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This year the Chambers Canada Guide Launch and Awards were held on Wednesday, September 27th at the Toronto Marriott City Centre Hotel.
The awards were based on the research for the 2018 edition of Chambers Canada: Canada's Leading Lawyers for Business. These awards reflect a law firm's pre-eminence in key practice areas. They also reflect notable achievements over the past 12 months including outstanding work, impressive strategic growth and excellence in client service.
Click here for photo gallery of the event
For future sponsorship opportunities, please contact Jess Lindsay-Lonton at [email protected].
Blake, Cassels & Graydon LLPFasken Martineau DuMoulin LLPMcCarthy Tétrault LLPStikeman Elliott LLPTorys LLP
Winner: Fasken Martineau DuMoulin LLPFasken Martineau DuMoulin LLP continues to go from strength to strength across various banking-related categories including capital markets, financial services regulation and insurance. It offers notable strength in project, mining and infrastructure finance and wins praise for its focus on the mid market.
Bennett Jones LLPBlake, Cassels & Graydon LLPDavies Ward Phillips & Vineberg LLPMcMillan LLPStikeman Elliott LLP
Winner: Davies Ward Phillips & Vineberg LLPDavies Ward Phillips & Vineberg LLP is home to some of the most highly regarded competition and antitrust lawyers in the country, many of whom offer significant experience working within federal enforcement agencies – including a former commissioner and deputy commissioner at the Competition Bureau. In addition to its prominent mergers practice, the firm has extensive experience of competition litigation and white collar defence.
Blake, Cassels & Graydon LLPDavies Ward Phillips & Vineberg LLPMcCarthy Tétrault LLPOsler, Hoskin & Harcourt LLPStikeman Elliott LLP
Winner: Stikeman Elliott LLPStikeman Elliott LLP operates a premium practice with a distinguished track record on both high-end and mid-market transactions. Important deals in which it was involved this year include the proposed merger of Potash Corporation of Saskatchewan and Agrium and the acquisition of Manitoba Telecom Services by BCE, to name just but a few.
Borden Ladner Gervais LLPFasken Martineau DuMoulin LLPHicks Morley Hamilton Stewart Storie LLPKoskie Minsky LLPNorton Rose Fulbright
Winner: Borden Ladner Gervais LLPBorden Ladner Gervais LLP has a dominant practice in labour and employment in Eastern Canada coupled with an up-and-coming reputation in BC and Alberta. It has a particularly strong reputation in Quebec and offers complementary expertise in pensions & benefits, immigration and human rights law.
Bennett Jones LLPDentons Canada LLPMcCarthy Tétrault LLPNorton Rose FulbrightStikeman Elliott LLP
Winner: Dentons Canada LLPDentons Canada LLP stands out for its energy regulatory practice and can regularly be seen advising on the regulatory, commercial, environmental and Aboriginal aspects of major linear projects, many of which have significant implications for future resource development work in Canada.
Bereskin & Parr LLPGowling WLG (Canada) LLPNorton Rose FulbrightSmart & Biggar/Fetherstonhaugh
Winner: Smart & Biggar/FetherstonhaughThis leading IP practice has offices across the country and is acclaimed for its prosecution and litigation expertise across diverse industry sectors, such as pharma, chemical and industrial biotechnology. It is the largest specialised firm of its kind in Canada.
Blake, Cassels & Graydon LLPLenczner Slaght Royce Smith Griffin LLPMcCarthy Tétrault LLPNorton Rose FulbrightOsler, Hoskin & Harcourt LLPTorys LLP
Winner: Norton Rose FulbrightNorton Rose Fulbright stands out for its transnational litigation practice in Canada and for its experience of multi-jurisdictional disputes. The department demonstrates significant capabilities in commercial and securities litigation, as well as offering key expertise in other areas such as Aboriginal law, IP and appellate advocacy.
Blake, Cassels & Graydon LLPBorden Ladner Gervais LLPGoodmans LLPMcCarthy Tétrault LLPStikeman Elliott LLP
Winner: McCarthy Tétrault LLPThis national firm wins widespread recognition for its sophisticated real estate practice with large clients in all of Canada's major markets. The department was also involved in several large hospitality deals this year, including the acquisition by Bluesky Hotels and Resorts of InnVest REIT.
Aird & Berlis LLPLavery, de BillyLawson Lundell LLPMLT Aikins LLPTorkin Manes LLP
Winner: Lavery, de BillyLavery, de Billy is one of the most well-established commercial firms in Quebec. With four offices spread throughout the province and a multi-talented bench of lawyers that includes a former premier of Quebec, it is a regular destination for referral work for firms and clients with operations in the province.
Melissa Kennedy, Sun Life FinancialMelissa Kennedy is the Executive Vice-President and Chief Legal Officer of Sun Life Financial. She is responsible for the company's global legal, compliance, corporate secretarial and public affairs functions. Ms. Kennedy impresses many with her work relating to the inclusion of environmental social governance structures in corporate behaviour. Her extremely accomplished career trajectory has seen her work in private practice as well as in regulatory and in-house roles at financial institutions. Prior to joining Sun Life, Ms. Kennedy was the Senior Vice President, General Counsel & Corporate Affairs at Ontario Teachers' Pension Plan, a position which saw her both oversee major transactions as well as influence the expansion of OTPP. According to sources who reflect on her period at Teachers, "she is inextricably linked to the maturity of that organisation." She joined Sun Life in 2014, the year in which the company became the first major Canadian life insurer to sign on to the United Nations supported Principles for Responsible Investment. In 2017, the organisation was recognised as one of Canada's top employers for young people. Ms. Kennedy is also the former head of the prosecution team at the Ontario Securities Commission. She is a member of the Executive Committee of the Legal Leaders for Diversity and Inclusion and is described by the market as an "honest, deliberate, focused and successful GC" who is "philanthropic and community-minded." Both private practice and other in-house lawyers agree: "What people identify about her is she is a mentor, she is a beacon, and a role model for others. She's somebody who projects that she is able to succeed in the role at a very high level in a very large and demanding organisation and at the same time seems to be a well-balanced and well-adjusted person – she has not burned other bridges to get to this result." "The arc of her professional career is in and of itself quite remarkable and shows great achievement."
To view the 2016 Award winners click here


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James G. Van Straten served as a U.S. Army senior medical advisor with the South Vietnamese Army throughout I Corps in 1966-67. During that period, he wrote three hundred fifty-two letters to his wife and six children in Texas. Forty-five years later, he has assembled the letters into A Different Face of War: Memories of a Medical Service Corps Officer in Vietnam (University of North Texas Press, 497 pp.; $34.95 hardcover; $15.99 Kindle). The book takes the reader through Van Straten's year, nearly day by day.
American advisors had no power of command within the Vietnamese military hierarchy; their leadership depended solely on persuasive talent. Advisors worked under the authority of Vietnamese commanders who made the final decisions on policies and actions. Nevertheless, advisors were accountable to their American regional commanders for overall results.
Recently, I read Bob Andretta's Brown Water Runs Red: My Year as an Advisor to the Vietnamese Navy Junk Force, which describes how an American lieutenant immersed himself in the Vietnamese culture to influence the Vietnamese regional commander's thinking. Maj. Van Straten employed a similar strategy. Both men, despite working long hours, including occasional around-the-clock days, attended countless Vietnamese town meetings and social functions to build relationships. Open hostility between Buddhists and Catholics complicated Van Straten's task of remaining politically neutral.
Andretta's experience centered on Navy combat actions. Van Straten performed Army medical service tasks. Their introspection about how to deal with cross-cultural differences makes these books valuable.
Under the supervision of Maj. Pham Viet Tu, the head doctor of Da Nang's Duy Tan hospital, Van Straten constantly traveled across I Corps. The United States provided much of the material medical support, and Van Straten performed many minor miracles to ensure that its delivery was timely and in adequate quantity for Vietnamese soldiers and civilians. He faced the endless task of suppressing rampant outbreaks of tuberculosis, bubonic plague, malaria, cholera, rubella (measles), lice, dysentery, and diarrhea.
He also supervised movements of the wounded. Sometimes hospitals had to handle double the number of patients they could handle. During his time in Vietnam, I Corps suffered more casualties than the rest of the country combined. He helped to sort the wounded from the dead and the Americans from the Vietnamese.
In his ten-year Army career before the war, Van Straten had never been assigned to a hospital of any type. "The number of civilian casualties produced by the war was appallingly high," he writes. "As a younger man, I had so wanted to become a physician, but exposure to trauma of that magnitude convinced me that I was not equipped to handle it. Sometimes I got mildly depressed. I was encountering heart-rending trauma almost on a daily basis."
From this experience, Van Straten developed an overwhelming compassion for the Vietnamese people. In 1967, to counter the high civilian casualty rate, he helped relocate eighteen thousand civilians away from the DMZ to safer homes southward.
In their occasional free time, he and American surgeons corrected deformities, such as cleft palates and clubfeet among civilians, mainly children. Dr. John Henry Giles was the prime motivator for this program.
Beyond describing his everyday activities, Van Straten tells stories about revelatory encounters with a long list of people, from the famous to the unknown. He explains nuances of Vietnamese culture overlooked in many other Vietnam War memoirs. He provides memorable word pictures of scenes such as the differences between American and Vietnamese casualty wards. He editorializes against the tactic of search-and-destroy.
He also subtly argues that the NVA won the war in I Corps while he was there and that an NVA final victory was inevitable. Many photographs, mostly taken by the author, supplement the text.
For James Van Straten, remorse for events that ended unsatisfactorily transcends the long interval since they occurred. He repeatedly apologizes for his mistakes—mainly minor and unavoidable—and wishes that any outcomes he left unresolved have had no negative repercussions on the people involved.
His conscience and wartime experience have locked the Vietnamese people in his mind forever.
—Henry Zeybel

