Investigating the Role of the Host Multidrug Resistance Associated Protein Transporter Family in Burkholderia cepacia Complex Pathogenicity Using a Caenorhabditis elegans Infection Model

This study investigated the relationship between host efflux system of the non-vertebrate nematode Caenorhabditis elegans and Burkholderia cepacia complex (Bcc) strain virulence. This is the first comprehensive effort to profile host-transporters within the context of Bcc infection. With this aim, two different toxicity tests were performed: a slow killing assay that monitors mortality of the host by intestinal colonization and a fast killing assay that assesses production of toxins. A Virulence Ranking scheme was defined, that expressed the toxicity of the Bcc panel members, based on the percentage of surviving worms. According to this ranking the 18 Bcc strains were divided in 4 distinct groups. Only the Cystic Fibrosis isolated strains possessed profound nematode killing ability to accumulate in worms’ intestines. For the transporter analysis a complete set of isogenic nematode single Multidrug Resistance associated Protein (MRP) efflux mutants and a number of efflux inhibitors were interrogated in the host toxicity assays. The Bcc pathogenicity profile of the 7 isogenic C. elegans MRP knock-out strains functionality was classified in two distinct groups. Disabling host transporters enhanced nematode mortality more than 50% in 5 out of 7 mutants when compared to wild type. In particular mrp-2 was the most susceptible phenotype with increased mortality for 13 out 18 Bcc strains, whereas mrp-3 and mrp-4 knock-outs had lower mortality rates, suggesting a different role in toxin-substrate recognition. The use of MRP efflux inhibitors in the assays resulted in substantially increased (>40% on average) mortality of wild-type worms.     

Flavin Adenine Dinucleotide Rescues the Phenotype of Frataxin Deficiency

Background

Friedreich ataxia is a neurodegenerative disease caused by the lack of frataxin, a mitochondrial protein. We previously demonstrated that frataxin interacts with complex II subunits of the electronic transport chain (ETC) and putative electronic transfer flavoproteins, suggesting that frataxin could participate in the oxidative phosphorylation.

Methods and Findings

Here we have investigated the effect of riboflavin and its cofactors flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) in Saccharomyces cerevisiae and Caenorhabditis elegans models of frataxin deficiency. We used a S. cerevisiae strain deleted for the yfh1 gene obtained by homologous recombination and we assessed growth in fermentable and non-fermentable cultures supplemented with either riboflavin or its derivates. Experiments with C. elegans were performed in transient knock-down worms (frh-1[RNAi]) generated by microinjection of dsRNA frh-1 into the gonads of young worms. We observed that FAD rescues the phenotype of both defective organisms. We show that cell growth and enzymatic activities of the ETC complexes and ATP production of yfh1Δ cells were improved by FAD supplementation. Moreover, FAD also improved lifespan and other physiological parameters in the C. elegans knock-down model for frataxin.

Conclusions/Significance

We propose that rescue of frataxin deficiency by FAD supplementation could be explained by an improvement in mitochondrial respiration. We suggest that riboflavin may be useful in the treatment of Friedreich ataxia.     

The Zinc Transporter,Slc39a7 (Zip7) Is Implicated in Glycaemic Control in Skeletal Muscle Cells

Dysfunctional zinc signaling is implicated in disease processes including cardiovascular disease, Alzheimer''s disease and diabetes. Of the twenty-four mammalian zinc transporters, ZIP7 has been identified as an important mediator of the ‘zinc wave’ and in cellular signaling. Utilizing siRNA targeting Zip7 mRNA we have identified that Zip7 regulates glucose metabolism in skeletal muscle cells. An siRNA targeting Zip7 mRNA down regulated Zip7 mRNA 4.6-fold (p = 0.0006) when compared to a scramble control. This was concomitant with a reduction in the expression of genes involved in glucose metabolism including Agl, Dlst, Galm, Gbe1, Idh3g, Pck2, Pgam2, Pgm2, Phkb, Pygm, Tpi1, Gusb and Glut4. Glut4 protein expression was also reduced and insulin-stimulated glycogen synthesis was decreased. This was associated with a reduction in the mRNA expression of Insr, Irs1 and Irs2, and the phosphorylation of Akt. These studies provide a novel role for Zip7 in glucose metabolism in skeletal muscle and highlight the importance of this transporter in contributing to glycaemic control in this tissue.     

