A mouse model of human TLR4 D299G/T399I SNPs reveals mechanisms of altered LPS and pathogen responses

Two cosegregating single-nucleotide polymorphisms (SNPs) in human TLR4, an A896G transition at SNP rs4986790 (D299G) and a C1196T transition at SNP rs4986791 (T399I), have been associated with LPS hyporesponsiveness and differential susceptibility to many infectious or inflammatory diseases. However, many studies failed to confirm these associations, and transfection experiments resulted in conflicting conclusions about the impact of these SNPs on TLR4 signaling. Using advanced protein modeling from crystallographic data of human and murine TLR4, we identified homologous substitutions of these SNPs in murine Tlr4, engineered a knock-in strain expressing the D298G and N397I TLR4 SNPs homozygously, and characterized in vivo and in vitro responses to TLR4 ligands and infections in which TLR4 is implicated. Our data provide new insights into cellular and molecular mechanisms by which these SNPs decrease the TLR4 signaling efficiency and offer an experimental approach to confirm or refute human data possibly confounded by variables unrelated to the direct effects of the SNPs on TLR4 functionality.

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The impact of FGF19/FGFR4 signaling inhibition in antitumor activity of multi-kinase inhibitors in hepatocellular carcinoma

Scientific Reports ◽

10.1038/s41598-021-84117-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Hiroaki Kanzaki ◽

Tetsuhiro Chiba ◽

Junjie Ao ◽

Keisuke Koroki ◽

Kengo Kanayama ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Molecular Mechanisms ◽

Kinase Inhibitors ◽

Progression Free Survival ◽

Erk Signaling ◽

Enzyme Linked Immunosorbent Assay ◽

Antitumor Effects ◽

The Impact

AbstractFGF19/FGFR4 autocrine signaling is one of the main targets for multi-kinase inhibitors (MKIs). However, the molecular mechanisms underlying FGF19/FGFR4 signaling in the antitumor effects to MKIs in hepatocellular carcinoma (HCC) remain unclear. In this study, the impact of FGFR4/ERK signaling inhibition on HCC following MKI treatment was analyzed in vitro and in vivo assays. Serum FGF19 in HCC patients treated using MKIs, such as sorafenib (n = 173) and lenvatinib (n = 40), was measured by enzyme-linked immunosorbent assay. Lenvatinib strongly inhibited the phosphorylation of FRS2 and ERK, the downstream signaling molecules of FGFR4, compared with sorafenib and regorafenib. Additional use of a selective FGFR4 inhibitor with sorafenib further suppressed FGFR4/ERK signaling and synergistically inhibited HCC cell growth in culture and xenograft subcutaneous tumors. Although serum FGF19high (n = 68) patients treated using sorafenib exhibited a significantly shorter progression-free survival and overall survival than FGF19low (n = 105) patients, there were no significant differences between FGF19high (n = 21) and FGF19low (n = 19) patients treated using lenvatinib. In conclusion, robust inhibition of FGF19/FGFR4 is of importance for the exertion of antitumor effects of MKIs. Serum FGF19 levels may function as a predictive marker for drug response and survival in HCC patients treated using sorafenib.

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Multicenter Study on Differential Human Neutrophil Antigen 2 Expression and Underlying Molecular Mechanisms

Transfusion Medicine and Hemotherapy ◽

10.1159/000505523 ◽

2020 ◽

Vol 47 (5) ◽

pp. 385-395

Author(s):

Brigitte K. Flesch ◽

Angelika Reil ◽

Núria Nogués ◽

Carme Canals ◽

Peter Bugert ◽

...

