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Following the 2015 Zika virus (ZIKV) outbreaks in the South Pacific, Caribbean, and Americas, ZIKV has emerged as a serious threat due to its association with infantile microcephaly and other neurologic disorders. Despite an international effort to develop a safe and effective vaccine to combat congenital Zika syndrome and ZIKV infection, only DNA and mRNA vaccines encoding the precursor membrane (prM) and envelope (E) proteins, an inactivated-ZIKV vaccine, and a measles virus-based ZIKV vaccine are currently in phase I or II (prM/E DNA) clinical trials. A ZIKV vaccine based on a nonreplicating, recombinant subunit platform offers a higher safety profile than other ZIKV vaccine candidates but is still highly immunogenic, inducing high virus-neutralizing antibody titers. Here, we describe the production and purification of Drosophila melanogaster S2 insect cell-derived, soluble ZIKV E protein and evaluate its immunogenicity and efficacy in three different mouse strains. As expected, significant virus-specific antibody titers were observed when using formulations containing clinically relevant adjuvants. Immunized mice challenged with live virus demonstrate inhibition of virus replication. Importantly, plaque reduction neutralization tests (PRNTs) indicate the high-titer production of neutralizing antibodies, a correlate of protection in the defense against ZIKV infection. ZIKV challenge of immunocompetent mice led to full protection against viremia with two doses of adjuvanted vaccine candidates. These data demonstrate a proof of concept and establish recombinant subunit immunogens as an effective vaccine candidate against ZIKV infection.
IMPORTANCE The recent outbreaks of Zika virus (ZIKV) infection in French Polynesia, the Caribbean, and the Americas have highlighted the severe neuropathological sequelae that such an infection may cause. The development of a safe, effective ZIKV vaccine is critical for several reasons: (i) the difficulty in diagnosing an active infection due to common nonspecific symptoms, (ii) the lack of a specific antiviral therapy, and (iii) the potentially devastating pathological effects of in utero infection. Moreover, a vaccine with an excellent safety profile, such as a nonreplicating, noninfectious vaccine, would be ideal for high-risk people (e.g., pregnant women, immunocompromised patients, and elderly individuals). This report describes the development of a recombinant subunit protein vaccine candidate derived from stably transformed insect cells expressing the ZIKV envelope protein in vitro, the primary antigen to which effective virus-neutralizing antibodies are engendered by immunized animals for several other flaviviruses; the vaccine candidate elicits effective virus-neutralizing antibodies against ZIKV and provides protection against ZIKV infection in mice.
In 1947, Zika virus (ZIKV) was initially isolated from a febrile rhesus macaque in Uganda, followed by its isolation in Aedes africanus mosquitoes in 1948 (1). Serological evidence showed human exposure to the virus (2, 3), but no human disease associated with infection was reported until 1954, when symptomatic infection was reported in three patients in Nigeria (4). No major human outbreaks were evident until 2007, when an illness causing rash, conjunctivitis, and arthralgia was observed on Yap Island, Federated States of Micronesia. ZIKV was identified as the causative agent, and an estimated 5,000 people were infected (5). In 2013, a large outbreak was reported in French Polynesia, resulting in 19,000 suspected infections. An increase in Guillain-Barré syndrome concurrent with the outbreak suggested a causal link between the two, and this was the first time that such an association was noted (6, 7). This was followed by a large outbreak in Brazil in 2015, which was the first incidence in the Western Hemisphere (8, 9).
During the Brazilian outbreak in 2015, an increased incidence of newborns with microcephaly was observed, and examination of microcephalic cases showed the presence of virus in infants shortly after birth, indicating transmission of the virus in utero (10, 11). Further research found a strong association between maternal ZIKV infection during the first trimester of pregnancy and the risk of microcephaly during the Brazilian outbreak (12), although the current data suggest infection during any trimester of pregnancy may result in ZIKV-associated birth defects (13). Experimental models using mice have shown that ZIKV infection during pregnancy can transmit virus to the fetus through the placenta and cause intrauterine growth restrictions that include cortical deformations (14). The virus has also been shown to infect neural progenitor cells in mice, leading to apoptosis (15, 16). In human cell models that mimic first-trimester brain development in vitro, ZIKV infection has been shown to cause cell death in neural stem cells and to reduce the growth of brain organoids, as well as to disrupt the development of neurospheres (17).
Currently, there are no commercially available vaccines against ZIKV, but vaccine candidates from a variety of different platforms are being explored. A purified, inactivated ZIKV based on the Puerto Rican ZIKV strain has been successfully demonstrated to protect against challenge with both homologous and nonhomologous strains in murine and nonhuman primate (NHP) models (18). The DNA vaccine platform using the ZIKV precursor membrane and envelope (prM-E) genes has also been tested in mice and NHPs (18–20). One vaccine candidate uses constructs from the prM-E gene sequences of the French Polynesian strain to create subviral particles (20). Parts of the ZIKV prM or E gene were replaced with analogous portions of Japanese encephalitis virus (JEV) to improve expression and secretion (19, 21). Immunization of C57BL/6J and BALB/c mice with VRC5283, containing the JEV prM signal sequence, and VRC5288, a modified VRC5283 strain with a JEV C-terminus substitution, yields ZIKV-specific neutralizing antibodies (19). Other DNA vaccines have used the prM-E sequences from the Brazilian strain and have shown protection against both homologous and nonhomologous ZIKV strains in BALB/c, SJL, and C57BL/6J mice after two doses (20). A third vaccine platform using a rhesus adenovirus vector has been shown to elicit antibody responses against a wide array of ZIKV E protein epitopes and protect against challenge with a homologous strain (22). There are several ongoing clinical trials that use either a purified, inactivated-ZIKV vaccine (ZPIV), a modified mRNA, a measles virus-based ZIKV vaccine (MV-ZIKA), or a DNA vaccine platform.
In this study, we describe the production of a novel recombinant subunit vaccine candidate comprised of the ZIKV envelope protein (E) ectodomain and two clinically relevant adjuvants and evaluate its immunogenicity in Swiss Webster (SW), BALB/c, and C57BL/6 mice. We also assess efficacy against intravenous ZIKV challenge in immunocompetent, adult BALB/c mice as a ZIKV infection model (20). Vaccines based on this platform have been developed previously against other flaviviruses, e.g., dengue virus (DENV) (24) and West Nile virus (WNV) (25), and have been shown to have excellent safety profiles, robust immunogenicity, and high protective efficacy (24–28).
Expression and purification of ZIKV E protein.The supernatant from stably transformed S2 cell lines yielded approximately 5 mg/liter of protein using the WAVE bioreactor. Subsequent purification from the S2 supernatant by immunoaffinity chromatography (IAC), using the anti-flavivirus E protein antibody 4G2, isolated a soluble protein of approximately 45 kDa and indicated a purity of >90% homogeneity, as shown by SDS-PAGE (Fig. 1A). Recognition by the monoclonal antibody (MAb) 4G2 indicated successful purification of the recombinant protein (Fig. 1B, lane 1). To gauge the immunoreactivity of ZIKV E protein, the membrane was probed with convalescent-phase sera from PRVABC59 ZIKV-infected mice and NHPs (Fig. 1B, lanes 2 to 3). Purified ZIKV E protein was detected by both mouse and NHP sera.
Recombinant ZIKV E protein expressed in Drosophila S2 cells after purification using 4G2 MAb. (A) Coomassie blue-stained SDS PAGE gel (4 to 12% polyacrylamide) featuring ZIKV E protein. The molecular weights of the standard marker sizes are shown in kilodaltons (kDa). Purified ZIKV E of approximately 1 μg migrated as a single band. (B) Western blot of purified ZIKV E protein loaded at the same concentration as in panel A. Replicate Western membranes were probed with 4G2 MAb, convalescent ZIKV-infected mouse, or NHP sera.
Recombinant ZIKV E antigen is immunogenic in mice.The immunogenicity of purified ZIKV E protein was initially tested in Swiss Webster (SW) mice by immunizing four groups of mice with either three doses (10 μg per dose) of ZIKV E alone, ZIKV E in formulations with the adjuvant aluminum hydroxide (2% Alhydrogel adjuvant or Imject alum, both of which are referred to as alum hereinafter) or CoVaccine HT, or alum alone as a control (Fig. 2A and B). Serum anti-ZIKV E IgG titers were measured by microsphere immunoassay (MIA). We first assessed whether the value of median fluorescence intensity (MFI) at a single dilution in the MIA correlated with the IgG antibody titers calculated as 50% effective concentrations (EC50s). Linear regression between the MFI at a 1:100 serum dilution and the EC50s of samples with different IgG titers showed good correlation (R2 = 0.9817) (Fig. 2C), allowing single-point MFI values to be used as an accurate measurement of IgG titers. Therefore, IgG antibody titers are shown as MFIs at the 1:100 dilution for all subsequent assays.