FBXO7 Y52C Polymorphism as a Potential Protective Factor in Parkinson's Disease

Mutations in the F-box only protein 7 gene (FBXO7), the substrate-specifying subunit of SCF E3 ubiquitin ligase complex, cause Parkinson''s disease (PD)-15 (PARK15). To identify new variants, we sequenced FBXO7 cDNA in 80 Taiwanese early onset PD patients (age at onset ≤50) and only two known variants, Y52C (c.155A>G) and M115I (c.345G>A), were found. To assess the association of Y52C and M115I with the risk of PD, we conducted a case–control study in a cohort of PD and ethnically matched controls. There was a nominal difference in the Y52C G allele frequency between PD and controls (p = 0.045). After combining data from China [1], significant difference in the Y52C G allele frequency between PD and controls (p = 0.012) and significant association of G allele with decreased PD risk (p = 0.017) can be demonstrated. Upon expressing EGFP-tagged Cys52 FBXO7 in cells, a significantly reduced rate of FBXO7 protein decay was observed when compared with cells expressing Tyr52 FBXO7. In silico modeling of Cys52 exhibited a more stable feature than Tyr52. In cells expressing Cys52 FBXO7, the level of TNF receptor-associated factor 2 (TRAF2) was significantly reduced. Moreover, Cys52 FBXO7 showed stronger interaction with TRAF2 and promoted TRAF2 ubiquitination, which may be responsible for the reduced TRAF2 expression in Cys52 cells. After induced differentiation, SH-SY5Y cells expressing Cys52 FBXO7 displayed increased neuronal outgrowth. We therefore hypothesize that Cys52 variant of FBXO7 may contribute to reduced PD susceptibility in Chinese.     

Cocaine Modulates Locomotion Behavior in C. elegans

Cocaine, a potent addictive substance, is an inhibitor of monoamine transporters, including DAT (dopamine transporter), SERT (serotonin transporter) and NET (norepinephrine transporter). Cocaine administration induces complex behavioral alterations in mammals, but the underlying mechanisms are not well understood. Here, we tested the effect of cocaine on C. elegans behavior. We show for the first time that acute cocaine treatment evokes changes in C. elegans locomotor activity. Interestingly, the neurotransmitter serotonin, rather than dopamine, is required for cocaine response in C. elegans. The C. elegans SERT MOD-5 is essential for the effect of cocaine, consistent with the role of cocaine in targeting monoamine transporters. We further show that the behavioral response to cocaine is primarily mediated by the ionotropic serotonin receptor MOD-1. Thus, cocaine modulates locomotion behavior in C. elegans primarily by impinging on its serotoninergic system.     

MUC4 and MUC1 Expression in Adenocarcinoma of the Stomach Correlates with Vessel Invasion and Lymph Node Metastasis: An Immunohistochemical Study of Early Gastric Cancer

