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Xianting Ding1, David Jesse Sanchez2,3, Arash Shahangian2, Ibrahim Al-Shyoukh1,4, Genhong Cheng2, Chih-Ming Ho11Department of Mechanical and Aerospace Engineering, UCLA, Los Angeles, CA, USA; 2Department of Microbiology, Immunology, and Molecular Genetics, UCLA, Los Angeles, CA, USA; 3Department of Pharmaceutical Sciences, College of Pharmacy, Western University of Health Sciences, Pomona, CA, USA; 4Molecular and Medical Pharmacology Department, David Geffen School of Medicine, UCLA, Los Angeles, CA, USABackground: Infectious diseases cause many molecular assemblies and pathways within cellular signaling networks to function aberrantly. The most effective way to treat complex, diseased cellular networks is to apply multiple drugs that attack the problem from many fronts. However, determining the optimal combination of several drugs at specific dosages to reach an endpoint objective is a daunting task.Methods: In this study, we applied an experimental feedback system control (FSC) method and rapidly identified optimal drug combinations that inhibit herpes simplex virus-1 infection, by only testing less than 0.1% of the total possible drug combinations.Results: Using antiviral efficacy as the criterion, FSC quickly identified a highly efficacious drug cocktail. This cocktail contained high dose ribavirin. Ribavirin, while being an effective antiviral drug, often induces toxic side effects that are not desirable in a therapeutic drug combination. To screen for less toxic drug combinations, we applied a second FSC search in cascade and used both high antiviral efficacy and low toxicity as criteria. Surprisingly, the new drug combination eliminated the need for ribavirin, but still blocked viral infection in nearly 100% of cases.Conclusion: This cascade search provides a versatile platform for rapid discovery of new drug combinations that satisfy multiple criteria.Keywords: drug combination, HSV-1, combinatorial drug optimization, feedback system control, FSC, drug screening

The colon is the largest compartment of the immune system, with innate immune cells exposed to antigens in the environment. However, the mechanisms by which the innate immune system is instigated are poorly defined in colorectal cancer (CRC). Here, a population of CD16 + neutrophils that specifically accumulate in CRC tumor tissues by imaging mass cytometry (IMC), immune fluorescence, and flow cytometry, which demonstrated pro-tumor activity by disturbing natural killer (NK) cells are identified. It is found that these CD16 + neutrophils possess abnormal cholesterol accumulation due to activation of the CD16/TAK1/NF-κB axis, which upregulates scavenger receptors for cholesterol intake including CD36 and LRP1. Consequently, these region-specific CD16 + neutrophils not only competitively inhibit cholesterol intake of NK cells, which interrupts NK lipid raft formation and blocks their antitumor signaling but also release neutrophil extracellular traps (NETs) to induce the death of NK cells. Furthermore, CD16-knockout reverses the pro-tumor activity of neutrophils and restored NK cell cytotoxicity. Collectively, the findings suggest that CRC region-specific CD16 + neutrophils can be a diagnostic marker and potential therapeutic target for CRC., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Modulation of innate immunity is critical for virus persistence in a host. In particular, viral-encoded disruption of type I interferon, a major antiviral cytokine induced to fight viral infection, is a key component in the repertoire of viral pathogenicity genes. We have identified a previously undescribed open reading frame within the Kaposi's sarcoma-associated herpesvirus (KSHV) genome that encodes a homologue of the human IPS-1 (also referred to as MAVS) protein that we have termed viral-IPS-1 (v-IPS-1). This protein is expressed during the lytic replication program of KSHV, and expression of v-IPS-1 blocks induction of type I interferon upstream of the TRAF3 signaling node including signaling initiated via both the RLR and TLR3/4 signaling axes. This disruption of signaling coincides with destabilization of the cellular innate signaling adaptors IPS-1 and TRIF along with a concatenate stabilization of the TRAF3 protein. Additionally, expression of v-IPS-1 leads to decreased antiviral responses indicating a blot to type I interferon induction during viral infection. Taken together, v-IPS-1 is the first described viral homologue of IPS-1 and this viral protein leads to reprogramming of innate immunity through modulation of type I interferon signaling during KSHV lytic replication.

Antiviral vaccine is essential for preventing and controlling virus spreading, along with declining morbidity and mortality. A major challenge in effective vaccination lies in the ability to enhance both the humoral and cellular immune responses by adjuvants. Herein, self-assembled nanoparticles based on graphene oxide quantum dots with components of carnosine, resiquimod and Zn 2+ ions, namely ZnGC-R, are designed as a new adjuvant for influenza vaccine. With its high capability for antigen-loading, ZnGC-R enhances antigen utilization, improves DC recruitment, and activates antigen-presenting cells. Single cell analysis of lymphocytes after intramuscular vaccination revealed that ZnGC-R generated multifaceted immune responses. ZnGC-R stimulated robust CD4 + CCR7 lo PD-1 hi Tfh and durable CD8 + CD44 hi CD62L - T EM immune responses, and simultaneously promoted the proliferation of CD26 + germinal center B cells. Besides, ZnGC-R elicited 2.53-fold higher hemagglutination-inhibiting antibody than commercial-licensed aluminum salt adjuvant. ZnGC-R based vaccine induced 342% stronger IgG antibody responses compared with vaccines with inactivated virus alone, leading to 100% in vivo protection efficacy against the H1N1 influenza virus challenge., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Fragile X Messenger Ribonucleoprotein 1 (FMR1) gene mutations lead to fragile X syndrome, cognitive disorders, and, in some individuals, scoliosis and craniofacial abnormalities. Four-month-old (mo) male mice with deletion of the FMR1 gene exhibit a mild increase in cortical and cancellous femoral bone mass. However, consequences of absence of FMR1 in bone of young/aged male/female mice and the cellular basis of the skeletal phenotype remain unknown. We found that absence of FMR1 results in improved bone properties with higher bone mineral density in both sexes and in 2- and 9-mo mice. The cancellous bone mass is higher only in females, whereas, cortical bone mass is higher in 2- and 9-mo males, but higher in 2- and lower in 9-mo female FMR1-knockout mice. Furthermore, male bones show higher biomechanical properties at 2mo, and females at both ages. Absence of FMR1 increases osteoblast/mineralization/bone formation and osteocyte dendricity/gene expression in vivo/ex vivo/in vitro, without affecting osteoclasts in vivo/ex vivo. Thus, FMR1 is a novel osteoblast/osteocyte differentiation inhibitor, and its absence leads to age-, site- and sex-dependent higher bone mass/strength., (© 2023. The Author(s).)

