Quantifying Protein-mRNA Interactions in Single Live Cells.

Specific binding proteins are crucial for the correct spatiotemporal expression of mRNA. To understand this process, a method is required to characterize RNA-protein interactions in single living cells with subcellular resolution. We combined endogenous single RNA and protein detection with two-photon fluorescence fluctuation analysis to measure the average number of proteins bound to mRNA at specific locations within live cells. We applied this to quantify the known binding of zipcode binding protein 1 (ZBP1) and ribosomes to ß-actin mRNA within subcellular compartments of primary fibroblasts and neurons. ZBP1-mRNA binding did not occur in nuclei, contrary to previous conclusions. ZBP1 interaction with ß-actin mRNA was enhanced perinuclearly in neurons compared to fibroblasts. Cytoplasmic ZBP1 and ribosome binding to the mRNA were anti-correlated depending on their location in the cell. These measurements support a mechanism whereby ZBP1 inhibits translation of localizing mRNA until its release from the mRNA peripherally, allowing ribosome binding.

Exploring heterogeneous expression of beta-actin (ACTB) in bladder cancer by producing a monoclonal antibody 6D6.

BACKGROUND: To predict outcomes and identify potential therapeutic targets for cancers, it is critical to find novel specific biomarkers. The objective of this study was to search for and explore novel bladder cancer-associated protein biomarkers. METHODS: A library of monoclonal antibodies (mAbs) against the JAM-ICR cell line was first generated, and clones with high affinity were selected. Hybridomas were screened using bladder cancer (BLCA) cell lines and normal cells. The target of the selected mAb was then characterized through immunoaffinity purification, western blotting, and mass spectrometry analysis. Expression of the target antigen was assessed by flow cytometry and IHC methods. Several databases were also used to evaluate the target antigen in BLCA and other types of cancers. RESULTS: Based on screenings, a 6D6 clone was selected that recognized an isoform of beta-actin (ACTB). Our data showed that ACTB expression on different cell lines was heterogeneous and varied significantly from low to high intensity. 6D6 bound strongly to epithelial cells while showing weak to no reactivity to stromal, endothelial, and smooth muscle cells. There was no association between ACTB intensity and related prognostic factors in BLCA. In silico evaluations revealed a significant correlation between ACTB and overexpressed genes and biomarkers in BLCA. Additionally, the differential expression of ACTB in tumor and healthy tissue as well as its correlation with survival time in a number of cancers were shown. CONCLUSIONS: The heterogeneous expression of ACTB may suggest the potential value of this marker in the diagnosis or prognosis of cancer.

The Role of Trp79 in ß-Actin on Histidine Methyltransferase SETD3 Catalysis.

Nτ -methylation of His73 in actin by histidine methyltransferase SETD3 plays an important role in stabilising actin filaments in eukaryotes. Mutations in actin and overexpression of SETD3 have been related to human diseases, including cancer. Here, we investigated the importance of Trp79 in ß-actin on productive human SETD3 catalysis. Substitution of Trp79 in ß-actin peptides by its chemically diverse analogues reveals that the hydrophobic Trp79 binding pocket modulates the catalytic activity of SETD3, and that retaining a bulky and hydrophobic amino acid at position 79 is important for efficient His73 methylation by SETD3. Molecular dynamics simulations show that the Trp79 binding pocket of SETD3 is ideally shaped to accommodate large and hydrophobic Trp79, contributing to the favourable release of water molecules upon binding. Our results demonstrate that the distant Trp79 binding site plays an important role in efficient SETD3 catalysis, contributing to the identification of new SETD3 substrates and the development of chemical probes targeting the biomedically important SETD3.

Post-zygotic ACTB mutations underlie congenital smooth muscle hamartomas.