subset: commoncrawl


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Neisseria gonorrhoeae (GC) is a human-specific pathogen, and the agent of a sexually transmitted disease, gonorrhea. There is a critical need for new approaches to study and treat GC infections because of the growing threat of multidrug-resistant isolates and the lack of a vaccine. Despite the implied role of the GC cell envelope and membrane vesicles in colonization and infection of human tissues and cell lines, comprehensive studies have not been undertaken to elucidate their constituents. Accordingly, in pursuit of novel molecular therapeutic targets, we have applied isobaric tagging for absolute quantification coupled with liquid chromatography and mass spectrometry for proteome quantitative analyses. Mining the proteome of cell envelopes and native membrane vesicles revealed 533 and 168 common proteins, respectively, in analyzed GC strains FA1090, F62, MS11, and 1291. A total of 22 differentially abundant proteins were discovered including previously unknown proteins. Among those proteins that displayed similar abundance in four GC strains, 34 were found in both cell envelopes and membrane vesicles fractions. Focusing on one of them, a homolog of an outer membrane protein LptD, we demonstrated that its depletion caused loss of GC viability. In addition, we selected for initial characterization six predicted outer membrane proteins with unknown function, which were identified as ubiquitous in the cell envelopes derived from examined GC isolates. These studies entitled a construction of deletion mutants and analyses of their resistance to different chemical probes. Loss of NGO1985, in particular, resulted in dramatically decreased GC viability upon treatment with detergents, polymyxin B, and chloramphenicol, suggesting that this protein functions in the maintenance of the cell envelope permeability barrier. Together, these findings underscore the concept that the cell envelope and membrane vesicles contain crucial, yet under-explored determinants of GC physiology, which may represent promising targets for designing new therapeutic interventions.
Neisseria gonorrhoeae (GC)1 is an obligate human pathogen and the etiological agent of gonorrhea, a sexually transmitted disease. GC infection remains a significant health and economic burden worldwide (1). It is also the second most commonly reported infectious disease in the United States (2). Gonorrhea ranges from clinically asymptomatic to local genital infections to disseminated bloodstream infections. Asymptomatic infections often have devastating consequences on women's health including pelvic inflammatory disease, ectopic pregnancy, and infertility (3). Additionally, GC infections facilitate transmission and acquisition of HIV (4). For all of these reasons it is critical to provide effective treatments against gonorrhea. Currently, a dual therapy with cephalosporin and either azithromycin or doxycycline is recommended (5). However, over the past several years treatment failures associated with GC strains displaying decreased susceptibility to extended spectrum cephalosporins have been reported from various parts of the world (6⇓⇓–9). This is especially concerning because no other antibiotics are clinically useful in these cases, and because no appropriate vaccine exists (10). The escalating problem of the spread of antimicrobial resistance in GC, and the importance of the development of new approaches to study, treat, and prevent GC infection, have been recognized by the World Health Organization and by the Centers for Disease Control and Prevention (11, 12).
We propose that largely unexplored proteins localized to bacterial cell envelope and naturally released membrane vesicles are particularly promising as potential novel molecular targets for therapeutic interventions against gonorrhea. The small fraction of known components of the GC cell envelope (outer membrane, periplasm, cytoplasmic membrane) plays a fundamental role in establishing infection by enabling the microbes to adhere to and invade host cells, facilitating nutrient acquisition, host tissue destruction, and suppression of immune responses (3, 13⇓–15). Further, GC, like many other Gram-negative bacteria, produces membrane vesicles (MVs), which are nano-sized bilayered proteolipids (16). Naturally produced MVs are potentially an effective way to deliver toxins, enzymes, and other effectors to host tissues. Additionally, evidence from various studies support that MVs participate in intercellular communication and horizontal gene transfer (16⇓⇓⇓⇓–21). In GC, MVs are necessary for biofilm formation, which is thought to play an important role in asymptomatic infection in women, resistance to antimicrobial agents, and suppression of host immune defenses (22⇓–24). MVs may also contribute to the serum resistance by providing an enhanced ability to bind and eliminate bactericidal factors (17).
Although the potential importance of proteins localized to the GC cell envelope and MVs has been reported previously (25, 26), only two proteomic studies have been published addressing GC membrane composition (27, 28). Most studies have focused on extensive characterization of factors involved in direct host cell interaction: protruding surface proteins (pili), outer membrane adhesins Opa, porins P.IA and P.IB, lipooligosaccharide, and several iron utilization proteins (3, 4, 15, 29⇓⇓–32). Many of these vital virulence factors undergo phase and/or antigenic variation, making them poor drug or vaccine targets. Therefore, the pursuit for novel and constitutively expressed proteins—therapeutic targets in GC—is of utmost importance.
Accordingly, in this study we applied global and unbiased proteomics to compare the composition of both the cell envelopes and MVs isolated from four GC strains: FA1090, F62, MS11, and 1291. Specifically, we used isotope tagging for relative and absolute quantification (iTRAQ) coupled with multidimensional liquid chromatography and tandem mass spectrometry (2D-LC/MS/MS). This approach allowed us to determine a uniformly and differentially expressed repertoire of proteins. Focusing on a homolog of LPS transport protein, LptD (OstA, Imp), which was identified in both the cell envelopes and MVs fractions, and ubiquitously expressed among analyzed strains, we showed that its depletion led to loss of GC viability. Finally, we selected for initial characterization six predicted outer membrane proteins, which were present at similar levels in the GC cell envelopes. We generated Δngo1344, Δngo1955, Δngo1985, Δngo2111, Δngo2121, and Δngo2139 mutant strains and examined their sensitivity toward different cell envelope-perturbing agents as well as chloramphenicol. These studies showed that the lack of NGO1985 resulted in dramatically decreased GC viability, suggesting that this protein functions in the maintenance of the cell envelope permeability barrier. Overall, these findings further support our hypothesis that the conserved proteins may represent promising targets for designing new therapeutic interventions.
Bacterial Strains and Growth Conditions
GC strains FA1090, F62, MS11, and 1291 used in this study are described in detail in Table I. Additionally we used strain FA19 (33), and a few isolates (LGB1, LG14, LG20, and LG26) collected from two public health clinics in Baltimore between 1991–1994. The strains were grown on either solid media, GCB agar in 5% CO2 atmosphere at 37 °C, or in liquid media, GCBL, at 37 °C as previously described (34⇓–36). GCB agar is GC Medium Base agar (Difco, Detroit, MI) with Kellogg's supplements, whereas GCBL is a GC Medium Base broth containing Kellogg's supplements and sodium bicarbonate at a final concentration of 0.042%. For anaerobic growth, GC was grown as described previously in an anaerobic jar using GasPak Plus anaerobic system with palladium catalyst (BD) and indicator strip (BBL) at 37 °C for 24 h in the presence of 5 μl of 20% sodium nitrite spotted on a paper disc placed in the middle of the GCB plates (37, 38).
To examine the proteome of the cell envelope and MVs, GC strains FA1090, F62, MS11, and 1291 were streaked at the same time on GCB plates and incubated for 20 h in 5% CO2 at 37 °C. Subsequently, bacteria were swabbed from plates and suspended in prewarmed GCBL medium to an optical density at 600 nm (OD600) of 0.1 and cultured in Fernbach flasks with shaking at 220 rpm. Growth was monitored every hour by determining the turbidity of the cultures (OD600). All cultures were harvested at the late-logarithmic phase of growth (at OD600 about 0.8). Cells were separated from culture supernatants by low speed centrifugation (6000 × g), and both membrane fractions and MVs were prepared for proteomic analysis as described below.
Preparation of the GC Cell Envelope Fraction
Preparation of the cell envelope fraction was accomplished according to (39, 40) with the following modifications. Pellet of bacterial cells obtained from 1.0 L of each GC strain cultured as described above was suspended in phosphate buffered saline pH 7.5, (PBS, Li-Cor, Lincoln, NE) supplemented with Complete Protease inhibitor mixture tablets EDTA-free (buffer A, Roche). Then, GC cells were lysed by three passages through French pressure cell at 12,000 psi, and the unbroken bacterial cells and debris were removed by centrifugation at 7000 × g for 30 min at 4 °C. Cell lysates obtained from respective GC cultures were subsequently diluted four times with freshly prepared ice-cold 0.1 m sodium carbonate (pH 11 without adjustment). The suspensions were sonicated (2 × 20 s, 5 Watts) and incubated for 1 h at 4 °C with a gentle stirring. Following another two cycles of sonication (20 s/cycle, 5 Watts), the crude membrane fractions were collected by ultracentrifugation in a Beckman Type 70 Ti rotor at 170,000 × g, 1 h, 4 °C. To remove the residual membrane associated proteins, the pellets were washed with ice-cold buffer A, sonicated (3 × 15 s, 5 Watts), and separated at 100,000 × g, 2 h, 4 °C in a Beckman SW41Ti rotor. These procedures were repeated twice and finally the pellets containing membrane proteins were suspended in 1 ml of PBS containing 0.2% SDS. The total concentration of proteins in each sample was assessed with 2-D Quant Kit (GE Health Care).
Preparation of Naturally Released MVs
The naturally released MVs were prepared using optimized protocol for GC (18). Briefly, supernatants (about 1.0 L per each culture) obtained from the same GC cultures used for cell envelope extractions, were first passed through the 0.22 μm filter units (ThermoFisher Scientific) and to prevent proteolysis, Complete Protease inhibitor mixture tablet EDTA-free was added. Subsequently, the supernatants were subjected to ultracentrifugation at 210,000 × g, 3 h, 4 °C in a Beckman Type 45 Ti rotor. The pellets containing MVs fraction were washed with PBS and finally reconstituted in PBS containing 0.2% SDS. The total protein concentration in MVs was measured using 2D Quant Kit.
Transmission Electron Microscopy
Native MVs isolated from culture supernatants of GC strains FA1090, F62, MS11, and 1291 as described above, were reconstituted in PBS, adsorbed on a formvar/carbon coated copper grid for 5 min, and stained with 2% ammonium molybdate for 1 min. Grids were air-dried and MVs were observed using FEI Titan 80–200 transmission electron microscope at the Oregon State University Electron Microscopy Facility.
iTRAQ Labeling, Two-Dimensional Liquid Chromatography, and Mass Spectrometry
