Adenovirus small E1A directs activation of Alu transcription at YAP/TEAD- and AP-1-bound enhancers through interactions with the EP400 chromatin remodeler.

Alu retrotransposons, which form the largest family of mobile DNA elements in the human genome, have recently come to attention as a potential source of regulatory novelties, most notably by participating in enhancer function. Even though Alu transcription by RNA polymerase III is subjected to tight epigenetic silencing, their expression has long been known to increase in response to various types of stress, including viral infection. Here we show that, in primary human fibroblasts, adenovirus small e1a triggered derepression of hundreds of individual Alus by promoting TFIIIB recruitment by Alu-bound TFIIIC. Epigenome profiling revealed an e1a-induced decrease of H3K27 acetylation and increase of H3K4 monomethylation at derepressed Alus, making them resemble poised enhancers. The enhancer nature of e1a-targeted Alus was confirmed by the enrichment, in their upstream regions, of the EP300/CBP acetyltransferase, EP400 chromatin remodeler and YAP1 and FOS transcription factors. The physical interaction of e1a with EP400 was critical for Alu derepression, which was abrogated upon EP400 ablation. Our data suggest that e1a targets a subset of enhancer Alus whose transcriptional activation, which requires EP400 and is mediated by the e1a-EP400 interaction, may participate in the manipulation of enhancer activity by adenoviruses.

Single-stranded pre-methylated 5mC adapters uncover the methylation profile of plasma ultrashort Single-stranded cell-free DNA.

Whole-genome bisulfite sequencing (BS-Seq) measures cytosine methylation changes at single-base resolution and can be used to profile cell-free DNA (cfDNA). In plasma, ultrashort single-stranded cfDNA (uscfDNA, ∼50 nt) has been identified together with 167 bp double-stranded mononucleosomal cell-free DNA (mncfDNA). However, the methylation profile of uscfDNA has not been described. Conventional BS-Seq workflows may not be helpful because bisulfite conversion degrades larger DNA into smaller fragments, leading to erroneous categorization as uscfDNA. We describe the '5mCAdpBS-Seq' workflow in which pre-methylated 5mC (5-methylcytosine) single-stranded adapters are ligated to heat-denatured cfDNA before bisulfite conversion. This method retains only DNA fragments that are unaltered by bisulfite treatment, resulting in less biased uscfDNA methylation analysis. Using 5mCAdpBS-Seq, uscfDNA had lower levels of DNA methylation (∼15%) compared to mncfDNA and was enriched in promoters and CpG islands. Hypomethylated uscfDNA fragments were enriched in upstream transcription start sites (TSSs), and the intensity of enrichment was correlated with expressed genes of hemopoietic cells. Using tissue-of-origin deconvolution, we inferred that uscfDNA is derived primarily from eosinophils, neutrophils, and monocytes. As proof-of-principle, we show that characteristics of the methylation profile of uscfDNA can distinguish non-small cell lung carcinoma from non-cancer samples. The 5mCAdpBS-Seq workflow is recommended for any cfDNA methylation-based investigations.

Human DNA methylation signatures differentiate persistent from resolving MRSA bacteremia.

Persistent methicillin-resistant Staphylococcus aureus (MRSA) bacteremia is life threatening and occurs in up to 30% of MRSA bacteremia cases despite appropriate antimicrobial therapy. Isolates of MRSA that cause antibiotic-persistent methicillin-resistant S. aureus bacteremia (APMB) typically have in vitro antibiotic susceptibilities equivalent to those causing antibiotic-resolving methicillin-resistant S. aureus bacteremia (ARMB). Thus, persistence reflects host-pathogen interactions occurring uniquely in context of antibiotic therapy in vivo. However, host factors and mechanisms involved in APMB remain unclear. We compared DNA methylomes in circulating immune cells from patients experiencing APMB vs. ARMB. Overall, methylation signatures diverged in the distinct patient cohorts. Differentially methylated sites intensified proximate to transcription factor binding sites, primarily in enhancer regions. In APMB patients, significant hypomethylation was observed in binding sites for CCAAT enhancer binding protein-ß (C/EBPß) and signal transducer/activator of transcription 1 (STAT1). In contrast, hypomethylation in ARMB patients localized to glucocorticoid receptor and histone acetyltransferase p300 binding sites. These distinct methylation signatures were enriched in neutrophils and achieved a mean area under the curve of 0.85 when used to predict APMB using a classification model. These findings validated by targeted bisulfite sequencing (TBS-seq) differentiate epigenotypes in patients experiencing APMB vs. ARMB and suggest a risk stratification strategy for antibiotic persistence in patients treated for MRSA bacteremia.

