Martes pennanti), a candidate for listing under the federal Endangered Species Act in the Pacific states. In an investigation of threats to fisher population persistence in the two isolated California populations, we investigate the magnitude of this previously undocumented threat to fishers, we tested 58 carcasses for the presence and quantification of ARs, conducted spatial analysis of exposed fishers in an effort to identify potential point sources of AR, and identified fishers that died directly due to AR poisoning. We found 46 of 58 (79%) fishers exposed to an AR with 96% of those individuals having been exposed to one or more second-generation AR compounds. No spatial clustering of AR exposure was detected and the spatial distribution of exposure suggests that AR contamination is widespread within the fisher’s range in California, which encompasses mostly public forest and park lands Additionally, we diagnosed four fisher deaths, including a lactating female, that were directly attributed to AR toxicosis and documented the first neonatal or milk transfer of an AR to an altricial fisher kit. These ARs, which some are acutely toxic, pose both a direct mortality or fitness risk to fishers, and a significant indirect risk to these isolated populations. Future research should be directed towards investigating risks to prey populations fishers are dependent on, exposure in other rare forest carnivores, and potential AR point sources such as illegal marijuana cultivation in the range of fishers on California public lands.Anticoagulant rodenticide (AR) poisoning has emerged as a significant concern for conservation and management of non-target wildlife. The purpose for these toxicants is to suppress pest populations in agricultural or urban settings. The potential of direct and indirect exposures and illicit use of ARs on public and community forest lands have recently raised concern for fishers ( Fisher carcasses were submitted from the two isolated California populations by three fisher monitoring projects . CarcassDeceased fishers were collected by project personnel whenever a fisher was determined to be inactive for >24 hours, a mortality signal from the VHF collar was detected or when unmarked fisher carcasses were opportunistically observed at the project sites or adjacent areas. Fisher carcasses were stored in a −20 °C freezer until a complete necropsy to determine causes of mortality was performed by a board-certified pathologist specializing in wildlife at the California Animal Health and Food Safety Laboratory System (CAHFS) or the University of California Davis Veterinary Medical Teaching Hospital in Davis, CA, USA. Liver samples were collected during necropsy and submitted for screening and quantification of seven ARs at CAHFS by liquid chromatography-tandem mass spectrometry for screening presence of ARs and high-performance liquid chromatography to quantitate positive samples. The AR compounds tested for included first-generation ARs, warfarin (WAF), diphacinone (DIP), chlorophacinone (CHL), and coumachlor (COM); and second-generation ARs, brodifacoum (BRD), bromodiolone (BRM), and difethialone (DIF). The reporting limits were 0.01 ppm for BRD, 0.05 for WAF, BRM, and COM, and 0.25 ppm for DIP, CHL, and DIF. Detectable compound concentrations that were below quantitate limits were labeled as “trace” concentrations. All results were reported on a tissue wet weight basis and reviewed by a board-certified toxicologist Age classification was determined by tooth wear, sagittal crest or testicular/teat development, field and laboratory observation, and monitoring of individual animals p = 0.05.Prevalence of AR exposure among fishers was calculated for the total sample, each sex and each age class. We compared the AR exposure prevalence between sexes within and between the two California populations using two-tailed heterogeneity chi-square tests of association For monitored fishers, telemetry locations were used to generate 95% minimum convex polygon (MCP) home-range centroids to represent a centralized point within the core area of movement within each individual fisher home-range within each project area st and 2nd) of ARs, or exposure to individual compounds of ARs were distributed uniformly or spatially clustered in each of the two California populations Centroids were analyzed by spatial scan statistics to determine whether exposure to ARs, exposure to different generation classes (1Forty-six of the 58 fisher carcasses tested (79%) were exposed to one or more compound of AR . Frequendetected . No sampThirteen of 18 (72%) fishers from the northern California population were exposed to an AR compound . BrodifaThirty-three of 40 (83%) fishers from the southern Sierra Nevada were exposed to an AR compound . BrodifaComplete centroids were generated for 42 monitored fishers, 12 fishers from the northwestern California population  and 30 from the southern Sierra Nevada population . Of these fishers, 3-month MCP centroids were generated for 39 fishers, and 6-month centroids for 27 . SpatialCause-specific mortality factors for all 58 fishers sampled ranged widely and included predation, infectious and non-infectious disease processes and vehicular strikes (M.W. Gabriel unpublished data). The cause of death for four of these fishers was attributed to lethal toxicosis, indicated by AR exposure with simultaneous coagulopathy and bleeding into tissues or cavities and ruling out any concurrent processes that might cause hemorrhaging An adult male fisher was recovered on 15 April 2009, in the southern Sierra Nevada at the SNAMP project area. The fisher showed no signs of predation or scavenging . Gross nThe second fisher mortality was a lactating adult female recovered on 2 May 2010 in the center of a paved rural highway in the SNAMP project area approximately 3.7 km from Yosemite National Park. Vehicular strike was initially suspected as the cause of mortality due to the location of the carcass but lacerations, abrasions and visual evidence of trauma were not seen on gross examination of the intact carcass. The post-mortem state of the carcass was good and the nutritional state was poor (2.54 kg). Shallow subcutaneous hemorrhage was noted over the hindquarters and spinal column with no associated fractures, punctures or abrasions. There was approximately 20 ml of frank blood within the thoracic cavity. There was no evidence of pneumothorax, vessel ruptures, or visceral tearing. No blood or visceral damage was seen in the abdominal cavity. Stomach contents contained various rodent parts with formed feces in the descending colon. Histopathologically, no significant changes were observed in any tissues. Brodifacoum and BRM were detected and quantified at 0.60 ppm and 0.17 ppm, while one first generation AR, DIP was detected at a trace level within the liver tissue . No evidDermacentor variabilis) and 8 female and 2 male western black-legged ticks (Ixodes pacificus) removed from various regions of the fisher. The liver sample from this fisher had quantifiable levels of BRD at 0.04 ppm as well as a trace level of CHL  was in poor nutritional condition with no subcutaneous or visceral fat. Frank blood was present in the right external ear canal, nasal and oral cavities, within the lumen of the trachea and within the periorbital tissue with no associated skull fractures or punctures. The stomach was devoid of prey. The colon only contained semi-formed feces. Ectoparisitism was severe with approximately 48 female and 10 male American dog ticks  had no body fat present in any of the tissues. Frank blood was present in both thoracic and abdominal cavities. The stomach contained red and black fluid but no prey. Ectoparasitism was severe with 204 female and 27 male adult American dog ticks in both replete and non-replete stages on areas of the muzzle, chest, tops of fore-and hind-limbs as well as inguinal sections. Severe nematodiasis was seen in skeletal muscle throughout the body (trichinosis). Pulmonary nematodiasis (lungworm) was also noted in the marginal portions of the lungs. Histopathologically, no notable disease processes were seen but severe parasitism was noted. The liver sample for this fisher had quantifiable levels of BRD at 0.61 ppm and trace levels of BRM .Necropsies and AR testing was performed on four kits who were all still dependent on mother’s milk when they died following maternal abandonment from their mothers death. One kit, a female fisher (0.32 kg) from KRFP tested positive for AR exposure. This kit was approximately six weeks of age and was recovered within a monitored maternal den tree shortly after maternal abandonment. Cause of death was determined to be acute starvation and dehydration. The liver tissue contained trace level of BRD but there was no associated hemorrhaging in any tissues, body cavities or lumina, suggesting that this finding was not clinically significant.Our findings demonstrate that anticoagulant rodenticides, which were not previously investigated in fishers or other remote forest carnivores, are a cause of mortality and may represent a conservation threat to these isolated California populations. This is the first documentation of exposure to ARs and of direct mortality from ARs in fishers anywhere in their geographic range. Earlier studies suggest ARs posed little or no additive mortality effects on non-target populations Rattus norvegicus) was given a choice between BRD bait and untreated food and another group had access only to the BRD bait 50) on the first day and 40 to 80 LD50 doses by day 6.5, respectively The high rate of exposure to second generation AR compounds (96% of exposed fishers) in these populations is surprising and cause for concern. This generation of ARs are not only more acutely toxic, but have long retention  through biphasic elimination in mammal tissues Many manufactures use “flavorizers” since the AR compound may be bitter and unpalatable to rodent pests In addition to the risk from lethal toxicosis, sub-lethal AR exposure may compromise fishers through a reduction in the function of normal clotting High levels of tick infestations were noted in two of the AR mortalities when compared to other sympatric species within the same project area Furthermore, sublethal AR exposure may decrease an animal’s resilience to environmental stressors. In a study on rabbits and rats subjected to stressors such as severe decreases in ambient temperature (i.e. frostbite), approximately 10% of test animals died; however when animals were exposed to low non-lethal doses of anticoagulants and subjected to the same stressors, mortality rates increased to 40–70% The documentation of neonatal or lactational transfer of AR to a dependent fisher kit was unexpected, and the effects of AR exposure to a kit during fetal development or shortly after birth are unstudied. AR exposure in pregnant or whelping domestic canids varied, causing no clinical signs in some cases Mustela ermine) Mustela nivalis) Mustela furo) Mustela vision) 50 doses among genera, where minute amounts of brodifacoum