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Lo-fi alt-rock pioneers Sebadoh are touring in support of the reissue of their landmark 1994 release Bakesale (which boasts the unforgettable cover art of a naked baby looking in a toilet). Among their most easily accessible albums, Bakesale benefited from some standout tales of disenchantment and escapism, such as "Not Too Amused" and "Skull," respectively. Fittingly, the group's current lineup consists of original members guitarist-vocalist Lou Barlow (also of Dinosaur Jr. fame) and bassist Jason Loewenstein, alongside touring drummer and Loewenstein's Fiery Furnaces bandmate Bob D'Amico. Original Sebadoh drummer Eric Gaffney, who was on some recent reunion tours, missed out on recording Bakesale, giving this tour some irony. With Richard Buckner.
Sun., April 10, 8 p.m., 2011


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Knife Sharpening in Portland, ME
Does anyone have a place they recommend for knife sharpening in or around Portland, Maine? I'm coming up with nothing in the yellow pages or online. Sorry if this isn't quite about food, but without good sharp knifes, the Home Cooking section is only dangerous to me!
I'm sure someone does. I would contact Leroux Kitchen on Commercial Street. If they don't do it, I'm sure they know a place that does.
I don't know of one in Portland itself, but the Freeport Knife Co. in Freeport is supposed to offer the best local sharpening services.
Most Hannaford meat departments will sharpen knives for free if asked. They use an in-house grinder that gives a very sharp edge but does tend to take a fair amount of metal in the process.
Shaw's doesn't do a bad job for free. They have asked me not to bring all my knives at once.
I've always sharpened my own knives but for the past year or so I have not had a nice tri-stone to use. I broke down and went to Freeport Knife Co. and was not happy with the out come at all. It's the first and last time I will let some one else sharpen my babies! Your best bet is getting your own tri-stone. Restaurant quality will cost you but its well worth it if you can swing it.
Was told this Place in Bath does it:
Must call to confirm. I Have no comment to the service as of yet
re: Passadumkeg
I would love to see someone offer a class/seminar on this. I have not had good results with Freeport Knife or the electric doo-dad I bought years ago. Would really like to just do it at home properly.
re: johnnydj
Its so easy to sharpen knives go to a Job Lot type store purchase a 2 sided stonee one a bit course one fine. Then use the penny under the blade and use water not oil if you are interested let me know and I will help you through it. Not to take anything from a sharpening place but you have razor sharp knives right there. Hope this helps a bit.
re: Frank Terranova
...or you could do it right and buy my pal Chad Ward's book, called "An Edge In The Kitchen - The Ultimate Guide to Kitchen Knives -- How to Buy Them, Keep Them Razor Sharp, and Use Them Like a Pro"

subset: commoncrawl


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Hello friends! :D I haven't been posting in a while.... but the holiday season is among us! Getting caught up with family and friends is a MUST, but it's also amazing too that I do not have to work everyday and I can take major breaks and STILL make money :D
Gotta love it!
Well, my team mate Polina Cango started a video challenge which consists of making a video for 50 days. I couldn't let her go in this alone, so I joined her! We are able to talk about anything we want (our lives, experiences, hobbies, events, funny stuff, etc.) Whatever it is, all that matters is that we share and SHOW the world that EVERYONE has a voice.
Joining this video challenge was a step for me for self- improvement, and maybe it was just an excuse to push myself. (I love challenges :D) hehe
We strive to make differences in people's lives, and we hope that our videos will help even ONE person make a change within themselves because every human being deserves to live their dreams :D
Remember that everyone has a voice AND anyone can join us in this video challenge!
1. Make a video
2.Upload to Facebook, Youtube, Vimeo, Instagram, etc.
3. Use #StretchBeyondYourVision
4. Make sure you share it with us! (We want to hear the amazing things you have to say!)
5. Have fun! :D
Want to work personally with us? See your life transform and Click Here: http://www.bit.ly/18E0n52
Connect with me on Facebook here: http://www.facebook.com/MoneyMeg
Or Twitter: @Meg'sEnvironment


text:
- freely available
- re-usable
Pharmaceuticals 2013, 6(6), 728-758; doi:10.3390/ph6060728
Published: 27 May 2013
Abstract: Antimicrobial peptides (AMPs), small host defense proteins, are indispensable for the protection of multicellular organisms such as plants and animals from infection. The number of AMPs discovered per year increased steadily since the 1980s. Over 2,000 natural AMPs from bacteria, protozoa, fungi, plants, and animals have been registered into the antimicrobial peptide database (APD). The majority of these AMPs (>86%) possess 11–50 amino acids with a net charge from 0 to +7 and hydrophobic percentages between 31–70%. This article summarizes peptide discovery on the basis of the APD. The major methods are the linguistic model, database screening, de novo design, and template-based design. Using these methods, we identified various potent peptides against human immunodeficiency virus type 1 (HIV-1) or methicillin-resistant Staphylococcus aureus (MRSA). While the stepwise designed anti-HIV peptide is disulfide-linked and rich in arginines, the ab initio designed anti-MRSA peptide is linear and rich in leucines. Thus, there are different requirements for antiviral and antibacterial peptides, which could kill pathogens via different molecular targets. The biased amino acid composition in the database-designed peptides, or natural peptides such as θ-defensins, requires the use of the improved two-dimensional NMR method for structural determination to avoid the publication of misleading structure and dynamics. In the case of human cathelicidin LL-37, structural determination requires 3D NMR techniques. The high-quality structure of LL-37 provides a solid basis for understanding its interactions with membranes of bacteria and other pathogens. In conclusion, the APD database is a comprehensive platform for storing, classifying, searching, predicting, and designing potent peptides against pathogenic bacteria, viruses, fungi, parasites, and cancer cells.
1. Introduction
The identification of antimicrobial peptides (AMPs), small proteins important in host defense, can be traced to the discovery of lysozyme by Alex Fleming in 1921 [1]. Another well-known peptide antibiotics, gramicidin, was also discovered as early as 1939 [2]. Some polypeptides, initially discovered for other purposes or functions, were established as AMPs later on. For example, kalata B1, a typical cyclotide from plants, was identified initially in 1973 to have uterotonic activity [3]. Its antimicrobial activity was not established until 1999 [4]. In the 1980s, Hans Boman identified cecropins in insects [5], Robert Lehrer isolated defensins from humans [6], and Michael Zasloff discovered magainins in frogs [7]. These pioneering discoveries ignited the interest of many other investigators in these host defense peptides, leading to the purification and characterization of hundreds of new AMPs.
