This study investigated genetic mutations associated with chloroquine resistance in Plasmodium falciparum samples from the Brazilian Amazon region. The study analyzed 40 samples from 4 localities for mutations in the pfmdr1, cg2, and pfcrt genes. It found 100% of samples contained mutations in pfmdr1 codons 184, 1042, and the cg2 gamma region associated with chloroquine resistance. Most samples also contained the pfcrt K76T mutation, except some from Porto Velho which matched a Thai resistant genotype. This research contributes to understanding the molecular basis of widespread chloroquine resistance in this region.
This study examined 51 Brazilian Plasmodium falciparum isolates for polymorphisms in the Pfmdr1 gene thought to be associated with chloroquine resistance. 49 of the isolates were found to be resistant to chloroquine in vitro, while all were sensitive to mefloquine, amodiaquine, and quinine. The isolates were analyzed for three Pfmdr1 polymorphisms: Asn86Tyr, Asn1042Asp, and Asp1246Tyr. Asn86Tyr was not detected in any isolates, while Asn1042Asp was found in 50 isolates and Asp1246Tyr was found in all 51 isolates. This provides support that As
Yr10 encodes for stripe rust resistance in wheat. Two related genomic sequences, 4B and 4E, were isolated from Moro wheat. Sequence 4B corresponds to the Yr10 resistance gene and is located on chromosome 1B. Silencing of sequence 4B using VIGS rendered Moro wheat susceptible to stripe rust, demonstrating that 4B is responsible for Yr10-mediated resistance. Yr10 was the first CC-NBS-LRR resistance gene cloned from wheat.
The Lr34 gene in wheat has provided effective resistance against rust for over 100 years. It confers partial and durable resistance by prolonging the latency period and reducing spore production. Lr34 is not race-specific and also confers resistance to other pathogens. It encodes an ATP-binding cassette (ABC) transporter protein and differs by only two amino acids from susceptible alleles. Transgenic wheat with Lr34 shows complementation of resistance, though the genetic background can influence effects. Lr34's durable resistance is likely due to its ability to transport antimicrobial compounds or prime defense responses through its function as an ABC transporter.
The document summarizes research on pleiotropic adult plant resistance (PAPR) loci in wheat. Key points:
1. CIMMYT has conducted PAPR research since the 1970s, identifying loci such as Lr34, Lr46, and Lr67 that confer resistance to multiple diseases.
2. Studies mapped additional PAPR QTL in various wheat populations and identified markers for genes like Lr46, Sr2, and Yr54 useful for marker-assisted selection.
3. Research involves fine mapping genes, identifying deletion mutants, and understanding resistance mechanisms to improve durability and pyramide genes in wheat breeding.
4. An international shuttle breeding program
Isolation of genes differentially expressed during the defense response of Ca...CIAT
- The document describes research isolating genes differentially expressed in the cassava plant Manihot esculenta during defense response to attack by the whitefly Aleurotrachelus socialis.
- Researchers used subtractive cDNA libraries and microarray expression profiling to identify genes up- or down-regulated in a resistant cassava genotype compared to a susceptible one when infested with whiteflies.
- Over 550 genes were found to be significantly regulated during the cassava-whitefly interaction, including many involved in defense response, oxidative stress, and cell wall modification.
This document summarizes the discovery of duplicated VegfA and KDR receptor genes in zebrafish that mediate vascular development. Specifically:
- The researchers identified a duplicated zebrafish VegfA gene (VegfAb) that encodes 171- and 210-amino acid isoforms not found in the single VegfA gene.
- They also found a duplicated KDR receptor gene (Kdrb) that encodes a receptor similar to mammalian KDR.
- Knockdown experiments in zebrafish showed that both VegfAb and the duplicated KDR receptor genes play important roles in vascular development.
- Further experiments demonstrated that the VegfAb isoforms are poorly secreted compared to VegfA isoforms
Cloning and sequence analysis of banana streak virus dna. harper 1998Paloma Susan
Banana streak virus (BSV) causes severe problems for banana cultivation. The researchers cloned and sequenced the genome of a Nigerian isolate of BSV. The genome was found to be 7,389 base pairs and organized similarly to other badnaviruses, with three open reading frames. Comparison showed BSV is distinct from but closely related to sugarcane bacilliform virus. PCR primers designed from the sequence data detected BSV sequences in banana plants, indicating portions of the BSV genome may integrate into the banana genome. The BSV sequence provides a basis for more sensitive PCR-based detection methods.
The study characterized the Campylobacter jejuni IAL 2383 strain isolated from humans in Brazil. They found that the strain harbored important virulence genes and expressed major virulence factor transcripts. It grew better at 41°C than 37°C, indicating ability to colonize avian hosts. The strain was sensitive to most antibiotics tested and could serve as an experimental model for interactions with host cells and acquisition of antibiotic resistance.
This study examined 51 Brazilian Plasmodium falciparum isolates for polymorphisms in the Pfmdr1 gene thought to be associated with chloroquine resistance. 49 of the isolates were found to be resistant to chloroquine in vitro, while all were sensitive to mefloquine, amodiaquine, and quinine. The isolates were analyzed for three Pfmdr1 polymorphisms: Asn86Tyr, Asn1042Asp, and Asp1246Tyr. Asn86Tyr was not detected in any isolates, while Asn1042Asp was found in 50 isolates and Asp1246Tyr was found in all 51 isolates. This provides support that As
Yr10 encodes for stripe rust resistance in wheat. Two related genomic sequences, 4B and 4E, were isolated from Moro wheat. Sequence 4B corresponds to the Yr10 resistance gene and is located on chromosome 1B. Silencing of sequence 4B using VIGS rendered Moro wheat susceptible to stripe rust, demonstrating that 4B is responsible for Yr10-mediated resistance. Yr10 was the first CC-NBS-LRR resistance gene cloned from wheat.
The Lr34 gene in wheat has provided effective resistance against rust for over 100 years. It confers partial and durable resistance by prolonging the latency period and reducing spore production. Lr34 is not race-specific and also confers resistance to other pathogens. It encodes an ATP-binding cassette (ABC) transporter protein and differs by only two amino acids from susceptible alleles. Transgenic wheat with Lr34 shows complementation of resistance, though the genetic background can influence effects. Lr34's durable resistance is likely due to its ability to transport antimicrobial compounds or prime defense responses through its function as an ABC transporter.
The document summarizes research on pleiotropic adult plant resistance (PAPR) loci in wheat. Key points:
1. CIMMYT has conducted PAPR research since the 1970s, identifying loci such as Lr34, Lr46, and Lr67 that confer resistance to multiple diseases.
2. Studies mapped additional PAPR QTL in various wheat populations and identified markers for genes like Lr46, Sr2, and Yr54 useful for marker-assisted selection.
3. Research involves fine mapping genes, identifying deletion mutants, and understanding resistance mechanisms to improve durability and pyramide genes in wheat breeding.
4. An international shuttle breeding program
Isolation of genes differentially expressed during the defense response of Ca...CIAT
- The document describes research isolating genes differentially expressed in the cassava plant Manihot esculenta during defense response to attack by the whitefly Aleurotrachelus socialis.
- Researchers used subtractive cDNA libraries and microarray expression profiling to identify genes up- or down-regulated in a resistant cassava genotype compared to a susceptible one when infested with whiteflies.
- Over 550 genes were found to be significantly regulated during the cassava-whitefly interaction, including many involved in defense response, oxidative stress, and cell wall modification.
This document summarizes the discovery of duplicated VegfA and KDR receptor genes in zebrafish that mediate vascular development. Specifically:
- The researchers identified a duplicated zebrafish VegfA gene (VegfAb) that encodes 171- and 210-amino acid isoforms not found in the single VegfA gene.
- They also found a duplicated KDR receptor gene (Kdrb) that encodes a receptor similar to mammalian KDR.
- Knockdown experiments in zebrafish showed that both VegfAb and the duplicated KDR receptor genes play important roles in vascular development.
- Further experiments demonstrated that the VegfAb isoforms are poorly secreted compared to VegfA isoforms
Cloning and sequence analysis of banana streak virus dna. harper 1998Paloma Susan
Banana streak virus (BSV) causes severe problems for banana cultivation. The researchers cloned and sequenced the genome of a Nigerian isolate of BSV. The genome was found to be 7,389 base pairs and organized similarly to other badnaviruses, with three open reading frames. Comparison showed BSV is distinct from but closely related to sugarcane bacilliform virus. PCR primers designed from the sequence data detected BSV sequences in banana plants, indicating portions of the BSV genome may integrate into the banana genome. The BSV sequence provides a basis for more sensitive PCR-based detection methods.
The study characterized the Campylobacter jejuni IAL 2383 strain isolated from humans in Brazil. They found that the strain harbored important virulence genes and expressed major virulence factor transcripts. It grew better at 41°C than 37°C, indicating ability to colonize avian hosts. The strain was sensitive to most antibiotics tested and could serve as an experimental model for interactions with host cells and acquisition of antibiotic resistance.