Human Astrocytes Exhibit Tumor Microenvironment-, Age-, and Sex-Related Transcriptomic Signatures.

Astrocytes are critical for the development and function of synapses. There are notable species differences between human astrocytes and commonly used animal models. Yet, it is unclear whether astrocytic genes involved in synaptic function are stable or exhibit dynamic changes associated with disease states and age in humans, which is a barrier in understanding human astrocyte biology and its potential involvement in neurologic diseases. To better understand the properties of human astrocytes, we acutely purified astrocytes from the cerebral cortices of over 40 humans across various ages, sexes, and disease states. We performed RNA sequencing to generate transcriptomic profiles of these astrocytes and identified genes associated with these biological variables. We found that human astrocytes in tumor-surrounding regions downregulate genes involved in synaptic function and sensing of signals in the microenvironment, suggesting involvement of peritumor astrocytes in tumor-associated neural circuit dysfunction. In aging, we also found downregulation of synaptic regulators and upregulation of markers of cytokine signaling, while in maturation we identified changes in ionic transport with implications for calcium signaling. In addition, we identified subtle sexual dimorphism in human cortical astrocytes, which has implications for observed sex differences across many neurologic disorders. Overall, genes involved in synaptic function exhibit dynamic changes in the peritumor microenvironment and aging. These data provide powerful new insights into human astrocyte biology in several biologically relevant states that will aid in generating novel testable hypotheses about homeostatic and reactive astrocytes in humans.SIGNIFICANCE STATEMENT Astrocytes are an abundant class of cells playing integral roles at synapses. Astrocyte dysfunction is implicated in a variety of human neurologic diseases. Yet our knowledge of astrocytes is largely based on mouse studies. Direct knowledge of human astrocyte biology remains limited. Here, we present transcriptomic profiles of human cortical astrocytes, and we identified molecular differences associated with age, sex, and disease state. We found that peritumor and aging astrocytes downregulate genes involved in astrocyte-synapse interactions. These data provide necessary insight into human astrocyte biology that will improve our understanding of human disease.

Deregulated intracellular pathways define novel molecular targets for HBV-specific CD8 T cell reconstitution in chronic hepatitis B.

BACKGROUND & AIMS: In chronic HBV infection, elevated reactive oxygen species levels derived from dysfunctional mitochondria can cause increased protein oxidation and DNA damage in exhausted virus-specific CD8 T cells. The aim of this study was to understand how these defects are mechanistically interconnected to further elucidate T cell exhaustion pathogenesis and, doing so, to devise novel T cell-based therapies. METHODS: DNA damage and repair mechanisms, including parylation, CD38 expression, and telomere length were studied in HBV-specific CD8 T cells from chronic HBV patients. Correction of intracellular signalling alterations and improvement of antiviral T cell functions by the NAD precursor nicotinamide mononucleotide and by CD38 inhibition was assessed. RESULTS: Elevated DNA damage was associated with defective DNA repair processes, including NAD-dependent parylation, in HBV-specific CD8 cells of chronic HBV patients. NAD depletion was indicated by the overexpression of CD38, the major NAD consumer, and by the significant improvement of DNA repair mechanisms, and mitochondrial and proteostasis functions by NAD supplementation, which could also improve the HBV-specific antiviral CD8 T cell function. CONCLUSIONS: Our study delineates a model of CD8 T cell exhaustion whereby multiple interconnected intracellular defects, including telomere shortening, are causally related to NAD depletion suggesting similarities between T cell exhaustion and cell senescence. Correction of these deregulated intracellular functions by NAD supplementation can also restore antiviral CD8 T cell activity and thus represents a promising potential therapeutic strategy for chronic HBV infection. IMPACT AND IMPLICATIONS: Correction of HBV-specific CD8 T cell dysfunction is believed to represent a rational strategy to cure chronic HBV infection, which however requires a deep understanding of HBV immune pathogenesis to identify the most important targets for functional T cell reconstitution strategies. This study identifies a central role played by NAD depletion in the intracellular vicious circle that maintains CD8 T cell exhaustion, showing that its replenishment can correct impaired intracellular mechanisms and reconstitute efficient antiviral CD8 T cell function, with implications for the design of novel immune anti-HBV therapies. As these intracellular defects are likely shared with other chronic virus infections where CD8 exhaustion can affect virus clearance, these results can likely also be of pathogenetic relevance for other infection models.

