Viral infections of the central nervous system (CNS) are caused by a variety of viruses, namely, herpesviruses, enteroviruses, and flaviviruses. The similar clinical signs provoked by these viruses make the diagnosis difficult. We report on the simultaneous detection of these major CNS pathogens using amplification by PCR and detection of amplified products using DNA microarray technology. Consensus primers were used for the amplification of all members of each genus. Sequences specific for the identification of each virus species were selected from the sequence alignments of each target gene and were synthesized on a high-density microarray. The amplified products were pooled, labeled, and cleaved, followed by hybridization on a single array. This method was successfully used to identify herpesviruses, namely, herpes simplex virus type 1 (HSV-1), HSV-2, and cytomegalovirus; all serotypes of human enteroviruses; and five flaviviruses (West Nile virus, dengue viruses, and Langat virus). This approach, which used highly conserved consensus primers for amplification and specific sequences for identification, would be extremely useful for the detection of variants and would probably help solve some unexplained cases of encephalitis. The analytical sensitivity of the method was shown to be 500 genome equivalents ml−1 for HSV-1, 0.3 50% tissue culture infectious doses (TCID50s) ml−1 for the enterovirus coxsackievirus A9, and 200 TCID50s ml−1 for West Nile virus. The clinical sensitivity of this method must now be evaluated.
Viral infections of the central nervous system (CNS), such as encephalitis, meningitis, and meningoencephalitis, are due to a wide range of viruses mostly belonging to the genera Herpesvirus, Enterovirus, and Flavivirus. Human herpesviruses, herpes simplex virus type 1 (HSV-1), HSV-2, cytomegalovirus (CMV), Epstein-Barr virus (EBV), varicella-zoster virus (VZV), and human herpesvirus 6 (HHV-6) have a DNA genome and are important causes of encephalitis, myelitis, and meningitis (17, 40). The most serious disease caused is herpes simplex virus encephalitis (10, 40). HSV-1 is believed to be responsible for 95% of cases of this disease, and HSV-2 is responsible for the remaining 5% (3). The high rates of morbidity and mortality from herpes simplex virus encephalitis that occur without early antiviral therapy emphasize the need for the development of a rapid and accurate diagnostic method (39), especially as virus-specific therapy with acyclovir, ganciclovir, and foscarnet is available.
Between 80 and 92% of aseptic meningitis cases with an identified etiologic agent are currently attributed to human enteroviruses (29). These viruses belong to the Enterovirus genus of the Picornaviridae family, which includes 64 nonpoliovirus serotypes and 3 poliovirus serotypes. Other clinical manifestations due to these single-stranded positive-sense RNA viruses are varied and include acute respiratory illness; meningoencephalitis; myocarditis; hand, foot, and mouth disease; neonatal multiorgan failure; and acute flaccid paralysis (24).
The genus Flavivirus of the family Flaviviridae comprises over 70 single-stranded positive-sense RNA viruses that are transmitted by arthropods. Of the 40 flaviviruses associated with human illness, tick-borne encephalitis virus (TBE), West Nile encephalitis virus (WN), Japanese encephalitis virus (JE), Murray Valley encephalitis virus (MV), and St. Louis encephalitis virus (SLE) cause infections of the CNS. These viruses used to have restricted niches but are now spreading across the world, as demonstrated, for example, by the West Nile virus outbreak in New York City (2), which subsequently spread to the whole of the United States and Canada.
Nucleic acid amplification techniques, and especially PCR, have revolutionized the diagnosis of CNS viral infections (10, 11). While virus isolation is efficient for the diagnosis of meningitis due to enteroviruses, this method is insensitive for cases due to herpes simplex virus and arboviruses (9). Furthermore, virus isolation takes time and requires costly facilities and trained personnel. Many different amplification strategies that mostly use virus-specific primers in a series of independent PCRs or consensus primers have been reported. Human herpesviruses have also been detected by multiplex PCR (5, 13, 25, 34).
The use of consensus PCR primers that exploit sequence homology between related viruses has been reported for all three families of viruses. The amplified product is then identified by DNA sequencing, hybridization with specific probes (1) or restriction enzyme analysis (34). All known human herpesviruses were amplified in two assays (16) and in one assay (30, 34) by using primers localized in the DNA polymerase gene. Enterovirus RNA has been detected with primers localized within the highly conserved 5′ noncoding region (NCR) (18). Many universal flavivirus assays targeting the NS5 region have been published (21, 26, 31, 33), with virus-specific PCRs available for JE and WN (8, 33) and for SLE (21, 22).
Different CNS infections result in similar neurological symptoms, which make diagnosis difficult. While there are numerous publications on the separate molecular detection of each of these virus families, only a few authors have reported on the simultaneous detection of herpesviruses and enteroviruses (7, 27, 28), and no method for the detection of all three families has yet been published. Flaviviruses could explain some of the undetermined etiology of neurological infections, as reported for encephalitis (32 to 75%) (12, 14, 19, 32).
This paper describes a diagnostic tool that uses a DNA probe array for the simultaneous detection of viruses from three different families whose members cause CNS diseases that sometimes have similar clinical manifestations and its analytical validation with prototype species from the three families. The approach that we report here first uses broadly reactive primers for each family and then uses the DNA probe array technology to differentiate between virus species within each family.
Virus.Virus cultures were obtained from different sources: HSV-1 strain MacIntyre, HSV-2 strain G2, and Rhinovirus strains RH16 and RH18 were from the American Type Culture Collection (ATCC; Manassas, Va.); and CMV strain AD169 was from bioMerieux (Marcy l'Etoile, France). Enterovirus serotypes were kindly provided by H. G. M. Niesters (Erasmus MC, Rotterdam, The Netherlands) and B. Lina (Universite Claud Bernard, Lyon, France). The concentrations of nonpoliovirus enterovirus serotypes were determined by H. G. M. Niesters, as described by van Doornum et al. (37). The flaviviruses West Nile virus strain Eg101, Langat virus strain Langat, dengue virus serotype 2 (DEN-2) strain NGC, and DEN-4 (strain YUNH) were obtained from Pasteur Institute, Paris, France, while dengue virus serotype 1 strain Hawaii was obtained from Pasteur Institute, Dakar, Senegal; these viruses were propagated in Vero cells.
Nucleic acid extraction.Viral nucleic acids were extracted by use of a DNA Blood mini kit (QIAGEN, Courtaboeuf, France) for DNA viruses and a QiAmp Viral RNA mini kit (QIAGEN) for RNA viruses, according to the manufacturer's instructions. Briefly, for DNA viruses, 100 μl of sample was mixed with 100 μl of phosphate-buffered saline and 20 μl of proteinase K. Lysis was then performed at 56°C for 10 min with 200 μl of lysis buffer. After addition of an equal volume of absolute ethanol, the mixture was loaded onto a spin column, which was then successively washed with 500 μl of wash buffers provided with the kit. The purified nucleic acids were then eluted with 100 μl of the elution buffer provided with the kit. The protocol was similar for RNA viruses, except that lysis was done at room temperature for 10 min.
