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.
The document discusses a study on MRSA 252, a methicillin-resistant Staphylococcus aureus strain. It aims to construct a prophage-free host strain of MRSA 252 that can propagate lytic bacteriophages. 31 mutant strains of MRSA 252 were created using mitomycin C to induce prophages. Prophage screening using PCR found that mutations 7 and 8 did not contain the φSa2 and φSa3 prophages. Most mutant strains were susceptible to lytic phages in spot tests, but were resistant to some phages and high dilutions of others. Mutations 7 and 8 were resistant to all tested phages. The attempt to mobilize prophages from M
A novel marker, ARM58, confers antimony resistance to Leishmania spp.Carola Schäfer
This document describes the identification of a novel genetic marker, LbrARM58, that confers antimony resistance to Leishmania species. Using a functional cloning strategy with genomic DNA libraries from antimony-resistant and sensitive L. braziliensis clinical isolates, researchers repeatedly selected a region on chromosome 20. This region contains the gene LbrM20.0210, which when overexpressed increases resistance to antimony (SbIII) in L. braziliensis and L. infantum in vitro and enhances parasite survival inside macrophages under antimony pressure. LbrM20.0210 encodes a previously uncharacterized protein with four repeats of a domain of unknown function that is unique to the Leishmania genus.
1) The drug MMV006169 inhibits the growth of Toxoplasma gondii and Cryptosporidium parvum. It is hypothesized that MMV006169 interacts with the protein CDC48 in T. gondii.
2) Both forms of the CDC48 protein from T. gondii were cloned into the pGADT7 vector to test for interaction with MMV006169 using a yeast 3-hybrid system.
3) An analog of MMV006169, called FT14.026.2, selectively inhibits the cytoplasmic form of CDC48 in Cryptosporidium, suggesting it interacts specifically with that version.
This document describes the development of a method to detect human CD4+ T cells specific for influenza A virus using soluble human MHC class II tetramers. Key points:
1) Soluble MHC class II tetramers containing an immunodominant peptide from influenza hemagglutinin were constructed and used to identify antigen-specific T cells from influenza-immune individuals.
2) After 7 days of proliferation in response to stimulation by influenza peptide or vaccine, tetramer-positive cells had undergone extensive division and expressed markers of activated CD4+ T cells.
3) The influenza peptide-specific T cells represented a major subset of proliferating CD4+ T cells responding to whole influenza vaccine.
4)
This document describes the development of 21 new nuclear genetic markers for the wall lizard genus Podarcis. DNA from one individual of P. vaucheri was used to generate anonymous sequence fragments which were sequenced and screened for repetitive elements. Primers were designed for fragments over 300bp without repeats. These markers showed high cross-amplification among closely related P. vaucheri, P. bocagei and P. liolepis, but lower success in more distant P. muralis and P. tiliguerta. Nucleotide diversity across the 5 species ranged from 0.35-3.5%, demonstrating their utility for population genetics and phylogenetics in Podarcis.
The document discusses developing new shuttle vector technologies for Thermococcus kodakarensis, a hyperthermophilic archaeon. Currently only one shuttle vector exists, limiting genetic techniques. Environmental plasmids from related organisms contain replicative origins and are potential new shuttle vectors, but purifying large amounts has been difficult due to low copy numbers. A transposon-based method is proposed, where the transposon inserts randomly into environmental plasmid DNA. This allows purification in E. coli and assessment of replication in T. kodakarensis, overcoming prior limitations and furthering plasmid biology understanding in archaea.
PCR-OLA is a technique that combines polymerase chain reaction with oligonucleotide ligation assay to detect single nucleotide polymorphisms by distinguishing between ligation and non-ligation of oligonucleotides. It involves two phases - a multiplex PCR amplification to amplify the target DNA, followed by a multiplex oligonucleotide ligation assay where detection probes hybridize adjacent to each other and are ligated if complementary, which can identify normal vs. mutant genotypes. Ligation is regulated by the specificity of oligonucleotide hybridization, the need for adjacent hybridization of probes, and perfect complementarity of two bases.
1. The study aims to analyze gene expression differences between near-isogenic cotton lines that differ at two major root-knot nematode resistance QTLs (qMi-C11 and qMi-C14) through RNA sequencing.
2. RNA was extracted from roots of resistant and susceptible lines infected with root-knot nematodes at different time points. Over 800 million RNA-seq reads were obtained and over 80% were mapped to the cotton reference genome.
3. Preliminary analysis found differences in the development of nematodes between the two resistance QTLs. qMi-C11 affected early nematode stages while qMi-C14 affected later stages. Variant calling also identified differences in the int
The document discusses a study on MRSA 252, a methicillin-resistant Staphylococcus aureus strain. It aims to construct a prophage-free host strain of MRSA 252 that can propagate lytic bacteriophages. 31 mutant strains of MRSA 252 were created using mitomycin C to induce prophages. Prophage screening using PCR found that mutations 7 and 8 did not contain the φSa2 and φSa3 prophages. Most mutant strains were susceptible to lytic phages in spot tests, but were resistant to some phages and high dilutions of others. Mutations 7 and 8 were resistant to all tested phages. The attempt to mobilize prophages from M
A novel marker, ARM58, confers antimony resistance to Leishmania spp.Carola Schäfer
This document describes the identification of a novel genetic marker, LbrARM58, that confers antimony resistance to Leishmania species. Using a functional cloning strategy with genomic DNA libraries from antimony-resistant and sensitive L. braziliensis clinical isolates, researchers repeatedly selected a region on chromosome 20. This region contains the gene LbrM20.0210, which when overexpressed increases resistance to antimony (SbIII) in L. braziliensis and L. infantum in vitro and enhances parasite survival inside macrophages under antimony pressure. LbrM20.0210 encodes a previously uncharacterized protein with four repeats of a domain of unknown function that is unique to the Leishmania genus.
1) The drug MMV006169 inhibits the growth of Toxoplasma gondii and Cryptosporidium parvum. It is hypothesized that MMV006169 interacts with the protein CDC48 in T. gondii.
2) Both forms of the CDC48 protein from T. gondii were cloned into the pGADT7 vector to test for interaction with MMV006169 using a yeast 3-hybrid system.
3) An analog of MMV006169, called FT14.026.2, selectively inhibits the cytoplasmic form of CDC48 in Cryptosporidium, suggesting it interacts specifically with that version.
This document describes the development of a method to detect human CD4+ T cells specific for influenza A virus using soluble human MHC class II tetramers. Key points:
1) Soluble MHC class II tetramers containing an immunodominant peptide from influenza hemagglutinin were constructed and used to identify antigen-specific T cells from influenza-immune individuals.
2) After 7 days of proliferation in response to stimulation by influenza peptide or vaccine, tetramer-positive cells had undergone extensive division and expressed markers of activated CD4+ T cells.
3) The influenza peptide-specific T cells represented a major subset of proliferating CD4+ T cells responding to whole influenza vaccine.
4)
This document describes the development of 21 new nuclear genetic markers for the wall lizard genus Podarcis. DNA from one individual of P. vaucheri was used to generate anonymous sequence fragments which were sequenced and screened for repetitive elements. Primers were designed for fragments over 300bp without repeats. These markers showed high cross-amplification among closely related P. vaucheri, P. bocagei and P. liolepis, but lower success in more distant P. muralis and P. tiliguerta. Nucleotide diversity across the 5 species ranged from 0.35-3.5%, demonstrating their utility for population genetics and phylogenetics in Podarcis.
The document discusses developing new shuttle vector technologies for Thermococcus kodakarensis, a hyperthermophilic archaeon. Currently only one shuttle vector exists, limiting genetic techniques. Environmental plasmids from related organisms contain replicative origins and are potential new shuttle vectors, but purifying large amounts has been difficult due to low copy numbers. A transposon-based method is proposed, where the transposon inserts randomly into environmental plasmid DNA. This allows purification in E. coli and assessment of replication in T. kodakarensis, overcoming prior limitations and furthering plasmid biology understanding in archaea.
PCR-OLA is a technique that combines polymerase chain reaction with oligonucleotide ligation assay to detect single nucleotide polymorphisms by distinguishing between ligation and non-ligation of oligonucleotides. It involves two phases - a multiplex PCR amplification to amplify the target DNA, followed by a multiplex oligonucleotide ligation assay where detection probes hybridize adjacent to each other and are ligated if complementary, which can identify normal vs. mutant genotypes. Ligation is regulated by the specificity of oligonucleotide hybridization, the need for adjacent hybridization of probes, and perfect complementarity of two bases.
1. The study aims to analyze gene expression differences between near-isogenic cotton lines that differ at two major root-knot nematode resistance QTLs (qMi-C11 and qMi-C14) through RNA sequencing.
2. RNA was extracted from roots of resistant and susceptible lines infected with root-knot nematodes at different time points. Over 800 million RNA-seq reads were obtained and over 80% were mapped to the cotton reference genome.
3. Preliminary analysis found differences in the development of nematodes between the two resistance QTLs. qMi-C11 affected early nematode stages while qMi-C14 affected later stages. Variant calling also identified differences in the int
Candidemia in HIV-positive patients in Dschang District Hospital (West Region...Claude Nangwat
Candidemia has been identified as a public health problem in HIV-infected patients. The evaluation of CD4 count, transaminases and blood glucose, are being used as a means to monitor the health of HIV-infected patients, without excluding the diagnosis of candidemia and other opportunistic infections. In order to contribute in improving the care of HIV-infected patients attending Dschang District Hospital and later on, in other hospitals in Cameroon, we conducted from June to September 2014 a cross-sectional study, with general objective; to determine the association between candidemia and selected biochemical and haematological parameter changes in HIV-infected patients, as a possible indicator in monitoring HIV disease progression.
To do this, blood samples were collected from HIV-infected patients assigned to the UPEC of Dschang District Hospital for follow up, and haemogram report, CD4 counts, ALAT level, ASAT level, and glucose level in blood were evaluated by cytometric and spectrophotometric assays. Candida species were isolated from some blood samples, and then identified using CHROMagar Candida culture medium. The broth microdilution method was afterwards used to test the susceptibility of the fungal isolates vis-a-vis three conventional antifungal agents.
Mycological analysis of blood samples showed that eight (08) patients had candidemia, a prevalence of 6.11%. Eight (08) isolates were obtained from these eight (08) candidemic HIV-infected patients; this consisted of 4(50%) Candida albicans, 3(37.5%) Candida parapsilosis and 1(12.5%) Candida glabrata. All these isolates were resistant (MICs ranged from 2 to >256 µg/mL) to the antifungals used, that is, ketoconazole, amphotericin B and nystatin.
A significant correlation was found between candidemia and white blood cell count, with a correlation coefficient of r = 0.240 (p < 0.05). Based on the results obtained, the systematic diagnosis of candidemia should be performed in patients infected with HIV in Cameroon in order to improve on their care.
