Results 1-25 of about 1,000
  1. Abstract Background : Accelerated cell apoptosis is a crucial pathogenic mechanism in lupus nephritis (LN) with dysregulated expression levels of long noncoding RNAs (lncRNAs). The expression of pro-apoptotic lincRNA-p21 and its competing endogenous RNA target miR-181a were studied in LN patients, human kidney cell and T-lymphocyte lines with CRISPR interference-conducted repression and lentiviral vector-mediated overexpression of lincRNA-p21, and a mouse LN model. Methods : Clinical samples were collected from LN patients with higher disease activity and control subjects including lupus patients without renal involvement and age/sex-matched healthy controls (HCs). The expression of lincRNA-p21, H19 (anti-apoptotic lncRNA) and miR-181a were examined in peripheral blood mononuclear cells (PBMNCs) and urine cells, and analyzed for clinical correlation. Cell lines were treated with doxorubicin (Dox) to induce apoptosis and evaluate for the expression of lincRNA-p21, caspase 3 and p21. LincRNA-p21-silened HEK 293T and Jurkat transfectants were examined for apoptosis and miR-181a expression. LincRNA-p21-overexpressed HK-2 cells were examined for apoptosis and p53-related down-stream molecules levels. Female Balb/C mice were injected with pristane to induce LN, and examined for the expression of anti-DNA, proteinuria, lincRNA-p21, caspase 3 and p21 as well as in situ apoptosis. Results : Up-regulated expression of lincRNA-p21 rather than H19 were identified in PBMNCs from LN patients, positively correlated with disease activity and proteinuria amount. Higher lincRNA-p21 levels were identified in LN CD4+T cells than other subpopulations. LN urine cells had greater lincRNA-p21 levels than HCs. There were lower miR-181a levels in PBMNCs from LN patients, negatively correlated with disease activity. Dox-induced apoptotic cell lines had up-regulated levels of lincRNA-p21, caspase 3 and p21, whereas down-regulated miR-181a expression with decreased TCRζchain and IL-2 levels was identified in Jurkat cells. LincRNA-p21-silenced transfectants displayed reduced apoptosis with up-regulated miR-181a expression. LincRNA-p21-overexpressed HK-2 cells revealed enhanced apoptosis with up-regulated expression of downstream PUMA and Bax molecules. LN mice had in situ apoptosis and progressively increased anti-dsDNA, proteinuria and renal lincRNA-p21 levels with up-regulated expression of caspase 3 and p21. Conclusions : By using clinical samples, human cell lines and a mouse model, we demonstrate up-regulated expression of lincRNA-p21 in LN, implicating a potential activity biomarker and therapeutic target.
    Date: 2020-09-15
    Authors: Chen Y, Kuo P, Wu C, Chou Y, Chong H, Yang M, Shiau A, Wang C.
    Ref: Research Square
  2. Abstract Background: Larval development in an intermediate host gastropod snail of the genus Biomphalaria is an obligatory component of the life cycle of Schistosoma mansoni . Understanding of the mechanism(s) of host defense may hasten the development of tools that block transmission of schistosomiasis. The allograft inflammatory factor 1, AIF, which is evolutionarily conserved and expressed in phagocytes, is a marker of macrophage activation in both mammals and invertebrates. AIF enhances cell proliferation and migration. The embryonic cell line, termed Bge, from Biomphalaria glabrata is a versatile resource for investigation of the snail-schistosome relationship since Bge exhibits a hemocyte-like phenotype . Hemocytes perform central roles in innate and cellular immunity in gastropods and in some cases can kill the parasite. However, the Bge cells do not kill the parasite in vitro . Methods: Bge cells were transfected by electroporation with plasmid pCas -Bg AIFx4, encoding the Cas9 nuclease and a guide RNA specific for exon 4 of the B. glabrata AIF ( Bg AIF) gene. Transcript levels for Cas9 and for Bg AIF were monitored by quantitative reverse-transcription-PCR and, in parallel, adhesion of gene-edited Bge cells during co-culture with of schistosome sporocysts was assessed. Results: Gene knockout manipulation induced gene-disrupting indels, frequently 1-2 bp insertions and/or 8-30 bp deletions, at the programmed target site; a range from 9 to 17% of the copies of the Bg AIF gene in the Bge population of cells were mutated. Transcript levels for Bg AIF were reduced by up to 73% (49.5±20.2% S.D, P ≤ 0.05, n =12). Adherence by Bg AIF gene-edited (Δ Bg AIF) Bge to sporocysts diminished in comparison to wild type cells, although cell morphology did not change. Specifically, as scored by a semi-quantitative cell adherence index (CAI), fewer Δ Bg AIF than control wild type cells adhered to sporocysts; control CAI, 2.66±0.10, Δ Bg AIF, 2.30±0.22 ( P ≤ 0.01). Conclusion: The findings supported the hypothesis that Bg AIF plays a role in the adherence of B. glabrata hemocytes to sporocysts during schistosome infection in vitro . This demonstration of the activity of programmed gene editing will enable functional genomics approaches using CRISPR/Cas9 to investigate additional components of the snail-schistosome host-parasite relationship.
    Date: 2020-09-14
    Authors: Coelho FS, Rodpai R, Miller A, Karinshak SE, Mann VH, Carvalho OdS, Cladeira RL, Mourao MdM, Brindley PJ, Ittiprasert W.
