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This study investigates the effect of shield tunnel grouting on excess pore pressure distributions around tunnel and ground consolidation settlement. First, it presents half-plane excess pore pressure around an unlined tunnel caused by grouting pressure based on the complex variable theory and the conformal mapping method. Second, the analytical solution for the soil consolidation is derived using the conformal transformation method and the consolidation theory of Terzaghi–Rendulic. The drainage conditions of the tunnel lining are idealized into three cases: fully drained, partially drained, and impermeable along the tunnel circumference. The impacts of the burial depth of the tunnel, the permeability coefficient of the tunnel lining and the surrounding soil, the Poisson’s ratio of the soil, the lateral Earth pressure coefficient of the soil, and the shear modulus are investigated.

Pyrometallurgical processes have been widely recognised as the preferred technology for E-scrap recycling, and E-scrap smelting is usually accompanied by the formation of high-Al2O3-content slag. However, this slag can cause problems, such as refractory wear and even accelerated failure. Therefore, the corrosion mechanism of magnesia-chrome and alumina-chrome refractories in contact with FeOx-SiO2-CaO-Al2O3 smelting slag was experimentally studied in the range of 1300–1400 °C, in air (pO2 = 0.21 atm) and at pO2 = 10-7 atm. The results showed that refractory-component dissolution and new phases formation were the major causes of refractory degradation. The dissolution of the refractory components increased along with both increasing temperature and decreasing Al2O3 concentration in the initial slag. When decreasing the pO2, the dissolution of MgO and Al2O3 into the slag slowed down. New phases such as forsterite, anorthite, and spinel were detected at the interface between the slag and the bulk refractory. The formation of forsterite accelerated the refractory wear, whereas the newly formed (MgO,FeO)·(Cr2O3,Fe2O3,Al2O3) spinel phase acted as a protective layer against further corrosion. A lower Al2O3 concentration in the slag and a higher smelting temperature both densified the formed spinel layer. Additionally, a high oxygen partial pressure was beneficial to the formation of spinel in the magnesia-chrome refractory corrosion experiments. However, a low oxygen partial pressure was favourable for the formation of spinel on the surface of the alumina-chrome refractory materials. This study provides useful information for designing refractory materials and optimizing the processing parameters of E-scrap smelting.

Elevated serum cytokine production in COVID-19 patients associates with disease progression and severity. However, the stimulus that initiates cytokine production in patients remains to be fully revealed. Virus-infected cells can release virus-associated exosomes, extracellular vesicles (EVs) of endocytic origin, into the blood to deliver viral cargoes able to regulate immune responses. Here, we report that plasma exosomes from COVID-19 patients contain SARS-CoV-2 RNA. COVID-19 plasma exosomes stimulated robust production of interleukin-6 (IL-6), IL-8, tumor necrosis factor-α (TNF-α), and other inflammatory cytokines and chemokines in human peripheral mononuclear cells (PBMCs). Exosome depletion abolished these stimulated responses. COVID-19 plasma exosomes induced pro-inflammatory responses in CD4+ T cells, CD8+ T cells, and CD14+ monocytes, but not significantly in regulatory T cells, Th17 T cells, or memory T cells. COVID-19 plasma exosomes carry viral double-stranded RNA (dsRNA) intermediates, protect the dsRNA cargo from RNase, and deliver the dsRNA to recipient cells. COVID-19 plasma exosomes significantly increase expression of endosomal toll-like receptors 3 (TLR3), TLR7, TLR8, and TLR9 in peripheral T cells and monocytes. Inhibition of TLR3 by a specific pharmacological inhibitor considerably reduced production of cytokines and chemokines in CD4+ and CD8+ T cells, but not in CD14+ monocytes, highlighting divergent signaling pathways of immune cells in response to COVID-19 plasma exosomes. Our results indicate a novel model of crosstalk between SARS-CoV-2 infection and immune responses able to contribute to elevated cytokine production associated with COVID-19 progression, severity, and long-haul symptoms.

Single particle experiments were carried out in the present study. An industrial copper matte powder, containing 68.04 wt.% Cu, 0.50 wt.% Se and 0.08 wt.% Te was taken as the starting material. Se and Te phase assemblies and distribution behaviors were systematically investigated in the air atmosphere. It was found that Se reported to blister copper, matte, and oxide phase, whilst Te will enrich in oxide phases, including phases in the Fe-Si-Te-O, Ca-Fe-Te-O, and Ca-Te-O-(S) systems. As the temperature increases, the selenium and tellurium content in the copper and matte phases decreases. The introduction of CaO flux, and typical impurities of Pb and Si, had significant influence on Se and Te distribution. The results revealed that CaO addition and Si impurity accelerated the removal of Se and Te from the copper and matte phases, but Pb impurity increased Se and Te in Cu-rich phases.