Proteins obtained from the cell envelopes and MVs extractions (100 μg and 40 μg, respectively) were precipitated overnight at −20 °C in 90% acetone and washed twice with ice-cold acetone. iTRAQ (AB Sciex) labeling was performed according to the manufacturer's recommendations. The following iTRAQ tags were used to label peptides in appropriate GC strains: 114 for FA1090, 115 for F62, 116 for MS11, and 117 for 1291. The reactions were carried out for 1 h at room temperature and were stopped by the addition of 250 μl of 0.1% Trifluoroacetic acid (TFA). Labeled peptides were pooled and purified on C18 column (Honeywell Burdick & Jackson). Subsequent procedures were performed at the Proteomic Core at Fred Hutchinson Cancer Research Center, Seattle, WA. iTRAQ labeled peptides were separated by strong cation exchange (SCX) using a Paradigm MG4 HPLC (Michrom Bioresources/Bruker). Peptide separations were performed using a Zorbax300 SCX column (2.1 mm × 150 mm; Agilent Technologies, Santa Clara, CA) with an in-line guard cartridge. Peptides were eluted using SCX A and SCX B buffers with a flow rate of 200 μl/min using the following gradient: 2% B from 0 to 5 min, 8% B to 5.1 min, 18% B to 20 min, 34% B to 32 min, 60% B to 40 min, 98% B to 40.1 min and held to 50 min, 2% B to 50.1 min and held to the end of the run at 60 min. SCX A buffer contained 5 mm KH2PO4 pH 2.7, 30% (v/v) acetonitrile (ACN) and SCX B buffer contained SCX A buffer with 500 mm KCl. Fractions were collected at 2 min intervals throughout the run and pooled to a reduced number of fractions for reversed-phase (RP-HPLC-MS) analysis based on ultraviolet absorption at 220 nm. The pooled fractions were dried in a vacuum concentrator. Each pooled fraction was reconstituted with 0.1% TFA (100 μl) and desalted using SepPak C18 cartridges (Waters, Milford, MA). Peptides were eluted with 50% ACN/0.1% TFA and concentrated in a vacuum concentrator. Subsequently, peptides were separated using Eksigent nano-two-dimensional high-pressure liquid chromatography (2D HPLC, AB Sciex). In-line de-salting was accomplished using a reversed-phase trap column (100 μm × 20 mm) packed with Magic C18AQ (5-μm 200 Å resin; Michrom BioResources) followed by peptide separations on a reversed-phase column (75 μm × 250 mm) packed with Magic C18AQ (5-μm 100 Å resin; Michrom BioResources) directly mounted to the Dionex Probot MALDI Spotter (ThermoFisher Scientific). A 90 min gradient from 7% to 35% ACN in 0.1% TFA at a flow rate of 500 nL/min was used for chromatographic separations. The gradient of solvent B was as follows: 0 min, 5% B; 2 min, 7% B; 92 min, 35% B; 93 min, 50% B; 102 min 50% B; 103 min, 95% B; 108 min, 95% B; 109 min, 5% B; 129 min, 5% B; and 130 min, 5% B. The column effluent was mixed in a micro Tee with matrix (5 mg/ml alpha-cyano 4-hydroxy cinnamic acid (Sigma-Aldrich) with 2% Ammonium Citrate (Sigma-Aldrich) and 40 fmol/μl Glu-Fibrinopeptide B (in-house synthesis) delivered at 1 μl/min. Fractions were spotted at 10 s intervals onto a stainless steel MALDI target plate (AB Sciex). MS and MS/MS spectra were acquired on an Applied Biosystems 4800 Proteomics Analyzer (TOF/TOF) (AB Sciex) in positive ion reflection mode with a 200 Hz Nd:YAG laser operating at 355 nm, and accelerating voltage with 400 ns delay. MS spectra were obtained with minimal laser energy in order to maintain the best resolution. MS spectra for the entire sample set were collected first, and on each sample spot MS/MS spectra were collected for the 20 most intense peaks above the signal-to-noise ratio threshold of 20 using a collision energy of 2 keV and air as the collision gas. Both MS and MS/MS data were acquired on the sample spots using an internal calibration with Glu-Fibrinopeptide B.
Proteomic Data Analysis
Data analysis was performed using the Paradigm search algorithm, which is a part of the ProteinPilot software (version 4.0) (AB Sciex) against the SwissProt target-decoy database for N. gonorrhoeae FA1090 with 1963 protein entries (downloaded on January 17, 2012). The user-defined search parameters were selected as follows: Sample Type: iTRAQ 4plex, Cys. Alkylation: MMTS, Digestion: Trypsin, Instrument: 4800, ID Focus: Biological Modifications, Search Effort: Thorough, FDR Analysis: Yes, User Modified Parameter Files: No. All other search parameters, such as number of missed and/or nonspecific cleavages allowed and the mass tolerance of the precursor and fragment ions, are hard-coded into the ProteinPilot software for analyzing MALDI 4800 data. The ProGroup Algorithm built within ProteinPilot software was used to perform the statistical analysis on the identified peptides to determine the minimal set of identifications. Data were normalized by bias correction built in ProteinPilot. The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository ProteomeXchange with the dataset identifier PXD000549. The annotated spectra can be examined using MS-Viewer, which is available on the public Protein Prospector website at the following uniform resource locator: http://prospector2.ucsf.edu/prospector/cgi-bin/msform.cgi?form=msviewer. The search keys for the individual data sets are as follows: ikgoxmdesw (cell envelope Exp.1; x5616y5bsg (cell envelope Exp.2); nyiveb6jjz (MVs Exp1); and zv4dvvldqm (MVs Exp. 2). Unprocessed and unfiltered results from Protein Pilot can be found in Microsoft Excel files in the supplemental Material S1–S4. All statistical analyses were performed as we described previously (41). Briefly, only proteins identified with 1% FDR with at least one peptide of 95% confidence or more were recorded. For protein quantification, only proteins with associated p values corresponding to the calculated iTRAQ ratios were included. Proteins were considered ubiquitously expressed if the iTRAQ ratios were between 0.5 and 2, and were designated as differentially expressed when the values were below 0.5 or above 2 with the corresponding p values < 0.05 in both biological experiments. The relative protein abundance heat maps were constructed using MultiExperiment Viewer (MeV version 4.8) (42).
Bioinformatics Analyses
Subcellular localization of identified proteins was assessed using PSORTb 3.0.2 (43), SOSUIGramN (44), and CELLO 2.5 (45, 46), and majority-votes strategy was used for protein assignment. In cases where PSORTb, SOSUIGramN, and CELLO 2.5 predicted different subcellular localizations for a particular protein, the protein was allocated to a group of "unknown subcellular localization." Furthermore, the predicted amino acid sequences of identified proteins were analyzed for the presence and location of signal peptides cleavage sites using both SignalP v.4.1 (47) and TatP v1.0 (48) servers. To phylogenetically classify identified proteins the Clusters of Orthologous Groups (COGs) functional categories were assigned using COGnitor, WebMGA (49, 50), and J. Craig Venter Institute-Comprehensive Microbial Resource (http://cmr.jcvi.org/tigr-scripts/CMR/). The predicted amino acid sequences of selected, newly identified proteins were also analyzed using protein BLAST similarity searches (www.ncbi.nlm.gov.blast).
Genetic Manipulations
The genome sequence of GC strain FA1090 was used as a template to design primers. Chromosomal DNA purified from FA1090 using Promega Wizard Genomic DNA Purification Kit was used in PCR reactions. Reactions were performed using Q5 High-Fidelity DNA Polymerase (New England BioLabs, Ipswich, MA) with oligonucleotides synthesized by Integrated DNA Technologies. PCR products were verified by sequencing at the Center for Genome Research and Biocomputing at OSU. The sequences of all oligonucleotides designed and used in this study are listed in the supplemental Table S1.
Construction of GC Strains Expressing RpsM-His, NGO1985-His, and NGO2054-His From Their Native Chromosomal Loci
Splicing by overlap extension PCR (SOE PCR) (51) was used to engineer an epitope-encoding tail, 6×His-tag, at the 3′ end of individual genes ngo1821, ngo1985, and ngo2054 in their respective loci of the FA1090 chromosome. The nucleotide sequence encoding 6×His-tag followed by a stop codon was included within the corresponding oligonucleotides. PCR reactions were used to amplify 500 bp upstream and downstream from the stop codon of each gene. Both fragments were spliced using a set of the external primers. The individual SOE PCR products were used to transform strain FA1090 in liquid culture as described (52). The presence of the His-tag-encoding sequence at the 3′ end of the particular gene was confirmed by PCR reaction using a forward primer specific to the His-tag, NGO-His, and a reverse primer designed to the individual gene; RpsM-His-down-R, NGO1985-His-down-R, or NGO2054-His-down-R.
Construction of Conditional lptD Mutant Strain
The conditional lptD mutant strain, FA1090 PlaclptD, was constructed as follows. First, a gene encoding LptD (NGO1715) including an upstream region encoding native ribosome binding site was amplified with primers NGO1715-C-F and NGO1715-C-R. The 2422 bp PCR product was subjected to digestion with endonuclease FseI and subcloned into ScaI/FseI-pGGC4, to yield pGCC4-lptD, resulting in the placement of the lptD gene behind the Plac promoter. This vector uses the Neisserial Complementation System and contains an isopropyl-β-d-thiogalactoside (IPTG)-inducible promoter, thus allowing controlled expression of a cloned gene (53). Subsequently, the fragment containing lacI repressor gene, Plac promoter and 630 bp of lptD gene was amplified with primers NGO1715-Down-F and NGO1715-Down-R, digested with BamHI and SalI and ligated into pUC18K, creating pUC18K-PlaclptD (54). Then the upstream region of lptD was amplified using primers NGO1715-Up-F and NGO1715-Up-R. The resulting 516 bp product was digested with EcoRI and KpnI, and cloned into pUC18K-PlaclptD. This final construct containing nonpolar kanamycin resistance cassette apha-3 (54) flanked by homologous regions for recombination and allelic exchange (upstream region of lptD, and PlaclptD) was used to introduce the mutation onto the FA1090 chromosome. The plasmid was linearized by digestion with NdeI and transformation was conducted as described previously with the exception that the growth media were supplemented with IPTG (100 μm final concentration) (55). Transformants were plated on GCB agar with 40 μg/ml kanamycin and 100 μm IPTG and selected FA1090 PlaclptD clones were verified by PCR.
Construction of In-frame Deletion Mutants Δngo1985, Δngo1344, Δngo1955, Δngo2111, Δngo2121, and Δngo2139
All strains containing in-frame deletions of individual genes were constructed using a scheme of genetic manipulations briefly described below. The length of individual PCR products as well as restriction enzymes used in cloning procedures are listed in the supplemental Table S1. The upstream regions of particular genes (about 500 bp) were amplified with primers designated as NGO-up-F and NGO-up-R (where F-forward and R-reverse). Subsequently, PCR products were digested and cloned into pUC18K vector, yielding pUC18K-ngo-up. Similarly, the corresponding downstream regions of individual genes (about 500 bp) were first amplified using primers NGO-down-F and NGO-down-R. The resulting PCR products were subjected to digestion with appropriate restriction enzymes and cloned into pUC18K-ngo-up. Thus, these final constructs, pUC18K-Δngo, contained kanamycin resistance cassette (54) flanked by homologous regions for recombination and allelic exchange. The specific pUC18K-Δngo was linearized with NdeI and transformation of GC was conducted as described (55). The presence of desired mutation on the FA1090 chromosome was examined with respective primers listed in the supplemental Table S1. In all PCR reactions, chromosomal DNA isolated from wild-type bacteria was used as a control.
To Construct the Complemented Strain, Δngo1985/Placngo1985, first, the 643 bp fragment of ngo1985 with a native ribosome-binding site was amplified with primers NGO1985-C-F and NGO1985-C-R. The PCR product was subjected to digestion with endonuclease FseI and subcloned into ScaI/FseI-pGGC4, to yield pGCC4-Plac ngo1985, which was subsequently used to complement the Δngo1985 strain as described above.
Effect of LptD Depletion on GC Physiology
To examine the effect of LptD depletion on GC physiology, FA1090 PlaclptD were plated from freezer stocks onto GCB plates supplemented with 100 μm IPTG. The following day, single nonpiliated colonies were passaged onto fresh GCB plates containing 100 μm IPTG and incubated ∼18 h in 5% CO2 atmosphere at 37 °C. A sterile Dacron swab was used to collect the bacteria from plates and suspend them to OD600 = 0.1 in a prewarmed to 37 °C GCBL. After three washes in GCBL, bacteria were divided and cultured in the presence or absence of IPTG (as indicated) for 5 h with shaking (220 rpm) at 37 °C in GCBL. Bacterial growth was monitored every hour by both measuring absorbance at OD600 and assessing cell viability by spotting 10 μl of 10-fold serial dilutions of GC cultures onto GCB agar plates with or without IPTG. The number of colonies was examined after 22 h, and colony-forming units, CFU, were calculated per ml of bacterial culture. These experiments were performed on four separate occasions and mean values and standard deviations of the mean (S.E.) are presented. Bacterial colonies were visualized with a Zeiss AxioObserver.D1 microscope at A-Plan 10× magnification 0.25 P hase Contrast 1.
