cometadata/datacite-titles-descriptions-related-identifiers huggingface dataset:
DOI: 10.6084/m9.figshare.18866727
title: Regulation of host factor γ-H2AX level and location by enterovirus A71 for viral replication

description: Numerous viruses manipulate host factors for viral production. We demonstrated that human enterovirus A71 (EVA71), a primary causative agent for hand, foot, and mouth disease (HFMD), increased the level of the DNA damage response (DDR) marker γ-H2AX. DDR is primarily mediated by the ataxia telangiectasia mutated (ATM), ATM and Rad3-related (ATR), or DNA-dependent protein kinase (DNA-PK) pathways. Upregulation of γ-H2AX by EVA71 was dependent on the ATR but not the ATM or DNA-PK pathway. As a nuclear factor, there is no previous evidence of cytoplasmic distribution of γ-H2AX. However, the present findings demonstrated that EVA71 encouraged the localization of γ-H2AX to the cytoplasm. Of note, γ-H2AX formed a complex with structural protein VP3, non-structural protein 3D, and the viral genome. Treatment with an inhibitor or CRISPR/Cas9 technology to decrease or silence the expression of γ-H2AX decreased viral genome replication in host cells; this effect was accompanied by decreased viral protein expression and virions. In animal experiments, caffeine was used to inhibit DDR; the results revealed that caffeine protected neonatal mice from death after infection with EVA71, laying the foundation for new therapeutic applications of caffeine. More importantly, in children with HFMD, γ-H2AX was upregulated in peripheral blood lymphocytes. The consistent <i>in vitro</i> and <i>in vivo</i> data on γ-H2AX from this study suggested that caffeine or other inhibitors of DDR might be novel therapeutic agents for HFMD.
DOI: 10.6084/m9.figshare.18866727.v1
title: Regulation of host factor γ-H2AX level and location by enterovirus A71 for viral replication

description: Numerous viruses manipulate host factors for viral production. We demonstrated that human enterovirus A71 (EVA71), a primary causative agent for hand, foot, and mouth disease (HFMD), increased the level of the DNA damage response (DDR) marker γ-H2AX. DDR is primarily mediated by the ataxia telangiectasia mutated (ATM), ATM and Rad3-related (ATR), or DNA-dependent protein kinase (DNA-PK) pathways. Upregulation of γ-H2AX by EVA71 was dependent on the ATR but not the ATM or DNA-PK pathway. As a nuclear factor, there is no previous evidence of cytoplasmic distribution of γ-H2AX. However, the present findings demonstrated that EVA71 encouraged the localization of γ-H2AX to the cytoplasm. Of note, γ-H2AX formed a complex with structural protein VP3, non-structural protein 3D, and the viral genome. Treatment with an inhibitor or CRISPR/Cas9 technology to decrease or silence the expression of γ-H2AX decreased viral genome replication in host cells; this effect was accompanied by decreased viral protein expression and virions. In animal experiments, caffeine was used to inhibit DDR; the results revealed that caffeine protected neonatal mice from death after infection with EVA71, laying the foundation for new therapeutic applications of caffeine. More importantly, in children with HFMD, γ-H2AX was upregulated in peripheral blood lymphocytes. The consistent <i>in vitro</i> and <i>in vivo</i> data on γ-H2AX from this study suggested that caffeine or other inhibitors of DDR might be novel therapeutic agents for HFMD.
DOI: 10.6084/m9.figshare.21324321
title: Arginine 58 is indispensable for proper function of the <i>Francisella tularensis</i> subsp. <i>holarctica</i> FSC200 HU protein, and its substitution alters virulence and mediates immunity against wild-type strain

