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Jatropha curcas L. (J. curcas), a shrub plant of the Euphorbiaceae family, has received enormous attention as a promising biofuel plant for the production of biodiesel and medical potential in ethnopharmacology. However, the tumor-promoter toxin phorbol esters present in J. curcas raise concerns for health and environmental risk as its large-scale cultivation limits the use of meal obtained after oil extraction for animal feed. Here, we determined the variation of phorbol ester profiles and contents in eight J. curcas tissues by high-performance liquid chromatography (HPLC) and found phorbol esters present in all parts of the plant except the seed shell. We showed tissue-specific patterns of accumulation of phorbol esters and associated terpenoids at the transcriptional level with high transcript levels in reproductive and young tissues. Genes involved in the same module of terpenoids biosynthesis were positively correlated. We further present diverse abiotic and biotic stresses that had different effects on the accumulation of transcripts in terpenoids shared and branched terpenoid pathways in plant seedlings. The fine-tuning of terpenoids biosynthesis may link with ecological functions in plants under extreme environments and defense against pathogens. [ABSTRACT FROM AUTHOR]

As the development of chronic wound therapeutics continues to expand, the demand for advanced assay systems mimicking the inflammatory wound microenvironment in vivo increases. Currently, this is performed in animal models or in in vitro cell-based models such as cell culture scratch assays that more closely resemble acute wounds. Here, we describe for the first time a delayed scratch closure model that mimics some features of a chronic wound in vitro. Chronic wounds such as those suffered by later stage diabetic patients are characterised by degrees of slowness to heal caused by a combination of continued localised physical trauma and pro-inflammatory signalling at the wound. To recreate this in a cell-based assay, a defined physical scratch was created and stimulated by combinations of pro-inflammatory factors, namely interferon, the phorbol ester PMA, and lipopolysaccharide, to delay scratch closure. The concentrations of these factors were characterised for commonly used human keratinocyte (HaCaT) and dermal fibroblast (HDF) cell lines. These models were then tested for scratch closure responsiveness to a proprietary healing secretome derived from human Wharton's jelly mesenchymal stem cells (MSCs) previously validated and shown to be highly effective on closure of acute wound models both in vitro and in vivo. The chronically open scratches from HaCaT cells showed closure after exposure to the MSC secretome product. We propose this delayed scratch closure model for academic and industrial researchers studying chronic wounds looking for responsiveness to drugs or biological treatments prior to testing on explanted patient material or in vivo. [ABSTRACT FROM AUTHOR]

Neutrophils are the first immune cells to reach inflamed sites and contribute to the pathogenesis of chronic inflammatory skin diseases. Yet, little is known about the pattern of neutrophil infiltration in inflamed skin in vivo and the mechanisms mediating their recruitment. Here, we provide insight into the dynamics of neutrophil infiltration in skin in response to acute or repeated inflammatory stress, highlighting a novel keratinocyte- and keratin 17 (K17)-dependent mechanism that regulates neutrophil recruitment to inflamed skin. We used the phorbol ester TPA and UVB, alone or in combination, to induce sterile inflammation in mouse skin. A single TPA treatment results in a neutrophil influx in the dermis that peaks at 12 h and resolves within 24 h. A subsequent TPA treatment or a UVB challenge, when applied 24 h but not 48 h later, accelerates, amplifies, and prolongs neutrophil infiltration. This transient amplification response (TAR) is mediated by local signals in inflamed skin, can be recapitulated in ex vivo culture, and involves the K17-dependent sustainment of protein kinase Cα (PKCα) activity and release of chemoattractants by stressed keratinocytes. K17 binds RACK1, a scaffold protein essential for PKCα activity. The N-terminal head domain of K17 is crucial for its association with RACK1 and regulation of PKCα activity. Analysis of RNAseq data reveals a signature consistent with TAR and PKCα activation in inflammatory skin diseases. These findings uncover a novel, keratin-dependent mechanism that amplifies neutrophil recruitment in skin under stress, with direct implications for inflammatory skin disorders. Neutrophils contribute to the pathogenesis of chronic inflammatory diseases, but the mechanisms underlying their recruitment to inflamed skin are unclear. This study shows that repeated acute stress to the skin amplifies a neutrophil infiltration response involving keratin 17 and PKCα. [ABSTRACT FROM AUTHOR]

Jatropha curcas L. (J. curcas), a shrub plant of the Euphorbiaceae family, has received enormous attention as a promising biofuel plant for the production of biodiesel and medical potential in ethnopharmacology. However, the tumor-promoter toxin phorbol esters present in J. curcas raise concerns for health and environmental risk as its large-scale cultivation limits the use of meal obtained after oil extraction for animal feed. Here, we determined the variation of phorbol ester profiles and contents in eight J. curcas tissues by high-performance liquid chromatography (HPLC) and found phorbol esters present in all parts of the plant except the seed shell. We showed tissue-specific patterns of accumulation of phorbol esters and associated terpenoids at the transcriptional level with high transcript levels in reproductive and young tissues. Genes involved in the same module of terpenoids biosynthesis were positively correlated. We further present diverse abiotic and biotic stresses that had different effects on the accumulation of transcripts in terpenoids shared and branched terpenoid pathways in plant seedlings. The fine-tuning of terpenoids biosynthesis may link with ecological functions in plants under extreme environments and defense against pathogens.

Euphorbia damarana L.C.Leach , also known as Damara milk bush, grows only in the north-western desert areas of Namibia and southern Angola. It is often the only vegetation in these desert regions and the local inhabitants know very well that it is extremely poisonous to humans and animals. During the 1960s, twenty-seven migrating mineworkers used its dead branches as firewood for their barbecue with deadly consequences. They all passed away after consuming meat that was infused with toxic volatiles in the smoke. We report in this study, on the isolation of the toxic triterpenoid, euphol that is present in high concentrations in the stems and smoke of E. damarana. Small concentrations of compounds with phorbol ester skeletons were also detected in this species. The cytotoxicity of euphol in the smoke and stem extracts of E. damarana was determined by the sulfo-rhodamine-B stain (SRB) assay on eight human cell lines (A549, PC-3, HeLa, HepG2, MCF-7, MCF-12A, MRC-5 and HaCaT). Significant cytotoxic activity was observed from the purified euphol, stem and smoke extracts with IC 50 values ranging from 1.99 to 3.99 μg/mL, 5.00 to 20.00 μg/mL and from 11.75 to 40.00 μg/mL respectively, on all the tested human cell lines. Since euphol is the primary compound (concentration 10.3 mg/g) in the smoke extract and toxic to all cell lines tested at a significantly low IC 50 level, it can be considered as the main toxin in E. damarana and responsible for the deaths of the miners. [Display omitted] • Euphorbia species were traditionally used for arrowhead poisoning. • Euphorbia damarana demonstrated cytotoxic activity in vitro against all tested human cell lines. • Euphol was isolated as the main toxin. • Euphol demonstrated significant cytotoxic activity in vitro against all tested human cell lines. • Euphorbia damarana has the potential to be considered for anticancer drug development. [ABSTRACT FROM AUTHOR]

