Cholera Toxin B Subunit Research Articles

Screening of a Glycopolymer Library of GM1 Mimics Containing Hydrophobic Units Using Surface Plasmon Resonance Imaging.

Effective screening methods for the development of glycopolymers as molecular recognition materials are desirable for the discovery of novel biofunctional materials. A glycopolymer library was prepared to obtain guidelines for the design of glycopolymers for the recognition of cholera toxin B subunits (CTB). Glycopolymers with varying ratios of hydrophobic and sugar units were synthesized by reversible addition fragmentation chain transfer polymerization. N-tert-Butylacrylamide, N-phenylacrylamide, and N-cyclohexylacrylamide as hydrophobic units were copolymerized in the polymer backbone, and galactose, which contributes to CTB recognition, was introduced into the side chains by “post-click” chemistry. The thiol-terminated glycopolymers were immobilized on a gold surface. The polymer immobilization substrate was analyzed in terms of interaction with galactose recognition proteins (CTB, peanut agglutinin, and Ricinus communis agglutinin I) using surface plasmon resonance imaging. The polymers with high ratios of sugar and hydrophobic units had the strongest interactions with the CTB, which was different from the trend with peanut agglutinin and Ricinus communis agglutinin I. The binding constant of the CTB with the glycopolymer with hydrophobic units was 4.1 × 106 M–1, which was approximately eight times larger than that of the polymer without hydrophobic units. A correlation was observed between the log P value and the binding constant, indicating that the hydrophobic interaction played an important role in binding. New guidelines for the design of recognition materials were obtained by our screening method.

Repeated Oral Administration of a KDEL-tagged Recombinant Cholera Toxin B Subunit Effectively Mitigates DSS Colitis Despite a Robust Immunogenic Response.

Cholera toxin B subunit (CTB), a non-toxic homopentameric component of Vibrio cholerae holotoxin, is an oral cholera vaccine antigen that induces an anti-toxin antibody response. Recently, we demonstrated that a recombinant CTB variant with a Lys-Asp-Glu-Leu (KDEL) endoplasmic reticulum retention motif (CTB-KDEL) exhibits colon mucosal healing effects that have therapeutic implications for inflammatory bowel disease (IBD). Herein, we investigated the feasibility of CTB-KDEL for the treatment of chronic colitis. We found that weekly oral administration of CTB-KDEL, dosed before or after the onset of chronic colitis, induced by repeated dextran sodium sulfate (DSS) exposure, could significantly reduce disease activity index scores, intestinal permeability, inflammation, and histological signs of chronicity. To address the consequences of immunogenicity, mice (C57BL/6 or C3H/HeJ strains) were pre-exposed to CTB-KDEL then subjected to DSS colitis and CTB-KDEL treatment. While the pre-dosing of CTB-KDEL elicited high-titer anti-drug antibodies (ADAs) of the immunoglobin A (IgA) isotype in the intestine of C57BL/6 mice, the therapeutic effects of CTB-KDEL were similar to those observed in C3H/HeJ mice, which showed minimal ADAs under the same experimental conditions. Thus, the immunogenicity of CTB-KDEL does not seem to impede the protein’s mucosal healing efficacy. These results support the development of CTB-KDEL for IBD therapy.

Needle- and Cold Chain- Free Rice-Based Oral Vaccine, MucoRice-CTB Shows Microbiota-Dependent Immunogenicity in Humans