Immunogenicity of recombinant ZIKV E protein in SW mice. (A) Vaccination schedule. The drawings of drops at days 14, 35, and 56 indicate blood draws. (B) Experimental groups. Alum, Imject alum. (C) Median fluorescence intensity (MFI) correlation of EC50s with single-point MFIs. The log EC50 values of seven different sera were determined by MIA. These values were plotted against the MFI values obtained from these sera at a dilution of 1:100 for each serum sample. (D) Geometric mean titers (GMTs) with 95% confidence intervals (CI) of ZIKV E-specific IgG MFI of sera from all groups of mice immunized with each formulation. S.D., standard deviations. *, P < 0.05; **, P < 0.01; ****, P < 0.0001.
Serum was collected 2 weeks after each immunization and was evaluated for ZIKV E-specific IgG titers. After three doses of the vaccine, mice receiving ZIKV E alone failed to generate a significant humoral response, indicating the need for coadministration with an adjuvant (Fig. 2D). The IgG MFI against ZIKV E protein in mice given the formulation with CoVaccine HT was not significantly above the assay cutoff 2 weeks after the first immunization but was significantly elevated following the second dose and remained high after the third dose. Only a slight increase in titer over that after the second dose was seen after the third immunization. The formulation with alum appeared to yield increased antibody titers after each dose of vaccine; however, the differences in titers after each subsequent vaccine dose were not statistically significant. Only the difference between titers following the first dose and the third dose was significant. These results suggest that two doses of the ZIKV E protein with adjuvant, especially with CoVaccine HT, may be sufficient for eliciting an optimal humoral response.
Recombinant ZIKV E protein elicits antibody responses in various strains of mice.To characterize the antibody responses generated by the recombinant subunit ZIKV E vaccine candidate in mice with different immunological backgrounds, SW (outbred), BALB/c (Th2-dominant), and C57BL/6 (Th1-dominant) mice were immunized with two doses of 5 μg ZIKV E formulated with either alum or CoVaccine HT at 3-week intervals (Fig. 3A and B). The dose was reduced to 5 μg of E protein in order to determine whether immunogenicity can be sustained with minimal antigen and to accentuate the differences between mouse strains that might otherwise be masked at higher doses. IgG antibody titers in mouse sera were measured 2 weeks following each immunization. Compared to control mice immunized with adjuvant alone, both BALB/c and C57BL/6 mice developed robust humoral responses after the first and second immunization with the recombinant subunit ZIKV E candidate vaccines. No difference in the IgG antibody titers was observed between these two mouse strains (Fig. 3C). An approximate 5-fold increase in IgG titers after the second immunization was seen in both BALB/c and C57BL/6 mice given the CoVaccine HT-adjuvanted vaccine, while the increase was about 20- to 26-fold in mice administered the alum-adjuvanted vaccine. This suggests that CoVaccine HT elicits a stronger immune response to the antigen than alum upon the initial dose but that after the second dose, the alum-adjuvanted formulation matches the titers observed in groups given CoVaccine HT. Although the IgG MFI values obtained from SW mouse antisera were lower than the values obtained from BALB/c and C57BL/6 mice (Fig. 3C), particularly after dose 1, the titers in sera after dose 1 and dose 2 increased nearly 9-fold and about 22-fold for the CoVaccine HT and alum formulations, respectively.
Antibody responses to recombinant ZIKV E protein in three strains of mice. (A) Vaccination schedule. (B) Experimental groups. Alum, 2% Alhydrogel adjuvant. (C) ZIKV E-specific IgG antibodies in the sera from three strains of mice were measured after each immunization using MIA and are plotted as GMTs with 95% CI of total IgG titers. S.D., standard deviations. (D) The neutralizing titers in mouse sera at 2 weeks after the second immunization were measured by PRNT and plotted as the PRNT50, the calculated dilution of serum that gave 50% inhibition. Statistical analysis was conducted using unpaired t tests and Prism (GraphPad) software. PRNT50 values were generated using a fixed, or variable-slope, sigmoidal dose-response model. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
The presence of neutralizing antibodies was assessed after dose 2 by a plaque reduction neutralization test (PRNT) using Vero cells. The results revealed that regardless of their immunological backgrounds, the majority of mice receiving the adjuvanted ZIKV E protein generated good neutralizing antibody titers. Although the serum dilutions yielding 50% virus neutralization (PRNT50 titers) were somewhat lower in SW mice than in BALB/c or C57BL/6 mice with both adjuvants, all candidate vaccine groups generated neutralizing antibody titers statistically higher than those in their controls receiving the same adjuvant, suggesting broad efficacy and protective potential (Fig. 3D). Both CoVaccine HT and alum formulations generated comparable titers of neutralizing antibodies.
Protective efficacy of ZIKV E vaccination in BALB/c mice.A viremia model of immunocompetent, adult mice was used to assess the protective efficacy provided by immunization with the E protein vaccine candidate formulation. Since BALB/c mice may be resistant to peripheral ZIKV infection (29), mice were inoculated systemically with 100 PFU of ZIKV intravenously (i.v.; through the tail vein) without immunosuppressants. This model had been used previously by Larocca et al. (20) to assess Zika vaccine efficacy. Viremia was determined by plaque assay daily for 5 days after infection. All inoculated animals showed detectable viremia on day 3, which established immunocompetent BALB/c mice as a permissive ZIKV challenge model (Fig. 4A) useful for further experiments.
Immunization of mice with recombinant ZIKV E protein vaccine inhibits viral replication postchallenge. (A) Kinetics of viremia in naive BALB/c mice challenged intravenously (i.v.) with 100 PFU of ZIKV Puerto Rican strain PRVABC59. Six animals of both sexes were euthanized daily after infection, and total serum was collected from each animal. Viremia was measured for each serum sample using a standard plaque assay on Vero cells. The limit of detection was 101.7 PFU/ml. (B) Vaccination schedule for the BALB/c mouse challenge study. (C) Experimental groups. Alum, 2% Alhydrogel adjuvant. (D) Viremia in vaccinated and control mice on day 3 after infection. Mice vaccinated once or twice were challenged as described for panel A. Viremia on day 3 postchallenge was measured as described for panel A. The limit of detection was 25 PFU/ml. (E) GMTs expressed as the MFIs (with 95% CI) of ZIKV E-specific IgG titers observed in serum samples collected 2 weeks after immunization and 2 weeks after ZIKV challenge measured by MIA. (F) Anti-NS1 antibody titers in vaccinated and control mice pre- and postchallenge. Antibody titers were measured by MIA on individual serum samples at a 1:100 dilution. **, P < 0.01; ****, P < 0.0001.
To examine the protective potential in this challenge model, 4- to 6-week-old BALB/c mice were immunized with one or two doses of 10 μg ZIKV E protein with either CoVaccine HT or alum and challenged 4 weeks after the last vaccination, as depicted in Fig. 4B and C. The presence of viremia was determined by plaque assay on Vero cells using sera collected on the third day after ZIKV challenge by cardiac puncture. This experiment indicated full protection (the absence of viremia) in all 6 mice receiving two 10-μg doses of ZIKV E with either adjuvant (Fig. 4D). Groups receiving a single immunization showed breakthrough viremia in one mouse per group, while the mice in the control group demonstrated viremia in five of the six mice (P < 0.01 compared to twice-immunized groups).
To provide additional support for the protective efficacy of the ZIKV E protein vaccine candidate, in a separate experiment, the antibody response postchallenge in vaccinated mice was assessed to determine whether an anamnestic response (4-fold increase in IgG titers) would be observed. The lack of such a response has been regarded as evidence for the inhibition of viral replication postchallenge, indicative of effective protection, which has recently also been used as a secondary outcome measure in various clinical dengue virus vaccine studies (for example, in a recently completed study by Durbin et al. [ClinicalTrials registration no. NCT02317900]). A single dose of ZIKV E protein with CoVaccine HT generated a greater antibody response than the antigen formulation with alum (Fig. 4E), consistent with the study results shown in Fig. 3C.
Two immunizations of ZIKV E with either adjuvant yielded higher IgG titers than one immunization. Although the CoVaccine HT-adjuvanted vaccine induced higher IgG antibody titers after the first immunization, the magnitude of increase in serum IgG titers between the first and second immunizations was higher in mice that received alum-adjuvanted vaccine (26-fold increase) than in mice receiving the CoVaccine HT-adjuvanted vaccine (5-fold increase), consistent with previous results also shown in Fig. 3C. After the second dose, both formulations elicited comparable IgG antibody titers.
To evaluate the humoral response after ZIKV challenge as an indirect marker of protection from viremia, mice were infected i.v. with 100 PFU of ZIKV via tail vein injection, without immunosuppressants, 4 weeks after the last immunization, and sera from all groups were collected 2 weeks postchallenge. In mice immunized with a single dose of the antigen formulation, ZIKV challenge resulted in an approximately 19-fold increase in IgG MFIs when mice were immunized with ZIKV E and alum and in a 3-fold increase when mice were immunized with the antigen plus CoVaccine HT compared to prechallenge titers, effectively demonstrating an anamnestic response in these animals (Fig. 4E). In contrast, no significant difference in post- versus prechallenge IgG titers was observed in mice receiving two doses of either candidate vaccine formulation. The lack of an anamnestic response in these animals suggests that two vaccinations successfully prevented systemic viral replication.