Human individual differences in brain cytochrome P450 (CYP) metabolism, including induction, inhibition, and genetic variation, may influence brain sensitivity to neurotoxins and thus participate in the onset of neurodegenerative diseases. The aim of this study was to explore the modulation of CYPs in neuronal cells. The experimental approach was focused on differentiating human neuroblastoma SH-SY5Y cells into a phenotype resembling mature dopamine neurons and investigating the effects of specific CYP isoform induction. The results demonstrated that the differentiation protocols using retinoic acid followed by phorbol esters or brain-derived neurotrophic factor successfully generated SH-SY5Y cells with morphological neuronal characteristics and increased neuronal markers (NeuN, synaptophysin, β-tubulin III, and MAO-B). qRT-PCR and Western blot analysis showed that expression of the CYP 1A1, 3A4, 2D6, and 2E1 isoforms was detectable in undifferentiated cells, with subsequent increases in CYP 2E1, 2D6, and 1A1 following differentiation. Further increases in the 1A1, 2D6, and 2E1 isoforms following β-naphthoflavone treatment and 1A1 and 2D6 isoforms following ethanol treatment were evident. These results demonstrate that CYP isoforms can be modulated in SH-SY5Y cells and suggest their potential as an experimental model to investigate the role of CYPs in neuronal processes involved in the development of neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Determining the balance between DNA double strand break repair (DSBR) pathways is essential for understanding treatment response in cancer. We report a method for simultaneously measuring non-homologous end joining (NHEJ), homologous recombination (HR), and microhomology-mediated end joining (MMEJ). Using this method, we show that patient-derived glioblastoma (GBM) samples with acquired temozolomide (TMZ) resistance display elevated HR and MMEJ activity, suggesting that these pathways contribute to treatment resistance. We screen clinically relevant small molecules for DSBR inhibition with the aim of identifying improved GBM combination therapy regimens. We identify the ATM kinase inhibitor, AZD1390, as a potent dual HR/MMEJ inhibitor that suppresses radiation-induced phosphorylation of DSBR proteins, blocks DSB end resection, and enhances the cytotoxic effects of TMZ in treatment-naïve and treatment-resistant GBMs with TP53 mutation. We further show that a combination of G2/M checkpoint deficiency and reliance upon ATM-dependent DSBR renders TP53 mutant GBMs hypersensitive to TMZ/AZD1390 and radiation/AZD1390 combinations. This report identifies ATM-dependent HR and MMEJ as targetable resistance mechanisms in TP53-mutant GBM and establishes an approach for simultaneously measuring multiple DSBR pathways in treatment selection and oncology research. New strategies are needed to address treatment resistance in glioblastoma. Here the authors show that TP53-mutant glioblastomas rely upon ATM-dependent double strand break repair to resist DNA-damaging therapy, rendering them vulnerable to drug combinations employing ATM inhibitors. [ABSTRACT FROM AUTHOR]

The interaction of indotricarbocyanine dyes with lipid-transfer protein Ns-LTP1 in Dulbecco's phosphate-buffered saline (PBS) was studied. An orthophenylene bridge with a chlorine in the meso-position of the dye was shown to be ineffective as a linker for protein macromolecules without a free cysteine residue. The indotricarbocyanine dye was modified into the N-hydroxysuccinimide ester to obtain a complex of the indotricarbocyanine dye with the Ns-LTP1 protein. A complex of dye molecules with the Ns-LTP1 protein was obtained by mixing a two-phase system of the protein in PBS and the dye activated ester in 1,2-dichloroethane. The half-width of the absorption spectrum increased from 56 to 61 nm; the fluorescence decay time, from 0.3 to 0.5 ns; and the degree of polarization, from 27 to 31% upon formation of the complex. It was established that HCl at concentrations equivalent to its content in gastric juice (0.1–0.5%) led to irreversible destruction of the dye. Capsules that can withstand stomach acid and dissolve in the intestines could be used to treat erosive and ulcerative lesions with peroral administration of the complex. [ABSTRACT FROM AUTHOR]

Objective:To investigate effect of Mycobacterium tuberculosis BCG-infected macrophages on damage and repair of renal tubular epithelial cells during development of renal tuberculosis. Methods: A co-culture model of BCG-infected M0 macrophages (upper chamber) and HK-2 cells (lower chamber) was established by Transwell, and THP-1 human monocyte macrophages were induced by 100 ng/ml phorbol ester (PMA) 24 h to become M0 macrophages, and BCG infection cell model was established. Total cell protein was collected at 12 h and 24 h of infection, respectively. Western blot was used to detect expressions of M1 macrophage marker CD86 and M2 macrophage marker CD206 protein. M1 macrophage polarization marker cytokines IL-6 and TNF-α and M2 macrophage polarization marker cytokine TGF-β expressions in cell culture supernatant were detected by ELISA; experiment was divided into HK-2 group, BCG+HK-2 group, BCG+M0+HK-2 group and M0+HK-2 group, CCK-8 was used to detect viability of HK-2 cells in each group, and Hoechst test was used to detect HK-2 cells apoptosis in each group. Epithelial cell marker E-cadhren and fibroblast marker α-SMA expressions in HK-2 cells of each group were detected by Western blot. Results: After BCG infection of M0 macrophages, Ml macrophage viability was higher than 24 h at 12 h (P<0.05), and M2 macrophage was higher than 12 h at 24 h (P<0.05). After two cells co-culture, HK-2 cell viability was higher than 12 h at 24 h (P<0.001), apoptosis level was higher than 24 h at 12 h, epithelial cell marker protein E-cadherin protein level was higher than 12 h at 24 h (P<0.001), fibroblast level of cell marker protein α-SMA protein at 12 h was higher than that at 24 h (P<0.01). Conclusion: During development of renal tuberculosis, early BCG-infected macrophages may promote inflammatory injury of renal tubular epithelial cells through M1-type polarization; with prolongation of infection time, they may repair renal tubular epithelial cells through M2-type polarization and plays an important protective role. [ABSTRACT FROM AUTHOR]

Chagas disease is caused by the single-flagellated protozoan Trypanosoma cruzi, which affects several million people worldwide. Understanding the signal transduction pathways involved in this parasite's growth, adaptation, and differentiation is crucial. Understanding the basic mechanisms of signal transduction in T. cruzi could help to develop new drugs to treat the disease caused by these protozoa. In the present work, we have demonstrated that Fetal Calf Serum (FCS) can quickly increase the levels of both phosphorylated and unphosphorylated forms of T. cruzi DNA polymerase beta (TcPolβ) in tissue-cultured trypomastigotes. The in vitro phosphorylation sites on TcPolβ by protein kinases TcCK1, TcCK2, TcAUK1, and TcPKC1 have been identified by Mass Spectrometry (MS) analysis and with antibodies against phosphor Ser-Thr-Tyr. MS analysis indicated that these protein kinases can phosphorylate Ser and Thr residues on several sites on TcPolβ. Unexpectedly, it was found that TcCK1 and TcPKC1 can phosphorylate a different Tyr residue on TcPolβ. By using a specific anti-phosphor Tyr monoclonal antibody, it was determined that TcCK1 can be in vitro autophosphorylated on Tyr residues. In vitro and in vivo studies showed that phorbol 12-myristate 13-acetate (PMA) can activate the PKC to stimulate the TcPolβ phosphorylation and enzymatic activity in T. cruzi epimastigotes. [ABSTRACT FROM AUTHOR]

Jatropha seed cake (JSC) derived from Jatropha curcas seeds is a by-product of biodiesel production and, due to its high protein content, has been considered as a potential animal feed ingredient. However, the presence of toxic compounds such as phorbol esters and other anti-nutritional factors limits its use in animal feeding. Several detoxification approaches have been used to tackle these constraints and this review aims to summarize the recent advances in JSC treatment aiming to enhance its potential as an animal feedstuff. The review first provides an overview of the structure and composition of phorbol esters and other anti-nutritional compounds, discussing its toxic effects on different animal species. It then explores several detoxification methodologies giving special emphasis to its effects on the nutritional composition of JSC and on the use of the treated substrate as a feed ingredient in fish, poultry, pigs, and ruminants, highlighting their growth performance, nutrient utilization, and animal health issues. Overall, the review concludes that these treatments hold great potential for the detoxification and utilization of JSC as an animal feed ingredient. However, further research is needed to optimize the treatment conditions, evaluate the economic feasibility, and assess the long-term effects of treated JSC on animal health and product quality. [ABSTRACT FROM AUTHOR]

Activation of the Wnt pathway lies at the core of many human cancers. Wnt and macropinocytosis are often active in the same processes, and understanding how Wnt signaling and membrane trafficking cooperate should improve our understanding of embryonic development and cancer. Here, we show that a macropinocytosis activator, the tumor promoter phorbol 12-myristate 13-acetate (PMA), enhances Wnt signaling. Experiments using the Xenopus embryo as an in vivo model showed marked cooperation between the PMA phorbol ester and Wnt signaling, which was blocked by inhibitors of macropinocytosis, Rac1 activity, and lysosome acidification. Human colorectal cancer tissue arrays and xenografts in mice showed a correlation of cancer progression with increased macropinocytosis/multivesicular body/lysosome markers and decreased GSK3 levels. The crosstalk between canonical Wnt, focal adhesions, lysosomes, and macropinocytosis suggests possible therapeutic targets for cancer progression in Wnt-driven cancers.