Despite potent suppression of HIV-1 viral replication in the central nervous system (CNS) by antiretroviral therapy (ART), between 15% and 60% of HIV-1-infected patients receiving ART exhibit neuroinflammation and symptoms of HIV-1-associated neurocognitive disorder (HAND) - a significant unmet challenge. We propose that the emergence of HIV-1 from latency in microglia underlies both neuroinflammation in the CNS and the progression of HAND. Recent molecular studies of cellular silencing mechanisms of HIV-1 in microglia show that HIV-1 latency can be reversed both by proinflammatory cytokines and by signals from damaged neurons, potentially creating intermittent cycles of HIV-1 reactivation and silencing in the brain. We posit that anti-inflammatory agents that also block HIV-1 reactivation, such as nuclear receptor agonists, might provide new putative therapeutic avenues for the treatment of HAND.

Human immune deficiency virus (HIV) infection of microglial cells in the brain leads to chronic neuroinflammation, which is antecedent to the development of HIV-associated neurocognitive disorders (HAND) in the majority of patients. Productively HIV infected microglia release multiple neurotoxins including proinflammatory cytokines and HIV proteins such as envelope glycoprotein (gp120) and transactivator of transcription (Tat). However, powerful counteracting silencing mechanisms in microglial cells result in the rapid shutdown of HIV expression to limit neuronal damage. Here we investigated whether the Nerve Growth Factor IB-like nuclear receptor Nurr1 (NR4A2), which is a repressor of inflammation in the brain, acts to directly restrict HIV expression. HIV silencing was substantially enhanced by Nurr1 agonists in both immortalized human microglial cells (hµglia) and induced pluripotent stem cells (iPSC)-derived human microglial cells (iMG). Overexpression of Nurr1 led to viral suppression, whereas by contrast, knock down (KD) of endogenous Nurr1 blocked HIV silencing. Chromatin immunoprecipitation (ChIP) assays showed that Nurr1 mediates recruitment of the CoREST/HDAC1/G9a/EZH2 transcription repressor complex to HIV promoter resulting in epigenetic silencing of active HIV. Transcriptomic studies demonstrated that in addition to repressing HIV transcription, Nurr1 also downregulated numerous cellular genes involved in inflammation, cell cycle, and metabolism, thus promoting HIV latency and microglial homoeostasis. Thus, Nurr1 plays a pivotal role in modulating the cycles of proviral reactivation by cytokines and potentiating the proviral transcriptional shutdown. These data highlight the therapeutic potential of Nurr1 agonists for inducing HIV silencing and microglial homeostasis and amelioration of the neuroinflammation associated with HAND.AUTHOR SUMMARYHIV enters the brain almost immediately after infection where it infects perivascular macrophages, microglia and, to a less extent, astrocytes. In previous work using an immortalized human microglial cell model, we observed that integrated HIV constantly underwent cycles of reactivation and subsequent silencing. In the present study, we found that the Nurr1 nuclear receptor is a key mediator of HIV silencing. The functional activation of Nurr1 by specific agonists, or the over expression of Nurr1, resulted in rapid silencing of activated HIV in microglial cells. Global gene expression analysis confirmed that Nurr1 not only repressed HIV expression but also regulated numerous genes involved in microglial homeostasis and inflammation. Thus, Nurr1 is pivotal for HIV silencing and repression of inflammation in the brain and is a promising therapeutic target for treatment of HAND.

Human immune deficiency virus (HIV) infection in the brain leads to chronic neuroinflammation due to the production of pro-inflammatory cytokines, which in turn promotes HIV transcription in infected microglial cells. However, powerful counteracting silencing mechanisms in microglial cells result in the rapid shutdown of HIV expression after viral reactivation to limit neuronal damage. Here we investigated whether the Nerve Growth Factor IB-like nuclear receptor Nurr1 (NR4A2), which is a repressor of inflammation in the brain, acts directly to restrict HIV expression. HIV silencing following activation by TNF-α, or a variety of toll-like receptor (TLR) agonists, in both immortalized human microglial cells (hμglia) and induced pluripotent stem cells (iPSC)-derived human microglial cells (iMG) was enhanced by Nurr1 agonists. Similarly, overexpression of Nurr1 led to viral suppression, while conversely, knock down (KD) of endogenous Nurr1 blocked HIV silencing. The effect of Nurr1 on HIV silencing is direct: Nurr1 binds directly to the specific consensus binding sites in the U3 region of the HIV LTR and mutation of the Nurr1 DNA binding domain blocked its ability to suppress HIV-1 transcription. Chromatin immunoprecipitation (ChIP) assays also showed that after Nurr1 binding to the LTR, the CoREST/HDAC1/G9a/EZH2 transcription repressor complex is recruited to the HIV provirus. Finally, transcriptomic studies demonstrated that in addition to repressing HIV transcription, Nurr1 also downregulated numerous cellular genes involved in inflammation, cell cycle, and metabolism, further promoting HIV latency and microglial homoeostasis. Nurr1 therefore plays a pivotal role in modulating the cycles of proviral reactivation by potentiating the subsequent proviral transcriptional shutdown. These data highlight the therapeutic potential of Nurr1 agonists for inducing HIV silencing and microglial homeostasis and ultimately for the amelioration of the neuroinflammation associated with HIV-associated neurocognitive disorders (HAND).

