Clinical expression and antigenic profiles of a Plasmodium vivax vaccine candidate: merozoite surface protein 7 (PvMSP-7).

BACKGROUND: Vivax malaria is the predominant form of malaria outside Africa, affecting about 14 million people worldwide, with about 2.5 billion people exposed. Development of a Plasmodium vivax vaccine is a priority, and merozoite surface protein 7 (MSP-7) has been proposed as a plausible candidate. The P. vivax genome contains 12 MSP-7 genes, which contribute to erythrocyte invasion during blood-stage infection. Previous analysis of MSP-7 sequence diversity suggested that not all paralogs are functionally equivalent. To explore MSP-7 functional diversity, and to identify the best vaccine candidate within the family, MSP-7 expression and antigenicity during bloodstream infections were examined directly from clinical isolates. METHODS: Merozoite surface protein 7 gene expression was profiled using RNA-seq data from blood samples isolated from ten human patients with vivax malaria. Differential expression analysis and co-expression cluster analysis were used to relate PvMSP-7 expression to genetic markers of life cycle stage. Plasma from vivax malaria patients was also assayed using a custom peptide microarray to measure antibody responses against the coding regions of 12 MSP-7 paralogs. RESULTS: Ten patients presented diverse transcriptional profiles that comprised four patient groups. Two MSP-7 paralogs, 7A and 7F, were expressed abundantly in all patients, while other MSP-7 genes were uniformly rare (e.g. 7J). MSP-7H and 7I were significantly more abundant in patient group 4 only, (two patients having experienced longer patency), and were co-expressed with a schizont-stage marker, while negatively associated with liver-stage and gametocyte-stage markers. Screening infections with a PvMSP-7 peptide array identified 13 linear B-cell epitopes in five MSP-7 paralogs that were recognized by plasma from all patients. CONCLUSIONS: These results show that MSP-7 family members vary in expression profile during blood infections; MSP-7A and 7F are expressed throughout the intraerythrocytic development cycle, while expression of other paralogs is focused on the schizont. This may reflect developmental regulation, and potentially functional differentiation, within the gene family. The frequency of B-cell epitopes among paralogs also varies, with MSP-7A and 7L consistently the most immunogenic. Thus, MSP-7 paralogs cannot be assumed to have equal potential as vaccines. This analysis of clinical infections indicates that the most abundant and immunogenic paralog is MSP-7A.

A Complex Interplay Between Melatonin and RORß: RORß is Unlikely a Putative Receptor for Melatonin as Revealed by Biophysical Assays.

A nuclear retinoic acid receptor (RAR)-related orphan receptor ß (RORß) is strictly expressed in the brain, particularly in the pineal gland where melatonin is primarily synthesized and concentrated. The controversial issues regarding the direct interaction of melatonin toward ROR receptors have prompted us to investigate the potential melatonin binding sites on different ROR isoforms. We adopted computational and biophysical approaches to investigate the potential of melatonin as the ligand for RORs, in particular RORß. Herein, possible melatonin binding sites were predicted by molecular docking on human RORs. The results showed that melatonin might be able to bind within the ligand-binding domain (LBD) of all RORs, despite their difference in sequence homology. The predicted melatonin binding scores were comparable to binding energies with respect to those of melatonin interaction to the well-characterized membrane receptors, MT1 and MT2. Although the computational analyses suggested the binding potential of melatonin to the LBD of RORß, biophysical validation failed to confirm the binding. Melatonin was unable to alter the stability of human RORß as shown by the unaltered melting temperatures upon melatonin administration in differential scanning fluorometry (DSF). A thermodynamic isothermal titration calorimetry (ITC) profile showed that melatonin did not interact with human RORß in solutions, even in the presence of SRC-1 co-activator peptide. Although the direct interaction between the LBD of RORß could not be established, RORα and RORß gene expressions were increased upon 24 h treatment with µM-range melatonin. Our data, thus, support the studies that the nuclear effects of melatonin may not be directly mediated via its interaction with the RORß. These findings warrant further investigation on how melatonin interacts with ROR signaling and urge the melatonin research community for a paradigm shift in the direct interaction of melatonin toward RORs. The quest to identify nuclear receptors for melatonin in neuronal cells remains valid for the community to achieve.

Tissue transcriptome profiling and pathway analyses revealed novel potential biomarkers in the tumor progression of canine oral melanoma.

