News Article | May 10, 2017
LA Connection Comedy Theatre Celebrates 40th Anniversary. Comedy Pioneer Kent Skov Founder says, “SHORT FORM COMEDY IMPROV is stronger and more popular than ever. They are the longest running Short Form IMPROV THEATRE in Los Angeles. 40th Anniv Celebration takes place May 13th 2017. Kent Skov, who created and founded THE LA CONNECTION COMEDY THEATRE also made a genre of comedy that is as popular today as it was way back in 1977 when he started and performed on the sidewalks in Venice Ca. In 1978 he opened the first brick and mortar theatre at the Crossroads of The World complex in Hollywood, CA. http://www.laconnectioncomedy.com By 1980 he opened a second location in Sherman Oaks, where its popularity gained, Kent and his troupe would appear on national TV on a regular basis making this genre of comedy nationally a HUGE HIT! When they opened, dubbing movies live it was called Improvision and they toured the state of California. This led to their daily segment on Thicke of the Night called Flicke of the night. The popularity of the segment led to their own TV series called Mad Movies with the LA Connection. It ran from 1985-1993: 2 years in syndication, 2 years on Nick at Night where it reached over a million viewers weekly then onto MTV for 3 years. Their dubbing of a Sherlock Holmes movie on A&E Network led to a CableAce award for "Best Comedy Special." Like an Emmy, while all the time, drawing big crowds to the 2 locations in Los Angeles. By 1998 they were taking the show on the road and had 178 company players. And the comedy was spreading all over the world. And even THE COMEDY STORE brings in their troupe to a sellout crowd multiple times. In 1993 LA CONNECTION COMEDY THEATRE stretches again with KIDS IMPROV which is still running today. Stars get their start at the LA Connection Comedy Theatre: Over the years stars got their start in comedy by joining the LCCT before they were stars they included: WILL FERRELL, MATTHEW PERRY, JON LOVITZ, HANK AZARIA, VICTORIA JACKSON, MINDY STERLING, VIC DUNLOP, to name a few. Today, LA CONNECTION COMEDY THEATRE is located in Burbank, CA on W. Magnolia Blvd, still drawing sellout crowds weekly, their comedy troupe is better than ever and funnier than ever. Skov announced today that this year Their 40th Anniversary will have a lot of special events to celebrate, one such event will take place in May with star studded party to celebrate their 40th Anniversary seeing many of its alum returning to pay homage to this LA Institution. Kent and his most popular comedy rep company are avail to appear on your show to perform. The alumni and all-star veteran cast show is Saturday May 13th from 8-10pm with after party. The CableAce nominated movie dubbing of Sherlock Holmes "The Woman in Green" will screen before at 7pm with guest stars from the TV show that appeared on A&E network. Wed. May 10 8pm Long form Kent Skov introduces 4 new long form games rarely scene that he created over the years. Thurs. May 11, 8:30pm Beyond Stand-up features top comedians from Steve and Barb North's Rep company Fri. May 12, 7pm Comedy Improv for Kids by Teens features are top teens from our LA Connection teen group 8-10:30pm Five improv groups from LAC's top veteran all-star casts. Features Less than Flattering 8pm Temporary Insanity 8:30pm Stranger than Fiction 9pm So Funny it Squirts 9:30pm Split Decision 10pm Sat May 13 5:30pm Comedy Improv for Kids by Teens 7pm Sherlock Holmes "Woman in Green" dubbed by LA Connection with a brand new hilarious comedy soundtrack. Nominated for a CableAce award for "Best Comedy Special" as seen on A&E Network. Live appearances by some of the cast. 8-10pm 40th anniversary celebration featuring alumni and celebrity guests performing with LAC's award winning veteran cast 2001 an Improv Odyssey show. Followed by after party. Sun May 14 Comedy Improv for Kids by Kids performed by kids 5-12 years old celebrating our 24th year. This is subject to a Mother's Day cancellation. Call first for reservations. 8pm Sunday Funny Sunday LAC's JV casts performs 2 improv sets back to back.
News Article | May 17, 2017
Fermentation specialists, Lallemand provide applied fermentation technical support and share their knowledge of fermentation application, the market needs, and commercialisation of new R&D innovations in the field of microbiology. With representatives all around the country, there’s a rep in every major wine region from the Hunter to Margaret River. Following a recent new partnership deal, Winequip are now the sole distributor for Lallemand biological products in WA, NSW, ACT, QLD, VIC, and TAS. In this blog series, we introduce you to the faces behind the Winequip and Lallemand, so you know who to look to in your part of the wine world. Next up, we meet Winequip Sales Representative for Western Victoria Rebecca Hellweg, a winemaker turned rep who loves being on the open road and is a keen Gridiron player! What is your role with Lallemand? As the Winequip Sales Representative for Western Victoria, my role with Lallemand is to provide product knowledge, insight and support to the clients located within my region. I am constantly looking out for the key products which will allow my clients to get the best results from their grapes, and to produce wines which are reflective of their regional style, and the winemakers individual vison. What do you love about your job? I love being on the road and getting to meet clients on their sites. Every winery is different, and every winemakers end goal is individual. It’s interesting to see how clients utilise the Lallemand tools they have at hand to tell the story of their vintage and their site. It also makes the job easier as Western Victoria (in my opinion) has some of the most beautiful landscapes in Australia. What makes Lallemand unique in your eyes? During my time winemaking, I had seen Lallemand products used worldwide, as they were trusted and reliable. Now on the other side of things, I am constantly impressed by the range of products, but as well as the way in which Lallemand keeps on innovating. Products such as the QTL yeasts are beyond innovative, and have such a useful application for the industry, as winemakers move towards a desire for lower sulphur wines, and cleaner ferments. Lallemand products are unique as they allow our clients to do their job with confidence. How do you think Lallemand’s role has changed in the wine landscape over the years? When I think Lallemand, I think innovation. The company is constantly reacting to the needs of the winemaker. The amount of technical support within Lallemand for us as representatives is amazing, as well as the information which we are able to pass onto our clients. Lallemand has helped to develop the conversation and sharing of ideas within the industry. When you’re not at work, where might we find you? When not at work, you’ll probably find me on Ranger Field, home of the Croydon Rangers Gridiron Club. Want to meet more of the team at Lallemand? Click here for Lallemand commercial director Jason Amos.
