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Execution of our strategic three year development plan previously announced in January 2017 began in earnest during the first quarter of 2017, marking the resumption of Bellatrix's long term profitable growth.  The Company delivered on its objectives, exceeding the previously set full year 2017 production guidance target and providing clear line of sight to deliver +/-15% compound annual growth in production volumes over the next three years.  First quarter 2017 performance included the following operational and financial achievements: Bellatrix has built a strong and sustainable business anchored by three pillars that provide the foundation for long term profitable growth: 1) high quality assets and acreage, 2) infrastructure ownership and control and 3) takeaway capacity and market egress. Bellatrix is positioned to accelerate profitable growth of our large asset base as evidenced by our first quarter 2017 operational performance.  The core foundational assets for the Company reside in a proven area of the Deep Basin in west central Alberta, known for its exceptional geologic and hydrocarbon bearing characteristics.  Bellatrix maintains a dominant core acreage position along the Deep Basin fairway with decades of development ready opportunities anchored by our large inventory of net identified Spirit River and Cardium well locations and the Company retains significant torque to a higher crude oil price environment through its Cardium position. The Spirit River liquids rich natural gas play represents one of North America's lowest supply cost natural gas plays and delivers strong rates of return at current natural gas prices.  Rate of return expectations for the Spirit River continue to rank among the highest within our portfolio of investment opportunities thereby attracting the majority of anticipated capital investment in 2017.  Bellatrix has proven itself as a premier operator within the Spirit River play, consistently delivering industry leading well productivity results which drive strong rates of return for every capital dollar invested.  To that end, the first two wells completed in early February of Bellatrix's 2017 Spirit River program ranked as two of the best wells in Alberta during the month, followed by the remaining first quarter program wells being completed and placed on stream at or above internal expectations.  Our top tier acreage position and material running room provide a key long term competitive advantage for the Company. Infrastructure ownership, operatorship and control creates significant barriers to competition within our core area thus ensuring operational flexibility and reliability to profitably process our production volumes and extract maximum value from each product stream. Since 2013, Bellatrix has invested approximately $350 million in strategic infrastructure assets within its core west central Alberta area providing above ground control of the region and creating significant barriers to industry competition. The capital build out for our long term growth strategy is nearly complete with investment in the Bellatrix O'Chiese Nees-Ohpawganu'ck deep-cut gas plant at Alder Flats (the "Alder Flats Plant"), ownership in two other major natural gas processing plants, operatorship and control of nine major compressor stations, and direct operatorship in over 1,000 kilometres of strategic gathering systems and pipelines.  Bellatrix's historic investment in key strategic infrastructure and facilities provide the processing capacity and capability to grow net Company production volumes beyond 60,000 boe/d, with minimal future facility related capital.  With the proportion of capital to facilities projects expected to materially reduce in 2018 and beyond, Bellatrix anticipates directing incremental capital directly to the drill bit, thereby enhancing already industry leading corporate capital efficiency metrics. The third pillar that provides a key competitive advantage and underpins the Company's long term profitable growth profile is ensuring ample takeaway capacity and market egress for our production volumes.  Bellatrix maintains several long term firm transportation ("FT") agreements, ensuring market egress for current and forecast production, currently representing approximately 120% of current gross operated natural gas volumes at multiple receipt points on the Nova Gas Transmission Ltd. (the "NGTL") system.  The NGTL system has experienced, and is expected to experience further curtailments of both interruptible and firm service capacity as the operator continues work through 2017 to expand capacity along the system.  With excess FT relative to current production levels, Bellatrix is well positioned to deliver volumes with minimal impacts during periods of system curtailments.  Bellatrix previously negotiated additional FT capacity to facilitate increased growth volumes from Phase 2 of its Alder Flats Plant which provides additional strategic long term value for the Company. Bellatrix also maintains firm service contracts through a number of third party processing plants in its greater core Ferrier region to ensure unfettered delivery capability for current and planned production growth, with staggered contract maturity dates to align with the in-service date of Phase 2 of the Alder Flats Plant.  Finally, Bellatrix has secured fractionation capacity for its natural gas liquids ("NGL") volumes by way of long term agreements providing 100% coverage for current and forecast NGL volumes from both Phase 1 and Phase 2 of its Alder Flats Plant.  The foresight to obtain and control firm transportation along the main transmission system for not only current but also forecast growth volumes provides a key competitive long term advantage for the Company. Bellatrix continued to protect its long term strategic plan by adding to its already strong risk management position during the first quarter of 2017.  Bellatrix maintains approximately 64% of forecast gross natural gas volumes in 2017 hedged at an average fixed price of approximately $3.36/mcf (based on the mid-point of the updated 2017 average gross production guidance of 34,500 boe/d; 76% natural gas weighted).  In addition, the Company has in place material risk management protection in 2018 with a total of 65.6 MMcf/d of 2018 natural gas volumes hedged at an average fixed price of approximately $3.08/mcf; this represents approximately 42% of volumes compared to the mid-point of the updated 2017 full year average guidance. Strong propane prices in the first quarter of 2017 provided an attractive opportunity for Bellatrix to hedge 1,500 bbl/d of propane volumes at an average price of 51% of WTI light oil prices from February through December of 2017, and 1,000 bbl/d of propane volumes at an average price of 47% of WTI light oil prices in 2018, both meaningfully above long term historical averages.  Bellatrix's hedging program is part of its overall risk management strategy focused on providing reduced commodity price volatility and greater assurance over future revenue and cash flows which help drive the capital and reinvestment decisions within our business. STRONG OPERATIONAL PERFORMANCE ACHIEVED IN THE FIRST QUARTER OF 2017 Bellatrix delivered strong operational performance through the first quarter of 2017. First quarter average production of 34,750 boe/d (75% natural gas weighted) surpassed the Company's 2017 prior average annual guidance (mid-point) estimate of 33,500 boe/d.  With first quarter objectives solidly met, Bellatrix remains favourably positioned to deliver strong results through 2017 with second quarter production levels currently exceeding management expectations.  With strong operational performance and momentum, Bellatrix is increasing its 2017 production guidance target as discussed in the Outlook section of this release. Our focus on operational execution and delivery of our three year growth strategy was evident by the strong results achieved during the first quarter of 2017.  