Molecular Wiring of a Mitochondrial Translational Feedback Loop.

The mitochondrial oxidative phosphorylation system comprises complexes assembled from subunits derived from mitochondrial and nuclear gene expression. Both genetic systems are coordinated by feedback loops, which control the synthesis of specific mitochondrial encoded subunits. Here, we studied how this occurs in the case of cytochrome b, a key subunit of mitochondrial complex III. Our data suggest the presence of a molecular rheostat consisting of two translational activators, Cbp3-Cbp6 and Cbs1, which operates at the mitoribosomal tunnel exit to connect translational output with assembly efficiency. When Cbp3-Cbp6 is engaged in assembly of cytochrome b, Cbs1 binds to the tunnel exit to sequester the cytochrome b-encoding mRNA, repressing its translation. After mediating complex III assembly, binding of Cbp3-Cbp6 to the tunnel exit replaces Cbs1 and the bound mRNA to permit cytochrome b synthesis. Collectively, the data indicate the molecular wiring of a feedback loop to regulate synthesis of a mitochondrial encoded protein.

Computational exploration of compounds in Xylocarpus granatum as a potential inhibitor of Plasmodium berghei using docking, molecular dynamics, and DFT studies.

Malaria remains a global health concern, with the emergence of resistance to the antimalarial drug atovaquone through cytochrome b (cyt b) being well-documented. This study was prompted by the presence of this mutation in cyt b to enable new drug candidates capable of overcoming drug resistance. Our objective was to identify potential drug candidates from compounds of Xylocarpus granatum by computationally assessing their interactions with Plasmodium berghei cyt b. Using computational methods, we modeled cyt b (GenBank: AF146076.1), identified the binding cavity, and analyzed the Ramachandran plot against cyt b. Additionally, we conducted drug-likeness and absorption, distribution, metabolism, excretion, and toxicity (ADMET) studies, along with density functional theory (DFT) analysis of the compounds. Molecular docking and molecular dynamics simulation (MDS) were used to evaluate the binding energy and stability of the cyt b-ligand complex. Notably, our investigation highlighted kaempferol as a promising compound due to its high binding energy of 7.67 kcal/mol among all X. granatum compounds, coupled with favorable pharmacological properties (ADMET) and antiprotozoal properties at Pa 0.345 > Pi 0.009 (PASS value). DFT analysis showed that kaempferol has an energy gap of 4.514 eV. MDS indicated that all tested ligands caused changes in bonding and affected the structural conformation of cyt b, as observed before MDS (0 ns) and after MDS (100 ns). The most notable differences were observed in the types of hydrogen bonds between 0 and 100 ns. Nevertheles, MDS results from a 100 ns simulation revealed consistent behavior for kaempferol across various parameters including root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), solvent-accessible surface area (SASA), molecular mechanics-Poisson Boltzmann surface area (MM-PBSA), and hydrogen bonds. The cyt b-kaempferol complex demonstrated favorable energy stability, as supported by the internal energy distribution values observed in principal component analysis (PCA), which closely resembled those of the atovaquone control. Additionally, trajectory stability analysis indicated structural stability, with a cumulative eigenvalue of 24.7 %. Dynamic cross-correlation matrix (DCCM) analysis revealed a positive correlation among catalytic cytochrome residues within the amino acid residues range 119-268. The results of our research indicate that the structure of kaempferol holds promise as a potential candidate against Plasmodium.

Environmental DNA unveils deep phylogeographic structure of a freshwater fish.

Phylogeography bears an important part in ecology and evolution. However, current phylogeographic studies are largely constrained by limited numbers of individual samples. Using an environmental DNA (eDNA) assay for phylogeographic analyses, this study provides detailed information regarding the history of Siberian stone loach Barbatula toni, a primary freshwater fish across the whole range of Hokkaido, Japan. Based on an eDNA metabarcoding on 293 river water samples, we detected eDNA from B. toni in 189 rivers. A total of 51 samples, representing the entire island, were then selected from the B. toni eDNA-positive sample set for the subsequent analyses. To elucidate the phylogeographic structure of B. toni, newly developed eDNA metabarcoding primers (Barba-cytb-F/R) were applied to these samples, specifically targeting their haplotypic variation in cytochrome b. After a bioinformatic processing to mitigate haplotypic false positives, a total of 50 eDNA haplotypes were identified. Two regionally restricted, genetically distinct lineages of the species were revealed as a result of phylogeographic analyses on the haplotypes and tissue-derived DNA from B. toni. According to a molecular clock analysis, they have been genetically isolated for at least 1.5 million years, suggesting their ancient origin and colonisation of Hokkaido, presumably in the glacial periods. These results demonstrate how freshwater fishes can alter their distributions over evolutionary timescales and how eDNA assay can deepen our understanding of phylogeography.

