Nociceptive afferents from spinal dorsal root ganglia and vagal nodose ganglia innervate the stomach and send sensory signals centrally to the brain and locally to the enteric nervous system. Such nociceptive afferents are detected with a variety of markers and can be visualized using immunohistochemistry under the confocal laser scanning microscope. Calcitonin gene-related peptide (CGRP) is one of the main neurotransmitters of nociceptive sensory fibers and was used as a marker in this study to label afferent axons and terminals in the whole stomach flat-mount. Previous investigations in the sectioned stomach have shown the presence of these sensory nerve fibers, however, their topographical distribution across the whole stomach has not been well established. Using mice and rats as two rodent models, we combined the immunohistochemistry labeling with the latest advanced neuroimaging technologies to illustrate the topographical organization and morphological structure of CGRP-IR (immunoreactive) axons and terminals in the whole stomach across animals as well as investigate the variability of their innervation pattern based on laterality. These axons were found densely innervating the vasculature, myenteric ganglia, longitudinal muscles, and circular muscles. With MBF's novel software Neurolucida 360®, these axons and terminals were digitally marked, traced, and reconstructed into a map of a fully innervated stomach, then these topographical maps were mathematically integrated into a 3D stomach scaffold. For the first time, a wholistic map of the nociceptive afferent CGRP-IR axon innervation of a whole stomach at single cell/axon/synapse resolution is provided and integrated into the stomach-brain atlas. This map also improves the understanding of the functional roles that CGRP has in relaxation of the smooth muscles, vasodilation, and protective mechanisms that are crucial to physiological and pathological conditions.

Huntington disease (HD) is a neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin (HTT) gene. HD manifests as a triad of psychiatric, cognitive, and motor symptoms, the latter of which defines symptomatic onset of the disease. Psychiatric symptoms including aggression and depression often emerge decades prior to motor symptom onset. Psychiatric disorders are closely linked with dysregulated neural plasticity, the ability of the brain to form new synaptic connections. Neural plasticity is impaired in HD, potentially playing a role in psychiatric symptoms. However, the association between plasticity and psychopathology in HD has yet to be thoroughly investigated. Psychoplastogens constitute a class of psychoactive compounds that induce neuroplasticity in key regions of emotional regulation, including the prefrontal cortex, hippocampus, and amygdala. In clinical settings, psychoplastogens promote lasting improvements in depression and anxiety scores following a single administration, suggesting an attractive therapeutic profile for HD-related psychopathology. Our lab has recently established the first preclinical model of HD-related aggression in Hu97/18 humanized HD model mice. This model further recapitulates other psychiatric-like aspects of HD, including depressive-like behavior, providing an opportunity to study mechanisms and explore therapeutic strategies for these psychiatric aspects of disease. In this study, we explore the behavioral response of Hu97/18 mice following treatment with psychoplastogens. A single hallucinogenic dose of psychoplastogens did not alter depressive-like behavior or aggression, though a trend towards reduced aggressive behaviors was observed. Encouragingly, psychoplastogens induced structural plasticity in the PFC, striatum, and hippocampus of Hu97/18 mice, suggesting that earlier intervention may be required for significant behavioral benefits. Our work furthers the investigation of interventions for HD psychopathology and expands our understanding of the relationship between plasticity and psychiatric symptoms in HD. This work implicates psychoplastogens as a potential therapeutic intervention for psychiatric dysfunction and impaired plasticity in HD.

Staphylococcus aureus (SA) is an opportunistic pathogen and a commensal member of the human microbiota that frequently colonizes the upper respiratory tract and skin. While SA can cause disease ranging from minor skin infections to life-threatening septicemia, it can also be carried asymptomatically. As carriage is the most significant risk factor for SA disease, surveillance is important for the prevention of outbreaks in vulnerable communities. Native Americans have a greater risk of infectious disease than the general US population, and Native Americans in the Southwestern United States have been shown to experience high rates of invasive SA disease. Here we explore the dynamics of SA carriage among the Native Americans by performing high density sampling from multiple anatomical sites and whole-genome sequencing on 310 SA isolates across 60 participants. We assess the richness and diversity of SA carriage isolates via differences in multi-locus sequence type (MLST), core-genome single-nucleotide-polymorphisms (SNPs), and gene content. We find that a considerable proportion (41.7%, n=60) of studied SA carriers exhibit co-carriage with multiple distinct SA strains. Among a small number of participants, we further find considerable genetic variation even among SA isolates belonging to the same MLST. Lastly, we find unequal distribution of clonal complex (CC) by body site, suggesting that certain lineages may be adapted to specific anatomical sites. Together, these findings reveal that co-carriage may occur at a higher rate than previously appreciated and contribute to our understanding of SA intrahost diversity during carriage, which has implications for clinical management and epidemiological investigations.

