Optimization of Methods for the Accurate Characterization of Whole Blood Neutrophils

Abstract Neutrophils are the most abundant circulating leukocyte population with critical roles in immune defense, regulation of innate and adaptive immune systems, and disease pathogenesis. Our progress in understanding precise mechanisms of neutrophil activation, recruitment, and function has been hampered by the lack of optimized and standardized methods for the characterization and phenotyping of this readily activated population. By comparing eight methods of neutrophil characterization, we demonstrate that the level of neutrophil activation and degranulation is associated with specific experimental conditions and the number and type of manipulation steps employed. Staining whole blood at 4ºC and removal of remaining unbound antibodies prior to one-step fixation and red blood cell lysis minimizes neutrophil activation, decreases phenotypic alterations during processing, and prevents nonspecific antibody binding. The effects of anticoagulants used for collection, processing delays, and time and temperature during sample analysis on neutrophil phenotype are addressed. The presented data provide a foundation for higher quality standards of neutrophil characterization improving consistency and reproducibility among studies.

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Characterization of Cas12a nucleases reveals diverse PAM profiles between closely-related orthologs

Nucleic Acids Research ◽

10.1093/nar/gkaa272 ◽

2020 ◽

Vol 48 (10) ◽

pp. 5624-5638

Author(s):

Thomas Jacobsen ◽

Fani Ttofali ◽

Chunyu Liao ◽

Srinivas Manchalu ◽

Benjamin N Gray ◽

...

Keyword(s):

Selective Pressure ◽

Amino Acid Identity ◽

Human Cells ◽

High Sequence Identity ◽

Guide Rnas ◽

Adaptive Immune ◽

Translation Systems ◽

Adaptive Immune Systems ◽

Single Effector

Abstract CRISPR-Cas systems comprise diverse adaptive immune systems in prokaryotes whose RNA-directed nucleases have been co-opted for various technologies. Recent efforts have focused on expanding the number of known CRISPR-Cas subtypes to identify nucleases with novel properties. However, the functional diversity of nucleases within each subtype remains poorly explored. Here, we used cell-free transcription-translation systems and human cells to characterize six Cas12a single-effector nucleases from the V-A subtype, including nucleases sharing high sequence identity. While these nucleases readily utilized each other's guide RNAs, they exhibited distinct PAM profiles and apparent targeting activities that did not track based on phylogeny. In particular, two Cas12a nucleases encoded by Prevotella ihumii (PiCas12a) and Prevotella disiens (PdCas12a) shared over 95% amino-acid identity yet recognized distinct PAM profiles, with PiCas12a but not PdCas12a accommodating multiple G’s in PAM positions -2 through -4 and T in position -1. Mutational analyses transitioning PiCas12a to PdCas12a resulted in PAM profiles distinct from either nuclease, allowing more flexible editing in human cells. Cas12a nucleases therefore can exhibit widely varying properties between otherwise related orthologs, suggesting selective pressure to diversify PAM recognition and supporting expansion of the CRISPR toolbox through ortholog mining and PAM engineering.

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Cas13b is a Type VI-B CRISPR-associated RNA-Guided RNase differentially regulated by accessory proteins Csx27 and Csx28

10.1101/092577 ◽

2016 ◽

Cited By ~ 1

Author(s):

Aaron Smargon ◽

David B.T. Cox ◽

Neena Pyzocha ◽

Kaijie Zheng ◽

Ian M. Slaymaker ◽

...

