Cutting Edge: Neutrophils License the Maturation of Monocytes into Effective Antifungal Effectors.

Neutrophils are critical for the direct eradication of Aspergillus fumigatus conidia, but whether they mediate antifungal defense beyond their role as effectors is unclear. In this study, we demonstrate that neutrophil depletion impairs the activation of protective antifungal CCR2+ inflammatory monocytes. In the absence of neutrophils, monocytes displayed limited differentiation into monocyte-derived dendritic cells, reduced formation of reactive oxygen species, and diminished conidiacidal activity. Upstream regulator analysis of the transcriptional response in monocytes predicted a loss of STAT1-dependent signals as the potential basis for the dysfunction seen in neutrophil-depleted mice. We find that conditional removal of STAT1 on CCR2+ cells results in diminished antifungal monocyte responses, whereas exogenous administration of IFN-γ to neutrophil-depleted mice restores monocyte-derived dendritic cell maturation and reactive oxygen species production. Altogether, our findings support a critical role for neutrophils in antifungal immunity not only as effectors but also as important contributors to antifungal monocyte activation, in part by regulating STAT1-dependent functions.

An essential domain of an early-diverged RNA polymerase II functions to accurately decode a primitive chromatin landscape.

A unique feature of RNA polymerase II (RNA pol II) is its long C-terminal extension, called the carboxy-terminal domain (CTD). The well-studied eukaryotes possess a tandemly repeated 7-amino-acid sequence, called the canonical CTD, which orchestrates various steps in mRNA synthesis. Many eukaryotes possess a CTD devoid of repeats, appropriately called a non-canonical CTD, which performs completely unknown functions. Trypanosoma brucei, the etiologic agent of African Sleeping Sickness, deploys an RNA pol II that contains a non-canonical CTD to accomplish an unusual transcriptional program; all protein-coding genes are transcribed as part of a polygenic precursor mRNA (pre-mRNA) that is initiated within a several-kilobase-long region, called the transcription start site (TSS), which is upstream of the first protein-coding gene in the polygenic array. In this report, we show that the non-canonical CTD of T. brucei RNA pol II is important for normal protein-coding gene expression, likely directing RNA pol II to the TSSs within the genome. Our work reveals the presence of a primordial CTD code within eukarya and indicates that proper recognition of the chromatin landscape is a central function of this RNA pol II-distinguishing domain.

Insulin-like growth factor receptor signaling in breast tumor epithelium protects cells from endoplasmic reticulum stress and regulates the tumor microenvironment.

BACKGROUND: Early analyses of human breast cancer identified high expression of the insulin-like growth factor type 1 receptor (IGF-1R) correlated with hormone receptor positive breast cancer and associated with a favorable prognosis, whereas low expression of IGF-1R correlated with triple negative breast cancer (TNBC). We previously demonstrated that the IGF-1R acts as a tumor and metastasis suppressor in the Wnt1 mouse model of TNBC. The mechanisms for how reduced IGF-1R contributes to TNBC phenotypes is unknown. METHODS: We analyzed the METABRIC dataset to further stratify IGF-1R expression with patient survival and specific parameters of TNBC. To investigate molecular events associated with the loss of IGF-1R function in breast tumor cells, we inhibited IGF-1R in human cell lines using an IGF-1R blocking antibody and analyzed MMTV-Wnt1-mediated mouse tumors with reduced IGF-1R function through expression of a dominant-negative transgene. RESULTS: Our analysis of the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) dataset revealed association between low IGF-1R and reduced overall patient survival. IGF-1R expression was inversely correlated with patient survival even within hormone receptor-positive breast cancers, indicating reduced overall patient survival with low IGF-1R was not due simply to low IGF-1R expression within TNBCs. Inhibiting IGF-1R in either mouse or human tumor epithelial cells increased reactive oxygen species (ROS) production and activation of the endoplasmic reticulum stress response. IGF-1R inhibition in tumor epithelial cells elevated interleukin (IL)-6 and C-C motif chemokine ligand 2 (CCL2) expression, which was reversed by ROS scavenging. Moreover, the Wnt1/dnIGF-1R primary tumors displayed a tumor-promoting immune phenotype. The increased CCL2 promoted an influx of CD11b+ monocytes into the primary tumor that also had increased matrix metalloproteinase (MMP)-2, MMP-3, and MMP-9 expression. Increased MMP activity in the tumor stroma was associated with enhanced matrix remodeling and collagen deposition. Further analysis of the METABRIC dataset revealed an increase in IL-6, CCL2, and MMP-9 expression in patients with low IGF-1R, consistent with our mouse tumor model and data in human breast cancer cell lines. CONCLUSIONS: Our data support the hypothesis that reduction of IGF-1R function increases cellular stress and cytokine production to promote an aggressive tumor microenvironment through infiltration of immune cells and matrix remodeling.

