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References

Screenshots from:

Qiao, Y., Yang, J., Liu, L., Zeng, Y., Ma, J., Jia, J., … Wang, Y. (2018). Successful treatment with pazopanib plus PD-1 inhibitor and RAK cells for advanced primary hepatic angiosarcoma: a case report. BMC Cancer, 18, 212. http://doi.org/10.1186/s12885-018-3996-3

Stabell, A. C., Meyerson, N. R., Gullberg, R. C., Gilchrist, A. R., Webb, K. J., Old, W. M., … Sawyer, S. L. (2018). Dengue viruses cleave STING in humans but not in nonhuman primates, their presumed natural reservoir. eLife, 7, e31919. http://doi.org/10.7554/eLife.31919

Both articles made available under a CC-BY license.

On March 18, 2019, PMC will no longer provide bulk packages of Open Access (OA) Subset text and XML at the top level directory of the FTP Service. These files were superseded in August 2016 by the Commercial Use and Non-Commercial Use bulk packages located in the oa_bulk subdirectory. One set comprises articles that may be used for commercial purposes (the Commercial Use Collection); the other contains articles that can be used only for non-commercial purposes. Anyone planning to use OA subset content for non-commercial purposes will need to download both “non_comm_use.*.tar.gz” and “comm_use.*.tar.gz” to access the complete collection. See the Open Access Subset page for additional details. Questions should be directed to pubmedcentral@ncbi.nlm.nih.gov.

Title: AHA News: Estrogen Therapy in Early Menopause May Help Keep Arteries Clear
Category: Health News
Created: 3/3/2020 12:00:00 AM
Last Editorial Review: 3/4/2020 12:00:00 AM

The number of citations an article receives is an important indicator to quantify its influence in its field. The aim of this study was to identify and analyze the characteristics of the 50 top-cited articles addressing dental education published in two journals dedicated to dental education (European Journal of Dental Education and Journal of Dental Education). The Web of Science database was searched to retrieve the 50 most-cited articles from the two journals in December 2018. The top-cited articles were analyzed for journal of publication, number of citations, institution and country of origin, year of publication, study type, keywords, theme and subtheme, and international collaborations. The results showed the 50 top-cited articles were cited between 24 and 146 times each. The majority of these top-cited articles (n=34) were published in the Journal of Dental Education. Half (n=25) of the articles were by authors in the U.S. The most common study types were surveys (n=26) and reviews (n=10). The main themes of these top-cited articles were curriculum and learner characteristics. This bibliometric analysis can serve as a reference for recognizing studies with the most impact in the scholarship of dental education.

Purpose: The purpose of this study was to determine strength of muscles involved with instrumentation (scaling) by dental hygienists and the additive effects of cellular (mobile) phone usage, as indicated by measurements of muscular force generation.Methods: A convenience sample of licensed dental hygienists currently in clinical practice (n=16) and an equal number of individuals not currently using devices/tools repetitively for work (n=16), agreed to participate in this pilot study. All participants completed a modified cell phone usage questionnaire to determine their use pattern and frequency. Upon completion of the questionnaire, participants' force production in six muscle groups was measured using a hand-held dynamometer. Descriptive statistics were used to analyze the data.Results: A total of 16 licensed dental hygienists (n=16) and 16 participants with no history of using tools/devices repetitively for work (n=16), comprised the experimental and control groups, repectively. The control group generated greater muscle force than the experimental group for the abductor pollicis longus (p=0.045). Significant differences were identified when comparing the low mobile phone users in the experimental group to the control group for the flexor pollicis brevis (p=0.031), abductor pollicis longus (p=0.031), and flexor digitorum (p=0.006), with the control group demonstrating higher muscle force. Years in clinical practice and mobile phone use was shown to have a significant effect on muscular force generation for the flexor pollicis brevis (F=3.645, df=3, p=0.020) and flexor digitorum (F=3.560, df=3, p=0.022); subjects who practiced dental hygiene the longest produced the least amount of muscle force.Conclusion: Results from this pilot study indicate there are no significant additive effects of cell phone use and dental hygiene practice on finger muscles used for instrumentation. However, results indicate that dental hygiene practice demonstrated significant effects on muscular strength as compared to individuals who do not use tools/devices repetitively for work. The small sample size may have impacted results and the study should be repeated with a larger sample.

Membrane-bound proteins have been proposed to mediate the transport of long-chain FA (LCFA) transport through the plasma membrane (PM). These proposals are based largely on reports that PM transport of LCFAs can be blocked by a number of enzymes and purported inhibitors of LCFA transport. Here, using the ratiometric pH indicator (2',7'-bis-(2-carboxyethyl)-5-(and-6-)-carboxyfluorescein and acrylodated intestinal FA-binding protein-based dual fluorescence assays, we investigated the effects of nine inhibitors of the putative FA transporter protein CD36 on the binding and transmembrane movement of LCFAs. We particularly focused on sulfosuccinimidyl oleate (SSO), reported to be a competitive inhibitor of CD36-mediated LCFA transport. Using these assays in adipocytes and inhibitor-treated protein-free lipid vesicles, we demonstrate that rapid LCFA transport across model and biological membranes remains unchanged in the presence of these purported inhibitors. We have previously shown in live cells that CD36 does not accelerate the transport of unesterified LCFAs across the PM. Our present experiments indicated disruption of LCFA metabolism inside the cell within minutes upon treatment with many of the "inhibitors" previously assumed to inhibit LCFA transport across the PM. Furthermore, using confocal microscopy and a specific anti-SSO antibody, we found that numerous intracellular and PM-bound proteins are SSO-modified in addition to CD36. Our results support the hypothesis that LCFAs diffuse rapidly across biological membranes and do not require an active protein transporter for their transmembrane movement.

Geranylgeranoic acid (GGA) originally was identified in some animals and has been developed as an agent for preventing second primary hepatoma. We previously have also identified GGA as an acyclic diterpenoid in some medicinal herbs. Recently, we reported that in human hepatoma-derived HuH-7 cells, GGA is metabolically labeled from ¹³C-mevalonate. Several cell-free experiments have demonstrated that GGA is synthesized through geranylgeranial by oxygen-dependent oxidation of geranylgeraniol (GGOH), but the exact biochemical events giving rise to GGA in hepatoma cells remain unclear. Monoamine oxidase B (MOAB) has been suggested to be involved in GGOH oxidation. Here, using two human hepatoma cell lines, we investigated whether MAOB contributes to GGA biosynthesis. Using either HuH-7 cell lysates or recombinant human MAOB, we found that: 1) the MAO inhibitor tranylcypromine dose-dependently downregulates endogenous GGA levels in HuH-7 cells; and 2) siRNA-mediated MAOB silencing reduces intracellular GGA levels in HuH-7 and Hep3B cells. Unexpectedly, however, CRISPR/Cas9-generated MAOB-KO human hepatoma Hep3B cells had GGA levels similar to those in MAOB-WT cells. A sensitivity of GGA levels to siRNA-mediated MAOB downregulation was recovered when the MAOB-KO cells were transfected with a MAOB-expression plasmid, suggesting that MAOB is the enzyme primarily responsible for GGOH oxidation and that some other latent metabolic pathways may maintain endogenous GGA levels in the MAOB-KO hepatoma cells. Along with the previous findings, these results provide critical insights into the biological roles of human MAOB and provide evidence that hepatic MAOB is involved in endogenous GGA biosynthesis via GGOH oxidation.

