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"Akata Witch" Review
Posted on March 2, 2011 by Omenka Egwuatu Nwa-Ikenga
"Akata Witch" by Nnedi Okarafor
Imagine if Harry Potter were a she instead of a he? What if he was Nigerian instead of British? What if he were learning at Leopard Knocks instead of at the Hogwarts School of Witchcraft and Wizardry? Enter Sunny, Nnedi Okorafor's latest anti-Disney heroine. I say that she's anti-Disney because unlike most Disney films, the main females in her stories serve not as damsels that need to be saved, but as the ones that do the saving. Furthermore, they have physical features that are typically considered to be undesirable by society, such as the protagonist Onyesonwu being biracial in her most previous work Who Fears Death, and Sunny being an albino in this one.
Same Script, Different Cast
Before we get into the contents of the book, let's start with the name of the book first. What is an Akata? When I was growing up, I was taught that it was a slang (and somewhat derogatory term) that Nigerians used for African Americans. That was until I was referred to as one by a native born Nigerian. I then began to see that it could also be a term used to describe any black person born and raised in America. Like me, the protagonist Sunny was born and raised in the United States. At the age of nine, her family decides to move back to Nigeria. Like any girl, she feels out of place and spends the next couple of years trying to find her place, when she finds out that her place is nothing like she imagined it to be. She discovers that she is a "Leopard person", a person that is born with mystical abilities and she is has to undergo intense training to learn how to utilize her powers while still living amongst the "Lamb people", or regular folks. One of the best things I loved about this book was the name dropping by the characters themselves of the books that had a large role in influencing this novel, including In the Shadow of the Bush by P. Amaury Talbot, Her Stories by Virginia Hamilton, and The Witches by Roald Dalhl.
"The Witches" by Roald Dahl
Different locations were also named, the significance of which will go unnoticed to the general population. One such location is referred to as being the African American "Leopard person" headquarters. This place is referred to as the Gullah Islands off the coast of South Carolina. This area is usually overshadowed by New Orleans, even though it has had a much larger impact on African American history, and mainstream American history. It also has retained more African culture and tradition (including the folk magic) than any other region in North America, but thats a discussion for another day.
The most creative literary element used by the author is the technique of having a "book within a book" (another very popular example being the Necronomicon). As Sunny begins to learn and master her abilities, she reads from a book entitled Fast Agents for Free Agents by Isong Abong Effiong Isong. The wonderful part is that the reader gets to also read from this short book. So instead of doing a typical review of Akata Witch, I will actually do a review of the book within the book, and explain some of the terms used:
What is a Leopard Person?
"A leopard person goes by many names around the world. The term "Leopard person" is a West African coinage, derived from the Efik term "ekpe", "leopard." All people of mystical true ability are Leopard people. And as humankind has evolved, so have Leopard folk around the world organized…from Fast Agents for Free Agents"
One of the real life "Leopard people" that the writer is referring to are the Ekpe society of southeastern Nigeria. This society, which was started by the Ekik people, spread through to the rest of the Ibibio, Oron, Igbo and Ekoi peoples.
Ekpe Society members during a procession in Arochukwu
The Ekpe Society was introduced to Igboland by the people of Arochukwu. In fact, their lodge is in my home village of Atani. In Igboland, the Ekpe Society usually went by the name Okonko.
Ekpe Lodge in Atani Village, Arochukwu
They were the premiere secret society in Southeastern Igboland, and the highest grades held alot of the secrets of the mystical arts, including the magical Nsibidi script. An entire topic will be dedicated to discussing the Ekpe society and its legacy in the near future.
What is a Free Agent
"A free agent is one who isn't privileged with even one pure Leopard spiritline from the survivors of the Great Attempt. She or he is a random of nature, a result of mixed up and confused spiritual genetics. Free agents are the hardest to understand, predict or explain. Learning will not come easy to you. you are a Leopard person only by the will of the Supreme Creator and as we all know, She isn't very concerned with Her own creations.
After your initiation, make sure that someone is there to help you, for you will not be able to help yourself, so new the world will be to you and so fragile your ego. You're likely an infant. You will be dumbfounded and disorientated. What's most important is…from Fast Agents for Free Agents"
Initiation, which simply means "beginning", plays a huge role in Igbo culture, as well as with other cultures all over Africa. Traditionally, both men and women would receive multiple initiations at different points in their life. A very good metaphor is employed by Okorafor when one of the character states: "Imagine that you are a computer that came with programs and applications already installed. In order to use them, they have to be activated; you have to , in a sense, wake it up. That's what initiation is."
Igbo boy being healed by a Dibia
What is chittim?
"Chittim is the currency of the Leopard people. Chittim are always made of metal (copper, bronze, silver, and gold) and always shaped like curved rods. The most valuable are the large copper ones, which are about the size of a dove's egg. Least valuable are chittim made of gold. When chittim fall, they never do harm. So one can stand in a rain of chittim, and never get hit. There is only way to earn chittim; by gaining knowledge and wisdom. The smarter you become, the better you process knowledge into wisdom, the more chittim will fall and thus the richer you will be…from Fast Agents for Free Agents"
The real life chittim that the author refers to are commonly known as manillas. The most popular African name for manillas, Okpoho, comes from the Igbo language. They were used as currency (as well as worn as jewelry) all over West Africa, but particularly in southeastern Nigeria, Ghana and Guinea.
Okpohos
What are the Masquerades?
