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The genus Flavivirus contains emerging arthropod-borne viruses (arboviruses) infecting vertebrates, as well as insect-specific viruses (ISVs) (i.e., viruses whose host range is restricted to insects). ISVs are evolutionary precursors to arboviruses. Knowledge of the nature of the ISV infection block in vertebrates could identify functions necessary for the expansion of the host range toward vertebrates. Mapping of host restrictions by complementation of ISV and arbovirus genome functions could generate knowledge critical to predicting arbovirus emergence. Here we isolated a novel flavivirus, termed Niénokoué virus (NIEV), from mosquitoes sampled in Côte d'Ivoire. NIEV groups with insect-specific flaviviruses (ISFs) in phylogeny and grows in insect cells but not in vertebrate cells. We generated an infectious NIEV cDNA clone and a NIEV reporter replicon to study growth restrictions of NIEV in comparison to yellow fever virus (YFV), for which the same tools are available. Efficient RNA replication of the NIEV reporter replicon was observed in insect cells but not in vertebrate cells. Initial translation of the input replicon RNA in vertebrate cells was functional, but RNA replication did not occur. Chimeric YFV carrying the envelope proteins of NIEV was recovered via electroporation in C6/36 insect cells but did not infect vertebrate cells, indicating a block at the level of entry. Since the YF/NIEV chimera readily produced infectious particles in insect cells but not in vertebrate cells despite efficient RNA replication, restriction is also determined at the level of assembly/release. Taking the results together, the ability of ISF to infect vertebrates is blocked at several levels, including attachment/entry and RNA replication as well as assembly/release.
IMPORTANCE Most viruses of the genus Flavivirus, e.g., YFV and dengue virus, are mosquito borne and transmitted to vertebrates during blood feeding of mosquitoes. Within the last decade, an increasing number of viruses with a host range exclusively restricted to insects in close relationship to the vertebrate-pathogenic flaviviruses were discovered in mosquitoes. To identify barriers that could block the arboviral vertebrate tropism, we set out to identify the steps at which the ISF replication cycle fails in vertebrates. Our studies revealed blocks at several levels, suggesting that flavivirus host range expansion from insects to vertebrates was a complex process that involved overcoming multiple barriers.
Arthropods can be infected with a wide taxonomic variety of viruses (1). Most of these viruses are transmitted only among arthropods and do not infect other hosts (arthropod-specific viruses). Interestingly, several virus families also contain viruses that are transmitted to vertebrate hosts during the blood-feeding process of the arthropod (arthropod-borne viruses; briefly, arboviruses). Arboviruses have the unique ability to circulate between invertebrate and vertebrate hosts. Infections with arboviruses such as dengue virus (DENV), Chikungunya virus, and Rift Valley fever virus can cause severe disease in humans and animals (2). Coancestral representation of arboviruses and arthropod-specific viruses is known in the RNA virus families Flaviviridae, Togaviridae, Bunyaviridae, Rhabdoviridae, Orthomyxoviridae, and Reoviridae (3, 4).
The evolutionary relationships between arthropod-specific viruses and arboviruses have been unclear. Recent studies on the evolution of bunyavirus host associations suggested that ancestral taxa in this family may have been arthropod specific (5). Host range expansion to vertebrates (arboviral emergence) would have occurred by convergence during the evolution of the four vertebrate-pathogenic genera (genera Hantavirus, Orthobunyavirus, Phlebovirus, and Nairovirus). An evolution of arboviruses from arthropod-specific viruses is also assumed for members of other taxa such as the genus Flavivirus (family Flaviviridae) (6, 7). The vertebrate-infecting flaviviruses, including mosquito-borne flaviviruses (MBF) and tick-borne flaviviruses (TBF) as well as flaviviruses with no known vector (NKV), are all placed in a phylogenetic sister relationship with the classical insect-specific flaviviruses (cISF) (8). However, we and others have recently discovered a novel group of ISFs, named dual-host-affiliated ISFs (dISF), that phylogenetically group within the MBF, making arbo-flaviviruses a paraphyletic group (8–11). Paraphily suggests that vertebrate tropism has been convergently acquired at least two times in flaviviruses.
Flaviviruses are enveloped, positive-stranded RNA viruses. Their genome encodes a single open reading frame (ORF) that is flanked by a 5′ untranslated region (5′ UTR) and a 3′ UTR of about 100 and 350 to 1,200 nucleotides (nt), respectively (12). The ORF encodes one large polyprotein that is co- and posttranslationally cleaved to release the viral proteins, namely, the three structural proteins capsid (C) protein, premembrane/membrane (prM/M) protein, and envelope (E) protein, as well as the nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5.
In spite of extensive knowledge of flavivirus replication, little is known about the viral proteins and the stages of the replication cycle involved in ISF host range restriction. So far, the host range restriction of ISVs has been studied using Kamiti River virus (KRV), an ISF, and Eilat virus (EILV), an ISV of the genus Alphavirus within the family Togaviridae. Studies on the ISF Kamiti River virus suggest that the vertebrate innate immune system does not seem to represent a major block, as only a low level of viral RNA and infectious particle shedding was observed in IRF3,5,7−/− mouse embryonic fibroblasts (13). EILV is in a sister relationship with vertebrate-infecting alphaviruses of the Western equine encephalitis virus (WEEV) complex (14). Studies of the replication of an EILV replicon, as well as chimerized constructs in which EILV protein domains were replaced by homologous domains of the arbo-alphavirus Sindbis virus (SINV), revealed that EILV is blocked at two independent stages of the replication cycle, namely, RNA replication and viral entry (14, 15). The latter findings were further supported by in vivo infection experiments in mosquitoes and newborn mice. Understanding the restrictions may reveal novel antiviral targets in vertebrates and open novel approaches for virus attenuation and vaccine development, as well as identify determinants of arboviral pathogenicity in humans and livestock.
As insect-specific viruses are described within different virus families, restrictions in host range may occur at different levels of replication cycles depending on the virus taxon. To further understanding of vertebrate tropism restriction in flaviviruses, we provide for the first time results of a replication study on an ISF chimerized with arbo-flavivirus genome elements. These studies were done on a novel flavivirus termed Niénokoué virus (NIEV) that groups with cISF and is a contemporary isolate which has not undergone passage-derived adaptation to cell culture. Chimerization of NIEV with the prototypic arbo-flavivirus yellow fever virus (YFV) is based on a novel NIEV reverse genetics system.
NIEV isolation and sequence analysis.Three strains of a novel flavivirus termed NIEV were isolated from Culex species mosquitoes collected in the Taï National Park in Côte d'Ivoire. The NIEV genome (strain B51/CI/2004) comprised 10,878 nt. Polyprotein sequence identity with other ISF was 38% to 57%. NIEV shows a genome organization typical for flaviviruses, with three putative structural and seven putative nonstructural proteins (see Fig. 2A). Protein cleavage sites were found to be conserved between NIEV and other flaviviruses. ISFs express an additional protein, named fifo (from fairly interesting flavivirus ORF), by a programed −1 ribosomal frameshift in the NS2A-NS2B gene regions (16). A slippery heptanucleotide was identified for NIEV in this region (G UUU UUU), suggesting a putative fifo protein of 221 amino acids (aa) (8). Phylogenetic analyses based on the complete polyprotein showed that NIEV groups within the primarily Culex-associated viruses of the cISF clade (Fig. 1). NIEV defines a new species in basal relationship to Nakiwogo virus from Uganda and to Palm Creek virus from Australia.
In addition to the C6/36 cells used for initial virus isolation, common vertebrate cell lines (Vero, BHK, and HEK-293) that are permissive for all or most arbo-flaviviruses were inoculated with NIEV. No growth was observed in any experiment (data not shown).
Establishment of a NIEV reverse genetics system.As a prerequisite to analysis to determine at which level(s) the vertebrate host range restriction of NIEV occurs, we established a NIEV reverse genetics system. Five reverse transcriptase (RT)-PCR fragments covering the complete NIEV genome were amplified from total RNA of infected cells and cloned into individual plasmids. During initial RT-PCR, an SP6 promoter sequence was added upstream of the 5′ end of the viral cDNA and an EagI linearization side was introduced downstream of the 3′ end of the viral cDNA. The individual plasmids were fused stepwise, resulting in two plasmids covering the complete viral sequence. Full-length templates were generated by in vitro ligation of the two plasmids (17) (Fig. 2A). RNA transcribed from the EagI-linearized ligation product yielded recombinant (rec) NIEV upon electroporation into C6/36 cells. Recombinant virus produced plaques on C6/36 cells similar to those produced by wild-type (wt) NIEV (Fig. 2B). In growth curve analyses, wt and rec NIEV grew comparably on C6/36 cells (Fig. 2C). The production of rec NIEV was demonstrated by the loss of an EcoRI site through an engineered silent A-to-G mutation at position 5018 (Fig. 2D).
Host range restriction at the level of RNA replication.In addition to an infectious full-length cDNA clone, we established a NIEV reporter replicon expressing Renilla luciferase (Fig. 3A). The latter allows circumventing the entry step, thereby enabling separate analyses of viral RNA replication and translation. The functionality of the NIEV replicon was confirmed by Renilla luciferase expression upon electroporation in insect cells, generating levels of luminescence that exceeded those of an established YFV replicon (Fig. 3A) (18). While the analogous YFV replicon efficiently replicated in different vertebrate cells (BHK, A549, Vero), only a slight increase in Renilla luciferase levels was observed at 6 h postelectroporation (p.e.) for the NIEV replicon (Fig. 3B) before a continuous decrease was observed. NIEV RNA replication could not be rescued in Dicer- and microRNA (miRNA)-deficient 293T cells (Fig. 3C) (19), while YFV RNA replication was still supported by these cells. These results indicate a restriction of NIEV in vertebrate cells at the level of RNA replication and suggest that vertebrate cell-specific miRNAs are unlikely to constitute a sufficient factor for the observed NIEV host range restriction.
To further evaluate whether the slight increase of luciferase levels observed at 6 h p.e. is due to a low level of RNA replication or to initial translation, we created a replication-incompetent version of the NIEV replicon in which the catalytic GDD motif of the RNA-dependent RNA polymerase had been mutated to GAA. Renilla luciferase expression levels were measured in short intervals immediately after electroporation. Similar extents of signal increase were observed for the two versions of the replicon, indicating that luciferase expression was caused by direct translation of incoming RNA rather than by RNA replication (Fig. 3D). For comparison, while a YFV replicon GAA variant also showed a slight initial increase in Renilla luciferase signal, the luciferase signal from the replicating wt YFV replicon increased exponentially from 8 h p.e. (Fig. 3D). It was concluded that NIEV replication was blocked in vertebrate cells at a stage downstream of initial genome translation.
