Characterization of murine amniotic fluid B cells in normal pregnancy and in preterm birth

The amniotic fluid provides mechanical protection and immune defense against pathogens to the fetus. Indeed, components of the innate and adaptive immunity, including B cells, have been described in the amniotic fluid. However, limited information concerning phenotype and functionality of amniotic fluid B cells is available. Hence, we aimed to perform a full phenotypical and functional characterization of amniotic fluid B cells in normal pregnancy and in a mouse model of preterm birth. Phenotypic analysis depicted the presence of two populations of amniotic fluid B cells: an immature population, resembling B1 progenitor cells and a more mature population. Further isolation and in vitro co-culture with a bone marrow stroma cell line demonstrated the capacity of the immature B cells to mature. This was further supported by spontaneous production of IgM, a feature of the B1 B cell sub-population. An additional in vitro stimulation with lipopolysaccharide induced the activation of amniotic fluid B cells as well as the production of pro and anti-inflammatory cytokines. Furthermore, amniotic fluid B cells were expanded in the acute phase of LPS-induced preterm birth. Overall our data add new insight not only on the phenotype and developmental stage of the amniotic fluid B1 B cells but especially on their functionality. This provides important information for a better understanding of their role within the amniotic fluid as immunological protective barrier, especially with regard to intraamniotic infection and preterm birth.

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Functional identification of ygiP as a positive regulator of the ttdA-ttdB-ygjE operon

Microbiology ◽

10.1099/mic.0.28753-0 ◽

2006 ◽

Vol 152 (7) ◽

pp. 2129-2135 ◽

Cited By ~ 17

Author(s):

Taku Oshima ◽

Francis Biville

Keyword(s):

Functional Characterization ◽

Anaerobic Growth ◽

Functional Identification ◽

New Members ◽

E Coli ◽

Inner Membrane Protein ◽

Tartrate Dehydratase

Functional characterization of unknown genes is currently a major task in biology. The search for gene function involves a combination of various in silico, in vitro and in vivo approaches. Available knowledge from the study of more than 21 LysR-type regulators in Escherichia coli has facilitated the classification of new members of the family. From sequence similarities and its location on the E. coli chromosome, it is suggested that ygiP encodes a lysR regulator controlling the expression of a neighbouring operon; this operon encodes the two subunits of tartrate dehydratase (TtdA, TtdB) and YgiE, an integral inner-membrane protein possibly involved in tartrate uptake. Expression of tartrate dehydratase, which converts tartrate to oxaloacetate, is required for anaerobic growth on glycerol as carbon source in the presence of tartrate. Here, it has been demonstrated that disruption of ygiP, ttdA or ygjE abolishes tartrate-dependent anaerobic growth on glycerol. It has also been shown that tartrate-dependent induction of the ttdA-ttdB-ygjE operon requires a functional YgiP.

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Functional Characterization of the mazEF Toxin-Antitoxin System in the Pathogenic Bacterium Agrobacterium tumefaciens

Microorganisms ◽

10.3390/microorganisms9051107 ◽

2021 ◽

Vol 9 (5) ◽

pp. 1107

Author(s):

Wonho Choi ◽

Yoshihiro Yamaguchi ◽

Ji-Young Park ◽

Sang-Hyun Park ◽

Hyeok-Won Lee ◽

...

Keyword(s):

Agrobacterium Tumefaciens ◽

Physiological Function ◽

Functional Characterization ◽

Site Directed Mutagenesis ◽

In Vitro Assays ◽

Cell Growth Inhibition ◽

The Right

Agrobacterium tumefaciens is a pathogen of various plants which transfers its own DNA (T-DNA) to the host plants. It is used for producing genetically modified plants with this ability. To control T-DNA transfer to the right place, toxin-antitoxin (TA) systems of A. tumefaciens were used to control the target site of transfer without any unintentional targeting. Here, we describe a toxin-antitoxin system, Atu0939 (mazE-at) and Atu0940 (mazF-at), in the chromosome of Agrobacterium tumefaciens. The toxin in the TA system has 33.3% identity and 45.5% similarity with MazF in Escherichia coli. The expression of MazF-at caused cell growth inhibition, while cells with MazF-at co-expressed with MazE-at grew normally. In vivo and in vitro assays revealed that MazF-at inhibited protein synthesis by decreasing the cellular mRNA stability. Moreover, the catalytic residue of MazF-at was determined to be the 24th glutamic acid using site-directed mutagenesis. From the results, we concluded that MazF-at is a type II toxin-antitoxin system and a ribosome-independent endoribonuclease. Here, we characterized a TA system in A. tumefaciens whose understanding might help to find its physiological function and to develop further applications.