Keyword(s):

Expression Pattern ◽

Human Neutrophil ◽

Molecular Mechanisms ◽

Normal Population ◽

Regulatory Elements ◽

Population Exposure ◽

Nucleotide Polymorphisms ◽

Single Nucleotide ◽

Immune Neutropenia ◽

The Impact

Background: The human neutrophil antigen 2 (HNA-2), which is expressed on CD177, is undetectable in 3–5% of the normal population. Exposure of these HNA-2null individuals to HNA-2-positive cells can cause immunization and production of HNA-2 antibodies, which can induce immune neutropenia and transfusion-related acute lung injury. In HNA-2-positive individuals, neutrophils are divided into a CD177pos. and a CD177neg. subpopulation. The molecular background of HNA-2 deficiency and the bimodal expression pattern, however, are not completely decoded. Study Design: An international collaboration was conducted on the genetic analysis of HNA-2-phenotyped blood samples, including HNA-2-deficient individuals, mothers, and the respective children with neonatal immune neutropenia and regular blood donors. Results: From a total of 54 HNA-2null individuals, 43 were homozygous for the CD177*787A>T substitution. Six carried the CD177*c.1291G>A single nucleotide polymorphism. All HNA-2-positive samples with >40% CD177pos. neutrophils carried the *787A wild-type allele, whereas a lower rate of CD177pos. neutrophils was preferentially associated with *c.787AT heterozygosity. Interestingly, only the *c.787A allele sequence was detected in complementary DNA (cDNA) sequence analysis carried out on all *c.787AT heterozygous individuals. However, cDNA analysis after sorting of CD177pos. and CD177neg. neutrophil subsets from HNA-2-positive individuals showed identical sequences, which makes regulatory elements within the promoter unlikely to affect CD177 gene transcription in different CD177 neutrophil subsets. Conclusion: This comprehensive study clearly demonstrates the impact of single nucleotide polymorphisms on the expression of HNA-2 on the neutrophil surface but challenges the hypothesis of regulatory epigenetic effects being implicated in the bimodal CD177 expression pattern.

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Current translational potential and underlying molecular mechanisms of necroptosis

Cell Death and Disease ◽

10.1038/s41419-019-2094-z ◽

2019 ◽

Vol 10 (11) ◽

Cited By ~ 14

Author(s):

Tamás Molnár ◽

Anett Mázló ◽

Vera Tslaf ◽

Attila Gábor Szöllősi ◽

Gabriella Emri ◽

...

Keyword(s):

Cell Death ◽

Protein Kinase ◽

Posttranslational Modifications ◽

Molecular Mechanisms ◽

Inflammatory Diseases ◽

Immune Surveillance ◽

Kinase Domain ◽

Tumor Formation

Abstract Cell death has a fundamental impact on the evolution of degenerative disorders, autoimmune processes, inflammatory diseases, tumor formation and immune surveillance. Over the past couple of decades extensive studies have uncovered novel cell death pathways, which are independent of apoptosis. Among these is necroptosis, a tightly regulated, inflammatory form of cell death. Necroptosis contribute to the pathogenesis of many diseases and in this review, we will focus exclusively on necroptosis in humans. Necroptosis is considered a backup mechanism of apoptosis, but the in vivo appearance of necroptosis indicates that both caspase-mediated and caspase-independent mechanisms control necroptosis. Necroptosis is regulated on multiple levels, from the transcription, to the stability and posttranslational modifications of the necrosome components, to the availability of molecular interaction partners and the localization of receptor-interacting serine/threonine-protein kinase 1 (RIPK1), receptor-interacting serine/threonine-protein kinase 3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL). Accordingly, we classified the role of more than seventy molecules in necroptotic signaling based on consistent in vitro or in vivo evidence to understand the molecular background of necroptosis and to find opportunities where regulating the intensity and the modality of cell death could be exploited in clinical interventions. Necroptosis specific inhibitors are under development, but >20 drugs, already used in the treatment of various diseases, have the potential to regulate necroptosis. By listing necroptosis-modulated human diseases and cataloging the currently available drug-repertoire to modify necroptosis intensity, we hope to kick-start approaches with immediate translational potential. We also indicate where necroptosis regulating capacity should be considered in the current applications of these drugs.