Since it was previously reported that insertion of a SINV 3′ UTR motif into the 3′ UTR of sleeping disease virus (SDV) increased SDV translation efficiency in a cell-specific manner (20), we performed similar analyses by inserting the YFV 3′ UTR downstream of the NIEV ORF stop codon and analyzing the result for enhanced translation in BHK cells. Although the chimeric NIEV Renilla replicon (NIEVR)/YFV 3′ UTR was still unable to replicate in BHK cells, its translation efficiency increased about 2-fold faster in BHK cells within the first 6 h p.e. than that of the wt NIEV replicon (Fig. 3E). This suggests that the arbo-flavivirus 3′ UTR might be involved in vertebrate tropism.
Host range restriction at the level of cell entry.To analyze whether the NIEV host range restriction is also manifested at the level of cell entry, we replaced the envelope protein prM and E genes with the homologous genes of NIEV in a YFV infectious cDNA clone, resulting in a chimeric construct termed YF/NIEV (Fig. 4A). Our construct contains the YFV capsid protein along with the YFV nonstructural proteins to ensure cleavage at the C protein anchor site by the NS2B-3 protease, which is a prerequisite for signalase-mediated cleavage at the C-prM junction (21). Electroporation of the in vitro-transcribed YF/NIEV RNA into C6/36 cells (22) resulted in formation of plaques in an infectious center assay (ICA), indicating the production of infectious particles (Fig. 4B). Infectious particle production was further proven in growth curve analyses after infection of C6/36 cells (Fig. 4C). Immunofluorescence analysis using a YFV anti-NS1 antibody showed that C6/36 cells were infected with both YFV and YF/NIEV (Fig. 4D). In contrast, no positive immunofluorescence signal was observed after infection of BHK cells with YF/NIEV (Fig. 4D), indicating that the structural proteins of NIEV are unable to mediate infection of BHK cells. To comparatively test further vertebrate cells for infection with YFV and YF/NIEV, we established YFV and YF/NIEV Renilla reporter viruses (YFV_Ren and YF/NIEV_Ren). To this end, a Renilla gene cassette was inserted in frame in the N-terminal region of the YFV or YF/NIEV polyprotein in a manner similar to that previously described for DENV (23) (Fig. 5A). After infection of insect cells (C6/36 and U4.4) with the reporter viruses YFV_Ren and YF/NIEV_Ren at a multiplicity of infection (MOI) of 1, luciferase levels increased over time as expected for viable reporter viruses (Fig. 5B). In several different vertebrate cell cultures, including cultures of BHK, HEK-293, and Vero cells, YFV_Ren but not YF/NIEV_Ren caused luciferase expression (MOI of 1) (Fig. 5C). Infection of vertebrate cells with YF/NIEV_Ren was also not observed at an increased MOI of 10 (Fig. 5D). The structural proteins of NIEV appeared to be unable to mediate entry into vertebrate cells.
Host range restriction at the level of assembly and release.Next, we analyzed whether the NIEV envelope proteins are able to mediate assembly and release of infectious virus in vertebrate cells. To circumvent the level of cell entry, RNA transcribed from reporter virus cDNA clones was directly electroporated into target cells. In BHK cells, YF/NIEV_Ren RNA yielded luciferase expression levels similar to those seen with YFV_Ren, indicating efficient RNA replication and protein expression (Fig. 6A). However, no plaques were observed for YF/NIEV_Ren in an infectious center assay (Fig. 6B), suggesting a lack of infectious particle production. Absence of released infectious viral progeny was confirmed by passaging of supernatants, obtained upon electroporation of BHK cells, in C6/36 cells. While an increase of luciferase values over time demonstrated passage of infectious particles for YFV_Ren, no luciferase expression was seen for YF/NIEV_Ren (Fig. 6C).
To further investigate whether infectious virus exists in intracellular locations, C6/36 and BHK cells were electroporated with YF_Ren or YF/NIEV_Ren in vitro transcripts and titers of infectious virus contained in cells were measured in addition to levels of released particles by endpoint dilution assay on C6/36 cells. YF/NIEV_Ren(GAA) bearing the exchange of the polymerase GDD motif to GAA was included as a negative control. Due to different replication rates of YFV in C6/36 and BHK cells and the occurrence of a strong cytopathic effect in BHK cells at day 3 (d3) postinfection (p.i.), titers of infectious virus were determined at different time points. As shown in Fig. 6D, intracellular particles were detected for YF_Ren in BHK and C6/36 cells at d2 and d6, respectively. While YF/NIEV_Ren also produced infectious intracellular particles in C6/36 cells, no intracellular infectivity was measured in BHK cells. Prolonged incubation of BHK cells until d6 p.e. also did not result in the recovery of infectious intracellular particles (data not shown). In accordance with the results presented in Fig. 6C, infectious extracellular particles were released in all cases where infectious intracellular particles were observed. Hence, NIEV envelope proteins appeared to be unable to mediate assembly of infectious intracellular particles and, consequently, no infectious particles were released in vertebrate cells.
While arboviruses show dual-host tropism with adaptation to insect and vertebrate hosts, insect-specific viruses, including ISF, have a single-host tropism. Here we have discovered NIEV, a new ISF, in mosquitoes from West Africa. NIEV provides a favorable model to study vertebrate host restriction due to its known and limited history of cell culture passages after primary isolation. Vertebrate host restriction may occur on the levels of viral attachment and entry, genome replication, and virus assembly and release. Experiments in the present study resembled earlier work on the replacement of surface proteins in two different arbo-flaviviruses, YFV and DENV, with the surface proteins of the NKV flavivirus Modoc virus (MODV) (24). These virus chimeras replicated efficiently in vertebrate and insect cells, whereas wt MODV does not replicate in insect cells, suggesting that host tropism is not determined by surface proteins (24). Similarly, it was demonstrated that a chimeric DENV genome carrying the envelope genes from Langat virus (LGTV), a tick-borne virus unable to infect mosquito cells, retained the ability to infect mosquito cells irrespective of the type of envelope proteins (25). Hence, the restriction to replicate in mosquito cells is at a postentry event for both MODV and LGTV, which is somewhat surprising considering the known mechanism of receptor-dependent entry for many flaviviruses.
These experiments were directed at permissiveness in insect cells. However, how ISFs are hindered from infecting vertebrate cells is of special interest in studying virus emergence. The only reverse genetics-based data available in this field have been generated recently from EILV, a novel alphavirus related to the WEEV serocomplex of arbo-alphaviruses, where restriction occurs at the levels of both entry and replication (14, 15).
In the present work, reverse genetics of an ISF, namely, NIEV, was used for the first time to study vertebrate restriction, revealing a clear block of entry and RNA replication and assembly in vertebrate cells. For other flaviviruses, several host factors have been described to be involved in RNA replication, either via binding to the viral RNA genome or via binding to viral proteins involved in the replication complex. Among those are proteins such as AUF1 p45 that bind to the viral RNA functioning as RNA chaperons (26). RNA chaperon activity was also assumed for the La autoantigen, whereas a function as an RNA helicase was suggested for the viral RNA binding translation elongation factor-1α (EF-1α) (27, 28). For cyclophilin A, an interaction with both viral RNA and the viral NS5 protein in the replication complex was previously described (29). Similarly, TIA/TIAR colocalize with viral replication complex components, namely, double-stranded RNA (dsRNA) and the NS3 protein (30). A lack of or a difference between such host factors (or their genetic homologs) in the vertebrate cell might account for the observed lack of NIEV RNA replication. Interestingly, for the tomato spotted wilt virus (TSWV), a plant- and insect-infecting Tospovirus belonging to the family Bunyaviridae, a viral polymerase-bound host factor was identified that rendered human cells permissive with respect to this virus (31).
In addition to proteins, host small noncoding RNAs can influence viral RNA replication (32). Recently, further miRNA interaction studies using cross-linking immunoprecipitation (CLIP) based on Argonaute protein have been performed for 15 different viruses (33). Although that study also included arboviruses, functional miRNA interaction studies have been performed so far only in vertebrate cells. Analogous studies for arboviruses and also ISFs in insect cells are awaited. Also, it was described that cellular miRNAs can target viral RNAs during infections, resulting in inhibition of virus replication (34–36). In the context of the present study, we found that vertebrate cell-specific miRNAs are unlikely to represent a factor sufficient for the observed NIEV host range restriction, since NIEV RNA replication did not occur in Dicer- and miRNA-deficient 293T cells also (19), while YFV RNA replication was still supported by these cells.
While RNA replication in vertebrate cells was inhibited for NIEV, translation of incoming RNA still occurred. Recently, it was demonstrated for SINV that its 3′ UTR contains an RNA motif that increases translation of SINV genomic and subgenomic mRNAs in insect cells but not in mammalian cells (20). This SINV RNA motif was also able to increase the replication efficiency of a replicon derived from sleeping disease virus (SDV), an alphavirus without known arthropod vector, in insect cells upon insertion into the SDV replicon 3′ UTR. These findings imply a possible role of the 3′ UTR motif in the virus host range. Similarly, when we inserted the 3′ UTR of YFV directly downstream of the ORF stop codon into the NIEV replicon, its translation efficiency in BHK cells was increased early after electroporation compared to that of the wt NIEV replicon, also suggesting a possible role of the YFV 3′ UTR in host range specificity. This would be in accordance with the findings for DENV, which indicated that certain RNA structure elements in the flavivirus 3′ UTR, like RNA structure duplications, play a role in host adaptation (37, 38). Furthermore, the flavivirus 3′ UTR is the origin of subgenomic flavivirus RNAs (sfRNAs) (39–41). It has been reported that sfRNAs exhibit interferon-antagonistic activities (41, 42). In comparison to, e.g., wt West Nile virus, growth of a West Nile virus mutant deficient in producing sfRNA was reduced in wt mouse embryonic fibroblasts (MEFs), while the mutant replicated like the wt virus in IRF3−/− × IRF7−/− MEFs as well as in Vero and BHK cells, which are also described to be interferon deficient (39, 42, 43). As NIEV did not grow in Vero or BHK cells, the interferon-antagonistic function of an sfRNA does not seem to be a crucial factor with regard to host range restriction. However, sfRNAs have also been described to inhibit the cellular 5′-to-3′ exonuclease XRN1, resulting in increased stability of many cellular mRNAs, or to counteract RNA interference mediated by small interfering RNAs (siRNAs) or miRNAs (44–46). It therefore will be interesting to analyze whether modulation of the host cell/host antiviral response via those mechanisms contributes to host range restriction.
Nevertheless, our data determined using the YF/NIEV chimera expressing the NIEV envelope proteins demonstrate that the first barrier against infection of vertebrate cells by ISF is viral entry. While the YF/NIEV chimera readily grew in insect cells, the recovered virus was unable to infect BHK cells. Also, additional vertebrate cells proved to be resistant to infection, as was shown using a reporter YF/NIEV chimera expressing Renilla luciferase. Several studies have been performed to elucidate factors and receptors involved in flavivirus attachment and entry. Still, detailed knowledge of interaction of the flaviviral envelope proteins with bona fide receptors is limited. Among the factors involved in attachment and entry are glycosaminoglycans, different C-type lectins (DC-SIGN/L-SIGN, mannose receptor, CLEC5A, and mosGCTL-1), phosphatidylserine receptors (TIM and TAM), and vitronectin receptor integrin ανβ3 (47). The majority of the factors have been determined in mammalian cells. Prohibitin and mosGCTL-1 have been described solely in the context of mosquito cells (47). Looking at the available data, it is obvious that individual flaviviruses use several different attachment and entry factors. On the other hand, differential expression of entry factors is already observed among different human cells, indicating the complex interplay between virus and cells involved in tropism. Hence, in-depth studies will be necessary to elucidate what restricts attachment and/or entry of ISFs to vertebrate cells.
