Malaria Antifolate Resistance With Contrasting Plasmodium Falciparum Dihydrofolate Reductase (DHFR) Polymorphisms in Humans and Anopheles Mosquitoes

Surveillance for drug-resistant parasites in human blood is a major effort in malaria control. Here we report contrasting antifolate resistance polymorphisms in Plasmodium falciparum when parasites in human blood were compared with parasites in Anopheles vector mosquitoes from sleeping huts in rural Zambia. DNA encoding P. falciparum dihydrofolate reductase (EC 1.5.1.3) was amplified by PCR with allele-specific restriction enzyme digestions. Markedly prevalent pyrimethamine-resistant mutants were evident in human P. falciparum infections - S108N (\u3e90%), with N51I, C59R, and 108N+51I+59R triple mutants (30-80%). This resistance level may be from selection pressure due to decades of sulfadoxine/pyrimethamine use in the region. In contrast, cycloguanil-resistant mutants were detected in very low frequency in parasites from human blood samples - S108T (13%), with A16V and 108T+16V double mutants (∼4%). Surprisingly, pyrimethamine-resistant mutants were of very low prevalence (2-12%) in the midguts of Anopheles arabiensis vector mosquitoes, but cycloguanil-resistant mutants were highly prevalent - S108T (90%), with A16V and the 108T+16V double mutant (49-57%). Structural analysis of the dihydrofolate reductase by in silico modeling revealed a key difference in the enzyme within the NADPH binding pocket, predicting the S108N enzyme to have reduced stability but the S108T enzyme to have increased stability. We conclude that P. falciparum can bear highly host-specific drug-resistant polymorphisms, most likely reflecting different selective pressures found in humans and mosquitoes. Thus, it may be useful to sample both human and mosquito vector infections to accurately ascertain the epidemiological status of drug-resistant alleles

Aquaporins in Saccharomyces: Genetic and functional distinctions between laboratory and wild-type strains

Aquaporin water channel proteins mediate the transport of water across cell membranes in numerous species. The Saccharomyces genome data base contains an open reading frame (here designated AQY1) that encodes a protein with strong homology to aquaporins. AQY1 from laboratory and wild-type strains of Saccharomyces were expressed in Xenopus oocytes to determine the coefficients of osmotic water permeability (Pf). Oocytes injected with wild-type AQY1 cRNAs exhibit high Pf values, whereas oocytes injected with AQY1 cRNAs from laboratory strains exhibit low Pf values and have reduced levels of Aqy1p due to two amino acid substitutions. When the AQY1 gene was deleted from a wild-type yeast and cells were cultured in vitro with cycled hypo-osmolar or hyper-osmolar stresses, the AQY1 null yeast showed significantly improved viability when compared with the parental wild-type strain. We conclude that Saccharomyces cerevisiae contains at least one aquaporin gene, but it is not functional in laboratory strains due to apparent negative selection pressures resulting from in vitro methods

Genome-wide association studies for sex determination and cross-compatibility in water yam (Dioscorea alata L.)

Open Access Journal; Published online: 10 Jul 2021Yam (Dioscorea spp.) species are predominantly dioecious, with male and female flowers borne on separate individuals. Cross-pollination is, therefore, essential for gene flow among and within yam species to achieve breeding objectives. Understanding genetic mechanisms underlying sex determination and cross-compatibility is crucial for planning a successful hybridization program. This study used the genome-wide association study (GWAS) approach for identifying genomic regions linked to sex and cross-compatibility in water yam (Dioscorea alata L.). We identified 54 markers linked to flower sex determination, among which 53 markers were on chromosome 6 and one on chromosome 11. Our result ascertained that D. alata is characterized by the male heterogametic sex determination system (XX/XY). The cross-compatibility indices, average crossability rate (ACR) and percentage high crossability (PHC), were controlled by loci on chromosomes 1, 6 and 17. Of the significant loci, SNPs located on chromosomes 1 and 17 were the most promising for ACR and PHC, respectively, and should be validated for use in D. alata hybridization activities to predict cross-compatibility success. A total of 61 putative gene/protein families with direct or indirect influence on plant reproduction were annotated in chromosomic regions controlling the target traits. This study provides valuable insights into the genetic control of D. alata sexual reproduction. It opens an avenue for developing genomic tools for predicting hybridization success in water yam breeding programs

Optimum time for hand pollination in yam (Dioscorea spp.)

