Pub Date : 2023-11-14DOI: 10.1101/2023.11.13.566858
Aliakbar Khalili Yazdi, Sumera Perveen, Xiaosheng Song, Aiping Dong, Magdalena Szewczyk, Matthew Calabrese, Agustin Casimiro-Garcia, Chakrapani Subramanyam, Matthew S Dowling, Emel Ficici, Jisun Lee, Justin I Montgomery, Thomas N O'Connell, Grzegorz J Skrzypek, Tuan P Tran, Matthew D Troutman, Feng Wang, Jennifer A Young, Jinrong Min, Dalia Barsyte-Lovejoy, Peter J Brown, Vijayaratnam Santhakumar, Cheryl Arrowsmith, Masoud Vedadi, Dafydd R Owen
We have developed a novel chemical handle (PFI-E3H1) and a chemical probe (PFI-7) as ligands for the Gid4 subunit of the human E3 ligase CTLH degradation complex. Through an efficient initial hit-ID campaign, structure-based drug design (SBDD) and leveraging the sizeable Pfizer compound library, we identified a 500 nM ligand for this E3 ligase through file screening alone. Further exploration identified a vector that is tolerant to addition of a linker for future chimeric molecule design. The chemotype was subsequently optimized to sub-100 nM Gid4 binding affinity for a chemical probe. These novel tools, alongside the suitable negative control also identified, should enable the interrogation of this complex human E3 ligase macromolecular assembly.
{"title":"Chemical Tools for the Gid4 Subunit of the Human E3 Ligase C-terminal to LisH (CTLH) Degradation Complex","authors":"Aliakbar Khalili Yazdi, Sumera Perveen, Xiaosheng Song, Aiping Dong, Magdalena Szewczyk, Matthew Calabrese, Agustin Casimiro-Garcia, Chakrapani Subramanyam, Matthew S Dowling, Emel Ficici, Jisun Lee, Justin I Montgomery, Thomas N O'Connell, Grzegorz J Skrzypek, Tuan P Tran, Matthew D Troutman, Feng Wang, Jennifer A Young, Jinrong Min, Dalia Barsyte-Lovejoy, Peter J Brown, Vijayaratnam Santhakumar, Cheryl Arrowsmith, Masoud Vedadi, Dafydd R Owen","doi":"10.1101/2023.11.13.566858","DOIUrl":"https://doi.org/10.1101/2023.11.13.566858","url":null,"abstract":"We have developed a novel chemical handle (PFI-E3H1) and a chemical probe (PFI-7) as ligands for the Gid4 subunit of the human E3 ligase CTLH degradation complex. Through an efficient initial hit-ID campaign, structure-based drug design (SBDD) and leveraging the sizeable Pfizer compound library, we identified a 500 nM ligand for this E3 ligase through file screening alone. Further exploration identified a vector that is tolerant to addition of a linker for future chimeric molecule design. The chemotype was subsequently optimized to sub-100 nM Gid4 binding affinity for a chemical probe. These novel tools, alongside the suitable negative control also identified, should enable the interrogation of this complex human E3 ligase macromolecular assembly.","PeriodicalId":486943,"journal":{"name":"bioRxiv (Cold Spring Harbor Laboratory)","volume":"38 6","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"134993134","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-11-14DOI: 10.1101/2023.11.10.566538
Luis Irastorza, Jose Maria Benitez, Francisco Montans, Luis Saucedo-Mora
The human brain is arguably the most complex ``machine'' to ever exist. Its detailed functioning is yet to be fully understood, let alone modeled. Neurological processes have logical signal-processing aspects and biophysical aspects, and both affect the brain structure, functioning and adaptation. Mathematical approaches based on both information and graph theory have been extensively used in an attempt to approximate its biological functioning, along with Artificial Intelligence approaches inspired by its logical functioning. In this article, we present an approach to model some aspects of the brain learning and signal processing, mimicking the metastability and backpropagation found in the real brain while also accounting for neuroplasticity. Several simulations are carried out with this model, to demonstrate how dynamic neuroplasticity, neural inhibition and neurons migration can remodel the brain logical connectivity to syncronize signal processing and obtain target latencies. This work demonstrates the importance of dynamic logical and biophysical remodelling in brain plasticity.