    Ref: Research Square
  3. Abstract Background: The CRISPR/Cas9 system derived from bacteria especially Streptococcus pyogenes ( Spy Cas9) is currently considered as the most advanced tool used for numerous areas of biological study in which it is useful to target or modify specific DNA sequences. However, low on-target cleavage efficiency and off-target effects impede its wide application. Results: Several different sgRNA design tools for Spy Cas9 by using various algorithms have been developed, including linear regression model, support vector machine (SVM) model and convolutional neuron network model. While the deep insight into the sgRNA features contributing for both on-target activity and off-target still remains to be determined. Here, with public large-scale CRISPR screen data, we evaluated contribution of different features influence sgRNA activity and off-target effects, and developed models for sgRNA off-target evaluation and on-target activity prediction. In addition, we combined both activity and off-target prediction models and packaged them as an online sgRNA design tool, OPT-sgRNA. This tool is freely available via github repositories (https://github.com/Jie-lan/OPT-sgRNA).
    Date: 2020-09-14
    Authors: Lan J, Cui Y, Wang X, Song G, Lou J.
    Ref: Research Square
  4. In vitro culture systems which structurally recapitulate human myogenesis and promote PAX7+ myogenic progenitor maturation have not been established. Here we report human skeletal muscle organoids differentiated from induced pluripotent stem cell lines that contain paraxial mesoderm and neuromesodermal progenitors and develop into organized structures, reassembling neural plate border and dermomyotome formation. Culture conditions causes neural lineage arrest and promotes fetal hypaxial myogenesis towards limb axial anatomical identity and generates sustainable uncommitted PAX7 myogenic progenitors, as well as fibroadipogenic (PDGFRa+) progenitor populations equivalent to those from second trimester of human gestation. Single cell comparison to human fetal and adult myogenic progenitors, reveals distinct molecular signatures for myogenic progenitors in activated (CD44+, CD98+, MYOD1+, MYF5+) and in dormant (PAX7+, FBN1+, SPRY1+, CHODL1+) states, as well as indicated developmental trajectories associated to myogenic commitment and differentiation or self-renewal. Our approach, further validated with Duchenne and CRISPR/Cas9 genome-edited Limb-girdle muscular dystrophy (LGMD2A) patient iPSC lines, provides a robust in vitro developmental system for investigating muscle tissue morphogenesis and homeostasis.
    Date: 2020-09-14
    Authors: Mavrommatis L, Jeong H, Gomez-Giro G, Stehling M, Kienitz M, Psathaki OE, Bixel G, Morosan-Puopolo G, Gerovska D, Arauzo-Bravo MJ, Schwamborn JC, Scholer HR, Adams RH, Vorgerd M, Brand-Saberi B, Zaehres H.
    Ref: bioRxiv
  5. Phototropins, the UVA-blue light photoreceptors, endow plants to detect the direction of light and optimize photosynthesis by regulating chloroplasts positioning and stomatal gas exchange. Little is known about their functions in other developmental responses. A tomato Non-phototropic seedling1 (Nps1) mutant, bearing an Arg495His substitution in the vicinity of LOV2 domain in phototropin1, dominant-negatively blocks phot1 and phot2 responses. The fruits of Nps1 mutant were enriched in carotenoids, particularly lycopene, than its parent, Ailsa Craig. Contrarily, CRISPR/CAS9-edited loss of function phototropin1 mutants displayed subdued carotenoids than the parent. The enrichment of carotenoids in Nps1 fruits is genetically linked with the mutation and exerted in a dominant-negative fashion. Nps1 also altered volatile profiles with high levels of lycopene-derived 6-methyl 5-hepten2-one. The transcript levels of several MEP and carotenogenesis pathways genes were upregulated in Nps1. Nps1 fruits showed altered hormonal profiles with subdued ethylene emission and reduced respiration. Proteome profiles showed a causal link between higher carotenogenesis and increased levels of protein protection machinery, which may stabilize proteins contributing to MEP and carotenogenesis pathways. Given the enhancement of carotenoid content by Nps1 in a dominant-negative fashion, it offers a potential tool for high lycopene-bearing hybrid tomatoes.
    Date: 2020-09-13
    Authors: Kilambi HV, Dindu A, Sharma K, Nizampatnam NR, Gupta N, Thazath NP, Dhanya AJ, Tyagi K, Sharma S, Kumar S, Sharma R, Sreelakshmi Y.
    Ref: bioRxiv
  6. Objective: White adipose tissue (WAT) expansion regulates energy balance and overall metabolic homeostasis. WAT absence or loss occurring through lipodystrophy and lipoatrophy contributes to the development of dyslipidemia, hepatic steatosis, and insulin resistance. We previously demonstrated the sole small ubiquitin-like modifier (SUMO) E2- conjuguating enzyme Ubc9 represses human adipocyte differentiation. Germline and other tissue-specific deletions of Ubc9 frequently cause lethality in mice. As a result, the role of Ubc9 during WAT development remains unknown. Methods: To determine how Ubc9 impacts body composition and energy balance, we generated adipocyte-specific Ubc9 knockout mice (Ubc9a-KO). CRISPR/Cas9 gene editing inserted loxP sites flanking exons 3 and 4 at the Ubc9 locus. Subsequent genetic crosses to AdipoQ-Cre transgenic mice allowed deletion of Ubc9 in white and brown adipocytes. We measured multiple metabolic endpoints that describe energy balance and carbohydrate metabolism in Ubc9a-KO and littermate controls during postnatal growth. Results: To our surprise, Ubc9a-KO mice developed hyperinsulinemia and hepatic steatosis. Global energy balance defects emerged from dysfunctional WAT marked by pronounced local inflammation, loss of serum adipokines, hepatomegaly, and near absence of major adipose tissue depots. We observed progressive lipoatrophy that commences in the early adolescent period. Conclusions: Our results demonstrate that Ubc9 expression in mature adipocytes is essential for maintaining WAT expansion. Deletion of Ubc9 in fat cells compromised and diminished adipocyte function that provoked WAT inflammation and ectopic lipid accumulation in the liver. Our findings reveal an indispensable role for Ubc9 during white adipocyte expansion and endocrine control of energy balance.