"Up to now you've known masquerades to be mere symbolic manifestations of the ancestors or spirits. Men and boys dress up in elaborate cloth and raffia costumes and dance, jeer, or joke depending on who they are manifesting. Up to now, you've believed masquerades to be nothing more than myth, folklore and tradition. Now that you are a Leopard person, know that your world has just become more real. Creatures are real. Ghosts, witches, demons, shape-shifters, and masquerades, all real. Masquerades are always dangerous. They can kill, steal your soul, take your mind, take your past, rewrite your future, bring the end of the world, even. As a free agent you will have nothing to do with the real thing, otherwise you face certain death. If you are smart you leave true masquerades up to those who know what to do with juju…from Fast Agents for Free Agents"
The above statements say it all. Masquerades in Igboland are known as mmanwu. Here are some pictures of some of them. Some of them are people in costumes, but the older pictures might be real ones. We will never know for sure:
Agbogho Mmuo (Maiden Spirits)
Masquerade at Igbo Farm Village
Ijele Masquerade
The Ijele masquerade originated in Anambra state is known as the "King of all masquerades." In ancient times, it had 45 masquerades perform on top of it, which are now represented by 45 figures. It was also used to scare away some of the early missionaries in Igboland. Masquerades made their way into the Diaspora, and can still be seen in modified forms in the Carribean Carnival celebrations:
Carnival Masquerade in Brooklyn, NY
Chapter 4: Your Abilities
"How to discover your ability: Its doubtful that you have the intelligence to figure out something so important. But here is something to think about: one's ability lies with those things that mark him or her. They can be talents, like an affinity towards gardening or being able to play the guitar well. Often they are things that Lambs make fun of, imperfections. They can be physical, psychological, behavioral. And I do not mean things that are a result of your actions like being fat because you eat too much and sit and play video games all day…from Fast Agents for Free Agents"
I really felt this was the most important chapter in the book. I truly feel that if more people did what they were naturally gifted at instead of trying to conform to the standards imposed on them by society, the world would be a drastically better place. By following these instructions, one could unlock their latent abilities, as Sunny and her cohorts were able to do. Who knows, you might even discover yourself to be a Leopard person 😉
"…So there you have it. All you need to know to get started. As I have repeated incessantly throughout this book, there is no direction you can turn that does not face you toward certain death. The best thing to do is be who you've been, don't move, stay where you are, drop all ambition as a Leopard person. Relax. Don't strive too high. Learn but do not use. And only learn the basics. It is best to remain in your protective shell. Ambition is not your friend. Be glad the Leopard world has been opened to you, but remain a mere spectator. And for the hundredth time, I repeat: "KEEP YOUR SECRET LIFE FROM YOUR LAMB RELATIONS AND ACQUAINTANCES. not only are there dire consequences for breaching secrecy, but you risk upsetting a very delicate, crucial hard earned balance. Now go well, free agent. Be well. And again I saw: Welcome…from Fast Facts for Free Agents"
This book is an essential read for Leopard people who know that they are free agents, free agents that think they are Lamb people, or for Lamb people who want to get a glimpse into the mystical and secret world of Leopard people. As of now, I cannot find any copies of Fast Facts for Free Agents, so the best way to get a glimpse of it would be by purchasing a copy of Akata Witch. It has an excellent story too 😀
Be sure to be on the lookout for an interview with the author on Igbo Kwenu Radio in the near future.
Filed under History, Reviews and tagged Akata, Akata Witch, anambra state, Anyanwu, Arochukwu, Brooklyn Carnival, Carribean Carnival, chittim, Dibia Initiation, Disney, Efik people, Ekoi people, Ekpe Society, Ekwensu, Gullah, Gullah Islands, H. P. Lovecraft, Harry Potter, Her Stories, Hogwarts School of Witchcraft and Wizardry, Ibibio people, Ibo Landing, Igbo masquerades, Igbo Religion, Igbo Spirituality, Ijele masquerade, In the Shadow of the Bush, Juju, Manilla, Masquerades, mmanwu, music, Necronomicon, New Orleans, Nnedi Okarafor, Nsibidi, Okonko, Okpoho, Oron people, P. Amaury Talbo, precolonial African money, Roald Dalhl, The Witches, Veve, Virginia Hamilton, Voodoo |	18 Comments
Ms. Ezi on March 2, 2011 at 3:35 pm said:
I ruola oru ukwu nwanne m nwoke!
The Divine Prince on March 2, 2011 at 4:10 pm said:
Divine All Blessed Peace and Community, Education and Edification!
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Dr. Uwa Osimiri on March 2, 2011 at 6:23 pm said:
Magnificent work! This is an impressive compilation of information to motivate and inspire learning about African genius. Thank you for sharing. The African community wins. My Chi honors yours!
George Kwame on March 3, 2011 at 7:39 am said:
ugo-chinyere chibuzor on March 3, 2011 at 9:02 am said:
i must confess, mazi Desta did a great job here, the review is so well detailed , i commend his efforts…
ths piece left me asking for more, and i pray the book is soon made available to the public.
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Henrietta Vinton Davis on March 4, 2011 at 3:45 pm said:
My first thought in reading this was of "The Bottled Leopard." My wife told me about it several years ago after we discussed "shapeshifters."
Thanks for this informative article. I plan to purchase a copy of "Akata Witch" because of it.
James Ihekwoab on March 4, 2011 at 5:03 pm said:
Nice Job Mazi Onyemobi, our Ancestors are really on your elbow …
Gin on March 17, 2011 at 4:11 pm said:
Nice, this was very informative.
I just noticed that the cover is full of nsibidi!
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Amah Jones on November 3, 2012 at 5:00 am said:
OKPOHO as in the old MANILLA currency shown above is not an IGBO word. Okpoho is Ibibio-Efik word for MONEY. The IGBO word for MONEY is EGO (EH-GOH).
nnamdi rowland dimkpa on August 2, 2017 at 7:31 am said:
d igboes are not only those fives in nigeria ….nauturally igboes are those that occupy The sharply equatorial belt of africa .. inhabitants with one culture/traditions e.t.c's but just d largest single group of people in equatorial belt
Chukwunweike Izuogu on March 6, 2019 at 12:57 pm said:
True , but the Ohaffia and Abiriba Igbo of current Abia state in Nigeria also call money Okpogho in their Igbo dialet.
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Amphotericin primarily kills yeast by simply binding ergosterol
Kaitlyn C. Gray, Daniel S. Palacios, Ian Dailey, Matthew M. Endo, Brice E. Uno, Brandon C. Wilcock, and Martin D. Burke
PNAS February 14, 2012 109 (7) 2234-2239; https://doi.org/10.1073/pnas.1117280109
Kaitlyn C. Gray
