text:
The Glycine-Phenylalanine-Rich Region Determines the Specificity of the Yeast Hsp40 Sis1
Wei Yan, Elizabeth A. Craig
Wei Yan
Department of Biomolecular Chemistry, University of Wisconsin—Madison, Madison, Wisconsin 53706
Elizabeth A. Craig
Hsp40s are ubiquitous, conserved proteins which function with molecular chaperones of the Hsp70 class. Sis1 is an essential Hsp40 of the cytosol of Saccharomyces cerevisiae, thought to be required for initiation of translation. We carried out a genetic analysis to determine the regions of Sis1 required to perform its key function(s). A C-terminal truncation of Sis1, removing 231 amino acids but retaining the N-terminal 121 amino acids encompassing the J domain and the glycine-phenylalanine-rich (G-F) region, was able to rescue the inviability of a Δsis1 strain. The yeast cytosol contains other Hsp40s, including Ydj1. To determine which regions carried the critical determinants of Sis1 function, we constructed chimeric genes containing portions of SIS1 and YDJ1. A chimera containing the J domain of Sis1 and the G-F region of Ydj1 could not rescue the lethality of the Δsis1 strain. However, a chimera with the J domain of Ydj1 and the G/F region of Sis1 could rescue the strain's lethality, indicating that the G-F region is a unique region required for the essential function of Sis1. However, a J domain is also required, as mutants expected to cause a disruption of the interaction of the J domain with Hsp70 are inviable. We conclude that the G-F region, previously thought only to be a linker or spacer region between the J domain and C-terminal regions of Hsp40s, is a critical determinant of Sis1 function.
Hsp70s and Hsp40s (DnaJs) are two families of molecular chaperones which work together in a variety of cellular processes, including protein translation, translocation, and folding, as well as in the regulation of the biological activity of signalling molecules (5). Hsp70s bind unfolded or partially folded polypeptides, preventing aggregation and assisting the folding of these polypeptide substrates (4, 14). The cycle of binding and release of substrate polypeptides to Hsp70 is nucleotide dependent. ATP-bound Hsp70 has a low affinity for substrates, while ADP-bound Hsp70 has a relatively high affinity (30). Therefore, the Hsp40s, which stimulate the weak intrinsic ATPase activity of Hsp70s, play critical roles in regulating substrate binding (23). Some Hsp40s also prevent aggregation by binding unfolded polypeptide substrates and therefore can be considered molecular chaperones in their own right (10). In some cases, Hsp40s may transfer bound substrates to Hsp70 (19).
Multiple Hsp40s have been discovered in both prokaryotic and eukaryotic cells. All contain a signature J domain of about 70 amino acids. Genetic and biochemical evidence indicates that the J domain of Hsp40s interacts with the ATPase domain of Hsp70 (9, 18). The structures of the J domains of Escherichia coli DnaJ and Hdj1, a mammalian Hsp40, have been solved by nuclear magnetic resonance (NMR) (16, 25, 27). The tertiary structures of the two different J domains are remarkably similar even though there is only 54% sequence similarity between them. Both consist of four α-helices. Helix II and helix III are antiparallel. Hydrophobic residues on the interior faces of helices I, II, and III form a hydrophobic core which stabilizes the structure. Amino acids on the outer surface of helices II and III and in the loop between them, which contains the highly conserved HPD tripeptide, are thought to be important in determining the affinity and selectivity of the interaction between a particular J domain and its Hsp70 partner (13, 25). Mutations within the HPD tripeptide lead to a loss of both J domain function and interaction with Hsp70 (11, 12, 36,37, 39).
The Hsp40 class of proteins is divided into three subgroups based on the presence of conserved domains in addition to the J domain (9). Class I Hsp40s have a glycine-phenylalanine-rich (G-F) region adjacent to the N-terminal J domain, followed by a cysteine-rich region which forms a zinc finger motif and a poorly conserved C-terminal region. DnaJ of E. coli and Ydj1 ofSaccharomyces cerevisiae are class I Hsp40s. Class II Hsp40s, which include Sis1 of S. cerevisiae and Hdj1 of mammalian cells, lack the zinc finger motif. Class III Hsp40s lack both the G-F region and the zinc finger motif. Thus, the J domain is the only conserved structure among these Hsp40s. While the conserved J domain is involved in interactions with Hsp70s, the polypeptide binding site(s) has been located in the zinc finger and/or poorly conserved C-terminal regions of representatives of class I and II Hsp40s. So far, no class III Hsp40 has been shown to bind unfolded proteins. The function of the G-F regions of class I and II Hsp40s has not been established. It has been proposed that the G-F region is a flexible linker between the J domain and other regions of the type I and II Hsp40s (34) but may be important for interactions with Hsp70s (17, 38).
In the yeast S. cerevisiae, 16 Hsp40s have been identified by their sequence similarities to E. coli DnaJ. Although they have not all been analyzed, some have been localized to major cellular compartments: three in the endoplasmic reticulum (ER) (Sec63, Scj1, and Jem1), three in the mitochondria (Mdj1, Mdj2, and Jac1), and at least four in the cytosol (Ydj1, Sis1, Zuo1, and Djp1) (10, 15,35, 40, 41) (37a). This report focuses on the yeast cytosolic Hsp40 Sis1. Sis1 and Ydj1, another yeast cytosolic Hsp40, have similar biochemical properties in vitro. Both can stimulate the ATPase activity of the yeast cytosolic Hsp70 Ssa1, can bind unfolded polypeptides, and can function with Ssa1 to refold denatured luciferase (20). However, they appear to carry out different functions in vivo. Ydj1 has been implicated in the folding of proteins and the translocation of proteins into organelles (1, 3, 6, 21). Sis1, on the other hand, appears to be required for the initiation of translation (42). Overexpression of YDJ1 cannot suppress the lethal phenotype of the sis1 disruption mutant; overexpression of SIS1 can only suppress the slow-growth phenotype of the ydj1-null mutant at low temperatures (7, 22) and the translocation defect of a temperature-sensitive ydj1 mutant (6).
To understand the domain structure of Sis1 required for its essential function within the cell, we carried out a genetic analysis. The J domain and G-F region of Sis1 alone are sufficient to support cell growth. A functional J domain is required to maintain cell viability, but the J domain of Ydj1 can substitute. However, the G-F region of Sis1 is specifically required, as the G-F region of Ydj1 cannot substitute for it. We conclude that the unique specificity of some Hsp40s is determined by their G-F regions.
Yeast strains and culturing methods.The yeast strains used in this study were as follows: PJ43-2B (MATα, trp1-1 ura3-1 leu2-3,112 his3-11,15 ade2-1 can1-100 GAL2+ met2-Δ1 lys2-Δ2), PJ53 (a/α, rp1-1/trp1-1 ura3-1/ura3-1 leu2-3,112/leu2-3,112 his3-11,15/his3-11,15 ade2-1/ade2-1 can1-100/can1-100 GAL2+/GAL2+met2-Δ1/met2-Δ1 lys2-Δ2/lys2-Δ2), and WY26 (MATα,trp1-1 ura3-1 leu2-3,112 his3-11,15 ade2-1 can1-100 GAL2+ met2-Δ1 lys2-Δ2 Δsis1::LEU2)/pYW17 (SIS1-YCP50).
The Δsis1::LEU2 mutation in the yeast strain WY26 used in this study contains the LEU2 gene in place of sequence −13 to +1,056 bp of the SIS1 gene. ASmaI-NotI fragment containing the entireLEU2 gene was cloned into the BstZ17I site (13 bp upstream of the ATG of the SIS1 coding region) and theNotI site (introduced immediately before the stop codon) ofSIS1 to replace the entire SIS1 coding region. AnEcoRI-SalI fragment from this construct containing the upstream and downstream noncoding regions ofSIS1 gene with an inserted LEU2 gene in place of the coding region was transformed into the yeast strain PJ53 to make a heterozygous diploid Δsis1 strain by one-step gene replacement (28). This heterozygous diploid Δsis1 strain carrying a wild-type SIS1 gene in YCp50 plasmid (pYW17) was sporulated and dissected to obtain a haploid Δsis1::LEU2 strain (WY26) whose survival is maintained by the pYW17 plasmid.
Yeast cultures were grown on either yeast extract-peptone-dextrose (YEPD) medium containing 2% glucose (31) or synthetic complete medium containing 5-fluoroorotic acid (5-FOA) (32). For drop test, cells were grown to an optical density at 600 nm (OD600) of 0.5 to 1.0. About 0.4 OD600 U of cells was resuspended in 5 ml of sterile water, and a 1:10 serial dilution was prepared. Seven microliters of each dilution was spotted on YEPD or 5-FOA plates which were then incubated at the temperatures indicated for the number of days indicated (see Fig. 1 and 5). To prepare cell lysates to analyze protein expression, about 1.5 OD600 U of cells was resuspended in 100 μl of 2× sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer to make cell lysates by beating with glass beads (2), and 12 μl of lysates was subjected to electrophoresis and immunoblot with the indicated antibodies.
PCR mutagenesis of SIS1 gene.ABamHI-SalI fragment of the SIS1 gene from plasmid CB371 (a generous gift from Kim Ardnt's lab) was cloned into YCp50 and used as a wild-type plasmid (pYW17, SIS1-YCP50). This fragment was also cloned into plasmid pRS314 (33) to be used as the backbone plasmid (pYW65, SIS1-pRS314) to constructsis1 mutants. An error-prone PCR was carried out in buffer A (10 mM Tris-HCl [pH 9], 1.5 mM MgCl2, 25 mM KCl, and 0.1 mg of gelatin per ml) to synthesize a NsiI-SalI fragment of the SIS1 gene. This mutant PCR fragment was cloned into the pYW65 plasmid to replace the wild-typeNsiI-SalI fragment and was transformed into the Δsis1 strain WY26 which carries SIS1-YCP50. Transformants were screened for temperature-sensitive alleles of the SIS1gene. Three mutants which grew slightly more slowly than the wild-type strain at 38.5°C were obtained from this screen. Sequencing of these mutated genes indicated that they encode three C-terminal truncated fragments of Sis1 containing the first 297, 262, and 206 amino acids, respectively.
To construct truncations 1–68, 1–121, and 1–172, a NotI site was introduced by PCR immediately after amino acid 68, 121, or 172, respectively. Taking advantage of another NotI site which was introduced immediately before the stop codon (see above),NotI fragments containing amino acids 69 to 352, 122 to 352, or 173 to 352 of Sis1 were removed to construct the truncations 1–68, 1–121, and 1–172, respectively. Consequently, truncations 1–68 and 1–172 encode the first 68 and 172 amino acids of Sis1, respectively, plus two extra amino acids (Gly and Arg) at the C terminus, and truncation 1–121 (SS) encodes the first 121 amino acid of Sis1 plus an Arg residue at the C terminus. A similar strategy was used to construct the internal deletion Δ71–121; a XhoI site was introduced immediately prior to amino acid 122. Then a XhoI fragment (from the native XhoI site of the SIS1 gene at codon 69 to this introduced XhoI site) containing amino acids 71 to 121 was removed by molecular cloning to construct a Sis1 protein carrying an internal deletion of the entire G-F region.
Two-point mutants of SIS1, H34Q, and HPD34-36AAA were constructed by site-directed PCR mutagenesis and were cloned into the pRS314 plasmid. The mutated sequences were synthesized in the oligonucleotide primers and were incorporated into the context of theSIS1 gene.
Construction of Sis1-Ydj1 chimeras.A Ydj1 truncation which encodes the first 104 amino acids of YDJ1 (YY) was kindly provided by Jill Johnson of this laboratory. Based on the YY construct and the SS (1–121) construct described above, chimeric genes encoding the J domain of Sis1 and the G-F region of Ydj1 (SY) and the J domain of Ydj1 and the G-F region of Sis1 (YS) were constructed. To create these chimeras, a BclI site was introduced intoYDJ1 gene (at the end of the J domain) without changing the amino acid sequence by substituting the C residue (+200 bp, A of ATG is counted as 1) with T through PCR to allow swapping the G-F region between SS (containing a native BclI site at the analogous position) and YY at the BclI site. To compare expression levels of YS and YY constructs using antibody against the same epitope, an NcoI site was introduced by PCR at the ATG of either YS or YY to allow addition of an Xpress and polyhistidine tag (XbaI-NcoI fragment of the pRSET B vector; Invitrogen Co., Carlsbad, Calif.) to the N terminus of the YS or YY coding region (encompassed in an NcoI-SalI fragment). This N-terminally tagged YS or YY (XbaI-NcoI-SalI fragment) region was then cloned into the SpeI and SalI sites of the p414GPD vector under the control of the glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter (24). To express the N-terminally tagged YS construction under the control of different promoters, such as the promoters of translation elongation factor 1α (TEF) and alcohol dehydrogenase 1 (ADH), a XbaI-SalI fragment from the above YS-p414GPDHisB construct was moved into p414TEF or p414ADH vectors at the SpeI and SalI sites. Similarly, aXbaI-SalI fragment from the YY-p414GPDHisB was cloned into the SpeI-SalI site of the multicopy 2μm version of the GPD vector, p424GPD, to overexpress the N-terminally tagged YY.
To construct the YYS fusion, an EcoRV site was introduced by PCR into the SIS1 gene close to the end of the G-F region, thereby replacing asparagine 113 with aspartic acid. This construct can rescue the lethal phenotype of the Δsis1 strain as well as the wild-type SIS1 (data not shown). Using this introducedEcoRV site, the glycine-methionine-rich (G-M) C-terminal region of Sis1 (EcoRV-SalI fragment of thisSIS1 construct) was fused to the C terminus of the Ydj1 G-F region (a SacI-EcoRV fragment of YDJ1containing the promoter, J domain, and G-F region of Ydj1) to make a YYS fusion. To make the truncated fusion YYS-292, YYS-262 and YYS-206, the wild-type parts of the YYS construction, were replaced with theNsiI-SalI fragments of SIS1 containing these sis1 truncation mutations.
Protein purification and antibody generation.Ydj1 was purified as described previously from a T7 expression construct inE. coli (43). Sis1, a generous gift from Tara Beck of this laboratory, was expressed from a T7 expression construct (pET11a-SIS1) in E. coli and was purified by chromatographic separations with Q-Sepharose and hydroxyapatite. Purified Ydj1 and Sis1 were used as immunogens to raise the count of polyclonal antibodies in rabbits.
Quantitation of protein expression by immunoblot.Yeast lysates from wild-type strain PJ43-2B were prepared as described previously (26). Serial dilutions of purified Ydj1 and Sis1 proteins were used as standards to determine the relative protein levels in the lysate. The cell lysate and purified proteins were run on SDS-PAGE, were transferred to nitrocellulose (Hybond-C; Amersham Corp., Arlington Heights, Ill.), and were immunoblotted for Ydj1 and Sis1 using the ECL detection kit (Amersham). Exposed film (BioMax-AR; Eastman Kodak Co., Rochester, N.Y.) was densitometrically analyzed using Ofoto (Light Source Computer Images, Inc.) and Scan Analysis (Biosoft) software packages. Quantification of the cellular amount of Ydj1 or Sis1 in the cell lysate was achieved by comparison of the densitometric signals from the serial dilutions of purified proteins to those from the cell lysate.
