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AKI pathology involves a rapid decline in kidney function and occurs in 8–16% of hospitalized patients. There is currently no therapeutic for AKI. AKI results in mitochondria dysfunction, microvasculature injury, and loss of renal tubular function. In an I/R-induced AKI mouse model, treatment with the FDA-approved 5-HT1F receptor-selective agonist lasmiditan induced mitochondrial biogenesis, improved vascular integrity, reduced fibrosis, and reduced proximal tubule damage. These data support repurposing lasmiditan for the treatment of AKI.

The β

The PI3Kinase/Akt/mTOR pathway has important roles in cancer development for multiple tumor types, including UV-induced nonmelanoma skin cancer. Immunosuppressed populations are at increased risk of aggressive cutaneous squamous cell carcinoma (SCC). Individuals who are treated with rapamycin (sirolimus, a classical mTOR inhibitor) have significantly decreased rates of developing new cutaneous SCCs compared with those that receive traditional immunosuppression. However, systemic rapamycin use can lead to significant adverse events. Here, we explored the use of topical rapamycin as a chemopreventive agent in the context of solar-simulated light (SSL)-induced skin carcinogenesis. In SKH-1 mice, topical rapamycin treatment decreased tumor yields when applied after completion of 15 weeks of SSL exposure compared with controls. However, applying rapamycin during SSL exposure for 15 weeks, and continuing for 10 weeks after UV treatment, increased tumor yields. We also examined whether a combinatorial approach might result in more significant tumor suppression by rapamycin. We validated that rapamycin causes increased Akt (S473) phosphorylation in the epidermis after SSL, and show for the first time that this dysregulation can be inhibited in vivo by a selective PDK1/Akt inhibitor, PHT-427. Combining rapamycin with PHT-427 on tumor prone skin additively caused a significant reduction of tumor multiplicity compared with vehicle controls. Our findings indicate that patients taking rapamycin should avoid sun exposure, and that combining topical mTOR inhibitors and Akt inhibitors may be a viable chemoprevention option for individuals at high risk for cutaneous SCC. Cancer Prev Res; 9(3); 215–24. ©2016 AACR.

An urgent need exists for the development of more efficacious molecular strategies targeting nonmelanoma skin cancer (NMSC), the most common malignancy worldwide. Inflammatory signaling downstream of Toll-like receptor 4 (TLR4) has been implicated in several forms of tumorigenesis, yet its role in solar UV-induced skin carcinogenesis remains undefined. We have previously shown in keratinocyte cell culture and SKH-1 mouse epidermis that topical application of the specific TLR4 antagonist resatorvid (TAK-242) blocks acute UV-induced AP-1 and NF-κB signaling, associated with downregulation of inflammatory mediators and MAP kinase phosphorylation. We therefore explored TLR4 as a novel target for chemoprevention of UV-induced NMSC. We selected the clinical TLR4 antagonist resatorvid based upon target specificity, potency, and physicochemical properties. Here, we confirm using ex vivo permeability assays that topical resatorvid can be effectively delivered to skin, and using in vivo studies that topical resatorvid can block UV-induced AP-1 activation in mouse epidermis. We also report that in a UV-induced skin tumorigenesis model, topical resatorvid displays potent photochemopreventive activity, significantly suppressing tumor area and multiplicity. Tumors harvested from resatorvid-treated mice display reduced activity of UV-associated signaling pathways and a corresponding increase in apoptosis compared with tumors from control animals. Further mechanistic insight on resatorvid-based photochemoprevention was obtained from unsupervised hierarchical clustering analysis of protein readouts via reverse-phase protein microarray revealing a significant attenuation of key UV-induced proteomic changes by resatorvid in chronically treated high-risk SKH-1 skin prior to tumorigenesis. Taken together, our data identify TLR4 as a novel molecular target for topical photochemoprevention of NMSC. Cancer Prev Res; 11(5); 265–78. ©2018 AACR.See related editorial by Sfanos, p. 251

Staining for Ki67 in mouse tumors.

Schema representing UV-induced signaling, rapamycin and PHT-427-regulated pathways in keratinocytes.

