Tetanus Toxin Research Articles

VAMPs sensitive to tetanus toxin are required for cortical granule exocytosis in mouse oocytes

Fusion of cortical granules with oocyte plasma membrane is one of the most significant secretory events to prevent polyspermy during oocyte activation. Cortical granule exocytosis (CGE) is distinct from most other exocytosis because cortical granules are not renewed after secretion. However, it is thought to be mediated by SNARE complex, which mediates membrane fusion in other exocytoses. SNAREs proteins are divided into Q (glutamine)- and R (arginine)-SNAREs. Q-SNAREs include Syntaxins and SNAP25 family, and R-SNAREs include VAMPs family. In mouse oocytes, Syntaxin4 and SNAP23 have been involved in CGE; nevertheless, it is unknown if VAMP is required. Here, we demonstrated by RT-PCR and immunoblotting that VAMP1 and VAMP3 are expressed in mouse oocyte, and they localized in the cortical region of this cell. Using a functional assay to quantify CGE, we showed that tetanus toxin –which specifically cleavages VAMP1, VAMP2 or VAMP3– inhibited CGE suggesting that at least one VAMP was necessary. Function blocking assays demonstrated that only the microinjection of anti-VAMP1 or anti-VAMP3 antibodies abolished CGE in activated oocytes. These findings demonstrate that R-SNAREs sensitive to tetanus toxin, VAMP1 and VAMP3 –but not VAMP2-, are required for CGE and demonstrate that CGE is mediated by the SNARE complex.

Posterior subthalamic nucleus (PSTh) mediates innate fear-associated hypothermia in mice

The neural mechanisms of fear-associated thermoregulation remain unclear. Innate fear odor 2-methyl-2-thiazoline (2MT) elicits rapid hypothermia and elevated tail temperature, indicative of vasodilation-induced heat dissipation, in wild-type mice, but not in mice lacking Trpa1–the chemosensor for 2MT. Here we report that Trpa1−/− mice show diminished 2MT-evoked c-fos expression in the posterior subthalamic nucleus (PSTh), external lateral parabrachial subnucleus (PBel) and nucleus of the solitary tract (NTS). Whereas tetanus toxin light chain-mediated inactivation of NTS-projecting PSTh neurons suppress, optogenetic activation of direct PSTh-rostral NTS pathway induces hypothermia and tail vasodilation. Furthermore, selective opto-stimulation of 2MT-activated, PSTh-projecting PBel neurons by capturing activated neuronal ensembles (CANE) causes hypothermia. Conversely, chemogenetic suppression of vGlut2+ neurons in PBel or PSTh, or PSTh-projecting PBel neurons attenuates 2MT-evoked hypothermia and tail vasodilation. These studies identify PSTh as a major thermoregulatory hub that connects PBel to NTS to mediate 2MT-evoked innate fear-associated hypothermia and tail vasodilation.

Structural basis of tetanus toxin neutralization by native human monoclonal antibodies.

Four potent native human monoclonal antibodies (mAbs) targeting distinct epitopes on tetanus toxin (TeNT) are isolated with neutralization potency ranging from approximately 17mg to 6mg each that are equivalent to 250 IU of human anti-TeNT immunoglobulin. TT0170 binds fragment B, and TT0069 and TT0155 bind fragment AB. mAb TT0067 binds fragment C and blocks the binding of TeNT to gangliosides. The co-crystal structure of TT0067 with fragment C of TeNT at a 2.0-Å resolution demonstrates that mAb TT0067 directly occupies the W pocket of one of the receptor binding sites on TeNT, resulting in blocking the binding of TeNT to ganglioside on the surface of host cells. This study reveals at the atomic level the mechanism of action by the TeNT neutralizing antibody. The key neutralization epitope on the fragment C of TeNT identified in our work provides the critical information for the development of fragment C of TeNT as a better and safer tetanus vaccine.

