Genetic control of generative cell shape by DUO1 in Arabidopsis.

KEY MESSAGE: The main features of generative cell morphogenesis, formation of a cytoplasmic projection and elongation of the GC body, operate through independent genetic pathways. Male gametogenesis in developing angiosperm pollen involves distinctive changes in cell morphogenesis. Re-shaping and elongation of the generative cell (GC) are linked to the formation of a GC cytoplasmic projection connected to the vegetative cell nucleus. Although genetic control of GC morphogenesis is unknown, we suspected the involvement of the germline-specific MYB transcription factor DUO POLLEN1 (DUO1). We used light and fluorescence microscopy to examine male germline development in pollen of wild-type Arabidopsis and in four allelic duo1 mutants expressing introduced cell markers. Our analysis shows that the undivided GC in duo1 pollen forms a cytoplasmic projection, but the cell body fails to elongate. In contrast GCs of cyclin-dependent kinase function mutants, which fail to divide like duo1 mutants, achieve normal morphogenesis. We conclude that DUO1 has an essential role in the elongation of the GC, but DUO1-independent pathways control the development of the GC cytoplasmic projection. The two main features of GC morphogenesis therefore operate through independently regulated genetic pathways.

A conserved zinc-binding site in Acinetobacter baumannii PBP2 required for elongasome-directed bacterial cell shape.

Acinetobacter baumannii is a gram-negative bacterial pathogen that causes challenging nosocomial infections. ß-lactam targeting of penicillin-binding protein (PBP)-mediated cell wall peptidoglycan (PG) formation is a well-established antimicrobial strategy. Exposure to carbapenems or zinc (Zn)-deprived growth conditions leads to a rod-to-sphere morphological transition in A. baumannii, an effect resembling that caused by deficiency in the RodA-PBP2 PG synthesis complex required for cell wall elongation. While it is recognized that carbapenems preferentially acylate PBP2 in A. baumannii and therefore block the transpeptidase function of the RodA-PBP2 system, the molecular details underpinning cell wall elongation inhibition upon Zn starvation remain undefined. Here, we report the X-ray crystal structure of A. baumannii PBP2, revealing an unexpected Zn coordination site in the transpeptidase domain required for protein stability. Mutations in the Zn-binding site of PBP2 cause a loss of bacterial rod shape and increase susceptibility to ß-lactams, therefore providing a direct rationale for cell wall shape maintenance and Zn homeostasis in A. baumannii. Furthermore, the Zn-coordinating residues are conserved in various ß- and γ-proteobacterial PBP2 orthologs, consistent with a widespread Zn-binding requirement for function that has been previously unknown. Due to the emergence of resistance to virtually all marketed antibiotic classes, alternative or complementary antimicrobial strategies need to be explored. These findings offer a perspective for dual inhibition of Zn-dependent PG synthases and metallo-ß-lactamases by metal chelating agents, considered the most sought-after adjuvants to restore ß-lactam potency against gram-negative bacteria.

Optimized separation of anhydrosafflor yellow B from safflower by high-speed counter-current chromatography and evaluation of its cardio-protective effect.

Anhydrosafflor yellow B (AHSYB) is a major active water-soluble pigment in Safflower, but it has not received enough attention yet. In this study, high-speed counter-current chromatography (HSCCC) was used to prepare AHSYB from safflower. The parameters of the separation process were optimized by response surface methodology for the first time. The entropy weight method (EWM) was applied to calculate the information entropy and the weight of five indexes, and then figure out a comprehensive index of the HSCCC separation effect. Under the optimized separation conditions, a HSCCC apparatus speed of 850 rpm, a flow rate of 2 mL min-1 for the mobile phase and a separation temperature of 40 °C for AHSYB were achieved with a purity of 98%. Furthermore, AHSYB was found to have cardio-protective effects by inhibiting apoptosis via the mitochondrial-mediated pathway in oxygen-glucose deprivation/reoxygenation-induced H9c2 cells. This research provides good method guides for the rapid and efficient separation of active compounds from food-grade Chinese herb medicines.

