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Rxivist combines biology preprints from bioRxiv and medRxiv with data from Twitter to help you find the papers being discussed in your field. Currently indexing 131,285 papers from 561,734 authors.

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in category molecular biology

3,933 results found. For more information, click each entry to expand.

3721: Exploration of endogenous miRNA-200b/c activity and regulation through a functional dual fluorescence reporter
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Posted 21 Jul 2020

Exploration of endogenous miRNA-200b/c activity and regulation through a functional dual fluorescence reporter
93 downloads bioRxiv molecular biology

Paradesi Gollavilli, Beatrice Parma, Aarif Siddiqui, Hai Yang, Vignesh Ramesh, Francesca Napoli, Annemarie Schwab, Ramakrishnan Natesan, Irfan Ahmed Asangani, Thomas Brabletz, Christian Pilarsky, Paolo Ceppi

Since their discovery, microRNAs (miRNA)s have been widely studied in almost every aspect of biology and medicine, leading to the identification of important gene regulation circuits and cellular mechanisms. However, investigations are generally focused on the analysis of their downstream targets and biological functions in overexpression and knockdown approaches, while miRNAs endogenous levels and activity remain poorly understood. Here, we used the cellular plasticity-regulating process of epithelial-to-mesenchymal transition (EMT) as a model to show the efficacy of a fluorescent sensor to separate cells with distinct EMT signatures, based on miR-200b/c activity. The system was further combined with a CRISPR-Cas9 screening platform to unbiasedly identify miR-200b/c upstream regulating genes. The sensor allows to infer miRNAs fundamental biological properties, as profiling of sorted cells indicated miR-200b/c as a molecular switch between EMT differentiation and proliferation, and suggested a role for metabolic enzymes in miR-200/EMT regulation. Analysis of miRNAs endogenous levels and activity could lead to a better understanding of their biological role in physiology and disease. ### Competing Interest Statement The authors have declared no competing interest.

3722: Identification of qPCR reference genes suitable for normalising gene expression in the developing mouse embryo
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Posted 29 Oct 2020

Identification of qPCR reference genes suitable for normalising gene expression in the developing mouse embryo
93 downloads bioRxiv molecular biology

John C.W. Hildyard, Dominic J. Wells, Richard J. Piercy

Mammalian embryogenesis is an intricate, tightly orchestrated process. Progression from zygote through somitogenesis and on to organogenesis and maturity involves many interacting cell types and multiple differentiating cell lineages. Quantitative PCR analysis of gene expression in the developing embryo is a valuable tool for deciphering these interactions and tracing lineages, but normalisation of qPCR data to stably expressed reference genes is essential. Patterns of gene expression change globally and dramatically as embryonic development proceeds, rendering identification of appropriate reference genes challenging at both the whole embryo- and individual tissue-level. We have investigated expression stability in mouse embryos from mid to late gestation (E11.5-E18.5), both at the whole-embryo level, and within more restricted tissue domains (head, developing forelimb), using 15 candidate reference genes ( ACTB, 18S, SDHA, GAPDH, HTATSF1, CDC40, RPL13A, CSNK2A2, AP3D1, HPRT1, CYC1, EIF4A, UBC, B2M and PAK1IP1 ), and four complementary algorithms (geNorm, Normfinder, Bestkeeper and deltaCt). Unexpectedly, all methods suggest that many genes within our candidate panel are acceptable references, and despite disagreement over highest-scoring candidates, AP3D1, RPL14A and PAK1IP1 are the strongest performing genes overall. Conversely, HPRT1 and B2M are consistently poor choices: these genes show strong developmental regulation. We further show that use of AP3D1, RPL13A and PAK1IP1 can reveal subtle patterns of developmental expression even in genes ostensibly ranked as acceptable ( CDC40, HTATSF1 ), and thus these three represent universally suitable reference genes for the mouse embryo. ### Competing Interest Statement The authors have declared no competing interest.

