The end of an era, and the beginning of several new ones…

This spring there have been a lot of changes around the lab…

– Our technician Bill Urciuoli graduated his MBA from the Simon School of Business at UR (tuition benefits FTW), and left us in April to start a new job in Williamsport PA.  We will have a few weeks without a technician (oh joy!) and our new Tech’ will hopefully be starting on June 1.

– Long-term colleague Dr. Chad Galloway has started a new job as a staff scientist in the Department of Ophthalmology here at URMC. Here’s a picture of Bill and Chad at their leaving party – the bucket on the floor is part of a beer brewing kit they each got as a leaving gift.

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– We’ve also had an RIT co-op student in the lab’ for the past 3 months.  Nick Gulati has been doing a lot of 3D printing, and we recently obtained some electrically-conductive printer filament from Proto-Pasta, which he’s been using to print custom electrodes (more news on this soon).

– Our multi-PI R01 (with Keith Nehrke from Medicine and Cole Haynes from Sloan Kettering) got funded!  The project is entitled “Role of the mitochondrial UPR in ischemic protection”, and as the name suggests, will seek to characterize the key players in the mitochondrial unfolded protein response, and how activating this pathway might be able to protect the heart from ischemia-reperfusion injury.

– Andrew Wojtovich (former psot-doc’ and now an R.A.P. in the Department of Medicine) got a fundable score on his first R01, entitled “Optogenetic control of mitochondrial ROS generation”. Andrew was also selected to give a talk at the annual Biochemistry Department retreat.

– We were privileged to host Aubrey DeGrey of the SENS research foundation, for a seminar. I’ve known Aubrey since >20 years ago from the UK, so it was great to catch up on old times and hear all about his exciting recent advances in aging research.

– We had a visit from former grad’ student Dr. Lindsay Burwell, who reports that she’s going to start a new position as an Assistant Professor of Chemistry at Wells College this fall.

Congratulations!

3D printed gel combs

One of the ideas out there in the “3Dprintosphere”, is that seemingly common plastic doo-dads are way too expensive, and would be a lot cheaper if they could be custom fabricated on-site.  A classic example is gel combs – those little plastic things we all use to form wells in our SDS-PAGE gels. For the privilege of owning one of these small pieces of plastic, a reputable manufacturer of such mini-gel apparatus will charge $37 for a pack of 5. Add in shipping costs and you’re looking at $10 a pop, for something that costs maybe 10c to make.

So, SketchUp, Repetier, and PrintrBot to the rescue…

GelComb

That’s a custom 7-well 1.5mm comb. The reason we did this is to load more sample.  A regular 10-well comb has 10mm deep wells, 5mm wide, so they hold ~75μl each. These wells are 12mm deep and 7mm wide, so they hold 126μl each (previously we had to use tape to join together 2 wells of a 15 well comb to make a wide lane).

This one was printed at 0.15mm layer height using MakerBot PLA filament at 225C with solid rectilinear infill. It took about 10 minutes to design and 16 minutes to print, and uses about 900mm of filament, so assuming a cost of 13c per meter** that’s a 12c material cost. If it breaks or wears out, who cares?  We could print a brand new one every month for 6 years still be ahead on cost. And we can customize the size for whatever sample is needed.

The STL file for this, plus those for standard 10 well and 15 well combs (for the mini-gel box maker known as “big green”), are in this zip folder. I also threw in the Sketchup file with a blank comb-body so you can draw in the lines and use the push/pull tool to design custom well sizes, as we did here for the 7 well version.

Enjoy.

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**PLA density = 1.24kg per liter
A 1kg roll costs ~$45 depending on where you source it
Diameter=1.75mm, radius 0.875mm, pi R squared and all that malarky, so 1 meter = 2.405 cm3
This 2.405cm3 weighs 2.982 grams.
So a 1kg spool has 335 meters, i.e. 13.4c per meter

Fun with 3D printing

One of the latest things we have in the lab’ is a 3D printer – a PrintrBot Simple Metal. I bought it over the summer and assembled it from a kit, which took about 5 hours. There are lots of things available to download and print from Thingiverse, or the NIH 3D print exchange, but if you want to make things from scratch, you need to learn to design in 3D using a program such as Sketchup. It’s free to download but there’s a steep learning curve and this was a big obstacle for me.  Now I’m over that initial hump however, we can make things for the lab…