Neuroprotective effects of macamide from maca (Lepidium meyenii Walp.) on corticosterone-induced hippocampal impairments through its anti-inflammatory, neurotrophic, and synaptic protection properties.

The present study aims to investigate the protective effects of N-(3-methoxybenzyl)-(9Z,12Z,15Z)-octadecatrienamide (M 18:3) on corticosterone-induced neurotoxicity. A neurotoxic model was established by subcutaneous injection of corticosterone (40 mg per kg bw) for 21 days. Depressive behaviors (the percentage of sucrose consumption, the immobility time in the forced swimming test, and the total distance in the open field test) were observed. The levels of the brain-derived neurotrophic factor, the contents of tumor necrosis factor-α and interleukin-6, and the numbers of positive cells of doublecortin and bromodeoxyuridine in the hippocampus were measured. The density of hippocampal neurons was calculated. The morphological changes of hippocampal neurons (the density of dendritic spines, the dendritic length, and the area and volume of dendritic cell bodies) were observed. The expression levels of synaptophysin, synapsin I, and postsynaptic density protein 95 were measured. Behavioral experiments showed that M 18:3 (5 and 25 mg per kg bw) could remarkably improve the depressive behaviors. The enzyme-linked immunosorbent assay showed that M 18:3 could considerably reduce hippocampal neuroinflammation and increase hippocampal neurotrophy. Nissl staining showed that M 18:3 could remarkably improve the corticosterone-induced decrease in the hippocampal neuron density. Immunofluorescence analysis showed that M 18:3 could considerably promote hippocampal neurogenesis. Golgi staining showed that M 18:3 could remarkably improve the corticosterone-induced changes in the hippocampal dendritic structure. Western blotting showed that M 18:3 could considerably increase the expression levels of synaptic-structure-related proteins in the hippocampus. In conclusion, the protective effects of M 18:3 may be attributed to the anti-inflammatory, neurotrophic, and synaptic protection properties.

A Novel In Vitro Assay Using Human iPSC-Derived Sensory Neurons to Evaluate the Effects of External Chemicals on Neuronal Morphology: Possible Implications in the Prediction of Abnormal Skin Sensation.

Neuronal morphological changes in the epidermis are considered to be one of causes of abnormal skin sensations in dry skin-based skin diseases. The present study aimed to develop an in vitro model optimised for human skin to test the external factors that lead to its exacerbation. Human-induced pluripotent stem cell-derived sensory neurons (hiPSC-SNs) were used as a model of human sensory neurons. The effects of chemical substances on these neurons were evaluated by observing the elongation of nerve fibers, incidence of blebs (bead-like swellings), and the expression of nicotinamide mononucleotide adenylyl transferase 2 (NMNAT2). The nerve fiber length increased upon exposure to two common cosmetic preservatives-methylparaben and phenoxyethanol-but not to benzo[a]pyrene, an air pollutant at the estimated concentrations in the epidermis. Furthermore, the incidence of blebs increased upon exposure to benzo[a]pyrene. However, there was a decrease in the expression of NMNAT2 in nerve fibers, suggesting degenerative changes. No such degeneration was found after methylparaben or phenoxyethanol at the estimated concentrations in the epidermis. These findings suggest that methylparaben and phenoxyethanol promote nerve elongation in hiPSC-SNs, whereas benzo[a]pyrene induces nerve degeneration. Such alterations may be at least partly involved in the onset and progression of sensitive skin.

Dendrobium officinale polysaccharide-induced neuron-like cells from bone marrow mesenchymal stem cells improve neuronal function a rat stroke model.

