Salmonid alphavirus (SAV) is a widespread pathogen in European aquaculture of salmonid fish. Distinct viral subtypes have been suggested based on sequence comparisons and some of these have different geographical distributions. In Norway, only SAV subtype 3 have so far been identified. Little is known about viral mechanisms important for pathogenesis and transmission. Tools for detailed exploration of SAV genomes are therefore needed.
Infectious cDNA clones in which a genome of subtype 3 SAV is under the control of a CMV promoter were constructed. The clones were designed to express proteins that are putatively identical to those previously reported for the SAVH20/03 strain. A polyclonal antiserum was raised against a part of the E2 glycoprotein in order to detect expression of the subgenomic open reading frame (ORF) encoding structural viral proteins. Transfection of the cDNA clone revealed the expression of the E2 protein by IFAT, and in serial passages of the supernatant the presence of infectious recombinant virus was confirmed through RT-PCR, IFAT and the development of a cytopathic effect similar to that seen during infection with wild type SAV. Confirmation that the recovered virus originated from the infectious plasmid was done by sequence identification of an introduced genetic tag. The recombinant virus was infectious also when an additional ORF encoding an EGFP reporter gene under the control of a second subgenomic alphavirus promoter was added. Finally, we used the system to study the effect of selected point mutations on infectivity in Chinook salmon embryo cells. While introduced mutations in nsP2197, nsP3263 and nsP3323 severely reduced infectivity, a serine to proline mutation in E2206 appeared to enhance the virus titer production.
We have constructed infectious clones for SAV based on a subtype 3 genome. The clones may serve as a platform for further functional studies.
The genus Alphavirus (Family Togaviridae) consists of viruses with positive sense, single stranded, capped and polyadenylated RNA genomes . Of the 29 recognized species in the genus, 28 are pathogens of higher vertebrates in the terrestrial environment, and the transmission cycle of these viruses includes an arthropode vector. The only species of the genus that infects fish, Salmon pancreas disease virus, today more commonly designated Salmonid alphavirus (SAV), is genetically distinct and a pathogen of farmed salmonids in Europe [2–4]. The genome of SAV is 11.9 kb long and has a genomic structure homologous to terrestrial alphaviruses with two large open reading frames (ORFs) of 8 and 4 kb length that are flanked by three untranslated regions (UTRs). The first ORF encodes non-structural proteins 1-4 (nsP1-4) and the second one encodes the structural proteins capsid, E3, E2, 6K, TF and E1 [5, 6], of which capsid, E2 and E1 have been demonstrated to be expressed during replication [7, 8]. The nsPs, possibly together with host proteins, make up the replicase complex (RC) that replicates the viral genome and transcribes the second ORF [9, 10]. The structural proteins are translated from the subgenomic mRNA of the second ORF, that is controlled by a SAV RC specific promoter [2, 3, 10]. Following its translation in the cytoplasm, the capsid protein cleaves itself from the adjacent structural proteins. Studies from terrestrial alphaviruses have demonstrated that the cleaved capsid interacts with viral genomic RNA to form nucleocapsids . It has also been suggested to have additional non-structural functions as the capsid of several alphaviruses, including SAV, may localize to the nucleus during infection [8, 11–15]. The remaining structural proteins are translated into the membrane of the endoplasmatic reticulum, where they undergo glycosylation and proteolytic cleavage, before they are transported to the cell membrane. Budding of viral particles is induced by interactions between the capsid protein and the cytosolic parts of viral glycoproteins . The glycoproteins are functional in the recognition and binding to receptors on the cell surface (E2), and fusion of the viral membrane with the host cellular membrane (E1) . It is likely that SAV uses the same route of budding, entry and glycoprotein maturation, based on homology in sequence motifs between alphaviruses and the observed intracellular localization of the glycoproteins during infection [2, 3, 7].
The SAV replication cycle can be reproduced and modified using reverse genetics systems . In these systems the complete or partial viral genome is cloned as cDNA where a promoter for RNA transcription controls the expression. Such systems may be designed as replicons, where the viral structural ORF is replaced with a gene of interest (GOI). Alternatively, the structural ORF is intact and addition of a second alphaviral promoter controls expression of the GOI. Expression of the GOI will then follow the same kinetics as the viral structural proteins.
Sequence analyses of SAV strains have suggested that at least six genetically distinct subtypes of the virus, SAV1-6, have evolved . Some of the observed sequence differences also lead to variations in antigenic epitopes . Of these subtypes, SAV1, 2 and 3 are the best studied, and full-length genomes of strains belonging to these subtypes have been sequenced [5, 9, 18]. Strains grouping to subtype 1 have primarily been associated with disease in the marine production phase of Atlantic salmon (Salmo salar) in Ireland, those grouping to subtype 2 with disease in Rainbow trout (Oncorhynchus mykiss) in the freshwater phase in continental Europe and UK, and recently reported from marine Atlantic salmon in Scotland, and subtype 3 strains with disease in Atlantic salmon and Rainbow trout in the marine production phase in Norway [17–19]. Experimental infection trials have demonstrated that strains belonging to the different subtypes cause similar clinical signs and pathologies in the target species . With the exception of SAV2 infections in rainbow trout fingerlings , the mortality rates observed in laboratory experimental transmission of SAV are typically low, if any mortality at all [5, 20–22]. Higher mortalities have sometimes been reported from field outbreaks [23, 24]. Although these variations in mortality could be explained by differences in virulence between viral strains, the contribution from factors other than SAV, such as fish strain, environmental variations and other pathogens, could be equally important. Different transmission routes of SAV have been suggested to be of importance in aquaculture [17, 25–28], but transmission mechanisms remain poorly understood. In Norway, SAV appears to be genetically homogenous, suggesting one or few original common sources for all outbreaks . After an outbreak, SAV may remain in the fish population in the form of a possibly lifelong, persistent infection in some individuals [20, 29, 30]. Information about viral and host mechanisms that regulate the persistency is not available, but could be useful for proper management of infected fish populations.
Although viral factors that contribute to the different outcomes of a SAV infection remain largely unresolved, we previously hypothesised based on sequence analyses that a mutation in E2, the viral receptor protein, could be of importance for pathogenicity . In general, outbreaks of several alphaviral epidemics have been associated with such point-mutations in E2 and E1 [31, 32]. Recently, studies of the epidemic caused by the alphavirus Chikungunya virus (CHIKV), have demonstrated how reverse genetics systems can be powerful tools for understanding evolution, mechanisms of pathogenesis and genetic factors contributing to epidemics [32–34]. Reverse genetics systems addressing such topics should express viral proteins that are closely related to those of the naturally occurring virus. A reverse genetics system has been developed for an attenuated strain of SAV2 originating from rainbow trout . Here we report the construction of a reverse genetics system that enables the recovery of a recombinant SAV subtype 3 with putatively identical proteins to those previously reported from the SAVH20/03 strain originally isolated from diseased Atlantic salmon in Norway . The system can be particularly useful for studies of evolution and mechanisms of pathogenesis and transmission of SAV.
Proper tools for detection of structural proteins from SAV3 strains are essential for confirmation of a functional reverse genetics system. We therefore obtained an antiserum against the E2 protein. A partial E2 coding sequence of SAVH20/03 was chosen based on a nucleotide alignment with a known antigenic region in Sindbis virus (SINV) and Ross River virus (RRV) (Figure 1a). The fragment was successfully expressed as a His-tagged peptide in E. coli (results not shown) and used to immunize a rabbit. Specificity of the obtained antiserum was verified by IFAT on Chinook salmon embryo (CHSE) cell cultures infected with the wild type SAV3 strain SAVH20/03. A positive signal was localized to the cell membrane (Figure 1b) and to cytoplasmic structures believed to be ER and Golgi (Figure 1b, arrow) as visualized by confocal microscopy. This corresponds well with the previously reported localization of E2 during infections with SAV [7, 35], as well as with the localization of E2 of terrestrial alphaviruses . No staining could be observed in mock treated cells (Figure 1b).
Development of polyclonal antiserum against E2 and detection of SAVH20/03 by IFAT. (A) A region of E2 was identified based on alignment of SAV3 with antigenic regions of the SINV and RRV E2 proteins. The peptide corresponded to amino acids 184 to 236 of the putative SAV3 E2 protein was cloned and expressed in E. coli, and used for immunization of a rabbit. (B) IFAT using the obtained E2 antiserum on CHSE cells infected with SAVH20/03 demonstrated specific staining located to the plasma membrane and cytoplasmic structures, possibly ER and Golgi (arrow). No staining could be observed in mock treated cells.
We have recently reported the construction of the DNA replicon-construct pmSAV3, where a SAV3 RC expresses an EGFP ORF from an alphavirus promoter . In order to obtain a full-length infectious clone, we replaced the EGFP ORF with the structural ORF of SAV through an introduced XbaI site (Figure 2a). The XbaI site was kept in the final construct and functioned as a genetic tag that could be used to verify the origin of progeny virus from infectious clones. Sequencing of the obtained full-length cDNA clone, prSAV3, revealed amino acid sequence identity, but 10 nucleotide mutations, not leading to amino acid changes, were present compared to previously reported consensus sequences of SAVH20/03 (accession nos. AY604235 and DQ149204). Of these, four were located in the non-structural ORF, two constituted the genetic tag in the junction, two were located in E2 and two were located in the 3'UTR (Table 1). Selection of a proper E. coli strain (XL10 Gold, Stratagene) was crucial for the propagation of these plasmids since proliferation of E. coli strain Top10 (Invitrogen) was dramatically reduced after transformation with plasmids harbouring the full-length sequence.
