DNA Demethylation Dynamics in the Human Prenatal Germline.

Global DNA demethylation in humans is a fundamental process that occurs in pre-implantation embryos and reversion to naive ground state pluripotent stem cells (PSCs). However, the extent of DNA methylation reprogramming in human germline cells is unknown. Here, we performed whole-genome bisulfite sequencing (WGBS) and RNA-sequencing (RNA-seq) of human prenatal germline cells from 53 to 137 days of development. We discovered that the transcriptome and methylome of human germline is distinct from both human PSCs and the inner cell mass (ICM) of human blastocysts. Using this resource to monitor the outcome of global DNA demethylation with reversion of primed PSCs to the naive ground state, we uncovered hotspots of ultralow methylation at transposons that are protected from demethylation in the germline and ICM. Taken together, the human germline serves as a valuable in vivo tool for monitoring the epigenome of cells that have emerged from a global DNA demethylation event.

A draft human pangenome reference.

Here the Human Pangenome Reference Consortium presents a first draft of the human pangenome reference. The pangenome contains 47 phased, diploid assemblies from a cohort of genetically diverse individuals1. These assemblies cover more than 99% of the expected sequence in each genome and are more than 99% accurate at the structural and base pair levels. Based on alignments of the assemblies, we generate a draft pangenome that captures known variants and haplotypes and reveals new alleles at structurally complex loci. We also add 119 million base pairs of euchromatic polymorphic sequences and 1,115 gene duplications relative to the existing reference GRCh38. Roughly 90 million of the additional base pairs are derived from structural variation. Using our draft pangenome to analyse short-read data reduced small variant discovery errors by 34% and increased the number of structural variants detected per haplotype by 104% compared with GRCh38-based workflows, which enabled the typing of the vast majority of structural variant alleles per sample.

Retrotransposon activation contributes to neurodegeneration in a Drosophila TDP-43 model of ALS.

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are two incurable neurodegenerative disorders that exist on a symptomological spectrum and share both genetic underpinnings and pathophysiological hallmarks. Functional abnormality of TAR DNA-binding protein 43 (TDP-43), an aggregation-prone RNA and DNA binding protein, is observed in the vast majority of both familial and sporadic ALS cases and in ~40% of FTLD cases, but the cascade of events leading to cell death are not understood. We have expressed human TDP-43 (hTDP-43) in Drosophila neurons and glia, a model that recapitulates many of the characteristics of TDP-43-linked human disease including protein aggregation pathology, locomotor impairment, and premature death. We report that such expression of hTDP-43 impairs small interfering RNA (siRNA) silencing, which is the major post-transcriptional mechanism of retrotransposable element (RTE) control in somatic tissue. This is accompanied by de-repression of a panel of both LINE and LTR families of RTEs, with somewhat different elements being active in response to hTDP-43 expression in glia versus neurons. hTDP-43 expression in glia causes an early and severe loss of control of a specific RTE, the endogenous retrovirus (ERV) gypsy. We demonstrate that gypsy causes the degenerative phenotypes in these flies because we are able to rescue the toxicity of glial hTDP-43 either by genetically blocking expression of this RTE or by pharmacologically inhibiting RTE reverse transcriptase activity. Moreover, we provide evidence that activation of DNA damage-mediated programmed cell death underlies both neuronal and glial hTDP-43 toxicity, consistent with RTE-mediated effects in both cell types. Our findings suggest a novel mechanism in which RTE activity contributes to neurodegeneration in TDP-43-mediated diseases such as ALS and FTLD.

Distinct and concurrent pathways of Pol II- and Pol IV-dependent siRNA biogenesis at a repetitive trans-silencer locus in Arabidopsis thaliana.