Background: Diarrheal disease remains a global health issue. It is estimated that there are 1.3 to 4.0 million cases of cholera and 20,000 to 140,000 cholera-related deaths worldwide each year. In addition, pandemic diarrhea and travelers’ diarrhea caused by enterotoxigenic Escherichia coli heat-labile enterotoxin (LT) are estimated to affect approximately 200 million and 27 million people, respectively, worldwide each year. Our previous studies showed that rice-based cholera toxin B subunit (CTB) vaccine, MucoRice-CTB is a cold-chain and needle-syringe free oral vaccine candidate which will be benefited to less infrastructure conditions. Methods: We conducted a double-blind, randomized, placebo-controlled, three-cohort, dose-escalation phase I study to evaluate the safety, tolerability, and efficacy of MucoRice-CTB (clinical study code name: IMSUT-MR1501) administered as four oral doses at 2-week intervals to healthy Japanese adults. In addition to vaccine evaluation, metagenomic analysis of study participants was performed by using bacterial DNA from fecal samples. Interpretation: MucoRice-CTB induces cross-reactive antigen-specific antibodies against CTB and B subunit of LT in a dose-dependent manner, without inducing serious adverse events. Antibodies isolated from responders to the vaccine inhibited the binding of CT and LT to GM1 ganglioside receptor and the elongation of Chinese hamster ovary cells caused by the both enterotoxins. Metagenome analysis of subjects’ feces collected prior to the start of vaccination revealed that responders had a gut microbiota of higher diversity that was characterized by larger populations of E. coli and Shigella spp. compared with those in non-responders. Findings: This phase I clinical trial showed that MucoRice-CTB induces neutralizing antibodies against diarrheal toxins in a gut microbiota-dependent manner without major adverse events. Trial Registration: (clinical trial registration: UMI18001) Funding Statement: The costs related to this study are covered by support of the “Translational Research Network Program” of the Ministry of Education, Culture, Sports, Science and Technology. Declaration of Interests: The authors stated that: None of the items involve any conflicts of interest. Ethics Approval Statement: The clinical research protocols were approved and received ethical clearance (permit #27-41- 0917 and #27-48-0128) from the IRB of the Institute of Medical Science, The University of Tokyo (IMSUT), Japan.

Booster vaccination with a fractional dose of an oral cholera vaccine induces comparable vaccine-specific antibody avidity as a full dose: A randomised clinical trial

Antibody avidity is an important measure of the quality of vaccine-induced immune responses. Murine and human studies suggest that antibody avidity may be augmented by limiting access to antigen. The primary objective of this study was to evaluate in primed Swedish adults if booster vaccination with fractional doses (1/5th and 1/25th) of a model oral vaccine, the cholera vaccine Dukoral®, results in higher avidity antibody responses compared to boosting with a full vaccine dose. We also evaluated if fractional booster vaccination elicited similar magnitudes of antibody response compared to a full dose, and if the previously observed increase in antibody avidity after booster vaccination 1–2 years later occurred when boosting after a shorter interval.To this end, a randomised, open-label, exploratory Phase-II trial was performed. Swedish adults (n = 44), primed with two full doses of Dukoral®, were randomised into three groups and given a booster dose at either full (n = 14), 1/5th (n = 17) or 1/25th (n = 13) dose four months later. Antibody responses to cholera toxin B-subunit (CTB) were measured in serum and mucosal antibody in lymphocyte secretions (ALS).We found that the 1/5th and 1/25th booster doses had similar abilities as the full dose to induce significantly higher avidity anti-CTB antibody responses in both ALS and serum samples, as compared to after priming vaccination. There was a non-significant trend to lower magnitudes of ALS and serum IgA responses after the 1/5th compared to the full booster dose, and responses after the 1/25th dose were significantly lower.Our findings suggest fractional booster doses of Dukoral® four months after priming result in anti-toxoid mucosal antibody responses with increased antibody avidity compared to after priming vaccinations.ISRCTN registry identifier 11806026.

Development of an oral vaccine using recombinant viral haemorrhagic septicaemia virus glycoproteins produced in tobacco

The viral haemorrhagic septicaemia virus (VHSV) causes high mortality in many marine and freshwater fish species, resulting in heavy economic losses in fish farming. Previously, cholera toxin B subunit (CTB)-fused recombinant viral haemorrhagic septicaemia virus glycoproteins (rec-VHSV-GPs) have been successfully expressed in tobacco, Nicotiana benthamiana. Here, we evaluated the potential of rec-VHSV-GPs as an oral vaccine against a live viral challenge. After immunisation of mice and fish (olive flounder, Paralichthys olivaceous) with those antigenic proteins in a feed additive form, the antibody titres were increased statistically, especially in the primed groups (P < 0.0001) in both the mouse and fish. After the viral challenge under low water temperature culture conditions (below 18 °C), the immunised fish were protected successfully against the challenge, showing a significantly lower mortality rate (P < 0.05). This result suggests that this plant-based immunisation system could induce an effective immune response. It could be used as a candidate to develop an oral vaccine for fish.

