Non-Invasive and Confirmatory Differentiation of Hermaphrodite from Both Male and Female Cannabis Plants Using a Hand-Held Raman Spectrometer.

Cannabis (Cannabis sativa L.) is a dioecious plant that produces both male and female inflorescences. In nature, male and female plants can be found with nearly equal frequency, which determines species out-crossing. In cannabis farming, only female plants are preferred due to their high yield of cannabinoids. In addition to unfavorable male plants, commercial production of cannabis faces the appearance of hermaphroditic inflorescences, species displaying both pistillate flowers and anthers. Such plants can out-cross female plants, simultaneously producing undesired seeds. The problem of hermaphroditic cannabis triggered a search for analytical tools that can be used for their rapid detection and identification. In this study, we investigate the potential of Raman spectroscopy (RS), an emerging sensing technique that can be used to probe plant biochemistry. Our results show that the biochemistry of male, female and hermaphroditic cannabis plants is drastically different which allows for their confirmatory identification using a hand-held Raman spectrometer. Furthermore, the coupling of machine learning approaches enables the identification of hermaphrodites with 98.7% accuracy, whereas both male and female plants can be identified with 100% accuracy. Considering the label-free, non-invasive and non-destructive nature of RS, the developed optical sensing approach can transform cannabis farming in the U.S. and overseas.

Promotion of lung tumor growth by interleukin-17.

Recent findings demonstrate that inhaled cigarette smoke, the predominant lung carcinogen, elicits a T helper 17 (Th17) inflammatory phenotype. Interleukin-17A (IL-17), the hallmark cytokine of Th17 inflammation, displays pro- and antitumorigenic properties in a manner that varies according to tumor type and assay system. To investigate the role of IL-17 in lung tumor growth, we used an autochthonous tumor model (K-Ras(LA1) mice) with lung delivery of a recombinant adenovirus that expresses IL-17A. Virus-mediated expression of IL-17A in K-Ras(LA1) mice at 8-10 wk of age doubled lung tumor growth in 3 wk relative to littermates that received a green fluorescent protein-expressing control adenovirus. IL-17 induced matrix metalloproteinase-9 (MMP-9) expression in vivo and in vitro. In accord with this finding, selective and specific inhibitors of MMP-9 repressed the increased motility and invasiveness of IL-17-treated lung tumor cells in culture. Knockdown or mutation of p53 promoted the motility of murine lung tumor cells and abrogated the promigratory role of IL-17. Coexpression of siRNA-resistant wild-type, but not mutant, human p53 rescued both IL-17-mediated migration and MMP-9 mRNA induction in p53 knockdown lung tumor cells. IL-17 increased MMP-9 mRNA stability by reducing interaction with the mRNA destabilizing serine/arginine-rich splicing factor 1 (SRSF1). Taken together, our results indicate that IL-17 stimulates lung tumor growth and regulates MMP-9 mRNA levels in a p53- and SRSF1-dependent manner.

Post-transcriptional up-regulation of miR-21 by type I collagen.

Composition of extracellular matrix (ECM) is crucial to the establishment and maintenance of epithelial apical-basolateral polarity. Increased ECM rigidity caused by deposition of fibrillar collagen, for example, collagen type I (Col-1), promotes loss of epithelial polarity and tumor progression. microRNAs are small non-coding RNAs that regulate gene expression and fundamental cellular processes. The current study explored a link between microRNAs and Col-1 using organotypic three-dimensional culture in which epithelial cells are embedded within Matrigel, a mimic of basement membrane matrix (Matrigel 3-D). Matrigel 3-D culture of A549, MCF-7, and mK-ras-LE cells (lung and mammary epithelial cell lines) gave rise to acinus, an in vitro equivalent of apical-basolateral polarity that consists of a polarized monolayer of epithelial cells facing a central lumen. Supplementation of Col-1 disrupted acinus. Moreover, Col-1 up-regulated the expression of miR-21, a well-documented oncogenic microRNA, via a post-transcriptional mechanism. Similar post-transcriptional up-regulation of miR-21 correlated with deposition of Col-1 in a murine model of lung fibrogenesis. In summary, our findings link altered ECM composition/rigidity and the expression of oncogenic microRNAs. The current study also suggests a novel post-transcriptional mechanism for regulation of miR-21 expression at maturation from pre-miR-21 to mature miR-21.

Modulation of lung inflammation by the Epstein-Barr virus protein Zta.

Several studies have implicated gamma-herpesviruses, particularly Epstein-Barr virus (EBV), in the progression of idiopathic pulmonary fibrosis. The data presented here examine the possible role that EBV plays in the potentiation of this disease by evaluating the pulmonary response to expression of the EBV lytic transactivator protein Zta. Expression of Zta in the lungs of mice via adenovirus-mediated delivery (Adv-Zta) produced profibrogenic inflammation that appeared most pronounced by day 7 postexposure. Relative to mice exposed to control GFP-expressing adenovirus (Adv-GFP), mice exposed to Adv-Zta displayed evidence of lung injury and a large increase in inflammatory cells, predominantly neutrophils, recovered by bronchoalveolar lavage (BAL). Cytokine and mRNA profiling of the BAL fluid and cells recovered from Adv-Zta-treated mice revealed a Th2 and Th17 bias. mRNA profiles from Adv-Zta-infected lung epithelial cells revealed consistent induction of mRNAs encoding Th2 cytokines. Coexpression in transient assays of wild-type Zta, but not a DNA-binding-defective mutant Zta, activated expression of the IL-13 promoter in lung epithelial cells, and detection of IL-13 in Adv-Zta-treated mice correlated with expression of Zta. Induction of Th2 cytokines in Zta-expressing mice corresponded with alternative activation of macrophages. In cell culture and in mice, Zta repressed lung epithelial cell markers. Despite the profibrogenic character at day 7, the inflammation resolves by 28 days postexposure to Adv-Zta without evidence of fibrosis. These observations indicate that the EBV lytic transactivator protein Zta displays activity consistent with a pathogenic role in pulmonary fibrosis associated with herpesvirus infection.