Analysis of domains of Sis1 required for essential functions.To determine the sequences of Sis1 which play critical roles in its function, a series of C-terminal truncation mutants of SIS1were isolated (Fig. 1A and Materials and Methods). These constructs were transformed into strain WY26, which contains a complete deletion of the SIS1 coding region on the chromosome (Δsis1::LEU2) and carries a wild-type SIS1 gene on a low-copy-number centromeric plasmid harboring the URA3 gene (SIS1-YCp50) to allow cell viability. To test whether these Sis1 truncations could substitute for the wild-type Sis1, the transformants were spotted onto plates containing 5-FOA to select for cells having lost the URA3-based plasmid carrying the wild-type SIS1 gene (32). As can be seen in Fig.1A, all of the truncations rescued the Δsis1 strain on 5-FOA plates with the exception of the shortest truncation, containing the first 68 amino acids of the protein. This 68-amino-acid fragment encompassing only the J domain is expressed as a stable protein (data not shown). The ability of all but one of the constructs to rescue indicates that much of the C terminus of Sis1 can be deleted, and the remaining fragments still retain sufficient activity to support growth.
Growth phenotype of sis1 truncation mutants. (A) Diagram of Sis1 truncations used in this study and their ability to rescue the lethal phenotype of the Δsis1 strain. All of the sis1-truncated mutants were cloned into the pRS314 plasmid and transformed into the strain WY26. Transformed cells were grown and spotted onto 5-FOA plates (see Materials and Methods) and were incubated at 30°C for 3 days. (B) Immunodetection of mutant Sis1 proteins. Cells surviving on the 5-FOA plates shown in A were grown in YEPD medium. Cell lysates were prepared as described in Materials and Methods and were subjected to SDS-PAGE and immunoblotted using a Sis1-specific antibody. (C) Cell cultures used in panel B were diluted and spotted on YEPD plates. Plates were incubated at the indicated temperatures for 2 days.
Those transformants which were viable on 5-FOA plates were analyzed by immunoblotting to ensure that the observed growth was caused by truncated Sis1 proteins rather than by the presence of any full-length Sis1 protein resulting from plasmid recombination (Fig. 1B). All truncation mutants showed bands of the expected size and lacked detectable levels of the wild-type protein. These cells were also tested for their ability to grow at a variety of temperatures on nutrient-rich media (Fig. 1C). Strains containing truncations 1–297, 1–262, 1–206, and 1–171, all of which lack part or all of the C-terminal region, grew as well as the wild-type strain at 30°C, and nearly as well as the wild type at 37°C. These results suggest that the C-terminal region (amino acids 172 to 353) of Sis1 may not play an essential role for cell survival, at least under normal laboratory conditions. Interestingly, the strain containing a truncated Sis1 (1–121), retaining only the J domain and the G-F region, allowed growth of the Δsis1 strain at 30 and 37°C, even though such cells grew somewhat more slowly than the wild type. Therefore, we conclude that the J domain and the G-F region of Sis1 are sufficient to carry out the essential function of Sis1 at optimal growth temperatures.
The G-F region of Sis1 is specifically required for its essential function.The lethal phenotype of the Δsis1 strain can be suppressed by a fragment containing the J domain and the G-F region of Sis1 (Fig. 1C) but not by overexpression of Ydj1 (22) (data not shown). We investigated why Ydj1, which also contains a similar J domain and G-F region, cannot substitute for the absence of Sis1. We reasoned that differential localization caused by the farnesylation signal at the C-terminal end of Ydj1 which results in a portion of Ydj1 being associated with the membrane might be responsible (8). A ydj1 mutant,ydj1-C406S, in which the farnesylation signal CASQ has been changed to SASQ has been shown to have reduced membrane association (8). To test if the inability to rescue Δsis1was due to farnesylation, ydj1-C406S (a gift from Avrom J. Caplan) was transformed into the Δsis1 strain. Thisydj1 mutant was unable to support cell growth (data not shown), suggesting the targeting of the membrane of Ydj1 caused by farnesylation does not prevent Ydj1 from substituting for Sis1.
We then tested if the J domain and G-F region of Ydj1 lacking the C-terminal regions could suppress the lethality of the Δsis1 strain. The Ydj1 truncation containing only the J domain and the G-F region (YY, amino acids 1 to 104) was transformed into the Δsis1 strain (Fig.2A). The YY truncation did not rescue the Δsis1 strain. This YY construction made functional protein as it rescued the slow-growth phenotype of the Δydj1strain (16a). These results suggest that a unique structure within the J domain and/or the G-F region of Sis1, which cannot be replaced by the analogous domains of Ydj1, is required to rescue the lethality of the Δsis1 strain.
Evidence for the importance of the G-F region of Sis1. (A) Truncated fusion constructs containing various combinations of the J domain and G-F region from either Ydj1 or Sis1 are shown in the diagram. The capitalized S or Y stands for domains from Sis1 and Ydj1, respectively. The s or y in parentheses indicates the individual promoter of each construct as being from either SIS1 orYDJ1, respectively. Cell cultures were diluted, spotted on 5-FOA plates, and incubated at 30°C for 4 days. (B) The YS or YY construct was cloned into either a low copy (CEN) or multiple-copy (2μm) plasmid under the control of the GPD, TEF, or ADH promoters to express the proteins tagged with Xpress epitope at different levels. Drop tests were performed as described in panel A using cells carrying these constructs. (C) Cell lysates from cells shown in panel B were separated by SDS-PAGE and were immunoblotted by using the anti-Xpress antibody.
To further dissect whether the J domain or the G-F region from Sis1 is important for the essential function, two fusion proteins were constructed: one in which the J domain from Sis1 was fused to the G-F region of Ydj1 (SY) and one in which the J domain from Ydj1 was fused to the G-F region of Sis1 (YS) (Fig. 2A). These two fusion constructions (YS and SY) were transformed in the Δsis1strain to test their ability to provide Sis1 function. YS was able to rescue as well as SS, while neither SY nor YY permitted growth (Fig.2A). This result suggests that it is the G-F region from Sis1 which is specifically required for that protein's essential function and that the J domain is not responsible for the functional specificity.
Differences in the extent of lethality suppression by the chimeric proteins could possibility be attributed to different expression levels. To eliminate this possibility, we wanted to compare the levels of chimeric proteins in the transformants. Unfortunately, available antibodies against either Sis1 or Ydj1 do not recognize the YS-YY pair equally and thus could not be used for this purpose. To circumvent this problem, the N termini of both YS and YY were tagged with an Xpress epitope (Invitrogen Co.) so that protein expression levels could be detected using anti-Xpress antibody for both proteins without epitope bias. The N-terminally tagged YS and YY were cloned into either low-copy-number (CEN) or multiple-copy (2-μm) plasmids under control of the GPD, TEF, or ADH promoters to allow differential expression levels of each construction. All YS constructs were able to rescue growth of Δsis1 cells (Fig. 2B). However, YY was unable to rescue even when expressed at a higher level than YS (compare lanes 3 and 5 in Fig. 2C). We conclude that the differential abilities of YS and YY to sustain growth of Δsis1 cells represent a functional difference between the two proteins and not merely different levels of expression.
Since overexpression of Sis1 partly suppresses the slow-growth phenotype of Δydj1 cells, but overexpression of Ydj1 does not allow growth of Δsis1 cells at any temperature (7, 22), we wanted to compare the normal levels of expression of these two Hsp40s in wild-type cells. The relative amounts of Ydj1 and Sis1 were determined by immunoblot analysis by using serial dilutions of cell extracts, purified Ydj1, and purified Sis1 as described in Materials and Methods. Ydj1 and Sis1 were detected by immunoblot analysis using antibodies specific to Ydj1 or Sis1, respectively. By comparing the signal from cell extracts with that from purified proteins, we determined the relative amounts of Ydj1 and Sis1 in cell extracts. The results of one experiment are shown in Fig.3. From the results of a number of experiments, we calculated that Ydj1 is between 10 and 15 times more abundant than Sis1 inside the cell. The fact that the expression of Ydj1 is higher than the expression of Sis1 in vivo, together with the fact that the loss of the farnesylation signal does not affect the rescue of the Δsis1 strain, supports the idea that Sis1 is functionally distinct from Ydj1.
Determination of the relative levels of Sis1 and Ydj1 in wild-type cells. Wild-type cells (PJ43-2B) were grown at 30°C in YEPD media to an OD600 of 0.5. Approximately 0.3 OD600 U of cells was collected, and 200-μl cell lysates were made from these cells. Ten microliters of these cell lysates and the indicated amounts of purified Sis1 and Ydj1 were run on SDS-PAGE and immunoblotted with anti-Sis1 and anti-Ydj1 antibodies. Immunoblot signals from cell lysates were compared with those from the predetermined amounts of Sis1 or Ydj1. The cell lysate was calculated to contain about 19 fmol of Sis1 and 285 fmol of Ydj1.
J domain function is required for the rescue of the Δsis1 strain.Since constructs containing the J domains from either Sis1 or Ydj1 are equally capable of rescuing aSIS1 deletion, we tested whether J domain function is required for Sis1 activity. Two different point mutations, H34Q and HPD34-36AAA, were introduced into the region of SIS1encoding the highly conserved HPD loop region. Similar mutations in several Hsp40 homologues such, as DnaJ, Ydj1, Sec63, and the large T antigen of simian virus 40, have been shown to disrupt the J domain functions (11, 12, 36, 37, 39). Plasmids containing H34Q or HPD34-36AAA in the context of either the SS truncation or full-length Sis1 were transformed into the Δsis1 strain. None of the constructs were able to rescue the lethality of the Δsis1strain (Fig. 4), even though the mutant genes expressed stable proteins (data not shown). We conclude that J domain function is required for Sis1 activity.
Amino acid changes in the HPD loop disrupt Sis1 function. Diagram of sis1 point mutations H34Q (single star) and HPD34-36AAA (triple stars) in truncated (amino acids 1 to 121) or full-length Sis1 (WT). WY26 cells containing these mutant constructs were diluted and spotted on 5-FOA plates; the plates were incubated at 30°C for 2 days (truncation constructs) and 3 days (full-length constructs), respectively.
Function of the G-F region is redundant to the functions of the G-M and C-terminal regions.Having determined that the essential Sis1 function requires a functional J domain and G-F region, we wanted to investigate the roles of the G-M and the C-terminal regions for Sis1 function. Since the N-terminal J domain is absolutely required for Sis1 function, we made a deletion mutant in which the segment encoding the G-F region was deleted, but the reading frame was maintained, and we tested the ability of this deletion mutant to support cell growth. This construct (S_S) rescued the Δsis1 strain as well as wild-type Sis1 (Fig. 5A). This rescue indicates that the G-M region and the C-terminal region from Sis1 can substitute for the G-F region of Sis1 to maintain cell survival in the absence of wild-type Sis1. Consistent with this result, a chimeric fusion protein, YYS, in which the J domain and the G-F region from Ydj1 were fused to the G-M and C-terminal regions of Sis1, efficiently rescued growth of the Δsis1 strain (Fig. 5A).
The G-F region is dispensable in the context of the full-length protein. (A) Diagram of the G-F region-deletedsis1 mutant SXS and YYS chimeric fusion. Cells carrying these constructs were created for drop test on 5-FOA plates and were incubated at 30°C for 3 days. (B) Full-length and truncated YYS chimeric fusions are shown in the diagram. A similar drop test was performed with cells containing these constructs, and cells were spotted onto YEPD plates which were incubated at the indicated temperatures for 3 days.
The above results suggest a functional redundancy between the G-F region and the C-terminal 231 amino acids of Sis1. To investigate which sequences within this C-terminal 231 amino acids are minimally required to duplicate the function of the G-F region, we introduced oursis1 C-terminal truncated mutations into the YYS chimeric construct (Fig. 5B). Among the four truncated fusion genes, only YYS-206, YYS-262, and YYS-297 expressed stable proteins (data not shown). As shown in Fig. 5B, these three truncated fusion proteins each allowed growth of the Δsis1 strain. The smallest truncation, YYS-206, which removes amino acids 207 to 352 of the C terminus, allowed wild-type growth rates, while growth of transformants carrying the longer fusions grew more slowly at 37°C. We conclude that the region from amino acid 122 to 206, which includes the G-M region and part of the C-terminal region, is redundant with the G-F region for the essential function of Sis1.
In this study, we have analyzed the domains of Sis1 needed for its essential house-keeping roles. Analysis of mutants encoding amino acid substitutions showed the Sis1 J domain to be essential, as is the case with many Hsp40s which have been tested. Our data showing that the J domain of Sis1 plays a critical role is consistent with the previously published observation that a 22-amino-acid deletion within the J domain of Sis1 which includes the conserved HPD sequence resulted in a lethal phenotype (22). Since the J domain has been shown to be the interactive site with the ATPase domain of Hsp70s, these results suggest that the site of interaction of Sis1 with an Hsp70 is essential for its in vivo function. Although the J domain is critical for Sis1 to support cell growth, it is not necessary that the J domain be from Sis1. The J domain from Ydj1 can provide the same essential function, indicating that the J domain structures of these two cytosolic Hsp40s are functionally similar.
As is the case with the J domains of cytosolic Ydj1 and Sis1, the J domains of two ER Hsp40s, Sec63 and Scj1, were found to be interchangeable. However, the J domain of Sis1 could not substitute for the J domain of Sec63 (29). Therefore, it was proposed that the specificity of J domain interaction with Hsp70s is determined by the amino acids on the outside of helices II and III of the J domain, as they were very similar between these two Hsp40s of the ER (9,25, 29). The Sis1 J domain was able to substitute if three amino acids on the surfaces of helix II or III were altered to more closely resemble those of the ER J domains (29). After modeling structures of Sis1 and Ydj1, we compared the amino acids on the outer surfaces of Sis1 and Ydj1 helices II and III. We found that the sequences of Sis1 and Ydj1 at these locations are more similar to each other than to other Hsp40s (Fig. 6A). Whether these similarities between the Hsp40s of the same cellular compartment (Sec63/Scj1 and Sis1/Ydj1) are simply the result of the concerted evolution of Hsp70s and Hsp40s that function together or whether these similarities play a more critical role is not known at this time.