3723: Genetic dissection of the mitochondrial lipoylation pathway in yeast
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Posted 24 Nov 2020

Genetic dissection of the mitochondrial lipoylation pathway in yeast
93 downloads bioRxiv molecular biology

Laura P. Pietikainen, M. Tanvir Rahman, J. Kalervo Hiltunen, Carol L. Dieckmann, Alexander Johannes Kastaniotis

Background: Lipoylation of 2-ketoacid dehydrogenases is essential for mitochondrial function in eukaryotes. While the basic principles of the lipoylation processes have been worked out, we still lack a thorough understanding of the details of this important post-translational modification pathway. Here we used yeast as a model organism to characterize substrate usage by the highly conserved eukaryotic octanoyl/lipoyl transferases in vivo and queried how amenable the lipoylation system is to supplementation with exogenous substrate. Results: We show that the requirement for mitochondrial fatty acid synthesis to provide substrates for lipoylation of the 2-ketoacid dehydrogenases can be bypassed by supplying the cells with free lipoic acid (LA) or octanoic acid (C8) and a mitochondrially targeted fatty acyl/lipoyl activating enzyme. We also provide evidence that the S. cerevisiae lipoyl transferase Lip3, in addition to transferring LA from the glycine cleavage system H protein to the pyruvate dehydrogenase (PDH) and -ketoglutarate dehydrogenase (KGD) E2 subunits, can transfer this cofactor from the PDH complex to the KGD complex. In support of yeast as a model system for human metabolism, we demonstrate that the human octanoyl/lipoyl transferases can substitute for their counterparts in yeast to support respiratory growth and protein lipoylation. Like the wild-type yeast enzyme, the human lipoyl transferase LIPT1 responds to LA supplementation in the presence of the activating enzyme LplA. Conclusions: In the yeast model system, the eukaryotic lipoylation pathway can use free LA and C8 as substrates when fatty/lipoic acid activating enzymes are targeted to mitochondria. Lip3 LA transferase has a wider substrate specificity than previously recognized. We show that these features of the lipoylation mechanism in yeast are conserved in mammalian mitochondria. Our findings have important implications for the development of effective therapies for the treatment of LA or mtFAS deficiency-related disorders.

3724: Structural determination of Streptococcus pyogenes M1 protein interactions with human immunoglobulin G using integrative structural biology
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Posted 21 Jul 2020

Structural determination of Streptococcus pyogenes M1 protein interactions with human immunoglobulin G using integrative structural biology
93 downloads bioRxiv molecular biology

Hamed Khakzad, Lotta Happonen, Yasaman Karami, Michael Nilges, Guy Tran Van Nhieu, Johan Malmström, Lars Malmström

Streptococcus pyogenes (Group A streptococcus; GAS) is an important human pathogen responsible for mild to severe, life-threatening infections. GAS expresses a wide range of virulence factors, including the M family proteins. The M proteins allow the bacteria to evade parts of the human immune defenses by triggering the formation of a dense coat of plasma proteins surrounding the bacteria, including IgGs. However, the molecular level details of the M1-IgG interaction have remained unclear. Here, we characterized the structure and dynamics of this interaction interface in human plasma on the surface of live bacteria using integrative structural biology, combining cross-linking mass spectrometry and molecular dynamics (MD) simulations. We show that the primary interaction is formed between the S-domain of M1 and the conserved IgG Fc-domain. In addition, we show evidence for a so far uncharacterized interaction between the A-domain and the IgG Fc-domain. Both these interactions mimic the protein G-IgG interface of group C and G streptococcus. These findings underline a conserved scavenging mechanism used by GAS surface proteins that block the IgG-receptor (FcγR) to inhibit phagocytic killing. We additionally show that we can capture Fab-bound IgGs in a complex background and identify the specific M1 epitopes targeted on live bacteria. Our results elucidate the M1-IgG interaction network involved in inhibition of phagocytosis and reveal important M1 peptides that can be further investigated as future vaccine targets.

3725: Whole genome bisulfite sequencing dataset of mycelium and spore of chalkbrood disease pathogen Ascosphaera apis
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Posted 25 Mar 2020

Whole genome bisulfite sequencing dataset of mycelium and spore of chalkbrood disease pathogen Ascosphaera apis
93 downloads bioRxiv molecular biology

Yu Du, Haibin Jiang, Huazhi Chen, Jie Wang, Yuanchan Fan, Xiaoxue Fan, Cuiling Xiong, Yanzhen Zheng, Dafu Chen, Rui Guo

Chalkbrood, a widespread fungal disease of bee larvae, is caused by the fungus Ascosphaera apis. In this article, mecylia and spores of A. apis were respectively collected followed by DNA isolation, bisulfite conversion, cDNA library construction and next-generation sequencing. Using whole genome bisulfite sequencing (WGBS), 69,844,360 and 60,570,990 raw reads were yielded from Aam and Aas, and after quality control, 9,982,386,951 and 8,825,601,434 clean reads were obtained, respectively. In addition, 67,685,866 and 58,886,072 clean reads were mapped to the reference genome of A. apis, including 37,643,592 and 31,568,442 unique mapped clean reads, and 49,686 and 13,348 multiple mapped clean reads. Furthermore, after bisulfite treatment, the conversion ratio of clean reads from Aam and Aas were 99.38% and 99.51%, respectively. The WGBS data ducumented here contributes to genome-wide identification of 5mC methylation sites in A. apis and comparison of methylomes between mycelium and spore.

