Chikungunya virus non-structural protein 2-mediated host shut-off disables the unfolded protein response
Fros, J.J. ; Major, L.D. ; Scholte, F.E. ; Gardner, J. ; Hemert, M.J. van; Suhrbier, A. ; Pijlman, G.P. - \ 2015
Journal of General Virology 96 (2015)3. - ISSN 0022-1317 - p. 580 - 589.
endoplasmic-reticulum stress - semliki-forest-virus - messenger-rna - mammalian-cells - er stress - translational shutoff - transcription factor - gene-expression - insect cells - infection
The unfolded protein response (UPR) is a cellular defence mechanism against high concentrations of misfolded protein in the endoplasmic reticulum (ER). In the presence of misfolded proteins, ER-transmembrane proteins PERK and IRE1a become activated. PERK phosphorylates eIF2a leading to a general inhibition of cellular translation, whilst the expression of transcription factor ATF4 is upregulated. Active IRE1a splices out an intron from XBP1 mRNA, to produce a potent transcription factor. Activation of the UPR increases the production of several proteins involved in protein folding, degradation and apoptosis. Here, we demonstrated that transient expression of chikungunya virus (CHIKV) (family Togaviridae, genus Alphavirus) envelope glycoproteins induced the UPR and that CHIKV infection resulted in the phosphorylation of eIF2a and partial splicing of XBP1 mRNA. However, infection with CHIKV did not increase the expression of ATF4 and known UPR target genes (GRP78/BiP, GRP94 and CHOP). Moreover, nuclear XBP1 was not observed during CHIKV infection. Even upon stimulation with tunicamycin, the UPR was efficiently inhibited in CHIKV-infected cells. Individual expression of CHIKV non-structural proteins (nsPs) revealed that nsP2 alone was sufficient to inhibit the UPR. Mutations that rendered nsP2 unable to cause host-cell shut-off prevented nsP2-mediated inhibition of the UPR. This indicates that initial UPR induction takes place in the ER but that expression of functional UPR transcription factors and target genes is efficiently inhibited by CHIKV nsP2.
An acute intake of plant stanol esters alters immune-related pathways in the jejunum of healthy volunteers
Smet, E. de; Mensink, M.R. ; Boekschoten, M.V. ; Ridder, R. de; Germeraad, W.T.V. ; Wolfs, T.G.A.M. ; Plat, J. - \ 2015
The British journal of nutrition 113 (2015)5. - ISSN 0007-1145 - p. 794 - 802.
atp-binding cassette - triglyceride transfer protein - niemann-pick c1-like-1 - cholesterol-metabolism - dietary phytosterols - beta-sitosterol - sterol-metabolism - messenger-rna - expression - abcg8
Plant sterols and stanols inhibit intestinal cholesterol absorption and consequently lower serum LDL-cholesterol (LDL-C) concentrations. The underlying mechanisms are not yet known. In vitro and animal studies have suggested that changes in intestinal sterol metabolism are attributed to the LDL-C-lowering effects of plant stanol esters. However, similar studies in human subjects are lacking. Therefore, we examined the effects of an acute intake of plant stanol esters on gene expression profiles of the upper small intestine in healthy volunteers. In a double-blind cross-over design, fourteen healthy subjects (eight female and six male; age 21–55 years), with a BMI ranging from 21 to 29 kg/m2, received in random order a shake with or without plant stanol esters (4 g). At 5 h after consumption of the shake, biopsies were taken from the duodenum (around the papilla of Vater) and from the jejunum (20 cm distal from the papilla of Vater). Microarray analysis showed that the expression profiles of genes involved in sterol metabolism were not altered. Surprisingly, the pathways involved in T-cell functions were down-regulated in the jejunum. Furthermore, immunohistochemical analysis showed that the number of CD3 (cluster of differentiation number 3), CD4 (cluster of differentiation number 4) and Foxp3+ (forkhead box P3-positive) cells was reduced in the plant stanol ester condition compared with the control condition, which is in line with the microarray data. The physiological and functional consequences of the plant stanol ester-induced reduction of intestinal T-cell-based immune activity in healthy subjects deserve further investigation.
Comprehensive insights into transcriptional adaptation of intracellular mycobacteria by microbe-enriched dual RNA sequencing
Rienksma, R.A. ; Suarez Diez, M. ; Mollenkopf, H.J. ; Dolganov, G.M. ; Dorhoi, A. ; Schoolnik, G.K. ; Martins Dos Santos, V.A.P. ; Kaufmann, S. ; Schaap, P.J. ; Gengenbacher, M. - \ 2015
BMC Genomics 16 (2015). - ISSN 1471-2164 - 31 p.
