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.

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.

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.

Acinar metaplasia is an initial step in a series of events that can lead to pancreatic cancer. Here we perform single-cell RNA-sequencing of mouse pancreas during the progression from preinvasive stages to tumor formation. Using a reporter gene, we identify metaplastic cells that originated from acinar cells and express two transcription factors, Onecut2 and Foxq1. Further analyses of metaplastic acinar cell heterogeneity define six acinar metaplastic cell types and states, including stomach-specific cell types. Localization of metaplastic cell types and mixture of different metaplastic cell types in the same pre-malignant lesion is shown. Finally, single-cell transcriptome analyses of tumor-associated stromal, immune, endothelial and fibroblast cells identify signals that may support tumor development, as well as the recruitment and education of immune cells. Our findings are consistent with the early, premalignant formation of an immunosuppressive environment mediated by interactions between acinar metaplastic cells and other cells in the microenvironment.

OBJECTIVE: Failing to properly repair damaged DNA drives the ageing process. Furthermore, age-related inflammation contributes to the manifestation of ageing. Recently, we demonstrated that the efficiency of repair of diethylnitrosamine (DEN)-induced double-strand breaks (DSBs) rapidly declines with age. We therefore hypothesised that with age, the decline in DNA damage repair stems from age-related inflammation. DESIGN: We used DEN-induced DNA damage in mouse livers and compared the efficiency of their resolution in different ages and following various permutations aimed at manipulating the liver age-related inflammation. RESULTS: We found that age-related deregulation of innate immunity was linked to altered gut microbiota. Consequently, antibiotic treatment, MyD88 ablation or germ-free mice had reduced cytokine expression and improved DSBs rejoining in 6-month-old mice. In contrast, feeding young mice with a high-fat diet enhanced inflammation and facilitated the decline in DSBs repair. This latter effect was reversed by antibiotic treatment. Kupffer cell replenishment or their inactivation with gadolinium chloride reduced proinflammatory cytokine expression and reversed the decline in DSBs repair. The addition of proinflammatory cytokines ablated DSBs rejoining mediated by macrophage-derived heparin-binding epidermal growth factor-like growth factor. CONCLUSIONS: Taken together, our results reveal a previously unrecognised link between commensal bacteria-induced inflammation that results in age-dependent decline in DNA damage repair. Importantly, the present study support the notion of a cell non-autonomous mechanism for age-related decline in DNA damage repair that is based on the presence of 'inflamm-ageing' cytokines in the tissue microenvironment, rather than an intrinsic cellular deficiency in the DNA repair machinery.

BACKGROUND: Mutations in GNE cause a recessive, adult onset myopathy characterized by slowly progressive distal and proximal muscle weakness. Knock-in mice carrying the most frequent mutation in GNE myopathy patients, GneM743T/M743T, usually die few days after birth from severe renal failure, with no muscle phenotype. However, a spontaneous sub-colony remains healthy throughout a normal lifespan without any kidney or muscle pathology. OBJECTIVE: We attempted to decipher the molecular mechanisms behind these phenotypic differences and to determine the mechanisms preventing the kidney and muscles from disease. METHODS: We analyzed the transcriptome and proteome of kidneys and muscles of sick and healthy GneM743T/M743T mice. RESULTS: The sick GneM743T/M743T kidney was characterized by up-regulation of extra-cellular matrix degradation related processes and by down-regulation of oxidative phosphorylation and respiratory electron chain pathway, that was also observed in the asymptomatic muscles. Surprisingly, the healthy kidneys of the GneM743T/M743T mice were characterized by up-regulation of hallmark muscle genes. In addition the asymptomatic muscles of the sick GneM743T/M743T mice showed upregulation of transcription and translation processes. CONCLUSIONS: Overexpression of muscle physiology genes in healthy GneM743T/M743T mice seems to define the protecting mechanism in these mice. Furthermore, the strong involvement of muscle related genes in kidney may bridge the apparent phenotypic gap between GNE myopathy and the knock-in GneM743T/M743T mouse model and provide new directions in the study of GNE function in health and disease.