In comparison to host factors involved in flavivirus entry, even less is known about host factors important for flavivirus assembly/release. Also, for the other insect-specific virus analyzed with regard to host range restriction, namely, the alphavirus EILV, no data are described analyzing a possible block in assembly and/or release. The respective data would have allowed judging whether an assembly/release block is independent of the budding site, since flaviviruses bud into the endoplasmic reticulum whereas alphaviruses bud from the cell membrane.
Taking the results together, our analyses show that the NIEV vertebrate host range restriction occurs at several levels of the viral life cycle. This suggests that flavivirus host range expansion from insects to vertebrates was a complex process that involved overcoming multiple barriers. The apparent complexity of adaptive barriers makes it unlikely that sudden host range expansions, such as by acquisition of single-site mutations, will occur in ISF. While NIEV belongs to the so-called cISF, which are phylogenetically distinct from all other flaviviruses, dISF have also been described (8). Since the latter phylogenetically group close to the mosquito-borne flaviviruses, it will be of interest to analyze whether those viruses are also restricted at the same levels or whether barriers restricting dISF infection of vertebrate cells might be determined by fewer adaptive steps.
Virus isolation and genome sequencing and analyses.Mosquito sampling and virus isolation was performed as described before (9, 48).
Three cultures of C6/36 cells that had been inoculated with pools of Culex species mosquitoes collected in the Taï National Park in Côte d'Ivoire induced mild cytopathic effects (CPE) 5 days postinfection (p.i.). A fragment of the NS5 gene was amplified and sequenced from each isolate. The obtained sequences showed 99% identity among themselves and less than 60% identity to cISF, suggesting the identification of three strains (strains NIEV-B21/CI/2004, NIEV-B51/CI/2004, and NIEV-C64/CI/2004) of a novel flavivirus, termed NIEV. The complete genome of strain NIEV-B51/CI/2004 and the complete ORF of NIEV-B21/CI/2004 (10,171 nt) as well as a subcomplete ORF of NIEV-C64/CI/2004 (9,864 nt) were sequenced using primer-walking techniques based on genome-specific and generic pan-flavivirus primers. Genome termini were sequenced by rapid amplification of cDNA ends (RACE)-PCR according to the instructions of the manufacturer (Invitrogen, Germany). As the three strains were more than 98% identical, further work was focused on strain NIEV-B51/CI/2004. For phylogenetic analyses, polyprotein ORFs were aligned in geneious using MAFFT and the E-INS-I algorithm. Alignment columns were stripped to contain gaps of less than 20%. Maximum likelihood analyses were inferred with the LG substitution matrix and 1,000 bootstrap replicates using PhyML.
Generation of recombinant NIEV and NIEV reporter replicon.C6/36 cells were infected with NIEV, and total cellular RNA was isolated using peqGOLD TriFast (Peqlab). cDNA synthesis was performed with genome-specific primers using SuperScript III reverse transcriptase (Invitrogen). The complete genome of NIEV was amplified in five PCR fragments using Phusion High-Fidelity polymerase and cloned into individual plasmids (Fig. 2A). At the 5′ end, an SP6 promoter sequence was fused together with an upstream AatII and MluI restriction site for cloning. At the 3′ end, an EagI site for linearization was added. The five amplified fragments were individually subcloned using standard procedures. Fragments 1 and 2 were further assembled into the pACYC177 background, resulting in pNIEV1-2, whereas fragments 3 to 5 were assembled into pSmart-LC-Kan, resulting in pNIEV3-5. As we failed to stably fuse all five individual plasmids into the two low-copy-number vectors pSMART and pACYC177, we generated a full-length template by in vitro ligation (Fig. 2A). To this end, the MluI-KpnI fragment excised from pNIEV1-2 was ligated into pNIEV3-5 cut with KpnI and MluI.
To generate a NIEV reporter replicon, a cassette encoding the sequences for ubiquitin–Renilla luciferase–foot-and-mouth disease virus 2A was amplified from the previously published YFV Renilla replicon (18). The resulting fragment was N-terminally fused to the first 30 codons of the NIEV capsid protein, including the SP6 promoter preceded by an MluI restriction site sequence, and C-terminally fused to a fragment encoding the last 23 codons of the E protein up to the genomic KpnI restriction site. To obtain the NIEV reporter replicon, the fusion product was cut with MluI and KpnI and inserted into a Mlu-KpnI-cut pACYC177 vector encoding NIEV fragments 3 to 5. Insertion of the YFV 3′ UTR downstream of the stop codon of the NIEV ORF was done using Fusion PCR technology. Plasmids were verified by sequencing. Primer sequences and further details on cloning can be obtained upon request.
Generation of reporter and chimeric viruses.To establish a YFV Renilla reporter virus (YF_Ren), two PCR fragments were amplified from pACNR/FLYF-17D using Bo617 (5′ CCAATCCCGGGCCCATGTCTGGTCGTAAAGCTC 3′) and Bo620 (5′ GAACTCCTCGTCGTACCATGTTAACGCCCAGGGTTTTTC 3′) or from Bo619 (5′ GAAAAACCCTGGGCGTTAACATGGTACGACGAGGAGTTC 3′) and Bo618 (5′ GATAGATCCATCGCAGTCTATGGTGTA 3′). The two PCR fragments were fused using Bo617 and Bo618 as amplifying primers. The resulting PCR fragment encoding the FMDV2A cleavage site followed by a full-length capsid gene encoding a silently mutated cyclization sequence to limit long-range interactions with the 3′ cyclization and the 5′ terminal region of the NS1 gene was cut with XmaI and MluI and inserted into the YFV Renilla replicon plasmid (18) cut with XmaI and MluI.
For generation of the YF/NIEV chimera, the NIEV prM-E genes were amplified from pNIEV1-2 using primers Bo320 (5′ GGGAATGCTGTTGATGACGGGTGGAGTTGTGGACGTGAACATGAGTTTGG 3′) and Bo323 (5′ CAAAGTTGATGGCGCATCCTTGATCTGCTCGTACATAGTAAATCATGCCC 3′). The resulting fragment was fused N-terminally with a pACNR/FLYF-17D-derived fragment amplified with Bo286 (5′ ACGAGAGAGATGATAGGGTCTGC 3′) and Bo321 (5′ CCAAACTCATGTTCACGTCCACAACTCCACCCGTCATCAACAGCATTCCC 3′) and C-terminally with a pACNR/FLYF-17D-derived fragment amplified with Bo322 (5′ GGGCATGATTTACTATGTACGAGCAGATCAAGGATGCGCCATCAACTTTG 3′) and Bo88 (5′ TTGGAGAGCCATGGGCACTC 3′) via fusion PCR technology using primers Bo286 and Bo88 as amplifying end primers. The obtained fragment was cut with NotI and MluI and inserted into pACNR/FLYF-17D cut with the same enzymes.
The YF/NIEV_Ren reporter virus was established according to the method used with the YF_Ren reporter virus, except that the template for amplification via Bo619 and Bo618 was pYF/NIEV. The integrity of the plasmids was verified by sequencing.
In vitro transcription and electroporation.The NIEV full-length template obtained via in vitro ligation of pNIEV1-2 and pNIEV3-5 and the NIEV reporter replicon plasmid (2 µg) amplified in MC1061 bacteria were linearized with EagI. Plasmids pACNR/FLYF and derivatives were linearized with XhoI. In vitro transcriptions were performed using an SP6 mMessage mMachine kit (Applied Biosystems). Electroporation of BHK-21J, A549, and Vero cells was performed as described previously for BHK cells except that the preset HeLa settings were used for the Vero cells (18). C6/36 and U4.4 cells were electroporated as previously described (22).
Infectious center assays.ICAs in BHK cells were performed as previously described (49). For ICAs in C6/36 cells, 10-fold serial dilutions of transfected cells were mixed with 2 × 106 untransfected C6/36 cells and seeded in 35-mm-diameter dishes. Following attachment for 4 h, the medium was replaced by Leibovitz 15 media containing 2.5% fetal calf serum (FCS) and 1% tragacanth. Plates were incubated for 7 days and fixed and stained as previously described (49).
Virus stocks, infectivity assays, and growth kinetics.YFV17D was derived from infectious full-length clone pACNR/FLYF-17D (kindly provided by Charles M. Rice, NY) by electroporation of in vitro-transcribed RNA into BHK cells (50). The NIEV wild-type strain and the YF/NIEV chimeras were cultured in C6/36 cells.
Plaque titration on BHK cells was performed as previously described (49). Plaque assays on C6/36 cells were performed accordingly using 2 × 106 cells seeded the day before in 35-mm-diameter dishes and proceeded using a 1% tragacanth overlay as described for ICAs. For the Renilla reporter viruses, viral titers were determined by 50% tissue culture infective dose (TCID50) titration using C6/36 cells seeded the day before with 4 × 104 cells/well. Virus-positive wells were identified by Renilla luciferase assay at d7 p.i.
To determine intracellular infectivity titers, electroporated cells were disrupted after extensive washing with phosphate-buffered saline (PBS) using three freeze-thaw cycles as previously described (51), except that BHK cell pellets were resuspended in minimum essential medium (MEM) (containing 7.5% FCS and nonessential amino acids [NEAA]) and C6/36 cell pellets in Leibovitz 15 media (containing 5% FCS). For the intracellular infectivity experiment, virus-containing culture supernatants from electroporated cells were subjected to the same three cycles of freezing and thawing and infectivity was determined in parallel by endpoint dilution assay as described above for Renilla reporter viruses.
For growth kinetic experiments, C6/36, U4.4., BHK-21J, Vero CCL81, and HEK-293 cells were infected at the indicated multiplicity of infection (MOI) for 1 h at 37°C or 28°C, depending on the cell type/temperature experiment performed. At defined time points after infection, either cell culture supernatants or lysed cells were collected and analyzed via real-time PCR (NIEV) or Renilla assay (reporter viruses), respectively.
RNA preparation and real-time PCRs.Viral RNA from cell culture supernatants was prepared using a NucleoSpin RNA virus kit (Macherey-Nagel). For NIEV RNAs, cDNA was synthesized using a SuperScript III RT system (Life Technologies, Inc.) and random hexamer primers. Viral genome copy numbers were quantified by real-time RT-PCR using primers B51_TM_F (5′ CATGTGGAGTGGGCGGAATA 3′) and B51_TM_R (5′ TGGGCCAGCTCTAACAGGAA 3′) and probe B51_TM (5′ 6-carboxyfluorescein [FAM]-CCAACCAGTGTTCTTTCCTAGCGATTTCTTC-6-carboxytetramethylrhodamine [TAMRA] 3′).
Quantification of YFV RNA was performed using a One Step RT-PCR protocol essentially as previously described (52).
Renilla assay.Renilla luciferase was measured from lysed infected cells using the Renilla luciferase assay system (Promega). Luciferase activity was measured in relative light units (RLU). Measurements were performed using a Junior LB 9509 luminometer (Berthold Technologies).
Immunofluorescence.Indirect immunofluorescence analyses were performed using monoclonal antibody 1A5 directed against the YFV NS1 protein (kindly provided by J. J. Schlesinger) and Alexa Fluor 555 goat anti-mouse serum (Thermo Fisher Scientific). Nuclei were stained using 4′,6-diamidin-2-phenylindol (DAPI).
We thank Pascal Trippner for excellent technical assistance. We are grateful to Charles M. Rice for providing the infectious YFV cDNA clone and to Bryan R. Cullen for providing Dicer- and miRNA-deficient 293T cells.
This work was financially supported by the Deutsche Forschungsgemeinschaft (grant agreement KU 1201/4-1 to B.M.K. and JU 2857/1-2 to S.J.) and by the Deutsches Zentrum für Infektionsforschung (infrastructural support to C.D.; grant agreement 301-4-8-01.801).
- Received December 21, 2016.
- Accepted December 22, 2016.
- Copyright © 2017 Junglen et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.