Hematite Reduction Buffers Acid Generation and Enhances Nutrient Uptake by a Fermentative Iron Reducing Bacterium, Orenia metallireducens Strain Z6.

Fermentative iron-reducing organisms have been identified in a variety of environments. Instead of coupling iron reduction to respiration, they have been consistently observed to use ferric iron minerals as an electron sink for fermentation. In the present study, a fermentative iron reducer, Orenia metallireducens strain Z6, was shown to use iron reduction to enhance fermentation not only by consuming electron equivalents, but also by generating alkalinity that effectively buffers the pH. Fermentation of glucose by this organism in the presence of a ferric oxide mineral, hematite (Fe2O3), resulted in enhanced glucose decomposition compared with fermentation in the absence of an iron source. Parallel evidence (i.e., genomic reconstruction, metabolomics, thermodynamic analyses, and calculation of electron transfer) suggested hematite reduction as a proton-consuming reaction effectively consumed acid produced by fermentation. The buffering effect of hematite was further supported by a greater extent of glucose utilization by strain Z6 in media with increasing buffer capacity. Such maintenance of a stable pH through hematite reduction for enhanced glucose fermentation complements the thermodynamic interpretation of interactions between microbial iron reduction and other biogeochemical processes. This newly discovered feature of iron reducer metabolism also has significant implications for groundwater management and contaminant remediation by providing microbially mediated buffering systems for the associated microbial and/or chemical reactions.

Dorsal root ganglion transcriptome analysis following peripheral nerve injury in mice.

BACKGROUND: Peripheral nerve injury leads to changes in gene expression in primary sensory neurons of the injured dorsal root ganglia. These changes are believed to be involved in neuropathic pain genesis. Previously, these changes have been identified using gene microarrays or next generation RNA sequencing with poly-A tail selection, but these approaches cannot provide a more thorough analysis of gene expression alterations after nerve injury. METHODS: The present study chose to eliminate mRNA poly-A tail selection and perform strand-specific next generation RNA sequencing to analyze whole transcriptomes in the injured dorsal root ganglia following spinal nerve ligation. Quantitative real-time reverse transcriptase polymerase chain reaction assay was carried out to verify the changes of some differentially expressed RNAs in the injured dorsal root ganglia after spinal nerve ligation. RESULTS: Our results showed that more than 50 million (M) paired mapped sequences with strand information were yielded in each group (51.87 M-56.12 M in sham vs. 51.08 M-57.99 M in spinal nerve ligation). Six days after spinal nerve ligation, expression levels of 11,163 out of a total of 27,463 identified genes in the injured dorsal root ganglia significantly changed, of which 52.14% were upregulated and 47.86% downregulated. The largest transcriptional changes were observed in protein-coding genes (91.5%) followed by noncoding RNAs. Within 944 differentially expressed noncoding RNAs, the most significant changes were seen in long interspersed noncoding RNAs followed by antisense RNAs, processed transcripts, and pseudogenes. We observed a notable proportion of reads aligning to intronic regions in both groups (44.0% in sham vs. 49.6% in spinal nerve ligation). Using quantitative real-time polymerase chain reaction, we confirmed consistent differential expression of selected genes including Kcna2, Oprm1 as well as lncRNAs Gm21781 and 4732491K20Rik following spinal nerve ligation. CONCLUSION: Our findings suggest that next generation RNA sequencing can be used as a promising approach to analyze the changes of whole transcriptomes in dorsal root ganglia following nerve injury and to possibly identify new targets for prevention and treatment of neuropathic pain.

