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3,582 results found. For more information, click each entry to expand.

3481: Human genetic variants disrupt RGS14 nuclear shuttling and regulation of LTP in hippocampal neurons
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Posted 10 Sep 2020

Human genetic variants disrupt RGS14 nuclear shuttling and regulation of LTP in hippocampal neurons
65 downloads bioRxiv molecular biology

Katherine E. Squires, Kyle J Gerber, Matthew C. Tillman, Daniel J Lustberg, Carolina Montañez-Miranda, Meilan Zhao, Suneela Ramineni, Christopher D. Scharer, Feng-jue Shu, Jason P. Schroeder, Eric Ortlund, David Weinshenker, Serena M. Dudek, John R Hepler

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. ### Competing Interest Statement The authors have declared no competing interest.

3482: 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
65 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.

3483: Cas9-mediated Genome Editing Reveals a Significant Contribution of Calcium Signaling Pathways to Anhydrobiosis in Pv11
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Posted 15 Oct 2020

Cas9-mediated Genome Editing Reveals a Significant Contribution of Calcium Signaling Pathways to Anhydrobiosis in Pv11
65 downloads bioRxiv molecular biology

Yugo Miyata, Hiroto Fuse, Shoko Tokumoto, Yusuke Hiki, Ruslan Deviatiiarov, Yuki Yoshida, Takahiro G Yamada, Richard Cornette, Oleg Gusev, Elena Shagimardanova, Akira Funahashi, Takahiro Kikawada

Pv11 is an insect cell line established from the midge Polypedilum vanderplanki that exhibits an extreme desiccation tolerance known as anhydrobiosis. Pv11 has also an anhydrobiotic ability which is induced by trehalose treatment. Here we report the successful construction of the genome editing system for Pv11 cells and its application for identifying the signaling pathways in the anhydrobiosis. Using the Cas9-mediated gene knock-in system, we established GCaMP3-stably expressing Pv11 cells to monitor intracellular Ca2+ mobilization. Intriguingly, trehalose treatment evoked a transient increase of cytosolic Ca2+ concentration, and further experiments indicated the contribution of the calmodulin - calcineurin - NFAT pathway to the tolerance for trehalose treatment as well as the desiccation tolerance, while the calmodulin - calmodulin Kinase - CREB pathway conferred only the desiccation tolerance on Pv11 cells. Thus, our results show the critical contribution of the trehalose-induced Ca2+ surge to the anhydrobiosis and the temporal different roles of each signaling pathway. ### Competing Interest Statement The authors have declared no competing interest.

3484: Enterococcal PrgU mitigates PrgB overexpression toxicity by binding to intergenic RNA downstream of the PQ promoter
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Posted 07 Jul 2020

Enterococcal PrgU mitigates PrgB overexpression toxicity by binding to intergenic RNA downstream of the PQ promoter
64 downloads bioRxiv molecular biology

Lena Lassinantti, Martha I Camacho, Rebecca J B Erickson, Julia L. E. Willett, Nicholas R De Lay, Josy ter Beek, Gary M. Dunny, Peter J. Christie, Ronnie Per-Arne Berntsson

Efficient horizontal gene transfer of the conjugative plasmid pCF10 from Enterococcus faecalis depends on the sex pheromone cCF10, which induces the expression of the Type 4 Secretion System (T4SS) genes controlled by the PQ promoter. The pheromone responsive PQ promoter is strictly regulated to prevent overproduction of the prgQ operon, which contains the T4SS, and to limit the cell toxicity caused by overproduction of PrgB, a T4SS adhesin involved in cellular aggregation. PrgU plays an important role in regulating this toxicity by decreasing PrgB production. PrgU has an RNA-binding fold, prompting us to test whether PrgU exerts its regulatory control through binding of prgQ transcripts. With a combination of lacZ reporter fusion, northern blot, and RNAseq analyses, we provide evidence that PrgU binds a specific RNA sequence within the intergenic region (IGR), ca 400 bp downstream of the PQ promoter. PrgU-IGR binding reduces levels of downstream transcripts, with the strongest decrease seen for prgB messages. Consistent with these findings, we determined that pCF10-carrying cells expressing prgU decreased transcript levels more rapidly than isogenic cells deleted of prgU. Finally, purified PrgU bound RNA in vitro, but without sequence specificity, suggesting that PrgU requires a specific RNA structure or one or more host factors to bind its RNA target in vivo. Together, our results support a working model where PrgU binding to the IGR serves to recruit RNase(s) for targeted degradation of downstream transcripts.

