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Peptides from American alligator plasma are antimicrobial against multi-drug resistant bacterial pathogens including Acinetobacter baumannii
Stephanie M. Barksdale1,
Evelyn J. Hrifko2,
Ezra Myung-Chul Chung3,4 &
Monique. L. van Hoek1,3
Our group has developed a new process for isolating and identifying novel cationic antimicrobial peptides from small amounts of biological samples. Previously, we identified several active antimicrobial peptides from 100 μl of plasma from Alligator mississippiensis. These peptides were found to have in vitro antimicrobial activity against Pseudomonas aeruginosa and Staphylococcus aureus. In this work, we further characterize three of the novel peptides discovered using this process: Apo5, Apo6, and A1P.
We examined the activity of these peptides against multi-drug resistant strains and clinical isolates of common human pathogens. We investigated their structural characteristics using circular dichroism and tested for membrane disruption and DNA binding. These peptides were found to have strong in vitro activity against multi-drug resistant and clinically isolated strains of S. aureus, Escherichia coli, P. aeruginosa, and Acinetobacter baumannii. Apo5 and Apo6, peptides derived from alligator apolipoprotein C-1, depolarized the bacterial membrane. A1P, a peptide from the serpin proteinase inhibitor, did not permeabilize membranes. Performing circular dichroism analysis, Apo5 and Apo6 were found to be predominantly helical in SDS and TFE buffer, while A1P has significantly different structures in phosphate buffer, SDS, and TFE. None of these peptides were found to be hemolytic to sheep red blood cells or significantly cytotoxic up to 100 μg/ml after 24 h exposure.
Overall, we suggest that Apo5 and Apo6 have a different mode of action than A1P, and that all three peptides make promising candidates for the treatment of drug-resistant bacteria, such as A. baumannii.
Cationic antimicrobial peptides (CAMPs) are short proteins produced by the innate immune system of virtually all eukaryotic organisms [1]. CAMPs have a variety of structures, such as helical, beta-sheet, or mixed structures [2], and antibacterial mechanisms, including membrane perturbation [3], DNA binding [4], and enzyme interference [5]. There has been strong interest in the discovery and exploration of CAMPs due to rising drug resistance in pathogenic bacteria and the subsequent need for new therapeutics [1]. Traditionally, new CAMPs are identified through either bioinformatics or fractionation. CAMPS are a diverse group of peptides that can differ by nucleic acid sequence, amino acid sequence, and secondary structure [2], which can complicate the process of locating new CAMPs by analysis of gene and protein databases. Fractionation through HPLC or other methods typically requires large amounts of biological sample, usually a liter or more [6], which can be problematic when working with small or endangered animals. Fractionation and testing can also be challenging due to dilution of samples, as CAMPs can be present in very small amounts [6, 7].
Our group recently developed a novel and powerful process for discovering and identifying new and potentially useful CAMPs from biological samples, called the Bioprospecter process [8]. Our CAMP discovery process is sample-agnostic and requires very small sample volumes for analysis. We have successfully employed this process to discover a large number of new peptides from 100 μl of plasma from the American alligator, Alligator mississippiensis; five of these show antibacterial activity against Gram-positive and/or Gram-negative bacteria. Moreover, it is unique in its approach to CAMP identification in that it directly mines the native antimicrobial peptidome through custom-made hydrogel particles whose properties complement the physico-chemical properties of CAMPs [8]. This bioprospecting approach provides us access to the CAMP peptidomes of some of the world's most remarkable species, to dramatically expand the current CAMP library and potentially unlock the key to overcoming antibiotic resistance via the discovery of new antimicrobial peptides.
Using this tool, we previously described three novel peptides from A. mississippiensis (Apo5, Apo6, and A1P) with activity against laboratory strains of E. coli, P. aeruginosa, S. aureus, and Bacillus cereus [8]. Apo5 and Apo6 are highly related peptides; nested fragments of a purported apolipoprotein. Apolipoproteins have antimicrobial activity against a variety of pathogenic bacteria [9–14] (see Discussion). A1P is the C-terminal fragment of alpha-1-proteinase inhibitor of the serpin family; this protease inhibitor has broad protease inhibiting activity, as well as immunomodulatory effects [15]. In this work, we further characterize the antimicrobial activity of these peptides against bacterial pathogens resistant to multiple antibiotics as well as clinical isolates, including multi-drug resistant A. baumannii, which was not previously tested. In addition, we sought to determine the secondary structure of these peptides in order to understand the mechanism by which they exert their antibacterial activity.
Staphylococcus aureus ATCC 33592 (MDR) and BAA-1718, Escherichia coli ATCC 51659 (MDR) and 4157, Pseudomonas aeruginosa ATCC BAA-2110 (MDR), and Acinetobacter baumannii ATCC BAA-1794 (MDR) and 9955 were purchased from the American Type Culture Collection (Manassas, VA). Pseudomonas aeruginosa strain PAO1 was generously provided by Karin Sauer from Binghamton University (Binghamton, NY). All strains are clinical isolates except for E. coli ATCC 4157. Bacteria were grown in Nutrient Broth (Difco 234000) overnight in a shaking incubator (37 °C). Bacteria were aliquoted and frozen at -80 °C and enumerated via serial dilution and plating prior to experimentation.
All peptides were synthesized to order by ChinaPeptides, Inc (Shanghai, China) using Fmoc chemistry. Peptides were provided at >95 % purity, and purity and structure were confirmed with RP-HPLC and ESI-MS.
Physiochemical properties were calculated using the Antimicrobial Peptide Database (APD2) [16]. The percent hydrophobicity is defined as the ratio of hydrophobic residues to total residues. The full-length sequences for the apolipoprotein C-1 containing Apo5 and Apo6 (Accession XP_006276575.1) and for the alpha-1-proteinase containing A1P (Accession XP_006266331.1) were found on the BLAST NCBI database [17]. Ribbon models displaying the full proteins were created using SWISS MODEL [18–20]. The A. mississippiensis apolipoprotein C-1 was modeled on the human apolipoprotein C-1 (SMTL id: 1ioj.1.A, Sequence identity = 43.40 %), and the A. mississippiensis alpha-1-proteinase was modeled on alpha1-antitrypsin (SMTL id: 3dru.1.A, Sequence identity = 51.41 %) [18–23]. The secondary structure of Apo5, Apo6, and A1P was predicted using I-TASSER [24] and visualized with Chimera [25]. Helical wheel projections and hydrophobic moment were calculated using HeliQuest [26].
Circular dichroism spectroscopy
Circular dichroism (CD) was performed using a Jasco J-1500 spectropolarimeter. 100 μg/ml of peptide was used in each experiment. Samples were allowed to equilibrate for 3 min prior to data collection at 25 °C in a 1 mm path length cuvette. Spectra were collected from 190 to 260 nm at 0.2-nm intervals, with a data integration time of 4 s and a 1 nm bandwidth. Data presented is an average of four spectra. Peptides were analyzed in 10 mM sodium phosphate buffer (6.12 mM sodium monohydrogen phosphate heptahydrate; 3.92 mM monosodium phosphate anhydrous; pH 7.4), 50 % (v/v) trifluoroethanol (TFE) in phosphate buffer, or 60 mM sodium dodecyl sulfate (SDS) in phosphate buffer. Percent contribution to secondary structure was measured using methods determined by Raussens et al. [27].
Antimicrobial assays
The antimicrobial MIC activity of the peptides was first determined in cation-adjusted Mueller Hinton Broth (BD 212322) in a 96 well plate following the CLSI protocol. Enumerated bacteria were diluted in broth and 105 CFU was added to each well with varying dilutions of peptide. The plate was incubated for 24 h at 37 °C and then read on a spectrophotometer at OD600 nm.
