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Identification of tandem duplicate regulatory small RNAs in Pseudomonas aeruginosa involved in iron homeostasis
Contributed by Susan Gottesman, May 13, 2004
In many bacteria, iron homeostasis is controlled primarily by the ferric uptake regulator (Fur), a transcriptional repressor. However, some genes, including those involved in iron storage, are positively regulated by Fur. A Fur-repressed regulatory small RNA (sRNA), RyhB, has been identified in Escherichia coli, and it has been demonstrated that negative regulation of genes by this sRNA is responsible for the positive regulation of some genes by Fur. No RyhB sequence homologs were found in Pseudomonas aeruginosa, despite the identification of genes positively regulated by its Fur homolog. A bioinformatics approach identified two tandem sRNAs in P. aeruginosa that were candidates for functional homologs of RyhB. These sRNAs (PrrF1 and PrrF2) are >95% identical to each other, and a functional Fur box precedes each. Their expression is induced under iron limitation. Deletion of both sRNAs is required to affect the iron-dependent regulation of an array of genes, including those involved in resistance to oxidative stress, iron storage, and intermediary metabolism. As in E. coli, induction of the PrrF sRNAs leads to the rapid loss of mRNAs for sodB (superoxide dismutase), sdh (succinate dehydrogenase), and a gene encoding a bacterioferritin. Thus, the PrrF sRNAs are the functional homologs of RyhB sRNA. At least one gene, bfrB, is positively regulated by Fur and Fe2+, even in the absence of the PrrF sRNAs. This work suggests that the role of sRNAs in bacterial iron homeostasis may be broad, and approaches similar to those described here may identify these sRNAs in other organisms.
While virtually all organisms require iron for survival, they also must manage the iron-catalyzed production of reactive oxygen intermediates that could lead to severe cellular damage (1). Consequently, they have evolved tightly regulated systems for both uptake and sequestration of this essential element. The ferric uptake regulator (Fur), a transcriptional repressor, is fundamental for maintaining iron homeostasis in many prokaryotes. Fur and its corepressor, Fe2+, inhibit the transcription of an array of genes that are crucial to iron-acquisition systems (e.g., siderophore synthesis and their uptake) by means of binding to a specific target sequence (Fur box) in their promoters.
Fur also acts as a positive regulator and affects the production of factors [e.g., superoxide dismutase (SodB) and bacterioferritins] that can mitigate iron toxicity under iron-replete conditions, as well as nonessential proteins that contain iron (e.g., aconitase, fumarase, and succinate dehydrogenase) (2, 3). The mechanism for this type of regulation is not as well understood as Fur-mediated repression. Massé and Gottesman (4) identified a Fur-regulated small RNA (sRNA), RyhB, which provides an explanation for the positive regulatory effects of Fur on gene expression in Escherichia coli and other enterobacteriae. RyhB RNA negatively regulates sodB, some tricarboxylic acid cycle genes, and genes encoding bacterioferritins by pairing with their mRNA and causing rapid degradation of the mRNA (5-7). Transcription of ryhB is in turn repressed by Fur. As a result, Fur-mediated inhibition of RyhB synthesis allows for the expression of certain genes (i.e., sodB) under iron-replete conditions but not under iron-limiting conditions. The nucleotide sequences of ryhB, its promoter (i.e., -35 and -10), and its operator (i.e., Fur box) are well conserved in E. coli, Salmonella, Klebsiella, Shigella, Photorhabdus luminescens (insect pathogen), and, to a lesser degree, Yersinia pestis.
In Pseudomonas aeruginosa, Fur is an essential regulator that has many of the properties of its homologs in the above organisms (8). That is, Fur negatively regulates a large assortment of genes involved in iron acquisition, as well as those that contribute to virulence (9, 10). Ochsner et al. (11) recently reported that P. aeruginosa Fur also may act as a positive regulatory factor. Yet, no sequence homologous to ryhB could be identified in the annotated genome of P. aeruginosa PAO1.
In this report, we investigate whether sRNAs cause the positive regulation by Fur of certain genes in P. aeruginosa. We describe a bioinformatics approach that led to the identification of two functional homologs of RyhB [PrrF1 and PrrF2, for Pseudomonas regulatory RNA involving iron (Fe)] in this opportunistic pathogen. Notably, their nucleotide sequences are not similar to ryhB. The data suggest that PrrF1 and PrrF2 provide overlapping roles in the negative regulation of genes involved in diverse functions including iron storage, defense against oxidative stress, and intermediary metabolism.
Materials and Methods
Bacterial Strains, Media, and Growth Conditions. P. aeruginosa PAO1 (www.pseudomonas.com, updated January 14, 2004) was the WT strain used in this study (12, 13). The PAO1 C6 mutant encodes Fur with an A10G mutation (14). Deletions were as follows: PAO1 ΔprrF1, base pairs 5283933-5284120 of PAO1 sequence; PAO1 ΔprrF2, base pairs 5284121-5284353; and PAO1 ΔprrF1-F2, base pairs 5283933-5284353. Two double mutants were made: ΔprrF1-F2, which does not have a gentamicin cassette in place of the deletion, and ΔprrF1-F2 *, which does. Complementation of the ΔprrF1-F2 mutant was by transformation with pVLT31 containing prrF1-F2 sequence (base pairs 5283788-5284517). The complemented ΔprrF1-F2 strain is termed ΔF1-F2::F1-F2. Chelexed and dialyzed tryptic soy broth containing 1% glycerol and 50 mM glutamate was used as the low-iron medium and supplemented with 50 μg/ml FeCl3 to serve as the high-iron medium (15).
For experiments to examine the kinetics of sRNA action, cultures were diluted in fresh LB to OD600 = 0.02 and grown at 37°C to OD600 = 0.80. The iron chelator 2,2′-dipyridyl was added to a final concentration of 300 μM. Samples were stabilized and collected by using RNAprotect Bacteria Reagent and the RNeasy Mini Kit (Qiagen, Valencia, CA).
Deletion/insertion mutations in PAO1 were constructed as described in refs. 16 and 17. Genetic manipulations were verified by using PCR. Antibiotics were used at the following concentrations: for E. coli, 100 μg/ml ampicillin, 15 μg/ml gentamicin, 100 μg/ml kanamycin, and 15 μg/ml tetracycline; for P. aeruginosa, 750 μg/ml carbenicillin, 75 μg/ml gentamicin, and 150 μg/ml tetracycline.
Identification of the sRNAs. The P. aeruginosa PAO1 genome (GenBank accession no. NC_002516) was partitioned into two data sets, one that included all known (annotated) ORFs and a second that comprised the intergenic (IG) regions defined by the annotated ORFs. The IG data set was queried, by using the program patscan (18), for the presence of sequences that included a consensus Fur box, a spacer of 0-200 bases, and a potential stem-loop structure immediately followed by a series of at least three T nucleotides (U nucleotides in RNA). The Fur-box query required an identity match of at least 14 bases of the 19-base consensus sequence, GATAATGATAATCATTATC. The stem-loop parameters were set to require a stem of 7-12 complementary bases with a loop of 5-9 bases.
Translational Fusions to the lacZ Reporter Gene and β-Galactosidase Assays. PCR products containing regions upstream and within the coding region of pvdS, bfrB, and PA4880 were cloned into pCR2.1 (Invitrogen) and sequenced. Translational fusions to LacZ were made by cloning the DNA products into pPZ20 or pPZ30 (19). P. aeruginosa PAO1 and deletion strains were transformed with the resulting plasmids and grown at 32°C for 12 h in low-iron medium (see above). β-Galactosidase activities in soluble cell extracts were determined by using ONPG (Sigma) as the substrate and expressed as units per milligram, as described in ref. 20.
Gel Mobility-Shift Assays. The DNA fragments were end-labeled with [32P]dATP and gel-purified. Various amounts (0-200 nM) of purified Fur were added to 0.1 ng of DNA in binding buffer [10 mM Bis-Tris, pH 7.5/40 mM KCl/0.1 mM MnSO4/1 mM MgSO4/100 μg/ml BSA/50 μg/ml poly(dI-dC)/10% glycerol] and incubated at room temperature. After 30 min, the reaction was resolved in acrylamide, and the results were viewed by using a phosphorimager.
RNA Isolation, RNase Protection, Northern Blot, and GeneChip Analysis. Total RNA was isolated by using the hot phenol method, followed by DNase I treatment (14), and RNA integrity was confirmed by RNase protection analysis with a riboprobe specific for the constitutively expressed omlA gene (21). Quantification of the image generated by a Bio-Rad phosphorimager was performed with quantity one software (Version 4.5, Bio-Rad), and expression of PA4880 was normalized to omlA expression. Probes for Northern analysis in Fig. 3 were as follows: PrrF1, GAGTCCGACTGCGTGGGTCTCTCAGCTTACCGGCTG; PrrF2, GAGTCCGACTGCTTGGTCTCTCAGCTTACCTGCTGGCCT.
For the Northern blots, 8 μg of RNA was resolved in acrylamide or agarose. The RNA was transferred to membrane, cross-linked, and prehybridized for 1 h at 42°C in ULTRAhyb hybridization solution (Ambion, Austin, TX). Probes were added to a final concentration of 200 ng/ml and hybridized overnight at 42°C. Blots were washed, and the nonisotopic probes were visualized by using the BrightStar Biodetect Kit (Ambion) according to the manufacturer's protocol. Probes were as follows: PrrFs, AAACCGTGATTAGCCTGATGAGGAGATAATCTGAA; SodB, TTCGGGCTCAGGCAGTTCCAGTAGAAGGTGTGGTT; and BfrB, TTCAGGTCGCACTGCAGCATTTCCTGGGTGTTCTC.
The GeneChip (Affymetrix, Santa Clara, CA) probes were prepared according to manufacturer's protocol with the modifications described in ref. 11. Analysis of microarray data was performed with microarray suite (Version 5.0, Affymetrix).