Adenosine 2A receptor inhibition protects phrenic motor neurons from cell death induced by protein synthesis inhibition

Respiratory motor neuron survival is critical for maintenance of adequate ventilation and airway clearance, preventing dependence to mechanical ventilation and respiratory tract infections. Phrenic motor neurons are highly vulnerable in rodent models of motor neuron disease versus accessory inspiratory motor pools (e.g. intercostals, scalenus). Thus, strategies that promote phrenic motor neuron survival when faced with disease and/or toxic insults are needed to help preserve breathing ability, airway defense and ventilator independence. Adenosine 2A receptors (A2A) are emerging as a potential target to promote neuroprotection, although their activation can have both beneficial and pathogenic effects. Since the role of A2A receptors in the phrenic motor neuron survival/death is not known, we tested the hypothesis that A2A receptor antagonism promotes phrenic motor neuron survival and preserves diaphragm function when faced with toxic, neurodegenerative insults that lead to phrenic motor neuron death. We utilized a novel neurotoxic model of respiratory motor neuron death recently developed in our laboratory: intrapleural injections of cholera toxin B subunit (CtB) conjugated to the ribosomal toxin, saporin (CtB-Saporin). We demonstrate that intrapleural CtB-Saporin causes: 1) profound phrenic motor neuron death (~5% survival); 2) ~7-fold increase in phrenic motor neuron A2A receptor expression prior to cell death; and 3) diaphragm muscle paralysis (inactive in most rats; ~7% residual diaphragm EMG amplitude during room air breathing). The A2A receptor antagonist istradefylline given after CtB-Saporin: 1) reduced phrenic motor neuron death (~20% survival) and 2) preserved diaphragm EMG activity (~46%). Thus, A2A receptors contribute to neurotoxic phrenic motor neuron death, an effect mitigated by A2A receptor antagonism.

Immunoinformatics Approach to Design a Novel Epitope-Based Oral Vaccine Against Helicobacter pylori.

Helicobacter pylori is an infectious agent that colonizes the gastric mucosa of half of the population worldwide. This bacterium has been recognized as belonging to group 1 carcinogen by the World Health Organization for the role in development of gastritis, peptic ulcers, and cancer. Due to the increase in resistance to antibiotics used in the anti-H. pylori therapy, the development of an effective vaccine is an alternative of great interest, which remains a challenge. Therefore, a rational, strategic, and efficient vaccine design against H. pylori is necessary where the use of the most current bioinformatics tools could help achieve it. In this study, immunoinformatics approach was used to design a novel multiepitope oral vaccine against H. pylori. Our multiepitope vaccine is composed of cholera toxin subunit B (CTB) that is used as a mucosal adjuvant to enhance vaccine immunogenicity for oral immunization. CTB fused to 11 epitopes predicted of pathogenic (UreB170–189, VacA459–478, CagA1103–1122, GGT106–126, NapA30–44, and OipA211–230) and colonization (HpaA33–52, FlaA487–506, FecA437–456, BabA129–149, and SabA540–559) proteins from H. pylori. CKS9 peptide (CKSTHPLSC) targets epithelial microfold cells to enhance vaccine uptake from the gut barrier. All sequences were joined to each other by proper linkers. The vaccine was modeled and validated to achieve a high-quality three-dimensional structure. The vaccine design was evaluated as nonallergenic, antigenic, soluble, and with an appropriate molecular weight and isoelectric point. Our results suggest that our newly designed vaccine could serve as a promising anti-H. pylori vaccine candidate.

A modified cholera toxin B subunit containing an ER retention motif enhances colon epithelial repair via an unfolded protein response.