tuberculosis gene-expression - human macrophage infection - complete genome sequence - cholesterol catabolism - regulated genes - messenger-rna - acyl-coenzyme - in-vitro - host - pathogen
BackgroundThe human pathogen Mycobacterium tuberculosis has the capacity to escape eradication by professional phagocytes. During infection, M. tuberculosis resists the harsh environment of phagosomes and actively manipulates macrophages and dendritic cells to ensure prolonged intracellular survival. In contrast to other intracellular pathogens, it has remained difficult to capture the transcriptome of mycobacteria during infection due to an unfavorable host-to-pathogen ratio.ResultsWe infected the human macrophage-like cell line THP-1 with the attenuated M. tuberculosis surrogate M. bovis Bacillus Calmette¿Guérin (M. bovis BCG). Mycobacterial RNA was up to 1000-fold underrepresented in total RNA preparations of infected host cells. We employed microbial enrichment combined with specific ribosomal RNA depletion to simultaneously analyze the transcriptional responses of host and pathogen during infection by dual RNA sequencing. Our results confirm that mycobacterial pathways for cholesterol degradation and iron acquisition are upregulated during infection. In addition, genes involved in the methylcitrate cycle, aspartate metabolism and recycling of mycolic acids were induced. In response to M. bovis BCG infection, host cells upregulated de novo cholesterol biosynthesis presumably to compensate for the loss of this metabolite by bacterial catabolism.ConclusionsDual RNA sequencing allows simultaneous capture of the global transcriptome of host and pathogen, during infection. However, mycobacteria remained problematic due to their relatively low number per host cell resulting in an unfavorable bacterium-to-host RNA ratio. Here, we use a strategy that combines enrichment for bacterial transcripts and dual RNA sequencing to provide the most comprehensive transcriptome of intracellular mycobacteria to date. The knowledge acquired into the pathogen and host pathways regulated during infection may contribute to a solid basis for the deployment of novel intervention strategies to tackle infection
Effects of dry period length and dietary energy source on metabolic status and hepatic gene expression of dairy cows in early lactation
Chen, J.C. ; Gross, J.J. ; Dorland, H.A. van; Remmelink, G.J. ; Bruckmaier, R.M. ; Kemp, B. ; Knegsel, A.T.M. van - \ 2015
Journal of Dairy Science 98 (2015)2. - ISSN 0022-0302 - p. 1033 - 1045.
organic nutrient metabolism - messenger-rna - transition period - somatotropic axis - milk-production - fatty-acids - liver - balance - system - performance
In a prior study, we observed that cows with a 0-d dry period had greater energy balance and lower milk production compared with cows with a 30- or 60-d dry period in early lactation. The objective of the current study was to evaluate the influence of dry period length on metabolic status and hepatic gene expression in cows fed a lipogenic or glucogenic diet in early lactation. Holstein-Friesian dairy cows (n = 167) were assigned randomly to 3 × 2 factorial design with 3 dry period lengths (n = 56, 55, and 56 for 0-, 30-, and 60-d dry, respectively) and 2 early lactation diets (n = 84 and 83 for glucogenic and lipogenic diet, respectively). Cows were fed a glucogenic or lipogenic diet from 10 d before the expected calving date and onward. The main ingredient for a glucogenic concentrate was corn, and the main ingredients for a lipogenic concentrate were sugar beet pulp, palm kernel, and rumen-protected palm oil. Blood was sampled weekly from 95 cows from wk 3 precalving to wk 8 postcalving. Liver samples were collected from 76 cows in wk -2, 2, and 4 relative to calving. Liver samples were analyzed for triacylglycerol concentrations and mRNA expression of 12 candidate genes. Precalving, cows with a 0-d dry period had greater plasma ß-hydroxybutyrate, urea, and insulin concentrations compared with cows with a 30- or 60-d dry period. Postcalving, cows with a 0-d dry period had lower liver triacylglycerol and plasma nonesterified fatty acids concentrations (0.20, 0.32, and 0.36 mmol/L for 0-, 30-, and 60-d dry period, respectively), greater plasma glucose, insulin-like growth factor-I, and insulin (24.38, 14.02, and 11.08 µIU/mL for 0-, 30-, and 60-d dry period, respectively) concentrations, and lower hepatic mRNA expression of pyruvate carboxylase, compared with cows with a 30- or 60-d dry period. Plasma urea and ß-hydroxybutyrate concentrations were greater in cows fed a lipogenic diet compared with cows fed a glucogenic diet. In conclusion, cows with a 0-d dry period had an improved metabolic status in early lactation, indicated by lower plasma concentrations of nonesterified fatty acids, greater plasma concentrations of glucose, insulin-like growth factor-I, and insulin, and lower mRNA expression of pyruvate carboxylase in the liver, compared with cows with a 30- or 60-d dry period. Independent of dry period length, the glucogenic diet also improved the metabolic status compared with the lipogenic diet.