    Date: 2020-09-12
    Authors: Cox AR, Chernis N, Kim KH, Masschelin PM, Saha PK, Briley SM, Sharp R, Felix JB, Moore DD, Pangas SA, Hartig SM.
    Ref: bioRxiv
  7. SUMMARY The ongoing SARS-CoV-2 pandemic has devastated the global economy and claimed nearly one million lives, presenting an urgent global health crisis. To identify host factors required for infection by SARS-CoV-2 and seasonal coronaviruses, we designed a focused high-coverage CRISPR-Cas9 library targeting 332 members of a recently published SARS-CoV-2 protein interactome. We leveraged the compact nature of this library to systematically screen four related coronaviruses (HCoV-229E, HCoV-NL63, HCoV-OC43 and SARS-CoV-2) at two physiologically relevant temperatures (33 °C and 37 °C), allowing us to probe this interactome at a much higher resolution relative to genome scale studies. This approach yielded several new insights, including unexpected virus and temperature specific differences in Rab GTPase requirements and GPI anchor biosynthesis, as well as identification of multiple pan-coronavirus factors involved in cholesterol homeostasis. This coronavirus essentiality catalog could inform ongoing drug development efforts aimed at intercepting and treating COVID-19, and help prepare for future coronavirus outbreaks. HIGHLIGHTS Focused CRISPR screens targeting host factors in the SARS-CoV-2 interactome were performed for SARS-CoV-2, HCoV-229E, HCoV-NL63, and HCoV-OC43 coronaviruses. Focused interactome CRISPR screens achieve higher resolution compared to genome-wide screens, leading to the identification of critical factors missed by the latter. Parallel CRISPR screens against multiple coronaviruses uncover host factors and pathways with pan-coronavirus and virus-specific functional roles. The number of host proteins that interact with a viral bait protein is not proportional to the number of functional interactors. Novel SARS-CoV-2 host factors are expressed in relevant cell types in the human airway.
    Date: 2020-09-11
    Authors: Hoffmann H, Schneider WM, Sánchez-Rivera FJ, Luna JM, Ashbrook AW, Soto-Feliciano YM, Leal AA, Le Pen J, Ricardo-Lax I, Michailidis E, Hao Y, Stenzel AF, Peace A, Allis CD, Lowe SW, MacDonald MR, Poirier JT, Rice CM.
    Ref: bioRxiv
  8. ABSTRACT SATB homeobox proteins are important regulators of developmental gene expression. Among the stem cell lineages determined during early embryonic development, trophoblast stem (TS) cells exhibit robust SATB expression. Both SATB1 and SATB2 act to maintain trophoblast stem-state. However, the molecular mechanisms that regulate TS-specific Satb expression are not yet known. We identified Satb1 variant 2 as the predominant transcript in trophoblasts. Histone marks, and RNA polymerase II occupancy in TS cells indicated active state of the promoter. A novel cis-regulatory region with active histone marks was identified ∼21kbp upstream of variant 2 promoter. CRISPR/Cas9 mediated disruption of this sequence decreased Satb1 expression in TS cells and chromatin conformation capture confirmed looping of this regulatory region into the promoter. Scanning position weight matrices across the enhancer predicted two ELF5 binding sites in close vicinity of SATB1 sites, which were confirmed by chromatin immunoprecipitation. Knockdown of ELF5 downregulated Satb1 expression in TS cells and overexpression of ELF5 increased the enhancer-reporter activity. Interestingly, ELF5 interacts with SATB1 in TS cells, and the enhancer activity was upregulated following SATB overexpression. Our findings indicate that trophoblast-specific Satb1 expression is regulated by long-range chromatin looping of an enhancer that interacts with ELF5 and SATB proteins.
    Date: 2020-09-11
    Authors: Yu W, Chakravarthi VP, Borosha S, Ratri A, Dalal K, Wolfe MW, Starks RR, Tuteja G, Rumi MK.
