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News Article | May 26, 2017
Site: www.techrepublic.com

Smart cities must be sustainable in order to survive. Sustainability is a key component of a truly smart city. If you add connectivity to a city without any connection to sustainability issues, you haven't really created a smart city. To be truly innovative, a city needs to consider elements including renewable energy, smart grids, smart parking and smart transportation. There must be a balance between economy, environment and society to build a strong, resilient city that survives the test of time. At IoT World 2017 (#IOTW17) in Santa Clara, CA last week, I hosted a panel of leaders who are working within the smart city realm. I asked them about what they've learned in regards to sustainability and smart cities and to share details on some of their projects. The information they shared with me was highly relevant to smart cities and is useful for any city seeking to become more sustainable. Here is a summary of the top five takeaways: Acosta said cities must start first with improving their infrastructure. In San Leandro, a city of 90,000 residents covering 15 square miles, the city entered the 1980's as a manufacturing powerhouse but lost jobs overseas throughout the decade. Manufacturing is still the city's lifeblood, as it is home to food giants such as Ghiradelli and Coca-Cola, as well as tech company OSIsoft. There was a risk the companies would leave without the proper infrastructure in place, so in 2012, San Leandro installed a 20-mile fiber optic loop to provide the foundation for IoT. In another move to stay relevant, last year the city embarked on a $5.2 million project with Climatec for smart city technologies such as smart streetlights and streetpoles on a mesh Wi-FI network. And earlier this month, the city closed an RFP for a vendor to help it develop a smart city strategy. Infrastructure, Acosta said, "is what cities do. Cities need to start with their infrastructure to make sure they're ready to create alternative energy paths." The role of city officials, she said, is "making sure that their communities are prepared for this crazy scary new world we are entering. You have to create safe ways for them to be actually be able to engage. Not only by saving money, but we have to create a world where they are 'prosumers' not just consumers. If we can create a world where energy is created by an individual and sold on the market, which we're doing in California by creating the CCA's [Community Choice Aggregation], which are competitors to our incumbent utilities, we believe we can accelerate that world." Jain said there are three essential components to the infrastructure of a city that can survive throughout the centuries, and that is having the ability to provide emergency services, essential services and entertainment. "If you don't create infrastructure for all these essentials, people cannot live in cities. It's happened before. Cities have been abandoned for these reasons." Baig said that when the California economy tanked in 2008, the budgets for the cities were decimated in 2009 and 2010, particilarly in IT. Meager funds were redirected to essential services. "The lesson I learned when it comes to IoT and making a city sustainable is you need to have capital, and you need to have resources, the people who are trained in the proper processes and tools." "It's mission critical to have infrastructure to support making a city truly resilient and sustainable," Baig said. SEE: IoT World 2017: HPE and Platform as a Service in a smart city (TechRepublic) Acosta said a flat "no" when asked if a sustainable city could exist without renewable energy. "The current way that we generate electricity in the United States through hydropower, through nuclear energy, through burning coal, is not sustainable. With coal of course being the most dangerous of all of them. We have to go the path of renewable energy." She said that not only is renewable energy clean, but it's an economical decision. In the book "Climate of Hope" by Carl Pope and Michael Bloomberg, she said the authors say that clean energy pays back a community. "Right now their estimate is there are six times more people employed in clean energies and sustainability than there are in oil, gas and coal combined in this country. That's an astonishing figure. It's all about the money. It's all about the economics. That's the reason we can't avoid this any more other than just the technological and the reasons we should be doing it for our planet. It's because it's an economic development issue." Baig said sustainability is a critical component for Oakland as well, and is a major point in the mayor's budget. "In the City of Oakland we have the Port of Oakland, which is the fourth largest port in the country. We have about 70,000 jobs associated with the port of Oakland." The port did $49 billion in trade last year and it helps reduce the number of tractor-trailers on the roads when products are transported through the port. SEE: IoT World 2017: How a smart city can use data analytics to improve services (TechRepublic) Illegal dumping is a big problem in Oakland. The city has installed IoT devices for license plate recognition embedded within cameras and to help with identifying hot spots for dumping. "Our target is to make sure we have a sustainable program with a policy so that we can take care of this whole illegal dumping issue," Baig said. In San Leandro, IoT devices create data points to give the city information to use to make better decisions about priorities. The recently closed RFP is part of that, with the desire for a smart city strategy. "We have to be able to prioritize first before we figure out how we can invest the return on investment. Our smart city lights will give a tremendous return on investment. The first few years we will be paying off our bond, but after that what can we invest in? We don't know. Remember cities are coming out of the Dark Ages, especially after the economic downturn. We don't know what we don't know, so we have to figure out what that best ROI is for us and how we can invest it," Acosta said. SEE: IoT World 2017: How to make big data useful for cities (TechRepublic) "Sustainability is an important part of a smart city. On our side, I think there are a few things we are doing that are part of the how to effort. One is to be a good backend support for demand management. Smart meters and smart grids, all of these great devices are coming online, especially for the consumer," Miri said. Connecting consumers with resources, and giving them the ability to know how much they're consuming, and when they're consuming it, is one way to combat the energy problem. When people can see how much they're using, and are given incentives to use it during low-demand hours, then it can make sustainability not only environmentally successful, but also financially and socially