Deletion of <Emphasis Type="Italic">ldh</Emphasis>A and <Emphasis Type="Italic">ald</Emphasis>H genes in <Emphasis Type="Italic">Klebsiella</Emphasis><Emphasis Type="Italic">pneumoniae</Emphasis> to enhance 1,3-propanediol production

Objectives

To improve 1,3-propanediol (1,3-PD) production and reduce byproduct concentration during the fermentation of Klebsiella pneumonia.

Results

Klebsiella. pneumonia 2-1ΔldhA, K. pneumonia 2-1ΔaldH and K. pneumonia 2-1ΔldhAΔaldH mutant strains were obtained through deletion of the ldhA gene encoding lactate dehydrogenase required for lactate synthesis and the aldH gene encoding acetaldehyde dehydrogenase involved in the synthesis of ethanol. After fed-batch fermentation, the production of 1,3-PD from glycerol was enhanced and the concentrations of byproducts were reduced compared with the original strain K. pneumonia 2-1. The maximum yields of 1,3-PD were 85.7, 82.5 and 87.5 g/l in the respective mutant strains.

Conclusion

Deletion of either aldH or ldhA promoted 1,3-PD production in K. pneumonia.

     

Inhibitory effects of YCW and MOS from <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> on <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Salmonella pullorum</Emphasis> adhesion to Caco-2 cells

Background

For many years, yeast cell walls (YCW) and mannan oligosaccharides (MOS) have been used as alternatives to antibiotics and health feed additives to enhance the growth performance and health of food animals. In the present study, the inhibitory effects of YCWand MOS on the adhesion of enteropathogenic bacteria to intestinal epithelial cells were tested.

Methods

YCW and MOS were extracted from Saccharomyces cerevisiae (XM 0315), and the morphology of YCW and MOS bound to pathogenic bacteria was observed by scanning electron microscopy (SEM). Real-time fluorescent quantitative PCR was used to quantitatively analyze the effects of YCW and MOS on the adhesion of Escherichia coli (CVCC3367) and Salmonella pullorum (CVCC520) to Caco-2 cells.

Results

The results showed that YCW inhibited E. coli and S. pullorum binding to Caco-2 cells by 95% and 74%, respectively, whereas MOS prevented E. coli and S. pullorum binding by 67% and 50%, respectively.

Conclusions

These data suggest that YCW has a stronger ability than MOS to inhibit pathogenic bacteria from adhering to Caco-2 cells in vitro.

     

Mutation analysis of the <Emphasis Type="Italic">COL1A1</Emphasis> and <Emphasis Type="Italic">COL1A2</Emphasis> genes in Vietnamese patients with osteogenesis imperfecta

Background

The genetics of osteogenesis imperfecta (OI) have not been studied in a Vietnamese population before. We performed mutational analysis of the COL1A1 and COL1A2 genes in 91 unrelated OI patients of Vietnamese origin. We then systematically characterized the mutation profiles of these two genes which are most commonly related to OI.

Methods

Genomic DNA was extracted from EDTA-preserved blood according to standard high-salt extraction methods. Sequence analysis and pathogenic variant identification was performed with Mutation Surveyor DNA variant analysis software. Prediction of the pathogenicity of mutations was conducted using Alamut Visual software. The presence of variants was checked against Dalgleish’s osteogenesis imperfecta mutation database.

Results

The sample consisted of 91 unrelated osteogenesis imperfecta patients. We identified 54 patients with COL1A1/2 pathogenic variants; 33 with COL1A1 and 21 with COL1A2. Two patients had multiple pathogenic variants. Seventeen novel COL1A1 and 10 novel COL1A2 variants were identified. The majority of identified COL1A1/2 pathogenic variants occurred in a glycine substitution (36/56, 64.3 %), usually serine (23/36, 63.9 %). We found two pathogenic variants of the COL1A1 gene c.2461G?>?A (p.Gly821Ser) in four unrelated patients and one, c.2005G?>?A (p.Ala669Thr), in two unrelated patients.

Conclusion

Our data showed a lower number of collagen OI pathogenic variants in Vietnamese patients compared to reported rates for Asian populations. The OI mutational profile of the Vietnamese population is unique and related to the presence of a high number of recessive mutations in non-collagenous OI genes. Further analysis of OI patients negative for collagen mutations, is required.