    Ref: bioRxiv
  9. Summary Drugs that block the activity of the methyltransferase EZH2 are in clinical development for the treatment of non-Hodgkin lymphomas harboring gain-of-function EZH2 mutations that enhance its polycomb repressive function. In contrast, in castration-resistant prostate cancer (CRPC) we have previously reported that EZH2 plays a non-canonical role as a transcriptional activator. In this setting, we now show that EZH2 inhibitors can also block the non-canonical activity of EZH2 and inhibit the growth of CRPC cells. Gene expression and epigenomic profiling of cells treated with EZH2 inhibitors demonstrated that rather than de-repressing tumor suppressor genes silenced by PRC2, EZH2 inhibitors downregulate a set of DNA repair genes that are directly regulated by EZH2. In addition, genome-wide CRISPR/Cas9-mediated loss-of-function screens in the presence of EZH2 inhibitors identified these DNA repair genes to underlie the growth-inhibitory function of these compounds. Interrogation of public data from diverse solid tumor types expressing wild-type EZH2 showed that expression of DNA damage repair genes is significantly correlated with cellular sensitivity to EZH2 inhibitors. Consistent with these findings, treatment of CRPC cells with EZH2 inhibitors dramatically enhanced their sensitivity to genotoxic stress. These studies reveal a previously unappreciated mechanism of action of EZH2 inhibitors and provide a mechanistic basis for potential new combination cancer therapies.
    Date: 2020-09-11
    Authors: Liao Y, Chen C, Shah N, Xiao T, Feit A, Yang M, Cai C, Gao S, Xue P, Liu Z, Xu H, Lee J, Li W, Mei S, Pierre RS, Shu S, Fei T, Duarte M, Zhao J, Bradner JE, Polyak K, Kantoff PW, Long H, Balk SP, Liu XS, Brown M, Xu K.
    Ref: bioRxiv
  10. Abstract Background: The C lustered R egularly I nterspaced S hort P alindromic R epeats (CRISPR)/Cas9 system has become a powerful tool for functional genomics in plants. The RNA-guided nuclease can be used to not only generate precise genomic mutations, but also to manipulate gene expression when present as a deactivated protein (dCas9). Results: In this study, we describe a vector toolkit for analyzing dCas9-mediated activation (CRISPRa) or inactivation (CRISPRi) of gene expression in maize protoplasts. An improved maize protoplast isolation and transfection method is presented, as well as a description of dCas9 vectors to enhance or repress maize gene expression. Conclusions: We anticipate that this maize protoplast toolkit will streamline the analysis of gRNA candidates and facilitate genetic studies of important trait genes in this transformation-recalcitrant plant.
    Date: 2020-09-11
    Authors: Gentzel I, Park CH, Bellizzi M, Xiao G, Gadharve K, Murphree C, Yang Q, LaMantia J, Redinbaugh M, Balint-Kurti P, Sit T, Wang G.
    Ref: Research Square
  11. Abstract The human genome contains vast genetic diversity in the form of naturally occurring coding variants, yet the impact of these variants on protein function and physiology is poorly understood. RGS14 is a multifunctional signaling protein that suppresses synaptic plasticity in dendritic spines of hippocampal neurons. RGS14 also is a nucleocytoplasmic shuttling protein, suggesting that balanced nuclear import/export and dendritic spine localization are essential for RGS14 functions. We identified genetic variants L505R (LR) and R507Q (RQ) located within the nuclear export sequence (NES) of human RGS14 . Here we report that RGS14 carrying LR or RQ profoundly impacts protein functions in hippocampal neurons and brain. Following nuclear import, RGS14 nuclear export is regulated by Exportin 1 (XPO1/CRM1). Remarkably, LR and RQ variants disrupt RGS14 binding to Gαi1-GDP and XPO1, nucleocytoplasmic equilibrium, and capacity to inhibit LTP. Variant LR accumulates irreversibly in the nucleus, preventing RGS14 binding to G proteins, localization to dendritic spines, and inhibitory actions on LTP induction, while variant RQ exhibits a mixed phenotype. When introduced into mice by CRISPR/Cas9, RGS14-LR protein expression was detected predominantly in the nuclei of neurons within hippocampus, central amygdala, piriform cortex, and striatum, brain regions associated with learning and synaptic plasticity. Whereas mice completely lacking RGS14 exhibit enhanced spatial learning, mice carrying variant LR exhibit normal spatial learning, suggesting that RGS14 may have distinct functions in the nucleus independent from those in dendrites and spines. These findings show that naturally occurring genetic variants can profoundly alter normal protein function, impacting physiology in unexpected ways.
    Date: 2020-09-10
    Authors: Squires KE, Gerber KJ, Tillman MC, Lustberg DJ, Montañez-Miranda C, Zhao M, Ramineni S, Scharer CD, Shu F, Schroeder JP, Ortlund EA, Weinshenker D, Dudek SM, Hepler JR.
    Ref: bioRxiv
  12. ABSTRACT The mitotic spindle functions as a molecular micromachine that evenly distributes chromosomes into two daughter cells during cell division. A major mechanical element of the spindle are kinetochore fibers attached to sister kinetochores on each chromosome and laterally linked by a bundle of antiparallel microtubules called the bridging fiber. Spindle microtubules are mainly nucleated at the centrosome and on the lateral surface of existing microtubules by the augmin complex. However, it is unknown how the augmin-mediated nucleation affects functionally distinct microtubule bundles and thus the architecture and forces within the spindle. Here we show, by using siRNA depletion and CRISPR knock-out of the augmin complex subunits HAUS6 and HAUS8 in human cells, that augmin is a major contributor to the nucleation of bridging microtubules. Augmin depletion resulted in a ∼70% reduction of the microtubule number in bridging fibers and ∼40% in kinetochore fibers, suggesting that the bridging microtubules are largely nucleated at the surface of present microtubules. In augmin-depleted cells, the interkinetochore distance decreases preferentially for kinetochores that lack a bridging fiber, independently of the thickness of their k-fibers, indicating that augmin affects forces on kinetochores largely via bridging fibers. Without augmin the number of bridging fibers decreases, with the remaining ones mostly confined to the spindle periphery with an increased overlap length. The reduced number of microtubules also results in a slower poleward flux. Our results demonstrate a critical role of augmin in the formation of bridging microtubules and proper architecture of the metaphase spindle, suggesting a model where sliding of augmin-nucleated bridging microtubules promotes poleward flux of k-fibers and thus tension on kinetochores.
