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Summary: Sterile inflammation contributes to many pathological states associated with mitochondrial injury. Mitochondrial injury disrupts calcium homeostasis and results in the release of CpG‐rich mitochondrial DNA. The role of CpG‐stimulated TLR9 innate immune signalling and sterile inflammation is well studied; however, how calcium dyshomeostasis affects this signalling is unknown. Therefore, we interrogated the relationship beτween intracellular calcium and CpG‐induced TLR9 signalling in murine macrophages. We found that CpG‐ODN‐induced NFκB‐dependent IL1α and IL1β expression was significantly attenuated by both calcium chelation and calcineurin inhibition, a finding mediated by inhibition of degradation of the NFκB inhibitory protein IκBβ. In contrast, calcium ionophore exposure increased CpG‐induced IκBβ degradation and IL1α and IL1β expression. These results demonstrate that through its effect on IκBβ degradation, increased intracellular Ca2+ drives a pro‐inflammatory TLR9‐mediated innate immune response. These results have implications for the study of innate immune signalling downstream of mitochondrial stress and injury. [ABSTRACT FROM AUTHOR]

The DNA barcodes are generally interpreted using distance-based and character-based methods. The former uses clustering of comparable groups, based on the relative genetic distance, while the latter is based on the presence or absence of discrete nucleotide substitutions. The distance-based approach has a limitation in defining a universal species boundary across the taxa as the rate of mt DNA evolution is not constant throughout the taxa. However, character-based approach more accurately defines this using a unique set of nucleotide characters. The character-based analysis of full-length barcode has some inherent limitations, like sequencing of the full-length barcode, use of a sparse-data matrix and lack of a uniform diagnostic position for each group. A short continuous stretch of a fragment can be used to resolve the limitations. Here, we observe that a 154-bp fragment, from the transversion-rich domain of 1367 COI barcode sequences can successfully delimit species in the three most diverse orders of freshwater fishes. This fragment is used to design species-specific barcode motifs for 109 species by the character-based method, which successfully identifies the correct species using a pattern-matching program. The motifs also correctly identify geographically isolated population of the Cypriniformes species. Further, this region is validated as a species-specific mini-barcode for freshwater fishes by successful PCR amplification and sequencing of the motif (154 bp) using the designed primers. We anticipate that use of such motifs will enhance the diagnostic power of DNA barcode, and the mini-barcode approach will greatly benefit the field-based system of rapid species identification. [ABSTRACT FROM AUTHOR]

Background The environmental and genetic factors are known to be associated with head and neck squamous cell carcinoma ( HNSCC). Here, we investigated the GSTM1 and GSTT1 polymorphisms and tobacco uses in patients and in their first-degree relatives to evaluate susceptibility toward HNSCC. Further, we explored high-risk interaction between genetic and environmental risk factors. Subjects and methods Genotyping of GSTM1 and GSTT1 was performed in 170 patients, 300 first-degree relatives of patients and 300 controls using multiplex PCR. Multifactor dimensionality reduction ( MDR) and logistic regression approach were applied for statistical analysis. Results Analysis revealed that GSTM1 and GSTT1 null genotype frequencies were significantly higher in patients (adjusted odds ratio ( OR) = 2.18; P < 0.001 and OR = 1.61; P = 0.031, respectively). Also, the GSTM1 and GSTT1 null genotype frequencies were significantly higher in first-degree relatives of patients compared with controls ( P = 0.004 and P = 0.041, respectively). In MDR analysis, the best model for HNSCC risk was four-factors model of tobacco, betel quid chewing, smoking and GSTM1 null genotypes (cross-validation consistency = 10/10 and P < 0.0001), whereas in interaction entropy graphs, tobacco chewing and GSTM1 null genotype further showed strongest synergistic interaction. Conclusion GSTM1 and GSTT1 null genotypes may act as markers to determine the genetic susceptibility in HNSCC patients and in their first-degree relatives. Furthermore, tobacco chewing and GSTM1 null genotype interaction identified as the strongest gene-environment model to predict HNSCC. [ABSTRACT FROM AUTHOR]