Orenia metallireducens sp. nov. Strain Z6, a Novel Metal-Reducing Member of the Phylum Firmicutes from the Deep Subsurface.

A novel halophilic and metal-reducing bacterium, Orenia metallireducens strain Z6, was isolated from briny groundwater extracted from a 2.02 km-deep borehole in the Illinois Basin, IL. This organism shared 96% 16S rRNA gene similarity with Orenia marismortui but demonstrated physiological properties previously unknown for this genus. In addition to exhibiting a fermentative metabolism typical of the genus Orenia, strain Z6 reduces various metal oxides [Fe(III), Mn(IV), Co(III), and Cr(VI)], using H2 as the electron donor. Strain Z6 actively reduced ferrihydrite over broad ranges of pH (6 to 9.6), salinity (0.4 to 3.5 M NaCl), and temperature (20 to 60°C). At pH 6.5, strain Z6 also reduced more crystalline iron oxides, such as lepidocrocite (γ-FeOOH), goethite (α-FeOOH), and hematite (α-Fe2O3). Analysis of X-ray absorption fine structure (XAFS) following Fe(III) reduction by strain Z6 revealed spectra from ferrous secondary mineral phases consistent with the precipitation of vivianite [Fe3(PO4)2] and siderite (FeCO3). The draft genome assembled for strain Z6 is 3.47 Mb in size and contains 3,269 protein-coding genes. Unlike the well-understood iron-reducing Shewanella and Geobacter species, this organism lacks the c-type cytochromes for typical Fe(III) reduction. Strain Z6 represents the first bacterial species in the genus Orenia (order Halanaerobiales) reported to reduce ferric iron minerals and other metal oxides. This microbe expands both the phylogenetic and physiological scopes of iron-reducing microorganisms known to inhabit the deep subsurface and suggests new mechanisms for microbial iron reduction. These distinctions from other Orenia spp. support the designation of strain Z6 as a new species, Orenia metallireducens sp. nov. IMPORTANCE: A novel iron-reducing species, Orenia metallireducens sp. nov., strain Z6, was isolated from groundwater collected from a geological formation located 2.02 km below land surface in the Illinois Basin, USA. Phylogenetic, physiologic, and genomic analyses of strain Z6 found it to have unique properties for iron reducers, including (i) active microbial iron-reducing capacity under broad ranges of temperatures (20 to 60°C), pHs (6 to 9.6), and salinities (0.4 to 3.5 M NaCl), (ii) lack of c-type cytochromes typically affiliated with iron reduction in Geobacter and Shewanella species, and (iii) being the only member of the Halanaerobiales capable of reducing crystalline goethite and hematite. This study expands the scope of phylogenetic affiliations, metabolic capacities, and catalytic mechanisms for iron-reducing microbes.

Normalization of TAM post-receptor signaling reveals a cell invasive signature for Axl tyrosine kinase.

BACKGROUND: Tyro3, Axl, and Mertk (TAMs) are a family of three conserved receptor tyrosine kinases that have pleiotropic roles in innate immunity and homeostasis and when overexpressed in cancer cells can drive tumorigenesis. METHODS: In the present study, we engineered EGFR/TAM chimeric receptors (EGFR/Tyro3, EGFR/Axl, and EGF/Mertk) with the goals to interrogate post-receptor functions of TAMs, and query whether TAMs have unique or overlapping post-receptor activation profiles. Stable expression of EGFR/TAMs in EGFR-deficient CHO cells afforded robust EGF inducible TAM receptor phosphorylation and activation of downstream signaling. RESULTS: Using a series of unbiased screening approaches, that include kinome-view analysis, phosphor-arrays, RNAseq/GSEA analysis, as well as cell biological and in vivo readouts, we provide evidence that each TAM has unique post-receptor signaling platforms and identify an intrinsic role for Axl that impinges on cell motility and invasion compared to Tyro3 and Mertk. CONCLUSION: These studies demonstrate that TAM show unique post-receptor signatures that impinge on distinct gene expression profiles and tumorigenic outcomes.