Sequence alignment of the J domains and G-F region. (A) Alignment of the sequences of the J domains of DnaJ, Hdj1, and Hsp40s of S. cerevisiae carried out by the Jotun-Hein method using MegAlign software of the DNA* program package (DNASTAR Inc., Madison, Wis.). Assignments of the helices was based on the NMR structures of Hdj1 (1HDJ) and DnaJ (1XBL) in the PDB from the Brookhaven National Laboratory. The asterisks indicate residues predicted to be on the outside of helix II and III. The residues of Ydj1 and Sis1 were marked with asterisks based on the predicted structures of the proteins modeled from the DnaJ structure using SYBYL molecular modeling software (Tripos Inc., St. Louis, Mo.). The three amino acids of Sis1 which, when replaced with the corresponding residues of Sec63, enable this J domain to substitute for the J domain of Sec63 are underlined. (B) The entire amino acid sequences of Sis1 and Ydj1 were aligned and compared by the same alignment method described for panel A. Only the alignment of the G-F region is shown.
While the J domain is critical and interchangeable between Sis1 and Ydj1, our results indicate that it is the G-F region that differentiates Sis1 from Ydj1. The minimal 121-amino-acid fragment of Sis1 containing only the G-F region and the J domain allows nearly wild-type growth rates at 30°C. The G-F region of Sis1 cannot be replaced by the G-F region of Ydj1, even when such a fusion is expressed at levels higher than the analogous Sis1 fragment. This is a surprising result, as no specific role has been assigned to the G-F region. It has been suggested that the G-F region serves as a linker or spacer region between the J domain and the more C-terminal regions of type I and II Hsp40s (17, 34). However, our results show that in the case of Sis1 the G-F region plays a much more fundamental role in the absence of C-terminal sequences, as it is specifically required.
Recently published results suggest that the interaction of the J domain of Sec63 with the ER Hsp70 BiP broadens the range of peptides which stably bind to BiP and may determine the peptide binding specificity of BiP (23). The G-F region may affect substrate binding of an Hsp70 by directly contacting Hsp70 or by regulating the conformation of the J domain. Recently Huang et al. showed that the NMR structure of the J domain in DnaJ is affected by the G-F region (16). It is possible that different Hsp40 partners are able to differentially affect the specificity of Hsp70 interaction with substrates by causing slightly altered conformations which in turn affect substrate binding affinities. In this regard, it is interesting that the only defect observed for a DnaJ mutant protein deleted for the G-F region was a defect in the targeting of the ς32 protein substrate to Hsp70/DnaK (38).
Previously reported data suggests that both Ydj1 and Sis1 may function with the cytosolic chaperone Ssa. Both are able to stimulate its ATPase activity. Both Sis1 and Ssa are involved in initiation of translation (15a, 42), while Ydj1 and Ssa play roles in protein translocation and folding (1, 3, 6, 21). Specificity of those roles in which Ssa functions could be determined by the Hsp40 partner. Such specificity could be conveyed by structural difference between Sis1 and Ydj1 within the G-F region. Comparison of the G-F sequences between these two Hsp40s indicates that there are two stretches of sequences (of 12 and 11 amino acids, respectively) in the Sis1 G-F region which are absent in the Ydj1 G-F region (Fig. 6B). These two extra stretches, which contain amino acids other than glycine and phenylalanine, might be responsible for the functional difference between the G-F regions of these two Hsp40s.
While the G-F region in conjunction with the J domain is sufficient to supply essential Sis1 function, the role of the G-F region of Sis1 can be carried out by more C-terminal sequences, as indicated by the ability of a protein containing a deletion of the G-F region or replacement of the Sis1 G-F region with the Ydj1 G-F region to allow growth of Δsis1 cells. This functionally redundant region was narrowed down to the segment between amino acids 122 and 206. This region includes the G-M region, which is encompassed within amino acids 122 to 171, and part of the more C-terminal region. Since both the G-F and G-M regions are rich in glycine, we suspect that the G-M region itself is sufficient. However, we were unable to test this idea directly, as the fusion protein containing the J domain and G-F region of Ydj1 linked directly to amino acids 122 to 171 of Sis1 was unstable.
The function of the most C-terminal segment of Sis1 remains unclear. Cells containing a truncated Sis1 in which the entire region between amino acids 207 and 352 has been deleted grow as well as wild-type cells at both 30 and 37°C (as demonstrated by the growth of truncation 1-206 shown in Fig. 1C and the growth of YYS-206 shown in Fig. 5B). However, removing part of the sequence from amino acid 207 to 352, as indicated by truncation YYS-262 and truncations 1-297 and YYS-297, resulted in a slow growth at 37°C (Fig. 1C, 5B). The growth defect caused by these truncations is consistent with previously published results. Four sis1 mutants, including the mutantsis1-85 which has been used for all the genetic analysis of Sis1 function, each having a 22-amino-acid deletion within this region, have either lethal or severe temperature-sensitive phenotypes (22). It is possible that the four internal deletions as well as our partial truncations within this region from amino acid 207 to 353 may produce structural alterations which affect the ability of Sis1 to function normally, while deletion of the entire region is not problematic.
In any case, our analysis of the deletions indicates that the C-terminal region is not required for robust growth under the conditions tested. Interestingly, the C-terminal region of Sis1 containing amino acids 171 to 353 of Sis1 is able to bind unfolded proteins (20). Together with the fact that the 1-172 truncation is able to perform the critical functions of Sis1, this result suggests that the ability to bind unfolded proteins is not an essential aspect of Sis1 function. Although it is still formally possible that the G-F region can bind unfolded polypeptides, to our knowledge no indication of such a function exists.
In summary, we suggest that the J domain and G-F region of Hsp40s of classes I and II may be sufficient for the basic functions of DnaJs in vivo, while the more C-terminal regions are important under suboptimal growth conditions (for example, at the upper limits of the temperature range allowing growth). This idea is supported by the data presented here. In addition, a Ydj1 truncation containing only the J domain and the G-F supports normal growth rates of a Δydj1 strain at 30°C (16a). A similar truncation of DnaJ also permits λ phage replication, albeit at a reduced efficiency (38). The challenge will be to determine the mechanistic role of the G-F region in determining the specificity of its function.
We thank J. Johnson, C. Pfund, and N. Lopez for critical reading of the manuscript, Tara Beck for providing antibodies to Sis1 and Ydj1 and purified Sis1 and Ydj1 proteins, A. Caplan for providingydj1 mutant C406S, J. Johnson for providing the Ydj1 truncation, and K. Arndt for providing a wild-type SIS1plasmid.
This work was supported by NIH grant 5RO1 GM31107 to E.A.C.
Received 14 June 1999.
Returned for modification 28 July 1999.
Accepted 10 August 1999.
Atencio D. P.,
Yaffe M. P.
(1992) MAS5, a yeast homolog of DnaJ involved in mitochondrial protein import. Mol. Cell. Biol. 12:283–291.
Ausubel F.,
Brent R.,
Kingston R.,
Moore D.,
Seidman J. G.,
Smith J.,
Struhl K.
(1997) Current protocols in molecular biology. (John Wiley and Sons, New York, N.Y).
Becker J.,
Walter W.,
Yan W.,
Craig E. A.
(1996) Functional interaction of cytosolic Hsp70 and DnaJ-related protein, Ydj1p, in protein translocation in vivo. Mol. Cell. Biol. 16:4378–4386.
Buchberger A.,
Reinstein J.,
Bukau B.
(1999) The DnaK chaperone system: mechanism and comparison with other Hsp70 systems. in Molecular chaperones and folding catalysts: regulation, cellular function and mechanisms. ed Bukau B. (Harwood Academic Publishers, Amsterdam, The Netherlands), pp 609–635.
Bukau B.,
Schmid F. X.,
Buchner J.
(1999) Assisted protein folding. in Molecular chaperones and folding catalysts: regulation, cellular function and mechanisms. ed Bukau B. (Harwood Academic Publishers, Amsterdam, The Netherlands), pp 3–10.
Caplan A. J.,
Cyr D. M.,
Douglas M. G.
(1992) YDJ1p facilitates polypeptide translocation across different intracellular membranes by a conserved mechanism. Cell 71:1143–1155.
(1991) Characterization of YDJ1: a yeast homologue of the bacterial DnaJ protein. J. Cell Biol. 114:609–621.
Tsai J.,
Casey P. J.,
(1992) Farnesylation of YDJ1p is required for function at elevated growth temperatures in S. cerevisiae. J. Biol. Chem. 267:18890–18895.
Cheetham M.,
Caplan A.
(1998) Structure, function and evolution of DnaJ: conservation and adaption of chaperone function. Cell Stress Chaperones 3:28–36.
Craig E.,
(1999) Genetic dissection of the Hsp70 chaperone system of yeast. in Molecular chaperones and folding catalysts: regulation, cellular function and mechanisms. ed Bukau B. (Harwood Academic Publishers, Amsterdam, The Netherlands), pp 139–162.
Dey B.,
Boschelli F.
(1996) The Ydj1 molecular chaperone facilitates formation of active p60v-src in yeast. Mol. Biol. Cell 7:91–100.
Feldheim D.,
Rothblatt J.,
Schekman R.
(1992) Topology and functional domains of Sec63p, an endoplasmic reticulum membrane protein required for secretory protein translocation. Mol. Cell. Biol. 12:3288–3296.
Greene M.,
Maskos K.,
Landry S.
(1998) Role of the J-domain in the cooperation of Hsp40 with Hsp70. Proc. Natl. Acad. Sci. USA 95:6108–6113.
Hartl F. U.
(1996) Molecular chaperones in cellular protein folding. Nature 381:571–580.
Hettema E. H.,
Ruigrok C. C. M.,
Koerkamp M. G.,
v. d. Berg M.,
Tabak H. F.,
Distel B.,
Braakman I.
(1998) The cytosolic DnaJ-like protein Djp1p is involved specifically in peroxisomal protein import. J. Cell Biol. 142:421–434.
15a.↵
Horton, L., and J. Hensold. Personal communication.
Huang K.,
Flanagan J. M.,
Prestegard J. H.
(1999) The influence of C-terminal extension on the structure of the "J-domain" in E. coli DnaJ. Protein Sci. 8:203–214.
Johnson, J., and E. Craig. Unpublished data.
Karzai A. W.,
McMacken R.
(1996) A bipartite signaling mechanism involved in DnaJ-mediated activation of the Escherichia coli DnaK protein. J. Biol. Chem. 271:11236–11246.
Kelley W. L.
(1998) The J-domain family and the recruitment of chaperone power. Trends Biochem. 23:222–227.
Laufen T.,
Mayer M.,
Beisel C.,
Klostermeier D.,
Mogk A.,
(1999) Mechanism of regulation of Hsp70 chaperones by DnaJ co-chaperones. Proc. Natl. Acad. Sci. USA 96:5452–5457.
Lu Z.,
Cyr D.
(1998) Protein folding activity of Hsp70 is modified differently by the Hsp40 co-chaperones Sis1 and Ydj1. J. Biol. Chem. 273:27824–27830.
Cyr D. M.
(1998) The conserved carboxyl terminus and zinc finger-like domain of the co-chaperone Ydj1 assist Hsp70 in protein folding. J. Biol. Chem. 273:5970–5978.
Luke M.,
Suttin A.,
Arndt K.
(1991) Characterization of SIS1, a Saccharomyces cerevisiae homologue of bacterial dnaJ proteins. J. Cell Biol. 114:623–638.
Misselwitz B.,
Staeck O.,
Rapoport T.
(1998) J proteins catalytically activate Hsp70 molecules to trap a wide range of peptide sequences. Mol. Cell 2:593–603.
Mumberg D.,
Muller R.,
Funk M.
(1995) Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene 156:119–122.
Pellecchia M.,
Szyperski T.,
Wall D.,
Georgopoulos C.,
Wuthrich K.
(1996) NMR structure of the J-domain and the Gly/Phe-rich region of the Escherichia coli DnaJ chaperone. J. Mol. Biol. 260:236–250.
Pfund C.,
Lopez-Hoyo N.,
Ziegelhoffer T.,
Schilke B. A.,
Lopez-Buesa P.,
Walter W. A.,
Wiedmann M.,
(1998) The molecular chaperone SSB from S. cerevisiae is a component of the ribosome-nascent chain complex. EMBO J. 17:3981–3989.
Qian Y. Q.,
Patel D.,
Hartl F. U.,
McColl D. J.
(1996) Nuclear magnetic resonance solution structure of the human Hsp40 (HDJ-1) J-domain. J. Mol. Biol. 260:224–235.
Rothstein R.
(1991) Targeting, disruption, replacement and allele rescue: intergrative DNA transformation in yeast. Methods Enzymol. 194:281–301.
Schlenstedt G.,
Harris S.,
Risse B.,
Lill R.,
Silver P. A.
(1995) A yeast DnaJ homologue, Scj1p, can function in the endoplasmic reticulum with BiP/Kar2p via a conserved domain that specifies interactions with Hsp70s. J. Cell Biol. 129:979–988.
Schmid D.,
Baici A.,
Gehring H.,
Christen P.
(1994) Kinetics of molecular chaperone action. Science 263:971–973.
Sherman F.,
Fink G. R.,
Hicks J. B.
(1986) Laboratory course manual for methods in yeast genetics. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y).
Sikorski R. S.,
Boeke J. D.
(1991) In vitro mutagenesis and plasmid shuffling: from cloned gene to mutant yeast. Methods Enzymol. 194:302–318.
Hieter P.
(1989) A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122:19–27.
Silver P. A.,
Way J.
(1993) Eukaryotic DnaJ homologs and the specificity of Hsp70 activity. Cell 74:5–6.
Strain J.,
Lorenz C. R.,
Bode J.,
Garland S.,
Smolen G. A.,
Ta D. T.,
Vickery L. E.,
Culotta V. C.
(1998) Suppressors of superoxide dismutase (SOD1) deficiency in Saccharomyces cerevisiae. J. Biol. Chem. 273:31138–31144.
Suh W.-C.,
Burkholder W.,
Lu C. Z.,
Zhao X.,
Gottesman M.,
Gross C.
(1998) Interaction of the Hsp70 molecular chaperone, DnaK, with its cochaperone DnaJ. Proc. Natl. Acad. Sci. USA 95:15223–15228.
(1996) A conserved HPD sequence of the J-domain is necessary for YDJ1 stimulation of Hsp70 ATPase activity at a site distinct from substrate binding. J. Biol. Chem. 271:9347–9354.
Voisine, C., and E. Craig. Unpublished results.
Zylicz M.,
Georgopoulos C.
(1995) The conserved G/F motif of the DnaJ chaperone is necessary for the activation of the substrate binding properties of the DnaK chaperone. J. Biol. Chem. 270:2139–2144.
(1994) The NH2-terminal 108 amino acids of the Escherichia coli DnaJ protein stimulate the ATPase activity of DnaK and are sufficient for lambda replication. J. Biol. Chem. 269:5446–5451.
Westermann B.,
Neupert W.
(1997) Mdj2p, a novel DnaJ homolog in the mitochondrial inner membrane of the yeast. J. Mol. Biol. 272:477–483.
Schilke B.,
Kim S.,
(1998) Zuotin, a ribosome-associated DnaJ molecular chaperone. EMBO J. 17:4809–4817.
Zhong T.,
Arndt K. T.
(1993) The yeast SIS1 protein, a DnaJ homolog, is required for initiation of translation. Cell 73:1175–1186.
(1995) The dissociation of ATP from hsp70 of Saccharomyces cerevisiae is stimulated by both Ydj1p and peptide substrates. J. Biol. Chem. 270:10412–10419.
Molecular and Cellular Biology Nov 1999, 19 (11) 7751-7758; DOI: 10.1128/MCB.19.11.7751
You are going to email the following The Glycine-Phenylalanine-Rich Region Determines the Specificity of the Yeast Hsp40 Sis1


