Polymorphisms in Plasmodium falciparum dihydropteroate synthetase and dihydrofolate reductase genes in Nigerian children with uncomplicated malaria using high-resolution melting technique

AbstractIn 2005, the Nigerian Federal Ministry of Health revised the treatment policy for uncomplicated malaria with the introduction of artemisinin-based combination therapies (ACTs). This policy change discouraged the use of Sulphadoxine-pyrimethamine (SP) as the second-line treatment of uncomplicated falciparum malaria. However, SP is used as an intermittent preventive treatment of malaria in pregnancy (IPTp) and seasonal malaria chemoprevention (SMC) in children aged 3–59 months. There have been increasing reports of SP resistance especially in the non-pregnant population in Nigeria, thus, the need to continually monitor the efficacy of SP as IPTp and SMC by estimating polymorphisms in dihydropteroate synthetase (dhps) and dihydrofolate reductase (dhfr) genes associated with SP resistance. The high resolution-melting (HRM) assay was used to investigate polymorphisms in codons 51, 59, 108 and 164 of the dhfr gene and codons 437, 540, 581 and 613 of the dhps gene. DNA was extracted from 271 dried bloodspot filter paper samples obtained from children (< 5 years old) with uncomplicated malaria. The dhfr triple mutant I51R59N108, dhps double mutant G437G581 and quadruple dhfr I51R59N108 + dhps G437 mutant haplotypes were observed in 80.8%, 13.7% and 52.8% parasites, respectively. Although the quintuple dhfr I51R59N108 + dhps G437E540 and sextuple dhfr I51R59N108 + dhps G437E540G581 mutant haplotypes linked with in-vivo and in-vitro SP resistance were not detected, constant surveillance of these haplotypes should be done in the country to detect any change in prevalence.

Download Full-text

Low prevalence of highly sulfadoxine‐resistant dihydropteroate synthase alleles in Plasmodium falciparum isolates in Benin

Malaria Journal ◽

10.1186/s12936-021-03605-5 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Samaly Souza Svigel ◽

Adicath Adeothy ◽

Augustin Kpemasse ◽

Ernest Houngbo ◽

Antoine Sianou ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Intermittent Preventive Treatment ◽

Preventive Treatment ◽

Nucleotide Polymorphisms ◽

Triple Mutant ◽

Single Nucleotide ◽

Dihydropteroate Synthase ◽

Seasonal Malaria Chemoprevention ◽

Study Sites ◽

Low Prevalence

Abstract Background In 2004, in response to high levels of treatment failure associated with sulfadoxine-pyrimethamine (SP) resistance, Benin changed its first-line malaria treatment from SP to artemisinin-based combination therapy for treatment of uncomplicated Plasmodium falciparum malaria. Resistance to SP is conferred by accumulation of single nucleotide polymorphisms (SNPs) in P. falciparum genes involved in folate metabolism, dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps), targeted by pyrimethamine and sulfadoxine, respectively. Because SP is still used for intermittent preventive treatment in pregnant women (IPTp) and seasonal malaria chemoprevention (SMCP) in Benin, the prevalence of Pfdhfr and Pfdhps SNPs in P. falciparum isolates collected in 2017 were investigated. Methods This study was carried out in two sites where the transmission of P. falciparum malaria is hyper-endemic: Klouékanmey and Djougou. Blood samples were collected from 178 febrile children 6–59 months old with confirmed uncomplicated P. falciparum malaria and were genotyped for SNPs associated with SP resistance. Results The Pfdhfr triple mutant IRN (N51I, C59R, and S108N) was the most prevalent (84.6%) haplotype and was commonly found with the Pfdhps single mutant A437G (50.5%) or with the Pfdhps double mutant S436A and A437G (33.7%). The quintuple mutant, PfdhfrIRN/PfdhpsGE (A437G and K540E), was rarely observed (0.8%). The A581G and A613S mutant alleles were found in 2.6 and 3.9% of isolates, respectively. Six isolates (3.9%) were shown to harbour a mutation at codon I431V, recently identified in West African parasites. Conclusions This study showed that Pfdhfr triple IRN mutants are near fixation in this population and that the highly sulfadoxine-resistant Pfdhps alleles are not widespread in Benin. These data support the continued use of SP for chemoprevention in these study sites, which should be complemented by periodic nationwide molecular surveillance to detect emergence of resistant genotypes.