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We train salespeople. Primarily for real estate. We've been doing this for over thirty years with hundreds of thousands of past graduates who are now a part of our Wickman family.
We are the Floyd Wickman Team. We're happy you are here.
Phone: 734-637-4030
Email: email@example.com
We'd love to hear from you! Your questions, your feedback and your results. Contact us today and we'll be sure to get back to you right away! To contact Trainers directly, please visit Our Team.
We look forward to hearing from you!
Your Floyd Wickman Team

subset: commoncrawl


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578 Sawdust Rd, The Woodlands TX 77380 281.715.6924
578 Sawdust Rd The Woodlands TX 77380 281.715.6924
Our Blog on Computer & Web Technology
Have a new business you want to advertise for? Ready for customers to come to your store and website? We can help you with all of that. At Computer Network Center, our experienced web design and development team our experts in the field, staying at the forefront of new code developments to ensure our customers the most user-friendly websites that their potential customers will love to use over and over again.
Call us today for any web design or development questions you may have. Call us to schedule a free pre-sales appointment with our web design team. We will go over your website, your vision, and your competitors. We will discuss our process and benefits with you at your one-on-one appointment.
At Computer Network Center we go a step further by offering you all pieces of the pie for your company. We can help you with Hosting and Email Services to ensure your company is squared away and ready to have sales skyrocket.
See our Website Design & Development Brochure below.


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St. Sabina's Father Pfleger to speak at St. Thomas
Michael Pfleger, senior pastor of the Faith Community at St. Sabina Church, will be the guest speaker at St. Thomas the Apostle Church's 11th Annual Prayer Breakfast.
The breakfast, which is sponsored by the Ladies Auxiliary, will take place at 10 a.m. Saturday, April 5 at St. Thomas the Apostle School, 5467 S. Woodlawn Ave.
During the breakfast Pflager will give a speech titled "What Will it Take to End the Violence Snatching Our Future?" He will engage the audience in a question and answer period after his speech. There will also be musical entertainment by Luciano Laurentiu, director of music and pianist at St. Thomas the Apostle Church, and Derrick Davis, pianist and a St. Thomas the Apostle School alum that now attends Mt. Carmel High School.
The breakfast will be catered by Lee & Eddie's and proceeds from the event will fund special projects at the church and the school.
Call Gloria Palmer Pitts at 312-339-4499 or Enid Collins 312-972-2230 for tickets or send donations to St. Thomas the Apostle School, 5467 S. Woodlawn, Ave., Chicago, IL 60615.


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UniGroup Logistics provides precision last mile services for even the most specialized supply chain needs.
Our specialists will provide you with cost-effective, comprehensive last mile solutions to help you mitigate the risks associated with the hand-offs among suppliers, carriers and service providers. This is done by streamlining all of your last mile transportation, warehousing, delivery and installation services.
We are dedicated to servicing your specialized transportation needs with highly-coordinated shipping, warehousing and project management services. We offer premium air-ride trailers with options for climate control and liftgate service when necessary.


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A cooling trend that will start today should bring relief to the inland valleys, many of which topped 90 degrees yesterday.
The Wild Animal Park reached 99 yesterday, one degree short of the record set in 1997. Santee hit 95, and Ramona and Fallbrook recorded highs of 94.
Lindbergh Field, San Diego's official weather station, had a maximum of 81, seven degrees warmer than normal but well below the record of 97 for the date set in 1958.
Today's high temperatures should drop to the mid-80s inland, then to the upper 70s to low 80s tomorrow. The coast also should cool down by several degrees during the weekend.
Fog and morning and evening low clouds are expected to thicken the next few days, bringing cooler-than-normal weather by Monday. A heavy marine layer could bring a chance of drizzle everywhere west of the mountains Monday.
But after a few days of cooler weather, a repeat of the heat is expected again by the middle of next week. Conditions should be similar to those from yesterday, with a little bit of wind tossed in, said National Weather Service forecaster Brandt Maxwell.
No significant rain is on the horizon, he said.


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Product Description
Bin cups nest inside shelf bins and provide segregated storage for smaller items and are sold in packs of 24 pieces. Cups are available in two sizes (3 1/4"W and 5 1/4"W) and two colors (Blue and Yellow)
Additional Information
Product Tags
Add Your Tags:
Use spaces to separate tags. Use single quotes (') for phrases.


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Bad Street Brawler is a 1987 video game by Beam Software Pty., Ltd. It was distributed worldwide by Mindscape Inc. and Mattel. It was released for the Commodore Amiga, the Commodore 64, MS-DOS, and the NES. The NES version was one of only two NES games specifically designed for use with Mattel's Power Glove.
The player plays the character Duke Davis, who goes from stage to stage beating up gangsters that get in his way, dressed in a yellow tank top, sunglasses, and yellow pants. He is described as a former punk rocker and the "world's coolest" martial artist.
Before the start of the next stage, quotes are introduced to entertain the player (such as "Never trouble trouble until trouble troubles you"). The player fights a variety of enemies, such as gorillas and old ladies who throw pocketbooks at the player (resembling weightlifter dwarves who throw dumbbells at the player).
Source: Wikipedia, "Bad Street Brawler", available under the CC-BY-SA License.


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This is probably my second to last post before I head off for the summer, which makes me really sad. As promised though, here is my top five reasons I love being involved with WAUG:
1) You will always know someone who is just as stoked as you are that your favorite band just came out with a new album.
2) Friends from home miss your voice? Can't afford to call them all the time? Why not have them call into your radio show? You'll look cool (since you have callers on your radio show) and you get to chat with them about how their life is going.
3) Not only does WAUG do radio but we also do sports broadcasting.
4) WAUG has a weird collection of people. I honestly can say we are a weird spectrum of students who were all pulled in together by one thing we had in common – our love for music. Some of my friends I probably wouldn't have met if it wasn't for WAUG.
5) There's no other place (maybe besides the Observer) where I can just be myself and share my passions with the world (okay, my listeners).
Posted on May 14th, 2012 by aliciaoken09
Filed under: Alicia Oken


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The grasslands are currently under extremely dry conditions on the Eastern Plains. Please be aware where you're parking!
Tall grass and a hot muffler can start a fire under these conditions so park on the dirt roads whenever possible.
If this isn't feasible or will inconvenience other drivers try to park on a bare spot or at least over very short grass. There have been numerous fires in the area over the last few weeks, one yesterday was only 8 miles from CPER HQ, so please be careful.
Prescribed burns are one thing, respect the fire danger and don't start a wild fire!


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I've always loved words—reading them, writing them, and as my friends and family will wearily attest, speaking them. I dabbled in poetry during my angst-ridden teenage years, but it wasn't until college that I truly came to comprehend the potency of the English language.
That appreciation materialised into something more tangible one dark wintry evening whilst I was making a papier-mâché Octonauts Gup-A(Google it—you'll be impressed) for my son, and watching a rerun of Pride and Prejudice on TV. Fired up by the remembrance of Austen's genius with words, I dug out my copy of the novel and in short order had been inspired to set my mind to writing in earnest. I began work on a Regency romance based on Austen's timeless classic, and my debut novel Mistaken is the result.
The Regency period continues to fascinate me, and I spend a good deal of my time cavorting about there in my daydreams, imagining all manner of misadventures. The rest of the time I can be found at home in Hertfordshire, where I live with my husband, two children, and an out-of-tune piano.