{"title":"An agent based model (ABM) to reproduce the boolean logic behaviour of neuronal self organized communities through pulse delay modulation and generation of logic gates","authors":"Luis Irastorza, Jose Maria Benitez, Francisco Montans, Luis Saucedo-Mora","doi":"10.1101/2023.11.10.566538","DOIUrl":"https://doi.org/10.1101/2023.11.10.566538","url":null,"abstract":"The human brain is arguably the most complex ``machine'' to ever exist. Its detailed functioning is yet to be fully understood, let alone modeled. Neurological processes have logical signal-processing aspects and biophysical aspects, and both affect the brain structure, functioning and adaptation. Mathematical approaches based on both information and graph theory have been extensively used in an attempt to approximate its biological functioning, along with Artificial Intelligence approaches inspired by its logical functioning. In this article, we present an approach to model some aspects of the brain learning and signal processing, mimicking the metastability and backpropagation found in the real brain while also accounting for neuroplasticity. Several simulations are carried out with this model, to demonstrate how dynamic neuroplasticity, neural inhibition and neurons migration can remodel the brain logical connectivity to syncronize signal processing and obtain target latencies. This work demonstrates the importance of dynamic logical and biophysical remodelling in brain plasticity.","PeriodicalId":486943,"journal":{"name":"bioRxiv (Cold Spring Harbor Laboratory)","volume":"37 24","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"134993138","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-11-14DOI: 10.1101/2023.11.14.566966
Yi-Ru Chen, Shun-Cheng Tseng, Eric Hwang
A functional nervous system is built upon the proper morphogenesis of neurons to establish the intricate connection between them. The microtubule cytoskeleton is known to play various essential roles in this morphogenetic process. While many microtubule-associated proteins (MAPs) have been demonstrated to participate in neuronal morphogenesis, the function of many more remains to be determined. This study focuses on a MAP called HMMR, which was originally identified as a hyaluronan binding protein and later found to possess microtubule and centrosome binding capacity. HMMR exhibits high abundance on neuronal microtubules and altering the level of HMMR significantly affects the morphology of neurons. Instead of confining to the centrosome(s) like cells in mitosis, HMMR localizes to microtubules along axons and dendrites. Furthermore, transiently expressing HMMR enhances the stability of neuronal microtubules and increases the formation frequency of growing microtubules along the neurites. HMMR regulates the microtubule localization of a non-centrosomal microtubule nucleator TPX2 along the neurite, offering an explanation for how HMMR contributes to the promotion of growing microtubules. This study sheds light on how progenitor cells utilize proteins involved in mitosis for non-mitotic functions.
{"title":"The non-mitotic role of HMMR in regulating the localization of TPX2 and the dynamics of microtubules in neurons","authors":"Yi-Ru Chen, Shun-Cheng Tseng, Eric Hwang","doi":"10.1101/2023.11.14.566966","DOIUrl":"https://doi.org/10.1101/2023.11.14.566966","url":null,"abstract":"A functional nervous system is built upon the proper morphogenesis of neurons to establish the intricate connection between them. The microtubule cytoskeleton is known to play various essential roles in this morphogenetic process. While many microtubule-associated proteins (MAPs) have been demonstrated to participate in neuronal morphogenesis, the function of many more remains to be determined. This study focuses on a MAP called HMMR, which was originally identified as a hyaluronan binding protein and later found to possess microtubule and centrosome binding capacity. HMMR exhibits high abundance on neuronal microtubules and altering the level of HMMR significantly affects the morphology of neurons. Instead of confining to the centrosome(s) like cells in mitosis, HMMR localizes to microtubules along axons and dendrites. Furthermore, transiently expressing HMMR enhances the stability of neuronal microtubules and increases the formation frequency of growing microtubules along the neurites. HMMR regulates the microtubule localization of a non-centrosomal microtubule nucleator TPX2 along the neurite, offering an explanation for how HMMR contributes to the promotion of growing microtubules. This study sheds light on how progenitor cells utilize proteins involved in mitosis for non-mitotic functions.","PeriodicalId":486943,"journal":{"name":"bioRxiv (Cold Spring Harbor Laboratory)","volume":"19 7","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"134953770","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-11-14DOI: 10.1101/2023.11.14.566999
Aishwarya A Makam, Abhimanyu Dubey, Shovamayee Maharana, Nikhil Gandasi