We have previously reported that MUC4 expression is a poor prognostic factor in various carcinomas. Our previous study also showed that MUC1 expression in gastric cancers, including the early and advanced stages is a poor prognostic factor. In the present study, the expression profiles of MUC4 and MUC1 were examined by immunohistochemistry (IHC) using two anti-MUC4 monoclonal antibodies (MAbs), 8G7 and 1G8, and anti-MUC1 MAb DF3 in 104 gastrectomy specimens of early gastric adenocarcinoma with submucosal invasion (pT1b2), including 197 histological subtype lesions. Before the IHC study of the human specimens, we evaluated the specificity of the two MAbs by Western blotting and IHC of two MUC4 mRNA expressing gastric cancer cell lines. MAb 8G7 reacted clearly, whereas MAb 1G8 did not show any reactivity, in either Western blotting or IHC. In the IHC of the gastric cancers, the expression rates of MUC4/8G7 detected by MAb 8G7, MUC4/1G8 detected by MAb 1G8 and MUC1/DF3 detected by MAb DF3 in well differentiated types (70%, 38/54; 67%, 36/54; 52%, 28/54) were significantly higher than those in poorly differentiated types (18%, 10/55; 36%, 20/55; 13%, 7/55) (P<0.0001; P = 0.0021; P<0.0001), respectively. The MUC4/8G7 expression was related with lymphatic invasion (r = 0.304, P = 0.033). On the other hand, the MUC4/1G8 expression was related with lymphatic invasion (r = 0.395, P = 0.001) and lymph node metastasis (r = 0.296, P = 0.045). The MUC1/DF3 expression was related with lymphatic invasion (r = 0.357, P = 0.032) and venous invasion (r = 0.377, P = 0.024). In conclusion, the expression of MUC4 as well as MUC1 in early gastric cancers is a useful marker to predict poor prognostic factors related with vessel invasion.     

Bioinformatics Analysis Identify Novel OB Fold Protein Coding Genes in C. elegans

Background

The C. elegans genome has been extensively annotated by the WormBase consortium that uses state of the art bioinformatics pipelines, functional genomics and manual curation approaches. As a result, the identification of novel genes in silico in this model organism is becoming more challenging requiring new approaches. The Oligonucleotide-oligosaccharide binding (OB) fold is a highly divergent protein family, in which protein sequences, in spite of having the same fold, share very little sequence identity (5–25%). Therefore, evidence from sequence-based annotation may not be sufficient to identify all the members of this family. In C. elegans, the number of OB-fold proteins reported is remarkably low (n = 46) compared to other evolutionary-related eukaryotes, such as yeast S. cerevisiae (n = 344) or fruit fly D. melanogaster (n = 84). Gene loss during evolution or differences in the level of annotation for this protein family, may explain these discrepancies.

Methodology/Principal Findings

This study examines the possibility that novel OB-fold coding genes exist in the worm. We developed a bioinformatics approach that uses the most sensitive sequence-sequence, sequence-profile and profile-profile similarity search methods followed by 3D-structure prediction as a filtering step to eliminate false positive candidate sequences. We have predicted 18 coding genes containing the OB-fold that have remarkably partially been characterized in C. elegans.

Conclusions/Significance

This study raises the possibility that the annotation of highly divergent protein fold families can be improved in C. elegans. Similar strategies could be implemented for large scale analysis by the WormBase consortium when novel versions of the genome sequence of C. elegans, or other evolutionary related species are being released. This approach is of general interest to the scientific community since it can be used to annotate any genome.     

Association of Three Common Single Nucleotide Polymorphisms of ATP Binding Cassette G8 Gene with Gallstone Disease: A Meta-Analysis

Background

In this study, we evaluated the association between these polymorphisms and gallstone disease using meta-analysis and compared the hepatic ABCG5/G8 mRNA expression and biliary lipids composition in patients with different genotypes of T400K and Y54C.

Methods

Data were analyzed using the Stata/SE 11.0 software and a random- effects model was applied irrespective of between-study heterogeneity. Hepatic mRNA expression of ABCG5/G8 genes in 182 patients with gallstone disease and 35 gallstone-free patients who underwent cholecystectomy were determined using real-time PCR. Genotypes of Y54C and T400K in the ABCG8 gene were determined by allelic discrimination using either genomic DNA or hepatic cDNA as template by Taqman assays. Biliary compostion in gallbladder bile was assayed in these patients as well.

