Publications by authors named "Martin Walsh"

140 Publications

An automatic pipeline for the design of irreversible derivatives identifies a potent SARS-CoV-2 M inhibitor.

Cell Chem Biol 2021 Jun 22. Epub 2021 Jun 22.

Department of Chemical and Structural Biology, Weizmann Institute of Science, 7610001 Rehovot, Israel. Electronic address:

Designing covalent inhibitors is increasingly important, although it remains challenging. Here, we present covalentizer, a computational pipeline for identifying irreversible inhibitors based on structures of targets with non-covalent binders. Through covalent docking of tailored focused libraries, we identify candidates that can bind covalently to a nearby cysteine while preserving the interactions of the original molecule. We found ∼11,000 cysteines proximal to a ligand across 8,386 complexes in the PDB. Of these, the protocol identified 1,553 structures with covalent predictions. In a prospective evaluation, five out of nine predicted covalent kinase inhibitors showed half-maximal inhibitory concentration (IC) values between 155 nM and 4.5 μM. Application against an existing SARS-CoV M reversible inhibitor led to an acrylamide inhibitor series with low micromolar IC values against SARS-CoV-2 M. The docking was validated by 12 co-crystal structures. Together these examples hint at the vast number of covalent inhibitors accessible through our protocol.
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http://dx.doi.org/10.1016/j.chembiol.2021.05.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8228784PMC
June 2021

Bispecific repurposed medicines targeting the viral and immunological arms of COVID-19.

Sci Rep 2021 06 24;11(1):13208. Epub 2021 Jun 24.

Diamond Light Source Ltd., Harwell Science and Innovation Campus, Didcot, OX11 0DE, UK.

Effective agents to treat coronavirus infection are urgently required, not only to treat COVID-19, but to prepare for future outbreaks. Repurposed anti-virals such as remdesivir and human anti-inflammatories such as barcitinib have received emergency approval but their overall benefits remain unclear. Vaccines are the most promising prospect for COVID-19, but will need to be redeveloped for any future coronavirus outbreak. Protecting against future outbreaks requires the identification of targets that are conserved between coronavirus strains and amenable to drug discovery. Two such targets are the main protease (M) and the papain-like protease (PL) which are essential for the coronavirus replication cycle. We describe the discovery of two non-antiviral therapeutic agents, the caspase-1 inhibitor SDZ 224015 and Tarloxotinib that target M and PL, respectively. These were identified through extensive experimental screens of the drug repurposing ReFRAME library of 12,000 therapeutic agents. The caspase-1 inhibitor SDZ 224015, was found to be a potent irreversible inhibitor of M (IC 30 nM) while Tarloxotinib, a clinical stage epidermal growth factor receptor inhibitor, is a sub micromolar inhibitor of PL (IC 300 nM, K 200 nM) and is the first reported PL inhibitor with drug-like properties. SDZ 224015 and Tarloxotinib have both undergone safety evaluation in humans and hence are candidates for COVID-19 clinical evaluation.
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http://dx.doi.org/10.1038/s41598-021-92416-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8225628PMC
June 2021

The 2019 practice analysis of hand therapy and the use of orthoses by certified hand therapists.

J Hand Ther 2021 Apr 15. Epub 2021 Apr 15.

Hand Therapy Certification Commission, Sacramento, CA. Electronic address:

Background: In 2019, the Hand Therapy Certification Commission (HTCC), in consultation with Scantron Corporation, performed a practice analysis study of hand therapy, the sixth in a series of similar studies performed byHTCC over a 35-year period.

Purpose: The primary goal of this study was to update and validate the definition and delineation of hand therapy and to ensure that the test content outline for the Hand Therapy Certification Examination (HTCE) reflects the critical tasks, knowledge, and skills required in the practice of hand therapy. Additionally, HTCC explored specific trends in hand therapy practice, compared findings with previous studies, and gathered data about the frequency, criticality, and performance expectations for the use and fabrication of orthoses by hand therapists.

Study Design: Quantitative Descriptive.

Methods: More than 40 subject matter experts from the United States and Canada, representing a broad range of experiences and perspectives, developed an updated delineation of the domains, tasks, knowledge, and techniques and tools used in hand therapy practice. A large-scale online survey of all certified hand therapists from the United States, Canada, and other countries was completed to test the profile within the practice of hand therapy.

Results: This large-scale online survey overwhelmingly validated the profile of hand therapy. The results affirmed the test specifications for the Hand Therapy Certification Examination; affirmed the definition of hand therapy; and refined the scope of hand therapy practice. New data was gathered regarding the use of orthotics in hand therapy.

Conclusions: This study establishes content validity for the HTCE. It highlights that the specialty of hand therapy is a mature and stable specialty field of occupational therapy and physical therapy. Certified Hand Therapists frequently issue pre-fabricated and fabricate custom orthoses in the course of rehabilitation for clients with hand and arm injuries, and overall consider this a highly critical task in hand therapy practice.

Level Of Evidence: N/A.
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http://dx.doi.org/10.1016/j.jht.2021.04.008DOI Listing
April 2021

CRISPRi screens reveal a DNA methylation-mediated 3D genome dependent causal mechanism in prostate cancer.

Nat Commun 2021 03 19;12(1):1781. Epub 2021 Mar 19.

Changhai Hospital, Shanghai, China.