Keyword(s):

Rna Interference ◽

Essential Gene ◽

Sequence Database ◽

Flanking Sequence ◽

E Coli ◽

Adaptive Immune ◽

Associated Proteins ◽

Adaptive Immune Systems ◽

Target Rna

CRISPR-Cas adaptive immune systems defend microbes against foreign nucleic acids via RNA-guided endonucleases. Using a computational sequence database mining approach, we identify two Class 2 CRISPR-Cas systems (subtype VI-B) that lack Cas1 and Cas2 and encompass a single large effector protein, Cas13b, along with one of two previously uncharacterized associated proteins, Csx27 or Csx28. We establish that these CRISPR-Cas systems can achieve RNA interference when heterologously expressed. Through a combination of biochemical and genetic experiments, we show that Cas13b processes its own CRISPR array with short and long direct repeats, cleaves target RNA, and exhibits collateral RNase activity. Using an E. coli essential gene screen, we demonstrate that Cas13b has a double-sided protospacer-flanking sequence and elucidate RNA secondary structure requirements for targeting. We also find that Csx27 represses, whereas Csx28 enhances, Cas13b-mediated RNA interference. Characterization of these CRISPR systems creates opportunities to develop tools to manipulate and monitor cellular transcripts.

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Pathogenesis of polymyalgia rheumatica

Reumatismo ◽

10.4081/reumatismo.2018.1048 ◽

2018 ◽

Vol 70 (1) ◽

pp. 10 ◽

Cited By ~ 5

Author(s):

G. Guggino ◽

A. Ferrante ◽

F. Macaluso ◽

G. Triolo ◽

F. Ciccia

Keyword(s):

Polymyalgia Rheumatica ◽

Treg Cells ◽

Inflammatory Disorder ◽

Infectious Agents ◽

Chronic Inflammatory Disorder ◽

Adaptive Immune ◽

Age Related ◽

Immune Alterations ◽

And Function ◽

Adaptive Immune Systems

Polymyalgia rheumatica (PMR) is a chronic, inflammatory disorder of unknown cause, almost exclusively occurring in people aged over 50 and often associated with giant cell arteritis. The evidence that PMR occurs almost exclusively in individuals aged over 50 may indicate that age-related immune alterations in genetically predisposed subjects contribute to development of the disease. Several infectious agents have been investigated as possible triggers of PMR even though the results are inconclusive. Activation of the innate and adaptive immune systems has been proved in PMR patients as demonstrated by the activation of dendritic cells and monocytes/macrophages and the altered balance between Th17 and Treg cells. Disturbed B cell distribution and function have been also demonstrated in PMR patients suggesting a pathogenesis more complex than previously imagined. In this review we will discuss the recent findings regarding the pathogenesis of PMR.

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Control of Neutrophil activation through Semaphorin 7A-Plexin C1 is essential for immune defense during pulmonary sepsis.

10.1101/2021.11.08.467692 ◽

2021 ◽

Author(s):

Peter Rosenberger ◽

Tiago Granja

Keyword(s):

Early Phase ◽

Defense Mechanisms ◽

Neutrophil Migration ◽

Serum Levels ◽

Neutrophil Activation ◽

Immune Defense ◽

And Function ◽

Neutrophil Surface

Pulmonary defense mechanisms are critical for host integrity during the early phase of pneumonia and sepsis. These processes are fundamentally dependent on the activation of neutrophils during the early phase of the innate immune response. Recent work has shown that semaphorin 7A (Sema7A) holds significant impact on platelet activation, yet its role in neutrophil migration and function is not well known. We report here that Sema7A binds to neutrophil PlexinC1, increasing integrins and L-selectin on the neutrophil surface. Sema7A-induced neutrophil activation also prompted neutrophil chemotaxis in vitro and the formation of platelet-neutrophil complexes in vivo. We also observed altered adhesion and transmigration of neutrophils in Sema7A-/- animals in the lung. Sema7A-/- animals also showed altered crawling properties of neutrophils. This resulted in increased number of neutrophils in the interstitial space of Sema7A-/- animals but reduced numbers of neutrophils in the alveolar space during pneumonia-induced pulmonary sepsis. This was associated with significantly worse outcome of Sema7A-/- animals in a model of Klebsiella pneumoniae. Furthermore, we were able to show a correlation between serum levels of Sema7A in patients with ARDS and oxygenation levels. Thus, we show here that Sema7A has an immunomodulatory effect though which might influence patient outcome during pulmonary sepsis.

