IFN-stimulated metabolite transporter ENT3 facilitates viral genome release.

An increasing amount of evidence emphasizes the role of metabolic reprogramming in immune cells to fight infections. However, little is known about the regulation of metabolite transporters that facilitate and support metabolic demands. In this study, we found that the expression of equilibrative nucleoside transporter 3 (ENT3, encoded by solute carrier family 29 member 3, Slc29a3) is part of the innate immune response, which is rapidly upregulated upon pathogen invasion. The transcription of Slc29a3 is directly regulated by type I interferon-induced signaling, demonstrating that this metabolite transporter is an interferon-stimulated gene (ISG). Suprisingly, we unveil that several viruses, including SARS-CoV-2, require ENT3 to facilitate their entry into the cytoplasm. The removal or suppression of Slc29a3 expression is sufficient to significantly decrease viral replication in vitro and in vivo. Our study reveals that ENT3 is a pro-viral ISG co-opted by some viruses to gain a survival advantage.

SUMO Modification of Histone Demethylase KDM4A in Kaposi's Sarcoma-Associated Herpesvirus-Induced Primary Effusion Lymphoma.

Primary effusion lymphoma (PEL) is a fatal B-cell lymphoma caused by Kaposi's sarcoma-associated herpesvirus (KSHV) infection. Inducing KSHV lytic replication that causes the death of host cells is an attractive treatment approach for PE; however, combination therapy inhibiting viral production is frequently needed to improve its outcomes. We have previously shown that the KSHV lytic protein K-bZIP can SUMOylate histone lysine demethylase 4A (KDM4A) at lysine 471 (K471) and this SUMOylation is required for virus production upon KSHV reactivation. Here, we demonstrate that SUMOylation of KDM4A orchestrates PEL cell survival, a major challenge for the success of PEL treatment; and cell movement and angiogenesis, the cell functions contributing to PEL cell extravasation and dissemination. Furthermore, integrated ChIP-seq and RNA-seq analyses identified interleukin-10 (IL-10), an immunosuppressive cytokine, as a novel downstream target of KDM4A. We demonstrate that PEL-induced angiogenesis is dependent on IL-10. More importantly, single-cell RNA sequencing (scRNA-seq) analysis demonstrated that, at the late stage of KSHV reactivation, KDM4A determines the fates of PEL cells, as evidenced by two distinct cell populations; one with less apoptotic signaling expresses high levels of viral genes and the other is exactly opposite, while KDM4A-K417R-expressing cells contain only the apoptotic population with less viral gene expression. Consistently, KDM4A knockout significantly reduced cell viability and virus production in KSHV-reactivated PEL cells. Since inhibiting PEL extravasation and eradicating KSHV-infected PEL cells without increasing viral load provide a strong rationale for treating PEL, this study indicates targeting KDM4A as a promising therapeutic option for treating PEL. IMPORTANCE PEL is an aggressive and untreatable B-cell lymphoma caused by KSHV infection. Therefore, new therapeutic approaches for PEL need to be investigated. Since simultaneous induction of KSHV reactivation and apoptosis can directly kill PEL cells, they have been applied in the treatment of this hematologic malignancy and have made progress. Epigenetic therapy with histone deacetylase (HDAC) inhibitors has been proved to treat PEL. However, the antitumor efficacies of HDAC inhibitors are modest and new approaches are needed. Following our previous report showing that the histone lysine demethylase KDM4A and its SUMOylation are required for lytic reactivation of KSHV in PEL cells, we further investigated its cellular function. Here, we found that SUMOylation of KDM4A is required for the survival, movement, and angiogenesis of lytic KSHV-infected PEL cells. Together with our previous finding showing the importance of KDM4A SUMOylation in viral production, KDM4A can be a potential therapeutic target for PEL.

Long non-coding RNA KIKAT/LINC01061 as a novel epigenetic regulator that relocates KDM4A on chromatin and modulates viral reactivation.