The rapid increase in natural AMPs makes it a challenging task to manage such information manually. As a consequence, several databases, including APD [8,9], DAMPD [10], CAMP [11], and YADAMP [12] were constructed to store AMPs from both prokaryotes and eukaryotes. Other databases, including the two earlier databases (AMSDb and peptaibols) [13,14], have a narrower scope. While AMSDb contains only AMPs from eukaryotes [13], others are even more specialized databases for plant AMPs (PhytAMP) [15], shrimp AMPs (PenBase) [16], amphibian peptides (DADP) [17], fungal peptides (peptaibols database) [14], bacteriocins (BACTIBASE and BAGEL) [18,19]. There are also databases for special classes of natural AMPs: bacterial thiopeptides (THIOBASE) [20], circular polypeptides (Cybase) [21], defensins [22], and large lytic proteins (EnzyBase) [23]. In addition, SAPD is an old database with only ~200 synthetic peptides [24] and RAPD is another small database for recombinant AMPs, which may, and may not, correspond to natural sequences [25]. Ideally, newer databases should avoid duplicated efforts and provide complementary features to existing database. For example, CAMP collected 1153 peptides predicted to be AMPs and 1651 peptides from patents [11]. Some information about AMP patents and grants can also be found in Defensins KnowledgeBase [22]. DADP contain sequences for the signal and pro-regions of amphibian peptides, which can have many different functions [17]. Cybase is a collection of all circular proteins (i.e., polypeptides with a peptide bond formation between the N- and C-termini), which can possess various functions such as antimicrobial, insecticidal and anticancer activities [21]. Although not yet demonstrated, YADAMP was created to facilitate QSAR studies of AMPs. These databases are chronically listed in the "links" of the APD and are further described in a recent book chapter [26].
The antimicrobial peptide database (APD) was developed for multiple purposes, including peptide nomenclature, classification, statistics, information search, peptide calculation, prediction and design [8,9,26]. In particular, the pipeline design of the APD enables users to search peptide activity and parameters either individually or in combination. Such a feature facilitates database guided peptide discovery. In the following, we first highlight the selected features of the APD and then describe its applications in peptide identification and design. To our knowledge, the use of other databases [10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25] in peptide design has not been demonstrated.
2. The APD Database Features Useful for Peptide Design
2.1. Peptide Sources
The APD database was initially built in 2003 with 525 entries [8]. These peptide entries were manually collected and registered into the database. Since 2007, this database has been further expanded and regularly updated. The second version of the database reported 1228 AMPs in 2009 [9]. As of March 2013, there were 2183 such peptides. For a natural peptide to be an APD entry, it must have demonstrated antimicrobial activity and known amino acid sequence, at least partially. The sources of the AMPs are annotated, and can be searched, in the name field. In total, there are 1656 AMPs from animals, 293 from plants, 181 from bacteria, five from protozoa, and 10 from fungi (Figure 1). It is clear that the AMPs in the APD originate from a variety of sources and the majority (76%) are identified from animal sources [26], especially amphibians [27,28,29,30,31]. The APD also collected a small number of synthetic peptides (2%). One of the reasons for the small number is that in many cases synthetic peptides are treated as derivatives of natural AMPs and are described under the parent entries. Although this practice reduces the number of AMPs, it does facilitate users to get a more complete view about a particular peptide.
Many natural AMPs were isolated by chromatographic methods [5,6,7,32,33,34]. Based on the APD [8,9], the total numbers of natural AMPs discovered annually from 1970 to 2012 are plotted in Figure 2. While only a few AMPs were isolated during 1970–1984, up to 29 AMPs were reported between 1985 and 1992, and 41–82 between 1993 and 1999. In the 2000s, AMPs discovered reached ~100–150 per year. A sharp increase in such peptides in 2011 was primarily due to large-scale peptide discoveries from amphibians by using genomic and proteomic techniques [27,28,29]. Hundreds of the newly identified peptides listed in those papers have not been registered into the APD due to the lack of antimicrobial activity data. Our peptide registration practice [26] is supported by the observation that many of the predicted peptides synthesized for antimicrobial assays were found to be inactive or poorly active against the testing bacterial strains [28].
2.2. AMP Activity Annotated in the APD
It is now appreciated that natural AMPs could play multiple functional roles in host defense. As key effector molecules of innate immunity, they are produced to ward off invading microbes [31,35,36,37]. In insects, different types of AMPs can be expressed via different pathways in response to a variety of invading pathogens such as Gram-positive, Gram-negative bacteria, viruses, or fungi. Many of the insect defense elements appear to be conserved in mammals [38]. For example, the Toll pathway, initially found in insects, also exists in humans [39,40]. Some AMPs also have anti-cancer, anti-HIV, or spermicidal activities [7,31,32,33,34,41]. In addition, AMPs can modulate the immune system and elucidation of the mechanisms of immune modulation may provide yet another avenue for therapeutic peptide development [37,42,43]. The antimicrobial and chemotactic activities of AMPs are now annotated in the APD [9], allowing users to obtain a list of AMPs with defined activity. Table 1 lists the number of AMPs with various activities. In addition, our database also annotated anti-HIV peptides as well as antibacterial peptides that only active against Gram-negative or Gram-positive bacteria. These peptides can be used as the starting templates for designing peptides with desired properties.
|Table 1. The number and abundant amino acids of antimicrobial peptides with defined biological activity 1.
|Peptide activity
|Peptide number
|Abundant amino acids
1 Data obtained from the APD (http://aps.unmc.edu/AP) [8,9]. In the table, there are more peptides with antibacterial (1768) or antifungal (777) activity due to the common use of E. coli, S. aureus, and C. albicans in antimicrobial activity assays.
2.3. Calculations of AMP Parameters and Amino Acid Composition
The APD also enables users to obtain the parameters (e.g., length, net charge, hydrophobic content, and structure type) for a single peptide or a group of candidates [8]. The distribution of all the AMPs as a function of peptide length is given in Figure 3A. It is clear that 88% the AMPs possess 11–50 amino acids with the peak in the range of 21–30 residues. Assuming pH 7, the calculation of the net charge of an AMP involves acidic amino acids D, E, and basic R and K. A distribution of all the AMPs as a function of peptide net charge is given in Figure 3B. There are 86% of AMPs with a net charge between 0 and +7 (maxima at +2 and +3). The hydrophobic percentage is the ratio between the sum of the amino acids in the hydrophobic group and the total number of amino acids. A distribution of all the AMPs as a function of peptide hydrophobic% is given in Figure 3C. The majority of AMPs (89%) contain a hydrophobic% in the range of 31–70% (the maximum at 41–50%). In the APD database, the 20 standard amino acids are classified into four groups: hydrophobic (L, I, V, M, F, W, A, and C), PG (P and G), polar (S, T, N, Q, and Y), and charged (D, E, K, R, and H). To a large extent, the amino acid composition of AMPs determines peptide activity [44]. The APD is the first AMP database that allows users to calculate the amino acid composition profile for each peptide or a selected group of AMPs with a specific activity or property [8,9,26]. Figure 3D is such a profile for all the AMPs, where on average amino acids L, G, and K have the highest frequencies. Here we define the abundant amino acid as the one that has the highest frequency or percentage in each of the four amino acid groups defined above. The abundant amino acids for a group of AMPs with defined antimicrobial activity are listed in Table 1. For example, the abundant amino acids for the 1768 antibacterial peptides are L, G, S, and K (i.e., identical to those in Figure 3D). However, C is the abundant hydrophobic residue for AMPs with antiviral, antifungal, or antiparasitic activity, indicative of the existence of a large population of cysteine-containing peptides in these activity groups. Because C/L, G, S/T, and K are consistently dominant in various activity groups in Table 1, they are preferred by nature in designing AMPs. In contrast, amino acids such as M, W, Y, Q, E, D, and H have the lowest frequencies on average. The biased use of amino acids in AMP design could be important for host defense since no active peptides were obtained in the 200 sequences assembled by choosing amino acids randomly [45].
