Loss of Dihydroxyacid Dehydratase Induces Auxotrophy in Bacillus anthracis

2021 ◽  
Author(s):  
Joseph Jelinski ◽  
Madeline Cortez ◽  
Austen Terwilliger ◽  
Justin Clark ◽  
Anthony Maresso
Keyword(s):  
Serum Albumin ◽  
Bacillus Anthracis ◽  
Human Blood ◽  
De Novo ◽  
Specific Effect ◽  
Current Treatment ◽  
Mammalian Host ◽  
Human Hemoglobin ◽  
New Targets ◽  
Species Specific

Anthrax disease is caused by infection with the bacteria Bacillus anthracis which, if left untreated, can result in fatal bacteremia and toxemia. Current treatment for infection requires prolonged administration of antibiotics. Despite this, inhalational and gastrointestinal anthrax still result in lethal disease. By identifying key metabolic steps that B. anthracis uses to grow in host-like environments, new targets for antibacterial strategies can be identified. Here, we report that the ilvD gene, which encodes dihydroxyacid dehydratase in the putative pathway for synthesizing branched chain amino acids, is necessary for B. anthracis to synthesize isoleucine de novo in an otherwise limiting microenvironment. We observed that Δ ilvD B. anthracis cannot grow in media lacking isoleucine, but growth is restored when exogenous isoleucine is added. In addition, ΔilvD bacilli are unable to utilize human hemoglobin or serum albumin to overcome isoleucine auxotrophy, but can when provided with the murine forms. This species-specific effect is due to the lack of isoleucine in human hemoglobin. Furthermore, even when supplemented with physiological levels of human serum albumin, apotransferrin, fibrinogen, and IgG, the ilvD knockout strain grew poorly relative to non-supplemented wild-type. In addition, comparisons upon infecting humanized mice suggest that murine hemoglobin is a key source of isoleucine for both WT and Δ ilvD bacilli. Further growth comparisons in murine and human blood show that the auxotrophy is detrimental for growth in human blood, not murine. This report identifies ilvD as necessary for isoleucine production in B. anthracis , and that it plays a key role in allowing the bacilli to effectively grow in isoleucine poor hosts. Importance Anthrax disease, caused by B. anthracis , can cause lethal bacteremia and toxemia, even following treatment with antibiotics. This report identifies the ilvD gene, which encodes a dihydroxyacid dehydratase, as necessary for B. anthracis to synthesize the amino acid isoleucine in a nutrient-limiting environment, such as its mammalian host. The use of this strain further demonstrated a unique species-dependent utilization of hemoglobin as an exogenous source of extracellular isoleucine. By identifying mechanisms that B. anthracis uses to grow in host-like environments, new targets for therapeutic intervention are revealed.

scholarly journals In Search of Species-Specific SNPs in a Non-Model Animal (European Bison (Bison bonasus))—Comparison of De Novo and Reference-Based Integrated Pipeline of STACKS Using Genotyping-by-Sequencing (GBS) Data

Animals ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 2226
Author(s):  
Sazia Kunvar ◽  
Sylwia Czarnomska ◽  
Cino Pertoldi ◽  
Małgorzata Tokarska
Keyword(s):  
Reference Genome ◽  
De Novo ◽  
Bos Taurus ◽  
Model Organism ◽  
Genotyping By Sequencing ◽  
Model Organisms ◽  
European Bison ◽  
Model Animal ◽  
Pcr Duplicates ◽  
Species Specific

The European bison is a non-model organism; thus, most of its genetic and genomic analyses have been performed using cattle-specific resources, such as BovineSNP50 BeadChip or Illumina Bovine 800 K HD Bead Chip. The problem with non-specific tools is the potential loss of evolutionary diversified information (ascertainment bias) and species-specific markers. Here, we have used a genotyping-by-sequencing (GBS) approach for genotyping 256 samples from the European bison population in Bialowieza Forest (Poland) and performed an analysis using two integrated pipelines of the STACKS software: one is de novo (without reference genome) and the other is a reference pipeline (with reference genome). Moreover, we used a reference pipeline with two different genomes, i.e., Bos taurus and European bison. Genotyping by sequencing (GBS) is a useful tool for SNP genotyping in non-model organisms due to its cost effectiveness. Our results support GBS with a reference pipeline without PCR duplicates as a powerful approach for studying the population structure and genotyping data of non-model organisms. We found more polymorphic markers in the reference pipeline in comparison to the de novo pipeline. The decreased number of SNPs from the de novo pipeline could be due to the extremely low level of heterozygosity in European bison. It has been confirmed that all the de novo/Bos taurus and Bos taurus reference pipeline obtained SNPs were unique and not included in 800 K BovineHD BeadChip.