Construction of a full-length infectious SAV3 cDNA and recovery of infectious virus. (A) A cDNA fragment containing the EGFP ORF and 3'UTR of a modified pmSAV3 plasmid was exchanged with the structural ORF and 3'UTR through an introduced XbaI site in the junction region. (B) IFAT on CHSE cells infected with supernatant from cells transfected with the prSAV3 plasmid. (C) Cytopathic effect in CHSE cells infected with supernatant containing recombinant SAV3 passage 3. (D) Identification of the introduced XbaI genetic tag by sequencing of an RT-PCR product obtained from RNA extracted from infected cell layers (passage 3) showing cytopathic effect.
Nucleotide differences between prSAV and SAVH20/03 (positions refer to accession no. AY604236).
1This position has previously been reported to be a G (accession no. DQ149204) or an A (accession no. AY604236) in SAVH20/03.
2The complete 3'UTR is not reported in AY604236 and positions are given relative to the 3'UTR start nucleotide .
The ability of the full-length clone to express viral structural proteins was addressed using the obtained antiserum against E2. IFAT with anti-E2 on transfected Blue gill fry 2 (BF2) and CHSE cells demonstrated a positive signal 12 days post transfection (d.p.t.) similar to that seen during infection with wild type SAV (not shown). Passage of supernatant from transfected cells onto non-infected CHSE and BF2 cells followed by IFAT on these, confirmed presence of infectious particles in the supernatant (Figure 2b). Following 3 passages in CHSE cells, a cytopathic effect similar to that of wild type SAV infection was evident (Figure 2c). The CPE included elongated, vacuolated cells with pseudopodia-like extensions and curled up, dead cells. The authenticity of the recombinant virus (rSAV3) was verified by RT-PCR and sequencing on passage 3 virus of the unique XbaI site that had been introduced in the junction region of the rSAV3 genome (Figure 2d).
A recombinant infectious virus with two subgenomic promoters was obtained by introducing the structural polyprotein ORF of SAV into a modified pmSAV3 replicon plasmid, making the final pEGFPrSAV3 plasmid (Figure 3a). The sequence of pEGFPrSAV3 was identical to that of prSAV3 with the exception of the additional subgenomic cassette expressing EGFP from the subgenomic promoter of SAV located upstream of a transcription cassette expressing the structural ORF. The exact start of the subgenomic promoter in the nsP4 sequence is not known for SAV, but the length of the fragment chosen was based on a previously reported SAV2 promoter that had been shown to be functional . The EGFP ORF is flanked by AgeI and AscI sites that allow exchange of the EGFP ORF with other cDNAs. Transfection of pEGFPrSAV3 into BF2 cells produced EGFP expression 5 days p.t. Foci of cells expressing EGFP were evident and the foci increased in size the following days. Passage of supernatant from transfected cells 8 d.p.t. onto new cells confirmed the presence of infectious particles, and packaging of genomes with the EGFP transgene into these (Figure 3b). CHSE cells infected with EGFPrSAV3 showed expression of EGFP already at 24 hours post infection (h.p.i.). The EGFPrSAV3 virus gave weak or no CPE compared to rSAV3, although the majority of cells were infected (EGFP positive). In one experiment, passage 3 of EGFPrSAV3 was grown in CHSE and BF2 cells for 30 days. After this prolonged incubation period less than 1% of cells still expressed EGFP, suggesting either gradual loss of the virus from cell culture or loss of/disruption of the transgene when incubation time is very long. During the work with constructing pEGFPrSAV3, a clone, pEGFPrSAV3nsP3263M323G was obtained that had accumulated mutations in nsP3 positions 263 (K to M) and 323 (E to G), likely a coincidence occurring during the cloning process. This clone was studied further in order to test the relative sensitivity of SAV to random mutations in this conserved region of nsP3. Although replicating and infectious recombinant virus expressing EGFP could be recovered after transfection (not shown), these clones appeared to spread slowly in infected cell cultures and was no longer detectable after two passages (Table 2).
Construction and validation of recombinant SAV3 with a double subgenomic promoter. (A) The full-length SAV3 was engineered to contain an additional ORF encoding the EGFP protein under the control of a SAV specific promoter. The construct was obtained by AscI/NotI cleavage and ligation of a PCR amplified fragment encoding SAV3 structural proteins and the SAV3 subgenomic promoter, into a modified pmSAV3 plasmid. (B) The obtained construct was transfected into BF2 cells and expression of EGFP was visualized by fluorescence microscopy. Presence of infectious virus containing the EGFP ORF was verified by serial passage of supernatant from transfected cells onto naïve CHSE cells followed by fluorescence microscopy of live cells.
Detection of different SAVs in cell culture.
BF2 cells were transfected with 5 μg of plasmid encoding the respective viruses, or infected with SAVH20/03. Supernatant was passed to new CHSE cells three times. All viruses were detected by IFAT using anti E2 antiserum, except EGFPrSAVnsP3mut and EGFPrSAV3 which were directly detected by fluorescence microscopy. The relative amount of positive cells for each virus is indicated (-/+/++/+++). SAVH20/03 served as positive control. ND = Not done.
Previously, we have associated a serine to proline mutation in E2206 of SAVH20/03 with cell culture adaptation based on sequence comparison studies . We also recently reported a replicon based on the SAVH20/03 genome to differ from the wild type sequence through an alanine to aspartic acid mutation in nsP2197 . Through genetic modifications of the prSAV3 plasmid, the nsP2197A or nsP2197 D was expressed in combination with E2206 S or E2206P and all four viral mutants were recovered and shown to be infectious by serial passage in CHSE cells followed by IFAT (Table 2). A visible CPE indistinguishable from wild type CPE was produced by both mutants containing nsP2197A, while no CPE could be seen for mutants containing nsP2197 D (Figure 4a). IFAT on infected cells suggested that foci of infection were small with the nsP2197 D mutants and that the majority of cells remained uninfected. After three passages in cell culture, infection by the two rSAV3nsP2197 D virus variants could not be detected by IFAT, while cells infected with the clones containing the nsP2197A remained readily detectable (Table 2). The E2S206P mutation did not cause lack of recognition by the E2 antiserum since indistinguishable staining was obtained with the two virus mutants (Figure 4b). Sequencing of the partial E2 coding sequence verified that both these mutants were genetically stable during three passages in CHSE cells (Figure 4c). Endpoint titration of supernatant from infected cell layers confirmed that the virus titer was reduced in the nsP2A197 D mutant while for the E2S206P mutant the production of infectious particles appeared to increase by almost one log (Table 3). Recombinant SAV with E2206P produced similar titers as the wild type SAVH20/03 isolate also carrying the E2206P.
Evaluation of mutations in nsP2 and E2. The plasmid prSAV3 was modified to express either serine or proline in E2206 and either alanine or aspartic acid in nsP2197. (A) Infectious viruses were recovered from all plasmids after transfection into BF2 cells. The variants expressing the nsP2197A produced CPE in CHSE cells, while viruses expressing nsP2197 D did not. The E2206 mutation did not appear to affect CPE. Representative images of CPE are shown for each mutant. (B) The E2206 mutation did not affect antigenicity of the E2 protein notably, since both the serine and proline mutants were readily detected by IFAT. (C) RT-PCR and sequencing of passage 3 of recombinant viruses confirmed that both the serine and the proline codons were stable.
Tissue culture infective dose (TCID50/ml) produced by wild type SAVH20/03 and different mutants 7 d.p.i. in CHSE cells.
Structural predictions of E2 using the PredictProtein server  and TMpred suggested that the transmembrane helix of SAVH20/03 E2 consists of amino acids E2385A to E2404A, while E2206 appears to be located in a loop area flanked by two sheet areas. Accessibility plots of the protein further predicted the E2206 position to be highly accessible (Figure 5a). Moreover, alignment with E2 from other alphaviruses supported the idea of SAV E2206 being located on the surface of the virus particle (Figure 5b). Finally, a reconstruction of the evolutionary history of the partial E2 protein of SAV suggested that the proline is the ancient aa in this position and that the serine has evolved in subtype 3 only (Figure 6). Some SAV3 isolates also still carry the proline.
In silico analyses of E2 structure and accessibility. (A) Secondary structure and solvent accessibility of the SAV3 E2 protein was estimated using the PredictProtein server and TMpred. Relative solvent accessibility is illustrated by black bars and predicted secondary structures are shown as red (sheet), green (loop) or blue (helix) lines. The predicted transmembrane helix is indicated. (B) Alignment of a partial SAV3 E2 sequence with a region of E2 from SINV and RRV previously suggested to be located on the protein surface. E2206 is indicated for SAV by red colour. E2200, E2216 and E2218 are indicated for RRV since they previously have been shown to be located on the very distal part of the spike .
Bayesian phylogenetic analysis of partial E2 sequences. Clusters corresponding to the SAV1-6 subtypes are indicated. Sequences containing the E2206 serine are indicated by red branches, while black branches indicate proline. Posterior probabilities are given for key nodes.
SAV is a virus that is associated with mortality and significant economical loss for salmonid fish farmers in Europe . Still, important questions related to viral and host mechanisms that contribute to transmission and pathogenesis remain unanswered. Here we report the development of infectious clones for a subtype 3 strain where the viral genome is transcribed by cellular RNA polymerase II from a CMV promoter in a pVAX1 backbone. The recombinant virus was also engineered to express non-viral RNA from an introduced, additional subgenomic promoter. Subtype 3 appears to be genetically homogenous, indicating a relatively recent common source for all known isolates . We therefore designed the system to recover viruses that express proteins identical to those reported previously from consensus sequences of SAVH20/03, the only subtype 3 strain where both ORFs have been completely sequenced [9, 18, 25]. Such alphaviral reverse genetics systems are suitable for studies of evolution and mechanisms of emergence and pathogenesis. For CHIKV, development of infectious clones with high similarity to wild type CHIKV has led to identification of point-mutations important for the emergence of epidemic strains [32–34]. Using the obtained SAV3 infectious clones, we demonstrated that point-mutations indeed may alter the replication properties of SAV dramatically.