The amino acids composition can be utilized to predict whether a natural peptide is potentially an AMP. This is the basis for programming the AMP prediction interface in the first version of the APD. The input peptide sequence is predicted as "rich" in certain amino acids, disulfide-linked (usually β-sheet structure), or α-helix [8]. Recently, Wang et al. showed that, among the multiple parameters used in peptide prediction, the contribution (weighting) of the amino acid composition accounts for 60% [46].
The amino acid composition profiles may also contain mechanistic information. For example, the amino acid profiles of bacterial lantibiotics and plant cyclotides are similar [26], although the sulfurs in these two families of peptides bridge side chains in a different manner: disulfide bonds in cyclotides but thioethers in lantibiotics. Interestingly, peptides from these two families can also share the same mechanism of action—both recognizing phosphatidylethanolamines (PEs) in membranes [47,48,49]. All these examples indicate that the amino acid composition calculated in the APD is an important feature of natural AMPs.
3. Peptide Discovery Based on the APD Database
Based on the APD database, various approaches have been developed for peptide discovery [50,51,52,53,54,55,56]. These methods, including linguistic model, database screening, de novo design, and template-based design, enabled the identification of potent peptides against human immunodeficiency virus type 1 (HIV-1) or methicillin-resistant Staphylococcus aureus (MRSA).
3.1. The Linguistic Model
Loose et al. developed a linguistic model for rational design of antibacterial peptides [51] based on the initial 525 peptide entries in the APD [8]. In this approach, the AMP sequences were regarded as a language. Over 700 sequence patterns (grammars) were identified from those AMPs and each grammar consisted of 10 amino acids. Just like the creation of a new sentence by grammar, they generated new AMPs by combining two grammars. This is reminiscent of the hybrid approach used by others to generate new peptides [57,58,59]. Two antibacterial peptides were identified for further optimization by first making the sequences more different from natural sequences and later more active by increasing cationic and hydrophobic amino acids. This method can clearly generate new sequences not yet found in nature.
3.2. Database Screening
For a particular natural peptide in the APD, antimicrobial activity assays are usually conducted with a limited number of microbial strains (typically E. coli, S. aureus, and C. albicans) and the strain which the peptide was expressed for or most active against might not have been uncovered. Hence, we hypothesized that the APD database (i.e., a library of mainly natural peptides) contains useful candidates and can be screened to identify most potent peptide templates with desired activity. Ideally, the entire database with various templates should be experimentally screened. In reality, this is impractical due to many challenges in obtaining all these compounds at a reasonable cost and within a required time frame. Therefore, we utilized the database to help select a representative group of templates. Two separate screens were performed [52,53]. In one screening, our goal was to identify compounds against HIV-1. In another screening, we aimed to identify most potent peptides against MRSA USA300, a USA community-associated superbug. Database screening should be applicable to the identification of peptides with other types of activity as well.
3.2.1. Identification of HIV-1 Inhibitory Peptides
The acquired immunodeficiency syndrome (AIDS) is one of the leading causes of death worldwide. The United Nations estimates that 1.5 to 1.9 million people died from HIV/AIDS worldwide in 2011 alone. Although the deaths dropped by 24% compared to those in 2005, continued efforts are needed to further reduce HIV-1 infections. In particular, effective HIV-1 vaccines are not yet available, making the development of topical microbicides desirable to prevent the HIV-1 transmission during sexual intercourses [41,60,61]. In collaboration with ImQuest Biosciences, we evaluated anti-HIV activity of 30 candidates using the established protocols (see ref. [56] for details). These peptides were selected from the APD database based on the following criteria: (1) length < 25 amino acid residues because shorter peptides are easier to synthesize in chemical labs; (2) net charge > 0, since anionic peptides tend to be inactive; (3) no cysteines, since the synthesis of peptides with multiple disulfide bonds are more time-consuming; (4) nontoxic to mammalian cells, because cytotoxicity is an undesired property; (5) not annotated as "synthetic" (i.e., man-made peptides) in the database, as such peptides might have been patented by other laboratories; and (6) anti-HIV-1 activity unknown, since we want to identify new candidates. Of the 30 candidates, 11 peptides showed EC50 concentrations in the range of 0.63–7.1 μM [52]. The success rate of our database screening (37%) appeared to be high. The top six anti-HIV peptides are given in Table 2. These peptides originated from diverse sources: amphibians (ascaphin-8 and brevinin-2 related), insects (ponericin L2), fish (piscidin 1), and human engineered (DASamP1 and DASamP5). The names of the last two engineered peptides were derived from "database screened antimicrobial peptides 1 and 5). While DASamP1 (amino acid sequence: FFGKVLKLIRKIF-amide) is a variant of temporin-PTa [62], and DASamP5 (amino acid sequence: SLSRFLRFLKIVYRRAF-amide) is a variant of temporin-LTc [63]. Our screening identified new peptides with anti-HIV activity. As of March 2013, there are 92 anti-HIV peptides in our database. This only accounts for 4% of the database entries [9]. Thus, one may anticipate that more anti-HIV peptides could be identified in this manner.
|Table 2. Top six potent antimicrobial peptides against Staphylococcus aureus USA300 LAC or HIV-1 identified from database screening 1.
|Peptide Name
|S. aureus USA300 (MIC, μM)
|Peptide Name
|HIV-1 (EC50, μM)
