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Histones represent a class of proteins ideally suited to analyses by top-down mass spectrometry due to their relatively small size, the high electron transfer dissociation-compatible charge states they exhibit, and the potential to gain valuable information concerning combinatorial post-translational modifications and variants. We recently described new methods in mass spectrometry for the acquisition of high-quality MS/MS spectra of intact proteins (Anderson, L. C., English, A. M., Wang, W., Bai, D. L., Shabanowitz, J., and Hunt, D. F. (2015) Int. J. Mass Spectrom. 377, 617–624). Here, we report an extension of these techniques. Sequential ion/ion reactions carried out in a modified Orbitrap Velos Pro/EliteTM capable of multiple fragment ion fills of the C-trap, in combination with data-dependent and targeted HPLC-MS experiments, were used to obtain high resolution MS/MS spectra of histones from butyrate-treated HeLa cells. These spectra were used to identify several unique intact histone proteoforms with up to 81% sequence coverage. We also demonstrate that parallel ion parking during ion/ion proton transfer reactions can be used to separate species of overlapping m/z that are not separated chromatographically, revealing previously indiscernible signals. Finally, we characterized several truncated forms of H2A and H2B found within the histone fractions analyzed, achieving up to 93% sequence coverage by electron transfer dissociation MS/MS. Results of follow-up in vitro experiments suggest that some of the truncated histone H2A proteoforms we observed can be generated by cathepsin L, an enzyme known to also catalyze clipping of histone H3.
Chromatin is the structural framework that packages DNA into chromosomes within the nucleus of a cell (2). Histones comprise the principal protein component of chromatin and are involved in the regulation of gene expression (3, 4). This epigenetic regulation is achieved through complex patterns of post-translational modifications (PTMs),1 the incorporation of histone variants, and through controlled histone proteolysis (5⇓⇓⇓⇓–10). Comprehensive characterization of histones by mass spectrometry (MS) has proven technically difficult for a number of reasons. Traditional methods (bottom-up MS) of sequence determination and PTM site localization are not practical. Histone N-terminal regions are rich in lysine and arginine residues, and thus proteolysis using trypsin generates peptides that are too small or that are poorly retained on reverse-phase HPLC C18 resins for subsequent MS detection (11). With the advent of electron transfer dissociation (ETD) and more efficient electron capture dissociation fragmentation methods, which are better suited for larger, more highly charged peptides (12, 13), several studies utilizing other endoproteases to generate longer peptides have emerged (14⇓–16). Although these methodologies do well to preserve the combinatorial PTM profiles of histone tails, in some cases it is still impossible to identify the proteoforms from which these peptides originate. This is why analyzing histones intact, as they exist in the cells from which they are derived, is the best method for identifying unique histone proteoforms.
The results of several recent studies involving top-down analyses of histones highlight the complexity of the histone proteome as well as important biological implications that are not easily captured unless the proteoforms are analyzed intact (17⇓⇓–20). Although these results are very promising, this approach is not routinely utilized because of the many challenges with which it is associated. In a 2014 Proteomics article authored by members of the Consortium for Top-Down Proteomics, Dang et al. concluded that analyses of intact proteins could be greatly improved by the development of higher resolution isolation capabilities and by the ability to acquire rich sequence informative MS/MS spectra on a time scale more compatible with chromatography (18).
We have previously described modifications to a Thermo Fisher Scientific Orbitrap Velos ProTM mass spectrometer that make it possible to perform multiple fills of the C-trap with product ions from sequential ion/ion reactions of intact proteins prior to high resolution mass analysis (1, 21). The result is a dramatic enhancement of the observed fragment ion current and significant improvement of signal-to-noise ratios (SNRs) in MS/MS spectra. This eliminates the need for the acquisition of several time-consuming transients and allows for the acquisition of high quality spectra on a time scale compatible with chromatography. The front-end ETD (FETD) source can also be used to ionize ion/ion proton transfer (IIPT) reagents. IIPT reactions are used to disperse fragment ions over the available m/z range in a controlled manner and to simplify ETD fragment ion spectra (1, 22, 23). Additionally, we have enabled parallel ion parking, first reported by McLuckey and co-workers (24, 25), which involves harmonic excitation of selected ions within the ion trap to reduce their reactivity in gas-phase ion/ion reactions. This allows us to perform IIPT without reacting the precursor outside of the targeted product m/z range. Here, we demonstrate the power of these new techniques through their coupling with on-line, nano-LC chromatography and application to the analysis of intact histones derived from butyrate-treated HeLa cells.
Unless otherwise stated, all MS analyses were carried out on an in-house modified (21) hybrid Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, San Jose, CA). In the time that the experiments described below were performed, the instrument was upgraded to include a high-field Orbitrap mass analyzer (26). Experiments involving recombinant H4 and parallel ion parking were performed following the upgrade and so they make use of enhanced FT (27). Otherwise, spectra are acquired in magnitude mode. All spectra were acquired in reduced profile mode, using instrument default noise reduction.
Direct Infusion of Recombinant H4
Recombinant H4 was purchased from New England Biolabs (Ipswich, MA), desalted using an Amicon® Ultra-0.5 3K centrifugal filter (Millipore, Billerica, MA), and reconstituted in a solution containing 40% acetonitrile and 60% of 0.1% acetic acid in water (all percentages expressed as v/v). The sample was directly infused at a concentration of 5 pmol/μl and ionized using microelectrospray ionization as described previously (28) into the front end of an in-house modified ETD/IIPT-enabled Orbitrap EliteTM. MS/MS spectra were acquired using the following instrument parameters: resolution (r) = 60,000 at 400 m/z; 190–2000 m/z scan range; 1E5 ion counts precursor automatic gain control (AGC) target; 3E5 reagent AGC target; 1–20 fragment ion fills of the C-trap; 1 μscan/spectrum; 2 m/z precursor isolation window centered at 708.4 m/z ([M + 16H]+16 charge state of H4); 6–8-ms ETD; 0–20-ms IIPT.
Histone Extraction from HeLa S3 Cells
Cultured HeLa S3 cells were treated with 10 mm sodium butyrate overnight and harvested, and the nuclei were isolated as described previously (29). The nuclei pellet was resuspended in 0.4 m NaCl containing 1 mm dithiothreitol (DTT), 0.3 mm 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, 10 mm sodium butyrate, and 5 μm Halt protease inhibitor mixture at a ratio of 10:1 (v/v), salt buffer/pellet. This was incubated with constant rotation for 2 h at 4 °C and then spun at 3000 × g for 5 min. Supernatant was discarded, and the pellet was resuspended in a 2.5 m NaCl solution containing DTT, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, sodium butyrate, and protease inhibitors as above in a ratio of 5:1 (v/v), salt buffer/pellet. The histones were then acid-extracted and TCA-precipitated as described previously (29).
Off-line Histone Fractionation
Histones purified from HeLa S3 cells (∼100 μg) were dissolved in water and chromatographically separated on a Vydac C18 column (4.6-mm inner diameter, 250-mm length, 5-μm particle size) at a flow rate of 0.8 ml/min using a gradient of 30–60% solvent B in 100 min, 60–100% B in 20 min, and 100–30% B in 10 min (solvent A = 5% (v/v) acetonitrile, 0.2% (v/v) trifluoroacetic acid in HPLC-grade water; solvent B = 95% (v/v) acetonitrile, 0.19% (v/v) trifluoroacetic acid in HPLC-grade water). Fractions were collected using an automatic sample collector in 1-min intervals between 15 and 80 min from the start of the gradient. The UV detector was set to monitor absorbance at 214 nm, and fractions containing histones H1, H2A, H2B, and H4 were identified and appropriately combined based on their known chromatographic retention times (29). These were transferred to microcentrifuge tubes, taken to dryness using a SpeedVac concentrator, and stored at −35 °C.
On-line RP-HPLC for Mass Spectrometry
Dried samples were reconstituted in water at concentrations of 5–10 pmol/μl. Aliquots of 1–2.5 pmol of protein along with 100 fmol of peptide standards were pressure-loaded onto a 360-μm outer diameter × 75-μm inner diameter fused-silica micro-capillary analytical column (packed 11-cm of POROSHELL 300SB-C18 resin (Agilent Technologies, Palo Alto, CA) (30) equipped with a laser-pulled electrospray emitter tip (31). The column was washed with ∼20 column volumes of 0.3% (v/v) formic acid in water. Samples were eluted at 60 nl/min using a gradient of 0–33% B in 10 min, 33–48% B in 60 min, 48–100% B in 10 min and 100–0% B in 10 min. The elution gradient utilized solvent A, 0.3% formic acid in water, and solvent B, 0.3% formic acid, 72% acetonitrile, 18% isopropyl alcohol, and 9.7% water (all v/v). Following reverse-phase HPLC separation, samples were directly ionized using microelectrospray ionization as described (28). For some samples, initial screens revealed that better chromatography might be achieved by enacting simple changes in the elution gradient. These changes included doubling the 0–33% B portion of the gradient to a time of 20 min for samples containing truncated histones and altering the gradient used for H1 to 0–20% B in 10 min, 20–40% B in 60 min, and then 40–100% B in 10 min. All other aspects of the RP-HPLC separation and ionization for mass spectrometry remained the same across all analyses.
On-line MS Analyses
Samples were ionized by microelectrospray into the front-end of an in-house modified ETD/IIPT-enabled Orbitrap Velos ProTM. Except for the occasional targeted analyses, the instrument was operated in data-dependent mode in which the MS1 scan was taken from m/z 300–2000 in the Orbitrap mass analyzer (r = 60,000 at m/z 400) followed by a second MS1 scan acquired in the linear ion trap over a limited m/z range, upon which data-dependent selection was based. In this way, same species/different charge state reselection was reduced. A single ETD/IIPT MS/MS scan was also taken in the Orbitrap. MS/MS scan parameters were set as follows: r = 30k, 60k or 100k at m/z 400; 200–2000 m/z scan range; 1E5 ion counts AGC target; 3E5 ion counts reagent AGC target; 5–30 fragment ion fills of the C-trap; 1–3 μscans; 2–3 m/z precursor isolation window; 4–25-ms ETD (fluoranthene reagent); and 20–40-ms IIPT (sulfur hexafluoride reagent). The instrument was operated using charge state exclusion (+1, +2, and +3) and dynamic exclusion with a repeat count of 1–2, repeat duration of 60 s, and an exclusion duration of 90 s. Analytes of different sizes and charge densities require different ETD/IIPT reaction times, C-trap fill counts, and resolving power; these were varied depending on the fraction being analyzed. For example, when analyzing H1, a 21.9-kDa protein, shorter ETD and IIPT reaction times, more multiple fills, and higher resolution settings were used than those used for the analysis of H4, an 11.4-kDa protein. Duty cycle times also varied accordingly, but in general they were within 6–15 s/cycle.
Some fractions contained larger proteins that required MS/MS scan ranges over 2000 m/z for sequence determination and PTM site mapping. However, the instrument control software did not allow the acquisition of spectra with scan ranges exceeding 2000 m/z for ETD/IIPT MS/MS scan types in data-dependent mode. This prompted us to perform "on-the-fly" targeted analyses based upon full MS data acquired in the initial sample screenings. Instrument method parameters were varied at our discretion based on our evaluation of the MS/MS spectra generated as data were acquired.
High resolution full MS and MS/MS data were manually inspected using Qual Browser software (Thermo Fisher Scientific). Interpretation of all ETD/IIPT MS/MS data was performed manually on the unprocessed raw spectra. Theoretical fragment ion masses were calculated using an in-house-developed fragment mass calculator. Theoretical isotopic distributions were modeled using Isotope Pattern Calculator software (Pacific Northwest National Laboratories, omics.pnl.gov/). Percent sequence coverage was calculated by dividing the number of observed N–Cα bond cleavages by the total number of predicted N–Cα bond cleavages (note that cleavage of the N–Cα bond that is N-terminal to proline does not produce an observable fragment). Masses for all intact proteoforms identified agreed to within 5 ppm and were confirmed by MS/MS. Relative abundance information for each truncated histone species was determined by taking the area under the curve of the extracted ion chromatogram for the most abundant isotope for every charge state present utilizing a 10-ppm mass window (32).
Parallel Ion Parking during IIPT