Results

Ten papers including 13 cohorts were included for the final analysis. In the genotype model, the overall association between genotype with gallstone was significant for D19H (OR = 2.43, 95%CI: 2.23–2.64, P<0.001), and for Y54C (OR = 1.36, 95%CI: 1.01–1.83, P = 0.044), or T400K (OR = 1.17, 95%CI: 0.96–1.43. P = 0.110). In allele model, minor alleles of D19H polymorphism (allele D: OR = 2.25, 95%CI: 2.10–2.42, P<0.001) and of T400K polymorphism (allele K: OR = 1.18, 95%CI: 1.06–1.31, P<0.001) were related with an increased risk of gallstone disease. However, minor allele of Y54C polymorphism (allele Y, OR = 1.08, 95%CI: 0.96–1.21, P = 0.146) was not related with gallstone disease. I ² statistics indicated no significant between-study heterogeneity for all genetic models for any of the three polymorphisms. Funnel plot and Egger’s test suggested the absence of publication bias as well. However, no association of T400K and Y54C polymorphism with hepatic ABCG8/G5 mRNA expression or biliary lipids composition was found.

Conclusions

Our study showed strong association of D19H polymorphism with gallstone disease. T400K and Y54C polymorphism, though to a less extent, may also relate with gallstone disease.     

Evidence for the High Importance of Co-Morbid Factors in HFE C282Y/H63D Patients Cared by Phlebotomies: Results from an Observational Prospective Study

Despite type I haemochromatosis (HC) is mainly associated with the HFE C282Y/C282Y genotype, a second genotype -C282Y/H63D- has mostly been described in other patients. Its association with HC, apart from any associated co-morbid factors, remains unclear and complex to interpret for physicians. This study assesses the weight of this genotype and the role of co-morbid factors in the occurrence of iron overload. This prospective study included the C282Y/C282Y (n = 172) and C282Y/H63D (n = 58) patients enrolled in a phlebotomy program between 2004 and 2007 in a blood centre of western Brittany (Brest, France), where HC is frequent. We compared prevalence of these two genotypes, as well as patients’ profile regarding degree of iron overload and prevalence of co-morbid factors. First, we confirmed the obvious deficit of C282Y/H63D compound heterozygotes among patients cared by phlebotomies. This genotype was 3.0 times less frequent than the C282Y/C282Y genotype among those patients (18.9% vs. 56.0%) whereas it was 4.9 times more frequent in the general population (4.3% vs. 0.9%; p<0.0001). Despite a similar level of hyperferritinaemia, the C282Y/H63D patients who came to medical attention had a milder plasma iron overload, reflected by a lower transferrin saturation median (52.0% vs. 84.0%; p<0.0001). They also exhibited more frequently co-morbid factors, as heavy drinking (26.0% vs. 13.9%; p = 0.0454), overweight (66.7% vs. 39.4%; p = 0.0005) or both (21.3% vs. 2.6%; p<0.0001). Ultimately, they required a lower amount of iron removed to reach depletion (2.1 vs. 3.4 g; p<0.0001), clearly reflecting their lower tissue iron. This study confirms that H63D is a discrete genetic susceptibility factor whose expression is most visible in association with other co-factors. It highlights the importance of searching for co-morbidities in these diagnostic situations and of providing lifestyle and dietary advice.     

Expression and Immune Responses to MAGE Antigens Predict Survival in Epithelial Ovarian Cancer

