OBJECTIVE: Leishmaniasis, caused by the protozoan parasite poses a significant health burden globally. With a very few specific drugs, increased drug resistance it is important to look for drug repurposing along with the identification of pre-clinical candidates against visceral leishmaniasis. This study aims to identify potential drug candidates against visceral leishmaniasis by targeting leishmanial MAP kinases and screening FDA approved protein kinase inhibitors.
METHODS: MAP kinases were identified from the Leishmania genome. 12 FDA approved protein kinase inhibitors were screened against Leishmania MAP kinases. Binding affinity, ADME and toxicity of identified drug candidates were profiled. The anti-proliferative effects and mechanism of action were assessed in Leishmania, including changes in cell morphology, flagellar length, cell cycle progression, reactive oxygen species (ROS) generation, and intra-macrophage parasitic burden.
RESULTS: 23 MAP kinases were identified from the Leishmania genome. Sorafenib and imatinib emerged as repurposable drug candidates and demonstrated excellent anti-proliferative effects in Leishmania. Treatment with these inhibitors resulted in significant changes in cell morphology, flagellar length, and cell cycle arrest. Furthermore, sorafenib and imatinib promoted ROS generation and reduced intra-macrophage parasitic burden, and elicited anti-leishmanial activity in in vivo experimental VL models.
CONCLUSIONS: Collectively, these results imply involvement of MAP kinases in infectivity and survival of the parasite and can pave the avenue for repurposing sorafenib and imatinib as anti-leishmanial agents. These findings contribute to the exploration of new treatment options for visceral leishmaniasis, particularly in the context of emerging drug resistance.

Naja naja snake bite causes thousands of deaths worldwide in a year. N. naja envenomed victims exhibit both local and systemic reactions that potentially lead to death. In clinical practice, pulmonary complications in N. naja envenomation are commonly encountered. However, the molecular mechanisms underlying N. naja venom-induced lung toxicity remain unknown. Here, we reasoned that N. naja venom-induced lung toxicity is prompted by NLRP3 inflammasome and MAPKs activation in mice. Treatment with dimethyl ester of bilirubin (BD1), significantly inhibited the N. naja venom-induced activation of NLRP3 inflammasome and MAPKs both in vivo and in vitro (p < 0.05). Further, BD1 reduced N. naja venom-induced recruitment of inflammatory cells, and hemorrhage in the lung toxicity examined by histopathology. BD1 also diminished N. naja venom-induced local toxicities in paw edema and myotoxicity in mice. Furthermore, BD1 was able to enhance the survival time against N. naja venom-induced mortality in mice. In conclusion, the present data showed that BD1 alleviated N. naja venom-induced lung toxicity by inhibiting NLRP3 inflammasome and MAPKs activation. Small molecule inhibitors that intervene in venom-induced toxicities may have therapeutic applications complementing anti-snake venom.

Toll-like receptors (TLRs) induce innate immune responses through activation of intracellular signaling pathways, such as MAP kinase and NF-κB signaling pathways, and play an important role in host defense against bacterial or viral infections. Meanwhile, excessive activation of TLR signaling leads to a variety of inflammatory disorders, including autoimmune diseases. TLR signaling is therefore strictly controlled to balance optimal immune response and inflammation. However, its balancing mechanisms are not fully understood. In this study, we identified the E3 ubiquitin ligase LINCR/ NEURL3 as a critical regulator of TLR signaling. In LINCR-deficient cells, the sustained activation of JNK and p38 MAPKs induced by the agonists for TLR3, TLR4, and TLR5, was clearly attenuated. Consistent with these observations, TLR-induced production of a series of inflammatory cytokines was significantly attenuated, suggesting that LINCR positively regulates innate immune responses by promoting the activation of JNK and p38. Interestingly, our further mechanistic study identified MAPK phosphatase-1 (MKP1), a negative regulator of MAP kinases, as a ubiquitination target of LINCR. Thus, our results demonstrate that TLRs fine-tune the activation of MAP kinase pathways by balancing LINCR (the positive regulator) and MKP1 (the negative regulator), which may contribute to the induction of optimal immune responses.

