Identity Noise and Adipogenic Traits Characterize Dermal Fibroblast Aging.

During aging, stromal functions are thought to be impaired, but little is known whether this stems from changes of fibroblasts. Using population- and single-cell transcriptomics, as well as long-term lineage tracing, we studied whether murine dermal fibroblasts are altered during physiological aging under different dietary regimes that affect longevity. We show that the identity of old fibroblasts becomes undefined, with the fibroblast states present in young skin no longer clearly demarcated. In addition, old fibroblasts not only reduce the expression of genes involved in the formation of the extracellular matrix, but also gain adipogenic traits, paradoxically becoming more similar to neonatal pro-adipogenic fibroblasts. These alterations are sensitive to systemic metabolic changes: long-term caloric restriction reversibly prevents them, whereas a high-fat diet potentiates them. Our results therefore highlight loss of cell identity and the acquisition of adipogenic traits as a mechanism underlying cellular aging, which is influenced by systemic metabolism.

Pulmonary glycogen deficiency as a new potential cause of respiratory distress syndrome.

The glycogenin knockout mouse is a model of Glycogen Storage Disease type XV. These animals show high perinatal mortality (90%) due to respiratory failure. The lungs of glycogenin-deficient embryos and P0 mice have a lower glycogen content than that of wild-type counterparts. Embryonic lungs were found to have decreased levels of mature surfactant proteins SP-B and SP-C, together with incomplete processing of precursors. Furthermore, non-surviving pups showed collapsed sacculi, which may be linked to a significantly reduced amount of surfactant proteins. A similar pattern was observed in glycogen synthase1-deficient mice, which are devoid of glycogen in the lungs and are also affected by high perinatal mortality due to atelectasis. These results indicate that glycogen availability is a key factor for the burst of surfactant production required to ensure correct lung expansion at the establishment of air breathing. Our findings confirm that glycogen deficiency in lungs can cause respiratory distress syndrome and suggest that mutations in glycogenin and glycogen synthase 1 genes may underlie cases of idiopathic neonatal death.

Dietary-Induced Bacterial Metabolites Reduce Inflammation and Inflammation-Associated Cancer via Vitamin D Pathway.

Environmental factors, including westernised diets and alterations to the gut microbiota, are considered risk factors for inflammatory bowel diseases (IBD). The mechanisms underpinning diet-microbiota-host interactions are poorly understood in IBD. We present evidence that feeding a lard-based high-fat (HF) diet can protect mice from developing DSS-induced acute and chronic colitis and colitis-associated cancer (CAC) by significantly reducing tumour burden/incidence, immune cell infiltration, cytokine profile, and cell proliferation. We show that HF protection was associated with increased gut microbial diversity and a significant reduction in Proteobacteria and an increase in Firmicutes and Clostridium cluster XIVa abundance. Microbial functionality was modulated in terms of signalling fatty acids and bile acids (BA). Faecal secondary BAs were significantly induced to include moieties that can activate the vitamin D receptor (VDR), a nuclear receptor richly represented in the intestine and colon. Indeed, colonic VDR downstream target genes were upregulated in HF-fed mice and in combinatorial lipid-BAs-treated intestinal HT29 epithelial cells. Collectively, our data indicate that HF diet protects against colitis and CAC risk through gut microbiota and BA metabolites modulating vitamin D targeting pathways. Our data highlights the complex relationship between dietary fat-induced alterations of microbiota-host interactions in IBD/CAC pathophysiology.

Defects in efferent duct multiciliogenesis underlie male infertility in GEMC1-, MCIDAS- or CCNO-deficient mice.

GEMC1 and MCIDAS are geminin family proteins that transcriptionally activate E2F4/5-target genes during multiciliogenesis, including Foxj1 and Ccno Male mice that lacked Gemc1, Mcidas or Ccno were found to be infertile, but the origin of this defect has remained unclear. Here, we show that all three genes are necessary for the generation of functional multiciliated cells in the efferent ducts that are required for spermatozoa to enter the epididymis. In mice that are mutant for Gemc1, Mcidas or Ccno, we observed a similar spectrum of phenotypes, including thinning of the seminiferous tubule epithelia, dilation of the rete testes, sperm agglutinations in the efferent ducts and lack of spermatozoa in the epididymis (azoospermia). These data suggest that defective efferent duct development is the dominant cause of male infertility in these mouse models, and this likely extends to individuals with the ciliopathy reduced generation of multiple motile cilia with mutations in MCIDAS and CCNO.

