Dihydrofolate Reductase

Background A priori analysis of the activity of drugs on the target protein by computational approaches can be useful in narrowing down drug candidates for further experimental tests. Currently, there are a large number of computational methods that predict the activity of drugs on proteins. In this study, we approach the activity prediction problem as a classification problem and, we aim to improve the classification accuracy by introducing an algorithm that combines partial least squares regression with mixed-integer programming based hyper-boxes classification method, where drug molecules are classified as low active or high active regarding their binding activity (IC50 values) on target proteins. We also aim to determine the most significant molecular descriptors for the drug molecules. Results We first apply our approach by analyzing the activities of widely known inhibitor datasets including Acetylcholinesterase (ACHE), Benzodiazepine Receptor (BZR), Dihydrofolate Reductase (D...

Molecular markers provide a rapid and relatively inexpensive approach for assessing antimalarial drug susceptibility. We collected 884 Plasmodium falciparum-infected blood samples from 17 Lao provinces. Each sample was genotyped for 11 codons in the chloroquine resistance transporter (pfcrt), dihydrofolate reductase (pfdhfr), and dihydropteroate synthase (pfdhps) genes. The samples included 227 collected from patients recruited to clinical trials. The pfcrt K76T mutation was an excellent predictor of treatment failure for both chloroquine and chloroquine plus sulfadoxine-pyrimethamine, and mutations in both pfdhfr and pfdhps were predictive of sulfadoxine-pyrimethamine treatment failure. In multivariate analysis, the presence of the pfdhfr triple mutation (51 + 59 + 108) was strongly and independently correlated with sulfadoxine-pyrimethamine failure (odds ratio ‫ס‬ 9.1, 95% confidence interval ‫ס‬ 1.4-60.2, P ‫ס‬ 0.017). Considerable geographic heterogeneity in allele frequencies occurred at all three loci with lower frequencies of mutant alleles in southern than in northern Laos. These findings suggest that chloroquine and sulfadoxinepyrimethamine are no longer viable therapy in this country.

Loop-mediated isothermal amplification (LAMP) has been used to detect several pathogens including malaria parasites from field and clinical samples. In this protocol, the malaria LAMP technology is developed to differentiate between Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) species by targeting the dihydrofolate reductase thymidylate synthase (dhfr-ts) gene, a known target for the antifolate class of drugs such as Pyrimethamine. LAMP primer sets are designed and validated for species specific amplification. Additionally, specific probes help improve detection and visualization of the products when combined with lateral flow dipstick-based (LFD) detection. The protocols are further simplified to eliminate tedious sample preparation steps, such that crude lysis prepared simply by diluting few microliter (μL) of blood sample with distilled water is sufficient. The LAMP-LFD malaria dhfr-ts protocols are sensitive and can detect as little as 1 picogram (pg) of PfDNA and 1 nanogram (ng) of PvDNA, or a few microliters of crude lysate from infected blood samples (Yongkiettrakul et al., Parasitol Int 63: 777-784, 2014). These simplified steps not only reduce cost but also increase the potential for large application in the fields and clinical settings.

We have examined the time required for mouse 3T6 cells to become resistant to 200 nM methotrexate by three selection protocols: (a) a single-step 0 to 200 nM dose; (b) a two-step 0 to 80 to 200 nM dose; and (c) a multistep 0 to 40 to 80 to 120 to 160 to 200 nM dose. An initial inoculum of 5 X 10(5) cells was grown to 10(6) cells at each increment of methotrexate, reduced to 5 X 10(5) cells, and again grown to 10(6) cells at the next increment. The total elapsed time required for an initial inoculum of 5 X 10(5) cells to grow to 1 X 10(6) cells resistant to 200 nM methotrexate was 45, 21, and 6.5 days, respectively, for the three drug dosage schedules. The single-step resistant variants did not contain amplified dihydrofolate reductase genes, whereas cells resistant to 200 nM methotrexate by the two stepwise selections were resistant as a result of a 6-fold amplification of the dihydrofolate reductase gene. We conclude that the resistance to 200 nM methotrexate resulting from gene am...

ABSTRACTIt has been an established idea in recent years that protein is a physiochemically connected network. Allostery, understood in this new context, is a manifestation of residue communicating between remote sites in this network, and hence a rising interest to identify functionally relevant communication pathways and the frequent communicators within. However, there have been limited computationally trackable general methods to discover proteins’ allosteric sites in atomistic resolution with good accuracy. In this study, we devised a time-dependent linear response theory (td-LRT) integrating intrinsic protein dynamics and perturbation forces that excite protein’s temporary reconfiguration at the non-equilibrium state, to describe atom-specific time responses as the propagating mechanical signals and discover that the most frequent remote communicators can be important allosteric sites, mutation of which could deteriorate the hydride transfer rate in DHFR by 3 orders. The prefer...

We assessed the effect of daily cotrimoxazole, essential for HIV care, on development of antifolateresistant Plasmodium falciparum, naso-pharyngeal Streptococcus pneumoniae (pneumococcus), and commensal Escherichia coli. HIV-positive subjects with CD4 cell count < 350 cells/L (lower-CD4; N ‫ס‬ 692) received cotrimoxazole; HIV-positive with CD4 cell count Ն 350 cells/L (higher-CD4; N ‫ס‬ 336) and HIV-negative subjects (N ‫ס‬ 132) received multivitamins. Specimens were collected at baseline, 2 weeks, monthly, and at sick visits during 6 months of follow-up to compare changes in resistance, with higher-CD4 as referent. P. falciparum parasitemia incidence density was 16 and 156/100 person-years in lower-CD4 and higher-CD4, respectively (adjusted rate ratio [ARR] ‫ס‬ 0.11; 95% confidence interval [CI] ‫ס‬ 0.06-0.15; P < 0.001) and 97/100 person-years in HIV-negative subjects (ARR ‫ס‬ 0.62; 95% CI ‫ס‬ 0.44-0.86; P ‫ס‬ 005). Incidence density of triple and quintuple dihydrofolate-reductase/dihydropteroate-synthetase mutations was 90% reduced in lower-CD4 compared with referent. Overall, cotrimoxazole non-susceptibility was high among isolated pneumococcus (92%) and E. coli (76%) and increased significantly in lower-CD4 subjects by Week 2 (P < 0.005). Daily cotrimoxazole prevented malaria and reduced incidence of antifolate-resistant P. falciparum but contributed to increased pneumococcus and commensal Escherichia coli resistance.

