Biology subjects

Baptista, R.

Publications and source records attributed to Baptista, R..

5 recordsLinked to original sources

bioRxiv2021

Isolation and characterisation of quercitrin as a potent anti-sickle cell anaemia agent from Alchornea cordifolia.

Alchornea cordifolia Mull. Arg. (commonly known as Christmas Bush) has been used traditionally in Africa to treat sickle cell anaemia (a recessive disease, arising from the S haemoglobin [Hb] allele) but the active compounds are yet to be characterised. Herein we describe the use of sequential fractionation coupled with in vitro anti-sickling assays to purify the active component. Sickling was induced in HbSS genotype blood samples using sodium metabisulphite (Na2S2O5) or incubation in 100 % N2. Methanol extracts of A. cordifolia leaves and its sub-fractions showed >70 % suppression of HbSS erythrocyte sickling. Purified compound demonstrated 87.2 {+/-} 2.39 % significant anti-sickling activity and 93.1 {+/-} 2.69 % erythrocyte sickling-inhibition at 0.4 mg/mL. Nuclear magnetic resonance (NMR) spectra and high-resolution mass spectroscopy identified it as quercitrin (quercetin 3-rhamnoside). Purified quercitrin also inhibited the polymerisation of isolated HbS and stabilized sickle erythrocytes membranes. Metabolomic comparisons of blood samples using flow-infusion electrospray-high resolution mass spectrometry indicated that quercitrin could convert HbSS erythrocyte metabolomes to be similar to HbAA. Sickling was associated with changes in anti-oxidants, anaerobic bioenergy and arachidonic acid metabolism, all of which were reversed by quercitrin. The findings described could inform efforts directed to the development of an anti-sickling drug or quality control assessments of A. cordifolia preparations.

pharmacology and toxicology↗

bioRxiv2021

Escaping the Fate of Sisyphus: Assessing Resistome Hybridization Baits for Antimicrobial Resistance Gene Capture

Finding, characterizing, and monitoring reservoirs for antimicrobial resistance (AMR) is vital to protecting public health. Hybridization capture baits are an accurate, sensitive, and cost-effective technique used to enrich and characterize DNA sequences of interest, including antimicrobial resistance genes (ARGs), in complex environmental samples. We demonstrate the continued utility of a set of 19,933 hybridization capture baits designed from the Comprehensive Antibiotic Resistance Database (CARD)v1.1.2 and Pathogenicity Island Database (PAIDB)v2.0, targeting 3,565 unique nucleotide sequences that confer resistance. We demonstrate the efficiency of our bait set on a custom-made resistance mock community and complex environmental samples to increase the proportion of on-target reads as much as >200-fold. However, keeping pace with newly discovered ARGs poses a challenge when studying AMR, because novel ARGs are continually being identified and would not be included in bait sets designed prior to discovery. We provide imperative information on how our bait set performs against CARDv3.3.1, as well as a generalizable approach for deciding when and how to update hybridization capture bait sets. This research encapsulates the full life cycle of baits for hybridization capture of the resistome from design and validation (both in silico and in vitro) to utilization and forecasting updates and retirement. Originality-Significance StatementThis work is applicable to a wide range of research. It helps to define conditions under which hybridization capture is useful regarding not only antimicrobial resistance specifically, but also more generally how to assess the ongoing utility of existing bait sets - giving objective criteria for when and by what strategies baits should be updated. We also provide a method for quantifying and comparing antimicrobial resistance genes (ARGs) similar to what is used for RNAseq experiments. This approach improves comparison of ARGs across environments. Thus, the work provides an improved foundation for ARG future studies, while cutting across traditional areas of microbiology and extending beyond.

microbiology↗

bioRxiv2020

The anti-mycobacterial activity of Artemisia annua L is based on deoxyartemisinin and artemisinic acid

