Domain specific mutations in dyskerin disrupt 3' end processing of scaRNA13.

Mutations in DKC1 (encoding dyskerin) cause telomere diseases including dyskeratosis congenita (DC) by decreasing steady-state levels of TERC, the non-coding RNA component of telomerase. How DKC1 mutations variably impact numerous other snoRNAs remains unclear, which is a barrier to understanding disease mechanisms in DC beyond impaired telomere maintenance. Here, using DC patient iPSCs, we show that mutations in the dyskerin N-terminal extension domain (NTE) dysregulate scaRNA13. In iPSCs carrying the del37L NTE mutation or engineered to carry NTE mutations via CRISPR/Cas9, but not in those with C-terminal mutations, we found scaRNA13 transcripts with aberrant 3' extensions, as seen when the exoribonuclease PARN is mutated in DC. Biogenesis of scaRNA13 was rescued by repair of the del37L DKC1 mutation by genome-editing, or genetic or pharmacological inactivation of the polymerase PAPD5, which counteracts PARN. Inspection of the human telomerase cryo-EM structure revealed that in addition to mediating intermolecular dyskerin interactions, the NTE interacts with terminal residues of the associated snoRNA, indicating a role for this domain in 3' end definition. Our results provide mechanistic insights into the interplay of dyskerin and the PARN/PAPD5 axis in the biogenesis and accumulation of snoRNAs beyond TERC, broadening our understanding of ncRNA dysregulation in human diseases.

A Murine Model for Enhancement of Streptococcus pneumoniae Pathogenicity upon Viral Infection and Advanced Age.

Streptococcus pneumoniae (pneumococcus) resides asymptomatically in the nasopharynx (NP) but can progress from benign colonizer to lethal pulmonary or systemic pathogen. Both viral infection and aging are risk factors for serious pneumococcal infections. Previous work established a murine model that featured the movement of pneumococcus from the nasopharynx to the lung upon nasopharyngeal inoculation with influenza A virus (IAV) but did not fully recapitulate the severe disease associated with human coinfection. We built upon this model by first establishing pneumococcal nasopharyngeal colonization, then inoculating both the nasopharynx and lungs with IAV. In young (2-month-old) mice, coinfection triggered bacterial dispersal from the nasopharynx into the lungs, pulmonary inflammation, disease, and mortality in a fraction of mice. In aged mice (18 to 24 months), coinfection resulted in earlier and more severe disease. Aging was not associated with greater bacterial burdens but rather with more rapid pulmonary inflammation and damage. Both aging and IAV infection led to inefficient bacterial killing by neutrophils ex vivo. Conversely, aging and pneumococcal colonization also blunted alpha interferon (IFN-α) production and increased pulmonary IAV burden. Thus, in this multistep model, IAV promotes pneumococcal pathogenicity by modifying bacterial behavior in the nasopharynx, diminishing neutrophil function, and enhancing bacterial growth in the lung, while pneumococci increase IAV burden, likely by compromising a key antiviral response. Thus, this model provides a means to elucidate factors, such as age and coinfection, that promote the evolution of S. pneumoniae from asymptomatic colonizer to invasive pathogen, as well as to investigate consequences of this transition on antiviral defense.

miR-21 antagonism reprograms macrophage metabolism and abrogates chronic allograft vasculopathy.

Despite much progress in improving graft outcome during cardiac transplantation, chronic allograft vasculopathy (CAV) remains an impediment to long-term graft survival. MicroRNAs (miRNAs) emerged as regulators of the immune response. Here, we aimed to examine the miRNA network involved in CAV. miRNA profiling of heart samples obtained from a murine model of CAV and from cardiac-transplanted patients with CAV demonstrated that miR-21 was most significantly expressed and was primarily localized to macrophages. Interestingly, macrophage depletion with clodronate did not significantly prolong allograft survival in mice, while conditional deletion of miR-21 in macrophages or the use of a specific miR-21 antagomir resulted in indefinite cardiac allograft survival and abrogated CAV. The immunophenotype, secretome, ability to phagocytose, migration, and antigen presentation of macrophages were unaffected by miR-21 targeting, while macrophage metabolism was reprogrammed, with a shift toward oxidative phosphorylation in naïve macrophages and with an inhibition of glycolysis in pro-inflammatory macrophages. The aforementioned effects resulted in an increase in M2-like macrophages, which could be reverted by the addition of L-arginine. RNA-seq analysis confirmed alterations in arginase-associated pathways associated with miR-21 antagonism. In conclusion, miR-21 is overexpressed in murine and human CAV, and its targeting delays CAV onset by reprogramming macrophages metabolism.

