Comparison of the Efficacy of Sterile Silicone Studs Versus Lidocaine for the Attenuation of the Hemodynamic Response to Skull Pin Insertion: A Randomized Controlled Trial.

BACKGROUND: Skull pin insertion causes hypertension and tachycardia that adversely affects cerebral hemodynamics. We compared the efficacy of sterile silicone studs (SS) and pin site infiltration with lidocaine in attenuation of the sympathetic response to skull pin insertion. METHODS: Adult patients (N = 120) undergoing supratentorial craniotomy under general anesthesia were randomized to receive either medical-grade sterile SS or 2 mL of 2% plain lidocaine infiltration at each pin site. Hemodynamic (heart rate and mean arterial pressure) response to skull pin insertion at baseline and at 0, 1, 2, 3, and 5 minutes after skull pin insertion was compared. Requirement of rescue analgesia (fentanyl), complications such as pin-site bleeding, and surgeon satisfaction score were assessed. RESULTS: Heart rate in the lidocaine group was significantly greater at 0, 1, 2, 3, and 5 minutes after pin insertion compared with the SS group (P < 0.05). Mean arterial pressure was also significantly higher in the lidocaine group at 0, 1, 2, and 3 minutes after pin insertion (P = 0.001, P = 0.01, P = 0.034, and P = 0.042) compared with the SS group. The number of patients requiring fentanyl [17/60 (28.3%) vs. 40/60 (66%), P = 0.001] was lower in the SS group. The incidence of pin site bleeding was also lower in the SS group, and surgeon satisfaction score was greater. CONCLUSIONS: Sterile SS appear to be more effective than lidocaine infiltration in attenuating the hemodynamic response to skull pin insertion with minimal adverse effects. Further multicenter studies are necessary to conclusively establish the safety and efficacy of sterile SS.

Sterile Silicone Studs-A Nonpharmacologic Modality for Prevention of Hemodynamic Response to Skull Pin Insertion: A Pilot Study.

OBJECTIVE: Application of surgical skull pins causes hemodynamic fluctuations in neurosurgical procedures. To reduce this response, we describe the use of a novel nonpharmacologic method in the form of medical-grade sterile silicone studs to cushion the pressure of the skull pin in the adult population. This study aimed to evaluate the use of conventionally used fentanyl and medical-grade sterile silicone studs for the prevention of hemodynamic response to skull pin insertion. METHODS: A prospective randomized pilot study was conducted of 20 adult patients categorized as American Society of Anesthesiologists class I and II scheduled for elective craniotomy in November 2022 in a tertiary-care hospital in Chandigarh, India. Patients were randomized into 2 groups: fentanyl only (FO group; n = 10) and medical-grade silicone studs (SS group; n = 10). Heart rate and mean arterial pressure were recorded at the following intervals: T1, baseline; T2, before induction; T3, after intubation; T4, before skull pin insertion; T5, T6, T7, T8, T9, and T10 at 0, 1, 3, 4, and 5 minutes after skull pin insertion. RESULTS: Demographic data (e.g., sex, age, disease pathology) were comparable between the groups. Although changes in heart rate between the 2 groups were comparable, there was a statistically significant decrease in mean arterial pressure from 1 minute to 5 minutes after pinning in patients with silicone studs compared with patients who received only fentanyl. CONCLUSIONS: The use of medical-grade silicone studs causes fewer hemodynamic fluctuations compared with fentanyl on skull pinning. Further studies with larger sample sizes are required to confirm the findings of this pilot study.

Crystallographic and Compositional Evolution of Ordered B2 and Disordered BCC Phases During Isothermal Annealing of Refractory High-Entropy Alloys.