The infectious clones were generated with focus on expressing proteins that were identical in amino acid sequence to those previously reported from SAVH20/03. However, compared to previously reported sequences from SAVH20/03, the prSAV3 plasmid contains 10 mutations that do not lead to amino acid change (Table 1). Although such mutations not leading to amino acid changes certainly have the potential to affect viral replication, the obtained recombinant virus had titer production comparable to SAVH20/03 and other wild type SAV isolates in cell culture . Similar to some other DNA-launched positive stranded RNA virus systems [38, 39] the exact rSAV3 3'-end is uncertain since transcription is stopped by the BGH signal in the pVAX1 vector, leaving some nonsense (non-viral) sequence downstream of the introduced polyA tail. This nonsense sequence will most likely be lost during replication, since alphaviruses are believed to maintain and repair their polyadenylated 3'-ends by the use of their RC [40–42]. Still, future work could benefit from investigating the true 3'end of the obtained transcripts, and potentially developing a more authentic 3' end, for example by inserting a Hepatitis delta virus ribozyme immediately downstream of the polyA tail . Such studies could also focus on understanding the impact of different lengths of polyA tails, as this has been reported to affect replication of alphaviruses .
Single point mutations in the RC have previously been shown to affect the replication efficiency of an attenuated strain of SAV2 . When we recovered an infectious recombinant SAV3 strain, we made a similar observation, as a mutation in nsP2197 dramatically reduced the ability of the virus to infect and replicate in CHSE cells. This mutation is located in a conserved region of nsP2 in alphaviruses, believed to include an NTP binding motif that is essential for RNA triphosphatase, helicase and NTPase activity of the protein . Thus, it is likely that an aspartic acid compared to the wild type alanine in this position reduces efficiency of the viral capping apparatus as well as helicase activity. Similarly, the virus with two mutations in nsP3 had a clearly reduced ability to replicate, and resulted in the loss of the virus from infected cells after two passages. Both mutations were located in a region of nsP3 that is conserved among alphaviruses, but outside the described macro domain [45, 46]. Although these mutations demonstrate that SAV can tolerate single amino acid mutations in highly conserved motifs of the RC, they also address the relatively large impact such single mutations may have on the viral phenotype. This suggests that SAV, like other alphaviruses [31, 32], has the potential of major changes in some phenotypic features as a result of minor genetic change. A similar observation has been done for a SAV2 strain which adapted to replication at an elevated temperature through six mutations in the structural ORF .
Recently, studies of CHIKV have shown the power of reverse genetics systems that are based on clones with genomic sequences similar to wild-type/field isolate sequences [31–33]. Such systems are suitable for identifying mutations that contribute to the emergence of outbreaks. Alphavirus epizootics appear to be particularly associated with point mutations in the viral receptor protein E2 or the fusion protein E1 [31, 32]. These mutations could lead to changes in cell tropism and affinity for host species . We utilized the rSAV3 system to confirm that the previously reported E2S206P mutation  enhances the viral titer production in CHSE cells. This is in agreement with the hypothesis that this mutation may be a beneficial adaptation for replication in CHSE cells. Our phylogenetic analyses suggested, however, that a proline is the ancient aa residue in this position of E2 in natural populations of SAV, and that the mutation towards serine is a relatively recent event that has happened in the SAV3 subtype only. Alignments of sequences from SAV3 isolates have previously demonstrated that several SAV3 isolates still carry the proline [17, 25]. This suggests that a proline codon might still be part of a viral quasispecies in SAV3 strains. If so, it could indicate a regulator of pathogenesis. In further support of this idea, in silico analyses predicted the E2206 to be located on the surface of the protein, probably in an area used for receptor recognition. It appears, however, that the mutation does not have any major effect on the antigenic properties, since both the serine and proline mutants were readily detected by the antiserum that was raised against a short peptide containing this region. Clearly, data from experimental transmission studies in fish are needed in order to better understand the role, if any, of the E2S206P mutation during an in vivo infection.
The pEGFPrSAV3, having a second subgenomic alphavirus promoter, could be a potential tool for delivery and expression of foreign RNA in fish, e. g. an RNA coding for an antigen or a reporter protein. Like other alphavirus vectors, they could provide several advantages over traditional vaccine vectors, since expression of the antigen occurs in the context of a viral infection . Another powerful future aspect of the pEGFPrSAV3 plasmid could be to address tissue tropism of SAV3 mutants through bioluminiscence imaging, as has been done for other alphaviruses [33, 48, 49]. Such studies could help clarify the role of tissue tropism in SAV pathogenesis. We observed, however, that rSAV3 with double subgenomic promoter is unstable similar to what has been shown with such constructs for other alphaviruses . It is possible that the virus is able to sustain in only a small fraction of cells when it contains a transgene, but it is more likely that the transgene itself is gradually lost or mutated as the infection progresses due to the lack of selection pressure. Further development of these SAV based expression systems should therefore aim at incorporating selection pressure on the transgene, as has been done for SINV .
We have developed a functional reverse genetics system that recovers recombinant SAV with identical proteins as those reported from the genetically distinct SAVH20/03 strain that was isolated in Norway in 2003 and suggested to represent a new viral subtype (SAV3) . The system is suitable for studies of viral replication, evolution and pathogenesis. It may also be used as a gene delivery system in fish cells and for the development of live attenuated vaccines.
Infected and non-infected Chinook salmon embryo (CHSE) and Bluegill fry (BF2) cells were grown as previously described . SAVH20/03 was used to infect CHSE or BF2 cells that were incubated at 14°C. Cells used for transfection were grown to approximately 80% confluence prior to transfection. Transfected cells were incubated at 20°C for 24 h before transfer to 14°C due to increased effect of the CMV promoter at the elevated temperature.
The coding sequence of the SAV3 E2 protein was aligned with known immunogenic motifs of alphaviruses SINV, RRV. Based on this alignment, a 348 bp cDNA fragment containing a partial coding sequence of the E2 protein, was amplified by RT-PCR from CHSE cells infected with SAVH20/03 using primers Fc1_E2 (CACCAAGGGCCACCACTTGTCCGA) and Rc1_E2 (TCACTTCACTTCCTTGCCTCCGC). The fragment was cloned into prokaryotic expression vector pET200/D-TOPO, to the C-terminus of a His-tag, using the Champion pET TOPO Directional Expression kit (Invitrogen) as recommended by the manufacturer. BL21 Star One Shot E. coli cells (Invitrogen) were transformed with the pET200/E2 plasmid and expression of the His-tagged peptide was induced using 1 mM isopropyl beta-D-thiogalacto-pyranoside (IPTG) for four hours. Expression was verified by SDS-PAGE gel and Western blot with the provided anti-his-G-HRP antibody. Purified and dialysed protein was used for immunization of a rabbit. The obtained anti-serum was pre-adsorbed onto monolayers of CHSE cells fixed with 4% paraformaldehyde in PBS and methanol.
CHSE-214 or BF2 cells were grown on cover-slips in 24 well plates or in 96 well plates and infected with different SAV strains as indicated in the results section. The cells were fixed and permeabilized with 4% paraformaldehyde in PBS and methanol. The cells were then washed twice with PBS-FBS (PBS with 5% fetal bovine serum), before blocking using 5% skimmed milk in PBS-FBS for 30 min. The primary antibody anti-E2 was then diluted 1:400 and incubated on the cells in PBS-FBS with 5% skimmed milk for 60 min at room temperature. Following antibody incubation, cells were washed three times, and incubated with Alexa Fluor 488 goat anti-rabbit secondary antibody (Invitrogen) in PBS-FBS with 5% skimmed milk for 60 min at room temperature. Finally, the cells were washed three times with PBS and directly examined in an Olympus IXI fluorescence microscope, or mounted using ProLong Gold with DAPI (Invitrogen) and visualized using a Leica TCS SP2 AOBS confocal microscope. Mock infected cells and cells not treated with primary antibody served as negative controls.
Total RNA was extracted from infected CHSE cells and reverse transcribed into cDNA using random hexamer primers according to standard techniques. Three PCR fragments, covering the entire structural ORF, were obtained and assembled by ligation into the pVAX1 vector (Invitrogen), leading to the pVAX1/SAV3 SP construct. The structural polyprotein ORF was further subcloned into a pCI vector (Promega) using the restriction sites EcoRI and NotI, after the correction of some mutations introduced during cloning, leading to the construct pCI/SAV3 SP*. In order to clone the 3'UTR sequence at the extremity of the SAV3 SP ORF sequence, the 3'UTR was obtained by RT-PCR using the primers PDuniF (CCGCACGGTTGTAAGATCAGT) and NotIXbaIPolyAR  and inserted into pCI/SAV3 SP* by StuI/NotI digestion and ligation, leading to the construct pCI/SAV3 SP+3'UTR.
An XbaI site was introduced by site-directed mutagenesis into the junction region within a previously reported replicon construct that contains a SAV3 replicon expressing EGFP in a pVAX1 backbone . The construct was further engineered to express the wild type nsP2197A, leading to the plasmid pmSAV3wt. The EGFP ORF and 3'UTR was then exchanged with an XbaI fragment from the pCI/SAV3 SP+3'UTR plasmid. The authenticity of the obtained full-length construct, prSAV3, was verified by REN analysis and DNA sequencing as previously described . All plasmids were replicated in XL10 Gold competent E. coli cells (Stratagene), and purified using Qiagen Endofree plasmid maxi kit according to the manufacturer's protocol.
