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Establishing Restricted Expression of Caveolin-1 in HIV Infected Cells and Inhibition of Virus Replication
Yung-Tsun Lo, Peter E Nadeau, Shanshan Lin , Ayalew Mergia*
Department of Infectious Disease and Pathology, University of Florida, Gainesville, FL 32611, USA
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Publisher Id: TOMICROJ-8-114
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Received Date: 16/6/2014
Revision Received Date: 22/9/2014
Acceptance Date: 2/10/2014
Electronic publication date: 31 /10/2014
Collection year: 2014
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© Lo et al.; Licensee Bentham Open.
open-access license: This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.
* Address correspondence to this author at the Department of Infectious Disease and Pathology, University of Florida, Gainesville, FL 32611, USA; Tel: (352)-294-4139; Fax: (352)-392-9704; E-mail: mergiaa@ufl.edu
Caveolin-1 (Cav-1) is the major protein of the caveolae and plays a role in multiple cellular functions and implicated to have anti-HIV activity. Regulated expression of Cav-1 is important for safe and effective use in order to exploit Cav-1 for HIV therapeutic applications.
A series of Cav-1 and GFP expression vectors were constructed under the control of the HIV LTR for conditional expression or CMV promoter and the expression of Cav-1 was monitored in the presence or absence of Tat or HIV infection in order to establish the restricted expression of Cav-1 to HIV infected cells.
Cav-1 expression was evident under the control of the HIV LTR in the absence of Tat or HIV infection as demonstrated by immunoblot. Placing two internal ribosomal entry sequences (IRES) and a Rev response element, RRE (5'~ LTR-IRES-GFP-RRE-IRES-Cav-1~3') resulted in no expression of Cav-1 in the absence of Tat with effective expression in the presence of Tat. Transduction of HIV permissive cells with this construct using a foamy virus vector show that Cav-1 was able to inhibit HIV replication by 82%. Cells that received LTR-IRES-GFP-RRE-IRES-Cav-1 remain healthy in the absence of Tat or HIV infection.
These results taken together reveal the inclusion of two IRES establishes a significant reduction of leak through expression of Cav-1 in the absence of Tat or HIV infection. Such regulated expression will have therapeutic application of Cav-1 for HIV infection as well as broad applications which can be beneficial for other host-targeted interventions as therapeutics.
Keywords: Caveolin-1, HIV, LTR, Tat.
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Caveolin-1 (Cav-1) is the major protein of the caveolae organelle, a specialized membrane lipid raft that is highly enriched in cholesterol, phospholipids and sphingolipids [1-7]. Caveolae serves as scaffolding for compartmentalized cellular processes and participates in multiple cellular functions, including endocytosis, cholesterol transport, and signal transduction [7-16]. Cav-1 is abundant in various cell types and is highly expressed in quiescent or terminally differentiated cells including dendritic cells and monocytes/ macrophages [3-7, 17-21]. The caveolin scaffolding domain (CSD), residues 82-101 in Cav-1, is essential for both caveolin oligomerization and the interaction of caveolins with other proteins [22]. The CSD serves as receptor for binding proteins containing the sequence with a motif φXφXXXXφ, φXXXXφXXφ, or φXφXXXXφXXφ (φ representing any aromatic amino acids and X any other amino acids) [22]. The human immunodeficiency virus (HIV) envelope (Env) gp41 has one such motif (WNNMTWMQW) [23, 24] located in the target cells [25, 26]. Through the CSD Cav-1 can interact with ectodomain of gp41 that is required for membrane fusion of with HIV transmembrane gp41 Env [23, 24, 27]. Our group has demonstrated interaction of Cav-1 and HIV Env in the lipid raft (caveolae) of the plasma membrane [28]. Such an interaction at the plasma membrane blocks HIV envelope mediated fusion with target cells and innocent bystander cell killing. Furthermore, the association of Cav-1 with HIV Env reduces virus replication [28]. In addition, Cav-1 can inhibit HIV by reducing nuclear factor κappa B (NF-κB) mediated transcription and enhancing cholesterol efflux [29-31]. The potential contribution of Cav-1 by suppressing HIV replication can be exploited for the development of a novel therapeutic strategy in treating HIV-1 infected patients. Cav-1, however, has multiple functions including regulating signal transduction, vesicle trafficking and cholesterol metabolism as well as being linked to multiple diseases including cancer [14, 21]. Therefore, overexpressing Cav-1 for therapeutic applications could have an adverse effect due to unregulated expression. To overcome such a problem we established a strategy that significantly restricts Cav-1 expression in HIV infected cells while effectively inhibiting virus replication.
Cell Cultures
The 293T (human embryonic kidney fibroblasts)cell line was obtained from American Type Culture Collection (Manassas, VA). Glial derived human U87MG-CD4 cells stably transfected with CXCR4 (U87-CD4-CXCR4) and an indicator cell line for tittering HIV (TZM-bl) was kindly provided by the NIH AIDS Reagent Program. U87MG-CD4 derived cells were maintained in Dulbecco's modified Eagle medium (DMEM) containing 15% FBS, penicillin-streptomycin (100 μg/mL), glutamine (584 µg/mL), puromycin (1 μg/ml; Sigma Chemical), and neomycin (G418; 300 μg/ml; Sigma). TZM-bl and 293T cells were grown in DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin (100 μg/ml).
