NIR Light-Fuse Drug-Free Photothermal Armor-Piercing Microcapsule for Femoral Vein Thrombosis Therapy.

Acute thrombosis and its complications are leading global causes of disability and death. Existing thrombolytic drugs, such as alteplase and urokinase (UK), carry a significant bleeding risk during clinical treatments. Thus, the development of a novel thrombolysis strategy is of utmost urgency. Based on the previous work, the hollow structure of microcapsules (MC) is fabricated. Subsequently, armor-piercing MC, known as Fucoidan/S-Nitrosoglutathione/Melanin@MC (FGM@MC) is obtained, using a layer-by-layer (LBL) self-assembly method. Utilizing near-infrared (NIR) light as a trigger, the FGM@MC demonstrated photothermal thrombolysis at the site of thrombus due to its stable and outstanding photothermal properties. Simultaneously, photothermal stimulation leads to the release of a significant amount of nitric oxide from the FGM@MC, resulting in cavitation effects for mechanical thrombolysis. In vivo experiments confirmed the stable release of nitric oxide under NIR light irradiation. Treatment of femoral vein thrombosis in rats revealed that the thrombolytic effectiveness of FGM@MC+NIR (53.71%) is comparable to that of UK (59.70%). Notably, FGM@MC does not interfere with the coagulation function of rats and exhibits a favorable safety profile. In conclusion, this study demonstrates that the drug-free armor-piercing microcapsule has significant potential in the treatment of thrombosis, offering a safe and effective alternative to traditional thrombolytic therapies.

All-in-One Self-Powered Microneedle Device for Accelerating Infected Diabetic Wound Repair.

Diabetic wound healing remains a significant clinical challenge due to the complex microenvironment and attenuated endogenous electric field. Herein, a novel all-in-one self-powered microneedle device (termed TZ@mMN-TENG) is developed by combining the multifunctional microneedle carried tannin@ZnO microparticles (TZ@mMN) with the self-powered triboelectric nanogenerator (TENG). In addition to the delivery of tannin and Zn2+, TZ@mMN also effectively conducts electrical stimulation (ES) to infected diabetic wounds. As a self-powered device, the TENG can convert biomechanical motion into exogenous ES to accelerate the infected diabetic wound healing. In vitro experiment demonstrated that TZ@mMN shows excellent conductive, high antioxidant ability, and effective antibacterial properties against both Staphylococcus aureus and Escherichia coli (>99% antibacterial rates). Besides, the TZ@mMN-TENG can effectively promote cell proliferation and migration. In the diabetic rat full-thickness skin wound model infected with Staphylococcus aureus, the TZ@mMN-TENG can eliminate bacteria, accelerate epidermal growth (regenerative epidermis: ≈303.3 ± 19.1 µm), enhance collagen deposition, inhibit inflammation (lower TNF-α and IL-6 expression), and promote angiogenesis (higher CD31 and VEGF expression) to accelerate infected wound repair. Overall, the TZ@mMN-TENG provides a promising strategy for clinical application in diabetic wound repair.

Protocol to generate fast-dissociating recombinant antibody fragments for multiplexed super-resolution microscopy.

Multiplexed high-density label super-resolution microscopy image reconstruction by integrating exchangeable single-molecule localization (IRIS) enables elucidating fine structures and molecular distribution in cells and tissues. However, fast-dissociating binders are required for individual targets. Here, we present a protocol for generating antibody-based IRIS probes from existing antibody sequences. We describe steps for retrieving antibody sequences from databases. We then detail the construction, purification, and evaluation of recombinant probes after site-directed mutagenesis at the base of complementarity-determining region loops. The protocol accelerates dissociation rates without compromising the binding specificity. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2022).1.

Alginate/polyacrylamide host-guest supramolecular hydrogels with enhanced adhesion.