A VLP for validation of the Plasmodium falciparum circumsporozoite protein junctional epitope for vaccine development

Malaria continues to be a pressing global health issue, causing nearly half a million deaths per year. An effective malaria vaccine could radically improve our ability to control and eliminate this pathogen. The most advanced malaria vaccine, RTS,S, confers only 30% protective efficacy under field conditions, and hence the search continues for improved vaccines. New antigens and formulations are always first developed at a pre-clinical level. This paper describes the development of a platform to supplement existing tools of pre-clinical malaria vaccine development, by displaying linear peptides on a virus-like particle (VLP). Peptides from PfCSP, particularly from outside the normal target of neutralizing antibodies, the central NANP repeat region, are screened for evidence of protective efficacy. One peptide, recently identified as a target of potent neutralizing antibodies and lying at the junction between the N-terminal domain and the central repeat region of PfCSP, is found to confer protective efficacy against malaria sporozoite challenge in mice when presented on the Qβ VLP. The platform is also used to explore the effects of increasing numbers of NANP unit repeats, and including a universal CD4+ T-cell epitope from tetanus toxin, on immunogenicity and protective efficacy. The VLP-peptide platform is shown to be of use in screening malaria peptides for protective efficacy and answering basic vaccinology questions in a pre-clinical setting.

Long-term seizure dynamics are determined by the nature of seizures and the mutual interactions between them

The seemingly random and unpredictable nature of seizures is a major debilitating factor for people with epilepsy. An increasing body of evidence demonstrates that the epileptic brain exhibits long-term fluctuations in seizure susceptibility, and seizure emergence seems to be a consequence of processes operating over multiple temporal scales. A deeper insight into the mechanisms responsible for long-term seizure fluctuations may provide important information for understanding the complex nature of seizure genesis. In this study, we explored the long-term dynamics of seizures in the tetanus toxin model of temporal lobe epilepsy. The results demonstrate the existence of long-term fluctuations in seizure probability, where seizures form clusters in time and are then followed by seizure-free periods. Within each cluster, seizure distribution is non-Poissonian, as demonstrated by the progressively increasing inter-seizure interval (ISI), which marks the approaching cluster termination. The lengthening of ISIs is paralleled by: increasing behavioral seizure severity, the occurrence of convulsive seizures, recruitment of extra-hippocampal structures and the spread of electrographic epileptiform activity outside of the limbic system. The results suggest that repeated non-convulsive seizures obey the ‘seizures-beget-seizures’ principle, leading to the occurrence of convulsive seizures, which decrease the probability of a subsequent seizure and, thus, increase the following ISI. The cumulative effect of repeated convulsive seizures leads to cluster termination, followed by a long inter-cluster period. We propose that seizures themselves are an endogenous factor that contributes to long-term fluctuations in seizure susceptibility and their mutual interaction determines the future evolution of disease activity.

Neutralization of Lethal Potency of Tetanus Toxin using Phage Display Produced scFv Antibody

Background and Aim: Phage display technology provides a new approach for making human antibody fragments that could be applicable in passive immune therapy. We applied the use of this technology to make human single-chain variable fragments (scFvs) specific for tetanus toxin. Tetanus toxin is a neurotoxin constituted by the association of two subunits, mediates its lethal action by blocking neuromuscular vesicle docking. Methods: We previously found that six Human scFv clones inhibit toxin binding to ganglioside GT1b. This is the final report of human tetanus scFvs (scFv 8 and scFv 13) isolated from an immunized library of more than 106 scFv clones with in vivo neutralizing activity. Results: Only scFv 13 can reduce the in vivo toxicity induced by tetanus toxin. Also, scFv 8 has a weak capability of reducing the in vivo toxicity of the toxin. Conclusion: These selected ScFvs can be considered as a possible option to substitute the human tetanus immunoglobulin (HTIG) which is extensively current immunotherapy for tetanus patients. Taken together, our results suggest that the use of human tetanus scFvs may lead to a less aggressive passive immune therapy against tetanus. *Corresponding Author: Mahdi Aminian; Email: amminian@tums.ac.ir Please cite this article as: Khalili E, Abbasi E, Aminian M. Neutralization of Lethal Potency of Tetanus Toxin using Phage Display Produced ScFv Antibody.Arch Med Lab Sci. 2021;7:(e3). https://doi.org/10.22037/amls.v7.33786

Recombinant Protein Stability in Cyanobacteria.

