Dormant bacterial spores encrypt a long-lasting transcriptional program to be executed during revival.

Sporulating bacteria can retreat into long-lasting dormant spores that preserve the capacity to germinate when propitious. However, how the revival transcriptional program is memorized for years remains elusive. We revealed that in dormant spores, core RNA polymerase (RNAP) resides in a central chromosomal domain, where it remains bound to a subset of intergenic promoter regions. These regions regulate genes encoding for most essential cellular functions, such as rRNAs and tRNAs. Upon awakening, RNAP recruits key transcriptional components, including sigma factor, and progresses to express the adjacent downstream genes. Mutants devoid of spore DNA-compacting proteins exhibit scattered RNAP localization and subsequently disordered firing of gene expression during germination. Accordingly, we propose that the spore chromosome is structured to preserve the transcriptional program by halting RNAP, prepared to execute transcription at the auspicious time. Such a mechanism may sustain long-term transcriptional programs in diverse organisms displaying a quiescent life form.

Recycled melanoma-secreted melanosomes regulate tumor-associated macrophage diversification.

Extracellular vesicles (EVs) are important mediators of communication between cells. Here, we reveal a new mode of intercellular communication by melanosomes, large EVs secreted by melanocytes for melanin transport. Unlike small EVs, which are disintegrated within the receiver cell, melanosomes stay intact within them, gain a unique protein signature, and can then be further transferred to another cell as "second-hand" EVs. We show that melanoma-secreted melanosomes passaged through epidermal keratinocytes or dermal fibroblasts can be further engulfed by resident macrophages. This process leads to macrophage polarization into pro-tumor or pro-immune cell infiltration phenotypes. Melanosomes that are transferred through fibroblasts can carry AKT1, which induces VEGF secretion from macrophages in an mTOR-dependent manner, promoting angiogenesis and metastasis in vivo. In melanoma patients, macrophages that are co-localized with AKT1 are correlated with disease aggressiveness, and immunotherapy non-responders are enriched in macrophages containing melanosome markers. Our findings suggest that interactions mediated by second-hand extracellular vesicles contribute to the formation of the metastatic niche, and that blocking the melanosome cues of macrophage diversification could be helpful in halting melanoma progression.

Hindbrain boundaries as niches of neural progenitor and stem cells regulated by the extracellular matrix proteoglycan chondroitin sulphate.

The interplay between neural progenitors and stem cells (NPSCs), and their extracellular matrix (ECM) is a crucial regulatory mechanism that determines their behavior. Nonetheless, how the ECM dictates the state of NPSCs remains elusive. The hindbrain is valuable to examine this relationship, as cells in the ventricular surface of hindbrain boundaries (HBs), which arise between any two neighboring rhombomeres, express the NPSC marker Sox2, while being surrounded with the membrane-bound ECM molecule chondroitin sulphate proteoglycan (CSPG), in chick and mouse embryos. CSPG expression was used to isolate HB Sox2+ cells for RNA-sequencing, revealing their distinguished molecular properties as typical NPSCs, which express known and newly identified genes relating to stem cells, cancer, the matrisome and cell cycle. In contrast, the CSPG- non-HB cells, displayed clear neural-differentiation transcriptome. To address whether CSPG is significant for hindbrain development, its expression was manipulated in vivo and in vitro. CSPG manipulations shifted the stem versus differentiation state of HB cells, evident by their behavior and altered gene expression. These results provide further understanding of the uniqueness of hindbrain boundaries as repetitive pools of NPSCs in-between the rapidly growing rhombomeres, which rely on their microenvironment to maintain their undifferentiated state during development.

Isoforms of the TAL1 transcription factor have different roles in hematopoiesis and cell growth.

