scholarly journals Transcriptional Profiling of the Bacillus anthracis Life Cycle In Vitro and an Implied Model for Regulation of Spore Formation

2006 ◽  
Vol 188 (17) ◽  
pp. 6092-6100 ◽  
Author(s):  
Nicholas H. Bergman ◽  
Erica C. Anderson ◽  
Ellen E. Swenson ◽  
Matthew M. Niemeyer ◽  
Amy D. Miyoshi ◽  
...  
Keyword(s):  
Life Cycle ◽  
Bacillus Anthracis ◽  
De Novo ◽  
Transcriptional Profiling ◽  
Expression Patterns ◽  
Temporal Expression ◽  
Bioinformatics Analyses ◽  
Microbial Forensics ◽  
Comprehensive Survey

ABSTRACT The life cycle of Bacillus anthracis includes both vegetative and endospore morphologies which alternate based on nutrient availability, and there is considerable evidence indicating that the ability of this organism to cause anthrax depends on its ability to progress through this life cycle in a regulated manner. Here we report the use of a custom B. anthracis GeneChip in defining the gene expression patterns that occur throughout the entire life cycle in vitro. Nearly 5,000 genes were expressed in five distinct waves of transcription as the bacteria progressed from germination through sporulation, and we identified a specific set of functions represented within each wave. We also used these data to define the temporal expression of the spore proteome, and in doing so we have demonstrated that much of the spore's protein content is not synthesized de novo during sporulation but rather is packaged from preexisting stocks. We explored several potential mechanisms by which the cell could control which proteins are packaged into the developing spore, and our analyses were most consistent with a model in which B. anthracis regulates the composition of the spore proteome based on protein stability. This study is by far the most comprehensive survey yet of the B. anthracis life cycle and serves as a useful resource in defining the growth-phase-dependent expression patterns of each gene. Additionally, the data and accompanying bioinformatics analyses suggest a model for sporulation that has broad implications for B. anthracis biology and offer new possibilities for microbial forensics and detection.

scholarly journals Transcriptional Profiling of Bacillus anthracis during Infection of Host Macrophages

2007 ◽  
Vol 75 (7) ◽  
pp. 3434-3444 ◽  
Author(s):  
Nicholas H. Bergman ◽  
Erica C. Anderson ◽  
Ellen E. Swenson ◽  
Brian K. Janes ◽  
Nathan Fisher ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
Host Cell ◽  
Transcriptional Profiling ◽  
Expression Patterns ◽  
Survival Strategies ◽  
Reverse Transcription Pcr ◽  
Bacterial Rna ◽  
Definition Of

ABSTRACT The interaction between Bacillus anthracis and the mammalian phagocyte is one of the central stages in the progression of inhalational anthrax, and it is commonly believed that the host cell plays a key role in facilitating germination and dissemination of inhaled B. anthracis spores. Given this, a detailed definition of the survival strategies used by B. anthracis within the phagocyte is critical for our understanding of anthrax. In this study, we report the first genome-wide analysis of B. anthracis gene expression during infection of host phagocytes. We developed a technique for specific isolation of bacterial RNA from within infected murine macrophages, and we used custom B. anthracis microarrays to characterize the expression patterns occurring within intracellular bacteria throughout infection of the host phagocyte. We found that B. anthracis adapts very quickly to the intracellular environment, and our analyses identified metabolic pathways that appear to be important to the bacterium during intracellular growth, as well as individual genes that show significant induction in vivo. We used quantitative reverse transcription-PCR to verify that the expression trends that we observed by microarray analysis were valid, and we chose one gene (GBAA1941, encoding a putative transcriptional regulator) for further characterization. A deletion strain missing this gene showed no phenotype in vitro but was significantly attenuated in a mouse model of inhalational anthrax, suggesting that the microarray data described here provide not only the first comprehensive view of how B. anthracis survives within the host cell but also a number of promising leads for further research in anthrax.