Previous studies have shown that cGMP increases mitochondrial biogenesis (MB). Our laboratory has determined that formoterol and LY344864, agonists of the β2-adrenergic receptor and 5-HT1F receptor, respectively, signal MB in a soluble guanylyl cyclase (sGC)-dependent manner. However, the pathway between cGMP and MB produced by these pharmacological agents in renal proximal tubule cells (RPTCs) and the kidney has not been determined. In the present study, we showed that treatment of RPTCs with formoterol, LY344864, or riociguat, a sGC stimulator, induces MB through protein kinase G (PKG), a target of cGMP, and p38, an associated downstream target of PKG and a regulator of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) expression in RPTCs. We also examined if p38 plays a role in PGC-1α phosphorylation in vivo. Administration of l-skepinone, a potent and specific inhibitor of p38α and p38β, to naïve mice inhibited phosphorylated PGC-1α localization in the nuclear fraction of the renal cortex. Taken together, we demonstrated a pathway, sGC/cGMP/PKG/p38/PGC-1α, for pharmacological induction of MB and the importance of p38 in this pathway.

Colon cancer (CC) is the third most commonly diagnosed cancer in the USA. While the overall incidence is declining, it is rising alarmingly in young patients (EOCC). CC in young patients tends to be more aggressive and often diagnosed at more advanced stages and portend poorer prognosis. Our recently published data showed that EOCC is a distinct disease with unique molecular features compared to late-onset CC (LOCC). The Cartilage Oligomeric Matrix Protein (COMP) was shown to be significantly upregulated in EOCC and correlated with poor survival. However, the role of COMP in CC tumorigenesis, especially in young patients, is not well understood. Thus, the aim of this study was to elucidate the role of COMP in CC tumorigenesis by modulating COMP levels in vitro and test how it affects proliferation. Then, patient samples were evaluated by testing the levels of proliferation marker Ki67. In addition, this study investigates whether higher transcriptional mRNA levels of COMP seen in more aggressive early-onset CC correlate with protein levels compared to late-onset CC. COMP mRNA levels in fresh frozen colon tumors (young: n = 5; old: n = 5) were assessed by quantitative PCR (qPCR). Additionally, CC cell lines were profiled for COMP expression to choose an in vitro model to study the role of COMP in CC tumorigenesis. HT-29 (low COMP expression) and CaCo-2 (high COMP expression) cells were used for in vitro proliferation studies. Immunohistochemical (IHC) analysis was conducted to assess COMP and Ki67 protein levels in formalin-fixed paraffin-embedded (FFPE) colon tumors. Significantly higher COMP expression levels were observed in fresh frozen EOCC compared to LOCC tumors. This observation confirmed our previously reported results from NanoString gene expression assay using FFPE samples. Cell proliferation was significantly increased in HT-29 and CaCo-2 cells upon treatment with human recombinant COMP protein after 48 and 72 h (P

Background: Although tunneled dialysis catheters (TDC) are far from ideal, they still represent the main form of vascular access for most patients beginning dialysis. Catheters are easy to place and allow patients instant access to dialysis, but regardless of these benefits, catheters are associated with a high incidence of significant complications like bloodstream infections, central venous stenosis, thrombosis, and dysfunction. In the present study, we aim to describe and characterize a swine model of catheter dysfunction and bloodstream infection, that recreates the clinical scenario, to help to serve as a platform to develop therapeutic innovations for this important clinical problem. Methods: Six Yorkshire cross pigs were used in this study. Non-coated commercial catheters were implanted in the external jugular recreating the main features of common clinical practice. Catheters were aseptically accessed twice a week for a mock dialysis procedure (flushing in and out) to assess for and identify catheter dysfunction. Animals were monitored daily for infections; once detected, blood samples were collected for bacterial culture and antibiograms. Study animals were euthanized when nonresponsive to treatment. Tissue samples were collected in a standardized fashion for macroscopic inspection and histological analysis. Results: The data analysis revealed an early onset of infection with a median time to infection of 9 days, 40% of the isolates were polymicrobial, and the average time to euthanasia was 20.16 ± 7.3 days. Median time to catheter dysfunction onset was 6 days post-implantation. Postmortem dissection revealed external fibrin sheath and internal thrombosis as the main causes of catheter dysfunction. There was also evidence of central venous stenosis with positive cells for αSMA, CD68, Ki67, Smoothelin, and Vimentin within the venous neointima. Conclusions: The described model represents a reliable and reproducible large animal model of catheter dysfunction and bloodstream infection, which recreates all the main complications of TDC’s and so could be used as a validated large animal model to develop new therapies for TDC related infection, thrombosis/dysfunction and central venous stenosis.

