Expression Patterns of Clock Gene mRNAs and Clock Proteins in Human Psoriatic Skin Samples

Psoriasis is a systemic inflammatory skin disorder that can be associated with sleep disturbance and negatively influence the daily rhythm. The link between the pathomechanism of psoriasis and the circadian rhythm has been suggested by several previous studies. However, there are insufficient data on altered clock mechanisms in psoriasis to prove these theories. Therefore, we investigated the expression of the core clock genes in human psoriatic lesional and non-lesional skin and in human adult low calcium temperature (HaCaT) keratinocytes after stimulation with pro-inflammatory cytokines. Furthermore, we examined the clock proteins in skin biopsies from psoriatic patients by immunohistochemistry. We found that the clock gene transcripts were elevated in psoriatic lesions, especially in non-lesional psoriatic areas, except for rev-erbα, which was consistently downregulated in the psoriatic samples. In addition, the REV-ERBα protein showed a different epidermal distribution in non-lesional skin than in healthy skin. In cytokine-treated HaCaT cells, changes in the amplitude of the bmal1, cry1, rev-erbα and per1 mRNA oscillation were observed, especially after TNFα stimulation. In conclusion, in our study a perturbation of clock gene transcripts was observed in uninvolved and lesional psoriatic areas compared to healthy skin. These alterations may serve as therapeutic targets and facilitate the development of chronotherapeutic strategies in the future.

Undetectable Production of the VIM-1 Carbapenemase in an Atlantibacter hermannii Clinical Isolate

The differential expression of VIM-1 in Atlantibacter hermannii WEB-2 and Enterobacter hormaechei ssp. hoffmannii WEB-1 clinical isolates from a rectal swab of a hospitalized patient in France was investigated. A. hermannii WEB-2 was resistant to all β-lactams except carbapenems. It produced ESBL SHV-12, but the Carba NP test failed to detect any carbapenemase activity despite the production of VIM-1. Conversely, E. hormaechei WEB-1, previously recovered from the same patient, was positive for the detection of carbapenemase activity. The blaVIM–1 gene was located on a plasmid and embedded within class 1 integron. Both plasmids were of the same IncA incompatibility group and conferred the same resistance pattern when electroporated in Escherichia coli TOP10 or Enterobacter cloacae CIP7933. Quantitative RT-PCR experiments indicated a weaker replication of pWEB-2 in A. hermannii as compared to E. hormaechei. An isogenic mutant of A. hermannii WEB-2 selected after sequential passages with increased concentrations of imipenem possessed higher MICs for carbapenems and cephalosporins including cefiderocol, higher levels of the blaVIM–1 gene transcripts, and detectable carbapenemase activity using the Carba NP test. Assessment of read coverage demonstrated that a duplication of the region surrounding blaVIM–1 gene occurred in the A. hermannii mutant with detectable carbapenemase activity. The lack of detection of the VIM-1 carbapenemase activity in A. hermannii WEB-2 isolate was likely due to a weak replication of the IncA plasmid harboring the blaVIM–1 gene. Imipenem as selective pressure led to a duplication of this gene on the plasmid and to the restoration of a significant carbapenem-hydrolyzing phenotype.

Cyclin F drives proliferation through SCF-dependent degradation of the retinoblastoma-like tumor suppressor p130/RBL2

Cell cycle gene expression programs fuel proliferation and are universally dysregulated in cancer. The retinoblastoma (RB)-family of proteins, RB1, RBL1/p107 and RBL2/p130, coordinately repress cell cycle gene expression, inhibiting proliferation and suppressing tumorigenesis. Phosphorylation of RB-family proteins by cyclin dependent kinases is firmly established. Like phosphorylation, ubiquitination is essential to cell cycle control, and numerous proliferative regulators, tumor suppressors, and oncoproteins are ubiquitinated. However, little is known about the role of ubiquitin signaling in controlling RB-family proteins. A systems genetics analysis of CRISPR/Cas9 screens suggested the potential regulation of the RB-network by cyclin F, a substrate recognition receptor for the SCF family of E3 ligases. We demonstrate that RBL2/p130 is a direct substrate of SCFcyclin F. We map a cyclin F regulatory site to a flexible linker in the p130 pocket domain, and show that this site mediates binding, stability, and ubiquitination. Expression of a mutant version of p130, which cannot be ubiquitinated, severely impaired proliferative capacity and cell cycle progression. Consistently, we observed reduced expression of cell cycle gene transcripts, as well a reduced abundance of cell cycle proteins, analyzed by quantitative, iterative immunofluorescent imaging. These data suggest a key role for SCFcyclin F in the CDK-RB network and raise the possibility that aberrant p130 degradation could dysregulate the cell cycle in human cancers.