To confirm the successful infection of all mice, IgG titers against ZIKV NS1 were also measured pre- and postchallenge using an in-house-developed anti-ZIKV NS1 IgG MIA. A marked increase in anti-NS1 IgG titers (as MFI) postchallenge (Fig. 4F) suggests that all mice were productively infected with the ZIKV. The MFIs against NS1 were below the cutoff for most of the mice prechallenge, but a few mice, mainly from the CoVaccine HT-containing group, showed low reactivity, possibly due to an immune response against a low level of common S2-derived proteins carried through purification of both E and NS1.
Sera from ZIKV E-immunized mice protect against ZIKV challenge.To evaluate whether antiserum from mice immunized with the ZIKV E vaccine formulation with CoVaccine HT or alum can provide passive protection, 100 μl of pooled sera with high or low antibody titers obtained from vaccinated SW mice (administered 10 μg ZIKV E with either CoVaccine HT or alum) (Fig. 2D) were transferred intraperitoneally (i.p.) to naive BALB/c mice one day before i.v. (tail vein) ZIKV challenge (Fig 5A and B). Antibody titers were measured 14 days after challenge to determine efficacy against productive virus infection. Both groups receiving either the low- or high-titer serum showed a decrease in anti-ZIKV E IgG of approximately 6- and 3-fold, respectively, compared to prechallenge titers. Due to the time point early after infection, mice receiving naive serum or no serum generated only low titers of ZIKV E-binding IgG antibodies (Fig. 5C); however, they showed consistent seroconversion. The relatively high MFIs observed from the high-titer serum transfer group postchallenge suggest residual IgG from the transfer remaining even 2 weeks after injection. Seroconversion is a sign of productive virus infection in unprotected animals, and therefore, the presence of anti-ZIKV NS1 titers postchallenge in all mice, including those receiving high-titer antisera (data not shown), suggests a low level of virus replication rather than sterilizing immunity; nevertheless, passive immunization appears to result in protection sufficient to prevent generation of significant antibody responses against the E protein, which otherwise indicate widespread virus dissemination.
The presence of neutralizing antibodies postchallenge (Fig. 5D) was determined in mice receiving high-titer, low-titer, or naive-mouse sera or in control mice (no serum transfer), and the data comparing the neutralizing antibody titers and the MIA titers for the pre- and postchallenge sera are shown in Table 1. Determination of prechallenge neutralizing antibody titers in individual mice was not possible due to standard Institutional Animal Care and Use Committee (IACUC)-imposed restrictions on the volume of blood drawn from live animals, which prevented sufficient volumes required for PRNT assays to be drawn. The difference in neutralizing titers in the transferred sera between the de novo high-titer (EC50 = 1,153) and low-titer (EC50 = 35) antisera was >30-fold. We believe that the relatively low level of neutralizing antibodies postchallenge in mice receiving high-titer antisera compared to levels in all other groups suggests that transfer of high-titer antisera provided passive protection by inhibition of virus replication, resulting in no primary antibody response in this group (Fig. 5D), consistent with no or very limited virus replication in these animals.
The simulated high-titer SW mouse serum pool (estimated by mixing SW mouse serum pools with naive BALB/c mouse sera at a 1:10 ratio) exhibited a PRNT50 of 1,153 (Table 1), while the anti-ZIKV E titer, measured by MIA, was 4,732 (Table 1). The simulation using this dilution is based on the proportion achieved by adding SW mouse sera (100 μl) to the total serum volume of a BALB/c mouse (~900 μl). In contrast, assays of the postchallenge sera from individual mice receiving the high-titer sera yielded a geometric mean titer (GMT) of PRNT50s of 153 (Fig. 5D), while the GMT of IgG titers (expressed as MFIs) was 1,422 (Table 1). Thus, the ratio of antigen-binding antibody to neutralizing antibody in the transferred serum, 4.1 (4,732/1,153), is half the value of 9.3 (1421/153) seen in the postchallenge sera from the same group, suggesting that neutralizing antibody activity after challenge declined much faster than the antigen-binding activity.
The results reported herein describe the development of a ZIKV recombinant subunit vaccine produced in Drosophila melanogaster S2 cells, its immunogenicity in three mouse strains, and its protective efficacy in a viremia model. Stably transformed S2 cell lines yielded high quantities of truncated ZIKV E protein at a relatively high purity after single-step purification by IAC and is reactive with convalescent-phase sera. The crystal structures of soluble ZIKV and DENV E proteins expressed using this platform have been elucidated previously (31, 32) and appear to be "native-like"; however, these soluble proteins did not form virus-like particles (VLPs) due to the truncation of the membrane anchor. Upon administration of two doses of ZIKV E protein formulated with two clinically relevant adjuvants, maximum antibody titers were observed in mice 2 weeks after the second dose, which is consistent with the humoral response from other adjuvanted subunit vaccines (27). ZIKV E with CoVaccine HT elicited higher antibody titers than E protein with alum upon the initial dose, although antibody titers equilibrate after subsequent booster immunizations.
The immunogenicities of our ZIKV E vaccine candidates were compared using three different mouse strains with different immunological backgrounds. IgG titers for the vaccinated BALB/c (Th2-dominant) and C57BL/6 (Th1-dominant) mice were consistent and displayed no difference between the two adjuvants after the second dose. Thus, the vaccine candidate was found to be equally immunogenic in a strain with a humoral bias (BALB/c) or a cell-mediated bias (C57BL/6). However, a wide variance in IgG titers from immunized SW (outbred) mice was observed from sera collected after both the first and the second dose for both adjuvanted formulations. This could be attributable to a suboptimal antigen dose (5 versus 10 μg) and to the outbred status of this strain. For immunization of heterogeneous populations (e.g., humans), a dose-response study is warranted in order to optimize the immunizing dose for maximal seroconversion. Such a study could be conducted with NHPs prior to initiating a human clinical trial. High neutralizing antibody titers have been correlated with protection from flavivirus infections (33, 34). We have demonstrated that two doses of the ZIKV E vaccine candidate induce high levels of neutralizing antibodies in BALB/c and C57BL/6 mice and somewhat lower, but still highly significant, titers in SW mice. For strains administered the same vaccine formulation, no difference was observed among the vaccinated groups, indicating a favorable humoral response regardless of the Th1 or Th2 dominance of the mouse strain. While high antigen-binding IgG titers are not always correlated with induction of high neutralizing antibody levels, the ZIKV E vaccine candidate used in this study appears to stimulate both high antigen-binding and neutralizing antibody titers, suggesting that the recombinant subunit ZIKV E antigen is presented to the immune system in a proper conformation.
The protective efficacy of the recombinant subunit vaccine candidate was evaluated in a mouse model of systemic viremia. BALB/c mice were chosen for this model based on previous work by Larocca et al. (20), who showed robust ZIKV replication in these mice when they were inoculated intravenously. Results from ZIKV challenge with the PRVABC59 strain in immunized BALB/c mice show complete protection against viremia after a two-dose regimen, assessed by plaque assay and corroborated by a lack of anamnestic antibody responses postchallenge. Protection from high-level virus replication is inferred based on the lack of increased IgG titers postchallenge in mice receiving two doses of either of the adjuvanted formulations. The rapid increase in IgG titers after challenge in groups receiving one dose of ZIKV E with either adjuvant is consistent with limited virus replication in these animals and reactivation of memory B cells. A single dose of antigen with CoVaccine HT generated higher IgG titers than with alum. These observations support the protective efficacy of the vaccine candidate, especially the formulation containing CoVaccine HT, as it is able to consistently produce higher initial IgG titers than the formulation using alum and maintains high IgG levels after subsequent immunizations.
A passive-protection experiment showed that sufficiently high anti-ZIKV E IgG and PRNT50 titers alone protected individuals from virus dissemination. These results are consistent with the protection from the Brazil ZIKV2015 strain seen in BALB/c mice receiving an i.v. adoptive transfer of purified IgG from prM-E DNA vaccine-immunized mice and rhesus macaques (20). While the transfer of low ZIKV E-specific antibody titer serum to naive mice generated slightly lower PRNT50 titers (GMT = 713) after virus challenge than were seen in naive-mouse serum recipients (GMT = 934) or in the control group (GMT = 939), these differences were not statistically significant. The depletion of neutralizing antibodies postchallenge in the high-titer serum transfer group was likely the result of potent neutralization upon challenge, yielding virus-antibody complexes which are subsequently cleared by the host and thus not available for further virus neutralization. Nonneutralizing, but antigen-reactive, antibodies might persist and continue to be observed in the MIA, consistent with successful passive immunoprophylaxis. In contrast, recipients of low-titer sera showed a significant increase in PRNT50 titers coupled with a drastic decrease in the ratio of antigen-binding antibody to neutralizing antibody titers (from 6.3 to 0.1). This sign of seroconversion subsequent to virus challenge indicates an absence of protection and mirrored the response seen in recipients of the naïve-mouse serum pool and the control group (postchallenge ratios of 0.05 for both groups). Since the PRNT50 titer of the low-titer serum transfer group did not exceed the PRNT50 titers of groups not administered immunoprophylaxis, we conclude that the presence of high anti-ZIKV titers protects against viremia but that low titers may provide an incremental degree of protection, although not sufficient to prevent viremia.
