Molecular characterization of Plasmodium vivax clinical isolates in Pakistan and Iran using pvmsp-1, pvmsp-3α and pvcsp genes as molecular markers

In this study, the diversity of Plasmodium vivax populations circulating in Pakistan and Iran has been investigated by using circumsporozoite protein (csp) and merozoite surface proteins 1 and 3α (msp-1 and msp-3α) genes as genetic markers. Infected P. vivax blood samples were collected from Pakistan (n = 187) and Iran (n = 150) during April to October 2008, and were analyzed using nested-PCR/RFLP and sequencing methods. Genotyping pvmsp-1 (variable block 5) revealed the presence of type 1, type 2 and recombinant type 3 allelic variants, with type 1 predominant, in both study areas. The sequence analysis of 33 P. vivax isolates from Pakistan and 30 from Iran identified 16 distinct alleles each, with one allele (R-8) from Iran which was not reported previously. Genotyping pvcsp gene also showed that VK210 type is predominant in both countries. Moreover, based on the size of amplified fragment of pvmsp-3α, three major types: type A (1800 bp), type B (1500 bp) and type C (1200 bp), were distinguished among the examined isolates that type A was predominant among Pakistani (72.7%) and Iranian (77.3%) parasites. PCR/RFLP products of pvmsp-3α with HhaI and AluI have detected 40 and 39 distinct variants among Pakistani and Iranian examined isolates, respectively. Based on these three studied genes, the rate of combined multiple genotypes were 30% and 24.6% for Pakistani and Iranian P. vivax isolates, respectively. These results indicate an extensive diversity in the P. vivax populations in both studies.     

A single-round multiplex PCR assay for the identification of Anopheles minimus related species infected with Plasmodium falciparum and Plasmodium vivax

This study aimed to develop a single-round multiplex PCR method for the identification of Anopheles minimus complex (An. minimus and Anopheles harrisoni) and Anopheles aconitus subgroup (An. aconitus and Anopheles varuna), and for the simultaneous detection of Plasmodium falciparum and Plasmodium vivax in these vectors. Five primers were created for a single-round multiplex PCR assay to identify four anopheline mosquitoes combined with three Plasmodium primers for the detection of P. falciparum and P. vivax in vectors. The four species of anopheline vectors and two Plasmodium species, P. falciparum and P. vivax, could be identified by the combination of eight primers in the single-round multiplex PCR assay. The amplified species-specific products were 380 bp for An. minimus, 180 bp for An. harrisoni, 150 bp for An. aconitus, 310 bp for An. varuna, 276 bp for P. falciparum, and 300 bp for P. vivax. The sensitivities were 0.5 pg/μl (25 sporozoites/μl) for P. falciparum DNA and between 0.5 and 5 pg/μl (25–250 sporozoites/μl) for P. vivax DNA. Furthermore, this developed method could be used to identify field caught An. minimus complex, An. aconitus subgroup from Thailand and Lao PDR. Also, it was successfully used to identify the species An. minimus, An. harrisoni, An. aconitus and An. varuna and to detect and identify P. falciparum and P. vivax in caught anopheline mosquitoes. The sensitivity of this method was high for simultaneous detection of P. falciparum and P. vivax in anopheline mosquitoes.     

Application of synthetic peptides for detection of anti-citrullinated peptide antibodies