Prophage induction, but not production of phage particles, is required for lethal disease in a microbiome-replete murine model of enterohemorrhagic E. coli infection.

Enterohemorrhagic Escherichia coli (EHEC) colonize intestinal epithelium by generating characteristic attaching and effacing (AE) lesions. They are lysogenized by prophage that encode Shiga toxin 2 (Stx2), which is responsible for severe clinical manifestations. As a lysogen, prophage genes leading to lytic growth and stx2 expression are repressed, whereas induction of the bacterial SOS response in response to DNA damage leads to lytic phage growth and Stx2 production both in vitro and in germ-free or streptomycin-treated mice. Some commensal bacteria diminish prophage induction and concomitant Stx2 production in vitro, whereas it has been proposed that phage-susceptible commensals may amplify Stx2 production by facilitating successive cycles of infection in vivo. We tested the role of phage induction in both Stx production and lethal disease in microbiome-replete mice, using our mouse model encompassing the murine pathogen Citrobacter rodentium lysogenized with the Stx2-encoding phage Φstx2dact. This strain generates EHEC-like AE lesions on the murine intestine and causes lethal Stx-mediated disease. We found that lethal mouse infection did not require that Φstx2dact infect or lysogenize commensal bacteria. In addition, we detected circularized phage genomes, potentially in the early stage of replication, in feces of infected mice, confirming that prophage induction occurs during infection of microbiota-replete mice. Further, C. rodentium (Φstx2dact) mutants that do not respond to DNA damage or express stx produced neither high levels of Stx2 in vitro or lethal infection in vivo, confirming that SOS induction and concomitant expression of phage-encoded stx genes are required for disease. In contrast, C. rodentium (Φstx2dact) mutants incapable of prophage genome excision or of packaging phage genomes retained the ability to produce Stx in vitro, as well as to cause lethal disease in mice. Thus, in a microbiome-replete EHEC infection model, lytic induction of Stx-encoding prophage is essential for lethal disease, but actual phage production is not.

Transposon Mutagenesis Screen of Klebsiella pneumoniae Identifies Multiple Genes Important for Resisting Antimicrobial Activities of Neutrophils in Mice.

Klebsiella pneumoniae is a Gram-negative bacterial pathogen that causes a range of infections, including pneumonias, urinary tract infections, and septicemia, in otherwise healthy and immunocompromised patients. K. pneumoniae has become an increasing concern due to the rise and spread of antibiotic-resistant and hypervirulent strains. However, its virulence determinants remain understudied. To identify novel K. pneumoniae virulence factors needed to cause pneumonia, a high-throughput screen was performed with an arrayed library of over 13,000 K. pneumoniae transposon insertion mutants in the lungs of wild-type (WT) and neutropenic mice using transposon sequencing (Tn-seq). Insertions in 166 genes resulted in K. pneumoniae mutants that were significantly less fit in the lungs of WT mice than in those of neutropenic mice. Of these, mutants with insertions in 51 genes still had significant defects in neutropenic mice, while mutants with insertions in 52 genes recovered significantly. In vitro screens using a minilibrary of K. pneumoniae transposon mutants identified putative functions for a subset of these genes, including in capsule content and resistance to reactive oxygen and nitrogen species. Lung infections in mice confirmed roles in K. pneumoniae virulence for the ΔdedA, ΔdsbC, ΔgntR, Δwzm-wzt, ΔyaaA, and ΔycgE mutants, all of which were defective in either capsule content or growth in reactive oxygen or nitrogen species. The fitness of the ΔdedA, ΔdsbC, ΔgntR, ΔyaaA, and ΔycgE mutants was higher in neutropenic mouse lungs, indicating that these genes encode proteins that protect K. pneumoniae against neutrophil-related effector functions.

