Epstein-Barr virus envelope glycoprotein 110 inhibits NF-κB activation by interacting with NF-κB subunit p65.

Epstein-Barr virus (EBV) is a member of the lymphotropic virus family and is highly correlated with some human malignant tumors. It has been reported that envelope glycoprotein 110 (gp110) plays an essential role in viral fusion, DNA replication, and nucleocapsid assembly of EBV. However, it has not been established whether gp110 is involved in regulating the host's innate immunity. In this study, we found that gp110 inhibits tumor necrosis factor α-mediated NF- κB promoter activity and the downstream production of NF- κB-regulated cytokines under physiological conditions. Using dual-luciferase reporter assays, we showed that gp110 might impede the NF-κB promoter activation downstream of NF-κB transactivational subunit p65. Subsequently, we used coimmunoprecipitation assays to demonstrate that gp110 interacts with p65 during EBV lytic infection, and that the C-terminal cytoplasmic region of gp110 is the key interaction domain with p65. Furthermore, we determined that gp110 can bind to the N-terminal Rel homologous and C-terminal domains of p65. Alternatively, gp110 might not disturb the association of p65 with nontransactivational subunit p50, but we showed it restrains activational phosphorylation (at Ser536) and nuclear translocation of p65, which we also found to be executed by the C-terminal cytoplasmic region of gp110. Altogether, these data suggest that the surface protein gp110 may be a vital component for EBV to antagonize the host's innate immune response, which is also helpful for revealing the infectivity and pathogenesis of EBV.

Design of substituted tetrahydrofuran derivatives for HIV-1 protease inhibitors: synthesis, biological evaluation, and X-ray structural studies.

Substituted tetrahydrofuran derivatives were designed and synthesized to serve as the P2 ligand for a series of potent HIV-1 protease inhibitors. Both enantiomers of the tetrahydrofuran derivatives were synthesized stereoselectivity in optically active forms using lipase-PS catalyzed enzymatic resolution as the key step. These tetrahydrofuran derivatives are designed to promote hydrogen bonding and van der Waals interactions with the backbone atoms in the S2 subsite of the HIV-1 protease active site. Several inhibitors displayed very potent HIV-1 protease inhibitory activity. A high-resolution X-ray crystal structure of an inhibitor-bound HIV-1 protease provided important insight into the ligand binding site interactions in the active site.

[Distribution and Antibiotic Resistance Analysis of Ocular Bacterial Pathogens at a Tertiary Hospital From 2012 to 2021]. / 某三甲医院2012­2021å¹´çœ¼éƒ¨ç»†èŒæ„ŸæŸ“ç— åŽŸèŒåˆ†å¸ƒåŠè€è¯æ€§åˆ†æž.

Objective: To analyze the distribution of ocular bacterial pathogens and their antibiotic resistance status at a tertiary-care hospital and to provide a reference for the appropriate use of antibiotics. Methods: Retrospective analysis was conducted with bacteria isolated from the ophthalmic samples sent for lab analysis at a tertiary-care hospital from 2012 to 2021. The suspected bacterial strains were identified with automated systems for microbial identification and susceptibility analysis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometer. VITEK 2 Compact, an automated microbial identification and antibiotic susceptibility analysis system, was used for antimicrobial susceptibility testing. Results: A total of 1556 ophthalmology bacteria culture samples were collected, 574 of which showed bacterial growth, presenting an overall positive rate of 36.89%. Of the isolated bacteria, Gram-positive cocci, Gram-positive bacilli, Gram-negative bacilli, and Gram-negative cocci accounted for 63.15% (377/597), 18.76% (112/597), 17.09% (102/597), and 1.00% (6/597), respectively. Among the bacteria isolated in different years over the course of a decade, Gram-positive cocci always turned out to be the main cause of eye infections. Of the Gram-positive cocci, 73.47% (277/377) were isolated from patients with endophthalmitis, with the most important species being Staphylococcus epidermidis, which was followed by Streptococcus viridans. The rest, or 26.53% (100/377), of the Gram-positive cocci were isolated from patients with external eye infections, with the main isolated strains being Staphylococcus epidermidis, Streptococcus viridans, and Staphylococcus aureus. More than 70% of Staphylococcus epidermidis isolated from both endophthalmitis and external eye infections were resistant to methicillin. No strains resistant to vancomycin, linezolid, or tigecycline were detected. Staphylococcus epidermidis isolated from patients with external eye infections had a low rate of resistance to levofloxacin (2/27 or 7.41%), whereas those isolated from patients with endophthalmitis had a higher resistance rate (43/127 or 33.86%). The difference in drug resistance rate between the two groups was statistically significant (P<0.05). Conclusion: The chief ocular bacterial pathogens identified in a tertiary-care hospital were Gram-positive cocci, among which, Staphylococcus epidermidis was the most common species. The Staphylococcus epidermidis identified in the hospital had a high rate of resistance to oxacillin, but remained highly sensitive to vancomycin, linezolid, and tigecycline. The endophthalmitis caused by Staphylococcus epidermidis in the hospital can be treated empirically with vancomycin and then the treatment plan can be further adjusted according to the results of the drug susceptibility test. However, the establishment of the breakpoint of drug susceptibility test is mainly based on the model of bloodstream infection and has limited reference value for the treatment of eye infection. The required drug distribution concentration at the infection site can be achieved by dose increase or local administration.