KDM4A is a histone lysine demethylase that has been described as an oncogene in various types of cancer. The importance of KDM4A-mediated epigenetic regulation in tumorigenesis is just emerging. Here, by using Kaposi's sarcoma associated herpesvirus (KSHV) as a screening model, we identified 6 oncogenic virus-induced long non-coding RNAs (lncRNAs) with the potential to open chromatin. RNA immunoprecipitation revealed KSHV-induced KDM4A-associated transcript (KIKAT)/LINC01061 as a binding partner of KDM4A. Integrated ChIP-seq and RNA-seq analysis showed that the KIKAT/LINC01061 interaction may mediate relocalization of KDM4A from the transcription start site (TSS) of the AMOT promoter region and transactivation of AMOT, an angiostatin binding protein that regulates endothelial cell migration. Knockdown of AMOT diminished the migration ability of uninfected SLK and iSLK-BAC16 cells in response to KIKAT/LINC01061 overexpression. Thus, we conclude that KIKAT/LINC01061 triggered shifting of KDM4A as a potential epigenetic mechanism regulating gene transactivation. Dysregulation of KIKAT/LINC01061 expression may represent a novel pathological mechanism contributing to KDM4A oncogenicity.

HIV-1 Tat Interacts with a Kaposi's Sarcoma-Associated Herpesvirus Reactivation-Upregulated Antiangiogenic Long Noncoding RNA, LINC00313, and Antagonizes Its Function.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma (KS), an AIDS-defining cancer with abnormal angiogenesis. The high incidence of KS in human immunodeficiency virus (HIV)-infected AIDS patients has been ascribed to an interaction between HIV type 1 (HIV-1) and KSHV, focusing on secretory proteins. The HIV-1 secreted protein HIV Tat has been found to synergize with KSHV lytic proteins to induce angiogenesis. However, the impact and underlying mechanisms of HIV Tat in KSHV-infected endothelial cells undergoing viral lytic reactivation remain unclear. Here, we identified LINC00313 as a novel KSHV reactivation-activated long noncoding RNA (lncRNA) that interacts with HIV Tat. We found that LINC00313 overexpression inhibits cell migration, invasion, and tube formation, and this suppressive effect was relieved by HIV Tat. In addition, LINC00313 bound to polycomb repressive complex 2 (PRC2) complex components, and this interaction was disrupted by HIV Tat, suggesting that LINC00313 may mediate transcription repression through recruitment of PRC2 and that HIV Tat alleviates repression through disruption of this association. This notion was further supported by bioinformatics analysis of transcriptome profiles in LINC00313 overexpression combined with HIV Tat treatment. Ingenuity Pathway Analysis (IPA) showed that LINC00313 overexpression negatively regulates cell movement and migration pathways, and enrichment of these pathways was absent in the presence of HIV Tat. Collectively, our results illustrate that an angiogenic repressive lncRNA, LINC00313, which is upregulated during KSHV reactivation, interacts with HIV Tat to promote endothelial cell motility. These results demonstrate that an lncRNA serves as a novel connector in HIV-KSHV interactions.IMPORTANCE KS is a prevalent tumor associated with infections with two distinct viruses, KSHV and HIV. Since KSHV and HIV infect distinct cell types, the virus-virus interaction associated with KS formation has focused on secretory factors. HIV Tat is a well-known RNA binding protein secreted by HIV. Here, we revealed LINC00313, an lncRNA upregulated during KSHV lytic reactivation, as a novel HIV Tat-interacting lncRNA that potentially mediates HIV-KSHV interactions. We found that LINC00313 can repress endothelial cell angiogenesis-related properties potentially by interacting with chromatin remodeling complex PRC2 and downregulation of cell migration-regulating genes. An interaction between HIV Tat and LINC00313 contributed to the dissociation of PRC2 from LINC00313 and the disinhibition of LINC00313-induced repression of cell motility. Given that lncRNAs are emerging as key players in tissue physiology and disease progression, including cancer, the mechanism identified in this study may help decipher the mechanisms underlying KS pathogenesis induced by HIV and KSHV coinfection.

Treatment Outcomes of Pulpectomy in Primary Maxillary Incisors Filled with ZOE and Metapex: A Two-year Retrospective Study.