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FC-98 Regulates TLR9-Mediated of CXCL-10 Expression in Dendritic Cells via MAPK and STAT1 Signaling Pathway

BioMed Research International ◽

10.1155/2014/926130 ◽

2014 ◽

Vol 2014 ◽

pp. 1-10 ◽

Cited By ~ 3

Author(s):

Yonghong Yang ◽

Huan Dou ◽

Xiaoqin Li ◽

Yuxian Song ◽

Wei Gong ◽

...

Keyword(s):

Dendritic Cells ◽

Surface Markers ◽

Toll Like Receptor ◽

Cpg Dna ◽

Bacterial Dna ◽

Adaptive Immune ◽

Stat1 Signaling ◽

And Function ◽

Antigen Presenting ◽

Adaptive Immune Systems

Dendritic cells (DCs), as the most potent professional antigen presenting cells, play a crucial role in both innate and adaptive immune systems. Genomic bacterial DNA mimicked by unmethylated CpG motifs is discovered to possess immunostimulatory effects. CpG-DNA recognized by Toll-like receptor 9 (TLR9) on DCs arouses many immune diseases (such as cancer, viral infection, and autoimmune disorders). In this study we investigated the effects of FC-98 on CpG-induced bone marrow-derived DCs (BMDCs). The results showed that FC-98 significantly inhibited the CpG-induced BMDCs maturation and function by suppressing the expression of surface markers (CD40, CD80, CD86, and MHCII). Moreover, FC-98 downregulated the expression of C-X-C motif chemokine 10 (CXCL-10) both at the mRNA and protein level after CpG induction. Meanwhile, FC-98 markedly affected the migration of BMDCs to T cells without affecting their endocytosis capacity. Furthermore, FC-98 was confirmed to decrease CXCL-10 expression by inhibiting CpG-induced activation of MAPKs (ERK, JNK, and p38) and STAT1 signaling. Overall, these results suggested that FC-98 was a potential molecule in the treatment of CXCL-10-mediated immune diseases.

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Lack of Cas13a inhibition by anti-CRISPR proteins from Leptotrichia prophages

10.1101/2021.05.27.445852 ◽

2021 ◽

Author(s):

Alexander J Meeske ◽

Matthew C Johnson ◽

Logan T Hille ◽

Benjamin P Kleinstiver ◽

Joseph Bondy-Denomy

Keyword(s):

Nucleic Acid ◽

Rna Cleavage ◽

Nucleic Acid Binding ◽

Bacterial Strains ◽

Bioinformatics Analyses ◽

Immune Systems ◽

Adaptive Immune ◽

Bona Fide ◽

Adaptive Immune Systems

CRISPR systems are prokaryotic adaptive immune systems that use RNA-guided Cas nucleases to recognize and destroy foreign genetic elements, like bacteriophages and plasmids. To overcome CRISPR immunity, phages have evolved diverse families of anti-CRISPR proteins (Acrs), each of which inhibits the nucleic acid binding or cleavage activities of specific Cas protein families. Recently, Lin et al. (2020) described the discovery and characterization of 7 different Acr families (AcrVIA1-7) that inhibit type VI-A CRISPR systems, which use the nuclease Cas13a to perform RNA-guided RNA cleavage. In this Matters Arising article, we detail several inconsistencies that question the results reported in the Lin et al. (2020) study. These include inaccurate bioinformatics analyses, as well as reported experiments involving bacterial strains that are impossible to construct. The authors were unable to provide their published strains with which we might reproduce their experiments. We independently tested the Acr sequences described in Lin et al. (2020) in two different Cas13 inhibition assays, but could not detect anti-CRISPR activity. Taken together, our data and analyses prompt us to question the claim that AcrVIA1-7 reported in Lin et al. are bona fide type VI anti-CRISPR proteins.

