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The oral bacterium Porphyromonas gingivalis is a key etiological agent of human periodontitis, a prevalent chronic disease that affects up to 80% of the adult population worldwide. P. gingivalis exhibits neuraminidase activity. However, the enzyme responsible for this activity, its biochemical features, and its role in the physiology and virulence of P. gingivalis remain elusive. In this report, we found that P. gingivalis encodes a neuraminidase, PG0352 (SiaPg). Transcriptional analysis showed that PG0352 is monocistronic and is regulated by a sigma70-like promoter. Biochemical analyses demonstrated that SiaPg is an exo-α-neuraminidase that cleaves glycosidic-linked sialic acids. Cryoelectron microscopy and tomography analyses revealed that the PG0352 deletion mutant (ΔPG352) failed to produce an intact capsule layer. Compared to the wild type, in vitro studies showed that ΔPG352 formed less biofilm and was less resistant to killing by the host complement. In vivo studies showed that while the wild type caused a spreading type of infection that affected multiple organs and all infected mice were killed, ΔPG352 only caused localized infection and all animals survived. Taken together, these results demonstrate that SiaPg is an important virulence factor that contributes to the biofilm formation, capsule biosynthesis, and pathogenicity of P. gingivalis, and it can potentially serve as a new target for developing therapeutic agents against P. gingivalis infection.
Sialic acid is a generic term to describe a group of over 40 naturally occurring nine-carbon keto sugar acids (52, 67, 74). Among these molecules, the most abundant and best-studied sialic acid is N-acetylneuraminic acid (Neu5Ac). As such, the term "sialic acid" in the literature primarily means this particular compound. Sialic acids typically occupy the terminal positions within glycan molecules on the surfaces of eukaryotic cells, where they play important roles in a wide range of biological processes, including cell-cell interactions and small molecule-cell recognitions (70–73). Several pathogenic bacteria have evolved to utilize sialic acids either as nutrients or as decorating molecules to modify their surface-exposed macromolecules, including lipopolysaccharides (LPS) and polysialic acid (PSA) capsules (11, 53, 74). These sialic acid modifications allow bacterial pathogens to disguise themselves as host cells and thus circumvent and/or counteract the host's immune responses.
Bacteria have two primary routes to acquire sialic acids: by de novo biosynthesis and by means of a scavenger pathway (53, 72, 74). For the first route, the metabolite UDP-GlcNAc can be converted to Neu5Ac. In Escherichia coli, UDP-GlcNAc is first converted to ManNAc by NeuC (UDP-GlcNAc 2-epimerase) and then to Neu5Ac by NeuB (Neu5Ac synthase). For the second route, bacteria can directly uptake free sialic acids from the environments via transporters. They may also utilize neuraminidases to release sialic acids from a diverse range of host sialoglycoconjugates. The released sialic acids can then be transported into the cells. Neuraminidase is a family of enzymes that hydrolyze the glycosidic linkage between a terminal sialic acid and an adjacent glycosyl residue of certain glycoproteins, glycolipids, and oligosaccharides (1, 49, 50, 73). There are two types of neuraminidases. One is exo-α-neuraminidase, which hydrolyzes α-(2-3)-, α-(2-6)-, and α-(2-8)-glycosidic linkages of terminal sialic acids; the other is endo-neuraminidase, which cleaves α-(2-8)-sialosyl linkages in oligo- or poly-sialic acids.
Neuraminidases have been identified in several pathogenic bacteria (53, 73), such as Vibrio cholerae (20), Streptococcus pneumoniae (47), and Pseudomonas aeruginosa (29, 38). In these pathogens, neuraminidases are often associated with virulence. The neuraminidase of V. cholerae enhances the activity of cholera toxin (20). The two neuraminidases of S. pneumoniae contribute to the progression of infection (e.g., promoting pneumococcal brain endothelial cell invasion) (59, 69), and the neuraminidase of P. aeruginosa increases the binding of this organism to host cells (8). Neuraminidase activity has also been detected in several oral bacteria, such as Tannerella forsythia and Porphyromonas gingivalis (44, 66). T. forsythia possesses two neuraminidases, and one of them (NanH) is associated with bacterial colonization and invasion (28, 51).
P. gingivalis, a black-pigmented, asaccharolytic, Gram-negative anaerobe (4), is considered a major pathogen in human periodontitis, which is a prevalent chronic disease that affects ca. 80% of the adult population (27, 48, 58). P. gingivalis produces several virulence factors, including LPS, capsule, fimbriae and a group of proteolytic enzymes that cleave host proteins such as immunoglobulin and complement components (6, 36, 54, 57). In addition, P. gingivalis is highly invasive and is able to invade a variety of host cells (35). The de novo biosynthesis pathway of Neu5Ac is not evident in the genome of P. gingivalis (45); however, previous studies suggest that it produces neuraminidase (3, 44). For instance, Moncla et al. examined 25 P. gingivalis isolates and found that they all had neuraminidase activity, suggesting that neuraminidase commonly exists in P. gingivalis strains and that it may play a very important role in the biology and pathogenesis of P. gingivalis. In the present study, a gene encoding a neuraminidase was identified, and its enzymatic activity was analyzed. Its roles in biofilm formation, capsule synthesis, serum killing resistance, and virulence were explored using an approach consisting of biochemistry, targeted mutagenesis, cryoelectron microscopy (cryo-EM) and cryoelectron tomography (cryo-ET), and both in vitro and in vivo virulence studies.
Ethics statement.All animal experimentation was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. Human serum samples were collected from two healthy volunteers in the laboratory, and a written informed consent was provided by two participants. The protocols for animal studies and the use of human serum were approved by the Institutional Animal Care and Use Committee (IACUC; permit number: ORB23068Y) and the Human Subjects Review Committee (HSIRB; permit number: ORB0321006E) of the State University of New York at Buffalo, respectively.
Bacterial strains and growth conditions.P. gingivalis W83 (Pg83) (45), ATCC 33277 (Pg33277) (26), and FDC381 (Pg381) (9) strains were grown either in Trypticase soy broth (TSB; BD Diagnostic Systems, Sparks, MD) supplemented with vitamin K (1 μg ml−1) and hemin (5 μg ml−1) or on TSB agar plates containing 5% defibrinated sheep blood, vitamin K, and hemin (PML Microbiologicals, Wilsonville, OR) at 37°C in an anaerobic atmosphere of 80% N2, 10% H2, and 10% CO2 as previously described (9). E. coli TOP10 (Invitrogen, Carlsbad, CA) was used for DNA cloning, strain DH5α was used for β-galactosidase assays, and strain M15 used for preparing recombinant proteins. E. coli strains were cultured in lysogeny broth (LB) supplemented with appropriate concentrations of antibiotics.
Reverse transcription-PCR (RT-PCR) and RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE).Total RNA was extracted from 100 ml of exponential-phase P. gingivalis cultures using TRI reagent (Sigma-Aldrich, St. Louis, MO) according to the manufacturer's instructions. The resultant samples were treated with Turbo DNase I (Ambion, Austin, TX) at 37°C for 2 h to eliminate genomic DNA contamination. The obtained RNA samples were then extracted using acid-phenol-chloroform, precipitated in isopropanol, and washed with 70% ethanol. Finally, the RNA pellets were resuspended in RNase-free water. RNA (1 μg) was reverse transcribed using avian myeloblastosis virus reverse transcriptase (Promega, Madison, WI) to generate cDNA.
For RT-PCR analysis, 1 μl of cDNA was PCR amplified using Taq DNA polymerase (Qiagen, Valencia, CA). The P. gingivalis 16S rRNA gene was used as a positive control. The primers for RT-PCR are listed in Table 1. To determine the transcription start site upstream of PG0352, 5′-RACE analysis was carried out using the FirstChoice RLM-RACE Kit (Ambion), according to the manufacturer's protocol. Purified P. gingivalis RNA (10 μg) was reverse transcribed to cDNA with a 5′-RACE adapter, followed by PCR amplifications with primers P1/P2 (Table 1). The resultant PCR products were cloned into pGEM-T-easy vector (Promega) and sequenced (Roswell Park Cancer Institute DNA Sequencing Laboratory, Buffalo, NY).
β-Galactosidase activity assay.A fragment spanning from nucleotides −425 to +3 of PG0352 was PCR amplified from Pg83 genomic DNA with primers P3/P4, generating a fragment with engineered EcoRI and BamHI cut sites at the 5′ and 3′ ends, respectively. The obtained fragment was in-frame fused to the promoterless lacZ gene in the pRS415 plasmid (56), creating a reporter vector, pRSCLPg352. The resultant plasmid was transformed into E. coli DH5α. The β-galactosidase activity was measured as previously described (25), and the activity was expressed as the average Miller units of triplicate samples from two independent experiments.
Preparations of PG0352 recombinant protein and antiserum.The entire open reading frame of PG0352 was PCR amplified with the primers P18/P19 using Vent DNA polymerase (New England Biolabs, Ipswich, MA). The resultant PCR product was cloned into pGEM-T vector (Promega) and then subcloned into pQE30 expression vector (Qiagen) at engineered SalI and PstI cut sites. The obtained plasmid was transformed into E. coli M15 strain. The overexpression of PG0352 was induced with 1 mM IPTG (isopropyl-β-d-thiogalactopyranoside). The recombinant protein was purified using Ni-NTA agarose (Qiagen) under native conditions as previously described (65). The purified protein (rSiaPg) was dialyzed overnight and then concentrated using a 6.0-kDa molecular cutoff spectra membrane (Spectrum Laboratories Inc., Rancho Dominguez, CA). The concentration of rSiaPg was determined using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA). Antiserum against rSiaPg was raised as previously described (65).
Detection of neuraminidase activity.The neuraminidase activity was detected by using a filter paper spot test as previously described (43, 44). Briefly, rSiaPg or the whole-cell lysates of P. gingivalis were coincubated with 4-methylumbelliferyl-α-d-N-acetylneuraminic acid (4-MUNANA; Sigma-Aldrich), a fluorogenic neuraminidase substrate. Fluorescence was detected using the ChemiDoc XRS System (Bio-Rad) with an excitation wavelength of 302 nm and an emission wavelength of 548 nm.