Bayesian GWAS and network analysis revealed new candidate genes for number of teats in pigs
Verardo, L.L. ; Silva, F.F. ; Varona, L. ; Resende, R. ; Bastiaansen, J.W.M. ; Lopes, P.S. ; Guimaraes, S.E.F. - \ 2015
Journal of Applied Genetics 56 (2015)1. - ISSN 1234-1983 - p. 123 - 132.
quantitative trait loci - stress-syndrome gene - divergent crosses - messenger-rna - mixed models - r-package - association - snp - receptor - identification
The genetic improvement of reproductive traits such as the number of teats is essential to the success of the pig industry. As opposite to most SNP association studies that consider continuous phenotypes under Gaussian assumptions, this trait is characterized as a discrete variable, which could potentially follow other distributions, such as the Poisson. Therefore, in order to access the complexity of a counting random regression considering all SNPs simultaneously as covariate under a GWAS modeling, the Bayesian inference tools become necessary. Currently, another point that deserves to be highlighted in GWAS is the genetic dissection of complex phenotypes through candidate genes network derived from significant SNPs. We present a full Bayesian treatment of SNP association analysis for number of teats assuming alternatively Gaussian and Poisson distributions for this trait. Under this framework, significant SNP effects were identified by hypothesis tests using 95 % highest posterior density intervals. These SNPs were used to construct associated candidate genes network aiming to explain the genetic mechanism behind this reproductive trait. The Bayesian model comparisons based on deviance posterior distribution indicated the superiority of Gaussian model. In general, our results suggest the presence of 19 significant SNPs, which mapped 13 genes. Besides, we predicted gene interactions through networks that are consistent with the mammals known breast biology (e.g., development of prolactin receptor signaling, and cell proliferation), captured known regulation binding sites, and provided candidate genes for that trait (e.g., TINAGL1 and ICK).
Thirty years of baculovirus-insect cell protein expression: From dark horse to mainstream technology
Oers, M.M. van; Pijlman, G.P. ; Vlak, J.M. - \ 2015
Journal of General Virology 96 (2015)1. - ISSN 0022-1317 - p. 6 - 23.
nuclear polyhedrosis-virus - late gene-expression - human fibroblast interferon - n-glycosylation pathway - large-scale production - non-hr origin - spodoptera-frugiperda - escherichia-coli - messenger-rna - mammalian-cells
In December 1983 a seminal paper appeared on the overexpression of human interferon-ß in insect cells with a genetically engineered baculovirus. The finding that baculoviruses produce massive amounts of two proteins (polyhedrin and p10) by means of two very strong promoters and that the corresponding genes are dispensable for virus propagation in insect cells was crucial in the development of this expression system. During the next 30 years major improvements have been achieved over the original baculovirus expression vector (BEV) system, facilitating the engineering of the baculovirus vectors, the modification of the sugar moieties of glycoproteins expressed in insect cells, and the scale-up of the cell culture process. To date, thousands of recombinant proteins have been produced in this successful expression system, including several protein-based human and veterinary vaccines that are currently on the market. Viral vectors based on adeno-associated virus are being produced using recombinant baculovirus technology and the first gene therapy treatment based on this method has been registered. Specially adapted baculovirus expression vectors are used to deliver and express heterologous genes in mammalian cells and may find applications for gene therapy and cancer treatment in the future. The purpose of this paper is to highlight the 30-years 'anniversary' of this expression system by summarizing the fundamental research that allowed the development of this expression system and by indicating the major technological advances since 1983. Finally, attention will be paid to the future challenges to further optimize this amazing technology.
Creation of Rift Valley Fever Viruses with four-segmented Genomes reveals flexibility in Bunyavirus Genome Packaging.
Wichgers Schreur, P.J. ; Oreshkova, N. ; Moormann, R.J.M. ; Kortekaas, J.A. - \ 2014
Journal of Virology 88 (2014)18. - ISSN 0022-538X - p. 10883 - 10893.
defective interfering particles - noncoding regions - immune-response - golgi retention - messenger-rna - nsm protein - replication - segment - glycoprotein - expression
Bunyavirus genomes comprise a small (S), medium (M) and a large (L) RNA segment of negative polarity. Although the untranslated regions (UTRs) have been shown to comprise signals required for transcription, replication and encapsidation, the mechanisms that drive the packaging of at least one S, M and L segment into a single virion to generate infectious virus are largely unknown. One of the most important members of the Bunyaviridae family that causes devastating disease in ruminants and occasionally humans is the Rift Valley fever virus (RVFV). Here we studied the flexibility of RVFV genome packaging by splitting the glycoprotein precursor gene, encoding the (NSm)GnGc polyprotein, into two individual genes encoding either (NSm)Gn or Gc. Using reverse-genetics, six viruses with a segmented glycoprotein precursor gene were rescued, varying from a virus comprising 2 S-type segments in the absence of an M-type segment to a virus consisting of 4 segments (RVFV-4s) of which 3 are M-type. Despite that all virus variants were able to grow in mammalian cell lines, they were unable to spread efficiently in cells of mosquito origin. Moreover, in vivo studies demonstrated that RVFV-4s is unable to cause disseminated infection and disease in mice, even in the presence of the main virulence factor NSs, but induced a protective immune response against a lethal challenge with wild-type virus. In summary, splitting bunyavirus glycoprotein precursor genes provides new opportunities to study bunyavirus genome packaging and offers new methods to develop next-generation live-attenuated bunyavirus vaccines.
