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APOBEC3A (A3A) is a cytidine deaminase that inactivates a variety of viruses through introduction of lethal mutations to the viral genome. Additionally, A3A can suppress HIV-1 transcription in a deaminase-independent manner by binding to the long terminal repeat of proviral HIV-1. However, it is unknown whether A3A targets additional host genomic loci for repression. In this study, we found that A3A suppresses gene expression by binding TTTC doublets that are in close proximity to each other. However, one TTTC motif is sufficient for A3A binding. Because TTTC doublets are present in interferon (IFN)-stimulated response elements (ISRE), we hypothesized that A3A may impact IFN-stimulated gene (ISG) expression. After scanning the human genome for TTTC doublet occurrences, we discovered that these motifs are enriched in the proximal promoters of genes associated with antiviral responses and type I IFN (IFN-I) signaling. As a proof of principle, we examined whether A3A can impact ISG15 expression. We found that A3A binding to the ISRE inhibits phosphorylated STAT-1 binding and suppresses ISG15 induction in response to IFN-I treatment. Consistent with these data, our RNA-sequencing analyses indicate that A3A loss results in increased IFN-I–dependent induction of several ISGs. This study revealed that A3A plays an unexpected role in ISG regulation and suggests that A3A contributes to a negative feedback loop during IFN signaling.

Saliva Specimens to Detect SARS-CoV-2 Infection In this letter, the investigators report that saliva specimens and nasopharyngeal swab specimens had similar sensitivity in the detection of SARS-CoV...

Prader-Willi syndrome (PWS) is often related to severe obesity and diabetes mellitus (DM). Clinical findings suggesting the benefits of glucagon-like peptide-1 (GLP-1) receptor agonists for glycemic control of DM in PWS have been recently increasing. However, there are only a few reports describing the effects of sodium-glucose cotransporter 2 (SGLT2) inhibitors for PWS. We present a diabetic female with PWS, whose glycemic control was deteriorated at the age of 19 but improved to a certain extent by introducing the GLP-1 analog liraglutide. At the age of 20, the SGLT2 inhibitor empagliflozin was administered. Subsequently, her HbA1c level and body weight markedly decreased. Improvement in both insulin resistance and secretion was observed during the subsequent six months. In addition to GLP-1 receptor agonists, SGLT2 inhibitors may be a potential approach for the management of DM in PWS, especially in young patients whose pancreatic insulin secretion capabilities are still preserved.

Previous studies have revealed that dysregulation of long interspersed nuclear element 1 (LINE-1), a dominant class of transposable elements in the human genome, correlates with neurodegeneration1–3. Yet whether LINE-1 dysregulation is causal to disease pathogenesis has not been proven directly. Here, we demonstrate that expression of evolutionarily younger LINE-1 families is elevated in the cerebella of ataxia telangiectasia (AT) patients, which was correlated with extensive downregulation of epigenetic silencers. To examine whether LINE-1 activation causes neurologic disease, we established an approach to directly target and activate the promoter of a young family of LINE-1 in mice. LINE-1 activation in the cerebellum was sufficient to lead to robust progressive ataxia. Purkinje cells in the diseased mice exhibited marked electrophysiological dysfunctions and degeneration with a significant accumulation of cytoplasmic ribonucleoprotein LINE-1Orf1p aggregates, endoplasmic reticulum (ER) stress, and DNA damage. Treatment with lamivudine, a nucleoside reverse transcriptase inhibitor, blunted the disease progression by reducing DNA damage, attenuating gliosis and interferon gene signature, and recovering the loss of key functional molecules for calcium homeostasis in Purkinje cells. This study provides direct evidence that young LINE-1 activation drives ataxia phenotype, and points to its pleiotropic effects leading to DNA damage, inflammation, and dysfunction and degeneration of neurons.

Dysregulation of long interspersed nuclear element 1 (LINE-1, L1), a dominant class of transposable elements in the human genome, has been linked to neurodegenerative diseases, but whether elevated L1 expression is sufficient to cause neurodegeneration has not been directly tested. Here, we show that the cerebellar expression of L1 is significantly elevated in ataxia telangiectasia patients and strongly anti-correlated with the expression of epigenetic silencers. To examine the role of L1 in the disease etiology, we developed an approach for direct targeting of the L1 promoter for overexpression in mice. We demonstrated that L1 activation in the cerebellum led to Purkinje cell dysfunctions and degeneration and was sufficient to cause ataxia. Treatment with a nucleoside reverse transcriptase inhibitor blunted ataxia progression by reducing DNA damage, attenuating gliosis, and reversing deficits of molecular regulators for calcium homeostasis in Purkinje cells. Our study provides the first direct evidence that L1 activation can drive neurodegeneration.

