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Article ; Online: Two-step protocol for preparing adherent cells for high-throughput flow cytometry.

Kaur, Mandeep / Esau, Luke

2015 Volume 59, Issue 3, Page(s) 119–126

Abstract: We have developed a simple, cost-effective, and labor-efficient two-step protocol for preparing adherent cells for high-throughput flow cytometry. Adherent cells were grown on microplates, detached with 2.9 mM EDTA (pH 6.14) added directly to wells ... ...

Abstract	We have developed a simple, cost-effective, and labor-efficient two-step protocol for preparing adherent cells for high-throughput flow cytometry. Adherent cells were grown on microplates, detached with 2.9 mM EDTA (pH 6.14) added directly to wells containing cell culture medium, stained, and then analyzed on a flow cytometer. This protocol bypasses washing, centrifugation, and transfer between plates, reducing the cell loss that occurs in standard multistep protocols. The method has been validated using six adherent cell lines, four commercially available dyes, and two antibodies; the results have been confirmed using two different flow cytometry (FC) instruments. Our approach has been used for estimating apoptosis, mitochondrial membrane potential, reactive oxygen species, and autophagy in response to exposure to pure compounds as well as plant and bacterial extracts.
MeSH term(s)	Apoptosis ; Cell Culture Techniques/methods ; Cell Line ; Cell Line, Tumor ; Edetic Acid ; Female ; Fibroblasts/cytology ; Flow Cytometry/instrumentation ; Flow Cytometry/methods ; Fluoresceins/chemistry ; Fluorescent Dyes/chemistry ; High-Throughput Screening Assays/methods ; Humans ; Male
Chemical Substances	Fluoresceins ; Fluorescent Dyes ; diacetyldichlorofluorescein (2044-85-1) ; Edetic Acid (9G34HU7RV0)
Language	English
Publishing date	2015-09
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	48453-2
ISSN	1940-9818 ; 0736-6205
ISSN (online)	1940-9818
ISSN	0736-6205
DOI	10.2144/000114325
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Article ; Online: Mining the deep Red-Sea brine pool microbial community for anticancer therapeutics

Esau, Luke / Zhang, Guishan / Sagar, Sunil / Stingl, Ulrich / Bajić, V. B. / Kaur, Mandeep

BMC Complement Altern Med. 2019 Dec., v. 19, no. 1 p.142-142

2019

Abstract: BACKGROUND: Microbial species in the brine pools of the Red Sea and the brine pool-seawater interfaces are exposed to high temperature, high salinity, low oxygen levels and high concentrations of heavy metals. As adaptations to these harsh conditions ... ...

Abstract	BACKGROUND: Microbial species in the brine pools of the Red Sea and the brine pool-seawater interfaces are exposed to high temperature, high salinity, low oxygen levels and high concentrations of heavy metals. As adaptations to these harsh conditions require a large suite of secondary metabolites, these microbes have a huge potential as a source of novel anticancer molecules. METHODS: A total of 60 ethyl-acetate extracts of newly isolated strains from extreme environments of the Red-Sea were isolated and tested against several human cancer cell lines for potential cytotoxic and apoptotic activities. RESULTS: Isolates from the Erba brine-pool accounted for 50% of active bacterial extracts capable of inducing 30% or greater inhibition of cell growth. Among the 60 extracts screened, seven showed selectivity towards triple negative BT20 cells compared to normal fibroblasts. CONCLUSION: In this study, we identified several extracts able to induce caspase-dependent apoptosis in various cancer cell lines. Further investigations and isolation of the active compounds of these Red Sea brine pool microbes may offer a chemotherapeutic potential for cancers with limited treatment options.
Keywords	apoptosis ; cancer therapy ; cell growth ; complement ; cytotoxicity ; drug therapy ; ethyl acetate ; fibroblasts ; humans ; microbial communities ; neoplasm cells ; oxygen ; salinity ; secondary metabolites ; temperature ; Red Sea
Language	English
Dates of publication	2019-12
Size	p. 142.
Publishing place	BioMed Central
Document type	Article ; Online
Note	Resource is Open Access
ZDB-ID	2050429-9
ISSN	1472-6882
ISSN	1472-6882
DOI	10.1186/s12906-019-2554-0
Database	NAL-Catalogue (AGRICOLA)

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Article: Identification of

Esau, Luke / Sagar, Sunil / Bangarusamy, Dhinoth / Kaur, Mandeep

Genes & cancer

2016 Volume 7, Issue 9-10, Page(s) 309–322

Abstract: Cholesterol and its metabolites act as steroid hormone precursors, which promote estrogen receptor positive (ER+) breast cancer (BC) progression. Development of cholesterol targeting anticancer drugs has been hindered due to the lack of knowledge of ... ...