To improve the sulfuric-acid leaching of germanium-bearing secondary zinc oxide, the occurrence states and sulfuric-acid leaching behavior of germanium in secondary zinc oxide were investigated experimentally via inductively coupled plasma optical emission spectrometry (ICP–OES), X-ray diffraction (XRD), scanning electron microscopy and energy-dispersive spectroscopy (SEM–EDS), electron probe microanalysis (EPMA), and chemical phase analysis combined with leaching tests. The germanium-bearing secondary zinc oxide was produced via a fuming process of lead smelting slag and zinc leaching residue in a lead and zinc smelter. The results show that zincite, wurtzite, galena, and anglesite are the major minerals containing zinc and lead in the secondary zinc oxide. Further, these zinc- and lead-bearing materials contain germanium (without independent minerals). In the sulfuric-acid leaching process without oxidant, wurtzite and galena cannot be leached, which results in a loss of germanium. Anglesite, which exists originally in the secondary zinc oxide or was formed in the leaching process, contains a low amount of germanium and exhibits a loss of germanium in the residue. Moreover, the leaching efficiencies of germanium and zinc increased to 99.7% and 99.4% in the leaching process under oxygen pressure, respectively.

Phase relations in the REM2O3-SiO2 (REM = Y/Yb/La) systems have been studied using the equilibration/quenching/scanning electron microscopy (SEM)-electron probe microanalysis (EPMA) technique. The solvus and liquidus between 1400°C and 1650°C have been determined. The results obtained in this study show that a maximum of 0.57 wt.% Yb2O3 can dissolve in SiO2 phase, while the maximum solubility of La2O3 and Y2O3 in SiO2 phase was 0.22 wt.% and 0.07 wt.%, respectively. Correspondingly, SiO2 shows low solubility in La2Si2O7, and nearly does not dissolve in Y2Si2O7 or Yb2Si2O7. No liquid phase was detected in the Y2O3-SiO2 or Yb2O3-SiO2 system in the temperature range of interest. One oxide liquid phase comprising 39.27 wt.% SiO2 and 59.07 wt.% La2O3 was found in the La2O3-SiO2 system sample equilibrated at 1653°C, revealing that the eutectic point temperature between La2Si2O7 and SiO2 is located between 1600°C and 1653°C. Results of this study were compared with previous observations, revealing significant differences.

Additional file 1: Table S1. Composition and nutrient levels of basal diets for broilers. Table S2. Statistics of sample sequencing data processing result. Figure S1. Rarefaction curve and Shannon index curve drawn based on sequencing data.

HIV-1 infects lymphoid and myeloid cells, which can harbor a latent proviral reservoir responsible for maintaining lifelong infection. Glycolytic metabolism has been identified as a determinant of susceptibility to HIV-1 infection, but its role in the development and maintenance of HIV-1 latency has not been elucidated. By combining transcriptomic, proteomic and metabolomic analysis, we here show that transition to latent HIV-1 infection downregulates glycolysis, while viral reactivation by conventional stimuli reverts this effect. Decreased glycolytic output in latently infected cells is associated with downregulation of NAD+/NADH. Consequently, infected cells rely on the parallel pentose phosphate pathway and its main product, the antioxidant NADPH, fueling antioxidant pathways maintaining HIV-1 latency. Of note, blocking NADPH downstream effectors, thioredoxin and glutathione, favors HIV-1 reactivation from latency in lymphoid and myeloid cellular models. This provides a “shock and kill effect” decreasing proviral DNA in cells from people-living-with-HIV/AIDS. Overall, our data show that downmodulation of glycolysis is a metabolic signature of HIV-1 latency that can be exploited to target latently infected cells with eradication strategies.

Kaposi sarcoma-associated herpesvirus (KSHV) stimulates proliferation, angiogenesis, and inflammation to promote Kaposi sarcoma (KS) tumor growth, which involves various growth factors and cytokines. Previously, we found that KSHV infection of human umbilical vein endothelial cells (HUVECs) induces a transcriptional induction of the proangiogenic and proinflammatory cytokine angiopoietin-2 (Ang-2). Here, we report that KSHV induces rapid release of Ang-2 that is presynthesized and stored in the Weibel-Palade bodies (WPB) of endothelial cells upon binding to its integrin receptors. Blocking viral binding to integrins inhibits Ang-2 release. KSHV binding activates the integrin tyrosine kinase receptor signaling pathways, leading to tyrosine phosphorylation of focal adhesion kinase (FAK), the tyrosine kinase Src, and the Calα2 subunit of the l-type calcium channel to trigger rapid calcium (Ca(2+)) influx. Pretreatment of endothelial cells with specific inhibitors of protein tyrosine kinases inhibits KSHV-induced Ca(2+) influx and Ang-2 release. Inhibition of Ca(2+) mobilization with calcium channel blockers also inhibits Ang-2 release. Thus, the interaction between KSHV and its integrin receptors plays a key role in regulating rapid Ang-2 release from endothelial cells. This finding highlights a novel mechanism of viral induction of angiogenesis and inflammation, which might play important roles in the early event of KS tumor development.

