Glabridin Functions as a Quorum Sensing Inhibitor to Inhibit Biofilm Formation and Swarming Motility of Multidrug-Resistant Acinetobacter baumannii.

Objective: Acinetobacter baumannii is a hazardous bacterium that causes hospital-acquired nosocomial infections, and the advent of multidrug-resistant A. baumannii (MDR-AB) strains is concerning. Novel antibacterial therapeutic strategies must be developed. The biological effects of glabridin on MDR-AB were investigated in this study. Methods: The minimum inhibitory concentrations (MICs) of glabridin against eight clinical MDR-AB strains were determined using the broth microdilution technique. Crystal violet staining was used to assess biofilm development, which has significant contribution to bacterial resistance. Swarming motility was measured according to surface growth zone of MDR-AB on LB agar medium. qRT-PCR was used to evaluate the expression of quorum sensing genes abaI and abaR. Glabridin and routinely used therapeutic antimicrobial agents were tested for synergistic action using the checkerboard method. Results: According to our findings, glabridin suppressed MDR-AB growth at high doses (512-1024 µg/mL). The 1/4 MIC of glabridin significantly decreased MDR-AB biofilm formation by 19.98% (P < 0.05), inhibited MDR-AB motility by 44.27% (P < 0.05), whereas the 1/2 MIC of glabridin dramatically reduced MDR-AB biofilm development by 27.43% (P < 0.01), suppressed MDR-AB motility by 50.64% (P < 0.05). Mechanistically, glabridin substantially downregulated the expression of quorum sensing-related genes abaI and abaR by up to 39.12% (P < 0.001) and 25.19% (P < 0.01), respectively. However, no synergistic effect between glabridin and antibacterial drugs was found. Conclusion: Glabridin might be a quorum sensing inhibitor that inhibits MDR-AB biofilm development and swarming motility.

Effects of high NaHCO3 alkalinity on growth, tissue structure, digestive enzyme activity, and gut microflora of grass carp juvenile.

With the gradual decrease in freshwater resources, the available space for freshwater aquaculture is diminishing. As a result, saline-alkaline water aquaculture has emerged as a crucial method to fulfill the increasing demand. This study investigates the impact of alkaline water on the growth performance, tissues (gill, liver, and kidney), digestive enzyme activity, and intestinal microbiology in grass carp (Ctenopharyngodon idella). The aquarium conditions were set with sodium bicarbonate (18 mmol/L (LAW), 32 mmol/L (HAW)) to simulate the alkaline water environment. A freshwater group was the control (FW). The experimental fish were cultured for 60 days. The findings revealed that NaHCO3 alkaline stress significantly reduced growth performance, caused alterations in the structural morphology of gill lamellae, liver, and kidney tissues, and led to decreased activity of intestinal trypsin and lipase amylase (P < 0.05). Analysis of 16S rRNA sequences demonstrated that alkalinity influenced the abundance of dominant bacterial phyla and genera. Proteobacteria showed a significant decrease under alkaline conditions, while Firmicutes exhibited a significant increase (P < 0.05). Furthermore, alkalinity conditions significantly reduced the abundance of bacteria involved in protein, amino acid, and carbohydrate metabolism, cell transport, cell decomposition, and environmental information processing. Conversely, the abundance of bacteria associated with lipid metabolism, energy metabolism, organic systems, and disease functional flora increased significantly under alkalinity conditions (P < 0.05). In conclusion, this comprehensive study indicates that alkalinity stress adversely affected the growth performance of juvenile grass carp, likely due to tissue damage, reduced activity of intestinal digestive enzymes, and alterations in intestinal microorganisms.

Corneal endothelial cells and acoustic cavitation in phacoemulsification.