DNA barcoding of southern African mammal species and construction of a reference library for forensic application.

Combating wildlife crimes in South Africa requires accurate identification of traded species and their products. Diagnostic morphological characteristics needed to identify species are often lost when specimens are processed and customs officials lack the expertise to identify species. As a potential solution, DNA barcoding can be used to identify morphologically indistinguishable specimens in forensic cases. However, barcoding is hindered by the reliance on comprehensive, validated DNA barcode reference databases, which are currently limited. To overcome this limitation, we constructed a barcode library of cytochrome c oxidase subunit 1 and cytochrome b sequences for threatened and protected mammals exploited in southern Africa. Additionally, we included closely related or morphologically similar species and assessed the database's ability to identify species accurately. Published southern African sequences were incorporated to estimate intraspecific and interspecific variation. Neighbor-joining trees successfully discriminated 94%-95% of the taxa. However, some widespread species exhibited high intraspecific distances (>2%), suggesting geographic sub-structuring or cryptic speciation. Lack of reliable published data prevented the unambiguous discrimination of certain species. This study highlights the efficacy of DNA barcoding in species identification, particularly for forensic applications. It also highlights the need for a taxonomic re-evaluation of certain widespread species and challenging genera.

Larger particle size distribution of environmental RNA compared to environmental DNA: a case study targeting the mitochondrial cytochrome b gene in zebrafish (Danio rerio) using experimental aquariums.

Environmental RNA (eRNA) analysis is conventionally expected to infer physiological information about organisms within their ecosystems, whereas environmental DNA (eDNA) analysis only infers their presence and abundance. Despite the promise of eRNA application, basic research on eRNA characteristics and dynamics is limited. The present study conducted aquarium experiments using zebrafish (Danio rerio) to estimate the particle size distribution (PSD) of eRNA in order to better understand the persistence state of eRNA particles. Rearing water samples were sequentially filtered using different pore-size filters, and the resulting size-fractioned mitochondrial cytochrome b (CytB) eDNA and eRNA data were modeled with the Weibull complementary cumulative distribution function (CCDF) to estimate the parameters characterizing the PSDs. It was revealed that the scale parameter (α) was significantly higher (i.e., the mean particle size was larger) for eRNA than eDNA, while the shape parameter (ß) was not significantly different between them. This result supports the hypothesis that most eRNA particles are likely in a protected, intra-cellular state, which mitigates eRNA degradation in water. Moreover, these findings also imply the heterogeneous dispersion of eRNA relative to eDNA and suggest an efficient method of eRNA collection using a larger pore-size filter. Further studies on the characteristics and dynamics of eRNA particles should be pursued in the future.

Study of Altenia wagneriella (rebel) as a predator associated with Forda marginata from Iran.

BACKGROUND: The larvae of Altenia spp. and gall aphids are known to feed on plants related to Anacardiaceae. This study documents the aphidophagous habit of Altenia wagneriella, which was verified by molecular techniques, subsequently by the gut dissection test, and direct observation. MATERIALS AND METHODS: To identify the moth larvae and adult aphids, two mitochondrial genes, cytochrome c oxidase I (COI) and cytochrome b (Cytb), were amplified by polymerase chain reaction (PCR). Nested PCR with the aphid-specific primer pairs AphidF and AphidR was used to detect aphids in the body of moth larvae. The specificity of the primers was verified by PCR analysis of DNA from moth larvae and adult aphids. RESULTS: The method detected aphids in moth larvae, and a band of approximately 200 bp was amplified from moth larvae feeding on aphids. No cross reactions with moth larvae were observed. In the laboratory, all moth larvae feeding on aphids (Forda marginata) were also PCR positive for aphids. CONCLUSIONS: Gall-inducing insects are microhabitat engineers that manipulate their host to obtain a better nutrient supply, as well as protection from natural enemies and abiotic factors. This is the first recorded instance worldwide of the carnivorous larva of the moth A. wagneriella acting as an aphid predator, as well as the first record of a host insect for this species. Additionally, it is the first effort to molecularly analyze the predator-prey relationship between the moth larvae and the aphids inside the wild pistachio gall.