Anti-citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis (RA) and represent an important tool for the serological diagnosis of RA.In this study, we describe ACPA reactivity to overlapping citrullinated Epstein-Barr virus nuclear antigen-1 (EBNA-1)-derived peptides and analyze their potential as substrates for ACPA detection by streptavidin capture enzyme-linked immunosorbent assay. Using systematically overlapping peptides, containing a 10 amino acid overlap, labelled with biotin C-terminally or N-terminally, sera from 160 individuals (RA sera (n = 60), healthy controls (n = 40), systemic lupus erythematosus (n = 20), Sjögren’s syndrome (n = 40)) were screened for antibody reactivity.Antibodies to a panel of five citrullinated EBNA-1 peptides were found in 67% of RA sera, exclusively of the IgG isotype, while 53% of the patient sera reacted with a single peptide, ARGGSRERARGRGRG-Cit-GEKR, accounting for more than half of the ACPA reactivity alone. Moreover, these antibodies were detected in 10% of CCP2-negative RA sera. In addition, 47% of the RA sera reacted with two or three citrullinated EBNA-1 peptides from the selected peptide panel. Furthermore, a negative correlation between the biotin attachment site and the location of citrulline in the peptides was found, i.e. the closer the citrulline was located to biotin, the lower the antibody reactivity.Our data suggest that citrullinated EBNA-1 peptides may be considered a substrate for the detection of ACPAs and that the presence of Epstein-Barr virus may play a role in the induction of these autoantibodies.     

Role of IL6, IL12B and VDR gene polymorphisms in Plasmodium vivax malaria severity,parasitemia and gametocytemia levels in an Amazonian Brazilian population

ObjectiveTo investigate the influence of IL6, IL12B and VDR single nucleotide polymorphisms (SNPs) in uncomplicated Plasmodium vivax infection symptoms intensity, parasitemia and gametocytemia levels in a Brazilian Amazonian population.MethodsA total of 167 malaria patients infected by P. vivax have parasitemia and gametocytemia levels estimated before treatment. Fourteen clinical symptoms were evaluated and included in a principal component analysis to derive a clinical symptom index. Patients were genotyped for IL6-174C > G, IL12B 735T > C, 458A > G, 159A > C, and VDR FokI, TaqI, BsmI SNPs by Taqman 5’ nuclease assays. A General Linear Model analysis of covariance with age, gender, exposure period and infection history and genetic ancestry was performed to investigate the association of genotypes with parasitemia and gametocytemia levels and with a clinical symptom index.ResultsHigher parasitemia levels were observed in IL6-174C carriers (p = 0.02) whereas IL12B CGT haplotype carriers presented lower parasitemia levels (p = 0.008). VDR TaqIC/BsmIA haplotype carriers showed higher gametocyte levels than non-carriers (p = 0.013). Based on the clinical index values the IL6-174C > G polymorphism was associated with malaria severity. The IL6-174C carriers presented a more severe clinical index when compared to GG homozygotes (p = 0.001).ConclusionThe present study suggests that IL6, IL12 and VDR influence severity, parasitemia and gametocytemia clearance in P. vivax infections, and highlights their potential role in malaria immune response in an Amazonian population.     

Genetic variations in CTLA-4, TNF-α, and LTA and susceptibility to T-cell lymphoma in a Chinese population

ObjectiveT-cell lymphoma is a highly aggressive malignant lymphoma that is rare in Caucasians but relatively common in Asian populations. Factors regulating T-cell proliferation and function may play an important role in the pathogenesis of T-cell lymphoma.MethodsA total of 8 single nucleotide polymorphisms in cytotoxic T lymphocyte antigen-4 (CTLA-4), tumor necrosis factor-α (TNF-α), and lymphotoxin-α (LTA) genes were detected by polymerase chain reaction–ligation detection reaction analysis in a Chinese population of 291 patients with T-cell lymphoma and 300 healthy controls. Logistic regression was used to determine the odds ratios (ORs) and 95% confidence intervals for the associations of genotypes and haplotypes with T-cell lymphoma risk.ResultsAmong these polymorphisms, the LTA +252AA genotype was significantly associated with T-cell lymphoma risk (OR, 2.3; P = 0.002). Furthermore, the TNF-α/LTA haplotype C-G-G-A (TNF-α ?857C, ?308G, and ?238G and LTA +252A) showed a significantly increased risk for T-cell lymphoma (OR, 1.6; P = 0.001).ConclusionOur study suggested that the LTA +252G>A polymorphism may influence susceptibility to T-cell lymphoma in the Chinese population.     