Download Full-text

Molecular Basis of In Vivo Resistance to Sulfadoxine-Pyrimethamine in African Adult Patients Infected withPlasmodium falciparum Malaria Parasites

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.42.7.1811 ◽

1998 ◽

Vol 42 (7) ◽

pp. 1811-1814 ◽

Cited By ~ 45

Author(s):

Leonardo K. Basco ◽

Rachida Tahar ◽

Pascal Ringwald

Keyword(s):

Dihydrofolate Reductase ◽

Point Mutations ◽

Gene Mutations ◽

Adult Patients ◽

Wild Type ◽

Dihydropteroate Synthase ◽

Reductase Gene ◽

Parasite Populations

ABSTRACT In vitro sulfadoxine and pyrimethamine resistance has been associated with point mutations in the dihydropteroate synthase and dihydrofolate reductase domains, respectively, but the in vivo relevance of these point mutations has not been well established. To analyze the correlation between genotype and phenotype, 10 Cameroonian adult patients were treated with sulfadoxine-pyrimethamine and followed up for 28 days. After losses to follow-up (n = 1) or elimination of DNA samples due to mixed parasite populations with pyrimethamine-sensitive and pyrimethamine-resistant profiles (n = 3), parasite genomic DNA from day 0 blood samples of six patients were analyzed by DNA sequencing. Three patients who were cured had isolates characterized by a wild-type or mutant dihydrofolate reductase gene (with one or two mutations) and a wild-type dihydropteroate synthase gene. Three other patients who failed to respond to sulfadoxine-pyrimethamine treatment carried isolates with triple dihydrofolate reductase gene mutations and either a wild-type or a mutant dihydropteroate synthase gene. Three dihydrofolate reductase gene codons (51, 59, and 108) may be reliable genetic markers that can accurately predict the clinical outcome of sulfadoxine-pyrimethamine treatment in Africa.

Download Full-text

Arabidopsis ADF5 Acts as a Downstream Target Gene of CBFs in Response to Low-Temperature Stress

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.635533 ◽

2021 ◽

Vol 9 ◽

Author(s):

Pan Zhang ◽

Dong Qian ◽

Changxin Luo ◽

Yingzhi Niu ◽

Tian Li ◽

...

Keyword(s):

Low Temperature ◽

Actin Cytoskeleton ◽

Temperature Stress ◽

Regulatory Element ◽

Low Temperature Stress ◽

Knockout Mutant ◽

Triple Mutant ◽

Freezing Resistance

Low temperature is a major adverse environment that affects normal plant growth. Previous reports showed that the actin cytoskeleton plays an important role in the plant response to low-temperature stress, but the regulatory mechanism of the actin cytoskeleton in this process is not clear. C-repeat binding factors (CBFs) are the key molecular switches for plants to adapt to cold stress. However, whether CBFs are involved in the regulation of the actin cytoskeleton has not been reported. We found that Arabidopsis actin depolymerizing factor 5 (ADF5), an ADF that evolved F-actin bundling function, was up-regulated at low temperatures. We also demonstrated that CBFs bound to the ADF5 promoter directly in vivo and in vitro. The cold-induced expression of ADF5 was significantly inhibited in the cbfs triple mutant. The freezing resistance of the adf5 knockout mutant was weaker than that of wild type (WT) with or without cold acclimation. After low-temperature treatment, the actin cytoskeleton of WT was relatively stable, but the actin cytoskeletons of adf5, cbfs, and adf5 cbfs were disturbed to varying degrees. Compared to WT, the endocytosis rate of the amphiphilic styryl dye FM4-64 in adf5, cbfs, and adf5 cbfs at low temperature was significantly reduced. In conclusion, CBFs directly combine with the CRT/DRE DNA regulatory element of the ADF5 promoter after low-temperature stress to transcriptionally activate the expression of ADF5; ADF5 further regulates the actin cytoskeleton dynamics to participate in the regulation of plant adaptation to a low-temperature environment.

Download Full-text

Germ Tubes and Proteinase Activity Contribute to Virulence of Candida albicans in Murine Peritonitis

Infection and Immunity ◽

10.1128/iai.67.12.6637-6642.1999 ◽

1999 ◽

Vol 67 (12) ◽

pp. 6637-6642 ◽

Cited By ~ 56

Author(s):

Marianne Kretschmar ◽

Bernhard Hube ◽

Thomas Bertsch ◽

Dominique Sanglard ◽

Renate Merker ◽

...

Keyword(s):

