplified ELISA on the barcode, the detection signal of tumor markers has been enhanced. Based on these features, it is demonstrated that the hydrogel PhC barcodes with tyramine deposition amplified ELISA could realize highly sensitive and multiplexed detection of HCC-related biomarkers. It was found that this method is flexible, sensitive and accurate, suitable for multivariate analysis of low abundance tumor markers and future cancer diagnosis. These features make the newly developed PhC barcodes an innovation platform, which possesses tremendous potential for practical application of low abundance targets.

Ultrasensitive fluorescence microRNA biosensor by coupling hybridization-initiated exonuclease I protection and tyramine signal amplification.

Tyramine signal amplification (TSA) has made its mark in immunoassay due to its excellent signal amplification ability and short reaction time, but its application in nucleic acid detection is still very limited. Herein, an ultrasensitive microRNA (miRNA) biosensor by coupling hybridization-initiated exonuclease I (Exo I) protection and TSA strategy was established. Target miRNA is complementarily hybridized to the biotin-modified DNA probe to form a double strand, which protects the DNA probe from Exo I hydrolysis. Subsequently, horseradish peroxidase (HRP) is attached to the duplex via the biotin-streptavidin reaction and catalyzes the deposition of large amounts of biotin-tyramine in the presence of hydrogen peroxide (H2O2), followed by the conjugation of signal molecule streptavidin-phycoerythrin (SA-PE), which generates an intense fluorescence signal upon laser excitation. This method gave broad linearity in the range of 0.1 fM - 10 pM, yielding a detection limit as low as 74 aM. An increase in sensitivity of 4 orders of magnitude was observed compared to the miRNA detection without TSA amplification. This biosensor was successfully applied to the determination of miR-21 in breast cancer cells and human serum. By further design of specific DNA probes and coupling with the Luminex xMAP technology, it could be easily extended to multiplex miRNA assay, which possesses great application potential in clinical diagnosis.

[Metabolic Activities Catalyzed by Human Cytochrome P450 (CYP) 2D6 and CYP3A Subfamily Members and Effect of Various Compounds, Including Endogenous Steroid Hormones, on These Activities].

My research focused on the effects of various drugs on (1) dopamine formation from p-tyramine catalyzed by polymorphic cytochrome P450 (CYP or P450) 2D6 variants and (2) endogenous steroid hormone hydroxylation catalyzed by CYP3A subfamily members (CYP3A4, CYP3A5, CYP3A7). The activation (cooperativity) of metabolic reactions catalyzed by P450s was especially emphasized. The effects of various psychotropic agents on dopamine formation from p-tyramine, catalyzed by wild-type CYP2D6.1 and CYP2D6 variants, including CYP2D6.2 (Arg296Cys;Ser486Thr), CYP2D6.10 (Pro34Ser;Ser486Thr), and CYP2D6.39 (Ser486Thr) were compared. Michaelis (Km) and inhibition (Ki) constants of the psychotropic agents in the presence of CYP2D6.10 were higher than those observed in the presence of other CYP2D6 variants. Fluvoxamine, fluoxetine, milnacipran, and haloperidol activated CYP2D6-catalyzed dopamine formation [decreasing the Km and/or increasing the maximal velocity (kcat)], and this activation was CYP2D6 variant-dependent. Regarding the CYP3A subfamily, the effects of various compounds including endogenous steroid hormones on the 6ß-hydroxylation of steroid hormones, such as testosterone, progesterone, and cortisol, were determined; it was found that testosterone, dehydroepiandrosterone, and/or α-naphthoflavone activated 6ß-hydroxylation of cortisol and/or progesterone, but the effects varied in the presence of different CYP3A subfamily members. Further studies are required to confirm the mechanisms and therapeutic relevance of these activation phenomena.

Tyramine-Invertase Bioconjugate-Amplified Personal Glucose Meter Signaling for Ultrasensitive Immunoassay.

