Research Articles (Biochemistry)

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This collection contains some of the full text peer-reviewed/ refereed articles published by researchers from the Department of Biochemistry

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Nutritional composition and cytoyoxicity studies of black monkey (Strychnos madagascariensis) ripe fruit
(African Scholarly Science Communications Trust (ASSCAT), 2023-02) Oboh, M.; Zharare, G.; Osunsanmi, F.; Mosa, Rebamang Anthony; Opoku, A.R.
Strychnos madagascariensis also known as black monkey orange is found in tropical and subtropical Africa including South Africa, Lesotho, Zimbabwe, and Swaziland. The fruit is consumed traditionally as food in the northern coastal region of Kwazulu-Natal in South Africa and the Southern part of Zimbabwe. This study investigated the nutritional, anti-nutritional composition, and cytotoxicity of S. madagascariensis ripe fruit. Fruits were randomly selected, and the parameters of each experiment were measured in triplicates. The seed coat and fruit pulp were analysed for proximate, mineral and anti-nutrients (phytic and oxalic acid) composition using standard protocols. The cytotoxic effect of methanolic extracts from the fruit parts of S. madagascariensis was tested on human embryonic kidney (HEK293) and cervical cancer (HeLa) cell lines using the MTT [3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide] cell proliferation assay. The fruit pulp’s moisture (6%), ash (11%), protein (5%), fat (1%), fibre (12%), and carbohydrate (12%) contents were significantly different (P<0.05) from the compositions of the fruit pulp (5% moisture, 5% ash, 4% protein, 33% fat, 8% fibre and 11% carbohydrate). The minerals analysed were significantly (P<0.05) higher in the fruit pulp compared to the seed coat (testa). The fruit's seed coat and fruit pulp's methanolic extracts did not show any significant toxicity in the HEK293 cell line. The seed coat's methanolic extract exhibited moderate toxicity in the HeLa cell line. The methanolic extracts from both fruit parts showed a dose-dependent effect in HEK293 and HeLa cell lines. The results of the cytotoxicity analysis revealed the safe nature of the fruit pulp while caution needs to be taken when consuming the seed coat. The seed coat contained 1.39±0.01% phytic acid, but oxalic acid and phytic acid were not detected in the fruit pulp. This study suggests that the fruit parts could serve as a source of potassium, magnesium, and nitrogen, but poor sources of protein, carbohydrates, and fat (pulp).
Mitochondrial genes from 18 angiosperms fill sampling gaps for phylogenomic inferences of the early diversification of flowering plants
(Wiley, 2022-07) Xue, Jia-Yu; Dong, Shan-Shan; Wang, Ming-Qiang; Song, Tian-Qiang; Zhou, Guang-Can; Li, Zhen; Van de Peer, Yves; Shao, Zhu-Qing; Wang, Wei; Chen, Min; Zhang, Yan-Mei; Sun, Xiao-Qin; Chen, Hong-Feng; Zhang, Yong-Xia; Zhang, Shou-Zhou; Chen, Fei; Zhang, Liang-Sheng; Cox, Cymon; Liu, Yang; Wang, Qiang; Hang, Yue-Yu
The early diversification of angiosperms is thought to have been a rapid process, which may complicate phylogenetic analyses of early angiosperm relationships. Plastid and nuclear phylogenomic studies have raised several conflicting hypotheses regarding overall angiosperm phylogeny, but mitochondrial genomes have been largely ignored as a relevant source of information. Here we sequenced mitochondrial genomes from 18 angiosperms to fill taxon-sampling gaps in Austrobaileyales, magnoliids, Chloranthales, Ceratophyllales, and major lineages of eudicots and monocots. We assembled a data matrix of 38 mitochondrial genes from 107 taxa to assess how well mitochondrial genomic data address current uncertainties in angiosperm relationships. Although we recovered conflicting phylogenies based on different data sets and analytical methods, we also observed congruence regarding deep relationships of several major angiosperm lineages: Chloranthales were always inferred to be the sister group of Ceratophyllales, Austrobaileyales to mesangiosperms, and the unplaced Dilleniales was consistently resolved as the sister to superasterids. Substitutional saturation, GC compositional heterogeneity, and codon-usage bias are possible reasons for the noise/conflict that may impact phylogenetic reconstruction; and angiosperm mitochondrial genes may not be substantially affected by these factors. The third codon positions of the mitochondrial genes appear to contain more parsimony-informative sites than the first and second codon positions, and therefore produced better resolved phylogenetic relationships with generally strong support. The relationships among these major lineages remain incompletely resolved, perhaps as a result of the rapidity of early radiations. Nevertheless, data from mitochondrial genomes provide additional evidence and alternative hypotheses for exploring the early evolution and diversification of the angiosperms.
