scholarly journals Functional Characterization of Multiple Ehrlichia chaffeensis Sodium (Cation)/Proton Antiporter Genes Involved in the Bacterial pH Homeostasis

2021 ◽  
Vol 22 (16) ◽  
pp. 8420
Author(s):  
Lanjing Wei ◽  
Huitao Liu ◽  
Kimia Alizadeh ◽  
Maria D. Juarez-Rodriguez ◽  
Roman R. Ganta
Keyword(s):  
Functional Characterization ◽  
Sodium Cation ◽  
Ehrlichia Chaffeensis ◽  
Ph Homeostasis ◽  
Protein Coding ◽  
Extracellular Milieu ◽  
Diffusion Analysis ◽  
Wide Range ◽  
Human Monocytic Ehrlichiosis

Ehrlichia chaffeensis causes human monocytic ehrlichiosis. Little is known about how this and other related tick-borne rickettsia pathogens maintain pH homeostasis in acidified phagosomes and the extracellular milieu. The membrane-bound sodium (cation)/proton antiporters are found in a wide range of organisms aiding pH homeostasis. We recently reported a mutation in an antiporter gene of E. chaffeensis (ECH_0379) which causes bacterial in vivo attenuation. The E. chaffeensis genome contains 10 protein coding sequences encoding for predicted antiporters. We report here that nine of these genes are transcribed during the bacterial growth in macrophages and tick cells. All E. chaffeensis antiporter genes functionally complemented antiporter deficient Escherichia coli. Antiporter activity for all predicted E. chaffeensis genes was observed at pH 5.5, while gene products of ECH_0179 and ECH_0379 were also active at pH 8.0, and ECH_0179 protein was complemented at pH 7.0. The antiporter activity was independently verified for the ECH_0379 protein by proteoliposome diffusion analysis. This is the first description of antiporters in E. chaffeensis and demonstrates that the pathogen contains multiple antiporters with varying biological functions, which are likely important for the pH homeostasis of the pathogen’s replicating and infectious forms.

scholarly journals Tunneling Nanotubes and the Eye: Intercellular Communication and Implications for Ocular Health and Disease

2020 ◽  
Vol 2020 ◽  
pp. 1-15 ◽  
Author(s):  
Holly R. Chinnery ◽  
Kate E. Keller
Keyword(s):  
Eye Diseases ◽  
Cellular Communication ◽  
Tunneling Nanotubes ◽  
Extracellular Milieu ◽  
Wide Range ◽  
Aqueous Environments ◽  
Health And Disease ◽  
Ocular Health

Cellular communication is an essential process for the development and maintenance of all tissues including the eye. Recently, a new method of cellular communication has been described, which relies on formation of tubules, called tunneling nanotubes (TNTs). These structures connect the cytoplasm of adjacent cells and allow the direct transport of cellular cargo between cells without the need for secretion into the extracellular milieu. TNTs may be an important mechanism for signaling between cells that reside long distances from each other or for cells in aqueous environments, where diffusion-based signaling is challenging. Given the wide range of cargoes transported, such as lysosomes, endosomes, mitochondria, viruses, and miRNAs, TNTs may play a role in normal homeostatic processes in the eye as well as function in ocular disease. This review will describe TNT cellular communication in ocular cell cultures and the mammalian eye in vivo, the role of TNTs in mitochondrial transport with an emphasis on mitochondrial eye diseases, and molecules involved in TNT biogenesis and their function in eyes, and finally, we will describe TNT formation in inflammation, cancer, and stem cells, focusing on pathological processes of particular interest to vision scientists.

scholarly journals Long non-coding RNAs in plants: emerging modulators of gene activity in development and stress responses

Planta ◽  
2020 ◽  
Vol 252 (5) ◽  
Author(s):  
Li Chen ◽  
Qian-Hao Zhu ◽  
Kerstin Kaufmann
Keyword(s):  
Stress Responses ◽  
Translational Regulation ◽  
Molecular Mechanisms ◽  
Functional Characterization ◽  
Reproductive Organs ◽  
Gene Activity ◽  
Protein Coding ◽  
Wide Range ◽  
Non Coding Rnas ◽  
Coding Potential

Abstract Main conclusion Long non-coding RNAs modulate gene activity in plant development and stress responses by various molecular mechanisms. Abstract Long non-coding RNAs (lncRNAs) are transcripts larger than 200 nucleotides without protein coding potential. Computational approaches have identified numerous lncRNAs in different plant species. Research in the past decade has unveiled that plant lncRNAs participate in a wide range of biological processes, including regulation of flowering time and morphogenesis of reproductive organs, as well as abiotic and biotic stress responses. LncRNAs execute their functions by interacting with DNA, RNA and protein molecules, and by modulating the expression level of their targets through epigenetic, transcriptional, post-transcriptional or translational regulation. In this review, we summarize characteristics of plant lncRNAs, discuss recent progress on understanding of lncRNA functions, and propose an experimental framework for functional characterization.