text:
6 Most Underrated Fast-Food Orders, According to Chefs
There's no denying it—sometimes fast food really hits the spot. Whether your pick up a Big Mac meal on the way home from a long day at work or treat yourself to a couple of tacos or a stuffed burrito, fast food can be both delicious and satisfying. In fact, even chefs are known to seek out fast food from time to time.
You might think chefs only eat meals made at fancy restaurants or whip up their own specialties, but that's not always the case. Sometimes chefs just need something quick and satisfying so they turn to fast food, too.
From, cheesy In-N-Out burgers to healthy vegetarian burrito bowls and spicy tacos, here are 6 of the most underrated fast-food orders that chefs actually eat.
RELATED: 8 Fast-Food Chains That Never Freeze Their Burgers
In-N-Out's Classic Hamburger
In-N-Out, the famous Southwest burger chain, is a favorite among many food industry experts, including Jessica Randhawa, owner and head chef of The Forked Spoon.
"Easily the most underrated fast food burger is In-N-Out's classic hamburger or cheeseburger," says Randhawa. "With its consistently fresh and high-quality vegetables, the flavors from each topping do not overpower one another, leaving every bite wanting more."
Del Taco's Del Taco with Del Scorcho Hot Sauce
Kevin Templeton, executive chef of Barleymash and consultant for The Smoking Gun and Spill the Beans, is a fan of Del Taco, the fast-food chain that specializes in Mexican dishes. "I like their plain quesadillas, fries, and the Del Taco—an OG on the menu," Templeton says. Templeton shares that the Del Taco, which comes either crunchy or soft, is made up of seasoned beef, fresh house-grated cheddar, shredded lettuce, and diced tomatoes.
Although he's a fan of Del Taco's dishes, he also praises the chain's must-try hot sauce. "My favorite menu item is the Del Scorcho Hot Sauce," Templeton adds, "It's the best hot sauce to ever come in a packet, hands down."
Chipotle's Veggie Burrito Bowl
Who can say no to Chipotle? Toni Okamoto, cookbook author and founder of Plant-Based on a Budget, shares that when she is road-tripping or needs fast food after a long day at work Chipotle is her go-to. "It's incredibly vegan-friendly and a good budget-conscious option for the amount of food you get," Okamoto says.
Her usual order is a veggie burrito bowl with a side of two tortillas, extra rice, both black and pinto beans, extra fajitas, pico de gallo, corn salsa, lettuce, and guacamole on the side. "If I'm feeling fancy, I'll also get sofritas," Okamoto adds. "It's easily enough for two meals—and the best thing is that there are no upcharges for the extras I mentioned!"
Whataburger's Honey Butter Chicken Biscuit
Whataburger's Honey Butter Chicken Biscuit, the chain's honey-glazed chicken sandwich made with a warm biscuit, is a must-have for Chef Rafael Gonzalez. "It tastes just like how it sounds and hits the spot," Gonzalez mentions. "The first time I tried it was before donating blood," he adds. "They asked me to go back after eating something and Whataburger was the closest fast food."
Gonzalez points out that while Whataburger is open 24 hours a day, this delicious menu item is available from 11 p.m. to 11 a.m. "Perfect for late-night munchies or coating your stomach after a night out," Gonzalez says.
McDonald's Chicken Nuggets with a fountain Coke
Sometimes you can't go wrong with a tried and true McDonald's classic—chicken nuggets and, of course, a fountain soda. "I'm a sucker for McDonald's chicken nuggets, they've been my favorite since I was a kid," says Diana Manalang, chef and owner of Little Chef, Little Café in NYC. "There's something about them that's addicting," Manalang adds, "Maybe it's the nostalgic flavors or the BBQ sauce, but it is my go-to hangover cure or a 'having a terrible awful no-good day need a pick-me-up' meal."
Manalang shares that while the chicken nuggets are her favorite, she also never leaves the chain without picking up a fountain soda. "Always, always, always a fountain coke," Manalang says, "McDonald's has the best fountain Coca-Cola. Period. End."
RELATED: Video Shows Camel (Yes, a Camel) Ordering Fries at In-N-Out Drive-Thru
White Castle's Double Jalapeno Cheese Sliders
Since 1921, White Castle has been whipping up delicious sliders that have made quite an impression on people, including Adam Richman, TV food personality and host of History Channel's "Adam Eats the 80s" and "Modern Marvels."
"The dark horse I think might be the most highly underrated is the double jalapeño cheese slider from White Castle," says Richman. "This burger has enough umami to knock you on your butt and enough heat and spice to cut against the richness," he adds.
Richman shares that when the sliders come out of the oven fresh they melt in your mouth, just like a Krispy Kreme donut. "Do not sleep on the mighty Castle!" he says.
The post 6 Most Underrated Fast-Food Orders, According to Chefs appeared first on Eat This Not That.
Work in These States? Y