Early Changes in Microbial Colonization Selectively Modulate Intestinal Enzymes, but Not Inducible Heat Shock Proteins in Young Adult Swine
Arnal, M.E. ; Zhang, J. ; Messori, S. ; Bosi, P. ; Smidt, H. ; Lallès, J.P. - \ 2014
PLoS ONE 9 (2014)5. - ISSN 1932-6203 - 14 p.
alkaline-phosphatase - epithelial-cells - gut microbiota - gastrointestinal-tract - gene-expression - messenger-rna - piglets - growth - diet - rat
Metabolic diseases and obesity are developing worldwide in a context of plethoric intake of high energy diets. The intestine may play a pivotal role due to diet-induced alterations in microbiota composition and increased permeability to bacterial lipopolysaccharide inducing metabolic inflammation. Early programming of metabolic disorders appearing in later life is also suspected, but data on the intestine are lacking. Therefore, we hypothesized that early disturbances in microbial colonization have short- and long-lasting consequences on selected intestinal components including key digestive enzymes and protective inducible heat shock proteins (HSP). The hypothesis was tested in swine offspring born to control mothers (n = 12) or mothers treated with the antibiotic amoxicillin around parturition (n = 11), and slaughtered serially at 14, 28 and 42 days of age to assess short-term effects. To evaluate long-term consequences, young adult offspring from the same litters were offered a normal or a fat-enriched diet for 4 weeks between 140 and 169 days of age and were then slaughtered. Amoxicillin treatment transiently modified both mother and offspring microbiota. This was associated with early but transient reduction in ileal alkaline phosphatase, HSP70 (but not HSP27) and crypt depth, suggesting a milder or delayed intestinal response to bacteria in offspring born to antibiotic-treated mothers. More importantly, we disclosed long-term consequences of this treatment on jejunal alkaline phosphatase (reduced) and jejunal and ileal dipeptidylpeptidase IV (increased and decreased, respectively) of offspring born to antibiotic-treated dams. Significant interactions between early antibiotic treatment and later diet were observed for jejunal alkaline phosphatase and sucrase. By contrast, inducible HSPs were not affected. In conclusion, our data suggest that early changes in bacterial colonization not only modulate intestinal architecture and function transiently, but also exert site- and sometimes diet-specific long-term effects on key components of intestinal homeostasis.
REDUCED DORMANCY5 Encodes a Protein Phosphatase 2C That Is Required for Seed Dormancy in Arabidopsis
Xiang, Y. ; Nakabayashi, K. ; Ding, J. ; He, F. ; Bentsink, L. ; Soppe, W.J.J. - \ 2014
The Plant Cell 26 (2014)11. - ISSN 1040-4651 - p. 4362 - 4375.
rna-binding proteins - abscisic-acid - messenger-rna - pp2c phosphatases - germination - thaliana - aba - reveals - gene - mutants
Seed dormancy determines germination timing and contributes to crop production and the adaptation of natural populations to their environment. Our knowledge about its regulation is limited. In a mutagenesis screen of a highly dormant Arabidopsis thaliana line, the reduced dormancy5 (rdo5) mutant was isolated based on its strongly reduced seed dormancy. Cloning of RDO5 showed that it encodes a PP2C phosphatase. Several PP2C phosphatases belonging to clade A are involved in abscisic acid signaling and control seed dormancy. However, RDO5 does not cluster with clade A phosphatases, and abscisic acid levels and sensitivity are unaltered in the rdo5 mutant. RDO5 transcript could only be detected in seeds and was most abundant in dry seeds. RDO5 was found in cells throughout the embryo and is located in the nucleus. A transcriptome analysis revealed that several genes belonging to the conserved PUF family of RNA binding proteins, in particular Arabidopsis PUMILIO9 (APUM9) and APUM11, showed strongly enhanced transcript levels in rdo5 during seed imbibition. Further transgenic analyses indicated that APUM9 reduces seed dormancy. Interestingly, reduction of APUM transcripts by RNA interference complemented the reduced dormancy phenotype of rdo5, indicating that RDO5 functions by suppressing APUM transcript levels.