Abstract	Cholesterol and its metabolites act as steroid hormone precursors, which promote estrogen receptor positive (ER+) breast cancer (BC) progression. Development of cholesterol targeting anticancer drugs has been hindered due to the lack of knowledge of viable molecular targets. Till now, Cholesteryl ester transfer protein (CETP) has been envisaged as a feasible molecular target in atherosclerosis, but for the first time, we show that
Language	English
Publishing date	2016-12-23
Publishing country	United States
Document type	Journal Article
ZDB-ID	2538519-7
ISSN	1947-6027 ; 1947-6019
ISSN (online)	1947-6027
ISSN	1947-6019
DOI	10.18632/genesandcancer.122
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Article ; Online: Mining the deep Red-Sea brine pool microbial community for anticancer therapeutics.

Esau, Luke / Zhang, Guishan / Sagar, Sunil / Stingl, Ulrich / Bajic, Vladimir B / Kaur, Mandeep

BMC complementary and alternative medicine

2019 Volume 19, Issue 1, Page(s) 142

Abstract: Background: Microbial species in the brine pools of the Red Sea and the brine pool-seawater interfaces are exposed to high temperature, high salinity, low oxygen levels and high concentrations of heavy metals. As adaptations to these harsh conditions ... ...

Abstract	Background: Microbial species in the brine pools of the Red Sea and the brine pool-seawater interfaces are exposed to high temperature, high salinity, low oxygen levels and high concentrations of heavy metals. As adaptations to these harsh conditions require a large suite of secondary metabolites, these microbes have a huge potential as a source of novel anticancer molecules. Methods: A total of 60 ethyl-acetate extracts of newly isolated strains from extreme environments of the Red-Sea were isolated and tested against several human cancer cell lines for potential cytotoxic and apoptotic activities. Results: Isolates from the Erba brine-pool accounted for 50% of active bacterial extracts capable of inducing 30% or greater inhibition of cell growth. Among the 60 extracts screened, seven showed selectivity towards triple negative BT20 cells compared to normal fibroblasts. Conclusion: In this study, we identified several extracts able to induce caspase-dependent apoptosis in various cancer cell lines. Further investigations and isolation of the active compounds of these Red Sea brine pool microbes may offer a chemotherapeutic potential for cancers with limited treatment options.
MeSH term(s)	Antineoplastic Agents/isolation & purification ; Antineoplastic Agents/pharmacology ; Apoptosis/drug effects ; Bacteria/chemistry ; Bacteria/classification ; Bacteria/genetics ; Bacteria/isolation & purification ; Cell Line, Tumor ; Humans ; Indian Ocean ; Microbiota ; Salts/chemistry ; Seawater/microbiology
Chemical Substances	Antineoplastic Agents ; Salts ; brine
Language	English
Publishing date	2019-06-20
Publishing country	England
Document type	Journal Article
ZDB-ID	2050429-9
ISSN	1472-6882 ; 1472-6882
ISSN (online)	1472-6882
ISSN	1472-6882
DOI	10.1186/s12906-019-2554-0
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Article ; Online: Impact of the SARS-CoV-2 nucleocapsid 203K/204R mutations on the inflammatory immune response in COVID-19 severity.

Shuaib, Muhammad / Adroub, Sabir / Mourier, Tobias / Mfarrej, Sara / Zhang, Huoming / Esau, Luke / Alsomali, Afrah / Alofi, Fadwa S / Ahmad, Adeel Nazir / Shamsan, Abbas / Khogeer, Asim / Hashem, Anwar M / Almontashiri, Naif A M / Hala, Sharif / Pain, Arnab

Genome medicine

2023 Volume 15, Issue 1, Page(s) 54

Abstract: Background: The excessive inflammatory responses provoked by SARS-CoV-2 infection are critical factors affecting the severity and mortality of COVID-19. Previous work found that two adjacent co-occurring mutations R203K and G204R (KR) on the ... ...

