The vacuolar Ca2+ transporter CATION EXCHANGER 2 regulates cytosolic calcium homeostasis, hypoxic signaling, and response to flooding in Arabidopsis thaliana.

Flooding represents a major threat to global agricultural productivity and food security, but plants are capable of deploying a suite of adaptive responses that can lead to short- or longer-term survival to this stress. One cellular pathway thought to help coordinate these responses is via flooding-triggered Ca2+ signaling. We have mined publicly available transcriptomic data from Arabidopsis subjected to flooding or low oxygen stress to identify rapidly upregulated, Ca2+ -related transcripts. We then focused on transporters likely to modulate Ca2+ signals. Candidates emerging from this analysis included AUTOINHIBITED Ca2+ ATPASE 1 and CATION EXCHANGER 2. We therefore assayed mutants in these genes for flooding sensitivity at levels from growth to patterns of gene expression and the kinetics of flooding-related Ca2+ changes. Knockout mutants in CAX2 especially showed enhanced survival to soil waterlogging coupled with suppressed induction of many marker genes for hypoxic response and constitutive activation of others. CAX2 mutants also generated larger and more sustained Ca2+ signals in response to both flooding and hypoxic challenges. CAX2 is a Ca2+ transporter located on the tonoplast, and so these results are consistent with an important role for vacuolar Ca2+ transport in the signaling systems that trigger flooding response.

Variation in the transcriptome of different ecotypes of Arabidopsis thaliana reveals signatures of oxidative stress in plant responses to spaceflight.

PREMISE OF THE STUDY: Spaceflight provides a unique environment in which to dissect plant stress response behaviors and to reveal potentially novel pathways triggered in space. We therefore analyzed the transcriptomes of Arabidopsis thaliana plants grown on board the International Space Station to find the molecular fingerprints of these space-related response networks. METHODS: Four ecotypes (Col-0, Ws-2, Ler-0 and Cvi-0) were grown on orbit and then their patterns of transcript abundance compared to ground-based controls using RNA sequencing. KEY RESULTS: Transcripts from heat-shock proteins were upregulated in all ecotypes in spaceflight, whereas peroxidase transcripts were downregulated. Among the shared and ecotype-specific changes, gene classes related to oxidative stress and hypoxia were detected. These spaceflight transcriptional response signatures could be partly mimicked on Earth by a low oxygen environment and more fully by oxidative stress (H2 O2 ) treatments. CONCLUSIONS: These results suggest that the spaceflight environment is associated with oxidative stress potentially triggered, in part, by hypoxic response. Further, a shared spaceflight response may be through the induction of molecular chaperones (such as heat shock proteins) that help protect cellular machinery from the effects of oxidative damage. In addition, this research emphasizes the importance of considering the effects of natural variation when designing and interpreting changes associated with spaceflight experiments.

Quantitative ROS bioreporters: A robust toolkit for studying biological roles of ROS in response to abiotic and biotic stresses.

While the accumulation of reactive oxygen species (ROS) through spontaneous generation or as the by-products of aerobic metabolism can be toxic to plants, recent findings demonstrate that ROS act as signaling molecules that play a critical role in adapting to various stress conditions. Tight regulation of ROS homeostasis is required to adapt to stress and survive, yet in vivo spatiotemporal information of ROS dynamics are still largely undefined. In order to understand the dynamics of ROS changes and their biological function in adapting to stresses, two quantitative ROS transcription-based bioreporters were developed. These reporters use ROS-responsive promoters from RBOHD or ZAT12 to drive green fluorescent protein (GFP) expression. The resulting GFP expression is compared to a constitutively expressed mCherry that is contained on the same cassette with the ROS-responsive promoter: This allows for the generation of ratiometric images comparing ROS changes (GFP) to the constitutively expressed mCherry. Both reporters were used to assess ROS levels to oxidative stress, salt stress, and the pathogen defense elicitor flg22. These bioreporters showed increases in the ratio values of GFP to mCherry signals between 10 and 30 min poststress application. Such stress-associated ROS signals correlated with the induction of abiotic/biotic stress responsive markers such as RbohD, ZAT12, SOS2 and PR5 suggesting these ROS bioreporters provide a robust indicator of increased ROS related to stress responses. Based upon the spatiotemporal response patterns of signal increase, ZAT12 promoter-dependent ROS (Zat12p-ROS) bioreporter appears to be suitable for cellular mapping of ROS changes in response to abiotic and biotic stresses.