The MAGE cancer-testis antigens (CTA) are attractive candidates for immunotherapy. The aim of this study was to determine the frequency of expression, humoral immunity and prognostic significance of MAGE CTA in human epithelial ovarian cancer (EOC). mRNA or protein expression frequencies were determined for MAGE-A1, -A3, -A4, -A10 and -C1 (CT7) in tissue samples obtained from 400 patients with EOC. The presence of autologous antibodies against the MAGE antigens was determined from 285 serum samples. The relationships between MAGE expression, humoral immunity to MAGE antigens, and clinico-pathologic characteristics were studied. The individual frequencies of expression were as follows: A1: 15% (42/281), A3: 36% (131/390), A4: 47% (186/399), A10: 52% (204/395), C1: 16% (42/267). Strong concordant expression was noted with MAGE-A1:–A4, MAGE-A1:–C1 and MAGE-A4:–A10 (p<0.0005). Expression of MAGE-A1 or -A10 antigens resulted in poor progression free survival (PFS) (OR 1.44, CI 1.01–2.04, p = 0.044 and OR 1.3, CI 1.03–1.64, p = 0.03, respectively); whereas, MAGE-C1 expression was associated with improved PFS (OR 0.62, CI 0.42–0.92, p = 0.016). The improved PFS observed for MAGE-C1 expression, was diminished by co-expression of MAGE-A1 or -A10. Spontaneous humoral immunity to the MAGE antigens was present in 9% (27/285) of patients, and this predicted poor overall survival (log-rank test p = 0.0137). These findings indicate that MAGE-A1, MAGE-A4, MAGE-A3, and MAGE-A10 are priority attractive targets for polyvalent immunotherapy in ovarian cancer patients.     

Two Golgi-resident 3′-Phosphoadenosine 5′-Phosphosulfate Transporters Play Distinct Roles in Heparan Sulfate Modifications and Embryonic and Larval Development in Caenorhabditis elegans

Synthesis of extracellular sulfated molecules requires active 3′-phosphoadenosine 5′-phosphosulfate (PAPS). For sulfation to occur, PAPS must pass through the Golgi membrane, which is facilitated by Golgi-resident PAPS transporters. Caenorhabditis elegans PAPS transporters are encoded by two genes, pst-1 and pst-2. Using the yeast heterologous expression system, we characterized PST-1 and PST-2 as PAPS transporters. We created deletion mutants to study the importance of PAPS transporter activity. The pst-1 deletion mutant exhibited defects in cuticle formation, post-embryonic seam cell development, vulval morphogenesis, cell migration, and embryogenesis. The pst-2 mutant exhibited a wild-type phenotype. The defects observed in the pst-1 mutant could be rescued by transgenic expression of pst-1 and hPAPST1 but not pst-2 or hPAPST2. Moreover, the phenotype of a pst-1;pst-2 double mutant were similar to those of the pst-1 single mutant, except that larval cuticle formation was more severely defected. Disaccharide analysis revealed that heparan sulfate from these mutants was undersulfated. Gene expression reporter analysis revealed that these PAPS transporters exhibited different tissue distributions and subcellular localizations. These data suggest that pst-1 and pst-2 play different physiological roles in heparan sulfate modification and development.     

Induction of stress resistance and extension of lifespan in Chaenorhabditis elegans serotonin-receptor knockout strains by withanolide A

IntroductionApproximately 300 million people worldwide suffer from depression. The COVID-19 crisis may dramatically increase these numbers. Severe side effects and resistance development limit the use of standard antidepressants. The steroidal lactone withanolide A (WA) from Withania somnifera may be a promising alternative. Caenorhabditis elegans was used as model to explore WA's anti-depressive and anti-stress potential.MethodsC. elegans wildtype (N2) and deficient strains (AQ866, DA1814, DA2100, DA2109 and MT9772) were used to assess oxidative, osmotic or heat stress as measured by generation of reactive oxygen species (ROS), determination of lifespan, and mRNA expression of serotonin receptor (ser-1, ser-4, ser-7) and serotonin transporter genes (mod-5). The protective effect of WA was compared to fluoxetine as clinically established antidepressant. Additionally, WA's effect on lifespan was determined. Furthermore, the binding affinities and pKi values of WA, fluoxetine and serotonin as natural ligand to Ser-1, Ser-4, Ser-7, Mod-5 and their human orthologues proteins were calculated by molecular docking.ResultsBaseline oxidative stress was higher in deficient than wildtype worms. WA and fluoxetine reduced ROS levels in all strains except MT9772. WA and fluoxetine prolonged survival times in wildtype and mutants under osmotic stress. WA but not fluoxetine increased lifespan of all heat-stressed C. elegans strains except DA2100. Furthermore, WA but not fluoxetine extended lifespan in all non-stressed C. elegans strains. WA also induced mRNA expression of serotonin receptors and transporters in wildtype and mutants. WA bound with higher affinity and lower pKi values to all C. elegans and human serotonin receptors and transporters than serotonin, indicating that WA may competitively displaced serotonin from the binding pockets of these proteins.ConclusionWA reduced stress and increased lifespan by ROS scavenging and interference with the serotonin system. Hence, WA may serve as promising candidate to treat depression.     