Cultivated hawthorn (Crataegus pinnatifida var. major) genome sheds light on the evolution of Maleae (apple tribe)
(Wiley, 2022-08) Zhang, Ticao; Qiao, Qin; Du, Xiao; Zhang, Xiao; Hou, Yali; Wei, Xin; Sun, Chao; Zhang, Rengang; Yun, Quanzheng; Crabbe, M. James C.; Van de Peer, Yves; Dong, Wenxuan
Cultivated hawthorn (Crataegus pinnatifida var. major) is an important medicinal and edible plant with a long history of use for health protection in China. Herein, we provide a de novo chromosomelevel genome sequence of the hawthorn cultivar “Qiu Jinxing.” We assembled an 823.41Mb genome encoding 40 571 genes and further anchored the 779.24Mb sequence into 17 pseudo‐chromosomes, which account for 94.64% of the assembled genome. Phylogenomic analyses revealed that cultivated hawthorn diverged from other species within the Maleae (apple tribe) at approximately 35.4 Mya. Notably, genes involved in the flavonoid and triterpenoid biosynthetic pathways have been significantly amplified in the hawthorn genome. In addition, our results indicated that the Maleae share a unique ancient tetraploidization event; however, no recent independent whole‐genome duplication event was specifically detected in hawthorn. The amplification of non‐specific long terminal repeat retrotransposons contributed the most to the expansion of the hawthorn genome. Furthermore, we identified two paleo‐sub‐genomes in extant species of Maleae and found that these two sub‐genomes showed different rearrangement mechanisms. We also reconstructed the ancestral chromosomes of Rosaceae and discussed two possible paleopolyploid origin patterns (autopolyploidization or allopolyploidization) of Maleae. Overall, our study provides an improved context for understanding the evolution of Maleae species, and this new highquality reference genome provides a useful resource for the horticultural improvement of hawthorn.
Protective immunity of plant-produced African horse sickness virus serotype 5 chimaeric virus-like particles (VLPs) and viral protein 2 (VP2) vaccines in IFNAR-/- mice
(Elsevier, 2022-08) O'Kennedy, Martha Magaretha; Coetzee, Peter; Koekemoer, Otto; Du Plessis, Lissinda; Lourens, Carina W.; Kwezi, Lusisizwe; Du Preez, Ilse; Mamputha, Sipho; Mokoena, Nobalanda B.; Rutkowska, Daria A.; Verschoor, J.A. (Jan Adrianus), 1953-; Lemmer, Yolandy
Next generation vaccines have the capability to contribute to and revolutionise the veterinary vaccine industry. African horse sickness (AHS) is caused by an arbovirus infection and is characterised by respiratory distress and/or cardiovascular failure and is lethal to horses. Mandatory annual vaccination in endemic areas curtails disease occurrence and severity. However, development of a next generation AHSV vaccine, which is both safe and efficacious, has been an objective globally for years. In this study, both AHSV serotype 5 chimaeric virus-like particles (VLPs) and soluble viral protein 2 (VP2) were successfully produced in Nicotiana benthamiana ΔXT/FT plants, partially purified and validated by gel electrophoresis, transmission electron microscopy and liquid chromatography-mass spectrometry (LC-MS/MS) based peptide sequencing before vaccine formulation. IFNAR-/- mice vaccinated with the adjuvanted VLPs or VP2 antigens in a 10 µg prime-boost regime resulted in high titres of antibodies confirmed by both serum neutralising tests (SNTs) and enzyme-linked immunosorbent assays (ELISA). Although previous studies reported high titres of antibodies in horses when vaccinated with plant-produced AHS homogenous VLPs, this is the first study demonstrating the protective efficacy of both AHSV serotype 5 chimaeric VLPs and soluble AHSV-5 VP2 as vaccine candidates. Complementary to this, coating ELISA plates with the soluble VP2 has the potential to underpin serotype-specific serological assays.