A global regulatory system links virulence and antibiotic resistance to envelope homeostasis in Acinetobacter baumannii.

The nosocomial pathogen Acinetobacter baumannii is a significant threat due to its ability to cause infections refractory to a broad range of antibiotic treatments. We show here that a highly conserved sensory-transduction system, BfmRS, mediates the coordinate development of both enhanced virulence and resistance in this microorganism. Hyperactive alleles of BfmRS conferred increased protection from serum complement killing and allowed lethal systemic disease in mice. BfmRS also augmented resistance and tolerance against an expansive set of antibiotics, including dramatic protection from ß-lactam toxicity. Through transcriptome profiling, we showed that BfmRS governs these phenotypes through global transcriptional regulation of a post-exponential-phase-like program of gene expression, a key feature of which is modulation of envelope biogenesis and defense pathways. BfmRS activity defended against cell-wall lesions through both ß-lactamase-dependent and -independent mechanisms, with the latter being connected to control of lytic transglycosylase production and proper coordination of morphogenesis and division. In addition, hypersensitivity of bfmRS knockouts could be suppressed by unlinked mutations restoring a short, rod cell morphology, indicating that regulation of drug resistance, pathogenicity, and envelope morphogenesis are intimately linked by this central regulatory system in A. baumannii. This work demonstrates that BfmRS controls a global regulatory network coupling cellular physiology to the ability to cause invasive, drug-resistant infections.

Interactions between the colonic transcriptome, metabolome, and microbiome in mouse models of obesity-induced intestinal cancer.

Obesity is a significant risk factor for colorectal cancer (CRC); however, the relative contribution of high-fat (HF) consumption and excess adiposity remains unclear. It is becoming apparent that obesity perturbs both the intestinal microbiome and metabolome, and each has the potential to induce protumorigenic changes in the epithelial transcriptome. The physiological consequences and the degree to which these different biologic systems interact remain poorly defined. To understand the mechanisms by which obesity drives colonic tumorigenesis, we profiled the colonic epithelial transcriptome of HF-fed and genetically obese (DbDb) mice with a genetic predisposition to intestinal tumorigenesis (Apc(1638N)); 266 and 584 genes were differentially expressed in the colonic mucosa of HF and DbDb mice, respectively. These genes mapped to pathways involved in immune function, and cellular proliferation and cancer. Furthermore, Akt was central within the networks of interacting genes identified in both gene sets. Regression analyses of coexpressed genes with the abundance of bacterial taxa identified three taxa, previously correlated with tumor burden, to be significantly correlated with a gene module enriched for Akt-related genes. Similarly, regression of coexpressed genes with metabolites found that adenosine, which was negatively associated with inflammatory markers and tumor burden, was also correlated with a gene module enriched with Akt regulators. Our findings provide evidence that HF consumption and excess adiposity result in changes in the colonic transcriptome that, although distinct, both appear to converge on Akt signaling. Such changes could be mediated by alterations in the colonic microbiome and metabolome.

Predominance of Lactobacillus spp. Among Patients Who Do Not Acquire Multidrug-Resistant Organisms.