OBJECTIVES: To address a literature gap by evaluating, in a larger set of samples, the clinical and radiographic outcomes of pulpectomy in primary maxillary incisors using ZOE and calcium hydroxide/iodoform paste. To also identify the predisposing factors for treatment outcomes. STUDY DESIGN: Medical charts of 124 patients (aged 16 to 60 months) and radiographs of their incisors (309 incisors) were reviewed (128 with ZOE and 181 with Metapex). All incisors were restored with composite resin crowns. RESULTS: The radiographic success rates for ZOE and Metapex were: 85.9% and 82.9% at the 12-month recall, and 69.2% and 64% at the 24-month recall, with no statistically significant difference between the two groups. Clinical failures occurred more frequently in the Metapex group (P = 0.006), as clinical signs of pain and soft tissue pathosis were found in 6.2% of the Metapex cases at 24 months but none in the ZOE group. Significant predisposing factors for radiographic success were type of tooth, degree of obturation at recalls, and preoperative root resorption. CONCLUSIONS: The radiographic success rates are comparable between the ZOE and Metapex groups. Clinical pathological manifestations such as pain and soft tissue pathosis are seen in the Metapex group at recalls, but none in the ZOE group. Predisposing factors such as type of incisor, preoperative root resorption, and extent of filling at recalls are associated with the radiographic success rate.

High Amplification of the Antiviral Activity of Curcumin through Transformation into Carbon Quantum Dots.

It is demonstrated that carbon quantum dots derived from curcumin (Cur-CQDs) through one-step dry heating are effective antiviral agents against enterovirus 71 (EV71). The surface properties of Cur-CQDs, as well as their antiviral activity, are highly dependent on the heating temperature during synthesis. The one-step heating of curcumin at 180 °C preserves many of the moieties of polymeric curcumin on the surfaces of the as-synthesized Cur-CQDs, resulting in superior antiviral characteristics. It is proposed that curcumin undergoes a series of structural changes through dehydration, polymerization, and carbonization to form core-shell CQDs whose surfaces remain a pyrolytic curcumin-like polymer, boosting the antiviral activity. The results reveal that curcumin possesses insignificant inhibitory activity against EV71 infection in RD cells [half-maximal effective concentration (EC50 ) >200 µg mL-1 ] but exhibits high cytotoxicity toward RD cells (half-maximal cytotoxic concentration (CC50 ) <13 µg mL-1 ). The EC50 (0.2 µg mL-1 ) and CC50 (452.2 µg mL-1 ) of Cur-CQDs are >1000-fold lower and >34-fold higher, respectively, than those of curcumin, demonstrating their far superior antiviral capabilities and high biocompatibility. In vivo, intraperitoneal administration of Cur-CQDs significantly decreases mortality and provides protection against virus-induced hind-limb paralysis in new-born mice challenged with a lethal dose of EV71.

SUMO modification of a heterochromatin histone demethylase JMJD2A enables viral gene transactivation and viral replication.

Small ubiquitin-like modifier (SUMO) modification of chromatin has profound effects on transcription regulation. By using Kaposi's sarcoma associated herpesvirus (KSHV) as a model, we recently demonstrated that epigenetic modification of viral chromatin by SUMO-2/3 is involved in regulating gene expression and viral reactivation. However, how this modification orchestrates transcription reprogramming through targeting histone modifying enzymes remains largely unknown. Here we show that JMJD2A, the first identified Jumonji C domain-containing histone demethylase, is the histone demethylase responsible for SUMO-2/3 enrichment on the KSHV genome during viral reactivation. Using in vitro and in vivo SUMOylation assays, we found that JMJD2A is SUMOylated on lysine 471 by KSHV K-bZIP, a viral SUMO-2/3-specific E3 ligase, in a SUMO-interacting motif (SIM)-dependent manner. SUMOylation is required for stabilizing chromatin association and gene transactivation by JMJD2A. These finding suggest that SUMO-2/3 modification plays an essential role in the epigenetic regulatory function of JMJD2A. Consistently, hierarchical clustering analysis of RNA-seq data showed that a SUMO-deficient mutant of JMJD2A was more closely related to JMJD2A knockdown than to wild-type. Our previous report demonstrated that JMJD2A coated and maintained the "ready to activate" status of the viral genome. Consistent with our previous report, a SUMO-deficient mutant of JMJD2A reduced viral gene expression and virion production. Importantly, JMJD2A has been implicated as an oncogene in various cancers by regulating proliferation. We therefore further analyzed the role of SUMO modification of JMJD2A in regulating cell proliferation. Interestingly, the SUMO-deficient mutant of JMJD2A failed to rescue the proliferation defect of JMJD2A knockdown cells. Emerging specific inhibitors of JMJD2A have been generated for evaluation in cancer studies. Our results revealed that SUMO conjugation mediates an epigenetic regulatory function of JMJD2A and suggests that inhibiting JMJD2A SUMOylation may be a novel avenue for anti-cancer therapy.