Construction of Plasmids
The NIH AIDS Research Program kindly provided pNL4-3 a plasmid construct containing the HIV provirus of NL 4-3. All expression constructs used for the studies are depicted in Fig. (1). The CMV-Cav-1plasmid contains the Cav-1 coding sequence followed by an internal ribosomal entry sequence (IRES) and then green fluorescent protein (GFP) under the control of the human cytomegalovirus (CMV) immediate early gene promoter. CMV-IR is the same as CMV-Cav-1 but lacks the coding sequence of Cav-1. The constructs LTR-IR, LTR-Cav-1, were derived from CMV-IR and CMV-Cav-1, respectively, by replacing the CMV promoter with the HIV NL4-3 LTR. In constructing LTR-GFP-IR-Cav-1 we placed the GFP upstream of Cav-1 while placing an IRES in between the two coding sequences with expression under the control of the HIV LTR. An additional IRES was inserted upstream GFP in LTR-GFP-IR-Cav-1 generating the construct LTR-IR-GFP-IR-Cav-1. The plasmid LTR-IR-GFP-RRE-IR-Cav-1 was derived from LTR-IR-GFP-IR-Cav-1 by inserting the HIV Rev response element sequence between GFP and the IRES located upstream Cav-1. RRE was also placed in LTR-IR-GFP upstream IRES and GFP generating plasmid LTR-RRE-IR-GFP. To deliver the Cav-1/GFP expression cassettes into HIV target cells foamy virus vectors were constructed using the simian foamy virus type 1, isolated from rhesus macaque (SFVmac). An SFVmac vector containing GFP (pEGFPD) was used as the backbone to construct foamy virus vectors to deliver into HIV target cells. The construction of pEGFPD and a foamy virus vector expressing a microRNA based siRNA targeting the HIV rev/env (SFV/HL-R) as well as foamy virus vector packaging plasmids pCIgag, pCIpol, and pCIenv expressing gag, polymerase and envelope, respectively were described previously [32]. The GFP expression cassette was removed in pEGFPD and replaced with the expression cassette of GFP and Cav-1 with two IRES and RRE under the control of LTR from LTR-IR-GFP-RRE-IR-Cav-1 or a CMV promoter expressing Cav-1 and GFP from CMV-Cav-1 generating SFV/LTR-IR-GFP-RRE-IR-Cav-1 or SFV/CMV-Cav-1, respectively. A Tat expression plasmid pTatz was described previously [32].
Fig. (1).
Schematic representation of Cav-1 and GFP expression constructs. Cav-1 and GFP expression cassettes were generated in the presence of one or two internal ribosomal entry sequence (IRES) (A) plus the Rev response element (RRE) (B). The Cav-1 and GFP expression under the control of the CMV promoter or the LTR for conditional expression were placed in the simian foamy virus isolated from rhesus macaque (SFVmac) vector to deliver into HIV target cells (C).
HIV LTR driven Cav-1 expression and inhibition of HIV replication. Cav-1 was placed under the control of CMV or the HIV LTR promoter for expression and each was co-transfected with the NL4-3 provirus DNA into 293T cells. HIV replication was monitored by reverse transcriptase activity (A) and the expression of Cav-1 by Western blot analysis (B). A series of Cav-1 and GFP expression cassettes were constructed as depicted in the Figure by placing one IRES (LTR-GFP-IR-Cav1) (C) or two IRES (LTR-IR-GFP-IR-Cav-1) (D). Each of these constructs were co-transfected into 293T cells with or without the Tat expression plasmid, pTatz. The expression levels of Cav-1 and GFP were examined by Western blot analysis 48 hours post transfection. Results are expressed as the means ± standard deviations (SD) from three determinations (P< 0.05).
Reduction of LTR driven GFP basal expression in in the absence of Tat. The IRES sequence alone or in combination with the HIV Rev response element (RRE) was placed between the HIV LTR and GFP. Each of these constructs were transfected into 293T cells and the level of GFP expression was examined by FACS measuring mean fluorescent intensity.
Inducible expression of Cav-1 with significant reduction in basal level leakage expression. A Cav-1 and GFP expression cassette was constructed as depicted in the Figure by using 2 IRES and an RRE (LTR-IR-GFP-RRE-IR-Cav-1) sequences (A). This construct was cotransfected into 293T cells with or without Tat expression plasmid, pTatz. The expression levels of Cav-1 and GFP were examined by Western blot analysis. (B) Cav-1 expression from LTR-IR-GFP-RRE-IR-Cav-1 was examined for efficacy of inhibition of HIV replication by co-transfecting with NL4-3 and monitoring HIV replication by reverse transcriptase assay. (C) The XTT cell viability assay (Cell Signaling Technology, Danvers MA) was used to determine the nontoxicity and safety of LTR-IR-GFP-RRE-IR-Cav-1. Results are expressed as the means ± standard deviations (SD) from three determinations (P< 0.05).
Inhibition of virus replication in HIV target cells by conditionally expressed Cav-1. The LTR-IR-GFP-RRE-IR-Cav-1 construct was placed in the foamy virus vector for transduction of HIV target cells to determine inhibition of virus replication by conditionally expressing Cav-1 in infected cells. Foamy virus vector mediated transductions of positive (SFV/HL-R and SFV/CMV-Cav-1) and negative (pEGFPD and pNL4-3) controls are included for comparisons. Results are expressed as the means ± standard deviations (SD) from three determinations (P < 0.05).
DNA Transfection, Cav-1 Expression, and HIV Inhibition Assay
Transfection of each of the plasmid constructs was performed in six well plates by the calcium phosphate method as described previously [33]. Briefly, 293T cells were cultured at a density of 4.5 x 105 in 6 well plates and transfected with 1 ug of each expression construct carrying the Cav-1 and GFP or GFP alone along with 1 ug of plasmid DNA lacking an expression cassette. For induction of LTR promoter mediated expression the Tat expression construct pTatz was co-transfected (1 ug) along with 1ug of each of the expression cassettes. Twenty-four hours post transfection the culture medium was removed and replaced with fresh media. The transfected cells were kept in culture for 3-4 days while monitoring for GFP expression under a microscope with ultraviolet source. The numbers of fluorescent cells were scored in the presence and absence of Tat. In addition, the levels of expression of Cav-1 and GFP were examined by Western blot and GFP by the intensity of fluorescence using FACS analysis. Immunoblotting was performed as described previously [30]. The primary antibodies used for immuno-blotting were rabbit polyclonal anti-Cav-1(Santa Cruz Biotechnology Inc, Dallas, Texas), anti-GFP (Santa Cruz Biotechnology Inc), and ß-actin protein antibody (Sigma, St. Louis, MO). The secondary antibodies were HRP-linked anti mouse or rabbit (Cell Signaling Technology Danvers, MA). To assay for HIV inhibition 1ug of plasmid constructs containing Cav-1 were co-transfected along with 1ug of NL4-3 provirus DNA plasmid (pNL4-3) into 293T cells. Virus production was monitored by reverse transcriptase activity assay in triplicate as described previously using 10ul supernatant of cell culture samples [33]. Cell viability was monitored using Cell Signaling Technology's XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2 H-tetrazolium-5-carbo-xanilide) Cell Viability Kit (Danvers MA) according to manufactures described protocol.
