Many animals are dependent on microbial partners that provide essential nutrients lacking from their diet. Ticks, whose diet consists exclusively on vertebrate blood, rely on maternally inherited bacterial symbionts to supply B vitamins. While previously studied tick species consistently harbor a single lineage of those nutritional symbionts, we evidence here that the invasive tick Hyalomma marginatum harbors a unique dual-partner nutritional system between an ancestral symbiont, Francisella, and a more recently acquired symbiont, Midichloria. Using metagenomics, we show that Francisella exhibits extensive genome erosion that endangers the nutritional symbiotic interactions. Its genome includes folate and riboflavin biosynthesis pathways but deprived functional biotin biosynthesis on account of massive pseudogenization. Co-symbiosis compensates this deficiency since the Midichloria genome encompasses an intact biotin operon, which was primarily acquired via lateral gene transfer from unrelated intracellular bacteria commonly infecting arthropods. Thus, in H. marginatum, a mosaic of co-evolved symbionts incorporating gene combinations of distant phylogenetic origins emerged to prevent the collapse of an ancestral nutritional symbiosis. Such dual endosymbiosis was never reported in other blood feeders but was recently documented in agricultural pests feeding on plant sap, suggesting that it may be a key mechanism for advanced adaptation of arthropods to specialized diets.

BACKGROUND: Maintenance of the corpus luteum (CL) beyond the time of luteolysis is essential for establishing pregnancy. Identifying the distinct features of early pregnancy CL remains unresolved, hence we analyzed here the transcriptome of CL on day 18 pregnant (P) and non-pregnant (NP) cows using RNA-Seq. CL of P cows expressed ISGs, verifying exposure to the pregnancy recognition signal, interferon-tau (IFNT), whereas the CL of NP cows had elevated luteal progesterone levels, implying that luteolysis had not yet commenced. RESULTS: The DEGs, IPA, and metascape canonical pathways, along with GSEA analysis, differed markedly in the CL of P cows from those of NP cows, at the same day of the cycle. Both metascape and IPA identified similar significantly enriched pathways such as interferon alpha/beta, sonic hedgehog pathway, TNFA, EDN1, TGFB1, and PDGF. However, type-1 interferon and sonic hedgehog pathways were positively enriched whereas most of the enriched pathways were downregulated in the P compared to NP samples. Thirty-four % of these pathways are known to be elevated by PGF2A during luteolysis. Notably, selective DEGs in luteinized granulosa cells were modulated by IFNT in vitro in a similar manner to their regulation in the CL of P cows. CONCLUSION: This study unraveled the unique transcriptomic signature of the IFNT-exposed, early pregnancy CL, highlighting the abundance of downregulated pathways known to be otherwise induced during luteolysis. These and IFNT-regulated in vitro pregnancy-specific DEGs suggest that IFNT contributes to the characteristics and maintenance of early pregnancy CL.

The current study aimed at investigating the long-term biological mechanisms governing bone regeneration in osseous defects filled with bovine bone (BB). Tooth extraction sockets were filled with BB or left unfilled for natural healing in a C57BL/6 mouse alveolar regeneration bone model (n = 12). Seven weeks later, the alveolar bone samples were analyzed histologically with hematoxylin/eosin and tartrate-resistant acid phosphatase staining. A separate group (n = 10) was used for RNA sequencing. Osteoclast inhibition was induced by zoledronic acid (ZA) administration at 2 wk postextraction in a third group (n = 28) for examination of osseous changes and cellular functions with micro-computed tomography and quantitative reverse transcription polymerase chain reaction, respectively. Histological and radiological osseous healing was observed in both BB-filled and normal-healing sockets. However, BB regenerated bone showed significant robust expression of genes associated with bone homeostasis and osteoclasts' function. Osteoclasts' inhibition in BB-filled sockets led to decreased bone resorption markers and reduced bone formation to a greater extent than that observed in osteoclasts' inhibition with natural healing. BB displays long-term biologically active properties, despite a naive osseous histological appearance. These include activation of osteoclasts, which in turn promotes osseous remodeling and maturation of ossified bone.