    Date: 2020-09-10
    Authors: Manenica M, Koprivec I, Štimac V, Simunić J, Tolić IM.
    Ref: bioRxiv
  13. Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy, as 90% of patients do not survive beyond five years from diagnosis. This dismal prognosis is largely due to the advanced stage of the disease at diagnosis, which precludes potentially curative surgical resection. Although early detection strategies hold significant promise for improving patient outcomes, there is still no accurate diagnostic tool to detect incipient PDAC. Here, we sought to develop antibodies for the early detection of PDAC by positron-emission tomography (PET) imaging. Accordingly, we establish a pipeline to generate novel tumor-specific monoclonal antibodies (mAbs) against cell-surface proteins of PDAC patient-derived organoids (PDOs). We identify a panel of 16 tumor organoid-binding antibodies (TOBi-bodies) that display high reactivity to human PDAC tissues but not to matched adjacent normal pancreas. We then employ biochemical, flow cytometric, mass spectrometric, and CRISPR/Cas9-mediated knockout methods to determine the cognate antigens of these TOBi-bodies. We identify two mAbs that bind to tumor-specific variants of the surface protein CEACAM6 and show minimal binding to normal tissues. PET imaging in mouse models using these TOBi-bodies enables the detection of incipient human organoid-derived PDAC tumors that are rather undetectable by palpation or high-resolution ultrasound imaging techniques. We propose that further development of these mAbs as PET radiotracers could facilitate the early detection and accurate staging of PDAC.
    Date: 2020-09-10
    Authors: Oni TE, Bautista C, Merrill JR, Goos JA, Rivera KD, Miyabayashi K, Biffi G, Garcia L, Plenker D, Patel H, Elyada E, Samaritano M, Yu KH, Pappin DJ, Goggins MG, Hruban RH, Lewis JS, Lyons SK, Yeh JT, Tuveson DA.
    Ref: bioRxiv
  14. Abstract Mutations in SPG11 constitute the major cause of autosomal recessive Hereditary Spastic Paraplegia (HSP) with thinning of the corpus callosum. Previous studies showed that SPG11/spatacsin orchestrates cellular traffic events through the formation of a coat-like complex and its loss of function results in lysosomal impairments. However, the upstream mechanisms that regulate SPG11/spatacsin trafficking are unknown. Here, using proteomics and CRISPR/Cas9-mediated tagging of endogenous SPG11/spatacsin, we identified a subset of 14-3-3 proteins as physiological interactors of SPG11. The interaction is modulated by PKA-dependent phosphorylation of SPG11 at Ser1955, which initiates SPG11 trafficking from the plasma membrane. Our study provides novel insight in understanding SPG11 physio-pathological roles with mechanistic dissection of its associated pathways.
    Date: 2020-09-10
    Authors: Cogo S, Tomkins JE, Vavouraki N, Forcellato F, Franchin C, Tessari I, Arrigoni G, Cendron L, Manzoni C, Civiero L, Lewis PA, Greggio E.
    Ref: bioRxiv
  15. Abstract Fibronectin (Fn1) is an essential ECM glycoprotein important for embryonic development and homeostasis. The functions of Fn1 in regulating cell fate decisions, morphogenesis and cellular responses to injury are intimately linked to the process of Fn1 fibrillogenesis. Therefore, understanding the mechanisms by which Fn1 proteins assemble into fibrils is necessary to gain insights into diverse functions of Fn1. Using CRISPR/Cas9 mutagenesis, we generated mice and cell lines wherein a sequence encoding a fluorescent protein (FP) was knocked into the Fn1 locus replacing the termination codon, resulting in the expression of Fn1-FP proteins subject to endogenous regulation. Live imaging and super-resolution microscopy revealed that Fn1 fibrils are not continuous fibers as was thought before, instead, they are comprised of a discontinuous array of small nanodomains. Live imaging showed that Fn1 nanodomains are mobile and that they become arranged into progressively longer linear arrays as they move toward the nucleus in parallel with the rearward actin flow. The organization of Fn1 nanodomains into linear fibrillar arrays but not the formation of Fn1 nanodomains is regulated by the interactions mediated by the Fn1 N-terminal assembly domain. The nanodomain architecture of Fn1 fibrils is observed in multiple contexts: in three-dimensional ECM in vivo , on substrata of different composition and stiffness, and is retained when the linkage of Fn1 fibrils to cells is disrupted. The modular assembly and structure of Fn1 fibrils bears important implications for mechanisms of ECM remodeling and signal transduction.
    Date: 2020-09-10
    Authors: Tomer D, Munshi S, Alexander BE, French B, Vedula P, House A, Guvendiren M, Kashina A, Schwarzbauer JE, Astrof S.