To study the total particulate matter (TPM) in flue gas emitted by coking plants, a sampling system that could be used to collect filterable particulate matter (FPM) and condensable particulate matter (CPM) was designed and developed based on Method 202 recommended by the U.S. Environmental Protection Agency in 2017 and HJ 836-2017 issued by China. Using this system, FPM and CPM in flue gas emitted by four coking furnaces named A, B, C, and D were tested in China. Further, 9 water-soluble ions, 20 elements, and organic matter present in the CPM were simultaneously examined to determine their formation mechanisms. Statistical data suggested that the FPM emission level in the coking flue gas was low and the average mass concentration was less than 10 mg/m3. However, with high CPM and TPM emission levels, the TPM mass concentrations of A, B, C, and D were 130 ± 11.1, 84.4 ± 6.36, 35.1 ± 17.0, and 63.8 ± 13.0 mg/m3, respectively. The main component of TPM was CPM, and the average mass concentration of CPM accounted for 98%, 95%, 68%, and 95% of TPM in furnaces A, B, C, and D, respectively. Water-soluble ions were the important components of CPM, and the total concentration of water-soluble ions accounted for 70%, 87%, 42%, and 66% of CPM in furnaces A, B, C, and D, respectively. Toxic and harmful heavy metals, such as Mn, Cr, Ni, Cu, Zn, As, Cd, and Pb, were detected in CPM. The formation mechanism of CPM was analyzed in combination with flue-gas treatment. It was shown that the treatment process "activated carbon– flue-gas countercurrent-integrated purification technology + ammonia spraying" used in furnaces A and B was less effective in removing CPM, water-soluble ions, metals, and compounds than that of "selective catalytic reduction denitrification + limestone–gypsum wet desulfurization (spraying NaOH solution)" in furnaces C and D. Hence, different flue-gas treatment technologies and operation levels played vital roles in the formation, transformation, and removal of CPM from flue-gas. Organic components in CPM discharged from furnace A were determined via gas chromatography–mass spectrometry, and the top 15 organic components in CPM were obtained using the area normalization method. N-alkanes accounted for the highest proportion, followed by esters and phenols, and most of them were toxic and harmful to humans and ecosystems. Therefore, advanced CPM treatment technologies should be developed to reduce atmospheric PM2.5 and its precursors to improve ambient air quality in China. [ABSTRACT FROM AUTHOR]

Aim: In neuroendocrine cells, large dense‐core vesicles (LDCVs) undergo highly regulated pre‐fusion processes before releasing hormones via membrane fusion. Significant heterogeneity has been found for LDCV population based on the dynamics of membrane fusion. However, how the pre‐fusion status impacts the heterogeneity of LDCVs still remains unclear. Hence, we explored pre‐fusion determinants of heterogeneous membrane fusion procedure of LDCV subpopulations. Methods: We assessed the pre‐fusion motion of two LDCV subpopulations with distinct membrane fusion dynamics individually, using total internal reflection fluorescence microscopy. These two subpopulations were isolated by blocking Rho GTPase‐dependent actin reorganization using Clostridium difficile toxin B (ToxB), which selectively targets the fast fusion vesicle pool. Results: We found that the fast fusion subpopulation was in an active motion mode prior to release, termed "active" LDCV pool, while vesicles from the slow fusion subpopulation were also moving but in a significantly more confined status, forming an "inert" pool. The depletion of the active pool by ToxB also eliminated fast fusion vesicles and was not rescued by pre‐treatment with phorbol ester. A mild actin reorganization blocker, latrunculin A, that partially disrupted the active pool, only slightly attenuated the fast fusion subpopulation. Conclusion: The pre‐fusion motion state of LDCVs also exhibits heterogeneity and dictates the heterogeneous fusion pore dynamics. Rearrangement of F‐actin network mediates vesicle pre‐fusion motion and subsequently determines the membrane fusion kinetics. [ABSTRACT FROM AUTHOR]

Exposure to hypoxia results in the development of pulmonary arterial hypertension (PAH). An increase in the intracellular Ca2+ concentration ([Ca2+]i) in pulmonary artery smooth muscle cells (PASMCs) is a major trigger for pulmonary vasoconstriction and proliferation. This study investigated the mechanism by which KMUP-1, a xanthine derivative with phosphodiesterase inhibitory activity, inhibits hypoxia-induced canonical transient receptor potential channel 1 (TRPC1) protein overexpression and regulates [Ca2+]i through store-operated calcium channels (SOCs). Ex vivo PASMCs were cultured from Sprague-Dawley rats in a modular incubator chamber under 1% O2/5% CO2 for 24 h to elucidate TRPC1 overexpression and observe the Ca2+ release and entry. KMUP-1 (1 μM) inhibited hypoxia-induced TRPC family protein encoded for SOC overexpression, particularly TRPC1. KMUP-1 inhibition of TRPC1 protein was restored by the protein kinase G (PKG) inhibitor KT5823 (1 μM) and the protein kinase A (PKA) inhibitor KT5720 (1 μM). KMUP-1 attenuated protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA, 1 μM)-upregulated TRPC1. We suggest that the effects of KMUP-1 on TRPC1 might involve activating the cyclic guanosine monophosphate (cGMP)/PKG and cyclic adenosine monophosphate (cAMP)/PKA pathways and inhibiting the PKC pathway. We also used Fura 2-acetoxymethyl ester (Fura 2-AM, 5 μM) to measure the stored calcium release from the sarcoplasmic reticulum (SR) and calcium entry through SOCs in hypoxic PASMCs under treatment with thapsigargin (1 μM) and nifedipine (5 μM). In hypoxic conditions, store-operated calcium entry (SOCE) activity was enhanced in PASMCs, and KMUP-1 diminished this activity. In conclusion, KMUP-1 inhibited the expression of TRPC1 protein and the activity of SOC-mediated Ca2+ entry upon SR Ca2+ depletion in hypoxic PASMCs. [ABSTRACT FROM AUTHOR]

Background: Currently five types of phorbol esters are reported in the root of Balliospermum montanum Muell. Here we observed the presence of 12-myristate 13-acetate (C36 H56O8) in the root, an additional phorbol ester which restricted its use in food or medicine due to its carcinogenic property. No specific method is reported that can remove or disintegrate the phorbol esters in Balliospermum montanum Muell. root. Aim: Identification of 12 myristate13 acetate phorbol ester (C36 H56O8) in B. montanum Muell. root and its purification and degradation by traditional method recommended by Acharya Charak. Materials and Methods: The traditional 'putpak' (similar to traditional oven method) method was employed to detoxify the raw sample of B. montanum root. Ethanolic extract of root of B. montanum. unprocessed and processed samples were analysed on the Camag HPTLC system. Results: The HPTLC analysis gave a band at Rf 0.49 corresponding to the standard 12 myristate 13acetate (C36H56O8). Further the band corresponding to the identified phorbol ester (biomarker standard, 12 myristate 13acetate, Sigma Aldrich, USA) was scraped out, the phorbol ester was separated by vortexing with methanol. The resulting sample was then subjected to LC-MS analysis using Impact HD system employing Electrospary Ionization (ESI) in positive ion mode to identify the isolated phorbol ester. Conclusion: The LC-MS analysis indicated the presence of this phorbol ester. The present 'putpak (similar to traditional oven method)' method of detoxification was able to reduce the identified new phorbol ester by 75% when compared to the raw/unprocessed sample. [ABSTRACT FROM AUTHOR]

