Anti-HIV Potential of Beesioside I Derivatives as Maturation Inhibitors: Synthesis, 3D-QSAR, Molecular Docking and Molecular Dynamics Simulations.

HIV-1 maturation is the final step in the retroviral lifecycle that is regulated by the proteolytic cleavage of the Gag precursor protein. As a first-in-class HIV-1 maturation inhibitor (MI), bevirimat blocks virion maturation by disrupting capsid-spacer peptide 1 (CA-SP1) cleavage, which acts as the target of MIs. Previous alterations of beesioside I (1) produced (20S,24S)-15ꞵ,16ꞵ-diacetoxy-18,24; 20,24-diepoxy-9,19-cyclolanostane-3ꞵ,25-diol 3-O-3',3'-dimethylsuccinate (3, DSC), showing similar anti-HIV potency compared to bevirimat. To ascertain the binding modes of this derivative, further modification of compound 1 was conducted. Three-dimensional quantitative structure−activity relationship (3D-QSAR) analysis combined with docking simulations and molecular dynamics (MD) were conducted. Five new derivatives were synthesized, among which compound 3b showed significant activity against HIV-1NL4-3 with an EC50 value of 0.28 µM. The developed 3D-QSAR model resulted in great predictive ability with training set (r2 = 0.99, q2 = 0.55). Molecular docking studies were complementary to the 3D-QSAR analysis, showing that DSC was differently bound to CA-SP1 with higher affinity than that of bevirimat. MD studies revealed that the complex of the ligand and the protein was stable, with root mean square deviation (RMSD) values <2.5 Å. The above results provided valuable insights into the potential of DSC as a prototype to develop new antiviral agents.

Calix[4]arene Bridge Mononitration with tert-Butyl Nitrite: Synthesis of Bridging Chiral p-tert-Butylcalix[4]arene with a Mononitro Bridge Substituent.

To explore the reaction universality of bridge nitration, the mononitration of different p-tert-butylcalix[4]arene derivatives was executed with tert-butyl nitrite as a nitration reagent. The effects of calix[4]arene conformations, substituents on the lower rim, and reaction conditions on bridge mononitration are systematically studied. The bridge nitration of p-tert-butylcalix[4]arene derivatives in 1,3-alternate, 1,2-alternate, and partial cone conformations can be smoothly executed while that of p-tert-butylcalix[4]arene derivatives strictly regulated in a cone conformation cannot. The nitration product complexity shows a positive correlation with the bridge-hydrogen types, and the optimal bridge-mononitrated substrate is calix[4]arene with only one bridge-hydrogen type. The electron-withdrawing substituent on the lower rim is apparently beneficial for the bridge mononitration. As a result, a variety of bridging chiral p-tert-butylcalix[4]arenes with a mononitro bridge substituent have been successfully synthesized. The highest bridge-mononitrated yield can reach 27% from 1,3-alternate p-tert-butylcalix[4]arene biscrown-5 under optimal reaction conditions.

Mononitration of a Calix[4]arene Methylene Bridge: Synthesis and Preliminary Catalysis Performances of Bridging Chiral p-tert-Butylcalix[4]arenes with a Monoamino Bridge Substituent in a 1,3-Alternate Conformation.

In order to prepare bridging chiral p-tert-butylcalix[4]crown-5 with a mononitro bridge substituent in a 1,3-alternate conformation, a mononitration method of calix[4]arene bridging methylene has been first developed with tert-butyl nitrite as a nitration reagent. The effects of solvent, reaction temperature, reaction time, and nitration reagent dosage on bridge mononitration have been deeply explored to obtain an optimal nitration condition. The facile nitration presents a new key for calix[4]arene bridge derivatization. After further modification and diastereoisomeric resolution, a pair of bridging chiral p-tert-butylcalix[4]arenes with a monoamino bridge substituent were produced from the bridge-mono-nitrated calix[4]arene. Their preliminary catalysis results in the Henry reaction show good catalytic activities (up to 95% yield) and still low but obviously enhanced enantioselectivities (up to 22.3% ee from 7a, 6% ee from 1), which confirms that the structural transformation indeed improves asymmetric catalysis performances of bridging chiral calix[4]crown-5 amines in a 1,3-alternate conformation.