Molecular assays for monitoring sulfadoxine-pyrimethamine-resistant Plasmodium falciparum have not been implemented because of the genetic and statistical complexity of the parasite mutations that confer resistance and their relation to treatment outcomes. This study analyzed pretreatment dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) genotypes and treatment outcomes in a double-blind, placebo-controlled trial of sulfadoxine-pyrimethamine and chlorproguanildapsone treatment for uncomplicated P. falciparum malaria. Multiple logistic regression was used to identify mutations that were predictive of treatment failure and to identify interactions and confounding factors. Infections caused by parasites with 3 DHFR mutations and 2 DHPS mutations (the "quintuple mutant") were associated with sulfadoxine-pyrimethamine treatment failure but not with chlorproguanil-dapsone treatment failure. The presence of a single DHFR mutation (Arg-59) with a single DHPS mutation (Glu-540) accurately predicted the presence of the quintuple mutant. If this model is validated in other populations, it will finally be possible to use molecular markers for surveillance of antifolate-resistant P. falciparum malaria in Africa.

Dihydropteroate synthase (DHPS) can metabolise sulfa drugs into sulfa-dihydropteroate (sulfa-DHP), which inhibits cell growth through competition with dihydrofolate (DHF), possibly indicating dihydrofolate reductase (DHFR) as the target of sulfa-DHP. The effect of over-production of DHFR on sulfa-DHP resistance was examined in Saccharomyces cerevisiae using a strain that requires DHF for growth. This strain was transformed with a plasmid which encodes over-production of DHFR in the presence of CuSO 4 . Over-production led to resistance to sulfa-DHP suggesting that sulfa-DHP targets DHFR. Spontaneous mutants hyperresistant to sulfa-DHP did not show any changes within DHFR.

We have transferred a mutant hamster gene coding for an altered dihydrofolate reductase to wild-type cultured mouse cells by using total genomic DNA from methotrexate-resistant Chinese hamster ovary A29 cells as donor. By demonstrating the presence of hamster gene sequences in transformants we have provided direct evidence for gene transfer. Transformants selected for increased resistance to methotrexate contain increased amounts of the newly transferred gene. We have used this mutant dhfr gene to introduce the Escherichia coli antibiotic resistance plasmid pBR322 into animal cells. Amplification of the dhfr sequences results in amplification of the pBR322 sequences as well. The use of this gene may allow the introduction and amplification of virtually any genetic element in various new cellular environments.

We have previously demonstrated that mTOR inhibitors (MTIs) are active in preclinical models of acute lymphoblastic leukemia (ALL). MTIs may increase degradation of cyclin D1, a protein involved in dihydrofolate reductase (DHFR) synthesis. Because resistance to methotrexate may correlate with high DHFR expression, we hypothesized MTIs may increase sensitivity of ALL to methotrexate through decreasing DHFR by increasing turn-over of cyclin D1. We tested this hypothesis using multiple ALL cell lines and nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice xenografted with human ALL. We found MTIs and methotrexate were synergistic in combination in vitro and in vivo. Mice treated with both drugs went into a complete and durable remission whereas single agent treatment caused an initial partial response that ultimately progressed. ALL cells treated with MTIs had markedly decreased expression of DHFR and cyclin D1, providing a novel mechanistic explanation for a combined eff...

January 2001 for CML at our institution were retrospectively reviewed. Imatinib-refractory CML was defined as either lack of any cytogenetic response (CGR) after at least 6mths of imatinib, loss of CGR or progression to a more advanced disease stage (accelerated or blast phase) during imatinib therapy. Using the EBMT risk score (Lancet 1998; 352: 1087), transplant outcomes for imatinib refractory CML were compared with all other CML transplants performed during the same time period. Survival analysis was performed using the Kaplan-Meier product-limit and comparison of survival data via the log-rank test. Results: Of 31 allogeneic transplants (19M; 12F) performed for CML, 12 had been performed for imatinib refractory CML (no CGR to imatinib nϭ3; loss of CGR nϭ3; progression to AP nϭ3; progression to BC nϭ3), 5 in patients with imatinib responsive CML, and 14 in patients never exposed to imatinib. Median age at SCT was 40yrs (range 19-63yrs). Donor source included HLAmatched unrelated donors in 14 cases, HLA-identical siblings in 16 and other matched family donors in 1. Graft source consisted of PBSC (17), BM (8) and G-BM (6). Conditioning regimens included Cy/TBI (20 cases), Bu/Cy (8 cases), Flu/Mel (2 cases) and Flu/Cy (1 case). CsA ϩ MTX was used as standard GVHD prophylaxis ( ), CsA alone (1) and Tacrolimus/Mycophenolate (1). EBMT risk scores were 1 (4 cases), 2 (6 cases), 3 (8 cases), 4 (5 cases), 5 (3 cases) and 6 (5 cases). At median follow-up post-SCT of 37mths (range 6-64mths), median PFS and OS are not reached; at 2yrs PFS, EFS and OS are 81%, 58% and 61% respectively. For patients with EBMT risk scores of 1-2 versus 3-4 versus 5-6, OS at 2 yrs post-SCT is 80%, 62% and 38% respectively (pϭ0.03). Based on EBMT risk score, no significant differences in PFS, EFS or OS were observed when comparing SCT for imatinib-refractory versus imatinib-responsive / imatinib-naive CML. Conclusion: Our experience suggests that survival post-SCT for imatinib-refractory CML is similar to SCT for imatinib-responsive / imatinib-naive CML. The EBMT risk score remains useful in predicting survival post-SCT in imatinib-refractory CML.

Background Sulphadoxine and pyrimethamine are anti-folate drugs that show synergistic anti-malarial effect. Point mutations in dihydrofolate reductase (dhfr) and dihydropteorate synthatase (dhps) cause anti-folate drug resistance phenotype in human malaria parasites. This study presents pattern of point mutations in dhfr/dhps genes among Indian sub-continent. Methods Microscopically diagnosed one hundred Plasmodium vivax field isolates were collected from five widely separated geographical regions of India. Dhfr and dhps genes were PCR amplified and sequenced. Previously published mutations data were collected and analyzed using Chi square test to identify geographical cluster of mutant/wild type genotypes. Results Sequence analysis revealed single (S58R), double (S58R/S117N) and quadruple (F57L/S58R/T61M/S117T/) point mutations at dhfr and single (A383G) to double (A383G/A553G) mutations at dhps in P. vivax field isolates. In addition, three new mutations were also observed at dhfr...