The uptake of cytotoxic free fatty acids (FFA) and their conversion to physiologically expedient triglycerides (TAG) which are later on assimilated to very low density lipoproteins (VLDL) is of utmost value among the multifarious tasks performed by the liver. Inflated concentration of VLDL in the blood stream directly correlates with the reinforcement of atherosclerosis. VLDL is synthesized in the hepatic endoplasmic reticulum (ER) and transported to the Golgi where it encounters several alterations. It is then enclosed in distinct post-Golgi VLDL transport vesicles (PG-VTVs) and released into the blood. The data generated in our lab has proved the requirement of ATP for PG-VTV biogenesis however, ATP substitution with non-hydrolyzable ATP analogue (ATPγS) had no effect on this process. Therefore, the present study is based on the hypothesis that ATP mediated protein phosphorylation regulates PG-VTV biogenesis. First, hepatic subcellular organelles were isolated and their purity was determined by performing Western blot. A cell-free in vitro budding assay was performed in presence or absence of ATP, GTP and cytosol using 3[H]-TAG labelled hepatic Golgi to generate PG-VTVs. We performed Western blotting to confirm distinct protein phosphorylation at tyrosine residue during PG-VTV formation however, protein phosphorylation event did not occur when PG-VTV budding was blocked. Two-dimensional gel electrophoresis identified Syntaxin 17 (STX17) as the phosphorylated protein required for PG-VTV formation. ATP mediated phosphorylation of STX17 during biogenesis of PG-VTVs was confirmed by its presence on PG-VTVs. PG-VTV budding was found to be significantly reduced on performing budding assay using STX17 immunodepleted cytosol compared to positive control. RNAi mediated knockdown of STX17 in McA-RH7777 cells resulted in increased VLDL secretion as measured by 3[H]-TAG liquid scintillation counter. Based on these results, it can be justified that STX17 plays a vital role in regulating PG-VTV budding and overall VLDL secretion from hepatocytes.

Atherosclerosis is a cardiovascular disease where plaques made up of lipids in the form of cholesterol ester build up in the carotid and innominate arteries that supply blood to the brain. Accumulation of the plaques limit the flow of blood and nutrients to the brain, leading to diminished oxygen supply, increased oxidative stress and cell death. All these have been implicated in Alzheimer's disease (AD). Alzheimer's disease, a chronic, progressive, age related neurodegenerative disorder is the most common form of dementia in the elderly accounting for 60-80% of the cases. Clinically, Alzheimer's disease is characterized by loss of memory, damage of brain tissues, and neuronal and synaptic loss. Pathologically, it is delineated by accumulation of amyloid beta and tau proteins forming senile plaques and neurofibrillary tangles respectively. Apolipoprotein E (ApoE) polymorphism, increased oxidative stress and products of lipid peroxidation are associated with atherosclerosis and Alzheimer's disease. ApoE is a glycosylated protein that mediates plasma lipoprotein metabolism. ApoE isoforms have differential effect on amyloid beta aggregation and clearance, thus playing an important role in Alzheimer's pathology. Serum paraoxonase 1 (PON1) is a lipoprotein associated antioxidant enzyme that prevents lipid peroxidation. S100B protein is a plasma biomarker, altered expression of which has been implied in AD. We propose the hypothesis that combined deficiencies in apolipoprotein E and antioxidant defense (established by the lack of PON1), together with dyslipidemia and development of carotid atherosclerosis in aging mice would reflect Alzheimer's pathology. The brains of young and old ApoE-PON1 double knockout (DKO) mice and control C57BL/6J mice were harvested. Atherosclerotic lesions were quantified by Image J. RNA was isolated, cDNA was synthesized and quantitative RT-PCR was performed to detect mRNA levels of S100B. Blood levels of S100B protein was measured by ELISA. Brain tissues were stained with Hematoxylin and Eosin stain and 4G8 immunostain to detect histopathological changes. The blood brain barrier (BBB) is altered in AD resulting in increased permeability and vascular dysfunction. The vascular permeability of BBB was analyzed by Evans Blue Dye (EBD) assay. The results showed that the older DKO mice had severe carotid atherosclerosis, increased levels of serum S100B protein and elevated mRNA levels of S100B. Histological examination showed the presence of characteristic hallmarks of AD. The leakage of EBD into brain parenchyma indicated disruption of BBB. The results suggest that diminished blood flow and nutrient supply to the brain due to atherosclerosis and increased oxidative stress might contribute to Alzheimer's pathology. We suggest that older ApoE-PON1 DKO mice may serve as a model of Alzheimer's disease and prevention of atherosclerosis might promote regression of Alzheimer's disease.