     

Iridoid and phenylethanoid/phenylpropanoid metabolite profiles of <Emphasis Type="Italic">Scrophularia</Emphasis> and <Emphasis Type="Italic">Verbascum</Emphasis> species used medicinally in North America

Introduction

Botanicals containing iridoid and phenylethanoid/phenylpropanoid glycosides are used worldwide for the treatment of inflammatory musculoskeletal conditions that are primary causes of human years lived with disability, such as arthritis and lower back pain.

Objectives

We report the analysis of candidate anti-inflammatory metabolites of several endemic Scrophularia species and Verbascum thapsus used medicinally by peoples of North America.

Methods

Leaves, stems, and roots were analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) and partial least squares-discriminant analysis (PLS-DA) was performed in MetaboAnalyst 3.0 after processing the datasets in Progenesis QI.

Results

Comparison of the datasets revealed significant and differential accumulation of iridoid and phenylethanoid/phenylpropanoid glycosides in the tissues of the endemic Scrophularia species and Verbascum thapsus.

Conclusions

Our investigation identified several species of pharmacological interest as good sources for harpagoside and other important anti-inflammatory metabolites.

     

Haplotype-specific <Emphasis Type="Italic">MAPT</Emphasis> exon 3 expression regulated by common intronic polymorphisms associated with Parkinsonian disorders

Background

Genome wide association studies have identified microtubule associated protein tau (MAPT) H1 haplotype single nucleotide polymorphisms (SNPs) as leading common risk variants for Parkinson’s disease, progressive supranuclear palsy and corticobasal degeneration. The MAPT risk variants fall within a large 1.8 Mb region of high linkage disequilibrium, making it difficult to discern the functionally important risk variants. Here, we leverage the strong haplotype-specific expression of MAPT exon 3 to investigate the functionality of SNPs that fall within this H1 haplotype region of linkage disequilibrium.

Methods

In this study, we dissect the molecular mechanisms by which haplotype-specific SNPs confer allele-specific effects on the alternative splicing of MAPT exon 3. Firstly, we use haplotype-hybrid whole-locus genomic MAPT vectors studies to identify functional SNPs. Next, we characterise the RNA-protein interactions at two loci by mass spectrometry. Lastly, we knockdown candidate splice factors to determine their effect on MAPT exon 3 using a novel allele-specific qPCR assay.

Results

Using whole-locus genomic DNA expression vectors to express MAPT haplotype variants, we demonstrate that rs17651213 regulates exon 3 inclusion in a haplotype-specific manner. We further investigated the functionality of this region using RNA-electrophoretic mobility shift assays to show differential RNA-protein complex formation at the H1 and H2 sequence variants of SNP rs17651213 and rs1800547 and subsequently identified candidate trans-acting splicing factors interacting with these functional SNPs sequences by RNA-protein pull-down experiment and mass spectrometry. Finally, gene knockdown of candidate splice factors identified by mass spectrometry demonstrate a role for hnRNP F and hnRNP Q in the haplotype-specific regulation of exon 3 inclusion.

Conclusions

We identified common splice factors hnRNP F and hnRNP Q regulating the haplotype-specific splicing of MAPT exon 3 through intronic variants rs1800547 and rs17651213. This work demonstrates an integrated approach to characterise the functionality of risk variants in large regions of linkage disequilibrium.

     

<Emphasis Type="Italic">Rhizobium</Emphasis> strains in the biological control of the phytopathogenic fungi <Emphasis Type="Italic">Sclerotium</Emphasis> (<Emphasis Type="Italic">Athelia</Emphasis>) <Emphasis Type="Italic">rolfsii</Emphasis> on the common bean

Aims

To identify Rhizobium strains’ ability to biocontrol Sclerotium rolfsii, a fungus that causes serious damage to the common bean and other important crops, 78 previously isolated rhizobia from common bean were assessed.

Methods

Dual cultures, volatiles, indole-acetic acid (IAA), siderophore production and 16S rRNA sequencing were employed to select strains for pot and field experiments.