Although injectable hydrogels with minimally invasive delivery have garnered significant interest, their potential applications have been restricted by a singular property. In this study, a supramolecular hydrogel system with improved adhesion was constructed through host-guest interactions between alginate and polyacrylamide. The maximum tensile adhesion strength between the ß-cyclodextrin and dopamine-grafted alginate/adamantane-grafted polyacrylamide (Alg-ßCD-DA/PAAm-Ad, namely AßCDPA) hydrogels and pigskin reached 19.2 kPa, which was 76 % stronger than the non-catechol-based control hydrogel (ß-cyclodextrin-grafted alginate/adamantane-grafted polyacrylamide, Alg-ßCD/PAAm-Ad). Moreover, the hydrogels demonstrated excellent self-healing, shear-thinning, and injectable properties. The required pressure to extrude the AßCDPA2 hydrogel from a 16G needle at a rate of 2.0 mL/min was 67.4 N. As the polymer concentration and adamantane substitution degree increased, the hydrogels exhibited higher modulus, stronger network structure, and lower swelling ratio and degradation rate. Encapsulating and culturing cells within these hydrogels demonstrated good cytocompatibility. Therefore, this hydrogel can serve as a viscosity extender or bioadhesive, and as a carrier material to deliver encapsulated therapeutic substances into the body through minimally invasive injection methods.

Layer-by-Layer Microneedle-Mediated rhEGF Transdermal Delivery for Enhanced Wound Epidermal Regeneration and Angiogenesis.

Appropriate treatments for acute traumas tend to avoid hemorrhages, vascular damage, and infections. However, in the homeostasis-imbalanced wound microenvironment, currently developed therapies could not precisely and controllably deliver biomacromolecular drugs, which are confronted with challenges due to large molecular weight, poor biomembrane permeability, low dosage, rapid degradation, and bioactivity loss. To conquer this, we construct a simple and effective layer-by-layer (LBL) self-assembly transdermal delivery patch, bearing microneedles (MN) coated with recombinant human epidermal growth factor (LBL MN-rhEGF) for a sustained release to wound bed driven by typical electrostatic force. Pyramidal LBL MN-rhEGF patches hold so enough mechanical strength to penetrate the stratum corneum, and generated microchannels allow rhEGF direct delivery in situ. The administrable delivery of biomacromolecular rhEGF through hierarchically coated MN arrays follows the diffusion mechanism of Fick's second law. Numerous efforts further have illustrated that finger-pressing LBL MN-rhEGF patches could not only promote cell proliferation of normal human dermal fibroblasts (NHDF) and human umbilical vein endothelial cells (HUVEC) in vitro but also take significant effects (regenerative epidermis: ∼144 µm; pro-angiogenesis: higher CD31 expression) in accelerating wound healing of mechanically injured rats, compared to the traditional dressing, which relies on passive diffusion. Our proof-of-concept features novel LBL biomacromolecular drug-delivery systems and self-administrated precision medicine modes at the point of care.

Polypropylene composite hernia mesh with anti-adhesion layer composed of PVA hydrogel and liposomes drug delivery system.

Polypropylene (PP) mesh has been widely used in hernia repair as prosthesis material owing to its excellent balanced biocompatibility and mechanical properties. However, abdominal adhesion between the visceral and PP mesh is still a major problem. Therefore, anti-adhesive PP mesh was designed with poly(vinyl alcohol) (PVA) hydrogel and liposomes drug delivery system. First, PVA hydrogel coating was formed on the surface of PP mesh with freezing-thawing processing cycles (FTP). Subsequently, the lyophilized PVA10-c-PP was immersed in rapamycin (RPM)-loaded liposome solution until swelling equilibrated to obtain the anti-adhesion mesh RPM@LPS/PVA10-c-PP. It was demonstrated that the hydrogel coating can stably fix on the surface of PP mesh even after immersed in PBS solution at 37 °C or 40 °C for up to 30 days. In vitro cell tests revealed the excellent cytocompatibility and the potential to inhibit cell adhesion of the modified PP mesh. Moreover, the anti-adhesive effects of the RPM@LPS/PVA10-c-PP mesh was evaluated through in vivo experiments. The RPM@LPS/PVA10-c-PP mesh exhibited less adhesion than original PP mesh throughout the duration of implantation. At 30 days, the adhesion score of RPM@LPS/PVA10-c-PP mesh was 1.37 ± 0.75, however the original PP was 3 ± 0.71. Furthermore, the results of H&E and Masson trichrome staining proved that the RPM@LPS/PVA10-c-PP mesh showed slighter inflammation response and significant looser fibrous tissue surrounded the PP filaments as compared to the native PP. The current findings manifested that this type of RPM@LPS/PVA10-c-PP might be a potential candidate for anti-adhesion treatment. DATA AVAILABILITY: Data will be made available on request.

