Juniperus communis extract induces cell cycle arrest and apoptosis of colorectal adenocarcinoma in vitro and in vivo.

Juniperus communis (JCo) is a well-known traditional Chinese medicinal plant that has been used to treat wounds, fever, swelling, and rheumatism. However, the mechanism underlying the anticancer effect of JCo extract on colorectal cancer (CRC) has not yet been elucidated. This study investigated the anticancer effects of JCo extract in vitro and in vivo as well as the precise molecular mechanisms. Cell viability was evaluated using the MTT assay. Cell cycle distribution was examined by flow cytometry analysis, and cell apoptosis was determined by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Protein expression was analyzed using western blotting. The in vivo activity of the JCo extract was evaluated using a xenograft BALB/c mouse model. The tumors and organs were examined through hematoxylin-eosin (HE) staining and immunohistochemistry. The results showed that JCo extract exhibited higher cytotoxicity against CRC cells than against normal cells and showed synergistic effects when combined with 5-fluorouracil. JCo extract induced cell cycle arrest at the G0/G1 phase via regulation of p53/p21 and CDK4/cyclin D1 and induced cell apoptosis via the extrinsic (FasL/Fas/caspase-8) and intrinsic (Bax/Bcl-2/caspase-9) apoptotic pathways. In vivo studies revealed that JCo extract suppressed tumor growth through the inhibition of proliferation and induction of apoptosis. In addition, there was no obvious change in body weight or histological morphology of normal organs after treatment. JCo extract suppressed CRC progression by inducing cell cycle arrest and apoptosis in vitro and in vivo, suggesting the potential application of JCo extract in the treatment of CRC.

Cedrus atlantica Extract Suppress Glioblastoma Growth through Promotion of Genotoxicity and Apoptosis: In Vitro and In Vivo Studies.

Glioblastoma (GBM) is the most common malignant primary brain tumor in humans, exhibiting highly infiltrative growth and drug resistance to conventional chemotherapy. Cedrus atlantica (CAt) extract has been shown to decrease postoperative pain and inhibit the growth of K562 leukemia cells. The aim of this study was to assess the anti-GBM activity and molecular mechanism of CAt extract in vitro and in vivo. The results showed that CAt extract greatly suppressed GBM cells both in vitro and in vivo and enhanced the survival rate in subcutaneous and orthotopic animal models. Moreover, CAt extract increased the level of ROS and induced DNA damage, resulting in cell cycle arrest at the G0/G1 phase and cell apoptosis. Western blotting results indicated that CAt extract regulates p53/p21 and CDK4/cyclin D1 protein expression and activates extrinsic and intrinsic apoptosis. Furthermore, CAt extract enhanced the cytotoxicity of Temozolomide and decreased AKT/mTOR signaling by combination treatment. In toxicity assays, CAt extract exhibited low cytotoxicity toward normal cells or organs in vitro and in vivo. CAt extract suppresses the growth of GBM by induction of genotoxicity and activation of apoptosis. The results of this study suggest that CAt extract can be developed as a therapeutic agent or adjuvant for GBM treatment in the future.

Positive end-expiratory pressure titration with electrical impedance tomography and pressure-volume curve: a randomized trial in moderate to severe ARDS.

OBJECTIVE: The aim of the study was to compare titration of positive end-expiratory pressure (PEEP) with electrical impedance tomography (EIT) and with ventilator-embedded pressure-volume (PV) loop in moderate to severe acute respiratory distress syndrome (ARDS). APPROACH: Eighty-seven moderate to severe ARDS patients (arterial oxygen partial pressure to fractional inspired oxygen ratio, PaO2/FiO2 ≤ 200 mmHg) were randomized to either EIT group (n = 42) or PV group (n = 45). All patients received identical medical care using the same general support guidelines and protective mechanical ventilation. In the EIT group, the selected PEEP equaled the airway pressure at the intercept between cumulated collapse and overdistension percentages curves and in the PV group, at the pressure where maximal hysteresis was reached. MAIN RESULTS: Baseline characteristics and settings were comparable between the groups. After optimization, PEEP was significantly higher in the PV group (17.4 ± 1.7 versus 16.2 ± 2.6 cmH2O, PV versus EIT groups, p = 0.02). After 48 h, driving pressure was significantly higher in the PV group (12.4 ± 3.6 versus 10.9 ± 2.5 cmH2O, p = 0.04). Lung mechanics and oxygenation were better in the EIT group but did not statistically differ between the groups. The survival rate was lower in the PV group (44.4% versus 69.0%, p = 0.02; hazard ratio 2.1, confidence interval 1·1-3.9). None of the other pre-specified exploratory clinical endpoints were significantly different. SIGNIFICANCE: In moderate to severe ARDS, PEEP titration guided with EIT, compared with PV curve, might be associated with improved driving pressure and survival rate. TRIAL REGISTRATION: NCT03112512, 13 April, 2017.