redpajama_set_name: RedPajamaCommonCrawl


Running a WordPress arcade can be very difficult especially if you want to use BuddyPress to start a community. Your primary issue when starting a site like this is overcoming spam. Spam comes in two main forms on a WordPress website comments and registration spam and there are solid ways you can go about limiting this (or even completely stopping it).
Captcha's are annoying they add a lot of resources to your front-end that hurt load time and they are a pain to integrate properly on your website (especially into ajax login windows). I personally don't like or use captcha's ever unless I need to and the reason is they are just a pain to both your users and yourself. While the latest version of Google's reCaptcha is as simple as a mouse click it's still distracting so what can we do?
Your second option is to install Askimet it will reduce the total amount of spam to near none the advantage here is Askimet is learning from millions of other websites virtually all spam will be blocked. If not you can use this in conjunction with the above comment blacklist rules.
Another interesting tip is to only allow comments from logged in users (which should reduce it to near 0 levels with the next plugin).
Registration spam is spam where there are bots logging into your site and are likely posting on a BuddyPress profile in order to generate backlinks to their website. The issue here is that they waste resources, are hurting your on-page SEO (for posting malicious links), and they are able to use this as a vector to launch attacks.
WordPress has no built-in way to prevent registration spam like the blacklist function for comments. To stop Registration Spam I recommend another free plugin WangGuard. Activate this plugin by signing up for an API key and your website registration spam should decrease dramatically (for me it goes down to 0). The plugin also has the ability to check currently registered users.
If you have 5k users and you are sure some of them are producing spam WangGuard can go through and check each of the users and will tell you the ones that are spammers and will delete them for you.
Spam is a difficult subject it's constantly changing and it's very difficult to check for it properly and there isn't much we can do from our end (theme-wise). There are tools such as honeypot, stop forum spam, etc, etc and you can check all of them but when you find generic spam that its brand new it's going to be near impossible for it to be detected. Remember to properly manage your arcade, don't enable registration if you aren't going to use something such as BuddyPress for it to be meaningful and continue to be an awesome webmaster!
I am trying re-captcha and it went well. lets see how it goes.


text:
Ijeoma Uchegbu is a Nigerian-British Professor of Pharmacy at University College London where she held the position of Pro-Vice Provost for Africa and the Middle East. She is the Chief Scientific Officer of Nanomerics, a pharmaceutical nanotechnology company specialising in drug delivery solutions for poorly water-soluble drugs, nucleic acids and peptides. She is also a Governor of the Wellcome, a large biomedical research charity.
Apart from her highly cited scientific research in Pharmaceutical Nanoscience, Uchegbu is also known for her work in science public engagement and equality and diversity in Science, Technology, Engineering and Mathematics (STEM).

Education and early career
Uchegbu grew up in Hackney and South East Nigeria. She studied pharmacy at the University of Benin, graduating in 1981, and earned her master's degree at the University of Lagos. She could not complete a PhD in Nigeria due to infrastructure difficulties. She moved back to the UK and completed her postgraduate studies at the University of London, graduating with a PhD in 1997. She was supervised by Alexander (Sandy) Florence, Dean of the School of Pharmacy. She was appointed a lecturer at the University of Strathclyde from 2002-2004.