    Ref: bioRxiv
  16. Connexin 43 (Cx43) forms gap junctions that mediate the direct intercellular diffusion of ions and small molecules between adjacent cells. Cx43 displays both pro- and anti-tumorigenic properties, but the mechanisms underlying these characteristics are not fully understood. Tunneling nanotubes (TNTs) are long and thin membrane projections that connect cells, facilitating the exchange of not only small molecules, but also larger proteins, organelles, bacteria, and viruses. Typically, TNTs exhibit increased formation under conditions of cellular stress and are more prominent in cancer cells, where they are generally thought to be pro-metastatic and to provide growth and survival advantages. Cx43 has been described in TNTs, where it is thought to regulate small molecule diffusion through gap junctions. Here, we developed a high-fidelity CRISPR/Cas9 system to knockout (KO) Cx43. We found that loss of Cx43 expression was associated with significantly reduced TNT length and number in breast cancer cell lines. Notably, secreted factors present in conditioned medium stimulated TNTs more potently when derived from Cx43-expressing cells than from KO cells. Moreover, TNT formation was significantly induced by inhibition of several key cancer signaling pathways that both regulate Cx43 and are regulated by Cx43, including RhoA kinase (ROCK), protein kinase A (PKA), focal adhesion kinase (FAK), and p38. Intriguingly, drug-induced stimulation of TNTs was more potent in Cx43 KO cells than in wild-type cells. In conclusion, this work describes a novel non-canonical role for Cx43 in regulating TNTs, identifies key cancer signaling pathways that regulate TNTs in this setting, and provides mechanistic insight into a pro-tumorigenic role of Cx43 in cancer.
    Date: 2020-09-09
    Authors: Tishchenko A, Domínguez Azorín D, Vidal-Brime L, Muñoz MJ, Arenas PJ, Pearce C, Girao H, Ramón y Cajal S, Aasen T.
    Ref: Preprints.org
  17. Abstract The R47H variant of the microglial membrane receptor TREM2 is linked to increased risk of late onset Alzheimer’s disease. Human induced pluripotent stem cell derived microglia (iPS-Mg) from patient iPSC lines expressing the AD-linked R47H het TREM2 variant, common variant (Cv) or an R47H hom CRISPR edited line and its isogeneic control, demonstrated that R47H-expressing iPS-Mg expressed a deficit in signal transduction in response to the TREM2 endogenous ligand phosphatidylserine with reduced pSYK-pERK1/2 signalling and a reduced NLRP3 inflammasome response, (including ASC speck formation, Caspase-1 activation and IL-1beta secretion). Apoptotic cell phagocytosis and soluble TREM2 shedding were unaltered, suggesting a disjoint between these pathways and the signalling cascades downstream of TREM2 in R47H-expressing iPS-Mg, whilst metabolic deficits in glycolytic capacity and maximum respiration were reversed when R47H expressing iPS-Mg were exposed to PS+ expressing cells. These findings suggest that R47H-expressing microglia are unable to respond fully to cell damage signals such as phosphatidylserine, which may contribute to the progression of neurodegeneration in late-onset AD.
    Date: 2020-09-09
    Authors: Cosker K, Mallach A, Limaye J, Piers TM, Staddon J, Neame SJ, Hardy J, Pocock JM.
    Ref: bioRxiv
  18. Abstract Asparagine deprivation by L-Asparaginase (L-ASNase) is an effective therapeutic strategy in Acute Lymphoblastic Leukemia, with resistance occurring due to upregulation of ASNS, the only human enzyme synthetizing Asparagine 1 . L-Asparaginase efficacy in solid tumors is limited by dose-related toxicities 2 . Large-scale loss of function genetic in vitro screens identified ASNS as a cancer dependency in several solid malignancies 3,4 . Here we evaluate the therapeutic potential of targeting ASNS in melanoma cells. While we confirm in-vitro dependency on ASNS silencing, this is largely dispensable for in vivo tumor growth, even in face of asparagine deprivation, prompting us characterize such resistance mechanism to devise novel therapeutic strategies. Using ex vivo quantitative proteome and transcriptome profiling, we characterize the compensatory mechanism elicited by ASNS knockout melanoma cells allowing their survival. Mechanistically, a genome-wide CRISPR screen revealed that such resistance mechanism is elicited by a dual axis: GCN2-ATF4 aimed at restoring amino acids levels and MAPK-BCLXL to promote survival. Importantly, pharmacological inhibition of such nodes synergizes with L-Asparaginase-mediated Asparagine deprivation in ASNS deficient cells suggesting novel potential therapeutic combinations in melanoma.
    Date: 2020-09-09
    Authors: Apfel V, Begue D, Cordo V, Holzer L, Martinuzzi L, Buhles A, Kerr G, Barbosa I, Naumann U, Piquet M, Ruddy D, Weiss A, Ferretti S, Almeida R, Bonenfant D, Tordella L, Galli GG.