Cholera toxin B subunit (CTB) exhibits broad-spectrum biologic activity upon mucosal administration. Here, we found that a recombinant CTB containing an endoplasmic reticulum (ER) retention motif (CTB-KDEL) induces colon epithelial wound healing in colitis via the activation of an unfolded protein response (UPR) in colon epithelial cells. In a Caco2 cell wound healing model, CTB-KDEL, but not CTB or CTB-KDE, facilitated cell migration via interaction with the KDEL receptor, localization in the ER, UPR activation, and subsequent TGF-β signaling. Inhibition of the inositol-requiring enzyme 1/X-box binding protein 1 arm of UPR abolished the cell migration effect of CTB-KDEL, indicating that the pathway is indispensable for the activity. CTB-KDEL's capacity to induce UPR and epithelial restitution or wound healing was corroborated in a dextran sodium sulfate-induced acute colitis mouse model. Furthermore, CTB-KDEL induced a UPR, up-regulated wound healing pathways, and maintained viable crypts in colon explants from patients with inflammatory bowel disease (IBD). In summary, CTB-KDEL exhibits unique wound healing effects in the colon that are mediated by its localization to the ER and subsequent activation of UPR in epithelial cells. The results provide implications for a novel therapeutic approach for mucosal healing, a significant unmet need in IBD treatment.-Royal, J. M., Oh, Y. J., Grey, M. J., Lencer, W. I., Ronquillo, N., Galandiuk, S., Matoba, N. A modified cholera toxin B subunit containing an ER retention motif enhances colon epithelial repair via an unfolded protein response.

Preparation of Cholera Toxin Subunit B Functionalized Nanoparticles for Targeted Therapy of Glioblastoma.

Cholera toxin subunit B (CTB) is the nontoxic moiety of cholera toxin. It can target the glycosphingolipid GM1 expressed in the blood-brain barrier (BBB), neovasculature, and glioblastoma cells. Thus, CTB has been utilized as a multifunctional molecule for targeted therapy of glioblastoma. Here, we describe a detailed method for preparation of CTB functionalized paclitaxel (PTX)-loaded poly (lactic-co-glycolic acid) (PLGA) nanoparticles. This unique modification can guide nanoparticles across the BBB and target glioblastoma cells. The characterization of nanoparticles such as size, zeta potential, morphology, drug loading, and encapsulation efficiency is shown in this chapter.

Optimization of the method for analyzing endocytosis of fluorescently tagged molecules: Impact of incubation in the cell culture medium and cell surface wash with glycine-hydrochloric acid buffer

To obtain the therapeutic effect of biological medicines, such as proteins and nucleic acids, these medicines must achieve their intracellular target, such as the cytoplasm, and pass through biological membrane barriers. Endocytosis is an attractive route for the intracellular delivery of such drugs, and various endocytosis inhibitors have been used as tools to study the involvement of endocytosis in the cell internalization of delivery carriers. However, the specificity of these inhibitors has been insufficiently studied, and our preliminary tests could not detect the expected effect of the well-known endocytosis inhibitors. Therefore, the present study aimed to optimize the experimental conditions to precisely analyze cellular internalization via endocytosis. We first found that incubation of model molecules, such as transferrin (Tf) and cholera toxin subunit B (CTB), in cell culture medium (DMEM) could efficiently induce their internalization to HeLa cells compared to that in transport buffer (HBSS). Moreover, we clarified that cell surface wash with glycine-hydrochloric acid buffer before confocal microscopy and flow cytometry strengthened the intracellular fluorescence of Tf, CTB, and dextran tagged with fluorescent probes possibly via the neutralization of endosomal pH. Even under the optimized condition, however, the specificity of endocytosis inhibitors was disputable. The present study suggested the importance of the optimization of the study design with endocytosis inhibitors in analyzing cellular internalization.

Cholera Toxin B Subunit and Peptide LKEKK Inhibit TNF-α Signaling in Intestinal Epithelial Cells and Reduce Inflammation in a Mouse Model of Colitis

Cholera toxin B subunit (CT-B) and synthetic peptide LKEKK corresponding to the sequence 16-20 of thymosin-α1 and the sequence 131-135 of interferon-α2 (the concentration range of 100-5000 µM) significantly reduced TNF-α-stimulated pro-inflammatory cytokine expression and increases the expression of the anti-inflammatory cytokine IL-10 in human Caco-2 intestinal epithelial cells. In a mouse model of dextran sodium sulfate-induced colitis CT-B and peptide, LKEKK (20 mg/kg body weight orally for 14 days) decreased the production of TNF-α and IL-6, as well as the severity of inflammation. Thus, CT-B and peptide LKEKK are able to suppress inflammation in vitro and in vivo.