Suppression of glycogen synthesis as a treatment for Lafora disease: Establishing the window of opportunity.

Lafora disease (LD) is a fatal adolescence-onset neurodegenerative condition. The hallmark of LD is the accumulation of aberrant glycogen aggregates called Lafora bodies (LBs) in the brain and other tissues. Impeding glycogen synthesis from early embryonic stages by genetic suppression of glycogen synthase (MGS) in an animal model of LD prevents LB formation and ultimately the pathological manifestations of LD thereby indicating that LBs are responsible for the pathophysiology of the disease. However, it is not clear whether eliminating glycogen synthesis in an adult animal after LBs have already formed would halt or reverse the progression of LD. Herein we generated a mouse model of LD with inducible MGS suppression. We evaluated the effect of MGS suppression at different time points on LB accumulation as well as on the appearance of neuroinflammation, a pathologic trait of LD models. In the skeletal muscle, MGS suppression in adult LD mice blocked the formation of new LBs and reduced the number of glycogen aggregates. In the brain, early but not late MGS suppression halted the accumulation of LBs. However, the neuroinflammatory response was still present, as shown by the levels of reactive astrocytes, microglia and inflammatory cytokines. Our results confirm that MGS as a promising therapeutic target for LD and highlight the importance of an early diagnosis for effective treatment of the disease.

Effects of Rifaximin on Luminal and Wall-Adhered Gut Commensal Microbiota in Mice.

Rifaximin is a broad-spectrum antibiotic that ameliorates symptomatology in inflammatory/functional gastrointestinal disorders. We assessed changes in gut commensal microbiota (GCM) and Toll-like receptors (TLRs) associated to rifaximin treatment in mice. Adult C57BL/6NCrl mice were treated (7/14 days) with rifaximin (50/150 mg/mouse/day, PO). Luminal and wall-adhered ceco-colonic GCM were characterized by fluorescent in situ hybridization (FISH) and microbial profiles determined by terminal restriction fragment length polymorphism (T-RFLP). Colonic expression of TLR2/3/4/5/7 and immune-related markers was assessed (RT-qPCR). Regardless the period of treatment or the dose, rifaximin did not alter total bacterial counts or bacterial biodiversity. Only a modest increase in Bacteroides spp. (150 mg/1-week treatment) was detected. In control conditions, only Clostridium spp. and Bifidobacterium spp. were found attached to the colonic epithelium. Rifaximin showed a tendency to favour their adherence after a 1-week, but not 2-week, treatment period. Minor up-regulation in TLRs expression was observed. Only the 50 mg dose for 1-week led to a significant increase (by 3-fold) in TLR-4 expression. No changes in the expression of immune-related markers were observed. Rifaximin, although its antibacterial properties, induces minor changes in luminal and wall-adhered GCM in healthy mice. Moreover, no modulation of TLRs or local immune systems was observed. These findings, in normal conditions, do not rule out a modulatory role of rifaximin in inflammatory and or dysbiotic states of the gut.

Inflammasome Signaling Regulates the Microbial-Neuroimmune Axis and Visceral Pain in Mice.

Interactions between the intestinal microbiota, immune system and nervous system are essential for homeostasis in the gut. Inflammasomes contribute to innate immunity and brain-gut interactions, but their role in microbiota-neuro-immune interactions is not clear. Therefore, we investigated the effect of the inflammasome on visceral pain and local and systemic neuroimmune responses after antibiotic-induced changes to the microbiota. Wild-type (WT) and caspase-1/11 deficient (Casp1 KO) mice were orally treated for 2 weeks with an antibiotic cocktail (Abx, Bacitracin A and Neomycin), followed by quantification of representative fecal commensals (by qPCR), cecal short chain fatty acids (by HPLC), pathways implicated in the gut-neuro-immune axis (by RT-qPCR, immunofluorescence staining, and flow cytometry) in addition to capsaicin-induced visceral pain responses. Abx-treatment in WT-mice resulted in an increase in colonic macrophages, central neuro-immune interactions, colonic inflammasome and nociceptive receptor gene expression and a reduction in capsaicin-induced visceral pain. In contrast, these responses were attenuated in Abx-treated Casp1 KO mice. Collectively, the data indicate an important role for the inflammasome pathway in functional and inflammatory gastrointestinal conditions where pain and alterations in microbiota composition are prominent.