description: HU protein, a member of the nucleoid-associated group of proteins, is an important transcription factor in bacteria, including in the dangerous human pathogen <i>Francisella tularensis</i>. Generally, HU protein acts as a DNA sequence non-specific binding protein and it is responsible for winding of the DNA chain that leads to the separation of transcription units. Here, we identified potential HU protein binding sites using the ChIP-seq method and two possible binding motifs in <i>F. tularensis</i> subsp. <i>holarctica</i> FSC200 depending upon growth conditions. We also confirmed that FSC200 HU protein is able to introduce negative supercoiling of DNA in the presence of topoisomerase I. Next, we showed interaction of the HU protein with a DNA region upstream of the <i>pigR</i> gene and inside the <i>clpB</i> gene, suggesting possible regulation of PigR and ClpB expression. Moreover, we showed that arginine 58 and partially arginine 61 are important for HU protein’s DNA binding capacity, negative supercoiling induction by HU, and the length and winding of FSC200 chromosomal DNA. Finally, in order to verify biological function of the HU protein, we demonstrated that mutations in arginine 58, arginine 61, and serine 74 of the HU protein decrease virulence of FSC200 both <i>in vitro</i> and <i>in vivo</i> and that immunization using these mutant strains is able to protect as many as 100% of mice against wild-type challenge. Taken together, our findings deepen knowledge about the role of the HU protein in tularaemia pathogenesis and suggest that HU protein should be addressed in the context of tularaemia vaccine development.
DOI: 10.6084/m9.figshare.21324321.v1
title: Arginine 58 is indispensable for proper function of the <i>Francisella tularensis</i> subsp. <i>holarctica</i> FSC200 HU protein, and its substitution alters virulence and mediates immunity against wild-type strain

description: HU protein, a member of the nucleoid-associated group of proteins, is an important transcription factor in bacteria, including in the dangerous human pathogen <i>Francisella tularensis</i>. Generally, HU protein acts as a DNA sequence non-specific binding protein and it is responsible for winding of the DNA chain that leads to the separation of transcription units. Here, we identified potential HU protein binding sites using the ChIP-seq method and two possible binding motifs in <i>F. tularensis</i> subsp. <i>holarctica</i> FSC200 depending upon growth conditions. We also confirmed that FSC200 HU protein is able to introduce negative supercoiling of DNA in the presence of topoisomerase I. Next, we showed interaction of the HU protein with a DNA region upstream of the <i>pigR</i> gene and inside the <i>clpB</i> gene, suggesting possible regulation of PigR and ClpB expression. Moreover, we showed that arginine 58 and partially arginine 61 are important for HU protein’s DNA binding capacity, negative supercoiling induction by HU, and the length and winding of FSC200 chromosomal DNA. Finally, in order to verify biological function of the HU protein, we demonstrated that mutations in arginine 58, arginine 61, and serine 74 of the HU protein decrease virulence of FSC200 both <i>in vitro</i> and <i>in vivo</i> and that immunization using these mutant strains is able to protect as many as 100% of mice against wild-type challenge. Taken together, our findings deepen knowledge about the role of the HU protein in tularaemia pathogenesis and suggest that HU protein should be addressed in the context of tularaemia vaccine development.
DOI: 10.6084/m9.figshare.19317876
title: Extracellular trap can be trained as a memory response

description: Extracellular trap (ET) appears as a double-edged sword for the host since it participates in host immune defense by entrapping pathogens, while excessive ET release also contributes to various diseases progression including atherosclerosis, cancer, and autoimmune disorders. A better understanding of ET formation and regulation will be beneficial for developing strategies for infection control and ET-associated disease treatment. There is some evidence indicating that prior infection can enhance extracellular killing. Neutrophils from cancer or sepsis are predisposed to generate ET. It is reasonable to suspect that ET may be trained to form as a memory response, just like cytokine memory response termed “trained immunity.” The mice were intraperitoneally injected with heat-killed <i>Candida albicans</i> (HK-<i>C. albicans</i>), 3 days later bone marrow-derived macrophages (BMDM) were isolated and challenged with <i>Clostridium perfringens</i> as a second stimulation. We found that HK-<i>C. albicans</i> priming enhanced ET formation upon <i>Clostridium perfringens</i> infection, accompanied by increased extracellular killing capacity. Mannan priming also enhanced ET formation. Since ETs memory was induced in chicken PBMC, ETs memory may be evolutionarily conserved. Moreover, mTOR was required for ETs memory response. Collectively, this study showed that ETs can be trained as a memory response and indicated that memory property of ETs should be considered during the understanding of recurrent infection and ET-associated disorders.
DOI: 10.6084/m9.figshare.19317876.v1
title: Extracellular trap can be trained as a memory response