BACKGROUND: Congenital smooth muscle hamartomas (CSMHs) are benign lesions that share clinical and histopathological features with Becker nevus, a mosaic disorder associated with post-zygotic ACTB mutations. Given the clinical and histopathological overlap between CSMH and Becker nevus, we hypothesized that post-zygotic mutations in ACTB may underlie CSMH. METHODS: Direct sequencing of ACTB gene in affected and unaffected tissue isolated from one case of hemihypertrichosis and hemihypertrophy corresponding to giant segmental CSMH and hemihypertrophy. This was followed by direct sequencing with and without enrichment assay for hotspot ACTB mutations in affected tissue from 12 samples of isolated CSMH from unrelated individuals. RESULTS: In total we identified somatic missense ACTB mutations in 9 out of 13 CSMHs (69%). Mutations were either novel or previously reported in Becker nevi and Becker nevus syndrome. CONCLUSIONS: CSMHs result from post-zygotic ACTB mutations. This study proves that CSMHs and Becker nevi are nosologically related, and expand the phenotypic spectrum of ACTB mutations.

Knockout of ACTB and ACTG1 with CRISPR/Cas9(D10A) Technique Shows that Non-Muscle ß and γ Actin Are Not Equal in Relation to Human Melanoma Cells' Motility and Focal Adhesion Formation.

Non-muscle actins have been studied for many decades; however, the reason for the existence of both isoforms is still unclear. Here we show, for the first time, a successful inactivation of the ACTB (CRISPR clones with inactivated ACTB, CR-ACTB) and ACTG1 (CRISPR clones with inactivated ACTG1, CR-ACTG1) genes in human melanoma cells (A375) via the RNA-guided D10A mutated Cas9 nuclease gene editing [CRISPR/Cas9(D10A)] technique. This approach allowed us to evaluate how melanoma cell motility was impacted by the lack of either ß actin coded by ACTB or γ actin coded by ACTG1. First, we observed different distributions of ß and γ actin in the cells, and the absence of one actin isoform was compensated for via increased expression of the other isoform. Moreover, we noted that γ actin knockout had more severe consequences on cell migration and invasion than ß actin knockout. Next, we observed that the formation rate of bundled stress fibers in CR-ACTG1 cells was increased, but lamellipodial activity in these cells was impaired, compared to controls. Finally, we discovered that the formation rate of focal adhesions (FAs) and, subsequently, FA-dependent signaling were altered in both the CR-ACTB and CR-ACTG1 clones; however, a more detrimental effect was observed for γ actin-deficient cells. Our research shows that both non-muscle actins play distinctive roles in melanoma cells' FA formation and motility.

Fabry disease: characterisation of the plasma proteome pre- and post-enzyme replacement therapy.

BACKGROUND: Fabry disease is characterised by the progressive accumulation of globotriaosylceramide (Gb3) and related glycosphingolipids in vascular endothelial cells. Enzyme replacement therapy (ERT) clears this accumulation. We analysed plasma proteome profiles before and after ERT to characterise its molecular pathology. METHODS: Two-dimensional electrophoresis and matrix-assisted laser desorption/ionisation-time of flight tandem mass spectrometry (MALDI-TOF MS) and tandem mass spectrometry (MS/MS) were done using plasma samples before and after ERT in eight patients with classical Fabry disease RESULTS: After short-term ERT (4-12 months), the levels of 15 plasma proteins involved in inflammation, oxidative and ischaemic injury, or complement activation were reduced significantly. Among them, ß-actin (ACTB), inactivated complement C3b (iC3b), and C4B were elevated significantly in pre-ERT Fabry disease plasma compared with control plasma. After longer-term ERT (46-96 months), iC3b levels gradually decreased, whereas the levels of other proteins varied. The gradual reduction of iC3b was comparable to that of Gb3 levels. In addition, iC3b increased significantly in pre-ERT Fabry disease mouse plasma, and C3 deposits were notable in renal tissues of pre-enzyme replacement therapy patients. CONCLUSION: These results indicated that C3-mediated complement activation might be altered in Fabry disease and ERT might promote its stabilisation.

Dystonia-deafness syndrome caused by a ß-actin gene mutation and response to deep brain stimulation.

