Alpha globin variation in the long-tailed macaque suggests malaria selection

Human haemoglobin variants, such as sickle, confer protection against death from malaria; consequently, frequencies of such variants are often greatly elevated in humans from malaria endemic regions. Among non-human primates, the long-tailed macaque, Macaca fascicularis, also displays substantial haemoglobin variation. Almost all M. fascicularis haemoglobin variation is in the alpha globin chain, encoded by two linked genes: HBA1 and HBA2. We demonstrate that alpha globin variation in M. fascicularis correlates with the strength of malaria selection. We identify a range of missense mutations in M. fascicularis alpha globin and demonstrate that some of these exhibit a striking HBA1 or HBA2 specificity, a pattern consistent with computational simulations of selection on genes exhibiting copy number variation. We propose that M. fascicularis accumulated amino acid substitutions in its alpha globin genes under malaria selection, in a process that closely mirrors, but does not entirely converge with, human malaria adaptation.

A comprehensive RNA handling and transcriptomics guide for high-throughput processing of Plasmodium blood-stage samples

Background Sequencing technology advancements opened new opportunities to use transcriptomics for studying malaria pathology and epidemiology. Even though in recent years the study of whole parasite transcriptome proved to be essential in understanding parasite biology there is no compiled up-to-date reference protocol for the efficient generation of transcriptome data from growing number of samples. Here, a comprehensive methodology on how to preserve, extract, amplify, and sequence full-length mRNA transcripts from Plasmodium-infected blood samples is presented that can be fully streamlined for high-throughput studies. Results The utility of various commercially available RNA-preserving reagents in a range of storage conditions was evaluated. Similarly, several RNA extraction protocols were compared and the one most suitable method for the extraction of high-quality total RNA from low-parasitaemia and low-volume blood samples was established. Furthermore, the criteria needed to evaluate the quality and integrity of Plasmodium RNA in the presence of human RNA was updated. Optimization of SMART-seq2 amplification method to better suit AT-rich Plasmodium falciparum RNA samples allowed us to generate high-quality transcriptomes from as little as 10 ng of total RNA and a lower parasitaemia limit of 0.05%. Finally, a modified method for depletion of unwanted human haemoglobin transcripts using in vitro CRISPR-Cas9 treatment was designed, thus improving parasite transcriptome coverage in low parasitaemia samples. To prove the functionality of the pipeline for both laboratory and field strains, the highest 2-hour resolution RNA-seq transcriptome for P. falciparum 3D7 intraerythrocytic life cycle available to date was generated, and the entire protocol was applied to create the largest transcriptome data from Southeast Asian field isolates. Conclusions Overall, the presented methodology is an inclusive pipeline for generation of good quality transcriptomic data from a diverse range of Plasmodium-infected blood samples with varying parasitaemia and RNA inputs. The flexibility of this pipeline to be adapted to robotic handling will facilitate both small and large-scale future transcriptomic studies in the field of malaria.

A comprehensive RNA handling and transcriptomics guide for high-throughput processing of Plasmodium blood-stage samples

BackgroundSequencing technology advancements opened new opportunities to use transcriptomics for studying malaria pathology and epidemiology. Even though in recent years the study of whole parasite transcriptome proved to be essential in understanding parasite biology there is no compiled up-to-date reference protocol for the efficient generation of transcriptome data from growing number of samples. Here, a comprehensive methodology on how to preserve, extract, amplify, and sequence full-length mRNA transcripts from Plasmodium-infected blood samples is presented that can be fully streamlined for high-throughput studies.ResultsThe utility of various commercially available RNA-preserving reagents in a range of storage conditions was evaluated. Similarly, several RNA extraction protocols were compared and the one most suitable method for the extraction of high-quality total RNA from low-parasitaemia and low-volume blood samples was established. Furthermore, the criteria needed to evaluate the quality and integrity of Plasmodium RNA in the presence of human RNA was updated. Optimization of SMART-seq2 amplification method to better suit AT-rich Plasmodium falciparum RNA samples allowed us to generate high-quality transcriptomes from as little as 10ng of total RNA and a lower parasitaemia limit of 0.05%. Finally, a modified method for depletion of unwanted human haemoglobin transcripts using in vitro CRISPR-Cas9 treatment was designed, thus improving parasite transcriptome coverage in low parasitaemia samples. To prove the functionality of the pipeline for both laboratory and field strains, the highest 2-hour resolution RNA-seq transcriptome for P. falciparum 3D7 intraerythrocytic life cycle available up-to-date was generated and the entire protocol was applied to create the largest transcriptome data from Southeast Asian field isolates.ConclusionsOverall, the presented methodology is an inclusive pipeline for generation of good quality transcriptomic data from a diverse range of Plasmodium-infected blood samples with varying parasitaemia and RNA inputs. The flexibility of this pipeline to be adapted to robotic handling will facilitate both small and large-scale future transcriptomic studies in the field of malaria.