The initial events of viral infection at the primary mucosal entry site following horizontal person-to-person transmission have remained ill defined. Our limited understanding is further underscored by the absence of animal models in the case of human-restricted viruses, such as human cytomegalovirus (HCMV), a leading cause of congenital infection and a major pathogen in immunocompromised individuals. Here, we established a novel ex vivo model of HCMV infection in native human nasal turbinate tissues. Nasal turbinate tissue viability and physiological functionality were preserved for at least 7 days in culture. We found that nasal mucosal tissues were susceptible to HCMV infection, with predominant infection of ciliated respiratory epithelial cells. A limited viral spread was demonstrated, involving mainly stromal and vascular endothelial cells within the tissue. Importantly, functional antiviral and proleukocyte chemotactic signaling pathways were significantly upregulated in the nasal mucosa in response to infection. Conversely, HCMV downregulated the expression of nasal epithelial cell-related genes. We further revealed tissue-specific innate immune response patterns to HCMV, comparing infected human nasal mucosal and placental tissues, representing the viral entry and the maternal-to-fetal transmission sites, respectively. Taken together, our studies provide insights into the earliest stages of HCMV infection. Studies in this model could help evaluate new interventions against the horizontal transmission of HCMV.IMPORTANCE HCMV is a ubiquitous human pathogen causing neurodevelopmental disabilities in congenitally infected children and severe disease in immunocompromised patients. The earliest stages of HCMV infection in the human host have remained elusive in the absence of a model for the viral entry site. Here, we describe the establishment and use of a novel nasal turbinate organ culture to study the initial steps of viral infection and the consequent innate immune responses within the natural complexity and the full cellular repertoire of human nasal mucosal tissues. This model can be applied to examine new antiviral interventions against the horizontal transmission of HCMV and potentially that of other viruses.

p16(INK4a) (CDKN2A) is a central tumor suppressor, which induces cell-cycle arrest and senescence. Cells expressing p16(INK4a) accumulate in aging tissues and appear in premalignant lesions, yet their physiologic effects are poorly understood. We found that prolonged expression of transgenic p16(INK4a) in the mouse epidermis induces hyperplasia and dysplasia, involving high proliferation rates of keratinocytes not expressing the transgene. Continuous p16(INK4a) expression increases the number of epidermal papillomas formed after carcinogen treatment. Wnt-pathway ligands and targets are activated upon prolonged p16(INK4a) expression, and Wnt inhibition suppresses p16(INK4a)-induced hyperplasia. Senolytic treatment reduces p16(INK4a)-expressing cell numbers, and inhibits Wnt activation and hyperplasia. In human actinic keratosis, a precursor of squamous cell carcinoma, p16(INK4a)-expressing cells are found adjacent to dividing cells, consistent with paracrine interaction. These findings reveal that chronic p16(INK4a) expression is sufficient to induce hyperplasia through Wnt-mediated paracrine stimulation, and suggest that this tumor suppressor can promote early premalignant epidermal lesion formation.

Sepsis is an excessive, dysregulated immune response to infection that activates inflammatory and coagulation cascades, which may lead to tissue injury, multiple organ dysfunction syndrome and death. Millions of individuals die annually of sepsis. To date, the only treatment available is antibiotics, drainage of the infection source when possible, and organ support in intensive care units. Numerous previous attempts to develop therapeutic treatments, directed at discreet targets of the sepsis cascade, could not cope with the complex pathophysiology of sepsis and failed. Here we describe a novel treatment, based on empty capsids of SV40 (nanocapsids - NCs). Studies in a severe rat sepsis model showed that pre-treatment by NCs led to a dramatic increase in survival, from zero to 75%. Transcript analyses (RNAseq) demonstrated that the NC treatment is a paradigm shift. The NCs affect multiple facets of biological functions. The affected genes are modified with time, adjusting to the recovery processes. The NCs effect on normal control rats was negligible. The study shows that the NCs are capable of coping with diseases with intricate pathophysiology. Further studies are needed to determine whether when applied after sepsis onset, the NCs still improve outcome.

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.

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.

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.

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.

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.