INTRODUCTION: Dystonia-deafness syndrome is a distinct clinical presentation within the dystonia-spectrum. Although several genetic and acquired causes have been reported, etiology remains unknown in the majority of patients. OBJECTIVES: To describe two patients with dystonia-deafness syndrome due to a beta-actin gene mutation. METHODS: We report on disease course, genetic testing, and management of 2 patients, mother and daughter, presenting with dystonia-deafness syndrome. RESULTS: After exclusion of known dystonia-deafness syndrome causes, whole-exome sequencing revealed a beta-actin gene mutation (p.Arg183Trp) in both patients. Although beta-actin gene mutations are generally associated with developmental Baraitser-Winter syndrome, dystonia-deafness syndrome has been reported once in identical twin brothers. Bilateral GPi-DBS led to a significant decrease of dystonia and regain of independency in our patients. CONCLUSION: The p.Arg183Trp mutation in the beta-actin gene is associated with the clinical presentation of dystonia-deafness syndrome, even with only minimal or no developmental abnormalities of Baraitser-Winter syndrome. GPi-DBS should be considered to ameliorate the invalidating dystonia in these patients. © 2016 International Parkinson and Movement Disorder Society.

Peri-conceptional obesogenic exposure induces sex-specific programming of disease susceptibilities in adult mouse offspring.

Vulnerability of the fetus upon maternal obesity can potentially occur during all developmental phases. We aimed at elaborating longer-term health outcomes of fetal overnutrition during the earliest stages of development. We utilized Naval Medical Research Institute (NMRI) mice to induce pre-conceptional and gestational obesity and followed offspring outcomes in the absence of any postnatal obesogenic influences. Male adult offspring developed overweight, insulin resistance, hyperleptinemia, hyperuricemia and hepatic steatosis; all these features were not observed in females. Instead, they showed impaired fasting glucose and a reduced fat mass and adipocyte size. Influences of the interaction of maternal diet∗sex concerned offspring genes involved in fatty liver disease, lipid droplet size regulation and fat mass expansion. These data suggest that a peri-conceptional obesogenic exposure is sufficient to shape offspring gene expression patterns and health outcomes in a sex- and organ-specific manner, indicating varying developmental vulnerabilities between sexes towards metabolic disease in response to maternal overnutrition.

DDX6 post-transcriptionally down-regulates miR-143/145 expression through host gene NCR143/145 in cancer cells.

In various human malignancies, widespread dysregulation of microRNA (miRNA) expression is reported to occur and affects various cell growth programs. Recent studies suggest that the expression levels of miRNAs that act as tumor suppressors are frequently reduced in cancers because of chromosome deletions, epigenetical changes, aberrant transcription, and disturbances in miRNA processing. MiR-143 and -145 are well-recognized miRNAs that are highly expressed in several tissues, but down-regulated in most types of cancers. However, the mechanism of this down-regulation has not been investigated in detail. Here, we show that DEAD-box RNA helicase 6, DDX6 (p54/RCK), post-transcriptionally down-regulated miR-143/145 expression by prompting the degradation of its host gene product, NCR143/145 RNA. In human gastric cancer cell line MKN45, DDX6 protein was abundantly expressed and accumulated in processing bodies (P-bodies). DDX6 preferentially increased the instability of non-coding RNA, NCR143/145, which encompasses the miR-143/145 cluster, and down-regulated the expression of mature miR-143/145. In human monocytic cell line THP-1, lipopolysaccharide treatment promoted the assembly of P-bodies and down-regulated the expression of NCR143/145 and its miR-143/145 rapidly. In these cells, cycloheximide treatment led to a loss of P-bodies and to an increase in NCR143/145 RNA stability, thus resulting in up-regulation of miR-143/145 expression. These data demonstrate that DDX6 contributed to the control of NCR143/145 RNA stability in P-bodies and post-transcriptionally regulated miR-143/145 expression in cancer cells.

S-nitrosylation and S-glutathionylation of GAPDH: Similarities, differences, and relationships.