Salt stress-induced Ca2+ waves are associated with rapid, long-distance root-to-shoot signaling in plants.

Their sessile lifestyle means that plants have to be exquisitely sensitive to their environment, integrating many signals to appropriate developmental and physiological responses. Stimuli ranging from wounding and pathogen attack to the distribution of water and nutrients in the soil are frequently presented in a localized manner but responses are often elicited throughout the plant. Such systemic signaling is thought to operate through the redistribution of a host of chemical regulators including peptides, RNAs, ions, metabolites, and hormones. However, there are hints of a much more rapid communication network that has been proposed to involve signals ranging from action and system potentials to reactive oxygen species. We now show that plants also possess a rapid stress signaling system based on Ca(2+) waves that propagate through the plant at rates of up to ∼ 400 µm/s. In the case of local salt stress to the Arabidopsis thaliana root, Ca(2+) wave propagation is channeled through the cortex and endodermal cell layers and this movement is dependent on the vacuolar ion channel TPC1. We also provide evidence that the Ca(2+) wave/TPC1 system likely elicits systemic molecular responses in target organs and may contribute to whole-plant stress tolerance. These results suggest that, although plants do not have a nervous system, they do possess a sensory network that uses ion fluxes moving through defined cell types to rapidly transmit information between distant sites within the organism.

Cytoplasmic Ca2+ influx mediates iron- and reactive oxygen species-dependent ferroptotic cell death in rice immunity.

Iron- and reactive oxygen species (ROS)-dependent ferroptosis occurs in plant cells. Ca2+ acts as a conserved key mediator to control plant immune responses. Here, we report a novel role of cytoplasmic Ca2+ influx regulating ferroptotic cell death in rice immunity using pharmacological approaches. High Ca2+ influx triggered iron-dependent ROS accumulation, lipid peroxidation, and subsequent hypersensitive response (HR) cell death in rice (Oryza sativa). During Magnaporthe oryzae infection, 14 different Ca2+ influx regulators altered Ca2+, ROS and Fe2+ accumulation, glutathione reductase (GR) expression, glutathione (GSH) depletion and lipid peroxidation, leading to ferroptotic cell death in rice. High Ca2+ levels inhibited the reduction of glutathione isulphide (GSSG) to GSH in vitro. Ca2+ chelation by ethylene glycol-bis (2-aminoethylether)-N, N, N', N'-tetra-acetic acid (EGTA) suppressed apoplastic Ca2+ influx in rice leaf sheaths during infection. Blocking apoplastic Ca2+ influx into the cytoplasm by Ca2+ chelation effectively suppressed Ca2+-mediated iron-dependent ROS accumulation and ferroptotic cell death. By contrast, acibenzolar-S-methyl (ASM), a plant defense activator, significantly enhanced Ca2+ influx, as well as ROS and iron accumulation to trigger ferroptotic cell death in rice. The cytoplasmic Ca2+ influx through calcium-permeable cation channels, including the putative resistosomes, could mediate iron- and ROS-dependent ferroptotic cell death under reduced GR expression levels in rice immune responses.

Icilin inhibits E2F1-mediated cell cycle regulatory programs in prostate cancer.

