Myelin basic protein mRNA levels affect myelin sheath dimensions, architecture, plasticity, and density of resident glial cells.

Myelin basic protein (Mbp) is essential for both elaboration and maintenance of CNS myelin, and its reduced accumulation results in hypomyelination. How different Mbp mRNA levels affect myelin dimensions across the lifespan and how resident glial cells may respond to such changes are unknown. Here, to investigate these questions, we used enhancer-edited mouse lines that accumulate Mbp mRNA levels ranging from 8% to 160% of wild type. In young mice, reduced Mbp mRNA levels resulted in corresponding decreases in Mbp protein accumulation and myelin sheath thickness, confirming the previously demonstrated rate-limiting role of Mbp transcription in the control of initial myelin synthesis. However, despite maintaining lower line specific Mbp mRNA levels into old age, both Mbp protein levels and myelin thickness improved or fully normalized at rates defined by the relative Mbp mRNA level. Sheath length, in contrast, was affected only when mRNA levels were very low, demonstrating that sheath thickness and length are not equally coupled to Mbp mRNA level. Striking abnormalities in sheath structure also emerged with reduced mRNA levels. Unexpectedly, an increase in the density of all glial cell types arose in response to reduced Mbp mRNA levels. This investigation extends understanding of the role Mbp plays in myelin sheath elaboration, architecture, and plasticity across the mouse lifespan and illuminates a novel axis of glial cell crosstalk.

Past, present, and future of microbiome-based therapies.

Technological advances in studying the human microbiome in depth have enabled the identification of microbial signatures associated with health and disease. This confirms the crucial role of microbiota in maintaining homeostasis and the host health status. Nowadays, there are several ways to modulate the microbiota composition to effectively improve host health; therefore, the development of therapeutic treatments based on the gut microbiota is experiencing rapid growth. In this review, we summarize the influence of the gut microbiota on the development of infectious disease and cancer, which are two of the main targets of microbiome-based therapies currently being developed. We analyze the two-way interaction between the gut microbiota and traditional drugs in order to emphasize the influence of gut microbial composition on drug effectivity and treatment response. We explore the different strategies currently available for modulating this ecosystem to our benefit, ranging from 1st generation intervention strategies to more complex 2nd generation microbiome-based therapies and their regulatory framework. Lastly, we finish with a quick overview of what we believe is the future of these strategies, that is 3rd generation microbiome-based therapies developed with the use of artificial intelligence (AI) algorithms.

Aptamer-based electrochemical nanobiosensor for research and monitoring of multiple sclerosis in mice models.

Multiple sclerosis (MS) is a severe progressive autoimmune-inflammatory, demyelinating process in the central nervous system (CNS) with heterogeneous neurological symptoms appearing as a consequence of myelin break down. Myelin basic protein (MBP) makes up to 30 % of the CNS myelin [1] and it is known to be released into the cerebrospinal fluid (CSF) as a bioindicator of MS. Autoimmune encephalomyelitis (EAE) is a mice model of MS widely used for research and development of new treatments [2]. Herein, MBP specific aptamer developed for possible therapeutic purposes in mouse model [3] was applied as a bioreceptor for MBP recognition. A nanobiosensor for MBP detection and monitoring was developed by using graphene oxide (GO) nanoparticles integrated onto the screen-printed carbon electrodes (SPCE) and aptamer immobilized to create a bioactive layer on the sensor surface for MBP binding. The measurements were carried out using electrochemical impedance spectrometry (EIS). Validation studies were carried out in a biological matrix (artificial CSF) containing MBP, and MSA. The aptasensor had LOD in artificial CSF 0.01 ng/mL and showed its usability in the concentration range of 0.01 … 64 ng/mL.

Structural studies of intrinsically disordered MLL-fusion protein AF9 in complex with peptidomimetic inhibitors.