Open Access Journal; Published online: 18 Aug 2022Hand pollination success rate is low in yam (Dioscorea spp.), due partly to suboptimal weather conditions. Thus, determining the most suitable time for pollination could improve the pollination success in yam breeding programs. We performed continuous hand pollination within flowering windows of D. rotundata and D. alata for two consecutive years to determine the most appropriate month, week, and hours of the day allowing maximum pollination success. In D. alata crossing block, we observed significant differences among crossing hours for pollination success (p = 0.003); morning hours (8–12 a.m.) being more conducive than afternoons (12–5 p.m.). No significant differences existed between crossing hours in D. rotundata, though the mid-day seemed optimal. For both species, the time interval 11–12 a.m. was more appropriate for crossing while 4–5 p.m. was the poorest. However, in vitro pollen germination tests showed that mid-day pollen collection (12 noon–2 p.m.) had better results than both extremes, though there were strong genotypic effects on outcomes. Pollination success rates differed significantly among months for D. alata (p 0.05). Differences in pollination success existed across weeks within flowering windows of both D. alata (p < 0.001) and D. rotundata (p = 0.004). The seed production efficiency (SPE) had a similar trend as the pollination success rate. No clear pattern existed between the pollination time and the seed setting rate (SSR) or seed viability (SV), though their dynamics varied with weeks and months. This study provided an insight on the dynamics of pollination outcomes under the influence of pollination times and allows detecting months, weeks, and hours of the day when hybridization activities should be focused for better results

Highly selective water channel activity measured by voltage clamp: Analysis of planar lipid bilayers reconstituted with purified AqpZ

Aquaporins are membrane channels selectively permeated by water or water plus glycerol. Conflicting reports have described ion conductance associated with some water channels, raising the question of whether ion conductance is a general property of the aquaporin family. To clarify this question, a defined system was developed to simultaneously measure water permeability and ion conductance. The Escherichia coli water channel aquaporin-Z (AqpZ) was studied, because it is a highly stable tetramer. Planar lipid bilayers were formed from unilamellar vesicles containing purified AqpZ. The hydraulic conductivity of bilayers made from the total extract of E. coli lipids increased 3-fold if reconstituted with AqpZ, but electric conductance was unchanged. No channel activity was detected under voltage-clamp conditions, indicating that less than one in 10(9) transport events is electrogenic. Microelectrode measurements were simultaneously undertaken adjacent to the membrane. Changes in sodium concentration profiles accompanying transmembrane water flow permitted calculation of the activation energies: 14 kcal/mol for protein-free lipid bilayers and 4 kcal/mol for lipid bilayers containing AqpZ. Neither the water permeability nor the electric conductivity exhibited voltage dependence. This sensitive system demonstrated that AqpZ is permeated by water but not charged ions and should permit direct analyses of putative electrogenic properties of other aquaporins