Angiopoietin/tyrosine protein kinase receptor Tie-2 signaling in endothelial cells plays an essential role in angiogenesis and wound healing. Angiopoietin-1 (Ang-1) is crucial for blood vessel maturation while angiopoietin-2 (Ang-2), in collaboration with vascular endothelial growth factor (VEGF), initiates angiogenesis by destabilizing existing blood vessels. In healthy people, the Ang-1 level is sustained while Ang-2 expression is restricted. In cancer patients, Ang-2 level is elevated, which correlates with poor prognosis. Ang-2 not only drives tumor angiogenesis but also attracts infiltration of myeloid cells. The latter rapidly differentiate into tumor stromal cells that foster tumor angiogenesis and progression, and weaken the host’s anti-tumor immunity. Moreover, through integrin signaling, Ang-2 induces expression of matrix metallopeptidases (MMPs) to promote tumor cell invasion and metastasis. Many oncogenic viruses induce expression of Ang-2 to promote development of neoplasia associated with viral infection. Multiple Ang-2 inhibitors exhibit remarkable anti-tumor activities, further highlighting the importance of Ang-2 in cancer development.

Despite effective antiretroviral therapy (ART), HIV-associated neurocognitive disorders (HAND) are found in nearly one-third of patients. Using a cellular co-culture system including neurons and human microglia infected with HIV (hμglia/HIV), we investigated the hypothesis that HIV-dependent neurological degeneration results from the periodic emergence of HIV from latency within microglial cells in response to neuronal damage or inflammatory signals. When a clonal hμglia/HIV population (HC69) expressing HIV, or HIV infected human primary and iPSC-derived microglial cells, were cultured for a short-term (24 h) with healthy neurons, HIV was silenced. The neuron-dependent induction of latency in HC69 cells was recapitulated using induced pluripotent stem cell (iPSC)-derived GABAergic cortical (iCort) and dopaminergic (iDopaNer), but not motor (iMotorNer), neurons. By contrast, damaged neurons induce HIV expression in latently infected microglial cells. After 48–72 h co-culture, low levels of HIV expression appear to damage neurons, which further enhances HIV expression. There was a marked reduction in intact dendrites staining for microtubule associated protein 2 (MAP2) in the neurons exposed to HIV-expressing microglial cells, indicating extensive dendritic pruning. To model neurotoxicity induced by methamphetamine (METH), we treated cells with nM levels of METH and suboptimal levels of poly (I:C), a TLR3 agonist that mimics the effects of the circulating bacterial rRNA found in HIV infected patients. This combination of agents potently induced HIV expression, with the METH effect mediated by the σ1 receptor (σ1R). In co-cultures of HC69 cells with iCort neurons, the combination of METH and poly(I:C) induced HIV expression and dendritic damage beyond levels seen using either agent alone, Thus, our results demonstrate that the cross-talk between healthy neurons and microglia modulates HIV expression, while HIV expression impairs this intrinsic molecular mechanism resulting in the excessive and uncontrolled stimulation of microglia-mediated neurotoxicity., Author summary Now that HIV patients are living longer due to improved anti-retroviral therapy, the prevalence of HIV-associated neurocognitive disorders (HAND) is rising. HAND is linked to chronic inflammation and microgliosis and is exacerbated by substances of abuse such as methamphetamine (METH). Using co-culture models of neurons and microglia, we demonstrate that healthy neurons can suppress HIV transcription in infected microglia over a short-term (24 h). By contrast, damaged neurons reactivate latent HIV expression. Thus, over a longer term (72 h), neurons lose the ability to suppress HIV expression, resulting in enhanced neural injury. This damaging cycle of latency reversal and neuronal degeneration can be exacerbated by METH and inflammation. Our results support the hypothesis that the dendritic simplification and neuronal injury associated with HAND results from viral reactivation induced by neuronal and microglial cross-talk and exogenous inflammatory stimuli.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causal agent for Kaposi’s sarcoma (KS), the most common malignancy in HIV/AIDS patients. Oral transmission through saliva is considered the most common route for spreading the virus among HIV/AIDS patients. However, the role of HIV-specific components in the cotransfection of KSHV is unclear. We demonstrate that exosomes purified from the saliva of HIV-positive patients and secreted by HIV-infected T-cell lines promote KSHV infectivity in immortalized and primary oral epithelial cells. HIV-associated exosomes promote KSHV infection, which depends on HIV trans-activation response element (TAR) RNA and EGFR of oral epithelial cells, which can be targeted for reducing KSHV infection. These results reveal that HIV-associated exosomes are a risk factor for KSHV infection in the HIV-infected population., Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causal agent for Kaposi’s sarcoma (KS), the most common malignancy in people living with human immunodeficiency virus (HIV)/AIDS. The oral cavity is a major route for KSHV infection and transmission. However, how KSHV breaches the oral epithelial barrier for spreading to the body is not clear. Here, we show that exosomes purified from either the saliva of HIV-positive individuals or the culture supernatants of HIV-1-infected T-cell lines promote KSHV infectivity in immortalized and primary human oral epithelial cells. HIV-associated saliva exosomes contain the HIV trans-activation response element (TAR), Tat, and Nef RNAs but do not express Tat and Nef proteins. The TAR RNA in HIV-associated exosomes contributes to enhancing KSHV infectivity through the epidermal growth factor receptor (EGFR). An inhibitory aptamer against TAR RNA reduces KSHV infection facilitated by the synthetic TAR RNA in oral epithelial cells. Cetuximab, a monoclonal neutralizing antibody against EGFR, blocks HIV-associated exosome-enhanced KSHV infection. Our findings reveal that saliva containing HIV-associated exosomes is a risk factor for the enhancement of KSHV infection and that the inhibition of EGFR serves as a novel strategy for preventing KSHV infection and transmission in the oral cavity. IMPORTANCE Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causal agent for Kaposi’s sarcoma (KS), the most common malignancy in HIV/AIDS patients. Oral transmission through saliva is considered the most common route for spreading the virus among HIV/AIDS patients. However, the role of HIV-specific components in the cotransfection of KSHV is unclear. We demonstrate that exosomes purified from the saliva of HIV-positive patients and secreted by HIV-infected T-cell lines promote KSHV infectivity in immortalized and primary oral epithelial cells. HIV-associated exosomes promote KSHV infection, which depends on HIV trans-activation response element (TAR) RNA and EGFR of oral epithelial cells, which can be targeted for reducing KSHV infection. These results reveal that HIV-associated exosomes are a risk factor for KSHV infection in the HIV-infected population.