scholarly journals SOX2 Plays a Critical Role in the Pituitary, Forebrain, and Eye during Human Embryonic Development

2008 ◽  
Vol 93 (5) ◽  
pp. 1865-1873 ◽  
Author(s):  
Daniel Kelberman ◽  
Sandra C. P. de Castro ◽  
Shuwen Huang ◽  
John A. Crolla ◽  
Rodger Palmer ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Anterior Pituitary ◽  
De Novo ◽  
Hypogonadotropic Hypogonadism ◽  
Expression Patterns ◽  
Critical Role ◽  
Loss Of Function ◽  
De Novo Mutations ◽  
Direct Role

Abstract Context: Heterozygous, de novo mutations in the transcription factor SOX2 are associated with bilateral anophthalmia or severe microphthalmia and hypopituitarism. Variable additional abnormalities include defects of the corpus callosum and hippocampus. Objective: We have ascertained a further three patients with severe eye defects and pituitary abnormalities who were screened for mutations in SOX2. To provide further evidence of a direct role for SOX2 in hypothalamo-pituitary development, we have studied the expression of the gene in human embryonic tissues. Results: All three patients harbored heterozygous SOX2 mutations: a deletion encompassing the entire gene, an intragenic deletion (c.70_89del), and a novel nonsense mutation (p.Q61X) within the DNA binding domain that results in impaired transactivation. We also show that human SOX2 can inhibit β-catenin-driven reporter gene expression in vitro, whereas mutant SOX2 proteins are unable to repress efficiently this activity. Furthermore, we show that SOX2 is expressed throughout the human brain, including the developing hypothalamus, as well as Rathke’s pouch, the developing anterior pituitary, and the eye. Conclusions: Patients with SOX2 mutations often manifest the unusual phenotype of hypogonadotropic hypogonadism, with sparing of other pituitary hormones despite anterior pituitary hypoplasia. SOX2 expression patterns in human embryonic development support a direct involvement of the protein during development of tissues affected in these individuals. Given the critical role of Wnt-signaling in the development of most of these tissues, our data suggest that a failure to repress the Wnt-β-catenin pathway could be one of the underlying pathogenic mechanisms associated with loss-of-function mutations in SOX2.

scholarly journals Autoregulation Is Essential for Precise Temporal and Steady-State Regulation by the Bordetella BvgAS Phosphorelay

2006 ◽  
Vol 189 (5) ◽  
pp. 1974-1982 ◽  
Author(s):  
Corinne L. Williams ◽  
Peggy A. Cotter
Keyword(s):  
Gene Expression ◽  
Steady State ◽  
Response Regulator ◽  
Expression Patterns ◽  
State Regulation ◽  
Temporal Expression ◽  
Multiple Gene ◽  
Insight Into

ABSTRACT The Bordetella BvgAS virulence control system is prototypical of phosphorelays that use a polydomain sensor and a response regulator to control gene expression in response to environmental cues. BvgAS controls the expression of at least three distinct phenotypic phases (Bvg−, Bvgi, and Bvg+) by differentially regulating the expression of at least four classes of genes. Among the loci regulated by BvgAS is bvgAS itself. We investigated the role of autoregulation in the ability of BvgAS to control multiple gene expression patterns in a temporal and steady-state manner by constructing Bordetella bronchiseptica strains in which the bvgAS promoter was replaced with constitutively active promoters. Our results show that positive autoregulation of bvgAS transcription is required for the temporal expression of multiple phenotypic phases that occurs in response to a shift from Bvg−-phase conditions to Bvg+-phase conditions. Autoregulation was also shown to contribute to steady-state regulation; it influences the sensitivity of the system in response to subtle differences in signal intensity. In addition, considered in relation to BvgA and BvgS activities demonstrated in vitro, our results provide insight into how BvgA and BvgS function mechanistically.

scholarly journals Engineering of α-PD-1 antibody-expressing long-lived plasma cells by CRISPR/Cas9-mediated targeted gene integration