Many clinical studies and epidemiological investigations have clearly demonstrated that women are twice as likely to develop cholesterol gallstones as men, and oral contraceptives and other estrogen therapies dramatically increase that risk. Further, animal studies have revealed that estrogen promotes cholesterol gallstone formation through the estrogen receptor (ER) α, but not ERβ, pathway. More importantly, some genetic and pathophysiological studies have found that G protein-coupled estrogen receptor (GPER) 1 is a new gallstone gene, Lith18, on chromosome 5 in mice and produces additional lithogenic actions, working independently of ERα, to markedly increase cholelithogenesis in female mice. Based on computational modeling of GPER, a novel series of GPER-selective antagonists were designed, synthesized, and subsequently assessed for their therapeutic effects via calcium mobilization, cAMP, and ERα and ERβ fluorescence polarization binding assays. From this series of compounds, one new compound, 2-cyclohexyl-4-isopropyl-N-(4-methoxybenzyl)aniline (CIMBA), exhibits superior antagonism and selectivity exclusively for GPER. Furthermore, CIMBA reduces the formation of 17β-estradiol-induced gallstones in a dose-dependent manner in ovariectomized mice fed a lithogenic diet for 8 weeks. At 32 μg/day/kg CIMBA, no gallstones are found, even in ovariectomized ERα (^–/–) mice treated with 6 μg/day 17β-estradiol and fed the lithogenic diet for 8 weeks. In conclusion, CIMBA treatment protects against the formation of estrogen-induced cholesterol gallstones by inhibiting the GPER signaling pathway in female mice. CIMBA may thus be a new agent for effectively treating cholesterol gallstone disease in women.­

The formation and properties of liquid-ordered (Lo) lipid domains (rafts) in the plasma membrane are still poorly understood. This limits our ability to manipulate ordered lipid domain-dependent biological functions. Giant plasma membrane vesicles (GPMVs) undergo large-scale phase separations into coexisting Lo and liquid-disordered lipid domains. However, large-scale phase separation in GPMVs detected by light microscopy is observed only at low temperatures. Comparing Förster resonance energy transfer-detected versus light microscopy-detected domain formation, we found that nanodomains, domains of nanometer size, persist at temperatures up to 20°C higher than large-scale phases, up to physiologic temperature. The persistence of nanodomains at higher temperatures is consistent with previously reported theoretical calculations. To investigate the sensitivity of nanodomains to lipid composition, GPMVs were prepared from mammalian cells in which sterol, phospholipid, or sphingolipid composition in the plasma membrane outer leaflet had been altered by cyclodextrin-catalyzed lipid exchange. Lipid substitutions that stabilize or destabilize ordered domain formation in artificial lipid vesicles had a similar effect on the thermal stability of nanodomains and large-scale phase separation in GPMVs, with nanodomains persisting at higher temperatures than large-scale phases for a wide range of lipid compositions. This indicates that it is likely that plasma membrane nanodomains can form under physiologic conditions more readily than large-scale phase separation. We also conclude that membrane lipid substitutions carried out in intact cells are able to modulate the propensity of plasma membranes to form ordered domains. This implies lipid substitutions can be used to alter biological processes dependent upon ordered domains.

The autosomal dominant disorder Schnyder corneal dystrophy (SCD) is caused by mutations in UbiA prenyltransferase domain-containing protein-1 (UBIAD1), which uses geranylgeranyl pyrophosphate (GGpp) to synthesize the vitamin K₂ subtype menaquinone-4 (MK-4). SCD is characterized by opacification of the cornea, owing to aberrant build-up of cholesterol in the tissue. We previously discovered that sterols stimulate association of UBIAD1 with ER-localized HMG-CoA reductase, which catalyzes a rate-limiting step in the synthesis of cholesterol and nonsterol isoprenoids, including GGpp. Binding to UBIAD1 inhibits sterol-accelerated ER-associated degradation (ERAD) of reductase and permits continued synthesis of GGpp in cholesterol-replete cells. GGpp disrupts UBIAD1-reductase binding and thereby allows for maximal ERAD of reductase as well as ER-to-Golgi translocation of UBIAD1. SCD-associated UBIAD1 is refractory to GGpp-mediated dissociation from reductase and remains sequestered in the ER to inhibit ERAD. Here, we report development of a biochemical assay for UBIAD1-mediated synthesis of MK-4 in isolated membranes and intact cells. Using this assay, we compared enzymatic activity of WT UBIAD1 with that of SCD-associated variants. Our studies revealed that SCD-associated UBIAD1 exhibited reduced MK-4 synthetic activity, which may result from its reduced affinity for GGpp. Sequestration in the ER protects SCD-associated UBIAD1 from autophagy and allows intracellular accumulation of the mutant protein, which amplifies the inhibitory effect on reductase ERAD. These findings have important implications not only for the understanding of SCD etiology but also for the efficacy of cholesterol-lowering statin therapy, which becomes limited, in part, because of UBIAD1-mediated inhibition of reductase ERAD.

Adipocytes take up long chain FAs through diffusion and protein-mediated transport, whereas FA efflux is considered to occur by diffusion. To identify potential membrane proteins that are involved in regulating FA flux in adipocytes, the expression levels of 55 membrane transporters without known function were screened in subcutaneous adipose samples from obese patients before and after bariatric surgery using branched DNA methodology. Among the 33 solute carrier (SLC) transporter family members screened, the expression of 14 members showed significant changes before and after bariatric surgery. One of them, Slc43a3, increased about 2.5-fold after bariatric surgery. Further investigation demonstrated that Slc43a3 is highly expressed in murine adipose tissue and induced during adipocyte differentiation in primary preadipocytes and in OP9 cells. Knockdown of Slc43a3 with siRNA in differentiated OP9 adipocytes reduced both basal and forskolin-stimulated FA efflux, while also increasing FA uptake and lipid droplet accumulation. In contrast, overexpression of Slc43a3 decreased FA uptake in differentiated OP9 cells and resulted in decreased lipid droplet accumulation. Therefore, Slc43a3 seems to regulate FA flux in adipocytes, functioning as a positive regulator of FA efflux and as a negative regulator of FA uptake.

Acne is one of the most common dermatological conditions, but the details of its pathology are unclear, and current management regimens often have adverse effects. Cutibacterium acnes is known as a major acne-associated bacterium that derives energy from lipase-mediated sebum lipid degradation. C. acnes is commensal, but lipase activity has been observed to differ among C. acnes types. For example, higher populations of the type IA strains are present in acne lesions with higher lipase activity. In the present study, we examined a conserved lipase in types IB and II that was truncated in type IA C. acnes strains. Closed, blocked, and open structures of C. acnes ATCC11828 lipases were elucidated by X-ray crystallography at 1.6–2.4 Å. The closed crystal structure, which is the most common form in aqueous solution, revealed that a hydrophobic lid domain shields the active site. By comparing closed, blocked, and open structures, we found that the lid domain-opening mechanisms of C. acnes lipases (_CAlipases) involve the lid-opening residues, Phe-179 and Phe-211. To the best of our knowledge, this is the first structure-function study of _CAlipases, which may help to shed light on the mechanisms involved in acne development and may aid in future drug design.