Key words: Candidemia, HIV, biochemical parameters, hematological parameters, Antifungals activities.
This document summarizes information about plastid transformation. Plastids are organelles found in plant cells that perform photosynthesis and store compounds. They have their own circular genome. Plastid transformation involves introducing foreign genes into the plastid genome using homologous recombination. It is preferred over nuclear transformation for traits because it allows high-level transgene expression and containment of transgenes within the plastid. The document discusses chloroplast structure, common vectors, selection markers, traits of interest for transformation including stress tolerance and pharmaceutical production, and limitations of plastid transformation technology.
This document discusses various methods of genetic recombination including transformation, conjugation, transduction, and protoplast fusion. It provides definitions and examples of key terms. Transformation involves the direct uptake of exogenous DNA by a cell. Conjugation is the transfer of genetic material between bacterial cells through direct contact. Transduction is the transfer of genes between bacteria mediated by bacteriophages. Protoplast fusion involves fusing plant protoplasts using electric shock or chemicals to produce somatic hybrid plants. The document also discusses applications of these techniques such as gene cloning, production of monoclonal antibodies, and genetic engineering.
This document summarizes previous research on the Epstein-Barr virus (EBV) and describes a study that mapped interactions between EBV proteins and human proteins. EBV infects 95% of humans and can cause cancers. The study used a yeast two-hybrid method to screen 216 EBV proteins against 15,483 human proteins, identifying 188 interacting pairs. Mapping these virus-host interactions may reveal how EBV disrupts cellular pathways to cause disease.
Hybridoma technology involves fusing antibody-producing B cells (splenocytes) with myeloma cells to produce immortal hybridoma cell lines that secrete monoclonal antibodies of a single specificity. Georges Köhler and César Milstein developed the technique in 1975 and were awarded the 1984 Nobel Prize for this discovery. The process involves immunizing an animal to generate B cells that produce antibodies against the target antigen. The B cells are isolated from the spleen and fused with myeloma cells using polyethylene glycol or electrofusion. The fused cells are selected in HAT medium, which allows only hybridomas expressing the hypoxanthine-guanine phosphoribosyl transferase enzyme to survive and multiply indefinitely. The resulting stable monoclonal antibody-
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
Plastids like chloroplasts contain their own circular DNA and are capable of expressing foreign genes at high levels. This document describes research where the human serum albumin (HSA) gene was successfully transformed into the tobacco chloroplast genome. Up to 11.1% of total soluble protein consisted of HSA, over 500 times higher than when introduced into the nuclear genome. The chloroplast transformation resulted in HSA accumulating in inclusion bodies, facilitating its purification. This demonstrated chloroplasts as an effective system for the high-level production of recombinant human therapeutic proteins.
This document discusses advances in HLA typing technologies over the past 25 years. It begins by outlining the evolution from low to high resolution typing methods like PCR-SSO, Sanger sequencing, and next generation sequencing using Illumina and PacBio platforms. Whole gene and long-range sequencing is now being done on over 80,000 samples using PacBio to type HLA Class I and II at high resolution without ambiguity. This has revealed over 5,000 novel variants, including insertions, deletions and nonsense mutations across exons and introns. Whole gene sequencing provides a more comprehensive view of variation than targeting only antigen recognition sites.
CRISPR/Cas9 is an advanced genome editing technology that can be used to develop plant disease resistance. It involves a Cas9 enzyme that acts like molecular scissors to cut DNA at specific locations guided by CRISPR RNA. This triggers DNA repair that can introduce changes to genes. Researchers have used CRISPR/Cas9 to develop resistance in plants against viruses, fungi, and bacteria by editing genes involved in host-pathogen interaction and disease susceptibility. It provides a precise and efficient way to edit plant genomes to improve crop resistance compared to previous tools. Scientists continue working to enhance the specificity and control of CRISPR/Cas9 for genome editing applications in agriculture.
The document provides a history of genomics, beginning with Mendel's work in 1866 establishing the gene concept and laws of genetics. Key developments include the discovery of DNA in 1871, the chromosomal theory of inheritance in 1902, and the discovery of linkage in 1910. The structure of DNA was elucidated in the 1950s, leading to the development of recombinant DNA technology in the 1970s and DNA sequencing techniques in the 1970s-80s. The first whole genome of an organism was sequenced in 1995. More recent developments include next generation sequencing starting in 2005 and the advent of CRISPR/Cas9 genome editing in 2012. The document concludes with discussing the potential use of CRISPR to target genes in the diamondb
Culture independent methods for detection & enumeration of gut microfloraAmna Jalil
This document discusses various culture-independent methods for detecting and enumerating gut microflora, including: 1) Design of PCR primers and hybridization probes to target specific species or groups; 2) PCR-ELISA to combine PCR amplification with ELISA detection; 3) Sequence analysis of randomly amplified 16S rRNA genes; 4) Temperature gradient gel electrophoresis (TGGE) and denaturing high performance liquid chromatography (DHPLC) to separate amplified DNA fragments by size; 5) Terminal restriction fragment length polymorphism (T-RFLP) to generate terminal restriction fragments for profiling; 6) Oligonucleotide arrays and quantitative real-time PCR for rapid, accurate detection of thousands of bacteria; 7) Fluorescence in
Karyotype variability in plant pathogenic fungi palm7016chaithram11
This document summarizes a seminar presentation on karyotype variability in plant pathogenic fungi. It discusses types of karyotype variability including chromosome number polymorphisms and chromosome length polymorphisms. Mechanisms that can lead to karyotype variability are described, such as meiotic recombination, DNA repair pathways, parasexual recombination, transposon-associated rearrangements, and lateral DNA transfer. Case studies on fungi like Mycosphaerella graminicola and Alternaria alternata are provided that demonstrate karyotype changes through these mechanisms. The importance of karyotype variability in the evolution and emergence of new virulence strains in fungi is also noted.
Agrobacterium and plant viruses can be used as biological vectors for plant transformation. Agrobacterium mediates transformation via its tumor-inducing plasmid, using virulence genes to transfer T-DNA containing the gene of interest into the plant genome. Plant viruses can also act as gene vectors by engineering viral genomes to contain and deliver foreign genes. Retroviruses have been developed as viral vectors for animal gene transfer due to their ability to stably integrate into the host chromosome.
Activation-of-human-immunodeficiency-virus-type-1-expression-by-Gardnerella-v...Farhad B. Hashemi, PhD
This study found that lysates from Gardnerella vaginalis, a bacteria commonly associated with bacterial vaginosis, significantly stimulated HIV expression in monocytoid cells and certain T cell lines. G. vaginalis lysates activated HIV long terminal repeat transcription and increased NF-kB binding activity in HIV-infected cells, indicating an effect on HIV transcription. The activation of HIV production by G. vaginalis suggests that G. vaginalis infection in the genital tract may increase the risk of HIV transmission by enhancing HIV expression levels in the genital tract. This could help explain the link between bacterial vaginosis and increased sexual transmission of HIV.
Single Cell Insights: Studying Environmental Microbial Communities Cell by CellQIAGEN
Dissecting complex microbial communities has incredible potential in the quest to decipher the world around us and find new sources of enzymes, antibiotics and other drugs. The challenge that remains is how to gain a deeper understanding of the roles and interactions of individual microbes. Single cell sequencing has emerged as an innovative investigational approach that provides a view of the cell-by-cell community by separating out individual microbes prior to sequencing. This issue of Single Cell Insights reviews peer-reviewed journal articles that describe applications and methodologies for single cell sequencing of environmental microbial communities.
Engineering plant immunity using crispr cas9 to generate virus resistanceSheikh Mansoor
Targeted genome editing by use of artificial nucleases has the plausible potential to speed basic research as well as plant breeding by providing the means to modify genomes quickly in a specific and predictable manner but advanced CRISPR-Cas9 based technologies first confirmed in mammalian cell systems are quickly being fitted for use in plants. These new technologies increase CRISPR-Cas9’s utility and effectiveness by diversifying cellular capabilities through expression construct system evolution and enzyme orthogonality, as well as enhanced efficiency through delivery and expression mechanisms. RNA-guided genome editing using Streptococcus pyogenes CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) has renewed the concept of genome editing in plants. CRISPR-associated surveillance complexes are easily programmable molecular sleds that can target any sequence of choice. These complexes offer new opportunities for implementation in biotechnology. Recent studies have used CRISPR/Cas9 to engineer virus resistance in plants, either by directly targeting and cleaving the viral genome, or by modifying the host plant genome to introduce viral immunity. The CRISPR/Cas9 platform could also be used for targeted mutagenesis to identify host factors that control plant resistance and susceptibility to viral infection. Thus, CRISPR/Cas9 technology offers a promising approach for under- standing and engineering resistance to single and multiple viral infections in plants.
This document describes a new method for specifically regulating genes between bacterial species using the CRISPR interference (CRISPRi) system. Researchers engineered a CRISPRi system on a conjugative plasmid to target and repress a fluorescent reporter gene (mRFP) in a recipient E. coli strain. The CRISPRi plasmid was transferred from a donor E. coli strain to the recipient strain through bacterial conjugation. When induced in the recipient, the CRISPRi system specifically repressed mRFP expression by 330-fold without affecting expression of another fluorescent reporter (sfGFP), demonstrating targeted gene regulation between bacterial cells via a natural horizontal gene transfer mechanism.
This document summarizes a seminar presentation on mixotrophy in land plants. Mixotrophy refers to the dual capability of both photosynthesis and organic carbon uptake. The presentation discusses the evolutionary pathways and diversity of mixotrophy strategies across eukaryotes. Tools for studying mixotrophy like isotopic tracing and genomic analysis are also reviewed. A case study examining the nuclear genomes of three mycoheterotrophic plants found profound reductions in photosynthesis and plastid-related genes, as well as increased substitution rates, demonstrating convergent evolution to heterotrophy.
In Vitro Analog of the Primitive Streak (ANIMATED)Nikolay Turovets
PLEASE DOWNLOAD TO SEE ANIMATION.
In vitro analog of the primitive streak: efficient derivation of highly enriched populations of hepatocytes from various types of human pluripotent stem cells.
May, 2011
1. The document discusses gene expression methods used to study the var2 and var3 genes of Plasmodium falciparum, which cause malaria.
2. Microarray, northern blotting, and RT-qPCR methods are described for analyzing var gene expression at different parasite stages. Studies found var2 mRNA is expressed at higher levels, associated with more severe malaria.
3. Understanding var gene expression is important as it leads to production of PfEMP1, which allows the parasite to bind red blood cells and evade the immune system, causing infection. Interfering with var gene expression may help circumvent malaria's dangers.