The EC50 antibacterial activity of the peptides was determined in 10 mM phosphate buffer using resazurin as an indicator of CFU as previously described and validated by Bishop et al. [8] or by colony counting. The sequences and net charges of the peptides tested are shown in Table 1. In a polypropylene 96 well plate, 105 bacteria were incubated in 10 mM phosphate buffer with various dilutions of peptide (3 h; 37 °C) (1:5). For the resazurin method, cation-adjusted Mueller Hinton Broth (BD 212322, final concentration 2.2 %) and resazurin (final concentration 109 μM) dissolved in PBS were added to each well, and the plate was read kinetically overnight on a Tecan Safire2 spectrofluorometer (37 °C; excitation = 540 nm; emission = 590 nm). Percent survival after treatment was then calculated with previously determined growth curves (Table 2). For colony counting, experimental wells were serially diluted after incubation, and 8 μl of each dilution was spotted onto cation-adjusted Mueller Hinton Agar (BD 211438) and allowed to dry. Agar plates were then incubated overnight at 37 °C. After incubation, colonies were counted and percent survival was calculated based on untreated bacteria. GraphPad Prism 6.0 was used to calculate EC50 using log concentration vs. percent survival. LL-37, polymyxin B (against Gram-negative bacteria), and vancomycin (against Gram-positive bacteria) are used as controls. LL-37 is a well-studied human CAMP with broad-spectrum antimicrobial activity, polymyxin B is a peptide antibiotic effective against Gram-negative bacteria, and vancomycin is a peptide antibiotic used against Gram-positive bacteria. Each experiment was repeated at least twice, and a representative experiment is shown. 95 % confidence intervals (CI) are reported to indicate the error of each EC50 determination.
Table 1 Amino acid sequences and molecular weights of selected peptides (identified in [8])
Table 2 Antimicrobial assay bacterial reduction rate equations used to determine CFU after incubation with peptide
Ethidium bromide uptake assay
The ethidium bromide uptake assay was performed as previously detailed [28, 29] with some modifications. E. coli ATCC 51659 was grown overnight in cation-adjusted Mueller Hinton Broth in a shaking incubator (37 °C). Bacteria was centrifuged, washed with PBS, and then adjusted to an OD600 nm of 0.1 in 10 mM phosphate buffer. 180 μl of bacteria was added to 10 μl ethidium bromide (10 mM final concentration) and 10 μl peptide in various concentrations. The plate was read in a Tecan Safire2 spectrofluorometer every 2 min for 30 min (37 °C; excitation = 540 nm; emission = 590 nm). Peak RFU at 50 μg/ml was used in Fig. 5.
Membrane depolarization study
Membrane depolarization was studied using DiSC3(5) as has previously been reported [30]. Depolarization of a membrane can be visualized by a drop in fluorescence. Enumerated frozen bacteria were pelleted and washed twice in 10 mM phosphate buffer and then resuspended to 4x107 CFU/ml in 10 mM phosphate buffer containing 50 μg/ml DiSC3(5). 100 μl of this suspension was added to wells of a black 96 well plate. The plate was incubated in a Tecan Safire2 spectrofluorometer and monitored until fluorescence leveled off. 100 μl of various concentrations of peptide in 10 mM phosphate buffer was added to each well. Bacteria without peptide and peptide without bacteria served as controls. The plate was immediately returned to the spectrofluorometer. Readings were taken every 15 s for 5 min (excitation = 622 nm; emission = 670 nm). Peak RFU at each concentration was used in Fig. 6.
Gel shift assay
Non-specific DNA-peptide binding was determined by a gel-retardation experiment as described by Park et al. [4]. Briefly, 200 ng of plasmid DNA (pTrcHis, Invitrogen V36020) was incubated with increasing concentrations of peptides in 20 μl of binding buffer (5 % glycerol, 10 mM Tris–HCl [pH 8.0], 1 mM EDTA, 1 mM DTT, 20 mM KCl and 50 μg/ml BSA) at room temperature for 20 min and subjected to electrophoresis on a 1.0 % agarose gel. DNA bands were visualized by ethidium bromide staining.
Hemolysis and MTT assays
To measure the hemolytic activity of peptides, 2 % sheep red blood cells were added to various dilutions of peptide reconstituted in PBS in a sterile U-bottom 96 well plate. 2 % RBCs with PBS 1X alone served as the negative control, and 2 % RBC in water as the positive control. The plate was incubated for 1 h at 37 °C and then centrifuged at 1000 rpm for 2 min. The supernatant was transferred to a fresh plate and read at OD540 [31].
Cytotoxicity assays were performed using the Vybrant MTT Cell Proliferation Assay Kit (Life Technologies) according to manufacturer's instructions. Assays were performed using human lung epithelial lung carcinoma line A549 (ATCC CCL-185), which were maintained at a low passage in Dulbecco's Minimal Essential Media (Life Technologies 11995073) with 10 % heat-inactivated fetal bovine serum and 13 U/ml penicillin-streptomycin. 1–100 μg/ml of peptide was used for each experimental well. Each experiment was performed in triplicate two times. A representative experiment is shown.
Statistical analyses were performed using GraphPad Prism 6.0. To determine statistical significance, the one-way ANOVA with Tukey's multiple comparisons was performed in all instances.
The native, plasma-derived peptide sequences from the de novo sequencing [8] were used to predict the secondary structure and placement within the parent proteins. Apo5 and Apo6 are both part of the C-terminus of an apolipoprotein, apolipoprotein C-1. Apo5 comprises amino acids (aa) 64-86, while Apo6 is the smaller fragment (aa 67-86, shown in Fig. 1a). Both peptides have a +4 charge and a hydrophobic ratio of just over 30 % as determined using APD2 [16]. Apo5 is cleaved at a Glu-Phe site and Apo6 at a Thr-Lys site. In the full apolipoprotein, both cleavages sites are located in a disordered hinge preceding a C-terminal alpha helix as seen in Fig. 1c.
Primary and tertiary structure of parental proteins and peptide placement within. a Amino acid sequence of A. mississippiensis apolipoprotein C-1 and the Apo5 and Apo6 fragments. b Amino acid sequence of A. mississippiensis alpha-1-proteinase 2-like protein and the A1P fragment. c Ribbon model of apolipoprotein C-1 showing cleavages points of Apo5 (long dashes) and Apo6 (short dashes). d Ribbon model of alpha-1-proteinase showing cleavage point of A1P
A1P is also the cleaved C-terminus of its parent protein, a serpin proteinase inhibitor (aa 394-428, shown in Fig. 1b). It carries a +4 charge and a 45 % hydrophobicity ratio. The peptide is cleaved from the parent protein at Asp-Pro site in a disordered region on the exterior of the folded protein, as shown in Fig. 1d. The cleaved peptide itself consists of the two β-sheet regions that run through the interior of the proteinase.
Secondary structure determination
To determine general secondary structure of Apo5, Apo6, and A1P, CD spectroscopy was used. CD was performed in 10 mM phosphate buffer, 60 mM SDS, and 50 % TFE in phosphate buffer. Many CAMPs, such as LL-37, maintain a random or disordered structure until associated with a membrane or micelle. SDS forms micelles with a negatively charged surface [32], mimicking the bacterial membrane and forcing the CAMP into a more ordered conformation [33, 34].
TFE is used in CD to promote a helical structure [35] and stabilize secondary structure [36].
As expected, Apo5 (Fig. 2a) and Apo6 (Fig. 2b) had nearly identical CD spectra. Both peptides have random coil and β-sheet characteristics in 10 mM phosphate buffer, and are primarily α-helical when CD is measured in buffers with SDS and TFE. Interestingly, Apo5, the longer of the two peptides, is calculated to have more α-helical character than Apo6 in both SDS (63.3 % vs. 57.0 %) and TFE (51.0 % vs. 50.3 %), shown in Table 3. When evaluating the α-helical properties of these two peptides by simple intensity at 208 nm and 222 nm, it is notable that the peaks at these wavelengths are more intense for Apo5 than Apo6, though the two Apo peptides maintain 12.5 % turn no matter the buffer used. Apo5 and Apo6 likely have a primarily α-helical structure with some random coil portions. Based on helical wheel projections, it appears that both Apo5 and Apo6 have significant amphipathic character, with several hydrophobic residues on one face and several basic amino acids on the other. The structure of Apo5 and Apo6 were predicted using I-TASSER and the resulting.pdb file was visualized using Chimera, shown in Fig. 3a and b. Consistent with the CD spectra, both are predicted to be α-helical structures. Apo5, the longer peptide, has a longer random coil portion at the N-terminus. This is consistent with the predicted structure of the C-terminal portion of the parental protein, apolipoprotein C-1 (Fig. 1c). Because Apo5 and Apo6 are predicted to helical, helical wheels were produced using Heliquest and modified (Fig. 4). Apo5 and Apo6 have a similar hydrophobic moment (0.436 vs 0.484); Apo6 likely has a slightly stronger hydrophobic moment due to the loss of polar Ser in the hydrophobic face of the helix. In 10 mM phosphate buffer, TFE, and SDS, A1P has significantly different spectra (Fig. 2c).
