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An activator of G protein-coupled receptor and MEK1/2-ERK1/2 signaling inhibits HIV-1 replication by altering viral RNA processing.

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An activator of G protein-coupled receptor and MEK1/2-ERK1/2 signaling inhibits HIV-1 replication by altering viral RNA processing.

PLoS Pathog. 2020 02;16(2):e1008307

Authors: Wong RW, Balachandran A, Cheung PK, Cheng R, Pan Q, Stoilov P, Harrigan PR, Blencowe BJ, Branch DR, Cochrane A

Abstract
The ability of HIV-1 to evolve resistance to combined antiretroviral therapies (cARTs) has stimulated research into alternative means of controlling this infection. We assayed >60 modulators of RNA alternative splicing (AS) to identify new inhibitors of HIV-1 RNA processing-a segment of the viral lifecycle not targeted by current drugs-and discovered compound N-[4-chloro-3-(trifluoromethyl)phenyl]-7-nitro-2,1,3-benzoxadiazol-4-amine (5342191) as a potent inhibitor of both wild-type (Ba-L, NL4-3, LAI, IIIB, and N54) and drug-resistant strains of HIV-1 (IC50: ~700 nM) with no significant effect on cell viability at doses tested. 5342191 blocks expression of four essential HIV-1 structural and regulatory proteins (Gag, Env, Tat, and Rev) without affecting total protein synthesis of the cell. This response is associated with altered unspliced (US) and singly-spliced (SS) HIV-1 RNA accumulation (~60% reduction) and transport to the cytoplasm (loss of Rev) whereas parallel analysis of cellular RNAs revealed less than a 0.7% of host alternative splicing (AS) events (0.25-0.67% by ≥ 10-20%), gene expression (0.01-0.46% by ≥ 2-5 fold), and protein abundance (0.02-0.34% by ≥ 1.5-2 fold) being affected. Decreased expression of Tat, but not Gag/Env, upon 5342191 treatment was reversed by a proteasome inhibitor, suggesting that this compound alters the synthesis/degradation of this key viral factor. Consistent with an affect on HIV-1 RNA processing, 5342191 treatment of cells altered the abundance and phosphorylation of serine/arginine-rich splicing factor (SRSF) 1, 3, and 4. Despite the activation of several intracellular signaling pathways by 5342191 (Ras, MEK1/2-ERK1/2, and JNK1/2/3), inhibition of HIV-1 gene expression by this compound could be reversed by pre-treatment with either a G-protein α-subunit inhibitor or two different MEK1/2 inhibitors. These observations demonstrate enhanced sensitivity of HIV-1 gene expression to small changes in host RNA processing and highlights the potential of modulating host intracellular signaling as an alternative approach for controlling HIV-1 infection.

PMID: 32069328 [PubMed - indexed for MEDLINE]



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Systematic genetics and single-cell imaging reveal widespread morphological pleiotropy and cell-to-cell variability.

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Systematic genetics and single-cell imaging reveal widespread morphological pleiotropy and cell-to-cell variability.

Mol Syst Biol. 2020 Feb;16(2):e9243

Authors: Mattiazzi Usaj M, Sahin N, Friesen H, Pons C, Usaj M, Masinas MPD, Shuteriqi E, Shkurin A, Aloy P, Morris Q, Boone C, Andrews BJ

Abstract
Our ability to understand the genotype-to-phenotype relationship is hindered by the lack of detailed understanding of phenotypes at a single-cell level. To systematically assess cell-to-cell phenotypic variability, we combined automated yeast genetics, high-content screening and neural network-based image analysis of single cells, focussing on genes that influence the architecture of four subcellular compartments of the endocytic pathway as a model system. Our unbiased assessment of the morphology of these compartments-endocytic patch, actin patch, late endosome and vacuole-identified 17 distinct mutant phenotypes associated with ~1,600 genes (~30% of all yeast genes). Approximately half of these mutants exhibited multiple phenotypes, highlighting the extent of morphological pleiotropy. Quantitative analysis also revealed that incomplete penetrance was prevalent, with the majority of mutants exhibiting substantial variability in phenotype at the single-cell level. Our single-cell analysis enabled exploration of factors that contribute to incomplete penetrance and cellular heterogeneity, including replicative age, organelle inheritance and response to stress.

PMID: 32064787 [PubMed - in process]



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Potential Therapeutic Targets for Lung Repair During Human Ex Vivo Lung Perfusion.

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Potential Therapeutic Targets for Lung Repair During Human Ex Vivo Lung Perfusion.