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Role of macrophage CD44 in the disposal of inflammatory cell corpses*

Clinical Science ◽

10.1042/cs1030441 ◽

2002 ◽

Vol 103 (5) ◽

pp. 441-449 ◽

Cited By ~ 1

Author(s):

Sharon VIVERS ◽

Ian DRANSFIELD ◽

Simon P. HART

Keyword(s):

Molecular Mechanisms ◽

Inflammatory Diseases ◽

Surrounding Tissue ◽

Human Macrophage ◽

Neutrophil Granulocytes ◽

Apoptotic Cells ◽

Tissue Destruction

Understanding the cellular and molecular mechanisms that determine whether inflammation resolves or progresses to scarring and tissue destruction should lead to the development of effective therapeutic strategies for inflammatory diseases. Apoptosis of neutrophil granulocytes is an important determinant of the resolution of inflammation, providing a mechanism for down-regulation of function and triggering clearance by macrophages without inducing a pro-inflammatory response. However, if the rate of cell death by apoptosis is such that the macrophage clearance capacity is exceeded, apoptotic cells may progress to secondary necrosis, resulting in the release of harmful cellular contents and in damage to the surrounding tissue. There are many possible ways in which the rate and capacity of the macrophage-mediated clearance of apoptotic cells may be enhanced or suppressed. Ligation of human macrophage surface CD44 by bivalent monoclonal antibodies rapidly and profoundly augments the capacity of macrophages to phagocytose apoptotic neutrophils in vitro. The molecular mechanism behind this effect and its potential significance in vivo is a current focus of research.

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Single-Nucleotide-Polymorphism-Specific PCR for Quantification and Discrimination of Chlamydia pneumoniae Genotypes by Use of a “Locked” Nucleic Acid

Applied and Environmental Microbiology ◽

10.1128/aem.72.5.3785-3787.2006 ◽

2006 ◽

Vol 72 (5) ◽

pp. 3785-3787 ◽

Cited By ~ 11

Author(s):

Jan Rupp ◽

Werner Solbach ◽

Jens Gieffers

Keyword(s):

Single Nucleotide Polymorphism ◽

Chlamydia Pneumoniae ◽

Locked Nucleic Acid ◽

Nucleotide Polymorphisms ◽

Nucleotide Polymorphism ◽

Single Nucleotide ◽

Specific Pcr ◽

Intraspecies Diversity

ABSTRACT Single-nucleotide polymorphisms (SNPs) are targets to discriminate intraspecies diversity of bacteria and to correlate a genotype with a potential pathotype. Quantification of polygenotypic populations supports this task for in vitro and in vivo applications. We present a novel assay capable of quantifying mixtures of two genotypes differing by only one SNP.

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Strain and Virulence Diversity in the Mouse Pathogen Chlamydia muridarum

Infection and Immunity ◽

10.1128/iai.00147-09 ◽

2009 ◽

Vol 77 (8) ◽

pp. 3284-3293 ◽

Cited By ~ 50

Author(s):

Kyle H. Ramsey ◽

Ira M. Sigar ◽

Justin H. Schripsema ◽

Cecele J. Denman ◽

Anne K. Bowlin ◽

...

Keyword(s):

Model Organism ◽

Nucleotide Polymorphisms ◽

Single Nucleotide ◽

Deletion Mutations ◽

Chlamydia Muridarum ◽

Adenocarcinoma Cells ◽

Tract Infections ◽

Virulence Diversity

ABSTRACT The mouse chlamydial pathogen Chlamydia muridarum has been used as a model organism for the study of human Chlamydia trachomatis urogenital and respiratory tract infections. To date, two commonly used C. muridarum isolates have been used interchangeably and are essentially taken to be identical. Herein, we present data that indicate that this is not the case. The C. muridarum Weiss isolate and C. muridarum Nigg isolate varied significantly in their virulences in vivo and possessed different growth characteristics in vitro. Distinct differences were observed in intravaginal 50% infectious doses and in challenge infections, with the Weiss isolate displaying greater virulence. Respiratory infection by the intranasal route also indicated a greater virulence of the Weiss isolate. In vitro, morphometric analysis revealed that the Weiss isolate produced consistently smaller inclusions in human cervical adenocarcinoma cells (HeLa 229) and smaller plaques in monolayers of mouse fibroblasts (L929) than did the Nigg isolate. In addition, the Weiss isolate possessed significantly higher replicative yields in vitro than did the Nigg isolate. In plaque-purified isolates derived from our stocks of these two strains, total genomic sequencing identified several unique nonsynonymous single nucleotide polymorphisms and insertion/deletion mutations when our Weiss (n = 4) and Nigg (n = 5) isolates were compared with the published Nigg sequence. In addition, the two isolates shared 11 mutations compared to the published Nigg sequence. These results prove that there is genotypic and virulence diversity among C. muridarum isolates. These findings can be exploited to determine factors related to chlamydial virulence and immunity.

