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Would autoclaving (steam) affect D/H analysis of keratin.
Would UV sterilization be adequate?
I use a cheap hair salon type UV cabinet for sterilizing water and anything else that might be needed (weigh paper, disposable gloves, drill bits centrifuge tubes and plastic bags) for samples that may be used for ancient DNA analysis. This should kill any DNA, but I don't know if will kill bugs.

After UV treatment, dirty, unfiltered water samples from natural water holes in Kenya sat on my lab counter for a year in screw cap centrifuge tubes (irradiated in the tubes) and were completely stable. No algae 9or grew, no biogenic gas pressure.

Stanley H. Ambrose, Professor 
Department of Anthropology
University of Illinois, Urbana
Urbana, IL 61801 USA
217 244-3504
http://www.anthro.illinois.edu/people/ambrose
[log in to unmask]
 

On 11/27/16, 2:29 PM, "Stable Isotope Geochemistry on behalf of Robert Van Hale" <[log in to unmask] on behalf of [log in to unmask]> wrote:

Hi Travis,
There is a mechanical advantage to steaming of feathers - that is to make them subsequently easy to ball mill and indicating that this treatment causes structural change. Without steaming, the feather will be recovered undamaged from our rolling ball mills. I milled a large amount of feather for laboratory control materials after boiling 30 minutes at 1.5 bar, obtaining a 100 um powder.
See you
Robert

-----Original Message-----
From: Stable Isotope Geochemistry [mailto:[log in to unmask]] On Behalf Of Travis Horton
Sent: Sunday, 27 November 2016 11:32 a.m.
To: [log in to unmask]
Subject: Re: [ISOGEOCHEM] Autoclaving feathers for bulk N and C stable isotope analysis?

Thank you Wolfram, Sergei, Len and others!

We will be exploring alternative 'sterilisation' approaches that don't involve autoclaving but remain acceptable to New Zealand biosecurity.  In the meantime, our plan will be to analyse some autoclaved and non-autoclaved feathers that are not subject to biosecurity sterilisation requirements.  

I hope to send a post with a general summary of our 'autoclaving experiment' results in the coming month.  It seems to me that it would be very interesting to also run the compound specific stable isotopes on these same autoclaved vs. non-autoclaved feathers, perhaps resulting in an RCMS manuscript.  I'll get in touch with my fellow NZ isotopists to see if any of them are interested in running the compound specifics as my lab is not able to do those analyses.

I wonder also if different feathers with different pigmentations and different feather structures/types/positions/ages would respond differently considering the fact that autoclaving will change protein structures?  Perhaps another can of worms for another day, but I've always been curious about how differences in pigmentation might influence individual feather isotopic compositions (and how these individual differences compare to homogenates derived from multiple feathers collected from the same individual).  Such information would be important to isotope tracing of bird movements/migrations between systems with distinctly different foodchain isotope architectures. These are not the primary topics of the current study (which is on Antarctic penguin feathers and Hg pathways through Ross Sea foodchains), but still very worth pursuing in my opinion.

Thanks for all your thoughts and knowledge sharing!  You guys are the best!

Travis





________________________________________
From: Stable Isotope Geochemistry [[log in to unmask]] on behalf of Wolfram Meier-Augenstein (aps) [[log in to unmask]]
Sent: Saturday, November 26, 2016 10:10 PM
To: [log in to unmask]
Subject: Re: [ISOGEOCHEM] Autoclaving feathers for bulk N and C stable isotope analysis?

There is no might about it.  The whole point of sterilization by autoclaving is to break down proteins.

Steam hydrolysis will not just break up intra-molecular hydrogen bonds or disulfide bonds between adjacent Cys residues thus changing protein structure.  Hydrolysis also breaks up peptide bonds. This process will result in at least partial formation of small oligopeptide pieces, and even in loss of individual AA residues. Terminal AAs of unravelled protein chains (or peptide fragments) would be most vulnerable.

Individual AAs and small oligopeptides could easily be lost (not on account of volatility but on account of solubility) thus changing THE BULK C and N isotopic composition in a measurable way.

Incidentally, steam hydrolyzed poultry feather meal is used as feed for livestock to replace more expensive protein sources.  Steam hydrolysis increases digestibility of feather protein.



-----Original Message-----
From: Stable Isotope Geochemistry [mailto:[log in to unmask]] On Behalf Of Sergei Verenitch
Sent: 25 November 2016 23:00
To: [log in to unmask]
Subject: Re: [ISOGEOCHEM] Autoclaving feathers for bulk N and C stable isotope analysis?

Even if autoclaving might change structural composition of some proteins, I don¹t see how it can change THE BULK C and N isotopic values of feathers, unless the process breaks it down to light volatile compounds, which highly unlikely.