TFIIIC as a Potential Epigenetic Modulator of Histone Acetylation in Human Stem Cells.

Regulation of histone acetylation dictates patterns of gene expression and hence cell identity. Due to their clinical relevance in cancer biology, understanding how human embryonic stem cells (hESCs) regulate their genomic patterns of histone acetylation is critical, but it remains largely to be investigated. Here, we provide evidence that acetylation of histone H3 lysine-18 (H3K18ac) and lysine-27 (H3K27ac) is only partially established by p300 in stem cells, while it represents the main histone acetyltransferase (HAT) for these marks in somatic cells. Our analysis reveals that whereas p300 marginally associated with H3K18ac and H3K27ac in hESCs, it largely overlapped with these histone marks upon differentiation. Interestingly, we show that H3K18ac is found at "stemness" genes enriched in RNA polymerase III transcription factor C (TFIIIC) in hESCs, whilst lacking p300. Moreover, TFIIIC was also found in the vicinity of genes involved in neuronal biology, although devoid of H3K18ac. Our data suggest a more complex pattern of HATs responsible for histone acetylations in hESCs than previously considered, suggesting a putative role for H3K18ac and TFIIIC in regulating "stemness" genes as well as genes associated with neuronal differentiation of hESCs. The results break ground for possible new paradigms for genome acetylation in hESCs that could lead to new avenues for therapeutic intervention in cancer and developmental diseases.

Epigenetic regulation of human non-coding RNA gene transcription.

Recent investigations on the non-protein-coding transcriptome of human cells have revealed previously hidden layers of gene regulation relying on regulatory non-protein-coding (nc) RNAs, including the widespread ncRNA-dependent regulation of epigenetic chromatin states and of mRNA translation and stability. However, despite its centrality, the epigenetic regulation of ncRNA genes has received relatively little attention. In this mini-review, we attempt to provide a synthetic account of recent literature suggesting an unexpected complexity in chromatin-dependent regulation of ncRNA gene transcription by the three human nuclear RNA polymerases. Emerging common features, like the heterogeneity of chromatin states within ncRNA multigene families and their influence on 3D genome organization, point to unexplored issues whose investigation could lead to a better understanding of the whole human epigenomic network.

Genetic structure and historic demography of endangered unarmoured threespine stickleback at southern latitudes signals a potential new management approach.

Habitat loss, flood control infrastructure, and drought have left most of southern California and northern Baja California's native freshwater fish near extinction, including the endangered unarmoured threespine stickleback (Gasterosteus aculeatus williamsoni). This subspecies, an unusual morph lacking the typical lateral bony plates of the G. aculeatus complex, occurs at arid southern latitudes in the eastern Pacific Ocean and survives in only three inland locations. Managers have lacked molecular data to answer basic questions about the ancestry and genetic distinctiveness of unarmoured populations. These data could be used to prioritize conservation efforts. We sampled G. aculeatus from 36 localities and used microsatellites and whole genome data to place unarmoured populations within the broader evolutionary context of G. aculeatus across southern California/northern Baja California. We identified three genetic groups with none consisting solely of unarmoured populations. Unlike G. aculeatus at northern latitudes, where Pleistocene glaciation has produced similar historical demographic profiles across populations, we found markedly different demographics depending on sampling location, with inland unarmoured populations showing steeper population declines and lower heterozygosity compared to low armoured populations in coastal lagoons. One exception involved the only high elevation population in the region, where the demography and alleles of unarmoured fish were similar to low armoured populations near the coast, exposing one of several cases of artificial translocation. Our results suggest that the current "management-by-phenotype" approach, based on lateral plates, is incidentally protecting the most imperilled populations; however, redirecting efforts toward evolutionary units, regardless of phenotype, may more effectively preserve adaptive potential.

Targeted bisulfite sequencing for biomarker discovery.

