Cholera Toxin Research Articles

Environmental Vibrio cholerae Strains Harboring Cholera Toxin and Vibrio Pathogenicity Island 1, Nigeria, 2008-2015.

Analysis of clinical and environmental Vibrio cholerae O1 strains obtained during 2008-2015 in Nigeria showed that lineages Afr9 and Afr12 carrying cholera toxin and Vibrio pathogenicity island 1 can be isolated from water. Our findings raise concerns about the role of the environment in maintenance and emergence of cholera outbreaks in Nigeria.

Fucosylation of glycoproteins and glycolipids: opposing roles in cholera intoxication.

Cholera toxin (CT) is the etiological agent of cholera. Here we report that multiple classes of fucosylated glycoconjugates function in CT binding and intoxication of intestinal epithelial cells. In Colo205 cells, knockout (KO) of B3GNT5, which encodes an enzyme required for synthesis of lacto and neolacto series glycosphingolipids (GSLs), reduces CT binding but sensitizes cells to intoxication. Overexpressing B3GNT5 to generate more fucosylated GSLs confers protection against intoxication, indicating that fucosylated GSLs act as decoy receptors for CT. KO of B3GALT5 causes increased production of fucosylated O-linked and N-linked glycoproteins and leads to increased CT binding and intoxication. KO of B3GNT5 in B3GALT5-KO cells eliminates production of fucosylated GSLs but increases intoxication, identifying fucosylated glycoproteins as functional receptors for CT. These findings provide insight into the molecular determinants regulating CT sensitivity of host cells.

Vibrio cholerae virulence is blocked by chitosan oligosaccharide-mediated inhibition of ChsR activity.

Vibrio cholerae causes cholera, an important cause of death worldwide. A fuller understanding of how virulence is regulated offers the potential for developing virulence inhibitors, regarded as efficient therapeutic alternatives for cholera treatment. Here we show using competitive infections of wild-type and mutant bacteria that the regulator of chitosan utilization, ChsR, increases V. cholerae virulence in vivo. Mechanistically, RNA sequencing, chromatin immunoprecipitation with sequencing and molecular biology approaches revealed that ChsR directly upregulated the expression of the virulence regulator, TcpP, which promoted expression of the cholera toxin and the toxin co-regulated pilus, in response to low O2 levels in the small intestine. We also found that chitosan degradation products inhibit the ChsR-tcpP promoter interaction. Consistently, administration of chitosan oligosaccharide, particularly when delivered via sodium alginate microsphere carriers, reduced V. cholerae intestinal colonization and disease severity in mice by blocking the chsR-mediated pathway. These data reveal the potential of chitosan oligosaccharide as supplemental therapy for cholera treatment and prevention.

Dnajc3 (HSP40) Modulates Axon Regeneration in the Mouse Optic Nerve.

The present study is designed to identify the genes modulating optic nerve regeneration in the mouse. Using the BXD mouse strains as a genetic mapping panel, we examined differential responses to axon regeneration in order to map genomic loci modulating axonal regeneration. To study regeneration in the optic nerve, Pten was knocked down in the retinal ganglion cells using adeno-associated virus (AAV) delivery of an shRNA, followed by the induction of a mild inflammatory response by an intravitreal injection of Zymosan with CPT-cAMP. The axons of the retinal ganglion cells were damaged by optic nerve crush (ONC). Following a 12-day survival period, regenerating axons were labeled by Cholera Toxin B. Two days later, the regenerating axons within the optic nerve were examined to determine the number of regenerating axons and the distance traveled down the optic nerve. An integral genomic map was made using the regenerative response. Candidate genes were tested by knocking down expression using shRNA or by overexpressing the gene in AAV vectors. The analysis revealed a considerable amount of differential axonal regeneration across all 33 BXD strains, demonstrated by the number of axons regenerating and the length of the regenerating axons. Some strains (BXD99, BXD90, and BXD29) demonstrated significant axonal regeneration; while other strains (BXD13, BXD18, and BXD34) had very little axon regrowth. Within the regenerative data, there was a 4-fold increase in distance regenerated and a 7.5-fold difference in the number of regenerating axons. These data were used to map a quantitative trait locus modulating axonal regeneration to Chromosome 14 (115 to 119 Mb). Within this locus were 16 annotated genes. Subsequent testing revealed that one candidate gene, Dnajc3 , modulates axonal regeneration. Knocking down of Dnajc3 led to a decreased regeneration response in the high regenerative strains (BXD90), while overexpression of Dnajc3 resulted in an increased regeneration response in C57BL/6J and a low regenerative strain (BXD34). In this study, Dnajc3 (encodes Heat Shock Protein 40, HSP40, a molecular chaperone) was identified as a modulator of axon regeneration in mice. This is the first report defining the role of Dnajc3 (HSP40) in axon regeneration.