Inflammatory Bowel Disease and Crohn's disease represent chronic intestinal inflammatory diseases. It is suspected that bacterial infection is one of the causes of gut inflammation. Studies from others as well as from our laboratory have indicated that peroxidized lipids and their decomposition products are pro-inflammatory. As we consume considerable amounts of dietary oxidized lipids (arising from deep frying of vegetable oils), we hypothesize that dietary peroxidized lipids may also lead to intestinal inflammation. To test this hypothesis, intestine from C57BL/6J mice were collected and used in this study. The intestinal epithelial tissue as well as intestinal lymphoid tissues [Peyer's Patches (PP)] were identified and harvested. Both the tissue samples were incubated with 13-Hydroperoxyoctadecadienoic acid (HPODE, a simple form of peroxidized fatty acid) or oxidized phosphatidyl choline (Ox-PL) or minimally modified LDL (mmLDL) or bacterial lipopolysaccharide (LPS) at 37°C. After 6 hours of incubation, RNA was extracted and RT-PCR analysis was performed to determine inflammatory markers using mouse primers for the gene expression of cytokines. We noted an increased basal gene expressions of inflammatory cytokines in PP tissues as opposed to the epithelial tissue. An increase in inflammatory cytokines gene expression was observed in LPS/POL treated intestinal tissues as compared to untreated tissues. Overall, our findings might suggest additional potential sources of gut inflammation as well as an active participation of epithelial cells in the inflammatory process. These might also offer novel targets for the control of inflammation of the gut in patients suffering from gut inflammatory diseases.

Doxorubicin (Doxo) is one of multiple anthracycline drugs used to effectively treat various forms of cancer. Unfortunately, Doxo treatment, as a side effect, induces cardiomyopathy and subsequent heart failure. We have previously demonstrated that transplanted embryonic stem (ES) cells and their conditioned medium (CM) modulate the PTEN pathway and reduce apoptosis, fibrosis and hypertrophy in a Doxo induced cardiomyopathy (DIC) model. However, mechanisms of inhibited apoptosis mediated through PTEN pathway are completely unknown. Therefore, we used VO-OHpic (VO), a potent PTEN inhibitor to understand the mechanism of apoptosis as well as its effect on cardiac remodeling in DIC. Animals were divided into three groups; Group 1: Control (Saline), Group 2: Doxo (12 mg/kg, cumulative dose) and Group 3: Doxo+VO (30ug/kg cumulative dose). Animals were studied at one week and eight weeks post-DIC. Mice were subjected to echocardiography to examine cardiac function, sacrificed and hearts were harvested for further analysis. Immunohistochemistry staining revealed a significant (p lt& 0.05) decrease in apoptotic cardiomyocytes in Doxo+VO treated hearts compared with Doxo group. Furthermore, Hematoxylin and Eosin (H&E) and Masson's Trichrome histological stains confirmed reduced hypertrophy and fibrosis in Doxo+VO treated subjects compared to Doxo group. Western Blotting confirmed the reduction of p-PTEN levels and the increase in p-AKT cell survival protein expression in Doxo+VO subjects. In addition, VO-OHpic administration was shown to reduce the number of pro-inflammatory macrophages and increase the number of anti-inflammatory M2 macrophages that may further be involved in reduced apoptosis and fibrosis. Finally, heart function was improved in mice treated with VO compared to Doxo group. Collectively, our data suggests that VO-OHpic treatment reduces apoptosis, cardiac fibrosis and the process is mediated through the PTEN/AKT and inflammatory mechanisms with improved heart function in the DIC heart.

Inflamed tissue in Crohn’s disease (CD) are continuously producing toxic oxygen metabolites leading to cellular injury and apoptosis. Here, we are evaluating the role of Mycobacterium avium subspecies paratuberculosis (MAP) in oxidative stress in CD by evaluation of lipid peroxidation and antioxidant defense activity. Specifically, we measured malondialdehyde (MDA) level and selenium-dependent glutathione peroxidase (GPx) activity in the plasma from patients and cattle infected with MAP. The level of MAP antibodies in bovine sera was determined by IDEXX kit whereas detection of MAP DNA was performed by IS900-based nPCR. A total of 42 cattle (21 infected with MAP and 21 healthy controls), 27 CD subjects, 27 of CD-healthy relatives, 66 subjects with various diseases and 34 non-related healthy subjects were investigated. Overall, GPx activity was significantly higher in MAP infected humans (0.80941±0.521) versus MAP (-ve) samples (0.42367±0.229 units/ml), P lt& 0.01. Similarly, the average of GPx activity in cattle infected with MAP was 1.59±0.65 units/ml compared to 0.46907±0.28 units/ml in healthy cattle (P lt& 0.01). Although it was not statistically significant, MDA average level was higher in MAP infected human samples versus MAP (-ve) controls (1.11±0.185 nmol/ml versus 0.805±0.151 nmol/ml, respectively). Similarly, MDA average level in CD samples that are MAP+ (1.703±0.231 nmol/ml) was higher than CD samples that are MAP (-ve) (1.429±0.187 nmol/ml). In cattle, MDA average level in MAP infected samples was significantly higher at 3.818±0.45 nmol/ml compared to 0.538±0.18 nmol/ml in healthy cattle (P lt& 0.01). Clearly, the data demonstrated that MAP infection is associated with oxidative stress and resulting in the pathophysiology of worsening of the condition of CD patients.