BACKGROUND: The emergence and dissemination of multidrug-resistant organisms (MDROs) is a global threat. Characterizing the human microbiome among hospitalized patients and identifying unique microbial signatures among those patients who acquire MDROs may identify novel infection prevention strategies. METHODS: Adult patients admitted to 5 general medical-surgical floors at a 649-bed, tertiary care center in Boston, Massachusetts, were classified according to in-hospital antimicrobial exposure and MDRO colonization status. Within 48 hours of hospital admission (baseline) and at discharge (follow-up), rectal swab samples were obtained, and compared with samples from an external control group of healthy persons from the community. DNA was extracted from samples, next-generation sequencing performed, and microbial community structure and taxonomic features assessed, comparing those who acquired MDROs and those who had not, and the external controls. RESULTS: Hospitalized patients (n = 44) had reduced microbial diversity and a greater abundance of Escherichia spp. and Enterococcus spp. than healthy controls (n = 26). Among hospitalized patients, 25 had no MDROs at the time of the baseline sample and were also exposed to antimicrobials. Among this group, 7 (28%) acquired ≥1 MDRO; demographic and clinical characteristics were similar between MDRO-acquisition and MDRO-nonacquisition groups. Patients in the nonacquisition group had consistently higher Lactobacillus spp. abundance than those in the acquisition group (linear discriminant score, 3.97; P = .04). CONCLUSIONS: The fecal microbiota of the hospitalized subjects had abnormal community composition, and Lactobacillus spp. was associated with lack of MDRO acquisition, consistent with a protective role.

Intestinal microbiota, microbial translocation, and systemic inflammation in chronic HIV infection.

BACKGROUND: Despite effective antiretroviral therapy (ART), patients with chronic human immunodeficiency virus (HIV) infection have increased microbial translocation and systemic inflammation. Alterations in the intestinal microbiota may play a role in microbial translocation and inflammation. METHODS: We profiled the fecal microbiota by pyrosequencing the gene encoding 16S ribosomal RNA (rRNA) and measured markers of microbial translocation and systemic inflammation in 21 patients who had chronic HIV infection and were receiving suppressive ART (cases) and 16 HIV-uninfected controls. RESULTS: The fecal microbial community composition was significantly different between cases and controls. The relative abundance of Proteobacteria, Gammaproteobacteria, Enterobacteriales, Enterobacteriaceae, Erysipelotrichi, Erysipelotrichales, Erysipelotrichaceae, and Barnesiella was significantly enriched in cases, whereas that of Rikenellaceae and Alistipes was depleted. The plasma soluble CD14 level (sCD14) was significantly higher and the endotoxin core immunoglobulin M (IgM) level lower in cases, compared with controls. There were significant positive correlations between the relative abundances of Enterobacteriales and Enterobacteriaceae and the sCD14 level; the relative abundances of Gammaproteobacteria, Enterobacteriales, and Enterobacteriaceae and the interleukin 1ß (IL-1ß) level; the relative abundances of Enterobacteriales and Enterobacteriaceae and the interferon γ level; and the relative abundances of Erysipelotrichi and Barnesiella and the TNF-α level. There were negative correlations between endotoxin core IgM and IL-1ß levels. CONCLUSIONS: Patients who have chronic HIV infection and are receiving suppressive ART display intestinal dysbiosis associated with increased microbial translocation and significant associations between specific taxa and markers of microbial translocation and systemic inflammation. This was an exploratory study, the findings of which need to be confirmed.

Monocyte transcriptomic profile following EPA and DHA supplementation in men and women with low-grade chronic inflammation.

BACKGROUND: Recent data indicate considerable variability in response to very long chain omega-3 fatty acid supplementation on cardiovascular disease risk. This inconsistency may be due to differential effects of EPA vs DHA and/or sex-specific responses. METHODS: Sixteen subjects (eight men and eight women) 50-75 y and with low-grade chronic inflammation participated in a randomized controlled crossover trial comparing 3 g/d EPA, 3 g/d DHA, and placebo (3 g/d high oleic acid sunflower oil). Blood monocytes were isolated at the end of each phase for RNA-sequencing. RESULTS: Sex dimorphism in monocyte gene expression was observed, therefore, data for men and women were analyzed separately. 1088 genes were differentially expressed in men and 997 in women (p < 0.05). In both men and women, EPA and DHA repressed genes involved in protein turnover and mitochondrial energy metabolism, relative to placebo. In men only, EPA and DHA upregulated genes related to wound healing and PPARα activation. In women only, EPA and DHA activated genes related to ER stress response. Relative to DHA, EPA resulted in lower expression of genes involved in inflammatory processes in men, and lower expression of genes involved in ER stress response in women. CONCLUSIONS: EPA and DHA supplementation elicited both similar and differential effects on monocyte transcriptome, some of which were sex specific. The observed variability in response to EPA and DHA in men and women could in part explain the conflicting results from previous cardiovascular clinical trials using omega-3 fatty acids.