An endoplasmic reticulum-localized cytochrome b5 regulates high-affinity K+ transport in response to salt stress in rice.

Potassium (K+) is an essential element for growth and development in both animals and plants, while high levels of environmental sodium (Na+) represent a threat to most plants. The uptake of K+ from high-saline environments is an essential mechanism to maintain intracellular K+/Na+ homeostasis, which can help reduce toxicity caused by Na+ accumulation, thereby improving the salt tolerance of plants. However, the mechanisms and regulation of K+-uptake during salt stress remain poorly understood. In this study, we identified an endoplasmic reticulum-localized cytochrome b5 (OsCYB5-2) that interacted with a high-affinity K+ transporter (OsHAK21) at the plasma membrane. The association of OsCYB5-2 with the OsHAK21 transporter caused an increase in transporter activity by enhancing the apparent affinity for K+-binding but not Na+-binding. Heme binding to OsCYB5-2 was essential for the regulation of OsHAK21. High salinity directly triggered the OsHAK21-OsCYB5-2 interaction, promoting OsHAK21-mediated K+-uptake and restricting Na+ entry into cells; this maintained intracellular K+/Na+ homeostasis in rice cells. Finally, overexpression of OsCYB5-2 increased OsHAK21-mediated K+ transport and improved salt tolerance in rice seedlings. This study revealed a posttranslational regulatory mechanism for HAK transporter activity mediated by a cytochrome b5 and highlighted the coordinated action of two proteins to perceive Na+ in response to salt stress.

High Genetic Differentiation and Genetic Diversity in Endangered Mahseer Tor khudree (Sykes, 1839) as Revealed from Concatenated ATPase 6/8 and Cyt b Mitochondrial Genes.

The Deccan mahseer, Tor khudree (Sykes 1839), belonging to family Cyprinidae is an important food and a game fish distributed in peninsular India. Due to overfishing and habitat destruction, the species is declared endangered and placed on the IUCN red list. Therefore, a well-designed conservation program may be essential to get this species protected in its natural habitat. We used a total of 152 samples from four rivers of peninsular India to assess the genetic diversity and structure of the mahseer using concatenated sequences of two mitochondrial genes, ATPase 6/8 (790 bp) and Cyt b (1000 bp). High haplotypic diversity was seen with 44 haplotypes. Individual gene wise haplotypes included 10 and 21 haplotypes for ATPase6/8 and Cyt b, respectively. AMOVA revealed most of the genetic variations (71.02%) to be within the populations. Significant genetic differentiation was observed between all population pairs, with FST values ranging from 0.121 to 0.372, with minimum between Tunga and Tungabhadra population and maximum between Tunga and Periyar population. Haplotype network showed one ancestral haplotype (TKACH04). Significant negative Fu's F and unimodal mismatch distribution suggested recent demographic expansion. The results of the present study would serve as a useful resource for further research on population genetics and conservation programs of the species.

First report on the occurrence of psoroptic mange in llamas (Lama glama) of the Andean region.

An outbreak of Psoroptes sp.-caused mange was detected in a llama herd of Larcas, Jujuy province, Argentina. Infested llamas showed alopecia, erythema, hyperpigmentation, hyperkeratosis, and inflammation of the ear pinnae, as well as crusts and serous, serosanguineous, or purulent drainage with unpleasant smell in the external ear canal. Microscopic evaluation of skin scrapings revealed 0.5- to 0.7-mm-long acari identified as Psoroptes sp. based on their morphology. Histology showed a typical allergic reaction with perivascular to periadnexal mixed inflammatory infiltrate. Phylogenetic tree analysis showed that the cytochrome c oxidase subunit I gene sequences analyzed from the sampled acari clustered into a single P. ovis clade including sequences isolated from rabbits and bighorn sheep, with P. natalensis as a sister taxon that infested bighorn sheep from the USA. Phylogenetic analysis of cytochrome b sequences showed three well-supported clades, one of which contained the sequences of the Larcas llamas and US bighorn sheep isolates. This is the first report on P. ovis infestation of llamas raised in their original location. Investigations on mange etiological agents acting on South American camelids and their distribution are necessary to implement control strategies to mitigate the negative impacts of these parasitic infections.