Image processing and analysis are two significant areas that are highly important for interpreting enormous amounts of data obtained from microscopy-based experiments. Several image analysis tools exist for the general detection of fundamental cellular processes, but tools to detect highly distinct cellular functions are few. One such process is exocytosis, which involves the release of vesicular content out of the cell. The size of the vesicles and the inherent differences in the imaging parameters demand specific analysis platforms for detecting exocytosis. In this direction, we have developed an image-processing algorithm based on Lagrangian particle tracking. The tool was developed to ensure that there is efficient detection of punctate structures initially developed by mathematical equations, fluorescent beads and cellular images with fluorescently labelled vesicles that can exocytose. The detection of these punctate structures using the tool was compared with other existing tools, such as find maxima in ImageJ and manual detection. The tool not only met the precision of existing solutions but also expedited the process, resulting in a more time-efficient solution. During exocytosis, there is a sudden increase in the intensity of the fluorescently labelled vesicles that look like punctate structures. The algorithm precisely locates the vesicles coordinates and quantifies the variations in their respective intensities. Subsequently, the algorithm processes and retrieves pertinent information from large datasets surpassing that of conventional methods under our evaluation, affirming its efficacy. Furthermore, the tool exhibits adaptability for the image analysis of diverse cellular processes, requiring only minimal modifications to ensure accurate detection of exocytosis.
{"title":"Algorithm for automatic detection of insulin granule exocytosis in human beta-cells","authors":"Aishwarya A Makam, Abhimanyu Dubey, Shovamayee Maharana, Nikhil Gandasi","doi":"10.1101/2023.11.14.566999","DOIUrl":"https://doi.org/10.1101/2023.11.14.566999","url":null,"abstract":"Image processing and analysis are two significant areas that are highly important for interpreting enormous amounts of data obtained from microscopy-based experiments. Several image analysis tools exist for the general detection of fundamental cellular processes, but tools to detect highly distinct cellular functions are few. One such process is exocytosis, which involves the release of vesicular content out of the cell. The size of the vesicles and the inherent differences in the imaging parameters demand specific analysis platforms for detecting exocytosis. In this direction, we have developed an image-processing algorithm based on Lagrangian particle tracking. The tool was developed to ensure that there is efficient detection of punctate structures initially developed by mathematical equations, fluorescent beads and cellular images with fluorescently labelled vesicles that can exocytose. The detection of these punctate structures using the tool was compared with other existing tools, such as find maxima in ImageJ and manual detection. The tool not only met the precision of existing solutions but also expedited the process, resulting in a more time-efficient solution. During exocytosis, there is a sudden increase in the intensity of the fluorescently labelled vesicles that look like punctate structures. The algorithm precisely locates the vesicles coordinates and quantifies the variations in their respective intensities. Subsequently, the algorithm processes and retrieves pertinent information from large datasets surpassing that of conventional methods under our evaluation, affirming its efficacy. Furthermore, the tool exhibits adaptability for the image analysis of diverse cellular processes, requiring only minimal modifications to ensure accurate detection of exocytosis.","PeriodicalId":486943,"journal":{"name":"bioRxiv (Cold Spring Harbor Laboratory)","volume":"5 6","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"134954392","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Background: The leaf of Rumex nepalensis has historically been employed to treat urinary retention and as a diuretic. Despite these assertions, there has been very little research addressing the diuretic effect of the leaves of R. nepalensis. Therefore, this study was done to examine the diuretic properties of R. nepalensis leaves. Methods: Cold maceration with 80% methanol was used to extract the coarsely powdered leaves of R. nepalensis. The extract was separated using increasing polarity solvents, beginning with n-hexane, ethyl acetate, and water. Mice were split into sections to test the plant's diuretic properties. The negative control groups were given either distilled water or 2% tween 80; the positive control groups were given furosemide (10 mg/kg); and the test groups were given the 80% methanol extract and solvent fractions orally at dosages of 100, 200, and 400 mg/kg. The urine volume was determined, and urine analysis was performed on each extract. Results: At dosage levels of 200 mg/kg and 400 mg/kg, the 80% methanol extract, ethyl acetate fraction, and aqueous fraction all produced substantial diuresis (p<0.001) as compared to the negative control. Similarly, mice given the 80% methanol extract, ethyl acetate fraction, and aqueous fraction demonstrated substantial natriuresis (p<0.001) and kaliuresis (p<0.001) at dosages of 200 mg/kg and 400 mg/kg, respectively, in comparison to the negative control. Conclusion: The diuretic activity of R. nepalensis was significantly seen in the 80% methanol extract, ethyl acetate, and aqueous fractions, which corroborated the plant's traditional use.