Phenotypic Analysis of Knockout Strains for Sensitivity to Different Chemical Probes
The individual knockout strains Δngo1985, Δngo1344, Δngo1955, Δngo2111, Δngo2121, and Δngo2139, the complemented strain Δngo1985/Plac1985, as well as isogenic parent strain, FA1090, were plated from freezer stocks and nonpiliated colonies were passaged as described above. All GC strains were collected from plates, and suspended to 5 × 105 CFU/ml in a prewarmed to 37 °C GCBL supplemented with 100 μm IPTG. Subsequently, 10 μl of undiluted and 10-fold serial dilutions of cell suspensions were spotted on solid media containing 100 μm IPTG and with or without the addition of various compounds (as indicated in the text). Bacterial growth was examined after 22 h. The relative viability of each GC strain was calculated by comparing CFUs/ml on GCB plates supplemented with different chemicals to the CFUs/ml on control plates. These experiments were performed in biological triplicates and mean values with calculated S.E. are presented.
Purification of Recombinant Soluble Domain of AniA and Generation of Rabbit Polyclonal Antibody
The gene encoding AniA (NGO1276) lacking the N-terminal palmitoylation signal was amplified, cloned, and purified as described previously in a study by Boulanger et al., where the three-dimensional crystal structure of AniA was resolved (56). Subsequently, 3 mg (1 mg/ml) of purified, recombinant soluble form of AniA was submitted to Pacific Immunology Corporation, where it was used to immunize two rabbits to generate polyclonal antibodies.
SDS-PAGE, Coomassie Staining and Immunoblotting Analyses
Cell envelopes, MVs, and cytoplasmic proteins were extracted from GC strains as described in the sections above. Whole cell lysates were derived from GC grown on GCB plates either anaerobically or aerobically, as specified in the text. When bacterial colonies reached approximately the same size, all strains were harvested, suspended in prewarmed GCBL, and the cell density was assessed turbidometrically at OD600. Cytoplasmic proteins, cell envelopes, and MVs (15 μg of proteins loaded per lane), or whole cell lysates matched by equivalent OD600 units, were boiled in SDS sample buffer in the presence of 50 mm dithiotreitol and resolved in 4–20% Tris-glycine gradient gels (Bio-Rad, Hercules, CA). When indicated, purified recombinant AniA (250 ng) was loaded as a positive control. The gels were either stained with Colloidal Coomassie Blue G-250, and imaged using GelDoc MP (Bio-Rad) or subjected to electrotransfer of proteins onto 0.2 μm nitrocellulose membrane (Bio-Rad) using Trans-blot Turbo (Bio-Rad). The membranes were blocked in 5% milk in PBS supplemented with 0.1% Tween 20, followed by incubation with polyclonal rabbit antisera against either AniA (1:5000) or Ng-MIP (1:10,000), monoclonal mouse antisera against MtrE (1:1000), or monoclonal mouse antibody against 6×His epitope tag (1:1000; ThermoFisher Scientific). The horseradish peroxidase conjugate of either goat anti-rabbit IgG antibody (BioRad) or goat anti-mouse IgG antibody (ThermoFisher Scientific) was used as secondary antisera at 1:10,000 dilution. Immunoblots were developed using Clarity Western ECL-Substrate (BioRad) on Chemi-DocTM MP System (BioRad).
Rationale and Experimental Design
To identify the potential target proteins for development of new pharmacological interventions against GC infections, we compared the proteomes of cell envelopes and MVs derived from four GC isolates: FA1090, F62, MS11, and 1291. We chose these strains for the following reasons: (1) they display several notable differences that might contribute to their fitness in a human host including resistance to human serum, ability to use iron from human transferrin and lactoferrin for growth, and presence of the GC genetic island (Table I) (30, 34, 57⇓⇓⇓–61); (2) their genomes have been sequenced (62); and (3) they have been used in many laboratories and human volunteer studies over the past 20 years (61).
Our experimental design for profiling of the GC cell envelope and MVs is outlined in Fig. 1. Bacterial cultures (1.0 L volume) of FA1090, F62, MS11, and 1291 were propagated simultaneously under standard laboratory growth conditions as described under "Experimental Procedures." The culture supernatants containing naturally elaborated MVs were separated from bacterial cells by low speed centrifugation at the late-logarithmic phase of growth (Fig. 1A). This stage of growth has been proven for GC to accumulate native MVs (17). Subsequently, cell pellets were used for isolation of membrane proteins by a sodium carbonate extraction procedure (40). This method facilitates efficient enrichment of membrane proteins and ensures the suitability of the samples for mass spectrometry analysis (39, 40, 63). To provide enough material for proteomic analyses of MVs, the entire amount of each cell-free culture supernatant (about 1.0 L) was filtered to remove any remaining bacterial cells and subjected to ultracentrifugation using optimized protocol for GC (18). Transmission electron microscopy of the harvested MVs fractions demonstrated the presence of spherical, tubular, and lobed MVs with wide variation in dimensions (Fig. 1B). Similar morphological types of MVs released by GC were reported previously (64). The examination of the purified cell envelopes and MVs by 1 D SDS-PAGE revealed, as expected, the presence of common and distinct proteins, and the differences were particularly apparent in the MVs fraction (Fig. 1C). The most prominent proteins migrated at about 35 and 25 kDa, likely corresponding to the GC major porins PorIB (P.IB) and PorIII, respectively (65, 66).
Comprehensive identification of cell envelope and MVs proteins represents a challenge because they are commonly hydrophobic, have a basic charge, and are often of high molecular weight. Thus, it is widely accepted that two-dimensional gel electrophoresis has very limited capabilities for the separation of integral membrane proteins (21, 67). Instead, a multidimensional protein identification technology that separates proteins using a combination of two different kinds of liquid chromatography (2D-LC) prior to protein identification significantly reduces the complexity of the sample at the peptide level, resulting in the identification of a greater number of proteins. Accordingly, we used a comprehensive proteomic platform including iTRAQ coupled with 2D-LC/MS/MS to identify and quantitatively compare proteins in both the GC cell envelope and naturally elaborated MVs. This proteomic approach allows for simultaneous comparison of up to eight samples of complex protein mixtures (68). In a four-plex experiment, proteins in each sample are subjected to digestion by trypsin or other proteolytic enzymes, and the obtained peptides are labeled at their free amine group with one of the four available mass tag labels: 114, 115, 116, and 117 (Fig. 1D). While, in an eight-plex experimental approach, in addition to the latter tags, 113, 118, 119, and 121 are also used. Comparison of the intensity of these reporter labels permits the relative quantification of identical peptides in each digest and thus the proteins from which they originate (68). In our studies, the four-plex iTRAQ was used on two separate occasions to label either 100 or 40 μg of cell envelope or MVs proteins, respectively, obtained from each GC strain (Fig. 1D). Thus, four independent iTRAQ experiments were performed in total. The following iTRAQ tags were used for respective GC strains: 114 for FA1090, 115 for F62, 116 for MS11, and 117 for 1291. After labeling, the samples were pooled and subjected to SCX fractionation followed by a reversed-phase separation and protein identification, and quantification using MALDI-TOF MS/MS (Fig. 1D).
Strategy for Rigorous Proteomic Profiling of the GC Cell Envelopes and MVs
The proteome mining of the GC cell envelopes revealed 765 and 568 proteins in two independent iTRAQ experiments (Fig. 2A and supplemental Table S2). Further, 308 and 213 proteins were detected in the MVs fractions (Fig. 2B and supplemental Table S3). All of these proteins were identified with <1% false discovery rate. There were 533 and 168 common proteins in the cell envelopes and MVs, respectively, identified in all four GC strains in both proteomic studies (Fig. 2A and 2B).
To quantify the relative abundance of identified proteins, strain FA1090 was arbitrarily selected as the reference strain and the iTRAQ ratios were calculated as follows: F62/FA1090 (115/114), MS11/FA1090 (116/114), and 1291/FA1090 (117/114). Similarly to our previous study (41) a >2.0-fold change in the iTRAQ ratios was chosen as a criterion for differential protein abundance (Fig. 2C and 2D). Subsequently, proteins without associated iTRAQ ratios, quantified in only one experiment, and displaying variable reporter ratios were eliminated (Fig. 2E). Hence, only these proteins were considered, which in two independent experiments were consistently identified as either ubiquitously (iTRAQ ratios of 0.5<proteins<2) or differentially expressed (down- and up-regulated with calculated p < 0.05 and the reporters ratios of either <0.5 or >2, respectively). This rigorous strategy eliminated nearly 40 and 68% of proteins identified in the cell envelope and MVs fractions, and the total number of quantified protein species decreased to 316 and 57, respectively (Fig. 2E, Tables II⇓⇓–V and supplemental Tables S4–S5). These proteins were subjected to further computational analysis.
Bioinformatic Analyses
Coupling bioinformatics prediction with experimental subcellular proteomics is particularly important because cytoplasmic proteins are repeatedly identified as constituents of the cell envelopes and MVs in both Gram-negative and Gram-positive bacteria (21, 63, 67, 69⇓–71). Many of the cytoplasmic proteins co-purify as components of large protein complexes that are localized to the membrane, whereas others are likely present as a function of cell lysis occurring during culture (63). We reasoned that the latter group might be higher in autolytic bacteria like GC (13). To analyze the subcellular distribution of identified proteins, a combination of bioinformatics tools was used including PSORTb, CELLO 2.5, and SOSUIGramN (43⇓⇓–46). A majority-votes strategy was chosen for the cellular assignment of proteins, and in these cases where the three engines assigned different localizations, a particular protein was allocated to a group of "unknown subcellular localization." Additionally, SignalP and TatP servers were employed to analyze the predicted amino acid sequences of individual proteins for the presence of either N-terminal signal peptide (47) or specifically Twin-arginine signal peptide cleavage sites (48), respectively (Tables II⇑⇑–V). These analyses revealed that in the cell envelope fractions the total numbers of 1, 21, 43, 45, 171, and 43 proteins were localized either extracellularly, to the outer membrane, periplasm, inner membrane, cytoplasm, or unknown, correspondingly (Tables II and IV, and supplemental Table S4). The predicted subcellular distribution of proteins identified in the MVs fraction was as follows: 9-outer membrane, 5-periplasmic, 1-inner membrane, 32-cytoplasmic, and 10-unknown (Tables III and V, and supplemental Table S5).
In addition, the identified proteins were subjected to classification based on predicted major cellular function (Fig. 3). All phylogenetic Clusters of Orthologous Groups (COG) of proteins were represented within the cell envelopes (Fig. 3), with the majority of proteins predicted to be involved in cell motility and secretion (17%), cell envelope biogenesis (11%), and ribosomal function (17%). The latter COG was the most represented in the MVs fraction, followed by the functional category of proteins involved in cell envelope biogenesis (Fig. 3). COGs could not be assigned for 7 and 12% of proteins associated with the cell envelopes and MVs, respectively.