Significance Influenza virus causes seasonal outbreaks in temperate regions, with an increase in disease and mortality in the winter months. Dry air combined with cold temperature is known to enable viral transmission. In this study, we asked whether humidity impacts the host response to influenza virus infections. Exposure of mice to low humidity conditions rendered them more susceptible to influenza disease. Mice housed in dry air had impaired mucociliary clearance, innate antiviral defense, and tissue repair function. Moreover, mice exposed to dry air were more susceptible to disease mediated by inflammasome caspases. Our study provides mechanistic insights for the seasonality of the influenza virus epidemics, whereby inhalation of dry air compromises the host’s ability to restrict influenza virus infection., In the temperate regions, seasonal influenza virus outbreaks correlate closely with decreases in humidity. While low ambient humidity is known to enhance viral transmission, its impact on host response to influenza virus infection and disease outcome remains unclear. Here, we showed that housing Mx1 congenic mice in low relative humidity makes mice more susceptible to severe disease following respiratory challenge with influenza A virus. We find that inhalation of dry air impairs mucociliary clearance, innate antiviral defense, and tissue repair. Moreover, disease exacerbated by low relative humidity was ameliorated in caspase-1/11–deficient Mx1 mice, independent of viral burden. Single-cell RNA sequencing revealed that induction of IFN-stimulated genes in response to viral infection was diminished in multiple cell types in the lung of mice housed in low humidity condition. These results indicate that exposure to dry air impairs host defense against influenza infection, reduces tissue repair, and inflicts caspase-dependent disease pathology.

[Purpose] We investigated whether an increase or decrease in subcutaneous fat mass secondary to cardiac cachexia can be evaluated using diagnostic ultrasonography in patients with heart failure. [Participant and Methods] We report a case of cardiac cachexia in a patient in whom cachexia was confirmed by weight loss, decreased dietary intake, and biochemical indicators measured by blood tests. We measured the subcutaneous fat mass in the patient’s thigh using ultrasonic diagnostic equipment during the cachectic state, as well as 1 and 2 months later. [Results] An increase in weight and ultrasonographically documented femoral subcutaneous fat mass was confirmed by improvement in heart failure-induced cachexia. [Conclusion] Clinically convenient ultrasonic diagnostic equipment is useful to assess subcutaneous fat mass, which serves as an indicator of the degree of cachexia.

The recent spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exemplifies the critical need for accurate and rapid diagnostic assays to prompt clinical and public health interventions. Currently, several quantitative reverse transcription–PCR (RT–qPCR) assays are being used by clinical, research and public health laboratories. However, it is currently unclear whether results from different tests are comparable. Our goal was to make independent evaluations of primer–probe sets used in four common SARS-CoV-2 diagnostic assays. From our comparisons of RT–qPCR analytical efficiency and sensitivity, we show that all primer–probe sets can be used to detect SARS-CoV-2 at 500 viral RNA copies per reaction. The exception for this is the RdRp-SARSr (Charite) confirmatory primer–probe set which has low sensitivity, probably due to a mismatch to circulating SARS-CoV-2 in the reverse primer. We did not find evidence for background amplification with pre-COVID-19 samples or recent SARS-CoV-2 evolution decreasing sensitivity. Our recommendation for SARS-CoV-2 diagnostic testing is to select an assay with high sensitivity and that is regionally used, to ease comparability between outcomes. This is a comparative analysis of the performance of the primer–probe sets from four open-source molecular diagnostic assays for SARS-CoV-2 recommended by the World Health Organization.

The current quantitative reverse transcription PCR (RT-qPCR) assay recommended for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing in the United States requires analysis of 3 genomic targets per sample: 2 viral and 1 host. To simplify testing and reduce the volume of required reagents, we devised a multiplex RT-qPCR assay to detect SARS-CoV-2 in a single reaction. We used existing N1, N2, and RP primer and probe sets by the Centers for Disease Control and Prevention, but substituted fluorophores to allow multiplexing of the assay. The cycle threshold (Ct) values of our multiplex RT-qPCR were comparable to those obtained by the single assay adapted for research purposes. Low copy numbers (≥500 copies/reaction) of SARS-CoV-2 RNA were consistently detected by the multiplex RT-qPCR. Our novel multiplex RT-qPCR improves upon current single diagnostics by saving reagents, costs, time, and labor., The current RT-qPCR assay recommended for SARS-CoV-2 testing in the United States requires analysis of three genomic targets per sample: two viral and one host. This study describes a multiplex PCR that saves both reagents and labor while accurately detecting SARS-CoV-2 RNA.