Foamy Virus Vector Mediated Cav-1 Transduction and HIV Challenges
To prepare foamy virus vector for Cav-1 transduction into HIV target cells 20 ug of each of the foamy vectors were co-transfected with the packaging constructs pCIgag (20 ug), pCIpol (4 ug), and pCIenv (2 ug) into 293T cells per T-75 culture flask as described previously [32]. Transfected cells were maintained in culture for 4 days and supernatants harvested by centrifugation at 5000 rpm for 20 minutes and then by filtering through a 0.45 um filter. Vector particles were concentrated by 100-fold using 150 kDa Apollo Centrifugal Spin Concentrators (Orbital Biosciences, Topsfield, MA). Concentrated virus particles were tittered by infecting fresh 293T cells seeded at a density of 2.5x104 per well in 24 wells and counting GFP positive cells. NL4-3 HIV target cells U87-CD4-CXCR4 were seeded at a density of 5×105 per well and infected with each of packaged foamy virus vectors at multiplicity of infection (moi) of 10 by spin inoculation twice as described previously [34]. The HIV NL4-3 strain was tittered using the TZM-bl indicator cells and counting Tat induced β-Gal gene expression before challenge. Foamy virus transduced U87-CD4-CXCR4 cells were challenged 48 hours later with HIV NL4-3 at an moi of 0.005 using the DEAE-dextran method as reported previously [33]. The level of HIV production was monitored from foamy virus vector transduced cells using reverse transcriptase assay.
Inhibition of HIV Replication by Cav-1 Expressed Under the Control of the HIV LTR
The cav-1 coding sequence was placed under the control of the HIV LTR (LTR-Cav-1), Fig. (1) for regulated Cav-1 expression in HIV infected cells to determine its ability to inhibit HIV replication. For comparison cav-1 was also placed under a CMV promoter for constitutive expression (CMV-Cav-1). 293T cells were transfected with LTR-Cav-1 or CMV-Cav-1 along with NL4-3 provirus DNA (pNL4-3) and the replication of HIV was monitored for 4 days by harvesting supernatants and assaying for reverse transcriptase. Controls lacking the coding sequence of cav-1 (LTR-IR or CMV-IR) were also co-transfected with pNL4-3 and the level of HIV replication was determined in the same manner. Virus replication was compared in Cav-1 expressing cells transfected with LTR-Cav-1 or CMV-Cav-1 with that of cells transfected with corresponding control vectors LTR-IR or CMV-IR, respectively. As shown in Fig. (2A), inhibition of HIV by Cav-1 under the control of the CMV promoter reached 86% compared to the control CMV-IR. Similarly Cav-1 expression by the HIV LTR was able to reduce virus replication by 90% as compared with the corresponding control. These results suggest that regulated expression of Cav-1 in HIV infected cells that effectively inhibits virus replication can be achieved with the level of Cav-1 expression being comparable to that of the expression of Cav-1 directed by a constitutive CMV promoter. Such a level of reduction of HIV replication in lieu of Cav-1 suppresses LTR mediated expression indicates that it is an effective inhibitor of virus replication.
Reducing LTR Driven Basal Expression of Cav-1 in the Absence of Tat
The level of LTR driven background expression of Cav-1 in uninfected cells is critical whether Cav-1 can be used for HIV therapeutics since alterations in Cav-1 expression can be pathogenic [14, 21]. As show in Fig. (2B) Cav-1 expression is present in cells where LTR-Cav-1 was transfected in the absence of pNL4-3 and such levels of Cav-1 expression could be toxic to uninfected cells. Since the cells we used to test our constructs do not express Cav-1, as demonstrated by the control construct, the amount of Cav-1 seen by immunoblot with LTR-Cav-1 in the absence of pNL4-3 represents the level of expression above endogenous levels of cells expressing Cav-1. To overcome this problem we constructed a series of cav-1 expression cassettes and examined the level of Cav-1 in the presence and absence of Tat. First, we placed GFP upstream of Cav-1 while inserting an IRES in between GFP and Cav-1 for LTR regulated expression (LTR-GFP-IR-Cav-1, Fig. (1A). This construct was co-transfected with or without pTatz, a Tat expressing plasmid, and the level of Cav-1 and GFP expression was determined by immunoblots. As shown in Fig. (2C), a significant level of Cav-1 expression was observed in the absence of Tat. A second IRES sequence was included upstream GFP (Fig. (1A), LTR-IR-GFP-IR-Cav-1). The LTR-IR-GFP-IR-Cav-1 construct revealed a significant reduction in Cav-1 expression in the absence of Tat compared to LTR-GFP-IR-Cav-1 while maintaining a high level of expression in the presence of Tat (Fig. 2D). Although placing an additional IRES upstream GFP significantly reduced background expression, there is still some Cav-1 present in LTR-IR-GFP-IR-Cav-1 transfected cells in the absence of Tat. We hypothesized extending the gap between GFP and the second IRES with DNA that will produce RNA with secondary structure would the reby reduce leak through expression of Cav-1. To test our hypothesis first we generated a construct where the rev response element (RRE) from NL4-3 HIV is placed upstream of the IRES-GFP sequence LTR-RRE-IR-GFP (Fig. 1B) and monitored GFP expression by FACs analysis and compared with, the same construct but lacking the RRE (LTR-IR, Fig. 1A) in the presence or absence of Tat. As shown in Fig. (3) fluorescent intensity was significantly reduced in the absence of Tat in cells transfected with LTR-RRE-IR-GFP compared to that of LTR-IR. Based on this information the RRE sequence was placed between IRES-GFP and the second IRES upstream Cav-1 as shown in Fig. (1B), (LTR-IR-GFP-RRE-IR-Cav-1) to further help reduce leak through expression. This construct was tested in the presence or absence of Tat for Cav-1 expression. As shown in Fig. (4A) no Cav-1 expression was observed in the absence of Tat as determined by Western blot analysis while maintaining the ability of high level Cav-1 expression in cells receiving pTatz. The LTR-IR-GFP-RRE-IR-Cav-1 construct was tested for its' ability to inhibit HIV replication by co-transfecting with pNL4-3 into 293T cells (Fig. 4B). Inhibition of HIV replication reached up to 90% in cells transfected with LTR-IR-GFP-RRE-IR-Cav-1 which is comparable to that of CMV-Cav-1. Cells transfected with LTR-IR-GFP-RRE-IR-Cav-1 remained healthy similar to mock transfected cells in the absence of HIV infection establishing there is no toxicity associated when Cav-1 is placed under a restrictive expression system (Fig. 4C). These results suggest that using a LTR-IR-GFP-RRE-IR-Cav-1 construct, undetectable levels of LTR driven Cav-1 expression can be established in the absence of Tat and HIV infection as determined by Western blots which allows efficient expression of Cav-1 mediated by Tat subsequently effectively inhibiting HIV replication.