Molecular genetic basis for complex flagellar antigen expression in a tripha...Yusriani Mangarengi
This document describes a study examining the molecular genetic basis for complex flagellar antigen expression in a triphasic strain of Salmonella rubislaw. The researchers discovered that this triphasic strain contains three flagellin genes - the normal chromosomal fliC and fljB genes, as well as a third locus (designated flpA) located on a large plasmid. The flpA gene encodes a type d flagellin. When the triphasic strain is grown with d antiserum, it loses the ability to express the type d flagellin due to deletion of part or all of the flpA gene on the plasmid. Thus, the presence of the plasmid-borne flpA gene provides
Pyramiding of bacterial blight resistance genes in riceawareswapnil1111
This document describes the materials and methods used to pyramid genes for resistance to bacterial blight in rice. It involves using four near-isogenic lines and their recurrent parents that contain different resistance genes. The plants were grown in fields and screened for resistance to different races of the bacterial blight pathogen. DNA markers linked to the resistance genes were used to select plants in the F2 and subsequent generations that were homozygous for multiple resistance genes, resulting in lines with different combinations of two or more genes conferring bacterial blight resistance. Southern analysis and PCR were used with the DNA markers to select targeted plants at different stages of the breeding program.
This master's dissertation aimed to demonstrate gene expression in Rat1 fibroblast cells transformed by EVI1 and the relationship between EVI1 levels and CAIII gene expression. Real-time PCR and western blotting showed higher CAIII gene and protein expression in Rat1neo cells compared to Rat15.6 cells, which overexpress EVI1. Luciferase assays also demonstrated higher activity in Rat1neo cells, indicating higher CAIII expression. Silencing CAIII in Rat1neo cells increased caspase 3 activity after hydrogen peroxide treatment, showing CAIII protects against apoptosis. The results suggest EVI1 overexpression represses CAIII expression, reducing protection against oxidative stress. Therefore, oxidative stress agents may selectively target cancer cells overexpressing
This study compared ELISA and PCR-ELISA techniques for detecting human Plasmodium parasites in Anopheles mosquitoes from the Amazon region of Brazil. The PCR-ELISA technique confirmed all positive and negative ELISA results but detected additional Plasmodium species in 5 of the 32 positive mosquitoes that were not detected by ELISA alone. The PCR-ELISA is more sensitive than ELISA for detecting human malaria parasites in mosquitoes.
This document reports on a study of the chromosome numbers and nuclear types of 44 species from 20 genera of Cymbidioid orchids occurring in Brazil. Chromosome numbers ranged from 2n=12 in Psygmorchis pusilla to approximately 2n=168 in Oncidium aff. flexuosum. Interphase nuclear types varied widely. Chromosome numbers observed included 2n=54 for subtribe Eulophiinae, 2n=44, 46, 92 for subtribe Cyrtopodiinae, 2n=54, approximately 108 for subtribe Catasetinae, 2n=52, approximately 96 for subtribe Zygopetalinae, 2n=40, 80 for subtribe Lycast
Activation of surrogate death receptor signalling triggers peroxynitrite depe...Saurabh Shekhar
includes information about cisplatin resistance cancer cells and their execution through peroxynitrite triggered apoptosis due to death signaling receptors basedon the findings of research article published in cell death and diseases.
Naturally acquired plasmodium knowlesi malaria in human, thailand[1]Prasit Chanarat
1) A 38-year-old Thai man contracted Plasmodium knowlesi malaria after spending time in a forested area of southern Thailand near the Thai-Myanmar border.
2) Microscopic examination of blood smears showed malaria parasites consistent with P. malariae. However, PCR and sequencing of the small subunit rRNA and mitochondrial cytochrome b genes confirmed the species as P. knowlesi.
3) This is the first reported case of naturally acquired P. knowlesi malaria in humans in Thailand, indicating that wild primate populations may serve as reservoirs for simian malaria parasites capable of infecting humans.
This document summarizes the complete genomic sequence of a novel betanucleorhabdovirus identified from a Cnidium officinale plant and tentatively named Cnidium virus 1 (CnV1). The genome of CnV1 was sequenced and found to be 13,994 nucleotides in length. BLAST searches showed CnV1 is most closely related to betanucleorhabdoviruses. Several viruses have previously been reported to infect C. officinale including two secoviruses and an alphaflexivirus. This represents the first report of a betanucleorhabdovirus infecting C. officinale.
This document describes research aimed at identifying a transcription factor that links activation of the synapsis checkpoint during meiosis in C. elegans to the apoptotic pathway. The researcher performed an RNAi screen of 18 candidate transcription factors in a synapsis checkpoint mutant background. This identified nhr-84 as a potential factor, as its knockdown reduced apoptosis levels. Further experiments showed that nhr-84 knockdown only reduced apoptosis in checkpoint mutants, not in wild-type, indicating it specifically promotes apoptosis in response to synapsis checkpoint activation. This suggests nhr-84 may be the transcription factor that links the synapsis checkpoint to apoptosis.
This proposal aims to develop an assay to screen for inhibitors of West Nile virus (WNV) protease. The assay will utilize a Gal4 fusion system where the WNV protease is inserted between the Gal4 DNA binding and activation domains. Protease cleavage of the fusion would prevent GFP expression, while inhibition would allow GFP expression. Preliminary data shows the mutated, non-cleavable fusion induces GFP, while the wild type fusion does not, demonstrating proof of concept. Stable cell lines will be generated expressing all assay components to enable high-throughput screening for WNV protease inhibitors.
C. trachoamtis detection and genotypings assayquint
This document describes the development of assays for amplifying, detecting, and genotyping Chlamydia trachomatis (Ct). It includes a Ct amplification assay using multiplex PCR, a Ct detection assay using a DNA enzyme immunoassay to detect conserved probes, and a Ct genotyping assay using reverse hybridization to identify Ct serovars. Clinical testing showed the assays had sensitivity comparable to other tests and allowed identification of multiple Ct infections and determination of serovar distribution patterns.
Plasmids are extrachromosomal DNA molecules found in bacteria that can replicate independently of the bacterial chromosome. They contain an origin of replication and may have genes for antibiotic resistance, virulence factors, or degradation of molecules. Plasmids can be classified based on their ability to integrate into chromosomes, copy number, ability to conjugate, and compatibility with other plasmids. Common plasmids used for cloning include pBR322, pUC, and pGEM3Z. These vectors contain antibiotic resistance genes and can be used to select for recombinant plasmids using antibiotic selection or blue/white screening of beta-galactosidase activity. pGEM3Z also allows for in vitro transcription of cloned genes.
Recombinant DNA technology uses restriction enzymes and DNA ligase to cut and join DNA from different species, generating recombinant molecules that are multiplied in host cells like bacteria. This allows mass production of human proteins like insulin and creation of genetically engineered crops with traits like insect or herbicide resistance. Restriction enzymes recognize short DNA sequences and cut DNA at or near these sites, leaving sticky or blunt ends. DNA can be cut, joined, and modified using various enzymes and techniques to recombine DNA from different sources. Gel electrophoresis is used to separate and analyze DNA fragments by size.
This document discusses advances in molecular techniques for identifying Phytophthora, Pythium, and related genera. It describes the challenges of morphological identification and outlines desired characteristics for molecular markers. Several nuclear and mitochondrial molecular loci used for species identification are discussed, including rDNA, Î2-tubulin, elicitin genes, and mitochondrial genes like cox1 and cox2. Techniques for molecular identification of isolates and subpopulations are summarized, such as DNA sequencing, PCR-RFLP, and development of species-specific PCR for pathogen detection.
Human Leukocyte Antigen (HLA) typing involves determining the presence of HLA antigens on white blood cells. HLA antigens are encoded by genes in the major histocompatibility complex located on chromosome 6. HLA typing was originally done using serology to detect antibodies binding to HLA antigens, but now molecular techniques are more commonly used. HLA antigens are highly polymorphic and inherited as haplotypes from each parent, contributing to diversity in transplantation compatibility.
This study characterized 10 novel human monoclonal antibodies (Mabs) against the Plasmodium falciparum protein CelTOS, which facilitates parasite invasion of hepatocytes. Western blot, ELISA, dot blot, and immunofluorescence assays were used to test antibody binding and specificity. The results showed that Mabs 907 and 909 strongly bound the C- and N-termini, respectively, of PfCelTOS. Fine mapping located 907's epitope to peptide 4 of the C-terminus and 909's to peptide 1 of the N-terminus. None of the Mabs cross-reacted with other Plasmodium species. Mabs 907, 916, 920 and 957 recognized
1) The study investigated using PCR-RFLP analysis of mitochondrial 16S rRNA and cytochrome b genes to distinguish between 7 species of clown anemonefish.
2) Restriction digestion of the 16S rRNA fragment yielded 3 patterns that separated the genus Premnas from Amphiprion species.
3) Restriction digestion of the cytochrome b fragment produced patterns that uniquely identified each of the 7 anemonefish species, providing an accurate identification method.