Cutaneous exposure to solar ultraviolet (UV) radiation is a major causative factor in skin carcinogenesis, and improved molecular strategies for efficacious chemoprevention of non-melanoma skin cancer (NMSC) are urgently needed. Toll-like receptor 4 (TLR4) signaling has been shown to drive skin inflammation, photoimmunosuppression and chemical carcinogenesis. Here we have examined the feasibility of genetic and pharmacological antagonism targeting cutaneous TLR4 for the suppression of UV-induced NF-κB and AP-1 signaling in keratinocytes and mouse skin. Using immunohistochemical and proteomic microarray analysis of human skin, we demonstrate for the first time that a significant increase in expression of TLR4 occurs in keratinocytes during the progression from normal skin to actinic keratosis (AK), also detectible during further progression to squamous cell carcinoma. Next, we demonstrate that siRNA-based genetic TLR4 inhibition blocks UV-induced stress signaling in cultured keratinocytes. Importantly, we observed that resatorvid (TAK-242), a molecularly-targeted clinical TLR4 antagonist, blocks UV-induced NF-κB and MAP kinase/AP-1 activity and cytokine expression (Il-6, Il-8, and Il-10) in cultured keratinocytes and in topically treated murine skin. Taken together, our data reveal that pharmacological TLR4 antagonism can suppress UV-induced cutaneous signaling, and future experiments will explore the potential of TLR4-directed strategies for prevention of NMSC.

An urgent need exists for the development of more efficacious molecular strategies targeting nonmelanoma skin cancer (NMSC), the most common malignancy worldwide. Inflammatory signaling downstream of Toll-like receptor 4 (TLR4) has been implicated in several forms of tumorigenesis, yet its role in solar UV-induced skin carcinogenesis remains undefined. We have previously shown in keratinocyte cell culture and SKH-1 mouse epidermis that topical application of the specific TLR4 antagonist resatorvid (TAK-242) blocks acute UV-induced AP-1 and NF-κB signaling, associated with downregulation of inflammatory mediators and MAP kinase phosphorylation. We therefore explored TLR4 as a novel target for chemoprevention of UV-induced NMSC. We selected the clinical TLR4 antagonist resatorvid based upon target specificity, potency, and physicochemical properties. Here, we confirm using ex vivo permeability assays that topical resatorvid can be effectively delivered to skin, and using in vivo studies that topical resatorvid can block UV-induced AP-1 activation in mouse epidermis. We also report that in a UV-induced skin tumorigenesis model, topical resatorvid displays potent photochemopreventive activity, significantly suppressing tumor area and multiplicity. Tumors harvested from resatorvid-treated mice display reduced activity of UV-associated signaling pathways and a corresponding increase in apoptosis compared with tumors from control animals. Further mechanistic insight on resatorvid-based photochemoprevention was obtained from unsupervised hierarchical clustering analysis of protein readouts via reverse-phase protein microarray revealing a significant attenuation of key UV-induced proteomic changes by resatorvid in chronically treated high-risk SKH-1 skin prior to tumorigenesis. Taken together, our data identify TLR4 as a novel molecular target for topical photochemoprevention of NMSC. Cancer Prev Res; 11(5); 265–78. ©2018 AACR. See related editorial by Sfanos, p. 251

The CyP40 protein encoded by PPID gene is a member of the peptidyl-prolyl cis–trans isomerase (PPIase) family. PPIases catalyze the cis–trans isomerization of proline imidic peptide bonds in oligopeptides and accelerate the folding of proteins. The CyP40 protein has been shown to possess PPIase activity and, similar to other family members, can bind to the immunosuppressant drug cyclosporin A (CsA). In this study, we created keratinocyte cell lines with CyP40 being stably knocked down using viral particles containing shRNA for CyP40 which knocked down the expression level of CyP40 transcripts by 90–99%. The proliferation rates of the cell lines with silenced CyP40 were decreased compared to the control cells. After UVA irradiation, the rate of apoptosis was found to be significantly lower in CyP40 silenced cell lines than it was in control cells. Moreover, mitochondrial membrane potential (MMP) was found to be less dissipated and mitochondrial permeability transition pore (MPTP) less active in cells with knocked down CyP40 than in control cells after UVA irradiation. Also, less mitochondrial superoxide was detected in the cells with silenced CyP40 compared to control cells after UVA exposure. Moreover, silencing of CyP40 partially modulates expression of key genes involved in mitochondrial pore formation including CyPD, ANTs and VDAC family members. The ability of CyP40 to regulate UV induced apoptosis implicates this protein as a potential target for therapy in cancer cells.