3485: Disproportionate presence of adenosine in mitochondrial and chloroplast DNA of Chlamydomonas reinhardtii
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Posted 27 Aug 2020

Disproportionate presence of adenosine in mitochondrial and chloroplast DNA of Chlamydomonas reinhardtii
64 downloads bioRxiv molecular biology

Waleed M. M. El-Sayed, Alli L. Gombolay, Penghao Xu, Taehwan Yang, Youngkyu Jeon, Sathya Balachander, Gary Newnam, Sijia Tao, Nicole E. Bowen, Raymond F. Schinazi, Baek Kim, Yongsheng Chen, Francesca Storici

Ribonucleoside monophosphates (rNMPs) represent the most common non-standard nucleotides found in the genomic DNA of cells. The distribution of rNMPs in DNA has been studied only in limited genomes, such as yeast nuclear and mitochondrial DNA, as well as human mitochondrial DNA. In this study, we used the ribose-seq protocol and the Ribose-Map bioinformatics toolkit to reveal the distribution of rNMPs incorporated into the whole genome of a photosynthetic unicellular green alga, Chlamydomonas reinhardtii. The study presents the discovery of a disproportionate incorporation of adenosine in the mitochondrial and chloroplast DNA, in contrast to the nuclear DNA, relative to the nucleotide content of these C. reinhardtii genomes. Our results demonstrate that the rNMP content in the DNA of the algal organelles reflects an elevated ATP level present in the algal cells. In addition, we reveal specific rNMP biases and patterns in the mitochondrial, chloroplast and nuclear DNA of C. reinhardtii. ### Competing Interest Statement The authors have declared no competing interest.

3486: Curcumin promotes progression of AApoAII amyloidosis and peroxisome proliferation in mice by activating the PPARα signaling pathway
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Posted 23 Sep 2020

Curcumin promotes progression of AApoAII amyloidosis and peroxisome proliferation in mice by activating the PPARα signaling pathway
64 downloads bioRxiv molecular biology

Jian Dai, Ying Li, Fuyuki Kametani, Xiaoran Cui, Yuichi Igarashi, Jia Huo, Hiroki Miyahara, Masayuki Mori, Keiichi Higuchi

Curcumin is a polyphenol compound that exhibits multiple physiological activities. To elucidate the mechanisms by which curcumin affects systemic amyloidosis, we investigated amyloid deposition and molecular changes in a mouse model of amyloid apolipoprotein A-II (AApoAII) amyloidosis, in which mice were fed a curcumin-supplemented diet. Curcumin supplementation for 12 weeks significantly increased AApoAII amyloid deposition relative to controls, especially in the liver and spleen. Liver weights and plasma ApoA-II and high-density lipoprotein concentrations were significantly elevated in curcumin-supplemented groups. RNA-sequence analysis revealed that curcumin intake affected hepatic lipid metabolism via the peroxisome proliferator-activated receptor (PPAR) pathway, especially PPARα activation, resulting in increased Apoa2 mRNA expression. The increase in liver weights was due to activation of PPARα and peroxisome proliferation. Taken together, these results demonstrate that curcumin is a PPARα activator and may affect expression levels of proteins involved in amyloid deposition to influence amyloidosis and metabolism in a complex manner. ### Competing Interest Statement The authors have declared no competing interest.