Fatty liver involves ectopic lipid accumulation and dysregulated hepatic oxidative metabolism, which can progress to a state of elevated inflammation and fibrosis referred to as nonalcoholic steatohepatitis (NASH). The factors that control progression from simple steatosis to NASH are not fully known. Here, we tested the hypothesis that dietary vitamin E (VitE) supplementation would prevent NASH progression and associated metabolic alterations induced by a Western diet (WD). Hyperphagic melanocortin-4 receptor-deficient (MC4R^–/–) mice were fed chow, chow+VitE, WD, or WD+VitE starting at 8 or 20 weeks of age. All groups exhibited extensive hepatic steatosis by the end of the study (28 weeks of age). WD feeding exacerbated liver disease severity without inducing proportional changes in liver triglycerides. Eight weeks of WD accelerated liver pyruvate cycling, and 20 weeks of WD extensively upregulated liver glucose and oxidative metabolism assessed by ²H/¹³C flux analysis. VitE supplementation failed to reduce the histological features of NASH. Rather, WD+VitE increased the abundance and saturation of liver ceramides and accelerated metabolic flux dysregulation compared with 8 weeks of WD alone. In summary, VitE did not limit NASH pathogenesis in genetically obese mice, but instead increased some indicators of metabolic dysfunction.

The pregnane X receptor (PXR) is a nuclear receptor that can be activated by numerous drugs and xenobiotic chemicals. PXR thereby functions as a xenobiotic sensor to coordinately regulate host responses to xenobiotics by transcriptionally regulating many genes involved in xenobiotic metabolism. We have previously reported that PXR has pro-atherogenic effects in animal models, but how PXR contributes to atherosclerosis development in different tissues or cell types remains elusive. In this study, we generated an LDL receptor-deficient mouse model with myeloid-specific PXR deficiency (PXR^MyeLDLR^–/–) to elucidate the role of macrophage PXR signaling in atherogenesis. The myeloid PXR deficiency did not affect metabolic phenotypes and plasma lipid profiles, but PXR^MyeLDLR^–/– mice had significantly decreased atherosclerosis at both aortic root and brachiocephalic arteries compared with control littermates. Interestingly, the PXR deletion did not affect macrophage adhesion and migration properties, but reduced lipid accumulation and foam cell formation in the macrophages. PXR deficiency also led to decreased expression of the scavenger receptor CD36 and impaired lipid uptake in macrophages of the PXR^MyeLDLR^–/– mice. Further, RNA-Seq analysis indicated that treatment with a prototypical PXR ligand affects the expression of many atherosclerosis-related genes in macrophages in vitro. These findings reveal a pivotal role of myeloid PXR signaling in atherosclerosis development and suggest that PXR may be a potential therapeutic target in atherosclerosis management.

Cellular membranes are not homogenous mixtures of proteins; rather, they are segregated into microdomains on the basis of preferential association between specific lipids and proteins. These microdomains, called lipid rafts, are well known for their role in receptor signaling on the plasma membrane (PM) and are essential to such cellular functions as signal transduction and spatial organization of the PM. A number of disease states, including atherosclerosis and other cardiovascular disorders, may be caused by dysfunctional maintenance of lipid rafts. Lipid rafts do not occur only in the PM but also have been found in intracellular membranes and extracellular vesicles (EVs). Here, we focus on discussing newly discovered functions of lipid rafts and microdomains in intracellular membranes, including lipid and protein trafficking from the ER, Golgi bodies, and endosomes to the PM, and we examine lipid raft involvement in the production and composition of EVs. Because lipid rafts are small and transient, visualization remains challenging. Future work with advanced techniques will continue to expand our knowledge about the roles of lipid rafts in cellular functioning.

ABSTRACT

The cell cycle is a critical component of cellular proliferation, differentiation, and response to stress, yet its role in the regulation of intracellular symbioses is not well understood. To explore host-symbiont cell cycle coordination in a marine symbiosis, we employed a model for coral-dinoflagellate associations: the tropical sea anemone Aiptasia (Exaiptasia pallida) and its native microalgal photosymbionts (Breviolum minutum and Breviolum psygmophilum). Using fluorescent labeling and spatial point-pattern image analyses to characterize cell population distributions in both partners, we developed protocols that are tailored to the three-dimensional cellular landscape of a symbiotic sea anemone tentacle. Introducing cultured symbiont cells to symbiont-free adult hosts increased overall host cell proliferation rates. The acceleration occurred predominantly in the symbiont-containing gastrodermis near clusters of symbionts but was also observed in symbiont-free epidermal tissue layers, indicating that the presence of symbionts contributes to elevated proliferation rates in the entire host during colonization. Symbiont cell cycle progression differed between cultured algae and those residing within hosts; the endosymbiotic state resulted in increased S-phase but decreased G₂/M-phase symbiont populations. These phenotypes and the deceleration of cell cycle progression varied with symbiont identity and host nutritional status. These results demonstrate that host and symbiont cells have substantial and species-specific effects on the proliferation rates of their mutualistic partners. This is the first empirical evidence to support species-specific regulation of the symbiont cell cycle within a single cnidarian-dinoflagellate association; similar regulatory mechanisms likely govern interpartner coordination in other coral-algal symbioses and shape their ecophysiological responses to a changing climate.

IMPORTANCE Biomass regulation is critical to the overall health of cnidarian-dinoflagellate symbioses. Despite the central role of the cell cycle in the growth and proliferation of cnidarian host cells and dinoflagellate symbionts, there are few studies that have examined the potential for host-symbiont coregulation. This study provides evidence for the acceleration of host cell proliferation when in local proximity to clusters of symbionts within cnidarian tentacles. The findings suggest that symbionts augment the cell cycle of not only their enveloping host cells but also neighboring cells in the epidermis and gastrodermis. This provides a possible mechanism for rapid colonization of cnidarian tissues. In addition, the cell cycles of symbionts differed depending on nutritional regime, symbiotic state, and species identity. The responses of cell cycle profiles to these different factors implicate a role for species-specific regulation of symbiont cell cycles within host cnidarian tissues.

ABSTRACT

While there is no effective vaccine against Chlamydia trachomatis infection, previous work has demonstrated the importance of C. trachomatis-specific CD4⁺ T cells (NR1 T cells) in pathogen clearance. Specifically, NR1 T cells have been shown to be protective in mice, and this protection depends on the host’s ability to sense the cytokine gamma interferon (IFN-). However, it is unclear what role NR1 production or sensing of IFN- plays in T cell homing to the genital tract or T cell-mediated protection against C. trachomatis. Using two-photon microscopy and flow cytometry, we found that naive wild-type (WT), IFN-^–/–, and IFN-R^–/– NR1 T cells specifically home to sections in the genital tract that contain C. trachomatis. We also determined that protection against infection requires production of IFN- from either NR1 T cells or endogenous cells, further highlighting the importance of IFN- in clearing C. trachomatis infection.

IMPORTANCE Chlamydia trachomatis is an important mucosal pathogen that is the leading cause of sexually transmitted bacterial infections in the United States. Despite this, there is no vaccine currently available. In order to develop such a vaccine, it is necessary to understand the components of the immune response that can lead to protection against this pathogen. It is well known that antigen-specific CD4⁺ T cells are critical for Chlamydia clearance, but the contexts in which they are protective or not protective are unknown. Here, we aimed to characterize the importance of gamma interferon production and sensing by T cells and the effects on the immune response to C. trachomatis. Our work here helps to define the contexts in which antigen-specific T cells can be protective, which is critical to our ability to design an effective and protective vaccine against C. trachomatis.