Resveratrol (3, 5, 4′-trihydroxy- trans- stilbene), a polyphenol compound, is derived from natural products such as the skin of red grapes, blueberries and cranberries. Resveratrol not only exhibits antioxidant, cardioprotection, and anti-aging properties, but can also inhibit cancer cell growth and induce apoptosis. It has been shown that resveratrol inhibits the activation of Nf-kB and subsequently down regulates the expression of Nf-kB regulated genes such as interleukin-2 and Bcl-2, leading to cell cycle arrest and increased apoptosis in multiple myeloma cells. In the skin, resveratrol has been reported to sensitize keratinocytes to UVA induced apoptosis. However, the effect of resveratrol on opening of the mitochondrial permeability transition pore has not been previously examined. Our data show that UVA (14J/cm2) along with resveratrol causes massive oxidative stress in mitochondria. As a consequence of oxidative stress, the mitochondrial membrane potential decreases which results in opening of the mitochondrial pores ultimately leading to apoptosis in human keratinocytes. These results may have clinical implications for development of future chemotherapeutic treatment for tumors of the skin.

There is increasing awareness of the role of mtDNA alterations in the development of cancer, as mtDNA point mutations are found at high frequency in a variety of human tumors. To determine the biological effects of mtDNA mutations in UV-induced skin tumors, hairless mice were irradiated to produce tumors, and the tumor mtDNAs were screened for single-nucleotide changes using temperature gradient capillary electrophoresis (TGCE), followed by direct sequencing. A mutation hot spot (9821insA) in the mitochondrially encoded tRNA arginine (mt-Tr) locus (tRNA(Arg)) was discovered in approximately one-third of premalignant and malignant skin tumors. To determine the functional relevance of this particular mutation in vitro, cybrid cell lines containing different mt-Tr (tRNA(Arg)) alleles were generated. The resulting cybrid cell lines contained the same nuclear genotype and differed only in their mtDNAs. The biochemical analysis of the cybrids revealed that the mutant haplotype is associated with diminished levels of complex I protein (CI), resulting in lower levels of baseline oxygen consumption and lower cellular adenosine triphosphate (ATP) production. We hypothesize that this specific mtDNA mutation alters cellular biochemistry, supporting the development of keratinocyte neoplasia.

Summary The physiological role of a vacuolar ATPase subunit c1 (SaVHAc1) from a halophyte grass Spartina alterniflora was studied through its expression in rice. The SaVHAc1-expressing plants showed enhanced tolerance to salt stress than the wild-type plants, mainly through adjustments in early stage and preparatory physiological responses. In addition to the increased accumulation of its own transcript, SaVHAc1 expression led to increased accumulation of messages of other native genes in rice, especially those involved in cation transport and ABA signalling. The SaVHAc1-expressing plants maintained higher relative water content under salt stress through early stage closure of the leaf stoma and reduced stomata density. The increased K+/Na+ ratio and other cations established an ion homoeostasis in SaVHAc1-expressing plants to protect the cytosol from toxic Na+ and thereby maintained higher chlorophyll retention than the WT plants under salt stress. Besides, the role of SaVHAc1 in cell wall expansion and maintenance of net photosynthesis was implicated by comparatively higher root and leaf growth and yield of rice expressing SaVHAc1 over WT under salt stress. The study indicated that the genes contributing toward natural variation in grass halophytes could be effectively manipulated for improving salt tolerance of field crops within related taxa.

Mitochondria are the major source of reactive oxygen species (ROS) in fibroblasts which are thought to be crucial regulators of wound healing with a potential to affect the expression of nuclear genes involved in this process. ROS generated by mitochondria are involved in all stages of tissue repair process but the regulation of ROS-generating system in fibroblasts still remains poorly understood. The purpose of this study was to better understand molecular mechanisms of how the regulation of ROS levels generated by mitochondria may influence the process of wound repair. Cybrid model system of mtDNA variations was used to study the functional consequences of altered ROS levels on wound healing responses in a uniform nuclear background of cultured ρ(0) fibroblasts. Mitochondrial ROS in cybrids were modulated by antioxidants that quench ROS to examine their ability to close the wound. Real-time PCR arrays were used to investigate whether ROS generated by specific mtDNA variants have the ability to alter expression of some key nuclear-encoded genes central to the wound healing response and oxidative stress. Our data suggest levels of mitochondrial ROS affect expression of some nuclear encoded genes central to wound healing response and oxidative stress and modulation of mitochondrial ROS by antioxidants positively affects in vitro process of wound closure. Thus, regulation of mitochondrial ROS-generating system in fibroblasts can be used as effective natural redox-based strategy to help treat non-healing wounds.