Postoperative complications of phacoemulsification, such as corneal edema caused by human corneal endothelial cell (CEC) injury, are still a matter of concern. Although several factors are known to cause CEC damage, the influence of ultrasound on the formation of free radicals during surgery should be considered. Ultrasound in aqueous humor induces cavitation and promotes the formation of hydroxyl radicals or reactive oxygen species (ROS). ROS-induced apoptosis and autophagy in phacoemulsification have been suggested to significantly promote CEC injury. CEC cannot regenerate after injury, and measures must be taken to prevent the loss of CEC after phacoemulsification or other CEC injuries. Antioxidants can reduce the oxidative stress injury of CEC during phacoemulsification. Evidence from rabbit eye studies shows that ascorbic acid infusion during operation or local application of ascorbic acid during phacoemulsification has a protective effect by scavenging free radicals or reducing oxidative stress. Both in experiments and clinical practice, hydrogen dissolved in the irrigating solution can also prevent CEC damage during phacoemulsification surgery. Astaxanthin (AST) can inhibit oxidative damage, thereby protecting different cells from most pathological conditions, such as myocardial cells, luteinized granulosa cells of the ovary, umbilical vascular endothelial cells, and human retina pigment epithelium cell line (ARPE-19). However, existing research has not focused on the application of AST to prevent oxidative stress during phacoemulsification, and the related mechanisms need to be studied. The Rho related helical coil kinase inhibitor Y-27632 can inhibit CEC apoptosis after phacoemulsification. Rigorous experiments are required to confirm whether its effect is realized through improving the ROS clearance ability of CEC.

A rhabdovirus accessory protein inhibits jasmonic acid signaling in plants to attract insect vectors.

Plant rhabdoviruses heavily rely on insect vectors for transmission between sessile plants. However, little is known about the underlying mechanisms of insect attraction and transmission of plant rhabdoviruses. In this study, we used an arthropod-borne cytorhabdovirus, Barley yellow striate mosaic virus (BYSMV), to demonstrate the molecular mechanisms of a rhabdovirus accessory protein in improving plant attractiveness to insect vectors. Here, we found that BYSMV-infected barley (Hordeum vulgare L.) plants attracted more insect vectors than mock-treated plants. Interestingly, overexpression of BYSMV P6, an accessory protein, in transgenic wheat (Triticum aestivum L.) plants substantially increased host attractiveness to insect vectors through inhibiting the jasmonic acid (JA) signaling pathway. The BYSMV P6 protein interacted with the constitutive photomorphogenesis 9 signalosome subunit 5 (CSN5) of barley plants in vivo and in vitro, and negatively affected CSN5-mediated deRUBylation of cullin1 (CUL1). Consequently, the defective CUL1-based Skp1/Cullin1/F-box ubiquitin E3 ligases could not mediate degradation of jasmonate ZIM-domain proteins, resulting in compromised JA signaling and increased insect attraction. Overexpression of BYSMV P6 also inhibited JA signaling in transgenic Arabidopsis (Arabidopsis thaliana) plants to attract insects. Our results provide insight into how a plant cytorhabdovirus subverts plant JA signaling to attract insect vectors.

MAPKs trigger antiviral immunity by directly phosphorylating a rhabdovirus nucleoprotein in plants and insect vectors.

Signaling by the evolutionarily conserved mitogen-activated protein kinase or extracellular signal-regulated kinase (MAPK/ERK) plays critical roles in converting extracellular stimuli into immune responses. However, whether MAPK/ERK signaling induces virus immunity by directly phosphorylating viral effectors remains largely unknown. Barley yellow striate mosaic virus (BYSMV) is an economically important plant cytorhabdovirus that is transmitted by the small brown planthopper (SBPH, Laodelphax striatellus) in a propagative manner. Here, we found that the barley (Hordeum vulgare) MAPK MPK3 (HvMPK3) and the planthopper ERK (LsERK) proteins interact with the BYSMV nucleoprotein (N) and directly phosphorylate N protein primarily on serine 290. The overexpression of HvMPK3 inhibited BYSMV infection, whereas barley plants treated with the MAPK pathway inhibitor U0126 displayed greater susceptibility to BYSMV. Moreover, knockdown of LsERK promoted virus infection in SBPHs. A phosphomimetic mutant of the N Ser290 (S290D) completely abolished virus infection because of impaired self-interaction of BYSMV N and formation of unstable N-RNA complexes. Altogether, our results demonstrate that the conserved MAPK and ERK directly phosphorylate the viral nucleoprotein to trigger immunity against cross-kingdom infection of BYSMV in host plants and its insect vectors.

