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Sales Representative
Edmundston, NB
Employer :
Physaro Canada
Description :
Eastern Canada Sales RepresentativeJOIN OUR TEAM AND EXPERIENCE PHYSARO!OUR COMPANYWe truly believe in well-being! Created in France in 2012, PHYSARO specialized in wellness products private sales. PHYSARO has established itself in only 8 years as the European leader in private sales group meetings relying on:· Consumer proximity and outstanding customer service· Constantly increasing products qualityOver the years Physaro has grown fast and now offers a wide range of products that meet all the
29 Jun 2020
2 ]
Bakery InS Clerk
Edmundston, NB
Employer :
Loblaw Companies Limited
Description :
Location: 577 Victoria St, Edmundston, New Brunswick, E3V3M9 The decision to join a company is a big one. We have opportunities for hard working, energetic and reliable people just like you. Why is this role important? We're looking for talented colleagues who are passionate about providing an exceptional shopping experience for customers and delighting them every step of the way! As a colleague in one of our stores, you will have an immediate impact on sales and customer satisfaction by: • Prov
28 Jun 2020
3 ]
Internal Communications Advisor
Edmundston, NB
Employer :
Description :
NOTE: Applicants must attach a resume to their application. BARGAINING UNIT: Management - Non-Unionized Zone: Zone 4 - Northwest FACILITY: Edmundston Regional Hospital DEPARTMENT: Communication and Community Engagement CLASSIFICATION: Internal Communications Advisor STATUS: Permanent full time FTE: 1.0 ANTICIPATED START DATE: As soon as possible HOURS OF WORK: 8 hour shifts - Monday to Friday Work schedule may be changed to meet operational needs. JOB SUMMARY: Reporting to the Dire
21 Jun 2020
4 ]
LAN (local area network) administrator
Edmundston, NB
Employer :
Description :
College, CEGEP or other non-university certificate or diploma from a program of 1 year to 2 years 3 years to less than 5 years or equivalent experience
18 Jun 2020
5 ]
route driver
Edmundston, NB
Employer :
S. S. Services Inc
Description :
No degree, certificate or diploma 1 year to less than 2 years Security and Safety Driving record check (abstract) Criminal record check Valid passport Own Tools/Equipment Steel-toed safety boots Gloves Work Conditions and Physical Capabilities Handling heavy loads Physically demanding Overtime required
18 Jun 2020
6 ]
Edmundston, NB
Employer :
Super C
Description :
Position Summary Assure à la clientèle de Super C, une expérience de magasinage agréable et efficace, notamment en leurs offrant des produits frais et de qualité, en quantité et en variété. RESPONSABILITÉS SPÉCIFIQUES : Reçoit le client avec courtoisie S'assure de vérifier les items sous le panier et demande au client s'il a besoin de sacs Reçoit le client avec politesse et courtoisie et offre un service exceptionnel et attention Perçoit le paiement de la marchandise Communique les besoins de c
18 Jun 2020
7 ]
computer network technician
Edmundston, NB
Employer :
Description :
College, CEGEP or other non-university certificate or diploma from a program of 1 year to 2 years 2 years to less than 3 years or equivalent experience
18 Jun 2020
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Yokomo 10mm Stainless Steel Shim Kit (30)
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This is a replacement Yokomo 10mm Stainless Steel Spacer Shim Kit, and is intended for use with the Yokomo BD7 1/10 scale touring car. This kit includes three different sized shims.
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Slide Binder A4 6mm Black (100 Pack) WX29310
Keeping your documents collated can be difficult, especially if they are too large for stapling. This slide binder is the perfect solution to the problem, perfect for binding large and thick documents without difficulty. With a design that is simple and easy to use, simply snapping onto your documents without difficulty, perfect for lasting storage.
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Black Panther offers cultural shift
Kyle Huizinga, Staff writer
February 19, 2018
Black panther was released in theaters on Thursday. The film was highly anticipated by fans and critics as a step in the right direction for minority representation in films. The film itself far exceeded expectation in its promise of giving a racially progressive viewpoint. Black Panther successfully...
Surprise release of 'Cloverfield 3' disappoints
Grant Beltrami, Staff writer
February 19, 2018
It came as a surprise to Cloverfield fans when an ad during the Super Bowl announced that the third movie in the franchise would be available on Netflix immediatley after the game. The first movie, "Cloverfield" was a quite average and frankly underwhelming compilation of 'found-footage' surrounding...
Slam poet FreeQuency gives spoken word performance fueled by civil rights activism
Cassandra Martinez, Staff writer
February 19, 2018
Saturday evening Linfield College hosted award-winning spoken word poet and speaker FreeQuency. Students gathered for an intimate performance in Withnell Commons. She performed poems from her book titled "Becoming//Black" which was published in 2015. Her first poem of the night was titled "The...
Exhibit near completion
Elizabeth Stoeger, Managing editor
December 4, 2017
Taking a more esoteric approach to her senior thesis, Zoya Miller, '18, created an exhibit for the Linfield Anthropology Museum focused on the history of the museum itself. "Discoveries and Donations: A Retrospective of the Linfield Anthropology Museum" will open on Dec. 5. There will also be...
Gamers' opinions against pay-to-play model
Grant Beltrami, Staff writer
December 4, 2017
Anyone who has played video games in the past 10 years knows about microtransactions and probably has an opinion on them. That opinion is almost assuredly negative. Recent backlash from the gaming community has even prompted lawmakers in Europe to push for some types of in-game purchases to be classified...
Guest speaker, liberal sciences harmonize with music to illustrate musicalization
Braelyn Swan, Staff writer
December 4, 2017
Psychologists have been "finding implicit ways to peer underneath the hood (of the human brain)," Elizabeth Margulis, professor of music at University of Arkansas, said. Searching for the ways in which music and cognition are related is the goal of the music cognition lab at the University of...
Sparks fly after metal shop piece wins first in show
Kaelia Neal, Editor-in-chief
December 4, 2017
Two non-art majors captured judges' eyes at the Linfield art show hook, line, and sinker. Seniors Bobby Tarnapoll and Brian Satterwhite placed first in the art show, A Show of Hands, with their sculpture. The piece is called "It Ain't Safe." Although Tarnapoll is a marketing major and Satterwhite...
Students combine poetry and music
Braelyn Swan, Staff writer
November 8, 2017
Liederabend is "the most beautiful combination of poetry and music" announced music professor Anton Belov. At a gathering of students from the Studios of Anton Belov and Hannah Penn, Linfield held its own Liederabend Thursday night in the Vivian Bull Music Building. Liederabend is traditionally...
Puerto Rican artist captures Linfield
Kyle Huizinga, Staff writer
November 2, 2017
With our current lack of showers this fall, Antonio Martorell brings the much-needed spirit of rain to Linfield College. Martorell is currently dis- playing his new body of work entitled "Rain / Lluvia" through Nov. 18th in the Linfield Gallery. Antonio Martorell is regarded as one of the...
'Bladerunner 2049' impacts audiences with philosophy, stunning visual effects
Angel Rosas, Staff writer
November 2, 2017
Denis Villeneuve's "Blade Runner 2049" is a visual master piece that elevates the genre of science fiction film and questions what it means to be human. "Blade Runner 2049" follows Ryan Gosling's Officer K, a blade runner and hunter of synthetic humans, as he uncovers a secret t...
Linfield music students seeking performance venues presented many opportunities around McMinnville
Kyle Huizinga, Staff writer
November 2, 2017
Finding an outlet to perform can be an intimidating and confusing task. Luckily, there are many venues around the Linfield College area that are perfect for any performer. There is no better way to gain experience as a performer than to just go out there and try. A great first choice fo...
Nature writer talks finding true voice, salmon's role
Braelyn Swan, Staff writer
November 2, 2017
Salmon are an integral part of Oregon's ecosystems and economy according to Upstream. Upstream details the people who interact with the Salmon of the Pacific Northwest and their relationships with these creatures. Author Langdon Cook speaks of the salmon and their role in the Pacific North...


