LENG8 regulation of mRNA processing is responsible for the control of mitochondrial activity

The processing of mRNA is essential for the maintenance of cellular and tissue homeostasis. However, the precise regulation of this process in mammalian cells, remains largely unknown. Here we have found that LENG8 represents the mammalian orthologue of the yeast mRNA processing factor Thp3 and Sac3. We go on to demonstrate that LENG8 binds to mRNAs, associates with components of mRNA processing machinery (the TREX complex) and contributes to mRNA nuclear export to the cytoplasm. Loss of LENG8 , leads to aberrant accumulation of poly (A) + RNA in the nucleus, in both Hela cells and murine fibroblasts. Furthermore, the precipitation of LENG8, is associated with an enrichment of both mRNAs and lncRNAs, and approximately half of these are also bound by the TREX component, THOC1. However, LENG8 preferentially binds mRNAs encoding for mitochondrial proteins and depletion of this processing factor, causes a dramatic breakdown in mitochondrial ultrastructure and a reduction in mitochondrial respiratory activity. Conditional deletion of Leng8 in mouse adipose tissues lead to a decreased body weight, and increased adipose thermogenesis. Our work has found an evolutionarily conserved mRNA processing factor that can control mitochondrial activity.

Investigation of an injectable gold nanoparticle extracellular matrix

Post-traumatic osteoarthritis (PTOA) is a progressive articular degenerative disease that degrades articular cartilage and stimulates apoptosis in chondrocyte cells. An injectable decellularized, extracellular matrix (ECM) scaffold, that might be able to combat the effects of PTOA, was developed where the ECM was conjugated with 20 nm gold nanoparticles (AuNP) and supplemented with curcumin and hyaluronic acid (HA). Porcine diaphragm ECM was decellularized and homogenized; AuNPs were conjugated using chemical crosslinking followed by mixing with curcumin and/or HA. Injection force testing and scanning electron microscopy with energy-dispersive X-ray spectroscopy were utilized to characterize the ECM scaffolds. In vitro testing with L929 murine fibroblasts, equine synovial fibroblasts, and Human Chondrocytes were used to determine biocompatibility, reactive oxygen species (ROS) reduction, and chondroprotective ability. The results demonstrated that conjugation of 20 nm AuNPs to the ECM was successful without significantly altering the physical properties as noted in the low injection force. In vitro work provided evidence of biocompatibility with a propensity to reduce intracellular ROS and an ability to mitigate apoptosis of chondrocyte cells stimulated with IL-1β, a known apoptosis inducing cytokine. It was concluded that an injectable AuNP-ECM may have the ability to mitigate inflammation and apoptosis.

K15 promoter-driven enforced expression of NKIRAS exhibits tumor suppressive activity against the development of DMBA/TPA-induced skin tumors

NKIRAS1 and NKIRAS2 (also called as κB-Ras) were identified as members of the atypical RAS family that suppress the transcription factor NF-κB. However, their function in carcinogenesis is still controversial. To clarify how NKIRAS acts on cellular transformation, we generated transgenic mice in which NKIRAS2 was forcibly expressed using a cytokeratin 15 (K15) promoter, which is mainly activated in follicle bulge cells. The ectopic expression of NKIRAS2 was mainly detected in follicle bulges of transgenic mice with NKIRAS2 but not in wild type mice. K15 promoter-driven expression of NKIRAS2 failed to affect the development of epidermis, which was evaluated using the expression of K10, K14, K15 and filaggrin. However, K15 promoter-driven expression of NKIRAS2 effectively suppressed the development of skin tumors induced by treatment with 7,12-dimethylbenz(a)anthracene (DMBA)/12-O-tetradecanoylphorbol 13-acetate (TPA). This observation suggested that NKIRAS seemed to function as a tumor suppressor in follicle bulges. However, in the case of oncogenic HRAS-driven cellular transformation of murine fibroblasts, knockdown of NKIRAS2 expression drastically suppressed HRAS-mutant-provoked cellular transformation, suggesting that NKIRAS2 was required for the cellular transformation of murine fibroblasts. Furthermore, moderate enforced expression of NKIRAS2 augmented oncogenic HRAS-provoked cellular transformation, whereas an excess NKIRAS2 expression converted its functional role into a tumor suppressive phenotype, suggesting that NKIRAS seemed to exhibit a biphasic bell-shaped enhancing effect on HRAS-mutant-provoked oncogenic activity. Taken together, the functional role of NKIRAS in carcinogenesis is most likely determined by not only cellular context but also its expression level.

