Cytosine methylation is a stable epigenetic modification of DNA that plays an important role in both normal physiology and disease. Most diseases exhibit some degree of sexual dimorphism, but the ...extent to which epigenetic states are influenced by sex is understudied and poorly understood. To address this deficit we studied DNA methylation patterns across multiple reduced representation bisulphite sequencing datasets (from liver, heart, brain, muscle and spleen) derived from isogenic male and female mice.
DNA methylation patterns varied significantly from tissue to tissue, as expected, but they also varied between the sexes, with thousands of sexually dimorphic loci identified. The loci affected were largely autonomous to each tissue, even within tissues derived from the same germ layer. At most loci, differences between genders were driven by females exhibiting hypermethylation relative to males; a proportion of these differences were independent of the presence of testosterone in males. Loci harbouring gender differences were clustered in ontologies related to tissue function.
Our findings suggest that gender is underwritten in the epigenome in a tissue-specific and potentially sex hormone-independent manner. Gender-specific epigenetic states are likely to have important implications for understanding sexually dimorphic phenotypes in health and disease.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Xenon (Xe) has shown great potential as a stroke treatment due to its exceptional ability to protect brain tissue without inducing side effects. We have previously developed Xe-loaded liposomes for ...the ultrasound-activated delivery of Xe into the cerebral region and demonstrated their therapeutic efficacy. At present, the sole FDA-approved thrombolytic agent for stroke treatment is recombinant tissue plasminogen activator (rtPA). In this study, we aimed to investigate the potential of combining Xe-liposomes with an intravenous rtPA treatment in a clinically relevant embolic rat stroke model. We evaluated the combinational effect using an in vitro clot lysis model and an in vivo embolic middle cerebral artery occlusion (eMCAO) rat model. The treatment groups received intravenous administration of Xe-liposomes (20 mg/kg) at 2 h post-stroke onset, followed by the administration of rtPA (10 mg/kg) at either 2 or 4 h after the onset. Three days after the stroke, behavioral tests were conducted, and brain sections were collected for triphenyltetrazolium chloride (TTC) and TUNEL staining. Infarct size was determined as normalized infarct volume (%). Both in vitro and in vivo clot lysis experiments demonstrated that Xe-liposomes in combination with rtPA resulted in effective clot lysis comparable to the treatment with free rtPA alone. Animals treated with Xe-liposomes in combination with rtPA showed reduced TUNEL-positive cells and demonstrated improved neurological recovery. Importantly, Xe-liposomes in combination with late rtPA treatment reduced rtPA-induced hemorrhage, attributing to the reduction of MMP9 immunoreactivity. This study demonstrates that the combined therapy of Xe-liposomes and rtPA provides enhanced therapeutic efficacy, leading to decreased neuronal cell death and a potential to mitigate hemorrhagic side effects associated with late rtPA treatment.
Methionine and homocysteine are metabolites in the transmethylation pathway leading to synthesis of the methyl-donor S-adenosylmethionine (SAM). Most cancer cells stop proliferating during methionine ...stress conditions, when methionine is replaced in the growth media by its immediate metabolic precursor homocysteine (Met-Hcy+). Non-transformed cells proliferate in Met-Hcy+ media, making the methionine metabolic requirement of cancer cells an attractive target for therapy, yet there is relatively little known about the molecular mechanisms governing the methionine stress response in cancer cells. To study this phenomenon in breast cancer cells, we selected methionine-independent-resistant cell lines derived from MDAMB468 breast cancer cells. Resistant cells grew normally in Met-Hcy+ media, whereas their parental MDAMB468 cells rapidly arrest in the G
1
phase. Remarkably, supplementing Met-Hcy+ growth media with S-adenosylmethionine suppressed the cell proliferation defects, indicating that methionine stress is a consequence of SAM limitation rather than low amino acid concentrations. Accordingly, mTORC1 activity, the primary effector responding to amino acid limitation, remained high. However, we found that levels of the replication factor Cdc6 decreased and pre-replication complexes were destabilized in methionine-stressed MDAMB468 but not resistant cells. Our study characterizes metabolite requirements and cell cycle responses that occur during methionine stress in breast cancer cells and helps explain the metabolic uniqueness of cancer cells.