Inhibition of Virus Replication by Inducible Cav-1 Expression in HIV Target Cells
The LTR-IR-GFP-RRE-IR-Cav-1 cassette was placed into foamy virus vector (SFV/LTR-IR-GFP-RRE-IR-Cav-1) to determine its' ability to inhibit virus replication in HIV target cells. A recombinant foamy vector containing Cav-1 with CMV promoter was also constructed as a control SFV/CMV-Cav-1. An additional control of a foamy virus vector containing a microRNA based siRNA targeting rev (SFV/HL-R) that effectively inhibits HIV replication and a negative foamy virus control expressing GFP (pEGFPD) were included in our studies. Also included, as a control, was HIV infected cells with no foamy virus vector transduction [mock (NL4-4-3 only)]. Each of these constructs were transfected with the packaging plasmids pCIgag, pCIpol, and pCIenv to generate vector particles and infect the HIV target U87-CD4-CXCR4 cells. The level of HIV replication was analyzed at intervals for 17 days by harvesting supernatants and assaying for reverse transcriptase activity. As shown in Fig. (5), in the mock transduced cells HIV replication was evident at day 3 and rising significantly at day 5 then plateauing at days 7, 9 and 11 until cell lysis began. HIV replication in the pEGFPD transduced control had a slow start, peaked at day 9 and 11. In Cav-1 transduced cells whether Cav-1 expression was under the CMV or the HIV LTR promoter the levels of HIV replication were significantly reduced as compared to that of the controls that were mock or pEGFPD transduced (days 5-11). The level of inhibition of HIV replication reached 86% and 82% in cells transduced with SFV/CMV-Cav-1 and SFV/LTR-IR-GFP-RRE-IR-Cav-1, respectively, compared to that of cells receiving pEGFPD or mock. This level of inhibition is comparable to that of cells transduced with our positive control SFV/HLR foamy virus vector (days 5-11), a reduction in HIV replication reaching 91%. The Cav-1 expression in HIV infected cells under the control of the HIV LTR is, therefore, an effective inhibitor of HIV replication.
The use of cellular proteins against HIV are an upcoming and evolving genetic approach that can be included in gene therapy combinations to control HIV infection to the point of a cure. Anti-HIV cellular proteins are important mainly for the reason that their targets are not viral genome that constantly evolve and therefore can safeguard against escape mutants. Cav-1 is one such protein which effectively inhibits HIV replication. Regulated expression of Cav-1 is important for safe and effective use of Cav-1 for HIV therapeutics. We have placed the cav-1 gene under the HIV LTR promoter and determined that it efficiently inhibits HIV replication. The expression of Cav-1 in these studies were Tat dependent and the effective inhibition of virus replication suggests that any negative regulation on the HIV LTR driven expression of the antiviral genes is not sufficient enough to have significant impact on the role of Cav-1 in the inhibition of HIV replication. Similar levels of inhibition of HIV replication were observed when cav-1 was placed under the control of a CMV promoter. Our results show no LTR driven Cav-1 expression by Western blot analysis in the absence of Tat or HIV infection revealing a significant reduction of leak through expression in the absence of Tat. This was accomplished by inserting an IRES sequence upstream the coding sequences of each of GFP and Cav-1 and adding a Rev response element between GFP and the IRES placed at the 5'end of cav-1.
The absence of basal Cav-1 expression as determined by Western blot analysis with LTR-IR-GFP-RRE-IR-Cav-1 as compared to LTR-GFP-IR-Cav-1 and LTR-IR-GFP-IR-Cav-1 could be due to distance spacing with RRE, the RRE structure, and/or weaker ribosomal entry for Cav-1 expression as compared to that of the one that is placed upstream the GFP. Since the amount of leak through of long transcripts is significantly low in the absence of Tat the expression of Cav-1 protein can be extremely low with the additional IRES sequence and the RRE. Cav-1 expression could be below the detection level of Western blots in the absence of Tat. If that is the case, this level of expression is therefore significantly lower than endogenous levels in cells expressing Cav-1 which can easily be seen by Western blots [28-30, 35]. The observation that Cav-1 was not detected by Western blot analysis with our constructs whereas endogenous expression of Cav-1 is easily observed in the absence of Tat or HIV infection indicates that the strategy applied for Cav-1 expression could significantly reduce any safety concerns of toxicity due to transgene expression in uninfected cells. Furthermore, such a strategy will have broad applications and can be beneficial for other host-targeted interventions as therapeutics.
As described above one of the mechanisms of Cav-1's ability of inhibiting HIV replication is through suppressing gene expression by modulating the NF-êB pathway and the two NF-κB sites in the U3 region of the LTR are critical for Cav-1 mediated inhibition of viral gene expression [29]. Interestingly, although Cav-1 can suppress its own expression through this mechanism our results show that the level of LTR driven expression remains effective enough to inhibit HIV replication. This result suggests the major inhibition of HIV replication is perhaps mediated through the other two mechanisms of Cav-1's ability of promoting cholesterol efflux and blocking of envelope fusion to target cells subsequently arresting the replication cycle. The inhibition mediated by the NF-êB pathway, therefore, is not sufficient enough to significantly impact its ability to inhibit HIV replication. Alternatively, there may always be adequate levels of Cav-1 expression that results in effective inhibition of viral expression even under a possibility of feed back suppression of its own expression. In support of this notion, LTR driven siRNA expression targeting the R region of the LTR is shown to effectively inhibit HIV replication [32]. Furthermore, several studies show RNA based anti-HIV approaches such as ribozymes, shRNAs, and antisense RNA expressed from the HIV LTR to effectively inhibit viral replication in HIV infected cells [36-39].
Innate immune antiviral factors have important implications in HIV therapeutics and there is a continual interest in understanding and transferring these molecules into preclinical therapeutic interventions. To this endeavor steps for preclinical studies have already been the case for HIV restriction factors APOBEC3G, TRIMa and tetherin [40]. The expressions of innate immune antiviral vectors do get activated during viral protection for the first line of defense. Cav-1 expression is also augmented by HIV infection in macrophages and microglial HIV targets and similar to innate antiviral factors Cav-1 suppresses HIV replication. In fact, such bidirectional association of Cav-1 with HIV can contribute to macrophage persistent infection [28-30, 35]. Therefore, like the HIV restriction factors and other antiviral innate immune molecules Cav-1 can be important for clinical therapeutic intervention. The results presented here using a cellular protein, Cav-1, as a gene therapy target in a cell culture system will be a basis to test its therapeutic potential in preclinical settings using animal models.