The living cell possesses extraordinary molecular and biochemical mechanisms by which to recognize and efficiently remove foreign, damaged, or denatured proteins. This essential function has been a barrier to the overexpression of recombinant proteins in most expression systems. A notable exception is the overexpression in E. coli of recombinant proteins, most of which, however, end-up as "inclusion bodies", i.e., cytoplasmic aggregates of proteins that are inaccessible to the cell's proteasome. "Fusion constructs as protein overexpression vectors" proved to be unparalleled in their ability to cause substantial accumulation of recombinant proteins from plants, animals, and bacteria, as soluble proteins in unicellular cyanobacteria. Recombinant protein levels in the range of 10-20% of the total cellular protein can be achieved. The present work investigated this unique property in the context of recombinant protein stability in Synechocystis sp. PCC 6803 by developing and applying an in vivo cellular tobacco etch virus cleavage system with the objective of separating the target heterologous proteins from their fusion leader sequences. The work provides new insights about the overexpression, cellular stability, and exploitation of transgenes with commercial interest, highly expressed in a cyanobacterial biofactory. The results support the notion that eukaryotic plant- and animal-origin recombinant proteins are unstable, when free in the cyanobacterial cytosol but stable when in a fusion configuration with a highly expressed cyanobacterial native or heterologous protein. Included in this analysis are recombinant proteins of the plant isoprenoid biosynthetic pathway (isoprene synthase, β-phellandrene synthase, geranyl diphosphate synthase), the human interferon protein, as well as prokaryotic proteins (tetanus toxin fragment C and the antibiotic resistance genes kanamycin and chloramphenicol). The future success of synthetic biology approaches with cyanobacteria and other systems would require overexpression of pathway enzymes to attain product volume, and the work reported in this paper sets the foundation for such recombinant pathway enzyme overexpression.

Crystallization of a nonreplicating rotavirus vaccine candidate.

Nonreplicating rotavirus vaccine (NRRV) candidates are being developed with the aim of serving the needs of developing countries. A significant proportion of the cost of manufacturing such vaccines is the purification in multiple chromatography steps. Crystallization has the potential to reduce purification costs and provide new product storage modality, improved operational flexibility, and reduced facility footprints. This communication describes a systematic approach for the design of the crystallization of an NRRV candidate, VP8 subunit proteins fused to the P2 epitope of tetanus toxin, using first‐principles models and preliminary experimental data. The first‐principles models are applied to literature data to obtain feasible crystallization conditions and lower bounds for nucleation and growth rates. Crystallization is then performed in a hanging‐drop vapor diffusion system, resulting in the nucleation and growth of NRRV crystals. The crystals obtained in a scaled‐up evaporative crystallization contain proteins truncated in the P2 region, but have no significant differences with the original samples in terms of antibody binding and overall conformational stability. These results demonstrate the promise of evaporative crystallization of the NRRV.

Identification of a non-coding RNA and its putative involvement in the regulation of tetanus toxin synthesis in Clostridium tetani