Various methods have been used to induce the neuronal differentiation of marrow mesenchymal stem cells (MSCs). However, the limited induction efficiency of cells in vitro has restricted their use. Therefore, identifying a simple and efficient treatment method is necessary. Dendrobium officinale is an important traditional Chinese medicine, and its main component, polysaccharides, has many pharmacological activities. However, the effects of D. officinale polysaccharide (DOP) on the neuronal differentiation of bone marrow mesenchymal stem cells (BMSCs) and treatment of ischaemic stroke remain unknown. We found that DOP promoted the neuronal differentiation of BMSCs by increasing the expression levels of neural markers, and the optimal concentration of DOP was 25 µg/mL. Additionally, the Notch signalling pathway was inhibited during the neuronal differentiation of BMSCs induced by DOP, and this effect was strengthened using an inhibitor of this pathway. The Wnt signalling pathway was activated during the differentiation of BMSCs, and inhibition of the Wnt signalling pathway downregulated the expression of neuronal genes. Furthermore, the transplantation of neuron-like cells induced by DOP improved neuronal recovery, as the brain infarct volume, neurologic severity scores and levels of inflammatory factors were all significantly reduced in vivo. In conclusion, DOP is an effective inducer of the neuronal differentiation of BMSCs and treatment option for ischaemic stroke.

MAPK and Notch-Mediated Effects of Meso-Xanthin F199 Compounds on Proliferative Activity and Apoptosis of Human Melanocytes in Three-Dimensional Culture.

Meso-Xanthin (Meso-Xanthin F199™) is a highly active antiaging injection drug of the latest generation. The main acting compound is fucoxanthin, supplemented with several growth factors, vitamins, and hyaluronic acid. Previous examination of fucoxanthin on melanocytes showed its ability to inhibit skin pigmentation through different signaling pathways focused on suppression of melanogenic-stimulating receptors. In turn, the anticancer property of fucoxanthin is realized through MAPK and PI3K pathways. We aimed to evaluate the effect of fucoxanthin and supplemented growth factors on melanocyte growth and transformation at a proteomic level. The effect of fucoxanthin on melanocytes cultivated in three-dimensional (3D) condition was examined using high-throughput proteomic and system biology approaches to disclose key molecular events of the targeted action. Our results demonstrated significant inhibition of cell differentiation and ubiquitination processes. We found that the negative regulation of PSME1 and PTGIS largely determines the inhibition of NF-κB and MAPK2. Besides, fucoxanthin selectively inhibits cell differentiation via negative regulation of Raf signaling and the upstream activation of IL-1 signaling. It is assumed that inhibition of Raf influences the Notch-4 signaling and switches off the MAPK/MAPK2 cascade. Blockage of MAPK/MAPK2 is feasible due to suppression of Ras and NF-κB by the addressed action of IKKB, IKK2, and TRAF6. Suggestively, Meso-Xanthin F199™ can manage processes of proliferative activity and inhibition of apoptosis due to composition of fucoxanthin and growth-stimulating factors, which may increase the risk of skin cancer development under certain condition.

Precision genetic cellular models identify therapies protective against ER stress.

Rare monogenic disorders often share molecular etiologies involved in the pathogenesis of common diseases. Congenital disorders of glycosylation (CDG) and deglycosylation (CDDG) are rare pediatric disorders with symptoms that range from mild to life threatening. A biological mechanism shared among CDG and CDDG as well as more common neurodegenerative diseases such as Alzheimer's disease and amyotrophic lateral sclerosis, is endoplasmic reticulum (ER) stress. We developed isogenic human cellular models of two types of CDG and the only known CDDG to discover drugs that can alleviate ER stress. Systematic phenotyping confirmed ER stress and identified elevated autophagy among other phenotypes in each model. We screened 1049 compounds and scored their ability to correct aberrant morphology in each model using an agnostic cell-painting assay based on >300 cellular features. This primary screen identified multiple compounds able to correct morphological phenotypes. Independent validation shows they also correct cellular phenotypes and alleviate each of the ER stress markers identified in each model. Many of the active compounds are associated with microtubule dynamics, which points to new therapeutic opportunities for both rare and more common disorders presenting with ER stress, such as Alzheimer's disease and amyotrophic lateral sclerosis.