Lectin blot assay.To determine the substrate specificity of rSiaPg, the lectin blot analysis described before (23) was carried out. Briefly, 1 μg of human α-1 acid glycoprotein (AGP) was coincubated with rSiaPg (0.6 nM) or with the Clostridium perfringens neuraminidase (NanH; Sigma-Aldrich) in a reaction buffer (10 mM potassium phosphate buffer [pH 7.0]) for 12 h. After the incubation, the samples were separated by SDS–12% PAGE and transferred to polyvinylidene difluoride membrane (Bio-Rad). A DIG glycan differentiation kit (Roche, Mannheim, Germany) was used to detect carbon hydrates within AGP according to the manufacturer's instructions. Lectins from three plants—Sambucus nigra (SNA), Maackia amurensis (MAA), and Datura stramonium (DSA)—were used to detect terminal α-(2-6)-linked sialic acid, α-(2-3)-linked sialic acid, and galactose linked to GlcNAc (14), respectively.
Constructing a P. gingivalis PG0352 deletion mutant.To inactivate PG0352, its upstream and downstream regions were PCR amplified from Pg83 genomic DNA using the primer pairs P5/P6 and P7/P8 (see Fig. S1 in the supplemental material), respectively. A previously described ermF/AM erythromycin resistance (Ermr) cassette was PCR amplified using the primer pair P9/P10 (39). The amplicons were individually cloned into pGEM-T Easy vector (Promega). The downstream fragment was released by BglII and NdeI digestion and then ligated to the 3′ end of the upstream fragment that had been digested at the same cut sites. The Ermr cassette was then released by BglII digestion and inserted into the above obtained fragment, generating PG0352-erm plasmid in which the entire open reading frame of PG0352 was deleted and replaced with the Ermr cassette. For the allelic-exchange mutagenesis, PG0352-erm plasmid was linearized with NdeI. Approximately 10 μg of the linearized plasmid was electroporated into the Pg83 cells, and the transformed cells were selected on the blood TSB agar plates containing clindamycin (5 μg ml−1) as described previously (18).
Measuring the growth rates and biofilm formation of P. gingivalis.To measure the growth rates, 50 μl of overnight P. gingivalis cultures (optical density at 600 nm [OD600] = 0.5) were inoculated into 5 ml of normal TSB or TSB medium supplemented with hemin (5 μg ml−1) and vitamin K (1 μg ml−1) without dextrose (BD Diagnostic Systems) and cultured at 37°C in an anaerobic chamber. The absorbance (OD600) of cultures at given times was measured using a Genesys spectrometer (Thermo Scientific, Waltham, MA). Biofilm formation was measured as previously described (24, 46, 77) with slight modifications. Briefly, Pg83 and ΔPG352 strains were grown to mid-log phase, and then the cell densities (OD600) were measured to ensure that the same amounts of Pg83 and ΔPG352 cells were used for biofilm measurements. These cultures were diluted 1:100 in phosphate-buffered saline (PBS; pH 7.4)-balanced TSB broth (50% PBS), and then 100-μl portions of the diluted cultures were added to 96-well flat-bottom polystyrene plates. The plates were then incubated anaerobically at 37°C for 3 days, allowing time for biofilms to develop. The biofilms were stained with 25 μl of 1% crystal violet for 15 min, washed with water three times, and then air dried for 30 min. To quantify the amount of biofilms, 150 μl of 95% ethanol was added to each well and shaken for 15 min. The absorbance (OD570) was measured with an xMark microplate spectrophotometer (Bio-Rad). The data are expressed as the average absorbance of 12 parallel samples. The data were statistically analyzed by one-way analysis of variance (ANOVA), followed by Tukey's multiple comparison at P < 0.01.
Cryo-EM and cryo-ET of frozen-hydrated P. gingivalis.Cryoelectron microscopy (cryo-EM) and cryoelectron tomography (cryo-ET) were carried out as described previously (40, 83). Briefly, the P. gingivalis cultures (5 μl) were mixed with 15-nm gold particles, deposited onto a freshly glow-discharged Holey carbon grid, blotted, and rapidly frozen in liquid ethane. The frozen-hydrated specimens were imaged at −170°C using a Polara G2 electron microscope (FEI Company, Hillsboro, OR) equipped with a field emission gun and a 4K×4K charge-coupled device (TVIPS, GmbH, Germany). The microscope was operated at 300 kV, and cryo-EM images were recorded at the magnification of ×23,000 (0.77 nm/pixel). A low-dose single-axis tilt series was collected from each bacterium at −8 μm defocus with a cumulative dose of ∼100 e−/Å2 distributed over 65 images with an angular increment of 2°, covering a range from −64° to +64°. The tilt series images were aligned and reconstructed using the IMOD software package (32). In all, 20 and 10 cryo-tomograms from Pg83 and ΔPG352 were reconstructed, respectively. Tomographic reconstructions were visualized using IMOD. One representative reconstruction from Pg83 and ΔPG352 was segmented manually using Amira 3-D modeling software (Visage Imaging, San Diego, CA). The 3-D segmentations of capsule (CPS), peptidoglycan (PG), outer membrane (OM), and inner membrane (IM) were manually constructed.
India ink staining.The staining was carried out as previously described (15) with slight modifications. P. gingivalis cells were taken from 4-day-old TSB agar plates and resuspended in 1 ml of PBS. Then, 10 μl of suspended cells was dropped on a glass slide, and a thin film was made by another slide. The film was air dried. A drop of 0.2% fuchsine (Sigma-Aldrich) was carefully added to the film and removed after 2 min by decanting. After air drying for 10 min, 10 μl of India ink (American MasterTech Scientific, Inc., Lodi, CA) was dropped on one end of the film, scraped by a clean slide to the other end, and then air dried again. A drop of 40% glycerol was added to the film and then covered with a new glass slip. Images were taken under a Zeiss Imager A2 microscope and processed using the Axiovision program (Zeiss, Germany).
Serum resistance assay.Serum resistance assays were carried out as previously described (57). Serum samples were collected from healthy laboratory donors. After venipuncture, the blood was allowed to clot, and the serum was separated by centrifugation. Serum samples were dispensed in aliquots and frozen at −80°C within 2 h of blood collection. Each aliquot was thawed once only. Heat-inactivated serum (used as a control) was prepared by incubating aliquots of the serum at 56°C for 30 min. Overnight cultures of P. gingivalis were diluted 1:10 in fresh TSB medium and grown to early exponential phase (5 h). Aliquots containing 75-μl bacterial cultures were mixed with 25 μl of serum (for a final concentration of 25% serum) and anaerobically coincubated for 1 or 3 h. After the incubations, the samples were removed, serially diluted, and plated on blood agar plates. The plates were incubated for 6 days before the colonies were counted. The average percentage of surviving bacteria (the number of living cells in fresh serum/the number of living cells in heat-inactivated serum) was calculated from triplicate samples of two independent experiments.
Animal studies and histopathology.Pg83, ΔPG352, and Pg381(a noncapsular strain) were tested for the virulence in a mouse model as previously described (18, 34). P. gingivalis strains were grown in TSB medium overnight, and cultures (10 ml at OD600 = 1.0) were harvested by centrifugation. The obtained cell pellets were washed twice with PBS under anaerobic conditions, counted in a Petroff-Hausser counting chamber (Hausser Scientific, Horsham, PA), and adjusted to 1011 ml−1 in PBS. Four-week-old BALB/c mice (female) were subcutaneously injected with 100 μl of bacterial suspension (∼1010 cells) on the dorsal surface. A group of mice was injected with PBS as a sham control. The general health, weight, appearance, and location of lesions were monitored daily until either the animals were dead or 10 days postinfection. Surviving mice were euthanized 10 days after infection by CO2 asphyxiation. Various tissues (skin with abscess, lung, liver, heart, spleen, and kidney) were collected and fixed in 10% neutral buffered formalin. Hematoxylin and eosin slides were prepared by standard pathological techniques. These experiments were approved by the IACUC at the State University of New York at Buffalo.
PG0352 is a neuraminidase.Neuraminidase activity has been detected in different isolates of P. gingivalis (44). To identify the enzyme(s) responsible for the activity, neuraminidases from other bacterial species such as Clostridium perfringens (NanH) were used as queries to search the genome of Pg83 (45). It was found that the gene PG0352 encodes a well-conserved neuraminidase. PG0352 (hereafter designated SiaPg) consists of 526 amino acids (aa) and has a predicted molecular mass of 58.5 kDa. The N terminus (1 to 30 aa) of SiaPg contains a putative signal peptide (see Fig. S2 in the supplemental material), suggesting that this protein is probably secreted. Following the signal peptide is a peptidoglycan-binding domain (30 to 180 aa). The C terminus (180 to 526 aa) is a conserved neuraminidase domain (12, 13, 84). Sequence alignment analysis further revealed that the C terminus of SiaPg has a conserved catalytic domain of neuraminidase, which consists of one RIP motif and three Asp-box motifs (S/T-X-d-[X]-G-X-T-W/F) (Fig. 1), suggesting that SiaPg is a neuraminidase.
To confirm these observations, the filter paper spot assay was carried out to test the enzymatic activity of recombinant SiaPg protein (rSiaPg). As shown in Fig. 2, rSiaPg was able to cleave the fluorogenic neuraminidase substrate, 4-MUNANA, and produced fluorescence (spot 1). This activity was completely abolished when the protein was denatured (spot 2). In addition, a neuraminidase specific inhibitor (82), 2,3-didehydro-2-deoxy-N-acetylneuraminic acid (Neu5Ac2en), inhibited the activity of rSiaPg (spots 3 and 4). Collectively, these results demonstrate that SiaPg is a neuraminidase.
SiaPg has an exo-α-neuraminidase activity.To determine whether SiaPg is an exo-neuraminidase or an endo-neuraminidase, the lectin blot assay was carried out. For this assay, C. perfringens neuraminidase (NanH, designated here as SiaCp), a well-studied exo-α-neuraminidase (33), was used as a positive control. As shown in Fig. 3, for the samples treated with PBS (lane 1), three lectins (DSA, SNA, and MMA [described in Materials and Methods]) bound to AGP. After treatment with SiaCp (lane 3), SNA and MMA failed to bind AGP, indicating that SiaCp removed the terminal α-(2-6)-linked and α-(2-3)-linked sialic acid. The treatment of rSiaPg had the same pattern as SiaCp (lane 2), showing that SiaPg is an exo-α-neuraminidase like its counterpart of C. perfringens.