Evaluation of darunavir-derived HIV-1 protease inhibitors incorporating P2' amide-derivatives: Synthesis, biological evaluation and structural studies.

We report here the synthesis and biological evaluation of darunavir derived HIV-1 protease inhibitors and their functional effect on enzyme inhibition and antiviral activity in MT-2 cell lines. The P2' 4-amino functionality was modified to make a number of amide derivatives to interact with residues in the S2' subsite of the HIV-1 protease active site. Several compounds exhibited picomolar enzyme inhibitory and low nanomolar antiviral activity. The X-ray crystal structure of the chloroacetate derivative bound to HIV-1 protease was determined. Interestingly, the active chloroacetate group converted to the acetate functionality during X-ray exposure. The structure revealed that the P2' carboxamide functionality makes enhanced hydrogen bonding interactions with the backbone atoms in the S2'-subsite.

Transcriptomic analysis reveals the regulatory role of quorum sensing in the Acinetobacter baumannii ATCC 19606 via RNA-seq.

BACKGROUND: Acinetobacter baumannii has emerged as the major opportunistic pathogen in healthcare-associated infections with high-level antibiotic resistance and high mortality. Quorum sensing (QS) system is a cell-to-cell bacterial communication mediated by the synthesis, secretion, and binding of auto-inducer signals. It is a global regulatory system to coordinate the behavior of individual bacteria in a population. The present study focused on the QS system, aiming to investigate the regulatory role of QS in bacterial virulence and antibiotic resistance. METHOD: The auto-inducer synthase gene abaI was deleted using the A. baumannii ATCC 19606 strain to interrupt the QS process. The RNA-seq was performed to identify the differentially expressed genes (DEGs) and pathways in the mutant (△abaI) strain compared with the wild-type (WT) strain. RESULTS: A total of 380 DEGs [the adjusted P value < 0.05 and the absolute value of log2(fold change) > log21.5] were identified, including 256 upregulated genes and 124 downregulated genes in the △abaI strain. The enrichment analysis indicated that the DEGs involved in arginine biosynthesis, purine metabolism, biofilm formation, and type VI secretion system (T6SS) were downregulated, while the DEGs involved in pathways related to fatty acid metabolism and amino acid metabolism were upregulated. Consistent with the expression change of the DEGs, a decrease in biofilm formation was observed in the △abaI strain compared with the WT strain. On the contrary, no obvious changes were found in antimicrobial resistance following the deletion of abaI. CONCLUSIONS: The present study demonstrated the transcriptomic profile of A. baumannii after the deletion of abaI, revealing an important regulatory role of the QS system in bacterial virulence. The deletion of abaI suppressed the biofilm formation in A. baumannii ATCC 19606, leading to decreased pathogenicity. Further studies on the role of abaR, encoding the receptor of auto-inducer in the QS circuit, are required for a better understanding of the regulation of bacterial virulence and pathogenicity in the QS network.

Intracellular distribution of pseudorabies virus UL2 and detection of its nuclear import mechanism.