News Article | May 17, 2017
Fermentation specialist Lallemand provides applied fermentation technical support and shares its knowledge of fermentation application, the market needs and commercialisation of new R&D innovations in the field of microbiology. With representatives all around the country, there’s one in every major wine region from the Hunter to Margaret River. Following a recent new partnership deal, Winequip is now the sole distributor for Lallemand biological products in WA, NSW, ACT, QLD, VIC and TAS. In this blog series, we introduce you to the faces behind Winequip and Lallemand, so you know who to look to in your part of the wine world. Next up, we chat to Thomas Hamann of Winequip and Lallemand’s brewing sales representative. What is your role with Lallemand? I’m responsible for sales, national distribution and technical advice for the Lallemand Brewing Yeast portfolio. What do you love about your job? Free beer! And introducing new brewers to the fabulous diversity of strains. What makes Lallemand unique in your eyes? Lallemand offer excellent and prompt technical back-up for their products. Quite an achievement for a global company. How do you think Lallemand’s role has changed in the beer landscape over the years? The large range of brewing yeasts are great to see in this rapidly expanding craft beer market. With more strains on the way and with brewers being able to brew all year round, there’s a lot to look forward to in the next few years. When you’re not at work, where might we find you? At Leon’s Wine Bar… Yeah right! Most Fridays I’ll be leaning on the local brewpub bar debating a grist and a yeast selection with the brewer (or emptying their mash tun!). Want to meet more of the team at Lallemand? Click here for Lallemand commercial director Jason Amos, here for Winequip Sales rep Rebecca Hellweg, here for Sales Manager for WA Amanda Kramer or here for Winequip's Peter Kopiec, here for microbiologist Eveline Bartowsky, here for Technical Sales Manager Simon Kinley or here for Tanya Worontschak.
News Article | May 16, 2017
SAN JOSE, Calif.--(BUSINESS WIRE)--Lumileds today announced that George Craford, Lumileds Solid State Lighting Fellow, was selected for the IEEE Edison Medal for “a lifetime of pioneering contributions to the development and commercialization of visible LED materials and devices.” Craford will be presented with the medal at the IEEE Honors Ceremony in San Francisco on May 25, 2017, during the IEEE Vision, Innovations, and Challenges (IEEE VIC) Summit. Craford’s career spans from the early days when LEDs were first developed to delivery of high brightness LEDs suitable for commercial use in a variety of applications, including LED bulbs. He is best known for his invention of the yellow LED in 1972. Craford then led the development of increasingly brighter red, orange and amber LEDs. In 1979, Craford began work at Hewlett-Packard, where his team pioneered the development of AlInGaP LEDs using metalorganic chemical vapor deposition (MOCVD). MOCVD was then a relatively expensive lower volume process and had not been utilized for the high volume commercial production of LEDs. AlInGaP LEDs increased the performance of red and yellow LEDs by more than 10 times. Craford’s team continued to achieve technology breakthroughs in AlInGaP LEDs, eventually reaching 100 lm/W. “Not only was George responsible for substantial breakthroughs in technology, but with his team, ensured that the technology could be reliably and cost effectively manufactured,” said Mark Karol, 2017 IEEE Awards Board Chair. One can see the impact of Craford’s early work in the color LEDs now ubiquitous in traffic signals, emergency and automotive lighting. Craford’s later work focused on making white LED light cost effective for retail, office, architectural, outdoor and industrial lighting markets. In the early 2000s, his team’s work enabled commercialization of the first high power LEDs in the 10-20 lumen range. Such LEDs contributed to the creation of the first LED bulbs to meet the high efficiency and long lifecycle requirements to win the U.S. Department of Energy’s “L Prize” for a 60W-equivalent LED bulb. “George has terrific instinct for what will work, but at the same time he’s got that practical engineering side that drives a solution until it produces the best results,” said Jy Bhardwaj, Chief Technology Officer of Lumileds. Today, Craford is Lumileds Solid State Lighting Fellow at Lumileds. He is an IEEE Life Fellow and a member of the National Academy of Engineering. He has received numerous awards including the 2002 National Medal of Technology and the 2015 U.S. National Academy of Engineering Charles Stark Draper Prize. He has also been awarded the International SSL Alliance Global Solid State Lighting Development Award, the Strategies in Light LED Pioneer Award, the University of Illinois Alumni Distinguished Service Award, the IEEE Morris N. Liebmann Award, the IEEE Third Millennium Medal, the Optical Society of America Nick Holonyak Jr. Award, the International Symposium on Compound Semiconductors Welker Award, the Materials Research Society MRS medal, the Electrochemical Society Electronic Division Award and the Economist Innovation Award. Lumileds is the global leader in light engine technology. The company develops, manufactures and distributes groundbreaking LEDs and automotive lighting products that shatter the status quo and help customers gain and maintain a competitive edge. With a rich history of industry “firsts,” Lumileds is uniquely positioned to deliver lighting advancements well into the future by maintaining an unwavering focus on quality, innovation and reliability. To learn more about our portfolio of light engines, visit lumileds.com.