Transcriptional analysis of PG0352.The genes adjacent to PG0352 are divergently transcribed (see Fig. S1 in the supplemental material), suggesting that this gene is monocistronic. To understand its regulation, RLM-RACE analysis was conducted to determine its transcription start site. The start site was mapped to an adenosine that is 101 nucleotides from the start codon of PG0352 (Fig. 4). A promoter-like consensus was identified at the −10 (TCTATT) and −35 (TTGGGA) regions (Fig. 4A), which is similar to those of the σ70 promoter of E. coli (68), suggesting that PG0352 is probably regulated by a σ70-like promoter. The identified promoter was named P0352. Transcription analysis using lacZ gene as a reporter further showed that P0352 was functional in E. coli. The β-galactosidase activity (the average value = 5190 ± 284 Miller units) of a plasmid containing the P0352 promoter region (pRSCLP0352) is ∼100-fold greater than that of pRS415 (average activity = 54 ± 11 Miller units), a promoterless vector (56). Collectively, these results indicate that P0352 is a promoter that can be recognized by the RNA polymerases and possibly by other transcription factors of E. coli.
Isolation and characterization of PG0352 deletion mutant.To study the function of SiaPg, the PG0352 gene was inactivated by targeted mutagenesis as illustrated in Fig. S1 in the supplemental material. Erythromycin-resistant (Ermr) colonies appeared 7 days after plating, and 10 colonies were selected and screened by PCR with specific primers to Ermr cassette. All of the examined colonies contained the Ermr cassette. One clone (ΔPG352) was further analyzed by PCR with different pairs of primers located at the flanking regions of PG0352 and the Ermr cassette, and the results showed that the PG0352 gene was deleted and replaced with Ermr cassette as expected (see Fig. S1 in the supplemental material). RT-PCR analysis showed that the PG0352 transcript was abolished in the mutant (Fig. 5A). Western blot analysis using a specific antibody against SiaPg showed that the cognate gene product was abrogated in the mutant (Fig. 5B).
The filter paper spot assay was repeated using the whole-cell lysates of Pg83 and ΔPG352. As expected, the neuraminidase activity was detected in Pg83 but was not in the mutant (Fig. 5C). Collectively, these results showed that the PG0352 gene was totally disrupted in the ΔPG352 mutant. In addition, the neuraminidase activity was also detected in Pg33277 and Pg381 strains (see Fig. S3 in the supplemental material). Taken together with previous reports (44), these results suggest that neuraminidase genes are broadly distributed in P. gingivalis isolates.
Inactivation of PG0352 does not influence the planktonic growth of P. gingivalis.To determine the influence of sialic acid on the growth of P. gingivalis, Pg83 and ΔPG352 were first cultured in normal TSB medium and then in the medium supplemented with exogenous Neu5Ac (30 μg ml−1). As shown in Fig. 6, the mutant grew at rates similar to the wild type with or without the sialic acid, indicating that SiaPg is not required for the planktonic growth of P. gingivalis. The experiment was repeated using a modified TSB medium in which dextrose was removed and supplemented with Neu5Ac (30 μg ml−1), and it was found that both Pg83 and ΔPG352 had a similar growth pattern (Fig. 6B), highlighting that P. gingivalis probably does not use sialic acid as a nutrient at the in vitro culture conditions.
SiaPg influences the biofilm formation of P. gingivalis.To test the effect of sialic acids on the biofilm growth of P. gingivalis, the normal biofilm growth medium was supplemented with four different concentrations of Neu5Ac (3, 8, 16, and 30 μg/ml). As shown in Fig. 7, for the wild type, the addition of exogenous sialic acid slightly reduced biofilm formation, but the effect was not significant. Compared to Pg83, ΔPG352 significantly decreased (∼2.5-fold reduction) the biofilm formation. Interestingly, the supplement of exogenous Neu5Ac restored the biofilm formation, and this restoration was in a dose-dependent manner (Fig. 7). For example, the addition of 3 μg of Neu5Ac/ml only partially restored the biofilm formation of the ΔPG352 mutant; however, biofilm formation in the mutant was fully restored at the concentration of 30 μg of Neu5A/ml. Taken together, these results suggest that SiaPg is involved in the biofilm formation of P. gingivalis.
The ΔPG352 mutant fails to produce an intact capsule.To further explore the role of SiaPg, cryo-ET was used to dissect the cellular structures of P. gingivalis. As shown in Fig. 8, the P. gingivalis cell is rod-shaped, and it consists of an inner membrane (IM), a peptidoglycan layer (PG), and an outer membrane (OM). The outermost part of the cell is a thick layer of capsule-like materials (Fig. 8A and C). This layer could not be detected in Pg381, a noncapsular strain (see Fig. S4 in the supplemental material), indicating that it is capsule (CPS). The CPS layer in Pg83 has a defined shape and is evenly distributed on the cell surface. Its average width is 37 ± 2.76 nm (n = 10 cells). Compared to Pg83, the CPS layer observed in the ΔPG352 mutant was uneven and substantially thinner (average thickness = 13.72 ± 1.36 nm, n = 10 cells), and it lost the defined shape due to the reduced thickness and density (Fig. 8B and D).
To rule out the possibility that the observed CPS-like structure in ΔPG352 is another type of surface structure that is composed of proteins (e.g., fimbriae), the mutant was treated with proteinase K prior to the cryo-ET analysis. After the treatment, this layer still could be observed in the mutant (Fig. 8E), indicating that the observed structure is indeed a thin layer of capsule. In addition, when the ΔPG352 mutant was cultured in the medium supplemented with exogenous Neu5Ac (30 μg/ml), the mutant had a layer of CPS that was similar to the wild-type CPS (Fig. 8F and G), suggesting that the defective CPS in the mutant is simply due to the lack of sialic acid. India ink staining further confirmed that the remaining CPS layer in the ΔPG352 mutant was defective. As expected, the encapsulated P83 strain displayed colorless halos around individual cells, indicative the layer of CPS (Fig. 9, left panel). In contrast, the ΔPG352 mutant and Pg381 were stained by the dye (Fig. 9, middle and right panels), indicating that the mutant is not resistant to the dye due to lack of intact capsule. Collectively, these results demonstrate that SiaPg is required for the synthesis and/or assembly of capsule of P. gingivalis.
The ΔPG352 mutant is less resistant to complement killing.P. gingivalis is resistant to killing by host complement, and it is believed that capsule plays a central role in the resistance (57, 63). Since ΔPG352 has a defective capsule, we hypothesize that the mutant might become sensitive to the complement killing. To test this hypothesis, Pg83 and ΔPG352 were coincubated with 25% fresh human serum for 1 and 3 h. As expected, Pg83 was resistant to the killing, since its survival rate at the 1-h incubation point was almost 100%. In contrast, after a 1-h exposure to the serum, the survival rate of ΔPG352 was decreased to 22.4% (Fig. 10), indicating that the mutant is more vulnerable to the killing. Interestingly, after the 3-h exposure, the survival rate (26.2%) of the mutant was almost identical to that of the 1-h incubation (Fig. 10), suggesting that there might be another factor(s) also contributing to the serum resistance of P. gingivalis. Taken together, these results indicate that the presence of SiaPg is able to enhance the serum resistance of P. gingivalis.
The ΔPG352 mutant has decreased virulence in vivo.A previously described mouse abscess model (18, 34) was used to further evaluate the role of SiaPg in the virulence of P. gingivalis. In the present study, Pg83, ΔPG352, Pg381, and a sham control (PBS) were included. Each mouse was subcutaneously injected with ∼1010 cells of P. gingivalis or an equal volume of PBS at the dorsal sites. As shown in Table 2, for the Pg83 strain, no localized abscesses were observed at the injection sites. Instead, all three mice developed secondary lesions (average size of secondary lesions was 18.7 mm2, ranging from 10 to 24 mm2) on their ventral sides. All three mice died within 6 days after the injections. In contrast to Pg83, Pg381 only induced localized abscesses at the injection sites (the average size of abscesses was 147 mm2, ranging from 60 to 260 mm2), but no secondary lesions were observed. All animals survived. Compared to these two strain, for the ΔPG352 mutant, two mice developed localized abscess at the injection sites (44.2 and 28.3 mm2), one mouse had a tiny secondary lesion (4 mm2), and all three mice survived. These results showed that the virulence of ΔPG352 was substantially reduced.
Histological studies confirmed that Pg83 was able to cause a spreading type of infection, which had been previously reported (30, 34). Inflammation could be observed in multiple organs, including the lungs, liver, spleen, and kidneys. The inflammation was more prominent in the lung and liver tissues of infected animals (e.g., severe intrasinusoidal inflammatory infiltrates were observed in the lung and liver tissues [Fig. 11]). In contrast, the ΔPG352 mutant failed to cause the spreading type of infection, and no obvious infiltrates were observed in the lung and liver tissues (Fig. 11). Taken together, these results indicate that the virulence of the ΔPG352 mutant is substantially reduced compared to that of the wild type, highlighting that SiaPg is essential for the pathogenicity of P. gingivalis.
Is SiaPg the sole neuraminidase of P. gingivalis?The evidence presented in this report (Fig. 1, 2, and 3) clearly demonstrates that SiaPg is a neuraminidase that is similar to its counterparts from other bacteria; inactivation of PG0352 completely abolished the neuraminidase activity (Fig. 5), suggesting that SiaPg may be the sole neuraminidase of P. gingivalis. However, Aruni et al. recently reported that in addition to PG0352, P. gingivalis has another two sialidases (PG0778 and PG1724), and the inactivation of PG0352 reduces total sialidase activity by only 5% (3). This discrepancy is most likely due to different detection methods. In the previous report, an Amplex Red neuraminidase assay kit was used to measure general sialidase and sialoglycoprotease activity; here, we used the filter paper spot assay—a specific method for testing neuraminidase activity (43, 44). PG0778 is annotated as a hypothetical protein and PG1724 as a putative DNA-binding/iron metalloprotein/AP endonuclease or O-sialoglycoprotein endopeptidase (45). BLAST analysis reveals that these two proteins do not belong to the neuraminidase Pfam (12). In addition, both PG0778 and PG1724 lack the F/YRIP motif. Crystal structural and site-directed mutagenesis analyses have shown that this motif is essential for the enzymatic activity of neuraminidases (10, 13). In this regard, it seems unlikely that PG0778 and PG1724 function as a neuraminidase. Instead, they may function as other enzymes, e.g., acting as a sialoglycoprotease, and their enzymatic activities can be defined by biochemical, genetic, and structural studies.