L'identification du rôle principal du gène pfcrt dans le mécanisme de chloroquino-résistance chez Plasmodium falciparum - Conférence de la 2e édition du Cours international « Atelier Paludisme » - FIDOCK David - Albert Einstein College of Medicine - USA - dfidock@aecom.yu.edu
Molecular genetic basis for complex flagellar antigen expression in a tripha...Yusriani Mangarengi
This document describes a study examining the molecular genetic basis for complex flagellar antigen expression in a triphasic strain of Salmonella rubislaw. The researchers discovered that this triphasic strain contains three flagellin genes - the normal chromosomal fliC and fljB genes, as well as a third locus (designated flpA) located on a large plasmid. The flpA gene encodes a type d flagellin. When the triphasic strain is grown with d antiserum, it loses the ability to express the type d flagellin due to deletion of part or all of the flpA gene on the plasmid. Thus, the presence of the plasmid-borne flpA gene provides
Pyramiding of bacterial blight resistance genes in riceawareswapnil1111
This document describes the materials and methods used to pyramid genes for resistance to bacterial blight in rice. It involves using four near-isogenic lines and their recurrent parents that contain different resistance genes. The plants were grown in fields and screened for resistance to different races of the bacterial blight pathogen. DNA markers linked to the resistance genes were used to select plants in the F2 and subsequent generations that were homozygous for multiple resistance genes, resulting in lines with different combinations of two or more genes conferring bacterial blight resistance. Southern analysis and PCR were used with the DNA markers to select targeted plants at different stages of the breeding program.
This master's dissertation aimed to demonstrate gene expression in Rat1 fibroblast cells transformed by EVI1 and the relationship between EVI1 levels and CAIII gene expression. Real-time PCR and western blotting showed higher CAIII gene and protein expression in Rat1neo cells compared to Rat15.6 cells, which overexpress EVI1. Luciferase assays also demonstrated higher activity in Rat1neo cells, indicating higher CAIII expression. Silencing CAIII in Rat1neo cells increased caspase 3 activity after hydrogen peroxide treatment, showing CAIII protects against apoptosis. The results suggest EVI1 overexpression represses CAIII expression, reducing protection against oxidative stress. Therefore, oxidative stress agents may selectively target cancer cells overexpressing
This study compared ELISA and PCR-ELISA techniques for detecting human Plasmodium parasites in Anopheles mosquitoes from the Amazon region of Brazil. The PCR-ELISA technique confirmed all positive and negative ELISA results but detected additional Plasmodium species in 5 of the 32 positive mosquitoes that were not detected by ELISA alone. The PCR-ELISA is more sensitive than ELISA for detecting human malaria parasites in mosquitoes.
This document reports on a study of the chromosome numbers and nuclear types of 44 species from 20 genera of Cymbidioid orchids occurring in Brazil. Chromosome numbers ranged from 2n=12 in Psygmorchis pusilla to approximately 2n=168 in Oncidium aff. flexuosum. Interphase nuclear types varied widely. Chromosome numbers observed included 2n=54 for subtribe Eulophiinae, 2n=44, 46, 92 for subtribe Cyrtopodiinae, 2n=54, approximately 108 for subtribe Catasetinae, 2n=52, approximately 96 for subtribe Zygopetalinae, 2n=40, 80 for subtribe Lycast
Activation of surrogate death receptor signalling triggers peroxynitrite depe...Saurabh Shekhar
includes information about cisplatin resistance cancer cells and their execution through peroxynitrite triggered apoptosis due to death signaling receptors basedon the findings of research article published in cell death and diseases.
Naturally acquired plasmodium knowlesi malaria in human, thailand[1]Prasit Chanarat
1) A 38-year-old Thai man contracted Plasmodium knowlesi malaria after spending time in a forested area of southern Thailand near the Thai-Myanmar border.
2) Microscopic examination of blood smears showed malaria parasites consistent with P. malariae. However, PCR and sequencing of the small subunit rRNA and mitochondrial cytochrome b genes confirmed the species as P. knowlesi.
3) This is the first reported case of naturally acquired P. knowlesi malaria in humans in Thailand, indicating that wild primate populations may serve as reservoirs for simian malaria parasites capable of infecting humans.
This document summarizes the complete genomic sequence of a novel betanucleorhabdovirus identified from a Cnidium officinale plant and tentatively named Cnidium virus 1 (CnV1). The genome of CnV1 was sequenced and found to be 13,994 nucleotides in length. BLAST searches showed CnV1 is most closely related to betanucleorhabdoviruses. Several viruses have previously been reported to infect C. officinale including two secoviruses and an alphaflexivirus. This represents the first report of a betanucleorhabdovirus infecting C. officinale.
This document describes research aimed at identifying a transcription factor that links activation of the synapsis checkpoint during meiosis in C. elegans to the apoptotic pathway. The researcher performed an RNAi screen of 18 candidate transcription factors in a synapsis checkpoint mutant background. This identified nhr-84 as a potential factor, as its knockdown reduced apoptosis levels. Further experiments showed that nhr-84 knockdown only reduced apoptosis in checkpoint mutants, not in wild-type, indicating it specifically promotes apoptosis in response to synapsis checkpoint activation. This suggests nhr-84 may be the transcription factor that links the synapsis checkpoint to apoptosis.
This proposal aims to develop an assay to screen for inhibitors of West Nile virus (WNV) protease. The assay will utilize a Gal4 fusion system where the WNV protease is inserted between the Gal4 DNA binding and activation domains. Protease cleavage of the fusion would prevent GFP expression, while inhibition would allow GFP expression. Preliminary data shows the mutated, non-cleavable fusion induces GFP, while the wild type fusion does not, demonstrating proof of concept. Stable cell lines will be generated expressing all assay components to enable high-throughput screening for WNV protease inhibitors.
C. trachoamtis detection and genotypings assayquint
This document describes the development of assays for amplifying, detecting, and genotyping Chlamydia trachomatis (Ct). It includes a Ct amplification assay using multiplex PCR, a Ct detection assay using a DNA enzyme immunoassay to detect conserved probes, and a Ct genotyping assay using reverse hybridization to identify Ct serovars. Clinical testing showed the assays had sensitivity comparable to other tests and allowed identification of multiple Ct infections and determination of serovar distribution patterns.
Plasmids are extrachromosomal DNA molecules found in bacteria that can replicate independently of the bacterial chromosome. They contain an origin of replication and may have genes for antibiotic resistance, virulence factors, or degradation of molecules. Plasmids can be classified based on their ability to integrate into chromosomes, copy number, ability to conjugate, and compatibility with other plasmids. Common plasmids used for cloning include pBR322, pUC, and pGEM3Z. These vectors contain antibiotic resistance genes and can be used to select for recombinant plasmids using antibiotic selection or blue/white screening of beta-galactosidase activity. pGEM3Z also allows for in vitro transcription of cloned genes.
Recombinant DNA technology uses restriction enzymes and DNA ligase to cut and join DNA from different species, generating recombinant molecules that are multiplied in host cells like bacteria. This allows mass production of human proteins like insulin and creation of genetically engineered crops with traits like insect or herbicide resistance. Restriction enzymes recognize short DNA sequences and cut DNA at or near these sites, leaving sticky or blunt ends. DNA can be cut, joined, and modified using various enzymes and techniques to recombine DNA from different sources. Gel electrophoresis is used to separate and analyze DNA fragments by size.
This document discusses advances in molecular techniques for identifying Phytophthora, Pythium, and related genera. It describes the challenges of morphological identification and outlines desired characteristics for molecular markers. Several nuclear and mitochondrial molecular loci used for species identification are discussed, including rDNA, Î2-tubulin, elicitin genes, and mitochondrial genes like cox1 and cox2. Techniques for molecular identification of isolates and subpopulations are summarized, such as DNA sequencing, PCR-RFLP, and development of species-specific PCR for pathogen detection.
Human Leukocyte Antigen (HLA) typing involves determining the presence of HLA antigens on white blood cells. HLA antigens are encoded by genes in the major histocompatibility complex located on chromosome 6. HLA typing was originally done using serology to detect antibodies binding to HLA antigens, but now molecular techniques are more commonly used. HLA antigens are highly polymorphic and inherited as haplotypes from each parent, contributing to diversity in transplantation compatibility.
This study characterized 10 novel human monoclonal antibodies (Mabs) against the Plasmodium falciparum protein CelTOS, which facilitates parasite invasion of hepatocytes. Western blot, ELISA, dot blot, and immunofluorescence assays were used to test antibody binding and specificity. The results showed that Mabs 907 and 909 strongly bound the C- and N-termini, respectively, of PfCelTOS. Fine mapping located 907's epitope to peptide 4 of the C-terminus and 909's to peptide 1 of the N-terminus. None of the Mabs cross-reacted with other Plasmodium species. Mabs 907, 916, 920 and 957 recognized
1) The study investigated using PCR-RFLP analysis of mitochondrial 16S rRNA and cytochrome b genes to distinguish between 7 species of clown anemonefish.
2) Restriction digestion of the 16S rRNA fragment yielded 3 patterns that separated the genus Premnas from Amphiprion species.
3) Restriction digestion of the cytochrome b fragment produced patterns that uniquely identified each of the 7 anemonefish species, providing an accurate identification method.
L'identification du rôle principal du gène pfcrt dans le mécanisme de chloroquino-résistance chez Plasmodium falciparum - Conférence de la 2e édition du Cours international « Atelier Paludisme » - FIDOCK David - Albert Einstein College of Medicine - USA - dfidock@aecom.yu.edu
P-glycoprotein is an ATP-binding cassette transporter that protects the body by pumping various molecules, including drugs and toxins, out of cells and preventing their accumulation. It is located on the membranes of excretory organs like the liver, kidneys, and intestines. P-gp recognizes substrates in the cell and uses ATP hydrolysis to flip them out through a central pore. Its physiological role and ability to efflux various drugs has important implications for pharmacokinetics and clinical outcomes like resistance to cancer treatments. Genetic polymorphisms in P-gp may also influence the effectiveness of drugs it transports like HIV protease inhibitors.