3D printed door-handle extension

The lab’ door opens outward, so opening it from the inside is easy when you’re laden with stuff or just wearing gloves – simply push and your elbow.  BUT, if you’re outside and want to get in, you can’t pull the door handle with your elbow.  So, I designed an extension to bolt onto the handle, providing a hook so you can pull the door open with your forearm, without touching anything with your hands.  Here’s the finished article…Doorpull1

The .STL file is available here (in a zip folder because web host will not allow direct hosting of STL files) if you want to print your own. The screws used were standard 3/4″ steel sheet metal screws, the kind used for air duct work and available in packs of 100 at Home Depot.  There’s a tab on the lower piece which grips against the ell in the metal handle, preventing it from spinning around. This one is for right handed opening doors, but in the next few days we plan to make one for left-handed doors too, and will update the STL then. The STL also contains some support material that has to be cut away after printing.

Trough for multi-pipeting small amounts

We use a Seahorse XF96 in the lab’. Each well has 4 drug-loading ports, but they only hold about 25 ul each. If you’re loading small amounts of expensive mitochondrial inhibitors (rotenone, FCCP etc.) the troughs typically available from lab-ware suppliers are just too big – twice as long as necessary and way too deep…

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Our 3D printed version is a lot smaller, holding about 2ml instead of the usual 20-30 ml. Below is a picture, and the STL file is available here.

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We’ve also played around with a few small dishes and chambers for patch clamping, various tube racks and holders, and some accessories for the PrintrBot itself. One issue we’ve come across is the water-tightness of flat printed surfaces – even with solid infill we seem to always end up with some leaks (hence the trough above was designed with angled walls that provide a much tighter seal).  Also we’re not sure yet about the long-term durability of PLA in a lab setting.  It’s a biodegradable corn plastic (polylactic acid), so might not hold up well to autoclaving etc. (we already had a tube rack deform when someone accidentally left the water bath cranked up to 70C).

We’ll be posting more STL models as they come.

 

Our new lab space!

Well, new to us at least.

Over the past couple of months, we’ve been lucky enough to absorb an adjacent lab’. Up to now, our main lab’ was 7420, at the bottom center on the plan shown below, with a small cell culture room in the back (20A). We also used the lab’ next door (7424) on the same corridor, but this was problematic because there’s no connecting door. For security reasons the adjacent lab had to be locked when unoccupied, so to get there meant removing gloves and fiddling with keys while carrying an ice bucket. Another problem – we were using the back room (20A) for animal work, but because it’s a dedicated cell culture room, it has positive air pressure. So, all the dander and allergens and other gunk would float out into the main lab. Not good when you’re allergic to mice!

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The cost to open a doorway between the 2 big labs was prohibitive, but for a while we’d been aware there was an old connecting door to the lab behind us (7512 on the plan). That room was used by Alan Smrcka ‘s group as a student office and general storage room, and the door was simply blocked on both sides with drywall and a few wooden shelves/cabinets. The lab’ once belonged to a former Dept. chair, but was never officially decommissioned when he retired a few years ago, so the cupboards were full of old supplies, chemicals and defunct equipment. So, we approached Alan and he very generously agreed to a swap – room 7512 in exchange for 7424. After a few weeks of construction dust, we now have a new lab, and finally 3 rooms of contiguous space. It wasn’t quite like this but you get the idea…

funny gifs

The new space is nothing short of a revelation for the way we work. We now have a simple arrangement with in-vivo stuff in the new room (7512), cell stuff in the middle, and biochemistry in the main lab at the front. The great thing is there’s a fume hood in the new room, so we can keep allergen exposures to a minimum elsewhere in the lab. Also now the cell culture room (20A) with positive air pressure can finally serve its intended purpose.

Here’s what’s left of the connecting door, looking through from the new lab’ into the cell culture room. On the right is our cardiomyocyte preparation rig, making it easy to transfer the freshly prepared cells into the cell culture hood and incubator. There’s still some finish work to be done on the floor and the bench-edges.

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This is now our perfusion bench. On the left is the surgical area with dissecting scope, ventilator, EKG, thermal pad etc.  Then on the right are two separate Langendorff heart perfusion rigs. There’s a third rig out of view, on the opposite side of the new lab. Due to fire regulations we’re not allowed to use the top shelves near the ceiling for storage, so those are blocked off.