Triggering Receptor Expressed on Myeloid cells-1 (TREM-1) has been a potential target in the management of pathophysiology and clinical sequelae of atherosclerosis. LR12 peptide effectively blocks ligand-TREM-1 interaction; however, the short half-life of LR12 is a major hurdle in its translational application in atherosclerosis management warranting new methods for sustained bioavailability in clinical applications. The present study reports a novel method of packing the coding sequence of LR12 in a lentiviral system to ensure a sustained expression and bioavailability for effective TREM-1 inhibition. Lentivirus vector systems (LV-LR12 and LV-SP) for the expression of LR12 peptide and SP (scrambled peptide) were successfully designed, constructed, and tested in vitro in smooth muscle cells (SMCs). Viral amounts obtained were 703.6 ± 145.12 and 609.3 ± 145.93 ng/ml p24 for LV-LR12 and LV-SP, respectively which correspond to ~ 107 IFU/ml for both vectors. Dot blot assay revealed significantly increased expression of LR12-FLAG and SP-FLAG in 125 μg total protein which was doubled in 250 μg protein with respect to un-transduced SMCs suggesting the sustained release of LR12/SP as confirmed by ELISA. Cellular expression of LR12-FLAG and SP-FLAG displayed 8.44-fold and 7.55-fold increase, respectively compared to the control SMCs. The findings demonstrated a promising strategy for packing the LR12 coding sequence in lentiviral vector for TREM-1 inhibition for the management of atherosclerosis and other inflammatory diseases., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The role of PPAR gamma (PPARγ) has been well characterized in the developmental process of adipogenesis, yet its aberrant expression patterns and functions in cancer subtypes are less understood. Although PPARγ has been recently demonstrated to play non-cell-autonomous roles in promoting bladder urothelial carcinoma (UC) progression, underlying mechanisms of the cell-intrinsic oncogenic activity remain unknown. Here, we report robust expression and nuclear accumulation of PPARγ in 47% of samples of patients with UC, exceeding mRNA expression patterns published by The Cancer Genome Atlas. In vitro assays revealed for the first time that treatment of UC cells with PPARγ inverse agonist or PPARG knockout by CRISPR-Cas9 reduces proliferation, migration, and invasion of multiple established UC cell lines, most strongly in those characterized by PPARG genomic amplification or activating mutations of RXRA , the obligate heterodimer of PPARγ. Through genome-wide approaches including chromatin immunoprecipitation sequencing and RNA sequencing, we define a novel set of PPARγ-regulated genes in UC, including Sonic Hedgehog ( SHH ). Similar to PPARγ, genetic inhibition of SHH reduces proliferation and motility. Finally, we demonstrate the PPARγ dependency of UC tumors in vivo by genetic and pharmacologic PPARγ inhibition in subcutaneous xenografts. Collectively, our data indicate that PPARγ promotes UC progression in a subset of patients, at least in part, through cell-autonomous mechanisms linked to SHH signaling. IMPLICATIONS: Genome-wide analysis of DNA-binding sites for oncogenic factor PPARγ revealed SHH as a novel downstream target involved in UC progression, providing important insight into the tumorigenic nature and molecular mechanism of PPARγ signaling in UC., (©2021 American Association for Cancer Research.)

Progressive globalization of our society brings not only worldwide integration, it also increases and promotes our exposure to new viral pathogens with evident impacts on our global health. Especially with the emergence of SARS-CoV-2, our biomedical research infrastructure has never been more compelled to rapidly develop antiviral regimens that demonstrate improved efficacy against these pathogens. Here, we showcase 3 poignant antivirals against the lucrative target, RNA-dependent RNA polymerase (RdRP) of RNA viruses - a timely and relevant topic given the present efforts against COVID-19. While effective drug designs against RdRP are important, their benefit and potential as a standard of care truly relies on them standing out in well-designed clinical trials., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

As antiviral drug resistance develops and new viruses emerge there is a pressing need to develop strategies to rapidly develop antiviral therapeutics. Here we use phospho-specific flow cytometry to assess perturbations of many different cellular signaling pathways during treatment with drug combinations that are highly effective in blocking Herpes simplex virus type 1 (HSV-1) infection. We discovered two antiviral drug combinations act on distinct signaling pathways, either STAT1 or S6 phosphorylation, to block HSV-1 infection. We focused on upregulation of S6 phosphorylation by HSV-1 infection, and our subsequent finding that ribavirin antagonizes this upregulation of S6 phosphorylation. We go on to show that the S6 kinase inhibitor SL0101 blocks HSV-1 replication in vitro and in an in vivo animal model of HSV-1 infection. Overall, we have used an unbiased analysis of cellular signaling pathways during treatment by antiviral drug combinations to discover a novel antiviral drug target against HSV-1 infection. The outcomes of the approach we present highlight the importance of analyzing how antiviral drugs modulate cellular and pathogen-induced signaling as a method to discover new drug therapy targets., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Introduction: Burnout is considered to be at the opposite end of the continuum from engagement. People who experience burnout first go through various intermediate patterns that lead to burnout, which in medical students is associated with reduced empathy, intention to leave school, and suicidal ideation. Thus, understanding how to mitigate burnout is of primary importance. In this study, we investigate if students' positive perceptions of the educational program's alignment with adult education principles decreased symptoms suggestive of typical patterns of intermediate burnout., Methods: We conducted a cross-sectional survey study of all currently enrolled Duke-NUS Medical School students in Singapore ( n = 238). An electronic questionnaire contained demographic questions and additional measures for factors known to be associated with burnout, including depression, anxiety, social support, and workload. In addition, we measured students' perceptions of how well the educational program aligned with adult learning principles by using a modified version of the Andragogical Practices Inventory (API) to suit medical education. An intermediate pattern of burnout was measured using the Maslach Burnout Inventory (MBI). Using logistic regression, we then assessed the unique association between the presence of an intermediate pattern of burnout with students' perceptions of the educational program's alignment with adult learning principles., Results: The survey response rate was 52%. Overall, 76% (95% CI 67-84%) displayed symptoms suggestive of an intermediate pattern of burnout. Perceptions of the educational program's alignment with adult learning principles were found to be inversely related to the pattern of burnout after controlling for depression, anxiety, and subjective workload., Discussion and Conclusion: Though adult learning theory is the subject of rich debate, the results of this study suggest that promoting educational activities that are aligned with adult learning principles may help to ultimately reduce the risk of burnout in medical school students., Competing Interests: Conflict of interestNone., (© International Association of Medical Science Educators 2019.)

Clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. While the localized form of this disease can be treated surgically, advanced and metastatic stages are resistant to chemotherapies. Although more innovative treatments, such as targeted or immune-based therapies, exist, the need for new therapeutic options remains. ccRCC presents unique metabolic signatures and multiple studies have reported a significant increase in levels of reduced glutathione (GSH) and its precursors in ccRCC tumor samples compared with normal kidney tissues. These observations led us to investigate the effects of blocking the GSH pathway, particularly the gamma-glutamyltransferase 1 (GGT1) enzyme, in multiple ccRCC cell lines. In this study, we provide in vitro and in vivo evidence that GGT1/GSH pathway inhibition impacts ccRCC cell growth, through increased cell-cycle arrest. Of note, GGT1 inhibition also impairs ccRCC cell migration. Finally, pharmacologic GSH pathway inhibition decreases ccRCC cell proliferation and increases sensitivity to standard chemotherapy. Our results suggest that GGT1/GSH pathway inhibition represents a new strategy to overcome ccRCC chemoresistance. IMPLICATIONS: GGT1/GSH pathway inhibition represents a promising therapeutic strategy to overcome chemoresistance and inhibit progression of ccRCC tumors., (©2019 American Association for Cancer Research.)

Slow-wave sleep (SWS) is important for overall health since it affects many physiological processes including cardio-metabolic function. Sleep and autonomic nervous system (ANS) activity are closely coupled at anatomical and physiological levels. Sleep-related changes in autonomic function are likely the main pathway through which SWS affects many systems within the body. There are characteristic changes in ANS activity across sleep stages. Notably, in non-rapid eye-movement sleep, the progression into SWS is characterized by increased parasympathetic activity, an important measure of cardiovascular health. Experimental manipulations that enhance slow-wave activity (SWA, 0.5-4 Hz) can improve sleep-mediated memory and immune function. However, effects of SWA enhancement on autonomic regulation have not been investigated. Here, we employed an adaptive algorithm to deliver 50 ms sounds phase-locked to slow-waves, with regular pauses in stimulation (~5 s ON/~5 s OFF), in healthy young adults. We sought to determine whether acoustic enhancement of SWA altered parasympathetic activity during SWS assessed with heart rate variability (HRV), and evening-to-morning changes in HRV, plasma cortisol, and blood pressure. Stimulation, compared with a sham condition, increased SWA during ON versus OFF intervals. This ON/OFF SWA enhancement was associated with a reduction in evening-to-morning change of cortisol levels and indices of sympathetic activity. Furthermore, the enhancement of SWA in ON intervals during sleep cycles 2-3 was accompanied by an increase in parasympathetic activity (high-frequency, HRV). Together these findings suggest that acoustic enhancement of SWA has a positive effect on autonomic function in sleep. Approaches to strengthen brain-heart interaction during sleep could have important implications for cardiovascular health., (© Sleep Research Society 2019. Published by Oxford University Press on behalf of the Sleep Research Society. All rights reserved. For permissions, please e-mail journals.permissions@oup.com.)

Type I Interferons were first described for their profound antiviral abilities in cell culture and animal models, and later, they were translated into potent antiviral therapeutics. However, as additional studies into the function of Type I Interferons progressed, it was also seen that pathogenic viruses have coevolved to encode potent mechanisms allowing them to evade or suppress the impact of Type I Interferons on their replication. For chronic viral infections, such as HIV and many of the AIDS-associated viruses, including HTLV, HCV, KSHV, and EBV, the clinical efficacy of Type I Interferons is limited by these mechanisms. Here, we review some of the ways that HIV and AIDS-associated viruses thrive in Type I Interferon-rich environments via mechanisms that block the function of this important antiviral cytokine. Overall, a better understanding of these mechanisms creates avenues to better understand the innate immune response to these viruses as well as plan the development of antivirals that would allow the natural antiviral effect of Type I Interferons to manifest during these infections.

This study investigates the modulation of Type I IFN induction of an antiviral state by HIV. IFNs, including IFN-α, are key innate immune cytokines that activate the JAK/STAT pathway leading to the expression of IFN-stimulated genes. IFN-stimulated gene expression establishes the antiviral state, limiting viral infection in IFN-α-stimulated microenvironments. Our previous studies have shown that HIV proteins disrupt the induction of IFN-α by degradation of IFN-β promoter stimulator-1, an adaptor protein for the up-regulation and release of IFN-α into the local microenvironment via the retinoic acid-inducible gene 1-like receptor signaling pathway. However, IFN-α is still released from other sources such as plasmacytoid dendritic cells via TLR-dependent recognition of HIV. Here we report that the activation of the JAK/STAT pathway by IFN-α stimulation is disrupted by HIV proteins Vpu and Nef, which both reduce IFN-α induction of STAT1 phosphorylation. Thus, HIV would still be able to avoid antiviral protection induced by IFN-α in the local microenvironment. These findings show that HIV blocks multiple signaling points that would lead to the up-regulation of IFN-stimulated genes, allowing more effective replication in IFN-α-rich environments.

