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Milly's Kitchen blog is a celebration of some of the most valuable things my grandma taught me. The secrets of a great pie crust. That there\u2019s always time to walk barefoot through the summer grass. That a warm bowl of soup can cure a bad case of the blues. That cooking for those you love is its own reward.
I'm super excited to introduce you to Jeffrey Knott, the owner of Blue Sparrow Coffee in Denver, CO. Jeffrey contacted me last year to shoot some images for his website. When I set foot inside BSC, I fell in love with the space. This cozy coffee shop has a beautiful decor, anchored by a deep cyan blue banquette and studded with brass accents throughout. They also serve first-rate coffee (we're talking homemade nitro cold brew on tap!) and have some of the friendliest baristas around. (Having lived in Seattle for over a decade, where baristas sometimes take themselves a little too seriously, I always give massive bonus points for friendly baristas.)
When Jeffrey told me about his unconventional path to owning his own coffee shop, I knew I wanted to share his story with you. If you've been dreaming of opening your own small business, read on for Jeffrey's excellent advice. And if you live in Denver or are planning a trip there, don't miss this gem of a coffee shop.
If you could get in a time machine, zoom back into the past and give yourself one piece of advice before starting your business, what would it be?
Start social media sooner. This time around I decided to keep quite on the new caf until we were open. It took much longer for people to find out about us than expected. Create your social media accounts early, reach out to the press, be active, build the hype and hope you can measure up to it!
Anyway, after 15 years in music, and five or six breakups with Molly, I realized it was time to quit music and transition to a more reasonable lifestyle for both of us, if Molly would have me. We got married at Treehouse Point in Fall City in 2011, we had a little girl (Named February Moon Reinig), and I studied law to get some of my logical mind back, having lost it from so many years on the road. We now live in a little house five minutes from the coffee shop.
How/when did you get the idea for your current venture?
A few years ago, I was kicking around various small commercial development ideas that resembled places I had been to in Holland and Japan, where a coffee shop was the center piece to the build-out of a space. I went down the road on a few projects only to be turned away during the final bit of negotiation. I decided that it would make more sense to simplify and focus on what I loved most about the project - coffee, music, and architecture in one small space where I can hang out all day and chat with friends. I knew the coffee and customer experience would have to be outstanding and I wanted to value my baristas as quality craftspeople, because a great barista is definitely the most important piece to pulling a great shot. So I hired, and continue to hire extremely carefully and offer full health benefits, the ability to tour and pursue art, and hopefully one day even paid family leave to baristas working over 28 hours per week, even though I know that would be expensive for a new small business. I designed the space to be very different from anything in Seattle, and did not compromise much at all from my original thought on the build - which took a ton of confidence in my vision.
If you could get in a time machine, zoom back in time and give yourself one piece of advice before starting your business, what would it be?
That ceiling is going to be expensive!!
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During early ischemic brain injury, glutamate receptor hyperactivation mediates neuronal death via osmotic cell swelling. Here we show that ischemia and excess NMDA receptor activation cause actin to rapidly and extensively reorganize within the somatodendritic compartment. Normally, F-actin is concentrated within dendritic spines. However,
Although apoptosis accounts for much of the delayed neuronal death that occurs over days and weeks following a stroke, most of the neuronal cell death that occurs in the early hours is due to a pathological swelling of neurons that leads to disruption of plasma membrane integrity6,7,8,9. Neuronal swelling, which is also called cytotoxic edema, is triggered when catastrophic ATP depletion perturbs ionic balance, leading to a massive influx of ions through multiple entry routes, with cation entry through N-methyl-d-aspartate receptors (NMDARs) and voltage-dependent channels and chloride entry through the SLC26A11 ion exchanger playing major roles10. Neuronal depolarization spreads in waves from the site of initial ischemia via local release of glutamate, thereby exacerbating and spreading the initial damage11. NMDA receptor hyperactivation plays an especially critical role, as administration of NMDA receptor antagonists before or even just after the onset of ischemia results in significantly reduced infarct volume in experimental models11,12,13.