Clostridium tetani produces the tetanus toxin (TeNT), one of the most powerful bacterial toxins known to humankind and responsible for tetanus. The regulation of toxin expression is complex and involves the alternative sigma factor TetR as well as other regulators. Here, a transcriptional analysis of the TeNT-encoding large plasmid of C. tetani identified a putative non-coding small RNA (sRNA), located in close vicinity of the 3′ untranslated region of the tent gene. A northern blot experiment could identify a respective sRNA with a size of approx. 140 nucleotides. Sequence analysis showed that the sRNA contains a 14-nucleotide region that is complementary to a 5′ located region of tent. In order to investigate the function of the sRNA, we applied a RNA interference approach targeting the sRNA in two C. tetani wild-type strains; the constructed antisense C. tetani strains showed an approx. threefold increase in both extracellular and total TeNT production compared to the respective wild-type strains. In addition, recombinant C. tetani strains were constructed that contained tent-locus harboring plasmids with and without the sRNA. However, the introduction of the tent-locus without the sRNA in a C. tetani strain lacking the wild-type TeNT-encoding large plasmid resulted in a lower TeNT production compared to the same strain with recombinant tent-locus with the sRNA. This suggests that the expression or the effect of the sRNA is modulated by the C. tetani genetic background, notably that of the wild-type TeNT-encoding large plasmid. In addition, some recombinant strains exhibited modulated growth patterns, characterized by premature bacterial cell lysis. Taken together, our data indicate that the sRNA acts as a negative regulator of TeNT synthesis, with a possible impact on the growth of C. tetani. We hypothesize that the role of this sRNA is to limit toxin levels in the exponential growth phase in order to prevent premature bacterial lysis.

An observational study of antibody responses to a primary or subsequent pertussis booster vaccination in Australian healthcare workers

Adult pertussis vaccination is increasingly recommended to control pertussis in the community. However, there is little data on the duration and kinetics of immunity to pertussis boosters in adults. We compared IgG responses to vaccination with a tetanus, low-dose diphtheria, low-dose acellular pertussis (Tdap) booster at 1 week, 1 month and 1 year post-vaccination in whole-cell (wP)-primed Australian paediatric healthcare workers who had received an adult Tdap booster 5–12 years previously, to those who received their first Tdap booster.Tdap vaccination was well tolerated in both groups. Previously boosted adults had significantly higher pre-vaccination IgG concentrations for all vaccine-antigens, and more were seropositive for pertussis toxin (PT)-specific IgG (≥ 5 IU/mL) (69.5%; 95% confidence interval (CI) 59.5–79.5) than adults in the naïve group (45.2%; 95% CI 32.8-57.5). Tdap vaccination significantly increased IgG responses 1 month post-vaccination in both groups. This increase was more rapid in previously boosted than in naïve adults, with geometric mean fold-increases in PT-IgG at 1 week post vaccination of 3.6 (95% CI 2.9–4.3) and 2.6 (95% CI 2.2–3.2), respectively. Antibody waning between 1 month and 1 year post-vaccination was similar between groups for IgG specific to PT and filamentous haemagglutinin (FHA), but was faster for IgG against pertactin (PRN) in the naïve group (GMC ratio 0.36; 95% CI 0.31–0.42) than the previously boosted group (GMC ratio 0.45; 95% CI 0.39–0.50). At baseline, all but one adult had protective IgG titres against tetanus toxin (TT) (≥ 0.1 IU/mL), and 75.6% in the previously boosted and 61.3% in the naïve group had protective IgG titres against diphtheria toxoid (DT) of ≥ 0.1 IU/mL.This study shows that pertussis immune memory is maintained up to 12 years after Tdap vaccination in wP-primed Australian adults. There was no evidence that pertussis immune responses waned faster after a booster dose. These findings support current recommendations of repeating Tdap booster vaccination in paediatric healthcare workers at least every 10 years. Clinical trials registry: ACTRN12615001262594.

The recombinant protein combined vaccine based on the fragment C of tetanus toxin and the cross-reacting material 197.

Diphtheria and tetanus toxoids and acellular pertussis (DTaP) vaccines were widely used since 1940s. The exceptional success of childhood vaccination is undisputed. However, the anti-diphtheria and tetanus antibody will decrease with the increase of age in human body. A boosting vaccine for tetanus and diphtheria in adult is recommended by WHO. Recombinant protein vaccine has the advantages of single component and high safety, which is one of the directions to develop boosting vaccines. Therefore, in this study, we evaluated a recombinant TTc and CRM197 combination vaccine (RTCV) that uses the fragment C (TTc) of tetanus toxin and the cross-reacting material 197 (CRM197) of the diphtheria toxin mutant. Our results displayed that RTCV (composed of 10 μg/mL TTc, 20 μg/mL CRM197 antigens, and 500 μg/mL aluminum adjuvants) could induce high levels of IgG and IgG1 antibody in mice, which were similar as those induced by DTaP. These results will provide technical support for a novel boosting vaccine against diphtheria and tetanus. KEY POINTS: • We successfully expressed CRM197 protein in E. coli BL21 (DE3) using pET26b (+) vector. • The anti-TTc and anti-CRM197 antibody titer (IgG) of RTCV was similar with DTaP.