Postoperative dental morbidity in children following dental treatment under general anesthesia.

BACKGROUND: General anesthesia has been widely used in pediatric dentistry in recent years. However, there remain concerns about potential postoperative dental morbidity. The goal of this study was to identify the frequency of postoperative dental morbidity and factors associated with such morbidity in children. METHODS: From March 2012 to February 2013, physically and mentally healthy children receiving dental treatment under general anesthesia at the Department of Pediatric Dentistry of the Chang Gung Memorial Hospital in Taiwan were recruited. This was a prospective and observational study with different time evaluations based on structured questionnaires and interviews. Information on the patient demographics, anesthesia and dental treatment performed, and postoperative dental morbidity was collected and analyzed. Correlations between the study variables and postoperative morbidity were analyzed based on the Pearson's chi-square test. Correlations between the study variables and the scale of postoperative dental pain were analyzed using the Mann-Whitney U test. RESULTS: Fifty-six pediatric patients participated in this study, with an average age of 3.34 ± 1.66 years (ranging from 1 to 8 years). Eighty-two percent of study participants reported postoperative dental pain, and 23% experienced postoperative dental bleeding. Both dental pain and bleeding subsided 3 days after the surgery. Dental pain was significantly associated with the total number of teeth treated, while dental bleeding, with the presence of teeth extracted. Patients' gender, age, preoperative dental pain, ASA classification, anesthesia time, and duration of the operation were not associated with postoperative dental morbidity. CONCLUSION: Dental pain was a more common postoperative dental morbidity than bleeding. The periods when parents reported more pain in their children were the day of the operation (immediately after the procedure) followed by 1 day and 3 days after the treatment.

K-bZIP Mediated SUMO-2/3 Specific Modification on the KSHV Genome Negatively Regulates Lytic Gene Expression and Viral Reactivation.

SUMOylation is associated with epigenetic regulation of chromatin structure and transcription. Epigenetic modifications of herpesviral genomes accompany the transcriptional switch of latent and lytic genes during the virus life cycle. Here, we report a genome-wide comparison of SUMO paralog modification on the KSHV genome. Using chromatin immunoprecipitation in conjunction with high-throughput sequencing, our study revealed highly distinct landscape changes of SUMO paralog genomic modifications associated with KSHV reactivation. A rapid and widespread deposition of SUMO-2/3, compared with SUMO-1, modification across the KSHV genome upon reactivation was observed. Interestingly, SUMO-2/3 enrichment was inversely correlated with H3K9me3 mark after reactivation, indicating that SUMO-2/3 may be responsible for regulating the expression of viral genes located in low heterochromatin regions during viral reactivation. RNA-sequencing analysis showed that the SUMO-2/3 enrichment pattern positively correlated with KSHV gene expression profiles. Activation of KSHV lytic genes located in regions with high SUMO-2/3 enrichment was enhanced by SUMO-2/3 knockdown. These findings suggest that SUMO-2/3 viral chromatin modification contributes to the diminution of viral gene expression during reactivation. Our previous study identified a SUMO-2/3-specific viral E3 ligase, K-bZIP, suggesting a potential role of this enzyme in regulating SUMO-2/3 enrichment and viral gene repression. Consistent with this prediction, higher K-bZIP binding on SUMO-2/3 enrichment region during reactivation was observed. Moreover, a K-bZIP SUMO E3 ligase dead mutant, K-bZIP-L75A, in the viral context, showed no SUMO-2/3 enrichment on viral chromatin and higher expression of viral genes located in SUMO-2/3 enriched regions during reactivation. Importantly, virus production significantly increased in both SUMO-2/3 knockdown and KSHV K-bZIP-L75A mutant cells. These results indicate that SUMO-2/3 modification of viral chromatin may function to counteract KSHV reactivation. As induction of herpesvirus reactivation may activate cellular antiviral regimes, our results suggest that development of viral SUMO E3 ligase specific inhibitors may be an avenue for anti-virus therapy.

