IL-6/STAT3 axis is hijacked by GCRV to facilitate viral replication via suppressing type â IFN signaling.

Grass carp reovirus (GCRV) infections and hemorrhagic disease (GCHD) outbreaks are typically seasonally periodic and temperature-dependent, yet the molecular mechanism remains unclear. Herein, we depicted that temperature-dependent IL-6/STAT3 axis was exploited by GCRV to facilitate viral replication via suppressing type Ⅰ IFN signaling. Combined multi-omics analysis and qPCR identified IL-6, STAT3, and IRF3 as potential effector molecules mediating GCRV infection. Deploying GCRV challenge at 18 °C and 28 °C as models of resistant and permissive infections and switched to the corresponding temperatures as temperature stress models, we illustrated that IL-6 and STAT3 expression, genome level of GCRV, and phosphorylation of STAT3 were temperature dependent and regulated by temperature stress. Further research revealed that activating IL-6/STAT3 axis enhanced GCRV replication and suppressed the expression of IFNs, whereas blocking the axis impaired viral replication. Mechanistically, grass carp STAT3 inhibited IRF3 nuclear translocation via interacting with it, thus down-regulating IFNs expression, restraining transcriptional activation of the IFN promoter, and facilitating GCRV replication. Overall, our work sheds light on an immune evasion mechanism whereby GCRV facilitates viral replication by hijacking IL-6/STAT3 axis to down-regulate IFNs expression, thus providing a valuable reference for targeted prevention and therapy of GCRV.

Helicase-independent function of RIG-I against murine gammaherpesvirus 68 via blocking the nuclear translocation of viral proteins.

Innate immunity is the first line of defense against viral pathogens. Retinoic Acid-Inducible Gene 1 (RIG-I) is a pattern recognition receptor that recognizes virus-associated double-stranded RNA and initiates the interferon responses. Besides signal transduction, RIG-I exerts direct antiviral functions to displace viral proteins on dsRNA via its Helicase activity. Nevertheless, this effector-like activity of RIG-I against herpesviruses remains largely unexplored. It has been previously reported that herpesviruses deamidate RIG-I, resulting in the abolishment of its Helicase activity and signal transduction. In this study, we discovered that RIG-I possessed signaling-independent antiviral activities against murine gamma herpesviruses 68 (γHV68, murid herpesvirus 4). Importantly, a Helicase-dead mutant of RIG-I (K270A) demonstrated comparable inhibition on herpesviruses lytic replication, indicating that this antiviral activity is Helicase-independent. Mechanistically, RIG-I bound the Replication and Transcription Activator (RTA) and diminished its nuclear localization to repress viral transcription. We further demonstrated that RIG-I blocked the nuclear translocation of ORF21 (Thymidine Kinase), ORF75c (vGAT), both of which form a nuclear complex with RTA and RNA polymerase II (Pol II) to facilitate viral transcription. Moreover, RIG-I retained ORF59 (DNA processivity factor) in the cytoplasm to repress viral DNA replication. Altogether, we illuminated a previously unidentified, Helicase-independent effector-like function of RIG-I against γHV68, representing an exquisite host strategy to counteract viral manipulations on innate immune signaling. IMPORTANCE: Retinoic acid-inducible gene I (RIG-I), a member of DExD/H box RNA helicase family, functions as a key pattern recognition receptor (PRR) responsible for the detection of intracellular double-stranded RNA (dsRNA) from virus-infected cells and induction of type I interferon (IFN) responses. Nevertheless, our understanding of the helicase-independent effector-like activity of RIG-I against virus infection, especially herpesvirus infection, remains largely unknown. Herein, by deploying murine gamma herpesviruses 68 (γHV68) as a model system, we demonstrated that RIG-I possessed an interferon and helicase-independent antiviral activity against γHV68 via blocking the nuclear trafficking of viral proteins, which concomitantly repressed the viral early transcription and genome replication thereof. Our work illuminates a previously unidentified antiviral strategy of RIG-I against herpesvirus infection.

Correction: An aquatic virus exploits the IL6-STAT3-HSP90 signaling axis to promote viral entry.

[This corrects the article DOI: 10.1371/journal.ppat.1011320.].

An Aquareovirus Exploits Membrane-Anchored HSP70 To Promote Viral Entry.