bait caused death in domestic canids but domestic felids required doses 5 to 40 times higher The quantity (ppm) of AR we observed in fisher liver tissues varied and overlapped extensively in both sublethal and lethal cases with no clear indication of a numeric threshold that might indicate an amount leading to morbidity or mortality. This lack of predictive ability has been shown in numerous wildlife cases Lynx rufus) and mountain lion (Felis concolor) total quantification levels of AR exposure were associated with human-developed areas. Numerous studies have documented that secondary poisoning cases are closely associated with recent agricultural or urban pest eradication efforts Spatial analyses did not reveal any obvious point sources of AR exposure. Instead, these analyses suggested that exposure is widespread across the landscape. Previous studies expected that exposure to AR compounds would be clustered near areas of human activity or inhabitations and that exposure would not be common outside of these areas The majority of habitat that fishers in California and fishers throughout the DPS currently and historically occupied is not within or near agricultural or urban settings A likely source of AR exposure to fishers is the emerging spread of illegal marijuana cultivation within California public and private lands It is noteworthy that the AR fisher mortalities we documented occurred in different areas of their California range but within a relatively short seasonal period between mid-April to mid-May. We cannot specify the exact explanation or source contributing to all AR mortalities that occurred within this short temporal period. This period is when females are providing for offspring as well as males searching for mates; however, preliminary spatial data for fishers in California document that females have more confined home-ranges during this period, while males have slightly larger home-ranges .Additionally, several books available to the general public identify the optimal time for planting marijuana outdoors is during mid to late spring, and seedlings are especially vulnerable to rodent pests Vulpes vulpes necator), American marten (Martes americana), wolverine (Gulo gulo), gray wolf (Canis lupus) or raptors such as northern spotted owls , California spotted owls  and great gray owls (Strix nebulosa). Future research should be directed to investigating potential risks to prey populations as well as other sympatric species that may allow a better understanding of the potential AR sources contributing to these exposure and mortality rates from anticoagulant rodenticides.In conclusion, this study has demonstrated that fishers in the western DPS, which are of conservation concern and a candidate for protection under the Endangered Species Act, are not only being exposed to ARs, but ARs are a direct cause of mortality and indirect mortality (i.e. kit abandonment) in both of California’s isolated populations. Consequently, these toxicants may not only pose a mortality risk to fishers but could also pose significant indirect risks by depleting rodent prey populations upon which fishers depend. The lack of spatial clustering of exposed individuals suggests that AR contamination is widespread within this species’ range and illegal or irresponsible use of ARs continues despite recent regulatory changes regarding their use. Because we do not know the long-term ecological ramifications of these toxicants left on site long after marijuana grows are dismantled, heightened efforts should be focused on the removal of these toxicants at these and adjacent areas at the time of dismantling. Further regulation restricting the use of ARs to only pest management professionals as well as continued public outreach through state wide Integrated Pest Management programs may be warranted. In addition, promotion of compounds that do not possess the propensity for secondary poisoning (i.e. zinc phosphide) should be considered in non-professional use settings. Furthermore, ARs in these habitats may pose equally grave risks to other rare and isolated California carnivores such as the Sierra Nevada red fox  populations and sex on the number of anticoagulant rodenticides found per individual.(DOCX)Click here for additional data file.Table S2Results of spatial scan statistics to detect clusters of anticoagulant rodenticide (AR) exposed fishers within each California fisher project. Number of individual fisher minimum convex polygon (MCP) centroids used for each temporal period, specific AR types, generation class of AR and distribution of numbers of ARs per fisher (number of AR positive fishers per test in parentheses) are shown.(DOCX)Click here for additional data file.


Androgen activity plays a key role in prostate cancer progression. Androgen receptor (AR) is the main mediator of androgen activity in the prostate, through its ability to act as a transcription mediator. Here we performed a genome-wide analysis of human AR binding to promoters in the presence of an agonist or antagonist in an androgen dependent prostate cancer cell line. Many of the AR bound promoters are bound in all examined conditions while others are bound only in the presence of an agonist or antagonist. Several motifs are enriched in AR bound promoters, including the AR Response Element (ARE) half-site and recognition elements for the transcription factors OCT1 and SOX9. This suggests that these 3 factors could define a module of co-operating transcription factors in the prostate. Interestingly, AR bound promoters are preferentially located in AT rich genomic regions. Analysis of mRNA expression identified chicken ovalbumin upstream promoter-transcription factor 1 (COUP-TF1) as a direct AR target gene that is downregulated upon binding by the agonist liganded AR. COUP-TF1 immunostaining revealed nucleolar localization of COUP-TF1 in epithelium of human androgen dependent prostate cancer, but not in adjacent benign prostate epithelium. Stromal cells both in human and mouse prostate show nuclear COUP-TF1 staining. We further show that there is an inverse correlation between COUP-TF1 expression in prostate stromal cells and the rising levels of androgen with advancing puberty. This study extends the pool of recognized putative AR targets and identifies a negatively regulated target of AR – COUP-TF1 – which could possibly play a role in human prostate cancer. Prostate cancer is the most common non-skin cancer in males in the US, with an estimated number of 217,730 new cases in the US in 2010 5′-TGTTCT-3′, arranged as inverted repeats, separated by three nucleotides Androgen's effect on normal and malignant prostate cells is mediated through its ability to enter cells and bind its receptor – the AR. In the absence of a ligand the AR is located in the cytoplasm in a complex with heat-shock proteins (HSP) and co-chaperones In the presence of an AR antagonist, such as flutamide, the AR transcriptional complex still forms, yet transcription of well known AR target genes does not occur possibly via the recruitment of co-repressors. For example, upon addition of the antagonist bicalutamide, AR shifts into the nucleus, binds the promoter of its well known target gene PSA and recruits co-repressors such as SMRT and NCoR We have utilized genome-wide location analysis of AR in the presence of agonist, antagonist or no ligand to study the differences and similarities between AR target genes in those conditions. We have seen several promoters that are constitutively bound in the presence of an agonist and antagonist, as well as promoters that are bound only in the presence of either one. We further characterize one novel AR negatively regulated target gene COUP-TF1, which promoter is bound only in the presence of the antagonist.LAPC4 prostate cancer cells express wild type AR µM flutamide for 16 hours. The lengths of activation and R1881 concentration were chosen according to the time of maximal AR recruitment to its best studied target gene PSA To identify the direct target genes of AR in prostate cancer cells in the presence of an AR agonist, AR antagonist, or in the absence of both, LAPC4 cells were first androgen ablated for three days. The cells were then incubated with vehicle alone, 10 nM R1881 or 40 An analysis of AR target genes with vehicle alone, R1881 and flutamide revealed three groups of ARBs. There is some overlap between target genes in those three conditions, as well as ARBs specific to each condition . The lisGenome-wide location analysis results were validated using gene specific chromatin IP for eight of the ARBs in all three treatments see . Chromatsox5 . B3gnt5 promoter is bound by the AR only in the presence of an agonist  analysis was performed to find functional groups that are enriched within ARBs. In all ligand settings examined, despite great variation in target genes, the enriched categories, were those categories involved in DNA binding and transcription activity .We looked for the prevalence of the canonical androgen recognition element in the ARB set we identified, compared with all 18,051 probes on the array. We allowed for up to two mismatches in the 15 bp androgen response element (ARE) sequence. The ARE was found in 4% of all probes on the array. When scanning for ARE in the three lists of ARBs there was only mild enrichment of ARE compared to the background in the R1881 and flutamide groups . When sc5′-AGAACA-3′) is enriched in any of the groups, without mismatches. The half-site was searched in ARBs compared with the array's background. The half-site ARE was found to be highly enriched in all three groups of ARBs . Interestingly, SOX9 was recently described as active in prostate carcinogenesis and embryogenesis de novo motif search. Weeder de novo motif search algorithm, revealed the motif GCAAATCA to be significantly enriched in the agonist bound group, and further analysis revealed it to be enriched in all ARB groups (We further wished to look for novel motifs that were enriched in ARBs through −13) and 48.5% (p = 3.4*10−5), respectively. In order to confirm that this phenomenon is not a selection bias of the array, we compared it to the GC content of MLL1 bound probes that were published using the same array et al. data set that analyzed androgen bound promoters in the presence of androgen In order to further characterize the ARBs we calculated the GC content of all ARBs, and compared it to the array's background GC content. Surprisingly we found that the ARBs are highly and significantly AT rich. The GC content of the entire array is 54.3%, while the GC content of the agonist and antagonist ARBs are 46.4%  is a vesicle transporter protein that was recently shown to be regulated by p53 and required for cancer cell adhesion and survival As expected, the well known AR target PSA is upregulated by androgen and down regulated by flutamide.Thus, AR binding to promoter sites can be associated with either upregulation, downregulation or no change of transcription.in vivo binding analysis suggested that AR binds the genomic sequence upstream to the