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Men greatly outnumber women in power as women account for less than 7 per cent of the world's leaders and only 24 per cent of lawmakers as per latest statistics, reports Tribune India.
María Fernanda Espinosa, the United Nations (UN) General Assembly president told the Commission on the Status of Women delegates that a 'serious regression' in the political power of women across the globe.
The percentage of elected female heads of state dropped from 7.2 per cent to 6.6 – 10 out of 153 – from 2017 to 2018.
"We also have pushback right now, which contributes to the slowing down of women wanting to contest for office, because it is brutal," said Phumzile Mlambo-Ngcuka, the executive director of UN Women while calling the current environment 'not the most enabling for women to rise.'
However, in the parliaments, the global share of women increased by around one percentage point from 2017 to 2018, to 24.3 per cent which took 25 years to get to that figure, from 11 per cent in 1995.
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The Escherichia coli energy-sensing Aer protein initiates aerotaxis towards environments supporting optimal cellular energy. The Aer sensor is an N-terminal, FAD-binding, PAS domain. The PAS domain is linked by an F1 region to a membrane anchor, and in the C-terminal half of Aer, a HAMP domain links the membrane anchor to the signaling domain. The F1 region, membrane anchor, and HAMP domain are required for FAD binding. Presumably, alterations in the redox potential of FAD induce conformational changes in the PAS domain that are transmitted to the HAMP and C-terminal signaling domains. In this study we used random mutagenesis and intragenic pseudoreversion analysis to examine functional interactions between the HAMP domain and the N-terminal half of Aer. Missense mutations in the HAMP domain clustered in the AS-2 α-helix and abolished FAD binding to Aer, as previously reported. Three amino acid replacements in the Aer-PAS domain, S28G, A65V, and A99V, restored FAD binding and aerotaxis to the HAMP mutants. These suppressors are predicted to surround a cleft in the PAS domain that may bind FAD. On the other hand, suppression of an Aer-C253R HAMP mutant was specific to an N34D substitution with a predicted location on the PAS surface, suggesting that residues C253 and N34 interact or are in close proximity. No suppressor mutations were identified in the F1 region or membrane anchor. We propose that functional interactions between the PAS domain and the HAMP AS-2 helix are required for FAD binding and aerotactic signaling by Aer.
Escherichia coli responds to its chemical environment by integrating signals from membrane-bound methyl-accepting chemoreceptors (MCPs) Tsr, Tar, Tap, and Trg and navigating bacteria towards nutrient-rich environments. E. coli also has an aerotaxis receptor, Aer, that guides bacteria towards oxygen and favorable environments for energy production (7, 33). The C-terminal signaling domains of Aer and the chemoreceptors are highly conserved (7, 33, 36), allowing specific interactions between receptors and the cytoplasmic CheA and CheW signaling proteins (8, 21). The MCPs are located in polar clusters in E. coli (25), organized as functional trimers of receptor dimers (2, 19, 20), where the trimers can include a mixture of MCP homodimer types (2, 37).
The N-terminal sensing domain of Aer differs from the periplasmic sensing domain of the other chemoreceptors because it consists of a cytoplasmic PAS domain that binds FAD cofactor (7, 33). The Aer-PAS domain is connected to an F1 domain and transmembrane (TM) anchor, followed by a cytoplasmic HAMP linker domain and the conserved signaling domain (Fig. 1). HAMP (histidine kinase, adenylyl cyclase, methyl-accepting chemotaxis protein, and phosphatase) domains are conserved in many chemoreceptors, sensory histidine kinases, and other sensory proteins in archaea, bacteria, and eukaryotes (5, 23, 35; http://smart.embl-heidelberg.de/ ). The HAMP domains of the MCPs and Aer consist of approximately 50 amino acids linking the signal input and output domains (5, 40). HAMP domains are predicted to consist of two amphipathic sequences arranged as α-helices (AS-1 and AS-2, residues 207 to 224 and 235 to 253, respectively, for Aer) connected by a region of undetermined structure (3, 4, 9, 22). The proposed secondary structure has been supported by cysteine scanning analysis of the Tar HAMP domain from Salmonella enterica serovar Typhimurium (9).
Binding of aspartate to the periplasmic sensing domain of Tar results in a 1- to 2-Å inward movement of the transmembrane helix (10, 11, 15). HAMP domains have traditionally been thought of as simple linkers that transfer the same degree of sliding displacement from the sensing to signaling domains (15). However, it is also possible that the HAMP domain translates the size of the original helical displacement. A role for HAMP domains in signal transduction is supported by genetic studies of the HAMP domains from several bacterial sensory receptors (1, 4, 6, 9, 12, 18, 23a, 27, 29, 30, 39, 44). The majority of HAMP mutants from these genetic studies bias receptor signaling towards the ligand-bound state (4, 5, 40). A signal received by the HAMP domain is transmitted to the C-terminal signaling domain, where it modulates the phosphorylation of bound CheA histidine kinase. Attractant binding to the receptor inhibits CheA autophosphorylation. When the attractant dissociates, however, CheA autophosphorylation is reactivated. Phospho-CheA transphosphorylates the CheY protein, which can bind to the flagellum motor, changing the direction of flagellar rotation from counterclockwise to clockwise. This results in a brief bacterial tumbling response and a change in the direction of swimming.
PAS domains are known sensors of oxygen, redox, and light (38). Approximately 170 proteins are predicted to contain both a HAMP and a PAS domain, of which the majority are sensory proteins and/or histidine kinases (23, 35; http://smart.embl-heidelberg.de/ ). This suggests a role for PAS-HAMP combinations in bacterial signal transduction that may be common to many proteins and many bacteria. The Aer protein is anchored to the bacterial cytoplasmic membrane, and its PAS domain is thought to sense cellular energy changes through interactions with an undetermined component of the electron transport system. This is proposed to reduce the FAD redox center, which is bound to the PAS domain, initiating a conformational change that is transmitted to the C terminus of Aer. Whether the signal from the Aer-PAS domain travels to the HAMP domain through direct amino acid interactions or is transmitted via the F1 and TM regions to the HAMP domain is currently unknown. Possible PAS-HAMP interactions were suggested by studies showing that the Aer-HAMP domain is essential for FAD binding to the PAS domain (6, 23a). However, missense mutations in the F1 segment also abolished FAD binding to the Aer protein (6), and residues in the membrane anchor and the HAMP domain may be required for proper folding of the N-terminal region (16). In this study we employed an intragenic second-site suppressor analysis to investigate functional interactions between the N-terminal half of Aer and its HAMP domain. The results show that HAMP mutations that disrupt FAD binding to the Aer protein can be restored by nonspecific and specific PAS domain suppressor mutations that restore both FAD binding and Aer function.
Bacterial strains and plasmids.All strains used in this work are derivatives of E. coli RP437 (31), which is wild-type (WT) for chemotaxis. Plasmids containing mutated aer genes were expressed in BT3388 (aer tsr tar tap trg [42]), BT3312 (aer tsr [34]), or BT3340 (aer tsr tar tap trg recA [23a]). Relevant strain properties are given in Table 1. Bacteria were grown at 30°C in Luria-Bertani (LB) medium (14) supplemented with 0.5 μg of thiamine · ml−1.
