Contribution of Massive Mitochondrial Fusion and Subsequent Fission in the Plant Life Cycle to the Integrity of the Mitochondrion and Its Genome

Plant mitochondria have large genomes to house a small number of key genes. Most mitochondria do not contain a whole genome. Despite these latter characteristics, the mitochondrial genome is faithfully maternally inherited. To maintain the mitochondrial genes—so important for energy production—the fusion and fission of mitochondria are critical. Fission in plants is better understood than fusion, with the dynamin-related proteins (DRP 3A and 3B) driving the constriction of the mitochondrion. How the endoplasmic reticulum and the cytoskeleton are linked to the fission process is not yet fully understood. The fusion mechanism is less well understood, as obvious orthologues are not present. However, there is a recently described gene, MIRO2, that appears to have a significant role, as does the ER and cytoskeleton. Massive mitochondrial fusion (MMF or hyperfusion) plays a significant role in plants. MMF occurs at critical times of the life cycle, prior to flowering, in the enlarging zygote and at germination, mixing the cells’ mitochondrial population—the so-called “discontinuous whole”. MMF in particular aids genome repair, the conservation of critical genes and possibly gives an energy boost to important stages of the life cycle. MMF is also important in plant regeneration, an important component of plant biotechnology.     

The Neurotrophin Receptor TrkC as a Novel Molecular Target of the Antineuroblastoma Action of Valproic Acid

Neurotrophins and their receptors are relevant factors in controlling neuroblastoma growth and progression. The histone deacetylase (HDAC) inhibitor valproic acid (VPA) has been shown to downregulate TrkB and upregulate the p75NTR/sortilin receptor complex. In the present study, we investigated the VPA effect on the expression of the neurotrophin-3 (NT-3) receptor TrkC, a favorable prognostic marker of neuroblastoma. We found that VPA induced the expression of both full-length and truncated (TrkC-T1) isoforms of TrkC in human neuroblastoma cell lines without (SH-SY5Y) and with (Kelly, BE(2)-C and IMR 32) MYCN amplification. VPA enhanced cell surface expression of the receptor and increased Akt and ERK1/2 activation by NT-3. The HDAC inhibitors entinostat, romidepsin and vorinostat also increased TrkC in SH-SY5Y, Kelly and BE(2)-C but not IMR 32 cells. TrkC upregulation by VPA involved induction of RUNX3, stimulation of ERK1/2 and JNK, and ERK1/2-mediated Egr1 expression. In SH-SY5Y cell monolayers and spheroids the exposure to NT-3 enhanced the apoptotic cascade triggered by VPA. Gene silencing of both TrkC-T1 and p75NTR prevented the NT-3 proapoptotic effect. Moreover, NT-3 enhanced p75NTR/TrkC-T1 co-immunoprecipitation. The results indicate that VPA upregulates TrkC by activating epigenetic mechanisms and signaling pathways, and sensitizes neuroblastoma cells to NT-3-induced apoptosis.      

Exploring Yeast as a Study Model of Pantothenate Kinase-Associated Neurodegeneration and for the Identification of Therapeutic Compounds

Mutations in the pantothenate kinase 2 gene (PANK2) are the cause of pantothenate kinase-associated neurodegeneration (PKAN), the most common form of neurodegeneration with brain iron accumulation. Although different disease models have been created to investigate the pathogenic mechanism of PKAN, the cascade of molecular events resulting from CoA synthesis impairment is not completely understood. Moreover, for PKAN disease, only symptomatic treatments are available. Despite the lack of a neural system, Saccharomyces cerevisiae has been successfully used to decipher molecular mechanisms of many human disorders including neurodegenerative diseases as well as iron-related disorders. To gain insights into the molecular basis of PKAN, a yeast model of this disease was developed: a yeast strain with the unique gene encoding pantothenate kinase CAB1 deleted, and expressing a pathological variant of this enzyme. A detailed functional characterization demonstrated that this model recapitulates the main phenotypes associated with human disease: mitochondrial dysfunction, altered lipid metabolism, iron overload, and oxidative damage suggesting that the yeast model could represent a tool to provide information on pathophysiology of PKAN. Taking advantage of the impaired oxidative growth of this mutant strain, a screening for molecules able to rescue this phenotype was performed. Two molecules in particular were able to restore the multiple defects associated with PKAN deficiency and the rescue was not allele-specific. Furthermore, the construction and characterization of a set of mutant alleles, allowing a quick evaluation of the biochemical consequences of pantothenate kinase (PANK) protein variants could be a tool to predict genotype/phenotype correlation.     