Candida Albicans ◽

Bowel Perforation ◽

Tube Length ◽

Triple Mutant ◽

Liver And Pancreas ◽

Organ Specific ◽

Secreted Aspartyl Proteinase ◽

Pepstatin A

ABSTRACT Peritonitis with Candida albicans is an important complication of bowel perforation and continuous ambulatory peritoneal dialysis. To define potential virulence factors, we investigated 50 strains of C. albicans in a murine peritonitis model. There was considerable variation in their virulence in this model when virulence was measured as release of organ-specific enzymes into the plasma of infected mice. Alanine aminotransferase (ALT) and α-amylase (AM) were used as parameters for damage of the liver and pancreas, respectively. The activities of ALT and AM in the plasma correlated with invasion into the organs measured in histologic sections and the median germ tube length induced with serum in vitro. When the activity of proteinases was inhibited in vivo with pepstatin A, there was a significant reduction of ALT and AM activities. This indicates that proteinases contributed to virulence in this model. Using strains ofC. albicans with disruption of secreted aspartyl proteinase gene SAP1, SAP2, SAP3, orSAP4 through SAP6 (collectively referred to asSAP4-6), we showed that only a Δsap4-6 triple mutant induced a significantly reduced activity of ALT in comparison to the reference strain. In contrast to the Δsap1, Δsap2, and Δsap3 mutants, the ALT induced by the Δsap4-6 mutant could not be further reduced by pepstatin A treatment, which indicates that Sap4-6 may contribute to virulence in this model.

Download Full-text

STABILIZATION OF DIHYDROFOLATE REDUCTASE BY INHIBITORS IN VIVO AND IN VITRO

Annals of the New York Academy of Sciences ◽

10.1111/j.1749-6632.1971.tb46972.x ◽

1971 ◽

Vol 186 (1 Folate Antago) ◽

pp. 187-205 ◽

Cited By ~ 4

Author(s):

Brian L. Hillcoat ◽

L. Marshall ◽

J. Gauldie ◽

M. Hiebert

Keyword(s):

Dihydrofolate Reductase

Download Full-text

In-Silico Analysis to Identify Potent Quinoline Analogues Against Multi-Targets of SARS-CoV-2

International Journal of Quantitative Structure-Property Relationships ◽

10.4018/ijqspr.2021100102 ◽

2021 ◽

Vol 6 (4) ◽

pp. 25-37

Author(s):

Shahanas Naisam ◽

Viji V.S. ◽

Suvanish Kumar ◽

Nidhin Sreekumar

Keyword(s):

Interaction Analysis ◽

In Silico Analysis ◽

Preventive Treatment ◽

Drug Molecule ◽

Current Outbreak ◽

In Vivo Analysis ◽

Effective Drugs ◽

Silico Analysis

In the current outbreak of COVID-19, various studies have been conducted all over the world to develop effective drugs against the virus. Recent studies have shown that hydroxychloroquine, chloroquine (antimalarial drugs), isoflavones, flavonoids, etc. have potent antiviral properties, and few have been proven as effective drugs for the preventive treatment of COVID-19. But their exact action against SARS-CoV-2 is still unknown. The strategy of this study is the virtual screening of quinoline analogues, design new ligand molecules, perform molecular interaction analysis, their MD validation against multi targets (Spike-ACE2, TMPRSS2, and Spike Protein) of SARS-CoV-2, and to suggest the most promising and effective drug molecule. Hydroxychloroquine and chloroquine were considered as the reference molecules in this study. A ligand N-[4-(3-Benzylideneazetidine-1-carbonyl)phenyl]quinoline-8-sulfonamide interacting with TMPRSS2 shows better interaction among the list even after MD validation. Further in-vitro and in-vivo analysis of this study is needed for future validation.

Download Full-text

From local to global matrix organization by fibroblasts: a 4D laser-assisted bioprinting approach

Biofabrication ◽

10.1088/1758-5090/ac40ed ◽

2021 ◽

Author(s):

Camille Douillet ◽

Marc Nicodeme ◽

Loïc Hermant ◽

Vanessa Bergeron ◽

Fabien Guillemot ◽

...

Keyword(s):

High Resolution ◽

Soft Tissue ◽

Dynamic Properties ◽

Unmet Need ◽

Specific Pattern ◽

Matrix Remodeling ◽

Collagen Gels ◽

Lattice Contraction

Abstract Fibroblasts and myofibroblasts play a central role in skin homeostasis through dermal organization and maintenance. Nonetheless, the dynamic interactions between (myo)fibroblasts and the extracellular matrix (ECM) remain poorly exploited in skin repair strategies. Indeed, there is still an unmet need for soft tissue models allowing to study the spatial-temporal remodeling properties of (myo)fibroblasts. In vivo, wound healing studies in animals are limited by species specificity. In vitro, most models rely on collagen gels reorganized by randomly distributed fibroblasts. But biofabrication technologies have significantly evolved over the past ten years. High-resolution bioprinting now allows to investigate various cellular micropatterns and the emergent tissue organizations over time. In order to harness the full dynamic properties of cells and active biomaterials, it is essential to consider “time” as the 4th dimension in soft tissue design. Following this 4D bioprinting approach, we aimed to develop a novel model that could replicate fibroblast dynamic remodeling in vitro. For this purpose, (myo)fibroblasts were patterned on collagen gels with laser-assisted bioprinting (LAB) to study the generated matrix deformations and reorganizations. First, distinct populations, mainly composed of fibroblasts or myofibroblasts, were established in vitro to account for the variety of fibroblastic remodeling properties. Then, LAB was used to organize both populations on collagen gels in even isotropic patterns with high resolution, high density and high viability. With maturation, bioprinted patterns of fibroblasts and myofibroblasts reorganized into dispersed or aggregated cells, respectively. Stress-release contraction assays revealed that these phenotype-specific pattern maturations were associated with distinct lattice tension states. The two populations were then patterned in anisotropic rows in order to direct the cell-generated deformations and to orient global matrix remodeling. Only maturation of anisotropic fibroblast patterns, but not myofibroblasts, resulted in collagen anisotropic reorganizations both at tissue-scale, with lattice contraction, and at microscale, with embedded microbead displacements. Following a 4D bioprinting approach, LAB patterning enabled to elicit and orient the dynamic matrix remodeling mechanisms of distinct fibroblastic populations and organizations on collagen. For future studies, this method provides a new versatile tool to investigate in vitro dermal organizations and properties, processes of remodeling in healing, and new treatment opportunities.