Primers.For the herpesviruses, the DNA polymerase gene was targeted. A pair of consensus primers that allowed the amplification of three herpesviruses (HSV-1, HSV-2, and CMV) were modified from the primers of Johnson et al. (16), HVF (GTGTTGGACTTTGCCAGCCT) and HVR (GTCCGTGTCCCCGTAGATGA). A pair of primers specific to the 5′ NCR of enteroviruses, ENTB (GGTACCTTTGTRCGCCTG) and ENTC (CCAAAGTAGTCGGTTCCG) (P. Renaud, E. Guillot, C. Mabilat, C. Vachon, B. Lacroix, G. Vernet, M.-A. Charvieu, P. Laffaire, April 2004, France, patent number WO0202811), that allow the amplification of a region of 500 bp were used. Typing of enteroviruses was done by amplification of part of the 2A and the VP1 genes, as described by Caro et al. (6) Flaviviruses were amplified with a pair of primers specific to the NS5 region, cFD2 (GTGTCCCAGCCGGCGGTGTCATCAGC) and MA (CATGATGGGRAARAGRGARRAG) (31).
RT-PCR.Reverse transcription (RT)-PCR was performed with a Titan one-tube RT-PCR system (Roche Molecular Biochemicals, Mannheim, Germany). Amplification of the three virus families was achieved in three separate reaction tubes, each of which contained primers specific for one family. DNA was added to the herpesvirus tube, and RNA was added to the enterovirus and flavivirus tubes. Each reaction contained 1× reaction buffer, 1.5 mM MgCl2, 5 U RNase inhibitor, 5 U avian myeloblastosis virus enzyme mix and Expand High Fidelity enzyme blend, 0.2 mM deoxynucleoside triphosphates dNTPs, 5 mM dithiothreitol, 27.5 μl of template, and molecular-grade water to a volume of 50 μl. The concentrations of primers were as follows: 0.2 μM for herpesvirus consensus primers and enterovirus primers and 0.15 μM for flaviviruses primers. The same PCR cycling conditions were used for the amplification of DNA and RNA viruses. RT-PCR started with a reverse transcription step at 50°C for 30 min, followed by denaturation at 94°C for 5 min and then 40 cycles each consisting of 30 s denaturation at 94°C, 1 min annealing at 59°C with a touchdown of 0.1°C per cycle, and 1 min extension at 68°C. A final extension of 7 min at 68°C was done. PCR was performed in a GeneAmp System 9700 thermocycler. Typing of enteroviruses was done as described by Caro et al. (6), except that a single-step RT-PCR was performed with the cycling conditions described above with an extension time of 90 s.
PCR product quantitation.Amplicons were quantified with an Agilent 2100 bioanalyzer and a DNA 1000 LabProbe array (Agilent Technologies, Massy, France), according to the manufacturer's instructions. Quantitation was performed with 1 μl of amplification products.
Labeling and cleavage of PCR products.Forty microliters of each of the three PCR products was mixed and first biotin labeled in a mixture containing 2.5 μl of RNase-free and DNase-free water (Sigma, St. Louis, Mo.) and 75 μl of 0.1 M meta-biotinphenylmethyldiazomethyl (bioMerieux) at 95°C for 25 min in a dry bath (4). DNA fragmentation was then performed by incubation of the reaction mixture with 36 mM HCl at 95°C for 5 min in a final volume of 250 μl. Fragmented labeled DNA was purified with a QIAquick 8 PCR purification kit, according to the manufacturer's protocol (QIAGEN), except for the PB buffer, which was replaced by PN buffer provided by the same company.
DNA probe array design.Sequence alignments of the target gene used for the detection of each virus family were constructed with the CLUSTALW program (version 1.4) (35) and included all available sequences of the relevant virus strains. This allowed the selection of probes necessary for the identification of each virus species in the case of herpesviruses and flaviviruses and the genus in the case of enteroviruses. This selection was performed in four steps. First, the amplified portions of targeted viruses sequences were chopped into probes of identical lengths that overlapped by 1 base. Then, probes that were not shared by a high enough proportion of strains from targeted viruses (typically 90%) were discarded. Probes which cross-hybridized with nonrelated viruses likely to be found in clinical samples were also eliminated. Finally, probes which did not cross-hybridize with related viruses within the same family were selected. In this way, species-specific sequences were obtained for herpesviruses and flaviviruses, and both genus-specific sequences and serotype-specific sequences (for typing purposes) were selected for enteroviruses. The repertoire of selected probes was then synthesized on the array by using a 2-L array tiling strategy, which allows the detection of a probe in a sample (36). This strategy consists of the synthesis of two probes of length (20-mers), one perfectly complementary to the original probe, with the other containing a mismatch at a specified position (typically, position 12 for 20-mers) (Fig. 1). The instability of probe-target mismatches relative to the stability of perfect matches is used to discriminate differences between nucleic acid targets through the identification of the probe which gives the highest fluorescence intensity (the match probe). If the signature sequence is present in the specimen, the fluorescence intensity of a majority of match probes will be higher than that of the corresponding mismatch probes. A total of 40,588 different probes have been synthesized on the final array of 0.8 by 0.8 cm with cells of 20 by 20 μm. Table 1 shows the number of probes representing the viruses validated in this study (herpesviruses, flaviviruses, and enteroviruses) and other viruses that have not yet been validated.
2-L tiling strategy. To determine if the reference sequence of a virus is present in the amplification product, two probes are used for each nucleotide of the sequence. These probes, usually 20-mer oligonucleotides, have the same sequence at all except one position, where one probe has the expected base (match probe) and the other one has one of the three other bases (mismatch probe). If the sequence is present, the fluorescence intensity of a majority of match probes will be higher that that of the corresponding mismatch probes.
DNA probe array hybridization and analysis.Labeled and cleaved amplicons were denatured at 95°C for 10 min in a 500-μl final volume containing 10× SSPE (1× SSPE is 0.18 M NaCl, 10 mM NaH2PO4, and 1 mM EDTA [pH 7.7]), 0.1% Triton X-100, 0.06% antifoam, and 0.09% sodium azide. Hybridization was done at 45°C for 45 min on an Affymetrix GeneChip Fluidics Station 400. After the probe array was washed with 6× SSPE, 0.005% Triton X-100, 0.001% antifoam, and 0.03% sodium azide and with 4× SSPE, 0.005% Triton X-100, 0.001% antifoam, and 0.03% sodium azide, the probe array was stained with 500 μl buffer containing 0.01 mg streptavidin-RPE (Dako, Glostrup, Denmark), 0.5 mg bovine serum albumin, and Tris-Cl (pH 7.2) at 35°C for 25 min. After the array was washed, the fluorescent signal emitted by the target bound to the probes was detected by a GeneArray scanner at a wavelength of 570 nm and with a pixel resolution of 3 μm. The highest signals came from the probes that best matched the target viral sequence. Probe array fluorescence intensities, nucleotide base call, sequence determinations, and reports were generated by functions available on the GeneChip 3.2 software. The ratio between the median intensity obtained for the sequence tiled and the median background intensity (It/b) was considered a criterion for identification. The background intensity was calculated as the mean for 16 probes in representative regions of the array. The percent base right score was determined by the percent homology between the experimentally derived sequence and the reference sequence tiled on the array. This criterion was used in addition to It/b to differentiate between the two closely related herpesviruses HSV-1 and HSV-2.