The authors confirm that this article content has no conflict of interest.
This research was supported by a grant from the National Institutes of Health (AI39126) to A.M.
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[31] Simmons GE Jr, Taylor HE, Hildreth JE. Caveolin-1 suppresses human immunodeficiency virus-1 replication by inhibiting acetylation of NF-kappaB. Virology 2012; 432: 110-9.
[32] Park J, Nadeau PE, Mergia A. Activity of TAR in inducible inhibition of HIV replication by foamy virus vector expressing siRNAs under the control of HIV LTR. Virus Res 2009; 140: 112-20.
[33] Park J, Nadeau P, Zucali Jr, Johnson CM, Mergia A. Inhibition of simian immunodeficiency virus by foamy virus vectors expressing siRNAs. Virology 2005; 343: 275-82.
[34] Mergia A, Soumya C, Kolson DL, Goodenow MM, Ciccarone T. The efficiency of simian foamy virus vector type-1 (SFV-1):in non-dividing cells and in human PBLs. Virology 2001; 280: 243-52.
[35] Lin S X-MW, Nadeau PE, Mergia A. HIV infection upregulates Caveolin 1 (Cav-1):expression to restrict virus production. J Virol 2010; 84: 9487-96.
[36] Dropulic B, Hermankova M, Pitha P. A conditionally replicating HIV-1 vector interferes with wild-type HIV-1 replication and spread. Proc Natl Acad Sci USA 1996; 93: 11103-8.
[37] Paik SY, Banerjea A, Chen CJ, Ye Z, Harmison GG, Schubert M. Defective HIV-1 provirus encoding a multi-target ribozyme inhibits accumulation of spliced and unspliced HIV-1 mRNAs, reduces infectivity of viral progeny, and protects the cells from pathogenesis. Hum Gene Ther 1997; 8: 1115-23.
[38] Unwalla HJ, Li HT, Bahner I, Li MJ, Kohn D, Rossi JJ. Novel pol ii fusion promoter directs human immunodeficiency virus type 1-inducible coexpression of a short hairpin rna and protein. J Virol 2006; 80: 1863-73.
[39] Unwalla HJ, Li MJ, Kim JD , et al. Negative feedback inhibition of HIV-1 by TAT-inducible expression of siRNA. Nat Biotechnol 2004; 22: 1573-8.
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Published Contents
Volume 15, 2021Volume 14, 2020Volume 13, 2019Volume 12, 2018Volume 11, 2017Volume 10, 2016Volume 9, 2015Volume 8, 2014Volume 7, 2013Volume 6, 2012Volume 5, 2011Volume 4, 2010Volume 3, 2009Volume 2, 2008Volume 1, 2007
Irina V. Kiseleva
Department of Virology
Institute of Experimental Medicine
Biography of Irina V. Kiseleva
Dr. Kiseleva obtained her MS in Microbiology from St Petersburg (SPb) State University, RF, PhD in Virology from Institute of Influenza, SPb, RF and DSci in Virology in Institute of Experimental Medicine (IEM), SPb, RF. She holds a position of Section Chief in IEM. She is also a Professor of SPb University, RF. She published over 280 publications including 40 RF patents. Currently she is the Editor-in-Chief of TOMICROJ, published by Bentham Science Publishers.
Department of Virology, Institute of Experimental Medicine, St. Petersburg, Russia
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The Open Microbiology Journal
Volume: 15, 2021
The Guest Edited Thematic Issues are published free of charge
Processing Time: Average publication time is 18 days between the final acceptance of revised manuscript and its publication
The state of multiple antibiotic resistant Enterobacteriaceae in Cape Coast, Ghana
This article by Dr. Kwame K. Asare et al. is published in The Open Microbiology Journal, Volume 13, 2019
Passing on dangerous naked explosives: the state of multiple antibiotic resistant Enterobacteriaceae in Cape Coast, Ghana.
The dynamisms involved in bacterial drug resistance, emergence and spread of multiple antibiotic resistant Enterobacteriaceae in Ghana are poorly understood. The current state of antibiotic resistance is gradually becoming alarming. To make matters worse, the factors that drive the transmission of antibiotic resistance among organisms in Ghana are still poorly understood. The isolation of multi-antibiotic resistant Enterobacteriaceae to ß-lactams, the first, second and third generation cephalosporins from circulating coin currencies and meat sources in Cape Coast, Ghana, is a major concern as it poses a serious threat to the entire healthcare system. E. coli, Enterobacter spp, Shigella spp, Klebsiellaspp, Pseudomonas spp, and Proteus spp showed resistance to second and third generation cephalosporins, an effective antibiotic against Enterobacteriaceae species. There is a possibility that this extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae in circulation will spread rapidly with limited timely treatment to curb the infections. These microbes cause a range of infections including acute diarrhoea, respiratory infection, urinary tract infections (UTI), bacteremia, sepsis, pneumonia, cystitis, pyelonephritis, and lung abscess. The easy spread of this multi-antibiotic resistant Enterobacteriaceae through contact with contaminated surfaces and by person-to-person contact makes it major public health concern as it has a high implication on the health facilities of the region which have limited resources. The isolation of MDR Enterobacteriaceae from circulating coin currencies and meat sources gives an indication of an individual's multiple points of contact with these pathogens during daily routines, risk of infection and possible transmission to another person. The impending danger of possible outbreak of multiple ESBL-E resistant strains requires immediate public health measures to circumvent an epidemic outbreak in Ghana. Understanding the underlying mechanisms of ESBL-E transmission would be essential in providing permanent control measures to deal with drug resistance in Ghana. Personal hygiene and proper handling of currencies are encouraged to mitigate the spread of drug-resistant pathogens.
To access the full text article, please visit: https://benthamopen.com/ABSTRACT/TOMICROJ-13-138
Asare Kwame Kumi, MPhil, PhD
Department of Protozoology, Institute of Tropical Medicine (NEKKEN), Nagasaki University
Kwame.asare@nagasaki-u.ac.jp
Epidemiological review on emerging & re-emerging parasitic infectious diseases in Malaysia
This article by Dr. Saad Musbah Alasil and Dr. Khairul Anuar Abdullah is published in The Open Microbiology Journal, Volume 13, 2019
Malaysia is facing many challenges caused by various parasitic pathogens. The lack of awareness among disadvantaged populations such as the Orang Asli community and the dependency on foreign workers has led to an influx of immigrants to Malaysia from countries endemic with various parasitic diseases. Amoebiasis is mainly encountered in poor rural areas in Malaysia, however it has the potential to re-emerge. Routine mass-drug administration on newly arriving foreign workers and health education programs are needed to prevent its re-emergence.