Characterization of the mammalian miRNA turnover landscape.

Steady state cellular microRNA (miRNA) levels represent the balance between miRNA biogenesis and turnover. The kinetics and sequence determinants of mammalian miRNA turnover during and after miRNA maturation are not fully understood. Through a large-scale study on mammalian miRNA turnover, we report the co-existence of multiple cellular miRNA pools with distinct turnover kinetics and biogenesis properties and reveal previously unrecognized sequence features for fast turnover miRNAs. We measured miRNA turnover rates in eight mammalian cell types with a combination of expression profiling and deep sequencing. While most miRNAs are stable, a subset of miRNAs, mostly miRNA*s, turnovers quickly, many of which display a two-step turnover kinetics. Moreover, different sequence isoforms of the same miRNA can possess vastly different turnover rates. Fast turnover miRNA isoforms are enriched for 5' nucleotide bias against Argonaute-(AGO)-loading, but also additional 3' and central sequence features. Modeling based on two fast turnover miRNA*s miR-222-5p and miR-125b-1-3p, we unexpectedly found that while both miRNA*s are associated with AGO, they strongly differ in HSP90 association and sensitivity to HSP90 inhibition. Our data characterize the landscape of genome-wide miRNA turnover in cultured mammalian cells and reveal differential HSP90 requirements for different miRNA*s. Our findings also implicate rules for designing stable small RNAs, such as siRNAs.

Ataxia telangiectasia mutated kinase mediates NF-κB serine 276 phosphorylation and interferon expression via the IRF7-RIG-I amplification loop in paramyxovirus infection.

UNLABELLED: Respiratory syncytial virus (RSV) is a primary etiological agent of childhood lower respiratory tract disease. Molecular patterns induced by active infection trigger a coordinated retinoic acid-inducible gene I (RIG-I)-Toll-like receptor (TLR) signaling response to induce inflammatory cytokines and antiviral mucosal interferons. Recently, we discovered a nuclear oxidative stress-sensitive pathway mediated by the DNA damage response protein, ataxia telangiectasia mutated (ATM), in cytokine-induced NF-κB/RelA Ser 276 phosphorylation. Here we observe that ATM silencing results in enhanced single-strand RNA (ssRNA) replication of RSVand Sendai virus, due to decreased expression and secretion of type I and III interferons (IFNs), despite maintenance of IFN regulatory factor 3 (IRF3)-dependent IFN-stimulated genes (ISGs). In addition to enhanced oxidative stress, RSV replication enhances foci of phosphorylated histone 2AX variant (γH2AX), Ser 1981 phosphorylation of ATM, and IKKγ/NEMO-dependent ATM nuclear export, indicating activation of the DNA damage response. ATM-deficient cells show defective RSV-induced mitogen and stress-activated kinase 1 (MSK-1) Ser 376 phosphorylation and reduced RelA Ser 276 phosphorylation, whose formation is required for IRF7 expression. We observe that RelA inducibly binds the native IFN regulatory factor 7 (IRF7) promoter in an ATM-dependent manner, and IRF7 inducibly binds to the endogenous retinoic acid-inducible gene I (RIG-I) promoter. Ectopic IRF7 expression restores RIG-I expression and type I/III IFN expression in ATM-silenced cells. We conclude that paramyxoviruses trigger the DNA damage response, a pathway required for MSK1 activation of phospho Ser 276 RelA formation to trigger the IRF7-RIG-I amplification loop necessary for mucosal IFN production. These data provide the molecular pathogenesis for defects in the cellular innate immunity of patients with homozygous ATM mutations. IMPORTANCE: RNA virus infections trigger cellular response pathways to limit spread to adjacent tissues. This "innate immune response" is mediated by germ line-encoded pattern recognition receptors that trigger activation of two, largely independent, intracellular NF-κB and IRF3 transcription factors. Downstream, expression of protective antiviral interferons is amplified by positive-feedback loops mediated by inducible interferon regulatory factors (IRFs) and retinoic acid inducible gene (RIG-I). Our results indicate that a nuclear oxidative stress- and DNA damage-sensing factor, ATM, is required to mediate a cross talk pathway between NF-κB and IRF7 through mediating phosphorylation of NF-κB. Our studies provide further information about the defects in cellular and innate immunity in patients with inherited ATM mutations.