transcription start site of coup-tf1 in several locations and the 5′ untranslated region (UTR) of the coup-tf1 gene. In order to study the regulation of COUP-TF1 by AR we first further validated binding of the AR to the genomic sequences surrounding the coup-tf1 transcription start site. In the presence of the AR antagonist flutamide the AR is bound in the area 1–2 KB upstream to the TSS, in the coup-tf1 promoter and in the 5′ UTR of the gene. However in the presence of androgen the AR binds the area 1–2 kb upstream to the TSS, but not the promoter or 5′UTR . Coup-TF1 is an orphan nuclear receptor which acts mainly as a transcription repressor or 5′UTR .As described above, COUP-TF1 mRNA levels are negatively regulated by androgen and positively regulated by flutamide. We further validated this observation at the protein level by western blot analysis. COUP-TF1 protein levels in LAPC4 cells do not change following addition of R1881 for 48 hours to androgen deprived cells. However, in the presence of flutamide for 48 hours, COUP-TF1 protein levels rise . InteresTo examine the expression pattern of COUP-TF1 in human prostate and prostate cancer we used immunohistochemistry to detect COUP-TF1 in 28 human tumor samples. We detected COUP-TF1 in the malignant epithelium of 21 out of 28 primary prostate cancer samples examined. We could not find a correlation between COUP-TF1 staining and Gleason score or disease recurrence in either the epithelial or stromal cells. However, we did find significantly higher levels of COUP-TF1 staining in neoplastic prostate epithelium and in the pre-malignant prostatic intraepithelial neoplasia (PIN) compared with staining of adjacent benign prostate epithelium . This suIn order to confirm COUP-TF1 antibody specificity we have stained LAPC4 xenografts for COUP-TF1 and found a nucleolar distribution of COUP-TF1  similar In order to validate the negative regulation between AR and COUP-TF1 in the prostate we used pre-pubertal Balb/c mice. Pre-pubertal mice have a low testosterone level at three weeks, with levels increasing as the mouse reaches puberty The transition of prostate cancer to the hormone refractory state is a major turning point in the progression of prostate cancer, and AR plays a major role in this transition. Currently used AR antagonists such as flutamide and bicalutamide, assume the role of receptor agonists when the disease becomes hormone refractory. The first evidence of this transition from antagonist to agonist was inferred from the clinical observation that prostate cancer patients had a 30% meaningful response to the withdrawal of a steroid hormone antagonist as the first maneuver after primary hormonal therapy failure The binding of AR to DNA, although ligand dependent, is not dependent on co-activators. Upon induction with flutamide, AR binds to the PSA promoter. The PSA promoter was also shown in different experiments to be bound by AR without any ligand , while tin vivo binding analysis, were validated in gene specific chromatin immunoprecipitation analysis; thus it is less likely that ligand binding induces AR dissociation from chromatin.In our genome-wide location analysis of AR target genes we have discovered promoters that are bound constitutively with agonistic ligand, antagonist ligand or no ligand at all . None ofSeveral genes were differentially bound by AR, in the presence of the agonist or antagonist . In ordeThis differential binding in the presence of flutamide can be of special importance when considering antagonist to agonist conversion in hormone refractory prostate cancer. It is reasonable to assume that those genes can be easily induced upon transition to hormone refractoriness since they are already bound by an AR, and need only to further recruit co-activators and the basal transcription machinery. This finding could also have therapeutic significance utilizing the concept of synthetic lethality: Those target genes that are only activated in flutamide treated cells could serve as therapeutic targets in combination with flutamide.In order to gain information on AR's activity in androgen dependent prostate cancer a bioinformatical analysis of AR target genes was performed. GO analysis of ARBs suggests that AR exerts its cellular effect by binding to promoters of other transcription factors in the presence of all the various ligands . This sude novo motifs in ARBs. This analysis could identify transcription factors that act as co-regulators of transcription together with the AR. Our analysis revealed a non-consensus OCT1 motif that was reported previously to be involved in AR transcription Next we looked for known and Brachyuri is a member of the T-box protein family that is extensively involved in embryogenesis SOX9, is a Sry-related High Mobility Group (HMG) factor that was previously reported to play a role in prostate development. It is expressed at developing prostate epithelial buds with strongest expression in the distal tips of the buds. A severe defect in the development of the ventral prostate was observed in SOX9 mutant animals Of further interest is the finding that AR preferentially binds AT-rich genomic areas. The biological significance of such binding should be further validated. This preferential AR binding to AT rich DNA could be explained by the relative lack of nucleosomes in AT rich regions, and specifically in areas containing poly  sites In the second part of our work we wished to focus on AR regulation of one promoter, COUP-TF1. We chose COUP-TF1 for two reasons. First, because COUP-TF1 is a promoter that is occupied only by flutamide bound AR and not by androgen bound AR. It is important to show that such promoter occupancy serves a functional role. Second, as transcriptional repression by the AR is less well studied, we chose to further evaluate one novel AR target gene – COUP-TF1. COUP-TF1 is itself a transcription repressor known to play a role in development We have demonstrated that COUP-TF1 is negatively regulated by AR in LAPC4 cells, both at the mRNA and protein levels. COUP-TF1 is down regulated upon treatment by the androgen R1881 and upregulated by treatment with flutamide.We further wished to define the expression pattern of COUP-TF1 in prostate cancer. Immunohistochemical staining of COUP-TF1 in human prostate tumor tissue from radical prostatectomy specimens of various Gleason grades, and the adjacent normal epithelium showed higher staining for COUP-TF1 in nucleoli of malignant prostate epithelium compared to adjacent normal epithelium . This stThe important role of AR in prostate mesenchyme, and the role of AR in regulation of COUP-TF1 have led us to investigate the expression of COUP-TF1 in prostate mesenchyme. We found that COUP-TF1 levels in prostate mesenchyme are inversely correlated with androgen levels in prepubertal mice  in agreeThe results presented in this work further promote our understanding of the importance of AR antagonist bound target genes, which are expected to play a major role in HRPC, and are possibly targets for therapy. These results, specifically with their bioinformatic analysis, provide a basis for further study on AR's role and mechanism of activity. We explored one AR target and analyzed its expression in both malignant and normal tissue. Other targets revealed in this work can be explored in a similar way, in order to investigate their role in androgen dependent and hormone refractory prostate cancer. The AR co-activators we suggested, and the AT rich environment of AR binding, should be further evaluated in biological experiments to validate their role and significance in prostate cancer.Experiments with human tissues were approved by the Institutional Review Board (IRB), at the Hadassah-Hebrew University Medical Center. Due to the retrospective nature of this study, the fact that tissues were unidentified and according to the declaration of Helsinki, our IRB waived the need for written informed consent. IRB approval number HMO0416-08. All mice experiments were approved by the Hadassah-Hebrew University Medical Center Institutional Animal Care and Use Committee - approval number MD 78.06-3.The LAPC4 cell line was generated from an androgen dependent human prostate cancer 3 cells per well. All treatments were performed on the same plate, and each treatment was repeated in 8 wells. Cells were plated in RPMI 1640 without phenol red supplemented with charcoal striped serum (CSS) with either ethanol (as vehicle), DHT 10 nM, flutamide 40 µM or the combination of DHT and flutamide . Seven days after plating XTT reagent  was added to the plate and absorbance was determined at 460 nm.LAPC4 cells were cultured in 96 well plates, 5×10Absorbance of wells containing medium alone was subtracted, and fold change compared to vehicle was calculated, as an average of 8 replicates. P value was calculated using student's t-test compared either to vehicle (for DHT and flutamide treatment groups) or to DHT treatment (for the combined treatment group).7 LAPC4 cells were used for each reaction. Cells were grown in RPMI 1640 without phenol red with CSS for three days. The media was then supplemented with ethanol (as vehicle), R1881 10 nM or flutamide 40 µM. The cells were fixed with 1% Formaldehyde for 10 minutes. Then cells were lysed in lysis buffer containing 50 mM Tris pH 8, 1% SDS, 10 mM EDTA and Protease inhibitor cocktail  and sonicated to yield fragments of 200–500 bp. 1/30 of the input sample was taken for further standardization. Lysates were pre-incubated with 60 µl salmon sperm coated protein G beads  in 50% slurry. Than lysates were incubated with 10 µg of monoclonal anti-AR antibody clone AR441  over-night. 