Candidemia in HIV-positive patients in Dschang District Hospital (West Region...Claude Nangwat
Candidemia has been identified as a public health problem in HIV-infected patients. The evaluation of CD4 count, transaminases and blood glucose, are being used as a means to monitor the health of HIV-infected patients, without excluding the diagnosis of candidemia and other opportunistic infections. In order to contribute in improving the care of HIV-infected patients attending Dschang District Hospital and later on, in other hospitals in Cameroon, we conducted from June to September 2014 a cross-sectional study, with general objective; to determine the association between candidemia and selected biochemical and haematological parameter changes in HIV-infected patients, as a possible indicator in monitoring HIV disease progression.
To do this, blood samples were collected from HIV-infected patients assigned to the UPEC of Dschang District Hospital for follow up, and haemogram report, CD4 counts, ALAT level, ASAT level, and glucose level in blood were evaluated by cytometric and spectrophotometric assays. Candida species were isolated from some blood samples, and then identified using CHROMagar Candida culture medium. The broth microdilution method was afterwards used to test the susceptibility of the fungal isolates vis-a-vis three conventional antifungal agents.
Mycological analysis of blood samples showed that eight (08) patients had candidemia, a prevalence of 6.11%. Eight (08) isolates were obtained from these eight (08) candidemic HIV-infected patients; this consisted of 4(50%) Candida albicans, 3(37.5%) Candida parapsilosis and 1(12.5%) Candida glabrata. All these isolates were resistant (MICs ranged from 2 to >256 µg/mL) to the antifungals used, that is, ketoconazole, amphotericin B and nystatin.
A significant correlation was found between candidemia and white blood cell count, with a correlation coefficient of r = 0.240 (p < 0.05). Based on the results obtained, the systematic diagnosis of candidemia should be performed in patients infected with HIV in Cameroon in order to improve on their care.
Key words: Candidemia, HIV, biochemical parameters, hematological parameters, Antifungals activities.
This document summarizes information about plastid transformation. Plastids are organelles found in plant cells that perform photosynthesis and store compounds. They have their own circular genome. Plastid transformation involves introducing foreign genes into the plastid genome using homologous recombination. It is preferred over nuclear transformation for traits because it allows high-level transgene expression and containment of transgenes within the plastid. The document discusses chloroplast structure, common vectors, selection markers, traits of interest for transformation including stress tolerance and pharmaceutical production, and limitations of plastid transformation technology.
This document discusses various methods of genetic recombination including transformation, conjugation, transduction, and protoplast fusion. It provides definitions and examples of key terms. Transformation involves the direct uptake of exogenous DNA by a cell. Conjugation is the transfer of genetic material between bacterial cells through direct contact. Transduction is the transfer of genes between bacteria mediated by bacteriophages. Protoplast fusion involves fusing plant protoplasts using electric shock or chemicals to produce somatic hybrid plants. The document also discusses applications of these techniques such as gene cloning, production of monoclonal antibodies, and genetic engineering.
This document summarizes previous research on the Epstein-Barr virus (EBV) and describes a study that mapped interactions between EBV proteins and human proteins. EBV infects 95% of humans and can cause cancers. The study used a yeast two-hybrid method to screen 216 EBV proteins against 15,483 human proteins, identifying 188 interacting pairs. Mapping these virus-host interactions may reveal how EBV disrupts cellular pathways to cause disease.
Hybridoma technology involves fusing antibody-producing B cells (splenocytes) with myeloma cells to produce immortal hybridoma cell lines that secrete monoclonal antibodies of a single specificity. Georges Köhler and César Milstein developed the technique in 1975 and were awarded the 1984 Nobel Prize for this discovery. The process involves immunizing an animal to generate B cells that produce antibodies against the target antigen. The B cells are isolated from the spleen and fused with myeloma cells using polyethylene glycol or electrofusion. The fused cells are selected in HAT medium, which allows only hybridomas expressing the hypoxanthine-guanine phosphoribosyl transferase enzyme to survive and multiply indefinitely. The resulting stable monoclonal antibody-
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
Plastids like chloroplasts contain their own circular DNA and are capable of expressing foreign genes at high levels. This document describes research where the human serum albumin (HSA) gene was successfully transformed into the tobacco chloroplast genome. Up to 11.1% of total soluble protein consisted of HSA, over 500 times higher than when introduced into the nuclear genome. The chloroplast transformation resulted in HSA accumulating in inclusion bodies, facilitating its purification. This demonstrated chloroplasts as an effective system for the high-level production of recombinant human therapeutic proteins.
This document discusses advances in HLA typing technologies over the past 25 years. It begins by outlining the evolution from low to high resolution typing methods like PCR-SSO, Sanger sequencing, and next generation sequencing using Illumina and PacBio platforms. Whole gene and long-range sequencing is now being done on over 80,000 samples using PacBio to type HLA Class I and II at high resolution without ambiguity. This has revealed over 5,000 novel variants, including insertions, deletions and nonsense mutations across exons and introns. Whole gene sequencing provides a more comprehensive view of variation than targeting only antigen recognition sites.
CRISPR/Cas9 is an advanced genome editing technology that can be used to develop plant disease resistance. It involves a Cas9 enzyme that acts like molecular scissors to cut DNA at specific locations guided by CRISPR RNA. This triggers DNA repair that can introduce changes to genes. Researchers have used CRISPR/Cas9 to develop resistance in plants against viruses, fungi, and bacteria by editing genes involved in host-pathogen interaction and disease susceptibility. It provides a precise and efficient way to edit plant genomes to improve crop resistance compared to previous tools. Scientists continue working to enhance the specificity and control of CRISPR/Cas9 for genome editing applications in agriculture.
The document provides a history of genomics, beginning with Mendel's work in 1866 establishing the gene concept and laws of genetics. Key developments include the discovery of DNA in 1871, the chromosomal theory of inheritance in 1902, and the discovery of linkage in 1910. The structure of DNA was elucidated in the 1950s, leading to the development of recombinant DNA technology in the 1970s and DNA sequencing techniques in the 1970s-80s. The first whole genome of an organism was sequenced in 1995. More recent developments include next generation sequencing starting in 2005 and the advent of CRISPR/Cas9 genome editing in 2012. The document concludes with discussing the potential use of CRISPR to target genes in the diamondb
Culture independent methods for detection & enumeration of gut microfloraAmna Jalil
This document discusses various culture-independent methods for detecting and enumerating gut microflora, including: 1) Design of PCR primers and hybridization probes to target specific species or groups; 2) PCR-ELISA to combine PCR amplification with ELISA detection; 3) Sequence analysis of randomly amplified 16S rRNA genes; 4) Temperature gradient gel electrophoresis (TGGE) and denaturing high performance liquid chromatography (DHPLC) to separate amplified DNA fragments by size; 5) Terminal restriction fragment length polymorphism (T-RFLP) to generate terminal restriction fragments for profiling; 6) Oligonucleotide arrays and quantitative real-time PCR for rapid, accurate detection of thousands of bacteria; 7) Fluorescence in
Karyotype variability in plant pathogenic fungi palm7016chaithram11
This document summarizes a seminar presentation on karyotype variability in plant pathogenic fungi. It discusses types of karyotype variability including chromosome number polymorphisms and chromosome length polymorphisms. Mechanisms that can lead to karyotype variability are described, such as meiotic recombination, DNA repair pathways, parasexual recombination, transposon-associated rearrangements, and lateral DNA transfer. Case studies on fungi like Mycosphaerella graminicola and Alternaria alternata are provided that demonstrate karyotype changes through these mechanisms. The importance of karyotype variability in the evolution and emergence of new virulence strains in fungi is also noted.
Agrobacterium and plant viruses can be used as biological vectors for plant transformation. Agrobacterium mediates transformation via its tumor-inducing plasmid, using virulence genes to transfer T-DNA containing the gene of interest into the plant genome. Plant viruses can also act as gene vectors by engineering viral genomes to contain and deliver foreign genes. Retroviruses have been developed as viral vectors for animal gene transfer due to their ability to stably integrate into the host chromosome.
Activation-of-human-immunodeficiency-virus-type-1-expression-by-Gardnerella-v...Farhad B. Hashemi, PhD
This study found that lysates from Gardnerella vaginalis, a bacteria commonly associated with bacterial vaginosis, significantly stimulated HIV expression in monocytoid cells and certain T cell lines. G. vaginalis lysates activated HIV long terminal repeat transcription and increased NF-kB binding activity in HIV-infected cells, indicating an effect on HIV transcription. The activation of HIV production by G. vaginalis suggests that G. vaginalis infection in the genital tract may increase the risk of HIV transmission by enhancing HIV expression levels in the genital tract. This could help explain the link between bacterial vaginosis and increased sexual transmission of HIV.
Single Cell Insights: Studying Environmental Microbial Communities Cell by CellQIAGEN
Dissecting complex microbial communities has incredible potential in the quest to decipher the world around us and find new sources of enzymes, antibiotics and other drugs. The challenge that remains is how to gain a deeper understanding of the roles and interactions of individual microbes. Single cell sequencing has emerged as an innovative investigational approach that provides a view of the cell-by-cell community by separating out individual microbes prior to sequencing. This issue of Single Cell Insights reviews peer-reviewed journal articles that describe applications and methodologies for single cell sequencing of environmental microbial communities.
Engineering plant immunity using crispr cas9 to generate virus resistanceSheikh Mansoor
Targeted genome editing by use of artificial nucleases has the plausible potential to speed basic research as well as plant breeding by providing the means to modify genomes quickly in a specific and predictable manner but advanced CRISPR-Cas9 based technologies first confirmed in mammalian cell systems are quickly being fitted for use in plants. These new technologies increase CRISPR-Cas9’s utility and effectiveness by diversifying cellular capabilities through expression construct system evolution and enzyme orthogonality, as well as enhanced efficiency through delivery and expression mechanisms. RNA-guided genome editing using Streptococcus pyogenes CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) has renewed the concept of genome editing in plants. CRISPR-associated surveillance complexes are easily programmable molecular sleds that can target any sequence of choice. These complexes offer new opportunities for implementation in biotechnology. Recent studies have used CRISPR/Cas9 to engineer virus resistance in plants, either by directly targeting and cleaving the viral genome, or by modifying the host plant genome to introduce viral immunity. The CRISPR/Cas9 platform could also be used for targeted mutagenesis to identify host factors that control plant resistance and susceptibility to viral infection. Thus, CRISPR/Cas9 technology offers a promising approach for under- standing and engineering resistance to single and multiple viral infections in plants.
This document describes a new method for specifically regulating genes between bacterial species using the CRISPR interference (CRISPRi) system. Researchers engineered a CRISPRi system on a conjugative plasmid to target and repress a fluorescent reporter gene (mRFP) in a recipient E. coli strain. The CRISPRi plasmid was transferred from a donor E. coli strain to the recipient strain through bacterial conjugation. When induced in the recipient, the CRISPRi system specifically repressed mRFP expression by 330-fold without affecting expression of another fluorescent reporter (sfGFP), demonstrating targeted gene regulation between bacterial cells via a natural horizontal gene transfer mechanism.