Identification of Haemoproteus infection in an imported grey crowned crane (Balearica regulorum) in China.

Blood parasites from the order Haemosporida infect many vertebrates and cause malaria-like diseases. In this study, a haemosporidian infection was detected in a sick grey crowned crane imported into China using a combination of morphological and molecular approaches. Blood samples were collected from the jugular vein and processed for morphological identification of infective parasites using stained blood smears and microscopy. No merogony occurs in the blood cells, and sporadic pigment granules were observed. Nested-PCR assays were employed for a molecular examination, which indicated that the cytb gene of this parasite had 94.1-94.9% identity to Haemoproteus antigonis. Subsequently, its mitochondrial genome structure was determined by high-throughput sequencing using the DNBSEQ-T7 platform. The determined structure was confirmed by the Sanger sequencing using amplicons. The mitochondrial genome obtained for this parasite exhibited a low CG content (32.0%) and possessed three protein-coding genes, encoding 1068 amino acids, which constituted 53.7% of the genome. Phylogenetic analysis indicated that this parasite clustered with Haemoproteus sp. is detected in grey crowned cranes from Africa. This parasite was likely acquired during importation of this animal; thus, strict quarantine of imported ornamental animals is required to prevent the entry of new pathogens.

Avian haemosporidians of the genera Plasmodium and Haemoproteus from resident and Neotropical migrant birds in Colombia.

Avian haemosporidians of the genera Plasmodium and Haemoproteus are a group of widely distributed blood parasites that can negatively affect the fitness of their hosts. Colombia contains the greatest diversity of birds on the planet, but knowledge about the associations between haemosporidian and its avifauna is scarce and fragmented. We collected blood samples from 255 birds (203 residents and 52 neotropical migrants) belonging to 27 families and 108 species. The study was conducted in six localities in the inter-Andean valleys of the Cauca and Magdalena rivers. Parasites of the genera Plasmodium and Haemoproteus were identified in the samples by morphological and molecular analysis of a fragment of the mitochondrial gene cyt b. Among the samples, 9.3% (n = 24) were positive for Plasmodium or Haemoproteus. Co-infection with Plasmodium and Haemoproteus was found in Red-eyed Vireo. Seventeen haemosporidian lineages were identified, five of which were reported for the first time in resident birds (Common Ground Dove, Checker-throated Stipplethroat, Tropical Kingbird, Pale-breasted Thrush, and Ruddy-breasted Seedeater) and one in the Summer Tanager (neotropical migrant). The research results confirm the wide diversity of haemosporidian present in tropical lowlands and the possible role of neotropical migratory birds in dissemination on haemosporidian along their migratory routes.

Prevalence of FRAC Group 11 Fungicide Resistance in Stemphylium vesicarium Isolates, but Not S. beticola Isolates, Causing Stemphylium Leaf Spot of Spinach (Spinacia oleracea).

Stemphylium leaf spot of spinach, caused by Stemphylium beticola and S. vesicarium, is a disease of economic importance in fresh market, processing, and seed production. There have been increasing reports of difficulty managing the disease in the southern United States using fungicides in Fungicide Resistance Action Committee (FRAC) group 11. Isolates of S. beticola and S. vesicarium obtained from spinach leaves and seed from 2001 to 2020 were screened for resistance to azoxystrobin and pyraclostrobin in vitro, in vivo, and using PCR assays to detect mutations in cytochrome b associated with resistance in other fungi (F129L, G137R, and G143A). EC50 values for mycelial growth and conidial germination of S. vesicarium isolates in vitro were significantly less (mean of 0.35 µg/ml) than that of S. vesicarium (mean of 14.17 µg/ml) with both fungicides. All isolates were slightly more sensitive to pyraclostrobin than azoxystrobin in both assays. In vivo assays of plants inoculated with the isolates of S. vesicarium demonstrated poor efficacy of fungicides with each of the two active ingredients. Only the G143A mutation was detected in all spinach isolates of S. vesicarium, including an isolate of S. vesicarium collected in 2003 and 82.9% of isolates from spinach seed lots harvested from crops grown in or after 2017 in Europe, New Zealand, and the United States. The FRAC 11 mutations were not detected in any isolates of S. beticola. The in vitro, in vivo, and DNA mutation assays suggest FRAC group 11 fungicide resistance is widespread in spinach isolates of S. vesicarium but not S. beticola.