Success with passive immunoprophylaxis by antiserum generated using the insect cell-expressed recombinant vaccine subunit antigen highlights the quality of the IgG response generated using ZIKV E as a lead antigen. Passive immunization with E-specific antibodies has also been shown to provide protection against other flavivirus diseases (35, 36). Treatment of BALB/c mice with E-specific antibodies provided protection from a lethal i.p. challenge against tick-borne encephalitis virus (TBEV) (37), similar to our observations, while increased NS1-, and lower E-specific antibody titers were observed postchallenge. Recovery of infectious TBEV after passive immunization and challenge of mice indicated a low level of virus replication, but nevertheless, protection against highly lethal disease was observed. Likewise, treatment with E-specific monoclonal antibodies reduced morbidity and increased survival against intracerebral (i.c.) challenge with JEV, WNV, and DENV-2 (35, 36, 38) in SW mice. Clearly, the presence of E-specific antibodies is positively associated with flavivirus disease protection.
While antigen-specific antibodies protect against homoserotypic infections, progression to severe disease is a concern with preexisting flavivirus immunity to a different serotype or different flavivirus. Although high virus-neutralizing titers are associated with protection, generation of antibody with suboptimal neutralizing levels may promote antibody-dependent enhancement (ADE) observed in other flaviviruses, such as DENV, which can complicate vaccine safety in areas where cocirculation is endemic. Several studies have shown that ZIKV-induced antibodies have the ability to enhance DENV and other flavivirus infection in vitro (39–41). Conversely, many more studies have documented the reverse, namely, DENV sero-cross-reactive antibodies driving ADE of ZIKV infection both in vitro (41–44) and in murine models (41, 45), while other studies have shown similar enhancement with WNV convalescent-phase serum (45–47). Yet despite the mounting in vitro evidence, preexisting flavivirus immunity appears not to cause severe ZIKV disease in rhesus macaques (48).
A contemporary study examined the immunogenicity of E protein domain III (EDIII) as a protein subunit vaccine candidate purified from transformed Escherichia coli inclusion bodies (49). This vaccine candidate induced high IgG1 antibody titers as well as ZIKV-neutralizing antibodies. Sera from immunized C57BL/6 mice showed no indication of eliciting ADE, which is notable considering that EDIII is known to contain many neutralizing antibody epitopes (49). Whether recombinant ZIKV E protein alone can promote heterologous virus enhancement remains to be investigated, although another study of a ZIKV VLP vaccine candidate expressing a full-length ZIKV E protein has shown no indication of enhancement from immunized mouse sera in U937 cells (50), a finding that further validates in vivo observations using the recombinant ZIKV E subunit vaccine candidate presented here.
Recombinant subunit vaccines have an excellent safety profile, as the immunogen is nonreplicative and there are no concerns regarding prior vaccine vector immunity. These advantages are especially beneficial considering stringent precautions required for the demographic most severely affected by this flavivirus infection (e.g., pregnant women and the elderly). Previous candidate recombinant subunit vaccines have been developed for WNV, DENV serotypes 1 to 4, Chikungunya virus (CHKV), and Ebola Virus (EBOV) and have been shown to be protective in mice (25, 27, 51, 52) and in NHPs (28, 53). WNV and DENV vaccine candidates based on the same platform have been evaluated in phase 1 clinical trials and have been shown to be well tolerated and immunogenic (54, 55).
In summary, we present an immunogenic recombinant ZIKV subunit vaccine candidate containing the E protein adjuvanted with either alum or CoVaccine HT. Two immunizations of the vaccine induced a robust antigen binding IgG titer and high levels of neutralizing antibodies in three different mouse strains. Furthermore, twice-immunized mice did not develop viremia after live-virus infection and did not mount an anamnestic antibody response after virus challenge, suggesting that immunization was sufficient to prevent virus replication. Passive transfer of immune mouse sera to naive mice corroborated the results seen in the virus challenge study of actively immunized mice and demonstrated the importance of the humoral response in protection. Based on the results reported herein, further development of the ZIKV E protein vaccine candidate is warranted.
Ethical statement.Mouse experiments were approved by the University of Hawai'i Institutional Animal Care and Use Committee (IACUC) and conducted in strict accordance with local, state, federal, and institutional policies established by the National Institutes of Health and the University of Hawai'i IACUC. The University of Hawai'i's John A. Burns School of Medicine (JABSOM) Laboratory Animal Facility is accredited by the American Association for Accreditation of Laboratory Animal Care (AAALAC). All animal experiments were conducted in consultation with veterinary and animal care staff at the University of Hawai'i. Mice were bred in colonies at JABSOM from original stocks obtained from Taconic Biosciences Inc. (Hudson, NY) (SW, BALB/c) or the Jackson Laboratory (Bar Harbor, ME) (C57/BL6).
Antigens were produced in a WAVE bioreactor (GE Healthcare, Piscataway, NJ) using 2- or 10-liter bag sizes (and 1- to 5-liter culture volumes) and were subsequently purified by IAC. The mouse monoclonal antibody 4G2 was coupled to N-hydroxysuccinimide (NHS)-Sepharose (GE Healthcare, Piscataway, NJ) at 10 mg/ml. For ZIKV E purification, S2 cell culture medium containing recombinant protein was clarified by centrifugation and sterile filtered (0.2-µm pore size). The material was then loaded onto the IAC column at a linear flow rate of approximately 1 ml/min. After the supernatant was loaded, the matrix was washed with 10 mM phosphate-buffered saline (PBS), pH 7.4, containing 0.05% (vol/vol) Tween 20 (PBST, 140 mM NaCl), followed by a wash with PBS, pH 7.4. Bound protein was eluted from the IAC column with 20 mM glycine buffer, pH 2.5. The eluent was neutralized with 1 M phosphate buffer, pH 7.4, and the buffer was exchanged with PBS and concentrated using Centricon Plus-20 devices (EMD Millipore, Burlington, MA).
Similar procedures were followed for polyhistidine-tagged NS1 purification using a HisTrap FF column (GE Healthcare, Piscataway, NJ) per the manufacturer's instructions. The recombinant protein was loaded at a linear flow rate of approximately 1 ml/min and washed with PBS containing 300 mM NaCl, pH 7.4. Bound protein was eluted on a linear gradient of up to 0.5 M imidazole in the same buffer, and then the buffer was exchanged with PBS and concentrated as described above.
The purified products were analyzed by SDS-PAGE by Coomassie blue staining followed by Western blotting and quantified by UV absorption. Purified recombinant proteins were filter-sterilized and stored at −80°C. A control "null" antigen was prepared by buffer exchange and concentrating supernatants from untransformed S2 cells grown under conditions identical to those used for the S2 cell lines expressing recombinant proteins and was used as a control to represent potential insect cell-derived contaminants (refer to "Luminex-based MIA" below).
Western blotting.The supernatant of an S2 cell culture collected one week postinduction was mixed with NuPAGE lithium dodecyl sulfate sample buffer (Thermo Fisher, Waltham, MA), boiled for 10 min, and run on NuPAGE 4 to 12% bis-Tris protein gels (Thermo Fisher, Waltham, MA). Protein was transferred to a nitrocellulose membrane with a Bolt miniblot module (Thermo Fisher, Waltham, MA) and blocked with 1% nonfat dry milk (Nestlé United States Inc., Solon, OH) in PBST, pH 7.4, with 0.01% sodium azide for 1 h at room temperature or overnight at 4°C. Membranes were probed with a 1:3,000 dilution of flavivirus E-specific 4G2 antibody (1.6 mg/ml) or convalescent-phase PRVABC59 ZIKV-infected mouse or NHP serum at a dilution of 1:1,000 for 1 h at room temperature. After being washed with PBST, membranes were incubated with alkaline phosphatase-conjugated, goat anti-mouse or anti-human IgG antibody (Southern Biotech, Birmingham, AL) for another hour. The membranes were washed three times with PBST and developed with a nitroblue tetrazolium (NBT)-BCIP (5-bromo-4-chloro-3-indolylphosphate) (Promega, Madison, WI) solid-phase alkaline phosphatase substrate solution.
Coupling of microspheres with recombinant ZIKV-E and NS1 antigens.The coupling of microspheres with ZIKV E or NS1 proteins was performed as described previously (56, 57). Internally dyed, carboxylated, magnetic microspheres (MagPlex-C) were obtained from Luminex Corporation (Austin, TX, USA). A two-step carbodiimide process recommended by Luminex was used to link 10 µg of purified ZIKV E or NS1 to the surfaces of 1.25 × 106 microspheres. The antigen-conjugated microspheres were stored in 250 µl of PBN buffer (PBS with 1% bovine serum albumin fraction V [OmniPur] and 0.05% Ultra sodium azide [Sigma-Aldrich, St. Louis, MO]) at 4°C. Microspheres dyed with spectrally different fluorophores were also coupled with the untransformed S2 cell culture supernatant (null), bovine serum albumin, and PBS for controls.