The aim of this work was to compare the effect of reversible post-translational modifications, S-nitrosylation and S-glutathionylation, on the properties of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and to reveal the mechanism of the relationship between these modifications. Comparison of S-nitrosylated and S-glutathionylated GAPDH showed that both modifications inactivate the enzyme and change its spatial structure, decreasing the thermal stability of the protein and increasing its sensitivity to trypsin cleavage. Both modifications are reversible in the presence of dithiothreitol, however, in the presence of reduced glutathione and glutaredoxin 1, the reactivation of S-glutathionylated GAPDH is much slower (10% in 2 h) compared to S-nitrosylated GAPDH (60% in 10 min). This suggests that S-glutathionylation is a much less reversible modification compared to S-nitrosylation. Incubation of HEK 293 T cells in the presence of H2O2 or with the NO donor diethylamine NONOate results in accumulation of sulfenated GAPDH (by data of Western blotting) and S-glutathionylated GAPDH (by data of immunoprecipitation with anti-GSH antibodies). Besides GAPDH, a protein of 45 kDa was found to be sulfenated and S-glutathionylated in the cells treated with H2O2 or NO. This protein was identified as beta-actin. The results of this study confirm the previously proposed hypothesis based on in vitro investigations, according to which S-nitrosylation of the catalytic cysteine residue (Cys152) of GAPDH with subsequent formation of cysteine sulfenic acid at Cys152 may promote its S-glutathionylation in the presence of cellular GSH. Presumably, the mechanism may be valid in the case of beta-actin.

Cytoplasmic Beta and Gamma Actin Isoforms Reorganization and Regulation in Tumor Cells in Culture and Tissue.

The cytoplasmic actin isoforms (ß- and γ-actins) contribute greatly to cellular processes such as cel-cell and cell-matrix interactions, as well as cell polarization, motility and division. Distinct isoforms modulations are linked to serious pathologies, so investigations of underlying mechanisms would be of major relevance not only for fundamental research but also for clinical applications. Therefore, the study of the relevant mechanisms of change in the isoform's balance is important for basic research and for clinical studies. The disruption of actin cytoskeleton and intercellular adhesions contribute to the neoplastic transformation, as it is important for the tumor growth, invasiveness and metastasis. Cytoplasmic actins display the functional diversity: ß-actin is responsible for contractility, whereas γ-actin participates in the submembrane flexible cortex organization and direction cell motility. The involvement of ß- and γ-actin in cell architecture, motility, division, and adhesion junctions in normal cells is not equivalent, and the major question was following: whether isoform ratio and the distribution in the cell corresponds to pathological function. Significant data were obtained in the study of tumor and normal cells in culture, as well as on clinical material of human tissues, and via selective regulation of ß- and γ-actin's expression. Investigation of the actins' diversity and function in cancers may help to choose the benefit treatment strategies, and to design new therapies.

Characterization of the mercury-binding proteins in tuna and salmon sashimi: Implications for health risk of mercury in food.

Fish consumption is one of the major ways through which humans receive exposure to mercury (Hg). The existing forms of Hg in food, particularly Hg bound to proteins, may affect the absorption of Hg by humans and subsequently its potentially toxic effects. However, the knowledge regarding Hg-binding proteins in edible fish muscle is scarce. In the present study, salmon and tuna fish muscles, collected from seven different regions and countries, were analyzed using metallomics- and proteomics-based techniques. The concentration of Hg in sashimi samples ranged from 4.4 to 317.4 ng/g. Size exclusion chromatography (SEC) coupled with inductively coupled plasma mass spectrometer (ICP-MS) showed that beta-actin was a novel Hg-binding protein from the fish muscles, and this protein could also bind bismuth (Bi), silver (Ag), and copper (Cu). Hg bound to beta-actin accounted for approximately 30.2-37.6% of the total Hg in the tuna muscles and was significantly correlated to total Hg in the fish muscles (r = 0.98, p < 0.01) and in the fraction of soluble proteins (r = 0.94, p < 0.01). These findings suggest that proteins act as the main Hg accumulation sites in edible fish; thus, increasing human exposure to Hg following gastrointestinal digestion.

ACTB and GAPDH appear at multiple SDS-PAGE positions, thus not suitable as reference genes for determining protein loading in techniques like Western blotting.