News Article | May 25, 2017
-- As one of the fastest growing sectors in home flooring popularity, Parquetry flooring allows home owners to create a look of timeless charm in any given space.O'Brien Timber Floors and their team of flooring professionals are proud to announce that "De Marque European Oak Engineered Parquetry", by Australian based flooring company Preference Floors, is now available to Melbourne residents who wish to enhance the appeal and beauty of their home or business.Parquetry flooring has been a part of history since 17th century France.The word "Parquet" was derived from the French term "Parchet" which means "a small enclosed space". 'Parquet de Versailles' are large diagonal squares which were introduced as 'Parquet De Menuiserie', meaning "parquet woodwork", which was generally used to replace Marble the most common form of flooring back then, as Marble required regular washing which resulted in the joints underneath the floor to rot.These days Parquetry floors are used to showcase creativity and opulent beauty in both palaces and private homes alike. Known also for their structural strength and freedom of design, this particular flooring has often been used to replace outdated marble flooring, (for a fraction of the price) and has been hailed for not only its beauty but it's ease of cleaning and upkeep.Parquetry flooring is now readily available in the modern world in either 15mm or 21mm thick planks and will be offered in a variety of colours and finishes, like the traditional herringbone and basket weave patterns, the timeless favourites of both aristocracy and the modern public.For those that prefer a more traditional look and feel, O'Brien's flooring specialists would be happy to install raw timber that can be sanded and stained to your specifications and lacquered in-home. The flooring also features micro bevelling on all four sides that ensure a perfect fit and flawless design.Parquetry flooring offers stability and original beauty, and customers can be assured that O'Brien's team of professionals will be proud to install your new flooring with the utmost attention to detail and your own specifications.With the flooring's tongue and groove locking mechanism O'Brien's installation team will be in and out before you notice, giving you quick access to your newly revamped space. This particular flooring also boasts a 25 year structural warranty and 20 year residential wear warranty, ensuring that your new floors will be a part of your home or space for decades to come.ABOUT O'BRIEN TIMBER FLOORSO'Brien Timber Floors ( http://www.obrientimberfloors.com.au/ ) is a premium flooring retailer located in Notting Hill, VIC, who offer "supply only" services for DIY projects as well as "supply with professional installation", to families and businesses Melbourne wide.If you would like more information about Parquetry flooring, or have questions for O'Brien Timber Floors team of experts, regarding a quote or installation, please feel free to contact them via their website, www.obrientimberfloors.com.au or by phone or email.Charlie O'Brien ( http://www.obrientimberfloors.com.au/ about-us 51 Howleys Road,Notting Hill, VIC, 31681300 500 firstname.lastname@example.orgSOURCE O'BRIEN TIMBER FLOORS
News Article | April 18, 2017
Sophia Antipolis, 18 April 2017. The use of smartphones to assist the continuity of care between hospital and community is set to be discussed at EuroHeartCare 2017, which will be held 18 to 20 May in Jonkoping, Sweden, at the Spira Cultural Centre. EuroHeartCare is the official annual meeting of the Council on Cardiovascular Nursing and Allied Professions (CCNAP) of the European Society of Cardiology (ESC). This year's meeting is held in collaboration with the Swedish Association on Cardiovascular Nursing and Allied Professions (VIC). The full scientific programme is available here EuroHeartCare is the event to attend for cardiovascular nurses and allied professionals. More than 500 delegates are expected from over 40 countries. During the two-and-a-half day event, members of the press can get the results of cutting edge research from original scientific abstracts and gain insights into topical areas of nursing and cardiovascular care in state-of-the art lectures by renowned experts. Journalists at the conference will hear about smartphone innovations for patients with cardiovascular disease. "Sophisticated wireless technologies are being developed for many aspects of care in the cardiac patient," said Dr Gabrielle McKee, programme chair of EuroHeartCare. "Remote ECG and rhythm monitoring have the potential for patients to alert the hospital when arrhythmias or a heart attack occur. These technologies are easy to use, yet safe, efficient and accurate, and should lead to patients responding and receiving treatment more promptly." "The amount of time patients stay in hospital is much, much shorter than it used to be," said Dr McKee. "After a major cardiac event we give substantial educational information to patients but the chances are that they don't take it all in. Smartphone technologies are enabling us to continue to educate patients, to monitor their risk factors and help promote goal setting, behavioural change and self-care in the long-term." The origins of obesity will be explored by international leaders in a dedicated session. Dr McKee said: "It is thought that when we became farmers instead of hunters and gatherers this increased the risk of famine, and those with the genetic make up to lay down fat survived better. In today's world, this adaptation plus the fact that we need less than half the calories of our forefathers, and that food has become more varied, appealing and calorific leave us at risk of eating more calories than we expend." Members of the press will discover how behaviour change can address the obesity crisis. "We need to shift the focus away from just the weighing scales and kilograms towards developing new habits," said Dr McKee. "For example not rewarding yourself with something to eat when something good happens. At the congress we'll discuss exciting ways that are helping people to change their behaviour which leads to a healthier lifestyle and subsequent weight loss that can be maintained long term." The use of mindfulness to improve mental health and wellbeing in cardiac patients is another hot topic. Journalists will get the most up-to-date research findings on this and other mental health strategies such as cognitive behavioural therapy for treating depression in patients with cardiac disease and heart failure. The theme "Team Work for Excellence in Cardiovascular Care" will be featured throughout the congress. A session on how to build a golden team strategy will show journalists how the multidisciplinary team delivers treatment for a heart attack within an hour from the onset of chest pain, referred to as the golden hour. One of the consequences of good health care and innovation is that we are living longer. Frailty is a growing issue that requires team work between cardiology and gerontology and will be discussed in a session on the new challenges in prevention and rehabilitation. Dr McKee said: "There are many challenges in today's care that mean that team work including cardiologists, surgeons, nurses, physiotherapists and nutritionists is needed to reinforce self-care and prevention messages. This will help patients not only to feel 'fixed' after a treatment in hospital but aware and educated with a care plan to help prevent recurrence."
