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Research Article Free access | 10.1172/JCI64416
1School of Optometry, University of California, Berkeley, Berkeley, California, USA. 2College of Pharmacy, Touro University California, Vallejo, California, USA. 3Programs in Vision Science, Infectious Disease and Immunity, and Microbiology, University of California, Berkeley, Berkeley, California, USA.
Address correspondence to: Suzanne M.J. Fleiszig, University of California, Berkeley, 688 Minor Hall, Berkeley, California 94720-2020, USA. Phone: 510.643.0990; Fax: 510.643.5109; E-mail: email@example.com.
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1School of Optometry, University of California, Berkeley, Berkeley, California, USA. 2College of Pharmacy, Touro University California, Vallejo, California, USA. 3Programs in Vision Science, Infectious Disease and Immunity, and Microbiology, University of California, Berkeley, Berkeley, California, USA.
Address correspondence to: Suzanne M.J. Fleiszig, University of California, Berkeley, 688 Minor Hall, Berkeley, California 94720-2020, USA. Phone: 510.643.0990; Fax: 510.643.5109; E-mail: firstname.lastname@example.com.
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1School of Optometry, University of California, Berkeley, Berkeley, California, USA. 2College of Pharmacy, Touro University California, Vallejo, California, USA. 3Programs in Vision Science, Infectious Disease and Immunity, and Microbiology, University of California, Berkeley, Berkeley, California, USA.
Address correspondence to: Suzanne M.J. Fleiszig, University of California, Berkeley, 688 Minor Hall, Berkeley, California 94720-2020, USA. Phone: 510.643.0990; Fax: 510.643.5109; E-mail: email@example.com.
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1School of Optometry, University of California, Berkeley, Berkeley, California, USA. 2College of Pharmacy, Touro University California, Vallejo, California, USA. 3Programs in Vision Science, Infectious Disease and Immunity, and Microbiology, University of California, Berkeley, Berkeley, California, USA.
Address correspondence to: Suzanne M.J. Fleiszig, University of California, Berkeley, 688 Minor Hall, Berkeley, California 94720-2020, USA. Phone: 510.643.0990; Fax: 510.643.5109; E-mail: firstname.lastname@example.com.
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Published September 24, 2012 - More info
In addition to its role in refraction, the cornea forms a barrier between the eye and environmental and infectious insults. Corneal infections are surprisingly rare, suggesting that multiple aspects of the immune system are at play in mediating protection. In this issue of the JCI, Tam et al. describe the unexpected role of a structural protein, cytokeratin 6A, in this process.
Michael Zasloff
Epithelial cells express antimicrobial proteins in response to invading pathogens, although little is known regarding epithelial defense mechanisms during healthy conditions. Here we report that epithelial cytokeratins have innate defense properties because they constitutively produce cytoprotective antimicrobial peptides. Glycine-rich C-terminal fragments derived from human cytokeratin 6A were identified in bactericidal lysate fractions of human corneal epithelial cells. Structural analysis revealed that these keratin-derived antimicrobial peptides (KDAMPs) exhibited coil structures with low α-helical content. Synthetic analogs of these KDAMPS showed rapid bactericidal activity against multiple pathogens and protected epithelial cells against bacterial virulence mechanisms, while a scrambled peptide showed no bactericidal activity. However, the bactericidal activity of a specific KDAMP was somewhat reduced by glycine-alanine substitutions. KDAMP activity involved bacterial binding and permeabilization, but the activity was unaffected by peptide charge or physiological salt concentration. Knockdown of cytokeratin 6A markedly reduced the bactericidal activity of epithelial cell lysates in vitro and increased the susceptibility of murine corneas to bacterial adherence in vivo. These data suggest that epithelial cytokeratins function as endogenous antimicrobial peptides in the host defense against infection and that keratin-derived antimicrobials may serve as effective therapeutic agents.
Exposed tissue surfaces, including skin and the mucosa, are under constant threat of attack by opportunistic and pathogenic microorganisms. Multilayers of epithelial cells lining this host-microbial interface form a defense barrier to prevent microbe penetration (1). Significant effort has been focused on understanding how epithelial cells participate in inflammatory responses when infectious disease occurs or during other forms of tissue challenge (2). It has been shown that Toll-like and Nod-like receptor-mediated innate defense systems allow epithelial detection of extracellular or intracellular microbes via conserved antigens, with resultant expression of cytokines and chemotactic signaling to recruit and activate cellular immune defenses (3–7). Less is known about how epithelia resist infection during health, i.e., under "constitutive" circumstances, except that they provide a formidable barrier.
As an example, the healthy human cornea resists epithelial penetration by virtually all microbes, and infection requires significant (deep-penetrating) damage or contact lens wear (8, 9). In animal models, even large inocula of potentially pathogenic bacteria are rapidly cleared from the ocular surface when it is healthy (10–12). Various antimicrobial peptides are expressed at ocular, and other, surfaces of humans and animals. While some (e.g., hBD-1) are constitutively expressed, many, including most β-defensins (hBD-2, hBD-3, and hBD-4) and cathelicidin LL-37, require the activation of innate defense responses for expression and secretion (13, 14).
Here we tested the hypothesis that human corneal epithelial cells contained undiscovered antimicrobials that were expressed constitutively and involved in defense against infection. Systematic fractionation of these cells, combined with mass spectrometry, revealed a series of glycine-rich C-terminal peptides of human cytokeratin 6A (hK6A) (13– to 26–amino acids). Synthetic analogs of the naturally occurring peptides were bactericidal against multiple bacterial pathogens and cytoprotective against Pseudomonas aeruginosa. Accordingly, knockdown of cytokeratin 6A (K6A) significantly reduced bactericidal activity of corneal epithelial cell lysates, and it increased bacterial adherence to the murine ocular surface. Structure-activity studies showed that glycine residues were required for activity and that the mechanism involved direct binding to bacteria and cell wall/membrane permeabilization. We believe that these data suggest a novel function for keratin proteins in innate defense against bacterial challenge.
K6A contributes to the endogenous antimicrobial activity of corneal epithelial cells. A telomerase-immortalized human corneal epithelial (hTCEpi) cell line, capable of differentiation, stratification, and desquamation similar to normal epithelium (15), was used to mine for constitutively expressed antimicrobial peptides. Calcium-induced differentiated cells were lysed and fractionated by molecular weight, and fractions were screened for bactericidal activity against Pseudomonas aeruginosa, a common Gram-negative opportunistic pathogen. For the laboratory isolate PAC1R, lysate fractions containing molecules of less than 3 kDa were most effective in killing bacteria (Figure 1A). For clinical isolates 6294 and 6206, both the <3-kDa and the 3- to 10-kDa fractions were found to be bactericidal in a concentration-dependent manner (Figure 1, B and C).
Keratin peptide derivatives are major constituents of the antimicrobial fractions of human corneal epithelial cell lysates. (A) Crude lysates were serially fractionated by size to yield 4 fractions: >100 kDa, 10–100 kDa, 3–10 kDa, and <3 kDa (black). Crude lysates and the small-molecule <3-kDa lysate fraction showed bactericidal activity against P. aeruginosa (strain PAC1R). Culture media was fractionated in parallel to serve as controls for the corresponding lysate fractions in the bacterial killing assays (white). (B and C) Similar results were observed for P. aeruginosa clinical isolate (B) 6294 (invasive strain) and (C) 6206 (cytotoxic strain) except that 3- to 10-kDa lysate fractions were also found to be bactericidal. Dilution (1:3) (gray) reduced activity, demonstrating that antimicrobial activity was concentration dependent. (D) The <3-kDa and 3- to 10-kDa fractions both protected epithelial cells against P. aeruginosa internalization. (E) The <3-kDa fraction protected epithelia from bacterial-induced cell death (indicated by trypan blue–staining).
To examine the functional significance of antimicrobial activity in epithelial cell lysates, lysate fractions were tested for their capacity to impact virulence mechanisms of P. aeruginosa. Both the <3-kDa and the 3- to 10-kDa small-molecule lysate fractions protected cultured human corneal epithelial cells against invasion by P. aeruginosa in vitro, again in a concentration-dependent manner (Figure 1D). The <3-kDa fraction also protected cells against the cytotoxic activity of P. aeruginosa strain 6206 (Figure 1E).
Since previously identified endogenously expressed antimicrobial peptides are mostly approximately 3 to 10 kDa or larger in size (16), the bactericidal and cytoprotective activity of the <3-kDa fraction suggested the presence of novel antimicrobials. Thus, lysate fractions were analyzed for their peptide content using LC MS/MS (Table 1). To identify native peptides in the <3-kDa fraction, a nontrypsin-digested fraction was initially analyzed. The majority of detected fragments were derivatives of human keratins, including K5, K6A, K15, and K19. Among these, a 17–amino acid K6A peptide fragment (AIGGGLSSVGGGSSTIK, aa 534–550) was consistently detected, with or without trypsin processing (Supplemental Table 1; supplemental material available online with this article; doi: 10.1172/JCI64416DS1). Interestingly, both the N and C termini of this fragment (Ala and Lys, respectively) were flanked by a theoretical trypsin cleavage site (Arg-Ala and Lys-Tyr, respectively), suggesting that this fragment could be proteolytically released from full-length K6A, e.g., via endogenous trypsin-like serine proteases. The same 17-mer fragment was detected in the 3- to 10-kDa fraction, along with a series of overlapping peptides comprising its full or partial sequence (variants) derived from the C-terminal region (aa 515–559) of K6A. Together these peptides represented the majority of detected fragments in this fraction (Supplemental Table 2).
Mass spectrometric analysis of small-molecule cell lysate fractions
K6A contributes to ocular defense against P. aeruginosa in vivo. Consistent with a previous proteome study of the healthy human cornea (17), Western immunoblot confirmed the presence of full-length (~60 kDa) and fragmented K6A (≤1.7 kDa) in the crude lysate of these immortalized human corneal epithelial cells (Figure 2A). Interestingly, fragments of K6A were not observed in lysates of cells grown in KGM-2 containing lower calcium (0.15 mM CaCl2) (Figure 2A). The knockdown of hK6A expression using siRNA resulted in an increase of P. aeruginosa growth in the crude lysate, suggesting reduced bactericidal activity (Figure 2B). Controls showed that hK6A knockdown did not significantly impact cell density, morphology, or viability, as demonstrated by trypan blue exclusion assays (data not shown). In vivo knockdown of endogenous K6A in murine corneas (murine K6A) by approximately 25% (assessed by quantitative real-time PCR) using subconjunctival injection of siRNA was found to increase P. aeruginosa adherence to the surface of intact corneas ex vivo by approximately 5 fold (Figure 2, C and E). Controls revealed that this knockdown did not impact general barrier function, as indicated by the absence of penetration by fluorescein (Figure 2D). Collectively these data support the hypothesis that endogenous K6A and keratin-derived antimicrobial peptides (KDAMPs) serve an antimicrobial role in epithelial innate defense.
K6A contributes to endogenous antimicrobial activity of corneal epithelial cells. (A) Western blot of whole cell lysates (from direct lysis with SDS) confirmed constitutive expression of K6A and its fragments (<2 kDa) in high-calcium–treated (1.15 mM CaCl2) human corneal epithelial (HCE) cells. Fragments of K6A were not observed in lysates of cells grown in KGM-2 with lower calcium (0.15 mM CaCl2). FL, full length. (B) Antimicrobial activity of crude cell lysates against P. aeruginosa strain 6206 was attenuated by siRNA knockdown of hK6A in vitro. Knockdown efficiency was confirmed by qRT-PCR. *P = 0.007. (C) Ex vivo bacterial adherence on the corneal epithelium of intact mouse eyeballs (3-hour incubation with 1011 CFU/ml of P. aeruginosa strain PAO1-GFP) was increased significantly by in vivo knockdown of mouse K6A (mK6A) in the corneas. Knockdown efficiency was confirmed by qRT-PCR. *P = 0.0005. (D) Slit-lamp examination of mouse eyes showed that integrity of the physical barrier was not impacted by K6A knockdown, as indicated by the absence of corneal staining with fluorescein. A superficially injured (by tissue paper blotting) mouse cornea served as the positive control of fluorescein staining. (E) Combined 2-photon confocal imaging showed increased adherence of P. aeruginosa (PAO1-GFP, green) to whole corneas previously treated with siRNA for murine K6A compared with those treated with scrambled control. Without P. aeruginosa, siRNA treatments alone did not visibly affect corneal morphology. Z stacks of 1.0-μm step size (100-μm total thickness) were taken with a ×60 water-dipping lens to give an xy field of 210 μm × 210 μm.