description: Extracellular trap (ET) appears as a double-edged sword for the host since it participates in host immune defense by entrapping pathogens, while excessive ET release also contributes to various diseases progression including atherosclerosis, cancer, and autoimmune disorders. A better understanding of ET formation and regulation will be beneficial for developing strategies for infection control and ET-associated disease treatment. There is some evidence indicating that prior infection can enhance extracellular killing. Neutrophils from cancer or sepsis are predisposed to generate ET. It is reasonable to suspect that ET may be trained to form as a memory response, just like cytokine memory response termed “trained immunity.” The mice were intraperitoneally injected with heat-killed <i>Candida albicans</i> (HK-<i>C. albicans</i>), 3 days later bone marrow-derived macrophages (BMDM) were isolated and challenged with <i>Clostridium perfringens</i> as a second stimulation. We found that HK-<i>C. albicans</i> priming enhanced ET formation upon <i>Clostridium perfringens</i> infection, accompanied by increased extracellular killing capacity. Mannan priming also enhanced ET formation. Since ETs memory was induced in chicken PBMC, ETs memory may be evolutionarily conserved. Moreover, mTOR was required for ETs memory response. Collectively, this study showed that ETs can be trained as a memory response and indicated that memory property of ETs should be considered during the understanding of recurrent infection and ET-associated disorders.
DOI: 10.6084/m9.figshare.20381586
title: SARS-CoV-2 shedding dynamics and transmission in immunosuppressed patients

description: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern have been emerging. However, knowledge of temporal and spatial dynamics of SARS-CoV-2 is limited. This study characterized SARS-CoV-2 evolution in immunosuppressed patients with long-term SARS-CoV-2 shedding for 73–250 days, without specific treatment. We conducted whole-genome sequencing of 27 serial samples, including 26 serial samples collected from various anatomic sites of two patients and the first positive sample from patient 2‘s mother. We analysed the intrahost temporal dynamics and genomic diversity of the viral population within different sample types. Intrahost variants emerging during infection showed diversity between individual hosts. Remarkably, N501Y, P681R, and E484K, key substitutions within spike protein, emerged <i>in vivo</i> during infection and became the dominant population. P681R, which had not yet been detected in the publicly available genome in Korea, appeared within patient 1 during infection. Mutually exclusive substitutions at residues R346 (R346S and R346I) and E484 (E484K and E484A) of spike protein and continuous turnover of these substitutions occurred. Unique genetic changes were observed in urine samples. A household transmission from patient 2 to his mother, at least 38 days after the diagnosis, was characterized. Viruses may differently mutate and adjust to the host selective pressure, which could enable the virus to replicate efficiently for fitness in each host. Intrahost variants could be candidate variants likely to spread to the population eventually. Our findings may provide new insights into the dynamics of SARS-CoV-2 in response to interactions between the virus and host.
DOI: 10.6084/m9.figshare.20381586.v1
title: SARS-CoV-2 shedding dynamics and transmission in immunosuppressed patients

description: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern have been emerging. However, knowledge of temporal and spatial dynamics of SARS-CoV-2 is limited. This study characterized SARS-CoV-2 evolution in immunosuppressed patients with long-term SARS-CoV-2 shedding for 73–250 days, without specific treatment. We conducted whole-genome sequencing of 27 serial samples, including 26 serial samples collected from various anatomic sites of two patients and the first positive sample from patient 2‘s mother. We analysed the intrahost temporal dynamics and genomic diversity of the viral population within different sample types. Intrahost variants emerging during infection showed diversity between individual hosts. Remarkably, N501Y, P681R, and E484K, key substitutions within spike protein, emerged <i>in vivo</i> during infection and became the dominant population. P681R, which had not yet been detected in the publicly available genome in Korea, appeared within patient 1 during infection. Mutually exclusive substitutions at residues R346 (R346S and R346I) and E484 (E484K and E484A) of spike protein and continuous turnover of these substitutions occurred. Unique genetic changes were observed in urine samples. A household transmission from patient 2 to his mother, at least 38 days after the diagnosis, was characterized. Viruses may differently mutate and adjust to the host selective pressure, which could enable the virus to replicate efficiently for fitness in each host. Intrahost variants could be candidate variants likely to spread to the population eventually. Our findings may provide new insights into the dynamics of SARS-CoV-2 in response to interactions between the virus and host.
DOI: 10.6084/m9.figshare.20252657
title: RIOK3-Mediated Akt phosphorylation facilitates synergistic replication of Marek’s disease and reticuloendotheliosis viruses