Identification of Potential Fur-Regulated sRNAs in P. aeruginosa. Massé and Gottesman (4) demonstrated that the expression of a Fur-regulated sRNA (RyhB) is responsible for the regulation of various genes in E. coli that are expressed under iron-replete conditions. Sequences homologous to these sRNAs were also identified in other Enterobacteriaceae (e.g., Y. pestis). However, this homology did not extend to the genus Pseudomonas. Because the vast majority of sRNAs that have been described in E. coli are encoded in IG regions, one approach to searching for functional homologs to RyhB in Pseudomonas would have been to assay for expression of a transcript within the IG regions under iron-limiting conditions by using microarrays. However, this procedure requires the presence of microarray probes for all of the IG regions, which are not available for P. aeruginosa; only 199 IG regions are present on the available microarray. Instead, we used a bioinformatics approach to look for functional homologs by searching all of the IG regions of P. aeruginosa PAO1 for two predicted properties of such a homolog: regulation by Fur and a ρ-independent terminator, found in many E. coli sRNAs (22, 23). The IG regions of the P. aeruginosa genome were queried for sequences that included a consensus Fur box (with an identity match of at least 14 bases of the 19-base consensus sequence, GATAATGATAATCATTATC), a spacer of 0-200 bases, and a potential stem loop structure (7-12 complementary bases with a loop of 5-9 bases), immediately followed by a series of at least three T nucleotides (U nucleotides in RNA). This analysis yielded only three candidates from the IG data set. One potential sRNA was located between PA1321 and PA1322, and two candidates were located in tandem between PA4704 and phuW (PA4705) (Fig. 1A ). By Northern blotting, no transcript was detected from the PA1321/2 IG region (data not shown). However, iron-regulated transcripts from the IG region between PA4704 and phuW had been previously detected (24). In that study, these transcripts' functions were not identified, and they had no detectable effect on the expression of the flanking genes, PA4704 and phuW. Herein, the transcript proximal to PA4704 is termed PrrF1 and the one proximal to phuW, PrrF2. Remarkably, PrrF1 and PrrF2 are >95% identical (Fig. 1B ). As required by the algorithm, we identified a consensus Fur box preceding each transcript. Gel mobility-shift assays (Fig. 1C ) indicated that Fur bound each Fur box at a biologically relevant concentration of Fur.
PrrF Homologs in Other Organisms. Various clinical and environmental isolates of P. aeruginosa (n = 49) were examined for sequences similar to prrF. Related sequences were detected in all strains examined, and identically sized PCR products were amplified by using primers within the flanking genes (data not shown). These data indicate that prrF1 and prrF2 are likely to be in the same location in all P. aeruginosa strains examined and that they all contain tandem copies of these genetic elements. Because blast searches revealed that sequences homologous to ryhB are only found in a relatively narrow range of organisms (e.g., Enterobacteriaceae), we asked whether prrF sequence homologs are present in other bacteria. A blast search of completed genomes in the National Center for Biotechnology Information database revealed that sequences closely related to prrF are only found in Pseudomonas spp. Moreover, although two putative prrF sequence homologs were found in Pseudomonas putida, Pseudomonas fluorescens, and Pseudomonas syringae, they are considerably distal to each other in these organisms in contrast with their tandem location in P. aeruginosa. However, all copies are preceded by a promoter sequence and conserved Fur box and have a core of identical sequence (shown in green in Fig. 1B for the P. aeruginosa sRNAs).
Expression of PrrF1 and PrrF2. PrrF1 and PrrF2 differ by five nucleotides, and a functional Fur-binding site precedes each (Fig. 1 B and C ). The iron-regulated expression of these transcripts was confirmed by evaluating their relative levels in iron-replete and -deficient cells. Transcripts of ≈110 nt (Fig. 2) were detected in WT cells cultured in iron-limiting conditions but not in cells cultured under iron-replete conditions. Deletions in prrF1, prrF2, or both were constructed. These mutants were used to further examine the regulation and function of these sRNAs. Detectable transcripts were still produced from growth of ΔprrF1 or ΔprrF2 in low iron, but not in high iron (Fig. 2). The PrrF2 transcript (present in ΔF1) consistently migrated faster than PrrF1 (present in ΔF2), thereby suggesting that PrrF1 is a slightly longer transcript, a conclusion that is consistent with other data (24). No PrrF-related transcripts were detected in ΔprrF1-F2 (Fig. 2).
PrrF-Regulated Genes in P. aeruginosa. Previously, by using DNA microarray technologies, an assortment of P. aeruginosa genes were identified that are responsive to either iron-limiting or -replete conditions (11). Table 1 lists a subset of the genes found to be positively affected by iron (PAO1 high Fe vs. low Fe). Genes expressed at higher levels under iron-replete conditions than under iron-limiting conditions included bfrB (see below), a gene encoding a probable bacterioferritin (PA4880), sodB, and sdh (encoding succinate dehydrogenase) (Table 1), similar to the set of genes regulated by RyhB in E. coli. In addition, some genes indicated strong regulation in the arrays. For example, expression of the probable transcriptional regulator PA2511 is increased 46-fold when cultured in iron-replete conditions, as compared with when it is cultured in iron-limiting conditions; the adjacent and divergently transcribed genes (PA2512-PA2514) are similarly regulated.
The contributions of prrF1 and prrF2 individually and collectively were determined by comparing global expression in prrF mutants and the PAO1 parental WT. Cells carrying the single and double mutants were grown under iron-limiting conditions, and the level of expression in the microarrays was compared with the level of expression for WT under the same growth conditions. Data in Table 1 indicate that the prrF1 and prrF2 sequences encode sRNAs with overlapping function because only minimal or no change was detected in the expression of the genes listed when either prrF1 or prrF2 alone was deleted (Table 1, ΔF1 vs. PAO1 and ΔF2 vs. PAO1). When both prrF1 and prrF2 sequences were deleted, expression of some, but not all, of the genes in Table 1 was substantially increased under iron-limiting conditions (Table 1, ΔF1-F2 vs. PAO1), consistent with the loss of iron regulation. In another experiment, the level of expression of the ΔprrF1-F2 mutant in iron-replete and iron-limiting media was compared in microarrays (data not shown). For nearly all of the genes listed in Table 1, the transcript levels were not as highly regulated by iron in the ΔprrF1-F2 mutant as they are in WT PAO1, as expected if the PrrF RNAs are necessary for this regulation. Introduction of a plasmid carrying prrF1-F2 sequences restored expression of all of the genes affected in the ΔprrF1-F2 mutant to the level detected in the WT strain (Table 1).
One exception to this pattern is bfrB. Either in a comparison between the ΔprrF1-F2 mutant and WT (Table 1) or in a comparison of expression in iron-replete and iron-limiting media in the ΔprrF1-F2 mutant (data not shown), bfrB regulation is not perturbed by the deletion of the PrrF sRNAs (see below).
These observations from microarray experiments were confirmed and examined further for two specific targets. SodB mRNA, which is positively regulated by Fur and iron (Table 1), rapidly disappeared after iron chelation in PAO1 (Fig. 3A ). A single mutant deleted for PrrF2 (Fig. 3A ) or PrrF1 (data not shown) still had iron-regulated expression of sodB, although it was not as stringent as when both PrrF RNAs were present. An examination of the possible pairing of sodB and PrrF indicated a reasonable region of complementarity just before the start of the coding region (Fig. 3B ). In experiments done with a probe for sdh, similar effects were observed (data not shown). These results confirm the finding from the microarrays that PrrF1 and PrrF2 both contribute to the regulation of target mRNAs.
The cultures examined by microarrays were grown under different conditions than those used for the Northern blots in Fig. 3. In the Northern blots, cells were grown in LB broth and chelator was added, then samples were taken in the hour immediately after addition of chelator. Cells growing under these conditions lose induction of the PrrF RNAs after 1 h (i.e., the target messages reaccumulate at that time), possibly as a consequence of the limited chelating ability of the 2,2′-dipyridyl. Chelation could be reversed by induction of the Fur-repressed siderophores (e.g., pyoverdine) of P. aeruginosa and removal of iron from 2,2′-dipyridyl and/or by the iron-sparing effect of expressing the PrrF sRNAs and therefore shutting down synthesis of other iron-binding proteins.
For the microarray experiments, cells were grown to the stationary phase in iron-limiting or iron-replete conditions as described in ref. 11. The regulatory effects of the PrrF sRNAs on expression of a specific gene, PA4880, were examined in more detail under the growth conditions used in the microarray experiments. PA4880 encodes a protein with homology to bacterioferritins. As shown in Fig. 4A , when both prrF1 and prrF2 are deleted (lane 4), the level of PA4880 transcript under iron-limiting conditions is ≈5-fold higher than when either one or both PrrF1-F2 transcripts are present (lanes 1-3). A constitutively expressed gene (omlA) served as a control for total RNA loading. Complementation of ΔprrF1-F2 with a plasmid carrying prrF1-F2 sequences (lane 6) resulted in transcript levels of PA4880 similar to those of WT (lane 1). When the vector alone (lane 5) was used, the relative level of PA4880 transcript remained as it was in the ΔprrF1-F2 mutant. A possible complementarity between PA4880 and PrrF is shown in Fig. 4B . The pattern of regulation we observed from RNase protection assays and from DNA microarray data for PA4880 in WT and the ΔprrF1-F2 mutants also was seen from a PA4880::LacZ translational fusion, even when only the region between the promoter and the ATG was included in the fusions (Fig. 4C ). The reason for the decreased levels of β-galactosidase observed in the ΔprrF1-F2 mutant grown in high iron (300 μM) as compared with either the WT or single mutants (Fig. 4C ) is unclear at this time, but it may be caused by an increased susceptibility of the double mutant to the toxic effects of iron. Such an effect of this iron concentration was not seen with the PAO1 parental WT or with the single mutants. Moreover, when lower concentrations of iron (i.e., 100 μM) were used, the decreased levels of β-galactosidase observed in the ΔprrF1-F2 double mutant grown in high iron relative to the WT were not observed. The microarray data (Table 1), the RNase protection assays and the translational fusion data with PA4880 confirm that this gene encoding a probable bacterioferritin is regulated by these sRNAs.
PrrF-Independent Regulation of bfrB Encoding Bacterioferritin. Fig. 5A illustrates the typical negative regulatory effect of Fe2+ on the transcription of genes (i.e., pvdS) involved in iron-acquisition systems. In contrast, characterization of a gene encoding bacterioferritin (bfrB), a subunit of the major iron-storage protein in bacteria, revealed that its regulation is characteristic for those positively regulated by Fur (Fig. 5A ). Moreover, this positive regulation depends on a functional Fur (Fig. 5B ). Although Fur is essential in P. aeruginosa, a missense mutant (A10G) has been shown to be defective in its ability to specifically bind to its operator (14). This increased expression under iron-replete conditions was confirmed with Northern blots in which the bfrB transcript level was measured in PAO1 after addition of chelator (Fig. 5C ). Thus, bfrB is another example of a gene positively regulated by Fur and iron.