{"title":"Evaluation of the Diuretic Activities of 80% Methanol Leaf Extract and Solvent Fractions of Rumex nepalensis in Mice","authors":"Fasika Argaw Tafesse, Assefa Belay Asrie, Tafere Mulaw Belete","doi":"10.1101/2023.11.10.566525","DOIUrl":"https://doi.org/10.1101/2023.11.10.566525","url":null,"abstract":"Background: The leaf of Rumex nepalensis has historically been employed to treat urinary retention and as a diuretic. Despite these assertions, there has been very little research addressing the diuretic effect of the leaves of R. nepalensis. Therefore, this study was done to examine the diuretic properties of R. nepalensis leaves. Methods: Cold maceration with 80% methanol was used to extract the coarsely powdered leaves of R. nepalensis. The extract was separated using increasing polarity solvents, beginning with n-hexane, ethyl acetate, and water. Mice were split into sections to test the plant's diuretic properties. The negative control groups were given either distilled water or 2% tween 80; the positive control groups were given furosemide (10 mg/kg); and the test groups were given the 80% methanol extract and solvent fractions orally at dosages of 100, 200, and 400 mg/kg. The urine volume was determined, and urine analysis was performed on each extract. Results: At dosage levels of 200 mg/kg and 400 mg/kg, the 80% methanol extract, ethyl acetate fraction, and aqueous fraction all produced substantial diuresis (p<0.001) as compared to the negative control. Similarly, mice given the 80% methanol extract, ethyl acetate fraction, and aqueous fraction demonstrated substantial natriuresis (p<0.001) and kaliuresis (p<0.001) at dosages of 200 mg/kg and 400 mg/kg, respectively, in comparison to the negative control. Conclusion: The diuretic activity of R. nepalensis was significantly seen in the 80% methanol extract, ethyl acetate, and aqueous fractions, which corroborated the plant's traditional use.","PeriodicalId":486943,"journal":{"name":"bioRxiv (Cold Spring Harbor Laboratory)","volume":"28 12","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"134954559","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-11-14DOI: 10.1101/2023.11.10.564582
Elisabeth Roider, Alexandra I.T. Lakatos, Alicia M. McConnell, Poguang Wang, Alina Mueller, Akinori Kawakami, Jennifer Tsoi, Botond L. Szabolcs, Anna A. Ascsillan, Yusuke Suita, Vivien Igras, Jennifer A. Lo, Jennifer J. Hsiao, Rebecca Lapides, Dorottya M.P. Pal, Anna S. Lengyel, Alexander Navarini, Arimichi Okazaki, Othon Iliopoulos, Istvan Nemeth, Thomas G. Graeber, Leonard Zon, Roger W. Giese, Lajos V. Kemeny, David E. Fisher
Microphthalmia-associated transcription factor (MITF) plays pivotal roles in melanocyte development, function, and melanoma pathogenesis. MITF amplification occurs in melanoma and has been associated with resistance to targeted therapies. Here, we show that MITF regulates a global antioxidant program that increases survival of melanoma cell lines by protecting the cells from reactive oxygen species (ROS)-induced damage. In addition, this redox program is correlated with MITF expression in human melanoma cell lines and patient-derived melanoma samples. Using a zebrafish melanoma model, we show that MITF decreases ROS-mediated DNA damage in vivo. Some of the MITF target genes involved, such as IDH1 and NNT, are regulated through direct MITF binding to canonical enhancer box (E-BOX) sequences proximal to their promoters. Utilizing functional experiments, we demonstrate the role of MITF and its target genes in reducing cytosolic and mitochondrial ROS. Collectively, our data identify MITF as a significant driver of the cellular antioxidant state.
