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Author(s):  
Marissa J Metz ◽  
Caitlin M Daimon ◽  
Connie M. King ◽  
Andrew R. Rau ◽  
Shane T Hentges

Proopiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus (ARH) are a diverse group of neurons that project widely to different brain regions. It is unknown how this small population of neurons organizes its afferent projections. In this study, we hypothesized that individual ARH POMC neurons exclusively innervate select target regions. To investigate this hypothesis, we first verified that only a fraction of ARH POMC neurons innervate the lateral hypothalamus (LH), the paraventricular nucleus of the hypothalamus (PVN), the periaqueductal gray (PAG), or the ventral tegmental area (VTA) using the retrograde tracer cholera toxin B (CTB). Next, two versions of CTB conjugated to distinct fluorophores were injected bilaterally into two of the regions such that PVN and VTA, PAG and VTA, or LH and PVN received tracers simultaneously. These pairs of target sites were chosen based on function and location. Few individual ARH POMC neurons projected to two brain regions at once, suggesting that there are ARH POMC neuron subpopulations organized by their afferent projections. We also investigated whether increasing the activity of POMC neurons could increase the number of ARH POMC neurons labeled with CTB, implying an increase in new synaptic connections to downstream regions. However, chemogenetic enhancement of POMC neuron activity did not increase retrograde tracing of CTB back to ARH POMC neurons from either the LH, PVN, or VTA. Overall, subpopulations of ARH POMC neurons with distinct afferent projections may serve as a way for the POMC population to organize its many functions.


F1000Research ◽  
2021 ◽  
Vol 10 ◽  
pp. 303
Author(s):  
Maysaa El Sayed Zaki ◽  
Dina Elhammady ◽  
Mona Foda Salama ◽  
Mostafa Abdelsalam ◽  
Asmaa Osama Bakr Osman

Background: Irritable bowel syndrome (IBS) is a common gastrointestinal disorder, categorized into various subtypes. Post-infection IBS may be attributed to the release of cytolethal distending toxin B (CdtB), which cross-reacts with the adhesion protein vinculin responsible for normal intestinal contractility. Objective: This study aims to identify anti-CdtB and anti-vinculin levels in IBS patients compared to healthy control. Subjects and methods: This retrospective case-control study was conducted on 100 subjects with IBS, as determined by a questionnaire based on Rome III criteria, recruited from the outpatient clinics of the Tropical Medicine at Mansoura University Hospital from January 2019 to January 2020. Results: The optical density (OD) results of the anti-vinculin and anti-CdtB levels were significantly elevated in patients with IBS (1.58±0.496 OD, 2.47±0.60 OD)  when compared to control subjects (1.13±0.249 OD, 2.1±0.24 OD), respectively with P=0.001 for both.  Anti-vinculin level was significantly higher in the IBS-D subtype than the other subtypes (P=0.001) while, Anti-CdtB was significantly elevated in IBS-C, IBS-D subgroups compared to control subjects (P=0.001). Conclusion: Findings of the present study support the hypothesis that IBS results from post-infectious disorders initiated by bacterial enteritis. A hypothesis could be applied to all IBS subgroups. On the other hand. These biomarkers might reflect the post-infectious state's severity.


2021 ◽  
Author(s):  
John-Paul Fuller-Jackson ◽  
Peregrine B Osborne ◽  
Janet R Keast

This protocol details the 3D reconstruction of the lumbosacral spinal cord using alternating cryosections, and then goes through the steps required to quantify lower urinary tract afferents. Using TissueMaker (MBF Bioscience), images of alternating sections can be ordered and aligned prior to the production of a single image stack. In Neurolucida 360 (MBF Bioscience), regions of interest can be defined within the image stack, and the bouton-like immunolabelling of cholera toxin B can be segmented. Once saved, this data can then be extracted using Neurolucida Explorer (MBF Bioscience).


2021 ◽  
Author(s):  
Janet R Keast ◽  
Peregrine B Osborne ◽  
John-Paul Fuller-Jackson

This protocol is used for immunohistochemical visualisation of cholera toxin subunit B within afferents innervating the lower urinary tract in cryosections of rat lumbosacral spinal cord. Free-floating sections are processed in a double labelling protocol to distinguish regions of innervation by these afferents. Cholera toxin B antibody [lower urinary tract afferents] Choline acetyltransferase antibody [preganglionic autonomic neurons and motoneurons]


2021 ◽  
Author(s):  
Jung-Hyun Byun ◽  
Dongeun Yong ◽  
Heejung Kim

Abstract In the pediatric population, severe Clostridioides difficile infection sometimes occurs, but most cases are asymptomatic. Since the asymptomatic carriage rate is reportedly high in pediatric populations, diagnosis of CDI is difficult. Here, we analyzed 960 results of gastrointestinal pathogen multiplex PCR to estimate the positive rate of toxigenic C. difficile in pediatric populations aged between 0 and 18 years. The overall rate of C. difficile toxin B positivity was 10.1% in the stool samples. The positive rate peaked in 1-year-old infants (29/153, 19.0%), and decreased continually thereafter. The positive rate we observed was lower than the rates described in the literature. Remarkably, no C. difficile was detected in neonates. Antibiotic usage was inversely related to the positive rate, especially in infants < 2 years of age. The odds ratio of antibiotics was 0.44 (95% confidence interval (CI) 0.28–0.68; P < 0.001). The presence of concomitant gastrointestinal pathogens was not associated with toxigenic C. difficile positivity. Even though toxigenic C. difficile infection is neither an important nor a common cause of pediatric diarrhea, children can spread it to adults who are at risk of developing CDI. Pediatric population can act as hidden reservoirs for pathogenic strains in the community.