Abstract	Background: The excessive inflammatory responses provoked by SARS-CoV-2 infection are critical factors affecting the severity and mortality of COVID-19. Previous work found that two adjacent co-occurring mutations R203K and G204R (KR) on the nucleocapsid (N) protein correlate with increased disease severity in COVID-19 patients. However, links with the host immune response remain unclear. Methods: Here, we grouped nasopharyngeal swab samples of COVID-19 patients into two cohorts based on the presence and absence of SARS-CoV-2 nucleocapsid KR mutations. We performed nasopharyngeal transcriptome analysis of age, gender, and ethnicity-matched COVID-19 patients infected with either SARS-CoV-2 with KR mutations in the N protein (KR patients n = 39) or with the wild-type N protein (RG patients n = 39) and compared to healthy controls (n = 34). The impact of KR mutation on immune response was further characterized experimentally by transcriptomic and proteomic profiling of virus-like-particle (VLP) incubated cells. Results: We observed markedly elevated expression of proinflammatory cytokines, chemokines, and interferon-stimulated (ISGs) genes in the KR patients compared to RG patients. Using nasopharyngeal transcriptome data, we found significantly higher levels of neutrophils and neutrophil-to-lymphocyte (NLR) ratio in KR patients than in the RG patients. Furthermore, transcriptomic and proteomic profiling of VLP incubated cells confirmed a similar hyper-inflammatory response mediated by the KR variant. Conclusions: Our data demonstrate an unforeseen connection between nucleocapsid KR mutations and augmented inflammatory immune response in severe COVID-19 patients. These findings provide insights into how mutations in SARS-CoV-2 modulate host immune output and pathogenesis and may contribute to more efficient therapeutics and vaccine development.
MeSH term(s)	COVID-19/immunology ; Inflammation/immunology ; Humans ; HEK293 Cells ; SARS-CoV-2/genetics ; Mutation ; Severity of Illness Index
Chemical Substances	N protein, SARS-CoV
Language	English
Publishing date	2023-07-21
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2484394-5
ISSN	1756-994X ; 1756-994X
ISSN (online)	1756-994X
ISSN	1756-994X
DOI	10.1186/s13073-023-01208-0
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Article ; Online: Refining the migration and engraftment of short-term and long-term HSCs by enhancing homing-specific adhesion mechanisms.

Al-Amoodi, Asma S / Li, Yanyan / Al-Ghuneim, Arwa / Allehaibi, Hanaa / Isaioglou, Ioannis / Esau, Luke E / AbuSamra, Dina B / Merzaban, Jasmeen S

Blood advances

2022 Volume 6, Issue 15, Page(s) 4373–4391

Abstract: In contrast to the short-term (ST) CD34+ stem cells, studies have suggested that long-term (LT) hematopoietic stem cells (HSCs) found in the CD34- stem cell pool have trouble migrating and engrafting when introduced through IV. To understand why these ... ...

Abstract	In contrast to the short-term (ST) CD34+ stem cells, studies have suggested that long-term (LT) hematopoietic stem cells (HSCs) found in the CD34- stem cell pool have trouble migrating and engrafting when introduced through IV. To understand why these deficiencies exist, we set out to fully elucidate the adhesion mechanisms used by ST and LT-HSCs to migrate to the bone marrow(BM). Specifically focusing on murine ST-HSCs (Flk2-CD34+) and LT-HSCs (Flk2-CD34-), we observed a distinctive expression pattern of BM homing effectors necessary for the first step, namely sialyl Lewis-X (sLex) (ligand for E-selectin), and the second step, namely CXCR4 chemokine receptor (receptor for SDF-1). sLex expression was higher on Flk2-CD34+ ST-HSCs (>60%) compared with Flk2-CD34- LT-HSCs (<10%), which correlated to binding to E-selectin. Higher concentrations of CXCR4 were observed on Flk2-CD34+ ST-HSCs compared with Flk2-CD34- LT-HSCs. Interestingly, the expression of CD26, a peptidase known to deactivate chemokines (ie, SDF-1), was higher on Flk2-CD34- LT-HSCs. Given that both E-selectin-binding and CXCR4-mediated migration are compromised in Flk2-CD34- LT-HSCs, we aimed to enhance their ability to migrate using recombinant human fucosyltransferase 6 (rhFTVI) and the CD26 inhibitor, Dip A (diprotin A). To this end, we observed that although LT-HSCs expressed low concentrations of sLex, they were able to engraft when transplanted into recipient mice. Moreover, although both CD26 inhibition and fucosylation enhanced migration of both HSC populations in vitro, only pretreatment of LT-HSCs with Dip A enhanced engraftment in vivo after transplantation into recipient mice. Remarkably, fucosylation of Flk2-CD34+ ST-HSCs consistently led to their ability to transplant secondary recipients. These data suggest that using fucosylation and Dip A to overcome the molecular disparity in adhesion mechanisms among ST-HSCs and LT-HSCs differentially influences their abilities to migrate and engraft in vivo and promotes the ability of ST-HSCs to engraft secondary recipient mice, the gold standard for testing functionality of LT-HSCs.
MeSH term(s)	Animals ; Antigens, CD34/metabolism ; Bone Marrow/metabolism ; Dipeptidyl Peptidase 4/metabolism ; E-Selectin/metabolism ; Hematopoietic Stem Cells/metabolism ; Humans ; Mice
Chemical Substances	Antigens, CD34 ; E-Selectin ; Dipeptidyl Peptidase 4 (EC 3.4.14.5)
Language	English
Publishing date	2022-06-09
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2915908-8
ISSN	2473-9537 ; 2473-9529
ISSN (online)	2473-9537
ISSN	2473-9529
DOI	10.1182/bloodadvances.2022007465
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Article ; Online: Molecular insights into the Darwin paradox of coral reefs from the sea anemone Aiptasia.