Does P. gingivalis utilize Neu5Ac as a nutrient?Several bacteria use Neu5Ac as an alternative nutrient (53, 74). In this case, the mutants, which fail to synthesize or use Neu5Ac, have growth defects when the sialic acid serves as a sole carbon source. Several lines of evidence suggest that P. gingivalis probably does not use Neu5Ac as a nutrient. First, P. gingivalis, as an asaccharolytic bacterium, primarily utilizes short peptides instead of carbohydrates for energy production (4). Second, in the catabolic pathway of sialic acid, NanA first cleaves Neu5Ac to ManNAc and pyruvate. ManNAc is ultimately converted to fructose-6-phosphate and ammonia via a series of reactions catalyzed by NanK, NanE, NagB, and NagA (16, 53, 74). However, the genes encoding these enzymes have not yet been annotated in the genome of P. gingivalis (45), suggesting that the catabolic pathway of Neu5Ac most likely does not exist in P. gingivalis. Third, supplementing sialic acid did not stimulate the growth of P. gingivalis (Fig. 6A). Finally, the inactivation of PG0352 did not influence the growth of P. gingivalis (Fig. 6B).
How does SiaPg influence the biofilm formation of P. gingivalis?The impact of neuraminidases on biofilm formation has been described in several pathogenic bacteria (47, 51, 59, 64), even though the mechanism involved remains obscure. In S. pneumoniae, the expression of nanA (encoding a neuraminidase) is upregulated when it grows under biofilm conditions and NanA inhibitors block its biofilm formation. In P. aeruginosa, the nanA deletion mutant has diminished biofilm production, and the mutant is unable to establish respiratory infection. Here, we also found that the biofilm formation was substantially reduced in the ΔPG352 mutant (Fig. 7). Interestingly, the ΔPG352 mutant restored the biofilm formation in the addition of exogenous sialic acid (Fig. 7), e.g., at the concentration of 30 μg of Neu5Ac/ml, the mutant almost produced the same amount of biofilms as Pg83. At the same condition, the ΔPG352 mutant also restored capsule synthesis (Fig. 8F). Thus, it is possible that the regaining biofilm production could be due to the restoration of capsule in the mutant. However, a recent report from Davey et al. does not support this assumption, since they found that the loss of capsule enhances the biofilm formation of P. gingivalis (15). Since sialic acids are often used to modify bacterial cell surface molecules (e.g., LPS and capsule), it is also possible that the presence of sialic acids may change the cell surface hydrophobicity and consequently influence the biofilm formation of P. gingivalis. Fimbria is required for the biofilm formation of P. gingivalis, and its expression level can be influenced by different environmental cues (80, 81). In E. coli, sialic acids often act as a signaling molecule to modulate the production of fimbriae (for recent review, see reference 74). Thus, a similar scenario can also exist in P. gingivalis, i.e., exogenous sialic acids may function as an environmental cue to regulate the fimbrial gene expression, which further influences the biofilm production of P. gingivalis. At this point, we are unable to rule out which one of these possibilities is accountable for the observed phenotype; however, the evidence described here highlights the role of neuraminidases in the biofilm formation of P. gingivalis, and it provides us a solid foundation to further unveil the potential mechanism involved.
How does SiaPg influence the capsule synthesis of P. gingivalis?The majority of P. gingivalis isolates are encapsulated. Based on the serotypes of K antigens, P. gingivalis isolates can be divided into at least six groups (K1 to K6) (7, 34), highlighting the complexity of carbohydrates presented in P. gingivalis capsular polysaccharides. Little is known about the structure and composition of carbohydrates in the capsule of P. gingivalis. It is possible that P. gingivalis (at least Pg83 strain) utilizes sialic acid either to directly synthesize the PSA-type capsule or to modify sugar molecules on other types of capsules. P. gingivalis lacks the de novo synthesis pathway of sialic acid. For example, NeuB (Neu5Ac synthase) (53, 74), a key enzyme that converts UDP-GlcNAc to Neu5Ac, is absent in the genome of Pg83 (45). As such, the scavenge pathway mediated by Siapg might be the only route for P. gingivalis to acquire sialic acid. If this is the case, we expect that the deletion of SiaPg would completely block the scavenge route and the mutant would fail to produce the capsule due to the lack of sialic acid. This proposition is consistent with the phenotype of the ΔPG352 mutant (Fig. 8 and 9).
The E. coli K1 and K92 capsules use polysialic acid (PSA) as the nucleotide sugars (17, 78, 79). In the biosynthesis pathway of PSA capsule, NeuA (CMP-Neu5Ac synthetase) and NeuS are two signature enzymes. NeuA converts Neu5Ac to its active form CMP-Neu5Ac (31, 42), which is then added to appropriate acceptors by NeuS, a linkage-specific sialyl-transferase (60, 61). PSA is exported through the Kps system (78). However, the homologs of NeuA and NeuS are not annotated in the genome of Pg83 (45), suggesting that P. gingivalis may have a unique system to utilize sialic acids to either directly synthesize PSA-type capsule or modify its capsule via sialylation. Two recent studies reveal that capsule biosynthesis in P. gingivalis is quite unique (2, 15). The findings in the present study could provide a new angle for studying the capsule biosynthesis of P. gingivalis. In addition, sialic acids can serve as signaling molecules (74). Thus, it is also possible that the lack of sialic acids in ΔPG352 may have an impact on other factors that are required for the capsule biosynthesis and/or assembly.
Why is ΔPG352 more sensitive to the serum killing?P. gingivalis is resistant to complement killing (63). Compared to the wild type, the ΔPG352 mutant became more vulnerable to serum killing (Fig. 9). Previous studies have suggested that the possible mechanisms for serum killing resistance of P. gingivalis include protease production and capsule formation (22, 57, 62). P. gingivalis is able to degrade the complement factors C3 and C5, and the degradation is dependent on the activity of gingipains. A recent report describes that the P. gingivalis sialidase/sialoglycoprotease activity may be involved in regulating gingipain activity (3). Thus, the loss of serum resistance in the ΔPG352 mutant could be due to the impact on the activity of gingipains. However, Slaney et al. report that gingipains are not required for the serum resistance, since the gingipain-deficient mutants are still as resistant as the wild type to serum killing. Instead, they found that the capsule plays an essential role in complement resistance. Since the ΔPG352 mutant has defected capsule (Fig. 8), it seems more likely that the vulnerability to serum killing in the mutant is ascribed to the defected capsule. In addition, previous studies from Neisseria show that sialyated surface molecules bind to factor H and enhance the complement resistance (76). Thus, it is also possible that the loss of SiaPg may influence the sialylation of other surface molecules and consequently interfere with the serum resistance. In future studies, we will attempt to pinpoint which mechanism(s) is responsible for the observed phenotype.
Role of SiaPg in the pathogenicity of P. gingivalis.As a member of the "red-complex" bacteria, P. gingivalis primarily inhabits the gingival crevices (27, 37). The major component of crevicular fluid is plasma (41). The total sialic acid in the plasma is quite abundant (∼2 mM). Under normal physiological conditions, almost all sialic acid (99.9%) is bound to a diverse range of proteins or lipids (55, 74). In addition to the plasma, salivary mucins and fibronectins are also rich in sialic acids (19). In this unique niche, a microorganism armed with a neuraminidase will be able to acquire sialic acid from the host via the scavenge pathway and strategically position itself to compete with other organisms or to overcome host immune attacks by camouflaging their surface molecules (e.g., LPS and capsules) with sialic acids. The studies described here reveal an important role that SiaPg plays in the pathogenicity of P. gingivalis, e.g., influencing the capsule biosynthesis.
The capsule is highly associated with the virulence of P. gingivalis (34). Animal studies have demonstrated that the noncapsular (K−) P. gingivalis strains are much less virulent than the encapsulated isolates. Gonzales et al. reported that immunization with purified K1 capsule prevents mice from P. gingivalis-elicited oral bone loss (21). Slaney et al. recently revealed that the capsule plays a key role in the resistance to serum killing (57). The studies reported here show that the ΔPG352 mutant has a defective capsule. Compared to the wild-type strain, the mutant is sensitive to complement killing and much less virulent in the mouse model (Fig. 10 and 11 and Table 2). The observed phenotype is similar to that of the noncapsular P. gingivalis isolates or mutants, suggesting that SiaPg most likely contributes to the pathogenicity of P. gingivalis by influencing capsule formation. Besides being used in capsule formation, sialic acids can also be utilized to modify LPS and other virulence factors, which have been observed in Haemophilus influenzae, N. meningitidis, and several other pathogenic bacteria (5, 53, 74, 75). Thus, the observed in vivo phenotype could also be ascribed to the change of other virulence factors in the ΔPG352 mutant. The existence of this possibility cannot be ruled out until a genome-wide glycomic analysis is conducted. Nevertheless, the studies described here provide us a solid starting point to further explore the role of sialylation in the biology and pathogenicity of P. gingivalis.
We thank H. Sojar, K. Homma, and A. Sharma for providing P. gingivalis strains and antibodies and E. Haase for providing pRS415 plasmid.
This study was supported by NIH/NIDCR and NIH/NIAID grants DE019667 (Chunhao Li), AI078958 (Chunhao Li), and AI087946 (J.L.), along with grant from the Welch Foundation (AU-1714 to J.L.). Maintenance of the Polara electron microscope facility is partially supported by the Structural Biology Center at the UT Houston Medical School.
- Received 8 August 2011.
- Returned for modification 7 September 2011.
- Accepted 18 October 2011.
- Accepted manuscript posted online 24 October 2011.
Supplemental material for this article may be found at http://dx.doi.org/10.1128/IAI.05773-11.