Metaproteomics of a gutless marine worm and its symbiotic microbial community reveal unusual pathways for carbon and energy use.

Low nutrient and energy availability has led to the evolution of numerous strategies for overcoming these limitations, of which symbiotic associations represent a key mechanism. Particularly striking are the associations between chemosynthetic bacteria and marine animals that thrive in nutrient-poor environments such as the deep sea because the symbionts allow their hosts to grow on inorganic energy and carbon sources such as sulfide and CO(2). Remarkably little is known about the physiological strategies that enable chemosynthetic symbioses to colonize oligotrophic environments. In this study, we used metaproteomics and metabolomics to investigate the intricate network of metabolic interactions in the chemosynthetic association between Olavius algarvensis, a gutless marine worm, and its bacterial symbionts. We propose previously undescribed pathways for coping with energy and nutrient limitation, some of which may be widespread in both free-living and symbiotic bacteria. These pathways include (i) a pathway for symbiont assimilation of the host waste products acetate, propionate, succinate and malate; (ii) the potential use of carbon monoxide as an energy source, a substrate previously not known to play a role in marine invertebrate symbioses; (iii) the potential use of hydrogen as an energy source; (iv) the strong expression of high-affinity uptake transporters; and (v) as yet undescribed energy-efficient steps in CO(2) fixation and sulfate reduction. The high expression of proteins involved in pathways for energy and carbon uptake and conservation in the O. algarvensis symbiosis indicates that the oligotrophic nature of its environment exerted a strong selective pressure in shaping these associations.

PRAP: an ab initio software package for automated genome-wide analysis of DNA repeats for prokaryotes.

MOTIVATION: Prokaryotic genome annotation has been focused mainly on identifying all genes and their protein functions. However, <30% of the prokaryotic genomes submitted to GenBank contain partial repeat features of specific types and none of the genomes contain complete repeat annotations. Deciphering all repeats in DNA sequences is an important and open task in genome annotation and bioinformatics. Hence, there is an immediate need of a tool capable of identifying full spectrum repeats in the whole genome. RESULTS: We report the PRAP (Prokaryotic Repeats Annotation Program software package to automate the analysis of repeats in both finished and draft genomes. It is aimed at identifying full spectrum repeats at the scale of the prokaryotic genome. Compared with the major existing repeat finding tools, PRAP exhibits competitive or better results. The results are consistent with manually curated and experimental data. Repeats can be identified and grouped into families to define their relevant types. The final output is parsed into the European Molecular Biology Laboratory (EMBL)/GenBank feature table format for reading and displaying in Artemis, where it can be combined or compared with other genome data. It is currently the most complete repeat finder for prokaryotes and is a valuable tool for genome annotation. AVAILABILITY: https://sites.google.com/site/prapsoftware/ CONTACT: hsuehc@ntu.edu.tw. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Response of intestinal microbiota to antibiotic growth promoters in chickens.