Short interfering RNAs (siRNAs) homologous to transcriptional regulatory regions can induce RNA-directed DNA methylation (RdDM) and transcriptional gene silencing (TGS) of target genes. In our system, siRNAs are produced by transcribing an inverted DNA repeat (IR) of enhancer sequences, yielding a hairpin RNA that is processed by several Dicer activities into siRNAs of 21-24 nt. Primarily 24-nt siRNAs trigger RdDM of the target enhancer in trans and TGS of a downstream GFP reporter gene. We analyzed siRNA accumulation from two different structural forms of a trans-silencer locus in which tandem repeats are embedded in the enhancer IR and distinguished distinct RNA polymerase II (Pol II)- and Pol IV-dependent pathways of siRNA biogenesis. At the original silencer locus, Pol-II transcription of the IR from a 35S promoter produces a hairpin RNA that is diced into abundant siRNAs of 21-24 nt. A silencer variant lacking the 35S promoter revealed a normally masked Pol IV-dependent pathway that produces low levels of 24-nt siRNAs from the tandem repeats. Both pathways operate concurrently at the original silencer locus. siRNAs accrue only from specific regions of the enhancer and embedded tandem repeat. Analysis of these sequences and endogenous tandem repeats producing siRNAs revealed the preferential accumulation of siRNAs at GC-rich regions containing methylated CG dinucleotides. In addition to supporting a correlation between base composition, DNA methylation and siRNA accumulation, our results highlight the complexity of siRNA biogenesis at repetitive loci and show that Pol II and Pol IV use different promoters to transcribe the same template.

MethGo: a comprehensive tool for analyzing whole-genome bisulfite sequencing data.

BACKGROUND: DNA methylation is a major epigenetic modification regulating several biological processes. A standard approach to measure DNA methylation is bisulfite sequencing (BS-Seq). BS-Seq couples bisulfite conversion of DNA with next-generation sequencing to profile genome-wide DNA methylation at single base resolution. The analysis of BS-Seq data involves the use of customized aligners for mapping bisulfite converted reads and the bioinformatic pipelines for downstream data analysis. RESULTS: Here we developed MethGo, a software tool designed for the analysis of data from whole-genome bisulfite sequencing (WGBS) and reduced representation bisulfite sequencing (RRBS). MethGo provides both genomic and epigenomic analyses including: 1) coverage distribution of each cytosine; 2) global cytosine methylation level; 3) cytosine methylation level distribution; 4) cytosine methylation level of genomic elements; 5) chromosome-wide cytosine methylation level distribution; 6) Gene-centric cytosine methylation level; 7) cytosine methylation levels at transcription factor binding sites (TFBSs); 8) single nucleotide polymorphism (SNP) calling, and 9) copy number variation (CNV) calling. CONCLUSIONS: MethGo is a simple and effective tool for the analysis of BS-Seq data including both WGBS and RRBS. It contains 9 analyses in 5 major modules to profile (epi)genome. It profiles genome-wide DNA methylation in global and in gene level scale. It can also analyze the methylation pattern around the transcription factor binding sites, and assess genetic variations such as SNPs and CNVs. MethGo is coded in Python and is publically available at http://paoyangchen-laboratory.github.io/methgo/.

Post-Operative Sleep Endoscopy with Target-Controlled Infusion After Palatopharyngoplasty for Obstructive Sleep Apnea: Anatomical and Polysomnographic Outcomes.

INTRODUCTION: The findings of drug-induced sleep endoscopy (DISE) are not always correlated with the outcome of upper airway surgery for obstructive sleep apnea (OSA), and whether multilevel surgery is truly required in treating multilevel obstruction identified in preoperative DISE remains an issue. We attempted to compare DISE findings before and after palatopharyngoplasty in patients with OSA because changes in DISE may be beneficial to better understand polysomnographic and anatomical outcomes. METHODS: This was a prospective cohort study for 34 patients with moderate to severe OSA who underwent palatopharyngoplasty at a tertiary care center from 2016 to 2018. We recorded the patients' demographic characteristics, procedures, and surgical outcomes and compared the preoperative and postoperative DISE staging patterns. RESULTS: The apnea-hypopnea index (AHI) values of 34 adults improved significantly after surgery (40.6 ± 23.3 versus 25.6 ± 20.6, P < 0.001). The majority of patients, 26/34, had preoperative complete concentric collapse at the velum, and for most (20/26, 77%) there was a change of the collapse pattern into anteroposterior collapse postoperatively. Patients with postoperative velar collapse had higher follow-up AHI values than those who without (27.8 ± 21.9 versus 15.2 ± 7.7, P = 0.023). Patients with preoperative complete tongue base collapse had higher follow-up AHI values than did those with no or partial collapse (40.6 ± 21.0 versus 21.0 ± 18.6, P = 0.017). Patients with postoperative complete tongue base collapse also had higher follow-up AHI values than the others (42.7 ± 22.1 versus 18.5 ± 15.4, P = 0.001). CONCLUSION: Palatopharyngoplasty could change the collapse pattern at the velum in most patients. Preoperative and postoperative complete tongue base collapse and postoperative velar collapse identified in TCI-DISE were associated with relatively poor outcomes.