Role of hydrogen sulfide in paramyxovirus infections.

UNLABELLED: Hydrogen sulfide (H2S) is an endogenous gaseous mediator that has gained increasing recognition as an important player in modulating acute and chronic inflammatory diseases. However, its role in virus-induced lung inflammation is currently unknown. Respiratory syncytial virus (RSV) is a major cause of upper and lower respiratory tract infections in children for which no vaccine or effective treatment is available. Using the slow-releasing H2S donor GYY4137 and propargylglycin (PAG), an inhibitor of cystathionine-γ-lyase (CSE), a key enzyme that produces intracellular H2S, we found that RSV infection led to a reduced ability to generate and maintain intracellular H2S levels in airway epithelial cells (AECs). Inhibition of CSE with PAG resulted in increased viral replication and chemokine secretion. On the other hand, treatment of AECs with the H2S donor GYY4137 reduced proinflammatory mediator production and significantly reduced viral replication, even when administered several hours after viral absorption. GYY4137 also significantly reduced replication and inflammatory chemokine production induced by human metapneumovirus (hMPV) and Nipah virus (NiV), suggesting a broad inhibitory effect of H2S on paramyxovirus infections. GYY4137 treatment had no effect on RSV genome replication or viral mRNA and protein synthesis, but it inhibited syncytium formation and virus assembly/release. GYY4137 inhibition of proinflammatory gene expression occurred by modulation of the activation of the key transcription factors nuclear factor κB (NF-κB) and interferon regulatory factor 3 (IRF-3) at a step subsequent to their nuclear translocation. H2S antiviral and immunoregulatory properties could represent a novel treatment strategy for paramyxovirus infections. IMPORTANCE: RSV is a global health concern, causing significant morbidity and economic losses as well as mortality in developing countries. After decades of intensive research, no vaccine or effective treatment, with the exception of immunoprophylaxis, is available for this infection as well as for other important respiratory mucosal viruses. This study identifies hydrogen sulfide as a novel cellular mediator that can modulate viral replication and proinflammatory gene expression, both important determinants of lung injury in respiratory viral infections, with potential for rapid translation of such findings into novel therapeutic approaches for viral bronchiolitis and pneumonia.

Distal Junctional Failures in Degenerative Thoracolumbar Hyperkyphosis.

OBJECTIVE: Degenerative thoracolumbar hyperkyphosis (DTH) is a disease that negatively affects individual health and requires surgical intervention, yet the ideal surgical approach and complications, especially distal junctional failures (DJF), remain poorly understood. This study aims to investigate DJF in DTH and to identify the risk factors for DJF so that we can improve surgical decision-making, and advance our knowledge in the field of spinal surgery to enhance patient outcomes. METHODS: This study retrospectively reviewed 78 cases (late osteoporotic vertebral compression fracture [OVCF], 51; Scheuermann's kyphosis [SK], 17; and degenerative disc diseases [DDD], 10) who underwent corrective surgery in our institute from 2008 to 2019. Clinical outcomes were assessed using health-related quality of life (HRQOL) measures, including the visual analogue scale (VAS) scores for back and leg pain, the Oswestry disability index (ODI), and the Japanese Orthopaedic Association (JOA) scoring system. Multiple radiographic parameters, such as global kyphosis (GK) and thoracolumbar kyphosis (TLK), were assessed to determine radiographic outcomes. Multivariate logistic regression analysis was employed to identify the risk factors associated with DJF. RESULTS: HRQOL improved, and GK, TLK decreased at the final follow-up, with a correction rate of 67.7% and 68.5%, respectively. DJF was found in 13 of 78 cases (16.7%), two cases had wedging in the disc (L3-4) below the instrumentation, one case had a fracture of the lowest instrumented vertebrae (LIV), one case had osteoporotic fracture below the fixation, nine cases had pull-out or loosening of the screws at the LIV and three cases (23.1%) required revision surgery. The DJF group had older age, lower computed tomography Hounsfield unit (CT HU), longer follow-up, more blood loss, greater preoperative sagittal vertical axis (SVA), and poorer postoperative JOA and VAS scores (back). The change in TLK level was larger in the non-DJF group. Post-sagittal stable vertebrae (SSV) moved cranially compared with pre-SSV. CONCLUSION: Age, CT HU, length of follow-up, estimated blood loss, and preoperative SVA were independent risk factors for DJF. We recommend fixation of the two vertebrae below the apex vertebrae for DTH to minimize surgical trauma.