3726: Early blood transcriptomic markers of necrotizing enterocolitis in preterm pigs
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Posted 21 Sep 2020

Early blood transcriptomic markers of necrotizing enterocolitis in preterm pigs
93 downloads bioRxiv molecular biology

Xiaoyu Pan, Tik Muk, Shuqiang Ren, Fei Gao, Per Sangild

Preterm infants frequently develop necrotizing enterocolitis (NEC), a severe intestinal disorder associated with high mortality. Early detection of NEC is difficult due to poor specificity and sensitivity of clinical signs. We hypothesized that early development of NEC, before clear clinical symptoms appear, might affect expression of blood genes, potentially related to early systemic immune responses. Using preterm pigs as models for preterm infants, a retrospective analysis was performed on 129 infant formula fed pigs that had NEC diagnosis at necropsy on day 5. Clinical data including growth, activity, hematology, gastric residuals, incidence of diarrhea, bloody stool and abdominal distention were retrospectively reviewed. During this early postnatal period, except that bloody stool was observed in 19% of NEC pigs and absent in healthy pigs, no other clinical outcomes showed difference between NEC and healthy pigs. Whole blood transcriptome was compared between NEC pigs (NEC, n=20) and their litter-mate healthy controls (CON, n=19) on day 5, and revealed 344 differentially expressed genes (DEGs). PubMed literature search identified 123 genes that co-occurred with at least one of 9 NEC-related keywords (NEC, colitis, necrotic, hemorrhage, epithelial apoptosis, intestinal inflammation, inflammatory bowel disease, Crohn’s disease, ulcerative colitis). Co-expression network analysis suggested PAK2 as one of the hub genes. Using whole blood and dried blood spots (DBS) from another group of preterm pigs for validation, up-regulation of PAK2 and genes that co-occurred with NEC and other keywords in PubMed literatures (AOAH, ARG2, FKBP5 and STAT3) was confirmed in severe NEC cases. Specifically, ex vivo stimulation of cord blood with S . epidermidis increased ARG2. Our results show that whole blood gene expressions are affected in preterm pigs at an early stage when NEC is suspicious. Expression of target genes may be used to indicate NEC severity and associated bacterial infection. Routinely collected neonatal DBS may be used to develop early biomarkers for identifying infants with severe NEC lesions, thus providing better intervention strategy. ### Competing Interest Statement The authors have declared no competing interest.

3727: Development of microsatellite markers for a giant water bug, Appasus japonicus, distributed in East Asia
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Posted 28 May 2020

Development of microsatellite markers for a giant water bug, Appasus japonicus, distributed in East Asia
92 downloads bioRxiv molecular biology

Tomoya Suzuki, Akira S. Hirao, Masaki Takenaka, Koki Yano, Koji Tojo

We developed microsatellite markers for Appasus japonicus (Hemiptera: Belostomatidae). This belostomatid bug is distributed in East Asia (Japanese Archipelago, Korean Peninsula, and Mainland China), and often listed as endangered species in the 'Red List' or the 'Red Data Book' at the national and local level in Japan. Here we describe twenty novel polymorphic microsatellite loci developed for A. japonicus , and marker suitability was evaluated on 56 individuals from four A. japonicus populations (Nagano, Hiroshima, and Yamaguchi prefecture, Japan, and Chungcheongnam-do, Korea). The number of alleles per locus ranged 1-12 (mean = 2.5), and average observed and expected heterozygosity, and fixation index per locus were 0.270, 0.323, and 0.153, respectively. The 20 markers described here will be useful for investigating the genetic structure of A. japonicus populations, which can contribute in population genetics studies of this species. ### Competing Interest Statement The authors have declared no competing interest.