Refractory high-entropy alloys (RHEAs) are promising candidates for next-generation high-temperature materials. RHEAs containing Al, often exhibit a checkered pattern microstructure comprising a combination of disordered BCC and ordered B2 phases. Since the ordered B2 phase is based on the BCC parent matrix, distinguishing these two phases can be rather challenging. Advanced characterization techniques are necessary for a reliable qualitative and quantitative analysis of BCC and B2 phases in RHEAs. Additionally, there is a tendency for transformation of the ordered B2 phase into more complex ordered-omega type phases that are usually deleterious to mechanical properties. The current study focuses on the phase stability of a candidate RHEA, Al0.5Mo0.5NbTa0.5TiZr. Correlative transmission electron microscopy (TEM) and atom probe tomography (APT) have been employed to investigate the phase stability and transformation pathway of this RHEA when isothermally annealed at 800°C. The results show that a metastable two-phase BCC + B2 microstructure formed at the early stages of decomposition, eventually transforming into a three-phase BCC + B2 + hP18 microstructure. The hP18 phase is an ordered omega derivative of the ordered B2 phase. The correlative microscopy techniques (TEM and APT) reveal a very interesting interplay of compositional partitioning between the different phases and their respective stability.

Hierarchical Eutectoid Nano-lamellar Decomposition in an Al0.3CoFeNi Complex Concentrated Alloy.

This paper reports a novel eutectoid nano-lamellar (FCC + L12)/(BCC + B2) microstructure that has been discovered in a relatively simple Al0.3CoFeNi high entropy alloy (HEA) or complex concentrated alloy (CCA). This novel eutectoid nano-lamellar microstructure presumably results from the complex interplay between Al-mediated lattice distortion (due to its larger atomic radius) in a face-centered cubic (FCC) CoFeNi solid solution, and a chemical ordering tendency leading to precipitation of ordered phases such as L12 and B2. This eutectoid microstructure is a result of solid-state decomposition of the FCC matrix and therefore distinct from the commonly reported eutectic microstructure in HEAs which results from solidification. This novel nano-lamellar microstructure exhibits a tensile yield strength of 1074 MPa with a reasonable ductility of 8%. The same alloy can be tuned to form a more damage-tolerant FCC + B2 microstructure, retaining high tensile yield stress (~900 MPa) with appreciable tensile ductility (>20%), via annealing at 700 °C. Such tunability of microstructures with dramatically different mechanical properties can be effectively engineered in the same CCA, by exploiting the complex interplay between ordering tendencies and lattice distortion.

Influence of ordered L12 precipitation on strain-rate dependent mechanical behavior in a eutectic high entropy alloy.

Recent studies indicate that eutectic high-entropy alloys can simultaneously possess high strength and high ductility, which have potential industrial applications. The present study focuses on Al0.7CoCrFeNi, a lamellar dual-phase (fcc + B2) precipitation-strengthenable eutectic high entropy alloy. This alloy exhibits an fcc + B2 (B2 with bcc nano-precipitates) microstructure resulting in a combination of the soft and ductile fcc phase together with hard B2 phase. Low temperature annealing leads to the precipitation of ordered L12 intermetallic precipitates within the fcc resulting in enhanced strength. The strengthening contribution due to fine scale L12 is modeled using Orowan dislocation bowing and by-pass mechanism. The alloy was tested under quasi-static (strain-rate = 10-3 s-1) tensile loading and dynamic (strain-rate = 103 s-1) compressive loading. Due to the fine lamellar microstructure with a large number of fcc-bcc interfaces, the alloy show relatively high flow-stresses, ~1400 MPa under quasi-static loading and in excess of 1800 MPa under dynamic loading. Interestingly, the coherent nano-scale L12 precipitate caused a significant rise in the yield strength, without affecting the strain rate sensitivity (SRS) significantly. These lamellar structures had higher work hardening due to their capability for easily storing higher dislocation densities. The back-stresses from the coherent L12 precipitate were insufficient to cause improvement in twin nucleation, owing to elevated twinning stress under quasi-static testing. However, under dynamic testing high density of twins were observed.

Shape-sensitive crystallization in colloidal superball fluids.

Guiding the self-assembly of materials by controlling the shape of the individual particle constituents is a powerful approach to material design. We show that colloidal silica superballs crystallize into canted phases in the presence of depletants. Some of these phases are consistent with the so-called "Λ1" lattice that was recently predicted as the densest packing of superdisks. As the size of the depletant is reduced, however, we observe a transition to a square phase. The differences in these entropically stabilized phases result from an interplay between the size of the depletants and the fine structure of the superball shape. We find qualitative agreement of our experimental results both with a phase diagram computed on the basis of the volume accessible to the depletants and with simulations. By using a mixture of depletants, one of which is thermosensitive, we induce solid-to-solid phase transitions between square and canted structures. The use of depletant size to leverage fine features of the shape of particles in driving their self-assembly demonstrates a general and powerful mechanism for engineering novel materials.