Pseudorabies virus (PRV) UL2 (pUL2) is a multifunctional protein, which is homologous with herpes simplex virus 1 early protein UL2 (hUL2) and crucial for the viral propagation. Yet, how pUL2 executes its roles in the viral life cycle remain inadequately understood. In order to uncover its effect on the procedure of PRV infection, investigation was performed to examine the subcellular distribution of pUL2 and establish its trafficking mechanism. In the present study, enhanced yellow fluorescent protein or Myc tag fused pUL2 was transiently overexpressed in transfected cells and exhibited an absolutely nuclear accumulation without the existence of other PRV proteins. Additionally, the nuclear trafficking of pUL2 was proved to rely on Ran-, transportin-1, importin ß1, importin α1, α3 and α5. Accordingly, these data will benefit the knowledge of pUL2-mediated biological effects in PRV infection cycle.

Epstein-Barr virus tegument protein BGLF2 inhibits NF-κB activity by preventing p65 Ser536 phosphorylation.

Epstein-Barr virus (EBV), a ubiquitous gammaherpesvirus, can regulate the antiviral response of NF-κB signaling, which is critical for cell survival, growth transformation, and virus latency. Here, we showed that tegument protein BGLF2 could inhibit TNF-α-induced NF-κB activity. BGLF2 was shown to interplay with the NF-κB subunits p65 and p50, and the Rel homology domain of p65 was the pivotal region to interact with BGLF2. Nonetheless, BGLF2 did not influence the development of p65-p50 dimerization. Yet, overexpression of BGLF2 inhibited the phosphorylation of p65 Ser536 (but not Ser276) and blocked the nuclear translocation of p65. In addition, knockdown of BGLF2 during EBV lytic replication elevated NF-κB activity and the phosphorylation of p65 Ser536. Taken together, these results suggest that the inhibition of NF-κB activation may serve as a strategy to escape the host's antiviral innate immunity to EBV during its lytic infection.-Chen, T., Wang, Y., Xu, Z., Zou, X., Wang, P., Ou, X., Li, Y., Peng, T., Chen, D., Li, M., Cai, M. Epstein-Barr virus tegument protein BGLF2 inhibits NF-κB activity by preventing p65 Ser536 phosphorylation.

Polystyrene Nanoparticles Reduced ROS and Inhibited Ferroptosis by Triggering Lysosome Stress and TFEB Nucleus Translocation in a Size-Dependent Manner.

Though plastic nanoparticles have already raised much concern for their potential impact on health, our understanding of their biological effects is still utterly limited. Here we demonstrate for the first time that carboxyl-modified polystyrene nanoparticles (CPS) could effectively inhibit ferroptosis as a result of reduced cellular ROS which was triggered by transcription factor EB (TFEB) nucleus translocation. In this process, CPS first entered cells via macropinocytosis, then CPS-containing macropinosomes fused with lysosomes and expanded into abnormal lysosome-like large vacuoles in vacuolar-type H+-ATPase (V-ATPase) and aquaporins (AQPs) in a dependent way. These large vacuoles were detected both in vitro and in vivo, which was found to be a sign of lysosome stress. The lysosome stress induced deactivation of mammalian target of rapamycin (mTOR) which led to nucleus translocation of TFEB. Then, TFEB-dependent enhanced expression of lysosomal proteins and superoxide dismutase (SOD) which ultimately led to ROS reduction and inhibition of ferroptosis. Knockout of TFEB-enhanced ferroptosis was triggered by Erastin and abolished the effect of CPS on ROS and ferroptosis. In summary, our results reveal a novel mechanism whereby CPS reduced ROS and inhibited ferroptosis in a TFEB-dependent way. These findings have important implications for understanding the biological effects of polystyrene nanoparticles and searching for new anti-ROS and antiferroptosis particles or reagents.

Structural studies of antiviral inhibitor with HIV-1 protease bearing drug resistant substitutions of V32I, I47V and V82I.