Bellatrix participated in 13 gross (10.6 net) wells in the first quarter which included 8 gross (7.6 net) operated Spirit River wells, 2 gross (2.0 net) operated Cardium wells and also 3 gross (1.0 net) non-operated wells in the Spirit River and Ellerslie formations.  Of the ten gross operated wells drilled during the first quarter, eight were brought on-stream during the first three months of the year, with the remaining operated Spirit River well subsequently brought on-stream in early April, and the one remaining Cardium operated well drilled and planned for completion and on-stream delivery in the third quarter of 2017. Bellatrix completed its planned first quarter capital program with exploration and development expenditures of $44.0 million and an unchanged expectation to spend approximately half of the full year's net capital expenditure budget of $105 million within the first six months of the year. First quarter production expenses averaged $9.37/boe, representing a marked reduction of $1.20/boe from the $10.57/boe production expenditure level in the fourth quarter of 2016.  First quarter production expenditure levels and anticipated production and expense levels position Bellatrix to maintain its full year average production expenditure guidance of approximately $9.00/boe. Previously set year 2017 guidance expectations refer to Bellatrix's guidance as announced on January 5, 2017; updated guidance metrics are included in the forward guidance section of this press release. With the conclusion of all joint venture drilling in 2016, Bellatrix has demonstrated the flexibility in 2017 to balance infill development drilling and expanded core area development.  The Company remains focused on growing production, adding reserves, and increasing our inventory of development drilling locations.  Bellatrix's working interest in operated wells drilled in the first quarter of 2017 averaged 96%.  Well results from the first quarter program continue to meet and exceed management expectations. To that end, Bellatrix is pleased to provide enhanced transparency for its operated 2017 development program results including the: Bellatrix completed the majority of its first half program in the first three months of the year (in advance of the seasonal spring break-up period) with exploration and development capital expenditures invested during the first quarter of $44.0 million.  The Company's capital expenditure plans remain on target and in line with previously stated guidance levels for $105 million in 2017 with approximately 50% of capital expenditures to be invested within the first half of the year. ALDER FLATS PHASE 2 EXPANSION NEARING COMPLETION WHICH REPRESENTS THE FINAL STAGE OF THE COMPANY'S LONG TERM STRATEGIC INFRASTRUCTURE BUILD-OUT The Alder Flats Plant represents a highly strategic asset for the Company as we continue to execute on our three year development plan to profitably grow production, expand netbacks and grow cash flow.  Phase 1 of the Alder Flats Plant has been on-stream for 21 continuous months delivering an average 96% capacity utilization rate over that period and firmly establishing the Bellatrix Alder Flats Plant as the most efficient plant in our greater west central Alberta core area. The Phase 2 expansion project, which will more than double gross throughput capacity at the plant to 230 MMcf/d (from 110 MMcf/d currently) remains on time and on budget for completion in the second quarter of 2018.  The Plant expansion is anticipated to drive improved revenue generation through additional higher margin NGL extraction, and provide further reductions to corporate operating costs, driving expanded corporate profit margins and cash flow.  Fabrication of all major equipment for Phase 2 is complete including compressors and propane bullets.  Fabrication and packaging of other material equipment including the condensate stabilizer, production tanks, heat medium package, and electrical equipment continues to progress according to plan and is expected to arrive on site for installation over the next several quarters.  Site construction activity will recommence late in the second quarter with scheduled pile driving activity anticipated to begin in June.  Major equipment will begin being delivered to site, with installation activities and mechanical work planned to begin in the third quarter of 2017. Bellatrix's investment in strategic infrastructure assets within the greater Ferrier and Willesden Green areas of west central Alberta provide the above ground control of the region and create significant barriers to industry competition.  The capital build out for our long term growth strategy is nearly complete given prior period investment in the Alder Flats Plant, major compressor stations, and strategic gathering systems and pipelines.  Bellatrix forecasts net capital expenditures of approximately $13 million in 2017 and $3 million in 2018 (excluding received partner prepayment) required to complete Phase 2 of the Alder Flats Plant which will solidify our infrastructure control, and provide the facilities and processing capacity to grow net production volumes beyond 60,000 boe/d, with minimal future facility related capital. Completion of Phase 2 of the Alder Flats Plant, which will add an incremental 30 MMcf/d ownership capacity net to Bellatrix's 25% working interest, is expected to deliver a favourable step change reduction in operating costs down by approximately $1.00/boe relative to current full year 2017 average production expense guidance. Bellatrix has established itself as a premier operator in west central Alberta, continuously delivering top tier well results from its Spirit River development program through the first quarter of 2017, coupled with continued capital cost reductions which in combination delivered another extremely capital efficient quarter.  All-in (drill, complete, equip and tie-in) estimated well costs in the first quarter of 2017 averaged $3.8 million, unchanged from average costs of $3.8 million achieved in 2016 despite modest upward pressure on select completion service costs in 2017.  Bellatrix continues to supress costs and mitigate inflationary pressure on capital costs, given structural and sustainable improvements achieved within both drilling and completion practices. Subsequent to the end of the first quarter, Bellatrix amended and restated the terms of its syndicated revolving credit facilities (the "Credit Facilities"). Effective May 9, 2017, the total commitments under the Credit Facilities were set at $120 million, comprised of a $25 million operating facility provided by a Canadian bank and a $95 million syndicated facility provided by four financial institutions.  Total commitments under the Credit Facilities were increased 20% relative to total commitments at year end 2016.  The borrowing base increase provides enhanced liquidity relative to prior levels, while maintaining Bellatrix's financial resources at a level that minimizes standby fees.  The Company remains committed to continued fiscal prudence, and achieving near term growth objectives within current capital spending guidance levels. Other than the $41.5 million outstanding as at March 31, 2017 on the Credit Facilities, the Company has no debt maturities until 2020 and 2021. Subsequent to the end of the first quarter 2017, Bellatrix completed two separate transactions whereby it monetized its $15 million vendor take back loan receivable and divested its marketable securities for combined net proceeds of approximately $20 million.  Bellatrix utilized proceeds from these two transactions to reduce the amount outstanding on its bank credit facilities to approximately $21 million as at April 30, 2017. Management's acute focus on operational execution during the first quarter of 2017 resulted in production volume outperformance relative to previously set full year 2017 average guidance levels, enabling management to increase our full year guidance expectations as described below. The majority of our first half capital investment program was completed during the first three months of the year, as capital investment activities are customarily curtailed during the second quarter, given the seasonal spring break-up period.  