Aims: Phorbol esters (PE) are toxic diterpenoids accumulated in physic nut (Jatropha curcas L.) seed tissues. Their biosynthetic pathway remains unknown, and the participation of roots in this process may be possible. Thus, we set out to study the deposition pattern of PE and other terpenoids in roots and leaves of genotypes with detected (DPE) and not detected (NPE) phorbol esters based on previous studies. Outline of data resources: We analyzed physic nut leaf and root organic extracts using LC-HRMS. By an untargeted metabolomics approach, it was possible to annotate 496 and 146 metabolites in the positive and negative electrospray ionization modes, respectively. Key results: PE were detected only in samples of the DPE genotype. Remarkably, PE were found in both leaves and roots, making this study the first report of PE in J. curcas roots. Furthermore, untargeted metabolomic analysis revealed that diterpenoids and apocarotenoids are preferentially accumulated in the DPE genotype in comparison with NPE, which may be linked to the divergence between the genotypes concerning PE biosynthesis, since sesquiterpenoids showed greater abundance in the NPE. Utility of the resource: The LC-HRMS files, publicly available in the MassIVE database (identifier MSV000092920), are valuable as they expand our understanding of PE biosynthesis, which can assist in the development of molecular strategies to reduce PE levels in toxic genotypes, making possible the food use of the seedcake, as well as its potential to contain high-quality spectral information about several other metabolites that may possess biological activity. [ABSTRACT FROM AUTHOR]

Skin exposure to noxious agents leads to cutaneous lesion marked by an increase in inflammation, cellular proliferation, and hyperplasiogenic reactions. Studies have demonstrated that these damages breach the skin integrity resulting in the aetiology of various cutaneous disorders like atopic dermatitis, eczema, psoriasis, and development of non-melanoma skin cancer. Celecoxib, a cyclooxygenase-2 (COX-2) inhibitor, is an effective treatment for a variety of inflammatory diseases. Its importance in the therapy of skin problems, however, remains under appreciated. We tested efficacy of topically applied celecoxib in mitigating skin inflammation, cellular proliferation, and hyperplasia induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) in Swiss albino mice. Celecoxib (5 and 10 μmol) markedly reduced TPA (10 nmol) induced prostaglandin E2 (PGE2) production, oedema formation, myeloperoxidase (MPO) activity, and levels of pro-inflammatory cytokines such as tumour necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6). It also resulted in a considerable decrease in ornithine decarboxylase (ODC) activity and the incorporation of [3H]-thymidine into DNA. In addition, there was a significant reduction in histoarchitectural abnormalities such as epidermal thickness, number of epidermal cell layers, neutrophil infiltration, intercellular oedema, and vasodilation. Our results demonstrate that topical celecoxib can reduce the inflammation, hyperproliferation, and hyperplasiogenic events of skin insults suggesting that it may prove to be a valuable management option for cutaneous lesion and associated illnesses such as atopic dermatitis, eczema, and psoriasis, as well as the emergence of non-melanoma cancer. [ABSTRACT FROM AUTHOR]

Staudinger reaction on the solid phase between an electronodeficit organic azide, such as sulfonyl azide, and the phosphite triester formed upon phosphoramidite coupling is a convenient method for the chemical modification of oligonucleotides at the internucleotidic phosphate position. In this work, 4-carboxybenzenesulfonyl azide, either with a free carboxy group or in the form of an activated ester such as pentafluorophenyl, 4-nitrophenyl, or pentafluorobenzyl, was used to introduce a carboxylic acid function to the terminal or internal internucleotidic phosphate of an oligonucleotide via the Staudinger reaction. A subsequent treatment with excess primary alkyl amine followed by the usual work-up, after prior activation with a suitable peptide coupling agent such as a uronium salt/1-hydroxybenzotriazole in the case of a free carboxyl, afforded amide-linked oligonucleotide conjugates in good yields including multiple conjugations of up to the exhaustive modification at each phosphate position for a weakly activated pentafluorobenzyl ester, whereas more strongly activated and, thus, more reactive aryl esters provided only single conjugations at the 5′-end. The conjugates synthesized include those with di- and polyamines that introduce a positively charged side chain to potentially assist the intracellular delivery of the oligonucleotide. [ABSTRACT FROM AUTHOR]

An innovative amphiphilic alternating copolymer with benzoxaborole ester‐linkages (P(NODGA‐alt‐PBA)) is synthesized in one step through an amination reduction reaction involving N‐octyl‐d‐glucamine and 2‐formylphenylboronic acid. Under CO2, lactic acid (LA) and H2O2 stimulations, nanoaggregates self‐assembled by P(NODGA‐alt‐PBA) can undergo cleavage via the breaking of benzoxaborole ester bonds, which is confirmed by analysis of UV–vis absorption spectra, dynamic light scattering, and transmission electron microscopy. As triggered by these stimuli, doxorubicin‐loaded nanocarriers (DOX‐PNs) are disrupted and quickly release drug, afterward only leaving some small molecular residues with low toxicity that can be absorbed and utilized by biological tissues. In vitro results indicate that P(NODGA‐alt‐PBA) is non‐toxic but DOX‐PN exhibits a markly enhanced therapeutic effect of DOX, compared to free DOX, ascribing to the tertiary amino protonated. This approach provides a promising candidate for biocompatible, biodegradable, and safe drug carriers for cancer therapy. [ABSTRACT FROM AUTHOR]

A sustainable system for the carbocarboxylation of olefins with redox-active esters and CO2via visible-light-induced photoredox catalysis has been developed using metal-free boron carbonitride (BCN) as the photocatalyst. The scheme introduces versatile alkyl groups (including primary, secondary, and tertiary) and CO2 into the C–C double bond in a fully regioselective manner to produce valuable carboxylic derivatives with excellent yields. [ABSTRACT FROM AUTHOR]

A sustainable system for the carbocarboxylation of olefins with redox-active esters and CO2via visible-light-induced photoredox catalysis has been developed using metal-free boron carbonitride (BCN) as the photocatalyst. The scheme introduces versatile alkyl groups (including primary, secondary, and tertiary) and CO2 into the C–C double bond in a fully regioselective manner to produce valuable carboxylic derivatives with excellent yields. [ABSTRACT FROM AUTHOR]

In this study, 37 derivatives of phorbol esters were synthesized and their anti-HIV-1 activities evaluated, building upon our previous synthesis of 51 phorbol derivatives. 12-Para-electron-acceptor- trans -cinnamoyl-13-decanoyl phorbol derivatives stood out, demonstrating remarkable anti-HIV-1 activities and inhibitory effects on syncytia formation. These derivatives exhibited a higher safety index compared with the positive control drug. Among them, 12-(trans -4-fluorocinnamoyl)-13-decanoyl phorbol, designated as compound 3c , exhibited the most potent anti-HIV-1 activity (EC 50 2.9 nmol·L−1, CC 50 /EC 50 11 117.24) and significantly inhibited the formation of syncytium (EC 50 7.0 nmol·L−1, CC 50 /EC 50 4891.43). Moreover, compound 3c is hypothesized to act both as an HIV-1 entry inhibitor and as an HIV-1 reverse transcriptase inhibitor. Isothermal titration calorimetry and molecular docking studies indicated that compound 3c may also function as a natural activator of protein kinase C (PKC). Therefore, compound 3c emerges as a potential candidate for developing new anti-HIV drugs. [ABSTRACT FROM AUTHOR]