Orthodontic tooth movement (OTM) is a "sterile" inflammatory process. The present study aimed to reveal the underlying biological mechanisms, by studying the force associated-gene expression changes, in a time-dependent manner. Ni-Ti springs were set to move the upper 1(st)-molar in C57BL/6 mice. OTM was measured by muCT. Total-RNA was extracted from tissue blocks at 1,3,7 and 14-days post force application, and from two control groups: naive and inactivated spring. Gene-expression profiles were generated by next-generation-RNA-sequencing. Gene Set Enrichment Analysis, K-means algorithm and Ingenuity pathway analysis were used for data interpretation. Genes of interest were validated with qRT-PCR. A total of 3075 differentially expressed genes (DEGs) were identified, with the greatest number at day 3. Two distinct clusters patterns were recognized: those in which DEGs peaked in the first days and declined thereafter (tissue degradation, phagocytosis, leukocyte extravasation, innate and adaptive immune system responses), and those in which DEGs were initially down-regulated and increased at day 14 (cell proliferation and migration, cytoskeletal rearrangement, tissue homeostasis, angiogenesis). The uncovering of novel innate and adaptive immune processes in OTM led us to propose a new term "Immunorthodontics". This genomic data can serve as a platform for OTM modulation future approaches.

Many Gram-negative plant and animal pathogenic bacteria employ a type III secretion system (T3SS) to secrete protein effectors into the cells of their hosts and promote disease. The plant pathogen Acidovorax citrulli requires a functional T3SS for pathogenicity. As with Xanthomonas and Ralstonia spp., an AraC-type transcriptional regulator, HrpX, regulates expression of genes encoding T3SS components and type III-secreted effectors (T3Es) in A. citrulli. A previous study reported eleven T3E genes in this pathogen, based on the annotation of a sequenced strain. We hypothesized that this was an underestimation. Guided by this hypothesis, we aimed at uncovering the T3E arsenal of the A. citrulli model strain, M6. We carried out a thorough sequence analysis searching for similarity to known T3Es from other bacteria. This analysis revealed 51 A. citrulli genes whose products are similar to known T3Es. Further, we combined machine learning and transcriptomics to identify novel T3Es. The machine learning approach ranked all A. citrulli M6 genes according to their propensity to encode T3Es. RNA-Seq revealed differential gene expression between wild-type M6 and a mutant defective in HrpX. Data combined from these approaches led to the identification of seven novel T3E candidates, that were further validated using a T3SS-dependent translocation assay. These T3E genes encode hypothetical proteins, do not show any similarity to known effectors from other bacteria, and seem to be restricted to plant pathogenic Acidovorax species. Transient expression in Nicotiana benthamiana revealed that two of these T3Es localize to the cell nucleus and one interacts with the endoplasmic reticulum. This study not only uncovered the arsenal of T3Es of an important pathogen, but it also places A. citrulli among the “richest” bacterial pathogens in terms of T3E cargo. It also revealed novel T3Es that appear to be involved in the pathoadaptive evolution of plant pathogenic Acidovorax species.Author summary Acidovorax citrulli is a Gram-negative bacterium that causes bacterial fruit blotch (BFB) disease of cucurbits. This disease represents a serious threat to cucurbit crop production worldwide. Despite the agricultural importance of BFB, the knowledge about basic aspects of A. citrulli-plant interactions is rather limited. As many Gram-negative plant and animal pathogenic bacteria, A. citrulli employs a complex secretion system, named type III secretion system, to deliver protein virulence effectors into the host cells. In this work we aimed at uncovering the arsenal of type III-secreted effectors (T3Es) of this pathogen by combination of bioinformatics and experimental approaches. We found that this bacterium possesses at least 51 genes that are similar to T3E genes from other pathogenic bacteria. In addition, our study revealed seven novel T3Es that seem to occur only in A. citrulli strains and in other plant pathogenic Acidovorax species. We found that two of these T3Es localize to the plant cell nucleus while one partially interacts with the endoplasmic reticulum. Further characterization of the novel T3Es identified in this study may uncover new host targets of pathogen effectors and new mechanisms by which pathogenic bacteria manipulate their hosts.

The genus Bartonella (Family: Bartonellaceae; Order: Rhizobiales; Class: Alphaproteobacteria) comprises facultative intracellular Gram-negative, haemotropic, slow-growing, vector-borne bacteria. Wild rodents and their fleas harbor a great diversity of species and strains of the genus Bartonella, including several zoonotic ones. This genetic diversity coupled with a fastidious nature of the organism results in a taxonomic challenge that has led to a massive collection of uncharacterized strains. Here, we report the genomic and phenotypic characterization of two strains, members of the genus Bartonella (namely Tel Aviv and OE 1-1), isolated from Rattus rattus rats and Synosternus cleopatrae fleas, respectively. Scanning electron microscopy revealed rod-shaped bacteria with polar pili, lengths ranging from 1.0 to 2.0 microm and widths ranging from 0.3 to 0.6 microm. OE 1-1 and Tel Aviv strains contained one single chromosome of 2.16 and 2.23 Mbp and one plasmid of 29.0 and 41.5 Kbp, with average DNA G+C contents of 38.16 and 38.47 mol%, respectively. These strains presented an average nucleotide identity (ANI) of 89.9 %. Bartonella elizabethae was found to be the closest phylogenetic relative of both strains (ANI=90.9-93.6 %). The major fatty acids identified in both strains were C18:1omega7c, C18 : 0 and C16 : 0. They differ from B. elizabethae in their C17 : 0 and C15 : 0 compositions. Both strains are strictly capnophilic and their biochemical profiles resembled those of species of the genus Bartonella with validly published names, whereas differences in arylamidase activities partially assisted in their speciation. Genomic and phenotypic differences demonstrate that OE 1-1 and Tel Aviv strains represent novel individual species, closely related to B. elizabethae, for which we propose the names Bartonella kosoyi sp. nov. and Bartonella krasnovii sp. nov.