Structural analysis of KDAMPs. ExPASy proteomic tools were used to predict peptide secondary structures, transmembrane helices, amphipathicity (hydrophobic moment), and net charges (18–21) for 8 variants of K6A (Table 2). Notably, none of the peptides tested showed sequence homology with any known mammalian antimicrobial peptides (22), but they all exhibited a predominance of glycine repeats and a common sequence, GGLSSVGGGS. Variants of the originally identified 17-mer fragment with an additional N- and/or C-terminal residue(s) (19-mer and two 18-mer peptides) were predicted to be favorable for antimicrobial activity based on predicted coil structure, potential for transmembrane helices, a hydrophobic face, and cationic charge. For example, the 19-mer peptide (aa 533–551) was predicted to have features associated with other well-recognized antimicrobial peptides, including a positive net charge and a coil structure able to form transmembrane helices. In contrast, other active KDAMPs showed variability in each of these properties except predicted coil structure. A scrambled (inactive) control of the 19-mer peptide was predicted to retain similar properties to the 19-mer, e.g., positive net charge and coil structure, but not transmembrane helix formation. The scrambled 19-mer peptide also had a lower hydrophobic moment than the active 19-mer and other active KDAMPs (Table 2).
Sequence and predicted structural characteristics of K6A-derived peptides
Antipseudomonal activity of synthetic KDAMPs. The bactericidal activity of synthetic K6A fragments was tested using a physiological salt (NaCl) concentration (150 mM) and a "lawn"/biofilm-like culture of bacteria. These conditions are known to reduce the activity of known cationic antimicrobial peptides, such as human β-defensins-1, -2, and -4 and cathelicidin LL-37 (23, 24). The 19-mer KDAMP was almost equally effective in killing the cytotoxic P. aeruginosa clinical isolate 6206 in 150 mM NaCl solution or in water, in both cases reducing bacterial viable counts by approximately 3 to 4 logs after 3 hours at 200 μg/ml (P < 0.005 in each instance) (Figure 3A). This 19-mer peptide also showed bactericidal activity against the invasive clinical isolate 6294 in physiological saline, a 97.5% reduction in bacterial viability after 3 hours at 200 μg/ml, and against a laboratory isolate PAO1, a 96.1% reduction in viability under the same conditions (P < 0.005 in each instance) (Figure 3B). A scrambled sequence control of the 19-mer KDAMP (IRGSVTISGYSGGLKGSAG) showed little or no bactericidal activity against all 5 P. aeruginosa isolates tested compared with that of the natural peptide sequence (P < 0.01) (Figure 3C). Real-time video phase-contrast microscopy revealed that 19-mer KDAMP–treated P. aeruginosa strain 6206 lost capacity for swimming motility and some appeared to aggregate, compared with untreated bacteria or bacteria treated with a scrambled peptide control (100 μg/ml, 15 minutes), in the presence of hTCEpi cells, which appeared healthy (Supplemental Videos 1–3).
Synthetic analogs of K6A-derived peptides found in corneal epithelial cell lysates are bactericidal and cytoprotective against P. aeruginosa. (A) The 19-mer was tested against P. aeruginosa strain 6206 (clinical isolate) in physiological saline (0.9% NaCl) or water and was found to be salt-tolerant (*P < 0.004, versus no peptide control). (B) The 19-mer peptide showed dose-dependent bactericidal activity against resuspended lawn culture of P. aeruginosa invasive strains 6294 (clinical isolate) and PAO1 (laboratory strain), with optimal activity at 200 μg/ml under high-salt conditions. (C) At 200 μg/ml, a scrambled control of the 19-mer peptide was inactive against P. aeruginosa. (D) Invasion of epithelial cells (strain 6294) (*P = 0.0008, versus no peptide control) and (E) cytotoxicity toward epithelial cells (strain 6206) were prevented by the 19-mer K6A-derived peptide (*P = 0.0009, versus no peptide control). (F) Synthetic variants of the 19-mer also showed dose-dependent bactericidal activity against P. aeruginosa strain 6206 (clinical isolate). Peptide as small as 10–amino acids retained bactericidal activity, despite a predicted loss of cationic charge (at pH 7.0), hydrophobic face, and ability to form a transmembrane helix (e.g., 13-mer, 10-mer). (G) Bactericidal activity of 19-mer (200 μg/ml) was only slightly affected by mutations in LPS for P. aeruginosa (strain PAC1R, serotype 03). PAC1RalgC- and PAC605 have incomplete LPS core oligosaccharide and are O antigen deficient (27), and PAC557 has a complete core but is O antigen deficient, while PAC611 has mutations only in the core (64).
Treatment of cultured human corneal epithelial cells with the 19-mer KDAMP (200 μg/ml) during challenge with P. aeruginosa protected the epithelial cells against bacterial invasion and bacteria-induced cytotoxicity as compared with controls. Bacterial invasion was reduced by 89.1%, while cytotoxicity was reduced by 85.1% (P < 0.001 in each instance) (Figure 3, D and E). Epithelial cells were not visibly impacted by peptide treatment alone, since they showed normal morphology and excluded trypan blue, which only enters epithelial cells if their membrane is permeabilized (data not shown). These data show that KDAMPs have potential to protect epithelial cells against bacterial virulence mechanisms at concentrations not toxic to host cells.
Synthetic analogs of other K6A-derived peptides detected in corneal epithelial cell lysates were also found to be active against P. aeruginosa strain 6206. The 18-mer N-terminal fragment (18-mer-N), 18-mer C-terminal fragment (18-mer-C), 17-mer, 14-mer, and 13-mer (fragments within the 19-mer) each showed concentration-dependent bactericidal activity (90% or greater killing at 200 μg/ml, P < 0.0005) comparable to that of the 19-mer peptide (Figure 3F). Lower concentrations (100 μg/ml) reduced bactericidal efficacy for most KDAMPs except the 18-mer-N and the 14-mer. These results were somewhat surprising, since several of these peptides (e.g., 14-mer and 13-mer) had predicted features less favorable for antimicrobial function, including less or no positive charge, no transmembrane helix, or hydrophobic face. However, other examples exist of truncated antimicrobial peptides retaining activity, including some of the defensins and LL-37 (25, 26). Two other variants of the 19-mer with longer or shorter peptide sequences were tested: a 36-mer, comprising all previously tested variants, and a 10-mer, a sequence contained within both the 14-mer and 13-mer KDAMPs (Table 2). Both of these peptides showed dose-dependent bactericidal activity against P. aeruginosa (Figure 3F). At 200 μg/ml, killing of P. aeruginosa strain 6206 by the 19-mer, 36-mer, and 10-mer was 99.1%, 96.9%, and 99.2% (P < 0.0005).
In other experiments, mutations in P. aeruginosa LPS, a negatively charged cell wall component of Gram-negative bacteria, had only a minor impact on susceptibility of P. aeruginosa strain PAC1R (27) to the 19-mer KDAMP (Figure 3G).
Together these data suggest that KDAMP antimicrobial activity is retained under physiological conditions and requires a specific amino acid sequence(s) and a coil secondary structure but is independent of potential for transmembrane α-helix formation, ability to form a hydrophobic face, and cationic charge.
KDAMP structure and bactericidal activity. Most known antimicrobial peptides require a specific secondary structure (e.g., α-helix, β-sheet) for antimicrobial and other functions (28). However, we tested the hypothesis that this was not the case for these short K6A-derived fragments, given their predicted structural characteristics and their common anti-Pseudomonas activity, combined with a high glycine composition (35% to 50%) that would be expected to confer minimal conformational constraints. Circular dichroism (CD) spectroscopy was used to analyze the secondary structure of the 19-mer KDAMP, its scrambled control, and the 17-mer, 36-mer, and 10-mer derivatives. Peptides were analyzed after mixing with water or 2,2,2 trifluoroethanol (TFE), a helix-promoting solvent that mimics the hydrophobic microenvironment of cytoplasmic membranes. CD spectra showed that, in water or 50% TFE, each of the peptides displayed negative mean residue ellipticity values at the 200-nm region, suggesting a random coil-like structure (Figure 4A), which was consistent with previous predictions (Table 2). When the TFE concentration was increased to 100%, only the 36-mer adopted a helical confirmation; all others remained unstructured (Figure 4, A and B). Analysis of the spectra using the K2D2 program (29) confirmed that each of these bactericidal K6A-derived fragments (19-mer, 17-mer, and 10-mer) and the nonbactericidal scrambled 19-mer control exhibit a low to negligible helical content of regardless of environment hydrophobicity, suggesting that α-helicity is not required for KDAMP bactericidal actions.
Synthetic analogs of K6A-derived peptides demonstrate conformational flexibility and target selectivity. (A) CD spectra of scrambled 19-mer (nonbactericidal control) as well as 19-mer, 17-mer, 10-mer, and 36-mer (bactericidal) in water, water/TFE (50:50), or TFE. Only 36-mer showed induced helical structure in TFE, others (with or without bactericidal activity) remained unstructured random coil-like peptides. (B) Analysis of the spectra using the K2D2 program (29) confirmed low to negligible helical content of scrambled 19-mer, active 19-mer, 17-mer, and 10-mer. (C) Glycine-to-alanine mutations (G2A and G8A) in the 10–amino acid peptide resulted in marked decrease of bactericidal activity against P. aeruginosa strain 6206 (*P < 0.0001). (D) Activity spectra of the K6A-derived peptides against various Gram-positive and Gram-negative pathogenic or commensal bacteria. The 19-mer K6A-derived peptide showed even greater bactericidal activity against S. pyogenes than P. aeruginosa (200 μg/ml, 3 hours), but activity was lost with removal of C- and N-terminal amino acids from 19-mer (17-mer peptide). E. coli was found to be susceptible to killing by the 14-mer and 18-mer-N, while S. marcescens was only minimally susceptible. S. aureus and S. epidermidis were both susceptible to killing by the 14-mer.
Since glycine residues were a common feature of naturally occurring KDAMPs (Table 2), and dominance of glycine has been identified in some nonmammalian antimicrobial peptides (22, 30), the contribution of 2 glycine residues to the bactericidal activity of synthetic KDAMPs was investigated. Two glycine residues (G-2 and G-8) of 10-mer peptide (50% glycine content) were substituted with alanine to interrupt consecutive glycine stretches. Thus, GGLSSVGGGS became GALSSVGAGS, while maintaining similar amphipathicity and physiochemical properties. The site-directed mutant (G2A/G8A) showed reduced bactericidal activity against P. aeruginosa (~86% killing versus >98% killing of control after 3 hours at 200 μg/ml, P < 0.0001) (Figure 4C), showing that these glycine residues contribute to activity.
KDAMP activity against other pathogens. The activity of synthetic KDAMPs was evaluated against other clinically important bacteria (Figure 4D). As expected, all K6A-derived peptides were active against P. aeruginosa. Antimicrobial activity was also observed toward other bacterial pathogens, but levels of activity varied between bacteria and peptides. The group A Streptococcus (Streptococcus pyogenes) was particularly susceptible. The 14-mer, the 19-mer, the two 18-mers (the 18-mer-N and the 18-mer-C, lacking the N- or C-terminal residue of the 19-mer, respectively), and the 36-mer were each highly effective against this clinically important pathogen (e.g., >99.9% killing for the 14-mer, 18-mer-N, and 19-mer, after 3 hours at 200 μg/ml, P < 0.001). The 14-mer (>99.9% killing, P < 0.001) and the 18-mer-N (98.1%, P < 0.0001) peptide also showed bactericidal activity against E. coli but less against Serratia marcescens (e.g., 14-mer, 32.4% killing, P < 0.001). However, the 14-mer was highly effective in killing Staphylococcus aureus (97.5%, P < 0.0005) and Staphylococcus epidermidis (>99.7%, P < 0.0001). These data show that KDAMP bactericidal activity extends beyond P. aeruginosa to other clinically important pathogens, suggesting that compositional flexibility may allow optimal activity against different pathogens. Indeed, it is possible that naturally occurring or synthetic KDAMPs may complement each other in situ to provide a broad-spectrum antimicrobial defense.