Cytosine methylation is one of the best studied epigenetic modifications. In mammals, DNA methylation patterns vary among cells and is mainly found in the CpG context. DNA methylation is involved in important processes during development and differentiation and its dysregulation can lead to or is associated with diseases, such as cancer, loss-of-imprinting syndromes and neurological disorders. It has been also shown that DNA methylation at the cellular, tissue and organism level varies with age. To overcome the costs of Whole-Genome Bisulfite Sequencing, the gold standard method to detect 5-methylcytosines at a single base resolution, DNA methylation arrays have been developed and extensively used. This method allows one to assess the status of a fraction of the CpG sites present in the genome of an organism. In order to combine the relatively low cost of Methylation Arrays and digital signals of bisulfite sequencing, we developed a Targeted Bisulfite Sequencing method that can be applied to biomarker discovery for virtually any phenotype. Here we describe a comprehensive step-by-step protocol to build a DNA methylation-based epigenetic clock.

An in situ high-throughput screen identifies inhibitors of intracellular Burkholderia pseudomallei with therapeutic efficacy.

Burkholderia pseudomallei (Bp) and Burkholderia mallei (Bm) are Tier-1 Select Agents that cause melioidosis and glanders, respectively. These are highly lethal human infections with limited therapeutic options. Intercellular spread is a hallmark of Burkholderia pathogenesis, and its prominent ties to virulence make it an attractive therapeutic target. We developed a high-throughput cell-based phenotypic assay and screened ∼220,000 small molecules for their ability to disrupt intercellular spread by Burkholderia thailandensis, a closely related BSL-2 surrogate. We identified 268 hits, and cross-species validation found 32 hits that also disrupt intercellular spread by Bp and/or Bm Among these were a fluoroquinolone analog, which we named burkfloxacin (BFX), which potently inhibits growth of intracellular Burkholderia, and flucytosine (5-FC), an FDA-approved antifungal drug. We found that 5-FC blocks the intracellular life cycle at the point of type VI secretion system 5 (T6SS-5)-mediated cell-cell spread. Bacterial conversion of 5-FC to 5-fluorouracil and subsequently to fluorouridine monophosphate is required for potent and selective activity against intracellular Burkholderia In a murine model of fulminant respiratory melioidosis, treatment with BFX or 5-FC was significantly more effective than ceftazidime, the current antibiotic of choice, for improving survival and decreasing bacterial counts in major organs. Our results demonstrate the utility of cell-based phenotypic screening for Select Agent drug discovery and warrant the advancement of BFX and 5-FC as candidate therapeutics for melioidosis in humans.

An RNA Polymerase III General Transcription Factor Engages in Cell Type-Specific Chromatin Looping.

Transcription factors (TFs) bind DNA in a sequence-specific manner and are generally cell type-specific factors and/or developmental master regulators. In contrast, general TFs (GTFs) are part of very large protein complexes and serve for RNA polymerases' recruitment to promoter sequences, generally in a cell type-independent manner. Whereas, several TFs have been proven to serve as anchors for the 3D genome organization, the role of GTFs in genome architecture have not been carefully explored. Here, we used ChIP-seq and Hi-C data to depict the role of TFIIIC, one of the RNA polymerase III GTFs, in 3D genome organization. We find that TFIIIC genome occupancy mainly occurs at specific regions, which largely correspond to Alu elements; other characteristic classes of repetitive elements (REs) such as MIR, FLAM-C and ALR/alpha are also found depending on the cell's developmental origin. The analysis also shows that TFIIIC-enriched regions are involved in cell type-specific DNA looping, which does not depend on colocalization with the master architectural protein CTCF. This work extends previous knowledge on the role of TFIIIC as a bona fide genome organizer whose action participates in cell type-dependent 3D genome looping via binding to REs.

Early adaptive chromatin remodeling events precede pathologic phenotypes and are reinforced in the failing heart.

The temporal nature of chromatin structural changes underpinning pathologic transcription are poorly understood. We measured chromatin accessibility and DNA methylation to study the contribution of chromatin remodeling at different stages of cardiac hypertrophy and failure. ATAC-seq and reduced representation bisulfite sequencing were performed in cardiac myocytes after transverse aortic constriction (TAC) or depletion of the chromatin structural protein CTCF. Early compensation to pressure overload showed changes in chromatin accessibility and DNA methylation preferentially localized to intergenic and intronic regions. Most methylation and accessibility changes observed in enhancers and promoters at the late phase (3 weeks after TAC) were established at an earlier time point (3 days after TAC), before heart failure manifests. Enhancers were paired with genes based on chromatin conformation capture data: while enhancer accessibility generally correlated with changes in gene expression, this feature, nor DNA methylation, was alone sufficient to predict transcription of all enhancer interacting genes. Enrichment of transcription factors and active histone marks at these regions suggests that enhancer activity coordinates with other epigenetic factors to determine gene transcription. In support of this hypothesis, ChIP-qPCR demonstrated increased enhancer and promoter occupancy of GATA4 and NKX2.5 at Itga9 and Nppa, respectively, concomitant with increased transcription of these genes in the diseased heart. Lastly, we demonstrate that accessibility and DNA methylation are imperfect predictors of chromatin structure at the scale of A/B compartmentalization-rather, accessibility, DNA methylation, transcription factors and other histone marks work within these domains to determine gene expression. These studies establish that chromatin reorganization during early compensation after pathologic stimuli is maintained into the later decompensatory phases of heart failure. The findings reveal the rules for how local chromatin features govern gene expression in the context of global genomic structure and identify chromatin remodeling events for therapeutic targeting in disease.