- Copyright © 2012, American Society for Microbiology. All Rights Reserved.
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Prescott Wins 2012 Sydney Harbor Regatta
12 March 2012
The fleet of Melges 32's were showcased at the weekends seventh Sydney Harbour Regatta hosted by Middle Harbour Yacht Club boasting 237 entries.
In the Melges 32 fleet, it was hard to get past Greg Prescott's 2 Unlimited Seven Star from Tasmania, which was skippered this weekend by gun Farr 40 owner, Martin Hill, who dragged Olympian and fellow Farr 40 sailor Bobby Wilmot along for the ride to trim mainsheet, and world champion one-design sailor and coach, Darren 'Twirler' Jones to call tactics. The crew pulled a win either side of a second place for an early series lead at the end of day one.
"It was fantastic; we were doing 15 knots in 19 knots of breeze and in a building easterly we went so fast downwind – you can really stretch out downwind – and we did," Hill said after having his first ride on a Melges, the Middle Harbour YC sailor described steering the boat as "light and responsive."
Of the regatta in general, Hill, the past commodore of the host club said: "It's great to see such a hot and huge fleet out on the Harbour – it's a real carnival of sail.
By the end of racing, day two, the Melges 32 One-Design fleet was dominated by Tasmanian entry 2 Unlimited Seven Star, five wins from six races. There were three Tassies aboard; Darren 'Twirler' Jones, who has called Sydney home for many years, Mark Jeffrey and Ollie Nicholas. And although the crew had not sailed together before, they dominated from go to whoa and are thrilled to be heading to the National Championship at Pittwater next Friday as 'the boat to beat'.
Ollie Nicholas admitted he was surprised by their domination in the hot fleet: "It's been good to come up to Sydney and sail with a good bunch of blokes and to finish this well – we had no idea how we'd go – to win so many races was a bit of a surprise," he said.
Angus Reid's Maxstar from Canberra Yacht Club stole a win from 2 Unlimited in race two and sailed consistently to take second place, followed three points behind by Middle Harbour Yacht Club representative Geoff Masters Funnelweb third place overall.
Day one of the regatta MHYC Club Captain John McCuaig skippered Funnelweb positioning them well for Masters who flew in from the USA this morning and was dropped onto the boat prior to racing.
The Melges 32 fleet will be heading to Pittwater for next weekend's Australian Championship.
Di Pearson
MHYC Media

subset: commoncrawl


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Skyline Releases Women's Basketball Report #7
- Kimberly Blakney named Player of the Week
Clifton Park, N.Y. - The Skyline Conference released the seventh Women's Basketball report for the 2009-10 season this afternoon, including Player of the Week, Rookie of the Week, Honor Roll and Conference statistics.
Senior guard Kimberly Blakney (Brooklyn, N.Y./Benjamin Banneker) was named Skyline Conference Player of the Week. Blakney averaged 28 points, 17.5 rebounds and seven steals a game, leading Farmingdale to wins over Connecticut College and SUNYIT in the ECAC Holiday Festival. Blakney was named the Bracket "C" Most Outstanding Player for the Tournament. She had 24 points, 16 rebounds and seven steals in a 69-55 win over Connecticut College. In the next game, Blakney set tournament single-game records with 32 points and 19 rebounds, while adding seven steals in a 61-54 win over SUNYIT.
To view the full report, click HERE.


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Enrichment: I have labeled this an enrichment lesson because in our unit we were working with only whole number estimation. While students struggling with whole number estimation worked on exercises to support them, those who mastered the skill were challenged by this rich real life task!
Setting the Stage: To help these students practice MP4 and apply critical thinking skills to a real world scenario using their mastered rounding/estimating skills, I decided to have some fun with a creative project. They also would be extending their rounding skills to the nearest dollar and thinking about how to use the decimal point. ( Lower students were working on MAPS based computer program and some RTI work.)
I pulled my middle to high level students aside to tell them the news!
I told them they each had an Italian Uncle who had just died and left them his restaurant! In the Will, he said that he required them to create new menus for a mid- price ranged restaurant. It could NOT be fast food! In the Will, he stipulated that a person should not have to spend over fifty dollars to eat a meal of appetizer, drink, entree, and dessert. The foods should be organic and locally grown if possible and therefore, it will be a little more expensive to supply, but it will keep up with the current competition and consumer trend.
I took a minute to explain all of that and what "competition" and "consumer trend" meant. I explained to them that I was their business agent who would help them figure out a good menu by showing them how to research the criteria for their menus.
Materials: Drawing paper & art supplies & iPad
I listed the requirements for their menu on the white board.
1. The menu had to consist of 3-5 appetizer choices, 3-5 entree choices, 3-5 dessert choices and drinks ( no alcohol)
2. The menu prices needed to be rounded to the nearest dollar and you must add combinations to be sure a customer can never go over $50.00 ( This exercises MP2 and reinforces and extends 4.NBCT.A.3 )
3. A person should not have to spend more than $50.00 to have a complete meal.
4. You must research and choose up to two websites to find examples of the items on your menu. (Otherwise it gets too time consuming).
5. You must describe the items on your menu. ( Except for drinks)
6. You must create it on the iPad or on your home computer.
7. You can print it!
Rather than just turning them loose to research restaurants, we made a list together of restaurants in the area that they knew. We discussed what makes a fast food restaurant. What is the difference between the mid- priced restaurant and a fast food and its menu.
After we made the list. I brought up a website. We discussed the menu design and how prices were listed. We looked at a few high priced restaurants ( Betony in NY City where my nephew is the Executive Chef. This added a personal touch to the lesson that they really liked) All of the prices have no cents. We discussed that mid- priced restaurants often have the cents with the dollars, but most of the time, the amounts are rounded.
After we looked at a few choices in our area, they went back to their desks and I let them research on their own for 10 minutes. I roved the classroom discussing the menu research and understanding the prices to help support student's understanding. There were wonderful discoveries about how restaurants list their prices. We learned that chain restaurants don't advertise prices on the internet. When I asked them why, they didn't know that prices vary from coast to coast.
They continually came to me to ask if what they were looking at was a fast food restaurant. I stopped them and talked about prices and how they could tell by the prices if it was fast food. Then we discussed the nutrition. What were the differences there?
Students worked in their assignment notebooks to develop rough drafts of their menus.
This real life exposure to thinking about cost of food, organizing a menu designed to fit a budget and then the chance to use that information to create a menu is rich and engaging!
Students presented their menus to the rest of the class either on drawing paper or on Apple TV on their iPad. I asked each one of them upon their presentation what the largest amount of money a person could spend in their restaurant for a meal including beverages, appetizer, entree and dessert. Each student easily added in their head and everyone's menu was under $50.00. Their menus were entertaining on Educreations with narrations and on Haiku Deck with backgrounds they had chosen. Menu 3 page 1,Menu 3 page 2, Menu 3 page 3 's sense of humor was fun. "Where the customer is always right." cracked me up. Menu 2 page 1,& Menu 2 page 1's restaurant's name is "Ducks". He claims that duck is not served there.
It's fun...It's all just fun and the rounding and estimation application is just great.


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EXETER — A desire to protect patients and comply with New Hampshire's medical privacy laws is behind Exeter Hospital's request for "judicial guidance" regarding whether it can share patient records with investigators probing a hepatitis C outbreak.
EXETER — A desire to protect patients and comply with New Hampshire's medical privacy laws is behind Exeter Hospital's request for "judicial guidance" regarding whether it can share patient records with investigators probing a hepatitis C outbreak.
An attorney representing the hospital speaking at a press conference Wednesday said he was "astonished" by the Attorney General's accusation they were obstructing the investigation by seeking guidance.
The hospital filed a petition in Merrimack Superior Court on Aug. 29 asking a judge to grant a protective order shielding the medical records of its patients from state health officials.
The state is still trying to determine the scope of the outbreak, which surfaced in May, and has led to infections in at least 32 Exeter Hospital patients.
A former Exeter Hospital technician has been charged with spreading the virus. He is accused of stealing hospital drugs, injecting himself, then placing syringes contaminated with his blood back into the laboratory, where they were used on patients.
The hospital says the state's Division of Public Health Services has requested broad access to medical records that contain confidential health information. Releasing the information would constitute a breach of both state and federal laws, according to the hospital.
In motions filed Tuesday in Merrimack Superior Court, staffers from the attorney general's office refuted that claim. They argue state and federal laws expressly provide health officials with the power to review medical records amid a viral outbreak.
In response, attorney Scott O'Connell, of the legal firm Nixon Peabody LLP, said Exeter Hospital has nothing to hide, and is eager to assist the state's investigation, but it was forced to seek help from a judge because "as time has gone on, the state has asked for more than it's legally entitled to have."
"Exeter wants to know what happened as much as anybody, but at the same time, we are fiduciaries for our clients' information," O'Connell said Wednesday at a press conference on the campus of the hospital.
"Exeter maintains sacrosanct, confidential, private information about the care it has given its population of patients, and we take that responsibility of maintaining that information seriously," he said.
According to O'Connell, under New Hampshire law, the hospital is prohibited from furnishing patient records to anyone — including state health officials — unless certain "threshold requirements" are met. Among those requirements is the need to spell out why the patient records being sought are linked with the hepatitis C outbreak, he said.
In some instances, the hospital must also obtain the consent of patients before releasing the records, O'Connell said. For example, New Hampshire law protects records regarding treatment for substance abuse, genetic testing, HIV status, sexual assault and psychiatric evaluation, he said.
O'Connell said the state wants access to Exeter Hospital's entire medical record system, and is unwilling to narrow its request or to allow the hospital to remove protected information from the records.
"We want to help the state, but the state is not meeting us in the middle," he said. "They are not making their requests surgical, discrete, tailored to get only that which is minimally necessary for their legitimate interests."
O'Connell also categorically denied an assertion that Exeter Hospital is using its litigation in Merrimack Superior Court as "an excuse to absolutely refuse to provide any information to Public Health." That charge was leveled in a sworn affidavit from State Epidemiologist Dr. Sharon-Alroy Preis.
O'Connell said he has attempted to establish new ground rules with the state regarding patient records, but the state has refused to identify which patient records, or which portions of those records, it is seeking.