The discovery of antimalarial artemisinin from Artemisia annua L. is an example of how Traditional Chinese Medicine (TCM) may be exploited to meet a recognized need. In this study, we systemically investigated A. annua L. for its antimicrobial activity and assessed it as a source of bioactive natural products for anti-mycobacterial activity. We used a silica gel column to perform antimicrobial activity-guided purification of the A. annua leaf, whose identity was confirmed by rbcL DNA barcoding, and used UHPLC-HRMS and NMR to elucidate the structure of purified active compounds. The antimicrobial activity of crude extracts, isolated compounds and the control artemisinin (Apollo Scientific Ltd) was assessed against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), Mycobacterium smegmatis strains by serial micro dilution method (31.25-1000 g/mL). The isolated compounds were tested for synergistic effects against mycobacterium. Bioactive compounds were purified and identified as deoxyartemisinin and artemisinic acid. Artemisinic acid (MIC 250 g/mL) was more effective in comparison to deoxyartemisinin (MIC 500 g/mL) and artemisinin (MIC 1000 g/mL) against M. smegmatis. We used a molecular docking approach to investigate the interactions between selected anti-mycobacterial compounds and proteins involved in vital physiological functions in M. tuberculosis, namely MtPks13, MtPknB, MtPanK, MtKasA, MtInhA and MtDprE1 and found artemisinic acid showed docking scores superior to the control inhibiters for MtKasA, suggesting it to be a potential nick for further in vitro biological evaluation and anti-TB drug design.

pharmacology and toxicology↗

bioRxiv2020

Stage-Specific Long Non-coding RNAs in Cryptosporidium parvum as Revealed by Stranded RNA-Seq

Cryptosporidium is a protist parasite that has been identified as the second leading cause of moderate to severe diarrhea in children younger than two and a significant cause of mortality worldwide. Cryptosporidium has a complex, obligate, intracellular but extra cytoplasmic lifecycle in a single host. How genes are regulated in this parasite remains largely unknown. Long non-coding RNAs (lncRNAs) play critical regulatory roles, including gene expression across a broad range of organisms. Cryptosporidium lncRNAs have been reported to enter the host cell nucleus and affect the host response. However, no systematic study of lncRNAs in Cryptosporidium has been conducted to identify additional lncRNAs. In this study, we analyzed a C. parvum in vitro strand-specific RNA-seq developmental time series covering both asexual and sexual stages to identify lncRNAs associated with parasite development. In total, we identified 396 novel lncRNAs 86% of which are differentially expressed. Nearly 10% of annotated mRNAs have an antisense lncRNA. lncRNAs also appear to occur most often at the 3 end of their corresponding sense mRNA. Putative lncRNA regulatory regions were identified and many appear to encode bidirectional promoters. A positive correlation trend between lncRNA and the upstream mRNA expression was observed. Evolutionary conservation and expression of lncRNA candidates was observed between C. parvum, C. hominis and C. baileyi. Ten C. parvum protein-encoding genes with antisense transcripts have P. falciparum orthologs that also have antisense transcripts. Three C. parvum lncRNAs with exceptional properties (e.g., intron splicing) were experimentally validated using RT-PCR and RT-qPCR. We provide an initial characterization of the C. parvum non-coding transcriptome to facilitate further investigations into the roles of lncRNAs in parasite development and host-pathogen interactions.

genomics↗

bioRxiv2020

A druggable oxidative folding pathway in the endoplasmic reticulum of human malaria parasites

Malaria remains a major global health problem, and there exists a constant need to identify druggable weaknesses in P. falciparum biology. The endoplasmic reticulum (ER) has many essential roles in the asexual lifecycle and may offer new drug targets, but it remains critically understudied. We generated conditional mutants of the putative redox-active, ER chaperone PfJ2, and show that it is essential for parasite survival. Using a redox-active cysteine crosslinker, we identify its substrates to be other mediators of oxidative folding, PfPDI8 and PfPDI11, suggesting a redox-regulatory role for PfJ2. Knockdown of these protein disulfide isomerases in PfJ2 conditional mutants show that PfPDI11 is not essential, while PfPDI8 is essential for asexual growth and may work in a complex with PfJ2 and other ER chaperones. Finally, we show that these redox interactions in the parasite ER are sensitive to small molecule inhibition. Together these data build a model for how oxidative folding occurs in the P. falciparum ER and demonstrate its suitability for antimalarial drug development.

microbiology↗