Research
Uchegbu was made a Chair in Drug Delivery at the University of Strathclyde in 2002. Here she worked on polymer self-assembly, identifying materials that could form stable nanosystems. She demonstrated that polymer molecular weight could be used to control the size of vesicles. She joined University College London in 2006 as a Chair in Pharmaceutical Nanoscience at the School of Pharmacy. Uchegbu leads a research group that investigate molecular design and dosage of pharmaceuticals. She has designed polymers that self-assemble into nanoparticles with the appropriate properties to transport drugs. She explores how nanoparticles can be used for drug delivery. Uchegbu holds several patents for drug delivery, and biocompatible polymers. Her pharmaceuticals deliver genes and siRNA to tumours and peptides to the brain as well as encouraging the absorption of hydrophobic drugs using nanoparticles. She is exploring how nanomedicine can be used to treat brain tumours. In 2018 she was part of a £5.7 million Engineering and Physical Sciences Research Council grant, Raman Nanotheranostics, that will use gold nanoparticles to identify disease and light to destroy diseased cells. She also works with magnetic nanoparticles.

Nanomerics
In 2010 Uchegbu founded Nanomerics with Andreas Schätzlein. Nanomerics is a pharmaceutical company that uses nanotechnology platforms to develop medicine. Uchegbu is the Chief Scientific Officer of Nanomerics. Nanomerics are developing structures that can transport antibodies that can cross the blood–brain barrier. Nanomerics develop molecular envelope technology nanoparticles from amphiphilic polymers that self-assemble. She won the Royal Society of Chemistry Emerging Technologies prize for their molecular envelope technology in 2017. She licensed the medicine NM133 to Iacta Pharmaceuticals in 2017. NM133 contains cyclosporine A and can be used to treat dry eye.

Professional service
Uchegbu serves on the editorial board of the Journal of Controlled Release. She has served as the scientific secretary of the Controlled Release Society. She is editor-in-chief of Pharmaceutical Nanotechnology. She is on the healthcare strategy advisory team of the Engineering and Physical Sciences Research Council. She was involved in the University College London celebrations of the National Health Service turning seventy. In 2007 she was chosen for Women of Outstanding Achievement in SET Photographic Exhibition which was displayed at the Science Museum and the British Museum.

In 2015 Uchegbu was appointed Pro-Vice Provost for Africa and the Middle East. She chairs the Africa and Middle East regional network at University College London, building partnerships and starting collaborative teams, welcoming international visitors and supporting student recruitment.

Public Engagement and Equality and Diversity
Uchegbu is involved in public engagement and science communication and is featured in BBC Woman's Hour discussing her research into how nano particles can be used to help deliver drugs to the body and taken part in Soapbox Science an international science outreach programme promoting women scientists and the work they do to members of the public.

Uchegbu is also involved in equality and diversity activities and programmes, acting as the UCL Provost's Envoy for Race Equality and featuring as the only Black British Role Model for the Women's Engineering Society. She also serves on the University College London Race Equality Charter self-assessment team. She is part of the Black Female Professors Forum, representing 1 of the 55 female professors of colour and 1 of the 25 Black female professors in the UK in 2017.

Books

 2000 Synthetic Surfactant Vesicles: Niosomes and Other Non-phospholipid Vesicular Systems: 11 (Drug Targeting and Delivery)
 2006 Polymers in Drug Delivery
 2013 Fundamentals of Pharmaceutical Nanoscience

Awards and honours

 2007 UK Department for Business, Innovation and Skills Women of Outstanding Achievement in Science Engineering and Technology
 2012 Royal Pharmaceutical Society Pharmaceutical Scientist of the Year
 2013 Academy of Pharmaceutical Sciences Eminent Fellow
 2013 Controlled Release Society College of Fellows
 2016 Academy of Pharmaceutical Sciences Innovative Science Award

References

Nigerian scientists
Academics of University College London
Academics of the University of Strathclyde
University of Benin (Nigeria) alumni
University of Lagos alumni
Year of birth missing (living people)
Living people

redpajama_set_name: RedPajamaWikipedia


If you're looking for a Trucker Hat that you can wear on vacation, or a performance hat you can wear at the gym, then this Grand Teton National Park Performance Trucker might just be the perfect choice. This snapback trucker is made of our microfiber and stretch mesh fabrics, making it lightweight and breathable, and we know it will fit comfortably and dry quickly every time you wear it. The terry sweatband wrapped in Eventure knit will help keep the sweat out of your eyes, and you'll love wearing this hat at the park, on the trail, or wherever life takes you. We support our National Parks!


Content marketing is the latest buzzword for creating SEO and user targeted articles for traffic. It's not new, there's nothing unique about the 2013 version. It just happens that search engines have decided they prefer high quality web text-based content. Based on this demand, SEO consultants recommend websites create original articles to generate fans, rank better, and build links.
However, simply writing articles isn't enough to achieve your goals. The content should be targeted and engaging. Social media has created a demand for higher quality content as real people share real articles. Content could be an article, video, infographic, or just an image. Successful content draws the audience in and makes them want to tell people about it. Once you figure out a good flow for creating content, these tips will help you get them viral.
Generating good quality content is essential for any website. People will always read something they're interested in. Once they become interested in you and your company, the only way to get them to stay interested is to provide them with fresh content on a daily basis. Even weekends.
Social networks are the best place to interact with your followers. Create profiles on every social network and start building your followers. Social networks are the best tool for content marketing because they reach the largest audience. Once your website becomes active on the social networks, the idea is to constantly generate high quality content so people can talk about you. Use the networks to engage your fans and let them know they're important.
Once people hooked on your content, you can use that to get them to read more. By creating good internal links on your website, you can get readers to click and follow related content. Readers should be able to navigate through your website easily.
Images are always important when it comes to attracting readers. Some people won't even bother to click on a story if the image is not interesting enough. Choose images that are interesting, powerful and convey ideas.
Content marketing should offer something for the reader, not push direct sales. Pushy sales tactics don't work anymore. People are constantly bombarded with thousands of advertisements on a daily basis and they are generally fed up with it. Content should inform and educated people to decide for themselves.
The content on your website should always engage your readers. Write about news, popular trend, or anything currently in the media. The world is moving forward and so should you.
Choosing the right niche market is essential for any business strategy. Who will your target audience be? By analyzing the market, you can determine the best method of marketing. Figure out your demographic group and target your content for that audience.
Once you start interacting with your readers, you will start understanding what they like, dislike, and want to read about. As you get to know your audience, it will be easier to create the content for them. Be open to any of their suggestions and try to build a relationship with your fans. Make them know they matter and offer engaging feedback.
Design is always important. If you are unable to create the website by yourself, invest and have it done by professionals. Make sure your website is user-friendly so people can navigate through it easily. Add widgets for social media networks you support and watch your fans grow.
If things don't start running smoothly from the very beginning, don't give up. Content marketing is not easy let alone running an entire website. You must be patient in this business and stick to your program. Good content will always attract followers. In some cases it may take longer than in others. But it will happen.


text:
The London Clinic
Home/Consultants
Mr Peter Andrews
Consultant Rhinologist and Anterior Skull Base Surgeon
BSc, MD, FRCS (ORL-HNS)
Peter Andrews BSc.(Hons), MD, FRCS (ORL-HNS) is the Senior Consultant Surgeon in Rhinology and Facial Plastic Reconstruction at the Royal National Throat, Nose and Ear Hospital and Consultant Anterior Skull Base Surgeon at the National Hospital for Neurology and Neurosurgery. He is the Associate Professor of Rhinology at University College London and Academic Lead for Rhinology.
Specialties and sub-specialties
Rhinoplasty and Septorhinoplasty Surgery
Endoscopic Sinus Surgery and Anterior Skull Base Surgery
Facial plastic reconstructive surgery and Facial reanimation surgery
Peter Andrews has over 20 years experience in rhinoplasty surgery and runs a tertiary referral service in rhinology at the Royal National Throat Nose and Ear Hospital (RNTNEH).
He also runs one of the largest facial reanimation services in the UK and has been an anterior skull base surgeon at the National Hospital for Neurology and Neurosurgery (NHNN) since 2008.
He trained at UCH and Middlesex Medical School and qualified in 1993. He received his Specialist Higher Surgical Training in the North Thames Deanery which included the RNTNE Hospital. He is a Fellow of the European Academy of Facial Plastic Surgery (EAFPS) and was awarded the EAFPS Joseph Travelling Fellowship in 2005 and trained and worked at the Academic Medical Centre, Amsterdam for 6 months during this time. He was appointed to the RNTNEH in 2005.
His surgical areas of interest include nasal plastic surgery, nasal airway reconstruction, facial reanimation surgery and anterior skull base surgery including complex frontal sinus surgery.
As the Associate Professor of Rhinology at UCL, his research interests include nasal airflow reconstruction, facial reanimation surgery and surgical treatments for the correction of loss of smell. He has recently patented a new medical device which measures and diagnoses nasal disease, in collaboration with the biomedical engineering department at UCL.
He specialises in Aesthetic and Functional Rhinoplasty Surgery as well as Facial Reanimation Surgery. He's an expert in Sinonasal Disease Management and Complex Endoscopic Sinus Surgery.
Consultant Rhinologist and Facial Plastic Reconstructive Surgeon
Royal National Throat Nose and Ear Hospital (RNTNEH), University College London Hospitals (UCLH)
Consultant Anterior Skull Base Surgeon
National Hospital of Neurology and Neurosurgery, UCLH
Current teaching posts
Associate Professor of Rhinology
Year qualified 1993
University College London 1987 to 1993
Personal interests
Dad of 4 kids and happily married in Harpenden.
Call or email the consultant's secretary.
Sladjana Stancovic
peterandrews2@gmail.com
Tweets by thelondonclinic


text:
Q: How do I find the index of the max value in a specific range of np.array? I have intensity values over time that I saved in a one-dimensional numpy array called cell. I calculate a maximum during a certain time window by:
peak = max(cell[750:791])

Now I want to have the index of this specific peak value but only in the same time window. I tried:
idx_peak = np.where(cell[750:791] == peak)

But this seems to give me all the indices from the entire array. How can I apply np.where in my specific time window? I hope my question is clear.