A high prevalence of Kaposi's sarcoma (KS) is seen in diabetic patients. It is unknown if the physiological conditions of diabetes contribute to KS development. We found elevated levels of viral lytic gene expression when Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells were cultured in high-glucose medium. To demonstrate the association between high glucose levels and KSHV replication, we xenografted telomerase-immortalized human umbilical vein endothelial cells that are infected with KSHV (TIVE-KSHV cells) into hyperglycemic and normal nude mice. The injected cells expressed significantly higher levels of KSHV lytic genes in hyperglycemic mice than in normal mice. We further demonstrated that high glucose levels induced the production of hydrogen peroxide (H 2 O 2 ), which downregulated silent information regulator 1 (SIRT1), a class III histone deacetylase (HDAC), resulting in the epigenetic transactivation of KSHV lytic genes. These results suggest that high blood glucose levels in diabetic patients contribute to the development of KS by promoting KSHV lytic replication and infection. IMPORTANCE Multiple epidemiological studies have reported a higher prevalence of classic KS in diabetic patients. By using both in vitro and in vivo models, we demonstrated an association between high glucose levels and KSHV lytic replication. High glucose levels induce oxidative stress and the production of H 2 O 2 , which mediates the reactivation of latent KSHV through multiple mechanisms. Our results provide the first experimental evidence and mechanistic support for the association of classic KS with diabetes.

We have developed models of HIV latency using microglia derived from adult human patient brain cortex and transformed with the SV40 T large and hTERT antigens. Latent clones infected by HIV reporter viruses display high levels of spontaneous HIV reactivation in culture. BrainPhys, a medium highly representative of the CNS extracellular environment, containing low glucose and 1% FBS, reduced, but did not prevent, HIV reactivation. We hypothesized that spontaneous HIV reactivation in culture was due to the expression of pro-inflammatory genes, such as TNF-α, taking place in the absence of the natural inhibitory signals from astrocytes and neurons. Indeed, expression and secretion of TNF-α is strongly reduced in HIV-latently infected microglia compared to the subset of cells that have undergone spontaneous HIV reactivation. Whereas inhibitors of NF-κB or of macrophage activation only had a short-term silencing effect, addition of dexamethasone (DEXA), a glucocorticoid receptor (GR) agonist and mediator of anti-inflammation, silenced the HIV provirus in a long-term, and shRNA-mediated knock-down of GR activated HIV. DEXA also decreased secretion of a number of cytokines, including TNF-α. Chromatin immunoprecipitation analysis revealed that DEXA strongly increased GR occupancy at the HIV promoter, and reduced histone 3 acetylated levels. Moreover, TNF-α expression inhibitors in combination with DEXA induced further HIV silencing and increased the histone 3 lysine 27 tri-methylated epigenetic mark of repression at the HIV promoter region. We conclude that GR is a critical repressor of HIV transcription in microglia, and a novel potential pharmacological target to restrict HIV expression in the CNS. Electronic supplementary material The online version of this article (10.1007/s11481-018-9798-1) contains supplementary material, which is available to authorized users.