LBL Noninvasively Peelable Biointerfacial Adhesives for Cutaneo-Inspired pH/Tactility Artificial Receptors.

Besides barrier functions, skin possesses multiple sentiences to external stimuli (e.g., temperature, force, and humidity) for human-outside interaction. Thus, skincare should be taken very seriously, especially by patients with sensory disorders. However, currently available skin-mimicking devices are always limited by so much insufficient response functions and nontunable interface behaviors so as not to realize precise health monitoring and self-defense against injury. Herein, a bioinspired cutaneous receptor-perceptual system (CRPS) patch is presented, integrating hybrid pH indicators and triboelectric nanogenerators into biointerface film-adhesives that are fabricated through facile layer-by-layer (LBL) self-assembly of amide and Schiff-base linkages between alginate grafted with N-hydroxysuccinimide ester (AN), tannic acid (TA), and polyethylenimine (PEI). This CRPS patch is adhered robustly to the soft-curved skin surface without failure via "molecular suturing," and amino acid enables its benign peel-on-demand from tissue interfaces. Postdamage self-healing brings it without surgical reoperation, avoiding extra cost, pain, as well as infection risks. Significantly, CRPS patches as artificial chemo/mechanoreceptors can remotely visualize skin physiological status by pH-induced chromism using smartphones and prevent skin contact injury by tactility-driven self-powered electrical signals. Overall, the LBL-based strategy to create controllably biointerface-adhesive CRPS patches will usher in a new era of the mobihealth care platform supporting smart diagnosis and self-protection.

Biointerfacial giant capsules with high paclitaxel loading and magnetic targeting for breast tumor therapy.

High drug loading, targeted delivery, prolonged drug release, and low systemic toxicity are effective weapons for hydrophobic drug delivery systems to solve serious concerns in poor water-solubility and toxicity of paclitaxel (PTX). Herein, we reported that biointerfacial giant multilayer microcapsules (BGMs) with the feature of high-density drug loading and high-efficiency magnetic delivery were fabricated templated by PTX-liposome-microbubble complex using the layer-by-layer self-assembly (LbL) technique. The drug loading capacity of BGMs was improved by optimizing the structure of microbubbles and capsules to increase the PTX-contained layers, and the resultant BGMs exhibited high drug loading content (50.56 ± 0.09 %) and sustained drug release properties. The BGMs with an average diameter of 74.1 ± 12.1 µm and an average thickness of 275.5 ± 48.4 nm contained abundant magnetic nanoparticles (MNPs) in their cavity, which endowed these capsules with outstanding magnetic properties and fast magnetophoretic velocity in the blood (∼0.3 mm/s, ▽B = 1 T/mm). Moreover, both in vitro and in vivo studies demonstrated that the biocompatible PTX-loaded magnetic BGMs (Capsule@PLMPPL) caused notable death (71.3 ± 2.9 %) of 4 T1 breast cancer cells through PTX diffusion, capsules degradation, and subsequent endocytosis by cancer cells, and ultimately effectively inhibited tumor growth. In general, the developed BGM with good deformability and degradation was the first reported giant polyelectrolyte capsule to be used in tumor therapy, which could notably improve the therapeutic efficacy of PTX while reducing its side effects.