T-cell acute lymphoblastic leukemia (T-ALL) protein 1 (TAL1) is a central transcription factor in hematopoiesis. The timing and level of TAL1 expression orchestrate the differentiation to specialized blood cells and its overexpression is a common cause of T-ALL. Here, we studied the 2 protein isoforms of TAL1, short and long, which are generated by the use of alternative promoters as well as by alternative splicing. We analyzed the expression of each isoform by deleting an enhancer or insulator, or by opening chromatin at the enhancer location. Our results show that each enhancer promotes expression from a specific TAL1 promoter. Expression from a specific promoter gives rise to a unique 5' UTR with differential regulation of translation. Moreover, our study suggests that the enhancers regulate TAL1 exon 3 alternative splicing by inducing changes in the chromatin at the splice site, which we demonstrate is mediated by KMT2B. Furthermore, our results indicate that TAL1-short binds more strongly to TAL1 E-protein partners and functions as a stronger transcription factor than TAL1-long. Specifically TAL1-short has a unique transcription signature promoting apoptosis. Finally, when we expressed both isoforms in mice bone marrow, we found that while overexpression of both isoforms prevents lymphoid differentiation, expression of TAL1-short alone leads to hematopoietic stem cell exhaustion. Furthermore, we found that TAL1-short promoted erythropoiesis and reduced cell survival in the CML cell line K562. While TAL1 and its partners are considered promising therapeutic targets in the treatment of T-ALL, our results show that TAL1-short could act as a tumor suppressor and suggest that altering TAL1 isoform's ratio could be a preferred therapeutic approach.

UV-Protection Timer Controls Linkage between Stress and Pigmentation Skin Protection Systems.

Skin sun exposure induces two protection programs: stress responses and pigmentation, the former within minutes and the latter only hours afterward. Although serving the same physiological purpose, it is not known whether and how these programs are coordinated. Here, we report that UVB exposure every other day induces significantly more skin pigmentation than the higher frequency of daily exposure, without an associated increase in stress responses. Using mathematical modeling and empirical studies, we show that the melanocyte master regulator, MITF, serves to synchronize stress responses and pigmentation and, furthermore, functions as a UV-protection timer via damped oscillatory dynamics, thereby conferring a trade-off between the two programs. MITF oscillations are controlled by multiple negative regulatory loops, one at the transcriptional level involving HIF1α and another post-transcriptional loop involving microRNA-148a. These findings support trait linkage between the two skin protection programs, which, we speculate, arose during furless skin evolution to minimize skin damage.

A nuclear redox sensor modulates gene activation and var switching in Plasmodium falciparum.

The virulence of Plasmodium falciparum, which causes the deadliest form of human malaria, is attributed to its ability to evade the human immune response. These parasites "choose" to express a single variant from a repertoire of surface antigens called PfEMP1, which are placed on the surface of the infected red cell. Immune evasion is achieved by switches in expression between var genes, each encoding a different PfEMP1 variant. While the mechanisms that regulate mutually exclusive expression of var genes are still elusive, antisense long-noncoding RNAs (lncRNAs) transcribed from the intron of the active var gene were implicated in the "choice" of the single active var gene. Here, we show that this lncRNA colocalizes with the site of var mRNA transcription and is anchored to the var locus via DNA:RNA interactions. We define the var lncRNA interactome and identify a redox sensor, P. falciparum thioredoxin peroxidase I (PfTPx-1), as one of the proteins associated with the var antisense lncRNA. We show that PfTPx-1 localizes to a nuclear subcompartment associated with active transcription on the nuclear periphery, in ring-stage parasite, when var transcription occurs. In addition, PfTPx-1 colocalizes with S-adenosylmethionine synthetase (PfSAMS) in the nucleus, and its overexpression leads to activation of var2csa, similar to overexpression of PfSAMS. Furthermore, we show that PfTPx-1 knockdown alters the var switch rate as well as activation of additional gene subsets. Taken together, our data indicate that nuclear PfTPx-1 plays a role in gene activation possibly by providing a redox-controlled nuclear microenvironment ideal for active transcription.

Muscle injury causes long-term changes in stem-cell DNA methylation.

Injury to muscle brings about the activation of stem cells, which then generate new myocytes to replace damaged tissue. We demonstrate that this activation is accompanied by a dramatic change in the stem-cell methylation pattern that prepares them epigenetically for terminal myocyte differentiation. These de- and de novo methylation events occur at regulatory elements associated with genes involved in myogenesis and are necessary for activation and regeneration. Local injury of one muscle elicits an almost identical epigenetic change in satellite cells from other muscles in the body, in a process mediated by circulating factors. Furthermore, this same methylation state is also generated in muscle stem cells (MuSCs) of female animals following pregnancy, even in the absence of any injury. Unlike the activation-induced expression changes, which are transient, the induced methylation profile is stably maintained in resident MuSCs and thus represents a molecular memory of previous physiological events that is probably programmed to provide a mechanism for long-term adaptation.