Role of ERK Pathway in the Pathogenesis of Atopic Dermatitis and Its Potential as a Therapeutic Target

Atopic dermatitis (AD) is an eczematous skin disorder characterized by type 2 inflammation, barrier disruption, and intense itch. In addition to type 2 cytokines, many other cytokines, such as interferon gamma (IFN-γ), interleukin 17 (IL-17), and interleukin 22 (IL-22), play roles in the pathogenesis of AD. It has been reported that the extracellular signal-regulated kinase (ERK) is downstream of such cytokines. However, the involvement of the ERK pathway in the pathogenesis of AD has not yet been investigated. We examined the expression of p-ERK in mouse and human AD skin. We also investigated the effects of the topical application of an ERK inhibitor on the dermatitis score, transepidermal water loss (TEWL), histological change, and expression of filaggrin, using an AD-like NC/Nga murine model. The effects of an ERK inhibitor on filaggrin expression in normal human epidermal keratinocytes (NHEKs) and on chemokine production from bone marrow-derived dendritic cells (BMDCs) were also evaluated. p-ERK was highly expressed in mouse and human AD skin. Topical application of an ERK inhibitor alleviated the clinical symptoms, histological changes, TEWL, and decrease in expression of filaggrin in the AD-like NC/Nga murine model. The ERK inhibitor also restored the IL-4 induced reduction in the expression of filaggrin in NHEK, and inhibited chemokine production from BMDC induced by IL-4. These results indicate that the ERK pathway is involved in the pathogenesis of AD, and suggest that the ERK pathway has potential as a therapeutic target for AD in the future.     

Nano‐Explosions of Nanoparticles for Sudden Release of Substances by Embedded Azo‐Components as Obtained via the Miniemulsion Process

The solid and thermally instable azoinitiators V‐65 and VR‐110 were embedded within a polymer particle by using the miniemulsion process and afterwards quickly decomposed by thermal treatment below the glass temperature of the polymer. The resulting nitrogen gas overpressure inside the particles leads to a disruption of the polymer particle and a possible sudden release of encapsulated substances. It is shown, by electron microscopic measurements, that the number of burst particles correlates with the applied temperatures as well as the heating time. The surface deformation could be verified by scanning electron microscope analyses.

     

Beyond platinum: synthesis,characterization, and in vitro toxicity of Cu(II)-releasing polymer nanoparticles for potential use as a drug delivery vector

The field of drug delivery focuses primarily on delivering small organic molecules or DNA/RNA as therapeutics and has largely ignored the potential for delivering catalytically active transition metal ions and complexes. The delivery of a variety of transition metals has potential for inducing apoptosis in targeted cells. The chief aims of this work were the development of a suitable delivery vector for a prototypical transition metal, Cu²⁺, and demonstration of the ability to impact cancer cell viability via exposure to such a Cu-loaded vector. Carboxylate-functionalized nanoparticles were synthesized by free radical polymerization and were subsequently loaded with Cu²⁺ via binding to particle-bound carboxylate functional groups. Cu loading and release were characterized via ICP MS, EDX, XPS, and elemental analysis. Results demonstrated that Cu could be loaded in high weight percent (up to 16 wt.%) and that Cu was released from the particles in a pH-dependent manner. Metal release was a function of both pH and the presence of competing ligands. The toxicity of the particles was measured in HeLa cells where reductions in cell viability greater than 95% were observed at high Cu loading. The combined pH sensitivity and significant toxicity make this copper delivery vector an excellent candidate for the targeted killing of disease cells when combined with an effective cellular targeting strategy.     

Inhibitors of Mitochondrial Human Carbonic Anhydrases VA and VB as a Therapeutic Strategy against Paclitaxel-Induced Neuropathic Pain in Mice