This document summarizes a seminar presentation on mixotrophy in land plants. Mixotrophy refers to the dual capability of both photosynthesis and organic carbon uptake. The presentation discusses the evolutionary pathways and diversity of mixotrophy strategies across eukaryotes. Tools for studying mixotrophy like isotopic tracing and genomic analysis are also reviewed. A case study examining the nuclear genomes of three mycoheterotrophic plants found profound reductions in photosynthesis and plastid-related genes, as well as increased substitution rates, demonstrating convergent evolution to heterotrophy.
In Vitro Analog of the Primitive Streak (ANIMATED)Nikolay Turovets
PLEASE DOWNLOAD TO SEE ANIMATION.
In vitro analog of the primitive streak: efficient derivation of highly enriched populations of hepatocytes from various types of human pluripotent stem cells.
May, 2011
1. The document discusses gene expression methods used to study the var2 and var3 genes of Plasmodium falciparum, which cause malaria.
2. Microarray, northern blotting, and RT-qPCR methods are described for analyzing var gene expression at different parasite stages. Studies found var2 mRNA is expressed at higher levels, associated with more severe malaria.
3. Understanding var gene expression is important as it leads to production of PfEMP1, which allows the parasite to bind red blood cells and evade the immune system, causing infection. Interfering with var gene expression may help circumvent malaria's dangers.
Insilico Comprehension of Stop Codon Readthrough in Human Virusesijtsrd
Readthrough is an event in which stop codon is misread, resulting in elongation of polypeptides. Stop codon suppression or termination codon readthrough, is a mechanism of expression of many disorder proteins. Many important cellular functions are carried out by way of the Readthrough process. This could alter the gene function which thereon produces either destructive or constructive effects. Hence, this study aims to diagnose this recoding mechanism in certain selected humans infecting pathogenic viruses through insilico approach. For this target, the 3'UnTranslated Regions of the selected viruses were retrieved from the Genbank database. Each of these 3'UTRs were translated into all their reading frames. Motif search using Interproscan in each of the frames, followed by homology search using BLASTX, and were achieved to identify stop codon readthrough candidates in each of the selected viruses. Finally, the secondary structure of RNA was predicted using RNAFold web server to ensure the stability of the RNA. The 3'UTRs from Aichi Virus 1, Cosa Virus A, Dengue Virus 1, Duvenhage Lyssavirus, Enterovirus A, HepatitisGB Virus B, Human Cosavirus A, Human Pegivirus 2, Langat Virus, Parechovirus A, WestNile Virus and Zika Virus were retrieved. A total of 48 motifs were identified in different reading frames of 3'UTR of the selected viruses. BlastX search recognized 9 homologs in the reading frames of 3'UTR. The secondary structure analysis and search of motifs and homologs resulted in the confirmation of 5 candidates with strong evidence for the readthrough event. These candidates showed homology with proteins of prime importance such as Imidazole glycerol Phosphate synthase protein, 50S ribosomal protein L27, DNA replication, and repair protein, replication origin binding protein, and adenosine deaminase. Hence, we proved that the 3'untranslated regions would undergo translation. This strongly suggests that many such readthrough events are to be determined to exactly unravel the pathogenicity behind Viruses. To design anti viral drugs to impede this viral machinery, it is essential to analyse their 3'UTR regions. Arockiyajainmary M | Balaji S | Sivashankari Selvarajan "Insilico Comprehension of Stop Codon Readthrough in Human Viruses" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-4 | Issue-4 , June 2020, URL: https://www.ijtsrd.com/papers/ijtsrd31550.pdf Paper Url :https://www.ijtsrd.com/biological-science/biotechnology/31550/insilico-comprehension-of-stop-codon-readthrough-in-human-viruses/arockiyajainmary-m
1) The study examined the effects of infliximab treatment on Crohn's disease patients infected with Mycobacterium avium ssp. paratuberculosis (MAP), which has been associated with Crohn's disease.
2) Patients treated with infliximab showed significantly decreased levels of antibodies against two MAP proteins compared to untreated patients, suggesting infliximab decreases secretion of these proteins.
3) The study also found infliximab treatment was detrimental to the survival of MAP within infected macrophages, with significantly decreased survival of MAP in macrophages exposed to infliximab.
This document summarizes the potential of RNA interference (RNAi) technology for crop improvement. It discusses how RNAi was discovered through early experiments in plants in the 1990s. The mechanism of RNAi involves long double-stranded RNA being cleaved by an enzyme called Dicer into small interfering RNAs (siRNAs) that are incorporated into a protein complex called RISC that targets and degrades complementary mRNAs, preventing gene expression. The document outlines several successful applications of RNAi technology for increasing biotic stress tolerance in crops against viruses, bacteria, fungi and insects by silencing key pathogen genes. It also discusses using RNAi to modify other crop traits like nutritional quality and abiotic stress resistance.
This ppts is based upon the recent adavancement and methodology about mitochondrial transformation. What is organellar transformation and what is the importance in contemporary time.
RNA interference (RNAi) is a biological process in which RNA molecules inhibit gene expression, typically by causing the destruction of specific mRNA molecules. Historically, it was known by other names, including co-suppression, post-transcriptional gene silencing (PTGS), and quelling. Only after these apparently unrelated processes were fully understood did it become clear that they all described the RNAi phenomenon. Andrew Fire and Craig C. Mello shared the 2006 Nobel Prize in Physiology or Medicine for their work on RNA interference in the nematode worm Caenorhabditis elegans, which they published in 1998. Since the discovery of RNAi and its regulatory potentials, it has become evident that RNAi has immense potential in suppression of desired genes. RNAi is now known as precise, efficient, stable and better than antisense technology for gene suppression. Two types of small ribonucleic acid (RNA) molecules – microRNA (miRNA) and small interfering RNA (siRNA) – are central to RNA interference. RNAs are the direct products of genes, and these small RNAs can bind to other specific messenger RNA (mRNA) molecules and either increase or decrease their activity, for example by preventing an mRNA from producing a protein. RNA interference has an important role in defending cells against parasitic nucleotide sequences – viruses and transposons. It also influences development.
Fernando de la Cruz presented a document on plasmid conjugation systems and controlling the dissemination of antibiotic resistance. The document contained the following key points:
1. The only element common among all conjugatively transmissible plasmids is the relaxase protein. Conjugative plasmids also contain VirB4 proteins.
2. Different types of plasmids - conjugative, mobilizable, and non-transmissible - were analyzed based on size. IncF and IncI plasmids frequently transfer antibiotic resistance.
3. COINs (conjugation inhibitory compounds) show potential to control the dissemination of multidrug resistance by inhibiting bacterial conjugation without affecting growth.
This document provides information on CRISPR Cas9 genome editing. It discusses the history and discovery of CRISPR dating back to 1987. It describes the key components of the CRISPR Cas9 system including Cas9 proteins, CRISPR RNA, protospacers, and PAM sequences. The mechanisms of how CRISPR Cas9 edits genomes through double strand breaks is explained. Finally, applications of CRISPR Cas9 are summarized, including using it to correct genetic mutations causing diseases in animals and potential applications in humans.
This document describes the development of a multiplex PCR assay targeting the cgcA gene, which encodes a diguanylate cyclase, to differentiate between species within the genus Cronobacter. Analysis of 12 Cronobacter genomes identified 7 conserved diguanylate cyclase-encoding genes, one of which, cgcA, showed species-specific divergence that matched known phylogenetic relationships between Cronobacter species. Primers were designed for this gene and tested in a multiplex PCR assay on 305 Cronobacter isolates representing 6 species. The assay correctly identified the species of all isolates tested and did not identify any of 20 non-Cronobacter species, demonstrating high specificity and sensitivity for rapid identification of Cronobacter.
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 study analyzed 264 gastric cancers for mutations in exons 9 and 20 of the PIK3CA gene. PIK3CA mutations were found in 42 cases (16%), all heterozygous missense mutations. The most common mutation was H1047R in exon 20 (62% of mutations) and the second most common was Q546K in exon 9 (9.5% of mutations). A meta-analysis of 27 publications found that the ratio of exon 20 to exon 9 mutations varied by cancer type, being highest in gastric cancer. The exon mutation selectivity is a signature of the cancer type.
1) Researchers mapped a genetic locus (belr1) that controls resistance to malaria liver stage infection in mice to a region containing the Trem2 gene.
2) They found that Trem2 expression closely correlates with resistance, and that Kupffer cells (liver macrophages) are the main cells expressing TREM2.
3) Trem2-deficient mice showed increased susceptibility to liver stage infection. TREM2 expression on Kupffer cells enables them to control parasite expansion in hepatocytes in vitro.
This document summarizes a study that identified genes in the extracellular matrix that regulate the susceptibility of cultured cells to prion infection. The study compared gene expression in prion-susceptible and resistant cell lines. They identified 9 genes, including fibronectin 1 and integrin α8, that were upregulated in resistant cells. Knockdown of these genes increased susceptibility by altering the extracellular matrix structure and deposition of prion proteins. The results suggest the extracellular matrix plays a key role in controlling prion infection by influencing how prion proteins interact and convert forms.
Rose and Desmolaize et al 2012_AAC Publication for Puneet JajuPuneet Jaju
This document describes a multiplex PCR assay that can rapidly and reliably detect three macrolide resistance genes - erm(42), msr(E), and mph(E) - in the bacterial pathogens Mannheimia haemolytica and Pasteurella multocida. The assay also distinguishes between these two bacteria by amplifying distinct fragments of their 23S rRNA genes. The multiplex PCR was tested on over 40 resistant isolates and correlated with their macrolide MICs and whole genome sequencing results, demonstrating it can accurately determine macrolide resistance genotypes and the bacterial genus in a single test.
The CDKN2A/CDKN2B locus on chromosome 9p21 encodes three tumor suppressor proteins - p16INK4A, p15INK4b, and p14ARF - that regulate the cell cycle and inhibit tumor growth. A long non-coding RNA called ANRIL overlaps this locus and induces epigenetic silencing of CDKN2A/CDKN2B by recruiting polycomb repressive complexes. Genetic variants in ANRIL are associated with increased risk of several diseases by impacting the expression of these tumor suppressors.
This research project aims to identify potential drug candidates for COVID-19 through computational molecular docking of secondary metabolites from natural products against SARS-CoV-2 viral proteins, followed by immunochemistry experiments. Over 100 secondary metabolites isolated from plants are being evaluated based on their known antiviral activities. Preliminary molecular docking and dynamics simulations have identified several compounds including Cordifolioside-A, Palmitoside-G and Tinosinenoside-A that show strong binding to SARS-CoV-2 spike and envelope proteins.
1. The document discusses plant viral RNA synthesis in cell-free systems using alfalfa mosaic virus (AMV) as a model. Natural minus-strand RNAs of AMV were tested as templates for viral RNA polymerase in vitro but did not support full-size plus-strand synthesis, with only minus-strand RNA 3 producing a small amount.