so that it "just becomes the way we generate power," Miri said. SEE: IoT World 2017: The role of smart grids in a connected city (TechRepublic) Acosta said transportation and mobility are critical to smart cities across the country. "Traffic is choking us. And it is compounded by applications like Waze which actually directs traffic around the freeway jams and into your cities, causing impact to your roads which are very expensive to maintain." In California, all cities have call to action plans. Most California cities, including San Leandro, have a goal of reducing their carbon impact by 25% by 2020. "For a city like San Leandro, the fiber optics of the infrastructure provides the basis that we need for the Internet of Things that is in turn going to allow us to be able to solve some of these serious sustainability problems and to be able to move towards zero net energy," she said. Miri said, "If you want to help us ... solve the public transportation problem, and not just our buses, but how people move around through cities. If you want to solve a problem for the ages, give us connected, autonomous driving cars and a shared economy. Game over." SEE: IoT World 2017: Sustainability and smart transportation with Zipcar (TechRepublic)


SACRAMENTO, Calif., May 25, 2017 (GLOBE NEWSWIRE) -- Anderson Chiropractic Clinic of Sacramento is pleased to announce that chiropractor Dr. Gregg Anderson, D.C. will be presenting at the upcoming 2017 California Chiropractic Association (CCA) Conference. At the event, he will showcase his own patented, innovative chiropractic equipment called the VDP-PRO. The 2017 California Chiropractic Association’s annual conference will be held from June 1–4 this year in San Diego, California at the Paradise Point Resort & Spa. Dr. Anderson, D.C. looks forward to sharing the innovative VDP-PRO tool with his peers and colleagues so that even more patients can benefit from it. The VDP-PRO is a revolutionary healthcare tool that can be used to enhance a range of existing chiropractic adjusting techniques, including replenishing nutrients to the IVDs, working on the spine to reduce IVD herniations, and relieving facet syndrome. Patients who suffer from back and neck issues, as well as nerve pain in the extremities that arise from their back or neck conditions can benefit from the VDP-PRO.  This conservative alternative to opioid prescriptions and surgery has provided relief to Anderson Chiropractic Clinic patients with disc herniation, disc degeneration, and facet syndrome. The VDP-PRO is constructed from very strong but extremely lightweight die-cast aluminum as well as steel and nylon. The tool fits the chiropractor’s hand comfortably for easy application in a range of techniques to meet each client’s specific health and pain relief needs. It is effective in treating many common spinal conditions including disc compression, disc herniation, disc degeneration, myofasciitis, and facet syndrome. Dr. Gregg Anderson, D.C. has been in practice in Sacramento since 1996. He specializes in the treatment of herniated disk injuries and other injury related conditions. He was selected as a Top Chiropractor by his peers in 2014, 2015, and 2016 for Sacramento Magazine. “I created the VDP-PRO to make my job easier, and I feel it has revolutionized the way I practice chiropractic. I look forward to introducing other professionals to this tool at the upcoming CCA conference so that others can benefit from it as well,” says Dr. Gregg Anderson, D.C. About Anderson Chiropractic Clinic Anderson Chiropractic Clinic features a Chirosight digital X-ray system, state-of-the-art electronic health records, and the most thoroughly researched and effective chiropractic strategies available today. Comprehensive chiropractic care, corrective exercises, and lifestyle advice are also holistic options available to patients. Those in the area who would like to learn more or book an appointment may do so by calling (916) 249-0579. Additional information about the practice is also available at the Anderson Chiropractic Clinic website at http://andersonchiro.com/.


SACRAMENTO, Calif., May 25, 2017 (GLOBE NEWSWIRE) -- Anderson Chiropractic Clinic of Sacramento is pleased to announce that chiropractor Dr. Gregg Anderson, D.C. will be presenting at the upcoming 2017 California Chiropractic Association (CCA) Conference. At the event, he will showcase his own patented, innovative chiropractic equipment called the VDP-PRO. The 2017 California Chiropractic Association’s annual conference will be held from June 1–4 this year in San Diego, California at the Paradise Point Resort & Spa. Dr. Anderson, D.C. looks forward to sharing the innovative VDP-PRO tool with his peers and colleagues so that even more patients can benefit from it. The VDP-PRO is a revolutionary healthcare tool that can be used to enhance a range of existing chiropractic adjusting techniques, including replenishing nutrients to the IVDs, working on the spine to reduce IVD herniations, and relieving facet syndrome. Patients who suffer from back and neck issues, as well as nerve pain in the extremities that arise from their back or neck conditions can benefit from the VDP-PRO.  This conservative alternative to opioid prescriptions and surgery has provided relief to Anderson Chiropractic Clinic patients with disc herniation, disc degeneration, and facet syndrome. The VDP-PRO is constructed from very strong but extremely lightweight die-cast aluminum as well as steel and nylon. The tool fits the chiropractor’s hand comfortably for easy application in a range of techniques to meet each client’s specific health and pain relief needs. It is effective in treating many common spinal conditions including disc compression, disc herniation, disc degeneration, myofasciitis, and facet syndrome. Dr. Gregg Anderson, D.C. has been in practice in Sacramento since 1996. He specializes in the treatment of herniated disk injuries and other injury related conditions. He was selected as a Top Chiropractor by his peers in 2014, 2015, and 2016 for Sacramento Magazine. “I created the VDP-PRO to make my job easier, and I feel it has revolutionized the way I practice chiropractic. I look forward to introducing other professionals to this tool at the upcoming CCA conference so that others can benefit from it as well,” says Dr. Gregg Anderson, D.C. About Anderson Chiropractic Clinic Anderson Chiropractic Clinic features a Chirosight digital X-ray system, state-of-the-art electronic health records, and the most thoroughly researched and effective chiropractic strategies available today. Comprehensive chiropractic care, corrective exercises, and lifestyle advice are also holistic options available to patients. Those in the area who would like to learn more or book an appointment may do so by calling (916) 249-0579. Additional information about the practice is also available at the Anderson Chiropractic Clinic website at http://andersonchiro.com/.