The prSAV3 plasmid (5 μg) was transfected into BF2 cells (passage 0) using Amaxa nucleofector kit T (Lonza) as recommended by the manufacturer. Transfected cells were then cultured as previously described . Twelve d.p.t., supernatant from transfected cells was passed to freshly prepared CHSE cells. Transfected and infected cell layers were tested by IFAT for expression of the E2 glycoprotein. Infected CHSE cells (passage 3) were harvested 7 days p.i., and extracted RNA was used as template for RT-PCR using SAV3 specific primers 544F (ATGATATGATGGTGGCCAGG) and 1238R (GCTCCCTTTCTTCTCCTGTTG). The obtained PCR product was sequenced and analysed for the presence of the introduced XbaI site.
A plasmid pEGFPrSAV3 where an additional ORF encoding EGFP under the SAV3 subgenomic promoter is placed upstream of the SAV3 structural transcription unit, was constructed based on the pmSAV3wt plasmid. A cDNA containing the structural ORF, 3' UTR, polyA tail and a presumed SAV3 subgenomic promoter which includes the junction region and the 3'-terminal 92 nucleotides of nsP4 (including the stop codon), was amplified from the prSAV3 plasmid using primers nsP4pro (GGGGCGCGCCGCGTGAATCACCTGCCGTTAGCCAC) and NotIXbaIPolyAR. The obtained cDNA was inserted into pmSAV3wt by AscI/NotI digestion and ligation. Authenticity of the plasmid was verified by sequencing of the complete transcription unit. One of the obtained clones, named pEGFPrSAV3nsP3263M323G, contained two amino acid mutations in nsP3 that probably were introduced as artefacts during cloning. Transfection and recovery of infectious virus was done as described above, and expression of the EGFP transgene was verified by direct fluorescence microscopy of infected cells and photographed using an Olympus IXI camera.
Infectious clones were engineered to test the effect of the nsP2D197A and E2S206P mutations. The clone prSAV3E2206P was made by site-directed mutagenesis using a QuickChange XL Mutagenesis kit (Stratagene). The clones prSAV3nsP2197DE2206 S and prSAV3 nsP2197DE2206P were generated by replacing the BstBI/NhiI fragments in prSAV3 and prSAV3E2206P with one from the previously reported pmSAV3 plasmid. The transcription units of all plasmids were sequenced completely prior to transfection. Transfections and recovery of infectious virus was done as described above. Recovered viruses were passaged in CHSE cells and supernatant from day 7 p.i. was titrated in 96 well plates using endpoint titration. Titers were calculated using the Kärber formula . In addition to cytopathic effect, IFAT with anti-E2 was done in all wells as described above in order to confirm the presence or absence of infection. Finally, a cDNA containing the partial E2 gene was amplified by RT-PCR using primers F2234 and SAV20R  and sequenced from infected cell layers in passage three in order to verify that the E2206P and E2206 S mutations were stable.
Predictions of protein secondary structure and solvent accessibility were obtained by submitting the complete E2 sequence of SAVH20/03 to the PredictProtein server . Transmembrane helices were predicted using TMpred http://www.ch.embnet.org/software/TMPRED_form.html. A nucleotide alignment (357 nt) was generated based on 67 previously reported SAV sequences covering the E2 position 206 (accession numbers AJ316244, AJ316246, EF675551 to EF675577, EF675579 to EF675594 and DQ122127 to DQ122146) [17, 25]. Sequences were aligned by AlignX in the VectorNTI package (Invitrogen). The alignment was imported into the software package BEAST for Bayesian reconstruction of phylogeny . The GTR model of nucleotide evolution with a four category gamma rate, and a constant population size assumption, was chosen. Trees were constructed using a relaxed lognormal clock assumption. A prior in lognormal distribution was set on the mean rate parameter, based on a previously reported substitution rate . The MCMC chains were run for 20 million generations and the results were inspected in Tracer 1.4. ESS values were higher than 200 for all parameters. A maximum clade credibility tree was viewed using FigTree, and the tree was obtained using a 10% burnin in TreeAnnotator.
MK, KFO and AN are funded by the University of Bergen and the Norwegian Research Council grant 185188/S40. SV is employed by Intervet/Schering-Plough animal health. ER is funded by Norwegian School of Veterinary Science. The authors are grateful to Dr. Petter Frost (Intervet/Schering-Plough animal health) for critically reading the manuscript.
MK planned the study, conducted all laboratory and bioinformatical work except production of E2 antiserum and cloning of the structural polyprotein ORF, analysed the results and wrote the manuscript. SV contributed to experimental design, performed the cloning of the structural polyprotein ORF and critically revised the manuscript. KFO developed the E2 antiserum and contributed to the revision of the manuscript. ER has contributed with discussions throughout the study and reading and contributing to the writing of the manuscript. AN has contributed during planning and with discussions throughout the study. All authors read and approved the final manuscript.


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Semi Final Preview: Sydney FC
By Sarah Groube Jan 24 2016 6:41AM
By Sarah Groube
Jan 24 2016 6:41AM
Sydney FC head down the Hume Highway to Canberra on Sunday carrying the proud banner of being the only team in the W-League to have made every semi-final round in the competition.
And they are riding the wave of seriously challenging Melbourne City last round, perhaps the first side to really do so throughout an entire match this season.
It was a far cry from the opening to their season when the Sky Blues were left in the dust behind the rest of the pack after after copping a 6-0 loss to red hot debutants Melbourne City, and 3-0 to the champions Canberra United.
But they are a professional outfit, and they worked their way back into finals contention with a steady set of wins over Western Sydney Wanderers, Adelaide United and Perth Glory.
They stumbled to a narrow loss against Canberra United at home, but perhaps their biggest wake up call following the opening rounds was a 3-0 annihilation by Brisbane Roar in December. Sydney FC looked lacklustre, unable to connect passes, and without intent to score. The match gave Brisbane Roar the taste of success they needed to kick on and make a run at the top four which they made, just, last weekend.
It was Sydney FC's victory over the Newcastle Jets in January that propelled the Sky Blues into the semi finals, keeping the Jets teetering on the edge of the four, with Adelaide United coming ever so close but slipping at the business end.
Sydney FC enter this round with a stream of confidence they haven't enjoyed all season, off the back of the Jets win and a narrow loss to Melbourne City that by all accounts, felt like a win.
Kyah Simon spoke to Sydney FC's website team after the loss: "We have taken a lot of confidence away from our performance against Melbourne City. It is the first time that we have really played to our potential.
"We walked away from that match with our heads held high, and take a lot of confidence going from that into our semi against Canberra. We are feeling confident at the moment, and ready for the challenge ahead.
"It was funny because on the weekend it brought back a lot of memories of the last time we took out a Grand Final (in 2013). How joyful and happy we were, and proud of ourselves as a team.
"That was exciting in itself, and has added an extra bit of hunger to experience that again. If we can do that again it would be a massive achievement for the team and the Sydney FC club."
There is daylight between Sydney and Canberra in the stats with Sydney scoring only 15 goals and conceding 21, while Canberra scored 26 and dropped 8.
Winning just half of the matches they played this season, it's the right time for Sydney FC to be striking a hot patch, and they'll be hoping it translates in the nation's capital this weekend.
Their lack of goals despite the high calibre strike force of Kyah Simon and US import Jasmyne Spencer has been a weakness, with only Leena Khamis consistently breaching defences.
The Sky Blues have averaged 1.45 goals per game. In comparison, Canberra United have scored an average of 2.3 goals per game.
Sydney haven't yet managed to score past Canberra's line this year, clean sheets prized as highly as goals this season by this week's home team that is putting all their energy in becoming the first back-to-back title winners in the league.
At the other end, Sydney FC have suffered blow outs against each of their fellow finalists, leaking 9 goals in the first two rounds to Melbourne City and Canberra, and another 3 against Brisbane Roar late in the season.
Last season, Sydney FC's attack was accused of appearing lacklustre in high profile fixtures and the defence encountering off days against their fellow 2014 semi-finalists. Sydney FC were bundled out of last year's finals by Perth Glory. They won't want history to repeat this season.
Michelle Betos (Defence)
Photo: Emily Mogic Photography
The USA's National Women's Soccer League's (NWSL) 2015 Goal Keeper of the Year, Michelle Betos, landed with a thud in Australia with a load of goals from Melbourne City and Canberra United to contemplate. She has settled in nicely since then, performing strongly through the rest of the season.
Betos certainly knows how to perform on big occasions, having played for high profile clubs Seattle Reign and Portland Thorns. Canberra will have to keep a close eye on Betos in dead ball plays – this header off a corner from Betos in stoppage time against eventual NWSL champions FC Kansas City is a (magnificent) case in point https://www.youtube.com/watch?v=8EgNf0tCnuQ
Teresa Polias (Midfield)
Photo: Joseph Mayers Photography
Sydney FC lost the midfield battle the last time they met, so Teresa Polias will be looking to flip the result.
It is Midfielder T who is the heart and soul of this Sydney FC side, and when she is on song, she can be the connector that unleashes Sydney FC's strikeforce
Still without a goal to her name in the W-League, the whole stadium might just erupt if the little general can sneak one past Lydia Williams.
Leena Khamis (Forward)
The biggest threat for Sydney FC this season, and since the inception of the W-League, has been Leena Khamis. The 29-year-old is a smart veteran currently sits on fourth in the All-time leading goalscorers table,
Her dynamic running, lightning quick reflexes and passion in front of goals makes her an unpredictable danger that the Canberra United defence will need to be prepared for.