Highly sensitive and facile detection of low levels of protein markers is of great significance for the early diagnosis and efficacy monitoring of diseases. Herein, aided by an efficient tyramine-signal amplification (TSA) mechanism, we wish to report a simple but ultrasensitive immunoassay with signal readout on a portable personal glucose meter (PGM). In this study, the bioconjugates of tyramine and invertase (Tyr-inv), which act as the critical bridge to convert and amplify the protein concentration information into glucose, are prepared following a click chemistry reaction. Then, in the presence of a target protein, the sandwich immunoreaction between the immobilized capture antibody, the target protein, and the horseradish peroxidase (HRP)-conjugated detection antibody is specifically performed in a 96-well microplate. Subsequently, the specifically loaded HRP-conjugated detection antibodies will catalyze the amplified deposition of a large number of Tyr-inv molecules onto adjacent proteins through highly efficient TSA. Then, the deposited invertase, whose dosage can faithfully reflect the original concentration of the target protein, can efficiently convert sucrose to glucose. The amount of finally produced glucose is simply quantified by the PGM, realizing the highly sensitive detection of trace protein markers such as the carcinoembryonic antigen and alpha fetoprotein antigen at the fg/mL level. This method is simple, cost-effective, and ultrasensitive without the requirement of sophisticated instruments or specialized laboratory equipment, which may provide a universal and promising technology for highly sensitive immunoassay for in vitro diagnosis of diseases.

Tyramine, one quorum sensing inhibitor, reduces pathogenicity and restores tetracycline susceptibility in Burkholderia cenocepacia.

Burkholderia cenocepacia is an opportunistic respiratory pathogen of particular relevance to patients with cystic fibrosis (CF), primarily regulating its biological functions and virulence factors through two quorum sensing (QS) systems (CepI/R and CciI/R). The highly persistent incidence of multidrug resistant Burkholderia cenocepacia poses a global threat to public health. In this study, we investigated the effects of tyramine, one biogenic amine, on the QS systems of Burkholderia cenocepacia. Genetic and biochemical analyses revealed that tyramine inhibited the production of N-hexanoyl-homoserine (AHL) signaling molecules (C8-HSL and C6-HSL) by blocking the CepI/R and CciI/R systems. As a result, the inhibition of QS systems leads to reduced production of various virulence factors, such as biofilm formation, extracellular polysaccharides, lipase, and swarming motility. Notably, as a potential quorum sensing inhibitor, tyramine exhibits low toxicity in vivo in Galleria mellonella larvae and is well characterized by Lipinski's five rules. It also shows high gastrointestinal absorption and the ability to cross the blood-brain barrier according to SwissADME database and ProTox-II server. Additionally, tyramine was found to enhance the efficacy of tetracycline in reducing the infectivity of Burkholderia cenocepacia in Galleria mellonella larvae infection model. Therefore, tyramine could be a promising candidate for combination therapy with traditional antimicrobials to improve their effectiveness against Burkholderia cenocepacia.

In situ photo-crosslinkable hyaluronic acid-based hydrogel embedded with GHK peptide nanofibers for bioactive wound healing.