Unresponsiveness of Mycobacterium w vaccine in managing acute and chronic Leishmania donovani infections in mouse and hamster.

The role of Mycobacterium w (Mw) vaccine as an immunomodulator and immunoprophylactant in the treatment of mycobacterial diseases (leprosy and pulmonary tuberculosis) is well established. The fact that it shares common antigens with leishmanial parasites prompted its assessment as an immunostimulant and as an adjunct to known anti-leishmanials that may help in stimulating the suppressed immune status of Leishmania donovani-infected individuals. The efficacy of Mw vaccine was assessed as an immunomodulator, prophylactically either alone or in combination with anti-leishmanial vaccine, as well as therapeutically as an adjunct to anti-leishmanial treatment in L. donovani-infected hamsters, representing a chronic human Visceral Leishmaniasis (VL) model. Similarly, its efficacy was also evaluated in L. donovani-infected BALB/c mice, representing an acute VL model. The preliminary studies revealed that Mw was ineffective as an immunostimulant and/or immunoprophylactant in hamsters infected with L. donovani, as estimated by T-cell immunological responses. However, in the BALB/c mice-VL model it appeared as an effective immunostimulant but a futile prophylactic agent. It is therefore inferred that, contrary to its role in managing tuberculosis and leprosy infections, Mw vaccine has not been successful in controlling VL infection, emphasizing the need to find detailed explanations for the failure of this vaccine against the disease.

Recombinant trehalose-6-phosphate phosphatase of Brugia malayi cross-reacts with human Wuchereria bancrofti immune sera and engenders a robust protective outcome in mice.

Trehalose-6-phosphate phosphatase of Brugia malayi (Bm-TPP) represents an attractive vaccine candidate because it is present in all the major life stages of parasite, but is absent in mammals. We have previously cloned, purified and biochemically characterized Bm-TPP. In the present study, we investigated the cross-reactivity of recombinant Bm-TPP (r-Bm-TPP) with the sera of human bancroftian patients belonging to different disease categories. In silico study using bioinformatics tool demonstrated that Bm-TPP is highly immunogenic in nature. BALB/c mice administered with r-Bm-TPP alone or in combination with Freund's complete adjuvant (FCA) generated a strong IgG response. Further investigations on the antibody isotypes showed generation of a mixed T helper cell response which was marginally biased towards Th1 phenotype. r-Bm-TPP with or without adjuvant lead to significantly increased accumulation of CD4+ and CD8+ T cells in the spleen of infected mice and increased the activation of peritoneal macrophages. Additionally, r-Bm-TPP enhanced the production of both proinflammatory (IL-2, IFN-γ) and anti-inflammatory (IL-4, IL-10) cytokines and mice immunized with r-Bm-TPP alone or in combination with FCA showed 54.5% and 67% protection respectively against B. malayi infective larvae challenge. Taken together, our findings suggest that Bm-TPP is protective in nature and might be a potential candidate for development of vaccine against lymphatic filarial infections.

Genome sequence of a novel actinophage PIS136 isolated from a strain of Saccharomonospora sp.

A wide-host-range bacteriophage (phage) PIS136 was isolated from PA136, a strain of Saccharomonospora belonging to the group actinomycetes. Here, we present the genome sequence of the PIS136 phage, which is 94,870 bp long and contains 132 putative coding sequences and one tRNA gene. An IS element-like region with two genes for putative transposases was identified in the genome. The presence of IS element-like sequences suggests that PIS136 is still under active evolution.

N-Methyl-6, 7-dimethoxyisoquinolone in Annona squamosa twigs is the major immune modifier to elicit polarized Th1 immune response in BALB/c mice.