News Article | May 24, 2017
Site: www.nature.com

A step-by step protocol describing the HSPC conversion of human PSCs can be found at Protocol Exchange48. All experiments were performed with H9 hESC (NIHhESC-10-0062), PB34 iPS49, MSC-iPS150, 1045-iPSC, and 1157-iPSC established by the hES Core Facility at Boston Children’s Hospital. Human ESCs and iPSCs were maintained on hESC-qualified Matrigel (BD) in mTeSR1 media (Stem Cell Technologies) or mouse embryonic fibroblasts (GlobalStem) feeders in DMEM/F12 + 20% KnockOutSerum Replacement (Invitrogen), 1 mM l-glutamine, 1 mM NEAA, 0.1 mM β-mercaptoethanol, and 10 ng ml−1 bFGF on 10 cm gelatinized culture dishes. Medium was changed daily, and cells were passaged 1:4 onto fresh feeders every 7 days using standard clump passaging with dispase. Morphology of PSCs was checked by microscopy daily. As a quality control, only dishes with more than 70% of typical PSC colonies were processed for embryoid body formation. Cell lines were tested for mycoplasma routinely. Embryoid body differentiation was performed as previously described19. Briefly, hPSC colonies were dissociated with 0.05% trypsin for 5 min at 37 °C, pipetted thoroughly with p1000 to form small aggregates, washed twice with PBS + 2% FBS, and resuspended in StemPro-34 (Invitrogen, 10639-011) supplemented with l-glutamine (2 mM), penicillin/streptomycin (10 ng ml−1), ascorbic acid (1 mM), human holo-Transferrin (150 μg ml−1, Sigma T0665), monothioglycerol (MTG, 0.4 mM) (referred to as ‘supplemented StemPro-34’), BMP4 (10 ng ml−1), and Y-27632 (10 μM). Cells were then seeded into non-adherent spheroid formation 10 cm plates (Ezsphere, Asahi Glass; well size diameter 400–500 μm, depth 100–200 μm; number of wells 14,000 per dish) at a density of 5 million per dish. Twenty-four hours later, bFGF (5 ng ml−1) and BMP4 (10 ng ml−1) were added to the medium. On day 2, the developing embryoid bodies were collected and resuspended in supplemented StemPro-34 with SB431542 (6 μM), CHIR99021 (3 μM), bFGF (5 ng ml−1), and BMP4 (10 ng ml−1). The formation of embryoid bodies was checked by microscopy on day 4 and the decision was made to continue embryoid body formation on the basis of the size and morphology of aggregations (quality control; >100 μM, compaction-like tight contact of cells). On day 4, medium was replaced by supplemented StemPro-34 with VEGF (15 ng ml−1) and bFGF (10 ng ml−1). At day 6, medium was replaced by supplemented StemPro-34 with bFGF (10 ng ml−1), VEGF (15 ng ml−1), interleukin (IL)-6 (10 ng ml−1), IGF-1 (25 ng ml−1), IL-11 (5 ng ml−1), and SCF (50 ng ml−1). Cultures were maintained in a 5% CO /5% O /90% N environment. All recombinant factors were human and purchased from Peprotech. To avoid potential damage resulting from hydrodynamic pressure and contamination through fluorescence-activated cell sorting (FACS), for functional assay, isolation of haemogenic endothelium was performed by magnetic cell isolation. Freshly dissociated embryoid body cells (at the day 8 time point) by 0.05% trypsin were filtered through a 70 μm filter and stained with CD34 microbeads (Miltenyi) for 30 min at 4 °C. CD34+ cells were isolated with LS columns (Miltenyi). Around 0.3 × 105 to 1.0 × 105 cells were obtained per 10 cm dish of embryoid body formation. A sample from each batch was analysed by FACS to validate its purity of haemogenic endothelium with the panel CD34 PE-Cy7 (8G12; BD), FLK1 AF647 (89106; BD), CD235a/glycophorin (GLY)-A FITC (11E4B-7-6; Coulter), CD43 PE (1G10; BD), and 4′,6-diamidino-2-phenylindole (DAPI). For expression profiling by microarray and qRT–PCR, isolation of haemogenic endothelium was performed by FACS. Dissociated embryoid bodies (at the day 8 time point) were resuspended at 1 × 106 to 3 × 106 per 100 μl of staining buffer (PBS + 2% FBS). Cells were stained with a 1:50 dilution of CD34 PE-Cy7 (8G12; BD), FLK1 AF647 (89106; BD), CD235a/glycophorin (GLY)-A FITC (11E4B-7-6; Coulter), CD43 PE (1G10; BD), and DAPI for 30 min at 4 °C in the dark. All FACS sorting was performed on a BD FACS Aria II cell sorter using an 85 μm nozzle to avoid potential damage to haemogenic endothelium. All the samples used for microarray analysis were FACS-sorted. Haemogenic endothelium panel: CD34 PE-Cy7 (8G12; BD), FLK1 AF647 (89106; BD), CD235a/glycophorin (GLY)-A FITC (11E4B-7-6; Coulter), and CD43 PE (1G10; BD). Fetal-liver HSCs were purchased from StemCell Technologies and stained with HSC panel: CD38 PE-Cy5 (LS198-4-3; Clontech), CD34 PE-Cy7 (8G12; BD), and CD45 PE (HI30; BD). Between 10,000 and 50,000 cells were sorted for each cell type with two or three biological replicates. An RNAeasy Microkit (Qiagen) was used to collect and prepare total RNA for microarray analysis. The Ovation Picokit (Nugen) was used for preamplification, where required. Gene expression profiling was performed on Affymetrix 430 2.0 gene chips according to standard protocol. Microarray data were analysed according to standard protocol using R/Bioconductor. Embryoid bodies were dissociated on day 8 by digestion with 0.05% trypsin for 5 min at 37 °C, pipetted thoroughly with p1000 to generate a single-cell suspension and washed with PBS + 2%FBS. Dissociated embryoid bodies were immediately processed for isolation of haemogenic endothelium. Cells were resuspended in 1mL PBS+2%FBS and incubated with human CD34 MicroBead kit for 1 h (Miltenyl Biotec, 130-046-702). After incubation, cells were washed with PBS+2%FBS and isolated by magnetic cell isolation using LS columns (Miltenyl Biotec, 130-042-401). Sorted CD34+ cells were resuspended in supplemented StemPro-34 medium, containing Y-27632 (10 μM), TPO (30 ng ml−1), IL-3 (10 ng ml−1), SCF (50 ng ml−1), IL-6 (10 ng ml−1), IL-11 (5 ng ml−1), IGF-1 (25 ng ml−1), VEGF (5 ng ml−1), bFGF (5 ng ml−1), BMP4 (10 ng ml−1), and FLT3 (10 ng ml−1) as