Single-Molecule Detection with Lightguiding Nanowires: Determination of Protein Concentration and Diffusivity in Supported Lipid Bilayers.

Determining the surface concentration and diffusivity of cell-membrane-bound molecules is central to the understanding of numerous important biochemical processes taking place at cell membranes. Here we use the high aspect ratio and lightguiding properties of semiconductor nanowires (NWs) to detect the presence of single freely diffusing proteins bound to a lipid bilayer covering the NW surface. Simultaneous observation of light-emission dynamics of hundreds of individual NWs occurring on the time scale of only a few seconds is interpreted using analytical models and employed to determine both surface concentration and diffusivity of cholera toxin subunit B (CTxB) bound to GM1 gangliosides in supported lipid bilayer (SLB) at surface concentrations down to below one CTxB per μm2. In particular, a decrease in diffusivity was observed with increasing GM1 content in the SLB, suggesting increasing multivalent binding of CTxB to GM1. The lightguiding capability of the NWs makes the method compatible with conventional epifluorescence microscopy, and it is shown to work well for both photostable and photosensitive dyes. These features make the concept an interesting complement to existing techniques for studying the diffusivity of low-abundance cell-membrane-bound molecules, expanding the rapidly growing use of semiconductor NWs in various bioanalytical sensor applications and live cell studies.

Innervated Properties of Acupuncture Points LI 4 and LR 3 in the Rat: Neural Pathway Tracing with Cholera Toxin Subunit B.

Objective: Increasing evidence from acupuncture research suggests that the nervous system corresponds closely with classical acupuncture points. The aim of this research was to provide neuroanatomical evidence for revealing the innervated properties of different acupuncture points through comparing the sensory and motor pathways associated with Hegu (LI 4) and Taichong (LR 3) in rat extremities. Materials and Methods: Cholera toxin subunit B (CTB) was injected into LI 4 and LR 3 in different rats, and CTB neural labeling was examined using fluorescent immunohistochemistry and observed under fluorescent microscopy in the corresponding areas from the peripheral nervous system to the central nervous system, including the dorsal root ganglia (DRG), spinal cord, and brainstem. Results: When LI 4 was injected with CTB, CTB-labeled sensory neurons ranged from C-5 to T-1 DRG, and their transganglionic axons terminated in the C-5 to C-8 spinal dorsal horn as far as the cuneate nucleus, while labeled motor neurons were located in the C-7 to T-1 spinal ventral horn. In contrast, similar neural labeling was observed for LR 3 CTB injection, with an orderly arrangement in the L-3 to L-5 DRG, L-3 to L-5 spinal dorsal horn, gracile nucleus, and L-4 to L-6 spinal ventral horn. Conclusions: The present results provide further evidence to aid understanding of the differential innervation of acupuncture points LI 4 and LR 3. This innervation establishes its connection with the nervous system in a distinct segmental and regional pattern through the spinal sensory and motor pathways.

Intranasal immunization with recombinant Trichinella spiralis serine protease elicits protective immunity in BALB/c mice

The aim of this study was to observe the intestinal mucosal/systemic responses triggered by intranasal vaccination using recombinant Trichinella spiralis serine protease (rTsSP) and its capacity to elicit immune protection against larva challenge in a murine model. rTsSP coupled with cholera toxin B subunit (CTB) was used to vaccinate mice via intranasal route. The results revealed that intranasal vaccination with rTsSP plus CTB elicited significantly intestinal local sIgA response and a TsSP-specific systemic antibody response in vaccinated mice. Furthermore, more goblet cells/acidic mucins and IgA-secreting cells were observed in jejunum from vaccinated mice. Anti-rTsSP immune serum strongly recognized the cuticle of various worm stages (muscle larva, intestinal infective larva and adult worm). The level of IFN-γ, IL-4 and IL-10 of rTsSP-vaccinated mice was significantly elevated relative to CTB and PBS control groups. The vaccinated mice exhibited a 71.10% adult reduction at 9 days pi and a 62.10% muscle larva reduction at 42 days pi following larva challenge. Additionally, vaccination with rTsSP also dampened intestinal T. spiralis development and decreased the female fecundity. Our results showed that intranasal vaccination using rTsSP adjuvanted with CTB triggered significantly local sIgA response and systemic concurrent Th1/Th2 response that induced an obvious protection against Trichinella infection.