Download Full-text

Increased Prevalence of Mutant Allele Pfdhps 437G and Pfdhfr Triple Mutation in Plasmodium falciparum Isolates from a Rural Area of Gabon, Three Years after the Change of Malaria Treatment Policy

Malaria Research and Treatment ◽

10.1155/2016/9694372 ◽

2016 ◽

Vol 2016 ◽

pp. 1-6 ◽

Cited By ~ 2

Author(s):

Jacques-Mari Ndong Ngomo ◽

Denise Patricia Mawili-Mboumba ◽

Noé Patrick M’Bondoukwe ◽

Rosalie Nikiéma Ndong Ella ◽

Marielle Karine Bouyou Akotet

Keyword(s):

Plasmodium Falciparum ◽

Dihydrofolate Reductase ◽

Mutant Allele ◽

Continuous Monitoring ◽

Intermittent Preventive Treatment ◽

Point Mutations ◽

Preventive Treatment ◽

Malaria Treatment ◽

Pcr Rflp ◽

Elevated Frequency

In Gabon, sulfadoxine-pyrimethamine (SP) is recommended for intermittent preventive treatment during pregnancy (IPTp-SP) and for uncomplicated malaria treatment through ACTs drug. P. falciparum strains resistant to SP are frequent in areas where this drug is highly used and is associated with the occurrence of mutations on Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and dihydropteroate synthetase (Pfdhps) genes. The aim of the study was to compare the proportion of mutations on Pfdhfr and Pfdhps genes in isolates collected at Oyem in northern Gabon, in 2005 at the time of IPTp-SP introduction and three years later. Point mutations were analyzed by nested PCR-RFLP method. Among 91 isolates, more than 90% carried Pfdhfr 108N and Pfdhfr 59R alleles. Frequencies of Pfdhfr 51I (98%) and Pfdhps 437G (67.7%) mutant alleles were higher in 2008. Mutations at codons 164, 540, and 581 were not detected. The proportion of the triple Pfdhfr mutation and quadruple mutation including A437G was high: 91.9% in 2008 and 64.8% in 2008, respectively. The present study highlights an elevated frequency of Pfdhfr and Pfdhps mutant alleles, although quintuple mutations were not found in north Gabon. These data suggest the need of a continuous monitoring of SP resistance in Gabon.

Download Full-text

Antiherpesvirus Activity of the Combination of 5-iodo-2′-deoxycytidine with methotrexate: An in vitro and in vivo Study

Antiviral Chemistry and Chemotherapy ◽

10.1177/095632029400500501 ◽

1994 ◽

Vol 5 (5) ◽

pp. 283-289

Author(s):

C. Cremonesi ◽

C. Scarpini ◽

R. Bianchi ◽

A. Radaelli ◽

M. Gimelli ◽

...

Keyword(s):

Antiviral Activity ◽

Dihydrofolate Reductase ◽

Reductase Inhibitor ◽

Cellular Viability ◽

Cutaneous Lesions ◽

Guinea Pig Model ◽

Simplex Virus ◽

Hsv 1

We evaluated the in vitro and in vivo antiviral activity of the deoxyribonucleoside analogue 5-iodo-2′-deoxycytidine (IDC) combined with the dihydrofolate reductase inhibitor methotrexate (MTX) on herpes simplex virus types 1 and 2 (HSV-1, HSV-2). The IDC-MTX combination synergistically inhibited HSV-1 and HSV-2 replication in vitro at concentrations that did not reduce cellular viability and was very effective in reducing the severity of cutaneous lesions in the experimental guinea pig model in vivo. The antiviral activity of the IDC-MTX combination in guinea pigs was also compared with that of acyclovir and was demonstrated to be higher.

Download Full-text