A series of substituted N,2-diphenylquinazolin-4-amines were synthesized by cyclization of methyl anthranilate and various substituted benzamide giving corresponding 2-phenylquinazolinone followed by replacement of ketonic oxygen with various aryl amino group via chlorination gateway to afford title compounds. The homogeneity and purity of compound was ascertained by Physical constant determination and chromatographic methods. The structure of the synthesized derivative was further confirmed by spectral (FTIR, NMR, MS) and elemental (C, H, N) analysis. These synthesized compounds were screened for anti-inflammatory and analgesic activities using, Rat paw edema method and Hot plate method respectively. All compounds showed average to better activity when compared with Diclofenac and Tramadol respectively as standard

We previously reported (J. Galivan et al., Proc. Natl. Acad. Sci. USA, 82: 2598-2602, 1985) the synthesis and characterization of DL-erythro,threo-gamma-fluoromethotrexate (FMTX). The individual diastereomers, DL-erythro-FMTX (eFMTX) and DL-threo-FMTX (tFMTX), and their radiolabeled counterparts have now been prepared and characterized. Transport of eFMTX (Km = 9.3 microM; Vmax = 7.5 pmol/min/10(7) cells) was similar to that of methotrexate (MTX: Km = 6.6-9.9 microM; Vmax = 11.4-14.2 pmol/min/10(7) cells), while tFMTX (Km = 65.1 microM; Vmax = 8.4 pmol/min/10(7) cells) was transported less efficiently. Both isomers were able to saturate intracellular dihydrofolate reductase and accumulate further as unbound intracellular drug. Based on competition experiments and studies with MTX transport-defective cell lines, both isomers utilized the reduced folate/MTX transport system. Efflux half-times for the isomers were similar to those of MTX. Each isomer was equivalent to MTX in its ability to inhibit dihydrofolate reductase activity and bind to intracellular dihydrofolate reductase when the intracellular drug concentration was limiting. Both isomers had drastically diminished capacity to be metabolized to poly(gamma-glutamyl) metabolites by isolated folylpolyglutamate synthetase and in whole cells; tFMTX was metabolized to a slightly lesser extent than eFMTX. Using the CCRF-CEM human leukemia and H35 rat hepatoma cell lines, the growth-inhibitory effects of eFMTX were almost the same as those of MTX during continuous exposure, while tFMTX was slightly less potent. This difference in growth-inhibitory potency of the two isomers correlated with their ability to inhibit de novo thymidylate synthesis in the H35 cell line. These results indicate that both diastereomers of FMTX are similar in their properties to MTX, except that both are incapable of being readily converted to polyglutamate derivatives. As a result of these properties, both isomers could be used under appropriate conditions in comparative studies with MTX to define the roles of MTX polyglutamates.

Allosteric signaling in biological molecules, which may be viewed as specific action at a distance due to localized perturbation upon binding of ligands or changes in environmental cues, is pervasive in biology. Insightful phenomenological MWC and KNF models galvanized research in describing allosteric transitions for over five decades, and these models continue to be the basis for describing the mechanisms of allostery in a bewildering array of systems. However, understanding allosteric signaling and the associated dynamics between distinct allosteric states at the molecular level is challenging, and requires novel experiments complemented by computational studies. In this review, we first describe symmetry and rigidity as essential requirements for allosteric proteins or multisubunit structures. The general features, with MWC and KNF as two extreme scenarios, emerge when allosteric signaling is viewed from an energy landscape perspective. To go beyond the general theories, we describe computational tools that are either based solely on multiple sequences or their structures to predict the allostery wiring diagram. These methods could be used to predict the network of residues that carry allosteric signals. Methods to obtain molecular insights into the dynamics of allosteric transitions are briefly mentioned. The utility of the methods is illustrated by applications to systems ranging from monomeric proteins in which there is little conformational change in the transition between two allosteric states to membrane bound G-protein coupled receptors, and multisubunit proteins. Finally, the role allostery plays in the functions of ATP-consuming molecular machines, bacterial chaperonin GroEL and molecular motors, is described. Although universal molecular principles governing allosteric signaling do not exist, we can draw the following general conclusions from a survey of different systems. (1) Multiple pathways connecting allosteric states are highly heterogeneous. (2) Allosteric signaling is exquisitely sensitive to the specific architecture of the system, which implies that the capacity for allostery is encoded in the structure itself. (3) The mechanical modes that connect distinct allosteric states are robust to sequence variations. (4) Extensive investigations of allostery in Hemoglobin and more recently GroEL, show that to a large extent a network of salt-bridge rearrangements serves as allosteric switches. In both these examples the dynamical changes in the allosteric switches are related to function.

Dihydrofolate reductase (DHFR) enzyme catalyzes tetrahydrofolate regeneration by reduction of dihydrofolate using NADPH as a cofactor. Tetrahydrofolate and its one carbon adducts are required for de novo synthesis of purines and thymidylate, as well as glycine, methionine and serine. DHFR inhibition causes disruption of purine and thymidylate biosynthesis and DNA replication, leading to cell death. Therefore, DHFR has been an attractive target for chemotherapy of many diseases including cancer. Over the following years, in order to develop better antifolates, a detailed understanding of DHFR at every level has been undertaken such as structure-functional analysis, mechanisms of action, transcriptional and translation regulation of DHFR using a wide range of technologies. Because of this wealth of information created, DHFR has been used

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Ruthenium trichloride complexes featuring η²-bound alkyne ligands are suggested as intermediates in the early stages of Ru and alkyne reactions. Our research has demonstrated that these unsaturated complexes, with the general formula [RuCl(η⁵-Cp) (η²-RC≡CR')], can be effectively stabilized by using a sterically demanding cyclopentadienyl ligand. Specifically, we have employed the η⁵-1-methoxy-2,4ferrocenyl-3-neopentyl-cyclopentadienyl ligand (abbreviated as Cp). Additionally, we have found that trimerization of alkynes, demonstrating significant potential for applications in inorganic synthesis. In addition to our studies on the chemical properties and of ruthenium complexes, we have also conducted a thorough investigation of their biological activity. Specifically, we examined their potential as antifungal, antibacterial, and antimalarial agents. This comprehensive analysis aimed to explore the applicability of these complexes in medicinal chemistry, potentially leading to the development of new treatments for various infectious diseases.