Phylogenetic and phylogeographic insights into Sri Lankan killifishes (Teleostei: Aplocheilidae).

Three nominal species of the killifish genus Aplocheilus are reported from the lowlands of Sri Lanka. Two of these, Aplocheilus dayi and Aplocheilus werneri, are considered endemic to the island, whereas Aplocheilus parvus is reported from both Sri Lanka and Peninsular India. Here, based on a collection from 28 locations in Sri Lanka, also including a dataset of Asian Aplocheilus downloaded from GenBank, we present a phylogeny constructed from the mitochondrial cytochrome b (cytb), mitochondrial cytochrome c oxidase subunit 1 (cox1), and nuclear recombination activating protein 1 (rag1), and investigate the interrelationships of the species of Aplocheilus in Sri Lanka. The endemic Sri Lankan aplocheilid clade comprising A. dayi and A. werneri is recovered as the sister group to the clade comprising A. parvus from Sri Lanka and Aplocheilus blockii from Peninsular India. The reciprocal monophyly of A. dayi and A. werneri is not supported in our molecular phylogeny. A. dayi and A. werneri display strong sexual dimorphism, but species-level differences are subtle, explained mostly by pigmentation patterns. Their phenotypes exhibit a parapatric distribution and may represent locally adapted forms of a single species. Alternatively, the present study does not rule out the possibility that A. dayi and A. werneri may represent an incipient species pair or that they have undergone introgression or hybridization in their contact zones. We provide evidence that the Nilwala-Gin region of southwestern Sri Lanka may have acted as a drought refugium for these fishes.

A new cavefish of Sinocyclocheilus (Teleostei: Cypriniformes: Cyprinidae) from the Nanpanjiang River in Guizhou, China.

A new species, Sinocyclocheilus xingyiensis, is described based on specimens collected from a karst cave in Guizhou Province, China. The authors used an integrated taxonomic approach, including morphological and molecular data, to identify the new species as a member of the Sinocyclocheilu angularis group, and it can be distinguished from all other members of this group by a combination of the following features: two pairs of long barbels and long pectoral fins, 42-46 lateral-line scales, 7 (13-14) on outer (inner) side of the first gill arch and 35 (14-15 + 4 + 16 - 17) vertebrae. Phylogenetic analyses based on the cytochrome b (cyt b) gene fragment suggest that S. xingyiensis is a sister lineage to Sinocyclocheilus flexuosdorsalis. The genetic distance (Kimura 2-parameter) between the S. xingyiensis and S. angularis groups of Sinocyclocheilus species based on cyt b gene fragment ranged from 1.2% to 15.4%.

From Caves to the Savannah, the Mitogenome History of Modern Lions (Panthera leo) and Their Ancestors.

Lions (Panthera leo) play a crucial ecological role in shaping and maintaining fragile ecosystems within Africa. Conservation efforts should focus on genetic variability within wild populations when considering reintroduction attempts. We studied two groups of lions from two conservation sites located in Zambia and Zimbabwe to determine their genetic make-up, information that is usually unknown to the sites. In this study, we analysed 17 specimens for cytb and seven microsatellite markers to ascertain family relationships and genetic diversity previously obtained by observational studies. We then produced a standardised haplogroup phylogeny using all available entire mitogenomes, as well as calculating a revised molecular clock. The modern lion lineage diverged ~151 kya and was divided into two subspecies, both containing three distinct haplogroups. We confirm that Panthera leo persica is not a subspecies, but rather a haplogroup of the northern P.l. leo that exited Africa at least ~31 kya. The progenitor to all lions existed ~1.2 Mya, possibly in SE Africa, and later exited Africa and split into the two cave lion lineages ~175 kya. Species demography is correlated to major climactic events. We now have a detailed phylogeny of lion evolution and an idea of their conservation status given the threat of climate change.