In 10 mM phosphate buffer, A1P is calculated to have primarily β-sheet contributions (57.9 %), as well as some random coil characteristics (34.7 %), shown in Table 3. By our calculations, A1P also has a negative percentage of contribution from the α-helix, which may be an artifact of the equations used. In TFE, A1P maintains its random coil nature (31.2 %), but also becomes strongly α-helical (38.2 %), with only 16.3 % contribution from the β-sheet. In SDS, A1P is calculated to be nearly equal parts random coil (36.9 %), turn (21.5 %), β-sheet (24.2 %), and α-helix (21.6 %). It has been shown by several groups that TFE can induce and support α-helical structures in peptides [37, 38]. By qualitatively evaluating the spectra, we conclude that only in TFE does A1P have a notable α-helical structure, though this conformation may be interrupted by several proline residues found along the polypeptide chain. It is likely that A1P has a mixed structure that may change dramatically based on environmental factors. The secondary structure of A1P was predicted by I-TASSER and visualized using Chimera. I-TASSER predicts that A1P is primarily random coil with two anti-parallel β-sheet in a hairpin formation (Fig. 3c), which corresponds with the structure of the full alpha-1-proteinase structure.
CD spectra of peptides to determine secondary structure. a Apo5, b Apo6, c A1P. All spectra were taken with peptide concentrations of 100 μg/ml in a 1 mm pathway cuvette. Spectra were read in 10 mM phosphate buffer (long dashes), 50 % TFE in 10 mM phosphate buffer (dash dot dash), and 60 mM SDS in 10 mM phosphate buffer (solid line)
Table 3 Percent secondary structure contribution as calculated by a method described by Raussens et al. [27]
Secondary structure prediction by I-TASSER [24], visualized by Chimera [25]. a Apo5, b Apo6, c A1P
Helical wheels of a Apo5 and b Apo6, modified from Heliquest
Antimicrobial activity
Previously, we reported that Apo5 and Apo6 had strong broad-spectrum antimicrobial activity against E. coli and B. cereus, as well as S. aureus and P. aeruginosa [8]. It was also found that A1P had greater activity against S. aureus than P. aeruginosa [8], indicating it may have stronger activity against Gram-positive organisms.
Antibiotic resistance has been increasing steadily for the past several decades, and CAMPs are considered a possible basis for novel antimicrobials. Because of this, these new peptides were tested against clinically isolated and multi-drug resistant strains of S. aureus, E. coli, P. aeruginosa and A. baumannii. Numerical and statistical data can be found in Table 4. Sharing a salt-sensitive phenotype with LL-37 [34, 39, 40], these peptides had no effect in MIC experiments as high as 250 μg/ml, so experiments were performed to determine the EC50 in 10 mM phosphate buffer. We performed these low-salt experiments using vancomycin as a control for Gram-positive bacteria and polymyxin B as a control for Gram-negative bacteria. Polymyxin B was found to be very effective against all strains tested, with all EC50 values being under 1 μg/ml, except against E. coli ATCC 4157 (2.50 μg/ml). In the case of vancomycin, we found that this antibiotic did not kill either S. aureus strain under 100 μg/ml, though the MIC for both strains was 1 μg/ml, indicating that vancomycin is bacteriostatic but not reliably bactericidal.
Table 4 Antimicrobial activity and statistical data
In general, it was found that the EC50 values of Apo5 and Apo6 were statistically similar and showed broad-spectrum activity. We found that both apolipoprotein-derived peptides had strong activity against a clinical isolate of S. aureus ATCC BAA-1718 (EC50 < 5 μg/ml). Both Apo5 and Apo6 were found to have very strong activity (EC50 < 1 μg/ml) against drug sensitive and MDR A. baumannii (ATCC BAA-1794), MRSA (ATCC 33592), and MDR P. aeruginosa (ATCC BAA-2110). It was also found that Apo5 and Apo6 were somewhat less active against both strains of E. coli tested, with EC50 values ranging from 4 to 20 μg/ml, though our previous work demonstrated that these peptides were extremely effective against E. coli ATCC 25922 [8]. Apo5 and Apo6 had differing activities against P. aeruginosa PAO1; Apo5 was found to have stronger activity against this strain than Apo6 (0.0878 μg/ml vs 1.17 μg/ml).
A1P was found to have stronger broad-spectrum activity than anticipated from our previous study [8]. Although A1P had weak activity against P. aeruginosa PAO1 (EC50 = 38.6 μg/ml), it was not effective at concentrations tested against MDR P. aeruginosa (ATCC BAA-2110) (Table 4). A1P had stronger antimicrobial activity against MDR strains of E. coli (ATCC 51659), S. aureus (ATCC 33592), and A. baumannii (ATCC BAA-1794), with EC50 values between 2 and 3 μg/ml, than against the antibiotic sensitive strains tested, which had EC50 values ranging from 9 to 36 μg/ml.
Membrane permeabilization and depolarization by peptides
To determine whether Apo5, Apo6, and A1P interacted with the bacterial membrane, each peptide's ability to disrupt or permeabilize the membrane was measured by the ethidium bromide uptake assay, while membrane depolarization was measured with the fluorescent chemical DiSC3(5), which is sensitive to the polarization of membranes.
When the ethidium bromide uptake assay was performed (Fig. 5a), it was found that Apo5 and Apo6 permeabilized the E. coli membrane at concentrations at 50 μg/ml quickly, comparable to control peptide LL-37, a known pore-forming peptide. Neither Apo peptide permeabilized membranes at lower concentrations. Like LL-37, these peptides permeabilized the membrane in a significant manner (p < 0.001), with peak fluorescence occurring within 3 min. A1P also permeabilized the membrane at 50 μg/ml significantly higher than the untreated control (p < 0.001), but displayed very different and slower kinetics. With this peptide, fluorescence gradually increased over the 20 min experimental time frame, until reaching nearly equivalent maximum fluorescence as Apo5 and Apo6 by the end of the experiment (Fig. 5b).
Pore-forming activity by Apo5, Apo6, and A1P. An increase in fluorescence demonstrates greater binding of DNA by ethidium bromide, which indicates the formation of pores in the bacterial membrane. a. Kinetics of permeabilization and binding of ethidium bromide to DNA after treatment with Apo5 (dashes), Apo6 (solid line), A1P (dots), and LL-37 (dash dot) b. Permeabilization of membrane after 20 min
Depolarization of a bacterial membrane indicates transient membrane disruption that allows for ion leakage, which damages the proton motive force and other gradients that store chemical energy. As shown in Fig. 6, within 1 min it was found that Apo5 and Apo6 depolarized bacterial membranes at concentrations as low as 0.5 μg/ml (p < 0.001), with depolarization showing a clear dose-dependent response to peptide concentration. A1P did not depolarize membranes except at the highest concentration tested, 50 μg/ml (p < 0.05). Depolarization signals at this concentration were well below those achieved at the lowest concentrations used for Apo5, Apo6, and LL-37 (p < 0.001).
Membrane depolarization activity by Apo5, Apo6, and A1P. Depolarization determined using DiSC3(5) for each peptide at 50 μg/ml (■), 5 μg/ml ( ), 0.5 μg/ml ( ), as well as no treatment ( )
These results indicate that Apo5 and Apo6 depolarize the bacterial membrane quickly, suggesting membrane disruption is the mechanism by which these peptides kill bacteria. A1P does not depolarize the membrane, nor does it form pores quickly except at high concentrations. This implies that the primary mechanism of A1P is not related to membrane disruption.
Some peptides, such as LL-37 or histone-derived Buforin II, bind nucleic acids [4, 41], inhibiting translation and transcription or promoting mutagenesis. In general, this binding mechanism has been shown to be non-specific [41, 42]. To determine if any of the CAMPs were able to bind DNA, a gel shift assay was performed, shown in Fig. 7. A1P bound DNA only at very high ratios, needing at least 20 times more mass of peptide than DNA to inhibit DNA movement. Apo6 did not bind DNA at any concentration, while Apo5 bound DNA only at the highest concentration tested. It is unlikely that the primary mechanism of action of these three peptides is related to DNA binding.