A versatile hydrogel was developed for enhancing bioactive wound healing by introducing the amphiphilic GHK peptide (GHK-C16) into a photo-crosslinkable tyramine-modified hyaluronic acid (HA-Ty). GHK-C16 self-assembled into GHK nanofibers (GHK NF) in HA-Ty solution, which underwent in situ gelation after the wound area was filled with precursor solution. Blue light irradiation (460-490 nm), with riboflavin phosphate as a photoinitiator, was used to trigger crosslinking, which enhanced the stability of the highly degradable hyaluronic acid and enabled sustained release of the nanostructured GHK derivatives. The hydrogels provided a microenvironment that promoted the proliferation of dermal fibroblasts and the activation of cytokines, leading to reduced inflammation and increased collagen expression during wound healing. The complexation of Cu2+ into GHK nanofibers resulted in superior wound healing capabilities compared with non-lipidated GHK peptide with a comparable level of growth factor (EGF). Additionally, nanostructured Cu-GHK improved angiogenesis through vascular endothelial growth factor (VEGF) activation, which exerted a synergistic therapeutic effect. Furthermore, in vivo wound healing experiments revealed that the Cu-GHK NF/HA-Ty hydrogel accelerated wound healing through densely packed remodeled collagen in the dermis and promoting the growth of denser fibroblasts. HA-Ty hydrogels incorporating GHK NF also possessed improved mechanical properties and a faster wound healing rate, making them suitable for advanced bioactive wound healing applications. STATEMENT OF SIGNIFICANCE: By combining photo-crosslinkable tyramine-modified hyaluronic acid with self-assembled Cu-GHK-C16 peptide nanofibers (Cu-GHK NF), the Cu-GHK NF/HA-Ty hydrogel offers remarkable advantages over conventional non-structured Cu-GHK for wound healing. It enhances cell proliferation, migration, and collagen remodeling-critical factors in tissue regeneration. The incorporation of GHK nanofibers complexed with copper ions imparts potent anti-inflammatory effects, promoting cytokine activation and angiogenesis during wound healing. The Cu-GHK NF/hydrogel's unique properties, including in situ photo-crosslinking, ensure high customization and potency in tissue regeneration, providing a cost-effective alternative to growth factors. In vivo experiments further validate its efficacy, demonstrating significant wound closure, collagen remodeling, and increased fibroblast density. Overall, the Cu-GHK NF/HA-Ty hydrogel represents an advanced therapeutic option for wound healing applications.

Beneficial Effects of Hordenine on a Model of Ulcerative Colitis.

Hordenine, a phenethylamine alkaloid, is found in a variety of plants and exhibits a broad array of biological activities and pharmacological properties, including anti-inflammatory and anti-fibrotic effects. However, the efficacy and underlying mechanisms of hordenine in treating ulcerative colitis (UC) remain unclear. To address this, we examined the therapeutic effects of hordenine on dextran sodium sulphate (DSS)-induced UC by comparing disease activity index (DAI), colon length, secretion of inflammatory factors, and degree of colonic histological lesions across diseased mice that were and were not treated with hordenine. We found that hordenine significantly reduced DAI and levels of pro-inflammatory factors, including interleukin (IL)-6, IL-1ß, and tumor necrosis factor alpha (TNF-α), and also alleviated colon tissue oedema, colonic lesions, inflammatory cells infiltration and decreased the number of goblet cells. Moreover, in vitro experiments showed that hordenine protected intestinal epithelial barrier function by increasing the expression of tight junction proteins including ZO-1 and occludin, while also promoting the healing of intestinal mucosa. Using immunohistochemistry and western blotting, we demonstrated that hordenine reduced the expression of sphingosine kinase 1 (SPHK1), sphingosine-1-phosphate receptor 1 (S1PR1), and ras-related C3 botulinum toxin substrate 1 (Rac1), and it inhibited the expression of phosphorylated signal transducer and activator of transcription 3 (p-STAT3) in colon tissues. Thus, hordenine appears to be effective in UC treatment owing to pharmacological mechanisms that favor mucosal healing and the inhibition of SPHK-1/S1PR1/STAT3 signaling.

An electrochemical immunosensor for the detection of Glypican-3 based on enzymatic ferrocene-tyramine deposition reaction.

An ultrasensitive electrochemical immunosensor based on signal amplification of the deposition of the electroactive ferrocene-tyramine (Fc-Tyr) molecule, catalyzed by horseradish peroxidase (HRP), was constructed for the detection of the liver cancer marker Glypican-3 (GPC3). Functional electroactive molecule Fc-Tyr is reported to exhibit both the enzymatic cascade catalytic activity of tyramine signal amplification (TSA) and the excellent redox properties of ferrocene. In terms of design, the low matrix effects inherent in using the magnetic bead platforms, a quasi-homogeneous system, allowed capturing the target protein GPC3 without sample pretreatment, and loading HRP to trigger the TSA, which induced a large amount of Fc-Tyr deposited on the electrode surface layer by layer as a signal probe for the detection of GPC3. The concept of Fc-Tyr as an electroactive label was validated, GPC3 biosensor exhibited high selectivity and sensitivity to GPC3 in the range of 0.1 ng mL-1-1 µg mL-1. Finally, the sensor was used simultaneously with ELISA to assess GPC3 levels in the serum of clinical liver cancer patients, and the results showed consistency, with a recovery of 98.33-105.35% and a relative standard deviation (RSD) of 4.38-8.18%, providing a theoretical basis for achieving portable, rapid and point of care testing (POCT) of tumor markers.