Eur Respir J. 2020 Feb 14;:

Authors: Wong A, Zamel R, Yeung J, Bader GD, Dos Santos CC, Bai X, Wang Y, Keshavjee S, Liu M

Abstract
INTRODUCTION: The ex vivo lung perfusion (EVLP) technique has been developed to assess the function of marginal donor lungs which has significantly increased donor lung utilisation. EVLP has also been explored as a platform for donor lung repair through injury specific treatments such as antibiotics or fibrinolytics. We hypothesised that actively expressed pathways shared between transplantation and EVLP may reveal common mechanisms of injury and potential therapeutic targets for lung repair prior to transplantation.
MATERIALS AND METHODS: A retrospective transcriptomics analyses were performed with peripheral tissue biopsies from "donation after brain death" lungs, with 46 pre/post-transplant pairs and 49 pre/post-EVLP pairs. Pathway analysis was used to identify and compare the responses of donor lungs to transplantation and to EVLP.
RESULTS: Twenty-one pathways were enriched predominantly in transplantation, including upregulation of lymphocyte activation and cell death, and downregulation of metabolism and protein synthesis. Seven pathways were enriched predominantly in EVLP, including downregulation of leukocyte functions and upregulation of vascular processes. Twenty-three pathways were commonly enriched, including activation of innate inflammation, cell death, heat stress and downregulation of metabolism. Of the inflammatory clusters, TLR/MYD88 signalling had the greatest number of nodes and was central to inflammation. These mechanisms have been previously speculated as major mechanisms of acute lung injury in animal models.
CONCLUSION: EVLP and transplantation share common molecular features of injury including innate inflammation and cell death. Blocking these pathways during EVLP may allow for lung repair prior to transplantation.

PMID: 32060066 [PubMed - as supplied by publisher]



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The evolutionary history of 2,658 cancers.

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The evolutionary history of 2,658 cancers.

Nature. 2020 02;578(7793):122-128

Authors: Gerstung M, Jolly C, Leshchiner I, Dentro SC, Gonzalez S, Rosebrock D, Mitchell TJ, Rubanova Y, Anur P, Yu K, Tarabichi M, Deshwar A, Wintersinger J, Kleinheinz K, Vázquez-García I, Haase K, Jerman L, Sengupta S, Macintyre G, Malikic S, Donmez N, Livitz DG, Cmero M, Demeulemeester J, Schumacher S, Fan Y, Yao X, Lee J, Schlesner M, Boutros PC, Bowtell DD, Zhu H, Getz G, Imielinski M, Beroukhim R, Sahinalp SC, Ji Y, Peifer M, Markowetz F, Mustonen V, Yuan K, Wang W, Morris QD, PCAWG Evolution & Heterogeneity Working Group, Spellman PT, Wedge DC, Van Loo P, PCAWG Consortium

Abstract
Cancer develops through a process of somatic evolution1,2. Sequencing data from a single biopsy represent a snapshot of this process that can reveal the timing of specific genomic aberrations and the changing influence of mutational processes3. Here, by whole-genome sequencing analysis of 2,658 cancers as part of the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA)4, we reconstruct the life history and evolution of mutational processes and driver mutation sequences of 38 types of cancer. Early oncogenesis is characterized by mutations in a constrained set of driver genes, and specific copy number gains, such as trisomy 7 in glioblastoma and isochromosome 17q in medulloblastoma. The mutational spectrum changes significantly throughout tumour evolution in 40% of samples. A nearly fourfold diversification of driver genes and increased genomic instability are features of later stages. Copy number alterations often occur in mitotic crises, and lead to simultaneous gains of chromosomal segments. Timing analyses suggest that driver mutations often precede diagnosis by many years, if not decades. Together, these results determine the evolutionary trajectories of cancer, and highlight opportunities for early cancer detection.

PMID: 32025013 [PubMed - indexed for MEDLINE]



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A deep learning system accurately classifies primary and metastatic cancers using passenger mutation patterns.

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A deep learning system accurately classifies primary and metastatic cancers using passenger mutation patterns.