For parallel ion parking experiments, the MS1 scan was taken from m/z 300 to 2000 in the high-field Orbitrap mass analyzer (1E6 ion counts AGC target, r = 120,000 at 400 m/z). A 40 m/z window centered at m/z 810 (790–830 m/z) was selected and subject to 140 ms of IIPT. The instrument control software was modified to apply a custom RF waveform to the x-rods of the linear ion trap during IIPT. The waveform included frequencies mapping to m/z 2300–3900, corresponding to the desired final charge state of the precursor. For the sample analyzed (fraction H2B), the [M + 5H]+5 charge state was the desired product (∼m/z 2770). The amplitude of the waveform was adjusted such that maximum activation of the target product ions was achieved without dissociation or ejection. Product ions were analyzed in the Orbitrap (1E5 ion counts AGC target, r = 120,000 at 400 m/z) after five fills of the C-trap.
In Vitro H2A Proteolysis and nano-HPLC-MS/MS Analysis
The in vitro proteolysis of recombinant histone H2A type 2 (Uniprot ID, Q6FI13) was carried out in 50 mm MES, pH 6.0, and 5 mm DTT. Recombinant cathepsin L (R&D Systems 1515-CY-010) was used in a 5000:1 (histone/enzyme) ratio during a 1-h time course reaction. 20 μg of the sample reaction was quenched with 10% acetic acid after 15, 30, and 60 min. Samples were desalted using in-house packed Stage tips as described previously (29). After Stage tip desalting, samples were resuspended in 0.1% formic acid prior to nano-LC-MS/MS analysis. 1 μg of sample was loaded onto a 75-μm inner diameter × 17-cm Reprosil-Pur C18-AQ (3 μm; Dr. Maisch GmbH, Germany) column using an EASY-nLC nano-HPLC (Thermo Fisher Scientific). The HPLC gradient was 0–100% solvent B (A = 0.1% formic acid; B = 95% acetonitrile, 0.1% formic acid) over 60 min at a flow rate of 250 nl/min. nano-LC was coupled to an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific). MS1 spectra (m/z 300–1200) were acquired in the Orbitrap with a resolution of 120,000 (at 200 m/z) and an AGC target of 5E5. Data-dependent selection was performed using TopSpeed with a cycle time of 3 seconds. MS/MS for charge states +2–5 was performed using higher-energy collisional dissociation (HCD), with a normalized collision energy of 27%, a maximum injection time of 150 ms, and an AGC target of 5E4. Higher charge states were fragmented using ETD, using a reaction time of 20 ms, a maximum injection time of 400 ms, 3 μscans, and an AGC target of 5E5. Both types of MS/MS fragmentation were acquired in the Orbitrap with a resolution of 30,000 (at 200 m/z). Data were analyzed using Proteome Discoverer (version 1.4, Thermo Scientific, Bremen, Germany) with Mascot (Matrix Science, London, UK) as the database search engine (database: human histones, Uniprot, March, 2014, 58 entries). Search parameters were as follows: HCD spectra were searched using a precursor ion mass tolerance of 10 ppm and a fragment ion mass tolerance of 0.05 Da; ETD spectra were deconvoluted using Xtract (Thermo Fisher Scientific) and searched using a precursor ion mass tolerance of 10 ppm and a fragment ion mass tolerance of 0.02 Da. Unspecific cleavage (no enzyme) and no modifications were specified. Only identifications with a false discovery rate of <1% (calculated using fixed value PSM validator) were accepted. Chromatograms of the relevant peptides were manually extracted using Xcalibur (Thermo Fisher Scientific) after deconvolution for peptide quantification.
Multiple Fills and IIPT
A Thermo Fisher Scientific Orbitrap Velos ProTM was modified to introduce reagent anions from the front by adding a glow discharge ionization source between the stacked ring ion guide and multipole 00, as described previously (21). The discharge is used to ionize ETD and IIPT reagents (fluoranthene and sulfur hexafluoride, respectively) for ion/ion reactions. By placing the reagent ion source in the front of the instrument, the C-trap is made available to store fragment ions from multiple iterations of ion/ion reactions. Once sufficient amounts of fragment ions are collected, they are sent to the Orbitrap for high resolution mass analysis.
Fig. 1, A and B, shows 8-ms ETD/20-ms IIPT MS/MS spectra of intact recombinant H4 using 1 and 20 fragment ion fills of the C-trap, respectively. SNRs for fragments shared between the two spectra are indicated in parentheses. By increasing the number of fragment ion volumes analyzed from 1 to 20, total fragment ion current is increased by an order of magnitude. This is also demonstrated in supplemental Fig. 1, which shows a trace of total ion current as the number of multiple fills is varied. The spectrum shown in Fig. 1C is an average of 20 spectra like that shown in Fig 1A and represents a cumulative acquisition time of 11.2 s (0.56 s/spectrum). Elapsed scan time for the spectrum shown in Fig. 1B is 6.24 s. Average SNR gains for the fragments indicated are 10.4 in Fig. 1B versus 3.5 in Fig. 1C. Hand-annotated spectra indicating the most abundant fragments observed are shown in supplemental Fig. 2.
The use and benefits of IIPT to charge reduce ETD-produced fragment ions have been described previously (1, 23). Briefly, following fragmentation by ETD, fragment ions are allowed to react with a proton transfer reagent. These reagents act as a base and abstract protons from highly charged fragments. The amount of charge reduction is tuned by varying the amount of time the reaction is allowed to take place. By extending IIPT reaction time such that fragment ions are significantly charge-reduced, the fragment ion signals are increasingly distributed over the full scan range (example shown in supplemental Fig. 3).
Characterization of Intact Histones Derived from Butyrate-treated HeLa Cells
HeLa cells were treated with sodium butyrate prior to histone isolation and separation. Butyrate treatment of cells results in histone hyperacetylation; butanoate acts as an inhibitor of histone deacetylase activity (33). The histones were acid-extracted from the nuclei and purified by off-line reverse-phase HPLC fractionation. The histones were identified based on their well known chromatographic retention times (34). Intact protein was directly loaded onto a reverse-phase C18 Poros-shell column and gradient-eluted into the mass spectrometer.
Fig. 2 summarizes the data that can be obtained from a single LC-MS experiment. Fig. 2A shows the base-peak chromatogram of the H2A2 fraction revealing three distinct regions of intact protein elution. Peaks 1 and 2 correspond to H4 in various modified forms containing oxidized and unoxidized methionines, respectively, whereas peak 3 corresponds to various proteoforms of intact H2A. Displayed in Fig. 2, B and C, are the averaged high resolution ESI spectra recorded on peaks 2 and 3 in the base peak chromatogram, respectively. Signals in the observed charge state distribution carry 7–18 and 8–23 positive charges for H4 and H2A, respectively. An ETD/IIPT MS/MS spectra recorded on an [M + 19H]19+ ion of an intact form of H2A is shown in Fig. 2D. This spectrum was obtained using a 4-ms ETD and a 25-ms IIPT and is an average of six spectra, each taking 11.7 s to acquire utilizing 3 μscans and 15 multiple fills with r = 30,000 at 400 m/z. Note that equivalent spectra taken as the sum of 45 μscans (transients) of single fragment ion fills would take ∼19.8 s/scan. Residues identified by ions of types c and z observed in Fig. 2D are indicated in Fig. 2E (hand annotated version shown in supplemental Fig. 4). These define 80% sequence coverage and identify the intact H2A as H2A.1 (Q93077).
Depicted in supplemental Fig. 5 is a full MS spectrum indicating signals for intact H2A proteoforms that were identified and confirmed by ETD/IIPT MS/MS. In addition to H2A.1, also detected were H2A.1-C and H2A.1-H (Uni Parc Q93077 and Q96KK5, respectively). H2A.1-C differs from H2A.1 by the substitution of Thr-16 with a serine residue and by substitution of Lys-99 with an arginine residue. These substitutions give rise to a δ mass of +13.9904 Da from H2A.1, which is very close to the δ mass that would be exhibited in the event of a monomethylation (+14.0157 Da). The calculated and observed mass differences for intact H2A.1-C and the monomethylated form of H2A.1 are 3.5 and 5.3 ppm, respectively, making H2A.1-C the better fit by mass accuracy. However, based on this information alone, neither could be conclusively excluded. The MS/MS spectra contained c ions that exhibited a δ mass of −14 Da following Thr-16 and z ions that exhibited a mass shift of +28 Da following Lys-99. The +28-Da mass shift is also indicative of a dimethylated lysine, but this possibility was ruled out based on the mass accuracy of the fragment ions observed (z313+̣, 13C m/z 1101.9823: 0.2 ppm for Lys → Arg, 7.8 ppm for Lys → di-methylation Lys). Thus, we were able to conclude that this is H2A.1-C. This demonstrates the importance of obtaining high quality MS/MS spectra for the confirmation of proteoform identity. H2A.1-H differs from H2A.1 by the deletion of the last two C-terminal residues, emphasizing the importance of analyzing the intact forms of these proteins, as digestions would produce identical peptides with the exception of those containing the C termini. This difference was easily discerned upon examination of the z fragment ion series in the MS/MS spectra. H2A.1-C and H2A.1-H are present at levels roughly 20–25 and 15–20%, respectively, relative to H2A.1. Eluting later in the gradient and also confirmed by MS/MS are the respective mono-Lys-acetylated forms of these proteins. The acetylation is localized to Lys-5 in all H2A types.
Fig. 3 displays a spectrum recorded on [M + 16H]16+ (m/z 718.0) from intact H4 using a 4-ms ETD reaction time and 40-ms IIPT. This is an average of eight spectra, each taking ∼8.75 s to acquire utilizing 3 μscans at r = 30,000 (at 400 m/z) with 10 multiple fills of the C-trap per μscan (∼13.9-s acquisition for single fill/transient equivalent spectrum). Fig. 3B offers an expanded view of the 1250–1500 m/z region of the spectrum. With the exception of the extreme C terminus, which is devoid of charge, a near contiguous series of both c′ and z•′ can be observed for ∼60 residues from either end of the protein, yielding 81% sequence coverage and site localization of five acetylations as well as Arg-20 dimethylation (fully annotated spectrum shown in supplemental Fig. 6).
Compared with other histones, H1 presents a more difficult situation for on-line LC-MS analysis of the intact form. The most common human isoforms of H1 range from 194 to 226 amino acids in length and, due to their larger size, require higher resolution, shorter gas-phase ion/ion reaction times, more multiple fills of the C-trap, and an extended mass range. Initial sample runs revealed the presence of at least two forms of H1, H1.4 and H1.2. MS/MS spectra acquired in data-dependent mode using 2-ms ETD/25-ms IIPT are shown in supplemental Fig. 7 along with the observed sequence coverage (H1.4, 72%, and H1.2, 67%, P16403 and P10412, respectively). Additional targeted analyses elucidated several additional unique H1 proteoforms. One of these was a form of H1.2 containing a previously uncharacterized single amino acid substitution, A141T. The spectrum and coverage are shown in Fig. 4 (hand-annotated version shown in supplemental Fig. 8).
Parallel Ion Parking during IIPT
Histone fraction H2B presented the most difficult challenge due to the number of unique proteoforms it contained. Fig. 5, A and B, displays the averaged (five spectra, r = 120,000 at 400 m/z) high resolution ESI spectra recorded on intact H2B proteoforms eluting over ∼45 s. Isolation of the region containing the [M + 17H]+17 (Fig. 5C, m/z 790–830) reveals several distinct isotopic clusters, each spaced less than 1 m/z apart. Using a 2 m/z isolation window, all MS/MS spectra acquired on precursors possessing ETD-compatible charge states contained fragment ions unique to at least two H2B proteoforms. Upon close inspection of Fig. 5C, two +8 precursors can be seen between m/z 825–830 (labeled D and E, also see supplemental Fig. 9, D and E). This same 40 m/z window was isolated and subjected to 140-ms IIPT. During the IIPT reaction, a waveform, including frequencies mapping to m/z 2300–3900, was applied. As the precursor ions are charge-reduced, their m/z values increase, and their frequencies of oscillation come into resonance with the applied waveform. This occurs when an ion's m/z reaches the 2300–3900 m/z range. At this point, these ions are activated, increasing their velocity relative to the reagent ions and slowing the rate of further proton transfer.
Fig. 5D displays the product ion spectrum following 140-ms IIPT/parking. The vast majority of the ion current lies within 2730–2800 m/z range where the "parked" intact H2B [M + 5H]+5 ions lie. An enlarged inset of this region is shown in supplemental Fig. 10. Signals corresponding to precursor ions that were not fully reacted to the +5 charge state or that were not fully parked and underwent further proton transfer to the +4 are indicated. Close inspection of the spectrum shown in Fig. 5D reveals several signals of lesser charged species (labeled A–E). Their observed charge state and m/z as well as their calculated m/z and charge state prior to IIPT are given in Table I. Comparisons of the observed masses to calculated theoretical values suggest that these signals are derived from truncated forms of H2B that co-eluted with intact H2B during the time these spectra were acquired (identity and mass accuracy of the most abundant isotopologue in each envelope are shown in Table I). Peptides containing residues 66–125, 67–125, and 68–125 derived from two isoforms of H2B match within 5 ppm mass error. Expected signals for these species A–C are not observed in Fig. 5C (also see supplemental Fig. 9), presumably due to the density of unique species within the window. Note that the assigned identities for these truncated H2Bs are not confirmed by MS/MS and are based on mass accuracy and on MS/MS-confirmed identities of the intact H2Bs observed in this fraction. These truncated H2Bs could be derived from several other proteoforms of intact H2B due to their sequence similarity.