Identifying potential Q-markers for quality evaluation of Zhenyuan capsule by integrating chemical analysis, network pharmacology, molecular docking, and molecular dynamics simulations.

Quality markers (Q-markers) are of great significance for quality evaluation of herbal medicines. Zhenyuan Capsule (ZYC) is a kind of Chinese patent medicine used to treat cardiovascular diseases. However, reliable and effective Q-markers for ZYC are still lacking. Herein, a UHPLC-Q/Orbitrap-MS/MS was performed to characterise the preliminary chemical profile of ZYC. A total of 86 components were characterised among which 20 constituents were unambiguously identified by reference compounds. Based on network pharmacology, seven major ginsenosides with great importance in the network were identified as Q-markers among which ginsenoside Re with the highest betweenness was screened to inhibit the development of coronary heart disease (CHD) by binding with vascular endothelial growth factor A (VEGFA). Docking and molecular dynamics simulation studies suggested that ginsenoside Re stably bound to VEGFA. Quantitative determination and chemical fingerprinting analysis were performed using HPLC-DAD. The results showed that ginsenosides screened might function as potential Q-markers for ZYC.

Jak/Stat3 signaling promotes somatic cell reprogramming by epigenetic regulation.

Although leukemia inhibitory factor (LIF) maintains the ground state pluripotency of mouse embryonic stem cells and induced pluripotent stem cells (iPSCs) by activating the Janus kinase/signal transducer and activator of transcription 3 (Jak/Stat3) pathway, the mechanism remained unclear. Stat3 has only been shown to promote complete reprogramming of epiblast and neural stem cells and partially reprogrammed cells (pre-iPSCs). We investigated if and how Jak/Stat3 activation promotes reprogramming of terminally differentiated mouse embryonic fibroblasts (MEFs). We demonstrated that activated Stat3 not only promotes but also is essential for the pluripotency establishment of MEFs during reprogramming. We further demonstrated that during this process, inhibiting Jak/Stat3 activity blocks demethylation of Oct4 and Nanog regulatory elements in induced cells, which are marked by suppressed endogenous pluripotent gene expression. These are correlated with the significant upregulation of DNA methyltransferase (Dnmt) 1 and histone deacetylases (HDACs) expression as well as the increased expression of lysine-specific histone demethylase 2 and methyl CpG binding protein 2. Inhibiting Jak/Stat3 also blocks the expression of Dnmt3L, which is correlated with the failure of retroviral transgene silencing. Furthermore, Dnmt or HDAC inhibitor but not overexpression of Nanog significantly rescues the reprogramming arrested by Jak/Stat3 inhibition or LIF deprivation. Finally, we demonstrated that LIF/Stat3 signal also represents the prerequisite for complete reprogramming of pre-iPSCs. We conclude that Jak/Stat3 activity plays a fundamental role to promote pluripotency establishment at the epigenetic level, by facilitating DNA demethylation/de novo methylation, and open-chromatin formation during late-stage reprogramming.

How to make a more optimal surgical plan for Lenke 5 adolescent idiopathic scoliosis patients: a comparative study based on the changes of the sagittal alignment and selection of the lowest instrumented vertebra.