We have demonstrated that nanostructures, and in particular nanorings incorporating a homodimeric enzyme, can be prepared by chemically induced self-assembly of dihydrofolate reductase (DHFR)histidine triad nucleotide binding 1(Hint1) fusion proteins. The dimensions of the nanorings were found by static light scattering and atomic force microscopy studies to be dependent on the length and composition of the peptide linking the fusion proteins, ranging in size from 10 to 70 nm in diameter and 64 to 740 kDa. The catalytic efficiency of the nanorings was found to be dependent on ring size, thus suggesting that the arrangement of supermolecular assemblies of enzymes maybe be used to control their catalytic parameters.

Pemetrexed (Alimta ® ) is a novel, multitargeted antifolate that inhibits at least three of the enzymes involved in folate metabolism, and purine and pyrimidine synthesis. These enzymes are thymidylate synthase, dihydrofolate reductase and glycinamide ribonucleotide formyltransferase. Pemetrexed has demonstrated broad antitumor activity in Phase II trials in a wide variety of solid tumors, including mesothelioma, non-small cell lung, breast, cervical, colorectal, head and neck, and bladder cancers. Promising activity has also been demonstrated when pemetrexed is combined with cisplatin and gemcitabine (Gemzar ® ). A pivotal Phase III study in mesothelioma has been presented. This study indicates the superiority of pemetrexed in combination with cisplatin versus cisplatin alone in this disease. The most significant toxicities of pemetrexed, myelossuprresion and mucositis have been significantly ameliorated by folate and vitamin B12 supplementation. More importantly, vitamin supplementation has not demonstrated any adverse efficacy. This review discusses the biochemistry and clinical activity of pemetrexed.

Background: Antimalarial drug resistance is one of the important challenges for governments in the fight against malaria. Molecular surveillance of antimalarial drug resistance supports early detection of how the recommended treatments work. This allows immediate action to reduce any threat and prevent it from spreading. Therefore, the aim of this study was to evaluate the frequency of dihydrofolate reductase (dhfr) mutants in Plasmodium falciparum resistance to pyrimethamine in Iranian malaria patients. In 2020, 27 patients (21 males and 5 females) with imported P. falciparum cases were studied. The nested-PCR technique first confirmed the species in all samples and then amplification was done by the semi-nested-PCR method in order to detect single nucleotide polymorphisms (SNPs) in dhfr gene related to pyrimethamine resistance. Results: All samples in the 18S rRNA gene had species-specific bands for P. falciparum strains. In the sequence analysis of pfdhfr gene amplification after comparison with the standard strain (wild type), 21 patients had a double mutation (C59R+S108N) and six patients had a triple mutation (N51I+C59R+S108N) of pyrimethamine resistance. The results of this study showed that the susceptibility of P. falciparum to pyrimethamine in the treatment of malaria is significantly reducing. These findings can raise concerns about pyrimethamine resistance in P. falciparum. Due to the high emergence of double and triple mutants related to pyrimethamine resistance, the malaria surveillance and treatment systems in Iran, the use of pyrimethamine should be considered.

SummarySulphadoxine/pyrimethamine (SP) has become the first‐line treatment of uncomplicated malaria in a number of African countries. Molecular surveillance of resistance‐mediating mutations in Plasmodium falciparum dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) has been proposed as a means of predicting SP treatment outcomes, but optimal methods of surveillance in different populations have not been well established. To investigate the relationship between molecular markers of SP resistance, host immunity, and response to therapy, we evaluated the association between the presence of five key dhfr and dhps mutations at enrollment and clinical outcome in children and adults treated with SP for uncomplicated malaria in Kampala, Uganda. Clinical treatment failure was 11% at 14 days, increasing to 30% at 28 days, after excluding new infections. Outcomes varied markedly based on the number of dhfr and dhps mutations and on the age of treated subjects. All infections w...

Much attention has been paid recently to bistability and switch-like behavior that might be resident in important biochemical reaction networks. There is, in fact, a great deal of subtlety in the relationship between the structure of a reaction network and its capacity to engender bistability. In common physicochemical settings, large classes of extremely complex networks, taken with mass action kinetics, cannot give rise to bistability no matter what values the rate constants take. On the other hand, bistable behavior can be induced in those same settings by certain very simple and classical mass action mechanisms for enzyme catalysis of a single overall reaction. We present a theorem that distinguishes between those mass action networks that might support bistable behavior and those that cannot. Moreover, we indicate how switch-like behavior results from a well-studied mechanism for the action of human dihydrofolate reductase, an important anti-cancer target.

Background: Plasmodium falciparum is the predominant human malaria species in Mozambique and a lead cause of mortality among children and pregnant women nationwide. Sulphadoxine/ pyrimethamine (S/P) is used as first line antimalarial treatment as a partner drug in combination with artesunate. Methods: A total of 92 P. falciparum-infected blood samples, from children with uncomplicated malaria attending the Centro de Saude de Bagamoyo in the Province of Maputo-Mozambique, were screened for S/P resistance-conferring mutations in the pfdhfr and pfdhps genes using a nested mutation-specific polymerase chain reaction and restriction digestion (PCR-RFLP). The panel of genetic polymorphisms analysed included the pfdhfr 164L mutation, previously reported to be absent or rare in Africa. The frequency of the S/P resistance-associated pfdhfr triple mutants (51I/59R/108N) and of pfdhfr/pfdhps quintuple mutants (51I/59R/108N + 437G/540E) was 93% and 47%, respectively. However, no pfdhfr 164L mutants were detected. The observation that a considerably high percentage of P. falciparum parasites contained S/P resistance-associated mutations raises concerns about the validity of this drug as first-choice treatment in Mozambique. On the other hand, no pfdhfr 164L mutant was disclosed, corroborating the view that that this allele is still rare in Africa.

Among various 150-nM methotrexate-resistant (MTX r ) V79 clones isolated, we found that two near-tetraploid clones as well as a near-diploid clone with amplification in the dihydrofolate reductase (dhfr) gene readily developed resistance to 40 000 nM MTX within 3 months during stepwise increased MTX selection, while two near-diploid clones without gene amplification could not acquire resistance beyond 5000 nM MTX. Then, we studied how the clones increased the resistance to MTX, and compared the propensity for gene amplification among three types of clones. Dot blot analysis showed that the acquisition of the high levels of resistance to MTX observed in two neartetraploid clones and a near-diploid clone with gene amplification was associated with amplification in the dhfr gene. The amplified dhfr gene was overexpressed at mRNA and protein levels in the clones. Southern blot analysis of Hind III-and Eco Rl-digested DNA in the clones at the time when they became resistant to 10 000 nM MTX indicated that they amplified the dhfr gene fragments which existed in low amounts in parental V79 cells, and that no gross rearrangement of the amplified dhfr gene was detected. Furthermore, fluorescence in situ hybridization analysis showed that the amplified dhfr gene was located on one chromosome as cluster(s). On the other hand, two neardiploid clones without gene amplification did not show any amplification of the dhfr gene even at 5000 nM-MTX resistant stage. These combined results suggest that the near-tetraploid clone as well as the near-diploid clone with the dhfr gene amplification have genomic instability with the propensity for gene amplification during stepwise MTX selection, and have a similar process for the development of the dhfr gene amplification.