Prostate cancer (PCa) risk-associated SNPs are enriched in noncoding cis-regulatory elements (rCREs), yet their modi operandi and clinical impact remain elusive. Here, we perform CRISPRi screens of 260 rCREs in PCa cell lines. We find that rCREs harboring high risk SNPs are more essential for cell proliferation and H3K27ac occupancy is a strong indicator of essentiality. We also show that cell-line-specific essential rCREs are enriched in the 8q24.21 region, with the rs11986220-containing rCRE regulating MYC and PVT1 expression, cell proliferation and tumorigenesis in a cell-line-specific manner, depending on DNA methylation-orchestrated occupancy of a CTCF binding site in between this rCRE and the MYC promoter. We demonstrate that CTCF deposition at this site as measured by DNA methylation level is highly variable in prostate specimens, and observe the MYC eQTL in the 8q24.21 locus in individuals with low CTCF binding. Together our findings highlight a causal mechanism synergistically driven by a risk SNP and DNA methylation-mediated 3D genome architecture, advocating for the integration of genetics and epigenetics in assessing risks conferred by genetic predispositions.
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http://dx.doi.org/10.1038/s41467-021-21867-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7979745PMC
March 2021

Mass spectrometry reveals potential of β-lactams as SARS-CoV-2 M inhibitors.

Chem Commun (Camb) 2021 Feb;57(12):1430-1433

Chemistry Research Laboratory, Department of Chemistry, 12 Mansfield Road, Oxford, OX1 3TA, UK.

The main viral protease (Mpro) of SARS-CoV-2 is a nucleophilic cysteine hydrolase and a current target for anti-viral chemotherapy. We describe a high-throughput solid phase extraction coupled to mass spectrometry Mpro assay. The results reveal some β-lactams, including penicillin esters, are active site reacting Mpro inhibitors, thus highlighting the potential of acylating agents for Mpro inhibition.
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http://dx.doi.org/10.1039/d0cc06870eDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8006714PMC
February 2021

Diamond Light Source: contributions to SARS-CoV-2 biology and therapeutics.

Biochem Biophys Res Commun 2021 01 19;538:40-46. Epub 2020 Nov 19.

Diamond Light Source Ltd., Harwell Science and Innovation Campus, Didcot, OX11 0DE, UK; Division of Structural Biology, The Nuffield Department of Medicine, University of Oxford, Headington, Oxford, OX3 7BN, UK; Instruct-ERIC, Oxford House, Parkway Court, John Smith Drive, Oxford, OX4 2JY, UK. Electronic address:

The impact of COVID-19 on public health and the global economy has led to an unprecedented research response, with a major emphasis on the development of safe vaccines and drugs. However, effective, safe treatments typically take over a decade to develop and there are still no clinically approved therapies to treat highly pathogenic coronaviruses. Repurposing of known drugs can speed up development and this strategy, along with the use of biologicals (notably monoclonal antibody therapy) and vaccine development programmes remain the principal routes to dealing with the immediate impact of COVID-19. Nevertheless, the development of broadly-effective highly potent antivirals should be a major longer term goal. Structural biology has been applied with enormous effect, with key proteins structurally characterised only weeks after the SARS-CoV-2 sequence was released. Open-access to advanced infrastructure for structural biology techniques at synchrotrons and high-end cryo-EM and NMR centres has brought these technologies centre-stage in drug discovery. We summarise the role of Diamond Light Source in responses to the pandemic and note the impact of the immediate release of results in fuelling an open-science approach to early-stage drug discovery.
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http://dx.doi.org/10.1016/j.bbrc.2020.11.041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7676326PMC
January 2021

Super-Resolution Fluorescence Microscopy Reveals Clustering Behaviour of Major Outer Membrane Protein.

Biology (Basel) 2020 Oct 20;9(10). Epub 2020 Oct 20.

School of Biological Sciences, University of Reading, Berkshire RG6 6AS, UK.

is a Gram-negative bacterium responsible for a number of human respiratory diseases and linked to some chronic inflammatory diseases. The major outer membrane protein (MOMP) of is a conserved immunologically dominant protein located in the outer membrane, which, together with its surface exposure and abundance, has led to MOMP being the main focus for vaccine and antimicrobial studies in recent decades. MOMP has a major role in the chlamydial outer membrane complex through the formation of intermolecular disulphide bonds, although the exact interactions formed are currently unknown. Here, it is proposed that due to the large number of cysteines available for disulphide bonding, interactions occur between cysteine-rich pockets as opposed to individual residues. Such pockets were identified using a MOMP homology model with a supporting low-resolution (~4 Å) crystal structure. The localisation of MOMP in the membrane was assessed using direct stochastic optical reconstruction microscopy (dSTORM), which showed a decrease in membrane clustering with cysteine-rich regions containing two mutations. These results indicate that disulphide bond formation was not disrupted by single mutants located in the cysteine-dense regions and was instead compensated by neighbouring cysteines within the pocket in support of this cysteine-rich pocket hypothesis.
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http://dx.doi.org/10.3390/biology9100344DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7589890PMC
October 2020

Crystallographic and electrophilic fragment screening of the SARS-CoV-2 main protease.

Nat Commun 2020 10 7;11(1):5047. Epub 2020 Oct 7.

Cancer Research UK Drug Discovery Unit, Newcastle University Centre for Cancer, Chemistry, School of Natural and Environmental Sciences, Bedson Building, Newcastle University, Newcastle upon Tyne, NE1 7RU, UK.

COVID-19, caused by SARS-CoV-2, lacks effective therapeutics. Additionally, no antiviral drugs or vaccines were developed against the closely related coronavirus, SARS-CoV-1 or MERS-CoV, despite previous zoonotic outbreaks. To identify starting points for such therapeutics, we performed a large-scale screen of electrophile and non-covalent fragments through a combined mass spectrometry and X-ray approach against the SARS-CoV-2 main protease, one of two cysteine viral proteases essential for viral replication. Our crystallographic screen identified 71 hits that span the entire active site, as well as 3 hits at the dimer interface. These structures reveal routes to rapidly develop more potent inhibitors through merging of covalent and non-covalent fragment hits; one series of low-reactivity, tractable covalent fragments were progressed to discover improved binders. These combined hits offer unprecedented structural and reactivity information for on-going structure-based drug design against SARS-CoV-2 main protease.
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http://dx.doi.org/10.1038/s41467-020-18709-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7542442PMC
October 2020

Molecular Transducers of Physical Activity Consortium (MoTrPAC): Mapping the Dynamic Responses to Exercise.