description: Co-infection of Marek’s disease virus (MDV) and reticuloendotheliosis virus (REV) synergistically drives disease progression, yet little is known about the mechanism of the synergism. Here, we found that co-infection of REV and MDV increased their replication via the RIOK3-Akt pathway. Initially, we noticed that the viral titres of MDV and REV significantly increased in REV and MDV co-infected cells compared with single-infected cells. Furthermore, tandem mass tag peptide labelling coupled with LC/MS analysis showed that Akt was upregulated in REV and MDV co-infected cells. Overexpression of Akt promoted synergistic replication of MDV and REV. Conversely, inhibition of Akt suppressed synergistic replication of MDV and REV. However, PI3K inhibition did not affect synergistic replication of MDV and REV, suggesting that the PI3K/Akt pathway is not involved in the synergism of MDV and REV. In addition, we revealed that RIOK3 was recruited to regulate Akt in REV and MDV co-infected cells. Moreover, wild-type RIOK3, but not kinase-dead RIOK3, mediated Akt phosphorylation and promoted synergistic replication of MDV and REV. Our results illustrate that MDV and REV activated a novel RIOK3-Akt signalling pathway to facilitate their synergistic replication.
DOI: 10.6084/m9.figshare.20252657.v1
title: RIOK3-mediated Akt phosphorylation facilitates synergistic replication of Marek’s disease and reticuloendotheliosis viruses

description: Co-infection of Marek’s disease virus (MDV) and reticuloendotheliosis virus (REV) synergistically drives disease progression, yet little is known about the mechanism of the synergism. Here, we found that co-infection of REV and MDV increased their replication via the RIOK3-Akt pathway. Initially, we noticed that the viral titers of MDV and REV significantly increased in REV and MDV co-infected cells compared with single-infected cells. Furthermore, tandem mass tag peptide labeling coupled with LC/MS analysis showed that Akt was upregulated in REV and MDV co-infected cells. Overexpression of Akt promoted synergistic replication of MDV and REV. Conversely, inhibition of Akt suppressed synergistic replication of MDV and REV. However, PI3K inhibition did not affect synergistic replication of MDV and REV, suggesting that the PI3K/Akt pathway is not involved in the synergism of MDV and REV. In addition, we revealed that RIOK3 was recruited to regulate Akt in REV and MDV co-infected cells. Moreover, wild-type RIOK3, but not kinase-dead RIOK3, mediated Akt phosphorylation and promoted synergistic replication of MDV and REV. Our results illustrate that MDV and REV activated a novel RIOK3-Akt signaling pathway to facilitate their synergistic replication.
DOI: 10.6084/m9.figshare.20252657.v2
title: RIOK3-Mediated Akt phosphorylation facilitates synergistic replication of Marek’s disease and reticuloendotheliosis viruses

description: Co-infection of Marek’s disease virus (MDV) and reticuloendotheliosis virus (REV) synergistically drives disease progression, yet little is known about the mechanism of the synergism. Here, we found that co-infection of REV and MDV increased their replication via the RIOK3-Akt pathway. Initially, we noticed that the viral titres of MDV and REV significantly increased in REV and MDV co-infected cells compared with single-infected cells. Furthermore, tandem mass tag peptide labelling coupled with LC/MS analysis showed that Akt was upregulated in REV and MDV co-infected cells. Overexpression of Akt promoted synergistic replication of MDV and REV. Conversely, inhibition of Akt suppressed synergistic replication of MDV and REV. However, PI3K inhibition did not affect synergistic replication of MDV and REV, suggesting that the PI3K/Akt pathway is not involved in the synergism of MDV and REV. In addition, we revealed that RIOK3 was recruited to regulate Akt in REV and MDV co-infected cells. Moreover, wild-type RIOK3, but not kinase-dead RIOK3, mediated Akt phosphorylation and promoted synergistic replication of MDV and REV. Our results illustrate that MDV and REV activated a novel RIOK3-Akt signalling pathway to facilitate their synergistic replication.
DOI: 10.6084/m9.figshare.20252660
title: Using <i>in vivo</i> transcriptomics and RNA enrichment to identify genes involved in virulence of <i>Candida glabrata</i>