Researchers from MAHSA University in Malaysia have published a review on the epidemiology of various parasitic diseases in the country over the last 20 years. The report has been published in The Open Microbiology Journal. The report stresses that Malaysian authorities must implement strategies that provide better water treatment to avoid the emergence of blastocystis. Lymphatic filariasis has the potential to re-emerge due to its easy mode of transmission as well as the presence of a large number of immigrant workers in Malaysia from endemic countries. To prevent the emergence of giardiasis in Malaysia, a multidisciplinary approach is required to determine the level of water contamination with Giardia as well as Cryptosporidium. The epidemiology of malaria is becoming more complex in Malaysia. There is a shift towards infections among men and adults rather than women and children. Therefore, the ministry of health should raise public awareness and the use of point-of-care diagnostics.
Malaysia harbors a large variety of ecological niches that favor the transmission of Toxoplasma spp. Which has led to an increase in the incidence of toxoplasmosis. Therefore, surveillance programs should be initiated to facilitate early diagnosis and treatment. The existence of human trypanosomiasis from neighboring Thailand and reporting cases in cattle from neighboring Indonesia, can all lead to its emergence in Malaysia. Parasitic infectious diseases will continue to appear in Malaysia leading to unpredictable outbreaks that challenge healthcare personnel and emphasize the urgent need for effective surveillance and control measures. Despite the challenges, Malaysia is strongly committed to curb the spread of these diseases.
To access the entire article please visit: https://benthamopen.com/ABSTRACT/TOMICROJ-13-112
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The Open Microbiology Journal is an Open Access online journal, which publishes research articles, reviews, letters, case reports and guest-edited single topic issues in all areas of theoretical and practical aspects of Microbial systematics, evolutionary microbiology, immunology, virology, parasitology , bacteriology, mycology, phycology, protozoology, microbial ecology, molecular biology, microbial physiology, biochemistry, microbial pathogenesis, host-microbe interaction, systems microbiology, synthetic microbiology, bioinformatics. Bentham Open ensures speedy peer review process and accepted papers are published within 2 weeks of final acceptance.
The Open Microbiology Journal is committed to ensuring high quality of research published. We believe that a dedicated and committed team of editors and reviewers make it possible to ensure the quality of the research papers. The overall standing of a journal is in a way, reflective of the quality of its Editor(s) and Editorial Board and its members.
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Experience in theoretical and practical aspects of Microbial systematics, evolutionary microbiology, immunology, virology, parasitology , bacteriology, mycology, phycology, protozoology, microbial ecology, molecular biology, microbial physiology, biochemistry, microbial pathogenesis, host-microbe interaction, systems microbiology, synthetic microbiology, bioinformatics with an academic degree.
At least 20 publication records of articles and /or books related to the field of theoretical and practical aspects of Microbial systematics, evolutionary microbiology, immunology, virology, parasitology , bacteriology, mycology, phycology, protozoology, microbial ecology, molecular biology, microbial physiology, biochemistry, microbial pathogenesis, host-microbe interaction, systems microbiology, synthetic microbiology, bioinformatics or in a specific research field.
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text:
Ex Premier League midfielder Danny Pugh speaks about his career in an exclusive interview.
Danny Pugh has been around playing professionally for 15 years and has seen it all in football. Experiencing extreme both promotion and relegation, playing in both the Champions League and League 2 Danny Pugh's career has been an interesting one.
In an exclusive interview, he talks about a multitude of topics such as; his time as a youth player at Manchester United, being Blackpool captain under the ownership of the controversial Oystons, studying for his coaching badges and much more. Danny Pugh has played for some of the biggest clubs in England but currently, plays at Vale Park for Port Vale.
CJ: "You have played under a lot of managers but who is the best manager you ever worked under and why?"
DP: "Although I only played 7 first team games for United I have to obviously say, Sir Alex Ferguson.
His winning mentality and his ability to achieve continued success for so many years was unbelievable for me to witness"
CJ: "Whilst in the youth setup at Manchester United which players stood out from a young age?"
DP: "Danny Webber was the year above me and he was always a stand out player from a very young age.
Daniel Nardiello was in my year and always scored a lot of goals
Darren Fletcher was the year below me and it was clear he was going to be a top player & he's gone on to have a great career."
CJ: "Who did you look up to in the Manchester United dressing room?"
DP: " Ryan Giggs, Paul Scholes and Roy Keane were the 3 I probably looked up to the most."
CJ: "You have played for a lot of clubs famed for their passionate fan bases (Leeds, Stoke, etc.) who were your favourite fans to play in front of"
DP: "I've been lucky to play in front of some fantastic fans. The Leeds fans were unbelievable on their day."
I think I'd have to say Stoke though, due to the success we had during that period.
CJ: "How is studying for your coaching badges going?"
DP: "I'm really enjoying studying for my badges. It makes you start looking at the game from a completely different angle"
6. "What was the best moment of your career?"
DP: "Really hard to just pick one moment.
My debut for Manchester United was obviously a massive moment for me.
Getting promoted with Stoke and playing in an FA Cup final were definite highlights as well"
CJ: "7. What was the worst moment of your career?"
DP: "Getting relegated this Season with Port Vale"
CJ: "What is the funniest story you have from the dressing room at any club?"
DP: "A lot of funny things that happen in dressing rooms should stay in dressing rooms so I'll leave this question haha"
CJ: "How do you think Port Vale will bounce back from being relegated?"
DP: "I'm confident we will have a good season. Some players have already left & the manager has started bringing some new faces in so I'm really looking to getting started"
CJ: "11. How was playing for Blackpool in arguably their worst spell in recent history? And What do you make of the Oystons running of Blackpool? Did it have an effect on the players? and did the fan protests affect your game?
DP: "To be honest as a player you stay away from the politics of a Football Club as much as possible as it is not something you can effect. It's very sad what's happening with the fans etc and unfortunately, I can't see it being redeemed anytime soon.
It shouldn't effect the players as their job is to concentrate on playing and as you can see the team did exceptionally well to get promoted with the issues going on in the background."