Cell 2020 06;181(7):1464-1474

Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA. Electronic address:

Exercise provides a robust physiological stimulus that evokes cross-talk among multiple tissues that when repeated regularly (i.e., training) improves physiological capacity, benefits numerous organ systems, and decreases the risk for premature mortality. However, a gap remains in identifying the detailed molecular signals induced by exercise that benefits health and prevents disease. The Molecular Transducers of Physical Activity Consortium (MoTrPAC) was established to address this gap and generate a molecular map of exercise. Preclinical and clinical studies will examine the systemic effects of endurance and resistance exercise across a range of ages and fitness levels by molecular probing of multiple tissues before and after acute and chronic exercise. From this multi-omic and bioinformatic analysis, a molecular map of exercise will be established. Altogether, MoTrPAC will provide a public database that is expected to enhance our understanding of the health benefits of exercise and to provide insight into how physical activity mitigates disease.
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http://dx.doi.org/10.1016/j.cell.2020.06.004DOI Listing
June 2020

Sedentary and Trained Older Men Have Distinct Circulating Exosomal microRNA Profiles at Baseline and in Response to Acute Exercise.

Front Physiol 2020 10;11:605. Epub 2020 Jun 10.

Department of Neurology, Center for Advanced Research on Diagnostic Assays, Icahn School of Medicine at Mount Sinai, New York, NY, United States.

Exercise has multi-systemic benefits and attenuates the physiological impairments associated with aging. Emerging evidence suggests that circulating exosomes mediate some of the beneficial effects of exercise via the transfer of microRNAs between tissues. However, the impact of regular exercise and acute exercise on circulating exosomal microRNAs (exomiRs) in older populations remains unknown. In the present study, we analyzed circulating exomiR expression in endurance-trained elderly men ( = 5) and age-matched sedentary males ( = 5) at baseline (Pre), immediately after a forty minute bout of aerobic exercise on a cycle ergometer (Post), and three hours after this acute exercise (3hPost). Following the isolation and enrichment of exosomes from plasma, exosome-enriched preparations were characterized and exomiR levels were determined by sequencing. The effect of regular exercise on circulating exomiRs was assessed by comparing the baseline expression levels in the trained and sedentary groups. The effect of acute exercise was determined by comparing baseline and post-training expression levels in each group. Regular exercise resulted in significantly increased baseline expression of three exomiRs (miR-486-5p, miR-215-5p, miR-941) and decreased expression of one exomiR (miR-151b). Acute exercise altered circulating exomiR expression in both groups. However, exomiRs regulated by acute exercise in the trained group (7 miRNAs at Post and 8 at 3hPost) were distinct from those in the sedentary group (9 at Post and 4 at 3hPost). Pathway analysis prediction and reported target validation experiments revealed that the majority of exercise-regulated exomiRs are targeting genes that are related to IGF-1 signaling, a pathway involved in exercise-induced muscle and cardiac hypertrophy. The immediately post-acute exercise exomiR signature in the trained group correlates with activation of IGF-1 signaling, whereas in the sedentary group it is associated with inhibition of IGF-1 signaling. While further validation is needed, including measurements of IGF-1/IGF-1 signaling in blood or skeletal muscle, our results suggest that training status may counteract age-related anabolic resistance by modulating circulating exomiR profiles both at baseline and in response to acute exercise.
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http://dx.doi.org/10.3389/fphys.2020.00605DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7298138PMC
June 2020

Fucosidases from the human gut symbiont Ruminococcus gnavus.

Cell Mol Life Sci 2021 Jan 24;78(2):675-693. Epub 2020 Apr 24.

The Gut Microbes and Health Institute Strategic Programme, Quadram Institute Bioscience, Norwich Research Park, Norwich, NR4 7UQ, UK.

The availability and repartition of fucosylated glycans within the gastrointestinal tract contributes to the adaptation of gut bacteria species to ecological niches. To access this source of nutrients, gut bacteria encode α-L-fucosidases (fucosidases) which catalyze the hydrolysis of terminal α-L-fucosidic linkages. We determined the substrate and linkage specificities of fucosidases from the human gut symbiont Ruminococcus gnavus. Sequence similarity network identified strain-specific fucosidases in R. gnavus ATCC 29149 and E1 strains that were further validated enzymatically against a range of defined oligosaccharides and glycoconjugates. Using a combination of glycan microarrays, mass spectrometry, isothermal titration calorimetry, crystallographic and saturation transfer difference NMR approaches, we identified a fucosidase with the capacity to recognize sialic acid-terminated fucosylated glycans (sialyl Lewis X/A epitopes) and hydrolyze α1-3/4 fucosyl linkages in these substrates without the need to remove sialic acid. Molecular dynamics simulation and docking showed that 3'-Sialyl Lewis X (sLeX) could be accommodated within the binding site of the enzyme. This specificity may contribute to the adaptation of R. gnavus strains to the infant and adult gut and has potential applications in diagnostic glycomic assays for diabetes and certain cancers.
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http://dx.doi.org/10.1007/s00018-020-03514-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7872956PMC
January 2021

Differential impact on motility and biofilm dispersal of closely related phosphodiesterases in Pseudomonas aeruginosa.

Sci Rep 2020 04 10;10(1):6232. Epub 2020 Apr 10.

National Biofilms Innovation Centre, University of Southampton, Southampton, SO17 1BJ, UK.