2. Two studies examined interactions between subunits of the AMV RNA-dependent RNA polymerase (RdRp). One found that coat protein is not required for minus-strand synthesis but inhibits it, and stimulates plus-strand synthesis. The other showed RdRp can unwind double-stranded RNA templates.
3. Replication complexes of AMV were found
The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
All manuscripts are subject to rapid peer review. Those of high quality (not previously published and not under consideration for publication in another journal) will be published without delay.
Identification and Characterization of Saccharomyces cerevisiae Cdc6 DNA-bind...gan-navi
This document summarizes a study that identified Saccharomyces cerevisiae Cdc6 as a DNA-binding protein. It was found that purified Cdc6 protein can bind double-stranded DNA with a dissociation constant of about 1 x 10-7 M. The minimal DNA-binding domain was mapped to the N-terminal 47 amino acids of Cdc6. Mutating the 29KRKK region abolished Cdc6's DNA-binding activity and its ability to complement cdc6 mutant cells. The results suggest Cdc6 may initiate DNA replication by interacting with DNA at replication origins through its DNA-binding activity.
Adhd Medication Shortage Uk - trinexpharmacy.comreignlana06
The UK is currently facing a Adhd Medication Shortage Uk, which has left many patients and their families grappling with uncertainty and frustration. ADHD, or Attention Deficit Hyperactivity Disorder, is a chronic condition that requires consistent medication to manage effectively. This shortage has highlighted the critical role these medications play in the daily lives of those affected by ADHD. Contact : +1 (747) 209 – 3649 E-mail : sales@trinexpharmacy.com
Muktapishti is a traditional Ayurvedic preparation made from Shoditha Mukta (Purified Pearl), is believed to help regulate thyroid function and reduce symptoms of hyperthyroidism due to its cooling and balancing properties. Clinical evidence on its efficacy remains limited, necessitating further research to validate its therapeutic benefits.
Osteoporosis - Definition , Evaluation and Management .pdfJim Jacob Roy
Osteoporosis is an increasing cause of morbidity among the elderly.
In this document , a brief outline of osteoporosis is given , including the risk factors of osteoporosis fractures , the indications for testing bone mineral density and the management of osteoporosis
- Video recording of this lecture in English language: https://youtu.be/kqbnxVAZs-0
- Video recording of this lecture in Arabic language: https://youtu.be/SINlygW1Mpc
- Link to download the book free: https://nephrotube.blogspot.com/p/nephrotube-nephrology-books.html
- Link to NephroTube website: www.NephroTube.com
- Link to NephroTube social media accounts: https://nephrotube.blogspot.com/p/join-nephrotube-on-social-media.html
These lecture slides, by Dr Sidra Arshad, offer a quick overview of the physiological basis of a normal electrocardiogram.
Learning objectives:
1. Define an electrocardiogram (ECG) and electrocardiography
2. Describe how dipoles generated by the heart produce the waveforms of the ECG
3. Describe the components of a normal electrocardiogram of a typical bipolar lead (limb II)
4. Differentiate between intervals and segments
5. Enlist some common indications for obtaining an ECG
6. Describe the flow of current around the heart during the cardiac cycle
7. Discuss the placement and polarity of the leads of electrocardiograph
8. Describe the normal electrocardiograms recorded from the limb leads and explain the physiological basis of the different records that are obtained
9. Define mean electrical vector (axis) of the heart and give the normal range
10. Define the mean QRS vector
11. Describe the axes of leads (hexagonal reference system)
12. Comprehend the vectorial analysis of the normal ECG
13. Determine the mean electrical axis of the ventricular QRS and appreciate the mean axis deviation
14. Explain the concepts of current of injury, J point, and their significance
Study Resources:
1. Chapter 11, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 9, Human Physiology - From Cells to Systems, Lauralee Sherwood, 9th edition
3. Chapter 29, Ganong’s Review of Medical Physiology, 26th edition
4. Electrocardiogram, StatPearls - https://www.ncbi.nlm.nih.gov/books/NBK549803/
5. ECG in Medical Practice by ABM Abdullah, 4th edition
6. Chapter 3, Cardiology Explained, https://www.ncbi.nlm.nih.gov/books/NBK2214/
7. ECG Basics, http://www.nataliescasebook.com/tag/e-c-g-basics
One health condition that is becoming more common day by day is diabetes.
According to research conducted by the National Family Health Survey of India, diabetic cases show a projection which might increase to 10.4% by 2030.
Integrating Ayurveda into Parkinson’s Management: A Holistic ApproachAyurveda ForAll
Explore the benefits of combining Ayurveda with conventional Parkinson's treatments. Learn how a holistic approach can manage symptoms, enhance well-being, and balance body energies. Discover the steps to safely integrate Ayurvedic practices into your Parkinson’s care plan, including expert guidance on diet, herbal remedies, and lifestyle modifications.
1. International Journal of Pharmaceutical Science Invention
ISSN (Online): 2319 – 6718, ISSN (Print): 2319 – 670X
www.ijpsi.org Volume 3 Issue 3‖ March 2014 ‖ PP.38-46
www.ijpsi.org 38 | P a g e
CarD-a reliable target in M.tuberculosis
1
V.G.ShanmugaPriya , 2
Uday M. Muddapur , 3
Kailas D.Sonawane , 4
Megha
Mehta
1,2,4
R & D Center, Department of Biotechnology, KLE Dr.M.S.Shesgiri College of Engineering and
Technology, Belgaum-8, Karnataka, India.
3
Department of Microbiology, Shivaji University, Kolhapur-4,Maharashtra, India
Corresponding author: priyaabhirajan@gmail.com
ABSTRACT :Tuberculosis remains a major health problem being the second leading cause of death from an
infectious disease worldwide. The largest increases between 2011 and 2012 were in India, South Africa and
Ukraine. (WHO Global tuberculosis report - 2013). Because most TB drugs are less efficient in killing slowly
replicating or dormant bacilli in the chronic phase of TB infection, a key challenge for identifying sterilizing
drugs stands before humanity. Identifying potential targets in the organism M.tuberculosis will aide in rapid
drug finding process. One recommended target is CarD. CarD proteins are highly conserved in many eubacteria
and are absent from archaebacteria and eukaryotes (Cayuela et al., 2003), but its exact function is yet to be
discovered. It is revealed to be an transcription factor protein in M.tuberculosis and has been found to be
essential for the organisms survival in the stringent host environment. Of the two domains –the N-terminal
domain interacts with RNA polymerase and is said to regulate transcription of few ribosomal proteins and
rRNA. The C-terminal domain, whose function was not known for a long time is suspected to interact with
DNA and has a unique fold. The predictions and analysis of this protein, regarding its importance in survival of
the organism, its structure, along with its known and suspected function in various studies are reviewed in this
paper.
KEYWORDS: CarD protein, M.tuberculosis, transcription regulation
I. INTRODUCTION
During Tuberculosis infection, in the lungs, within the granuloma, the infected macrophages are
activated to destroy the bacteria- M. tuberculosis with which they are infected (Kaufmann SH., 2002). The
infected cells restrain mycobacteria from proliferating by imposing an arsenal of oxidative stress, hypoxia, acid
stress, genotoxic stress, cell surface stress, and starvation. Despite this onslaught of attacks, M. tuberculosis is
able to persist for the lifetime of the host, indicating that this pathogen has substantial molecular mechanisms for
resisting host defenses. To persist in this hostile environment M.tuberculosis enters a state of dormancy and
rapidly down regulates ribosome biogenesis to match declining translational need. Generally, Bacteria
accomplishes this via the stringent response, a global regulatory mechanism in which transcription of stable
RNAs is inhibited, in part by the production of the hyperphosphorylated guanine nucleotides ppGpp and
pppGpp (Avarbock et al., 2000; Magnusson et al.,2005). DksA protein potentiates the effect of (p)ppGpp by
directly binding the RNAP (Paul et al.,2004). DksA homologs are absent in some divisions of bacteria, whereas
(p)ppGppsynthetases are broadly distributed. Recent work (Stallings et al., 2009) has identified CarD protein
interacts with RNAP β subunit to control rRNA transcription in Mycobacterium tuberculosis and this interaction
is vital for Mtb’s survival during the persistent infection state. It was found that CarD is essential for viability,
in that declining carD transcript levels directly correlated with cell death. CarD depletion leads to an up
regulation of 16s rRNA and rpsH ribosomal protein transcripts in the organism. Mutant cells lacking CarD were
found to be highly susceptible to antibiotics, when compared to wild strains. Hence CarD is suggested to be a
potential drug target (Stallings et al., 2009).
II. CarD
M. tuberculosis carD (Rv3583c) has earlier been identified as a transcriptionally upregulated gene in
microarray experiments after exposure to the DNA-damaging agents mitomycin C, UV radiation, H2O2, and
quinolone DNA gyrase inhibitors (Boshoff et al., 2004).Also earlier work in M. xanthus had identified a paralog
of CarD that contains an HMGA domain and is said to be involved in fruiting body formation and
carotenogenesis but which is nonessential (Padmanabhan,2001). The same research group examined the
function of the M. xanthus ortholog of mycobacterial CarD called CdnL (CarD N-terminal Like).
2. CarD-A Reliable Target In M.tuberculosis
www.ijpsi.org 39 | P a g e
(Garcia-Moreno,2010) to distinguish this protein from the HMGA domain containing CarD. Studies
in B.burgdorferi have also identified a CarD homolog, LtpA, that is induced at low temperatures(Yang
XF.,2008).