Developing reverse genetics systems of northern cereal mosaic virus to reveal superinfection exclusion of two cytorhabdoviruses in barley plants.

Recently, reverse genetics systems of plant negative-stranded RNA (NSR) viruses have been developed to study virus-host interactions. Nonetheless, genetic rescue of plant NSR viruses in both insect vectors and monocot plants is very limited. Northern cereal mosaic virus (NCMV), a plant cytorhabdovirus, causes severe diseases in cereal plants through transmission by the small brown planthopper (SBPH, Laodelphax striatellus) in a propagative manner. In this study, we first developed a minireplicon system of NCMV in Nicotiana benthamiana plants, and then recovered a recombinant NCMV virus (rNCMV-RFP), with a red fluorescent protein (RFP) insertion, in SBPHs and barley plants. We further used rNCMV-RFP and green fluorescent protein (GFP)-tagged barley yellow striate mosaic virus (rBYSMV-GFP), a closely related cytorhabdovirus, to study superinfection exclusion, a widely observed phenomenon in dicot plants rarely studied in monocot plants. Interestingly, cellular superinfection exclusion of rBYSMV-GFP and rNCMV-RFP was observed in barley leaves. Our results demonstrate that two insect-transmitted cytorhabdoviruses are enemies rather than friends at the cellular level during coinfections in plants.

Host casein kinase 1-mediated phosphorylation modulates phase separation of a rhabdovirus phosphoprotein and virus infection.

Liquid-liquid phase separation (LLPS) plays important roles in forming cellular membraneless organelles. However, how host factors regulate LLPS of viral proteins during negative-sense RNA (NSR) virus infection is largely unknown. Here, we used barley yellow striate mosaic virus (BYSMV) as a model to demonstrate regulation of host casein kinase 1 (CK1) in phase separation and infection of NSR viruses. We first found that the BYSMV phosphoprotein (P) formed spherical granules with liquid properties and recruited viral nucleotide (N) and polymerase (L) proteins in vivo. Moreover, the P-formed granules were tethered to the ER/actin network for trafficking and fusion. BYSMV P alone formed droplets and incorporated the N protein and the 5' trailer of genomic RNA in vitro. Interestingly, phase separation of BYSMV P was inhibited by host CK1-dependent phosphorylation of an intrinsically disordered P protein region. Genetic assays demonstrated that the unphosphorylated mutant of BYSMV P exhibited condensed phase, which promoted viroplasm formation and virus replication. Whereas, the phosphorylation-mimic mutant existed in diffuse phase state for virus transcription. Collectively, our results demonstrate that host CK1 modulates phase separation of the viral P protein and virus infection.

A cytorhabdovirus-based expression vector in Nilaparvata lugens, Laodelphax striatellus, and Sogatella furcifera.

The brown planthopper (BPH, Nilaparvata lugens), the small brown planthopper (SBPH, Laodelphax striatellus), and the white-backed planthopper (WBPH, Sogatella furcifera) are problematic insect pests and cause severe yield losses through phloem sap-sucking and virus transmission. Barley yellow striate mosaic virus (BYSMV), a plant cytorhabdovirus, has been developed as versatile expression platforms in SBPHs and cereal plants. However, bio-safe overexpression vectors based on recombinant BYSMV (rBYSMV) remain to be developed and applied to the three kinds of planthoppers. Here, we found that rBYSMV was able to infect SBPHs, BPHs and WBPHs through microinjection with crude extracts from rBYSMV-infected barley leaves. To ensure bio-safety of the rBYSMV vectors, we generated an rBYSMV mutant by deleting the accessory protein P3, a putative viral movement protein. As expected, the resulting mutant abolished viral systemic infection in barley plants but had no effects on BYSMV infectivity in insect vectors. Subsequently, we used the modified rBYSMV vector to overexpress iron transport peptide (ITP) in the three kinds of planthoppers and revealed the potential functions of ITP. Overall, our results provide bio-safe overexpression platforms to facilitate functional genomics studies of planthoppers.