In Pseudomonas aeruginosa, the transition between planktonic and biofilm lifestyles is modulated by the intracellular secondary messenger cyclic dimeric-GMP (c-di-GMP) in response to environmental conditions. Here, we used gene deletions to investigate how the environmental stimulus nitric oxide (NO) is linked to biofilm dispersal, focusing on biofilm dispersal phenotype from proteins containing putative c-di-GMP turnover and Per-Arnt-Sim (PAS) sensory domains. We document opposed physiological roles for the genes ΔrbdA and Δpa2072 that encode proteins with identical domain structure: while ΔrbdA showed elevated c-di-GMP levels, restricted motility and promoted biofilm formation, c-di-GMP levels were decreased in Δpa2072, and biofilm formation was inhibited, compared to wild type. A second pair of genes, ΔfimX and ΔdipA, were selected on the basis of predicted impaired c-di-GMP turnover function: ΔfimX showed increased, ΔdipA decreased NO induced biofilm dispersal, and the genes effected different types of motility, with reduced twitching for ΔfimX and reduced swimming for ΔdipA. For all four deletion mutants we find that NO-induced biomass reduction correlates with increased NO-driven swarming, underlining a significant role for this motility in biofilm dispersal. Hence P. aeruginosa is able to differentiate c-di-GMP output using structurally highly related proteins that can contain degenerate c-di-GMP turnover domains.
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http://dx.doi.org/10.1038/s41598-020-63008-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7148300PMC
April 2020

Inactivation of foodborne pathogens based on synergistic effects of ultrasound and natural compounds during fresh produce washing.

Ultrason Sonochem 2020 Jun 22;64:104983. Epub 2020 Jan 22.

Department of Nutrition and Food Science, University of Maryland, College Park, MD 20740, USA. Electronic address:

Ultrasound has potential to be used for disinfection, and its antimicrobial effectiveness can be enhanced in presence of natural compounds. In this study, we compared the antimicrobial effects of ultrasound at 20 kHz (US 20 kHz) or 1 MHz (US 1 MHz) in combination with carvacrol, citral, cinnamic acid, geraniol, gallic acid, lactic acid, or limonene against E. coli K12 and Listeria innocua at a constant power density in water. Compared to the cumulative effect of the individual treatments, the combined treatment of US 1 MHz and 10 mM citral generated >1.5 log CFU/mL additional inactivation of E. coli K12. Similarly, combined treatments of US 1 MHz and 2 mM carvacrol (30 min), US 20 kHz and 2 mM carvacrol, 10 mM citral, or 5 mM geraniol (15 min) generated >0.5-2.0 log CFU/mL additional inactivation in L. innocua. The synergistic effect of citral, as a presentative compound, and US 20 kHz treatment was determined to be a result of enhanced dispersion of insoluble citral droplets in combination with physical impact on bacterial membrane structures, whereas the inactivation by US 1 MHz was likely due to generation of oxidative stress within the bacteria. Combined ultrasound and citral treatments improved the bacterial inactivation in simulated wash water in presence of organic matter or during washing of inoculated blueberries but only additive antimicrobial effects were observed. Findings in this study will be useful to enhance fresh produce safety and shelf-life and design other alternative ultrasound based sanitation processes.
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http://dx.doi.org/10.1016/j.ultsonch.2020.104983DOI Listing
June 2020

Elucidation of a sialic acid metabolism pathway in mucus-foraging Ruminococcus gnavus unravels mechanisms of bacterial adaptation to the gut.

Nat Microbiol 2019 12 21;4(12):2393-2404. Epub 2019 Oct 21.

The Gut Microbes and Health Institute Strategic Programme, Quadram Institute Bioscience, Norwich Research Park, Norwich, UK.

Sialic acid (N-acetylneuraminic acid (Neu5Ac)) is commonly found in the terminal location of colonic mucin glycans where it is a much-coveted nutrient for gut bacteria, including Ruminococcus gnavus. R. gnavus is part of the healthy gut microbiota in humans, but it is disproportionately represented in diseases. There is therefore a need to understand the molecular mechanisms that underpin the adaptation of R. gnavus to the gut. Previous in vitro research has demonstrated that the mucin-glycan-foraging strategy of R. gnavus is strain dependent and is associated with the expression of an intramolecular trans-sialidase, which releases 2,7-anhydro-Neu5Ac, rather than Neu5Ac, from mucins. Here, we unravelled the metabolism pathway of 2,7-anhydro-Neu5Ac in R. gnavus that is underpinned by the exquisite specificity of the sialic transporter for 2,7-anhydro-Neu5Ac and by the action of an oxidoreductase that converts 2,7-anhydro-Neu5Ac into Neu5Ac, which then becomes a substrate of a Neu5Ac-specific aldolase. Having generated an R. gnavus nan-cluster deletion mutant that lost the ability to grow on sialylated substrates, we showed that-in gnotobiotic mice colonized with R. gnavus wild-type (WT) and mutant strains-the fitness of the nan mutant was significantly impaired, with a reduced ability to colonize the mucus layer. Overall, we revealed a unique sialic acid pathway in bacteria that has important implications for the spatial adaptation of mucin-foraging gut symbionts in health and disease.
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http://dx.doi.org/10.1038/s41564-019-0590-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6881182PMC
December 2019

Multiscale network analysis reveals molecular mechanisms and key regulators of the tumor microenvironment in gastric cancer.

Int J Cancer 2020 03 11;146(5):1268-1280. Epub 2019 Oct 11.

Department of Genetics and Genomic Sciences, Icahn Institute of Genomics and Multiscale Biology, Icahn School of Medicine at Mount Sinai, New York, NY.

Gastric cancer (GC) is the third leading cause of cancer deaths and the fourth most prevalent malignancy worldwide. The high incidence and mortality rates of gastric cancer result from multiple factors such as ineffective screening, diagnosis, and limited treatment options. In our study, we sought to systematically identify predictive molecular networks and key regulators to elucidate complex interacting signaling pathways in GC. We performed an integrative network analysis of the transcriptomic data in The Cancer Genome Atlas (TCGA) gastric cancer cohort and then comprehensively characterized the predictive subnetworks and key regulators by the matched genetic and epigenetic data. We identified 221 gene subnetworks (modules) in GC. The most prognostic subnetworks captured multiple aspects of the tumor microenvironment in GC involving interactions among stromal, epithelial and immune cells. We revealed the genetic and epigenetic underpinnings of those subnetworks and their key transcriptional regulators. We computationally predicted and experimentally validated specific mechanisms of anticancer effects of GKN2 in gastric cancer proliferation and invasion in vitro. The network models and the key regulators of the tumor microenvironment in GC identified here pave a way for developing novel therapeutic strategies for GC.
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http://dx.doi.org/10.1002/ijc.32643DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7004118PMC
March 2020

LINE-1 Retrotransposition Promotes the Development and Progression of Lung Squamous Cell Carcinoma by Disrupting the Tumor-Suppressor Gene FGGY.