ABSTRACT

Human adenoviruses (HAdVs) have developed mechanisms to manipulate cellular antiviral measures to ensure proper DNA replication, with detailed processes far from being understood. Host cells repress incoming viral genomes through a network of transcriptional regulators that normally control cellular homeostasis. The nuclear domains involved are promyelocytic leukemia protein nuclear bodies (PML-NBs), interferon-inducible, dot-like nuclear structures and hot spots of SUMO posttranslational modification (PTM). In HAdV-infected cells, such SUMO factories are found in close proximity to newly established viral replication centers (RCs) marked by the adenoviral DNA binding protein (DBP) E2A. Here, we show that E2A is a novel target of host SUMOylation, leading to PTMs supporting E2A function in promoting productive infection. Our data show that SUMOylated E2A interacts with PML. Decreasing SUMO-E2A protein levels by generating HAdV variants mutated in the three main SUMO conjugation motifs (SCMs) led to lower numbers of viral RCs and PML-NBs, and these two structures were no longer next to each other. Our data further indicate that SUMOylated E2A binds the host transcription factor Sp100A, promoting HAdV gene expression, and represents the molecular bridge between PML tracks and adjacent viral RCs. Consequently, E2A SCM mutations repressed late viral gene expression and progeny production. These data highlight a novel mechanism used by the virus to benefit from host antiviral responses by exploiting the cellular SUMO conjugation machinery.

IMPORTANCE PML nuclear bodies (PML-NBs) are implicated in general antiviral defense based on recruiting host restriction factors; however, it is not understood so far why viruses would establish viral replication centers (RCs) juxtaposed to such "antiviral" compartments. To understand this enigma, we investigate the cross talk between PML-NB components and viral RCs to find the missing link connecting both compartments to promote efficient viral replication and gene expression. Taken together, the current concept is more intricate than originally believed, since viruses apparently take advantage of several specific PML-NB-associated proteins to promote productive infection. Simultaneously, they efficiently inhibit antiviral measures to maintain the viral infectious program. Our data provide evidence that SUMOylation of the viral RC marker protein E2A represents the basis of this virus-host interface and regulates various downstream events to support HAdV productive infection. These results are the basis of our current attempts to generate and screen for specific E2A SUMOylation inhibitors to constitute novel therapeutic approaches to limit and prevent HAdV-mediated diseases and mortality of immunosuppressed patients.

ABSTRACT

The malaria parasite Plasmodium falciparum traffics the virulence protein P. falciparum erythrocyte membrane protein 1 (PfEMP1) to the surface of infected red blood cells (RBCs) via membranous organelles, known as the Maurer’s clefts. We developed a method for efficient enrichment of Maurer’s clefts and profiled the protein composition of this trafficking organelle. We identified 13 previously uncharacterized or poorly characterized Maurer’s cleft proteins. We generated transfectants expressing green fluorescent protein (GFP) fusions of 7 proteins and confirmed their Maurer’s cleft location. Using co-immunoprecipitation and mass spectrometry, we generated an interaction map of proteins at the Maurer’s clefts. We identified two key clusters that may function in the loading and unloading of PfEMP1 into and out of the Maurer’s clefts. We focus on a putative PfEMP1 loading complex that includes the protein GEXP07/CX3CL1-binding protein 2 (CBP2). Disruption of GEXP07 causes Maurer’s cleft fragmentation, aberrant knobs, ablation of PfEMP1 surface expression, and loss of the PfEMP1-mediated adhesion. GEXP07 parasites have a growth advantage compared to wild-type parasites, and the infected RBCs are more deformable and more osmotically fragile.

IMPORTANCE The trafficking of the virulence antigen PfEMP1 and its presentation at the knob structures at the surface of parasite-infected RBCs are central to severe adhesion-related pathologies such as cerebral and placental malaria. This work adds to our understanding of how PfEMP1 is trafficked to the RBC membrane by defining the protein-protein interaction networks that function at the Maurer’s clefts controlling PfEMP1 loading and unloading. We characterize a protein needed for virulence protein trafficking and provide new insights into the mechanisms for host cell remodeling, parasite survival within the host, and virulence.

ABSTRACT

Satellite viruses, most commonly found in plants, rely on helper viruses to complete their replication cycle. The only known example of a human satellite virus is the hepatitis D virus (HDV), and it is generally thought to require hepatitis B virus (HBV) to form infectious particles. Until 2018, HDV was the sole representative of the genus Deltavirus and was thought to have evolved in humans, the only known HDV host. The subsequent identification of HDV-like agents in birds, snakes, fish, amphibians, and invertebrates indicated that the evolutionary history of deltaviruses is likely much longer than previously hypothesized. Interestingly, none of the HDV-like agents were found in coinfection with an HBV-like agent, suggesting that these viruses use different helper virus(es). Here we show, using snake deltavirus (SDeV), that HBV and hepadnaviruses represent only one example of helper viruses for deltaviruses. We cloned the SDeV genome into a mammalian expression plasmid, and by transfection could initiate SDeV replication in cultured snake and mammalian cell lines. By superinfecting persistently SDeV-infected cells with reptarenaviruses and hartmaniviruses, or by transfecting their surface proteins, we could induce production of infectious SDeV particles. Our findings indicate that deltaviruses can likely use a multitude of helper viruses or even viral glycoproteins to form infectious particles. This suggests that persistent infections, such as those caused by arenaviruses and orthohantaviruses used in this study, and recurrent infections would be beneficial for the spread of deltaviruses. It seems plausible that further human or animal disease associations with deltavirus infections will be identified in the future.

IMPORTANCE Deltaviruses need a coinfecting enveloped virus to produce infectious particles necessary for transmission to a new host. Hepatitis D virus (HDV), the only known deltavirus until 2018, has been found only in humans, and its coinfection with hepatitis B virus (HBV) is linked with fulminant hepatitis. The recent discovery of deltaviruses without a coinfecting HBV-like agent in several different taxa suggested that deltaviruses could employ coinfection by other enveloped viruses to complete their life cycle. In this report, we show that snake deltavirus (SDeV) efficiently utilizes coinfecting reptarena- and hartmaniviruses to form infectious particles. Furthermore, we demonstrate that cells expressing the envelope proteins of arenaviruses and orthohantaviruses produce infectious SDeV particles. As the envelope proteins are responsible for binding and infecting new host cells, our findings indicate that deltaviruses are likely not restricted in their tissue tropism, implying that they could be linked to animal or human diseases other than hepatitis.

ABSTRACT

Streptococcus pneumoniae (or pneumococcus) is a highly prevalent human pathogen. Toll-like receptors (TLRs) function as immune sensors that can trigger host defenses against this bacterium. Defects in TLR-activated signaling pathways, including deficiency in the adaptor protein myeloid differentiation factor 88 (MyD88), are associated with markedly increased susceptibility to infection. However, the individual MyD88-dependent TLRs predominantly involved in antipneumococcal defenses have not been identified yet. Here we find that triple knockout mice simultaneously lacking TLR7, TLR9, and TLR13, which sense the presence of bacterial DNA (TLR9) and RNA (TLR7 and TLR13) in the phagolysosomes of phagocytic cells, display a phenotype that largely resembles that of MyD88-deficient mice and rapidly succumb to pneumococcal pneumonitis due to defective neutrophil influx into the lung. Accordingly, TLR7/9/13 triple knockout resident alveolar macrophages were largely unable to respond to pneumococci with the production of neutrophil-attracting chemokines and cytokines. Mice with single deficiencies of TLR7, TLR9, or TLR13 showed unaltered ability to control lung infection but were moderately more susceptible to encephalitis, in association with a decreased ability of microglia to mount cytokine responses in vitro. Our data point to a dominant, tissue-specific role of nucleic acid-sensing pathways in innate immune recognition of S. pneumoniae and also show that endosomal TLRs are largely capable of compensating for the absence of each other, which seems crucial to prevent pneumococci from escaping immune recognition. These results may be useful to develop novel strategies to treat infections by antibiotic-resistant pneumococci based on stimulation of the innate immune system.