RT-PCR.PCR products of the expected size were generated for each target (535 bp for HSV-1 and HSV-2, 600 bp for CMV, 500 bp for enteroviruses, and 250 bp for flaviviruses) (Fig. 2). DNA and RNA viruses were amplified by using the same amplification kit and the same cycling conditions to simplify the diagnostic procedure. No deleterious effect of the reverse transcription step was observed on herpesvirus amplification (data not shown).
Amplification of HSV-1 (lane 2), HSV-2 (lane 3), CMV (lane 4), West Nile virus (lane 5), and poliovirus 1 (lane 6). Lane 1, molecular weight markers. The PCR products were separated by using an Agilent 2100 bioanalyzer.
DNA probe array hybridization and analysis.Specific identification of each virus was achieved on the array by using several regions within the sequence amplified for herpesviruses and flaviviruses. The number of regions per amplicon varied between the viruses (Table 1). A single signature sequence was used for enterovirus genus identification. Serotyping could be achieved by using VP1-specific probes, which were also present on the array.
The threshold for identification was determined to be an It/b of 1. This was done by analyzing data from arrays hybridized with negative cerebrospinal fluid (CSF) from patients with cranial trauma (three arrays) and with water (two arrays). The mean It/b of all herpesviruses, enteroviruses, and flaviviruses probes was calculated from the five arrays to be 0.33 (0.20 to 0.80). The threshold was given as the mean ratio added to three times the standard deviation (0.06), which gave a value of 0.5. It was, however, reasonable to use a value of 1 and not below, as it means that the median intensity of the sequence tiled is at least equal to and not below the background intensity. When a particular virus was hybridized on the array, the It/bs obtained for closely related viruses were similar to those obtained for nonspecific hybridization (Table 2). This shows that the sequences represented on the array are virus specific. It should be noted that an additional criterion, the percent base right score, was used to differentiate between HSV-1 and HSV-2 (see below).
Moreover, when a single DNA probe array was hybridized with equal amounts of amplicons obtained from the three targets HSV-1, human poliovirus type 1 (PV-1), and WN, each target was correctly identified with an It/b's of 1.90 for HSV-1, 4.57 for WN, and 2.03 for PV-1 (Table 3). This suggests that the hybridization of each type of amplicon is an independent event and that several nonrelated sequences can be correctly identified on the same probe array.
Herpesviruses.HSV-1, HSV-2, and CMV were all amplified with the herpesvirus consensus primer pair and were distinguished by the probes on the probe array. Culture supernatants of HSV-1 and HSV-2 and of CMV patient samples which were characterized by real-time PCR (38) were tested. All samples of the three viruses were correctly identified (Table 4). HSV-1 and HSV-2, being very closely related, were subjected to the two criteria ratio and percent base right score. Both viruses were first identified as the herpes simplex type by using the It/b threshold of 1, and then the two viruses were differentiated by using the percent base right score. The threshold for the score was calculated from the 16 HSV-1 and HSV-2 samples from Table 4 as the mean percent base right score (72.98%) obtained with heterologous probes for the samples added to three times the standard deviation (4.3%), which gave a value of 85.88%. A score above the threshold of 86% allows the identification of the herpes simplex virus present. A score below the threshold means that a herpes simplex type is present but that it cannot be further identified. Table 4 shows that 15 of 16 samples can thus be correctly identified by using this threshold, suggesting that 93.75% of HSV-1 and HSV-2 samples can be correctly differentiated.
The analytical sensitivity of this method was evaluated for HSV-1 to be 500 genome equivalents (Geq)/ml of CSF spiked with virus (Table 5). This was done by using virus material from various sources (virus culture quantified by electron microscopy and virus culture quantified by the TaqMan assay). Similar results were obtained for HSV-2 (data not shown).
The dynamic range of detection was tested by hybridizing the probe array with nucleic acids extracted from CSF samples containing 108, 106, and 103 genomes ml−1 of HSV-2 (Table 6). These three concentrations of virus were correctly identified by the DNA microarray. This shows that large amounts of material on the array do not affect the base right scores and that no major saturation effects hinder detection.
Enteroviruses.Fifty-six of 61 nonpoliovirus serotypes and 3 poliovirus serotypes of enteroviruses were successfully identified as enteroviruses by the enterovirus genus-specific signature sequence of 22 nucleotides present on the array (Table 7). Five enteroviruses (CV-A1, CV-A5, CV-A14, CV-A19, and CVA-22) were not tested due to the nonavailability of the viruses. No cross-reaction with rhinoviruses RH16 and RH18 was observed during either amplification or hybridization, showing that the primers are specific to the enterovirus genus but still amplify human parechovirus enterovirus type 1 (HPeV-1) and HPeV-2.
The analytical sensitivity was determined with the Quality Control for Molecular Diagnostics (QCMD) Enterovirus Proficiency Panel 2003 (Table 8), since no sample quantified in genome equivalents/ml was available. The sensitivity limit is 0.3 50% tissue culture infectious doses (TCID50s) ml−1 for coxsackievirus A9. The panel also confirmed that rhinovirus RH16 was indeed not detected by our protocol. Parechovirus 1 was not detected from this panel. However, it is interesting that parechoviruses 1 and 2 (previously echoviruses 22 and 23, respectively) were successfully amplified and detected by the probe array when they were tested at high titers (Table 7), while no sequence of these two viruses was included in the probe array design.
Flaviviruses.Probes specific for a large number of flaviviruses were represented on the probe array. WN, DEN-1, DEN-2, and DEN-4, which may be considered representatives of mosquito-borne flaviviruses, and Langat virus, which is a tick-borne virus, were chosen for this initial validation. RNA extracted from these five viruses was correctly amplified by the flavivirus group primers, and the viruses were typed to the species level by the probes selected for these flaviviruses. No cross-hybridization between the different viruses was observed (Table 9).
The sensitivity of West Nile virus detection was 200 TCID50s ml−1 (Table 5). This method of combining amplification with flavivirus group primers and hybridization on a microarray is more sensitive than that involving the use of the group primers only (105 TCID50s ml−1) and is as sensitive as the seminested method with an internal primer, both reported by Scaramozzino et al. (31). While hybridization of this sample on the array gave a base right score of 100% and an It/b of 4.5, detection by the Agilent array was negative.
All amplicons were quantified by using Agilent probe arrays prior to hybridization on the DNA probe array. It is interesting that detection by the DNA probe array was 10-fold more sensitive than that by the Agilent array, as illustrated in Table 5 for HSV-1 and WN.