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Dissociated Steroids

The Scientific World JOURNAL ◽

10.1100/tsw.2007.97 ◽

2007 ◽

Vol 7 ◽

pp. 421-430 ◽

Cited By ~ 11

Author(s):

Matthew C. Catley

Keyword(s):

Side Effects ◽

Molecular Mechanisms ◽

Inflammatory Diseases ◽

Therapeutic Index ◽

Clinical Use ◽

Response Elements ◽

Activation Of Transcription

Glucocorticoids (GCs) are some of the most important drugs in clinical use today. They are mainly used to suppress disease-related inflammation and are widely used for the treatment of many inflammatory diseases including asthma and arthritis. However, GCs are also associated with debilitating side effects that place limitations on the long-term use of these drugs. The development of a GC with reduced side effects would allow more effective treatments for patients who require long-term suppression of inflammation. GCs exert their effects by binding and activating the GC receptor (GR). The activated receptor then binds GC response elements (GREs) in the promoter of genes, and activates transcription (transactivation) or interferes with the activation of transcription by inhibiting the transactivating function of other transcription factors, such as AP-1 and NF-ĸB (transrepression). Transrepression is believed to be responsible for the majority of the beneficial anti-inflammatory effects of GCs, whereas transactivation is believed to play a bigger role in the unwanted side effects of GCs. Compounds that can dissociate the transactivation function of GCs from the transrepression function may, therefore, have an improved therapeutic index. A number of these dissociated corticosteroids have been developed.In vitroassays using these compounds appear to show good dissociation. However,in vivo, the dissociation appears to be lost and these compounds still produce many of the side effects associated with conventional GCs. A better understanding of the molecular mechanisms behind GC-induced effects would allow the design of novel selective GR modulators with an improved therapeutic index.

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A Comprehensive In Silico Analysis of the Functional and Structural Impact of Nonsynonymous SNPs in the ABCA1 Transporter Gene

Cholesterol ◽

10.1155/2014/639751 ◽

2014 ◽

Vol 2014 ◽

pp. 1-19 ◽

Cited By ~ 6

Author(s):

Francisco R. Marín-Martín ◽

Cristina Soler-Rivas ◽

Roberto Martín-Hernández ◽

Arantxa Rodriguez-Casado

Keyword(s):

In Silico ◽

Rare Variants ◽

Experimental Studies ◽

In Silico Analysis ◽

Nucleotide Polymorphisms ◽

Single Nucleotide ◽

Experimental Approaches ◽

And Function

Disease phenotypes and defects in function can be traced to nonsynonymous single nucleotide polymorphisms (nsSNPs), which are important indicators of action sites and effective potential therapeutic approaches. Identification of deleterious nsSNPs is crucial to characterize the genetic basis of diseases, assess individual susceptibility to disease, determinate molecular and therapeutic targets, and predict clinical phenotypes. In this study using PolyPhen2 and MutPred in silico algorithms, we analyzed the genetic variations that can alter the expression and function of the ABCA1 gene that causes the allelic disorders familial hypoalphalipoproteinemia and Tangier disease. Predictions were validated with published results from in vitro, in vivo, and human studies. Out of a total of 233 nsSNPs, 80 (34.33%) were found deleterious by both methods. Among these 80 deleterious nsSNPs found, 29 (12.44%) rare variants resulted highly deleterious with a probability >0.8. We have observed that mostly variants with verified functional effect in experimental studies are correctly predicted as damage variants by MutPred and PolyPhen2 tools. Still, the controversial results of experimental approaches correspond to nsSNPs predicted as neutral by both methods, or contradictory predictions are obtained for them. A total of seventeen nsSNPs were predicted as deleterious by PolyPhen2, which resulted neutral by MutPred. Otherwise, forty two nsSNPs were predicted as deleterious by MutPred, which resulted neutral by PolyPhen2.