IMPORTANCE The pneumococcus is a bacterium that frequently causes infections in the lungs, ears, sinus cavities, and meninges. During these infections, body defenses are triggered by tissue-resident cells that use specialized receptors, such as Toll-like receptors (TLRs), to sense the presence of bacteria. We show here that pneumococci are predominantly detected by TLRs that are located inside intracellular vacuoles, including endosomes, where these receptors can sense the presence of nucleic acids released from ingested bacteria. Mice that simultaneously lacked three of these receptors (specifically, TLR7, TLR9, and TLR13) were extremely susceptible to lung infection and rapidly died after inhalation of pneumococci. Moreover, tissue-resident macrophages from these mice were impaired in their ability to respond to the presence of pneumococci by producing inflammatory mediators capable of recruiting polymorphonuclear leucocytes to infection sites. This information may be useful to develop drugs to treat pneumococcal infections, particularly those caused by antibiotic-resistant strains.

ABSTRACT

Legionella pneumophila governs its interactions with host cells by secreting >300 different "effector" proteins. Some of these effectors contain eukaryotic domains such as the RCC1 (regulator of chromosome condensation 1) repeats promoting the activation of the small GTPase Ran. In this report, we reveal a conserved pattern of L. pneumophila RCC1 repeat genes, which are distributed in two main clusters of strains. Accordingly, strain Philadelphia-1 contains two RCC1 genes implicated in bacterial virulence, legG1 (Legionella eukaryotic gene 1), and ppgA, while strain Paris contains only one, pieG. The RCC1 repeat effectors localize to different cellular compartments and bind distinct components of the Ran GTPase cycle, including Ran modulators and the small GTPase itself, and yet they all promote the activation of Ran. The pieG gene spans the corresponding open reading frames of legG1 and a separate adjacent upstream gene, lpg1975. legG1 and lpg1975 are fused upon addition of a single nucleotide to encode a protein that adopts the binding specificity of PieG. Thus, a point mutation in pieG splits the gene, altering the effector target. These results indicate that divergent evolution of RCC1 repeat effectors defines the Ran GTPase cycle targets and that modulation of different components of the cycle might fine-tune Ran activation during Legionella infection.

IMPORTANCE Legionella pneumophila is a ubiquitous environmental bacterium which, upon inhalation, causes a life-threatening pneumonia termed Legionnaires’ disease. The opportunistic pathogen grows in amoebae and macrophages by employing a "type IV" secretion system, which secretes more than 300 different "effector" proteins into the host cell, where they subvert pivotal processes. The function of many of these effector proteins is unknown, and their evolution has not been studied. L. pneumophila RCC1 repeat effectors target the small GTPase Ran, a molecular switch implicated in different cellular processes such as nucleocytoplasmic transport and microtubule cytoskeleton dynamics. We provide evidence that one or more RCC1 repeat genes are distributed in two main clusters of L. pneumophila strains and have divergently evolved to target different components of the Ran GTPase activation cycle at different subcellular sites. Thus, L. pneumophila employs a sophisticated strategy to subvert host cell Ran GTPase during infection.

ABSTRACT

Intracellular bacterial pathogens remodel cellular functions during their infectious cycle via the coordinated actions of effector molecules delivered through dedicated secretion systems. While the function of many individual effectors is known, how they interact to promote pathogenesis is rarely understood. The zoonotic bacterium Brucella abortus, the causative agent of brucellosis, delivers effector proteins via its VirB type IV secretion system (T4SS) which mediate biogenesis of the endoplasmic reticulum (ER)-derived replicative Brucella-containing vacuole (rBCV). Here, we show that T4SS effectors BspB and RicA display epistatic interactions in Brucella replication. Defects in rBCV biogenesis and Brucella replication caused by deletion of bspB were dependent on the host GTPase Rab2a and suppressed by the deletion of ricA, indicating a role of Rab2-binding effector RicA in these phenotypic defects. Rab2a requirements for rBCV biogenesis and Brucella intracellular replication were abolished upon deletion of both bspB and ricA, demonstrating that the functional interaction of these effectors engages Rab2-dependent transport in the Brucella intracellular cycle. Expression of RicA impaired host secretion and caused Golgi fragmentation. While BspB-mediated changes in ER-to-Golgi transport were independent of RicA and Rab2a, BspB-driven alterations in Golgi vesicular traffic also involved RicA and Rab2a, defining BspB and RicA’s functional interplay at the Golgi interface. Altogether, these findings support a model where RicA modulation of Rab2a functions impairs Brucella replication but is compensated by BspB-mediated remodeling of Golgi apparatus-associated vesicular transport, revealing an epistatic interaction between these T4SS effectors.

IMPORTANCE Bacterial pathogens with an intracellular lifestyle modulate many host cellular processes to promote their infectious cycle. They do so by delivering effector proteins into host cells via dedicated secretion systems that target specific host functions. While the roles of many individual effectors are known, how their modes of action are coordinated is rarely understood. Here, we show that the zoonotic bacterium Brucella abortus delivers the BspB effector that mitigates the negative effect on bacterial replication that the RicA effector exerts via modulation of the host small GTPase Rab2. These findings provide an example of functional integration between bacterial effectors that promotes proliferation of pathogens.

ABSTRACT

Packaging of genomic RNA (gRNA) by retroviruses is essential for infectivity, yet the subcellular site of the initial interaction between the Gag polyprotein and gRNA remains poorly defined. Because retroviral particles are released from the plasma membrane, it was previously thought that Gag proteins initially bound to gRNA in the cytoplasm or at the plasma membrane. However, the Gag protein of the avian retrovirus Rous sarcoma virus (RSV) undergoes active nuclear trafficking, which is required for efficient gRNA encapsidation (L. Z. Scheifele, R. A. Garbitt, J. D. Rhoads, and L. J. Parent, Proc Natl Acad Sci U S A 99:3944–3949, 2002, https://doi.org/10.1073/pnas.062652199; R. Garbitt-Hirst, S. P. Kenney, and L. J. Parent, J Virol 83:6790–6797, 2009, https://doi.org/10.1128/JVI.00101-09). These results raise the intriguing possibility that the primary contact between Gag and gRNA might occur in the nucleus. To examine this possibility, we created a RSV proviral construct that includes 24 tandem repeats of MS2 RNA stem-loops, making it possible to track RSV viral RNA (vRNA) in live cells in which a fluorophore-conjugated MS2 coat protein is coexpressed. Using confocal microscopy, we observed that both wild-type Gag and a nuclear export mutant (Gag.L219A) colocalized with vRNA in the nucleus. In live-cell time-lapse images, the wild-type Gag protein trafficked together with vRNA as a single ribonucleoprotein (RNP) complex in the nucleoplasm near the nuclear periphery, appearing to traverse the nuclear envelope into the cytoplasm. Furthermore, biophysical imaging methods suggest that Gag and the unspliced vRNA physically interact in the nucleus. Taken together, these data suggest that RSV Gag binds unspliced vRNA to export it from the nucleus, possibly for packaging into virions as the viral genome.

IMPORTANCE Retroviruses cause severe diseases in animals and humans, including cancer and acquired immunodeficiency syndromes. To propagate infection, retroviruses assemble new virus particles that contain viral proteins and unspliced vRNA to use as gRNA. Despite the critical requirement for gRNA packaging, the molecular mechanisms governing the identification and selection of gRNA by the Gag protein remain poorly understood. In this report, we demonstrate that the Rous sarcoma virus (RSV) Gag protein colocalizes with unspliced vRNA in the nucleus in the interchromatin space. Using live-cell confocal imaging, RSV Gag and unspliced vRNA were observed to move together from inside the nucleus across the nuclear envelope, suggesting that the Gag-gRNA complex initially forms in the nucleus and undergoes nuclear export into the cytoplasm as a viral ribonucleoprotein (vRNP) complex.