Neuropathy development is a major dose-limiting side effect of anticancer treatments that significantly reduces patient’s quality of life. The inadequate pharmacological approaches for neuropathic pain management warrant the identification of novel therapeutic targets. Mitochondrial dysfunctions that lead to reactive oxygen species (ROS) increase, cytosolic Ca²⁺ imbalance, and lactate acidosis are implicated in neuropathic pain pathogenesis. It has been observed that in these deregulations, a pivotal role is played by the mitochondrial carbonic anhydrases (CA) VA and VB isoforms. Hence, preclinical studies should be conducted to assess the efficacy of two novel selenides bearing benzenesulfonamide moieties, named 5b and 5d, and able to inhibit CA VA and VB against paclitaxel-induced neurotoxicity in mice. Acute treatment with 5b and 5d (30–100 mg/kg, per os – p.o.) determined a dose-dependent and long-lasting anti-hyperalgesic effect in the Cold plate test. Further, repeated daily treatment for 15 days with 100 mg/kg of both compounds (starting the first day of paclitaxel injection) significantly prevented neuropathic pain development without the onset of tolerance to the anti-hyperalgesic effect. In both experiments, acetazolamide (AAZ, 100 mg/kg, p.o.) used as the reference drug was partially active. Moreover, ex vivo analysis demonstrated the efficacy of 5b and 5d repeated treatments in reducing the maladaptive plasticity that occurs to glia cells in the lumbar portion of the spinal cord and in improving mitochondrial functions in the brain and spinal cord that were strongly impaired by paclitaxel-repeated treatment. In this regard, 5b and 5d ameliorated the metabolic activity, as observed by the increase in citrate synthase activity, and preserved an optimal mitochondrial membrane potential (ΔΨ) value, which appeared depolarized in brains from paclitaxel-treated animals. In conclusion, 5b and 5d have therapeutic and protective effects against paclitaxel-induced neuropathy without tolerance development. Moreover, 5b and 5d reduced glial cell activation and mitochondrial dysfunction in the central nervous system, being a promising candidate for the management of neuropathic pain and neurotoxicity evoked by chemotherapeutic drugs.     

Seroma after Simple Mastectomy in Breast Cancer—The Role of CD4+ T Helper Cells and the Evidence as a Possible Specific Immune Process

Seroma development after breast cancer surgery is the most common postoperative complication seen after mastectomy but neither its origin nor its cellular composition is known. To investigate the assumption of immunological significance, one of the first aims of this pilot study is to describe the cellular content of collected seroma fluids and its corresponding serum in patients with simple mastectomy after needle aspiration, as well as the serum of healthy controls. The content of red blood cells (RBC) was measured by haemato-counter analyses, and the lymphocyte identification/quantification was conducted by flow cytometry analyses in seroma fluid (SFl) and the sera of patients (PBp) as well as controls (PBc). Significantly lower numbers of RBCs were measured in SFl. Cytotoxic T cells are significantly reduced in SFl, whereas T helper (Th) cells are significantly enriched compared to PBp. Significantly higher numbers of Th2 cells were found in SFl and PBp compared to PBc. The exact same pattern is seen when analyzing the Th17 subgroup. In conclusion, in contrast to healthy controls, significantly higher Th2 and Th17 cell subgroup-mediated immune responses were measured in seroma formations and were further confirmed in the peripheral blood of breast cancer (including DCIS) patients after simple mastectomy. This could lead to the assumption of a possible immunological cause for the origin of a seroma.     

Generation of Rho Zero Cells: Visualization and Quantification of the mtDNA Depletion Process

Human mitochondrial DNA (mtDNA) is located in discrete DNA-protein complexes, so called nucleoids. These structures can be easily visualized in living cells by utilizing the fluorescent stain PicoGreen^®. In contrary, cells devoid of endogenous mitochondrial genomes (ρ⁰ cells) display no mitochondrial staining in the cytoplasm. A modified restriction enzyme can be targeted to mitochondria to cleave the mtDNA molecules in more than two fragments, thereby activating endogenous nucleases. By applying this novel enzymatic approach to generate mtDNA-depleted cells the destruction of mitochondrial nucleoids in cultured cells could be detected in a time course. It is clear from these experiments that mtDNA-depleted cells can be seen as early as 48 h post-transfection using the depletion system. To prove that mtDNA is degraded during this process, mtDNA of transfected cells was quantified by real-time PCR. A significant decline could be observed 24 h post-transfection. Combination of both results showed that mtDNA of transfected cells is completely degraded and, therefore, ρ⁰ cells were generated within 48 h. Thus, the application of a mitochondrially-targeted restriction endonuclease proves to be a first and fast, but essential step towards a therapy for mtDNA disorders.     

颗粒溶素活性肽与增强型绿色荧光蛋白融合基因真核表达质粒的构建及其在小鼠黑色素瘤细胞中的表达

目的构建人颗粒溶素(GLS)活性肽(9ku-GLS)与增强型绿色荧光蛋白(EGFP)融合基因的真核表达载体,并检测其在小鼠黑色素瘤细胞B16中的表达。方法经PCR扩增9ku-GLS基因,插入质粒pBudCE4.1中,经酶切及测序鉴定正确后,亚克隆至质粒pEGFP-C1中,将融合基因真核表达质粒pEGFP-C1/S9K转染B16细胞,采用荧光显微镜及RT-PCR法检测融合蛋白的表达。结果融合基因真核表达质粒pEGFP-C1/S9K经双酶切鉴定证明构建正确,转染B16细胞24h后,荧光显微镜下可观察到绿色荧光,且经RT-PCR可扩增出267bp的目的基因片段。结论已成功构建9ku-GLS与EGFP融合基因的真核表达质粒,且在B16细胞中表达了融合蛋白。     