{"title":"MITF regulates IDH1 and NNT and drives a transcriptional program protecting cutaneous melanoma from reactive oxygen species","authors":"Elisabeth Roider, Alexandra I.T. Lakatos, Alicia M. McConnell, Poguang Wang, Alina Mueller, Akinori Kawakami, Jennifer Tsoi, Botond L. Szabolcs, Anna A. Ascsillan, Yusuke Suita, Vivien Igras, Jennifer A. Lo, Jennifer J. Hsiao, Rebecca Lapides, Dorottya M.P. Pal, Anna S. Lengyel, Alexander Navarini, Arimichi Okazaki, Othon Iliopoulos, Istvan Nemeth, Thomas G. Graeber, Leonard Zon, Roger W. Giese, Lajos V. Kemeny, David E. Fisher","doi":"10.1101/2023.11.10.564582","DOIUrl":"https://doi.org/10.1101/2023.11.10.564582","url":null,"abstract":"Microphthalmia-associated transcription factor (MITF) plays pivotal roles in melanocyte development, function, and melanoma pathogenesis. MITF amplification occurs in melanoma and has been associated with resistance to targeted therapies. Here, we show that MITF regulates a global antioxidant program that increases survival of melanoma cell lines by protecting the cells from reactive oxygen species (ROS)-induced damage. In addition, this redox program is correlated with MITF expression in human melanoma cell lines and patient-derived melanoma samples. Using a zebrafish melanoma model, we show that MITF decreases ROS-mediated DNA damage in vivo. Some of the MITF target genes involved, such as IDH1 and NNT, are regulated through direct MITF binding to canonical enhancer box (E-BOX) sequences proximal to their promoters. Utilizing functional experiments, we demonstrate the role of MITF and its target genes in reducing cytosolic and mitochondrial ROS. Collectively, our data identify MITF as a significant driver of the cellular antioxidant state.","PeriodicalId":486943,"journal":{"name":"bioRxiv (Cold Spring Harbor Laboratory)","volume":"22 4","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"134957016","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-11-14DOI: 10.1101/2023.11.10.566590
Romane Junker, Florence Valence, Michel-Yves Mistou, Stéphane Chaillou, Helene Chiapello
The management of food fermentation is still largely based on empirical knowledge, as the dynamics of microbial communities and the underlying metabolic networks that produce safe and nutritious products remain beyond our understanding. Although these closed ecosystems contain relatively few taxa, they have not yet been thoroughly characterized with respect to how their microbial communities interact and dynamically evolve. However, with the increased availability of metataxonomic datasets on different fermented vegetables, it is now possible to gain a comprehensive understanding of the microbial relationships that structure plant fermentation. In this study, we present a bioinformatics approach that integrates public metataxonomic 16S datasets targeting fermented vegetables. Specifically, we developed a method for exploring, comparing, and combining public 16S datasets in order to perform meta-analyses of microbiota. The workflow includes steps for searching and selecting public time-series datasets and constructing association networks of amplicon sequence variants (ASVs) based on co-abundance metrics. Networks for individual datasets are then integrated into a core network of significant associations. Microbial communities are identified based on the comparison and clustering of ASV networks using the "stochastic block model" method. When we applied this method to 10 public datasets (including a total of 931 samples), we found that it was able to shed light on the dynamics of vegetable fermentation by characterizing the processes of community succession among different bacterial assemblages.