Covalent addition of a methyl group to the adenosine N6 (m6A) is an evolutionarily conserved and common RNA modification that is thought to modulate several aspects of RNA metabolism. While the presence of multiple m6A editing sites on diverse viral RNAs was reported starting almost 40 years ago, how m6A editing affects virus replication has remained unclear. Here, we used photo-crosslinking-assisted m6A sequencing techniques to precisely map several m6A editing sites on the HIV-1 genome and report that they cluster in the HIV-1 3’ untranslated region (3'UTR). Viral 3'UTR m6A sites or analogous cellular m6A sites strongly enhanced mRNA expression in cis by recruiting the cellular YTHDF m6A “reader” proteins. Reducing YTHDF expression inhibited, while YTHDF overexpression enhanced, HIV-1 protein and RNA expression, and virus replication in CD4+ T cells. These data identify m6A editing, and the resultant recruitment of YTHDF proteins, as major positive regulators of HIV-1 mRNA expression.

N 6 -adenosine methylation (m 6 A) is the most common posttranscriptional RNA modification in mammalian cells. We found that most transcripts encoded by the Kaposi's sarcoma-associated herpesvirus (KSHV) genome undergo m 6 A modification. The levels of m 6 A-modified mRNAs increased substantially upon stimulation for lytic replication. The blockage of m 6 A inhibited splicing of the pre-mRNA encoding the replication transcription activator (RTA), a key KSHV lytic switch protein, and halted viral lytic replication. We identified several m 6 A sites in RTA pre-mRNA crucial for splicing through interactions with YTH domain containing 1 (YTHDC1), an m 6 A nuclear reader protein, in conjunction with serine/arginine-rich splicing factor 3 (SRSF3) and SRSF10. Interestingly, RTA induced m 6 A and enhanced its own pre-mRNA splicing. Our results not only demonstrate an essential role of m 6 A in regulating RTA pre-mRNA splicing but also suggest that KSHV has evolved a mechanism to manipulate the host m 6 A machinery to its advantage in promoting lytic replication. IMPORTANCE KSHV productive lytic replication plays a pivotal role in the initiation and progression of Kaposi's sarcoma tumors. Previous studies suggested that the KSHV switch from latency to lytic replication is primarily controlled at the chromatin level through histone and DNA modifications. The present work reports for the first time that KSHV genome-encoded mRNAs undergo m 6 A modification, which represents a new mechanism at the posttranscriptional level in the control of viral replication.

Tumor associated macrophages (TAMs) promote angiogenesis, tumor invasion and metastasis, and suppression of anti-tumor immunity. These myeloid cells originate from monocytes, which differentiate into TAMs upon exposure to the local tumor microenvironment. We previously reported that Kaposi's sarcoma-associated herpes virus (KSHV) infection of endothelial cells induces the cytokine angiopoietin-2 (Ang-2) to promote migration of monocytes into tumors. Here we report that KSHV infection of endothelial cells induces additional cytokines including interleukin-6 (IL-6), interleukin-10 (IL-10), and interleukin-13 (IL-13) that drive monocytes to differentiate and polarize into TAMs. The KSHV-induced TAMs not only express TAM-specific markers such as CD-163 and legumain (LGMN) but also display a gene expression profile with characteristic features of viral infection. More importantly, KSHV-induced TAMs enhance tumor growth in nude mice. These results are consistent with the strong presence of TAMs in Kaposi's sarcoma (KS) tumors. Therefore, KSHV infection of endothelial cells generates a local microenvironment that not only promotes the recruitment of monocytes but also induces their differentiation and polarization into TAMs. These findings reveal a new mechanism of KSHV contribution to KS tumor development.