Rhododendron molle G. Don Extract Induces Apoptosis and Inhibits Migration in Human Colorectal Cancer Cells and Potential Anticancer Components Analysis.

Rhododendron molle G. Don is one example of traditional Chinese medicine with important medicinal value. In this study, the effects of methanol extract of R. molle leaves (RLE) on colorectal cancer HT-29 cells and its potential molecular mechanism were investigated. MTT analysis showed that RLE could significantly inhibit the cell viability and migration of HT-29 cells in a concentration-dependent manner. Cell cycle analyses via flow cytometer suggested that RLE induced DNA fragmentation, indicative of apoptosis, and arrest at the S phase in HT-29 cells. Quantitative real-time PCR (qRT-PCR) analysis showed that RLE could upregulate the mRNA expression of p53 and p21 in HT-29 cells, which would result in HT-29 cells being blocked in S phase. Meanwhile, RLE could upregulate the expression of Bax, and downregulate the expression of Bcl-2, which would induce cell apoptosis. Further western blot analysis showed that the protein expression changes of Bax and P53 were basically consistent with the results of qRT-PCR. In addition, GC-MS analysis detected 17 potential anticancer components in R. molle. These results indicate that R. molle has significant anticancer activity, which provides some useful information for further study and clinical application for R. molle.

Optimally designed theranostic system based folic acids and chitosan as a promising mucoadhesive delivery system for encapsulating curcumin LbL nano-template against invasiveness of breast cancer.

Curcumin is a potential candidate in cancer therapy due to its ability to inhibit many signalling pathways at the same time of exposure because of its unique content of aromatic ring, B diketone, olefinic linker, and O methoxy phenolic groups. Its applications in biomedical therapy is limited because of its sensitivity, and its rapid degradation. In the current study, curcumin inserted into polyelectrolyte pairs (protamine and dextran) and then was functionalized by folic acid conjugated chitosan used for the first time, as theranostic system. Such this strategy allows to improve its mucoadhesion and penetration that increases their accumulation inside cancer cells. CUR-LbL NPs were then used to investigate drug release inside Human Mammary Carcinoma (MCF-7 cell lines) after their incubations for 3 h, 6 h and 24 h. Flow cytometry indicated that the percentages of apoptosis, necrosis and cell cycle arrest were increased significantly in MCF-7 cell lines treated by CUR-LbL NPs. Furthermore, SEM image showed many debris in the section of MCF-7 treated by CUR-LbL NPs. Here, it can be summarized that curcumin functionalized by multi-layered polyelectrolyte capsules can be used as a model to study the fate of the adsorbed nanocarriers and to investigate the drug release inside cells.

Distinct Epigenetic Reprogramming, Mitochondrial Patterns, Cellular Morphology, and Cytotoxicity after Bee Venom Treatment.

BACKGROUND: Bee venom is a promising agent for cancer treatment due to its selective cytotoxic potential for cancer cells through apoptotic pathways. However, there is no evidence for changes in the epigenome and mitochondrial DNA copy numbers after bee venom application. The purpose of this study was to determine the impact of bee venom on cytosine modifications and mitochondrial DNA copy number variation. METHODS: A broad range of methods was applied to elucidate the impact of bee venom on neoplastic cells. These included MTT assay for detection of cytotoxicity, immunostaining of cytosine modifications and mitochondria, assessment of cellular morphology by flow cytometry, and quantification of mitochondrial DNA copy numbers using QPCR. RESULTS: Bee venom-induced cell death was selective for cancer cells, where it triggered a response characterized by alteration of cytosine modification. In contrast, normal cells were more resistant to DNA modifications. Furthermore, application of the venom resulted in variation of mitochondrial membrane permeability and mitochondrial DNA copy numbers, together with alterations in cell morphology, manifesting as reduced affected cell size. CONCLUSION: The study findings suggest that bee venom can be used as a selective DNA (de)methylating agent in cancer. Various agents (such as decitabine and 5-azacytidine) have been synthesized and developed for cancer treatment, and a range of syntheses and preparation and application methods have been described for these patented drugs. However, to the best of our knowledge, no previous research has investigated the use of bee venom or any component thereof for epigenetic therapy in cancer cells.