An urgent need exists for the development of more efficacious molecular strategies targeting non-melanoma skin cancer (NMSC), the most common malignancy worldwide. Inflammatory signaling downstream of Toll-like receptor 4 (TLR4) is a causative factor in several forms of tumorigenesis, yet its role in solar UV-induced skin carcinogenesis remains undefined. In our recently published work based upon immunohistochemical (IHC) analysis of NMSC tissue microarrays and proteomic analysis of reverse-phase protein microarrays (RPPA) from banked human tissue, we observed increased TLR4 expression in association with tumorigenic progression from normal skin to actinic keratosis and cutaneous squamous cell carcinoma. In immortalized keratinocytes expressing luciferase reporter constructs, resatorvid potently inhibits UV-induced AP-1 and NF-κB signaling, associated with downregulation of inflammatory mediators (IL-6, IL-8, IL-10) and MAP Kinase phosphorylation. In a subsequent acute exposure model in SKH-1 mice, topical resatorvid efficiently antagonized UV-induced stress signaling while potentiating UV-induced epidermal apoptosis. In the current report, we show for the first time that pharmacological inhibition of TLR4 using the specific antagonist resatorvid (TAK-242) blocks UV-induced tumorigenesis in SKH-1 mice. After assessing photostability, efficient cutaneous delivery, and skin residence time of topical resatorvid, we then tested the feasibility of TLR4-directed inhibition of UV-induced tumorigenesis. To this end, SKH-1 mice were split into three groups (n = 20). Each group was exposed to solar-simulated UV (three times a week; 15 weeks duration), followed by another 10 weeks in the absence of UV exposure (sacrifice at week 25). The control group received topical vehicle (acetone) on their backs 1 hour prior to UV exposure (three times a week until sacrifice). The “Prevention” group received topical resatorvid following an analogous dosing regimen. The “Intervention” group received vehicle 1hr prior to each UV treatment but switched to topical resatorvid three times a week only after UV was terminated. In the Prevention model, topical treatment with resatorvid significantly inhibited both tumor area (66% reduction, p = 0.0052) and tumor multiplicity (32% reduction, p = 0.0329; cross sectional Kruskal-Wallis test). In the Intervention model, a marked inhibition of both measures of tumor yield was observed, yet they did not reach statistical significance. Likewise, suppression of UV-induced TLR4-driven signaling was also confirmed in murine skin harvested at week 14 (prior to tumor onset), as assessed by IHC and RPPA analysis. Taken together, these data generated using resatorvid in a murine photocarcinogenesis model suggest that pharmacological TLR4 antagonism may represent a novel molecular strategy for topical prevention of solar UV-induced NMSC. Citation Format: Sally E. Dickinson, Karen Blohm-Mangone, Nichole B. Burkett, Shekha Tahsin, Paul B. Myrdal, Kelly L. Karlage, Jaroslav Janda, Kathylynn Saboda, Denise J. Roe, Zigang Dong, Ann M. Bode, Emanuel F. Petricoin, Valerie S. Calvert, Clara Curiel-Lewandrowski, David S. Alberts, Georg T. Wondrak. Pharmacological TLR4 antagonism using topical resatorvid blocks solar UV-induced skin tumorigenesis in SKH-1 mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2244. doi:10.1158/1538-7445.AM2017-2244

mtDNA mutations are common in human cancers and are thought to contribute to the process of neoplasia. We examined the role of mtDNA mutations in skin cancer by generating fibroblast cybrids harboring a mutation in the gene encoding the mitochondrial tRNA for arginine. This somatic mutation (9821insA) was previously reported in UV-induced hyperkeratotic skin tumors in hairless mice and confers specific tumorigenic phenotypes to mutant cybrids. Microarray analysis revealed and RT-PCR along with Western blot analysis confirmed the up-regulation of CCL20 and its receptor CCR6 in mtBALB haplotype containing the mt-Tr 9821insA allele compared to wild type mtB6 haplotype. Based on reported role of CCL20 in cancer progression we examined whether the hyper-proliferation and enhanced motility of mtBALB haplotype would be associated with CCL20 levels. Treatment of both genotypes with recombinant CCL20 (rmCCL20) resulted in enhanced growth and motility of mtB6 cybrids. Furthermore, the acquired somatic alteration increased the in vivo tumor growth of mtBALB cybrids through the up-regulation of CCL20 since neutralizing antibody significantly decreased in vivo tumor growth of these cells; and tumors from anti-CCL20 treated mice injected with mtBALB cybrids showed significantly decreased CCL20 levels. When rmCCL20 or mtBALB cybrids were used as chemotactic stimuli, mtB6 cybrids showed increased motility while anti-CCL20 antibody decreased the migration and in vivo tumor growth of mtBALB cybrids. Moreover, the inhibitors of MAPK signaling and NF-κB activation inhibited CCL20 expression in mtBALB cybrids and decreased their migratory capabilities. Thus, acquired mtDNA mutations may promote tumorigenic phenotypes through up-regulation of chemokine CCL20.