Tyramine-induced gastrointestinal dysregulation is attenuated via estradiol associated mechanisms in a zebrafish larval model.

Development of targeted therapeutics to alleviate gastrointestinal (GI) inflammation and its debilitating consequences are required. In this context, the trace aminergic system may link together sex, diet and inflammation. Utilising a zebrafish larval model of GI inflammation, the current study aimed to investigate mechanisms by which excess amounts of trace amines (TAs) may influence GI health. In addition, we probed the potential role of 17ß-estradiol (E2) and its receptors, given the known female-predominance of many GI disorders. To assess GI functionality and integrity, live imaging techniques (neutral red staining) and post-mortem immunofluorescent staining of tight junction proteins (occludin and ZO-1) were analyzed respectively. In addition, behavioural assays, as an indication of overall wellbeing, as well as whole body H2O2 and prostaglandin E2 assays were performed to inform on oxidative and inflammatory status. Excess ß-phenethylamine (PEA), tryptamine (TRP) and ρ-tyramine (TYR) resulted in adverse GI and systemic effects. In this regard, clear beneficial effects of E2 to modulate the effects of PEA, TRP and TYR was evident. Moreover, agmatine displayed potential protective effects on GI epithelium and whole body oxidative status, however, potential to induce systemic inflammation suggests the importance of dosage and administration optimisation. Taken together, TYR seems like the most prominent TA to have damaging GI effects, feasibly exacerbating GI inflammation. In this context, the relative lack of E2 may provide mechanistic insights into the reported female-predominance of GI disorders. Moreover, an effective therapeutic in this context may be required to maintain GI TA load despite fluctuating E2 levels.

Injectable enzyme-catalyzed crosslinking hydrogels as BMSCs-laden tunable scaffold for osteogenic differentiation.

Bone defects caused by trauma or tumor are a significant challenge in clinical practice. Hydrogel-based tissue engineering has been considered an effective strategy. This study successfully formed a series of injectable hydrogels by enzyme-catalyzed crosslinking hyaluronic acid-tyramine (HA-TA) and sodium alginate-tyramine (ALG-TA) under physiological conditions in the presence of both horseradish peroxidase and hydrogen peroxide. The morphology, mechanical properties, swelling properties, and biodegradation properties of hydrogels were investigated. The results showed that the mechanical properties, swelling properties and biodegradation of HA/ALG hydrogels varied with the precursor solution concentration. Furthermore, the proliferation and osteogenic differentiation of BMSCs within the HA/ALG hydrogels were evaluated in vitro. The results illustrated that the hydrogels could offer an excellent microenvironment for BMSCs growth and promote osteogenic differentiation. Therefore, the injectable hydrogels can be used as an effective 3 D scaffold for bone repair and regeneration.

De novo biosynthesis of N-acetyltyramine in engineered Escherichia coli.

N-acetyltyramine as a tyramine alkaloid has drawn great attention because of its excellent anti-free radical, antithrombotic, and antitumour activity. Therefore, it is an attractive compound. In this study, we reported for the first time the construction a synthetic pathway of N-acetyltyramine in engineered Escherichia coli. First, the tyrosine decarboxylase tdc gene and arylalkylamine N-acyltransferase aanat gene were introduced into E. coli to generate a recombinant N-acetyltyramine producer with L-tyrosine as substrate. Subsequently, overexpressing aroGfbr and TyrAfbr enhance the availability of L-tyrosine to achieve de novo biosynthesis of N-acetyltyramine from glucose. Finally, overexpressing the transketolase I tktA and phosphoenolpyruvate synthase ppsA genes improved the N-acetyltyramine production to 854 mg/L.