2020 ◽  
Vol 11 (11) ◽  
Author(s):  
Baohong Luo ◽  
Yikang Zhan ◽  
Minqi Luo ◽  
Huimin Dong ◽  
Jun Liu ◽  
...  
Keyword(s):  
B Lymphocytes ◽  
Plasma Cells ◽  
De Novo ◽  
Lentiviral Vector ◽  
Transcriptional Profiling ◽  
Cellular Immunotherapy ◽  
B Virus ◽  
Antitumor Immunotherapy

Abstract Long-lived plasma cells (LLPCs) are robust specialized antibody-secreting cells that mainly stay in the bone marrow and can persist a lifetime. As they can be generated by inducing the differentiation of B-lymphocytes, we investigated the possibility that human LLPCs might be engineered to express α-PD-1 monoclonal antibody to substitute recombinant α-PD-1 antitumor immunotherapy. To this end, we inserted an α-PD-1 cassette into the GAPDH locus through Cas9/sgRNA-guided specific integration in B-lymphocytes, which was mediated by an integrase-defective lentiviral vector. The edited B cells were capable of differentiating into LLPCs both in vitro and in vivo. Transcriptional profiling analysis confirmed that these cells were typical LLPCs. Importantly, these cells secreted de novo antibodies persistently, which were able to inhibit human melanoma growth via an antibody-mediated checkpoint blockade in xenograft-tumor mice. Our work suggests that the engineered LLPCs may be utilized as a vehicle to constantly produce special antibodies for long-term cellular immunotherapy to eradicate tumors and cellular reservoirs for various pathogens including human immunodeficiency virus type 1 (HIV-1) and hepatitis B virus (HBV).

99 EXTRACELLULAR VESICLES OF BOVINE OVIDUCTAL FLUID MODIFY THE GENE EXPRESSION ON BOVINE IN VITRO-DERIVED EMBRYOS

2016 ◽  
Vol 28 (2) ◽  
pp. 179 ◽  
Author(s):  
R. Lopera-Vasquez ◽  
M. Hamdi ◽  
V. Maillo ◽  
C. Nunez ◽  
M. Yanez-Mo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Embryo Development ◽  
Extracellular Vesicles ◽  
Membrane Trafficking ◽  
Dna Methyltransferase ◽  
De Novo ◽  
Expression Patterns ◽  
Water Channel ◽  
Developmental Competence

Extracellular vesicles (EVs) act as intercellular communicators through their protein, lipid, and mRNA content. The interaction of EVs from oviducal environment and the first stages of embryo development is currently an enigma. The aim of the present study was to evaluate the developmental competence and the expression profile of bovine blastocysts cultured with previously purified EVs recovered from ampullary and isthmic oviducal fluid (OF) under different centrifugal forces. OF-EVs recovered from oviducts of slaughtered heifers in early luteal phase were quantified with a nanoparticle tracking analysis system, and their integrity and size were assessed by electron microscopy. In vitro-produced zygotes were cultured in SOF+3 mg mL–1 BSA (C–), C– with 3 × 105 OF-EVs/mL from the ampulla (A) and isthmus (I) isolated at 1 × 103 (A10k and I10k, respectively) and 1 × 105 (A100k and I100k, respectively) × g. A control culture group of SOF+5% FCS (C+) was included. Blastocyst development was recorded on Day 7, 8, and 9 (D0: day of fertilization). Blastocysts on Days 7/8 cultured in C–, C+, I10k, and I100k were used to measure the relative mRNA expression of genes related with membrane trafficking (AQP3, AQP11, and ATP1A1), metabolism (LDLR and LDHA), and epigenetics (DNMT3A, IGF2R, GRB10, and SNRPN) by RT-qPCR. One-way ANOVA was used for statistical analysis. The size of ampullary and isthmic OF-EVs was similar with a mean of 220 nm. The concentration of I10k was significantly lower compared with A100k (3.6 × 108 v. 10.5 × 108 EVs/mL, respectively; P < 0.05); however, no differences were found in the rest of the groups with a mean concentration of 7.6 × 108 EVs/mL. EVs and C– groups showed a delayed embryo development at Day 7 compared with C+ (range: 12.0–13.8 v. 20.6%, respectively, P < 0.05); however, it was compensated at Days 8 and 9 (Day 9 range: 28.5–30.8%). The water channel related protein AQP3, associated with blastocoel formation, water, and cryoprotectant movement during cryopreservation, was up-regulated in I10k and I100k blastocysts compared with C+. The lipid receptor LDLR, proposed as a regulator of lipid uptake in blastocysts, was significantly down-regulated in C+ compared with the other groups, a possible consequence of a higher concentration of lipids in the C+ group. The de novo DNA methyltransferase DNMT3A and the imprinting gene SNRPN were down-regulated in the C+ compared with I100k, suggesting alterations in imprinting. In conclusion, bovine isthmic OF-EVs supplementation in in vitro embryo culture has a positive effect on gene expression patterns of developmental related genes compared with serum supplementation, suggesting an association between the oviducal environment and the developing embryo. Funded by the Spanish Ministry of Science and Innovation (AGL2012–37510 and AGL2012–39652-C02–01).