ABSTRACT

The Escherichia coli microcin C (McC) and related compounds are potent Trojan horse peptide-nucleotide antibiotics. The peptide part facilitates transport into sensitive cells. Inside the cell, the peptide part is degraded by nonspecific peptidases releasing an aspartamide-adenylate containing a phosphoramide bond. This nonhydrolyzable compound inhibits aspartyl-tRNA synthetase. In addition to the efficient export of McC outside the producing cells, special mechanisms have evolved to avoid self-toxicity caused by the degradation of the peptide part inside the producers. Here, we report that histidine-triad (HIT) hydrolases encoded in biosynthetic clusters of some McC homologs or by standalone genes confer resistance to McC-like compounds by hydrolyzing the phosphoramide bond in toxic aspartamide-adenosine, rendering them inactive.

IMPORTANCE Uncovering the mechanisms of resistance is a required step for countering the looming antibiotic resistance crisis. In this communication, we show how universally conserved histidine-triad hydrolases provide resistance to microcin C, a potent inhibitor of bacterial protein synthesis.

ABSTRACT

Polymorphonuclear granulocytes (PMNs) are indispensable for controlling life-threatening fungal infections. In addition to various effector mechanisms, PMNs also produce extracellular vesicles (EVs). Their contribution to antifungal defense has remained unexplored. We reveal that the clinically important human-pathogenic fungus Aspergillus fumigatus triggers PMNs to release a distinct set of antifungal EVs (afEVs). Proteome analyses indicated that afEVs are enriched in antimicrobial proteins. The cargo and the release kinetics of EVs are modulated by the fungal strain confronted. Tracking of afEVs indicated that they associated with fungal cells and even entered fungal hyphae, resulting in alterations in the morphology of the fungal cell wall and dose-dependent antifungal effects. To assess as a proof of concept whether the antimicrobial proteins found in afEVs might contribute to growth inhibition of hyphae when present in the fungal cytoplasm, two human proteins enriched in afEVs, cathepsin G and azurocidin, were heterologously expressed in fungal hyphae. This led to reduced fungal growth relative to that of a control strain producing the human retinol binding protein 7. In conclusion, extracellular vesicles produced by neutrophils in response to A. fumigatus infection are able to associate with the fungus, limit growth, and elicit cell damage by delivering antifungal cargo. This finding offers an intriguing, previously overlooked mechanism of antifungal defense against A. fumigatus.

IMPORTANCE Invasive fungal infections caused by the mold Aspergillus fumigatus are a growing concern in the clinic due to the increasing use of immunosuppressive therapies and increasing antifungal drug resistance. These infections result in high rates of mortality, as treatment and diagnostic options remain limited. In healthy individuals, neutrophilic granulocytes are critical for elimination of A. fumigatus from the host; however, the exact extracellular mechanism of neutrophil-mediated antifungal activity remains unresolved. Here, we present a mode of antifungal defense employed by human neutrophils against A. fumigatus not previously described. We found that extracellular vesicles produced by neutrophils in response to A. fumigatus infection are able to associate with the fungus, limit growth, and elicit cell damage by delivering antifungal cargo. In the end, antifungal extracellular vesicle biology provides a significant step forward in our understanding of A. fumigatus host pathogenesis and opens up novel diagnostic and therapeutic possibilities.

Pedestrian fatalities from vehicle impacts in 2019 were the highest in the U.S. in over three decades, a February report finds.

Consider consumption of annonacin-containing plant products, including pawpaw, as a possible environmental risk factor for atypical parkinsonism.

Systemic sclerosis sine scleroderma (ssSSc) is a rare variant of systemic sclerosis, with only one pediatric case reported in the medical literature to date. Pulmonary arterial hypertension as the presenting feature of ssSSc is extremely rare, even in adults, and so far has never been reported in children. We report, for the first time, a case of pediatric ssSSc in a 3-year-old girl, who presented with interstitial lung disease and pulmonary hypertension. The patient was prescribed early aggressive pulmonary vasodilators combined with anti-inflammatory medications. The clinical response was good, and her current condition at 12 years of age is remarkable, considering the high mortality rates reported in adults. We underscore the importance of early aggressive treatment in future cases of similar presentation.

Secondary branch number per panicle plays a crucial role in regulating grain number and yield in rice. Here, we report the positional cloning and functional characterization for SECONDARY BRANCH NUMBER7 (qSBN7), a quantitative trait locus affecting secondary branch per panicle and grain number. Our research revealed that the causative variants of qSBN7 are located in the distal promoter region of FRIZZLE PANICLE (FZP), a gene previously associated with the repression of axillary meristem development in rice spikelets. qSBN7 is a novel allele of FZP that causes an ~56% decrease in its transcriptional level, leading to increased secondary branch and grain number, and reduced grain length. Field evaluations showed that qSBN7 increased grain yield by 10.9% in a temperate japonica variety, TN13, likely due to its positive effect on sink capacity. Our findings suggest that incorporation of qSBN7 can increase yield potential and improve the breeding of elite rice varieties.

Key Points

A Yersinia pestis mutant synthesizing an adjuvant form of lipid A (monophosphoryl lipid A, MPLA) displayed increased biogenesis of bacterial outer membrane vesicles (OMVs). To enhance the immunogenicity of the OMVs, we constructed an Asd-based balanced-lethal host-vector system that oversynthesized the LcrV antigen of Y. pestis, raised the amounts of LcrV enclosed in OMVs by the type II secretion system, and eliminated harmful factors like plasminogen activator (Pla) and murine toxin from the OMVs. Vaccination with OMVs containing MPLA and increased amounts of LcrV with diminished toxicity afforded complete protection in mice against subcutaneous challenge with 8 x 10⁵ CFU (80,000 50% lethal dose [LD₅₀]) and intranasal challenge with 5 x 10³ CFU (50 LD₅₀) of virulent Y. pestis. This protection was significantly superior to that resulting from vaccination with LcrV/alhydrogel or rF1-V/alhydrogel. At week 4 postimmunization, the OMV-immunized mice showed more robust titers of antibodies against LcrV, Y. pestis whole-cell lysate (YPL), and F1 antigen and more balanced IgG1:IgG2a/IgG2b-derived Th1 and Th2 responses than LcrV-immunized mice. Moreover, potent adaptive and innate immune responses were stimulated in the OMV-immunized mice. Our findings demonstrate that self-adjuvanting Y. pestis OMVs provide a novel plague vaccine candidate and that the rational design of OMVs could serve as a robust approach for vaccine development.

The Bcl-2 (B cell lymphoma 2)-related protein Nr-13 plays a major role in the regulation of cell death in developing avian B cells. With over 65% sequence similarity to the chicken Nr-13, herpesvirus of turkeys (HVT) vNr-13, encoded by the HVT079 and HVT096 genes, is the first known alphaherpesvirus-encoded Bcl-2 homolog. HVT-infected cells were reported to be relatively more resistant to serum starvation, suggested that vNr-13 could be involved in protecting the cells. Here, we describe CRISPR/Cas9-based editing of exon 1 of the HVT079 and HVT096 genes from the HVT genome to generate the mutant HVT-vNr-13 to gain insights into its functional roles. Overall, wild-type HVT and HVT-vNr-13 showed similar growth kinetics; however, at early time points, HVT-vNr-13 showed 1.3- to 1.7-fold-lower growth of cell-associated virus and 3- to 6.2-fold-lower growth of cell-free virus. In transfected cells, HVT vNr-13 showed a mainly diffuse cytoplasmic distribution with faint nuclear staining. Further, vNr-13 localized to the mitochondria and endoplasmic reticulum (ER) and disrupted mitochondrial network morphology in the transfected cells. In the wild-type HVT-infected cells, vNr-13 expression appeared to be directly involved in the disruption of the mitochondrial network, as the mitochondrial network morphology was substantially restored in the HVT-vNr-13-infected cells. IncuCyte S3 real-time apoptosis monitoring demonstrated that vNr-13 is unequivocally involved in the apoptosis inhibition, and it is associated with an increase of PFU, especially under serum-free conditions in the later stages of the viral replication cycle. Furthermore, HVT blocks apoptosis in infected cells but activates apoptosis in noninfected bystander cells.