Juniperus communis extract induces cell cycle arrest and apoptosis of colorectal adenocarcinoma in vitro and in vivo

Juniperus communis (JCo) is a well-known traditional Chinese medicinal plant that has been used to treat wounds, fever, swelling, and rheumatism. However, the mechanism underlying the anticancer effect of JCo extract on colorectal cancer (CRC) has not yet been elucidated. This study investigated the anticancer effects of JCo extract in vitro and in vivo as well as the precise molecular mechanisms. Cell viability was evaluated using the MTT assay. Cell cycle distribution was examined by flow cytometry analysis, and cell apoptosis was determined by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Protein expression was analyzed using western blotting. The in vivo activity of the JCo extract was evaluated using a xenograft BALB/c mouse model. The tumors and organs were examined through hematoxylin-eosin (HE) staining and immunohistochemistry. The results showed that JCo extract exhibited higher cytotoxicity against CRC cells than against normal cells and showed synergistic effects when combined with 5-fluorouracil. JCo extract induced cell cycle arrest at the G0/G1 phase via regulation of p53/p21 and CDK4/cyclin D1 and induced cell apoptosis via the extrinsic (FasL/Fas/caspase-8) and intrinsic (Bax/Bcl-2/caspase-9) apoptotic pathways. In vivo studies revealed that JCo extract suppressed tumor growth through the inhibition of proliferation and induction of apoptosis. In addition, there was no obvious change in body weight or histological morphology of normal organs after treatment. JCo extract suppressed CRC progression by inducing cell cycle arrest and apoptosis in vitro and in vivo, suggesting the potential application of JCo extract in the treatment of CRC.

Silencing of PhLA, a CIN-TCP gene, causes defected petal conical epidermal cell formation and results in reflexed corolla lobes in petunia.

BACKGROUND: TCP-domain proteins, plant specific transcription factors, play important roles in various developmental processes. CIN-TCPs control leaf curvature in simple leaf species while regulate leaf complexity in compound leaf species. However, the knowledge was largely based on findings in few model species. To extend our knowledge on this group of proteins in Solanaceae species, we identified a CIN-TCP gene from petunia, and studied its functions using virus-induced gene silencing (VIGS). RESULTS: Consistently, silencing of CIN-TCPs increases complexity of tomato leaves, and enhances leaf curvature in Nicotiana benthamiana. However, in petunia (Petunia hybrida), silencing of petunia LA, a CIN-TCP, through VIGS did not obviously affect leaf shape. The silencing, however, enhanced petal curvature. The event was associated with petal expansion at the distal portion where epidermal cell size along the midribs was also increased. The enlarged epidermal cells became flattened. Although shapes of PhLA-silenced flowers largely resemble phmyb1 mutant phenotype, PhMYB1 expression was not affected when PhLA was specifically silenced. Therefore, both PhLA and PhMYB1 are required to regulate flower morphology. In corolla, PhLA and miR319 deferentially express in different regions with strong expressions in limb and tube region respectively. CONCLUSIONS: In conclusion, unlike LA-like genes in tomato and N. benthamiana, PhLA plays a more defined role in flower morphogenesis, including petal curvature and epidermal cell differentiation.

Silencing of CrNPR1 and CrNPR3 Alters Plant Susceptibility to Periwinkle Leaf Yellowing Phytoplasma.

Phytoplasmas are prokaryotic plant pathogens that cause considerable loss in many economically important crops, and an increasing number of phytoplasma diseases are being reported on new hosts. Knowledge of plant defense mechanisms against such pathogens should help to improve strategies for controlling these diseases. Salicylic acid (SA)-mediated defense may play an important role in defense against phytoplasmas. Here, we report that SA accumulated in Madagascar periwinkle (Catharanthus roseus) infected with periwinkle leaf yellowing (PLY) phytoplasma. CrPR1a expression was induced in both symptomatic and non-symptomatic tissues of plants exhibiting PLY. NPR1 plays a central role in SA signaling, and two NPR1 homologs, CrNPR1 and CrNPR3, were identified from a periwinkle transcriptome database. Similar to CrPR1a, CrNPR1 expression was also induced in both symptomatic and non-symptomatic tissues of plants exhibiting PLY. Silencing of CrNPR1, but not CrNPR3, significantly repressed CrPR1a induction in Tobacco rattle virus-infected periwinkle plants. In addition, symptoms of PLY progressed fastest in CrNPR1-silenced plants and slowest in CrNPR3-silenced plants. Consistently, expression of CrNPR1, but not CrNPR3, was induced by phytoplasma infection as well as SA treatment. This study highlights the importance of NPR1- and SA-mediated defense against phytoplasma in periwinkle and offers insight into plant-phytoplasma interactions to improve disease control strategies.