Temperature dependency of viral diseases in ectotherms has been an important scientific issue for decades, while the molecular mechanism behind this phenomenon remains largely mysterious. In this study, deploying infection with grass carp reovirus (GCRV), a double-stranded RNA aquareovirus, as a model system, we demonstrated that the cross talk between HSP70 and outer capsid protein VP7 of GCRV determines temperature-dependent viral entry. Multitranscriptomic analysis identified HSP70 as a key player in the temperature-dependent pathogenesis of GCRV infection. Further biochemical, small interfering RNA (siRNA) knockdown, pharmacological inhibition, and microscopic approaches revealed that the primary plasma membrane-anchored HSP70 interacts with VP7 to facilitate viral entry during the early phase of GCRV infection. Moreover, VP7 functions as a key coordinator protein to interact with multiple housekeeping proteins and regulate receptor gene expression, concomitantly facilitating viral entry. This work illuminates a previously unidentified immune evasion mechanism by which an aquatic virus hijacks heat shock response-related proteins to enhance viral entry, pinpointing targeted preventives and therapeutics for aquatic viral diseases. IMPORTANCE The seasonality of viral diseases in ectotherms is a prevailing phenomenon in the aquatic environment, which causes huge economic losses every year worldwide and hinders sustainable development of the aquaculture industry. Nevertheless, our understanding of the molecular mechanism of how temperature determines the pathogenesis of aquatic viruses remains largely unexplored. In this study, by deploying grass carp reovirus (GCRV) infection as a model system, we demonstrated that temperature-dependent, primarily membrane-localized HSP70 interacts with major outer capsid protein VP7 of GCRV to bridge the virus-host interaction, reshape the host's behaviors, and concomitantly facilitate viral entry. Our work unveils a central role of HSP70 in the temperature-dependent pathogenesis of aquatic viruses and provides a theoretical basis for the formulation of prevention and control strategies for aquatic viral diseases.

An aquatic virus exploits the IL6-STAT3-HSP90 signaling axis to promote viral entry.

Viral seasonality in the aquaculture industry is an important scientific issue for decades. While the molecular mechanisms underpinning the temperature-dependent pathogenesis of aquatic viral diseases remain largely unknown. Here we report that temperature-dependent activation of IL6-STAT3 signaling was exploited by grass carp reovirus (GCRV) to promote viral entry via increasing the expression of heat shock protein 90 (HSP90). Deploying GCRV infection as a model system, we discovered that GCRV induces the IL6-STAT3-HSP90 signaling activation to achieve temperature-dependent viral entry. Further biochemical and microscopic analyses revealed that the major capsid protein VP7 of GCRV interacted with HSP90 and relevant membrane-associated proteins to boost viral entry. Accordingly, exogenous expression of either IL6, HSP90, or VP7 in cells increased GCRV entry in a dose-dependent manner. Interestingly, other viruses (e.g., koi herpesvirus, Rhabdovirus carpio, Chinese giant salamander iridovirus) infecting ectothermic vertebrates have evolved a similar mechanism to promote their infection. This work delineates a molecular mechanism by which an aquatic viral pathogen exploits the host temperature-related immune response to promote its entry and replication, instructing us on new ways to develop targeted preventives and therapeutics for aquaculture viral diseases.

Lanostane-type triterpenoids from the mycelial mat of Ganoderma lucidum and their hepatoprotective activities.

Ganoderma lucidum (G. lucidum), a well-known Polyporaceae family fungus, is valued for its edibility and medicinal properties. It is a rich source of active polysaccharides and triterpenoids. However, obtaining material for medicinal purposes relies on artificial cultivation in a greenhouse, which requires large amounts of tree trunk due to the low biomass transformation rate. Therefore, an effective and environment-friendly culture method should be developed and the chemical compounds in the cultured material should be studied. Here we report the isolation and structural elucidation of 10 undescribed lanostane triterpenoids and 21 known compounds from statically cultured mycelial mat of G. lucidum. The hepatoprotective activity of these compounds in H2O2-induced HepG2 cells was evaluated. The structure-activity relationship is discussed. Our results demonstrated that twelve ganoderic acid derivatives possess significant hepatoprotective activities, as judged by suppressed activities of ALT, AST and LDH and increased GSH levels in H2O2-injured HepG2 cells.

Circumferential Decompression Technique of Posterior Endoscopic Cervical Foraminotomy.