Extra- and intra-cellular ice formation in Stage I and II Xenopus laevis oocytes.

We are currently investigating factors that influence intracellular ice formation (IIF) in mouse oocytes and oocytes of the frog Xenopus. A major reason for choosing these two species is that while their eggs normally do not possess aquaporin channels in their plasma membranes, these channels can be made to express. We wish to see whether IIF is affected by the presence of these channels. The present Xenopus study deals with control eggs not expressing aquaporins. The main factor studied has been the effect of a cryoprotective agent [ethylene glycol (EG) or glycerol] and its concentration. The general procedure was to (a) cool the oocytes on a cryostage to slightly below the temperatures at which extracellular ice formation occurs, (b) warm them to just below the melting point, and (c) then re-cool them to -50 degrees C at 10 degrees C/min. In the majority of cases, IIF occurs well into step (c), but a sizeable minority undergo IIF in steps (a) or (b). The former group we refer to as low-temperature flashers; the latter as high-temperature flashers. IIF is manifested as abrupt blackening of the egg, which we refer to as "flashing." Observations on the Linkam cryostage are restricted to Stage I and II oocytes, which have diameters of 200 300 microm. In the absence of a cryoprotective agent, that is in frog Ringers, the mean flash temperature for the low-temperature freezers is -11.4 degrees C, although a sizeable percentage flash at temperatures much closer to that of the EIF (-3.9 degrees C). When EG is present, the flash temperature for the low-temperatures freezers drops significantly to approximately -20 degrees C for EG concentrations ranging from 0.5 to 1.5 M. The presence of 1.5 M glycerol also substantially reduces the IIF temperature of the low-temperature freezers; namely, to -29 degrees C, but 0.5 and 1 M glycerol exert little or no effect. The IIF temperatures observed using the Linkam cryostage agree well with those estimated by calorimetry [F.W. Kleinhans, J.F. Guenther, D.M. Roberts, P. Mazur, Analysis of intracellular ice nucleation in Xenopus oocytes by differential scanning calorimetry, Cryobiology 52 (2006) 128-138]. The IIF temperatures in Xenopus are substantially higher than those observed in mouse oocytes [P. Mazur, S. Seki, I.L. Pinn, F.W. Kleinhans, K. Edashige, Extra- and intracellular ice formation in mouse oocytes, Cryobiology 51 (2005) 29-53]. Perhaps that is a reflection of their much larger size.

Phosphorylation of soybean nodulin 26 on serine 262 enhances water permeability and is regulated developmentally and by osmotic signals.

Soybean nodulin 26 is expressed and targeted to the symbiosome membrane of nitrogen-fixing nodules, where it forms an aquaporin channel with a modest water transport rate. In this study, we show that the phosphorylation of nodulin 26 on Ser-262, which is catalyzed by a symbiosome membrane-associated calcium-dependent protein kinase, stimulates its intrinsic water transport rate. Furthermore, using a phosphospecific antibody, we have elucidated the developmental appearance and regulation of nodulin 26 phosphorylation in vivo. Although nodulin 26 protein is detected first in differentiating infected cells (16 days), phosphorylated nodulin 26 does not become pronounced until infected cell maturation (25 days). Phosphorylation is sustained at steady state levels until entry into senescence. Nodulin 26 phosphorylation is enhanced further by osmotic stresses (water deprivation and salinity). Thus, the phosphorylation of nodulin 26 coincides with the establishment of mature nitrogen-fixing symbiosomes, is regulated by osmotic stresses that induce calcium-signaling pathways, and appears to be part of the adaptive responses of infected cells to osmotic challenge.

Functional selectivity for glycerol of the nodulin 26 subfamily of plant membrane intrinsic proteins.

The nodulin-like intrinsic protein (NIP) subfamily of water and solute channels in plants is named for nodulin 26 of legume nodules. Two NIPs, soybean nodulin 26 and Lotus japonicus LIMP2, show a distinct functional profile with a low intrinsic osmotic water permeability (P(f)) and the ability to flux uncharged polyols such as glycerol. NIPs have a conserved signature sequence within the 'aromatic/arginine' region that forms the selectivity filter for major intrinsic proteins. This sequence is a hybrid of glyceroporin and aquaporin residues as well as exhibiting substitutions unique to the NIP subfamily. Site-directed mutagenesis of a conserved tryptophan in helix 2 of LIMP2 shows that this is a major determinant of glycerol selectivity.