Casein Kinase 1 Regulates Cytorhabdovirus Replication and Transcription by Phosphorylating a Phosphoprotein Serine-Rich Motif.

Casein kinase 1 (CK1) family members are conserved Ser/Thr protein kinases that regulate important developmental processes in all eukaryotic organisms. However, the functions of CK1 in plant immunity remain largely unknown. Barley yellow striate mosaic virus (BYSMV), a plant cytorhabdovirus, infects cereal crops and is obligately transmitted by the small brown planthopper (SBPH; Laodelphax striatellus). The BYSMV phosphoprotein (P) exists as two forms with different mobilities corresponding to 42 kD (P42) and 44 kD (P44) in SDS-PAGE gels. Mass spectrometric analyses revealed a highly phosphorylated serine-rich (SR) motif at the C-terminal intrinsically disordered region of the P protein. The Ala-substitution mutant (PS5A) in the SR motif stimulated virus replication, whereas the phosphorylation-mimic mutant (PS5D) facilitated virus transcription. Furthermore, PS5A and PS5D associated preferentially with nucleocapsid protein-RNA templates and the large polymerase protein to provide optimal replication and transcription complexes, respectively. Biochemistry assays demonstrated that plant and insect CK1 protein kinases could phosphorylate the SR motif and induce conformational changes from P42 to P44. Moreover, overexpression of CK1 or a dominant-negative mutant impaired the balance between P42 and P44, thereby compromising virus infections. Our results demonstrate that BYSMV recruits the conserved CK1 kinases to achieve its cross-kingdom infection in host plants and insect vectors.

CCR4, a RNA decay factor, is hijacked by a plant cytorhabdovirus phosphoprotein to facilitate virus replication.

Carbon catabolite repression 4 (CCR4) is a conserved mRNA deadenylase regulating posttranscriptional gene expression. However, regulation of CCR4 in virus infections is less understood. Here, we characterized a pro-viral role of CCR4 in replication of a plant cytorhabdovirus, Barley yellow striate mosaic virus (BYSMV). The barley (Hordeum vulgare) CCR4 protein (HvCCR4) was identified to interact with the BYSMV phosphoprotein (P). The BYSMV P protein recruited HvCCR4 from processing bodies (PBs) into viroplasm-like bodies. Overexpression of HvCCR4 promoted BYSMV replication in plants. Conversely, knockdown of the small brown planthopper CCR4 inhibited viral accumulation in the insect vector. Biochemistry experiments revealed that HvCCR4 was recruited into N-RNA complexes by the BYSMV P protein and triggered turnover of N-bound cellular mRNAs, thereby releasing RNA-free N protein to bind viral genomic RNA for optimal viral replication. Our results demonstrate that the co-opted CCR4-mediated RNA decay facilitates cytorhabdovirus replication in plants and insects.

Rescue of a plant cytorhabdovirus as versatile expression platforms for planthopper and cereal genomic studies.

Plant viruses have been used as rapid and cost-effective expression vectors for heterologous protein expression in genomic studies. However, delivering large or multiple foreign proteins in monocots and insect pests is challenging. Here, we recovered a recombinant plant cytorhabdovirus, Barley yellow striate mosaic virus (BYSMV), for use as a versatile expression platform in cereals and the small brown planthopper (SBPH, Laodelphax striatellus) insect vector. We engineered BYSMV vectors to provide versatile expression platforms for simultaneous expression of three foreign proteins in barley plants and SBPHs. Moreover, BYSMV vectors could express the c. 600-amino-acid ß-glucuronidase (GUS) protein and a red fluorescent protein stably in systemically infected leaves and roots of cereals, including wheat, barley, foxtail millet, and maize plants. Moreover, we have demonstrated that BYSMV vectors can be used in barley to analyze biological functions of gibberellic acid (GA) biosynthesis genes. In a major technical advance, BYSMV vectors were developed for simultaneous delivery of CRISPR/Cas9 nuclease and single guide RNAs for genomic editing in Nicotiana benthamiana leaves. Taken together, our results provide considerable potential for rapid screening of functional proteins in cereals and planthoppers, and an efficient approach for developing other insect-transmitted negative-strand RNA viruses.