OBJECTIVE: Cervical osseous foraminal stenosis (COFS) results from the uncinate process and facet hyperostosis. Currently, the optimal surgical technique for the treatment of COFS remains controversial. MATERIALS AND METHODS: Patients with COFS presenting radiculopathy underwent posterior endoscopic cervical foraminotomy by the circumferential decompression technique. The neck disability index (NDI), the visual analogue scale (VAS), and the modified MacNab criteria were used to evaluate the outcomes. In addition, the range of motion (ROM) and the slippage distance between the operated vertebrae in flexion-extension position were measured to evaluate the stability of the cervical spine. RESULTS: There were 24 consecutive patients in the study. The mean follow-up period was 16.2 months (range: 12-26 months). The NDI and VAS scores for arm/neck pain improved significantly from preoperatively to the last follow-up. The satisfaction rate by modified MacNab criteria was 91.7% on the third postoperative day and 100% on the day of final follow-up. There were no significant differences in intervertebral ROM or slippage distance between the last follow-up and preoperatively (P = 0.968, P = 0.394). Arm pain occurred in one patient, and sustained fingers numbness in two patients, but these symptoms resolved at the last follow-up. CONCLUSIONS: Posterior endoscopic cervical foraminotomy by the circumferential decompression technique is a safe and effective treatment for COFS. Moreover, it preserves the stability and physiological mobility of the cervical spine.

Heterotrophically Ultrahigh-Cell-Density Cultivation of a High Protein-Yielding Unicellular Alga Chlorella With a Novel Nitrogen-Supply Strategy.

The unicellular green alga Chlorella is an ideal protein source. However, the high production cost and low production capability of the current main photoautotrophic culture mode limit its application especially as an alternative protein source for food and feed, which might be overcome through high-cell-density cultivation in fermenters. In this study, a Chlorella sorokiniana strain CMBB276 with high protein content was selected from five Chlorella strains by comprehensive evaluation of their growth rates, protein contents, and yields. The optimal cultural temperature, pH, and mole ratio of carbon and nitrogen (C/N) for C. sorokiniana CMBB276 growth were found to be 30°C, 6.5, and 18, respectively. Ammonium chloride was proved to be the best nitrogen (N) source for C. sorokiniana CMBB276 growth, whereas growth inhibition caused by the accumulation of salts was observed under fed-batch cultivation when maintaining a constant C/N ratio of 18 by controlling pH with sodium hydroxide solution. By simultaneously reducing the concentration of ammonium chloride in the feeding medium and controlling pH with ammonium hydroxide, we finally achieved the ultrahigh-cell-density cultivation of C. sorokiniana CMBB276. The highest biomass concentration and protein yield reached 232 and 86.55 g l-1, respectively, showing the great potential of culturing C. sorokiniana CMBB276 in fermenters for economic and large-scale protein source production.

Ultrahigh-cell-density heterotrophic cultivation of the unicellular green alga Chlorella sorokiniana for biomass production.

Heterotrophic cultivation of Chlorella has achieved commercial success, but the application of Chlorella biomass is still limited due to the high cost of biomass production. In this study, an effective and industrially scalable heterotrphic cultivation technology has been developed for a production strain Chlorella sorokiniana GT-1. Under the optimized culturing conditions, the ultrahigh biomass concentration of 271 and 247 g L-1 was achieved in 7.5 L bench-scale and 1000 L pilot-scale fermenters, respectively. Technoeconomic (TE) analysis indicated that the production cost of C. sorokiniana GT-1 could be reduced to $1601.27 per ton of biomass if the biomass concentration reached 200 g L-1 , which is 24.2% lower than that of the reported highest Chlorella biomass production through fermentation with the same TE model. Under the same growth conditions, the maximum biomass concentration of a low-starch mutant SLM2 was reduced to 93 g L-1 , which was 54% lower than that of the wild type, indicating the capabilities of C. sorokiniana GT-1 cells in accumulating large amounts of starch are essential for achieving the ultrahigh-cell-density under the heterotrophic conditions. In addition, the ultrahigh-cell-density growth potential of C. sorokiniana GT-1 cells was inferred to be related to the intrinsic biological characteristics including the tolerance to low dissolved oxygen and a moderate doubling time under the heterotrophic conditions as well. The breakthrough in cultivation technology is promising for Chlorella industry and would expand its applications in food and feed.

Ultrahigh-cell-density heterotrophic cultivation of the unicellular green microalga Scenedesmus acuminatus and application of the cells to photoautotrophic culture enhance biomass and lipid production.

Although production of biodiesels from microalgae is proved to be technically feasible, a commercially viable system has yet to emerge. High-cell-density fermentation of microalgae can be coupled with photoautotrophic cultivation to produce oils. In this study, by optimizing culturing conditions and employing a sophisticated substrate feed control strategy, ultrahigh-cell-density of 286 and 283.5 g/L was achieved for the unicellular alga Scenedesmus acuminatus grown in 7.5-L bench-scale and 1,000-L pilot-scale fermenters, respectively. The outdoor scale-up experiments indicated that heterotrophically grown S. acuminatus cells are more productive in terms of both biomass and lipid accumulation when they are inoculated in photobioreactors for lipid production as compared to the cells originally grown under photoautotrophic conditions. Technoeconomic analysis based on the pilot-scale data indicated that the cost of heterotrophic cultivation of microalgae for biomass production is comparable with that of the open-pond system and much lower than that of tubular PBR, if the biomass yield was higher than 200 g/L. This study demonstrated the economic viability of heterotrophic cultivation on large-scale microalgal inocula production, but ultrahigh-productivity fermentation is a prerequisite. Moreover, the advantages of the combined heterotrophic and photoautotrophic cultivation of microalgae for biofuels production were also verified in the pilot-scale.