AF9 (MLLT3) and its paralog ENL(MLLT1) are members of the YEATS family of proteins with important role in transcriptional and epigenetic regulatory complexes. These proteins are two common MLL fusion partners in MLL-rearranged leukemias. The oncofusion proteins MLL-AF9/ENL recruit multiple binding partners, including the histone methyltransferase DOT1L, leading to aberrant transcriptional activation and enhancing the expression of a characteristic set of genes that drive leukemogenesis. The interaction between AF9 and DOT1L is mediated by an intrinsically disordered C-terminal ANC1 homology domain (AHD) in AF9, which undergoes folding upon binding of DOT1L and other partner proteins. We have recently reported peptidomimetics that disrupt the recruitment of DOT1L by AF9 and ENL, providing a proof-of-concept for targeting AHD and assessing its druggability. Intrinsically disordered proteins, such as AF9 AHD, are difficult to study and characterize experimentally on a structural level. In this study, we present a successful protein engineering strategy to facilitate structural investigation of the intrinsically disordered AF9 AHD domain in complex with peptidomimetic inhibitors by using maltose binding protein (MBP) as a crystallization chaperone connected with linkers of varying flexibility and length. The strategic incorporation of disulfide bonds provided diffraction-quality crystals of the two disulfide-bridged MBP-AF9 AHD fusion proteins in complex with the peptidomimetics. These successfully determined first series of 2.1-2.6 Å crystal complex structures provide high-resolution insights into the interactions between AHD and its inhibitors, shedding light on the role of AHD in recruiting various binding partner proteins. We show that the overall complex structures closely resemble the reported NMR structure of AF9 AHD/DOT1L with notable difference in the conformation of the ß-hairpin region, stabilized through conserved hydrogen bonds network. These first series of AF9 AHD/peptidomimetics complex structures are providing insights of the protein-inhibitor interactions and will facilitate further development of novel inhibitors targeting the AF9/ENL AHD domain.

SHU9119 and MBP10 are biased ligands at the human melanocortin-4 receptor.

The melanocortin-4 receptor (MC4R), a G protein-coupled receptor, is critically involved in regulating energy homeostasis as well as modulation of reproduction and sexual function. Two peptide antagonists (SHU9119 and MBP10) were derived from the endogenous agonist α-melanocyte stimulating hormone. But their pharmacology at human MC4R is not fully understood. Herein, we performed detailed pharmacological studies of SHU9119 and MBP10 on wild-type (WT) and six naturally occurring constitutively active MC4Rs. Both ligands had no or negligible agonist activity in Gαs-cAMP signaling on WT MC4R, but stimulated extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation on WT and mutant MC4Rs. Mechanistic studies revealed that SHU9119 and MBP10 stimulated ERK1/2 signaling of MC4R by different mechanisms, with SHU9119-stimulated ERK1/2 signaling mediated by phosphatidylinositol 3-kinase (PI3K) and MBP10-initiated ERK1/2 activation through PI3K and ß-arrestin. In summary, our studies demonstrated that SHU9119 and MBP10 were biased ligands for MC4R, preferentially activating ERK1/2 signaling through different mechanisms. SHU9119 acted as a biased ligand and MBP10 behaved as a biased allosteric modulator.

Use of pure recombinant human enzymes to assess the disease-causing potential of missense mutations in urea cycle disorders, applied to N-acetylglutamate synthase deficiency.

N-acetylglutamate synthase (NAGS) makes acetylglutamate, the essential activator of the first, regulatory enzyme of the urea cycle, carbamoyl phosphate synthetase 1 (CPS1). NAGS deficiency (NAGSD) and CPS1 deficiency (CPS1D) present identical phenotypes. However, they must be distinguished, because NAGSD is cured by substitutive therapy with the N-acetyl-L-glutamate analogue N-carbamyl-L-glutamate, while curative therapy of CPS1D requires liver transplantation. Since their differentiation is done genetically, it is important to ascertain the disease-causing potential of CPS1 and NAGS genetic variants. With this goal, we previously carried out site-directed mutagenesis studies with pure recombinant human CPS1. We could not do the same with human NAGS (HuNAGS) because of enzyme instability, leading to our prior utilization of a bacterial NAGS as an imperfect surrogate of HuNAGS. We now use genuine HuNAGS, stabilized as a chimera of its conserved domain (cHuNAGS) with the maltose binding protein (MBP), and produced in Escherichia coli. MBP-cHuNAGS linker cleavage allowed assessment of the enzymatic properties and thermal stability of cHuNAGS, either wild-type or hosting each one of 23 nonsynonymous single-base changes found in NAGSD patients. For all but one change, disease causation was accounted by the enzymatic alterations identified, including, depending on the variant, loss of arginine activation, increased Km Glutamate, active site inactivation, decreased thermal stability, and protein misfolding. Our present approach outperforms experimental in vitro use of bacterial NAGS or in silico utilization of prediction servers (including AlphaMissense), illustrating with HuNAGS the value for UCDs of using recombinant enzymes for assessing disease-causation and molecular pathogenesis, and for therapeutic guidance.