Cui, Guoxin / Konciute, Migle K / Ling, Lorraine / Esau, Luke / Raina, Jean-Baptiste / Han, Baoda / Salazar, Octavio R / Presnell, Jason S / Rädecker, Nils / Zhong, Huawen / Menzies, Jessica / Cleves, Phillip A / Liew, Yi Jin / Krediet, Cory J / Sawiccy, Val / Cziesielski, Maha J / Guagliardo, Paul / Bougoure, Jeremy / Pernice, Mathieu /

Hirt, Heribert / Voolstra, Christian R / Weis, Virginia M / Pringle, John R / Aranda, Manuel

Science advances

2023 Volume 9, Issue 11, Page(s) eadf7108

Abstract: Symbiotic cnidarians such as corals and anemones form highly productive and biodiverse coral reef ecosystems in nutrient-poor ocean environments, a phenomenon known as Darwin's paradox. Resolving this paradox requires elucidating the molecular bases of ... ...

Abstract	Symbiotic cnidarians such as corals and anemones form highly productive and biodiverse coral reef ecosystems in nutrient-poor ocean environments, a phenomenon known as Darwin's paradox. Resolving this paradox requires elucidating the molecular bases of efficient nutrient distribution and recycling in the cnidarian-dinoflagellate symbiosis. Using the sea anemone Aiptasia, we show that during symbiosis, the increased availability of glucose and the presence of the algae jointly induce the coordinated up-regulation and relocalization of glucose and ammonium transporters. These molecular responses are critical to support symbiont functioning and organism-wide nitrogen assimilation through glutamine synthetase/glutamate synthase-mediated amino acid biosynthesis. Our results reveal crucial aspects of the molecular mechanisms underlying nitrogen conservation and recycling in these organisms that allow them to thrive in the nitrogen-poor ocean environments.
MeSH term(s)	Animals ; Sea Anemones/genetics ; Coral Reefs ; Ecosystem ; Anthozoa/genetics ; Symbiosis ; Dinoflagellida/genetics ; Nitrogen
Chemical Substances	Nitrogen (N762921K75)
Language	English
Publishing date	2023-03-15
Publishing country	United States
Document type	Journal Article
ZDB-ID	2810933-8
ISSN	2375-2548 ; 2375-2548
ISSN (online)	2375-2548
ISSN	2375-2548
DOI	10.1126/sciadv.adf7108
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Article ; Online: Proteome profiling of nasopharynx reveals pathophysiological signature of COVID-19 disease severity

Ooi, Amanda / Esau, Luke E. / Pugachev, Artyom / Groen, Arnoud / Mfarrej, Sara / Salunke, Rahul P. / Subudhi, Amit K. / Ben-Rached, Fathia / Alofi, Fadwa / Alsomali, Afrah / Alquthami, Khaled / Khogeer, Asim / Hashem, Anwar M. / Almontashiri, Naif / Magistretti, Pierre J. / Hala, Sharif / Pain, Arnab

bioRxiv

Abstract: An aberrant innate immune system caused by the beta coronavirus SARS-CoV-2 is a characteristic manifestation of severe coronavirus disease 2019 (COVID-19). Here, we performed proteome profiling of nasopharyngeal (NP) swabs from 273 hospitalized patients ... ...