Sudden cardiac death (SCD) is the most devastating complication in hypertrophic cardiomyopathy (HCM). The implantable cardioverter-defibrillator (ICD) has proven to be effective in SCD prevention in several clinical scenarios. In HCM population, it has demonstrated to successfully abort life-threatening ventricular arrhythmias despite the extreme morphology characteristic of HCM, often with massive degrees of left ventricular hypertrophy and/or LV outflow tract obstruction. Studies showed a high rate of appropriate intervention in secondary prevention and in primary prevention of patients considered at high risk. This appropriate intervention rate is even more significant considering the young and otherwise healthy patients that compose HCM population. Since SCD incidence in HCM is relatively low, optimal identification of patients at high risk is crucial. Classical strategy of risk stratification based on clinical risk factors has several limitations and has proven to overestimate risk. A new risk prediction model that provides individual 5-year estimated risk appears to be superior to traditional models based on bivariate risk factors. Perioperative complications seem to be similar to those related to the implant of other cardiac devices, while long-term complications have been traditionally in the spotlight. HCM patients are considered more vulnerable to ICD-related complications and inappropriate ICD therapy because of their young age at implant and increased prevalence of atrial fibrillation, but long-term follow-up data on ICD-related complications in general practice is limited. The subcutaneous implantable cardioverter defibrillator seems to be a safe and effective alternative in HCM, although long-term data are scarce.

Clear cell renal cell carcinoma (ccRCC) is a malignancy characterized by deregulated hypoxia-inducible factor signaling, mutation of several key chromatin modifying enzymes, and numerous alterations in cellular metabolism. Pre-clinical studies have historically been limited to cell culture models, however, the identification of critical tumor suppressors and oncogenes from large-scale patient sequencing data has led to several new genetically engineered mouse models with phenotypes reminiscent of ccRCC. In this review, we summarize recent literature on these topics and discuss how they inform targeted therapeutic approaches for the treatment of ccRCC., (Copyright © 2018. Published by Elsevier B.V.)

Objective: Clear cell renal cell carcinoma (ccRCC) is a subtype of kidney cancer defined by robust lipid accumulation, which prior studies have indicated plays an important role in tumor progression. We hypothesized that the peroxisome proliferator-activated receptor gamma (PPARγ), detected in both ccRCC tumors and cell lines, promotes lipid storage in ccRCC and contributes to tumorigenesis in this setting. PPARγ transcriptionally regulates a number of genes involved in lipid and glucose metabolism in adipocytes, yet its role in ccRCC has not been described. The objective of this study was to elucidate endogenous PPARγ function in ccRCC cells., Methods and Results: Using chromatin immunoprecipitation followed by deep sequencing (ChIP-seq), we found that PPARγ and its heterodimer RXR occupy the canonical DR1 PPAR binding motif at approximately 1000 locations throughout the genome that can be subdivided into adipose-shared and ccRCC-specific sites. CRISPR-Cas9 mediated, loss-of-function studies determined that PPARγ is dispensable for viability, proliferation, and migration of ccRCC cells in vitro and in vivo. Also, surprisingly, PPARγ deletion had little effect on the robust lipid accumulation that typifies the "clear cell" phenotype of kidney cancer., Conclusion: Our results suggest that PPARγ plays neither a tumor suppressive nor oncogenic role in advanced ccRCC, and thus single-agent therapeutics targeting PPARγ are unlikely to be effective for the treatment of this disease. The unique cistrome of PPARγ in ccRCC cells demonstrates the importance of cell type in determining the functions of PPARγ., (Copyright © 2018 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Myc activation is a primary oncogenic event in many human cancers; however, these transcription factors are difficult to inhibit pharmacologically, suggesting that Myc-dependent downstream effectors may be more tractable therapeutic targets. Here, we show that Myc overexpression induces endoplasmic reticulum (ER) stress and engages the inositol-requiring enzyme 1α (IRE1α)/X-box binding protein 1 (XBP1) pathway through multiple molecular mechanisms in a variety of c-Myc- and N-Myc-dependent cancers. In particular, Myc-overexpressing cells require IRE1α/XBP1 signaling for sustained growth and survival in vitro and in vivo, dependent on elevated stearoyl-CoA-desaturase 1 (SCD1) activity. Pharmacological and genetic XBP1 inhibition induces Myc-dependent apoptosis, which is alleviated by exogenous unsaturated fatty acids. Of note, SCD1 inhibition phenocopies IRE1α RNase activity suppression in vivo. Furthermore, IRE1α inhibition enhances the cytotoxic effects of standard chemotherapy drugs used to treat c-Myc-overexpressing Burkitt's lymphoma, suggesting that inhibiting the IRE1α/XBP1 pathway is a useful general strategy for treatment of Myc-driven cancers.

Background: Comparative studies of chloroplast genomes (plastomes) across the Chlorophyceae are revealing dynamic patterns of size variation, gene content, and genome rearrangements. Phylogenomic analyses are improving resolution of relationships, and uncovering novel lineages as new plastomes continue to be characterized. To gain further insight into the evolution of the chlorophyte plastome and increase the number of representative plastomes for the Sphaeropleales, this study presents two fully sequenced plastomes from the green algal family Hydrodictyaceae (Sphaeropleales, Chlorophyceae), one from Hydrodictyon reticulatum and the other from Pediastrum duplex ., Methods: Genomic DNA from Hydrodictyon reticulatum and Pediastrum duplex was subjected to Illumina paired-end sequencing and the complete plastomes were assembled for each. Plastome size and gene content were characterized and compared with other plastomes from the Sphaeropleales. Homology searches using BLASTX were used to characterize introns and open reading frames (orfs) ≥ 300 bp. A phylogenetic analysis of gene order across the Sphaeropleales was performed., Results: The plastome of Hydrodictyon reticulatum is 225,641 bp and Pediastrum duplex is 232,554 bp. The plastome structure and gene order of H. reticulatum and P. duplex are more similar to each other than to other members of the Sphaeropleales. Numerous unique open reading frames are found in both plastomes and the plastome of P. duplex contains putative viral protein genes, not found in other Sphaeropleales plastomes. Gene order analyses support the monophyly of the Hydrodictyaceae and their sister relationship to the Neochloridaceae., Discussion: The complete plastomes of Hydrodictyon reticulatum and Pediastrum duplex , representing the largest of the Sphaeropleales sequenced thus far, once again highlight the variability in size, architecture, gene order and content across the Chlorophyceae. Novel intron insertion sites and unique orfs indicate recent, independent invasions into each plastome, a hypothesis testable with an expanded plastome investigation within the Hydrodictyaceae., Competing Interests: The authors declare there are no competing interests.