Results

Thirty-three antagonistic strains were detected in dual cultures, 16 of which were able to inhibit ≥84% fungus mycelial growth. Antagonistic strains produced up to 36.5 μg mL^?1 of IAA, and a direct correlation was verified between IAA production and mycelium inhibition. SEMIA 460 inhibited 45% of mycelial growth through volatile compounds. 16S rRNA sequences confirmed strains as Rhizobium species. In pot condition, common bean plants grown on S. rolfsii-infested soil and inoculated with SEMIA 4032, 4077, 4088, 4080, 4085, or 439 presented less or no disease symptoms. The most efficient strains under field conditions, SEMIA 439 and 4088, decreased disease incidence by 18.3 and 14.5% of the S. rolfsii-infested control.

Conclusions

Rhizobium strains could be strong antagonists towards S. rolfsii growth. SEMIA 4032, 4077, 4088, 4080, 4085, and 439 are effective in the biological control of the collar rot of the common bean.

     

Seroprevalence of <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis> sensu lato and <Emphasis Type="Italic">Anaplasma phagocytophilum</Emphasis> in Danish horses

Background

Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum are able to infect horses. However, the extend to which Danish horses are infected and seroconvert due to these two bacteria is unknown. The aim of the present study was to evaluate the seroprevalence of B. burgdorferi sensu lato and A. phagocytophilum in Danish horses.

Methods

A total of 390 blood samples collected from all major regions of Denmark and with a geographical distribution corresponding to the density of the Danish horse population were analyzed. All samples were examined for the presence of antibodies against B. burgdorferi sensu lato and A. phagocytophilum by the use of the SNAP^®4DX ^® ELISA test.

Results

Overall, 29.0% of the horses were seropositive for B. burgdorferi sensu lato whereas 22.3% were seropositive for A. phagocytophilum.

Conclusions

Antibodies against B burgdorferi sensu lato and A. phagocytophilum are commonly found among Danish horses thus showing that Danish horses are frequently infected by these organisms.

     

Enhancement of crystallinity of cellulose produced by <Emphasis Type="Italic">Escherichia coli</Emphasis> through heterologous expression of <Emphasis Type="Italic">bcsD</Emphasis> gene from <Emphasis Type="Italic">Gluconacetobacter xylinus</Emphasis>

Objectives

To evaluate the crystallinity index of the cellulose produced by Escherichia coli Nissle 1917 after heterologous expression of the cellulose synthase subunit D (bcsD) gene of Gluconacetobacter xylinus BPR2001.

Results

The bcsD gene of G. xylinus BPR2001 was expressed in E. coli and its protein product was visualized using SDS-PAGE. FTIR analysis showed that the crystallinity index of the cellulose produced by the recombinants was 0.84, which is 17% more than that of the wild type strain. The increased crystallinity index was also confirmed by X-ray diffraction analysis. The cellulose content was not changed significantly after over-expressing the bcsD.

Conclusion

The bcsD gene can improve the crystalline structure of the bacterial cellulose but there is not any significant difference between the amounts of cellulose produced by the recombinant and wild type E. coli Nissle 1917.

     

<Emphasis Type="Italic">PiggyBac</Emphasis> transposon-mediated mutagenesis and application in yeast <Emphasis Type="Italic">Komagataella phaffii</Emphasis>

Objective

Around one-fourth of the Komagataella phaffii genes encode hypothetical proteins with unknown functions. However, lack of powerful tools for genetic screening in K. phaffii significantly limits the functional analysis of these unknown genes. Transposon mutagenesis has been utilized as an insertional mutagenesis tool in many other organisms and would be extremely valuable if it could be applied in K. phaffii.

Results

In this study, we investigated in K. phaffii the transposition activity and efficiency of piggyBac (PB) transposon, a DNA transposon from the cabbage looper moth Trichoplusia ni through the integrated-plasmid system. We also designed a binary-plasmid system which could generate stable mutants. Finally we evaluated the quality of this mutagenesis system by a simple screening for functional genes involved in K. phaffii carbon catabolite repression.

Conclusions

Our results demonstrate that PB-mediated mutagenesis could be a feasible and useful tool for functional gene screening in K. phaffii.

     

A constitutive expression system for <Emphasis Type="Italic">Pichia pastoris</Emphasis> based on the <Emphasis Type="Italic">PGK1</Emphasis> promoter

Objectives

To develop a new vector for constitutive expression in Pichia pastoris based on the endogenous glycolytic PGK1 promoter.