Association of naturally acquired IgG antibodies against Plasmodium falciparum serine repeat antigen-5 with reduced placental parasitemia and normal birth weight in pregnant Ugandan women: A pilot study

Plasmodium falciparum infection during pregnancy contributes substantially to malaria burden in both mothers and offspring. Analysis of naturally acquired immune responses that confer protection against parasitemia and clinical disease is important to guide vaccine evaluation as well as identify immune correlates. Unfortunately, few studies have addressed the relationship between immune responses to malaria vaccine candidate antigens and protection against adverse effects on pregnant women and newborn birth weight. This study examines the relationship of maternal antibody responses to serine repeat antigen-5 (SE36) and merozoite surface protein-1 (MSP1₁₉ and MSP1₄₂) with placental parasitemia and birth weight. In a peri-urban setting in Uganda, pregnant women without placental parasites have high median ODs for antibodies against SE36 (P < 0.001). Naturally acquired anti-SE36 IgG was most prevalent in women without placental parasitemia (P < 0.001). Furthermore, pregnant women with significantly high levels of anti-SE36 IgG delivered babies with normal birth weights (P < 0.001). That antibody to SE36 was associated with both a reduced risk of placental parasitemia and resulting normal birth weight in newborns suggests some protective role. In contrast, although antibody to MSP1₄₂ was also associated with reduced placental parasitemia and immune responses to both MSP1₁₉ and MSP1₄₂ may be of importance, there was no association between anti-MSP1₁₉ antibodies and infant birth weight outcomes. This study highlights the need for conducting further studies to investigate the association of antibodies against SE36 and outcomes of malaria infection in pregnant women.     

PCSK2-null mice exhibit delayed intestinal motility, reduced refeeding response and altered plasma levels of several regulatory peptides

AimsTo examine the effects of global lack of proprotein convertase subtilisin/kexin 2 (PCSK2) in mouse on intestinal motility, post-fast refeeding response and levels of several PCSK-generated peptides known to regulate food intake and processing.Main methodsUsing male and female PCSK2 knockout (KO) and wild-type (WT) mice, intestinal motility was assessed by determining the percent of intestinal length travelled by a charcoal-dyed meal following an oral gavage; the refeeding response by measuring the amount of meal consumed following an overnight fast; the levels of the regulatory peptides by enzyme immunoassays or immunoblotting.Key findingsRelative to same-gender WT mice, KO mice exhibited delayed intestinal transit (P < 0.001 in females; P < 0.05 in males). Their post-fast feeding response was reduced in females during the first hour of refeeding (P < 0.05). The circulating level of substance P (SP) was lower (P < 0.001 in females; P < 0.05 in males); it was higher for somatostatin (SS) (P < 0.001 in females; P < 0.05 in males) and GLP-1 (P < 0.001 in females; P < 0.01 in males) and GLP-2 (P < 0.001 in both genders); it was higher for peptide YY (PYY) in female mice only (P < 0.01). Processing of brain proneuropeptide Y was impaired in both genders.SignificanceThe alterations in intestinal motility and post-fast refeeding response observed in PCSK2-KO mice correlate with changes in the circulating and tissue levels of the regulatory peptides tested, suggesting that PCSK2 is needed for normal food intake and processing.     

Antimicrobial properties of two novel peptides derived from Theobroma cacao osmotin