However, unlike sodB and PA4880, iron regulation of bfrB does not depend on PrrF1 and PrrF2. In Table 1, neither the single nor double prrF mutants change the expression of bfrB at low iron, compared with WT cells. This observation was confirmed by two other approaches. In a Northern blot in the double-deletion mutant, addition of chelator still leads to loss of bfrB message within 10 min (Fig. 5C ) (compare with behavior of sodB transcripts in Fig. 3B ). The expression of bfrB also was examined under conditions similar to those used for the microarray experiments of reporter constructs with bfrB fused to lacZ. LacZ activity in the ΔprrF1-F2 mutant was still regulated by iron, as it is in the WT PAO1 (Fig. 5D ). No β-galactosidase activity was detected when the strains were transformed with the vector alone (data not shown). Therefore, PrrF1 and PrrF2 are not sufficient to explain all positive regulation by Fur and iron in P. aeruginosa.
Studies during the last few years have demonstrated that the IG regions in both prokaryotes and eukaryotes, formerly classified as "spacer" or "junk" DNA, frequently harbor sRNAs that play remarkable roles in critical cellular processes [e.g., oncogenesis (25), apoptosis (26), and iron homeostasis (4, 6)]. A large class of these sRNAs act by complementary base-pairing with a target mRNA, leading to a change in the target structure that can positively or negatively affect translation of the target (27, 28).
One of the challenges in understanding the contribution of sRNAs to regulation has been finding them. Comparative sequence analysis alone is not sufficient, and mutations have not been detected in these bacterial sRNAs (reviewed in refs. 27-29), possibly because of the small target size. Various computational and experimental methods have been developed (30-36) and tested in E. coli. Many of the 50 sRNAs now known in E. coli are conserved in near neighbors (Salmonella, Klebsiella, and Yersinia), but very few can be found by sequence comparisons in more distant organisms. In this study, we use the characteristics of one of these E. coli sRNAs to develop a strategy for seeking functional homologs. Two such homologs were found in P. aeruginosa, and the approach should be easily applicable to other organisms and other sRNAs.
Massé and Gottesman (4) identified a role for one well conserved E. coli sRNA, RyhB, in iron metabolism and potentially in defense against oxidative stress. RyhB is repressed by the Fur repressor under iron-replete conditions. Under iron-starvation conditions, RyhB is made and negatively regulates the expression of genes encoding iron-binding proteins (e.g., sdh, sodB, and bfrB) at the posttranscriptional level. This mechanism provided an explanation for the puzzling observation that Fur positively regulates the expression of these genes by repressing the expression of another repressor.
Many studies suggested that pseudomonads also positively regulate some of their iron-binding proteins. Ochsner et al. (11) examined global gene regulation by iron in P. aeruginosa. Although many iron-starvation-induced genes were identified in this study, there were also a significant number of genes specifically induced under iron-replete conditions, including those involved in iron storage, oxidative stress defenses, and intermediary metabolism. P. putida was found to down-regulate sodB transcripts and protein under iron-deficient conditions (37, 38). These data suggested parallels between this regulatory process in P. aeruginosa and P. putida and the positive regulation of gene expression in E. coli and other organisms by Fur via RyhB as described above. Although no RyhB sequence homologs could be detected in the P. aeruginosa genome, we proposed that some of the structural features of RyhB might be conserved and could thereby be identified by a computer algorithm. Thus, we asked whether equivalent sRNAs could be found in Pseudomonas by using three characteristics, two of them common to many sRNAs and the third specific to RyhB. Those characteristics are as follows: (i) RyhB and other sRNAs are present in IG regions; (ii) RyhB and many other sRNAs end with a ρ-independent terminator, a stem-loop followed by a run of T nucleotides; and (iii) RyhB is regulated directly by the Fur repressor. This approach led to the identification of two tandem RyhB-like sRNAs we termed PrrF1 and PrrF2. We demonstrated that both are Furand iron-regulated and are functional homologs of RyhB. This approach can easily be extended to look for sRNAs regulated by any well defined regulatory protein in any sequenced organism that is known to use ρ-independent terminators.
In P. aeruginosa, the PrrF sRNAs are duplicated and tandemly located. In P. putida, P. fluorescens, and P. syringae, two copies are also present, but only one copy is in a context with some similarity to that of P. aeruginosa; the second copy is distal in location in the genome. Intriguingly, duplicated versions of RyhB and its regulatory sequences also have been detected in the genomes of Salmonella and Y. pestis (4). In these organisms, the second copies are not tandemly located. This finding suggests that bacteria frequently have use for two copies of RyhB/PrrF. The basis for the requirement for two copies of PrrF is not clear. Both participate in the functions we have reported here; only deletion of both relieves the positive regulation by Fur and iron (Figs. 3 and 4 and Table 1). However, it seems likely that the two sRNAs are under somewhat different regulation and also may have different preferential targets. Ochsner et al. (24) showed differential regulation by heme of prrF1, but not by prrF2. This finding may reflect a differential sensitivity to low iron levels. We know of one other case of tandem sRNAs, the rygA and rygB RNAs in E. coli. These sRNAs are clearly under somewhat different regulation, although the RNAs themselves are highly conserved (32, 34).
Many organisms respond to iron deprivation by rearranging their metabolism to bypass iron-dependent enzymes, such as sodB and tricarboxylic acid cycle enzymes, and to dispense with iron-binding proteins, such as ferritins. Our study extends the role of sRNAs in mediating this change in metabolism from E. coli and its relatives to the pseudomonads. Our findings also demonstrate that the PrrF RNAs do not explain all positive regulation by Fur and iron in P. aeruginosa; bfrB regulation was not affected by mutations in either or both PrrF sRNAs (Fig. 5). Therefore, at least one other mechanism of Fur-mediated positive regulation must exist in this organism. One possibility would be a third, undetected PrrF RNA. A second possibility would be direct regulation by Fur, as is seen in Neisseria (39). A third possibility would be a regulatory protein, rather than a regulatory RNA, repressed by Fur and itself capable of repressing bfrB. We have previously identified such a candidate, PA4570, during a global GeneChip analysis of iron-regulated genes (11). PA4570 expression showed an ≈403-fold expression increase by iron and possessed a strong Fur-box in its promoter region. Although the protein encoded by PA4570 is of unknown function, we noticed a weak similarity to the IclR repressor family. Expression studies with PA4570 mutants and strains hyperexpressing PA4570 may clarify whether PA4570 plays a mediatory role in positive regulation of bfrB by Fur.
In other organisms, positive regulation by Fur has not been investigated sufficiently to identify the mechanism of regulation. Certainly, our findings suggest that sRNAs carry out this important function in various prokaryotic organisms and should be considered potential candidates for positive regulation by Fur in other organisms.
The PrrF sRNAs are previously unrecognized examples in Pseudomonas of a major class of sRNAs that may act by complementary base-pairing. In E. coli, this class of small RNAs uses an RNA chaperone, Hfq (40). P. aeruginosa also carries Hfq, which has been shown to be functional in E. coli (41). Therefore, we would predict that the PrrF sRNAs also will use Hfq and that the many other Hfq-using sRNAs found in E. coli also will have orthologs in Pseudomonas. Another class of regulatory RNAs has previously been described in P. fluorescens; RsmY and RsmZ bind to and inhibit the activity of a translational regulatory protein, RsmA (42, 43); this target protein and these sRNAs are similar to the CsrA protein in E. coli and its two inhibitory RNAs, CsrB and CsrC. Thus, pseudomonads are likely to have all of the major classes of sRNAs defined in E. coli.
We thank members of our laboratories, Gisela Storz, and Peter Greenberg for their comments on the manuscript and Cystic Fibrosis Foundation Therapeutics for subsidizing the Affymetrix Gene-Chips. This work was supported by National Institutes of Allergy and Infectious Diseases Grant AI15940 (to M.L.V.).
↵ § To whom correspondence may be addressed. E-mail: mike.vasil{at}uchsc.edu or djpf{at}helix.nih.gov.
↵ ∥ Present address: Replidyne Inc., 1450 Infinite Drive, Louisville, CO 80027.
Abbreviations: Fur, ferric uptake regulator; IG, intergenic; SodB, superoxide dismutase; sRNA, small RNA.
Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession nos. NC_002516, BK005485, and BK005486).
- Copyright © 2004, The National Academy of Sciences
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Hershberg, R., Altuvia, S. & Margalit, H. (2003) Nucleic Acids Res. 31 , 1813-1820. pmid:12654996
Ochsner, U. A., Johnson, Z. & Vasil, M. L. (2000) Microbiology 146 , 185-198. pmid:10658665
Martinez, L. A., Naguibneva, I., Lehrmann, H., Vervisch, A., Tchenio, T., Lozano, G. & Harel-Bellan, A. (2002) Proc. Natl. Acad. Sci. USA 99 , 14849-14854. pmid:12403821
Zender, L., Hutker, S., Liedtke, C., Tillmann, H. L., Zender, S., Mundt, B., Waltemathe, M., Gosling, T., Flemming, P., Malek, N. P., et al. (2003) Proc. Natl. Acad. Sci. USA 100 , 7797-7802. pmid:12810955
Gottesman, S. (2002) Genes Dev. 16 , 2829-2842. pmid:12435626
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Gottesman, S. (2004) Annu. Rev. Microbiol. 58 , in press.
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Rivas, E. & Eddy, S. R. (2001) BMC Bioinformatics 2, 8. Available at www.biomedcentral.com/1471-2105/2/8.
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Sonnleitner, E., Moll, I. & Blasi, U. (2002) Microbiology 148 , 883-891. pmid:11882724
Heeb, S., Blumer, C. & Haas, D. (2002) J. Bacteriol. 184 , 1046-1056. pmid:11807065
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text:
While I am still not sewing much, I may as well catch up what has been going on in my guild's 12 x 12 group. Sunshine was the theme in (ahem) March–so many weeks ago, I'll just let the quilts speak for themselves.
Mine was not very inspired and I hated it until I added the pleated ribbon and beads.
Melinda's, front and back…
Kathy's…I hid some facial features because her husband joked that he was wanted in several states.
Sharyn's was an exquisite embroidery of the Mayan Sun God, inspired by a trip she and I took took in February. (Note the corn border!)
Next theme (for May) was "Seasons." Coming soon.