Aberrant expression of cell cycle regulators have been implicated in prostate cancer development and progression. Therefore, understanding transcriptional networks controlling the cell cycle remain a challenge in the development of prostate cancer treatment. In this study, we found that icilin, a super-cooling agent, down-regulated the expression of cell cycle signature genes and caused G1 arrest in PC-3 prostate cancer cells. With reverse-engineering and an unbiased interrogation of a prostate cancer-specific regulatory network, master regulator analysis discovered that icilin affected cell cycle-related transcriptional modules and identified E2F1 transcription factor as a target master regulator of icilin. Experimental analyses confirmed that icilin reduced the activity and expression levels of E2F1. These results demonstrated that icilin inactivates a small regulatory module controlling the cell cycle in prostate cancer cells. Our study might provide insight into the development of cell cycle-targeted cancer therapeutics.

Single-center study on clinicopathological and typical molecular pathologic features of metastatic brain tumor.

BACKGROUND: The metastatic brain tumor is the most common brain tumor. The aim of this study was to demonstrate the clinicopathological and molecular pathologic features of brain metastases (BM). METHODS: A total of 269 patients were diagnosed with BM through surgical resection at Seoul St. Mary's Hospital from January 2010 to March 2020. We reviewed the clinicopathological features and molecular status of primary and metastatic brain tissues using immunohistochemistry and molecular pathology results. RESULTS: Among 269 patients, 139 males and 130 females were included. The median age of primary tumor was 58 years (range, 13 to 87 years) and 86 patients (32.0%) had BM at initial presentation. Median BM free interval was 28.0 months (range, 1 to 286 months). The most frequent primary site was lung 46.5% (125/269), and followed by breast 15.6% (42/269), colorectum 10.0% (27/269). Epidermal growth factor receptor (EGFR) mutation was found in 50.8% (32/63) and 58.0% (40/69) of lung primary and BM, respectively. In both breast primary and breast cancer with BM, luminal B was the most frequent subtype at 37.9% (11/29) and 42.9% (18/42), respectively, followed by human epidermal growth factor receptor 2 with 31.0% (9/29) and 33.3% (14/42). Triple-negative was 20.7% (6/29) and 16.7% (7/42), and luminal A was 10.3% (3/29) and 7.1% (3/42) of breast primary and BM, respectively. In colorectal primary and colorectal cancer with BM, KRAS mutation was found in 76.9% (10/13) and 66.7% (2/3), respectively. CONCLUSIONS: We report the clinicopathological and molecular pathologic features of BM that can provide useful information for understanding the pathogenesis of metastasis and for clinical trials based on the tumor's molecular pathology.

SK&F 96365 induces apoptosis and autophagy by inhibiting Akt-mTOR signaling in A7r5 cells.

SK&F 96365 has been widely used as an inhibitor of transient receptor potential (TRP) calcium channels in various physiological settings. However, growing evidence suggests that SK&F 96365 affects several cellular and molecular processes via uncharacterized off-target mechanisms. In this study, we showed that SK&F 96365 induces apoptosis and autophagy in A7r5 vascular smooth muscle cells. The combined suppression of apoptosis and autophagy provoked necrosis rather than rescued cell death in the cells treated with SK&F 96365. In addition, we found that SK&F 96365 inhibits Akt-mTOR signaling pathways, which is comparable with the efficacy of other known Akt inhibitors. Our findings suggest that SK&F 96365 can be a useful agent for delineating the molecular mechanisms underlying crosstalk among cell death pathways.

Menthol induces cell-cycle arrest in PC-3 cells by down-regulating G2/M genes, including polo-like kinase 1.

Menthol, a naturally occurring monoterpene, is used in foods, cosmetic products, and topical therapeutic preparations. It also exerts cytotoxic activity against several cancer cell types, including prostate cancer cells. However, little is known about the mechanism of menthol action against prostate cancer cells. In this study, we investigated the effect of menthol on the gene expression profile of PC-3 prostate cancer cells using DNA microarray analyses. Gene set enrichment analysis revealed that menthol primarily affects the expression of cell cycle-related genes. Subsequent experimental analyses validated that menthol induces G2/M arrest. Particularly, menthol markedly down-regulated polo-like kinase 1 (PLK1), a key regulator of G2/M phase progression and inhibited its downstream signaling. Our computational analyses and experimental validation provide a basis for future investigations that are aimed at elucidating the action of menthol on cell cycle control in prostate cancer cells.