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Dendritic Cells as Arbiters of Peritoneal Immune Responses

Peritoneal Dialysis International ◽

10.1177/089686080602600102 ◽

2006 ◽

Vol 26 (1) ◽

pp. 8-25 ◽

Cited By ~ 6

Author(s):

Michelle L. McCully ◽

Joaquín Madrenas

Keyword(s):

Immune Response ◽

Dendritic Cells ◽

Immune Responses ◽

Adaptive Immune Response ◽

Immune Defense ◽

Dendritic Cell Differentiation ◽

Adaptive Immune ◽

The Past ◽

Key Players ◽

And Function

During the past few years, there has been a substantial increase in the understanding of innate immunity. Dendritic cells are emerging as key players in the orchestration of this early phase of immune responses, with a role that will translate into the subsequent type of adaptive immune response against infection. Here we provide an overview of dendritic cell differentiation and function, with particular emphasis on those features unique to the immune defense of the peritoneal cavity and in the context of peritoneal dialysis-associated immune responses. The reader is referred to the primary references included in the accompanying list for specific details in this fascinating field.

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Serpins: Purification and characterization of potent protease inhibitors from Clostridium thermocellum

10.1101/2020.04.21.053413 ◽

2020 ◽

Author(s):

Maria Mushtaq ◽

Muhammad Javaid Asad ◽

Muhammad Zeeshan Hyder ◽

Syed Muhammad Saqlan Naqvi ◽

Saad Imran Malik ◽

...

Keyword(s):

Protease Inhibitors ◽

Clostridium Thermocellum ◽

Multiprotein Complex ◽

Purification And Characterization ◽

Ligation Independent Cloning ◽

One Step ◽

The One ◽

And Function ◽

Extracellular Location

AbstractClostridium thermocellum produces an extracellular cellulosome (a multiprotein complex produced by firmicutes bacteria), which, owing to its extracellular location, is open to protease attack. Serine protease inhibitors (serpins) protect bacteria against protease attack. However, their structure and function are poorly characterized. This study identified and amplified the serpin 1270 gene from the C. thermocellum genome. Purified serpins were cloned into the pTXB1 vector using the one-step sequence and ligation-independent cloning reaction and transformed into Escherichia coli BL21 DE3 cells. Enzyme overexpression and purification and enzyme inhibitory assays were performed. The results showed that serpin 1270 has 89% inhibition against Bacillus subtilisin and 64% inhibition against trypsin, chymotrypsin, and papain.

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Functional characterization of human brown adipose tissue metabolism

Biochemical Journal ◽

10.1042/bcj20190464 ◽

2020 ◽

Vol 477 (7) ◽

pp. 1261-1286 ◽

Cited By ~ 3

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.

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Parasite defense mechanisms in bees: behavior, immunity, antimicrobials, and symbionts

Emerging Topics in Life Sciences ◽

10.1042/etls20190069 ◽

2019 ◽

Vol 4 (1) ◽

pp. 59-76 ◽

Cited By ~ 2

Author(s):

Alison E. Fowler ◽

Rebecca E. Irwin ◽

Lynn S. Adler

Keyword(s):

Defense Mechanisms ◽

Poor Quality ◽

Future Research ◽

Immune Priming ◽

Social Bees ◽

Bee Health ◽

Landscape Scales ◽

And Function ◽

Solitary Species

Parasites are linked to the decline of some bee populations; thus, understanding defense mechanisms has important implications for bee health. Recent advances have improved our understanding of factors mediating bee health ranging from molecular to landscape scales, but often as disparate literatures. Here, we bring together these fields and summarize our current understanding of bee defense mechanisms including immunity, immunization, and transgenerational immune priming in social and solitary species. Additionally, the characterization of microbial diversity and function in some bee taxa has shed light on the importance of microbes for bee health, but we lack information that links microbial communities to parasite infection in most bee species. Studies are beginning to identify how bee defense mechanisms are affected by stressors such as poor-quality diets and pesticides, but further research on this topic is needed. We discuss how integrating research on host traits, microbial partners, and nutrition, as well as improving our knowledge base on wild and semi-social bees, will help inform future research, conservation efforts, and management.

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