KDAMP bactericidal activity involves bacterial cell wall/membrane disruption. To begin to understand how KDAMPs kill bacteria, P. aeruginosa were treated with a 19-mer peptide tagged with the fluorochrome TAMRA (5-(and-6)-carboxytetramethylrhodamine) (Invitrogen), and treated bacteria were stained with SYTOX Blue, a cell-impermeant dye. Fluorescence microscopy showed that untreated control bacteria and those treated with a scrambled 19-mer or TAMRA alone did not label with SYTOX, indicating that their cell walls/membranes remained intact, i.e., impermeable to the dye (Figure 5A). Those bacteria also remained motile (Supplemental Videos 1 and 2). In contrast, 19-mer–treated bacteria labeled with SYTOX (blue) and colabeled with TAMRA when the peptide was tagged (red) (Figure 5B) and were nonmotile (Supplemental Video 3). Bactericidal assays (viable counts) confirmed that TAMRA-tagged, or non-tagged, 19-mer–treated bacteria were dead, that killing activity of tagged-19-mer was similar to that of the untagged peptide, and that TAMRA alone was inactive (Figure 5C). Interestingly, while TAMRA tagging did not affect activity of 19-mer against P. aeruginosa, it did impact 19-mer activity against other bacteria. For example, the TAMRA-tag enhanced 19-mer activity against S. marcescens and E. coli relative to unlabeled peptide (P < 0.005) but reduced activity against S. pyogenes (P < 0.005) (Supplemental Figure 1). Both of those results may reflect steric effects of the TAMRA-tag on the N-terminal residue, but further studies will be needed to understand their significance. At present, the data show that the K6A-derived 19-mer peptide binds bacterial cells and that it causes bacterial cell wall/membrane permeabilization, loss of swimming motility, and bacterial cell death.
The K6A-derived 19-mer binds to bacterial cells and permeabilizes their cell membranes. (A) P. aeruginosa strain 6206 was either untreated (control) or treated with an equimolar concentration (110 μM each) of scrambled 19-mer (200 μg/ml), TAMRA (50 μg/ml), or 19-mer (200 μg/ml) for 3 hours and then stained with a cell-impermeant dye SYTOX Blue. Only the 19-mer–treated bacteria were stained with SYTOX Blue. TAMRA itself also did not bind to bacteria. Original magnification, ×1,000. BF, bright field. (B) Strain 6206 was incubated with TAMRA-tagged (N-terminal) 19-mer (110 μM, equivalent to 250 μg/ml) for 3 hours then stained with SYTOX Blue. Fluorescence microscopy showed that the bacteria were labeled with the TAMRA-tagged 19-mer peptide (red) and SYTOX (blue) and that these labels colocalized. Original magnification, ×1,000. Higher-magnification images are shown in inset panels (original magnification, ×~2,000). (C) TAMRA-tagged 19-mer had similar bactericidal activity to that of unlabeled peptide (93.2% and 98.4%, respectively, P < 0.05) after 3 hours at 110 μM against P. aeruginosa (strain 6206). TAMRA alone was inactive. (D) Rapid and extensive killing action of the 19-mer against P. aeruginosa strain 6294 and strain 6206. (E) SYTOX staining of P. aeruginosa 6206 at 5 and 15 minutes after treatment with the 19-mer KDAMP at 75 μg/ml. Bacteria incubated with the scrambled 19-mer or untreated served as controls. DIC, differential interference contrast. Original magnification, ×400.
KDAMP-induced bacterial cell permeabilization could directly cause cell death, as has been shown for cationic antimicrobial peptides (31). Alternatively, permeabilization may be secondary to peptide-induced cell death via another mechanism. Thus, the temporal profile of killing by the 19-mer peptide was examined. This was done using log-phase bacterial cultures and a concentration of 108 CFU/ml of P. aeruginosa suspended in saline. The 19-mer peptide killed 71.7% and 91.7% of strain 6206 after 5 and 15 minutes, respectively, and 98% of strain 6294 after 15 minutes (Figure 5D). Antimicrobial proteins or peptides that exert killing effects via inhibition of macromolecular synthesis, i.e., DNA, RNA, and/or protein, generally require at least 30 minutes of exposure for maximal impact on bacterial viability (>90% killing) (32, 33). The rapid action of the 19-mer suggested that direct membrane disruption was involved in the mechanism by which it killed P. aeruginosa. That hypothesis was supported by the observation of SYTOX-labeled P. aeruginosa at 5 and 15 minutes after treatment with the 19-mer KDAMP (Figure 5E).
Keratins have long been recognized as indispensable structural materials that maintain cell integrity and resilience. Here we show a role for human keratins in epithelial innate immunity as a source of endogenous antimicrobial peptides. Specifically, the data show that short peptide fragments derived from the C terminus of hK6A are found within differentiated human corneal epithelial cells, that these peptides exert antimicrobial activity against multiple bacterial pathogens in vitro, and that depletion of K6A in vivo reduces corneal defense against bacterial attachment. Antimicrobial activity of these coiled, glycine-rich peptides (termed KDAMPs) is not constrained by stringent secondary structure or net charge requirements. Direct visualization showed that KDAMPs bind to P. aeruginosa, that they induce cell wall/membrane permeabilization, and that they inhibit bacterial motility. Together, we believe these data establish a new role for cytokeratin 6 in protecting mammalian epithelia from bacterial infection.
Structure-activity and imaging studies with K6A-derived peptide fragments and P. aeruginosa provided initial insights into KDAMP mechanism(s) of action. Firstly, antimicrobial activity of K6A-derived peptides was unaffected by changes in charge or predicted hydrophobic moment, and all but one (36-mer) lacked an α-helical secondary structure in conditions mimicking the environment of bacterial cell membranes. This contrasts with many other previously identified types of antimicrobial peptide for which cationic charge, hydrophobicity, and α-helix formation are important criteria in their antimicrobial function (25, 34). Proposed mechanisms for these requirements include binding to anionic bacterial cell membranes with disruption involving pore formation. However, amphipathicity of antimicrobial peptides can have a more significant role in determining bactericidal activity than hydrophobicity or α-helical content, as shown against E. coli (35). Moreover, rapid action of some antimicrobial peptides involves membrane disintegration via the "carpet" mechanism, in which peptides accumulate to critical thresholds on bacterial membrane surfaces (36). Selectivity between host and microbial cells can also be based on electrochemical potential across the bacterial cytoplasmic membrane due to the proton flux (transmembrane potential) (37). Studies have also suggested that more specific interactions within bacterial cell walls or membranes help to define antimicrobial peptide activity (38). For example, binding of the antimicrobial peptide nisin can be mediated by peptidoglycan-associated lipid II (39), and salivary histatins bind a 67-kDa surface protein of Candida albicans (40). Specific cell wall/membrane targeting may explain some of the differences in the spectrum of K6A-derived peptide activity, e.g., the 19-mer is effective against S. pyogenes but not S. aureus or E. coli, both of which are highly susceptible to the 14-mer. Scrambling the sequence of the 19-mer all but eliminated activity against P. aeruginosa, even though predicted charge and coil structure were similar to those of the active peptide. Although the scrambled 19-mer lost ability to form a transmembrane helix, that predicted property was not evident for other smaller KDAMPs (e.g., 13-mer, 10-mer) that showed similar activity against P. aeruginosa as the 19-mer. The scrambled 19-mer did show a reduced hydrophobic moment (μH) of 0.138 μH compared with that of the active 19-mer (0.279 μH) and other active KDAMPs (0.219 μH or greater). Studies with fallaxin-based antimicrobial peptides have shown that active and inactive peptides could be grouped according to hydrophobic moment data, although these data alone were not helpful in predicting activity (41). However, for citropin-based antimicrobial peptides, increased antimicrobial activity and improved bacterial membrane binding (using lipid extracts) were associated with increased hydrophobic moment (42). Thus, differences in the hydrophobic moment of KDAMPs may be helpful in further analysis of their structure-activity relationships.
Use of TAMRA-labeled K6A-derived peptides and Sytox showed these that peptides bind P. aeruginosa and that they cause cell wall/membrane permeabilization. The specific mechanism for cell wall/membrane disruption by K6A-derived peptides is not yet known. Differences between predicted KDAMP structural features (e.g., charge, hydrophobicity, secondary structure, etc.), compared with those of other known antimicrobial peptides, suggest the possibility that the mechanism is novel.
Glycine predominates the amino acid composition of the K6A-derived peptides (up to 50% of the 10-mer), and substitutions of glycine residues with alanine at positions 2 and 8 reduced bactericidal activity of keratin-derived (K6A-derived) fragments. This contrasts with early magainin antimicrobial peptides, for which glycine-alanine substitution promotes antimicrobial activity by increasing α-helix formation (43). However, it is consistent with the results of structure-activity experiments that revealed that α-helical structure was not critical for KDAMP activity. A potential role of glycine repeats in KDAMP structure would be that they confer high structural flexibility (degrees of freedom) to these molecules to enable their antimicrobial action. Further studies will be needed to elucidate the role of glycine in the antimicrobial activity of K6A-derived peptides and whether peptides derived from keratins K5, K15, and K19, which were also detected in bactericidal cell lysate fractions and which also show glycine-rich tandem repeats in N- or C-terminal regions (acidic or basic keratins, respectively) (Supplemental Figures 2 and 3), are also antimicrobial.
Keratins are an ancient and extremely diverse set of proteins (44). In humans, keratins comprise a large family (54 functional genes) distributed intracellularly and extracellularly among multiple tissues, including skin, teeth, hair, nails, and mucosal surfaces (45, 46). Within epithelial cells, basic (type II) and acidic (type I) cytokeratins work as complementary pairs to form heteropolymeric intermediate filaments of the cytoskeleton and are key structural components of human cells and tissues. Cytokeratins also play roles in wound healing and tissue regeneration (47) in intracellular signaling and apoptosis (46). Discovery of antimicrobial and cytoprotective properties for K6A-derived peptides could advance our understanding of the functional significance of cytokeratin 6, and other keratins, in general. For example, it might explain the universal presence of keratins in cells and tissues of virtually all species and their diversity within species. Interestingly, a previous study reported that TR65, an antibacterial, pore-forming glycoprotein found in the skin mucous of the rainbow trout, shows strong homology to the trout type II epidermal cytokeratin E1 and is rich in glycine and serine residues (48). Moreover, the authors of that paper suggested that there may be a role for human cytokeratins in innate immunity, given the previously reported increased expression of several cytokeratins in respiratory epithelial cells in response to P. aeruginosa strain PAO1 (49). Another study showed that PiBP-5, a 5.7-kDa glycine-, serine-, and threonine-rich antibacterial peptide derived from porcine bladder mucosa, has strong homology to human cytokeratin 7 (50). The abundance of cytokeratin cytoskeletal proteins, and the constant and rapid manner in which full-length cytokeratin monomers are released from their filamentous assembly (51), could support a cellular demand for antimicrobial peptides to help defend human mucosal epithelia against microbial pathogens. It is possible that antimicrobial activity contributes to the demonstrated role of cytokeratin 6 in wound healing and tissue repair (52, 53). It is also of interest that dysfunctional intermediate filament turnover has been implicated in the pathogenesis of various internal conditions, including infectious hepatitis, pancreatitis, and inflammatory bowel disease (54).
Turnover of intermediate filaments, and soluble keratin proteins, in epithelial cells is a highly regulated and complex process that is known to involve the ubiquitin-proteasome pathway (UPP) (55, 56). It is not yet known whether the UPP contributes to the generation or degradation of KDAMPs. Our results (Figure 2A) showed a calcium-dependent effect on the generation of fragments from full-length K6A in human corneal epithelial cells, and UPP degradation of host proteins can be increased by elevated intracellular calcium (57). However, generation and degradation of KDAMPs could also reflect the activities of calcium-dependent proteases, e.g., calpains, which are known to target keratins (58, 59) and are present in corneal epithelial cells and other ocular tissues (60). Further studies are underway to investigate the mechanisms of KDAMP generation and regulation in epithelial cells.
