scholarly journals SILAC-based quantitative proteomics reveals pleiotropic, phenotypic modulation in primary murine macrophages infected with the protozoan pathogen Leishmania donovani

2019 ◽  
Author(s):  
Despina Smirlis ◽  
Florent Dingli ◽  
Pascale Pescher ◽  
Eric Prina ◽  
Damarys Loew ◽  
...  
Keyword(s):  
Quantitative Proteomics ◽  
Leishmania Donovani ◽  
Ex Vivo ◽  
Mononuclear Phagocytes ◽  
Mammalian Host ◽  
Primary Metabolism ◽  
Strong Anion Exchange ◽  
Significant Enrichment ◽  
Bona Fide ◽  
Underlying Mechanisms

ABSTRACTLeishmaniases are major vector-borne tropical diseases responsible for great human morbidity and mortality, caused by protozoan, trypanosomatid parasites of the genus Leishmania. In the mammalian host parasites survive and multiply within mononuclear phagocytes, especially macrophages. However, the underlying mechanisms by which Leishmania spp affect their host, are not fully understood. Herein, proteomic alterations of primary bone marrow-derived, BALB/c macrophages are documented after 72 h of infection with Leishmania donovani insect-stage promastigotes, with the use of a SILAC-based, quantitative proteomics approach. The protocol was optimised by combining strong anion exchange and gel electrophoresis fractionation that displayed similar depth of analysis (>5500 proteins). Our analyses revealed 86 differentially modulated proteins (35 showing increased and 51 decreased abundance) in response to Leishmania donovani infection. The proteomics results were validated by analysing the abundance of selected proteins. Intracellular Leishmania donovani infection led to changes in various host cell biological processes, including primary metabolism and catabolic process, with a significant enrichment in lysosomal organisation. Overall, our analysis allows new technical insight into the challenges of quantitative proteomics applied on primary cells, and establishes the first proteome of bona fide primary macrophages infected ex vivo with Leishmania donovani, revealing new mechanisms acting at the host/pathogen interface.

scholarly journals Non-viral ex vivo genome-editing in mouse bona fide hematopoietic stem cells with CRISPR/Cas9

2021 ◽  
Vol 20 ◽  
pp. 451-462
Author(s):  
Suvd Byambaa ◽  
Hideki Uosaki ◽  
Tsukasa Ohmori ◽  
Hiromasa Hara ◽  
Hitoshi Endo ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Genome Editing ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Bona Fide

scholarly journals Modulation of Intestinal Phosphate Transport in Young Goats Fed a Low Phosphorus Diet

2021 ◽  
Vol 22 (2) ◽  
pp. 866
Author(s):  
Joie L. Behrens ◽  
Nadine Schnepel ◽  
Kathrin Hansen ◽  
Karin Hustedt ◽  
Marion Burmester ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Phosphate Transport ◽  
Ussing Chambers ◽  
Intestinal Epithelia ◽  
Pi Transport ◽  
Low Phosphorus ◽  
Small Intestinal ◽  
Underlying Mechanisms ◽  
Growing Goats ◽  
1,25 Dihydroxy Vitamin D3

The intestinal absorption of phosphate (Pi) takes place transcellularly through the active NaPi-cotransporters type IIb (NaPiIIb) and III (PiT1 and PiT2) and paracellularly by diffusion through tight junction (TJ) proteins. The localisation along the intestines and the regulation of Pi absorption differ between species and are not fully understood. It is known that 1,25-dihydroxy-vitamin D3 (1,25-(OH)2D3) and phosphorus (P) depletion modulate intestinal Pi absorption in vertebrates in different ways. In addition to the apical uptake into the enterocytes, there are uncertainties regarding the basolateral excretion of Pi. Functional ex vivo experiments in Ussing chambers and molecular studies of small intestinal epithelia were carried out on P-deficient goats in order to elucidate the transepithelial Pi route in the intestine as well as the underlying mechanisms of its regulation and the proteins, which may be involved. The dietary P reduction had no effect on the duodenal and ileal Pi transport rate in growing goats. The ileal PiT1 and PiT2 mRNA expressions increased significantly, while the ileal PiT1 protein expression, the mid jejunal claudin-2 mRNA expression and the serum 1,25-(OH)2D3 levels were significantly reduced. These results advance the state of knowledge concerning the complex mechanisms of the Pi homeostasis in vertebrates.