HIV-1 protease inhibitors are effective in HIV/AIDS therapy, although drug resistance is a severe problem. This study examines the effects of four investigational inhibitors against HIV-1 protease with drug resistant mutations of V32I, I47V and V82I (PRTri) that model the inhibitor-binding site of HIV-2 protease. These inhibitors contain diverse chemical modifications on the darunavir scaffold and form new interactions with wild type protease, however, the measured inhibition constants for PRTri mutant range from 17 to 40 nM or significantly worse than picomolar values reported for wild type enzyme. The X-ray crystal structure of PRTri mutant in complex with inhibitor 1 at 1.5 Šresolution shows minor changes in interactions with inhibitor compared with the corresponding wild type PR complex. Instead, the basic amine at P2 of inhibitor together with mutation V82I induces two alternate conformations for the side chain of Arg8 with new interactions with inhibitor and Leu10. Hence, inhibition is influenced by small coordinated changes in hydrophobic interactions.

Identification of the molecular determinants for nuclear import of PRV EP0.

Pseudorabies virus (PRV) early protein EP0 is a homologue of the herpes simplex virus 1 (HSV-1) immediate-early protein ICP0, which is a multifunctional protein and important for HSV-1 infection. However, the definite function of EP0 during PRV infection is not clear. In this study, to determine if EP0 might localize to the nucleus, as it is shown for its homologue in HSV-1, the subcellular localization pattern and molecular determinants for the nuclear import of EP0 were investigated. EP0 was demonstrated to predominantly target the nucleus in both PRV infected- and plasmid-transfected cells. Furthermore, the nuclear import of EP0 was shown to be dependent on the Ran-, importin α1-, α3-, α7-, ß1- and transportin-1-mediated multiple pathways. Taken together, these data will open up new horizons for portraying the biological roles of EP0 in the course of PRV lytic cycle.

Crystal structure of yeast nitronate monooxygenase from Cyberlindnera saturnus.

Nitronate monooxygenase (NMO) is an FMN-dependent enzyme that oxidizes the neurotoxin propionate 3-nitronate (P3N) and represents the best-known system for P3N detoxification in different organisms. The crystal structure of the first eukaryotic Class I NMO from Cyberlindnera saturnus (CsNMO) has been solved at 1.65 Å resolution and refined to an R-factor of 14.0%. The three-dimensional structures of yeast CsNMO and bacterial PaNMO are highly conserved with the exception of three additional loops on the surface in the CsNMO enzyme and differences in four active sites residues. A PEG molecule was identified in the structure and formed extensive interactions with CsNMO, suggesting a specific binding site; however, 8% PEG showed no significant effect on the enzyme activity. This new crystal structure of a eukaryotic NMO provides insight into the function of this class of enzymes.

Characterization of the Nucleocytoplasmic Transport Mechanisms of Epstein-Barr Virus BFLF2.

BACKGROUND/AIMS: Epstein-Barr virus (EBV) BFLF2, the homologue of herpes simplex virus 1 (HSV-1) UL31, is crucial for the efficient viral DNA packaging and primary egress across the nuclear membrane. However, we still do not know its subcellular transport mechanisms. METHODS: Interspecies heterokaryon assays were utilized to detect the nucleocytoplasmic shuttling of BFLF2, and mutation analysis, plasmid transfection and fluorescence microscopy assays were performed to identify the functional nuclear localization sequence (NLS) and nuclear export sequence (NES) of BFLF2 in live cells. Furthermore, the nuclear import and export of BFLF2 were assessed by confocal microscopy, co-immunoprecipitation and immunoblot assays. RESULTS: BFLF2 was confirmed to shuttle between the nucleus and cytoplasm. Two predicted NESs were shown to be nonfunctional, yet we proved that the nuclear export of BFLF2 was mediated through transporter associated with antigen processing (TAP), but not chromosomal region maintenance 1 (CRM1) dependent pathway. Furthermore, one functional NLS, 22RRLMHPHHRNYTASKASAH40, was identified, and the aa22-23, aa22-25, aa28-30 and aa37-40 had an important role in the nuclear localization of BFLF2. Besides, the nuclear import of BFLF2 was demonstrated through Ran-, importin α7-, importin ß1- and transportin-1-dependent mechanism that does not require importin α1, α3 and α5. CONCLUSION: These works are of significance for the further study of the functions of BFLF2 during EBV infection, as well as for further insights into the design of new antiviral drug target and vaccine development against EBV.

[Effects of Tongbi capsule on joint lesions in rabbits with rheumatoid arthritis].