Full wwPDB X-ray Structure Validation Report 4IQB 1 Overall quality at a glance i ○ The following experimental techniques were used to determine the structure: X-RAY DIFFRACTION The reported resolution of this entry is 1.13 Å. Percentile scores (ranging between 0-100) for global validation metrics of the entry are shown in the following graphic. The table shows the number of entries on which the scores are based. Similar resolution (#Entries, resolution range(Å))

Previous work has shown that the presence of a phorbol ester tumor promoter, phorbol 12-myristate 13-acetate (PMA), during a single-step selection for methotrexate (MTX)-resistant mouse 3T6 cells results in an up to 100-fold increase in the incidence of MTX-resistant, colony-forming cells. MTX resistance of most of these cells is due to amplification of the gene for dihydrofolate reductase (DHFR), the target enzyme for MTX. We show here that other active, noncytotoxic phorbol ester tumor promoters, such as phorbol 12, 13-didecanoate and 20-phorbol 12,13-butyrate, at their optimal concentrations (approximately equal to 0.1 microM) are approximately equal to PMA in increasing the incidence of MTX-resistant 3T6 colonies. Mezerein, a potent second-stage tumor promoter, but a weak complete promoter, increases the incidence of MTX resistance up to 350-fold, the strongest effect for any of the agents so far tested. PMA analogs that are inactive as tumor promoters, such as phorbol or phorbo...

Heading Ion, Oxford 0 A3 9 R eco m b in an t D N A techniques have been used to express the proteins of influenza virus individually. T a rg e t cells expressing single viral proteins were then used to identify the molecules recognized by cytotoxic T lym phocytes (c t l s ). R esults have shown th at, co n trary to expectation, the m ajority of the proteins recognized by class I m ajor histocom patibility com plex-restricted ctls are not tran sm em b ran e glycoproteins. E xperim ents w ith deletion m u tan ts of the nucleoprotein (NP) gene showed th a t tran sp o rt of epitopes to the m em b ran e for recognition by ctls was in d ep en d en t of a definable signal sequence. In ad d itio n , the epitopes recognized were contained w ithin short linear sequences of am ino acids, and rapid d eg rad atio n of large N P fragm ents w ithin the target cell did not prevent recognition by c t l s . T hese results led to the suggestion th a t the epitopes recognized by class-I-restricted ctls resulted from d eg rad atio n of viral proteins. If so, the epitopes should, like those for class-II-restricted T cells, be replaceable in vitro w ith short synthetic peptides. Five different epitopes of N P have now been d em onstrated th a t can be defined w ith short peptides in vitro. Each peptide is recognized w ith a specific class I m olecule (D h, K k, K d and H LA B37). This has been extended to the influenza m atrix protein, and a peptide epitope defined th a t is recognized by h u m an ctls in association w ith H L A -A 2. T h e question arose as to w hether a sim ilar phenom enon w ould be found w ith viral proteins which are n atu rally inserted in the target cell m em brane. A m u ta n t haem agglutinin has been produced th a t lacks a hydrophobic signal sequence. T his protein is expressed as a short-lived, unglycosylated, in tracellu lar protein. H ow ever, target cells expressing this molecule were recognized efficiently by ctls raised to the w ild-type haem agglutinin and vice versa. These and m ore recent results w ith nonviral glycoproteins are consistent w ith the existence of a m echanism for d egrading viral (and perhaps host) proteins and exposing them at the cell surface for recognition by cytotoxic T cells in association w ith class I molecules of the m ajor histocom patibility complex.

In Saudi Arabia, Origanum vulgare is widely disseminated. In the present work, we used GC-MS analysis to identify the components of Origanum vulgare essential oil. The disc diffusion assay was used to assess the essential oil's in vitro antibacterial efficacy against Gram-positive and Gramnegative Staphylococcus aureus and Escherichia coli. The antimicrobial efficacy against many microbial proteins, including tyrosyl-tRNA synthetase (TyrRS), DNA gyrase, and dihydrofolate reductase (DHFR), was further evaluated using molecular docking. Eighteen compounds were identified using GC-MS analysis, which constituted 99.81% of the total essential oil content. Terpinen-4-ol (34.3%), Beta-Terpineol (16.96%), 3-Epimoretenol (11.84%), and Alpha-Terpineol (3.86%) were the main substances identified. According to the antibacterial investigation, the inhibition zone against Staphylococcus aureus was 8 mm and 6 mm against Escherichia coli. High affinities were found between 3-Epimoretenol and tyrosyl-tRNA synthetase (TyrRS) and dihydrofolate reductase (DHFR) compared to positive controls (Clorobiocin, SCHEMBL2181345); the affinity values were -8.3 Kcal/mol and -9.2, respectively. The results of the present study indicate that Origanum vulgare essential oil can be used as a nutraceutical to treat infectious diseases.

In Saudi Arabia, Origanum vulgare is widely disseminated. In the present work, we used GC-MS analysis to identify the components of Origanum vulgare essential oil. The disc diffusion assay was used to assess the essential oil’s in vitro antibacterial efficacy against Gram-positive and Gram-negative Staphylococcus aureus and Escherichia coli. The antimicrobial efficacy against many microbial proteins, including tyrosyl-tRNA synthetase (TyrRS), DNA gyrase, and dihydrofolate reductase (DHFR), was further evaluated using molecular docking. Eighteen compounds were identified using GC-MS analysis, which constituted 99.81% of the total essential oil content. Terpinen-4-ol (34.3%), Beta-Terpineol (16.96%), 3-Epimoretenol (11.84%), and Alpha-Terpineol (3.86%) were the main substances identified. According to the antibacterial investigation, the inhibition zone against Staphylococcus aureus was 8 mm and 6 mm against Escherichia coli. High affinities were found between 3-Epimoretenol and tyros...