Author Charles JonesPosted on June 3, 2017 June 3, 2017 Categories Blackpool, Champions League, Championship, English Football, Exclusive, Football, Interview, Leeds, Manchester United, Port Vale, SportsTags Exclusive, Football, Interview, Manchester United, Soccer, Sport
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redpajama_set_name: RedPajamaCommonCrawl


(2) Appearance by attorney. PROVIDED. Where a civil litigant is indigent (defined as below 200% of the federal poverty guidelines), the court shall provide counsel at public expense where the assistance of counsel is needed to protect the litigant's rights to basic human needs, including sustenance, shelter, clothing, heat, medical care, safety and child custody and placement. In making the determination as to whether the assistance of counsel is needed, the court may consider the personal characteristics of the litigant, such as age, mental capacity, education and knowledge of the law and of legal proceedings, and the complexity of the case.
The draft order denying the petition is here (pdf) and the final order should be posted here once several changes from the open administrative conference are incorporated. Video of the administrative conference is available on Wisconsin Eye (discussion of Petition 10-08 begins at the 1:19 mark).
Consideration of this issue now shifts to the Access to Justice Commission. As reflected in the Commission's recent letter to the Supreme Court, a working group has been formed to examine potential pilot projects that could be developed to evaluate the costs and benefits of providing legal counsel for low income litigants in certain types of civil legal matters. The working group held an initial meeting to discuss the project and is scheduled to meet again on January 30 at 1 pm (contact us for more information about the working group).


text:
titulní stranu
Integrace
Servisní
Vývojářské nástroje
FM Bench
BenchTest
Xgode
fmRESTor
LogMonitor
Plug-iny pro FileMaker
Bridge for Arduino
Bridge for Phidgets
Gonector
Phone Companion
SimpleDialog
SimpleFile
SimpleTalk
SimpleHASP
SimpleSound
Aplikace na míru
Konzultace FileMaker
Integrace hardware
FileMaker pluginy
Omlouváme se, informace na této stránce jsou k dispozici pouze anglicky. Použít překladač Přepnout do angličtiny
News, tips, advice, ideas, experience…
Product Testing for Constant Improvements
We've been developing FileMaker plug-ins since the release of FileMaker Pro 4, so some of our products have been there for our users for over a decade. We would like to share with you a few notes on how to make a product successful.
(This post is written about developing FileMaker plug-ins, but it is applicable to all software development in general.)
No matter how precisely you plan the future of your product, there will aways be things you can't predict, especially in software development. A lot has changed since we've released our first plug-ins. Operating systems have changed, hardware has changed, the FileMaker platform has changed and even our user requirements have changed. We also have changed, and so therefore our products have changed.
We grew and our product became better. I say "only better", because an ideal version of our product would contain all the features our clients ask for, would contain no bugs and we would make the product compatible with current and future environments and systems. But that is hard to achieve and often not economically viable. So we are pushing our products closer to this ideal with this motto: "Every new version of a product has to be better than the previous one."That is much more realistic and measurable.
There are two key processes to achieving this:
Keeping track of all the issues/tasks.
Testing all the current and previous versions' issues before the product is released.
We use Redmine to keep track of all the bugs, issues and feature requests. It doesn't matter if they are reported by us (we do use our plug-ins, too), through our helpdesk system) or through other channels. They are all recorded in one system, categorized, assigned a status and either included in some version or held for the future.
Whenever a new plug-in version initiates from this list containing new features, bug fixes or both, there has to be comprehensive testing to ensure that nothing else breaks by those changes. And that can't be accomplished by random testing, it has to be systematic, it has to be written somewhere and it has to be easily repeated with the current and every future version of the product. So our "acceptance tests list" was born.
It is a combination of positive tests, negative tests, boundary tests, UI tests, integration tests and so on. This list evolves with the product as new features are added or removed, as new issues are found and fixed. Nevertheless having one such list for each product is sufficient to determine which version of a product is better and passes more tests.
To make it easier for us, we have developed a FileMaker solution for our acceptance tests list. No paperwork, no complex requirements from the tester, just one database with all the tests. The process of testing then consists of these simple steps:
click the "execute test" button
compare the actual result with the expected result
mark the test as done
go to the next test
To keep the testing as simple as possible, a lot of tests could be written in a way that the tester just compares the resulted output with an "ok" string. However a lot of plug-in functions can't be tested that easily. For example in our SimpleDialog plug-in almost all of the tests are related to creating new dialogs and windows. When testing this, the tester has to be fully focused on what appears and sometimes even do a few steps as addressed in the test. The tester then has to evaluate if it looked and behaved as written in the test.
So there is no general solution to make all the tests simple, but at least all the tests can be divided into 2 lists. Those that require tester interaction and those that do not. And the latter ones can be evaluated all at once with a single button click. And that is a real time saver. Then the only slow down is creating the tests in the beginning. But there are ways to automate that, too.
For example in our SimpleFile plug-in we tried to think of all the combinations for our file handling functions (functions as simple as moving a file from one folder to another) and it was plentiful. All those combinations of relative and absolute paths, file existence, folder existence, folder permissions, file permissions… and it grew exponentially with each factor we tried to cover. So we ended up with a script that generates hundreds of tests for us. It prepares all the testing folders and files and generates all the tests for our testing solution. Then the only remaining challenge is to automatically generate the expected outputs, especially the error codes it should return when a condition (or more of them at once) fails. After that all those tests can be tested by a single click and the test statistics will be available.
We always have ideas as to how to move our development and testing processes one step further. And as we focus on developing better and better products, we have to apply our ever-improving motto to our testing process too. Only that way we can both learn from the past and be prepared for future changes.
That's our testing approach in a nutshell. And how do you test your products?
Odkaz byl zkopírován do schránky.
nám
Obvykle jsme dostupní v pracovní dny mezi 8. a 18. hodinou
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12/3/2018 -- Climatewire -- Policy. Science. Business.
The birth of the Sunrise Movement started with a small grant, a lot of Monster Energy drinks and a little help from the Sierra Club. Now it's the most talked-about environmental group in Washington.
Can wildfires shift climate change politics?


text:
Конференція зі скорочення та обмеження озброєнь 1932—1934 ().