reported20 and seeded at a density of 25 × 103 to 50 × 103 cells per well onto thin-layer Matrigel-coated 24-well plates. All recombinant factors were human and most were purchased from Peprotech. Plasmids for the transcription factor library were obtained as Gateway plasmids (Harvard Plasmid Service; GeneCopoeia). Open reading frames were cloned into lentiviral vectors using LR Clonase (Invitrogen). Two vectors were used, pSMAL-GFP (constitutive) and pINDUCER-21 (ORF-EG)51. pINDUCER21 (ORF-EG) was a gift from S. Elledge and T. Westbrook (Addgene plasmid 46948). Lentiviral particles were produced by transfecting 293T-17 cells (ATCC) with the second-generation packaging plasmids (pMD2.G and psPAX2 from Addgene). Virus were harvested 36 and 60 h after transfection and concentrated by ultracentrifugation at 23,000 r.p.m. for 2 h 15 min at 4 °C. Viruses were reconstituted with 50 μl of EHT culture medium. Constructs were titred by serial dilution on 293T cells using GFP as an indicator. Polycistronic vectors were made as follows: LCOR-P2A-HOXA9-T2A-HOXA5 and RUNX1-P2A-ERG DNA fragments were synthesized and cloned into pENTR-D/TOPO cloning vector by GenScript, then Gateway-recombined with pINDUCER-21 (ORF-EG). At day 3 of EHT culture, haemogenic endothelium cells were beginning to produce potentially haematopoietic ‘round’ cells; the occurrence of this phenomenon was used as quality control of haemogenic endothelium induction and transition to haematopoietic cells for each batch of experiments. The infection medium was EHT culture medium supplemented with Polybrene (8 μg ml−1, Sigma). Lentiviral infections were performed in a total volume of 250 μl (24-well plate). The multiplicity of infection for the factors was as follows: Library 3.0 for each, ERG 5.0, HOXA5 5.0, HOXA9 5.0, HOXA10 5.0, LCOR 5.0, RUNX1 5.0, SPI1 5.0, LCOR–HOXA9–HOXA5 2.0, and RUNX1–ERG 2.0. Haemogenic endothelium was vulnerable to damage during spinoculation, thus infections were performed static for 12 h, then 250 μl of fresh EHT medium was supplemented to dilute Polybrene. Parallel wells were cultured for an additional 3 days to measure infection efficiency by the percentage of GFP+ DAPI cells by FACS, achieving 30–70% of infection efficiency. Followed by lentiviral gene transfer, cells were maintained for 5 days in EHT culture medium supplemented with doxycycline (2 μg ml−1, Sigma) to induce transgene expression in vitro. Fifty thousand cells were plated into 3 ml complete methylcellulose (H4434; StemCell Technologies). Additional cytokines added were 10 ng ml−1 FLT3, 10 ng ml−1 IL6, and 50 ng ml−1 TPO (R&D Systems). The mixture was distributed into two 60 mm dishes and maintained in a humidified chamber at 37 °C for 14 days. Colonies were scored manually or using a BD Pathway 855 fluorescent imager. At 14 days, granulocyte, erythrocyte, monocyte, megakaryocyte (GEMM) colonies were picked up by P20 pipette. Between 10 and 20 GEMM colonies were picked with 2 or 3 biological replicates. A QIAamp DNA Micro Kit (Qiagen) was used to collect and prepare total genomic DNA for PCR detection of transgenes. Nested PCR reaction was as follows: first round with LNCX forward primer (5′-AGC TCG TTT AGT GAA CCG TCA GAT C-3′) and EGFP N reverse primer (5′-CGT CGC CGT CCA GCT CGA CCA G-3′), 95 °C 5 min, 36 cycles of (95 °C for 30 s, 60 °C for 30 s, 72 °C for 5 min), 72 °C for 5 min, 4 °C hold; second round with forward primer for each gene and HA reverse primer (5′-TCT GGG ACG TCG TAT GGG TA-3′), 95 °C 5 min, 36 cycles of (95 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s), 72 °C for 5 min, 4 °C hold. Twelve hours after lentiviral gene transfer, cells were recovered by dispase for 5 min at 37 °C, and washed by PBS three times to ensure no carry-over of virus. Cells were resuspended at 0.3 × 105 to 3.0 × 105 cells per 25 μl buffer (PBS + 2% FBS from StemCell Technologies) and kept on ice until injection. Thirty thousand to 3.0 × 105 cells were intrafemorally injected in to NOD/LtSz-scidIL2Rgnull (NSG) mice and treated with doxycycline as described below (see section on ‘Mouse transplantation’). Up to 100 μl peripheral blood was collected every 2–4 weeks, to 14 weeks. Mice were euthanized and bone marrow and thymus removed at 8–14 weeks. For transgene detection in engrafted cells, each lineage of cells was FACS-sorted from bone marrow. Myeloid cells: CD33 APC (P67.6; BD), CD45 PE-Cy5 (J33; Coulter). B cells: CD19 PE (HIB19; BD), CD45 PE-Cy5 (J33; Coulter). T cells: CD3 PE-Cy7 (SK7; BD), CD45 PE-Cy5 (J33; Coulter). Between 10,000 and 50,000 cells were isolated with 2 or 3 biological replicates for multiple cell lines (iPSCs and ESCs). The QIAamp DNA Micro kit (Qiagen) was used to collect and prepare total genomic DNA for PCR detection of transgenes. Nested PCR reaction was performed similarly to the in vitro screening described in the above section. NOD/LtSz-scidIL2Rgnull (NSG) mice (The Jackson Laboratory) were bred and housed at the Boston Children’s Hospital animal care facility. Animal experiments were performed in accordance with institutional guidelines approved by Boston Children’s Hospital Animal Care Committee. Intrafemoral transplantations were conducted with 6- to 10-week-old female mice irradiated (250 rad) 12 h before transplantation. Before transplantation, mice were temporarily sedated with isoflurane. A 26-half-gauge needle was used to drill the femur and a 0.3 × 105 to 3.0 × 105 range of cells was transplanted in a 25 μl volume using a 28.5-gauge insulin needle. Sulfatrim was administered in drinking water to prevent infections after irradiation. Doxycycline Rodent Diet (Envigo-Teklad Diets; 625 p.p.m.) and doxycycline (1.0 mg ml−1) were added to the drinking water to maintain transgene expression in vivo for 2 weeks (ref. 2). Secondary transplantation was performed with 1,000–3,000 human CD34+ cells (isolated from bone marrow by magnetic cell isolation with CD34 microbeads) at 8 weeks. Isolated cells were resuspended at 1,000–3,000 cells per 25 μl buffer (PBS + 2% FBS from StemCell Technologies) and kept on ice until injection. Cells were intrafemorally injected in to NSG mice. Sorted CD34+CD43+CD45+ (25,000 cells) or CD34+CD43−CD45− (25,000 cells) HE-7TF cells were either intrafemorally or intravenously injected. For non-irradiated c-Kit-deficient immune-deficient recipients, the NOD.Cg-KitW-41J Tyr + Prkdcscid Il2rgtm1Wjl/ThomJ model was used (The Jackson Laboratory). Investigators were blinded for the analysis of mice. The experiments were not randomized. No statistical methods were used to predetermine sample size. For this analysis, multi-lineage engraftment was defined as chimaerism of human CD45+ cells in bone marrow encompassing four distinct lineages (myeloid, erythroid, B- and T-lymphoid, each comprising more than 1% of engrafted human CD45+ cells) in this study. Cells grown in EHT culture or harvested animal tissues were stained with the following antibody panels. Haemogenic endothelium panel: CD34 PE-Cy7 (8G12; BD), FLK1 AF647 (89106; BD), CD235a/glycophorin (GLY)-A FITC (11E4B-7-6; Coulter), and CD43 PE (1G10; BD). HSPC panel: CD38 PE-Cy5 (LS198-4-3; Clontech), CD34 PE-Cy7, and CD45 PE (HI30; BD). Lineage panel: CD235a/glycophorin (GLY)-A PE-Cy7 or FITC (11E4B-7-6; Coulter), CD33 APC (P67.6; BD), CD19 PE (HIB19; BD), IgM BV510 (G20-127; BD), CD4 PE-Cy5 (13B8.2; Coulter), CD3 PE-Cy7 (SK7; BD), CD8 V450 (RPA-T8; BD), TCRαβ BV510 (T10B9; BD), TCRγδ APC (B1; BD), CD45 PE-Cy5 (J33; Coulter), CD15 APC (HI98; BD), and CD31/PECAM PE (WM59; BD). All stains were performed with fewer than 1 × 106 cells per 100 μl staining buffer (PBS + 2% FBS) with 1:100 dilution of each antibody, for 30 min at 4 °C in the dark. Compensation was performed by automated compensation with anti-mouse Igk negative beads (BD) and cord blood MNC stained with individual antibodies. All acquisitions were performed on a BD Fortessa cytometer. For detection of engraftment, human cord-blood-engrafted mouse marrow was used as a control to set gating; sorting was performed on a BD FACS Aria II cell sorter. Five thousand to 10,000 FACS-sorted erythroid cells (CD235a/glycophorin (GLY)-A PE-Cy7 or FITC (11E4B-7-6; Coulter)), plasmacytoid lymphocytes (CD19 PE (HIB19; BD), IgM BV510 (G20-127; BD), CD38 PE-Cy5 (LS198-4-3; Clontech)), neutrophils (CD15 APC (HI98; BD), CD31/PECAM PE (WM59; BD) and CD45 PE-Cy5 (J33; Coulter)) were cytospun onto slides (500 r.p.m. for 10 min), air dried, and stained with May-Grunwald and Giemsa stains (both from Sigma), washed with water, air dried, and mounted, followed by examination by light microscopy. RNA extraction was performed using an RNAeasy Microkit (Qiagen). Reverse transcription was performed using Superscript III (>5,000 cells) or VILO reagent (<5,000 cells) (Invitrogen). Quantitative PCR was performed in triplicate with SYBR Green (Applied Biosystems). Transcript abundance was calculated using the standard curve method. Primers used for globin genes were as follows52: huHbB F (5′-CTG AGG AGA AGT CTG CCG TTA-3′), huHbB R (5′-AGC ATC AGG AGT GGA CAG AT-3′), huHbG F (5′-TGG ATG ATC TCA AGG GCA C-3′), huHbG R (5′-TCA GTG GTA TCT GGA GGA CA-3′), huHbE F (5′-GCA AGA AGG TGC TGA CTT CC-3′), and huHbE R (5′-ACC ATC ACG TTA CCC AGG AG-3′). FACS-isolated neutrophils (CD15+PECAM+CD45+) and T cells (CD3+CD45+) were cultured in IMDM + 10%FBS overnight in 96-well plates (flat-bottom), seeding 5,000–20,000 cells per well obtained from mice engrafted over 10% in primary recipients, or pooled mice engrafted less than 5% in primary recipients. Then supernatant was taken and analysed by MPO- or IFN-γ-ELISA –Ready-SET-Go! Kit (eBioscience) according to the manufacturer’s protocol. The amount of IFN-γ was normalized per 1,000 cells. PMA (20 ng ml−1) and ionomycin (1 μg ml−1) were added to either neutrophils or T cells, then cells were cultured overnight (6–18 h). Human Ig production was measured from 50 μl of serum from NSG mice at 8 weeks (IgM) and 14 weeks after engraftment (IgG). Immunization of mice was done with OVA (F5503, Sigma) with Freund’s complete adjuvant (F5881, Sigma), followed by booster doses of Freund’s incomplete adjuvant (F5506, Sigma) according to the manufacturer’s instructions. Six to 14 weeks after engraftment, mice were injected with antigen OVA (0.1%) emulsified in complete adjuvant subcutaneously at two sites on the back, injecting 100 μl at each site. A booster injection of antigen OVA (0.1%) emulsified in incomplete adjuvant was administered 14 days after immunization. The booster was given as a single subcutaneous injection with 100 μl at one site on the back. A serum sample was isolated from mice 7 days after the first booster dose, and human ova-specific antibody concentration was tested with an ovalbumin-specific IgG, OVA sIgG, ELISA Kit (Mybiosource, MB S700766) for human Ova-specific IgG and a Human Anti-Ovalbumin (Gal d 2) IgM ELISA Kit (Alpha Diagnostic, 670-145-OVM) to detect human Ova-specific IgG and IgM, respectively. The technical replicates were done with three measurements of the same experimental setup. Human CD3+ T cells were FACS-isolated from thymus of engrafted NSG mice. Purified DNA was subjected to next-generation sequencing of CDR3 using immunoSEQ (Adaptive Biotechnology, Seattle, Washington, USA) and analysed with immunoSEQ Analyzer software (Adaptive Biotechnology). Aliquots (250 ng) of genomic DNA from human CD45+ bone marrow cells (CD45 PE-Cy5 (J33; Coulter)) from engrafted NSG mice and original PSCs (two biological replicates) were digested with either Nsp1 or Sty1. A universal adaptor oligonucleotide was then ligated to the digested DNAs. The ligated DNAs were diluted with water and three 10 μl aliquots from each