description: <i>Candida</i> species are the most commonly isolated opportunistic fungal pathogens in humans. <i>Candida albicans</i> causes most of the diagnosed infections, closely followed by <i>Candida glabrata</i>. <i>C. albicans</i> is well studied, and many genes have been shown to be important for infection and colonization of the host. It is however less clear how <i>C. glabrata</i> infects the host. With the help of fungal RNA enrichment, we here investigated for the first time the transcriptomic profile of <i>C. glabrata</i> during urinary tract infection (UTI) in mice. In the UTI model, bladders and kidneys are major target organs and therefore fungal transcriptomes were addressed in these organs. Our results showed that, next to adhesins and proteases, nitrogen metabolism and regulation play a vital role during <i>C. glabrata</i> UTI. Genes involved in nitrogen metabolism were upregulated and among them we show that <i>DUR1,2</i> (urea amidolyase) and <i>GAP1</i> (amino acid permease) were important for virulence. Furthermore, we confirmed the importance of the glyoxylate cycle in the host and identified <i>MLS1</i> (malate synthase) as an important gene necessary for <i>C. glabrata</i> virulence. In conclusion, our study shows with the support of <i>in vivo</i> transcriptomics how <i>C. glabrata</i> adapts to host conditions.
DOI: 10.6084/m9.figshare.17706111
title: <i>Aeromonas hydrophila</i> inhibits autophagy triggering cytosolic translocation of mtDNA which activates the pro-apoptotic caspase-1/IL-1β-nitric oxide axis in headkidney macrophages

description: The molecular mechanisms underlying <i>Aeromonas hydrophila</i>-pathogenesis are not well understood. Using head kidney macrophages (HKM) of <i>Clarias gariepinus</i>, we previously reported the role of ER-stress in <i>A. hydrophila</i>-induced pathogenesis. Here, we report that PI3K/PLC-induced cytosolic-Ca<sup>2+</sup> imbalance induces the expression of pro-apoptotic ER-stress marker, CHOP in <i>A. hydrophila-</i>infected HKM. CHOP promotes HKM apoptosis by inhibiting AKT activation and enhancing JNK signaling. Elevated mitochondrial ROS (mtROS) was recorded which declined significantly by ameliorating ER-stress and in the presence of ER-Ca<sup>2+</sup> release modulators (2-APB and dantrolene) and mitochondrial-Ca<sup>2+</sup> uptake inhibitor, Ru360, together suggesting the role of ER-mitochondrial Ca<sup>2+</sup> dynamics in mtROS generation. Inhibiting mtROS production reduced HKM death implicating the pro-apoptotic role of mtROS in <i>A. hydrophila</i>-pathogenesis. The expression of autophagic proteins (LC3B, beclin-1, and atg 5) was suppressed in the infected HKM. Our results with autophagy-inducer rapamycin demonstrated that impaired autophagy favored the cytosolic accumulation of mitochondrial DNA (mtDNA) and the process depended on mtROS levels. Enhanced caspase-1 activity and IL-1β production was detected and transfection studies coupled with pharmacological inhibitors implicated mtROS/mtDNA axis to be crucial for activating the caspase-1/IL-1β cascade in infected HKM. RNAi studies further suggested the involvement of IL-1β in generating pro-apoptotic NO in <i>A. hydrophila</i>-infected HKM. Our study suggests a novel role of ER-mitochondria cross-talk in regulating <i>A. hydrophila</i> pathogenesis. Based on our observations, we conclude that <i>A. hydrophila</i> induces ER-stress and inhibits mitophagy resulting in mitochondrial dysfunction which leads to mtROS production and translocation of mtDNA into cytosol triggering the activation of caspase-1/IL-1β-mediated NO production, culminating in HKM apoptosis.