Characterization of C9orf72 haplotypes to evaluate the effects of normal and pathological variations on its expression and splicing.

Expansion of the hexanucleotide repeat (HR) in the first intron of the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) in Caucasians. All C9orf72-ALS/FTD patients share a common risk (R) haplotype. To study C9orf72 expression and splicing from the mutant R allele compared to the complementary normal allele in ALS/FTD patients, we initially created a detailed molecular map of the single nucleotide polymorphism (SNP) signature and the HR length of the various C9orf72 haplotypes in Caucasians. We leveraged this map to determine the allelic origin of transcripts per patient, and decipher the effects of pathological and normal HR lengths on C9orf72 expression and splicing. In C9orf72 ALS patients' cells, the HR expanded allele, compared to non-R allele, was associated with decreased levels of a downstream initiated transcript variant and increased levels of transcripts initiated upstream of the HR. HR expanded R alleles correlated with high levels of unspliced intron 1 and activation of cryptic donor splice sites along intron 1. Retention of intron 1 was associated with sequential intron 2 retention. The SNP signature of C9orf72 haplotypes described here enables allele-specific analysis of transcriptional products and may pave the way to allele-specific therapeutic strategies.

KDM3A regulates alternative splicing of cell-cycle genes following DNA damage.

Changes in the cellular environment result in chromatin structure alteration, which in turn regulates gene expression. To learn about the effect of the cellular environment on the transcriptome, we studied the H3K9 demethylase KDM3A. Using RNA-seq, we found that KDM3A regulates the transcription and alternative splicing of genes associated with cell cycle and DNA damage. We showed that KDM3A undergoes phosphorylation by PKA at serine 265 following DNA damage, and that the phosphorylation is important for proper cell-cycle regulation. We demonstrated that SAT1 alternative splicing, regulated by KDM3A, plays a role in cell-cycle regulation. Furthermore we found that KDM3A's demethylase activity is not needed for SAT1 alternative splicing regulation. In addition, we identified KDM3A's protein partner ARID1A, the SWI/SNF subunit, and SRSF3 as regulators of SAT1 alternative splicing and showed that KDM3A is essential for SRSF3 binding to SAT1 pre-mRNA. These results suggest that KDM3A serves as a sensor of the environment and an adaptor for splicing factor binding. Our work reveals chromatin sensing of the environment in the regulation of alternative splicing.

Amyloidogenic light chains impair plasma cell survival.

Systemic light chain amyloidosis (AL) is a clonal plasma cell disorder characterized by the deposition of misfolded immunoglobulin light chains (LC) as insoluble fibrils in organs. The lack of suitable models has hindered the investigation of the disease mechanisms. Our aim was to establish AL LC-producing plasma cell lines and use them to investigate the biology of the amyloidogenic clone. We used lentiviral vectors to generate cell lines expressing LC from patients suffering from AL amyloidosis. The AL LC-producing cell lines showed a significant decrease in proliferation, cell cycle arrest, and an increase in apoptosis and autophagy as compared with the multiple myeloma LC-producing cells. According to the results of RNA sequencing the AL LC-producing lines showed higher mitochondrial oxidative stress, and decreased activity of the Myc and cholesterol pathways. The neoplastic behavior of plasma cells is altered by the constitutive expression of amyloidogenic LC causing intracellular toxicity. This observation may explain the disparity in the malignant behavior of the amyloid clone compared to the myeloma clone. These findings should enable future in vitro studies and help delineate the unique cellular pathways of AL, thus expediting the development of specific treatments for patients with this disorder.

Mesenchymal Stromal Cell-Derived Small Extracellular Vesicles Modulate Apoptosis, TNF Alpha and Interferon Gamma Response Gene mRNA Expression in T Lymphocytes.