The document provides information on the management of chloroquine resistant malaria. It discusses the life cycle of malaria parasites, various antimalarial drugs including their mechanisms of action and treatment of chloroquine sensitive and resistant malaria. It summarizes that malaria is caused by Plasmodium parasites and transmitted by Anopheles mosquitoes. It affects over 500 million people annually, especially children in developing countries. Resistance to chloroquine, previously the first-line treatment, has emerged and led to the use of alternative antimalarial drugs.
The document summarizes the classification, life cycle, and key characteristics of Plasmodium, the malarial parasite. It belongs to the phylum Protozoa. It has a complex life cycle involving two hosts - humans and female Anopheles mosquitoes. In humans, it replicates asexually in the liver and blood, causing the symptoms of malaria. In mosquitoes, it undergoes sexual reproduction resulting in sporozoites that can infect other humans and continue the life cycle. P. falciparum is the most lethal species and can cause severe complications like cerebral malaria if left untreated.
Plasmodium is a protozoan parasite that causes malaria in humans. It has a complex life cycle involving two hosts. In humans, it infects liver cells and red blood cells in an asexual reproductive cycle. In mosquitoes, it undergoes sexual reproduction resulting in sporozoites that can infect other humans and continue the cycle. The four main Plasmodium species that infect humans are P. vivax, P. falciparum, P. malariae, and P. ovale, with P. falciparum being the most lethal.
Malaria is caused by Plasmodium parasites and transmitted via mosquito bites. It affects 500 million people annually and kills over 1 million. The lifecycle involves two stages: 1) A sexual phase in mosquitoes where sporozoites develop. 2) An asexual phase in humans where the sporozoites infect the liver and then red blood cells, undergoing schizogony to produce merozoites that infect more cells, until some differentiate into transmissible gametocytes.
This document discusses antimalarial drugs and their classification, mechanisms of action, and therapeutic uses. It begins by identifying the four main Plasmodium species that infect humans. It then covers individual drugs like chloroquine, primaquine, mefloquine, and artemisinin derivatives. It classifies drugs based on their therapeutic effects and chemical structures. Key points include how each drug works against the malaria parasite, their pharmacokinetics, adverse effects, and indications. Artemisinin-based combination therapy is highlighted as the recommended treatment for acute uncomplicated malaria.
This document defines extraction as the removal of soluble constituents from a solid or liquid with a suitable solvent. It discusses various types of extraction including solid-liquid extraction, liquid-liquid extraction, and expression. Key terms like menstruum, marc, and extractives are defined. Important solvents for extraction like water, alcohol, ether, and chloroform are described. The document also covers the theory of extraction and importance of extraction in quantitative control of drugs and producing more stable, palatable forms.
The document discusses various general methods used for the isolation and separation of plant constituents, including extraction processes, separation techniques, and analytical methods. Extraction methods covered include maceration, infusion, digestion, decoction, percolation, soxhlet extraction, ultrasound extraction, and supercritical fluid extraction. Separation techniques include fractional crystallization, fractional distillation, thin layer chromatography, column chromatography, and paper chromatography. Analytical methods for identification discussed are gas chromatography, high performance liquid chromatography, and qualitative chemical reactions.
This study evaluated the distribution of Plasmodium vivax variants (VK210, VK247, and P. vivax-like) in three areas of Brazil using a new GFM-PCR-ELISA technique. All variants were found in all three areas. VK210 was most commonly found as a single infection while the others occurred in mixed infections. VK210 was associated with the highest parasitemia levels while P. vivax-like had the lowest. Parasitemia clearance times did not differ based on variant or treatment schedule. The new technique was accurate for epidemiological surveys of the vivax complex.
This document analyzes genetic variation at the Pfs48/45 gene and microsatellite loci in 255 Plasmodium falciparum isolates from 5 populations. It finds:
1) Alleles and haplotypes of 5 SNPs in the Pfs48/45 gene varied extremely between populations, much more so than alleles at 11 neutral microsatellite loci.
2) Measurements of between-population allele frequency variation (FST) were 4-7 times higher for Pfs48/45 than microsatellites, both within and between continents.
3) The highly skewed Pfs48/45 patterns suggest divergent selection on the protein's amino acid sequence between populations, indicating it may determine game
This document discusses molecular approaches for studying Fasciola species. It begins by introducing molecular methods like PCR that are useful for species identification and population genetics studies by targeting divergent genes. It then discusses Fascioliasis, caused by the liver flukes Fasciola hepatica, F. gigantica, and F. jaksoni. Common molecular markers for Fasciola species identification include mitochondrial DNA and ribosomal DNA. Specific PCR assays have been developed to differentiate Fasciola species using markers like ITS-2 sequences. The document concludes that molecular genetics studies have improved understanding of Fasciola taxonomy and enabled accurate identification of species.
Presentation 6: Vibrio parahaemolyticus: genome plasticity, mobile genetic el...ExternalEvents
http://www.fao.org/documents/card/en/c/28b6bd62-5433-4fad-b5a1-8ac61eb671b1/
International Technical Seminar/Workshops on Acute hepatopancreatic necrosis disease (AHPND)
This document describes the development of a PCR-RFLP assay to identify Plasmodium species and variants of P. vivax infecting Anopheles mosquitoes. Specific primers were designed that target regions of the circumsporozoite gene to distinguish P. falciparum, P. malariae, and P. vivax variants VK210, VK247, and P. vivax-like. The assay was tested on artificially infected mosquitoes and showed good agreement with nested PCR. The PCR-RFLP method provides a sensitive way to detect Plasmodium species and variants, which can help understand malaria transmission dynamics.
This study aimed to detect Mycoplasma gallisepticum (MG) strains in chicken flocks in Egypt with respiratory disease. Nasal swabs from 49 flocks were tested by semi-nested PCR and restriction fragment length polymorphism (RFLP) of the pvpA gene. MG was detected in 37 flocks (75.5% incidence). RFLP analysis identified genetic similarity between some field isolates and the F vaccine strain. Experimental infection of chickens found that field isolates and the F strain were transmitted between infected and contact birds based on PCR testing, with some contact birds developing antibodies. This highlights the need for improved diagnostics and control of MG variant strains.
This document summarizes information about malaria vaccines. It discusses how malaria is caused by Plasmodium parasites and transmitted by mosquitoes. Four species can infect humans. Current vaccines target different stages of the parasite's life cycle, including pre-erythrocytic, blood, and sexual stages. Challenges to vaccine development include the parasite's ability to evade the immune system through antigenic variation. Several candidate vaccines are discussed that target different stages, but none have achieved high levels of efficacy and durability.
This study evaluated antibody responses to the Pv200L fragment of Plasmodium vivax merozoite surface protein-1 (PvMSP-1) in individuals from 4 malaria-endemic regions in Brazil. Plasma samples from 261 P. vivax infected individuals were tested for antibodies to Pv200L by ELISA. The frequency of antibody responders ranged from 71.9-98.7% between regions and correlated with malaria transmission intensity. Higher antibody levels were also associated with greater past exposure to malaria parasites. Results provide evidence that Pv200L elicits naturally acquired antibodies and could be a potential vaccine candidate.
RAPD is commonly used to genotype organisms but has disadvantages like poor reproducibility. Newer techniques like gene-targeted sequencing and rRNA intergenic spacer region sequencing appear more sensitive and reproducible. The document reports using RAPD to genotype 7 recent Mycoplasma gallisepticum isolates from house finches. Results showed the 7 isolates were similar to a known house finch strain, supporting the hypothesis that the outbreak was caused by a single strain introduction.
1) The study evaluated the performance of the OptiMal malaria rapid diagnostic test under different storage conditions of 25°C, 30°C, and 39°C for 24, 48, and 72 hours.
2) The test detected all 111 positive blood samples except for 2 low parasitemia Plasmodium malariae samples.
3) The study suggests that the OptiMal test can be used for malaria diagnosis in Brazilian regions, though further research is needed to evaluate its performance under different environmental conditions like humidity.
This study investigated the relationship between variants of the glycophorin B (GPB) gene and susceptibility to Plasmodium falciparum infection in the Brazilian Amazon. The researchers found that individuals carrying the GYPB*S allele were more likely to be infected with P. falciparum than those without this allele. Sequence analysis of GYPB also suggested it has been under natural selection due to malaria. This study provides evidence that genetic variation in GPB receptor influences the ability of P. falciparum to invade red blood cells in this population.
This study investigated the relationship between variants of the glycophorin B (GPB) gene and susceptibility to Plasmodium falciparum infection in the Brazilian Amazon. The researchers found that individuals carrying the GYPB*S allele were more likely to be infected with P. falciparum than those without this allele. Additionally, population genetics analysis suggested that natural selection has shaped patterns of genetic diversity at the GYPB locus. This study provides evidence that genetic variation in GPB influences susceptibility to P. falciparum infection in this population.
This study examined how aging affects AICD gene expression in zebrafish. The researchers analyzed AICD RNA levels in the gut tissue of 5-, 11-, and 13-month old zebrafish using qPCR and found no significant difference, indicating that AICD expression is not altered during this period. They also found that changing the zebrafish housing conditions did not affect AICD levels. Future work will involve analyzing AICD and Tcf3a gene expression in 20-month old zebrafish to further study the effects of aging, requiring the isolation of B-cells from the zebrafish.