Comparative study of the chemical composition, larvicidal, antimicrobial and cytotoxic activities of volatile oils from E. punicifolia leaves from Minas Gerais and Goiás

Eugenia punicifolia (Kunth) D.C., Myrtaceae, known as “pedra-ume-caá”, is popularly used in the treatment of inflammation, infections, fever, flu, diabetes, and diarrhea. This study aimed to carry out a comparative study of the chemical composition of volatile oil from E. punicifolia leaves collected in Goiás and Minas Gerais, as well as to evaluate the larvicidal activity against Aedes aegypti L3 larvae, the antimicrobial activity against bacteria, pathogenic fungi, and environmental, and cytotoxic activity to Balb 3T3 cells (murine fibroblasts). Volatile oils were obtained by hydrodistillation in a Clevenger apparatus and analyzed by Gas Chromatography Coupled to Mass Spectrometry (CG/MS). A total of 60 compounds were identified, the main components found in the leaves of Goiás being Germacrene D, bicyclogermacrene and β-longipenene and in the leaves collected in Minas Gerais they were (Z)-caryophyllene, γ-cadinene, spathulenol, caryophyllene oxide, and α-cadinol. The larvicidal effect was moderate against Ae. aegypti, with LC50 of 85.53 µg / mL for samples from Goiás and LC50 of 91.52 µg / mL for samples from Minas Gerais. Both oils showed moderate bactericidal activity against K. rhiziphyla (ATCC 9341), M. luteus (ATCC 10240), and S. aureus (ATCC 29737). The oils from Goiás (IC50 706.7 µg / mL) and Minas Gerais (IC50 160.7 µg / mL) had a lower cytotoxic concentration than the toxic action for larvae and bacteria, evidencing a safety profile and an interesting therapeutic potential, mainly concerning to volatile oil from Goiás. Therefore, the volatile oils from E. punicifolia leaves collected in Goiás and Minas Gerais that presented moderate larvicidal activity for Ae. aegypti also presented a bactericide activity and less cytotoxicity against murine fibroblasts. This is the first study of the larvicidal, antimicrobial and cytotoxic activity of volatile oils from E. punicifolia leaves.

LENG8 regulation of mRNA processing, is responsible for the control of mitochondrial activity

The processing of mRNA is essential for the maintenance of cellular and tissue homeostasis. However, the precise regulation of this process in mammalian cells, remains largely unknown. Here we have found that LENG8 represents the mammalian orthologue of the yeast mRNA processing factor Thp3 and Sac3. We go on to demonstrate that LENG8 binds to mRNAs, associates with components of mRNA processing machinery (the TREX complex) and contributes to mRNA nuclear export to the cytoplasm. Loss of LENG8, leads to aberrant accumulation of poly (A)+ RNA in the nucleus, in both Hela cells and murine fibroblasts. Furthermore, the precipitation of LENG8, is associated with an enrichment of both mRNAs and lncRNAs, and approximately half of these are also bound by the TREX component, THOC1. However, LENG8 preferentially binds mRNAs encoding for mitochondrial proteins and depletion of this processing factor, causes a dramatic breakdown in mitochondrial ultrastructure and a reduction in mitochondrial respiratory activity. Conditional deletion of Leng8 in mouse adipose tissues lead to a decreased body weight, and increased adipose thermogenesis. Our work has found an evolutionarily conserved mRNA processing factor that can control mitochondrial activity.

Cytotoxic Effect of Addition of Different Concentrations of Nanohydroxyapatite to Resin Modified and Conventional Glass Ionomer Cements on L929 Murine Fibroblasts

Objectives: In this study we assessed the cytotoxic effect of nanohydroxyapatite (NHA) incorporated into resin modified and conventional glass ionomer cements (RMGICs and CGICs) on L929 murine fibroblasts. Materials and Methods: In this in vitro study, 0wt%, 1wt%, 2wt%, 5wt%, 7wt% and 10wt% concentrations of NHA were added to Fuji II LC RMGIC and Fuji IX CGIC powders. Eighteen samples (5×3mm) were fabricated from each type of glass ionomer, in six experimental groups (n=3): CG0, CG1, CG2, CG5, CG7, CG10, RMG0, RMG1, RMG2, RMG5, RMG7, and RMG10. Samples were incubated for 72h. The overlaying solution was removed and added to L929 fibroblasts. The methyl thiazolyl tetrazolium bromide (MTT) assay was performed at 24, 48 and 72h. The wavelength was read by a spectrophotometer. Data were analyzed by ANOVA and Tukey’s test. Results: There was no significant difference in cytotoxicity of the two types of glass ionomers, with and without NHA, except for CG0 and RMG0 groups after 72h. RMG0 group was significantly more cytotoxic than the CG0 group (P<0.05). In CG groups during the first 24h, the cytotoxicity of CG5 and CG7 groups was significantly higher than that of CG1; while, there was no significant difference between the RMG groups. Cytotoxicity significantly decreased in all groups after 24h (P<0.05). Conclusion: Incorporation of NHA into Fuji II LC RMGIC and Fuji IX CGIC did not affect their biocompatibility and therefore its addition to these materials can provide favorable biological properties, especially considering its beneficial effects on the other properties of GICs.