Customized m-RCNN and hybrid deep classifier for liver cancer segmentation and classification.

Diagnosing liver disease presents a significant medical challenge in impoverished countries, with over 30 billion individuals succumbing to it each year. Existing models for detecting liver abnormalities suffer from lower accuracy and higher constraint metrics. As a result, there is a pressing need for improved, efficient, and effective liver disease detection methods. To address the limitations of current models, this method introduces a deep liver segmentation and classification system based on a Customized Mask-Region Convolutional Neural Network (cm-RCNN). The process begins with preprocessing the input liver image, which includes Adaptive Histogram Equalization (AHE). AHE helps dehaze the input image, remove color distortion, and apply linear transformations to obtain the preprocessed image. Next, a precise region of interest is segmented from the preprocessed image using a novel deep strategy called cm-RCNN. To enhance segmentation accuracy, the architecture incorporates the ReLU activation function and the modified sigmoid activation function. Subsequently, a variety of features are extracted from the segmented image, including ResNet features, shape features (area, perimeter, approximation, and convex hull), and enhanced median binary pattern. These extracted features are then used to train a hybrid classification model, which incorporates classifiers like SqueezeNet and DeepMaxout models. The final classification outcome is determined by averaging the scores obtained from both classifiers.

Generation and characterization of cerebellar granule neurons specific knockout mice of Golli-MBP.

Golli-myelin basic proteins, encoded by the myelin basic protein gene, are widely expressed in neurons and oligodendrocytes in the central nervous system. Further, prior research has shown that Golli-myelin basic protein is necessary for myelination and neuronal maturation during central nervous system development. In this study, we established Golli-myelin basic protein-floxed mice to elucidate the cell-type-specific effects of Golli-myelin basic protein knockout through the generation of conditional knockout mice (Golli-myelin basic proteinsfl/fl; E3CreN), in which Golli-myelin basic proteins were specifically deleted in cerebellar granule neurons, where Golli-myelin basic proteins are expressed abundantly in wild-type mice. To investigate the role of Golli-myelin basic proteins in cerebellar granule neurons, we further performed histopathological analyses of these mice, with results indicating no morphological changes or degeneration of the major cellular components of the cerebellum. Furthermore, behavioral analysis showed that Golli-myelin basic proteinsfl/fl; E3CreN mice were healthy and did not display any abnormal behavior. These results suggest that the loss of Golli-myelin basic proteins in cerebellar granule neurons does not lead to cerebellar perturbations or behavioral abnormalities. This mouse model could therefore be employed to analyze the effect of Golli-myelin basic protein deletion in specific cell types of the central nervous system, such as other neuronal cells and oligodendrocytes, or in lymphocytes of the immune system.

Deciphering the Association of Epstein-Barr Virus and Its Glycoprotein M Peptide with Neuropathologies in Mice.