subset: commoncrawl


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Committee votes to issue lease termination to arts center
February 3, 2014
City officials moved Monday to issue a notice of lease termination to the current tenant of the Harley Clarke Mansion, moving closer to a resolution in the longstanding and heated debate about who should occupy the historic lakefront property moving forward.
At a packed and tense meeting Monday night, Evanston's Human Services Committee tackled two hot-button topics, both of which have elicited community outrage. In addition to discussing the future use of the Harley Clarke Mansion, committee members heard about 25 people speak on the future of the Evanston animal shelter.
If City Council approves the committee's recommendation, Evanston Art Center, the mansion's current tenant, will have 240 days to evacuate the space. Prior to the committee's decision, representatives from and supporters of the art center spoke in support of extending the time the center would have to find a new location. The center's director, Norah Diedrich, requested that the nonprofit be allowed to stay in the building, located at 2603 Sheridan Road, for up to two years to allow for adequate time.
Committee members ultimately denied the center's request for additional time to vacate the space, standing by the lease's original stipulation that the center have 240 days to evacuate following the termination of their lease.
"The Art Center has had a very long time to make a decision on what to do," said Ald. Coleen Burrus (9th). "This is not somehow a big surprise to the Art Center."
The committee also directed the city manager to continue discussions with the Illinois Department of Natural Resources, which introduced plans last month to use the mansion as a public coastal education center.
(Illinois Department of Natural Resources proposes offices, coastal education center for Harley Clarke Mansion)
The committee then sat through nearly two hours of public comment on the future of the Evanston animal shelter and the shelter's current tenant, Community Animal Rescue Effort. Representatives from CARE and those opposed to CARE's current policies engaged in a heated and, at times, vitriolic debate about how the nonprofit handles dog adoptions and behavioral evaluations. Several CARE volunteers approached city officials in 2012 in protest of the nonprofit's high euthanasia rate, which they claim is between 45 and 50 percent.
(Community pressures Evanston animal shelter to decrease its dog euthanasia rate)
After hearing just five of the 30 scheduled commenters, Ald. Jane Grover (7th) moved to form a subcommittee within Human Services to continue the discussion around CARE's policies. Grover's proposal passed unanimously. Alds. Judy Fiske (1st) and Mark Tendam (6th) will be the city officials on the committee.
Although the committee had already acted on the issue, people continued to discuss CARE for the rest of the evening. The two opposing groups, composed of the center's supporters, who all donned red CARE volunteer shirts, and people criticizing and questioning CARE, often clapped or cheered after a member of their group had spoken, and could be seen vigorously shaking their heads when an opposing speaker was at the podium.
City manager Wally Bobkiewicz said the subcommittee will be comprised of three representatives from CARE and three critics of CARE, in addition to Fiske and Tendam. Bobkiewicz said the subcommittee will work to determine the future of the Evanston animal shelter and to create a renewed one-year lease between Evanston and CARE. Because the lease will be for 2014, he said he hopes the subcommittee will work quickly to resolve the issue.
Email: [email protected]
Twitter: @mccarthy_ciara
Ciara is a Weinberg senior majoring in American Studies. Her past positions at The Daily include editor in chief, managing editor, city editor and reporter....


text: Holiday Giving, Kindness Counts Helping the Homeless in Virginia Beach November 24, 2018 Pay It Forward Kids Leave a comment Was a successful Thanksgiving with 3 days of handing out Bags for the Homeless in Virginia Beach. Someone is cold, tired and hungry. Get out there and do your part.#payitforward Share this:TwitterFacebookPinterestEmailLike this:Like Loading... Related


text: Imur6 20 Posted August 31, 2014 Hi folks, I have X-55 rhino, and sometimes, it squeak when I move stick very slowly. I found root cause of this noise, and need to apply some lubricant to stick. So, what kind of lubricant can i use? the area that I plan to apply is plastic. looks like I need lubricant that can use to plastic. what about this? please let me know if you know better stuff http://www.ebay.com/itm/SHIN-ETSU-SILICONE-GREASE-G-40M-100-FOR-LUBRICATING-SANWA-SEIMITSU-JOYSTICKS-/390848546565?pt=UK_Video_Games_Coin_Operated_MJ&hash=item5b00638705 Share this post Link to post Share on other sites


text:
Faculty Advisor
Kinsel, Mary E.
Natural estrogens are endocrine disrupting compounds and common pollutants in municipal wastewater. The concentration of 17β-estradiol was monitored in effluent from both the southeast and northwest Carbondale wastewater treatment plants (WWTPs) and their receiving waters for nine weeks. The analysis was performed using gas chromatography tandem mass spectrometry (GC/MS/MS) and the internal standard estrone 3-methyl ether. Recoveries were 60.0±3.9%, and significant loss of analyte was found after storage greater than one week. The northwest effluent (NWE) had higher 17β-estradiol levels of 7.1-76.2ng/L than the southeast effluent (SEE) between Below Detection Limits (BDL)-54.0ng/L, which suggests 17β-estradiol was carried with colloidal organic particulates. River water had very similar 17β-estradiol concentrations compared to the effluent despite dilution. The university exhibited no measurable effect on 17β-estradiol levels when samples from in-session were compared with samples from out-of-session. Future ecological studies are recommended to determine the effect of estrogenic pollution on fish populations of receiving waters.


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The Chicago Board of Education sold three vacant school buildings for about $8.5 million and approved up to $1.2 billion in borrowing at Wednesday's monthly meeting.
Typically, the school board approves a budget in July, but principals were given individual school budgets just last week. A complete budget must be passed before students go back to class in September.
"The budget process this year has been delayed for a variety of reasons," said David Vitale, outgoing president of the Board of Education. Those reasons include a push for pension reform in Springfield and ongoing contract talks with the Chicago Teachers Union.
But Chicago Public Schools has been dealing with a structural deficit for several years, a truth not lost on Vitale during his last meeting on the school board.
"For what it's worth, that funding problem was identifiable from the day I sat down in this chair," Vitale said, listing some of the contributing factors: ballooning pension payments, decreasing federal and state funding, and skyrocketing debt payments.
Vitale said the school board hasn't been "ignorant" or "sitting back, waiting for disaster to happen," but he argued its power is limited, saying, "all we can do is advocate to others to, frankly, give us more authority to tax."
The board, under Vitale's leadership, voted to raise property taxes to the legal limit every year for the past four. But it still hasn't been enough.
In June, the board approved a $1.1 billion line of credit that will expire at the end of August. The move was done to ensure CPS could make payroll. This week, the board approved another $1.2 billion in long-term borrowing.
CPS Chief Financial Officer Ginger Ostro insisted the amount of money was simply an estimate and should be considered "a cap" or "limit" to what the district can issue in bonds.
The Board will have to approve actual bond sales in September or October, Ostro said.
Revenue from school sales
Board members on Wednesday also approved the sale of three vacant schools that were shuttered in 2013, providing a small windfall of cash for its struggling budget.
Liza Balistreri is in charge of real estate at Chicago Public Schools and outlined the sales of Near North Elementary in Noble Square, Overton Elementary in Bronzeville, and Von Humboldt Elementary in Humboldt Park.
Near North is being purchased by Svigos Asset Management for $5.1 million. It will be used for residential and commercial development, Balistreri said. Svigos also purchased the old Peabody Elementary for $3.5 million. There was some controversy around Peabody earlier this year when a public charter school rented space in the building. Previously, district officials had said no closed school would be used as a public school.
Von Humboldt is being bought by IFF Von Humboldt LLC for $3.1 million and will be redeveloped to include a day care, housing for current and retired public school teachers, office space, and a cafe. There was a higher bid for the massive school, but it did not line up with what the community wanted, CPS officials said.
Overton is being sold to Washington Park Development Group for $325,000 and will be used for counseling, career training, housing or retail space.
Newly-seated board member and former CEO of BMO Harris Bank Mark Furlong told Balistreri he wants to see the district pick up the pace when it comes to selling its vacant property.
"It's been a couple years," Furlong said. "We've gotta find a way to accelerate the sale of these buildings so that we can bring cash into the classrooms."
Balistreri said seven or eight more buildings are almost ready to go out for bid.
Shuffling at the top
There were several new faces in the board chamber on Wednesday.
David Vitale remained in the president's chair, but was flanked by new mayoral appointees Furlong, Dominique Jordan Turner, Gail Ward, and longstanding member Mahalia Hines. Ward and Hines are both former CPS principals, and Jordan Turner runs the Chicago Scholars Foundation, which works with first generation, low-income, college-bound public school students in the city.
Rev. Michael Garanzini is also officially a new member of the board, but was not present Wednesday because he was travelling, Vitale said. Incoming Board President Frank Clark sat in the audience.
Interim CEO Jesse Ruiz remains in his post through the end of the week, and then will return to serving as vice president of the school board. He took a moment to thank Mayor Rahm Emanuel for giving him the responsibility to manage CPS, and, in Spanish, he thanked the community for its support.
"Muchísimas gracias a todos," he said.
The son of Mexican immigrants, Ruiz is the only Latino among the top leadership at CPS, despite the fact that Latino students are the largest ethnic group in the district — at 46 percent and growing.
Replacing Ruiz as schools CEO is mayoral confidant and former head of the Chicago Transit Authority Forrest Claypool. Janice Jackson will serve as Chief Education Officer and Denise Little will take on the role of senior adviser to the CEO.
Claypool will earn $250,000 annually, while Jackson and Little will make $195,000 and $180,000, respectively. Claypool and Jackson's salaries are comparable to those of their predecessors, but Little's is a new position with no precedent for salary.
Becky Vevea is an education reporter for WBEZ. You can reach her at email@example.com and follow her @WBEZeducation.


text:
Happy Thursday everyone! I don't know about you, but I am ready for the weekend. I have grand plans to binge watch Big Little Lies tonight, and I can't wait.
So Tuesday, I shared some of my favorite shoes for Spring. And today it is all about some of my favorite spring trends + pieces!
Pink is EVERYWHERE. Have you noticed? Especially baby pink. I am not complaining at all.
Off the Shoulder / One Shoulder
The cold shoulder was everywhere for fall, and continues to be popular for spring.
I LOVE gingham! It is one of my very favorite prints. So seeing it everywhere for spring makes me really happy!
Bell Sleeves
Another trend from fall that is sticking around for spring. I love the short sleeve blouses with the bell sleeves!
So what are you loving for spring? What are your favorite trends?
PS: Check the SHOP tab for more of my favorites for Spring!