Supramolecular linear coordination polymers of human serum albumin and haemoglobin

Human serum albumin and human haemoglobin having two-terpyridyl arms were readily polymerized via a bis(terpyridyl)–Fe2+ complex to generate watersoluble, uniform, and linear nanofibres.

Genetically engineered haemoglobin wrapped covalently with human serum albumins as an artificial O2 carrier

Recombinant human haemoglobin expressed in Pichia yeast was wrapped covalently with recombinant human serum albumins, yielding a core–shell structured rHbA(X)–rHSA3 cluster as an entirely synthetic O2 carrier used for a red blood cell substitute.

In vitro-Modulation of HbS Erythrocyte Parameters By Prednisolone Testing For Fe2+/Fe3+ Ratio, HbS Gelation and Osmotic Fragility

Aim: The study is targeted at the sickle cell disease. It has been discovered that some drugs or medications taken for certain ailments are either pro-sickling or anti-sickling in nature. In this study, acorticosteroid by the name of prednisolone was investigated to determineits possible effects on human haemoglobin-S gelation, erythrocyte fragility and Fe2+ and Fe3+ concentrations. Materials and Method: The blood sample of 5ml was collected from adult male and female donors by vein puncture using a 5 ml syringe and needle. The blood samples were confirmed as HbSS using standard haemoglobin electrophoresis. Various concentrations of the drug (0.05, 0.1, 0.3, 0.5 and 1 mg/ml) were used to determine the effects on human haemoglobin-S, gelation rate, erythrocyte fragility, Fe2+& Fe3+ concentrations. Absorbance reading was taken at 540 nm using a spectrophotometer. Results: The results showed that Prednisolone increased haemoglobin S gelation at all concentrations (p< 0.05) when compared to the control. The Fe2+/Fe3+ ratio showed a reduction in haemoglobin values at 0.3, 0.5 and 1.0 mg/ml concentrations when compared to the control and a slight increase at 0.05 and 0.1 mg/ml. For Erythrocyte Fragility, there was destabilization of red cell in all concentrations. Conclusion: This study suggests that this drug could have some undesirable effects on sickle cell subjects.

The ALGINATE MICROSPHERES CONTAINING CURCUMIN: PREPARATION, CHARACTERIZATION, AND IN VITRO HUMAN HAEMOGLOBIN GLYCOSYLATION ASSAY

Objective: The objective of the present study was to formulate, evaluate alginate microspheres of curcumin, and to investigate the inhibitory effect on glycosylated hemoglobin. Methods: All formulations were prepared by an ionotropic gelation technique using sodium alginate as a polymer and calcium chloride as a crosslinker in varying concentrations. The formulation batches (F1–F6) were evaluated for physical properties such as compatibility studies, percentage entrapment efficiency (%EE), microsphere yield, particle size, and polydispersity index. In vitro, drug release was studied and surface morphology was characterized by scanning electron microscopy. Results: The microspheres showed %EE, microsphere yield, particle size in the ranges of 44.86%–84.24%, 43.05%–81.4%, and 352–559 μm, respectively. In vitro, drug release and release kinetics showed that the developed curcumin microspheres system is a promising delivery system for controlled drug release. Scanning electron micrographs indicate porous and rough surface. The inhibitory properties of curcumin and microspheres (F4) on glycosylation formation were investigated in hemoglobin using quercetin as standard. The decreased in hemoglobin concentration after incubation of hemoglobin with a graded concentration of glucose over a specified time was used as an index for in vitro human hemoglobin glycosylation assay. Glycosylation inhibition was about 75% for standard quercetin, 60% for curcumin microspheres, and 38.74% for curcumin suspension occurred after 72 h. Conclusion: From these results, it can be concluded that curcumin in microsphere formulation has better therapeutic potential and could prove to be useful in the development of antidiabetic formulation.