Results

P. pastoris plasmids bearing at least 415 bp of PGK1 promoter sequences can be used to drive plasmid integration by addition at this locus without affecting cell growth. Based on this result, a new P. pastoris integrative vector, pPICK2, was constructed bearing some features that facilitate protein production in this yeast: a ~620 bp PGK1 promoter fragment with three options of restriction sites for plasmid linearization prior to yeast transformation: a codon-optimized α-factor secretion signal, a new polylinker, and the kan marker for vector propagation in bacteria and selection of yeast transformants.

Conclusions

A new constitutive vector for P. pastoris represents an alternative platform for recombinant protein production and metabolic engineering purposes.

     

<Emphasis Type="Italic">C</Emphasis>-Terminal carbohydrate-binding module 9_2 fused to the <Emphasis Type="Italic">N</Emphasis>-terminus of GH11 xylanase from <Emphasis Type="Italic">Aspergillus niger</Emphasis>

Objective

The 9_2 carbohydrate-binding module (C2) locates natively at the C-terminus of the GH10 thermophilic xylanase from Thermotoga marimita. When fused to the C-terminus, C2 improved thermostability of a GH11 xylanase (Xyn) from Aspergillus niger. However, a question is whether the C-terminal C2 would have a thermostabilizing effect when fused to the N-terminus of a catalytic module.

Results

A chimeric enzyme, C2-Xyn, was created by step-extension PCR, cloned in pET21a(+), and expressed in E. coli BL21(DE3). The C2-Xyn exhibited a 2 °C higher optimal temperature, a 2.8-fold longer thermostability, and a 4.5-fold higher catalytic efficiency on beechwood xylan than the Xyn. The C2-Xyn exhibited a similar affinity for binding to beechwood xylan and a higher affinity for oat-spelt xylan than Xyn.

Conclusion

C2 is a thermostabilizing carbohydrate-binding module and provides a model of fusion at an enzymatic terminus inconsistent with the modular natural terminal location.

     

Time-Kill Curves Studies with Amphotericin B Against <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis>/<Emphasis Type="Italic">C</Emphasis>. <Emphasis Type="Italic">gattii</Emphasis> Species Complex Clinical Isolates

Purpose of Review

We reviewed data on amphotericin B (AmB) tolerance among Cryptococcus neoformans/C. gattii species complex clinical isolates and present our results of large recent study on this issue.

Recent Findings

The standard method to detect antifungal susceptibility is based on MIC (minimal inhibitory concentration) determination; however, there is no interpretative clinical breakpoints defined for antifungal agents against Cryptococcus species, and to date, there is no correlation of MIC and clinical response. The time-kill curves (TKC) methodology seems to provide some correlation with outcome and it could identify distinct profiles of AmB-fungicidal activity.

Summary

Our group analyzed 83 human isolates from cryptococcosis cases. The isolates were tested by TKC and showed up 8.3% of tolerance to AmB. Importantly, the AmB-MIC was low for all isolates, including tolerant ones. Our findings are similar to other authors, due the ability of TKC to identify distinct AmB-fungicidal activity and detecting low susceptible isolates.

     

Homologous gene targeting of a carotenoids biosynthetic gene in <Emphasis Type="Italic">Rhodosporidium toruloides</Emphasis> by <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation

Objectives

To target a carotenoid biosynthetic gene in the oleaginous yeast Rhodosporidium toruloides by using the Agrobacterium-mediated transformation (AMT) method.

Results

The RHTO_04602 locus of R. toruloides NP11, previously assigned to code the carotenoid biosynthetic gene CRTI, was amplified from genomic DNA and cloned into the binary plasmid pZPK-mcs, resulting in pZPK-CRT. A HYG-expression cassette was inserted into the CRTI sequence of pZPK-CRT by utilizing the restriction-free clone strategy. The resulted plasmid was used to transform R. toruloides cells according to the AMT method, leading to a few white transformants. Sequencing analysis of those transformants confirmed homologous recombination and insertional inactivation of CRTI. When the white variants were transformed with a CRTI-expression cassette, cells became red and produced carotenoids as did the wild-type strain NP11.

Conclusions

Successful homologous targeting of the CrtI locus confirmed the function of RHTO_04602 in carotenoids biosynthesis in R. toruloides. It provided valuable information for metabolic engineering of this non-model yeast species.