Daniel S. Palacios
Ian Dailey
Matthew M. Endo
Brice E. Uno
Brandon C. Wilcock
Martin D. Burke
For correspondence: burke@scs.uiuc.edu
Edited by* Jerrold Meinwald, Cornell University, Ithaca, NY, and approved December 8, 2011 (received for review October 19, 2011)
Amphotericin B (AmB) is a prototypical small molecule natural product that can form ion channels in living eukaryotic cells and has remained refractory to microbial resistance despite extensive clinical utilization in the treatment of life-threatening fungal infections for more than half a century. It is now widely accepted that AmB kills yeast primarily via channel-mediated membrane permeabilization. Enabled by the iterative cross-coupling-based synthesis of a functional group deficient derivative of this natural product, we have discovered that channel formation is not required for potent fungicidal activity. Alternatively, AmB primarily kills yeast by simply binding ergosterol, a lipid that is vital for many aspects of yeast cell physiology. Membrane permeabilization via channel formation represents a second complementary mechanism that further increases drug potency and the rate of yeast killing. Collectively, these findings (i) reveal that the binding of a physiologically important microbial lipid is a powerful and clinically validated antimicrobial strategy that may be inherently refractory to resistance, (ii) illuminate a more straightforward path to an improved therapeutic index for this clinically vital but also highly toxic antifungal agent, and (iii) suggest that the capacity for AmB to form protein-like ion channels might be separable from its cytocidal effects.
protein-like functions
N-methyliminodiacetic acid boronates
Amphotericin B (AmB) represents an important prototype for small molecules that can self-assemble into membrane-permeabilizing ion channels in eukaryotic cells (1). An advanced understanding of how this natural product interacts with living systems thus stands to enable efforts to develop small molecules that serve as surrogates for deficient or dysfunctional protein ion channels that underlie currently incurable human diseases (2⇓⇓⇓⇓–7). In addition, in stark contrast to most antimicrobial agents (8, 9), resistance to AmB has remained exceptionally rare despite extensive worldwide utilization to treat life-threatening systemic fungal infections for more than half a century (10). Understanding how this natural product kills yeast cells therefore also stands to guide the vital search for new types of resistance-refractory antimicrobial agents. Moreover, although AmB remains the last line of defense in the treatment of life-threatening systemic fungal infections, it suffers from serious dose-limiting side effects (11). Efforts to improve the therapeutic index of this drug would benefit substantially from a more complete molecular understanding of its mode of action.
Despite extensive investigation for the past 50 years, however, the mechanism(s) of action of AmB have remained unclear. In the now widely accepted leading model, AmB kills yeast via ion channel-mediated membrane permeabilization (12⇓⇓⇓⇓⇓–18). This channel model has been extended to also rationalize the unique lack of resistance to AmB as well as its dose-limiting toxicity (12⇓⇓⇓–16). This perceived understanding has stimulated extensive research toward the goals of developing new channel-forming compounds as putative resistance-refractory antimicrobial agents (19) and/or less toxic derivatives of AmB that more selectively form channels in fungal cells versus human cells (12⇓⇓–15). Moreover, this model suggests that the capacity of AmB to form ion channels and exert cytocidal effects are inextricable, which challenges the notion that ion channels formed by AmB or one of its derivatives might have therapeutic benefit in the setting of protein ion channel deficiencies.
Importantly, it has long been known that lipid bilayers containing sterols are uniquely vulnerable to permeabilization by AmB, but it had remained unclear if this effect was due to indirect sterol-mediated global changes in membrane properties or direct sterol binding. We recently determined that AmB directly binds membrane-embedded ergosterol, the main sterol found in yeast cells, in a manner that requires the mycosamine appendage at C19 (2). Deletion of this appendage yields a derivative, amphoteronolide B (AmdeB) (Fig. 1A), which cannot bind ergosterol, is unable to form ion channels, and has no antifungal activity (2, 3). These findings caused us to consider two possible conclusions regarding the mechanism(s) by which AmB kills yeast: (i) ergosterol binding-dependent channel formation is required for antifungal activity, or (ii) ergosterol binding alone is a critical mechanism of antifungal activity, and channel formation represents a second complementary mode of action (Fig. 1B).
Chemistry and biology of polyene macrolides. (A) Molecular structures of the polyene macrolide natural products AmB, natamycin, and a series of their functional group-deficient derivatives prepared via chemical synthesis. (B) Representations of two mechanisms for the antifungal activity of AmB: ergosterol binding and membrane permeabilization. (C) Representations of the two leading models for the structure of the AmB ion channel. Both models predict that the hydroxyl group at C35 is critical for ion channel self-assembly. (D) Iterative cross-coupling, a small molecule synthesis strategy, analogous to iterative peptide coupling, in which bifunctional building blocks are sequentially linked using only one reaction recursively.
Albeit in stark contrast with the leading model, several lines of evidence led us to the hypothesis that the latter dual mechanism is operative and that ergosterol binding alone is the primary mechanism of action. First, a dissociation between the membrane-permeabilizing and antifungal activities of AmB has been observed in some strains of yeast (20). To rationalize these findings, a second mechanism involving oxidative membrane damage caused by the putatively redox active heptaene motif of AmB is often invoked (9, 10, 12). However, we found that AmdeB (Fig. 1A), which contains the complete heptaene motif found in AmB and binds very readily to lipid bilayers, is devoid of antifungal activity (2). Thus, an alternative and previously unidentified major mechanism of action for AmB seemed likely. Second, another antifungal polyene macrolide natural product, natamycin (Fig. 1A), was recently found to directly bind ergosterol but not to cause membrane permeabilization (21). Third, ergosterol is critical for many aspects of yeast cell physiology, including vacuole fusion (22), endocytosis (23), pheromone signaling (24), membrane compartmentalization (25), and the proper functioning of membrane proteins (26). Thus, binding alone of this multifunctional lipid should be sufficient for killing yeast cells. Fourth, structural modification and/or decreased expression of ergosterol via mutations in the sterol biosynthesis pathway substantially diminish fungal pathogenicity in vivo (27). Therefore, a primary mechanism involving direct ergosterol binding and sequestration would help explain why clinically significant resistance to AmB is so rare.