Overlapping recombinant X1059 phages carrying regions of the dhfr locus from the amplified Chinese hamster ovary (CHO) cell clone MK42 have been iso- lated. In addition, dhfr cDNAs from this cell line have been cloned into plasmid pBR322. Restriction analysis of these recombinant molecules has led to a map of the Chinese hamster dhfr gene. This gene has a minimum size of 26 kb and contains six exons as defined by hybridization to a combination of mouse and CHO cDNA probes. The latter probes reveal 3' exonic sequences that are not present in mouse cDNA. The CHO dhfr gene thus extends about 700 bp further 3' than in the mouse, consistent with the larger size of the hamster mRNA. At least five intervening sequences are present, of approximate sizes: 0.3, 2.5, 8.6, 2.6 and 9.4 kb. Four sequences from highly re- peated families are situated in introns within the dhfr gene. The overall structure of this gene is strikingly similar to that of the mouse. Evolu- tionary conservation of interrupted gene structure among mammals thus extends to genes that code for household enzymes as well as specialized or structural proteins.

Overlapping recombinant X1059 phages carrying regions of the dhfr locus from the amplified Chinese hamster ovary (CHO) cell clone MK42 have been iso- lated. In addition, dhfr cDNAs from this cell line have been cloned into plasmid pBR322. Restriction analysis of these recombinant molecules has led to a map of the Chinese hamster dhfr gene. This gene has a minimum size of 26 kb and contains six exons as defined by hybridization to a combination of mouse and CHO cDNA probes. The latter probes reveal 3' exonic sequences that are not present in mouse cDNA. The CHO dhfr gene thus extends about 700 bp further 3' than in the mouse, consistent with the larger size of the hamster mRNA. At least five intervening sequences are present, of approximate sizes: 0.3, 2.5, 8.6, 2.6 and 9.4 kb. Four sequences from highly re- peated families are situated in introns within the dhfr gene. The overall structure of this gene is strikingly similar to that of the mouse. Evolu- tionary conservation of interrupted gene structure among mammals thus extends to genes that code for household enzymes as well as specialized or structural proteins.

In this work, co-crystal screening was carried out for two important dihydrofolate reductase (DHFR) inhibitors, trimethoprim (TMP) and pyrimethamine (PMA), and for 2,4-diaminopyrimidine (DAP), which is the pharmacophore of these active pharmaceutical ingredients (API). The isomeric pyridinecarboxamides and two xanthines, theophylline (THEO) and caffeine (CAF), were used as co-formers in the same experimental conditions, in order to evaluate the potential for the pharmacophore to be used as a guide in the screening process. In silico co-crystal screening was carried out using BIOVIA COSMOquick and experimental screening was performed by mechanochemistry and supported by (solid + liquid) binary phase diagrams, infrared spectroscopy (FTIR) and X-ray powder diffraction (XRPD). The in silico prediction of low propensities for DAP, TMP and PMA to co-crystallize with pyridinecarboxamides was confirmed: a successful outcome was only observed for DAP + nicotinamide. Successful synthesis of multicomponent solid forms was achieved for all three target molecules with theophylline, with DAP co-crystals revealing a greater variety of stoichiometries. The crystalline structures of a (1:2) TMP:THEO co-crystal and of a (1:2:1) DAP:THEO:ethyl acetate solvate were solved. This work demonstrated the possible use of the pharmacophore of DHFR inhibitors as a guide for co-crystal screening, recognizing some similar trends in the outcome of association in the solid state and in the molecular aggregation in the co-crystals, characterized by the same supramolecular synthons.

One of the mechanism actions of antimalarial drugsis by an inhibiting on the enzyme dihydrofolate reductase (DHFR), an enzyme target antifolate drug. Epi-croomine and croomine, are alkaloids isolated from Stemonatuberosa showed DHFR inhibition with K i of 61.14 and 100.59 µM and K M values of30.68 and 27.06 µM at 10 ppm. The IC 50 to the DHFR of croomine and pyrimethamine were 5.29 and 7.71 µM, respectively. Tuberostemonine is not active to the enzyme. The kinetic analysis showed that both epi-croomine and croomine competitively inhibited to the human DHFR recombinant. The molecular modeling of the compounds to the human DHFR was estimate depi-croomine and croomine&#39;s binding free energy of -6.66 and -7.60 kcal/mol. The docking showed that both epi-croomine and croomine could possibly form hydrogen bonds with the amino acid residue of theAla9, which residues on the active site of the enzyme.

The structure, genomic organization and transcrlptlon of the gene encoding hlstone H2B In the protozoan parasite Trypansosoma cruzl have been studied. This gene consists of a 746~nucleotide unit, tandemly repeated at least 18 times in each of two clusters. DNA probes corresponding to hlstones H2B and H3 hybridized to different chromosomes revealing that the genes coding for these two hlstones are not physlcally linked in the genome of r. cruzl. The primary transcription product of the H2B gene Is processed by trans-splicing and polyadenylatlon. lnhibltlon of DNA synthesis with aphidicolin resulted in the reductlon of histone H2B mRNA to undetectable levels in about two hours, suggesting that Its abundance is regulated throughout the cell cycle as it occurs in other eukaryotes. in addition, a concomitant inhibition of translation by cyclohexfmide reverted this effect indicating that de novo protein synthesis is required for RNA instability. Hlstone mRNA abundance was dependent on the life-cycle stage of r. cruzl: abundant in amastigotes and epimastlgotes, the dividing forms in the host cell and the Insect vector, respectively, while undetected in trypomastigotes, the parasite's nondlvidlng life stage.

The slightly water-soluble 5-nitroindazole derivative (5-NI) and its inclusion with either b-cyclodextrin (bCD) or Heptakis (2,6-di-O-methyl)-b-cyclodextrin (DMbCD) were investigated. The stoichiometric ratios and stability constants describing the extent of formation of the complexes were determined by phase-solubility measurements obtaining type-A L diagrams in both cases. According to the continuous variation method (Job's plot) a 1:1 stoichiometry has been proposed for the complexes. Also electrochemical studies were carried out on both CDs complexes, where the observed change in the E PC value for DMbCD indicated a lower feasibility of the nitro group reduction. The detailed spatial configuration is proposed based on two-dimensional NMR methods. These results are further interpreted using molecular modeling studies. The latter results are in good agreement with the experimental data.

