Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem. 1951;193:265–75. Su GD, Huang DF, Han SY, Zheng SP, Lin Y. Display of Candida antarctica lipase B on Pichia pastoris and its application to flavor ester synthesis. Appl Microbiol Biotechnol. 2010;86:1493–501. MacDonald, Cheyenne (September 7, 2020). "Sharing a video of a person's suicide doesn't make you edgy. It makes you a POS". www.inputmag.com. Input . Retrieved March 25, 2022. For scale-up cutinase production, the transformant showing the highest cutinase activity in shake-flask culture was cultivated in a 5-L fermentor with 1.5 L working volume at 30 °C. The fermentation process including glycerol batch phase, glycerol fed-batch phase, and methanol fed-batch phase was performed according to Pichia Fermentation Guidelines (Version B, 053002, Invitrogen Inc.). The fermentation medium for cutinase production in the fermentor was FBS medium containing 4.35 mL/L of PTM 1 trace salts. Throughout the cultivation period, the dissolved oxygen level was maintained above 20% by adjusting the air flow rate and agitation speed. The initial pH was controlled at 4.0 by ammonium hydroxide. Upon depletion of initial glycerol, fed-batch fermentation was initiated with the addition of 50% (w/v) glycerol containing 12 mL/L PTM 1 trace salts at a rate of 18.4 mL/h/L (initial medium volume). After a 6-h glycerol fed-batch phase, the methanol induction phase was started with the addition of 100% methanol containing 12 mL/L PTM 1 trace salts at a flow rate of 10 mL/h/L (initial medium volume), and the medium pH was adjusted to 6.0. During the methanol induction phase, samples were withdrawn every 12 h, and dcw, cutinase activity, and protein content were determined. The cell-surface cutinase activity on P. pastoris cell wall was measured by the method of Su et al. [ 48]. The cells were collected by centrifugation at 11, 510 g for 10 min, washed three times with distilled water, and resuspended in 50 mM Tris–HCl pH 8.0, and the activity of the suspended solution was then assayed according to the standard method. The dry cell weight (dcw) was quantified by drying the washed cells until a constant weight was achieved. Enzyme assay and protein determination However, Mal Young, the BBC's controller of drama series at the time, admitted they 'fell out' but said the show refused to be her 'safety net'.

Bedo, Stephanie. "Ronnie McNutt suicide video leaves kids traumatised after platforms struggle to remove it". www.news.com.au. News.com.AU . Retrieved March 25, 2022. Prabhu AA, Veeranki VD, Dsilva SJ. Improving the production of human interferon gamma (hIFN-γ) in Pichia pastoris cell factory: an approach of cell level. Process Biochem. 2016;51:709–18.Martine McCutcheon suffers from 'migraines from hell' amid fibromyalgia flare-ups". 13 August 2021. Archived from the original on 12 August 2021 . Retrieved 13 August 2021. Ronnie's death happened in Mississippi on August 31, with TikTok desperately now trying to remove any clips of the incident and banning anyone who re-uploads it. Dhake KP, Thakare DD, Bhanage BM. Lipase: a potential biocatalyst for the synthesis of valuable flavor and fragrance ester compounds. Flavour Frag J. 2012;28:71–83. is officially the Year of TLC – Tummy Loving Care". Activia promotion. Danone Plc. Archived from the original on 21 February 2010 . Retrieved 11 February 2010.

The campaign calls on social media networks to accept responsibility for "all content created, posted, and distributed" on their platforms, adding: "You must immediately stop passing the blame on to the users when content you distribute actively harms them. Vile trolls have harassed Mr McNutt's family online, set up fake accounts under his name and claimed the suicide was faked, and created fake fundraising campaigns. Calls for social media reform In 2000, McCutcheon released her second album, Wishing, which was less successful, charting at number 25 in the UK. The album spawned two singles – the number two hit "I'm Over You" and the follow-up song, a cover of the Donna Summer classic "On the Radio" which peaked at number seven. Despite peaking at only number 25, the album still managed to sell around 250,000 copies, and received a gold certification from BPI. McCutcheon released her third album, Musicality, a Broadway-influenced cover album, in 2002. It reached number 55 in the UK and was certified silver by BPI. Due to poor sales, her recording contract was cancelled and her pop career stalled. [20] Her two-CD best-of-album called The Collection was released in September 2012. [29] Cutinase activity was assayed by the method of Xu et al. [ 11] using pNPB as a substrate. The reaction mixture (500 μL) contained 400 μL 50 mM Tris–HCl buffer pH 8.0, 50 μL suitably diluted enzyme, and 50 μL 20 mM pNPB dissolved in isopropanol. After reaction for 10 min at 45 °C, the released pNP was quantified by measuring the absorbance at 410 nm. One unit of enzyme activity was defined as the amount of enzyme liberating 1 μmol pNP per min under the above conditions. Protein concentration was determined according to the method of Lowry et al. [ 49], using bovine serum albumin as the standard. Purification of McCut Kitadokoro K, Thumarat U, Nakamura R, Nishimura K, Karatani H, Suzuki H, Kawai F. Crystal structure of cutinase Est119 from Thermobifida alba AHK119 that can degrade modified polyethylene terephthalate at 1.76 angstrom resolution. Polym Degrad Stab. 2012;97:771–5.They kind of suggested that I was lazy and didn't want to do the job. You know, I'd never in my life cancelled anything," she told the Guardian in 2003 ahead of her lauded return to the spotlight in Christmas movie Love Actually, which fans continue to love to this day. After reporting the stream, Mr Steen said he received a reply from Facebook about 90 minutes after Mr McNutt died, stating: “This post will remain on Facebook because we only remove content that goes against our Community Standards. Martins AB, Friedrich JLR, Cavalheiro JC, Garcia-Galan C, Barbosa O, Ayub MAZ, Fernandez-Lafuente R, Rodrigues RC. Improved production of butyl butyrate with lipase from Thermomyces lanuginosus immobilized on styrene–divinylbenzene beads. Bioresour Technol. 2013;134:417–22. We are banning accounts that repeatedly try to upload clips, and we appreciate our community members who've reported content and warned others against watching, engaging, or sharing such videos on any platform out of respect for the person and their family.

Crystallization experiments were set up using the sitting-drop vapor diffusion method according to Qin et al. [ 50]. Crystals suitable for diffraction were grown in drops containing 0.2 M KCl and 20% polyethylene glycol 3350. Diffraction data of McCut were collected at beamline BL17U at Shanghai Synchrotron Research Facility (SSRF). The data were indexed, integrated, and scaled using the program HKL-2000 [ 51]. Roth C, Wei R, Oeser T, Then J, Föllner C, Zimmermann W, Sträter N. Structural and functional studies on a thermostable polyethylene terephthalate degrading hydrolase from Thermobifida fusca. Appl Microbiol Biotechnol. 2014;98:7815–23. Martine McCutcheon declared bankrupt". The Guardian. London, UK. 12 February 2013 . Retrieved 13 February 2013. a b c Cortés, Michelle Santiago. "Let's Talk About The Graphic Video Going Viral On TikTok". www.refinery29.com. Refinery29 . Retrieved March 24, 2022.In an interview with Heavy, Mr Steen claimed "Facebook could’ve stopped this and didn’t", and said in his opinion the social media giant is “directly responsible” for the video being shared online and going viral. The Love Actually star said in the post that there is "no medical explanation" behind her brother's death, adding that the family are still looking into the cause. Mr McNutt, a former Army veteran who served in Iraq, worked at a Toyota plant in Blue Springs, New Albany. There are unconfirmed reports that he had lost his job and broken up with his girlfriend before his suicide.