{"title":"Integration of metataxonomic datasets into microbial association networks highlights shared bacterial community dynamics in fermented vegetables","authors":"Romane Junker, Florence Valence, Michel-Yves Mistou, Stéphane Chaillou, Helene Chiapello","doi":"10.1101/2023.11.10.566590","DOIUrl":"https://doi.org/10.1101/2023.11.10.566590","url":null,"abstract":"The management of food fermentation is still largely based on empirical knowledge, as the dynamics of microbial communities and the underlying metabolic networks that produce safe and nutritious products remain beyond our understanding. Although these closed ecosystems contain relatively few taxa, they have not yet been thoroughly characterized with respect to how their microbial communities interact and dynamically evolve. However, with the increased availability of metataxonomic datasets on different fermented vegetables, it is now possible to gain a comprehensive understanding of the microbial relationships that structure plant fermentation. In this study, we present a bioinformatics approach that integrates public metataxonomic 16S datasets targeting fermented vegetables. Specifically, we developed a method for exploring, comparing, and combining public 16S datasets in order to perform meta-analyses of microbiota. The workflow includes steps for searching and selecting public time-series datasets and constructing association networks of amplicon sequence variants (ASVs) based on co-abundance metrics. Networks for individual datasets are then integrated into a core network of significant associations. Microbial communities are identified based on the comparison and clustering of ASV networks using the \"stochastic block model\" method. When we applied this method to 10 public datasets (including a total of 931 samples), we found that it was able to shed light on the dynamics of vegetable fermentation by characterizing the processes of community succession among different bacterial assemblages.","PeriodicalId":486943,"journal":{"name":"bioRxiv (Cold Spring Harbor Laboratory)","volume":"11 6","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"134957457","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-11-14DOI: 10.1101/2023.11.13.566888
Anouk Lkharrazi, Kurt Tobler, Sara Marti, Anna Bratus-Neuenschwander, Bernd Vogt, Cornel Fraefel
Adeno-associated virus type 2 (AAV2) is a small, non-pathogenic, helper virus-dependent parvovirus with a single-stranded (ss) DNA genome of approximately 4.7 kb. AAV2 DNA replication requires the presence of a helper virus such as adenovirus type 5 (AdV5) or herpes simplex virus type 1 (HSV-1) and is generally assumed to occur as a strand-displacement rolling hairpin (RHR) mechanism initiated at the AAV2 3’ inverted terminal repeat (ITR). We have recently shown that AAV2 replication supported by HSV-1 leads to the formation of double-stranded head-to-tail concatemers, which provides evidence for a rolling circle replication (RCR) mechanism. We have revisited AAV2 DNA replication and specifically compared the formation of AAV2 replication intermediates in presence of either HSV-1 or AdV5 as the helper virus. The results confirmed that the AAV2 DNA replication mechanism is helper virus-dependent and follows a strand-displacement RHR mechanism when AdV5 is the helper virus and primarily an RCR mechanism when HSV-1 is the helper virus. We also demonstrate that recombination plays a negligible role in AAV2 genome replication. Interestingly, the formation of high molecular weight AAV2 DNA concatemers in presence of HSV-1 as the helper virus was dependent on an intact HSV-1 DNA polymerase.
{"title":"Adeno-associated virus (AAV2) can replicate its DNA by a rolling hairpin or rolling circle mechanism, depending on the helper virus.","authors":"Anouk Lkharrazi, Kurt Tobler, Sara Marti, Anna Bratus-Neuenschwander, Bernd Vogt, Cornel Fraefel","doi":"10.1101/2023.11.13.566888","DOIUrl":"https://doi.org/10.1101/2023.11.13.566888","url":null,"abstract":"Adeno-associated virus type 2 (AAV2) is a small, non-pathogenic, helper virus-dependent parvovirus with a single-stranded (ss) DNA genome of approximately 4.7 kb. AAV2 DNA replication requires the presence of a helper virus such as adenovirus type 5 (AdV5) or herpes simplex virus type 1 (HSV-1) and is generally assumed to occur as a strand-displacement rolling hairpin (RHR) mechanism initiated at the AAV2 3’ inverted terminal repeat (ITR). We have recently shown that AAV2 replication supported by HSV-1 leads to the formation of double-stranded