The osmotin proteins of several plants display antifungal activity, which can play an important role in plant defense against diseases. Thus, this protein can be useful as a source for biotechnological strategies aiming to combat fungal diseases. In this work, we analyzed the antifungal activity of a cacao osmotin-like protein (TcOsm1) and of two osmotin-derived synthetic peptides with antimicrobial features, differing by five amino acids residues at the N-terminus. Antimicrobial tests showed that TcOsm1 expressed in Escherichia coli inhibits the growth of Moniliophthora perniciosa mycelium and Pichia pastoris X-33 in vitro. The TcOsm1-derived peptides, named Osm-pepA (H-RRLDRGGVWNLNVNPGTTGARVWARTK-NH₂), located at R23-K49, and Osm-pepB (H-GGVWNLNVNPGTTGARVWARTK-NH₂), located at G28-K49, inhibited growth of yeasts (Saccharomyces cerevisiae S288C and Pichia pastoris X-33) and spore germination of the phytopathogenic fungi Fusarium f. sp. glycines and Colletotrichum gossypi. Osm-pepA was more efficient than Osm-pepB for S. cerevisiae (MIC = 40 μM and MIC = 127 μM, respectively), as well as for P. pastoris (MIC = 20 μM and MIC = 127 μM, respectively). Furthermore, the peptides presented a biphasic performance, promoting S. cerevisiae growth in doses around 5 μM and inhibiting it at higher doses. The structural model for these peptides showed that the five amino acids residues, RRLDR at Osm-pepA N-terminus, significantly affect the tertiary structure, indicating that this structure is important for the peptide antimicrobial potency. This is the first report of development of antimicrobial peptides from T. cacao. Taken together, the results indicate that the cacao osmotin and its derived peptides, herein studied, are good candidates for developing biotechnological tools aiming to control phytopathogenic fungi.     

Attempts to vaccinate ewes and their lambs against natural infection with Haemonchus contortus in a tropical environment

A vaccine containing integral membrane glycoproteins from the intestine of Haemonchus contortus was evaluated in three groups of grazing sheep each containing 13 ewes and their 16 lambs naturally infected with gastrointestinal nematodes. Two groups were vaccinated with either 5 or 50 μg of the antigen per immunisation, while the third, the control group, received adjuvant alone. The sheep were immunised six times at 3 week intervals, partly because the vaccine antigens are hidden and thus no immunological boost would be delivered by subsequent infection and partly because the level of Haemonchus spp. challenge was expected to be high. The vaccinated ewes, first immunised approximately 1 month before lambing, showed a circulating antibody response but no signs of reduced anaemia or Haemonchus spp. egg counts, compared with control ewes. Several ewes with severe haemonchosis in all three groups had to be given precautionary treatment with anthelmintic drugs. In contrast, vaccinating their lambs with either 5 or 50 μg of the antigen per immunisation resulted in 10 fold higher antibody titres. In the case of the lower antigen dose this was associated with significantly less anaemia, 72% reduction in the overall number of Haemonchus spp. eggs produced and significantly fewer worms compared with control lambs. It is hypothesised that the heavily pregnant or lactating ewes did not have sufficient physiological reserves to mount a protective response following vaccination in the tropical weather and high challenge conditions that prevailed. Nevertheless, the vaccine could afford useful protection for lambs against H. contortus.     

Surface behavior of peptides from E1 GBV-C protein: Interaction with anionic model membranes and importance in HIV-1 FP inhibition

The interaction between a peptide sequence from GB virus C E1 protein (E1P8) and its structural analogs (E1P8-12), (E1P8-13), and (E1P8-21) with anionic lipid membranes (POPG vesicles and POPG, DPPG or DPPC/DPPG (2:1) monolayers) and their association with HIV-1 fusion peptide (HIV-1 FP) inhibition at the membrane level were studied using biophysical methods. All peptides showed surface activity but leakage experiments in vesicles as well as insertion kinetics in monolayers and lipid/peptide miscibility indicated a low level of interaction: neither E1P8 nor its analogs induced the release of vesicular content and the exclusion pressure values (π_e) were clearly lower than the biological membrane pressure (24–30 mN m^− 1) and the HIV-1 FP (35 mN m^− 1). Miscibility was elucidated in terms of the additivity rule and excess free energy of mixing (G^E). E1P8, E1P8-12 and E1P8-21 (but not E1P8-13) induced expansion of the POPG monolayer. The mixing process is not thermodynamically favored as the positive G^E values indicate. To determine how E1 peptides interfere in the action of HIV-1 FP at the membrane level, mixed monolayers of HIV-1 FP/E1 peptides (2:1) and POPG were obtained. E1P8 and its derivative E1P8-21 showed the greatest HIV-1 FP inhibition. The LC-LE phase lipid behavior was morphologically examined via fluorescence microscopy (FM) and atomic force microscopy (AFM). Images revealed that the E1 peptides modify HIV-1 FP–lipid interaction. This fact may be attributed to a peptide/peptide interaction as indicated by AFM results. Finally, hemolysis assay demonstrated that E1 peptides inhibit HIV-1 FP activity.     