This paper describes the specific detection of viruses from three major families that cause CNS diseases, human herpesviruses (HSV-1, HSV-2, and CMV), human enteroviruses (59 of 64 existing serotypes), and five flaviviruses, by using a consensus approach. The detection of these three families from a single sample has not yet been reported, with most papers describing the multiplex detection of herpesviruses and enteroviruses. Such simultaneous investigation of several potential agents is very appropriate to CNS diseases, as very often, small volumes of CSF are available for molecular biology diagnosis and the clinical symptoms provoked by these viruses are not clear-cut. Flaviviruses are pathogens of the CNS with a growing importance in both industrialized and developing countries and should be considered in the panel of viruses to be searched for. Despite extensive testing, the majority of encephalitis cases remain unexplained (14).
The importance of high-density microarrays in microbiology has been demonstrated in Mycobacterium species identification and antibiotic resistance determination (36), in multilocus typing of Staphylococcus aureus (39), and genotyping of human immunodeficiency virus (15, 20). Oligonucleotide probes, synthesized on the microarray, have been designed to determine every single nucleotide of target sequences in the virus present in a sample being tested. The assay established the percent homology between the microarray signature sequences that are specific to a virus or a subtype and the corresponding sequences of the virus being analyzed. For reliable detection, several regions of the target gene were used for the identification of each virus except enteroviruses, where only one region of 22 nucleotides was found to be a consensus sequence among all strains of all serotypes for which sequences are available. Maximum sensitivity was provided by the use of several overlapping probes for each target sequence. Specificity has also been optimized through the careful selection of probe sequences by excluding sequences that could cross-hybridize with nucleic acids generated by closely related viruses (exclusion perimeter). The assay has allowed the use of consensus primers, which reduces or eliminates the need for multiplexing for the specific identification of each species. In this paper, 3 herpesviruses, 59 serotypes of enteroviruses, and 5 flaviviruses have been detected by using consensus primers.
The concept of the microarray-based reagent allows flexibility in the detection of more viruses than those mentioned here. A single probe array can be synthesized with a large number of signature sequences from many viruses of interest. For instance, other pathogens that infect the CNS, especially in immunocompromised patients (polyomaviruses [JC and BK], measles, EBV, mumps virus, and Toxoplasma gondii), can be amplified in different combinations, depending on the clinical history of the patient, and can be hybridized on the same microarray. Furthermore, the multiplex approach can also be used to reduce the consumption of precious CSF. Such flexibility would have considerable impact on the cost of such a diagnostic tool.
We suggest that this tool be used in the following manner: nucleic acid is extracted from a sample of 200 μl of CSF and used for three amplification tubes. The amplicons are pooled at the step of purification of labeled DNA. Finally, the amplicons resulting from the three tubes are hybridized on a single probe array so that the clinician can know whether the patient is infected with one of these viruses. Mixed infections are rare but can be detected by this method, where hybridization of more than one type of amplicon is possible. Amplicons resulting from WN, PV-1, and HSV-1 have been successfully identified after their simultaneous hybridization on a single array in this study. The availability of an efficient system for the extraction of both RNA and DNA would further reduce the volume of CSF required.
The analytical sensitivity of this method for HSV-1, HSV-2, and CMV detection is 500 Geq ml−1. This respects the limit suggested by Linde et al. (23) of 10 to 20 genomes μl−1 for HSV-1 detection in CSF. The sensitivities for enterovirus and flavivirus detection were 0.3 TCID50s ml−1 and 200 TCID50s ml−1, respectively. Detection by this assay was, as expected, more sensitive than agarose gel detection and also than Agilent array detection. Samples that were probably not quantified by the Agilent array because they had a DNA content lower than the limit of 0.5 ng μl−1 were successfully identified by the assay. This can be explained by the larger amounts of amplicons analyzed by the DNA microarray than by the Agilent array.
The sequence selected for the DNA array as well as the threshold for It/b determined from negative controls allowed the specific detection of HSV-1, HSV-2, CMV, 59 human enteroviruses, and 5 flaviviruses. No cross-hybridization between viruses of the same family was observed. The two closely related herpesviruses HSV-1 and HSV-2 were identified as herpes simplex type by using the ratio threshold and were then differentiated by using the additional criterion of percent base right score. The threshold for this score was determined by using the percent base right scores for 16 samples of HSV-1 and HSV-2. Primers specific to enteroviruses did not allow the amplification of two strains of rhinoviruses but amplified, probably less efficiently than enteroviruses, parechoviruses 1 and 2. All serotypes of enteroviruses were successfully detected by using a genus-specific sequence. The array also allows the typing of these serotypes by using the VP1 gene. As an example, coxsackieviruses B6 and A17, PV-1, and PV-3 were typed and were correctly identified (data not shown). The typing of four serotypes was given as an example in this paper. The amplicons resulting from the identification primers (5′ NCR) and typing primers (2A and VP1) can be pooled and hybridized on the same probe array. Such an approach would confirm the presence of an enterovirus in the case of the emergence of a new serotype.
A few viruses from the Flaviviridae family that may be considered representatives of mosquito-borne and tick-borne viruses were tested and successfully typed on the array. Any cross-hybridization was well below the threshold determined. The use of the highly conserved NS5 region was very appropriate for the selection of consensus primers but also allowed distinction of the different flaviviruses. Three serotypes of dengue virus were tested and could be differentiated by using this region of the flavivirus genome. The use of genus-specific consensus primers and the presence of specific probes on the array would allow the detection of any of the flaviviruses in the case of obscure nonspecific symptoms. Also, this assay can be used for the diagnosis of hemorrhagic fevers. The sensitivity was evaluated with West Nile virus only due to the limited availability of virus samples. However, the lack of cross-reaction between the probes specific for each flavivirus shows that the microarray is very promising for the detection of members of this virus family, which contains many emerging and reemerging members.
The data in this paper show that in the case of herpesviruses and flaviviruses, virus-specific sequences from highly conserved regions of the genome can be obtained. This approach would allow the detection of variants, as the primers would always cover a highly conserved region. The analytical sensitivity of the assay has been evaluated by using selected virus species which are major concerns for public health. Specimens collected in various geographical areas from patients in different clinical settings must now be tested to evaluate the clinical sensitivity of the assay and to confirm its pertinence as a clinical diagnostic tool. We believe that this assay will be useful in explaining the vast numbers of cases of encephalitis with unknown etiologies. Moreover, with the emergence of new pathogens, the determination of causal agents of CNS diseases is becoming increasingly important. This DNA probe array technique, which provides exhaustive typing capabilities for CNS viruses, is especially designed for reference centers, which will be able to use it to confirm infections and to further characterize virus isolates. In that respect, it complements rather than replaces easy-to-use, low-cost reagents that are essential for field detection of these viruses, especially in less developed countries.
This work benefited from a grant from the European Community (Marie Curie Industry Fellowship).