"These are the minimum requirements under state law and federal law to make it available to the state epidemiologist and her team," O'Connell said. "In the absence of some level of cooperation and some understanding in Concord that they've got an obligation to help us maintain the privacy of these patient records, we are left with the only option of going to state court for assistance."
O'Connell said he was "astonished" to read an assertion from the state attorney general's office that the hospital has obstructed investigators. Nothing could be further from the truth, he said. Exeter has worked cooperatively with the state, and was willing to accept an expedited court ruling regarding patient records, O'Connell said. The fact that it's taken the state a month to respond to Exeter Hospital's petition is evidence that the issues involved are complex, he said.
"We did what our system requires," O'Connell said. "We sought a judge to help us. Ladies and gentleman, that's not obstructing justice — that's using justice. That's using the system of laws that we have in this state and this nation to get a ruling from a judge authorized to tell us where the balancing has to be struck for these competing concerns."
Mark Whitney, vice president for strategy and community relations at Exeter Health Resources, said the hospital anticipates a judge will rule that the state's request for patient records far outreaches its legal rights. Whitney said the judge is likely to rule that the state must make a clearer connection between the records sought and the hepatitis C outbreak.
Whitney said New Hampshire citizens have high regard for medical privacy, and the state has adopted a patients' bill of rights that has more stringent disclosure requirements than federal law. Exeter Hospital administrators wish to continue assisting the state's investigation, Whitney said, but they also have a responsibility to protect the privacy of tens of thousands of patients.
"Exeter Hospital absolutely wants to work with state officials to get to the bottom of what's happened here," Whitney said, "but that's why we actually asked a judge for guidance to help us balance the hospital's responsibility related to protecting patient privacy and the state's request for getting more information from our electronic medical record systems."


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Air tankers and helicopters flew repeatedly into a deep rim rock canyon to douse the spearhead of the massive Ponderosa wildfire in Northern California and stop it from driving into a nearby mountain community and Lassen Volcanic National Park.
"This is the critical spot we have on the fire now," CalFire Division Chief Scott Lindgren said Thursday as he watched the air drops of fire retardant and water on flames burning the steep timbered sides of the canyon, about 6 miles west of Mineral.
He was standing in a backyard on the edge of a cliff, where the brush and dirt were colored orange-pink by a recent drop of retardant.
IN PICTURES: Wildfires sweeping the West
"The further east we go, the harder it is to stop this thing," he said. "The problem is, we catch it up on top (of the basalt rim rock) at night, but we can't catch it on the bottom (of the canyon) because of the cliff."
It has helped to slow the fire's spread so far, reducing the threat to miles of commercial timberland, the mountain community of Mineral, and the park. A logging road to the east was cleared to serve as a contingency line if the air attacks don't stop the fire from running up the canyon.
Sixty-four homes and 20 other buildings have been destroyed, mostly in the Manton area, since lightning ignited the blaze Saturday, state fire spokesman Don Camp said. It was threatening 900 other homes as it burned a new front to the south.
About 2,500 firefighters were at the blaze, which has grown to 44 square miles in the piney hills about 25 miles southeast of Redding.
Watching the drops with the fire chief were Jane Carney, Kelly Strong and Sherill Jenkins, who said they were ready to leave their vacation home if necessary.
"We'll get out if we're told to get out," Strong said.
They stayed because, Jenkins said, "It's beautiful up here."
Mineral is a town of 187 vacation and year-round homes that serves as the gateway to the national park.
Beth Glenn, whose family owns area commercial buildings dating to 1896 said she was not nervous, having grown up in a firefighting family and living with the frequent fires in Southern California.
Elsewhere in the state, a large wildfire in Plumas National Forest expanded with help from gusty winds. The blaze, about 120 miles north of Sacramento, has consumed 99 square miles since it started at the end of July and threatens about 900 homes.
The California fires were among several stubborn wildfires in the West. In Washington, crews were still working on a week-old wildfire that has destroyed 51 homes and 26 outbuildings and damaged at least six other homes, authorities said.
The fire, about 75 miles east of Seattle, has caused an estimated $8.3 million in property damage.
In central Idaho, firefighters were burning and removing vegetation near the town of Featherville in hopes of protecting hundreds of evacuated homes from an approaching wildfire. Fire managers said that blaze already has charred 164 square miles, forcing the residents to evacuate due to heavy smoke.
IN PICTURES: Wildfires sweeping the West
Collins reported from San Francisco.
Copyright 2012 The Associated Press.


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Why this course is perfect for you?
This Skills Programme is part of the IBS National Certificate: Management and Administration NQF Level 4 and has been designed for any individual who is interested in the administration function in any organisation or business in any sector, including non-commercial organisations such as clubs and charitable organisations. Administration refers to secretaries, administrative assistants, administrators and clerks.
Through building day-to-day administration skills, it teaches the learner to meet standards of service excellence.
This qualification is recorded against the Professional Qualification: Business Administration (PQMA), with SAQA ID No. 60152 (120 credits).
Careers you'd be great at
Learners who complete the qualification could pursue a career as a/an:
- Receptionist
- Secretary
- Office/Admin Assistant
- Office Administrator
How to get in?
- Knowledge, comprehension and application of the English language at NQF 3/Grade 11 or equivalent
- Knowledge, comprehension and application of Mathematical Literacy at NQF 3/Grade 11 or equivalent
- Admission may be subject to an interview with an academic staff member.
Time you will spend on the course
The programme duration is 65 hours and the tuition is face-to-face based on the NQF notional hours. This excludes time to complete assignments, research and self study. Each Damelin campus has a different tuition schedule. Programmes are offered during the week, in the evenings, or on Saturdays.
Course content and modules
Click here for quick access to the fact sheet for more info.
The strength in your qualification
Upon successful completion of each skills programme, IBS will issue learners with a Results Letter outlining the modules and credits achieved. Only on completion of 3 modules towards the full qualification will the learner be awarded a Certificate with a FASSET endorsement. Learner results will be recorded on the IBS National Learners' Records Database within 6 months of completion.
**Whenever Damelin offers training towards a programme that will be awarded by another provider or institute, it is called an Agent Programme. In the case of an Agent Programme, Damelin registers the learners, provides tuition and conducts internal assessments. External examinations with the agent body will result in learners receiving the certificate from them.


text:
Happy to see Minka back again.
Full Scene Downloads
Pop Your Cork With Minka
It's cork popping time with Minka, still the #1 Asian big-bust star on the planet. Minka opens a bottle of champagne and that cork shoots out like a rocket followed by a geyser of foamy liquid, kinda like the guys in her XXX scenes.
SCORELAND: So, Minka, how was shooting again after being away for so long?
MINKA: Fun, but I am pretty tired! Also, fucking so much makes me hungry. When I'm done, I go back to the hotel and eat Chinese food in the hotel, then I go out to Burger King and have a double Whopper and chicken nuggets. Tenpiece. Then I shower and go to bed.
SCORELAND: Why are you so hungry?
MINKA: I don't know. I'm hungry all the time. Fucking makes me horny and hungry and tired.
If you've seen Minka demolish a buffet at a Las Vegas resort, you know how much she can eat yet she burns it all off with intense tennis playing. The managers try to close the doors when they see her coming for brunch and always fail. At this point, they've given up. Read More »
SCORELAND: So, Minka, how was shooting again after being away for so long?
MINKA: Fun, but I am pretty tired! Also, fucking so much makes me hungry. When I'm done, I go back to the hotel and eat Chinese food in the hotel, then I go out to Burger King and have a double Whopper and chicken nuggets. Tenpiece. Then I shower and go to bed.
SCORELAND: Why are you so hungry?
MINKA: I don't know. I'm hungry all the time. Fucking makes me horny and hungry and tired.
If you've seen Minka demolish a buffet at a Las Vegas resort, you know how much she can eat yet she burns it all off with intense tennis playing. The managers try to close the doors when they see her coming for brunch and always fail. At this point, they've given up. Read More »
Featuring: Minka
Date: December 17th, 2016
Duration: 08:53
Member Comments
More Big Tit Videos
Jan 20
19:23 Mins.
Good Neighbor Minka
There is only one Minka. She is an icon. There are few places where Minka isn't known. Tajikistan may be one of those countries. Peter steps outside his house when he spots a woman with super-mega-giant knockers taking boxes out of a car and bringing them into the house next door. He recognizes Minka immediately. The odds of Minka moving in next door are equal to being hit by a lightning bolt. He runs over, losing his mind but seizing the moment. Minka is about to get more boxes when she hears Peter knocking on the door. Tripping out, he stammers that he's a huge fan and does she need any help moving in. Kidding him, Minka asks him if he means the boxes or with "these," putting her hands on her tits. She tells him to move the boxes to another room and also tells him to keep his eyes on the boxes, not her chest. Of course, he looks at her tits while he's carrying the boxes and trips. "Concentrate," Minka admonishes. "Look at the boxes, not this," she says, pointing at her tight tank-top. With a wicked smile, Minka has fun teasing and taunting him. "Do you want some water? Get some here," Minka says, lifting her top and patting her nipple. She totally controls him like one of her puppies. She exposes both super-breasts and lets him feel them and bury his face in them. Then she tells him to take his clothes off. "Your brain is not here," says Minka, pointing at his head. "Your brain is down here," patting his junk. Peter takes off his clothes like they're on fire. Minka sits down and puts some lube on his cock. She's going to suck him, tit-sex him and fuck him and drain his balls. Being a good neighbor can pay off.
Oct 7
15:28 Mins.
Mega-Boobs Office
Minka should have taken the executive job offered her at SCORE. Instead, she opted for a job at an office closer to home. The only problem is the manager has the personality of a high school vice principal and doesn't understand why Minka is making photocopies of her mega-boobs and working at her desk with her 44KK bra exposed, revealing yards of cleavage. In her own special, funny, horny way, she straightens him out and teaches him about her style of office politics. This office now belongs to Minka. SCORELAND: Hi, Minka. It's great to have you back. The fans never stop asking for you. The last time you were in our studio was when? Minka: Almost five years ago. 2011. My fans wanted to see me, so I am happy to be back. SCORELAND: Five long years, Minka. We know they're happy to see you back. What do your fans tell you they want to see? Minka: They want to see fucking. That's what they want to see most. They want to see me fucking big dicks! That is all they ask for! SCORELAND: What do you want? Minka: Find a small dick. Then I can cum large! I have a very small pussy, so I like small dicks. SCORELAND: Minka, you're one in 50 million. And with that, we'll say, welcome back!