Annexins A2, A6 and Fetuin-A Affect the Process of Mineralization in Vesicles Derived from Human Osteoblastic hFOB 1.19 and Osteosarcoma Saos-2 Cells.

The mineralization process is initiated by osteoblasts and chondrocytes during intramembranous and endochondral ossifications, respectively. Both types of cells release matrix vesicles (MVs), which accumulate Pi and Ca2+ and form apatites in their lumen. Tissue non-specific alkaline phosphatase (TNAP), a mineralization marker, is highly enriched in MVs, in which it removes inorganic pyrophosphate (PPi), an inhibitor of apatite formation. MVs then bud from the microvilli of mature osteoblasts or hypertrophic chondrocytes and, thanks to the action of the acto-myosin cortex, become released to the extracellular matrix (ECM), where they bind to collagen fibers and propagate mineral growth. In this report, we compared the mineralization ability of human fetal osteoblastic cell line (hFOB 1.19 cells) with that of osteosarcoma cell line (Saos-2 cells). Both types of cells were able to mineralize in an osteogenic medium containing ascorbic acid and beta glycerophosphate. The composition of calcium and phosphate compounds in cytoplasmic vesicles was distinct from that in extracellular vesicles (mostly MVs) released after collagenase-digestion. Apatites were identified only in MVs derived from Saos-2 cells, while MVs from hFOB 1.19 cells contained amorphous calcium phosphate complexes. In addition, AnxA6 and AnxA2 (nucleators of mineralization) increased mineralization in the sub-membrane region in strongly mineralizing Saos-2 osteosarcoma, where they co-localized with TNAP, whereas in less mineralizing hFOB 1.19 osteoblasts, AnxA6, and AnxA2 co-localizations with TNAP were less visible in the membrane. We also observed a reduction in the level of fetuin-A (FetuA), an inhibitor of mineralization in ECM, following treatment with TNAP and Ca channels inhibitors, especially in osteosarcoma cells. Moreover, a fraction of FetuA was translocated from the cytoplasm towards the plasma membrane during the stimulation of Saos-2 cells, while this displacement was less pronounced in stimulated hFOB 19 cells. In summary, osteosarcoma Saos-2 cells had a better ability to mineralize than osteoblastic hFOB 1.19 cells. The formation of apatites was observed in Saos-2 cells, while only complexes of calcium and phosphate were identified in hFOB 1.19 cells. This was also evidenced by a more pronounced accumulation of AnxA2, AnxA6, FetuA in the plasma membrane, where they were partly co-localized with TNAP in Saos-2 cells, in comparison to hFOB 1.19 cells. This suggests that both activators (AnxA2, AnxA6) and inhibitors (FetuA) of mineralization were recruited to the membrane and co-localized with TNAP to take part in the process of mineralization.

2D exfoliated black phosphorus influences healthy and cancer prostate cell behaviors.

Nowadays, prostate cancer is the most widespread tumour in worldwide male population. Actually, brachytherapy is the most advanced radiotherapy strategy for the local treatment of prostate cancer. It consists in the placing of radioactive sources closed to the tumour side thus killing cancer cells. However, brachytherapy causes the same adverse effects of external-beam radiotherapy. Therefore, alternative treatment approaches are required for enhancing radiotherapy effectiveness and reducing toxic symptoms. Nanostructured exfoliated black phosphorus (2D BP) may represent a strategic tool for local cancer therapy because of its capability to induce singlet oxygen production and act as photosensitizer. Hence, we investigated 2D BP in vitro effect on healthy and cancer prostate cell behavior. 2D BP was obtained through liquid exfoliation. 2D BP effect on healthy and cancer prostate cell behaviors was analyzed by investigating cell viability, oxidative stress and inflammatory marker expression. 2D BP inhibited prostate cancer cell survival, meanwhile promoted healthy prostate cell survival in vitro by modulating oxidative stress and immune response with and without near-infrared light (NIR)-irradiation. Nanostructured 2D BP is able to inhibit in vitro prostate cancer cells survival and preserve healthy prostate cell vitality through the control of oxidative stress and immune response, respectively.