Tdap booster to adolescents with juvenile idiopathic arthritis on and off anti-TNF agents is safe and immunogenic

Introduction: The response to vaccines in juvenile idiopathic arthritis (JIA) patients on and off anti-tumor necrosis factor (anti-TNF) agents remains highly discussed. There are no published studies on the immune response following a Tdap booster dose in JIA patients so far. ObjectiveTo evaluate the immune response and safety after a Tdap booster in JIA patients and in healthy adolescents. MethodsNineteen adolescents with JIA according to the ILAR criteria on anti-TNF medication, 19 adolescents with JIA off anti-TNF medication, and 27 healthy adolescents (control group) were compared after a Tdap booster. Adverse events and disease activity were evaluated. Lymphocyte immunophenotyping was performed by flow cytometry. Tetanus, diphtheria and pertussis toxin antibodies were assessed by ELISA; whole blood was stimulated with whole-cell pertussis, and supernatants were assessed for cytokines by xMAP. ResultsThe three groups showed a similar frequency of adverse events. There was no disease reactivation after the Tdap booster. Tetanus, diphtheria and pertussis antibodies showed a significant response when D0 and D14 concentrations were compared in both JIA groups and controls. Over time, a different pattern of response to the Tdap booster was observed among the groups for tetanus antibodies (p = 0.005) but not for diphtheria and pertussis antibodies. In contrast to the protection attained for tetanus and diphtheria, in the three groups, not all individuals showed pertussis seroconversion at either D14 or D28. In addition, the seroconversion of three subjects with JIA on anti-TNF medication was not maintained at D28. JIA patients off anti-TNF showed a higher percentage of naive CD8 + T cells (p = 0.007) and central memory CD8 + cells (p = 0.003) and a lower percentage of effector CD8 + T cells (p = 0.003) and NK cell numbers (p = 0.018) than the control group. The JIA group off anti-TNF medication had fewer B lymphocytes than both the JIA group on anti-TNF medication and the control group (p = 0.016). Cellular immunity to Bordetella pertussis showed that IFNγ levels were significantly lower in both JIA groups than in the control group (p = 0.003), IL10 levels were higher in the JIA off anti-TNF group (p = 0.009), IL17A and IL5 levels were lower in the JIA on anti-TNF group than in the control group (p = 0.018 and p = 0.016, respectively); however, an increase in IFNγ (p = 0.008), IL17A (p = 0.030) and TNFα (p = 0.041) levels was observed at D14 in both patient groups. Both JIA groups showed higher levels of IL21 than the control group (p = 0.023). ConclusionWe conclude that individuals with JIA on or off anti-TNF agents showed a good response to a booster dose for the three antigens studied in the absence of major adverse events and without the reactivation of the disease.