The recent spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exemplifies the critical need for accurate and rapid diagnostic assays to prompt clinical and public health interventions. Currently, several quantitative reverse-transcription polymerase chain reaction (qRT-PCR) assays are being used by clinical, research, and public health laboratories. However, it is currently unclear if results from different tests are comparable. Our goal was to evaluate the primer-probe sets used in four common diagnostic assays available on the World Health Organization (WHO) website. To facilitate this effort, we generated RNA transcripts to be used as assay standards and distributed them to other laboratories for internal validation. We then used these (1) RNA transcript standards, (2) full-length SARS-CoV-2 RNA, and (3) pre-COVID-19 nasopharyngeal swabs, and (4) clinical samples from COVID-19 patients to determine analytical efficiency and sensitivity of the qRT-PCR primer-probe sets. We show that all primer-probe sets can be used to detect SARS-CoV-2, but there are clear differences in the ability to differentiate between true negatives and positives with low amounts of virus. We found that several primer-probe sets cross-react with SARS-CoV-2-negative nasopharyngeal swabs. However, background cross-reactivity by the 2019-nCoV_N2 set issued by the US Centers for Disease Control and Prevention did not interfere with outcomes of the combined 9N19 and 9N29 assay when testing COVID-19 clinical samples. Our findings characterize the limitations of currently used primer-probe sets and can assist other laboratories in selecting appropriate assays for the detection of SARS-CoV-2.

Current therapies for human immunodeficiency virus type 1 (HIV-1) do not completely eliminate viral reservoirs in cells, such as macrophages. The HIV-1 accessory protein viral protein R (Vpr) promotes virus production in macrophages, and the maintenance of Vpr is essential for HIV-1 replication in these reservoir cells. We identified two novel Vpr-binding proteins, i.e., protein arginine N-methyltransferases (PRMTs) 5 and 7, using human monocyte-derived macrophages (MDMs). Both proteins found to be important for prevention of Vpr degradation by the proteasome, in the context of PRMT5 and PRMT7 knockdowns, degradation of Vpr could be prevented using a proteasome inhibitor. In MDMs infected with a wild-type strain, knockdown of PRMT5/PRMT7 and low expression of PRMT5 resulted in inefficient virus production like Vpr-deficient strain infections. Thus, our findings suggest that PRMT5 and PRMT7 support HIV-1 replication via maintenance of Vpr protein stability.

This paper reports research results of the proposed experiential learning using a game-based educational material. The educational material was an application running on a mobile device and was supported for learners to study university library instruction. During the experiential learning, the learners answered several quizzes provided from the educational material. The quizzes were including experiential contents such as actually to find an academic journal or to reserve a learning room. In order to evaluate learning effectiveness of the experiential learning, this research made a comparative experiment. The experimental group used the educational material and the control group used another educational material of e-learning (non-experiential) but the contents was almost same as the contents of the experiential learning. The comparative experiment took several evaluation elements such as pre-/post-test scores, delayed-test scores and learning motivation scores based on IMMS of ARCS model. The experimental results revealed that experiential learning was superior for the several elements of these metrics. This research, furthermore, analyzed learner's operation data to the educational material and learner's behavior data during the learning in the experimental group. The analyzing results showed three types of the learning behaviors and indicated that an instructor needs to adopt suitable instructional design to each type to bring superior learning effectiveness.

Influenza A virus (IAV) infection-associated morbidity and mortality are a key global health care concern, necessitating the identification of new therapies capable of reducing the severity of IAV infections. In this study, we show that the consumption of a low-carbohydrate, high-fat ketogenic diet (KD) protects mice from lethal IAV infection and disease. KD feeding resulted in an expansion of γδ T cells in the lung that improved barrier functions, thereby enhancing antiviral resistance. Expansion of these protective γδ T cells required metabolic adaptation to a ketogenic diet because neither feeding mice a high-fat, high-carbohydrate diet nor providing chemical ketone body substrate that bypasses hepatic ketogenesis protected against infection. Therefore, KD-mediated immune-metabolic integration represents a viable avenue toward preventing or alleviating influenza disease.

N(6) methyladenosine (m(6)A) is the most abundant RNA modification, but little is known about its role in mammalian hematopoietic development. Here, we show that conditional deletion of the m(6)A writer METTL3 in murine fetal liver resulted in hematopoietic failure and perinatal lethality. Loss of METTL3 and m(6)A activated an aberrant innate immune response, mediated by the formation of endogenous double-stranded RNAs (dsRNAs). The aberrantly formed dsRNAs were long, highly m(6)A modified in their native state, characterized by low folding energies, and predominantly protein-coding. We identified coinciding activation of pattern recognition receptor pathways normally tasked with the detection of foreign dsRNAs. Disruption of the aberrant immune response via abrogation of downstream Mavs or Rnasel signaling partially rescued the observed hematopoietic defects in METTL3-deficient cells in vitro and in vivo. Our results suggest that m(6)A modification protects against endogenous dsRNA formation and a deleterious innate immune response during mammalian hematopoietic development.