The reactivation of ubiquitously present Epstein-Barr virus (EBV) is known to be involved with numerous diseases, including neurological ailments. A recent in vitro study from our group unveiled the association of EBV and its 12-amino acid peptide glycoprotein M146-157 (gM146-157) with neurodegenerative diseases, viz., Alzheimer's disease (AD) and multiple sclerosis. In this study, we have further validated this association at the in vivo level. The exposure of EBV/gM146-157 to mice causes a decline in the cognitive ability with a concomitant increase in anxiety-like symptoms through behavioral assays. Disorganization of hippocampal neurons, cell shrinkage, pyknosis, and apoptotic appendages were observed in the brains of infected mice. Inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were found to be elevated in infected mouse brain tissue samples, whereas TNF-α exhibited a decline in the serum of these mice. Further, the altered levels of nuclear factor-kappa B (NF-kB) and neurotensin receptor 2 affirmed neuroinflammation in infected mouse brain samples. Similarly, the risk factor of AD, apolipoprotein E4 (ApoE4), was also found to be elevated at the protein level in EBV/gM146-157 challenged mice. Furthermore, we also observed an increased level of myelin basic protein in the brain cortex. Altogether, our results suggested an integral connection of EBV and its gM146-157 peptide to the neuropathologies.

Myelin Basic Protein Attenuates Furin-Mediated Bri2 Cleavage and Postpones Its Membrane Trafficking.

Myelin basic protein (MBP) is the second most abundant protein in the central nervous system and is responsible for structural maintenance of the myelin sheath covering axons. Previously, we showed that MBP has a more proactive role in the oligodendrocyte homeostasis, interacting with membrane-associated proteins, including integral membrane protein 2B (ITM2B or Bri2) that is associated with familial dementias. Here, we report that the molecular dynamics of the in silico-generated MBP-Bri2 complex revealed that MBP covers a significant portion of the Bri2 ectodomain, assumingly trapping the furin cleavage site, while the surface of the BRICHOS domain, which is responsible for the multimerization and activation of the Bri2 high-molecular-weight oligomer chaperone function, remains unmasked. These observations were supported by the co-expression of MBP with Bri2, its mature form, and disease-associated mutants, which showed that in mammalian cells, MBP indeed modulates the post-translational processing of Bri2 by restriction of the furin-catalyzed release of its C-terminal peptide. Moreover, we showed that the co-expression of MBP and Bri2 also leads to an altered cellular localization of Bri2, restricting its membrane trafficking independently of the MBP-mediated suppression of the Bri2 C-terminal peptide release. Further investigations should elucidate if these observations have physiological meaning in terms of Bri2 as a MBP chaperone activated by the MBP-dependent postponement of Bri2 membrane trafficking.

The restoration of hippocampal nerve de-myelination by methylcobalamin relates with the enzymatic regulation of homocysteine level in a rat model of moderate grade hepatic encephalopathy.

This article describes how methylcobalamin (MeCbl) restores nerve myelination in a moderate- grade hepatic encephalopathy (MoHE) model of ammonia neurotoxicity. The comparative profiles of myelin basic protein (MBP), homocysteine (Hcy) and methionine synthase (MS: a MeCbl- dependent enzyme) activity versus nerve myelination status were studied in the hippocampus of the control, the MoHE (developed by administering 100 mg/kg bw thioacetamide i.p. for 10 days) and the MoHE rats treated with MeCbl (500 µg/kg BW i.p.) for 7 days. Compared to those of control rats, the hippocampal CA1 and CA3 regions of the MoHE rats showed significantly lower myelinated areas and MBP immunostaining. This coincided with the deranged myelin layering in TEM images, decreased MBP protein and its transcript levels in hippocampus of MoHE rats. However, all these parameters recovered to normal levels after MeCbl treatment. MeCbl is a cofactor of MS that catalyzes the conversion of Hcy to methionine as a feeder step of methylation reactions. We observed significantly increased serum and hippocampal Hcy levels in MoHE rats, however, these levels were restored to control values with a concordant activation of MS due to MeCbl treatment. A significant recovery in neurobehavioral impairments in the MoHE rats due to MeCbl treatment was also observed. These findings suggest that MoHE pathogenesis is associated with deranged nerve myelination in the hippocampus and that MeCbl treatment is able to restore it mainly by activating MS, a MeCbl-dependent Hcy-metabolizing enzyme.

Effects of acrylamide exposure during pregnancy and lactation on the development of myelin sheath of corpus callosum in offspring rats.