Hypertonic induction of aquaporin-5 expression through an ERK-dependent pathway

Aquaporin-5 (AQP5) is a water channel protein expressed in lung, salivary gland, and lacrimal gland epithelia. Each of these sites may experience fluctuations in surface liquid osmolarity; however, osmotic regulation of AQP5 expression has not been reported. This study demonstrates that AQP5 is induced by hypertonic stress and that induction requires activation of extracellular signal-regulated kinase (ERK). Incubation of mouse lung epithelial cells (MLE-15) in hypertonic medium produced a dose-dependent increase in AQP5 expression; AQP5 protein peaked by 24 h and returned to baseline levels within hours of returning cells to isotonic medium. AQP5 induction was observed only with relatively impermeable solutes, suggesting an osmotic pressure gradient is required for induction. ERK was selectively activated in MLE-15 cells by hypertonic stress, and inhibition of ERK activation with two distinct mitogen-activated extracellular regulated kinase kinase (MEK) inhibitors, U0126 and PD98059, blocked AQP5 induction. AQP5 induction was also observed in the lung, salivary, and lacrimal glands of hyperosmolar rats, suggesting potential physiologic relevance for osmotic regulation of AQP5 expression. This report provides the first example of hypertonic induction of an extrarenal aquaporin, as well as the first association between mitogen-activated protein kinase signaling and aquaporin expression

Structural basis for conductance by the archaeal aquaporin AqpM at 1.68 Å

To explore the structural basis of the unique selectivity spectrum and conductance of the transmembrane channel protein AqpM from the archaeon Methanothermobacter marburgensis, we determined the structure of AqpM to 1.68-A resolution by x-ray crystallography. The structure establishes AqpM as being in a unique subdivision between the two major subdivisions of aquaporins, the water-selective aquaporins, and the water-plus-glycerol-conducting aquaglyceroporins. In AqpM, isoleucine replaces a key histidine residue found in the lumen of water channels, which becomes a glycine residue in aquaglyceroporins. As a result of this and other side-chain substituents in the walls of the channel, the channel is intermediate in size and exhibits differentially tuned electrostatics when compared with the other subfamilies

Aquaporin-4 and brain edema.

Aquaporin-4 (AQP4) is a water-channel protein expressed strongly in the brain, predominantly in astrocyte foot processes at the borders between the brain parenchyma and major fluid compartments, including cerebrospinal fluid (CSF) and blood. This distribution suggests that AQP4 controls water fluxes into and out of the brain parenchyma. Experiments using AQP4-null mice provide strong evidence for AQP4 involvement in cerebral water balance. AQP4-null mice are protected from cellular (cytotoxic) brain edema produced by water intoxication, brain ischemia, or meningitis. However, AQP4 deletion aggravates vasogenic (fluid leak) brain edema produced by tumor, cortical freeze, intraparenchymal fluid infusion, or brain abscess. In cytotoxic edema, AQP4 deletion slows the rate of water entry into brain, whereas in vasogenic edema, AQP4 deletion reduces the rate of water outflow from brain parenchyma. AQP4 deletion also worsens obstructive hydrocephalus. Recently, AQP4 was also found to play a major role in processes unrelated to brain edema, including astrocyte migration and neuronal excitability. These findings suggest that modulation of AQP4 expression or function may be beneficial in several cerebral disorders, including hyponatremic brain edema, hydrocephalus, stroke, tumor, infection, epilepsy, and traumatic brain injury

Human-centered Explainable AI: Towards a Reflective Sociotechnical Approach

Explanations--a form of post-hoc interpretability--play an instrumental role in making systems accessible as AI continues to proliferate complex and sensitive sociotechnical systems. In this paper, we introduce Human-centered Explainable AI (HCXAI) as an approach that puts the human at the center of technology design. It develops a holistic understanding of "who" the human is by considering the interplay of values, interpersonal dynamics, and the socially situated nature of AI systems. In particular, we advocate for a reflective sociotechnical approach. We illustrate HCXAI through a case study of an explanation system for non-technical end-users that shows how technical advancements and the understanding of human factors co-evolve. Building on the case study, we lay out open research questions pertaining to further refining our understanding of "who" the human is and extending beyond 1-to-1 human-computer interactions. Finally, we propose that a reflective HCXAI paradigm-mediated through the perspective of Critical Technical Practice and supplemented with strategies from HCI, such as value-sensitive design and participatory design--not only helps us understand our intellectual blind spots, but it can also open up new design and research spaces.Comment: In Proceedings of HCI International 2020: 22nd International Conference On Human-Computer Interactio