Background Scaling SARS-CoV-2 testing to meet demands of safe reopenings continues to be plagued by assay costs and supply chain shortages. In response, we developed SalivaDirect, which received Emergency Use Authorization (EUA) from the U.S. Food and Drug Administration (FDA). Methods We simplified our saliva-based diagnostic test by (1) not requiring collection tubes with preservatives, (2) replacing nucleic acid extraction with a simple enzymatic and heating step, and (3) testing specimens with a dualplex qRT-PCR assay. Moreover, we validated SalivaDirect with reagents and instruments from multiple vendors to minimize supply chain issues. Findings From our hospital cohort, we show a high positive agreement (94%) between saliva tested with SalivaDirect and nasopharyngeal swabs tested with a commercial qRT-PCR kit. In partnership with the National Basketball Association (NBA) and National Basketball Players Association (NBPA), we tested 3,779 saliva specimens from healthy individuals and detected low rates of invalid (0.3%) and false-positive (, Context and Significance Frequent testing is critical to limit SARS-CoV-2 transmission. In response to this need, we developed SalivaDirect, a sensitive, simplified, and flexible testing framework, which received Emergency Use Authorization (EUA) from the U.S. Food and Drug Administration (FDA). We tested saliva collected from a hospital cohort and showed a high positive agreement (94%) as compared to paired nasopharyngeal swabs tested with a commercial diagnostic kit. Then, we partnered with the National Basketball Association (NBA) to test a large cohort of mostly healthy individuals, and we detected low rates of invalid (0.3%) and false-positive (0.03%–0.05%) results. Our study shows that SalivaDirect can help to increase testing capacity by providing access to an affordable framework that is less prone to supply chain shortages., Graphical Abstract, Highlights SalivaDirect is a simplified saliva-based test for detection of SARS-CoV-2 The testing framework is flexible to minimize the risk of supply chain issues SalivaDirect is sensitive, with low rates of invalid and false-positive results Laboratories can be designated to use SalivaDirect to increase testing capacity, SalivaDirect is a sensitive saliva-based COVID-19 diagnostic test, which received Emergency Use Authorization from the U.S. FDA. With the ability to designate other laboratories, the flexible and simplified framework helps to increase the capacity of existing laboratory infrastructure for SARS-CoV-2 testing.

Severe immunodeficient mice have become invaluable tools in human stem cell and tumor research. In this study, we compared the phagocytic activity of peritoneal macrophages against primary effusion lymphoma (PEL) among Rag-2/Jak3 double-deficient (Rag-2(-/-)Jak3(-/-)) mice with NOD and non-NOD (Balb/c and C57/BL6). We also evaluated lymphomatous effusion and infiltration in a PEL xenograft mouse model using these severe immunodeficient mice. In the phagocytic assay, peritoneal macrophages in the NOD background phagocytosed CFSE-labeled BCBL-1, a PEL cell line, less efficiently than those in the non-NOD background. BCBL-1 cells were successfully engrafted into both the NOD background and non-NOD background; however, the volume of ascites of the NOD background was significantly higher than that of the non-NOD background. Moreover, the organ invasion of PEL cells was suppressed in non-NOD background mice. Thus, the NOD genetic background is considered to contribute to more lymphomatous effusion and the infiltration of PEL cells than a non-NOD background. Our results showed that the NOD background allowed more lymphomatous effusion and infiltration than other backgrounds and peritoneal macrophages played a critical role in preventing the growth and infiltration of PEL cells.

The transcription factor NF-κB is important for HIV-1 transcription initiation in primary HIV-1 infection and reactivation in latently HIV-1-infected cells. However, comparative analysis of the regulation and function of NF-κB in latently HIV-1-infected cells has not been done. Here we show that the expression of IκB-α, an endogenous inhibitor of NF-κB, is enhanced by latent HIV-1 infection via induction of the host-derived factor COMMD1/Murr1 in myeloid cells but not in lymphoid cells by using four sets of latently HIV-1-infected cells and the respective parental cells. IκB-α protein was stabilized by COMMD1, which attenuated NF-κB signaling during Toll-like receptor ligand and tumor necrosis factor alpha treatment and enhanced HIV-1 latency in latently HIV-1-infected cells. Activation of the phosphoinositol 3-kinase (PI3K)–JAK pathway is involved in COMMD1 induction in latently HIV-1-infected cells. Our findings indicate that COMMD1 induction is the NF-κB inhibition mechanism in latently HIV-1-infected cells that contributes to innate immune deficiency and reinforces HIV-1 latency. Thus, COMMD1 might be a double-edged sword that is beneficial in primary infection but not beneficial in latent infection when HIV-1 eradication is considered. IMPORTANCE HIV-1 latency is a major barrier to viral eradication in the era of combination antiretroviral therapy. In this study, we found that COMMD1/Murr1, previously identified as an HIV-1 restriction factor, inhibits the proteasomal degradation of IκB-α by increasing the interaction with IκB-α in latently HIV-1-infected myeloid cells. IκB-α protein was stabilized by COMMD1, which attenuated NF-κB signaling during the innate immune response and enhanced HIV-1 latency in latently HIV-1-infected cells. Activation of the PI3K-JAK pathway is involved in COMMD1 induction in latently HIV-1-infected cells. Thus, the host-derived factor COMMD1 is beneficial in suppressing primary infection but enhances latent infection, indicating that it may be a double-edged sword in HIV-1 eradication.