We thank François Fournel and Mylène Ribes for their technical assistance, Fanny Poyet, and Hélène Savelli.
Returned for modification 1 February 2005.
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Launch event will be LEGENDARY, are Tesla events better than Apple's ?
Discussion in 'Model 3' started by Lex, Mar 23, 2016.
... so much new tech jammed into such a short delivery ! Such a dramatic contrast to the Apple event this week that was a whole lot of very nice Earth-positive and privacy stuff which is 100% great, but only contained a few new tech related announcements.
Other than the promised prototype rides for select few journalists, what else might be in store ?
I wonder if they will have some produced "bits" for this one, to stretch it out a bit... it will be a front-page-news event around the world.
Who said the rides were for a "few select journalists"?
No contest really. Apple does one thing right (and only one thing) and that's marketing. Their hardware is over-priced, and lacks the most basic of features from their competitors, but they know how to sell them.
Apple launch events start on time, and you can buy the actual product almost immediately afterwards, and it will have the features they talk about, and they'll work.
Tesla launch events start late, very late, and reveal a product that you can't have in your hands/garage for a long time, and often doesn't even do what they promise it will.
Green1 is very much correct about this. However, the first test drive event for the Model S was a lot of fun.
The Model X launch event was disaster ... 2 hours late and weak presentation.
I think Tesla will have learned from the Model X reveal. My money says they're saving lots of surprises for 3/31 and many more for later.
Not just the reveal, but the product itself.
Sure, once it came to market, it was to rave reviews, but man....they took an absolute beating getting to the point where it was finally being delivered.
I think the Model 3 will be a massive departure from the Model X in many ways.
I wouldn't expect too much. The biggest surprise has already been spoiled for a very long time -- the price. It won't be possible to cram much else in at that price point. Maybe they will give us some nifty battery factory details.
Apple wins this hands down. I am absolutely blown away that a multi-million dollar corporation can have product releases that have less organization and tech than a high school science fair. I love Tesla, but they desperately need a new events coordinator.
LOL almost read like you were scolding Tesla lol.. Well they will get better over time and hopefully this is one of those times!
I'm hoping Tesla does learn from the last event. There are often comparisons to Apple. And there is that little competition they have (who got whose employees this week?) . All I know is I'll be pretty excited for this reveal, just like the last one.
Apple presentation might be better, but they need to use big words and colourful videos because their products are same rehashed marginally upgraded stuff. They use their presentation to compensate for lack of innovation. Tesla knocks every one out in the area of innovation. Tesla's products are its best presentation. They sell their products on word of mouth. They spend money on R&D instead of marketing.
I just hope they start on time and get to the point. It will at least show they've learned from their prior mistakes and might be a sign of things to come for Model 3.
I was going to let this thread disappear, but now that it's back, having watched the iPhone SE/iPad Pro event this week, while also choosing to rewatch the Model X and "D" reveal events, there's really no contest. Apple's presentations are a focus-grouped, microanalyzed art form. Tesla just puts Elon and a car on stage and let's him say words.
Sure, Tesla maybe makes more impressive products these days, and reveals more significant upgrades, but to compare the two or even say Tesla presentations are better, is ridiculous. No one on that Apple stage has ever said "um" once. Elon, for all his strengths, leaves a lot to be desired in his presentations.
The goals and the style are completely different. Tesla does unveilings of the prototype and the production model well before for they finalized them and in general doesn't really put a lot of polish into it.
Apple does unveilings only when the product is completely finalized and ready to sell, and their presentations are meticulously designed/polished (in order to maximize impact even for what may in general be less significant features).
Who cares if it's better or worse than Apple's events? You all know you want to be there. And who cares about Elon's presenting style? It's ... Elon.
He is heading two most important endeavors of mankind. He doesn't have time for anything else. He is inspirational and he likes to be on point instead of smothering cheese all over his words. His words are important, his delivery might not be the best but it doesn't matter to me.
are Tesla events better than Apple's?
IMO that's irrelevant. It's all about the content (what's more important, new iphone features or a compelling affordable EV?).
You are going to be really surprised (it will be compelling)!
If I want amazing delivery style, I tune to late night stand up comedy shows. For real stuff that would make an impact on my lifestyle, Elon's presentations are great.


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STB hosts SingapoReimagine @Expo to discuss future of travel & tourism
The Singapore Tourism Board (STB) is partnering with leading forum organiser Intelligence Squared to stage a forum at Expo 2020 Dubai. Presented under the STB's SingapoReimagine Global Conversations series, it aims to spur thought-leadership conversations on the future of travel and tourism. The SingapoReimagine Global Conversations which was launched in Singapore last year, encourages global industry leaders to exchange ideas and explore collaboration opportunities to reshape travel. It also highlights changing consumer behaviours as well as the latest trends and observations on travel and tourism.
The Dubai edition is titled Reimagined Cities: Can cities leverage urban design and technology to attract the next generation traveller?. Taking place from 3.30pm to 5.00pm on Saturday, January 22, 2022 in the Terra Auditorium at Expo 2020 Dubai, the expert panel will dive into the shifts in global travel trends and what the next generation would want from a destination. Placing the spotlight on urban design and technology, these conversations will challenge traditional ways of destination marketing and discuss exciting trends within the travel and tourism industry.
Starring a line-up of industry experts, the discussion will include insightful inputs from Dr. Barkathunnisha, Co-Founder, World Women Tourism and Founder of Elevated Consultancy & Training; Michael Magill, Managing Director, RSP Architects Panners & Engineers (Pte) Ltd Middle East; and Xavier Anglada, Managing Director at Accenture – Innovation, Digital Transformation, Energy Transition.
The session will be moderated by Sarah Khan, recently appointed Editor-in-Chief of Condé Nast Traveller Middle East. Representing a new generation of modern travellers, Sarah brings a contemporary and global perspective to the travel industry and will be leading discussions on future of travel with a special focus on sustainability.
The panel will cover a variety of topics, including how the tourism sector can adapt to changing consumer preferences by employing artificial intelligence and technological innovations. For example, touchless technology can be used to facilitate a seamless visitor journey, while augmented reality can provide immersive navigation services and bridge language barriers.
The panel will also share how cities are creatively deploying architecture to design tourist attractions of the future – from championing sustainable, green buildings to incorporating bold and contemporary urban design features. It will also explore how sustainability is influencing where people choose to travel to, where they stay and how destinations are transforming in tandem with these needs.
Beverly Au Yong, the Singapore Tourism Board's Area Director for the Middle East, said: "We are pleased to partner with Intelligence Squared for this edition of the SingapoReimagine Global Conversations at Expo 2020 Dubai. We look forward to hearing more from the panel and tap into their expertise in sustainable travel, technological innovations and urban design. We hope these conversations can provide useful insights to help businesses shape their recovery strategies, so that the travel industry can bounce back stronger than ever."