Challenges and Considerations in Selecting Animal Models for Evaluating a Live, Cold-Chain-Free, Dual-Use Vaccine (MyChol) for Diarrhoeal Diseases: A Comprehensive Review.

Diarrhoeal diseases are the second leading cause of death for children under 5 years old in 69 low- and middle-income countries, with an annual economic burden of US$ 4 billion and over 525,000 lives lost. Cholera and enterotoxigenic Escherichia coli (ETEC) traveller's diarrhoea are major diarrhoeal diseases caused by Vibrio cholerae (O1 and O139 serogroups) and ETEC, which have similar pathogeneses and can co-infect. There is no exclusive vaccine for ETEC, but cholera vaccines containing the cholera toxin B (CT-B) component offer short-term cross-protection. However, licensed oral cholera vaccines are expensive due to cold-chain supplies and the need for multiple doses. A cost-effective, dual-protection, live, cold-chain-free vaccine is, therefore, required for vaccination campaigns in low-resource settings, and MyChol - a prototype cold-chain-free live attenuated cholera vaccine, targeting V. cholerae O139 and ETEC H10407 - was developed in this context. The vaccine was evaluated in three animal models (Sprague Dawley [SD] rats, BALB/c mice and New Zealand white rabbits) for safety, colonisation capacity, reactogenicity and immunogenicity against challenge strains. In suckling mice, MyChol displayed high colonisation potential compared to unformulated VCUSM14P (the vaccine candidate) and wild-type O139. In the acute toxicity assessment, the SD rats with the highest MyChol dose (1 × 107 colony-forming unit [CFU]/kg) demonstrated no adverse effects or mortality. Mice vaccinated with MyChol exhibited elevated antibody levels, including anti-CT, anti-heat-labile enterotoxin (LT), anti-CT-B and anti-LT-B. Anti-CT antibodies neutralised LT toxin in ETEC H10407 in challenge studies and cross-protected against ETEC H10407 in both mice and rabbits, preventing weight loss and diarrhoea. Ileal loop experiments in rabbits and BALB/c mice showed no reactogenicity. This review, based on our previous research, therefore provides valuable insights into improving the selection of animal models to advance preclinical evaluations of diarrhoeal vaccines.

Chronic hyperglycemia alters retinal astrocyte microstructure and uptake of cholera toxin B in a murine model of diabetes.

Astrocytes are the principle glial cells of the central nervous system and play an active role in maintaining proper metabolism in surrounding neurons. Because of their involvement in metabolic control, it is likely that their physiology changes in response to metabolic diseases such as diabetes and associated diabetic retinopathy. Here, we investigated whether microstructural changes in astrocyte morphology occur during the early stages of chronic hyperglycemia that may be indicative of early pathogenic programs. We used MORF3 mice in conjunction with streptozotocin-induced hyperglycemia to investigate the morphology of single retinal astrocytes at an early timepoint in diabetic disease. We report that astrocytes initiate a morphological remodeling program, which depends on both the glycemic background and the presence of intravitreal injury, to alter the amount of the neuronal-associated pad and bristle microstructural motifs. Additionally, hyperglycemia increases astrocyte uptake of cholera toxin B, possibly reflecting changes in glycolipid and glycoprotein biosynthesis. Chronic hyperglycemia coupled with intravitreal injection of cholera toxin B also causes extensive leukocyte infiltration into the retina. Our results have important clinical relevance as current therapies for diabetic retinopathy involve intravitreal injection of pharmaceuticals in individuals with often poorly controlled blood glucose levels.