An epitope tag introduced to a gene of interest (GOI) greatly increases the ease of studying cellular proteins. Rapid PCR‐based strategies for epitope tagging a protein's C‐terminus at its native ...gene locus are widely used in yeast. C‐terminal epitope tagging is not suitable for all proteins, however. Epitope tags fused to the C‐terminus can interfere with function of some proteins or can even be removed by C‐terminal protein processing. To overcome such problems, proteins can be tagged with epitopes at their amino‐termini, but generating yeast strains expressing N‐terminal epitope tagged genes under control of the endogenous promoter at the native locus is comparatively more difficult. Strategies to introduce N‐terminal epitope tags have been reported previously but often introduce additional sequences other than the epitope tag into the genome. Furthermore, N‐terminal tagging of essential genes by current methods requires formation of diploid strains followed by tetrad dissection or expression of an additional copy of the GOI from a plasmid. The strategies described here provide a quick, facile means of epitope tagging the N‐terminus of both essential and nonessential genes in a two‐step PCR‐based procedure. The procedure has the significant advantage of leaving tagged genes under the control of their endogenous promoters, and no additional sequences other than the epitope tag encoding nucleotides are inserted into the genome.
Abstract only Introduction: Epigenetic markers including 5-metylcytosine (5mC) can reflect disease severity and treatment effect. Stroke is a multifactorial disease causing aberrant DNA methylation ...(DNAm) and memory damage. Hypothesis: Models built for 5mC biomarker panel signatures may be useful for quantitative prediction of post-stroke long-term memory recovery in response to treatment. Methods: Stroke was induced with a middle cerebral artery occlusion (MCAO)(18 rats: sham(6), stroke(6), and stroke with treatment (6)). Xe-liposomes were intravenously administrated repetitively for 3 days after MCAO. At day 35, memory was assessed and scaled using a novel objective recognition test. Brain hippocampus and cortex tissue samples were prepared for whole-genome bisulfite sequencing (WGBS) to generate quantitative DNA methylation datasets in response to repetitive intermittent Xe administration. The resulting WGBS data were evaluated and quantitatively associated with memory recovery. Results: A post-stroke long-term memory outcome model was generated with scaled data using the following inputs: (1) good recovery, (2) moderate recovery, (3) edge, (4) light damage, and (5) severe damage. Differential DNAm 5mC analysis was performed comparing MCAO affected hemisphere with sham and Xe-treated samples. Initial 5mC differential analysis identified 6,175 (Cortex), and 9,037 (Hippocampus) CpG sites that were different between groups. Subsequently the LASSO algorithm was applied and selected 22 CpG sites that correlated with the long-term memory recovery scale. PCA analysis using the 22 CpGs demonstrated separation of memory outcomes. We have also built a predictive linear regression model of the 22 CpGs to assess memory loss or recovery in response to treatment. Analysis showed 7 out of 22 CpGs are conserved in humans (orthologs) for 30 bp regions. Conclusions: Epigenetic 5mC marker panels have the potential for quantitating post-stroke memory damages/recovery.