well of the Sty 1 plate and four 10 μl aliquots from each well of the Nsp 1 plate were transferred to fresh 96-well plates. PCR master mix was added to each well and the reactions cycled as follows: 94 °C for 3 min; 30 cycles of 94 °C for 30 s, 60 °C for 45 s, 68 °C for 15 s; 68 °C for 7 min; 4 °C hold. After PCR, the seven reactions for each sample were combined and purified by precipitation from 2-propanol/7.5 M ammonium acetate. The ultraviolet absorbance of the purified PCR products was measured to ensure a yield ≥4 μg μl−1. Forty-five microlitres (≥180 μg) of each PCR product were fragmented with DNase 1 so the largest fragments were <185 base pairs. The fragmented PCR products were then end-labelled with a biotinylated nucleotide using terminal deoxynucleotidyl transferase. For hybridization, the end-labelled PCR products were combined with hybridization cocktail, denatured at 95 °C for 10 min and incubated at 49 °C. Two hundred microlitres of each mixture was loaded on a GeneChip and hybridized overnight at 50 °C and 60 r.p.m. After 16–18 h of hybridization, the chips were washed and stained using the GenomeWideSNP6_450 fluidics protocol with the appropriate buffers and stains. After washing and staining, the GeneChips were scanned on a GeneChip Scanner 3000 using AGCC software. Genotype calls (chp files) were generated in Affymetrix Genotyping Console using the default parameters. The resulting chp files were analysed for familial relationships using the identity by state algorithm implemented in Partek Genomics Suite. Engrafted human CD34+CD38−CD45+ HSCs were isolated from bone marrow from either iPS-derived haemogenic endothelium- or cord-blood-injected NSG mice, then RNA was purified with an RNeasy Micro kit (Qiagen). Quality control of RNA was done by Bioanalyzer and qubit analysis. Samples that passed quality control were converted into libraries and sequenced by a Nextseq PE75 kit. Raw reads were aligned to the human genome/transcriptome using TopHat2 software53. Gene expression levels and reads per kilobase per million (RPKM) values were estimated using a htseq-count tool54 and the edgeR package55. For a legitimate transcriptome-wide comparison, we retrieved raw RNA-seq data of two published from the Gene Expression Omnibus database (Long non-coding RNA profiling of human lymphoid progenitors reveals transcriptional divergence of B-cell and T-cell lineages, accession number GSE69239; Distinct routes of lineage development reshape the human blood hierarchy across ontogeny, accession number GSE76234) and calculated RPKM values using a same analysis pipeline. Engrafted human CD34+CD38−CD45+ HSCs were isolated from bone marrow from either iPS-derived haemogenic endothelium- or cord-blood-injected NSG mice, then processed for in-droplet barcoding according to a previous report29. The library was QCed with Bioanalyzer and sequenced by Nextseq PE 75 kit. The t-SNE algorithm was used to visualize transcriptome similarities and population heterogeneity of cord blood HSCs and iPSC-derived HSCs. The t-SNE algorithm performs a dimensionality reduction of multidimensional single-cell RNA-seq data into a low-dimensional space, preserving pairwise distances between data points as well as possible, allowing a global visualization of subpopulation structure and cell–cell similarities. We used the R package tsne in our analyses. The t-SNE map was initialized with point-to-point distances computed by classical multidimensional scaling, and the R plot function was used to visualize t-SNE maps annotated by cord blood or iPSC-derived HSCs. Plots showing t-SNE maps coloured by expression of selected genes were created using the ggplot2 package. For subpopulation identification, we used the top 500 genes with the highest variance to elucidate global differences among single cells. To assess transcriptome similarities in terms of induction of haematopoietic genes in iPSC-derived HSCs, we used 62 haematopoietic genes for t-SNE analysis in Supplementary Table 2. Gene set enrichment analysis was performed with the desktop client version (javaGSEA, http://software.broadinstitute.org/gsea/downloads.jsp) with default parameters. RPKM values from the 7F-HSPC were obtained from the RNA-seq (described previously). These values were normalized to a terminally differentiated cell and the normalized values were used to rank the most differentially expressed genes. These differentially expressed genes were used to run gene set enrichment analysis with gene sets obtained from mSigDB (KEGG, Hallmark, immunological, transcription factors, and chemical and genetic perturbations gene sets were used). In addition, gene sets specific to progenitors, cord blood, or fetal-liver HSC were obtained from previous reports16, 56. FDR < 0.25 with P < 0.05 was considered significant. CD33+ myeloid cells, CD19+ B cells, and CD3+ T cells were isolated from bone marrow from haemogenic-endothelium-injected NSG mice. Genomic DNA was purified with a QIAamp DNA Micro kit (Qiagen). Ligation-mediated PCR-based detection of lentiviral integration sites was done with a Lenti-X Integration Site Analysis Kit (Clontech) according to the manufacturer’s instructions. Sequencing-based detection (integration sequencing) was done as previously described57. RNA-seq from this study have been deposited in the Gene Expression Omnibus under accession number GSE85112. We retrieved raw RNA-seq data of two published from the Gene Expression Omnibus database (Long non-coding RNA profiling of human lymphoid progenitors reveals transcriptional divergence of B-cell and T-cell lineages, accession number GSE69239; Distinct routes of lineage development reshape the human blood hierarchy across ontogeny, accession number GSE76234). The data are all in the paper, or are available from the corresponding author upon reasonable request if not.


