Cholera Toxin Subunit Research Articles

Brain-Wide Mapping of Afferent Inputs to Accumbens Nucleus Core Subdomains and Accumbens Nucleus Subnuclei.

The nucleus accumbens (NAc) is the ventral part of the striatum and the interface between cognition, emotion, and action. It is composed of three major subnuclei: i.e., NAc core (NAcC), lateral shell (NAcLS), and medial shell (NAcMS), which exhibit functional heterogeneity. Thus, determining the synaptic inputs of the subregions of the NAc is important for understanding the circuit mechanisms involved in regulating different functions. Here, we simultaneously labeled subregions of the NAc with cholera toxin subunit B conjugated with multicolor Alexa Fluor, then imaged serial sections of the whole brain with a fully automated slide scanning system. Using the interactive WholeBrain framework, we characterized brain-wide inputs to the NAcC subdomains, including the rostral, caudal, dorsal, and ventral subdomains (i.e., rNAcC, cNAcC, dNAcC, and vNAcC, respectively) and the NAc subnuclei. We found diverse brain regions, distributed from the cerebrum to brain stem, projecting to the NAc. Of the 57 brain regions projecting to the NAcC, the anterior olfactory nucleus (AON) exhibited the greatest inputs. The input neurons of rNAcC and cNAcC are two distinct populations but share similar distribution over the same upstream brain regions, whereas the input neurons of dNAcC and vNAcC exhibit slightly different distributions over the same upstream regions. Of the 55 brain regions projecting to the NAcLS, the piriform area contributed most of the inputs. Of the 72 brain regions projecting to the NAcMS, the lateral septal nucleus contributed most of the inputs. The input neurons of NAcC and NAcLS share similar distributions, whereas the NAcMS exhibited brain-wide distinct distribution. Thus, the NAcC subdomains appeared to share the same upstream brain regions, although with distinct input neuron populations and slight differences in the input proportions, whereas the NAcMS subnuclei received distinct inputs from multiple upstream brain regions. These results lay an anatomical foundation for understanding the different functions of NAcC subdomains and NAc subnuclei.

Biochemical and immunological characterization of an ETEC CFA/I adhesin cholera toxin B subunit chimera.

Surface-expressed colonization factors and their subunits are promising candidates for inclusion into a multivalent vaccine targeting enterotoxigenic Escherichia coli (ETEC), a leading cause of acute bacterial diarrhea in developing regions. However, soluble antigens are often poorly immunogenic in the absence of an adjuvant. We show here that the serum immune response to CfaE, the adhesin of the ETEC colonization factor CFA/I, can be enhanced in BALB/c mice by immunization with a chimeric antigen containing CfaE and pentameric cholera toxin B subunit (CTB) of cholera toxin from Vibrio cholerae. We constructed this antigen by replacing the coding sequence for the A1 domain of the cholera toxin A subunit (CTA) with the sequence of donor strand complemented CfaE (dscCfaE) within the cholera toxin operon, resulting in a dscCfaE-CTA2 fusion. After expression, via non-covalent interactions between CTA2 and CTB, the fusion and CTB polypeptides assemble into a complex containing a single dscCfaE-CTA2 protein bound to pentameric CTB (dscCfaE-CTA2/CTB). This holotoxin-like chimera retained the GM1 ganglioside binding activity of CTB, as well as the ability of CfaE to mediate the agglutination of bovine red blood cells when adsorbed to polystyrene beads. When administered intranasally to mice, the presence of CTB in the chimera significantly increased the serum immune response to CfaE compared to dscCfaE alone, stimulating a response similar to that obtained with a matched admixture of dscCfaE and CTB. However, by the orogastric route, immunization with the chimera elicited a superior functional immune response compared to an equivalent admixture of dscCfaE and CTB, supporting further investigation of the chimera as an ETEC vaccine candidate.

Incidence of Bacterial Enteropathogens among Diarrhea Patients from Tribal Areas of Odisha.