scholarly journals Total flavonoids from Astragalus alleviate endothelial dysfunction by activating the Akt/eNOS pathway

2017 ◽  
Vol 46 (6) ◽  
pp. 2096-2103 ◽  
Author(s):  
Qian Liu ◽  
Ling Zhang ◽  
Qiyuan Shan ◽  
Yuxia Ding ◽  
Zhaocai Zhang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Vascular Endothelial Cells ◽  
Ex Vivo ◽  
Umbilical Vein ◽  
Cell Counting ◽  
Total Flavonoids ◽  
Vascular Endothelial ◽  
Protective Effects ◽  
Hypoxic Conditions ◽  
Underlying Mechanisms

Objective To investigate the vasodilative and endothelial-protective effects and the underlying mechanisms of total flavonoids from Astragalus (TFA). Methods The vasodilative activities of TFA were measured with a myograph ex vivo using rat superior mesenteric arterial rings. The primary human umbilical vein endothelial cell (HUVEC) viabilities were assayed using the cell counting kit-8 after hypoxia or normoxia treatment with or without TFA. Akt, P-Akt, eNOS, P-eNOS, Erk, P-Erk, Bcl-2 and Bax expression were analyzed using western blotting. Results TFA showed concentration-dependent vasodilative effects on rat superior mesenteric arterial rings, but had no effects on normal or potassium chloride precontracted arterial rings. TFA did not affect HUVEC viabilities in normoxia, but dramatically promoted cell proliferation in the concentration range of 1 to 30 µg/mL under hypoxia. Moreover, TFA significantly increased the ratios of P-Akt/Akt and P-eNOS/eNOS in vascular endothelial cells under hypoxic conditions, but did not change the P-Erk/Erk or Bcl-2/Bax ratios. Conclusions TFA might exhibit vasorelaxant and endothelial-protective effects via the Akt/eNOS signaling pathway.

scholarly journals Lymphatic Vascular Structures: A New Aspect in Proliferative Diabetic Retinopathy

2018 ◽  
Vol 19 (12) ◽  
pp. 4034 ◽  
Author(s):  
Erika Gucciardo ◽  
Sirpa Loukovaara ◽  
Petri Salven ◽  
Kaisa Lehti
Keyword(s):  
Diabetic Retinopathy ◽  
Ex Vivo ◽  
Tissue Level ◽  
Clinical Samples ◽  
Related Factors ◽  
Disease Etiology ◽  
Working Age ◽  
Vascular Structures ◽  
Underlying Mechanisms ◽  
End Stage

Diabetic retinopathy (DR) is the most common diabetic microvascular complication and major cause of blindness in working-age adults. According to the level of microvascular degeneration and ischemic damage, DR is classified into non-proliferative DR (NPDR), and end-stage, proliferative DR (PDR). Despite advances in the disease etiology and pathogenesis, molecular understanding of end-stage PDR, characterized by ischemia- and inflammation-associated neovascularization and fibrosis, remains incomplete due to the limited availability of ideal clinical samples and experimental research models. Since a great portion of patients do not benefit from current treatments, improved therapies are essential. DR is known to be a complex and multifactorial disease featuring the interplay of microvascular, neurodegenerative, metabolic, genetic/epigenetic, immunological, and inflammation-related factors. Particularly, deeper knowledge on the mechanisms and pathophysiology of most advanced PDR is critical. Lymphatic-like vessel formation coupled with abnormal endothelial differentiation and progenitor cell involvement in the neovascularization associated with PDR are novel recent findings which hold potential for improved DR treatment. Understanding the underlying mechanisms of PDR pathogenesis is therefore crucial. To this goal, multidisciplinary approaches and new ex vivo models have been developed for a more comprehensive molecular, cellular and tissue-level understanding of the disease. This is the first step to gain the needed information on how PDR can be better evaluated, stratified, and treated.

scholarly journals Cell-Based Regeneration and Treatment of Liver Diseases

2021 ◽  
Vol 22 (19) ◽  
pp. 10276
Author(s):  
Julia Hofmann ◽  
Verena Hackl ◽  
Hannah Esser ◽  
Andras T. Meszaros ◽  
Margot Fodor ◽  
...  
Keyword(s):  
Liver Diseases ◽  
Ex Vivo ◽  
Organ Shortage ◽  
Clinical Implementation ◽  
Organ Function ◽  
Novel Technologies ◽  
Recent Developments ◽  
Underlying Mechanisms ◽  
The Moment ◽  
End Stage