Canberra United v Sydney FC
McKellar Park, Canberra
Local kick-off: 2pm
Head to Head: 12 played | 6 wins (CBR) | 4 wins (SYD) | 2 Draws
Canberra United squad: 1. Lydia Williams (gk), 2. Catherine Brown, 3. Julia De Angelis, 4. Kendall Fletcher, 5. Jenna McCormick, 6. Caitlin Munoz, 7. Ellie Brush, 10. Grace Maher, 11. Michelle Heyman, 12. Emma Kete, 13. Nicole Begg (c), 14. Ashleigh Sykes, 16. Tegan Riding, 17. Veronica Perez, 18. Rebecca Kiting, 19. Nickoletta Flannery, 20. Melissa Maizels (gk)
Ins: Catherine Brown (promoted), Emma Kete (returns from New Zealand international duty)
Unavailable: Emma Checker (knee – season)
Sydney FC squad: 1. Michelle Betos (gk), 2. Elizabeth Ralston, 3. Ellyse Perry, 4. Sunny Franco, 5. Jasmyne Spencer, 6. Teresa Polias (c), 7. Nicola Bolger, 9. Princess Ibini, 10. Renee Rollason, 11. Natalie Tobin, 12. Olivia Price, 14. Alanna Kennedy, 15. Teigen Allen, 17. Kyah Simon, 18. Sham Khamis (gk), 19. Leena Khamis
Ins: Ellyse Perry (return from cricket commitments)
Unavailable: Amy Harrison (knee - indefinite)
canberra unitedfootballmatch previewsemi finalsgroubesydney fcw-league
Updated Again: A-League Women Round 5 and rescheduled games schedule
Perth Glory fixtures changes affect five A-League Women's teams
Canberra United announce new A-League head coach

redpajama_set_name: RedPajamaCommonCrawl


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Similar to a roll cage in a race car, a ROPS is made by bars that attach to the frame of the machinery that maintain a space that's large enough for the body of the operator in the case of the machine rolling over.
Generally, a ROPS is a steel structure that surrounds the cab area of a piece of heavy machinery, such as a tractor. Depending on the machine, the ROPS may have been installed at the factory, or it could have been purchased from an aftermarket dealer.
Tractor rollover has become one of the leading causes of occupational death in the agricultural industry, and that is why ROPS have become essential for many businesses within the heavy-lifting industries.
The steel bars used in a ROPS prevent the cab area of heavy-lifting machinery becoming crushed in the event of a rollover or overturn. However, it's important to remember that a ROPS will not stop an operator from being ejected – meaning seatbelts are still a crucial safety aspect even when working in a machinery that features ROPS.
Are there ROPS regulations in Alberta?
A ROPS must also be designed with a material that meets a minimum cold weather toughness. Using standard tubing can be a concern since most tubing does not necessarily meet the required toughness. That's why you will notice most ROPS manufacturers use plate material in their designs.
and machine make and model for which the ROPS is designed.
How does a Rollover Protective Structure differ to a FOPS?
A ROPS and a FOPS tend to have similar design features, which tends to mislead people into thinking they have a ROPS. However, A FOPS is a falling object protective structure and is only intended to protect the occupant from objects falling from overhead. It is not intended to protect the person in a rollover.
ROPS and FOPS can sometimes be combined into one design, but if you have any doubt whether the piece of machinery you are using has a ROPS we strongly recommend that you contact a professional engineer as soon as possible to confirm if it is.
According to the Occupational Health and Safety Code 2009, a ROPS must either be supplied by the manufacturer or be certified by a professional engineer as being suited to that equipment.
In-depth knowledge and experience is crucial when it comes to the safety of your employees. With over 12 years in the industry, Sparta Engineering knows exactly what to look for when it comes to the stability and quality of your ROPS.
Our weld inspectors know exactly what to look for when it comes to safety issues, and know exactly how to fix them when we do find one. Because of our experience we see frequent and repeated issues, meaning we can resolve them quickly and to the highest standards.
Want more information on a ROPS or our inspection services? Contact us today.
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What is a question stem?
I am trying to create a Quiz / Test in Canvas.
ANA, are you possibly using the new quizzing engine? If so, then the question stem is "contains the question and, if necessary, additional instructions and information." You can see more about this on the following guide - How do I create a Formula question in Quizzes.Next?
Mona, thank you so much!
I'm sorry to be daft but my mind isn't wrapping around the purpose of a stem. The sample in the link appears to be for teachers. Does someone have a sample that would be helpful to students? Do students see the stem? Unrelated, why have a RTE in the stem and not the question?
"A question stem is the part of the survey question that presents the issue about which the question is asking. With closed-ended questions, the stem can perhaps best be defined as the first half of a survey question that consists of two parts: (1) the wording or text that presents the issue the respondent is being asked to consider (along with any instructions, definitions, etc.) and (2) the answer options (response alternatives) from which a respondent may choose."
So yes, students do see the stem but it's not referred to as being the stem; it's simply the text displayed as the question. It uses the RTE so that you can include formatting and any rich media you want in the question itself. The question Title is NOT seen by students. This was the same in the old Quizzing tool and I also see it in most question authoring tools I've looked at. I think the idea of a title is a shortened version of the Stem (question) that makes is easier for the author or teacher to identify. I've often seen titles of questions from textbook publishes include numbers like 13.1A which presumably somehow tie back to the textbook or content area.


Go Caribbean without leaving your kitchen. All you need are four things — fresh pineapple rings, butter, rum, and balsamic vinegar. Mix it all together in a bowl and brush it on the pineapple slices. Broil them in the oven for seven minutes on each side, until the slices are beautifully caramelized. Sprinkle them with a dash of cinnamon and serve with vanilla ice cream. I like to toss on chopped macadamia nuts and turn up the Hawaiian music. If you're serving to guests who don't drink alcohol, skip the rum.


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Fukushima is Not Alone
There are certain proper names of places — Fukushima, Hiroshima, Auschwitz, Timbuktu, Chernobyl, Marichjhapi, Hong Kong, Tulsa, Palmyra, Gujarat, Kilvenmani, others — which lift off from the land to hover over other words while resisting to be just another word; they haunt other places to remind them to never become them. They ask us to remain alert to the call of another place without locus, which has no name yet for it. The question being asked ten years after Fukushima in this special issue of March 2021 through the unusual writings gathered here—certainly unusual for philosophical discourse which speaks often without restraint— is the meaning of what it means to say "Fukushima is not alone".
here are certain dates which detach from the binding of time and move across the tenses—1917, 1921, 1776, 347 BCE, 19 March 2003, May 1968, 1st November. These dates without places could mean many things across the regions of the world, as we continue to mistake the world for them — regions!
In the same way, the places—Hiroshima, Auschwitz, Timbuktu, Chernobyl, Marichjhapi, Hong Kong, Tulsa, Nagasaki, Palmyra, Fukushima, Gujarat, Kilvenmani, others. Certain proper names of places lift off from the land to hover over other words while resisting to be just another word; they haunt other places to remind them to never become them. There is still no end to the spectres and ghosts of our own making, as if there is still room here for more. As if, in the words of Jacques Derrida, "the dissociation between the place where competence is exercised and the place where the stakes are located" has not "seemed more rigorous, more dangerous, more catastrophic." (1) These places shatter our gravely false sense of the regional confinement of man.
Fukushima is not alone, and this is the quiet terror which is animating this month, March 2021. Today three places hover over Japan and together they haunt everywhere else, beginning in 6th August 1945. Through the name Fukushima, through the discipline of the act of mourning, two other names are recalled without being called out loudly. It is in the cold of this quiet terror witnessing the quiver of the pen that these texts of this month should be read.
As Jean-Luc Nancy remarked, Fukushima is also the twin experience of limits—of the limits set by nature and the limits of our technical mastery—a natural nuclear disaster.(2) Fukushima is with us, haunting without seeking to make a home anywhere, asking us only to think. To think in order to make an interval to think; it asks of "we" who have fallen in the desert of time. To ask, as Osamu Nishitani did in 2011, where is our future? (3) This is all that it wants which is however not the easiest task to achieve compared to the ease with which we still lurch for new wars which were anticipated by Hannah Arendt when she observed that with nuclear technology "the Kantian statement that nothing should happen in a war to make a later peace impossible has likewise been set on its head, so that we live in a peace in which nothing may be left undone to make a future war still possible." (4)
The question being asked ten years after Fukushima in this special issue of March 2021 through the unusual writings gathered here—certainly unusual for philosophical discourse which speaks often without restraint— is the meaning of what it means to say "Fukushima is not alone". Is it not-alone in the sense of Fukushima speaking to Chernobyl and the sites of nuclear tests, sites of barren time? Or is it that the people of Fukushima, offering in that name the other two names—Hiroshima and Nagasaki—to think with, are asking all of us to come over our terror to join in their decades long mourning?
These filamental places which can no longer host a sparrow nor a song are powerless to comprehend our world, either to save us from the calamities we nurture by closing our eyes to evil, or to comprehend us towards the end, the eschaton. They ask us to remain alert to the call of another place without locus, which has no name yet for it. We have to be near it when it calls our name; it will have to call us in order that we may be raised again.
What will it call us?
DIVYA DWIVEDI
1. Jacques Derrida, "No Apocalypse, Not Now (Full Speed Ahead, Seven Missiles, Seven Missives)", trans. Catherine Porter and Philip Lewis, Diacritics 14.2, 1984: 20-31.
2. Jean-Luc Nancy, After Fukushima: The Equivalence of Catastrophes, trans. Charlotte Mandell, New York: Fordham University Press, 2015.
3. Osamu Nishitani, "Où est notre avenir ?", Ebisu, 47, printemps-été 2012, https://doi.org/10.4000/ebisu.256 .
4. Hannah Arendt, The Promise of Politics, New York: Shocken Books, 2005.


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Malta-registered oil tanker collides with Norwegian warship
Royal Norwegian Navy frigate collided with Maltese oil tanker in a fjord in Norway as it was returning from NATO exercise Trident Juncture
9 November 2018, 6:46am
by MaltaToday Staff
A Royal Norwegian Navy frigate has collided with a Malta-registered oil tanker in a fjord in Norway as it was returning from NATO exercise Trident Juncture.