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Legislative Update-Veto Session
Tax Policy for the State of Kansas: Senate Substitute for House Bill 2109
Friday, June 12, marked the 113th day of the 2015 Legislative Session, the longest session in history for the state of Kansas. Early on Friday morning, the House passed Senate Substitute for House Bill 2109 by a margin of 63 votes, the amount needed to pass out of the House of Representatives. This tax bill was not the tax bill that I desired to address the financial issues with the state of Kansas, however given our lengthy session, we were approaching deadlines that were unprecedented and needed to meet our constitutional obligation of balancing the Kansas state budget. The details of HB 2109 are: raising the state sales tax rate from the current rate of 6.15% to 6.50%; tax amnesty for certain delinquent taxes; guaranteed payments would be subjected to income tax rates; income tax rate freeze until the tax year of 2018; the Rural Opportunity Zones extension until 2022; with the exception of Charitable (100% deduction), Mortgage Interest, and Property Taxes (50% deduction), all other deductions would be repealed; and Cigarette Taxes would increase by $0.50 a pack. There were also some policy provisions that were added by the Senate such as Sales Tax Exemptions, Property Taxes imposed by local units of government, and tax credits for scholarships. However, these policy pieces were changed by the House in Senate Bill 270 to not have the effect that the Senate had initially desired.
My true conviction is that this tax bill that passed the legislature, and now awaits the Governor's signature, is not a long-term, sustainable tax policy. In order to generate long-term stability for our state, we must readdress the tax policy that was passed by the 2012 legislature. That is the cause of the financial duress that our state is currently experiencing and the plan that passed will only be short-term. Due to the fiscal deadlines that our state was approaching and allotments that would need to be made to the budget, for example Fort Hays State University would experience a $32 million reduction, I voted "yes" thinking of the best interest of our state, residents, educational institutions, and our disabled citizens.
House Bill 2135
Senate Substitute for House Bill 2135 would authorize the Governor's Director of the Budget, if the Director determines the unencumbered ending balance of the State General Fund will be less than $100 million, to lapse appropriations or transfer funding from special revenue funds to the SGF from Executive Branch agencies, up to a total of $100 million, for FY 2016. This authority would not apply to appropriations for debt service; employer contributions to the Kansas Public Employees Retirement System; the Department of Education, except the operating expenditures account of the SGF; or demand transfers to the school district capital improvements fund. This bill was passed to protect these funding measures from an allotment from the Governor. I voted "yes" on this measure and it passed both the House and Senate on Friday, June 12.
The Group of 28
During the final two weeks of the session, and with no solid tax plan in place, there was a group of 28 conservative Republican House members, I included, that contend we must address the 2012 tax plan. The leader of our group was Representative Mark Hutton from Wichita and we met regularly to discuss our position and drafted plans that we wanted to be included in the final tax policy that we hoped would be passed.
We were able for the tax conference committee between the House and Senate to allow for our plan to be debated and voted on the House floor, however it only received 27 affirmative votes. Later, we tried to meet with other groups within the House to compromise and hopefully have enough votes for passage.
There were five identifiable groups within the Kansas House of Representatives: The conservative base was split between two factions, ours included, moderate Republicans, Democrats, and then the legislators that were absent during the latter part of the session.
These meetings with the other groups, with the exception of the absent legislators, could not materialize because of differing ideologies, and the threat of non-passage of our position by the Senate and a veto by the Governor if components of our plan of reinstating LLC's, sole proprietorships, and S Corps back on the income tax rolls, even at the lowest income bracket rate, we acknowledged that at this moment there would not be enough votes in our chamber to get to the magic number of 63 for passage.
Even though we were at an impasse, our group fought for our position until late Thursday evening. However, with the looming fiscal deadlines and the latest proposals of cutting our regent institutions, we had no choice but to accept the plan before us.
Later, we did hear from others in the chamber that voted "no" on our plan when it was debated and voted on the House floor said that our plan was the best solution for our state's fiscal crisis. Although we do identify that the plan that has passed is only a short-term solution, our resolve is to revisit our plan at the beginning of the 2016 Legislative Session.
Explanation of Vote and Contact Information
As your public servant, I feel that it is my duty that you are aware of my explanation of vote that I submitted to the House Clerk after voting "yes" and having it read before the entire chamber.
"Madam Speaker, I vote yes on House Bill 2109 in order to protect the solvency of our higher educational institutions for the state of Kansas. This committee report does not address all the possible revenue streams in order to resolve our financial calamity that our state currently faces, however as a representative of the Kansas House, we need to claim responsibility and make certain that we provide funding for our government and its needs. In extreme situations as in these, we need to make certain that we do not jeopardize our citizens, institutions, and our state."
If you have any concerns, feel free to contact my office at (785) 296-7672, visit www.troywaymaster.com or email me at email@example.com
The honor to serve you in the 109th Kansas House District and the state of Kansas is one I do not take lightly. Do not hesitate to contact me with your thoughts, concerns and questions. I appreciate hearing from the residents of the 109th House District and others from the state of Kansas.
Troy L. Waymaster,
State Representative
109th Kansas House
300 SW 10thTopeka, KS 66612


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UWA Transfer Scholarship Info Table
About this event:
Created by mpartain
Transfer your associate degree from a partner community college and earn a scholarship for up to $2000 at the University of West Alabama. It will be distributed to your online bachelor's degree program at $50 per credit hour for the first 40 consecutive credits.
4600 Valleydale Road
Birmingham, Alabama 35242
Fred Ellis
(502) 322-7505


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I'm a sucker for all things vintage and everyday I have my little trifting hour on Etsy. Yesterday I've spotted some amazing decorating objects for your home. Let's have a peek at some wonderful objects I've found !
Timbertoys. I love how all of the items are photographed. So pretty !
The Old Crow Farm, they are currently on vacantion so here are some of the sold items in their shop.
ClockworkUniverse, all the way from Latvia.


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(click here for live demo)
I don't design this template from scratch :) , so I use pattern from designfruit.com , changed the color combo and put on Blogspot's classic minima layout. Somehow I just love its simplicity..thank you Douglas Bowman!
**download the code, copy all the texts & paste on "Edit HTML box"


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Batman Jump is an Action game. You can play Batman Jump in your browser for free.
Description: Sort My Tiles Dora The Expoler is a Puzzle game. You can play Sort My Tiles Dora The Expoler in your browser for free. Dora and the gang needs some sorting out in the forest. <br>
All copyrights and trademarks of this game are held by their respective owners and their use is allowed under the fair use clause of the relevant Copyright Law. If you believe we are violating your copyright, please refer to our DMCA statement for more information.


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Hi guys got a problem with two separate form fields.
one is a search field and the other is a donation form which users can give the amount of the donation.
now the search field is working properly without any problem. but the donation form has some issues which i have not found a fix for.
the problem is when ever u press the donate button it does noting now i have checked this with the firefox console and it seems the donation button is interfering with the search field. So when i have typed in something in the first form which is the search form field and pressing the donate button in the donation form the search form is getting its results. Now i have narrowed the problem down to which lines of the two form are conflicting with each other and those lines are if i am correct here:
Hope you guys understand my problem.Code:<input id="search" name="search" class="searchinput" placeholder="Search..." type="text">
<input class="donationbutton" name="submit" value="donate »" type="submit">

is there anyway to fix please let me know.Code:

 <input id="search" name="search" class="searchinput" placeholder="Search..." type="text">
 <button type="submit" id="searchbutton"></button>

 <form action="idealcheckout/checkout.php" method="post"> <fieldset style="border: 0px none;">

Amount:

$ <input name="amount" value="" type="text">

<input class="donationbutton" name="submit" value="donate »" type="submit">

 </fieldset> </form>
  </html
thanks in advance


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For inquiries and comments to IIC, contact:
IIC's Communications Group
E-mail: email@example.com
For project inquiries, including environmental and social questions related to an IDB Invest investment, please contact the client or IDB Invest using the contact information provided above. As a last resort, affected communities have access to the IDB Invest Independent Consultation and Investigation Mechanism.
Tel:+1 (202) 623-3952
Fax: +1 (202) 312-4057
Address: 1300 New York Ave. NW Washington, DC. USA. 20577
E-mail: email@example.com or email@example.com
Project Name: Inversiones Amalgamadas, S.A. de C.V.
Project Number: HO3732A-01
Country: EN_COUNTRY_HN-Banco Central de Honduras
Sector: Food and Beverages
Approval Date: 11/17/2014
Date of First Disbursement: 12/03/2014
Total Project Cost: $ 1,400,000
Financing Currency: United States Dollar
IIC Financing Amount: $ 1,200,000
Syndicated / Co-financing Amount: $ 315,000
Investment Officers:
Fonseca,Juan Reyes
Beckham Jr.,John
Project Attorney: Botto,Angel
Environmental Engineer: Factor,Seth
Additionality Officer: Penedo, Maryline
Project Name: Inalma
Expected Consideration Date by the Board: 10/28/2014
Date Posted: 09/26/2014
Company: Inversiones Amalgamadas S.A. de C.V. ("Inalma")
Scope Objective:
Inalma is an agroindustry exports company, founded in 1987, whose area of business is the production of precooked and frozen products, mainly based on banana and cassava, ready to heat and serve.
The project has two objectives: ensuring the supply of raw materials through joint investments in plantain plantations with several suppliers, and refinancing medium-term liabilities to improve the company's debt maturity profile.
Environmental Review:
Environmental and Labor Issues:Environmental and Social Risk Categorization
According to the IIC Environmental and Social Sustainability Policy, this is a Category B project: potential environmental and social risks and impacts are limited to the project site, largely reversible, and can be mitigated via measures that are readily available and feasible to implement in the context of the operation.
Environmental and Social Appraisal
The IIC's Environmental and Social Appraisal focused on the project's current and anticipated performance in four key areas: (i) the corporate environmental and social assessment and management system; (ii) labor and working conditions; (iii) resource efficiency and pollution prevention; and (iv) corporate social responsibility.
(i) Environmental and Social Assessment and Management System
Inalma has a corporate quality policy (Política de Calidad) that affirms the company's commitment to complying with the laws and regulations of Honduras and of the countries to which the Company exports its products. The policy explicitly mentions the Company's respect for environmental preservation and its commitment to being a socially responsible corporation. Furthermore, the policy states that the company will select suppliers with the capacity to comply with its corporate standards. Inalma also has a corporate health and safety policy (Política de Salud y Seguridad) which clearly states that it is the responsibility of each manager and supervisor to ensure that all personnel have the protective equipment and training necessary to be able to perform their duties free from risk of accident or illness. New employees are introduced to these policies during their induction training, and all employees are thereafter provided with semi-annual refresher courses on health and safety management.
Environmental and social risks and impacts at Inalma are identified and managed primarily by a Health and Safety Committee which meets every two months. The committee reports directly to top-level management (e.g., the Operations Manager), and is comprised of the manager of the Environment and Safety Department, the manager of the Human Resources Department, the Production Supervisor (for emergency response matters), and several representatives from the general workforce.
Food safety and quality risks are management by a separate committee made up of all the department heads. This committee meets monthly. Inalma has been in possession since 2012 of Food Safety Certification 22000 (FSSC:22000). The FSSC 22000 system is officially recognized by the Global Food Safety Initiative, and this certification is awarded by NSF International.
Emergency response procedures are contained within a Contingency Plan (Plan de Contingencies). Inalma conducts semiannual evacuation simulations, in which all personnel participate and the emergency procedures described in the contingency plan are tested. After each simulation exercise, opportunities for improvements are identified and resolved by the responsible Inalma staff.
Inalma is operating under two relevant licenses: a health license emitted by the Secretary of Public Health (Food Control Section); and an environmental license emitted by the Ministry of Natural Resources and Environment and by the Direction of Environmental Control. To obtain both of these licenses, Inalma's operation was inspected by the authorities and the Company was required to present acceptable management and monitoring plans. The plant is inspected/audited annually by the following entities: the Municipal Environment Unit (Unidad Municipal Ambiental), the Fire Department, the Ministry of Environment (Secretaría de Medio Ambiente), inspectors for the FSSC 220000 certification, and the Ministry of Public Health (Secretaría de Salud Pública).
(ii) Labor and Working Conditions
Inalma currently has 292 employees, 30 percent of whom are females. At the supervisory level, 19 (49 percent) of the 39 employees are females.
In accordance with the commitment stated in its human resource policy, the Company operates in compliance with Honduran labor laws. These include the Honduran Labor Code of the Ministry of Labor (Secretaría de Trabajo y Seguridad Social) and with the Occupational Health Code of the Ministry of Health (Secretaría de Salud Pública). All employees undergo an induction training process, during which they are presented with the Company's human resources policies and rules, which reference these labor laws. During the induction process, new employees are given basic occupational health and safety training, which is later tailored to specific work functions by the employees' direct supervisors.
Inalma does not employ minors. Employees are free to join labor associations. The maximum length of dayshifts is 10 hours; nightshifts are limited to 9 hours. The employee grievance mechanism currently consists of suggestion boxes located in various locations around the plant. The IIC's Environmental and Social Action Plan (ESAP) for the Project will include a requirement for the Company to implement a more robust grievance mechanism which, among other considerations, will define the responsibilities, formal procedures, and timing for resolving any employee grievances.
Beyond the benefits that the Company is required to provide by law, Inalma offers the following benefits to its employees: subsidized meals in the company cafeteria; merit-based bonuses; a savings club (Club de Ahorro) to help employees with personal or familial emergencies; a minimum salary which is currently 8 percent above the legal minimum; a commissary offering household staples to employees at cost; and an onsite medical clinic for attention to basic health needs.
(iii) Resource Efficiency and Pollution Prevention
Inalma mitigates environmental and social risks related to its supply chain in several ways. These include contractual clauses, the provision of direct technical assistance to all of its suppliers of fruits and vegetables, and regular monitoring of the activities of these suppliers. For example, Inalma ensures the appropriate use of agrochemicals by its suppliers in three ways: by contractually requiring its suppliers to use only those agrochemicals that are preapproved by the Company; by training its suppliers in the correct handling of these agrochemicals; and by performing random tests of the fruits and vegetables received from suppliers.
Organic wastes from the processing plant are disposed of at a municipal incinerator. Liquid wastes are pre-treated by the Company and then sent for final treatment to a contracted third party. The used vegetable oil from production processes (approximately 60 gallons per month) is sold to a reseller for use as biofuel.
The Company is currently embarking on an analysis of options for converting the organic wastes from its production processes into methane which could then be used to as fuel for those same production processes. IIC is in discussions with the Company regarding the possibility of providing technical assistance funds to support this effort.
(iv) Corporate Social Responsibility
Inalma supports the local educational system by providing the salaries of four teachers in the municipality of Choloma. In addition, in 2014 alone the Company has already donated over 26 thousand pounds of food to various local organizations, including churches, nursery schools, and nursing homes.
Monitoring and Compliance
To verify the project's ongoing compliance with local regulations as well as with the IIC Environmental and Social Sustainability Policy, each year Inalma will submit to the IIC an environmental and social monitoring report. The IIC will review these annual reports and conduct periodic site visits throughout the life of the project in order to verify continued compliance.
© 2018 Inter-American Investment Corporation