Expression of DNMT Isoforms Is Differentially Associated with Aberrant Promotor Methylation in MDS Hematopoietic Progenitor Cells during Lineage Specific Differentiation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2628-2628 ◽  
Author(s):  
Olaf J. Hopfer ◽  
Martina Komor ◽  
Ina S. Koehler ◽  
Matthias Schulze ◽  
Claudia Freitag ◽  
...  
Keyword(s):  
High Risk ◽  
Dna Methyltransferase ◽  
De Novo ◽  
Disease Risk ◽  
Expression Patterns ◽  
Survivin Expression ◽  
Low Risk ◽  
Dnmt1 Expression ◽  
Time Point

Abstract Recent findings suggest that in myelodysplastic syndrome (MDS) several key regulatory genes are affected by aberrant promotor methylation. To explore the molecular basis of this impairment we have generated an in vitro model of MDS lineage-specific hematopoietic differentiation by culturing CD34+ cells from healthy donors (n=7) and MDS patients (low risk: RA/n=6, RARS/n=3; high risk: RAEB/n=4, RAEB-T/n=2) with EPO, TPO and GCSF. Cell harvest was at days 0, 4, 7 and 11. Promotor methylation analysis of key genes involved in the control of apoptosis (p73, survivin, DAPK), DNA-repair (hMLH1), differentiation (RARb, WT1) and cell cycle control (p14, p15, p16, CHK2) was performed by methylation specific PCR of bisulfite treated genomic DNA for each lineage at each time point. In addition, expression of DNMT1 (maintenance DNA methyltransferase), DNMT3a and DNMT3b (both de novo DNA methyltransferase) was analyzed by real time RT-PCR and correlated with gene promotor methylation at any time point. DNMT1 expression was increased during erythropoiesis in both, normal controls and MDS patients. On the other hand, expression of de novo DNMTs was elevated during thrombopoiesis at all time points. During erythropoiesis hypermethylation of p73, hMLH1 and RARb was associated with elevated DNMT1, hypermethylation of p15, p16, p73 and survivin was positively associated with increasing DNMT3 expression. Interestingly, DNMT1 was only elevated in low risk MDS, but not further increased in high risk MDS patients. Surprisingly, MDS specific survivin promotor methylation was inverse correlated with DNTM1 and DNMT3a expression. However, a negative correlation of DNMT3a with survivin expression was found in low risk MDS but not in high risk MDS. In summary our data indicate that all mammalian DNMT isoforms may be involved in the aberrant DNA-methylation phenotype in MDS. Elevated DNMT1 expression may in particular contribute to ineffective erythropoiesis in low risk MDS. DNMT3a and 3b were elevated during megakaryopoiesis and their expression was inversely correlated with MDS disease risk (IPSS). We conclude that the knowledge about distinct expression patterns of DNMT isoforms in hematopoiesis may be of help for further strategies to implicate DNMT-inhibitors in the treatment of patients with MDS.