text:
GFG Investigates the Panama Canal Expansion – in person …
"The GFG investigates the world's most logistic location – the Panama Canal – for its annual conference …
GFG Group Administrator, Rachel Marchant, reports on the Panama conference, 25 – 28 September 2016
With the build up to #GFGPanama16 growing, I'm sure there were many outside the GFG who were waiting to see what this hashtag related to. Well, we're pleased to report it was a resounding success! Read below for the full report…
Panama – a cultural, welcoming country at a cross-road of canal, continents and even climate!
"The weather in Panama is bi-polar" was possibly the best local introduction to a country that I've experienced. It encapsulated the seasonal variations and proved to be absolutely true.
Admittedly #GFGPanama16 landed right in the middle of the rainy season. However, 'rainy' was an understatement. The rain literally blocked out buildings, trees and even the sea. It was a wall of water, not just rain. Then the sun came out again and Panama's alter ego returned – sun sparkling everywhere as it the city had just been spring cleaned.
The #GFGPanama16 says it all – the GFG's annual conference for 2016 was held in Panama. It brought together over 60 top class freight forwarding companies to take part in the Global Freight Group event.
Many of the original founding members still remain in the membership, proud to have founded a group which values quality over quantity. At our conferences members are not lost amongst the masses but are each valued individually in what has become a gathering of friends who do business with each other. Trust and friendship provides the reliability of service required.
This conference we welcomed two new members in key locations – Los Angeles and Guatemala. Over the years, the GFG has sought to increase its presence in Central and South American and was delighted to welcome Lina Vinchira of ASE Logyca for her first conference. Although originally from Colombia, Lina moved to Guatemala where she set up her own freight forwarding business. She loves Guatemala but was also happy to catch up with three fellow Colombians representing MAP Cargo at our conference. (Linna is lower left in the image below)
Karen West (lower right in above image) represented her company Earth Cargo Inc, attending as a prospective member, wishing to experience the conference and meet the members to weigh up the benefits of joining the GFG. We were delighted that her positive experience and the resulting potential for business encouraged her to join us as a member. Below: during a break in the meetings, members gather to compare opinions and comment.
Below: The cocktail party is the first social non-work event and members look forward to welcoming back old friends and meeting new ones. This year, along with many friends, we welcomed newbies from Guatemala – Linna Vinchira – and Los Angeles – Karen West.
The face-to-face sales meetings are where the members get down to serious business. It's a chance to keep up with each other's services, the latest trends and regulations in each other's countries and generally develop business between their companies.
Below you can see meetings between Helka (China) and MAP (Colombia) on top right, Leo (Thailand) and CLand (Sweden) bottom right and Hansaeng (South Korea) and DAC (Panama) bottom left.
Below: With windows offering a marvellous panoramic view across the Panama Bay, members enjoyed their lunch with a view, along with great company and delicious food.
The GFG members naturally congregate so, even if you start off alone, you won't be alone for long.
Below: Members join each other in conversation and business plans in various scenarios – formal, informal and social. For this reason the GFG also stands for the Global Friends Group – famous for its welcoming attitude.
Members gather on the stairs for a quick group photo – oh, and to shout 'Happy Birthday' to one member's daughter!
We're a family after all so we appreciate the importance of keeping in touch even from the other side of the world.
Below: As you can see below, the GFG members were fascinated by the canal. If you put 100 freight forwarders in a restaurant overlooking the biggest most famous canal in the world, you to have to expect them to ignore the food – at least for an hour or so! Above: the Miraflores Locks on the Pacific end of the canal.
Below: Ronaldo Buchanan of DAC Transport, the local host for the GFG conference, addressed the GFG members, thanking them for visiting his country.
Over the years the GFG has developed two awards to recognise worthy members and their employees.
The President's Award is chosen by the incumbent President and this year Ilan Fidler presented it to the GFG's member for North China, Headwin Logistics.
Another very popular award, created by a former GFG President, Les Allan, is the Operator of the Year Award. It recognises the employees within the member companies who, on a daily basis, work hard to provide an excellent service to other member companies. Helka Global Logistics have the most highly and frequently nominated employees and so, when their very popular owner, PK Ki, sadly passed away, the award was renamed in honour of him, and his company's achievements. Thus we now have the PK Ki Operator of the Year Award.
This year, the award was shared between two equally voted upon employees: Ida Cheung of Helka Global Logistics and Wanderson H Pereira of ARI Shipping Florida. Ida had been invited to attend the Panama conference by her boss, Ms Sharon Ki, as a reward for her previous nomination and subsequent win of the award in 2016. It was perfect for her to be able to receive the award in person in front of the appreciative GFG members.
In 2014 the GFG celebrated 20 years of membership, business and friendship. At that point it became apparent how many companies had joined and stayed with the GFG during much of that time. As recognition for this loyalty, we designed and presented the first of a now annual recognition award. Members were presented with 5 year, 10 year, 15 year and 20 year awards at the 2016 conference in Seoul. (read Seoul conference report)
In Panama, we awarded the second round of awards, covering members who had now reached the 5, 10 and 20 year milestone.
The awards were presented to the following:
5 Year Award for:
– MAP Cargo, Colombia
– Meacher Global Logistics, UK
– Posey International, Texas USA
– ScanGlobal Finland OY, Finland
10 Year Award for:
– Universal Logistics, Illinois USA
20 Year Award for:
– Eagle Express Lines, Philippines
– Del Corona & Scardigli Srl, Italy (sea freight)
– Esse Emme Srl, Italy (air freight)
– Globe Cargo, Germany
– TFI Global, Australia and New Zealand
– Vogel Transport, Brazil
Following the conference I always arrange a day trip for members so they have a chance to appreciate their surroundings outside the hotel and conference events. We had a record number of attendees this year. It was perhaps due to the subject matter – a trip to the Agua Claras locks of the extended Panama Canal at Colón, at the Atlantic end of the canal.
Having appreciated the Pacific Ocean end of the canal at the closing dinner, members now viewed the other end of the canal.
Our timing was great in that a ship was passing through the canal as we watched, enabling us to fully admired the mechanisms and engineering of the lock system and its water management and flow. Admittedly, immediately after that ship passed through a torrential rain storm rolled in and visibility was reduced to zero. Another example of Panama's 'bi-polar weather'.
After returning to Panama City, we visited various culturally interesting sites, including Casco Viejo and the Bio Museum. Panama's divide once more evident when comparing the fascinating vintage buildings in the streets of Casco Viejo against the backdrop of modern Panama City.
During the conferences, there is always time for GFG members to enjoy each other's company and share the experience of travel in an interesting location. Panama was no different as members joined each other to enjoy socialising, exploring, being Barbie and even gate-crashing the owner's birthday party in the renowned Diablicos restaurant – he seemed delighted!
Of course the main impetus of the conference is to promote business and join forces to increase efforts to build business. However, the strength of the GFG is that this is all done in a spirit of trust and genuine friendship. They say that those who "play together, stay together". The GFG has proven this by the fact that over half the original founding members, from 1994, still maintain their membership and involvement with the Global Freight Group. Longevity and loyalty are good traits in an ever- changing world – long may it continue.
#GFGPanama16 may have passed but follow us via #GFGGreece17 and sign up for 'business in friendship' with the GFG in Greece, 14-17 October 2017.
Now looking forward to #GFGGreece17
Link up with the GFG and 'go global', click here…
We're extending our special offer – find out what it is here…


text:
The Plantation City Council has granted its first set of approvals to pave the way for one of the largest residential-retail projects in Broward.
The yet-unnamed project, expected to rise on the site of the defunct Fashion Mall, at 321 N. University Drive, will be a self-contained, downtown-style neighborhood where people can enjoy stores and restaurants just a short walk from their homes.
It calls for: 700 homes, office space, and 200,000 square feet of retail and restaurants with outdoor seating and walkways to encourage pedestrians.
The City Council on Wednesday night signed off on changing the designation of the area to allow for residences; Some apartments will be built on top of retail space for a total of seven stories, and two stand-alone apartment complexes would range from 8 to 12 stories.
And 346 units, the first batch of 700 total rental apartments, were approved, too. Rent is expected to range from $1,800 to $3,000 per month.
The developer, Art Falcone, said the market demands new housing. "When was the last time an apartment complex got built in the area?" he said. "We're in a built-out market, there really is no land left in Broward County and there's a growing population."
It's a landmark move for a towering eyesore that had starts and stops for more than a decade.
In 2004, after years of decline, a Chinese investment firm bought the property, promising office and shopping space and a movie theater. That didn't happen, and then Hurricane Wilma slammed South Florida in 2005. Macy's was damaged, and closed. The rest of the mall closed in 2007 and by 2013 the city was faulting the property for numerous code violations.
More projects were proposed and then the Chinese investors filed for bankruptcy. A real estate development firm, an affiliate of Boca Raton-based Encore Capital Management, bought the mall for $37.7 million at a bankruptcy auction in 2015 and pledged to invest $300 million to turn it into a self-contained mixed-use project.
Falcone, chief executive and managing principal of Encore Capital Management, said the hotel on site, as well as the 2,600-parking space garage and an office building, will remain. The inside of the office building, however, has been gutted and is being remodeled.
In addition to the apartments and retail stores, a second, 100,000-square-foot office building will be built.
Falcone said he has another month left of demolition - work that took longer than expected because 911 antennas had to be removed from the roof, beams that support the parking garage needed to be worked around, and electrical lines for the hotel couldn't be touched.
Demolition "was like having surgery, you open the person up and [say] it's a little bit different than what I was thinking," Falcone said.
Falcone said he has about a dozen prospective tenants who have signed letters of intent to lease space for retail and restaurants.
"There will be a little bit of everything, from fashion to service to different types of entertainment" and sit-down and quick-serve restaurants, he said. "The whole idea is to keep people there."
He expects to break ground in the second quarter of next year.
email@example.com or 954-572-2008


text:
Gold Star License Needed For Travel In 2020September 17, 2019
A standard driver's license will no longer be adequate for domestic air travel or entering certain federal buildings after October 1, 2020. You will need a "verified" driver's license (AKA REAL ID), state-issued ID card or license or be required to show additional proof of identification, such as a passport. You can easily tell if you have a REAL ID; it has a gold star on it.
You can wait to renew as long as you don't plan on flying without a passport, or you can head to the Department of Motor Vehicles and pay a $30 duplicate license fee. Just know that you will need to bring specific forms of ID to get REAL ID.
Click HERE for more information.