Modulation of immune responses by Plasmodium falciparum infection in asymptomatic children living in the endemic region of Mbita,western Kenya

Individuals living in malaria endemic areas become clinically immune after multiple re-infections over time and remain infected without apparent symptoms. However, it is unclear why a long period is required to gain clinical immunity to malaria, and how such immunity is maintained. Although malaria infection is reported to induce inhibition of immune responses, studies on asymptomatic individuals living in endemic regions of malaria are relatively scarce. We conducted a cross-sectional study of immune responses in asymptomatic school children aged 4–16 years living in an area where Plasmodium falciparum and Schistosoma mansoni infections are co-endemic in Kenya. Peripheral blood mononuclear cells were subjected to flow cytometric analysis and cultured to determine proliferative responses and cytokine production. The proportions of cellular subsets in children positive for P. falciparum infection at the level of microscopy were comparable to the negative children, except for a reduction in central memory-phenotype CD8⁺ T cells and natural killer cells. In functional studies, the production of cytokines by peripheral blood mononuclear cells in response to P. falciparum crude antigens exhibited strong heterogeneity among children. In addition, production of IL-2 in response to anti-CD3 and anti-CD28 monoclonal antibodies was significantly reduced in P. falciparum-positive children as compared to -negative children, suggesting a state of unresponsiveness. These data suggest that the quality of T cell immune responses is heterogeneous among asymptomatic children living in the endemic region of P. falciparum, and that the responses are generally suppressed by active infection with Plasmodium parasites.     

Characterization of two peptides isolated from the venom of social wasp Chartergellus communis (Hymenoptera: Vespidae): Influence of multiple alanine residues and C-terminal amidation on biological effects

Chatergellus communis is a wasp species endemic to the neotropical region and its venom constituents have never been described. In this study, two peptides from C. communis venom, denominated Communis and Communis-AAAA, were chemically and biologically characterized. In respect to the chemical characterization, the following amino acid sequences and molecular masses were identified:Communis: Ile-Asn-Trp-Lys-Ala-Ile-Leu-Gly-Lys-Ile-Gly-Lys-COOH (1340.9 Da)Communis-AAAA: Ile-Asn-Trp-Lys-Ala-Ile-Leu-Gly-Lys-Ile-Gly-Lys-Ala-Ala-Ala-Ala-Val-Xle-NH₂ (1836.3 Da).Furthermore, their biological effects were compared, accounting for the differences in structural characteristics between the two peptides. To this end, three biological assays were performed in order to evaluate the hyperalgesic, edematogenic and hemolytic effects of these molecules. Communis-AAAA, unlike Communis, showed a potent hemolytic activity with EC₅₀ = 142.6 μM. Moreover, the highest dose of Communis-AAAA (2nmol/animal) induced hyperalgesia in mice. On the other hand, Communis (10nmol/animal) was able to induce edema but did not present hemolytic or hyperalgesic activity. Although both peptides have similarities in linear structures, we demonstrated the distinct biological effects of Communis and Communis-AAAA. This is the first study with Chartegellus communis venom, and both Communis and Communis-AAAA are unpublished peptides.     