The impact of size on particle drainage dynamics and antibody response

Vaccine-induced immune response can be greatly enhanced by mimicking pathogen properties. The size and the repetitive geometric shape of virus-like particles (VLPs) influence their immunogenicity by facilitating drainage to secondary lymphoid organs and enhancing interaction with and activation of B cells and innate humoral immune components. VLPs derived from the plant Bromovirus genus, specifically cowpea chlorotic mottle virus (CCMV), are T = 3 icosahedral particles. (T) is the triangulation number that refers to the number and arrangements of the subunits (pentamers and hexamers) of the VLPs. CCMV-VLPs can be easily expressed in an E. coli host system and package ssRNA during the expression process. Recently, we have engineered CCMV-VLPs by incorporating the universal tetanus toxin (TT) epitope at the N-terminus. The modified CCMVTT-VLPs successfully form icosahedral particles T = 3, with a diameter of ~30 nm analogous to the parental VLPs. Interestingly, incorporating TT epitope at the C-terminus of CCMVTT-VLPs results in the formation of Rod-shaped VLPs, ~1 μm in length and ~ 30 nm in width. In this study, we have investigated the draining kinetics and immunogenicity of both engineered forms (termed as Round-shaped CCMVTT-VLPs and Rod-shaped CCMVTT-VLPs) as potential B cell immunogens using different in vitro and in vivo assays. Our results reveal that Round-shaped CCMVTT-VLPs are more efficient in draining to secondary lymphoid organs to charge professional antigen-presenting cells as well as B cells. Furthermore, compared to Rod-shaped CCMVTT-VLPs, Round-shaped CCMVTT-VLPs led to more than 100-fold increased systemic IgG and IgA responses accompanied by prominent formation of splenic germinal centers. Round-shaped CCMVTT-VLPs could also polarize the induced T cell response toward Th1. To our knowledge, this is the first study investigating and comparing the draining kinetics and immunogenicity of one and the same VLP monomer forming nano-sized icosahedra or rods in the micrometer size.

Soluble \u03b1Klotho downregulates Orai1-mediated store-operated Ca2+ entry via PI3K-dependent signaling

αKlotho is a type 1 transmembrane anti-aging protein. αKlotho-deficient mice have premature aging phenotypes and an imbalance of ion homeostasis including Ca2+ and phosphate. Soluble αKlotho is known to regulate multiple ion channels and growth factor-mediated phosphoinositide-3-kinase (PI3K) signaling. Store-operated Ca2+ entry (SOCE) mediated by pore-forming subunit Orai1 and ER Ca2+ sensor STIM1 is a ubiquitous Ca2+ influx mechanism and has been implicated in multiple diseases. However, it is currently unknown whether soluble αKlotho regulates Orai1-mediated SOCE via PI3K-dependent signaling. Among the Klotho family, αKlotho downregulates SOCE while βKlotho or γKlotho does not affect SOCE. Soluble αKlotho suppresses serum-stimulated SOCE and Ca2+ release-activated Ca2+ (CRAC) channel currents. Serum increases the cell-surface abundance of Orai1 via stimulating vesicular exocytosis of the channel. The serum-stimulated SOCE and cell-surface abundance of Orai1 are inhibited by the preincubation of αKlotho protein or PI3K inhibitors. Moreover, the inhibition of SOCE and cell-surface abundance of Orai1 by pretreatment of brefeldin A or tetanus toxin or PI3K inhibitors prevents further inhibition by αKlotho. Functionally, we further show that soluble αKlotho ameliorates serum-stimulated SOCE and cell migration in breast and lung cancer cells. These results demonstrate that soluble αKlotho downregulates SOCE by inhibiting PI3K-driven vesicular exocytosis of the Orai1 channel and contributes to the suppression of SOCE-mediated tumor cell migration.

Characterization and evaluation of the enzymatic activity of tetanus toxin submitted to cobalt-60 gamma radiation.

BackgroundTetanus toxin blocks the release of the inhibitory neurotransmitters in the central nervous system and causes tetanus and its main form of prevention is through vaccination. The vaccine is produced by inactivation of tetanus toxin with formaldehyde, which may cause side effects. An alternative way is the use of ionizing radiation for inactivation of the toxin and also to improve the potential immunogenic response and to reduce the post-vaccination side effects. Therefore, the aim of this study was to characterize the tetanus toxin structure after different doses of ionizing radiation of 60Co.MethodsIrradiated and native tetanus toxin was characterized by SDS PAGE in reducing and non-reducing conditions and MALD-TOF. Enzymatic activity was measured by FRET substrate. Also, antigenic properties were assessed by ELISA and Western Blot data.ResultsCharacterization analysis revealed gradual modification on the tetanus toxin structure according to doses increase. Also, fragmentation and possible aggregations of the protein fragments were observed in higher doses. In the analysis of peptide preservation by enzymatic digestion and mass spectrometry, there was a slight modification in the identification up to the dose of 4 kGy. At subsequent doses, peptide identification was minimal. The analysis of the enzymatic activity by fluorescence showed 35 % attenuation in the activity even at higher doses. In the antigenic evaluation, anti-tetanus toxin antibodies were detected against the irradiated toxins at the different doses, with a gradual decrease as the dose increased, but remaining at satisfactory levels.ConclusionIonizing radiation promoted structural changes in the tetanus toxin such as fragmentation and/or aggregation and attenuation of enzymatic activity as the dose increased, but antigenic recognition of the toxin remained at good levels indicating its possible use as an immunogen. However, studies of enzymatic activity of tetanus toxin irradiated with doses above 8 kGy should be further analyzed.