3487: The soluble glutathione transferase superfamily: Role of Mu class in Triclabendazole sulphoxide challenge in Fasciola hepatica
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Posted 22 Jul 2020

The soluble glutathione transferase superfamily: Role of Mu class in Triclabendazole sulphoxide challenge in Fasciola hepatica
63 downloads bioRxiv molecular biology

Rebekah B Stuart, Suzanne Zwaanswijk, Neil D MacKintosh, Boontarikaan Witikornkul, Mark Prescott, Peter M. Brophy, Russell M. Morphew

Fasciola hepatica (liver fluke), a significant threat to food security, causes global economic loss for the livestock production industry and is re-emerging as a food borne disease of humans. In the absence of vaccines the commonly used method of treatment control is by anthelmintics; with only Triclabendazole (TCBZ) currently effective against all stages of F. hepatica in livestock and humans. There is widespread resistance to TCBZ and detoxification by flukes might contribute to the mechanism. However, there is limited Phase I capacity in adult parasitic helminths and the major Phase II detoxification system in adults is the soluble Glutathione transferases (GST) superfamily. Previous global proteomic studies have shown that the levels of Mu class GST from pooled F. hepatica parasites respond under TCBZ-Sulphoxide (TCBZ-SO), the likely active metabolite, challenge during in vitro culture ex-host. We have extended this finding by using a sub-proteomic lead approach to measure the change in the total soluble GST profile (GST-ome) of individual TCBZ susceptible F. hepatica on TCBZ-SO-exposure in vitro culture. TCBZ-SO exposure demonstrated a FhGST-Mu29 and FhGST-Mu26 response following affinity purification using both GSH and S-hexyl GSH affinity resins. Furthermore, a low affinity Mu class GST (FhGST-Mu5) has been identified and recombinantly expressed and represents a novel low affinity mu class GST. Low affinity GST isoforms within the GST-ome was not limited to FhGST-Mu5 with second likely low affinity sigma class GST (FhGST-S2) uncovered through genome analysis. This study represents the most complete Fasciola GST-ome generated to date and has supported the sub proteomic analysis on individual adult flukes. ### Competing Interest Statement The authors have declared no competing interest.

3488: 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
63 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.

3489: Biomolecular models of EPI-X4 binding to CXCR4 allow the rational optimization of peptides with therapeutic potential
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Posted 24 Oct 2020

Biomolecular models of EPI-X4 binding to CXCR4 allow the rational optimization of peptides with therapeutic potential
63 downloads bioRxiv molecular biology

Pandian Sokkar, Mirja Harms, Christina M Stuerzel, Andrea Gilg, Goenuel Kizilsavas, Martina Raasholm, Nico Preising, Manfred Wagner, Ludger Staendker, Gilbert Weidinger, Jan Muench, Elsa Sanchez-Garcia,

The Endogenous Peptide Inhibitor of CXCR4 (EPI-X4) is a body-own fragment of albumin and specific antagonist of the CXC-motif-chemokine receptor 4 (CXCR4). CXCR4 signaling is induced by its sole chemokine ligand CXCL12 and is involved in a plethora of functions including cell homing, differentiation, survival and angiogenesis. Consequently, dysregulation of CXCR4 is involved in a variety of disorders, such as cancer or inflammatory diseases, making CXCR4 an attractive drug target. EPI-X4 and derivatives with increased CXCR4 binding affinities represent promising leads as CXCR4 antagonists and have shown therapeutic activity in mouse models of inflammatory diseases. However, it is currently unclear how EPI-X4 and its derivatives interact with CXCR4. Here, by combining biomolecular simulations with experimental mutagenesis and activity studies we investigated the binding behavior of EPI-X4 to CXCR4 at the molecular level. Our work allowed us to show that the EPI-X4 peptide interacts primarily in the minor pocket of CXCR4 through its N-terminal residues. The biomolecular interactions highlighted by the computational studies are in good agreement with the experimental mutagenesis data. Moreover, we found that the N-terminal seven amino-acids of EPI-X4 (a 16-mer) and its improved derivatives (12-mers) are sufficient for CXCR4 binding, which led to the development of shorter leads with optimized CXCR4 antagonizing properties. Collectively, we here established how EPI-X4 binds to its receptor and used this knowledge for rational drug design. The new peptide variants developed by us are more potent in terms of inhibiting CXCR4-downstream signaling and cancer cell migration, without toxic effects. ### Competing Interest Statement Authors P.S., M.H., L.S., J.M. and E.S.-G. are inventors of granted and filed patents that claim tu use EPI-X4 and its derivatives for therapy of CXCR4 linked diseases.