Interferon inducible pseudouridine modification in human mRNA by quantitative nanopore profiling

Pseudouridine (Ψ) is an abundant mRNA modification in mammalian transcriptome, but its functions have remained elusive due to the difficulty of transcriptome-wide mapping. We develop a nanopore native RNA sequencing method for quantitative Ψ prediction (NanoPsu) that utilizes native content training, machine learning modeling, and single-read linkage analysis. Biologically, we find interferon inducible Ψ modifications in interferon-stimulated gene transcripts which are consistent with a role of Ψ in enabling efficacy of mRNA vaccines.

Ion Channel and Ubiquitin Differential Expression during Erythromycin-Induced Anhidrosis in Foals

Macrolide drugs are the treatment of choice for Rhodococcus equi infections, despite severe side-effects temporary anhidrosis as a. To better understand the molecular biology leading to macrolide induced anhidrosis, we performed skin biopsies and Quantitative Intradermal Terbutaline Sweat Tests (QITSTs) in six healthy pony-cross foals for three different timepoints during erythromycin administration—pre-treatment (baseline), during anhidrosis and post-recovery. RNA sequencing of biopsies followed by differential gene expression analysis compared both pre and post normal sweating timepoints to the erythromycin induced anhidrosis episode. After Bonferroni correction for multiple testing, 132 gene transcripts were significantly differentially expressed during the anhidrotic timepoint. Gene ontology analysis of the full differentially expressed gene set identified over-represented biological functions for ubiquitination and ion-channel function, both biologically relevant to sweat production. These same mechanisms were previously implicated in heritable equine idiopathic anhidrosis and sweat gland function and their involvement in macrolide-induced temporary anhidrosis warrants further investigation.

Distribution of Immune Cells Including Macrophages in the Human Cochlea

Background: The human cochlea was earlier believed to lack capacity to mount specific immune responses. Recent studies established that the human cochlea holds macrophages. The cells appear to surveil, dispose of, and restore wasted cells to maintain tissue integrity. Macrophage activities are believed to be the central elements in immune responses and could swiftly defuse invading microbes that enter via adjacent infection-prone areas. This review updates recent human studies in light of the current literature and adds information about chemokine gene expression.Materials and Methods: We analyzed surgically obtained human tissue using immunohistochemistry, confocal microscopy, and multichannel super-resolution structured illumination microscopy. The samples were considered representative of steady-state conditions. Antibodies against the ionized calcium-binding adaptor molecule 1 were used to identify the macrophages. CD68 and CD11b, and the major histocompatibility complex type II (MHCII) and CD4 and CD8 were analyzed. The RNAscope technique was used for fractalkine gene localization.Results: Many macrophages were found around blood vessels in the stria vascularis but not CD4 and CD8 lymphocytes. Amoeboid macrophages were identified in the spiral ganglion with surveilling “antennae” projecting against targeted cells. Synapse-like contacts were seen on spiral ganglion cell bodies richly expressing single CXC3CL gene transcripts. Branching neurite-like processes extended along central and peripheral axons. Active macrophages were occasionally found near degenerating hair cells. Some macrophage-interacting T lymphocytes were observed between the scala tympani wall and Rosenthal's canal. CD4 and CD8 cells were not found in the organ of Corti.Conclusions: The results indicate that the human cochlea is equipped with macrophages and potentially lymphocytes, suggesting both an innate and adaptive immune capacity. A rich expression of fractalkine gene transcripts in spiral ganglion neurons suggest an essential role for auditory nerve protection, as has been demonstrated experimentally. The findings provide further information on the important role of the immune machinery present in the human inner ear and its potential to carry adverse immune reactions, including cytotoxic and foreign body responses. The results can be used to form a rationale for therapies aiming to modulate these immune activities.