VarSome: the human genomic variant search engine.

SUMMARY: VarSome.com is a search engine, aggregator and impact analysis tool for human genetic variation and a community-driven project aiming at sharing global expertise on human variants. AVAILABILITY AND IMPLEMENTATION: VarSome is freely available at http://varsome.com. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Whey protein effects on energy balance link the intestinal mechanisms of energy absorption with adiposity and hypothalamic neuropeptide gene expression.

We tested the hypothesis that dietary whey protein isolate (WPI) affects the intestinal mechanisms related to energy absorption and that the resulting energy deficit is compensated by changes in energy balance to support growth. C57BL/6 mice were provided a diet enriched with WPI with varied sucrose content, and the impact on energy balance-related parameters was investigated. As part of a high-sucrose diet, WPI reduced the hypothalamic expression of pro-opiomelanocortin gene expression and increased energy intake. The energy expenditure was unaffected, but epididymal weight was reduced, indicating an energy loss. Notably, there was a reduction in the ileum gene expression for amino acid transporter SLC6a19, glucose transporter 2, and fatty acid transporter 4. The composition of the gut microbiota also changed, where Firmicutes were reduced. The above changes indicated reduced energy absorption through the intestine. We propose that this mobilized energy in the adipose tissue and caused hypothalamic changes that increased energy intake, acting to counteract the energy deficit arising in the intestine. Lowering the sucrose content in the WPI diet increased energy expenditure. This further reduced epididymal weight and plasma leptin, whereupon hypothalamic ghrelin gene expression and the intestinal weight were both increased. These data suggest that when the intestine-adipose-hypothalamic pathway is subjected to an additional energy loss (now in the adipose tissue), compensatory changes attempt to assimilate more energy. Notably, WPI and sucrose content interact to enable the component mechanisms of this pathway.

Enhanced cough reflex in a model of bleomycin-induced lung fibrosis in guinea pigs.

Fibrotic lung diseases, such as idiopathic pulmonary fibrosis, are associated with spontaneous dry cough and hypersensitivity to tussive agents. Understanding the pathophysiology driving enhanced cough may help us to define better therapies for patients. We hypothesized that lung fibrosis induced by intratracheal bleomycin would exacerbate the cough reflex induced by tussive agents in guinea pigs. Disease progression in the lungs was characterized at days 1, 7, 14, 21 and 28 after bleomycin administration. Inflammatory and fibrotic markers, as well as neurotrophin levels, were assessed in bronchoalveolar lavage fluid and/or lung tissue. Cough sensitivity to citric acid, capsaicin and allylisothiocyanate was evaluated in conscious animals at days 14 and 21 after bleomycin administration. Pulmonary lesions evolved from an early inflammatory phase (from day 1 to day 7) to a fibrotic stage (between days 14 and 28). Fibrosis was related to increased levels of matrix metalloproteinase-2 in bronchoalveolar lavage fluid (day 21: saline, 0.26 ng/ml; bleomycin, 0.49 ng/ml). At day 14, we also observed increased cough reflexes to citric acid (163%), capsaicin (125%) and allylisothiocyanate (178%). Cough exacerbation persisted, but at a lower extent, by day 21 for capsaicin (100%) and allylisothiocyanate (54%). Moreover, bronchoalveolar lavage fluid concentrations of brain-derived neurotrophic factor, suggested to induce nerve remodelling in chronic cough, were also enhanced (day 1: saline, 14.21 pg/ml; bleomycin, 30.09 pg/ml). In summary, our model of bleomycin-induced cough exacerbation may be a valuable tool to investigate cough hypersensitivity and develop antitussive therapies for fibrotic lung diseases.

Genomic variation and its impact on gene expression in Drosophila melanogaster.