|Lycotoxin I
|Ponericin L2
|Maculatin 1.3
|Brevinin-2 related
|Piscidin 1
|Piscidin 1
1 Data obtained from refs [52,53]. See the text for additional information of these peptides.
3.2.2. Identification of Anti-Staphylococcal Peptides
The spread of MRSA from the clinical setting to the communities is worrisome. In fact, the annual frequency of deaths from MRSA is rapidly increasing and has surpassed those caused by HIV/AIDS. Therefore, there is a need to develop new treatments to control MRSA. To identify new compounds against MRSA USA300 LAC, we also assayed the antibacterial activity of the same 30 peptides used for screening anti-HIV-1 peptides above [52]. Six peptides, including frog ascaphin-8, DASamP1, DASamP2, spider lycotoxin I, frog maculatain 1.3, and fish piscidin 1 (Table 2), were found to kill S. aureus with the minimal inhibitory concentrations (MIC) of 3.1–6.2 µM. DASamP2 (amino acid sequence: IKWKKLLRAAKRIL-amide) is a derivative of polybia-MPI [64]. To better gauge antimicrobial ability of these peptides, we also tested their antibacterial activity against Gram-positive Bacillus subtilis, and Gram-negative Escherichia coli and Pseudomonas aeruginosa. Aascaphin-8, DASamP2, lycotoxin I, and piscidin 1 are thus established as broad-spectrum AMPs that are active against S. aureus USA300, B. subtilis, E. coli, and P. aeruginosa. On contrary, maculatin 1.3 is only active against the two Gram-positive bacteria S. aureus USA300 and B. subtilis, but not the two Gram-negative bacteria E. coli and P. aeruginosa. DASamP1 appears to be species-specific since it is active only against S. aureus, but not other bacteria tested. Therefore, these six peptides with varying activity spectrum are useful templates for developing different antibacterial agents. For example, DASamP1 was able to prevent Staphylococcal biofilm formation in the catheter embedded in mice [53]. Considering the pain in replacing medical devices and the difficulty in treating biofilm infection, this short peptide may be developed into antimicrobial agents for topical use.
3.3. Database-Guided Design of Antimicrobial Agents
3.3.1. De Novo Design of Anti-HIV Peptides
We also explored database approaches to designing antimicrobial compounds against HIV-1 [9,54]. Our initial attempt was a stepwise design based on the bioinformatics results from our database. In particular, we observed that AMPs from different kingdoms or families have distinct amino acid composition profiles [9,26,44]. When all the AMPs were statistically analyzed, amino acids L, G, and K were found to be abundant (Figure 3D). These three amino acids appear to contain sufficient information for peptide design, since we succeeded in designing an antibacterial peptide GLK-19, a 19-residue peptide containing only G, L, and K (Table 3). This peptide possessed good activity against E. coli (MIC 10 μM) but not HIV-1. Next, we intended to confer anti-HIV activity to this peptide template based on our database observation that arginines, rather than lysines, are preferred in antiviral peptides (Table 1) [8]. Indeed, GLR-19, an arginine analog of GLK-19, became HIV-1 inhibitory, although it lost activity against E. coli (Table 3).
The APD also suggests that antiviral peptides are rich in cysteines [8]. This finding inspired us to introduce a pair of cysteines into GLR-19 at varying positions along the polypeptide chain, leading to a series of 19-residue peptides with various loop sizes [54]. These GLRC peptides (Table 3) are made of only amino acids G, L, R, and C. Of the four such peptides, two showed higher anti-HIV activity than GLR-19. GLRC-2, the most potent peptide (EC50 0.8 μM), has a disulfide bond between C4 and C16 (Table 3). It also has an inhibitory effect on herpes simplex virus type-2 (HSV-2) [54]. Our work illustrates that one can improve peptide activity step by step based on database-derived knowledge [8].
While GLRC-2 is most effective against HIV-1, the most potent antibacterial peptide is GLRC-3 (MIC 7.5 μM). It is evident that the best antibacterial and antiviral peptides possess different loop structures. Interestingly, the most potent anti-HIV peptide GLRC-2 is also most stable to the action of chymotrypsin (Table 3). One possible interpretation is that the polypeptide chain of GLRC-2 could be more compact than GLRC-3.
|Table 3. Database-guided design of anti-HIV peptides 1.
|Peptide name
|Amino acid sequence
|Design strategy
|EC50 (μM)
|MIC (μM)
|Stability (% left)
|K to R
1 The changed amino acids are in bold. Data taken from refs. [9,52,54]. EC50 and MIC are used as a measure of peptide potency against HIV-1 and E. coli K12. NE stands for "not evaluated". For additional description of the table, see the text.
3.3.2. Ab Initio Design of Anti-MRSA Peptides
It is also possible to design novel peptides based entirely on the APD [55]. We referred to this unique method as ab initio design to distinguish it from the de novo methods above. Our design comprises two major modules. The first module consists of an activity filter that allows us to obtain a list of peptides with desired activity (e.g., antibacterial, antifungal, antiviral, antiparasitic, or anticancer). To obtain compounds to kill S. aureus, we retrieved all AMPs with activity against Gram-positive bacteria as model peptides. The second module is composed of a series of parameter filters. In each step, we followed the most probable principle to derive the needed peptide parameters from the set of the model candidates. For example, a plot of peptide entries as a function of net charge showed that the largest peptide group in the 268 AMPs had a net charge of +1. This was translated into one lysine for the peptide. Likewise, the hydrophobic content of the peptide (61–65%) was also determined, leading to eight leucines for a 13-residue peptide. This peptide length was also determined by the database. These operations led to 1K, 2G, 2S, and 8L. Although such an amino acid composition could generate many different sequences, the possible candidates reduced dramatically once most probable peptide motifs came into consideration. In this way, we arrived at a unique peptide sequence dubbed DFTamP1, the first AMP designed based on the database filtering technology [55]. DFTamP1 possessed the activity and structure as designed. It is active against S. aureus, but not E. coli, B. subtilis, or P. aeruginosa. DFTamP1 is able to kill MRSA USA300 rapidly, suggesting membrane targeting. The membrane targeting is consistent with our observation [55] that the two versions of a similar peptide synthesized in all L- or d-amino acids are equally active [66,67]. These results set the stage for determining the active conformation in membrane-mimetic micelles by NMR spectroscopy (see below). DFTamP1 has indeed an amphipathic helical structure, ideal for membrane binding. In particular, the hydrophobic surface, consisting of a cluster of eight leucines, is critical for bacterial killing as substitutions of leucines with either valines or isoleucines led to poor activity and reduced solubility in the isoleucine case [55]. It seems that there is a good reason for nature to choose those abundant amino acids for AMP design (Table 1).
3.4. Template-Based Design and Optimization
It is feasible to design new AMPs based on known peptide templates collected in the APD. In one example, we succeeded in converting a non-toxic membrane anchor to an antibacterial peptide [50]. The starting peptide template commences with GLFD and can form a three-turn amphipathic helix covering residues 1–10 [68,69]. A search of the APD database led to a family of AMPs that share the N-terminal GLFD sequence with non-toxic membrane anchor. A representative member is the antimicrobial and anticancer peptide aurein 1.2 isolated from an Australian frog [70]. Aurein 1.2 with 13 residues was found to be entirely helical in membrane-mimetic organic solvents [70] or micelles [50]. In analog to aurein 1.2, the non-toxic bacterial membrane anchor gained antibacterial activity when D13 was changed to F13. It seems that tuning peptide length offers a mechanism for controlling peptide function.