An Intermediate Concentration of Calcium with Antioxidant Supplement in Culture Medium Enhances Proliferation and Decreases the Aging of Bone Marrow Mesenchymal Stem Cells.

Human bone marrow stem cells (HBMSCs) are isolated from the bone marrow. Stem cells can self-renew and differentiate into various types of cells. They are able to regenerate kinds of tissue that are potentially used for tissue engineering. To maintain and expand these cells under culture conditions is difficult-they are easily triggered for differentiation or death. In this study, we describe a new culture formula to culture isolated HBMSCs. This new formula was modified from NCDB 153, a medium with low calcium, supplied with 5% FBS, extra growth factor added to it, and supplemented with N-acetyl-L-cysteine and L-ascorbic acid-2-phosphate to maintain the cells in a steady stage. The cells retain these characteristics as primarily isolated HBMSCs. Moreover, our new formula keeps HBMSCs with high proliferation rate and multiple linage differentiation ability, such as osteoblastogenesis, chondrogenesis, and adipogenesis. It also retains HBMSCs with stable chromosome, DNA, telomere length, and telomerase activity, even after long-term culture. Senescence can be minimized under this new formulation and carcinogenesis of stem cells can also be prevented. These modifications greatly enhance the survival rate, growth rate, and basal characteristics of isolated HBMSCs, which will be very helpful in stem cell research.

Levels of Cyclooxygenase 2, Interleukin-6, and Tumour Necrosis Factor-α in Fibroblast Cell Culture Models after Photobiomodulation at 660 nm.

Chemicals and signaling molecules released by injured cells at the beginning of wound healing prompt inflammation. In diabetes, prolonged inflammation is one of the probable causes for delayed wound healing. Increased levels of cyclooxygenase-2 (cox-2), interleukin-6 (IL-6), and tumour necrosis factor-alpha (TNF-α) are associated with the inflammatory response and in diabetes, and increased levels of these contribute to chronic wounds that do not heal. Rising levels of cox-2, IL-6, and TNF-α have also been associated with increased oxidative stress. Photobiomodulation (PBM) may impact wound healing processes by affecting the signaling pathways and molecules pertinent to tissue repair. In the present study, the effect of PBM (wavelength: 660 nm; energy density: 5 J/cm2) on levels of cox-2, IL-6, and TNF-α was determined in fibroblast cell culture models. Four WS1 models (normal, normal wounded, diabetic, and diabetic wounded) were irradiated at 660 nm, and the culture media was collected at 0, 24, and 48 h postirradiation. Cells that were not irradiated (0 J/cm2) served as the controls. The following parameters were determined postirradiation: cell morphology using light microscopy, cell viability using the Trypan Blue exclusion assay, and levels of the inflammatory markers cox-2, IL-6, and TNF-α were measured using ELISA. Cell migration increased in the wounded groups over the 48 h interval after PBM; viability improved postirradiation in the diabetic wounded groups at 0 and 24 h (P ≤ 0.05 and P ≤ 0.01, respectively); levels of cox-2 decreased in normal and diabetic wounded groups at 0 h (P ≤ 0.001) and increased in the diabetic and diabetic wounded groups at 48 h postirradiation (P ≤ 0.05 and P ≤ 0.01, respectively), while levels of IL-6 decreased in the normal (P ≤ 0.01), diabetic (P ≤ 0.05), and diabetic wounded (P ≤ 0.001) groups at 24 h and in the diabetic and diabetic wounded groups at 48 h (P ≤ 0.05) postirradiation. TNF-α was decreased in the normal wounded groups (P ≤ 0.05) at 48 h. Through its effect on decreased IL-6 levels in diabetic cell models, PBM at 660 nm may be successful at decreasing oxidative stress; however, the present study also found an increase in cox-2 levels at 48 h postirradiation.