Membrane fusion between host cells and HIV-1 is the initial step in HIV-1 infection, and plasma membrane fluidity strongly influences infectivity. In the present study, we demonstrated that GUT-70, a natural product derived from Calophyllum brasiliense, stabilized plasma membrane fluidity, inhibited HIV-1 entry, and down-regulated the expression of CD4, CCR5, and CXCR4. Since GUT-70 also had an inhibitory effect on viral replication through the inhibition of NF-κB, it is expected to be used as a dual functional and viral mutation resistant reagent. Thus, these unique properties of GUT-70 enable the development of novel therapeutic agents against HIV-1 infection.

Primary effusion lymphoma (PEL) is an aggressive subtype of non-Hodgkin lymphoma that shows malignant effusion most commonly seen in advanced AIDS patients. In this study, we clarified the potential role of VEGF and IL-6 in PEL fluid retention and evaluated the efficacy of humanized anti-VEGF monoclonal antibody (mAb), bevacizumab, and humanized anti-IL-6 receptor mAb, tocilizumab, against PEL. The production of VEGF and IL-6, and the expression of IL-6Rα in PEL cell lines were examined. The antiproliferative effect of bevacizumab and tocilizumab on PEL cells was evaluated in vitro. The effect of tocilizumab on VEGF was also examined. An intraperitoneal xenograft mouse model was used for in vivo efficacy. Although we found the production of VEGF and IL-6, and the expression of IL-6Rα in PEL cell lines, bevacizumab and tocilizumab did not inhibit the proliferation of PEL cells in vitro. Tocilizumab decreased VEGF mRNA and VEGF production by inhibiting Stat3 phosphorylation and Stat3 binding to VEGF promoter. In a PEL xenograft mouse model that showed profuse ascites, bevacizumab suppressed ascites formation completely, indicating the critical role of VEGF for PEL fluid retention. Tocilizumab also significantly inhibited ascites formation in vivo. Moreover, these mAbs improved the overall survival of treated mice. IL-6-VEGF axis contributed to fluid retention, and bevacizumab and tocilizumab could be effective molecular targeting therapies for PEL.

Hematopoietic stem cell (HSC) self-renewal and lineage output are orchestrated by multiple regulatory layers, including RNA modifications. N6-methyladenosine (m6A) is an abundant modification found in RNAs which affects the translation and stability of modified transcripts. The effects of m6A are determined by m6A writers (install m6A), erasers (remove m6A) and readers (recognize m6A). In embryonic stem cells, deletion of the m6A writer METTL3 enforces a naïve pluripotent state. This raises the question of whether m6A RNA methylation analogously regulates stem cell self-renewal and differentiation in somatic stem cells such as HSCs. Using a Vav-Cre/Mettl3 (VCM3) hematopoietic-specific knockout mouse model, we show that loss of the RNA m6A writer METTL3 in fetal HSCs results in hematopoietic failure and perinatal lethality. At E14.5 (FL) hematopoiesis loss of Mettl3/m6A results in hematopoietic failure with expansion of Lin-Sca-1+c-Kit+ (LSK) hematopoietic stem and progenitor cells (HSPCs) that are defective in the production of progenitors and mature blood cells, as evidenced by failure to rescue lethally irradiated congenic recipient mice in transplant experiments. The relative defect I hematopoiesis was further demonstrated by competitive transplant experiments, in which transplanted KO FL were consistently out-competed by WT FL. Interestingly, BrdU/7AAD labeling reveals a significant proliferative defect with reduced BrdU uptake in VCM3 KO FL cells, and specifically in Lin-c-Kit+Sca-1- (LK) progenitor cells. RNA-seq analysis of FL LSK cells reveals that loss of m6A results in upregulation of multiple 2'-5'-oligoadenylate synthetase (OAS) family genes. Interestingly, the majority of OAS family genes are not m6A modified in several m6A sequencing data sets. We therefore hypothesized that the OAS genes might be regulated at the transcriptional level. We performed cleavage under targets and release using nuclease (CUT&RUN) analysis and found that OAS family genes are transcriptionally activated, as evidenced by significant increase in H3K4 trimethylation (H3K4me3) at their respective promoter regions. The OAS family genes are activated by the presence of double stranded RNA (dsRNA), which can arise either endogenously or as a pathogen-associated trigger of the innate immune system in the context of viral infection. The dsRNA response includes three major response mechanisms. First, OAS genes facilitate RNase L dimerization, which mediates cleavage of cellular tRNA and rRNA, resulting in translation and proliferation arrest. Second, activation of the PKR/eIF2a pathway also results in translation arrest. Lastly, activation of the MDA-5/RIG-I/MAVS response induces interferon signaling, which further limits cellular proliferation. Staining of VCM3 WT & KO FL cells with the dsRNA-specific J2 antibody directly demonstrated significant accumulation of dsRNA in KO FL cells. We further demonstrated activation of the OAS/RNaseL axis in KO cells by quantification of tRNA-His-36 cleavage, which acts as a sensitive marker of RNase L activity. Phosphorylation of both PKR and eIF2a in KO cells demonstrated enhanced activity of this pathway. Lastly, we found a significant upregulation of interferon pathway genes in KO cells, and demonstrated that CRISPR mediated knockout of Mavs in VCM3 KO FL cells diminishes the activity of interferon response genes and rescues the Mettl3 KO phenotype in colony forming unit (CFU) assays. These results suggest that in the absence of m6A, endogenous dsRNA formation is enhanced, resulting in an associated inflammatory response which partially accounts for the hematopoietic defect observed in Mettl3 KO mice. In conclusion, our study suggests a novel, protective role for the m6A RNA modification in preventing endogenous dsRNA formation and aberrant activation of a detrimental innate immune response during mammalian hematopoietic development. Disclosures Fan: IsoPlexis: Other: served on the scientific discovery board, Patents & Royalties; Singleron Biotechnologies: Other: served on the Scientific Advisory Board; BioTechne: Other: served on the Scientific Advisory Board. Flavell:Rheos Biomedicines: Equity Ownership; GSK: Consultancy; Artizan Biosciences: Equity Ownership; Troy: Equity Ownership; SMOC: Equity Ownership; Zai labs: Consultancy.