As a second example, we also aimed to identify the minimal active region in a longer peptide. Human cathelicidin LL-37 is a 37-residue peptide starting with a pair of leucines. Our studies produced a library of model peptides with distinct antimicrobial activity. KR-12 is identified as the minimal antibacterial peptide corresponding to residues 18–29 of human LL-37. It is active against E. coli but not HIV-1 [56,71]. However, FK-13, a core antimicrobial peptide with one more phenylalanine at the N-terminus than KR-12 [72], is active against both HIV-1 and E. coli. Interestingly, the reverse sequence of FK-13 (retro-FK13) [73] is active against E. coli, but not HIV-1 [56]. These results underscore the importance of both F17 and sequence order for HIV inhibitory activity. While LL-23, the N-terminal fragment of LL-37, is inactive against HIV-1, GF-17, a central fragment of human LL-37 corresponding to residues 17–32 [72], is both bactericidal and virucidal [56,74]. Actually, GF-17 is the major antimicrobial peptide of LL-37. However, introduction of 1 to 2 d-amino acids to GF-17 abolished its anti-HIV activity [56]. In contrast, the same sequence remained antibacterial even after incorporation of three d-amino acids (i.e., GF-17d3). NMR analysis revealed a novel non-canonical amphipathic structure for GF-17d3 [72]. Thus, anti-HIV activity of these LL-37 fragments requires a specific sequence as well as the helical structure [56]. Further extending the sequence of GF-17 to GI-20 (corresponding to residues 13–32 with the positions between I13 and G14 swapped) generated an anti-HIV peptide with the best therapeutic index [56,75]. Our observation is consistent with subsequent mechanistic studies using similar LL-37 fragments, which suggest the association of these fragments with HIV-1 reverse transcriptase in vitro [76]. In contrast, antibacterial activity of those LL-37 fragments can be attributed to membrane disruption, which does not require a defined peptide sequence or structure. This example, as well as the results from our database screening and de novo design discussed above, indicates the requirements of different peptides to target HIV-1 or pathogenic bacteria.
4. Differences in Binding Targets and Mechanisms of Action of AMPs
AMPs can be broadly classified into two families: cell surface targeting peptides and intracellular targeting peptides [26]. Cell surface-targeting peptides, including both membrane-targeting and non-membrane targeting peptides, can be further classified based on specific targets such as cell wall/carbohydrates, lipids/membranes, and proteins/receptors. Likewise, intracellular targeting AMPs can be further classified based on the specific target molecules (e.g., heat shock proteins, DNA, and RNA).
In the APD, 332 and 163 entries were annotated as AMPs only against Gram-positive and Gram-negative bacteria, respectively. This species specificity of AMPs to a large extent can be ascribed to the cellular differences (cell wall, membranes, and other molecules) between these two types of bacteria [77,78]. While lipopolysaccharides (LPS) are one important component in the outer membranes of Gram-negative bacteria, lipid II is an important precursor in the cell wall synthesis of Gram-positive bacteria. Our APD search identified 16 lipid-II targeting AMPs [9] that are able to inhibit cell wall synthesis [79,80,81]. These cell-wall active peptides may and may not be active against Gram-negative bacteria. If they do, they usually work by a different mechanism. For example, HNP-1 targets S. aureus cell wall but membranes of E. coli [82]. In line with this observation, two mirror-imaged peptides (i.e., synthesized using either all l- or all d-amino acids) [66,67] showed identical activity against E. coli but not S. aureus. Interestingly, HNP-1 binds to HIV-1 gp-120 glycoprotein (a lectin), but shows inhibitory effects after HIV-1 entry [83,84]. Thus, the demonstration of the association of an AMP with certain molecule from a pathogen in vitro may not necessarily mean that it is also responsible for in vivo killing [26].
The fundamental differences between bacteria and human cells, at least at the membrane level, are clearly significant from the standpoint of cell selectivity of AMPs. While bacterial membranes are rich in anionic lipids such as phosphatidylglycerols (PGs), human cell membranes are dominated by phosphocholines (PCs) and also contain cholesterol [85,86,87,88]. This explains why cationic AMPs are emphasized in the current research because they have preference for anionic bacterial membranes.
There are numerous biophysical techniques that can lead to useful mechanistic information. Confocal microscopy can be used to follow the location of a fluorescent tagged peptide in cells. Surface or intracellular locations can be judged directly from the images. Using this technique, magainin 2 was found to be located on the bacterial surface to form a pore, while buforin II was found to be within the cell. Further in vitro studies by gel retardation demonstrated its association with DNA [89]. For this cell-penetrating peptide, DNA binding could be important for killing.
The mode of peptide-membrane interactions appears to play an important role in bacterial killing [90]. Lipid vesicles are useful models to provide insight into peptide-membrane interactions [74,91,92]. While magainin 2 induced lipid flip-flop and caused dye leakage in lipid vesicles, buforin II did not [92]. In particular, the proline in buforin II is critical for membrane penetration [93]. Based on this observation, it may not be surprising that proline-rich peptides such as drosocin, pyrrhocoricin, and apidaecin can also cross the membranes of Gram-negative bacteria and associate with heat shock proteins as shown by mass spectrometry, Western blot, and fluorescence polarization experiments [65]. However, these proline-rich peptides appeared to bind to LPS first. Consistent with protein binding, a peptide analog of pyrrhocoricin synthesized in all d-amino acid was inactive [65]. Electron microscopy (EM) is also a powerful tool that can be used to view whether the cell is intact, damaged or lysed [55,94,95]. Indicator molecules can be used to report membrane permeation or disruption by flow cytometry [96]. For example, GF-17, the major antimicrobial region of human antimicrobial peptide LL-37, can disrupt bacterial membranes based on the fluorescence increase due to the association of non-permeable dye with DNA [74].
Human LL-37 is an important cationic defense peptide that has been extensively studied by various biophysical techniques. To provide insight into the action of the peptide, especially on membranes, polarized FT-IR and solid-state NMR techniques were utilized to indicate a surface location of the peptide on lipid bilayers [97,98]. Interestingly, Huang detected two states for LL-37 using oriented circular dichroism (OCD) and neutron in-plane scattering [99]. In the first state, LL-37 is parallel to the membrane surface as also observed by other techniques; in the second state, the peptide is perpendicular to the membrane corresponding to a pore formation state. A peptide orientation parallel to the membrane surface would support the carpet model [87,88], while an orientation perpendicular to the membrane surface suggests pore formation [99]. However, such results obtained by different techniques under different conditions are not always easily reconciled. In addition, prior to our work, outstanding structural questions remain. Which region of LL-37 is involved in membrane binding? Is there any basic residue important for this binding? What is the basis for cooperative binding of LL-37 to LPS? We addressed these questions by high-resolution NMR studies (see below) [100].