Genetic diversity and structure of mongolian gazelle (Procapra gutturosa) populations in fragmented habitats.

BACKGROUND: The Mongolian gazelle (Procapra gutturosa) population has shown a considerable range of contractions and local extinctions over the last century, owing to habitat fragmentation and poaching. A thorough understanding of the genetic diversity and structure of Mongolian gazelle populations in fragmented habitats is critical for planning effective conservation strategies. RESULT: In this study, we used eight microsatellite loci and mitochondrial cytochrome b (Cytb) to compare the levels of genetic diversity and genetic structure of Mongolian gazelle populations in the Hulun Lake National Nature Reserve (HLH) with those in the China-Mongolia border area (BJ). The results showed that the nucleotide diversity and observed heterozygosity of the HLH population were lower than those of the BJ population. Moreover, the HLH and BJ populations showed genetic differentiation. We concluded that the HLH population had lower genetic diversity and a distinct genetic structure compared with the BJ population. CONCLUSION: The genetic diversity of fragmented Mongolian gazelle populations, can be improved by protecting these populations while reinforcing their gene exchange with other populations. For example, attempts can be made to introduce new individuals with higher genetic diversity from other populations to reduce inbreeding.

Mitochondrial Genomes in Perkinsus Decode Conserved Frameshifts in All Genes.

Mitochondrial genomes of apicomplexans, dinoflagellates, and chrompodellids that collectively make up the Myzozoa, encode only three proteins (Cytochrome b [COB], Cytochrome c oxidase subunit 1 [COX1], Cytochrome c oxidase subunit 3 [COX3]), contain fragmented ribosomal RNAs, and display extensive recombination, RNA trans-splicing, and RNA-editing. The early-diverging Perkinsozoa is the final major myzozoan lineage whose mitochondrial genomes remained poorly characterized. Previous reports of Perkinsus genes indicated independent acquisition of non-canonical features, namely the occurrence of multiple frameshifts. To determine both ancestral myzozoan and novel perkinsozoan mitochondrial genome features, we sequenced and assembled mitochondrial genomes of four Perkinsus species. These data show a simple ancestral genome with the common reduced coding capacity but disposition for rearrangement. We identified 75 frameshifts across the four species that occur as distinct types and that are highly conserved in gene location. A decoding mechanism apparently employs unused codons at the frameshift sites that advance translation either +1 or +2 frames to the next used codon. The locations of frameshifts are seemingly positioned to regulate protein folding of the nascent protein as it emerges from the ribosome. The cox3 gene is distinct in containing only one frameshift and showing strong selection against residues that are otherwise frequently encoded at the frameshift positions in cox1 and cob. All genes lack cysteine codons implying a reduction to 19 amino acids in these genomes. Furthermore, mitochondrion-encoded rRNA fragment complements are incomplete in Perkinsus spp. but some are found in the nuclear DNA suggesting import into the organelle. Perkinsus demonstrates further remarkable trajectories of organelle genome evolution including pervasive integration of frameshift translation into genome expression.

Phylogenetic relationships and the origin of New World soles (Teleostei: Pleuronectiformes: Achiridae): The role of estuarine habitats.

Even though the monophyletic status of Achiridae has been supported by morphological and molecular data, the interrelationships within the representatives of this family are poorly resolved. In the present study, we carried out the most complete molecular phylogenetic analysis of this group, encompassing all genera and employing both nuclear (Rhodopsin, Recombination activator [Rag 1], Mixed - lineage Leukemia [MLL] and Early Growth Response Protein 3 [EGR3]) and mitochondrial (Cytochrome C Oxidase Subunit I [COI], Cytochrome B [CytB], ATPase 6.8, 16S and 12S RNAr) genes. All topologies based on Maximum Likelihood, Bayesian inferences and Bayesian Inference of the Multispecies Coalescent confirmed the monophyletism of Achiridae, in spite of some incongruences in relation to Achirus mucuri, A. lineatus, Apionichthys finis and Trinectes microphthalmus. In fact, Achirus and Trinectes proved to be non-monophyletic genera while Hypoclinemus mentalis was closely related to A. achirus, suggesting this species should be reevaluated. We provided evidence that Achiridae has first arisen in estuaries (about 23.5 million years ago) and some lineages have evolved independently to either marine or freshwater habitats. Furthermore, we propose a diversification scenario of New World soles involving at least two events of marine incursions during Miocene and Pliocene - Pleistocene associated with natural geographic barriers (Victoria-Trindade chain), the width and exposure of continental shelf and headwater capture along the Amazon basin. Finally, the evolutionary dependence of Achirid soles on estuaries, characterized as highly dynamic environments, has probably driven the recent divergence of many species of Achiridae.