Icilin induces G1 arrest through activating JNK and p38 kinase in a TRPM8-independent manner.

Aberrant regulation of cell cycle confers a limitless replicative potential, which is a hallmark of cancer. Currently, the compounds targeting the cell cycle are undergoing cancer clinical trials. In this study, we demonstrated that icilin, a cooling compound, induces G1 arrest in PC-3 prostate cancer cells without cell death. Icilin modulated the expression level of various cell cycle regulators at transcription or post-translational levels. In addition, icilin activated JNK and p38 kinase pathways, but not ERK. Both JNK and p38 kinases cooperatively mediated icilin-induced G1 arrest, which was rescued by pharmacologic inhibition of these kinases. The action of icilin on G1 arrest was unrelated to the activation of TRPM8 calcium channel. Our findings suggest that icilin is a valuable chemical probe for future investigation aiming at delineating the molecular mechanisms of cell cycle regulation in prostate cancer.

Geraniol inhibits prostate cancer growth by targeting cell cycle and apoptosis pathways.

The progression of prostate cancer is associated with escape from cell cycle arrest and apoptosis under androgen-depleted conditions. Here, we found that geraniol, a naturally occurring monoterpene, induces cell cycle arrest and apoptosis in cultured cells and tumor grafted mice using PC-3 prostate cancer cells. Geraniol modulated the expression of various cell cycle regulators and Bcl-2 family proteins in PC-3 cells in vitro and in vivo. Furthermore, we showed that the combination of sub-optimal doses of geraniol and docetaxel noticeably suppresses prostate cancer growth in cultured cells and tumor xenograft mice. Therefore, our findings provide insight into unraveling the mechanisms underlying escape from cell cycle arrest and apoptosis and developing therapeutic strategies against prostate cancer.

Recapitulation of the Function and Role of ROS Generated in Response to Heat Stress in Plants.

In natural ecosystems, plants are constantly exposed to changes in their surroundings as they grow, caused by a lifestyle that requires them to live where their seeds fall. Thus, plants strive to adapt and respond to changes in their exposed environment that change every moment. Heat stress that naturally occurs when plants grow in the summer or a tropical area adversely affects plants' growth and poses a risk to plant development. When plants are subjected to heat stress, they recognize heat stress and respond using highly complex intracellular signaling systems such as reactive oxygen species (ROS). ROS was previously considered a byproduct that impairs plant growth. However, in recent studies, ROS gained attention for its function as a signaling molecule when plants respond to environmental stresses such as heat stress. In particular, ROS, produced in response to heat stress in various plant cell compartments such as mitochondria and chloroplasts, plays a crucial role as a signaling molecule that promotes plant growth and triggers subsequent downstream reactions. Therefore, this review aims to address the latest research trends and understandings, focusing on the function and role of ROS in responding and adapting plants to heat stress.

A Ratiometric Calcium Reporter CGf Reveals Calcium Dynamics Both in the Single Cell and Whole Plant Levels Under Heat Stress.

Land plants evolved to quickly sense and adapt to temperature changes, such as hot days and cold nights. Given that calcium (Ca2+) signaling networks are implicated in most abiotic stress responses, heat-triggered changes in cytosolic Ca2+ were investigated in Arabidopsis leaves and pollen. Plants were engineered with a reporter called CGf, a ratiometric, genetically encoded Ca2+ reporter with an mCherry reference domain fused to an intensiometric Ca2+ reporter GCaMP6f. Relative changes in [Ca2+]cyt were estimated based on CGf's apparent K D around 220 nM. The ratiometric output provided an opportunity to compare Ca2+ dynamics between different tissues, cell types, or subcellular locations. In leaves, CGf detected heat-triggered cytosolic Ca2+ signals, comprised of three different signatures showing similarly rapid rates of Ca2+ influx followed by differing rates of efflux (50% durations ranging from 5 to 19 min). These heat-triggered Ca2+ signals were approximately 1.5-fold greater in magnitude than blue light-triggered signals in the same leaves. In contrast, growing pollen tubes showed two different heat-triggered responses. Exposure to heat caused tip-focused steady growth [Ca2+]cyt oscillations to shift to a pattern characteristic of a growth arrest (22%), or an almost undetectable [Ca2+]cyt (78%). Together, these contrasting examples of heat-triggered Ca2+ responses in leaves and pollen highlight the diversity of Ca2+ signals in plants, inviting speculations about their differing kinetic features and biological functions.