Dysregulation of hsa-miR-34a and hsa-miR-449a leads to overexpression of PACS-1 and loss of DNA damage response (DDR) in cervical cancer.

We have observed overexpression of PACS-1, a cytosolic sorting protein in primary cervical tumors. Absence of exonic mutations and overexpression at the RNA level suggested a transcriptional and/or posttranscriptional regulation. University of California Santa Cruz genome browser analysis of PACS-1 micro RNAs (miR), revealed two 8-base target sequences at the 3' terminus for hsa-miR-34a and hsa-miR-449a. Quantitative RT-PCR and Northern blotting studies showed reduced or loss of expression of the two microRNAs in cervical cancer cell lines and primary tumors, indicating dysregulation of these two microRNAs in cervical cancer. Loss of PACS-1 with siRNA or exogenous expression of hsa-miR-34a or hsa-miR-449a in HeLa and SiHa cervical cancer cell lines resulted in DNA damage response, S-phase cell cycle arrest, and reduction in cell growth. Furthermore, the siRNA studies showed that loss of PACS-1 expression was accompanied by increased nuclear γH2AX expression, Lys382-p53 acetylation, and genomic instability. PACS-1 re-expression through LNA-hsa-anti-miR-34a or -449a or through PACS-1 cDNA transfection led to the reversal of DNA damage response and restoration of cell growth. Release of cells post 24-h serum starvation showed PACS-1 nuclear localization at G1-S phase of the cell cycle. Our results therefore indicate that the loss of hsa-miR-34a and hsa-miR-449a expression in cervical cancer leads to overexpression of PACS-1 and suppression of DNA damage response, resulting in the development of chemo-resistant tumors.

Omega-3 fatty acids increase OXPHOS energy for immune therapy of Alzheimer disease patients.

Sporadic late-onset Alzheimer disease (LOAD) preceded by mild cognitive impairment (MCI) is the most common type of dementia. Long-term studies of immunity to pathogenic amyloid-ß (Aß) in LOAD are lacking. Innate immunity of LOAD patients is malfunctioning in phagocytosis and degradation of Aß and LOAD patients' macrophage transcriptome and metabolome are deregulated. We previously showed omega-3 fatty acid (ω-3)-mediated repair of unfolded protein response and here we show much broader transcriptomic effects. ω-3 treatment in vitro and ω-3 supplementation by the drink Smartfish (SMF) in vivo increased the transcripts of the genes and pathways of immunity, glycolysis, tricarboxylic acid cycle, OX-PHOS, nicotinamide dinucleotide (NAD+ ) synthesis, and reversed the defects in Aß phagocytosis. In both peripheral blood mononuclear cells (PBMC) and macrophages, ω-3 increased ATP-linked oxygen consumption rate (OCR) and ω-3 with carnitine was superior to ω-3. ω-3 treatment in vitro and supplementation by the ω-3 drink SMF in vivo rescued macrophage phagocytosis when glycolysis or glycosylation were blocked. ω-3 provide flexible energy for immune clearance of the brain throughout the diurnal cycle, even in hypo- or hyper-glycemia. In certain LOAD patients, ω-3 may delay progression to dementia.

A randomized, phase 1, placebo-controlled trial of APG-157 in oral cancer demonstrates systemic absorption and an inhibitory effect on cytokines and tumor-associated microbes.