Recent studies have highlighted the therapeutic potential of small extracellular bodies derived from mesenchymal stem cells (MSC-sEVs) for various diseases, notably through their ability to alter T-cell differentiation and function. The current study aimed to explore immunomodulatory pathway alterations within T cells through mRNA sequencing of activated T cells cocultured with bone marrow-derived MSC-sEVs. mRNA profiling of activated human T cells cocultured with MSC-sEVs or vehicle control was performed using the QIAGEN Illumina sequencing platform. Pathway networks and biological functions of the differentially expressed genes were analyzed using Ingenuity pathway analysis (IPA)® software, KEGG pathway, GSEA and STRING database. A total of 364 differentially expressed genes were identified in sEV-treated T cells. Canonical pathway analysis highlighted the RhoA signaling pathway. Cellular development, movement, growth and proliferation, cell-to-cell interaction and inflammatory response-related gene expression were altered. KEGG enrichment pathway analysis underscored the apoptosis pathway. GSEA identified enrichment in downregulated genes associated with TNF alpha and interferon gamma response, and upregulated genes related to apoptosis and migration of lymphocytes and T-cell differentiation gene sets. Our findings provide valuable insights into the mechanisms by which MSC-sEVs implement immunomodulatory effects on activated T cells. These findings may contribute to the development of MSC-sEV-based therapies.

Combined hepatocellular-cholangiocarcinoma derives from liver progenitor cells and depends on senescence and IL-6 trans-signaling.

BACKGROUND & AIMS: Primary liver cancers include hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (CCA) and combined HCC-CCA tumors (cHCC-CCA). It has been suggested, but not unequivocally proven, that hepatic progenitor cells (HPCs) can contribute to hepatocarcinogenesis. We aimed to determine whether HPCs contribute to HCC, cHCC-CCA or both types of tumors. METHODS: To trace progenitor cells during hepatocarcinogenesis, we generated Mdr2-KO mice that harbor a yellow fluorescent protein (YFP) reporter gene driven by the Foxl1 promoter which is expressed specifically in progenitor cells. These mice (Mdr2-KOFoxl1-CRE;RosaYFP) develop chronic inflammation and HCCs by the age of 14-16 months, followed by cHCC-CCA tumors at the age of 18 months. RESULTS: In this Mdr2-KOFoxl1-CRE;RosaYFP mouse model, liver progenitor cells are the source of cHCC-CCA tumors, but not the source of HCC. Ablating the progenitors, caused reduction of cHCC-CCA tumors but did not affect HCCs. RNA-sequencing revealed enrichment of the IL-6 signaling pathway in cHCC-CCA tumors compared to HCC tumors. Single-cell RNA-sequencing (scRNA-seq) analysis revealed that IL-6 is expressed by immune and parenchymal cells during senescence, and that IL-6 is part of the senescence-associated secretory phenotype. Administration of an anti-IL-6 antibody to Mdr2-KOFoxl1-CRE;RosaYFP mice inhibited the development of cHCC-CCA tumors. Blocking IL-6 trans-signaling led to a decrease in the number and size of cHCC-CCA tumors, indicating their dependence on this pathway. Furthermore, the administration of a senolytic agent inhibited IL-6 and the development of cHCC-CCA tumors. CONCLUSION: Our results demonstrate that cHCC-CCA, but not HCC tumors, originate from HPCs, and that IL-6, which derives in part from cells in senescence, plays an important role in this process via IL-6 trans-signaling. These findings could be applied to develop new therapeutic approaches for cHCC-CCA tumors. LAY SUMMARY: Combined hepatocellular carcinoma-cholangiocarcinoma is the third most prevalent type of primary liver cancer (i.e. a cancer that originates in the liver). Herein, we show that this type of cancer originates in stem cells in the liver and that it depends on inflammatory signaling. Specifically, we identify a cytokine called IL-6 that appears to be important in the development of these tumors. Our results could be used for the development of novel treatments for these aggressive tumors.

Human Nasal and Lung Tissues Infected Ex Vivo with SARS-CoV-2 Provide Insights into Differential Tissue-Specific and Virus-Specific Innate Immune Responses in the Upper and Lower Respiratory Tract.