Oxidation processes in mitochondria and different environmental insults contribute to unwarranted accumulation of reactive oxygen species (ROS). These, in turn, rapidly damage intracellular lipids, proteins, and DNA, ultimately causing aging and several human diseases. Cells have developed different and very effective systems to control ROS levels. Among these, removal of excessive amounts is guaranteed by upregulated expression of various antioxidant enzymes, through activation of the NF-E2-Related Factor 2 (NRF2) protein. Here, we show that Mitogen Activated Protein Kinase 15 (MAPK15) controls the transactivating potential of NRF2 and, in turn, the expression of its downstream target genes. Specifically, upon oxidative stress, MAPK15 is necessary to increase NRF2 expression and nuclear translocation, by inducing its activating phosphorylation, ultimately supporting transactivation of cytoprotective antioxidant genes. Lungs are continuously exposed to oxidative damages induced by environmental insults such as air pollutants and cigarette smoke. Interestingly, we demonstrate that MAPK15 is very effective in supporting NRF2-dependent antioxidant transcriptional response to cigarette smoke of epithelial lung cells. Oxidative damage induced by cigarette smoke indeed represents a leading cause of disability and death worldwide by contributing to the pathogenesis of different chronic respiratory diseases and lung cancer. Therefore, the development of novel therapeutic strategies able to modulate cellular responses to oxidative stress would be highly beneficial. Our data contribute to the necessary understanding of the molecular mechanisms behind such responses and identify new potentially actionable targets.

The mechanisms underlying chronic inflammatory diseases remain unclear. Therefore, researchers have explored the mechanisms underlying colitis using diverse materials. Recently, there has been an increasing interest in fermented products and bioconversion materials, their potential efficacy is being actively studied. Gochujang, a traditional Korean fermented product, is crafted by blending fermented Meju powder, gochu (Korean chili) powder, glutinous rice, and salt. In our study, we explored the effectiveness of Gochujang (500 mg/kg; Cheongju and Hongcheon, Korea) in dextran sulfate sodium (DSS)-induced colitis mice model. Gochujang was orally administered for 2 weeks, followed by the induction of colitis using 3% DSS in the previous week. During our investigation, Gochujang variants (TCG22-25, Cheongju and TCG22-48, Hongcheon) did not exhibit significant inhibition of weight reduction (p = 0.061) but notably (p = 0.001) suppressed the reduction in large intestine length in DSS-induced colitis mice. In the serum from colitis mice, TCG22-48 demonstrated reduced levels of the inflammatory cytokines interleukin (IL)-6 (p = 0.001) and tumor necrosis factor (TNF)-α (p = 0.001). Additionally, it inhibited the phosphorylation of Erk (p = 0.028), p38, and NF-κB (p = 0.001) the inflammatory mechanism. In our study, TCG22-25 demonstrated a reduction in the IL-6 level (p = 0.001) in serum and inhibited the phosphorylation of p38 and NF-κB (p = 0.001). Histological analysis revealed a significant (p = 0.001) reduction in the pathological score of the large intestine from TCG22-25 and TCG22-48. In conclusion, the intake of Gochujang demonstrates potent anti-inflammatory effects, mitigating colitis by preventing the large intestine length reduction of animals with colitis, lowering serum levels of TNF-α and IL-6 cytokines, and inhibiting histological disruption and inflammatory mechanism phosphorylation.

Guanylate-binding proteins (GBPs) are a family of interferon (IFN)-inducible GTPases and play a pivotal role in the host immune response to microbial infections. These are upregulated in immune cells after recognizing the lipopolysaccharides (LPS), the major membrane component of Gram-negative bacteria. In the present study, the expression pattern of GBP1-7 was initially mapped in phorbol 12-myristate 13-acetate-differentiated human monocytes THP-1 and mouse macrophages RAW 264.7 cell lines stimulated with LPS. A time-dependent significant expression of GBP1-7 was observed in these cells. Moreover, among the various GBPs, GBP1 has emerged as a central player in regulating innate immunity and inflammation. Therefore, to study the specific role of GBP1 in LPS-induced inflammation, knockdown of the Gbp1 gene was carried out in both cells using small interfering RNA interference. Altered levels of different cytokines (interleukin [IL]-4, IL-10, IL-12β, IFN-γ, tumor necrosis factor-α), inducible nitric oxide synthase, histocompatibility 2, class II antigen A, protein kinase R, and chemokines (chemokine (C-X-C motif) ligand 9 [CXCL9], CXCL10, and CXCL11) in GBP1 knockdown cells were reported compared to control cells. Interestingly, the extracellular-signal-regulated kinase 1/2 mitogen-activated protein (MAP) kinases and signal transducer and activator of transcription 1 (STAT1) transcription factor levels were considerably induced in knockdown cells compared to the control cells. However, no change in the level of phosphorylated nuclear factor-kB, c-Jun, and p38 transcription factors was observed in GBP1 knockdown cells compared to the control cells. This study concludes that GBP1 may alter the expression of cytokines, chemokines, and effector molecules mediated by MAP kinases and STAT1 transcription factors.