Transcriptome Analysis Reveals Higher Levels of Mobile Element-Associated Abnormal Gene Transcripts in Temporal Lobe Epilepsy Patients

Mesial temporal lobe epilepsy (MTLE) is the most common form of epilepsy, and temporal lobe epilepsy patients with hippocampal sclerosis (TLE-HS) show worse drug treatment effects and prognosis. TLE has been shown to have a genetic component, but its genetic research has been mostly limited to coding sequences of genes with known association to epilepsy. Representing a major component of the genome, mobile elements (MEs) are believed to contribute to the genetic etiology of epilepsy despite limited research. We analyzed publicly available human RNA-seq-based transcriptome data to determine the role of mobile elements in epilepsy by performing de novo transcriptome assembly, followed by identification of spliced gene transcripts containing mobile element (ME) sequences (ME-transcripts), to compare their frequency across different sample groups. Significantly higher levels of ME-transcripts in hippocampal tissues of epileptic patients, particularly in TLE-HS, were observed. Among ME classes, short interspersed nuclear elements (SINEs) were shown to be the most frequent contributor to ME-transcripts, followed by long interspersed nuclear elements (LINEs) and DNA transposons. These ME sequences almost in all cases represent older MEs normally located in the intron sequences. For protein coding genes, ME sequences were mostly found in the 3′-UTR regions, with a significant portion also in the coding sequences (CDSs), leading to reading frame disruption. Genes associated with ME-transcripts showed enrichment for the mRNA splicing process and an apparent bias in epileptic transcriptomes toward neural- and epilepsy-associated genes. The findings of this study suggest that abnormal splicing involving MEs, leading to loss of functions in critical genes, plays a role in epilepsy, particularly in TLE-HS, thus providing a novel insight into the molecular mechanisms underlying epileptogenesis.

MUC4 is not expressed in cell lines used for live cell imaging

Background: The ability to visualise specific mammalian gene loci in living cells is important for understanding the dynamic processes linked to transcription. However, some of the tools used to target mammalian genes for live cell imaging, such as dCas9, have been reported to themselves impede processes linked to transcription. The MUC4 gene is a popular target for live cell imaging studies due to the repetitive nature of sequences within some exons of this gene. Methods: We set out to compare the impact of dCas9 and TALE-based imaging tools on MUC4 expression, including in human cell lines previously reported as expressing MUC4. Results: We were unable to detect MUC4 mRNA in these cell lines. Moreover, analysis of publicly available data for histone modifications associated with transcription, and data for transcription itself, indicate that neither MUC4, nor any of the mucin gene family are significantly expressed in the cell lines where dCas9 targeting has been reported to repress MUC4 and MUC1 expression, or in the cell lines where dCas13 has been used to report MUC4 RNA detection in live cells. Conclusions: Methods for visualising specific gene loci and gene transcripts in live human cells are very challenging. Our data suggest that care should be given to the choice of the most appropriate cell lines for these analyses and that orthogonal methods of assaying gene expression be carefully compared.

Legume plant defenses and nutrients mediate indirect interactions between soil rhizobia and chewing herbivores

Soil bacteria that form mutualisms with plants, such as rhizobia, affects susceptibility of plants to herbivores and pathogens. Soil rhizobia also promote nitrogen fixation, which mediates host nutrient levels and defenses. However, whether aboveground herbivores affect the function of soil rhizobia remains poorly understood. We assessed reciprocal interactions between Sitona lineatus, a chewing herbivore, and pea (Pisum sativum) plants grown with or without rhizobia (Rhizobium leguminosarum biovar viciae). We also examined the underlying plant-defense and nutritional mechanisms of these interactions. In our experiments, soil rhizobia influenced feeding and herbivory by chewing herbivores. Leaf defoliation by S. lineatus was lower on plants treated with rhizobia, but these insects had similar amino acid levels compared to those on un-inoculated plants. Plants grown with soil rhizobia had increased expression of gene transcripts associated with phytohormone-mediated defense, which may explain decreased susceptibility to S. lineatus. Rhizobia also induced expression of gene transcripts associated with physical and antioxidant-related defense pathways in P. sativum. Conversely, S. lineatus feeding reduced the number of root nodules and nodule biomass, suggesting a disruption of the symbiosis between plants and rhizobia. Our study shows that aboveground herbivores can engage in mutually antagonistic interactions with soil microbes mediated through a multitude of plant-mediated pathways.