Annona squamosa (AS) has traditionally been used as ethnomedicine. We have earlier extracted and fractionated the twigs of AS based upon its bioactivity and observed its immune potentiating activity that was localized in its three fractions. Present communication deals with the phytochemical analysis and pharmacological investigation of the most active chloroform fraction that led to isolation and identification of a number of compounds whose structures were elucidated using 1D and 2D NMR spectroscopic analysis. Amongst the twelve pure compounds isolated, five compounds Lanuginosine (1), (+)-O-methylarmepavine (2), (+)-anomuricine (3), Isocorydine (4), and N-methyl-6, 7-dimethoxyisoquinolone (5) were evaluated in vivo for their immune modifier activities in BALB/c mice after oral administration at three log doses of 0.3, 1.0 and 3.0mg/kg for 14 consecutive days. Of these, three compounds (1, 2 and 5) showed dose dependent immune stimulating activity. However, the uppermost activity was noted in the compound N-methyl-6, 7-dimethoxyisoquinolone at the 3.0mg/kg oral dose. The activity was assessed in the form of increased splenic T and B cellular proliferation, up-regulated CD4+, CD8+ and CD19+ cell population and accentuation in the peritoneal macrophage function. The compound possibly acted modifying the expression of Th1- and Th2- cytokines via stimulation of pro-inflammatory Th1 cytokines IL-2 and IFN-γ. These results warrant the use of the above compounds as an efficient immune-stimulant or immune-adjuvant against diseases with immune suppression. The analogs of the compound may further be chemically synthesized to achieve desired immune modifying activity.

Pharmacological properties of Datura stramonium L. as a potential medicinal tree: an overview.

India has a great wealth of various naturally occurring plant drugs which have great potential pharmacological activities. Datura stramonium (D. stramonium) is one of the widely well known folklore medicinal herbs. The troublesome weed, D. stramonium is a plant with both poisonous and medicinal properties and has been proven to have great pharmacological potential with a great utility and usage in folklore medicine. D. stromonium has been scientifically proven to contain alkaloids, tannins, carbohydrates and proteins. This plant has contributed various pharmacological actions in the scientific field of Indian systems of medicines like analgesic and antiasthmatic activities. The present paper presents an exclusive review work on the ethnomedical, phytochemical, pharmacological activities of this plant.

Chemotypical variations in Withania somnifera lead to differentially modulated immune response in BALB/c mice.

Withania somnifera (Ashwagandha) is a plant with known ethnomedicinal properties and its use in Ayurvedic medicine in India is well documented. The present investigation reports on immunomodulatory efficacy of aqueous-ethanol extracts of roots of three selected Withania somnifera chemotypes designated as NMITLI 101R, NMITLI 118R and NMITLI 128R. Each chemotype was administered 10-100 mg/kg orally to BALB/c mice once daily for 14 days. The immunomodulatory consequences were recorded by determining the humoral immune response with the help of hemagglutination, plaque forming cell assay and cellular response by measuring delayed type hypersensitivity reaction. Additionally, other immune parameters such as proliferation of T and B cells, intracellular and secreted Th1 and Th2 cytokines along with modulation in ROS production by peritoneal macrophages were monitored after feeding with lower doses (3-30 mg/kg/day) of these three chemotypes individually. NMITLI 101R incited both humoral and cellular immune response in terms of higher number of antibody producing cells and enhanced foot pad swelling at the 10mg dose as also dose dependent B and T cell proliferations. Levels of intracellular and secreted cytokines post-NMITLI 101R treatment illustrated generation of mixed Th1/Th2 response that remained more polarized towards Th1. This chemotype also generated maximum reactive oxygen species. NMITLI 118R provoked comparatively reduced immune response in all humoral and cellular parameters at lower doses but induced highly polarized Th1 cytokine response. In contrast, NMITLI 128R led to enhanced antibody production with minimal cellular response demonstrating marginally Th2 dominance at a lower dose. Taken together, it may therefore be concluded that there were distinct modulation in the immune response exhibited by the three chemotypes of Withania somnifera and NMITLI 101R appeared to possess a better immunostimulatory activity than the other chemotypes at lower doses.

Roscovitine inhibits EBNA1 serine 393 phosphorylation, nuclear localization, transcription, and episome maintenance.