A: Just use
idx_peak = cell[750:791].argmax()

redpajama_set_name: RedPajamaStackExchange


text:
Email. info@bookbeanandicecream.co.uk
Fast Forward by Stephen Morris
Back to Stocking Fillers
Harry Potter and the Philosopher's Stone: the Illustrated edition
Prepare to be spellbound by Jim Kay's dazzling depiction of the wizarding world and much loved characters in this full-colour illustrated hardback edition of the nation's favourite children's book – Harry Potter and the Philosopher's Stone. Brimming with rich detail and humour that perfectly complements J.K. Rowling's timeless classic, Jim Kay's glorious illustrations will captivate fans and new readers alike.
When a letter arrives for unhappy but ordinary Harry Potter, a decade-old secret is revealed to him that apparently he's the last to know. His parents were wizards, killed by a Dark Lord's curse when Harry was just a baby, and which he somehow survived. Leaving his unsympathetic aunt and uncle for Hogwarts School of Witchcraft and Wizardry, Harry stumbles upon a sinister mystery when he finds a three-headed dog guarding a room on the third floor. Then he hears of a missing stone with astonishing powers, which could be valuable, dangerous – or both. An incredible adventure is about to begin!
Harry Potter and the Philosopher's Stone: the Illustrated edition quantity
SKU: 978-1408845646 Categories: Bestsellers, Children's, Gifts, Stocking Fillers Tags: J K Rowling, Jim Kay
Pirate Stew
Meet LONG JOHN McRON, SHIP'S COOK . . . and the most unusual babysitter you've ever seen. Long John has a whole crew of wild pirates in tow, and - for two intrepid children - he's about to transform a perfectly ordinary evening into a riotous adventure beneath a pirate moon. It's time to make some PIRATE STEW. Pirate Stew! Pirate Stew! Pirate Stew for me and you! Pirate Stew, Pirate Stew Eat it and you won't be blue You can be a pirate too!
Harry Potter and the Goblet of Fire: Illustrated Edition
Dragons! Daring! Danger! The first fully illustrated edition of Harry Potter and the Goblet of Fire is an extraordinary creative achievement by two extraordinary talents. Jim Kay's inspired reimagining of J.K. Rowling's classic series has captured a devoted following worldwide, and the drama just gets bigger as the series progresses. With over 150 illustrations, Jim Kay's unique vision delivers breathtaking scenes and unforgettable characters - including fan favourites Cedric Diggory, Fleur Delacour and Viktor Krum illustrated by Jim Kay for the first time. Fizzing with magic and brimming with humour, this full-colour edition will captivate fans and new readers alike as Harry, now in his fourth year at Hogwarts School of Witchcraft and Wizardry, finds himself competing in the legendary Triwizard Tournament and facing death-defying tasks, dragons and Dark wizards . Making magic in paint, pencil and pixels, this is the Wizarding World as we have never seen it before.
Harry Potter and the Prisoner of Azkaban: illustrated edition
An extraordinary creative achievement by an extraordinary talent, Jim Kay's inspired reimagining of J.K. Rowling's classic series has captured a devoted following worldwide. This stunning new fully illustrated edition of Harry Potter and the Prisoner of Azkaban brings more breathtaking scenes and unforgettable characters - including Sirius Black, Remus Lupin and Professor Trelawney. With paint, pencil and pixels, Kay conjures the wizarding world as we have never seen it before. Fizzing with magic and brimming with humour, this full-colour edition will captivate fans and new readers alike as Harry, now in his third year at Hogwarts School of Witchcraft and Wizardry, faces Dementors, death omens and - of course - danger.
The Book of Dust: The Secret Commonwealth
From the author of the phenomenal His Dark Materials comes the next chapter in the story of Lyra Silvertongue . . . Lyra is now studying at St Sophia's College, Oxford, with her daemon Pantalaimon. But, for the first time there are serious divisions between the two. Lyra is questioning everything she once held dear. Pan misses the impulsiveness of their youth.
Book, Bean and Ice Cream
61 Poulton Street
PR4 2AJ
Andrew Brookes Anthony Wright Astrid Lindgren Bernadine Evaristo Britta Teckentrup Charlie Mackesy Chris Wormell Daniel Egneus Daniel Start David Walliams diary Fiona Waters Good Housekeeping Graham Pitchfork Henry Firth Ian Theasby Isabel Thomas Ixta Belfrage Jackie Morris Jamie Oliver J B Priestley Jeff Kinney Jim Kay J K Rowling John Lewis-Stempel John Nichol Lauren Child Maggie Mason Mary Berry Michael Townsend Olivia Lomenech Gill Peter Shackleton Peter Townsend Philip Pullman Raynor Winn Richard Pike Robert MacFarlane Sarah Maycock Steve Bond Susie Dent Suzanna Cruickshank Tom Kerridge Tony Blackburn Tony Blackman Yotam Ottolenghi
Monday – 8.30AM to 5.30PM
Tuesday – 8.30AM to 5.30PM
Wednesday – 8.30AM to 5.30PM
Thursday – 8.30AM to 5.30PM
Friday – 8.30AM to 5.30PM
Saturday – 8.30AM to 5.00PM
Silverwood Events
For all our great book events check out our sister site.
© 2021 Book, Bean and Ice Cream.


PRESQUE ISLE is located in PRESQUE ISLE county, Michigan at latitude 45.311011 and longitude -83.498787. The population of PRESQUE ISLE is 1,541 as of the year 2000. The most common zip code of PRESQUE ISLE is 49777.


Thank you, Easter, for providing us with a long weekend (Friday and Monday are Bank Holidays here in the UK). F and I wanted to enjoy some lamb as an ode to spring, so we invited friends to join us for a pre-Easter dinner on Friday. We could've done a traditional roast with the usual carrots and new potatoes, but in a fit of experimentation (and knowing we'd have the whole day to prepare — thank you again, Bank Holiday), F suggested we make Moroccan-style lamb. I suggested that we might as well go all-out and make Moroccan sides, too.
Needless to say, Googling commenced. I went straight to NYT Cooking, the New York Times' great hub for all the recipes they publish in their Food and other sections. I searched "Moroccan" and loads of vibrant, delicious looking dishes appeared. I was drawn to the Moroccan Cooked Carrot Salad; Spicy Orange Salad, Moroccan Style; and this couscous. Meanwhile, F found a recipe for Moroccan Lamp with Apricots, Almonds & Mint from BBC Good Food; it is a stew rather than a roast, which made it more attractive as it required less cooking time.
Perhaps it is a bit monochrome, but boy was it delicious. The stew had such depth of flavor, thanks to cinnamon, apricots, and orange, and the ground almonds gave it a deceptively "creamy" texture. The carrot salad — dressed with lemon juice, spiced with cumin and garlic, and balanced with olives — turned out beautifully. We really liked the pearl couscous laced with cumin, golden raisins, and sautéed onions. The orange salad packed a bit of heat from cayenne, although I left out the garlic, parsley, and olives, as those were already present in the carrot salad.
All in all, a great and delicious success. Will we make these recipes again? Definitely. I'm already looking forward to enjoying the leftovers for lunch.
Do you like Moroccan food? Ever cooked any of it? Post your thoughts and experiences in the comments!
This entry was posted in celebrations/holidays, culture, food, full meals, health, Highlights, pictures, recipes and tagged almonds, BBC Good Food, carrots, couscous, Easter, Friday dinner, ground almonds, lamb, leftovers, life, Martha Rose Shulman, mint, Moroccan, NYT Cooking, parsley, salad, spring, stew on 04.04.2015 by taplatt.


Because no two individuals or problems are alike, we offer a wide range of services and can design a treatment program suited to your personal needs and goals. After an initial assessment, you and your therapist will decide what type of treatment will be most beneficial to you.
Treatment options include individual, couples, and family therapy; child and adolescent therapy; group therapy; psychiatric consultation, and medication management. We have psychiatrists on staff at Central Dupage Hospital who are available to assist with hospitalization or admission to a partial hospital program if appropriate. Our psychiatrists can also help facilitate admission to other hospitals if needed.
In addition to the mainstay of individual, couples, and family therapy, several of our therapists also offer group therapy. Group therapy offers opportunities for growth and interaction within the context of a caring and supportive group, facilitated by a skilled therapist. Group therapy fees are typically lower than individual session fees, making it an economical as well as effective therapy choice.


text:
Now The Mistaken available at audible.co.uk and amazon.com
A little about the plot and what a plot it is.
Tyler Karras is an honest man, a transplanted Brit living the American dream, but his charmed life takes an unexpected turn when his brother, Nick, is coerced into joining ranks with San Francisco's Russian mafia. Ty intervenes to secure Nick's freedom, yet only succeeds in incurring their wrath. With no choice but to accept Nick's new life, Ty returns to his own, but his dreams are dashed when his wife - pregnant with their first child - is killed, the victim of a reckless crime. Despondent and bitter, Ty macerates his grief in alcohol. From the depths of the bottle screams a voice, howling for vengeance. His target is a stranger, the woman who drew his wife toward her death. He doesn't know her, but he'll find her, and when he does, he will make her pay, for a deal has been struck with Nick's Russian associates, enslaving her into a life of bondage.
A fast paced and high energy story about guilt, anger and learning how to forgive. At times harrowing and very unpredictable. It will have you on the edge of your seat. With an intricate plot, the twists and turns will get you until the very end. Their are links below to the audiobook.
The Mistaken on Audible.co.uk
The Mistaken on Amazon.com
The Mistaken in many formats and now in audiobook format, don't miss out on this cracking read.


As crude prices plummeted last summer, Steve Pruett, chief executive of a small west Texas oilfield developer, idled a drilling rig, opting to pay $21,000 a day to store it rather than dig more wells and risk bigger losses.
Now as oil prices rise again, the third-generation oil man is offering his only rig crew bonuses to drill wells as fast as they can, but says his company, Elevation Resources, will wait until next year to deploy a second rig.
Last year's rollercoaster when crude prices rallied in the first half of the year only to come crashing down in the second half, burned many producers and Pruett and his peers remain cautious.
But there are signs of guarded optimism that this time the industry has really seen the worst of the nearly two-year downturn that cost hundreds of thousands of jobs, pushed dozens of firms into bankruptcy and led to an investment slump so severe that it weighed on the whole U.S. economy.
With crude prices now nearly double their February lows near $26 per barrel, new wells in the Permian Basin – North America's richest source of shale oil – are again becoming profitable and producers are taking baby steps to crank up output again.
The number of rigs in the shale oil basin that spreads across more than 50 counties near the border with New Mexico rose by 17 to 146 on May 31 after bottoming in late April, according to data shared with Reuters by Drillinginfo, a consultancy.
The Permian rig count remains far below its November 2014 peak of 467 rigs recorded by oil services company Baker Hughes, and many areas and new wells less productive than those in "sweet spots" will need prices to climb further to be profitable again. But the stirrings suggest some relief is on its way. Job losses in Midland and Odessa, the region's main cities, have slowed, local oil executives talk about plans to drill and some are already arranging new financing.
Pruett says with oil near $50, wells on three of Elevation Resources' seven fields are already profitable and he expects prices to climb $60 next year, which would call for a second rig.
There are some signs of activity elsewhere. In North Dakota, permit requests rose by 10 to 66 in April, according to the latest readout and the rig count inched up to 28 last month before slipping back to a decade-long low of 27.
To be sure, it will take a broad, lasting recovery to stem the slide in business investment that shaved more than half a percentage point off the U.S. economic growth in the first quarter.
Drillers are also starting from a low base. For example, Elevation Resources has 30 employees now, compared with several hundred during the shale oil boom.
The fear of another "false dawn" is also keeping expansion plans in check. Last year, when crude prices climbed as far as $60 in late June, rig counts and drilling permits picked up too.
At this time of last year, the U.S. crude production was still close to its peak of 9.69 million barrels per day and the global supply overhang showed no signs of abating.
Today, U.S. production is falling and global supply growth is no longer outpacing demand. The U.S. government forecasts domestic output will bottom at 8.1 million bpd in September, then edge up by just 150,000 bpd through December.
The downturn also forced service companies to slash prices on everything from the trailers that house oil workers to lease rates for the drilling rigs. Pruett says he expects to lease his second rig for $15,000 a day, compared with $25,000 it costs him now under a lease signed during the boom.
He also says drilling crews that are still in the game are more experienced and it now takes them half the time to drill a new well as it used to. It means that many producers can find ways to be profitable even if prices do not recover that much.
For Kirk Edwards, CEO of Odessa-based Latigo Petroleum, $55 per barrel will be enough to wade back in and take advantage of a perceived tightening of the market.
"We are looking to buy properties and we are looking to start drilling immediately if we hit our number." Like many, he remains a bit wary though.
Some analysts warn of a repeat of last year's reversal saying hefty global inventories will keep weighing on prices while temporary factors such as wildfires in Canada's oil sands might have been behind a perceived tightening of supply.
Stephen Robertson, the executive vice president of Permian Basin Petroleum Association, a trade group, said oil executives are mindful of committing too soon like they did in 2015, but also concerned about missing out on a nascent recovery.
"A lot of people are optimistic about $50 oil," he said.
Karr Ingham, a petroleum economist based in Amarillo, Texas, said another slump was possible, but noted a shift in psychology.