Genetic Analysis of Floral Symmetry Transition in African Violet Suggests the Involvement of Trans-acting Factor for CYCLOIDEA Expression Shifts.

With the growing demand for its ornamental uses, the African violet (Saintpaulia ionantha) has been popular owing to its variations in color, shape and its rapid responses to artificial selection. Wild type African violet (WT) is characterized by flowers with bilateral symmetry yet reversals showing radially symmetrical flowers such as dorsalized actinomorphic (DA) and ventralized actinomorphic (VA) peloria are common. Genetic crosses among WT, DA, and VA revealed that these floral symmetry transitions are likely to be controlled by three alleles at a single locus in which the levels of dominance are in a hierarchical fashion. To investigate whether the floral symmetry gene was responsible for these reversals, orthologs of CYCLOIDEA (CYC) were isolated and their expressions correlated to floral symmetry transitions. Quantitative RT-PCR and in situ results indicated that dorsal-specific CYCs expression in WT S. ionantha (SiCYC and SiCYC1B) shifted in DA with a heterotopically extended expression to all petals, but in VA, SiCYC1s' dorsally specific expressions were greatly reduced. Selection signature analysis revealed that the major high-expressed copy of SiCYC had been constrained under purifying selection, whereas the low-expressed helper SiCYC1B appeared to be relaxed under purifying selection after the duplication into SiCYC and SiCYC1B. Heterologous expression of SiCYC in Arabdiopsis showed petal growth retardation which was attributed to limited cell proliferation. While expression shifts of SiCYC and SiCYC1B correlate perfectly to the resulting symmetry phenotype transitions in F1s of WT and DA, there is no certain allelic combination of inherited SiCYC1s associated with specific symmetry phenotypes. This floral transition indicates that although the expression shifts of SiCYC/1B are responsible for the two contrasting actinomorphic reversals in African violet, they are likely to be controlled by upstream trans-acting factors or epigenetic regulations.

Collagen microsphere production on a chip.

We have developed an integrated microfluidic material processing chip and demonstrated the rapid production of collagen microspheres encapsulating cells with high uniformity and cell viability. The chip integrated three material processing steps. Monodisperse microdroplets were generated at a microfluidic T junction between aqueous and mineral oil flows. The flow was heated immediately to 37 °C to initiate collagen fiber assembly within a gelation channel. Gelled microspheres were extracted from the mineral oil phase into cell culture media within an extraction chamber. Collagen gelation immediately after microdroplet generation significantly reduced coalescence among microdroplets that led to non-uniform microsphere production. The microfluidic extraction approach led to higher microsphere recovery and cell viability than when a conventional centrifugation extraction approach was employed. These results indicate that chip-based material processing is a promising approach for cell-ECM microenvironment generation for applications such as tissue engineering and stem cell delivery.

Dependence of cyclic stretch-induced stress fiber reorientation on stretch waveform.

Cyclic uniaxial stretching of adherent nonmuscle cells induces the gradual reorientation of their actin stress fibers perpendicular to the stretch direction to an extent dependent on stretch frequency. By subjecting cells to various temporal waveforms of cyclic stretch, we revealed that stress fibers are much more sensitive to strain rate than strain frequency. By applying asymmetric waveforms, stress fibers were clearly much more responsive to the rate of lengthening than the rate of shortening during the stretch cycle. These observations were interpreted using a theoretical model of networks of stress fibers with sarcomeric structure. The model predicts that stretch waveforms with fast lengthening rates generate greater average stress fiber tension than that generated by fast shortening. This integrated approach of experiment and theory provides new insight into the mechanisms by which cells respond to matrix stretching to maintain tensional homeostasis.

Stretch-induced stress fiber remodeling and the activations of JNK and ERK depend on mechanical strain rate, but not FAK.

BACKGROUND: Cells within tissues are subjected to mechanical forces caused by extracellular matrix deformation. Cells sense and dynamically respond to stretching of the matrix by reorienting their actin stress fibers and by activating intracellular signaling proteins, including focal adhesion kinase (FAK) and the mitogen-activated proteins kinases (MAPKs). Theoretical analyses predict that stress fibers can relax perturbations in tension depending on the rate of matrix strain. Thus, we hypothesized stress fiber organization and MAPK activities are altered to an extent dependent on stretch frequency. PRINCIPAL FINDINGS: Bovine aortic endothelial cells and human osteosarcoma cells expressing GFP-actin were cultured on elastic membranes and subjected to various patterns of stretch. Cyclic stretching resulted in strain rate-dependent increases in stress fiber alignment, cell retraction, and the phosphorylation of the MAPKs JNK, ERK and p38. Transient step changes in strain rate caused proportional transient changes in the levels of JNK and ERK phosphorylations without affecting stress fiber organization. Disrupting stress fiber contractile function with cytochalasin D or Y27632 decreased the levels of JNK and ERK phosphorylation. Previous studies indicate that FAK is required for stretch-induced cell alignment and MAPK activations. However, cyclic uniaxial stretching induced stress fiber alignment and the phosphorylation of JNK, ERK and p38 to comparable levels in FAK-null and FAK-expressing mouse embryonic fibroblasts. CONCLUSIONS: These results indicate that cyclic stretch-induced stress fiber alignment, cell retraction, and MAPK activations occur as a consequence of perturbations in fiber strain. These findings thus shed new light into the roles of stress fiber relaxation and reorganization in maintenance of tensional homeostasis in a dynamic mechanical environment.