Abstract only Acute ischemic stroke induces widespread cellular necrosis and apoptosis and activation of inflammation. These mediate release of cell-free DNA (cfDNA) into the circulation. Although ...increased cfDNA concentrations have been associated with clinical outcome in stroke, data concerning neuronal cfDNA is rare. In this study, we assessed the association of cfDNA with neuron biomarkers in patients with acute ischemic stroke. Methods: Thirteen patients without stroke and four patients with middle cerebral artery occlusion were recruited at UT Memorial Hermann Hospital. Blood plasma samples were collected and cfDNA samples were prepared for whole-genome bisulfite sequencing (WGBS) in order to generate quantitative DNA methylation datasets. The resulting WGBS data were analyzed for epigenetic biomarker identification using bioinformatic statistical approaches. CelFiE was used for cell deconvolution, designed to accurately estimate the relative abundances of cell types and tissues present in plasma cfDNA from methylation sequencing, a technique frequently referred to as cell deconvolution. Results: Differential methylation statistical analysis comparing the stroke against the non-stroke group of samples found 3493 DNA methylation differences. Pathway Analysis discovered greater enrichment for neuronal function with the top hit being “Neuronal System” followed by activity surrounding synapses, demonstrating that a clear enrichment of methylation change in neuronal pathways can be reflected from blood plasma samples following the stroke. Cell deconvolution showed trends of increased megakaryocyte and neutrophils which are involved in the post-stroke immune response. It may be that the secreted neutrophil nuclear DNA in blood plasma drives the increase in the observed proportion of neutrophil cell type in the stroke group samples. Principal Component Analysis demonstrated good separation between the stroke and normal groups. Conclusion: Blood sample can be used for circulating cfDNA analysis and neuron biomarker discovery for acute ischemic stroke prediction/recovery.
Abstract only
Introduction:
Ischemic stroke is one of the main causes of long-term morbidity/mortality and early treatment is key. Xenon (Xe) has demonstrated powerful neuroprotective effects on both ...ischemic and hemorrhagic stroke, but its clinical and long-term outpatient administration is limited by currently available delivery methods. Development of an oral Xe formulation is an attractive strategy for stroke treatment in the field. We developed a Xenon (Xe)-cyclodextrin (CD) oral formulation.
Hypothesis:
Xe-CD formulation can be delivered orally for acute stroke treatment.
Methods:
A stable, oral Xe-CD clathrate (Xe solid gas) was formed by pressurizing Xe (3 atm) into 14% alpha-cyclodextrin at room temperature. Xe concentration was measured by GC-MS. Rat middle cerebral artery occlusion (MCAO) was induced by intraluminar suture. The animals were divided into groups: stroke (n=4); stroke with CD (n=3); stroke with 0.5 ml Xe-CD (n=3); stroke with 1.0 ml Xe-CD (n=3); and stroke with 1.5 ml Xe-CD (n=3). Treatments were administered by gavage once a day for 3 days. At day 3, neurological behavior testing was conducted. The infarct size and neuronal death were assessed and normalized by total brain volume.
Results:
There are 1.56 mM Xe clathrate per 0.6 mM alpha-cyclodextrin. MCAO for 2 hours induced 21% ± 3.6% infarct volume. Xe-CD treatment dose-dependently reduced the infarct size to 18.9% ± 2.3% by 0.5 ml Xe-CD, 6.7% ± 1.8% by 1.0 ml Xe-CD, and 4.8% ± 1.2% by 1.5 ml Xe-CD (Fig. 1A). Behavioral test assessment using forelimb placement rate and Rotarod score matched infarct size. TUNEL staining demonstrated significant decreases in apoptosis in stroke rats treated with 1.5 ml Xe-CD (Fig. 1B).
Conclusions:
We have demonstrated oral Xe-CD formulation ameliorates neuronal apoptosis and reduces infarct size. Xe-CD represents a promising therapeutic for ischemic stroke treatment.
Background
Effective treatments for Alzheimer’s disease (AD) are classically designed to canonical disease players (e.g., misfolded proteins, neurodegeneration, and brain inflammation). Importantly, ...treatments must be able to cross the blood‐brain barrier (BBB) to cause meaningful therapeutic effects. Sufficient evidence has demonstrated that xenon (Xe), a bioactive gas, has profound neuroprotective effects with advantages of rapid diffusion across the BBB into hypoperfused brains without side effects. For chronic use, a long‐term and effective Xe delivery method is needed. Here, we present novel Xe formulations with effective cerebral delivery strategies for extended endogenous neuroprotection and neurorestoration.