The incident occurred at 4:26am (local time) on 8 November in Hjeltefjorden, north of Sotra, Norwegian Armed Forces said.
After colliding with the Malta-flagged tanker Sola TS and the tug Tenax, the frigate started taking on water.
Eight sailors suffered minor injuries and have been taken care of. All 137 crew members of the frigate HNoMS Helge Ingstad were evacuated.
Aerial footage taken by Norwegian coastal administration aircraft during the day shows the frigate partially submerged sitting on her starboard side on the rocks. The ship's captain decided to beach the ship to prevent it from sinking in deep water.
The navy said the frigate is still not stable and that efforts are underway to secure her. Damages on the ship were also recorded with underwater cameras and all actions, which include recovery plans and oil spill containment, are coordinated with the Norwegian coastal administration.
No oil spill has been reported from the tanker, which was sailing from Equinor's Sture terminal in Øygarden. In connection with the collision, Equinor decided to "gradually shut down operations at Sture."
The 62,000-ton tanker suffered minor damages and was towed to the terminal.
The Sture terminal and the Kollsnes plant resumed operations Thursday afternoon after being shut down in connection with the collision between the frigate and the tanker.
An investigation into the causes of the incident will be launched today, in collaboration with the Defense Accident Investigation Board Norway (DAIBN) and the Marine Safety Investigation Unit (MSIU) from Malta.
Helge Ingstad, the Fridtjof-Nansen class frigate commissioned in 2007, was en route to Haakonsvern naval base.
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Sleep disorders don't just affect marriages, they harm the quality of a good night's rest and can cause some serious health risks. Now some dentists are playing a key role in the treatment of certain sleep disorders, including snoring and sleep apnea, according to a recent report in General Dentistry, the peer-reviewed journal of the Academy of General Dentistry, an organization of general dentists dedicated to continuing education.
"Your dentist should be able to complete an initial consultation to determine the problem, such as how you are feeling upon wakening or if you wake up in the middle of the night," says T. Bob Davis, DMD, FAGD, spokesperson for the Academy. Once the initial consultation is completed, your dentist might consult with a physician or a sleep clinic to determine a diagnosis.
Snoring is caused by vibration at the back of the throat, and dental appliances help to minimize or eliminate the problem. "The diagnosis is key because snoring alone doesn't automatically signify a serious problem. For basic snorers, a "snoreguard" (a plastic bite plate) can provide relief because it repositions the jaw and muscles, tissue and tongue. The apparatus helps breathing become less labored giving a person more room to breath quietly," says Dr. Davis.
For those with sleep apnea, a much more serious condition, Dr. Davis refers patients to a sleep clinic to make the diagnosis. People with sleep apnea cannot get enough oxygen, and breathing actually stops for a short period of time, predisposing them to a collapsed airway and other health problems.
Most dentists can help if you snore or refer you to a physician to determine if there is an underlying medical problem. If you want to try to reduce the symptoms of snoring yourself, Dr. Davis recommends losing weight and reducing alcohol consumption when appropriate.


The Hubbard Glacier calves with the roar of a thousand canons. Diamond-blue icebergs float on a glassy bay as a humpback whale breaches in a spectacular display. Meanwhile, a grissly bear catches his first salmon of the season.
Lofty mountain peaks, ice-sculptured fjords, an abundance of marine wildlife and massive tidewater glaciers make the Top 10 list of things to see in Alaska . The most memorable of experiences is a helicopter flight to one of the many glaciers; a journey into these giant and majestic mountains will take you to the dog sledding camps and the ride of your life.
The best way to see the water's edge is on a cruise. To ensure your experience is up close and personal, we recommend the smaller cruises. If you are a nature lover the un-cruise option is a great choice, or if you are a pure luxurist then a Silversea option will suit.


Employees at the Environmental Protection Agency were so worried that a fancy desk that their former boss Scott Pruitt had ordered for his office might contain high levels of a known carcinogen that they arranged to set it up in a warehouse and let it air out for a week.
The problem is that the same officials later blocked the release of a draft report prepared before President Donald Trump took office on the dangers of exposure to the same substance, formaldehyde, to the general public.
LOS ANGELES (Reuters) – In Sacha Baron Cohen's provocative new comedy show, American politicians are filmed backing a fictitious program to teach kindergartners how to use guns to defend themselves in school shootings.
While Donald Trump and his surrogates were loudly campaigning against Robert Mueller on Twitter and on TV, the special counsel and his team of F.B.I. investigators has been quietly building their case, the details of which burst into public view Wednesday when The Washington Post reported that the bureau searched the home of Trump's former campaign chairman Paul Manafort in a dramatic pre-dawn raid at the end of last month.


A highly sensitive liquid flow reaction-solution microcalorimetry system based on the heat conduction principle with multi-thermomodules, applicable to suspended solutions was developed. This calorimetry system was well suited for measurements on the biochemical and solution chemical studies. Two concrete examples; binding and reaction studies in Na+, K+-ATPase- ATP system by using suspended Jørgensen's sample, and studies on the interactions of the oxyethylene moiety of poly (oxyethylene) glycole alkylether with benzene, by means of this microcalorimetry were introduced and explained.
Adriamycin (ADR) was covalently conjugated with a copolymer of divinyl ether and maleic anhydride (DIVEMA). Either sole ADR or DIVEMA-ADR conjugate was injected intraperitoneally into mice, and the concentration of ADR in the serum and peritoneal fluid (PF) was measured by HPLC (high-performance liquid chromatography). A reversed phase liquid chromatography column enabled to measure the concentration of ADR as low as 6 ng/ml. In sole ADR administration, the ADR concentration in the PF reached 179 ng/ml at one hour then gradually decreased to 13 ng/ml at 24 hours after i.p. injection. In contrast, the concentration in the serum fell rapidly from 16.8 ng/ml at one hour to 6.6 ng/ml at 2 hours after the injection. In DIVEMA-ADR conjugate, the ADR concentration in the PF was elevated to 4, 057 ng/ml at one hour and decreased more slowly than in sole ADR. Even at 24 hours after i.p. administration of the conjugate, ADR was kept as high as 90 ng/ml in the PF. On the other hand, the serum concentration remained under the limit of detection at all times measured. Pharmacokinetic parameters were obtained based on the time-concentration curves of either sole ADR or DIVEMA-ADR administration, using two-compartment model. The results showed that the conjugate increased the AUC(area under the curve) of ADR in the PF by 15.4 times higher than that of sole ADR. These remarkable ability of DIVEMA conjugate on pharmacokinetics of ADR would be greatly contributable for improvement of cancer chemotherapy, especially for the treatment of peritoneal dissemination from intraabdominal malignancies.


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Home News In France, a teenage gunman takes four women hostage in a Toulouse...
In France, a teenage gunman takes four women hostage in a Toulouse bar but is confirmed as not an act of terrorism
Damon Mitchell
Police in Blagnac just outside Toulouse Credit: Twitter
In France, a teenage gunman takes four women hostage in a Toulouse bar but is confirmed as not an act of terrorism.
A TEENAGER is being questioned by police after allegedly taking four women hostage at a bar on the outskirts of the southern French city of Toulouse.
The 17-year-old remained holed up alone in the establishment in the north-west Blagnac suburb of the city for a couple of hours afterwards before finally being arrested following lengthy negotiations.
The teen, "who is nearly 18, has a police record for incidents of violence, notably against police officers, for robbery, and also for participating in December in a 'yellow vest' protest during which he was arrested," said Toulouse chief prosecutor Dominique Alzeari.
The gunman, who has not been publicly named, burst into the bar in the afternoon, firing two shots and allegedly warning he would fire on police if they approached.
He left a letter at his home "in which he seemed fairly depressive, or at least worried about his state of health, and he made mention of the 'yellow vest' movement but stating that the act he was about to commit would not go beyond what turned out to be not so impressive," said Alzeari.
He was not "someone who was classed as dangerous" and the mayor's office in Blagnac pointed out there was "no suspicion of a terrorist motive."
The gunman released the female owner of the bar first, then a short time later the three women who some witnesses said included bar staff.
A delivery man who said he knew the father of the gunman told media outside the police cordon at the scene that the teen, named 'Yanis,' was a local resident from a nearby downmarket neighbourhood.
He said police had brought the mother to the scene, apparently to help with negotiations. The women were freed 'safe and sound.'
In France a teenage gunman takes four women hostage in a Toulouse bar but is confirmed as not an act of terrorism
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From the interviewed to the interviewer
As frontman of a rock band Damon used to court the British press, now he lives the quiet life in Spain and seeks to get to the heart of the community, scoring exclusive interviews with ex-pats about their successes and struggles during their new life in the sun.
Originally from Scotland but based on the coast for the last three years, Damon strives to bring the most heartfelt news stories from the spanish costas to the Euro Weekly News.


text:
State(s) featured in this episode: New Mexico
Rafting and fishing in the Rio Grande Del Norte National Monument, curious journalists learn the truth about monuments that protect national treasures and a wide range of public uses.
PrevPreviousRestoring a river
NextBalance in the Gunnison basinNext
Where the Colorado River approaches the Sea of Cortez, conservationists re-plant forests and promote wildlife habitat to revive the Delta after decades of neglect and desertification.
State(s) featured in this episode: Arizona / New Mexico
Farmland Runoff, Maintaining National Parks, Rafting Through A Monument
A farmer in southwestern Iowa has a mission to develop his farm as an example to others, using no-till seeding, multi-crop and pasture rotation, minimal fertilizing, and runoff filtering to keep the nutrients in his soil and prevent runoff. The backlog of deferred maintenance in national parks is a growing problem that needs Congress to act: we see the need for urgent maintenance and repairs at the Grand Canyon, the National Mall, and the Martin Luther King, Jr. Historic Site in Atlanta. Rafting down the river through the Rio Grande Del Norte National Monument.