Latent Epstein-Barr virus (EBV) infection causes human lymphomas and carcinomas. EBV usually persists as an episome in malignant cells. EBV episome persistence, replication, and gene expression are dependent on EBNA1 binding to multiple cognate sites in oriP. To search for inhibitors of EBNA1- and oriP-dependent episome maintenance or transcription, a library of 40,550 small molecules was screened for compounds that inhibit EBNA1- and oriP-dependent transcription and do not inhibit EBNA1- and oriP-independent transcription. This screening identified roscovitine, a selective inhibitor of cyclin-dependent kinase 1 (CDK1), CDK2, CDK5, and CDK7. Based on motif predictions of EBNA1 serine 393 as a CDK phosphorylation site and (486)RALL(489) and (580)KDLVM(584) as potential cyclin binding domains, we hypothesized that cyclin binding to EBNA1 may enable CDK1, -2, -5, or -7 to phosphorylate serine 393. We found that Escherichia coli-expressed EBNA1 amino acids 387 to 641 were phosphorylated in vitro by CDK1-, -2-, -5-, and -7/cyclin complexes and serine 393 phosphorylation was roscovitine inhibited. Further, S393A mutation abrogated phosphorylation. S393A mutant EBNA1 was deficient in supporting EBNA1- and oriP-dependent transcription and episome persistence, and roscovitine had little further effect on the diminished S393A mutant EBNA1-mediated transcription or episome persistence. Immunoprecipitated FLAG-EBNA1 was phosphorylated in vitro, and roscovitine inhibited this phosphorylation. Moreover, roscovitine decreased nuclear EBNA1 and often increased cytoplasmic EBNA1, whereas S393A mutant EBNA1 was localized equally in the nucleus and cytoplasm and was unaffected by roscovitine treatment. These data indicate that roscovitine effects are serine 393 specific and that serine 393 is important in EBNA1- and oriPCp-dependent transcription and episome persistence.

The n-hexane and chloroform fractions of Piper betle L. trigger different arms of immune responses in BALB/c mice and exhibit antifilarial activity against human lymphatic filarid Brugia malayi.

Modulation of immune functions by using herbal plants and their products has become fundamental regime of therapeutic approach. Piper betle Linn. (Piperaceae) is a widely distributed plant in the tropical and subtropical regions of the world and has been attributed as traditional herbal remedy for many diseases. We have recently reported the antifilarial and antileishmanial efficacy in the leaf extract of Bangla Mahoba landrace of P. betle which is a female plant. The present report describes the in vivo immunomodulatory efficacy of the crude methanolic extract and its n-hexane, chloroform, n-butanol fractions of the female plant at various dose levels ranging between 0.3 and 500 mg/kg in BALB/c. Attempts were also made to observe antifilarial activity of the active extracts and correlate it with the antigen specific immune responses in another rodent Mastomys coucha infected with human lymphatic filarial parasite Brugia malayi. The crude methanol extract and n-hexane fraction were found to potentiate significant (p<0.001) enhancement of both humoral (plaque forming cells, hemagglutination titre) as well as cell-mediated (lymphoproliferation, macrophage activation, delayed type hypersensitivity) immune responses in mice. The flow cytometric analysis of splenocytes of treated mice indicated enhanced population of T-cells (CD4(+), CD8(+)) and B-cells (CD19(+)). The n-hexane fraction (3 mg/kg) was found to induce biased type 2 cytokine response as revealed by increased IL-4(+) and decreased IFN-gamma(+) T-cell population while the chloroform fraction (10 mg/kg) produced a predominant type 1 cytokines. Crude methanolic extract (100 mg/kg) demonstrated a mixed type 1 and type 2 cytokine responses thus suggesting a remarkable immunomodulatory property in this plant. The induction of differential T-helper cell immune response appears ideal to overcome immunosuppression as observed in case of lymphatic, filarial Brugia malayi infection which may also be extended to other infections as well.

Immunomodulator effect of picroliv and its potential in treatment against resistant Plasmodium yoelii (MDR) infection in mice.