News Article | May 4, 2017
Fermentation specialist Lallemand provides applied fermentation technical support and shares its knowledge of fermentation application, the market needs and commercialisation of new R&D innovations in the field of microbiology. With representatives all around the country, there’s one in every major wine region from the Hunter to Margaret River. Following a recent new partnership deal, Winequip is now the sole distributor for Lallemand biological products in WA, NSW, ACT, QLD, VIC and TAS. In this blog series, we introduce you to the faces behind Winequip and Lallemand, so you know who to look to in your part of the wine world. Next up, we chat to Simon Kinley. What is your role with Lallemand? I’m the Technical Sales Manager for Lallemand and I provide technical support for our multi winery site corporate customers, the Riverland region and South East Asia. I also serve in an ambassador role with our distribution partner Winequip on the east coast of Australia to support their sales team in these regions. What do you love about your job? The diversity throughout the year and even on the same day. Wine and winemaking technology never stops moving so there is a steady stream of new products and innovations emerging from the global wine industry all the time. Lallemand is at the forefront of many of these technologies and it is really exciting to be involved in these projects. For instance, in V15 I coordinated the world’s first commercial scale trials of our new low alcohol yeast – IONYS – in two Australian wineries. I feel proud to be a part of that. What makes Lallemand unique in your eyes? The people within the company are everything. From the top down there are passionate, driven people trying to solve common problems faced by winemakers around the world with innovative, cutting edge research from all around the world. Being able to provide support for our customers based on world expert’s knowledge in bacteria, sparkling or whatever field of winemaking is incredibly valuable. How do you think Lallemand’s role has changed in the wine landscape over the years? I think Lallemand’s role is being valued more and more by our customers. We provide excellent technical support and innovative products which is something that a lot of large companies have let slip in recent years. When you’re not at work, where might we find you? Working on a project at home or in the garden, out fishing in my kayak, at SA Base Camp obstacle course training or playing with my two sons. Want to meet more of the team at Lallemand? Click here for Lallemand commercial director Jason Amos, here for Winequip Sales rep Rebecca Hellweg, here for Sales Manager for WA Amanda Kramer, here for Winequip's Peter Kopiec or here for microbiologist Eveline Bartowsky.
News Article | February 27, 2017
Harris Teeter is proud to welcome customers to its Quarterpath Crossing Fuel Center on Friday, March 3, 2017 as the company celebrates its grand opening with a $0.20 off per gallon fuel promotion. The Fuel Center, which is located in close proximity to the Quarterpath Crossing Harris Teeter, will offer customers $0.03 off per gallon every day with the use of a VIC card, but shoppers are encouraged to fill up during the grand opening when the Center will feature a special $0.20 off per gallon discount March 3-5, 2017. This location is Harris Teeter’s first fuel center in Virginia. The company operates 15 other fuel centers throughout North and South Carolina. At each of its Fuel Centers, Harris Teeter strives to provide customers an excellent experience through high-quality products and great customer service. Fast Facts Store Address: Quarterpath Crossing Fuel Center, 1530 Quarterpath Rd., Williamsburg, VA 23185 Grand Opening Date: Friday, March 3, 2017 Store Hours: Staffed daily from 6 a.m. – 10 p.m.; fuel available for purchase by debit/credit card 24 hours Square Footage: 240 Fuel Dispensers: Four
News Article | February 22, 2017
The study was approved by the ethics committee of the medical faculty and the university clinics of the University of Tübingen and strictly adhered to Good Clinical Practice and the principles of the Declaration of Helsinki. The study is registered at ClinicalTrials.gov (https://clinicaltrials.gov/ct2/show/NCT02115516) and in the EudraCT database, number 2013-003900-38. The study was carried out under FDA IND 15862 and with approval of the Paul-Ehrlich-Institute. Volunteers were healthy, 18–45 years old, malaria-naive adults. The full list of inclusion and exclusion criteria is given in the clinical trial protocol (Supplementary Information). All volunteers, except those in the second part who received the every 5-day regimen, received 10 mg kg−1 or 620 mg chloroquine base (Resochin, Bayer) loading dose 2 days before the first dose of PfSPZ Challenge, whichever dose was less, followed by weekly doses of 5 mg kg−1 or 310 mg through 5 days after the last dose of PfSPZ Challenge. Volunteers who were immunized on days 0, 5, and 10 received chloroquine on days 0 (loading dose), 5, 10, and 15. Randomization was performed on the day of first immunization by an independent party through provision of sealed envelopes to the syringe preparation team members, who diluted PfSPZ Challenge12, 13, 34 or loaded placebo (normal saline) into syringes at an allocation ratio of 9:5, PfSPZ:placebo. Only the syringe preparation team was aware of allocation to the intervention and had no other role in the trial. The rest of the team remained blinded until completion of CHMI of group III. PfSPZ Challenge dose escalation for groups I, II, and III was staggered by at least four weeks and in