Testing the hypothesis that sterol binding is paramount to the antifungal activity of AmB required a derivative of this natural product that retains the ability to bind ergosterol but lacks the capacity to form ion channels. The structures of the AmB/ergosterol complex and the multimeric AmB-based ion channel are unknown, thus making the rational design of such a derivative challenging. However, given its recently established critical role in sterol binding (2), it was clear that the mycosamine appendage should be retained. Alternatively, in both of the leading models for the structure of the AmB ion channel (Fig. 1C) the hydroxyl group at C35 (Fig. 1A) is predicted to be critical for channel formation. Specifically, in the single barrel model (Fig. 1C, Left), an aggregate of AmB molecules spans a dimpled membrane, and the C35 hydroxyl groups anchor the channel to one side of the lipid bilayer (28). In the competing double barrel model (Fig. 1C, Right), two molecular aggregates, each spanning one half of the lipid bilayer, are united into a membrane-spanning channel via hydrogen bonds between the C35 hydroxyl groups on AmB molecules in opposing leaflets (28). Supporting an important role for this hydroxyl group in channel formation, in vitro studies in model liposomes demonstrated that a doubly modified derivative of AmB, in which the C41 carboxylic acid is protected as a methyl ester and the C35 hydroxyl group is removed, caused only weak potassium efflux at high concentration (10 μM) and no efflux at low concentration (1 μM) (29). Our own studies suggested that the weak efflux observed in the former case was likely an artifact of the relatively sensitive nature toward membrane permeabilization of liposomes versus live yeast cells (SI Appendix, Fig. 1) and/or unique biophysical properties resulting from the net positive charge of C41 methyl ester derivatives (SI Appendix, Fig. 2). Thus, with the goal of retaining ergosterol binding, completely eliminating the capacity for membrane permeabilization, and enabling direct comparisons with the biophysical and biological properties of the zwitterionic natural product, we targeted the synthesis of a derivative of AmB that retains the mycosamine appendage at C19, lacks a hydroxyl group at C35, and retains the anionic carboxylate group at C41 (C35deOAmB) (Fig. 1A).
The synthesis of polyene macrolides is challenging because of the sensitivity of these molecules to light, oxygen, and many organic reagents, including the basic and/or acidic conditions often required at the end of a pathway to remove various protective groups. These sensitivities can be magnified considerably when specific functional groups have been deleted from the polyene macrolide skeleton, which has previously precluded the synthesis of some targeted AmB derivatives, including C35deOAmB (29). To improve the efficiency and flexibility with which such complex small molecules can be prepared, we have recently developed a simple and modular synthesis strategy, analogous to iterative peptide coupling, in which building blocks having all of the required functional groups preinstalled in the correct oxidation states and with the desired stereochemical relationships are sequentially linked via iterative application of one mild reaction (30⇓⇓⇓⇓⇓⇓⇓–38). Enabling this approach, we discovered that N-methyliminodiacetic acid (MIDA) is a highly effective ligand for reversibly attenuating the reactivity of a boronic acid and thereby permitting the controlled sequential assembly of bifunctional haloboronic acids (Fig. 1D).
To complete the synthesis of C35deOAmB via this iterative cross-coupling strategy, it was critical to develop a collection of chemically robust building blocks having all of the required functional groups preinstalled in such a way that they can be liberated at the end of the synthesis without affecting the structural or stereochemical integrity of the sensitive polyene macrolide skeleton. This problem turned out to be very challenging. However, after extensive experimentation this goal was achieved in the form of building blocks BB1, BB2, and BB3 (Fig. 2). These three building blocks were all efficiently constructed by using a suite of versatile MIDA boronate reagents and associated methods recently developed in our group (SI Appendix, Figs. 3–5) with the majority of complex building block BB1 obtained via selective degradation of the natural product (13, 39).
Synthesis of C35deOAmB via iterative cross-coupling. Inset shows three building blocks for the synthesis of C35deOAmB having all of the required functional groups preinstalled in the correct oxidation states and with the required stereochemical relationships. A single cross-coupling reaction was used in an iterative fashion to sequentially assemble BB1, BB2, and BB3 with complete stereocontrol. Reagents and conditions are, as follows: a, pinacol, NaHCO3, MeOH, 45 °C; b, BB2, 10 mol % PdCl2dppf, K3PO4, DMSO, 23 °C, 53% over two steps; c, BB3, 5 mol % Pd(OAc)2, 10 mol % XPhos, aqueous NaOH, THF, 45 °C; d, LiOH, THF : MeOH : H2O, 35 °C; e, MNBA, DMAP, DCM, 23 °C, 56% over three steps; f, TBAF, THF, 23 °C; g, HF/pyridine, MeOH, 40 °C, 15% over two steps; h, HCl, MeCN : H2O, 0 °C, 32%; and g, penicillin G amidase, H2O, 37 °C, 28%. Bn, benzyl; DCM, dichloromethane; DMAP, 4-dimethylaminopyridine; dppf, 1,1′-bis(diphenylphosphino)ferrocene; MNBA, 2-methyl-6-nitrobenzoic acid; PA, phenylacyl; PMP, para-methoxyphenyl; Pyr, pyridine; TBAF, tetrabutylammonium fluoride; TBS, tert-butyldimethylsilyl; THF, tetrahydrofuran; TMSE, trimethylsilylethyl; XPhos, 2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl.
With these three building blocks in hand, they were readily assembled using a single reaction in an iterative fashion (Fig. 2). Specifically, deprotection of BB1 followed by selective Suzuki–Miyaura cross-coupling with bifunctional iodotrienyl MIDA boronate BB2 provided intermediate 1. Subsequent in situ boronate deprotection and a second Suzuki–Miyaura cross-coupling with BB3, which lacks a C35 hydroxyl group, yielded advanced intermediate 2 possessing the full carbon skeleton of the targeted derivative. Finally, macrocyclization and deprotection of all protic functional groups completed the synthesis of C35deOAmB. In addition to enabling access to this key compound, the successful application of this iterative cross-coupling strategy to such a complex target suggests that this approach has substantial potential to improve synthetic access to a broad range of other complex molecules en route to advanced understanding and/or optimization of their functions.