Matt McAllester, CEO of Intelligence Squared, said: "We are excited to be launching this series and supporting Singapore Tourism's mission to become a thought leader in the field of sustainable travel and green tourism".
singaporeimagine at Expo	2022-01-11
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Review – The Station
Posted by Adam O'Connell | Feb 19, 2018 | Reviews | 0 |
The Kickstater campaign can be used for many things. From gathering money to help the sick pay their medicals, to helping people fund their education, to helping the creative and imaginative creators of the world fund their products in art, television, film and video games. Over the years the crowd funding service has gone on to fund many great games which include FTL, Superhot and Shovel Knight which helped gave this way of creating games legitimacy, leading more and more developers to give it a try. The latest example to make its way to me is The Station, a Kickstarter funded First Person/Sci-Fi/Murder Mystery game created by a team of veteran developers working out of Vancouver, Canada. Finishing its campaign with over 5000 Canadian Dollars over its goal with one insanely devoted backer donating over CA$2500, rewarding that person with creative sessions with the team as well as a cooked breakfast from them, a deluxe VR copy, access to testing, a copy of the script, a pancake party, getting their name in the game and a credit as Associate Producer, Backer and concept artist AMONG OTHER THINGS! This just went to show the kid of potential this title is capable of. The Station as a whole offers certifiably chilling experience with stimulating puzzles and a minimalist but harrowing story that slowly reveal itself as you progress, all in the space of a pretty short playtime.
In The Station you play as a Recon Specialist sent to uncover a mystery of three Scientists who have gone incommunicado on a space station while coming into contact with other sentient life. However, as they did they soon realise that this race of Aliens are in a state of civil war which is around the time that the team go silent. You are tasked in finding out what has happened to this research team, if they are dead or alive and what exactly happened on this space station. It's the mystery that really drives this game and the fact that the story gains more and more momentum as you discover more about the team's findings, their personal relationships and unveiling the events that lead to their disappearance that really urges you to keep playing. And the conclusion that it ultimately leads up to acts as somewhat of an ambiguous powder keg that could be interpreted in a number of different ways.
The Space Station Espial's interior and exterior is equal parts Enterprise, Sevastopol and Thunderbird 5 but still holds an ominous presence as you approach it at the beginning of the game with its impressive sci fi style and massive size. And for the most part the same can be said for the inside of it too, which features some impressive visuals brimming with futuristic technology that would satisfy any Trekkie or Alien fan and intense atmosphere which comes down to the path of destruction that you explore, the eerie lighting and the strange feeling that you are not alone in this space station. This is only amplified by finding fire suppressors on standby in the rooms and corridors and addition of a run button that gives you a feeling of unease. However, due to the short game span the inside of The Espial just doesn't feel as big as the outside, as a whole it features a pretty small map. Despite this the surroundings of The Station are still pretty easy on the eyes and there are some places that really make want to in VR, particularly the glass floors that make you feel like you're suspending in the endless void of space. Sadly, as I was making my way through unsettling environment I experienced a few frame rate drops which took me out of the experience, but this remedied by the sounds of malfunctioning hardware, distant explosions and calls for help, and the droning of the synthesiser heavy soundtrack really helps to pile on the ambience and the dark tone of this title.
With its gameplay, The Station has you discovering what happened to the three researchers through uncovering clues and audio logs that detail their findings and exactly what happened when they came across this alien species, which as I said earlier makes the game pretty exhilarating. But another thing that there is plenty of is puzzles, as you wander round the corridors and in the to the different rooms you come across certain puzzles (kinda like the Crystal Maze!) but as the developers have said they wanted the whole space station to feel like one big puzzle, and much to their credit they have achieved this. The puzzles you are faced stimulate the mind and requite some thought however there doesn't seem to be a difficulty curve to them. The Station also goes for a realistic approach when it comes to this mechanics which works in some places and falls a little short in others, the Augmented Reality Menu takes a futuristic approach to an in-game menu screen and making your choices from it by your playable characters sight is a good touch and picking up items feels clunky and unnatural.
Summary The Station is just one example of how the gaming community helps people create. I mean sure, this game is not perfect, and it can be clocked in a little under an hour on your first try, but The Station is an experience full of atmosphere, dramatic storytelling and thought-provoking gameplay all in an attractive setting in the serenity of space. And though it is over before it has really begun, this is a game take takes two different niches and puts them together almost effortlessly.
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access_time May 19, 2014 at 12:59 PM	in PC/Mac by Tim St. Croix
Review| The Last Tinker: City of Colors
Upon booting up the Last Tinker the first thing that went through my head was WOW. This game looks and feels different from so many others that I have played, its visuals are striking, and its gameplay is very much like an old school platformer of old. To say I was impressed is a slight understatement.
I was worried however, if it would suffer from many of the same tropes that platformers have suffered from, I am pretty pleased to say it does not. Frequent check points abound, plenty of health pickups,, and the only slight difficulty is in learning timing, you have to watch sometimes for a few seconds to learn a rhythm before you run across.
I think however this game plays better if you use the controller enabled feature. I myself found no issues using the traditional keyboard and mouse setup, but it just feels not the same, that's neither good nor bad it's just a fact.
The plot is relatively simple. You have to help bring color back to a world that is slowly having it leached out by an evil force called the bleakness. This is a force that you accidently helped set in motion and as a result you are the only one who can do anything about it. You are a legendary figure called a Tinker, and only you can right what has gone wrong.
Along the way you gain power ups unlocked via new clothing items, you unlock new combat moves through vending machines, and as usual more of the plot is revealed and your ultimate destiny revealed. The combat is quick but easy to learn, and I like how each time you defeat a mini-boss, suddenly what was grey and leeched of all color, blooms almost like a flower right in front of your eyes. With plenty of in-jokes like Rob Boss, the greatest painter in colortown, who just so happens to look like another afro'd painter of legend, this game is funny and fun in all the best ways.
I really like this game, it hearkens back to platformers of old and yet still feels surprisingly fresh, with stunning graphics, great platforming, and interesting story it is a game that gamers of any age should feel comfortable playing, by themselves or for the older gamers, with their kids. There are honestly too few games that truly feel like they were made for everyone and not a core group. This game is a welcome change to the lineup. The Last Tinker is currently available on Steam and get a very high rating of 4.5/5.


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The Cactus Revisited
3rd Bass :: The Cactus Revisited
Back to the LabEPReview
It pains me to tell you "The Cactus Revisited" is a pointless EP. That could be the entire review, but I'll go ahead and explain why anyway. One year earlier 3rd Bass released "The Cactus Album," a certified hip-hop classic from the duo of MC Serch and Pete Nice. Production by Sam Sever plus a few Prince Paul joints, and one by The Bomb Squad was the glue that held them together. We'd learn in subsequent years that the differences between the two artistically and personally would dissolve that bond. As a result of how short their career together would be, any remnant of that time together feels worth exploring, but "The Cactus Revisited" is a hint in hindsight that their union would ultimately fall apart.