Novel method for functional brain imaging in awake minimally restrained rats.

Functional magnetic resonance imaging (fMRI) in rodents holds great promise for advancing our knowledge about human brain function. However, the use of anesthetics to immobilize rodents during fMRI experiments has restricted the type of questions that can be addressed using this technique. Here we describe an innovative procedure to train rats to be constrained without the need of any anesthesia during the whole procedure. We show that with 8-10 days of acclimation rats can be conscious and remain still during fMRI experiments under minimal stress. In addition, we provide fMRI results of conscious rodents in a variety of commonly used fMRI experimental paradigms, and we demonstrate the improved quality of these scans by comparing results when the same rodents were scanned under anesthesia. We confirm that the awake scanning procedure permits an improved evaluation of brain networks and brain response to external stimuli with minimal movement artifact. The present study further advances the field of fMRI in awake rodents, which provide more direct, forward and reverse, translational opportunities regarding brain functional correspondences between human and rodent research.

A lytic viral long noncoding RNA modulates the function of a latent protein.

Latent Kaposi's sarcoma-associated herpesvirus (KSHV) episomes are coated with viral latency-associated nuclear antigen (LANA). In contrast, LANA rapidly disassociates from episomes during reactivation. Lytic KSHV expresses polyadenylated nuclear RNA (PAN RNA), a long noncoding RNA (lncRNA). We report that PAN RNA promotes LANA-episome disassociation through an interaction with LANA which facilitates LANA sequestration away from KSHV episomes during reactivation. These findings suggest that KSHV may have evolved an RNA aptamer to regulate latent protein function.

Counting injury deaths: a comparison of two definitions and two countries.

OBJECTIVES: This study sought to examine the discrepancy in counting injury deaths according to two definitions, that is, (a) external cause of injury as the underlying cause of death (UCOD), and (b) with mention of external cause of injury, and differences in certification practices between Taiwan and the USA. METHODS: We calculated the percentage (U/M%) in which external cause of injury was selected as the UCOD (U) among deaths with mention (M) of external cause of injury anywhere on the death certificate to assess the discrepancy between the two definitions in counting injury deaths. RESULTS: The discrepancy was small in Taiwan and in the USA for transport injuries (with U/M% 99% and 98%) and intentional self-harm (100% and 100%) and large for accidental suffocation (64% and 29%) as well as complications of medical and surgical care (61% and 10%). The magnitude of discrepancy was not consistent between countries in accidental falls (96% vs 81%) and accidental drowning (80% vs 97%). Certifiers in Taiwan were more likely to report an accidental fall in Part I of the death certificate and less likely to report medical conditions (such as stroke, Alzheimer's disease or Parkinson's disease, etc) with accidental suffocation than their counterpart US certifiers. CONCLUSIONS: The counting of injury deaths according to two definitions varied by external cause of injury as well as between countries. For some external causes of injury with high discrepancies, we suggest presenting the number of injury deaths according to two definitions.

An improved ChIP-seq peak detection system for simultaneously identifying post-translational modified transcription factors by combinatorial fusion, using SUMOylation as an example.