5. Structural Annotation, Classification and Determination of AMPs
5.1. Structural Annotation and Classification
Once the molecular target has been elucidated by various experiments above, one feels like to examine in details how the AMP in action looks like. While FT-IR, circular dichroism (CD), and fluorescence spectroscopies only provide low-resolution information, high-resolution techniques such as NMR spectroscopy and X-ray diffraction are able to reveal the atomic details of a peptide in water (e.g. distinctin and defensins), binding to membranes or in complex with other molecular targets. Structural information for each AMP is also annotated in the APD [8]. It is clear that AMPs can adopt a variety of structural scaffolds (Figure 4) [100,101,102]. By following the link for each peptide entry in the APD, one can view the 3D structure of the peptide if it is deposited in the Protein Data Bank. In March 2013, there were 288 peptides with known 3D structures in the APD database. Of these, 260 were solved by NMR and 28 by X-ray crystallography. It is evident that more AMP structures are determined by NMR spectroscopy than the X-ray method [8,9]. A possible reason for this may be the difficulty in obtaining crystals because many linear AMPs are disordered in water and only become ordered in the presence of membrane-mimetic models. For solution NMR studies, micelles are usually preferred, although organic solvents are also used. Rapid tumbling of small micelles in solution averages out chemical shift and dipolar coupling anisotropies, leading to high-resolution NMR spectra for structural determination. The commonly used micelle models are sodium dodecylsulfate (SDS) and dodecylphosphocholine (DPC) [75,103,104,105,106,107]. While dihexanoyl phosphatidylcholine (DHPC) is occasionally utilized [108,109], dioctanoyl phosphatidylglycerol (D8PG) should better mimic bacterial membranes than SDS [110,111,112,113]. This D8PG model is unique because arginine side chain amide signals are well resolved, allowing for the detection of arginine-PG interactions by 2D NMR spectroscopy [75]. A more complete description of membrane models and NMR methods can be found elsewhere [113].
In the APD, various three-dimensional structures [50,71,114,115,116,117,118,119,120,121,122,123] are classified into four families: α, β, αβ, and non-αβ (see Figure 4) based on the presence or absence of secondary structures such as α-helix and β-sheet [26]. The α family consists of AMPs with α-helical structures (Figure 4, Panels A–D). Only a few peptides (e.g., distinctin) possess a helical structure even in water due to the stabilization of disulfide bonds (Panels C and D). Many AMPs are only helical in complex with bacterial targets such as membranes (Panels A and B). The β family is characterized by the formation of β-sheet structures (panels E-H). The αβ family contains both α-helical and β-sheet structures (panels I-K). Multiple disulfide bonds in these molecules are responsible for the observed structure in aqueous solution. Examples are various defensin-like molecules from fungi, plants, mussels, monkeys, and humans (Figure 4). Although there are few examples, the non-αβ family consists of neither α-helices nor β-sheet structure (panel L). A more complete and updated list of AMPs from each structural family can be obtained in the APD database [8,9].
5.2. Structural Determination of Database-Designed Peptides by the Improved 2D NMR Method
Some types of AMPs are rich in amino acids such as prolines and histidines. These peptides could pose problem for structural determination by the traditional proton-only 2D NMR method. One alternative approach is to use the improved 2D NMR, which was first demonstrated in 2005 [50]. The major experiments for the traditional and improved 2D NMR methods are listed in Table 4. It is clear that additional experiments are conducted in the case of the improved 2D NMR method. In particular, heteronuclear correlated experiments are recorded at natural abundance to extend the measurements beyond 1H. Our measurements of 13C and 15N chemical shifts at natural abundance are cost effective because one can avoid the cost in producing isotope labeled polypeptides [100]. In the following, we use two recent examples to illustrate the merits of the improved 2D NMR method for structural determination of small AMPs.
|Table 4. Key experiments for traditional and improved 2D NMR methods.
|Traditional 2D NMR method [103]
|Improved 2D NMR method [50]
|TOCSY, DQF-COSY, and NOESY
|TOCSY, DQF-COSY, and NOESY
|Natural abundance (1H, 13C) HSQC
|Natural abundance (1H, 15N) HSQC
The first example is DFTamP1, an anti-MRSA peptide designed in 2011 based on our database [55]. Of the 13 amino acids in this peptide, there are eight leucines (61.5%), which appeared in similar spectral regions. The spectral overlap made some proton cross peaks unresolved, leading to a low-resolution structure (Figure 5, panels A and C; Wang, unpublished data). To improve spectral resolution, we recorded heteronuclear single quantum coherence (HSQC) spectra at natural abundance (only 1.1% for 13C and 0.36% for 15N). Both 13C and 15N heteronuclei have much broader chemical shift ranges than 1H, making it possible to measure different carbon or nitrogen chemical shifts even when the proton chemical shifts are similar. The inclusion of heteronuclear chemical shifts derived angle restraints into structural calculations further defined the structural ensemble, leading to consensus helical structures (Figure 5, panels B and D).
As a second example, another group also utilized the improved 2D NMR [50] to re-determine the structure of θ-defensins such as RTD-1 in 2012 [117]. RTD-1 is the first circular AMP found in mammals [32]. This peptide is rich in both arginines and cysteines (sequence: GFCRCLCRRGVCRCICTR). As anticipated, the new structure (Figure 6B,D) is better defined by using the improved 2D NMR method than that (Figure 6A,C) determined previously by the traditional proton 2D NMR spectroscopy [118]. Craik and colleagues discussed that "Earlier structure calculation on RTD-1 and HTD-2 did not have access to the 13C and 15N HSQC information, leading to the suggestion that the θ-defensins were flexible". In the abstract, they concluded that "the θ-defensins are more rigid and structurally defined than previously thought" [117]. The rigid structure of circular θ-defensins is stabilized by three pairs of parallel disulfide bonds, leading to a ladder-like structure.
These two examples in Figure 5, Figure 6 represent AMPs from different structural families: α-helix for DFTamP1 [55] and β-sheet for RTD-1 [117,118]. Furthermore, these structures were determined in different environments. While RTD-1 was determined in an aqueous solution, DFTamP1 was determined in the presence of membrane-mimetic SDS micelles. Although these two structures were determined by different laboratories, the outcomes are remarkably similar—both pointing to the advantage of the improved 2D method [50] that gave more precisely defined peptide structures. In both cases, the TALOS program [125] was utilized to convert a set of heteronuclear chemical shifts (1H, 15N, 13Cα, and 13Cβ) into backbone angles for structural refinement. Protein dynamics analysis confirmed that the well-defined structural regions are indeed rigid (i.e., less mobile) [71,117,126]. These examples indicate that the improved 2D NMR is able to generate the correct structures for these small AMPs with a biased amino acid composition, thereby avoiding the publication of misleading structure and dynamics.
5.3. Beyond 2D NMR
Although the improved 2D NMR method [50] led to impressive results (Figure 5, Figure 6), it is important to be aware of its limitations. When there is too much spectral overlap, it is necessary to use 3D heteronuclear NMR techniques. This is exactly the case with the structural determination of human cathelicidin LL-37 in membrane-mimetic micelles. Isotope labeling of LL-37 was achieved by expressing the peptide in E. coli in a chemically defined minimal medium containing 15N-NH4Cl and 13C-glucose followed by formic acid cleavage and chromatographic purification [127,128]. This isotope-labeled LL-37 enabled the collection of 3D triple-resonance NMR experiments [129], where the crowded proton cross peaks were resolved by separating them onto different 2D planes along the third 15N or 13C dimension. The excellent spectral resolution enabled us to determine the LL-37 structure to high quality [71]. Our high-quality structure of human LL-37 in membrane-mimetic micelles is entirely consistent with backbone dynamics (ps-ns time scale) of the polypeptide chain, further validating the structure. In addition, we found essentially the same structure for LL-37 in SDS and D8PG micelles. We also obtained a similar structure for human innate peptide LL-23 in SDS, DPC and D8PG micelles [107]. These results suggest that it is spectral resolution, rather than the micelle model, that led to the differences in the two structures of LL-37 determined by 2D [130] and 3D NMR [71] techniques.