text:
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Supreme Court of North Dakota
July 12, 2017
State of North Dakota, Plaintiff and Appelleev.Eybon Avyon Watkins, Defendant and Appellant
from the District Court of Burleigh County, South Central
Judicial District, the Honorable David E. Reich, Judge.
A. Lawyer, Burleigh County Assistant State's Attorney,
Bismarck, ND, for plaintiff and appellee.
Jackson Lofgren, Bismarck, ND, for defendant and appellant.
VandeWalle, Chief Justice.
1] Eybon Watkins appealed from a criminal judgment entered
after a jury found him guilty of robbery and the district
court imposed upon him a four-year mandatory minimum sentence
as an armed offender. Although the court did not instruct the
jury to make a finding whether Watkins possessed a firearm
during the robbery, we affirm because Watkins invited the
2] Watkins was charged with robbing a hotel in Bismarck
during August 2015. The charge was a class B felony under
N.D.C.C. § 12.1-22-01(2), which applies "if the
robber possesses or pretends to possess a firearm,
destructive device, or other dangerous weapon, or menaces
another with serious bodily injury, or inflicts bodily injury
upon another, ..." The State sought a four-year
mandatory minimum sentence under N.D.C.C. §
12.1-32-02.1(2)(a), which applies "only when possession
of a dangerous weapon, explosive, destructive device, or
firearm has been charged and admitted or found to be true in
the manner provided by law, ..."
3] Before the trial started, the State informed the district
MS. LAWYER [Prosecutor]:... In order for the mandatory
minimum to apply, since the State is alleging that he was in
possession of a firearm, the jury has to make a finding that
he was in possession of a firearm. So I would suggest that
after we have the guilty not guilty/guilty, that we have a
further paragraph that says if you find the defendant guilty,
something along the lines of, do you further find that the
defendant was in possession of a firearm at the time of the
offense? Yes or no. And have them check that as well. Because
I believe that the case law says that the jury has to make a
beyond a reasonable doubt finding for that factor to apply
for the mandatory minimum to apply, but, again, that's
just something we can think about for the next couple days.
court said it appreciated the "heads up" and gave
the jury a preliminary instruction on the essential elements
of the offense which included the requirement that the jury
find "[i]n the course of committing the theft the
defendant willfully possessed or pretended to possess a
firearm, destructive device, or other dangerous weapon."
4] During the trial, the State presented testimony of the
hotel's night auditor who was the only eyewitness to the
robbery. She testified that a man wearing a mask, glasses and
gloves approached her "holding something at me wrapped
in a garbage bag." The night auditor thought the man had
a gun because of the way he was pointing, but did not
actually see a gun in his possession.
5] At the close of the evidence, the district court and the
parties discussed the final jury instructions and the
State's request for a specific verdict question whether
the defendant used a firearm for the mandatory minimum
sentence to apply. The court said it did not believe a second
question was necessary on the verdict form because "[i]t
seems to me they already have to find that in order to find
him guilty." The discussion continued:
MS. LAWYER:... I'm fine with leaving the question out if
the Court is comfortable with that and I'm comfortable
with that interpretation of that case law as well.
THE COURT: I'm fine either way. It's just that when I
didn't do a lot of research, but I just looked at the
annotations. And it seems like the annotation or the primary
case at least where they talked about making a specific
finding was a reckless endangerment case and that
wouldn't have the element the essential element that
there is in this case. So I don't know if the question I
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text:
As a Christian, you hold certain values close to your heart. Christian drug rehab will allow you to embrace strategies for recovery that also embrace your Christian beliefs.
The Power Of Fellowship For Recovery
Let's face it: as addicts, we might have been the social butterflies and life of the party for a time, but as the addiction escalated, we often found ourselves becoming increasingly isolated. We didn't like ourselves and couldn't understand why anyone else would either. We felt safe in the arms of our addiction, and after a while didn't see the point in trying to engage others. It often felt like too much work.
The "Fellowship" Part Of Recovery May Feel Like An Overload, But It's Needed And Worth It
Thus, for many of us, getting into recovery can be a fellowship overload. There are these lively recovering people all around us wanting to help us, hug us, call us on the phone and send encouraging texts. They want to meet for coffee, they are inviting us to help set up chairs before the meeting and they are delving into the most private details of our lives. For the newcomer—the one who has been isolating in the dark corners of addiction—the program's focus on fellowship can feel like a hurdle.
Can't we just work the steps on our own? Can't we just come to the meeting a few minutes late and slip out a few minutes early to avoid the chit-chat? Why do we have to call our sponsors every day? Why do we have to pick up the phone when others call us? We barely know these people.
If you're a newcomer, you may not yet see the vital importance of fellowship. That's OK. You will. We are accustomed to thinking we can do everything on our own. But isn't that what we were trying to do before we got into recovery? And how was it working for us? We were full-blown addicts. Although all this fellowship feels foreign and uncomfortable, it's time to give this new way of living a chance.
While all the people around you hugging you and calling you may feel overwhelming, the truth is, you need these people and they need you. They are welcoming you into the fold with a level of open acceptance unlike anything you are likely to receive anywhere else. The rest of the world is evaluating and assessing us, loving us conditionally.
In the 12-step rooms, the performance and the distinctions and the conditions go out the window. We are nothing more than a humble group of addicts desperately trying to get well and leaning on each other to do it.
You may feel guilty at first that so many people are going out of their way to help you. That's because most of us don't know how to let others serve us. We believe we are unworthy or that something will be asked of us in return. But as we stick around the 12-step rooms, we see that service to others strengthens sobriety. And then it is easy to see why everyone is so happy to give and serve and sacrifice.
Power Of Letting Others Help You
If you are a newcomer, this is your chance to open yourself up and receive. It is not the time to ask how you can pay everyone back for all they have done for you. That day will come. As you begin working through the first steps, you are letting others help you. You are getting your head cleared out and you are reconnecting with God. These are rocky days and you have all you can handle to simply not take a drink or get high.
The most important service you do for your fellowship right now is to stay sober and to keep showing up at your meeting. You may not see how important this is now, but later you will understand.
As you continue working through the steps, the program will start to feel more natural. You will be getting your bearings and enjoying some solid sobriety. And soon you will feel yourself reaching out. Instead of letting others call you, you will pick up the phone. After some months you may be asked to sponsor. You may take on a simple service position.
Soon you find that these people are becoming your greatest friends and confidants. You look forward to going to your meeting and even welcome the phone calls. You have finally found a group of people who understand you, want to know you and like you. You like them, too.
And as you give back to this fellowship through service, through your continued sobriety and through simply sharing your own story of addiction and recovery, you are strengthening the group as a whole and you are practicing the 12th step. Newcomers walking through the doors hear your story of hope and dare to believe this program could work for them. This is the service we do for our fellowship and for the community. And as recovering addicts, it is our highest calling and our greatest joy.
Serve God & Serve Others & You Will Be Blessed!


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A vulnerability in Cisco Unified MeetingPlace could allow an authenticated, remote attacker to view passwords in plain text.
The vulnerability is due to the inclusion of sensitive information in the web page source code of the affected software. An attacker could exploit this vulnerability to view passwords in plain text format.
Cisco has confirmed the vulnerability and released software updates.
To exploit this vulnerability, an attacker must authenticate to the targeted device. This access requirement reduces the likelihood of a successful exploit.
Cisco indicates through the CVSS score that functional exploit code exists; however, the code is not known to be publicly available.


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As sturdy as they look, horses are actually fragile creatures that require a unique level of care. They cannot be left to their own devices for days on end without you checking up on them to make sure they are safe and well. Even with constant attention, a horse can develop debilitating illnesses that could shorten its life.
Your horse is more than just a monetary investment that you do not want to lose. It is also a valued family member about which you care deeply. You can research how to care for horses, pay close attention to your equine friend, and buy horse supplements online to address a variety of health concerns in these animals.
Horse Anxiety
Like humans, horses can be prone to anxiety if the circumstances are right. They may get nervous when they are left one for hours at a stretch. They also may be afraid of the weather, other animals, unfamiliar settings, and a host of other things that you may or may not be able to identify.
Even if you are not sure what is causing your horse's anxiety, it can still be critical for you to get it under control. Along with spending as much time as possible with your horse, you also may want to use safe and natural supplements designed just for these creatures. The supplement will help it remain calm while in the stable, during horse shows, while working on the farm, or anytime else it can get worked up and act out.
In unique cases, some horses can be hardwired to be anxious and nervous. Their chemical makeup inside of their bodies make it hard for them to keep calm. A nervous horse can be dangerous for you to be around, however. You need yours to remain calm especially if you plan on training it for riding.
The website sells a paste that you can apply to your horse. The paste has safe and natural ingredients that calm the horse and make it gentle to train and ride. You can use the paste as often as needed until your horse is gentled.
Equine Arthritis
Also like humans, horses can suffer from arthritis. As they grow older, they may develop this conditions in their legs and hips. They also may develop it after being injured in a fall or other accident.
A horse with arthritis may be reluctant to be ridden or run. It may also lie down more than usual. You can renew your horse's muscular and skeletal system by giving it supplements designed to heal its joints and bones. After a few days of using the product, you may notice that your horse has regained some of its former energy and flexibility.
The company knows that you do not want to give your horse anything that could harm it. It avoids using harsh ingredients in its products. Your horse will be able to tolerate the supplements without getting sick or suffering other side effects like hair loss.
A horse can be a fragile creature that requires you to pay close attention to it. You can keep yours in the best health by using natural supplements designed for equine use. They are made to fight illnesses like anxiety and arthritis from which horses can suffer frequently.
- 21Shares


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BATTLE-weary Auchinleck Talbot have been given a welcome boost in their quest for another extended run in the William Hill Scottish Cup.
Tommy Sloan's team kept their hopes alive by drawing 2-2 away to Fife cracks Dundonald Bluebell on Saturday and then reached the quarter-finals of the West Region Sectional Cup with a 3-0 home win against Kello Rovers.
However the last eight tie at home to Whitletts Vics was switched from tonight (Wednesday) until Monday, August 26 to allow Tucker's team to prepare for the hotly anticipated replay on Saturday at Beechwood Park.
Talbot fell behind late in the game before a late Graham Wilson penalty forced a rematch.
Sloan said: "To be fair Dundonald are a decent side and it was disappointing not to win the tie.
"But the positive thing for me is that we're still in the Scottish Cup. It's important for us to progress and hopefully we can do the business at Beechwood Park on Saturday. It was good the SJFA put back the quarter-final as we want to focus on the Dundonald game which is vital for us."
Dwayne Hyslop has a knock and is set to miss out and Bryan Boylan is recovering well after being sidelined from last month.
Meanwhile Glenafton Athletic were held at home by newcomers Benburb in the McBookie Premiership on Saturday. The game ended in a 1-1 draw.
After a period of sustained pressure from the home side the visitors began to threaten and in 33 minutes earned a free-kick on the outskirts of the box. Kyle McAusland's effort thundered into the wall before Ryan Nisbet rolled the loose ball back to Findlay Frye who rifled it from 18 yards to the top right hand corner. However in 58 minutes, Bens levelled when substitute Ciaran Diver picked up the ball on the halfway line and went on a mazy run to the edge of the box and drilled home a low shot.