Saengmaeksan (SMS), a representative oriental medicine that contains Panax ginseng Meyer, Liriope muscari, and Schisandra chinensis (1:2:1), is used to improve body vitality and enhance physical activity. However, there is limited scientific evidence to validate the benefits of SMS. Here, we investigated the in vitro and in vivo regulatory effects of SMS and its constituents on energy metabolism and the underlying molecular mechanisms. For this, quantitative real-time polymerase chain reaction, 3D holotomographic microscopy, western blotting, and glucose uptake experiments using 18F-fluoro-2-deoxy-D-glucose (18F-FDG) were performed using L6 cells to investigate in vitro energy metabolism changes. In addition, 18F-fluorocholine (18F-FCH) and 18F-FDG positron emission tomography/computed tomography (PET/CT) analyses, immunohistochemistry, and respiratory gas analysis were performed in mice post-endurance exercise on a treadmill. In the energy metabolism of L6 cells, a significant reversal in glucose uptake was observed in the SMS-treated group, as opposed to an increase in uptake over time compared to the untreated control group. Furthermore, P. ginseng alone and SMS significantly decreased the volume of lipid droplets. SMS also regulated the phosphorylation of extracellular signal-regulated kinase (ERK), phosphorylation of p38, mitochondrial morphology, and the expression of apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE/Ref-1) in H2O2-stimulated L6 cells. In addition, SMS treatment was found to regulate whole body and muscle energy metabolism in rats subjected to high-intensity exercise, as well as glucose and lipid metabolism in skeletal muscle. Therefore, SMS containing P. ginseng ameliorated imbalanced energy metabolism through oxidative stress-induced APE/Ref-1 expression. SMS may be a promising supplemental option for metabolic performance. [ABSTRACT FROM AUTHOR]

For power transformer applications, this study explores an alternative insulating liquid. With this aim, edible natural esters such as refined Olea europaea (olive oil), rice bran oil, soya bean oil, sunflower oil, and corn oil are investigated as suitable replacements for the mineral oil (MO) used in the transformer. In addition, olive oil and other natural esters are incorporated into the blend for further analysis to obtain a better insulating medium. Blended natural esters were also tested for performance enrichment by antioxidant inclusion. Butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) were chosen as antioxidants for this study. In this study, we aimed to investigate the role of key input factors [A-speed, B-time, and C-temperature] on the output response [Y-breakdown voltage]. It was determined that the optimal conditions for [Y] are [A-699.91 rpm, B-49.95 min, and C-88.75 °C]. In order to ensure the desirable properties, the natural esters were subjected to certain experimentations such as breakdown voltage (BDV), viscosity, fire point (FeP), and flash point (FhP). From the results, it is observed that the natural esters and blended natural esters can be used in the transformer as an alternate insulating medium and that the antioxidants have a significant effect on the properties of natural ester combinations. [ABSTRACT FROM AUTHOR]

Particularly expressed in the kidney, αKlotho is a transmembrane protein that acts together with bone hormone fibroblast growth factor 23 (FGF23) to regulate renal phosphate and vitamin D homeostasis. Soluble Klotho (sKL) is released from the transmembrane form and controls various cellular functions as a paracrine and endocrine factor. αKlotho deficiency accelerates aging, whereas its overexpression favors longevity. Higher αKlotho abundance confers a better prognosis in cardiovascular and renal disease owing to anti-inflammatory, antifibrotic, or antioxidant effects and tumor suppression. Serine/threonine protein kinase C (PKC) is ubiquitously expressed, affects several cellular responses, and is also implicated in heart or kidney disease as well as cancer. We explored whether PKC is a regulator of αKlotho. Experiments were performed in renal MDCK or NRK-52E cells and PKC isoform and αKlotho expression determined by qRT-PCR and Western Blotting. In both cell lines, PKC activation with phorbol ester phorbol-12-myristate-13-acetate (PMA) downregulated, while PKC inhibitor staurosporine enhanced αKlotho mRNA abundance. Further experiments with PKC inhibitor Gö6976 and RNA interference suggested that PKCγ is the major isoform for the regulation of αKlotho gene expression in the two cell lines. In conclusion, PKC is a negative regulator of αKlotho gene expression, an effect which may be relevant for the unfavorable effect of PKC on heart or kidney disease and tumorigenesis. [ABSTRACT FROM AUTHOR]

In this work, the effect on a defatted meal of Jatropha platyphylla subjected to hydrothermal treatment with solid state fermentation was evaluated in comparison with another batch of meal that was only fermented, using Saccharomyces cerevisiae. Yeast (6 × 109 CFU/g) with 64 and 60% humidity in the meals were used and incubated for 24, 72, and 144 h at 36 °C. The content of antinutritional compounds was evaluated by spectrophotometric methods, phorbol esters by High-Resolution Thin Layer Chromatography, and the nutritional composition by proximate analysis. A bifactorial design (fermentation time and type of treatment) was followed for the response variables of phytates, saponins, tannins, phorbol esters, and nutritional composition. The meal with the desired characteristics was the one that was fermented for 144 h and did not have a hydrothermal treatment because it showed a significant reduction of 53% of phytates, 80% of saponins, the tannin concentration is low of 21.57 mg cat/100 g of meal; in addition, phorbol esters were not detected. The protein content in the meal remains unchanged at 50.93%, and the ash content is still at 9.63%. However, there is a noticeable decrease in fat content from 13.41 to 9.1% and crude fiber from 9.63 to 4.9%. Based on these findings, it is recommended to include meal (treated by fermentation for 144 h) in balanced diets for Nile tilapia due to its favorable chemical composition, low fiber, antinutrient content, and lack of harmful substances. [ABSTRACT FROM AUTHOR]

NETosis, i.e., the formation of neutrophil extracellular traps (NET), and neutrophil autophagy are important elements in the pathogenesis and the development of complications of type 2 diabetes mellitus (T2DM). Therefore, the search of drugs that can regulate the level of NETosis and autophagy in T2DM is relevant. Here we studied an ex vivo NET formation and neutrophil death in whole blood from healthy subjects upon the addition of glucose up to a high concentration of 15 mM or/and the phorbol ester PMA (phorbol-12-myristate-13-acetate). Their individual and combined action caused neutrophil death and an increase in NET content. It can be hypothesized that this resulted from activation of NETosis and autophagy. It was also shown that this activation of NETosis and autophagy is completely prevented by daily intake of 1000 IU vitamin D3 for 14 days. Therefore, vitamin D3 supplementation can be considered as a preventive measure against the development of T2DM complications. [ABSTRACT FROM AUTHOR]

GPRC5A is the first member of a new class of orphan receptors coupled to G proteins, which also includes GPRC5B, GPRC5C, and GPRC5D. Since its cloning and identification in the 1990s, substantial progress has been made in understanding the possible functions of this receptor. GPRC5A has been implicated in a variety of cellular events, such as cytoskeleton reorganization, cell proliferation, cell cycle regulation, migration, and survival. It appears to be a central player in different pathological processes, including tumorigenesis, inflammation, immune response, and tissue damage. The levels of GPRC5A expression differ depending on the type of cancer, with increased expression in colon, pancreas, and prostate cancers; decreased expression in lung cancer; and varied results in breast cancer. In this review, we discuss the early discovery of GPRC5A as a phorbol ester-induced gene and later as a retinoic acid-induced gene, its regulation, and its participation in important canonical pathways related to numerous types of tumors and inflammatory processes. GPRC5A represents a potential new target for cancer, inflammation, and immunity therapies. [ABSTRACT FROM AUTHOR]