Antibiotic growth promoters (AGPs) have been used as feed additives to improve average daily weight gain and feed efficiency in food animals for more than five decades. However, use of AGPs is associated with the emergence of antibiotic-resistant human pathogens of animal origin, posing a significant threat to food safety and public health. Thus, development of novel alternatives to AGPs is important to mitigate antimicrobial resistance in foodborne pathogens. To achieve this goal, the mode of action of AGPs should be elucidated. In this study, the response of the chicken gut microbiota to AGPs was examined using two culture-independent approaches: phospholipid fatty acid (PLFA) biomarker analysis and 16S rDNA clone library sequencing. PLFA analysis showed that AGP tylosin treatment changed composition of the microbiota in various intestinal sites; however, total viable bacterial biomass in intestine was not affected by tylosin treatment. PLFA analysis also revealed an abundant viable fungal population in chicken microbiota. Eight 16S rDNA libraries (96 clones per library) were constructed using ileal samples from chickens that received either antibiotic-free or medicated feed. The 16S rDNA clone analysis of the growth-relevant samples showed the AGP treatment influenced the diversity of ileum microbiota in the chickens primarily in the Firmicutes division. In particular, Lactobacillus spp. populations in the ileum of AGP-treated chickens were significantly lower than those from chickens receiving antibiotic-free feed. Together, this study revealed novel features of the intestinal microbiota in response to AGP treatment and suggested approach to develop potential alternatives to AGPs for mitigation of antimicrobial resistance in foodborne pathogens.

Cholesterol biosynthesis defines oligodendrocyte precursor heterogeneity between brain and spinal cord.

Brain and spinal cord oligodendroglia have distinct functional characteristics, and cell-autonomous loss of individual genes can result in different regional phenotypes. However, a molecular basis for these distinctions is unknown. Using single-cell analysis of oligodendroglia during developmental myelination, we demonstrate that brain and spinal cord precursors are transcriptionally distinct, defined predominantly by cholesterol biosynthesis. We further identify the mechanistic target of rapamycin (mTOR) as a major regulator promoting cholesterol biosynthesis in oligodendroglia. Oligodendroglia-specific loss of mTOR decreases cholesterol biosynthesis in both the brain and the spinal cord, but mTOR loss in spinal cord oligodendroglia has a greater impact on cholesterol biosynthesis, consistent with more pronounced deficits in developmental myelination. In the brain, mTOR loss results in a later adult myelin deficit, including oligodendrocyte death, spontaneous demyelination, and impaired axonal function, demonstrating that mTOR is required for myelin maintenance in the adult brain.

Breast tumor IGF1R regulates cell adhesion and metastasis: alignment of mouse single cell and human breast cancer transcriptomics.

Introduction: The acquisition of a metastatic phenotype is the critical event that determines patient survival from breast cancer. Several receptor tyrosine kinases have functions both in promoting and inhibiting metastasis in breast tumors. Although the insulin-like growth factor 1 receptor (IGF1R) has been considered a target for inhibition in breast cancer, low levels of IGF1R expression are associated with worse overall patient survival. Methods: To determine how reduced IGF1R impacts tumor phenotype in human breast cancers, we used weighted gene co-expression network analysis (WGCNA) of Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) patient data to identify gene modules associated with low IGF1R expression. We then compared these modules to single cell gene expression analyses and phenotypes of mouse mammary tumors with reduced IGF1R signaling or expression in a tumor model of triple negative breast cancer. Results: WGCNA from METABRIC data revealed gene modules specific to cell cycle, adhesion, and immune cell signaling that were inversely correlated with IGF1R expression in human breast cancers. Integration of human patient data with single cell sequencing data from mouse tumors revealed similar pathways necessary for promoting metastasis in basal-like mammary tumors with reduced signaling or expression of IGF1R. Functional analyses revealed the basis for the enhanced metastatic phenotype including alterations in E- and P-cadherins. Discussion: Human breast and mouse mammary tumors with reduced IGF1R are associated with upregulation of several pathways necessary for promoting metastasis supporting the conclusion that IGF1R normally helps maintain a metastasis suppressive tumor microenvironment. We further found that reduced IGF1R signaling in tumor epithelial cells dysregulates cadherin expression resulting in reduced cell adhesion.

Downregulation of a Dorsal Root Ganglion-Specifically Enriched Long Noncoding RNA is Required for Neuropathic Pain by Negatively Regulating RALY-Triggered Ehmt2 Expression.