Average second quarter 2017 production levels are anticipated to commensurately meet the revised full year average guidance of 34,500 boe/d given strong well results and strong operational momentum achieved during the first quarter of 2017. Our capital investment plans for 2017 remain unchanged; Bellatrix plans to expand its drilling efforts across our core west central Alberta acreage including expanded development of the Spirit River formation in the Willesden Green area, following on our success in the Ferrier area. With the completion of joint venture programs in 2016, Bellatrix has strategically reviewed its drilling program to optimize capital investment, forecast rates of return, and long term net asset value and reserve growth potential.  Our current 2017 capital expenditure guidance anticipates drilling approximately 19 net wells during the year, of which approximately 10.6 wells were drilled during the first quarter of the year with one operated Cardium well currently uncompleted.  Our focused capital investment program is supported by an active risk management program, and will continue to target the low cost and high return Spirit River liquids rich natural gas play which delivers strong rates of return. INCREASED 2017 PRODUCTION GUIDANCE WITH NO CHANGE TO CAPITAL EXPENDITURES GIVEN STRONG OPERATIONAL PERFORMANCE Strong first quarter operational results and positive momentum into the second quarter provide visibility to meaningfully outperform prior forecast guidance expectation levels.  To that end, Bellatrix is increasing its full year 2017 average production guidance expectation to 34,500 boe/d, an increase of 1,000 boe/d from prior guidance announced on January 5, 2017.  Bellatrix has maintained its full year capital expenditure guidance at $105 million despite the increase in average production levels.  Bellatrix remains committed to providing sustainable long term growth for shareholders including delivery of our 2017 capital program providing +/-15% forecast production growth. As the Company embarks on its three year strategic plan outlined in January of 2017, we remain committed to maximizing capital resources, maintaining a laser sharp focus on operational execution, and most importantly delivering on our promises.  Bellatrix's three foundational pillars provide the long term competitive advantages required to thrive in the current commodity price environment and deliver profitable growth in production, cash flow, and net asset value.  I want to personally thank our employees for their unwavering efforts as we continue to deliver and achieve our business objectives.  To our shareholders and stakeholders, we thank you for your long term support and look forward to providing continued updates throughout the year, including enhanced transparency of our operational and financial performance, and delivery of our long term strategy delivering enhanced shareholder value. Sales volumes for the three months ended March 31, 2017 averaged 34,750 boe/d, a decrease of 10% from an average of 38,467 boe/d realized in the first quarter of 2016.  The weighting towards crude oil, condensate and NGLs for the three months ended March 31, 2017 was 25%, compared to 27% in the first quarter of 2016.  Total sales volumes between the three month periods ended March 31, 2016 and March 31, 2017 declined due to non-core dispositions completed in the fourth quarter of 2016 in the Pembina and Harmattan areas. First quarter 2017 average production levels exceeded prior 2017 annual guidance (mid-point) of 33,500 boe/d and increased from the fourth quarter of 2016 by 9%.  Utilization remained strong at the Bellatrix Alder Flats Plant in the first quarter of 2017, contributing to an average capacity utilization rate of 97% achieved in the first quarter 2017 and 96% over the trailing 21 month period. During the first quarter of 2017, Bellatrix drilled and/or participated in 9 gross (7.8 net) Spirit River liquids rich gas wells, two gross (2.0 net) Cardium wells and two gross (0.8 net) non-operated Ellerslie liquids rich natural gas wells.  The Company continues to focus capital investment in its low-cost Spirit River natural gas play, which continues to deliver strong returns at current natural gas and liquids prices. During the three months ended March 31, 2017, Bellatrix invested $44.0 million in exploration and development capital projects, excluding property acquisitions and dispositions, compared to $29.0 million in the same period in 2016. Bellatrix focused its capital activity in the first quarter of 2017 on drilling and completion activity within the Spirit River formation, as well as facilities and equipment expenditures related to the construction of Phase 2 of the Alder Flats Plant.  Bellatrix continues to advance the Phase 2 expansion project of the Alder Flats Plant which is expected to more than double the inlet capacity of the Plant from 110 MMcf/d currently to 230 MMcf/d.  The project remains on time and budget, and is scheduled for completion in the second quarter 2018. Subsequent to the first quarter of 2017, Bellatrix closed an agreement to complete the construction of, and transfer to a third party midstream company, certain production facilities and infrastructure in exchange for proceeds of $20 million.  Capital expenditures of $5.6 million were incurred in the first quarter 2017 and will be transferred under the agreement to the third party midstream company in the second quarter of 2017. Under the terms of the arrangement, Bellatrix will have exclusive access to, and operatorship of, the infrastructure. At March 31, 2017, Bellatrix had approximately 173,595 undeveloped acres of land in Alberta, British Columbia, and Saskatchewan. Management believes that, in addition to cash flow from operating activities, funds flow from operations is a useful supplemental measure as it demonstrates the Company's ability to generate the cash necessary to fund future capital investments and to repay debt.  Funds flow from operations is calculated as cash flow from operating activities, excluding decommissioning costs incurred and changes in non-cash working capital incurred. Bellatrix's cash flow from operating activities for the three months ended March 31, 2017 decreased by 19% to $8.3 million ($0.03 per basic and diluted share) from $10.3 million ($0.05 per basic and diluted share) generated in the first quarter of 2016.  Bellatrix generated funds flow from operations of $14.9 million ($0.06 per basic and $0.05 per diluted share) in the first quarter of 2017, an increase of 16% from $12.9 million ($0.07 per basic and diluted share) generated in the comparative 2016 period.  Funds flow increased in the first quarter of 2017 compared to the same period in 2016 primarily from the increased realized commodity prices over the comparable period. For the three months ended March 31, 2017, Bellatrix recognized net profit of $13.0 million ($0.05 per basic share and diluted share), compared to a net profit of $19.3 million ($0.10 per basic and diluted share) in the first quarter of 2016 due to a decrease in the unrealized foreign exchange gain in the first quarter of 2017, offset by an increase in the operating netbacks realized. The operating netback before commodity price risk management contracts for crude oil, condensate, NGLs, and natural gas during the first quarter of 2017 averaged $8.35/boe, an increase of 28% from the $6.50/boe realized during the same period in 2016 reflecting improved realized commodity prices over the comparable periods. Total revenue increased by 20% to $66.0 million for the three months ended March 31, 2017, compared to $55.2 million realized in the first quarter of 2016. The increase in total revenue was mainly attributable to an increase in commodity prices for oil, NGLs and natural gas from the comparative periods. Total revenue from crude oil, condensate, and NGLs contributed 38% of total first quarter 2017 revenue before other income, royalties, and commodity price risk management contracts, compared to 40% in the three months ended March 31, 2016. In the three months ended March 31, 2017, production expenses totaled $29.3 million ($9.37/boe), compared to $25.8 million ($7.37/boe) recorded in the same period of 2016.  