Acquired Antibody Responses against Plasmodium vivax Infection Vary with Host Genotype for Duffy Antigen Receptor for Chemokines (DARC)

Background

Polymorphism of the Duffy Antigen Receptor for Chemokines (DARC) is associated with susceptibility to and the severity of Plasmodium vivax malaria in humans. P. vivax uses DARC to invade erythrocytes. Individuals lacking DARC are ‘resistant’ to P. vivax erythrocytic infection. However, susceptibility to P. vivax in DARC+ individuals is reported to vary between specific DARC genotypes. We hypothesized that the natural acquisition of antibodies to P. vivax blood stages may vary with the host genotype and the level of DARC expression. Furthermore, high parasitemia has been reported to effect the acquisition of immunity against pre-erythrocytic parasites. We investigated the correlation between host DARC genotypes and the frequency and magnitude of antibodies against P. vivax erythrocytic stage antigens.

Methodology/Findings

We assessed the frequencies and magnitudes of antibody responses against P. vivax and P. falciparum sporozoite and erythrocytic antigens in Colombian donors from malaria-endemic regions. The frequency and level of naturally-acquired antibodies against the P. vivax erythrocytic antigens merozoite surface protein 1 (PvMSP1) and Duffy binding protein (PvDBP) varied with the host DARC genotypes. Donors with one negative allele (FY*B/FY*Bnull and FY*A/FY*Bnull) were more likely to have anti-PvMSP1 and anti-PvDBP antibodies than those with two positive alleles (FY*B/FY*B and FY*A/FY*B). The lower IgG3 and IgG1 components of the total IgG response may account for the decreased responses to P. vivax erythrocytic antigens with FY*A/FY*B and FY*B/FY*B genotypes. No such association was detected with P. falciparum erythrocytic antigens, which does not use DARC for erythrocyte invasion.

Conclusion/Significance

Individuals with higher DARC expression, which is associated with higher susceptibility to P. vivax infection, exhibited low frequencies and magnitudes of P. vivax blood-stage specific antibody responses. This may indicate that one of the primary mechanisms by which P. vivax evades host immunity is through DARC indirectly down-regulating humoral responses against erythrocytic invasion and development.     

Evaluation of the antigenicity of universal epitopes from PvDBPII in individuals exposed to Plasmodium vivax malaria

The Duffy-binding protein (PvDBP) mediates invasion of reticulocytes by the malaria parasite Plasmodium vivax. PvDBP has been recognized as a good vaccine candidate due to its ability to induce antibody responses capable of inhibiting target cell invasion after natural infections. For the development of subunit-based vaccines, it is important to identify universal epitopes that could be presented by different HLA-DR alleles to induce effective cellular and humoral immune responses. In this study, the antigenicity of universal epitopes from PvDBPII was evaluated by stimulating peripheral blood mononuclear cells (PBMCs) isolated from individuals with different degrees of P. vivax malaria exposure and distinct HLA-DR alleles. Peptides 1635 and 1638 induced lymphoproliferation and stimulated the production of IL-6 and IFN-γ. The results suggest that conserved peptides binding with high activity to red blood cells and with known affinity to HLA-DR proteins could be good components for a P. vivax vaccine.     

Characterization of the host-defense peptides from skin secretions of Merlin's clawed frog Pseudhymenochirus merlini: Insights into phylogenetic relationships among the Pipidae