Sep 10
09:05 Mins.
Minka Returns
It's been a long time since Minka visited SCORELAND. Not a week goes by without an email or a letter asking when Minka will appear again. Many of the writers are longtime SCORE guys like R.N. Minka's one of his go-to girls. The wait is finally over. "The thing that makes Minka different from most big-tit models is that most of them really excite me the first few times I see them," R.N. wrote. "But after several layouts, they become kind of ordinary and the excitement diminishes. Minka is just the opposite. She gets more and more arousing for me with every layout." An inspiration to such big-boob stars as Dolly Fox and Sandra Star, Minka keeps busy with her daily tennis practice and matches, taking care of her many dogs, her vegetable gardening, traveling to tennis tournaments and working on her website. In this video, she checks herself out in a sectional mirror.
Jan 21
Angel Eyes
48:25 Mins.
Boob Cruise Paradise Part 3
What did it mean to sail on the Boob Cruise? It meant staggering to bed every night at four a.m. and waking up two hours later for another day of action and a new island. It meant eating at a busy table of busty shipmates where there is never a silent moment. It meant dancing with a scantily-clad girl who laughed when her big tits fell out of her dress. It meant looking at a pure black sky and noticing the stars like never before. It meant the heady scent of suntan lotion and sweat emanating from suntanned girl-flesh. It meant living in your own separate world, and whatever was happening back home, well, that didn't matter. The ship was the world. This ship, the Star Clipper, sailed to Anguilla, Prickly Pear Island, St. Maarten, Tortola, Virgin Gorda and other islands that most people know about only from travel magazines and television. This leg of Boob Cruise Paradise kicks off with the morning aerobics class on deck. This daily workout was open to everyone although most of the guys were still sleeping off the previous night or on their second cup of coffee. A few were on the sidelines watching the big beautiful show as the girls in their skimpy gym clothes moved in unison. Morning and early afternoon photo shoots were the order of the day, both on the deck and on Virgin Gorda. The evening was for dinner followed by strip shows performed by the greatest exotic dancers and then climaxing with partying all over the ship until early morning. The booze and the boobs flowed freely. Wrote Chuck, returning passenger and the ship's log master that year, "To be appropriately blunt, and from talking to the guys, I think I speak for everyone who went on Boob Cruise '98, the only bad thing about this entire week is leaving! I swear, if somebody were to handcuff all of the passengers and girls to the ship and hand me the key, I'd toss it overboard!" "I tell everyone that Boob Cruise '98 was the wildest week of my life," recalled passenger P.R. "And that includes innumerable Mardi Gras, fraternity parties, bachelor parties and other parties. I came for the women, and they were everything I thought they'd be and more, but I also enjoyed my stay due to the great guys I met on the Cruise, the SCORE staff and the whole sailing experience!"
Nov 5
13:09 Mins.
Close Encounters of the Mega-Boobed Kind
Wearing a white mini-dress without a bra, Minka douses herself with a bottle of water, turning her dress see-through, the wet fabric clinging to her mega-boobs. Minka leaves the terrace and enters the house to get her soaked dress off, chat a bit and get naked on the bed to give her thick-lipped pussy a happy ending. "You guys call me, I'll come back again," said Minka when our photographer asks when we'll see her again. SCORELAND: What do you like to do when you have free time? Minka: Go on vacation to resorts in Jamaica and Mexico. You know, I play a lot of tennis. I'm going to start playing more in competition because I enjoy it. I'm coming back because every year I got suspended. SCORELAND: Why do you get suspended? Minka: I have a team. I coach, but I made them too strong. Mentally strong. We intimidate the other team. Psyche them out. If you're going to be good, you have to have a mental game. And I'm the coach, so every team is writing letters complaining about me, so I got suspended one year, and last year, again, a woman's team wrote letters to the headquarters, so I got suspended again. They do not like what I do with my partner on the court, chest bumps and kissing. It psyches out the other team. SCORELAND: They might as well throw their racquets on the court and leave!
Mar 17
18:15 Mins.
The Adventures of Super-Minka
"You like the big nipples, baby?" Minka asks Jim, who's preoccupied by Minka's famous protruding nips. Her nickname is "Nipple #1." She has nipples that can cut glass. Minka points out that there's some fruit on the table but he's not especially interested. He's absorbed by her equally famous mega-boobs. "I'm gonna feed you," Jim says as she rubs his junk through his pants. He runs a bunch of grapes over Minka's gigantic hooters, then holds it to her mouth so she can bite off a grape, then transfer it to him mouth-to-mouth. A strawberry is next. Jim holds up an orange to Minka's tits. Then some melons. Forget it. He'll need to find a giant watermelon to compare a piece of fruit to Minka's fruits. The fruits of their labors lead to Minka sucking Jim's cock and Jim eating Minka's neatly trimmed peach. He shoehorns his pussy-stretcher into Minka's very tight slit and starts pumping the pink. As she's banged hard in doggie, Minka's face says it all. As she put it, "Little dicks are better. A guy with a big dick can eat my pussy. That is good!" After fucking Minka under him and on top of him, Jim is close to losing his nut, and her verbal cum-encouragement and squeezing pussy make him blow his load all over her boobs. Nipple #1 wins again.
Jan 28
Angel Eyes
38:20 Mins.
Boob Cruise Paradise Part 4
Boob Cruise Paradise Part 4 begins on the island of Saint Barthélemy or St. Barths. This French territory is a picture perfect location for photo shoots. Busty Dusty, a beach bunny and avid SCUBA diver, loves this place. Following Dusty is Adele Stephens of England, on board for her only Boob Cruise. Adele began her career as a famous Page 3 Girl which brought her to the attention of SCORE's photo team. By sheer coincidence and timing, the famous Pandora Peaks was vacationing on St. Barths, heard the Boob Cruise was in town and came out to meet some of the shipmates. As the Cruise entered its final leg of the voyage, the parties and shows on board the Star Clipper went full blast. This was the end. The dancers on-deck included Minka (who would always grab a volunteer for some oily one-on-ones), Summer Cummings, Skye Blue and Summer Leigh. Cruise diarist Summer Leigh wrote, "I can see why everybody loves the Boob Cruise beach party. It's so relaxing and everybody just parties like crazy. There's something about being on an island at night, with the water lapping at the shore, the moon and stars shining bright. It's the perfect romantic setting and it really got me horny. So, what did I do? I went back to the ship after the party and masturbated in my cabin! Finally!" And on the last night before landfall, Summer wrote this entry, "Back on the ship, we're all getting ready for the Captain's dinner! The last dinner. I can't believe it's almost over. The dinner was great! It was nice seeing everybody dressed up. Such class! I myself dressed up every night except for one because I wanted to make a good impression. I don't get to dress up that often, so it was fun. I got a little drunk on wine. I hadn't been drunk all week except maybe the first day. "And of all nights, they put up the sails this last night, which made it hard to walk on the ship in my high heels. I was trying to spend as much time as I possibly could to say goodbye. It was sad. I felt like they were family after spending a whole week with them. However, I didn't get to know enough of the girls. I guess because I was too busy with the passengers." Chuck, writer of the ship's official log closed the book on Boob Cruise 1998. "It won't hit the passengers until they get back that it's all been a giant SCORE fantasy that lured them in for a magical week, then spit them back out into the real world: the office, the boss, the wives, children, what have you. And that's when they'll really start their vivid remembrances. That's what this fantasy does to you and those of us who know it all too well."
Jun 24
Casey James
41:29 Mins.
Mega-Boob Olympics Part 3
The Mega-Boob Olympics wraps up the biggest top of them all with Maxi Mounds at poolside, a group pool flotation with underwater footage, the tricky strap-on ring toss event, Plenty, Kayla and Minka getting messy and tit-crazy and the nipple erection competition. So who won? No one and everyone. That's show biz! Behind the scenes at this south Florida location, there was some worry. A tropical storm was threatening to approach land (you can see the palm trees bending if you can take your eyes off the ladies). Fortunately, it all ended well and the weather gave us a break. Almost the same circumstances affected the Busty Ladies of Oil Wrestling (B.L.O.W.) three years later in 2004 when an approaching storm also tried to butt in. The World Cup is every four years. The Mega-Boob Olympics is forever. The power of the bra triumphs again.


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This curriculum uses an inquiry-based Earth system science approach, and leverages Project BudBurst, a citizen science phenology project, to engage students in authentic research on plant and ecosystem responses to climate change. Students collect... (View More) local data then analyze that data in the context of NASA regional and global data sets and satellite imagery to understand their data in personal, regional, and global contexts. The curriculum is divided into four units: The Earth as a System; Identifying the key changing conditions of the Earth system; Earth system responses to natural and human induced changes; and Predicting the consequences of changes for human civilization. Each unit consists of several activities with accompanying teacher answer sheets. (View Less)
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This activity uses rain and flood events in the Midwest to engage students in an exploration of the collection, comparison, analysis and utilization of rainfall data. Students will access online precipitation data from both a ground-based station... (View More) (the Community Collaborative Rain Hail and Snow Network (CoCoRaHS) network) and a satellite (the Tropical Rainfall Measuring Mission (TRMM)). Explicit instructions are provided to allow students to graph, map and analyze that data. Instructions are also provided for setting up a school-based rain gauge to gather local rainfall data for analysis. (View Less)
This is a design challenge about heat transfer and insulation. Learners will apply the scientific method to design and build a container that will keep items cool when placed in boiling water. They will practice collaboration in team-building and in... (View More) teamwork. This is lesson 4 of 4 at the Grade 9-12 range of the module, Staying Cool. (View Less)
This is an activity about the Sun's position in the sky. Learners will use a clear plastic hemisphere to create a model of the sky and mark the position of the Sun in the sky on the hemisphere several times throughout the day. From this information,... (View More) learners can determine the location of sunrise, sunset, and the Sun's height at noon. This activity can be repeated over the course of several days, weeks, and/or months. (View Less)


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Arcoris Soho, Mont Kiara
Jalan Kiara 4, Mont Kiara, 50840, Kuala Lumpur
- Built-up : 847 sq. ft.