HIV-1 persists during antiretroviral therapy (ART) due to long-lived and proliferating latently-infected host cells, with the outcome being an incomplete cure. The latently-infected cells, or reservoir cells, are transcriptionally absent and invisible to the immune response. Elimination of latency is one strategy in activating virus production, making it visible to immune clearance. We previously showed that Ephedrae herba reactivated HIV-1 from latency. In this study, we used ephedrine, a major component of Ephedra herba, to reactivate HIV-1 from latency. The results showed that ephedrine enhances HIV-1 reactivation in the presence of TNFα. Combination treatment demonstrates a synergistic effect of HIV-1 reactivation compared to TNFα alone. Ephedrine treatment shows a higher TNFRII expression level, which is related to increased HIV-1 reactivation. However, the mechanism of ephedrine in HIV-1 reactivation is still unclear, and may be related to TNFRII receptor expression. Our results indicate that ephedrine enhances HIV-1 reactivation from latency in combination with TNFα treatment. This new reagent could be a promising latency reversal agent (LRA).

Influenza A virus (IAV) infection-associated morbidity and mortality are a key global healthcare concern, necessitating the identification of novel therapies capable of reducing the severity of IAV infections. In this study, we show that the consumption of a low-carbohydrate, high-fat ketogenic diet (KD) protects mice from lethal IAV infection and disease. KD feeding resulted in an expansion of γδ T cells in the lung that improved barrier functions, thereby enhancing anti-viral resistance. Expansion of these protective γδ T cells required metabolic adaptation to a ketogenic diet, as neither feeding mice a high-fat high-carbohydrate diet nor providing chemical ketone body substrate that bypasses hepatic ketogenesis protected against infection. Therefore, KD mediated immune-metabolic integration represents a viable avenue towards preventing or alleviating influenza disease.

HIV protease inhibitor (PI)-induced ER stress has been associated with adverse effects. Although it is a serious clinical problem for HIV/AIDS patients, comparative analyses of ER stress induction by clinically used PIs have rarely been done. Especially, there is no report on the differential ER stress response between lopinavir (LPV) and darunavir (DRV), although these PIs are the most clinically used PIs. We show here that LPV induces the most potent CHOP expression, ER stress marker, among the 9 Food and Drug Administration (FDA)-approved PIs in human peripheral blood mononuclear cells, several human epithelial cells, and mouse embryonic fibroblasts. LPV induced the most potent ROS production and JNK activation in 9 PIs. A comparison among the most clinically used PIs, ritonavir (RTV), LPV, and DRV, revealed that LPV potently and RTV moderately but not DRV induced ER stress via ROS-dependent JNK activation rather than proteasome inhibition. Finally, we analyzed ER stress induction in tissues of mice intraperitoneally injected with RTV, LPV, and DRV. RTV and LPV but not DRV showed ER stress induction in several mice tissues. In conclusion, we first identify LPV as the most potent ER stress inducing PI among 9 FDA-approved PIs in human cells, and although clinical verification is necessary, we show here that DRV has the advantage of less ROS and ER stress induction potential compared with LPV in vitro and in vivo.