Cancer Res 2019 09 9;79(17):4453-4465. Epub 2019 Jul 9.

Cancer Molecular Diagnostics Core, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center of Caner, Key Laboratory of Cancer Prevention and Therapy, Key Laboratory of Cancer Immunology and Biotherapy, Tianjin, China.

Somatic long interspersed element-1 () retrotransposition is a genomic process that relates to gene disruption and tumor occurrence. However, the expression and function of retrotransposition in lung squamous cell carcinoma (LUSC) remain unclear. We analyzed the transcriptomes of LUSC samples in The Cancer Genome Atlas and observed retrotransposition in 90% of tumor samples. Thirteen retrotranspositions of high occurrence were identified and further validated from an independent Chinese LUSC cohort. Among them, ( was identified as the most frequent retrotransposition in the Chinese cohort and significantly correlated with poor clinical outcome. occurred with smoke-induced hypomethylation of the promoter and contributed to the development of local immune evasion and dysfunctional metabolism. Overexpression of or knockdown of promoted cell proliferation and invasion , facilitated tumorigenesis , and dysregulated cell energy metabolism and cytokine/chemotaxin transcription. Importantly, specific reverse transcription inhibitors, nevirapine and efavirenz, dramatically countered abundance, inhibited tumor growth, recovered metabolism dysfunction, and improved the local immune evasion. In conclusion, hypomethylation-induced expression is a frequent genomic event that promotes the development and progression of LUSC and represents a promising predictive biomarker and therapeutic target in LUSC. SIGNIFICANCE: is a prognosis predictive biomarker and potential therapeutic target to overcome local immune evasion in lung squamous cell carcinoma.
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http://dx.doi.org/10.1158/0008-5472.CAN-19-0076DOI Listing
September 2019

The structure of Erwinia amylovora AvrRpt2 provides insight into protein maturation and induced resistance to fire blight by Malus × robusta 5.

J Struct Biol 2019 05 27;206(2):233-242. Epub 2019 Mar 27.

Bioorganic Chemistry and Bio-Crystallography Laboratory (B(2)Cl), Faculty of Science and Technology, Free University of Bolzano, Piazza Università 5, 39100 Bolzano, Italy. Electronic address:

The AvrRpt2 protein of the phytopathogenic bacterium Erwinia amylovora (AvrRpt2) is a secreted type III effector protein, which is recognised by the FB_MR5 resistance protein of Malus × robusta 5, the only identified resistance protein from a Malus species preventing E. amylovora infection. The crystal structure of the immature catalytic domain of AvrRpt2 a C70 family cysteine protease and type III effector, was determined to a resolution of 1.85 Å. The structure provides insights into the cyclophilin-dependent activation of AvrRpt2, and identifies a cryptic leucine of a non-canonical cyclophilin binding motif. The structure also suggests that residue Cys156, responsible for the gene induced resistance, is not involved in substrate determination, and hints that recognition by FB_MR5 is due to direct interaction.
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http://dx.doi.org/10.1016/j.jsb.2019.03.010DOI Listing
May 2019

Use of alcohol biomarkers to identify alcohol misuse in organ donors.

Alcohol 2018 12 31;73:67-72. Epub 2018 Mar 31.

Alcohol Research Program, Loyola University Chicago Health Science Division, Maywood, IL, United States; Division of Pulmonary and Critical Care, Department of Medicine, Loyola University Medical Center, Maywood, IL, United States; Loyola University Chicago Stritch School of Medicine, Maywood, IL, United States.

Phosphatidylethanol is a direct alcohol biomarker for identifying alcohol misuse. It carries several advantages over other alcohol biomarkers, including a detection half-life of several weeks and little confounding by patient characteristics or organ dysfunction. The aim of this study is to derive an optimal phosphatidylethanol cut point to identify organ donors with alcohol misuse, and to assess the impact of alcohol misuse on organ allocation. Discrimination of phosphatidylethanol was evaluated using the area under the ROC curve from a mixed effects logistic regression model. Phosphatidylethanol had an area under the ROC curve of 0.89 (95% CI 0.80-0.98). A phosphatidylethanol cut point of ≥84 ng/mL provided optimal discrimination for the identification of alcohol misuse with a sensitivity of 75% (95% CI 52.9%-89.4%) and a specificity of 97% (95% CI 91%-99%), a positive predictive value of 82% (95% CI 59%-94%), and a negative predictive value of 95% (95% CI 89%-98%). In deceased organ donors who had been critically ill, phosphatidylethanol had good test characteristics to discriminate alcohol misuse. Other alcohol biomarkers performed poorly in deceased organ donors. Liver allocation was decreased in donors with alcohol misuse by proxy history, but not in those with phosphatidylethanol >84 ng/mL, revealing possible information bias in liver allocation.
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http://dx.doi.org/10.1016/j.alcohol.2018.03.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6188836PMC
December 2018

Analysis of chromatin accessibility uncovers TEAD1 as a regulator of migration in human glioblastoma.

Nat Commun 2018 10 1;9(1):4020. Epub 2018 Oct 1.

Department of Pathology, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA.