BACKGROUND: The treatment of patients with Lenke 5 adolescent idiopathic scoliosis (AIS) is closely related to the pelvic because the spine-pelvis is an interacting whole. Besides, the choice of fusion segment is a significant issue; with the optimal choice, there will be fewer complications and restoring the pelvic morphology to some extent. This study aims to analyze the impact of changes in sagittal parameters and selection of the lowest instrumented vertebra (LIV) on spine and pelvic morphology for better surgical strategy. METHOD: Ninety-four patients with Lenke 5 AIS who underwent selective posterior thoracolumbar/lumbar (TL/L) curve fusion were included in the study and grouped according to pelvic morphology and position of LIV. Spinopelvic parameters were measured preoperatively, postoperatively, and at the latest follow-up. The patient's preoperative and last follow-up quality of life was assessed with the MOS item short-form health survey (SF-36) and scoliosis research society 22-item (SRS-22). RESULT: Patients being posterior pelvic tilt had the oldest mean age (P = 0.010), the smallest lumbar lordosis (LL) (P = 0.036), the smallest thoracic kyphosis (TK) (P = 0.399) as well as the smallest proximal junctional angle (PJA) while those being anterior pelvic tilt had the largest PJA. The follow-up TK significantly increased in both groups of anterior and normal pelvic tilt (P < 0.039, P < 0.006) while no significant changes were observed in the posterior pelvic tilt group. When LIV is above L4, the follow-up PJA was larger than other groups (P = 0.049, P = 0.006). When LIV is below L4, the follow-up TK and PT were larger and LL was smaller than other groups(P < 0.05). The SF-36 and SRS-22 scores were better in the LIV = L4 group than in other groups at the last follow-up (P < 0.05). CONCLUSION: The correction of TK and LL after surgery can improve pelvic morphology. Besides, LIV is best set at L4, which will facilitate the recovery of TK, the improvement of symptoms, and the prevention of complications and pelvic deformities. Level of evidence Level III.

High-efficiency siRNA-based gene knockdown in human embryonic stem cells.

Loss-of-function studies in human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) via nonviral approaches have been largely unsuccessful. Here we report a simple and cost-effective method for high-efficiency delivery of plasmids and siRNAs into hESCs and iPSCs. Using this method for siRNA delivery, we achieve >90% reduction in the expression of the stem cell factors Oct4 and Lin28, and observe cell morphological and staining pattern changes, characteristics of hESC differentiation, as a result of Oct4 knockdown.

Hepatitis C virus NS2 protein serves as a scaffold for virus assembly by interacting with both structural and nonstructural proteins.

Many aspects of the assembly of hepatitis C virus (HCV) remain incompletely understood. To characterize the role of NS2 in the production of infectious virus, we determined NS2 interaction partners among other HCV proteins during productive infection. Pulldown assays showed that NS2 forms complexes with both structural and nonstructural proteins, including E1, E2, p7, NS3, and NS5A. Confocal microscopy also demonstrated that NS2 colocalizes with E1, E2, and NS5A in dot-like structures near lipid droplets. However, NS5A did not coprecipitate with E2 and interacted only weakly with NS3 in pulldown assays. Also, there was no demonstrable interaction between p7 and E2 or NS3 in such assays. Therefore, NS2 is uniquely capable of interacting with both structural and nonstructural proteins. Among mutations in p7, NS2, and NS3 that prevent production of infectious virus, only p7 mutations significantly reduced NS2-mediated protein interactions. These p7 mutations altered the intracellular distribution of NS2 and E2 and appeared to modulate the membrane topology of the C-terminal domain of NS2. These results suggest that NS2 acts to coordinate virus assembly by mediating interactions between envelope proteins and NS3 and NS5A within replication complexes adjacent to lipid droplets, where virus particle assembly is thought to occur. p7 may play an accessory role by regulating NS2 membrane topology, which is important for NS2-mediated protein interactions and therefore NS2 function.

Lin28-mediated post-transcriptional regulation of Oct4 expression in human embryonic stem cells.

Lin28 acts as a repressor of microRNA processing and as a post-transcriptional regulatory factor for a subset of mRNAs. Here we report that in human embryonic stem cells Lin28 facilitates the expression of the pivotal pluripotency factor Oct4 at the post-transcriptional level. We provide evidence that Lin28 binds Oct4 mRNA directly through high affinity sites within its coding region and that an interaction between Lin28 and RNA helicase A (RHA) may play a part in the observed regulation. We further demonstrate that decreasing RHA levels impairs Lin28-dependent stimulation of translation in a reporter system. Taken together with previous studies showing that RHA is required for efficient translation of a specific class of mRNAs, these findings suggest a novel mechanism by which Lin28 may affect target mRNA expression and represent the first evidence of post-transcriptional regulation of Oct4 expression by Lin28 in human embryonic stem cells.