DNA binding by a Apo5, b Apo6, c A1P, and d LL-37 as measured using a gel shift assay. Ratio of DNA:CAMP is shown
The physiochemical properties of CAMPs preclude significant host-cell cytotoxicity; however, some CAMPs, such as SMAP-29, have been found to cause damage to eukaryotic cells at similar concentrations. Cytotoxicity against red blood cells and A549 lung epithelial cells was measured. For red blood cells, a spectrophotometric assay that measures free heme was used, while an MTT assay was used for A549 cells. As shown in Table 4, these peptides have EC50's between 0.07–39 μg/ml. In Fig. 8a, hemolytic activity is shown as percent hemolysis. All four peptides showed hemolysis of RBCs of less than 1 % at 300 μg/ml, a, comparable to the control peptide LL-37. No statistically significant difference was found between the untreated control and all peptides, indicating that Apo5, Apo6, and A1P are not hemolytic. The MTT assay was used to measure cytotoxicity of other cells lines. After 24 h exposure, concentrations of peptide up to 100 μg/ml were not significantly cytotoxic against A549 cells, as shown in Fig. 8b, while the EC50 against A. baumannii was less than 1 μg/ml of Apo peptide.
Peptide Cytotoxicity a. Hemolysis of sheep red blood cells was measured using a photometric assay after exposure to 300 μg/ml for 1 h and b. Inhibition of Proliferation of A549 human lung epithelial cells measured using the MTT assay after 24 h exposure. Apo5 (■), Apo6 ( ), A1P ( ), LL-37 ( )
Using our CAMP discovery process and only 100 μl of alligator plasma, we have previously identified five novel antimicrobial peptides from A. mississippiensis that exhibit antibacterial activity [8]. Our bioprospecting-based process provides a unique access to the antimicrobial peptidome, and is a significant advance in the effort to identify novel antimicrobial peptides in nature. In this study, we present detailed characterization of the structure and function of three alligator plasma-derived peptides: Apo5, Apo6, and A1P. We demonstrated that Apo5, Apo6, and A1P are potent antimicrobial peptides that extend their efficacy against multi-drug resistant and clinically relevant pathogens, such as A. baumannii.
The two peptides Apo5 and Apo6 are both derived from a predicted apolipoprotein C-1 in A. mississippiensis. Apolipoproteins bind lipids; apolipoprotein C-1 in particular is known to bind phospholipids and is a marker of apoptosis [11, 12, 43, 44]. Apo6 is a smaller derivative of Apo5; Apo5 contains residues 64-88 of predicted apolipoprotein C-1, while Apo6 contains residues 67-88. These peptides were very active against Gram-negative bacteria. Previously, Apo5 and Apo6 were each found to be strongly active against E. coli ATCC 25922 and P. aeruginosa ATCC 9027 while EC50 values were much higher for virulent Staphylococcus aureus (ATCC 25923). We discovered that Apo5 and Apo6 are predominantly helical peptides. Their mode of antibacterial activity appears to be primarily through membrane interaction. At low concentrations (approximately the EC50 for all bacteria), Apo5 and Apo6 depolarized the membrane of E. coli. At high concentrations, well above the EC50 for all bacteria tested, these peptides more strongly disrupted the bacterial membrane, possibly via pore formation, allowing for the escape of large cellular milieu. Based on these observations, it seems that the primary mode of action of these apolipoprotein-derived peptides is membrane perturbation and depolarization.
The antimicrobial activity of apolipoproteins has been highlighted in other organisms, although typically apolipoprotein A is studied [9–12, 14, 45]. Many whole apolipoproteins have been found to have antimicrobial activity against a variety of bacteria, as well as some viruses. Apolipoprotein A-1 from various fishes have been shown to have antimicrobial activity against E. coli [11, 12, 46], Streptococcus spp. [11] and fish pathogens such as Mycobacterium marinum [11] and Yersinia ruckeri [12]. The antimicrobial activity of human apolipoprotein A-1 has also been examined, and several groups have found that this protein is effective against E. coli, Klebsiella pneumoniae, and Yersinia enterocolitica [10, 44]. The primary mode of action of apolipoprotein A-1 seems to be as a detergent; it has been shown to bind lipids including LPS and to dissolve micelles [12, 44, 47]. The LPS-binding activity in particular has been shown to be reliant on the N-terminus of the protein [44]. The mechanism of action and antimicrobial activity of apolipoprotein C-1 has not yet been explored, but it likely binds lipids as some part of its mechanism.
Apo5 and Apo6 were not found to bind E. coli LPS as part of its mechanism (data not shown), but these two peptides are located on the C-terminus of apolipoprotein C-1. To our knowledge, fragments of apolipoproteins have not been characterized prior to this work. Other groups have studied synthetic mimetics of apolipoprotein E, which have been found to have anti-infective effects against HIV-1 and HSV-1 [48] and antimicrobial activity against MDR bacteria [14]. This characterization of the broad antimicrobial activity of apolipoprotein C-1-derived peptides in the present work adds to the understanding of this family of CAMPs.
A1P is a fragment of a predicted alpha-1-proteinase. A1P contains amino acids 394-428 of this proteinase inhibitor. Though the primary role of alpha-1-proteinase is the inhibition of human neutrophil elastase, it also inhibits a number of other proteases, such as proteinase 3 [49], some kallikreins [50], matriptase [51], and caspase-3 [52]. It has also been shown to have an important role in immune modulation. Alpha-1-proteinase is an acute-phase reactant; levels of this protein increase 3 to 4 fold in response to PAMPs or inflammation [53, 54]. Alpha-1-proteinase is involved in neutrophil degranulation [55]. It has also been shown to protect mice from LPS- and TNF-associated lethality, possibly by preventing the release of membrane-bound TNF [56]. Alpha-1-proteinase also interacts with human neutrophil peptides to reduce their cytotoxic affects towards lung cells; this has been demonstrated in vitro and in vivo [57].
This protein may play a role in the disease progression of some bacteria such as Francisella tularensis. Studies have shown that alpha-1-proteinase is decreased during a pulmonary F. tularensis challenge in mice. This could lead to unchecked neutrophil elastase activity, which damages the alveoli and lung tissue, allowing bacteria to quickly disseminate through the body and to the liver [58]. Though other protease inhibitors have been shown to have antimicrobial activity, previously α-1-proteinase and its fragments had only been shown to have anti-infective activity, not direct antimicrobial activity. The whole protein has shown to have protective activity against P. aeruginosa, by suppressing bacterial proliferation and tissue inflammation [59, 60]. Whole alpha-1-proteinase, as well as a fragment and cyclic derivatives, have demonstrated the ability to prevent HIV-1 infection, possibly by inhibiting production of the virus [61–63]. Interestingly, this anti-HIV-1 fragment is the C-terminal fragment of the protein, where A1P is also found. The antibacterial mechanism of action is not yet clear from the scope of this study. A1P was not found to depolarize the bacterial membrane or form larger pores except at concentrations much higher than calculated EC50 values. A1P was also not found to bind DNA effectively. However, considering that A1P does interact with the membrane and form pores in high concentrations slowly, as discussed in section 3.4, it is possible that A1P crosses the bacterial membrane and interacts with a non-nucleic acid target or inhibits bacterial proteases that are necessary for viability. It seems possible that the anti-HIV-1 function described by other groups is the same function that kills bacteria.
