Cloning and expression of transgenes using linear vectors in Trypanosoma cruzi

The identification of new targets for vaccine and drug development for the treatment of Chagas’ disease is dependent on deepening our understanding of the parasite genome. Vectors for genetic manipulation in Trypanosoma cruzi basically include those that remain as circular episomes and those that integrate into the parasite’s genome. Artificial chromosomes are alternative vectors to overcome problematic transgene expression often occurring with conventional vectors in this parasite. We have constructed a series of vectors named pTACs (Trypanosome Artificial Chromosomes), all of them carrying telomeric and subtelomeric sequences and genes conferring resistance to different selection drugs. In addition, one pTAC harbours a modified GFP gene (pTAC-gfp), and another one carries the ornithine decarboxilase gene from Crithidia fasciculata (pTAC-odc). We have encountered artificial chromosomes generated from pTACs in transformed T. cruzi epimastigotes for every version of the designed vectors. These extragenomic elements, in approximately 6–8 copies per cell, remained as linear episomes, contained telomeres and persisted after 150 and 60 generations with or without selection drugs, respectively. The linear molecules remained stable through the different T. cruzi developmental forms. Furthermore, derived artificial chromosomes from pTAC-odc could complement the auxotrophy of T. cruzi for polyamines. Our results show that pTACs constitute useful tools for reverse functional genetics in T. cruzi that will contribute to a better understanding of T. cruzi biology.     

Molecular Identification and Genetic Relationships of Palestinian Grapevine Cultivars

Palestine has a wide range of agro-ecological concerns and hosts a large variety of plants. Grapes are part of the cultural heritage and provide an indispensable food ingredient. Local cultivars have been traditionally identified on the basis of morphological traits, geographical origin, or names of the vineyard owner; therefore, the occurrence of homonymy, synonymy, and misnaming significantly prevents their valorization. DNA profiling by 22 common SSR markers was used to characterize 43 putative cultivars grown mainly for local table grape consumption at the southern highland regions of West-Bank, to further evaluate genetic diversity and relationships of the population. Consistent matching of SSR markers with grapevines cultivated in neighboring countries or maintained in European germplasm collections was found for 8 of the 21 different non-redundant genotypes discovered, suggesting possible synonyms as well as the occurrence of breeding selections formerly developed in the USA. Genetic relationships inferred from SSR markers clearly assigned Palestinian cultivars to the Proles orientalis subpr. Antasiatica ancestral population, and they even remarked the connection between local resources and cultivars generated from international table grape breeding. This study supports the value of collection and conservation of vines endemic to a region of immense historical importance for viticulture.     

Evaluation of in vitro anticancer activity of sphaeropsidins A–C,fungal rearranged pimarane diterpenes,and semisynthetic derivatives

Sphaeropsidins A (1), B (7) and C (10), three fungal phytotoxins, unrearranged pimarane diterpenes produced by Diplodia cupressi and 10 semisynthetic derivatives were evaluated for their in vitro anticancer activities. Among these 13 compounds, sphaeropsidin A and two derivatives (2 and 6) thereof display 50% growth-inhibitory concentration in the low micromolar range for all cell lines analyzed. Structure activity relationship paralleled the phytopathogenic and antimicrobial ones except regarding the vinyl group at C-13 that does not seems to be required as it is for their antipathogenic activity.     

Structure of nicotinamide mononucleotide adenylyltransferase: a key enzyme in NAD(+) biosynthesis