In conclusion, the subcellular distribution and biological functions of proteins identified in the GC cell envelope and MVs corroborated with previous studies assessing these proteome fractions in Gram-negative and Gram-positive bacteria (21, 63, 67, 69⇓⇓⇓⇓⇓⇓–76). Thus, our study adds to the growing amount of evidence that a large number of cytoplasmic proteins associates with cell envelope and MVs. Interestingly, similar observations come from proteomic profiling of membrane vesicles secreted from the plasma and endosomal membrane by various types of mammalian cells (77).
Ubiquitously Expressed Proteins Present in the GC Cell Envelopes and MVs
The quantitative analyses of relative protein abundance revealed that 305 and 46 proteins were uniformly present in the cell envelopes and MVs, respectively, in four GC strains (Tables II–III, supplemental Tables S4–S5). The large number of identified novel, and also previously described proteins precluded a detailed discussion, thus we focused only on several proteins assigned by bioinformatics analyses either to the cell envelope or with unknown localization (Tables II–III).
Among the stably expressed cell envelope proteins, several previously uncharacterized in GC have been identified including LPS-transport protein LptD (formerly OstA, Imp), a protein designated in the SwissProt database as adhesion MafA 1/4, a putative TonB-dependent receptor, and eight predicted outer membrane proteins NGO1205, NGO0834, NGO2121, NGO2111, NGO1985, NGO1956, NGO1955, and NGO1344. A plethora of predicted periplasmic and inner membrane proteins as well as proteins with unknown localization were also discovered (Table II). Moreover, our experiments revealed ubiquitous expression of a number of recognized proteins including an outer membrane-localized enzyme, Phospholipase A, and a surface-exposed lipoprotein Ng-MIP (Table II). Concurring with our data, these proteins were found invariably expressed in many different GC and meningococcal isolates (78, 79). Perhaps not surprisingly, proteins participating in the process of outer membrane biogenesis, BamA (Omp85) and BamD (ComL), were identified by iTRAQ experiments as equally present in GC strains (Table II). The pivotal role of Bam proteins comprising the β-barrel assembly machinery have been recognized in both model organisms E. coli and N. meningitidis, reviewed in (80, 81). In GC, a peptidoglycan-linked lipoprotein, BamD, although not essential for cell viability, was required for efficient transformation by species-related DNA and its depletion- caused growth defects and altered colony morphology (82). Finally, OmpA-like protein and NGO1686 quantified in our proteomic studies (Table II), could serve as additional examples of ubiquitously expressed proteins that play crucial functions in the GC biology. OmpA, characterized by Serino et al., is involved in the binding of GC to human cervical carcinoma and endometrial cells, entry into macrophages, intracellular survival in epithelial cells, and in vivo colonization (83). NGO1686 is a periplasmic, peptidoglycan-degrading peptidase and a virulence factor that protects GC against both nonoxidative polymorphonuclear leukocytes-mediated killing and oxidative-killing by hydrogen peroxide (84, 85). Together, these findings warrant further investigation of the newly discovered stably expressed proteins in the context of the GC general fitness and pathogenesis.
Our proteomic profiling of naturally shed MVs revealed a ubiquitous abundance of several outer membrane proteins including Phospholipase A, PorB, PilQ, adhesion MafA 1/4, and BamA (Table III). Sorting of these proteins into MVs was established in N. meningitidis and N. lactamica (75, 76, 78, 79, 86). One of the more fascinating features of a major GC porin, PorB (65) is its translocation from bacterial outer membrane and insertion into the membranes of eukaryotic cell organelles (87). It remains to be determined whether MVs play a role in this process, however, experimental data from different pathogenic bacteria suggest that such a mechanism could be mediated via a direct adherence and internalization of MVs to host cells (16). Further, we discovered that LptD was also uniformly distributed into the MVs (Table III). Consistent with these results, sorting of LptD into the naturally released MVs was observed in other examined Neisseriaceae, E. coli, and Pseudoaltermonas antarctica (21, 75). The localization of this organic solvent tolerance protein into MVs might contribute to the bacterial survival under hostile conditions (21). Among other proteins present at similar levels in GC MVs were catalase, DsbD, and newly identified proteins including a predicted lipoprotein NGO2139, a putative carboxy-terminal processing protease NGO0572, and an uncharacterized protein NGO2054 (Table III). The intriguing location of catalase in MVs has also been reported in Helicobacter pylori (88). Further studies will be required to determine whether catalase is enclosed within the MVs or decorates their surface, perhaps because of the release of the cell contents after bacterial cell death. However, it is tempting to speculate that the MVs localization of this potent enzyme contributes to the remarkable resistance of GC against the oxygen-dependent mechanisms of bacterial killing within the human host. In addition, our experiments suggested that a virulence factor, which specifically cleaves human IgA1, extracellular metalloprotease IgA1 (89), was uniformly present in MVs isolated from GC strains (Table III). Although protease IgA1 was the first autotransporter described in Neisseriaceae (89), its presence in MVs has also been observed in N. meningitidis (86, 90, 91), which further suggests that IgA1 might be associated with the MVs.
Differentially Abundant Proteins in the GC Cell Envelopes and MVs
Considering the recognized differences between GC strains (Table I), it was not surprising that the iTRAQ experiments revealed 22 differentially abundant proteins in the cell envelopes and MVs (Fig. 2E, Tables IV–V). Worth noting, among the significantly altered proteins, were well-characterized components of the multidrug efflux-pump complex, MtrC-MtrD-MtrE, where "Mtr" corresponds to multiple transferable resistance. The orchestrated work of this three-component machinery provides an increased resistance to macrolide antibiotics, and antimicrobial compounds that GC might encounter in the human host including bile salts, progesterone, and antimicrobial peptide, LL37 (92⇓–94). The relative abundance of the channel protein MtrE, the periplasmic accessory lipoprotein MtrC, and the inner membrane component MtrD was about threefold higher in the strain MS11 in comparison to that in FA1090 (Table IV). In contrast, strains F62 and 1291 displayed similar relative levels of Mtr constituents compared with FA1090. Consistently, the increased abundance of MtrE was observed in the MVs fraction isolated from strain MS11 (Table V). Warner et al. (94) revealed that the elevated expression of mtr operon in strain MS11 is because of a novel mutation, mtr120, which yields the highest reported levels of Mtr-based resistance. In the same report, the authors provide experimental evidence for the correlation between an increased gradient of antimicrobial resistance and the levels of in vivo fitness. Indeed, studies in both the male urethritis model and in the murine model for GC infection clearly demonstrated that strain MS11 was more infectious than FA1090 (61). Interestingly, however, introducing the mtr mutation into strain F62 did not increase its infectivity (93), suggesting that another strain-dependent factor(s) might contribute to the levels of pathogenicity. Our iTRAQ experiments revealed previously unrecognized discrepancies between compared GC strains including differential abundance of NGO1889 encoding a putative lipoprotein, NGO0926 coding for a putative peroxiredoxin family protein, as well as putative uncharacterized proteins NGO1438, NGO1056, NGO0861, and NGO1949 (Tables IV–V). Future studies will establish whether these proteins represent virulence determinants contributing to the differential fitness observed between GC isolates.
Immunoblotting Analyses Confirm the iTRAQ Data
We were surprised that iTRAQ analyses indicated that AniA, which is an anaerobically induced surface-exposed outer membrane glycoprotein (95⇓–97), was also ubiquitous in membrane fractions isolated from GC strains cultured aerobically (Table II). Therefore, we have chosen this protein as a first candidate for verification of our proteomic experiments. The gene encoding AniA (NGO1276) was cloned, overexpressed and a truncated soluble domain of AniA was purified to 95% purity as described under "Experimental Procedures." This purified antigen was used to immunize rabbits and obtain anti-AniA antibodies. Subsequently, the same amounts of cell envelope and MVs proteins (15 μg) extracted from GC strains FA1090, F62, MS11, and 1291 were separated by 4–20% gradient SDS-PAGE, transferred, and probed with anti-AniA antiserum. An example of such immunoblotting analysis is shown in Fig. 4A. As expected, there was a shift in migration of the purified truncated AniA (rAniA) in comparison to the native protein, which migrated at about 50 kDa. In agreement with the iTRAQ experiments, similar levels of AniA were present in the cell envelope fractions isolated from these four GC strains, and the protein was not detected in the MVs. Additionally tested GC strains including FA19 and a few clinical isolates (LG1, LG14, LG20, and LG26), expressed AniA at comparable levels during aerobic growth (Fig. 4B). As anticipated, the amounts of AniA were significantly decreased in comparison to that detected in bacteria cultured anaerobically in the presence of nitrite (Fig. 4B, FA1090 -O2) (98).
To further validate iTRAQ experiments, the immunoblotting analyses of cell envelopes and MVs fractions isolated from FA1090, F62, MS11, and 1291 were performed with anti-Ng-MIP and anti-MtrE antisera (Fig. 4A). Overall, these studies confirmed iTRAQ quantifications. Similar levels of Ng-MIP were detected in all GC cell envelopes, whereas the level of MtrE was increased in strain MS11 (Table II and Fig. 4A). In addition, Ng-MIP was detected in the MVs fractions (Fig. 2A). This protein was identified by MS/MS but failed our rigorous criteria for protein quantification and thus was not included in the final list of 57 MVs proteins (Fig. 2E and supplemental Table S3).
Lastly, we aimed to evaluate the subcellular localization of several newly identified proteins. We have chosen predicted outer membrane protein NGO1985, and proteins with unknown localization NGO2139 and NGO2054. A homolog of ribosomal protein RpsM, NGO1821, and a periplasmic catalase, NGO1767, were also selected. We engineered an epitope-encoding tail, 6×His-tag, at the 3′ end of individual genes in their respective loci of FA1090 chromosome. Subsequently, liquid cultures of GC FA1090 carrying C-terminal-tagged proteins were harvested, subjected to the fractionation procedures, and processed for the detection of His epitope. The monoclonal anti-His antisera recognized recombinant RpsM, NGO1985, and NGO2054, which migrated according to their predicted molecular weight of 13.4-, 21.7-, and 10.5-kDa (Fig. 4C). The analysis failed to detect NGO1767 and NGO2139 (data not shown). Furthermore, these experiments showed that RpsM localized predominantly to the soluble fraction containing cytoplasmic proteins and was also present in the GC cell envelope, whereas both NGO1985 and NGO2054 were detected in the cell envelope and MVs fractions (Fig. 4C). Together, these results provided additional validation of our proteomic studies.
A Minor Outer Membrane Protein, LptD, is Essential in GC