Understanding the relationship between genetic and phenotypic variation is one of the great outstanding challenges in biology. To meet this challenge, comprehensive genomic variation maps of human as well as of model organism populations are required. Here, we present a nucleotide resolution catalog of single-nucleotide, multi-nucleotide, and structural variants in 39 Drosophila melanogaster Genetic Reference Panel inbred lines. Using an integrative, local assembly-based approach for variant discovery, we identify more than 3.6 million distinct variants, among which were more than 800,000 unique insertions, deletions (indels), and complex variants (1 to 6,000 bp). While the SNP density is higher near other variants, we find that variants themselves are not mutagenic, nor are regions with high variant density particularly mutation-prone. Rather, our data suggest that the elevated SNP density around variants is mainly due to population-level processes. We also provide insights into the regulatory architecture of gene expression variation in adult flies by mapping cis-expression quantitative trait loci (cis-eQTLs) for more than 2,000 genes. Indels comprise around 10% of all cis-eQTLs and show larger effects than SNP cis-eQTLs. In addition, we identified two-fold more gene associations in males as compared to females and found that most cis-eQTLs are sex-specific, revealing a partial decoupling of the genomic architecture between the sexes as well as the importance of genetic factors in mediating sex-biased gene expression. Finally, we performed RNA-seq-based allelic expression imbalance analyses in the offspring of crosses between sequenced lines, which revealed that the majority of strong cis-eQTLs can be validated in heterozygous individuals.

The horizontal ladder test (HLT) protocol: a novel, optimized, and reliable means of assessing motor coordination in Sus scrofa domesticus.

Pigs can be an important model for preclinical biological research, including neurological diseases such as Alcohol Use Disorder. Such research often involves longitudinal assessment of changes in motor coordination as the disease or disorder progresses. Current motor coordination tests in pigs are derived from behavioral assessments in rodents and lack critical aspects of face and construct validity. While such tests may permit for the comparison of experimental results to rodents, a lack of validation studies of such tests in the pig itself may preclude the drawing of meaningful conclusions. To address this knowledge gap, an apparatus modeled after a horizontally placed ladder and where the height of the rungs could be adjusted was developed. The protocol that was employed within the apparatus mimicked the walk and turn test of the human standardized field sobriety test. Here, five Sinclair miniature pigs were trained to cross the horizontally placed ladder, starting at a rung height of six inches and decreasing to three inches in one-inch increments. It was demonstrated that pigs can reliably learn to cross the ladder, with few errors, under baseline/unimpaired conditions. These animals were then involved in a voluntary consumption of ethanol study where animals were longitudinally evaluated for motor coordination changes at baseline, 2.5, 5, 7.5, and 10% ethanol concentrations subsequently to consuming ethanol. Consistent with our predictions, relative to baseline performance, motor incoordination increased as voluntary consumption of escalating concentrations of ethanol increased. Together these data highlight that the horizontal ladder test (HLT) test protocol is a novel, optimized and reliable test for evaluating motor coordination as well as changes in motor coordination in pigs.

The efficacy of chemotherapy is limited by intratumoral senescent cells expressing PD-L2.

Chemotherapy often generates intratumoral senescent cancer cells that strongly modify the tumor microenvironment, favoring immunosuppression and tumor growth. We discovered, through an unbiased proteomics screen, that the immune checkpoint inhibitor programmed cell death 1 ligand 2 (PD-L2) is highly upregulated upon induction of senescence in different types of cancer cells. PD-L2 is not required for cells to undergo senescence, but it is critical for senescent cells to evade the immune system and persist intratumorally. Indeed, after chemotherapy, PD-L2-deficient senescent cancer cells are rapidly eliminated and tumors do not produce the senescence-associated chemokines CXCL1 and CXCL2. Accordingly, PD-L2-deficient pancreatic tumors fail to recruit myeloid-derived suppressor cells and undergo regression driven by CD8 T cells after chemotherapy. Finally, antibody-mediated blockade of PD-L2 strongly synergizes with chemotherapy causing remission of mammary tumors in mice. The combination of chemotherapy with anti-PD-L2 provides a therapeutic strategy that exploits vulnerabilities arising from therapy-induced senescence.

A retrospective, dual-isotope approach reveals individual predispositions to winter-drought induced tree dieback in the southernmost distribution limit of Scots pine.