Study on the Relationship between the Use of Bisphosphonates for Antiosteoporosis and Vertebral Re-Fracture after Vertebroplasty.

Objective: To explore the effect of bisphosphonates after vertebroplasty in patients with osteoporotic vertebral compression fractures (OVCF), and to analyze the relationship between the use of bisphosphonates and vertebral refracture. Methods: A total of 150 patients with OVCF were selected from the pain department of our hospital from January 2018 to May 2020. All patients received vertebroplasty after admission, and were divided into the surgery group (62 cases) and combined with the bisphosphonates group (combined group, 88 cases) according to whether patients had used bisphosphonates after surgery. Before surgery, 1 month, 3 months, 6 months, and 1 year after surgery, visual analogue scale (VAS), Oswestry disability index (ODI), vertebral body and femoral neck bone mineral density (BMD), and Cobb Angle were collected, and the differences among groups were compared to analyze the treatment effect. After the follow-up, patients were divided into two groups according to whether vertebral refracture occurred during the follow-up period. Clinical characteristics, general information, and surgical indicators of patients in the two groups were collected, and related factors of postoperative vertebral refracture were analyzed. Results: There were no significant differences in preoperative VAS score, ODI index, BMD value, and Cobb angle between the two groups (P > 0.05). At 12 months after surgery, VAS score, ODI index, and Cobb angle decreased, while BMD value increased in both groups. The VAS score, ODI index, and Cobb angle in the combined group were lower than those in the operation group, while the BMD value was higher than that in the operation group, and the difference was significant (P < 0.05). The results of multivariate regression analysis showed that in BMD, no postoperative antiosteoporosis treatment, bone cement leakage, and poor cement diffusion were independent risk factors for vertebral refracture after vertebroplasty in patients with vertebral compression fractures. Conclusion: In order to avoid recurrent fractures in OVCF patients, attention should be paid to BMD, whether patients take antiosteoporosis drugs, whether bone cement permeation occurs and the diffusion of bone cement, etc. The above factors are the main influencing factors leading to recurrent fractures after PKP and PVP in the clinic.

The role of FYCO1-dependent autophagy in lens fiber cell differentiation.

FYCO1 (FYVE and coiled-coil domain containing 1) is an adaptor protein, expressed ubiquitously and required for microtubule-dependent, plus-end-directed transport of macroautophagic/autophagic vesicles. We have previously shown that loss-of-function mutations in FYCO1 cause cataracts with no other ocular and/or extra-ocular phenotype. Here, we show fyco1 homozygous knockout (fyco1-/-) mice recapitulate the cataract phenotype consistent with a critical role of FYCO1 and autophagy in lens morphogenesis. Transcriptome coupled with proteome and metabolome profiling identified many autophagy-associated genes, proteins, and lipids respectively perturbed in fyco1-/- mice lenses. Flow cytometry of FYCO1 (c.2206C>T) knock-in (KI) human lens epithelial cells revealed a decrease in autophagic flux and autophagic vesicles resulting from the loss of FYCO1. Transmission electron microscopy showed cellular organelles accumulated in FYCO1 (c.2206C>T) KI lens-like organoid structures and in fyco1-/- mice lenses. In summary, our data confirm the loss of FYCO1 function results in a diminished autophagic flux, impaired organelle removal, and cataractogenesis.Abbreviations: CC: congenital cataracts; DE: differentially expressed; ER: endoplasmic reticulum; FYCO1: FYVE and coiled-coil domain containing 1; hESC: human embryonic stem cell; KI: knock-in; OFZ: organelle-free zone; qRT-PCR: quantitative real-time PCR; PE: phosphatidylethanolamine; RNA-Seq: RNA sequencing; SD: standard deviation; sgRNA: single guide RNA; shRNA: shorthairpin RNA; TEM: transmission electron microscopy; WT: wild type.

Trans-complementation of an NS2 defect in a late step in hepatitis C virus (HCV) particle assembly and maturation.