In conclusion, this study reveals what we believe to be a novel function for human keratins as a source of endogenous antimicrobial peptides contributing to antimicrobial activity of cell lysates and defense against bacterial adherence in vivo. Small synthetic analogs of these peptides exert broad-spectrum antimicrobial activity against diverse human pathogens, including P. aeruginosa, E. coli, S. pyogenes, and S. aureus. These peptides are active under physiological conditions and induce bacterial cell wall/membrane permeabilization after binding, which does not require cationic charge or α-helix formation but does require glycine residues. Further studies will be required to determine the mechanistic details of their bactericidal activity, whether other forms of keratin also give rise to KDAMPs, the mechanism(s) by which they are generated in vivo, and their functional significance in the innate defense of mucosal epithelia and epithelial homeostasis. KDAMPs may also be of value as robust, biocompatible, and efficacious therapeutic agents for combating infection.
Bacterial strains. P. aeruginosa corneal clinical isolates 6294 (invasive) and 6206 (cytotoxic), cystic fibrosis clinical isolate 2192, and laboratory strains PAO1 (invasive) and PA14 (cytotoxic) were used in this study. PAC1R and its series of isogenic LPS mutants (PAC1RalgC:tet, 557, 605, 611) were provided by Gerald B. Pier (Harvard Medical School, Boston, Massachusetts, USA). For antimicrobial spectrum analysis, E. coli (ATCC 25922), S. marcescens (ATCC 43862), S. epidermidis (ATCC 12228), S. aureus (ATCC 29213), and S. pyogenes (ATCC 19615) were used. For antimicrobial activity assays, lawn culture of P. aeruginosa was grown on tryptic soy agar plates at 37°C for 16 hours, while other bacteria were grown on nutrient agar plates for 16 hours at media and conditions specified by ATCC. Starting inoculum, at a concentration of approximately 108 CFU/ml, was prepared fresh by suspending bacteria in serum-free Keratinocyte Basal Medium (KBM) (Lonza Inc.) until OD650 reading was approximately 0.1. Specific bacterial concentration used in antimicrobial and cytoprotective assays was prepared by 100-fold dilution of this starting inoculum in the indicated test media (~106 CFU/ml). For experiments involving peptide effects on bacterial swimming motility or the time course of bacterial killing, broth cultures of P. aeruginosa were prepared by subculturing overnight culture in tryptic soy broth at 37°C until OD600 reading was approximately 0.3 (log phase). Bacteria were washed and resuspended in saline to an OD650 reading of approximately 0.1 (~108 CFU/ml).
Cell culture and lysate fractionation. hTCEpi cells (15) were maintained at 37°C/5% CO2 in regular keratinocyte growth medium (KGM-2) containing 0.15 mM CaCl2 (Lonza). hTCEpi cells in antibiotic-free KGM-2 media were grown to 90% confluence in 96-well tissue culture–treated plates and then switched high-calcium antibiotic-free KGM-2 media (containing 1.15 mM CaCl2 for 32 hours. Cells were then lysed in 25 μl KBM (Lonza) per well (for antimicrobial and cytoprotective activity assays) or 100 mM Tris-HCl (pH 8.5) (for mass spectrometry) by 3 cycles of freeze and thaw. Crude lysate was pooled and confirmed to have equal total protein concentration using a Bicinchoninic Acid Assay Kit (Pierce Biotechnology Inc., Thermo Scientific). Serial fractionation of crude lysate was performed at 4°C using sterile water–prerinsed Microcon centrifugal filter devices, with membrane cutoffs at 100, 10, and 3 kDa (Millipore). Fresh vehicle was added back to all lysate fractions to maintain equal volume to that of their original crude lysates.
Mass spectrometry. Mass spectrometry was performed at the Proteomics/Mass Spectrometry Laboratory at University of California, Berkeley. A nano LC column was packed in a 100-μm inner diameter glass capillary with an emitter tip. The column consisted of 10 cm of Polaris c18 5-μm packing material (Varian). The column was loaded by use of a pressure bomb and washed extensively with buffer A (see below). The column was then directly coupled to an electrospray ionization source mounted on a Thermo-Fisher LTQ XL linear ion trap mass spectrometer. An Agilent 1200 HPLC equipped with a split line so as to deliver a flow rate of 30 nl/min was used for chromatography. Peptides were eluted with a 90-minute gradient from 100% buffer A to 60% buffer B. Buffer A was 5% acetonitrile/0.02% heptafluorobutyric acid (HBFA); buffer B was 80% acetonitrile/0.02% HBFA. Collision-induced dissociation and electron transfer dissociation spectra were collected for each m/z. The SEQUEST and DTASELECT programs were used to identify peptides and proteins from the human database (61, 62).
Bioinformatics. Peptide sequences were analyzed using web-based tools hosted on the Expert Protein Analysis System (ExPASy) proteomics server of the Swiss Institute of Bioinformatics ( http://ca.expasy.org/tools/#proteome). Specifically, ProtParam was used to compute theoretical pI, NetSurfP was used for secondary structure prediction, TMpred was used for transmembrane helices prediction, HeliQuest was used for amphipathicity and net charge computation as well as helical wheel presentations. Clustal Omega was used to align protein sequences.
Western blotting. hTCEpi cells were lysed on plates directly by tricine gel loading buffer containing SDS and β-mercaptoethanol (Bio-Rad). Detection of full-length and fragmented hK6A and GADPH proteins in the crude lysate (containing 10 μg of total protein) were performed by standard Western blotting using rabbit anti–19-mer antiserum (prepared by New England Peptide) and rabbit polyclonal anti-GAPDH antibody (sc-25778; Santa Cruz Biotechnology Inc.), respectively, followed by goat anti-rabbit IgG-HRP secondary antibody (Bio-Rad). The rabbit anti–19-mer antiserum also recognized murine cytokeratin 6 (data not shown).
siRNA knockdown and real-time PCR. For in vitro knockdown, transfection (6 hours) in 96-well plate format was performed when hTCEpi cells were 80% confluent using Lipofectamine RNAi Max (Invitrogen) and siRNAs (10 pmol in 100 μl) against hK6A (5′-CGAAGGCGUUGGACAAGUC-3′, 5′-GAACAAGGUUGAACUGCAA-3′, 5′-GCAGUUCCACCAUCAAGUA-3′, and 5′-GAGAUCAACUUCCUGAGAG-3′; ON-TARGETplus SMARTpool L-012116-00-0005; Dharmacon) or scrambled control (ON-TARGETplus Non-targeting Pool D-001810-10-05; Dharmacon), followed by 16 hours of recovery in antibiotic-free KGM-2, and then 16 hours of differentiation in high-calcium antibiotic-free KGM-2 (containing 1.15 mM CaCl2). To collect lysate for antimicrobial assays, cells were lysed in 25 μl saline per well (0.9% NaCl) by 3 cycles of freeze and thaw, and crude lysate was pooled and confirmed to have equal total protein concentration prior to use. For in vivo knockdown, C57BL/6 wild-type mice (Charles River) were subconjunctivally injected with siRNAs in PBS (5 μl of 8 μM per eye) against mouse K6A (5′-GCUCUUGGCUUCCUCGUAA-3′, 5′-GCUCAGAGAAGUCGGGCUG-3′, 5′-GGUUGAAUGGUGAAGGUGU-3′, 5′-GAUGUAAGUUUCUGCUCAU-3′; ON-TARGETplus SMARTpool L-061465-01-0005; Dharmacon) or scrambled control (ON-TARGETplus Non-targeting Pool D-001810-10-05; Dharmacon). Injections were performed once a day for 2 days before mice were sacrificed by lethal injection of anesthetic cocktail (21 mg/ml ketamine, 2.4 mg/ml xylazine, and 0.3 mg/ml acepromazine). Whole eyes were enucleated and PBS-rinsed to exclude tear fluid, followed by incubation with 200 μl GFP-expressing P. aeruginosa strain PAO1 (i.e., 1011 CFU/ml of PAO1-GFP) for 3 hours at 35°C, and then imaged using combined 2-photon confocal microscopy as previously described (63). To confirm successful knockdown, total RNA was isolated using the RNeasy Mini Kit (Qiagen), and 1 μg was used for cDNA synthesis using the Verso cDNA Synthesis Kit (Dharmacon). K6A and GAPDH (internal control) were amplified from cDNA (0.5 μl of each cDNA sample per 10 μl of PCR reaction) using QuantiTect SYBR Green PCR Master Mix and Primer Assays (Qiagen). Samples were analyzed in triplicates in 3 independent runs using a real-time detection system (Bio-Rad). Transcript levels of K6A were normalized to those of GAPDH (human) or Rn18s (mouse).
Peptide synthesis. All peptides (with or without N-terminal TAMRA-tag) were synthesized by Biomatik to >95% purity and verified by HPLC and MS. Stock solutions were prepared in sterile water or DMSO, and aliquots were kept at –20°C and limited to 1 thaw prior to use.
Antimicrobial activity assay. Bacteria (60 μl; 106 CFU/ml of suspended lawn culture) in whole lysate, lysate fractions, and saline, with indicated peptides (as specified in figure legends) as well as the corresponding vehicle controls, were incubated in triplicates at 37°C for 3 hours. Serial dilutions of the samples at time 0 and 3 hours were plated on nutrient agar plates and incubated at 37°C overnight for viable bacterial counts (CFU/ml). The percentage of bacterial killing at 3 hours was determined as follows: (bacterial counts without peptide – bacterial counts with peptide)/bacterial counts without peptide × 100%. Shorter incubation times (from 5 to 60 minutes) were used in some experiments (see Figure 5).
Invasion and cytotoxicity assay. To assess protective activity against bacterial invasion, high-calcium–treated hTCEpi cells were incubated with P. aeruginosa clinical isolate 6294 (40 μl/well; 106 CFU/ml in crude lysates/lysate fractions versus KBM control for 3 hours or 108 CFU/ml in peptide-containing saline versus no-peptide saline control for 2 hours), followed by gentamicin treatment (200 μg/ml for 1 hour). Viable intracellular bacteria was collected with 0.25% Triton-X in PBS (100 μl/well) and quantified by plating on tryptic soy agar. To assay protection against bacterial cytotoxicity, P. aeruginosa clinical isolate 6206 was used instead (40 μl/well; 106 CFU/ml for 3 hours). Dead cells were quantified by trypan blue (0.04%) staining.
CD spectroscopy and analysis. CD spectra were recorded at 25°C with a spectropolarimeter (model 410, Biomedical Inc.) from 190 to 300 nm using a 0.1-cm path-length quartz cuvette. Peptides were dissolved in pure water or 50% water/50% TFE mixture or pure TFE to a final concentration of 0.1 mg/ml. Three scans were conducted at 1-nm intervals and 1-s intervals. Spectra were averaged and subtracted for blank control, and mean residue ellipticity values were presented. CD data were further analyzed using the web-based K2D2 program.
Phase-contrast and fluorescence microscopy. To assess bacterial motility, P. aeruginosa clinical isolate 6206 (700 μl; ~108 CFU/ml of log-phase culture in saline) was incubated with or without peptide (100 μg/ml synthetic 19-mer peptide or its scrambled control peptide) at 37°C for 15 minutes in the presence of hTCEpi cells in 12-well tissue culture plates. Real-time bacterial motility was visualized with an Olympus IX70 epifluorescent microscope using a ×40 objective and phase contrast (total magnification, ×400). To assess peptide binding and membrane permeabilization, bacteria (200 μl; 106 CFU/ml of suspended lawn culture) were incubated at 37°C for 2.5 hours with or without indicated peptides, including TAMRA-tag peptides and TAMRA tag control (Invitrogen) at the equimolar concentration of 110 μM, followed by 30 minutes of incubation with the impermeant SYTOX blue nucleic acid stain (5 μM; Invitrogen). Bacteria were pelleted by centrifugation, and supernatant was removed before resuspension in 10 μl saline. Bacteria were visualized by phase-contrast microscopy, SYTOX blue stain, and TAMRA fluorescence under a ×100 objective (total magnification, ×1,000).
Statistics. Data are expressed as mean ± SD, calculated from triplicate samples per group in each experiment. Statistical significance of differences between 2 groups was determined by the Student's t test (2 tailed). P < 0.05 was considered significant. All experiments were repeated at least 3 times, and representative experiments are shown in each figure.
Study approval. All animal procedures were performed in strict accordance with protocols approved by the Animal Care and Use Committee, University of California, Berkeley, which is an AAALAC-accredited institution.
This work was supported by a Grand Challenges Exploration Grant from the Bill and Melinda Gates Foundation and by NIH grant RO1-EY011221. We wish to thank Gerald B. Pier (Harvard Medical School) for providing P. aeruginosa strain PAC1R and its LPS-defective mutants; Lori Kohlstaedt (Proteomics/Mass Spectrometry Laboratory, University of California, Berkeley) and Susan Marqusee (QB3 Institute, University of California, Berkeley) for instrumentation and helpful advice; and Arjay Clemente, Jong Hun Kim, Gary Chan, Angela Wong, and Robin So for technical assistance.
Address correspondence to: Suzanne M.J. Fleiszig, University of California, Berkeley, 688 Minor Hall, Berkeley, California 94720-2020, USA. Phone: 510.643.0990; Fax: 510.643.5109; E-mail: firstname.lastname@example.com.
Conflict of interest: The authors are listed as coinventors on a US Provisional Patent Application filed April 27, 2011, No. 61/479,507, and a subsequent US Patent Application submitted April, 2012, both entitled "Antimicrobial Peptides and Methods of Use Thereof," that include data presented in this article.
Reference information: J Clin Invest. 2012;122(10):3665–3677. doi:10.1172/JCI64416.
See the related article at Defending the cornea with antibacterial fragments of keratin.