Vancomycin and polymyxin B are drugs of last resort in the treatment of extremely multi-drug-resistant bacteria, and yet resistance to even these two drugs is currently rising [64–66]. In addition, both vancomycin and polymyxin B are known to be hemolytic and cytotoxic to some cell lines [67, 68]. It is clear that new antimicrobials are needed, particularly drugs with low cytotoxicity. LL-37 is a well-studied peptide with broad-spectrum activity. In this study, we have used LL-37 as a peptide control. We found that the alligator CAMPs tested had comparable activity to LL-37 against some species. LL-37 was found to have very strong activity against all species tested, with EC50 values all below 3 μg/ml. Apo5 and Apo6 had comparable activity against strains of P. aeruginosa, A. baumannii, and MRSA (ATCC 33592), but were less active against strains of E. coli tested, with EC50 values ranging from 2–20 μg/ml. These peptides also had similar membrane perturbation activity as LL-37, with membrane depolarization at low concentrations and pore formation and disruption at very high concentrations. A1P had weaker activity than LL-37 against susceptible strains of bacteria tested (EC50 values between 9 and 40 μg/ml) but stronger and comparable activity to LL-37 against drug-resistant strains of E. coli, S. aureus, and A. baumannii, with EC50 values less than3 μg/ml. Interestingly, A1P was not active against drug-resistant P. aeruginosa. We performed experiments with ethidium bromide and Disc3(5) using A1P against both strains of P. aeruginosa at 40 μg/ml, the approximate EC50 of A1P against P. aeruginosa PAO1, to determine if this difference in activity was due to differences in membrane interaction (data not shown). However, there was no difference in either depolarization or disruption. In our previous work, we found that A1P was least effective against P. aeruginosa ATCC 9027 in a panel also including other strains of S. aureus, E. coli, and Bacillus cereus [8]. It is possible that P. aeruginosa is intrinsically resistant to this particular peptide.
The novel CAMPs characterized here are among the first discovered using our hydrogel bioprospecting technology, illustrating the power of this technology to capture naturally occurring small peptides with net cationic charge and antimicrobial activity [8]. Considering the strong and broad-spectrum activity of these CAMPs, it seems likely that there is a greater role for protein fragments in peptide-based innate immunity than previously thought. Apolipoproteins are a highly conserved class of proteins, and it is very likely that fragments are produced in similar patterns across species. For example, though the human apolipoprotein C-I (accession AAH55093) shares 41 % identity with the American alligator version, the C-terminal 25 aa on each protein, where Apo5 is found, shares 59 % identity. This theoretical fragment of the human apolipoprotein C-I, with the sequence ELSAKMREWFSETFQKVKEKLKIDS, is also predicted to be helical, with a weaker hydrophobic moment (0.375 μH) and similar hydrophobicity (36 %), but is less charged the alligator Apo5 (+1), according to Heliquest [26] and The Antimicrobial Peptide Database [16]. The human peptide is not predicted to be antimicrobial by CAMPR3, but neither is Apo5, and we have discussed previously the weaknesses of commonly used antimicrobial peptide predictors [8]. We are currently working to determine whether some of these fragmentation patterns are conserved across species.
In addition, these CAMPs are more effective against drug-resistant strains than normal laboratory strains, indicating these CAMPs may be valuable basis for treatments against multi-drug resistant bacteria. Considering the strength of Apo5 and Apo6 on MDR A. baumannii in particular and the low cytotoxicity of these peptides, these are strong candidates for in vivo testing, or to use as scaffolds for stronger synthetic CAMPs.
Apo5, Apo6 and A1P are antimicrobial peptides found the plasma of the American alligator. Apo5 and Apo6 are alpha-helical C-terminal fragments of apolipoprotein C-1. These peptides have strong activity against S. aureus and a number of Gram-negative bacteria, including MDR A. baumannii and P. aeruginosa. Apo5 and Apo6 primarily work by depolarizing the bacterial membrane. A1P is a C-terminal fragment of the alligator alpha-1-proteinase. It has strong activity against S. aureus and a number of Gram-negative bacteria, but not MDR P. aeruginosa. A1P has a mixed structure, and the mechanism of action was not clear based on experiments performed; however, it does seem to slowly disrupt the bacterial membrane.
These peptides are fragments of conserved proteins found in many animals, indicating that these peptides could play a role in immune response of many animals. In addition, new antimicrobials are desperately needed against MDR Gram-negative bacteria. Apo5 and Apo6 in particular have strong activity against MDR pathogens of clinical interest, and the native peptides or synthetic variations will make strong candidates for in vivo testing and pre-clinical trials.
ATCC, American type culture collection; BLAST, basic local alignment search tool; BSA, bovine serum albumin; CAMP, cationic antimicrobial peptide; CD, circular dichroism; CI, 95 % confidence intervals; DiSC3(5), 3,3'- dipropylthiadicarbocyanine iodide; DTT, dithiothreitol; EC50, half maximal effective concentration; EDTA, ethylenediaminetetraacetic acid; ESI-MS, electrospray ionization mass spectrometry; Fmoc, fluorenylmethyloxycarbonyl; HIV, human immunodeficiency virus; HPLC, high performance liquid chromatography; LPS, lipopolysaccharide; MDR, multi-drug resistant; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NCBI, National Center for Biotechnology Information; OD, optical density; PAMP, pathogen-associated molecular pattern; PBS, phosphate buffered saline; RBC, red blood cell; RFU, relative fluorescence units; SDS, sodium dodecyl sulfate; TFE, trifluoroethanol; TNF, tumor necrosis factor
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We thank Dr. Barney Bishop for continued collaboration and Dr. Joel Schnur and the "peptide team" for support and helpful discussions. Many thanks to Robert Ulrey and Dr. Kajal Gupta for technical assistance and to Dr. K. Sauer (Binghamton University) for PA01.
SMB, EJH, EMCC and MVH are supported by HDTRA1-12-C-0039 "Translational Peptide Research for Personnel Protection." Circular dichroism was performed on a CD instrument purchased with funds from the same grant (HDTRA1-12-C-0039). The funding body played no role in the design of the study, the collection, analysis, and interpretation of data, and in writing the manuscript.
The datasets during and/or analysed during the current study available from the corresponding author on reasonable request.
SMB performed antimicrobial assays, ethidium bromide uptake assays, membrane depolarization assays, CD spectroscopy, bioinformatics, and statistical analysis and drafted the manuscript. EJH performed hemolysis and cytotoxicity studies and antimicrobial assays. EMCC performed gel shift assays and assisted with ethidium bromide uptake assays and membrane depolarization assays. MVH developed the study and its design and helped draft the manuscript. All authors read and approved the final manuscript.
The authors declare they have no competing interests.
School of Systems Biology, George Mason University, Manassas, VA, USA
Stephanie M. Barksdale & Monique. L. van Hoek
College of Science, George Mason University, Manassas, VA, USA
Evelyn J. Hrifko
National Center of Biodefense and Infectious Diseases, George Mason University, 10920 George Mason Cir, 10920 George Mason Circle, MSN 1H8, Manassas, VA, 20110, USA
Ezra Myung-Chul Chung & Monique. L. van Hoek
Present Address: STCube Pharmaceuticals, Inc., 401 Professional Dr. Suite 108, Gaithersburg, MD, 20879-3429, USA
Ezra Myung-Chul Chung
Stephanie M. Barksdale
Monique. L. van Hoek
Correspondence to Monique. L. van Hoek.
Barksdale, S.M., Hrifko, E.J., Chung, E.MC. et al. Peptides from American alligator plasma are antimicrobial against multi-drug resistant bacterial pathogens including Acinetobacter baumannii . BMC Microbiol 16, 189 (2016). https://doi.org/10.1186/s12866-016-0799-z
Antimicrobial peptides
Alligator mississippiensis
Multi-drug resistant bacteria


A review paper by van Buul et al. has concluded that the current evidence does not link the consumption of fructose and fructose-containing sugars with the global obesity epidemic.
A recent review found no association between fructose and several factors such as body weight, glycemic control and blood pressure.
This research was intended to test whether a high-fructose diet would induce leptin resistance in rats.
The article appearing in The Environmental Magazine entitled, "Sugar Or Sweetener? Sucrose Has Its Problems, But So Do Artificial Substitutes," by Brian C. Howard is to be complemented for identifying excess calorie intake relative to caloric expenditure (exercise) as a dominant contributor to the current obesity.


If is a powerful little word. Some people are stuck in "if only," trudging through lives marked with regret. But God wants us to live lives marked with possibilities, with the "what if" attitude that looks forward to the future with confidence. God is always on our side. Every day, in every way. You will need to purchase the workbook which will be available in the RPC bookstore. We will also have them available the first night. Please join us for this 6 week series beginning Tuesday, March 22nd at 7:00 PM.


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The Antique Corsage Tiara
The Antique Corsage Tiara (Photo: Chris Jackson/Getty Images)
The vaults of the former Greek royal family are filled with heirloom jewels, but a number of the pieces they own today came from the Danish royal family through Queen Anne-Marie. Today's tiara, the Antique Corsage Tiara, was the first tiara she ever owned.