Production expenditures are expected to decline during 2017 and average $9.00/boe during the year, given cost suppression initiatives and increased production volumes which reduce overall production expenditures on a per unit of production basis. For the three months ended March 31, 2017, Bellatrix incurred royalties of $7.4 million, compared to $3.4 million in the first quarter of 2016.  Overall royalties as a percentage of revenue (after transportation costs) in the first quarter of 2017 were 12% compared to 7% in the comparative 2016 period.  Higher average corporate royalty rates period over period include the impact from higher commodity prices as well as decreased GCA credits in the current period than in the first quarter of 2016 when the Company had credits associated with significant infrastructure and facilities investments. Global crude oil prices fluctuated during the first quarter 2017 as the Organization of the Petroleum Exporting Countries ("OPEC") members worked to achieve production cuts and add stability to crude oil prices.  Despite the collaborative efforts between OPEC and non-OPEC producing countries in stabilizing prices, WTI oil prices exhibited volatility, opening the year at US$53.72/bbl, reaching a low of US$47.00/bbl during the first quarter of 2017, before recovering to US$50.60/bbl at March 31, 2017. Robust global crude inventories and weekly increases in the United States oil directed drilling rig count are factors that tempered oil prices strengthening during the first quarter of 2017. North American natural gas prices declined during February of 2017 as warmer than normal weather kept heating demand at lower than expected levels. United States natural gas storage inventories at March 31, 2017 were 427 Bcf below last year's record high level, while declining United States production levels have added support to natural gas prices, rebounding from US$2.44/MMBtu in late February to close the first quarter at US$3.10/MMBtu. Total United States natural gas production continues to decline despite increased drilling activity. The combination of lower production, higher exports of liquefied natural gas ("LNG") and increased gas supplies to Mexico improved supply/demand dynamics in the market. Overall, industry activity levels are causing a slower supply response given backwardation in the forward pricing market. In the first quarter of 2017 Bellatrix realized an average price of $67.30/bbl before commodity price risk management contracts for crude oil and condensate, an increase of 71% from the average price of $39.33/bbl received in the first quarter of 2016.  By comparison, Canadian Light crude blend price increased by 57% and the average WTI crude oil benchmark price increased by 54% between the first quarters of 2017 and 2016.  The WTI/Canadian Light sweet differential has remained in a historically tight range, averaging -US$3.54/bbl for the quarter. Bellatrix's average realized price for NGLs (excluding condensate) increased by 76% to $18.18/bbl during the first quarter of 2017, compared to $10.35/bbl received in the comparable 2016 period. NGL pricing in Western Canada improved significantly through the fourth quarter of 2016 given stronger underlying light oil prices and improved individual market conditions for propane and butane products.  Normal winter demand through the first quarter of 2017 kept North American propane demand firm, while exports materially reduced robust storage levels resulting in much stronger propane prices through the quarter.  Butane prices closely track the trend in WTI pricing and thus exhibited similar volatility to oil prices during the first quarter of 2017. Butane prices improved in the first quarter of 2017 compared to the first quarter of 2016 and Bellatrix's average realized NGL price reflected the improvement in butane prices by 75% year over year. Natural gas prices increased during the first quarter of 2017 given strong demand and lower United States production resulting in reduced storage levels.  Bellatrix's natural gas sales are priced with reference to the daily or monthly AECO indices.  Bellatrix's natural gas sold has a higher heat content than the industry average, which results in slightly higher realized prices per mcf than the AECO daily index.  During the first quarter of 2017, the AECO daily reference price increased by 47% and the AECO monthly reference price increased by approximately 39% compared to the first quarter of 2016.  Bellatrix's natural gas average sales price before commodity price risk management contracts for the first quarter of 2017 increased by 44% to $2.87/mcf compared to $1.99/mcf in the same period in 2016.  Bellatrix's natural gas average price after including commodity price risk management contracts for the three months ended March 31, 2017 averaged $3.09/mcf compared to $2.41/mcf in the comparative 2016 period. Bellatrix was active in the first quarter of 2017 increasing its 2017 risk management protection, with approximately 64% of 2017 gross natural gas volumes hedged at an average fixed price of approximately $3.36/mcf. At March 31, 2017, the Company had $41.5 million outstanding under its syndicated revolving Credit Facilities at a weighted average interest rate of 4.45%. As at March 31, 2017, the Company's Credit Facilities were available on an extendible revolving term basis and consisted of a $100 million facility provided by nine financial institutions, subject to a borrowing base test.  The maturity date of the Credit Facilities was October 1, 2017 as at March 31, 2017. Subsequent to March 31, 2017, Bellatrix entered into an amended and restated syndicated revolving credit facility agreement provided by four financial institutions increasing the borrowing base of the Credit Facilities to $120 million. Under the amended and restated terms, the Credit Facilities are available on an extendible revolving term basis and consists of a $25 million operated facility and a $95 million syndicated facility. Under the amended and restated terms, the Credit Facilities have an initial term of one year that is extendible annually at the option of the Company, subject to lender approval, with a 1 year term-out period if not renewed.  Availability under the Credit Facilities is subject to a borrowing base test, which will be subject to redetermination in May and November of each year, with the next regularly scheduled redetermination to occur in November 2017. At March 31, 2017, Bellatrix had approximately $58.5 million of available capacity based on outstanding bank debt as at such date of approximately $41.5 million (excluding letters of credit). The Credit Facilities include a single financial covenant being the Company's Senior Debt to EBITDA ratio must not exceed 3.5 times for the fiscal quarters ending on or before March 31, 2017 ("Senior Debt Covenant"). Under the amended and restated terms of the Credit Facilities, the maximum Senior Debt to EBITDA ratio has been reduced to 3.0 times (3.5 times for the two fiscal quarters immediately following a material acquisition).  As at March 31, 2017, the Senior Debt to EBITDA ratio was 1.81 times. At March 31, 2017, the Company has outstanding US$250 million of 8.50% senior unsecured notes maturing on May 15, 2020 (the "Senior Notes").  