3728: Evolution and origin of sliding clamp in bacteria, archaea and eukarya
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Posted 09 Oct 2020

Evolution and origin of sliding clamp in bacteria, archaea and eukarya
92 downloads bioRxiv molecular biology

Sandesh Acharya, Amol Dahal, Hitesh Kumar Bhattarai

Replication of DNA is an essential process in all domains of life. A protein often involved without exception in replication is the sliding clamp. The sliding clamp encircles the DNA and helps replicative polymerase stay attached to the replication machinery increasing the processivity of the polymerase. In eukaryotes and archaea the sliding clamp is called the Proliferating Cell Nuclear Antigen (PCNA) and consists of two domains. This PCNA forms a trimer encircling the DNA as a hexamer. In bacteria, the structure of the sliding clamp is highly conserved, but the protein itself, called beta clamp, contains three domains, which dimerize to form a hexamer. The bulk of literature touts a conservation of the structure of the sliding clamp, but fails to recognize conservation of protein sequence among sliding clamps. In this paper we have used PSI blast to the second interation in NCBI to show a statistically significant sequence homology between Pyrococcus furiosus PCNA and Kallipyga gabonensis beta clamp. The last two domains of beta clamp align with the two domains of PCNA. This homology data demonstrates that PCNA and beta clamp arose from a common ancestor. In this paper, we have further used beta clamp and PCNA sequences from diverse bacteria, archaea and eukarya to build maximum likelihood phylogenetic tree. Most, but not all, species in different domains of life harbor one sliding clamp from vertical inheritance. Some of these species that have two or more sliding clamps have acquired them from gene duplication or horizontal gene transfer events.

3729: Separation of coiled-coil structures in lamin A/C is required for elongation of the filament
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Posted 29 Sep 2020

Separation of coiled-coil structures in lamin A/C is required for elongation of the filament
92 downloads bioRxiv molecular biology

Jinsook Ahn, Soyeon Jeong, So-mi Kang, Inseong Jo, Bum-Joon Park, Nam-Chul Ha

Intermediate filaments (IFs) commonly have structural elements of a central α-helical coiled-coil domain consisting of coil 1a, coil 1b, coil 2, and their flanking linkers. Recently, crystal structure of a long lamin A/C fragment was determined and showed detailed features of a tetrameric unit. The structure further suggested a new binding mode between tetramers, designated eA22, where a parallel overlap of coil 1a and coil 2 is the key interaction. In this study, we investigated the biochemical effects of genetic mutations causing human diseases, focusing on the eA22 interaction. The mutant proteins exhibited either weakened or augmented interactions between coil 1a and coil 2. The ensuing biochemical results indicated that the interaction requires the separation of the coiled-coils in N-terminal of coil 1a and C-terminal of coil 2, coupled with the structural transition in the central α-helical rod domain. This study provides insight into the role of coil 1a as a molecular regulator in elongation of the IF proteins.

3730: The smORF-containing gene mille-pattes is required for moulting and Trypanosoma cruzi metacyclogenesis in the Chagas disease vector Rhodnius prolixus
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Posted 06 Oct 2020

The smORF-containing gene mille-pattes is required for moulting and Trypanosoma cruzi metacyclogenesis in the Chagas disease vector Rhodnius prolixus
92 downloads bioRxiv molecular biology

Carina Azevedo, Bruno Rodrigues, Sandy Alves, Lupis Ribeiro, Carlos Logullo, Jose Nepomuceno, Jose Roberto-Silva, Flavia Mury, Rodrigo Nunes-da-Fonseca

Chagas disease is estimated to affect 8 million people worldwide and is responsible for approximately 10,000 deaths in Latin America every year [1]. Control of the triatomine bugs that transmit the flagellated parasite Trypanosoma cruzi has been the most successful strategy to avoid disease spread. Genes containing small open reading frames (smORFs, < 100 amino acids) constitute a putative reservoir of new vector control targets, since hundreds of these genes are present in insect genomes [2]. Here, we show that the prototypic smORF-containing gene mille-pattes/polished-rice/tarsalless (mlpt/pri/tal) [3-6] is essential for postembryonic development of the kissing bug Rhodnius prolixus and for T. cruzi metacyclogenesis during the nymphal stages. Injection of double-stranded RNA against mlpt (Rp-dsmlpt) during the nymphal stages leads to a plethora of phenotypes, which impair postembryonic development. First, fourth or fifth stage nymphs injected with Rp-dsmlpt do not moult even in the presence of the ecdysone receptor (EcR) mRNA. Second, Rp-dsmlpt nymphs have defects in gut morphology, delayed haemoglobin digestion, and decreased defecation volume compared with those of the control nymphs. Third, Rp-mlpt knockdown inhibits T. cruzi differentiation to the trypomastigote infective stage (metacyclogenesis) inside the R. prolixus gut. Overall, our study is the first to provide evidence that a smORF-containing gene regulates vector physiology and parasitic cycle thus enabling the development of novel molecular strategies to eliminate Chagas disease transmission. ### Competing Interest Statement The authors have declared no competing interest.