60 µg Salmon sperm protein G beads were added for 1 hour. Beads were than washed twice with 1 ml of each of three wash buffers as recommended by upstate biotechnology ChIP protocol . Elution was done using TE buffer  and 1% SDS for 30 minutes. The immunoprecipitated and input samples were reverse cross linked by incubation at 65°C for 4–16 hours and then proteinase K was added for purification. The eluted and input samples were purified using Qiaquick PCR purification kit . A sample of input was run on agarose gel to ensure DNA fragmentation to 200–500 bp fragments and quantified using NanoDrop to about 400 ng/µl. The IP sample had a concentration of approximately 10 ng/µl.A total of 5×10Quantification of ChIP was done using a standard gene, either histone H2A promoter or GAPDH promoter. The input sample was diluted 1∶300 followed by four consecutive three-fold dilutions. IP was used as template in 0.06 µl and 0.18 µl per reaction. PCR for promoter of interest and reference gene were done using Abgene Reddymix  with annealing temperature of 56–60°C, 37 cycles. A sample of PCR product was resolved on a 1.5% Agarose- Ethidium Bromide gel, and documented using GelDoc-It imaging system.We wrote a Matlab  program to quantify specific promoter enrichment compared to a standard gene, in a non-biased way. Input samples were set as the standard and piecewise linear interpolation was done. IP samples were quantified according to that interpolation. Enrichment of the promoter in hand was calculated as specific promoter on standard divided by reference gene on standard. An average of two IP samples was used. Each PCR was repeated at least twice, and represents at least two independent ChIP reactions. For semi-quantitative PCR primers see 5′ -GCGGTGACCCGGGAGATCTGAATTC-3′, oJW103 5′- GAATTCAGATC-3′Protocol for ChIP amplification and hybridization was done as previously described Input and IP were purified using Qiaquick PCR purification kit , mixed and hybridized to HU19K spotted promoter array The array was scanned and analyzed with GenePix Pro software , and the fluorescence intensity in both channels was obtained for each spot.The Hu19K array was manufactured and kindly provided by Richard A. Young's lab For each probe the intensity was normalized according to whole chip error model which incorporates signal intensity and background noise to calculate p-value for the significance of enrichment for each probe. A probe with enrichment p<0.001 was considered as a bound promoter. A probe that was enriched p<0.001 in one array and p<0.01 in another was considered to be enriched in both arrays.The bound probes were analyzed with 300 bp added on each side to compensate for length of sonicated genomic DNA fragments (200–500 bp). We then applied a series of motif search algorithms. To search for well defined motifs, without enumeration , we used the motif search procedure FindPatterns from the GCG package . Motifs of 6 bp to 8 bp length were searched with no mismatches allowed, and 9 to12 bp sequences were searched with two mismatches allowed. P-value of the number of hits, compared to the array's background, was calculated using corrected hypergeometric distribution.We further performed Position Weight Matrix (PWM) based motif scanning from the TRANSFAC database, release 12.1 De novo motif search was done using Weeder Gene Ontology (GO) category analysis was performed using Webgestalt software For expression assays cells were grown in RPMI 1640 without phenol red with CSS for three days. The media was then supplemented with ethanol (as vehicle), R1881 10 nM or flutamide 40 µM for 24 hours. RNA was extracted from LAPC4 cells after various treatments using Trizol reagent , as stated in the manufacturer protocol. RNA was separated using chloroform, and purified using isopropanol and ethanol. The RNA was purified again using ethanol precipitation, and quantified using NanoDrop.Reverse transcription PCR was preformed using High capacity cDNA Reverse Transcription kit .Real time PCR was performed using Platinum SYBR green qPCR SuperMix-UDG with Rox , according to the manufacturer's instructions in an Applied Biosystems 7900HT real time PCR machine . See primers used in 7 cells were harvested for each reaction. Cells were lysed in Buffer A . 1% was taken for input sample.Co-immunoprecipitation was preformed according to Lysate was precleared with protein A sepharose beads and then either anti AR  or control Ab  were added overnight. Than samples were incubated with sepharose A beads for one hour and washed with buffer A three times. Samples were analyzed on SDS-PAGE gel. Western blot was preformed as described below using antibodies recognizing AR  or SOX9 .For protein expression assays cells were grown in RPMI 1640 without phenol red supplemented with CSS for three days. The media was then supplemented with ethanol (as vehicle), R1881 10 nM or flutamide 40 µM for 48 hours. Proteins were extracted using lysis buffer containing 50 mM Tris pH 7.6, 150 mM NaCl, 0.5% NP-40, 5 mM EDTA, protease inhibitor cocktail 1∶100. Protein concentrations were determined using Bradford assay, and 50 µg of total protein was loaded on 9% SDS-PAGE gel. Proteins were transferred to a nitrocellulose membrane and were blotted with polyclonal rabbit anti-AR  at 1∶1000 dilution, mouse monoclonal anti-COUP-TF1  at 1∶1000 dilution and mouse monoclonal anti-GAPDH  at 1∶30,000 dilution, all for one hour at room temperature. Secondary antibodies used were HRP conjugated goat anti-mouse or anti-rabbit  diluted 1∶10000 for 45 minutes at room temperature. ECL reagents were purchased from Amersham Pharmacia, NJ, USA and used according to manufacturer's recommendations.The study included 28 samples of human prostate carcinoma from radical prostatectomy samples from patients previously untreated. The Gleason score was obtained from the pathology report, and information regarding recurrence of disease after prostatectomy was obtained from the patients' records.Murine prostates were taken from Balb/c mice at defined ages. At least four mice in each group were used. Anterior, ventral and dorsolateral prostate samples were obtained.6 cells were injected subcutaneously to 6–7 weeks old nude male mice together with Matrigel . A total of 4 mice were injected. Tumors were harvested when reached measurable size and formalin fixed.LAPC4 cells were harvested, washed and reconstituted in PBS. 10Four micrometer thick sections of paraffin embedded human or mouse tissue were de-paraffinized in xylene and rehydrated in graded alcohols. Endogenous peroxidase was blocked using 3% hydrogen peroxide for 5 minutes before and after antigen retrieval. Antigen retrieval was preformed using 50 mM EDTA pH 8.0 in a pressure cooker for 3 minutes (AR) or using 100 mM Glycine pH 9.0 in a pressure cooker for 3 minutes, twice (COUP-TF1). Primary antibodies were used as follows: polyclonal rabbit anti-AR  at 1∶500 dilution and mouse monoclonal anti- COUP-TF1  at 1∶150 dilution. Both were diluted in Cas block  and incubated with target tissues at room temperature for one hour. Detection was done using Mach2 and Mach3 AP polymer detection kits . Staining was done using DAB for two minutes and counter staining using hematoxylin for 30 seconds. Staining was quantified as percent of cells stained, by a pathologist (EP) who was unaware of the experimental group.Figure S1Effect of androgen modulation on growth of LAPC4 cells in vitro. LAPC4 cells were grown in the presence of vehicle, androgen (DHT 10 nM), the AR antagonist flutamide (40 µM) or the combination of androgen and flutamide. Cell growth was monitored on day 7 compared to vehicle using XTT assay. Each datapoint represents the average of 8 independent wells. Y axis – fold proliferation compared to vehicle. P-value was calculated using student's t-test compared either to vehicle (for DHT and flutamide treatment groups) or to DHT treatment (for the combined treatment group).(TIFF)Click here for additional data file.Figure S2Validation of novel AR target genes. LACP4 cells were androgen deprived for 72 hours and then treated with vehicle (ethanol), a synthetic androgen (R1881) or an AR antagonist (flutamide). Cells were fixed 16 hours after treatment and chromatin immunoprecipitation with an anti AR antibody was performed. PCR for the indicated target genes compared to non-bound gene are presented for 3 fold dilutions of input and immunoprecipitated fraction. Enrichment of each promoter compared to a non-bound promoter is quantified below each image using a Matlab procedure designed to calculate enrichment in an unbiased manner. Each experiment represents at least two different chromatin IP and at least two PCR reactions for each chromatin IP. a. PSA b. SOX5 c. DOCK1 d. B3GNT5 e. SLITRK3 f. IL1R2.(TIF)Click here for additional data file.Figure S3Chromosomal distribution of AR target genes. Chromosomal distribution of AR target genes in the three treatment groups. Red dots indicate chromosomal locations of AR bound promoters.(TIF)Click here for additional data file.Table S1Primers used for PCR reactions.(PDF)Click here for additional data file.Table S2Genomic locations (Hg19) and p value of enrichment upon treatment with different ligands.(XLS)Click here for additional data file.


Objectives. To demonstrate the feasibility of the prenatal diagnosis of partial androgen insensitivity syndrome by 3D-4D ultrasound. Methods. To report prenatal diagnosis of partial androgen insensitivity syndrome at 32nd week of gestation by 3D-4D ultrasound in a fetus with a 46XY karyotype, testing negative to the mutation analysis of SRY gene and the 5α-reductase 2 gene (SRD5A2). Results. 3D-4D surface rendering allows the detection of external and internal genital malformations and can address the prenatal diagnosis of PAIS and can exclude associated complications. Conclusions. Prenatal diagnosis of PAIS allows an adequate parental counseling and an early optimal management of the condition, not only for the psychological and social reflections but also for the avoidance of complications and postnatal morbidity due to misdiagnosis or delays in the treatment of the genital ambiguity. Androgen insensitivity syndrome  represents the most common identifiable cause of male pseudohermaphroditism, accounting for as many as 1 : 20.000 births [α-reductase 2 gene (SRD5A2) to exclude other causes of hermaphroditism or pseudohermaphroditism. Both analyses did not reveal any mutation. To clarify the discrepancy between the male karyotype and the female genital phenotype, an amniocentesis was performed and the fetal DNA extracted from the cultured amniocytes was tested by direct sequencing for mutations in the SRY gene and in the 5Therefore, the presence of female external genitalia without the uterus, in association with normal SRY and SRD5A2 genes, led us to the diagnostic hypothesis of  (CAIS/PAIS), that was tested through the sequence analysis of the AR gene which did not show any mutation. A multidisciplinary counseling session was scheduled with the parents to address the issue of the female sex assignment at birth. Delivery was at the 39th gestational week. The female external genitalia with clitoromegaly at birth are shown in In the present report, we show how the prenatal diagnosis of PAIS allows an adequate parental counseling and an optimal management of the condition, not only for the relevant psychological and social implications of an early diagnosis but also for the reduction of the chance of complications and postnatal morbidity due to misdiagnosis or delays in the treatment of the genital ambiguity. The contribution of US in prenatal sex determination is well known, and its role in detecting sexual differentiation disorders has been previously established . In addiα-reductase 2 deficiency), and the confirmation of the diagnosis through the identification of a molecular defect in AR gene. In our case, direct sequencing of all of the 8 exons of the AR gene did not detect any mutation, consistent with the mutation rate of 28% to 73% for the incomplete, milder phenotypes [The mainstay of the diagnosis of PAIS is the presence of a 46 XY karyotype, the determination of the normality (baseline or after hCG stimulation) of testosterone and dihydrotestosterone levels  has given the possibility of a reassuring prenatal counseling session about the excellent prognosis of the condition, contributing to improve the quality of life and health for both the child and the parents.