     

Species of family <Emphasis Type="Italic">Promicromonosporaceae</Emphasis> and family <Emphasis Type="Italic">Cellulomonadeceae</Emphasis> that produce cellulosome-like multiprotein complexes

Objectives

To screen the phylogenetically-nearest members of Cellulosimicrobium cellulans for the production of cellulosome-like multienzyme complexes and extracellular β-xylosidase activity against 7-xylosyltaxanes and to get corresponding molecular insights.

Results

Cellulosimicrobium (family Promicromonosporaceae) and all genera of the family Cellulomonadeceaec produced both cellulosome-like multienzyme complexes and extracellular β-xylosidase activity, while the other genera of the family Promicromonosporaceae did not. Multiple sequence alignments further indicated that hypothetic protein M768_06655 might be a possible key subunit.

Conclusion

This is the first report that many actinobacteria species can produce cellulosome-like multienzyme complexes. The production of cellulosome-like complexes and the extracellular β-xylosidase activity against 7-xylosyltaxanes might be used to differentiate the genus Cellulosimicrobium from other genera of the family Promicromonosporaceae.

     

Identification of <Emphasis Type="Italic">O</Emphasis>-GlcNAcylated proteins in <Emphasis Type="Italic">Plasmodium falciparum</Emphasis>

Background

Post-translational modifications (PTMs) constitute a huge group of chemical modifications increasing the complexity of the proteomes of living beings. PTMs have been discussed as potential anti-malarial drug targets due to their involvement in many cell processes. O-GlcNAcylation is a widespread PTM found in different organisms including Plasmodium falciparum. The aim of this study was to identify O-GlcNAcylated proteins of P. falciparum, to learn more about the modification process and to understand its eventual functions in the Apicomplexans.

Methods

The P. falciparum strain 3D7 was amplified in erythrocytes and purified. The proteome was checked for O-GlcNAcylation using different methods. The level of UDP-GlcNAc, the donor of the sugar moiety for O-GlcNAcylation processes, was measured using high-pH anion exchange chromatography. O-GlcNAcylated proteins were enriched and purified utilizing either click chemistry labelling or adsorption on succinyl-wheat germ agglutinin beads. Proteins were then identified by mass-spectrometry (nano-LC MS/MS).

Results

While low when compared to MRC5 control cells, P. falciparum disposes of its own pool of UDP-GlcNAc. By using proteomics methods, 13 O-GlcNAcylated proteins were unambiguously identified (11 by click-chemistry and 6 by sWGA-beads enrichment; 4 being identified by the 2 approaches) in late trophozoites. These proteins are all part of pathways, functions and structures important for the parasite survival. By probing clicked-proteins with specific antibodies, Hsp70 and α-tubulin were identified as P. falciparum O-GlcNAc-bearing proteins.

Conclusions

This study is the first report on the identity of P. falciparum O-GlcNAcylated proteins. While the parasite O-GlcNAcome seems close to those of other species, the structural differences exhibited by the proteomes provides a glimpse of innovative therapeutic paths to fight malaria. Blocking biosynthesis of UDP-GlcNAc in the parasites is another promising option to reduce Plasmodium life cycle.

     

Functional analysis of alcohol dehydrogenase (<Emphasis Type="Italic">ADH</Emphasis>) genes in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Objectives

To characterize the genes responsible for ethanol utilization in Pichia pastoris.

Results

ADH3 (XM_002491337) and ADH (FN392323) genes were disrupted in P. pastoris. The ADH3 mutant strain, MK115 (Δadh3), lost its ability to grow on minimal ethanol media but produced ethanol in minimal glucose medium. ADH3p was responsible for 92 % of total Adh enzyme activity in glucose media. The double knockout strain MK117 (Δadh3Δadh) also produced ethanol. The Adh activities of X33 and MK116 (Δadh) strains were not different. Thus, the ADH gene does not play a role in ethanol metabolism.

Conclusion

The PpADH3 is the only gene responsible for consumption of ethanol in P. pastoris.

     

<Emphasis Type="Italic">In silico</Emphasis> characterization and Molecular modeling of double-strand break repair protein <Emphasis Type="Italic">MRE11</Emphasis> from <Emphasis Type="Italic">Phoenix dactylifera</Emphasis> v deglet nour

Background

DNA double-strand breaks (DSBs) are highly cytotoxic and mutagenic. MRE11 plays an essential role in repairing DNA by cleaving broken ends through its 3′ to 5′ exonuclease and single-stranded DNA endonuclease activities.