A cytorhabdovirus phosphoprotein forms mobile inclusions trafficked on the actin/ER network for viral RNA synthesis.

As obligate parasites, plant viruses usually hijack host cytoskeletons for replication and movement. Rhabdoviruses are enveloped, negative-stranded RNA viruses that infect vertebrates, invertebrates, and plants, but the mechanisms of intracellular trafficking of plant rhabdovirus proteins are largely unknown. Here, we used Barley yellow striate mosaic virus (BYSMV), a plant cytorhabdovirus, as a model to investigate the effects of the actin cytoskeleton on viral intracellular movement and viral RNA synthesis in a mini-replicon (MR) system. The BYSMV P protein forms mobile inclusion bodies that are trafficked along the actin/endoplasmic reticulum network, and recruit the N and L proteins into viroplasm-like structures. Deletion analysis showed that the N terminal region (aa 43-55) and the remaining region (aa 56-295) of BYSMV P are essential for the mobility and formation of inclusions, respectively. Overexpression of myosin XI-K tails completely abolishes the trafficking activity of P bodies, and is accompanied by a significant reduction of viral MR RNA synthesis. These results suggest that BYSMV P contributes to the formation and trafficking of viroplasm-like structures along the ER/actin network driven by myosin XI-K. Thus, rhabdovirus P appears to be a dynamic hub protein for efficient recruitment of viral proteins, thereby promoting viral RNA synthesis.

Transmission Characteristics of Barley Yellow Striate Mosaic Virus in Its Planthopper Vector Laodelphax striatellus.

The most economically important plant viruses are specifically transmitted by phytophagous insects that significantly affect viral epidemiology. Barley yellow striate mosaic virus (BYSMV), a member of the genus Cytorhabdovirus, is transmitted by the small brown planthopper (SBPH, Laodelphax striatellus) in a persistent-propagative manner. However, the infection route of BYSMV in SBPHs is poorly understood. In this study, immunofluorescence confocal laser scanning microscopy (iCLSM) was performed to investigate the route of BYSMV in SBPHs. We unexpectedly found that BYSMV initially infected the hindgut epithelium of SBPHs, instead of the midgut epithelium initially infected by other persistent-propagative viruses. Subsequently, BYSMV disseminated to the hindgut visceral muscles and spread to other parts of alimentary canals, hemolymph, and salivary glands. Comparative analysis of gene expression on viral mRNAs and the BYSMV nucleoprotein by using different molecular detection and immunohistochemistry further demonstrated that BYSMV initially infected and replicated in the hindgut epithelial cells of SBPHs. Collectively, our study provides the first insight into that hindgut is initial infection site of BYSMV that represents a new dissemination route of persistent-propagative viruses.

Development and Validation of a Gas Chromatography Method for Quality Control of Residual Solvents in Azilsartan Bulk Drugs.

A new gas chromatographic method for the simultaneous determination of six organic residual solvents (acetonitrile, tetrahydrofuran, ethanol, acetone, 2-propanol and ethyl acetate) in azilsartan bulk drug is described. The chromatographic determination was achieved on an OV-624 capillary column employing programmed temperature within 21 min. The validation was carried out according to International Conference on Harmonization validation guidelines. The method was shown to be specific (no interference in the blank solution), sensitive (Limit of detection can achieve 1.5 µg/mL), precise (relative standard deviation of repeatability and intermediate precision ≤5.0%), linear (r≥ 0.999), accurate (recoveries range from 98.8% to 107.8%) and robust (carrier gas flow from 2.7 to 3.3 mL/min, initial oven temperature from 35°C to 45°C, temperature ramping rate from 19°C/min to 21°C/min, final oven temperature from 145°C to 155°C, injector temperature from 190°C to 210°C and detector temperature from 240°C to 260°C did not significantly affect the system suitability, test parameters and peak areas). This extensively validated method has been applied to the determination of residual solvents in real azilsartan bulk samples.