2021 ◽  
Vol 12 ◽  
Author(s):  
Florian Stieglitz ◽  
Ralf Gerhard ◽  
Andreas Pich

Clostridioides difficile is a major cause of nosocomial infection worldwide causing antibiotic-associated diarrhea and some cases are leading to pseudomembranous colitis. The main virulence factors are toxin A and toxin B. Hypervirulent strains of C. difficile are linked to higher mortality rates and most of these strains produce additionally the C. difficile binary toxin (CDT) that possesses two subunits, CDTa and CDTb. The latter is responsible for binding and transfer of CDTa into the cytoplasm of target cells; CDTa is an ADP ribosyltransferase catalyzing the modification of actin fibers that disturbs the actin vs microtubule balance and induces microtubule-based protrusions of the cell membrane increasing the adherence of C. difficile. The underlying mechanisms remain elusive. Thus, we performed a screening experiment using MS-based proteomics and phosphoproteomics techniques. Epithelial Hep-2 cells were treated with CDTa and CDTb in a multiplexed study for 4 and 8 h. Phosphopeptide enrichment was performed using affinity chromatography with TiO2 and Fe-NTA; for quantification, a TMT-based approach and DDA measurements were used. More than 4,300 proteins and 5,600 phosphosites were identified and quantified at all time points. Although only moderate changes were observed on proteome level, the phosphorylation level of nearly 1,100 phosphosites responded to toxin treatment. The data suggested that CSNK2A1 might act as an effector kinase after treatment with CDT. Additionally, we confirmed ADP-ribosylation on Arg-177 of actin and the kinetic of this modification for the first time.


F1000Research ◽  
2021 ◽  
Vol 10 ◽  
pp. 303
Author(s):  
Maysaa El Sayed Zaki ◽  
Dina Elhammady ◽  
Mona Foda Salama ◽  
Mostafa Abdelsalam ◽  
Asmaa Osama Bakr Osman

Background: Irritable bowel syndrome (IBS) is a common gastrointestinal disorder, categorized into various subtypes. Post-infection IBS may be attributed to the release of cytolethal distending toxin B (CdtB), which cross-reacts with the adhesion protein vinculin responsible for normal intestinal contractility. Objective: This study aims to identify anti-CdtB and anti-vinculin levels in IBS patients compared to healthy control. Subjects and methods: This retrospective case-control study was conducted on 100 subjects with IBS, as determined by a questionnaire based on Rome III criteria, recruited from the outpatient clinics of the Tropical Medicine at Mansoura University Hospital from January 2019 to January 2020. Results: Anti-vinculin and anti-CdtB levels were significantly elevated in patients with IBS (1.58±0.496, 2.47±0.60)  when compared to control subjects (1.13±0.249ng/ml, 2.1±0.24 ng/ml), respectively with P=0.001 for both.  Anti-vinculin level was significantly higher in the IBS-D subtype than the other subtypes (P=0.001) while, Anti-CdtB was significantly elevated in IBS-C, IBS-D subgroups compared to control subjects (P=0.001). Conclusion: Findings of the present study support the hypothesis that IBS results from post-infectious disorders initiated by bacterial enteritis. A hypothesis could be applied to all IBS subgroups. On the other hand. These biomarkers might reflect the post-infectious state's severity.


2021 ◽  
Author(s):  
Hayley Fisher ◽  
Hongyu Lin ◽  
Jensen May ◽  
Caitlin McLean ◽  
Charles L Pickens

Deficits in goal-directed action are reported in multiple neuropsychiatric conditions, including schizophrenia. However, dysfunction is not always apparent in early stages of schizophrenia, possibly due to neural compensation. We designed a novel devaluation task in which goal-directed action could be guided by stimulus-outcome (S-O) [presumably orbitofrontal cortex (OFC)-mediated] or response-outcome (R-O) associations [presumably prelimbic cortex (PL)-mediated]. We previously found suggestive evidence that OFC and PL could compensate for each other in this task, and we more directly assessed this potential compensation here. In Experiment 1, rats received OFC, PL, combined OFC+PL, or sham lesions and then completed our devaluation task. The OFC+PL lesion group exhibited impaired devaluation. In Experiment 2, rats received cholera-toxin-b (CTb) into OFC and either neurotoxic or sham PL lesions. Rats were then sacrificed on the last training day to double-label for Arc and CTb. We found increased Arc+CTb in mediodorsal thalamus (MD) and increased Arc+ neurons in OFC when PL was lesioned, suggesting that PL lesions lead to a compensatory increased activation of the MD->OFC circuit. Our results suggest that our devaluation task can model neural compensation between OFC and PL and this compensation may be regulated by MD.


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