BACKGROUND: Post-translational modification (PTM) of transcriptional factors and chromatin remodelling proteins is recognized as a major mechanism by which transcriptional regulation occurs. Chromatin immunoprecipitation (ChIP) in combination with high-throughput sequencing (ChIP-seq) is being applied as a gold standard when studying the genome-wide binding sites of transcription factor (TFs). This has greatly improved our understanding of protein-DNA interactions on a genomic-wide scale. However, current ChIP-seq peak calling tools are not sufficiently sensitive and are unable to simultaneously identify post-translational modified TFs based on ChIP-seq analysis; this is largely due to the wide-spread presence of multiple modified TFs. Using SUMO-1 modification as an example; we describe here an improved approach that allows the simultaneous identification of the particular genomic binding regions of all TFs with SUMO-1 modification. RESULTS: Traditional peak calling methods are inadequate when identifying multiple TF binding sites that involve long genomic regions and therefore we designed a ChIP-seq processing pipeline for the detection of peaks via a combinatorial fusion method. Then, we annotate the peaks with known transcription factor binding sites (TFBS) using the Transfac Matrix Database (v7.0), which predicts potential SUMOylated TFs. Next, the peak calling result was further analyzed based on the promoter proximity, TFBS annotation, a literature review, and was validated by ChIP-real-time quantitative PCR (qPCR) and ChIP-reChIP real-time qPCR. The results show clearly that SUMOylated TFs are able to be pinpointed using our pipeline. CONCLUSION: A methodology is presented that analyzes SUMO-1 ChIP-seq patterns and predicts related TFs. Our analysis uses three peak calling tools. The fusion of these different tools increases the precision of the peak calling results. TFBS annotation method is able to predict potential SUMOylated TFs. Here, we offer a new approach that enhances ChIP-seq data analysis and allows the identification of multiple SUMOylated TF binding sites simultaneously, which can then be utilized for other functional PTM binding site prediction in future.

The gamification and development of a chatbot to promote oral self-care by adopting behavior change wheel for Taiwanese children.

Background: Oral health is closely related to general health and quality of life. School-aged children are at a critical stage for developing their self-care ability in oral health. Digital interventions can encourage and facilitate oral self-care in children. Objective: This study aims to present the development of an educational chatbot for school-aged children to address their oral self-care and evaluate its usability. Methods: The development and evaluation of the chatbot for oral self-care consisted of four stages: target behavior analysis, intervention design, system development, and the chatbot evaluation. The target behavior analysis identified barriers to children's engagement in oral self-care based on dentists' clinical observations; hence, the requirements for achieving the desired behavior were categorized according to the capability-opportunity-motivation behavior model. Interventional functions were created following the behavior change wheel. A menu-driven chatbot was created and evaluated for usability as well as likeability. Results: The barriers and requirements for achieving good behavior in school-aged children's oral self-care were identified by the dental professionals. Intervention strategy incorporated specific functions enriched with gamification features to support school-aged children in developing their abilities for engaging in oral self-care. The intervention functions consist of capability establishment, motivation enhancement, and opportunity creation, which were designed to support children in their oral self-care practices. The designed chatbot was piloted with a convenient sample of 30 school-aged children and their accompanying parents at the pediatric dental clinic. The results indicated good usability, with a mean usability score of 79.91, and high likeability with a mean score of 4.32 out of 5 for the designed chatbot. Conclusions: The educational chatbot incorporated a combination of clinical dentistry practice and guidelines, aiming to promote oral self-care behavior in school-aged children. The designed chatbot achieved high scores for its usability and user likability.

The lncRNA TPT1-AS1 promotes the survival of neuroendocrine prostate cancer cells by facilitating autophagy.

The lncRNA tumor protein translationally controlled 1-antisense RNA 1 (TPT1-AS1) is known for its oncogenic role in various cancers, but its impact on the pathological progression of prostate cancer remains unclear. Our previous study demonstrated that the RE1-silencing transcription factor (REST) regulates neuroendocrine differentiation (NED) in prostate cancer (PCA) by derepressing specific long non-coding RNAs (lncRNAs), including TPT1-AS1. In this study, we revealed that TPT1-AS1 is overexpressed in LNCaP and C4-2B cells after IL-6 and enzalutamide treatment. By analyzing The Cancer Genome Atlas (TCGA) prostate adenocarcinoma dataset, we detected upregulated TPT1-AS1 expression in neuroendocrine-associated PCA but not in prostate adenocarcinoma. Single-cell RNA sequencing data further confirmed the increased TPT1-AS1 levels in neuroendocrine prostate cancer (NEPC) cells. Surprisingly, functional experiments indicated that TPT1-AS1 overexpression had no stimulatory effect on NED in LNCaP cells and that TPT1-AS1 knockdown did not inhibit IL-6-induced NED. Transcriptomic analysis revealed the essential role of TPT1-AS1 in synaptogenesis and autophagy activation in neuroendocrine differentiated PCA cells induced by IL-6 and enzalutamide treatment. TPT1-AS1 was found to regulate the expression of autophagy-related genes that maintain neuroendocrine cell survival through autophagy activation. In conclusion, our data expand the current knowledge of REST-repressed lncRNAs in NED in PCA and highlight the contribution of TPT1-AS1 to protect neuroendocrine cells from cell death rather than inducing NED. Our study suggested that TPT1-AS1 plays a cytoprotective role in NEPC cells; thus, targeting TPT1-AS1 is a potential therapeutic strategy.