The purpose of this experiment is to observe the effects of Tongbi capsule on joint lesions in rabbit with rheumatoid arthritis induced by ovalbumin and explore the mechanism in order to provide reference for clinical application of Tongbi capsule. Rheumatoid arthritis in rabbits was induced by subcutaneous injection of emulsions of ovalbumin and Freund's complete adjuvant and intra articular injection of ovalbumin. After successful modeling, 30 New Zealand rabbits with arthritis were randomly divided into model control group, the high, medium and low dose groups of Tongbi capsule (90, 45, 22.5 mg·kg⁻¹) and prednisone group (5 mg·kg⁻¹). Another six normal rabbits were used as normal control group. After 24 hours of modeling, the rabbits in Tongbi capsule groups received intragastric (i.g.) administrations of Tongbi capsule at 90, 45, 22.5 mg·kg⁻¹·d⁻¹, and the rabbits of prednisone group received i.g. administrations of prednisone at 5 mg·kg⁻¹·d⁻¹ for 2 weeks. The rabbits in normal and model groups received the same volume of distilled water at the same time. The swelling degree of rabbit knee joint and local skin temperature were observed daily. After two weeks of administration, pathological changes of rabbit knee joint were examined by magnetic resonance imaging (MRI); the morphological changes of articular cartilage and synovial membrane were observed by microscope; and the contents of interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-α) in serum were detected by enzyme linked immunosorbent assay (ELISA).The results showed that 24 h after modeling, the knee joints of the rabbits were swollen, with red or dark redlocal skin, and fever, elevated local skin temperature and increased diameters of knee joints. Two weeks after modeling, the swelling of rabbit knee joints was obvious in model group; the joint cavities were filled with purulent fluid; joint synovial membranes were obviously thickened, and even joint cavities were fibrotic and cartilage surfaces showed slight defect; the surface of articular cartilage was obvious fibrosis; synovial epithelial cell proliferation was obvious and accompanied by extensive inflammatory cell infiltration; the levels of IL-1 and TNF-α were significantly higher as compared with those seen in model rabbits (P<0.05, P<0.01). After 1 and 2 weeks of administration, knee joint diameters and local skin temperatures were smaller or lower than thosein model group (P<0.05, P<0.01); The lesions of joint cartilage and synovial of all rabbits in each group were less than those in model group; IL-1 and TNF-α levels in serum were also lower than those in model group (P<0.05, P<0.01). The results reveal that high and medium doses of Tongbi capsule can suppress rheumatoid arthritis induced by ovalbumin in rabbits, reduce joint swelling, inhibit synovial epithelial and fiber hyperplasia and inflammatory cell infiltration, and alleviate articular cartilage damage. The mechanism may be associated with decreasing IL-1 and TNF-α levels in serum.

Design, synthesis, X-ray studies, and biological evaluation of novel macrocyclic HIV-1 protease inhibitors involving the P1'-P2' ligands.

Design, synthesis, and evaluation of a new class of HIV-1 protease inhibitors containing diverse flexible macrocyclic P1'-P2' tethers are reported. Inhibitor 5a with a pyrrolidinone-derived macrocycle exhibited favorable enzyme inhibitory and antiviral activity (Ki=13.2nM, IC50=22nM). Further incorporation of heteroatoms in the macrocyclic skeleton provided macrocyclic inhibitors 5m and 5o. These compounds showed excellent HIV-1 protease inhibitory (Ki=62pM and 14pM, respectively) and antiviral activity (IC50=5.3nM and 2.0nM, respectively). Inhibitor 5o also remained highly potent against a DRV-resistant HIV-1 variant.

Design of novel HIV-1 protease inhibitors incorporating isophthalamide-derived P2-P3 ligands: Synthesis, biological evaluation and X-ray structural studies of inhibitor-HIV-1 protease complex.

Based upon molecular insights from the X-ray structures of inhibitor-bound HIV-1 protease complexes, we have designed a series of isophthalamide-derived inhibitors incorporating substituted pyrrolidines, piperidines and thiazolidines as P2-P3 ligands for specific interactions in the S2-S3 extended site. Compound 4b has shown an enzyme Ki of 0.025nM and antiviral IC50 of 69nM. An X-ray crystal structure of inhibitor 4b-HIV-1 protease complex was determined at 1.33Å resolution. We have also determined X-ray structure of 3b-bound HIV-1 protease at 1.27Å resolution. These structures revealed important molecular insight into the inhibitor-HIV-1 protease interactions in the active site.