Characterization of Truncated H2B and H2A Proteoforms Derived from Butyrate-treated HeLa Cells
Eluting after the intact H2B proteoforms were several fairly abundant forms of truncated H2B. The base peak chromatogram and the sequence of the most abundant intact H2B (H2B-1C, P62807) observed in the fraction are shown in Fig. 6. The solid black lines in Fig. 6 indicate "primary" cleavage sites mapped by MS. Less abundant cleavage sites in Fig. 6 are marked with gray lines. This is not a comprehensive account of all the cleavages that are observed, but rather only those that are most abundant. Complementary N-terminal peptides, 1–25 and 1–51, are present and elute near the front of the gradient. They are less abundant and are more difficult to characterize because their signals are split among multiple acetylated forms (+1–3 lysine-acetyl confirmed by MS/MS). The acetylations are shared among multiple sites, including Lys-11, Lys-12, Lys-15, Lys-16, and Lys-20, which are all sites that were confirmed by MS/MS in the intact form and that have previously been identified by MS in an earlier study (35). Truncations derived from two additional H2B isoforms (possibly H2B.2-E, Q16778, and H2B.1-K, O60814) were also present. Note that the sequences of the intact forms found in the sample differ by only one residue (V39I for H2B.2-E or S124A for H2B.1-K), and so distinction between truncated peptides can only be made if both unique residues are contained within the peptide. If only one of these residues is present, then only one of the two additional isoforms can be excluded. There is also no way to distinguish the modified (acetylated) form of the intact protein from which the C-terminal peptides are derived. Up to 93% sequence coverage is obtained by ETD/IIPT MS/MS of the three most abundant C-terminal containing peptides (90%, 26–125; 93%, 52–125; and 84%, 50–125). The spectrum and sequence coverage of the 26–125 peptide are shown in supplemental Fig. 11.
Additionally, dozens of truncated H2As were observed in the H2A1 fraction. These ranged in size from 10 to 75 residues in length, and each account for less than 1% of the total ion current relative to intact H2A from the same fraction. The base peak chromatogram is shown in Fig. 7, A–C, and indicates where truncated species elute relative to intact H2A. Fig. 7D shows the sequence of the most abundant intact H2A observed in the fraction (H2A2A, Q6FI13). Again, the solid black lines in Fig. 7 indicate "primary" cleavage sites, and the less abundant cleavage sites are indicated with gray lines. This is not a comprehensive account of all the truncated histone forms observed, only the most abundant. Supplemental Table 1 lists these truncated proteoforms along with their respective abundances. The most abundant of these was the N terminus-containing proteoform cleaved C-terminal to Gly-44. Intact forms of H2A identified on the fraction were H2A2A and H2A2C (Q16777), which differ by their last 5–6 C-terminal residues. Because of this, there is no way to determine whether the N terminus-containing truncations were derived from H2A2A or H2A2C. MS/MS spectra of truncated species are shown in supplemental Fig. 12.
Identification of Histone H2A Clipping Enzyme by in Vitro proteolytic Assay
Given the large variety of truncated histone proteoforms identified, we further investigated whether cathepsin L, a protease responsible for histone H3 and H3.3 clipping (9, 37, 38), is capable of cleaving histone H2A. To do so, we performed an in vitro assay incubating recombinant histone H2A with cathepsin L and characterized the cleavage specificity using nano-LC coupled to an Orbitrap Fusion with FETD. Supplemental Fig. 13 and supplemental Table 2 show that the signal of intact histone H2A decreased by ∼3 orders of magnitude after 1 h of incubation, while the clipped forms increased in abundance over time. Specifically, we determined the clipped proteoform Gly-44 (cleaved after glycine 44) as one of the most abundant cleaved proteoforms and confirmed its sequence by ETD MS/MS (supplemental Fig. 13C). We also observed several other C-terminal peptides (supplemental Table 2). Taken together, our data suggest that cathepsin L might have a larger variety of targets than currently described.
In the commercial configuration of the Orbitrap Velos Pro/EliteTM, the reagent ion source is located at the rear of the instrument, requiring that reagent anions traverse the C-trap to reach the ion trap, where ion/ion reactions are performed. By putting the reagent ion source in the front of the instrument, the C-trap is made available for the storage of fragments from multiple iterations of ion/ion reactions prior to transient acquisition in the Orbitrap. In this way, we are able to dramatically increase fragment ion current. The multiple fill strategy for improving SNRs has a number of advantages over transient summing or spectral averaging. Chief among these is that the time that it takes to acquire spectra of multiple C-trap fills is less than the time it takes to acquire transients from the same number of single C-trap fills to be averaged or summed. Specifically, if a transient takes 380 ms to acquire, an additional fragment ion fill can be accomplished in less than ½ that time, i.e. ∼130 ms for overhead plus time for ETD/IIPT activation. Additionally, SNRs improve faster using multiple fills compared with summing transients or averaging spectra and so fewer transients are needed to produce a useful spectrum for analysis. This enables the acquisition of high quality spectra on a time scale more compatible with chromatography.
The power of top-down mass spectrometry lies in its ability to provide an unambiguous view of protein expression dynamics by distinguishing closely related proteoforms from one another while also observing any combinations of PTMs. Bottom-up MS strategies are not capable of this, which is why there has been a significant push in the field of proteomics to develop instruments and methodologies that enable high throughput, top-down mass spectrometry of intact proteins. Here, we demonstrated the power of FETD-enabled sequential ion/ion reactions along with multiple fragment ion fills through their coupling with on-line HPLC and application to the analysis of intact histones. Data-dependent and targeted LC-MS experiments were employed to record high resolution MS/MS spectra of histones from butyrate-treated HeLa cells. Using these spectra, several unique intact histone proteoforms were identified with up to 81% sequence coverage. Examples shown here contain multiple modifications, including single amino acid substitutions, methylations, and acetylations. Additionally, many truncated forms of histones H2A and H2B were characterized with up to 93% sequence coverage.
Histone proteolysis has been implicated in several important regulatory processes, yet it remains poorly understood. Only eight histone-specific proteases are known, and their biological significance has not been extensively defined (39). This area of study is clearly poised for significant expansion, as evidenced by the identification of two H2A-specific proteases and several novel histone clipping events in just the past few years (39⇓⇓–42). Discerning whether any histone-cleaving event is the result of continuous histone turnover or sample degradation versus an alternative biological process with epigenetic implications is challenging (36). Our in vitro data suggest that cathepsin L might be a potential candidate to explain some of the truncated histone H2A proteoforms we observed in the butyrate-treated HeLa cell histone samples. We found that cleavage C-terminal to Gly-44 produced the most abundant truncated H2A proteoforms observed in both the in vitro experiments and those derived in vivo from HeLa cells. Differences between the in vitro and the HeLa cell data in the abundance of other truncated proteoforms might be explained by improper folding of the recombinant histone or by the absence of a proper nucleosome structure in the in vitro experiments. In the future, we hope that the tools we describe here will aid in deciphering whether or not these truncated histone proteoforms are biologically regulated and contribute to epigenetic mechanisms.
That we are able to observe these low level truncated histone proteoforms in simple top-down LC-MS experiments further demonstrates the utility of our methodologies as it is very likely that bottom-up- or middle-down-based proteomics approaches would have missed cleavage events like those we describe in our analyses. This could happen for a number of reasons. First, given the low levels at which these truncations were observed (which, for any single truncated form, is less than 1% relative to the intact histone), along with the added complexity that is generated when samples are digested for MS proteomic studies, peptides of this type may have not been detected or may have simply gone unnoticed by investigators. Several of the generated peptides derived from originally truncated species might have been too small to be retained by the HPLC column. Quantitative analysis of the relative abundance of unique truncated forms would be difficult to perform as evidence for some unique truncated species could be lost or could be below the limit of detection. Also, the ability to correlate the endogenous histone proteolysis with any specific histone variant or PTM state would not be possible unless those PTMs or sequence variations and the cleavage sites were contained within the same peptide produced in the sample preparative digestion. Only by determining the primary structures of these species, as they existed in the cell, can one draw these potentially important biological parallels.
Furthermore, the use of parallel ion parking during IIPT is demonstrated in an analysis of H2B proteoforms. Chromatographic separation is made difficult by the fact that several H2B proteoforms differ from one another by just a single amino acid residue. At high ETD-compatible charge states, multiple proteoforms are present within a relatively small m/z range. Upon data-dependent selection, often more than one proteoform is isolated and fragmented, resulting in mixed MS/MS spectra, making identification difficult. Additionally, as demonstrated here, overlapping signals make it difficult to tease out the number of unique species present. By using IIPT to charge-reduce the precursors contained within a wide isolation window and parking the products in a desired m/z range, we are able separate species that, prior to IIPT, possessed roughly the same m/z but a significantly different mass. This reveals signals that can be attributed to less abundant H2B proteoforms that were not observed in the MS1 spectrum nor the isolation-only spectrum due to their proximity to more abundant signals derived from other species. This is the first time ion parking has been demonstrated on a commercially available linear ion trap using ion cloud densities that lend themselves to high resolution MS/MS analyses. We believe that parallel ion parking will become a powerful tool in identifying proteins in a mixture when used in conjunction with MS/MS fragmentation methods and multiple fill technology. Most exciting is the potential for this technology to allow differentiation and identification of closely related proteoforms that are difficult to separate chromatographically, like H2B. As demonstrated in supplemental Fig. 10, following IIPT/parking, we are able to spread apart isotopic clusters such that their centers are ∼3 m/z apart (compared with <1 m/z prior to IIPT) without losing signal due to further proton transfer. This is a stepping stone to enabling higher resolution isolation on the MS3 level. However, doing so will require further instrument modification to increase the precursor ion capacity of the linear ion trap to be able to observe fragments in MS3 spectra following IIPT.
We thank John E. P. Syka, Christopher Mullen, and Lee Early of Thermo Fisher Scientific for aiding in the modification of the Orbitrap Velos Pro to include FETD and for helpful discussion. We also thank Dina L. Bai for critical reading of early manuscript revisions and Greg T. Blakney for helpful discussion.
Author contributions: L.C.A., J.S., B.A.G., and D.F.H. designed the research; L.C.A., K.R.K., S.A.U., M.C., and S.S. performed the research; A.M.E. contributed new reagents or analytic tools; L.C.A., S.A.U., M.C., S.S., and J.S. analyzed data; and L.C.A., K.R.K., S.A.U., M.C., S.S., and B.A.G. wrote the paper.
↵* This work was supported in part by National Institutes of Health Grants GM037537 and AI033993 (to D.F.H.). This work was also supported by NIH Grants GM110174 and AI118891 and a National Science Foundation Faculty Early Career Award (to B.A.G). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
↵ This article contains supplemental materials.
The authors declare that they have no conflicts of interest with the contents of this article.
↵1 The abbreviations used are:
- post-translational modification
- higher-energy collisional dissociation
- electron transfer dissociation
- electron capture dissociation
- tandem mass spectrometry
- signal-to-noise ratio
- front-end electron transfer dissociation
- ion/ion proton transfer
- m/z
- mass to charge ratio
- v/v
- volume/volume percent
- automatic gain control
- reverse-phase high-performance liquid chromatography
- ppm
- parts per million.
- Received July 20, 2015.
- Revision received January 11, 2016.
- © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.


