Three-dimensional genome architecture persists in a 52,000-year-old woolly mammoth skin sample.

Analyses of ancient DNA typically involve sequencing the surviving short oligonucleotides and aligning to genome assemblies from related, modern species. Here, we report that skin from a female woolly mammoth (†Mammuthus primigenius) that died 52,000 years ago retained its ancient genome architecture. We use PaleoHi-C to map chromatin contacts and assemble its genome, yielding 28 chromosome-length scaffolds. Chromosome territories, compartments, loops, Barr bodies, and inactive X chromosome (Xi) superdomains persist. The active and inactive genome compartments in mammoth skin more closely resemble Asian elephant skin than other elephant tissues. Our analyses uncover new biology. Differences in compartmentalization reveal genes whose transcription was potentially altered in mammoths vs. elephants. Mammoth Xi has a tetradic architecture, not bipartite like human and mouse. We hypothesize that, shortly after this mammoth's death, the sample spontaneously freeze-dried in the Siberian cold, leading to a glass transition that preserved subfossils of ancient chromosomes at nanometer scale.

The Energetics and Physiological Impact of Cohesin Extrusion.

Cohesin extrusion is thought to play a central role in establishing the architecture of mammalian genomes. However, extrusion has not been visualized in vivo, and thus, its functional impact and energetics are unknown. Using ultra-deep Hi-C, we show that loop domains form by a process that requires cohesin ATPases. Once formed, however, loops and compartments are maintained for hours without energy input. Strikingly, without ATP, we observe the emergence of hundreds of CTCF-independent loops that link regulatory DNA. We also identify architectural "stripes," where a loop anchor interacts with entire domains at high frequency. Stripes often tether super-enhancers to cognate promoters, and in B cells, they facilitate Igh transcription and recombination. Stripe anchors represent major hotspots for topoisomerase-mediated lesions, which promote chromosomal translocations and cancer. In plasmacytomas, stripes can deregulate Igh-translocated oncogenes. We propose that higher organisms have coopted cohesin extrusion to enhance transcription and recombination, with implications for tumor development.

Cohesin Loss Eliminates All Loop Domains.

The human genome folds to create thousands of intervals, called "contact domains," that exhibit enhanced contact frequency within themselves. "Loop domains" form because of tethering between two loci-almost always bound by CTCF and cohesin-lying on the same chromosome. "Compartment domains" form when genomic intervals with similar histone marks co-segregate. Here, we explore the effects of degrading cohesin. All loop domains are eliminated, but neither compartment domains nor histone marks are affected. Loss of loop domains does not lead to widespread ectopic gene activation but does affect a significant minority of active genes. In particular, cohesin loss causes superenhancers to co-localize, forming hundreds of links within and across chromosomes and affecting the regulation of nearby genes. We then restore cohesin and monitor the re-formation of each loop. Although re-formation rates vary greatly, many megabase-sized loops recovered in under an hour, consistent with a model where loop extrusion is rapid.

Large DNA Methylation Nadirs Anchor Chromatin Loops Maintaining Hematopoietic Stem Cell Identity.

Higher-order chromatin structure and DNA methylation are implicated in multiple developmental processes, but their relationship to cell state is unknown. Here, we find that large (>7.3 kb) DNA methylation nadirs (termed "grand canyons") can form long loops connecting anchor loci that may be dozens of megabases (Mb) apart, as well as inter-chromosomal links. The interacting loci cover a total of ∼3.5 Mb of the human genome. The strongest interactions are associated with repressive marks made by the Polycomb complex and are diminished upon EZH2 inhibitor treatment. The data are suggestive of the formation of these loops by interactions between repressive elements in the loci, forming a genomic subcompartment, rather than by cohesion/CTCF-mediated extrusion. Interestingly, unlike previously characterized subcompartments, these interactions are present only in particular cell types, such as stem and progenitor cells. Our work reveals that H3K27me3-marked large DNA methylation grand canyons represent a set of very-long-range loops associated with cellular identity.

The fundamental role of chromatin loop extrusion in physiological V(D)J recombination.