Adipocyte spliced form of X-box-binding protein 1 promotes adiponectin multimerization and systemic glucose homeostasis
Sha, H. ; Yang, L. ; Liu, M. ; Xia, S. ; Liu, Y. ; Liu, F. ; Kersten, A.H. ; Qi, L. - \ 2014
Diabetes 63 (2014)3. - ISSN 0012-1797 - p. 867 - 879.
endoplasmic-reticulum stress - plasma-cell differentiation - transcription factor xbp-1 - links er stress - down-regulation - messenger-rna - dsba-l - insulin sensitivity - adipose-tissue - obesity
The physiological role of the spliced form of X-box–binding protein 1 (XBP1s), a key transcription factor of the endoplasmic reticulum (ER) stress response, in adipose tissue remains largely unknown. In this study, we show that overexpression of XBP1s promotes adiponectin multimerization in adipocytes, thereby regulating systemic glucose homeostasis. Ectopic expression of XBP1s in adipocytes improves glucose tolerance and insulin sensitivity in both lean and obese (ob/ob) mice. The beneficial effect of adipocyte XBP1s on glucose homeostasis is associated with elevated serum levels of high-molecular-weight adiponectin and, indeed, is adiponectin-dependent. Mechanistically, XBP1s promotes adiponectin multimerization rather than activating its transcription, likely through a direct regulation of the expression of several ER chaperones involved in adiponectin maturation, including glucose-regulated protein 78 kDa, protein disulfide isomerase family A, member 6, ER protein 44, and disulfide bond oxidoreductase A–like protein. Thus, we conclude that XBP1s is an important regulator of adiponectin multimerization, which may lead to a new therapeutic approach for the treatment of type 2 diabetes and hypoadiponectinemia.
Noncoding subgenomic flavivirus RNA: multiple functions in West Nile virus pathogenesis and modulation of host responses
Roby, J.A. ; Pijlman, G.P. ; Wilusz, J. ; Khromykh, A.A. - \ 2014
Viruses 6 (2014)2. - ISSN 1999-4915 - p. 404 - 427.
japanese encephalitis-virus - double-stranded-rna - la protein binds - innate immune-response - 3 untranslated region - dengue virus - genomic rna - messenger-rna - viral-rna - 3'-untranslated region
Flaviviruses are a large group of positive strand RNA viruses transmitted by arthropods that include many human pathogens such as West Nile virus (WNV), Japanese encephalitis virus (JEV), yellow fever virus, dengue virus, and tick-borne encephalitis virus. All members in this genus tested so far are shown to produce a unique subgenomic flavivirus RNA (sfRNA) derived from the 3' untranslated region (UTR). sfRNA is a product of incomplete degradation of genomic RNA by the cell 5'–3' exoribonuclease XRN1 which stalls at highly ordered secondary RNA structures at the beginning of the 3'UTR. Generation of sfRNA results in inhibition of XRN1 activity leading to an increase in stability of many cellular mRNAs. Mutant WNV deficient in sfRNA generation was highly attenuated displaying a marked decrease in cytopathicity in cells and pathogenicity in mice. sfRNA has also been shown to inhibit the antiviral activity of IFN-a/ß by yet unknown mechanism and of the RNAi pathway by likely serving as a decoy substrate for Dicer. Thus, sfRNA is involved in modulating multiple cellular pathways to facilitate viral pathogenicity; however the overlying mechanism linking all these multiple functions of sfRNA remains to be elucidated.
Flavivirus RNAi suppression: decoding non-coding RNA
Pijlman, G.P. - \ 2014
Current Opinion in Virology 7 (2014). - ISSN 1879-6257 - p. 55 - 60.
west-nile-virus - aedes-aegypti mosquitos - double-stranded-rna - arbovirus infection - antiviral immunity - albopictus cells - subgenomic rna - messenger-rna - nss protein - interference
Flaviviruses are important human pathogens that are transmitted by invertebrate vectors, mostly mosquitoes and ticks. During replication in their vector, flaviviruses are subject to a potent innate immune response known as antiviral RNA interference (RNAi). This defense mechanism is associated with the production of small interfering (si)RNA that lead to degradation of viral RNA. To what extent flaviviruses would benefit from counteracting antiviral RNAi is subject of debate. Here, the experimental evidence to suggest the existence of flavivirus RNAi suppressors is discussed. I will highlight the putative role of non-coding, subgenomic flavivirus RNA in suppression of RNAi in insect and mammalian cells. Novel insights from ongoing research will reveal how arthropod-borne viruses modulate innate immunity including antiviral RNAi.