syntenet : an R/Bioconductor package for the inference and analysis of synteny networks
(Oxford University Press, 2023-01) Almeida-Silva, Fabricio; Zhao, Tao; Ullrich, Kristian K.; Schranz, M. Eric; Van de Peer, Yves
Interpreting and visualizing synteny relationships across several genomes is a challenging task. We previously proposed a network-based approach for better visualization and interpretation of large-scale microsynteny analyses. Here, we present syntenet, an R package to infer and analyze synteny networks from whole-genome protein sequence data. The package offers a simple and complete framework, including data preprocessing, synteny detection and network inference, network clustering and phylogenomic profiling, and microsynteny-based phylogeny inference. Graphical functions are also available to create publication-ready plots. Synteny networks inferred with syntenet can highlight taxon-specific gene clusters that likely contributed to the evolution of important traits, and microsynteny-based phylogenies can help resolve phylogenetic relationships under debate.
Novel 3-trifluoromethyl-1,2,4-oxadiazole analogues of astemizole with multi-stage antiplasmodium activity and in vivo efficacy in a Plasmodium berghei mouse malaria infection model
(American Chemical Society, 2022-12) Mambwe, Dickson; Korkor, Constance M.; Mabhula, Amanda; Ngqumba, Zama; Cloete, Cleavon; Kumar, Malkeet; Barros, Paula Ladeia; Leshabane, Meta Kgaogelo; Coertzen, Dina; Taylor, Dale; Gibhard, Liezl; Njoroge, Mathew; Lawrence, Nina; Reader, Janette; Moreira, Diogo Rodrigo; Birkholtz, Lyn-Marie; Wittlin, Sergio; Egan, Timothy J.; Chibale, Kelly
Please read abstract in the article.
Population genetics of a lethally managed medium-sized predator
(Wiley, 2022-09) Slater, K.; De Jager, Deon; Van Wyk, A.M.; Dalton, Desire Lee; Kropff, Anna S.; Du Preez, Ilse
Globally, levels of human–wildlife conflict are increasing as a direct consequence of the expansion of people into natural areas resulting in competition with wildlife for food and other resources. By being forced into increasingly smaller pockets of suitable habitat, many animal species are at risk of becoming susceptible to loss of genetic diversity, inbreeding depression and the associated inability to adapt to environmental changes. Predators are often lethally controlled due to their threat to livestock. Predators such as jackals (black backed, golden and side striped; Canis mesomelas, C. aureus and C. adustus, respectively), red foxes (Vulpes vulpes) and coyotes (C. latrans) are highly adaptable and may respond to ongoing persecution through compensatory reproduction such as reproducing at a younger age, producing larger litters and/or compensatory immigration including dispersal into vacant territories. Despite decades of lethal management, jackals are problematic predators of livestock in South Africa and, although considered a temporary measure, culling of jackals is still common. Culling may affect social groups, kinship structure, reproductive strategies and sex-biased dispersal in this species. Here, we investigated genetic structure, variation and relatedness of 178 culled jackals on private small-livestock farms in the central Karoo of South Africa using 13 microsatellites. Genetic variation was moderate to high and was similar per year and per farm. An absence of genetic differentiation was observed based on STRUCTURE, principal component analysis and AMOVA. Relatedness was significantly higher within farms (r = 0.189) than between farms (r = 0.077), a result corroborated by spatial autocorrelation analysis. We documented 18 occurrences of dispersal events where full siblings were detected on different farms (range: 0.78–42.93 km). Distance between identified parent–offspring varied from 0 to 36.49 km. No evidence for sex-biased dispersal was found. Our results suggest that in response to ongoing lethal management, this population is most likely able to maintain genetic diversity through physiological and behavioural compensation mechanisms.