New influenza vaccines that provide effective and broad protection are desperately needed. Live attenuated viruses are attractive vaccine candidates because they can elicit both humoral and cellular immune responses. However, recent formulations of live attenuated influenza vaccines (LAIVs) have not been protective. We combined high-coverage transposon mutagenesis of influenza virus with a rapid high-throughput screening for attenuation to generate W7-791, a live attenuated mutant virus strain. W7-791 produced only a transient asymptomatic infection in adult and neonatal mice even at doses 100-fold higher than the LD 50 of the parent strain. A single administration of W7-791 conferred full protection to mice against lethal challenge with H1N1, H3N2, and H5N1 strains, and improved viral clearance in ferrets. Adoptive transfer of T cells from W7-791-immunized mice conferred heterologous protection, indicating a role for T cell-mediated immunity. These studies present an LAIV development strategy to rapidly generate and screen entire libraries of viral clones., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Our main objective of this study was to determine how Human Immunodeficiency Virus (HIV) avoids induction of the antiviral Type I Interferon (IFN) system. To limit viral infection, the innate immune system produces important antiviral cytokines such as the IFN. IFN set up a critical roadblock to virus infection by limiting further replication of a virus. Usually, IFN production is induced by the recognition of viral nucleic acids by innate immune receptors and subsequent downstream signaling. However, the importance of IFN in the defense against viruses has lead most pathogenic viruses to evolve strategies to inhibit host IFN induction or responses allowing for increased pathogenicity and persistence of the virus. While the adaptive immune responses to HIV infection have been extensively studied, less is known about the balance between induction and inhibition of innate immune defenses, including the antiviral IFN response, by HIV infection. Here we show that HIV infection of T cells does not induce significant IFN production even IFN I Interferon production. To explain this paradox, we screened HIV proteins and found that two HIV encoded proteins, Vpu and Nef, strongly antagonize IFN induction, with expression of these proteins leading to loss of expression of the innate immune viral RNA sensing adaptor protein, IPS-1 (IFN-β promoter stimulator-1). We hypothesize that with lower levels of IPS-1 present, infected cells are defective in mounting antiviral responses allowing HIV to replicate without the normal antiviral actions of the host IFN response. Using cell lines as well as primary human derived cells, we show that HIV targeting of IPS-1 is key to limiting IFN induction. These findings describe how HIV infection modulates IFN induction providing insight into the mechanisms by which HIV establishes infection and persistence in a host.

The present study aimed to compare laparoscopic (LP) and ultrasound-guided (US) biopsy methods to obtain either liver or splenic tissue samples for ectopic gene expression analysis in transgenic goats. Tissue samples were collected from human granulocyte colony stimulating factor (hG-CSF)-transgenic bucks and submitted to real-time PCR for the endogenous genes (Sp1, Baff, and Gapdh) and the transgene (hG-CSF). Both LP and US biopsy methods were successful in obtaining liver and splenic samples that could be analyzed by PCR (i.e., sufficient sample sizes and RNA yield were obtained). Although the number of attempts made to obtain the tissue samples was similar (P > 0.05), LP procedures took considerably longer than the US method (P = 0.03). Finally, transgene transcripts were not detected in spleen or liver samples. Thus, for the phenotypic characterization of a transgenic goat line, investigation of ectopic gene expression can be made successfully by LP or US biopsy, avoiding the traditional approach of euthanasia.

Unlabelled: Two hallmarks of clear-cell renal cell carcinoma (ccRCC) are constitutive hypoxia-inducible factor (HIF) signaling and abundant intracellular lipid droplets (LD). However, regulation of lipid storage and its role in ccRCC are incompletely understood. Transcriptional profiling of primary ccRCC samples revealed that expression of the LD coat protein gene PLIN2 was elevated in tumors and correlated with HIF2α, but not HIF1α, activation. HIF2α-dependent PLIN2 expression promoted lipid storage, proliferation, and viability in xenograft tumors. Mechanistically, lipid storage maintained integrity of the endoplasmic reticulum (ER), which is functionally and physically associated with LDs. Specifically, PLIN2-dependent lipid storage suppressed cytotoxic ER stress responses that otherwise result from elevated protein synthetic activity characteristic of ccRCC cells. Thus, in addition to promoting ccRCC proliferation and anabolic metabolism, HIF2α modulates lipid storage to sustain ER homeostasis, particularly under conditions of nutrient and oxygen limitation, thereby promoting tumor cell survival., Significance: We demonstrate that HIF2α promotes lipid storage, ER homeostasis, and cell viability in ccRCC via upregulation of the LD coat protein PLIN2, revealing a novel function for the well-documented "clear-cell" phenotype and identifying ER stress as a targetable vulnerability created by HIF2α/PLIN2 suppression in this common renal malignancy., (©2015 American Association for Cancer Research.)

Studies of implicit perceptual-motor sequence learning have often shown learning to be inflexibly tied to the training conditions during learning. Since sequence learning is seen as a model task of skill acquisition, limits on the ability to transfer knowledge from the training context to a performance context indicates important constraints on skill learning approaches. Lack of transfer across contexts has been demonstrated by showing that when task elements are changed following training, this leads to a disruption in performance. These results have typically been taken as suggesting that the sequence knowledge relies on integrated representations across task elements (Abrahamse, Jiménez, Verwey, & Clegg, Psychon Bull Rev 17:603-623, 2010a). Using a relatively new sequence learning task, serial interception sequence learning, three experiments are reported that quantify this magnitude of performance disruption after selectively manipulating individual aspects of motor performance or perceptual information. In Experiment 1, selective disruption of the timing or order of sequential actions was examined using a novel response manipulandum that allowed for separate analysis of these two motor response components. In Experiments 2 and 3, transfer was examined after selective disruption of perceptual information that left the motor response sequence intact. All three experiments provided quantifiable estimates of partial transfer to novel contexts that suggest some level of information integration across task elements. However, the ability to identify quantifiable levels of successful transfer indicates that integration is not all-or-none and that measurement sensitivity is a key in understanding sequence knowledge representations.