    Ref: bioRxiv
  19. Abstract Background Engineered nuclease-mediated gene targeting through homology-directed repair (HDR) in autologous hematopoietic stem and progenitor cells (HSPCs) has the potential to cure β-thalassemia (β-thal). Although previous studies have precisely corrected site-specific HBB mutations by HDR in vitro and in vivo, targeting the various HBB mutations in β-thal is still challenging. Here, we devised a universal strategy to achieve repaired most types of HBB mutations through the CRISPR/Cas9 and the rAAV6 donor. Methods Using cord blood-derived HSPCs from health donors, we tested the strategy to achieved highly efficient targeted integration by optimizing design and delivery parameters of a ribonucleoprotein (RNP) complex comprising Cas9 protein and modified single guide RNA, together with a rAAV6 donor. We assessed the edited HSPCs function in vitro by methylcellulose colonies assay, CFU assay, differentiation experiment and Wright-Giemsa staining. Edited HSPCs transplanted into NSI mice to assess the long-term reconstitution in vivo. Whole-genome sequencing was used to analysis the off-target mutagenesis of edited HSPCs. Results Edited HSPCs exhibited normal multilineage formation and erythroid differentiation abilities without off-target mutagenesis and retained the ability to engraft. Moreover, we used the strategy to efficiently correct the β-CD41/42 mutation of patient-derived HSPCs, erythrocytes differentiation from which expressed more HBB mRNA than uncorrected cells. Conclusion This strategy demonstrated a universal approach to correct most types of HBB gene mutations in β-thal.
    Date: 2020-09-09
    Authors: He L, Yang Y, Cheng Y, Wu H, Lin S, Song B, Liu N, Chen D, Lu D, Yang Y, Zeng J, Fan Y, Xiaofang S.
    Ref: Research Square
  20. Abstract The evolution of multicellularity was accompanied by the emergence of processes to regulate cell fate, identity and differentiation in a robust and faithful manner. Chromatin regulation has emerged as a key process in development and yet its contribution to the evolution of such processes is largely unexplored. Chromatin is regulated by a diverse set of proteins, which themselves are tightly regulated in order to play cell/ tissue-specific functions. Using the cnidarian Nematostella vectensis , a model for basal metazoans, we explore the function of one such chromatin regulator, Lysine specific demethylase 1 (Lsd1). We generated an endogenously tagged allele and show that the expression of NvLsd1 is developmentally regulated and higher in differentiated neural cells than their progenitors. We further show, using a CRISPR/Cas9 generated mutant that loss of NvLsd1 leads to several distinct developmental abnormalities. Strikingly, NvLsd1 loss leads to the almost complete loss of differentiated cnidocytes, cnidarian-specific neural cells, which we show to be the result of a cell-autonomous requirement for NvLsd1 . Together this suggests that complex regulation of developmental processes by chromatin modifying proteins predates the split of the cnidarian and bilaterian lineages, approximately 600 million years ago, and may constitute an ancient feature of animal development.
    Date: 2020-09-08
    Authors: Gahan JM, Kouzel IU, Rentzsch F.
    Ref: bioRxiv
  21. ABSTRACT Downregulation of major histocompatibility complex I (MHC-I) on tumor cells is a primary means of immune evasion by many types of cancer. Additionally, MHC-I proteins are a primary target of humoral and cellular mechanisms associated with transplant rejection. Transmissible tumors that overcome allograft rejection mechanisms and evade anti-tumor immunity have killed thousands of wild Tasmanian devils ( Sarcophilus harrisii ). Interferon-gamma (IFNG) upregulates surface MHC-I expression on devil facial tumor (DFT) cells but is not sufficient to induce tumor regressions. Transcriptome analysis of IFNG-treated DFT cells revealed strong upregulation of NLRC5 , a master regulator of MHC-I in humans and mice. To explore the role of NLRC5 in transmissible cancers, we developed DFT cells lines that constitutively overexpress NLRC5. Transcriptomic results suggest that the role of NLRC5 as a master regulator of MHC-I is conserved in devils. Furthermore, NLRC5 was shown to drive the expression of many components of the antigen presentation pathway. To determine if MHC-I is a target of allogeneic immune responses, we tested serum from devils with natural DFT regressions against DFT cells. Antibody binding occurred with cells treated with IFNG and overexpressed NLRC5. However, CRISPR/Cas9-mediated knockout of MHC-I subunit beta-2-microglobulin ( B2M ) eliminated antibody binding to DFT cells. Consequently, MHC-I could be identified as a target for anti-tumor and allogeneic immunity and provides mechanistic insight into MHC-I expression and antigen presentation in marsupials. NLRC5 could be a promising target for immunotherapy and vaccines to protect devils from transmissible cancers and inform development of transplant and cancer therapies for humans.
    Date: 2020-09-07
    Authors: Ong CEB, Patchett AL, Darby JM, Chen J, Liu G, Lyons AB, Woods GM, Flies AS.
    Ref: bioRxiv
  22. Abstract CRISPR/Cas9 mutagenesis is a revolutionary tool for genetics in organismal and cell culture systems. One notable caveat with this system is the potential for phenotype-inducing off-target/background mutations. There has been considerable success in modifying the methodology to minimize these potential confounds. Here we have developed a tool to functionally demonstrate that a targeted mutation of interest is responsible for the phenotype observed. This approach creates revertable mutations in cell culture systems using CRISPR/Cas9-induced homology-directed repair (HDR) to insert a LoxP-flanked transcriptional stop sequence into an early intron of a target gene. This method has the potential to be used in multiplexed and inducible scenarios to restore gene function within a given experiment.
    Date: 2020-09-07
    Authors: Walsh J, Eggenschwiler J.