OBJECTIVE: To gain insights into how proteases signal to connective tissues cells in the periodontium.
BACKGROUND: The connective tissue degradation observed in periodontitis is largely due to matrix metalloproteinase (MMP) release by gingival fibroblasts. Granzyme B (GzmB) is a serine protease whose role in periodontitis is undefined.
METHODS: Human gingival crevicular fluid (GCF) samples were obtained from sites with periodontal disease and healthy control sites. GzmB was quantified in the GCF ([GzmB]GCF ) by ELISA. Gingival fibroblasts (GF) were cultured in the presence or absence of recombinant GzmB. Culture supernatants were analyzed by ELISA to quantify GzmB-induced release of interstitial collagenase (MMP-1). In some experiments, cells were pre-treated with the inhibitor PD98059 to block MEK/ERK signaling. The protease-activated receptor-1 (PAR-1) was blocked with ATAP-2 neutralizing antibody prior to GzmB stimulation. Systemic MMP-1 levels were measured in plasma from wild-type (WT) and granzyme-B-knockout (GzmB-/- ) mice.
RESULTS: The [GzmB]GCF in human samples was ~4-5 fold higher at sites of periodontal disease (gingivitis/periodontitis) compared to healthy control sites, suggesting an association between GzmB and localized matrix degradation. GzmB induced a ~4-5-fold increase in MMP-1 secretion by cultured fibroblasts. GzmB induced phosphorylation of Erk1/2, which was abrogated by PD98059. GzmB-induced upregulation of MMP-1 secretion was also reduced by PD98059. Blockade of PAR-1 function by ATAP-2 abrogated the increase in MMP-1 secretion by GF. Circulating MMP-1 was similar in WT and GzmB-/- mice, suggesting that GzmB\'s effects on MMP-1 release are not reflected systemically.
CONCLUSIONS: These data point to a novel GzmB-driven signaling pathway in fibroblasts in which MMP-1 secretion is upregulated in a PAR1- and Erk1/2-dependent manner.

Ribosomes that synthesize proteins are among the most central and evolutionarily conserved organelles. Given the key role of proteins in cellular functions, prokaryotic and eukaryotic pathogens have evolved potent toxins to inhibit ribosomal functions and weaken their host. Many of these ribotoxin-producing pathogens are associated with food. For example, food can be contaminated with bacterial pathogens that produce the ribotoxin Shiga toxin, but also with the fungal ribotoxin deoxynivalenol. Shiga toxin cleaves ribosomal RNA, while deoxynivalenol binds to and inhibits the peptidyl transferase center. Despite their distinct modes of action, both groups of ribotoxins hinder protein translation, but also trigger other comparable toxic effects, which depend or not on the activation of the ribotoxic stress response. Ribotoxic stress response-dependent effects include inflammation and apoptosis, whereas ribotoxic stress response-independent effects include endoplasmic reticulum stress, oxidative stress, and autophagy. For other effects, such as cell cycle arrest and cytoskeleton modulation, the involvement of the ribotoxic stress response is still controversial. Ribotoxins affect one organelle yet induce multiple toxic effects with multiple consequences for the cell. The ribosome can therefore be considered as the cellular \"Achilles heel\" targeted by food borne ribotoxins. Considering the high toxicity of ribotoxins, they pose a substantial health risk, as humans are highly susceptible to widespread exposure to these toxins through contaminated food sources.

E2F is the key transcription factor that determines the proliferative status of cells by regulating the G1/S phase of the cell cycle. In this study, we show that in rice (Oryza sativa), OsE2F2 is a phosphorylation target of MAP kinases. The MAP kinases OsMPK3, OsMPK4, and OsMPK6 interact with and phosphorylate OsE2F2. Next, we determined the serine and threonine residues that could play a role in the phosphorylation of OsE2F2. Subsequently, our study suggests a possible link between MAP kinase-mediated OsE2F2 phosphorylation and its impact on DNA proliferation in the roots of rice seedlings. Finally, we found positive feedback regulation of OsMPK4 by OsE2F2. Therefore, our study hints at the potential impact of MAP kinase signaling on the cell cycle of rice plants.

Wound healing is a complex process to restore skin. Plant-derived bioactive compounds might be a source of substances for the treatment of wounds stalled in a non-resolving stage of wound healing. Oleanolic acid (OA), a pentacyclic triterpene, has shown favorable wound healing properties both in vitro and in vivo. Unfortunately, OA cannot be solubilized in aqueous media, and it needs to be helped by the use of dimethyl sulfoxide (DMSO). In this paper, we have shown that cyclodextrins (CDs) are a good alternative to DMSO as agents to deliver OA to cells, providing better features than DMSO. Cyclodextrins are natural macromolecules that show a unique tridimensional structure that can encapsulate a wide variety of hydrophobic compounds. We have studied the cyclodextrin-encapsulated form of OA with OA/DMSO, comparing their stability, biological properties for cell migration, and cell viability. In addition, detailed parameters related to cell migration and cytoskeletal reorganization have been measured and compared. Our results show that OA-encapsulateds compound exhibit several advantages when compared to non-encapsulated OA in terms of chemical stability, migration enhancement, and preservation of cell viability.