New hyaluronan-based biomatrix for 3-D follicle culture yields functionally competent oocytes.

BACKGROUND: Encapsulation of follicles within a biomatrix is one approach to maintaining 3-D follicle architecture during culture. Hyaluronan is one component of the natural extracellular matrix (ECM) that provides support to cells in vivo. This report describes the application of a novel tyramine-linked hyaluronan for 3-D in vitro follicle culture and the production of developmentally competent metaphase II oocytes. MATERIALS AND METHODS: Enzymatically isolated mouse preantral follicles or follicle clusters (FL-C) from fresh or vitrified ovaries were encapsulated in 3 mg/ml of hyaluronan gel (HA). Follicle growth, antrum formation and meiotic maturation to metaphase II oocytes was monitored. Chromatin staining was used to assess GV oocyte progression towards meiotic competence. Functional competence of in vitro matured (IVM) oocytes was evaluated by in vitro fertilization and ability to develop to blastocyst. Modifying the HA gel by inclusion of laminin (HA-LM), mouse sarcoma extracellular matrix (Matrigel;HA-MG) or placental extracellular matrix (HA-PM) was also tested to see if this might further enhance IVM outcomes. RESULTS: A total of 402 preantral follicles were cultured in HA gel. After hCG trigger, 314 oocyte-cumulus complexes ovulated from the embedded follicles. Meiotic maturation rate to the metaphase II stage was 73% (228/314). After insemination 83% (188/228) of IVM oocytes fertilized with a subsequent blastulation rate of 46% (87/188). A pilot transfer study with 3 recipient mice resulted in the birth of a single pup. HA gel supported individually isolated follicles as well ovarian tissue fragments containing clusters of 6-8 preantral follicles. Meiotic maturation was lower with FL-clusters from vitrified versus fresh ovaries (34% and 55%, respectively; p < 0.007). Modification of the HA gel with ECMs or laminin affected antrum formation and follicle retention. Maturation rates to the metaphase II stage were however not significantly different: 74% for HA gel alone as compared to HA-LM (67%), HA-MG (56%) and HA-PM (58%). CONCLUSION: Hyaluronan gel is an effective and versatile extracellular matrix based biomaterial for 3-D culture of ovarian follicles. This culture model allowed ovulation of functionally competent metaphase II oocytes, capable of fertilization, genomic activation and blastulation. Future testing with human follicles that require longer in vitro culture times should be considered.

Chemical Synthesis and Biological Activities of Amaryllidaceae Alkaloid Norbelladine Derivatives and Precursors.

Amaryllidaceae alkaloids (AAs) are a structurally diverse family of alkaloids recognized for their many therapeutic properties, such as antiviral, anti-cholinesterase, and anticancer properties. Norbelladine and its derivatives, whose biological properties are poorly studied, are key intermediates required for the biosynthesis of all ~650 reported AAs. To gain insight into their therapeutic potential, we synthesized a series of O-methylated norbelladine-type alkaloids and evaluated their cytotoxic effects on two types of cancer cell lines, their antiviral effects against the dengue virus (DENV) and the human immunodeficiency virus 1 (HIV-1), and their anti-Alzheimer's disease (anti-cholinesterase and -prolyl oligopeptidase) properties. In monocytic leukemia cells, norcraugsodine was highly cytotoxic (CC50 = 27.0 µM), while norbelladine was the most cytotoxic to hepatocarcinoma cells (CC50 = 72.6 µM). HIV-1 infection was impaired only at cytotoxic concentrations of the compounds. The 3,4-dihydroxybenzaldehyde (selectivity index (SI) = 7.2), 3',4'-O-dimethylnorbelladine (SI = 4.8), 4'-O-methylnorbelladine (SI > 4.9), 3'-O-methylnorbelladine (SI > 4.5), and norcraugsodine (SI = 3.2) reduced the number of DENV-infected cells with EC50 values ranging from 24.1 to 44.9 µM. The O-methylation of norcraugsodine abolished its anti-DENV potential. Norbelladine and its O-methylated forms also displayed butyrylcholinesterase-inhibition properties (IC50 values ranging from 26.1 to 91.6 µM). Altogether, the results provided hints of the structure−activity relationship of norbelladine-type alkaloids, which is important knowledge for the development of new inhibitors of DENV and butyrylcholinesterase.