MakC and MakD are two proteins associated with a tripartite toxin of Vibrio cholerae.

Pathogenic serotypes of Vibrio cholerae, transmitted through contaminated water and food, are responsible for outbreaks of cholera, an acute diarrheal disease. While the cholera toxin is the primary virulence factor, V. cholerae also expresses other virulence factors, such as the tripartite toxin MakABE that is secreted via the bacterial flagellum. These three proteins are co-expressed with two accessory proteins, MakC and MakD, whose functions remain unknown. Here, we present the crystal structures of MakC and MakD, revealing that they are similar in both sequence and structure but lack other close structural relatives. Our study further investigates the roles of MakC and MakD, focusing on their impact on the expression and secretion of the components of the MakABE tripartite toxin. Through deletion mutant analysis, we found that individual deletions of makC or makD do not significantly affect MakA expression or secretion. However, the deletion of both makC and makD impairs the expression of MakB, which is directly downstream, and decreases the expression of MakE, which is separated from makCD by two genes. Conversely, MakA, encoded by the makA gene located between makB and makE, is expressed normally but its secretion is impaired. Additionally, our findings indicate that MakC, in contrast to MakD, exhibits strong interactions with other proteins. Furthermore, both MakC and MakD were observed to be localized within the cytosol of the bacterial cell. This study provides new insights into the regulatory mechanisms affecting the Mak protein family in V. cholerae and highlights the complex interplay between gene proximity and protein expression.

Wastewater-based surveillance of Vibrio cholerae: Molecular insights on biofilm regulatory diguanylate cyclases, virulence factors and antibiotic resistance patterns

Vibrio cholerae is an inherent inhabitant of aquatic ecosystems. The Indian state of West Bengal, especially the Gangetic delta region is the highest cholera affected region and is considered as the hub of Asiatic cholera. V. cholerae were isolated from publicly accessible wastewater of Midnapore, West Bengal, India. Serotyping determined all isolates to be of non-O1/non-O139 serogroups. Moderate biofilm-forming abilities were noticed in most of the isolates (74.7 %) while, high biofilm formation was recorded for only 6.3 % isolates and 19 % of isolates exhibited low/non-biofilm-forming abilities. PCR-based screening of crucial diguanylate cyclases (DGCs) involved in cyclic-di-GMP-mediated biofilm signaling was performed. cdgH and cdgM were the most abundant DGCs among 93.7 % and 91.5 % of isolates, respectively. Other important DGCs, i.e., cdgK, cdgA, cdgL, and vpvC were present in 84 %, 75.5 %, 72 % and 68 % of isolates, respectively. Besides, the non-O1/non-O139 isolates were screened for the occurrence of virulence factor encoding genes. Moreover, among these non-O1/non-O139 isolates, two strains (3.17 %) harbored both ctxA and ctxB genes, which encode the cholera toxin associated with epidemic cholera. ompU was the most prevalent virulence factor, present in 24.8 % of isolates. Other virulence factors like, zot and st were found in 4.7 % and 9.5 % of isolates. Genes encoding tcp and ace were found to be PCR-negative for the isolates. Additionally, crucial virulence factor regulators, toxT, toxR and hapR were found to be PCR-positive in all the isolates. Antibiotic resistance patterns displayed further vulnerabilities with decreased sensitivity towards commonly used antibiotics with multiple antibiotic resistance index ranging between 0.37 and 0.62. The presence of cholera toxin-encoding multi-drug resistant (MDR) V. cholerae strains in environmental settings is alarming. High occurrence of DGCs are considered to encourage further investigations to use them as alternative therapeutic targets against MDR cholera pathogen due to their unique presence in bacterial systems.

Utilizing Adenovirus Knob Proteins as Carriers in Cancer Gene Therapy Amidst the Presence of Anti-Knob Antibodies.