Growth inhibition assays indicated that the IC50 values for methotrexate (MTX) and 5-fluorodeoxyuridine (FdUrd) in HS-18, a liposarcoma cell line lacking retinoblastoma protein (pRB), and SaOS-2, an osteosarcoma cell line with a truncated and nonfunctional pRB, were 10- to 12-fold and 4- to 11-fold higher, respectively, than for the HT-1080 (fibrosarcoma) cell line, which has wild-type pRB. These Rb-/- cell lines exhibited a 2- to 4-fold increase in both dihydrofolate reductase (DHFR) and thymidylate synthase (TS) enzyme activities as well as a 3- to 4-fold increase in mRNA levels for these enzymes compared to the HT-1080 (Rb+/+) cells. This increase in expression was not due to amplification of the DHFR and TS genes. Growth inhibition by MTX and FdUrd was increased and DHFR and TS activities and expression were correspondingly decreased in Rb transfectants of SaOS-2 cells. In contrast, there was no significant difference in growth inhibition among these cell lines for the nonantime...

12 157 12. Mammalian Cell Culture ■ 159 higher manufacturing cost, and other challenges compared with microbial systems, cultured mammalian cells have become a widely used platform for producing recombinant proteins (250), and cell culture capacity has grown from less than 300,000 liters to around 3 million liters today, driven by the demands for MAbs (219). About one-half (212, 237) to two-thirds (152) of all the approved biopharmaceuticals are glycosylated proteins produced by mammalian cell culture, such as MAbs, blood factors, anticoagulants, thrombolytics, erythropoietin, specific interferons, hormones, and various therapeutic enzymes. In 2007, there were 15 biopharmaceuticals approved in the United States and European Union (238), including 6 erythropoietin-based products, 3 antibodies, 2 hormones, 1 recombinant vaccine, 1 growth factor, and a fusion product. Eleven of these 15 are produced in mammalian cell culture systems, 2 in E. coli, 1 in S. cerevisiae, and 1 in a baculovirus/ insect cell-based system, confirming the prominence of mammalian-based expression systems in the biopharmaceutical sector. The rapid increase of MAbs as product candidates in recent years has been enabled by improvements made in the recombinant expression technologies that use mammalian cells. While there are 2 nonglycosylated MAb Fab fragment products from E. coli fermentations, Lucentis (for macular degeneration) and Cimzia (for Crohn's disease), which recently were approved by the FDA (18, 180), the other 24 MAb products currently on the market are produced in mammalian cells (Table ) , of which all but 2 are produced by recombinant technology. In addition, in the last 2 decades, recombinant therapeutic protein yields from mammalian cells in batch and fed-batch processes have increased dramatically due to the utilization of highly productive cell lines, improved media, and better bioprocess conditions that enhance cell viabilities in high-density suspension cultures (94), thus easing the major concern of using mammalian production systems. It is obvious that mammalian cell culture will continue to be the cornerstone of large-scale production for biopharmaceuticals, especially for MAbs. This review will summarize the recent development of biopharmaceutical production using mammalian cell culture. Since it is not practical to attempt to cover all categories of biopharmaceutical products, this chapter will focus on MAbs, which is the most dominant and most rapidly developing sector in the biopharmaceutical industry, and its large-scale production the most established. The concepts and methods described below should nevertheless be applicable to the production of other biopharmaceutical molecules derived from mammalian cells. Specific development and manufacturing information for vaccines can be found in the published literature . Mammalian cells are fragile because they are approximately 10-to 50-fold larger than microbial cells, without the tough cell walls of microbes, and they are sensitive to impurities. Simply getting these cells to thrive and reproduce in culture is a challenge in itself. The fundamental and unique aspects of mammalian cell culture characteristics, growth kinetics, cell metabolism, bioreactor development, process monitoring and control, and process issues in largescale mammalian cell cultures were well reviewed in the last edition of this manual (174). The history of the discovery, development, and production of therapeutic antibodies is covered in a recent review (114). In the present review, the recently published literature from peer-reviewed journals, but not conference presentations or Web seminars, etc., is summarized, including each of the major mammalian culture process development steps for MAbs such as cell line, medium, and cell culture. Other important aspects

A series of antimalarial 4-aminoquinoline-pyrimidine hybrids incorporating modified anilines at the pyrimidine end was designed and synthesized through a facile synthetic route. The present hybrids displayed excellent equipotent in vitro antiplasmodial activities against both chloroquine-sensitive (D6) and chloroquine-resistant (W2) strains of P. falciparum with up to 11-fold (IC 50 = 0.033 mM) better activity than the reference drug, (chloroquine, IC 50 = 0.370 mM) against the resistant strain. The compounds were found to be non-cytotoxic towards the mammalian cells up to the highest tested concentration. The present hybrids displayed good binding interactions with both monomeric and dimeric heme, suggesting heme-detoxification as their mechanism of action. Furthermore, the most active hybrids were found to display good in silico docking interactions within the active site of plasmodial dihydrofolate reductase (DHFR) enzyme (wild and mutant type). Computational studies predicted favorable pharmacokinetic parameters and oral drugability for the synthesized molecules.

Dihydrofolate reductase (DHFR, EC 1.5.1.3) is found in both prokaryotes and eukaryotes, and is essential in the folate biosynthetic pathway . DHFR catalyzes NADPH-dependent reduction of dihydrofolate (FAH 2 ) to tetrahydrofolate (FAH 4 ) (Fig. ). Reduction of FAH 2 to FAH 4 is a universal requirement for the maintenance of an intracellular reduced folate pool, which is important in one-carbon transfer reactions that are necessary for the biosynthesis of DNA, RNA and protein . A common bacterial antifolate inhibitor of this enzyme is trimethoprim (TMP) (Fig. ). Previously, we identified the Mycobacterium avium folA gene and confirmed the functionality of its product, DHFR . Subsequently, we demonstrated its inherent resistance to TMP . Further studies revealed a group of 2,4-diamino-5-deazapteridines that

Brilliance of terbium: Heterodimeric conjugates of trimethoprim covalently linked to sensitized terbium chelates bind to Escherichia coli dihydrofolate reductase fusion proteins with nanomolar affinity (see picture). Terbium luminescence enables sensitive and time‐resolved detection of labeled proteins in vitro and on the surface of living mammalian cells.magnified image

Dihydrofolate reductase (DHFR) converts dihydrofolate (DHF) into tetrahydrofolate (THF) and plays an essential role in cell metabolism and cellular growth. Folic acid from multivitamins needs to be reduced by DHFR before it participates in cellular reactions. We examined the relation of a 19-base pair (bp) deletion polymorphism of the DHFR gene with the risk of breast cancer by using data from the Long Island Breast Cancer Study Project, a population-based case-control study. We also investigated the transcriptional effect of this deletion polymorphism. Dietary data and habitual use of multivitamins were assessed from a modified Block food-frequency questionnaire (FFQ). Genotypes of DHFR were ascertained from 1062 case subjects and 1099 control subjects by allele-specific polymerase chain reaction. Unconditional logistic regression was used to estimate odds ratios (ORs) and 95% CIs. Although the DHFR 19-bp deletion polymorphism was not associated with overall breast cancer risk, we ...