Tuning the Cell-Adhesive Properties of Two-Component Hybrid Hydrogels to Modulate Cancer Cell Behavior, Metastasis, and Death Pathways.

This work presents a polysaccharide and protein-based two-component hybrid hydrogel integrating the cell-adhesive gelatin-tyramine (G-Tyr) and nonadhesive hyaluronic acid-tyramine (HA-Tyr) through enzyme-mediated oxidative coupling reaction. The resulting HA-Tyr/G-Tyr hydrogel reflects the precise chemical and mechanical features of the cancer extracellular matrix and is able to tune cancer cell adhesion upon switching the component ratio. The cells form quasi-spheroids on HA-Tyr rich hydrogels, while they tend to form an invasive monolayer culture on G-Tyr rich hydrogels. The metastatic genotype of colorectal adenocarcinoma cells (HT-29) increases on G-Tyr rich hydrogels which is driven by the material's adhesive property, and additionally confirmed by the suppressed gene expressions of apoptosis and autophagy. On the other hand, HA-Tyr rich hydrogels lead the cells to necrotic death via oxidative stress in quasi-spheroids. This work demonstrates the ideality of HA-Tyr/G-Tyr to modulate cancer cell adhesion, which also has potential in preventing primary metastasis after onco-surgery, biomaterials-based cancer research, and drug testing.

Multiple Direct Effects of the Dietary Protoalkaloid N-Methyltyramine in Human Adipocytes.

Dietary amines have been the subject of a novel interest in nutrition since the discovery of trace amine-associated receptors (TAARs), especially TAAR-1, which recognizes tyramine, phenethylamine, tryptamine, octopamine, N-methyltyramine (NMT), synephrine, amphetamine and related derivatives. Alongside the psychostimulant properties of TAAR-1 ligands, it is their ephedrine-like action on weight loss that drives their current consumption via dietary supplements advertised for 'fat-burning' properties. Among these trace amines, tyramine has recently been described, at high doses, to exhibit an antilipolytic action and activation of glucose transport in human adipocytes, i.e., effects that are facilitating lipid storage rather than mobilization. Because of its close structural similarity to tyramine, NMT actions on human adipocytes therefore must to be reevaluated. To this aim, we studied the lipolytic and antilipolytic properties of NMT together with its interplay with insulin stimulation of glucose transport along with amine oxidase activities in adipose cells obtained from women undergoing abdominal surgery. NMT activated 2-deoxyglucose uptake when incubated with freshly isolated adipocytes at 0.01-1 mM, reaching one-third of the maximal stimulation by insulin. However, when combined with insulin, NMT limited by half the action of the lipogenic hormone on glucose transport. The NMT-induced stimulation of hexose uptake was sensitive to inhibitors of monoamine oxidases (MAO) and of semicarbazide-sensitive amine oxidase (SSAO), as was the case for tyramine and benzylamine. All three amines inhibited isoprenaline-induced lipolysis to a greater extent than insulin, while they were poorly lipolytic on their own. All three amines-but not isoprenaline-interacted with MAO or SSAO. Due to these multiple effects on human adipocytes, NMT cannot be considered as a direct lipolytic agent, potentially able to improve lipid mobilization and fat oxidation in consumers of NMT-containing dietary supplements.

Scar prevention through topical delivery of gelatin-tyramine-siSPARC nanoplex loaded in dissolvable hyaluronic acid microneedle patch across skin barrier.