We performed polyacrylamide gel electrophoresis of human proteins with sodium dodecyl sulfate, isolated proteins at multiple positions, and then used liquid chromatography and tandem mass spectrometry (LC-MS/MS) to determine the protein identities. Although beta-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are 41.7 and 36 kDa proteins, respectively, LC-MS/MS identified their peptides at all the positions studied. The National Center for Biotechnology Information (USA) database lists only one ACTB mRNA but five GAPDH mRNAs and one noncoding RNA. The five GAPDH mRNAs encode three protein isoforms, while our bioinformatics analysis identified a 17.6 kDa isoform encoded by the noncoding RNA. All LC-MS/MS-identified GAPDH peptides at all positions studied are unique, but some of the identified ACTB peptides are shared by ACTC1, ACTBL2, POTEF, POTEE, POTEI, and POTEJ. ACTC1 and ACTBL2 belong to the ACT family with significant similarities to ACTB in protein sequence, whereas the four POTEs are ACTB-containing chimeric genes with the C-terminus of their proteins highly similar to the ACTB. These data lead us to conclude that GAPDH and ACTB are poor reference genes for determining the protein loading in such techniques as Western blotting, a leading role these two genes have been playing for decades in biomedical research.

Responses of oral-microflora-exposed dental pulp to capping with a triple antibiotic paste or calcium hydroxide cement in mouse molars.

INTRODUCTION: Responses of oral-microflora-exposed dental pulp to a triple antibiotic paste (TAP), a mixture of ciprofloxacin, metronidazole, and minocycline in ointment with macrogol and propylene glycol, remain to be fully clarified at the cellular level. This study aimed to elucidate responses of oral-microflora-exposed dental pulp to capping with TAP in mouse molars. METHODS: A cavity was prepared on the first molars of 6-week-old mice to expose the dental pulp for 24 h. The exposed pulp was capped with TAP (TAP group) or calcium hydroxide cement (CH group), in addition to the combination of macrogol (M) and propylene glycol (P) (MP, control group), followed by a glass ionomer cement filling. The samples were collected at intervals of 1, 2, and 3 weeks, and immunohistochemistry for nestin and Ki-67 and deoxyuride-5'-triphosphate biotin nick end labeling (TUNEL) assay were performed in addition to quantitative real-time polymerase chain reaction (qRT-PCR) analyses. RESULTS: The highest occurrence rate of pulp necrosis was found in the control group followed by the CH group at Weeks 2 and 3, whereas the highest occurrence rate of healed areas in the dental pulp was observed in the TAP group at each time point. Tertiary dentin formation was first observed in the dental pulp of the TAP group at Week 2. In contrast, bone-like and/or fibrous tissues were frequently observed in the CH group. qRT-PCR analyses clarified that TAP activated the stem and dendritic cells at Weeks 1 and 2, respectively. CONCLUSIONS: The use of TAP as a pulp-capping agent improved the healing process of oral-microflora-exposed dental pulp in mouse molars.

Induction of intrinsic apoptotic signaling pathway in A549 lung cancer cells using silver nanoparticles from Gossypium hirsutum and evaluation of in vivo toxicity.

In the past decade, the research communities raised wide concerns on using medicinal plants for synthesis of nanomaterials due to its effective biological activity, lower side effects and also eco-friendly manner. Our previous report concentrated on the biomedical efficacy of fine characterized silver nanoparticles (AgNPs) from Gossypium hirsutum (cotton) leaf extract. Further, the current examination is planned to reveal the molecular mechanisms involving for activation of mitochondria-mediated signaling pathway by AgNPs in human lung cancer cells (A549) using various biological endpoints such as apoptotic induction by HOECHST 33342, AO/EtBr and Rhodamine 123 staining, cell cycle analysis using flow cytometry, gene and protein expressions by RT-PCR and immunoblotting respectively. This study was further extended to identify the toxicity of AgNPs using an animal model. Interestingly, we observed that A549 cells treated with AgNPs resulted in G2/M arrest and ultimately leads to induction of apoptosis cell death. Moreover, gene analysis demonstrated that diminished expression of anti-apoptotic (Bcl-2) and enhanced expression of pro-apoptotic (Bax) mitochondrial genes. The alterations in the gene pattern may interrupt of mitochondrial membrane potential which facilitates the releasing of cytochrome c (cyt c) into cytosol. The cyt c act as a key molecule for activation of caspases (9 and 3) to initiate intrinsic apoptotic signaling cell death process. The histological analysis proven the application of AgNPs in nanomedicine is quietly harmless and would not cause any discernible stress like swelling and inflammation to the organs of mice. Taken together, this investigation may provide solid evidence for cotton crop mediated AgNPs induced apoptosis cell death pathway and offer a novel approach for cancer therapy.