The document discusses malaria, which is caused by Plasmodium parasites transmitted via mosquito bites. It outlines the life cycle and species of Plasmodium that cause malaria in humans. The presentation summarizes techniques used to study malaria parasites in vitro, including culturing isolates, examining blood smears, and assessing drug sensitivity. Genetic analysis of field isolates by PCR targeting msp1 and msp2 genes showed changes in alleles present over time in culture. In vitro culture allows studying parasite growth and drug resistance profiles.
Malaria due to Plasmodium falciparum is a major public health concern in Cameroon and Africa, responsible for high rates of morbidity and mortality. While control measures have been effective, an effective vaccine is needed to achieve global malaria elimination goals. This study analyzed genetic diversity in the merozoite surface protein 1 (msp1) gene of P. falciparum isolates from Mount Cameroon. It found 45 distinct DNA fragments in samples from 81 of 173 infected individuals, indicating significant intragenic recombination contributing to parasite diversity. Understanding this diversity is important for vaccine development against P. falciparum.
This work package aims to understand genome evolution in Phytophthora by analyzing UK diversity, origins of emerging strains, and mechanisms of adaptation such as hybridization and horizontal gene transfer. The researchers will sequence genomes to understand how Phytophthora adapts to new hosts, woody hosts, and the role of hybridization and gene transfer in evolution.
International Journal of Pharmaceutical Science Invention (IJPSI) is an international journal intended for professionals and researchers in all fields of Pahrmaceutical Science. IJPSI publishes research articles and reviews within the whole field Pharmacy and Pharmaceutical Science, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online.
Characteristics of salmonella spp. isolated from wild birds confiscated in il...racheltrans
1) Salmonella was isolated from 3 of 109 wild birds confiscated from illegal wildlife trade in Rio de Janeiro, Brazil, including one strain of Salmonella Typhimurium and two strains of Salmonella Panama.
2) All Salmonella isolates showed resistance to multiple antimicrobial drugs. PFGE analysis found 100% similarity between the Salmonella Typhimurium strain isolated from a bird and strains from a human outbreak in southern Brazil, indicating potential spread between wildlife and humans.
3) The two Salmonella Panama strains isolated from birds in the same catch showed identical genetic fingerprints, suggesting a common source of infection. However, these strains did not match any isolates in reference databases.
1. The study characterized antibody responses to Plasmodium falciparum invasion ligands EBA-140 and EBA-181 in individuals from malaria-endemic areas of Brazil and Cameroon.
2. Responses differed between populations, with African individuals strongly reacting to most EBA fragments while Brazilian individuals from Mato Grosso reacted weakly and those from the Amazon had elevated but lower responses than Africans.
3. When compared to responses against other malaria proteins, the Brazilian population appeared to have more variable ability to recognize P. falciparum invasion ligands, distinct from the African population.
This document analyzes the frequency of two polymorphisms (C282Y and H63D) in the HFE gene in malaria patients and blood donors from four states in the Brazilian Amazon region. The study found:
1) No individuals were homozygous for the C282Y polymorphism, and only 5 heterozygous individuals were detected, all from Pará State.
2) The most common genotype for the H63D polymorphism was heterozygous in both patient groups.
3) Allele frequencies for the H63D polymorphism were higher than for the C282Y polymorphism, consistent with other studies on Brazilian populations.
This document summarizes a study that investigated the influence of HLA-DRB1 alleles on the production of antibodies against malaria antigens in individuals naturally infected with Plasmodium vivax in Brazil. The study found an association between HLA-DR3 and HLA-DR5 alleles and lack of antibody response to the amino terminal region of the circumsporozoite protein (CSP). It also found an association between HLA-DR3 and higher antibody response to merozoite surface protein 1 (MSP1). No significant associations were found between HLA-DRB1 alleles and antibody responses to other malaria antigens tested.
1. Researchers studied Plasmodium falciparum field isolates from Colombia, Peru, and Brazil to determine the invasion pathways and ligand polymorphisms used.
2. They found that most isolates from Colombia and Peru invade red blood cells through an atypical pathway that is resistant to all enzyme treatments, unlike known pathways.
3. This unusual pathway was associated with specific variants of PfRh2a, PfRh5, and EBA-181 ligands, suggesting these proteins play a major role in this pathway. The study demonstrates diversity in invasion mechanisms between regions.
This study evaluated four recombinant proteins representing the 19 kDa C-terminal region of the Plasmodium vivax merozoite surface protein-1 (MSP119) for detecting P. vivax antibodies in human serum samples. The sensitivity of an ELISA using the recombinant proteins to detect antibodies in 200 samples from individuals with P. vivax infection ranged from 90-93.5%. Specificity using control samples without P. vivax exposure was 98.3-100%. The study demonstrates the potential of an ELISA using recombinant MSP119 proteins for serological detection of P. vivax infection.
1) O documento analisa os casos de malária no estado de Santa Catarina entre 1996-2001, com 5,5% das 4.707 amostras sendo positivas.
2) Plasmodium vivax causou 69% dos casos, Plasmodium falciparum 25,6%, infecções mistas 5% e Plasmodium malariae 0,4%.
3) 67,4% dos casos foram importados e 32,6% autóctones, com aumento de casos importados nos anos subsequentes.
This study analyzed genetic diversity and population structure of Plasmodium falciparum in 5 populations in the Brazilian Amazon region. Microsatellite markers were analyzed in 196 parasite isolates. There was significant multilocus linkage disequificance within populations, particularly those with fewer mixed infections. However, most isolates had unique multilocus genotypes, indicating genetic diversity. Genetic divergence between populations was substantial but did not correlate simply with geographical distance. The findings suggest distinct population structures and minimal gene flow between foci in the region.
This study evaluated the frequency of asymptomatic Plasmodium carriers (APCs) among blood donors in four blood banks in the Brazilian Amazon region. Blood samples from 400 donors who passed screening were tested using PCR to detect Plasmodium DNA. The positivity rate varied from 1-3% between blood banks, with an overall rate of 2.3%. All positive samples contained mixed infections of P. vivax and P. falciparum. While screening methods used by the blood banks did not detect the infections, PCR revealed its superiority for detecting low levels of parasites. The results emphasize the need to improve screening for APCs in blood banks in malaria endemic areas to control transfusion-transmitted malaria.
1. The study developed a new PCR/RFLP technique to identify the 3 genotypes of Plasmodium vivax circumsporozoite protein (VK210, VK247, and P. vivax-like) using DNA extracted from blood samples.
2. The technique uses PCR amplification of the central immunodominant region of the CSP gene followed by restriction enzyme digestion and fragment analysis to distinguish the genotypes.
3. Testing demonstrated the technique could accurately identify the genotypes using plasmid controls for each variant, and that it had high sensitivity detecting parasitemia levels as low as 0.0069 parasites per microliter.
We present evidence of Plasmodium vivax infection in two homozygous FY*B-33 (Duffy-negative) individuals from the Brazilian Amazon region. Molecular analysis confirmed P. vivax infection through detection of P. vivax small subunit rRNA and circumsporozoite protein genes. One individual had a mixed infection of VK210 and P. vivax-like genotypes, while the other was infected with VK210. Both individuals also had antibodies to the P. vivax merozoite surface protein 1. This provides rare evidence that P. vivax may be capable of invading Duffy-negative red blood cells in some regions, though additional studies are needed to better understand this finding.
This document summarizes a study that analyzed Duffy blood group genotypes in Plasmodium vivax malaria patients and blood donors in four areas of the Brazilian Amazon region. The study found:
1) A high frequency of the FYA/FYB genotype in both patients and donors, followed by FYB/FYB.
2) Some Duffy genotypes showed significant differences in frequency between patients and donors.
3) Individuals with the FYA/FYB genotype may have higher susceptibility to malaria, while the presence of the FYB-33 allele could provide resistance.
Mixed Plasmodium falciparum infections were common in four areas of the Brazilian Amazon region. Molecular diagnosis found 73% of infections were single P. falciparum infections, while 27% were mixed infections, mostly double infections. Mixed infections were associated with weaker clinical malaria symptoms like lower rates of fever and headache compared to single P. falciparum infections. The study highlights the need for improved malaria diagnosis to better understand mixed infections and their impact on disease severity.
This study aimed to investigate the presence of arboviruses like dengue virus in clinical samples from 111 malaria patients in the Amazon region of Brazil.
Dengue virus serotype 2 was detected in two patients from Novo Repartimento, Pará who also had active Plasmodium vivax malaria infections. Despite limited data, concurrent dengue and malaria infections are likely more common in the Amazon region than detected, as both diseases co-circulate and have similar transmission vectors and clinical presentations, making dual diagnosis possible. Further research is needed to better understand the frequency of co-infection in this region.
This study investigated the frequencies of ABO blood group genotypes and alleles in Plasmodium falciparum malaria patients and blood donors from the Brazilian Amazon region. The researchers found that over half of individuals had the ABO*O01O01 genotype. The ABO*AO01 genotype was the second most common. No significant differences were detected in genotype or allele frequencies between the malaria patients and blood donors. Analysis of O alleles found the O1 variant allele to be most frequent in both groups, with no evidence of the homozygous O2 allele.
1. A study analyzed genetic and immune response differences between P. vivax circumsporozoite (CS) genotypes VK210 and P. vivax-like.