DARPP‐32 and Cdk5 Expression in the Ventral Cervical Spinal Cord of Rats

Dopamine‐ and cAMP‐regulated phosphoprotein, 32 kDa (DARPP‐32) is a phosphatase that integrates complex neuronal information, including dopamine responsive neurons in the striatum. Differential regulation of DARPP‐32 phosphorylation is an important CNS mechanism integrating neuronal responses to concurrent activation by diverse neurochemicals, including neurotransmitters, neuromodulators and hormones. When Gs protein‐coupled metabotropic receptors activate adenylyl cyclase and increase cAMP levels, PKA activation phosphorylates DARPP‐32 at Threonine 34; in this state, DARPP‐32 inhibits protein phosphatase‐1 (PP‐1). Conversely, when DARPP‐32 is phosphorylated at Threonine 75 by Cyclin‐dependent kinase 5 (Cdk5), DARPP‐32 becomes a PKA inhibitor. Thus, DARPP‐32 is a dual‐function enzyme that inhibits PP‐1 or PKA, depending on prevailing conditions. Since PKA (Hoffman & Mitchell, Neuroscience, 2013) and PP‐1 (Huxtable et. al., J Appl. Physiol., 2018) are both key regulators of plasticity in the phrenic motor system, we performed preliminary studies to determine DARPP‐32 and Cdk5 expression in phrenic motor neurons or nearby cells. Since cervical spinal injury and/or intermittent hypoxia preconditioning trigger plasticity in the phrenic motor system, we explored if they change expression patterns of DARPP‐32 (n = 3) and/or Cdk5 (n = 15). We used immunofluorescence to detect DARPP‐32 and Cdk5 expression in the cervical spinal cords of rats that received intrapleural cholera toxin B subunit (CtB) injections to identify phrenic motor neurons. We studied intact and chronic (~8 week) C2 spinal hemisection (C2Hx) rats exposed to 4 weeks of normoxia or repetitive acute intermittent hypoxia (10, 5 min episodes of 10.5% O2; 5 min intervals; 4 days/week, 4 weeks; rAIH). Spinal cords were harvested, sectioned, and processed with antibodies for CtB and either DARPP‐32 or Cdk5 (C3‐C5; 40 μm). In all rats, DARPP‐32 was not expressed in motor nuclei, including phrenic motor neurons, or in the substantia gelatinosa of the dorsal spinal cord. Dense labeling was observed in distinct cells distributed throughout the medial grey matter; the size, shape, and distribution of these cells is consistent with tanycytes. Cdk5 expression was observed in many cell types throughout the grey matter, including phrenic motor neurons. Using a custom MATLAB algorithm, phrenic motor neuron somatic and total ventral horn expression was quantified; there were no significant effects of C2Hx or rAIH on staining intensity or distribution. In separate rats (n = 12), daily AIH for 28 days had no detectable effect on Cdk5 mRNA levels in ventral spinal cord homogenates, as assessed by qPCR (p=0.116). Thus, DARPP‐32 is not expressed in motor nuclei of the mid‐cervical spinal cord, making a role in phrenic motor plasticity unlikely; the role of DARPP‐32 in putative spinal tanycytes is unknown. Conversely, Cdk5 expression in phrenic motor neurons suggests potential for roles in plasticity or other motor neuron functions.Support or Funding InformationSupported by: University of Florida Graduate School Preeminence Award (MNK), NIH T32 HD043730 (LLA), NIH OT2OD023854 (SPARC; GSM), NIH R01 69064 (GSM), and the McKnight Brain Institute.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Electroacupuncture decreases Netrin-1-induced myelinated afferent fiber sprouting and neuropathic pain through μ-opioid receptors.