    Ref: bioRxiv
  23. Abstract Animals are extremely useful genetic tools in science and global resources in agriculture. However, a single sex is often required in surplus, and current genetic methods for producing all-female or all-male litters are inefficient. Using the mouse as a model, we developed a synthetic, two-part bicomponent strategy for generating all-male litters. We achieved this using CRISPR-Cas9 genome editing technology to generate large stable knock-ins on the autosomes and X chromosome. The bicomponent system functions via the sex-specific co-inheritance of a Cas9 transgene and an sgRNA transgene targeting the essential Topoisomerase 1 gene. This technology proved to be highly efficient in generating on-target mutations, resulting in embryonic lethality of the target sex. Our study is the first to successfully generate all-male mammalian litters using a CRISPR-Cas9 bicomponent system and provides great strides towards generating single-sex litters for laboratory or agricultural research.
    Date: 2020-09-07
    Authors: Douglas C, Maciulyte V, Zohren J, Snell DM, Ojarikre OA, Ellis PJ, Turner JM.
    Ref: bioRxiv
  24. Abstract We present OpenAWSEM and Open3SPN2, new cross-compatible implementations of coarse-grained models for protein (AWSEM) and DNA (3SPN2) molecular dynamics simulations within the OpenMM framework. These new implementations retain the chemical accuracy and intrinsic efficiency of the original models while adding GPU acceleration and the ease of forcefield modification provided by OpenMM’s Custom Forces software framework. By utilizing GPUs, we achieve more than a 100-fold speedup in protein and protein-DNA simulations over the existing LAMMPS-based implementations running on a CPU. We showcase the benefits of OpenMM’s Custom Forces framework by devising and implementing two new potentials that allow us to address important aspects of protein folding and structure prediction and by testing the ability of the combined OpenAWSEM and Open3SPN2 to model protein-DNA binding. The first potential is used to describe the changes in effective interactions that occur as a protein becomes partially buried in a membrane. We also introduced an interaction to describe proteins with multiple disulfide bonds. Using simple pairwise disulfide bonding terms results in unphysical clustering of cysteine residues, posing a problem when simulating the folding of proteins with many cysteines. We now can computationally reproduce Anfinsen’s early Nobel prize winning experiments [1] by using OpenMM’s Custom Forces framework to introduce a multi-body disulfide bonding term that prevents unphysical clustering. Our protein-DNA simulations show that the binding landscape is funneled towards structures that are quite similar to those found using experiments. In summary, this paper provides a simulation tool for the molecular biophysics community that is both easy to use and sufficiently efficient to simulate large proteins and large protein-DNA systems that are central to many cellular processes. These codes should facilitate the interplay between molecular simulations and cellular studies, which have been hampered by the large mismatch between the time and length scales accessible to molecular simulations and those relevant to cell biology. Author summary The cell’s most important pieces of machinery are large complexes of proteins often along with nucleic acids. From the ribosome, to CRISPR-Cas9, to transcription factors and DNA-wrangling proteins like the SMC-Kleisins, these complexes allow organisms to replicate and enable cells to respond to environmental cues. Computer simulation is a key technology that can be used to connect physical theories with biological reality. Unfortunately, the time and length scales accessible to molecular simulation have not kept pace with our ambition to study the cell’s molecular factories. Many simulation codes also unfortunately remain effectively locked away from the user community who need to modify them as more of the underlying physics is learned. In this paper, we present OpenAWSEM and Open3SPN2, two new easy-to-use and easy to modify implementations of efficient and accurate coarse-grained protein and DNA simulation forcefields that can now be run hundreds of times faster than before, thereby making studies of large biomolecular machines more facile.
    Date: 2020-09-07
    Authors: Lu W, Bueno C, Schafer NP, Moller J, Jin S, Chen X, Chen M, Gu X, de Pablo JJ, Wolynes PG.
    Ref: bioRxiv
  25. Abstract Antimalarial drug which target more than one life stage of the parasite are valuable tools in the fight against malaria. Previous generation of antifolate drugs are able to inhibit replicative stages of drug-sensitive, but not resistant parasites in humans, and mosquitoes. The lack of reliable gametocyte-producing, antifolate resistant P. falciparum hindrance the development of new antifolate compounds against mosquito stages. We used CRISPR-Cas9 technology to develop transgenic gametocyte producing P. falciparum with quadruple mutations in dhfr gene, using NF54 as a parental strain. The transgenic parasites gained pyrimethamine resistance while maintaining the gametocyte producing activity. In contrast to pyrimethamine that cannot inhibit exflagellation of the quadruple dhfr mutant parasite, the novel antifolate P218 showed a good potency for exflagellation inhibition (exflagellation IC 50 10.74 ± 4.22 nM). The exflagellation IC 50 was 5.3 times lower than erythrocytic IC 50 suggesting that the human to mosquito transmission poses as a strong barrier to prevent P218 resistant parasite among population. This study demonstrates that P218 can be considered as a highly potent tool to prevent the spread of antifolate resistant parasites. Graphical Abstract Research Highlights - Transgenic gametocyte producing pyrimethamine resistant P. falciparum was generated. - P218 asexual stage IC 50 in NF54-4mut Pfdhfr was 56.94 ± 15.69 nM. - P218 exflagellation IC 50 in NF54-4mut Pfdhfr was 10.74 ± 4.22 nM. - P218 exflagellation IC 50 in NF54-4mut Pfdhfr is 5.3 times lower than erythrocytic IC 50 . - P218 is an invaluable tool for malaria treatment and transmission control.
    Date: 2020-09-06
    Authors: Posayapisit N, Pengon J, Prommana P, Shoram M, Yuthavong Y, Uthaipibull C, Kamchonwongpaisan S, Jupatanakul N.
    Ref: bioRxiv
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