The RAG endonuclease initiates Igh V(D)J assembly in B cell progenitors by joining D segments to JH segments, before joining upstream VH segments to DJH intermediates1. In mouse progenitor B cells, the CTCF-binding element (CBE)-anchored chromatin loop domain2 at the 3' end of Igh contains an internal subdomain that spans the 5' CBE anchor (IGCR1)3, the DH segments, and a RAG-bound recombination centre (RC)4. The RC comprises the JH-proximal D segment (DQ52), four JH segments, and the intronic enhancer (iEµ)5. Robust RAG-mediated cleavage is restricted to paired V(D)J segments flanked by complementary recombination signal sequences (12RSS and 23RSS)6. D segments are flanked downstream and upstream by 12RSSs that mediate deletional joining with convergently oriented JH-23RSSs and VH-23RSSs, respectively6. Despite 12/23 compatibility, inversional D-to-JH joining via upstream D-12RSSs is rare7,8. Plasmid-based assays have attributed the lack of inversional D-to-JH joining to sequence-based preference for downstream D-12RSSs9, as opposed to putative linear scanning mechanisms10,11. As RAG linearly scans convergent CBE-anchored chromatin loops4,12-14, potentially formed by cohesin-mediated loop extrusion15-18, we revisited its scanning role. Here we show that the chromosomal orientation of JH-23RSS programs RC-bound RAG to linearly scan upstream chromatin in the 3' Igh subdomain for convergently oriented D-12RSSs and, thereby, to mediate deletional joining of all D segments except RC-based DQ52, which joins by a diffusion-related mechanism. In a DQ52-based RC, formed in the absence of JH segments, RAG bound by the downstream DQ52-RSS scans the downstream constant region exon-containing 3' Igh subdomain, in which scanning can be impeded by targeted binding of nuclease-dead Cas9, by transcription through repetitive Igh switch sequences, and by the 3' Igh CBE-based loop anchor. Each scanning impediment focally increases RAG activity on potential substrate sequences within the impeded region. High-resolution mapping of chromatin interactions in the RC reveals that such focal RAG targeting is associated with corresponding impediments to the loop extrusion process that drives chromatin past RC-bound RAG.

Hybrid de novo genome assembly and centromere characterization of the gray mouse lemur (Microcebus murinus).

BACKGROUND: The de novo assembly of repeat-rich mammalian genomes using only high-throughput short read sequencing data typically results in highly fragmented genome assemblies that limit downstream applications. Here, we present an iterative approach to hybrid de novo genome assembly that incorporates datasets stemming from multiple genomic technologies and methods. We used this approach to improve the gray mouse lemur (Microcebus murinus) genome from early draft status to a near chromosome-scale assembly. METHODS: We used a combination of advanced genomic technologies to iteratively resolve conflicts and super-scaffold the M. murinus genome. RESULTS: We improved the M. murinus genome assembly to a scaffold N50 of 93.32 Mb. Whole genome alignments between our primary super-scaffolds and 23 human chromosomes revealed patterns that are congruent with historical comparative cytogenetic data, thus demonstrating the accuracy of our de novo scaffolding approach and allowing assignment of scaffolds to M. murinus chromosomes. Moreover, we utilized our independent datasets to discover and characterize sequences associated with centromeres across the mouse lemur genome. Quality assessment of the final assembly found 96% of mouse lemur canonical transcripts nearly complete, comparable to other published high-quality reference genome assemblies. CONCLUSIONS: We describe a new assembly of the gray mouse lemur (Microcebus murinus) genome with chromosome-scale scaffolds produced using a hybrid bioinformatic and sequencing approach. The approach is cost effective and produces superior results based on metrics of contiguity and completeness. Our results show that emerging genomic technologies can be used in combination to characterize centromeres of non-model species and to produce accurate de novo chromosome-scale genome assemblies of complex mammalian genomes.

An encyclopedia of enhancer-gene regulatory interactions in the human genome.