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Mechanism of DNA alkylation-induced transcriptional stalling, lesion bypass, and mutagenesis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1708748114 ◽

2017 ◽

Vol 114 (34) ◽

pp. E7082-E7091 ◽

Cited By ~ 15

Author(s):

Liang Xu ◽

Wei Wang ◽

Jiabin Wu ◽

Ji Hyun Shin ◽

Pengcheng Wang ◽

...

Keyword(s):

Rna Polymerase Ii ◽

Molecular Mechanisms ◽

Minor Groove ◽

Dna Lesions ◽

Dna Alkylation ◽

Lesion Bypass ◽

Pol Ii ◽

The Impact

Alkylated DNA lesions, induced by both exogenous chemical agents and endogenous metabolites, interfere with the efficiency and accuracy of DNA replication and transcription. However, the molecular mechanisms of DNA alkylation-induced transcriptional stalling and mutagenesis remain unknown. In this study, we systematically investigated how RNA polymerase II (pol II) recognizes and bypasses regioisomeric O2-, N3-, and O4-ethylthymidine (O2-, N3-, and O4-EtdT) lesions. We observed distinct pol II stalling profiles for the three regioisomeric EtdT lesions. Intriguingly, pol II stalling at O2-EtdT and N3-EtdT sites is exacerbated by TFIIS-stimulated proofreading activity. Assessment for the impact of the EtdT lesions on individual fidelity checkpoints provided further mechanistic insights, where the transcriptional lesion bypass routes for the three EtdT lesions are controlled by distinct fidelity checkpoints. The error-free transcriptional lesion bypass route is strongly favored for the minor-groove O2-EtdT lesion. In contrast, a dominant error-prone route stemming from GMP misincorporation was observed for the major-groove O4-EtdT lesion. For the N3-EtdT lesion that disrupts base pairing, multiple transcriptional lesion bypass routes were found. Importantly, the results from the present in vitro transcriptional studies are well correlated with in vivo transcriptional mutagenesis analysis. Finally, we identified a minor-groove–sensing motif from pol II (termed Pro-Gate loop). The Pro-Gate loop faces toward the minor groove of RNA:DNA hybrid and is involved in modulating the translocation of minor-groove alkylated DNA template after nucleotide incorporation opposite the lesion. Taken together, this work provides important mechanistic insights into transcriptional stalling, lesion bypass, and mutagenesis of alkylated DNA lesions.

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A Novel Role of Bergamottin in Attenuating Cancer Associated Cachexia by Diverse Molecular Mechanisms

Cancers ◽

10.3390/cancers13061347 ◽

2021 ◽

Vol 13 (6) ◽

pp. 1347

Author(s):

Young Yun Jung ◽

Jeong-Hyeon Ko ◽

Jae-Young Um ◽

Gautam Sethi ◽

Kwang Seok Ahn

Keyword(s):

Western Blot ◽

Cancer Cachexia ◽

Molecular Mechanisms ◽

Suppressive Effect ◽

In Vivo Experiment ◽

Hematoxylin And Eosin ◽

Loss Method ◽

The Impact

Purpose: The potential effects of bergamotiin (BGM) on the suppression of cancer cachexia was evaluated under in vitro and in vivo conditions to investigate its possible inhibitory effects on the muscle and fat loss. Method: The differentiated C2C12 and 3T3L1 cells were treated with BGM after the induction of cancer-cachexia with pancreatic cancer conditioned media (CM). The expression levels of the various molecules involved in the differentiation and loss of muscle and fat (MuRF-1, Atrogin-1, C/EBPα, and PPARγ) were analyzed by Western blot and oil red O staining. For in vivo experiment, MIA PaCa-2 cells were injected into the mice (n = 6), and then BGM (1 mg/kg) was intraperitoneally administered to analyze muscle and adipose tissue by Hematoxylin and Eosin staining and Western blot. Result: BGM displayed a significant effect on the inhibition of muscle and fat catabolism under both in vitro and in vivo conditions. The results of the in vivo experiment revealed a remarkable suppressive effect of BGM on the weight loss in mice. Conclusions: The potential effects of BGM on the inhibition of muscle and fat catabolism in vitro and in vivo were thus confirmed. Based on the results, the impact of BGM on cancer cachexia could be possibly analyzed in the future clinical studies.

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