Infectious diarrheal diseases remain a leading cause of morbidity and mortality in developing and underdeveloped countries. The present study documented the etiology of bacterial enteropathogens in three tribal districts of Odisha from July 2010 to September 2013. A total of 1427 rectal swabs were collected and bacteriologically analyzed by following standard procedure. Among the 930 (65.2%) culture positive samples, Escherichia coli (E. coli) constituted 636 (44.6%); Vibrio cholerae (V. cholerae) O1, 146 (10.2%); Salmonella species (spp.), 10 (0.7%); Shigella spp., 79 (5.5%); and Aeromonas spp., 59 (4.1%). Of the 729 environmental water samples taken from river, open well, Nala (a small stream), and Chua (a shallow pit on a river bed), 14 (1.9%) contained non-O1/non-O139 V. cholerae and 13 (1.8%) had V. cholerae O1 strains. An analysis of the demographics showed that people in the 14 to 40-year age group were highly susceptible to diarrhea caused by V. cholerae which occurred mainly during the rainy and post-rainy seasons. All enteropathogens were multidrug-resistant and found throughout the study period. The V. cholerae strains isolated were El Tor variants carrying the classical, El Tor, and Haitian cholera toxin subunit B (ctxB) genes. The classical ctxB was the dominant allele, and the prevalence of the Haitian ctxB allele increased during the test period. These findings indicate that active surveillance is needed to monitor the changing antibiotic resistance patterns of V. cholerae serogroups and biotypes present in this region.

B subunit of cholera toxin fused with VP7 from GCRV (grass carp reovirus) was expressed in E. coli and folds into an active protein

Grass carp reovirus (GCRV) is one of the most serious pathogens threatening grass carp (Ctenopharyngodon idella) production and results in high mortality in China. To obtain a genetically engineered oral vaccine against GCRV, the cholera toxin B subunit (CTB) of Vibrio cholerae was fused to VP7 (CTB-VP7) and transformed into BL21(DE3) for expression. SDS-PAGE and Western blotting showed that the purified rCTB-VP7 fusion protein (rCTB-VP7) was approximately 49.0 kDa. The monomeric nature of rCTB-VP7 through multistage purification showed a binding affinity for GM1, a receptor for biologically active CTB.rCTB-VP7 is not vulnerable to disassembly by SDS but is vulnerable to disassembly by 2-mercaptoethanol. rCTB-VP7 is stable and highly active at room temperature. The binding affinity experiment between rCTB-VP7 and GM1 also confirms the effects of acid and alkalinity in solution on the structure of rCTB-VP7. rCTB-VP7 bound to GM1 with different affinities under different temperatures and pH values.Prokaryotic expression of rCTB-VP7 was characterized by high expression and easy purification and had a strong binding force with GM1 at 37 °C and pH 7.4. Our results suggest that rCTB-VP7 has the potential as an oral vaccine for protection against GCRV in aquaculture.

Activating Transcription Factor 3 (ATF3) Protects Retinal Ganglion Cells and Promotes Functional Preservation After Optic Nerve Crush.

PurposeTo investigate the possible role of activating transcription factor 3 (ATF3) in retinal ganglion cell (RGC) neuroprotection and optic nerve regeneration after optic nerve crush (ONC).MethodsOverexpression of proteins of interest (ATF3, phosphatase and tensin homolog [PTEN], placental alkaline phosphatase, green fluorescent protein) in the retina was achieved by intravitreal injections of recombinant adenovirus-associated viruses (rAAVs) expressing corresponding proteins. The number of RGCs and αRGCs was evaluated by immunostaining retinal sections and whole-mount retinas with antibodies against RNA binding protein with multiple splicing (RBPMS) and osteopontin, respectively. Axonal regeneration was assessed via fluorophore-coupled cholera toxin subunit B labeling. RGC function was evaluated by recording positive scotopic threshold response.ResultsThe level of ATF3 is preferentially elevated in osteopontin+/RBPMS+ αRGCs following ONC. Overexpression of ATF3 by intravitreal injection of rAAV 2 weeks before ONC promoted RBPMS+ RGC survival and preserved RGC function as assessed by positive scotopic threshold response recordings 2 weeks after ONC. However, overexpression of ATF3 and simultaneous downregulation of PTEN, a negative regulator of the mTOR pathway, combined with ONC, only moderately promoted short distance RGC axon regeneration (200 μm from the lesion site) but did not provide additional RGC neuroprotection compared with PTEN downregulation alone.ConclusionsThese results reveal a neuroprotective effect of ATF3 in the retina following injury and identify ATF3 as a promising agent for potential treatments of optic neuropathies.