3490: Potential universal PCR method to detect decapod hepanhamaparvovirus (DHPV) in crustaceans
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Posted 03 Sep 2020

Potential universal PCR method to detect decapod hepanhamaparvovirus (DHPV) in crustaceans
63 downloads bioRxiv molecular biology

Jiraporn Srisala, Dararat Thaiue, Piyachat Sanguanrut, Diva January Aldama-Cano, Timothy W Flegel, Kallaya Sritunyalucksana

Parvoviruses that infect the hepatopancreas (HP) of the penaeid shrimp Penaeus chinensis , P. monodon , and P. merquiensis were previously called hepatopancreatic parvoviruses (HPV). They are now classified in the family Parvoviridae , sub-family Hamaparvovirinae as members of the same genus called Hepanhamaparvovirus and referred to as decapod hepanhamaparvovirus, designated here as DHPV. However, a virus that causes similar lesions in the HP of the giant river prawn Macrobrachium rosenbergii resembles hepanhamaparvoviruses by microscopy and histochemistry. Unfortunately, no genome information is yet available and PCR detection methods that work for DHPV in P. monodon do not work with M. rosenbergii . For hatchery samples of M. rosenbergii in Thailand with DHPV-like lesions, we hypothesized it might be possible to design primer pairs from 8 full DHPV genome sequences at GenBank for use in PCR detection of DHPV in M. rosenbergii . Using this strategy, we successfully designed a new set of primers and a PCR protocol called the DHPV-U method that gave an amplicon with DNA extracts from larvae of M. rosenberigii samples that showed DHPV-like lesions, while extracts from normal larvae gave none. DNA extracts from P. monodon infected with DHPV also gave amplicons. At the same time, the normal PCR method for DHPV in P. monodon gave no amplicon with the M. rosenbergii DNA extracts. The DHPV-U amplicons from P. monodon and M. rosenbergii shared 99% sequence identity, and in situ hybridization (ISH) assays using the DIG-labeled amplicon gave positive histochemical results in the HP tissue of both P. monodon and M. rosenbergii . The DHPV-U method is now being used in Thailand for detection of DHPV in both P. monodon and M. rosenbergii . Overall, the results support the proposal that the HP virus in M. rosenbergii is also a hepanhamaparvovirus . Based on 100% sequence identity of the target region in the currently published DHPV sequences at GenBank, the DHPV-U method may also work for detection of other DHPV isolates. ### Competing Interest Statement The authors have declared no competing interest.

3491: Vascular Endothelial Growth Factor as an Immediate-Early Activator of UV-induced Skin Injury
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Posted 14 Jul 2020

Vascular Endothelial Growth Factor as an Immediate-Early Activator of UV-induced Skin Injury
63 downloads bioRxiv molecular biology

Stella P. Hartono, Victoria M. Bedell, Sk Kayum Alam, Madelyn O’Gorman, MaKayla Serres, Stephanie R Hall, Krishnendu Pal, Rachel A Kudgus, Priyabrata Mukherjee, Davis M. Seelig, Alexander Meves, Debabrata Mukhopadhyay, Stephen C. Ekker, Luke H. Hoeppner

