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Materjali u Metodi
3.1. Shake Flask Cultivations
Pre-kulturi ta’ ċelluli ta’ sospensjoni MDCK.SUS2 tkabbru fi shaker flasks (125 mL polikarbonat Erlenmeyer flasks, #431143, Corning®, New York city, NY, USA) b’volum ta’ xogħol ta’ 50 mL (wv) , f'inkubatur Multitron Pro (Infors HT, Bottmingen, l-Isvizzera) f'atmosfera ta '37 °C u 5% CO2 bi frekwenza ta' tħawwad ta '180 rpm. Iċ-ċelluli ġew mgħoddija kull 3-4 ijiem b'densità ta 'żrigħ ta' 0.5-0.8 × 106 ċelluli/mL.
IAV A/Puerto Rico/8/34 (H1N1) intuża l-virus taż-żerriegħa għall-infezzjoni, iġġenerat f'ċelloli aderenti MDCK (ECACC # 84121903). Il-virus taż-żerriegħa kellu titru ta' 1.1 × 109 TCID50/mL.
Iċ-ċelloli ġew ikkultivati f'medju definit kimikament, ħieles mill-proteini u mingħajr komponenti mill-annimali, Smif8 (Smif8 PGD 2×, supplimentat b'5 mM glutamina, u 8 mM pyruvate), żviluppati speċifikament għall-kultivazzjoni ta’ ċelluli MDCK ta’ sospensjoni [95].
Esperimenti ta’ tkabbir u infezzjoni għall-validazzjoni tal-mudell saru bl-użu ta’ fliexken tat-tħawwid ta’ 500 mL (#4113-0500, Nalgene™, Thermo Scientific, Waltham, MA, USA) b'volum ta' kultivazzjoni inizjali ta' 200 mL f'150 rpm. Twettqu żewġ kultivazzjonijiet, infezzjoni waħda finta (Kultivazzjoni 1), fejn iċ-ċelloli kibru għal madwar 200 siegħa, u infezzjoni waħda (Kultivazzjoni 2), fejn iċ-ċelloli kienu infettati bl-IAV f'madwar 49 siegħa wara t-tilqim. Biex tinkiseb infezzjoni sinkronika tal-popolazzjoni taċ-ċelluli, intuża moi = 10. Minħabba l-konċentrazzjoni taċ-ċelluli relattivament baxxa fil-ħin tal-infezzjoni (2.1 × 106 ċelluli/mL) u l-moi għoli użat, la sostituzzjoni tal-medja u lanqas żieda ta 'trypsin (għall-attivazzjoni tal-virus) ma kienet meħtieġa. Fiż-żewġ kultivazzjonijiet, ġie miżjud ilma Milli-Q sterili qabel it-teħid tal-kampjuni biex jikkumpensa għall-evaporazzjoni tal-ilma (1-2 mL/jum) peress li l-esperiment sar f'inkubatur mhux idratat.
Sors minn: https:/ /www.mdpi.com/2218-1989/12/3/239/htm
The FAI climbed 5.9 percent year-on-year in the first 11 months of 2018, quickening from the 5.7-percent growth in Jan-Oct, the National Bureau of Statistics (NBS) said Friday in an online statement.
The key indicator of investment, dubbed a major growth driver, hit the bottom in August and has since started to rebound steadily.
In the face of emerging economic challenges home and abroad, China has stepped up efforts to stabilize investment, in particular rolling out measures to motivate private investors and channel funds into infrastructure.
Friday's data showed private investment, accounting for more than 60 percent of the total FAI, expanded by a brisk 8.7 percent.
NBS spokesperson Mao Shengyong said funds into weak economic links registered rapid increases as investment in environmental protection and agriculture jumped 42 percent and 12.5 percent respectively, much faster than the average.
In breakdown, investment in high-tech and equipment manufacturing remained vigorous with 16.1-percent and 11.6-percent increases respectively in the first 11 months. Infrastructure investment gained 3.7 percent, staying flat. Investment in property development rose 9.7 percent, also unchanged.