Review on application of neural tracing technique to experimental research of acupuncture

To collect literatures on the application of neural tracing technique in experimental acupuncture research, and summarize its application status in acupoints, meridians and Zang-fu organs. We collected papers published from databases of CNKI (1979-2017), Wan-Fang (1990-2017), VIP (1989-2017) and PubMed(1997-2017)by using keywords of "neural tracing technique" "neuroanatomic tracing" "neural tracers" and "acupuncture" "electroacupuncture" "auricular acupuncture" "eye acupuncture" "meridians" "acupuncture points" "acupoint injection", and made a summary about the current state of application of neural tracing technique in the fields of acupoint, meridian and Zang-fu organs (viscera). A total of 94 articles were collected, the most commonly used neural tracers were horseradish peroxidase and cholera toxin subunit B. The experimental animals used were rat, rabbit, cat, monkey, etc., and injection site was acupoint. After the injection of neural tracers, the survival time of animals was range from 1 day to 12 weeks, and the labelled tissues included neurons and nerve fibers of the sensory, motor, and autonomic systems. The outcomes of neural labeling mainly revealed the segmental pattern, neuroanatomical connection (neural pathways/circuits) and chemical features (shown by immunohistochemical staining) of neurons and nerve fibers innervating both the acupoints and visceral organs, suggesting their involvement in the effect of acupuncture and moxibustion treatment. This application of neural tracing technology help us understand the under-lying mechanisms of acupuncture and moxibustion interventions from different perspectives of neural pathways/circuits and related chemical properties, which also lays a greater role for this technology in future experimental acupuncture research.

Red fluorescent AuNDs with conjugation of cholera toxin subunit B (CTB) for extended-distance retro-nerve transporting and long-time neural tracing

A retrograde transportation nerve probe, Au nanodots-cholera toxin B subunit (AuNDs-CTB), are prepared and fully characterized, which emit bright red fluorescence and show high quantum yield (7.2%) and good stability. The fluorescence emitted by the AuNDs is constant across a wide pH range (4–10) and after prolonged UV irradiation (>4 h). Previously, CTB has shown targeting characteristic for nerve cells with high sensitivity and effectiveness. After linking CTB to AuNDs through amidation reactions, AuNDs-CTB are obtained with excellent fluorescence property, nerve target characteristic, and, particularly, neural retrograde transportation feature. The red emission of the AuNDs-CTB is well distinguished from the blue autofluorescence of normal tissues, which provides potential for detection by naked eyes. Further, the fluorescence emission intensity maintains for 10 days in vivo, suggesting great utility for long-time monitoring and sensing of the nerve tissue. Furthermore, the AuNDs-CTB with bright red fluorescence can travel through the peripheral nerve to the spinal cord rapidly by retrograde transportation. The transportation occurs for a long distance (>5 cm) within only 2 days after injection of the AuNDs-CTB into the sciatic nerve. The present study exhibits a novel method for nerve visualization and drug delivery. Statement of significanceAu nanodots (AuNDs) conjugated with cholera toxin subunit B (CTB) have been developed for nerve labeling and neural retro-transporting. The red fluorescence from AuNDs-CTB is stable in vitro (pH 4–10 and 4 h UV irradiation) and in vivo (for a long time, more than 10 days). When injecting AuNDs-CTB into the sciatic nerve located at the midpiece of the thigh, the targeted nerve emits bright red fluorescence under UV light. Furthermore, the nerve can retrograde transport the AuNDs-CTB to the spinal cord for a distance of more than 5 cm just in 2 days. This work exhibits a novel method for nerve visualization by naked eyes and demonstrates the potential for intraoperative navigation.