Interest on the Senior Notes is payable semi-annually and the Senior Notes are redeemable at the Company's option, in whole or in part, commencing on May 15, 2017 at specified redemption prices. At March 31, 2017, the Company has outstanding $50 million principal amount of 6.75% convertible subordinated debentures (the "Convertible Debentures") due on September 30, 2021 (the "Maturity Date"). Interest on the Convertible Debentures is payable semiannually in arrears on September 30 and March 31 of each year. A conference call to discuss Bellatrix's first quarter results will be held on May 10, 2017 at 9:00 am MT / 11:00 am ET. To participate, please call toll-free 1-800-319-4610 or 403-351-0324 or 416-915-3239.  The call can also be heard live through an internet webcast accessible via the investors section of Bellatrix's website at http://investors.bellatrixexploration.com/webcasts and will be archived on the website for approximately 30 days following the call. Bellatrix Exploration Ltd. is a Western Canadian based growth oriented oil and gas company engaged in the exploration for, and the acquisition, development, and production of oil and natural gas reserves in the provinces of Alberta, British Columbia, and Saskatchewan. Common shares of Bellatrix trade on the Toronto Stock Exchange and on the New York Stock Exchange under the symbol "BXE". Throughout this press release, the Company uses terms that are commonly used in the oil and natural gas industry, but do not have a standardized meaning presented by International Financial Reporting Standards ("IFRS") and therefore may not be comparable to the calculations of similar measures for other entities. Management believes that the presentation of these non-GAAP measures provide useful information to investors and shareholders as the measures provide increased transparency and the ability to better analyze performance against prior periods on a comparable basis. Operating netbacks are calculated by subtracting royalties, transportation, and operating expenses from total revenue.  Management believes this measure is a useful supplemental measure of the amount of total revenue received after transportation, royalties and operating expenses. The Company's calculation of total revenue includes petroleum and natural gas sales and other income, and excludes commodity price risk management. Total capital expenditures – net includes the cash impact of capital expenditures and property dispositions, as well as the non-cash capital impacts of corporate acquisitions, property acquisitions, adjustments to the Company's decommissioning liabilities, and share based compensation. Bellatrix's method of calculating this measure may differ from other entities, and accordingly, may not be comparable to measures used by other companies.  Total capital expenditures - net includes the cash impact of capital expenditures and property dispositions, as well as the non-cash capital impacts of corporate acquisitions, property acquisitions, adjustments to the Company's decommissioning liabilities, and share based compensation. These measures have been described and presented in this news release in order to provide shareholders and potential investors with additional information regarding Bellatrix's liquidity and its ability to generate funds to finance its operations. In addition to the non-GAAP measures described above, there are also terms that have been reconciled in the Company's financial statements to the most comparable IFRS measures. These terms do not have any standardized meaning prescribed by IFRS and therefore may not be comparable with the calculations of similar measures for other entities. These terms have been referenced in the Company's press release, MD&A and financial statements. These terms are used by management to analyze operating performance on a comparable basis with prior periods and to analyze the liquidity of the Company. This press release contains the term "funds flow from operations" which should not be considered an alternative to, or more meaningful than "cash flow from operating activities" as determined in accordance with GAAP as an indicator of the Company's performance. Therefore reference to funds flow from operations or funds flow from operations per share may not be comparable with the calculation of similar measures for other entities. Management uses funds flow from operations to analyze operating performance and leverage and considers funds flow from operations to be a key measure as it demonstrates the Company's ability to generate the cash necessary to fund future capital investments and to repay debt.  Funds flow from operations is calculated as cash flow from operating activities, excluding decommissioning costs incurred, changes in non-cash working capital incurred, and transaction costs.  The reconciliation between cash flow from operating activities and funds flow from operations can be found in the MD&A.  Funds flow from operations per share is calculated using the weighted average number of shares for the period. This press release also contains the terms "total net debt" and "adjusted working capital deficiency", which also are not recognized measures under GAAP. Therefore reference to total net debt and adjusted working capital deficiency, may not be comparable with the calculation of similar measures for other entities.  The Company's calculation of total net debt excludes other deferred liabilities, deferred capital obligations, long-term risk management contract liabilities, decommissioning liabilities, and deferred tax liabilities.  Total net debt includes the adjusted working capital deficiency, long term loans receivable, Convertible Debentures (liability component), current bank debt and long term bank debt.  The adjusted working capital deficiency is calculated as net working capital deficiency excluding current risk management contract assets and liabilities, current portion of other deferred liabilities, current portion of deferred capital obligation and the current bank debt.  Management believes these measures are useful supplementary measures of the total amount of current and long-term debt. Certain information contained in this press release may contain forward looking statements within the meaning of applicable securities laws. The use of any of the words "position", "continue", "opportunity", "expect", "plan", "maintain", "estimate", "assume", "target", "believe" "forecast", "intend", "strategy", "anticipate", "enhance" and similar expressions are intended to identify forward-looking statements. More particularly and without limitation, this document contains forward-looking statements concerning management's assessment of future plans, Bellatrix's intention to deliver 10% to 15% compound annual growth in production volumes over the next three years, the belief that Bellatrix has built a strong and sustainable business that provide the foundation for long term profitable growth, future drilling locations, the belief that the Company can benefit from a higher crude oil price environment through its Cardium position, the expectation that the majority of anticipated capital investment in 2017 will be in the Spirit River, the belief that the Company's acreage position provides a key long term competitive advantage for the Company, the belief that the Company's infrastructure ownership, operatorship and control will provide for operational flexibility and reliability to profitably process our production volumes and extract maximum value from each product stream, the expectation that historic investment in key strategic infrastructure and facilities may provide the processing capacity and capability to grow net Company production volumes beyond 60,000 boe/d with minimal future facility related capital, the expectation that Bellatrix will direct incremental capital directly to the drill bit in 2018 and beyond which may enhance corporate capital efficiency metrics,  the expectation that the NGTL system may experience further curtailments of both interruptible and firm