Highway proximity associations with blood markers of inflammation: evidence for a role for IL-1ß.

Cardiovascular disease is known to be associated with proximity to major roadways and highways. Thus, blood samples from 20 near highway and 20 urban background residents were analyzed for presence of cytokines and other biomarkers. Near-highway participants displayed significantly lower socioeconomic status (SES) and significantly higher occupational vehicle exhaust exposure and higher low-density lipoprotein (LDL) levels. Controlling for exposure to vehicle exhaust on the job, interleukin-6 (IL-6) was numerically higher in near highway participants. Using logistic regression analyses, IL-1ß was significantly elevated near highway. It is interesting that elevations were found in IL-1ß, a key cytokine linked to inflammation from particulate matter (PM). More studies are needed with larger sample sizes to assess the possible role of IL-1ß.

Identification of a Bile Acid-Binding Transcription Factor in Clostridioides difficile Using Chemical Proteomics.

Clostridioides difficile is a Gram-positive anaerobic bacterium that is the leading cause of hospital-acquired gastroenteritis in the US. In the gut milieu, C. difficile encounters microbiota-derived, growth-inhibiting bile acids that are thought to be a significant mechanism of colonization resistance. While the levels of certain bile acids in the gut correlate with susceptibility to C. difficile infection, their molecular targets in C. difficile remain unknown. In this study, we sought to use chemical proteomics to identify bile acid-interacting proteins in C. difficile. Using photoaffinity bile acid probes and chemical proteomics, we identified a previously uncharacterized MerR family protein, CD3583 (now BapR), as a putative bile acid-sensing transcription regulator. Our data indicate that BapR specifically binds to and is stabilized by lithocholic acid (LCA) in C. difficile. Although loss of BapR did not affect C. difficile's sensitivity to LCA, ΔbapR cells elongated more in the presence of LCA compared to wild-type cells. Transcriptomics revealed that BapR regulates several gene clusters, with the expression of the mdeA-cd3573 locus being specifically de-repressed in the presence of LCA in a BapR-dependent manner. Electrophoretic mobility shift assays revealed that BapR directly binds to the mdeA promoter region. Because mdeA is involved in amino acid-related sulfur metabolism and the mdeA-cd3573 locus encodes putative transporters, we propose that BapR senses a gastrointestinal tract-specific small molecule, LCA, as an environmental cue for metabolic adaptation.

Sexual dimorphism of monocyte transcriptome in individuals with chronic low-grade inflammation.

Sexual dimorphism in the immune system is evidenced by a higher prevalence of autoimmune diseases in women and higher susceptibility to infectious diseases in men. However, the molecular basis of these sex-based differences is not fully understood. We have characterized the transcriptome profiles of peripheral blood monocytes from males and postmenopausal females with chronic low-grade inflammation. We identified 41 sexually differentially expressed genes [adjusted p value (FDR) < 0.1], including genes involved in immune cell activation (e.g., CEACAM1, FCGR2B, and SLAMF7) and antigen presentation (e.g., AIM2, CD1E, and UBA1) with a higher expression in females than males. Moreover, signaling pathways of immune or inflammatory responses, including interferon (IFN) signaling [z-score = 2.45, -log(p) = 3.88], were found to be more upregulated in female versus male monocytes, based on a set of genes exhibiting sex-biased expression (p < 0.03). The contribution of IFN signaling to the sexual transcriptional differences was further confirmed by direct comparisons of the monocyte sex-biased genes with IFN signature genes (ISGs) that were previously curated in mouse macrophages. ISGs showed a greater overlap with female-biased genes than male-biased genes and a higher overall expression in female than male monocytes, particularly for the genes of antiviral and inflammatory responses to IFN. Given the role of IFN in immune defense and autoimmunity, our results suggest that sexual dimorphism in immune functions may be associated with more priming of innate immune pathways in female than male monocytes. These findings highlight the role of sex on the human immune transcriptome.