Luminex-based MIA.Microspheres coupled with ZIKV-E or NS1, the untransformed S2 supernatant, bovine serum albumin (BSA), and PBS were pooled in PBS-1% BSA (PBS-BSA) at a dilution of 1:200. Fifty microliters of the coupled microsphere immunoassay (MIA) suspension was added to each well of black-sided 96-well plates. Serum samples were diluted 1:100 in PBS-BSA, and 50 µl was added to the microspheres in duplicate and incubated for 30 min on a plate shaker set at 700 rpm in the dark at room temperature. The plates were then washed twice with 200 µl of PBS-BSA using a magnetic plate separator (Millipore Corp., Billerica, MA). Fifty microliters of red phycoerythrin (R-PE)-conjugated F(ab′)2 fragment goat anti-mouse IgG specific to the Fcγ fragment (Jackson ImmunoResearch, Inc., West Grove, PA) was added at 1 µg/ml to the wells and incubated another 45 min. The plates were washed twice, as described above, and microspheres were then resuspended in 100 µl of sheath fluid and analyzed on a Luminex 100 apparatus (Luminex Corporation, Austin, TX). Data acquisition detecting the MFI was set to 50 beads per spectral region. Antigen-coupled beads were recognized and quantified based on their spectral signature and signal intensity, respectively.
Cutoff values were calculated as the average MFI of 25 serum samples from mock-treated BALB/c mice plus three standard deviations (Microsoft Office Excel). Serum samples with MFI values greater than the cutoff were considered positive. Control beads were coupled with BSA and the S2 cell supernatant using the aforementioned coupling protocol (56) to check for nonspecific attachment of serum proteins to the microspheres. The signals generated with BSA-, S2 supernatant-, and PBS-coupled beads were below the cutoff.
To establish the correlation between MFI and antibody titer, single-point (1:100 dilution) MFI values from seven serum samples containing different anti-ZIKV E titers were plotted against their EC50s. EC50 titers were determined by MIA on mouse antisera derived by using six 4-fold serial dilutions of each serum sample, starting at 1:25, and measuring the MFI at each dilution. A sigmoidal dose-response computer model (Prism, GraphPad Software, Inc., San Diego, CA) was used to generate the values for the EC50s from these data.
Virus stock and cell culture.Vero cells were grown in Hanks balanced salt medium 199 (M199), containing 2 mM l-glutamine, 5% fetal bovine serum (FBS), and 100 U/ml penicillin-streptomycin (Invitrogen, Carlsbad, CA), maintained at 37°C, and humidified in 5% CO2. ZIKV Puerto Rican strain PRVABC59 was obtained from the American Type Culture Collection (ATCC; Manassas, VA) and amplified in Vero cells. Virus titers were determined by plaque assay as described previously (59).
Vaccination, serum collection, and challenge.All mice (ages at the first immunization ranged from 6 to 9, 6 to 10, and 15 to 18 weeks for BALB/c, C57/BL6, and SW mice, respectively) were immunized intramuscularly (i.m.) with 5 to 10 μg of ZIKV E protein (specified above) with or without adjuvants using an insulin syringe with a 29-gauge needle. The adjuvants used were alum (2% Alhydrogel adjuvant [InvivoGen, San Diego, CA] or Imject alum [Thermo Fisher, Waltham, MA]) or CoVaccine HT (Protherics Medicines Development Ltd, London, United Kingdom), a squalane-in-water emulsion containing sucrose fatty acid sulfate esters immobilized on submicron-sized oil droplets (60). Sera were collected 2 weeks postimmunization by tail bleeding or cardiac puncture for terminal bleeds. ZIKV E-specific IgG antibody titers in mouse sera were measured using the MIA as described above. Challenge with 100 PFU of the Puerto Rican ZIKV strain PRVABC59 was conducted via injection of 100 µl of diluted virus stock into the tail veins of mice 11 to 13 weeks old at the time of challenge. To determine the kinetics of viremia, blood was collected daily for 5 days postchallenge and a plaque assay of serum samples was performed as described previously (61).
PRNT.Sera from individual animals were heat inactivated by incubation at 56°C for 30 min. The titer of anti-ZIKV neutralizing antibody was measured in the serum using the plaque reduction neutralization test (PRNT) as described previously (59). Briefly, a series of 4-fold dilutions, starting at 1:20, of each sample was prepared using M199 medium, and the samples were incubated overnight with medium containing previously titrated virus at 4°C in a 1:1 (vol/vol) ratio to generate approximately 50 PFU of virus per well. The antibody-virus complex was then added to Vero cells in duplicate and incubated at 37°C for 1 h. Cells were overlaid with M199 containing 1% agarose and incubated for 72 h. A second overlay of 1% agarose in M199 containing 1.5% neutral red (Sigma, St. Louis, MO) was added, and plaques were counted 24 to 48 h later. PRNT50 values, the serum dilutions yielding 50% virus neutralization, were generated using a fixed, or variable-slope sigmoidal dose-response computer model (Prism; GraphPad Software, Inc., San Diego, CA).
Passive-transfer studies with BALB/c mice.Sera from SW mice immunized with ZIKV E with adjuvant were pooled and filter sterilized, and 100 μl of undiluted serum was injected i.p. into 11-week-old BALB/c mice 1 day prior to virus challenge. Serum collection by tail vein bleeding and cardiac puncture was performed 2 and 3 weeks after challenge, respectively. ZIKV E-specific IgG antibody titers in mouse sera were measured by MIA as described above. The presence of neutralizing antibodies was determined by PRNT.
Statistical analysis.Determination of significant differences in IgG antibody titers between groups of mice was performed using an unpaired t test. Significant differences in ZIKV PRNT50 values among sera from passively protected BALB/c mice (Fig. 5D) receiving high-titer, low-titer, or naive SW mouse serum or from the control mouse group were determined using a one-way analysis of variance (ANOVA), followed by Tukey's multiple-comparison test. The Fisher exact probability test was used to determine significance between the numbers of viremic mice in vaccinated or control (nonvaccinated) groups subsequently challenged with live virus. All statistical analyses were performed with a commercial statistical program (Prism; GraphPad Software, Inc., San Diego, CA). A P of <0.05 was considered significant in one-tailed tests comparing vaccinated and control groups and comparing responses to primary vaccination and responses to each subsequent vaccination and in two-tailed tests comparing different vaccinated groups (GraphPad Prism).
We thank Wei-Kung Wang and Wen Yang Tsai for providing the alum adjuvant used in the initial immunogenicity study and for their valuable advice. We also thank David Clements (Hawaii Biotech, Inc.) for providing some purified ZIKV E protein and acknowledge the gift of the adjuvant (CoVaccine HT) from Protherics, a BTG company.
Funding for this project was provided in part by the Pacific Center for Emerging Infectious Diseases Research (COBRE grant P30GM114737), by the National Institute of General Medical Sciences (NIGMS), and by institutional funds. K.O.M. was supported by a grant (D43TW009074) from the Global Infectious Diseases Training Program, Fogarty International Center, NIH.
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HomeTranslation and Tafsir of the Holy Qur'anTafsir of Sura Qaf - Shaykh Abdullah Sirajuddin al-Husayni (Author), Rashad Jameer (Translator)
Imam al-Sabuni's Tafsir of Surah al-Rahman - Rashad Jameer (Translator)Back to Translation and Tafsir of the Holy Qur'anImam Sabuni's : Tafsir of Surah Al-Waqi'ah Selections from Safwat al-Tafasir - Rashad Jameer (Translator)
Tafsir of Sura Qaf - Shaykh Abdullah Sirajuddin al-Husayni (Author), Rashad Jameer (Translator)
The Tafsir of Sura Qaf was written for seekers who wish to learn the meanings of the Book of Allah and deepen their connection to the Messenger of Allah s.w.t by...
SKU: N/ACategories: Best Selling, Translation and Tafsir of the Holy Qur'anTags: Surah, Tafsir
The Tafsir of Sura Qaf was written for seekers who wish to learn the meanings of the Book of Allah and deepen their connection to the Messenger of Allah s.w.t by the Syrian gnostic ('arifbillah) and lover of the Prophet ﷺ, Shaykh 'Abdullah Sirajuddin al-Husayni r.a. The blessings of the Quran are infused into the wisdom of its meanings. Sura Qaf comprises timeless messages focusing the reader on the hereafter, transporting him to the time of his own death (sakrat al-mawt), removing the veil from the Recording Angels (raqibun 'arid) on his shoulders, and placing him squarely in the middle of the Day of Judgement. Yet, despite this sura's heavy focus on the akhira, it is Shaykh 'Abdullah Sirajuddin's love of the Prophet s.a.w that surfaces throughout this work.
Shaykh 'Abdullah Sirajuddin al-Husayni r.a. was a descendant of Imam Husayn b. 'Ali b. Abu Talib a.s. from his father's side. Shaykh 'Abdullah was born into an honourable and pious family just prior to the collapse of the Ottoman Empire in 1923. During his childhood, the Shaykh was surrounded by the love and care of his father, Shaykh Muhammad Najib Sirajuddin, who was also a leading scholar of tafsir, fiqh, hadith and tasawwuf. Shaykh 'Abdullah Sirajuddin was known to see the Prophet ﷺ frequently in his dreams and whilst awake. In one such vision, the Prophet s.a.w told him: "you are our dedicated servant" after which the Shaykh would say: "The Prophet is pleased to have me as his servant." He passed in 2002 in Syria.