Nerve injury-induced maladaptive changes of gene expression in dorsal root ganglion (DRG) neurons contribute to neuropathic pain. Long non-coding RNAs (lncRNAs) are emerging as key regulators of gene expression. Here, a conserved lncRNA is reported, named DRG-specifically enriched lncRNA (DS-lncRNA) for its high expression in DRG neurons. Peripheral nerve injury downregulates DS-lncRNA in injured DRG due, in part, to silencing of POU domain, class 4, transcription factor 3, a transcription factor that interacts with the DS-lncRNA gene promoter. Rescuing DS-lncRNA downregulation blocks nerve injury-induced increases in the transcriptional cofactor RALY-triggered DRG Ehmt2 mRNA and its encoding G9a protein, reverses the G9a-controlled downregulation of opioid receptors and Kcna2 in injured DRG, and attenuates nerve injury-induced pain hypersensitivities in male mice. Conversely, DS-lncRNA downregulation increases RALY-triggered Ehmt2/G9a expression and correspondingly decreases opioid receptor and Kcna2 expression in DRG, leading to neuropathic pain symptoms in male mice in the absence of nerve injury. Mechanistically, downregulated DS-lncRNA promotes more binding of increased RALY to RNA polymerase II and the Ehmt2 gene promoter and enhances Ehmt2 transcription in injured DRG. Thus, downregulation of DS-lncRNA likely contributes to neuropathic pain by negatively regulating the expression of RALY-triggered Ehmt2/G9a, a key neuropathic pain player, in DRG neurons.

Critical role of interferons in gastrointestinal injury repair.

The etiology of ulcerative colitis is poorly understood and is likely to involve perturbation of the complex interactions between the mucosal immune system and the commensal bacteria of the gut, with cytokines acting as important cross-regulators. Here we use IFN receptor-deficient mice in a dextran sulfate sodium (DSS) model of acute intestinal injury to study the contributions of type I and III interferons (IFN) to the initiation, progression and resolution of acute colitis. We find that mice lacking both types of IFN receptors exhibit enhanced barrier destruction, extensive loss of goblet cells and diminished proliferation of epithelial cells in the colon following DSS-induced damage. Impaired mucosal healing in double IFN receptor-deficient mice is driven by decreased amphiregulin expression, which IFN signaling can up-regulate in either the epithelial or hematopoietic compartment. Together, these data underscore the pleiotropic functions of IFNs and demonstrate that these critical antiviral cytokines also support epithelial regeneration following acute colonic injury.

Axl and Mertk Receptors Cooperate to Promote Breast Cancer Progression by Combined Oncogenic Signaling and Evasion of Host Antitumor Immunity.

Despite the promising clinical benefit of targeted and immune checkpoint blocking therapeutics, current strategies have limited success in breast cancer, indicating that additional inhibitory pathways are required to complement existing therapeutics. TAM receptors (Tyro-3, Axl, and Mertk) are often correlated with poor prognosis because of their capacities to sustain an immunosuppressive environment. Here, we ablate Axl on tumor cells using CRISPR/Cas9 gene editing, and by targeting Mertk in the tumor microenvironment (TME), we observed distinct functions of TAM as oncogenic kinases, as well as inhibitory immune receptors. Depletion of Axl suppressed cell intrinsic oncogenic properties, decreased tumor growth, reduced the incidence of lung metastasis and increased overall survival of mice when injected into mammary fat pad of syngeneic mice, and demonstrated synergy when combined with anti-PD-1 therapy. Blockade of Mertk function on macrophages decreased efferocytosis, altered the cytokine milieu, and resulted in suppressed macrophage gene expression patterns. Mertk-knockout mice or treatment with anti-Mertk-neutralizing mAb also altered the cellular immune profile, resulting in a more inflamed tumor environment with enhanced T-cell infiltration into tumors and T-cell-mediated cytotoxicity. The antitumor activity from Mertk inhibition was abrogated by depletion of cytotoxic CD8α T cells by using anti-CD8α mAb or by transplantation of tumor cells into B6.CB17-Prkdc SCID mice. Our data indicate that targeting Axl expressed on tumor cells and Mertk in the TME is predicted to have a combinatorial benefit to enhance current immunotherapies and that Axl and Mertk have distinct functional activities that impair host antitumor response. SIGNIFICANCE: This study demonstrates how TAM receptors act both as oncogenic tyrosine kinases and as receptors that mediate immune evasion in cancer progression.