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Original Gillette Mach 3 Shaving Razor Blades Brand Mach3 For Men Beard Shave Blade Razor Travel Box
NOTE: Gillette series product packaging changes frequently, we only guarantee genuine original, does not guarantee the physical and picture exactly the same!
Address and Full Name:
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About Packing:
1. All products are in standardized packing, just as follows:
2. We will carefully check your order and the package to make sure everything is in good condition before shipping it out.
3. The packages may be slightly damaged during long-time transportation, but it doesn't affect the effect of our products.
4. If you want products packed perfectly (such as for gifts etc.), please contact us first, we can send the parcel with special package to avoid damage, but you need pay some extra fee.
Please click here to pay freight or extra fee. Thanks!
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We will send out the order in 2-5 working days, and it needs almost 7-40 days from China to you.
As you know, the delivery time is depend on the shipping company, the air line, the custom and the local post office, so we are not able to control it accurately.
If the parcel has not arrived in expected time, please feel free to contact us, we will track it and handle it with you.

subset: commoncrawl


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1990 College Football Recap
If NCAA officials were undecided as to whether some type of bowl alliance championship was necessary for Division I-A college football, 1990 was the year that may have swayed them. Six different teams held the No. 1 spot during the season and for the first time since Alabama and USC split the national championship in 1978, two teams shared the title. Bobby Ross' Georgia Tech squad, tied once but unbeaten, was named UPI's national champ by the narrowest of margins (847-846), while Colorado (11-1-1) was voted No. 1 by AP's panel of broadcasters and sportswriters.
Though Colorado's schedule was tougher, two of their wins were shrouded in controversy. In a now infamous game on October 6, the Buffaloes mistakenly were allowed five downs to score a last minute touchdown to beat Missouri 33-31. In the Orange Bowl, the Buffs were up 10-9 over Notre Dame late in the fourth when speedster Rocket Ismail took a punt and raced 91 yards for an apparent game-winning TD. Officials, however, called a clip on the play and the TD was disallowed, giving Colorado the win.
BYU junior QB Ty Detmer took home the Heisman Trophy, to the surprise of many, over Notre Dame's Ismail. Detmer decided to return for his final season at BYU while Ismail went for the riches of the CFL, signing a four-year, $18.2 million deal with the Toronto Argonauts.
1990 marked the final installment of the heated Notre Dame-Miami series with the Irish defeating Miami 29-20 in South Bend. Houston QB David Klingler had a game for the ages in late November, throwing for 572 yards and a record 11 TDs against Division I-AA Eastern Washington. The Cougars won 84-21 and Klingler was finally pulled late in the fourth with his team safely up by 63.
Here are the facts and trivia that people are buzzing about.