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NFB-01. FUNCTIONAL CHARACTERIZATION OF ATRX LOSS IN NF1-ASSOCIATED GLIOMA AND MPNST

Neuro-Oncology ◽

10.1093/neuonc/noaa222.605 ◽

2020 ◽

Vol 22 (Supplement_3) ◽

pp. iii417-iii418

Author(s):

Ming Yuan ◽

Karlyne Reilly ◽

Christine Pratilas ◽

Christopher Heaphy ◽

Fausto Rodriguez

Keyword(s):

Cell Lines ◽

Functional Characterization ◽

Tert Promoter ◽

Aggressive Tumors ◽

Sirna Knockdown ◽

Nih 3T3 ◽

Vitro Effect ◽

Murine Glioma

Abstract To identify the biologic relevance of ATRX loss in NF1-associated gliomagenesis, we studied the effects of Atrx loss using four previously characterized Nf1+/-Trp53+/- murine glioma lines. Lines 130G#3 and 158D#8 (corresponding to grade IV and III gliomas, respectively) displayed preserved ATRX protein expression compared to NIH-3T3 cells. We studied the effects of Atrx knockdown in these two lines in the presence and absence of the TERT inhibitor, BIRBR1532. Using a telomere-specific FISH assay, we identified increased signal intensity after Atrx knockdown, only in the presence of the TERT inhibitor. These features are reminiscent of ALT, although there were no significant alterations in cell growth. Next, we studied the effect of ATRX loss in MPNST lines ST88-14, NF90-8, STS-26T. These cell lines all expressed ATRX and DAXX. However, STS-26T contained a TERT promoter mutation and ST88-14 had a known SNP in the TERT promoter, while NF90-8 had no alterations. ATRX siRNA knockdown showed no significant effects in cell proliferation or apoptosis. However, ATRX knockdown resulted in rare ultra-bright foci, indicative of ALT. Next, we studied the in vitro effect of the ATR inhibitor VE-821 in MPNST cell lines. Only NF90-8 (lacking TERT alterations) demonstrated a decrease in growth after ATRX knockdown and VE-821 treatment. However, ATRX knockdown alone did not affect sensitivity to carboplatin. Our findings further support a role for ATRX loss with subsequent ALT activation in a biologic subset of NF1-associated malignancies, thereby opening an opportunity for therapeutic targeting of these aggressive tumors using specific classes of drugs.

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Functional Characterization of GABAA Receptor Ligands In Vitro

Molecular Neuropharmacology ◽

10.1007/978-1-59259-672-0_4 ◽

2004 ◽

pp. 85-94

Author(s):

Bjarke Ebert ◽

Sally Anne Thompson ◽

Signe Í. Stórustovu ◽

Keith A. Wafford

Keyword(s):

Gabaa Receptor ◽

Functional Characterization ◽

Receptor Ligands

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Biochemical and Functional Characterization of Anthocyanidin Reductase (ANR) from Mangifera indica L.

Molecules ◽

10.3390/molecules23112876 ◽

2018 ◽

Vol 23 (11) ◽

pp. 2876 ◽

Cited By ~ 2

Author(s):

Lin Tan ◽

Mei Wang ◽

Youfa Kang ◽

Farrukh Azeem ◽

Zhaoxi Zhou ◽

...

Keyword(s):

Enzyme Assay ◽

Molecular Mechanisms ◽

Mangifera Indica ◽

Functional Characterization ◽

Citrate Buffer ◽

Anthocyanidin Reductase ◽

High Amino Acid ◽

Optimum Ph

Mango (Mangifera indica L.) is abundant in proanthocyanidins (PAs) that are important for human health and plant response to abiotic stresses. However, the molecular mechanisms involved in PA biosynthesis still need to be elucidated. Anthocyanidin reductase (ANR) catalyzes a key step in PA biosynthesis. In this study, three ANR cDNAs (MiANR1-1,1-2,1-3) were isolated from mango, and expressed in Escherichia coli. In vitro enzyme assay showed MiANR proteins convert cyanidin to their corresponding flavan-3-ols, such as (−)-catechin and (−)-epicatechin. Despite high amino acid similarity, the recombinant ANR proteins exhibited differences in enzyme kinetics and cosubstrate preference. MiANR1-2 and MiANR1-3 have the same optimum pH of 4.0 in citrate buffer, while the optimum pH for MiANR1-1 is pH 3.0 in phosphate buffer. MiANR1-1 does not use either NADPH or NADH as co-substrate while MiANR1-2/1-3 use only NADPH as co-substrate. MiANR1-2 has the highest Km and Vmax for cyanidin, followed by MiANR1-3 and MiANR1-1. The overexpression of MiANRs in ban mutant reconstructed the biosynthetic pathway of PAs in the seed coat. These data demonstrate MiANRs can form the ANR pathway, leading to the formation of two types of isomeric flavan-3-ols and PAs in mango.