redpajama_set_name: RedPajamaCommonCrawl


All of the Thai translators are listed in the Treaty Times Thirty book. Translators who chose to be included on the website are also listed below.
Kamolwan Sonsomsook first started to work in Thai and English, translating news at the BBC World Service at Bush House, London. Through her journalistic career in Thailand, she also translated news for the Bangkok Post newspaper and the Bangkok Bureau of the Far Eastern Economic Review.
Arriving in New Zealand in the 1980s, she worked for the New Zealand Translation Centre as a translator and an interpreter. Later she worked as a freelance translator for the Translation Service of the Department of Internal Affairs. She also took translation work for major Thai companies.
Completing her Masters in Teaching English as a Second Language at Victoria University, Wellington, she now designs Thai language courses and teaches Thai children and New Zealanders wishing to learn the Thai language.
Kamolwan was educated at Kent College Junior school, Tonbridge Wells, England; Mater Dei Convent, Bangkok and Chiang Mai University, in Thailand.


text:
with Charles Christian
New Americana Music Radio Show now on Mixcloud...
First Americana Music Radio Show now available on Mixcloud
Submitting your music to the show
Yes, of course you are welcome to submit your own music to the Americana Music Radio Show – that's how we discover new talent although given the pressure on time (we only have 59 minutes) there's no guarantee we'll be able to play it. However, a few brief guidelines...
* You can physically submit music on a CD – use the Oak Lodge contact address you'll find in the footer of this page.
* You can also submit music digitally as an MP3 file (or files) to christian.uncut@icloud.com BUT PLEASE DO NOT send the MP3 as an email attachment (it will kill our internet) instead email it as a WeTransfer, Dropbox or similar link that we can then download. And NOTE: MP3 files only.
* Also include some short biographical notes about you, the track (is it a taster track from an album etc) and any social media links you have, so we can fully promote it.
New Americana show drops today!
The first episode of my new Americana Music Radio Show drops today on A1 Radio at 4pm UK (repeats 6pm Friday) – we've tracks from The Highwomen, Neil Young & Crazy Horse + an interview & tracks from Sam Coe – and much more. Listen online via TuneIn Radio & at www.a1radio.co.uk
First station signs up for new music show
I'm very happy to report that KCOR Radio in Las Vegas has signed up to carry the my new Americana Music Radio Show, with the first episode scheduled to air on Sunday 22 September – that's 7pm UK time.
I'm in conversation with several more US stations PLUS have three UK stations that have already committed to it (and with more in the pipeline). I'll be posting up station details, including show times and how to listen, over the next couple of days on this site. Full details will appear on the side-bar both here and on the home page. Now all I need to do is record the first show!
The Americana Music Radio Show is an hour-long, weekly syndicated radio show made in England for Americana lovers everywhere. We cover Roots, Alt-Country and Folk-Rock, along with occasional interviews and Americana albums news but always with an emphasis on more music and less chat.
Here's what the station manager of one of our US syndicates said about the show "Keep up the great work, people are loving it and so are the bands who don't normally have a voice." While one of the acts we played said "Thank you again for everything, your support really makes a huge difference in so many people's careers."
The Americana Music Radio Show is also available on Mixcloud – right here www.mixcloud.com/charles-christian/
Americana Music Radio Showtimes...
Blues & Roots Radio
Wednesdays 8:00pm EST & Sundays 6:00pm EST. Ways to listen online https://www.bluesandrootsradio.com/ways-to-listen
Radio Scarborough
Wednesdays 6:00pm & Saturdays 6:00pm UK. Listen online at
https://streams.radio.co/s3584039e7/listen
SG1 Radio
Wednesdays 7:00pm UK. Listen online via TuneIn Radio, iOS & Android app & at www.sg1radio.co.uk
Mod Radio
Thursdays 1:00pm & Sundays 8:00pm UK. Listen online via TuneIn Radio & at https://www.modradio.co.uk/listen
Cambrian Radio
Thursdays 6:00pm UK. Listen online & via apps full details here http://www.cambrianradio.co.uk/
SeaBird Radio
Thursdays 8:00pm UK. Listen online via iOS & Android app & at s3.radio.co/s554d67209/low
Thursdays 9:00pm UK. Listen online at http://fantasyradio.stream
Fridays 6:00pm, Sundays 4:00pm & Mondays 8:00am UK. Listen online via TuneIn Radio & at www.a1radio.co.uk
East Point Radio
Saturdays 3:00pm UK. Listen online on TuneIn Radio & at
https://www.eastpointradio.com
Cheeky FM
Saturdays 6:00pm UK. Listen online on TuneIn Radio & at https://www.cheekyfm.com
Faithful Radio
Saturdays 8:00pm & Wednesdays 1:00pm UK. Listen online via TuneIn Radio, iOS app & at www.faithfulradio.com
Saturdays 4:00pm CST - 10:00pm UK & Sundays 10:00am UK. Listen online via TuneIn Radio & at www.cmrnashville.com
KCOR Digital Radio Network
Sundays 11:00am PST - 2:00pm EST - 7:00pm UK. Listen online via TuneIn Radio & at https://kcorradio.com
Americana Music Radio Show Page
Mix Tape Radio International
Mondays 4:00am - 10:00am - 4:00pm UK. Listen online at www.mtri.co.uk
* By mail as a CD to the Oak Lodge address in the footer of this page
* Digitally as an MP3 file – send a downloadable link (WeTransfer, Dropbox etc – singles OK by email) to christian.uncut@icloud.com
* Please include information about you, the track & any socialmedia links
Email: christian.uncut@icloud.com
Tel: +44(0)1986 788666
Tel/Txt: +44(0)7786 738172
WhatsApp: +44(0)7786 738172
Skype: ChristianUncut
Twitter: @ChristianUncut
Charles Christian Radio & Writing
Tweets by ChristianUncut


Each spring for the past few years, I have given a cooking class called simply "Salads, Salads, Salads." Without fail the course is always the most popular of the season. The format remains the same every year; I choose four salads that can be used as first courses, as entrees or as side dishes.
The menu includes one salad with seafood, another with beef or poultry, one that features vegetables and one that highlights fresh fruit. Students tell me repeatedly that they find themselves using these salads over and over again when entertaining during the warm weather months, when lighter fare is in order.
Napa, Shrimp and Snow Pea Salad in Sesame Vinaigrette, a dish I created recently, would be a perfect addition to my cooking-class repertoire. The idea for this original salad came from Amy Mehaffey, one of my talented culinary assistants. She encouraged me to use thinly sliced Napa cabbage, the elongated Chinese cabbage with crisp ivory- and celadon-colored leaves, for this recipe rather than traditional lettuce greens. And, she suggested that coral-hued shrimp and bright-green snow peas would complement the cabbage visually. Together we decided that a sesame vinaigrette made with rice vinegar, soy sauce and dark toasted sesame oil would be the perfect dressing for these ingredients.
This salad works well in varying roles. It could be served in smaller portions to begin a spring or summer dinner party. It would be equally good to offer with a bowl of soup (asparagus or carrot soup comes to mind) and a basket of warm crusty French bread for a luncheon or light supper.
And, at this time of year when celebrations, including graduations, weddings and anniversaries abound, this salad would made a beautiful addition to a buffet table.
This recipe yields 6 servings when used as starter or accompaniment and 4 as a main course, but the ingredients can be doubled or tripled easily to feed a crowd.
For vinaigrette, place vinegar, soy sauce and sesame oil in nonreactive salad bowl. Cut off roots from green onions and mince white parts and add to dressing. Reserve green stems for garnish.
Cook snow peas in boiling salted water to cover until just tender, 1 to 2 minutes. Drain in colander and rinse under cold running water to cool. Pat dry with clean kitchen towel. Set aside.
Shell and devein shrimp, then cook in boiling salted water to cover until pink and curled, 2 to 3 minutes. Remove, drain in colander, and rinse under cold running water to cool. Pat dry and set aside.
Cut off about 4 inches from base of cabbage and discard. Then cut cabbage, crosswise, into 1/2-inch-wide slices.
To assemble salad, place snow peas in bowl with dressing and toss well; remove 1/3 of snow peas and arrange as border on serving platter.
Add shrimp and cabbage to salad bowl. Toss well to coat lightly with dressing. Mound salad on platter. Chop reserved green onion stems on diagonal to make about 2 tablespoons. Sprinkle over salad. Serve immediately.


This entry was posted on Thursday, June 21st, 2018 at 10:20 am and is filed under Gift Ideas. You can follow any responses to this entry through the RSS 2.0 feed. You can leave a response, or trackback from your own site.
Amazing and trending presentation of these gifts.
Thanks for sharing current trends in gifting.


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The Many Hats of a Business Owner
Tina Ellis 27 January 2020
As the saying goes, a business owner has to wear many hats. After you've owned a business for a while, you really come to understand how true this is. Even if you have employees who do a lot of your day-to-day marketing, ordering, and accounting, the responsibility for these tasks is ultimately your own. If you are starting to feel overwhelmed with the many hats you're required to wear, you have stumbled upon a good resource. We created this website to share knowledge about all of those hats — all of those roles you have to play as a business person. Start reading, and you'll not only gain helpful advice, but also realize that you're not alone.
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V-Ray Unreal: Phoenix FD 3ds Max Training
Today's digital rendering animation suites have made portable and accessible software that was once unconscionably expensive and difficult to use just a decade ago. If you're looking to learn a v-ray rendering suite for an application like the Phoenix FD 3ds Max program, a few shortcuts and tweaks can drastically reduce your learning curve.
Update Your Presets
Most v-ray programs come with a host of preset options to provide general ease of use. Although these presets might work for most of your projects, they can slow you down if you don't spend a little time modifying them for your specific uses.
Fluid Dynamics: programs like Phoenix FD 3ds Max were originally designed for post animation editing for major movie studios. You can unlock the potential of the fluid dynamic presets by considering the shading, lighting, and other ambient features that might dominate the project you're working on. For instance, if you're working on shots in low light, you might want to grab a number of still shots and adjust the fluid dynamic presets to give you a consistent look for the continuity of the project. Once you have you've customized the presets you can create a keyboard short cut to making editing as easy as a few keystrokes.
Smoke and Fire: smoke and fire remain among the most difficult to naturally replicate in post animation renderings. V-ray suites can help you not only add smoke and fire but make it look realistic too. The realism often stems from the adaptive grid built into the programs. If you've never worked with an adaptive grid, the process can feel a bit daunting. The key, however, is not to focus too acutely on the isolated details. When tweaking smoke and/or fire in the grid, you should adjust the saturation and lighting within the grid shots. These holistic changes can be more effective in capturing the feel of a shot or image. Once you've nailed down the lighting and/or saturation for your grid shots you should create a customized preset and keyboard short cut for efficient editing. Many v-ray software suites have galleries of customized presets created by editing professionals available through their website with the purchase of the licensing and software.
Optimize Before Final Editing
The most advanced v ray suites come with AI optimization to sharpen your project with powerful algorithms. Often, you're better off optimizing broadly before you spend time on your final editing touches.
Part to Whole: the best editing makes a shot or series of shots look unedited. You can capture an organic and seamless feel to your animation by blading several scenes, optimizing each bladed section individually, before combining and optimizing the shot collectively in the end. By optimizing each shot or scene independently, you're more likely to standardize the quality, lighting, and other fluid dynamic components, which will create a standardized baseline for the software's AI program. This is particularly true when cobbling together scenes with unique lighting elements like fog or smoke.
Sound Mixing: although most v-ray programs are built with visualization in mind, the best programs also offer advanced sound mixing. For instance, if you're scene calls for a shot of a turbulent ocean, you won't create an immersive experience unless the sound is mixed appropriately. To add a depth of complexity, consider optimizing the sound mixing of a shot to add additional ambient layers. For an ocean scene, this will not only be the sound of the waves lapping onto the shore, but also the reverberation of the small waves created by the rippling of the wind on the water or the distant call of birds in the sky.
© The Many Hats of a Business Owner


Surprise, AZ (March 28, 2019) The Surprise Tennis and Racquet Complex is proud to welcome the United States Tennis Association (USTA) National Tennis Rating Program (NTRP) 50 & Over Men's and Women's Championships this spring.
Information regarding match times, bracket play and results is available on the USTA NTRP official tournament site.
Due to the overwhelming success of the 2018 event, the 2019 NTRP National Championships have been expanded to include the 3.0 level for 18 & over and 50 & over age groups as well as the 5.0 level for the 18 & Over age group.
The Surprise Tennis and Racquet Complex was recently recognized as a USTA National and USTA Southwest Section Facility of the Year. The complex welcomes over 2,000 active USTA members throughout the winter/spring competitive season.
Visit www.surpriseaz.gov/tennis for information on tournaments, classes and more at Surprise Tennis & Racquet Complex. For information on registration or open court availability please call 623.222.2400.