2. Phylogenetic analysis of 18S rRNA and CytB genes found high similarity between the genotypes, with zero nucleotide diversity, placing them in the same clade.
3. Individuals infected with P. vivax-like had a lower antibody response against CS repetitive region peptides than those with VK210, suggesting variation is limited to the CS repetitive region.
This document summarizes a study that investigated the influence of HLA-DRB1 alleles on the production of antibodies against malaria antigens in individuals naturally infected with Plasmodium vivax in Brazil. The study found an association between HLA-DR3 and HLA-DR5 alleles and lack of antibody response to the amino terminal region of the circumsporozoite protein (CSP). It also found an association between HLA-DR3 and higher antibody response to merozoite surface protein 1 (MSP1). No significant associations were found between HLA-DRB1 alleles and antibody responses to other malaria antigens tested.
1) Field isolates of Plasmodium falciparum from Colombia and Peru were found to invade red blood cells through an atypical pathway that was resistant to all enzyme treatments, unlike what is typically seen.
2) The invasion pathways and ligand polymorphisms differed between Colombian, Peruvian, and Brazilian isolates, with Peruvian isolates showing a combination of Colombian and Brazilian characteristics.
3) The atypical resistant pathway was associated with specific variants of PfRh2a, PfRh5 and EBA-181, which may be major players in this pathway based on expression levels.
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The skin is the largest organ and its health plays a vital role among the other sense organs. The skin concerns like acne breakout, psoriasis, or anything similar along the lines, finding a qualified and experienced dermatologist becomes paramount.
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These lecture slides, by Dr Sidra Arshad, offer a simplified look into the mechanisms involved in the regulation of respiration:
Learning objectives:
1. Describe the organisation of respiratory center
2. Describe the nervous control of inspiration and respiratory rhythm
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4. Describe the influences of the Pneumotaxic and Apneustic centers
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2. Chapter 36, Ganong’s Review of Medical Physiology, 26th edition
3. Chapter 13, Human Physiology by Lauralee Sherwood, 9th edition
5-hydroxytryptamine or 5-HT or Serotonin is a neurotransmitter that serves a range of roles in the human body. It is sometimes referred to as the happy chemical since it promotes overall well-being and happiness.
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Cell Therapy Expansion and Challenges in Autoimmune DiseaseHealth Advances
There is increasing confidence that cell therapies will soon play a role in the treatment of autoimmune disorders, but the extent of this impact remains to be seen. Early readouts on autologous CAR-Ts in lupus are encouraging, but manufacturing and cost limitations are likely to restrict access to highly refractory patients. Allogeneic CAR-Ts have the potential to broaden access to earlier lines of treatment due to their inherent cost benefits, however they will need to demonstrate comparable or improved efficacy to established modalities.
In addition to infrastructure and capacity constraints, CAR-Ts face a very different risk-benefit dynamic in autoimmune compared to oncology, highlighting the need for tolerable therapies with low adverse event risk. CAR-NK and Treg-based therapies are also being developed in certain autoimmune disorders and may demonstrate favorable safety profiles. Several novel non-cell therapies such as bispecific antibodies, nanobodies, and RNAi drugs, may also offer future alternative competitive solutions with variable value propositions.
Widespread adoption of cell therapies will not only require strong efficacy and safety data, but also adapted pricing and access strategies. At oncology-based price points, CAR-Ts are unlikely to achieve broad market access in autoimmune disorders, with eligible patient populations that are potentially orders of magnitude greater than the number of currently addressable cancer patients. Developers have made strides towards reducing cell therapy COGS while improving manufacturing efficiency, but payors will inevitably restrict access until more sustainable pricing is achieved.
Despite these headwinds, industry leaders and investors remain confident that cell therapies are poised to address significant unmet need in patients suffering from autoimmune disorders. However, the extent of this impact on the treatment landscape remains to be seen, as the industry rapidly approaches an inflection point.
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1. ARTIGO ARTICLE 2703
Mutations in the pfmdr1, cg2, and pfcrt
genes in Plasmodium falciparum samples
from endemic malaria areas in Rondonia
and Pará State, Brazilian Amazon Region
Mutações nos genes pfmdr1, cg2 e pfcrt em
isolados de Plasmodium falciparum provenientes
de localidades malarígenas dos Estados de
Rondônia e Pará, Amazônia Legal Brasileira
Giselle Maria Rachid Viana 1
Ricardo Luís Dantas Machado 2
Vanja Sueli Pachiano Calvosa 1
Marinete Marins Póvoa 1
Abstract Introduction
1 Seção de Parasitologia,
The objectives of this study were to investigate The first references to malaria cases from chloro-
Instituto Evandro Chagas,
Belém, Brasil.
the molecular basis for Plasmodium falciparum quine-resistant Plasmodium falciparum date to
2 Departamento de Doenças resistance to chloroquine in isolates from the 1960 in South America and Southeast Asia, and
Dermatológicas, Infecciosas Brazilian Amazon and to identify polymor- since then drug resistance has been viewed as
e Parasitárias, Faculdade de
Medicina de São José do Rio
phisms in the pfmdr1 gene, codons 184, 1042, a public health problem in various countries of
Preto, São Paulo, Brasil. and 1246, the kappa and gamma regions of the the world 1.
cg2 gene, and the K76T mutation of the pfcrt In Africa, chloroquine-resistant P falciparum
.
Correspondence
M. M. Póvoa gene, in order to calculate the distribution of malaria has been expanding since the 1970s.
Laboratório de Pesquisas polymorphism within each target gene, com- Currently, resistance to this drug is found in near-
em Malária, Seção de
paring samples from distinct geographic areas, ly all the endemic countries, especially in East
Parasitologia, Instituto
Evandro Chagas. using allele-specific polymerase chain reaction Africa 2,3. In Brazil, the first reports of resistance
Rod. BR-316, km 7, s/n, (PCR) for the pfmdr gene and PCR plus restric- to anti-malarials date to the early 20th century,
Ananindeua, PA
tion fragment length polymorphism (RFLP) for when it was observed that quinine was losing its
67030-000, Brasil.
marinetepovoa@iec.pa.gov.br the cg2 and pfcrt genes. The sample consisted of efficacy 4. In relation to chloroquine, clinical fail-
40 human blood isolates, already collected and ure is already reaching nearly 100% in the major-
morphologically diagnosed as carriers of P. fal- ity of endemic areas in Brazil 1,5,6,7.
ciparum parasites, from four localities: Porto The molecular basis for chloroquine resis-
Velho in Rondonia State and Maraba, Itaituba, tance has been studied, but various aspects re-
and Tailandia in Pará State. Distribution of P. main to be elucidated. In 1989, two homologues
falciparum in vitro chloroquine resistance in of the multiple drug resistant gene (mdr) were
the isolates was 100% for pfmdr1, cg2 gamma identified and denominated pfmdr1 and pfmdr2,
region, and pfcrt, except for the polymorphism located respectively in chromosomes 5 and 14
in the cg2 kappa region, which was not found. of P falciparum. Polymorphisms in the pfmdr1
.
homologue have been associated with the chlo-
Plasmodium falciparum; Genetic Polymor- roquine resistance phenotype 8. Although some
phism; Chloroquine studies have indicated a lack of association be-
tween the chloroquine resistance phenotype and
point mutations in this gene 9,10, there is grow-
ing evidence of the role of the pfmdr1 gene in
P falciparum chloroquine resistance 5,6,11,12,13.
.
Cad. Saúde Pública, Rio de Janeiro, 22(12):2703-2711, dez, 2006
2. 2704 Viana GMR et al.
Polymorphism in the Asn86Tyr codon has been codons Asn1042Asp, Asp1246Tyr, and Tyr184Phe of the
related to chloroquine resistance in P falcipa-
. pfmdr1 gene, polymorphisms in κ (kappa) and γ
rum isolates in Nigeria 11, Guinea-Bissau 12, (gamma) in the cg2 gene, and the K76T mutation
Gambia 14, and Malaysia 15. An association has in the pfcrt gene.
also been demonstrated between polymorphism
in the Asn1042Asp and Asp1246Tyr codons in the
pfmdr1 gene and in vitro chloroquine resistance Material and methods
and absence of mutation in the Asn86Tyr codon in
samples in Brazil 5. Study area
After genetic crossing between a resistant
strain (W2) and one sensitive to chloroquine The selected study localities were Porto Vel-
(HB3), Su et al. 16 characterized the cg2 gene ho (longitude 63° 55’ 00’’; latitude: 8° 40’ 00’’),
(region 36-kb of chromosome 7) as responsible Rondônia; Marabá (longitude 49° 07’ 44.2’’; lati-
for chloroquine resistance. The cg2 genotype tude: 5° 23’ 27.5’’), Itaituba (longitude 55° 59’ 27’’;
is characterized by twelve point mutations and latitude: 4° 16’ 33.3’’), and Tailândia (longitude
three length polymorphisms (kappa, gamma, 48° 57’ 07.2’’; latitude: 2° 56’ 06.6’’), Pará, all lo-
and omega), which are associated with clones cated in the Brazilian Amazon.