The intrinsic drivers of migration in glioblastoma (GBM) are poorly understood. To better capture the native molecular imprint of GBM and its developmental context, here we isolate human stem cell populations from GBM (GSC) and germinal matrix tissues and map their chromatin accessibility via ATAC-seq. We uncover two distinct regulatory GSC signatures, a developmentally shared/proliferative and a tumor-specific/migratory one in which TEAD1/4 motifs are uniquely overrepresented. Using ChIP-PCR, we validate TEAD1 trans occupancy at accessibility sites within AQP4, EGFR, and CDH4. To further characterize TEAD's functional role in GBM, we knockout TEAD1 or TEAD4 in patient-derived GBM lines using CRISPR-Cas9. TEAD1 ablation robustly diminishes migration, both in vitro and in vivo, and alters migratory and EMT transcriptome signatures with consistent downregulation of its target AQP4. TEAD1 overexpression restores AQP4 expression, and both TEAD1 and AQP4 overexpression rescue migratory deficits in TEAD1-knockout cells, implicating a direct regulatory role for TEAD1-AQP4 in GBM migration.
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http://dx.doi.org/10.1038/s41467-018-06258-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6167382PMC
October 2018

High resolution annotation of zebrafish transcriptome using long-read sequencing.

Genome Res 2018 09 30;28(9):1415-1425. Epub 2018 Jul 30.

Department of Neurology, Icahn School of Medicine at Mount Sinai, New York, New York 10029, USA.

With the emergence of zebrafish as an important model organism, a concerted effort has been made to study its transcriptome. This effort is limited, however, by gaps in zebrafish annotation, which are especially pronounced concerning transcripts dynamically expressed during zygotic genome activation (ZGA). To date, short-read sequencing has been the principal technology for zebrafish transcriptome annotation. In part because these sequence reads are too short for assembly methods to resolve the full complexity of the transcriptome, the current annotation is rudimentary. By providing direct observation of full-length transcripts, recently refined long-read sequencing platforms can dramatically improve annotation coverage and accuracy. Here, we leveraged the SMRT platform to study the transcriptome of zebrafish embryos before and after ZGA. Our analysis revealed additional novelty and complexity in the zebrafish transcriptome, identifying 2539 high-confidence novel transcripts that originated from previously unannotated loci and 1835 high-confidence new isoforms in previously annotated genes. We validated these findings using a suite of computational approaches including structural prediction, sequence homology, and functional conservation analyses, as well as by confirmatory transcript quantification with short-read sequencing data. Our analyses provided insight into new homologs and paralogs of functionally important proteins and noncoding RNAs, isoform switching occurrences, and different classes of novel splicing events. Several novel isoforms representing distinct splicing events were validated through PCR experiments, including the discovery and validation of a novel 8-kb transcript spanning multiple elements, an important driver of early development. Our study provides a significantly improved zebrafish transcriptome annotation resource.
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http://dx.doi.org/10.1101/gr.223586.117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6120630PMC
September 2018

Spatially constrained tandem bromodomain inhibition bolsters sustained repression of BRD4 transcriptional activity for TNBC cell growth.

Proc Natl Acad Sci U S A 2018 07 16;115(31):7949-7954. Epub 2018 Jul 16.

Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029;

The importance of BET protein BRD4 in gene transcription is well recognized through the study of chemical modulation of its characteristic tandem bromodomain (BrD) binding to lysine-acetylated histones and transcription factors. However, while monovalent inhibition of BRD4 by BET BrD inhibitors such as JQ1 blocks growth of hematopoietic cancers, it is much less effective generally in solid tumors. Here, we report a thienodiazepine-based bivalent BrD inhibitor, MS645, that affords spatially constrained tandem BrD inhibition and consequently sustained repression of BRD4 transcriptional activity in blocking proliferation of solid-tumor cells including a panel of triple-negative breast cancer (TNBC) cells. MS645 blocks BRD4 binding to transcription enhancer/mediator proteins MED1 and YY1 with potency superior to monovalent BET inhibitors, resulting in down-regulation of proinflammatory cytokines and genes for cell-cycle control and DNA damage repair that are largely unaffected by monovalent BrD inhibition. Our study suggests a therapeutic strategy to maximally control BRD4 activity for rapid growth of solid-tumor TNBC cells.
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http://dx.doi.org/10.1073/pnas.1720000115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6077712PMC
July 2018

Structural and functional analysis of Erwinia amylovora SrlD. The first crystal structure of a sorbitol-6-phosphate 2-dehydrogenase.

J Struct Biol 2018 08 29;203(2):109-119. Epub 2018 Mar 29.

Bioorganic Chemistry and Bio-Crystallography Laboratory (B(2)Cl), Faculty of Science and Technology, Free University of Bolzano, Piazza Università 5, 39100 Bolzano, Italy. Electronic address:

Sorbitol-6-phosphate 2-dehydrogenases (S6PDH) catalyze the interconversion of d-sorbitol 6-phosphate to d-fructose 6-phosphate. In the plant pathogen Erwinia amylovora the S6PDH SrlD is used by the bacterium to utilize sorbitol, which is used for carbohydrate transport in the host plants belonging to the Amygdaloideae subfamily (e.g., apple, pear, and quince). We have determined the crystal structure of S6PDH SrlD at 1.84 Å resolution, which is the first structure of an EC 1.1.1.140 enzyme. Kinetic data show that SrlD is much faster at oxidizing d-sorbitol 6-phosphate than in reducing d-fructose 6-phosphate, however, equilibrium analysis revealed that only part of the d-sorbitol 6-phosphate present in the in vitro environment is converted into d-fructose 6-phosphate. The comparison of the structures of SrlD and Rhodobacter sphaeroides sorbitol dehydrogenase showed that the tetrameric quaternary structure, the catalytic residues and a conserved aspartate residue that confers specificity for NAD over NADP are preserved. Analysis of the SrlD cofactor and substrate binding sites identified residues important for the formation of the complex with cofactor and substrate and in particular the role of Lys42 in selectivity towards the phospho-substrate. The comparison of SrlD backbone with the backbone of 302 short-chain dehydrogenases/reductases showed the conservation of the protein core and identified the variable parts. The SrlD sequence was compared with 500 S6PDH sequences selected by homology revealing that the C-terminal part is more conserved than the N-terminal, the consensus of the catalytic tetrad (Y[SN]AGXA) and a not previously described consensus for the NAD(H) binding.
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http://dx.doi.org/10.1016/j.jsb.2018.03.010DOI Listing
August 2018

Where is crystallography going?