To further probe the putatively general primary role of ergosterol binding in the antifungal activity of the polyene macrolides, we also prepared via site-selective degradation of natamycin the corresponding natamycin aglycone (Fig. 1A and SI Appendix, Fig. 6) (40). As described above, AmdeB completely lacks the capacity to bind ergosterol, and we expected that the same would be true for this derivative of natamycin lacking its mycosamine appendage.
With all targeted probes in hand (Fig. 1A), we first determined the capacity of each compound to bind membrane-embedded ergosterol via an isothermal titration calorimetry-based assay (Fig. 3A). Consistent with our recent report (2), when a solution of AmB was titrated with either sterol-free or ergosterol-containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) large unilamellar vesicles (LUVs), a substantial increase in the net exotherm was observed in the latter experiment, indicating a direct binding interaction between AmB and ergosterol. This assay also confirmed that AmdeB, which lacks the mycosamine appendage, completely lacks the capacity to bind ergosterol. The same series of results were observed for natamycin and natamycin aglycone, demonstrating that the mycosamine appendage is also critical for the ergosterol binding capacity of this natural product. Most importantly, when this pair of titrations was repeated with C35deOAmB, an increase in exotherm equivalent to that observed with AmB was recorded. Thus, the capacity for ergosterol binding is retained in this C35 deoxygenated AmB derivative.
Ergosterol binding and membrane-permeabilizing activities of polyene macrolides. (A) Total exotherm, as measured by ITC, for solutions of each polyene macrolide titrated with either sterol-free or 10% ergosterol-containing POPC LUVs. AmB, natamycin, and C35deOAmB all bind ergosterol, whereas AmdeB and natamycin aglycone do not. Values represent the mean of at least three experiments ± SD. * p ≤ 0.035; NS, not significant. (B, C) Potassium efflux from 10% ergosterol-containing POPC LUVs (B) and live S. cerevisiae cells (C) after treatment with polyene macrolides. Percent of potassium release was monitored via a potassium-sensitive electrode and expressed relative to the total potassium released upon the addition of Triton X-100 (B) or digitonin (C) at the end of the experiment. In both LUVs and live yeast cells, robust membrane permeabilization is observed with AmB, whereas AmdeB, natamycin, natamycin aglycone, and C35deOAmB are all inactive.
We next determined whether each of these small molecules can permeabilize lipid bilayers (Fig. 3B). AmB at a concentration of 1 μM produced a rapid efflux of potassium ions from ergosterol-containing POPC liposomes (2). In contrast, AmdeB, natamycin, and natamycin aglycone all showed no ion permeabilizing activity at the same concentration. Importantly, C35deOAmB was also devoid of permeabilizing activity in this in vitro experiment, even at an elevated concentration of 10 μM. We further tested the capacity of all five of these compounds to permeabilize live Saccharomyces cerevisiae cells (Fig. 3C). Again, AmB produced rapid and substantial efflux of potassium ions, whereas AmdeB, natamycin, and natamycin aglycone were completely inactive. Most importantly, C35deOAmB also caused no membrane permeabilization in this in vivo experiment, even at the very elevated concentration of 30 μM.
Collectively, these studies revealed that C35deOAmB retains the capacity to bind ergosterol but completely lacks the capacity to permeabilize membranes, making this derivative a powerful probe for the hypothesis that sterol binding is paramount for the antifungal action of AmB. Moreover, the ability to eliminate sterol binding via deletion of the mycosamine appendages from both AmB and natamycin further enabled evaluation of the putatively dominant and general role of ergosterol binding in the antifungal activity of these polyene macrolide natural products.
We therefore next determined the relative capacities of our five probes to kill S. cerevisiae cells in a standardized broth microdilution assay (Fig. 4A). AmB demonstrated a minimum inhibitory concentration (MIC) of 0.5 μM, whereas its non-sterol-binding counterpart, AmdeB, was completely inactive (2, 3), even at 1,000-fold higher concentration (500 μM). Natamycin was only fourfold less potent than AmB (MIC = 2 μM), whereas, like AmdeB, the natamycin aglycone demonstrated no antifungal activity. Most importantly, C35deOAmB, which like natamycin binds ergosterol but cannot permeabilize membranes, demonstrated substantial antifungal activity (MIC = 3 μM), i.e., this derivative is only sixfold less potent than AmB. Moreover, the MICs for C35deOAmB and natamycin are remarkably similar. A very similar series of results were obtained when these same five compounds were tested against Candida albicans, the species of yeast that is the primary cause of systemic fungal infections in humans. Killing kinetics studies (41) further demonstrated that, like AmB, C35deOAmB is a powerful fungicidal agent against both strains of yeast with small decreases in killing rates (Fig. 4A). Finally, we determined via alkaline extraction and HPLC quantification (42) the number of ergosterol molecules per yeast cell employed in these assays. At the MICs for AmB, there is an order of magnitude more AmB relative to ergosterol for both S. cerevisiae and C. albicans (Fig. 4C). Therefore, there is an overwhelming amount of AmB available at the MICs to bind the ergosterol found in these lipid bilayer membranes and thereby kill the yeast.
Antifungal activities of polyene macrolides. (A) MIC as measured by broth dilution. Relative to AmB against S. cerevisiae, C35deOAmB, which cannot permeabilize membranes but retains the capacity to bind ergosterol, is only six times less active. Also note that the MICs for C35deOAmB and natamycin are very similar. A similar series of results is observed with C. albicans. (B) Killing kinetics of S. cerevisiae (Left) and C. albicans (Right) cells exposed to various antifungal agents. In contrast to the fungistatic agent ketoconazole, both AmB and C35deOAmB are powerful fungicidal agents. All compounds were tested at four times their MICs. (C) The number of molecules of AmB and ergosterol, as determined by HPLC, per yeast cell employed in the broth dilution experiments. At the MICs for both S. cerevisiae and C. albicans there is ample AmB available for binding ergosterol and thereby killing the yeast cells. CFU, colony forming units. * p ≤ 0.0003.