Cholangiocarcinoma (CCA) is a tumor of biliary ducts, which has a high mortality rate and dismal prognosis. Constitutively activation of the transcription factor nuclear factor kappa-B (NF-κB) has been previously demonstrated in CCA. It is therefore a potential target for CCA treatment. Effects of diethyldithiocarbamate (DDTC) on NF-κB-dependent apoptosis induction in cancer have been reported; however, anti-metastasis has never been addressed. Therefore, here the focus was on DDTC effects on CCA migration and adhesion. Anti-proliferation, anti-migration and anti-adhesion activities were determined in CCA cell lines, along with p65 protein levels and function. NF-κB target gene expression was determined by quantitative RT-PCR. DDTC inhibited CCA cell proliferation. Suppression of migration and adhesion were observed prior to anti-CCA proliferation. These effects were related to decreased p65, reduction in NF-κB DNA binding, and impaired activity. Moreover, suppression of ICAM-1 expression supported NF-kB-dependent anti-metastatic effects of DDTC. Taken together, DDTC suppression of CCA migration and adhesion through inhibition of NF-κB signaling pathway is suggested from the current study. This might be a promising treatment choice against CCA metastasis.

Autophagy plays a crucial role in cancer cell survival and the inhibition of autophagy is attracting attention as an emerging strategy for the treatment of cancer. Chloroquine (CQ) is an anti-malarial drug, and is also known as an inhibitor of autophagy. Recently, it has been found that CQ induces cancer cell death through the inhibition of autophagy; however, the underlying mechanism is not entirely understood. In this study, we identified the role of CQ-induced cancer cell death using Primary Effusion Lymphoma (PEL) cells. We found that a CQ treatment induced caspase-dependent apoptosis in vitro. CQ also suppressed PEL cell growth in a PEL xenograft mouse model. We showed that CQ activated endoplasmic reticulum (ER) stress signal pathways and induced CHOP, which is an inducer of apoptosis. CQ-induced cell death was significantly decreased by salbrinal, an ER stress inhibitor, indicating that CQ-induced apoptosis in PEL cells depended on ER stress. We show here for the first time that the inhibition of autophagy induces ER stress-mediated apoptosis in PEL cells. Thus, the inhibition of autophagy is a novel strategy for cancer chemotherapy.

Hypoxia-inducible factor (HIF)-1α is a transcription factor belonging to the HIF family that is activated in mammalian cells during conditions of low oxygen tension or hypoxia to induce an adaptive response and promote cell survival. Some of the genes targeted by HIF-1α are important for angiogenesis and proliferation. Here, we found that the E26 transformation-specific (ETS) transcription factor myeloid elf-1-like factor (MEF)/Elf4 is activated by HIF-1α. MEF induces genes such as human beta-defensin 2 (HβD2) and perforin (PRF1), and is known to affect the cell cycle. Treatment with hypoxia mimetic CoCl2 or low O2 incubation up-regulated MEF mRNA and protein levels in various cell lines. HIF-1α overexpression in HEK293 cells also increased MEF mRNA and protein levels. In contrast, HIF-1α knockdown by small interfering RNA (siRNA) suppressed the induction of MEF in response to hypoxia. HIF-1α binds to the hypoxia response element in the MEF promoter region (-200 bp) and activates MEF promoter under hypoxia condition. The induction of MEF by hypoxia/HIF-1α correlated with the increase of MEF target genes HβD2 and PRF1. Intriguingly, the hypoxia-induced expression of HIF-1α target gene vascular endothelial growth factor (VEGF) was enhanced by the exogenous addition of MEF. Overall, these data indicate that hypoxia or HIF-1α positively regulates MEF expression and function.

「Web 2.0」の概念が主流になりつつある現在，図書館の世界でも，ビジュアル化，情報の表紙イメージや内容情報によって情報を強化した「次世代OPAC」が注目を集めている。スペルチェック／サジェスト機能，絞り込み検索，適合度によるソート，利用者参加型機能，統合検索機能などを備えたシステムが注目され，世界各国で開発・運用され始めている。本稿では，まず現在一般的な大学図書館で導入されているOPACの現状を述べ，次に海外での導入事例をもとに，次世代OPACの特徴を具体的に説明する。さらに，現在開発されている主な次世代OPACシステムについてレビューを行う。最後に，これらのシステムを日本の大学図書館で導入する際の課題と，課題解決に向けたいくつかの方法を提示する。

