Fitness Trade-Offs in the Evolution of Dihydrofolate Reductase and Drug Resistance in Plasmodium falciparum

Background: Patterns of emerging drug resistance reflect the underlying adaptive landscapes for specific drugs. In Plasmodium falciparum, the parasite that causes the most serious form of malaria, antifolate drugs inhibit the function of essential enzymes in the folate pathway. However, a handful of mutations in the gene coding for one such enzyme, dihydrofolate reductase, confer drug resistance. Understanding how evolution proceeds from drug susceptibility to drug resistance is critical if new antifolate treatments are to have sustained usefulness. Methodology/Principal Findings: We use a transgenic yeast expression system to build on previous studies that described the adaptive landscape for the antifolate drug pyrimethamine, and we describe the most likely evolutionary trajectories for the evolution of drug resistance to the antifolate chlorcycloguanil. We find that the adaptive landscape for chlorcycloguanil is multi-peaked, not all highly resistant alleles are equally accessible by evolution, and there are both commonalities and differences in adaptive landscapes for chlorcycloguanil and pyrimethamine. Conclusions/Significance: Our findings suggest that cross-resistance between drugs targeting the same enzyme reflect the fitness landscapes associated with each particular drug and the position of the genotype on both landscapes. The possibl

Biomarkers of hepatic injury and function in neonatal hypoxic ischemic encephalopathy and with therapeutic hypothermia

Therapeutic hypothermia (TH) is now provided as standard care to infants with moderate-severe hypoxic ischemic encephalopathy (HIE). The role of TH in limiting neuronal injury is well recognized, but its effect on hepatic injury which occurs frequently in neonatal HIE is not known. Our objective was to characterize biomarkers of liver injury and function in the setting of neonatal HIE and to describe whether HIE severity and provision of TH influence these hepatic biomarkers. We performed a multicenter retrospective study and compared hepatic biomarkers obtained during the first postnatal week, according to the severity of HIE and whether treated with TH. Of a total of 361 infants with HIE, 223 (62%) received TH and 138 (38%) were managed at normal temperature. Most hepatic biomarkers and C-reactive protein (CRP) were significantly associated with the severity of HIE (p<0.001). Infants treated with TH had lower peak Alanine aminotransferase (ALT) concentrations (p=0.025) and delay in reaching peak CRP concentration (p<0.001).  Conclusion: We observed a significant association between the clinical grade of HIE and biomarkers of liver metabolism and function. Therapeutic hypothermia was associated with delayed CRP responses and with lower ALT concentrations and so may have the potential to modulate hepatic injury

The JWST Galactic Center Survey -- A White Paper

The inner hundred parsecs of the Milky Way hosts the nearest supermassive black hole, largest reservoir of dense gas, greatest stellar density, hundreds of massive main and post main sequence stars, and the highest volume density of supernovae in the Galaxy. As the nearest environment in which it is possible to simultaneously observe many of the extreme processes shaping the Universe, it is one of the most well-studied regions in astrophysics. Due to its proximity, we can study the center of our Galaxy on scales down to a few hundred AU, a hundred times better than in similar Local Group galaxies and thousands of times better than in the nearest active galaxies. The Galactic Center (GC) is therefore of outstanding astrophysical interest. However, in spite of intense observational work over the past decades, there are still fundamental things unknown about the GC. JWST has the unique capability to provide us with the necessary, game-changing data. In this White Paper, we advocate for a JWST NIRCam survey that aims at solving central questions, that we have identified as a community: i) the 3D structure and kinematics of gas and stars; ii) ancient star formation and its relation with the overall history of the Milky Way, as well as recent star formation and its implications for the overall energetics of our galaxy's nucleus; and iii) the (non-)universality of star formation and the stellar initial mass function. We advocate for a large-area, multi-epoch, multi-wavelength NIRCam survey of the inner 100\,pc of the Galaxy in the form of a Treasury GO JWST Large Program that is open to the community. We describe how this survey will derive the physical and kinematic properties of ~10,000,000 stars, how this will solve the key unknowns and provide a valuable resource for the community with long-lasting legacy value

Novel alleles of the Plasmodium falciparum dhfr highly resistant to pyrimethamine and chlorcycloguanil, but not WR99210.

We have expressed dhfr alleles of Plasmodium falciparum in the budding yeast, Saccharomyces cerevisiae, and used this yeast model to identify single amino acid substitutions that confer high level pyrimethamine resistance on the background of the triple mutant dhfr (I51+R59+N108). Mutations in three clusters were identified: codons 50-57, 187-193, and 213-214. Several mutations previously identified in field samples were also isolated, including codons 50 and 164. The I164L mutation is of particular interest, because the quadruple mutant genotype (N51I+C59R+S108N+I164L) encodes an enzyme that is no longer inhibited by pyrimethamine, rendering sulfadoxine/pyrimethamine (SP; Fansidar) clinically ineffective. Thirty-six novel alleles were tested to determine their sensitivity to chlorcycloguanil and WR99210, two DHFR inhibitors that are in clinical and pre-clinical development, respectively. Chlorcycloguanil is effective against parasites that carry the triple mutant allele, but in vitro analysis has suggested that chlorcycloguanil will be clinically ineffective against parasites that carry the quadruple mutant allele of dhfr. In our screen, 23 of 36 novel strains were as resistant to chlorcycloguanil as the quadruple mutant, and one strain was 10-fold more resistant. WR99210 is still effective in the nM range against parasites that carry the quadruple mutant allele. In the preliminary screen, 31 of 36 novel alleles were as sensitive to WR99210 as the quadruple mutant. Detailed analysis of the remaining five showed that four of the five had IC(50) values in the same range as the quadruple mutant, and one, N51I+C59R+S108N+E192G, had an IC(50) value about fivefold higher. This result suggests that WR99210 and related compounds will be clinically effective against quadruple mutants currently found in Southeast Asia and South America and against most novel alleles that could be selected on the background of the triple mutant genotype now prevalent in East Africa

The stepwise selection for ketoconazole resistance induces upregulation of C14-demethylase (CYP51) in Leishmania amazonensis

Ketoconazole is a clinically safe antifungal agent that also inhibits the growth of Leishmania spp. A study was undertaken to determine whether Leishmania parasites are prone to becoming resistant to ketoconazole by upregulating C14-demethylase after stepwise pharmacological pressure. Leishmania amazonensis promastigotes [inhibitory concentration (IC)50 = 2 µM] were subjected to stepwise selection with ketoconazole and two resistant lines were obtained, La8 (IC50 = 8 µM) and La10 (IC50 = 10 µM). As a result, we found that the resistance level was directly proportional to the C14-demethylase mRNA expression level; we also observed that expression levels were six and 12 times higher in La8 and La10, respectively. This is the first demonstration that L. amazonensis can up-regulate C14-demethylase in response to drug pressure and this report contributes to the understanding of the mechanisms of parasite resistance