BACKGROUND: Nicotinamide adenine dinucleotide (NAD(+)) is an essential cofactor involved in fundamental processes in cell metabolism. The enzyme nicotinamide mononucleotide adenylyltransferase (NMN AT) plays a key role in NAD(+) biosynthesis, catalysing the condensation of nicotinamide mononucleotide and ATP, and yielding NAD(+) and pyrophosphate. Given its vital role in cell life, the enzyme represents a possible target for the development of new antibacterial agents. RESULTS: The structure of NMN AT from Methanococcus jannaschii in complex with ATP has been solved by X-ray crystallography at 2.0 A resolution, using a combination of single isomorphous replacement and density modification techniques. The structure reveals a hexamer with 32 point group symmetry composed of alpha/beta topology subunits. The catalytic site is located in a deep cleft on the surface of each subunit, where one ATP molecule and one Mg(2+) are observed. A strictly conserved HXGH motif (in single-letter amino acid code) is involved in ATP binding and recognition. CONCLUSIONS: The structure of NMN AT closely resembles that of phosphopantetheine adenylyltransferase. Remarkably, in spite of the fact that the two enzymes share the same fold and hexameric assembly, a striking difference in their quaternary structure is observed. Moreover, on the basis of structural similarity including the HXGH motif, we identify NMN AT as a novel member of the newly proposed superfamily of nucleotidyltransferase alpha/beta phosphodiesterases. Our structural data suggest that the catalytic mechanism does not rely on the direct involvement of any protein residues and is likely to be carried out through optimal positioning of substrates and transition-state stabilisation, as is proposed for other members of the nucleotidyltransferase alpha/beta phosphodiesterase superfamily.     

Coagulation factor levels in non-metastatic colorectal cancer patients

There is evidence that high plasma levels of factor (F) VIII, FIX, FXI and fibrinogen are independent risk factors for venous thromboembolism. AIM: To determine the plasma concentrations of several coagulation factors and C4b-binding protein (C4BP) in a group of patients with non-metastatic colorectal cancer in order to investigate some aspects of cancer-acquired thrombophilia. METHODS: Plasma fibrinogen, FII, FV, FVII, FVIII, FIX, FX, FXI and FXII activity levels and C4BP concentrations were determined in 73 patients with non-metastatic colorectal cancer (48 colon and 25 rectum) and in 67 matched control subjects. No one in either group had had previous thrombotic events. RESULTS: Mean plasma concentrations of fibrinogen (functional and antigen), FVIII, FIX, FV and C4BP were significantly higher in colorectal cancer patients than in control subjects, while FVII and FXII levels were significantly decreased. Several correlations were found between the increased coagulation factors and C4BP concentrations, while FVII was highly correlated with FXII. CONCLUSIONS: In colorectal cancer patients high plasma fibrinogen, FVIII and FIX levels might represent further risk factors for venous thrombotic complications in the immediate post-surgery period, while decreased FVII and FXII concentrations may be an index of intravascular coagulation activation, still in a subclinical phase.     

Anoplin, a novel antimicrobial peptide from the venom of the solitary wasp Anoplius samariensis.

A novel antimicrobial peptide, anoplin, was purified from the venom of the solitary wasp Anoplius samariensis. The sequence was mostly analyzed by mass spectrometry, which was corroborated by solid-phase synthesis. Anoplin, composed of 10 amino acid residues, Gly-Leu-Leu-Lys-Arg-Ile-Lys-Thr-Leu-Leu-NH2, has a high homology to crabrolin and mastoparan-X, the mast cell degranulating peptides from social wasp venoms, and, therefore, can be predicted to adopt an amphipathic alpha-helix secondary structure. In fact, the circular dichroism (CD) spectra of anoplin in the presence of trifluoroethanol or sodium dodecyl sulfate showed a high content, up to 55%, of the alpha-helical conformation. A modeling study of anoplin based on its homology to mastoparan-X supported the CD results. Biological evaluation using the synthetic peptide revealed that this peptide exhibited potent activity in stimulating degranulation from rat peritoneal mast cells and broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria. Therefore, this is the first antimicrobial component to be found in the solitary wasp venom and it may play a key role in preventing potential infection by microorganisms during prey consumption by their larvae. Moreover, this peptide is the smallest among the linear alpha-helical antimicrobial peptides hitherto found in nature, which is advantageous for chemical manipulation and medical application.     