PURPOSE: The present study was envisaged to evaluate potential of combination therapy comprising of immunomodulator picroliv and antimalarial chloroquine against drug resistant Plasmodium yoelii (P. yoelii) infection in BALB/c mice. METHODS: The immunomodulatory potential of picroliv was established by immunizing animals with model antigen along with picroliv. Immune response was assessed using T-cell proliferation assay and also by determining the antibody isotype-profile induced in the immunized mice. In the next set of experiment, prophylactic potential of picroliv to strengthen antimalarial properties of chloroquine against P. yoelii (MDR) infection in BALB/c mice was assessed. RESULTS: T-cell proliferation as well as antibody production study reveals that picroliv helps in evoking strong immuno-potentiating response against model antigen in the immunized mice. Co-administration of picroliv enhances efficacy of CHQ against experimental murine malaria. CONCLUSION: The activation of host immune system can increase the efficacy of chloroquine for suppression of drug resistant malaria infection in BALB/c mice.

Epstein-Barr virus nuclear antigen 1 does not cause lymphoma in C57BL/6J mice.

To test whether transgenic Epstein-Barr virus nuclear antigen 1 (EBNA1) expression in C57BL/6 mouse lymphocytes causes lymphoma, EBNA1 expressed in three FVB lineages at two or three times the level of latent infection was crossed up to six successive times into C57BL/6J mice. After five or six crosses, 14/36, (38%) EBNA1 transgenic mice, 11/31 (36%) littermate EBNA1-negative controls, and 9/25 (36%) inbred C57BL/6J mice housed in the same facility had lymphoma. These data indicate that EBNA1 does not significantly increase lymphoma prevalence in C57BL/6J mice.

LMP1 TRAFficking activates growth and survival pathways.

Epstein-Barr Virus (EBV) Latent Infection Membrane Protein 1 (LMP1) is expressed in all the EBV related malignancies. LMP1 expression is critical for transformation of human B-cells by EBV. LMP1 expression in human B cells induces activation and adhesion molecule expression and cell dumping, which are characteristic of CD40 activated B lymphocytes. In immortalized fibroblasts, LMP1 mimics aspects of activated ras in enabling serum, contact, and anchorage independent growth. Reverse genetic analyses implicate six transmembrane domains (TM), TM1-6, and two C-terminal cytosolic domains, transformation effector sites 1 and 2 (TES1 and 2) or C-terminal activation regions 1 and 2 (CTAR1 and 2) as the essential domains for LMP1 effects. The 6 transmembrane domains cause intermolecular interaction, whereas the C-terminal domains signal through tumor necrosis factor receptor (TNFR) associated factors (TRAFs) or TNFR associated death domain proteins (TRADD) and activate NF-kappaB, JNK, and p38. LMP1 TES1/CTAR1 directly recruits TRAFs 1, 2, 3 and 5 whereas LMP1 TES2/CTAR2 indirectly recruits TRAF6 via BS69. LMP1 TES1/CTAR1 activates TRAF2, NIK, IKKalpha and p52 mediated noncanonical NF-KB pathway and LMP1 TES2/CTAR2 activates TRAF6, TAB1, TAK1, IKKalpha/ IKKbeta/ IKKgamma mediated canonical NF-KB pathway. Interestingly, TRAF3 is a negative regulator of noncanonical NF-kappaB activation, although a positive role in LMP1 signaling has also been described. LMP1 mediated JNK activation is predominantly TES2/CTAR2 dependent and requires TRAF6. LMP1 specifically increases TRAF3 partitioning into lipid rafts and interestingly does not induce degradation of any of the TRAFs upon NF-kappaB activation. Studies of the chemistry and biology of LMP1-TRAF interaction mediated activation of signaling pathways are important for controlling EBV infected cell survival and growth.

Characterization of Mycobacterium tuberculosis WhiB1/Rv3219 as a protein disulfide reductase.