head-to-tail concatemers, which provides evidence for a rolling circle replication (RCR) mechanism. We have revisited AAV2 DNA replication and specifically compared the formation of AAV2 replication intermediates in presence of either HSV-1 or AdV5 as the helper virus. The results confirmed that the AAV2 DNA replication mechanism is helper virus-dependent and follows a strand-displacement RHR mechanism when AdV5 is the helper virus and primarily an RCR mechanism when HSV-1 is the helper virus. We also demonstrate that recombination plays a negligible role in AAV2 genome replication. Interestingly, the formation of high molecular weight AAV2 DNA concatemers in presence of HSV-1 as the helper virus was dependent on an intact HSV-1 DNA polymerase.","PeriodicalId":486943,"journal":{"name":"bioRxiv (Cold Spring Harbor Laboratory)","volume":"10 7","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"134957464","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-11-14DOI: 10.1101/2023.11.14.567086
Adrian Arrieta, Douglas J Chapski, Anna Reese, Todd H Kimball, Kunhua Song, Manuel Rosa-Garrido, Thomas M Vondriska
Rationale: During postnatal cardiac hypertrophy, cardiomyocytes undergo mitotic exit, relying on DNA replication-independent mechanisms of histone turnover to maintain chromatin organization and gene transcription. In other tissues, circadian oscillations in nucleosome occupancy influence clock-controlled gene expression, suggesting an unrecognized role for the circadian clock in temporal control of histone turnover and coordinate cardiomyocyte gene expression. Objective: To elucidate roles for the master circadian transcription factor, Bmal1, in histone turnover, chromatin organization, and myocyte-specific gene expression and cell growth in the neonatal period. Methods and Results: Bmal1 knockdown in neonatal rat ventricular myocytes (NRVM) decreased myocyte size, total cellular protein, and transcription of the fetal hypertrophic gene Nppb following treatment with increasing serum concentrations or the α-adrenergic agonist phenylephrine (PE). Bmal1 knockdown decreased expression of clock-controlled genes Per2 and Tcap, and salt-inducible kinase 1 (Sik1) which was identified via gene ontology analysis of Bmal1 targets upregulated in adult versus embryonic hearts. Epigenomic analyses revealed co-localized chromatin accessibility and Bmal1 localization in the Sik1 promoter. Bmal1 knockdown impaired Per2 and Sik1 promoter accessibility as measured by MNase-qPCR and impaired histone turnover indicated by metabolic labeling of acid-soluble chromatin fractions and immunoblots of total and chromatin-associated core histones. Sik1 knockdown basally increased myocyte size, while simultaneously impairing and driving Nppb and Per2 transcription, respectively. Conclusions: Bmal1 is required for neonatal myocyte growth, replication-independent histone turnover, and chromatin organization at the Sik1 promoter. Sik1 represents a novel clock-controlled gene that coordinates myocyte growth with hypertrophic and clock-controlled gene transcription.
{"title":"Circadian Control of Histone Turnover During Cardiac Development and Growth","authors":"Adrian Arrieta, Douglas J Chapski, Anna Reese, Todd H Kimball, Kunhua Song, Manuel Rosa-Garrido, Thomas M Vondriska","doi":"10.1101/2023.11.14.567086","DOIUrl":"https://doi.org/10.1101/2023.11.14.567086","url":null,"abstract":"Rationale: During postnatal cardiac hypertrophy, cardiomyocytes undergo mitotic exit, relying on DNA replication-independent mechanisms of histone turnover to maintain chromatin organization and gene transcription. In other tissues, circadian oscillations in nucleosome occupancy influence clock-controlled gene expression, suggesting an unrecognized role for the circadian clock in temporal control of histone turnover and coordinate cardiomyocyte gene expression. Objective: To elucidate roles for the master circadian transcription factor, Bmal1, in histone turnover, chromatin organization, and myocyte-specific gene expression and cell growth in the neonatal period. Methods and Results: Bmal1 knockdown in neonatal rat ventricular myocytes (NRVM) decreased myocyte size, total cellular protein, and transcription of the fetal hypertrophic gene Nppb following treatment with increasing serum concentrations or the α-adrenergic agonist phenylephrine (PE). Bmal1 knockdown decreased expression of clock-controlled genes Per2 and Tcap, and salt-inducible kinase 1 (Sik1) which was identified via gene ontology analysis of Bmal1 targets