Acta Crystallogr D Struct Biol 2018 Feb 1;74(Pt 2):152-166. Epub 2018 Feb 1.

Science Division, Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE, England.

Macromolecular crystallography (MX) has been a motor for biology for over half a century and this continues apace. A series of revolutions, including the production of recombinant proteins and cryo-crystallography, have meant that MX has repeatedly reinvented itself to dramatically increase its reach. Over the last 30 years synchrotron radiation has nucleated a succession of advances, ranging from detectors to optics and automation. These advances, in turn, open up opportunities. For instance, a further order of magnitude could perhaps be gained in signal to noise for general synchrotron experiments. In addition, X-ray free-electron lasers offer to capture fragments of reciprocal space without radiation damage, and open up the subpicosecond regime of protein dynamics and activity. But electrons have recently stolen the limelight: so is X-ray crystallography in rude health, or will imaging methods, especially single-particle electron microscopy, render it obsolete for the most interesting biology, whilst electron diffraction enables structure determination from even the smallest crystals? We will lay out some information to help you decide.
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http://dx.doi.org/10.1107/S2059798317016709DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5947779PMC
February 2018

A complete structural characterization of the desferrioxamine E biosynthetic pathway from the fire blight pathogen Erwinia amylovora.

J Struct Biol 2018 06 8;202(3):236-249. Epub 2018 Feb 8.

Bioorganic Chemistry and Bio-Crystallography Laboratory (B(2)Cl), Faculty of Science and Technology, Free University of Bolzano, Piazza Università 5, 39100 Bolzano, Italy. Electronic address:

The Gram-negative bacterium Erwinia amylovora is the etiological agent of fire blight, a devastating disease which affects Rosaceae such as apple, pear and quince. The siderophore desferrioxamine E plays an important role in bacterial pathogenesis by scavenging iron from the host. DfoJ, DfoA and DfoC are the enzymes responsible for desferrioxamine production starting from lysine. We have determined the crystal structures of each enzyme in the desferrioxamine E pathway and demonstrate that the biosynthesis involves the concerted action of DfoJ, followed by DfoA and lastly DfoC. These data provide the first crystal structures of a Group II pyridoxal-dependent lysine decarboxylase, a cadaverine monooxygenase and a desferrioxamine synthetase. DfoJ is a homodimer made up of three domains. Each monomer contributes to the completion of the active site, which is positioned at the dimer interface. DfoA is the first structure of a cadaverine monooxygenase. It forms homotetramers whose subunits are built by two domains: one for FAD and one for NADP binding, the latter of which is formed by two subdomains. We propose a model for substrate binding and the role of residues 43-47 as gate keepers for FAD binding and the role of Arg97 in cofactors turnover. DfoC is the first structure of a desferrioxamine synthetase and the first of a multi-enzyme siderophore synthetase coupling an acyltransferase domain with a Non-Ribosomal Peptide Synthetase (NRPS)-Independent Siderophore domain (NIS).
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http://dx.doi.org/10.1016/j.jsb.2018.02.002DOI Listing
June 2018

Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092.

J Vis Exp 2017 10 2(128). Epub 2017 Oct 2.

Diamond Light Source, Harwell Science & Innovation Campus; Research Complex at Harwell, Harwell Science & Innovation Campus.

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant strains. Carbohydrate substrate binding proteins (SBPs) represent viable targets for the development of protein-based vaccines and new antimicrobials because of their extracellular localization and the centrality of carbohydrate import for pneumococcal metabolism, respectively. Described here is a rationalized integrated protocol to carry out a comprehensive characterization of SP0092, which can be extended to other carbohydrate SBPs from the pneumococcus and other bacteria. This procedure can aid the structure-based design of inhibitors for this class of proteins. Presented in the first part of this manuscript are protocols for biochemical analysis by thermal shift assay, multi angle light scattering (MALS), and size exclusion chromatography (SEC), which optimize the stability and homogeneity of the sample directed to crystallization trials and so enhance the probability of success. The second part of this procedure describes the characterization of the SBP crystals using a tunable wavelength anomalous diffraction synchrotron beamline, and data collection protocols for measuring data that can be used to resolve the crystallized protein structure.
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http://dx.doi.org/10.3791/56294DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5752355PMC
October 2017

The PRMT5/WDR77 complex regulates alternative splicing through ZNF326 in breast cancer.

Nucleic Acids Res 2017 Nov;45(19):11106-11120

Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.

We observed overexpression and increased intra-nuclear accumulation of the PRMT5/WDR77 in breast cancer cell lines relative to immortalized breast epithelial cells. Utilizing mass spectrometry and biochemistry approaches we identified the Zn-finger protein ZNF326, as a novel interaction partner and substrate of the nuclear PRMT5/WDR77 complex. ZNF326 is symmetrically dimethylated at arginine 175 (R175) and this modification is lost in a PRMT5 and WDR77-dependent manner. Loss of PRMT5 or WDR77 in MDA-MB-231 cells leads to defects in alternative splicing, including inclusion of A-T rich exons in target genes, a phenomenon that has previously been observed upon loss of ZNF326. We observed that the alternatively spliced transcripts of a subset of these genes, involved in proliferation and tumor cell migration like REPIN1/AP4, ST3GAL6, TRNAU1AP and PFKM are degraded upon loss of PRMT5. In summary, we have identified a novel mechanism through which the PRMT5/WDR77 complex maintains the balance between splicing and mRNA stability through methylation of ZNF326.
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http://dx.doi.org/10.1093/nar/gkx727DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5737218PMC
November 2017

Enhanced antimicrobial effect of ultrasound by the food colorant Erythrosin B.