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Find Insurance and Reinsurance expertise in Antigua and Barbuda
There are around 150 licensed insurance companies (2004) operating on Antigua and Barbuda, eight of which cover life insurance business, while 45 of them are committed to general insurance matters. The income of the annual insurance premium varies between 4 and 7% of the country's GDP, depending on the respective jurisdiction. Due to the International Business Corporations Act (1982), offshore banking is allowed, so that foreign insurance corporations also work on the islands. There is a part-funded national insurance system established in Antigua and Barbuda and plans for a National Health Insurance Scheme were made in 2011. The contribution rate to social security accounts for some 4% of the employee and 5% of the employer. The local Social Security Board is based in St. John's, Antigua.
|Insurance and Reinsurance organisations in Antigua and Barbuda
|Bryson's Insurance Agency
|Ira Archibald Insurance
|Travellers Life Insurance Co.
|Wilco Agencies

subset: commoncrawl


text:
AMC Bridge was formed by a team of experienced industry professionals who focus on computer-aided design, engineering, and manufacturing. Since 1999 we have been delivering innovative solutions for CAD, CAE, CAM, PDM, and PLM applications. We now help companies from all over the world because we believe that guaranteed high-quality solutions can only be realized through long-term cooperation with our clients and partners.
Our job is to build the Internet of Tools. We make industrial facilities safer, cleaner, and more productive by connecting workers, tools, and data. Our award-winning system has a wide application in all processing, liquefaction, and refining installations and allows our clients to save costs both directly and indirectly through reducing installation time.
2+ years of software development experience.
EDUCATION: BS/MS in Computer Science, Math, or Engineering.
Strong JavaScript development skills
Expert-level knowledge of HTML5 and CSS
Strong experience with React or React Native, Redux, Node.js
Strong object-oriented programming and design skills
Experience with RESTful web services
Ability to solve problems independently
Outstanding teamwork and communication skills
Intermediate speaking, reading, and writing skills in English
Build a web application front end using React Redux and component libraries and back end using Node.js
Attend daily stand-ups, planning sessions, and sprint reviews
Competitive salary
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Paid vacation, sick days, etc.
Medical insurance
Possibility of business travel abroad


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Remote Learning at CDM: Resources and FAQ | DePaul University: Fall Quarter 2020.
Academic policies protect the integrity of academic curricula and provide the checks and balances necessary to maintain the standards set forth by the University. All students are expected to be familiar with University policies that are documented in the University catalog and student handbooks, as well as college-level policies outlined on the college website. Knowing these policies and referencing them on a regular basis allow students, faculty, staff, and administrators the ability to operate under the same set of expectations as all parties work together to achieve the best possible student experience.
Exceptions to academic policy are rare. In cases where severe extenuating circumstances prevent adherence to a policy, students should first consult with an advisor in the CDM Academic Success Center to determine an appropriate course of action. If it is determined that a policy appeal should be submitted, the student must
submit the appeal online via MyCDM.
Graduate CDM students must
submit a request to change their program and/or concentration in MyCDM. Undergraduate students must use the Declarations and Inter-College Transfer Tool in
Campus Connect (My Academics > change College, Major or Minor) to officially change their college, major, minor and/or concentration.
Information on the policy, process and requirements to successfully complete your degree and graduate.
Learn more about managing your class schedule each term in accordance with enrollment, withdrawal and waitlist policies.
Details on the grading policy, including Incomplete Grades, Course Repeats, and Grade Challenges.
Information on transferring credit from a community college or four-year university, as well as inter-college transfers within DePaul.
Instructions on the steps necessary to apply to enroll in an independent study course.
Information on DePaul's academic probation policy.
Links to common forms current students might need over the course of your academic career.