Reciprocal connectivity between secondary auditory cortical field and amygdala in mice

Recent studies have examined the feedback pathway from the amygdala to the auditory cortex in conjunction with the feedforward pathway from the auditory cortex to the amygdala. However, these connections have not been fully characterized. Here, to visualize the comprehensive connectivity between the auditory cortex and amygdala, we injected cholera toxin subunit b (CTB), a bidirectional tracer, into multiple subfields in the mouse auditory cortex after identifying the location of these subfields using flavoprotein fluorescence imaging. After injecting CTB into the secondary auditory field (A2), we found densely innervated CTB-positive axon terminals that were mainly located in the lateral amygdala (La), and slight innervations in other divisions such as the basal amygdala. Moreover, we found a large number of retrogradely-stained CTB-positive neurons in La after injecting CTB into A2. When injecting CTB into the primary auditory cortex (A1), a small number of CTB-positive neurons and axons were visualized in the amygdala. Finally, we found a near complete absence of connections between the other auditory cortical fields and the amygdala. These data suggest that reciprocal connections between A2 and La are main conduits for communication between the auditory cortex and amygdala in mice.

Caveolin elastin-like polypeptide fusions mediate temperature-dependent assembly of caveolar microdomains.

Caveolae are membrane organelles formed by submicron invaginations in the plasma membrane, and are involved in mechanosensing, cell signaling, and endocytosis. Although implicated broadly in physiology and pathophysiology, better tools are required to elucidate the precise role of caveolar processes through selective activation and inactivation of their trafficking. Our group recently reported that thermally-responsive elastin-like polypeptides (ELPs) can trigger formation of 'genetically engineered protein microdomains (GEPMs)' functionalized with either Clathrin-light chain or the epidermal growth factor receptor. This manuscript is the first report of this strategy to modulate caveolin-1 (CAV1). By attaching different ELP sequences to CAV1, mild heating can be used to self-assemble CAV1-ELP microdomains inside of cells. The temperature of self-assembly can be controlled by tuning the ELP sequence. The formation of CAV1-ELP microdomains internalizes Cholera Toxin Subunit B, a commonly used marker of caveolae mediated endocytosis. CAV1-ELPs also colocalize with Cavin 1, an essential component of functional caveolae biogenesis. With the emerging significance of caveolae in health and disease and the lack of specific probes to rapidly and reversibly affect caveolar function, CAV1-ELP microdomains are a new tool to rapidly probe caveolae associated processes in endocytosis, cell signaling, and mechanosensing.

The roles of mesoporous silica and carbon nanoparticles in antigen stability and intensity of immune response against recombinant subunit B of cholera toxin in a rabbit animal model.

Vaccines are the front line in the fight against diseases. However, setbacks with existing cholera vaccines have ignited a considerable effort to develop more suitable vaccine formulations. In this study, we aim to investigate the effect of antigen stability and controlled release in inducing an immune response. Therefore, two types of silica and carbon mesoporous nanoparticles of the same size and shape but different pore architectures were synthesized and loaded with recombinant cholera toxin subunit B to serve as a model for antigen stability and controlled release of antigenic CTB. In order to evaluate immune response efficacy for these model formulations, IgG and IgA responses and fluid accumulation (FA) index were measured in immunized rabbits, which were challenged with wild-type Vibrio cholerae. Our result suggests that mesoporous silica nanoparticles have greater efficacy in inducing mucosal immune responses, and it proved more proficiency in overall immune responses in challenge experiments and FA index (p<0.05). These findings indicate that mesoporous nanoparticles and, in particular, mesoporous silica nanoparticles, could be used in oral vaccine formulation against cholera.