A20 has been suggested to limit NF-κB activation by removing regulatory ubiquitin chains from ubiquitinated substrates. A20 is a ubiquitin-editing enzyme that removes K63-linked ubiquitin chains from adaptor proteins, such as RIP1, and then conjugates them to K48-linked polyubiquitin chains to trigger proteasomal degradation. To determine the role of the deubiquitinase function of A20 in downregulating NF-κB signaling, we have generated a knock-in mouse that lacks the deubiquitinase function of A20 ( A20- OTU mice). These mice are normal and have no signs of inflammation, have normal proportions of B, T, dendritic, and myeloid cells, respond normally to LPS and TNF, and undergo normal NF-κB activation. Our results thus indicate that the deubiquitinase activity of A20 is dispensable for normal NF-κB signaling. [ABSTRACT FROM AUTHOR]

Recovery of DNA barcode sequences is often challenging from the archived specimens. However, short fragments of DNA may be recovered, which would significantly improve many unresolved taxonomic conflicts. Here, we designed a mini-barcode for catfishes comprising several species and many cryptic taxa. We analysed a data set of 3048 publicly available COI barcode sequences representing 547 worldwide catfish species and performed 152 628 interspecies comparisons. A significantly more positively correlated interspecies distance was detected with transversion (0.78, P < 0.001) than with transition (0.70, P < 0.001). This suggested that transversions were better diagnostics for species identification. In the aligned data set, two transversion-rich fragments (53 bp and 119 bp) were identified. Transition/transversion bias value was 1.04 in 53-bp fragment, 1.23 in 119-bp fragment and 1.50 in full-length barcode. The interspecies distance with full-length barcode was 0.212 ± 0.037, while that with 53-bp and 119-bp fragments was 0.325 ± 0.039 and 0.218 ± 0.045, respectively. Survey of 53-bp fragment showed a possibility of only 1144 barcodes, while that of 119-bp fragment showed >4 million barcodes. Thus, the 119-bp fragment is a viable mini-barcode for catfishes comprising >3000 extant species. Experiment with 82 archived catfishes showed successful recovery of this mini-barcode using the designed primer. The mini-barcode sequences showed species-specific similarity in the range of 98-100% with the global database. Therefore, survey of a transversion-rich fragment within the full-length barcode would be an ideal approach of mini-barcode design for biodiversity assessment. [ABSTRACT FROM AUTHOR]

The nuclear factor-κB ( NF-κB) family of transcription factors plays important roles in various biological processes including apoptosis, stress response, immunity, and inflammation. NF-κB signaling is involved in both immune cell development and function, and it is critical in modulation of the immune response through the transcriptional regulation of cytokine and chemokine expression. An area of great interest in T-cell-mediated adaptive immunity is the ability of naive CD4+ T cells generated in the thymus to differentiate into various subsets including T-helper 1 (Th1), Th2, Th17, Th9, follicular helper T (Tfh), Th22, and regulatory T (Treg) cells, upon encountering different pathogens and microenvironments. In this review, we discuss the role of NF-κB pathway in the development and functional divergence of the different helper T-cell subsets as well as in regulatory T cells. [ABSTRACT FROM AUTHOR]

CXCL2 (macrophage inflammatory protein-2 (MIP-2)), a critical chemokine for neutrophils, has been shown to be produced in the rat intestine in response to platelet-activating factor (PAF) and to mediate intestinal inflammation and injury. The intestinal epithelium, constantly exposed to bacterial products, is the first line of defence against micro-organisms. It has been reported that enterocytes produce proinflammatory mediators, including tumour necrosis factor (TNF) and PAF, and we showed that lipopolysaccharide (LPS) and TNF activate nuclear factor (NF)-κB in enterocytes. However, it remains elusive whether enterocytes release CXCL2 in response to LPS and TNF via a NF-κB-dependent pathway and whether this involves the endogenous production of TNF and PAF. In this study, we found that TNF and LPS markedly induced CXCL2 gene expression in IEC-6 cells, TNF within 30 min, peaking at 45 min, while LPS more slowly, peaking after 2 hr. TNF- and LPS- induced CXCL2 gene expression and protein release were completely blocked by pyrrolidine dithiocarbamate (PDTC) and helenalin, two potent NF-κB inhibitors. NEMO-binding domain peptide, a specific inhibitor of inhibitor protein κB kinase (IKK) activation, a major upstream kinase mediating NF-κB activation, significantly blocked CXCL2 gene expression and protein release induced by LPS. WEB2170 (PAF antagonist) and anti-TNF antibodies had no effect on LPS-induced CXCL2 expression. In conclusion, CXCL2 gene is expressed in enterocytes in response to both TNF and LPS. LPS-induced CXCL2 expression is dependent on NF-κB activation via the IKK pathway. The effect of LPS is independent of endogenous TNF and PAF. [ABSTRACT FROM AUTHOR]