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A 'possessive' mechanic launched a bloody attack on his lover as her two children screamed in fear because he thought she was cheating on him, a court heard today.
Lee Birks, 27, was jailed for 18 months for launching the frenzied assault on Natalie Ranson as she tried to keep him away from her children who were just feet away in the bath.
He had head-butted her twice in the face before pummeling her with his fists when they argued about her ex-lover and a man she was in contact with on Facebook. The beating left her needing surgery to repair her nose. Hew put her head in the oven and threatened to burn her alive under the grill.
During the attack at her home in Werrington, Staffordshire, on May 17 this year, Birks initially threw Miss Ranson's laptop against a wall and stamped on it before hurling a glass against the kitchen wall.
He then punched a television in the bedroom before head-butting helpless Miss Ranson twice in the face and thumping her in the right eye causing her to lose consciousness.
The attack, which left Miss Ranson with horrific facial injuries, happened just feet away from her children, aged four and 18 months.
Jailing Birks, Judge Paul Glenn told him: 'You had been in a relationship from 2008.
'She described you as jealous and possessive during that relationship.
Jealous: Despite being left bloodied Natalie Ranson, pictured right, was most concerned for her children who were in the bath when Lee Birks, left, flew into a rage
'On May 17 you argued, it was about one of her ex-partners and Facebook contact she allegedly had with someone you knew.
'You had drunk alcohol and became increasingly angry and ignored a request to leave.
'This young lady was a vulnerable victim, a single mother. Her injuries were unpleasant. It was a repeated assault.
'When you head-butt people, that equates to using a weapon. The aggravating features are a conviction for a previous assault on the same victim and the presence of two young children.'
After battering his lover, Birks fled the house before being arrested later that day when she came round and dialled 999.
Speaking after the case, Miss Ranson, who had to undergo surgery on her nose, described a sense of relief after her 'terrifying ordeal' came to an end.
The full-time mum said: 'Lee started going off on one. I just asked him to leave. I could see the anger in his eyes.
Guilty: Lee Birks, pictured outside Hanley Crown Court, Stoke-On-Trent, smashed up his lovers home before head-butting her and punching her in the face
'I was checking on the children who were in the bath. I heard a loud noise. My laptop had been slung on one side of the room.
'Lee was in a rage. I went up to the children. I came downstairs. Lee threw the glass at the kitchen wall.
'I was just asking him to leave. He then went upstairs, the children were still in the bath. I went upstairs because I did not want him anywhere near my children.
'Lee was in my bedroom. I told him to get out the house. He swung around and punched my television. It was in pieces.
'He came towards me and head-butted me. I think I lost consciousness. He head-butted me twice. My nose was pouring with blood.
'I was in a complete and utter daze. I did not know what had gone on. He then punched me to my right eye and cut me open where my eyebrow is.
'My children were screaming. I just wanted to make sure they were OK. He left. I went downstairs. I knocked on next door and asked for help. I had blood everywhere.'
Birks, from Meir Park, Stoke-on-Trent, Staffs., was jailed for 18 months at Stoke-on-Trent Crown Court after he was convicted of assault on December 5.
He was also banned from contacting Miss Ranson for three years.
Miss Ranson added: 'I have got my closure now. He put me through absolute hell that night.
'My kids were in the bath and all I could think about was getting to them and making sure they were okay.
'I am still suffering because I need surgery on my nose to replace the cartilage that was damaged. I am getting on with my life now.'
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Each year Fazenda features a special coffee for the holiday season; this year we are celebrating with a coffee from Finca La Pastora in the Santa Maria Valley of Costa Rica. This coffee was a hit through the holidays, so we're continuing it through the first few months of 2020!
La Pastora has been producing coffee under the ownership of Esquivel family for six decades. For most of its history, La Pastora supplied coffee cherry to other mills for processing. In 2011, the Esquivel's built their own wet mill on site to be able to more tightly control quality and be able to better feature single estate microlots.
This particular lot is processed in the "yellow honey" style. Unlike traditional washed coffees, a small amount of mucilage (the sugary, sticky fruit pulp from the coffee cherry) is left on the beans after pulping. The resemblance of the mucilage to honey is what gives the processing its name. The coffee is then dried with the mucilage remaining on the bean.
During the drying process, the fermentation of the mucilage produces a variety of aromatic compounds that affect the final flavor profile; additionally, the bean retains a higher sugar content compared to traditional fully washed coffees. Honey coffees tend to have heavier bodies while retaining sweetness and flavor clarity.
Region: Santa Maria de Dota, Tarrazu
Producer: Mainor Esquivel Picado
Process: Yellow Honey
Varietals: Caturra, Yellow Catuai, Typica
Elevation: 1700 - 2000 meters