Our high-quality LL-37 structure provides a solid basis for understanding the interactions of this important human cationic peptide with anionic bacterial membranes. The long amphipathic helix covering residues 2–31 of LL-37 (Figure 4B) is required for interactions with both LPS (one of the major component in Gram-negative bacterial outer membranes) and PGs (major anionic lipids in bacterial inner membranes) [71]. Our introduction of the D8PG model [110,111,112,113] allowed for the first detection of aromatic-PG and arginine-PG interactions by solution NMR spectroscopy, indicative of the importance of both electrostatic and hydrophobic interactions between the cationic peptide and anionic lipids [112]. Interestingly, the same arginine R23 (not all arginines) that contacts PGs is also the most important one for antibacterial activity, membrane permeation and lipid clustering [74]. A recent study suggests that R23 is also important for interactions with DNA quadruplex [131]. The cooperative binding of human LL-37 with LPS observed earlier by Lehrer and colleagues [132] can also be understood based on the high-quality 3D structure of LL-37—a unique serine at position 9 splits the hydrophobic surface of the long amphipathic helix into two domains [71,107]. These two domains can work together to better bind to LPS. Our high-quality structure also serves as a model to understand the structure-activity relationship of homologous primate cathelicidins. For example, a helical bend was mapped to residues 14–16 in the LL-37 structure. In the same region of homologous cathelicidins, as many as three glycines are possible [71]. Our studies also provide insight into the LL-37 oligomerization [97]. First, the elution times of a series of proteins on a size-exclusion column suggest a tetramer. Second, NMR data suggest the involvement of residues 2–36 in tetramer formation [100,127].
AMPs can use a variety of molecular targets and not all these targets are amenable to NMR studies. Under these circumstances, one may consider other biophysical techniques such as X-ray diffraction. For example, the structure of a Pro-rich peptide in complex with heat shock protein has recently been determined [133]. This structure laid a solid foundation for structure-based design of novel antibiotics.
In summary, this section emphasizes the importance of choosing a proper method for structural determination of AMPs to avoid structural artifacts. In many cases where the spectra are well resolved, the traditional 2D NMR method [103] is sufficient. For example, we found it redundant to include heteronuclear chemical shift-derived restraints into the structural calculations of aurein 1.2 (Figure 4A) because of many well-resolved proton NMR restraints [50]. However, under certain circumstances such as those amino acid-rich peptides in Figure 5, Figure 6, the improved 2D NMR method [50] is no doubt important to produce a correct structure. We recommend the use of the improved NMR method for structural determination of small AMPs because it helps improve and validate the structure [113]. Note that the high-quality structure of LL-37 was only made possible via 3D NMR studies, because 2D NMR was insufficient in this case [71,72]. Advanced NMR and/or X-ray techniques should be considered for more complicated structural problems involving AMPs.
6. Concluding Remarks
Nature keeps inspiring us in discovering new medicine from the ancient time to the modern era [134,135]. Antimicrobial peptides have been recognized as critical defense molecules to ward off invading pathogens thanks to the pioneering work of the forerunners who took tremendous efforts in isolating and characterizing many natural AMPs in the classic way [5,6,7,31,35,36,42,43]. Several databases have been built to manage this information [8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26]. The antimicrobial peptide database [8,9] is a leading resource for nomenclature, classification, information search, statistical analysis, prediction and design of AMPs. The APD database has been expanded substantially as a consequence of regular update and continued developments. The growing amount of information and extensive annotation increases the possibility of identifying therapeutic molecules of interest. Based on the APD, we have demonstrated various approaches for peptide discovery [52,53,54,55,56,136]. Our database screening [52,53] appears to have a higher success rate than combinatorial library screens in identifying antimicrobials [137,138,139]. This may not be surprising because AMPs have been optimized in nature as host defense molecules, whereas randomness has to be introduced in artificial libraries even though the amino acids used are biased. An outstanding feature of the APD database is that it is composed of multiple information filters that can be assembled in numerous ways to extract the needed information. Such a database feature enabled us to conduct both step-by-step database-guided design of anti-HIV peptides and ab initio design of anti-staphylococcal peptides based on the database filtering technology. The sequence space of the designed peptides can be further explored via sequence shuffling or amino acid mutations [51,52]. Alternatively, the identified peptide may be further optimized by screening a synthetic peptide library [56] or conducting in silico screening [45]. In silico screening is preferred as it is cost effective. However, the scope of its applications may be limited because each program only shows peak performance on sequences that resemble those being trained. Also, it remains experimentally validated whether the inactive sequences in the training set are truly inactive. Therefore, one can anticipate that experimental screening and validation of peptide candidates will continue, although it can be time consuming, labor extensive, and expensive.
Recently, there is a growing interest in AMP prediction. This is because it is cheaper to identify AMPs from the genome than to isolate AMPs from natural sources. The APD programmed a peptide calculation and prediction interface in 2003 [8]. Our database data set is frequently used in developing other prediction methods [140,141,142,143,144]. Some known prediction protocols are also programmed into recent databases such as CAMP and DAMPD [10,11]. In AMPer, the information from both the mature peptide and precursor sequences were utilized [145]. While it is pre-mature to correctly predict every possible AMP gene from a genome, such prediction programs may be helpful to locate potential genes followed by experimental validation. Here the prediction can be based on the sequence of conserved modifying enzymes or the genomic context depending on the amount of information involved in the program [18]. This approach plays an important role in identifying novel bacteriocins in bacterial genome [146]. There are also programs that predict AMPs by scanning known protein sequences [147]. Whether the predicted peptides are actually generated for biological defense is not clear.
Both peptide screening and de novo design suggest different requirements for AMPs depending on the target pathogens (or more exactly molecular targets). While sequence order and the helical structure of LL-37 fragments are less important in killing E. coli due to membrane targeting [74], such features are essential in inhibiting HIV-1 due to interactions with the viral reverse transcriptase [76]. It is evident that peptide length is also an important factor in determining peptide activity spectrum [50,56]. While the full length LL-37 is implicated in cancer and became less effective against parasites or superbugs such as MRSA USA in vitro, its central fragments are effective in killing cancer cells or pathogens [74,148,149]. In addition, one can also improve cell selectivity by maximizing bacterial targeting and minimizing toxic effects on human cells [150,151,152]. In our database design, it is possible to insert a selectivity filter after activity and parameter filters [55]. The basis for this design has been laid since the APD annotated the cytotoxicity data of AMPs from the first version [8]. Juretic and colleagues designed selective glycine-rich peptides based on a library of helical AMPs from amphibians [85]. As we proposed previously, modulating peptide hydrophobicity provides a general approach for improving peptide cell selectivity, although there are various methods to achieve incoherent hydrophobic packing [72].