The liver, in combination with a functional biliary system, is responsible for maintaining a great number of vital body functions. However, acute and chronic liver diseases may lead to irreversible liver damage and, ultimately, liver failure. At the moment, the best curative option for patients suffering from end-stage liver disease is liver transplantation. However, the number of donor livers required by far surpasses the supply, leading to a significant organ shortage. Cellular therapies play an increasing role in the restoration of organ function and can be integrated into organ transplantation protocols. Different types and sources of stem cells are considered for this purpose, but highly specific immune cells are also the focus of attention when developing individualized therapies. In-depth knowledge of the underlying mechanisms governing cell differentiation and engraftment is crucial for clinical implementation. Additionally, novel technologies such as ex vivo machine perfusion and recent developments in tissue engineering may hold promising potential for the implementation of cell-based therapies to restore proper organ function.

scholarly journals FLAMs: A self-replicating ex vivo model of alveolar macrophages for functional genetic studies

2021 ◽  
Author(s):  
Sean Thomas ◽  
Kathryn Wierenga ◽  
James Pestka ◽  
Andrew Olive
Keyword(s):  
Alveolar Macrophages ◽  
Ex Vivo ◽  
Fetal Liver ◽  
Expression Profiles ◽  
Surface Expression ◽  
Specific Gene ◽  
Ex Vivo Model ◽  
A Genome ◽  
Underlying Mechanisms ◽  
And Function

Alveolar macrophages (AMs) are tissue resident cells in the lungs derived from the fetal liver that maintain lung homeostasis and respond to inhaled stimuli. While the importance of AMs is undisputed, they remain refractory to standard experimental approaches and high-throughput functional genetics as they are challenging to isolate and rapidly lose AM properties in standard culture. This limitation hinders our understanding of key regulatory mechanisms that control AM maintenance and function. Here, we describe the development of a new model, fetal liver-derived alveolar-like macrophages (FLAMs), which maintains cellular morphologies, expression profiles, and functional mechanisms similar to murine AMs. FLAMs combine treatment with two key cytokines for AM maintenance, GM-CSF and TGFβ. We leveraged the long-term stability of FLAMs to develop functional genetic tools using CRISPR-Cas9-mediated gene editing. Targeted editing confirmed the role of AM-specific gene Marco and the IL-1 receptor Il1r1 in modulating the AM response to crystalline silica. Furthermore, a genome-wide knockout library using FLAMs identified novel genes required for surface expression of the AM marker Siglec-F, most notably those related to the peroxisome. Taken together, our results suggest that FLAMs are a stable, self-replicating model of AM function that enables previously impossible global genetic approaches to define the underlying mechanisms of AM maintenance and function.

scholarly journals Clopidogrel Resistance: Current Issues

2016 ◽  
Vol 6 (1) ◽  
pp. 38-46
Author(s):  
NS Neki
Keyword(s):  
Antiplatelet Therapy ◽  
Dual Antiplatelet Therapy ◽  
Ex Vivo ◽  
Platelet Reactivity ◽  
Antiplatelet Agents ◽  
Clinical Problem ◽  
Coronary Intervention ◽  
Platelet Activity ◽  
Clopidogrel Resistance ◽  
Underlying Mechanisms

Antiplatelet agents are mainly used in the prevention and management of atherothrombotic complications. Dual antiplatelet therapy, combining aspirin and clopidogrel, is the standard care for patients having acute coronary syndromes or undergoing percutaneous coronary intervention according to the current ACC/AHA and ESC guidelines. But in spite of administration of dual antiplatelet therapy, some patients develop recurrent cardiovascular ischemic events especially stent thrombosis which is a serious clinical problem. Antiplatelet response to clopidogrel varies widely among patients based on ex vivo platelet function measurements. Clopidogrel is an effective inhibitor of platelet activation and aggregation due to its selective and irreversible blockade of the P2Y12 receptor. Patients who display little attenuation of platelet reactivity with clopidogrel therapy are labeled as low or nonresponders or clopidogrel resistant. The mechanism of clopidogrel resistance remains incompletely defined but there are certain clinical, cellular and genetic factors including polymorphisms responsible for therapeutic failure. Currently there is no standardized or widely accepted definition of clopidogrel resistance. The future may soon be realised in the routine measurement of platelet activity in the same way that blood pressure, cholesterol and blood sugar are followed to help guide the therapy, thus improving the care for millions of people. This review focuses on the methods used to identify patients with clopidogrel resistance, the underlying mechanisms, metabolism, clinical significance and current therapeutic strategies to overcome clopidogrel resistance.J Enam Med Col 2016; 6(1): 38-46

scholarly journals Comparative Replication and Immune Activation Profiles of SARS-CoV-2 and SARS-CoV in Human Lungs: An Ex Vivo Study With Implications for the Pathogenesis of COVID-19