The iTRAQ approaches led to the identification of 34 proteins that were ubiquitously expressed in four GC strains and identified in both cell envelopes and MVs fractions (Tables II–III). We propose that these proteins may represent attractive therapeutic targets, particularly if they are conserved throughout different GC strains and play an important biological function. As proof of principle, we focused on LptD, which was uniformly present in the cell envelopes and MVs in analyzed GC strains. This protein has not been previously characterized in GC, whereas in E. coli, LptD is an essential low-abundant outer membrane β-barrel protein involved in the cell envelope biogenesis (99). LptD is highly conserved among Gram-negative bacteria and plays a crucial function in transporting lipopolysaccharide into the outer leaflet of the outer membrane (100). Its role in the transport of lipopolysaccharide was first discovered in N. meningitidis but interestingly LptD was not found essential for the survival of this bacterium (101). To verify the notion that this gene is essential in GC (102), we engineered a strain in which chromosomal expression of lptD was placed under the control of IPTG-regulatable promoter, PlaclptD (Fig. 5A). This strain grew robustly on GCB agar and in liquid media supplemented with 100 μm IPTG, whereas it was unable to form colonies upon direct plating from freezer stocks onto plates without IPTG (Fig. 5B and data not shown). Moreover, within three hours of culture in GCBL broth in the absence of IPTG there was a cessation of growth and at 5 hours the cell viability decreased almost 13-fold as compared with the strain expressing LptD (Fig. 5B and 5C). Consequently, upon depletion of LptD, the FA1090 PlaclptD failed to form colonies when plated on solid media lacking IPTG (Fig. 5D). We noted, however, that sometimes a few colonies, likely carrying suppressor(s) mutations, were able to arise on this media (Fig. 5C). These colonies had drastically changed morphology as compared with that producing LptD. Individual colonies were characterized by an irregular edge; increase opacity, and a granular, rugose appearance (Fig. 5D). Growth defect and increase in colony opacity were also observed in N. meningitidis lptD mutant strain (101). Together, our observations support that LptD plays a pivotal function in GC.
A Lack of NGO1985 Dramatically Affects GC Cell Envelope Permeability Barrier
Finally, we have begun to address whether the newly identified proteins are important for GC physiology, and thus could serve as potential pharmacological targets. Six predicted outer membrane proteins with unknown function were selected for initial characterization including NGO1985, NGO1344, NGO1955, NGO2111, NGO2121, and NGO2139. These proteins were identified by iTRAQ as uniformly present in the cell envelopes derived from examined GC isolates (Table II). In addition, our fractionation experiments coupled with immunoblotting analyses showed that NGO1985 was localized to both cell envelope and MVs (Fig. 4C). The BLAST searches of the predicted amino acid sequences against NCBI and JCVI CMR (103) databases, revealed that all ORFs encode conserved hypothetical proteins with up to 50% identity throughout different species belonging to Neisseriaceae. Further, a presence of a putative membrane binding BON domain, bacterial OsmY and nodulation, was identified in NGO1985. This domain is found in a family of osmotic shock protection proteins, a family of hemolysins, and a group of nodulation specificity proteins and secretory channels (104). The Omp85- and the AsmA-family domains, which are characteristics of proteins involved in the assembly of outer membrane, were identified in NGO1955 and NGO1344, respectively (105, 106).
The presence of these conserved domains suggested that these proteins function in the GC cell envelope homeostasis. To test this hypothesis, we first created in-frame deletions of individual genes. Subsequently the isogenic wild-type, FA1090, as well as Δngo1985, Δngo1344, Δngo1955, Δngo2111, Δngo2121, and Δngo2139 were examined for their susceptibility toward a variety of compounds. All strains were suspended to 5 × 105 CFUs/ml, serially diluted, and plated on GCB agar either with or without different membrane-perturbing agents as well as chloramphenicol. An absence of individual proteins had no effect on GC growth under permissive conditions; however, loss of NGO1985 and NGO2121 resulted in significant inhibition of bacterial growth in the presence of bile salts and antimicrobial peptide, polymyxin B (Fig. 6A). Additionally, when compared with the parent strain, Δngo1985 displayed dramatically decreased survival upon plating on media supplemented with detergents including Tween 20, SDS, and urea as well as chloramphenicol (Fig. 6B). This strong sensitivity phenotype was reversed in the complemented strain, Δngo1985/Plac1985, to the level observed in the wild-type bacteria (Fig. 6A and 6B). We concluded that loss of NGO1985, in particular, dramatically affected the general GC cell envelope permeability barrier. These studies further underscore the concept of ubiquitous cell envelope and MVs proteins as potential targets for new therapeutic interventions.
Final Remarks
Significant research in the GC field has been dedicated to elucidating the function and structure of several important virulence factors that are not stable because of both phase and antigenic variation. As such these proteins are not good candidates for developing therapeutic approaches. An example of this challenge was the monovalent pilus vaccine, which failed in a clinical trial (10). Our study began to establish a map of proteins in the cell envelope and MVs of a clinically relevant human pathogen. To the best of our knowledge, this is also the first time iTRAQ technology was used to elucidate the composition of bacterial MVs. Substantial research effort will be required to establish whether the proteins identified herein play important pathophysiological functions, to examine their phase and antigenic stability, and conservation throughout current GC clinical isolates, as well as their antigenic potency. Nevertheless, this study provides a framework for future research to elucidate the potential of the identified proteins as novel drug and vaccine targets.
We thank Dr. Josephine Bonventre for her excellent assistance, Dr. Stacey Harper (Department of Toxicology, OSU) for the use of the microscope, and Lisa Jones (Proteomics Facility, FHCRC) for assistance with performing proteomics experiments. We are grateful to Dr. Ann Jerse (Uniformed Services University, Bethesda, MD) for a generous gift of GC strains and anti-MtrE antisera. We thank Dr. Hank Seifert and Dr. Elizabeth Stohl (Northwestern University, Chicago, IL) for helpful discussion regarding GC complementation and plasmid pGGC4. We are thankful to Dr. Mariagrazia Pizza (Novartis Vaccines and Diagnostics, Siena, Italy) for providing the anti-Ng-MIP antisera. We also thank Dr. Robert Chalkley (University of California, San Francisco) and the PRIDE Team for their assistance in the mass spectrometry data deposition.
Author contributions: R.A.Z. and A.E.S. designed research; R.A.Z., I.H.W., J.V.W., P.R.G., and A.E.S. performed research; R.A.Z., P.R.G., and A.E.S. analyzed data; R.A.Z., P.R.G., and A.E.S. wrote the paper.
↵* This work was supported by the Medical Research Foundation of Oregon seed grant F0731A and the General Research Fund (OSU), as well as startup funds from OSU to AES. Additionally, we would like to acknowledge Undergraduate Research, Innovation, Scholarship and Creativity (OSU) for funding to J.V. Weber.
↵ This article contains supplemental Tables S1 to S5 and supplemental Material S1 to S4.
↵1 The abbreviations used are:
- Neisseria gonorrhoeae
- acetonitrile
- cell envelopes
- colony-forming units
- Clusters of Orthologous Groups
- GC agar plates
- GC broth liquid medium
- isopropyl-β-D-galactopyranoside
- iTRAQ
- isotope tagging for relative and absolute quantification
- lipopolysaccharide
- MVs
- membrane vesicles
- two-dimensional liquid chromatography
- strong cation exchange
- trifluoroacetic acid.
- Received March 26, 2013.
- Revision received February 28, 2014.
- © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
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|While walking to class, Jeff and Britta spot Annie flirting with Vaughn Miller. Jeff is still bothered by Britta's ex-boyfriend but Britta claims she is over him. Over in the study room, Abed invites Shirley and Pierce to join him and Troy for movie night in his dorm room. Outside the room, Annie meets up with Britta before the study group meeting and asks for her permission to date Vaughn. Annie explains that she has given up on Troy who seems more interested in Abed than her. Although Britta gives her blessing to Annie, she later finds Jeff in the cafeteria and alerts him to the situation. As she expected, Jeff strongly disapproves referring to Vaughn as a "gateway douchebag" and warning her that soon other similar douchebags will want to date Annie.
|Meanwhile at Abed's dorm room, Troy, Shirley, Pierce, and Chang gather to watch and ridicule a movie called "Kickpuncher". They all take turns mocking the film, but Pierce's lame attempts fail to amuse anyone. Afterwards, Abed invites Pierce for another movie night tomorrow oblivious to the others objections. On the campus quad, Jeff and Britta resolve to end Annie's new relationship with Vaughn after seeing them canoodling. They decide to manipulate her former crush Troy into helping them by convincing him Annie is in love with him. meanwhile, Pierce recruits the Greendale sketch comedy troupe to help him write a few good jokes before the next screening.
|Over in the student lounge, Troy finds Annie and tries to flirt with her. Annie rejects his advances just as Vaughn walks in. He assumes the worst when Troy blurts out that Annie is in love with him. Annie denies this but Vaughn doesn't believe her and immediately breaks up with Annie. She then learns from Troy that Jeff and Britta were responsible for what just happened. She goes to the study room and angrily tells them off for meddling in her love life. Later that night, Pierce attends Abed's movie screening but the others become suspicious when he immediately starts delivering rapid fire jokes. He confesses to coming prepared after failing to deflect their criticisms. Leaving in a huff, he trips and falls making a huge mess but is ecstatic when everyone laughs.
|Jeff and Britta arrive for the study group meeting the next day looking to apologize to Annie. She refuses, saying they need to treat her as an adult and not interfere in her love life. Britta lashes out and tells her if she wants to be treated as an adult she needs to stop dating her ex-boyfriend. Jeff realizes she lied about her reasons for wanting to break Annie and Vaughn and was jealous. Britta in turn questions Jeff's motivations, hinting at his own feelings for Annie. The conversation becomes heated as the other study group members join in. Jeff tries to put an end to the arguing by conceding that although this group is as close as a family, everyone could become a romantic prospect since they aren't actually related; this assessment causes the group to reflect and consider the possibilities.
|Annie stops the group's musings, feeling that they are getting off topic. She states that she genuinely likes Vaughn. The group then hears music playing, and they leave the study room to investigate. Outside, they find Vaughn, and he begins to serenade Annie with an original song he wrote. When he finishes singing he manages to win over the study group with the songs honesty and sincerity. Annie runs over and embraces Vaughn and after a brief chat goes back to the group. She asks if it's okay with them if she goes with Vaughn and both Jeff and Britta give Annie her their blessing. Pierce then grosses everyone out by asking if he has a shot with anyone in the study group.
End tagEdit
|Troy and Abed shoot their own fanvid of Kick Puncher. An abbreviated version of this was shown on the original broadcast airing. The full version is seen on the right.
Recurring themesEdit
|Continuity lists
Running gagsEdit
Pop culture referencesEdit
Meta referencesEdit
Community content is available under CC-BY-SA unless otherwise noted.