The tiara that was given to the future Greek queen began its life as another piece of jewelry: a corsage ornament (or stomacher) that belonged to Queen Victoria of Sweden, Anne-Marie's great-grandmother (hence the piece's usual name).
The corsage was inherited by Victoria's granddaughter (and Anne-Marie's mother), Queen Ingrid, who was born a Swedish princess. Ingrid wore the piece, which is made of diamonds and pearls, as a necklace and as a brooch; it was she who had the piece mounted on a tiara frame as a gift for her daughter.
Anne-Marie received the tiara on her eighteenth birthday in 1964. But not even three weeks later, she wasn't a Danish princess anymore — she turned eighteen on August 30 of that year and married King Constantine II of Greece in Athens on September 18.
Photo: Pascal Le Segretain/Getty Images
But although as queen she had access to many of the large and historical diadems of the Greek royal family, Anne-Marie still wore her birthday tiara on occasion. When her own daughters, Alexia and Theodora, grew to adulthood, they too wore their mother's tiara at various royal events. In the photo above, Princess Theodora wears the tiara at the wedding of her cousin, Prince Joachim of Denmark, in 2008.
Photo: ANDREW WINNING/AFP/Getty Images
In recent years, the tiara has also become something of a secondary wedding tiara for the family (with the Khedive tiara, of course, on reserve as the wedding tiara for all of Queen Ingrid's female descendants). Above, Marie-Chantal Miller wears the tiara at her wedding to Crown Prince Pavlos, Anne-Marie's eldest son, in 1995.
Photo: Nikolas Kominis - Pool/Getty Images
Anne-Marie's other daughter-in-law, Tatiana Blatnik, also wore the tiara on her wedding day; she married Anne-Marie's second son, Prince Nikolaos, in 2010. I imagine we might see it once more on a bride if Prince Philippos, her youngest son, marries in an elaborate ceremony.
Photo: Chris Jackson/Getty Images
We also saw this tiara on Princess Tatiana in 2013 at the wedding of Princess Madeleine of Sweden -- an event that brought the tiara back to one of its earlier royal homes.
Note: This is an updated version of an earlier post, with new text/images.
Labels: denmark , Greece

redpajama_set_name: RedPajamaCommonCrawl


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Gorokan Public School Newsletter - Week 8, Term 4, 2020
Principal's Report
At Gorokan Public School we believe that student achievements, efforts and talents must be acknowledged and celebrated. We also believe that opportunities for inspiration often come from these celebrations.
2020 has truly been a year like no other. As COVID-19 continues to spread around the world, it has transformed children's day-to-day lives. It has affected schooling in most countries, and whilst we still have some restrictions in place, we have fared well in comparison.
When schools were closed earlier this year, many roles changed to support learning from home. Our teachers became facilitators of learning, supporting parents and carers in their new role of teacher. Our dedicated teaching and non-teaching staff worked together to achieve a coordinated approach to support learning from home.
Our teachers developed an online learning program that would maintain a high standard of education for our students. As a team, we developed and continually refined a platform from which teachers could collaboratively timetable their lessons, making it easy for families to use from home. The platform was improved through regular feedback from the community and became an inspiration to other schools.
The school invested heavily in online educational subscriptions for students to access from home and we produced information videos to assist students and families in their use. Daily live streaming was available for students and parents to stay connected with myself and the staff, and proved to be a lot of fun in supporting home learning and promoting an authentic connection between home and school.
Regular recorded community updates were introduced to keep parents and carers up-to-date with what was happening at school. These updates provided important up-to-date information regarding any adjustments to routines that were applied in response to the pandemic. I would like to thank our supportive community for responding so well to the changes that have been put in place over the course of the year.
One of these measures was the exclusion of non-essential adults from our school. For a school which prides itself in working in partnership with its parents, carers and community, this was extremely difficult. But for the safety and wellbeing of the students, and staff, together we continue to make a difference working together from a distance.
Our dedicated and caring teachers have made regular contact with their students and families to ensure support for learning was provided throughout the remote learning phase and beyond. I know that this was greatly appreciated and worthwhile. I am very grateful for the wonderful staff that we have at our school and their efforts during this time and always.
Behind the scenes, the school has worked diligently to ensure the ongoing improvements of its facilities and some achievements this year include:
•	The construction of the K-2 sports shed,
•	The tennis court upgrade,
•	The resurfacing of the basketball courts and the installation of handball courts,
•	A major overhaul of the school's technology infrastructure and the purchasing of devices to support learning for all students,
•	The commissioning of two new murals designed by two of our talented students, and
•	Further enhancements to the Stephanie Alexander Kitchen Garden program which has provided our Year 6 classes with an opportunity to counteract some of the disappointment of cancelled events in their last year of primary school.
2020 was also a year when our great school shone in the broader community. Our efforts in technology over the past few years and in our response to remote learning, led to the school being awarded the Google Reference School status. A highly prestigious award that allows us to trial new programs and provide feedback directly to the Google team in relation to educational programs and tools.
Gorokan Public School was also featured twice in the Department of Education's Technology for Learning magazine this year and the school's response to the pandemic was reported to the community through the media via radio and newspaper articles.
Fostering a sense of pride in our school, has been the intention behind the establishment and maintenance of our stunning grounds and this has resulted in the school taking out two major awards in the 2020 Wyong District Garden Competition:
•	First Place - School Culinary Garden, and
•	First Place - School Garden.
The Kindergarten orientation restrictions provided another opportunity for the school to demonstrate our innovative approach towards adversity. Unable to hold our usual comprehensive program, the school produced a series of podcasts to inform and support our new students and their families which included:
•	a virtual tour of the school, and
•	information sharing videos covering important topics such as:
-	Learning and support,
-	Managing and promoting positive student behaviour,
-	How English and Mathematics are taught, and
-	How technology supports learning at our great school.
Throughout this year of many restrictions, the school looked for ways to support engagement through fun activities. This was certainly achieved by the Colour Explosion Fun Run, organised by our dedicated Stage 3 team. The day was a huge success and arguably a significant highlight for students and staff in this year of restrictions and compromise.
In my seven years as principal of this great school, my mantra has always been that together we can make a difference. I would now like to acknowledge some of the people who have worked in partnership with the school and myself to ensure the best possible outcomes for our students.
I will start with the 2020 student leadership team. These students served their school to the highest standard. They consistently set a standard of safe respectful learning and adjusted to the restrictions that were in place throughout their leadership time. They assisted me in the delivery of live streamed and recorded whole school assemblies keeping the school informed and celebrating student and school achievements, building their leadership skills each step of the way.
Congratulations to the 2020 student leadership team consisting of: School Captains - Hannah T and Kingston S, School Vice Captains - Aimee M and J J and Prefects - Thomas M, Chloe M, Gabriella D and Jorjah C.
Year 6 Gift - As you may know, this year marks the 40th anniversary of our school. What you may not know is that one of our teachers has worked diligently at Gorokan Public School since it opened its doors all those years ago. She is well respected by the school and broader community and has received the NSW State Award for Excellent Service to Sport. It is lovely that our Year 6 students chose to honour this service through their gift this year which is a beautiful sign naming our freshly resurfaced basketball/netball court - The Gail Farrell Courts in her honour. Thank you, Year 6, for this thoughtful gesture of appreciation.
Presentation Day Assembly – This year our Presentation Day Assembly will be broadcast to classrooms and the community at 10:00am on Tuesday 8th December. Congratulations to our deserving award recipients and to all of our great students for their personal achievements this year.
2021 Student Enrolments
If you have a child who will not be returning to our school next year (excluding Year 6) could you please inform the office. The number of students we have plays a vital role in our planning of class structures. As in previous years our classes will commence the new year with the students returning to their 2020 class, where they will remain for the shortest time possible until our final student numbers are confirmed. We have found this to be the best practice to ensure a smooth transition for the students. Please be reassured that all students will commence the new year being engaged in their grade appropriate work.
Drop Off and Pick Up Procedures – Due to the uncertainty of the world health situation, the current drop off and pick up arrangements will continue for the remainder of the year and the beginning of the new year until further guidelines are provided. A community update will be provided when school resumes in the new year to outline any changes to these procedures.