Messenger RNA expression patterns in bovine embryos derived from in vitro procedures and their implications for development

2005 ◽  
Vol 17 (2) ◽  
pp. 23 ◽  
Author(s):  
Christine Wrenzycki ◽  
Doris Herrmann ◽  
Andrea Lucas-Hahn ◽  
Karin Korsawe ◽  
Erika Lemme ◽  
...  
Keyword(s):  
Messenger Rna ◽  
De Novo ◽  
Culture Media ◽  
Expression Patterns ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Developmental Abnormalities ◽  
Embryonic Genome

The preimplantation bovine embryo is initially under the control of maternal genomic information that is accumulated during oogenesis. The genetic programme of development soon becomes dependent on new transcripts derived from activation of the embryonic genome. The early steps in development, including the timing of the first cleavage, activation of the embryonic genome, compaction and blastocyst formation, can be affected by the culture media and conditions, as well as the production procedure itself. These perturbations can possibly result in a marked decrease in the quality of the resulting blastocysts and may even affect the viability of offspring born after transfer. In vitro procedures such as in vitro production and somatic nuclear transfer of bovine embryos have been shown to be correlated with significant up- or downregulation, de novo induction or silencing of genes critical for undisturbed fetal and neonatal development. These alterations are likely to be caused by epigenetic modifications, such as DNA methylation and histone modifications. Analysis of perturbed epigenetic reprogramming and of the related phenomena, such as genomic imprinting and X-chromosome inactivation, in bovine embryos is promising for understanding the underlying mechanisms of developmental abnormalities, such as large offspring syndrome.

scholarly journals Transcriptome Analysis of Melocactus glaucescens (Cactaceae) Reveals Metabolic Changes During in vitro Shoot Organogenesis Induction

2021 ◽  
Vol 12 ◽  
Author(s):  
Gabriela Torres-Silva ◽  
Ludmila Nayara Freitas Correia ◽  
Diego Silva Batista ◽  
Andréa Dias Koehler ◽  
Sheila Vitória Resende ◽  
...  
Keyword(s):  
De Novo ◽  
Shoot Organogenesis ◽  
Average Length ◽  
Transcriptome Assembly ◽  
Expression Patterns ◽  
Genetic Regulation ◽  
Pairwise Comparison ◽  
Illumina Hiseq ◽  
Real Time Quantitative Pcr

Melocactus glaucescens is an endangered cactus highly valued for its ornamental properties. In vitro shoot production of this species provides a sustainable alternative to overharvesting from the wild; however, its propagation could be improved if the genetic regulation underlying its developmental processes were known. The present study generated de novo transcriptome data, describing in vitro shoot organogenesis induction in M. glaucescens. Total RNA was extracted from explants before (control) and after shoot organogenesis induction (treated). A total of 14,478 unigenes (average length, 520 bases) were obtained using Illumina HiSeq 3000 (Illumina Inc., San Diego, CA, USA) sequencing and transcriptome assembly. Filtering for differential expression yielded 2,058 unigenes. Pairwise comparison of treated vs. control genes revealed that 1,241 (60.3%) unigenes exhibited no significant change, 226 (11%) were downregulated, and 591 (28.7%) were upregulated. Based on database analysis, more transcription factor families and unigenes appeared to be upregulated in the treated samples than in controls. Expression of WOUND INDUCED DEDIFFERENTIATION 1 (WIND1) and CALMODULIN (CaM) genes, both of which were upregulated in treated samples, was further validated by real-time quantitative PCR (RT-qPCR). Differences in gene expression patterns between control and treated samples indicate substantial changes in the primary and secondary metabolism of M. glaucescens after the induction of shoot organogenesis. These results help to clarify the molecular genetics and functional genomic aspects underlying propagation in the Cactaceae family.

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