Anti-Aging Constituents from Pinus morrisonicola Leaves.

Pinus morrisonicola Hayata is a unique plant species found in Taiwan. Previous studies have identified its anti-hypertensive, anti-oxidative, and anti-inflammatory effects. In this study, a bioactivity-guided approach was employed to extract 20 compounds from the ethyl acetate fraction of the ethanol extract of Pinus morrisonicola Hayata's pine needles. The anti-aging effects of these compounds were investigated using HT-1080 cells. The structures of the purified compounds were confirmed through NMR and LC-MS analysis, revealing the presence of nine flavonoids, two lignans, one coumarin, one benzofuran, one phenylic acid, and six diterpenoids. Among them, PML18, PML19, and PML20 were identified as novel diterpene. Compounds 3, 4, and 5 exhibited remarkable inhibitory effects against MMP-2 and showed no significant cell toxicity at 25 µM. Although the purified compounds showed lower activity against Pro MMP-2 and Pro MMP-9 compared to the ethyl acetate fraction, we speculate that this is the result of synergistic effects.

Cytotoxicity Effect of Constituents of Pinus taiwanensis Hayata Twigs on B16-F10 Melanoma Cells.

Pinus taiwanensis Hayata (Pinaceae) is an endemic plant in Taiwan. According to the Chinese Materia Medica Grand Dictionary, the Pinus species is mainly used to relieve pain, and eliminate pus and toxicity. In this study, nineteen compounds were isolated from the ethyl acetate layer of the ethanolic extract of P. taiwanensis Hayata twigs using bioassay-guided fractionation, and their anti-melanoma effects were investigated through a B16-F10 mouse melanoma cell model. The structures of the purified compounds were identified by 2D-NMR, MS, and IR, including 1 triterpenoid, 9 diterpenoids, 2 lignans, 4 phenolics, 1 phenylpropanoid, 1 flavonoid, and 1 steroid. Among them, compound 3 was found to be a new diterpene. Some of the compounds (2, 5, 6, 17, 18) showed moderate cytotoxicity effects. On the other hand, the anti-melanoma effect was no better than that from the original ethyl acetate layer. We presumed it resulted from the synergistic effect, although further experimentation needs to be performed.

New Trichothecenes Isolated from the Marine Algicolous Fungus Trichoderma brevicompactum.

Eight trichothecenes, including four new compounds 1-4 and four known entities 5-8, together with one known cyclonerane (9) were isolated from the solid-state fermentation of Trichoderma brevicompactum NTU439 isolated from the marine alga Mastophora rosea. The structures of 1-9 were determined by 1D/2D NMR (nuclear magnetic resonance), MS (mass spectrometry), and IR (infrared spectroscopy) spectroscopic data. All of the compounds were evaluated for cytotoxic activity against HCT-116, PC-3, and SK-Hep-1 cancer cells by the SRB assay, and compound 8 showed promising cytotoxic activity against all three cancer cell lines with the IC50 values of 3.3 ± 0.3, 5.3 ± 0.3, and 1.8 ± 0.8 µM, respectively. Compounds 1-2, 4-6, and 7-8 potently inhibited LPS-induced NO production, and compounds 5 and 8 showed markedly inhibited gelatinolysis of MMP-9 in S1 protein-stimulated THP-1 monocytes.

The Risk of Postpartum Hemorrhage Following Prior Prelabor Cesarean Delivery Stratified by Abnormal Placentation: A Multicenter Historical Cohort Study.

Background: Prior prelabor cesarean delivery (CD) was associated with an increase in the risk of placenta previa (PP) in a second delivery, whether it may impact postpartum hemorrhage (PPH) independent of abnormal placentation. This study aimed to assess the risk of PPH stratified by abnormal placentation following a first CD before the onset of labor (prelabor) or intrapartum CD. Methods: This multicenter, historical cohort study involved singleton, pregnant women at 28 weeks of gestation or greater with a CD history between January 2017 and December 2017 in 11 public tertiary hospitals within 7 provinces of China. PPH was analyzed in the subsequent pregnancy between women with prior prelabor CD and women with intrapartum CD. Furthermore, PPH was analyzed in pregnant women stratified by complications with PP alone [without placenta accreta spectrum (PAS) disorders], complications with PP and PAS, complications with PAS alone (without PP), and normal placentation. We performed multivariate logistic regression to calculate adjusted odds ratios (aOR) and 95% CI controlling for predefined covariates. Results: Out of 10,833 pregnant women, 1,197 (11%) women had a history of intrapartum CD and 9,636 (89%) women had a history of prelabor CD. Prior prelabor CD increased the risk of PP (aOR 1.91, 95% CI 1.40-2.60), PAS (aOR 1.68, 95% CI 1.11-2.24), and PPH (aOR 1.33, 95% CI 1.02-1.75) in a subsequent pregnancy. After stratification by complications with PP alone, PP and PAS, PAS alone, and normal placentation, prior prelabor CD only increased the risk of PPH (aOR 3.34, 95% CI 1.35-8.23) in a subsequent pregnancy complicated with PP and PAS. Conclusion: Compared to intrapartum CD, prior prelabor CD increased the risk of PPH in a subsequent pregnancy only when complicated by PP and PAS.