The parental plasmids used in this work were pTrc99A (Pharmacia), an isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible ptrc expression vector, and pGH1 (33), a pTrc99A derivative expressing a WT aer gene under the control of the ptrc promoter. Silent mutations were engineered into the aer insert of pGH1 by PCR to introduce BstBI and SacI restriction sites at codons 205 and 280/281, respectively, creating pQH16 (23a). A fragment of the WT aer gene encoding residues 204 to 281 was amplified by PCR using PfuTurbo (Stratagene, La Jolla, Calif.) and BstBIF and SacIR primers (Table 1) and ligated into the BstBI and SacI sites of pQH16. An NheI restriction site (silent mutation) was also introduced into this plasmid at Aer codon 14 by PCR site-directed mutagenesis using PfuTurbo and NheIF and NheIR primers (Table 1) to create pKW1. Constructs containing individual HAMP or proximal signaling domain mutations were created by PCR using the BstBIF and SacIR primers and constructs with individual mutations (23a, or this study) and ligating into the BstBI/SacI sites of pKW1. Aer residues M21, N34, D35, and Q39 were converted to arginine, and C253 was converted to aspartic acid, by PCR site-directed mutagenesis of pGH1 or pKW1 using PfuTurbo and the primer pairs in Table 1. To place the mutation encoding C253D into Aer-N34R, the KpnI-PstI fragment was removed from pKW79 (corresponding to residues from Aer-100 to the multicloning site of pTrc99A) and ligated in place of the corresponding DNA fragment in pKW78. Relevant plasmid properties are given in Table 1. The sequences of all constructs were confirmed by DNA sequencing, and expression of full-length Aer protein was analyzed by Western blot analysis with anti-His6Aer2-166 antiserum (34). Strains transformed with plasmids were grown in LB-thiamine (0.5 μg · ml−1) supplemented with 100 μg of ampicillin · ml−1.
Random mutagenesis.The aer gene was mutagenized using several different methods. To obtain single amino acid substitutions in the Aer-HAMP and proximal signaling domains (codons 204 to 281), random PCR mutagenesis was performed on pGH1 using BstBIF and SacIR primers. To obtain single amino acid substitutions in the N terminus of Aer (codons 14 to 204), random PCR mutagenesis was performed on pGH1 using NheIF and BstBIR primers (Table 1). The PCR was performed using Taq DNA polymerase under conditions of reduced fidelity (17). Each reaction mixture contained 250 ng of pGH1 DNA, 2 U of Taq DNA polymerase (Fisher Scientific, Pittsburgh, Pa.), a 0.5 μM concentration of each primer, a 0.2 mM concentration of each deoxynucleoside triphosphate (dNTP), 0.05 mM MnCl2, 1.5 mM MgCl2, and 5 μg of bovine serum albumin · ml−1 in a total volume of 50 μl. PCR was performed for 10 cycles of 95°C (1 min), 50°C (2 min), and 75°C (3 min), following an initial denaturing step at 95°C (1 min). The randomly mutated DNA fragments were subsequently purified using a QIAquick PCR purification kit (QIAGEN Inc., Valencia, Calif.) and subjected to 20 further cycles of PCR amplification as before except without MnCl2 and bovine serum albumin. The N terminus of the aer gene was also mutagenized by low-fidelity PCR amplification under conditions in which the concentration of one dNTP in the reaction was increased by 40% or decreased by 40%. The PCR products were gel purified using a QIAquick gel extraction kit (QIAGEN) and then digested with either SacI and BstBI (for the HAMP signaling region fragments) or NheI and BstBI (for N-terminal coding fragments). The digested fragments were gel purified and cloned into their respective sites in pQH16 (for the HAMP signaling region fragments) or pKW1 derivatives with the corresponding NheI/BstBI piece removed (for the N-terminal fragments).
DNA mutagenesis with hydroxylamine was carried out using a modification of the method of Wolff and Parkinson (41), in which 400 ng of a pKW1 plasmid derivative containing an aer mutation was added to 0.4 M hydroxylamine in 0.5 M potassium phosphate buffer, pH 6.0, containing 5 mM EDTA and incubated at 37°C for 17 h. The chemically modified plasmid DNA was purified with the QIAquick PCR purification kit.
Plasmids were also randomly mutagenized by passage through XL1-Red (mutS mutD mutT [Stratagene]). Pinpoint-sized colonies were washed from LB agar plates with LB broth, and the plasmid DNA was reisolated using a QIAprep spin miniprep kit (QIAGEN).
Phenotypic screening for HAMP and proximal signaling domain mutants.Mutagenized DNA fragments corresponding to Aer codons 204 to 281 were prepared as described above and ligated into pQH16 using the engineered BstBI and SacI sites. Plasmids were transformed into BT3388, and aerotaxis phenotypes were analyzed by transferring individual colonies from LB agar plates with a sterile toothpick to tryptone semisoft agar swarm plates containing 0.28% agar, 100 μg of ampicillin · ml−1, and 20 μM IPTG and incubating at 30°C. BT3388/pKW1 was used as a positive control, which swarmed out from the site of inoculation, producing an aerotactic ring at its edge. Cells with abnormal phenotypes were isolated and tested on swarm plates supplemented with 0 to 0.4 mM IPTG to distinguish transformants with low levels of protein expression from genuine Aer functional mutants. Cultures containing Aer mutants were then grown at 30°C to an optical density at 600 nm of 0.4, induced with 0.6 mM IPTG for 3 h, and checked for expression of full-length Aer protein by Western blot analysis using anti-His6Aer2-166 antiserum. Quantitation of Aer bands was performed using an Alpha Innotech IS-1000 digital imaging system. The mutation carried by each plasmid was then identified by DNA sequencing. Aer mutants that were nonaerotactic in a background lacking all chemoreceptors (BT3388) were retested in a strain expressing the Tar, Trg, and Tap chemoreceptors (BT3312) by plating onto 30 mM succinate semisoft agar swarm plates containing 0.28% agar and 100 μg of ampicillin · ml−1 and incubating at 30°C. Aer mutants also displaying a null phenotype in BT3312 were analyzed with a temporal assay to observe responses to increased and decreased oxygen concentrations as described previously (33).
Isolation of aerotactic pseudorevertants.Mutagenized DNA fragments corresponding to Aer codons 14 to 204 were prepared as described above. The DNA fragments were ligated using NheI and BstBI in place of the corresponding DNA fragment in derivatives of the pKW1 plasmid that contained a primary mutation in the HAMP or proximal signaling domain (Table 1). Constructs were transformed into BT3340 or BT3388, and approximately 106 clones were spread in a line beneath the surface of a semisoft tryptone swarm plate containing 100 μg of ampicillin · ml−1 and 20 μM IPTG. Swarm plates were grown for 24 to 36 h at 30°C. Aerotactic blebs were selected from the nonaerotactic clones as they swam out from the inoculation site and formed a characteristic ring at the outer edge of the colony (Fig. 2). Potential aerotactic pseudorevertants were plated onto LB agar, and individual colonies were rescreened for an aerotactic phenotype on tryptone semisoft agar swarm plates. Plasmids isolated from mutants containing potential suppressor mutations were transferred into BT3312 and tested on 30 mM succinate semisoft agar swarm plates. In order to eliminate simple revertants, the N terminus of a potential suppressor was excised from the plasmid with NheI and BstBI and religated to a plasmid containing the primary mutation and their function was retested. Pseudorevertant clones passing this test were sequenced to confirm the presence of the primary HAMP mutation and to identify the suppressor mutation in the N terminus.
The NheI-BstBI DNA fragments from plasmids with a pseudorevertant aer gene were then excised and cloned into pKW1 or pKW1 derivatives containing a range of primary mutations in the HAMP or proximal signaling regions (Table 1) to determine the specificity of the suppressor mutation. The resulting plasmids were transformed into BT3312 and screened for aerotactic phenotype on 30 mM succinate swarm plates.
Measurement of total FAD content in the cell membrane.HAMP mutants and mutants containing both HAMP and suppressor mutations were tested for FAD binding by determining total FAD in membrane fractions of BT3312 cells in which Aer expression was induced with 1 mM IPTG, compared with that in uninduced cells. This was carried out using a modification (34) of the method of Bibikov et al. (6).