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We also relocated our HPLC to the new lab. Here it is next to the fume hood…

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In the cell culture room we now have our 2 seahorses – XF24 and XF96.  This used to be the bench where all our perfusion gear was. You can also see the gas perfusion system mounted on the shelf – we do some fun things involving flooding the plates inside the seahorse with argon gas, as reported here.

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Here’s another view of the cell culture room showing the hood, with the seahorse bench on the left…

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During the reshuffle we were able to divest some old equipment that was broken no longer being used.  A key piece was our Aviv Model 14 spectrophotometer, which Jack Aviv himself came to collect. It had served us well, and was the source of several publications, but had sat unused for many years and took up a lot of space. Here it is in the back of Jack’s van…

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The other thing this move allowed us to do, was consolidate our refrigeration in the front lab’, so now we have 4C, -20C and -80C all in one place.

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Most of the demo’ work was done by UR facilities, plus moving electrical panels, putting in new floor tiles to bridge the gap, some HVAC work to balance out the flows, removal of old telephone lines, replacing all the fluorescent light tubes etc. Some things we did ourselves – for example we used the opportunity to do some paint touch-up throughout the lab (the drywall was pretty beaten up in places). Here’s our technician Bill hard at work – this is how we roll!

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Environmental Health Sciences also helped us to dispose of the various chemicals through the hazardous waste program. We managed to fill 3 dumpsters with old equipment and supplies from the cupboards of the 7512 lab – a bonus for the Department is the lab is now clean and no longer a hazard liability.

Like all moves this one was pretty disruptive, but the new space really works well. The 3 Langendorff rigs are already cranking out data. Having all the cell culture stuff in one room means less contamination. No more messing with gloves and keys to get between labs.  The new lab has a door leading out to the elevator lobby, which gives us another fire escape route, plus that’s where the bathrooms are located so it’s easier to go answer the call of nature during experiments!  All in all a very worthwhile process.

Retraction? No problem just send it to Frontiers!

A while ago I documented some data problems in a JBC paper, including a failed attempt by the authors to correct the paper, followed ultimately by its retraction.

Well, guess what showed up at Frontiers this week… the exact same paper. The title is almost the same, huge blocks of the text are the same, and the majority of the data and figures (minus the mistakes during figure preparation) are the same. Without making any comments on the reliability of the data in the new paper, which I haven’t examined in detail yet, the following questions come to mind….

  • Did the editors at Frontiers know the paper had been retracted from another journal?  If so (or not), how would this have influenced the peer review process?
  • The authors listed on the retracted paper and the new one are identical. I don’t know how other people run their labs, but if my lab had a paper retracted for questionable data integrity, the person responsible for those problems would be out the door faster than you can say “Committee on Publication Ethics”. Keeping the same authors suggests that either Dr. Donmez is an extremely forgiving employer and chose not to fire the subordinate responsible for this mess, or that she was personally responsible for the problem data so there is no subordinate to fire.**
  • What about copyright? The original version of the paper is still up at the JBC site for all the world to see. Does Frontiers have the right to publish the same information, in clear in breach of JBC’s copyright?  How does copyright work on a retracted paper?
  • How should episodes such as this inform how we deal with retracted papers in the future?

Regarding the last point, perhaps a good starting point would be a check-box on the intake form for journals, stating whether the work is a republication of a previously retracted work. Perhaps such papers could then be accompanied by a statement with assurances that the original data have been scrutinized to ensure their integrity.

** I’ve run into this problem before – seeing a paper in the review stages with creative data presentation, and then seeing it appear elsewhere with the exact same list of authors. How many more papers are out there, where authors dodged a bullet in the review stages but made it to press somewhere else?

UPDATE (4pm 9/2/2014) – I just learned via PubPeer that a paper from Rui Curi (favorite South American dood who threatened to sue me) was re-published in an open access journal OmicsOnline, which is on Beall’s list of predatory (pay-4-glam) journals. Incidentally, the original paper was in J Lipid Res, also an ASBMB journal (like JBC), and so the original full-text is still up there on the JLR website. Again, the copyright question arises – does omicsonline own the paper now?  Has anyone done an analysis to see how much of the content of these new predatory open access journals is comprised of stuff retracted from other places?

UPDATE 2 (9am 9/3/2014) – Following an email last night, I have now heard back from the Frontiers in Aging Neuroscience EiC, Gemma Casadesus Smith. They were “unaware of this issue and will look closely into it”.  Hopefully that’s not a euphemism for try to forget about it until all washes over, as frequently seems to happen at other journals.  Now we play the waiting game…