The family Pipidae comprises the genera Hymenochirus, Pipa, Pseudhymenochirus, Silurana, and Xenopus but phylogenetic relationships within the family are unclear. Peptidomic analysis of norepinephrine-stimulated skin secretions from Pseudhymenochirus merlini Chabanaud, 1920, the single species within the genus Pseudhymenochirus, led to identification of 13 host-defense peptides with antimicrobial activity. Two peptides (hymenochirin-1Pa and -1Pb) show structural similarity to hymenochirin-1B from Hymenochirus boettgeri and eight peptides (hymenochirin-5Pa, -5Pb, -5Pc, -5Pd, -5Pe, -5Pf, 5Pg and -5Ph) are structurally similar to each other and to hymenochirin-5B from H. boettgeri. Two peptides differing by a single amino acid (IKIPSFFRNILKKVGKEAVSLM/I AGALKQS), termed pseudhymenochirin-1Pa and -1Pb, and pseudhymenochirin-2Pa (GIFPIFAKLLGKVIKVASSLISKGRTE) do not resemble host-defense peptides previously isolated from pipid frogs. Hymenochirin-5Pe was the most abundant peptide in the secretions and hymenochirin-1Pa the most potent against Staphylococcus aureus (MIC = 2.5 μM) and Escherichia coli (MIC = 10 μM). The data support a close phylogenetic relationship between Hymenochirus and Pseudhymenochirus that is distinct from the Xenopodinae (Xenopus + Silurana) clade with Pipa sister-group to all other extant pipids.     

Effect of Pichia membranaefaciens in combination with salicylic acid on postharvest blue and green mold decay in citrus fruits

The separate or combined effects of Pichia membranaefaciens and salicylic acid (SA) on the control of blue and green mold decay in citrus fruits were investigated. Results indicate that combining P. membranaefaciens (1 × 10⁸ CFU ml⁻¹) with SA (10 μg ml⁻¹) either in a point-inoculated or dipped treatment provided a more effective control of blue and green mold than separately applying yeast or SA. SA (10 μg ml⁻¹) did not significantly affect P. membranaefaciens growth in vitro but slightly increased the yeast population in fruit wounds. P. membranaefaciens plus SA effectively enhanced the phenylalanine ammonia-lyase, peroxidase, polyphenoloxidase, chitinase, and β-1,3-glucanase activities and stimulated the synthesis of phenolic compounds. The combined treatment did not impair quality parameters such as weight loss or titratable acidity, but resulted in low average natural infection incidence and increased total soluble solids and ascorbic acid contents in citrus fruits after 14 d at 20 °C.     

The effects of arsenic and induced-phytoextraction methods on photosynthesis in Pteris species with different arsenic-accumulating abilities

Arsenic (As) accumulation and photosynthesis occur simultaneously in plants under As exposure. We investigated the effects of As and induced-phytoextraction methods on photosynthesis in two As hyperaccumulators (Pteris vittata and Pteris cretica var. nervosa) and two non-hyperaccumulators (Pteris semipinnata and Pteris ensiformis) under soil culture conditions. Chlorophyll fluorescence parameters (the maximum [F_v/F_m] and actual quantum efficiency [F_PSII]) and the activities of three photosynthetic enzymes (ATPase, ribulose-1, 5-bisphosphate carboxylase [RuBPC] and glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) were measured. Arsenic accumulation and photosynthetic behaviours in response to enhanced-phytoextraction methods (trans-1, 2-cyclobexylenedinitrilotetraacetic acid [CDTA] and phosphorous [P] addition and soil pH adjustment) of P. cretica and P. semipinnata were monitored and compared under conditions of 100 mg As kg⁻¹. Significant decreases in the F_v/F_m (19.9%) and F_PSII (36.1%) were observed in P. vittata when exposed to 100 mg As kg⁻¹ in comparison to the control (0 mg As kg⁻¹). Compared to the control (0 mg As kg⁻¹), the activities of GAPDH increased by 0.5% in P. cretica var. nervosa and decreased by only 8.3% in P. vittata even when both of them were treated with 200 mg As kg⁻¹, whereas a significant decrease, 56.1% and 51.7%, of this enzyme was observed in P. semipinnata and P. ensiformis, respectively, when exposed to 50 mg As kg⁻¹. Compared to the control (0 mg CDTA kg⁻¹ or 0 mg P kg⁻¹), the activities of ATPase increased by 53.7% and 82.7% in P. cretica when exposed to 0.5 g CDTA kg⁻¹ and 50 mg P kg⁻¹, respectively, and an increase of up to 175% was also observed in P. semipinnata when exposed to 600 mg P kg⁻¹. The activities of GAPDH increased by 68.9% and 90.7% in P. cretica when exposed to 2 g CDTA kg⁻¹ and 600 mg P kg⁻¹, respectively, but a decrease of up to 60% was observed in P. semipinnata when exposed to 2 g CDTA kg⁻¹. The uptake of As in P. semipinnata increased by 80.9% and 73.3% when 1 g CDTA kg⁻¹ and 600 mg P kg⁻¹ were added, respectively, compared to the control (0 g CDTA kg⁻¹ or 0 mg P kg⁻¹). It was concluded that GAPDH played an important role in the photosynthesis of As hyperaccumulators under As treatments.     