In closing, I would like to farewell the families who are moving out of the area and our Year 6 students who will be going on to high school. I thank you for the contribution that you have made to our school and wish you a bright and happy future at your new schools.
The staff and I thank you for your ongoing support during a year that has truly been like no other.
Thank you for another wonderful year and I look forward to greeting the students when school resumes on Friday 29th January.
Monday 7th December Year 6 Taster Day to Gorokan High School
Tuesday 8th December 2020 Online Presentation Day 10:00am
Thursday 10th December Mingara Rewards Day
Tuesday 15th December Year 6 Graduation
Wednesday 16th December last day of school for students
Wednesday 27th and Thursday 28th January 2021 Staff Development days
Friday 29th January 2021 first day of school
Early Stage One
Current Learning
We will be exploring celebrations around the world and in particular Christmas. Students will be engaged in a variety of enjoyable tasks across all key learning areas. Please contact your child's teacher if you have any concerns.
Kindergarten students who would like to participate in Gorokan's Got Talent competition are invited to perform on Tuesday 8th December. Please complete the permission slip and return it to school asap and send your child to school on that day with their required props, if needed. Winners of the competition will be announced and filmed at the completion of Week 9.
Three students from each class have been selected to receive a class award. Parents/Carers have been notified via a letter if their child is a recipient. Please return the permission slip if you are happy for your child to be filmed receiving their award. All remaining Kindergarten students will be presented a certificate in Week 10 for completing their first year at school and making a fantastic contribution to Gorokan Public School. We congratulate each child in Kindergarten for their hard work and determination this year. Teachers and staff members have thoroughly enjoyed getting to know your children.
Congratulations to the following students for receiving class awards last fortnight.
KC – Lailahni, Haylen, Felicity and James
KDT – Ethan, Alex, Prince and Taliah
KFL – Noah, Ansley, Kanisha and Jacob
KL – Noah, Braxtyn, Kiara and Isabelle
KJ – Maddy, Kailee, Ava and Gunkirat
Mrs D Jeanpierre - ES1 Assistant Principal
This week Stage One students have been learning how to use questions and exclamation marks in their writing. In mathematics, students are discussing numbers in their daily number talks to demonstrate their understanding, while also working on their maths strategies with fun activities.
Class Picnic
We are encouraging all students to bring a special lunch for an end of year class picnic on Friday 11th December in Week 9. You could decorate your lunch box or pack something special to eat.
Decorate your lunch box
Pack something special to eat
Stage One Rewards Day
Students who have reached Bronze, Silver, Gold or Emerald Level will be rewarded with special activities on Monday 14th December in Week 10. Students who have reached Gold Level are encouraged to bring in their favourite board game.
1L are celebrating the completion of their colourful NAIDOC Week artwork. Each child was responsible for an individual piece of the artwork. They have also been developing their computer skills.
1L's collaborative artwork of an Echidna
1L students working hard at the computers
Mr R Johnson - Relieving S1 Assistant Principal
Throughout the past two weeks, students in Stage 2 have been learning about the structure and language features of procedure texts. We have written everything from how to build and fly a balloon rocket and paper plane to how to make a maze, reindeer biscuits and catapults. These highly engaging learning experiences had led to some very informative writing being completed by our students. Well done Stage 2 on your ingenuity and your writing.
Balloon rocket making
Reindeer biscuit making
Reindeer biscuit decorating
A reminder that even at this late stage of the year, it is still vital that students are completing reading activities whilst at home. Reading aloud every night can assist your child in becoming a more competent reader. Thank you for your support.
Congratulations to those students who have been nominated for awards at our Presentation Day assembly. We are so proud of you.
Mrs R Ranse - Acting S2 Assistant Principal
Students have been busy over the past fortnight with lots of teaching and learning occurring. In English we have had a focus on procedures with students following instructions, making and creating some wonderful artworks along the way. Dream catchers are hanging in every classroom and they are just beautiful to look at. These will be coming home with students in the coming weeks.
All Stage 3 classes have been revising and implementing our school expectations of being safe, being respectful and being a learner. This has included revisiting our Behaviour Management Plan and we encourage you to speak to your child about our school expectations. Please keep an eye on your child's Class Dojo points which are updated daily.
Year 5 Election of School Leaders for 2021
A huge congratulations to our successful school leaders for 2021 which were announced last Wednesday. They are: Lincoln and Ruby – School Captains
Riley and Elissa – School Vice Captains
Chloe, Lily, Dom and Tyler – School Prefects
We are looking forward to next year and seeing you lead the way here at Gorokan!
Year 6 Farewell
Plans are going ahead for our Year 6 farewell on Tuesday 15th December, 2020 here at Gorokan Public School. Our Year 6 farewell will be a little different this year and we are 'thinking outside the square' as we still need to ensure we are following the restriction guidelines. Unfortunately, parents and carers will not be allowed on school grounds. Please be assured that the evening will be captured and shared with the community. All students are to be dropped off and picked up from our Dudley Street entrance. Dress Code: Smart casual (with sensible enclosed shoes) and no technology. Staff will be taking photos. There will be a catered meal, photobooth, disco and presentation. We are so excited! 😊
Year 6 Transition to High School (Gorokan HS)
Gorokan High School Orientation Day was held on Tuesday 1st December. The Aboriginal student breakfast has unfortunately been cancelled this year due to COVID-19 restrictions.
Gorokan High School Taster Day
This event will be held on Monday 7th December. Buses have been organised to depart Gorokan Public School at 8:45am to travel to Gorokan High School, and will return to school for our normal school dismissal procedures. Please ensure your child is at school on time. Students must wear full school uniform and bring their lunch and recess with them. No students are to travel independently to or from Gorokan High School.
Please ensure your child brings a drink bottle and their own writing instruments (pens, pencils etc) to school every day.
A huge congratulations to all our students who continue to earn their class dojo's each and every day. We look forward to seeing how many you achieve for our Week 10 reward day! Keep aiming high!
If you have any questions or concerns please phone your child's class teacher.
Have a wonderful fortnight.
Mrs K Day - S3 Assistant Principal
K-6J have been learning about things that melt in science lessons with Miss O recently. They had fun testing to see what happened when they tried to melt some different cooking ingredients. I think the chocolate was the winner!
Kaia was a tidy chef
Leo is loving the chocolate
During Grade Gathering this week, we had many students receive rewards for their great behaviour and learning. They are as following:
Sienna, Kaia, Noah A, Claudia and Jarvis.
Mrs S Crook - Deputy Principal Student Wellbeing
P&C News
Thank you to our amazing community for all the donations towards the Salvation Army canned food drive. Your support is amazing and is much appreciated.
Just a reminder to return your dental permission notes as soon as possible. If you require another note please ask your child to ask at the school office.
We are always happy to receive second hand uniform donations for our emergency clothing pool.
I would like to wish our community a safe and happy Christmas.
Jasmine Daunt - Community Liaison Officer
School and Community Centre News
Central Coast Schools as Community Centres Facebook page CCSaCC Facebook supporting children transitioning to school in 2021. All live sessions are recorded and can be accessed later if you miss the live session.
Transition to School Sessions are held on Wednesdays at 11:00am
Transition to School Session Four
Let's talk language, screen-time & routines!
Yerin Eleanor Duncan Aboriginal Health sessions are held on Wednesdays at 11:30am
Yerin Eleanor Duncan Aboriginal Health Transition to School Session Two
Health checks and obtaining Immunisation records
Interrelate Live workshops are held on Mondays at 11:00am to hear their expert tips and advice
28th September: Building Resilience in Children
5th October: Self-care for Parents
SaCC Term 3 regular program parenting courses and speech group (all free and online)
Monday: Little Learners, Baby Massage;
Tuesday: Mother Goose/Baby Play;
Wednesday: Kids on the Move, Transition to School, Talkers Storytime (Speech);
Thursday: Busy Beats, and
Friday: Little Thinkers group, Talkers Play Pals (Speech).
Information: Phone Liz McMinn, SaCC Facilitator Gorokan Public School on 0467760712
Parenting Ideas
Managing the mother load
by Dr Jodi Richardson
"The mental load means always having to remember."