Winter-drought induced forest diebacks in the low-latitude margins of species' distribution ranges can provide new insights into the mechanisms (carbon starvation, hydraulic failure) underlying contrasting tree reactions. We analysed a winter-drought induced dieback at the Scots pine's southern edge through a dual-isotope approach (Δ(13) C and δ(18) O in tree-ring cellulose). We hypothesized that a differential long-term performance, mediated by the interaction between CO(2) and climate, determined the fates of individuals during dieback. Declining trees showed a stronger coupling between climate, growth and intrinsic water-use efficiency (WUEi) than non-declining individuals that was noticeable for 25 years prior to dieback. The rising stomatal control of water losses with time in declining trees, indicated by negative Δ(13) C-δ(18) O relationships, was likely associated with their native aptitude to grow more and take up more water (suggested by larger tracheid lumen widths) than non-declining trees and, therefore, to exhibit a greater cavitation risk. Freeze-thaw episodes occurring in winter 2001 unveiled such physiological differences by triggering dieback in those trees more vulnerable to hydraulic failure. Thus, WUEi tightly modulated growth responses to long-term warming in declining trees, indicating that co-occurring individuals were differentially predisposed to winter-drought mortality. These different performances were unconnected to the depletion of stored carbohydrates.

Genetic and Pharmacological Blockade of Sigma-1 Receptors Attenuates Inflammation-Associated Hypersensitivity during Acute Colitis in CD1 Mice.

Sigma-1 receptors (σ1Rs) are implicated in nociception, including pain sensitization, and inflammation. We assessed the role of σ1Rs on acute colitis-associated hypersensitivity using both genetic (constitutive knockout) and pharmacological blockade of the receptor. Colitis was induced in CD1 wild-type (WT) and σ1R KO mice (exposure to dextran sodium sulfate, 3%). A von Frey test was used to assess referred mechanosensitivity (abdominal and plantar withdrawal responses). The effects of the selective σ1R antagonists BD1063 and E-52862 were also assessed in WT animals. The expression of immune and sensory-related markers (RT-qPCR, Western blot) was assessed in the colon and lumbosacral spinal cord. The genetic ablation or pharmacological blockade of σ1Rs attenuated acute colonic inflammation in a similar manner. Mechanosensitivity was similar in WT and σ1R KO mice before colitis. In WT mice, but not in σ1R KO, colitis was associated with the development of referred mechanical hypersensitivity, manifested as a reduction in the withdrawal thresholds to mechanical probing (paw and abdominal wall). In WT mice, BD1063 and E-52862 blocked colitis-associated hypersensitivity. A genotype- and treatment-related differential regulation of sensory-related markers was detected locally (colon) and within the spinal cord. σ1Rs are involved in the development of acute intestinal inflammation and its associated referred mechanical hypersensitivity. The selective modulation of sensory-related pathways within the colon and spinal cord might be part of the underlying mechanisms. These observations support the pharmacological use of σ1R antagonists for the treatment of intestinal inflammation-induced hypersensitivity.

Targeting lymphoid-derived IL-17 signaling to delay skin aging.

Skin aging is characterized by structural and functional changes that contribute to age-associated frailty. This probably depends on synergy between alterations in the local niche and stem cell-intrinsic changes, underscored by proinflammatory microenvironments that drive pleotropic changes. The nature of these age-associated inflammatory cues, or how they affect tissue aging, is unknown. Based on single-cell RNA sequencing of the dermal compartment of mouse skin, we show a skew towards an IL-17-expressing phenotype of T helper cells, γδ T cells and innate lymphoid cells in aged skin. Importantly, in vivo blockade of IL-17 signaling during aging reduces the proinflammatory state of the skin, delaying the appearance of age-related traits. Mechanistically, aberrant IL-17 signals through NF-κB in epidermal cells to impair homeostatic functions while promoting an inflammatory state. Our results indicate that aged skin shows signs of chronic inflammation and that increased IL-17 signaling could be targeted to prevent age-associated skin ailments.

Human senescent fibroblasts trigger progressive lung fibrosis in mice.