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3 Dunbar Court, CooroySold September 28, 2017
- Property ID: 61642390
- Bedrooms: 4
- Bathrooms: 2
- Property type: HOUSE
- Land size: 647 Metres²
- Building size:
- Garage: 2
- Listing type: SOLD
Modern Home in Cul-D-Sac Position !!
Located in Cooroy's newest residential precinct is this 2010 built 4 bedroom, 2 bathroom ground level home. The rendered brick veneer home is set on a level 647 sqm approx. ¾ fenced allotment which backs onto a reserve. A short walk and you're in the Cooroy town centre is just one of the many lifestyle benefits that has made Greenwood Grove so popular. Solid low maintenance construction, a 5 kW solar array, plumbed water tank and garden shed add to the home's sustainability credentials.
All the bedrooms have ceiling fans and built in robes with the main bedroom having the ensuite bathroom, walk-in robe and air-conditioning. A big covered outdoor dining area off the air-conditioned open living room provides an extension of the living area and is great for entertaining. The big double garage and internal laundry complete the package.
The property is currently rented at $480 per week on a fixed term tenancy, so if you are contemplating a move to the Sunshine Coast and want the small town community atmosphere with all the benefits of a coastal lifestyle then Cooroy township is with worth more than a cursory look. The lower property values, the laid back lifestyle and the abundance of amenities make this area ideal for families. If you are a property investor then location, the improvements and the build quality make this property worthy of consideration.


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Kruithof Curve
"Kruithof's graph," as published in 1941 by Kruithof, that shows, for general lighting in interiors, what combinations of illuminance level and correlated color temperature result in a "pleasing" or "not pleasing" assessment by persons in that interior.
Preferred Correlated Color Temperature (CCT) for General Lighting in Interiors
Around the time of the introduction of tubular fluorescent lamps in the early 1940s, Kruithof was the first who carried out a study on preferred correlated color temperature (CCT) for interior lighting. He investigated the relationship between lighting level in an interior and the CCT of the light sources used, on the assessment of people in terms of pleasantness [1]. His study received much recognition, although many subsequent researchers got inconsistent results. It must be realized that not only the lighting level but aspects as climatic conditions and cultural background influence the preferred illuminance as well.
Lighting not only has a...
- 1.Kruithof, A.A.: Tubular luminescence lamps for general illumination. Philips Tech. Rev. 6, 65–96 (1941)Google Scholar
- 2.Van Bommel, W.J.M., van den Beld, G.J.: Lighting for work, a review of visual and biological effects. Light Res. Technol. 36, 255–269 (2004)CrossRefGoogle Scholar
- 3.De Boer, J.B., Fischer, D.: Interior Lighting, 2nd edn. Kluwer Technische Boeken, Deventer (1981)Google Scholar
- 4.Mills, P.R., Tomkins, S.C., Schlangen, L.J.M.: The effect of high correlated colour temperature office lighting on employee wellbeing and work performance. J. Circadian. Rhythm. 5, 2 (2007)CrossRefGoogle Scholar
- 5.Boyce, P.R., Cuttle, C.: Effect of correlated color temperature on the perception of interiors and color discrimination performance. Light Res. Technol. 22, 19–36 (1990)CrossRefGoogle Scholar
- 6.Vienot, F., Durand, M., Mahler, E.: Kruithof's rule revisited using LED illumination. J. Mod. Opt. 56, 1433–1446 (2009)ADSCrossRefGoogle Scholar
- 7.CIE Publication: Ocular Lighting Effects on Human Physiology and Behaviour, vol. 158. International Commission on Illumination, CIE, Vienna (2004)Google Scholar


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My passion is to deliver marketing insights based on in-depth qualitative and quantitative research
Over the past 30 years I have conducted almost 5,000 qualitative sessions, mostly with Hispanics in the US, both in Spanish and English. I have done research with women, men, and children of diverse ages and language preferences.
I conduct focus groups, ethnographies, and in-depth one-on-one interviewing anywhere in the United States, Mexico and other Latin American countries. My background as a marketing research practitioner, researcher, and social scientist allows me to deliver actionable insights that help build brands and solve marketing problems.
I have worked on most product and service categories including consumer packaged goods, soft drinks, alcoholic drinks, travel, insurance products and services, telecommunications, technology, health, beauty, and many others. Most of my clients have been Fortune 1000 companies.
My signature benefit is that I know how to obtain insights from consumers that others seldom can. The book we published in 2012 "Hispanic Marketing: Connecting with the New Latino Consumer" serves as a testimonial to the skills and knowledge I bring to the qualitative research context.
In the research I carry out I utilize varied approaches, including:
- Metaphor elicitation
- Guided imagery
- Laddering
- Role playing
- Scenario building
- Games and simulations
- Story telling
Please contact me to discuss your qualitative Hispanic research needs.
or call me at 850 583 0378


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Friday, April 25, 2014
Peace Out
3 comments:
- GnomeLover said... [Reply to comment]
You look great in all of your styles. I feel the same way. I like all different ways of dressing. And that is what is fun about being a girl. There are so many fun different styles to choose from!
That green sweater is perfect with that peace sign shirt. I am so bad. I forgot that it was Earth Day until the end of the day. We were at Disneyland and it was a long day!
That necklace is gorgeous, Heather. You are so talented. I can see why it was featured. It is a work of art.
I need to try my braids that way! Genius to pin it up!
- April 25, 2014 at 11:11 PM
- Kacie Ellis said... [Reply to comment]
I've given up in trying to braid my hair. I did a good job once but every time since then, it has looked horrible. lol. Great idea with the fake braid thing! So cute!
- April 26, 2014 at 7:00 PM
- Brooke said... [Reply to comment]
I love mixing up my style too. I could never commit to one sort of look! I love the peace sign tee. I could see myself wearing that ALL the time. Your hair looks great! I've had long hair for years and have only mastered a messy ponytail and fishtail braid. I'll have to check out the tutorial!
- April 28, 2014 at 6:13 PM


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Quantum Dot Eigenstate Scatterplot.
The figures above show the isosurfaces of the ground state computed in an sp3s* tightbinding model of a cubic quantum dot of dimensions 7x5x3.5 nm. Three different isosurfaces are plotted at 60%, 35% and 12% of the maximum wavefunction value. The lower plot shows some asymmtery we are currently still investigating.


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To hold your reservation, we require a 50% deposit for the total of your stay. The initial deposit is due within a week of your booking request. The remaining 50% is to be paid at least two weeks prior to your arrival. If you are making a last minute reservation, the total amount will need to be paid in full before your arrival. Once your final payment has been received, you will receive a final confirmation with all of the details of your reservation and how to enter the condo. We cannot hold reservations without a deposit.
• For cancellations more than 30 days prior to arrival, a full refund applies.
• For cancellations within 30 days of arrival, 50% of the total rate will be refunded.
• Any cancellation made within 48 hours of arrival will not be given a refund.
Cable, Internet & Other Amenities
All condos have basic cable in both the living room and bedroom, wireless interest access and other free amenities. While we offer these amenities free to our guests, we can not offer any refunds if the cable or internet network is down as sometimes outages happen and repairs are necessary.


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Yesterday we were sitting around the office (our new office rugs came!), and Kate said to me, "Grace, I have a submission I think you're going to love." The next words that came out of her mouth pretty much sealed the deal: "There's a bunny jumping on a trampoline!" That was all I needed to hear — my morning post decision was made. These funny new home designs are from a company called HAM. Launched last summer, the entire line follows "unexpected moments" from the lives of a pig, horse and rabbit. I love that concept for a collection, and frankly, if it results in more tea towels that deal with bouncing rabbits, I'm in. Click here to check out the full collection online and place an order. Every time I view the url (hammade.com), I think it's a funny animal-spin on "homemade." It doesn't take much to make me laugh this early in the morning. xo, grace
Pigs mowing the lawn? I love it!