The negative health consequences of acute ultraviolet (UV) exposure are evident, with reports of 30,000 emergency room visits annually to treat the effects of sunburn in the United States alone. Acute effects of sunburn include erythema, edema, and severe pain, and chronic overexposure to UV radiation can lead to skin cancer. While the pain associated with the acute effects of sunburn may be relieved by current interventions, existing post-sunburn treatments are not capable of reversing the cumulative and long-term pathological effects of UV exposure, an unmet clinical need. Here we show that activation of the vascular endothelial growth factor (VEGF) pathway is a direct and immediate consequence of acute UV exposure, and activation of VEGF signaling is necessary for the initiating the acute pathological effects of sunburn. In UV-exposed human subjects, VEGF signaling is activated within hours. Topical delivery of VEGF pathway inhibitors, targeted against the ligand VEGF-A (gold nanoparticles conjugated with anti-VEGF antibodies) and small molecule antagonists of VEGF receptor signaling, prevent the development of erythema and edema in UV-exposed mice. Collectively, these findings suggest targeting VEGF signaling may reduce the subsequent inflammation and pathology associated with UV-induced skin damage, which reveals a new post-exposure therapeutic window to potentially inhibit the known detrimental effects of UV on human skin. ### Competing Interest Statement The authors have declared no competing interest.

3492: Changes in cellular signaling patterns before and after stroke in the middle cerebral arteries of stroke prone spontaneously hypertensive rats
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Posted 11 Sep 2020

Changes in cellular signaling patterns before and after stroke in the middle cerebral arteries of stroke prone spontaneously hypertensive rats
62 downloads bioRxiv molecular biology

Killol Chokshi, Julie Warren, Krista Squires, Kayla A. Kitselman, Jules Doré, Noriko Daneshtalab

Background Hemorrhagic stroke is associated with loss of middle cerebral artery (MCA) autoregulation in the stroke-prone spontaneously hypertensive rat (SHRsp). The signaling mechanism associated with the functional loss has yet to be defined. We hypothesize that physiological alterations coincide with changes to cerebrovascular inflammatory and contractile signaling and altered calcium signaling. METHODS: SHRsp rats were fed a high salt (4% NaCl) diet and sacrificed at 9 weeks of age for pre-stroke and after evidence of stroke for post-stroke samples. The MCAs were isolated for measuring protein levels using immunofluorescence (IF) & western blot (WB) for inflammatory signaling and contractile proteins. Tissues surrounding the MCA were analyzed for neuro-inflammation, neuronal damage, total and activated inflammatory proteins (ERK1/2 and p38MAPK), cerebrovascular contraction (PKC and MLC), and transient receptor potential V4 (TRPV4) expression. RESULTS: Our data show increase in activated inflammatory proteins after stroke with an associated decrease in expression of activated contractile proteins and TRPV4 channel expression compared to pre-stroke MCA. The post-stroke samples also show significant increase in neuro-inflammation and neuronal damage compared to pre-stroke samples. CONCLUSION An increase in activated/total (p38 MAPK &ERK1/2) is accompanied by a decrease in activated/total PKC & TRPV4 channel expression in post-stroke SHRsps. The decrease in vessel structural integrity and altered vascular tone of the MCAs may affect its ability to contract in response to pressure. Significant neuro-inflammation and neuronal damage in the brain tissues surrounding the MCA in post-stroke samples suggest MCA dysfunction is accompanied with neuronal and neural damage during stroke. * BCA : Bicinchoninic acid COX-2 : Cyclooxygenase-2 EMILIN1 : Elastin microfibril interface–located protein 1 GAPDH : Glyceraldehyde 3-phosphate dehydrogenase GFAP : Glial fibrillary acidic protein HRP : Horseradish peroxidase Iba-1 : Ionized calcium binding adaptor molecule 1 iNOS : inducible nitric oxide synthase MCA : Middle Cerebral Artery MGV : Mean gray value MLC : Myosin Light Chain MMP-9 : Matrix metalloproteinases-9 NBF : Neutral Buffered Formalin NGS : Normal Goat Serum PBS : Phosphate Buffered Saline PDC : Pressure Dependent Constriction PFA : Paraformaldehyde PKC : Protein Kinase C PVDF : Polyvinylidene fluoride RIPA : Radioimmunoprecipitation assay SDS : Sodium Dodecyl Sulfate SHRsp : Stroke prone Spontaneously hypertensive rats TBS : Tris-buffered saline TBST : Tris-buffered Saline & Tween 20 TRPV4 : Transient Receptor Potential Cation Channel

3493: 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
62 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.