Biological properties the novel application of N-trimethyl chitosan nanospheres as a stabilizer and preservative in tetanus vaccine.

PurposeChitosan is a natural polymer that has excellent properties include biocompatibility, biodegradability, no cytotoxicity, high charge density, low cost, mucoadhesive, permeation enhancing (ability to cross tight junction), and immunomodulating ability that makes the spectrum of its applicability much broader. This study was conducted to investigate the stabilizing, preservative and immunogenicity properties of N-trimethyl chitosan nanospheres (N-TMCNS).Materials and MethodsThe tetanus toxoid (TT) was encapsulated into N-TMCNS and then characterized by scanning electron microscope, atomic force microscope, and dynamic light scattering. For stabilizer assay of N-TMCNS after storage of TT-N-TMCNS at different temperatures for 3 weeks, they were used for immunization of mice and different temperatures groups' anti-TT-N-TMCNS production compared with other groups. Finally, the immunized mice were challenged with tetanus toxin. The preservation activity of TT-N-TMCNS against Escherichia coli was compared with thimerosal formulated TT.ResultsOur results revealed that heat-treated TT-N-TMCNS could induce higher titer of neutralizing immunoglobulin G in compared to TT vaccine and was able to protect the mice better than TT vaccine in challenge test. Furthermore, N-TMCNS as a preservative inhibited the growth of E. coli more effective than thimerosal.ConclusionOverall, the obtained results indicated that the N-TMCNS is one of the best stabilizer and preservative agent that can be used in the formulation of TT vaccine.

VAMP4 Controls Constitutive Recycling and Sorting of Post-Synaptic Receptors in Neuronal Dendrites

The endosomal recycling system dynamically tunes synaptic strength which underlies synaptic plasticity. Post-synaptic AMPA receptors exchange continuously between synaptic, extrasynaptic and intracellular compartments through diffusion in dendrites, endocytosis and recycling. Exocytosis is involved in the expression of long term potentiation (LTP), as post-synaptic expression of tetanus toxin, which cleaves the SNARE protein VAMP2, blocks LTP. Moreover, induction of LTP increases the exocytosis of transferrin receptors (TfR), markers of recycling endosomes (REs), as well as AMPARs. However, the interplay between AMPAR and TfR exocytosis in basal conditions and during LTP remains unclear. Here we identify VAMP4 as a prominent marker and the key vesicular SNARE protein that mediates most dendritic RE exocytosis. In contrast, VAMP2 labels only a small subset of REs. Knock-down (KD) of VAMP4 decreases TfR recycling but increases AMPAR recycling. In addition, VAMP4 KD increases AMPAR-mediated synaptic transmission, which consequently occludes LTP expression. The opposing changes in AMPAR and TfR recycling upon VAMP4 KD reveal their sorting into separate endosomal populations.

Functional analysis of the tyrosine phosphatase LAR in the endocytosis of the tetanus toxin

Functional analysis of the tyrosine phosphatase LAR in the endocytosis of the tetanus toxin

A Discrete Glycinergic Neuronal Population in the Ventromedial Medulla That Induces Muscle Atonia during REM Sleep and Cataplexy in Mice.