High cytotoxicity of betulin towards fish and murine fibroblasts: Is betulin safe for nonneoplastic cells?

Background Betulin, a natural pentacyclic triterpene with the lupane structure that is present in significant amounts in the outer bark of birch, is known for its broad array of biological and pharmacological properties. Betulin has attracted attention as a potential, natural-origin antimicrobial substance. The literature describes it as selectively toxic to neoplastic cells but safe for normal cells. The research aim was to evaluate the basal cytotoxicity of betulin towards fish (BF-2) and murine (NIH/3T3) fibroblasts. We used four colorimetric tests that provide a preliminary evaluation of possible mechanisms of the cytotoxicity of a compound to assess the degree of the toxicity of betulin after 24, 48 and 72 h of incubation with cells: the MTT assay (mitochondrial activity assessment), the NRU assay (lysosomal membrane integrity assessment), the LDH assay (cellular membrane integrity assessment) and the SRB assay (total cellular protein content determination). Results The results revealed an exceptionally high sensitivity of mitochondria to the effect of betulin, with the other endpoints being less sensitive. Although murine fibroblasts were more vulnerable to the toxic effect of betulin than fish fibroblasts, the betulin CC50 values for both cell lines were comparable with analogous IC50 values determined by other researchers in studies involving cancerous cells. Conclusions The results indicate the need to verify the claim about the selective toxicity of betulin towards malignant cells and to conduct safety/toxicity tests before any potential therapeutic use of betulin in veterinary medicine.

Boosting Antimicrobial Activity of Ciprofloxacin by Functionalization of Mesoporous Silica Nanoparticles

Mesoporous silica nanoparticles (MSNs) are very promising nanomaterials for treating bacterial infections when combined with pharmaceutical drugs. Herein, we report the preparation of two nanomaterials based on the immobilization of ciprofloxacin in mesoporous silica nanoparticles, either as the counter-ion of the choline derivative cation (MSN-[Ch][Cip]) or via anchoring on the surface of amino-group modified MSNs via an amide bond (MSN-Cip). Both nanomaterials were characterized by TEM, FTIR and solution 1H NMR spectroscopies, elemental analysis, XRD and N2 adsorption at 77 K in order to provide the desired structures. No cytotoxicity from the prepared mesoporous nanoparticles on 3T3 murine fibroblasts was observed. The antimicrobial activity of the nanomaterials was determined against Gram-positive (Staphylococcus aureus and Bacillus subtilis) and Gram-negative (Klebsiella pneumoniae) bacteria and the results were promising against S. aureus. In the case of B. subtilis, both nanomaterials exhibited higher antimicrobial activity than the precursor [Ch][Cip], and in the case of K. pneumoniae they exhibited higher activity than neutral ciprofloxacin.

Pretreatment of aged mice with retinoic acid supports alveolar regeneration via upregulation of reciprocal PDGFA signalling

ObjectivesIdiopathic pulmonary fibrosis (IPF) primarily affects the aged population and is characterised by failure of alveolar regeneration, leading to loss of alveolar type 1 (AT1) cells. Aged mouse models of lung repair have demonstrated that regeneration fails with increased age. Mouse and rat lung repair models have shown retinoic acid (RA) treatment can restore alveolar regeneration. Herein, we seek to determine the signalling mechanisms that become activated on RA treatment prior to injury, which support alveolar differentiation.DesignPartial pneumonectomy lung injury model and next-generation sequencing of sorted cell populations were used to uncover molecular targets regulating alveolar repair. In vitro organoids generated from epithelial cells of mouse or patient with IPF co-cultured with young, aged or RA-pretreated murine fibroblasts were used to test potential targets.Main outcome measurementsKnown alveolar epithelial cell differentiation markers, including HOPX and AGER for AT1 cells, were used to assess outcome of treatments.ResultsGene expression analysis of sorted fibroblasts and epithelial cells isolated from lungs of young, aged and RA-pretreated aged mice predicted increased platelet-derived growth factor subunit A (PDGFA) signalling that coincided with regeneration and alveolar epithelial differentiation. Addition of PDGFA induced AT1 and AT2 differentiation in both mouse and human IPF lung organoids generated with aged fibroblasts, and PDGFA monoclonal antibody blocked AT1 cell differentiation in organoids generated with young murine fibroblasts.ConclusionsOur data support the concept that RA indirectly induces reciprocal PDGFA signalling, which activates regenerative fibroblasts that support alveolar epithelial cell differentiation and repair, providing a potential therapeutic strategy to influence the pathogenesis of IPF.