3731: Rapid conversion of replicating and integrating Saccharomyces cerevisiae plasmid vectors via Cre recombinase
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Posted 03 Nov 2020

Rapid conversion of replicating and integrating Saccharomyces cerevisiae plasmid vectors via Cre recombinase
92 downloads bioRxiv molecular biology

Daniel Nickerson, Monique A. Quinn, Joshua M. Milnes

Plasmid shuttle vectors capable of replication in both Saccharomyces cerevisiae and Escherichia coli and optimized for controlled modification in vitro and in vivo are a key resource supporting yeast as a premier system for genetics research and synthetic biology. We have engineered a series of yeast shuttle vectors optimized for efficient insertion, removal and substitution of plasmid yeast replication loci, allowing generation of a complete set of integrating, low copy and high copy plasmids via predictable operations as an alternative to traditional subcloning. We demonstrate the utility of this system through modification of replication loci via Cre recombinase, both in vitro and in vivo , and restriction endonuclease treatments.

3732: The Role of Aquaporin-4 in Optic Nerve Head Astrocytes in Experimental Glaucoma
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Posted 04 Dec 2020

The Role of Aquaporin-4 in Optic Nerve Head Astrocytes in Experimental Glaucoma
92 downloads bioRxiv molecular biology

Elizabeth Kimball, Julie Schaub, Sarah Quillen, Casey Keuthan, Mary E Pease, Arina Korneva, Harry Quigley

Purpose: To study aquaporin channel expression in astrocytes of the mouse optic nerve (ON) and the response to IOP elevation in mice lacking aquaporin 4 (AQP4 null). Methods: C57BL/6 (B6) and AQP4 null mice were exposed to bead-induced IOP elevation for 3 days (3D-IOP), 1 and 6 weeks. Mouse ocular tissue sections were immunolabeled against aquaporins 1(AQP1), 4(AQP4), and 9(AQP9). Ocular tissue was imaged to identify normal AQP distribution, ON changes, and axon loss after IOP elevation. Ultrastructure examination, cell proliferation, gene expression & transport block was also analyzed. Results: B6 mice presented abundant AQP4 in Muller cells, astrocytes of retina and myelinated ON (MON), but minimal expression in prelaminar and unmyelinated ON (UON). MON of AQP4 nulls had smaller ON area, smaller axon diameter, higher axon density, and larger proportionate axon area than B6 (all p[&le;]0.05). Bead-injection led to comparable 3D-IOP elevation (p=0.42) and axonal transport blockade in both strains. In B6, AQP4 distribution was unchanged after 3D-IOP. At baseline, AQP1 and AQP9 were present in retina, but not in UON and this was unaffected after IOP elevation in both strains. In 3D-IOP mice, ON astrocytes and microglia proliferated, more in B6 than AQP4 null. After 6 week IOP elevation, axon loss occurred equally in the two mouse types (24.6%, AQP4 null vs. 23.3%, B6). Conclusion: Lack of AQP4 was neither protective nor detrimental to the effects of IOP elevation. The minimal presence of AQP4 in UON may be a vital aspect of the regionally specific phenotype of astrocytes in the mouse optic nerve head.

3733: Pleiotropic Roles for the Plasmodium berghei RNA Binding Protein UIS12 in Transmission and Oocyst Maturation
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Posted 21 Dec 2020

Pleiotropic Roles for the Plasmodium berghei RNA Binding Protein UIS12 in Transmission and Oocyst Maturation
92 downloads bioRxiv molecular biology

Katja Müller, Olivier Silvie, Hans-Joachim Mollenkopf, Kai Matuschewski

Colonization of the mosquito host by Plasmodium parasites is achieved by sexually differentiated gametocytes. Gametocytogenesis, gamete formation and fertilization are tightly regulated processes, and translational repression is a major regulatory mechanism for stage conversion. Here, we present a characterization of a Plasmodium berghei RNA binding protein, UIS12, that contains two conserved eukaryotic RNA recognition motifs (RRM). Targeted gene deletion resulted in viable parasites that replicate normally during blood infection, but form fewer gametocytes. Upon transmission to Anopheles stephensi mosquitoes, both numbers and size of midgut-associated oocysts were reduced and their development stopped at an early time point. As a consequence, no salivary gland sporozoites were formed indicative of a complete life cycle arrest in the mosquito vector. Comparative transcript profiling in mutant and wild-type infected red blood cells revealed a decrease in transcript abundance of mRNAs coding for signature gamete-, ookinete- and oocyst-specific proteins in uis12(-) parasites. Together, our findings indicate multiple roles for UIS12 in regulation of gene expression after blood infection in good agreement with the pleiotropic defects that terminate successful sporogony and onward transmission to a new vertebrate host.