"The Gas Face (Remix)" is as good of a place as any to start. MC Serch has spoken at length about his bond with the late Daniel Dumile, who was better known at the time as Zev Love X from K.M.D., so his absence from this "lyrical remix" feels like a slap in the face. As much as I appreciate Serch offering a moment of silence for Yusef Hawkins, I can't help but think some form of disagreement between Nice and Serch resulted in Dumile not being included. If they could record new verses for the revisit so could Dumile, so the new version is interesting but SUBPAR compared to the original. The same can be said for Dave Dorrell's revision of "The Cactus."
You have to go three deep into "The Cactus Revisited" before anything of substance happens, and that's the Sam Sever remix to "Wordz of Wizdom." I don't favor it over the original, but as a so-called "club mix" I can actually picture it getting people onto the dancefloor. It has the same kind of energy as tracks Sever would produce for his own group Downtown Science with Bosco Money, and it honestly wouldn't have shocked me if Money had made a cameo on the song. I'm sure some clever mash-up artist could make that happen if they were motivated to do so.
Unfortunately no one would be because this album is ASS BACKWARD in so many ways. The version of "3 Strikes 5000" here is inferior to the one that ultimately wound up on "Derelicts of Dialect." The bloated CJ Mackintosh & Dave Dorrell reworking of "Brooklyn-Queens" removes all of the whimsical charm of Prince Paul's original version. The one lone shining star of this whole release is Marley Marl's revision of "Product of the Environment."
It's in every way the opposite of 3rd Bass' own "Steppin' to the A.M." remix that closes out this EP — a fun new take on an already good song as opposed to a poorly chosen one that makes matters worse. The more you listen to "The Cactus Revisited" the more apparent it becomes that 3rd Bass were already at loggerheads creatively about their direction, and as a result they resorted to trying to revise and re-release the material they had already done. It's a feeble effort that aside from historical footnotes is best left forgotten, and should NOT be considered as indicative of the talents of Pete Nice or MC Serch individually or collectively. This is a swing and a miss.
5Overall Score
Lyrics5
3rd BassColumbia RecordsDef JamThe Cactus Revisited
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Home DepartmentsSaturday Selects
A weekly Saturday recap to share with you our favorite links, discoveries, exhibitions, and more from the past seven days. This week: A look way back at — what else? — 1970s-era Italian design; a dip into the recent past in Milan; and a forecast of things to come at our OFFSITE event, debuting in just two weeks! Plus, the amazing risograph talents of Glasgow-based artist Gabriella Marcella, above.
Images of the swings Philippe Malouin created for Caesarstone have been all over the web in the weeks since Milan. But we had to go hunting for close-up pictures of the incredible planters the London designer debuted simultaneously in order to highlight the interesting material properties of the quartz rock. We love these colorful, textural examples, which demonstrate how Caesarstone can be used in inlay applications. Designboom has a full overview here.
An Instagram discovery this week was The Granite, a Portland, Oregon–based all-female collaborative who make ceramic wares in a perfect palette of black, white, pink, and electric blue. We love the styling of the vase above, but our favorite product might be this mismatched set of espresso cups, with one solid handle and one cutout. You might remember Italian designer Valentina Cameranesi from the amazing, Sottsass-inspired vases of hers we published last fall. Her latest is a series of angular, Plexiglas containers, commissioned by Yoox.com for its Made in Milano project, which asked nine Milan-based studios to create objects that could be fabricated entirely within the city's confines.
Speaking of Milanese designers, our obsession continues with the Italian designer and manufacturer Cleto Munari (whom, you'll recall, commissioned jewelry by architects like Arata Isozaki, Peter Shire, and Michele de Lucchi). De Lucchi is also responsible for this 1979 sterling silver and plastic pitcher for Munari, which we found this week on 1stdibs.
We got an email in our inboxes this week from Glasgow-based graphic artist Gabriella Marcella, introducing us to not one but two excellent new projects: The first is Risotto, a risograph print studio where you can upload your own design for reproduction, hire Marcella to create something for you, or purchase one of the pre-designed posters like those at the top of this post and above. The second project is Tappeti, a new series of artisan rugs made in Scotland and art-directed by Marcella. We'll be keeping an eye on this one! A nice interview on Design Milk with Maarten Baas, whose launch flew relatively under the radar in Milan last month. His Das Pop collection for Lasvit, shown here, marries the rough, textural fingerprints of his Clay collection with the smoothness of mouth-blown glass. Here, he talks about new work as well as passing the torch to a new generation of design students at Design Academy Eindhoven, where he teaches.
Ornate, historical environments were used to great effect in Milan this year (see Philippe Malouin's aforementioned swing set in palazzo) but sometimes the busyness of the interior actually detracts from the beauty of the works. Case in point: It was hard to discern what we loved in ECAL's exhibition of luxury goods made with Swiss craftsmen at the Museo Bagatti Valsecchi until we saw the pickup art. We especially love this brass and stone container by Stefano Panterotto.
Some epic Pinteresting this week led Monica to this 1945 Bruno Munari alarm clock, called L'Ora X, which had rotating half-discs instead of hands. It was produced in multiples in 1963 but we'd never seen it until now. As Monica said, "Someone needs to reissue — or remake — this STAT." This week we were introduced to Line, a cute new collection of furniture and lighting by New Zealand father-and-daughter team Douglas and Bec. Each piece is designed by Bec Dowie and then fabricated by her father, Douglas Snelling, with a team of local artisans. The new collection represents a move from a mostly wood aesthetic to a more expanded materials palette that includes blackened brass, glass, steel, and painted timber. This week we got a preview of something special that Indiana-based studio LUUR is producing for our Sight Unseen OFFSITE exhibition, which opens in two weeks. Made from Corian and neodymium magnets, the studio's North/South bracelets are a modern take on friendship jewelry, where you can buy several different halves and swap them out with others from your own collection or share them with friends. Speaking of OFFSITE, this year our production team is led by Jonathan Irons of the Brooklyn-based studio Cain Cain. While poking around his website, we found that the studio does not only event production but furniture as well, including this beautiful brass and walnut triangle mirror.
Artsy clued us in last year on the great work Brooklyn-based artist Kate Steciw is doing. Her newest exhibition is at Retrospective, the Hudson, New York gallery owned by Zach Feuer, and she's broadened her signature series of layered stock images, manipulated in Photoshop, with an increasingly complex system of frames. (Artsy also has a great primer on the artist here.)
LA artist Math Bass gets her first museum solo exhibition this week at MoMA PS1; "Off the Clock" will show most of the artist's recent work as well as a new video commissioned specially for this exhibition. What is the significance of the recurring cigarette in Bass's work? We want to know. "Bass's paintings deploy a personal lexicon that centers on possible actions or transitional spaces: cigarettes emit plumes of smoke; alligators emerge with mouths wide open; letters and punctuation marks twist and overlap; and archways, staircases, and zigzags suggest movement."
Tags: Art, graphic art, Italian design, lighting, vintage
A weekly Saturday recap to share with you our favorite links, discoveries, exhibitions, and more from the past seven days. This week: Three particularly timely design objects that launched in Milan, one peculiar woven-glass lamp that didn't, and a show by the design world's most beloved artist, Carol Bove, pictured above.