Viral replication kinetics and in vitro cytopathogenicity of parental and reassortant strains of bluetongue virus serotype 1, 6 and 8
Coetzee, M.P.A. ; Vuuren, M. van; Stokstad, M. ; Myrmel, M. ; Gennip, H.G.P. van; Rijn, P.A. van; Venter, E.H. - \ 2014
Veterinary Microbiology 171 (2014)1-2. - ISSN 0378-1135 - p. 53 - 65.
genome segment reassortment - newcastle-disease virus - nonstructural protein - mixed infection - transplacental transmission - culicoides-variipennis - genetic reassortment - avirulent strains - bovine fetuses - messenger-rna
Bluetongue virus (BTV), a segmented dsRNA virus, is the causative agent of bluetongue (BT), an economically important viral haemorrhagic disease of ruminants. Bluetongue virus can exchange its genome segments in mammalian or insect cells that have been co-infected with more than one strain of the virus. This process, may potentially give rise to the generation of novel reassortant strains that may differ from parental strains in regards to their phenotypic characteristics. To investigate the potential effects of reassortment on the virus’ phenotype, parental as well as reassortant strains of BTV serotype 1, 6, 8, that were derived from attenuated and wild type strains by reverse genetics, were studied in vitro for their virus replication kinetics and cytopathogenicity in mammalian (Vero) cell cultures. The results indicate that genetic reassortment can affect viral replication kinetics, the cytopathogenicity and extent/mechanism of cell death in infected cell cultures. In particular, some reassortants of non-virulent vaccine (BTV-1 and BTV-6) and virulent field origin (BTV-8) demonstrate more pronounced cytopathic effects compared to their parental strains. Some reassortant strains in addition replicated to high titres in vitro despite being composed of genome segments from slow and fast replicating parental strains. The latter result may have implications for the level of viraemia in the mammalian host and subsequent uptake and transmission of reassortant strains (and their genome segments) by Culicoides vectors. Increased rates of CPE induction could further suggest a higher virulence for reassortant strains in vivo. Overall, these findings raise questions in regards to the use of modified-live virus (MLV) vaccines and risk of reassortment in the field. To further address these questions, additional experimental infection studies using insects and/or animal models should be conducted, to determine whether these results have significant implications in vivo
Common and rare single nucleotide polymorphisms in the LDLR gene are present in a black South African population and associate with low-density lipoprotein cholesterol levels
Zyl, T. ; Jerling, J.C. ; Conradie, K.R. ; Feskens, E.J.M. - \ 2014
Journal of Human Genetics 59 (2014). - ISSN 1434-5161 - p. 88 - 94.
coronary-heart-disease - plasma-lipid levels - receptor gene - familial hypercholesterolemia - messenger-rna - human genome - mutations - risk - stabilization - expression
The LDL receptor has an essential role in regulating plasma LDL-C levels. Genetic variation in the LDLR gene can be associated with either lower or moderately raised plasma levels of LDL-C, or may cause familial hypercholesterolemia. The prevalence of single-nucleotide polymorphisms (SNPs) in the LDLR in the black South African population is not known and therefore, we aimed to determine the genotypic variation of the LDLR in the study population as well as to define the association of the different genotypes with plasma LDL-C levels. A random selection of 1860 apparently healthy black South African volunteers aged 35–60 years was made in a cross-sectional study. Novel SNPs were identified in a subset of 30 individuals by means of automated sequencing before screening the entire cohort by means of the Illumina VeraCode GoldenGate Genotyping Assay on a BeadXpress Reader system. Twenty-five SNPs were genotyped, two of which were novel. A very rare SNP, rs17249141, in the promoter region was significantly associated with lower levels of LDL-C. Four other SNPs (rs2738447, rs14158, rs2738465 and rs3180023) were significantly associated with increased levels of LDL-C. We can conclude that some of the various SNPs identified do indeed associate with LDL-C levels
Planting the seed: target recognition of short guide RNAs
Künne, T. ; Swarts, D.C. ; Brouns, S.J.J. - \ 2014
Trends in Microbiology 22 (2014)1. - ISSN 0966-842X - p. 74 - 83.
bacterial immune-system - staphylococcus-aureus reveals - binding small rnas - sm-like protein - messenger-rna - crispr rna - escherichia-coli - streptococcus-thermophilus - noncoding rnas - enzyme complex
Small guide RNAs play important roles in cellular processes such as regulation of gene expression and host defense against invading nucleic acids. The mode of action of small RNAs relies on protein-assisted base pairing of the guide RNA with target mRNA or DNA to interfere with their transcription, translation, or replication. Several unrelated classes of small noncoding RNAs have been identified including eukaryotic RNA silencing-associated small RNAs, prokaryotic small regulatory RNAs (sRNAs), and prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats) RNAs (crRNAs). All three groups identify their target sequence by base pairing after finding it in a pool of millions of other nucleotide sequences in the cell. In this complicated target search process, a region of 612 nucleotides (nt) of the small RNA termed the seed plays a critical role. We review the concept of seed sequences and discuss its importance for initial target recognition and interference
Immune adjuvants as critical guides directing immunity triggered by therapeutic cancer vaccines
Schijns, V.E.J.C. ; Tartour, E. ; Michalek, J. ; Stathopoulos, A. ; Dobrovolskiene, N.T. ; Strioga, M.M. - \ 2014
Cytotherapy 16 (2014)4. - ISSN 1465-3249 - p. 427 - 439.