Constituents of Aquilaria sinensis Leaves Upregulate the Expression of Matrix Metalloproteases 2 and 9.

In this novel study, we isolated 28 compounds from the leaves of Aquilaria sinensis (Lour.) Gilg based on a bioassay-guided procedure and also discovered the possible matrix metalloprotease 2 (MMP-2) and 9 (MMP-9) modulatory effect of pheophorbide A (PA). To evaluate the regulatory activity on MMP-2 and MMP-9, the HT-1080 human fibrosarcoma cells were treated with various concentrations of extracted materials and isolated compounds. PA was extracted by methanol from the leaves of A. sinensis and separated from the fraction of the partitioned ethyl acetate layer. PA is believed to be an active component for MMP expression since it exhibited significant stimulation on MMP-2 and proMMP-9 activity. When treating with 50 µM of PA, the expression of MMP-2 and MMP-9 were increased 1.9-fold and 2.3-fold, respectively. PA also exhibited no cytotoxicity against HT-1080 cells when the cell viability was monitored. Furthermore, no significant MMP activity was observed when five PA analogues were evaluated. This study is the first to demonstrate that C-17 of PA is the deciding factor in determining the bioactivity of the compound. The MMP-2 and proMMP-9 modulatory activity of PA indicate its potential applications for reducing scar formation and comparative medical purposes.

Determination of phenylalanine enantiomers in the plasma and urine of mammals and á´ -amino acid oxidase deficient rodents using two-dimensional high-performance liquid chromatography.

A two-dimensional (2D) HPLC system focusing on the determination of phenylalanine (Phe) enantiomers in mammalian physiological fluids has been developed. ᴅ-Phe is indicated to have potential values as a disease biomarker and therapeutic molecule in several neuronal and metabolic disorders, thus the regulation of ᴅ-Phe in mammals is a matter of interest. However, the precise determination of amino acid enantiomers is difficult in complex biological samples, and the development of an analytical method with practically acceptable sensitivity, selectivity and throughput is expected. In the present study, a 2D-HPLC system equipped with a reversed-phase column in the 1st dimension and an enantioselective column in the 2nd dimension has been designed, following the fluorescence derivatization of the target amino acid enantiomers with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). The analytical method was validated using both plasma and urine samples, and successfully applied to human, rat and mouse fluids. Trace levels of ᴅ-Phe were determined in the plasma, and the %ᴅ values were around 0.1% for all species. In the urine, relatively large amounts of ᴅ-Phe were observed, and the %ᴅ values for humans, rats and mice were 3.99, 1.76 and 5.25%, respectively. The relationships between the enzymatic activity of ᴅ-amino acid oxidase (DAO) and the amounts of intrinsic ᴅ-Phe have also been clarified, and high ᴅ-Phe amounts were observed (around 0.3% in the plasma and around 50% in the urine) in the DAO deficient rats and mice.

Seroepidemiological and molecular investigation of spotted fever group rickettsiae and Coxiella burnetii in Sao Tome Island: A One Health approach.