Numerous gene therapy drugs for cancer have received global approval, yet their efficacy against solid tumors remains inadequate. Our previous research indicated that the fiber protein, a component of the adenovirus capsid, can propagate from infected cells to neighboring cells that express the adenovirus receptor. We hypothesize that merging this fiber protein with an anti-cancer protein could enable the anti-cancer protein to disseminate around the transfected cells, presenting a novel approach to cancer gene therapy. In our study, we discovered that the knob region of the adenovirus type 5 fiber protein is the smallest unit capable of spreading to adjacent cells in a receptor-specific manner. We also showed that the recombinant knob protein infiltrates cells after dispersing to surrounding cells. To assess the potential of the knob protein to augment gene therapy for solid tumors in mice, we expressed a fusion gene of the A subunit of cytotoxic cholera toxin and the knob region in mouse tumors. We found that this fusion protein only inhibited tumor growth in receptor-expressing mouse melanomas, and this inhibitory effect persisted even in mice with anti-knob antibodies. Our study's findings propose a novel cancer gene therapy strategy that enhances therapeutic effects by specifically delivering therapeutic proteins, expressed from in vivo administered genes, to target molecules. This outcome offers a fresh perspective on gene therapy for solid cancers, and we anticipate that knob proteins will serve as a platform for this method.

Application of immunoinformatics to develop a novel and effective multiepitope chimeric vaccine against Variovorax durovernensis

Bloodstream infections pose a significant public health challenge caused by resistant bacteria such as Variovorax durovernensis, a recently reported Gram-negative bacterium, worsening the burden on healthcare systems. The design of a vaccine using chimeric peptides derived from a representative V. durovernensis strain holds significant promise for preventing disease onset. The current study aimed to employ reverse vaccinology (RV) approaches such as the retrieval of V. durovernensis proteomics data, removal of redundant proteins by CD-HIT, filtering of non-homologous proteins to humans and essential proteins, identification of outer membrane (OM) proteins by CELLO and PSORTb. Following these steps immunoinformatic approaches were applied, such as epitope prediction by IEDB, vaccine design using linkers and adjuvant andanalysis of antigenicity, allergenicity, safety and stability. Among the 4208 nonredundant proteins, an OmpA family protein (A0A940EKP4) was designated a potential candidate for the development of a multiepitope vaccine construct. Upon analysis of OM protein, six immunodominant (B cell) epitopes were found on the basis of the chimeric construct following the prediction of CTL stands cytotoxic T lymphocyte and HTL stands helper T lymphocyte epitopes. To ensure comprehensive population coverage globally, the CTL and HTL coverage rates were 58.18% and 46.56%, respectively, and 77.23% overall. By utilizing EAAAK, GPGPG, and AAY linkers, Cholera toxin B subunit adjuvants, and appropriate epitopes were smoothly incorporated into a chimeric vaccine effectively triggering both adaptive and innate immune responses. For example, the administered antigen showed a peak in counts on the fifthday post injection and then gradually declined until the fifteenth day. Elevated levels of several antibodies (IgG + IgM > 700,000; IgM > 600,000; IgG1 + IgG2; IgG1 > 500,000) were observed as decreased in the antigen concentration. Molecular dynamics simulations carried out via iMODS revealed strong correlations between residue pairs, highlighting the stability of the docked complex. The designed vaccine has promising potential in eliciting specific immunogenic responses, thereby facilitating future research for vaccine development against V. durovernensis.

LF-DBS of the Ventral Striatum Shortens Persistence for Morphine Place Preference and Modulates BDNF Expression in the Hippocampus

BackgroundDeep brain stimulation (DBS) of the ventral capsule/ventral striatum (VC/VS) represents a promising therapy for treatment-refractory patients with substance-use disorders. We previously found that low-frequency (LF) DBS aimed to the VC/VS during extinction training strengthens the extinction memory for morphine seeking under a partial extinction protocol. Objectives/HypothesisIn this study, animals were tested in a full extinction protocol to determine whether LF-DBS applied during extinction facilitates extinction while preventing drug reinstatement, and study the molecular mechanisms underlying the effects of LF-DBS, Methods/ResultsWe used a full extinction CPP paradigm combined with LF-DBS to assess behavior. Western blots for the pro-extinction molecule, brain-derived neurotrophic factor (BDNF) were then performed in corticomesolimbic regions of the brain. Lastly, to determine whether changes in BDNF expression elicited by LF-DBS were specific to the VS/NAc afferents from the hippocampus, amygdala, and medial prefrontal cortex, we performed BDNF-like immunohistochemistry, combined with the retrograde tracer cholera toxin B (CtB). ResultsWe showed a significant reduction in the number of days required to fully extinguish morphine CPP in animals exposed to LF-DBS during extinction training accompanied by a significant increase in BDNF expression in the hippocampus. However, LF-DBS applied during extinction did not prevent drug reinstatement. Lastly, no changes in BDNF/CtB double-labeled cells were found in VS/NAc projecting cells after one-day exposure to LF-DBS. Conclusion(s)These data suggest that LF-DBS can facilitate extinction of morphine CPP by decreasing drug seeking through potential synaptic plasticity changes in the hippocampus to strengthen extinction memories.