dendritic cells induce - human langerhans cells - cd8 t-cells - melanoma patients - messenger-rna - lymph-nodes - in-vivo - alpha-galactosylceramide - antigen - vaccination
Tumor growth is controlled by natural antitumor immune responses alone or by augmented immune reactivity resulting from different forms of immunotherapy, which has demonstrated clinical benefit in numerous studies, although the overall percentage of patients with durable clinical responses remains limited. This is attributed to the heterogeneity of the disease, the inclusion of late-stage patients with no other treatment options and advanced tumor-associated immunosuppression, which may be consolidated by certain types of chemotherapy. Despite variable responsiveness to distinct types of immunotherapy, therapeutic cancer vaccination has shown meaningful efficacy for a variety of cancers. A key step during cancer vaccination involves the appropriate modeling of the functional state of dendritic cells (DCs) capable of co-delivering four critical signals for proper instruction of tumor antigen–specific T cells. However, the education of DCs, either directly in situ, or ex vivo by various complex procedures, lacks standardization. Also, it is questioned whether ex vivo–prepared DC vaccines are superior to in situ–administered adjuvant-guided vaccines, although both approaches have shown success. Evaluation of these variables is further complicated by a lack of consensus in evaluating vaccination clinical study end points. We discuss the role of signals needed for the preparation of classic in situ and modern ex vivo DC vaccines capable of proper reprogramming of antitumor immune responses in patients with cancer.
Seasonal differences in cytokine expression in the skin of Shetland ponies suffering from insect bite hypersensitivity
Meulenbroeks, C. ; Meide, N.M.A. van der; Zaiss, D.M.W. ; Oldruitenborgh-Oosterbaan, M.M.S. ; Lugt, J.J. van der; Smak, J. ; Rutten, V.P.M.G. ; Willemse, T. - \ 2013
Veterinary Immunology and Immunopathology 151 (2013)1-2. - ISSN 0165-2427 - p. 147 - 156.
culicoides-hypersensitivity - messenger-rna - icelandic horses - igg antibodies - sweet itch - allergy - netherlands - cells - flea - identification
Insect bite hypersensitivity (IBH) in horses is a seasonal, IgE-mediated, pruritic skin disorder primarily caused by Culicoides spp. We hypothesize that a mixed Th2/Th1-type immune status, off season, alters into Th2-dominated immune reactivity in the skin of IBH-affected ponies in the IBM season. To study these immune response patterns Culicoides-specific IgE levels, skin histopathology and cytokine and transcription factor mRNA expression (IL4, IL10, IL13, IFN gamma, FoxP3 and CD3(zeta)) in lesional and non-lesional skin of ponies affected by IBH in the IBH season were compared with those of the same animals off season and those in skin of healthy ponies in both seasons. The present study revealed a significantly higher histopathology score in lesional skin of affected ponies than in non-lesional skin and skin of healthy ponies in the IBH season. Culicoides obsoletus-specific IgE serum levels of ponies with IBH were significantly higher than those in healthy ponies in both seasons. Interestingly, C. obsoletus-specific IgE serum levels within each group were the same in the IBH season and off season. The expression of IL4, IL13 and IFN gamma mRNA in skin biopsies in the IBH season showed a significant increase compared to off season in both skin derived from healthy control ponies (n = 14) as well as in lesional and in non-lesional skin from IBH-affected animals (n = 17). This apparently general up-regulation of cytokine expression during the IBH season directly correlated with an increased CD3(zeta) mRNA expression in the skin, indicating an overall increased T cell influx during the summer months. The only significant difference observed between lesional skin from IBH-affected animals as compared to skin from healthy control animals in the IBH season was a lower expression of IL13/CD3(zeta) in the affected animals. FoxP3 and IL10 levels were unaffected, except for a lower expression of FoxP3 in healthy control skin in the IBM season as compared to off season, In addition, the increased level of C obsoletus-specific IgE did not correlate with higher histological scores in LE skin. In summary, our data indicate a general immune activation in the skin of both healthy and IBH-affected ponies during the IBH season that potentially obscures the Culicoides-specific immune reaction pattern, even in lesional skin of IBH-affected animals. (C) 2012 Elsevier B.V. All rights reserved.