Thus, in stark contrast to the now widely accepted channel model, AmB like natamycin (21) primarily kills yeast by simply binding ergosterol. Membrane permeabilization via channel formation represents a second complementary mechanism that further increases potency and the rate of yeast cell killing. Conservation of the mycosamine appendage in the very large family of polyene macrolides combined with the finding that this subunit is required for the sterol binding and antifungal activities of both AmB and natamycin further suggests that mycosamine-mediated ergosterol binding accounts primarily for the antifungal activities of this entire family of natural products.
AmB represents the archetype for both ion channel-forming small molecules and resistance-refractory antimicrobial agents. For decades it has been widely believed that these two features are inextricably linked, i.e., that channel formation is a necessary condition for the fungicidal activity of this natural product. The herein disclosed discovery that sterol binding is actually paramount to the antifungal action of AmB has implications in several important areas.
First, microbial resistance to most antibiotics has been observed within a few years of their introduction into clinical medicine, resulting in an emergent global public health crisis (8, 9). Interestingly, microbial resistance long predates modern medicine (43), which suggests that the quest for resistance-refractory mechanisms of antimicrobial action also has a rich natural history. In this vein, despite extensive utilization in the treatment of life-threatening systemic fungal infections for more than half a century, clinically significant resistance to AmB remains exceptionally rare (10, 12, 13). Thus, clinically relevant antimicrobial mechanisms that can evade resistance exist, and the mode of action(s) of AmB is one of them.
In this context, the discovery that AmB kills yeast primarily by binding ergosterol suggests that the simple binding of a functionally vital lipid is an antimicrobial mode of action with substantial potential for evading resistance in the clinical setting. Interestingly, our results support the conclusion that sterol binding is also primarily responsible for the antifungal action of natamycin (21), and several antibacterial peptides exert their effects via specific lipid binding as well (44⇓⇓–47). Collectively, these findings suggest that the potential generality of this mode of action is substantial. Thus, as accelerating advances in lipid biology continue to illuminate many specific lipids that are required for microbial cell physiology (48), such lipids can be viewed as promising new targets in the search for superior antimicrobials that may be less vulnerable to resistance. It is also likely that the possession of a dual mode of action, i.e., lipid binding and membrane permeabilization, which also appears to be more general (46), further contributes to the resistance-refractory nature of AmB. Gaining a more advanced understanding of how all of these processes are related at the molecular level thus also represents a frontier challenge of substantial importance.
Second, the classic mechanistic model in which membrane permeabilization is paramount to the biological activities of AmB has focused extensive efforts to improve the therapeutic index of this clinically vital antifungal agent on increasing its capacity to selectively form ion channels in yeast cells versus human cells (12⇓–14, 16). The discovery that ergosterol binding is in fact primarily responsible for yeast cell killing reveals a much simpler path forward, i.e., increasing the selective binding of ergosterol versus cholesterol, the main sterol found in human cells to which AmB also binds (2), should be sufficient. To further enable this path, more advanced characterization of this small molecule/small molecule interaction, including determination of the binding stoichiometry, will be the subject of future investigations. Moreover, the herein demonstrated capacity for the iterative cross-coupling strategy to efficiently and stereospecifically assemble the polyene core of AmB from readily accessible MIDA boronate building blocks provides a powerful platform for systematic modifications of this substructure in the pursuit of more sterol-discriminating derivatives.
Finally, the prototypical capacity for AmB to form discrete ion channels in eukaryotic cells suggests that small molecules may possess untapped potential to replicate the functions of deficient protein ion channels that underlie a wide range of currently incurable human diseases. Realizing this potential, however, will require the development of ion channel-forming small molecules that are not cytocidal. The discovery that membrane permeabilization represents a relatively minor contributor to the fungicidal action of AmB suggests that such a goal might be achievable. For example, derivatives of AmB that do not bind endogenous sterols yet maintain the capacity for ion channel formation now represent particularly promising targets. Thus, the herein disclosed advanced understanding of how AmB interacts with living systems is cause for increased optimism regarding the prospect of replacing deficient or dysfunctional protein ion channels with small molecule surrogates.
All reactions were performed under an inert atmosphere using dry solvents in anhydrous conditions, unless otherwise noted. Full experimental details and characterization for all compounds are included in the SI Appendix. Isothermal titration calorimetry (ITC) studies were performed as described (2) using a Nano ITC instrument with a 190 μL reference cell. Potassium efflux (2), MIC (2), killing kinetics (41), and ergosterol quantification (42) assays were performed as described. Full experimental details and associated references are included in SI Appendix.
We thank Bristol–Myers Squibb for a gift of amphotericin B. This work was supported by the National Institutes of Health Grant GM080436. M.D.B. is an Howard Hughes Medical Institute Early Career Scientist.
↵1K.C.G. and D.S.P. contributed equally to this work.
↵2To whom correspondence should be addressed. E-mail: burke{at}scs.uiuc.edu.
Author contributions: K.C.G., D.S.P., I.D., M.M.E., B.C.W., and M.D.B. designed research; K.C.G., D.S.P., I.D., M.M.E., B.E.U., and B.C.W. performed research; K.C.G., D.S.P., I.D., B.C.W., and M.D.B. contributed new reagents/analytic tools; K.C.G., D.S.P., I.D., M.M.E., B.E.U., B.C.W., and M.D.B. analyzed data; and K.C.G., D.S.P., I.D., and M.D.B. wrote the paper.
*This Direct Submission article had a prearranged editor.
Freely available online through the PNAS open access option.
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You are going to email the following Amphotericin primarily kills yeast by simply binding ergosterol
Kaitlyn C. Gray, Daniel S. Palacios, Ian Dailey, Matthew M. Endo, Brice E. Uno, Brandon C. Wilcock, Martin D. Burke
Proceedings of the National Academy of Sciences Feb 2012, 109 (7) 2234-2239; DOI: 10.1073/pnas.1117280109