Перші спроби досягти на міжнародному рівні домовленостей про обмеження озброєнь були здійснені на Гаазьких конференціях 1899 та 1907. Країни Антанти, окрім США, взяли на себе зобов'язання розпочати процес роззброєння за Версальським мирним договором 1919. 1925 Ліга Націй створила підготовчу комісію з роззброєння (1926–32), завданням якої стало визначення переліку озброєнь, що підлягатимуть обмеженням, та розроблення заходів з роззброєння. До 1931 ця комісія розробила проект для дискусії, який став основою роботи конференції. Перше її спільне засідання відбулося 2–24 лютого 1932 в Женеві (Швейцарія) і проходило за участю представників держав-членів Ліги Націй, а також Афганістану, Бразилії, Єгипту, Еквадору, Коста-Рики, Мексики, СРСР, США та Туреччини. Франція висунула пропозицію про інтернаціоналізацію деяких видів озброєнь (зокрема, бомбардувальної авіації, лінкорів тоннажем понад 10 тис. т, далекобійної артилерії тощо) та створення міжнародних військових сил під егідою Ліги Націй. Водночас вона виступила проти обговорення загального скорочення сухопутних та повітряних озброєнь і наполягала на збереженні диференційованого підходу до різних держав залежно від їхньої ролі в європейській безпеці. Велика Британія і Японія виступили проти включення питання про скорочення морських озброєнь в обговорення в принципі. СРСР запропонував два плани – негайного повного роззброєння і пропорційно-послідовного скорочення всіх озброєнь. Німеччина пропонувала взяти за основу підходу щодо визначення параметрів скорочення озброєнь та ЗС кількісні обмеження, подібні до тих, що були накладені на неї Версальським мирним договором 1919; у разі незгоди з такою пропозицією Берлін залишав за собою право збільшити кількісні параметри власних ЗС для досягнення паритету з ін. великими д-вами. Принцип "рівності в озброєннях" разом з Німеччиною підтримала також Італія. 22 червня 1932 американська делегація висунула проект паритетного скорочення озброєнь на 1/3 всіма державами світу (план Г.-К.Гувера). Однак жоден із планів не дістав значної підтримки учасників конференції. Більшість делегацій наполягала на необхідності в першу чергу провести скорочення тих озброєнь, які могли використовуватися для наступальних дій і становили безпосередню загрозу міжнар. миру. У зв'язку з цим основне обговорення зосередилося навколо визначення понять "агресивна" та "неагресивна" зброя. Після того, як учасникам не вдалося дійти згоди і в цьому питанні, 23 лип. 1932 в роботі була оголошена перерва. Заг. сесії не проводилися, а обговорення проблем роззброєння відбувалося в комісіях та групах експертів. Відновлення роботи конференції блокувалося Німеччиною, яка наполягала на якомога швидшому вирішенні питання про рівні з ін. д-вами права щодо озброєнь. 14 верес. 1932 Німеччина офіційно припинила участь у конференції і повернулася до неї лише після підписання 11 груд. 1932 п'ятьма д-вами (Великою Британією, Італією, Німеччиною, США та Францією) протоколу про надання Німеччині рівних з ін. великими д-вами прав у рамках майбутньої єдиної системи безпеки. Одночасно Велика Британія, Італія, Німеччина та Франція підписали декларацію про відмову від застосування сили для вирішення міждерж. спорів.

Після відновлення обговорення проектів роззброєння ключовим планом став т. зв. план Е.Eppio-Ж.Поль-Бонкура, що передбачав організацію системи взаємних гарантій безпеки, заборону далекобійної артилерії та важких танків, встановлення єдиного 3-місячного терміну військової служби. У плані повторювалися французькі пропозиції про створення міжнародних військових формувань під проводом Ліги Націй, встановлення міжнародного контролю над європейськими повітряними шляхами та цивільною авіацією, а також пропонувалося скоротити морські озброєння. Оскільки план надавав однобічні переваги Франції, проти нього виступили представники Великої Британії, Італії та Німеччини, і в лютому 1933 сесія генеральної комісії конференції відхилила план Е.Eppio-Ж.Поль-Бонкура, формально вмотивувавши це невідповідністю плану до формату конференції, оскільки він передбачав заходи, спрямовані лише на скорочення, і жодних заходів щодо обмеження озброєнь. У березні 1933 британська делегація запропонувала план Дж.Макдональда, що передбачав встановлення граничних рівнів сухопутних озброєнь для д-в континентальної Європи, однакових рівнів повітряних озброєнь для всіх великих д-в, встановлення короткострокового (8–12 місяців) військ. обов'язку та створення постійної міжнар. комісії з роззброєння, яка мала перебрати на себе вирішення питань, що залишалися б нерозв'язаними після закриття конференції. 8 квіт. 1933 ген. комісія прийняла план Дж.Макдональда за основу майбутньої конвенції з роззброєння, але 14 жовт. 1933 Німеччина, яка взяла курс на ремілітаризацію, відкликала своїх представників із Женеви та офіційно припинила участь у конференції. Остання сесія ген. комісії конференції відбулася 29 трав. – 11 черв. 1934 і завершилася фактично без результату.

Джерела та література
 Константинов В.Ю. Конференція зі скорочення та обмеження озброєнь 1932—1934 //

Міжнародні конференції

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Produced and Directed the social media commercial for the launch of the LG V20.
A show about making cocktails and how to drink them.
Produced Hennessy's summer 2017 social media campaign featuring BJ The Chicago Kid and Tony Tixier as they explore jazz music in Paris.
'In The Zone' was an original mini-series created for Facebook Watch. It was produced through Cycle Media.
Produced the spot and directed the Miami segment for Hennessy US.


The mushroom and tomato pâté is delicious, but you can substitute any dip or filling of your choice.
Heat oil in a medium, heavy-based saucepan. Add onion and sauté. Add mushrooms and sun-dried tomatoes and fry for about 2 minutes until soft. Remove from heat.
Place in a food processor with remaining ingredients and blitz to a slightly coarse texture.
Use a potato peeler to cut long, thin strips of cucumber lengthways.
Spread filling onto each strip, roll and secure with toothpicks. Serve immediately.


U.S. Pacific Command has dispatched 36 service members from Okinawa to Thailand to assist with efforts to rescue 12 boys from a Thai soccer team and their coach, who have been trapped in a cave complex since June 23, a Pentagon spokesman told Task & Purpose.
The Marine Corps is opening a labor camp in Okinawa to "award" informally disciplined Devil Dogs an opportunity to redeem themselves through their sweat and tears.
The US military has banned the consumption of alcohol for servicemembers after a US Marine struck and killed an Okinawa man in a truck where "alcohol may have been a factor," on Sunday, according to a statement.
A Camp Hansen Marine was arrested last week after a bullet was found in his bag at Naha Airport, Japanese police said.


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