3494: MicroRNA840 accelerates leaf senescence by targeting the overlapping 3'UTRs of PPR and WHIRLY3 in Arabidopsis
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Posted 24 Oct 2020

MicroRNA840 accelerates leaf senescence by targeting the overlapping 3'UTRs of PPR and WHIRLY3 in Arabidopsis
62 downloads bioRxiv molecular biology

Yujun Ren, Wanzhen Wang, Wei Lan, Dirk Schenke, Daguang Cai, Ying Miao

MicroRNAs (miRNAs) negatively regulate gene expression by cleaving the target mRNA and/or impairing its translation, thereby playing a crucial role in plant development and environmental stress responses. In Arabidopsis, MIR840 is located within the overlapping 3'-UTR of PPR and WHIRLY3 ( WHY3 ), both being predicted targets of miR840. Gain- and loss-of-function of miR840 in Arabidopsis resulted in opposite senescent phenotypes. Highest expression of pri-miR840 is observed at senescence initiation, and is negatively correlated with a significant reduction of PPR transcripts but not of WHY3 . Although WHY3 transcript levels were not significantly affected by miR840 overexpression, its protein synthesis was strongly reduced. Mutating the target sites or replacing the target sequences abolishes the miR840-mediated degradation of PPR transcripts and inhibition of WHY3 translation. In support for this, concurrent knock-down of both PPR and WHY3 in the WT resulted in the senescent phenotype resembling that of the miR840-overexpressing mutant. This indicates that both PRR and WHY3 are targets in the miR840-regulated senescent pathway. Moreover, single knockout mutant of PPR or WHY3 shows a convergent up-regulated subset of senescence-associated genes, which are also found among those induced by miR840 overexpression. Our data provide evidences for a regulatory role of miR840 in plant senescence.

3495: The nuclear receptor RORα preserves cardiomyocyte mitochondrial function by regulating mitophagy through caveolin-3
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Posted 02 Oct 2020

The nuclear receptor RORα preserves cardiomyocyte mitochondrial function by regulating mitophagy through caveolin-3
62 downloads bioRxiv molecular biology

JY Beak, HS Kang, W. Huang, A Aghajanian, K Gerrish, AM Jetten, BC Jensen

Preserving optimal mitochondrial function is critical in the heart, which is the most ATP-avid organ in the body. Recently, we showed that global deficiency of the nuclear receptor RORα in the “staggerer” (RORαsg/sg) mouse exacerbates angiotensin II-induced cardiac hypertrophy and compromises cardiomyocyte mitochondrial function. The mechanisms underlying these observations have not been defined. Here we present evidence that RORα regulates cardiomyocyte mitophagy using pharmacological and genetic gain- and loss-of-function tools, including a novel cardiomyocyte-specific RORα knockout mouse. Cardiomyocyte RORα is upregulated by hypoxia and the loss of RORα blunts hypoxia-induced mitophagy and broadly compromises mitochondrial function. We show that RORα is a direct transcriptional regulator of the mitophagy mediator caveolin-3 in cardiomyocytes and that increased expression of RORα increases caveolin-3 abundance and enhances mitophagy. Knockdown of RORα impairs cardiomyocyte mitophagy, but this defect can be rescued by caveolin-3 overexpression. Collectively, these findings reveal a novel role for RORα in regulating mitophagy through caveolin-3 and expand our currently limited understanding of the mechanisms underlying RORα-mediated cardioprotection.

3496: 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
62 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.