scholarly journals Achieving tight control of a photoactivatable Cre recombinase gene switch: new design strategies and functional characterization in mammalian cells and rodent

2019 ◽  
Vol 47 (17) ◽  
pp. e97-e97 ◽  
Author(s):  
Kyle Meador ◽  
Christina L Wysoczynski ◽  
Aaron J Norris ◽  
Jason Aoto ◽  
Michael R Bruchas ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Cultured Cells ◽  
Functional Characterization ◽  
Cre Recombinase ◽  
Common Mechanism ◽  
Protein Fragments ◽  
Low Background ◽  
Wide Range ◽  
Split Protein

AbstractA common mechanism for inducibly controlling protein function relies on reconstitution of split protein fragments using chemical or light-induced dimerization domains. A protein is split into fragments that are inactive on their own, but can be reconstituted after dimerization. As many split proteins retain affinity for their complementary half, maintaining low activity in the absence of an inducer remains a challenge. Here, we systematically explore methods to achieve tight regulation of inducible proteins that are effective despite variation in protein expression level. We characterize a previously developed split Cre recombinase (PA-Cre2.0) that is reconstituted upon light-induced CRY2-CIB1 dimerization, in cultured cells and in vivo in rodent brain. In culture, PA-Cre2.0 shows low background and high induced activity over a wide range of expression levels, while in vivo the system also shows low background and sensitive response to brief light inputs. The consistent activity stems from fragment compartmentalization that shifts localization toward the cytosol. Extending this work, we exploit nuclear compartmentalization to generate light-and-chemical regulated versions of Cre recombinase. This work demonstrates in vivo functionality of PA-Cre2.0, describes new approaches to achieve tight inducible control of Cre DNA recombinase, and provides general guidelines for further engineering and application of split protein fragments.

scholarly journals Ehrlichia chaffeensis Infections in Drosophila melanogaster

2009 ◽  
Vol 77 (11) ◽  
pp. 4815-4826 ◽  
Author(s):  
Alison Luce-Fedrow ◽  
Tonia Von Ohlen ◽  
Stephen K. Chapes
Keyword(s):  
Drosophila Melanogaster ◽  
Amblyomma Americanum ◽  
Immune Defense ◽  
Ehrlichia Chaffeensis ◽  
Humoral Immune ◽  
Humoral Immune Responses ◽  
Defense Induction ◽  
Humoral Responses ◽  
Human Monocytic Ehrlichiosis

ABSTRACT Ehrlichia chaffeensis is an obligate, intracellular bacterium, transmitted by the tick Amblyomma americanum, and is the causative agent of human monocytic ehrlichiosis infections. We previously demonstrated that E. chaffeensis is capable of growing in Drosophila S2 cells. Therefore, we tested the hypothesis that E. chaffeensis can infect adult Drosophila melanogaster. Adult Drosophila organisms were experimentally challenged with intra-abdominal injections of bacteria. Ehrlichia-infected flies showed decreased survival compared to wild-type flies, and bacteria isolated from flies could reinfect mammalian macrophages. Ehrlichia infections activated both the cellular and humoral immune responses in the fly. Hemocytes phagocytosed bacteria after injection, and antimicrobial peptide pathways were induced following infection. Increased pathogenicity in flies carrying mutations in genes in both the Toll and Imd pathways suggests that both immune defense pathways participate in host defense. Induction of Drosophila cellular and humoral responses and the in vivo replication of E. chaffeensis suggests that D. melanogaster is a suitable host for E. chaffeensis. In the future, it will be a useful tool to unlock some of the in vivo mysteries of this arthropod-borne bacterium.

scholarly journals The long non-coding RNA landscape of Candida yeast pathogens

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Hrant Hovhannisyan ◽  
Toni Gabaldón
Keyword(s):  
Functional Characterization ◽  
Evolutionary Constraint ◽  
Sequence Motifs ◽  
Candida Auris ◽  
Protein Coding ◽  
Cellular Processes ◽  
Non Coding Rna ◽  
Wide Range ◽  
Eukaryotic Microbes ◽  
Genomic Regions