Acrylamide is an alkene known to induce neurotoxicity in humans and experimental animals. However, the effects of acrylamide on the development of myelin sheath are unclear. The present study was to explore the effects of acrylamide exposure during pregnancy and lactation on the development of myelin sheath in offspring rats. Four groups of thirty-two pregnant Sprague-Dawley rats were exposed to 0, 4.5, 9 and 18 mg/kg BW acrylamide by gavage from gestational day 15 to postnatal day 13. The corpus callosum of nine offspring rats per group were dissected in postpartum day 14. Structural changes and lipid contents in myelin sheaths were examined by transmission electron microscopy(TEM) and Luxol Fast Blue staining(LFB). The expression of MBP and PLP was evaluated by immunohistochemistry and Western blotting. TEM showed that the myelin sheaths in the 18 mg/kg group were disordered compared with control group. Luxol Fast Blue staining gradually decreased with increasing acrylamide maternal exposure. The immunohistochemistry and Western Blotting results showed that maternal exposure to acrylamide caused a decreasing trend in MBP and PLP in the corpus callosum of rats at postnatal day 14. Furthermore, these reduced protein levels may be neurodevelopmental toxicity's mechanism in response to maternal exposure to acrylamide.

Phase-Dependent Adsorption of Myelin Basic Protein to Phosphatidylcholine Lipid Bilayers.

The dense packing of opposite cytoplasmic surfaces of the lipid-enriched myelin membrane, responsible for the proper saltatory conduction of nerve impulses through axons, is ensured by the adhesive properties of myelin basic protein (MBP). Although preferentially interacting with negatively charged phosphatidylserine (PS) lipids, as an intrinsically disordered protein, it can easily adapt its shape to its immediate environment and thus adsorb to domains made of zwitterionic phosphatidylcholine (PC) lipids. As the molecular-level interaction pattern between MBP and PC lipid membranes suffers from scarce characterization, an experimental and computational study of multilamellar liposomes (MLVs) composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in the presence of bovine MBP is presented here. Calorimetric and temperature-dependent UV-Vis measurements identified DPPC pretransition temperature (Tp) and calorimetric enthalpy (ΔHcal) as the physicochemical parameters most responsive to the presence of MBP. Besides suggesting an increase in ß-sheet fractions of structured MBP segments as DPPC lipids undergo from the gel (20 °C) to the fluid (50 °C) phase, FTIR spectra unraveled the significant contribution of lysine (Lys) residues in the adsorption pattern, especially when DPPC is in the fluid (50 °C) phase. In addition to highlighting the importance of Lys residues in the MBP adsorption on DPPC lipid bilayer, employing salt bridges (SBs) and hydrogen bonds (HBs), MD data suggest the crucial importance of the orientation of MBP with respect to the surface of the DPPC lipid bilayer.

Expression and Purification of Recombinant Human Mitochondrial RNA Polymerase (POLRMT) and the Initiation Factors TFAM and TFB2M.

Human mitochondrial DNA (mtDNA) encodes several components of oxidative phosphorylation responsible for the bulk of cellular energy production. The mtDNA is transcribed by a dedicated human mitochondrial RNA polymerase (POLRMT) that is structurally distinct from its nuclear counterparts, instead closely resembling the single-subunit viral RNA polymerases (e.g., T7 RNA polymerase). The initiation of transcription by POLRMT is aided by two initiation factors: transcription factor A, mitochondrial (TFAM), and transcription factor B2, mitochondrial (TFB2M). Although many details of human mitochondrial transcription initiation have been elucidated with in vitro biochemical and structural studies, much remains to be addressed relating to the mechanism and regulation of transcription. Studies of such mechanisms require reliable, high-yield, and high-purity methods for protein production, and this protocol provides the level of detail and troubleshooting tips that are necessary for a novice to generate meaningful amounts of proteins for experimental work. The current protocol describes how to purify recombinant POLRMT, TFAM, and TFB2M from Escherichia coli using techniques such as affinity column chromatography (Ni2+ and heparin), how to remove the solubility tags with TEV protease and recover untagged proteins of interest, and how to overcome commonly encountered challenges in obtaining high yield of each protein. Key features • This protocol builds upon purification methods developed by Patel lab (Ramachandran et al., 2017) and others with greater detail than previously published works. • The protocol requires several days to complete as various steps are designed to be performed overnight. • The recombinantly purified proteins have been successfully used for in vitro transcription experiments, allowing for finer control of experimental components in a minimalistic system.