Chromosome-scale assembly of the Moringa oleifera Lam. genome uncovers polyploid history and evolution of secondary metabolism pathways through tandem duplication
(Wiley Open Access, 2022-09) Chang, Jiyang; Marczuk-Rojas, Juan Pablo; Waterman, Carrie; Garcia-Llanos, Armando; Chen, Shiyu; Ma, Xiao; Hulse-Kemp, Amanda; Van Deynze, Allen; Van de Peer, Yves; Carretero-Paulet, Lorenzo
The African Orphan Crops Consortium (AOCC) selected the highly nutritious, fast growing and drought tolerant tree crop moringa (Moringa oleifera Lam.) as one of the first of 101 plant species to have its genome sequenced and a first draft assembly was published in 2019. Given the extensive uses and culture of moringa, often referred to as the multipurpose tree, we generated a significantly improved new version of the genome based on long-read sequencing into 14 pseudochromosomes equivalent to n = 14 haploid chromosomes. We leveraged this nearly complete version of the moringa genome to investigate main drivers of gene family and genome evolution that may be at the origin of relevant biological innovations including agronomical favorable traits. Our results reveal that moringa has not undergone any additional whole-genome duplication (WGD) or polyploidy event beyond the gamma WGD shared by all core eudicots. Moringa duplicates retained following that ancient gamma events are also enriched for functions commonly considered as dosage balance sensitive. Furthermore, tandem duplications seem to have played a prominent role in the evolution of specific secondary metabolism pathways including those involved in the biosynthesis of bioactive glucosinolate, flavonoid, and alkaloid compounds as well as of defense response pathways and might, at least partially, explain the outstanding phenotypic plasticity attributed to this species. This study provides a genetic roadmap to guide future breeding programs in moringa, especially those aimed at improving secondary metabolism related traits.
Investigating the role of African horse sickness virus VP7 protein crystalline particles on virus replication and release
(MDPI, 2022-10-04) Bekker, Shani; Potgieter, Christiaan A.; Van Staden, Vida; Theron, Jacques; vida.vanstaden@up.ac.za
African horse sickness is a deadly and highly infectious disease of equids, caused by African horse sickness virus (AHSV). AHSV is one of the most economically important members of the Orbivirus genus. AHSV is transmitted by the biting midge, Culicoides, and therefore replicates in both insect and mammalian cell types. Structural protein VP7 is a highly conserved major core protein of orbiviruses. Unlike any other orbivirus VP7, AHSV VP7 is highly insoluble and forms flat hexagonal crystalline particles of unknown function in AHSV-infected cells and when expressed in mammalian or insect cells. To examine the role of AHSV VP7 in virus replication, a plasmid-based reverse genetics system was used to generate a recombinant AHSV that does not form crystalline particles. We characterised the role of VP7 crystalline particle formation in AHSV replication in vitro and found that soluble VP7 interacted with viral proteins VP2 and NS2 similarly to wild-type VP7 during infection. Interestingly, soluble VP7 was found to form uncharacteristic tubule-like structures in infected cells which were confirmed to be as a result of unique VP7-NS1 colocalisation. Furthermore, it was found that VP7 crystalline particles play a role in AHSV release and yield. This work provides insight into the role of VP7 aggregation in AHSV cellular pathogenesis and contributes toward the understanding of the possible effects of viral protein aggregation in other human virus-borne diseases.
Evolutionary history and pan-genome dynamics of strawberry (Fragaria spp.)
(National Academy of Sciences, 2021-10) Qiao, Qin; Edger, Patrick P.; Xue, Li; Qiong, La; Lu, Jie; Zhang, Yichen; Cao, Qiang; Yocca, Alan E.; Platts, Adrian E.; Knapp, Steven J.; Van Montagu, Marc; Van de Peer, Yves; Lei, Jiajun; Zhang, Ticao
Strawberry (Fragaria spp.) has emerged as a model system for various fundamental and applied research in recent years. In total, the genomes of five different species have been sequenced over the past 10 y. Here, we report chromosome-scale reference genomes for five strawberry species, including three newly sequenced species’ genomes, and genome resequencing data for 128 additional accessions to estimate the genetic diversity, structure, and demographic history of key Fragaria species. Our analyses obtained fully resolved and strongly supported phylogenies and divergence times for most diploid strawberry species. These analyses also uncovered a new diploid species (Fragaria emeiensis Jia J. Lei). Finally, we constructed a pan-genome for Fragaria and examined the evolutionary dynamics of gene families. Notably, we identified multiple independent single base mutations of the MYB10 gene associated with white pigmented fruit shared by different strawberry species. These reference genomes and datasets, combined with our phylogenetic estimates, should serve as a powerful comparative genomic platform and resource for future studies in strawberry.