Identifying transcriptional enhancers and their target genes is essential for understanding gene regulation and the impact of human genetic variation on disease1-6. Here we create and evaluate a resource of >13 million enhancer-gene regulatory interactions across 352 cell types and tissues, by integrating predictive models, measurements of chromatin state and 3D contacts, and largescale genetic perturbations generated by the ENCODE Consortium7. We first create a systematic benchmarking pipeline to compare predictive models, assembling a dataset of 10,411 elementgene pairs measured in CRISPR perturbation experiments, >30,000 fine-mapped eQTLs, and 569 fine-mapped GWAS variants linked to a likely causal gene. Using this framework, we develop a new predictive model, ENCODE-rE2G, that achieves state-of-the-art performance across multiple prediction tasks, demonstrating a strategy involving iterative perturbations and supervised machine learning to build increasingly accurate predictive models of enhancer regulation. Using the ENCODE-rE2G model, we build an encyclopedia of enhancer-gene regulatory interactions in the human genome, which reveals global properties of enhancer networks, identifies differences in the functions of genes that have more or less complex regulatory landscapes, and improves analyses to link noncoding variants to target genes and cell types for common, complex diseases. By interpreting the model, we find evidence that, beyond enhancer activity and 3D enhancer-promoter contacts, additional features guide enhancerpromoter communication including promoter class and enhancer-enhancer synergy. Altogether, these genome-wide maps of enhancer-gene regulatory interactions, benchmarking software, predictive models, and insights about enhancer function provide a valuable resource for future studies of gene regulation and human genetics.

Applying a Mixed-Method Approach to Improve On-the-Job Learning and Job Satisfaction in a Cohort of Interns at a University Hospital.

INTRODUCTION: Job satisfaction is vital for the optimal functioning of medical practitioners. Herein, we report our experience of restructuring the internship program by identifying the gaps, developing, implementing strategies to overcome gaps and sharing the results of the pre-implementation and post-implementation audit, as an example for establishing a system for improving intern's work-based learning and satisfaction in a university hospital setting. METHODS: Using Kern's six-step instructional model, a prospective mixed-method study was conducted at Aga Khan University Hospital. In phase 1 (2013) gaps were identified by evaluating various aspects of the internship program. Strategies were developed and implemented to overcome the identified gaps. In phase 2 (2014-2016) the impact of these developmental strategies was assessed. RESULTS: A total of 65 interns, 30 residents, and 22 faculty members participated in phase I, while 71 interns participated in phase II. The reformation of orientation sessions, including practical exposure and content of sessions, opportunities to enhance hands-on experience and supervision in inpatient areas, operating rooms, supervision by fellows, supervision for hands-on procedures, career counseling, and mentorship, led to significant improvement in satisfaction. It was identified that the lack of hands-on opportunities can be overcome by surgical skills-based workshops. These reforms led to an overall rise in intern satisfaction (50% vs 75.4%, p=0.02). CONCLUSION: Periodic restructuring of an existing program helps to improve the work-based learning experience and overall satisfaction among interns. This not only maximizes learning but also eases interns into their postgraduate life and workload subsequently enabling them to become more competent and well-rounded health practitioners.

MCPH1 inhibits Condensin II during interphase by regulating its SMC2-Kleisin interface.

Dramatic change in chromosomal DNA morphology between interphase and mitosis is a defining features of the eukaryotic cell cycle. Two types of enzymes, namely cohesin and condensin confer the topology of chromosomal DNA by extruding DNA loops. While condensin normally configures chromosomes exclusively during mitosis, cohesin does so during interphase. The processivity of cohesin's loop extrusion during interphase is limited by a regulatory factor called WAPL, which induces cohesin to dissociate from chromosomes via a mechanism that requires dissociation of its kleisin from the neck of SMC3. We show here that a related mechanism may be responsible for blocking condensin II from acting during interphase. Cells derived from patients affected by microcephaly caused by mutations in the MCPH1 gene undergo premature chromosome condensation. We show that deletion of Mcph1 in mouse embryonic stem cells unleashes an activity of condensin II that triggers formation of compact chromosomes in G1 and G2 phases, accompanied by enhanced mixing of A and B chromatin compartments, and this occurs even in the absence of CDK1 activity. Crucially, inhibition of condensin II by MCPH1 depends on the binding of a short linear motif within MCPH1 to condensin II's NCAPG2 subunit. MCPH1's ability to block condensin II's association with chromatin is abrogated by the fusion of SMC2 with NCAPH2, hence may work by a mechanism similar to cohesin. Remarkably, in the absence of both WAPL and MCPH1, cohesin and condensin II transform chromosomal DNAs of G2 cells into chromosomes with a solenoidal axis.