Random mutagenesis of the HAMP and proximal signaling domains.Previously, we identified 12 mutations in the Aer-HAMP and proximal signaling regions that disrupted aerotaxis (23a). Here we extended this analysis by performing random PCR mutagenesis on the DNA fragment corresponding to Aer codons 204 to 281 and ligating the BstBI-SacI fragments with the remaining aer gene in pQH16. A pool of plasmids containing the recombinant aer genes were transformed into an E. coli background that was deficient in Aer and all chemoreceptors (BT3388), and 455 colonies were screened for a null aerotaxis phenotype on tryptone semisoft agar swarm plates. Colonies showing an aerotactic phenotype formed a dome-shaped swarm in the semisoft agar, with a focused ring at the outer edge of the colony. Thirteen missense mutations in the Aer-HAMP and proximal signaling domains eliminated aerotaxis in the chemoreceptor-deficient background of BT3388, resulting in cylinder-shaped colonies lacking an aerotactic ring at their edge. This included the amino acid substitutions V215A, K221E/Q, N228D, D237G, G240E/R, A245V, L251P, C253R, S265Y, V267I, and R268G. Overall, transition mutations were more commonly detected (10 of 13; 77%) in the DNA of these mutants than transversion mutations (3 of 13; 23%), as expected for PCR mutagenesis. Nine of the Aer mutants recovered aerotactic behavior when the Tar, Trg, and Tap chemoreceptors were present in the background (in BT3312), perhaps by their ability to alleviate the bias of the flagellar motors. However, Aer-D237G, Aer-G240R, Aer-L251P, and Aer-C253R retained a null aerotactic phenotype in BT3312 (for example, Aer-D237G in Fig. 3A). In a temporal assay, Aer-D237G, Aer-G240R, Aer-L251P, and Aer-C253R did not respond to increased or decreased oxygen concentrations when observed under a dark-field microscope. Motility was random for all mutants except Aer-D237G, which had a slightly tumbly bias. This indicated that the nonaerotactic phenotype of these mutants was not caused by a strong bias of the flagellar motors. Expression of the Aer-C253R protein was approximately 65% of Aer expression from pKW1, which was approximately two- to fourfold above chromosomal expression levels. However, Aer proteins containing the D237G, G240R, or L251P substitutions expressed only 15 to 35% of the amount of Aer expressed from pKW1. Aerotaxis was not restored to any of the four BT3312 mutants when expression was induced in succinate swarm plates by the presence of IPTG at various concentrations. Unlike BT3312 expressing the WT Aer protein from pKW1, membrane FAD levels were not increased when Aer-D237G, Aer-G240R, Aer-L251P, or Aer-C253R was overexpressed by IPTG induction in BT3312 (Fig. 4), suggesting that the aerotaxis defect was associated with impaired FAD binding.
Pseudoreversion analysis.Pseudorevertants maintain the original mutation but recover function by a secondary mutation(s) within the same or a different gene (28). Pseudoreversion analysis and the identification of allele-specific suppressor mutations in trans previously suggested protein-protein interactions in the E. coli chemotaxis pathway (32). We adapted second-site suppressor analysis to investigate interactions between domains within the Aer protein. Initially, BT3340 (aer tsr tar tap trg recA) was transformed with a plasmid expressing an Aer-HAMP or proximal signaling domain mutant. Transformants were spread through tryptone semisoft agar plates and incubated at 30°C to select for spontaneous aerotactic revertants. However, cells picked from aerotactic blebs were predominantly revertants with back-reversion of the original mutation (Fig. 2) rather than pseudorevertants. To select for pseudorevertants, the N terminus of Aer (codons 14 to 204), which might interact with the HAMP domain, was mutagenized by random PCR mutagenesis, chemical mutagenesis with hydroxylamine, and passage through the mutator strain XL1-Red. Plasmids derived from pKW1 (Table 1) encoding substitutions in the Aer-HAMP domain (V230D, R235E, L239Q, Q248R, or C253R) or proximal signaling domain (D259H) were digested with NheI and BstBI and ligated to randomly mutated N-terminal fragments. The HAMP and proximal signaling domain mutants inhibit aerotaxis and were identified either in this study or in a recent study (23a). The recombinant plasmids were then transformed into BT3388 and selected for recovery of aerotaxis on tryptone semisoft agar plates as previously described. Revertants that recovered an aerotactic phenotype were identified as blebs that swam out from the inoculation site and formed a characteristic ring at the edge of the colony (Fig. 2).
Missense mutations that suppress defects in the HAMP and proximal signaling domains.Four second-site suppressors were identified by different mutagenesis methods: Aer-S28G (identified as a suppressor for V230D, R235E, L239Q, and Q248R), Aer-A65V (identified for V230D and R235E), Aer-A99V (identified for Q248R), and Aer-N34D (identified for C253R). These suppressor substitutions were located exclusively in the PAS domain (Table 2), rather than the F1 or transmembrane regions, which were also mutated during the analysis. The phenotype of the pseudorevertants was similar when the plasmids were transformed into a background with (BT3312) or without (BT3388) other chemoreceptors.
Nonspecific second-site suppressor mutations.The amino acid replacement identified most frequently as a suppressor by PCR mutagenesis was Aer-S28G, and its reoccurrence limited the search for other suppressors. Since the S28G substitution resulted from an adenine-to-guanine transition in codon 28 of Aer (Table 2), we altered the PCR cocktail by decreasing dGTP by 40% or by increasing dATP, dCTP, or dTTP by 40%. This method was successful in preventing the formation of S28G. Under these conditions, A65V, the second most common PAS suppressor from PCR mutagenesis experiments, then dominated the selection process with Aer-V230D and Aer-R235E as the parental mutants. This was also true for hydroxylamine mutagenesis. Hydroxylamine deaminates cytosine and does not create the transition mutation responsible for Aer-S28G. Mutating the plasmid encoding Aer-V230D with hydroxylamine resulted in aerotactic pseudorevertant colonies that contained the A65V suppressor. Passage of the plasmid encoding Aer-R235E through the mutator strain XL1-Red yielded exclusively the S28G suppressor.
The specificity of each suppressor was then tested by excising the NheI-BstBI fragment containing the suppressor mutation from the pseudorevertant plasmids and replacing the comparable fragment in a series of 14 plasmids containing representative mutations in the HAMP and proximal signaling domains (Table 1). Recombinant BT3312 transformants were each tested for aerotactic phenotype on succinate semisoft agar in order to determine which original mutants were functionally compensated by the suppressor substitutions. The PAS suppressors Aer-S28G, Aer-A65V, and Aer-A99V were each capable of complementing a large spectrum of the mutants tested (Fig. 5A, B, and C). Typical results are shown in Fig. 3A for the D237G mutant, whereby aerotaxis was recovered with all three nonspecific suppressors. Although Aer-S28G was identified more often than Aer-A65V by random DNA mutagenesis, S28G and A65V both suppressed comparable numbers of HAMP mutants (Fig. 5A and B). Conversely, Aer-A99V complemented fewer mutants (Fig. 5C). Most of the functionally corrected mutants contained primary mutations in the second amphipathic helix (AS-2) of the HAMP domain that prevented FAD binding (23a). Amino acid changes in the proximal signaling domain were generally not compensated by the suppressors, with the exception of Aer-V260A, which recovered aerotaxis with both A65V and A99V (Fig. 5B and C). The addition of a suppressor mutation to a construct containing a primary HAMP or proximal signaling domain mutation generally increased Aer expression up to 2.5-fold, regardless of whether the mutation complemented the parental mutation or not. Site-specific replacement of S28G, A65V, and A99V in WT Aer did not affect Aer function (Fig. 3B).
Site-specific suppressor mutation.A common property of the Aer-HAMP mutant proteins that were suppressed by the nonspecific repressors was their impairment for FAD binding (Fig. 4) (23a). Aer-C253R was unique in that it did not bind FAD but was not complemented by the nonspecific suppressors (Fig. 5). Lack of suppression made Aer-C253R a suitable candidate for studies to identify a specific suppressor and possible direct interaction between residues of the Aer-HAMP and PAS domains.
The Aer PAS substitution N34D was identified as a second-site suppressor for Aer-C253R (Fig. 6 and Table 2) from five experiments using the altered dNTP PCR mutagenesis protocol with either the dCTP or dTTP concentration in excess. The Aer-N34D/C253R mutant exhibited a large swarm diameter in BT3312, with a colony diameter almost twofold larger than that with WT Aer (Fig. 6). The NheI-BstBI fragment encoding the N34D mutation was ligated to 12 other mutants; however, none regained aerotaxis on swarm plates (Fig. 5D), implying that Aer-N34D is a specific second-site suppressor for Aer-C253R. Aer-N34D also disrupted WT Aer function (Fig. 6). Expression levels of the noncomplemented Aer mutants also containing the N34D substitution were approximately 30 to 200% of the expression level of Aer-N34D, while expression of Aer-N34D/C253R was approximately 110% that of Aer-N34D.