Development of an Anti-HER2 Single-Chain Variable Antibody Fragment Construct for High-Yield Soluble Expression in Escherichia coli and One-Step Chromatographic Purification.

Although single-chain variable fragment (scFv) is recognized as a highly versatile scaffold of recombinant antibody fragment molecules, its overexpression in Escherichia coli often leads to the formation of inclusion bodies. To address this issue, we devised and tested four different constructs, named v21, v22, v23 and v24, for producing anti-human epidermal growth factor receptor 2 (HER2) scFv. Among them, the v24 construct obtained from N-terminal fusion of maltose-binding protein (MBP) and subsequent tobacco etch virus protease (TEV) was identified as the most efficient construct for the production of anti-HER2 scFv. Aided by an MBP tag, high-yield soluble expression was ensured and soluble scFv was liberated in cells via autonomous proteolytic cleavage by endogenously expressed TEV. The isolated scFv containing a C-terminal hexahistidine tag was purified through a one-step purification via nickel-affinity chromatography. The purified scFv exhibited a strong (nanomolar Kd) affinity to HER2 both in vitro and in cells. Structural and functional stabilities of the scFv during storage for more than one month were also assured. Given the great utility of anti-HER2 scFv as a basic platform for developing therapeutic and diagnostic agents for cancers, the v24 construct and methods presented in this study are expected to provide a better manufacturing system for producing anti-HER2 scFv with various industrial applications.

Engineering chimeric autoantibody receptor T cells for targeted B cell depletion in multiple sclerosis model: An in-vitro study.

Background: Recent evidence suggests that B cells and autoantibodies have a substantial role in the pathogenesis of Multiple sclerosis. T cells could be engineered to express chimeric autoantibody receptors (CAARs), which have an epitope of autoantigens in their extracellular domain acting as bait for trapping autoreactive B cells. This study aims to assess the function of designed CAAR T cells against B cell clones reactive to the myelin basic protein (MBP) autoantigen. Methods: T cells were transduced to express a CAAR consisting of MBP as the extracellular domain. experimental autoimmune encephalomyelitis (EAE) was induced by injecting MBP into mice. The cytotoxicity, proliferation, and cytokine production of the MBP-CAAR T cells were investigated in co-culture with B cells. Results: MBP-CAAR T cells showed higher cytotoxic activity against autoreactive B cells in all effector-to-target ratios compared to Mock T cell (empty vector-transduced T cell) and Un-T cells (un-transduced T cell). In co-cultures containing CAAR T cells, there was more proliferation and inflammatory cytokine release as compared to Un-T and Mock T cell groups. Conclusion: Based on these findings, CAAR T cells are promising for curing or modulating autoimmunity and can be served as a new approach for clone-specific B cell depletion therapy in multiple sclerosis.

Non-Invasive and Minimally Invasive Biomarkers for the Management of Eosinophilic Esophagitis beyond Peak Eosinophil Counts: Filling the Gap in Clinical Practice.

Eosinophilic esophagitis (EoE) is a chronic esophageal disease that needs lifelong management and follow-up. The diagnosis requires an upper endoscopy with at least one esophageal biopsy demonstrating >15 eosinophils/high-power field, and often occurs with a diagnostic delay of up to ten years, partly due to the absence of valid non-invasive screening tools. In addition, serial upper endoscopies with esophageal biopsies are mandatory to assess the efficacy of any ongoing treatment in patients with EoE. These procedures are invasive, costly, and, when performed without sedation, are often poorly tolerated by patients. Therefore, there is the clinical need to identify reliable non-invasive or minimally invasive biomarkers that could be used to assess disease activity in clinical practice as a surrogate of peak eosinophil counts on esophageal biopsies. This review summarizes evidence on investigational non-invasive or minimally invasive biomarkers for the diagnosis and follow-up of EoE to report on the state of the art in the field and support future research. We discussed eosinophil-derived mediators including eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN, also known as eosinophil protein X), eosinophil peroxidase (EPO), and major basic protein (MBP) as well as other promising non-eosinophil-derived biomarkers. Although several studies have shown the utility of most biomarkers collected from the serum, esophageal luminal secretions, and feces of EoE patients, numerous limitations currently hamper the integration of such biomarkers in clinical practice. Future studies should aim at validating the utility of non-invasive and minimally invasive biomarkers using rigorous protocols and updated consensus criteria for EoE.