3497: Humanized Drosophila model of the Meier-Gorlin syndrome reveals conserved and divergent features of the Orc6 protein.
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Posted 01 May 2020

Humanized Drosophila model of the Meier-Gorlin syndrome reveals conserved and divergent features of the Orc6 protein.
61 downloads bioRxiv molecular biology

Maxim Balasov, Katarina Akhmetova, Igor Chesnokov

Meier-Gorlin syndrome (MGS) is a rare autosomal recessive disorder characterized by microtia, primordial dwarfism, small ears and skeletal abnormalities. Patients with MGS often carry mutations in the genes encoding the subunits of the Origin Recognition Complex (ORC), components of the pre-replicative complex (pre-RC) and replication machinery. Orc6 is an important component of ORC and has functions in both DNA replication and cytokinesis. A mutation in conserved C-terminal motif of Orc6 associated with MGS impedes the interaction of Orc6 with core ORC. Recently, new mutation in Orc6 was also identified however, it is localized in the N-terminal domain of the protein. In order to study the functions of Orc6 we used human gene to rescue the orc6 deletion in Drosophila. Using the humanized Orc6-based Drosophila model of the Meier-Gorlin syndrome we discovered that unlike previous Y225S MGS mutation in Orc6, the K23E substitution in the N-terminal TFIIB-like domain of Orc6 disrupts the protein ability to bind DNA. Our studies revealed the importance of evolutionary conserved and variable domains of Orc6 protein and allowed the studies of human protein functions and the analysis of the critical amino acids in live animal heterologous system as well as provided novel insights into the mechanisms underlying MGS pathology. ### Competing Interest Statement The authors have declared no competing interest.

3498: The importance of charge in perturbing the aromatic glue stabilizing the protein-protein interface of homodimeric tRNA-guanine transglycosylase
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Posted 01 Sep 2020

The importance of charge in perturbing the aromatic glue stabilizing the protein-protein interface of homodimeric tRNA-guanine transglycosylase
61 downloads bioRxiv molecular biology

Andreas Nguyen, Dzung Nguyen, Tran Xuan Phong Nguyen, Maurice Sebastiani, Stefanie Dörr, Oscar Hernandez-Alba, François Debaene, Sarah Cianférani, Andreas Heine, Gerhard Klebe, Klaus Reuter

Bacterial tRNA-guanine transglycosylase (Tgt) is involved in the biosynthesis of the modified tRNA nucleoside queuosine present in the anticodon wobble position of tRNAs specific for aspartate, asparagine, histidine and tyrosine. Inactivation of the tgt gene leads to decreased pathogenicity of Shigella bacteria. Therefore, Tgt constitutes a putative target for Shigellosis drug therapy. Since only active as homodimer, interference with dimer-interface formation may, in addition to active-site inhibition, provide further means to disable this protein. A cluster of four aromatic residues seems important to stabilize the homodimer. We mutated residues of this aromatic cluster and analyzed each exchange with respect to dimer and thermal stability or enzyme activity applying native mass spectrometry, thermal shift assay, enzyme kinetics, and X-ray crystallography. Our structural studies indicate strong influence of pH on homodimer stability. Obviously, protonation of a histidine within the aromatic cluster promotes the collapse of an essential structural motif within the dimer interface at slightly acidic pH. TOC Graphic For table of contents use only. ![Figure][1]</img> ### Competing Interest Statement The authors have declared no competing interest. [1]: pending:yes

3499: Functional characterization of RebL1 highlights the evolutionary conservation of oncogenic activities of the RBBP4/7 orthologue in Tetrahymena thermophila
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Posted 08 Nov 2020

Functional characterization of RebL1 highlights the evolutionary conservation of oncogenic activities of the RBBP4/7 orthologue in Tetrahymena thermophila
61 downloads bioRxiv molecular biology