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Summit Park Will Attend the Food Safety Summit
Recent News April 22, 2015
Summit Park invests capital in growing companies in the Food Safety Industry. We will be attending the Food Safety Summit next week and would welcome the opportunity to meet you there.
If you are planning to attend the 2015 Food Safety Summit next week in Baltimore, we would welcome the opportunity to set up a time to meet with you to introduce ourselves and learn more about your company. If you would like to set up a one-on-one meeting, please email Rachel Hannon or call her at 704-654-3400 x240.
Summit Park Investment Thesis
Our team's operational experience in the food processing industry utilizing food safety diagnostic vendors, as well as relevant experience in food policy and supply chain, gives us a unique and differentiated investment perspective. We believe diagnostic service providers and product vendors provide a mission critical value proposition to agricultural and food processing companies, which have a very high cost of failure with respect to food safety. The current regulatory climate, including FSMA, coupled with secular perceptions of food safety threats are driving new innovations in food safety diagnostics in the U.S. and internationally.
Our Food Safety Sector Focus
Food Safety Test Manufacturers (pathogen, allergen, authenticity, GMO)
Food Safety Laboratories
Food Safety Products
Traceability and Cold Chain Management
View our most recent Research Profile on the Food Safety Industry.
Summit Park is a Charlotte, North Carolina based private investment firm focused exclusively on lower middle market businesses. We seek to be the partner of choice for owners, entrepreneurs and superior management teams, and our capital can be used to facilitate a change in ownership, to support expansion and growth, to provide partial liquidity to existing owners or to support an industry consolidation plan. Summit Park targets businesses with revenue between $10 and $100 million and located in the Mid-South, Mid-Atlantic and Southeastern regions of the United States.
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Connect: 704-654-3400
info@summitparkllc.com
Office: 300 S. Tryon St.
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Online Counselling how do I start and is it effective?
Online Counselling is not something that we often associate with therapy. In this day and age, it makes sense to offer therapy online. It is a service that is becoming more widely available. Online Counselling offers flexibility, convenience, comfort and choice of a counsellor. I often get asked how do I start online counselling, and is this as effective as seeing a therapist in person? I hope to offer some answers to these questions.
How do I start Online Counselling?
Contact a therapist who specialises in Online Counselling and has had training in working online. The counsellor will assess if this is suitable for you, and you can discuss what medium you prefer to meet your counsellor.
Online Counselling is counselling via secure online mediums such as video, telephone, email and instant messaging. It is generally a 50-minute appointment or email exchange once a week with a counsellor. You can discuss how you are feeling and look at ways to manage or process what has brought you to counselling. This is similar to working in person with a counsellor. How long you work with a counsellor online is also your decision and one that you can also make together with your counsellor.
Is it as effective as seeing a therapist in person?
Research has found 'much support for the application of psychotherapeutic interventions through the Internet, using various approaches, methods, and online modalities……and on the average, is as effective as face-to-face intervention'. In my own professional and personal experience, online counselling has also proven to be as effective and the most important factor is how well you get along with your counsellor. I suggest you talk to me prior to our first session for 15 minutes and see how you feel about working together.
Online Counselling has proven to be effective if you are finding it challenging with anxiety or stress. Clients have found that they can be more open about how they are feeling online. They also feel supported in living the life they really want to live.


"With an Overall DxOMark Lens Metric Score of 35 the Nikon AF-S 85mm f/1.8G is the best 85mm lens in the DxOMark database and well exceeds the average DxOMark Overall Score of 28 for this type of lens."
This entry was posted in Nikon Lenses and tagged DxoMark test results, Nikkor AF-S 85mm f/1.8G lens. Bookmark the permalink. Trackbacks are closed, but you can post a comment.


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Huddersfield Town vs Swansea City kick-off times, live stream details and team news
Swansea City make their long-awaited return to championship action this afternoon as they take on Huddersfield Town.
Here's all the details about the game at John Smith's Stadium.
What time does Huddersfield Town vs Swansea City start?
Huddersfield Town vs Swansea City 3:00 on Saturday, January 15th.
Is Huddersfield Town vs Swansea City on TV?
No, the match will not be shown live on television.
Due to EFL broadcast regulations, SwansTV will not be streaming the game in the UK and Ireland, although fans in international territories can watch it for £10.
You can follow live updates on the match via our blog which will be available on our Swansea City page.
What's the latest team news?
Academy goalkeeper Nicolas Bilokapic is set to be included in the bench for the hosts due to an injury to Ryan Schofield.
Lee Nichols is next in line to make a Target debut after recovering from illness.
Huddersfield will also give a late fitness test to Chelsea loanee Levi Colville, who has been battling a knee problem.
Strikers Jordan Rhodes and Fraser Campbell are both available.
As for Swansea, the new signings from Cyrus Christie and Andy Fischer are terriers worth playing against.
Kyle Joseph – who was recently recalled from his loan spell with Cheltenham Town – is also set to be included in the squad on match day.
However, Russell Martin has confirmed that the Huns will be without "a couple" of players due to COVID-19.
What have the managers said?
Huddersfield Town head coach Carlos Corberon: "Swansea City are playing better than they are getting, so it will be a big challenge for us.
"We need to be ready to face whatever they do," he said.
"It will be a test of our capabilities in defense and attack."
Swansea City Boss Russell Martin: "They've been great, they've done really well. Benefit of giving the manager time."
"They had a tough season last season. You see it a lot. The change can be really messy. But they've come through it and they've recruited well with players that fit their system and style of play. are fit.
"They have some excellent players who have been at a really good level [Lewis] O'Brien and [Sorba] Thomas.
"They have spent a lot of time together as a group and they are benefiting from it, and I have no doubt that eventually we will be the same."
Huddersfield Town win – 10/17
Painting – 10/21
swansea city win – 7/4
(Courtesy of Sky Bet, correct at the time of publication)
Who is the referee?
David Webb will lead the proceedings in West Yorkshire.
The County Durham official was the man in the middle for Swansea's loss to Blackburn Rovers and victory at Barnsley.
He will be assisted by Adam Krisel and Matthew McGrath. Neil Heyer will be the fourth officer.
Swansea City fans, we want your thoughts!
You can join even more of WellsOnline's Swansea City coverage by signing up to comment on stories, deliver your verdict, discuss the biggest breaking news, and chat with our reporters in our dedicated Q&A.
match the facts
Huddersfield Town have lost one of their last nine home league games against Swansea (W5 D3), winning 4–1 in this exact fixture last season.
Swansea City are looking to complete a league double at Huddersfield under Frank Burroughs for the first time since the 1992–93 season.
Huddersfield are unbeaten in their last six league matches (W3 D3), their best run since scoring seven without a loss between September and November 2019.
Swansea have lost their last three league games, their worst run under Russell Martin. Indeed, Martin has not lost more than three consecutive league games in his managerial career so far (including three losses to MK Dons in March 2021).
Huddersfield's Harry Toffolo has helped score six league goals this season, his best tally in the Football League season since the 2018–19 Football League season for Lincoln City (also six). He last made more assists in a season for Swindon Town in 2014–15, when he scored seven league goals.
For the latest Swansea City news and analysis delivered straight to your inbox, you can sign up for our newsletter here.
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Brentford vs Man Utd kick-off time, TV Channels & Live Stream info


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Q: Java - error: cannot find symbol - switch / case public static void main(String[] args) {

    System.out.println("Bitte geben Sie einen Betrag und den Buchstaben der gewunschten Wahrung ein.");

    try {
        Scanner input = new Scanner(System.in);

        double betrag = input.nextInt();
        String wahrung = input.next();
        double result = 0;

        switch(wahrung.charAt(0)) {
            case d:
                result = betrag * 1.18;
                System.out.println(result);
                break;
            case p:
                result = betrag * 0.88;
                System.out.println(result);
                break;
            case f:
                result = betrag * 0.88;
                System.out.println(result);
                break;
        }
    } catch (Exception e) {
        System.out.println("Bitte geben Sie eine Zahl ein.");
    }
}

When I try to compile it, it says:
Rechner.java:18: error: cannot find symbol
                case d:
                     ^
  symbol:   variable d
  location: class Rechner
Rechner.java:22: error: cannot find symbol
                case p:
                     ^
  symbol:   variable p
  location: class Rechner
Rechner.java:26: error: cannot find symbol
                case f:
                     ^
  symbol:   variable f
  location: class Rechner

A: You use you switch with a char, so each case should also be a char value.
Try
switch(wahrung.charAt(0)) {
            case 'd':
                result = betrag * 1.18;
                System.out.println(result);
                break;
            case 'p':
                result = betrag * 0.88;
                System.out.println(result);
                break;
            case 'f':
                result = betrag * 0.88;
                System.out.println(result);
                break;
        }

redpajama_set_name: RedPajamaStackExchange


We are looking for a Primary Teacher to apply for a flexible and diverse supply teaching role that will cover classes across the EYFS, KS1 and KS2 in a multitude of Primary Schools that we are federated with in the Kings Lynn area.
This unique role will apply to those who are looking to work part-time hours across multiple sites and provide morning, afternoon and full days cover in mainstream Primary classes. A high level of prior experience is not essential for this position, but all applicants must be confident in their ability to teach and understand the curriculum for each class.
It is essential that your knowledge of the Primary Curriculum is extensive enough to work across multiple year groups. Planning is provided upon arrival for each class, making the role extremely accessible to those who choose to apply.
If you are interested in this unique opportunity, we would love to hear from you.