Identification of potential functional peptides involved in demyelinating injury in the central nervous system.

Multiple sclerosis (MS) is a chronic inflammatory neurologic disease characterized by the demyelinating injury of the central nervous system (CNS). It was reported that the mutant peptide came from myelin proteolipid protein (PLP) and myelin basic protein (MBP) might play a critical role in immunotherapy function of MS. However, endogenous peptides in demyelinating brain tissue of MS and their role in the pathologic process of MS have not been revealed. Here, we performed peptidomic analysis of freshly isolated corpus callosum (CC) from the brains of CPZ-treated mice and normal diet controls of male C57BL/6 mice by LC-MS/MS. Identified a total of 217 peptides were expressed at different levels in MS mice model compared with controls. By performed GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis, we found that the precursor protein of these differently expressed peptides (DEPs) were associated with myelin sheath and oxidative phosphorylation. Our study is the first brain peptidomic of MS mice model, revealing the distinct features of DEPs in demyelination brain tissue. These DPEs may provide further insight into the pathogenesis and complexity of MS, which would facilitate the discovery of the potential novel and effective strategy for the treatment of MS.

DBP exposure induces thyroid inflammatory impairment through activating AKT/NF-κB/NLRP3 signaling.

Previous studies exhibited reproductive and neurodevelopmental toxicity in rats exposed to Di-n-butyl phthalate (DBP). However, the effects of DBP exposure on the other endocrine organ are still unclear. This study aimed to assess the impact of DBP exposure on the thyroid of male rats and the associated mechanisms. Here, rats were respectively treated with DBP at 0 (control), 50 (low dose), 250 (medium dose), or 500 (high dose) mg/kg/day dissolved in 1 ml quantity of corn oil by intragastrical administration for two weeks. The results demonstrated that the proliferation and inflammatory response changes were significantly different compared to the control. In vivo DBP is mainly converted to mono-n-butyl phthalate (MBP), an active form producing untoward reactions of DBP exposure. Therefore, for in vitro experiments, we treated the thyroid follicular epithelial cell line (Nthy-ori 3-1) in a temporal gradient using 1 mM MBP. Further in vitro studies showed that MBP exposure upregulated tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), as well as interleukin-1ß (IL-1ß) by activating AKT/NF-κB/NLRP3 signaling. Meanwhile, we detected that Pellino2 (Peli2) played an essential role in promoting the activation of NLRP3 inflammasome. Briefly speaking, this study confirmed that DBP exposure caused impaired thyroid structure and thyroid inflammation in male rats, which offered new views into the harm of DBP exposure on the endocrine organ.

Expression and immunogenicity of recombinant porcine epidemic diarrhea virus Nsp9.

Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea, vomiting, dehydration, and high mortality in newborn piglets, which leads to significant economic losses. Coronavirus nonstructural protein 9 (Nsp9) is an essential RNA binding protein for coronavirus replication, which renders it a promising candidate for developing antiviral drugs and diagnosis targeting PEDV. In this study, PEDV Nsp9 protein fused with MBP protein and His-tag were expressed and purified in Escherichia coli. Furthermore, immunization of MBP-Nsp9 enhances both humoral and cellular immunity responses as compared with that of His-Nsp9 protein. Finally, the swine immunization showed that Nsp9 protein could stimulate the swine immunity system to carry out humoral immunity, and the generated antibody could inhibit the proliferation of PEDV in Vero cells. Altogether, our data provide direct evidence for the immunogenicity of PEDV Nsp9, which sheds light on the future developments of anti-PEDV drugs and vaccines for PED prevention.