III. ESSENTIABILITY
A broad range of experiments and assays were carried out by Stallings et al, which threw light on the
essentiability of this protein in M.tuberculosis and M.smegmatis. In one experiment, the transcriptional
induction of carD after I-SceI-generated double stranded break in M.smegmatis was quantified by DNA
microarray and verified by quantitative real-time PCR (qRT-PCR).Wild-type M. smegmatis cultures when
treated with double-strand DNA-damaging agents bleomycin and Ciprofloxacin, alkylating agent methyl
methanesulphonate (MMS), and the oxidizing agent hydrogen peroxide (H2O2), it was observed that carD
transcript increased with all genotoxins tested except high doses of H202, with the most dramatic induction
observed with Ciprofloxacin and 10 mM H2O2, which induced carD transcription 8-fold compared to untreated
cells. Survival assays and quantitative RT-PCR showed that declining carD transcript levels directly correlated
with cell death which indicated that CarD is essential for viability. In the other experiment, it was found that
mycobacterial cells lacking CarD were 50,000-fold more sensitive to 10 mM H2O2 and 30 times more sensitive
to Ciprofloxacin treatment compared to control cells producing CarD. also CarD depletion caused a 10-fold
reduction in M. smegmatis survival during starvation. These findings indicated that CarD is necessary for
survival during oxidative stress, double stranded DNA breaks and nutrient limitation. Also, with M. smegmatis
cells, the microarray analysis revealed that during CarD depletion, 193 genes were upregulated >2-fold and 176
genes were downregulated >2-fold. Later, the microarray and qRT-PCR experiments in M. smegmatis and M.
tuberculosis demonstrate that CarD depletion leads to transcriptional upregulation of stable RNAs and other
components of the translation machinery such as 16S rRNA and rpsH ribosomal protein transcripts. Also in
their studies it was found that M. tuberculosis CarD is necessary for replication and persistence during infection
of mice (Stallings et al.,2009). Chromatin immunoprecipitation (ChIP) experiments in M. smegmatis with
antibodies targeted against CarD precipitated the promoters regulating the rRNA operons or the rplN operon
(encoding ribosomal proteins L14, L24, L5, and S14p/S29e), thus confirming that CarD is physically present on
the RNAP complex at the rRNA and ribosomal protein loci. It was analyzed that the microorganisms in which
CarD is known or suspected to be essential (Mycobacteria, M. xanthus and B. burgdorferi) all contain 4 or fewer
rRNA operons (Stallings et al.,2009; Garcia-Moreno et al.,2010; Yang XF et al.,2008) Whereas, in organisms
like B. subtilis, where CarD is non essential 10 or more rRNA operons are found. (Kobayashi et al 2003)
IV. COMPARISON WITH DKSA
It was known that the universal protein (p)ppGpp participates in stringent control by downregulating
rRNA formation. In E. coli, the DksA protein potentiates the effect of (p)ppGpp by directly binding the RNAP.
DksA protein decreases the open complex half- life during transcription initiation and thereby known to
downregulate transcription from the P1 promoter of the E.coli rRNA rrnB operon. In a DksA deletion strain,
rRNA transcription is insensitive to (p)ppGpp accumulation, thus leaving rRNA promoters unresponsive to
changes in amino acid availability and growth rate (Paul et al., 2004). Deletion of DksA has pleiotropic effects
including DNA damage sensitivity which have not been clearly linked to its effect on transcription initiation
(Branny et al.,2001; Magnusson et al., 2007). Despite the intricate functional relationship between DksA and
(p)ppGpp in E. coli, obvious DksA homologs are absent from most nonproteobacteria, whereas (p)ppGpp
synthetases are broadly distributed, possibly indicating that other factors exist to control ribosomal biogenesis in
these organisms.In mycobacteria, a protein CarD was found to bind the RNAP to control rRNA
transcription.To test whether CarD could replace DksA in E. coli, M.smegmatis CarD is expressed in ΔdksA
strain (Magnusson et al., 2007) from an E. coli promoter. This restored ΔdksA growth on minimal media almost
to the extent as DksA itself. During nutrient limitation in the ΔdksA strain, rRNA levels was measured
during starvation and found that CarD was able to decrease the elevated rRNA levels indicating that CarD
could replace DksA in the stringent response.
Structural modeling and experimental evidence support a model in which DksA exerts its effects on the
RNAP through interactions within the secondary channel. In contrast, the CarD N-terminus has a TRCF
RID(Transcription repair coupling factor - RNA polymerase interacting Domain) module and interacts with the
N terminus of the RNAP β subunit, where TRCF binds. Accordingly, TRCF RID alone can mimic the effect of
CarD on rRNA transcription in an E.coli DdksA strain. These results suggest that CarD affects rRNA
transcription through a different mechanism than DksA. Thus CarD can complement an E. coli DdksA strain,
but in contrast to E. coli DksA, CarD is required for mycobacterial viability under all conditions, both in culture
and during all stages of mouse infection whereas E. coli DksA is dispensable in nutrient-rich media (Stallings et
al, 2009). Loss of DksA or CarD causes similar phenotypes despite having different interaction sites on RNAP.
In a recent paper, Tehranchi et al. show that, in addition to its role in regulating rRNA transcription initiation,
3. CarD-A Reliable Target In M.tuberculosis
www.ijpsi.org 40 | P a g e
DksA ensures progression of DNA replication in E.coli by removing transcription roadblocks during nutrient
deprivation. This leads to a speculation that CarD might play a role in resolving conflicts between transcription
and DNA replication and that this function is required for the viability of the mycobacterial cell (Stallings and
Glickman,2010).
V. COMPARISON WITH TRCF
TRCF (Transcription repair coupling factor), encoded by the mfd gene, is necessary for DNA strand-
specific repair during transcription. A lesion in the template strand blocks the RNA polymerase complex. The
RNAP-DNA-RNA complex is specifically recognized by TRCF which releases RNAP and the truncated
transcript. The TRCF may replace RNAP at the lesion site and then recruit the uvrA/B/C repair system
(Westblade et al.,2010;Chambers et al.,2003). Abundant evidence indicates that the TRCF RID(RNAP β
Interacting Domain) interacts directly with the N terminus of the RNAP βsubunit (Deaconescu et al.,2006).
Earlier,with bacterial two-hybrid assay, it was noticed that the N-terminal domain of T. thermophilus CarD,
which shares homology with the TRCF RID and M. tuberculosis CarD, interacts with the N-terminus of RNAP
β (amino acids 10–133) to the same degree as did the TRCF RID. Full-length T. thermophilus CarD also
interacted strongly with RNAP β amino acids. These data demonstrate that the mode of binding of CarD to
RNAP is the same as TRCF. Despite the similarities between the N terminus of CarD and the TRCF RID,
attempts to complement CarD depletion with the M. smegmatis TRCF RID were unsuccessful, indicating that
RNAP binding is not sufficient for CarD activity. Additionally, an M. smegmatis Dmfd strain does not
phenocopy depletion of CarD in that TRCF is not essential in mycobacteria, loss of TRCF does not affect
mycobacterial sensitivity to oxidative or genotoxic stress, and rRNA levels are regulated normally in Dmfd both
during normal growth and in conditions that elicit stringent control. Therefore, despite the similar mechanism of
interaction with the RNAP, CarD and TRCF have distinct cellular functions (Stallings et al.,2009).
Comparison of the predicted 3D structure of CarD of M.tuberculosis with TRCF RNAP-β interacting
domain (TRCF-RID; PDB ID: 3MLQ) and Thermus thermophilus CdnL-NTD [PDB ID: 2LQK) predicted that
one of the major differences between the structures of CarD-NTD and TRCF-RID/CdnL-NTD lies in the
curvature of β sheet formed by β1 and β2 strands. The reduced curvature in the β sheet of CarD-NTD may have
a physiological implication, as one of the key interactions predicted between the CarD R25 residue and RNAP-β
E1385 would require large conformational rearrangements. High B-factor in this region of the CarD structure,
suggests the possibility for such conformational rearrangements (Kaur et al.,2013).The X-ray crystal structure
(PDB ID 3MLQ) of the T. thermophilus TRCF RID complexed with the Thermus aquaticus RNAP β1 domain
revealed that T. thermophilus TRCF RID R341 forms a hydrogen bond with RNAP β1 Q99, as well as polar
interactions with RNAP β1 E110. The RNAP β1 residues I108 and K109 are also central to the protein/protein
interface, making extensive van der Waals contacts with the TRCF RID .To extrapolate these structural insights
to mycobacteria, the T.aquaticus β1 sequence was aligned with the β1 domain from M.tuberculosis and
concluded that residues E138, I147, K148, and S149 of M. tuberculosis RNAP β1 correspond to residues Q99,
I108, K109, and E110 in T.aquaticus RNAP β1, respectively. Bacterial two-hybrid experiments were performed
between the M. tuberculosis TRCF RID and RNAP β1 and between M.tuberculosis CarD RID and RNAP β1
harboring one or more mutations in these residues.
The M. tuberculosis RNAP β1E138R
and RNAP β1I147A
substitutions had dramatic effects on the strength
of the interaction with the TRCF RID and CarD RID by weakening the interaction. In contrast, alanine
substitutions at M. tuberculosis RNAP β1 K148 or S149 did not inhibit the association with the TRCF RID but
weakened CarD RID binding. They therefore concluded that, CarD and TRCF associate with the RNAP β
subunit in similar manners, although with subtle differences (Weiss et al.,2014).Later when M.tuberculosis
CarD/RNAP complex structure was solved, comparison of Mtb CarD/RNAP and Tth TRCF-RID/β1 complex
structures reveals that CarD and TRCF-RID display a similar set of interactions with RNAP, even though there
is no sequence conservation between the CarD β4- (43DLTVRVP49) and TRCF β4- (358EGKLYLP364)
strands that interact with the RNAP β1 domain except for the last proline residues(Gulten et al.,2013).In a
computational study, the proteins interacting with CarD and TRCF of Mycobacterium species was known by
sequence comparison against STRING database and were compared and analyzed. DNA directed RNA
polymerase subunit β and 50 s ribosomal protein L25 are common proteins who have functional association
with CarD and TRCF and relates to their common function. Two proteins 2-C-methyl –D-erythritol 2,4 –
cyclodiphosphate synthase and 2-c-methyl-D-erythritol 4-phosphate cytidylyl transferase, both of which are
involved in isoprenoid biosynthesis pathway and are already recognized as drug targets were found to be
functionally associated with CarD (based on its genomic neighborhood) and not with TRCF. As Isoprenoids are
essential for the survival of the organism (Heuston et al,2012;Beutow et al,2007) this gives a clue of possible
function of CarD other than RNAP interaction ,which makes it essential for the survival of the organism, when
compared with TRCF(Priya et al.,2012).
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VI. N-TERMINAL DOMAIN (NTD)
Earlier, being not able to obtain suitable crystals of M.tuberculosis CarD, Srivatsa et al, determined the
structure of CarD from Thermus thermophilus at 2.4 Å resolution, as it has 44% sequence similarity with
M.tuberculosis CarD. The T. thermophilus CarD structure, was found to comprise of Tudor-like fold of the N-
terminal domain, in common with the E.coli TRCF-RID. T.thermophilus CarD was crystallized with two
molecules in the asymmetric unit, so two crystallographically independent structures were refined. Despite
having unique crystal packing environments, the two molecules are nearly identical in structure (rmsd of 0.965
Å over 158 α-carbon positions), indicating that the relative orientation of the two CarD structural domains is
rigidly maintained. This is likely due to a small but significant interface between the two domains (∼810 Å2
buried accessible surface area) that includes a network of conserved interactions. Two conserved charged
residues (CarD-RID-R60 and CarD-CTD-E139) form a partially buried, inter domain salt bridge. CarD-RID-
R60 is also in position to hydrogen bond with the carbonyl-oxygen of conserved CarD-CTD-G100. Conserved
hydrophobic residues V11 and V17 make van derWaals interactions with the alkyl chains of R60 and E139 as
well as with the side chain of CarD-CTD(C-terminal domain) residue 142 (Q in T. thermophilus CarD, but
conserved as a hydrophobic residue in the larger family of CarD proteins) (Srivastava et al.,2013). Later with
the crystal structure (PDB ID:4ILU) it was confirmed that CarD of M.tuberculosis has two domains, where the
N-terminal domain (CarD-NTD) forms an antiparallel β sheet (β1–β3) and the C terminal domain (CarD-CTD)
adopts predominantly an all-α (α1-α5) conformation, a characteristic of (α+β) family of proteins ( Kaur et
al.,2013).Also in the structure (PDB ID: 4KBM) M.tuberculosis CarD is seen to composed of two distinct
domains: an all β-stranded N-terminal domain (residues 1–49) and an all α-helical C-terminal domain (residues
63–160). The N- and C-terminal domains are connected by a six residue twisted α helix (α1) and an eight
residue loop. The N-terminal domain has a Tudor-like fold (Selenko et al., 2001) consisting of four anti parallel
β strands. Residues Thr26, Ile27, Lys28, and Gly29, which lie on the β-turn connecting the β2 and β3 strands,
were the only residues disordered in the N-terminal domain(Gulten et al.,2013).