Molecular and radiological characterization of glioblastoma multiforme using magnetic resonance imaging.

BACKGROUND: Glioblastoma multiforme (GBM) is the most malignant, aggressive and common form of primary brain cancer. Currently, GBM is considered to be a homogenous mass as all its margins are treated equally at the time of resection. However, it is not known whether radiologically distinct regions of GBM are also distinct at molecular level. We conducted this study to see if radiologically distinct regions were also different at the molecular level. METHODS: In 20 patients, MRI derived variance known as Apparent Diffusion Coefficient (ADC) was plotted against Contrast Enhancement (CE). Four radiologically distinct regions were identified: 1) high ADC and low CE; 2) low ADC and low CE; 3) high ADC and high CE; and 4) low ADC and high CE. Biopsy samples were collected from these four regions of interest in each patient and immunohistochemistry was conducted to characterize cellular features and identify oncogene and stem cell marker expressing cells. RESULTS: Markedly increased nuclear pleomorphism, cellularity and necrosis were seen in region 2. Oncogene IDH was expressed in all regions, however, it was highest in region 4. Stem cell marker, CD44 expression was highest in region 1 and lowest in region 2 and 3. The expression of CD133 was highest in region 3. CONCLUSIONS: This study shows that ADC/CE plot can divide GBM into four regions, whose heterogeneity is evidenced by differential expression of nuclear pleomorphism, necrosis, cellularity and mitotic rate as well as the expression of oncogene and stem cell markers.

Low cost quality initiatives for management of neurosurgical patients in developing nations: Perspective from a tertiary care centre in Pakistan.

BACKGROUND: Targeting resources on cost-effective care strategies is pertinent for developing nations, specifically for already burdened specialties such as neurosurgery, where without state support and insurance companies, expenses are borne by the patient themselves. METHODS: This was a descriptive review of literary work published by the section of neurosurgery at Aga Khan University Hospital, Pakistan. We searched PubMed and CiNAHL databases to identify articles, which were then critically analyzed and discussed from a perspective of low-cost quality care. RESULTS: We demonstrate the cost effectiveness of three initiatives, namely, blood ordering protocol for elective spine arthrodesis, nonoperative management being superior to surgical stabilization in spine injury patients with complete neurological deficits, and early tracheostomy in patients with isolated severe traumatic brain injury. CONCLUSION: Initiation and implementation of such cost-effective strategies without compromising quality health standards must be emphasized by neurosurgical centers throughout the developing world for smart allocation and utilization of funds.

Perioperative steroids for lumbar disc surgery: A meta-analysis of randomized controlled trials.

BACKGROUND: Our review question was "Does perioperative steroids administration, in comparison with other treatments or placebo, improve either postoperative pain control, length of hospital stay, or return to work in patients undergoing lumbar disc surgery?" METHODS: We searched PubMed, CINAHL PLUS, and Cochrane databases for randomized control trials (RCTs) studying the role of steroids for lumbar disc surgery. Studies that compared perioperative steroids with other treatments or placebo were included. Study outcomes included postoperative back pain, leg pain, length of hospital stay, and return to work. Data was extracted through a proforma. Means and mean differences were calculated for continuous data, whereas odds ratios were calculated for dichotomous data. Data were analyzed with the help of Rev Man 5. RESULTS: Twenty RCTs were included in the review. Quantitative analysis could be performed on 19 RCTs. Intraoperative steroids improve control of back pain at 24-48 hours. Although there was some benefit of steroid administration in controlling postoperative leg pain, it disappeared at 1 year and in the overall pooled analysis. The length of hospital stay was much shorter in the steroid group. The frequency of adverse events and complications also favored steroid administration. CONCLUSION: Intraoperative epidural steroid administration offers some benefit in pain control with a significant reduction in the length of hospital stay. However, there is insufficient evidence to support the routine use of oral and intravenous steroids in the perioperative period.

De novo assembly of the Aedes aegypti genome using Hi-C yields chromosome-length scaffolds.