Purpose: We determined whether electroacupuncture (EA) reduces Netrin-1-induced myelinated primary afferent nerve fiber sprouting in the spinal cord and pain hypersensitivity associated with postherpetic neuralgia (PHN) through activation of μ-opioid receptors.Methods: PHN was induced by systemic injection of resiniferatoxin (RTX) in rats. Thirty-six days after RTX injection, a μ-opioid receptor antagonist, beta-funaltrexamine (β-FNA) or a κ-opioid receptor antagonist, nor Binaltorphimine (nor-BNI), was injected intrathecally 30 mins before EA, once every other day for 4 times. Mechanical allodynia was tested with von Frey filaments. The protein expression level of Netrin-1 and its receptors (DCC and UNC5H2) were quantified by using western blotting. The myelinated primary afferent nerve fiber sprouting was mapped with the transganglionic tracer cholera toxin B-subunit (CTB).Results: Treatment with 2 Hz EA at “Huantiao” (GB30) and “Yanglingquan” (GB34) decreased the mechanical allodynia at 22 days and the myelinated primary afferent nerve fiber preternatural sprouting into the lamina II of the spinal dorsal horn at 42 days after RTX injection. Also, treatment with 2 Hz EA reduced the protein levels of DCC and Netrin-1 and promoted the expression of UNC5H2 in the spinal dorsal horn 42 days after RTX injection. Furthermore, the μ-opioid receptor antagonist β-FNA, but not the κ-opioid receptor antagonist nor-BNI, reversed the effect of EA on neuropathic pain caused by RTX. In addition, morphine inhibited the Netrin-1 protein level induced by RTX in SH-SY5Y cells.Conclusions: Through activation of μ-opioid receptors, treatment with EA reduces the expression level of DCC and Netrin-1 and changes a growth-permissive environment in spinal dorsal horn into an inhibitory environment by increasing UNC5H2, thus decreasing RTX-caused primary afferent nerve sprouting in the spinal dorsal horn and neuropathic pain.

Oxytocinergic Neurons in the Hypothalamic Paraventricular Nucleus Stimulate Brown Adipose Tissue Thermogenesis through Rostral Medullary Raphe

Hypothalamic neurons in the paraventricular nucleus (PVN) produce several neuropeptides, including oxytocin and vasopressin, and send their axons to various brain regions. These PVN neurons have been considered to serve neuroendocrine and autonomic functions, and many studies have demonstrated the involvement of oxytocin in social and emotional behaviors (e.g. maternal and sexual behaviors, and stress responses). Although these behaviors are accompanied with autonomic responses, it is unclear how oxytocin modulates the activities of the autonomic nervous system. To investigate the roles of PVN oxytocinergic neurons in the central regulation of autonomic function, in this study, we first performed neural tract tracing in rats from PVN oxytocinergic neurons to the rostral medullary raphe region (rMR), which contains sympathetic premotor neurons controlling brown adipose tissue (BAT) thermogenesis. In the rMR, oxytocin‐immunoreactive fibers, but not vasopressin‐immunoreactive fibers, were distributed, and oxytocin‐containing terminals were apposed to tryptophan hydroxylase‐expressing (i.e., serotonergic) neurons, potentially BAT sympathetic premotor neurons. Retrograde tracing from the rMR with cholera toxin b‐subunit (CTb) labeled a portion of oxytocinergic neurons in the caudal PVN, but not in the supraoptic nucleus, suggesting that the PVN is a major source of oxytocinergic fibers in the rMR. Then, we performed in vivo BAT sympathetic nerve recording combining optogenetic stimulation of the PVN‐derived oxytocinergic axon terminals in the rMR. Recombinant adeno‐associated viral vectors were injected into the PVN to express the engineered channel rhodopsin variant, ChIEF, under the oxytocin promoter. We confirmed that most of PVN neurons expressing ChIEF‐mCherry contained oxytocin and that their axon fibers were densely distributed in the rMR. Optogenetic stimulation of these fibers evoked BAT sympathetic nerve activities as well as increased expired CO2 and heart rate. Similar thermogenic and cardiac responses were elicited by nanoinjection of oxytocin into the rMR, and these responses were blocked by prior injection of the oxytocin receptor antagonist, L‐368,899 into the rMR. In addition, oxytocin injection into the rMR also potentiated NMDA‐evoked BAT sympathetic nerve activities. These findings indicate that PVN oxytocinergic neurons stimulate thermogenic sympathetic outflow to BAT through activation of rMR neurons. This oxytocinergic pathway may play an important role in emotion‐related autonomic responses.Support or Funding InformationThis work was supported by JSPS KAKENHI Grant Numbers JP16H05128, JP15H05932, JP26118508 (KN) and by AMED under Grant Number JP18gm5010002 (KN)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Towards the first multi-epitope recombinant vaccine against Crimean-Congo hemorrhagic fever virus: A computer-aided vaccine design approach.