each group three sentinel volunteers (PfSPZ:placebo, 2:1) received injections one to seven days before the main group. For group I, II, and III the three immunizations were given at 28-day intervals and CHMI by DVI of 3.2 × 103 PfSPZ was done 8–10 weeks following the last immunization. In the second part the three immunizations were administered at 14-day and 5-day intervals and CHMI was done at 10 weeks post immunization. Chloroquine concentrations were measured by mass spectrometry in the blood of selected volunteers on the day before CHMI to exclude carry-over from the immunization period. Following CHMI volunteers were regularly visited for 20 weeks. The primary efficacy endpoint was the proportion of volunteers with thick blood smears positive for Pf within 21 days of CHMI, primary safety endpoint was occurrence of related grade 3 or serious adverse events from first chloroquine dose until the end of the follow-up. We performed a randomized, placebo-controlled, double-blind trial in healthy, malaria-naive, 18–45 year olds (TÜCHMI-002; ClinicalTrials.gov ID: NCT02115516). Between 1 May and 4 July 2014, 42 volunteers were randomized to receive either three doses of normal saline (placebo) or 3.2 × 103 PfSPZ (PfSPZ Challenge12, 13, 34) (group I), 1.28 × 104 PfSPZ (group II), or 5.12 × 104 PfSPZ (group III) by DVI12, 13 at four-week intervals (Extended Data Fig. 2). All volunteers received oral chemoprophylaxis with chloroquine starting with a loading dose (10 mg kg−1 chloroquine base or 620 mg, whichever dose was less) two days before first PfSPZ inoculation followed by weekly maintenance doses (5 mg kg−1) through five days after last PfSPZ inoculation; total of 10 doses. Chloroquine is not active against SPZ or liver-stage parasites35 and Pf asexual blood-stage parasites leave the liver between days 5 and 6 following inoculation36; hence dosing five days following inoculation ensured high drug levels upon liver egress. Dose-escalation was staggered in four-week intervals and at each dose escalation the ratio of placebo-immunized to PfSPZ-CVac-immunized subjects was 5:9. Following PfSPZ dose escalation, accelerated regimens (14- and 5-day intervals) were assessed using essentially the same procedures. A full report will be published separately. Eight to ten weeks after last vaccine or placebo dose (51–67 days after last chloroquine dose), protective efficacy was assessed by CHMI using DVI with PfSPZ Challenge12, 13. Daily thick blood smears were performed as previously described37 from day 6 to 21, following each DVI for immunization and CHMI, during antimalarial treatment and at each late follow-up visit. Slides were considered positive when at least two readers detected two unambiguous parasites, each. A negative slide was defined as no observed parasites in the volume of blood required to detect with 95% probability less than 10 parasites per μl (~0.5 μl). In case of discordance, a third reading was performed. In addition, 1.2 ml blood was sampled in EDTA tubes (Sarstedt) for nucleic acid extraction at the same time-points. DNA extraction control 610 (Bioline) was added and total nucleic acid (DNA and RNA) was isolated from 0.5 ml blood using the QIAamp DNA blood mini kit (Qiagen) according to the manufacturer’s specifications but without addition of RNase. Parasitaemia was calculated from a standard curve generated from serially diluted (2–20,000 parasites per ml) Pf 3D7 ring-stage synchronized cultured parasites, counted by microscopy and cytometry. All purified nucleic acid samples were stored at –20 °C until time of use. Reverse transcription quantitative polymerase chain reaction (RT–qPCR) was performed using TaqMan RNA-to-CT 1-Step Kit using published primers and probes38, with a different fluorophore and addition of a minor groove binder (probe: VIC-ATGGCCGTTTTTAGTTCGTG-NFQMGB; primers: 5′-GCTCTTTCTTGATTTCTTGGATG-3′ and 5′-AGCAGGTTAAGATCTCGTTCG-3′). Reactions were done in 384 wells at 48 °C for 20 min (reverse transcription), 96 °C for 10 min (enzyme activation), and 45 cycles of 95 °C for 15 s, 62 °C 1 min. Samples were run as triplicates with no-template control, no-RT control and positive controls in the same plate. Amplification controls were assessed manually and cycle values (C ) were calculated with the second derivative maximum method (LightCycler 480 software; version 126.96.36.199). The assay was validated in accordance to MIQE guidelines38, 39 and had a lower limit of quantification of 3 parasites per ml. qPCR results were not reported to the clinical and microscopy teams during the study period to maintain blinding of the study. Sample size was calculated with the intention to show, with a power of 80% and a two-tailed alpha of 5%, a difference in proportion of infected volunteers of 25% or less of immunized volunteers and 99% in controls, allocated in a 2:1 ratio (8 PfSPZ:4 controls), allowing for one dropout in each group (9:5). Clinical data were captured on paper case report forms and transferred to an electronic database (OpenClinica; version 3.2) by double data entry. Efficacy data were reported as proportions (primary) and time to parasitaemia. Safety and tolerability data were listed and reported as summary statistics. Results of immunological assays were explored by post hoc analyses and used to generate hypotheses about correlates and immunological mechanisms of protection. Analyses were coded in R (version 3.2.3)40 when not otherwise stated. Where possible, estimate and 95% confidence interval are given. Box plots display median (middle line), 25th (lower hinge) and 75th (upper hinge) quartile. Whiskers extend to values within 1.5× the inter-quartile