Canine oral melanoma (COM) is an aggressive oral malignancy in dogs, mostly with metastasis. However, the understanding of total gene expression of oral melanoma (OM) at different clinical stages has been limited. The objective of this study was to identify novel mRNA biomarkers of early-stage OM (EOM) and late-stage OM (LOM). Transcriptome sequencing of 3 EOM, 5 LOM and 4 normal gingival tissues (controls) was performed. Selected transcriptome results were validated by quantitative reverse transcription-PCR (qRT-PCR) using 12 LOM and 10 controls. We found 534 differentially expressed in EOM compared with controls, whereas 696 genes in LOM were differentially expressed compared with controls (P < 0.05). Moreover, 27 genes were differentially expressed in LOM compared with EOM (P < 0.05). The genes expressed in COM were involved in the molecular mechanism of cancer and melanocyte development pathways, promoting melanoma progression. qRT-PCR confirmed an increased expression of genes encoding an important protein in chemotherapy resistance (dopachrome tautomerase, DCT) and tumor progression (forkhead box M1, FOXM1), and decreased expression of a tumor suppression gene (N-myc downstream-regulated gene 2, NDRG2) in LOM, concordant with transcriptome results. In conclusion, this study revealed the comprehensive transcriptome from COM tissues, and increased DCT and FOXM1 and decreased NDRG2 gene expression indicated the potential candidate biomarkers in COM progression.

A parasite DNA binding protein with potential to influence disease susceptibility acts as an analogue of mammalian HMGA transcription factors.

Intracellular pathogens construct their environmental niche, and influence disease susceptibility, by deploying factors that manipulate infected host cell gene expression. Theileria annulata is an important tick-borne parasite of cattle that causes tropical theileriosis. Excellent candidates for modulating host cell gene expression are DNA binding proteins bearing AT-hook motifs encoded within the TashAT gene cluster of the parasite genome. In this study, TashAT2 was transfected into bovine BoMac cells to generate three expressing and three non-expressing (opposite orientation) cell lines. RNA-Seq was conducted and differentially expressed (DE) genes identified. The resulting dataset was compared with genes differentially expressed between infected cells and non-infected cells, and DE genes between infected cell lines from susceptible Holstein vs tolerant Sahiwal cattle. Over 800 bovine genes displayed differential expression associated with TashAT2, 209 of which were also modulated by parasite infection. Network analysis showed enrichment of DE genes in pathways associated with cellular adhesion, oncogenesis and developmental regulation by mammalian AT-hook bearing high mobility group A (HMGA) proteins. Overlap of TashAT2 DE genes with Sahiwal vs Holstein DE genes revealed that a significant number of shared genes were associated with disease susceptibility. Altered protein levels encoded by one of these genes (GULP1) was strongly linked to expression of TashAT2 in BoMac cells and was demonstrated to be higher in infected Holstein leucocytes compared to Sahiwal. We conclude that TashAT2 operates as an HMGA analogue to differentially mould the epigenome of the infected cell and influence disease susceptibility.

Inter-ankle Systolic Blood Pressure Difference Is a Marker of Increased Fasting Blood-Glucose in Asian Pregnant Women.

Objective: This cross-sectional study aimed to determine the relationship between clinical blood pressures and blood pressures measured using Doppler with blood glucose in pregnancy by ethnicity. Methods: We recruited 179 (52% White European, 48% Asian) pregnant women at 24-28 weeks of gestation who underwent a glucose tolerance test in an antenatal clinic in Bradford Royal Infirmary, the UK, from 2012 to 2013. Systolic blood pressures in the arm (left and right brachial) and ankle [left and right posterior tibial (PT) and dorsalis pedalis (DP)] blood pressures were measured using a Doppler probe. The inter-arm (brachial) and inter-ankle (PT and DP) systolic blood pressure differences were obtained. A multivariate linear regression model adjusted for age, body mass index, and diabetes risk was used to assess the relationship between blood pressures and blood glucose. Results: Asian pregnant women had higher blood glucose but lower ankle blood pressures than White Europeans. In White Europeans, brachial blood pressures and clinical blood pressures were positively associated with fasting blood glucose (FBG), but brachial blood pressures did not perform better as an indicator of FBG than clinical blood pressures. In Asians, increased inter-ankle blood pressure difference was associated with increased FBG. For each 10 mmHg increase in the inter-ankle blood pressure difference, FBG increased by 0.12 mmol/L (Beta=0.12, 95%CI: 0.01-0.23). Conclusion: The relationship between blood pressures with blood glucose differed by ethnicity. In Asians, inter-ankle systolic blood pressure difference was positively associated with blood glucose. This is first ever report on ankle blood pressures with blood glucose in pregnancy which suggests future potential as a non-invasive gestational diabetes risk screening tool.