Over the past years, professional students have had extensive exposure to clinical cases during basic science classes. With this in mind, we have taken this clinical case exposure moment to be an opportune time to introduce the ethics of working with patients during biomedical research. Our goal is to present a straightforward assignment that allows for active student research into the facts of the Tuskegee Experiment of the 1900s. The assignment provides the necessary background to allow for a student-centered discussion on the ethical issues of the events and ramifications of what happened. Thus, in educating a class on the event's happenings, one concomitantly creates a platform for meaningful discussion on the principles and ethics of patient care. We have found that an ethics-infused event such as the Tuskegee Experiment is an excellent way to introduce students to these topics.

Implicit skill learning occurs incidentally and without conscious awareness of what is learned. However, the rate and effectiveness of learning may still be affected by decreased availability of central processing resources. Dual-task experiments have generally found impairments in implicit learning, however, these studies have also shown that certain characteristics of the secondary task (e.g., timing) can complicate the interpretation of these results. To avoid this problem, the current experiments used a novel method to impose resource constraints prior to engaging in skill learning. Ego depletion theory states that humans possess a limited store of cognitive resources that, when depleted, results in deficits in self-regulation and cognitive control. In a first experiment, we used a standard ego depletion manipulation prior to performance of the Serial Interception Sequence Learning (SISL) task. Depleted participants exhibited poorer test performance than did non-depleted controls, indicating that reducing available executive resources may adversely affect implicit sequence learning, expression of sequence knowledge, or both. In a second experiment, depletion was administered either prior to or after training. Participants who reported higher levels of depletion before or after training again showed less sequence-specific knowledge on the post-training assessment. However, the results did not allow for clear separation of ego depletion effects on learning versus subsequent sequence-specific performance. These results indicate that performance on an implicitly learned sequence can be impaired by a reduction in executive resources, in spite of learning taking place outside of awareness and without conscious intent.

We here examined the potential liver toxicity in rat pups from dams exposed during the gestational and lactation periods to 2,2',4,4',5-pentabromodiphenyl ether (BDE-99). Dams were exposed to 0, 1, and 2mg/kg/day of BDE-99 from gestation day 6 to postnatal day 21. When the pups were weaning, the liver from 1 pup of each litter was excised to evaluate oxidative stress markers and the messenger RNA (mRNA) expression of multiple cytochrome P450 (CYP) isoforms. To determine whether thyroid hormone (TH) was disrupted, the protein and mRNA expressions of several TH receptor (TR) isoforms, as well as the protein levels of cyclin D1 and the phosphorylated protein kinases Akt and glycogen synthase kinase 3 beta (GSK3β), were evaluated. Perinatal exposure to BDE-99 produced decreased levels of cyclin D1 in rat pup livers. A decrease in the active form of Akt and an increase in the active form of GSK3β were observed. The decreased Akt pathway may be due to a potential disruption of the nongenomic actions of TH by BDE-99 and its metabolites. This possible TH disruption was noted as a decrease in TR isoforms expression. By contrast, we observed an upregulation of CYP2B1 gene expression, which is correlated with an increase in reactive oxygen species production. This outcome indicates activation of the nuclear constitutive androstane receptor, which could induce the expression of other enzymes capable of metabolizing TH. The present findings support the hypothesis that perinatal exposure to PBDEs, at levels found in humans, may have serious implications for metabolic processes in rat pup livers.

The Canindé breed of goats (Capra hircus) is currently endangered. The aims of this study were to characterize the estrus behavior, ovulatory responses and progesterone profiles, and to evaluate the in vitro embryo production (IVP) in this breed. In Experiment 1, ten nulliparous and seven pluriparous females received medroxyprogesterone acetate (MAP)-containing sponges (60mg) plus 75μg d-cloprostenol for estrus synchronization and their reproductive parameters were evaluated. In Experiment 2, oocytes obtained by laparascopy from hormonally stimulated females (n=15) were used for IVP. There was no difference (p>0.05) between nulliparous and pluriparous goats in terms of estrus response (40.0% vs. 85.7%), time from progestagen sponge removal to the onset of estrus (62.0±15.5 vs. 50.7±19.2h; mean±SEM), duration of estrus (25.0±16.1 vs. 30.0±15.1h), percentage of ovulating animals (60.0% vs. 85.7%), number of ovulations (1.2±0.4 vs. 1.3±0.8), and diameter of the preovulatory follicle (5.8±0.5 vs. 6.1±0.3mm). Progesterone concentrations were also similar (p>0.05) in both groups. During laparoscopic recovery, there were average 12.2 aspirated follicles and 9.1 oocytes per goat, resulting in a high recovery rate (74.3%, 182/245). A total of 78 embryos were produced (51.0%). The mean number of cells in the blastocysts at day 7 of in vitro culture was 170.3±12.5. In conclusion, nulliparous and pluriparous Canindé goats exhibited similar reproductive profiles. It was possible to produce embryos in vitro, allowing the instigation of an embryo bank for preservation of this breed., (Copyright © 2013 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.)

In previous work, we presented theory of how atmospheric turbulence can impart orbital angular momentum to propagating optical waves. In this paper we provide the first experimental demonstration of the detection of orbital angular momentum from distributed volume turbulence through the identification of well-defined, turbulence-induced, optical vortex trails in Shack-Hartmann wave front sensor measurements.