Method
Orally deliverable Xe cyclodextrin (Xe‐CD) and intravenous deliverable Xe‐liposome were prepared. Xe‐CD was orally administrated in aged (10 months old) ApoE knockout mice fed with high‐fat diet for 6 weeks. The brain and blood amyloid‐β (Aβ) levels were measured by ELISA. To extend therapeutic effects, Xe‐liposomes were intravenously administrated using an intermittent repetitive administration strategy in a rat stroke model. Xe effects on epigenetic regulation were evaluated in this rat stroke model using Whole‐genome bisulfite sequencing (WGBS) and gene pathway enrichment analyses. Neurocognitive recovery was also evaluated.
Result
Aged (10 months old) ApoE knockout mice fed with high‐fat diet for 6 weeks developed high Ab expression levels in the brain and blood. However, Xe oral administration for 6‐week prevented the high‐fat diet‐induced Aβ elevation. Repetitive intermittent administration of Xe formulations extended the protective effects over time as long‐term memory restoration post stroke was observed. Whole‐genome bisulfite sequencing (WGBS) and gene pathway enrichment analyses revealed enrichment of differential methylation in multiple biologic pathways associated with growth factor response, memory consolidation, synaptic plasticity, and brain aging in the hippocampus. Of interesting, Xe regulated endogenous β‐secretase (BACE1), and low‐density lipoprotein receptor‐related protein 1 (LRP1), both of which are known to be associated with Ab. Xe also regulated memory and age related genes such as Gadd45b, DMNT3a, and HDAC9; and growth factor related genes NTrK2 and NGF. Gadd45b and PFAa also regulate long‐term memory consolidation.
Conclusion
We have developed a novel Xe formulation and delivery strategy for long‐term neuroprotection that stabilizes the brain through multiple pathways.
Introduction More than half of patients with relapsed or refractory large B-cell lymphoma (R/R LBCL) will experience progressive disease after CD19 chimeric antigen receptor T-cell therapy (CART). ...Circulating tumor DNA profiling has been used to prognosticate patients before CART, but mainly has focused on identification of somatic genomic aberrations. Epigenetic changes, specifically aberrant DNA methylation, can alter transcriptional regulation of genes important for treatment resistance. Altered methylation features can be detected from cell-free DNA (cfDNA) in patients with LBCL and are associated with outcomes after frontline immunochemotherapy. In the current study, we used whole genome bisulfite sequencing (WGBS) to analyze the methylation landscape in cfDNA from patient plasma before CART and identified novel methylation signatures associated with poor treatment outcomes. Methods Patients with R/R LBCL who received standard-of-care CART with plasma samples drawn before lymphodepletion (LD) or at cell infusion were identified. The primary endpoint was 3-month overall response after CART. cfDNA was extracted from 3-5 ml of plasma and cfDNA libraries were prepared using a Zymo-Seq cell free WGBS kit. Each library was sequenced to 300 - 600 million read pairs at a read length of 150 bp, enabling an average CpG coverage of 7-13X per detected CpG. Unique alignment rate among all libraries were about or above 82%. Reads were adapter trimmed with Trim Galore and aligned to reference genome hg19 using Bismark. Methylation calling was performed by MethylDackel. Differentially methylated cytosines (DMC, FDR ≤0.05) and regions (DMR) from WGBS data with an absolute methylation difference of ≥ 0.1 between 3-month responders and non-responders were identified using DSS. Functional enrichment analysis of genes overlapped by DMRs was performed by gprofiler2. Results A total of 22 patients with plasma collected before LD (n=21) or at cell infusion (n=1) who received CART were identified. Median follow-up was 21.5 months (95% CI 12.5-NR). Three-month response was evaluable in 21 patients (1 pending evaluation). Overall response rate (ORR) was 77% (8 CR, 9 PR) and 55% (9 CR, 2 PR) at one- and 3-months post-CART, respectively. Median PFS was 6.5 months (95% CI 3.0-NR), and median OS was 57.7 months (95% CI 7.7-NR). Patient and disease characteristics are described in Table 1. Differential methylation analysis identified 22,815 DMCs, of which 1,043 were hypomethylated and 21,772 were hypermethylated in non-responders. The global average