BACKGROUND & AIMS: Development of nonalcoholic steatohepatitis (NASH) is associated with reductions in hepatic microRNA122 (MIR122); the RAR related orphan receptor A (RORA) promotes expression of MIR122. Increasing expression of RORA in livers of mice increases expression of MIR122 and reduces lipotoxicity. We investigated the effects of a RORA agonist in mouse models of NASH. METHODS: We screened a chemical library to identify agonists of RORA and tested their effects on a human hepatocellular carcinoma cell line (Huh7). C57BL/6 mice were fed a chow or high-fat diet (HFD) for 4 weeks to induce fatty liver. Mice were given hydrodynamic tail vein injections of a MIR122 antagonist (antagomiR-122) or a control antagomiR once each week for 3 weeks while still on the HFD or chow diet, or intraperitoneal injections of the RORA agonist RS-2982 or vehicle, twice each week for 3 weeks. Livers, gonad white adipose, and skeletal muscle were collected and analyzed by reverse-transcription polymerase chain reaction, histology, and immunohistochemistry. A separate group of mice were fed an atherogenic diet, with or without injections of RS-2982 for 3 weeks; livers were analyzed by immunohistochemistry, and plasma was analyzed for levels of aminotransferases. We analyzed data from liver tissues from patients with NASH included in the RNA-sequencing databases GSE33814 and GSE89632. RESULTS: Injection of mice with antagomiR-122 significantly reduced levels of MIR122 in plasma, liver, and white adipose tissue; in mice on an HFD, antagomiR-122 injections increased fat droplets and total triglyceride content in liver and reduced beta-oxidation and energy expenditure, resulting in significantly more weight gain than in mice given the control microRNA. We identified RS-2982 as an agonist of RORA and found it to increase expression of MIR122 promoter activity in Huh7 cells. In mice fed an HFD or atherogenic diet, injections of RS-2982 increased hepatic levels of MIR122 precursors and reduced hepatic synthesis of triglycerides by reducing expression of biosynthesis enzymes. In these mice, RS-2982 significantly reduced hepatic lipotoxicity, reduced liver fibrosis, increased insulin resistance, and reduced body weight compared with mice injected with vehicle. Patients who underwent cardiovascular surgery had increased levels of plasma MIR122 compared to its levels before surgery; increased expression of plasma MIR122 was associated with increased levels of plasma free fatty acids and levels of RORA. CONCLUSIONS: We identified the compound RS-2982 as an agonist of RORA that increases expression of MIR122 in cell lines and livers of mice. Mice fed an HFD or atherogenic diet given injections of RS-2982 had reduced hepatic lipotoxicity, liver fibrosis, and body weight compared with mice given the vehicle. Agonists of RORA might be developed for treatment of NASH.

BACKGROUND: In mammals, sex chromosomes pose an inherent imbalance of gene expression between sexes. In each female somatic cell, random inactivation of one of the X-chromosomes restores this balance. While most genes from the inactivated X-chromosome are silenced, 15-25% are known to escape X-inactivation (termed escapees). The expression levels of these genes are attributed to sex-dependent phenotypic variability. RESULTS: We used single-cell RNA-Seq to detect escapees in somatic cells. As only one X-chromosome is inactivated in each cell, the origin of expression from the active or inactive chromosome can be determined from the variation of sequenced RNAs. We analyzed primary, healthy fibroblasts (n = 104), and clonal lymphoblasts with sequenced parental genomes (n = 25) by measuring the degree of allelic-specific expression (ASE) from heterozygous sites. We identified 24 and 49 candidate escapees, at varying degree of confidence, from the fibroblast and lymphoblast transcriptomes, respectively. We critically test the validity of escapee annotations by comparing our findings with a large collection of independent studies. We find that most genes (66%) from the unified set were previously reported as escapees. Furthermore, out of the overlooked escapees, 11 are long noncoding RNA (lncRNAs). CONCLUSIONS: X-chromosome inactivation and escaping from it are robust, permanent phenomena that are best studies at a single-cell resolution. The cumulative information from individual cells increases the potential of identifying escapees. Moreover, despite the use of a limited number of cells, clonal cells (i.e., same X- chromosomes are coordinately inhibited) with genomic phasing are valuable for detecting escapees at high confidence. Generalizing the method to uncharacterized genomic loci resulted in lncRNAs escapees which account for 20% of the listed candidates. By confirming genes as escapees and propose others as candidates from two different cell types, we contribute to the cumulative knowledge and reliability of human escapees.