The genome sequence of Methanohalophilus mahii SLP(T) reveals differences in the energy metabolism among members of the Methanosarcinaceae inhabiting freshwater and saline environments.

Methanohalophilus mahii is the type species of the genus Methanohalophilus, which currently comprises three distinct species with validly published names. Mhp. mahii represents moderately halophilic methanogenic archaea with a strictly methylotrophic metabolism. The type strain SLP(T) was isolated from hypersaline sediments collected from the southern arm of Great Salt Lake, Utah. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 2,012,424 bp genome is a single replicon with 2032 protein-coding and 63 RNA genes and part of the Genomic Encyclopedia of Bacteria and Archaea project. A comparison of the reconstructed energy metabolism in the halophilic species Mhp. mahii with other representatives of the Methanosarcinaceae reveals some interesting differences to freshwater species.

Alternative Splicing of Nrcam Gene in Dorsal Root Ganglion Contributes to Neuropathic Pain.

NrCAM, a neuronal cell adhesion molecule in the L1 family of the immunoglobulin superfamily, is subjected to extensively alternative splicing and involved in neural development and some disorders. The aim of this study was to explore the role of Nrcam mRNA alternative splicing in neuropathic pain. A next generation RNA sequencing analysis of dorsal root ganglions (DRGs) showed the differential expression of two splicing variants of Nrcam, Nrcam+10 and Nrcam-10, in the injured DRG after the fourth lumbar spinal nerve ligation (SNL) in mice. SNL increased the exon 10 insertion, resulting in an increase in the amount of Nrcam+10 and a corresponding decrease in the level of Nrcam-10 in the injured DRG. An antisense oligonucleotide (ASO) that specifically targeted exon 10 of Nrcam gene (Nrcam ASO) repressed RNA expression of Nrcam+10 and increased RNA expression of Nrcam-10 in in vitro DRG cell culture. Either DRG microinjection or intrathecal injection of Nrcam ASO attenuated SNL-induced the development of mechanical allodynia, thermal hyperalgesia, or cold allodynia. Nrcam ASO also relieved SNL- or chronic compression of DRG (CCD)-induced the maintenance of pain hypersensitivities in male and female mice. PERSPECTIVE: We conclude that the relative levels of alternatively spliced Nrcam variants are critical for neuropathic pain genesis. Targeting Nrcam alternative splicing via the antisense oligonucleotides may be a new potential avenue in neuropathic pain management.

Dendritic morphology and inhibitory regulation distinguish dentate semilunar granule cells from granule cells through distinct stages of postnatal development.

Semilunar granule cells (SGCs) have been proposed as a morpho-functionally distinct class of hippocampal dentate projection neurons contributing to feedback inhibition and memory processing in juvenile rats. However, the structural and physiological features that can reliably classify granule cells (GCs) from SGCs through postnatal development remain unresolved. Focusing on postnatal days 11-13, 28-42, and > 120, corresponding with human infancy, adolescence, and adulthood, we examined the somato-dendritic morphology and inhibitory regulation in SGCs and GCs to determine the cell-type specific features. Unsupervised cluster analysis confirmed that morphological features reliably distinguish SGCs from GCs irrespective of animal age. SGCs maintain higher spontaneous inhibitory postsynaptic current (sIPSC) frequency than GCs from infancy through adulthood. Although sIPSC frequency in SGCs was particularly enhanced during adolescence, sIPSC amplitude and cumulative charge transfer declined from infancy to adulthood and were not different between GCs and SGCs. Extrasynaptic GABA current amplitude peaked in adolescence in both cell types and was significantly greater in SGCs than in GCs only during adolescence. Although GC input resistance was higher than in SGCs during infancy and adolescence, input resistance decreased with developmental age in GCs, while it progressively increased in SGCs. Consequently, GCs' input resistance was significantly lower than SGCs in adults. The data delineate the structural features that can reliably distinguish GCs from SGCs through development. The results reveal developmental differences in passive membrane properties and steady-state inhibition between GCs and SGCs which could confound their use in classifying the cell types.