Methicillin-resistant staphylococci among school children in Mariental, Namibia
(Elsevier, 2022-03) Walter, Sunette; Beukes, Mervyn; Mumbengegwi, Davis; Bock, Ronnie
Methicillin-resistant Staphylococcus aureus (MRSA) is resistant to beta-lactam antibiotics, while some strains are multi-drug resistant and may produce disease-causing toxins. Drug resistant strains are often responsible for chronic, persistent and recurrent infections, which pose a challenge for healthcare practitioners. Our study aimed to determine the prevalence of nasal MRSA and methicillin-resistant coagulase-negative staphylococci (MRCoNS) among school children in the Mariental community, southern Namibia. This was a cross-sectional study in the Mariental District. Nasal specimens (swabs) were collected from 272 randomly selected learners aged 6–14 years attending school in the area during the months of March, September and October 2016. Isolation and identification of staphylococci were performed using standard microbiological methods. Methicillin-resistant isolates were identified by their resistance towards cefoxitin (30 μg) using the Kirby-Bauer disk diffusion assay. Enterotoxin production among multi-drug resistant MRSA isolates was detected with a SET-RPLA toxin detection kit. Methicillin-resistant S. aureus was isolated from 48 (17.6%) learners and MRCoNS from only seven (2.6%). Methicillin-resistant S. aureus colonization was significantly higher (P = 0.003) in the age group 11–14 years than in the group 6–10 years. Among the 433 staphylococcal isolates screened for cefoxitin resistance, 51 (11.8%) were MRSA and seven (1.6%) were MRCoNS. From the 51 MRSA isolates, 22 (43.1%) were multi-drug resistant of which six were enterotoxigenic. This is the first report on MRSA and MRCoNS among school children in Namibia. The presence of multidrug resistant and potentially virulent staphylococci among school children in Mariental, Namibia, is of concern. Self-infection by these bacteria poses various health risks for the children. It is recommended that school health programmes improve current hygiene practices. Frequent handwashing can prevent staphylococcal disease and spread of resistant strains among learners and the wider community.
The genome of hibiscus hamabo reveals its adaptation to saline and waterlogged habitat
(Oxford University Press, 2022-03-23) Wang, Zhiquan; Xue, Jia-Yu; Hu, Shuai-Ya; Zhang, Fengjiao; Yu, Ranran; Chen, Dijun; Van de Peer, Yves; Jiang, Jiafu; Song, Aiping; Ni, Longjie; Hua, Jianfeng; Lu, Zhiguo; Yu, Chaoguang; Yin, Yunlong; Gu, Chunsun
Hibiscus hamabo is a semi-mangrove species with strong tolerance to salt and waterlogging stress. However, the molecular basis and mechanisms that underlie this strong adaptability to harsh environments remain poorly understood. Here, we assembled a high-quality, chromosome-level genome of this semi-mangrove plant and analyzed its transcriptome under different stress treatments to reveal regulatory responses and mechanisms. Our analyses suggested that H. hamabo has undergone two recent successive polyploidy events, a whole-genome duplication followed by a whole-genome triplication, resulting in an unusually large gene number (107 309 genes). Comparison of the H. hamabo genome with that of its close relative Hibiscus cannabinus, which has not experienced a recent WGT, indicated that genes associated with high stress resistance have been preferentially preserved in the H. hamabo genome, suggesting an underlying association between polyploidy and stronger stress resistance. Transcriptomic data indicated that genes in the roots and leaves responded differently to stress. In roots, genes that regulate ion channels involved in biosynthetic and metabolic processes responded quickly to adjust the ion concentration and provide metabolic products to protect root cells, whereas no such rapid response was observed from genes in leaves. Using co-expression networks, potential stress resistance genes were identified for use in future functional investigations. The genome sequence, along with several transcriptome datasets, provide insights into genome evolution and the mechanism of salt and waterlogging tolerance in H. hamabo, suggesting the importance of polyploidization for environmental adaptation.