Kaposi's sarcoma-associated herpesvirus (KSHV) establishes persistent latent infection in immunocompetent hosts. Disruption of KSHV latency results in viral lytic replication, which promotes the development of KSHV-related malignancies in immunocompromised individuals. While inhibitors of classes I and II histone deacetylases (HDACs) potently reactivate KSHV from latency, the role of class III HDAC sirtuins (SIRTs) in KSHV latency remains unclear. Here, we examined the effects of inhibitors of SIRTs, nicotinamide (NAM) and sirtinol, on KSHV reactivation from latency. Treatment of latently KSHV-infected cells with NAM or sirtinol induced transcripts and proteins of the master lytic transactivator RTA (ORF50), early lytic genes ORF57 and ORF59, and late lytic gene ORF65 and increased the production of infectious virions. NAM increased the acetylation of histones H3 and H4 as well as the level of the active histone H3 trimethyl Lys4 (H3K4me3) mark but decreased the level of the repressive histone H3 trimethyl Lys27 (H3K27me3) mark in the RTA promoter. Consistent with these results, we detected SIRT1 binding to the RTA promoter. Importantly, knockdown of SIRT1 was sufficient to increase the expression of KSHV lytic genes. Accordingly, the level of the H3K4me3 mark in the RTA promoter was increased following SIRT1 knockdown, while that of the H3K27me3 mark was decreased. Furthermore, SIRT1 interacted with RTA and inhibited RTA transactivation of its own promoter and that of its downstream target, the viral interleukin-6 gene. These results indicate that SIRT1 regulates KSHV latency by inhibiting different stages of viral lytic replication and link the cellular metabolic state with the KSHV life cycle. IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is the causal agent of several malignancies, including Kaposi's sarcoma, commonly found in immunocompromised patients. While latent infection is required for the development of KSHV-induced malignancies, viral lytic replication also promotes disease progression. However, the mechanism controlling KSHV latent versus lytic replication remains unclear. In this study, we found that class III histone deacetylases (HDACs), also known as SIRTs, whose activities are linked to the cellular metabolic state, mediate KSHV replication. Inhibitors of SIRTs can reactivate KSHV from latency. SIRTs mediate KSHV latency by epigenetically silencing a key KSHV lytic replication activator, RTA. We found that one of the SIRTs, SIRT1, binds to the RTA promoter to mediate KSHV latency. Knockdown of SIRT1 is sufficient to induce epigenetic remodeling and KSHV lytic replication. SIRT1 also interacts with RTA and inhibits RTA's transactivation function, preventing the expression of its downstream genes. Our results indicate that SIRTs regulate KSHV latency by inhibiting different stages of viral lytic replication and link the cellular metabolic state with the KSHV life cycle.

Wireless power transfer (WPT) technology are hot topic in current researches in SMPS. This paper focuses on the structure of magnetic coupling system, which regarded as the most significant part in WPT system. A new magnetic structure with good position robust and low flux leakage to nearby space was proposed comparing to the traditional spiral structure. The simulation results, magnetic evaluation results and test results verified the proposed magnetic structure is more suitable for the cases in which the receive devices could offset horizontally.

Heparinase I (HepI), which specifically cleaves heparin and heparan sulfate, is one of the most extensively studied glycosaminoglycan lyases. Low productivity of HepI has largely hindered its industrial and pharmaceutical applications. Loss of bacterial HepI enzyme activity through poor thermostability during its expression and purification process in production can be an important issue. In this study, using a thermostabilization strategy combining site-directed mutagenesis and calcium ion addition during its production markedly improved the yield of maltose-binding protein-fused HepI (MBP–HepI) from recombinant Escherichia coli. Substitution of Cys297 to serine in MBP–HepI offered a 30.6 % increase in the recovered total enzyme activity due to a mutation-induced thermostabilizing effect. Furthermore, upon addition of Ca2+ as a stabilizer at optimized concentrations throughout its expression, extraction, and purification process, purified mutant MBP–HepI showed a specific activity of 56.3 IU/mg, 206 % higher than that of the wild type obtained without Ca2+ addition, along with a 177 % increase in the recovered total enzyme activity. The enzyme obtained through this novel approach also exhibited significantly enhanced thermostability, as indicated by both experimental data and the kinetic modeling. High-yield production of thermostable MBP–HepI using the present system will facilitate its applications in laboratory-scale heparin analysis as well as industrial-scale production of low molecular weight heparin as an improved anticoagulant substitute.

Infections by viruses are associated with approximately 12% of human cancer. Kaposi's sarcoma-associated herpesvirus (KSHV) is causally linked to several malignancies commonly found in AIDS patients. The mechanism of KSHV-induced oncogenesis remains elusive, due in part to the lack of an adequate experimental system for cellular transformation of primary cells. Here, we report efficient infection and cellular transformation of primary rat embryonic metanephric mesenchymal precursor cells (MM cells) by KSHV. Cellular transformation occurred at as early as day 4 after infection and in nearly all infected cells. Transformed cells expressed hallmark vascular endothelial, lymphatic endothelial, and mesenchymal markers and efficiently induced tumors in nude mice. KSHV established latent infection in MM cells, and lytic induction resulted in low levels of detectable infectious virions despite robust expression of lytic genes. Most KSHV-induced tumor cells were in a latent state, although a few showed heterogeneous expression of lytic genes. This efficient system for KSHV cellular transformation of primary cells might facilitate the study of growth deregulation mechanisms resulting from KSHV infections.