Molecular characterization of the giant freshwater prawn (Macrobrachium rosenbergii) beta-actin gene promoter.

Constitutive promoters are important tools for gene function studies and transgenesis. The Beta-actin (actb1) gene promoter has been isolated from many species but remains to be cloned from the giant freshwater prawn (Macrobrachium rosenbergii). In this study, we cloned and characterized the Mractb1 gene promoter. Two alternative promoters were identified for the Mractb1 gene, which direct the generation of two transcripts with different 5' untranslated regions. Three CpG islands were predicted in the upstream sequence, which are intimately related to transcription initiation and promoter activity. In addition to the CCAAT-box and the CArG-box, molecular dissection of the flanking sequence revealed the existence of one negative and two positive elements in the upstream region and the first intron. Finally, the Mractb1 promoter demonstrated comparative activity to the carp (Cyprinus carpio) actb1 promoter. Our investigations provide a valuable genetic tool for gene function studies and shed light on the regulation of the Mractb1 gene.

Na+ , K+ -ATPase activity in children with autism spectrum disorder: Searching for the reason(s) of its decrease in blood cells.

Na+ , K+ -ATPase (NKA) activity, which establishes the sodium and potassium gradient across the cell membrane and is instrumental in the propagation of the nerve impulses, is altered in a number of neurological and neuropsychiatric disorders, including autism spectrum disorders (ASD). In the present work, we examined a wide range of biochemical and cellular parameters in the attempt to understand the reason(s) for the severe decrease in NKA activity in erythrocytes of ASD children that we reported previously. NKA activity in leukocytes was found to be decreased independently from alteration in plasma membrane fluidity. The different subunits were evaluated for gene expression in leukocytes and for protein expression in erythrocytes: small differences in gene expression between ASD and typically developing children were not apparently paralleled by differences in protein expression. Moreover, no gross difference in erythrocyte plasma membrane oxidative modifications was detectable, although oxidative stress in blood samples from ASD children was confirmed by increased expression of NRF2 mRNA. Interestingly, gene expression of some NKA subunits correlated with clinical features. Excess inhibitory metals or ouabain-like activities, which might account for NKA activity decrease, were ruled out. Plasma membrane cholesterol, but not phosphatidylcholine and phosphatidlserine, was slighty decreased in erythrocytes from ASD children. Although no compelling results were obtained, our data suggest that alteration in the erytrocyte lipid moiety or subtle oxidative modifications in NKA structure are likely candidates for the observed decrease in NKA activity. These findings are discussed in the light of the relevance of NKA in ASD. Autism Res 2018, 11: 1388-1403. © 2018 International Society for Autism Research, Wiley Periodicals, Inc. LAY SUMMARY: The activity of the cell membrane enzyme NKA, which is instrumental in the propagation of the nerve impulses, is severely decreased in erythrocytes from ASD children and in other brain disorders, yet no explanation has been provided for this observation. We strived to find a biological/biochemical cause of such alteration, but most queries went unsolved because of the complexity of NKA regulation. As NKA activity is altered in many brain disorders, we stress the relevance of studies aimed at understanding its regulation in ASD.

Effects of Sub-Chronic MPTP Exposure on Behavioral and Cognitive Performance and the Microbiome of Wild-Type and mGlu8 Knockout Female and Male Mice.