The aim of the work was to investigate the resistance to cavitation erosion and sliding wear of sprayed HVOF coatings. M(Ni,Co)CrAlY and Cr3C2-NiCr based coatings were deposited using HVOF method onto stainless steel substrate grade AISI 304. As-sprayed coatings' surface morphology was examined by SEM-EDS and profilometer methods. Cavitationerosion tests were conducted in distilled water with the use of vibratory rig and stationary specimen method. Cavitation erosion curves were plotted as well as cavitational wear mechanism was observed with the use of SEM microscope. Sliding wear tests were performed using the ball-on-disc tribotester with counter-specimen (ball) made of steel 100Cr6. Wearrates and coefficient of friction were computed. Normalized wear resistance with referenceto stainless steel reference sample AISI 304 was calculated. In addition, comparable analysis of wear resistance results was conducted. M(Ni,Co)CrAlY coating presented the highest cavitation erosion resistance, therefore Cr3C2-NiCr coating represented the highest sliding wear resistance of all tested materials.
N. Jegadeeswaran, M.R. Ramesh, K.U. Bhat, Combating Corrosion Degradation of Turbine Materials Using HVOF Sprayed 25% (Cr3C2-25(Ni20Cr)), International Journal of Corrosion (2013), DOI:10.1155/2013/824659.
J.R. Davis, Handbook of Thermal Spray Technology, ASM International, OH, USA, 2004.
A. Klimpel, Napawanie i natryskiwanie cieplne: technologie, WNT, 2009.
K. Szymański, A. Hernas, G. Moskal, H. Myalska, Thermally spray- ed coatings resistant to erosion and corrosion for power plant boilers - A review, Surface and Coatings Technology 268 (2015), 153-164, DOI:10.1016/j.surfcoat.2014.10.046.
P. Zhang, J.H. Jiang, A.B. Ma, Z.H. Wang, Y.P. Wu, P.H. Lin, Cavitation Erosion Resistance of WC-Cr-Co and Cr3C2-NiCr Coatings Prepared by HVOF, Advanced Materials Research (2007), DOI:10.4028/www.scientific.net/ AMR.15-17.199.
G. Taillon, F. Pougoum, S. Lavigne, L. Ton-That, R. Schulz, E. Bousser, S. Savoie, L. Martinu, J.-E. Klemberg-Sapieha, Cavitation erosion mechanisms in stainless steels and in composite metal-ceramic HVOF coatings, Wear. 364-365 (2016), 201-210, DOI:10.1016/j.wear.2016.07.015.
P. Zhang, R. Lin Peng, X.-H. Li, S. Johansson, P. Zhang, R. Lin Peng, X.- H. Li, S. Johansson, Investigation of Element Effect on High-Temperature Oxidation of HVOF NiCoCrAlX Coatings, Coatings 8 (2018), 129, DOI:10.3390/coatings8040129.
W. Zhou, K. Zhou, Y. Li, C. Deng, K. Zeng, High temperature wear performance of HVOF-sprayed Cr3C2-WC-NiCoCrMo and Cr3C2-NiCr hardmetal coatings, Applied Surface Science 416 (2017), 33-44, DOI:10.1016/j.ap- susc.2017.04.132.
S. Saeidi, K.T. Voisey, D.G. McCartney, Mechanical Properties and Microstructure of VPS and HVOF CoNiCrAlY Coatings, J Therm Spray Tech. 20 (2011), 1231-1243, DOI:10.1007/s11666-011-9666-5.
S. Lavigne, F. Pougoum, S. Savoie, L. Martinu, J.E. Klemberg-Sapieha, R. Schulz, Cavitation erosion behavior of HVOF CaviTec coatings, Wear. 386-387 (2017), 90-98, DOI:10.1016/j.wear.2017.06.003.
S. Hong, Y. Wu, Q. Wang, G. Ying, G. Li, W. Gao, B. Wang, W. Guo, Microstructure and cavitation-silt erosion behavior of high-velocity oxygen-fuel (HVOF) sprayed Cr3C2-NiCr coating, Surface and Coatings Technology. 225 (2013), 85-91, DOI:10.1016/j.surfcoat.2013.03.020.
M. Oksa, E. Turunen, T. Suhonen, T. Varis, S.-P. Hannula, M. Oksa, E. Turunen, T. Suhonen, T. Varis, S.-P. Hannula, Optimization and Characterization of High Velocity Oxy-fuel Sprayed Coatings: Techniques, Materials, and Applications, Coatings. 1 (2011), 17-52, DOI:10.3390/coatings1010017.
M. Szala, T. Hejwowski, Cavitation Erosion Resistance and Wear Mechanism Model of Flame-Sprayed Al2O3-40%TiO2/NiMoAl Cermet Coatings, Coatings. 8 (2018) 254, DOI:10.3390/coatings8070254.
L. Łatka, M. Szala, M. Michalak, P. Sokołowski, T. Pałka, Impact of atmospheric plasma spray parameters on cavitation erosion resistance of Al2O3-13%TiO2 coatings, Acta Physica Polonica A. w druku (2019).
A. Kumar, J. Boy, R. Zatorski, L.D. Stephenson, Thermal spray and weld repair alloys for the repair of cavitation damage in turbines and pumps: A technical note, (2005) 177-182, http://link.springer.com/article/10.1361/ 10599630523737 (accessed June 12, 2013).
M. Szkodo, Erozja kawitacyjna materiałów konstrukcyjnych metalowych, Wydawn. Politechniki Gdańskiej, 2008.
M. Szala, Powłoki zwiększające odporność na erozję kawitacyjną elementów maszyn i urządzeń, Rozprawa doktorska, Politechnika Lubelska, Lublin, Polska, 2016.
M. Szala, T.J. Hejwowski, Zwiększanie odporności kawitacyjnej stopów metali przez napawanie powłok, Welding Technology Review (2015), vol. 87 (9), 56-60.
M. Walczak, D. Pieniak, M. Zwierzchowski, The tribological characteristics of SiC particle reinforced aluminium composites, Archives of Civil and Mechanical Engineering 15 (2015), 116-123, DOI:10.1016/j.acme. 2014.05.003.
J.A. Cabral-Miramontes, C. Gaona-Tiburcio, F. Almeraya-Calderón, F.H. Estupiñan-Lopez, G.K. Pedraza-Basulto, C.A. Poblano-Salas, Parameter Studies on High-Velocity Oxy-Fuel Spraying of CoNiCrAlY Coatings Used in the Aeronautical Industry, International Journal of Corrosion (2014), DOI:10.1155/2014/703806.
M. Xie, Y. Lin, P. Ke, S. Wang, S. Zhang, Z. Zhen, L. Ge, M. Xie, Y. Lin, P. Ke, S. Wang, S. Zhang, Z. Zhen, L. Ge, Influence of Process Parameters on High Velocity Oxy-Fuel Sprayed Cr3C2-25%NiCr Coatings, Coatings 7 (2017), 98, DOI:10.3390/coatings7070098.
M. Szala, Application of computer image analysis software for determining incubation period of cavitation erosion – preliminary results, 15 (2017), 06003, DOI:10.1051/itmconf/20171506003.
J. Chmiel, R. Jasionowski, D. Zasada, Cavitation Erosion and Corrosion of Pearlitic Gray Cast Iron in Non-Standardized Cavitation Conditions, Solid State Phenomena (2015), DOI:10.4028/www.scientific.net/SSP.225.19.
based coatings, Adv. Sci. Technol. Res. J. 8 (2014), 36-42, DOI:10.12913/ 22998624.1091876.


text:
Q: Exportar html y mostrarlo en la pagina Buenas estoy itentando exportar es decir mediante la etiqueta inmput type se pueda seleccionar un archivo xml en la web y luego lo muestre, no se muy bien como se puede hacer por ahora solo he logrado importar xml es decir que el xml este en nuestra carpeta del servidor.

<!DOCTYPE html>

Pagina de XML

    <button type="button" onclick="loadDoc()">Importa un ejemplo de ficha de pez</button>

    <button onclick="esconder()">Esconde la importacion</button>


text:
 <meta http-equiv="Content-Type" content="text/html; charset=utf-8">
<title>Mooshak</title>
<link rel="stylesheet" href="../../styles/$Conf(style).css" type="text/css">

   <link REL="icon" HREF="../../icons/mooshak-16.gif" TYPE="image/gif">
   <link REL="SHORTCUT ICON" HREF="../../icons/mooshak-16.gif">

Bem vindo ao Mooshak, um sistema para gerir concursos de
programação na web. O Mooshak suporta avaliação automática
dos programas submetidos, resposta a questões a seguimento de
impressões.

<form method="POST" action="$Conf(controller)?login"><input type="submit" value="Login"></form>

Use este apontador para
entrar no Mooshak.
Há 4 perfis de utilização requerendo autenticação. Esta
distribuição inclui um concurso de teste com um utilizador definido em
cada perfil. Os nomes de utilizador e senhas são os mesmos do nome do perfil.

Pode (e deve) criar novos utilizadores e alterar estas senhas
posteriormente., especialmente a senha de admin.

Pode também utilizar outras funcionalidades do Mooshak, tais como

Resultados de concursos

Registar em concursos em-linha

Ajuda em linha

Listas de verificação

Pode também usar o Mooshak como um serviço web REST. Use
  esta página de teste para ver como o
  serviço é usado.

Comentários, sugestões ou relatórios de problemas? por favor envie-os para
José Paulo Leal

redpajama_set_name: RedPajamaGithub