On 2016-11-25, 6:21 AM, "Stable Isotope Geochemistry on behalf of Wolfram Meier-Augenstein (aps)" <[log in to unmask] on behalf of [log in to unmask]> wrote:

>Depending on material to be autoclaved, autoclaving conditions range 
>from
>4 minutes at 132°C to 30 minutes at 121°C.  More to the point, 
>autoclaving is carried out under excess pressure.  Flash sterilization 
>at 132°C for 4 minutes is carried out at a pressure of 30 psi (2.07 bar)!
>
>The only types of protein (relatively) immune to such treatment are 
>prions.  Breaking down prions requires 60 min at 132°C.
>
>So, while typical autoclaving time may be short, it more than makes up 
>for this by doubling the amount of pressure compared to ambient.  Hence 
>the term "steam hydrolysis" for the underlying chemical reaction.
>Breaking a peptide bond is a hydrolysis reaction.
>
>If these were my samples and if the biosecurity people insist on 
>sterilization, I would explore other options of sterilization such as 
>gamma-irradiation or UV radiation.  Having said that, if the main 
>concern is to get rid of avian flu viruses I doubt UV radiation will suffice.
>
>
>-----Original Message-----
>From: Stable Isotope Geochemistry [mailto:[log in to unmask]] On 
>Behalf Of Leonard Wassenaar
>Sent: 25 November 2016 10:27
>To: [log in to unmask]
>Subject: Re: [ISOGEOCHEM] Autoclaving feathers for bulk N and C stable 
>isotope analysis?
>
>The short answer below is not quite "fit for purpose".
>
>The problem with quoting that particular paper (aimed at archaeological
>issues) in this context, is their heated water exposure times - showing 
>minor and/or variable effects on C/H/N isotopes on non-homogenized wool 
>(not feathers) - is from 5-60 days!!!
>
>They show no data for minute-scale  preparative / sterilization 
>exposures, and even after 5 days of exposure 100 % of raw wool mass was 
>retained at 80 C.  They did not appear to control for residual moisture 
>content (for H or O), nor wool intra-sample isotopic heterogeneity for 
>all of the isotopes.  And they used median delta values with no 
>uncertainty at experimental exposure time=0, which suggests to me the 
>latter is a rather important point.  Nevertheless it is a nice work, 
>and does show important potential isotopic and structural changes that 
>are here particularly relevant to impacted archaeological wool and hair 
>samples.
>
>Autoclaving takes only 15-20 minutes, and will likely have a negligible 
>isotopic effect on whole feathers (I am NOT saying for all organics in 
>general).  Any autoclaving impact on whole feathers is likely to fall 
>within intra-sample CHNS isotopic variance, or even analytical 
>measurement uncertainty.  There lots of avian papers to show this, for 
>H at least.  I'd be interested to hear what others have found what 
>autoclaving does to CNS of feathers, if anything.
>
>Is alcohol sterilization an option?  (caveats for 13C).
>
>If in doubt, try before-and-after autoclaving snippets to see for 
>yourself.  I guess the problem is they won't give you the "before" sample.
>
>Len
>
>-----Original Message-----
>From: Stable Isotope Geochemistry [mailto:[log in to unmask]] On 
>Behalf Of [log in to unmask]
>Sent: Friday, 25 November 2016 09:27
>To: [log in to unmask]
>Subject: Re: [ISOGEOCHEM] Autoclaving feathers for bulk N and C stable 
>isotope analysis?
>
>Hi Travis,
>
>The short answer is yes; exposing proteins to steam under pressure and 
>temperatures >100°C will change their stable isotopic composition. The 
>whole point of autoclaving (aka steam hydrolysis) is to sterilise 
>samples by destroying (hydrolysing) or at least denaturising protein 
>structures so as to nullify any infectious properties they might have had.
>
>As for the effect of hot water on keratin, Isabella von Holstein has 
>published a nice paper looking at changes in stable isotopic signature 
>with increasing temperature; cf. Figure 6 in:  I.C.C von Holstein, K.E.H.
>Penkman, E.E. Peacock and M.J. Collins (2014): "Wet degradation of 
>keratin proteins: linking amino acid, elemental and isotopic 
>compositions", Rapid Commun. Mass Spectrom., 28, 2121-2133.
>
>I sympathize with your biosecurity dept's worries about avian flu and 
>the like, but I wonder if they would be amenable to a solution 
>involving secure transport of the samples to your lab on the basis that 
>samples will be destroyed completely during the analytical process.
>
>Best,
>
>Wolfram
>
>
>-----Original Message-----
>From: Stable Isotope Geochemistry [mailto:[log in to unmask]] On 
>Behalf Of Travis Horton
>Sent: 25 November 2016 00:51
>To: [log in to unmask]
>Subject: [ISOGEOCHEM] Autoclaving feathers for bulk N and C stable 
>isotope analysis?
>
>Howdy folks,
>
>New Zealand biosecurity would like us to autoclave imported feathers 
>prior to transfer to our stable isotope analytical facility for bulk C 
>and N stable isotope analyses.
>
>I'm not familiar with what autoclaving does to keratin and its isotopic 
>composition, thus I don't know if autoclaving the feathers would alter 
>the isotopic compositions.
>
>Any thoughts, comments, experiences or references to published works 
>would be immensely appreciated!
>
>Best,
>
>Travis
>
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