Motor dysfunction is a hallmark of Parkinson's disease (PD); however, non-motor symptoms such as gastrointestinal dysfunction often arise prior to motor symptoms. Alterations in the gut microbiome have been proposed as the earliest event in PD pathogenesis. PD symptoms often demonstrate sex differences. Glutamatergic neurotransmission has long been linked to PD pathology. Metabotropic glutamate receptors (mGlu), a family of G protein-coupled receptors, are divided into three groups, with group III mGlu receptors mainly localized presynaptically where they can inhibit glutamate release in the CNS as well as in the gut. Additionally, the gut microbiome can communicate with the CNS via the gut-brain axis. Here, we assessed whether deficiency of metabotropic glutamate receptor 8 (mGlu8), group III mGlu, modulates the effects of the neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), on behavioral and cognitive performance in female and male mice. We studied whether these effects are associated with changes in striatal tyrosine hydroxylase (TH) levels and the gut microbiome. Two-week sub-chronic MPTP increased activity of female and male wild-type (WT) and mGlu8 knockout (KO) mice in the open field. MPTP also showed genotype- and sex-dependent effects. MPTP increased the time WT, but not KO, females and males spent exploring objects. In WT mice, MPTP improved sensorimotor function in males but impaired it in females. Further, MPTP impaired cued fear memory in WT, but not KO, male mice. MPTP reduced striatal TH levels in WT and KO mice but these effects were only pronounced in males. MPTP treatment and genotype affected the diversity of the gut microbiome. In addition, there were significant associations between microbiome α-diversity and sensorimotor performance, as well as microbiome composition and fear learning. These results indicate that specific taxa may directly affect motor and fear learning or that the same physiological effects that enhance both forms of learning also alter diversity of the gut microbiome. MPTP's effect on motor and cognitive performance may then be, at least in part, be mediated by the gut microbiome. These data also support mGlu8 as a novel therapeutic target for PD and highlight the importance of including both sexes in preclinical studies.

A High-Throughput Organoid Microinjection Platform to Study Gastrointestinal Microbiota and Luminal Physiology.

Background & Aims: The human gut microbiota is becoming increasingly recognized as a key factor in homeostasis and disease. The lack of physiologically relevant in vitro models to investigate host-microbe interactions is considered a substantial bottleneck for microbiota research. Organoids represent an attractive model system because they are derived from primary tissues and embody key properties of the native gut lumen; however, access to the organoid lumen for experimental perturbation is challenging. Here, we report the development and validation of a high-throughput organoid microinjection system for cargo delivery to the organoid lumen and high-content sampling. Methods: A microinjection platform was engineered using off-the-shelf and 3-dimensional printed components. Microinjection needles were modified for vertical trajectories and reproducible injection volumes. Computer vision (CVis) and microfabricated CellRaft Arrays (Cell Microsystems, Research Triangle Park, NC) were used to increase throughput and enable high-content sampling of mock bacterial communities. Modeling preformed using the COMSOL Multiphysics platform predicted a hypoxic luminal environment that was functionally validated by transplantation of fecal-derived microbial communities and monocultures of a nonsporulating anaerobe. Results: CVis identified and logged locations of organoids suitable for injection. Reproducible loads of 0.2 nL could be microinjected into the organoid lumen at approximately 90 organoids/h. CVis analyzed and confirmed retention of injected cargos in approximately 500 organoids over 18 hours and showed the requirement to normalize for organoid growth for accurate assessment of barrier function. CVis analyzed growth dynamics of a mock community of green fluorescent protein- or Discosoma sp. red fluorescent protein-expressing bacteria, which grew within the organoid lumen even in the presence of antibiotics to control media contamination. Complex microbiota communities from fecal samples survived and grew in the colonoid lumen without appreciable changes in complexity. Conclusions: High-throughput microinjection into organoids represents a next-generation in vitro approach to investigate gastrointestinal luminal physiology and the gastrointestinal microbiota.

The Survival of Motor Neuron Protein Acts as a Molecular Chaperone for mRNP Assembly.

Spinal muscular atrophy (SMA) is a motor neuron disease caused by reduced levels of the survival of motor neuron (SMN) protein. SMN is part of a multiprotein complex that facilitates the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs). SMN has also been found to associate with mRNA-binding proteins, but the nature of this association was unknown. Here, we have employed a combination of biochemical and advanced imaging methods to demonstrate that SMN promotes the molecular interaction between IMP1 protein and the 3' UTR zipcode region of ß-actin mRNA, leading to assembly of messenger ribonucleoprotein (mRNP) complexes that associate with the cytoskeleton to facilitate trafficking. We have identified defects in mRNP assembly in cells and tissues from SMA disease models and patients that depend on the SMN Tudor domain and explain the observed deficiency in mRNA localization and local translation, providing insight into SMA pathogenesis as a ribonucleoprotein (RNP)-assembly disorder.