Multiple retrograde tracing methods compatible with 3DISCO clearing

Exploring the spatial relationship of various neuron pools in the spinal cord is crucial and difficult due to its complexity. The single-labelling tracing and sectioning were employed in previous studies exploring the distribution of spinal motor neuron pools, which could only delineate one single motor neuron pool in one specimen and could not achieve intact-tissue observation. Here, with combination of neuroanatomy tracing techniques and the optical clearing technique, we developed a multiple retrograde tracing method compatible with 3DISCO clearing. Fluoro-Gold, Fluoro-Ruby, Cholera Toxin Subunit B, Alexa Fluor 488 and 647 Conjugate were injected intramuscularly in hindlimbs of C57BL/6 adults. After labelling, the harvested spinal cords were optically cleared by 3DISCO method and imaged using confocal microscope. There were positive signals of all four tracers and four motor neurons pools targeting injected muscles were labelled. Three-dimension model of four motor neuron pools was successfully reconstructed based on tomography images showing the spatial relationship of different neuron pools. In conclusion, using this method, we first delineated the spatial relationship of four different motor neuron pools targeting four skeletal muscles in one spinal cord at the same time, which provide a holistic view of motor neuron pools in the spinal cord.

Synaptic Inputs to the Mouse Dorsal Vagal Complex and Its Resident Preproglucagon Neurons.

Stress responses are coordinated by widespread neural circuits. Homeostatic and psychogenic stressors activate preproglucagon (PPG) neurons in the caudal nucleus of the solitary tract (cNTS) that produce glucagon-like peptide-1; published work in rodents indicates that these neurons play a crucial role in stress responses. While the axonal targets of PPG neurons are well established, their afferent inputs are unknown. Here we use retrograde tracing with cholera toxin subunit b to show that the cNTS in male and female mice receives axonal inputs similar to those reported in rats. Monosynaptic and polysynaptic inputs specific to cNTS PPG neurons were revealed using Cre-conditional pseudorabies and rabies viruses. The most prominent sources of PPG monosynaptic input include the lateral (LH) and paraventricular (PVN) nuclei of the hypothalamus, parasubthalamic nucleus, lateral division of the central amygdala, and Barrington's nucleus (Bar). Additionally, PPG neurons receive monosynaptic vagal sensory input from the nodose ganglia and spinal sensory input from the dorsal horn. Sources of polysynaptic input to cNTS PPG neurons include the hippocampal formation, paraventricular thalamus, and prefrontal cortex. Finally, cNTS-projecting neurons within PVN, LH, and Bar express the activation marker cFOS in mice after restraint stress, identifying them as potential sources of neurogenic stress-induced recruitment of PPG neurons. In summary, cNTS PPG neurons in mice receive widespread monosynaptic and polysynaptic input from brain regions implicated in coordinating behavioral and physiological stress responses, as well as from vagal and spinal sensory neurons. Thus, PPG neurons are optimally positioned to integrate signals of homeostatic and psychogenic stress.SIGNIFICANCE STATEMENT Recent research has indicated a crucial role for glucagon-like peptide-1-producing preproglucagon (PPG) neurons in regulating both appetite and behavioral and autonomic responses to acute stress. Intriguingly, the central glucagon-like peptide-1 system defined in rodents is conserved in humans, highlighting the translational importance of understanding its anatomical organization. Findings reported here indicate that PPG neurons receive significant monosynaptic and polysynaptic input from brain regions implicated in autonomic and behavioral responses to stress, as well as direct input from vagal and spinal sensory neurons. Improved understanding of the neural pathways underlying the recruitment of PPG neurons may facilitate the development of novel therapies for the treatment of stress-related disorders.