BACKGROUND: Although curcumin's effect on head and neck cancer has been studied in vitro and in vivo, to the authors' knowledge its efficacy is limited by poor systemic absorption from oral administration. APG-157 is a botanical drug containing multiple polyphenols, including curcumin, developed under the US Food and Drug Administration's Botanical Drug Development, that delivers the active components to oromucosal tissues near the tumor target. METHODS: A double-blind, randomized, placebo-controlled, phase 1 clinical trial was conducted with APG-157 in 13 normal subjects and 12 patients with oral cancer. Two doses, 100 mg or 200 mg, were delivered transorally every hour for 3 hours. Blood and saliva were collected before and 1 hour, 2 hours, 3 hours, and 24 hours after treatment. Electrocardiograms and blood tests did not demonstrate any toxicity. RESULTS: Treatment with APG-157 resulted in circulating concentrations of curcumin and analogs peaking at 3 hours with reduced IL-1ß, IL-6, and IL-8 concentrations in the salivary supernatant fluid of patients with cancer. Salivary microbial flora analysis showed a reduction in Bacteroidetes species in cancer subjects. RNA and immunofluorescence analyses of tumor tissues of a subject demonstrated increased expression of genes associated with differentiation and T-cell recruitment to the tumor microenvironment. CONCLUSIONS: The results of the current study suggested that APG-157 could serve as a therapeutic drug in combination with immunotherapy. LAY SUMMARY: Curcumin has been shown to suppress tumor cells because of its antioxidant and anti-inflammatory properties. However, its effectiveness has been limited by poor absorption when delivered orally. Subjects with oral cancer were given oral APG-157, a botanical drug containing multiple polyphenols, including curcumin. Curcumin was found in the blood and in tumor tissues. Inflammatory markers and Bacteroides species were found to be decreased in the saliva, and immune T cells were increased in the tumor tissue. APG-157 is absorbed well, reduces inflammation, and attracts T cells to the tumor, suggesting its potential use in combination with immunotherapy drugs.

Novel features of telomere biology revealed by the absence of telomeric DNA methylation.

Cytosine methylation regulates the length and stability of telomeres, which can affect a wide variety of biological features, including cell differentiation, development, or illness. Although it is well established that subtelomeric regions are methylated, the presence of methylated cytosines at telomeres has remained controversial. Here, we have analyzed multiple bisulfite sequencing studies to address the methylation status of Arabidopsis thaliana telomeres. We found that the levels of estimated telomeric DNA methylation varied among studies. Interestingly, we estimated higher levels of telomeric DNA methylation in studies that produced C-rich telomeric strands with lower efficiency. However, these high methylation estimates arose due to experimental limitations of the bisulfite technique. We found a similar phenomenon for mitochondrial DNA: The levels of mitochondrial DNA methylation detected were higher in experiments with lower mitochondrial read production efficiencies. Based on experiments with high telomeric C-rich strand production efficiencies, we concluded that Arabidopsis telomeres are not methylated, which was confirmed by methylation-dependent restriction enzyme analyses. Thus, our studies indicate that telomeres are refractory to de novo DNA methylation by the RNA-directed DNA methylation machinery. This result, together with previously reported data, reveals that subtelomeric DNA methylation controls the homeostasis of telomere length.

Alu RNA Modulates the Expression of Cell Cycle Genes in Human Fibroblasts.

Alu retroelements, whose retrotransposition requires prior transcription by RNA polymerase III to generate Alu RNAs, represent the most numerous non-coding RNA (ncRNA) gene family in the human genome. Alu transcription is generally kept to extremely low levels by tight epigenetic silencing, but it has been reported to increase under different types of cell perturbation, such as viral infection and cancer. Alu RNAs, being able to act as gene expression modulators, may be directly involved in the mechanisms determining cellular behavior in such perturbed states. To directly address the regulatory potential of Alu RNAs, we generated IMR90 fibroblasts and HeLa cell lines stably overexpressing two slightly different Alu RNAs, and analyzed genome-wide the expression changes of protein-coding genes through RNA-sequencing. Among the genes that were upregulated or downregulated in response to Alu overexpression in IMR90, but not in HeLa cells, we found a highly significant enrichment of pathways involved in cell cycle progression and mitotic entry. Accordingly, Alu overexpression was found to promote transition from G1 to S phase, as revealed by flow cytometry. Therefore, increased Alu RNA may contribute to sustained cell proliferation, which is an important factor of cancer development and progression.

Omega-3 fatty acids increase the unfolded protein response and improve amyloid-ß phagocytosis by macrophages of patients with mild cognitive impairment.