Food Res Int 2017 10 6;100(Pt 1):344-351. Epub 2017 Jul 6.

Department of Nutrition and Food Science, University of Maryland, 4300 Chapel Lane, Skinner building Room 0112, College Park, MD 20742, United States. Electronic address:

The synergistic combination of the food colorant Erythrosin B (E-B, FD&C 3) (0, 25, and 50μM) and low-frequency ultrasound (20kHz, 0.86-0.90WmL) was evaluated against Listeria innocua. Although E-B was antibacterial by itself, the inactivation rate significantly increased in a concentration-dependent manner upon exposure to ultrasound and followed a sigmoidal behavior. The enhanced antimicrobial effect of E-B in the presence of ultrasound can be explained in part from a microbubble disappearance study in which it was confirmed that the presence of E-B enhances inertial cavitation, thereby enhancing the antimicrobial effect of ultrasound. The inactivation rate in a sequential treatment, where L. innocua was sonicated for 4min followed by exposure to 25μM Erythrosin B, was comparable to that obtained by the simultaneous treatment, indicating complementary mechanisms of inactivation. Fluorescence microscopy showed attachment of E-B to the cells, which may explain its intrinsic antimicrobial property. Other mechanism may include the confirmed decrease in the cavitation threshold of water by addition of E-B, resulting in more effective cavitation. The study offers a proof-of-concept of a novel approach to complement ultrasound treatment for enhanced microbial inactivation.
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http://dx.doi.org/10.1016/j.foodres.2017.07.012DOI Listing
October 2017

Interdomain conformational flexibility underpins the activity of UGGT, the eukaryotic glycoprotein secretion checkpoint.

Proc Natl Acad Sci U S A 2017 08 24;114(32):8544-8549. Epub 2017 Jul 24.

Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom;

Glycoproteins traversing the eukaryotic secretory pathway begin life in the endoplasmic reticulum (ER), where their folding is surveyed by the 170-kDa UDP-glucose:glycoprotein glucosyltransferase (UGGT). The enzyme acts as the single glycoprotein folding quality control checkpoint: it selectively reglucosylates misfolded glycoproteins, promotes their association with ER lectins and associated chaperones, and prevents premature secretion from the ER. UGGT has long resisted structural determination and sequence-based domain boundary prediction. Questions remain on how this single enzyme can flag misfolded glycoproteins of different sizes and shapes for ER retention and how it can span variable distances between the site of misfold and a glucose-accepting N-linked glycan on the same glycoprotein. Here, crystal structures of a full-length eukaryotic UGGT reveal four thioredoxin-like (TRXL) domains arranged in a long arc that terminates in two β-sandwiches tightly clasping the glucosyltransferase domain. The fold of the molecule is topologically complex, with the first β-sandwich and the fourth TRXL domain being encoded by nonconsecutive stretches of sequence. In addition to the crystal structures, a 15-Å cryo-EM reconstruction reveals interdomain flexibility of the TRXL domains. Double cysteine point mutants that engineer extra interdomain disulfide bridges rigidify the UGGT structure and exhibit impaired activity. The intrinsic flexibility of the TRXL domains of UGGT may therefore endow the enzyme with the promiscuity needed to recognize and reglucosylate its many different substrates and/or enable reglucosylation of N-linked glycans situated at variable distances from the site of misfold.
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http://dx.doi.org/10.1073/pnas.1703682114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5559018PMC
August 2017

The N-Methyladenosine RNA modification in pluripotency and reprogramming.

Curr Opin Genet Dev 2017 Oct 3;46:77-82. Epub 2017 Jul 3.

Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Mount Sinai Center for RNA Biology and Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA. Electronic address:

Chemical modifications of RNA provide a direct and rapid way to manipulate the existing transcriptome, allowing rapid responses to the changing environment further enriching the regulatory capacity of RNA. N-Methyladenosine (mA) has been identified as the most abundant internal modification of messenger RNA in eukaryotes, linking external stimuli to an intricate network of transcriptional, post-transcriptional and translational processes. MA modification affects a broad spectrum of cellular functions, including maintenance of the pluripotency of embryonic stem cells (ESCs) and the reprogramming of somatic cells into induced pluripotent stem cells (iPSCs). In this review, we summarize the most recent findings on mA modification with special focus on the different studies describing how mA is implicated in ESC self-renewal, cell fate specification and iPSC generation.
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http://dx.doi.org/10.1016/j.gde.2017.06.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5626584PMC
October 2017

Electron Bio-Imaging Centre (eBIC): the UK national research facility for biological electron microscopy.

Acta Crystallogr D Struct Biol 2017 Jun 31;73(Pt 6):488-495. Epub 2017 May 31.

Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE, England.

The recent resolution revolution in cryo-EM has led to a massive increase in demand for both time on high-end cryo-electron microscopes and access to cryo-electron microscopy expertise. In anticipation of this demand, eBIC was set up at Diamond Light Source in collaboration with Birkbeck College London and the University of Oxford, and funded by the Wellcome Trust, the UK Medical Research Council (MRC) and the Biotechnology and Biological Sciences Research Council (BBSRC) to provide access to high-end equipment through peer review. eBIC is currently in its start-up phase and began by offering time on a single FEI Titan Krios microscope equipped with the latest generation of direct electron detectors from two manufacturers. Here, the current status and modes of access for potential users of eBIC are outlined. In the first year of operation, 222 d of microscope time were delivered to external research groups, with 95 visits in total, of which 53 were from unique groups. The data collected have generated multiple high- to intermediate-resolution structures (2.8-8 Å), ten of which have been published. A second Krios microscope is now in operation, with two more due to come online in 2017. In the next phase of growth of eBIC, in addition to more microscope time, new data-collection strategies and sample-preparation techniques will be made available to external user groups. Finally, all raw data are archived, and a metadata catalogue and automated pipelines for data analysis are being developed.
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http://dx.doi.org/10.1107/S2059798317007756DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5458490PMC
June 2017
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