Protein arginine methyltransferase 1-directed methylation of Kaposi sarcoma-associated herpesvirus latency-associated nuclear antigen.

The Kaposi sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) is a multifunctional protein with roles in gene regulation and maintenance of viral latency. Post-translational modification of LANA is important for functional diversification. Here, we report that LANA is subject to arginine methylation by protein arginine methyltransferase 1 in vitro and in vivo. The major arginine methylation site in LANA was mapped to arginine 20. This site was mutated to either phenylalanine (bulky hydrophobic, constitutive methylated mimetic) or lysine (positively charged, non-arginine methylatable) residues. The significance of the methylation in LANA function was examined in both the isolated form and in the context of the viral genome through the generation of recombinant KSHV. In addition, authentic LANA binding sites on the KSHV episome in naturally infected cells were identified using a whole genome KSHV tiling array. Although mutation of the methylation site resulted in no significant difference in KSHV LANA subcellular localization, we found that the methylation mimetic mutation resulted in augmented histone binding in vitro and increased LANA occupancy at identified LANA target promoters in vivo. Moreover, a cell line carrying the methylation mimetic mutant KSHV showed reduced viral gene expression relative to controls both in latency and in the course of reactivation. These results suggest that residue 20 is important for modulation of a subset of LANA functions and properties of this residue, including the hydrophobic character induced by arginine methylation, may contribute to the observed effects.

The chromatin modification by SUMO-2/3 but not SUMO-1 prevents the epigenetic activation of key immune-related genes during Kaposi's sarcoma associated herpesvirus reactivation.

BACKGROUND: SUMOylation, as part of the epigenetic regulation of transcription, has been intensively studied in lower eukaryotes that contain only a single SUMO protein; however, the functions of SUMOylation during mammalian epigenetic transcriptional regulation are largely uncharacterized. Mammals express three major SUMO paralogues: SUMO-1, SUMO-2, and SUMO-3 (normally referred to as SUMO-1 and SUMO-2/3). Herpesviruses, including Kaposi's sarcoma associated herpesvirus (KSHV), seem to have evolved mechanisms that directly or indirectly modulate the SUMO machinery in order to evade host immune surveillance, thus advancing their survival. Interestingly, KSHV encodes a SUMO E3 ligase, K-bZIP, with specificity toward SUMO-2/3 and is an excellent model for investigating the global functional differences between SUMO paralogues. RESULTS: We investigated the effect of experimental herpesvirus reactivation in a KSHV infected B lymphoma cell line on genomic SUMO-1 and SUMO-2/3 binding profiles together with the potential role of chromatin SUMOylation in transcription regulation. This was carried out via high-throughput sequencing analysis. Interestingly, chromatin immunoprecipitation sequencing (ChIP-seq) experiments showed that KSHV reactivation is accompanied by a significant increase in SUMO-2/3 modification around promoter regions, but SUMO-1 enrichment was absent. Expression profiling revealed that the SUMO-2/3 targeted genes are primarily highly transcribed genes that show no expression changes during viral reactivation. Gene ontology analysis further showed that these genes are involved in cellular immune responses and cytokine signaling. High-throughput annotation of SUMO occupancy of transcription factor binding sites (TFBS) pinpointed the presence of three master regulators of immune responses, IRF-1, IRF-2, and IRF-7, as potential SUMO-2/3 targeted transcriptional factors after KSHV reactivation. CONCLUSION: Our study is the first to identify differential genome-wide SUMO modifications between SUMO paralogues during herpesvirus reactivation. Our findings indicate that SUMO-2/3 modification near protein-coding gene promoters occurs in order to maintain host immune-related gene unaltered during viral reactivation.