Menthol regulates TRPM8-independent processes in PC-3 prostate cancer cells.

Menthol, a naturally occurring compound from peppermint oil, binds and activates the TRPM8 Ca(2+)-permeable channel that exhibits abnormal expression patterns in prostate cancer, suggesting that TRPM8 links Ca(2+) transport pathways to tumor biology. We thus investigated the cellular responses of prostate cancer cells to menthol. Here we found that menthol increases [Ca(2+)](i) via Ca(2+) influx mechanism(s) independent of TRPM8 in PC-3 cells. We demonstrated that menthol induces cell death at supramillimolar concentrations in PC-3 cells and the cell death is not suppressed by low extracellular Ca(2+) condition which indicates that menthol-induced cell death is not associated with Ca(2+) influx pathways. In addition, we showed that menthol increases a phosphorylated form of c-jun N-terminal kinase (JNK) in PC-3 cells through TRPM8-independent mechanisms. Thus, our data indicate that there is an apparent lack of causality between TRPM8 activation and menthol-induced cell death and that menthol can regulate TRPM8-independent Ca(2+)-transport and cellular processes.

Altered biochemical properties of transient receptor potential vanilloid 6 calcium channel by peptide tags.

Ion channels are commonly expressed in recombinant forms with peptide tags, which facilitates their molecular and electrophysiological studies. However, peptide tags may alter ion channel properties. Here we describe the differential effect of peptide tags on the biochemical properties of transient receptor potential vanilloid 6 (TRPV6) channels. Yellow fluorescent protein (YFP)-tagged wild-type TRPV6 (YFP-TRPV6(WT)) showed much lower levels of aggregate-like bands in Western blots than those of Myc-TRPV6(WT). By contrast, the glycosylation level was higher with Myc-TRPV6(WT) than that with YFP-TRPV6(WT). We additionally demonstrate that peptide tags affect the protein integrity of TRPV6 channels. Myc-TRPV6(WT) was expressed as an intact channel, whereas the pore mutants Myc-TRPV6(D542A) and Myc-TRPV6(D542K) were observed to be partially fragmented. By contrast, all YFP-tagged channels were intact, although the YFP-tagged pore mutants were less glycosylated than YFP-TRPV6(WT). However, regardless of the peptide tag used, TRPV6(D542A) and TRPV6(D542K) electrophysiologically inhibited TRPV6(WT) which indicates that all pore mutants are equivalent electrophysiologically, not biochemically. Thus, our findings suggest that peptide tags can produce unintended biochemical changes of ion channels which highlight the necessity of careful biochemical evaluation to clarify the roles of ion channels.

Menthol Enhances an Antiproliferative Activity of 1alpha,25-Dihydroxyvitamin D(3) in LNCaP Cells.