Effect of 3-dimensional Collagen Fibrous Scaffolds with Different Pore Sizes on Pulp Regeneration.

INTRODUCTION: In this study, we generated a 3-dimensional (3D) collagen fibrous scaffold for potential pulp regeneration and investigated the influence of various pore sizes of these scaffolds on proliferation, odontoblastic differentiation of human dental pulp cells (hDPCs), and subsequent tissue formation during pulp regeneration. METHODS: Electrospinning followed by freeze-drying was used to fabricate 3D fibrous collagen scaffolds. hDPCs were cultured on these scaffolds. Cell growth was detected by a Cell Counting Kit-8 assay and observed via scanning electron microscopy. Odontogenic genes and protein expression were analyzed by real-time reverse transcription polymerase chain reaction and immunofluorescence staining. The formation of mineralized nodules was tested by von Kossa staining, scanning electron microscopy, and energy-dispersive X-ray microanalysis. Subcutaneous transplantation of the seeded scaffold/tooth fragments into nude mice was performed to observe tissue formation for pulp regeneration. RESULTS: Collagen 3D fibrous scaffolds with 3 distinct mean pore sizes (approximately 20 µm, 65 µm, and 145 µm) were fabricated, which showed good biocompatibility and bioactivity. Scaffolds with larger mean pore sizes of 65 and 145 µm improved hDPC ingrowth and proliferation, with the 65-µm scaffold group presenting the highest level of odontogenic gene expression (DSPP and DMP-1), protein expression (DMP-1), mineralized area ratio, and vascular pulplike tissue formation after 6 weeks of subcutaneous implantation. CONCLUSIONS: The pore size of collagen 3D fibrous scaffolds significantly affected cell adhesion, proliferation, odontoblastic differentiation, and tissue rehabilitation. Scaffolds with a mean pore size of 65 µm presented superior results and could be an alternative for pulp regeneration.

Engineered fast-dissociating antibody fragments for multiplexed super-resolution microscopy.

Image reconstruction by integrating exchangeable single-molecule localization (IRIS) achieves multiplexed super-resolution imaging by high-density labeling with fast exchangeable fluorescent probes. However, previous methods to develop probes for individual targets required a great amount of time and effort. Here, we introduce a method for generating recombinant IRIS probes with a new mutagenesis strategy that can be widely applied to existing antibody sequences. Several conserved tyrosine residues at the base of complementarity-determining regions were identified as candidate sites for site-directed mutagenesis. With a high probability, mutations at candidate sites accelerated the off rate of recombinant antibody-based probes without compromising specific binding. We were able to develop IRIS probes from five monoclonal antibodies and three single-domain antibodies. We demonstrate multiplexed localization of endogenous proteins in primary neurons that visualizes small synaptic connections with high binding density. It is now practically feasible to generate fast-dissociating fluorescent probes for multitarget super-resolution imaging.

A Connexin Gene (GJB3) Mutation in a Chinese Family With Erythrokeratodermia Variabilis, Ichthyosis and Nonsyndromic Hearing Loss: Case Report and Mutations Update.

Background: Gap junctions formed by connexins are channels on cytoplasm functioning in ion recycling and homeostasis. Some members of connexin family including connexin 31 are significant components in human skin and cochlea. In clinic, mutations of connexin 31 have been revealed as the cause of a rare hereditary skin disease called erythrokeratodermia variabilis (EKV) and non-syndromic hearing loss (NSHL). Objective: To determine the underlying genetic cause of EKV, ichthyosis and NSHL in three members of a Chinese pedigree and skin histologic characteristics of the EKV patient. Methods: By performing whole exome sequencing (WES), Sanger sequencing and skin biopsy, we demonstrate a Chinese pedigree carrying a mutation of GJB3 with three patients separately diagnosed with EKV, ichthyosis and NSHL. Results: The proband, a 6-year-old Chinese girl, presented with demarcated annular red-brown plaques and hyperkeratotic scaly patches on her trunk and limbs. Her mother has ichthyosis with hyperkeratosis and geographic tongue while her younger brother had NSHL since birth. Mutation analysis revealed all of them carried a heterozygous missense mutation c.293G>A of GJB3. Skin biopsy showed many grain cells with dyskeratosis in the granular layer. Acanthosis, papillomatosis, and a mild superficial perivascular lymphocytic infiltrate were observed. Conclusion: A mutation of GJB3 associated with EKV, ichthyosis and NSHL is reported in this case. The daughter with EKV and the son with NSHL in this Chinese family inherited the mutation from their mother with ichthyosis. The variation of clinical features may involve with genetic, epigenetic and environmental factors.