3734: Hypusinated eIF5A is required for the translation of collagen
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Posted 23 Feb 2021

Hypusinated eIF5A is required for the translation of collagen
91 downloads bioRxiv molecular biology

Marina Barba-Aliaga, Adriana Mena, Vanessa Espinoza, Nadezda Apostolova, Mercedes Costell, Paula Alepuz

The evolutionary conserved elongation factor eIF5A is required for the translation of mRNAs that encode protein sequences with consecutive prolines or combined with glycine and charged amino acids. Mammalian collagens are enriched in putative eIF5A-dependent Pro-Gly-containing tripeptides. Here, we show that eIF5A is needed for heterologous expression of collagen in yeast, and using a dual luciferase reporter system we confirmed that eIF5A depletion interrupts translation at Pro-Gly-collagenic motifs. Using mouse fibroblasts, we showed that depletion of active eIF5A reduced collagen 1 (Col1a1) content, which became concentrated around the nuclei, in contrast to a stronger and all over the cell collagen signal in untreated cells. Active eIF5A-depleted mouse fibroblast showed upregulation of endoplasmic reticulum (ER) stress markers, suggesting retention of partially synthesized Col1a1 in the ER. A dramatically lower level of Col11 protein was also observed in functional eIF5A-depleted human hepatic stellate cells treated with the profibrotic cytokine TGF-{beta}1. Our results show that collagen expression requires eIF5A and imply its potential as a target for regulating collagen production in fibrotic diseases.

3735: Intron-assisted, viroid-based production of insecticidal circular double-stranded RNA in Escherichia coli
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Posted 02 Dec 2020

Intron-assisted, viroid-based production of insecticidal circular double-stranded RNA in Escherichia coli
91 downloads bioRxiv molecular biology

Beltran Ortola, Teresa Cordero, Xu Hu, Jose-Antonio Daros

RNA interference (RNAi) is a natural mechanism for protecting against harmful genetic elements and regulating gene expression, which can be artificially triggered by the delivery of homologous double-stranded RNA (dsRNA). This mechanism can be exploited as a highly specific and environmentally friendly pest control strategy. To this aim, systems for producing large amounts of recombinant dsRNA are necessary. We describe a system to efficiently produce large amounts of circular dsRNA in Escherichia coli and demonstrate the efficient insecticidal activity of these molecules against Western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte), a highly damaging pest of corn crops. In our system, the two strands of the dsRNA are expressed in E. coli embedded within the very stable scaffold of Eggplant latent viroid (ELVd), a small circular non-coding RNA. Stability in E. coli of the corresponding plasmids with long inverted repeats was achieved by using a cDNA coding for a group-I autocatalytic intron from Tetrahymena thermophila as a spacer. RNA circularization and large-scale accumulation in E. coli cells was facilitated by co-expression of eggplant tRNA ligase, the enzyme that ligates ELVd during replication in the host plant. The inserted intron efficiently self-spliced from the RNA product during transcription. Circular RNAs containing a dsRNA moiety homologous to smooth septate junction 1 (DvSSJ1) gene exhibited excellent insecticide activity against WCR larvae. Finally, we show that the viroid scaffold can be separated from the final circular dsRNA product using a second T. thermophila self-splicing intron in a permuted form.

3736: CSF metabolites associate with CSF tau and improve prediction of Alzheimer's disease status
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Posted 01 Feb 2021

CSF metabolites associate with CSF tau and improve prediction of Alzheimer's disease status
91 downloads bioRxiv molecular biology

Ruocheng Dong, Burcu F Darst, Yuetiva Deming, Yue Ma, Qiongshi Lu, Henrik Zetterberg, Kaj Blennow, Cynthia M. Carlsson, Sterling C. Johnson, Sanjay Asthana, Corinne D. Engelman

INTRODUCTION: Cerebrospinal fluid (CSF) total tau (t-tau) and phosphorylated tau (p-tau) are biomarkers of Alzheimer's disease (AD), yet much is unknown about AD-associated changes in tau metabolism and tau tangle etiology. METHODS: We assessed the variation of t-tau and p-tau explained by 38 previously identified CSF metabolites using linear regression models in middle-age controls from the Wisconsin Alzheimer's Disease Research Center, and predicted AD/mild cognitive impairment (MCI) vs. an independent set of older controls using metabolites selected by the least absolute shrinkage and selection operator (LASSO). RESULTS: The 38 CSF metabolites explained 70.3% and 75.7% of the variance in t-tau and p-tau. Of these, 7 LASSO-selected metabolites improved the prediction ability of AD/MCI vs. older controls (AUC score increased from 0.92 to 0.97 and 0.78 to 0.93) compared to the base model. DISCUSSION: These tau-correlated CSF metabolites increase AD/MCI prediction accuracy and may provide insight into tau tangle etiology.