State(s) featured in this episode: Arizona / California / Georgia / Iowa / New Mexico / Washington D.C.
Restoring River Flows, Welcome Back Monarchs
Managing irrigation demand in the upper Colorado Basin: collaborating with landowners, water managers in western Colorado are developing innovative, more efficient systems to conserve water and restore flows to rivers. In Oklahoma, removing invasive cedars and reviving essential prairie habitat for migrating monarch butterflies. In White Sands, New Mexico, researchers study lizards to learn how changing habitats influence evolution.
State(s) featured in this episode: Colorado / New Mexico / Oklahoma
State(s) featured in this episode: New Mexico / South Carolina / Texas
In the southeastern corner of the state, the Brokeoff Mountains are a little-known stretch of rugged canyons and peaks that are still relatively untouched by development.
Navajos in Utah want protection for lost ancestral lands, A new way to save sport fishermen's bycatch, More wilderness protection proposed for remote mountains in New Mexico
The proposal for a national conservation area would preserve Cedar Mesa and adjacent areas that are filled with some of America's oldest archaeological treasures that need urgent protection
Off the coast of San Diego, marine biologists test an experimental device for increasing the survival rate of bottom-dwelling fish that are released at the surface as bycatch but are traumatized by changes in water pressure
In the southeastern corner of the state, the Brokeoff Mountains are a little-known stretch of rugged canyons and peaks that are still relatively untouched by development
State(s) featured in this episode: California / New Mexico / Utah
Rio Grande Del Norte, Climate Adaptation, Flying Aces of the Insect World, Peel Watershed, Indigo Snakes
Saving the Upper Rio Grande: In northern New Mexico the Rio Grande runs through a spectacular gorge formed by a rift in the Earth's crust. This river corridor is a critical flyway for migratory birds, and the arid plateau on either side of it is a major migration habitat for elk and deer. A pending bill in Congress would protect these areas as the Rio Grande del Norte National Conservation Area, in addition to designating two majestic cinder cone mountains east and west of the plateau as protected wilderness. The bill has widespread support among local Hispanic farmers and ranchers because it would allow their traditional hunting, grazing, fishing and wood-gathering to continue, preserving the culture that developed there over hundreds of years.
Facing Climate Change with Wind Power: Severe drought has taken a toll on farming and ranching communities in Eastern New Mexico. Residents are trying to adjust for prolonged dry times, and some are finding salvation in wind turbine projects that generate revenue for them as well as power for the Southwest.
Flying Aces of the Insect World: Just how do these insects pull off complex aerial feats, hunting and reproducing in midair? These four- winged insects pre-date dinosaurs, and can fly straight up, straight down, or hover like helicopters. Researchers are getting some inspiration from these insects, to improve small- scale aircraft design.
Peel Watershed: A hundred miles from the Alaska border in Canada's Yukon Territory, the Peel Watershed is a huge area of wild and pristine rivers, arboreal forests and mountain ranges. Caribou from Alaska migrate to and from the region, but they face threats from a modern day gold rush that also threatens other wildlife including grizzly bears and wolverines. Efforts are underway to protect this land, and these fragile ecosystems. But it looks like a fight is brewing with miners and developers.
Indigo Snakes: Known as the "Lord of the Forest", the eastern indigo snake is the largest native snake in North America, averaging six to seven feet in length. Endangered and in decline, this nonvenomous reptile is extinct from a third of its former range, the coastal plain of the Southeast. The Orianne Society is using cutting edge science, fire, and longleaf pine restoration to ensure the survival of not only indigo snakes, but an entire complex of animals that inhabit this unique landscape.
State(s) featured in this episode: Alaska / Florida / New Mexico
Critical Colorado, Checkups for Manatees, Appalachian Forests
The Colorado River brings drinking water, irrigation, recreation and livelihood to millions of people in the West. But it's clear now that there's not an unlimited supply of this precious resource. Business owners on and near the river are working to make sure their neighbors, and policy makers in Washington, get a complete picture of how critical this river is. Traveling through Arizona and northern Mexico, Bruce Burkhardt shows us there's a lot that needs to be done to protect these waters now and for the future.
Different hikers get different inspiration from the Cherokee National Forest in Tennessee. An artist takes photos that she will later paint; a woodworker studies how trees grow to get ideas for the furniture he builds; and a retired Marine gives back to his community by clearing fallen limbs from the trail. They all support a Congressional designation of this beautiful area as wilderness, so it will be preserved from development for future generations. From the amphibians to the wildflowers to the fishes—the array of diversity in the southern Appalachian forest is just astounding!
Shy, smart, curious and vulnerable: Manatees are slow-moving marine mammals that have not had it easy in recent decades. Diseases and red tide, but mostly strikes from boats and propellers, have killed and injured hundreds of them. Both Florida and federal authorities are stepping up protection of manatees, especially in their winter sanctuaries on the state's west coast. Veterinarians and volunteers conduct physicals on these gentle giants to gauge their health and long- term outlook. Our host Caroline Raville swims with some manatees to bring us the story!
Removing a dam can cause big changes to a community, and to the environment. Before cities make the decision to take down a dam that's either deteriorating or no longer needed, they must be prepared. Researchers at Dartmouth College use sophisticated tools to study river systems to help predict what will happen when the dam is gone. It's all about "shoring up" what we know about how rivers flow, in order to make smart choices when it is time for a dam to come down.
State(s) featured in this episode: Arizona / Colorado / New Mexico


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Garry Littman
OWNER OF THE LANGUAGE HOUSE
Garry Littman is the owner and director of The Language House in Geneva which organises English language training for professional people, companies and students. He was a radio and newspaper journalist in his native Australia and ran a restaurant in Kathmandu in his younger days. He is an English language trainer and an aficionado of pétanque.
24. novembre 2015 6 min
'A splendid income': The world's greatest drug cartel
England's national flower is the red Tudor rose. But the prickly truth is that the English owe much of their wealth to another blood-red flower; the poppy (pavot or coquelicot).
The poppy is much more than just the symbol of remembrance of fallen soldiers on the battlefields of Europe.
The British empire was bankrolled by the milky fluid of the poppy flower; opium.
During the 1800s the empire managed a massive drug cartel based in British India that was both state-sponsored and under Royal patronage.
The British controlled massive fields of poppy farmed by forced Indian labour and built industrial-scale opium factories. They then smuggled hundreds of thousands of tonnes of the highly addictive drug into China during much of the 19th century.
These lithographs below, published in the Scientific American in 1882, show the British opium factory in Patna in the eastern state of Bihar. The first is entitled The Drying Room. The football-like shapes in the foreground, stretching back as far as the eye can see, are tens of thousands of balls of opium. The second picture shows the Stacking Room and third, The Examining Hall.
Opium imports to China increased from around 200 chests (coffres) in 1729 to more than 40,000 smuggled chests (2,160 tonnes) in 1832. After the second Opium War this amount rose to as much as 80,000 chests (4,320 tonnes) per year.
The operation was managed by the British East India Company, a trading company owned by wealthy English merchants and aristocrats, which operated under Royal charter.
Each chest of opium (below) contained about 54 kilos of semi-processed opium. An average ship could transport about 1,000 chests from Calcutta to the port of Canton (Guangzhou) in 25 days.
Why opium trafficking?
The imbalance in trade was somewhat similar to today. European demand for Chinese tea, silk and porcelain was soaring. However, the Chinese were relatively self-sufficient and demand for European goods was almost non-existent. The Chinese demanded payment in silver which began to put pressure on the British coffers.
The idea of using a narcotic to redress the imbalance in trade was conceived by the first Governor General of British India, Warren Hastings, in 1780. Within 10 years, demand for the highly addictive drug had begun to spread and multiply.
The British East India Company circumvented a Chinese ban on opium by sub-contracting opium transportation to 'country traders'- a delightful euphemism for smugglers. These private traders were licensed by the company to take goods from India to China. They sold the opium to smugglers along the Chinese coast for silver and gold which was then paid to the British East India in China. The company then used the sliver and gold to purchase goods that could be sold profitably in England.
By the 1830s the balance of payments had swung back it favour of the British, but at a devastating cost for the Chinese. There were an estimated 12 million opium addicts in around the coastal regions, where an estimated 80 per cent of males under the age of 40 were addicted to opium. Society, business and government collapsed.
The reigning Emperor Dao Guand, whose three sons died from opium overdoses, denounced Britain as "a Christian nation devoid of four of the five Virtues". He appointed the respected statesman Lin Ze-Xu as the Canton regional Commissioner.
Commissioner Lin calculated that in the fiscal year 1839, Chinese opium smokers consumed 100 million taels' worth of the drug while the entire spending by the imperial government that year was only 40 million taels. He wrote "if we continue to allow this trade to flourish, in a few dozen years we will find ourselves not only with no soldiers to resist the enemy, but also with no money to equip the army".
Lin forced the merchants to handover nearly 1,200 tonnes of opium stocks. It took 500 workers 22 days to destroy the opium. The British reacted swiftly and went to war; the first of the two Opium Wars, which the British won easily with their superior arms and ships.
The spoils of war were immense. China was forced to give over the island of Hong Kong (it remained under British control until 1997), open five ports to Western trade and residence, grant Great Britain most-favoured nation status for trade, and compensate the merchants whose opium had been destroyed.
They were also were forced to legalise the opium trade. It was the start of what Chinese historians refer as the "century of humiliation".
In India alone, it is believed opium production accounted for about 17-20% of Indian revenues during much of the 1800s. Unquestionably, the lucrative narcotics trade and war concessions bankrolled the British for many years to come.