A molecular mechanism explaining mutated AE1-linked stomatocytosis involves changes in AE1 transport properties that become leaky to Na+ and K+. However, another explanation suggests that point-mutated AE1 could regulate a cation leak through other transporters. This short paper intends to discuss these two alternatives.Missense mutations in the erythroid band 3 protein (Anion Exchanger 1) have been associated with hereditary stomatocytosis. Features of cation leaky red cells combined with functional expression of the mutated protein led to the conclusion that the AE1 point mutations were responsible for Na It belongs to the Solute Carrier 4A family (SLC4A) grouping bicarbonate transporters [myocytes . In red myocytes . Besidesmyocytes , and hummyocytes , whereasmyocytes , 10. SLC4A1 gene coding for AE1 have been reported [SLC4A1 mutations. In this paper we will focus on red cell AE1, and the reader interested in kidney AE1 is therefore addressed to very exhaustive recent reviews on this subject [In human, many different mutations in reported . Some of subject –15. SLC4A1 mutations, the symptoms are hyperhaemolysis and anaemia, icterus, and splenomegaly. However, these symptoms may vary widely in intensity. It appears that the SLC4A1 mutations can be divided into two classes according to the way they impair AE1 function: (1) those that prevent correct folding of the protein so that it is not addressed to plasma membrane. This leads to a lower amount of AE1 in red cell membrane that impairs connection of skeleton and membrane, a feature of hereditary spherocytosis condition [When a red cell phenotype is associated with ondition , 17; (2)ondition , 19.SLC4A1 gene  were associated with increased red cell Na+ and K+ leak [+ and K+ transporters (or channels) in red cell membrane by mutated AE1 [Since the initial discovery that 5 point mutations in  K+ leak , 4 other K+ leak –24. It h K+ leak . Howeverated AE1 , 24, 26.AE1 polypeptide can be divided into 3 functional domains: a cytoplasmic amino-terminal domain, about 400 amino acids, interacting in red cells with various enzymes, haemoglobin, ankyrin, and band 4.2; a membrane spanning domain where anion exchange takes place and a short carboxy terminal end in the cytoplasm that associates with carbonic anhydrase II . The proThe permeability of red cells from patients bearing heterozygous mutation on AE1 has been investigated, and the transport features of the point-mutated AE1 have been studied by expression in amphibian oocytes. Thus, in most cases, it was possible to combine data from red cells to data from heterologous expression system. These data are presented here and will be discussed in the third part. + and K+ by an ouabain- and bumetanide-resistant mechanism that is temperature dependent; it is increased by temperatures below 37°C. This has been extensively studied in red cells of patients heterozygous for L687P, D705Y, S731P, H734R, R760Q, and S762R AE1 mutants [+ and Na+ according to their electrochemical gradients leads to osmotic fragility of the red cells. At body temperature, the cation leak can be more or less compensated depending on the mutants. An artefactual rise in plasma K+  can be observed after cooling blood to room temperature [+-K+-2Cl− cotransporter, K+-Cl− cotransporter, Na+/H+ exchanger, or K+/Na+/H+ exchanger has been reported [+/H+ exchanger and Na+/K+-pump, whereas the Gardos channel fluxes are reduced [+ and K+ movements. The regulation of these other carriers could differ between patients as well as how the body cope with cation leaky red cells. This could explain variations in patient's phenotypes  has long been known to inhibit anion exchange at micromolar concentrations , 36. Moran wtAE1 .+ and K+ leak induced by L687P, D705Y, S731P, H734R, and R760Q mutations has been observed in red cells with SITS, dipyridamole, and NS1652 also known to block anion exchanger [+ and K+ conductance induced by expression of mutated AE1 was observed with SITS and plurivalent cations such as Zn2+, La3+, and Gd3+ [Pharmacology of the cation leak induced by AE1 point mutations has also been investigated. Inhibition of the Naxchanger . Pharmacxchanger , 38. Howand Gd3+ , 25.86Rb+ uptake, and only a ≈2.5-fold increase in Li+ uptake is observed (to compare to ≈8-fold increase in oocytes expressing S731P mutant for instance). Moreover expression of R730C mutant is associated with an increased activity of the Na+/K+ ATPase [86Rb+ uptake in both systems. However, it appears that this Rb permeability is not correlated to the expression level of the mutant when expressed in ambystoma: the higher number of transporters when coexpressed with GPA does not induce a higher 86Rb uptake. The expression of E758K mutant also slightly increases xenopus oocyte conductance, but this conductance does not account for the observed Rb+ permeability as deduced from differences in pharmacological pattern [Amongst AE1 point mutations associated with cation leaky red cells, two mutants (R730C and E758K) exhibit peculiar transport features in amphibian oocytes. R730C AE1 mutant induces only a weak ouabain- and bumetanide-resistant cation leak in xenopus oocytes. It is not possible to measure a significant increase in ouabain- and bumetanide-resistant + ATPase . For E75 pattern .Point mutations in AE1 are associated with monovalent cation leak in red cells as in heterologous expression systems. This monovalent cation leak could be correlated to a nonselective cation conductance and to elevated activity of endogenous transport systems.+ and K+, or the mutated AE1 stimulates native transporters for Na+ and K+ in red cells as in heterologous expression systems.Two possibilities that are not exclusive could be envisioned: the missense mutations in AE1 polypeptide change the transport properties of the protein that becomes leaky to Na+-K+-2Cl− cotransporter by its carboxy terminal domain, stimulating the activity of this transporter in xenopus oocytes [The work on trout AE1 has shown that this protein could interact with Na oocytes . ReportsAs AE1 forms part of a macrocomplex, it functionally interacts with carbonic anhydrase , 40, and2+ permeable cation channels  or nonselective voltage-independent cation channels (NSVCCs) [2+ sensitive K+ channel (Gardos channel) [+ and K+ leak induced by AE1 point mutations could be mediated by one of these conductances. However, the features of the cation leak associated with AE1 point mutations do not point out any of the red cell channels described so far. In particular, this cation leak is insensitive to amiloride, known to block the NSVCC; it is insensitive to extracellular Cl− concentration, known to stimulate red cell cation channels; it is insensitive to extracellular Ca2+ concentration [Since pioneer work of electrophysiologists on red cells in the 80s , numerou(NSVCCs) –51 and Cchannel)  are wellchannel) . Howeverntration . Thus, i+-K+-2Cl− cotransporter, Na+/K+ ATPase, Na+(K+)/H+ exchanger, and K+-Cl+ cotransporter [Whatever the origin of the cation leak induced by AE1 point mutations, the consecutive alteration of cation permeability will impair red cell homeostasis and modulate the activity of different transporters. The permeability features of red cells with H734R, G796R, or R730C AE1 mutations show that the activity of different transporters could be stimulated: Nansporter , 23, 26.− slippage occurs occasionally (1 for 10 000 exchange). Crystallographic structures are not available yet with enough resolution to help understand the transport mechanism [+ and K+) [− concentration [In the absence of identified native transporter mediating the cation leak associated with AE1 point mutations, the hypothesis of a cation leaky AE1 is challenging and the simplest to propose. Moreover, it provides an attractive approach to understand the transport mechanism of this protein. Whereas it is depicted as a typical electroneutral anion exchanger, the anion exchange rate through AE1 is extremely fast (10 000 per second) and Clechanism . This me and K+) , 55. The and K+) . Thus, intration . The speA mutated state where no anion exchange occurs only cation leak exists and a B mutated state where anion exchange and cation leak coexist  . SAO AE1echanism .+ and K+ for which a high driving force exists. This leads to consider that the transport site is susceptible to structural changes that could be induced by diverse but specific amino acid substitutions. This change unmasks a conductance for monovalent cations that does not seem to interfere with the ability to exchange anions since some mutants exhibit both transport activities.In wtAE1, the anion exchange site involves transmembrane helix 8 (TM 8) and an anion selectivity filter including a region at the top of TM 12 and 13 and amino acids in the loop connecting TM 7 and 8 , 62. Our+-K+ pump, are other examples of ambiguous transport mechanisms between channels and transporters which strengthen our simple hypothesis of cation leaky AE1 [3/NH4+ transporter in red cells [The possibility for a carrier to function as a channel seems conflicting. Indeed channels are seen as structures that could simultaneously connect intra- and extracellular medium, what should never happen through a carrier. However, there are increasing examples of membrane proteins with ambiguous behaviour between channels and transporters. A historical example of transporter with channel activity is the glutamate transporter which is also a chloride channel –66. Chloeaky AE1 –69. In red cells .Whereas specific AE1 mutations are undoubtedly linked to cation leaky red cells responsible for hereditary haemolytic anaemia, it is observed that all the 9 AE1 mutations presented here do not impair AE1 transport features in a similar manner. Moreover, membrane permeability of cells expressing point-mutated AE1 shows some differences suggesting a complicated regulation of this permeability. The proposition of point mutations altering AE1 transport mechanism is an attractive hypothesis supported by experimental evidence. However, this does not exclude the possibility for some mutated AE1 to also regulate the activity of other transporters. Resolution of the 3D structure of AE1 would greatly help to understand the peculiar transport properties of this surprising protein. It would be of particular interest to know how the studied AE1 point mutations alter AE1 structure, if these different point mutations have a common mechanism of action on the structure. A better understanding of the mechanism of interactions between AE1 and its partners would also help to assign new regulatory function to AE1.