1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)], the most active form of vitamin D(3), and its analogues have therapeutic benefits for prostate cancer treatment. However, the development of hypercalcemia is an obstacle to clinical applications of 1alpha,25(OH)(2)D(3) for cancer therapy. In this study, we provide evidence that menthol, a key component of peppermint oil, increases an anti-proliferation activity of 1alpha,25(OH)(2)D(3) in LNCaP prostate cancer cells. We found that menthol per se does not exhibit antiproliferative activity, but it is able to enhance 1alpha,25(OH)(2)D(3)-mediated growth inhibition in LNCaP cells. Fluorometric assays using Fura-2 showed that 1alpha,25(OH)(2)D(3) does not induce acute Ca(2+) response, whereas menthol evokes an increase in [Ca(2+)](i), which suggests that cross-talks of menthol-induced Ca(2+) signaling with 1alpha,25(OH)(2)D(3)-mediated growth inhibition pathways. In addition, Western blot analysis revealed that 1alpha,25(OH)(2)D(3) and menthol cooperatively modulate the expression of bcl-2 and p21 which provides the insight into the molecular mechanisms underlying the enhanced 1alpha,25(OH)(2)D(3)-mediated growth inhibition by menthol. Thus, our findings suggest that menthol may be a useful natural compound to enhance therapeutic effects of 1alpha,25(OH)(2)D(3).

Sense and sensibility: the use of fluorescent protein-based genetically encoded biosensors in plants.

Fluorescent protein-based biosensors are providing us with an unprecedented, quantitative view of the dynamic nature of the cellular networks that lie at the heart of plant biology. Such bioreporters can visualize the spatial and temporal kinetics of cellular regulators such as Ca2+ and H+, plant hormones and even allow membrane transport activities to be monitored in real time in living plant cells. The fast pace of their development is making these tools increasingly sensitive and easy to use and the rapidly expanding biosensor toolkit offers great potential for new insights into a wide range of plant regulatory processes. We suggest a checklist of controls that should help avoid some of the more cryptic issues with using these bioreporter technologies.

Rapid, Long-Distance Electrical and Calcium Signaling in Plants.

Plants integrate activities throughout their bodies using long-range signaling systems in which stimuli sensed by just a few cells are translated into mobile signals that can influence the activities in distant tissues. Such signaling can travel at speeds well in excess of millimeters per second and can trigger responses as diverse as changes in transcription and translation levels, posttranslational regulation, alterations in metabolite levels, and even wholesale reprogramming of development. In addition to the use of mobile small molecules and hormones, electrical signals have long been known to propagate throughout the plant. This electrical signaling network has now been linked to waves of Ca(2+) and reactive oxygen species that traverse the plant and trigger systemic responses. Analysis of cell type specificity in signal propagation has revealed the movement of systemic signals through specific cell types, suggesting that a rapid signaling network may be hardwired into the architecture of the plant.

Geraniol suppresses prostate cancer growth through down-regulation of E2F8.

Geraniol, an acyclic dietary monoterpene, has been found to suppress cancer survival and growth. However, the molecular mechanism underlying the antitumor action of geraniol has not been investigated at the genome-wide level. In this study, we analyzed the microarray data obtained from geraniol-treated prostate cancer cells. Geraniol potently altered a gene expression profile and primarily down-regulated cell cycle-related gene signatures, compared to linalool, another structurally similar monoterpene that induces no apparent phenotypic changes. Master regulator analysis using the prostate cancer-specific regulatory interactome identified that the transcription factor E2F8 as a specific target molecule regulates geraniol-specific cell cycle signatures. Subsequent experiments confirmed that geraniol down-regulated E2F8 expression and the knockdown of E2F8 was sufficient to suppress cell growth by inducing G2 /M arrest. Epidemiological analysis showed that E2F8 is up-regulated in metastatic prostate cancer and associated with poor prognosis. These results indicate that E2F8 is a crucial transcription regulator controlling cell cycle and survival in prostate cancer cells. Therefore, our study provides insight into the role of E2F8 in prostate cancer biology and therapeutics.

Disseminated herpes zoster in an immunocompetent elderly patient.

Herpes zoster is a cutaneous infection that is characterized by an acute vesicobullous rash with ipsilateral one or two dermatomal distribution and painful allodynia, while predominantly being found in the elderly. Extensive cutaneous dissemination has been reported in immune-compromised patients, such as those who suffer from HIV infections, cancer, chemotherapy, and corticosteroid therapy patients. However, we report a case of disseminated herpes zoster infection in an immuno-competent elderly individual.