Evaluation of bile salt hydrolase inhibitor efficacy for modulating host bile profile and physiology using a chicken model system.

Gut microbial enzymes, bile salt hydrolases (BSHs) are the gateway enzymes for bile acid (BA) modification in the gut. This activity is a promising target for developing innovative non-antibiotic growth promoters to enhance animal production and health. Compelling evidence has shown that inhibition of BSH activity should enhance weight gain by altering the BA pool, host signalling and lipid metabolism. We recently identified a panel of promising BSH inhibitors. Here, we address the potential of them as alternative, effective, non-antibiotic feed additives, for commercial application, to promote animal growth using a chicken model. In this study, the in vivo efficacy of three BSH inhibitors (caffeic acid phenethylester, riboflavin, carnosic acid) were evaluated. 7-day old chicks (10 birds/group) were either untreated or they received one of the specific BSH inhibitors (25 mg/kg body weight) via oral gavage for 17 days. The chicks in treatment groups consistently displayed higher body weight gain than the untreated chicks. Metabolomic analysis demonstrated that BSH inhibitor treatment led to significant changes in both circulating and intestinal BA signatures in support of blunted intestinal BSH activity. Consistent with this finding, liver and intestinal tissue RNA-Seq analysis showed that carnosic acid treatment significantly altered expression of genes involved in lipid and bile acid metabolism. Taken together, this study validates microbial BSH activity inhibition as an alternative target and strategy to antibiotic treatment for animal growth promotion.

Paraspinous muscle gene expression profiling following simulated staged endovascular repair of thoracoabdominal aortic aneurysm: exploring potential therapeutic pathways.

OBJECTIVES: Thoracic endovascular techniques for aneurysm repair offer less invasive alternatives to open strategies. Both approaches, however, are associated with the risk for neurological complications. Despite adjuncts to maintain spinal cord perfusion, ischaemia and paraplegia continue to occur during thoracoabdominal aortic aneurysm (TAAA) repair. Staging of such extensive procedures has been proven to decrease the risk for spinal cord injury. Archived biopsy specimens may offer insight into the molecular signature of the reorganization and expansion of the spinal collateral network during staged endovascular interventions in the setting of TAAA. METHODS: Biological replicates of total RNA were isolated from existing paraspinous muscle samples from 22 Yorkshire pigs randomized to 1 of 3 simulated TAAA repair strategies as part of a previous study employing coil embolization of spinal segmental arteries within the thoracic and lumbar spine. Gene expression profiling was performed using the Affymetrix GeneChip Porcine array. RESULTS: Microarray analysis identified 649 differentially expressed porcine genes (≥1.3-fold change, P ≤ 0.05) when comparing paralysed and non-paralysed subjects. Of these, 355 were available for further analysis. When mapped to the human genome, 169 Homo sapiens orthologues were identified. Integrated interpretation of gene expression profiles indicated the significant regulation of transcriptional regulators (such as nuclear factor кB), cytokine (including CXCL12) elements contributing to hypoxia signalling in the cardiovascular system (vascular endothelial growth factor and UBE2) and cytoskeletal elements (like dystrophin (DMD) and matrix metallopeptidase (MMP)). CONCLUSIONS: This study demonstrates the ability of microarray-based platforms to detect the differential expression of genes in paraspinous muscle during staged TAAA repair. Pathway enrichment analysis detected subcellular actors accompanying the neuroprotective effects of staged endovascular coiling. These observations provide new insight into the potential prognostic and therapeutic value of gene expression profiling in monitoring and modulating the arteriolar remodelling in the collateral network.