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Additional information
|14000 lbs
2007 Freighliner CL120 Truck
This truck has been kept in good condition and runs/operates as it should.
It has about 870,500 miles on it with lots of life left in her.
No rust, lightweight, clean, Oklahoma truck.
It features a Mercedes-Benz/Detroit OM 460 12.8L diesel powered engine that produces 450 horsepower.
It also features an engine break, as well as an Eaton Fuller 10 speed auto-shift transmission.
It has airliner suspension, a 218 inch (18ft) wheelbase, a mid-roof sleeper and alum covers too.
It has almost new front tires and the drive tires are varying in condition from 20% to 70%. Brakes are in great shape.
This sale does not include the trailer or equipment in the photos.
We are open to offers!
|14000 lbs


text:
Welcome to Blox Services. We have been established for over 15 years and provide low cost mobile vehicle repairs for all makes and models. I am an auto-electrician/mechanic in Farnborough with 32 years' experience and work throughout the Hampshire and Surrey area including:
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I spent years making the same mistakes in night game over and over again. I realized recently that it doesn't take that long to get good at night game as long as you are making the most out of your nights.
My biggest growth spurts in game have come from times of full immersion; going out three to four nights a week to bars and clubs for hours at a time.
Here is a list of things I wish I knew from the beginning about getting good at night game. Some of what I suggest will sound extreme, but making up for years of not going out in a few months will be an extreme experience.
About the
Author: William Gupta
A mixed race (black and Indian) U.S.-born seducer with a dash of wanderlust, William Gupta has lived in the U.K. and throughout the United States. He is well versed both in meeting girls during the daytime in a variety of venues, as well as at night. When he's not meeting women on the street, in cafés, in libraries, or in bars, William performs as an improv comedy artist, up on stage.


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Just because you are enjoying a holiday beach vacation or an extended snowbird stay doesn't mean you have to miss out on all of the fun and excitement that Myrtle Beach has to offer. Sea Crest resort is conveniently located in the heart of Myrtle Beach, making it easy to find nearby events and attractions – even in the offseason!
Check out these top-rated Myrtle Beach attractions that stay open all year long for your enjoyment!
Ripley's Aquarium | (843) 916-0888
Explore the undersea world and make a few new nautical friends at Ripley's Aquarium. This Broadway at the Beach attraction is a favorite among guests of all ages and offers something for everyone. And, with two levels of exhibits, there's plenty to keep you busy for an afternoon. Popular exhibits include Rio Amazon, Rainbow Rock, Friendship Flats, and the famous Dangerous Reef glide-path that allows guests to get up close and personal with sharks. Other draws include the on-site café, interactive discovery center, and gift shop. Call (843) 916-0888 for more information.
Hollywood Wax Museum | (843) 444-0091
Located right across from the popular Broadway at the Beach venue, this recent addition to the Myrtle Beach attraction lineup is hard to miss! The Hollywood Wax Museum Entertainment Center is actually home to three unique attractions. The largest and most well known, as one might guess, is the Hollywood Wax Museum. Guests can post with popular famous figures like Jonny Depp, Brad Pitt, Michael Jackson, and Marilyn Monroe – to name a few. The other two attractions include Hannah's House of Mirrors and Breakout – a Zombie Apocalypse Experience. All attractions can be enjoyed with the All Access Pass, $29.99 for adults and $19.99 for children 4-11, or toured individually. Call (843) 462-5991 for more information.
EdVenture Myrtle Beach | (843) 839-0475
Explore, play, and learn with a variety of interactive exhibits built for young minds at Edventure Myrtle Beach. Interactive exhibits include an interactive space ship climbing structure with a mission control attached on top, a 60's theme child-size playhouse with a looping TV of the moon landing and interactive telescope, a flight simulator, two model airplanes and much more! The facility also holds birthday parties and camps. For more info, contact (843) 839-0475.
Legends in Concert | (843) 238-7827
See your idols live at Legends in Concert! This exciting live entertainment venue is home to the nation's most talented tribute artists. Authentic costumes, colorful props, and expert choreography are just a few things that guests can look forward to. Popular past performers include Michael Jackson, Dolly Parton, The Blues Brothers, Rod Stewart, and the king himself – Elvis. Shows are offered throughout the year and change seasonally to make sure you always have something new to look forward to. Call (843) 238-7827 for information or tickets.
Brookgreen Gardens | (843) 235-6000
With more than 9,000 square acres of professionally landscaped gardens and an impressive lineup of handmade sculptures, it's not hard to see why Brookgreen Gardens remains a popular destination throughout the year. Notable exhibits include Live Oak Allee, Fountain of the Muses, the Poetry Garden, and the Labyrinth. Additionally, Brookgreen offers a Lowcountry Zoo, on-site café, creek excursion boat rides, and lively events throughout the year like Brew in the Zoo and Night of a Thousand Candles. Call (843) 235-6000 for more information.
WonderWorks | (843) 626-9962
Experience more than 100 interactive exhibits that will spark the minds of guests young and old at WonderWorks Museum. Located at Broadway at the Beach, WonderWorks features six different "wonder zones" full of unique, hands-on exhibits. Popular attractions include the Hurricane Shack, Trivia Wall, Mission to Mars, Wonder Coaster, and Wonder Brite – to name just a few. Additionally, WonderWorks offers an indoor ropes course, outdoor obstacle course, 6D moving theatre, laser tag arena, and zip line over Lake Broadway. Call (843) 626-9962 for more information.
Rockin' Jump | (843) 242-3264
Jump, dunk, and challenge yourself to a slew of interactive attractions at Rockin' Jump, Myrtle Beach's ultimate indoor trampoline park! With eight entertaining attractions, including the Ninja Challenge Course, Dodgeball Arena, Slam Dunk Zone, Warped Wall, Wipe Out, Aerial Silks, Fidget Ladder and Open Jump Area. Rockin' Jump is an enjoyable experience for kids and adults. Rockin' Jump also features a handful of events throughout the year, an on-site cafe, and holds group events. Contact (843) 242-3264 for more details.
Myrtle Beach Boardwalk
Located along the bustling downtown area, the Myrtle Beach Boardwalk offers plenty of year-round entertainment to please guests of all ages. And, at over a mile in length, guests will find a wide array of attractions to keep busy. The Ripley's Museum, Myrtle Beach Skywheel, Sling Shot Thrill Ride, and Ripley's Moving Theater are just a few things one will find, in addition to a slew of arcades, local shops, and lively events. The Boardwalk also offers tons of decadent dining options, both casual and more upscale, to help visitors refuel. Just head to the Myrtle Beach Boardwalk to see what all the fun is about!
Broadway Grand Prix | (843) 839-4080
Located right across the street from the bustling Broadway at the Beach is the fast-moving Broadway Grand Prix. This family race park is home to seven different go-cart tracks, an arcade, mini-golf course, kiddie amusement rides, and a snack bar. Each track is segmented by skill level from the expert level Grand Prix Pro track all the way to the family track, where racers (40" and up) can ride alone or in doubles. All attractions can be available with the online purchase of the Grand Prix Day-Pass Wristband starting at $35 for adults and $28 for children.
Mini Golf
It's no secret that golf is one of the biggest draws to the Myrtle Beach area. Get the whole family involved by playing some of the year-round mini-golf courses along the Grand Strand. Some well-known mini-golf courses open year-round are;
- Jungle Lagoon Miniature Golf – 0.5 miles from Sea Crest Resort
- Cancun Lagoon Adventure Golf – 1.4 miles from Sea Crest Resort
- Mt. Atlanticus Minotaur Golf – 1.7 miles from Sea Crest Resort
- Captain Hooks Adventure Golf – 2.6 miles from Sea Crest Resort
Bring the family to one of the mini-golf courses listed above, conveniently located from the Sea Crest Resort.
Barefoot Landing | (843) 272-8349
Barefoot Landing is an enormous shopping center located in North Myrtle Beach (13 miles from Sea Crest Resort). It has over 100 stores and dining options to choose from, plus exciting events throughout the year. If bad weather happens during your vacation, then Barefoot Landing is a perfect place for the family to spend quality time together. Barefoot Landing is open from 10:00 a.m. to 6:00 p.m. every day.
Be sure to check our full Myrtle Beach Attractions page for more information on area things to do.


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Pak govt bans 11 organisations for having links with JuD, FIF, JeM
Islamabad, May 11: Pakistan government has banned 11 organisations for having links with the proscribed outfits Jaamat-ud-Dawa (JuD), Falah-e-Insaniat Foundation (FIF) and Jaish-e-Mohammed (JeM), an official statement said on Saturday.
The decision to ban these organisations was taken during a meeting between Pakistan's Prime Minister Imran Khan and Interior Minister Ijaz Shah on Friday.
After the February 14 Pulwama attack, in which 40 Indian security personnel were killed by a suicide bomber of the Pakistan-based JeM, Khan had said Islamabad would not spare any group involved in militancy or using Pakistani soil for any kind of terror activity against other countries.
Pakistan's National Counter Terrorism Authority (NACTA), which works under the Ministry of Interior, announced on its website that seven groups have been banned for their affiliation with the JuD, which was proscribed in March by the Pakistan government.
Terrorists storm 5-star hotel in Pak's Gwadar, gunshots heard
The organisation which have been proscribed are Al-Anfal Trust, Idara Khidmat-e-Khalaq, Al-Dawat ul Irshad, Mosques & Welfare Trust, Al-Medina Foundation, Mazz-Bin-Jabel Education Trust and Al-Hamad Trust, the statement said.
All these groups are Lahore-based.
Pakistan's Ministry of Interior took the action on the government's directive to speed up implementation of the National Action Plan of 2015 to eliminate militancy and extremism from the country's soil.
Apart from the seven, Lahore-based Al-Fazal Foundation/Trust and Al-Easar Foundation were also banned for having links with the FIF, the NACTA said.
The FIF, like JuD, was also banned in March by the Pakistan government.
According to the NACTA, Bahawalpur-based Al-Rehmat Trust Organization and Karachi-based Al-Furqan Trust were also banned on Friday for having links with the JeM, which was banned in January 2002.
Recently, the government announced to take control of more than 30,000 religious seminaries.