Parallel buprenorphine phMRI responses in conscious rodents and healthy human subjects.

Pharmacological magnetic resonance imaging (phMRI) is one method by which a drug's pharmacodynamic effects in the brain can be assessed. Although phMRI has been frequently used in preclinical and clinical settings, the extent to which a phMRI signature for a compound translates between rodents and humans has not been systematically examined. In the current investigation, we aimed to build on recent clinical work in which the functional response to 0.1 and 0.2 mg/70 kg i.v. buprenorphine (partial µ-opioid receptor agonist) was measured in healthy humans. Here, we measured the phMRI response to 0.04 and 0.1 mg/kg i.v. buprenorphine in conscious, naive rats to establish the parallelism of the phMRI signature of buprenorphine across species. PhMRI of 0.04 and 0.1 mg/kg i.v. buprenorphine yielded dose-dependent activation in a brain network composed of the somatosensory cortex, cingulate, insula, striatum, thalamus, periaqueductal gray, and cerebellum. Similar dose-dependent phMRI activation was observed in the human phMRI studies. These observations indicate an overall preservation of pharmacodynamic responses to buprenorphine between conscious, naive rodents and healthy human subjects, particularly in brain regions implicated in pain and analgesia. This investigation further demonstrates the usefulness of phMRI as a translational tool in neuroscience research that can provide mechanistic insight and guide dose selection in drug development.

NLRP12 is an innate immune checkpoint for repressing IFN signatures and attenuating lupus nephritis progression.

Signaling driven by nucleic acid sensors participates in interferonopathy-mediated autoimmune diseases. NLRP12, a pyrin-containing NLR protein, is a negative regulator of innate immune activation and type I interferon (IFN-I) production. Peripheral blood mononuclear cells (PBMCs) derived from systemic lupus erythematosus (SLE) patients expressed lower levels of NLRP12, with an inverse correlation with IFNA expression and high disease activity. NLRP12 expression was transcriptionally suppressed by runt-related transcription factor 1-dependent (RUNX1-dependent) epigenetic regulation under IFN-I treatment, which enhanced a negative feedback loop between low NLRP12 expression and IFN-I production. Reduced NLRP12 protein levels in SLE monocytes was linked to spontaneous activation of innate immune signaling and hyperresponsiveness to nucleic acid stimulations. Pristane-treated Nlrp12-/- mice exhibited augmented inflammation and immune responses; and substantial lymphoid hypertrophy was characterized in NLRP12-deficient lupus-prone mice. NLRP12 deficiency mediated the increase of autoantibody production, intensive glomerular IgG deposition, monocyte recruitment, and the deterioration of kidney function. These were bound in an IFN-I signature-dependent manner in the mouse models. Collectively, we reveal a remarkable link between low NLRP12 expression and lupus progression, which suggests the impact of NLRP12 on homeostasis and immune resilience.

Single-cell analysis of castration-resistant prostate cancers to identify potential biomarkers for diagnosis and prognosis of neuroendocrine prostate cancer.

The high heterogeneity and low percentage of neuroendocrine cells in prostate cancer limit the utility of traditional bulk RNA sequencing and even single-cell RNA sequencing to find better biomarkers for early diagnosis and stratification. Re-clustering of specific cell-type holds great promise for identification of intra-cell-type heterogeneity. However, this has not yet been used in studying neuroendocrine prostate cancer heterogeneity. Neuroendocrine cluster(s) were individually identified in each castration-resistant prostate cancer specimen and combined for trajectory analysis. Three neuroendocrine states were identified. Neuroendocrine state 2 with the highest AR score was considered the initial starting state of neuroendocrine transdifferentiation. State 1 and state 3 with distinct high neuroendocrine scores and marker genes enriched in N-Myc and REST target genes, respectively, were considered as two different types of neuroendocrine differentiated cancer cells. These two states contained distinct groups of prostate cancer biomarkers and a strong distinguishing ability of normal versus cancerous prostate across different pathological grading was found in the N-Myc-associated state. Our data highlight the central role of N-Myc and REST in mediating lineage plasticity and classifying neuroendocrine phenotypes.