Schistosomiasis is a vector-borne water-based disease caused by Schistosoma blood flukes. It mostly affects people in low-income regions, 90% of reported cases being in developing countries. Schistosoma has a complex lifecycle, alternately infecting mammalian hosts and snails. The snails hosting the parasite are the most viable targets. Selective preparations for reducing the parasite pool in snails and infected water are required as current molluscicides are also nontoxic to other organisms, including fish, and thus affect food supplies in infected areas. Plants (e.g. Annona crassiflora Mart., A. muricata L., and A. montana Macfad.) are attractive potential sources as alternative molluscicides and novel entity to treat the disease owned to their diverse biologically potent compounds including; saponins, alkaloids, terpenoids, and tannins. Additionally, they can be locally cultivated, providing income for farmers and reducing treatment costs. Here, we review plants, plant extracts and isolated compounds that have shown activities against the host snails or Schistosoma in various parts of its life cycle. Plants have a lot of potential and will continue to contribute feasible, effective medicines and/or pesticides; more research is warranted to fully explore their future applications. [ABSTRACT FROM AUTHOR]

1,3‐Bis(boronic) esters can be readily synthesized from alkylBpin precursors. Selective transformations of these compounds hold the potential for late‐stage functionalization of the remaining C−B bond, leading to a diverse array of molecules. Currently, there are no strategies available to address the reactivity and, more importantly, the controllable regiodivergent functionalization of 1,3‐bis(boronic) esters. In this study, we have achieved controllable regiodivergent alkynylation of these molecules. The regioselectivity has been clarified based on the unique chelation patterns observed with different organometallic reagents. Remarkably, this methodology effectively addresses the low reactivity of 1,3‐bis(boronic) esters and bridges the gap in radical chemistry, which typically yields only the classical products formed via stable radical intermediates. Furthermore, the compounds synthesized through this approach serve as potent building blocks for creating molecular diversity. [ABSTRACT FROM AUTHOR]

Post-tetanic potentiation (PTP) is a form of short-term plasticity that lasts for tens of seconds following a burst of presynaptic activity. It has been proposed that PTP arises from protein kinase C (PKC) phosphorylation of Munc18-1, an SM (Sec1/Munc-18 like) family protein that is essential for release. To test this model, we made a knock-in mouse in which all Munc18-1 PKC phosphorylation sites were eliminated through serine-to-alanine point mutations (Munc18-1SA mice), and we studied mice of either sex. The expression of Munc18-1 was not altered in Munc18-1SA mice, and there were no obvious behavioral phenotypes. At the hippocampal CA3-to-CA1 synapse and the granule cell parallel fiber (PF)-to-Purkinje cell (PC) synapse, basal transmission was largely normal except for small decreases in paired-pulse facilitation that are consistent with a slight elevation in release probability. Phorbol esters that mimic the activation of PKC by diacylglycerol still increased synaptic transmission in Munc18-1SA mice. In Munc18-1SA mice, 70% of PTP remained at CA3-to-CA1 synapses, and the amplitude of PTP was not reduced at PF-to-PC synapses. These findings indicate that at both CA3-to-CA1 and PF-to-PC synapses, phorbol esters and PTP enhance synaptic transmission primarily by mechanisms that are independent of PKC phosphorylation of Munc18-1.SIGNIFICANCE STATEMENT A leading mechanism for a prevalent form of short-term plasticity, post-tetanic potentiation (PTP), involves protein kinase C (PKC) phosphorylation of Munc18-1. This study tests this mechanism by creating a knock-in mouse in which Munc18-1 is replaced by a mutated form of Munc18-1 that cannot be phosphorylated. The main finding is that most PTP at hippocampal CA3-to-CA1 synapses or at cerebellar granule cell-to-Purkinje cell synapses does not rely on PKC phosphorylation of Munc18-1. Thus, mechanisms independent of PKC phosphorylation of Munc18-1 are important mediators of PTP.

We report the development of a C(sp3)−C(sp2) coupling reaction using styrene boronic acids and redox‐active esters under photoredox catalysis. The reaction proceeds through an unusual polarity‐mismatched radical addition mechanism that is orthogonal to established processes. Synergistic activation of the radical precursor and organoboron are critical mechanistic events. Activation of an N‐hydroxyphthalimide (NHPI) ester by coordination to boron enables electron transfer, with decomposition leading to a nucleofuge rebound, activating the organoboron to radical addition. The unique mechanism enables chemoselective coupling of styrene boronic acids in the presence of other alkene radical acceptors. The scope and limitations of the reaction, and a detailed mechanistic investigation are presented. [ABSTRACT FROM AUTHOR]

Naturally occurring protein kinase C (PKC) activators such as phorbol esters, teleocidins, and aplysiatoxins, have the potential to become anti-cancer agents, since they are anti-proliferative against specific cancer cell lines in vitro. However, their potent tumor-promoting and proinflammatory activities have hampered their clinical uses. Recently, we developed 10-methyl-aplog-1 (1), a simplified analog of tumor-promoting debromoaplysiatoxin (DAT), which retained anti-proliferative activity comparable to DAT, but induced neither tumorigenesis nor inflammation on mouse skin. Our previous study suggested that PKCα and δ were involved in the cell line-selective anti-proliferative activity of 1 , but the downstream signaling of PKC isozymes remained unknown. In this study, we confirmed that 1 inhibited the growth of three aplog-sensitive cancer cell lines (NCI–H460, HCC-2998, and HBC-4) without severe side effects in mice xenograft models. In addition, in vitro analysis using A549, one of the aplog-sensitive cell lines in vitro , revealed that PKCα induced PP2A-mediated attenuation of the Akt/S6 signaling axis. Since S6 inhibition in A549 was reported to result in G1 arrest, this pathway could be involved in the PKCα-dependent anti-proliferative activity of 1. • 10-Methyl-aplog-1 showed anti-cancer activity in vivo against NCI–H460, HCC2988, and HBC-4 cancer cell lines. • 10-Methyl-aplog-1 induced cell cycle arrest in G1 phase via PKCα activation in A549 cells. • PKCα induced PP2A-mediated attenuation of Akt/S6 signaling axis in A549 cells. • The crosstalk between PKC and Akt could be a factor that determines sensitivity to PKC ligands. [ABSTRACT FROM AUTHOR]

Physic nut Jatropha curcas cake/meal obtained after oil extraction has a high protein content, however, the presence of antinutrients (trypsin inhibitor, lectin and phytate) and toxic compounds (phorbol esters) limit their use as an alternative feedstuff. Thus, the detoxification process in cake/meal is necessary to allow their inclusion in fish diets. The present study aimed to evaluate the effects of solvent and extrusion-treated jatropha cake (SETJC) in Nile tilapia (Oreochromis niloticus) diets on growth, body composition, nutrient utilization, metabolic and hematological responses, and digestibility of experimental diets. Five experimental diets were formulated to be isonitrogenous (28.50% digestible protein) and isoenergetic (13.39 MJ/kg digestible energy) with graded levels of SETJC (0, 3, 6, 9, and 12%). The experimental design was completely randomized with five treatments and four replicates. The detoxification treatments reduced the phorbol esters (PE) of jatropha cake by 96% (0.58 mg/g of PE before and 0.023 mg/g of PE after treatments). Increased levels of SETJC depressed growth, feed efficiency, and protein digestibility. A similar trend was observed for hematological and biochemistry parameters. Aspartate and alanine aminotransferase, as well as phosphorus and magnesium concentrations in the fillets, increased at the highest levels of SETJC. Thus, the data of the present study suggests that the residual content, different structural forms of phorbol ester and its biological activity, as well as some antinutritional factors, can influence negatively the growth, metabolism and digestibility of experimental diets for Nile tilapia. [ABSTRACT FROM AUTHOR]