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The narrative of a westerner travelling to the East to find enlightenment – and a sense of spiritual calm and…
Self-help gurus love goals. "Success is goals, and all else is commentary," says one guru. Self-help masters proclaim their love for goal
setting in myriad places – books, audiotapes, box sets with titles like Write it Down, Make it Happen, and meditation exercises designed to achieve goals that really excite you, often involving hot property and fast cars.
It's true that knowing what you want – and writing it down – can help you to plan. You can write out a step-by-step action plan, for example, complete with boxes to tick. Goals can help stave off procrastination – and once specific goals have been ticked off, you can then replace them with even more goals, ensuring that your life journey is always on the straight and up. Up, up, up the ladder you go.
The underlying message here is that there's a gap to fill – the gap between how things are now and how you really want them to be. It's premised on the assumption that there's a shortfall in your life – a deficit that with commitment and labour you may eventually overcome, provided you follow your goals.
But one could argue that living a life of 'deficit', one of 'what's missing', is no way to live. You may habitually overlook your accomplishments and blessings. If you're forever climbing the ladder of goals you fail to appreciate the surrounding scenery.
And whose goals are these anyway? Are you chasing prefabricated dreams sold from television sets and books you read as a child? Perhaps what's needed is a final goal to every list you create, and that's to accept and appreciate things as they already are.


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11:20am PT by Borys Kit
'Game of Thrones' Actor Alfie Allen Joins Shane Black's 'The Predator' (Exclusive)
Alfie Allen, who plays Theon Greyjoy on HBO's Game of Thrones, has joined the cast of Shane Black's reboot of Predator.
The 20th Century Fox movie began shooting Monday in Vancouver, with Boyd Holbrook (Logan) leading an ensemble that also includes Room star Jacob Tremblay, Sterling K. Brown, Keegan-Michael Key, Trevante Rhodes, Thomas Jane and Olivia Munn.
Black co-wrote the script with Fred Dekker and is directing the movie, titled The Predator.
Allen will play an ex-Marine who teams with several other outsiders, including one played by Holbrook, to stop the human-hunting creatures in a setting that hasn't yet been seen in a Predator movie: suburbia.
On top of being known for his work on Thrones, Allen also memorably played a Russian gangster who kills the wrong dog in 2014's John Wick.
He is repped by UTA, Untitled and U.K.'s The Artists Partnership.
Borys email@example.com borys_kit


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These plugs are a favorite with our film industry customers, due to their neutral "flesh" coloring. We also get a lot of music industry orders for these due to the fact that they are more inconspicuous that other plugs.
But don't let their crossover appeal fool you, with a Noise Reduction Rating (NRR) of 29 these are a great match for a day at the range.


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I hope that you enjoy the birthday, we certainly did. I think that Piper celebrated all week. Soon I will have pictures of her first day of school.
Then, as soon as I get them the pictures of Austin and Alexis' first days of school will follow.
Now for effect:
Isn't she beautiful !!!! I have lots of pictures of this process, so check back for more on this later. Have a great week.


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Raw Silk w/ Leslie Lembo
Jan 18
, 2018
Doors: 6:30 PM
Show: 8:00 PM
All Ages
SOhO Restaurant and Music Club
1221 State Street
Santa Barbara, CA
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Publication - Strategy/plan
National Strategy for Community Justice
Published: 24 Nov 2016
- Directorate:
- Justice Directorate
- Part of:
- Communities and third sector, Law and order
- 9781786526168
This strategy provides a shared vision to help partners and communities work together effectively to improve community justice outcomes.
National Strategy for Community Justice
12 Annex B: National Strategy Steering Group Representative Organisations
12 Annex B: National Strategy Steering Group Representative Organisations
- Community Planning Partnership Managers
- Police Scotland
- Scottish Prison Service
- Skills Development Scotland
- Criminal Justice Voluntary Sector Forum
- Positive Prison? Positive Futures
- Office of the Chief Social Work Advisor
- Social Work Scotland
- Health Boards
- Academia
- Scottish Government Community Justice Division
- Community Justice Authorities


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School Counselor Advocacy Video
The Bridge to Student Success
Please share...
Virginia School Counselors! We hope you will share this video with your stakeholders for it embodies the message we want to convey to decision makers...school counselors are an invaluable resource to the educational system!!! The testimonies shared by students and adults support the belief statements held by the Virginia School Counselor Association and school counselors, alike. We are VSCA!
Virginia School Counselors: The Bridge to Student Success
VSCA would like to thank West Potomac High School in Fairfax County and Sutherland Media Productions for their assistance with the filming and production of the video.


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Nicola Bellini
Nicola Bellini is Professor of Economics and Management at the Scuola Superiore Sant'Anna in Pisa (www.idm.sssup.it - on leave) and Director of the Tourism Management Institute at the La Rochelle Business School (www.esc-larochelle.fr).
He was visiting or affiliate professor at the Stanford University Center in Florence, University of Sassari, University of Pisa and Grenoble Ecole de Management. From 2009 to 2011 he was the Director of the Regional Institute for Economic Planning of Tuscany – IRPET. From 2007 to 2014 he was Director of the Galileo Galilei Italian Institute in Chongqing and Co-Director of the Confucius Institute in Pisa.
Currently he also serves as an expert for the EU Commission on Regional strategies for smart specialisation. He is author and editor of books and articles on industrial policy, local and regional development, business support services and place branding.


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Pheasants Forever's fourth Pheasant Fest opens Friday at RiverCentre in St. Paul.
Here are 25 facts about Pheasants Forever, which was founded in St. Paul and is celebrating its 25th anniversary.
1 PF has 700 chapters, including 74 in Minnesota, and about 110,000 members nationwide, including 22,500 in Minnesota. Quail Forever, created in 2005, has 5,000 members nationwide. There is one chapter in Minnesota with 141 members.
2 The first PF banquet was held April 15, 1983, at the former Prom Center on University Avenue in St. Paul. About 800 attended. The event was, and still is, one of the biggest of its kind in Minnesota.
3 The speaker at the first PF banquet was the late Gov. Rudy Perpich, who later signed the state's first pheasant stamp bill -- which Pheasants Forever and supportive legislators had written and promoted.
4 The state pheasant stamp has raised $14.6 million for pheasant habitat and development on private and public lands, and for public land acquisition. The stamp costs $7.50 and is required of pheasant hunters age 18 and older.
5 Among attendees at the first banquet was famed waterfowler and outdoors writer Jimmy Robinson. Then 85, Robinson had been a supporter in the 1930s of the formation of Ducks Unlimited. He presented two $1,000 checks to PF banquet organizers, one from him and one from the late Robert Naegele Sr. A St. Louis Park resident, Robinson said he liked Perpich but didn't vote for him. "I voted for Harold Washington of Chicago,'' he said.
6 Here's how Pheasants Forever membership ranks with other conservation groups in Minnesota:
1. Ducks Unlimited, 42,000
2. National Wild Turkey Federation, 26,000
3. Pheasants Forever, 22,500
4. Minnesota Deer Hunters Association, 20,000
5. Minnesota Waterfowl Association, 4,000
7 The first outstate banquet was held in Willmar, in Kandiyohi County. Doug Lovander was principal organizer. When Lovander contacted Dennis Anderson, then outdoors columnist with the Pioneer Press, to express interest in forming a chapter, Anderson asked Lovander how serious he was about pheasants. "Put it this way,'' Lovander said. "I conduct my own spring roadside [population] surveys.''
8 The Kandiyohi chapter netted $16,000 from its banquet, drawing 500 people. Lovander and other chapter officers wondered how giving the money to St. Paul-based PF would help Willmar-area pheasants. A meeting beneath a circus tent on Lovander's lawn after a pheasant feed helped solidify a strategy that remains PF's hallmark. Namely, chapter money, exclusive of membership fees, remains with local chapters for habitat development.
9 PF says it and Quail Forever have affected about 5 million acres -- including planting grasslands, restoring wetlands, planting woody cover or food plots and purchasing lands -- since its formation. The total includes 370,262 habitat projects.
10 In Minnesota, the group says it has spent $33.7 million and affected about nearly 200,000 acres.
11 The formation of Pheasants Forever was not endorsed by all Minnesota wildlife groups. Some members of a group called the Minnesota Association of Farmers, Landowners and Sportsmen opposed the idea, viewing PF as competition. MAFLAS had encountered difficulty uniting landowners and hunters and eventually disbanded.
12 Iowa is the only state with a PF chapter in every county. Some counties have more than one chapter, so Iowa tallies 103 chapters in its 99 counties.
13 Minnesota has the most PF members, but Iowa isn't far behind:
1. Minnesota, 22,500
2. Iowa, 20,200
3. Nebraska, 10,200
4. Michigan, 7,900
5. Illinois and Wisconsin, 7,500
14 A key hire early in the group's history was that of Jim Wooley of Chariton, Iowa. Wooley was that state's upland bird biologist and his employment as a field representative/biologist smoothed the way for PF to expand in Iowa.
15 Early on, some chapters or would-be chapters wanted to stock pheasants. PF always has had a policy against stocking, arguing that habitat expansion is the key to higher ringneck numbers.
16 This is the fourth Pheasant Fest; the first was held in 2003 in Bloomington and was attended by about 12,000 people. Another was held in Omaha in 2005 and Des Moines in 2007, both attended by about 24,000 people. PF is hoping the St. Paul show will be the biggest yet.
17 At the first Pheasants Forever banquet in St. Paul, a Browning Citori 12 gauge shotgun was auctioned. The gun was engraved, "Pheasants Forever Number 1.'' Know the person who owns it? If so, e-mail email@example.com The average PF member is getting older, reflecting national trends on the aging of the hunting and angling community. In 1990, the average PF member was 40, and by 1999, it was 44. Today, the average age is 49.9 years old.
19 The number of PF employees has grown from a handful to 57 in 2004 to 150 today, including 45 in the national office in White Bear Lake and more than 100 in the field.
20 The most unusual PF chapter? There's the MN Lady Slippers, an all-female chapter. And the North Central States Regional Council of Carpenters chapter -- the first union chapter -- based in St. Paul. And there's the Royal Flush chapter in California, the nation's only chapter open only exclusively to life members.
21 PF has spent $260 million for its habitat and education programs, including $33.7 million in Minnesota.
22 PF has donated more than 5,500 acres and helped fund the purchase of another 22,000 acres of public hunting lands in Minnesota. Most are state wildlife management areas.
23 PF secretary Bob Larson of Wayzata and retired chief executive officer Jeff Finden of White Bear Lake are the only members of the original board of directors to remain in those positions today.
24 Howard Vincent, 51, of White Bear Lake, a Duluth native, has been president and CEO of PF for the past eight years, and has been with the group for 20 years.25 PF has 15,000 youth members; chapters pay their $5 membership fee, so the kids get to join for free. That program has boosted youth membership, which tallied 4,000 seven years ago.