Rumohra adiantiformis). The main symptom of FDS is distortion of fronds, making them unmarketable. Additional symptoms include stunting, irregular sporulation, decreased rhizome diameter, and internal discoloration of rhizomes. We previously reported an association of symptoms with increased endophytic rhizome populations of fluorescent pseudomonads (FPs). The aim of the current study was to determine if FPs from ferns in Costa Rica with typical FDS symptoms would recreate symptoms of FDS.Fern Distortion Syndrome (FDS) is a serious disease of Leatherleaf fern , and four different groups of FPs, each at a two concentrations. The four groups of FPs included one group from healthy ferns without symptoms (another control treatment), two groups isolated from inside rhizomes of symptomatic ferns, and one group isolated from inside roots of symptomatic ferns. Symptoms were assessed 12 and 17 months later, and populations of FPs inside newly formed rhizomes were determined after 17 months. Results showed that inoculation with mixtures of FPs from ferns with FDS symptoms, but not from healthy ferns, recreated the primary symptom of frond deformities and also the secondary symptoms of irregular sporulation, decreased rhizome diameter, and internal discoloration of rhizomes.These results suggest a model of causation of FDS in which symptoms result from latent infections by multiple species of opportunistic endophytic bacteria containing virulence genes that are expressed when populations inside the plant reach a minimum level. Rumohra adiantiformis) is a valuable ornamental plant used in cut flower arrangements because of its long shelf life and symmetrical pyramidal shaped fronds. Areas with substantial production of Leatherleaf fern include Florida, Costa Rica, and other countries in Central America. Since the 1980s, distortions of ferns and other symptoms of damage were reported by growers. The disease was termed fern distortion syndrome (FDS) in 2010 Leatherleaf fern . Rhizosphere bacteria (treatments 5A and 5B in The fluorescent pseudomonads used to inoculate rhizomes  were iso5′- AGAGTTTGATCCTGGCTCAG -3′) and 1492R (5′- ACGGCTACCTTGTTACGACTT- 3′). PCR was performed using Lucigen EconoTaq Plus Green 2× master mix (Lucigen Corp.) with the following cycling parameters: initial denaturation at 95°C for 5 min; 31 cycles of 94°C for 1 min, 57°C for 45 sec, 70°C for 2 min; and a final extension at 70°C for 10 min.Taxonomic classification of each strain of the fluorescent pseudomonads used to inoculate rhizomes was based on the partial sequence of 16S rRNA gene. DNA was extracted and amplified with universal bacterial primers: 8F , which has been used to determine the phytopathogenic potential of plant-associated bacteria The potato slice maceration test was used as a measure of pectinolytic enzyme activity. The procedure was modified from González et al. 3-HClO4 reagent, which was kept in the dark. Development of a pink color in 25 min indicated production of IAA. Production of IAA was quantified by inoculating 200 µl of a 24 hour nutrient broth culture into 20 ml of minimal salt medium amended with 5 mM/L of L-tryptophan and then shaking at room temperature for 48 hours. After centrifuging at 12,000 rpm for 5 min, 1.0 ml of the supernatant was added to 2 ml FeCl3-HClO4 reagent. After 25 min, absorbance at 530 nm was determined in a uv-spectrophotometer, and the concentration of IAA was determined in µg/ml using a standard curve.Bacterial production of indole-3 acetic acid (IAA) was determined qualitatively and quantitatively based upon previously described methods An experiment was designed to determine if fluorescent pseudomonads isolated from plants with typical symptoms of FDS in Costa Rica recreate the primary distortion symptoms upon inoculation into asymptomatic ferns. The experiment was a randomized complete block design with 9 treatments and 8 replications per treatment, for a total experimental number of 72 plants. Treatments, shown in Rhizomes of uniform size from ferns derived from tissue culture and grown 12 months in the greenhouse at Auburn University, as described above, were selected, with 8 rhizomes per treatment. Rhizomes were dipped into treatments for 5 minutes immediately prior to planting in the greenhouse. Pots were randomized in the greenhouse and were grown as described above.The effect of bacterial treatments on the development of symptoms of FDS was assessed at 12 months after inoculation in a non-destructive sampling of the fern plants. The mean FDS severity was determined for the youngest fully developed frond on each plant by using the four point scale developed previously At 17 months after inoculation, the effects of bacterial treatments on plant growth and the population densities of fluorescent pseudomonads inside rhizomes (described below) were determined by destructively sampling the inoculated plants. Measurement of plant growth included total plant fresh weight, number of rhizomes per plant, the caliper of three rhizomes per plant (as a way to assess rhizome size), and the dry weights of roots and fronds.P = 0.01.For each measurement conducted at 12 and 17 months after planting, data were analyzed for significant treatment effects using two-way analysis of variance with SAS. When a significant F value was determined, LSD values were calculated at An isolation of bacteria was conducted at 17 months after inoculation to quantify and characterize fluorescent pseudomonads inside rhizomes of plants that developed symptoms of FDS . From these treatments, three rhizomes from each replicate plant of each treatment were selected for sampling. After surface disinfestation as described above, a combined sample of 0.5–1.5 g of rhizome tissue was made for each replication. Samples were processed as described above, and serial 10-fold dilutions were prepared and plated on 50% KB. After incubation for 48 hours at 28°C, fluorescent colonies were counted under an ultraviolet light, and the mean log cfu/g of rhizome was calculated for each treatment. Data were analyzed for significant treatment effects using two-way analysis of variance as described above.A collection was made of 350 strains of fluorescent pseudomonads from symptomatic ferns. Combined collections of 100 strains from treatments 3A and 3B, 100 from treatment 4A and 4B, and 100 from treatments 5A and 5B were made from the dilution plates used to quantify the fluorescent pseudomonads inside rhizomes. In addition, a collection of 50 strains (labeled in results as 5INT) was made from inside rhizomes from treatments 5A and 5B that showed internal discoloration, which was previously reported inside rhizomes of ferns expressing symptoms of FDS in Costa Rica  , while 4 strains . In contP = 0.01) increases compared to the controls  in FDS severity, number of largest five fronds with a twisted rachis, and number of largest five fronds with pinnae twisted out of the plane of the frond. All treatments inoculated with bacteria from diseased ferns resulted in a significantly lower number of the largest five fronds with a pyramidal shape, compared to the controls. The corresponding photographs of deformed frond growth resulting from inoculation with pseudomonads from plants with FDS symptoms  reduced by inoculation with all six treatments of pseudomonads from diseased plants compared to inoculation with the two treatments of pseudomonads from healthy plants and the water control  . In addiIn addition to analyzing the 350 reisolated strains phylogenetically, we also compared their physiological profiles in comparison to the inoculated strains by evaluating production of HR, pectinolytic enzymes, and IAA. The results indicated that HR was elicited in 24–48 hours by 77% of the inoculated strains  increased one parameter of plant growth, frond width, compared to the water control Click here for additional data file.