Interaction with RNA polymerase:
To identify residues in CarD that are important for the CarD/RNAP interaction, a homology model of
the M. tuberculosis CarD RID interacting with the RNAP β1 domain was generated based on the structure of
the Thermus sp. TRCF RID/RNAP β1 complex (PDB ID 3MLQ). According to modeled
structure, M.tuberculosis CarD RID R25 interacts with M. tuberculosis RNAP β1 E138 (homologous to
the Thermus sp. TRCF-RID R341: RNAP β1 Q99 interaction). In addition to M. tuberculosis CarD RID R25,
the model structure predicts that another arginine that at position 47, would interact with M. tuberculosis RNAP
β1 E138. Arginine-to-glutamic acid substitutions at position 25 or 47 in CarD RID bacterial two-hybrid
constructs was made and CarD and RNAP β1 interaction was tested. Either substitution on its own greatly
diminished the ability of these proteins to interact, with CarD RIDR25E
having the most severe effect. A double
point mutation (CarD RIDR25E,R47E
) completely abolished the interaction. They were able to fully suppress the
effect of the CarD RIDR47E
mutation with the RNAP β1E138R
substitution, confirming the specific interaction
between these two residues predicted from the structural model. The RNAP β1E138R
mutation did not suppress
the effects of the CarD RIDR25E
mutation, suggesting that the CarD RIDR25E
substitution disrupts additional
contacts between these proteins. This is supported by the Thermus sp. TRCF RID/β1 structure,
where Thermus sp. TRCF RID R341 (corresponding to M. tuberculosis CarD RID R25) interacts with multiple
residues in the β1 subunit. The carD gene of M. smegmatis and M. tuberculosis was replaced with the alleles
encoding CarDR25E
, CarDR47E
, or the CarDR25E,R47E
double mutant using a gene-switching technique .It was not
possible to engineer a viable CarDR25E,R47E
double mutant, which is incapable of interacting with RNAP β1,
indicating that the interaction between CarD and the RNAP is important for viability. In fact, even the
CarDR25E
substitution was unattainable in M. tuberculosis, again emphasizing that attenuating the CarD/RNAP
β1 interaction compromises survival (Weiss et al., 2012).Co-immuno precipitation experiments in mycobacteria
demonstrated that even though the intracellular levels of CarD and RNAP β were unchanged, CarDR25E
and
CarDR47E
mutants co-precipitated less RNAP β than CarDWT
. These data confirmed that the R25E and R47E
substitutions in CarD compromise the association with RNAP and emphasize the importance of the CarD/RNAP
β1 interaction for mycobacterial growth and survival. In light of these findings, it was recommended that small
molecules that interfere with the CarD/RNAP β1 interaction could be promising novel chemotherapies for
killing M. tuberculosis (Weiss et al.,2012).
During infection, M. tuberculosis must withstand an arsenal of host-derived stresses, including the
production of reactive oxygen species by the oxidative burst. Log-phase cultures of M. tuberculosis strains
expressing different carD alleles were treated with 25 mM H2O2 before dilutions were plated to count the
surviving CFU and found that all CarD mutants were more sensitive to H2O2 treatment than were the wild type,
with the M. smegmatis CarDR25E
mutant being more sensitive than was M. smegmatis expressing CarDR47E
,
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demonstrating that the strength of the CarD/RNAP β1 interaction directly correlates to survival in the presence
of reactive oxygen species(Weiss et al., 2012).Both M.smegmatis and M.tuberculosis strains expressing
CarDWT
,CarDR25E
(M. smegmatis only), or CarDR47E
(M. smegmatis and M. tuberculosis) were transiently treated
with rifampin and monitored survival by plating for CFU. In both mycobacterial species, the strength of the
CarD interaction with RNAP directly correlated to survival during rifampin treatment, in that weakening the
association of CarD with RNAP increases the potency of rifampin. In a similar experiment, depleting
CarDWT
levels did not make M. smegmatis more sensitive to rifampin treatment. This implies that it is the
specific interference of the CarD/RNAP interaction that compromises the bacteria's survival during rifampin
treatment(Weiss et al., 2012).
It was previously known that depleting CarD increased the sensitivity of M. smegmatis to ciprofloxacin
treatment (Stallings et al., 2009). In contrast, it was found weakening the M. smegmatis CarD/RNAP β1
interaction had no effect on sensitivity to 10 μg/ml ciprofloxacin treatment in 2 h of transient-treatment liquid
culture assays. The finding that ciprofloxacin susceptibility is unresponsive to changes in the interaction of
CarD with RNAP β1 but is sensitive to CarD protein levels indicates that CarD has a function distinct from
binding RNAP. To test how CarD depletion and the CarD RID mutants would affect the sensitivity of
mycobacteria to other antibiotics used to treat M. tuberculosis infection, transient-treatment liquid culture
assays, disk zone of inhibition assays, etc were carried out.
The depletion of CarD or weakening of the CarD/RNAP β interaction had no effect on sensitivity to
pyrazinamide (PZA) and no reproducible effect on isoniazid(INH) sensitivity. However,it was found that the M.
smegmatis CarDR25E
strain, as well as a strain depleted for CarDWT
, were significantly more sensitive to
streptomycin treatment than control strains (Weiss et al., 2012). Also based on five replicate experiments it was
known that the doubling time of the M. smegmatis CarDR25E
mutant was 1.198 times (standard deviation, 0.067)
slower than that of the wild-type control strain. Likewise, the doubling time of the M.
tuberculosis CarDR47E
mutant was 1.181 times (standard deviation, 0.001) slower than that of the wild-type
control strain in three replicate experiments. The phenotypes associated with interfering with the CarD/RNAP β
interaction were dramatically more severe in M. tuberculosis than in M. smegmatis. This was evidenced by the
decrease in the growth rate of the M. tuberculosis CarDR47E
mutant compared to that in the wild-type CarD-
expressing strain, while in M. smegmatis the analogous mutant grew at wild-type rates. These findings suggest
that an optimal interaction between CarD and RNAP β is more critical for the growth of M. tuberculosis than
of M. smegmatis, and further comparison of these bacteria may shed light on the functions of CarD (Weiss et
al., 2012).
With the crystal structure(PDB ID:4KBM) of M.tb CarD /RNAP β-subunit(residues 47-433) Complex,
it is analysed that the CarD binding site of RNAP is located at the solvent exposed surface of the β1 domain,
which is approximately 70A° away from the RNAP active site Mg+2 (based on the th EC; PDB ID: 2O5I).
Despite the long distance between the binding site and the active site, this domain serves as an interaction
module for various regulatory proteins, including sigma factors at different stages of transcription, and is
important for RNAP -DNA binding and open complex stability (Trinh et al., 2006; Vassylyevet al., 2002). At
the CarD-RNAP interface, the primary four-stranded β-sheet of the CarD N-terminal domain forms an extended
eight stranded β sheet with the β1 domain of the RNAP β-subunit. Specifically, the β4 strand of CarD
comprising residues Leu44 to Pro49 forms an antiparallel β sheet with the β4 strand residues Thr138 to Gln144
of RNAP β subunit. This results in a mixed β sheet topology. Surprisingly, association of RNAP with CarD
results in only a 500 A°2 (otherwise solvent exposed) buried surface area, which is below average (1,500–2,000
A°2) for heteromeric protein-protein complexes (Kleanthous.,2000). While the buried surface is relatively small,
it is rich in intermolecular hydrogen bonds. There are eight hydrogen bonds and 69 non bonded contacts
between RNAP β subunit and CarD formed by the residues located on the β4 strands of both proteins, on the
loop connecting α12 and α13 of the RNAP β1 domain, and on the turn between the β1 and β2 strands of CarD.
Specifically, β1-Ile141 interacts with CarD-Arg47 (2.8A°), and β1-Ser143 interacts with CarD-Thr45 (2.7A°)
through four backbone-backbone hydrogen bonds. Interestingly, the side chain-specific hydrogen bonding
interactions are present only between β1-Lys142:CarD-His14 (2.9 A°),β1-Glu140:CarD-Tyr11 (2.4 A°), β1-
Thr138:CarD-Asn52 (2.8 A° ), and β1-Gln144:CarD-Gly42 (3.0 A°) (Gulten et al.,2013).The intermolecular
interface is also stabilized by electrostatic, hydrophobic, and van der Waals interactions. In fact, electrostatic
forces contribute significantly to the CarD/RNAP interaction because altering the local charge distribution at the
interface was reported to abolish CarD/RNAP interaction completely (Weiss et al., 2012) But in contrast to the
detail suggested by structural model generated by homology modeling (based on Tth TRCF-RID/β1 structure),
mutagenesis and two-hybrid assays by Weiss et al, that β1-Glu132 interacts with both Arg25 and Arg47
directly through hydrogen bonding and that these residues are critical for
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intermolecular interaction, it was observed from the Mtb CarD/RNAP structure(PDB ID:4KBM) that
β1-Glu132 is not in direct contact with CarD-Arg25 and CarD-Arg47 (5.0 A° and 6.1 A°, respectively). Arg25
interacts with b1-Ile141 only through van der Waals interactions and does not appear crucial for CarD/RNAP
interaction. Similarly, Glu132 and Arg47 interact only through a water molecule in the CarD/RNAP crystal
structure, and Arg47 is engaged in other hydrogen bonding and van der Waals interactions and does not appear
crucial for CarD/RNAP interaction. Similarly, Glu132 and Arg47 interact only through a water molecule in the
CarD/RNAP crystal structure, and Arg47 is engaged in other hydrogen bonding and van der Waals interactions
with β1-Ile141, β1-Glu140, and β1-Gly139. Therefore, loss of the CarD/RNAP interaction, upon E132R, R25E,
and R47E mutations, should be due to these factors rather than the disruption of the direct interaction between
Glu132-Arg25 and Glu132-Arg47(Gulten et al.,2013).