Plasmodium falciparum dihydrofolate reductase (pf-DHFR) is one of the several targets in the treatment of malaria. Double and quadruple mutations at residues 51, 59, 108, and 164 of pf-DHFR have been linked to antifolate resistance. Several efforts are underway to overcome this drug resistance and to produce potential inhibitors. In this regard, the quantitative structure-activity relationship (QSAR) and docking studies were performed for previously reported 4-anilinoquinoline and 1,3,5-triazines based molecular hybrids. The generated model showed good correlation coefficients (R 2 ¼ 0.70) and test set prediction coefficient (R 2 ¼ 0.74). These outcomes showed the good predictive competence of the established QSAR model. Based on these results we docked into active site of pf-DHFR protein with the most active (4) and the less active (5) compounds. The docking results revealed that these molecules interact specifically with SER108 and ILE164 in the pf-DHFR binding pocket as that of best active compound but also showed additional interactions with LEU40 and GLY44.

Malaria remains one of the most formidable parasitic diseases globally, exerting a substantial burden, particularly in tropical and subtropical regions. The clinical course of malaria varies significantly depending on the Plasmodium species, geographical factors, patient immune status, and timeliness of treatment initiation. Prognostic outcomes are influenced by the potential for relapse, recrudescence, and species-specific life cycles, with Plasmodium falciparum posing the greatest threat due to its rapid erythrocytic replication and severe complications. Cerebral malaria, severe malarial anemia, and nephrotic syndrome represent the most life-threatening complications, each with distinct pathophysiological mechanisms and clinical implications. The mortality associated with severe malaria remains high, particularly in pediatric populations and pregnant women. Effective management necessitates a multidisciplinary approach involving infectious disease specialists, pharmacists, and nurses to optimize treatment protocols and monitor for adverse outcomes. Preventive strategies, including vector control, patient education, and chemoprophylaxis, are pivotal in mitigating disease transmission and severity. Pre-travel consultations and tailored prophylaxis based on patient-specific risk factors are critical in non-endemic settings. This review consolidates current knowledge on malaria prognosis, complications, clinical management, and prevention, with an emphasis on improving patient outcomes through coordinated interprofessional care.

The inhibition activity on carbonic anhydrase II of forty substituted 1,3,4-thiadiazole-and 1,3,4thiadiazoline-disulfonamides has been studies using a molecular descriptor family on structure-activity relationship approach. The approach use the structure information of substituted 1,3,4-thiadiazole-and 1,3,4-thiadiazoline-disulfonamides for generating and calculating the molecular descriptors family. Computed molecular descriptors entered into a multiple linear regression analysis and the models with grater performance in modeling of inhibition activity on carbonic anhydrase II are analyzed. The analysis of the models was performed through correlation coefficients, squared correlation coefficients, and regressions parameters. The predictivity of the models was evaluated by cross-validation leave-one-out and training versus test (starting with 50% and up to 25% of compounds in training sets) analysis. The comparison between bi-and four-varied models was performed through a correlated correlation analysis and the comparison between the best performing model and previous reported models by Fisher Z test. The four-varied model allows acquiring some knowledge about the relationships between complex structural information of substituted 1,3,4-thiadiazole-and 1,3,4-thiadiazoline-disulfonamides and theirs inhibition activity on carbonic anhydrase II.

The therapeutic responses to the eight most widely used antimalarial drugs were assessed in 207 adult patients with Plasmodium vivax malaria. This parasite does not cause marked sequestration, so parasite clearance can be used as a direct measure of antimalarial activity. The activities of these drugs in descending order were artesunate, artemether, chloroquine, mefloquine, quinine, halofantrine, primaquine, and pyrimethamine-sulfadoxine (PS). Therapeutic responses to PS were poor; parasitemias did not clear in 5 of the 12 PS-treated patients, whereas all the other patients made an initial recovery. Of 166 patients monitored for ≥28 days, 35% had reappearance of vivax malaria 11 to 65 days later and 7% developed falciparum malaria 5 to 21 days after the start of treatment. There were no significant differences in the times taken for vivax malaria reappearance among the different groups except for those given mefloquine and chloroquine, in which all vivax malaria reappearances develo...

Blood smear-positive asymptomatic infections are an important
malaria parasite reservoir because they harbor gametocytes. However,
asymptomatic infections are often submicroscopic, can infect mosquitoes
and can only be detected by molecular methods. Malaria prevention
programs pursue mass treatment of asymptomatic individuals, which
may contribute to development and spread of drug resistance. Here, we
investigated the prevalence of molecular markers of drug resistance of
Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and P. falciparum
dihydropteroate synthase (Pfdhps) during subpatent parasitemia compared
to patent parasitemia episodes in Mali. From July to December 2019,
blood smears and filter paper blood spots were collected from children
with subpatent parasitemia (BS-) and patent parasitemia (BS+) in
Doneguebougou. Point mutations at codons (50, 51, 59, 108, 140 and
164) of the Pfdhfr gene and codons (431, 436, 437, 540, 581 and 613)
of the Pfdhps gene were evaluated by nested PCR amplification followed
by direct sequencing. A total of 84 children under five years of age were
evaluated (27 BS- and 57 BS+). When assessed as a binary endpoint
(any versus no detection of resistance allele), we found a significance
higher prevalence of Pfdhps 581G (51.9% vs. 12.3%, p=0.001) and
Pfdhfr at codons 50R (75.0% vs. 22.2%, p=0.001), 140L (66.7% vs.
25.0%, p=0.014) and 164L (88.9% vs. 21.2%, p=0.0004) in BS- vs. BS+,
respectively. In BS- the prevalence of Pfdhps at codons (431V, 437G and
540E) and Pfdhfr at codon (51I) were higher but not significant in BS- vs.
BS+. No Pfdhps 540E was detected in BS+. For all markers except one,
mutations were more prevalent in BS- samples in Mali. Asymptomatic
subpatent parasitemia can be a reservoir of drug-resistant parasites and
should be regularly surveyed (in addition to BS+ episodes) to accurately
monitor the spread of molecular markers of resistance.