EPA and DHA differentially modulate monocyte inflammatory response in subjects with chronic inflammation in part via plasma specialized pro-resolving lipid mediators: A randomized, double-blind, crossover study.

BACKGROUND AND AIMS: The independent effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on chronic inflammation through their downstream lipid mediators, including the specialized pro-resolving lipid mediators (SPM), remain unstudied. Therefore, we compared the effects of EPA and DHA supplementation on monocyte inflammatory response and plasma polyunsaturated fatty acids (PUFA) SPM lipidome. METHODS: After a 4-week lead-in phase (baseline), 9 men and 12 postmenopausal women (50-75 years) with chronic inflammation received two phases of 10-week supplementation with 3 g/day EPA and DHA in a random order, separated by a 10-week washout. RESULTS: Compared with baseline, EPA and DHA supplementation differently modulated LPS-stimulated monocyte cytokine expression. EPA lowered TNFA (p < 0.001) whereas DHA reduced TNFA (p < 0.001), IL6 (p < 0.02), MCP1 (p < 0.03), and IL10 (p < 0.01). DHA lowered IL10 expression relative to EPA (p = 0.03). Relative to baseline, EPA, but not DHA, decreased the ratios of TNFA/IL10 and MCP1/IL10 (both p < 0.01). EPA and DHA also significantly changed plasma PUFA SPM lipidome by replacing n-6 AA derivatives with their respective derivatives including 18-hydroxy-EPA (+5 fold by EPA) and 17- and 14-hydroxy-DHA (+3 folds by DHA). However, DHA showed a wider effect than EPA by also significantly increasing EPA derivatives and DPA-derived SPM at a greater expense of AA derivatives. Different groups of PUFA derivatives mediated the differential effects of EPA and DHA on monocyte cytokine expression. CONCLUSIONS: EPA and DHA had distinct effects on monocyte inflammatory response with a broader effect of DHA in attenuating pro-inflammatory cytokines. These differential effects were potentially mediated by different groups of PUFA derivatives, suggesting immunomodulatory activities of SPM and their intermediates.

Activation of endothelial Wnt/ß-catenin signaling by protective astrocytes repairs BBB damage in ischemic stroke.

The role of astrocytes in dysregulation of blood-brain barrier (BBB) function following ischemic stroke is not well understood. Here, we investigate the effects of restoring the repair properties of astrocytes on the BBB after ischemic stroke. Mice deficient for NHE1, a pH-sensitive Na+/H+ exchanger 1, in astrocytes have reduced BBB permeability after ischemic stroke, increased angiogenesis and cerebral blood flow perfusion, in contrast to wild-type mice. Bulk RNA-sequencing transcriptome analysis of purified astrocytes revealed that ∼177 genes were differentially upregulated in mutant astrocytes, with Wnt7a mRNA among the top genes. Using a Wnt reporter line, we confirmed that the pathway was upregulated in cerebral vessels of mutant mice after ischemic stroke. However, administration of the Wnt/ß-catenin inhibitor, XAV-939, blocked the reparative effects of Nhe1-deficient astrocytes. Thus, astrocytes lacking pH-sensitive NHE1 protein are transformed from injurious to "protective" by inducing Wnt production to promote BBB repair after ischemic stroke.

Contamination of human DNA samples with mouse DNA can lead to false detection of XMRV-like sequences.