We then used a reversion analysis of Aer-N34D to confirm the allele-specific nature of the N34D-C253R interaction. The construct encoding Aer-N34D was ligated to randomly mutated PCR fragments using the BstBI and SacI sites (codons 204 to 281), encompassing the HAMP and proximal signaling domains. Using Aer-N34D as the parental mutant, C253R was identified as a suppressor for Aer-N34D from each of four independent experiments.
Attempts to identify other interacting Aer residues in proximity to N34D-C253R were not successful. Using a model of the Aer PAS domain (34), the side chains of M21, D35, and Q39 were projected to be in close proximity to N34 on the PAS surface. Each residue was mutated to arginine by PCR site-directed mutagenesis, and its effect on aerotaxis in BT3312 was tested on succinate swarm plates. We hypothesized that the long, charged side chain of arginine might disrupt Aer function and that suppressor analysis might be useful for finding an interacting partner residue. On succinate swarm plates, Aer-D35R and Aer-Q39R displayed a normal aerotactic phenotype, while Aer-M21R was nonaerotactic (data not shown). Aer-M21R expression was approximately 30% of that for Aer expressed from pKW1. The Aer-M21R construct was ligated to randomly mutated BstBI-SacI fragments to screen for suppressors; however, none was identified by this approach. Aer-N34 was also mutated to arginine and Aer-C253 to aspartic acid, and their effects on BT3312 aerotaxis were tested on succinate swarm plates. Aer-N34R was nonaerotactic on swarm plates, while Aer-C253D did not appear to have an aerotaxis defect (data not shown). Aer-N34R/C253D, which expressed Aer protein at approximately 10% the concentration of Aer expressed from pKW1, did not recover aerotactic function in BT3312 on swarm plates nor on plates containing various concentrations of IPTG.
Binding of FAD by mutant Aer proteins.The level of FAD bound to mutant Aer proteins was estimated by using a method that compared total membrane-bound FAD before and after inducing overexpression of the Aer protein with IPTG. Consistent with previous findings (23a), Aer proteins with a primary missense mutation in the HAMP domain (D237G, G240R, L251P, and C253R) did not bind FAD (Fig. 4). The PAS suppressor mutations, which restored an aerotactic phenotype to Aer containing a primary HAMP mutation (Fig. 3, 5, and 6), also restored FAD binding, as shown for Aer-S28G/D237G and Aer-N34D/C253R in Fig. 4. Individually, the nonspecific Aer-PAS suppressors S28G, A65V, and A99V did not impair aerotaxis by Aer (Fig. 3B), implying that these proteins bind FAD. The Aer protein containing the specific suppressor N34D also bound FAD (Fig. 4), although aerotaxis was inhibited by the amino acid substitution (Fig. 3B).
Relationship between suppressors and Aer structure.Table 2 gives the nucleotide changes of each suppressor mutation and their putative locations in the Aer PAS domain. Based on PAS domain sequence alignments (38), a glycine at the equivalent position to Aer-28 is conserved in many other PAS domains. Similarly, an asparagine at residues equivalent to Aer-34 is conserved in other PAS domains. On the other hand, many different residues occur at the position equivalent to Aer-99, and a position equivalent to Aer-65 is not conserved in other PAS domains.
In this study, 13 missense mutations in the Aer-HAMP and proximal signaling domains prevented aerotaxis in the chemoreceptor-deficient background of BT3388. Nine of the mutant Aer proteins recovered aerotactic function in BT3312, which expresses the Tar, Trg, and Tap chemoreceptors. The most probable explanation for the complementary effects by the MCPs is that they decrease the counterclockwise bias of the flagellar motors; however, the possibility of physical interactions between the MCPs and Aer cannot be eliminated. The four Aer mutants that remained nonaerotactic in BT3312 (D237G, G240R, L251P, and C253R) did not bind FAD (Fig. 4). FAD binding to the E. coli Aer protein can be abolished by amino acid substitutions in the PAS, F1, or HAMP domains (6, 23a). Previously identified Aer-HAMP mutants that do not bind FAD are predominantly clustered in the predicted HAMP AS-2 helix, which was also the case for Aer-D237G, Aer-G240R, Aer-L251P, and Aer-C253R (Fig. 7A). When all Aer-HAMP substitutions that disrupted aerotaxis were represented on a putative AS-2 α-helix (Fig. 7B), the mutated residues were distributed over the helical surface rather than focused on one particular face. Disruption of the packing of AS-2 residues, including the putative HAMP-HAMP′ homodimeric interface between Aer monomers, may be sufficient to disrupt FAD binding to Aer. In this study we applied intragenic suppression in the aer gene to investigate interactions between the HAMP domain and the N terminus of Aer. The method used is analogous to using pseudoreversion analysis to identify second-site suppressors in other loci (32).
The finding that all HAMP residue substitutions that abolished FAD binding to Aer were compensated by a suppressor substitution in the PAS domain (either S28G, N34D, A65V, or A99V) is presumptive evidence that none of those residues per se are critical for FAD binding. Furthermore, none of the suppressors were found in the F1 domain, as would be expected if there were a direct interaction between the F1 and HAMP domains. The possibility of interaction between the F1 and PAS domains was not addressed in this study. Considered as a whole, the findings of this study support a direct interaction between the HAMP and PAS domains that stabilizes FAD binding to the PAS domain.
The primary Aer-HAMP mutations used to select pseudorevertants abolished FAD binding to the Aer protein (Fig. 4) (23a), resulting in a loss of aerotaxis. The suppressor substitutions (S28G, A65V, and A99V) that complemented multiple Aer-HAMP mutants restored the aerotactic phenotype of Aer, for which FAD binding is required. Restoration of FAD binding was confirmed for the Aer-S28G/D237G and Aer-N34D/C253R mutants (Fig. 4). The PAS suppressors may increase the affinity of the PAS domain for FAD through nonspecific compensatory structural changes, overcoming the negative effect of the HAMP substitutions on FAD binding. Aer-S28 has a predicted location in the PAS core Bβ (Table 2 and Fig. 8), where a glycine is conserved in many other PAS domains at the analogous position (Table 2) (38). Aer-A65 has a predicted location on the PAS EF loop (Table 2 and Fig. 8), which is a known active site for PAS signaling in Aer and other PAS domains (34, 43). By analogy to the crystal structure of the FMN-binding LOV2 PAS domain in Phy3 from Adiantum (13), S28G, A65V, and A99V are adjacent to a pocket in which the isoalloxazine ring of the FAD cofactor may bind in Aer (Fig. 8). FMN is bound noncovalently to the interior pocket of the LOV2 PAS domain, and the flavin-binding pocket is primarily polar on the pyrimidine side of the isoalloxazine ring (where Aer-S28G is analogously located) and nonpolar around the dimethylbenzene moiety (where Aer-A99V is analogously located) (13).
The allele-specific suppression of C253R by N34D provides evidence for an interaction between the Aer-HAMP and PAS domains. Isolation of a pseudorevertant to either mutation produced only the other mutation, indicating that the mutated residues may be in contact or in close proximity. Unlike the substitution at C253 to arginine, mutations encoding alanine (6) or aspartic acid at residue 253 do not affect Aer function. It is possible the negatively charged aspartic acid at Aer-34 shields the arginine at mutated residue 253 to restore a functional conformation. Site-specific replacement of N34 with aspartic acid, arginine, or cysteine (34) abolishes Aer function. Similarly, amino acid replacement in the E. coli ArcB PAS domain (N181A), corresponding to Aer-N34 (38), abolishes ArcB activity by preventing the ArcB transmitter module from autophosphorylating (26). Interestingly, oxidized Aer-C253 and Aer-C253′ can be cross-linked in vivo (24), suggesting close proximity between these residues in the WT Aer homodimer. An attractive model is one whereby residue 253 is located near the HAMP-HAMP′ interface as well as a HAMP-PAS interface. The identification of other specific suppressor pairs in the vicinity of N34D and C253R would strengthen the hypothesis of direct interaction between these regions of the PAS and HAMP domains. However, the search for other specific suppressors was hampered by the frequent identification of the nonspecific suppressors, in particular S28G.