Osteosarcoma (OS) is an aggressive malignancy affecting mostly children and adolescents. MicroRNAs (miRNAs) play important roles in OS development and progression. Here we found that miR-16-1-3p and miR-16-2-3p "passenger" strands, as well as the "lead" miR-16-5p strand, are frequently downregulated and possess strong tumor suppressive functions in human OS. Furthermore, we report different although strongly overlapping functions for miR-16-1-3p and miR-16-2-3p in OS cells. Ectopic expression of these miRNAs affected primary tumor growth, metastasis seeding and chemoresistance and invasiveness of human OS cells. Loss-of-function experiments verified tumor suppressive functions of these miRNAs at endogenous levels of expression. Using RNA immunoprecipitation (RIP) assays, we identify direct targets of miR-16-1-3p and miR-16-2-3p in OS cells. Moreover, validation experiments identified FGFR2 as a direct target for miR-16-1-3p and miR-16-2-3p. Overall, our findings underscore the importance of passenger strand miRNAs, at least some, in osteosarcomagenesis.

Mitophagy is an important quality-control mechanism in eukaryotic cells, and defects in mitophagy correlate with aging phenomena and neurodegenerative disorders. It is known that different mitochondrial matrix proteins undergo mitophagy with very different rates but, to date, the mechanism underlying this selectivity at the individual protein level has remained obscure. We now present evidence indicating that protein phosphorylation within the mitochondrial matrix plays a mechanistic role in regulating selective mitophagic degradation in yeast via involvement of the Aup1 mitochondrial protein phosphatase, as well as 2 known matrix-localized protein kinases, Pkp1 and Pkp2. By focusing on a specific matrix phosphoprotein reporter, we also demonstrate that phospho-mimetic and nonphosphorylatable point mutations at known phosphosites in the reporter increased or decreased its tendency to undergo mitophagy. Finally, we show that phosphorylation of the reporter protein is dynamically regulated during mitophagy in an Aup1-dependent manner. Our results indicate that structural determinants on a mitochondrial matrix protein can govern its mitophagic fate, and that protein phosphorylation regulates these determinants.

Differential exposure of tumor cells to blood-borne and angiocrine factors results in diverse metabolic microenvironments conducive for non-genetic tumor cell diversification. Here, we harnessed a methodology for retrospective sorting of fully functional, stroma-free cancer cells solely on the basis of their relative distance from blood vessels (BVs) to unveil the whole spectrum of genes, metabolites, and biological traits impacted by BV proximity. In both grafted mouse tumors and natural human glioblastoma (GBM), mTOR activity was confined to few cell layers from the nearest perfused vessel. Cancer cells within this perivascular tier are distinguished by intense anabolic metabolism and defy the Warburg principle through exercising extensive oxidative phosphorylation. Functional traits acquired by perivascular cancer cells, namely, enhanced tumorigenicity, superior migratory or invasive capabilities, and, unexpectedly, exceptional chemo- and radioresistance, are all mTOR dependent. Taken together, the study revealed a previously unappreciated graded metabolic zonation directly impacting the acquisition of multiple aggressive tumor traits.