Risk factors for infection with chikungunya and Zika viruses in southern Puerto Rico: A community-based cross-sectional seroprevalence survey

Chikungunya virus (CHIKV) caused a large outbreak in Puerto Rico in 2014, followed by a Zika virus (ZIKV) outbreak in 2016. Communities Organized for the Prevention of Arboviruses (COPA) is a cohort study in southern Puerto Rico, initiated in 2018 to measure arboviral disease risk and provide a platform to evaluate interventions. To identify risk factors for infection, we assessed prevalence of previous CHIKV infection and recent ZIKV and DENV infection in a cross-sectional study among COPA participants. Participants aged 1–50 years (y) were recruited from randomly selected households in study clusters. Each participant completed an interview and provided a blood specimen, which was tested by anti-CHIKV IgG ELISA assay and anti-ZIKV and anti-DENV IgM MAC-ELISA assays. We assessed individual, household, and community factors associated with a positive result for CHIKV or ZIKV after adjusting for confounders. During 2018–2019, 4,090 participants were enrolled; 61% were female and median age was 28y (interquartile range [IQR]: 16–41). Among 4,035 participants tested for CHIKV, 1,268 (31.4%) had evidence of previous infection. CHIKV infection prevalence was lower among children 1–10 years old compared to people 11 and older (adjusted odds ratio [aOR] 2.30; 95% CI 1.71–3.08). Lower CHIKV infection prevalence was associated with home screens (aOR 0.51; 95% CI 0.42–0.61) and air conditioning (aOR 0.64; 95% CI 0.54–0.77). CHIKV infection prevalence also varied by study cluster of residence and insurance type. Few participants (16; 0.4%) had evidence of recent DENV infection by IgM. Among 4,035 participants tested for ZIKV, 651 (16%) had evidence of recent infection. Infection prevalence increased with older age, from 7% among 1–10y olds up to 19% among 41–50y olds (aOR 3.23; 95% CI 2.16–4.84). Males had an increased risk of Zika infection prevalence compared with females (aOR 1.31; 95% CI 1.09–1.57). ZIKV infection prevalence also decreased with the presence of home screens (aOR 0.66; 95% CI 0.54–0.82) and air conditioning (aOR 0.69; 95% CI 0.57–0.84). Similar infection patterns were observed for recent ZIKV infection prevalence and previous CHIKV infection prevalence by age, and the presence of screens and air conditioners in the home decreased infection risk from both viruses by as much as 50%.     

Development of user-friendly functional molecular markers for VvDXS gene conferring muscat flavor in grapevine

High fruit and wine quality combined with good climatic adaptation and disease resistance are essential objectives of grape breeding. While several molecular markers are available for pyramiding resistance to fungal pathogens, molecular tools for predicting fruit composition are still scarce. Muscat flavor, caused by the accumulation of monoterpenoids in the berry, is an important target trait for breeding, sought after in both table grapes and wine. Four missense mutations in the VvDXS gene in grape germplasm have been shown to be tightly linked to muscat flavor. Here we present highly reproducible and breeder-friendly functional markers for each of the targeted polymorphisms developed by using either the multiplexed minisequencing SNaPshot? method, the high-resolution melting (HRM) assay or the cleaved amplified polymorphic sequence system. A total of 242 grapevine accessions were analyzed to optimize these different genotyping methods and to provide allele-specific markers for accurate selection of muscat flavor at early stages of grape breeding programs. The HRM and the minisequencing SNaPshot multiplex assays allow for high-throughput automated screening and are suitable for large-scale breeding programs and germplasm characterization.     

Bean common mosaic virus and cucumber mosaic virus naturally infecting Aristolochia spp. plants in Brazil

In April 2022, Aristolochia plants with symptoms of mosaic were observed in a garden at Jardim Botânico Plantarum, Nova Odessa, São Paulo State, Brazil. Potyviridae-like particles were observed by transmission electron microscopy in leaf extracts. Total RNA extracted from symptomatic plants used in RT-PCR with universal and BCMV-specific primers detected the potyvirus bean common mosaic virus (BCMV). The cucumovirus cucumber mosaic virus (CMV) was identified only in Aristolochia littoralis plants that tested negative by RT-PCR for BCMV. Phylogenetic analysis grouped samples of Aristolochia in a different clade among samples of Phaseolus vulgaris. Phylogenetic analysis indicated that the CMV isolate from Aristolochia belongs to the CMV group IA. BCMV was mechanically transmitted to healthy plants of A. fimbriata, Chenopodium quinoa, P. vulgaris cv. Jalo and Macroptilium lathyroides. CMV was mechanically transmitted to plants of A. fimbriata and C. quinoa. The BCMV and CMV were aphid transmitted only by Aphis gossypii to Aristolochia plants. This is the first report of BCMV and CMV infecting Aristolochia plants in Brazil.