S)-1) is reported. The route starts from α-(trifluoromethyl)acrylic acid (2). Conjugate addition of α-(p-methoxyphenyl)ethylamine ((S)-3b), generated an addition adduct 4b which was cyclised to β-lactam 5b. Separation of the diastereoisomers by chromatography gave -5b). N-Debenzylation afforded the desired α-(trifluoromethyl)-β-lactam ((S)-1). The absolute stereochemistry of diastereoisomers 5 was determined by X-ray crystallographic determination of a close structural analogue, -5c, and then 1H and 19F NMR correlation to the individual diastereoisomers of 5a and 5b.The first synthesis of α-(trifluoromethyl)-β-lactam (( The comparison of a racemic sample of 1 and then a sample after diastereoisomer separation of 5b as described above, indicated that β-lactam 1 was prepared in an enantiomerically pure form. There was no evidence that benzylic cleavage of the (S)-α-(p-methoxyphenyl)ethyl moiety with CAN resulted in epimerisation at the stereogenic centre of the β-lactam.Accordingly, -3c rendered the resultant β-lactam diastereoisomers 5c more crystalline, and a single crystal X-ray structure was solved for the major-diastereomer revealing the -5c configuration. The structure is shown in Finally, it was necessary to determine the absolute configuration of the resultant β-lactam. To this end, X-ray crystallography of the major diastereoisomers of 5b. The 1H and 19F NMR spectra of the major and minor diastereoisomers of 5a–c were now compared. Clear chemical shift trends are observed as illustrated and tabulated in 1H NMR chemical shifts of the C-3 hydrogens (Ha) of the β-lactam are shifted downfield in the major relative to the minor diastereoisomers. The non-equivalent faces of the planar β-lactam ring differentiates the two diastereotopic hydrogens at C-4 (Hb and Hc).With this structural information in hand it was necessary to correlate to the stereoisomers of 1H NMR signals for Hb and Hc have a larger chemical shift difference than that between the Hb and Hc signals of the minor isomers. Also the chemical shifts for Hb and Hc of the minor isomers lie within those of the major isomers. For the 19F NMR spectra shown in It is clear that for all three major isomers the R,3R)-5c, where the absolute stereochemistry was determined by X-ray crystallography, allows the absolute stereochemistry of diastereoisomers 5a and 5b to be deduced by correlation, and it follows that benzylic cleavage of (S)-α-(p-methoxyphenyl)ethylamine (3b) gave rise to β-lactam (S)-1. Ready access to the (R)-1 enantiomer would require starting the synthetic route from (S)-α-(p-methoxyphenyl)ethylamine (3b).On this basis, the NMR spectra of (αS)-1 is reported which took advantage of an aza-Michael addition between (S)-α-(p-methoxyphenyl)ethylamine (3b) and α-(trifluoromethyl)acrylic acid (2). Cyclisation and then chromatographic resolution of the β-lactam diastereoisomers 5b, followed by deprotection with ceric ammonium nitrate generated the β-lactam (S)-1. This contributes diversity to the known β-lactam motifs and incorporates the CF3 group.In conclusion, a short single enantiomer synthesis of β-lactam (2Cl2 was obtained from the Solvent Purification System MB SPS-800. Room temperature (RT) refers to 20–25 °C. Reaction temperatures of 0 °C were obtained in an ice/water bath. Reaction reflux conditions were obtained using an oil bath equipped with a contact thermometer. Solvent evaporations were carried out under reduced pressure on a Büchi rotary evaporator. Thin layer chromatography (TLC) was performed using Macherey-Nagel Polygram Sil G/UV254 plastic plates. Visualisation was achieved by inspection under UV light (255 nm). Column chromatography was performed using silica gel 60 (40–63 micron). NMR spectra were recorded on Bruker AVANCE 300, 400 or 500 MHz instruments. 1H and 13C NMR spectra were recorded in CDCl3 as solvent. 19F NMR spectra were referenced to CFCl3 as the external standard. Chemical shifts are reported in parts per million (ppm) and coupling constants (J) are given in Hertz (Hz). IR spectra were recorded on a Nicolet Avatar 360 FT-IR from a thin film (either neat or combined with nujol) supported between NaCl plates. Optical rotations [α]D are given in 10–1 deg cm2 g–1 and were measured using a Perkin Elmer Model 341 polarimeter. Mass spectrometric (m/z) data was acquired by electrospray ionisation (ESI). High resolution mass analyses were recorded on a Micromass LCT TOF mass spectrometer using ES ionisation in positive ion mode.All reagents were obtained from commercial sources and were used without further purification unless otherwise stated. Air- and moisture-sensitive reactions were carried out under a positive pressure of argon in flame-dried glassware using standard Schlenk-line techniques. Dry CH3  was added to a solution of α-(trifluoromethyl)acrylic acid  in methanol (10 mL), and then after 30 min stirring, a single enantiomer amine 3 (10.0 mmol) was added. The reaction was stirred at RT for 24 h. The solvent was evaporated under reduced pressure to afford the aza-Michael product as a colourless oil (quant) which solidified upon standing. Optionally, the sodium salt was converted to the amino acid HCl salt by treatment with aq HCl, followed by evaporation of the water and redissolving the residual in DCM.NaHCO2 , NaHCO3  and (S)-phenylethylamine  in methanol (20 mL), gave after 96 h 830 mg (95%) of the title compound as a mixture of diastereoisomers (93% purity). Product 4a was used without further purification. 1H NMR  7.42–7.25 , 4.02–3.90 , 3.30–3.13 , 3.10–2.91 , 2.90–3.76 , 1.44 ; 13C NMR  172.3 , 172.2 , 143.9 (C-Ar), 143.5 (C-Ar), 129.9 (C-Ar), 129.8 (C-Ar), 128.9 (C-Ar), 128.8 (C-Ar), 128.1 (C-Ar), 128.0 (C-Ar), 126.8 , 126.9 , 59.6 (PhCHCH3), 59.0 (PhCHCH3), 52.6 , 52.6 , 52.1 , 45.12 , 44.7 , 23.1 (CHCH3), 22.9 (CHCH3); 19F NMR  −69.0 , −69.1 ; HRMS–ESI (m/z): Calcd for C12H14NO2F3Na, 284.0874; found, 284.0869.General procedure with α-(trifluoromethyl)acrylic acid 2 , NaHCO3  and (S)-p-methoxyphenylethylamine  in methanol (20 mL), gave after 20 h 3.06 g (98%) of the title compound as a mixture of diastereoisomers (93% purity). The material was used without further purification. 1H NMR  7.17–7.12 , 6.85–6.80 , 4.86 , 3.74 , 3.73–3.65 , 3.27 , 3.01 , 1.52 ; 13C NMR  167.4 (NCOCH), 167.3 (NCOCH), 162.2 (C-Ar), 131.6 (C-Ar), 130.4 (C-Ar), 128.5 (C-Ar), 128.4 (C-Ar), 125.1 , 125.5 and 125.1 , 115.83 (C-Ar), 115.51 (C-Ar), 60.61 (PhCHCH3), 60.55 (PhCHCH3), 55.9 (PhOCH3), 48.2 , 48.1 , 42.6 (NCH2CH), 19.9 (CHCH3), 19.5 (CHCH3); 19F NMR  −68.8 , −68.9 ; HRMS–ESI (m/z): Calcd for C13H16NO3F3Na, 314.0980; found, 314.0975.General procedure with α-(trifluoromethyl)acrylic acid 2 , NaHCO3  and (R)-1-naphthylethylamine  in methanol (10 mL), gave after 96 h 550 mg (98%) of the title compound as a mixture of diastereoisomers (89% purity). The material was used without further purification. 1H NMR  8.20 , 7.91–7.86 , 7.83–7.79 , 7.72–7.68 , 7.59–7.46 , 4.99–4.88 , 3.35–3.22 , 3.21–3.09 , 3.03–2.94 , 1.57 and 1.56 ; 13C NMR  172.2 (COONa), 172.0 (COONa), 139.5 (C-Ar), 138.9 (C-Ar), 135.5 (C-Ar), 132.48 (C-Ar), 132.45 (C-Ar), 130.08 (C-Ar), 135.05 (C-Ar), 129.2 (C-Ar), 129.15 (C-Ar), 127.5 (C-Ar), 127.4 (C-Ar), 126.8 (C-Ar), 126.75 (C-Ar), 126.7 (C-Ar), 126.6 (C-Ar), 124.2 (C-Ar), 123.7 (C-Ar), 123.5 (C-Ar), 54.5 (PhCHCH3), 54.1 (PhCHCH3), 52.5 , 52.2 , 45.2 , 44.9 , 22.51 (CHCH3), 22.46 (CHCH3); 19F NMR  −69.0 , −69.1 ; HRMS–ESI (m/z): Calcd for C16H16NO2F3Na, 334.1031; found, 334.1031.General procedure with α-(trifluoromethyl)acrylic acid 4 (8.00 mmol) was dissolved in dry DCM (100 mL) and DMF (5 drops) was added. The mixture was cooled to 0 °C and then SOCl2  added dropwise. The reaction mixture was warmed to RT and stirred overnight. Solvent and excess of SOCl2 were removed under reduced pressure and the oily residue was redissolved in dry DCM (10 mL). The solution was heated to reflux while Et3N  was added. The reaction mixture was diluted with water (10 mL) and extracted into DCM (3 × 10 mL). The combined organic phases were washed with brine (10 mL), dried (Na2SO4) and solvent was removed under reduced pressure to give 4 as a mixture of two isomers, which were then separated by chromatography.The sodium salt of 4a  and SOCl2  in DCM (20 mL), followed by Et3N , gave 5 as a yellow oily solid.Sodium salt Major isomer -5a : [α]D20 −88.5 ; IR (neat): 2978 (s), 1753 (vs), 1180 (s), 1120 (s), 892 (s), 699 (s), 571 (s) cm−1; 1H NMR  7.42–7.27 , 4.97 , 3.85–3.75 , 3.39 , 3.16–3.11 , 1.64 ; 13C NMR  159.4 , 139.3 (C-Ar), 128.9 (C-Ar), 128.0 (C-Ar), 126.6 (C-Ar), 123.9 , 52.0 (PhCHCH3), 51.1 , 37.6 , 18.2 (CHCH3); 19F NMR  −68.72 ; HRMS–ESI (m/z): Calcd for C12H12NOF3Na, 266.0769; found, 266.0769.Minor isomer -5a : [α]D20 −36.7 ; IR (neat): 2976 (s), 1756 (vs), 1184 (s), 1119 (s), 700 (s) cm−1; 1H NMR  7.42–7.27 , 4.94 , 3.80–3.71 , 3.30 , 3.22 , 1.67 ; 13C NMR  159.3 , 139.3 (C-Ar), 129.0 (C-Ar), 128.1 (C-Ar), 126.7 (C-Ar), 123.8 , 52.5 (PhCHCH3), 51.3 , 37.7 , 