3737: Targeted delivery of a phosphoinositide 3-kinase γ inhibitor to restore organ function in sepsis through dye-functionalized lipid nanocarriers
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Posted 21 Jan 2021

Targeted delivery of a phosphoinositide 3-kinase γ inhibitor to restore organ function in sepsis through dye-functionalized lipid nanocarriers
91 downloads bioRxiv molecular biology

Adrian T Press, Petra Babic, Bianca Hoffmann, Tina Mueller, Wanling Foo, Walter Hauswald, Jovana Benecke, Martina Beretta, Zoltan Cseresnyes, Stephanie Hoeppner, Ivo Nischang, Sina M Coldewey, Markus H. Graeler, Reinhard Bauer, Falk Gonnert, Nikolaus Gassler, Reinhard Wetzker, Marc Thilo Figge, Ulrich S. Schubert, Michael Bauer

Jaundice, the clinical hallmark of infection-associated liver dysfunction, reflects altered membrane organization of the canalicular pole of hepatocytes and portends poor outcomes. Mice lacking phosphoinositide 3-kinase-{gamma} (PI3K{gamma}) are protected against membrane disintegration and hepatic excretory dysfunction. However, they exhibit a severe immune defect that hinders neutrophil recruitment to sites of infection. To exploit the therapeutic potential of PI3K{gamma} inhibition in sepsis, a targeted approach to deliver drugs to hepatic parenchymal cells without compromising other cells, in particular immune cells, seems warranted. Here we demonstrate that nanocarriers functionalized through DY-635, a fluorescent polymethine dye and a ligand of organic anion transporters can selectively deliver therapeutics to hepatic parenchymal cells. Applying this strategy to a murine model of sepsis, we observed PI3K{gamma}-dependent restoration of biliary canalicular architecture, maintained excretory liver function, and improved survival without impairing host defense mechanisms. This strategy carries the potential to expand targeted nanomedicines to disease entities with systemic inflammation and concomitantly impaired barrier functionality.

3738: Follow-up of a hospital cohort during the first 3,530 suspected cases of COVID-19 in Sao Jose do Rio Preto, Sao Paulo, Brazil
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Posted 04 Feb 2021

Follow-up of a hospital cohort during the first 3,530 suspected cases of COVID-19 in Sao Jose do Rio Preto, Sao Paulo, Brazil
91 downloads bioRxiv molecular biology

Carolina Colombelli Pacca, Nathalia Zini, Alice Versiani, Edoardo Lobl, Bruno Milhim, Guilherme Campos, Marilia Moraes, Thayza dos Santos, Fernanda Dourado, Beatriz Moraes, Leonardo Rocha, Andresa dos Santos, Leonardo Ruiz, Gislaine da Silva, Raphael Nicesio, Flavia Queiroz, Maria Lucia Salomão, Natal da Silva, Andreia Negri, Mauricio Nogueira, Cassia Estofolete

Introduction: In a global context, COVID-19 is the most significant health threat in the present days, evidenced by the fact that, in just over four months, SARS-CoV-2 has spread to 171 countries, reaching a Pandemic status. Most patients with COVID-19 have a mild course of the disease. However, approximately 20% develop severe illness with a high mortality rate which is associated with age, comorbidities, and immunosuppression. Epidemiological studies are used to reveal the extent of viral spread in homes, communities, and hospitals. Thus, preventive and control measures can be established by the authorities. Objective: In this study, patients with suspect COVID-19 symptoms who search for hospital care at the city of Sao Jose do Rio Preto (Sao Paulo, Brazil) were monitored, in order to identify the first case of this new disease in the region. In the first two months (March and April), more than 3000 individuals looked for the public and private health system with suspected respiratory symptoms, but only 164 (8.4%) were COVID-19 confirmed. Results: From those, males (56.1%) and patients of the age distribution of 16-59 (91.2%), with diarrhea (22.2%), runny nose (25%), altered taste (15.9%), and anosmia (11.6%) presented statistical significance, although none comorbidities were related with COVID-19 occurrence. The odds ratio analysis supports this finding. Days of onset of symptoms are positively associated with whit viral load, and the same happens with the occurrence of symptoms (dyspnea and low saturation).