Currently, there is no effective method to prevent the formation of hypertrophic scars and keloids, which can cause severe physical and psychological burdens to patients. Secreted protein acidic and cysteine-rich (SPARC) is involved in wound fibrosis by modulating fibroblast functions, causing excessive collagen deposition during wound healing. Thus, the reduction in SPARC gene expression after wounding can contribute to the downstream reduction in collagen production at the wound site and prevent scar formation. In this study, a dissolvable and biocompatible hyaluronic acid (HA) microneedle patch loaded with nanoplexes containing tyramine-modified gelatin and siRNA for SPARC (siSPARC/Gtn-Tyr) was investigated for topical scar prevention. Tyramine-modified gelatin (Gtn-Tyr) provides electrostatic protection and enhances cell internalization for siSPARC. In vitro studies using human dermal fibroblasts showed that both siSPARC/Gtn-Tyr nanoplexes and siSPARC/Gtn-Tyr-loaded microneedle patches can significantly reduce SPARC gene expression (P < 0.05) and do not cause discernable cytotoxic effects. Further studies using a mouse wound model demonstrate that the siSPARC/Gtn-Tyr-loaded microneedle patch can reduce collagen production during wound healing without triggering an immune response. When Gtn-Tyr-siSPARC is administered transdermally at the wound site, effective collagen reduction is achieved through silencing of the matricellular SPARC protein, thus promising the reduction of scar formation. Overall, the siSPARC/Gtn-Tyr loaded microneedle patch can potentially provide an effective transdermal anti-fibrotic treatment.

CRISPR/Cas12a-Triggered Chemiluminescence Enhancement Biosensor for Sensitive Detection of Nucleic Acids by Introducing a Tyramide Signal Amplification Strategy.

CRISPR-based biosensors have attracted increasing attention in accurate and sensitive nucleic acid detection. In this work, we report a CRISPR/Cas12a-triggered chemiluminescence enhancement biosensor for the ultrasensitive detection of nucleic acids by introducing tyramide signal amplification for the first time (termed CRICED). The hybrid chain DNA (crDNA) formed by NH2-capture DNA (capDNA) and biotin-recognition DNA (recDNA) was preferentially attached to the magnetic beads (MBs), and the streptavidin-HRP was subsequently introduced to obtain MB@HRP-crDNA. In the presence of the DNA target, the activated CRISPR/Cas12a is capable of randomly cutting initiator DNA (intDNA) into vast short products, and thus the fractured intDNA could not trigger the toehold-mediated DNA-strand displacement reaction (TSDR) event with MB@HRP-crDNA. After the addition of tyramine-AP and H2O2, abundant HRP-tyramine-AP emerges through the covalent attachment of HRP-tyramine, exhibiting enhanced chemiluminescence (CL) signals or visual image readouts. By virtue of this biosensor, we achieved high sensitivity of synthetic DNA target and amplified DNA plasmid using recombinase polymerase amplification (RPA) as low as 17 pM and single-copy detection, respectively. Our proposed CRICED was further evaluated to test 20 HPV clinical samples, showing a superior sensitivity of 87.50% and specificity of 100.00%. Consequently, the CRICED platform could be an attractive means for ultrasensitive and imaging detection of nucleic acids and holds a promising strategy for the practical application of CRISPR-based diagnostics.

Robust gelatin hydrogels for local sustained release of bupivacaine following spinal surgery.

Adequate treatment of pain arising from spinal surgery is a major clinical challenge. Opioids are the mainstay of current treatment methods, but the frequency and severity of their side effects display a clear need for opioid-free analgesia. Local anesthetics have been encapsulated into sustained-release drug delivery systems to provide postoperative pain relief. However, these formulations are limited by rapid diffusion out of the surgical site. To overcome this limitation, we synthesized ring-shaped hydrogels incorporating bupivacaine, designed to be co-implanted with pedicle screws during spinal surgery. Hydrogels were prepared by riboflavin-mediated crosslinking of gelatin functionalized with tyramine moieties. Additionally, oxidized ß-cyclodextrin was introduced into the hydrogel formulation to form dynamic bonds with tyramine functionalities, which enables self-healing behavior and resistance to shear. Feasibility of hydrogel implantation combined with pedicle screws was qualitatively assessed in cadaveric sheep as a model for instrumented spinal surgery. The in-situ crystallization of bupivacaine within the hydrogel matrix provided a moderate burst decrease and sustained release that exceeded 72 hours in vitro. The use of bupivacaine crystals decreased drug-induced cytotoxicity in vitro compared to bupivacaine HCl. Thus, the presented robust hydrogel formulation provides promising properties to enable the stationary release of non-opioid analgesics following spinal surgery. STATEMENT OF SIGNIFICANCE: Currently, postoperative pain following spinal surgery is mainly treated with opioids. However, the use of opioids is associated with several side effects including addiction. Here we developed robust and cytocompatible gelatin hydrogels, prepared via riboflavin-mediated photocrosslinking, that can withstand orthopedic implantation. The implantability was confirmed in cadaveric instrumented spinal surgery. Further, hydrogels were loaded with bupivacaine crystals to provide sustained release beyond 72 hours in vitro. The use of crystallized bupivacaine decreased cytotoxicity compared to bupivacaine HCl. The present formulation can aid in enabling opioid-free analgesia following instrumented spinal surgery.