MicroRNA-mediated post-transcriptional regulation of Pinus pinaster response and resistance to pinewood nematode
(Nature Research, 2022-03-25) Modesto, Ines; Inacio, Vera; Van de Peer, Yves; Miguel, Celia
Pine wilt disease (PWD), caused by the parasitic nematode Bursaphelenchus xylophilus, or pinewood nematode (PWN), is a serious threat to pine forests in Europe. Pinus pinaster is highly susceptible to the disease and it is currently the most affected European pine species. In this work, we investigated the role of small RNAs (sRNAs) in regulating P. pinaster–PWN interaction in an early stage of infection. After performing an artificial PWN inoculation assay, we have identified 105 plant microRNAs (miRNAs) responsive to PWN. Based on their predicted targets, part of these miRNAs was associated with roles in jasmonate-response pathway, ROS detoxification, and terpenoid biosynthesis. Furthermore, by comparing resistant and susceptible plants, eight miRNAs with putative functions in plant defence and resistance to PWN have been identified. Finally, we explored the possibility of bidirectional trans-kingdom RNA silencing, identifying several P. pinaster genes putatively targeted by PWN miRNAs, which was supported by degradome analysis. Targets for P. pinaster miRNAs were also predicted in PWN, suggesting a role for trans-kingdom miRNA transfer and gene silencing both in PWN parasitism as in P. pinaster resistance to PWD. Our results provide new insights into previously unexplored roles of sRNA post-transcriptional regulation in P. pinaster response and resistance to PWN.
Adapt or die : targeting unique transmission-stage biology for malaria elimination
(Frontiers Media S.A., 2022-06-09) Van der Watt, Mariette Elizabeth; Reader, Janette; Birkholtz, Lyn-Marie; lbirkholtz@up.ac.za
Plasmodium parasites have a complex life cycle that includes development in the human host as well as the Anopheles vector. Successful transmission of the parasite between its host and vector therefore requires the parasite to balance its investments in asexual replication and sexual reproduction, varying the frequency of sexual commitment to persist within the human host and generate future opportunities for transmission. The transmission window is extended further by the ability of stage V gametocytes to circulate in peripheral blood for weeks, whereas immature stage I to IV gametocytes sequester in the bone marrow and spleen until final maturation. Due to the low gametocyte numbers in blood circulation and with the ease of targeting such life cycle bottlenecks, transmission represents an efficient target for therapeutic intervention. The biological process of Plasmodium transmission is a multistage, multifaceted process and the past decade has seen a much deeper understanding of the molecular mechanisms and regulators involved. Clearly, specific and divergent processes are used during transmission compared to asexual proliferation, which both poses challenges but also opportunities for discovery of transmission-blocking antimalarials. This review therefore presents an update of our molecular understanding of gametocyte and gamete biology as well as the status of transmission-blocking activities of current antimalarials and lead development compounds. By defining the biological components associated with transmission, considerations for the development of new transmission-blocking drugs to target such untapped but unique biology is suggested as an important, main driver for transmissionblocking drug discovery.
SNP detection in Pinus pinaster transcriptome and association with resistance to pinewood nematode
(MDPI, 2022-06-17) Modesto, Ines; Inacio, Vera; Novikova, Polina; Carrasquinho, Isabel; Van de Peer, Yves; Miguel, Celia
Pinewood nematode (PWN, Bursaphelenchus xylophilus) is the causal agent of pine wilt disease (PWD), which severely affects Pinus pinaster stands in southwestern Europe. Despite the high susceptibility of P. pinaster, individuals of selected half-sib families have shown genetic variability in survival after PWN inoculation, indicating that breeding for resistance can be a valuable strategy to control PWD. In this work, RNA-seq data from susceptible and resistant plants inoculated with PWN were used for SNP discovery and analysis. A total of 186,506 SNPs were identified, of which 31 were highly differentiated between resistant and susceptible plants, including SNPs in genes involved in cell wall lignification, a process previously linked to PWN resistance. Fifteen of these SNPs were selected for validation through Sanger sequencing and 14 were validated. To evaluate SNP-phenotype associations, 40 half-sib plants were genotyped for six validated SNPs. Associations with phenotype after PWN inoculation were found for two SNPs in two different genes (MEE12 and PCMP-E91), as well as two haplotypes of HIPP41, although significance was not maintained following Bonferroni correction. SNPs here detected may be useful for the development of molecular markers for PWD resistance and should be further investigated in future association studies.