3739: Sialylation and fucosylation changes of cytidine monophosphate-Nacetylneuraminic acid hydroxylase (CMAH) and glycoprotein, alpha1,3-galactosyltransferase(GGTA1) knockout pig erythrocyte membranes
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Posted 07 Aug 2020

Sialylation and fucosylation changes of cytidine monophosphate-Nacetylneuraminic acid hydroxylase (CMAH) and glycoprotein, alpha1,3-galactosyltransferase(GGTA1) knockout pig erythrocyte membranes
91 downloads bioRxiv molecular biology

Hak Myong Choe, Zhao-Bo Luo, Mei-Fu Xuan, Biao-Hu Quan, Jin-Dan Kang, Myung Jin Oh, Hyun Joo An, Xi-jun Yin

The recent GGTA1 and CMAH DKO pigs have made it possible to resolve the immune barriers which are duo to xenoantigens on RBC such as αGal and Neu5Gc. Nevertheless, it still requires the detection of glycosylation alternation on the pig RBCs because even the minor changes would be unexpected xenoantigens. DKO RBC immune reactivity with human serum was assessed by hemagglutination assay. Glycosylation alteration of RBC membranes was characterized by NanoLC-Q-TOF-MS system and lectin blotting assay. Twelve GGTA1/CMAH DKO piglets were successfully produced. The immunoreactivity with human serum was remarkably reduced in DKO (less than 1:2 dilution), whereas wild type(WT) pigs showed agglutination (the least 1:256 dilution). The MS results showed that DKO increased neutral N-glycans as well as decreased total sialylated N-glycans, especially suggesting significant decrease of di-sialylated N-glycans (P < 0.05). Moreover, lectin blotting assay revealed that DKO pigs reduced the binding signals with AAL, AOL, LCA and SNA and increased the binding signal with MAL. DKO pigs decreased the expression of total fucosylation and sialylated N-glycans on the erythrocyte membrane. Our findings will support further investigation into DKO pig RBC glycosylation and contribute to uncover the roles of glycan changes for xenotransfusion.

3740: WSB1 Regulates c-Myc Expression Through β-catenin Signaling and Forms a Feedforward Circuit Promoting the Development of Cancer
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Posted 25 Sep 2020

WSB1 Regulates c-Myc Expression Through β-catenin Signaling and Forms a Feedforward Circuit Promoting the Development of Cancer
91 downloads bioRxiv molecular biology

Xiaomeng Gao, Yanling Gong, Jieqiong You, Meng Yuan, Haiying Zhu, Liang Fang, Hong Zhu, Meidan Ying, Qiaojun He, Bo Yang, Ji Cao

The dysregulation of transcription factors is widely associated with tumorigenesis. As the most well-defined transcription factor in multiple types of cancer, c-Myc can directly transform cells by transactivating various downstream genes. Given that there is no effective way to directly inhibit c-Myc, c-Myc targeting strategies based on its regulatory mechanism hold great potential for cancer therapy. In this study, we found that WSB1, a direct target gene of c-Myc, can positively regulate c-Myc expression, which forms a feedforward circuit promoting cancer development. Luciferase-based promoter activity assays and RNA sequencing results confirmed that WSB1 promoted c-Myc expression through the β-catenin pathway. Mechanistically, WSB1 affected β-catenin destruction complex-PPP2CA assembly and E3 ubiquitin ligase adaptor β-TRCP recruitment, which inhibited the ubiquitination of β-catenin and subsequently transactivated c-Myc. Of interest, the promoting effect of WSB1 on c-Myc was independent of its E3 ligase activity. Moreover, co-expression of WSB1 and c-Myc strongly enhanced the initiation and progression of tumours both in vitro and in vivo . Thus, our findings revealed a novel mechanism involved in tumorigenesis in which the WSB1/c-Myc feedforward circuit played an essential role, highlighting a potential c-Myc intervention strategy in cancer treatment. ### Competing Interest Statement The authors have declared no competing interest. * TFs : Transcription factors WSB1 : WD repeat and SOCS box containing 1 HIF1-α : Hypoxia induced factor 1-alpha RhoGDI2 : Rho GDP dissociation inhibitor 2 HCC : Hepatocellular carcinoma Wnt3a-CM : Wnt3a-conditioned medium CK1 : Casein kinase 1 GSK3β : Glycogen synthase kinase 3β EBP2 : EBNA1-binding protein 2 FBW7 : F-box/WD repeat-containing protein 7 eIF4F : Eukaryotic initiation factor 4F

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