TEsmall Identifies Small RNAs Associated With Targeted Inhibitor Resistance in Melanoma.

MicroRNAs (miRNAs) are small 21-22 nt RNAs that act to regulate the expression of mRNA target genes through direct binding to mRNA targets. While miRNAs typically dominate small RNA (sRNA) transcriptomes, many other classes are present including tRNAs, snoRNAs, snRNAs, Y-RNAs, piRNAs, and siRNAs. Interactions between processing machinery and targeting networks of these various sRNA classes remains unclear, largely because these sRNAs are typically analyzed separately. Here, we present TEsmall, a tool that allows for the simultaneous processing and analysis of sRNAs from each annotated class in a single integrated workflow. The pipeline begins with raw fastq reads and proceeds all the way to producing count tables formatted for differential expression analysis. Several interactive charts are also produced to look at overall distributions in length and annotation classes. We next applied the TEsmall pipeline to sRNA libraries generated from melanoma cells responding to targeted inhibitors of the MAPK pathway. Targeted oncogene inhibitors have emerged as way to tailor cancer therapies to the particular mutations present in a given tumor. While these targeted strategies are typically effective for short intervals, the emergence of resistance is extremely common, limiting the effectiveness of single-agent therapeutics and driving the need for a better understanding of resistance mechanisms. Using TEsmall, we identified several microRNAs and other sRNA classes that are enriched in inhibitor resistant melanoma cells in multiple melanoma cell lines and may be able to serve as markers of resistant populations more generally.

Prenatal Growth Patterns and Birthweight Are Associated With Differential DNA Methylation and Gene Expression of Cardiometabolic Risk Genes in Human Placentas: A Discovery-Based Approach.

Inherent genetic programming and environmental factors affect fetal growth in utero. Epidemiologic data in growth-altered fetuses, either intrauterine growth restricted (IUGR) or large for gestational age (LGA), demonstrate that these newborns are at increased risk of cardiometabolic disease in adulthood. There is growing evidence that the in utero environment leads to epigenetic modification, contributing to eventual risk of developing heart disease or diabetes. In this study, we used reduced representation bisulfite sequencing to examine genome-wide DNA methylation variation in placental samples from offspring born IUGR, LGA, and appropriate for gestational age (AGA) and to identify differential methylation of genes important for conferring risk of cardiometabolic disease. We found that there were distinct methylation signatures for IUGR, LGA, and AGA groups and identified over 500 differentially methylated genes (DMGs) among these group comparisons. Functional and gene network analyses revealed expected relationships of DMGs to placental physiology and transport, but also identified novel pathways with biologic plausibility and potential clinical importance to cardiometabolic disease. Specific loci for DMGs of interest had methylation patterns that were strongly associated with anthropometric presentations. We further validated altered gene expression of these specific DMGs contributing to vascular and metabolic diseases (SLC36A1, PTPRN2, CASZ1, IL10), thereby establishing transcriptional effects toward assigning functional significance. Our results suggest that the gene expression and methylation state of the human placenta are related and sensitive to the intrauterine environment, as it affects fetal growth patterns. We speculate that these observed changes may affect risk for offspring in developing adult cardiometabolic disease.

Driver Fusions and Their Implications in the Development and Treatment of Human Cancers.