Genomic and metabolomic analyses of the marine fungus Emericellopsis cladophorae: insights into saltwater adaptability mechanisms and its biosynthetic potential
(MDPI, 2022-01) Goncalves, Micael; Hilario, Sandra; Van de Peer, Yves; Esteves, Ana; Alves, Artur
The genus Emericellopsis is found in terrestrial, but mainly in marine, environments with a worldwide distribution. Although Emericellopsis has been recognized as an important source of bioactive compounds, the range of metabolites expressed by the species of this genus, as well as the genes involved in their production are still poorly known. Untargeted metabolomics, using UPLC- QToF–MS/MS, and genome sequencing (Illumina HiSeq) was performed to unlock E. cladophorae MUM 19.33 chemical diversity. The genome of E. cladophorae is 26.9 Mb and encodes 8572 genes. A large set of genes encoding carbohydrate-active enzymes (CAZymes), secreted proteins, transporters, and secondary metabolite biosynthetic gene clusters were identified. Our analysis also revealed genomic signatures that may reflect a certain fungal adaptability to the marine environment, such as genes encoding for (1) the high-osmolarity glycerol pathway; (2) osmolytes’ biosynthetic processes; (3) ion transport systems, and (4) CAZymes classes allowing the utilization of marine polysaccharides. The fungal crude extract library constructed revealed a promising source of antifungal (e.g., 9,12,13-Trihydroxyoctadec-10-enoic acid, hymeglusin), antibacterial (e.g., NovobiocinA), anticancer (e.g., daunomycinone, isoreserpin, flavopiridol), and anti-inflammatory (e.g., 2’-O-Galloylhyperin) metabolites. We also detected unknown compounds with no structural match in the databases used. The metabolites’ profiles of E. cladophorae MUM 19.33 fermentations were salt dependent. The results of this study contribute to unravel aspects of the biology and ecology of this marine fungus. The genome and metabolome data are relevant for future biotechnological exploitation of the species.
The artemiside-artemisox-artemisone-m1 tetrad : efficacies against blood stage p. falciparum parasites, dmpk properties, and the case for artemiside
(MDPI, 2021-12-03) Gibhard, Liezl; Coertzen, Dina; Reader, Janette; Van der Watt, Mariette Elizabeth; Birkholtz, Lyn-Marie; Wong, Ho Ning; Batty, Kevin T.; Haynes, Richard K.; Wiesner, Lubbe; dina.coertzen@up.ac.za
Because of the need to replace the current clinical artemisinins in artemisinin combination therapies, we are evaluating fitness of amino-artemisinins for this purpose. These include the thiomorpholine derivative artemiside obtained in one scalable synthetic step from dihydroartemisinin (DHA) and the derived sulfone artemisone. We have recently shown that artemiside undergoes facile metabolism via the sulfoxide artemisox into artemisone and thence into the unsaturated metabolite M1; DHA is not a metabolite. Artemisox and M1 are now found to be approximately equipotent with artemiside and artemisone in vitro against asexual P. falciparum (Pf ) blood stage parasites (IC50 1.5–2.6 nM). Against Pf NF54 blood stage gametocytes, artemisox is potently active (IC50 18.9 nM early-stage, 2.7 nM late-stage), although against the late-stage gametocytes, activity is expressed, like other amino-artemisinins, at a prolonged incubation time of 72 h. Comparative drug metabolism and pharmacokinetic (DMPK) properties were assessed via po and iv administration of artemiside, artemisox, and artemisone in a murine model. Following oral administration, the composite Cmax value of artemiside plus its metabolites artemisox and artemisone formed in vivo is some 2.6-fold higher than that attained following administration of artemisone alone. Given that efficacy of short half-life rapidly-acting antimalarial drugs such as the artemisinins is associated with Cmax, it is apparent that artemiside will be more active than artemisone in vivo, due to additive effects of the metabolites. As is evident from earlier data, artemiside indeed possesses appreciably greater efficacy in vivo against murine malaria. Overall, the higher exposure levels of active drug following administration of artemiside coupled with its synthetic accessibility indicate it is much the preferred drug for incorporation into rational new artemisinin combination therapies.