BACKGROUND: In 2006, a novel gammaretrovirus, XMRV (xenotropic murine leukemia virus-related virus), was discovered in some prostate tumors. A more recent study indicated that this infectious retrovirus can be detected in 67% of patients suffering from chronic fatigue syndrome (CFS), but only very few healthy controls (4%). However, several groups have published to date that they could not identify XMRV RNA or DNA sequences in other cohorts of CFS patients, while another group detected murine leukemia virus (MLV)-like sequences in 87% of such patients, but only 7% of healthy controls. Since there is a high degree of similarity between XMRV and abundant endogenous MLV proviruses, it is important to distinguish contaminating mouse sequences from true infections. RESULTS: DNA from the peripheral blood of 112 CFS patients and 36 healthy controls was tested for XMRV with two different PCR assays. A TaqMan qPCR assay specific for XMRV pol sequences was able to detect viral DNA from 2 XMRV-infected cells (~ 10-12 pg DNA) in up to 5 µg of human genomic DNA, but yielded negative results in the test of 600 ng genomic DNA from 100,000 peripheral blood cells of all samples tested. However, positive results were obtained with some of these samples, using a less specific nested PCR assay for a different XMRV sequence. DNA sequencing of the PCR products revealed a wide variety of virus-related sequences, some identical to those found in prostate cancer and CFS patients, others more closely related to known endogenous MLVs. However, all samples that tested positive for XMRV and/or MLV DNA were also positive for the highly abundant intracisternal A-type particle (IAP) long terminal repeat and most were positive for murine mitochondrial cytochrome oxidase sequences. No contamination was observed in any of the negative control samples, containing those with no DNA template, which were included in each assay. CONCLUSIONS: Mouse cells contain upwards of 100 copies each of endogenous MLV DNA. Even much less than one cell's worth of DNA can yield a detectable product using highly sensitive PCR technology. It is, therefore, vital that contamination by mouse DNA be monitored with adequately sensitive assays in all samples tested.

Small-Molecule PAPD5 Inhibitors Restore Telomerase Activity in Patient Stem Cells.

Genetic lesions that reduce telomerase activity inhibit stem cell replication and cause a range of incurable diseases, including dyskeratosis congenita (DC) and pulmonary fibrosis (PF). Modalities to restore telomerase in stem cells throughout the body remain unclear. Here, we describe small-molecule PAPD5 inhibitors that demonstrate telomere restoration in vitro, in stem cell models, and in vivo. PAPD5 is a non-canonical polymerase that oligoadenylates and destabilizes telomerase RNA component (TERC). We identified BCH001, a specific PAPD5 inhibitor that restored telomerase activity and telomere length in DC patient induced pluripotent stem cells. When human blood stem cells engineered to carry DC-causing PARN mutations were xenotransplanted into immunodeficient mice, oral treatment with a repurposed PAPD5 inhibitor, the dihydroquinolizinone RG7834, rescued TERC 3' end maturation and telomere length. These findings pave the way for developing systemic telomere therapeutics to counteract stem cell exhaustion in DC, PF, and possibly other aging-related diseases.

A conserved coccidian gene is involved in Toxoplasma sensitivity to the anti-apicomplexan compound, tartrolon E.

New treatments for the diseases caused by apicomplexans are needed. Recently, we determined that tartrolon E (trtE), a secondary metabolite derived from a shipworm symbiotic bacterium, has broad-spectrum anti-apicomplexan parasite activity. TrtE inhibits apicomplexans at nM concentrations in vitro, including Cryptosporidium parvum, Toxoplasma gondii, Sarcocystis neurona, Plasmodium falciparum, Babesia spp. and Theileria equi. To investigate the mechanism of action of trtE against apicomplexan parasites, we examined changes in the transcriptome of trtE-treated T. gondii parasites. RNA-Seq data revealed that the gene, TGGT1_272370, which is broadly conserved in the coccidia, is significantly upregulated within 4 h of treatment. Using bioinformatics and proteome data available on ToxoDB, we determined that the protein product of this tartrolon E responsive gene (trg) has multiple transmembrane domains, a phosphorylation site, and localizes to the plasma membrane. Deletion of trg in a luciferase-expressing T. gondii strain by CRISPR/Cas9 resulted in a 68% increase in parasite resistance to trtE treatment, supporting a role for the trg protein product in the response of T. gondii to trtE treatment. Trg is conserved in the coccidia, but not in more distantly related apicomplexans, indicating that this response to trtE may be unique to the coccidians, and other mechanisms may be operating in other trtE-sensitive apicomplexans. Uncovering the mechanisms by which trtE inhibits apicomplexans may identify shared pathways critical to apicomplexan parasite survival and advance the search for new treatments.