Neurons, like most other cells, respond to cellular stress and injury, including ischemic injury, by mounting a series of pro-survival responses, with changes in organelles, reduction in protein synthesis, and activation of pro-survival genes14. In contrast, the pro-survival functions of the cytoskeleton are less well characterized. In the present work we describe an extensive reorganization of neuronal filamentous actin (F-actin) that occurs in response to stroke, oxygen and glucose deprivation, or NMDA receptor hyperactivation. F-actin is rapidly depolymerized within dendritic spines14,15,16, and, as we demonstrate here, it simultaneously polymerizes extensively within the soma and dendrites. This result is surprising given the usual ATP dependence of actin polymerization and the reduced ATP availability during ischemia. We find that this F-actin response is pro-survival and selectively triggered by conditions that elicit neuronal cytotoxic edema. The F-actin build-up in the soma and dendrites results in long, slowly turning over actin filaments that persist while the stress is present. However, the F-actin reorganization spontaneously reverses if the stress is transient. We demonstrate that activation of inverted formin-2 (INF2) is a key mediator of this neuronal pro-survival cytoskeletal response.
Dendritic spine shrinkage and spine F-actin loss have been described previously as an early response to stroke, OGD, or to NMDA/glutamate receptor hyperactivation, and in several cases actin-related mechanisms contributing to spine shrinkage were investigated14,15,16,24,25,26,27,28,29,30,31,32. However, the significant accumulation of F-actin seen in the somatodendritic compartment is unexplored, and is surprising given that F-actin polymerization typically involves ATP consumption. Logically speaking, actin polymerization would be disfavored in conditions of hypoxic cellular stress, when energy supplies become severely limited. We therefore focused on understanding the mechanism and biological relevance of somatodendritic actinification.
Once formed, actin filaments in the actinified neuronal somata and dendrites appeared to be highly stable. We tested whether incubation with latrunculin A would accelerate the removal of the actinified F-actin in the dendrite shaft. Actin filaments within dendritic spines turnover with a half-time of
We examined whether manipulations of microtubule stability would alter the induction of actinification. Taxol had no effect on the propensity of neurons to actinify under control conditions (Fig. 3d); however, we observed a tendency for nocodazole to induce actinification of a small number of neurons under control conditions, but this effect was completely prevented in the presence of NMDA antagonists, indicating that nocodazole may slightly increase spontaneous glutamate release (Fig. 3e). Incubation with neither the microtubule stabilizing compound taxol (Fig. 3d) nor the microtubule polymerization inhibitor nocodazole prevented NMDA-induced actinification (Fig. 3e), indicating that stable microtubules are not essential to actinification.
We next tested whether water entry was necessary for induction of actinification. Incubation of cultures with the non-cell permeable sugar mannitol, to reduce the osmotic driving force for water entry, indicated that swelling was indeed contributing to NMDA-induced actinification (Fig. 4c).
We next asked whether calcium released from intracellular endoplasmic reticulum (ER) stores participates in actinificiation in neuronal cultures. However, we found that thapsigargin, an inhibitor of the ER Ca2+ ATPase, neither stimulated actinification on its own nor inhibited NMDA-induced actinification (Fig. 4f), indicating that release of ER calcium stores is neither sufficient nor necessary to induce actinification.
Overall, the requirement for sodium, chloride, and water entry together with calcium entry from extracellular sources suggests that actinification may be optimally be triggered by the convergence of both cell swelling and calcium influx.
We next turned to identifying the key actin mechanisms involved in catalyzing neuronal actinification. The polymerization of most actin filaments in cells are initiated via two distinct mechanisms. The Arp2/3 complex nucleates and elongates daughter filaments from the side of an existing actin filament, thereby forming branched F-actin networks, as seen in lamellipodia and dendritic spines39,40,41. Conversely, formin-driven F-actin polymerization induces formation of unbranched actin filaments, as seen in filopodia and other structures where straight filaments predominate39,42,43. We applied either SMIFH2 or CK666, small molecule inhibitors of formin-mediated and Arp2/3-mediated actin polymerization, respectively. Only the formin inhibitor prevented NMDA-induced actinification (Fig. 5a). This result is consistent with the ultrastructural observation that NMDA induced long, unbranched actin filaments (Fig. 2d).
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