Memory systems theory argues for separate neural systems supporting implicit and explicit memory in the human brain. Neuropsychological studies support this dissociation, but empirical studies of cognitively healthy participants generally observe that both kinds of memory are acquired to at least some extent, even in implicit learning tasks. A key question is whether this observation reflects parallel intact memory systems or an integrated representation of memory in healthy participants. Learning of complex tasks in which both explicit instruction and practice is used depends on both kinds of memory, and how these systems interact will be an important component of the learning process. Theories that posit an integrated, or single, memory system for both types of memory predict that explicit instruction should contribute directly to strengthening task knowledge. In contrast, if the two types of memory are independent and acquired in parallel, explicit knowledge should have no direct impact and may serve in a "scaffolding" role in complex learning. Using an implicit perceptual-motor sequence learning task, the effect of explicit pre-training instruction on skill learning and performance was assessed. Explicit pre-training instruction led to robust explicit knowledge, but sequence learning did not benefit from the contribution of pre-training sequence memorization. The lack of an instruction benefit suggests that during skill learning, implicit and explicit memory operate independently. While healthy participants will generally accrue parallel implicit and explicit knowledge in complex tasks, these types of information appear to be separately represented in the human brain consistent with multiple memory systems theory., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Trisomy 21 is associated with hematopoietic abnormalities in the fetal liver, a preleukemic condition termed transient myeloproliferative disorder, and increased incidence of acute megakaryoblastic leukemia. Human trisomy 21 pluripotent cells of various origins, human embryonic stem (hES), and induced pluripotent stem (iPS) cells, were differentiated in vitro as a model to recapitulate the effects of trisomy on hematopoiesis. To mitigate clonal variation, we isolated disomic and trisomic subclones from the same parental iPS line, thereby generating subclones isogenic except for chromosome 21. Under differentiation conditions favoring development of fetal liver-like, γ-globin expressing, definitive hematopoiesis, we found that trisomic cells of hES, iPS, or isogenic origins exhibited a two- to fivefold increase in a population of CD43(+)(Leukosialin)/CD235(+)(Glycophorin A) hematopoietic cells, accompanied by increased multilineage colony-forming potential in colony-forming assays. These findings establish an intrinsic disturbance of multilineage myeloid hematopoiesis in trisomy 21 at the fetal liver stage.

Previous work by Allen, demonstrated that optical beams possess orbital angular momentum. Other work has shown that a random, phase-only disturbance can impart ± 1 orbital angular momentum states to propagating waves. However, the field preceding the formation of these ± 1 states was unknown. In this paper, we identify the unique field that leads to the formation of a pair of branch points, indicators of orbital angular momentum. This field is then verified in a bench-top optical experiment.

The memory system that supports implicit perceptual-motor sequence learning relies on brain regions that operate separately from the explicit, medial temporal lobe memory system. The implicit learning system therefore likely has distinct operating characteristics and information processing constraints. To attempt to identify the limits of the implicit sequence learning mechanism, participants performed the serial interception sequence learning (SISL) task with covertly embedded repeating sequences that were much longer than most previous studies: ranging from 30 to 60 (Experiment 1) and 60 to 90 (Experiment 2) items in length. Robust sequence-specific learning was observed for sequences up to 80 items in length, extending the known capacity of implicit sequence learning. In Experiment 3, 12-item repeating sequences were embedded among increasing amounts of irrelevant nonrepeating sequences (from 20 to 80% of training trials). Despite high levels of irrelevant trials, learning occurred across conditions. A comparison of learning rates across all three experiments found a surprising degree of constancy in the rate of learning regardless of sequence length or embedded noise. Sequence learning appears to be constant with the logarithm of the number of sequence repetitions practiced during training. The consistency in learning rate across experiments and conditions implies that the mechanisms supporting implicit sequence learning are not capacity-constrained by very long sequences nor adversely affected by high rates of irrelevant sequences during training.

Type I and type II interferons (IFNs) are cytokines that establish the cellular antiviral state through the induction of IFN-stimulated genes (ISGs). We sought to understand the basis of the antiviral activity induced by type I and II IFNs in relation to the functions of their ISGs. Based on gene expression studies, we systematically identified antiviral ISGs by performing blinded, functional screens on 288 type I and type II ISGs. We assessed and validated the antiviral activity of these ISGs against an RNA virus, vesicular stomatitis virus (VSV), and a DNA virus, murine gammaherpes virus (MHV-68). Overall, we identified 34 ISGs that elicited an antiviral effect on the replication of either one or both viruses. Fourteen ISGs have uncharacterized antiviral functions. We further defined ISGs that affect critical life-cycle processes in expression of VSV protein and MHV-68 immediate-early genes. Two previously undescribed antiviral ISGs, TAP1 and BMP2, were further validated. TAP1-deficient fibroblasts were more susceptible to VSV infection but less so to MHV-68 infection. On the other hand, exogenous BMP2 inhibits MHV-68 lytic growth but did not affect VSV growth. These results delineate common and distinct sets of type I and type II IFN-induced genes as well as identify unique ISGs that have either broad or specific antiviral effects on these viruses.

Light propagating through atmospheric turbulence acquires spatial and temporal phase variations. For strong enough turbulence conditions, interference from these phase variations within the optical wave can produce branch points; positions of zero amplitude. Under the assumption of a layered turbulence model, our previous work has shown that these branch points can be used to estimate the number and velocities of atmospheric layers. Key to this previous demonstration was the property of branch point persistence. Branch points from a single turbulence layer persist in time and through additional layers. In this paper we extend persistence to include branch point pairs. We develop an algorithm for isolating persistent pairs and show that through experimental data that they exist through time and through additional turbulence.

This is the first in a series of papers demonstrating that photons with orbital angular momentum can be created in optical waves propagating through distributed turbulence. The scope of this first paper is much narrower. Here, we demonstrate that atmospheric turbulence can impart non-trivial angular momentum to beams and that this non-trivial angular momentum is highly localized. Furthermore, creation of this angular momentum is a normal part of propagation through atmospheric turbulence.

This is the second of two papers demonstrating that photons with orbital angular momentum can be created in optical waves propagating through distributed turbulence. In the companion paper, it is shown that propagation through atmospheric turbulence can create non-trivial angular momentum. Here, we extend the result and demonstrate that this momentum is, at least in part, orbital angular momentum. Specifically, we demonstrate that branch points (in the language of the adaptive optic community) indicate the presence of photons with non-zero OAM. Furthermore, the conditions required to create photons with non-zero orbital angular momentum are ubiquitous. The repercussions of this statement are wide ranging and these are cursorily enumerated., (© 2011 Optical Society of America)

The expression of expert motor skills typically involves learning to perform a precisely timed sequence of movements. Research examining incidental sequence learning has relied on a perceptually cued task that gives participants exposure to repeating motor sequences but does not require timing of responses for accuracy. In the 1st experiment, a novel perceptual-motor sequence learning task was used, and learning a precisely timed cued sequence of motor actions was shown to occur without explicit instruction. Participants learned a repeating sequence through practice and showed sequence-specific knowledge via a performance decrement when switched to an unfamiliar sequence. In the 2nd experiment, the integration of representation of action order and timing sequence knowledge was examined. When either action order or timing sequence information was selectively disrupted, performance was reduced to levels similar to completely novel sequences. Unlike prior sequence-learning research that has found timing information to be secondary to learning action sequences, when the task demands require accurate action and timing information, an integrated representation of these types of information is acquired. These results provide the first evidence for incidental learning of fully integrated action and timing sequence information in the absence of an independent representation of action order and suggest that this integrative mechanism may play a material role in the acquisition of complex motor skills.