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I AM in Midtown
I am a long way from the warm breeze of voices I heard a week ago on Easter Sunday.
"Glorify your name," the island women sang, as they swayed in a cut sandstone church. I was overwhelmed by a riot of color, an emotional swell that carried me to sea.
Christianity, it turns out, has a rhythm — and it crescendos this time of year. The rumba of Carnival gives way to the slow march of Lent, then to the staccato hymnals of the Easter parade. From revelry to reverie. After 40 days in the desert, sort of ...
Carnival — rock stars are good at that.
It's Lent I've always had issues with. I gave it up ... self-denial is where I come a cropper. My idea of discipline is simple — hard work — but of course that's another indulgence.Continue reading the main story
Then comes the dying and the living that is Easter.
It's a transcendent moment for me — a rebirth I always seem to need. Never more so than a few years ago, when my father died. I recall the embarrassment and relief of hot tears as I knelt in a chapel in a village in
Last Sunday, the choirmaster was jumping out of his skin ... stormy then still, playful then tender, on the most upright of pianos and melodies. He sang his invocations in a beautiful oaken tenor with a freckle-faced boy at his side playing conga and tambourine as if it was a full drum kit. The parish sang to the rafters songs of praise to a God that apparently surrendered His voice to ours.
I come to lowly church halls and lofty cathedrals for what purpose? I search the Scriptures to what end? To check my head? My heart? No, my soul. For me these meditations are like a plumb line dropped by a master builder — to see if the walls are straight or crooked. I check my emotional life with
The preacher said, "What good does it profit a man if he gains the whole world and loses his soul?" Hearing this, every one of the pilgrims gathered in the room asked, "Is it me, Lord?" In America, in
Well, yes. It is us.
Carnival is over. Commerce has been overheating markets and climates ... the sooty skies of the industrial revolution have changed scale and location, but now melt ice caps and make the seas boil in the time of technological revolution. Capitalism is on trial; globalization is, once again, in the dock. We used to say that all we wanted for the rest of the world was what we had for ourselves. Then we found out that if every living soul on the planet had a fridge and a house and an S.U.V., we would choke on our own exhaust.
Lent is upon us whether we asked for it or not. And with it, we hope, comes a chance at redemption. But redemption is not just a spiritual term, it's an economic concept. At the turn of the millennium, the debt cancellation campaign, inspired by the Jewish concept of Jubilee, aimed to give the poorest countries a fresh start. Thirty-four million more children in
A few weeks ago I was in
But I read recently that Americans are taking up public service in greater numbers because they are short on money to give. And, following a successful bipartisan Senate vote, word is that Congress will restore the money that had been cut from the aid budget — a refusal to abandon those who would pay such a high price for a crisis not of their making. In the roughest of times, people show who they are.
Your soul.
So much of the discussion today is about value, not values. Aid well spent can be an example of both, values and value for money. Providing
Strangely, as we file out of the small stone church into the cruel sun, I think of
Not all soul music comes from the church.Continue reading the main story


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HOMER final conference (Cyprus)
Click here to download the agenda of this event.
Day 1: 9th September 2014
08:30 - 09:00 Participants registration
09:10 - 09:30 Welcome speeches.
09:30 - 10:30 HOMER project: objectives, results and achievements
Overview of HOMER Project. Luca Guerretta. Project manager (Regione Piemonte)
HOMER Federation Nives Alciato, Alessandro Franceschetti, Saverino Reale (CSI Piemonte)
Open Data Action Plans Constantine J. Strataridakis (Decentralized Administration of Crete)
10:30 - 11:30 Round table: "Policies on Open Data in the context of the MED Programme and the European Neighbourhood Instrument – Experiences from the period 2007-2013 and the new programming period 2014-2020."
Open Data & the Re-Use of Public Sector Information. Constantia Constantinou, Directorate General for European Programmes Coordination and Development.
The MED Programme experience and Future Sophie Scarvelis, Project and Finance Officer (Joint Technical Secretariat of the MED Programme)
11:30 - 12:00 Coffee break
12:00 - 1:00 Round table: "Digital Agenda, policies and strategies and State of the Art in Europe and in Cyprus, assessing links with HOMER project"
Digital Strategy of Cyprus. George Komodromos, Acting Director of the Department of Electronic Communications of the Ministry of Communications and Works.
Public Sector Information. State of Play in Cyprus. Dimitris Michael, Public Administration and Personnel Department.
INSPIRE implementation in Cyprus. Andreas Hadjiraftis, Cartography Branch Senior Officer of the Department of Lands and Surveys.
13:00 - 14:00 Lunch Break
14:00 – 15:30 Round table: "The reuse of Open Data – Practical Examples"
The SBLA experience with HOMER. Iacovos Papaiacovou, General Manager, Sewerage Board of Limassol-Amathus - SBLA.
Project DIAVLOS Aristotelis Savva, Executive Engineer, Ministry of Communications & Works, Public Works Department.
Aragón Open Data. José Maria Subero, advisor in new technologies, Government of Aragón.
Pilot B: A good Example of Open Data utilization by citizens.. Mathilde Dioudonnat, AVITEM.
Open Rosetta and Km0Farms. Team participating in HACK4MED (Piemonte and Veneto Regions),
Implementing transparency and open government projects in Greece. Georgios Karamanolis (GFOSS)
15:30 - 16:00 Wrap-up: Discussion and evaluation about HOMER project
Day 2: 10th September
Project Partners' Meeting
09:00 - 09:30 Arrival of the Participants – Registration & Group Photo
09:30 - 11:00 Status of open and federated data, presentation of the federated portal and the future of the federation Claudia Simonato, CSI Piemonte.
Business Plan Overview Isaac Matamoros, FUNDITEC.
11:00 - 11:15 Coffee break
11:15 – 12:00 Steering Committee and Financial Group (Final Project meeting)Luca Guerretta, Emanuela Elia. Regione Piemonte
12:00 – 12:30 Communication and Dissemination of the Project (Planning Brussels InfoDays) Mª Antonia Cobacho, Head of Department of International Relationship. AGAPA
12:30 – 13:00 Wrap–up of Day 2
13:00 – 14:00 Lunch Break
Hits : 3394


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All 127 sound proofed renovated rooms up to 45 M2, provide free WiFi, free wired Internet and 24-hourroom service. Thoughtful touches include bathrobes and slippers. Guests will also find minibars and LCD TVs. Located in Providencia, this hotel is 1 mi (1,6km) from Mall Costanera Center and within 0,6 mi(1 km) of San Cristobal Hill and Santiago Town Square. Sculpture Park and Parque Arauco are also within 10 minutes. We want to make your stay an even better experience. We redecorated our spaces to surprise you with a Brand new look and feel. We invite you to our fully renovated restaurant "Buganvilia Restaurant" and our new "Bar 111." Outdoor pool and a fitness center are available at this smoke-free hotel. WiFi in public areas is free. Additionally, a bar/lounge, a sauna and a rooftop terrace are onsite.Show More
Four Points by Sheraton Santiago is at an Excellent Location.
Four Points by Sheraton Santiago includes free HSIA Access.
Four Points by Sheraton Sanitago includes 127 soundproofed renovated rooms.


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Payday Lenders Curtailed, For Now
BALTIMORE — While consumer rights groups are celebrating last week's decision by the Consumer Financial Protection Bureau to stop payday debt traps, they say there's still more work to be done.
The new rules require payday lenders to start verifying a borrower's ability to repay the loan before rolling it over into a new loan. Marceline White, executive director of the Maryland Consumer Rights Coalition, said Maryland has been taking extra steps to protect consumers for three decades, but there have been attempts to scrap those rules.
"In Maryland, last session during the General Assembly, we had to fight off another attempt by payday lenders to exploit a loophole in our law," White said. "And they were able to do that and charge 300 to 400 percent loans."
The General Assembly shot that attempt down, and Maryland's cap on payday loans remains at 33 percent. White called attempts by the payday loan industry to exploit hard-working families across the country every year "never ending."
White said she applauds the decision by the Consumer Financial Protection Bureau because she said other states have not taken steps to protect consumers.
"The CFPB cannot separate caps. They're not allowed to, but now any kind of lender will have to prove that somebody can pay back a 1,000 percent loan and meet all their other financial obligations," she said. "We strongly believe that they will fail that test. "
According to White, conservatives in Congress are expected to try to repeal the new rule using the Congressional Review Act before it even goes into effect. And in 2018, President Trump will get the chance to nominate a new head of the CFPB. Its current director, Richard Cordray, is a holdover from the Obama administration.


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NEW YORK (WFAN) — The lineup. The lineup. The lineup. It's all the rage these days when it comes to the Yankees.
Who will bat where in the order? Where will the home runs threats be slotted? How will the kids fit in?
The man who will eventually answer those questions and make the decisions is Aaron Boone. The Yankees' new manager called in to WFAN on Wednesday morning and talked about the state of the team with guest host John Sterling.
Boone admitted he has been tinkering with the batting order in his mind, a fun chore considering the Yankees now sport sluggers Giancarlo Stanton, Aaron Judge, and Gary Sanchez, in addition to a handful of others who can really mash the baseball.
"We're hoping to see Greg Bird take another giant leap this year and be one of those guys that really makes a pitcher work," Boone said. "We saw what Didi (Gregorius) did last year. Obviously, Gary Sanchez. When an opposing pitcher looks at our lineup and thinks about having to work through it, it's a team that you better make pitches against. Otherwise, this is a team that not only will make you pay with a base hit, but it will also make you pay with some damage."
MORE: Judge Thrilled To Have Stanton On Yankees: 'He'll Blend Right In To This Team'
As for the situation at second base and third base, positions where the Yankees could turn to rookies, Boone said he's leaving all of his options open.
"Ronald Torreyes showed us last year how valuable he is as that utility infielder, so there is certainly confidence that he can play a number of positions if we need him to," Boone said. "There's a number of guys knocking on the door at the big league level that we feel will be big league players that can fill in there. Obviously, Gleyber Torres is going to factor in at some point at one of those positions. (Miguel) Andujar is on the come, so we have a lot of guys that are knocking on that door."
Boone also discussed the Yankees' quest for a sixth starting pitcher. Various reports have linked them to trades involving Gerrit Cole, and others. Boone's team could also be a factor in free agency, given the power arms still available and the fact that New York's payroll is well below the $197 million luxury tax threshold next season.
"Well, I think it just depends on (getting the right) matchup and if it is something that will ultimately make sense for us as an organization not only in the short term, but also in the long term," Boone said of adding another starter. "I know it's something (GM Brian Cashman) is actively looking into, pursuing, kicking tires, having conversations with other teams. We don't feel like we're in a situation where we are desperate to go out and have to get something, but it is definitely something we would like to add."
To hear Boone's complete interview, please click on the audio player above.