2020 ◽  
Vol 71 (6) ◽  
pp. 1400-1409 ◽  
Author(s):  
Hin Chu ◽  
Jasper Fuk-Woo Chan ◽  
Yixin Wang ◽  
Terrence Tsz-Tai Yuen ◽  
Yue Chai ◽  
...  
Keyword(s):  
Immune Activation ◽  
Human Lung ◽  
Ex Vivo ◽  
Clinical Manifestations ◽  
Asymptomatic Infection ◽  
Replication Capacity ◽  
Cell Tropism ◽  
Human Lungs ◽  
Activation Profile ◽  
Underlying Mechanisms

Abstract Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging coronavirus that has resulted in more than 2 000 000 laboratory-confirmed cases including over 145 000 deaths. Although SARS-CoV-2 and SARS-CoV share a number of common clinical manifestations, SARS-CoV-2 appears to be highly efficient in person-to-person transmission and frequently causes asymptomatic or presymptomatic infections. However, the underlying mechanisms that confer these viral characteristics of high transmissibility and asymptomatic infection remain incompletely understood. Methods We comprehensively investigated the replication, cell tropism, and immune activation profile of SARS-CoV-2 infection in human lung tissues with SARS-CoV included as a comparison. Results SARS-CoV-2 infected and replicated in human lung tissues more efficiently than SARS-CoV. Within the 48-hour interval, SARS-CoV-2 generated 3.20-fold more infectious virus particles than did SARS-CoV from the infected lung tissues (P < .024). SARS-CoV-2 and SARS-CoV were similar in cell tropism, with both targeting types I and II pneumocytes and alveolar macrophages. Importantly, despite the more efficient virus replication, SARS-CoV-2 did not significantly induce types I, II, or III interferons in the infected human lung tissues. In addition, while SARS-CoV infection upregulated the expression of 11 out of 13 (84.62%) representative proinflammatory cytokines/chemokines, SARS-CoV-2 infection only upregulated 5 of these 13 (38.46%) key inflammatory mediators despite replicating more efficiently. Conclusions Our study provides the first quantitative data on the comparative replication capacity and immune activation profile of SARS-CoV-2 and SARS-CoV infection in human lung tissues. Our results provide important insights into the pathogenesis, high transmissibility, and asymptomatic infection of SARS-CoV-2.

scholarly journals Challenges and Strategies in Ascribing Functions to Long Noncoding RNAs

Cancers ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 1458 ◽  
Author(s):  
Yang Zhao ◽  
Hongqi Teng ◽  
Fan Yao ◽  
Shannon Yap ◽  
Yutong Sun ◽  
...  
Keyword(s):  
Regulation Of Gene Expression ◽  
Noncoding Rnas ◽  
Genetically Engineered ◽  
Long Noncoding Rnas ◽  
Genetically Engineered Mouse Models ◽  
Genomic Location ◽  
Bona Fide ◽  
Underlying Mechanisms ◽  
Post Transcriptional Regulation ◽  
In Cis

Long noncoding RNAs (lncRNAs) are involved in many physiological and pathological processes, such as development, aging, immunity, and cancer. Mechanistically, lncRNAs exert their functions through interaction with proteins, genomic DNA, and other RNA, leading to transcriptional and post-transcriptional regulation of gene expression, either in cis or in trans; it is often difficult to distinguish between these two regulatory mechanisms. A variety of approaches, including RNA interference, antisense oligonucleotides, CRISPR-based methods, and genetically engineered mouse models, have yielded abundant information about lncRNA functions and underlying mechanisms, albeit with many discrepancies. In this review, we elaborate on the challenges in ascribing functions to lncRNAs based on the features of lncRNAs, including the genomic location, copy number, domain structure, subcellular localization, stability, evolution, and expression pattern. We also describe a framework for the investigation of lncRNA functions and mechanisms of action. Rigorous characterization of cancer-implicated lncRNAs is critical for the identification of bona fide anticancer targets.

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