A family of seven NADPH oxidase enzymes (Nox1-5, Duox1-2) has been implicated in a variety of diseases, including inflammatory lung diseases, neurodegenerative diseases, cardiovascular diseases, and cancer. Here, we report the results of our studies aimed at developing novel brain-permeable Nox2 inhibitors with potential application as neuroprotective agents. Using cell-based assays, we identified a novel Nox2 inhibitor, TG15-132, that prevents PMA-stimulated oxygen consumption and reactive oxygen species (superoxide radical anion and hydrogen peroxide) formation upon acute treatment in differentiated HL60 cells. Long-term treatment with TG15-132 attenuates the induction of genes encoding Nox2 subunits, several inflammatory cytokines, and iNOS in differentiated THP-1 cells. Moreover, TG15-132 shows a relatively long plasma half-life (5.6 h) and excellent brain permeability, with a brain-to-plasma ratio (>5-fold) in rodent models. Additionally, TG15-132 does not cause any toxic effects on vital organs or blood biomarkers of toxicity in mice upon chronic dosing for seven days. We propose that TG15-132 may be used as a Nox2 inhibitor and a potential neuroprotective agent, with possible further structural modifications to increase its potency. [ABSTRACT FROM AUTHOR]

The presence of phthalic acid esters in wines presents a major risk to human health due to their very toxic metabolism. In this paper, aluminosilicate materials were used, with the aim of retaining various pollutants and unwanted compounds in wine. The pollutants tested were di-butyl and di-ethyl hexyl phthalates. They were tested and detected using the gas chromatography–mass spectrometry (CG-MS) analytical technique. Nanomaterials were prepared using sodium bentonite, and were chemically modified via impregnation using three types of Boltron dendrimers of second, third and fourth generations (NBtH20, NBtH30 and NBtH40). The synthesized nanomaterials were characterized using the Brunauer–Emmett–Teller (BET) method, Fourier-transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) analysis. In this paper, two aspects were addressed: the first related to the retention of phthalate-type pollutants (phthalic acid esters—PAEs) and the second related to the protein and polyphenol levels in the white wine of the Aligoté grape variety. The results obtained in this study have a major impact on PAEs in wine, especially after treatment with NBtH30 and NBtH40 (volumes of 250–500 μL/10 mL wine), with the retention of the pollutants being up to 85%. [ABSTRACT FROM AUTHOR]

Organic–inorganic metal halides (OIMHs) with high‐efficiency solar spectrum‐like emission are attracting broad and current interest. Here, five 0D Zn‐based hybrid halides are synthesized based on aromatic organic cations with different carbon‐chain lengths: C6H5CH2NH3+ (PMA+) and C6H5(CH2)4NH3+ (PBA+). (PMA)2ZnCl4 exhibits the highest photoluminescence quantum yield of 37.2% of reported Zn‐based white‐emission OIMHs. The emission spectrum of (PBA)2ZnI4 indicates a color rendering index of 98, which is the highest among single‐component white‐light‐emitting phosphors. Spectral characterizations and density functional theory calculations demonstrate that the extremely broad emission of (PBA)2ZnI4 originates from the synergistic emission of organic cations and self‐trapped excitons. The optical properties of the obtained (PMA)2ZnBr4, (PMA)2ZnI4·H2O, and (PBA)2ZnCl4 are also characterized for comparison, and with the same organic cations, the PLQY decreases from chloride to bromide to iodide. This work demonstrates that the selection of appropriate organics and halogens can enable fine tuning of single‐component white‐light emission, satisfying varying needs for solid‐state lighting. [ABSTRACT FROM AUTHOR]

The present study tested the hypothesis that protein kinase C-a (PKC-a) recruitment in the presence of the p38a/b MAPK inhibitor SB203580 facilitated the appearance and cell cycle re-entry of nestin(-)- neonatal rat ventricular cardiomyocytes (NNVMs) and induced a transcript profile delineating a proliferative phenotype. Phorbol 12,13-dibutyrate (PDBu) treatment did not induce de novo nestin expression or increase the cell cycle re-entry of 1-day-old NNVMs but significantly increased runt-related transcription factor 1 (Runx1) and p16 cell cycle inhibitor (CDKN2a) mRNA levels and downregulated epithelial cell transforming 2 (ECT2) mRNA expression. SB203580 administration to PDBu-treated NNVMs induced de novo nestin expression, preferentially increased the density (normalized to 500 NNVMs) of nestin(-)- NNVMs that incorporated 5-bromo-20- deoxyuridine (PDBu, 1.4 ± 3 vs. PDBu/SB203580, 128 ± 34; n = 5 independent litters), significantly inhibited CDKN2a and Runx1 mRNA upregulation and reversed ECT2 mRNA downregulation. PDBu treatment of NNVMs reduced PKC-a, protein kinase-d (PKC-d) and protein kinase-ɛ (PKC-ɛ) protein levels and GF109203X (conventional PKC isoform inhibitor) selectively attenuated PKC-a protein downregulation. GF109203X administration to PDBu/SB203580-treated NNVMs significantly reduced the density of nestin(-)- NNVMs that incorporated 5-bromo-20- deoxyuridine (PDBu/SB203580/GF109203X, 40 ± 46; n = 5). Moreover, GF109203X/PDBu/SB203580 treatment unmasked the predominant appearance of a separate NNVM population that incorporated 5-bromo-20- deoxyuridine (PDBu/ SB203580/GF109203X, 192 ± 42; n = 5) delineated by the absence of de novo nestin expression. Sotrastaurin (conventional/ novel PKC isoform inhibitor) administration to PDBu/SB203580-treated NNVMs significantly attenuated the density of nestin(-)- NNVMs (PDBu/SB203580/sotrastaurin, 8 ± 10; n = 4) and nestin()- NNVMs (PDBu/SB203580/sotrastaurin, 64 ± 30; n = 4) that incorporated 5-bromo-20- deoxyuridine. These data reveal that the neonatal rat heart contains at least two separate populations of NNVMs that re-enter the cell cycle and the preferential appearance of nestin(-)- or nestin()- NNVMs is driven by distinct PKC isoforms in the presence of SB203580. NEW & NOTEWORTHY The appearance of nestin(-)- neonatal rat ventricular cardiomyocytes that re-entered the cell cycle following phorbol ester stimulation in the presence of p38a/b MAPK inhibitor SB203580 was associated with the inhibition of Runx1 and CDKN2a mRNA upregulation. PKC-a selectively induced the cell cycle re-entry of nestin(-)- neonatal rat ventricular cardiomyocytes. Pharmacological inhibition of PKC-a with concomitant p38a/b MAPK suppression unmasked the cell cycle reentry of a second population of neonatal rat ventricular cardiomyocytes in the absence of nestin expression. [ABSTRACT FROM AUTHOR]

Transcription of the human papillomavirus (HPV) oncogenes, E6 and E7, is regulated by the long control region (LCR) of the viral genome. Although various transcription factors have been reported to bind to the LCR, little is known about the transcriptional cofactors that modulate HPV oncogene expression in association with these transcription factors. Here, we performed in vitro DNA-pulldown purification of nuclear proteins in cervical cancer cells, followed by proteomic analyses to identify transcriptional cofactors that bind to the HPV16 LCR via the transcription factor TEAD1. We detected the proinflammatory cytokine S100A9 that localized to the nucleus of cervical cancer cells and associated with the LCR via direct interaction with TEAD1. Nuclear S100A9 levels and its association with the LCR were increased in cervical cancer cells by treatment with a proinflammatory phorbol ester. Knockdown of S100A9 decreased HPV oncogene expression and reduced the growth of cervical cancer cells and their susceptibility to cisplatin, whereas forced nuclear expression of S100A9 using nuclear localization signals exerted opposite effects. Thus, we conclude that nuclear S100A9 binds to the HPV LCR via TEAD1 and enhances viral oncogene expression by acting as a transcriptional coactivator. [ABSTRACT FROM AUTHOR]