Znhit1 controls intestinal stem cell maintenance by regulating H2A.Z incorporation.

Lgr5+ stem cells are crucial to gut epithelium homeostasis; however, how these cells are maintained is not fully understood. Zinc finger HIT-type containing 1 (Znhit1) is an evolutionarily conserved subunit of the SRCAP chromosome remodeling complex. Currently, the function of Znhit1 in vivo and its working mechanism in the SRCAP complex are unknown. Here we show that deletion of Znhit1 in intestinal epithelium depletes Lgr5+ stem cells thus disrupts intestinal homeostasis postnatal establishment and maintenance. Mechanistically, Znhit1 incorporates histone variant H2A.Z into TSS region of genes involved in Lgr5+ stem cell fate determination, including Lgr5, Tgfb1 and Tgfbr2, for subsequent transcriptional regulation. Importantly, Znhit1 promotes the interaction between H2A.Z and YL1 (H2A.Z chaperone) by controlling YL1 phosphorylation. These results demonstrate that Znhit1/H2A.Z is essential for Lgr5+ stem cell maintenance and intestinal homeostasis. Our findings identified a dominant role of Znhit1/H2A.Z in controlling mammalian organ development and tissue homeostasis in vivo.

Effect of Ethanol Accumulation on Porcine Interferon-α Production by Pichia pastoris and Activities of Key Enzymes in Carbon Metabolism.

In production of porcine interferon α (pIFN-α) by Pichia pastoris, improper glycerol feeding strategy leads to ethanol accumulation in the last stage of growth phase. In the present study, taking two runs with low ethanol accumulation under 2 g/L as control group, effects of long-term (>4 h) and instantaneous high ethanol concentration (>10 g/L) on pIFN-α production, and activities of key enzymes in carbon metabolism were discussed. As a result, compared with control group, pIFN-α expression level was decreased about 4~12 folds under long-term high ethanol concentration, from the level above 3 g/L to the level under 1 g/L; pIFN-α expression level was decreased about 8 folds under instantaneous high ethanol concentration, reaching to the low level of 0.42 g/L. The low production of pIFN-α was caused by the severe inhibitory effect of ethanol on these enzymes.

dBrms1 acts as a positive regulator of notch signaling in Drosophila wing.

The highly conserved Notch signaling is precisely regulated at different steps in a series of developmental events. However, little is known about the regulation of Notch receptor at transcriptional level. Here, we demonstrate that dBrms1 is involved in regulating Notch signaling in Drosophila wing. We show that knockdown of dBrms1 by RNA interference (RNAi) in wing disc suppresses the expression of Notch signaling target genes wingless (wg), cut and Enhancer of split m8 [E(spl)m8]. Consistently, the levels of Wg and Cut are reduced in the dBrms1 mutant clones. Importantly, loss of dBrms1 leads to significant reduction of Notch proteins. Furthermore, depletion of dBrms1 results in apparent downregulation of Notch transcription in the wing disc. Moreover, we find that dBrms1 is functionally conserved with human Breast cancer metastasis suppressor 1 like (hBRMS1L) in the modulation of Notch signaling. Taken together, our data provide important insights into the biological function of dBrms1 in regulating Notch signaling.

Enhanced porcine circovirus Cap protein production by Pichia pastoris with a fuzzy logic DO control based methanol/sorbitol co-feeding induction strategy.

Porcine circovirus Cap protein production by P. pastoris with strong AOX promoter suffered with the problems with traditional pure methanol induction: (1) inefficient methanol metabolism; (2) extensive oxygen supply load; (3) difficulty in stable DO control; (4) low protein titer. In this study, based on the difference of DO change patterns in response to methanol and sorbitol additions, a novel fuzzy control system was proposed to automatically regulate the co-feeding rates of methanol and sorbitol for efficient Cap protein induction. With aid of the proposed control system when setting DO control level at 10%, overall fermentation performance was significantly improved: (1) DO could be stably controlled under mild aeration condition; (2) methanol consumption rate could be restricted at moderate level and the major enzymes involved with methanol metabolism were largely activated; (3) Cap protein concentration reached a highest level of 198mg/L, which was about 64% increase over the best one using the pure methanol induction strategies.