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N-Acetylglucosamine-1-Phosphate Transferase, WecA, as a Validated Drug Target in Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01310-17 ◽

2017 ◽

Vol 61 (11) ◽

Cited By ~ 6

Author(s):

Stanislav Huszár ◽

Vinayak Singh ◽

Alica Polčicová ◽

Peter Baráth ◽

María Belén Barrio ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Drug Target ◽

Functional Characterization ◽

Drug Candidate ◽

Content Type ◽

Chemical Libraries ◽

Whole Cells ◽

Encoding Gene

ABSTRACT The mycobacterial phosphoglycosyltransferase WecA, which initiates arabinogalactan biosynthesis in Mycobacterium tuberculosis, has been proposed as a target of the caprazamycin derivative CPZEN-45, a preclinical drug candidate for the treatment of tuberculosis. In this report, we describe the functional characterization of mycobacterial WecA and confirm the essentiality of its encoding gene in M. tuberculosis by demonstrating that the transcriptional silencing of wecA is bactericidal in vitro and in macrophages. Silencing wecA also conferred hypersensitivity of M. tuberculosis to the drug tunicamycin, confirming its target selectivity for WecA in whole cells. Simple radiometric assays performed with mycobacterial membranes and commercially available substrates allowed chemical validation of other putative WecA inhibitors and resolved their selectivity toward WecA versus another attractive cell wall target, translocase I, which catalyzes the first membrane step in the biosynthesis of peptidoglycan. These assays and the mutant strain described herein will be useful for identifying potential antitubercular leads by screening chemical libraries for novel WecA inhibitors.

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Phenotypic and functional characterization of T-cells and in vitro replication of FMDV serotypes in bovine lymphocytes

Vaccine ◽

10.1016/j.vaccine.2009.08.107 ◽

2009 ◽

Vol 27 (48) ◽

pp. 6656-6661 ◽

Cited By ~ 12

Author(s):

Gurudutt Joshi ◽

Ravindra Sharma ◽

Naresh Kumar Kakker

Keyword(s):

T Cells ◽

Functional Characterization ◽

In Vitro Replication ◽

Fmdv Serotypes ◽

Bovine Lymphocytes

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Synthetic repertoires derived from convalescent COVID-19 patients enable discovery of SARS-CoV-2 neutralizing antibodies and a novel quaternary binding modality

10.1101/2021.04.07.438849 ◽

2021 ◽

Author(s):

Jimmy D Gollihar ◽

Jason S McLellan ◽

Daniel R Boutz ◽

Jule Goike ◽

Andrew Horton ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Surface Display ◽

Functional Characterization ◽

Yeast Surface Display ◽

Spike Protein ◽

Emerging Pathogens ◽

Effective Interventions ◽

Yeast Surface

The ongoing evolution of SARS-CoV-2 into more easily transmissible and infectious variants has sparked concern over the continued effectiveness of existing therapeutic antibodies and vaccines. Hence, together with increased genomic surveillance, methods to rapidly develop and assess effective interventions are critically needed. Here we report the discovery of SARS-CoV-2 neutralizing antibodies isolated from COVID-19 patients using a high-throughput platform. Antibodies were identified from unpaired donor B-cell and serum repertoires using yeast surface display, proteomics, and public light chain screening. Cryo-EM and functional characterization of the antibodies identified N3-1, an antibody that binds avidly (Kd,app = 68 pM) to the receptor binding domain (RBD) of the spike protein and robustly neutralizes the virus in vitro. This antibody likely binds all three RBDs of the trimeric spike protein with a single IgG. Importantly, N3-1 equivalently binds spike proteins from emerging SARS-CoV-2 variants of concern, neutralizes UK variant B.1.1.7, and binds SARS-CoV spike with nanomolar affinity. Taken together, the strategies described herein will prove broadly applicable in interrogating adaptive immunity and developing rapid response biological countermeasures to emerging pathogens.

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Omics profiling identifies MAPK/ERK pathway as a gatekeeper of nephron progenitor metabolism

10.1101/2021.09.27.461969 ◽

2021 ◽

Author(s):

Hyuk Nam Kwon ◽

Kristen Kurtzeborn ◽

Xing Jin ◽

Bruno Reversade ◽

Sunghyouk Park ◽

...

Keyword(s):

Functional Characterization ◽

Mitogen Activated Protein Kinase ◽

Metabolic Effects ◽

Erk Pathway ◽

Double Knockout ◽

Nephron Progenitor ◽

Progenitor Maintenance

Nephron endowment is defined by fetal kidney growth and it critically dictates renal health in adults. Despite the advances in understanding the molecular regulation of nephron progenitor maintenance, propagation, and differentiation, the causes for low congenital nephron count and contribution of basic metabolism to nephron progenitor regulation remain poorly studied. Here we have analyzed the metabolic effects that depend on and are triggered by the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway, which is an essential intracellular cascade required for nephron progenitor maintenance. Our combined approach utilizing LC/MS-based metabolomics and transcriptional profiling of MAPK/ERK-deficient cells identified 18 out of total 46 metabolites (38 untargeted and 8 targeted) that were down-regulated. These represent glycolysis, gluconeogenesis, pentose phosphate, glycine, and proline pathways among others. We focused our functional characterization of identified metabolites on pyruvate and proline. Use of in vitro kidney cultures revealed dosage-specific functions for pyruvate in not only controlling ureteric bud branching but also determining progenitor and differentiated (tip-trunk) cell identities. Our in vivo characterization of Pycr1/2 double knockout kidneys revealed functional requirement for proline metabolism in nephron progenitor maintenance. In summary, our results demonstrate that MAPK/ERK cascade regulates energy and amino acid metabolism in developing kidney where these metabolic pathways specifically regulate progenitor preservation.

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