Syed Nabeel-Shah, Jyoti Garg, Alejandro Saettone, Kanwal Ashraf, Hyunmin Lee, Suzanne Wahab, Nujhat Ahmed, Jacob Fine, Joanna Derynck, Marcelo Ponce, Shuye Pu, Edyta Marcon, Zhaolei Zhang, Jack F. Greenblatt, Ronald E. Pearlman, Jean-Philippe Lambert, Jeffrey Fillingham

Retinoblastoma-binding proteins 4 and 7 (RBBP4 and RBBP7) are two highly homologous human histone chaperones. They function in epigenetic regulation as subunits of multiple chromatin-related complexes and have been implicated in numerous cancers. Due to their overlapping functions, our understanding of RBBP4 and 7, particularly outside of Opisthokonts, has remained limited. Here, we report that in the ciliate protozoan Tetrahymena thermophila a single orthologue of human RBBP4 and 7 proteins, RebL1, physically interacts with histone H4 and functions in multiple epigenetic regulatory pathways. Functional proteomics identified conserved functional links associated with Tetrahymena RebL1 protein as well as human RBBP4 and 7. We found that putative subunits of multiple chromatin-related complexes including CAF1, Hat1, Rpd3, and MuvB, co-purified with RebL1 during Tetrahymena growth and conjugation. Iterative proteomics analyses revealed that the cell cycle regulatory MuvB-complex in Tetrahymena is composed of at least five subunits including evolutionarily conserved Lin54, Lin9 and RebL1 proteins. Genome-wide analyses indicated that RebL1 and Lin54 (Anqa1) bind within genic and intergenic regions. Moreover, Anqa1 targets primarily promoter regions suggesting a role for Tetrahymena MuvB in transcription regulation. RebL1 depletion decreased cellular viability and altered the expression of selected targets. Consistent with observations in glioblastoma tumors, RebL1 depletion suppressed DNA repair protein Rad51 in Tetrahymena, thus underscoring the evolutionarily conserved functions of RBBP4/7 proteins. Our results suggest the essentiality of RebL1 functions in multiple epigenetic regulatory complexes in which it impacts transcription regulation and cellular viability. ### Competing Interest Statement The authors have declared no competing interest.

3500: Phylogenetic analysis by SNP and development of SSR marker in Passiflora
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Posted 15 Jul 2020

Phylogenetic analysis by SNP and development of SSR marker in Passiflora
60 downloads bioRxiv molecular biology

Yanyan Wu, Qinglan Tian, Weihua Huang, Jieyun Liu, Xiuzhong Xia, Xinghai Yang, Haifei Mou

Information of the Passiflora genome is still very limited. Understand the evolutionary relationship between different species of Passiflora, and develop a large number of SSR markers to provide a basis for the genetic improvement of Passiflora. Applying restriction site associated DNA sequencing (RAD-Seq) technology, we studied the phylogeny, simple sequence repeat (SSR) and marker transferability of 10 accessions of 6 species of Passiflora. Taking the partial assembly sequence of accessions P4 as the reference genome, we constructed the phylogenetic tree using the detected 46,451 high-quality single nucleotide polymorphisms (SNPs), showing that P6, P7, P8 and P9 were a single one while P5 and P10 were clustered together, and P1, P2, P3 and P4 were closer in genetic relationship. Using P8 as the reference genome, a total of 12,452 high-quality SNPs were used to construct phylogenetic tree. P3, P4, P7, P8, P9 and P10 were all single branch while P1 and P2 were clustered together, and P5 and P6 were clustered into one branch. A principal component analysis (PCA) revealed a similar population structure, which four cultivated passion fruits forming a tight cluster. A total of 2,614 SSRs were identified in the genome of 10 Passiflora accessions. The core motifs were AT, GA, AAG etc., 2-6 bases, 4-16 repeats, and 2,515 pairs of SSR primer were successfully developed. Tthe SSR transferability in cultivated passion fruits is the best. These results will contribute to the study of genomics and molecular genetics in passion fruit. ### Competing Interest Statement The authors have declared no competing interest.

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