Orbitofrontal cortex to dorsal striatum circuit is critical for incubation of oxycodone craving after forced abstinence.

Relapse is a major challenge in treating opioid addiction, including oxycodone. During abstinence, oxycodone seeking progressively increases, a phenomenon termed incubation of oxycodone craving. We previously demonstrated a causal role of orbitofrontal cortex (OFC) in this incubation. Here, we studied the interaction between glutamatergic projections from OFC and dopamine 1-family receptor (D1R) signaling in dorsal striatum (DS) in this incubation in male rats. We first examined the causal role of D1R signalling in DS in incubated oxycodone seeking. Next, we combined fluorescence-conjugated cholera toxin subunit B (CTb-555, a retrograde tracer) with Fos (a neuronal activity marker) to assess whether the activation of OFC→DS projections was associated with incubated oxycodone seeking. We then used a pharmacological asymmetrical disconnection procedure to examine the role of the interaction between projections from OFC and D1R signalling in DS in incubated oxycodone seeking. We also tested the effect of unilateral pharmacological inactivation of OFC or unilateral D1R blockade of DS on incubated oxycodone seeking. Finally, we assessed whether contralateral disconnection of OFC→DS projections impacted non-incubated oxycodone seeking on abstinence day 1. We found that D1R blockade in DS decreased incubated oxycodone seeking and OFC→DS projections were activated during incubated oxycodone seeking. Moreover, anatomical disconnection of OFC→DS projections, but not unilateral inactivation of OFC or unilateral D1R blockade in DS, decreased incubated oxycodone seeking. Lastly, contralateral disconnection of OFC→DS projections had no effect on oxycodone seeking on abstinence day 1. Together, these results demonstrated a causal role of OFC→DS projections in incubation of oxycodone craving.

Hypothalamic cerebrospinal fluid-contacting neurons project to the rostral agranular insular cortex: An immunofluorescence and ultrastructural analysis in the rat.

Cerebrospinal fluid-contacting neurons (CSF-cNS) are considered mechanoreceptors and chemoreceptors involved in detecting changes in CSF circulation. However, considering that recent data suggest that this type of cell could exert an active response when an external stimulus is sensed, identification of CSF-cNS may be relevant. In this regard, some data suggest that a neuronal connection exists between the ventral region of the hypothalamic paraventricular nucleus (PVN) and rostral agranular insular cortex (RAIC); indeed, a potential CSF-cNS is hypothesized. However, a detailed analysis of this connection has not been conducted. Thus, using neuronal tracers (Fluoro-Gold® (FG) and cholera toxin (ChT)) coupled with transmission electron microscopy and immunofluorescence assays against Fluoro-Gold®, oxytocin (OXT), vasopressin (AVP) and oxytocin receptors (OTR), we describe an oxytocinergic or vasopressinergic CSF-cNS between the PVN and RAIC. Our results showed that CSF-cNS along the PVN labelled with oxytocin and/or AVP were present in dendritic projections near the third ventricle. This CSF-cNS in the PVN seems to project to the RAIC. Inside the RAIC, ultrastructural analysis showed that axons immunopositive for oxytocin from the PVN sustained synaptic connections with neurons that expressed OTR. These findings show that the CSF-cNS from the PVN sends projections to the RAIC. To the best of our knowledge, the relevance of CSF-cNS has not been elucidated; however, we hypothesized that the activation of cells could concomitantly release neuropeptides (i.e., oxytocin and AVP) in the CSF and RAIC. Thus, further analysis of the impact of neuropeptides released into the third ventricle and RAIC is warranted.