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The day before Christmas I signed up for dictionary.com's word of the day e-mail. It's free (but given the amount of advertizing on that site, I'm sure they sell your information. Oh, well.)
Today's word is "Paltry"–of little worth or insignificant.
Did you know that the compartive and superlative forms are "paltrier" and "paltriest?" Did you know "paltry" had a comparitive and superlative form?
"This appetizer course is paltry. I've never seen paltrier!"
Or, "That's the paltriest excuse for a defense I've ever seen in the NFL!"
Okay, maybe that does work.


text: Lace up the 860 v9 and you get responsive cushioning and reliable support when you hit the road for your run. The 860 v9 features TruFuse midsole that delivers sustained cushioning, while a dual-density post helps ensure stability mile after mile, so you can go the distance. ONLY available for in store purchase.


text:
Street Improvements for Low Heaton
We worked with a group of 20-30 residents establish a new Community Association that is working to improve the area by addressing rubbish, the appearance of planters and back lanes, creating safe places to play, improving gardens and green spaces, and tackling traffic/parking issues. They are also creating more social and community activities for local people.
We are providing ongoing help to Low Heaton Community Association to secure external funding from charities and other sources.


text:
Have you ever thought of making your own Christmas cards?
This year I decided to give it a try. I got my watercolor paints out. Then looked around on Pintrest to give my creative side a kickstart. I painted a few cards like the ones that I found on Pintrest and then as I got into it I started creating my own designs. It was a lot of fun!


text:
$ 85.00
Explains the function and construction of evaporators. Covers direct-expansion, dry-expansion, and flooded evaporators, as well as systems using multiple evaporators. Discusses ways to boost evaporator performance. Explains evaporator defrosting, maintenance, and troubleshooting. Describes the function, operation, and maintenance of various metering devices, including hand-operated, automatic, thermostatic, thermal-electric devices.
TPC Training Systems is authorized by IACET to offer 0.5 CEUs for this program.


text:
In Android, ImageView class is used to display an image file in application. Image file is easy to use but hard to master in Android, because of the various screen sizes in Android devices. An android is enriched with some of the best UI design widgets that allows us to build good looking and attractive UI based application.
Important Note: ImageView comes with different configuration options to support different scale types. Scale type options are used for scaling the bounds of an image to the bounds of the imageview. Some of them scaleTypes configuration properties are center, center_crop, fit_xy, fitStart etc. You can read our ScaleType tutorial to learn all details on it.
Below is an ImageView code in XML:
Make sure to save lion image in drawable folder
<ImageView android:id="@+id/simpleImageView" android:layout_width="fill_parent" android:layout_height="wrap_content" android:src="@drawable/lion" />


text:
Running out of foundation (the kind you slap on your face, not the kind you wear under 16 layers of clothing now it's the end of 'summer'* ). Place almost-empty plastic bottle into clear plastic bag in case it discovers a hitherto hidden pocket of gloop once it's in my handbag.
Go into pharmacy store I won't name because you know how the internet hates any form of advertising, so let's just call it 'Shoes'.** In fact, only go into it myself because I have a gift voucher as compensation for standing in another branch of the chain last week for 1hr 20 minutes, after which time I *still* left without my medication, but that's another post.
Approach counter of cosmetics brand. Talk to plastic woman, whose epidermis arrived in position 5 minutes before the rest of her did, as she's wearing so many layers of slap, it's visible from the International Space Station.
"I'd like one of these, please," I say, as I hold up the diaphanous bag (it's also see-through) which contains the beige tube of gunk. (I have to take it with me, as there are so many adjectives in the description of what it does, it'll be the end of next summer* before I remember them all).
She takes the bag from me and looks at the front the of the bottle.
"I need to see the back of it for the shade," she tells me. And she makes to remove the bottle from the clear holdall.
"You don't need to take it out," I proffer helpfully, in a loving and caring, derisory sniggering kind of a way. "The bag's see-through on all sides."
She snorts. She turns the bag around. All on her own, but then, I am in a hysterical heap on the floor at this point. She gleans the information and finds the product in her drawers. (Now don't start). I say thank you, hand over my gift card and scarper to split my sides on the sidewalk. (What?)
What makes me think she's a Brexit voter?
* Old English word you may not have come across if you're under 108
** Other footwear names are available
Posted on September 17, 2017, in Uncategorized. Bookmark the permalink. Leave a comment.