The nasal mucosa constitutes the primary entry site for respiratory viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While the imbalanced innate immune response of end-stage coronavirus disease 2019 (COVID-19) has been extensively studied, the earliest stages of SARS-CoV-2 infection at the mucosal entry site have remained unexplored. Here, we employed SARS-CoV-2 and influenza virus infection in native multi-cell-type human nasal turbinate and lung tissues ex vivo, coupled with genome-wide transcriptional analysis, to investigate viral susceptibility and early patterns of local mucosal innate immune response in the authentic milieu of the human respiratory tract. SARS-CoV-2 productively infected the nasal turbinate tissues, predominantly targeting respiratory epithelial cells, with a rapid increase in tissue-associated viral subgenomic mRNA and secretion of infectious viral progeny. Importantly, SARS-CoV-2 infection triggered robust antiviral and inflammatory innate immune responses in the nasal mucosa. The upregulation of interferon-stimulated genes, cytokines, and chemokines, related to interferon signaling and immune-cell activation pathways, was broader than that triggered by influenza virus infection. Conversely, lung tissues exhibited a restricted innate immune response to SARS-CoV-2, with a conspicuous lack of type I and III interferon upregulation, contrasting with their vigorous innate immune response to influenza virus. Our findings reveal differential tissue-specific innate immune responses in the upper and lower respiratory tracts that are specific to SARS-CoV-2. The studies shed light on the role of the nasal mucosa in active viral transmission and immune defense, implying a window of opportunity for early interventions, whereas the restricted innate immune response in early-SARS-CoV-2-infected lung tissues could underlie the unique uncontrolled late-phase lung damage of advanced COVID-19. IMPORTANCE In order to reduce the late-phase morbidity and mortality of COVID-19, there is a need to better understand and target the earliest stages of SARS-CoV-2 infection in the human respiratory tract. Here, we have studied the initial steps of SARS-CoV-2 infection and the consequent innate immune responses within the natural multicellular complexity of human nasal mucosal and lung tissues. Comparing the global innate response patterns of nasal and lung tissues infected in parallel with SARS-CoV-2 and influenza virus, we found distinct virus-host interactions in the upper and lower respiratory tract, which could determine the outcome and unique pathogenesis of SARS-CoV-2 infection. Studies in the nasal mucosal infection model can be employed to assess the impact of viral evolutionary changes and evaluate new therapeutic and preventive measures against SARS-CoV-2 and other human respiratory pathogens.

Use of Nanocellulose to Produce Water-Based Conductive Inks with Ag NPs for Printed Electronics.

The need for more sustainable printed electronics has emerged in the past years. Due to this, the use of nanocellulose (NC) extracted from cellulose has recently been demonstrated to provide interesting materials such as functional inks and transparent flexible films due to its properties. Its high specific surface area together with the high content of reactive hydroxyl groups provide a highly tailorable surface chemistry with applications in ink formulations as a stabilizing, capping, binding and templating agent. Moreover, NC mechanical, physical and thermal properties (high strength, low porosity and high thermal stability, respectively) provide an excellent alternative for the currently used plastic films. In this work, we present a process for the production of water-based conductive inks that uses NC both as a template for silver nanoparticles (Ag NPs) formation and as an ink additive for ink formulation. The new inks present an electrical conductivity up to 2 × 106 S/m, which is in the range of current commercially available conductive inks. Finally, the new Ag NP/NC-based conductive inks have been tested to fabricate NFC antennas by screen-printing onto NC-coated paper, demonstrating to be operative.

Pax7, Pax3 and Mamstr genes are involved in skeletal muscle impaired regeneration of dy2J/dy2J mouse model of Lama2-CMD.

Congenital muscular dystrophy type-1A (Lama2-CMD) and Duchenne muscular dystrophy (DMD) result from deficiencies of laminin-α2 and dystrophin proteins, respectively. Although both proteins strengthen the sarcolemma, they are implicated in clinically distinct phenotypes. We used RNA-deep sequencing (RNA-Seq) of dy2J/dy2J, Lama2-CMD mouse model, skeletal muscle at 8 weeks of age to elucidate disease pathophysiology. This study is the first report of dy2J/dy2J model whole transcriptome profile. RNA-Seq of the mdx mouse model of DMD and wild-type (WT) mouse was carried as well in order to enable a novel comparison of dy2J/dy2J to mdx. A large group of shared differentially expressed genes (DEGs) was found in dy2J/dy2J and mdx models (1834 common DEGs, false discovery rate [FDR] < 0.05). Enrichment pathway analysis using ingenuity pathway analysis showed enrichment of inflammation, fibrosis, cellular movement, migration and proliferation of cells, apoptosis and necrosis in both mouse models (P-values 3E-10-9E-37). Via canonical pathway analysis, actin cytoskeleton, integrin, integrin-linked kinase, NF-kB, renin-angiotensin, epithelial-mesenchymal transition, and calcium signaling were also enriched and upregulated in both models (FDR < 0.05). Interestingly, significant downregulation of Pax7 was detected in dy2J/dy2J compared to upregulation of this key regeneration gene in mdx mice. Pax3 and Mamstr genes were also downregulated in dy2J/dy2J compared to WT mice. These results may explain the distinct disease course and severity in these models. While the mdx model at that stage shows massive regeneration, the dy2J/dy2J shows progressive dystrophic process. Our data deepen our understanding of the molecular pathophysiology and suggest new targets for additional therapies to upregulate regeneration in Lama2-CMD.