Methods

The present study aimed to in silico characterization and molecular modeling of MRE11 from Phoenix dactylifera L cv deglet nour (DnMRE11) by various bioinformatic approaches. To identify DnMRE11 cDNA, assembled contigs from our cDNA libraries were analysed using the Blast2GO2.8 program.

Results

The DnMRE11 protein length was 726 amino acids. The results of HUMMER show that DnMRE11 is formed by three domains: the N-terminal core domain containing the nuclease and capping domains, the C-terminal half containing the DNA binding and coiled coil region. The structure of DnMRE11 is predicted using the Swiss-Model server, which contains the nuclease and capping domains. The obtained model was verified with the structure validation programs such as ProSA and QMEAN servers for reliability. Ligand binding studies using COACH indicated the interaction of DnMRE11 protein with two Mn²⁺ ions and dAMP. The ConSurf server predicted that residues of the active site and Nbs binding site have high conservation scores between plant species.

Conclusions

A model structure of DnMRE11 was constructed and validated with various bioinformatics programs which suggested the predicted model to be satisfactory. Further validation studies were conducted by COACH analysis for active site ligand prediction, and revealed the presence of six ligands binding sites and two ligands (2 Mn²⁺ and dAMP).

     

Molecular Identification of <Emphasis Type="Italic">Malassezia</Emphasis> Species in Patients with <Emphasis Type="Italic">Malassezia</Emphasis> folliculitis in Sfax,Tunisia

Aim

Malassezia folliculitis is caused by the invasion of hair follicles by large numbers of Malassezia cells. Several Malassezia researches still use cultures, morphology and biochemical techniques. The aim of this study was to identify Malassezia species isolated from patients diagnosed with folliculitis, at the Parasitology and Mycology Laboratory of Sfax University Hospital, and to explore the genetic diversity of Malassezia by using PCR-RFLP and PCR-sequencing targeting the rDNA region of the Malassezia genome.

Patients and Methods

Specimens were taken from 27 patients with Malassezia folliculitis. For the molecular identification, PCR amplification of the 26S rDNAD1/D2 region was carried out using the Malup and Maldown primers and three restriction enzymes (BanI, MspI and HeaII) for RFLP analysis. The nucleotide sequences of each isolate were compared to those in the NCBI GenBank by using BLASTIN algorithm.

Results

Three species of Malassezia yeasts were identified among the 31 Malassezia strains isolated: M. globosa (83.9%), M. sympodialis (12. 9%) and M. furfur (3.2%). The sequence analysis of M. globosa showed six genotypes.

Conclusion

There is a high genotypic variability of M. globosa colonizing patients with folliculitis.

     

The asthma candidate gene <Emphasis Type="Italic">NPSR1</Emphasis> mediates isoform specific downstream signalling

Background

Neuropeptide S Receptor 1 (NPSR1, GPRA, GPR154) was first identified as an asthma candidate gene through positional cloning and has since been replicated as an asthma and allergy susceptibility gene in several independent association studies. In humans, NPSR1 encodes two G protein-coupled receptor variants, NPSR1-A and NPSR1-B, with unique intracellular C-termini. Both isoforms show distinct expression pattern in asthmatic airways. Although NPSR1-A has been extensively studied, functional differences and properties of NPSR1-B have not yet been clearly examined. Our objective was to investigate downstream signalling properties of NPSR1-B and functional differences between NPSR1-A and NPSR1-B.

Methods

HEK-293 cells transiently overexpressing NPSR1-A or NPSR1-B were stimulated with the ligand neuropeptide S (NPS) and downstream signalling effects were monitored by genome-scale affymetrix expression-arrays. The results were verified by NPS concentration-response and time series analysis using qRT-PCR, cAMP and Ca²⁺ assays, and cAMP/PKA, MAPK/JNK and MAPK/ERK pathway specific reporter assays.

Results

NPSR1-B signalled through the same pathways and regulated the same genes as NPSR1-A, but NPSR1-B yielded lower induction on effector genes than NPSR1-A, with one notable exception, CD69, a marker of regulatory T cells.

Conclusions

We conclude that NPSR1-B is regulating essentially identical set of genes as NPSR1-A, with few, but possibly important exceptions, and that NPSR1-A induces stronger signalling effects than NPSR1-B. Our findings suggest an isoform-specific link to pathogenetic processes in asthma and allergy.