methylation values across 25 million cytosines were 0.807 and 0.786 for non-responders and responders, respectively (p=0.265). A total of 582 DMRs were identified, of which 34 were hypomethylated and 548 were hypermethylated in non-responders (Figure 1). Of these, 281 (48%) overlapped intergenic regions, 265 (46%) gene bodies, and 33 (6%) promoter regions. DMRs overlapping DNA repair gene PARP1 (areaStat 88.2), B-cell receptor signaling genes SYK (27.2), FYN (21.4), and PTPN6 (20.9), polycomb repressor complex genes JARID2 (25.2) and BCOR (21.4), tumor suppressor WT1 (47.1), immune mediators NRP1 (80.0), TRAF3 (56.7), PAK3 (28.4), IRAK2 (28.7), and TNFS14 (23.6), and non-coding RNAs MIR4520 (95.8) and VTRNA-2 (23.1) were hypermethylated in non-responders and IL17RA (-33.2) hypomethylated. Functional enrichment analysis on overlapped genes also identified common molecular functions involving guanyl nucleotide exchange factors (p=0.0007), GTPase regulation (p=0.001), and cGMP binding (p=0.007). Discussion This proof-of-concept study is the first to perform WGBS on pretreatment plasma from patients with R/R LBCL to associate methylation signatures with post-CART outcomes. We identified that non-responders were enriched in hypermethylated DMRs compared to responders, overlapping with genes important to lymphomagenesis, immune function, and GTP energy metabolism. Further work will expand this cohort, compare with matched-tissue to find tumor-specific markers, and identify candidate DMR targets for targeted bisulfite sequencing from pre- and post-treatment plasma samples to assess response. By identifying high-risk methylation signatures in the plasma of CART recipients, we may be able to identify patients to be prioritized for novel treatment strategies such as combination with hypomethylating agents to improve outcomes.
Abstract only
Aging impacts many important attributes of human beings such as the onset of diseases and the length of lifespans. Previous research demonstrated that DNA methylation based epigenetic ...clocks can be used to estimate biological age in both humans and mice. Interestingly, an accelerated aging process was observed in a short‐lived laboratory mouse strain (AKR/J, median lifespan = 288 days). By comparison, the more common laboratory mouse strain C57BL/6J lives substantially longer (median lifespan = 901 days). To elucidate the underlying mechanisms linking the accelerated aging process to a shortened lifespan, we used reduced representation bisulfite sequencing (RRBS) to profile DNA methylation in the cortex and the hippocampus extracted from these two strains, respectively (4 biological replicates per brain region for the C57BL/6J strain, 6 biological replicates per brain region for the AKR/J strain). At a 5X read depth cutoff, we detected 2,376,777 ± 195,194 unique CpG sites in the C57BL/6J strain samples and 2,288,300 ± 137,243 unique CpG sites in the AKR/J strain samples. Samples from the same brain region of the same strain clustered tightly based on their distinctive DNA methylation patterns. Using a publication‐supported R package, “DSS”, to compare between the two strains, we identified 14,625 differentially methylated cytosines (DMCs, Benjamini‐Hochberg FDR <0.05; methylation difference ≥0.15) and 257 differentially methylation regions (DMRs, p<0.05; methylation difference ≥0.15) from the cortex samples. Similarly, we found 14,215 DMCs (Benjamini‐Hochberg FDR <0.05; methylation difference ≥0.15) and 304 DMRs (p<0.05; methylation difference ≥0.15) from the hippocampus samples. Subsequent annotation of the DMRs from both sample groups revealed that 106 genes had significantly different methylation levels in their promoter regions. Pathway analysis demonstrated differential promoter methylation of genes (for example,
Psat1, Smarca5, Rnf152 and Hspg2)
enriched in the regulation of chromatin remodeling, cellular development, and metabolism. Importantly,
Rnf152
and
Hspg2
are associated with mouse aging. In summary, our study discovered potential epigenetic features involved in the accelerated aging process of a short‐lived mouse model. Future work should focus on the detailed functions of the identified genes and further understand their link to altered mice lifespans, eventually leading to strategies for improved healthcare in human lives.
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