CYB561A3 is the key lysosomal iron reductase required for Burkitt B-cell growth and survival.

Epstein-Barr virus (EBV) causes endemic Burkitt lymphoma, the leading childhood cancer in sub-Saharan Africa. Burkitt cells retain aspects of germinal center B-cell physiology with MYC-driven B-cell hyperproliferation; however, little is presently known about their iron metabolism. CRISPR/Cas9 analysis highlighted the little-studied ferrireductase CYB561A3 as critical for Burkitt proliferation but not for that of the closely related EBV-transformed lymphoblastoid cells or nearly all other Cancer Dependency Map cell lines. Burkitt CYB561A3 knockout induced profound iron starvation, despite ferritinophagy ad plasma membrane transferrin upregulation. Elevated concentrations of ascorbic acid, a key CYB561 family electron donor, or the labile iron source ferrous citrate rescued Burkitt CYB561A3 deficiency. CYB561A3 knockout caused catastrophic lysosomal and mitochondrial damage and impaired mitochondrial respiration. Conversely, lymphoblastoid B cells with the transforming EBV latency III program were instead dependent on the STEAP3 ferrireductase. These results highlight CYB561A3 as an attractive therapeutic Burkitt lymphoma target.

In vitro activity of rhinacanthin analogues against drug resistant Plasmodium falciparum isolates from Northeast Thailand.

BACKGROUND: New anti-malarial drugs are needed urgently to address the increasing challenges of drug-resistant falciparum malaria. Two rhinacanthin analogues containing a naphthoquinone moiety resembling atovaquone showed promising in-vitro activity against a P. falciparum laboratory reference strain (K1). The anti-malarial activity of these 2 compounds was further evaluated for P. falciparum field isolates from an area of multi-drug resistance in Northeast Thailand. METHODS: Using a pLDH enzyme-linked immunosorbent assay, four P. falciparum isolates from Northeast Thailand in 2018 were tested for in vitro sensitivity to the two synthetic rhinacanthin analogues 1 and 2 as well as established anti-malarials. Mutations in the P. falciparum cytochrome b gene, a marker for atovaquone (ATQ) resistance, were genotyped in all four field isolates as well as 100 other clinical isolates from the same area using PCR-artificial Restriction Fragment Length Polymorphisms. Pfkelch13 mutations, a marker for artemisinin (ART) resistance, were also examined in all isolates. RESULTS: The 50% inhibitory concentrations (IC50) of P. falciparum field isolates for rhinacanthin analogue 1 was 321.9-791.1 nM (median = 403.1 nM). Parasites were more sensitive to analogue 2: IC50 48.6-63.3 nM (median = 52.2 nM). Similar results were obtained against P. falciparum reference laboratory strains 3D7 and W2. The ART-resistant IPC-5202 laboratory strain was more sensitive to these compounds with a median IC50 45.9 and 3.3 nM for rhinacanthin analogues 1 and 2, respectively. The ATQ-resistant C2B laboratory strain showed high-grade resistance towards both compounds (IC50 > 15,000 nM), and there was a strong positive correlation between the IC50 values for these compounds and ATQ (r = 0.83-0.97, P < 0.001). There were no P. falciparum cytochrome b mutations observed in the field isolates, indicating that P. falciparum isolates from this area remained ATQ-sensitive. Pfkelch13 mutations and the ring-stage survival assay confirmed that most isolates were resistant to ART. CONCLUSIONS: Two rhinacanthin analogues showed parasiticidal activity against multi-drug resistant P. falciparum isolates, although less potent than ATQ. Rhinacanthin analogue 2 was more potent than analogue 1, and can be a lead compound for further optimization as an anti-malarial in areas with multidrug resistance.