Bone metabolism is regulated by hormonal or local factors in the bone microenvironment, and recent studies have revealed that bone homeostasis is also influenced by immune system. The term ‘osteoimmunology’ has been proposed to explain the cross-talk between bone and the immune system. A critical element in this cross-talk is the inducible transcription factor nuclear factor-κB (NF-κB), which regulates gene expression during inflammatory and immune responses. However, NF-κB-signaling pathways are also important for bone homeostasis, in particular for osteoclast differentiation. By bridging inflammation and bone homeostasis, NF-κB also contributes to the onset and progression of arthritis. Several natural compounds, synthetic drugs, and gene-transfer technologies that lead to inhibition of the inhibitor of NF-κB kinase (IKK)/NF-κB activation pathway can prevent arthritis in animal models. In this review, we discuss the signaling pathway that leads to NF-κB activation and the role of NF-κB on osteoclast differentiation. Furthermore, we discuss the possibility that inhibition of NF-κB might provide novel therapeutic approach for inhibiting bone destruction in rheumatoid arthritis. [ABSTRACT FROM AUTHOR]

Dendritic cells (DC) are the only antigen-presenting cells for naive T cells and, therefore, they are crucial players in the initiation of immune responses. Because DC maturation and cytokine production are NF-κB dependent, we hypothesized that blocking NF-κB activity in DC by selectively targeting the inhibitor of κB (IκB) kinase (IKK) complex using the novel NF-κB inhibitor NEMO-binding domain (NBD) peptide could inhibit DC maturation and other functional characteristics, resulting in modulation of the immune response. We used human monocyte-derived DC to test the biological effects of the NBD peptide in vitro. NF-κB inhibition by the NBD peptide resulted in blockade of IKK-mediated IκBα phosphorylation and subsequent nuclear translocation and DNA binding of NF-κB p65 in DC. In addition, IL-6, IL-12, and TNF-α production was dose-dependently blocked and NBD peptide treatment also led to a strong reduction of LPS-induced maturation. Functional analysis of these DC showed marked inhibition of T cell proliferation in the allogeneic mixed lymphocyte reaction, accompanied by less Th1 and Th2 polarization. The current study reveals for the first time the unique properties of this novel, highly specific NF-κB inhibitor in DC. Also, these data indicate that the NBD peptide could be used as an elegant tool in DC based immunotherapy for unwanted cellular immune responses. [ABSTRACT FROM AUTHOR]

Objective: We used an experimental model of inflammation in mice, carrageenan-induced paw edema, to study the antiinflammatory effects of the NEMO-binding domain (NBD) peptide, which blocks activation of the inducible transcription factor NF-kappaB., Methods: Paw edema was induced by subplantar injection of 1% lambda-carrageenan into the mouse left hind paw. Test agents were given intraperitoneally immediately after carrageenan injection. The increase in footpad thickness was considered to be edema. In some experiments, the mice were killed and the paws were removed for histologic and molecular biology analysis. NF-kappaB DNA binding activity was evaluated in nuclear extracts by electrophoretic mobility shift assays. The expression levels of NF-kappaB-regulated cyclooxygenase 2 (COX-2) protein and tumor necrosis factor alpha (TNFalpha) messenger RNA (mRNA) were evaluated by immunoblot analysis and polymerase chain reaction amplification of reverse-transcribed mRNA, respectively., Results: We found that systemically administered NBD peptide significantly inhibited edema formation and cellular infiltration in inflamed mouse paws. This antiinflammatory activity was most likely due to inhibition of expression of proinflammatory mediators, such as TNFalpha and COX-2, in inflamed tissues., Conclusion: These studies further establish NF-kappaB as a target for antiinflammatory therapy and provide support for the use of the NBD peptide as a possible therapeutic agent for inflammatory diseases.