18.4 (CHCH3); 19F NMR  −68.85 ; HRMS–ESI (m/z): Calcd for C12H12NOF3Na, 266.0769; found, 266.0766.4b  and SOCl2  in DCM (100 mL) and DMF (5 drops), followed by Et3N , gave 5b as a colourless oil.Sodium salt Major isomer -5b : [α]D20 +96.5 ; IR (neat): 2980 (s), 1756 (vs), 1612 (s), 1515 (s), 1372 (s), 1180 (s), 1120 (s), 1031 (s), 1010 (s), 835 (s) cm−1; 1H NMR  7.30–7.25 , 6.95–6.90 , 4.94 , 3.85 , 3.84–3.73 , 3.36 , 3.11 , 1.61 ; 13C NMR  159.7 (NCOCH), 131.6 (C-Ar), 128.3 (C-Ar), 124.4  114.6 (C-Ar), 55.8 (PhCHCH3), 51.7 (PhOCH3), 51.4 , 37.9 , 18.6 (CHCH3); 19F NMR  −69.2 ; HRMS–ESI (m/z): Calcd for C13H14NO2F3Na, 296.0874; found, 296.0868.Minor isomer -5b : [α]D20 −42.1 ; IR (neat): 2975 (s), 1760 (vs), 1611 (s), 1515 (s), 1370 (s), 1244 (vs), 1121 (s), 1031 (s), 1010 (s), 833 (s) cm−1; 1H NMR  7.25–7.22 , 6.94–6.90 , 4.91 , 3.83 , 3.78–3.68 , 3.27 , 3.19 , 1.64 ; 13C NMR  159.3 (NCOCH), 131.3 (C-Ar), 127.9 (C-Ar), 123.9 , 114.2 (C-Ar), 55.3 (PhCHCH3), 51.9 (PhOCH3), 51.0 , 37.5 , 18.5 (CHCH3); 19F NMR  −69.35 ; HRMS–ESI (m/z): Calcd for C13H14NO2F3Na, 296.0874; found, 296.0883.4c , SOCl2  in DCM (20 mL), followed by Et3N  gave 5c as a yellow solid.From sodium salt Major isomer -5c : [α]D20 −40.9 ; IR (neat): 2981 (s), 1760 (vs), 1368 (s), 1260 (s), 1158 (s), 1122 (s), 1011 (s), 780 (s) cm−1; 1H NMR  8.10 , 7.92 , 7.88 , 7.64–7.58 , 7.58–7.46 , 5.78 , 3.82–3.73 , 3.31 , 2.77 , 1.80 ; 13C NMR  159.3 , 133.9 (C-Ar), 133.7 (C-Ar), 130.9 (C-Ar), 129.2 (C-Ar), 128.9 (C-Ar), 127.0 (C-Ar), 126.2 (C-Ar), 123.7 , 123.7 (C-Ar), 122.7 (C-Ar), 51.0 , 47.9 (PhCHCH3), 37.4 , 17.8 (CHCH3); 19F NMR  −68.6 ; HRMS–ESI (m/z): Calcd for C16H14NOF3Na, 316.0925; found, 316.0915.Minor isomer -5c : [α]D20 −35.7 ; IR (neat): 2978 (s), 1755 (vs), 1366 (s), 1191 (s), 1120 (s), 778 (s) cm−1; 1H NMR  8.15 , 7.92 , 7.87 , 7.63–7.58 , 7.58–7.48 , 5.79 , 3.69–3.60 , 3.25 , 2.86 , 1.82 ; 13C NMR  159.2 , 133.9 (C-Ar), 133.8 (C-Ar), 130.9 (C-Ar), 129.2 (C-Ar), 129.0 (C-Ar), 127.1 (C-Ar), 126.2 (C-Ar), 125.1 (C-Ar), 123.9 , 123.7 (C-Ar), 122.6 (C-Ar), 47.7 (PhCHCH3), 50.9 , 37.2 , 17.7 (CHCH3); 19F NMR  −68.8 ; HRMS–ESI (m/z): Calcd for C16H14NOF3Na, 316.0925; found, 316.0919.S,3S)-5b  in a mixture of MeCN (8 mL) and water (2 mL), ceric ammonium nitrate  was added portionwise. The reaction mixture was stirred at RT for 16 h and then quenched with sat. sodium bicarbonate (5 mL). After 10 min of stirring (when gas evolution ceased), the mixture was extracted into Et2O (3 × 10 mL), the combined organic phases were dried (Na2SO4) and the solvent was removed. Purification on silica gel (20% EtOAc in petrol) gave (S)-1 as a colourless oil which solidified upon standing . Mp 85 °C; [α]D20 +26.9 ; IR (neat): 1707 (s), 1652 (s), 1180 (s), 1120 (s) cm−1; MS–ESI (m/z): 161.98 (M + Na); 1H NMR  6.73 , 3.91–3.81 , 3.47 , 3.37 ; 13C NMR  161.3 , 123.7 , 53.7 , 36.7 ; 19F NMR  −69.38 ; HRMS–ESI (m/z): Calcd for C4H4NOF3Na, 162.0143; found, 162.0147.To a solution of , a = 9.226(11), b = 5.507(6), c = 11.229(12) Å, β = 99.401(13)°. V = 562.9(11) Å3, F(000) = 280, Z = 4, Dc = 1.641 Mg m−3, μ = 0.181 mm−1 . The data were collected at T = 93 (2) K, 5405 reflections (2.24 to 25.39°) were measured on a Rigaku Saturn 92 detector with 007 generator yielding 2016 unique data (Rint = 0.0777). Conventional R = 0.0194 for 1860 reflections with I ≥ 2σ, GOF = 1,011; 171 refined parameters. The largest peak in the residual map is 0.247 eÅ−3. Crystallographic data has been deposited with the Cambridge Crystallographic Data Centre as supplementary publication (CCDC 821176).R,3R)-5c C16H14F3NO, M = 293.28, Monoclinic, space group P2(1), a = 9.677(5), b = 6.094(3), c = 12.231(5) Å, β = 92.685(13)°. V = 720.5(5) Å3, F(000) = 304, Z = 2, Dc = 1.352 Mg m−3, μ = 0.949 mm−1 . The data were collected at T = 173 (2) K, 9149 independent reflections (3.62 to 25.35°) were measured on a Rigaku Saturn 92 detector with 007 generator yielding 2504 unique data (Rint = 0.0450). Conventional R = 0.0194 for 2416 reflections with I ≥ 2σ, GOF = 0.973; 191 refined parameters. The largest peak in the residual map is 0.124 eÅ−3. Flack parameter 0.08 (13). Crystallographic data has been deposited with the Cambridge Crystallographic Data Centre as supplementary publication (CCDC 821177).(α


The primary aim of the current management of diabetes mellitus is to achieve and/or maintain a glycated hemoglobin level of ≤6.5%. However, recent evidence indicates that intensive treatment of hyperglycemia is characterized by increased weight gain, severe hypoglycemia and higher mortality. Besides, evidence suggests that it is difficult to achieve and/or maintain optimal glycemic control in many diabetic patients; and that the benefits of intensively-treated hyperglycemia are restricted to microvascular complications only. In view of these adverse effects and limitations of intensive treatment of hyperglycemia in preventing diabetic complications, which is linked to oxidative stress, this commentary proposes a hypothesis that “simultaneous targeting of hyperglycemia and oxidative stress” could be more effective than “intensive treatment of hyperglycemia” in the management of diabetes mellitus. Diabetes mellitus is a metabolic disorder characterized by chronic hyperglycemia with impaired metabolism of carbohydrate, fat and protein resulting from defects in insulin secretion or insulin action or both . The priWhile increased weight gain could be due to some component drugs (such as sulphonylureas or insulin) of the intensive therapy regimens, hypoglycemia could be drug-induced or comorbidity-induced –6,11. CoCurrently, one of such complementary options is the potential of “concurrently targeting hyperglycemia and oxidative stress”. Oxidative stress is defined as an “imbalance between oxidants and antioxidants in favor of the oxidants, potentially leading to damage” . It is iEven though oxidative stress plays an important role in its pathogenesis and complications, unlike other diseases characterized by oxidative stress, diabetes mellitus is unique. Its cure  does not prevent oxidative stress and diabetic complications –18. ThisConsidering that intensive therapy of hyperglycemia is associated with increased weight gain, severe hypoglycemia and higher mortality than standard therapy of hyperglycemia –6,11, thA study that targeted hyperglycemia and oxidative stress with insulin and antioxidants (vitamin E and C), respectively in diabetic rats found that the combination of these two classes of agents normalized hyperglycemia, weight loss and oxidative stress parameters more considerably than either agent . SimilarIn humans with type 1 or type 2 diabetes mellitus, it was reported that maintenance of optimal glycemic control alone was less effective in ameliorating oxidative stress compared to when optimal glycemic control was combined with antioxidant (vitamin E) therapy ,27. A stBy and large, these studies indicate that co-administration of oral hypoglycemic drugs (and/or insulin) and antioxidants (honey or vitamins C and E) does not reduce or interfere with the hypoglycemic or antioxidant effect of either agent. Therefore, a combination of these two classes of agents is justifiable in the management of diabetes mellitus. With the combination, hypoglycemic drugs will target hyperglycemia to improve glycemic control. In view of evidence which implicates hyperglycemia as the sole contributor of ROS and oxidative stress in diabetes mellitus, targeting hyperglycemia may also be beneficial in reducing oxidative stress . On the Besides, targeting hyperglycemia and oxidative stress may also offer additional benefits. These include amelioration of oxidative stress and damage in the pancreas ,23,32. IIn a nutshell, in view of the compelling evidence that demonstrates an association between an intensive therapy of hyperglycemia, it would be worthwhile to reconsider any therapeutic goal that target “hyperglycemia or glycemic control alone” in diabetic patients. Instead, targeting hyperglycemia and oxidative stress simultaneously might be a better option. While this commentary does not underestimate the importance of optimal glycemic control, however, the data, views and explanations presented or posited, do strengthen the hypothesis that the prospect of managing diabetes mellitus more effectively by targeting hyperglycemia and oxidative stress concurrently holds much promise. This hypothesis is worth investigating in diabetic patients. Therefore, both small and large randomized clinical trials that investigate the effects of targeting hyperglycemia and oxidative stress simultaneously in patients with type 1 or type 2 diabetes mellitus are highly desirable. Also, clinical trials that compare the effects of a standard or conventional therapy of hyperglycemia and antioxidant therapy with those of intensive therapy of hyperglycemia alone are also recommended. These clinical trials would help to substantiate or perhaps refute this hypothesis in diabetic human subjects.