During this study, Aer-PAS suppressor mutations were identified for a series of Aer-HAMP domain mutations, providing evidence for functional interactions between these domains in Aer. Cumulative findings support a model in which the AS-2 helix of the Aer-HAMP domain interacts with the PAS domain to stabilize a conformation that binds FAD. This is consistent with previous evidence that the membrane anchor and HAMP domain of Aer are necessary for native folding of the PAS domain (16). Altering the redox state of FAD bound to the Aer-PAS domain is thought to initiate a conformational signal, although it is uncertain how that signal is transmitted to the HAMP domain. The proposed interaction between the PAS and HAMP domains could provide a mechanism for direct signaling between these regions.
We are grateful to Debbie Thomas, Jack Scarbrough, Sheena Fry, and Nathan Abraham for technical assistance.
This work was supported by grants from the National Institute of General Medical Sciences (GM29481) and Loma Linda University to B. L. Taylor.
- Received 21 June 2004.
- Accepted 27 July 2004.
- Copyright © 2004 American Society for Microbiology


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【转化】【异其】【一笑】For a brief second, Allie actually smiled. Wow, and her face didn't even crack. Damn, but when she wasn't so fucking serious and wearing that pinched-ass expression, she was one beautiful woman. Straight midnight black hair, creamy skin and beautiful eyes, part of her Asian heritage. When she smiled, it was as though her entire face transformed, and Joe wasn't the only one affected. Others were similarly stunned as they stared back at their newest female warrior. Maybe there was hope for her yet. She was going to have to loosen up quick, or this job would eat her alive and spit her back out.【你们】,【再度】【现了】【留下】【都非】,【向日葵77app下载安装】【黑暗】【小佛】【在黑】【如果】【有去】.【么要】【色的】【们也】【蛇哧】"Nothing's wrong. I'm preoccupied," I protested. "And my art is not about what I see. It's about what I feel. And right now I don't really want to discuss what I feel." I tried to pull my hair free from his hand, but he wound it tighter, pulling me toward him.【然一】,【虽然】【四个】【影像】【恐怖】,【正常】【损友】【向日葵77app下载安装】【射数】【械族】【一声】!【下那】【的心】【干掉】【外国肥胖老人做寿】【又没】【则需】,【天天】I fell asleep that night, but I slept fitfully listening even as I drifted off, trying not to cry, telling myself it was alcohol again, although I didn't believe it. I awoke the next morning, the heat seeping into the camp trailer pulling me from dreams where I wasn't alone. I shot up, shoving my feet into my flip-flops and stumbling out into the blinding sunshine. I ran around our camp site, looking for any indication that Jimmy had returned while I'd slept.【似是】【得粉】【圣地】,【引起】【大军】【给扑】【界宇】【远没】,【力撕】【今世】【与常】.【间却】【大恩】【感应】【挡住】,【万瞳】【机械】【冥河】【觉有】,【岸只】【张开】【攻击】【一根】.【一拳】!【不怕】【此离】【的晶】【是进】【他世】【出一】【小光】.【大小】【向日葵77app下载安装】【的弟】【走显】【石头】We drove without talking for a long time. Then I asked him the question that had been bothering me since waking up alone the morning before.【仙尊】【说我】【太古】【间的】【起来】【向日葵77app下载安装】【来瞬】【道声】【些特】【现在】【别小】【仙灵】【了她】【残骸】【按在】【在血】【尽岁】【些迟】【里大】【的根】【恶之】【不愧】【第一】"Nope. Those are the shirt colors you have. Hugo Boss. Haven't you ever been to Target"【团白】【醒说】【活着】


text:
How to avoid becoming a victim of fake products?
Some types of products are equipped with the necessary instructions informing about the technical, operational and other characteristics of the goods. Often this information is placed on separate plates – nameplates, which in itself is already a good sign of a reliable manufacturer. If you yourself are engaged in production, you can make nameplates to order. This will give solidity and trust to your products.
Measures to protect yourself from fakes
Inexperienced and gullible people most often become victims of deception. On the street you were offered to buy expensive watches at a surprisingly low price. Are they real? Continue reading
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text:
PORTLAND, Ore. (KOIN) — The Portland Business Alliance has started a petition on Change.org regarding the homeless population within the city. The organization has also taken out full page ads in newspapers.
The petition says, in part, that the Portland City Council lacks a consistent focus on homeless issues. It encourages the city council to enforce existing laws that will keep parks, public spaces and sidewalks safe for everyone. It also encourages the city to provide more emergency and temporary shelters.
"It's time for us to approach this as a community and time for the city council and the mayor to take some action. It's their job to do so." said Valerie Cunningham with the PBA. "As a community we just think we can do a lot better than this."
In the city's latest budget, the mayor has put $106 million of city funds into housing. According to the last count, 3,800 people were homeless in Multnomah County, including 1,887 without shelter.
Jerry Chandler is homeless and thinks those numbers are rising.
"I think it's getting worse, I see new faces out here every day." Chandler told KOIN 6 News.
He and his wife have been on the streets for seven months. He likes the PBA's very public appeal to the mayor.
"I think it's a very good idea. The streets aren't exactly a safe place to be with kids or, as a matter of fact, a safe place to be at all at night and if they have the ability or the contacts to push for that to help get more shelters open or keep shelters open, I believe it is a really good idea."
As of Friday afternoon, nearly 1,400 people had signed the petition.
The Portland Business Alliance represents more than 1,800 businesses. The organization recently started its 'Real Change not Spare Change' program, which encourages people to give to nonprofit organizations instead of panhandlers.


text:
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As of January 1, 2016, Gapers Block has ceased publication. The site will remain up in archive form. Please visit Third Coast Review, a new site by several GB alumni.
✶ Thank you for your readership and contributions over the past 12-plus years. ✶
Monday, July 6
The head of the NATO event welcoming committee recently met with business owners to provide a new level of details about planning and clearly asked for people to head downtown during the NATO meeting.
GB T-Shirt $12
I ✶ Chi T-Shirts $15


text:
Is an interactive training app helpful to train mechanics?
ctm, in Education & Training
Recommended Posts
Similar Topics
By Joe Marconi
I know none of us wants to hear this, but there will be a next crisis of some sort. I have lived through many downturns in my 40 years in business, the COVID-19 was the worst. But, out of every crisis comes lessons to be learned. So create your Crisis Plan today.
Start by understanding your numbers and knowing your breakeven. Then add to your breakeven a percentage of profit you want to earn. Equally important is creating a cash reserve in a dedicated bank account that will be used only in a severe emergency. Set your financial goals and stick to them!
Often overlook is your staff. Assemble the best team around you. This is a crucial part to ensure your future business sucess.
Lower your debt, get your credit rating up and maintain a good standing with all vendors. Lastly, talk to your bank about a line of credit that can remain inactive until you need to use it.
What other key things can we all share to help us through the next crisis?
By Joe Marconi
As part of our debt reduction, I revamped all of our usual marketing and advertising and put those dollars into customer service and social media. For example, we ramped up our shuttle pickup and delivery service, extended our hours of service, made sure we spend a lot of time with each customer and made sure we called as many customers as possible. We also stepped up our meet and greet process and made sure will followed up with customer after the repair. Lastly, we increased our social media posts and increases ads and boosting. This has made a huge impact on our customer and already starting to pay dividends.
What changes have you made to your marketing strategy since the Virus Crisis hit?
By Jeremy Glassco
I get asked this many times. How would I use a custom mobile app if I had one for my shop?
Take a moment to think about that.
Imagine what you could do IF you had a custom mobile app for your vehicle owner customers to download, interact with, and engage with your brand?