The Zika outbreak, spread by the Aedes aegypti mosquito, highlights the need to create high-quality assemblies of large genomes in a rapid and cost-effective way. Here we combine Hi-C data with existing draft assemblies to generate chromosome-length scaffolds. We validate this method by assembling a human genome, de novo, from short reads alone (67× coverage). We then combine our method with draft sequences to create genome assemblies of the mosquito disease vectors Aeaegypti and Culex quinquefasciatus, each consisting of three scaffolds corresponding to the three chromosomes in each species. These assemblies indicate that almost all genomic rearrangements among these species occur within, rather than between, chromosome arms. The genome assembly procedure we describe is fast, inexpensive, and accurate, and can be applied to many species.

Portfolios in Saudi medical colleges. Why and how?

Over recent decades, the use of portfolios in medical education has evolved, and is being applied in undergraduate and postgraduate programs worldwide. Portfolios, as a learning process and method of documenting and assessing learning, is supported as a valuable tool by adult learning theories that stress the need for learners to be self-directed and to engage in experiential learning. Thoughtfully implemented, a portfolio provides learning experiences unequaled by any single learning tool. The credibility (validity) and dependability (reliability) of assessment through portfolios have been questioned owing to its subjective nature; however, methods to safeguard these features have been described in the literature. This paper discusses some of this literature, with particular attention to the role of portfolios in relation to self-reflective learning, provides an overview of current use of portfolios in undergraduate medical education in Saudi Arabia, and proposes research-based guidelines for its implementation and other similar contexts.

Juicer Provides a One-Click System for Analyzing Loop-Resolution Hi-C Experiments.

Hi-C experiments explore the 3D structure of the genome, generating terabases of data to create high-resolution contact maps. Here, we introduce Juicer, an open-source tool for analyzing terabase-scale Hi-C datasets. Juicer allows users without a computational background to transform raw sequence data into normalized contact maps with one click. Juicer produces a hic file containing compressed contact matrices at many resolutions, facilitating visualization and analysis at multiple scales. Structural features, such as loops and domains, are automatically annotated. Juicer is available as open source software at http://aidenlab.org/juicer/.

Juicebox Provides a Visualization System for Hi-C Contact Maps with Unlimited Zoom.

Hi-C experiments study how genomes fold in 3D, generating contact maps containing features as small as 20 bp and as large as 200 Mb. Here we introduce Juicebox, a tool for exploring Hi-C and other contact map data. Juicebox allows users to zoom in and out of Hi-C maps interactively, just as a user of Google Earth might zoom in and out of a geographic map. Maps can be compared to one another, or to 1D tracks or 2D feature sets.

Factors affecting ventriculoperitoneal shunt survival in adult patients.

BACKGROUND: Ventriculoperitoneal (VP) shunt insertion remains the mainstay of treatment for hydrocephalus despite a high rate of complications. The predictors of shunt malfunction have been studied mostly in pediatric patients. In this study, we report our 11-year experience with VP shunts in adult patients with hydrocephalus. We also assess the various factors affecting shunt survival in a developing country setting. METHODS: A retrospective chart analysis was conducted for all adult patients who had undergone shunt placement between the years 2001 and 2011. Kaplan-Meier curves were used to determine the duration from shunt placement to first malfunction and log-rank (Cox-Mantel) tests were used to determine the factors affecting shunt survival. RESULTS: A total of 227 patients aged 18-85 years (mean: 45.8 years) were included in the study. The top four etiologies of hydrocephalus included post-cranial surgery (23.3%), brain tumor or cyst (22.9%), normal pressure hydrocephalus (15%), and intracranial hemorrhage (13.7%). The overall incidence of shunt malfunction was 15.4% with the median time to first shunt failure being 120 days. Etiology of hydrocephalus (P = 0.030) had a significant association with the development of shunt malfunction. Early shunt failure was associated with age (P < 0.001), duration of hospital stay (P < 0.001), Glasgow Coma Scale (GCS) score less than 13 (P = 0.010), excision of brain tumors (P = 0.008), and placement of extra-ventricular drains (P = 0.033). CONCLUSIONS: Patients with increased age, prolonged hospital stay, GCS score of less than 13, extra-ventricular drains in situ, or excision of brain tumors were more likely to experience early shunt malfunction.