having chloroquine resistance phenotypes 16. In each study area, ten human blood samples
Currently, the study of molecular markers for were used that were already available in the blood
resistance in malaria epidemiology focuses on samples bank at the Malaria Research Labora-
polymorphism in the protein coded by the pfcrt tory of the Evandro Chagas Institute (Instituto
gene (chloroquine-resistant P falciparum related
. Evandro Chagas), diagnosed microscopically as
to the transporter), which contains 13 exons and P falciparum malaria, totaling an overall sample
.
is located in chromosome 7 of the parasite 17,18. of forty isolates (n = 40). The study was approved
Point mutations in this gene have been associ- by the Research Ethics Committee of the Instituto
ated with in vitro chloroquine resistance in P fal-
. Evandro Chagas.
ciparum isolates from Africa, Southeast Asia, and
South America, in which a mutation, substitu- Laboratory methods
tion of lysine (K) with treonine (T) in position 76
(K76T), was present in all of the chloroquine-re- • Preparation and washing of samples
sistant samples and absent in all the isolates that
were sensitive to this drug according to in vitro From each total blood sample, five 20µL drops
and in vivo analysis 2,19,20,21,22,23. were added to a fiberglass membrane measur-
Despite the results of studies reporting flaws ing 2.5cm in diameter (Whatman International,
in the association between the K76T mutation of Maidstone, UK), placed on Whatman filter pa-
the pfcrt gene and chloroquine resistance 24,25, per measuring 5.5cm in diameter (Whatman
genetic transformation experiments with plas- International, Maidstone, UK). After drying the
mids expressing mutant forms of pfcrt conferred droplets at room temperature and properly iden-
resistance in the three different chloroquine- tifying the samples, the membranes were stored
sensitive clones tested, thus demonstrating this in plastic recipients (BHL Limited, Poole, UK) at
gene’s important role for in vitro chloroquine -20°C until submitting to washing according to
resistance 26. Warhurst et al. 27.
The high rate of chloroquine-resistant P fal-
.
ciparum cases in Brazil requires studies on the • Polymerase chain reaction (PCR)
molecular basis of this resistance through the
identification and characterization of genes One eighth of one drop of blood soaked into the
with pfmdr1, cg2, and pfcrt, given that the stud- membrane (the equivalent of 5µL of extracted
ies conducted to date in the region (localities in DNA) and obtained with a sterile scalpel was
the States of Amapá 5, Amazonas 7,22, Mato Gros- used as the source of DNA. The following re-
so 6,7,22, and Rondônia 7,22) are scarce in light of agents were added to this part of the membrane
the problem’s geographic extension. It is equally for analysis of the pfmdr1, cg2, and pfcrt genes:
important to produce knowledge allowing the 5µL of 10X buffer [tris-HCl 10mM, pH 8.3; gelatin
development of new drugs and the application 0.01%(p/v)]; KCl 10M; and MgCl2 15mM) (Bio-
of anti-malarial prophylactic measures. line, Massachusetts, USA – cat. M95801B); 2 to
The objective of the current study was to in- 2,5µL of each specific initiator for each region
vestigate the molecular basis for P falciparum
. of the target gene; 0.75 to 1.0µL of each dNTP
chloroquine resistance in isolates from the Bra- (2.5µmol – Pharmacia Biotech) (final concentra-
zilian Amazon, by identifying polymorphisms in tion 200µM); 0.25µL of Taq polymerase (Biotaq
Cad. Saúde Pública, Rio de Janeiro, 22(12):2703-2711, dez, 2006
3. Plasmodium Falciparum MUTATIONS 2705
5U/µL: 550 units – Bioline M95801B); and ster- ucts in each Eppendorf tube. The restriction frag-
ile distilled water q.s.p. 50µL/tube. The initiator ments had the sizes fractionated in agarose gel at
sequences and PCR conditions for codons 184, 2% at 100 volts for one hour. Later, the agarose gel
1042, and 1246 of the pfmdr1 gene were con- was stained with ethidium bromide (5µL/mL in
ducted according to Grobusch et al. 13 and Adagu TBE) for 30 minutes and the bands were viewed
& Warhurst 28, for the kappa and gamma of the under ultraviolet light (Fluo-Link, Flowgen) and
cg2 gene according to Adagu & Warhurst 28, and photographed in a photodocumentation system
for the K76T mutation of the pfcrt gene according (Kodak® Edas 290).
to the protocol by Christopher Plowe (University
of Maryland, USA) 26.
In addition to the test samples, size 50 or Results
100bp markers were used, and as controls, types
HB3 (standard sensitive clone, Honduras), 7G8 In relation to codons 184 and 1042 on the pfmdr1
(standard resistant clone, Brazil), DD2 (standard gene, all 40 isolates showed a profile similar to
resistant clone, Southeast Asia), and K1 (standard type 7G8 (the standard resistant clone for Brazil)
resistant clone, Thailand). according to the allele-specific PCR technique
The tubes were centrifuged rapidly at (Figures 1 and 2). For codon 1246 on the same
14,000rpm and placed in a thermal cycler (Hy- gene, the study samples showed a similar pro-
baid OmniGene® Thermal Cycler) to be submit- file to 7G8, except for samples 808/98, 809/98,
ted to the specific amplification conditions for 810/98, 813/98, 815/98, 816/98, and 817/98 from
each region of the target gene. the municipality of Porto Velho, which showed a
The PCR products were fractionated electro- profile similar to K1 (standard resistant clone for
phoretically in agarose gel at 2% for codons 184, Thailand). For the kappa region of the cg2 gene, all
1042, and 1246 on the pfmdr1 gene, at 3% for 40 isolates showed a profile similar to HB3 (stan-
the kappa and gamma regions of the cg2 gene, dard sensitive clone for Honduras) in both tests
and at 1% for the K76T mutation of the pfcrt gene (PCR and RFLP) (Figure 3), while for the gamma
(Ultra pure agarose, BRL 155517-014), at 100 volts region of this same gene, all 40 samples showed
for one hour. Later, the agarose gel was stained a profile similar to type 7G8 (standard resistant
with ethidium bromide (5µL/mL in TBE) for 30 clone for Brazil) in both tests (Figure 4).
minutes and the resulting bands were viewed The mutant allele K76T of the pfcrt gene was
under ultraviolet light (Fluo-Link, Flowgen) and found in all 40 samples. Amplification of a 134bp
photographed in a photodocumentation system region containing codon 76 was obtained in
(Kodak® Edas 290). these 40 isolates by nested PCR and confirmed by
RFLP using the APO 1 endonuclease, presenting
• Restricted fragment length a profile similar to 7G8 (standard resistant clone,
polymorphism (RFLP) Brazil) (Table 1) (Figure 5).
For the kappa region of the cg2 gene, the endo-
nuclease Tse I was used. The PCR products with Discussion
a pattern similar to DD2 or K1 produced three
fragments with 558bp, 90bp, and 68bp, while The molecular mechanisms for the development
the isolates with a pattern similar to HB3 pro- of chloroquine resistance by the parasite are still
duced two fragments with 632bp and 68bp. For not completely clear, but many candidate mo-
the gamma region of this same gene, the restric- lecular markers have been identified 20,21,29 and
tion enzyme was Rca 1. The PCR products with a there is evidence that P falciparum chloroquine
.
pattern similar to 7G8 produced two bands with resistance is a multigenic event 2,10,16.
194bp and 97bp, while the isolates with a pat- In relation to the pfmdr1 gene, the 40 iso-
tern similar to HB3 produced three bands with lates were tested for the TYR184PHE, ASN1042ASP,
119bp, 106bp, and 45bP For the pfcrt gene, the
. and ASP1246TYR mutations associated with the
APO 1 endonuclease was used, and the samples chloroquine resistance phenotype from samples
with a pattern similar to 7G8 produced a frag- in Africa, Asia, and South America 5,11,12,13,30,31.
ment with 134bp, and those with a pattern simi- Although all the isolates tested for the TYR184PHE
lar to the HB3 type produced two fragments, with mutation presented a resistance pattern simi-
100bp and 34bp. Digestions were conducted at lar to 7G8 (standard Brazilian resistant clone),
65ºC in 10µL volume, using 0.2µL of restriction changes in this codon appear not to be corre-
endonuclease (Tse I, Rca 1, or APO 1, according lated with chloroquine resistance 29. Meanwhile
to the target gene), 1µL of buffer solution, 4.8µL the presence of a profile similar to clone 7G8 in
of sterile distilled water, and 4µL of the PCR prod- the ASN1042ASP and ASP1246TYR mutations in the
Cad. Saúde Pública, Rio de Janeiro, 22(12):2703-2711, dez, 2006
4. 2706 Viana GMR et al.
Figure 1
PCR electrophoresis pattern for codon 184 of the pfmdr1 gene.
1 2 3 4 5 6 7 8 9 10 11 12 13 14
200
1 = 100bp size marker; 2 = 7G8 (standard resistant clone, Brazil); 3 to 13 = test samples; and 14 = sterile distilled water.
Figure 2
PCR electrophoresis pattern for codon 1042 of the pfmdr1 gene.
1 2 3 4 5 6 7 8 9 10 11 12 13
345
200
1 = 100bp size marker; 2 = 7G8 (standard resistant clone, Brazil); 3 = blank; 4 to 12 = test samples; and 13 = sterile distilled water.