Plasmodium vivax VIR Proteins Are Targets of Naturally-Acquired Antibody and T Cell Immune Responses to Malaria in Pregnant Women

P. vivax infection during pregnancy has been associated with poor outcomes such as anemia, low birth weight and congenital malaria, thus representing an important global health problem. However, no vaccine is currently available for its prevention. Vir genes were the first putative virulent factors associated with P. vivax infections, yet very few studies have examined their potential role as targets of immunity. We investigated the immunogenic properties of five VIR proteins and two long synthetic peptides containing conserved VIR sequences (PvLP1 and PvLP2) in the context of the PregVax cohort study including women from five malaria endemic countries: Brazil, Colombia, Guatemala, India and Papua New Guinea (PNG) at different timepoints during and after pregnancy. Antibody responses against all antigens were detected in all populations, with PNG women presenting the highest levels overall. P. vivax infection at sample collection time was positively associated with antibody levels against PvLP1 (fold-increase: 1.60 at recruitment -first antenatal visit-) and PvLP2 (fold-increase: 1.63 at delivery), and P. falciparum co-infection was found to increase those responses (for PvLP1 at recruitment, fold-increase: 2.25). Levels of IgG against two VIR proteins at delivery were associated with higher birth weight (27 g increase per duplicating antibody levels, p<0.05). Peripheral blood mononuclear cells from PNG uninfected pregnant women had significantly higher antigen-specific IFN-γ T_H1 responses (p=0.006) and secreted less pro-inflammatory cytokines TNF and IL-6 after PvLP2 stimulation than P. vivax-infected women (p<0.05). These data demonstrate that VIR antigens induce the natural acquisition of antibody and T cell memory responses that might be important in immunity to P. vivax during pregnancy in very diverse geographical settings.     

Synthesis,biological activity and structure–activity relationship of endomorphin-1/substance P derivatives

Endomorphins have been shown to produce potent analgesia in various rodent models of pain. However, their central administration led to the development of tolerance and physical dependence. Conjugation of C-terminal substance P (SP) fragments to opioids and opioid peptides was previously shown to produce hybrid peptides with strong analgesic activity, with low or no propensity to develop tolerance. In this study, four peptides (2–5) comprised of endomorphin-1 (1) and C-terminal fragments of SP (four or five amino acids, SP_8–11 (2) or SP_7–11 (4), respectively), with an overlapping Phe residue, were synthesized. To overcome low metabolic stability and poor membrane permeability of the peptide, the N-terminus of 2 and 4 was further modified with a C10-carbon lipoamino acid (C10LAA) achieving 3 and 5, respectively. LAA-modification of the hybrid peptides resulted in a significant increase in metabolic stability and membrane permeability compared to peptides 1, 2 and 4. Compound 5 showed potent μ-opioid receptor binding affinity (K_iμ = 3.87 ± 0.51 nM) with dose-dependent agonist activity in the nanomolar range (IC₅₀ = 45 ± 13 nM). In silico modeling was used to investigate the binding modes and affinities of compounds 1–5 in the active site of μ-opioid receptors. The docking scores were in agreement with the K_iμ values obtained in the receptor binding affinity studies. The more active LAA-modified hybrid peptide showed a lower total interaction energy and higher negative value of MolDock score.