Differential associations of ankle and brachial blood pressures with diabetes and cardiovascular diseases: cross-sectional study.

Increased brachial systolic blood-pressure (BP) predicts diabetes (T2DM) but is not fully effective. Value of absolute ankle systolic BP for T2DM compared to brachial systolic BP is not known. Our objectives were to assess independent relationships of ankle-systolic BP with T2DM and cardiovascular disease in Europeans and south Asians. Cross-sectional studies of anonymised data from registered adults (n = 1087) at inner city deprived primary care practices. Study includes 63.85% ethnic minority. Systolic BP of the left and right-brachial, posterior-tibial and dorsalis-pedis-arteries measured using a Doppler probe. Regression models' factors were age, sex, ethnicity, body mass index (BMI) and waist height ratio (WHtR). Both brachial and ankle systolic-BP increase with diabetes in Europeans and south Asians. We demonstrated that there was a significant positive independent association of ankle BP with diabetes, regardless of age and sex compared to Brachial. There was stronger negative association of ankle blood pressure with cardiovascular disease, after adjustment for BMI, WHtR and ethnicity. Additionally, we found that ankle BP were significantly associated with cardiovascular disease in south Asians more than the Europeans; right posterior tibial. Ankle systolic BPs are superior to brachial BPs to identify risks of Type 2DM and cardiovascular diseases for enhanced patient care.

Advantages of CEMiTool for gene co-expression analysis of RNA-seq data.

Gene co-expression analysis is widely applied to transcriptomics data to associate clusters of genes with biological functions or identify therapeutic targets in diseases. Recently, the emergence of high-throughput technologies for gene expression analyses allows researchers to establish connections through gene co-expression analysis to identify clinical disease markers. However, gene co-expression analysis is complex and may be a daunting task. Here, we evaluate three co-expression analysis packages (WGCNA, CEMiTool, and coseq) using published RNA-seq datasets derived from ischemic cardiomyopathy and chronic obstructive pulmonary disease. Results show that the packages produced consensus co-expression clusters using default parameters. CEMiTool package outperformed the other two packages and required less computational resource and bioinformatics experience. This evaluation provides a basis on which data analysts can select bioinformatics tools for gene co-expression analysis.

Polymorphism in merozoite surface protein-7E of Plasmodium vivax in Thailand: Natural selection related to protein secondary structure.

Merozoite surface protein 7 (MSP-7) is a multigene family expressed during malaria blood-stage infection. MSP-7 forms complex with MSP-1 prior to merozoite egress from erythrocytes, and could affect merozoite invasion of erythrocytes. To characterize sequence variation in the orthologue in P. vivax (PvMSP-7), a gene member encoding PvMSP-7E was analyzed among 92 Thai isolates collected from 3 major endemic areas of Thailand (Northwest: Tak, Northeast: Ubon Ratchathani, and South: Yala and Narathiwat provinces). In total, 52 distinct haplotypes were found to circulate in these areas. Although population structure based on this locus was observed between each endemic area, no genetic differentiation occurred between populations collected from different periods in the same endemic area, suggesting spatial but not temporal genetic variation. Sequence microheterogeneity in both N- and C- terminal regions was predicted to display 4 and 6 α-helical domains, respectively. Signals of purifying selection were observed in α-helices II-X, suggesting structural or functional constraint in these domains. By contrast, α-helix-I spanning the putative signal peptide was under positive selection, in which amino acid substitutions could alter predicted CD4+ T helper cell epitopes. The central region of PvMSP-7E comprised the 5'-trimorphic and the 3'-dimorphic subregions. Positive selection was identified in the 3' dimorphic subregion of the central domain. A consensus of intrinsically unstructured or disordered protein was predicted to encompass the entire central domain that contained a number of putative B cell epitopes and putative protein binding regions. Evidences of intragenic recombination were more common in the central region than the remainders of the gene. These results suggest that the extent of sequence variation, recombination events and selective pressures in the PvMSP-7E locus seem to be differentially affected by protein secondary structure.