Gene fusions represent an important class of somatic alterations in cancer. We systematically investigated fusions in 9,624 tumors across 33 cancer types using multiple fusion calling tools. We identified a total of 25,664 fusions, with a 63% validation rate. Integration of gene expression, copy number, and fusion annotation data revealed that fusions involving oncogenes tend to exhibit increased expression, whereas fusions involving tumor suppressors have the opposite effect. For fusions involving kinases, we found 1,275 with an intact kinase domain, the proportion of which varied significantly across cancer types. Our study suggests that fusions drive the development of 16.5% of cancer cases and function as the sole driver in more than 1% of them. Finally, we identified druggable fusions involving genes such as TMPRSS2, RET, FGFR3, ALK, and ESR1 in 6.0% of cases, and we predicted immunogenic peptides, suggesting that fusions may provide leads for targeted drug and immune therapy.

A termite symbiotic mushroom maximizing sexual activity at growing tips of vegetative hyphae.

BACKGROUND: Termitomyces mushrooms are mutualistically associated with fungus-growing termites, which are widely considered to cultivate a monogenotypic Termitomyces symbiont within a colony. Termitomyces cultures isolated directly from termite colonies are heterokaryotic, likely through mating between compatible homokaryons. RESULTS: After pairing homokaryons carrying different haplotypes at marker gene loci MIP and RCB from a Termitomyces fruiting body associated with Odontotermes formosanus, we observed nuclear fusion and division, which greatly resembled meiosis, during each hyphal cell division and conidial formation in the resulting heterokaryons. Surprisingly, nuclei in homokaryons also behaved similarly. To confirm if meiotic-like recombination occurred within mycelia, we constructed whole-genome sequencing libraries from mycelia of two homokaryons and a heterokaryon resulting from mating of the two homokaryons. Obtained reads were aligned to the reference genome of Termitomyces sp. J132 for haplotype reconstruction. After removal of the recombinant haplotypes shared between the heterokaryon and either homokaryons, we inferred that 5.04% of the haplotypes from the heterokaryon were the recombinants resulting from homologous recombination distributed genome-wide. With RNA transcripts of four meiosis-specific genes, including SPO11, DMC1, MSH4, and MLH1, detected from a mycelial sample by real-time quantitative PCR, the nuclear behavior in mycelia was reconfirmed meiotic-like. CONCLUSION: Unlike other basidiomycetes where sex is largely restricted to basidia, Termitomyces maximizes sexuality at somatic stage, resulting in an ever-changing genotype composed of a myriad of coexisting heterogeneous nuclei in a heterokaryon. Somatic meiotic-like recombination may endow Termitomyces with agility to cope with termite consumption by maximized genetic variability.

Morphine for Intravenous Patient-Controlled Analgesia May Inhibit Delirium Tremens: A Case Report and Literature Review.

Alcoholism is common among trauma patients and often lacks the appropriate monitoring. Alcohol withdrawal syndrome (AWS), including delirium tremens (DT), can be associated with significant postoperative morbidity and mortality. However, appropriate acute pain management may protect against delirium; the administration of intravenous patient-controlled analgesia (IV - PCA) may not only alleviate pain, but also reduce the incidence of post-operative delirium. IV-PCA is widely used today; however, little attention has been paid to its influence on the development of AWS or DT post-surgery. Here we present a case in which the administration of IV-PCA may have delayed the onset of DT that interfered with postoperative care and the initiation of psychiatric consultation. The literature was reviewed to determine the potential mechanisms behind the effects of IV-PCA on the onset of AWS or DT.IV-PCA may delay the onset of DT. It is imperative to take into consideration trauma patients' psychiatric history including answers to questions on alcoholism, so that when an IV-PCA is administered and then discontinued, adequate interventions to prevent further morbidity associated with AWS and DT can be initiated in sufficient time.

An Rtf2 Domain-Containing Protein Influences Pre-mRNA Splicing and Is Essential for Embryonic Development in Arabidopsis thaliana.