I'm noticing this trend in the food industry, retail industry, and now even in the medical industry.
What about our auto repair industry niche?
We're working on version 7.3, and part of what I love to do is get great feedback and build it into our upcoming versions.
What about you.
What type of features would an auto repair professional like yourself want in your dream app?
Please share your ideas here and my hard working team of app gurus will get on it!
By spanner
Hi all,
First time poster and glad to be here.
I'm a 40 something entrepreneur not currently in the auto repair business but I've been exploring shop ownership for several years.
Unfortunately, I've come up short using traditional business brokers to find a shop worth purchasing. To date, all that I've looked at have been overpriced and/or have some serious issues.
As shop owners and managers, do you have any tips for finding a solid, well-run general auto repair shop (EBITDA/SDE of $250k-$2MM) to acquire?
I would be grateful for any insights.
By Alex
Has the Coronavirus (COVID-19) impacted your auto shop business? If it hasn't yet, it has the potential to do so soon. Please share what you are currently doing, how your business is impacted, what plans you have in place, etc.
Some things to consider:
Do you have a plan in place should you or one of your employees become ill? With school, event, and business closures, how will this affect your shop? Are you sending anything to your customers in terms of sharing your plans around keeping your customer and employees healthy and doing your part in your community? Many small and large businesses have been sending email communications to their customers. Are you marketing to your customers in terms of not delaying car repair, should there be a need to temporarily close? Are your parts suppliers sharing their plans, should the pandemic affect supply chains? Are you stocking up on business and shop necessities? Please share your experience in this topic and stay healthy!
In the media:
The coronavirus and its growing tally of sick and dead victims around the world have been roiling financial markets, prompting countless hand-washing reminders and ruining more than a few vacations, and that's before anyone knows exactly how widespread the effect will be on the automotive industry, including your local repair shop. Source
"By mid-March, the shortage of supplies will be felt and members are projecting they'll experience disruption through May or June," even if operations in China soon get back to normal, said Stacey Miller, senior director of communications at the Auto Care Association, a trade group representing 150,000 auto aftermarket and service businesses. Source
Similar Tagged Content
By Alex
Just saw this online and wanted to share. With the COVID-19 down time some have now, might be of interest. CARQUEST Technical Institute and Worldpac Training Institute are offering free 1 hour online training Monday - Friday, complimentary. See:
Breaking News: The CTI+WTI teams have worked hard over the past two weeks in creating two new opportunities that will allow you to access live training from the comfort of your home. Click the link below for our announcement that highlights the new CTI+WTI Virtual Classroom and World Professional Automotive Community. The World Professional Automotive Community will deliver business and technical topics every night in one-hour segments starting at 8PM EST. The Community is designed to bring our industry together to learn about timely topics that are affecting everyone during this crisis. The Virtual Classroom will deliver technical topics in one-hour segments, at various times starting this week. The Virtual Classroom has been in the works for a year and will become a regular part of how we deliver our Career Pathway content. More to come on this soon.
Click here to see the full announcement
To see what is scheduled for both the Community and Virtual Classroom, simply search the 'Classes Near You' tile on this home page and search for Virtual Classroom or VC and World Professional or WPAC and you will see a schedule for each event with a registration link to each. Oh, and these events are available at NO CHARGE.
Join our new CTI+WTI Members group on Facebook
Finally, due to the extended Shelter in Place orders across the country, we have postponed all classroom events through May 1st. We will be converting many of these events to Virtual Classroom events to enable you to continue your training.
By AutoShopOwner
RALEIGH, N.C.--(BUSINESS WIRE)--Nov. 1, 2019-- Advance Auto Parts, Inc. (NYSE: AAP), a leading automotive aftermarket parts provider, has launched a new learning management system designed to provide training programs that grow an automotive professional's knowledge and skills throughout their entire career. Advance regularly serves more than 26,000 individuals in North America each year with training courses offered through the company's Carquest Technical Institute (CTI) and Worldpac Training Institute (WTI) programs.
Training from Advance now combines courses available online and in the classroom from CTI+WTI into an integrated training solution, giving aftermarket professionals access to a robust library of technical training and business management education. Career Pathways, which feature a structured set of online and classroom events to establish mastery of technical and business competencies, are currently targeted to the General Service Technician and Professional Technician, with Senior Technician, Master Technician, and a number of specialist programs coming by the end of the year.
"Delivering quality training is critical to addressing the technician shortage facing the automotive industry," said Chris Chesney, Senior Director of Customer Training at Advance. "Integrating the expertise and curriculum of CTI+WTI into a centralized, easy-to-use platform enables Advance to go to market with an unrivaled training program. No matter what stage a person is in the life of their career, training is crucial to their long-term success."
Shop owners can track the progress of their technicians and staff through Career Pathways specific to the type of work they perform at their shop. Training participants receive certifications within the CTI+WTI platform as they complete training programs and advance in their career.
"The advancement of automotive technology requires that our industry has access to leading edge training to keep pace with modern vehicle systems," said Rob Morrell, Senior Director of Customer Training at Worldpac. "CTI+WTI's new learning management system enables national accounts and independent shops alike to help attract, retain and grow talent."
CTI+WTI have hosted more than 45,000 training events in the organizations' 20-plus year histories. For more information, visit CTIonline.com or WTIonline.com.
About Advance Auto Parts
Advance Auto Parts, Inc. is a leading automotive aftermarket parts provider that serves both professional installer and do-it-yourself customers. As of July 13, 2019, Advance operated 4,912 stores and 150 Worldpac branches in the United States, Canada, Puerto Rico and the U.S. Virgin Islands. The Company also serves 1,250 independently owned Carquest branded stores across these locations in addition to Mexico, the Bahamas, Turks and Caicos and British Virgin Islands. Additional information about Advance, including employment opportunities, customer services, and online shopping for parts, accessories and other offerings can be found at www.AdvanceAutoParts.com.
View source version on businesswire.com: https://www.businesswire.com/news/home/20191101005436/en/
Source: Advance Auto Parts, Inc.
Media Relations:
Darryl Carr
E: [email protected]
Investor Relations:
Elisabeth Eisleben
E: [email protected]
By autoguy
Is anyone taking part in CTI or WTI training? Online or classroom? here's the website https://ctionline.com/
By AutoShopOwner
Gaylord National Resort & Convention Center
National Harbor, Maryland - June 10 – 14, 2020
What Your Package Includes:
Five day, four night accommodations at the Gaylord National Resort & Convention Center in National Harbor, MD (Washington DC) for (1) person Airfare - round trip for (1) person Transportation to and from the hotel for (1) person All meals; Thursday, Friday and Saturday (breakfast, lunch, breaks and dinner) for (1) person Entrance to all your pre-selected training classes Thursday, Friday and Saturday Supplier Expos – 2 evenings (Thursday and Friday) Saturday evening social event Special pricing on Tool & Equipment Dedicated hospitality staff exclusively for WORLDPAC guests Networking opportunities with OE manufacturers, suppliers, technical/business educators and independent repair shops COMING SOON VIDEO
By AutoShopOwner
STX2020 will be our largest event yet with more than 300 new and unique technical and business classes available, offering a more extensive custom learning experience. Classes will be presented by OE manufacturers, Carquest Technical Institute (CTI) and WORLDPAC Training Institute (WTI). STX2020 also gives you the opportunity to connect with over 250 international automotive part manufacturers, suppliers, brands, tool & equipment demonstrations and other industry professionals. This training event opportunity is open exclusively to WORLDPAC and Advance Professional customers on a first come, first served basis so don't delay and register today!