Regulation of alternative splicing by p300-mediated acetylation of splicing factors.

Splicing of precursor mRNA (pre-mRNA) is an important regulatory step in gene expression. Recent evidence points to a regulatory role of chromatin-related proteins in alternative splicing regulation. Using an unbiased approach, we have identified the acetyltransferase p300 as a key chromatin-related regulator of alternative splicing. p300 promotes genome-wide exon inclusion in both a transcription-dependent and -independent manner. Using CD44 as a paradigm, we found that p300 regulates alternative splicing by modulating the binding of splicing factors to pre-mRNA. Using a tethering strategy, we found that binding of p300 to the CD44 promoter region promotes CD44v exon inclusion independently of RNAPII transcriptional elongation rate. Promoter-bound p300 regulates alternative splicing by acetylating splicing factors, leading to exclusion of hnRNP M from CD44 pre-mRNA and activation of Sam68. p300-mediated CD44 alternative splicing reduces cell motility and promotes epithelial features. Our findings reveal a chromatin-related mechanism of alternative splicing regulation and demonstrate its impact on cellular function.

Human Nasal Turbinate Tissues in Organ Culture as a Model for Human Cytomegalovirus Infection at the Mucosal Entry Site.

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.

Gut microbiota shape 'inflamm-ageing' cytokines and account for age-dependent decline in DNA damage repair.

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.

Modulation of caveolae by insulin/IGF-1 signaling regulates aging of Caenorhabditis elegans.

Reducing insulin/IGF-1 signaling (IIS) extends lifespan, promotes protein homeostasis (proteostasis), and elevates stress resistance of worms, flies, and mammals. How these functions are orchestrated across the organism is only partially understood. Here, we report that in the nematode Caenorhabditis elegans, the IIS positively regulates the expression of caveolin-1 (cav-1), a gene which is primarily expressed in neurons of the adult worm and underlies the formation of caveolae, a subtype of lipid microdomains that serve as platforms for signaling complexes. Accordingly, IIS reduction lowers cav-1 expression and lessens the quantity of neuronal caveolae. Reduced cav-1 expression extends lifespan and mitigates toxic protein aggregation by modulating the expression of aging-regulating and signaling-promoting genes. Our findings define caveolae as aging-governing signaling centers and underscore the potential for cav-1 as a novel therapeutic target for the promotion of healthy aging.

Dual function of C/D box small nucleolar RNAs in rRNA modification and alternative pre-mRNA splicing.

C/D box small nucleolar RNAs (SNORDs) are small noncoding RNAs, and their best-understood function is to target the methyltransferase fibrillarin to rRNA (for example, SNORD27 performs 2'-O-methylation of A27 in 18S rRNA). Unexpectedly, we found a subset of SNORDs, including SNORD27, in soluble nuclear extract made under native conditions, where fibrillarin was not detected, indicating that a fraction of the SNORD27 RNA likely forms a protein complex different from canonical snoRNAs found in the insoluble nuclear fraction. As part of this previously unidentified complex,SNORD27 regulates the alternative splicing of the transcription factor E2F7p re-mRNA through direct RNA-RNA interaction without methylating the RNA, likely by competing with U1 small nuclear ribonucleoprotein (snRNP). Furthermore, knockdown of SNORD27 activates previously "silent" exons in several other genes through base complementarity across the entire SNORD27 sequence, not just the antisense boxes. Thus, some SNORDs likely function in both rRNA and pre-mRNA processing, which increases the repertoire of splicing regulators and links both processes.