Cad. Saúde Pública, Rio de Janeiro, 22(12):2703-2711, dez, 2006
5. Plasmodium Falciparum MUTATIONS 2707
Figure 3
PCR electrophoresis pattern for the kappa region of the cg2 gene.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
700
1 = 100bp size marker; 2 = HB3 (standard sensitive clone, Honduras); 3 to 7 = test samples; 8 = blank; 9 to 14 = test samples;
and 15 = sterile distilled water.
Figure 4
PCR electrophoresis pattern for gamma region of the cg2 gene.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
291
1 = 100bp size marker; 2 = 7G8 (standard resistant clone, Brazil); 3 = HB3 (standard sensitive clone, Honduras); and 4 to 16 = test samples.
Cad. Saúde Pública, Rio de Janeiro, 22(12):2703-2711, dez, 2006
6. 2708 Viana GMR et al.
Table 1
Percentage distribution of resistance associated with pfmdr1, cg2, and pfcrt genes in 40 samples from the Brazilian Amazon.
Genes Porto Velho (%) Marabá (%) Itaiatuba (%) Tailândia (%)
pfmdr1 100 100 100 100
cg2κ 0 0 0 0
cg2 γ 100 100 100 100
pfcrt 100 100 100 100
pfmdr1: gene 1 for multiple drug resistance in Plasmodium falciparum; cg2κ: kappa region of gene 2 for chloroquine
resistance; cg2γ: gamma region of gene 2 for chloroquine resistance; pfcrt: chloroquine-resistant
Plasmodium falciparum related to transporter.
Figure 5
RFLP electrophoresis pattern for mutation K76T on the pfcrt gene.
1 2 3 4 5 6 7 8 9 10 11 12 13
134
1 = 100bp size marker; 2 = 7G8 (standard resistant clone, Brazil); 3 to 12 = test samples; and 13 = sterile distilled water.
samples corroborates previous studies reporting In transfection studies, Reed et al. 32 report-
the association between polymorphisms in these ed that the switch from aspargine to tyrosine
codons and in vitro P falciparum chloroquine re-
. on codon 1246 of pfmdr1 increased the chlo-
sistance in South American samples 2,5,6. The sev- roquine resistance in a clone known to be re-
en samples from the municipality of Porto Velho sistant and carrying the pfcrt mutant. The as-
(808/98, 809/98, 810/98, 813/98, 815/98, 816/98, sociation between pfmdr1 and in vitro chloro-
and 817/98) with profile similar to K1 (standard quine resistance has not been confirmed by all
resistant clone, Thailand), indicate that previ- the studies 9,10, indicating that there are diverse
ously rare mutations in South America are now chloroquine resistance mechanisms 10 and
emerging on this continent 23,29. that the pfmdr1 gene does not play a primary
Cad. Saúde Pública, Rio de Janeiro, 22(12):2703-2711, dez, 2006
7. Plasmodium Falciparum MUTATIONS 2709
role in conferring chloroquine resistance in P . metabolic rates can also influence the type of re-
falciparum 2,26. sponse to chloroquine treatment. Another poten-
In the κ (kappa) region of cg2, the second can- tial factor is the possibility that other pfcrt muta-
didate gene for the resistance phenotype to this tions, like those in codons 72, 74, 75, 97, 144, 148,
4-aminoquinoline in P falciparum, a 100% sensi-
. 160, 194, 220, 271, 326, 333, 356, and 371, are also
tivity pattern was found in the isolates, although associated with the resistance phenotype 34,35,37,
studies report an association between repetitions in addition to the fact that P falciparum has more
.
in the kappa region and failures in sensitivity to transport proteins than those already known to
chloroquine in samples from Africa 28,29. These influence the pathogen’s physiology 30.
findings suggest that kappa repetitions in the There is evidence that chloroquine resistance
cg2 gene are not important genetic markers in is a multigenic phenomenon and that the K76T
isolates from the Brazilian Amazon, unlike the mutation in the pfcrt gene is necessary, but not
γ (gamma) region of this same gene, which pre- sufficient, to confer resistance 20. Therefore, oth-
sented a pattern similar to the 7G8 clone in the er studies are needed to evaluate the role of pfcrt
40 study samples. Thus, this result together with as a reliable genetic marker for in vivo response
that of Calvosa et al. 33, who found 100% in vitro to chloroquine 21.
chloroquine resistance in the same samples in The presence of polymorphisms in the
the municipality of Marabá, suggest a significant pfmdr1, cg2, and pfcrt genes in the same samples
correlation between gamma repetitions in cg2 indicates that these loci are important genetic
and in vitro chloroquine resistance in samples markers for in vitro chloroquine resistance 38 and
from the Brazilian Amazon. that this connection is maintained by the drug’s
The presence of mutations in the pfmdr1 and selective pressure 19.
cg2 genes in the same samples suggests a selec-
tive pressure for its installation and maintenance
in a given geographic area, in addition to other Conclusions
factors favoring this drug pressure, like the ma-
laria transmission patterns, the degree of popu- The presence of the ASN1042ASP and ASP1246TYR
lation immunity, population migration, pharma- mutations in the pfmdr1 gene in the samples
cokinetic factors, and indiscriminate use and/or from the Brazilian Amazon suggests that these
sub-therapeutic dosage of antimalarials 20,21. codons are important markers for in vitro chlo-
Mutations in the pfcrt gene are associated roquine resistance in P falciparum isolates from
.
with in vivo chloroquine resistance in Africa and South America; however, the κ (kappa) region of
in vitro resistance in South America 2,17,21,22,23,34 the cg2 gene is not a relevant marker for P falci-
.
and allele transfection studies with this gene, like parum resistance to 4-aminoquinoline in sam-
the K76T mutation, have shown that the latter ples from the Brazilian Amazon. Meanwhile, the
confers in vitro resistance to chloroquine 26,35. presence of polymorphism in the γ (gamma) re-
In this study, 100% of the study isolates in the gion of the cg2 gene in the study isolates suggests
four municipalities in the Brazilian Amazon pre- an association between in vitro P falciparum
.
sented the K76T genotype, similar to the 7G8 type chloroquine resistance and this mutation. The
(standard resistant clone for Brazil). This finding presence of the K76T mutation in the pfcrt gene
agrees with the work of Vieira et al. 22, who found in all the study samples suggests that this muta-
a high prevalence of this mutation in samples tion is an important genetic marker for in vitro P.
from the Brazilian Amazon, although this muta- falciparum resistance to chloroquine. The identi-
tion is not absolute, since it can be found both fication of the mutations in the pfmdr1, cg2, and
in chloroquine-resistant samples and those sen- pfcrt loci in samples from the Brazilian Amazon
sitive to this drug, suggesting that other factors suggests that they are important genetic markers
control the expression of the resistance pheno- for in vitro P falciparum chloroquine resistance
.
type 24,25,36. Possible reasons for this fact may be and that the chloroquine resistance phenotype
associated with the involvement of the host re- in P falciparum is a multigenic process, indicat-
.
sponse, like the immune status, which can cause ing a possible local clonal expansion of P falci-
.
negative conversion of parasitemia, regardless of parum chloroquine resistance, selected by the
whether the sample is resistant to chloroquine. indiscriminate or inadequate use of this drug in
In addition, the drug’s absorption and individual the Brazilian Amazon.
Cad. Saúde Pública, Rio de Janeiro, 22(12):2703-2711, dez, 2006
8. 2710 Viana GMR et al.
Resumo Contributors
O estudo foi desenvolvido para investigar a base mo- G. M. R. Viana, R. L. D. Machado, and V. S. P Calvosa
.
lecular da resistência do Plasmodium falciparum à collected the samples. M. M. Póvoa, G. M. R. Viana, and
cloroquina em isolados da região Amazônica brasilei- R. L. D. Machado participated in the tests, data analysis,
ra e identificar os polimorfismos nos códons TYR184PHE, discussion of the results, and drafting of the article.
ASN1042ASP e ASP1246TYR do gene pfmdr1, as regiões ka-
ppa e gamma do gene cg2 e a mutação K76T do gene
pfcrt, a fim de determinar a distribuição percentual dos Acknowledgments
alelos de cada gene estudado, comparando amostras de
áreas geográficas distintas, utilizando a reação em ca- The authors wish to thank technicians José Maria de
deia da polimerase (PCR) alelo-específica para o pfmdr1 Souza Nascimento and José Mario Veloso Peres from
e a PCR e o polimorfismo do comprimento do fragmento the Laboratório de Pesquisas em Malária, Seção de Pa-
de restrição (RFLP) para os genes cg2 e pfcrt. A amostra rasitologia, Instituto Evandro Chagas, and the Coorde-
foi constituída de quarenta isolados de sangue humano nação de Aperfeiçoamento de Pessoal de Nível Superior
já coletados e microscopicamente diagnosticados com for providing a Master’s grant to Giselle Maria Rachid
malária por P falciparum das localidades de Porto Velho
. Viana during her graduate studies in the Biology of In-
(Rondônia) e Marabá, Itaituba e Tailândia (Pará). A dis- fectious and Parasitic Agents at the Universidade Fede-
tribuição percentual da resistência in vitro do P falcipa-
. ral do Pará.
rum à cloroquina nas amostras estudadas foi de 100% de
resistência para os genes pfmdr1, região gamma do cg2 e
pfcrt. O polimorfismo na região kappa do gene cg2 não
foi encontrado nas amostras estudadas.
Plasmodium falciparum; Polimorfismo Genético; Clo-
roquina
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