Abstract	An aberrant innate immune system caused by the beta coronavirus SARS-CoV-2 is a characteristic manifestation of severe coronavirus disease 2019 (COVID-19). Here, we performed proteome profiling of nasopharyngeal (NP) swabs from 273 hospitalized patients with mild and severe COVID-19 symptoms, including non-survivors. We identified depletion in STAT1-mediated type I interferon response, retinol metabolism and NRF2 antioxidant system that are associated with disease severity in our patient demography. We found that the dysregulation of glucocorticoid signaling and renin-angiotensin-aldosterone system (RAAS) contribute to the pathophysiology of COVID-19 fatality. Hyperactivation of host innate immune system was observed in severe patients, marked by elevated proteins involved in neutrophil degranulation and platelet aggregation. Our study using high-throughput proteomics on the nasopharynx of COVID-19 patients provides additional evidence on the SARS-CoV-2-induced pathophysiological signatures of disease severity and fatality.
Keywords	covid19
Language	English
Publishing date	2023-07-10
Publisher	Cold Spring Harbor Laboratory
Document type	Article ; Online
DOI	10.1101/2023.07.09.548285
Database	COVID19

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Article ; Online: Proteome profiling of nasopharynx reveals pathophysiological signature of COVID-19 disease severity

Ooi, Amanda / Esau, Luke Emmanuel / Pugachev, Artyom / Groen, Arnoud / Sara Mfarrej, Rached / Salunke, Rahul P. / Subudhi, Amit Kumar / Ben-Rached, Fathia / Alofi, Fadwa / Alsomali, Afrah / Khogeer, Asim / Hashem, Anwar M / Almontashiri, Naif / Magistretti, Pierre J / Hala, Sharif / Pain, Arnab

bioRxiv

Abstract	An aberrant innate immune system caused by the beta coronavirus SARS-CoV-2 is a characteristic manifestation of severe coronavirus disease 2019 (COVID-19). Here, we performed proteome profiling of nasopharyngeal (NP) swabs from 273 hospitalized patients with mild and severe COVID-19 symptoms, including non-survivors. We identified depletion in STAT1-mediated type I interferon response, retinol metabolism and NRF2 antioxidant system that are associated with disease severity in our patient demography. We found that the dysregulation of glucocorticoid signaling and renin-angiotensin-aldosterone system (RAAS) contribute to the pathophysiology of COVID-19 fatality. Hyperactivation of host innate immune system was observed in severe patients, marked by elevated proteins involved in neutrophil degranulation and platelet aggregation. Our study using high-throughput proteomics on the nasopharynx of COVID-19 patients provides additional evidence on the SARS-CoV-2-induced pathophysiological signatures of disease severity and fatality.
Keywords	covid19
Language	English
Publishing date	2023-07-10
Publisher	Cold Spring Harbor Laboratory
Document type	Article ; Online
DOI	10.1101/2023.07.09.548285
Database	COVID19

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Article: A Guide to Transient Expression of Membrane Proteins in HEK-293 Cells for Functional Characterization.

Ooi, Amanda / Wong, Aloysius / Esau, Luke / Lemtiri-Chlieh, Fouad / Gehring, Chris

Frontiers in physiology

2016 Volume 7, Page(s) 300

Abstract: The human embryonic kidney 293 (HEK-293) cells are commonly used as host for the heterologous expression of membrane proteins not least because they have a high transfection efficiency and faithfully translate and process proteins. In addition, their ... ...

Abstract	The human embryonic kidney 293 (HEK-293) cells are commonly used as host for the heterologous expression of membrane proteins not least because they have a high transfection efficiency and faithfully translate and process proteins. In addition, their cell size, morphology and division rate, and low expression of native channels are traits that are particularly attractive for current-voltage measurements. Nevertheless, the heterologous expression of complex membrane proteins such as receptors and ion channels for biological characterization and in particular for single-cell applications such as electrophysiology remains a challenge. Expression of functional proteins depends largely on careful step-by-step optimization that includes the design of expression vectors with suitable identification tags, as well as the selection of transfection methods and detection parameters appropriate for the application. Here, we use the heterologous expression of a plant potassium channel, the Arabidopsis thaliana guard cell outward-rectifying K(+) channel, AtGORK (At5G37500) in HEK-293 cells as an example, to evaluate commonly used transfection reagents and fluorescent detection methods, and provide a detailed methodology for optimized transient transfection and expression of membrane proteins for in vivo studies in general and for single-cell applications in particular. This optimized protocol will facilitate the physiological and cellular characterization of complex membrane proteins.
Language	English
Publishing date	2016-07-19
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2564217-0
ISSN	1664-042X
ISSN	1664-042X
DOI	10.3389/fphys.2016.00300
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