VII. C-TERMINAL DOMAIN
In the homology model of M.tb CarD built with template of Thermus thermophilus CarD crystal
structure the CarD C-terminal domain (CarD-CTD) is a compact, all α-helical fold having 5 helices with no
apparent structural similarity to any previously described fold. A notable feature of the CarD-CTD is a nearly
universally conserved tryptophan (T. thermophilus CarD-W86) with a largely solvent exposed side chain. The
tryptophan is located at the end of the CarD-CTD distal to the CarD-RID and is surrounded by a basic
electrostatic surface formed from a cluster of basic residues. Within this basic surface patch, R89, R119, and
R126 are universally conserved; R91 and R131 are conserved between T. thermophilus and Mycobacterium sp.;
whereas K84 and R130 are found in T. thermophilus CarD but not Mycobacterium sp. CarD. Analysis of M.
smegmatis and M. tuberculosis CarD homology models revealed that the surface-exposed tryptophan and
surrounding basic surface patch are conserved features(Srivastava et al.,2013).Though on the basis of primary
sequence analysis, a leucine zipper motif was proposed to be present in CarD CTD ,it was reported that no such
motif was found in the crystal structure (PDB ID 4ILU ) studied by kaur et al; rather CarD CTD folds into a
five-helical bundle. Analysis of this helical bundle conformation using DALI server revealed that three of five
helices (α2α3α4) could superimpose well on the top hits, and the remaining two helices (α1 and α5) take a
unique conformation, suggesting that a five-helical bundle of CarD CTD adopts a novel five helical fold. The
function of such a novel fold remains to be elucidated ( Kaur et al.,2013).
But Gulten et al, with CarD/RNAP β structure complex (PDB ID:4KBM) has reported that the CarD
C-terminal domain is comprised of an α-helical bundle of five α helices(α2-6) that contains an unexpected
internal leucine zipper between helices α4 and α5. Helices α4 and α5 interact only through hydrophobic and van
der Waals interactions, afforded by the leucine zipper. Helices α2 and α3 are positioned parallel to each other,
whereas α4, α5, and α6 pack orthogonally to each other. Helices α3 and α4 are connected by a γ-turn, while α4-
α5 and α5-α6 are connected by β-turns. It is also reported that the three-helix bundle of α3, α4, and α5 is
involved in DNA binding. The structure of this three-helix bundle is unlike other DNA-binding proteins in the
PDB. The DNA-interacting region of M.tb CarD is mapped to the N termini of α3 and α5, the C terminus of α4,
and the β-turn connecting α4 and α5. The leucine zipper motif of CarD appears to stabilize the conformation of
α4 and α5 inside the hydrophobic core and is not involved directly in dimerization or DNA interaction. Further
PDBeFold, VAST, and DALI servers against the PDB and Structural Classification of Proteins database, did not
identify any significant structural homologs for the C-terminal domain(Gulten et al.,2013).
Binding with DNA:
Chromatin immunoprecipitation sequencing (ChIP-seq) was used to survey the distribution of CarD
throughout the M. smegmatis chromosome. Specific antibodies targeting core RNAP, σA, or a
hemagglutinin(HA) epitope fused to CarD (CarD-HA) were used to co immune precipitate associated DNA that
was then sequenced. It was found that CarD was never present on the genome in the absence of RNAP and was
primarily associated with promoter regions and highly correlated with σA. Compilation of the ChIP-seq data and
previous microarray expression profiling analyses (Stallings et al.,2009) indicated that CarD was broadly
distributed on promoters of most transcription units regardless of whether they were deregulated during CarD
depletion. This has lead to propose that in vivo, CarD associates with RNAP initiation complexes at most
promoters and is therefore a global regulator of transcription initiation (Srivastava et al.,2013). As CarD-RID
shares sequence and structural homology with the TRCF-RID, as well as a common binding mode to the RNAP
β1-lobe, the crystal structure of the Thermus TRCF-RID/RNAP β1-lobe complex [Protein Data Bank (PDB) ID
3MLQ] was used as a template to generate structural model of the complex between CarD and the RNAP
open promoter (RPo) complex. In the CarD/RPo model, the interaction of the CarD-RID with the RNAP β1-
lobe places the CarD-CTD in a position to interact directly with the promoter DNA at the upstream edge of the
−10 element on the opposite face of the DNA as σ.
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Consistent with a role for the CarD-CTD in DNA interaction, CarD alone, at high concentration, is
capable of non-sequence specific protein/DNA interactions, and these interactions are mediated by the CarD-
CTD. The CarD-CTD/DNA interactions are centered on W86 and the surrounding basic patch, both conserved
structural features of CarD. CarD potentially interacts with the DNA from about −11 to −15. The basic surface
of the CarD-CTD seems to closely approach the DNA from the widened minor groove, with CarD-W86 most
proximal to the DNA, roughly aligned with the −12 base pair in the open complex. To directly test the effect of
CarD on rRNA promoter activity in vivo, β-galactosidase (βgal) activity was measured from lacZ which was
fused to the M. smegmatis rrnA control region [comprising promoters rrnA-P1, -P2, and -P3 (rrnAP123)] and
transformed into M. smegmatis. Depletion of CarD resulted in increased βgal activity , comparable to the
increase in 16S rRNA amount observed previously during CarD depletion (Stallings et al.,2009). However,
weakening the CarD/RNAP protein/protein interaction resulted in decreased βgal activity. The findings that
weakening the CarD/RNAP protein/protein interaction decreases promoter activity and leads to lower 16S
rRNA levels suggest that CarD may activate M. smegmatis rRNA transcription initiation, in contrast to the
proposal that CarD acts as a repressor of rRNA transcription. It is possible that depletion of CarD,a global
regulator that is essential for M. smegmatis viability and present at most M. smegmatis promoters , might give
rise to pleiotropic effects that indirectly increase M. smegmatis rRNA promoter activity. (Srivastava et al.,2013).
VIII. DIMERIZATION
In the crystal structure PDB ID:4ILU, CarD was predicted to have two domains, where the N-terminal
domain (CarD-NTD) forms an antiparallel β sheet (β1–β3) and the C-terminal domain (CarD-CTD) adopts
predominantly an all-α (α1-α5) conformation. Structural and packing analyses suggested that the two monomers
of CarD related by crystallographic twofold symmetry assemble to form a tight and closed homo dimer by
swapping 21 N amino acids, resulting in a quasidomain-swapped (Liu et al.,2002) structure. Due to domain
swapping between the β1 and β1′ strands of the monomers, the topology of the CarDNTD in the dimer
constitutes (β1′β1β2β3) and (β1β1′β2′β3′) arrangements. The core of this dimeric interface includes a highly
curved 14 residue long antiparallel β-sheet, formed by β1 and β1′ strands of each monomer, which contributes
18 interchain hydrogen bonds, several nonbonded contacts and a buried surface area of ~1600 A°2, as revealed
by PDBe-PISA(Krissinel et al.,2007).Thus, the CarD-NTD plays an important role in dimerization. Analytical
gel filtration experiments performed also found CarD to be predominantly dimeric in solution. Also it was
known, Tth CdnLNTD, the homologue of CarD-NTD, exists as a monomer in solution, as judged by the NMR
structure (Gallego-Garcia et al.,2012). On the contrary, Mxa CdnL has been reported to exist as a monomer /
dimer mixture at the concentration range of 0.5–1 mM used for NMR studies(Mirassou et al.,2013). Unlike Tth
CdnL-NTD or Mxa CdnL, monomeric Mtb CarD was not found in the study( Kaur et al.,2013).Structural
superposition of the Mtb CarD structure on the TRCF-RID/RNAP-β1 complex (PDB ID 3MLQ; (Westblade et
al.,2010)),revealed that the RNAP binding site of CarD-NTD β3 in one monomer is occluded by interactions of
CarD β4′ (formed by non-native sequence) of the second monomer. Hence, suggesting that CarD crystallized in
a closed conformation. These CarD-NTD β3 /CarD β4′ interactions was believed to represent a snapshot of a
possible mode of CarD-NTD/RNAP-β1 interactions. The closed conformation of CarD also suggests that it is
inherently flexible molecule that may undergo large domain motion to perform function(Kaur et al.,2013).
IX. SEQUENCE HOMOLOGS IN HUMANS
Blastp search done against Human protein database has given 4 hits - pantothenate kinase 2,
mitochondrial isoform 1 preproprotein , plexin domain-containing protein 2 precursor, importin-9 and
telomeric repeat-binding factor. These hits are with very less score and high E-value. The aligned region
between the query and hits are less , with less percentage of sequence identity. This confirms the absence of
highly similar proteins in humans. The hits were also analysed and found that they do not possess the domain
CarD_TRCF which is present in CarD protein( Priya et al.,2012).
X. CONCLUSION
CarD protein is a bacterial transcriptional regulator and in M.tuberculosis is known to play a critical
role in survival of the organism by downregulating few rRNA and ribosomal proteins during dormant stage in
the stringent hostile environment. Also it is found to be necessary for replication and persistence during
infection in mice. carD transcripts are upregulated with DNA damaging agents and also lack of CarD makes the
organism more vulnerable to DNA damaging agents. This, along with CarD’s ability to restore Dksa depletion
strain suggests its possible role in replication. CarD is found to interact with RNA polymerase in a similar
manner like TRCF, which aides in DNA strand specific repair. But it is found that TRCF would not replace
CarD in that CarD might have other functions apart from RNAP interaction. Depletion of CarD confers
increased sensitivity to the fluoroquinolone ciprofloxacin but is unresponsive to changes in the interaction of
CarD with RNAP β1 indicates that CarD has a function distinct from binding RNAP. But it is confirmed that
8. CarD-A Reliable Target In M.tuberculosis
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mutants that weaken the CarD/RNAP interaction display an extensive antibiotic-susceptibility profile
particularly to rifampin. Moreover, CarD does not have a homolog in human proteome known by sequence
similarity search. This holds up the possibility of using CarD as a potential drug target in treatment against
Tuberculosis. Hence presently, small molecules which can block the CarD and RNAP interaction can be
promising leads against tuberculosis.
Many issues of CarD has yet to be solved. Though both M.tuberculosis and M.smegmatis show similar response
in many of the experimental conditions they also differ in some responses which infers that CarD is essential
for viability in M.tuberculosis but not so in M.smegmatis. The reason for it has to be studied in depth. Also the
presence of leucine zipper motif in C-terminal domain and its role in DNA binding, is still in debate and needs
to be confirmed. The function of novel fold in C-terminal domain, the dimerisation ability of CarD all need to
be confirmed. Its role in sensitivity response to varied antibiotics drugs has to be studied extensively. The
answers to these queries may help in designing even more efficient leads against this target.
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