AbstractLong non-coding RNAs (lncRNAs) constitute a poorly studied class of transcripts with emerging roles in key cellular processes. Despite efforts to characterize lncRNAs across a wide range of species, these molecules remain largely unexplored in most eukaryotic microbes, including yeast pathogens of the Candida clade. Here, we analyze thousands of publicly available sequencing datasets to infer and characterize the lncRNA repertoires of five major Candida pathogens: Candida albicans, Candida tropicalis, Candida parapsilosis, Candida auris and Candida glabrata. Our results indicate that genomes of these species encode hundreds of lncRNAs that show levels of evolutionary constraint intermediate between those of intergenic genomic regions and protein-coding genes. Despite their low sequence conservation across the studied species, some lncRNAs are syntenic and are enriched in shared sequence motifs. We find co-expression of lncRNAs with certain protein-coding transcripts, hinting at potential functional associations. Finally, we identify lncRNAs that are differentially expressed during infection of human epithelial cells for four of the studied species. Our comprehensive bioinformatic analyses of Candida lncRNAs pave the way for future functional characterization of these transcripts.

scholarly journals Mutations in Ehrlichia chaffeensis Genes ECH_0660 and ECH_0665 Cause Transcriptional Changes in Response to Zinc or Iron Limitation

2021 ◽  
Author(s):  
Ascención Torres-Escobar ◽  
María D. Juárez-Rodríguez ◽  
Roman R. Ganta
Keyword(s):  
Iron Limitation ◽  
Ehrlichia Chaffeensis ◽  
Wild Type ◽  
Gene Encoding ◽  
Transcriptional Changes ◽  
Rapid Clearance ◽  
Human Monocytic Ehrlichiosis ◽  
Phage Proteins ◽  
Type Infection

Ehrlichia chaffeensis causes human monocytic ehrlichiosis by replicating within phagosomes of monocytes/macrophages. A function disruption mutation within the pathogen’s ECH_0660 gene encoding a phage head-to-tail connector protein resulted in the rapid clearance of the pathogen in vivo, while aiding to induce sufficient immunity in a host to protect against wild-type infection challenge. In this study, we describe the characterization of a cluster of seven genes spanning from ECH_0659 to ECH_0665, which contained four genes encoding bacterial phage proteins, including the ECH_0660 gene. Assessment of the promoter region upstream to the first gene of the seven genes (ECH_0659) in Escherichia coli demonstrated transcriptional enhancement under zinc and iron starvation. Further, transcription of the seven genes was significantly higher for E. chaffeensis having a mutation in the ECH_0660 gene compared to the wild-type pathogen under zinc and iron starving conditions. In contrast, transcription from the genes was mostly similar to wild-type or moderately downregulated for the ECH_0665 gene mutant with the function disruption. Recently, we reported that this mutation caused a minimal impact on the pathogen’s in vivo growth, as it persisted similar to wild-type. The current study is the first in describing how zinc and iron contribute to E. chaffeensis biology. Specifically, we demonstrated that the functional disruption in the gene encoding the predicted head-to-tail connector protein in E. chaffeensis results in the enhanced transcription of seven genes including those encoding phage proteins during zinc and iron limitation. IMPORTANCE Ehrlichia chaffeensis, a tick-transmitted bacterium, causes human monocytic ehrlichiosis by replicating within phagosomes of monocytes/macrophages. A function disruption mutation within the pathogen’s gene encoding a phage head-to-tail connector protein resulted in the rapid clearance of the pathogen in vivo, while aiding to induce sufficient immunity in a host to protect against wild-type infection challenge. In the current study, we investigated if the functional disruption in the predicted head-to-tail connector protein gene caused transcriptional changes resulting from metal ion limitations. This is the first study describing how zinc and iron may contribute to E. chaffeensis replication.

scholarly journals Tracking calcium dynamics from individual neurons in behaving animals

2021 ◽  
Vol 17 (10) ◽  
pp. e1009432
Author(s):  
Thibault Lagache ◽  
Alison Hanson ◽  
Jesús E. Pérez-Ortega ◽  
Adrienne Fairhall ◽  
Rafael Yuste
Keyword(s):  
Visual Cortex ◽  
Calcium Imaging ◽  
Functional Characterization ◽  
Ground Truth ◽  
Time Lapse ◽  
Local Deformation ◽  
Neuron Activity ◽  
Imaging Data ◽  
Wide Range