Hormesis of low-dose inhibition of pAkt1 (Ser473) followed by a great increase of proline-rich inositol polyphosphate 5-phosphatase (PIPP) level in oocytes.

Hormesis describes a biphasic dose-response relationship generally characterized by a low-dose excitement and a high-dose inhibition. This phenomenon has been observed in the regulation of cell, organ, and organismic level. However, hormesis has not reported in oocytes. In this study, we observed, for the first time, hormetic responses of PIPP levels in oocytes by inhibitor of Akt1 or PKCδ. The expression of PIPP was detected by qPCR, immunofluorescent (IF), and Western Blot (WB). To observe the changes of PIPP levels, we used the inhibitors against pAkt1 (Ser473) or PKCδ, SH-6 or sotrastaurin with low and/or high-dose, treated GV oocytes and cultured for 4 h, respectively. The results showed that PIPP expression was significantly enhanced when oocytes were treated with SH-6 or sotrastaurin 10 µM, but decreased with SH-6 or sotrastaurin 100 µM. We also examined the changes of PIPP levels when GV oocytes were treated with exogenous PtdIns(3,4,5)P3 or LY294002 for 4 h. Our results showed that PIPP level was enhanced much higher under the treatment of 0.1 µM PtdIns(3,4,5)P3 than that of 1 µM PtdIns(3,4,5)P3, which is consistent with the changes of PIPP when oocytes were treated with inhibitors of pAkt1 (Ser473) or PKCδ. In addition, with PIPP siRNA, we detected that down-regulated PIPP may affect distributions of Akt, Cdc25, and pCdc2 (Tyr15). Taken together, these results show that the relationships between PIPP and Akt may follow the principle of hormesis and play a key role during release of diplotene arrest in mouse oocytes.

Gymnema inodorum (Lour.) Decne. Extract Alleviates Oxidative Stress and Inflammatory Mediators Produced by RAW264.7 Macrophages.

Gymnema inodorum (Lour.) Decne. (G. inodorum) is widely used in Northern Thai cuisine as local vegetables and commercial herb tea products. In the present study, G. inodorum extract (GIE) was evaluated for its antioxidant and anti-inflammatory effects in LPS plus IFN-γ-induced RAW264.7 cells. Major compounds in GIE were evaluated using GC-MS and found 16 volatile compounds presenting in the extract. GIE exhibited antioxidant activity by scavenging the intracellular reactive oxygen species (ROS) production and increasing superoxide dismutase 2 (SOD2) mRNA expression in LPS plus IFN-γ-induced RAW264.7 cells. GIE showed anti-inflammatory activity through suppressing nitric oxide (NO), proinflammatory cytokine production interleukin 6 (IL-6) and also downregulation of the expression of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and IL-6 mRNA levels in LPS plus IFN-γ-induced RAW264.7 cells. Mechanism studies showed that GIE suppressed the NF-κB p65 nuclear translocation and slightly decreased the phosphorylation of NF-κB p65 (p-NF-κB p65) protein. Our studies applied the synchrotron radiation-based FTIR microspectroscopy (SR-FTIR), supported by multivariate analysis, to identify the FTIR spectral changes based on macromolecule alterations occurring in RAW264.7 cells. SR-FTIR results demonstrated that the presence of LPS plus IFN-γ in RAW264.7 cells associated with the increase of amide I/amide II ratio (contributing to the alteration of secondary protein structure) and lipid content, whereas glycogen and other carbohydrate content were decreased. These findings lead us to believe that GIE may prevent oxidative damage by scavenging intracellular ROS production and activating the antioxidant gene, SOD2, expression. Therefore, it is possible that the antioxidant properties of GIE could modulate the inflammation process by regulating the ROS levels, which lead to the suppression of proinflammatory cytokines and genes. Therefore, GIE could be developed into a novel antioxidant and anti-inflammatory agent to treat and prevent diseases related to oxidative stress and inflammation.