Nat Commun. 2020 02 05;11(1):728

Authors: Jiao W, Atwal G, Polak P, Karlic R, Cuppen E, PCAWG Tumor Subtypes and Clinical Translation Working Group, Danyi A, de Ridder J, van Herpen C, Lolkema MP, Steeghs N, Getz G, Morris Q, Stein LD, PCAWG Consortium

Abstract
In cancer, the primary tumour's organ of origin and histopathology are the strongest determinants of its clinical behaviour, but in 3% of cases a patient presents with a metastatic tumour and no obvious primary. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, we train a deep learning classifier to predict cancer type based on patterns of somatic passenger mutations detected in whole genome sequencing (WGS) of 2606 tumours representing 24 common cancer types produced by the PCAWG Consortium. Our classifier achieves an accuracy of 91% on held-out tumor samples and 88% and 83% respectively on independent primary and metastatic samples, roughly double the accuracy of trained pathologists when presented with a metastatic tumour without knowledge of the primary. Surprisingly, adding information on driver mutations reduced accuracy. Our results have clinical applicability, underscore how patterns of somatic passenger mutations encode the state of the cell of origin, and can inform future strategies to detect the source of circulating tumour DNA.

PMID: 32024849 [PubMed - indexed for MEDLINE]



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Reconstructing evolutionary trajectories of mutation signature activities in cancer using TrackSig.

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Reconstructing evolutionary trajectories of mutation signature activities in cancer using TrackSig.

Nat Commun. 2020 02 05;11(1):731

Authors: Rubanova Y, Shi R, Harrigan CF, Li R, Wintersinger J, Sahin N, Deshwar A, PCAWG Evolution and Heterogeneity Working Group, Morris Q, PCAWG Consortium

Abstract
The type and genomic context of cancer mutations depend on their causes. These causes have been characterized using signatures that represent mutation types that co-occur in the same tumours. However, it remains unclear how mutation processes change during cancer evolution due to the lack of reliable methods to reconstruct evolutionary trajectories of mutational signature activity. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole-genome sequencing data from 2658 cancers across 38 tumour types, we present TrackSig, a new method that reconstructs these trajectories using optimal, joint segmentation and deconvolution of mutation type and allele frequencies from a single tumour sample. In simulations, we find TrackSig has a 3-5% activity reconstruction error, and 12% false detection rate. It outperforms an aggressive baseline in situations with branching evolution, CNA gain, and neutral mutations. Applied to data from 2658 tumours and 38 cancer types, TrackSig permits pan-cancer insight into evolutionary changes in mutational processes.

PMID: 32024834 [PubMed - indexed for MEDLINE]



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A proactive genotype-to-patient-phenotype map for cystathionine beta-synthase.

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A proactive genotype-to-patient-phenotype map for cystathionine beta-synthase.

Genome Med. 2020 Jan 30;12(1):13

Authors: Sun S, Weile J, Verby M, Wu Y, Wang Y, Cote AG, Fotiadou I, Kitaygorodsky J, Vidal M, Rine J, Ješina P, Kožich V, Roth FP

Abstract
BACKGROUND: For the majority of rare clinical missense variants, pathogenicity status cannot currently be classified. Classical homocystinuria, characterized by elevated homocysteine in plasma and urine, is caused by variants in the cystathionine beta-synthase (CBS) gene, most of which are rare. With early detection, existing therapies are highly effective.
METHODS: Damaging CBS variants can be detected based on their failure to restore growth in yeast cells lacking the yeast ortholog CYS4. This assay has only been applied reactively, after first observing a variant in patients. Using saturation codon-mutagenesis, en masse growth selection, and sequencing, we generated a comprehensive, proactive map of CBS missense variant function.
RESULTS: Our CBS variant effect map far exceeds the performance of computational predictors of disease variants. Map scores correlated strongly with both disease severity (Spearman's ϱ = 0.9) and human clinical response to vitamin B6 (ϱ = 0.93).
CONCLUSIONS: We demonstrate that highly multiplexed cell-based assays can yield proactive maps of variant function and patient response to therapy, even for rare variants not previously seen in the clinic.

PMID: 32000841 [PubMed - in process]



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Autism-Misregulated eIF4G Microexons Control Synaptic Translation and Higher Order Cognitive Functions.

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Autism-Misregulated eIF4G Microexons Control Synaptic Translation and Higher Order Cognitive Functions.

Mol Cell. 2020 03 19;77(6):1176-1192.e16

Authors: Gonatopoulos-Pournatzis T, Niibori R, Salter EW, Weatheritt RJ, Tsang B, Farhangmehr S, Liang X, Braunschweig U, Roth J, Zhang S, Henderson T, Sharma E, Quesnel-Vallières M, Permanyer J, Maier S, Georgiou J, Irimia M, Sonenberg N, Forman-Kay JD, Gingras AC, Collingridge GL, Woodin MA, Cordes SP, Blencowe BJ