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Job Description
At Steward Health Care System, we are committed to improving the health of our communities by delivering exceptional, personalized behavioral health care with dignity, compassion and respect. Our continued focus on the patient experience informs our caregivers in how to provide care that is respectful of and responsive to individual patient and family preferences, needs and values.
We dedicate ourselves in the communities we serve to delivering affordable health care to all and being responsible partners. No matter what your role, as a member of the Steward family, you are a specialist in the making every patient and family feel right at home, every co-worker a key to our success, and every referring practice, a team of prized colleagues.
In support of this, we commit ourselves to the following values:
If you are seeking a fast-paced, challenging position in an organization committed to achieving and maintaining a standard of excellence in all we do, our organization may be a good fit for you.
Full time 40 Hours per week
Performs functions of the PCA in caring for assigned adolescent, adult, and geriatric patients during his/her shift. Assists the RN/healthcare team in implementation of the Patient Plan of Care. Provides patient care as directed by RN.
Job Description:
Provides patient care based on standards of care, standards of practice, protocols and established policies and procedures. Assists with preparation for patient discharge. Appropriately prioritizes patient care assignment (e.g. seeks assistance as needed from appropriate resources, recognizes need for emergency interventions, etc.) Provides thorough, timely and accurate documentation of all pertinent data, according to established standards. Provides timely and thorough report on assigned patients to other health care team members to ensure integration of services and continuity of patient care. Promotes learning for colleagues and self (e.g. functions as preceptor, attends professional development programs where appropriate, etc.) Proactively identifies risk to patient safety and identifies opportunities to reduce medical/healthcare errors (i.e. appropriate reporting of errors/incidents). Identifies ethical issues in care and reports them to the RN. Reports patient pain level in a timely manner utilizing the appropriate scale (numeric, FACES).
High school graduate or equivalent; Basic Cardiac Life Support (BCLS) certification and Massachusetts CNA Certification required. Demonstrates clinical competency within the scope of practice of a CNA.
Important COVID message
Please note, Holy Family hospital is taking additional, necessary preparations to ensure patients can receive compassionate care in a safe, carefully managed environment - with confidence and without fear. Our Safe and Ready program consists of a rigorous [five-point] standard ensuring patient safety, confidence and convenience: Expanded hours will allow previously cancelled procedures to be scheduled as quickly as possible. Any COVID-19 related care takes place in designated areas away from other patients and their families. Emergency Departments are reorganized to be a safe place to treat all emergency patients. A stringent cleaning policy has been implemented throughout the hospital. A strictly controlled visitor and masking policy is required for patient safety. You can rest assured that we have made the necessary preparations to provide care in a safe, controlled and professional way.
Steward Health Care is an Equal Employment Opportunity (EEO) employer. Steward Health Care does not discriminate on the grounds of race, color, religion, sex, national origin, age, disability, veteran status, sexual orientation, gender identity and/or expression or any other non-job-related characteristic.
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Please click on the link below to apply for this position. A new window will open and direct you to apply at our corporate careers page. We look forward to hearing from you!Apply Online
Posted: 9/8/2023
Job Status: Part Time
Job Reference #: 131743


text: A mouth watering book that brings out Italian creativity through its art and cuisine, accompanied by a delicious food charm in sterling silver and enamel. A tribute to Italy through taste and beauty- a special gift based on the good and the beauty of Italy. "L'arte in cucina" (or "Art in the kitchen") (Editoriale Giorgio Mondadori) written by Domenico Monteforte, who is known and appreciated for his landscape paintings. Art in the Kitchen is an artistic recipe book, which takes you across beautiful Italy through its flavors, dishes and typical products. In this book, 50 contemporary artists share their favorite recipe from their region, certified from a high-end professional chef. Both the artists and companies based their work on Italian style. Among these there is Gioielli DOP who transformed the love between art and food in a successful brand. The Chili Pepper is a fiery ingredient in many Italian dishes- it adds a special kick. Apart from being the strong protagonist in many Italian dishes, especially in the Calabria region. It's a symbol of health, love, and luck. Keep this bracelet with you at all times! The Bow-Tie Pasta bracelet represents the metamorphasis- the distinction between the visible and the invisible, between dream and reality. The waxed cotton rope with nautical knots and silver ends, fits all wrist sizes. Check out the other bracelets in our Pasta collection and choose your favorite! Details: Charm bracelet, handmade in Italy. 925 Sterling Silver, hypoallergenic (nickel free). Scratch-resistant enamel. Every charm is handmade by passionate Italian craftsmen in Tuscany, 2 year guarantee.


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Chitlins Ain't Menudo
Dear Negrito: Why should Mexicans deviate from history? The American ethnic experience hews to a rigid trajectory that goes like this: Immigrants settle in the bad part of town because gabachos won't tolerate minorities near their homes. Said immigrants revitalize undesirable neighborhoods. The barrio/ghetto/'hood is born. It flourishes for a generation. Gabachos visit solely for hole-in-the-wall restaurants, prostitutes, drugs, gambling and cockfights. The immigrants sweat through life in order to get their children into college, only to see the kids repudiate their wabby parents and move to the suburbs. A new wave of immigrants living eight to a couch settle in the old neighborhood. The remaining pioneer immigrants despise the newbies for replacing the businesses, languages and culture of their once-familiar streets but can't stop the change. The old generation dies. The new immigrants prosper. And the circle of life begins again. You claim to understand this, Chitlins, so get over your lost catfish stands, and join Cleophus and LaKeisha in scaring the gabachos out of the suburbs and back into their hipster downtowns. But don't get too settled in: In a couple of years, Mexicans will relocate to your suburban 'hood to get away from the Guatemalans who are destroying our quaint barrios as we speak.
Dear Mexican: Why do Mexicans call people with curly hair chinos? Most chinos I know have very straight, hard-to-curl hair.
China Confundida
Dear Confused Chinita: The Mexican has discussed the word chino before, as in why Mexicans call all Asians chinos (same reason why gabachos call all Latinos "Mexican"). Chino is one of the more fascinating homographs (words with the same spelling but different meanings) in Spanish. Its Old World meaning specifically refers to a person of Chinese descent, but in his Dictionary of Latin American Racial and Ethnic Terminology, Rutgers linguist Thomas M. Stephens documents how chino assumed different connotations once the conquistadors pillaged the Americas and none of those connotations were positive. Stephens' book devotes an incredible seven pages to chino; some of its more peculiar Latin-American definitions include "female servant," "slave from Mozambique," "concubine," "young Indian female who served in a convent" and, yes, "curly-haired." Chino also was the category in the Spanish Empire's byzantine castas (caste) system designated for the offspring of parents with varying degrees of African and Amerindian blood. Stephens' only sin is that he doesn't explain why chino took on so many non-Chinese connotations, though he did write that china in Quechua signifies "female servant or animal," while Nahuatl speakers used chinoa ("toasted") to describe dark-skinned people. And he offers no insight into the chino-curly connection.
But it doesn't take a Ph-pinche-D to identify the common threads in chino's various meanings: African blood and servitude. Many blacks, of course, have naturally kinky hair, so at some point over the centuries, chino became an ethnicon (a term meant to comment on an ethnic group's prominent cultural characteristic that become popular shorthand for said characteristic) for both "black person" and "curly." Mexicans then went on to drop the black denotation and kept the curly connection. Such linguistic amnesia isn't unprecedented in Mexican Spanish: marrano, which many Mexicans use to call someone a "pig" or "filthy," comes from the Inquisition-era slur used against Jews who converted to Christianity. All this wordplay is further proof that Mexico is a country with a racial problem that makes America seem like Sesame Street. The proper Spanish word for "curly," by the way, is rizado.
Got a spicy question about Mexicans? Ask the Mexican at [email protected]. And those of you who do submit questions: include a hilarious pseudonym, por favor, or we'll make one up for you!