Target Hopping as a Useful Tool for the Identification of Novel EphA2 Protein–Protein Antagonists

Lithocholic acid (LCA), a physiological ligand for the nuclear receptor FXR and the G‐protein‐coupled receptor TGR5, has been recently described as an antagonist of the EphA2 receptor, a key member of the ephrin signalling system involved in tumour growth. Given the ability of LCA to recognize FXR, TGR5, and EphA2 receptors, we hypothesized that the structural requirements for a small molecule to bind each of these receptors might be similar. We therefore selected a set of commercially available FXR or TGR5 ligands and tested them for their ability to inhibit EphA2 by targeting the EphA2‐ephrin‐A1 interface. Among the selected compounds, the stilbene carboxylic acid GW4064 was identified as an effective antagonist of EphA2, being able to block EphA2 activation in prostate carcinoma cells, in the micromolar range. This finding proposes the “target hopping” approach as a new effective strategy to discover new protein–protein interaction inhibitors.     

Congo Red as a Supramolecular Carrier System for Doxorubicin: An Approach to Understanding the Mechanism of Action

The uptake and distribution of doxorubicin in the MCF7 line of breast-cancer cells were monitored by Raman measurements. It was demonstrated that bioavailability of doxorubicin can be significantly enhanced by applying Congo red. To understand the mechanism of doxorubicin delivery by Congo red supramolecular carriers, additional monolayer measurements and molecular dynamics simulations on model membranes were undertaken. Acting as molecular scissors, Congo red particles cut doxorubicin aggregates and incorporated them into small-sized Congo red clusters. The mixed doxorubicin/Congo red clusters were adsorbed to the hydrophilic part of the model membrane. Such behavior promoted transfer through the membrane.     

Polycaprolactone/Chitosan Composite Nanofiber Membrane as a Preferred Scaffold for the Culture of Mesothelial Cells and the Repair of Damaged Mesothelium

Mesothelial cells are specific epithelial cells lining the serosal cavity and internal organs. Nonetheless, few studies have explored the possibility to culture mesothelial cells in a nanostructure scaffold for tissue engineering applications. Therefore, this study aims to fabricate nanofibers from a polycaprolactone (PCL) and PCL/chitosan (CS) blend by electrospinning, and to elucidate the effect of CS on the cellular response of mesothelial cells. The results demonstrate that a PCL and PCL/CS nanofiber membrane scaffold could be prepared with a comparable fiber diameter (~300 nm) and porosity for cell culture. Blending CS with PCL influenced the mechanical properties of the scaffold due to interference of PCL crystallinity in the nanofibers. However, CS substantially improves scaffold hydrophilicity and results in a ~6-times-higher cell attachment rate in PCL/CS. The mesothelial cells maintain high viability in both nanofiber membranes, but PCL/CS provides better maintenance of cobblestone-like mesothelial morphology. From gene expression analysis and immunofluorescence staining, the incorporation of CS also results in the upregulated expression of mesothelial marker genes and the enhanced production of key mesothelial maker proteins, endorsing PCL/CS to better maintain the mesothelial phenotype. The PCL/CS scaffold was therefore chosen for the in vivo studies, which involved transplanting a cell/scaffold construct containing allograft mesothelial cells for mesothelium reconstruction in rats. In the absence of mesothelial cells, the mesothelium wound covered with PCL/CS showed an inflammatory response. In contrast, a mesothelium layer similar to native mesothelium tissue could be obtained by implanting the cell/scaffold construct, based on hematoxylin and eosin (H&E) and immunohistochemical staining.     

直接由SiO2合成有机硅单体及其应用

综述了直接从占地壳１／４的ＳｉＯ２合成有机硅单体的新方法,介绍了由此方法得出的有机硅单体在纳米复合、导电聚合物及高性能陶瓷等主要方面的应用,并对该方法的发展及潜在应用作了展望。     

Neuronal Nitric Oxide Synthase as a Shared Target for the Effects of Adiponectin and Resistin on the Mechanical Responses of the Mouse Gastric Fundus