redpajama_set_name: RedPajamaCommonCrawl


text:
The concept of an Office Desk has been with us since time immemorial. To say that this piece of furniture, and its companion piece the office chair, has symbolized all offices, ancient and modern, is an understatement. People who spend important hours of their life working from an Office Desk, often start with a new and glitzy piece of furniture, but in spite of periodic polishing up and the usual repairs, age begins to catch up with both the worker and his desk. But even when the office staff is retired, the old office desk remains as an eyesore. How to Get Most Out of your old office desk remains an unanswered question, and we try our best to answer it here.
Getting The Most
How to Get Most Out of your old office desk, is the kind of question to which there are no ready answers. A curt response would be simply, "Throw it away". It is the kind of instant answer that will please many people, particularly if they happen to be Philistines, with no regard for history or fondness for an old servant. It is the kind of soul-less bloodthirsty attitude that characterizes many of the new generations of self-seekers. But then, this Article is not for these classes of robotized humans, on a constant quest for what they call "progress". The old Office Desk represents our past, our station in a long working life. In saluting this sentiment, there are a growing number of paternal companies who not only gift their retiring workers of long-standing with the usual gold watches but allow these sentinels of their march to affluence to carry away the most significant reminder of their useful career, their old Office Desks. And here lies the catch. What do these old retirees do with their old Office Desks? Or even, in the case where the company opts not to hand out their old furniture to their departing employees, how can they make these relics of a fruitful but hoary past become standard-bearers of the future? So here are some ways to repurpose an old Office Desk, so that they can rejoin the society of Useful Artifacts once more:
Old Wooden Office Desks: Wood is a primitive but regal material, and is nearly ageless. The woods used for our ancient Office Desks were always of the best available technical quality, the Pines, Willows, Shaal and Sepeles. The best Desks were even made from Royal Woods like Rosewood, Oak, mahogany and teak. When properly treated at birth, this furniture was insect and termite-proof. It took to the natural polishes like a duck to water, and was, in the case of hardwoods, almost scratch-proof as well. The typical finishes are:
Paint: The use of Paint has traditionally been one of the easiest ways to update an old Office Desk. Comparatively less heavy on the pocket, it allows good cheer and comfort to the eyes with minimum effort. For the best results, a Primer coat must be applied first, with rough and smooth sanding in between the coats in order to ensure an even finish. For the base paint, a crisp white or deep black is recommended to give a neutral look. Or the piece could be made to look quite unusual with a stunning and unexpected color shade such as bright ruby red or lemon yellow. Highlighting the features and unique details can be done in different color combinations. Distressing the furniture deliberately during the updating process can add character to the Desk, forcing others to view the piece with real interest. Distressing can be done by scuffing the paint with sandpaper or steel wool to reveal the old wood underneath or even denting and scratching with hammer and chain.
Stain: The use of stains can retain the nostalgic old-world beauty of the Desk, or it may be used to match the other pieces in the room where it will be placed. Contrast stains are often used, to create an ensemble effect. First, the existing stains must be removed completely with a chemical stripper, or the applied stain must match the underlying older stains perfectly. Restoring old detailed engravings and trimmings of a really old Office Desk are an art in itself, and needs a dense brush, lots of clean rags and immense patience, to create an even finish.
Oil Finish: The existing finish must first be stripped off first using a chemical stripper, followed by a thorough sanding with sanding discs and sandpaper. Then comes the hydrating finish, with sufficient resting time allowed, before buffing motion is used to wipe off the excess. The results, in several coats of high shine, luxurious varnish, are a pleasure to see.
Hardware: It may not always be mandatory to refinish the old Office Desk in the paint. Replacing the old hardware to introduce modern fittings will make the piece not only a joy to behold, but a pleasure to use. The modern accessories to suit the Digital Age can all be accommodated, as well as some old world charming add-ons, and of course, some indoor greens for fresh oxygen. Powerbanks and sources, Routers, a sliding tabletop for the laptop, inbuilt lighting to focus on study matters, can all be built-in while refurbishing the old office desk. All this must be aesthetically planned before putting a hand to the job. The DIY enthusiast will carefully prepare several versions, and take opinions from his peers, before jumping off.
Additions: The scope for Additions is vast, such as hanging a sleek mirror or portrait, which can completely transform the old office desk. Tile, stone or glass can be added for super effect and can create an artistic tabletop. The old interiors can be gutted, to be replaced by modern digital steel lockers, cabinets, sliding table-tops and so on.
The possibilities are truly endless, and only the creative imagination of the owner can be the determinant of the final Office Desk.
12 must have office furniture accessories
7 life lessons learned when using a cheap office furniture
Dubai is the City in the Sun, the modern Shangr...


Arthur Hayes, the Co-Founder and CEO of BitMEX, a Peer-to-Peer Trading Platform that offers leveraged contracts that are bought and sold in Bitcoin predicts that the pioneer cryptocurrency will sell for at least $50,000 by the end of 2018. Hayes made his prediction while speaking on the CNBC Fast Money show on June 29 2018.
The Acer Nitro 5 is an outstanding entry-level gaming laptop and worthy of consideration if you have a more modest $900 budget.


Although peaceful today, Bembridge and Culver Downs were once a scene of much military activity. This short but energetic coastal walk will give you a fascinating insight into the Isle of Wight's role in wartime defence and intelligence. It is also rich in wildlife and offers spectacular views over Sandown and Whitecliff Bays.
Circular walk, with an ascent of 250ft (75m). Two kissing gates, one step-stile and a steep flight of concrete steps. Mostly well-defined grassy paths with some stony and uneven surfaces. Keep well clear of the cliff edge, it can be slippery in wet conditions. Dogs are welcome, but please keep your dog on a lead around wildlife and take any mess home with you. Sadly there have been incidents of dogs worrying the Hebridean sheep.
Military history – Beacons like the one on Culver Down were part of a Medieval south coast early warning system to respond to the threat of French invasion. A chain of beacons, each consisting of a mast with an access ladder and a fire bucket on top, stretched across the Island and were used to pass messages, by way of a flame, to and from the mainland. Culver Gun Battery built in 1893 was part of a series of batteries guarding the Solent approaches from attack by the French. All that remains today are the gun emplacements.
Look down on the Down Culver Down is rich in tender chalk grassland flowers, like rock rose, Bee orchid and birdsfoot trefoil, that live in the shorter turf. Look for fine displays of cowslip in spring. In summer you may see bush crickets and grasshoppers, plus many butterflies including the chalkhill blue. In addition to the wildlife on Culver Down, look out for gun platforms, War Department boundary stones, fence posts, trenches and radar mast anchor chains.
1. From the car park near Bembridge Fort, follow a grassy path on the right side of the road towards the coast. Keeping a hedge on your left and taking the left fork when the path splits, youll meet the road again by a cattle grid. Cross this road and emerge onto Culver Down. Head along the road towards the monument. Take care, this road can be busy at times.
2. Take a look at the Yarborough Monument (a memorial to Lord Yarborough, the first Commodore of the Royal Yacht Squadron), then return to the road. Continue past the Culver Haven Inn and the site of Culver Signal Station (now a car park) to Culver Beacon and Culver Battery.
3. Descend some steep concrete steps and cross Culver Battery car park. Continue in the direction of Whitecliff Bay, along a grassy path which bends to the right and follows the crest of the chalk ridge of Culver Down eastwards to the sea.
4. On reaching the cliff top fence, turn right and follow the path southwest, in the direction of Sandown, for the next 1.5 miles (2.5km). Pass through two kissing gates before reaching the natural amphitheatre of Red Cliff, shortly before a holiday park.
5. Marvel at the views before turning round and retracing your steps to the last gate you went through. 6. Take the left fork in the path and follow a grassy track away from the coast. Head across the down, back towards Bembridge Fort, which is visible on the horizon. Keep a fence on your left and at the way marker follow the path uphill. Within 50 yd (50m) of the Fort, branch right, under some electric wires, to reach the Fort entrance directly. The Fort is not open to the public. You can visit a viewpoint on the east side of the Fort on the way back to the car park.