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I've felt the need to make a new challenge (since I have 4…oh noes! ;m;)! What a perfect time to make one! XD This one should be a tad bit relaxing (maybe?):
Main Idea
Draw a picture of what somebody would wearing walking down the street, or just something casual. Something with that kind of atmosphere!
You can draw your OCs, yourself, characters from actual anime/manga, anybody you want in this style. Just make it as casual and comfy as can be~!
1. You can post more than once
2. Stick with theOtaku's rules
3. It can be traditional or CG, it doesn't matter
4. You get extra points for background, but you don't have to if you don't feel it needs one
5. You can not submit old art!
6. It's okay if it's not coloured.
7. You can draw more than one character
How You'll be Judged
Creativity - 20
Originality - 15
Quality - 10
Personal Preference – 5
1st Place: One full coloured picture (CG) of an OC or character in casual clothes
2nd Place: One lineart picture of an OC or character in casual clothes
3rd Place: One sketch picture of an OC or character in casual clothes
(To those I've invited, you're not obligated to join in the contest, I just like to spam the Member Invitations thingy! >:D)


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Comment on the Zimmerman Verdict from the UUA
Standing Against Racism and Oppression
The Rev. Peter Morales, president of the Unitarian Universalist Association (UUA), issued this statement on the George Zimmerman trial verdict.
July 15, 2013
A Florida jury has acquitted George Zimmerman in the killing of Trayvon Martin, an unarmed African American teenager. It is hard to imagine that if an unarmed white teenager had been shot and killed by an African American man that the verdict would have been the same. The legal system has had its say, but justice has not been served. As we search for meaning in the wake of these events, I remember the words of the Rev. Dr. Martin Luther King, Jr.: "We must accept finite disappointment, but never lose infinite hope."
At this year's General Assembly, delegates approved an Action of Immediate Witness (AIW) condemning racist mistreatment of young people of color by police. The AIW stems from the work of author Michelle Alexander whose book The New Jim Crow describes the institutional racism behind the mass incarceration of people of color. The resolution calls on congregations to condemn the pattern of mistreatment of people of color through practices such as "stop and frisk" by police. It asks congregations to work with other congregations and other groups to stop this practice.
As president of the Unitarian Universalist Association, an institution committed to anti-racism and anti-oppression, I am committed to partnering with our congregations to put this work into action. We must respond to our society's violence, hatred, and fear with compassion and justice. It has always been a matter of life and death. It always will be.
Read the statement by The Rev. Peter Morales, president of the Unitarian Universalist Association on uua.org here.
Watch the Youth Caucus statement in support of the AIW 2 on the racist mistreatment by police of young people of color here.
Learn more about work being done by the Unitarian Universalist Association and Unitarian Universalist congregations on Michelle Alexander's The New Jim Crow here.


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Burst pipe plumbing emergencies can strike at any time and catch us unaware. While we wait for our Toronto plumber water leaks out of the pipe and floods the floor at a rapid rate. Aside from wasting gallons of water, split pipes cause costly water related damage to walls, floors and your indoor belongings. Water related plumbing emergencies can be damaged controlled by closing the water shutoff valve to your Toronto home. Get to know where your water shutoff valve is located should you encounter water related plumbing emergency or even a running toilet. Close water supply and save on money and water.
Home water supply systems contains main and master shutoff valves for stopping water flow to entire house or to specific plumbing fixtures. Most water meters have a shutoff valve before the meter and one after meter. Cold climate properties have their water meter located in warm indoor spaces like basements to prevent freezing complications. Milder climate homes have their water meter attached to an exterior wall or nestled in an underground box with a removable lid. Between the street's water main and meter are located a buried curb stop valve and a corporation stop, where your Toronto home's water supply connects to water mains. Both the curb stop valve and corporation stop are inaccessible to the public, only to city workers with special tools.
In addition to the water meter, modern Toronto homes also have localized shutoff valves with handles, called fixture supply stops, and located on water supply pipes to toilets, faucets, washing machines, water heaters, dishwashers, and water softeners. Closing the water supply to one of the above fixtures is a matter of turning the handle clockwise two to four full turns. Older homes have one fixture supply stop on the water supply to the toilet only.
Your Toronto plumber highly recommends people to get to know their water shutoff valves and avoid expensive water related damages, high water bills, and water wastage.
Article Source: http://EzineArticles.com/?expert=Samantha_Richardson
Just call Priority Plumbing or send us an e-mail to Schedule your Plumbing Repair & Installation Service throughout Toronto! 416-907-7972


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22 November, 2013
Jack Meyer
Bespoke jewellery designer, and specialist in jewellery CAD/CAM and emergent technologies that affect jewellery.
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It's coming up on six full NFL seasons since Bill Cowher ended his long run as coach of the Pittsburgh Steelers.
Appearing Tuesday at an event to promote CBS' coverage of Super Bowl XLVII, Cowher acknowledged he plans to eventually return to the sideline.
"It would be a challenge," Cowher told Neil Best of Newsday. "But I think that's probably why I would get back into it, because of the challenge."
Cowher, 55, dismissed the idea the game has changed too much for him to still succeed.
"I did it for 27 years," he said. "You don't just forget things overnight. One thing about this job is it's been really good because it allows me to study the game and do features on the game. ... It's not like I'm out of it. I know how the game is changing."
We believe him on that. Cowher hasn't been growing an impressive beard in some remote jungle since the mid-aughts. He's been sitting in a studio, talking about the NFL, pretending to laugh at Shannon Sharpe jokes.
That said, each year away from the game pulls Cowher further off the NFL radar. Five years ago, Cowher was the dream target for any NFL franchise looking for a reboot. Now? Not so much.


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LOS ANGELES (AP) - Nine-year-old Celia Renteria was certain her father was still alive when she crawled out of a steep California canyon and walked in the desert darkness to find help after their car crashed.
Braving the cold and howling of coyotes, she hiked through rugged terrain to a nearby home. No one answered. Then she hiked up the rocky embankment and along the remote highway to a commuter rail station, where she flagged down a passing motorist early Sunday.
When officers responded, they found Celia's father, Alejandro Renteria, 35, dead. He was pinned in the driver's seat.
In all, the girl hiked up to 3.2 kilometres.
"She was very courageous, being able to walk through the dark, through bushes and very rough terrain to get help for her dad," said California Highway Patrol (CHP) Officer Gil Hernandez. "Had she just waited there, we probably would not have found her until the next day."
The Renterias' car was launched about 60 metres down into the canyon along an isolated stretch of the Sierra Highway in the high desert, the CHP said. The vehicle flipped several times.
Authorities said Celia freed herself from the mangled wreck to find help.
Law enforcement officials praised her bravery and tenacity.
"She walked quite a distance in a very, very threatening environment," CHP Sgt. Tom Lackey told KABC-TV. "It's very steep and it's brushy, and there's also coyotes in the background." She was treated for minor injuries, including bumps and bruises and a cut on her face.
"She's in good condition," Ms Lyndsay Hutchison, a hospital spokeswoman, said Monday. She could not provide any further details, saying parents or a legal guardian must permit the release of information under medical privacy laws.
On video shot by the family in the hospital, the girl told her family she was saddened to learn her father did not survive.
The girl's caregiver, Consuelo Amador, told the station the child didn't panic and turned off the car's engine after she smelled gasoline.
The CHP was investigating whether alcohol played a role in the crash. Toxicology tests are pending from the coroner.


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Happy New Year!
One of my resolutions for 2013 is to blog – regularly and purposefully. My blog started during preparations for my October's ACTEM conference presentation, "Just Imagine." I have continued it as a place to provide support and tech ideas to educators (not just librarians) based on our program here at Old Town Elementary.
Please take a peek. Hope you'll share the ideas, and perhaps comment and follow!
-- Lynn Lowell Mayer
Library Media Specialist
Old Town Elementary School
576 Stillwater Avenue
Old Town, Maine 04468
207-827-1544 x214


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An Android app and the ability to order a drink from a mobile phone are coming soon, an executive from Starbucks said as he shared lessons and tips from his company's experience offering mobile applications.
Starbucks claims to have developed the very first iPhone app, which was an alert that people using the first iPhone received showing the name of the song playing in a Starbucks store when they walked in, said K.C. MacLaren, director of mobile and emerging platforms at Starbucks. He spoke at the Mobile Northwest conference on Monday in Seattle.
Since then, Starbucks has offered mobile apps to iPhone and BlackBerry users that let people manage their Starbucks cards and pay for drinks in the stores. The in-store payment service has handled millions of transactions, MacLaren said.
Apps for Android phones are "in process," he said. The platform presents challenges because it supports multiple screen resolutions and sizes, and users have various versions of the operating system, he said.
One of the most common questions he gets is: When will Starbucks let people order drinks from their phones? MacLaren said that the capability is coming in some form. "We know how to do it, the question is: Does it create the behavior in the customer we really want?" he said.
There's been a "lively debate" over offering the capability, he said. It could encourage some people to buy a drink when they'd otherwise walk away because of a long line, but it could also annoy people who are waiting in line when they see people head to the front of the line to pick up a pre-ordered drink, he said. People might order a drink from their phone but arrive to the store late and complain that their drink is cold. "We'll try something with it. Stay tuned," he said.
MacLaren shared some tips from his company's experience building mobile apps. He suggests offering information that's particularly useful to mobile users. "There's nothing wrong with telling your story but you can't just take your website and stick it on the phone. There's a different use case than on your laptop," he said.
For instance, mobile users don't simply want to see a list of stores. They want to see stores that have drive-through windows or those that are open, he said.
IPads are an entirely separate category. "IPads are not mobile. You can't scan it and pay," he said. He thinks iPads are an important segment but they should be served differently from a mobile phone, he said.
He advised companies that are building mobile apps to think carefully about measuring usage. Starbucks has discovered that it cannot predict how people will use apps and that being able to collect data about usage is key in order to learn about what people want. It will often launch one new feature and find 10 ways to measure its usage.
Starbucks also recommends considering different ways to test applications. Starbucks has faced challenges testing features like mobile payment. "Once you put an app in the store, a zillion people have access to it. You can't just say I want to try this segment," he said. To test out the mobile payment feature, Starbucks decided to release an app with many functions that would appeal broadly and say that the payment feature would be available only to visitors to 16 specific stores.
In addition to Android and mobile ordering, Starbucks thinks that the next step for mobile apps are features that help consumers interact with each other. "There's us to you and you to us and the third generation will be how do consumers interact with each other around our brand. That's where the power will be," he said.
Nancy Gohring covers mobile phones and cloud computing for The IDG News Service. Follow Nancy on Twitter at @idgnancy. Nancy's e-mail address is email@example.com