Sulforaphane is a natural product found in broccoli, which is known to exert many different molecular effects in the cell, including inhibition of histone deacetylase (HDAC) enzymes. Here, we examine for the first time the potential for sulforaphane to inhibit HDACs in HaCaT keratinocytes and compare our results with those found using HCT116 colon cancer cells. Significant inhibition of HDAC activity in HCT116 nuclear extracts required prolonged exposure to sulforaphane in the presence of serum. Under the same conditions HaCaT nuclear extracts did not exhibit reduced HDAC activity with sulforaphane treatment. Both cell types displayed down-regulation of HDAC protein levels by sulforaphane treatment. Despite these reductions in HDAC family member protein levels, acetylation of marker proteins (acetylated Histone H3, H4, and tubulin) was decreased by sulforaphane treatment. Time-course analysis revealed that HDAC6, HDAC3, and acetylated histone H3 protein levels are significantly inhibited as early as 6 h into sulforaphane treatment. Transcript levels of HDAC6 are also suppressed after 48 h of treatment. These results suggest that HDAC activity noted in nuclear extracts is not always translated as expected to target protein acetylation patterns, despite dramatic inhibition of some HDAC protein levels. In addition, our data suggest that keratinocytes are at least partially resistant to the nuclear HDAC inhibitory effects of sulforaphane, which is exhibited in HCT116 and other cells. © 2014 The Authors. Molecular Carcinogenesis published by Wiley Periodicals, Inc.

One of the primary components of the East Indian sandalwood oil (EISO) is α-santalol, a molecule that has been investigated for its potential use as a chemopreventive agent in skin cancer. Although there is some evidence that α-santalol could be an effective chemopreventive agent, to date, purified EISO has not been extensively investigated even though it is widely used in cultures around the world for its health benefits as well as for its fragrance and as a cosmetic. In the current study, we show for the first time that EISO-treatment of HaCaT keratinocytes results in a blockade of cell cycle progression as well as a concentration-dependent inhibition of UV-induced AP-1 activity, two major cellular effects known to drive skin carcinogenesis. Unlike many chemopreventive agents, these effects were not mediated through an inhibition of signaling upstream of AP-1, as EISO treatment did not inhibit UV-induced Akt, or MAPK activity. Low concentrations of EISO were found to induce HaCaT cell death, although not through apoptosis as annexin V and PARP cleavage were not found to increase with EISO treatment. However, plasma membrane integrity was severely compromised in EISO-treated cells, which may have led to cleavage of LC3 and the induction of autophagy. These effects were more pronounced in cells stimulated to proliferate with bovine pituitary extract and EGF prior to receiving EISO. Together, these effects suggest that EISO may exert beneficial effects upon skin, reducing the likelihood of promotion of pre-cancerous cells to actinic keratosis (AK) and skin cancer.

Flow cytometry has been employed for the analysis of higher plants for approximately the last 30 years. For the angiosperms, ∼500,000 species, itself a daunting number, parametric measurements enabled through the use of flow cytometers started with basic descriptors of the individual cells and their contents, and have both inspired the development of novel cytometric methods that subsequently have been applied to organisms within other kingdoms of life, and adopted cytometric methods devised for other species, particularly mammals. Higher plants offer unique challenges in terms of flow cytometric analysis, notably the facts that their organs and tissues are complex three-dimensional assemblies of different cell types, and that their individual cells are, in general, larger than those of mammals.This chapter provides an overview of the general types of parametric measurement that have been applied to plants, and provides detailed methods for selected examples based on the plant model Arabidopsis thaliana. These illustrate the use of flow cytometry for the analysis of protoplasts and nuclear DNA contents (genome size and the cell cycle). These are further integrated with measurements focusing on specific cell types, based on transgenic expression of Fluorescent Proteins (FPs), and on analysis of the spectrum of transcripts found within protoplasts and nuclei. These measurements were chosen in particular to illustrate, respectively, the issues encountered in the flow analysis and sorting of large biological cells, typified by protoplasts; how to handle flow analyses under conditions that require processing of large numbers of samples in which the individual samples contain only a very small minority of objects of interest; and how to deal with exceptionally small amounts of RNA within the sorted samples.