Diversity and Complexity of CTXΦ and Pre-CTXΦ Families in Vibrio cholerae from Seventh Pandemic.

CTXΦ is a lysogenic filamentous phage that carries the genes encoding cholera toxin (ctxAB), the main virulence factor of Vibrio cholerae. The toxigenic conversion of environmental V. cholerae strains through CTXΦ lysogenic infection is crucial for the emergence of new pathogenic clones. A special allelic form of CTXΦ, called pre-CTXΦ, is a precursor of CTXΦ and without ctxAB. Different members of the pre-CTXΦ and CTXΦ families are distinguished by the sequence of the transcriptional repressor-coding gene rstR. Multiple rstR alleles can coexist within a single strain, demonstrating the diverse structure and complex genomic integration patterns of CTXΦ/pre-CTXΦ prophage on the chromosome. Exploration of the diversity and co-integration patterns of CTXΦ/pre-CTXΦ prophages in V. cholerae can help to understand the evolution of this phage family. In this study, 21 V. cholerae strains, which were shown to carry the CTXΦ/pre-CTXΦ prophages as opposed to typical CTXETΦ-RS1 structure, were selected from approximately 1000 strains with diverse genomes. We identified two CTXΦ members and six pre-CTXΦ members with distinct rstR alleles, revealing complex chromosomal DNA integration patterns and arrangements of different prophages in these strains. Promoter activity assays showed that the transcriptional repressor RstR protected against CTXΦ superinfection by preventing the replication and integration of CTXΦ/pre-CTXΦ phages containing the same rstR allele, supporting the co-integration of the diverse CTXΦ/pre-CTXΦ members observed. The numbers and types of prophages and their co-integration arrangements in serogroup O139 strains were more complex than those in serogroup O1 strains. Also, these CTXΦ/pre-CTXΦ members were shown to present the bloom period of the CTXΦ/pre-CTXΦ family during wave 2 of the seventh cholera pandemic. Together, these analyses deepen our comprehension of the genetic variation of CTXΦ and pre-CTXΦ and provide insights into the evolution of the CTXΦ/pre-CTXΦ family in the seventh cholera pandemic.

Scheduled feeding improves behavioral outcomes and reduces inflammation in a mouse model of Fragile X syndrome.

Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by the abnormal expansion of CGG repeats in the fragile X mental retardation 1 (FMR1) gene. Many FXS patients experience sleep disruptions, and we sought to explore these symptoms along with the possible benefits of a scheduled feeding intervention using the Fmr1 knockout (KO) mouse model. These mutants displayed clear evidence for sleep and circadian disturbances including delay in the onset of sleep and fragmented activity rhythms with increases in cycle-to-cycle variability. The Fmr1 KO mice exhibited deficits in their circadian behavioral response to light with reduced masking, longer time to resetting to shifts in the LD cycle, altered synchronization to a skeleton photoperiod and lower magnitude light-induced phase shifts of activity rhythms. Investigation of the retinal input to the surprachiasmatic nucleus (SCN) with the neurotracer cholera toxin (β subunit) and quantification of the light-evoked cFos expression in the SCN revealed an abnormal retinal innervation of the SCN in the Fmr1 KO, providing a possible mechanistic explanation for the observed behavioral deficits. Interestingly, disruptions in social and repetitive behavior correlated with sleep duration and fragmentation. Understanding the nature of the deficits, we decided to apply a scheduled feeding regimen (6-hr/18-hr feed/fast cycle) as a circadian-based strategy to boast circadian rhythms independently of light. This intervention significantly improved the activity rhythms and sleep in the mutants. Strikingly, the scheduled feeding ameliorated social interactions and reduced repetitive behaviors as well as the levels of Interferon-gamma and Interleukin-12 in the Fmr1 KO mutants, suggesting that timed eating may be an effective way to reduce inflammation. Collectively, this work adds support to efforts to develop circadian based interventions to help with symptoms of neurodevelopmental disorders.