A dynamic stochastic model of frequency-dependent stress fiber alignment induced by cyclic stretch.

BACKGROUND: Actin stress fibers (SFs) are mechanosensitive structural elements that respond to forces to affect cell morphology, migration, signal transduction and cell function. Cells are internally stressed so that SFs are extended beyond their unloaded lengths, and SFs tend to self-adjust to an equilibrium level of extension. While there is much evidence that cells reorganize their SFs in response to matrix deformations, it is unclear how cells and their SFs determine their specific response to particular spatiotemporal changes in the matrix. METHODOLOGY/PRINCIPAL FINDINGS: Bovine aortic endothelial cells were subjected to cyclic uniaxial stretch over a range of frequencies to quantify the rate and extent of stress fiber alignment. At a frequency of 1 Hz, SFs predominantly oriented perpendicular to stretch, while at 0.1 Hz the extent of SF alignment was markedly reduced and at 0.01 Hz there was no alignment at all. The results were interpreted using a simple kinematic model of SF networks in which the dynamic response depended on the rates of matrix stretching, SF turnover, and SF self-adjustment of extension. For these cells, the model predicted a threshold frequency of 0.01 Hz below which SFs no longer respond to matrix stretch, and a saturation frequency of 1 Hz above which no additional SF alignment would occur. The model also accurately described the dependence of SF alignment on matrix stretch magnitude. CONCLUSIONS: The dynamic stochastic model was capable of describing SF reorganization in response to diverse temporal and spatial patterns of stretch. The model predicted that at high frequencies, SFs preferentially disassembled in the direction of stretch and achieved a new equilibrium by accumulating in the direction of lowest stretch. At low stretch frequencies, SFs self-adjusted to dissipate the effects of matrix stretch. Thus, SF turnover and self-adjustment are each important mechanisms that cells use to maintain mechanical homeostasis.

A kinematic model of stretch-induced stress fiber turnover and reorientation.

A kinetic model based on constrained mixture theory was developed to describe the reorganization of actin stress fibers in adherent cells in response to diverse patterns of mechanical stretch. The model was based on reports that stress fibers are pre-extended at a "homeostatic" level under normal, non-perturbed conditions, and that perturbations in stress fiber length destabilize stress fibers. In response to a step change in matrix stretch, the model predicts that stress fibers are initially stretched in registry with the matrix, but that these overly stretched fibers are gradually replaced by new fibers assembled with the homeostatic level of stretch in the new configuration of the matrix. In contrast, average fiber stretch is chronically perturbed from the homeostatic level when the cells are subjected to cyclic equibiaxial stretch. The model was able to describe experimentally measured time courses of stress fiber reorientation perpendicular to the direction of cyclic uniaxial stretch, as well as the lack of alignment in response to equibiaxial stretch. The model also accurately described the relationship between stretch magnitude and the extent of stress fiber alignment in endothelial cells subjected to cyclic uniaxial stretch. Further, in the case of cyclic simple elongation with transverse matrix contraction, stress fibers orient in the direction of least perturbation in stretch. In summary, the model predicts that the rate of stretch-induced stress fiber disassembly determines the rate of alignment, and that stress fibers tend to orient toward the direction of minimum matrix stretch where the rate of stress fiber turnover is a minimum.

Dispersed fluorescence spectroscopy of the CBr2A1B1-X1A1 transition.

Dispersed fluorescence spectra following the excitation of the CBr2A1B1-X1A1 2 and 2 bands at visible wavelengths were acquired in a discharge supersonic free jet expansion using an intensified charge-coupled device (ICCD) detector. The dispersed fluorescence spectra show signal-to-noise ratios of up to 60 and extend out to a maximum red shift frequency of 6000 cm(-1). Complete and detailed vibrational structure of the ground singlet state (X1A1) was observed. Vibrational parameters including fundamental frequencies, anharmonicities, and coupling constants were determined for the CBr2 X1A1 state. Additional vibrational structure starting at approximately 3650 cm(-1) was observed and this indicates the singlet-triplet energy gap to be >10 kcal mol(-1).