IMPORTANCE B cell lymphoma 2 (Bcl-2) family proteins play important roles in regulating apoptosis during homeostasis, tissue development, and infectious diseases. Several viruses encode homologs of cellular Bcl-2-proteins (vBcl-2) to inhibit apoptosis, which enable them to replicate and persist in the infected cells and to evade/modulate the immune response of the host. Herpesvirus of turkeys (HVT) is a nonpathogenic alphaherpesvirus of turkeys and chickens that is widely used as a live vaccine against Marek’s disease and as recombinant vaccine viral vectors for protecting against multiple avian diseases. Identical copies of the HVT genes HVT079 and HVT096 encode the Bcl-2 homolog vNr-13. While previous studies have identified the potential ability of vNr-13 in inhibiting apoptosis induced by serum deprivation, there have been no detailed investigations on the functions of vNr-13. Using CRISPR/Cas9-based ablation of the vNr-13 gene, we demonstrated the roles of HVT vNr-13 in early stages of the viral replication cycle, mitochondrial morphology disruption, and apoptosis inhibition in later stages of viral replication.

Upon infection, the highly structured 5' untranslated region (5' UTR) of picornavirus is involved in viral protein translation and RNA synthesis. As a critical element in the 5' UTR, the internal ribosome entry site (IRES) binds to various cellular proteins to function in the processes of picornavirus replication. Foot-and-mouth disease virus (FMDV) is an important member in the family Picornaviridae, and its 5' UTR contains a functional IRES element. In this study, the cellular heterogeneous nuclear ribonucleoprotein L (hnRNP L) was identified as an IRES-binding protein for FMDV by biotinylated RNA pulldown assays, mass spectrometry (MS) analysis, and determination of hnRNP L-IRES interaction regions. Further, we found that hnRNP L inhibited the growth of FMDV through binding to the viral IRES and that the inhibitory effect of hnRNP L on FMDV growth was not due to FMDV IRES-mediated translation, but to influence on viral RNA synthesis. Finally, hnRNP L was demonstrated to coimmunoprecipitate with RNA-dependent RNA polymerase (3D^pol) in an FMDV RNA-dependent manner in the infected cells. Thus, our results suggest that hnRNP L, as a critical IRES-binding protein, negatively regulates FMDV replication by inhibiting viral RNA synthesis, possibly by remaining in the replication complex.

IMPORTANCE Picornaviruses, as a large family of human and animal pathogens, cause a bewildering array of disease syndromes. Many host factors are implicated in the pathogenesis of these viruses, and some proteins interact with the viral IRES elements to affect function. Here, we report for the first time that cellular hnRNP L specifically interacts with the IRES of the picornavirus FMDV and negatively regulates FMDV replication through inhibiting viral RNA synthesis. Further, our results showed that hnRNP L coimmunoprecipitates with FMDV 3D^pol in a viral RNA-dependent manner, suggesting that it may remain in the replication complex to function. The data presented here would facilitate further understanding of virus-host interactions and the pathogenesis of picornavirus infections.

Respiratory syncytial virus (RSV) is an enveloped RNA virus which is responsible for approximately 80% of lower respiratory tract infections in children. Current lines of evidence have supported the functional involvement of long noncoding RNA (lncRNA) in many viral infectious diseases. However, the overall biological effect and clinical role of lncRNAs in RSV infection remain unclear. In this study, lncRNAs related to respiratory virus infection were obtained from the lncRNA database, and we collected 144 clinical sputum specimens to identify lncRNAs related to RSV infection. Quantitative PCR (qPCR) detection indicated that the expression of lncRNA negative regulator of antiviral response (NRAV) in RSV-positive patients was significantly lower than that in uninfected patients, but lncRNA psoriasis-associated non-protein coding RNA induced by stress (PRINS), nuclear paraspeckle assembly transcript 1 (NEAT1), and Nettoie Salmonella pas Theiler’s (NeST) showed no difference in vivo and in vitro. Meanwhile, overexpression of NRAV promoted RSV proliferation in A549 and BEAS-2B cells, and vice versa, indicating that the downregulation of NRAV was part of the host antiviral defense. RNA fluorescent in situ hybridization (FISH) confirmed that NRAV was mainly located in the cytoplasm. Through RNA sequencing, we found that Rab5c, which is a vesicle transporting protein, showed the same change trend as NRAV. Subsequent investigation revealed that NRAV was able to favor RSV production indirectly by sponging microRNA miR-509-3p so as to release Rab5c and facilitate vesicle transportation. The study provides a new insight into virus-host interaction through noncoding RNA, which may contribute to exploring potential antivirus targets for respiratory virus.

IMPORTANCE The mechanism of interaction between RSV and host noncoding RNAs is not fully understood. In this study, we found that the expression of long noncoding RNA (lncRNA) negative regulator of antiviral response (NRAV) was reduced in RSV-infected patients, and overexpression of NRAV facilitated RSV production in vitro, suggesting that the reduction of NRAV in RSV infection was part of the host antiviral response. We also found that NRAV competed with vesicle protein Rab5c for microRNA miR509-3p in cytoplasm to promote RSV vesicle transport and accelerate RSV proliferation, thereby improving our understanding of the pathogenic mechanism of RSV infection.

Porcine reproductive and respiratory syndrome (PRRS) is a serious viral disease affecting the global swine industry. Its causative agent, PRRS virus (PRRSV), is an enveloped virus, and therefore membrane fusion between its envelope and host cell target membrane is critical for viral infection. Though much research has focused on PRRSV infection, the detailed mechanisms involved in its membrane fusion remain to be elucidated. In the present study, we performed confocal microscopy in combination with a constitutively active (CA) or dominant negative (DN) mutant, specific inhibitors, and small interfering RNAs (siRNAs), as well as multiple other approaches, to explore PRRSV membrane fusion. We first observed that PRRSV membrane fusion occurred in Rab11-recycling endosomes during early infection using labeled virions and subcellular markers. We further demonstrated that low pH and cathepsin E in Rab11-recycling endosomes are critical for PRRSV membrane fusion. Moreover, PRRSV glycoprotein 5 (GP5) is identified as being cleaved by cathepsin E during this process. Taken together, our findings provide in-depth information regarding PRRSV pathogenesis, which support a novel basis for the development of antiviral drugs and vaccines.

IMPORTANCE PRRS, caused by PRRSV, is an economically critical factor in pig farming worldwide. As PRRSV is a lipid membrane-wrapped virus, merging of the PRRSV envelope with the host cell membrane is indispensable for viral infection. However, there is a lack of knowledge on its membrane fusion. Here, we first explored when and where PRRSV membrane fusion occurs. Furthermore, we determined which host cell factors were involved in the process. Importantly, PRRSV GP5 is shown to be cleaved by cathepsin E during membrane fusion. Our work not only provides information on PRRSV membrane fusion for the first time but also deepens our understanding of the molecular mechanisms of PRRSV infection, which provides a foundation for future applications in the prevention and control of PRRS.