Retinal Input to Macaque Superior Colliculus Derives from Branching Axons Projecting to the Lateral Geniculate Nucleus.

The superior colliculus receives a direct projection from retinal ganglion cells. In primates, it remains unknown if the same ganglion cells also supply the lateral geniculate nucleus. To address this issue, a double-label experiment was performed in 2 male macaques. The animals fixated a target while injection sites were scouted in the superior colliculus by recording and stimulating with a tetrode. Once suitable sites were identified, cholera toxin - subunit B Alexa Fluor 488 was injected via an adjacent micropipette. In a subsequent acute experiment, cholera toxin subunit B - Alexa Fluor 555 was injected into the lateral geniculate nucleus at matching retinotopic locations. After a brief survival period, ganglion cells were examined in retinal flatmounts. The percentage of double-labeled cells varied locally, depending on the relative efficiency of retrograde transport by each tracer and the precision of retinotopic overlap of injection sites in each target nucleus. In counting boxes with extensive overlap, 76-98% of ganglion cells projecting to the superior colliculus were double-labeled. Cells projecting to the superior colliculus constituted 4.0 - 6.7% of the labeled ganglion cell population. In one particularly large zone, there were 5,746 cells labeled only by CTB-AF555, 561cells double-labeled by CTB-AF555 and CTB-AF488, but no cell labeled only by CTB-AF488. These data indicate that retinal input to the macaque superior colliculus arises from a collateral axonal branch supplied by about 5% of the ganglion cells that project to the lateral geniculate nucleus. Surprisingly, there exist no ganglion cells that project exclusively to the SC.Significance statement The retina contains a multitude of ganglion cell classes, projecting in parallel to different brain targets. The superior colliculus receives retinal input, but its source remains controversial. In two macaques, a green tracer was injected into the superior colliculus and an orange tracer into the lateral geniculate nucleus. When retinotopic overlap was optimal, ganglion cells containing the green tracer were also labeled by the orange tracer. This finding indicates that retinal input to the superior colliculus arises from axon collaterals of ganglion cells that project to the lateral geniculate nucleus, even though these two retinal targets serve utterly different functions. The next challenge is to determine the purpose of this projection and why it is shared.

Tongue exercise ameliorates structural and functional upper airway deficits in a rodent model of hypoglossal motor neuron loss.

Tongue weakness and atrophy can lead to deficits in the vital functions of breathing and swallowing in patients with motor neuron diseases (MNDs; e.g., amyotrophic lateral sclerosis (ALS) and pseudobulbar palsy), often resulting in aspiration pneumonia, respiratory failure, and death. Available treatments for patients with MNDs are largely palliative; thus, there is a critical need for therapies targeting preservation of upper airway function and suggesting a role for tongue exercise in patients with MNDs. Here, we leveraged our inducible rodent model of hypoglossal (XII) motor neuron degeneration to investigate the effects of a strength endurance tongue exercise program on upper airway structure and function. Our model was created through intralingual injection of cholera toxin B conjugated to saporin (CTB-SAP) into the genioglossus muscle of the tongue to induce targeted death of XII motor neurons. Rats in this study were allocated to 4 experimental groups that received intralingual injection of either CTB-SAP or unconjugated CTB + SAP (i.e., control) +/- tongue exercise. Following tongue exercise exposure, we evaluated the effect on respiratory function (via plethysmography), macrostructure [via magnetic resonance imaging (MRI) of the upper airway and tongue], and ultrafine structure [via ex vivo magnetic resonance spectroscopy (MRS) of the tongue] with a focus on lipid profiles. Results showed that sham exercise-treated CTB-SAP rats have evidence of upper airway restriction (i.e., reduced airflow) and structural changes present in the upper airway (i.e., airway compression) when compared to CTB-SAP + exercise rats and control rats +/- tongue exercise, which was ameliorated with tongue exercise. Additionally, CTB-SAP + sham exercise rats have evidence of increased lipid expression in the tongue consistent with previously observed tongue hypertrophy when compared to CTB-SAP + exercise rats or control rats +/- tongue exercise. These findings provide further evidence that a strength endurance tongue exercise program may be a viable therapeutic treatment option in patients with XII motor neuron degeneration in MNDs such as ALS. Future directions will focus on investigating the underlying mechanism responsible for tongue exercise-induced plasticity in the hypoglossal-tongue axis, particularly inflammatory associated factors such as BDNF.