Measuring the activity of neuronal populations with calcium imaging can capture emergent functional properties of neuronal circuits with single cell resolution. However, the motion of freely behaving animals, together with the intermittent detectability of calcium sensors, can hinder automatic monitoring of neuronal activity and their subsequent functional characterization. We report the development and open-source implementation of a multi-step cellular tracking algorithm (Elastic Motion Correction and Concatenation or EMC2) that compensates for the intermittent disappearance of moving neurons by integrating local deformation information from detectable neurons. We demonstrate the accuracy and versatility of our algorithm using calcium imaging data from two-photon volumetric microscopy in visual cortex of awake mice, and from confocal microscopy in behaving Hydra, which experiences major body deformation during its contractions. We quantify the performance of our algorithm using ground truth manual tracking of neurons, along with synthetic time-lapse sequences, covering a wide range of particle motions and detectability parameters. As a demonstration of the utility of the algorithm, we monitor for several days calcium activity of the same neurons in layer 2/3 of mouse visual cortex in vivo, finding significant turnover within the active neurons across days, with only few neurons that remained active across days. Also, combining automatic tracking of single neuron activity with statistical clustering, we characterize and map neuronal ensembles in behaving Hydra, finding three major non-overlapping ensembles of neurons (CB, RP1 and RP2) whose activity correlates with contractions and elongations. Our results show that the EMC2 algorithm can be used as a robust and versatile platform for neuronal tracking in behaving animals.

Functional characterization of human brown adipose tissue metabolism

2020 ◽  
Vol 477 (7) ◽  
pp. 1261-1286 ◽  
Author(s):  
Marie Anne Richard ◽  
Hannah Pallubinsky ◽  
Denis P. Blondin
Keyword(s):  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Functional Characterization ◽  
Human Infants ◽  
Positron Emission ◽  
Brown Adipose ◽  
Treatment Of Obesity ◽  
And Function

Brown adipose tissue (BAT) has long been described according to its histological features as a multilocular, lipid-containing tissue, light brown in color, that is also responsive to the cold and found especially in hibernating mammals and human infants. Its presence in both hibernators and human infants, combined with its function as a heat-generating organ, raised many questions about its role in humans. Early characterizations of the tissue in humans focused on its progressive atrophy with age and its apparent importance for cold-exposed workers. However, the use of positron emission tomography (PET) with the glucose tracer [18F]fluorodeoxyglucose ([18F]FDG) made it possible to begin characterizing the possible function of BAT in adult humans, and whether it could play a role in the prevention or treatment of obesity and type 2 diabetes (T2D). This review focuses on the in vivo functional characterization of human BAT, the methodological approaches applied to examine these features and addresses critical gaps that remain in moving the field forward. Specifically, we describe the anatomical and biomolecular features of human BAT, the modalities and applications of non-invasive tools such as PET and magnetic resonance imaging coupled with spectroscopy (MRI/MRS) to study BAT morphology and function in vivo, and finally describe the functional characteristics of human BAT that have only been possible through the development and application of such tools.

Evaluation of 99mTc-Label led Vitamin K4 as an Agent for Testicular Imaging

1991 ◽  
Vol 30 (01) ◽  
pp. 35-39 ◽  
Author(s):  
H. S. Durak ◽  
M. Kitapgi ◽  
B. E. Caner ◽  
R. Senekowitsch ◽  
M. T. Ercan
Keyword(s):  
Soft Tissue ◽  
Room Temperature ◽  
Normal Subjects ◽  
Left Testis ◽  
Wide Range ◽  
Activity Ratios ◽  
The Right ◽  
Radioactivity Levels

Vitamin K4 was labelled with 99mTc with an efficiency higher than 97%. The compound was stable up to 24 h at room temperature, and its biodistribution in NMRI mice indicated its in vivo stability. Blood radioactivity levels were high over a wide range. 10% of the injected activity remained in blood after 24 h. Excretion was mostly via kidneys. Only the liver and kidneys concentrated appreciable amounts of radioactivity. Testis/soft tissue ratios were 1.4 and 1.57 at 6 and 24 h, respectively. Testis/blood ratios were lower than 1. In vitro studies with mouse blood indicated that 33.9 ±9.6% of the radioactivity was associated with RBCs; it was washed out almost completely with saline. Protein binding was 28.7 ±6.3% as determined by TCA precipitation. Blood clearance of 99mTc-l<4 in normal subjects showed a slow decrease of radioactivity, reaching a plateau after 16 h at 20% of the injected activity. In scintigraphic images in men the testes could be well visualized. The right/left testis ratio was 1.08 ±0.13. Testis/soft tissue and testis/blood activity ratios were highest at 3 h. These ratios were higher than those obtained with pertechnetate at 20 min post injection.99mTc-l<4 appears to be a promising radiopharmaceutical for the scintigraphic visualization of testes.

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