Osteocytes, cells forming an elaborate network within the bones of most vertebrate taxa, are thought to be the master regulators of bone modeling, a process of coordinated, local bone-tissue deposition and removal that keeps bone strains at safe levels throughout life. Neoteleost fish, however, lack osteocytes and yet are known to be capable of bone modeling, although no osteocyte-independent modeling regulatory mechanism has so far been described. Here, we characterize a novel, to our knowledge, bone-modeling regulatory mechanism in a fish species (medaka), showing that although lacking osteocytes (i.e., internal mechanosensors), when loaded, medaka bones model in mechanically directed ways, successfully reducing high tissue strains. We establish that as in mammals, modeling in medaka is regulated by the SOST gene, demonstrating a mechanistic link between skeletal loading, SOST down-regulation, and intense bone deposition. However, whereas mammalian SOST is expressed almost exclusively by osteocytes, in both medaka and zebrafish (a species with osteocytic bones), SOST is expressed by a variety of nonosteocytic cells, none of which reside within the bone bulk. These findings argue that in fishes (and perhaps other vertebrates), nonosteocytic skeletal cells are both sensors and responders, shouldering duties believed exclusive to osteocytes. This previously unrecognized, SOST-dependent, osteocyte-independent mechanism challenges current paradigms of osteocyte exclusivity in bone-modeling regulation, suggesting the existence of multivariate feedback networks in bone modeling-perhaps also in mammalian bones-and thus arguing for the possibility of untapped potential for cell targets in bone therapeutics.

Cellular mechanisms that act in concert to maintain protein homeostasis (proteostasis) are vital for organismal functionality and survival. Nevertheless, subsets of aggregation-prone proteins form toxic aggregates (proteotoxicity) that in some cases, underlie the development of neurodegenerative diseases. Proteotoxic aggregates are often deposited in the vicinity of the nucleus, a process that is cytoskeleton-dependent. Accordingly, cytoskeletal dysfunction contributes to pathological hallmarks of various neurodegenerative diseases. Here, we asked whether the linker of nucleoskeleton and cytoskeleton (LINC) complex, which bridges these filaments across the nuclear envelope, is needed for the maintenance of proteostasis. Employing model nematodes, we discovered that knocking down LINC components impairs the ability of the worm to cope with proteotoxicity. Knocking down anc-1, which encodes a key component of the LINC complex, modulates the expression of transcription factors and E3 ubiquitin ligases, thereby affecting the rates of protein ubiquitination and impairing proteasome-mediated protein degradation. Our results establish a link between the LINC complex, protein degradation, and neurodegeneration-associated proteotoxicity.

At some point early in the vertebrate lineage, two whole genome duplication events (1R, 2R) took place that allowed for the diversification and sub-/neo-functionalization of the glycoprotein hormones (GpHs). All jawed vertebrates possess the GpHs luteinizing hormone (LH), follicle stimulating hormone (FSH), and thyroid stimulating hormone (TSH), each of which are heterodimers with a common alpha subunit and unique beta subunits. In 2002, a novel glycoprotein hormone named thyrostimulin was described to have unique GpA2 and GpB5 subunits that were homologous to the vertebrate alpha and beta subunits. The presence of GpA2 and GpB5 in representative protostomes and deuterostomes indicates their ancestry in the GpH family. There are several reports of GpH subunit evolution, but none have included GpA2 and GpB5 for species in each major vertebrate class. Thus, we addressed the ancestry of two paralogous GpB5 subunits (GpB5a and GpB5b) that were previously only recognized in two teleost species. Our search for orthologous GpB5a and GpB5b sequences in representative vertebrates and phylogenetic analysis, in addition to the currently published evolutionary scenarios of the GpH family, supports that GpB5a and GpB5b are paralogs that arose from the first vertebrate whole genome duplication event (1R). Syntenic analysis supports lineage specific losses of GpB5a in chondrichthyes, basal actinopterygians, and tetrapods, and retention in coelacanth and teleosts. Additionally, we were unable to identify GpA2 transcripts from tilapia mRNA, suggesting that this species does not produce heterodimeric thyrostimulin. While the conserved or even species-specific functional role of thyrostimulin or its individual subunits are still unknown in vertebrates, the analyses presented here provide context for future studies on the functional divergence of the GpH family.

RNA localization in eukaryotes is a mechanism to regulate transcripts fate. Conversely, bacterial transcripts were not assumed to be specifically localized. We previously demonstrated that E. coli mRNAs may localize to where their products localize in a translation-independent manner, thus challenging the transcription-translation coupling extent. However, the scope of RNA localization in bacteria remained unknown. Here, we report the distribution of the E. coli transcriptome between the membrane, cytoplasm, and poles by combining cell fractionation with deep-sequencing (Rloc-seq). Our results reveal asymmetric RNA distribution on a transcriptome-wide scale, significantly correlating with proteome localization and prevalence of translation-independent RNA localization. The poles are enriched with stress-related mRNAs and small RNAs, the latter becoming further enriched upon stress in an Hfq-dependent manner. Genome organization may play a role in localizing membrane protein-encoding transcripts. Our results show an unexpected level of intricacy in bacterial transcriptome organization and highlight the poles as hubs for regulation.