Crimean-Congo hemorrhagic fever (CCHF) is considered one of the major public health concerns with case fatality rates of up to 80%. Currently, there is no effective approved vaccine for CCHF. In this study, we used a computer-aided vaccine design approach to develop the first multi-epitope recombinant vaccine for CCHF. For this purpose, linear B-cell and T-cell binding epitopes from two structural glycoproteins of CCHF virus including Gc and Gn were predicted. The epitopes were further studied regarding their antigenicity, allergenicity, hydrophobicity, stability, toxicity and population coverage. A total number of seven epitopes including five T-cell and two B-cell epitopes were screened for the final vaccine construct. Final vaccine construct composed of 382 amino acid residues which were organized in four domains including linear B-cell, T-cell epitopes and cholera toxin B-subunit (CTxB) along with heat labile enterotoxin IIc B subunit (LT-IIc) as adjuvants. All the segments were joined using appropriate linkers. The physicochemical properties as well as the presence of IFN-γ inducing epitopes in the proposed vaccine, was also checked to determining the vaccine stability, solubility and its ability to induce cell-mediated immune responses. The 3D structure of proposed vaccine was subjected to the prediction of computational B-cell epitopes and molecular docking studies with MHC-I and II molecules. Furthermore, molecular dynamics stimulations were performed to study the vaccine-MHCs complexes stability during stimulation time. The results suggest that our proposed vaccine was stable, well soluble in water and potentially antigenic. Results also demonstrated that the vaccine can induce both humoral and cell-mediated immune responses and could serve as a promising anti-CCHF vaccine candidate.

Chemical Bioconjugation of Proteins in an Undergraduate Lab: One-Pot Oxidation and Derivatization of the N-Terminus

A laboratory experiment introducing the concept of chemical bioconjugation of proteins to undergraduate students in a therapeutically relevant context was developed. Initially, students installed an aldehyde functionality into a protein via the oxidation of the N-terminal threonine residue of the cholera toxin subunit B (CTB) protein, which was followed by subsequent modification via hydrazone addition under mild conditions with a chromophore bearing a distinct UV–vis-absorption peak. Students determined the yield of the reaction to be ca. 11% by HPLC coupled to UV–vis spectroscopy and developed key skills such as preparation of stock solutions, chemical manipulation of proteins, and analysis via HPLC. The reported experiment can be readily adapted for use with other proteins and may contribute to enhancing constructive alignment in interdisciplinary degree programs at the chemistry–biology interface.

Effects of a 15-amino-acid isoform of amyloid- β expressed by silkworm pupae on B6C3-Tg Alzheimer’s disease transgenic mice

Silkworms are an economically important insect.Silkworm pupae are also a nutrient-rich food and can be used as a pharmaceutical intermediate.The N-terminus of Aβ includes 1–15 amino acid residues with a B cell surface antigen that is necessary to produce antibody and prevent the adverse reactions observed in response to the full Aβ42 peptide. In this study, we used silkworm pupae to develop a safer vaccine for Alzheimer’s disease (AD) patients. Aβ15 peptide was fused with the cholera toxin B subunit (CTB) and expressed in silkworm pupae. Then, we tested an oral vaccine with the peptide expressed by silkworm pupae in a transgenic mouse model of AD. The results show that anti-Aβ antibodies were induced, Aβ deposition in the brain decreased, the content of malondialdehyde was lower than in the other group, and memory and cognition of the mice improved. These results suggest that the high-nutrient CTB-Aβ15 silkworm pupa vaccine has a potential clinical application for the prevention of AD.