Review of treatment and therapeutic targets in brain arteriovenous malformation

2021 ◽  
pp. 0271678X2110267
Author(s):  
Peipei Pan ◽  
Shantel Weinsheimer ◽  
Daniel Cooke ◽  
Ethan Winkler ◽  
Adib Abla ◽  
...  
Keyword(s):  
Animal Models ◽  
De Novo ◽  
Mapk Pathway ◽  
Treatment Options ◽  
Therapeutic Targets ◽  
Current Treatment ◽  
Mitogen Activated Protein Kinase ◽  
De Novo Mutations ◽  
Genetic Syndromes ◽  
Mitogen Activated Protein

Brain arteriovenous malformations (bAVM) are an important cause of intracranial hemorrhage (ICH), especially in younger patients. The pathogenesis of bAVM are largely unknown. Current understanding of bAVM etiology is based on studying genetic syndromes, animal models, and surgically resected specimens from patients. The identification of activating somatic mutations in the Kirsten rat sarcoma viral oncogene homologue (KRAS) gene and other mitogen-activated protein kinase ( MAPK) pathway genes has opened up new avenues for bAVM study, leading to a paradigm shift to search for somatic, de novo mutations in sporadic bAVMs instead of focusing on inherited genetic mutations. Through the development of new models and understanding of pathways involved in maintaining normal vascular structure and functions, promising therapeutic targets have been identified and safety and efficacy studies are underway in animal models and in patients. The goal of this paper is to provide a thorough review or current diagnostic and treatment tools, known genes and key pathways involved in bAVM pathogenesis to summarize current treatment options and potential therapeutic targets uncovered by recent discoveries.

Changes in the transferrin requirement of cultured chick embryo mesoderm cells during early differentiation

Development ◽  
1986 ◽  
Vol 95 (1) ◽  
pp. 81-93
Author(s):  
E. J. Sanders
Keyword(s):  
Chick Embryo ◽  
Type I Collagen ◽  
Specific Effect ◽  
Primitive Streak ◽  
Cellular Adhesion ◽  
Type I ◽  
Surface Binding ◽  
Surface Receptors ◽  
Early Differentiation ◽  
Species Specific

Mesodermal tissue from the chick embryo at various stages of early differentiation was cultured in hydrated gels of type I collagen in the presence and absence of transferrin. Primary mesoderm explants from primitive-streak-stage embryos responded to the presence of avian transferrin by significantly improved outgrowth which appeared to be related to the ability of the cells to attach to, and migrate in, the collagen. No evidence was obtained which suggested that this observation was dependent on increased cell proliferation. This outgrowth enhancement was not duplicated by transferrin of human origin. The avian transferrin did not produce this effect on cells cultured on plastic substrata, suggesting that the species-specific effect involves modulation by the extracellular matrix. Mesoderm explants from somite stages of development showed no increase in outgrowth in the presence of either avian or human transferrin as judged by counting the number of outwandering cells. Ultrastructural immunocytochemistry indicated surface binding of transferrin by cells in the gels, and the presence of endogenous transferrin on the surfaces of mesoderm cells in situ and in their extracellular environment. It is suggested that by binding to cell surface receptors, transferrin may be able to influence the strength of cellular adhesion to collagen and hence the capacity for cell locomotion.

scholarly journals Osteocalcin Production in Primary Osteoblast Cultures Derived from Normal and Hyp Mice