service capacity as the operator continues work through 2017 to expand capacity along the system, the belief that Bellatrix is well positioned to deliver volumes with minimal impacts during periods of system curtailments, the belief that previously negotiated additional FT capacity may facilitate increased growth volumes from Phase 2 of its Alder Flats Plant and provide additional strategic long term value for the Company, expectation of percentage of production hedged in 2017 and 2018, the expectation that Bellatrix's hedging program will provide reduced commodity price volatility and greater assurance over future revenue and cash flows, the expectations for timing for drilling, completing and bringing on-stream of certain wells, the expectation to spend approximately half of the full year's 2017 net capital expenditure budget of $105 million within the first six months of the year, full year average production expenditure guidance, the intent of the Company to remains focused on growing production, adding reserves, and increasing our inventory of development drilling locations, the expected capacity of Phase 2 of the Alder Flats Plant, the expectation that construction of Phase 2 of the Alder Flats Plant will be completed on time and on budget in the second quarter of 2018, expectation of timing of specific tasks required for construction and completion of Phase 2 of the Alder Flats Plant, expected net capital expenditures in 2017 and 2018 required to complete Phase 2 of the Alder Flats Plant, the expectation that completion of Phase 2 of the Alder Flats Plant may deliver a reduction in operating costs, Bellatrix's plans to expand its drilling efforts across our core west central Alberta acreage including expanded development of the Spirit River formation in the Willesden Green area, details of Bellatrix's current 2017 capital expenditure budget and the goals of such budget, guidance relating to 2017 average daily production and exit production (including the product mix) and the expectation that Bellatrix's business provide the long term competitive advantages required to thrive in the current commodity price environment and deliver profitable growth in production, cash flow, and net asset value, may constitute forward-looking statements under applicable securities laws. To the extent that any forward-looking information contained herein constitute a financial outlook, they were approved by management on May 9, 2017 and are included herein to provide readers with an understanding of the anticipated funds available to Bellatrix to fund its operations and readers are cautioned that the information may not be appropriate for other purposes.  Forward-looking statements necessarily involve risks, including, without limitation, risks associated with oil and gas exploration, development, exploitation, production, marketing and transportation, loss of markets, volatility of commodity prices, currency fluctuations, imprecision of reserve estimates, environmental risks, competition from other producers, inability to retain drilling rigs and other services, incorrect assessment of the value of acquisitions, failure to realize the anticipated benefits of acquisitions, delays resulting from or inability to obtain required regulatory approvals, actions taken by the Company's lenders that reduce the Company's available credit and ability to access sufficient capital from internal and external sources.  Events or circumstances may cause actual results to differ materially from those predicted, as a result of the risk factors set out and other known and unknown risks, uncertainties, and other factors, many of which are beyond the control of Bellatrix. In addition, forward looking statements or information are based on a number of factors and assumptions which have been used to develop such statements and information but which may prove to be incorrect and which have been used to develop such statements and information in order to provide shareholders with a more complete perspective on Bellatrix's future operations.  Such information may prove to be incorrect and readers are cautioned that the information may not be appropriate for other purposes.  Although the Company believes that the expectations reflected in such forward looking statements or information are reasonable, undue reliance should not be placed on forward looking statements because the Company can give no assurance that such expectations will prove to be correct.  In addition to other factors and assumptions which may be identified herein, assumptions have been made regarding, among other things: the impact of increasing competition; the general stability of the economic and political environment in which the Company operates; the timely receipt of any required regulatory approvals; the ability of the Company to obtain qualified staff, equipment and services in a timely and cost efficient manner; drilling results; the ability of the operator of the projects which the Company has an interest in to operate the field in a safe, efficient and effective manner; the ability of the Company to obtain financing on acceptable terms; field production rates and decline rates; the ability to replace and expand oil and natural gas reserves through acquisition, development of exploration; the timing and costs of pipeline, storage and facility construction and expansion and the ability of the Company to secure adequate product transportation; future commodity prices; currency, exchange and interest rates; the regulatory framework regarding royalties, taxes and environmental matters in the jurisdictions in which the Company operates; and the ability of the Company to successfully market its oil and natural gas products.  Readers are cautioned that the foregoing list is not exhaustive of all factors and assumptions which have been used.  As a consequence, actual results may differ materially from those anticipated in the forward-looking statements.  Additional information on these and other factors that could affect Bellatrix's operations and financial results are included in reports on file with Canadian and United States securities regulatory authorities and may be accessed through the SEDAR website (www.sedar.com), through the SEC website (www.sec.gov), and at Bellatrix's website (www.bellatrixexploration.com).  Furthermore, the forward looking statements contained herein are made as at the date hereof and Bellatrix does not undertake any obligation to update publicly or to revise any of the included forward looking statements, whether as a result of new information, future events or otherwise, except as may be required by applicable securities laws. The term barrels of oil equivalent ("boe") may be misleading, particularly if used in isolation. A boe conversion ratio of six thousand cubic feet of natural gas to one barrel of oil equivalent (6 mcf/bbl) is based on an energy equivalency conversion method primarily applicable at the burner tip and does not represent a value equivalency at the wellhead. All boe conversions in this press release are derived from converting gas to oil in the ratio of six thousand cubic feet of gas to one barrel of oil. Given that the value ratio based on the current price of crude oil as compared to natural gas is significantly different from the energy equivalency of 6:1, utilizing a conversion on a 6:1 basis may be misleading as an indication of value. References in this press release to initial production rates associated with certain wells are useful in confirming the presence of hydrocarbons, however such rates are not determinative of the rates at which such wells will commence production and decline thereafter and are not indicative of long term performance or of ultimate recovery. While encouraging, readers are cautioned not to place reliance on such rates in calculating the aggregate production for the Company. The Company cautions that such production rates should be considered to be preliminary.