text:
The Georgia Swarm today announced that it has signed Joel Tinney to a two-year deal. The Swarm has also signed free agents Bennett Drake and Zach Tomkinson to two-year contracts and Braden Wallace to a one-year contract.
Tinney, 23, was selected No. 18 overall in the 2018 NLL Entry Draft. The speedy midfielder was team captain of the Blue Jays during his senior season, leading the team to the NCAA Tournament Quarterfinals.
"Joel had a great career at Johns Hopkins University, and he was an extremely productive box lacrosse player in Canada prior to his college career," Swarm Owner and General Manager John Arlotta said. "He is known as an ultra-competitive player with great leadership qualities. He also brings great flexibility to the roster, as he is capable of playing out of both the front end and the back end. We're excited to have Joel signed and part of the Swarm organization."
That excitement is shared by Tinney, and with his contract signing out of the way, he is ready to join his fellow 2018 Swarm draftees and fellow JHU alum and new member of the Swarm, Holden Cattoni.
"Being a Canadian kid and growing up watching guys that are on the Swarm right now and seeing them win two years ago, it's obviously an organization that has that winning mentality, and that's the kind of mentality that I carry with everything I do," Tinney said. "It seems like a great fit for me, and I'm just excited that this is in the rear-view mirror now. Now I can focus on what goes on the floor."
With Johns Hopkins, Tinney became the fastest midfielder in Blue Jay history to reach the 30-goal career mark. Through 50 career games, the Innerkip, Ontario native recorded 119 points (68G, 51A), 107 ground balls, and 24 caused turnovers.
Tinney played in the Ontario Junior A Lacrosse League with the Toronto Beaches and Orangeville Northmen from 2015-16. He posted 76 points (28G, 48A) in 19 games.
"If you ask any guy on the Swarm, just for me barely being a part of this organization for a month, you can tell that it is a winning-mentality, and the championship is our main goal," Tinney said. "For me, I just want to get through a positive Training Camp, learning experience, and crack this roster and be able to help them out any way possible come Dec. 1."
Drake, 22, was drafted in the 2017 NLL Entry Draft by the Georgia Swarm (No. 47 overall). Graduating from the University of Albany, Drake participated in the Swarm's 2017-18 Training Camp. He finished his collegiate career with 111 points (80G, 31A). Drake also boasts a family connection on the Swarm. Dan Ladouceur, the Swarm's assistant coach, is his uncle.
Tomkinson, 27, graduated from Mars Hills University and was signed by the Buffalo Bandits as a free agent in 2017. In his senior season at Mars Hill, Tomkinson finished with 31 points (20G, 11A) in 14 games played.
Wallace, 24, was picked No. 60 overall by the Buffalo Bandits in the 2017 NLL Entry Draft. As a midfielder at Rochester Institute of Technology, Wallace played in 91 games and recorded 141 points (94G, 47A), grabbed 76 ground balls, and caused 20 turnovers.
Season Tickets for the Swarm's 2018-19 season are On Sale Now! Season Ticket packages include 10 games (nine regular season games and one postseason game) and a handful of VIP benefits. Join Sting City by reserving your membership today by calling 844-4-GASWARM.
Georgia released its full 2018-19 regular season schedule on Sept. 6. The 2018-19 NLL regular season runs from Dec. 1 to April 20, and fans can view the entire Swarm schedule here.


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text:
the first Young Socialist Conference, this Easter, can give only limited encouragement to those who hope for a movement of young people carrying into broader socialist terms the activity and idealism of CND. The Conference was dominated by a violent confrontation of left and right wingers, over the Bomb, Gaitskell's leadership, the paper "Keep Left", and the "guiding" role of Transport House in the Young Socialists' development.
Transport House itself was heavily represented in the proceedings. The Chairman, George Brinham, presided with a kindly, but fumbling incompetence—at times like Canute, waving hopelessly at the storm of hostility. Supporting Mr. Brinham was the Assistant National Agent, Reg. Underhill, smoothly effective, skilful enough to argue the shouting delegates into silence on several points of legality. Regional Youth Officers stewarded the meeting. Massed together, they looked like chuckers-out—which, at one point, they were.
Two major speeches were made from the platform for the National Executive. Ray Gunter, posing as an ordinary member of the Labour Party, neither right, left, nor centre, denounced Keep Left, and probably swung the narrow vote supporting the Transport House ban. He spoke with accomplished eloquence, evoking loyalty to the Party, Nye Bevan, and all the rest of it, to condemn the "Trotskyists". They have every right of free speech, he said, but no right to be in our Party. The hurt, angry faces of the many delegates who supportered Keep Left, but who think of themselves firstly as Young Socialists, showed one what Gunter was doing. Mr. Crossman spoke to the Conference, of the need for Protest and Power, and was uncertainly received.
Faced with this obstructive pressure from the Platform, the Young Socialists obviously had a difficult job to set any direction of their own. The 350-odd delegates (from 726 nominal branches) had relatively little contact beforehand, and if there were natural spokesmen for the majority present in the hall, they rarely reached the platform. But the first session was promising, with an important procedural battle fought and won by the delegates, in relatively good humour.
The second session, held in secret, concerned New Advance, the official Young Socialist paper run from Transport House; the right of the movement to discuss politics at Federation level, and choose its delegates and motions independent of constituency parties; and Keep Left. The debate on New Advance looked at one point as though it might break up the Conference. A large majority of delegates obviously opposed Transport House control, and a circulated statement by the editor, denouncing the restrictions imposed on him, made support for Transport House difficult. The Platform's refusal to let the Editor speak caused pandemonium, and though tempers rapidly subsided, the effect of the outbursts was serious. The heckling and shouting had been led by relatively small groups in the hall, and even delegates who voted with the left, became impatient with the concerted interruptions.


text:
Yesterday, St Mark's had their Christmas Dinner for all the staff and pupils.
The whole school pulled out all the stops and dressed in their best Christmas outfits, sang songs and ate a lovely traditional Christmas meal served up by our brilliant lunch staff.
A lovely time was had by all.
Merry Christmas!
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Click here for more information.


text:
Introduction: Emergency Sushi Rolling Mat.
Skewer sticks and duct tape.
Step 1: Ingredients.
Duct tape.
Helper (https://www.instructables.com/member/fastesthamster/).
Skewer sticks or chop sticks.
Flat surface.
Pair of scissors.
Step 2: Make.
Lay the sticks of choice out on your flat surface. Use one stick and push against the ends of the sticks to get the edge straight. Cut a piece of duct tape and tape the edge of the sticks so that half the tape sticks to the sticks and the other half can be folded round on to the other side, but don't do this yet. Stick tape to the other edge with half the tape off the edge again. Press tape firmly onto the sticks. turn this assembly around and fold the tape over and press flat. Trim tape.
Step 3: Roll.
Roll the mat to make it nice and flexible.
Be the First to Share
5 Discussions
I actually carry a sushi rolling mat with me at all times just in case.
Not bad though I can't imagine having an emergency where I'm suddenly needed to roll sushi...
Maybe if classy zombies attack you can placate them with some maki while waiting for the tempura to get hot... who says your zombie plan shouldn't involve catering?
Hahahaha! I like it!
Believe it or not I could have used this a couple weeks ago!


text: TRUNK OR TREAT IS BACK FOR OUR 5th YEAR IN 2018! SUNDAY, October 21st at BOTH locations! 1230-2PM – Franklin (10001 St Martins Road) 4:30-6PM – Oak Creek (9160 S Pennsylvania Ave) Join APA for some safe Halloween fun! Open to the public! Bounce House, Pumpkin Patch, Pumpkin Decorating, Glitter Tattoos and more!! $4/Non Members $2/APA Members REGISTER HERE–> Registration Now Open!! FRANKLIN APA MEMBER 12:30pm FRANKLIN NON MEMBER 12:30pm OAK CREEK APA MEMBER 4:30pm OAK CREEK NON MEMBER 4:30pm Trunks are hosted by local area businesses – If you are interested in hosting a trunk, please email Rachel at [email protected]


text: How Filipino Migrants Gave The Grape Strike Its Radical Politics Strategize! , AgricultureFarmworkersLeft PoliticsStrikesThe PhilippinesBy David Bacon, Dollarsandsense.org - May 17, 2018The great Delano grape strike started on September 8, 1965, when Filipino pickers stayed in their labor camps, and refused to go into the fields. Mexican workers joined them two weeks later. The [...] READ MORE


text:
I am happy ...yeah! i managed to get a tall and fluffy chiffon cake. Pandan flavour has been a popular choice when comes to chiffon cake...the aroma fills my house ...yumm...
Thanks to nasi lemak lover for sharing this wonderful recipe
Recipe : (makes one 21cm chiffon pan)
5 egg yolks
20g sugar
60g water + 20g homemade pandan paste
80g corn oil
125g cake flour
5 egg whites
90g sugar
1/2tsp lemon juices or 1/2tsp cream of Tartar
1. Lightly whisk egg yolks and sugar till combined.
2. Add in water, homemade pandan paste and corn oil, mix well.
3. Add in cake flour, stir and fold till well combine.
4. Whisk egg whites and lemon juices till foamy, slowly add in sugar and continue beat until soft peak formed.
5. Take 1/3 of meringue and use a hand whisk to mix well with egg yolk batter.
6. Fold in 2/3 or meringue and use a spatula, fold with egg yolk batter till combined.
7. Pour into chiffon tins, bake at pre-heated oven at 160c (fan-forced) for 40-45mins.
8. Once baked, turn your chiffon tin upside down and cool completely before remove from the tin.