A flower-like ionic molecularly imprinted membrane for the deglycosylation of rutin.

A kind of molecularly imprinted polymer based on ionic liquids (MIPIL) with flower-like shape was developed for the adsorption of rutin and its deglycosylated product. The MIPIL film was characterized by scanning electron microscope (SEM) and X-ray photo-electron spectroscopy (XPS). The adsorption capacity of quercetin on the proposed imprinted cavity of rutin in the presence of glucose and rhamnose was 3.7 ± 0.017 times as much as that in the absence of glucose and rhamnose. And the adsorption capacity of quercetin varied with the concentration of glucose and rhamnose changing. Thus, the proposed MIPIL film coupled with HPLC was used to explore the deglycosylation of rutin by tracking rutin and quercetin, which confirmed to the pseudo-first-order reaction kinetic with the constant of 0.044 ± 1.5 × 10-4 min-1 at 35 °C. The rutin and quercetin were quantified using the above MIPIL film in the two Ginkgo leaves extracted by pure water and pure ethanol, respectively. Because of lower solubility in water, the content of rutin in ethanol extraction solution was higher than in water solution. On the contrary, the content of quercetin in water extraction solution was higher than in ethanol solution, which resulted from the higher solubility of glucose and rhamnose in water. The RSD ranged from 2.8 to 4.5%, and the recovery rate ranged from 91.9 to 105.3%.

Alginate/gelatin-based hybrid hydrogels with function of injecting and encapsulating cells in situ.

Multi-network hydrogels with high strength and toughness have attracted increasing attention. Herein, a hybrid hydrogel consisting of alginate, gelatin, and polyacrylamide was constructed with the combination of advantages of natural and synthetic polymers. Alginate grafted with host-guest complex of ßCD/Ad-AAm was first prepared, namely Alg-ßCD/Ad-AAm, then further crosslink with gelatin methacryloyl (GelMA) to form hydrogel via one-step UV light initiation. The hydrogel produced by this method has more uniform and well-crosslinked networks. The hydrogels demonstrated uniform porosity, adjustable hydrophilicity (water contact angle within 32.7-91.5°), and desired mechanical properties (maximum tensile strain of 242.8%, tensile strength of 75.9 kPa, and Young's modulus of 28.5 kPa). The hydrogel also possessed self-healing ability and pH sensitivity, showing higher mechanical tensile strength at lower pH. The temperature-adjustable viscosity of pre-gel solution (sol-gel transition point of 20.4 °C) endowed it to be 3D printed as a bioink, and the printed scaffold exhibited good resilience and toughness. Moreover, HUVEC, L929, and 3T3 cells were cultured on hydrogel surfaces for 28 days and were enveloped within the hydrogels for 3D culture, indicating excellent cytocompatibility of the hydrogels. Therefore, this hybrid hydrogel system can be used potentially in cell culture scaffold and tissue engineering.

Amphiphilic Crosslinked Four-Armed Poly(lactic-co-glycolide) Electrospun Membranes for Enhancing Cell Adhesion.