Week of April 6, 2015
A weekly Saturday recap to share with you our favorite links, discoveries, exhibitions, and more from the past seven days. This week brought a bounty of our favorite things: oxidized metal, iridescent leather, lenticulars, mini-sized fruit and furniture, and a special appearance by a Bucky Fuller–inspired Jell-O mold.
A weekly Saturday recap to share with you our favorite links, discoveries, exhibitions, and more from the past seven days. This week: While the international design community (including Jill and Monica) convened in Milan for the Salone del Mobile, we put Ryland on the case to round up a few of his favorite finds, ranging from the dark, moody objects of Blackman Cruz at Wright (pictured above) to a Technicolor Instagram account involving pool noodles and sneakers.


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MOVIOCRITY
Putting the "Cult" Back in "Culture."
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The Films of 2017… and the Women That Made Them Work
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The central role taken by women in some 2017's best films made for a refreshing change that we hope is just the beginning. Continue reading The Films of 2017… and the Women That Made Them Work
March 6, 2017 March 6, 2017 Scott W. Davis
FINALLY! After a long delay, I finally present my picks for the twenty best films of 2016. Continue reading The Best Films of 2016
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Everybody Has Something to Say About DEADPOOL
February 27, 2016 February 27, 2016 Scott W. Davis
Reaction Shots takes a look at the by now infamous DEADPOOL. And then the guys from the Spoiler Room have something to say about it. Continue reading Everybody Has Something to Say About DEADPOOL
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FANTASTIC FOUR, or: It's Clobberin' Time
August 9, 2015 August 9, 2015 Scott W. Davis
At least it was short. Continue reading FANTASTIC FOUR, or: It's Clobberin' Time
I Live Tweeted STREET CORNER JUSTICE and I May Never Recover
July 11, 2015 July 15, 2015 Scott W. Davis
What happens when I live tweet a 1990s action film starring Marc Singer? Magic, that's what. Continue reading I Live Tweeted STREET CORNER JUSTICE and I May Never Recover
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THE WHOLE PICTURE: Come with Me If You Want to Live (Part 1 of 2)
July 6, 2015 July 6, 2015 Scott W. Davis
Part one of a two-part retrospective in which we look at all five TERMINATOR films, starting with James Cameron's original two-picture epic. Continue reading THE WHOLE PICTURE: Come with Me If You Want to Live (Part 1 of 2)
EpisodesLeave a comment
New MOVIOCRITY Episode Is Too Big For YouTube!!!
June 24, 2015 June 24, 2015 Scott W. Davis
Moviocrity looks at the MAD MAX ripoffs of yesteryear, shining a spotlight on the EMPIRE OF ASH series! Check out this new episode that is too big for YouTube! Continue reading New MOVIOCRITY Episode Is Too Big For YouTube!!!
JURASSIC WORLD, or: Put Down the Smartphone, We're Trying to Amaze You
JURASSIC WORLD is a film that acknowledges the fickle nature of the public and tries to overcome it anyway. Continue reading JURASSIC WORLD, or: Put Down the Smartphone, We're Trying to Amaze You
THE WHOLE PICTURE: A Walk in the Park
With JURASSIC WORLD hitting theaters, we look back at the first three films in the JURASSIC PARK franchise. Continue reading THE WHOLE PICTURE: A Walk in the Park
PodcastsLeave a comment
TOMORROWLAND and the Struggle Between Hope and Defeat
June 4, 2015 June 12, 2015 Scott W. Davis
Some films you want to write about and some films you want to talk about. TOMORROWLAND was definitely a film I wanted to talk about. Continue reading TOMORROWLAND and the Struggle Between Hope and Defeat


text:
Ereignisse

Politik und Weltgeschehen
 Nach dem Sieg der Römer über keltische Stämme im Rahmen der römisch-gallischen Kriege wird der Norden Italiens endgültig an das Römische Reich angeschlossen.
 190/189 v. Chr.: Schlacht bei Magnesia

Kultur
 Die Mittellatènezeit wird von der Spätlatènezeit abgelöst.

Geboren
 um 190 v. Chr.: Cornelia, Mutter der Gracchen († um 100 v. Chr.)
 um 190 v. Chr.: Hipparchos, griechischer Astronom († um 120 v. Chr.)
 um 190 v. Chr.: Seleukos von Seleukia, griechischer Astronom, der das heliozentrische Weltbild verteidigte

Gestorben
 Cao Can, chinesischer Politiker
 um 190 v. Chr.: Apollonios von Perge, griechischer Mathematiker (* um 262 v. Chr.)

redpajama_set_name: RedPajamaWikipedia


But this time, they've included a shot at The Rebel in their attempt to raise money.
Watch as I show you how Dogwood attacked us and also show you how small their grassroots support actually is.
After two weeks, they've managed just 200 supporters as compared to what we've done in our latest campaign at TheyCantStopUs.com.
Thanks to you, our true grassroots supporters, we've raised a lot since launching the crowdfunding campaign to create our free app on August 3, but we still need more help.
Unlike us, Dogwood have been hired by foreign donors including the Tides Foundation to attack Canadian jobs, while we're independent Canadian media who call it like we see it.
They're looking for funds to pay themselves but we're crowdfunding an app to end our reliance on YouTube due to threats of censorship.
We've only been around since 2015 and Dogwood has been around since 1999. They had a full 16 years of cashing US foundation cheques before we even came along, yet we're years ahead of them?
I guess all of that US foundation money just made Dogwood lazy and complacent.
Yee Haw! Ride 'em (urban) cowboy.
Spur 'em out! Make that fur fly! Keep jabbin' 'em with the truth!
Give 'em one for me…Ha ha ha ha ha.
The paradigm has shifted, the left WILL NOT prevail, because the truth will set us free – if only in hind sight.
My job is easy. All great posts again. You guys said what needs to be said.
My contribution…..Staple St. Suzuki and Gregor Robinson to a telephone pole….in the hot sun.
Great report , and of course the self important Dogwood hypocrites cannot even defend their BS with a reasonable lie. They assume everyone is a mindless progressive that does as they are told.
I would find a pile of dog sh*t to be more useful than dogwood.
The difference is that The Rebel is providing a product for the money it receives. I am purchasing that product. The Rebel provides jobs while producing that product, and purchases capital. In short, The Rebel contributes to the economy.
On the other hand, groups like the Dogpatch initiative do not.
I wonder how many Canadian Tax dollars Dogwood is getting from Trudeau?
Excellent report Christopher. Please continue to challenge the Soros puppet to a public debate.
Excellent Christopher, expose Tides, I mean Dogwood for the hypocritical bunch of globalist recruits they really are. They are right about one thing though, the Rebel is years ahead of them and for good reason.
Now let me see, who could that anonymous contributor who is matching Dogwood's pathetically small numbers be?


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