Label-free, real-time monitoring of membrane binding events at zeptomolar concentrations using frequency-locked optical microresonators

G-protein coupled receptors help regulate cellular function and communication, and are targets of small molecule drug discovery efforts. Conventional techniques to probe these interactions require labels and large amounts of receptor to achieve satisfactory sensitivity. Here, we use frequency-locked optical microtoroids for label-free characterization of membrane interactions in vitro at zeptomolar concentrations for the kappa opioid receptor and its native agonist dynorphin A 1-13, as well as big dynorphin (dynorphin A and dynorphin B) using a supported biomimetic membrane. The measured affinity of the agonist dynorphin A 1-13 to the κ-opioid receptor was also measured and found to be 3.1 nM. Radioligand assays revealed a dissociation constant in agreement with this value (1.1 nM). The limit of detection for the κOR/DynA 1-13 was calculated as 180 zM. The binding of Cholera Toxin B-monosialotetrahexosyl ganglioside was also monitored in real-time and an equilibrium dissociation constant of 1.53 nM was found. Our biosensing platform provides a method for highly sensitive real-time characterization of membrane embedded protein binding kinetics that is rapid and label-free, for drug discovery and toxin screening among other applications.

ER-transiting bacterial toxins amplify STING innate immune responses and elicit ER stress.

The cGAS/STING sensor system drives innate immune responses to intracellular microbial double-stranded DNA (dsDNA) and bacterial cyclic nucleotide second messengers (e.g., c-di-AMP). STING-dependent cell-intrinsic responses can increase resistance to microbial infection and speed pathogen clearance. Correspondingly, STING activation and signaling are known to be targeted for suppression by effectors from several bacterial pathogens. Whether STING responses are also positively regulated through sensing of specific bacterial effectors is less clear. We find that STING activation through dsDNA, by its canonical ligand 2'-3' cGAMP, or the small molecule DMXAA is potentiated following intracellular delivery of the AB5 toxin family member pertussis toxin from Bordetella pertussis or the B subunit of cholera toxin from Vibrio cholerae. Entry of pertussis toxin or cholera toxin B into mouse macrophages triggers markers of endoplasmic reticulum (ER) stress and enhances ligand-dependent STING responses at the level of STING receptor activation in a manner that is independent of toxin enzymatic activity. Our results provide an example in which STING responses integrate information about the presence of relevant ER-transiting bacterial toxins into the innate inflammatory response and may help to explain the in vivo adjuvant effects of catalytically inactive toxins.

Surface Display of Cholera Toxin B Subunit Recombinant Escherichia coli Ghosts Further Enhances Resistance to Chlamydia abortus Infection in Mice.

Chlamydia abortus (C. abortus) is an important zoonotic pathogen that seriously endangers the development of animal husbandry. Vaccination is the most effective approach to preventing C. abortus infection. We previously reported a recombinant Escherichia coli ghost (rECG)-based C. abortus vaccine that demonstrated outstanding protective efficacy. In this study, we further attempted to fuse the cholera toxin B subunit (CTB), a widely studied potent mucosal immune adjuvant, with macrophage infectivity potentiator (MIP), a candidate antigen of C. abortus, on the surface of the rECG and explore its protective effect against C. abortus infection. The MIP fusion protein was highly expressed in the rECGs, and the CTB-modified rECGs significantly induced the activation of mouse bone marrow-derived dendritic cells in vitro. Intranasal immunization with rECGs induced a Th1-biased cellular immune response. Compared to the rECGs without CTB, the CTB-modified rECGs induced higher concentrations of IgA in the serum and vaginal wash solution. Moreover, in a mouse infection model, the CTB-modified rECGs significantly improved the clearance efficiency of C. abortus and reduced the pathological damage to the uterus. This study demonstrates that incorporating CTB into rECGs significantly enhances the immunogenic potential of the rECG vaccine and can significantly enhance its protective efficacy against a C. abortus challenge.