Jatropha curcas is an oilseed crop with biorefinery applications. Whilst cake generated following oil extraction offers potential as a protein source for animal feed, inactivation of toxic phorbol esters present in the material is necessary. Pleurotus pulmonarius is a detoxifying agent for jatropha cake with additional potential as animal feed, edible mushroom and for enzyme production. For the characterization of fungal genes involved in phorbol ester degradation, together with other industrial applications, reverse transcription-quantitative PCR (RT-qPCR) is a tool that enables accurate quantification of gene expression. For this, reliable analysis requires reference genes for normalization of mRNA levels validated under conditions employed for target genes. The stability of potential reference genes β-TUB, ACTIN, GAPDH, PHOS, EF1α, TRPHO, LAC, MNP3, MYP and VP were evaluated following growth of P. pulmonarius on toxic, non-toxic jatropha cake and a combined treatment, respectively. NormFinder and geNorm algorithms for expression stability analysis identified PHOS, EF1α and MNP3 as appropriate for normalizing gene expression. Reference gene combinations contrasting in ranking were compared following normalization of relative expression of the CHU_2040 gene, encoding an esterase enzyme potentially involved in phorbol ester degradation. The reference genes for P. pulmonarius will facilitate the elucidation of mechanisms involved in detoxification of phorbol esters as well as analysis of target genes for application in biorefinery models. [ABSTRACT FROM AUTHOR]

Ormeloxifene (ORM) (3,4‐trans‐2,2‐dimethyl‐3‐phenyl‐4‐p‐(β‐pyrrolidinoethoxy) phenyl‐7‐methoxychroman), world's first nonsteroidal selective estrogen receptor modulator approved for contraception in India has been shown to have potential anticancer activities. Here, we show that ORM can induce megakaryocyte and myeloid (granulocytic) but not erythroid differentiation in multipotent human myeloid leukemia cell line K562. We show that ORM at an IC50 of 7.5 µM can induce morphological changes similar to megakaryocytes in K562 cells. ORM led to increase in levels of megakaryocytic differentiation markers (CD41 and CD61) as well as key transcription factors GATA1 and AML1. We further show that ORM induces megakaryocytic differentiation in K562 cells through ERK activation and induction of autophagy in a fashion similar to other known inducers of megakaryocytic differentiation such as phorbol esters. In addition, as shown earlier, we yet again observed that ORM led to activation of caspases since their inhibition through pan‐caspase inhibitor mitigated megakaryocytic differentiation as they led to significant decrease in CD41 and CD61. Because induction of megakaryocytic differentiation in K562 involves growth arrest and exit from cell cycle, we also observed an increase in levels of p21 and p27 with decrease in c‐Myc protein levels in K562 cells treated with 7.5 µM ORM for 24 and 48 h, respectively. Taken together, these findings indicate that ORM can markedly induce megakaryocytic differentiation in K562 cells. [ABSTRACT FROM AUTHOR]

A widely used approach for protein conjugation is through the lysine residues reacting with NHS- or other active esters. However, it is a challenge to precisely control the degree of labeling (DoL) due to the instability of active ester and variability of reaction efficiencies. Here, we provide a protocol for better control of aDoL using existing Copper-free Click Chemistry reagents. It is a two-step reaction with one purification in between. Briefly, proteins of interest were first activated with azide-NHS. After removing unreacted azide-NHS, the protein-N3 is then reacted with a limited amount of complementary click tag. Our studies have shown the click tag will fully react with the protein-N3 after 24 h' incubation, and therefore does not require additional purification steps. As such, the aDoL is equal to the input molar ratio of the click tag and the protein. Furthermore, this approach offers a much simpler and more economical way to perform parallel microscale labeling. Once a protein is pre-activated with N3-NHS, any fluorophore or molecule with the complementary click tag can be attached to the protein by mixing the two ingredients. Quantities of the protein used in the click reaction can be at any desired amount. In one example, we labeled an antibody in parallel with 9 different fluorophores using a total of 0.5 mg of antibody. In another example, we labeled Ab with targeted aDoL value from 2 to 8. In a stability comparison study, we have found the conjugated fluorophore using the suggested click protocol stayed attached to the protein longer than with standard NHS-fluorophore labeling. [ABSTRACT FROM AUTHOR]

CD248 (endosialin) belongs to a glycoprotein family that also includes thrombomodulin (CD141), CLEC14A, and CD93 (AA4) stem cell markers. We analyzed the regulated expression of CD248 in vitro using skin (HFFF) and synovial (FLS) mesenchymal stem cell lines, and in fluid and tissue samples of rheumatoid arthritis (RA) and osteoarthritis (OA) patients. Cells were incubated with either rhVEGF165, bFGF, TGF-β1, IL1-β, TNF-α, TGFβ1, IFN-γ, or PMA (Phorbol ester). There was no statistically significant change in membrane expression. A soluble (s) form of cleaved CD248 (sCD248) was detected after cell treatment with IL1-β and PMA. Matrix metalloprotease (MMP) MMP-1 and MMP-3 mRNAs were significantly up-regulated by IL1-β and PMA. A broad MMP inhibitor blocked the release of soluble CD248. In RA synovial tissue, we identified CD90+ perivascular MSCs double-stained for CD248 and VEGF. High sCD248 levels were detected in synovial fluid from RA. In culture, subpopulations of CD90+ CD14− RA MSCs were either identified as CD248+ or CD141+ cells but CD93−. CD248 is abundantly expressed by inflammatory MSCs and shed in an MMP-dependent manner in response to cytokines and pro-angiogenic growth factors. Both membrane-bound and soluble CD248 (acting as a decoy receptor) may contribute to RA pathogenesis. [ABSTRACT FROM AUTHOR]

Introduction: Dynamic physiological changes in brain extracellular calcium ([Ca2+]o) occur when high levels of neuronal activity lead to substantial Ca2+ entry via ion channels reducing local [Ca2+]o. Perturbations of the extracellular microenvironment that increase [Ca2+]o are commonly used to study how [Ca2+] regulates neuronal activity. At excitatory synapses, the Ca2+-sensing receptor (CaSR) and other G-protein coupled receptors link [Ca2+]o and spontaneous glutamate release. Phospholipase C (PLC) is activated by G-proteins and is hypothesized to mediate this process. Methods: Patch-clamping cultured neocortical neurons, we tested how spontaneous glutamate release was affected by [Ca2+]o and inhibition of PLC activity. We used hypertonic sucrose (HS) to evaluate the readily releasable pool (RRP) and test if it was affected by inhibition of PLC activity. Results: Spontaneous glutamate release substantially increased with [Ca2+]o, and inhibition of PLC activity, with U73122, abolished this effect. PLC-β1 is an abundant isoform in the neocortex, however, [Ca2+]o-dependent spontaneous release was unchanged in PLC-β1 null mutants (PLC-β1-/-). U73122 completely suppressed this response in PLC-β1-/- neurons, indicating that this residual [Ca2+]o-sensitivity may be mediated by other PLC isoforms. The RRP size was substantially reduced after incubation in U73122, but not U73343. Phorbol esters increased RRP size after PLC inhibition. Discussion: Together these data point to a strong role for PLC in mediating changes in spontaneous release elicited by [Ca2+]o and other extracellular cues, possibly by modifying the size of the RRP. [ABSTRACT FROM AUTHOR]

Phorbol esters are the major toxic compounds found in Jatropha seed cake and oil. Removal of phorbol esters from Jatropha seed cake would allow the utilisation of detoxified seed cake as a protein-rich supplement for animal feed. Research was performed to determine the effects of submerged fermentation (SMF) on the phorbol ester (PE) degradation rate. In Taguchi design of experiments, the L18 orthogonal array was used for optimising the SMF process for detoxification of Jatropha seed cake. Process parameters selected for the study were pH, percentage of seed cake, time, temperature and rpm. The response parameter for detoxification was measured in terms of phorbol ester degradation. Analysis of variance (ANOVA) was performed for predicting the optimum process parameters. The percentage contribution of the process parameters with reference to phorbol ester degradation has been predicted. The hierarchy of significant process parameters is in the order of seed cake (1%), temperature (35 °C), pH (7.5) and time (12 h). [ABSTRACT FROM AUTHOR]