High-Throughput Analysis of Gene Function in the Bacterial Predator Bdellovibrio bacteriovorus.

Bdellovibrio bacteriovorus is a bacterial predator capable of killing and replicating inside most Gram-negative bacteria, including antibiotic-resistant pathogens. Despite growing interest in this organism as a potential therapeutic, many of its genes remain uncharacterized. Here, we perform a high-throughput genetic screen with B. bacteriovorus using transposon sequencing (Tn-seq) to explore the genetic requirements of predation. Two hundred one genes were deemed essential for growth in the absence of prey, whereas over 100 genes were found to be specifically required for predative growth on the human pathogens Vibrio cholerae and Escherichia coli in both planktonic and biofilm states. To further this work, we created an ordered-knockout library in B. bacteriovorus and developed new high-throughput techniques to characterize the mutants by their stage of deficiency in the predator life cycle. Using microscopy and flow cytometry, we confirmed 10 mutants defective in prey attachment and eight mutants defective in prey rounding. The majority of these genes are hypothetical and previously uncharacterized. Finally, we propose new nomenclature to group B. bacteriovorus mutants into classes based on their stage of predation defect. These results contribute to our basic understanding of bacterial predation and may be useful for harnessing B. bacteriovorus to kill harmful pathogens in the clinical setting.IMPORTANCEBdellovibrio bacteriovorus is a predatory bacterium that can kill a wide range of Gram-negative bacteria, including many human pathogens. Given the global rise of antibiotic resistance and dearth of new antibiotics discovered in the past 30 years, this predator has potential as an alternative to traditional antibiotics. For many years, B. bacteriovorus research was hampered by a lack of genetic tools, and the genetic mechanisms of predation have only recently begun to be established. Here, we comprehensively identify and characterize predator genes required for killing bacterial prey, as well as genes that interfere in this process, which may allow us to design better therapeutic predators. Based on our study, we and other researchers may ultimately be able to genetically engineer strains that have improved killing rates, target specific species of prey, or preferentially target prey in the planktonic or biofilm state.

Dysregulation of valvular interstitial cell let-7c, miR-17, miR-20a, and miR-30d in naturally occurring canine myxomatous mitral valve disease.

Canine myxomatous mitral valve disease (MMVD) resembles the early stages of myxomatous pathology seen in human non-syndromic mitral valve prolapse, a common valvular heart disease in the adult human population. Canine MMVD is seen in older subjects, suggesting age-related epigenetic dysregulation leading to derangements in valvular cell populations and matrix synthesis or degradation. We hypothesized that valvular interstitial cells (VICs) undergo disease-relevant changes in miRNA expression. In primary VIC lines from diseased and control valves, miRNA expression was profiled using RT-qPCR and next generation sequencing. VICs from diseased valves showed phenotypic changes consistent with myofibroblastic differentiation (vimentinlow+, α-SMAhigh+), increases in senescence markers (p21, SA-ß-gαl), and decreased cell viability and proliferation potential. RT-qPCR and miRNA sequencing analyses both showed significant (p<0.05) downregulation of let-7c, miR-17, miR-20a, and miR-30d in VICs from diseased valves compared to controls. Decreased let-7c, miR-17, and miR-20a may contribute to myofibroblastic differentiation in addition to cell senescence, and decreased miR-30d may disinhibit cell apoptosis. These data support the hypothesis that epigenetic dysregulation plays an important role in age-related canine MMVD.