Facile Amidation of Non-Protected Hydroxycinnamic Acids for the Synthesis of Natural Phenol Amides.

Phenol amides are bioactive compounds naturally present in many plants. This class of compounds is known for antioxidant, anti-inflammatory, and anticancer activities. To better understand the reactivity and structure-bioactivity relationships of phenol amides, a large set of structurally diverse pure compounds are needed, however purification from plants is inefficient and laborious. Existing syntheses require multiple steps, including protection of functional groups and are generally overly complicated and only suitable for specific combinations of hydroxycinnamic acid and amine. Thus, to facilitate further studies on these promising compounds, we aimed to develop a facile general synthetic route to obtain phenol amides with a wide structural diversity. The result is a protocol for straightforward one-pot synthesis of phenol amides at room temperature within 25 h using equimolar amounts of N,N'-dicyclohexylcarbodiimide (DCC), amine, hydroxycinnamic acid, and sodium bicarbonate in aqueous acetone. Eight structurally diverse phenol amides were synthesized and fully chemically characterized. The facile synthetic route described in this work is suitable for a wide variety of biologically relevant phenol amides, consisting of different hydroxycinnamic acid subunits (coumaric acid, ferulic acid, and sinapic acid) and amine subunits (agmatine, anthranilic acid, putrescine, serotonin, tyramine, and tryptamine) with yields ranging between 14% and 24%.

Exercise Training Lowers Arterial Blood Pressure Independently of Pannexin 1 in Men with Essential Hypertension.

INTRODUCTION: Regular exercise training reduces arterial blood pressure, but the underlying mechanisms are unclear. Here, we evaluated the potential involvement of pannexin 1, an ATP releasing channel, in the blood pressure-reducing effect of training. METHODS: Middle-age men, 13 normotensive and 14 nonmedicated stage 1 hypertensive, completed 8 wk of intensive aerobic cycle training. Before and after training, blood pressure and changes in leg vascular conductance, induced by femoral arterial infusion of tyramine (induces endogenous noradrenaline release), acetylcholine, or sodium nitroprusside, were measured during control conditions and after acute pannexin 1 inhibition by probenecid. A skeletal muscle biopsy was obtained from the thigh, pre- and posttraining. RESULTS: Exercise training reduced mean systolic and diastolic blood pressure by ~5 ( P = 0.013) and 5 mm Hg ( P < 0.001), respectively, in the hypertensive group only. The reduction in blood pressure was not related to changes in pannexin 1 function because mean arterial blood pressure and tyramine-induced vasoconstriction remain unaltered by pannexin 1 inhibition after training in both groups. After training, pannexin 1 inhibition enhanced leg vascular conductance in the normo- and hypertensive groups at baseline (41.5%, P = 0.0036, and 37.7%, P = 0.024, respectively) and in response to sodium nitroprusside infusion (275%, P = 0.038, and 188%, P = 0.038, respectively). Training did not alter the pannexin 1 protein expression in skeletal muscle. Training enhanced the vasodilator response to acetylcholine infusion and increased the expression of microvascular function-relevant proteins. CONCLUSIONS: The exercise training-induced lowering of arterial blood pressure in nonmedicated hypertensive men does not involve an altered function of pannexin 1.