Abstract
Microexons represent the most highly conserved class of alternative splicing, yet their functions are poorly understood. Here, we focus on closely related neuronal microexons overlapping prion-like domains in the translation initiation factors, eIF4G1 and eIF4G3, the splicing of which is activity dependent and frequently disrupted in autism. CRISPR-Cas9 deletion of these microexons selectively upregulates synaptic proteins that control neuronal activity and plasticity and further triggers a gene expression program mirroring that of activated neurons. Mice lacking the Eif4g1 microexon display social behavior, learning, and memory deficits, accompanied by altered hippocampal synaptic plasticity. We provide evidence that the eIF4G microexons function as a translational brake by causing ribosome stalling, through their propensity to promote the coalescence of cytoplasmic granule components associated with translation repression, including the fragile X mental retardation protein FMRP. The results thus reveal an autism-disrupted mechanism by which alternative splicing specializes neuronal translation to control higher order cognitive functioning.

PMID: 31999954 [PubMed - indexed for MEDLINE]



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HK97 gp74 possesses an α-helical insertion in the ββα fold that affects its metal binding, cos site digestion, and in vivo activities.

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HK97 gp74 possesses an α-helical insertion in the ββα fold that affects its metal binding, cos site digestion, and in vivo activities.

J Bacteriol. 2020 Jan 27;:

Authors: Weiditch SA, Bickers SC, Bona D, Maxwell KL, Kanelis V

Abstract
The last gene in the genome of the bacteriophage HK97 encodes for gp74, an HNH endonuclease. HNH motifs contain two conserved His residues and an invariant Asn residue, and adopt a ββα structure. Gp74 is essential for phage head morphogenesis, likely because gp74 enhances the specific endonuclease activity of the HK97 terminase complex. Notably, the ability of gp74 to enhance the terminase-mediated cleavage of the phage cos site requires an intact HNH motif in gp74. Mutation of H82, the conserved metal-binding His residue in the HNH motif, to Ala abrogates gp74-mediated stimulation of terminase activity. Here we present NMR studies demonstrating gp74 contains an α-helical insertion in the Ω-loop, which connects the two β-strands of the ββα fold, and a disordered C-terminal tail. NMR data indicate that the Ω-loop insert makes contacts to the ββα fold and influences the ability of gp74 to bind divalent metal ions. Further, the Ω-loop insert and C-terminal tail contribute to gp74-mediated DNA digestion and to gp74 activity in phage morphogenesis. The data presented here enrich our molecular-level understanding of how HNH endonucleases enhance terminase-mediated digestion of the cos site and contribute to the phage replication cycle.Importance This study demonstrates that residues outside the canonical ββα fold, namely the Ω-loop α-helical insert and a disordered C-terminal tail, control the activity of the HNH endonuclease gp74. The increased divalent-metal ion binding when the Ω-loop insert is removed compared to reduced cos site digestion and phage formation indicates that the Ω-loop insert plays multiple regulatory roles. The data presented here provide insights into the molecular basis of the involvement of HNH proteins in phage DNA packing.

PMID: 31988081 [PubMed - as supplied by publisher]



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Injectable hydrogel enables local and sustained co-delivery to the brain: Two clinically approved biomolecules, cyclosporine and erythropoietin, accelerate functional recovery in rat model of stroke.

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Injectable hydrogel enables local and sustained co-delivery to the brain: Two clinically approved biomolecules, cyclosporine and erythropoietin, accelerate functional recovery in rat model of stroke.

Biomaterials. 2020 Jan 16;235:119794

Authors: Tuladhar A, Obermeyer JM, Payne SL, Siu RCW, Zand S, Morshead CM, Shoichet MS

Abstract
Therapeutic delivery to the brain is limited by the blood-brain barrier and is exacerbated by off-target effects associated with systemic delivery, thereby precluding many potential therapies from even being tested. Given the systemic side effects of cyclosporine and erythropoietin, systemic administration would be precluded in the context of stroke, leaving only the possibility of local delivery. We wondered if direct delivery to the brain would allow new reparative therapeutics, such as these, to be identified for stroke. Using a rodent model of stroke, we employed an injectable drug delivery hydrogel strategy to circumvent the blood-brain barrier and thereby achieved, for the first time, local and sustained co-release to the brain of cyclosporine and erythropoietin. Both drugs diffused to the sub-cortical neural stem and progenitor cell (NSPC) niche and were present in the brain for at least 32 days post-stroke. Each drug had a different outcome on brain tissue: cyclosporine increased plasticity in the striatum while erythropoietin stimulated endogenous NSPCs. Only their co-delivery, but not either drug alone, accelerated functional recovery and improved tissue repair. This platform opens avenues for hitherto untested therapeutic combinations to promote regeneration and repair after stroke.

PMID: 31981761 [PubMed - as supplied by publisher]



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