It has been reported that adiponectin (ADPN) and resistin are co-secreted by white mouse adipocytes and exert similar inhibitory effects in the mouse gastric fundus, in which resistin was observed to increase neuronal nitric oxide synthase (nNOS) expression. On these grounds, the present work aimed to investigate whether the effects of the two adipokines on the neurally-induced relaxant responses potentiate each other and whether there is a possible correlation with changes in nNOS expression in preparations from the mouse gastric fundus. In carbachol (CCh)-precontracted strips, electrical field stimulation elicited nitrergic relaxant responses, whose amplitude was increased by ADPN or resistin, but no additional enhancements were observed in their concomitant presence. Western blot and immunofluorescence analyses revealed that ADPN, like resistin, was able to up-regulate nNOS expression and to increase the percentage of nNOS-positive neurons in the myenteric plexus: co-treatment with the two adipokines did not induce additional changes. The results indicate that the two adipokines modulate nitrergic neurotransmission, and both do so by up-regulating nNOS expression. Therefore, nNOS appears to be a shared target for the two adipokines’ effects, which, rather than mutually reinforcing each other, may represent a dual physiological control mechanism to guarantee gastric fundus relaxation.     

软质聚氨酯泡沫塑料生产过程中自燃故障的原因及预防

对软质聚氨酯泡沫塑料生产过程中自燃故障产生的原因及其影响因素进行了分析,并介绍了预防自燃的措施。     

Influence of 6-Amino chrysene on the Kinetics of the Metabolic Process for 9-Hydroxybenzo(a)pyrene in 3T3 and RTG2 Cells

PAH metabolism is known to proceed in two successive steps, the first one resulting in the production of activated metabolites which are subsequently transformed by the different pathways involved in the second step. Microspectrofluorometry enables us to study the kinetics of these steps in living intact cells in which no imbalance has been introduced artificially. We have used this technique in order to check the influence of 6-Amino-chrysene (6 AC) on the kinetics of the metabolism of 9-Hydroxybenzo(a)pyrene (9-OH-B(a)P) which, in a previous study, had been shown to proceed mainly through the glucuronide pathway. We have first demonstrated that both 6 AC and 9-OH-B(a)P were good substrates for Rabbit Glucuronic Transferase. Then using RTG2 and 3T3 cells, we have verified that the glucuronide pathway was strongly involved in the metabolism of 6 AC. Finally we have demonstrated that 6 AC strongly inhibits the metabolism of 9-OH-B(a)P in both cell lines. Such results are consistent with a competition of both compounds for the same binding site of GTAse 1.     

The Phagocytosis of Lacticaseibacillus casei and Its Immunomodulatory Properties on Human Monocyte-Derived Dendritic Cells Depend on the Expression of Lc-p75, a Bacterial Peptidoglycan Hydrolase

The human gut symbiont Lacticaseibacillus (L.) casei (previously Lactobacillus casei) is under intense research due to its wide range of immunomodulatory effects on the human host. Dendritic cells (DCs) are crucial players in the direct and indirect communication with lactobacilli in the gastrointestinal tract. Here, we demonstrate that human monocyte-derived DCs (moDCs) are able to engulf L. casei BL23, in which the intact bacterial cell wall and morphology have a key role. The absence of the bacterial cell-wall-degrading enzyme, Lc-p75, in L. casei cells causes remarkable morphological changes, which have important consequences in the phagocytosis of L. casei by moDCs. Our results showed that the Lc-p75 mutation induced defective internalization and impaired proinflammatory and T-cell-polarizing cytokine secretion by bacteria-exposed moDCs. The T helper (Th) 1 and Th17 cell activating capacity of moDCs induced by the mutant L. casei was consequently reduced. Moreover, inhibition of the phagocytosis of wild-type bacteria showed similar results. Taken together, these data suggested that formation of short bacterial chains helps to exert the potent immunomodulatory properties of L. casei BL23.     

钛酸盐纳米纤维形貌控制及固体碱催化性能

研究了钛前驱体、碱种类和合成温度对水热法制备钛酸盐纳米纤维形貌的影响。结果显示,以无定形钛凝胶为前驱体和KOH为碱,均有利于形成无孔的钛酸盐纳米纤维,在90℃下得到了形貌均一,长径比为1 000,比表面积为311 m2/g的钛酸盐纳米纤维。以钛酸盐纳米纤维为载体,采用浸渍法负载了碱金属卤化物作为固体碱催化剂。该催化剂在环氧丙烷醇化制备丙二醇单甲醚的反应中,降低了反应温度、缩短了反应时间,表现出良好的碱催化活性。CO2-TPD结果显示,掺杂后的催化剂的碱含量是空白载体的两倍,并出现中强碱活性中心。     

Trifluoroacetic Acid Hydroxylamine System as Organocatalyst Reagent in a One-Pot Salt Free Process for the Synthesis of Caprolactam and Amides of Industrial Interest

Catalysis Letters - In this work we studied the reactivity of the Trifluoroacetic acid hydroxylamine system in the one step salt free synthesis of amides from ketones. A particular regards was paid...