"This event epitomizes one of the FCCA's main goals: to help destinations maximize their share of cruise tourism," said Michele Paige, President. "In addition to imparting necessary operational knowledge and tackling specific issues to the region, the event provides an invaluable resource -- knowing the right people -- by linking attendees with some of industry's most important decision makers." The biennial Summit will achieve this by linking attendees with executives from the FCCA's 19 Member Lines. Attendees were able to pre-schedule one-on-one meetings with executives of their choice, providing a chance to promote products and receive individualized input. Cruise executives from sectors like shore excursions, destination development, government relations, port operations and purchasing will be on-hand looking to take on business and help attendees fine-tune their products to best fit the cruise lines. Executives will also provide input through workshops aimed at addressing specific regional operations and improving general business with the cruise industry. Vina Adams, Procurement Manager, Food & Beverage, RCCL, will lead an interactive workshop focused on the cruise industry's purchasing guidelines, with panelists including other cruise executives, including Javonte Anyabwele, Vice President, Strategic Sourcing, Carnival Corporation & plc., and successful suppliers. David Candib, Vice President, Development & Operations, Carnival Corporation & plc, will moderate "The Destination Experience," revealing current trends in itinerary planning, how they affect Central America, and what destinations can do to improve the experience and overall region. Other panelists include Federico Gonzalez-Denton, Associate Vice President, Government Relations, Caribbean & Latin America, RCCL, and Arnaldo Zanonato, Senior Manager, Port Adventures, Disney Cruise Line. Plus, executives will be accessible during social functions fostering networking and relationship building. The functions will also introduce them to Honduras' friendly people and wide array of activities and destination products, with the attending cruise executives and stakeholders getting first-hand experiences of the destination's cruise tourism infrastructure and what passengers can see and do while visiting, as well as Honduras' ability to organize events and provide MICE tourism. About the Florida-Caribbean Cruise Association Created in 1972, the FCCA is a not-for-profit trade organization that provides a forum for discussion on tourism development, ports, safety, security, and other cruise industry issue and builds bilateral relationships with destinations' private and public sectors. By fostering an understanding of the cruise industry and its operating practices, the FCCA works with governments, ports and private sector representatives to maximize cruise passenger, crew and cruise line spending, as well as enhance the destination experience and increase the amount of cruise passengers returning as stay-over visitors. For more information, visit F-CCA.com, the FCCA on Facebook, and @FCCAupdates on Twitter. To view the original version on PR Newswire, visit:http://www.prnewswire.com/news-releases/local-stakeholders-improve-cruise-tourism-business-and-understanding-at-the-fcca-central-america-cruise-summit-in-honduras-300454139.html


News Article | May 12, 2017
Site: www.prweb.com

New Hampshire Businesses for Social Responsibility (NHBSR) is excited to announce the winners of this year’s Cornerstone and Partnership for Innovation Awards which were awarded at NHBSR’s16th annual conference on May 3. Applicants are judged on their Corporate Social Responsibility (CSR) practices within their workplace/organization, the ways in which they encourage others to engage in sustainability, and promote the value of CSR within the New Hampshire business community. The 2017 Cornerstone Award, sponsored this year by Service Credit Union, goes to Cirtronics of Milford. The Cornerstone is presented annually to an individual, organization, or company exemplifying the concepts of corporate responsibility within their organization and the greater business community in the state of New Hampshire. "We are truly honored to receive the NHBSR Cornerstone award," says George Mandragouras, CFO of Cirtronics. "Social responsibility and a service mentality are knit into the very fabric of our culture at Cirtronics. As a manufacturing company, we focus on continuous improvement in our methods and processes-- and we apply this same focus to our culture, and to the "6 we serve". We're proud of the culture we have built, but we don't take it for granted. We keep at it- we are constantly assessing how we are doing against our values, and asking how can we do better?" "I'm delighted to hear that Cirtronics has won the 2017 Cornerstone award," says local business consultant Kate Johnson, who submitted the nomination. "Service is truly central to the culture of Cirtronics. In fact, as part of their core values, Cirtronics has what they call the "6 we serve" -- supplier, company, employee owner, customer, community, and environment. And it's true: Every customer, every employee, and every supplier can feel their dedication to service in every interaction. And this commitment is also felt in the ongoing work of Cirtronics' environmentally sustainable initiatives, and their quiet support of a wide range of organizations in the local community from soup kitchens, to scout troops. The warmth of the employees, and their enthusiasm for fostering meaningful connections within the company and the community truly set them apart." The Partnership for Innovation Award, a new award introduced in 2016, recognizes a collaboration that created synergy and progress on a sustainability initiative where it might not otherwise have been possible. The 2017 Partnership for Innovation Award, sponsored this year by ReVision Energy, goes to CCA for Social Good, for leveraging business knowledge for positive community impact in partnership with Early Learning N.H. Denise Sayer, Vice President of CCA for Social Good, was present to receive the award. She shares, “CCA For Social Good is honored to be selected as this year’s recipient of the NHBSR Partnership for Innovation Award.  The CCA For Social Good work is rooted in CCA Global Partners’ innovative model that benefits communities in New Hampshire and across the United States by supporting independent business’ economic vitality and strengthening the fabric of our community. It truly is a privilege to have our work recognized for its social impact and shared value for business and society.” The following finalists were recognized: Ray Dube of Coca-Cola Bottling Company NNE of Bedford, ReVision Energy of Exeter and W.S. Badger of Gilsum, who was inducted into the Cornerstone Hall of Fame, which recognizes prior winners who finish as a top- three finalist. Photo 1: Cornerstone Award Recipient: Pictured left to right: Michelle Veasey (Executive Director, NH Businesses for Social Responsibility), Jeffrey Rose (Commissioner, Department of Resources of Economic Development); Jim Pellerin, George Mandragouras, Matt McCormack and Jessica Kinsey (Cirtronics); and Andrew McGeorge (Service Credit Union). Photo 2: Partnership for Innovation Award Recipient: Pictured left to right: Christina Zlotnick (ReVision Energy), Michelle Veasey (NH Businesses for Social Responsibility), Denise Sayer (CCA for Social Good), Jeffrey Rose (Commissioner, Department of Resources of Economic Development) Photo credit: Annie Card ABOUT NHSBR: New Hampshire Businesses for Social Responsibility is a member-based non-profit organization that fosters socially and environmentally responsible business in New Hampshire, realizing that people, principles and profits must be linked. As sustainable business becomes mainstream, NHBSR's network of CSR-focused business leaders provides the support to encourage the growth of sustainability throughout the state with value-driven results.   NHBSR is the business organization driving the social responsibility agenda in New Hampshire.  For more information, visit http://www.nhbsr.org.