Cell senescence has recently emerged as a potentially relevant pathogenic mechanism in fibrosing interstitial lung diseases (f-ILDs), particularly in idiopathic pulmonary fibrosis. We hypothesized that senescent human fibroblasts may suffice to trigger a progressive fibrogenic reaction in the lung. To address this, senescent human lung fibroblasts, or their secretome (SASP), were instilled into the lungs of immunodeficient mice. We found that: (1) human senescent fibroblasts engraft in the lungs of immunodeficient mice and trigger progressive lung fibrosis associated to increasing levels of mouse senescent cells, whereas non-senescent fibroblasts do not trigger fibrosis; (2) the SASP of human senescent fibroblasts is pro-senescence and pro-fibrotic both in vitro when added to mouse recipient cells and in vivo when delivered into the lungs of mice, whereas the conditioned medium (CM) from non-senescent fibroblasts lacks these activities; and, (3) navitoclax, nintedanib and pirfenidone ameliorate lung fibrosis induced by senescent human fibroblasts in mice, albeit only navitoclax displayed senolytic activity. We conclude that human senescent fibroblasts, through their bioactive secretome, trigger a progressive fibrogenic reaction in the lungs of immunodeficient mice that includes the induction of paracrine senescence in the cells of the host, supporting the concept that senescent cells actively contribute to disease progression in patients with f-ILDs.

The Horizontal Ladder Test (HLT) protocol: A novel, optimized, and reliable means of assessing motor coordination in Sus scrofa domesticus.

Pigs can be an important model for preclinical biological research, including neurological diseases such as Alcohol Use Disorder. Such research often involves longitudinal assessment of changes in motor coordination as the disease or disorder progresses. Current motor coordination tests in pigs are derived from behavioral assessments in rodents and lack critical aspects of face and construct validity. While such tests may permit for the comparison of experimental results to rodents, a lack of validation studies of such tests in the pig itself may preclude the drawing of meaningful conclusions. Here, we present a novel, optimized, and reliable horizontal ladder test (HLT) test protocol for evaluating motor coordination in pigs and an initial validation of its construct validity using voluntary alcohol consumption as an experimental manipulation.

Iron accumulation drives fibrosis, senescence and the senescence-associated secretory phenotype.

Fibrogenesis is part of a normal protective response to tissue injury that can become irreversible and progressive, leading to fatal diseases. Senescent cells are a main driver of fibrotic diseases through their secretome, known as senescence-associated secretory phenotype (SASP). Here, we report that cellular senescence, and multiple types of fibrotic diseases in mice and humans are characterized by the accumulation of iron. We show that vascular and hemolytic injuries are efficient in triggering iron accumulation, which in turn can cause senescence and promote fibrosis. Notably, we find that senescent cells persistently accumulate iron, even when the surge of extracellular iron has subdued. Indeed, under normal conditions of extracellular iron, cells exposed to different types of senescence-inducing insults accumulate abundant ferritin-bound iron, mostly within lysosomes, and present high levels of labile iron, which fuels the generation of reactive oxygen species and the SASP. Finally, we demonstrate that detection of iron by magnetic resonance imaging might allow non-invasive assessment of fibrotic burden in the kidneys of mice and in patients with renal fibrosis. Our findings suggest that iron accumulation plays a central role in senescence and fibrosis, even when the initiating events may be independent of iron, and identify iron metabolism as a potential therapeutic target for senescence-associated diseases.

Lack of p62 Impairs Glycogen Aggregation and Exacerbates Pathology in a Mouse Model of Myoclonic Epilepsy of Lafora.

Lafora disease (LD) is a fatal childhood-onset dementia characterized by the extensive accumulation of glycogen aggregates-the so-called Lafora Bodies (LBs)-in several organs. The accumulation of LBs in the brain underlies the neurological phenotype of the disease. LBs are composed of abnormal glycogen and various associated proteins, including p62, an autophagy adaptor that participates in the aggregation and clearance of misfolded proteins. To study the role of p62 in the formation of LBs and its participation in the pathology of LD, we generated a mouse model of the disease (malinKO) lacking p62. Deletion of p62 prevented LB accumulation in skeletal muscle and cardiac tissue. In the brain, the absence of p62 altered LB morphology and increased susceptibility to epilepsy. These results demonstrate that p62 participates in the formation of LBs and suggest that the sequestration of abnormal glycogen into LBs is a protective mechanism through which it reduces the deleterious consequences of its accumulation in the brain.