He took part in the creation of the production company Les Films du Tambour de Soie in 1987 and became its manager in 1996. Member of the CNC's Writing Grants Commission (Commission d'Aide à l'Ecriture) from 1999 to 2001. Member of the FACA (Regional Fund in Brittany, in 2004/2005), Languedoc-Roussillon region in 2006/2008, Rhône-Alpes region in 2009/2010 and currently member of the Documentary Commission in Region Nord – Pas de Calais. President of the PROCIREP's Television Commission in 2005 and 2006. Member of the EDN Executive committee since December 2008.
For several years, he has been a workshop tutor, developing documentary projects for the Femis, EDN, DocuRégio, Lussas and Eurodoc (in France, Portugal, Greece, Czech Republic…).
Alexandre was selected (2001, 2004, 2011) for the Prix du Producteur Français (French Producer's Award) granted by the PROCIREP. He hasn't yet won!


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Transgender Super PAC Bought Illinois School Board Election to Pass Radical Policy
By Megan Fox Nov 15, 2019 10:32 PM ET
Bloomfield High School transgender athlete Terry Miller wins the final of the 55-meter dash over transgender athlete Andraya Yearwood and other runners in the Connecticut girls Class S indoor track meet at Hillhouse High School in New Haven, Conn. (AP Photo/Pat Eaton-Robb)
In the most radical rule change to ever be implemented in Illinois high schools, the District 211 school board in Northwestern Illinois passed a new policy allowing any transgender student access to any private space or sport regardless of anatomy. This decision was hotly contested by the community. Supporters of the policy were dwarfed in number by those opposed. New information reported by Illinois Family Institute shows that a super PAC created by transgender activists in Chicago poured out-of-town money into the April 2018 election that led to the policy change.
The transgender super PAC, Trans United Fund, set up shop just 13 days before the April 2018 election and raised over $26,000 that they poured into another super PAC founded by Kim Cavill, a board member and far-left activist who hosts a show telling teens and tweens how to have anal sex, and promoting gender confusion. Cavill and her sister, Lindsey Christenson, launched a campaign with that money under their super PAC called Parents and Neighbors for Quality Education to beat out the board members who were running on locker room privacy.
The district had faced these challenges in the past and had been sued by transgender students demanding full access to all girls spaces. It's important to note that all the challenges have been from boys demanding access to female spaces. There are no lawsuits against D211 with biological girls suing to take over the boys' locker rooms or sports. The three candidates who were running on privacy, Jean Forrest, Katherine Jee Young David, and Ralph Bonatz were defeated by the smear campaign run by Cavill. The Windy City Times ran their propaganda.
Today, Trans United Fund, a national trans-led advocacy group, in partnership with local parents and trans youth in Illinois School District 211, announced a decisive and historic victory against a slate of candidates aligned with the Alliance Defending Freedom ( ADF ), which the Southern Poverty Law Center has designated as a hate group. Trans United Fund, working with local parents and youth, won all three of their targeted races, playing a decisive role in reelecting incumbents Anna Klimkowicz and Robert LeFevre and challenger Edward Yung.
"A mom from the district reached out to us for help," said LaSaia Wade, a Chicago-based board member of Trans United Fund. "She shared that local parents and students had been battling a network of national and state hate groups on their own. They were up against a 'hate slate' of candidates intent on taking over their local school board and rolling back protections for trans youth. But we knew that if the opposition won, it would mean more violence and bullying for LGBTQ students and any kid that's different. The timeline was short and our resources were already stretched thin, but we weren't going to let that happen."
Trans United Fund ( TUF ) and a group of local parents, youth, and allies, worked together to launch the first trans-led, trans-focused independent expenditure in history. TUF assembled a powerful team of thoughtful allies to quickly build and execute a research-informed and strategic plan to help the parents and youth get their message out. TUF supported the parents' efforts through digital, mail, phone banking and helping to train volunteers to reach their neighbors at the door.
There was no evidence that any of these allegations were true. No transgender students had been the subject of any violence and they were already given access to the girls' locker room but were asked to change behind a privacy screen. This was not good enough for the trans-activists whose goal it is to completely destroy privacy for women.
Twitter Bans Gay Man For Telling Inconvenient Truths About Transgender People
After winning the re-election, Robert Lefevre Jr., Anna Klimkowicz and Ed Young quickly went ahead with pushing through full access to the locker rooms and sports. During the board meeting Thursday evening, the board was asked to delay the vote until the matter could be put on a referendum so the community could record their say in an advisory capacity. While non-binding it would be a good way for the school board to test whether or not their community actually wanted to do away with sex-segregated facilities. The motion failed and the board refused to allow the community to have their say because this was never about what the community wanted but what far-left activists insist on forcing on communities against their will.
In every contentious board meeting that D211 had, the objectors outweighed the supporters two to one. Many of the supporters of the trans-agenda who showed up at meetings did not have children in the district, nor did they even live in the district. Chicago LGBT activists have a history of busing in people from other districts to make their voices seem loudest. Despite the vociferous opposition, the board voted 5-2 in favor of destroying privacy and fairness for girls.
Margolis & Cox Editorial Cartoons
In a particularly distasteful show of arrogance, five board members refused to stand and honor the slain students in the recent school shooting in Santa Clarita. One can only hope that it was due to the fact that a member of the parents who were opposed to the policy asked them to do it and not a callous disregard for the victims. Steve Rosenblum, Superintendent Cates, Anna Klimkowicz, Lisa Small, and Robert LeFevre Jr. all stayed seated and refused to honor the request. It's worth noting that both sides of citizens, who were moments before fighting, stood together in agreement for a moment of silence for the victims. You can see it happen at the 53:45 mark in the video below.
The far left in America have been diligently taking over local boards to implement unpopular, fringe ideologies in communities across this nation and no one has noticed because most are too distracted by what happens in Washington D.C. But the most powerful and dangerous people in the whole nation sit on your local school, library, town, or city boards.
Posted by D211 Parents for Privacy on Thursday, November 14, 2019
Megan Fox is the author of "Believe Evidence; The Death of Due Process from Salome to #MeToo." Follow on Twitter @MeganFoxWriter
Megan Fox is an award-winning journalist, broadcaster, and author of Believe Evidence; the death of due process from Salome to #MeToo. and Shut Up! The Bizarre War that One Public Library Waged Against the First Amendment, For media inquiries, praise, or hate mail please contact [email protected] or contact @MeganFoxWriter on Twitter, @MeganFox on Gab, and her Author Page on Facebook
Read more by Megan Fox
Tags: ACTIVISM EDUCATION TRANSGENDER
In Wake of Texas Synagogue Hostage-Taking, Anti-Defamation League Warns Against 'Islamophobia'
Wolves and Sheepdogs in Colleyville


She stood by the table, the light from a shaded lamp making her colour glow.
She made no reply, but opened the door into the cool and, shaded little parlor.
It was shaded by dark chestnut hair, just silvered with grey.
He shaded the dinner, cooled the wines, chilled the gravy, and blighted the vegetables.
She came into the parlor, clasping the bowl of a shaded lamp—the only light in the room.
It was so huge and shadowy, with only shaded lights here and there.
It will be more distinct if the ground glass be shaded from the light.
Before us was a driveway, shaded by great elms and bordered by rose hedges.
Bishop shaded his eyes with his hand to look in the direction in which she was pointing.


Deposition Author(s): Thomas, C.B., Scavetta, R.D., Gumport, R.I., Churchill, M.E.A.
Thomas, C.B., Scavetta, R.D., Gumport, R.I., Churchill, M.E.A.
The structures of RsrI DNA methyltransferase (M.RsrI) bound to the substrate S-adenosyl-l-methionine (AdoMet), the product S-adenosyl-l-homocysteine (AdoHcy), the inhibitor sinefungin, as well as a mutant apo-enzyme have been determined by x-ray crystallography. Two distinct binding configurations were observed for the three ligands. The substrate AdoMet adopts a bent shape that directs the activated methyl group toward the active site near the catalytic DPPY motif. The product AdoHcy and the competitive inhibitor sinefungin bind with a straight conformation in which the amino acid moiety occupies a position near the activated methyl group in the AdoMet complex. Analysis of ligand binding in comparison with other DNA methyltransferases reveals a small, common subset of available conformations for the ligand. The structures of M.RsrI with the non-substrate ligands contained a bound chloride ion in the AdoMet carboxylate-binding pocket, explaining its inhibition by chloride salts. The L72P mutant of M.RsrI is the first DNA methyltransferase structure without bound ligand. With respect to the wild-type protein, it had a larger ligand-binding pocket and displayed movement of a loop (223-227) that is responsible for binding the ligand, which may account for the weaker affinity of the L72P mutant for AdoMet. These studies show the subtle changes in the tight specific interactions of substrate, product, and an inhibitor with M.RsrI and help explain how each displays its unique effect on the activity of the enzyme.
Department of Pharmacology, University of Colorado Health Sciences Center, Denver Colorado 80262, USA.


This is it! The moment that we have been waiting for! Will Belial finally have his revenge or will his son save his soul from eternal damnation? Find out in this action packed (with a side of drama) clips from the final episode of Ultraman Geed!
"For the love of a son to his father"