Macrophages (Mϕs) of patients with Alzheimer's disease and mild cognitive impairment (MCI) are defective in amyloid-ß1-42 (Aß) phagocytosis and have low resistance to apoptosis by Aß. Omega-3 fatty acids (ω-3s) in vitro and in vivo and the ω-3 mediator, resolvin D1, in vitro increase Aß phagocytosis by Mϕs of patients with MCI. We have investigated the unfolded protein response (UPR) to endoplasmic reticulum (ER) stress by Mϕs in a longitudinal study of fish-derived, ω-3-supplemented patients with MCI. Patients in the apolipoprotein E (ApoE)e3/e3 subgroup over time exhibited an increase of protein kinase RNA-like ER kinase (PERK) expression, Aß phagocytosis, intermediate M1-M2 Mϕ type, and a Mini-Mental State Examination (MMSE) rate of change of +1.8 points per year, whereas patients in the ApoEe3/e4 subgroup showed individually divergent results with an MMSE rate of change of -3.2 points per year. In vitro treatment of Mϕs by fish-derived ω-3 emulsion increased Aß phagocytosis, PERK expression, and UPR RNA signature, and decreased ER stress signature. Augmented genes in the UPR signature included chaperones, lectins, foldases, and N-linked glycosylation enzymes. In summary, fish-derived ω-3s increase cytoprotective genes and decrease proapoptotic genes, improve immune clearance of Aß, and are associated with an improved MMSE rate of change in ApoEe3/e3 vs. ApoEe3/e4 patients.-Olivera-Perez, H. M., Lam, L., Dang, J., Jiang, W., Rodriguez, F., Rigali, E., Weitzman, S., Porter, V., Rubbi, L., Morselli, M., Pellegrini, M., Fiala, M. Omega-3 fatty acids increase the unfolded protein response and improve amyloid-ß phagocytosis by macrophages of patients with mild cognitive impairment.

Dynamic Changes in the Transcriptome and Methylome of Chlamydomonas reinhardtii throughout Its Life Cycle.

The green alga Chlamydomonas reinhardtii undergoes gametogenesis and mating upon nitrogen starvation. While the steps involved in its sexual reproductive cycle have been extensively characterized, the genome-wide transcriptional and epigenetic changes underlying different life cycle stages have yet to be fully described. Here, we performed transcriptome and methylome sequencing to quantify expression and DNA methylation from vegetative and gametic cells of each mating type and from zygotes. We identified 361 gametic genes with mating type-specific expression patterns and 627 genes that are specifically induced in zygotes; furthermore, these sex-related gene sets were enriched for secretory pathway and alga-specific genes. We also examined the C. reinhardtii nuclear methylation map with base-level resolution at different life cycle stages. Despite having low global levels of nuclear methylation, we detected 23 hypermethylated loci in gene-poor, repeat-rich regions. We observed mating type-specific differences in chloroplast DNA methylation levels in plus versus minus mating type gametes followed by chloroplast DNA hypermethylation in zygotes. Lastly, we examined the expression of candidate DNA methyltransferases and found three, DMT1a, DMT1b, and DMT4, that are differentially expressed during the life cycle and are candidate DNA methylases. The expression and methylation data we present provide insight into cell type-specific transcriptional and epigenetic programs during key stages of the C. reinhardtii life cycle.

Design and Construction of a Designed Ankyrin Repeat Protein (DARPin) Display Library.

Protein display systems are powerful techniques used to identify protein molecules that bind with high affinity to target proteins of interest. The initial challenge in implementing a display system is the construction of a high-diversity naïve library. Here, we describe the methods to generate a designed ankyrin repeat protein (DARPin) display library using degenerate oligonucleotides. Specifically described is the construction of a single DARPin repeat module by overlap extension PCR, concatenation of the module by restriction enzyme digestion and ligation, and incorporation of the concatenated modules into a full-length DARPin sequence in a bacterial cloning or display vector containing the hydrophilic N- and C-terminal capping domains. Protocols for PCR amplification of DARPin sequences to estimate diversity of naïve and enriched libraries via next-generation sequencing are included, as is a simple Linux-based program for analysis of naïve and enriched sequences. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of a single DARPin repeat by overlap extension PCR Basic Protocol 2: Concatenation of DARPin repeats Basic Protocol 3: Ligation of internal repeats into cloning/display vector containing N- and C-terminal capping repeats Basic Protocol 4: Estimation of library size and diversity by next-generation sequencing (NGS) Basic Protocol 5: NGS analysis of naïve and enriched libraries.