ranges of the lower and upper hinges, respectively. A two-sided P value less than 5% was considered statistically significant. Flow cytometry data were analysed with Pestle v1.7, SPICE v5.3 (ref. 41) and Prism 6 (GraphPad). Graphs were rendered in FlowJo, SPICE, and Prism. For vaccine immunogenicity, comparisons to pre-vaccine were performed using Wilcoxon signed rank test with Bonferroni correction for multiple comparisons or two-way ANOVA with Bonferroni correction, as specified in the text. Immune responses assessed at baseline, two weeks after final immunization, and at the time of CHMI were compared to outcome (parasitaemia or no parasitaemia) after CHMI. Assessment of immune responses that correlated with sterile protection was made using a stratified Wilcoxon test controlling for vaccine dose group as a covariate. Sera were assessed for vaccine-induced antibodies by ELISA (enzyme-linked immunosorbent assay), immunofluorescence assay, inhibition of sporozoite invasion assay and protein arrays representing 91% of the Pf proteome. ELISAs were performed for antigens first expressed in PfSPZ (PfCSP, PfSSP2/TRAP, PfCelTOS, PfMSP5, PfAMA1), early liver stages (PfEXP1 and PfLSA1) and late liver stages (PfMSP1 and PfEBA175). The ELISA methods for each antigen are described below. Recombinant (r) proteins used in ELISA assays are listed in Supplementary Table 7. 96-well plates (Nunc Maxisorp Immuno Plate) were coated overnight at 4 °C with 0.5 μg to 2.0 μg recombinant proteins (Supplementary Table 7) in 50 μl per well in coating buffer (KPL). Plates were washed three times with 2 mM imidazole, 160 mM NaCl, 0.02% Tween 20, 0.5 mM EDTA and blocked with 1% Bovine Serum Albumin (BSA) blocking buffer (KPL) containing 1% or 5% non-fat dry milk (Supplementary Table 7) for 1 h at 37 °C. Plates were washed three times and serially diluted serum samples (in triplicates) were added and incubated at 37 °C for 1 h. After three washes, peroxidase labelled goat anti-human IgG (KPL) was added at a dilution of 0.05 μg ml−1 to 0.2 μg ml−1 (Supplementary Table 7) and incubated at 37 °C for 1 h. Plates were washed three times, ABTS peroxidase substrate was added for plate development, and the plates were incubated for defined periods at 22 °C room temperature (Supplementary Table 7). The plates were read with a Spectramax Plus384 microplate reader (Molecular Devices) at 405 nm. The data were collected using Softmax Pro GXP v5 and fit to a 4-parameter logistic curve, to calculate the serum dilution at OD 1.0. A negative control (pooled serum from non-immune individuals from malaria free area) was included in all assays. The following positive control sera were used for the different antigens: serum from an individual with anti-PfCSP antibodies for PfCSP; pooled sera from individuals immunized with PfLSA-1 and PfEBA-175 subunit vaccines respectively for PfLSA1 and PfEBA175; pooled sera from volunteers from a malaria-endemic area (acquired from a blood bank in Kenya) for PfAMA1, PfEXP1, and PfMSP1. No positive control sera were available for PfMSP5, PfSSP2/TRAP or PfCelTOS. Samples were considered positive if the difference between the post-immunization OD 1.0 and the pre-immunization OD 1.0 (net OD 1.0) was ≥50 and the ratio of post-immunization OD 1.0 to pre-immunization OD 1.0 (ratio) was ≥3. Purified PfSPZ (NF54 strain) from aseptic Anopheles stephensi mosquitoes produced by Sanaria were resuspended in phosphate buffered saline (PBS (pH 7.4)) to obtain 5 × 103 PfSPZ per 40 μl. 40 μl (5 × 103 PfSPZ) were added to each well of Greiner cellstar clear-bottom black 96-well plates (Sigma-Aldrich). After addition of the suspension, plates were left at room temperature for 12–18 h for air-drying. 50 μl of sera diluted in PBS with 2% BSA were added to each well of the 96-well plate containing air-dried PfSPZ. Sera samples were added at twofold dilutions starting at 1:50. After adding samples, plates were incubated at 37 °C for 1 h. Plates were washed in PBS three times on an Aquamax Microplate washer. Alexa Fluor 488 conjugated goat anti-human IgG (Molecular Probes) was diluted to 1:250 in PBS with 2% BSA and 50 μl was added to each well. The plates were then incubated for 1 h at 37 °C. Plates were washed three times with PBS on an Aquamax Microplate washer. 100 μl PBS was added to each well. The plates were sealed using a plate sealer and stored in the dark at 4 °C until data acquisition. Samples were assessed by scanning the plates using an Acumen eX3 laser scanning imaging cytometer. The positive control was pooled human serum taken two weeks after the last immunization from 12 uninfected volunteers immunized 4 or 5 times with 1.35 × 105 PfSPZ Vaccine5. The Acumen image cytometer scans the entire surface area of each well in a 96-well plate and the fluorescence intensity values (arbitrary units) therefore represent the signal from all 5 × 103 PfSPZ that were seeded in each well. The data obtained from the Acumen image cytometer were plotted to fit a 4-parameter sigmoidal curve in GraphPad Prism software using serum dilution (log transformed) as the x axis variable and arbitrary fluorescence units (AFU) on the y axis. Over many iterations during development of this assay we have determined that sera from naive volunteers in the USA and Europe, including pre-immune sera, always register an arbitrary fluorescence value less than 2 × 105 even at the highest concentration (1:50 dilution, see above) used in this assay. Moreover, for sera that react to PfSPZ, 2 × 105 AFU falls in the exponential portion of their sigmoidal curves. Therefore, 2 × 105 was chosen as a threshold in the automated immunofluorescence