Emma, a French cartoonist summed up the mental load that most mothers carry in her 2017 viral comic, 'You should've asked'. In the opening scene a very hassled mother was preparing for a dinner party, while feeding her two young children, dealing with a noisy dog and answering the phone, all while she was hosting a colleague for dinner. As the dinner boils over the stove and onto the floor she looked at her partner imploringly. He unwittingly answered, "But you should've asked! I would have helped."
This scene artfully exemplifies the many invisible layers of responsibility that mothers carry including arranging most household tasks, managing the family home and being on top of the fine detail in every family member's life.
This is not the case in all families, but current research reveals that the great majority of women bear the load when it comes to housework (mums do twice as much as dads), caring for children (again, mums doing twice as much as dads) and carrying the mental load for their family.
There's only so much we can manage. For those of us who are anxious, there will be times our plates will fill, and overflow. While there's no single way to ease the mental load that mothers carry the following ideas will help to make life more manageable:
Stop making life easy for others
Anecdotal evidence suggests that many mums will overdo preparation rather than expect others to step up and help. I have been known in the past to cook and freeze up to a week's worth of meals prior to travelling for work. Not any more though. Magically, my husband and two school-aged children have become very capable in the kitchen as I've stepped back.
Delegate and let go
Delegate some household jobs to your children. Leah Ruppanner, professor of sociology at Melbourne University says, "Unless death is impending from poor domestic decisions, step back, support and allow others to learn from their mistakes." Letting go of jobs and allowing kids to do things in their own way is hard for perfectionist personality types, but it's essential if you are going to last the parenting journey. Surprisingly, both guilt and perfectionism ensure that many mums keep their children dependent upon them.
Diarise and check
One way to ease is the mental load is to lessen the number of mental notes and details that you carry around. Set up recurring electronic calendar entries for routine tasks and check regularly. This way you have less to remember and a place to find what you need, which is more efficient and mum-friendly way of being organised.
Lower the bar
Many mothers confess that they constantly feel guilt. if they don't put their children first. That's a heavy burden to carry. If anxiety is a constant companion, it's essential to lower the bar on your self-expectations. Unfulfilled expectations are proven stressors for mothers so take away tasks, rather than add to your mother load.
There's little doubt that many mothers carry an overwhelming mental load that adds to their anxiety and stress. I'm not suggesting that you abandon your job entirely but rather to look for ways to make the mental load you carry a little lighter. In doing so, life will become easier to manage. As with all behavioural change its easiest to start small. But whatever you do, if you're straining under the mother load make a start at releasing and sharing the load.
Parents can learn more about minimising anxiety as a mum in Dr. Jodi Richardson's book Anxious Mums: How mums can turn their anxiety into strength published by Penguin Random House.
Dr Jodi Richardson
Dr Jodi Richardson helps people to reduce their anxiety, strengthen their wellbeing, live by their personal values and achieve resilience and lasting happiness. Her research-based advice is grounded in a career of extensive university studies, clinical practice, education and endless compassion. Jodi is the co-author of the highly acclaimed book Anxious Kids. Her latest release is Anxious Mums: How mums can turn their anxiety into strength. For further details visit www.drjodirichardson.com.au


text:
Double-overtime loss stuns Gallaudet on home turf
Penn College (1-3, 1-1 NEAC) 0 1 0 1 2
Gallaudet (2-3, 1-1 NEAC) 0 1 0 0 1
2nd - 52:17 - Sean O'Callaghan (Gallaudet)
2nd - 60:38 - Team (Penn College)
OT2 - 107:58 - Colton Wartman (Penn College)
G: 2 Players (#21, #TM) - 1
A: Austin Dowrick - 1
Sh: 2 Players (#5, #8) - 4
Sv: Hunter Gosnell - 3
G: Sean O'Callaghan - 1
Sh: Chad Johnson - 4
Sv: Shane Angel - 3
WASHINGTON - Two minutes. That was the time left in the second overtime period Gallaudet University needed to kill off to salvage a tie here Saturday afternoon at Hotchkiss Field against Penn College in a North Eastern Athletic Conference (NEAC) men's soccer match.
Instead, Penn College (1-3, 1-1 NEAC) snuck in a low shot into the center of the Bison goal at the 107:58 mark to end the marathon match, as the Wildcats celebrated a 2-1 double-overtime victory.
Gallaudet (2-3, 1-1 NEAC) missed a golden opportunity to move up the conference standings against the winless Wildcats.
The two teams battled through a scoreless first half as tempers began to flare between the squads.
In the second half, Gallaudet got on the scoreboard when sophomore Sean O'Callaghan (San Diego, Calif.) buried his second goal of the season, off a free kick, at the 52:17 mark.
The lead was short-lived as the Bison defense faltered in the 60th minute.
Penn College's Chris McFarland booted a corner kick, from the nearside of the field, towards the Bison goal. The ball deflected off a Bison defender's foot back towards GU goalie Shane Angel (Mechanicsville, Md.), who got a hand on it but couldn't bring in the loose ball. A second Bison defender, positioned by the far post, leapt to try and get the ball out but ended up heading the ball into the goal.
The match continued to get chippy as the two teams combined for 21 fouls in the second half.
Gallaudet had a great scoring chance to start the first overtime period when freshman forward Chad Johnson (Gardena, Calif.) put a shot on goal. His shot from the right side of the Wildcat goal was saved by a sprawling Hunter Gosnell (three saves), who dove to make save. Off the rebound the Bison failed to get a second shot off with an empty net in front of them.
Colton Wartman scored the game-winning goal at the 107:58 mark off an assist from Austin Dowrick for the Wildcats in the second overtime period.
Johnson led the Bison with four shots (two on goal)
Angel made three saves
Penn College outshot Gallaudet, 13-12
GU held a 6-5 edge in corner kicks
The two teams had a combined 39 fouls, six cards
The loss ends GU's two-game win streak
Gallaudet will try and bounce back on Wednesday night (Sept. 19) at 7 p.m. with a conference match against Penn St.-Abington (4-2, 2-0 NEAC).


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The trade knowledge exam will be administered in one session via computer in the calm atmosphere of a convenient testing center at a time of your choosing.
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Florida Building Code "Test Protocols for High Velocity Hurricane Zones"


For the first time on vinyl is Sublime's Live album, Stand by Your Van. Stand by Your Van is a retrospective compilation live album. Tracks 1 to 11 were recorded live at Kommotion, San Francisco, on the 9th of September, 1994.


text:
Home Breaking Does Moon blame China for fine dust?
Does Moon blame China for fine dust?
Tim Kim
Seen above is a city blanketed by fine dust. Courtesy of Unsplash
President Moon Jae-in instructed officials to cooperate with China to grapple with the fine dust pollution that has blanketed the country throughout the past week.
The presidential office said on March 6 that Moon gave the order at a time when the country is suffering from the worst ultra-fine particulate pollution since data began to be recorded.
President Moon has come under fire because he blasted the previous administration for its failure to properly deal with fine dust pollution in early 2017 when he was a presidential candidate.
Back then he said, "I want to consume all the fine dust myself … The government's incapability and complacency drive people crazy. What they do is to just message the level of fine dust."
Moon comes under heavy criticism
President Moon Jae-in/Courtesy of Wikipedia
Critics point out that Moon's administration is also just messaging the people about the level of fine dust without coming up with appropriate measures to combat the pollution, which is choking the country.
Debate continues over the cause of the dust with many Koreans believing that a big portion comes from China. But Seoul has refrained from blaming Beijing for fear of creating a possible diplomatic dispute.
As the Moon administration seemed to have remained reluctant to point the finger at Beijing, some even claimed that Korean fine dust was being blown to China to "plague" Chinese people.
President Moon appears to have lost his patience in the face of the mounting criticism over the deteriorating fine dust conditions this month, and he finally referenced China.
Moon told his government to talk with China about measures that could effectively cut down the fine dust pollution.
"China argues that our dust flies into China's Shanghai region. Hence, making artificial rain over the Yellow Sea would help China as well," Moon was quoted as saying by his spokesman.
In addition, Moon asked his officials to check on whether a supplementary budget was needed for steps to reduce the fine dust.
fine dust
President Moon Jae-in
The publisher studied Korean history in Seoul and management of business administration in the United Kingdom. He has 20-year experiences in the media business. Kim can be reached at voc200@gmail.com or 82-2-6956-6698.