BACKGROUND: Crohn's disease (CD) is an inflammatory bowel disease (IBD) and has been associated with Mycobacterium avium subspecies paratuberculosis (MAP). MAP has been detected in stool, tissue and blood samples from patients with CD. Gamma interferon (γ-IFN) is an inflammatory cytokine that plays a crucial role in killing intracellular pathogens like MAP, and its receptor (IFNGR1) mutations cause immunodeficiency and severe disseminated mycobacterial infections. The role of MAP in association with IFNGR1 mutation in CD patients have not been investigated. METHODS: In this study, we investigated blood samples of 79 human subjects for MAP infection in association with IFNGR1 gene dysfunction. Samples were divided into 22 CD, 6 Ulcerative colitis (UC), 32 normal healthy and 19 non-inflammatory bowel disease (NIBD). Five variants of IFNGR1 single nucleotide polymorphisms (SNP) were investigated using Taqman Genotyping assay, then IFNGR1 expression measured by RT-PCR and serum IFNGR1 and γ-IFN levels were measured using ELISA. MAP infection was detected using nested PCR. RESULTS: Among 28 IBD patients, 4/6 (66.67%) of UC and 18/22 (81.82%) of CD are tested positive for at least one SNP homozygous minor form compared to 21.88% and 47.37%% in 32 healthy and 19 NIBD (P < 0.05). IFNGR1 gene expression was downregulated 1.4-fold in IBD patients (P =0.07) and 1.7-fold downregulated in MAP positive IBD patients compared to MAP negative IBD patients (P=0.06). Serum IFNGR1 protein levels were downregulated 1.53-fold in IBD patients compared to normal, and 1.4-fold downregulated in MAP positive IBD patients compared to MAP negative IBD patients. MAP infection is more common in rs2234711 SNP positive patients (5/7 =71.42%) (P < 0.05). Serum γ-IFN levels were not elevated in both groups. CONCLUSION: IFNGR1 SNP's, MAP infection and IFNGR1 downregulation were found in higher incidence in IBD, suggesting role of IFNGR1 in susceptibility of MAP infection in IBD patients.

The intrinsic cardiac nervous system (ICN) has been regarded as the "little brain" of the heart and is implicated to have a major role in the autonomic regulation of chronotropy, inotropy, and dromotropy. There has been an increasing interest in studying the neurons of the ICN as a region of interest for developing targeted therapeutics in the modulation of heart disease. Previous studies have characterized the intrinsic cardiac neurons through sectioned tissue in immunohistochemical studies, whole-mount heart preparations, and macroscopic inflation studies. However, these techniques show a limited perspective on the location of the intrinsic cardiac neurons with respect to the entire heart. To characterize a holistic distribution of the intrinsic cardiac neurons in the context of the whole heart, we have developed a scalable data acquisition workflow that consists of a set of tools and techniques that enable novel whole-organ imaging, precise 3D neuroanatomical mapping, and molecular phenotyping of the intrinsic cardiac neurons. A Knife Edge Scanning Microscope (KESM) was used for high-throughput whole-heart imaging of 9 rat hearts to assemble image stacks consisting of a range of 1400-2580 sections, where each section was 5 µm thick. The precise annotation of intrinsic cardiac neurons, delineation of cardiac anatomy on each section, and visualization of the spatial distribution of mapped neurons in 3D heart reconstructions was performed through the Tissue Mapper software. Laser capture microdissection was performed on 151 individual neurons for RNA sequence analysis, where the location of each cell was marked on the section they were lifted. The spatial location of each RNA sequenced neuron was mounted onto an interactive heart scaffold to visualize the molecular heterogeneity of the ICN in a 3D environment. For the first time, we have established a comprehensive large-scale atlas of the rat ICN at a single-cell resolution.