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Hillary's America: the Secret History of the Democratic Party
Jonathan Hoffman
July 28, 2016
32 comments
Author and film maker
Dinesh D'Souza's
latest film opened nationwide last Thursday. It is a history lesson in two parts.
It starts with a short and somewhat creepy sequence of swirling cartoon representations of different Democrat politicians to the tune of "Happy Days are Here Again". The movie then begins with a re-enactment of the sentencing phase of D'Souza's trial for violation of campaign finance laws. This was the beginning of part one.
Yes, it's true, Dinesh D'Souza had a friend who was running for office to whom he donated $20k. So far, so good, but he then had a third party donate another $20k which was reimbursed by D'Souza. He was charged with a felony. His lawyer said that this sort of case is common and that nobody suffers a felony and that he would get it reduced for him. After some time, his lawyer told him that the court was not budging, he could not get the charge reduced, and that somebody must really want to get him. This took place after the D'Souza movie
2016: Obama's America
which was critical of the president. He pleaded guilty to the felony and was sentenced to five years probation, eight months in a "community confinement center," eight hours a week of community service during the probation, and a thirty thousand dollar fine. It was sort of a "Lite" version of
G.Gordon Liddy's
sentence of 20 years in prison (commuted to eight years by President Carter) for a first offense breaking and entering where nothing was stolen—his punishment for not co-operating with Democrats after the
Watergate fiasco.
After the courtroom scene, there was a humorous sequence showing his induction to the "community confinement center" and getting used to the company of hardened criminals. He began to learn about the criminal subculture which had been totally foreign to him. Through speaking with his fellow inmates, he distilled the four major aspects of the criminal enterprise: 1, Develop a plan; 2, Recruit; 3, Make the pitch; 4, If caught, always deny, never give up the con. He uses his newfound understanding of criminality as a framework for explaining the success and ultimate goal of the
Democrat Party.
D'Souza dived back in history to the presidency of
Andrew Jackson
, the Democrat president who drove Native Americans off their land onto reservations, then sold the land cheaply to buy votes. The Republicans fought against the plan, but the Democrats got it passed. He proceeds through history to the Civil War, Reconstruction, the Ku Klux Klan, lynching, big city political machines,
Margaret Sanger,
and finally debunking the claim that Republicans under Nixon decided to appeal to Southern racists and that is why black people turned to the Democrats after the racists in the party became Republicans. It was given the term, "The Southern Strategy."
Unlike his movie
2016: Obama's America
, in which imitated
Charles Kuralt
traveling all over the world interviewing everybody, D'Souza used dramatic re-enactments to provide the cinematic imagery reifying the historical facts. The imagery was quite powerful because it was almost exclusively of individuals and their actions, which probably accounts for the PG-13 rating. Little time was wasted on sweeping scenes of landscapes and such, though there was a brief interview at a diner with
Jonah Goldberg
which was mildly interesting but seemed odd.
There was one particularly heart-wrenching scene in which a journalist named
Ida B. Wells
attempted to intervene at a lynching. Wells, like Harriet Tubman before her, was a gun toting Republican black woman. She devoted herself to ending the practices of lynching and segregation, mostly through her work as a reporter for a Republican newspaper. She was treated in the movie as one of the great heroes of yesteryear, reappearing a number of times toward the end of the film. The history lesson was part one.
Part two was a biography of
Hillary Clinton.
It began with her as a young woman and her association with
Saul Alinsky
(much of the material on Alinsky was drawn from a television interview he gave shortly before his death in 1972, it was revealing). Hillary Clinton's story covered most of her misdeeds from the Whitewater scandal, to her husband's victims, to the theft of money collected for Haitian earthquake relief. Many of the clips of Clinton can be sorted into one or more of the aspects of criminal enterprise.
The movie ends with an extended patriotic fanfare complete with fireworks, Civil War re-enactments, a choir, an orchestra, and the
Blue Angels,
that would touch the heart of all but the most jaded Americans.
This latest effort on the part of Dinesh D'Souza is a two part documentary that covers much of the dark side of American history. It is humorous at times, and occasionally hard to watch. Almost everyone will find one or two tidbits of information new to them. It flows well with pretty good production and acting for an indie film. However, if you are someone who would be greatly disturbed or offended by a bare knuckled criticism of Hillary Clinton and her party, you may want to give this one a pass.
Jonathan Hoffman is the
resident libertarian columnist.
Arts and Culture
Dinesh D'Souza
Hillary Clinton
Hillary's America:the Secret History of the Democrat Party
Ida B. Wells
Saul Alinsky
Andrew Jackson
Democrat Party
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A Perfect Storm in Cyberspace
Cyberspace is being re-shaped by technological changes, a growing underworld of cyber crime, a burgeoning cyber security industrial complex, and an increasingly intense geopolitical contest over the domain itself. These forces threaten to subvert cyberspace through over-reaction, the imposition of heavy-handed controls, and through partition or cantoning.
Ron Deibert is professor of Political Science and director of the Canada Centre for Global Security Studies and the Citizen Lab at the Munk School of Global Affairs at the University of Toronto.
Publication Date
cyberspace, technology, cyber crime, cyber security industrial complex, geopolitics, partition, cantoning
Information Security | International Relations
Recommended Citation
Deibert, Ron. "A Perfect Storm in Cyberspace." Ensemble video, 01:16:14. February 1, 2012. https://ensemble.dickinson.edu/Watch/Wj69PtLb


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Browse Businesses by NAICS code or description
NAICS Code: 332992
332992 NAICS Code for Small Arms Ammunition Manufacturing. These businesses listed in 332992 are owned by veterans, service disabled veterans (SDVOSB/DVBE) active duty military, reservists and military spouses of the United States Army, Air Force (USAF), Marine Corps (USMC), Navy (USN) and Coast Guard (USCG).
Search returned 3 Businesses.
GunnerLife | Sanford, NC 27332
GunnerLife is an Online store for firearm parts and accessories, tactical gear, and ammo.
US Reloading Supply | Englewood, FL 34223
RELOADING SUPPLIES and TRAINING - Reloading since 1972! We are a Veteran/LEO owned and operated small business. ** All products are ...
White Birch Armory | Dover, NH 03820
White Birch Armory is a Service Disabled Veteran Owned Small Business (SDVOSB) specializing in the distribution of firearms, ammunition, ...
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FIRST IN THEIR PROGRAM–Red Brick School held a ceremony last week recognizing five students in the school's first ever ranking ceremony for the Civil Air Patrol's Cadet Program. Pictured after receiving their pins at the ceremony are: Sean Edwards, Mason Pritchard, Rylan Delay, Nickelijah Duffin and Zachary Hornbostel.
On Patriots Day, Sept. 11, a special ceremony was held at the Career Center of Southern Illinois (CCSI) for five students in a new program.
Red Brick School, a "safe school", is located at CCSI and "provides education for students who, for a variety of reasons, are unable to be successful at their home school. The alternative education program serves the schools in Monroe and Randolph Counties." It serves students in sixth through tenth grades who are identified as "at-risk" of dropping out or failing to graduate on time.
Red Brick School is in its first year of participating in the Civil Air Patrol's Cadet Program, which is a program for youth ages 12-18.
The Civil Air Patrol (CAP) is America's premier public service organization for carrying out emergency services and disaster relief missions nationwide. CAP supports America's communities with emergency response, diverse aviation and ground services, youth development, and promotion of air, space and cyber power. CAP membership does not equate to membership in the military; it is a private, non-profit humanitarian organization, and is the official auxiliary of the United States Air Force.
For more on this story, please see this week's print edition.