Whatever the sources of the peptide templates (isolation from natural sources, de novo peptide design, or in silico prediction from the genome), they are all subject to stability tests as a requisite for practical use. The differences in amino acid composition and structural scaffold in the case of database-designed anti-HIV and anti-MRSA peptides required different strategies to improve peptide stability to proteases. We achieved this by introducing a disulfide bond in the case of anti-HIV peptides (Table 3) [54], and non-standard d-amino acids [55] in the case of database designed peptides. Our design was mainly guided by the APD database [9,136], which also annotates nature's post-translational modification strategies for AMPs (for a review, see ref. [153]). These chemical modification strategies should inspire peptide engineering to make natural AMPs more druggable. It is useful to point out that peptide stability and activity are not necessarily always programmed into the same molecular form. While the disulfide bond of distinctin is required for stability (peptide storage) but not activity [114], human beta defensin-1 (HBD-1) is more active after disruption of the disulfide bonds [154]. For those AMPs with a stable scaffold that is already protease-resistant (e.g., cyclotides), much effort is now spent on conferring the desired activity to the molecule by sequence mutation or grafting [155,156,157]. A careful structural determination is highly recommended because high-quality structures form the basis for us to interpret biology correctly and to conduct rational peptide design more effectively. In the end, it is useful to mention two nature's antibiotics. Gramicidin, with a mixture of L- and D-type amino acids, is the first peptide antibiotics approved for clinical use [2]. Daptomycin, a cyclic anionic lipopeptide discovered in the 1980s, was approved by the FDA in 2003 to treat skin infection [158,159,160]. These successful peptides with multiple chemical modifications should also inspire the development of natural compounds into a battery of new therapeutic molecules.
The author thanks Zhe Wang, Biswajit Mishra, Joseph Menousek (UNMC), Karen Buckheit, and Robert Buckheit (ImQuest Biosciences) for their contributions to the database-related work described here. Continued support from the author's Institution as well as the State of Nebraska is greatly appreciated.
Conflict of Interest
The author declares no conflict of interest.
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The CEO of our company just told a group of veteran employees that he is requiring them to register for a Six Sigma certification course at the EMPLOYEE'S expense. He wants the employees to be registered for their chosen course in two weeks.
This training has never been required before. Sure, it will benefit both the employee and the employer, but the deadline is very sudden and the cost to the employee is high (over $500 each, at least).
We are in Texas, and of course do not have any sort of employment contract. The employees in question are all hourly non-exempt. All of these employee have been employed at this company for over a year, and there are not expectations in our job descriptions, offer letters, etc. for this type of thing.
Bottom line, can the boss require this at the employee's expense?


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Off Topic Sunday: October 20
Yesterday I was asked to shoot one of the new choirs at Phoenix Children's Chorus. This is an elite group of singers that are available for gigs in the valley. This is one of my favorites from the shoot.
I am going through a simplification moment in my life. Looking to do more with less, and not being enamored of every shiny object that comes along. Whether photography or design, I am taking a more simple approach and looking to let go of things that are un-needed or simply take too much time and attention. This post at Unclutterer caught my attention.
"Using simple tools, when that's all you need.
I used to be intrigued by all the fancy apps for creating and managing to-do lists, and those apps certainly make sense for some people. But at some point, I realized that for me, a simple text file was sufficient, and going back to that basic tool made my life easier. Sometimes extra features are a distraction, not a benefit."
The Italian Opera composer, Puccini could never have known that one of his arias would become a world favorite, and THE audition piece for every male opera singer to ever hit a "talent" TV show. The great tenor Pavaortti made the song a household tune in the late 90's.
From Wiki:
" "Nessun dorma" achieved pop status after Luciano Pavarotti's 1972 recording of it was used as the theme song of BBC television's coverage of the 1990 FIFA World Cup in Italy. It subsequently reached #2 on the UK Singles Chart Although Pavarotti rarely sang the role of Calaf on stage, Nessun dorma became his signature aria and, in turn, a sporting anthem in its own right, especially for football.[5]Pavarotti gave a rendition of "Nessun dorma" at his final performance, the finale of the Opening Ceremony of the 2006 Torino Winter Olympics, although it was later revealed that he had lip-synched the specially pre-recorded performance (at the time of his Winter Olympics appearance Pavarotti was physically incapable of performing as he was suffering from pancreatic cancer to which he succumbed the year following). His Decca recording of the aria was played at his funeral during the flypast by the Italian Air Force.[7] In 2013, the track was certified gold by the Federation of the Italian Music Industry."
In 1998, Pavarotti was scheduled to sing at the Grammy's, but at the last moment became quite ill. The show's producers, knowing that Aretha Franklin was attending the show, asked her to sing in Pavarotti's stead. She agreed, and this magical moment occured – live on TV.
While some Opera "aficionados" decried the performance because of the liberties Aretha took, many others welcomed this expression and personally approached rendition as something the classical world could certainly embrace.
I agree. Why not allow the performers to bring something new, their own spin to the songs we have allowed to go unchanged for decades. Jazz players do it. Pop music does it. Why not classical?
I am not suggesting adding a disco beat to the Verdi Requiem, I am happy with letting voices of renown add their color and personal attention to the performance. Perhaps one day we could look forward to hearing Sting in Pagliaci… yeah, that would be cool.
One of John Coltrane's most celebrated masterpieces was "A Love Supreme". (Wiki) It is one of the most highly regarded jazz albums of all time, and sold over 500,000 copies in Trane's lifetime… and hundreds of thousands since then as well.
Here is the incredible Branford Marsalis doing "A Love Supreme" and doing it supremely well.
And this may be actually a bit on topic, but it is very nice little look at Photoshop's Patch Tool.


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Bella's Desserts
Serving Southeast PA, Southern NJ and Northern Delaware
Rabbit Run Lane
Glenmoore, PA 19343 | map
Glenmoore, PA 19343 | map
Website: Visit my Website »
Blog: Visit my blog »
Desserts: Cookies, Cupcakes, Dessert Buffet, Groom's Cake
Additional services: Consultations, On-site Delivery, On-site Setup, Pick...
Dietary options: Gluten Free, Lactose Free, Organic, Vegan
Health license: Yes
About Us
Welcome to Bella's, an artisan dessert company located in Chester County, Pennsylvania.
We are thrilled to offer wedding cakes, specialty cakes, and unique desserts to the discerning customer! From small and large wedding cakes, to cupcakes, mini desserts, and edible favors, we offer desserts that are unavailable anywhere else in our area! We are now offering Vegan, dairy free and gluten free cakes.
We also offer custom cookies, perfect for showers and favors! Come see our website today!
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