Chronic pain is a highly prevalent disease with poorly understood pathophysiology. In particular, the brain mechanisms mediating the transition from acute to chronic pain remain largely unknown. Here, we identify a subcortical signature of back pain. Specifically, subacute back pain patients who are at risk for developing chronic pain exhibit...

Neural networks have become the method of choice in surrogate modeling because of their ability to characterize arbitrary, high-dimensional functions in a data-driven fashion. This paper advocates for the training of surrogates that are 1) consistent with the physical manifold, resulting in physically meaningful predictions, and 2) cyclically consistent with...

Proteasomes are essential protease complexes that maintain cellular homeostasis, and aberrant proteasomal activity supports cancer development. The regulatory mechanisms and biological function of the ubiquitin-26S proteasome have been studied extensively, while those of the ubiquitin-independent 20S proteasome system remain obscure. Here, we show that the cap ’n’ collar (CNC) family transcription factor NRF3 specifically enhances 20S proteasome assembly in cancer cells and that 20S proteasomes contribute to colorectal cancer development through ubiquitin-independent proteolysis of the tumor suppressor p53 and retinoblastoma (Rb) proteins. The NRF3 gene is highly expressed in many cancer tissues and cell lines and is important for cancer cell growth. In cancer cells, NRF3 upregulates the assembly of the 20S proteasome by directly inducing the gene expression of the 20S proteasome maturation protein POMP. Interestingly, NRF3 knockdown not only increases p53 and Rb protein levels but also increases p53 activities for tumor suppression, including cell cycle arrest and induction of apoptosis. Furthermore, protein stability and cell viability assays using two distinct proteasome inhibitor anticancer drugs, the 20S proteasome inhibitor bortezomib and the ubiquitin-activating enzyme E1 inhibitor TAK-243, show that the upregulation of the NRF3-POMP axis leads to ubiquitin-independent proteolysis of p53 and Rb and to impaired sensitivity to bortezomib but not TAK-243. More importantly, the NRF3-POMP axis supports tumorigenesis and metastasis, with higher NRF3/POMP expression levels correlating with poor prognoses in patients with colorectal or rectal adenocarcinoma. These results suggest that the NRF3-POMP-20S proteasome assembly axis is significant for cancer development via ubiquitin-independent proteolysis of tumor suppressor proteins.

Cep57 has been characterized as a component of a pericentriolar complex containing Cep63 and Cep152. Interestingly, Cep63 and Cep152 self-assemble into a pericentriolar cylindrical architecture, and this event is critical for the orderly recruitment of Plk4, a key regulator of centriole duplication. However, the way in which Cep57 interacts with the Cep63-Cep152 complex and contributes to the structure and function of Cep63-Cep152 self-assembly remains unknown. We demonstrate that Cep57 interacts with Cep63 through N-terminal motifs and associates with Cep152 via Cep63. Three-dimensional structured illumination microscopy (3D-SIM) analyses suggested that the Cep57-Cep63-Cep152 complex is concentrically arranged around a centriole in a Cep57-in and Cep152-out manner. Cep57 mutant cells defective in Cep63 binding exhibited improper Cep63 and Cep152 localization and impaired Sas6 recruitment for procentriole assembly, proving the significance of the Cep57-Cep63 interaction. Intriguingly, Cep63 fused to a microtubule (MT)-binding domain of Cep57 functioned in concert with Cep152 to assemble around stabilized MTs in vitro. Thus, Cep57 plays a key role in architecting the Cep63-Cep152 assembly around centriolar MTs and promoting centriole biogenesis. This study may offer a platform to investigate how the organization and function of the pericentriolar architecture are altered by disease-associated mutations found in the Cep57-Cep63-Cep152 complex.

Recently, abundant evidence has clarified that long noncoding RNAs (lncRNAs) play an oncogenic or anticancer role in the tumorigenesis and development of diverse human cancers. Described as a crucial regulator in some cancers, MIR22HG has not yet been studied in laryngocarcinoma and therefore the underlying regulatory role of MIR22HG in laryngocarcinoma is worth detecting. In this study, MIR22HG expression in laryngocarcinoma cells was confirmed to be downregulated, and upregulated MIR22HG expression led to suppressive effects on laryngocarcinoma cell proliferation and migration. Molecular mechanism assays revealed that MIR22HG sponges miR-5000-3p in laryngocarcinoma cells. Besides, decreased expression of miR-5000-3p suppressed laryngocarcinoma cell proliferation and migration. Moreover, the FBXW7 gene was reported to be a downstream target gene of miR-5000-3p in laryngocarcinoma cells. More importantly, rescue assays verified that FBXW7 depletion or miR-5000-3p upregulation countervailed the repressive effects of MIR22HG overexpression on laryngocarcinoma progression. In addition, E2F6 was proved to be capable of inhibiting MIR22HG transcription in laryngocarcinoma cells. To sum up, E2F6-induced downregulation of MIR22HG promotes laryngocarcinoma progression through the miR-5000-3p/FBXW7 axis.

Microtubule-associated serine/threonine kinase like (MASTL), also known as Greatwall (Gwl) kinase, has an important role in the regulation of mitosis. By inhibiting protein phosphatase 2A (PP2A), it plays a crucial role in activating one of the most important mitotic kinases, known as cyclin-dependent kinase 1 (CDK1). MASTL has been seen to be upregulated in various types of cancers and is also involved in tumor recurrence. It is activated by CDK1 through phosphorylations in the activation/T-loop, but the complete mechanism of its activation is still unclear. Here, we report that AKT phosphorylates MASTL at residue T299, which plays a critical role in its activation. Our results suggest that AKT increases CDK1-mediated phosphorylation and hence the activity of MASTL, which, in turn, promotes mitotic progression through PP2A inhibition. We also show that the oncogenic potential of AKT is augmented by MASTL activation, since AKT-mediated proliferation in colorectal cell lines can be attenuated by inhibiting and/or silencing MASTL. In brief, we report that AKT plays an important role in the progression of mitosis in mammalian cells and that it does so through the phosphorylation and activation of MASTL.

The metabolic state of the brain can greatly impact neurologic function. Evidence of this includes the therapeutic benefit of a ketogenic diet in neurologic diseases, including epilepsy. However, brain lipid bioenergetics remain largely uncharacterized. The existence, capacity, and relevance of mitochondrial fatty acid β-oxidation (FAO) in the brain are highly controversial, with few genetic tools available to evaluate the question. We have provided evidence for the capacity of brain FAO using a pan-brain-specific conditional knockout (KO) mouse incapable of FAO due to the loss of carnitine palmitoyltransferase 2, the product of an obligate gene for FAO (CPT2^B–/–). Loss of central nervous system (CNS) FAO did not result in gross neuroanatomical changes or systemic differences in metabolism. Loss of CPT2 in the brain did not result in robustly impaired behavior. We demonstrate by unbiased and targeted metabolomics that the mammalian brain oxidizes a substantial quantity of long-chain fatty acids in vitro and in vivo. Loss of CNS FAO results in robust accumulation of long-chain acylcarnitines in the brain, suggesting that the mammalian brain mobilizes fatty acids for their oxidation, irrespective of diet or metabolic state. Together, these data demonstrate that the mammalian brain oxidizes fatty acids under normal circumstances with little influence from or on peripheral tissues.

JABFM seeks to widely disseminate its peer-reviewed publications, increasing article visibility for the purpose of advancing scientific knowledge. We describe the journal’s approach to dissemination and recommend a number of strategies for authors to implement, including press releases and social media. Providing the article’s digital object identifier (DOI) is most useful, compared with links that can break, or attaching the article PDF, which will depress reader metrics. All JABFM articles are freely accessible online worldwide.