Spotted fever group rickettsiae (SFGR) and Coxiella burnetii are intracellular bacteria that cause potentially life-threatening tick-borne rickettsioses and Q fever respectively. Sao Tome and Principe (STP), small islands located in the Gulf of Guinea, recently experienced a dramatic reduction in the incidence of malaria owing to international collaborative efforts. However, unexplained febrile illnesses persist. A One Health approach was adopted to investigate exposure to SFGR and C. burnetii in humans and examine the diversity of these bacteria in ticks parasitizing domestic ruminants. A cross-sectional human serological study was conducted in Agua Grande district in Sao Tome Island from January to March 2016, and ticks were collected from farmed domestic ruminants in 2012 and 2016. In total, 240 individuals varying in age were randomly screened for exposure to SFGR and C. burnetii by indirect immunofluorescence assay. Twenty of 240 individuals (8.3%) were seropositive for SFGR (4 for Rickettsia africae and 16 for R. conorii) and 16 (6.7%) were seropositive for C. burnetii. Amblyomma astrion were collected exclusively in 2012, as were A. variegatum in 2016 and Rickettsia spp. were detected in 22/42 (52.4%) and 49/60 (81.7%) respectively. Sequence analysis of multiple gene targets from Rickettsia spp. detected in ticks suggests the presence of a single divergent R. africae strain (Sao Tome). While no ticks were found positive for C. burnetii, Coxiella-like endosymbionts were detected in nearly all ticks. This is the first study in STP to provide serological evidence in humans of SFGR and C. burnetii and additional molecular evidence in ticks for SFGR, which may be responsible for some of the unexplained febrile illnesses that persist despite the control of malaria. Future epidemiological studies are needed to confirm the occurrence and risk factors associated with SFG rickettsioses and Q fever in both humans and animals.

Bupivacaine induces apoptosis through caspase-dependent and -independent pathways in canine mammary tumor cells.

Local anesthetics have been reported to induce apoptosis in various cell lines. In this study, we showed that bupivacaine also induced apoptosis in DTK-SME cells, a vimentin(+)/AE1(+)/CK7(+)/HSP27(+), tumorigenic, immortalized, canine mammary tumor cell line. Bupivacaine induced apoptosis in DTK-SME cells in a time- and concentration-dependent manner. Apoptosis-associated morphological changes, including cell shrinkage and rounding, chromatin condensation, and formation of apoptotic bodies, were observed in the bupivacaine-treated DTK-SME cells. Apoptosis was further confirmed with annexin V staining, TUNEL staining, and DNA laddering assays. At the molecular level, the activation of caspases-3, -8, and -9 corresponded well to the degree of DNA fragmentation triggered by bupivacaine. We also demonstrated that the pan-caspase inhibitor, z-VAD-fmk, only partially inhibited the apoptosis induced by bupivacaine. Moreover, treated cells increased expression of endonuclease G, a death effector that acts independently of caspases. Our data suggested that bupivacaine-induced apoptosis occurs through both caspase-dependent and caspase-independent apoptotic pathways.

Canine heat shock protein 27 promotes proliferation, migration, and doxorubicin resistance in the canine cell line DTK-F.

Canine mammary tumors (CMTs) are the most common type of tumors in female dogs. Heat shock proteins are highly expressed in many cancers and are involved in tumor progression and chemoresistance in CMTs; however, the biological role of canine heat shock protein 27 (cHSP27) in CMTs has not been thoroughly characterized. This study investigated the roles of cHSP27 in cell growth, migration, anchorage, and resistance to doxorubicin (DOX) using DTK-F cells, a CMT cell line that does not express cHSP27. DTK-F cells were transfected with cHSP27 and stable overexpression was established. A mouse monoclonal antibody against cHSP27 was also produced. The biological functions of cHSP27 in DTK-F cells were then evaluated using a variety of assays. Overexpression of cHSP27 was associated with increased cell proliferation, clone formation, migration, and decreased DOX sensitivity. In conclusion, these data provide evidence that cHSP27 overexpression can promote anchorage-independent growth, migration, and increased DOX resistance in CMT cells.

Critical involvement of ILK in TGFbeta1-stimulated invasion/migration of human ovarian cancer cells is associated with urokinase plasminogen activator system.