Recent studies using cell culture infection systems that recapitulate the entire life cycle of hepatitis C virus (HCV) indicate that several nonstructural viral proteins, including NS2, NS3, and NS5A, are involved in the process of viral assembly and release. Other recent work suggests that Ser-168 of NS2 is a target of CK2 kinase-mediated phosphorylation, and that this controls the stability of the genotype 1a NS2 protein. Here, we show that Ser-168 is a critical determinant in the production of infectious virus particles. Substitution of Ser-168 with Ala (or Gly) ablated production of infectious virus by cells transfected with a chimeric viral RNA (HJ3-5) containing core-NS2 sequences from the genotype 1a H77 virus within the background of genotype 2a JFH1 virus. An S168A substitution also impaired production of virus by cells transfected with JFH1 RNA. This mutation did not alter polyprotein processing or genome replication. This defect in virus production could be rescued by expression of wt NS2 in trans from an alphavirus replicon. The trans-complementing activities of NS2 from genotypes 1a and 2a demonstrated strong preferences for rescue of the homologous genotype. Importantly, the S168A mutation did not alter the association of core or NS5A proteins with host cell lipid droplets, nor prevent the assembly of core into particles with sedimentation and buoyant density properties similar to infectious virus, indicating that NS2 acts subsequent to the involvement of core, NS5A, and NS3 in particle assembly. Second-site mutations in NS2 as well as in NS5A can rescue the defect in virus production imposed by the S168G mutation. In aggregate, these results indicate that NS2 functions in trans, in a late-post assembly maturation step, perhaps in concert with NS5A, to confer infectivity to the HCV particle.

A peripheral neuroimmune link: glutamate agonists upregulate NMDA NR1 receptor mRNA and protein, vimentin, TNF-alpha, and RANTES in cultured human synoviocytes.

Human primary and clonal synovial cells were incubated with glutamate receptor agonists to assess their modulating influence on glutamate receptors N-methyl-d-aspartate (NMDA) NR1 and NR2 and inflammatory cytokines to determine potential for paracrine or autocrine (neurocrine) upregulation of glutamate receptors, as has been shown for bone and chondrocytes. Clonal SW982 synoviocytes constitutively express vimentin, smooth muscle actin (SMA), and NMDA NR1 and NR2. Coincubation (6 h) with glutamate agonists NMDA (5 microM), and the NMDA NR1 glycine site activator (+/-)1-aminocyclopentane-cis-1,3-dicarboxylic acid (5 muM), significantly increases cellular mRNA and protein levels of glutamate receptors, as well as increasing vimentin, SMA, tumor necrosis factor-alpha, and RANTES (regulated on activation, normal T-cell expressed and secreted), assessed qualitatively and quantitatively with nucleotide amplification, image analysis of immunocytochemical staining, fluorescein-activated cell sorting, Western blotting, and immunoassays. Human primary synovial cells harvested from patients with arthritic conditions also constitutively expressed NMDA NR1 with increases after agonist treatment. Glutamate receptor agonist-induced increases were blocked by the noncompetitive glutamate antagonist MK-801 (8 microg/ml) and NR1 blocking antibody. Coincubation with glutamate agonists and phorbol 12-myristate 13-acetate, a protein kinase C activator, significantly enhanced mean levels of TNF-alpha and RANTES in SW982 cell supernatants compared with incubation with either agent alone. Increases were diminished with protein kinase inhibitor and NR1 blocking antibody. The functional activation of glutamate receptors on human synoviocytes establishes a neurogenic cell signaling link between neurotransmitter glutamate released from nerve terminals and target cells in the joint capsule. The influence of glutamate on subsequent release of cellular proinflammatory mediators in non-neural tissue for activation of downstream immune events supports a peripheral neuroimmune link in arthritis.

NS3 helicase domains involved in infectious intracellular hepatitis C virus particle assembly.