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DESCRIPTION:SendEasy is a simple and clean e-mail template which is easy to adapt for any kind of campaign. It features 5 different layouts with 5 colors and 2 sets of backgrounds – light and dark.
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text:
Rare meningitis infects more than 100, kills 8
(CNN) -- Eight people have died from a rare, noncontagious form of meningitis linked to contaminated steroid injections, the Centers for Disease Control and Prevention said Monday.
In addition, 105 people in nine states have been infected with the illness as a result of the shots, the CDC said.
The number of reported cases has increased significantly in recent days as federal and state authorities continue to investigate the outbreak.
As many as 13,000 people may have received the medicine between May 21 and September 24, a spokesman for the CDC told CNN.
Tennessee is reporting the most number of overall cases, with 35 infections and four deaths, according to the CDC.
"In our state we estimate there are about 1,000 people who we believe are at risk," said Tennessee Commissioner of the Department of Health Dr. John Dreyzehner. "In other words they have potentially received some of the contaminated medication."
He told reporters Monday that because not enough was known about the incubation period for the disease, his department continues to stay in contact with people who might have been exposed.
Dreyzehner said there haven't been any new exposures since the department contacted clinics in the state two weeks ago about the contaminated injections. The number of cases grows as the state learns of more people with meningitis, he explained
The fourth death reported in Tennessee on Monday occurred in late September, he said.
The patients contracted the deadly fungal meningitis after being injected in their spines with a preservative-free steroid called methylprednisolone acetate that was contaminated by a fungus. The steroid is used to treat pain and inflammation.
The New England Compounding Center (NECC), the Massachusetts-based pharmacy that made the contaminated injections, voluntarily recalled three lots of the injected steroid last week.
On Saturday, the same pharmacy announced a voluntary nationwide recall of all its other products as well. NECC said that the new recall was being announced out of an abundance of caution and that there is no indication any of its other products are contaminated.
The Food and Drug Administration has already asked doctors, clinics and consumers to stop using any of the pharmacy's products. The pharmacy on Wednesday voluntarily surrendered its license to operate until the FDA investigation into the contamination is complete.
Health officials say 75 medical facilities in 23 states received the contaminated steroid injections from NECC.
Other than Tennessee deaths have been reported in Maryland, Michigan and Virginia.
There are also confirmed cases in Florida, Indiana, Minnesota, North Carolina, and Ohio.
The other states that received the contaminated products from NECC are California, Connecticut, Georgia, Idaho, Illinois, New Hampshire, New Jersey, Nevada, New York, Pennsylvania, Rhode Island, South Carolina, Texas and West Virginia.
Federal health inspectors began inspecting the NECC plant last Monday. Inspectors found foreign particles in unopened vials, and after testing one of the unopened vials, they determined the substance was a fungus.
The investigation is still under way.
Nearly 10% of drugs administered in the United States come from compound pharmacies, according to a 2003 Government Accountability Office report.
Drugs manufactured by compound pharmacies do not have to go through FDA-mandated premarket approval. Instead, state health pharmacy boards are responsible for the oversight and licensing of these pharmacies.
Compound pharmacists create customized medication solutions for patients for whom manufactured pharmaceuticals won't work, according to the International Academy of Compounding Pharmacists.
Meningitis is an inflammation of the protective membranes covering the brain and spinal cord. It is usually caused by an infection, frequently with bacteria or a virus, but it can also be caused by less common pathogens like fungi, according to the CDC.
Fungal meningitis is very rare and, unlike viral and bacterial meningitis, it is not contagious.
Symptoms of fungal meningitis are similar to symptoms from other forms of meningitis, but they often appear more gradually and can be very mild at first, the CDC says.
Dr. William Schaffner, chairman of the Department of Preventive Medicine at Vanderbilt University Medical Center, told CNN that fungal infections are not usually mild. He said when a fungus invades small blood vessels, it can cause them to clot or bleed, which can lead to symptoms of small strokes.
In addition to typical meningitis symptoms like headache, fever, nausea and stiffness of the neck, people with fungal meningitis may also experience confusion, dizziness and discomfort from bright lights. Patients might just have one or two of these symptoms, the CDC says.
Health officials say any patients who received an injection at one of the facilities beginning May 21 and who began showing symptoms between one and three weeks after being injected should see their doctor right away.
The earlier a patient gets treatment, the more likely he or she will survive.
Patients are treated with anti-fungal medication, which is given intravenously, so patients have to be admitted to the hospital, the CDC said. Patients may need to be treated for months.
The FDA is urging anyone who has experienced problems following an injection with the NECC product to report it to MedWatch, the FDA's voluntary reporting program, by phone at 1-800-FDA-1088 or online at www.fda.gov/medwatch/report.htm.
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Google on Friday confirmed that it has acquired Slide, an online entertainment company focused on virtual communities.
The acquisition may help Google boost its social networking aspirations and fuels speculation that it is developing a social networking platform centered on games.
In a blog post, Google said that with the Slide acquisition, it is investing in more ways to add social networking features to Google services. Gmail, Docs, Blogger, Picasa and YouTube already have "strong social elements," David Glazer, an engineering director at Google, wrote in the blog post.
Slide develops virtual community applications used on social networking sites like Facebook and MySpace. On its Website, Slide says its goal is to build communities that allow virtual goods to be created and distributed.
Google did not reveal the price of the acquisition. Earlier this week, TechCrunch, which first reported the deal, said it was worth $182 million while The New York Times later put the value at $228 million.
A variety of recent rumors have suggested that Google is pursuing social networking opportunities around gaming. Reports have suggested that Google invested in Zynga, the company that built Farmville, the popular Facebook game. Other reports say that Google has been talking to gaming companies to help launch a social networking platform around games.
Some past attempts by Google to break into social networking have floundered. Google Buzz, which lets Gmail users share status updates, photos and videos, met with an uproar when it launched. That's because users quickly discovered that their lists of followers, automatically generated based on people they e-mail, were publicly shared with others. Google changed its privacy policies in response.
Google's Orkut has been popular in some regions, notably Brazil, but hasn't been broadly successful at the level of MySpace and Facebook.


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Whether it's magnetic nanoparticles (mNPs) giving an army of 'therapeutically armed' white blood cells direction to invade a deadly tumour's territory, or the use of mNPs to target specific nerve channels and induce nerve-led behaviour (such as the life-dependant thumping of our hearts), mNPs have come a long way in the past decade.
The future for mNPs however appears even brighter. With the design of 'theranostic' molecules, mNPs could play a crucial role in developing one-stop tools to simultaneously diagnose, monitor and treat a wide range of common diseases and injuries.
Multifunctional particles, modelled on viral particles such as the flu and HIV, are being researched and developed to carry signal-generating sub-molecules and drugs, able to reach target areas through a safe sprinkling of tiny mNPs and external magnetic forces, creating a medical means to confirm specific ailments and automatically release healing drugs while inside a living system.
A landmark selection of review articles published this week in IOP Publishing's Journal of Physics D: Applied Physics, 'Progress in Applications of Magnetic Nanoparticles in Biomedicine', shows just how far magnetic nanoparticles for application in biomedicine have come and what exciting promise they hold for the future.
The magnetic component of the direction-giving nanoparticles is usually an iron-based compound called ferric oxide which is coated in a biocompatible surface, sometimes using, for example, fatty acids, to provide stability during the particles journey through one's body. For biomedicine, the particles are useful because you can add specific signal triggering molecules to identify certain conditions, or dyes to help in medical imaging, or therapeutic agents to remedy a wide-range of afflictions.
Already well documented, mNPs have sparked interest after being attached to stem cells and used in vivo to remedy heart injury in rats. On humans, in 2007, Berlin's Charité Hospital used a technique which involved mNPs, called hyperthermia, to destroy a particularly severe form of brain cancer in 14 patients. The technique, utilising well-tested knowledge that tumour cells are more sensitive to temperature increases than healthy cells, uses mNPs to direct nano-heaters towards the inoperable tumours and, essentially, cook them to death.
Dr Catherine Berry, one of the review paper's authors from the Centre for Cell Engineering in Glasgow, writes, "One of the main forerunners in the development of multifunctional particles for theranostics is magnetic nanoparticles. Following recent advances in nanotechnology, the composition, size, morphology and surface chemistry of particles can all be tailored which, in combination with their magnetic nanoscale phenomena, makes them highly desirable."
From Friday, 6 November, the selection of review articles can be found at http://stacks.iop.org/JPhysD/42/i=22.


text: |THE MAORI MESSENGER. TE KARERE MAORI. VOL. VII.l AUCKLAND, APRIL 30, I860.AKARANA, APERIRA 50, 1860. [No. 8. The fullest information with respect to the disturbed state of Taranaki has been published in the "Messenger" for the special perusal of our native readers. If those publications which contained an exact and true narrative of the origin of the present question between the Government and W. Kingi te Rangitake, have been carefully read by right thinking natives, the recent hostile demonstrations on the part of some of the Rangiaohia and Kawhia natives may well occasion them feelings of uneasiness. Calm and deliberate consideration must, how- ever, make it apparent to all that there can be little to gain but much to lose by countenancing W. King's folly, or endorsing the murders recently committed by Ngatiruanui and Taranaki natives. What harm had the pakehas done to those tribes? The answer is, not any. We repeat itnot any, and challenge contradiction. Without the slightest provocation those mean people invaded the pakeha territory, plundered and burnt their houses, stole their cattle, devas- tated their farms, and cruelly murdered three unarmed settlers and two little boys! The latter were literally hacked to pieces. By whom were the little boys hacked to pieces? By men? Yes, by men, nearly all of whom have hitherto regularly assembled for Christian worship on the Sabbath day. Ko nga korero mo nga mahi whakaoho- oho e mahia nei ki Taranaki, kua oti te korero ki te Karere Maori hei tirohanga ma o matou boa Maori. Kei ena nupepa e mau ana te korero pono me nga tikanga o tera whawhai, o te Kawanatanga o. Wire- mu Kingi Te Rangitake, me tona putake i tupu ake ai. Na, me he mea kua ata kore- rotia mariretia aua korero e nga tangata Maori whakaaro tika tena e awangawanga te ngakau ki te mahi whakatupu whawhai o etahi o nga tangata Maori o Rangiawhia o Kawhia, kua rangona nei i roto i enei ra. Ki te ata huri huri marire, ka marama tenei whakaaro i nga tangata katoa, ara, kahore he pai e hua mai i runga i te whakauru, ki te mahi kuare a Wiremu Kingi i runga ra- nei i te whakatika ki te mahi kohuru a Nga- tiruanui a Taranaki, engari he kina anake ona hua, He aha koia te hara o te Pakeha ki era iwi? Kahore kau, a ka tuaruatia ano e matou tenei kupu, kahore kau ra he hara o te Pakeha. Kahore he take kahore he aha, i whakauka pokanoa ai era iwi tutua. Heoi, whakateka noa ake, haere mai aha ki runga ki to te Pakeha whenua, ko nga whare o nga Pakeha tahuna ana e ratou ki te ahi, ko nga kau, riro atu te tango, whakamara-