News Article | May 9, 2017
Site: www.realwire.com

Announcement follows Cogeco Peer 1’s partnership with Jisc and connection to Janet Network London. 9 May, 2017: Cogeco Peer 1, a globally recognised specialist provider of colocation, network connectivity, managed hosting, cloud and managed services, today announced its partnership with DTP Group (DTP), a leading provider of information technology solutions and services whose client list includes Imperial College, British Library, University of Liverpool and University of Manchester. The partnership follows the recent announcement that Cogeco Peer 1 has partnered with JISC to provide higher education institutions on the Janet Network with access to IT services. Cogeco Peer 1’s addition to the Janet network provides a direct connection to flexible, high-performance, bespoke, managed IT infrastructure solutions including colocation, hosting and cloud. DTP provides intelligent, ethical solutions that are practical, achievable and affordable and deliver real outcomes. Together, the two companies will be focused on helping organisations including higher education and research institutions to access and maximise the value of Janet-connected cloud, colocation, hosting, security and network services, augmenting their existing range of IT services. Susan Bowen, Vice President and General Manager, EMEA at Cogeco Peer 1 said: “Cogeco Peer 1 is working with JISC to provide the infrastructure to higher education and research institutions. DTP’s partnership, which is one of a number we will be cultivating, reflects our strategic commitment to provide a comprehensive, tailored and flexible range of solutions to the sector.” Bowen added: “DTP has outstanding credentials in higher education and has established itself as a leading provider of IT services to this sector. We look forward to working with DTP and its customers as they take advantage of the partnership between Cogeco Peer 1 and JISC.” Howard Hall, Group Managing Director at DTP said; “We are very impressed by the range of services and the approach that Cogeco Peer 1 is offering the higher education sector, specifically through its partnership with JISC. Through this partnership we will be able to work with our customers to ensure that the advantages of the DTP, Cogeco Peer 1 and JISC offering can be applied to full effect.” Universities and research establishments can now work with DTP and use their existing high speed Janet network to connect directly to Cogeco Peer 1 and a suite of infrastructure options from colocation, to hosting, to cloud. This choice and flexibility, combined with the expertise of DTP and Cogeco Peer 1’s teams will allow the UK’s higher education and research facilities to put in place bespoke suites of technology infrastructure that meet the demands they face today, and make the right choices to future-proof their IT architecture for tomorrow too. About Cogeco Peer 1: Cogeco Peer 1 is a wholly-owned subsidiary of Cogeco Communications Inc. (TSX:CCA) and is a global provider of essential business-to-business products and services, such as colocation, network connectivity, managed hosting, cloud services and managed services, that allow customers across Canada, Mexico, the United States and Western Europe to focus on their core business. With 16 data centres, extensive FastFiber Network® and more than 50 points of presence in North America and Europe combined, Cogeco Peer 1 is a trusted partner to businesses small, medium and large, providing the ability to access, move, manage and store mission-critical data worldwide, backed by superior customer support. More information visit: www.cogecopeer1.com About DTP: DTP Group is an IT infrastructure and solutions specialist with expertise across cloud and infrastructure services, end user computing, and managed print and desktop services. As the sixth largest IT provider to Higher Education it provides innovative IT services and solutions to over 80% of the UKs Universities, as well as to the public sector and some of the largest UK businesses. With offices in Leeds, Central London, Wales and Scotland it offers nationwide coverage to its thousands of customers. For more information visit www.dtpgroup.co.uk.


News Article | May 12, 2017
Site: www.prweb.com

ABC-Amega is proud to announce that its Chief Financial Officer, Robert (Bob) States, has been promoted to the additional post of Executive Vice President. Bob States began his affiliation with ABC-Amega in 2011 serving in a consulting capacity for the company, before being named Chief Financial Officer in March of 2012. In his current role, Bob oversees the company’s Finance and IT divisions. He recently directed the implementation of state-of-the-art data security measures, and in 2015, he directly managed the company’s move to the historic 500 Seneca building in Buffalo’s Hydraulics District. “Bob’s accomplishments through the years make him an outstanding executive who enhances our standing in our marketplace, in our community, and among each other,” said David Herer, Chief Executive Officer, ABC-Amega. States holds an MBA in Finance and Accounting from St. Bonaventure University. He also earned a CPA, beginning his career in public accounting for industry leader KPMG, and held the position of CFO for Liazon Corporation before joining ABC-Amega. Founded in 1929 as The American Bureau of Collections, ABC-Amega is an award-winning commercial collections agency specializing in global debt collection and accounts receivable management solutions. ABC-Amega partners with clients to improve and manage credit, cash flow and customer retention with services in 3rd party commercial debt collection, 1st party accounts receivable outsourcing, industry credit group management, and credit and A/R management training and education. The firm is also a certified member of the CCA of A, dual-certified by the CLLA/IACC and is a platinum partner of the Credit Research Foundation (CRF). For additional information, please contact info(at)abc-amega(dot)com or visit http://www.abc-amega.com.

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