Common poly(lactide-co-glycolide) (i-PLGA) has emerged as a biodegradable and biocompatible material in tissue engineering. However, the poor hydrophilicity and elasticity of i-PLGA lead to its limited application in tissue engineering. To this end, an amphiphilic crosslinked four-armed poly(lactic-co-glycolide) was prepared. First, four-armed PLGA (4A-PLGA) was synthesized by polymerizing l-lactide (LA) and glycolide (GA) with pentaerythritol as the initiator. Then, the hydrophilic polymer poly(glutamate propylene ester) (PGPE) was prepared through the esterification of glutamic acid and 1,2-propanediol. The hydrophilic 4A-PLGA-PGPE was finally synthesized through the condensation reaction of 4A-PLGA and PGPE with the aid of triphosgene. 4A-PLGA-PGPE was then used to prepare amphiphilic membranes by electrospinning. It was demonstrated that the mechanical properties and biocompatibility of 4A-PLGA were improved after the introduction of PGPE. Furthermore, the introduction of glutamate improved the hydrophilicity of 4A-PLGA, thus effectively promoting cell entry and adhesion, which makes the electrospun 4A-PLGA-PGPE membranes promising for tissue engineering.

Hydrogel adhesive formed via multiple chemical interactions: from persistent wet adhesion to rapid hemostasis.

To date, the robust and durable adhesion capability of hydrogel adhesives in wet environments remains a huge challenge. Herein, a physicochemically double-network crosslinked hydrogel matrix was prepared by mixing acrylic acid (AAc), chitosan (CS) and tannic acid (TA) as the main components and the subsequent in situ polymerization of AAc. The abundant reactive sites on the surface of the hydrogel matrix facilitate rapid, strong and repeatable adhesion to different surfaces of engineering solids and biological tissues in an aquatic environment. The formation of amide covalent bonds resulting from the addition of the bridging agent further expands the long-term application of the hydrogel in tissue repair, and the constructed hydrogel-tissue adhesive interface still has robust adhesion energy after soaking in a physiological environment for up to one month. Moreover, the hydrogel showed fantastic hemostatic performance due to its characteristics of platelet adhesion and high burst pressure. Overall, the persistent adhesion and excellent cytocompatibility of the hydrogel adhesive make it potentially applicable in medical adhesives.

Novel mutation c.1210-3C > G in cis with a poly-T tract of 5T affects CFTR mRNA splicing in a Chinese patient with cystic fibrosis.

Cystic fibrosis (CF) is a rare autosomal recessive disease with only one pathogenic gene cystic fibrosis transmembrane conductance regulator (CFTR). To identify the potential pathogenic mutations in a Chinese patient with CF, we conducted Sanger sequencing on the genomic DNA of the patient and his parents and detected all 27 coding exons of CFTR and their flanking intronic regions. The patient is a compound heterozygote of c.2909G > A, p.Gly970Asp in exon 18 and c.1210-3C > G in cis with a poly-T of 5T (T5) sequence, 3 bp upstream in intron 9. The splicing effect of c.1210-3C > G was verified via minigene assay in vitro, indicating that wild-type plasmid containing c.1210-3C together with T7 sequence produced a normal transcript and partial exon 10-skipping-transcript, whereas mutant plasmid containing c.1210-3G in cis with T5 sequence caused almost all mRNA to skip exon 10. Overall, c.1210-3C > G, the newly identified pathogenic mutation in our patient, in combination with T5 sequence in cis, affects the CFTR gene splicing and produces nearly no normal transcript in vitro. Moreover, this patient carries a p.Gly970Asp mutation, thus confirming the high-frequency of this mutation in Chinese patients with CF.

Biomechanical Motion-Activated Endogenous Wound Healing through LBL Self-Powered Nanocomposite Repairer with pH-Responsive Anti-Inflammatory Effect.