upregulated in adult versus embryonic hearts. Epigenomic analyses revealed co-localized chromatin accessibility and Bmal1 localization in the Sik1 promoter. Bmal1 knockdown impaired Per2 and Sik1 promoter accessibility as measured by MNase-qPCR and impaired histone turnover indicated by metabolic labeling of acid-soluble chromatin fractions and immunoblots of total and chromatin-associated core histones. Sik1 knockdown basally increased myocyte size, while simultaneously impairing and driving Nppb and Per2 transcription, respectively. Conclusions: Bmal1 is required for neonatal myocyte growth, replication-independent histone turnover, and chromatin organization at the Sik1 promoter. Sik1 represents a novel clock-controlled gene that coordinates myocyte growth with hypertrophic and clock-controlled gene transcription.","PeriodicalId":486943,"journal":{"name":"bioRxiv (Cold Spring Harbor Laboratory)","volume":"48 22","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"134991316","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-11-14DOI: 10.1101/2023.11.13.566961
Bernadeta Dadonaite, Jack Brown, Teagan E McMahon, Ariana G Farrell, Daniel Asarnow, Cameron Stewart, Jenni Logue, Ben Murrell, Helen Y Chu, David Veesler, Jesse D Bloom
SARS-CoV-2 variants acquire mutations in spike that promote immune evasion and impact other properties that contribute to viral fitness such as ACE2 receptor binding and cell entry. Knowledge of how mutations affect these spike phenotypes can provide insight into the current and potential future evolution of the virus. Here we use pseudovirus deep mutational scanning to measure how >9,000 mutations across the full XBB.1.5 and BA.2 spikes affect ACE2 binding, cell entry, or escape from human sera. We find that mutations outside the receptor-binding domain (RBD) have meaningfully impacted ACE2 binding during SARS-CoV-2 evolution. We also measure how mutations to the XBB.1.5 spike affect neutralization by serum from individuals who recently had SARS-CoV-2 infections. The strongest serum escape mutations are in the RBD at sites 357, 420, 440, 456, and 473--however, the antigenic impacts of these mutations vary across individuals. We also identify strong escape mutations outside the RBD; however many of them decrease ACE2 binding, suggesting they act by modulating RBD conformation. Notably, the growth rates of human SARS-CoV-2 clades can be explained in substantial part by the measured effects of mutations on spike phenotypes, suggesting our data could enable better prediction of viral evolution.
{"title":"Full-spike deep mutational scanning helps predict the evolutionary success of SARS-CoV-2 clades","authors":"Bernadeta Dadonaite, Jack Brown, Teagan E McMahon, Ariana G Farrell, Daniel Asarnow, Cameron Stewart, Jenni Logue, Ben Murrell, Helen Y Chu, David Veesler, Jesse D Bloom","doi":"10.1101/2023.11.13.566961","DOIUrl":"https://doi.org/10.1101/2023.11.13.566961","url":null,"abstract":"SARS-CoV-2 variants acquire mutations in spike that promote immune evasion and impact other properties that contribute to viral fitness such as ACE2 receptor binding and cell entry. Knowledge of how mutations affect these spike phenotypes can provide insight into the current and potential future evolution of the virus. Here we use pseudovirus deep mutational scanning to measure how >9,000 mutations across the full XBB.1.5 and BA.2 spikes affect ACE2 binding, cell entry, or escape from human sera. We find that mutations outside the receptor-binding domain (RBD) have meaningfully impacted ACE2 binding during SARS-CoV-2 evolution. We also measure how mutations to the XBB.1.5 spike affect neutralization by serum from individuals who recently had SARS-CoV-2 infections. The strongest serum escape mutations are in the RBD at sites 357, 420, 440, 456, and 473--however, the antigenic impacts of these mutations vary across individuals. We also identify strong escape mutations outside the RBD; however many of them decrease ACE2 binding, suggesting they act by modulating RBD conformation. Notably, the growth rates of human SARS-CoV-2 clades can be explained in substantial part by the measured effects of mutations on spike phenotypes, suggesting our data could enable better prediction of viral evolution.","PeriodicalId":486943,"journal":{"name":"bioRxiv (Cold Spring Harbor Laboratory)","volume":"45 24","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"134991744","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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