Neural control of fasting-induced torpor in mice

Torpor is a peculiar mammalian behaviour, characterized by the active reduction of metabolic rate, followed by a drop in body temperature. To enter torpor, the activation of all thermogenic organs that could potentially defend body temperature must be prevented. Most of these organs, such as the brown adipose tissue, are controlled by the key thermoregulatory region of the Raphe Pallidus (RPa). Currently, it is not known which brain areas mediate the entrance into torpor. To identify these areas, the expression of the early gene c-Fos at torpor onset was assessed in different brain regions in mice injected with a retrograde tracer (Cholera Toxin subunit b, CTb) into the RPa region. The results show a network of hypothalamic neurons that are specifically activated at torpor onset and a direct torpor-specific projection from the Dorsomedial Hypothalamus to the RPa that could putatively mediate the suppression of thermogenesis during torpor.

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.

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.

The lateral neocortex is critical for contextual fear memory reconsolidation

Memories are a product of the concerted activity of many brain areas. Deregulation of consolidation and reprocessing of mnemonic traces that encode fearful experiences might result in fear-related psychopathologies. Here, we assessed how pre-established memories change with experience, particularly the labilization/reconsolidation of memory, using the whole-brain analysis technique of positron emission tomography in male mice. We found differences in glucose consumption in the lateral neocortex, hippocampus and amygdala in mice that underwent labilization/reconsolidation processes compared to animals that did not reactivate a fear memory. We used chemogenetics to obtain insight into the role of cortical areas in these phases of memory and found that the lateral neocortex is necessary for fear memory reconsolidation. Inhibition of lateral neocortex during reconsolidation altered glucose consumption levels in the amygdala. Using an optogenetic/neuronal recording-based strategy we observed that the lateral neocortex is functionally connected with the amygdala, which, along with retrograde labeling using fluorophore-conjugated cholera toxin subunit B, support a monosynaptic connection between these areas and poses this connection as a hot-spot in the circuits involved in reactivation of fear memories.

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.

A systematic topographical relationship between mouse lateral posterior thalamic neurons and their visual cortical projection targets.

Higher-order visual thalamus communicates broadly and bi-directionally with primary and extrastriate cortical areas in various mammals. In primates, the pulvinar is a topographically and functionally organized thalamic nucleus that is largely dedicated to visual processing. Still, a more granular connectivity map is needed to understand the role of thalamocortical loops in visually guided behavior. Similarly, the secondary visual thalamic nucleus in mice (the lateral posterior nucleus, LP) has extensive connections with cortex. To resolve the precise connectivity of these circuits, we first mapped mouse visual cortical areas using intrinsic signal optical imaging and then injected fluorescently tagged retrograde tracers (cholera toxin subunit B) into retinotopically-matched locations in various combinations of seven different visual areas. We find that LP neurons representing matched regions in visual space but projecting to different extrastriate areas are found in different topographically organized zones, with few double-labeled cells (~4-6%). In addition, V1 and extrastriate visual areas received input from the ventrolateral part of the laterodorsal nucleus of the thalamus (LDVL). These observations indicate that the thalamus provides topographically organized circuits to each mouse visual area and raise new questions about the contributions from LP and LDVL to cortical activity.

Sensitive and rapid detection of cholera toxin subunit B using magnetic frequency mixing detection.

Cholera is a life-threatening disease caused by the cholera toxin (CT) as produced by some Vibrio cholerae serogroups. In this research we present a method which directly detects the toxin’s B subunit (CTB) in drinking water. For this purpose we performed a magnetic sandwich immunoassay inside a 3D immunofiltration column. We used two different commercially available antibodies to capture CTB and for binding to superparamagnetic beads. ELISA experiments were performed to select the antibody combination. The beads act as labels for the magnetic frequency mixing detection technique. We show that the limit of detection depends on the type of magnetic beads. A nonlinear Hill curve was fitted to the calibration measurements by means of a custom-written python software. We achieved a sensitive and rapid detection of CTB within a broad concentration range from 0.2 ng/ml to more than 700 ng/ml.