Type I interferons (IFNs) are cytokines of the innate immune system that induce antiviral protein expression in response to viral infection. Various proteins and pathways have been shown to recognize nucleic acid ligands especially from RNA viruses. Here, we will review recent developments including transcription of DNA virus genomes into RNA ligands, and the recognition of viruses by TLR2 for interferon induction. The induced IFNs activate many interferon stimulated genes (ISGs) that have direct antiviral effects. Recent studies have identified IFITM proteins as the first ISG to inhibit viral entry processes and revealed mechanistic understanding of known antiviral ISGs such as ISG15 and Viperin., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Memory-impaired patients express intact implicit perceptual-motor sequence learning, but it has been difficult to obtain a similarly clear dissociation in healthy participants. When explicit memory is intact, participants acquire some explicit knowledge and performance improvements from implicit learning may be subtle. Therefore, it is difficult to determine whether performance exceeds what could be expected on the basis of the concomitant explicit knowledge. Using a challenging new sequence-learning task, robust implicit learning was found in healthy participants with virtually no associated explicit knowledge. Participants trained on a repeating sequence that was selected randomly from a set of five. On a performance test of all five sequences, performance was best on the trained sequence, and two-thirds of the participants exhibited individually reliable improvement (by chi-square analysis). Participants could not reliably indicate which sequence had been trained by either recognition or recall. Only by expressing their knowledge via performance were participants able to indicate which sequence they had learned.

Optical waves propagating through atmospheric turbulence develop spatial and temporal variations in their phase. For sufficiently strong turbulence, these phase differences can lead to interference in the propagating wave and the formation of branch points; positions of zero amplitude. Under the assumption of a layered turbulence model, we show that these branch points can be used to estimate the number and velocities of atmospheric layers. We describe how to carry out this estimation process and demonstrate its robustness in the presence of sensor noise.

A conserved herpesviral kinase, designated ORF36 in murine gamma-herpesvirus 68 (MHV-68), plays multiple vital roles in the viral life cycle. Here, we show by screening mutant viruses that ORF36 counteracts the antiviral type I interferon (IFN) response. ORF36 specifically binds to the activated form of interferon regulatory factor 3 (IRF-3) in the nucleus, inhibiting IRF-3 interaction with the cotranscriptional activator CBP and thereby suppressing the recruitment of RNA polymerase II to the interferon beta promoter. The anti-IFN function of ORF36 is conserved among herpesvirus subfamilies, although the conserved kinase activity is not absolutely required for this function. MHV-68 lacking ORF36 induces a greater interferon response and is attenuated in vitro and in vivo, where acute viral infection in the lung and latency in the spleen are compromised. Our data suggest that herpesviruses have evolved within their conserved kinase an anti-IFN activity critical for evasion of host immunity and for persistence.

Objective: To test the hypothesis that fluoride prophylactic agents do not affect the fracture strength and fracture morphology of the tie-wing complex of ceramic brackets., Materials and Methods: The fracture strength of the distal incisal tie-wing of two polycrystalline brackets, Clarity and Mystique, and a monocrystalline bracket, Inspire, was measured after the brackets were exposed to either Prevident, Phos-flur gel, or distilled water (control). Scanning electron microscopy was used to qualitatively evaluate the tie-wing intact and fractured surfaces., Results: A two-way analysis of variance and Fisher-Hayter post hoc test, alpha = .05, indicated a significant decrease in tie-wing fracture strength following both fluoride treatments when compared with the distilled water control only with the monocrystalline bracket. None of the bracket brands exhibited any qualitative differences in the tie-wing intact or fracture surfaces as a function of fluoride treatment., Conclusions: The hypothesis is rejected. Based on the results, using topical fluoride agents with monocrystalline brackets might be contraindicated because of increased tie-wing fracture susceptibility.

Innate immune responses against viral infection, especially the induction of type I interferon, are critical for limiting the replication of the virus. Although it has been shown that DNA can induce type I interferon, to date no natural DNA ligand of a virus that induces type I interferon has been described. Here we screened the genome of murine gammaherpesvirus 68 with mutations at various genomic locations to map the region of DNA that induces type I interferon. A repetitive region termed the 100-base-pair repeat region is a ligand that is both necessary and sufficient for the viral genomic DNA to induce type I interferon. A region colinear with this ligand in the genome of Kaposi's sarcoma-associated herpesvirus also induces type I interferon. We have thus defined a repetitive region of the genomes of gammaherpesviruses as the first natural DNA virus ligand that induces type I interferon.

Langerhans cells (LC) are a unique subset of dendritic cells (DC), present in the epidermis and serving as the first line of defense against pathogens invading the skin. To investigate the role of human LCs in innate immune responses, we examined TLR expression and function of LC-like DCs derived from CD34+ progenitor cells and compared them to DCs derived from peripheral blood monocytes (monocyte-derived DC; Mo-DC). LC-like DCs and Mo-DCs expressed TLR1-10 mRNAs at comparable levels. Although many of the TLR-induced cytokine patterns were similar between the two cell types, stimulation with the TLR3 agonist poly(I:C) triggered significantly higher amounts of the IFN-inducible chemokines CXCL9 (monokine induced by IFN-gamma) and CXCL11 (IFN-gamma-inducible T cell alpha chemoattractant) in LC-like DCs as compared with Mo-DCs. Supernatants from TLR3-activated LC-like DCs reduced intracellular replication of vesicular stomatitis virus in a type I IFN-dependent manner. Finally, CXCL9 colocalized with LCs in skin biopsy specimens from viral infections. Together, our data suggest that LCs exhibit a direct antiviral activity that is dependent on type I IFN as part of the innate immune system.