WhiB family of protein is emerging as one of the most fascinating group and is implicated in stress response as well as pathogenesis via their involvement in diverse cellular processes. Surprisingly, available in vivo data indicate an organism specific physiological role for each of these proteins. The WhiB proteins have four conserved cysteine residues where two of them are present in a C-X-X-C motif. In thioredoxins and similar proteins, this motif works as an active site and confers thiol-disulfide oxidoreductase activity to the protein. The recombinant WhiB1/Rv3219 was purified in a single step from Escherichia coli using Ni(2+)-NTA affinity chromatography and was found to exist as a homodimer. Mass spectrometry of WhiB1 shows that the four cysteine residues form two intramolecular disulfide bonds. Using intrinsic tryptophan fluorescence as a measure of redox state, the redox potential of WhiB1 was calculated as -236+/-2mV, which corresponds to the redox potential of many cytoplasmic thioredoxin-like proteins. WhiB1 catalyzed the reduction of insulin disulfide thus clearly demonstrating that it functions as a protein disulfide reductase. Present study for the first time suggests that WhiB1 may be a part of the redox network of Mycobacterium tuberculosis through its involvement in thiol-disulfide exchange with other cellular proteins.

LMP1 transmembrane domain 1 and 2 (TM1-2) FWLY mediates intermolecular interactions with TM3-6 to activate NF-kappaB.

The Epstein-Barr virus oncoprotein LMP1 has six transmembrane domains (TMs) that enable intermolecular aggregation and constitutive signaling through two C-terminal cytosolic domains. Expression of both TMs 1 and 2 without the C terminus (TM1-2DeltaC) and TMs 3 to 6 fused to the C terminus (TM3-6) results in partial association, which is substantially decreased by TM1 F38WLY41 mutation to A38ALA41. We now investigate whether TM1-2DeltaC can functionally interact with TM3-6. TM1-2DeltaC induced TM3-6 to mediate NF-kappaB activation at 59% of LMP1 levels, and the effect was dependent on TM1-2 F38WLY41. TM1-2DeltaC even induced TM3-4 C terminus-mediated NF-kappaB activation to 44% of LMP1 levels. Surprisingly, this effect was TM1 F38WLY41 independent, indicative of a role for TMs 5 and 6 in TM1 F38WLY41 effects. TM3 W98 was also important for TM1-2DeltaC induction of TM3-6-mediated NF-kappaB activation, for association, and for TM1 F38WLY41 dependence on C-terminal NF-kappaB activation. These data support models in which the TM1 F38WLY41 effects are at least partially dependent on TM3 W98 and a residue(s) in TMs 5 and 6.

IL-1 receptor-associated kinase 1 is critical for latent membrane protein 1-induced p65/RelA serine 536 phosphorylation and NF-kappaB activation.

Epstein-Barr virus latent infection integral membrane protein 1 (LMP1) mimics a constitutively active TNF receptor (TNFR). LMP1 has two C-terminal cytosolic domains, transformation effector sites (TES)1 and -2, that engage TNFR-associated factors (TRAFs) and the TNFR-associated death domain protein, respectively, and activate NF-kappaB. NF-kappaB activation is critical for Epstein-Barr virus-infected lymphoblast survival. TES1- and TES2-mediated NF-kappaB activations are IL-1 receptor-associated kinase 1 (IRAK1)-dependent. Because IRAK1 is upstream of TRAF6 in IL-1 activation of NF-kappaB, the potential role of IRAK1 in LMP1-mediated NF-kappaB activation through TRAF6 and inhibitor of kappaB (IkappaB) kinase (IKK) was initially investigated. Surprisingly, LMP1 expression activated TRAF6 ubiquitination, IKKbeta induction of IkappaB alpha phosphorylation, and p65 nuclear translocation in both WT and IRAK1-deficient I1A 293 cells. LMP1 also induced IKK alpha-mediated p100 processing and p52 nuclear localization in WT and IRAK1-deficient I1A 293 cells. Further, LMP1 TES1 and TES2 induced p65, p50, and p52 NF-kappaB DNA binding in WT and IRAK1-deficient I1A 293 cells. However, LMP1 induced p65/RelA S536 phosphorylation only in WT 293 cells or in IRAK1 kinase point mutant reconstituted I1A 293 cells but not in IRAK1-deficient I1A 293 cells. IRAK1 was also required for LMP1 activation of p38, one of the kinases that can mediate p65/RelA S536 phosphorylation and activate NF-kappaB-dependent transcription. Thus, the critical IRAK1 role in LMP1-induced NF-kappaB activation is in mediating p65/RelA S536 phosphorylation through an effect on p38 or other p65 S536 kinases.