In 1888 Rudyard Kipling, author of The Jungle Book, wrote a short essay about production at the Ghazipur Opium factory. He finished his essay with this sentence:
"And this is the way the drug, which yields such a splendid income to the Indian Government, is prepared".
Sea of Poppies (2008) is the first of three novels by Indian novelist Amitav Ghosh which is set in India at the time of the first Opium War
BBC interview with Amitav Ghost
Video: Poppy installation in the Tower of London –
The wearing of poppies on Remembrance Day, November 11 can be traced back to this poem called In Flanders Field by John McCrae:
That mark our place; and in the sky
We are the Dead. Short days ago
We lived, felt dawn, saw sunset glow,
Loved and were loved, and now we lie,
In Flanders fields.
Take up our quarrel with the foe:
To you from failing hands we throw
The torch; be yours to hold it high.
If ye break faith with us who die
We shall not sleep, though poppies grow
GENÈVE/Le Mamco brade des livres sur l'art ce samedi et ce dimanche
LISBONNE/Le Musée Gulbenkian revisite la sculpture du XIXe siècle
A monster from the deep
Scientists confirm Federer is not human
Thanks Donald
Meet Rose. Is it/she the future of banking?
Switzerland: Gateway to the Alps and Gateway to Hell
Smart-phone zombies are the new walking dead


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56 donkeys pulled up for sand mining!
DC | SHRUTI GANAPATYE
Published Aug 3, 2015, 4:07 pm IST
Updated Mar 28, 2019, 8:07 am IST
State minister tells Assembly the donkeys were carrying sand bags
Maharashtra revenue minister Eknath Khadse (Photo: PTI)
Mumbai: While bringing members of the sand mafia to book for illegal mining activities is not unusual, the state government has arrested 56 donkeys from Chandrabhaga river in Pandharpur because they were carrying sand bags. This came to light when revenue minister Eknath Khadse admitted to the arrests in the Legislative Assembly last week while answering a discussion on illegal sand mining in Pandharpur's Chandrabhaga river.
There was a serious debate on the illegal sand mining, which took a hilarious turn when the state brought up the arrests. The House witnessed an outburst of laughter as the minister explained how the government was taking care of the donkeys that were "caught" for illegal mining.
"We have taken action against the sand mafia and have also arrested 56 donkeys carrying sand bags. We have sent them to a government shelter home," Mr Khadse informed the Assembly.
Not wanting to miss an opportunity to create more mirth in the House, NCP MLA Dilip Sopal asked the government to take care of the donkeys. "The donkeys do not know whether they were carrying sand or gold. The government should not ignore their health, and good facilities should be provided to them," Mr Sopal said in a jovial manner, which resulted in enormous belly laughs among the House
members. The minister also replied to the MLA, saying adequate facilities were being given to them. "We have instructed that the donkeys be taken care off so that they are not troubled. In fact, we are trying to reach their owners, even if no one has come forward. We aren't saying their owners are here in the House," Khadse added.
Tags: maharashtra, sand mining
Ram Madhav on CAA: Even Austria amended law for Jews


Cayman National's calendar has become a collector's item for our customers and residents, as well as many visitors to the Cayman Islands. We are delighted to offer a compilation of all the past Cayman National calendar covers.
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Sibernita: A Call for Awareness among Women on Cybercrimes
By Murniati Abu Karim
Photo Shahiddan Saidi
BANGI, July 8 2021– Cybercrimes against working women, retirees and housewives have shown a worrying rise in Malaysia, said the Minister of Women, Family and Community Development Datuk Seri Rina Mohd Harun.
Speaking at the officiating ceremony of Sibernita – a seminar for women on cybersecurity, Datuk Seri Rina expressed concerns over the safety of women in the cyber space as they are exposed to serious threats such as online scams, phishing and espionage as well as data breaches.
There are also cases where women being tricked into having an online romance that end up with victims losing their money, she added.
"On top of that, women suffer from public humiliation over leaked private photos, blackmails and insults by unknown individuals on social media.
"The wide reach of the internet and rapid spread of information could put women at risk hence, it is important for them to be fully aware of the cybercriminals' modus operandi," she continued.
Themed 'Aware, Avoid, Act', the two-day seminar is a joint effort between Universiti Kebangsaan Malaysia (UKM), the Ministry of Women, Family and Community Development (KPWKM) and International Muslim Women's Union (IMWU).
The event was held from 7th to 8th July 2021 in a fully virtual format.
During the seminar, more than 200 participants were exposed to the cybersecurity skillsets which is essential to suppress the cybercrime rate in the country.
Also present at the closing ceremony was UKM Deputy Vice Chancellor (Academic and International) Prof. Dr. Mohammad Kassim.
He said: "The emergence of the Internet as the latest communication tool provides a platform for the dissemination of information that strengthens knowledge and information. However, there is no limitation on what kind of information goes online and that includes false news, propagandas and ideologies.
"I hope this seminar can provide exposure and awareness of cyber safety, especially to the general public, and in turn can combat the escalating numbers of cybercrimes."
The training modules which has been recognized as professional courses were developed by UKM experts in the area of Cyber Security, Law, Linguistics and Islamic Studies.
Towards the new narrative by UKM – 'Universiti Watan Kita', this seminar complements the university's initiatives and the strategies in the Malaysian Digital Economy Blueprint (2021-2030) which prioritizes the safety of the people by building a trusted, secure and ethical digital environment.
The forum sessions feature notable speakers from IMWU, Royal Malaysia Police (PDRM), Cyber Security Academia Malaysia (CSAM) and Universiti Malaya – all are co-organizers of Sibernita alongside IEEE Women in Engineering.
Participants who wish to delve deeper into the topic of cyber safety can look forward to obtaining a Cyber Protection Professional Certificate in the second series of the seminar in August. The modules will include in-depth understanding on the risks, responsibilities, best practices, prevention methods as well as guidance and victim support.
Equine Therapy for Children with Disabilities
A Platform to Discuss Law and Justice
Stakeholders Inception Workshop on LRGS Global Warming
CHILDREN ARE VULNERABLE TO CLIMATE CHANGE Joint study by UNICEF, UKM, UMS discovered the climate impact on children in Malaysia
UKM Creates Chippie Choc Cookies for a Healthy Diet
UKM Doctoral Gold Award Receiver Develops Ulam Raja Supplement to Prevent Dimentia
Media Experts Come Together to Discuss Challenges, Issues in Pandemic World
Tapping into The Mind of Young Researchers
UKM Co-hosted the International Symposium on Islam, Civilization, and Science
MoU to Strengthen Academic and Research in Contemporary Islamic Studies
Prof. Emeritus Dato' Dr. Mohamad Abd Razak Appointed Chairman of UKM Board of Directors
Making Palm Oil Industry More Sustainable
UKM Fresh Graduates Stand a Chance in MADC HyTalent Programme
UKM HADHARI Director Conferred with an Honorary Award from Uzbekistan
Citra UKM Hosted the First International Conference on Liberal Studies
Preparing Medium Small Micro Enterprises in Rural Tourism for Digitalisation
e-AYVP Philippines 2021: Gain Leadership and Volunteerism Skills while Serving the Communities
UKM Royal Fellow Speaks About Youth's Role in the ASEAN Community
Breast Cancer Screenings Plummet During Pandemic
Honing Leadership Qualities Trough Volunteerism and Community Engagement
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Fatema Al-zahraa L.
Experienced physics, math and language tutor
Physics (Electricity and Magnetism)
Is all radiation electromagnetic?
Electromagnetic is just one of several forms of radiation. We use the term "radiation" to describe waves or particles emanating from a source. Thus even a loudspeaker (or a person's mouth) radiates sound waves. Radioactive particles, while some are having an electric charge, are not purely electromagnetic. Those include electrons (beta radiation), protons, helium ions (alpha radiation), neutrons, neutrinos and a long list of other particles many of them are known as "cosmic rays." To put some order in the vast variety of types of radiation, one might describe them by the kind of force they carry. There are only four fundamental forces: gravity, electromagnetic, weak, and strong. Each radiation in nature comprises one or more of these three forces. For example, sound consists of three forces since it involves the electromagnetic interactions between molecules with each molecule possessing all three forces internally.
I hear that Arabic is a difficult language, is that true?
Absolutely not. Arabic is like any other language consists of just 28 letters that join up to form words. Arabic is a Sematic language that has a derivation system, whereby from a single root (defined as a three-letter combination), you can derive a whole array of related meanings. For example, from the root 'a-l-m we get the verbs 'alima (to know), 'allama(to teach),a'lama (to inform),ta'allama (to learn), ista'lama (to inquire). Besides, the way each of these verbs is related to the primary root 'a-l-m helps with vocabulary acquisition. Furthermore, Arabic is written phonetically; therefore, every word is spelled exactly as it sounds. Also, there is no correct intonation to learn in Arabic since all syllables are equally stressed. Of course, Arabic has its fair share of challenges; however, you might find that it is much easier to understand and get along with than you had thought.
When is calculus used in the real world?
Calculus is integrated into almost all disciplines and has many real-life applications. Such disciplines include physics, engineering, economics, statistics, and medicine. It is used to create mathematical models to arrive at an optimal explanation. For instance, calculus is used in most of the physics concepts such as motion, electricity, heat, light, harmonics, acoustics, astronomy, and dynamics. Even advanced physics concepts such as electromagnetism and Einstein's theory of relativity apply calculus. In the field of chemistry, calculus is used to predict functions such as reaction rates and radioactive decay. Meanwhile, in biology, it is utilized to formulate rates such as birth and death rates. In economics, calculus is used to compute marginal cost and marginal revenue, enabling economists to predict maximum profit in a specific setting. Besides, it is used to check answers for different mathematical disciplines such as statistics, analytical geometry, and algebra.
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