Genome-wide analysis of butterfly bush (Buddleja alternifolia) in three uplands provides insights into biogeography, demography and speciation
(Wiley, 2021-11) Ma, Yong-Peng; Liao, Rong-Li; Zhang, Ren-Gang; Yun, Quan-Zheng; Olmstead, Richard G.; Chau, John H.; Milne, Richard I.; Van de Peer, Yves; Sun, Wei-Bang
Understanding processes that generate and maintain large disjunctions within plant species can provide valuable insights into plant diversity and speciation. The butterfly bush Buddleja alternifolia has an unusual disjunct distribution, occurring in the Himalaya, Hengduan Mountains (HDM) and the Loess Plateau (LP) in China. We generated a high-quality, chromosome-level genome assembly of B. alternifolia, the first within the family Scrophulariaceae. Whole-genome re-sequencing data from 48 populations plus morphological and petal colour reflectance data covering its full distribution range were collected. Three distinct genetic lineages of B. alternifolia were uncovered, corresponding to Himalayan, HDM and LP populations, with the last also differentiated morphologically and phenologically, indicating occurrence of allopatric speciation likely to be facilitated by geographic isolation and divergent adaptation to distinct ecological niches. Moreover, speciation with gene flow between populations from either side of a mountain barrier could be under way within LP. The current disjunctions within B. alternifolia might result from vicariance of a once widespread distribution, followed by several past contraction and expansion events, possibly linked to climate fluctuations promoted by the Kunlun–Yellow river tectonic movement. Several adaptive genes are likely to be either uniformly or diversely selected among regions, providing a footprint of local adaptations. These findings provide new insights into plant biogeography, adaptation and different processes of allopatric speciation.
Streamlined and robust stage-specific profiling of gametocytocidal compounds against Plasmodium falciparum
(Frontiers Media, 2022-06) Reader, Janette; Van der Watt, Mariette Elizabeth; Birkholtz, Lyn-Marie; lbirkholtz@up.ac.za
Malaria elimination is dependent on the ability to target both the pathogenic and transmissible stages of the human malaria parasite, Plasmodium falciparum. These forms of the parasite are differentiated by unique developmental stages, each with their own biological mechanisms and processes. These individual stages therefore also respond differently to inhibitory compounds, and this complicates the discovery of multistage active antimalarial agents. The search for compounds with transmission-blocking activity has focused on screening for activity on mature gametocytes, with only limited descriptions available for the activity of such compounds on immature stage gametocytes. This therefore poses a gap in the profiling of antimalarial agents for pan-reactive, multistage activity to antimalarial leads. Here, we optimized an effective and robust strategy for the simple and cost-effective description of the stage-specific action of gametocytocidal antimalarial compounds.
A dynamic and combinatorial histone code drives malaria parasite asexual and sexual development
(Elsevier, 2022-03) Von Gruning, Hilde; Coradin, Mariel; Mendoza, Mariel R.; Reader, Janette; Sidoli, Simone; Garcia, Benjamin A.; Birkholtz, Lyn-Marie; lbirkholtz@up.ac.za
Histone posttranslational modifications (PTMs) frequently co-occur on the same chromatin domains or even in the same molecule. It is now established that these “histone codes” are the result of cross talk between enzymes that catalyze multiple PTMs with univocal readout as compared with these PTMs in isolation. Here, we performed a comprehensive identification and quantification of histone codes of the malaria parasite, Plasmodium falciparum. We used advanced quantitative middle-down proteomics to identify combinations of PTMs in both the proliferative, asexual stages and transmissible, sexual gametocyte stages of P. falciparum. We provide an updated, high-resolution compendium of 77 PTMs on H3 and H3.3, of which 34 are newly identified in P. falciparum. Coexisting PTMs with unique stage distinctions were identified, indicating that many of these combinatorial PTMs are associated with specific stages of the parasite life cycle. We focused on the code H3R17me2K18acK23ac for its unique presence in mature gametocytes; chromatin proteomics identified a gametocyte-specific SAGA-like effector complex including the transcription factor AP2-G2, which we tied to this specific histone code, as involved in regulating gene expression in mature gametocytes. Ultimately, this study unveils previously undiscovered histone PTMs and their functional relationship with coexisting partners. These results highlight that investigating chromatin regulation in the parasite using single histone PTM assays might overlook higher-order gene regulation for distinct proliferation and differentiation processes.

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