News Article | May 22, 2017
Site: www.businesswire.com

SOUTH SAN FRANCISCO, Calif.--(BUSINESS WIRE)--Genentech, a member of the Roche Group (SIX: RO, ROG; OTCQX: RHHBY), announced today that the U.S. Food and Drug Administration (FDA) has approved Actemra® (tocilizumab) subcutaneous injection for the treatment of GCA, a chronic and severe autoimmune condition. Actemra is the first therapy approved by the FDA for the treatment of adult patients with GCA. This is the sixth FDA approval for Actemra since the medicine was launched in 2010. “Today’s FDA decision means people living with giant cell arteritis will, for the first time, have an FDA-approved treatment option for this debilitating disease,” said Sandra Horning, M.D., chief medical officer and head of Global Product Development. “With no new treatments in more than 50 years, this approval could be transformational for people with GCA and for their physicians.” The approval is based on the positive outcome of the Phase III GiACTA study evaluating Actemra in patients with GCA. The results showed that Actemra, initially combined with a six-month steroid (glucocorticoid) regimen, more effectively sustained remission through 52 weeks (56 percent in the Actemra weekly group and 53.1 percent in the Actemra bi-weekly group) compared to placebo combined with a 26-week steroid taper (14 percent) and placebo combined with a 52-week steroid taper (17.6 percent).1 GiACTA (NCT01791153) is a Phase III, global, randomized, double-blind, placebo-controlled trial investigating the efficacy and safety of Actemra as a novel treatment for GCA. It is the first successful clinical trial conducted in GCA and the first to use blinded, variable-dose, variable-duration steroid regimens. The multicenter study was conducted in 251 patients across 76 sites in 14 countries. The primary endpoint was evaluated at 52 weeks. GCA, also known as temporal arteritis (TA), affects an estimated 228,000 people over the age of 50 in the U.S., and the disease is two to three times more likely to affect women than men.2,3 GCA is often difficult to diagnose because of the wide and variable spectrum of signs and symptoms. GCA can cause severe headaches, jaw pain and visual symptoms and if left untreated, can lead to blindness, aortic aneurysm or stroke.3 Treatment to date for people with GCA has been limited to high-dose steroids that play a role as an effective ‘emergency’ treatment option to prevent damage such as vision loss.3 Due to the variability of symptoms, complexity of the disease and disease complications, people with GCA are often seen by several physicians including rheumatologists, ophthalmologists and neurologists. Actemra is the first humanized interleukin-6 (IL-6) receptor antagonist approved for the treatment of adult patients with moderately to severely active rheumatoid arthritis (RA) who have used one or more disease-modifying antirheumatic drugs (DMARDs), such as methotrexate (MTX), that did not provide enough relief. The extensive Actemra RA IV clinical development program included five Phase III clinical studies and enrolled more than 4,000 people with RA in 41 countries. The Actemra RA subcutaneous clinical development program included two Phase III clinical studies and enrolled more than 1,800 people with RA in 33 countries. In addition, Actemra is also used as an IV formulation for patients with active polyarticular juvenile idiopathic arthritis (PJIA) or systemic juvenile idiopathic arthritis (SJIA) two years of age and older. Actemra is not approved for subcutaneous use in people with PJIA or SJIA. It is not known if Actemra is safe and effective in children with PJIA or SJIA under two years of age or in children with conditions other than PJIA or SJIA. Actemra is intended for use under the guidance of a healthcare practitioner. Actemra can cause serious side effects. Actemra changes the way a patient’s immune system works. This can make a patient more likely to get infections or make any current infection worse. Some people taking Actemra have died from these infections. Actemra can cause other serious side effects. These include: Patients should not receive Actemra if they are allergic to Actemra or if they have had a bad reaction to Actemra previously. Patients should tell their doctor if they have these or any other side effect that bothers them or does not go away: Patients should tell their doctor if they are planning to become pregnant, are pregnant, plan to breastfeed, or are breastfeeding. The patient and their doctor should decide if the patient will take Actemra or breastfeed. Patients should not do both. If a patient is pregnant and taking Actemra, they should join the pregnancy registry. To learn more, patients should call 1-877-311-8972 or talk to their doctor to register. Patients should tell their doctor right away if they are experiencing any side effects. Report side effects to the FDA at 1-800-FDA-1088 or http://www.FDA.gov/medwatch. Report side effects to Genentech at 1-888-835-2555. Please visit http://www.actemra.com for the full Prescribing Information, including Boxed Warning and Medication Guide, for additional Important Safety Information or call 1-800-ACTEMRA (228-3672). Actemra is part of a co-development agreement with Chugai Pharmaceutical Co. and has been approved in Japan since June 2005. Actemra is approved in the European Union, where it is known as RoActemra, and several other countries, including China, India, Brazil, Switzerland and Australia. Founded more than 40 years ago, Genentech is a leading biotechnology company that discovers, develops, manufactures and commercializes medicines to treat patients with serious or life-threatening medical conditions. The company, a member of the Roche Group, has headquarters in South San Francisco, California. For additional information about the company, please visit http://www.gene.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.