Flow cytometry has been employed for the analysis of higher plants for approximately the last 30 years. For the angiosperms, ∼500,000 species, itself a daunting number, parametric measurements enabled through the use of flow cytometers started with basic descriptors of the individual cells and their contents, and have both inspired the development of novel cytometric methods that subsequently have been applied to organisms within other kingdoms of life, and adopted cytometric methods devised for other species, particularly mammals. Higher plants offer unique challenges in terms of flow cytometric analysis, notably the facts that their organs and tissues are complex three-dimensional assemblies of different cell types, and that their individual cells are, in general, larger than those of mammals.This chapter provides an overview of the general types of parametric measurement that have been applied to plants, and provides detailed methods for selected examples based on the plant model Arabidopsis thaliana. These illustrate the use of flow cytometry for the analysis of protoplasts and nuclear DNA contents (genome size and the cell cycle). These are further integrated with measurements focusing on specific cell types, based on transgenic expression of Fluorescent Proteins (FPs), and on analysis of the spectrum of transcripts found within protoplasts and nuclei. These measurements were chosen in particular to illustrate, respectively, the issues encountered in the flow analysis and sorting of large biological cells, typified by protoplasts; how to handle flow analyses under conditions that require processing of large numbers of samples in which the individual samples contain only a very small minority of objects of interest; and how to deal with exceptionally small amounts of RNA within the sorted samples.

The narrow-leafed lupin possesses valuable traits for environment-friendly agriculture and for the production of unconventional agricultural products. Despite various genetic and environmental studies, the breeding of improved cultivars has been slow due to the limited knowledge of its genomic structure. Further advances in genomics require, among other things, the availability of a genomic DNA library with large inserts. We report here on the construction of the first DNA library cloned in a BAC (bacterial artificial chromosome) vector from diploid Lupinus angustifolius L. cv. Sonet. The high molecular weight DNA used for its preparation was isolated from interphase nuclei that were purified by flow cytometry. The library comprises 55,296 clones and is ordered in 144×384-well microtitre plates. With an average insert size of 100 kb, the library represents six haploid genome equivalents. Thanks to the purification of the nuclei by flow cytometry, contamination with chloroplast DNA and mitochondrial DNA was negligible. The availability of a BAC library opens avenues for the development of a physical contig map and positional gene cloning, as well as for the analysis of the plant’s genome structure and evolution.

Common wheat (Triticum aestivum L., 2n = 6x = 42) is a polyploid species possessing one of the largest genomes among the cultivated crops (1C is approximately 17 000 Mb). The presence of three homoeologous genomes (A, B and D), and the prevalence of repetitive DNA make sequencing the wheat genome a daunting task. We have developed a novel 'chromosome arm-based' strategy for wheat genome sequencing to simplify this task; this relies on sub-genomic libraries of large DNA inserts. In this paper, we used a di-telosomic line of wheat to isolate six million copies of the short arm of chromosome 1B (1BS) by flow sorting. Chromosomal DNA was partially digested with HindIII and used to construct an arm-specific BAC library. The library consists of 65 280 clones with an average insert size of 82 kb. Almost half of the library (45%) has inserts larger than 100 kb, while 18% of the inserts range in size between 75 and 100 kb, and 37% are shorter than 75 kb. We estimated the chromosome arm coverage to be 14.5-fold, giving a 99.9% probability of identifying a clone corresponding to any sequence on the short arm of 1B. Each chromosome arm in wheat can be flow sorted from an appropriate cytogenetic stock, and we envisage that the availability of chromosome arm-specific BAC resources in wheat will greatly facilitate the development of ready-to-sequence physical maps and map-based gene cloning.