Alternative splicing is prevalent in plants, but little is known about its regulation in the context of developmental and signaling pathways. We describe here a new factor that influences pre-messengerRNA (mRNA) splicing and is essential for embryonic development in Arabidopsis thaliana. This factor was retrieved in a genetic screen that identified mutants impaired in expression of an alternatively spliced GFP reporter gene. In addition to the known spliceosomal component PRP8, the screen recovered Arabidopsis RTF2 (AtRTF2), a previously uncharacterized, evolutionarily conserved protein containing a replication termination factor 2 (Rtf2) domain. A homozygous null mutation in AtRTF2 is embryo lethal, indicating that AtRTF2 is an essential protein. Quantitative RT-PCR demonstrated that impaired expression of GFP in atrtf2 and prp8 mutants is due to inefficient splicing of the GFP pre-mRNA. A genome-wide analysis using RNA sequencing indicated that 13-16% of total introns are retained to a significant degree in atrtf2 mutants. Considering these results and previous suggestions that Rtf2 represents an ubiquitin-related domain, we discuss the possible role of AtRTF2 in ubiquitin-based regulation of pre-mRNA splicing.

Delayed diagnosis of esophageal perforation following intraoperative transesophageal echocardiography during valvular replacement--a case report.

A 62 year-old man sustained esophageal perforation following intra-operative transesophageal echocardiography (TEE) in a valvular replacement surgery. Septic shock developed on the 12th postoperative day (POD) and the esophageal perforation was diagnosed with chest CT. Emergent operation together with intensive care saved the patient's life. We speculate that the mechanism of perforation was not due to manipulation of the probe, but rather due to ischemia of the esophagus resulting from the combination of probe compression, non-pulsatile flow and the distension of the atria during a lengthy procedure. It is advisable that in patients with operative risk factors, such as distension of atria, long cardiac procedure and likely ischemia of organs due to cardiopulmonary bypass, the monitoring probe of TEE should not constantly rest in the esophagus and be withdrawn when it is idle or not in actual use. In addition, if resistance has been met during the intraoperative manipulation of the probe in a patient without previous history of esophageal disease, perforation might suspected if he or she sustains postoperative fever with positive chest X-ray findings.

General anesthesia for patient with homocystinuria--a case report.

Homocystinuria is an autosomal recessive disease with multiple systemic disorders. Here we report a 15-year-old lad suffering from homocystinuria who required an ocular surgery including lentectomy and implant of plastic lens, OS and anterior retinal cryotherapy, OD under general anesthesia because of lens subluxation and lattice degeneration. It is the elective ocular procedure most commonly performed for homocystinuric children. Proper precautions should be taken during anesthetic management since this condition inspires some particular anesthetic complications that could be prevented by careful consideration and understanding of its pathophysiology. Providentially our patient stood the operation well and was discharged without subsequent thromboembolism or other complication as an aftermath.

A comprehensive resource of genomic, epigenomic and transcriptomic sequencing data for the black truffle Tuber melanosporum.

BACKGROUND: Tuber melanosporum, also known in the gastronomic community as "truffle", features one of the largest fungal genomes (125 Mb) with an exceptionally high transposable element (TE) and repetitive DNA content (>58%). The main purpose of DNA methylation in fungi is TE silencing. As obligate outcrossing organisms, truffles are bound to a sexual mode of propagation, which together with TEs is thought to represent a major force driving the evolution of DNA methylation. Thus, it was of interest to examine if and how T. melanosporum exploits DNA methylation to maintain genome integrity. FINDINGS: We performed whole-genome DNA bisulfite sequencing and mRNA sequencing on different developmental stages of T. melanosporum; namely, fruitbody ("truffle"), free-living mycelium and ectomycorrhiza. The data revealed a high rate of cytosine methylation (>44%), selectively targeting TEs rather than genes with a strong preference for CpG sites. Whole genome DNA sequencing uncovered multiple TE-enriched, copy number variant regions bearing a significant fraction of hypomethylated and expressed TEs, almost exclusively in free-living mycelium propagated in vitro. Treatment of mycelia with 5-azacytidine partially reduced DNA methylation and increased TE transcription. Our transcriptome assembly also resulted in the identification of a set of novel transcripts from 614 genes. CONCLUSIONS: The datasets presented here provide valuable and comprehensive (epi)genomic information that can be of interest for evolutionary genomics studies of multicellular (filamentous) fungi, in particular Ascomycetes belonging to the subphylum, Pezizomycotina. Evidence derived from comparative methylome and transcriptome analyses indicates that a non-exhaustive and partly reversible methylation process operates in truffles.