The present study investigated the role of integrin-linked kinase (ILK) in TGFbeta1-stimulated invasion/migration of human ovarian cancer cells. We investigated TGFbeta1 regulation of ILK, and effects of ILK knockdown on TGFbeta1-stimulated invasion/migration and the associated proteinase systems, urokinase plasminogen activator (uPA) and matrix metalloproteinases (MMPs) in SKOV3 cells. TGFbeta1 stimulated ILK kinase activity, and had no effect on ILK protein/mRNA levels. Transient transfection of an ILK-specific siRNA (ILK-H) reduced ILK protein level, mRNA level and kinase activity. ILK knockdown by ILK-H suppressed the basal and TGFbeta1-stimulated invasion and migration. Further, ILK-H reduced the basal and TGFbeta1-stimulated secretion of uPA, and increased the secretion of its inhibitor (PAI-1). Conversely, ILK-H did not affect TGFbeta1-stimulated secretion of MMP2 and its cell-associated activator MT1-MMP. Additionally, TGFbeta1 activated Smad2 phosphorylation, and this was not affected by ILK knockdown. Earlier reports indicate that Smad2 activation increased the expression of MMP2 and MT1-MMP. Thus, TGFbeta1 may act through ILK-independent and Smad2-dependent signaling in regulating MMP2 and MT1-MMP in SKOV3 cells. Collectively, this study suggests that ILK serves as a key mediator in TGFbeta1 regulation of uPA/PAI-1 system critical for the invasiveness of human ovarian cancer cells. And ILK is a potential target for cancer therapy.

Time of day modulates low-temperature Ca signals in Arabidopsis.

We tested the hypothesis that the circadian clock modulates Ca(2+)-based signalling pathways, using low-temperature (LT)-induced Ca(2+) signals. We investigated the relationship between diurnal and circadian modulation of LT-induced increases in cytosolic-free calcium ([Ca(2+)](cyt)), and regulation of [Ca(2+)](cyt)-dependent outputs of the LT-signalling network (RD29A transcript abundance and stomatal closure). We measured [Ca(2+)](cyt) non-invasively using aequorin, and targeted aequorin to the guard cell using a guard cell-specific GAL4-green fluorescent protein enhancer trap line. LT caused transient increases in whole plant and guard cell [Ca(2+)](cyt). In guard cells, the LT-induced [Ca(2+)](cyt) elevation preceded stomatal closure. In whole plants, the magnitude of LT-induced [Ca(2+)](cyt) transients, measured from the entire plant or specifically the guard cell, varied with the time of day: LT-induced [Ca(2+)](cyt) transients were significantly higher during the mid-photoperiod than at the beginning or end. Diurnal variation in LT-induced guard cell [Ca(2+)](cyt) increases was not correlated to diurnal variation in LT-induced stomatal closure. There was circadian modulation of LT-induced whole plant [Ca(2+)](cyt) increases, which were correlated to the circadian pattern of RD29A induction. In order to understand the significance of LT-induced [Ca(2+)](cyt) increases, we used a computer simulation to demonstrate that, in guard cells, LT-induced [Ca(2+)](cyt) increases measured from a population of cells are likely to represent the summation of cold-induced single-cell [Ca(2+)](cyt) oscillations.

The modulatory role of transforming growth factor beta1 and androstenedione on follicle-stimulating hormone-induced gelatinase secretion and steroidogenesis in rat granulosa cells.

To investigate the potential roles of matrix metalloproteinases (MMPs) in ovarian granulosa cell differentiation, we studied the interactive effects of FSH and local ovarian factors, transforming growth factor beta1 (TGFbeta1) and androstenedione, on gelatinase secretion and progesterone production in rat ovarian granulosa cells. Granulosa cells of eCG-primed immature rats were treated once with various doses of FSH and TGFbeta1 and androstenedione alone or in combinations for 2 days. Conditioned media were analyzed for gelatinase activity using gelatin-zymography/densitometry and progesterone levels using enzyme immunoassay. Cell lysates were analyzed for steroidogenic acute regulatory (StAR) and cholesterol side-chain-cleavage (P450scc) enzyme protein levels. This study demonstrates for the first time that FSH dose-dependently increased the secretion of a major 63-kDa gelatinase and minor 92- and 67-kDa gelatinases. TGFbeta1 also dose-dependently increased the secretion of 63-kDa gelatinase, while androstenedione alone had no effect. The 92-kDa gelatinase was identified as the pro-MMP9 that could be cleaved by aminophenylmercuric acetate into the 83-kDa active form. Importantly, we show that TGFbeta1 and androgen act in an additive manner to enhance FSH stimulatory effects both on the secretion of gelatinases and the production of progesterone. We further show by immunoblotting that the enhancing effect of TGFbeta1 and androstenedione on FSH-stimulated steroidogenesis is partly mediated through the increased level of StAR protein and/or P450scc enzyme. In conclusion, this study indicates that, during antral follicle development, TGFbeta1 and androgen act to enhance FSH promotion of granulosa cell differentiation and that the process may involve the interplay of modulating cell- to-matrix/cell-to-cell interaction and steroidogenic activity.