Effect of Temperature Pre-Exposure on the Locomotion and Chemotaxis of C. elegans

The effect of temperature pre-exposure on locomotion and chemotaxis of the soil-dwelling nematode Caenorhabditis elegans has been extensively studied. The behavior of C. elegans was quantified using a simple harmonic curvature-based model. Animals showed increased levels of activity, compared to control worms, immediately after pre-exposure to 30°C. This high level of activity in C. elegans translated into frequent turns by making ‘complex’ shapes, higher velocity of locomotion, and higher chemotaxis index () in presence of a gradient of chemoattractant. The effect of pre-exposure was observed to be persistent for about 20 minutes after which the behavior (including velocity and ) appeared to be comparable to that of control animals (maintained at 20°C). Surprisingly, after 30 minutes of recovery, the behavior of C. elegans continued to deteriorate further below that of control worms with a drastic reduction in the curvature of the worms'' body. A majority of these worms also showed negative chemotaxis index indicating a loss in their chemotaxis ability.     

In Vivo Imaging of Schistosomes to Assess Disease Burden Using Positron Emission Tomography (PET)

Background

Schistosomes are chronic intravascular helminth parasites of humans causing a heavy burden of disease worldwide. Diagnosis of schistosomiasis currently requires the detection of schistosome eggs in the feces and urine of infected individuals. This method unreliably measures disease burden due to poor sensitivity and wide variances in egg shedding. In vivo imaging of schistosome parasites could potentially better assess disease burden, improve management of schistosomiasis, facilitate vaccine development, and enhance study of the parasite''s biology. Schistosoma mansoni (S. mansoni) have a high metabolic demand for glucose. In this work we investigated whether the parasite burden in mice could be assessed by positron emission tomography (PET) imaging with 2-deoxy-2[¹⁸F]fluoro-D-glucose (FDG).

Methodology/Principal Findings

Live adult S. mansoni worms FDG uptake in vitro increased with the number of worms. Athymic nude mice infected with S. mansoni 5–6 weeks earlier were used in the imaging studies. Fluorescence molecular tomography (FMT) imaging with Prosense 680 was first performed. Accumulation of the imaging probe in the lower abdomen correlated with the number of worms in mice with low infection burden. The total FDG uptake in the common portal vein and/or regions of elevated FDG uptake in the liver linearly correlated to the number of worms recovered from infected animals (R² = 0.58, P<0.001, n = 40). FDG uptake showed a stronger correlation with the worm burden in mice with more than 50 worms (R² = 0.85, P<0.001, n = 17). Cryomicrotome imaging confirmed that most of the worms in a mouse with a high infection burden were in the portal vein, but not in a mouse with a low infection burden. FDG uptake in recovered worms measured by well counting closely correlated with worm number (R² = 0.85, P<0.001, n = 21). Infected mice showed a 32% average decrease in total FDG uptake after three days of praziquantel treatment (P = 0.12). The total FDG uptake in untreated mice increased on average by 36% over the same period (P = 0.052).

Conclusion

FDG PET may be useful to non-invasively quantify the worm burden in schistosomiasis-infected animals. Future investigations aiming at minimizing non-specific FDG uptake and to improve the recovery of signal from worms located in the lower abdomen will include the development of more specific radiotracers.