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RM 2,118 per month
Arcoris Soho, Mont Kiara
???? ????,???????????????????? 2. ???? ??????????,??????????????????????? 3. ???? ??0167760001????????????????,?????????????????(POS)??????(COS)?? 4. ??????? ?????,????????????????????????? 5. ?????? ???????????????(Reserve Price)?10%???????????????,???????????????????????10%?? 6. ?????? ???????????????(Reserve Price)?10%???????????????,???????????????????????10%?? 7. ????? ????????????????????????????????????????? 8. ?? ??????? (Memorandum). ??????????????????????????????????????????? 9. ?? ??????????,????????????????? Take note of the description, address and the relevant information of the property you are interested in. 2. Inspect the property Inspect the property for its external condition and surrounding area if it is not vacant. 3. Pre-registration Contact 0167760001 to check property background & hidden costs and submit your bidder registration to get a copy of POS & COS. 4. Relevant checking on the property Do a caveat search and to seek legal advice on the property. 5. Prepare bank draft Prepare a bank draft equivalent to 10% of the reserve price. Be prepared to top up the difference between the 10% of the final sold price immediately after the auction. 6. Register on the auction day On the day of auction, register yourself. A bidding card with number will be issued to identify the registered bidder during the auction. 7. Bidding Bidder is to raise his/her hand to signify his/her interest to bid for the property. The successful bidder is identified as the highest offer or during the bidding process. 8. Successful bid Sign the Sales Contract. Bidder will be advised to collect the stamped Contract at a later time. Contact any bank to arrange for bank loans. 9. Loss Bid Proceed to the registration counter to take back relevant document and bank draft without any charges.
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Reward doubled for information on slain AmeriCorps volunteer
Crimestoppers raise reward to $5,000
Crimestoppers announced on Monday morning that the reward for information leading to the arrest in the killing of an AmeriCorps volunteer was doubled.
The reward now stands at $5,000.
Police said Joseph Massenburg was gunned down at the corner of Eagle and Birch streets on April 1 about 11 p.m. as he was talking on his cell phone. Police said he was shot several times.
Eighteen-year-old Massenburg was rushed to a hospital where he was pronounced dead.
Massenburg, who joined AmeriCorps to give back to the community, had not been in town a month when he was gunned down in the West Carrollton neighborhood.
Anyone with information is asked to call Crimestoppers at 504-822-1111.
Previous story:
AmeriCorps mourns slain volunteer in Crescent City
Click to watch previous report


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At a leading specialty pharmaceutical company, the IT organization supported its commercial applications using numerous local staffing companies.
Together, the company and Accenture simplified commercial IT processes and supported commercial applications for the business units across 34 countries over 10 years. Accenture is delivering a streamlined and standardized application support model, using cost-efficient resources with the right skills to meet the company's requirements.
As a result, Accenture's blended offshore and onshore resources have saved 30 percent in support costs annually, while providing more consistent outcomes at a fixed cost. Process improvements have yielded savings of $500,000—approximately 3,000 man hours. The company can now refocus employees on more strategic initiatives that will help further improve performance.


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With the tiger stripes on your shoulder, you'll turn heads along urban road and park paths. And you can take to the streets at night with reflective dots at the front and back.
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Dennis Zotigh, a cultural specialist at the National Museum of the American Indian in Washington, won't be celebrating Thanksgiving today the way most of us are going to.
Neither will lots of other Native Americans.
Surprised? Then you're way too close to the papier mache, elementary school version of the Thanksgiving feast, presented as a Disneyesque love fest between the Pilgrims and the Indians.
Many American Indians don't see it that way at all.
"It makes me really mad — the Thanksgiving myth and what happens on Friday," said Zotigh, who is a Kiowa, Santee Dakota and Ohkay Owingeh Pueblo Indian.
Zotigh is tired of the stereotyping and romanticizing that comes with this day.
"The Thanksgiving myth has done so much damage and harm to the cultural self-esteem of generations of Indian people, including myself, by perpetuating negative and harmful images to both young Indian and non-Indian minds," Zotigh wrote on the Smithsonian museum's blog. "There are so many things wrong with the happy celebration that takes place in elementary schools and its association to American Indian culture; compromised integrity, stereotyping, and cultural misappropriation are three examples."
Think about it: Thanksgiving is a pretty grim day in Native American history. After the native Americans helped the ragged colonists survive, they let them in on their tradition of a harvest feast.
And what did the colonists do in response? Rape, pillage and nearly destroy a civilization.
We like to be all gooey about family and cranberry sauce and football.
But the day in 1621 when the Wampanoags feasted with the starving colonists was the beginning of one huge, bloody betrayal of the people who were here first.
Even today, some Native Americans refuse to acknowledge the holiday, as mainstream America celebrates it.
The National Day of Mourning is what the United American Indians of New England has called it since 1970, when they first led a march and protest to the area known as Plymouth Rock.
Their flier for this year's event urges marchers to "Help shatter the untrue glass image of the Pilgrims and the unjust system based on racism, sexism, homophobia and war."
Zotigh said he's heard from Native American parents who sign their kids out of school on the day of their Thanksgiving re-enactments, whose children have been punished in class for bringing up the American Indian's side of the story, from those who want "the national moral atrocity of genocide" to be acknowledged and from those who simply call that day of national gorging "The Last Supper."
He remembers having to bring a paper bag to school to make his costume for the re-enactment of the big feast, complete with cartoonish, construction paper feathers and headdresses.
This week? He's going to keep it simple and have some chili with friends.
The native Americans I talked to said they've all heard of someone who doesn't celebrate the holiday, food catalog style.
But all of people I talked to said they hold on to the original message that the Wampanoag had that day — a harvest feast to give thanks.
"Thanksgiving is like every day for us. Giving thanks is a big part of the native cultures. So the basic message of the holiday, that's still part of who we are," said Ben Norman, 32, a member of the Pamunkey tribe in Virginia.
His tribe's chief, Kevin Brown, said he travels to reservations all across America and he hears about folks who won't celebrate Thanksgiving.
"But most people I know, we love eating and we love being together with family. And that's what this day is about," said Brown, 58.
His favorite part of the meal? "Turkey. Not fried, just plain cooked, Betty Crocker style," he said. "And venison, we have venison, too."
"I'm too busy eating and watching football to spend my life worrying about the past," he said.
The Oneida Nation representative, Ray Halbritter, who has been the primary spokesman for the campaign to get the Redskins football team to change its name, takes a similar approach to Thanksgiving.
"Thanksgiving comes out of our culture," said Halbritter, who himself is a stuffing and squash kind of guy. "It's a wonderful time to reflect on being thankful, to be with family, to celebrate our blessings. It really comes from our harvest celebrations."
Halbritter's point, the one he makes when he's talking football, is that American Indians don't want to be thought of as relics or mascots.
And actively celebrating a harvest feast, rather than dwelling on the injustices by the colonists that came after that day, is one of those ways to keep a culture alive and relevant.
"That's one of the reasons we do the Thanksgiving parade," he said. The Oneida Nation has a big, turtle island float in the Macy's Thanksgiving Parade in New York every year. And they say it's to remind Americans of that first meeting, that day when they helped and trusted.
If it isn't enough that the rest of America has marred the harvest feast of thanks with family squabbles, idealizing of the pilgrims, jellied cranberry sauce in a can, marshmallows on yams (the worst),Turducken and football, what happens on Friday is the final slap in the face.
Black Friday — after supposedly giving thanks for all that we have — Americans are now compelled on this day to shove, fight and maim in order to buy even more!
"You know that's supposed to be our heritage day?" Zotigh said.
Yes, the Friday after Thanksgiving is designated as the official day in America to pay homage to the heritage and culture of the American Indian.
Somehow, I just don't think that hand-to-hand combat over a big screen is what the Wampanaog had in mind.
Please, for once, listen to the people who were on this land first.
Keep it simple, just give thanks.
Petula Dvorak is a columnist for The Washington Post.
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Zydeco comes from New Orleans, and is a musical gumbo with French, Creole, jazz, and rock influences. It's some of the best party music around, featuring accordion and electric guitar over a strong backbeat. The rhythm has a real bounce to it that makes you want to move your feet. You just can't sit still to this kind of bar room dance music.
If you are looking for a quirky and infectiously upbeat party background track, this is a great choice. It makes a perfect 34 second loop for fade out at your convenience.


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Preparing the public for a future in which the use of genetic and genomic information will become a routine part of medical care was the focus of a genomic literacy workshop organized by the National Human Genome Research Institute (NHGRI) in the fall of 2011. Fifty participants from state and federal governments, kindergarten to grade 12 educators, and advocacy groups gathered on Nov. 17-18, 2011, in Rockville, Md. to make recommendations on the scope and priorities for a genomic literacy research agenda. Now, a meeting report describing the results of that workshop has been published online in Genetics in Medicine, the official journal of the American College of Medical Genetics and Genomics.
"We know that the public's ability to integrate genomics into their personal health care lags behind the explosive scientific achievements in the field," said Belen Hurle, Ph.D., public health educator in NHGRI's Education and Community Involvement Branch. "But, at the same time, what constitutes a genomically literate public remains to be fully articulated."
To explore this topic, participants examined challenges facing educating the public from both the K-12 and adult perspectives. They also discussed the gaps and areas of opportunity for studying genomic literacy in both the short and long term, and the role of the media in disseminating scientific messages.
"The public encompasses many different communities, and the interface with genetics and genomics changes in each life cycle stage," said Dr. Hurle. "That is why involving diverse communities in decision-making and ensuring all voices are heard is essential to achieving a long-term, genomic literacy goal."
Participants also recommended more research on the use of family health history as a clinical tool. Family health history remains the most effective, least expensive tool available to the public to assess personal disease risk.
"The meeting was very exciting, especially because of its trans-disciplinary nature," said Dr. Hurle. "Professionals and lay communities came together to share their experiences and expertise on the challenges and the opportunities ahead."
To read the meeting report and recommendations in the Genetics in Medicine paper, go to: What does it mean to be genomically literate?: National Human Genome Research Institute Meeting Report [nature.com]
Posted: April 5, 2013