The analysis of the hexaploid wheat genome (Triticum aestivum L., 2 n=6 x=42) is hampered by its large size (16,974 Mb/1C) and presence of three homoeologous genomes (A, B and D). One of the possible strategies is a targeted approach based on subgenomic libraries of large DNA inserts. In this work, we purified by flow cytometry a total of 10(7) of three wheat D-genome chromosomes: 1D, 4D and 6D. Chromosomal DNA was partially digested with HindIII and used to prepare a specific bacterial artificial chromosome (BAC) library. The library (designated as TA-subD) consists of 87,168 clones, with an average insert size of 85 kb. Among these clones, 53% had inserts larger than 100 kb, only 29% of inserts being shorter than 75 kb. The coverage was estimated to be 3.4-fold, giving a 96.5% probability of identifying a clone corresponding to any sequence on the three chromosomes. Specificity for chromosomes 1D, 4D and 6D was confirmed after screening the library pools with single-locus microsatellite markers. The screening indicated that the library was not biased and gave an estimated coverage of sixfold. This is the second report on BAC library construction from flow-sorted plant chromosomes, which confirms that dissecting of the complex wheat genome and preparation of subgenomic BAC libraries is possible. Their availability should facilitate the analysis of wheat genome structure and evolution, development of cytogenetic maps, construction of local physical maps and map-based cloning of agronomically important genes.

The analysis of the complex genome of common wheat (Triticum aestivum, 2n = 6x = 42, genome formula AABBDD) is hampered by its large size ( approximately 17 000 Mbp) and allohexaploid nature. In order to simplify its analysis, we developed a generic strategy for dissecting such large and complex genomes into individual chromosomes. Chromosome 3B was successfully sorted by flow cytometry and cloned into a bacterial artificial chromosome (BAC), using only 1.8 million chromosomes and an adapted protocol developed for this purpose. The BAC library (designated as TA-3B) consists of 67 968 clones with an average insert size of 103 kb. It represents 6.2 equivalents of chromosome 3B with 100% coverage and 90% specificity as confirmed by genetic markers. This method was validated using other chromosomes and its broad application and usefulness in facilitating wheat genome analysis were demonstrated by target characterization of the chromosome 3B structure through cytogenetic mapping. This report on the successful cloning of flow-sorted chromosomes into BACs marks the integration of flow cytogenetics and genomics and represents a great leap forward in genetics and genomic analysis.

Background We report the development of a microarray platform for rapid and cost-effective genetic mapping, and its evaluation using rice as a model. In contrast to methods employing whole-genome tiling microarrays for genotyping, our method is based on low-cost spotted microarray production, focusing only on known polymorphic features. Results We have produced a genotyping microarray for rice, comprising 880 single feature polymorphism (SFP) elements derived from insertions/deletions identified by aligning genomic sequences of the japonica cultivar Nipponbare and the indica cultivar 93-11. The SFPs were experimentally verified by hybridization with labeled genomic DNA prepared from the two cultivars. Using the genotyping microarrays, we found high levels of polymorphism across diverse rice accessions, and were able to classify all five subpopulations of rice with high bootstrap support. The microarrays were used for mapping of a gene conferring resistance to Magnaporthe grisea, the causative organism of rice blast disease, by quantitative genotyping of samples from a recombinant inbred line population pooled by phenotype. Conclusion We anticipate this microarray-based genotyping platform, based on its low cost-per-sample, to be particularly useful in applications requiring whole-genome molecular marker coverage across large numbers of individuals.

Background Genomics of rye (Secale cereale L.) is impeded by its large nuclear genome (1C~7,900 Mbp) with prevalence of DNA repeats (> 90%). An attractive possibility is to dissect the genome to small parts after flow sorting particular chromosomes and chromosome arms. To test this approach, we have chosen 1RS chromosome arm, which represents only 5.6% of the total rye genome. The 1RS arm is an attractive target as it carries many important genes and because it became part of the wheat gene pool as the 1BL.1RS translocation. Results We demonstrate that it is possible to sort 1RS arm from wheat-rye ditelosomic addition line. Using this approach, we isolated over 10 million of 1RS arms using flow sorting and used their DNA to construct a 1RS-specific BAC library, which comprises 103,680 clones with average insert size of 73 kb. The library comprises two sublibraries constructed using Hin dIII and Eco RI and provides a deep coverage of about 14-fold of the 1RS arm (442 Mbp). We present preliminary results obtained during positional cloning of the stem rust resistance gene SrR, which confirm a potential of the library to speed up isolation of agronomically important genes by map-based cloning. Conclusion We present a strategy that enables sorting short arms of several chromosomes of rye. Using flow-sorted chromosomes, we have constructed a deep coverage BAC library specific for the short arm of chromosome 1R (1RS). This is the first subgenomic BAC library available for rye and we demonstrate its potential for positional gene cloning. We expect that the library will facilitate development of a physical contig map of 1RS and comparative genomics of the homoeologous chromosome group 1 of wheat, barley and rye.