Cholangiocarcinoma (CCA) is a unique liver cancer subtype with an increasing incidence globally. The lack of specific symptoms and definite diagnostic markers results in a delayed diagnosis and disease progression. Systemic chemotherapy is commonly selected for advanced CCA even though its advantages remain unknown. Targeted therapy, especially anti-vascular endothelial growth factor (VEGF) therapy, is promising for CCA; however, improvements in the therapeutic regimen are necessary to overcome subsequent resistance. We demonstrated VEGF expression was higher in CCA cell lines than in other liver cancer cells. Secreted VEGFs played roles in the induction of peri- and intra-tumoral vascularization. VEGF neutralization by bevacizumab effectively reduced tumor growth, mainly through the suppression of angiogenesis; however, increases in the expression of hypoxia-inducible factor 1α (HIF1α) and HIF1α-responsive genes (such as VEGF, VEGFR1, VEGFR2, carbonic anhydrase (CA) IX and CAXII) indicated the potential for subsequent therapeutic resistance. Supplementation with a carbonic anhydrase inhibitor, acetazolamide, enhanced the anti-CCA effects of bevacizumab. Anti-angiogenesis and anti-proliferation were observed with the combination treatment. These results suggested a novel treatment strategy to overcome anti-angiogenesis resistance and the importance of "induced essentiality" in the treatment of CCA.

This study involves the development and evaluation of a game-based learning environment for library instruction that is based on the ARCS model and designed to run on mobile platforms such as iOS and Android. A questionnaire and performance test was administered to compare game-based and e-learning performance, no significant difference in performance was found between either groups, although three ARCS factors were significantly higher for the experimental group when compared to the control group. Furthermore, subjects in the experimental group were more likely to recognize the effects of the ARCS design when compared to those who used e-learning.

This study reports an experiment that was conducted to reexamine self-serving bias for success and failure among Japanese participants. Previous research has generally failed to find that Japanese participants exhibit a self-serving bias. However, with careful procedures added to reduce evaluation apprehension of participants, the participants in the success condition made more internal attribution than those in the failure condition. The authors discuss self-serving bias and self-critical bias in terms of evaluation apprehension and social norms.

Introduction: Primary effusion lymphoma (PEL) is a subtype of aggressive non-Hodgkin lymphoma that shows serous lymphomatous effusion without tumor masses in body cavities especially in advanced acquired immunodeficiency syndrome (AIDS). PEL is universally associated with Kaposi sarcoma-associated herpesvirus (KSHV)/human herpesvirus-8 (HHV-8) infection. The prognosis of PEL is extremely poor due to its aggressiveness and resistance to conventional chemotherapy. Therefore, the understanding of the molecular pathogenesis of PEL is vital to develop novel therapeutic strategies. PU.1, one of the Ets family transcription factors, has been reported to be down-regulated in some B-cell malignancies and acute myeloid leukemia. In this study, we examined the methylation of the PU.1 promoter and enhancer regions in PEL cell lines and performed the restoration of PU.1 in PEL cells in vitro and in vivo. Methods: PU.1 mRNA expressions of eight PEL cell lines were examined by polymerase chain reaction (PCR). PU.1 was conditionally expressed using tet-on system in three PEL cell lines. The bisulfite sequencing was performed to analyze the methylation of the PU.1 promoter and enhancer regions in PEL cells. The DNA methyltransferase inhibitor, 5-Azacytidine, was used as an experimental tool for demethylation. The anti-proliferative activities of restoring PU.1 or 5-Azacytidine in PEL cell lines were measured by the methylthiotetrazole method. Apoptosis was quantified using Annexin V antibody. To evaluate the association of PU.1 with TRAIL promoter, we analyzed its recruitment to the promoter by luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay. BCBL-1 cells that expressed tet-inducible PU.1 were injected intraperitoneally into NOD/Rag-2/Jak3double-deficient mice to assess the in vivo efficacy of restoring PU.1. Organ invasion by BCBL-1 cells was evaluated by immunohistochemistry. Results: The expression of PU.1 mRNA in PEL cell lines was weaker than in the human monoblast leukemia cell line, U937, which expresses PU.1 mRNA. The promoter and enhancer regions of the PU.1 gene were highly methylated in PEL cells. The treatment with 5-Azacytidine up-regulated the expression of PU.1 mRNA. Restoration of PU.1 or treatment with 5-Azacytidine inhibited cell proliferation. PU.1 up-regulated TRAIL gene expression among apoptosis-related genes and caused apoptosis. PU.1 induced the transactivation of TRAIL promoter in the reporter assay. Disruption of the PU.1 binding site repressed its transactivation. PU.1 was recruited to TRAIL promoter in the ChIP assay. Although PU.1 induced the master regulator of viral replication, ORF50 expression, the expression of ORF57, which is essential for virus production as a posttranscriptional activator, decreased. PU.1 suppressed ORF 57 by the occupancy of IRF7 in the ORF57 promoter region. Restoration of PU.1 inhibited the ascites formation and organ invasion significantly in vivo compared with control mice. Conclusions : The up-regulation of PU.1 induces growth inhibition and apoptosis via the direct transactivation of TRAIL and modulates viral transactivation via the up-regulation of IRF7. PU.1 is a tumor suppressor in PEL and its down-regulation has a critical role in PEL development. The targeting of PU.1 could be a therapeutic approach in patients with PEL. Disclosures No relevant conflicts of interest to declare.