A mutation within subdomain 1 of the hepatitis C virus (HCV) NS3 helicase (NS3-Q221L) (M. Yi, Y. Ma, J. Yates, and S. M. Lemon, J. Virol. 81:629-638, 2007) rescues a defect in production of infectious virus by an intergenotypic chimeric RNA (HJ3). Although NS3-Gln-221 is highly conserved across HCV genotypes, the Leu-221 substitution had no effect on RNA replication or NS3-associated enzymatic activities. However, while transfection of unmodified HJ3 RNA failed to produce either extracellular or intracellular infectious virus, transfection of HJ3 RNA containing the Q221L substitution (HJ3/QL) resulted in rapid accumulation of intracellular infectious particles with release into extracellular fluids. In the absence of the Q221L mutation, both NS5A and NS3 were recruited to core protein on the surface of lipid droplets, but there was no assembly of core into high-density, rapidly sedimenting particles. Further analysis demonstrated that a Q221N mutation minimally rescued virus production and led to a second-site I399V mutation in subdomain 2 of the helicase. Similarly, I399V alone allowed only low-level virus production and led to selection of an I286V mutation in subdomain 1 of the helicase which fully restored virus production, confirming the involvement of both major helicase subdomains in the assembly process. Thus, multiple mutations in the helicase rescue a defect in an early-intermediate step in virus assembly that follows the recruitment of NS5A to lipid droplets and precedes the formation of dense intracellular viral particles. These data reveal a previously unsuspected role for the NS3 helicase in early virion morphogenesis and provide a new perspective on HCV assembly.

Identification of the Genome-wide Expression Patterns of Long Non-coding RNAs and mRNAs in Mice with Streptozotocin-induced Diabetic Neuropathic Pain.

Diabetic neuropathic pain (DNP), an early symptom of diabetic neuropathy, involves complex mechanisms. Long non-coding RNA (lncRNA) dysregulation contributes to the pathogenesis of various human diseases. Here, we investigated the genome-wide expression patterns of lncRNAs and genes in the spinal dorsal horn of mice with streptozotocin-induced DNP. Microarray analysis identified 1481 differentially expressed (DE) lncRNAs and 1096 DE mRNAs in DNP mice. Functional analysis showed that transforming growth factor-beta receptor binding was the most significant molecular function and retrograde endocannabinoid signaling was the most significant pathway of DE mRNAs. Calcium ion transport was the second most significant biological process of DE lncRNAs. Finally, we found 289 neighboring and 57 overlapping lncRNA-mRNA pairs, including ENSMUST00000150952-Mbp and AK081017-Usp15, which may be involved in DNP pathogenesis. Microarray data were validated through quantitative PCR of selected lncRNAs and mRNAs. These results suggest that aberrant expression of lncRNAs may contribute to the pathogenesis of DNP.

Comparative transcriptome analysis of hESC- and iPSC-derived lentoid bodies.

The ocular lens serves as an excellent system to investigate the intricate details of development and differentiation. Generation of lentoid bodies or lens-like structures using pluripotent stem cells is important for understanding the processes critical for lens morphogenesis and the mechanism of cataractogenesis. We previously reported the generation of peripheral blood mononuclear cell (PBMC)-originated, induced pluripotent stem cells (iPSCs). Here, we report generation of lentoid bodies from human embryonic stem cells (hESCs) and (PBMC)-originated, iPSCs employing the "fried egg" method with brief modifications. The ultrastructure analysis of hESC- and iPSC-derived lentoid bodies identified closely packed lens epithelial- and differentiating fiber-like cells. In addition, we performed RNA sequencing (RNA-Seq) based transcriptome profiling of hESC- and iPSC-derived lentoid bodies at differentiation day 25. Next-generation RNA sequencing (RNA-Seq) of hESC- and iPSC-derived lentoid bodies detected expression (≥0.659 RPKM) of 13,975 and 14,003 genes, respectively. Comparative transcriptome analysis of hESC- and iPSC-derived lentoid bodies revealed 13,563 (>96%) genes common in both datasets. Among the genes common in both transcriptome datasets, 12,856 (~95%) exhibited a quantitatively similar expression profile. Next, we compared the mouse lens epithelial and fiber cell transcriptomes with hESC- and iPSC-derived lentoid bodies transcriptomes and identified > 96% overlap with lentoid body transcriptomes. In conclusion, we report first-time comparative transcriptome analysis of hESC- and iPSC-derived lentoid bodies at differentiation day 25.