Whoever said "It's just business, not personal," never worked in the field of architecture. For more than 35 years, Manuel Cadrecha, Design Director for the Atlanta office of global architecture and design firm Perkins + Will, has taken a highly personal and accessible approach to design. Manuel's style comes from a deep sense of empathy and an embrace of interdisciplinary design. His work embodies a statement his father made when Manuel was young: "Architecture is the last humanist profession."
A current chronicle of Cadrecha's work has been recently published by ORO Editions: an internationally recognized, award-winning fine art and architecture publishing firm. In his career monograph, Manuel shares personal commentary about the design process and inspirations that were used. The book is also full of simple, clear photos and hand sketches that illustrate careful, detailed and accessible designs. These designs are clarified only with a frugality of words: mirroring the wordless feeling of awe Cadrecha experienced – also in his youth, fresh to America from Spain - sitting in Central Park with trees and buildings mingling in his view. He lets the design do the talking.
"In architecture, I feel a deep connection to my past and my future, to tradition and innovation, and to beauty and invention," said Cadrecha. "I love being an architect because, in architecture, I can have the remarkable effect of improving people's lives and bridging cultures together."
Projects in the book represent the last 15 years of Cadrecha's design work across a wide range of project types, clients and geographies. From city-scale international developments to neighborhood schools, they works are grounded in his abiding interest in materials and form, color and light, the old in the new, and the search for the inspiring in the every day. Projects highlighted in the book include several in Atlanta: 1315 Peachtree Street, Charles R. Drew Charter School, Junior and Senior Academy and those located in Centers for Disease Control and Prevention, Building 106 & 110, EVIVA Peachtree, Georgia Tech Chris Klaus Advanced Computing Center and Springdale Park Elementary School.
"Manuel Cadrecha's work is marked by who he is as a person—someone with a global education and experience, combined with tremendous agility, curiosity, and humility," said Phil Harrison, President and CEO of Perkins+Will. "Armed with broad experience and deeply humane capacity, Cadrecha is able to reach more complex levels of design synthesis in a natural and unforced manner."
Purchase this book at ORO Editions.


Buy some Orange Lanscaper's Marking Paint. Take it to the local school yards, Police Station lawns, Firehouses, Libraries, etc.
Paint your favorite 9/11 Truth slogan prominently on the lawn. It will last a few weeks depending on mowing habits.
Ideal for the rural activist. And can be done anonymously during the hours of the night if so desired.
The paint often comes in boxes, but I say we put a few resources here in order for people to buy them.
This action isn't going to give you the kind of fame or publicity as a YouTube clip at a Presidential Town Hall meeting, but I think it can be just as effective in educating the public, particularly those without internet access, and getting our goals met. Which I'm ready for.
9/11 blogger is the best, and don't hesitate to comment over at www.WeAreChange.org and www.TruthAction.org as well.


text:
Q: Is there a way to retrieve user specific information when running a cloud project locally? We have a few values which are user specific in our cscfg files like service bus namespaces, usernames and passwords etc. I wonder if we can retrieve those values upon build time when running the solution locally so the scenario would be:

*

*Get solution from repo

*Build locally (this will cause developer specific variables to replace either tokens like %username% or %password% or pre-defined values in config files

*Run solution without having to substitute anything in config files

I have seen this solution implemented with powershell, but I am looking to an alternative to that. Basically you run a ps script before building the solution and that replaces (de-tokenizes) any tokens in config files.
I wonder if I can do that with web.config transformation at all. But then again I suppose need to change values in the cscfg. Any thoughts?

redpajama_set_name: RedPajamaStackExchange


Savvier consumers and increased competition have long made commercial success, whether at market entry or during product maturity, more elusive. At the same time, new technologies have greatly expanded the reach and potential of cost-efficient marketing communication through Web sites, social media and traditional media sources.
Research and Advisory Services benchmarks and tools provides executives, directors, and managers in Marketing and Public Relations with actionable insights to better align PR and marketing activities according to product lifecycle stages. Marketing leaders will gain insights into performance measurements for marketing campaigns and what Web-based resources best engage various customers segments.
Demand generation - What are the best communication tools, channels and calls to action?


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redpajama_set_name: RedPajamaGithub


I never thought of Laguna Beach as anything other than that show on MTV about a group of teenagers whose lives were full of drama and yet admittedly it was one of my guilty pleasures. As I was planning my California itinerary, I wanted to visit one of their famous beaches. My choices were between Palm Springs or Laguna Beach. I ended up choosing the latter and it was the best decision I made. Laguna Beach was a dream! I couldn't help but smile the entire time while I was there because I was so happy. I even thought to myself, "I could totally live here!"
We checked-in at Capri Laguna for the weekend. It was right in front of the beach and walking distance from the best restaurants and small quaint shops.
We spent the day getting lost in the town. It's really small- it will take you just an hour by car to reach from start to end but it was filled with so much character. They believe in energies. You will see a lot of stores that sell crystals and do tarot reading.
Laguna Beach also has the most breathtaking sunsets. Just take a look at our view during dinner.
Rise and shine, it's a new day! We started our day with the most important meal breakfast. Capri Laguna had a Starbucks vending machine in the lobby. Of course- me being obsessed with coffee, I abused it just a little bit. I made three cups of coffee which left me feeling extra energetic the entire day.
On our last day, we headed to Oak to get brunch and sip on cocktails. Oak was one of the recommended restaurants on Visit Laguna's website. This was a helpful guide to maximize our short stay. You can see the reviews of the hotels, things to do, how to get around, and where to eat.
Look at my smile! If you haven't visited Laguna Beach, you're missing out. I highly recommend going here if you're in the California area.
I love your shots! Such a nice place! Definitely putting it on my bucket list!


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