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Just installed Alpha build 703 on my new Fiio X7 mark II... No Hi Res Audio support Was hoping it would work as the dac supports up to 32bit 384khz natively. If the new beta build ever gets released, hopefully with support for Fiio X7 & X5ii, then Ill give Poweramp another go. Until then, Neutron Player and Fiio Music will be my daily drivers.
It will never be released. But if you don't need Hi Res output, the current alpha version is brilliant anyways.
Neutron is nipping at the heels of Poweramp. Latest version of Neutron Player supports Hi-Res output on the Fiio X5iii and iBasso DX200 (Android based dedicated DAPs). Poweramp is still the best music player for Android, but with the extremely slow update cycle it's quite possible another player will soon overtake it.
True, my apologies. I was scanning over the replies, kind of misread yours I guess. You did say you'd like to hear a few words from Max, that he's still alive, which is fair enough. Problem is, once you log in, most people read would read through at least the last few replies.
Why on earth would he want to come on here and read all these negative comments? He's working hard, reading this crap is just gonna demotivate him. Regarding the alpha build, I did mention previously that alpha should have been updated prior to starting the beta project / UI revamp. But this was a tactical error at the time, and it is what it is. It's perfectly reasonable if Max wants to focus his efforts on the beta build, any work today on alpha couldn't be ported to beta, so it would just be a waste of time. I think the greatest fear people have is that Max has abandoned the project. But why would the moderator continue moderating this forum if its abandonwear? And why would the developer need to post here? That's not his job. At the end of the day we just need to be patient. Poweramp will continue to be the number 1 audio app for Android, if beta is released with minimal bugs.
July release date, for sure! Lolz, jking, never gonna happen... Realistically though, I think a lot of people here don't appreciate the complexity of building an app like this for Android. The challenge is in the fact that there are so many different manufacturers and devices, all of which have multiple builds and versions of Android. Poweramp uses its own audio engine which bypasses Android, essentially manipulating the device software to allow direct access to the built in Hi Res dac. Developing an app requires testing of all the most popular devices, and fixing a bug on one device can create a new bug on a different device... Large companies have whole teams for this type of work. Poweramp doesn't have these kinds of resources. The only negative thing I would say, is that the existing 704 build should have seen a 705 release to fix the most glaring bugs, most notably issues with the Hi Res output. People here would not be complaining anywhere near as much, if this approach had been taken. Practically everybody is on Android N by now, and thus stuck with 704 if they want to use Hi Res output (being a key feature of Poweramp). It's completely understandable and acceptable that a full UI rebuild is taking longer than expected. But you need to give the people a product that works, in the meanwhile. Making empty promises about the release date has only compounded the issue. Im coming back to this thread mostly for entertainment these days, secretly hoping that eventually the new build will be released... But I still think Poweramp is a fantastic app. I nearly rolled back to Marshmallow when I found out about this issue with build 704.
Im checking this thread almost daily for news of the beta release. Im not sure why..
FLAC and Hi-resolution Audio discussion
HiFiZ replied to Timmy Fox's topic in General Poweramp ChatterI get where your coming from, but what I am saying is that regardless of the frequencies, on the HTC 10 specifically, the Aqstic chip is simply a better DAC than the standard Qualcomm DAC. And I can also hear the difference when upscaling CD quality FLAC files, I've done A>B comparisons and 192 kHz sounds better than 44.1 kHz on the 24bit Aqstic. My headphones are Beyerdynamic T51, which are a pretty good match with the HTC 10 imo. I trust my ears more than any arguments pro or contra. The difference is subtle, but it's noticable. I would describe Hi Res vs CD audio as being fuller, more detailed, with more clarity and texture. This possibly has more to do with the mastering than the actual frequency etc., as digital Hi Res audio is more likely to be from a modern remastered source, and the mastering techniques used for Hi Res audio deliberately allow for greater dynamic range.
FLAC and Hi-resolution Audio discussion
HiFiZ replied to Timmy Fox's topic in General Poweramp ChatterRegardless if you believe there is no difference between Hi Res audio and CD quality audio (I disagree) either way the audio output on HTC Hi Res 24bit 192k is better quality than the standard 16bit 48k. But you will need good headphones to hear the difference. Mastering makes a huge difference too. Well mastered CD quality audio can sound better than Hi Res audio with poor mastering. Well mastered Hi Res audio can sound absolutely fantastic, but again is dependent on you equipment. Without top notch headphones and DAP / amp etc. you won't hear much difference. A lot of vinyl rips aren't that great, due to poor recording equipment and/or mastering, and this doesn't do much for promoting Hi Res audio. Also, CD quality lossless FLAC is much better than any lossy source.
Omg, this thread is hilarious. Everyone just calm down! I really am beginning to think that the next beta build is a myth, seems like it will never be released. Either way, bashing the dev won't achieve anything. I do wish, however, that the dev could have fixed the bugs of the last alpha build, before embarking on an ambitious overhaul of the entire UI. Most of us are on Nougat by now, and hi-res output is a core feature of this app. It's annoying to have buggy support for hi-res. Let's be realistic here. Neutron has a fantastic output, however, only supports hi-res via an external usb dac. So it's useless for owners of LG V20, HTC 10 etc. Plus the UI is rubbish... No other audio player app can match the output of Poweramp. The existing UI of Poweramp is fine, in fact I like it, so I'm a bit perplexed as to why a UI overhaul is required in the first place. But I will be very happy if and when it does get released, provided all bugs related to audio output are fixed. If anger is directed at anybody, it should be directed towards HTC. They have released a phone with high end audio capability without the required app to take advantage of the hardware. It ships with Google Play music... Can't comment on LG, as I am a HTC 10 owner, but it should be noted that Poweramp is the only app that can really take advantage of the dac & amp combo in this phone. For this I am thankful, regardless of the buggy alpha release and eternally delayed new release.


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Honda's new test track will host 10 MW solar project
This article was originally posted on www.cleanenergyauthority.com Written by Chris Meehan When Honda starts testing cars at its new test track in Sakura, Japan, its test models will zoom by...
Pool blankets are a great way to ensure and healthy and efficient pool all year round. They can cut energy consumption by up to 50%, practically eliminate surface evaporation and filtration cycles will be reduced. We supply and install, contact us today for a quote.
There are many benefits to using a pool blanket and below are just a few:
1. Will virtually eliminate evaporation, which is calculated by the Department of Water Affairs to be 5 – 6 liters per m² of surface area per day.
2. Will cut chemical consumption by 50%. For average pool, ½ cup of chlorine every other dayand in winter 1 cup per week should be adequate.
3. Filtration cycle may be reduced saving money on electricity and wear and tear on pool cleaner.
4. Life expectancy +/- two seasons if used continuously but experience has shown that with care the cover will last for anything up to 6 years.
5. Leaves falling on top of the cover may be brushed to the side using a pool brush and removed by hand.
6. Will raise pool temperature by 3ºC + in season giving more pleasurable swimming and extended season. A solar bubble cover is the cheapest way of raising the pool temperature by a significant amount.
7. Floats on the water, is an attractive colour, needs no sandbags or tie downs and will not blow away except under extreme conditions. It can also be made up to fit any size or shape of pool – pools with sundecks is no problem.
8. If one considers the saving in water, electricity, chemicals and wear and tear on the pool cleaner, a cover will pay for itself within 12-15 months and the warmer water will greatly enhance the pleasure of a swimming pool.


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How Much Does TEFL Certification Cost? Is It Worth It?By Bridge
January 2, 2019
To teach English as a foreign language, one of the qualifications employers ask for is certification. You know you need it, but how much does TEFL certification cost? Prices vary depending on some key factors, but expect to pay about $200 for very minimal online certification, and closer to $400-500 USD for online certification of enough hours to qualify for most TEFL jobs (at least 100). If you opt for university-level online training or an onsite TEFL course, the price, as you might expect, is higher– usually between $1,000 to $2,500 USD.
Here's a breakdown of how much a TEFL course is, depending on the type you choose. Find out what you are paying for vs. what you're getting with each option to decide which course is right for you.
First things first: Is a TEFL course worth it?
Almost all schools worldwide that hire English teachers require that they hold TEFL certification, among other qualifications; therefore, yes, it is definitely worth it to get certified if you plan to teach English. (Just starting out? Start here: What is TEFL?) Even if you are already a licensed teacher in the U.S. teaching another subject, for example, TEFL certification will still show employers you have the special skills to teach English to speakers of other languages.
Yet not all TEFL courses are created equal, and neither are all TEFL trainees, so to make certification worth your while, make sure to choose the right course for your background and teaching plans.
How much does an online TEFL certification cost?
The vast majority of people who get TEFL certified to prepare to teach English earn their certificates online, and online certification is accepted by most language schools around the world. That's great news since online TEFL certification courses cost significantly less than in-person courses and they are far more flexible and convenient, as well.
You'll see a range of prices when you browse online TEFL courses – from $150 to as much as $1500. The average 120-hour online TEFL course with a tutor typically falls in the middle range of this, at around $300-$600.
What factors affect the cost of online TEFL certification?
These variances in TEFL course cost are due to a number of factors. Let's break those down to clear up pricing confusion and to help you decide which one is right for you.
The number of hours of certification
Generally speaking, more TEFL hours = a higher price tag, and this is a major factor affecting TEFL course cost. Most teaching jobs require that you hold at least 100 hours of TEFL certification, so courses of fewer than 100 hours will be considered more introductory-level and will be less expensive. These courses are usually in the $150-$350 range. TEFL courses of 100 hours or more, on the other hand, will be more in the range of $300- $600, with the price going up as the hours do.
Amount of tutor involvement in the course
Another factor that affects TEFL certification cost is how much guidance you are provided in the course.
The TEFL course provider
As you might expect, TEFL courses offered by well-established and accredited training centers will be more expensive than courses offered by what are sometimes referred to as "TEFL cowboys" or fly-by-night online "shops" with no verifiable accreditation.
Choosing an accredited TEFL certification course gives you, as a consumer, a layer of assurance that you're buying a quality training, and also lets your future employers know you are well-trained and prepared to teach.
Take a look inside our online TEFL course in this video:
How much is an online TEFL course vs. an in-person course?
Now that you we've looked at online TEFL course tuition, what about in-person (or "onsite") courses? Is the $1,500- $2,500 price tag for onsite TEFL certification a good decision for you? Should you choose online or in-person TEFL certification?
Despite the convenience and affordability of online TEFL courses, there are still some situations in which taking an in-person TEFL course is a better choice.
Can I pay in installments for a TEFL course?
Whether you choose online or onsite TEFL certification, the answer to this will depend on each individual TEFL provider's policies. However, as a rule, online TEFL course tuition is usually paid up front, upon registration. That said, one way to "finance" TEFL certification of more hours is to begin with a course of minimal hours that you is in your budget, and later upgrade that course by adding hours when you can afford to.
With onsite TEFL courses, you usually pay an initial deposit, with the balance of your course tuition due closer to the start date.
Is the certificate itself included in the course? If not, how much does a TEFL certificate cost?
With online courses, you can typically download and print your TEFL certificate upon completion and submit this to schools when you apply. Some online TEFL providers may also provide an online portal where schools can go to verify your TEFL certificate by entering the unique number. These options are free.
Often a "hard copy" original version of your certificate will require a fee (around $50-$75) since the certificate will be printed on certificate-quality paper, embossed and signed, and mailed to graduates at locations around the world. You'll also have the option to have the certificate notarized or authenticated internationally (with an apostille), which would cost extra.