Anti-senescent drug screening by deep learning-based morphology senescence scoring.

Advances in deep learning technology have enabled complex task solutions. The accuracy of image classification tasks has improved owing to the establishment of convolutional neural networks (CNN). Cellular senescence is a hallmark of ageing and is important for the pathogenesis of ageing-related diseases. Furthermore, it is a potential therapeutic target. Specific molecular markers are used to identify senescent cells. Moreover senescent cells show unique morphology, which can be identified. We develop a successful morphology-based CNN system to identify senescent cells and a quantitative scoring system to evaluate the state of endothelial cells by senescence probability output from pre-trained CNN optimised for the classification of cellular senescence, Deep Learning-Based Senescence Scoring System by Morphology (Deep-SeSMo). Deep-SeSMo correctly evaluates the effects of well-known anti-senescent reagents. We screen for drugs that control cellular senescence using a kinase inhibitor library by Deep-SeSMo-based drug screening and identify four anti-senescent drugs. RNA sequence analysis reveals that these compounds commonly suppress senescent phenotypes through inhibition of the inflammatory response pathway. Thus, morphology-based CNN system can be a powerful tool for anti-senescent drug screening.

Seasonal changes in adenosine kinase in tanycytes of the Arctic ground squirrel (Urocitellus parryii).

Hibernation is a seasonal strategy to conserve energy, characterized by modified thermoregulation, an increase in sleep pressure and drastic metabolic changes. Glial cells such as astrocytes and tanycytes are the brain metabolic sensors, but it remains unknown whether they contribute to seasonal expression of hibernation. The onset of hibernation is controlled by an undefined endogenous circannual rhythm in which adenosine plays a role through the activation of the A1 adenosine receptor (A1AR). Seasonal changes in brain levels of adenosine may contribute to an increase in A1AR sensitivity leading to the onset of hibernation. The primary regulator of extracellular adenosine concentration is adenosine kinase, which is located in astrocytes. Using immunohistochemistry to localize and quantify adenosine kinase in Arctic ground squirrels' brain collected during different seasons, we report lower expression of adenosine kinase in the third ventricle tanycytes in winter compared to summer; a similar change was not seen in astrocytes. Moreover, for the first time, we describe adenosine kinase expression in tanycyte cell bodies in the hypothalamus and in the area postrema, both brain regions involved in energy homeostasis. Next we describe seasonal changes in tanycyte morphology in the hypothalamus. Although still speculative, our findings contribute to a model whereby adenosine kinase in tanycytes regulates seasonal changes in extracellular concentration of adenosine underling the seasonal expression of hibernation.

A GPAT1 Mutation in Arabidopsis Enhances Plant Height but Impairs Seed Oil Biosynthesis.

Glycerol-3-phosphate acyltransferases (GPATs) play an important role in glycerolipid biosynthesis, and are mainly involved in oil production, flower development, and stress response. However, their roles in regulating plant height remain unreported. Here, we report that Arabidopsis GPAT1 is involved in the regulation of plant height. GUS assay and qRT-PCR analysis in Arabidopsis showed that GPAT1 is highly expressed in flowers, siliques, and seeds. A loss of function mutation in GPAT1 was shown to decrease seed yield but increase plant height through enhanced cell length. Transcriptomic and qRT-PCR data revealed that the expression levels of genes related to gibberellin (GA) biosynthesis and signaling, as well as those of cell wall organization and biogenesis, were significantly upregulated. These led to cell length elongation, and thus, an increase in plant height. Together, our data suggest that knockout of GPAT1 impairs glycerolipid metabolism in Arabidopsis, leading to reduced seed yield, but promotes the biosynthesis of GA, which ultimately enhances plant height. This study provides new evidence on the interplay between lipid and hormone metabolism in the regulation of plant height.