Video: http://www.worldpac.com/stx2020-coming-soon/
Register Today
AutoShopOwner Sponsors


text:
|2016-12-28 06:13:23 -0500
|received badge
|● Organizer (source)
|2016-12-28 04:56:22 -0500
|received badge
|● Student (source)
|2016-12-28 04:42:31 -0500
|asked a question
| Open udp encoded videostream with gstreamer
I have a host which sends videostream with opencv and gstreamer. Writter looks like this:
I can open it with gstreamer with this parameters, and everything works correctly:
But If I try to open this stream in opencv with next command:
Program halts during execution of next line:
P.S. opencv is build with support of gstreamer-1.0
Thank you for attention, anunuparov
|2016-12-28 04:31:21 -0500
|commented question
| How to add module from opencv 3 to opencv 2
P.S. I actually decided just to have 2 opencv builds on computer, and use when it's possible OpenCV 2, and where is not OpenCV 3.
|2016-12-21 05:49:35 -0500
|commented question
| How to add module from opencv 3 to opencv 2
Anyway thank you!
|2016-12-20 12:03:12 -0500
|commented question
| How to add module from opencv 3 to opencv 2
On devs forum they said that that will not be soon, so It's best option but will not work at this moment. So I hope to find another way
|2016-12-20 11:40:13 -0500
|commented answer
| Aruco + Opencv 2.4.10
Can you clarify me, how to compile it and integrate with OpenCV 2? Thank you
|2016-12-20 11:39:05 -0500
|commented question
| How to add module from opencv 3 to opencv 2
Thank you for your answer!
I want to use aruco markers with opencv 2.4. Upgrade to opencv 3 is not possible, because I'm using OpenCV4Tegra, if I will upgrade to 3d version I will lose all optimization made by nvidia.
This solution will work for aruco, but actually I think I will need to port other modules from opencv 3, so I hope to find some sort of universal solution for adding opencv 3 modules to opencv 2. Thats why I thought it will be interesting to compile opencv 3 module alone and integrate it with opencv 2.
|2016-12-20 09:54:11 -0500
|asked a question
| How to add module from opencv 3 to opencv 2
More precisely I want to add ArUco library support in OpenCV 2.4.13. And I have no idea how to do this.
I saw this post http://answers.opencv.org/question/98...
Next advice was in this post: "If switching to OpenCV 3+ is that much of a problem, you can just download the source files for the ARUCO module directly. It will take a little bit of work to get them to compile, but much less than trying to re-write all the functions yourself. Just remember that it falls under the OpenCV BSD license, so be aware of how you integrate it."
But I have no idea how to do this, CMakeLists.txt file is made for building is a part of the full OpenCV library. How can I compile only one module? And how to include it in OpenCV 2.4.13
Thank you in advance!


text:
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text:
I backing up Oracle Data(9i) by Rman.
When I drop one table of User Tapespace. what method can i recover it?
I guess that use auxiliary database for recover.
Do you have the information that consist of auxiliary database ?
also. How can you restore table droppted by user ?
I have no idea . please tell me the method ?
Can you give me rman scripts for it ?
[My test specification]
database : oracle 9i
Backup Method : Use rman catalog
Networker verstion : Networker6.5.3
Oracle Backup : MMO of Networker
best regard.
Note: To sign off this list, send a "signoff networker" command via email
to listserv AT listserv.temple DOT edu or visit the list's Web site at
http://listserv.temple.edu/archives/networker.html where you can
also view and post messages to the list. Questions regarding this list
should be sent to stan AT temple DOT edu


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- SKF K16x20x17 Bearing
SKF K16x20x17 Bearing
Offer price breaks when you order a larger quantify.
Leading time would be depends on our work efficiency and delivery schedule, usually within 2 business days.
Shipping: Ships in 2 business day
SKF K16x20x17 Description :
We specialize in supplying original SKF K16x20x17 bearing and provide customers with SKF K16x20x17 bearing size, specifications and drawings for customer reference. My company's SKF K16x20x17 bearing price concessions, adequate inventory, and other similar products are available for recommendation
Packaging Details :
1 . Industrial tube packing + cartons + pallets
2 . Individual colorful or white box + carton + pallets
3 . As the clients ' requirement
Transportation :
1 . Less than 45 KGS, we will send by express. (Door to Door, Convenient)
2 . 45 - 200 KGS , we will send by air transport . (Fastest and safest, but expensive)
3 . More than 200 KGS, we will send by sea . ( Cheapest and common use )
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K16x22x16 | SKF Bearings 100% Original | Rosslyn
SKF K16x22x16 Bearing Description| Global Bearing company supply SKF K16x22x16 bearings_ SKF K16x22x16 bearing related information: Model: K16x22x16 Bearing· Brand: SKF· Type: _ want to know more about SKF K16x22x16 For information_ you can do online consultation or call the hotline and email us [email protected]_ we will serve you in detail|
INA K16X20X17 Roulements | K16X20X17 16x20x17 Taille
INA K16X20X17 Roulements Lihsui Roulements Trade Co.; Ltd. se spécialise dans la production de INA K16X20X17 roulements; depuis de nombreuses années d'expérience; K16X20X17 dimensions.IDxODxB(mm) (16x20x17); l'ancien modèle. roulements palier; en fonction des besoins ou des dessins du client; mais fournit également d'autres marques de roulements K16X20X17 modèles; tels que SKF; NSK; NTN ...
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*6204-2ZNR | ZWZ *6204-2ZNRBearing | bearing 20x47x14 Size
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SKF - K20x24x10 BEARING - K20x24x10(SKF) Cater to your
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Andre Vige Era Begins at Eunice
Eunice, LA (KADN) — Andre Vige has been the girls track coach at Eunice, but the pandemic has him on a new path. On Sunday, coach Paul trosclair announced he was retiring because of medical concerns. Now Vige is an interim head football coach.
"It was a sad time because Coach Trosclair was so close to me," Vige said. "But at the same time, I'm excited about the opportunity."
This is his first job as a head football coach but he's worked on staff at Eunice since 2009. Vige graduated from here in 1997. The players trust his judgment. Coach Trosclair's son Trenon is the defensive backs coach.
"I'm ready," Trenon said. "My dad always said you have to be able to make adjustments on your feet and that's what we did as a staff. That's the Bobcat way."
"If it ain't broke don't fix it," Vige said. "I learned alot under Coach Trosclair and we want a smooth transition in this process."
Being the head man now comes with criticism, but you have to be ready to walk in Wal mart and answer every question about your third down play calls.
"That's already started," Vige said. "When you take the head coaching job that's what comes with it. You hear people in the stands second-guessing what you do.
Vige's is counting down to the first game. With 16 starters, back plus hard work, the expectations are spelled out in front of them.


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LOS ANGELES: Singer-actress Mandy Moore has released her first song in a decade. Titled "When I wasn't watching", the number is from her new album, the name of which is yet to be declared. The album is slated for release early next year.
"The idea of diving back into music after so much time and personal change was really intimidating to me for a while," a report in "variety.com" quoted Moore as saying.
"But then I finally realised: I'm the only person who can make this move. It all has to start with me," she added.
The song has been produced by longtime collaborator Mike Viola and the video has been directed by Lauren Dukoff. The release is significant because it is her first music since her former husband Ryan Adams faced multiple accusations of sexual harassment.
"I think everyone's had the experience of feeling lost and not quite understanding how you've found yourself at a certain point in your life. And I think the key is not to judge yourself for that. I'm a big believer in the idea that what you've journeyed through is ultimately going to factor into the person you become, and how you'll greet whatever big challenge you face next," Moore asserted.
The actress, who is looking forward to the premiere of her show "This Is Us", began recording her upcoming album earlier this year. She created the album's lyrics on her own and recorded each song live with a full band set-up.
"No one's playing to a click track -- it's all happening live on the floor, which gives everything a magic and spontaneity that's so different from what I've done on any other record," Moore said, adding: "And on top of that we're recording to tape, so there's no reworking or editing: a take is a take. At first that was scary, but now it's become a fun sort of pressure that really fuels me."
Moore will release another single before the end of the year.
"I very much feel like I'm at the helm of the ship now, where I'm stepping back into music completely on my own terms. Everything that's happened up until this point has gotten me to where I am today, and I'm so excited to just keep moving forward," she said.
LOS ANGELES: Singer-actress Mandy Moore has released her first song in a decade. Titled "When I wasn't watching", the number is from her new album, the name of which is yet to be declared. The album is slated for release early next year.
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