During rapid eye movement (REM) sleep, anti-gravity muscle tone and bodily movements are mostly absent, because somatic motoneurons are inhibited by descending inhibitory pathways. Recent studies showed that glycine/GABA neurons in the ventromedial medulla (VMM; GlyVMM neurons) play an important role in generating muscle atonia during REM sleep (REM-atonia). However, how these REM-atonia-inducing neurons interconnect with other neuronal populations has been unknown. In the present study, we first identified a specific subpopulation of GlyVMM neurons that play an important role in induction of REM-atonia by virus vector-mediated tracing in male mice in which glycinergic neurons expressed Cre recombinase. We found these neurons receive direct synaptic input from neurons in several brain stem regions, including glutamatergic neurons in the sublaterodorsal tegmental nucleus (SLD; GluSLD neurons). Silencing this circuit by specifically expressing tetanus toxin light chain (TeTNLC) resulted in REM sleep without atonia. This manipulation also caused a marked decrease in time spent in cataplexy-like episodes (CLEs) when applied to narcoleptic orexin-ataxin-3 mice. We also showed that GlyVMM neurons play an important role in maintenance of sleep. This present study identified a population of glycinergic neurons in the VMM that are commonly involved in REM-atonia and cataplexy.SIGNIFICANCE STATEMENT We identified a population of glycinergic neurons in the ventral medulla that plays an important role in inducing muscle atonia during rapid eye movement (REM) sleep. It sends axonal projections almost exclusively to motoneurons in the spinal cord and brain stem except to those that innervate extraocular muscles, while other glycinergic neurons in the same region also send projections to other regions including monoaminergic nuclei. Furthermore, these neurons receive direct inputs from several brainstem regions including glutamatergic neurons in the sublaterodorsal tegmental nucleus (SLD). Genetic silencing of this pathway resulted in REM sleep without atonia and a decrease of cataplexy when applied to narcoleptic mice. This work identified a neural population involved in generating muscle atonia during REM sleep and cataplexy.

Zona incerta neurons projecting to the ventral tegmental area promote action initiation towards feeding.

Key points The zona incerta (ZI) and ventral tegmental area (VTA) are brain areas that are both implicated in feeding behaviour. The ZI projects to the VTA, although it has not yet been investigated whether this projection regulates feeding.We experimentally (in)activated the ZI to VTA projection by using dual viral vector technology, and studied the effects on feeding microstructure, the willingness to work for food, general activity and body temperature.Activity of the ZI to VTA projection promotes feeding by facilitating action initiation towards food, as reflected in meal frequency and the willingness to work for food reward, without affecting general activity or directly modulating body temperature.We show for the first time that activity of the ZI to VTA projection promotes feeding, which improves the understanding of the neurobiology of feeding behaviour and body weight regulation. Both the zona incerta (ZI) and the ventral tegmental area (VTA) have been implicated in feeding behaviour. The ZI provides prominent input to the VTA, although it has not yet been investigated whether this projection regulates feeding. Therefore, we investigated the role of ZI to VTA projection neurons in the regulation of several aspects of feeding behaviour. We determined the effects of (in)activation of ZI to VTA projection neurons on feeding microstructure, food‐motivated behaviour under a progressive ratio schedule of reinforcement, locomotor activity and core body temperature. To activate or inactivate ZI neurons projecting to the VTA, we used a combination of canine adenovirus‐2 in the VTA, as well as Cre‐dependent designer receptors exclusively activated by designer drugs (DREADD) or tetanus toxin (TetTox) light chain in the ZI. TetTox‐mediated inactivation of ZI to VTA projection neurons reduced food‐motivated behaviour and feeding by reducing meal frequency. Conversely, DREADD‐mediated chemogenetic activation of ZI to VTA projection neurons promoted food‐motivated behaviour and feeding. (In)activation of ZI to VTA projection neurons did not affect locomotor activity or directly regulate core body temperature. Taken together, ZI neurons projecting to the VTA exert bidirectional control overfeeding behaviour. More specifically, activity of ZI to VTA projection neurons facilitate action initiation towards feeding, as reflected in both food‐motivated behaviour and meal initiation, without affecting general activity.