Endocrinology ◽  
1998 ◽  
Vol 139 (1) ◽  
pp. 35-43 ◽  
Author(s):  
Thomas O. Carpenter ◽  
Kathleen C. Moltz ◽  
Bruce Ellis ◽  
Monica Andreoli ◽  
Thomas L. McCarthy ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Cultured Cells ◽  
Primary Cultures ◽  
Specific Effect ◽  
Continuous Exposure ◽  
Primary Osteoblast ◽  
Characteristic Features ◽  
Species Specific ◽  
Osteocalcin Production

Abstract Rickets and osteomalacia are characteristic features of the Hyp mouse model of human X-linked hypophosphatemia. Hyp mice demonstrate elevated circulating osteocalcin levels, as well as altered regulation of osteocalcin by 1,25(OH)2D3. Whether this osteocalcin abnormality is intrinsic to the osteoblast, or mediated by the in vivo milieu, has not been established. We therefore characterized osteocalcin production and its regulation by 1,25(OH)2D3 in primary cultures of murine osteoblasts and examined osteocalcin and its messenger RNA in response to 1,25(OH)2D3 in cultures of Hyp mouse-derived osteoblasts. Cell viability and osteocalcin production are optimal when murine cells are harvested within 36 h of age. Murine primary osteoblast cultures mineralize and produce osteocalcin in a maturation-dependent fashion (as demonstrated in other species), and continuous exposure to 1,25(OH)2D3, beginning at day 9 of culture, inhibits osteoblast differentiation and osteocalcin production and prevents mineralization of the culture. However, in contrast to other species, exposure to 1,25(OH)2D3, added later (days 17–25) in culture, does not stimulate osteocalcin but arrests osteocalcin production at current levels. Ambient media levels of osteocalcin were no different in cultures from Hyp mice and their normal litter mates, and the down-regulatory response to 1,25(OH)2D3 was comparable in cultures from normal and Hyp mice. Furthermore, expression of osteocalcin messenger RNA in murine cultures is reduced with exposure to 1,25(OH)2D3, and there is no difference between normal and Hyp cultures in this response. Thus, primary murine osteoblasts manifest a species-specific effect of 1,25(OH)2D3 on osteocalcin production. Furthermore, the increased serum osteocalcin production seen in intact Hyp mice, and the altered response to 1,25(OH)2D3 in Hyp mice, are not observed in osteoblast cultures derived from the mutant strain. These data indicate that abnormalities of osteocalcin described in intact Hyp mice require factors other than those present in cultured cells.

scholarly journals High light induces species specific changes in the membrane lipid composition of Chlorella

2020 ◽  
Vol 477 (13) ◽  
pp. 2543-2559
Author(s):  
Janka Widzgowski ◽  
Alexander Vogel ◽  
Lena Altrogge ◽  
Julia Pfaff ◽  
Heiko Schoof ◽  
...  
Keyword(s):  
Cell Membranes ◽  
De Novo ◽  
Algal Cell ◽  
Isotopic Labeling ◽  
Biochemical Properties ◽  
Membrane Lipid ◽  
Light Conditions ◽  
Hydrophobic Barrier ◽  
Glycerolipid Composition ◽  
Species Specific

Algae have evolved several mechanisms to adjust to changing environmental conditions. To separate from their surroundings, algal cell membranes form a hydrophobic barrier that is critical for life. Thus, it is important to maintain or adjust the physical and biochemical properties of cell membranes which are exposed to environmental factors. Especially glycerolipids of thylakoid membranes, the site of photosynthesis and photoprotection within chloroplasts, are affected by different light conditions. Since little is known about membrane lipid remodeling upon different light treatments, we examined light induced alterations in the glycerolipid composition of the two Chlorella species, C. vulgaris and C. sorokiniana, which differ strongly in their ability to cope with different light intensities. Lipidomic analysis and isotopic labeling experiments revealed differences in the composition of their galactolipid species, although both species likely utilize galactolipid precursors originated from the endoplasmic reticulum. However, in silico research of de novo sequenced genomes and ortholog mapping of proteins putatively involved in lipid metabolism showed largely conserved lipid biosynthesis pathways suggesting species specific lipid remodeling mechanisms, which possibly have an impact on the response to different light conditions.

scholarly journals Uptake of fluorescent-labeled BSA into root cells: endocytosis?