assay and the results for each volunteer for antibodies to PfSPZ are reported as the reciprocal serum dilution at which fluorescence intensity was equal to 2 × 105 AFU. HC-04 (1F9) (ref. 42) cells (hepatocytes) were obtained from the Naval Medical Research Center. Master and working cell banks were produced, and after establishing they were free of mycoplasma, were quality control released. For the assay they were cultured in complete medium (10% FBS in DMEM/F12 with 100 units per ml penicillin and 100 μg per ml streptomycin; Gibco by Life Technologies) in Entactic-Collagen IV-Laminin (ECL)-coated 96-well clear bottom black well plates (Greiner Bio-One North America) at a density of 2.5 × 104 cells per well, and incubated for 24 h at 37 °C, 5% CO with 85% relative humidity. Twenty-four hours later cells were infected with 104 aseptic, purified, cryopreserved PfSPZ per well, without or with sera diluted in a 12-point series from subjects immunized with PfSPZ Vaccine. The assay control included PfSPZ added with media alone. All subjects were assessed at pre-immunization (baseline), post-immunization and pre-CHMI time points. Three hours later, PfSPZ that had not invaded the HC-04 cells were removed by washing three times with Dulbecco’s phosphate-buffered saline (DPBS), and the cultures were fixed using 4% paraformaldehyde for 15 min at room temperature. Differential immunostaining was performed to differentiate between PfSPZ inside the hepatocytes versus PfSPZ outside the hepatocytes. PfSPZ outside the hepatocytes were stained with an anti-PfCSP mAb (2A10, 6.86 μg ml−1) (Protein Potential LLC, with permission from New York University School of Medicine) conjugated with Alexa Fluor 633 (far-red) (1 μg ml−1; custom-conjugated at GenScript). The hepatocytes were then permeabilized with 0.1% Triton X-100 for 10 min at room temperature, and the PfSPZ inside the hepatocytes were stained with the anti-PfCSP mAb (2A10, 6.86 μg ml−1) conjugated with Alexa Fluor 488 (green; 1 μg ml−1, conjugated from Genscript). The numbers and intensity of infected hepatocytes (green only) were counted by scanning the plates using an Acumen eX3 laser scanning imaging cytometer. The data obtained from the Acumen image cytometer were plotted to fit a 4-parameter sigmoidal curve in GraphPad Prism software using serum dilution (log transformed) as the x axis variable and arbitrary fluorescence units on the y axis. 75% inhibition was interpolated from the sigmoidal curves as the reciprocal serum dilution at which the fluorescent intensity of infected wells with serum was 25% of the negative control without serum. The number of invaded PfSPZ scored in this assay in the absence of sera ranged from 400–600 (intensity of 1–3 × 106 fluorescence units) (4% to 6% of those added to the wells). A whole-proteome microarray with 91% coverage of the Pf proteome (PfWPM) was produced by Antigen Discovery, Inc. (ADI). Proteins were expressed as previously described43 from a library of Pf partial or complete open reading frames (ORFs) cloned into a T7 expression vector pXI using an in vitro transcription and translation (IVTT) system, the Escherichia coli cell-free Rapid Translation System (RTS) kit (5 Prime). The library was created through an in vivo recombination cloning process with PCR-amplified Pf ORFs, and a complementary linearized expressed vector transformed into chemically competent E. coli was amplified by PCR and cloned into pXI vector using a high-throughput PCR recombination cloning method described elsewhere44. Each expressed protein includes a 5′ polyhistidine (HIS) epitope and 3′ haemagglutinin (HA) epitope. After expressing the proteins according to manufacturer’s instructions, translated proteins were printed onto nitrocellulose-coated glass AVID slides (Grace Bio-Labs) using an Omni Grid Accent robotic microarray printer (Digilabs, Inc.). Microarray chip printing and protein expression were quality checked by probing random slides with anti-HIS and anti-HA monoclonal antibodies with fluorescent labelling. PfWPM chips contained 7,455 Pf peptide fragments, representing proteins from 4,805 unique genes, 302 IgG positive control spots and 192 spotted IVTT reactions without Pf ORFs (IVTT controls). For each PfWPM chip, 3 replicates were printed per microarray slide on 3 nitrocellulose pads. IgG-positive control spots were included as an assay control, whereas IVTT control spots were included as a sample-level normalization factor. Serum samples were diluted 1:100 in a 3 mg ml−1 E. coli lysate solution in protein arraying buffer (Maine Manufacturing) and incubated at room temperature for 30 min. Chips were rehydrated in blocking buffer for 30 min. Blocking buffer was removed, and chips were probed with pre-incubated serum samples using sealed, fitted slide chambers to ensure no cross-contamination of sample between pads. Chips were incubated overnight at 4 °C with agitation. Chips were washed five times with TBS-0.05% Tween 20, followed by incubation with biotin-conjugated goat anti-human IgG (Jackson ImmunoResearch) diluted 1:200 in blocking buffer at room temperature. Chips were washed three times with TBS-0.05% Tween 20, followed by incubation with streptavidin-conjugated SureLight P-3 (Columbia Biosciences) at room temperature protected from light. Chips were washed three times with TBS-0.05% Tween 20, three times with TBS, and once with water. Chips were air dried by centrifugation at 1,000g for 4 min and scanned on a ScanArray Express HT spectral scanner (Perkin-Elmer), and spot and background intensities were measured using an annotated grid file (.GAL). Data were exported and normalized using the