Wound care is still worthy of concern, and effective measures such as electrical stimulating therapy (EST) have sparked compellingly for wound repair. Especially, portable and point-of-care EST devices get extremely desired but these are often limited by inevitable external power sources, lack of biological functions, and mechanical properties conforming to skin tissue. Herein, a dress-on-person self-powered nanocomposite bioactive repairer of wound is designed. As such, the cooperation of the film prepared by layer-by-layer self-assembling 2-hydroxypropyltrimethyl ammonium chloride chitosan (HTCC), alginate (ALG), and poly-dopamine/Fe3+ nanoparticles (PFNs), with a self-powered nanogenerator (SN) driven by motion into a nanocomposite repairer (HAP/SN-NR) is conducted. The HAP/SN-NR not only guides cell behavior (proliferation and migration rate ≈61.7%, ≈52.3%), but also facilitates neovascularization (enhanced CD31 expression >4-fold) through its self-powered EST, and the endogenous wound closure with no inflammatory in rats owing to reactive oxygen species (ROS)-clearance of HAP/SN-NR in vitro/vivo through responsively releasing poly-dopamine nanoparticles at wound pH. Enormous efforts illustrate that the repairer is endowed with high self-adhesion to tissue, self-healing, and biodegradation, accelerating wound healing (50% closure ≈5 days). This strategy sheds light on novel multifunctional portable sensor-type dressings and propels the development of intelligent medical devices.

Molecular cloning and characterization of the novel adropin from tilapia (Oreochromis niloticus): Involvement in the control of food intake.

Adropin has been shown to be involved in the regulation of food intake in mice. However, the mechanism of adropin in feeding regulation is still largely unknown. Using the tilapia, Oreochromis niloticus, we identified and characterized a novel form of adropin (designated adropin-b) encoding a 68-amino acid precursor. Although adropin-b shared low amino acid identities with its tilapia paralog (designated adropin-a), synteny analysis proved that tilapia adropin is orthologous to its human counterpart. The transcripts of adropin-b were ubiquitously expressed in various tissues with the highest levels in the olfactory bulb. A decrease in adropin-b mRNA levels was observed 1 h following a meal in the olfactory bulb, hypothalamus, and optic tectum, whereas fasting for 7 days induced an increase in adropin-b mRNA levels in the olfactory bulb, hypothalamus, and optic tectum of tilapia brain. However, no changes in adropin-a mRNA levels were observed in the postprandial and fasting state. Intraperitoneal injection of tilapia adropin-b was shown to increase food consumption, but adropin-a did not affect feeding. Co-treatment of the fish with adropin-b and neuropeptide Y (NPY) had no additive effects on appetite. The appetite stimulatory effects of adropin-b appeared to be mediated by upregulating the orexigenic Npy, Orexin, and Proapelin gene expression, paralleled by inhibition of the mRNA levels of anorexigenic proopiomelanocortin (Pomc) and cocaine-amphetamine-regulated transcript (Cart) in vivo and in vitro. These observations suggested that adropin-b participated in appetite control and gene regulation of central orexigenic and anorexigenic factors in a fish model.

Semi-automated single-molecule microscopy screening of fast-dissociating specific antibodies directly from hybridoma cultures.

Fast-dissociating, specific antibodies are single-molecule imaging probes that transiently interact with their targets and are used in biological applications including image reconstruction by integrating exchangeable single-molecule localization (IRIS), a multiplexable super-resolution microscopy technique. Here, we introduce a semi-automated screen based on single-molecule total internal reflection fluorescence (TIRF) microscopy of antibody-antigen binding, which allows for identification of fast-dissociating monoclonal antibodies directly from thousands of hybridoma cultures. We develop monoclonal antibodies against three epitope tags (FLAG-tag, S-tag, and V5-tag) and two F-actin crosslinking proteins (plastin and espin). Specific antibodies show fast dissociation with half-lives ranging from 0.98 to 2.2 s. Unexpectedly, fast-dissociating yet specific antibodies are not so rare. A combination of fluorescently labeled Fab probes synthesized from these antibodies and light-sheet microscopy, such as dual-view inverted selective plane illumination microscopy (diSPIM), reveal rapid turnover of espin within long-lived F-actin cores of inner-ear sensory hair cell stereocilia, demonstrating that fast-dissociating specific antibodies can identify novel biological phenomena.