2014 ◽  
Vol 66 (3-4) ◽  
pp. 319-324 ◽  
Author(s):  
A. Kaźmierczak ◽  
J. Maszewski
Keyword(s):  
Zea Mays ◽  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Bovine Serum ◽  
Cytochalasin B ◽  
Fluorescein Isothiocyanate ◽  
Cell Type ◽  
Root Cells ◽  
Root Zones ◽  
Species Specific

Incorporation of rhodamine- and fluorescein-isothiocyanate labeled bovine serum albumin (BSA-TRITC, BSA-FITC) was examined in different root zones of the 3-day-old seedlings in <em>Melandrium noctiflorum</em>, <em>Allium cepa</em> and <em>Zea mays</em>. The uptake of fluorescent-labeled BSA was found: (1) species-specific, (2) cell-type dependent, and (3) cytochalasin B-sensitive. The characteristic punctute distribution of vesicles within the cytoplasm suggests the internalization of labeled proteins by endocytosis.

Preliminary examinations aiming at confirming presence of human blood in biological traces by means of mRNA analysis

2017 ◽  
Vol 295 ◽  
pp. 70-79
Author(s):  
Marta Gorzkiewicz ◽  
◽  
Małgorzata Grabowska ◽  
Tomasz Grzybowski ◽  
◽  
...  
Keyword(s):  
Human Blood ◽  
Genetic Material ◽  
Mrna Analysis ◽  
Species Specific ◽  
Additional Reference ◽  
Mrna Markers ◽  
Optimal Quantity ◽  
Validation Experiments ◽  
Gene Mrna

The aim of the project was to develop a specific and at the same time economical method for detecting human blood in biological traces based on analysis of haemoglobin mRNA with use of PCR reaction in real time and non-specific SYBR Green detector. The test, which has eventually been developed enabled simultaneous analysis of melting curves for three fragments of various lengths: HBB61, HBA197 and HBB503, as well as an additional reference gene: mRNA β-actin. A definite identification was possible already for 0,1 μl of blood. The method is tissue and species specific. The analysed mRNA markers are characterized by high stability, as compared to haemoglobin detected by standard methods. The result of mRNA profiling shows the predictive value as regards quality of genetic material and occurrence of mixture of liquids. Results of analyses performed during the project indicate potential usefulness of HBB and HBA1 markers in routine forensic genetic examinations. However, it is necessary to carry out a broader spectrum of validation experiments, and particularly to analyse a larger number of actual biological casework and precisely determining an optimal quantity of RNA and identifying ontogenetic differences in the levels of expression.

scholarly journals Species-Specific Marker Discovery in Tilapia

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Mochamad Syaifudin ◽  
Michaël Bekaert ◽  
John B. Taggart ◽  
Kerry L. Bartie ◽  
Stefanie Wehner ◽  
...  
Keyword(s):  
Phylogenetic Trees ◽  
Restriction Site ◽  
De Novo ◽  
Snp Markers ◽  
Specific Marker ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Species Analysis ◽  
Species Specific ◽  
Marker Discovery

Abstract Tilapias (family Cichlidae) are of importance in aquaculture and fisheries. Hybridisation and introgression are common within tilapia genera but are difficult to analyse due to limited numbers of species-specific genetic markers. We tested the potential of double digested restriction-site associated DNA (ddRAD) sequencing for discovering single nucleotide polymorphism (SNP) markers to distinguish between 10 tilapia species. Analysis of ddRAD data revealed 1,371 shared SNPs in the de novo-based analysis and 1,204 SNPs in the reference-based analysis. Phylogenetic trees based on these two analyses were very similar. A total of 57 species-specific SNP markers were found among the samples analysed of the 10 tilapia species. Another set of 62 species-specific SNP markers was identified from a subset of four species which have often been involved in hybridisation in aquaculture: 13 for Oreochromis niloticus, 23 for O. aureus, 12 for O. mossambicus and 14 for O. u. hornorum. A panel of 24 SNPs was selected to distinguish among these four species and validated using 91 individuals. Larger numbers of SNP markers were found that could distinguish between the pairs of species within this subset. This technique offers potential for the investigation of hybridisation and introgression among tilapia species in aquaculture and in wild populations.

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