IVTT control spots for statistical analysis in R40. Raw spot and local background fluorescence intensities, spot annotations and sample phenotypes were imported and merged in R, where all subsequent procedures were performed40. Foreground spot intensities were adjusted by local background by subtraction, and negative values were converted to 1. Next, all foreground values were transformed using the base 2 logarithm (log ). The dataset was normalized to remove systematic effects by subtracting the median signal intensity of the IVTT controls for each sample. As the IVTT control spots carry the chip, sample and batch-level systematic effects, but also antibody background activity to the IVTT system, this procedure normalizes the data and provides a relative measure of the specific antibody binding to the non-specific antibody binding to the IVTT controls (a.k.a. background). With the normalized data, a value of 0.0 means that the intensity is no different than the background and a value of 1.0 indicates a doubling with respect to background. A seropositivity threshold was established for each protein on the chip using the top 2.5th percentile of the pre-immunization samples for each protein. Reactive antigens were defined as those that had seropositive responses after immunization and before CHMI, but which did not show seropositive responses in the mock-immunization group. PBMCs were isolated by density-gradient centrifugation from heparinized whole blood. Assessment of cellular immune responses using multi-parameter flow cytometry was performed on PBMCs from cryopreserved samples at the completion of the study, as described6. In brief, PBMCs were thawed and rested in complete RPMI for 8 h followed by stimulation for 17 h with: (1) 1.5 × 105 viable, irradiated, aseptic, purified, cryopreserved PfSPZ from a single production lot; (2) PfSPZ Vaccine diluent (1% human serum albumin, HSA, CSL Behring); (3) 2 × 105 lysed RBC, >90% infected with late-stage schizonts (PfRBC) from a single production lot; or (4) 2 × 105 donor-matched uninfected erythrocytes from a single production lot. For the last 5 h of the stimulation, 10 μg ml−1 Brefeldin A (BD) was added to the culture. After stimulation, cells were stained as previously described45. The staining panels are shown in Supplementary Table 8 and the antibody clones and manufacturers are shown in Supplementary Table 9. Briefly, cells were surface stained with CCR7 at 37 °C for 20 min. Dead cells were identified by Aqua Live-Dead dye (Invitrogen), as per manufacturer’s instructions. This was followed by 15 min surface staining at room temperature for CD4, CD8, CD14, CD38, CD45RA, CD56, CD57, CD127, CD161, TCR-γδ, TCR-Vδ1, TCR-Vδ2, TCR-Vγ9, TCR-Vα7.2, CXCR6, or PD-1. Cells were washed, fixed, and permeabilized using Cytofix/Cytoperm kit (BD) and stained intracellularly for CD3, IFN-γ, IL-2, TNF-α, IL-4, IL-10, perforin, or Ki-67. Cells were washed, fixed in 0.5% paraformaldehyde, and measured on a modified LSR II (BD). Flow cytometry data were analysed using FlowJo v9.9 (Tree Star) blinded to vaccination group and CHMI outcome. All antigen-specific cytokine frequencies are reported after background subtraction of identical gates from the same sample incubated with the control antigen stimulation (HSA or uninfected erythrocytes). The data that support the findings of these studies are available in part on request from the corresponding author (S.L.H.) subject to restrictions. Some data are not publicly available, as they contain information that could compromise research participant privacy/consent.
News Article | February 15, 2017
Venco Venturo Industries, LLC. has opened the Venturo Installation Center (VIC) at its manufacturing facility in Cincinnati, Ohio. The plant, which manufactures Venturo truck cranes and Venco dump hoists, will include a skilled team of mechanics to immediately start up-fitting all of the company’s products, including Venturo crane and service body packages, Ferrari articulating cranes, Boss air compressors and Venco dump hoists. “Our expansion into up-fitting allows us to offer an additional service to our network of up-fitters around the country. To those that currently do not have the capacity or capability to offer Venturo crane and body up-fits, this will provide that added resource.” says company President, Brett Collins. Factory up-fitting is a real bonus for both distributors and end users alike. Factory installation offers those distributors that lack capacity or capability, a resource that will save them time and money while increasing their opportunities to sell Venturo work ready trucks. With no need to tie up distributor’s valuable shop time, factory installations allow distributors to concentrate on the up-fits they know best. Factory installations assure the highest quality, often with less cost and shorter lead-times. Expensive cost over-runs and other problems caused by unfamiliar installation crews are eliminated. Technical competence and experience saves valuable time and money. With each installation, the equipment is installed, tested and fine-tuned by technicians to exacting factory specifications. After the installation, Venturo Training Services (VTS) is available as an additional resource to provide crane operator training, crane inspector training and train the trainer programs. Single responsibility - from engineering and manufacturing through installation and delivery - insures the complete satisfaction of distributors and end users alike. For more information, visit http://www.venturo.com/factory-installation/ contact your factory representative, or call the factory at 800-226-2238.