News Article | May 5, 2017
Site: globenewswire.com

At the 2017 Waterfront Conference on May 10, the Waterfront Alliance Previews the Region’s First-Ever Harbor Scorecard How protected are our coastal communities and our infrastructure? Are waterways meeting Clean Water Act standards? Where are opportunities for public access to the water? Turn to the Harbor Scorecard for answers and action, neighborhood by neighborhood NEW YORK, May 05, 2017 (GLOBE NEWSWIRE) -- At this year’s Waterfront Conference, titled “Measuring Our Harbor: Strong, Healthy, and Open,” the Waterfront Alliance will preview the region’s first-ever Harbor Scorecard. Comprehensive and user-friendly, the Scorecard compiles research in public access, ecology, and resiliency for a neighborhood-by-neighborhood evaluation of the waterfronts of New York City and northern New Jersey. Distilling extensive research into an easy-to-use tool, the Harbor Scorecard will be previewed at the Waterfront Alliance’s annual Waterfront Conference on Wednesday, May 10, and rolled out three weeks later in time for the start of hurricane season. The Waterfront Conference takes place aboard the Hornblower Infinity, dockside at Hudson River Park, Pier 40, in the morning, and cruising New York Harbor throughout the afternoon. Featured speakers include Rep. Nydia M. Velázquez, Member of Congress; Lauren Brand, U.S. Department of Transportation, Maritime Administration; Hon. Ras J. Baraka, Mayor, City of Newark; and Alicia Glen, Deputy Mayor for Housing and Economic Development, City of New York, along with dozens of expert panelists and workshop leaders, and hundreds of policy-makers, waterfront advocates, and professionals. Hornblower Cruises & Events is the venue sponsor. For the third year, Arcadis is the premier sponsor of the Waterfront Conference, and for the second year, the sponsor of Arcadis Waterfront Scholars, a program that invites more than 70 undergraduate and graduate students to participate in the conference and engage with professional mentors. On May 10, Arcadis Global Lead for Water Management Piet Dircke will discuss the Harbor Scorecard in a global context using the Arcadis Sustainable Cities Water Index, which assesses the water resources of 50 cities around the world. “New York City is making strides in protecting its coastline, and the Waterfront Conference is an important step in bringing together the best minds to help benchmark our harbors and waterways,” said Mr. Dircke. “Waiting until the next big storm to create safeguards against future disasters is not a sound resiliency strategy.” “As the Waterfront Alliance begins our milestone tenth year of work, we have put together the Harbor Scorecard, an essential tool to take stock of progress along our waterways,” said John Boulé, vice-chair of the Waterfront Alliance and senior vice president at Dewberry, where he is business manager for New York operations. “While we’ve got a lot to be proud of, it’s clear that we need to do better, and the Harbor Scorecard will give citizens and policy-makers alike the information they need to act.” “The Waterfront Alliance cannot solve all of New York City’s waterfront and sea level rise issues, but its Harbor Scorecard will indicate whether we are moving in the right direction, becoming more resilient while at the same time providing more and better access to the waterfront,” said Klaus Jacob of Columbia University’s Lamont-Doherty Earth Observatory. “Right now, New York City is making decisions that will affect clean water investments for the next generation,” said Larry Levine, a senior attorney at Natural Resources Defense Council. “Thanks to the Clean Water Act, our harbor is cleaner than it used to be. But far too often the water is still too polluted to touch. Sewage overflows still foul our waterways after it rains, making them unsafe for eight million New Yorkers to use recreationally. The Harbor Scorecard will provide a call to action for local, state and federal officials, shining a light on where we need to invest in our infrastructure for a cleaner, healthier future.” Learn more about the Waterfront Conference and purchase tickets ($150 regular ticket; $75 government agencies and nonprofits; $50 students). Registration and breakfast is 8am to 8:45am; the boat is dockside at Pier 40, Hudson River Park until 1pm; the afternoon harbor cruise returns at 5pm. Continuing education credits will be offered (AIA CES; with APA AICP CM, and LA CES credits pending). The Waterfront Conference is generously sponsored by: Venue Sponsor: Hornblower Cruises & Events Premier Sponsor and Waterfront Scholars: Arcadis Commander: AECOM, GCA , New York City Economic Development Corporation Supporter: Dewberry, ExxonMobil, GBX Gowanus Bay Terminal, Hudson River Foundation, Newtown Creek Group, Red Hook Container Terminal, Seastreak, Stantec, Studio V, Two Trees, United Metro Energy Champion: Entertainment Cruises, HDR, Industry City, Langan, New York Water Taxi, Park Tower Group, Queens Chamber of Commerce, Sims Metal Management/Sims Municipal Recycling, Friend: ARUP, Ecology and Environment, HATCH, Kyle Conti Construction, M.G. McLaren Engineering Group, Moffatt & Nichol, Mott MacDonald, NY Waterway, Perkins & Will, Scape Studio, Starr Whitehouse Landscape Architects and Planners, Steer Davies & Gleave, Williams, WSP/Parsons Brinckerhoff Continuing Education Partners: AIA New York, APA NY Metro Chapter, ASLA NY The Waterfront Alliance works to protect, transform, and revitalize our harbor and waterfront.

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