Single Mutation in Shine-Dalgarno-Like Sequence Present in the Amino Terminal of Lactate Dehydrogenase of Plasmodium Effects the Production of an Eukaryotic Protein Expressed in a Prokaryotic System
Cicek, M. ; Mutlu, O. ; Erdemir, A. ; Ozkan, E. ; Saricay, Y. ; Turgut-Balik, D. - \ 2013
Molecular Biotechnology 54 (2013)2. - ISSN 1073-6085 - p. 602 - 608.
human malaria parasite - escherichia-coli - messenger-rna - recombinant proteins - bacillus-subtilis - ribosomal-rna - q-beta - falciparum - genome - gene
One of the most important step in structure-based drug design studies is obtaining the protein in active form after cloning the target gene. In one of our previous study, it was determined that an internal Shine-Dalgarno-like sequence present just before the third methionine at N-terminus of wild type lactate dehydrogenase enzyme of Plasmodium falciparum prevent the translation of full length protein. Inspection of the same region in P. vivax LDH, which was overproduced as an active enzyme, indicated that the codon preference in the same region was slightly different than the codon preference of wild type PfLDH. In this study, 5'-GGAGGC-3' sequence of P. vivax that codes for two glycine residues just before the third methionine was exchanged to 5'-GGAGGA-3', by mimicking P. falciparum LDH, to prove the possible effects of having an internal SD-like sequence when expressing an eukaryotic protein in a prokaryotic system. Exchange was made by site-directed mutagenesis. Results indicated that having two glycine residues with an internal SD-like sequence (GGAGGA) just before the third methionine abolishes the enzyme activity due to the preference of the prokaryotic system used for the expression. This study emphasizes the awareness of use of a prokaryotic system to overproduce an eukaryotic protein.
Alterations in mucosal neuropeptides in patients with irritable bowel syndrome and ulcerative colitis in remission: A role in pain symptom generation?
Keszthelyi, D. ; Troost, F.J. ; Jonkers, D.M. ; Helyes, Z. ; Hamer, H.M. ; Ludidi, S. ; Vanhoutvin, S. ; Venema, K. ; Dekker, J. ; Szolcsanyi, J. ; Masclee, A.A. - \ 2013
European Journal of Pain 17 (2013)9. - ISSN 1090-3801 - p. 1299 - 1306.
vanilloid receptor vr1 - gastrointestinal-tract - abdominal-pain - substance-p - axonal-transport - trpv1 receptor - messenger-rna - rectal mucosa - expression - disease
Background Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder characterized by chronic abdominal pain. The transient receptor potential vanilloid 1 (TRPV1) channel, which is involved in visceral pain signalling, has been shown to be up-regulated in IBS. Activation of TRPV1 leads to the release of neuropeptides, such as somatostatin and substance P (SP). We hypothesized that increased pain perception in IBS could be explained by increased transcription in TRPV1 and/or altered levels of neuropeptides. We therefore assessed the transcription of TRPV1 and the mucosal concentration of somatostatin and SP in IBS in comparison to healthy volunteers and patients with ulcerative colitis (UC) in remission as disease controls, and to ascertain their relationship to pain symptoms. Method Sigmoid colonic mucosal samples were collected from 12 patients with IBS, 34 patients with UC in remission and 9 healthy volunteers, in which groups TRPV1 mRNA levels were determined using quantitative polymerase chain reaction and neuropeptide concentrations by radioimmunoassay. Pain symptom intensity was determined by questionnaires. Results Transcription of TRPV1 as well as the concentration of neuropeptides were significantly higher in IBS, but only the former correlated with pain symptom severity. Conclusion Increased transcription of TRPV1 may provide a possible explanation for pain generation in IBS. While the neuropeptides SP and somatostatin were both found to be increased in IBS, these changes are not sufficient to explain pain generation. Pain generation in IBS is probably explained by a complex redundancy in the regulation of local nociceptive mechanisms, which remains a subject of intensive investigation.
Mechanisms underlying the dualistic mode of action of major soy isoflavones in relation to cell proliferation and cancer risks
Rietjens, I.M.C.M. ; Sotoca, A.M. ; Vervoort, J. ; Louisse, J. - \ 2013
Molecular Nutrition & Food Research 57 (2013)1. - ISSN 1613-4125 - p. 100 - 113.
estrogen-receptor-beta - randomized controlled-trial - health initiative memory - mediated gene-regulation - tumor-suppressor genes - breast-cancer - prostate-cancer - postmenopausal women - in-vitro - messenger-rna
Isoflavones are phytoestrogens that have been linked to both beneficial as well as adverse effects in relation to cell proliferation and cancer risks. The present article presents an overview of these seemingly contradicting health effects and of mechanisms that could be involved in this dualistic mode of action. One mechanism relates to the different ultimate cellular effects of activation of estrogen receptor (ER) a, promoting cell proliferation, and of ERß, promoting apoptosis, with the major soy isoflavones genistein and daidzein activating especially ERß. A second mode of action includes the role of epigenetics, including effects of isoflavones on DNA methylation, histone modification and miRNA expression patterns. The overview presented reveals that we are only at the start of unraveling the complex underlying mode of action for effects of isoflavones, both beneficial or adverse, on cell proliferation and cancer risks. It is evident that whatever model system will be applied, its relevance to human tissues with respect to ERa and ERß levels, co-repressor and co-activator characteristics as well as its relevance to human exposure regimens, needs to be considered and defined