Paraventricular nucleus is involved in the central pathway of adipose afferent reflex in rats.

Increasing evidence indicates a link between sympathetic nervous system activation and obesity, but the underlying mechanisms remain elusive. The adipose afferent reflex (AAR) is a sympathoexcitatory reflex that is activated by afferent neurotransmission from the white adipose tissue (WAT). This study aimed to investigate whether the hypothalamic paraventricular nucleus (PVH) is an important component of the central neurocircuitry of the AAR. In anesthetized rats, the discharge activity of individual PVH neurons was recorded in vivo. Activation of WAT afferents was initiated by capsaicin injection, and the AAR was evaluated by monitoring renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) responses. The responses of PVH neurons to activation of WAT afferents were evaluated by c-fos immunoreactivity and the discharge activity of individual PVH neurons, which was recorded using extracellular single-unit recording. After activation of WAT afferents, both individual PVH neuron discharge activity and c-fos immunoreactivity increased. Bilateral selective lesions of the neurons in the PVH with kainic acid abolished the AAR. These results indicate that PVH is an important component of the central neurocircuitry of the AAR.

Structure-based design, synthesis, X-ray studies, and biological evaluation of novel HIV-1 protease inhibitors containing isophthalamide-derived P2-ligands.

We describe the design, synthesis and biological evaluation of a series of novel HIV-1 protease inhibitors bearing isophthalamide derivatives as the P2-P3 ligands. We have investigated a range of acyclic and heterocyclic amides as the extended P2-P3 ligands. These inhibitors displayed good to excellent HIV-1 protease inhibitory activity. Also, a number of inhibitors showed very good antiviral activity in MT cells. Compound 5n has shown an enzyme Ki of 0.17 nM and antiviral IC50 of 14 nM. An X-ray crystal structure of inhibitor 5o-bound to HIV-1 protease was determined at 1.11Å resolution. This structure revealed important molecular insight into the inhibitor-HIV-1 protease interactions in the active site.

Design, synthesis, biological evaluation and X-ray structural studies of HIV-1 protease inhibitors containing substituted fused-tetrahydropyranyl tetrahydrofuran as P2-ligands.

Design, synthesis, biological and X-ray crystallographic studies of a series of potent HIV-1 protease inhibitors are described. Various polar functionalities have been incorporated on the tetrahydropyranyl-tetrahydrofuran-derived P2 ligand to interact with the backbone atoms in the S2-subsite. The majority of the inhibitors showed very potent enzyme inhibitory and antiviral activity. Two high-resolution X-ray structures of 30b- and 30j-bound HIV-1 protease provide insight into ligand-binding site interactions. In particular, the polar functionalities on the P2-ligand appear to form unique hydrogen bonds with Gly48 amide NH and amide carbonyl groups in the flap region.

Quantum Chemical Study on ·Cl-Initiated Atmospheric Degradation of Monoethanolamine.

Recent findings on the formation of ·Cl in continental urban areas necessitate the consideration of ·Cl initiated degradation when assessing the fate of volatile organic pollutants. Monoethanolamine (MEA) is considered as a potential atmospheric pollutant since it is a benchmark and widely utilized solvent in a leading CO2 capture technology. Especially, ·Cl may have specific interactions with the N atom of MEA, which could make the MEA + ·Cl reaction have different pathways and products from those of the MEA + ·OH reaction. Hence, ·Cl initiated reactions with MEA were investigated by a quantum chemical method [CCSD(T)/aug-cc-pVTZ//MP2/6-31+G(3df,2p)] and kinetics modeling. Results show that the overall rate constant for ·Cl initiated H-abstraction of MEA is 5 times faster than that initiated by ·OH, and the tropospheric lifetimes of MEA will be overestimated by 6-46% when assuming that [·Cl]/[·OH] = 1-10% if the role of ·Cl is ignored. The MEA + ·Cl reaction exclusively produces MEA-N that finally transforms into several products including mutagenic nitramine and carcinogenic nitrosamine via further reactions with O2/NOx, and the contribution of ·Cl to their formation is about 25-250% of that of ·OH. Thus, it is necessary to consider ·Cl initiated tropospheric degradation of MEA for its risk assessment.