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Question: Dear Luise: We have a nice, older model, Monaco Motorhome. Our problem is that we have a very small income, so RVing is something we have to budget for. We know there are a lot of people who don't have this problem and many of the parks are set up with them in mind. That's OK, but how about the rest of us on minimum Social Security and/or small pensions? Do you have any suggestions besides staying home? Thanks for at least reading this. Bertha and Maude, (sisters)
Answer: Well, "Hi" and welcome to both of you. You bet I have some suggestions. There are a lot of us that must shop carefully for RVs and plan trips with excruciating budgetary limitations. We still want to extend our horizons beyond four walls. We like windshields better than TV screens! Viva la open road!
There is a web site called www.freecampgrounds.com That's the place I would suggest you get started on this project. They have almost 1000 campsites that are free or close to it. Every state can be accessed and researched for suitable RVing.
Large stores like Wal*Mart allow free overnight parking on the perimeter of their parking lots and some casinos offer the same thing. Fraternal Organizations like the Elks often offer free RV parking. (Yes, I know there are no "Elkesses"…but you might know someone who knows someone.) Are you widows? If so, do either of you have privileges you haven't checked out?
When we were buying our motorhome, we found that there was a huge difference in gas consumption. We got 18 to 20 mpg, while others often were getting 5 – 8 mpg. When you are thinking about going thousands of miles, that's no small consideration.
Food can be another place to save a lot of bucks. Eating in as opposed to eating out often cuts your expenditures in half. Volume buying and leaving most of it at home, keeps costs down on trips…and preparing your own recipes instead of getting packaged meals is not only cheaper but healthier eating.
Many towns along the way have Food Banks and Senior Centers offer tasty meals for less. Stop in, meet people and socialize.
By all means, take that Monaco Motorhome out as often and as long as you can! Talk with others, and find out what they do. Visit relatives and park in driveways! Go! Blessings, Luise


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Directed by Jun Robles Lana | 120 min | Tagalog | Philippines | Comedy, Drama
Trisha, a Filipino transgender woman, suddenly dies while being crowned in a beauty pageant. Her last wish was to be presented as a different celebrity on each night of her wake.
This film is presented with English subtitles.


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BRUGES (Belgium) • As Manchester United's 18-man squad flew to Belgium yesterday without want-away goalkeeper David de Gea for the second leg of their Champions League play-off against Club Brugge, the presence of Ed Woodward raised some eyebrows.
United's executive vice-chair was part of the travelling party despite speculation of an audacious move for Barcelona forward Neymar.
Woodward has reportedly claimed that United have bigger targets after missing out on Pedro, who moved from Barcelona to Chelsea last week. But the Spanish club yesterday rejected talk that Neymar could leave for Old Trafford in a world-record deal.
Barcelona president Josep Maria Bartomeu also revealed that the Catalan giants are keen to offer the 23-year-old Brazilian superstar a new contract.
"Those are just rumours and what people write but there is nothing to it," Bartomeu told Catalan radio station Catalunya Radio.
"Neymar is happy at Barca. We don't want Neymar to leave at all.
"In a few months' time, we will discuss his contract extension as he has three years left on his current deal. We want him to stay for many years, three, five, 10 or even 15 years more with us."
Neymar scored 29 goals last season as Barcelona won a treble.
United's problems in front of goal remain a concern ahead of tonight's clash against Club Brugge.
Louis van Gaal's side have scored only twice in their three league games to date. One was an own goal by Tottenham Hotspur's Kyle Walker, the other was a deflected shot by Adnan Januzaj against Aston Villa.
Captain Wayne Rooney has come to symbolise United's problems in the opposition penalty area, having gone 10 club games without finding the net.
United right-back Matteo Darmian is expecting a tough away test but is adamant the Red Devils can return to winning ways following their 0-0 home draw against Newcastle on Saturday.
"For us, 0-0 would be a good result but we always try to win and, when we go to Brugge, it will be no different," the Italian told MUTV.
"We know that they are a good team, particularly at home, but we'll be going out to win and we know what we have to do. We don't think that it will be easy but it's very important to all the people at the club for us to qualify."
United are strong favourites to book a spot in the lucrative group stage - after a first season out of the competition in 19 years - following last week's 3-1 win in the first leg.
Club Brugge manager Michel Preud'homme believes that his side require a perfect performance if they are to achieve the "miracle" of eliminating United.
The Belgian side overturned a 1-2 first-leg deficit against Panathinaikos in the previous qualifying round by winning 3-0 at the Jan Breydel Stadium.
However, Preud'homme struck a pessimistic tone at his pre-match press conference.
"It would be a great miracle," said the former Belgium goalkeeper.
"They are two goals in front, they have a very professional team, can make a decision by an action at each moment.
"And we have some problems with the number of players that were already out in the first leg, plus (captain) Timmy Simons and (centre-back) Brandon Mechele."
Mechele is suspended after being sent off for two bookable offences in the first leg.
CLUB BRUGGE V MANCHESTER UNITED StarHub Ch212, tomorrow, 2.30am


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Third Visit: Kate Fiori, Vero Beach Elementary School, Vero Beach - Visiting Vero Beach Elementary School was delightful! I was greeted with a cute sign welcoming me and happy smiles from the clerical staff. As I walked through the teachers' lounge area, there were huge charts posted on the walls. They showed the growth classes were making toward mastering their standards, and there was a pineapple chart for teachers...
Second Visit: Vanessa Ko, Palm Harbor Middle School, Palm Harbor - Palm Harbor Middle School is a beautiful school. Bright posters and student work adorned the walls showing their Panther Pride. The office staff was very friendly and employed students as office assistants to check-in and escort visitors across campus. The young man who accompanied me was very friendly and made polite conversation as we walked to...
This blog series is going to be a blast! I am visiting the classrooms of some amazing teachers, the other four 2018 Florida Teacher of the Year finalists. The five of us, Diego Fuentes, Vanessa Ko, Kate Fiori, Mike Miller and I instantly became the best of friends and devoted colleagues during our time spent together during the interview process and TOY roundtable week last summer. I have great respect and admiration for...
Since my children graduated from high school and left home to pursue their dreams, it has been hard to get everyone home at the same time. This year, all four of my children were able to travel home for spring break. Having them all together reminded me of how unique each child is and, for the three who are teachers, how unique their classrooms must be as well. Being intrigued by the many different classrooms I have visited ac...
This Valentine's Day, I am excited to serve up the Florida Standards in the sweetest way possible – PERFECT CHOCOLATE CAKE! My grandmother first made this recipe in 1982, and it quickly took the top spot in our hearts. I hope that your family enjoys this delicious dessert and the many great opportunities it provides for learning!
PERFECT CHOCOLATE CAKE (Serves 16)
1 cup cocoa
2 cups boiling water
1 cup but...
Today, I am thrilled to share two recipes that are perfect for holiday potlucks! They are portable and easy to save if there are leftovers, although I doubt there will be with my dad's favorite pineapple casserole. Try these easy-to-make and easy-to-take recipes with your children and WOW your loved ones! You may even want to add them to your New Year's Day Dinner.
PINEAPPLE CASSEROLE (Serves 10-12)
2 20-ounce...
The holidays are well underway, and I am excited to share two more of my prized family recipes. I hope that you and your families enjoy these delicious new menu items and that these tips help you incorporate learning into your food preparation.
The first recipe is JOY SALAD, from my Aunt Shirley.
JOY SALAD (Serves 10-12)
2 large heads of romaine lettuce
½ cup olive oil
¼ cup red wine vinegar
½ cup sugar
1 cup sliced almon...
As Christmas approaches and we put together our menu for the Jerkins family dinner, I can't help but think of dessert. Our family loves dessert and it is always the hardest part of the menu to choose. This year, we've decided on Hummingbird Cake. I am thrilled to share this recipe with you because it was one of my grandfather's favorites that we have been cooking for almost 40 years. Plus, it offers many great opportu...
One of my dear friends, Margaret Wesley, shared this fantastic recipe for GELATIN FRUIT SALAD with my family. Margaret made something special for every church potluck, and she made you feel special when you talked to her. Talking to your children is the same. When parents make the time to be completely present with their children and give them undivided attention, it helps them develop the self-esteem and confidence they ne...
Our family recipe book is a collection of delicious foods with recipes from so many different people. I hope you were able to try last week's green bean bundles! This week, I am excited to bring you two of our family favorites: marinated carrots and sweet potato casserole.
MARINATED CARROTS (Serves 8-10)
Getting your children to eat their vegetables can be a challenge, but my mother-in-law had the solution with this...
In the Jerkins home, the holiday season is a time for family. We love to cook and eat, but most of all, we love spending hours together in the kitchen preparing for a feast. As Thanksgiving and the winter holidays approach, families throughout Florida will gather together, presenting fun learning opportunities.
Over the next five weeks, I will share some of my family's favorite holiday recipes, along with tips to help you bri...
Excitement, enthusiasm, and a little nervous energy! The pre-service teachers at the Florida Gulf Coast University's (FGCU) Eagles Educate Annual Mini Conference reminded me of how I felt many years ago when I began teaching. We all feel these emotions, and it is important that we don't allow our nerves to keep us from acting in the best interests of our students.
I learned this lesson early in my career wit...
In part four of my first blog series Volunteering to Learn, I will introduce you to Mrs. Karyl Elton. Like all teachers, she absolutely loved her craft, but what set her apart was her ability to develop a community within her high school classroom.
Being the choral director at Leesburg High School, Mrs. Karyl Elton worked with students of many grade levels. She believed that all students could learn to sing,...
Volunteering in classrooms has greatly impacted my teaching practice. Last week I shared about Mrs. Kertz's ability to reinforce positive behaviors and differentiate instruction in her first grade class. In this post, I'll open the door to Ms. Whitaker's middle school English classroom.
Creative, enthusiastic, and engaging! Anyone who walked into Ms. Judie Whitaker's classroom at Carver Middle School i...
In last week's post, I blogged about how volunteering with different teachers exposed me to the unique strategies that make their classrooms successful. This week, I am excited to share about the elementary classroom that transformed my teaching.
Three of my children had the blessing of having Mrs. Nancy Kertz as their first grade teacher at Skeen Elementary School in Leesburg. I volunteered in her room severa...