Coadministration of glycogen-synthase kinase 3 inhibitor with morphine attenuates chronic morphine-induced analgesic tolerance and withdrawal syndrome.

BACKGROUND: Glycogen-synthase kinase 3 (GSK3) is involved in many signaling pathways and is associated with a host of high-profile pathophysiological states. However, its role in morphine tolerance, especially naloxone-precipitated withdrawal syndrome, has not been well investigated. The present study was undertaken to study the role of GSK3 in chronic morphine exposure. METHODS: Adult male Sprague-Dawley rats were subjected to intraperitoneal (i.p.) injections of morphine (10 mg/kg) twice daily for 6 consecutive days, and tail-flick tests were conducted to evaluate changes of morphine-induced antinociception. GSK3 inhibitor, SB216763 or SB415286, was i.p. injected prior to morphine to investigate the influences on morphine tolerance. There were four groups receiving morphine plus vehicle (2% dimethyl sulfoxide), morphine plus SB216763 (0.6 mg/kg) or SB415286 (1.0 mg/kg), GSK3 inhibitor alone, or dimethyl sulfoxide: as the control group. On Day 7, naloxone (i.p., 1 mg/kg) was administered and naloxone-precipitated withdrawal behaviors were individually compared between groups. RESULTS: Repeated morphine exposure in this study led to progressive shortening of tail-flick latencies and produced six of nine observed naloxone-precipitated withdrawal behaviors. Coadministration with SB216763 or SB415286 significantly prevented antinociceptive tolerance and alleviated parts of withdrawal syndrome. Both inhibitors could similarly reverse withdrawal behaviors including grooming, chewing, and ptosis, but did not affect withdrawal behaviors of penis licking and defecation. CONCLUSION: The results demonstrate the importance of GSK3 in reducing chronic morphine-induced tolerance and withdrawal syndrome. Although GSK3 is involved in diverse physiological functions, aiming at GSK3-related pathway could still be a potential tool to improve therapeutic quality in clinical morphine treatment.

The effect of cerebral monitoring on recovery after sevoflurane anesthesia in ambulatory setting in children: a comparison among bispectral index, A-line autoregressive index, and standard practice.

BACKGROUND: The bispectral index (BIS) and A-line autoregressive index (AAI) are electroencephalogram-derived monitoring indices of anesthesia. This study evaluated the efficacy of BIS- and AAI-guided sevoflurane anesthesia in children receiving ambulatory urologic surgeries. METHODS: One hundred sixty children (aged 3-12 years) undergoing ambulatory urologic surgery were randomized to receive sevoflurane anesthesia controlled either solely by clinical parameters (standard practice group), BIS-guided group within the BIS range of 40-60 (BIS group), or AAI-guided group within the AAI range of 15-30 (AAI group). The primary outcome was the recovery time, and the secondary outcome was the quality of recovery, including the incidence of emergency delirium measured by Pediatric Anesthesia Emergence Delirium score, incidence of postoperative nausea and vomiting, and parental satisfaction. RESULTS: Compared with the standard practice group, patients with BIS or AAI monitoring had shortened recovery time and consumed less sevoflurane. There were no significant differences in the incidences of emergence delirium, postoperative nausea and vomiting, or parental satisfaction among the three groups. CONCLUSION: BIS- and AAI- guided titration sevoflurane anesthesia could result in shortened recovery and reduced sevoflurane concentration and consumption without affecting the quality of recovery in children receiving ambulatory urologic surgery. The beneficial effects of AAI- and BIS-guided anesthesia in pediatric ambulatory surgeries are similar.

Myoclonic seizure in the postanesthesia care unit after thoracic laminectomy.

IMPLICATIONS: We report a patient who developed myoclonic seizure in the postanesthesia care unit after thoracic laminectomy. Expeditious diagnostic evaluation of unrecognized dura tear during surgery must be instituted immediately to avoid untoward sequelae. Specific treatment in addition to supportive care is required if the diagnosis is to be clearly identified.