Enzymatic degradation of heparin by heparin lyases has not only largely facilitated heparin structural analysis and contamination detection, but also showed great potential to be a green and cost-effective way to produce low molecular weight heparin (LMWH). However, the commercial use of heparinase I (HepI), one of the most studied heparin lyases, has been largely hampered by its low productivity and extremely poor thermostability. Here we report the thermal inactivation mechanism and strategic thermal stabilization of maltose-binding protein (MBP)-HepI, a fusion HepI produced in E. coli with high yield, solubility and activity. Biochemical studies demonstrated that the thermal inactivation of MBP-HepI involves an unfolding step that is temperature-dependently reversible, followed by an irreversible dimerization step induced by intermolecular disulfide bonds. A good consistency between the kinetic modeling and experimental data of the inactivation was obtained within a wide range of temperature and enzyme concentration, confirming the adequacy of the proposed inactivation model. Based on the inactivation mechanism, a comprehensive strategy was proposed for the thermal stabilization of MBP-HepI, in which Ca(2+) and Tween 80 were used to inhibit unfolding while site mutation at Cys297 and DTT were employed to suppress dimerization. The engineered enzyme exhibits remarkably improved storage and operational thermostability, for example, 16-fold increase in half-life at its optimum temperature of 30 °C and 8-fold increase in remaining activity of 95% after 1-week storage at 4 °C, and therefore shows great potential as a commercial biocatalyst for heparin degradation in the pharmaceutical industry.

Kaposi's sarcoma-associated herpesvirus (KSHV) is causally linked to several acquired immune deficiency syndrome-related malignancies, including Kaposi's sarcoma, primary effusion lymphoma (PEL) and a subset of multicentric Castleman's disease. Control of viral lytic replication is essential for KSHV latency, evasion of the host immune system and induction of tumours. Here, we show that deletion of a 14 microRNA (miRNA) cluster from the KSHV genome significantly enhances viral lytic replication as a result of reduced NF-kappaB activity. The miRNA cluster regulates the NF-kappaB pathway by reducing expression of IkappaBalpha protein, an inhibitor of NF-kappaB complexes. Computational and miRNA seed mutagenesis analyses were used to identify KSHV miR-K1, which directly regulates the IkappaBalpha protein level by targeting the 3'UTR of its transcript. Expression of miR-K1 is sufficient to rescue NF-kappaB activity and inhibit viral lytic replication, whereas inhibition of miR-K1 in KSHV-infected PEL cells has the opposite effect. Thus, KSHV encodes an miRNA to control viral replication by activating the NF-kappaB pathway. These results demonstrate an important role for KSHV miRNAs in regulating viral latency and lytic replication by manipulating the host survival pathway.

Low molecular weight heparin (LMWH) produced by the degradation of heparin has been widely used for clinical use, because it retains heparin's anticoagulant activity while has fewer side effects. In this study, a bioprocess for efficient production of LMWH was established by an ultrafiltration (UF) membrane bioreactor using maltose-binding protein (MBP)-heparinase I. The absorption of the reaction mixture at 235 nm (A235) was used as an indicator for monitoring the enzymatic reaction and product quality. When A235 of the reaction mixture reached 45, the produced LMWH reached a weight-average molecular weight (Mw) of approximately 5038 Da, a number-average molecular weight (Mn) of 3498 Da, the anti-factor Xa activity of 154.95 ± 5.80 IU/mg, and the ratio of anti-factor Xa activity to anti-factor IIa activity of 2.2, which all meet the standard of European Pharmacopoeia 5.0. The average molecular weight of LMWH showed a good linear relationship with the increment in A235. These results indicated that the qualified LMWH could be produced by the UF membrane bioreactor using the fusion enzyme.

Kaposi's sarcoma-associated herpesvirus (KSHV) replication and transcription activator (RTA) encoded by ORF50 is a lytic switch protein for viral reactivation from latency. The expression of RTA activates the expression of downstream viral genes, and is necessary for triggering the full viral lytic program. Using chromatin immunoprecipitation assay coupled with a KSHV whole-genome tiling microarray (ChIP-on-chip) approach, we identified a set of 19 RTA binding sites in the KSHV genome in a KSHV-infected cell line BCBL-1. These binding sites are located in the regions of promoters, introns, or exons of KSHV genes including ORF8, ORFK4.1, ORFK5, PAN, ORF16, ORF29, ORF45, ORF50, ORFK8, ORFK10.1, ORF59, ORFK12, ORF71/72, ORFK14/ORF74, and ORFK15, the two origins of lytic replication OriLyt-L and OriLyt-R, and the microRNA cluster. We confirmed these RTA binding sites by ChIP and quantitative real-time PCR. We further mapped the RTA binding site in the first intron of the ORFK15 gene, and determined that it is RTA-responsive. The ORFK15 RTA binding sequence TTCCAGGAA TTCCTGGAA consists of a palindromic structure of two tandem repeats, of which each itself is also an imperfect inverted repeat. Reporter assay and electrophoretic mobility shift assay confirmed the binding of the RTA protein to this sequence in vitro. Sequence alignment with other RTA binding sites identified the RTA consensus binding motif as TTCCAGGAT(N)0–16TTCCTGGGA. Interestingly, most of the identified RTA binding sites contain only half or part of this RTA binding motif. These results suggest the complexity of RTA binding in vivo, and the involvement of other cellular or viral transcription factors during RTA transactivation of target genes.