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A piece of the pie / From coastal farm to city cafe, Mission High students learn about baking and beyond
Easy to love, wholesome and sweet, smelling of home: That's pie. Even those of us whose mothers never mastered pie associate it with happy times. Now a new Bay Area venture is acting on the potential of pie to connect urbanites to local farms, to inspire inner-city youth, and to encourage more sustainable food choices. Call it Pie Power.
On 25th Street near Mission in San Francisco, the minuscule, 4-month-old Mission Pie fills its 14 seats with locals lured in for a slice. This being San Francisco, diners can have an organic latte with their pear-raspberry pie, served by streetwise Mission High kids working after school. It takes awhile, sometimes several visits, for customers to realize that the teens behind the counter are some of the same ones grinning in the farm photos on the wall, and to learn that those unassuming pies drive an ambitious social experiment.
In a cycle of production and consumption that is rarely so neat and direct, Mission Pie's proceeds help support a farm on the San Mateo coast, which in turn supplies ingredients -- eggs, wheat, pumpkins, berries -- for the pies.
The teens, all of them learning disabled, work on the farm one day a month, getting up-close exposure to the environmental issues they've explored in science class. The students who work in the store stay connected to those berries from farm to table and proudly recommend pies that contain fruit they helped to grow.
"It's this incredible opportunity to demonstrate that there's a need and a place for a city business making a proactive commitment to an agricultural producer," says Karen Heisler, a partner in both Mission Pie and Pie Ranch, a 14-acre parcel in Pescadero that she purchased with partners in late 2002.
Named for the wedge shape of the property and for the positive associations with farm and table, Pie Ranch has, from its inception, been a farm with a mission. Heisler and her ranch partners -- farmers Jered Lawson and Nancy Vail, who are husband and wife -- saw the property as a vehicle for social change, a working farm that could educate city dwellers, especially youth, about the sources of their food and consequently build urban support for local farms.
Not long after purchasing the farm, Heisler and her partners began hunting for a school that would collaborate on educational programming. They targeted San Francisco, intent on linking city dwellers to the coastal foodshed.
Matt Heller, a science teacher at Mission High School, saw the potential immediately. Heller and one of his colleagues, English teacher Corky Kern, had been talking about providing their special-education students with learning experiences outside the classroom, in an environment where they might excel.
Two years ago, the teachers took the first group of five students to Pie Ranch for a half-day of farm chores and cooking. Now, one morning a month, two vans navigate the long, eucalyptus-lined driveway at Pie Ranch and disgorge 12 to 15 students in jeans, hooded sweatshirts and sneakers.
They divide into small groups and spend the next few hours on collaborative tasks: harvesting berries; planting spinach and radishes; preparing lunch; baking pie; or -- nobody's favorite -- moving the portable chicken coop to fresh pasture. A half-hour of journal writing gets worked into the mix, and the outing ends with -- what else? -- a slice of warm pie all around.
"The first time, they wore really good shoes and their good clothes, worried that somebody might see them," says Kern. "They wanted to look cute. Now they dress down, ready to get dirty."
Pie Ranch nestles in a flat, sun-splashed clearing, embraced by towering eucalyptus, oaks and Douglas fir. When the students arrived on one recent chilly morning, the chickens were patrolling their pen, scratching the ground until the dirt flew. Inside the coop, the laying hens were squawking loudly enough to wake the next county. Despite a brisk wind, a brilliant sun illuminated the farm, its alternating bands of lime green, forest green and Kelly green signaling shifts between pasture and crops. Olallieberries and blackberries clambered up trellises, and strawberries poked up through plastic groundcover.
After climbing a hill to view the farm and note what had changed since their last visit -- a monthly ritual -- the students split into their work groups.
In Lawson and Vail's spacious yurt, Scott Chow and Mark Collins made whole-wheat piecrusts under the gentle direction of Krysten Rudin, a professional baker who oversees both young men in their counter jobs at the pie shop. Rudin showed them how to measure flour accurately by spooning it into a cup and leveling it off and how to work the chilled butter into the flour by hand. They filled their crusts with sugared berries frozen after last year's harvest, and made lattice tops.
In a primitive kitchen in another farm building, with only a two-burner propane stove, students Midget Beltran and Da-Ron Raynaldo prepared lunch for the whole group. Midget chopped fresh tomato and cilantro for salsa and scooped avocado into a bowl for guacamole, while Da-Ron stir-fried the chicken for tacos. Their adult guide, a young and energetic former restaurant cook named Megan Hanson, teaches an informal nutrition class at Mission High and has clearly made an impression.
"Da-Ron's saying is: Healthy food looks nasty but it tastes great,'" said Hanson as Da-Ron pushed his browning chicken around the skillet. "Midget used to sneak in Doritos and Cokes. "Now he's kind of a fruititarian." Midget mashed the avocado, seasoned it with salt, and offered a taste to Hanson.
Getting teens to eat well is "not that tricky," claims Hanson, a program coordinator for Nextcourse, a San Francisco nonprofit that teaches youth and low-income communities how to eat healthfully and support local agriculture. It helps to engage them with culturally appropriate food that they already like, she says, such as this day's taco lunch, a nutritionally balanced feast with shredded lettuce, steamed brown rice and black beans grown on the farm, beans the students had shelled the previous fall.
Several students said that they had cut out sodas or cut back on junk food after Hanson's classes, which highlight the difference between whole and processed foods and attempt to make the kids into competent label readers.
"It has changed how we eat," says Helen Chow, a San Francisco teacher's aide and Scott Chow's mother. "He talks a lot about organic food and is more picky about what he eats. He stopped drinking soda, and when we shop, he's looking more at labels."
The kids have planted apple trees on the farm, with the vision of apples for pie in the future. By hand, they have harvested and winnowed the farm's wheat, a low-protein variety suitable for piecrusts.
Lawson and Vail have struggled to find equipment to process the wheat efficiently as few American wheat farmers operate on a small scale. In the meantime, Mission Pie crusts contain what might be called a symbolic amount of Pie Ranch flour, with more to come.
Also on the wish list: a dairy cow that can provide the milk needed for butter. Until this young farm hits its stride, many of the pie ingredients must be purchased, with preference given to local organic sources.
"I'm not even sure our aspiration is to provide a certain amount of produce to the pie shop," says Keisler. More important, in the partners' view, is for the shop to demonstrate the contribution of local farms and to engage customers in supporting them.
Later this year, the tiny shop will undergo an expansion into an adjacent space and add a professional pastry kitchen. Currently, the pies are baked at Destination Baking Co., a Glen Park shop whose owner, Joe Schuver, is a Mission Pie partner.
When the new kitchen is up and running, the teens who work in the shop will get pastry training to supplement what they've already learned: how to make cappuccino, interact with customers, and show up for shifts on time.
Lawson and Vail say these learning-challenged youth have blossomed visibly since the farm visits began. "They were withdrawn when they first arrived," says Lawson. "Now they are gaining a sense of capacity and confidence."
They call themselves the Piesters now, a tight group initiated into the mysteries of pastry dough and pie filling. They are white, black, Hispanic, Asian, sophomores, juniors and seniors, and they have bonded in a way no one imagined.
"A sense of family has developed," says Kern, the English teacher. "They are talking to kids they wouldn't normally talk to, across color lines and class lines and all the ways they normally divide themselves at school. It's an amazing evolution."
In town, they are more competitive, she says. They have to maintain their street cred. But on the farm, there is no such pressure.
Last year, the adults initiated a conversation with the teens to solicit feedback about the farm program. "They stunned us," says Heisler. "They talked about love and community. They said, 'You don't have to watch your back here.' "
Aware that summer is a threatening time for these city kids, when gang activity soars, Heisler and her partners have tried to devise a way to stay in touch with them. Some teens will work in the shop over the summer; others will assist at the Pie Ranch farmstand, if transportation can be arranged.
As transformative as the project has been for the youth, the teachers say they have profited, too. Whether the subject is renewable versus nonrenewable energy in science class, or the immigrant experience in English class, the teachers link their classroom lessons to life at Pie Ranch.
Both students and teachers say that thanks to their time on the farm together, and especially their leisurely communal lunch, they have developed a rapport that rarely occurs inside a classroom.
"This has definitely been one of the best experiences I've had teaching in 15 years," says Heller, the science teacher. "Probably the best."
Where to buy pie
Mission Pie. 2901 Mission St. (entrance on 25th Street), San Francisco; (415) 282-4743. Open 7 a.m.-9 p.m. Monday-Thursday, 7 a.m.-10 p.m. Friday, 8 a.m.-10 p.m. Saturday, 8 a.m.-9 p.m. Sunday.
Pie Ranch Farmstand. 2080 Cabrillo Hwy. (Highway One), Pescadero (about 10 miles south of Pescadero Road; (650) 879-0971 or www.pieranch.org. Scheduled to open Memorial Day weekend, noon-6 p.m. Saturday-Sunday.
Operated in collaboration with neighboring Blue House Farm, the farmstand will have berries, eggs, beans, pumpkins, whole wheat (ground into flour on site) and other produce in season.
Pie Ranch's Mixed Berry Pie
Makes 1 double-crust pie Serves 8 to 12
The Crust
2 1/2 cups whole-wheat pastry flour
1/2 teaspoon kosher salt
1/2 pound (2 sticks) unsalted butter, chilled, cut into small pieces
Approximately 1/3 cup ice water
The Filling
2/3 cup sugar
3 tablespoons cornstarch
6 cups mixed berries (raspberries, blackberries, strawberries)
To make the crust: Put the flour and salt in the work bowl of a food processor and pulse to blend. Add the butter and pulse until all the pieces are coated with flour and about the size of peas. Transfer the mixture to a bowl.
Alternatively, you can incorporate the flour and butter by hand: Put the flour and salt in a bowl and stir well to blend. Add the butter and massage it into the flour with your fingers, working until all the butter is flattened into thin, flour-coated flakes. You should not see any large pieces of butter, but don't over blend the dough or it will be tough. The flakes of butter will produce a flaky pastry.
Add the water gradually, tossing with a fork until all the flour is moistened and the dough begins to come together. Divide the shaggy dough into 2 mounds, one slightly larger than the other. With your hands, gather each mound into a ball, kneading it gently if necessary to help it adhere. Wrap each ball in plastic wrap, then flatten each ball into an evenly thick disk resembling a hamburger patty, and refrigerate for 1 hour.
Preheat the oven to 400°.
To make the filling: Just before rolling out the pie dough, whisk the sugar and cornstarch together until well blended in a small bowl. Put the berries in a large bowl and add the sugar-cornstarch mixture. Toss gently but well.
Put the larger disk of dough on a lightly floured surface, or between 2 sheets of parchment paper. With a rolling pin, flatten the disk into a round large enough to fit your pie pan and overhang the pan by about 1/2 inch. Transfer the round to the pie pan. You may find it easier to transfer if you fold the circle in half first, then place it in the pie pan with the fold in the center and unfold it. Gently press it against the bottom and sides of the pan.
Top the dough with the sugared berries, spreading them evenly.
Repeat the rolling procedure with the second disk of dough, flattening it into a circle large enough to cover the pie. Place it over the berries. Fold the overhanging edges of dough under and use your thumb and forefinger to crimp the rim of the dough decoratively, sealing the top and bottom doughs together. With a paring knife, make 6 to 8 slits in the top of the dough to allow air to vent.
Put the pie in the oven and lower the temperature to 375°. Bake until the crust is golden brown and crisp and the filling is bubbly, about 1 hour. Transfer the pie pan to a rack to cool, and cool completely before slicing.
Per serving: 280 calories, 3 g protein, 34 g carbohydrate, 16 g fat (10 g saturated), 41 mg cholesterol, 92 mg sodium, 5 g fiber.
Rhubarb & Strawberry Galette
Makes 1 galette Serves 8 to 10
San Francisco's Destination Baking Co. makes a strawberry galette for Mission Pie but declined to share the recipe. This rhubarb and strawberry galette is a variation on the theme, inspired by the Rhubarb and Strawberry Pie in "Chez Panisse Desserts," by Lindsey Remolif Shere (Random House, 1985). Note that you need parchment paper for rolling out the dough.
The Dough
2 cups all-purpose flour
3/4 teaspoons kosher salt
15 tablespoons chilled unsalted butter, in small pieces
1/4 cup ice water
The Filling
Approximately 1 1/4 pounds rhubarb
1 pint strawberries
1 cup sugar
2 1/2 tablespoons all-purpose flour
1 egg yolk whisked with 1 teaspoon water
Coarse decorating sugar
To make the dough: Put the flour and salt in a food processor and pulse to blend. Add the butter and pulse just until all the butter pieces are coated with flour and are about the size of large peas. Do not overblend.
Transfer to a bowl and stir in the water with a fork, mixing until the dough just begins to come together. With one hand, knead it briefly, just until you can collect the dough in a single shaggy mass. Resist the temptation to add more water. Wrap the dough in plastic wrap and press it into the shape of a large round hamburger patty. Refrigerate at least 2 hours.
Preheat the oven to 425°. If you have a baking stone, put it on a rack in the lower third of the oven and preheat for 30 minutes.
Remove the galette dough from the refrigerator and let it soften for about 15 minutes so it will be easier to roll.
To make the filling: While the galette dough softens, trim the ends of the rhubarb stalks, then cut the stalks into 1/3-inch-wide slices. You should have about 3 3/4 cups. Hull the strawberries and chop medium-fine. In a large bowl, toss the rhubarb, strawberries, sugar and flour until well blended.
Roll the galette dough into a 15-inch circle between two large sheets of parchment paper. If the dough threatens to stick to the paper, lift off the paper and dust the dough lightly with flour. Replace the paper, flip the dough over and continue rolling.
Transfer the dough, still between sheets of parchment paper, to a pizza peel or rimless baking sheet. Remove the top sheet of paper. Top the dough with the filling, spreading it evenly but keeping it about 2 inches from the edge. Working quickly, slide a palette knife (the type used for frosting cakes) or a chef's knife under the edge of the dough and fold it over the filling to make a wide border all around. Be fastidious about patching any cracks in the dough or the filling will leak out.
Brush the rim of the dough with the egg wash (you may not need it all) and sprinkle the rim generously with coarse sugar. With scissors, trim away excess parchment paper. (It's OK if a little paper is exposed; it won't burn.)
If you are using a baking stone, slide the galette, still on parchment paper, directly onto the baking stone. If you are not using a baking stone, bake the galette on a rimless baking sheet.
Bake until the crust is well browned and the filling is bubbling, about 50 minutes. Slide the galette onto a rack to cool. Let cool for at least 30 minutes before slicing.
Per serving: 355 calories, 4 g protein, 45 g carbohydrate, 18 g fat (11 g saturated), 68 mg cholesterol, 166 mg sodium, 2 g fiber.


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Beth Chaim's annual gala helps support a "celebrating spiritual community." Come help bring light to the congregation inside and outside. Dinner, dancing, drinks & dessert plus raffles & a silent auction.
$75 per person.
To reserve a space and/or to learn more: 925.736.7146; https://bethchaim.ejoinme.org/LightTheWay.
Venue: Crow Canyon Country Club, Danville.


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This "natural blonde" bombshell is unafraid to take the perceived "lemons" of life and serve them up to your guest in an ice cold glass of lemonade in the form of laugh out loud jokes. Clean, clever, and relatable, Karen Mayer Cunningham has been bringing side-splitting comedy to crowds for thirty years.
In a culture seemingly ashamed by our mistakes, Karen puts hers on display disarming the hardest hearts. The truth of her humanity makes her relatable which also positioned her as a favorite in comedy competition, "Funniest Moms In America", and renowned TV show, America's Got Talent.


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"Consumers are making choices that have more positive impact on the world. When we think about the clothing of the future, it has to be sustainable, it has to be useful and it should be comfortable," —Nina Faulhaber, co-founder, Aday
Hear more from Nina. Buy your Sourcing Summit 2018 ticket today here.