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Comparative gene expression analysis of planktonic Porphyromonas gingivalis ATCC 33277 in the presence of a growing biofilm versus planktonic cells
María C. Sánchez1,2,
Patricia Romero-Lastra1,
Honorato Ribeiro-Vidal1,2,
Arancha Llama-Palacios1,2,
Elena Figuero2,
David Herrera2 &
Mariano Sanz2,3
BMC Microbiology volume 19, Article number: 58 (2019) Cite this article
Porphyromonas gingivalis, a microorganism residing in the oral cavity within complex multispecies biofilms, is one of the keystone pathogens in the onset and progression of periodontitis. In this in vitro study, using DNA microarray, we investigate the differential gene expression of Porphyromonas gingivalis ATCC 33277 when growing in the presence or in absence of its own monospecies biofilm.
Approximately 1.5% of genes (28 out of 1909 genes, at 1.5 fold change or more, p-value < 0.05) were differentially expressed by P. gingivalis cells when in the presence of a biofilm. These genes were predominantly related to the metabolism of iron, bacterial adhesion, invasion, virulence and quorum-sensing system. The results from microarrays were consistent with those obtained by RT-qPCR.
This study provides insight on the transcriptional changes of planktonic P. gingivalis cells when growing in the presence of a biofilm. The resulting phenotypes provide information on changes occurring in the gene expression of this pathogen.
Porphyromonas gingivalis, a Gram-negative anaerobe residing within the oral cavity, has been identified as one of the key pathogenic species implicated in the establishment and development of periodontal diseases [1,2,3], mainly through the expression of a broad range of virulence factors involved in tissue colonization, evasion of host defenses and stimulation of a chronic inflammatory response [3].
P. gingivalis is found within the oral cavity adopting a sessile biofilm lifestyle, predominately as a component of complex biofilms containing multiple microbial communities [4]. P. gingivalis has the capacity to adhere to mucosal and dental surfaces, including the teeth, gingiva, cheek and tongue, as well as to other oral bacteria, thus withstanding the host natural barriers, the host immune defenses and the presence of multiple antimicrobial agents [5,6,7,8]. These virulence factors are the consequence of specific gene expression, and it is, therefore, important to improve our knowledge on them in order to understand how these microorganisms can adapt to specific ecological determinants, and their role within the biofilm environment.
It is well known that microbial gene expression is significantly different within biofilms when compared to planktonic growth [9,10,11,12]. Recent investigations have studied the specific gene expression of P. gingivalis when growing in biofilms, and compared to planktonic growth, demonstrating the differential expression of a broad range of genes [13,14,15,16,17,18,19]. In biofilm growth, the cell replication and growth rate are decreased with repressed genes involved in cell envelope biogenesis, DNA replication, energy production, biosynthesis and phospholipid metabolism. By contrast, there is an important number of genes involved in regulatory mechanisms which are overexpressed, such as those encoding transport and binding proteins, proteins involved in signal transduction and transcriptional regulation and many others not well characterized. Within this research line, Ang et al. (2008) [20] conducted a proteomic study comparing the envelope proteins of P. gingivalis, either growing in planktonic or in biofilms, demonstrating an overexpression of proteins involved in hemin transport (HmuY and IhtB), in metabolic pathways, virulence factors or proteins of the cell envelope.
Other investigations have studied the overexpression of P. gingivalis genes affecting structural characteristics such as the presence of different fimbrial types, as well as specific polysaccharides involved in adhesion and colonization mechanisms, which may significantly contribute to biofilm formation [21,22,23]. Also, cell-cell signaling mechanisms has been the focus of study, and P. gingivalis cells have shown up-regulation of the LuxS-dependent signaling regulatory genes, involved in inter-species communication and quorum sensing but also involved in stress, protease modulation and haemagglutination or in hemin uptake [24,25,26,27].
In spite of this knowledge, the genetic and environmental determinants affecting P. gingivalis cells when transiting from free-floating cells to biofilm have not been fully elucidated [28,29,30]. It was, therefore, the purpose of this investigation to study the gene expression of free-floating P. gingivalis cells either growing in a pure planktonic environment or when they are placed in the presence of a P. gingivalis mono-species biofilm.
Bacterial strain and culture conditions
Porphyromonas gingivalis ATCC 33277 was used in this study and grown on supplemented blood agar plates [Blood Agar Oxoid No 2; Oxoid, Basingstoke, UK; with 5.0 mg/L hemin (Sigma, St. Louis, MO, USA), 1.0 mg/L menadione (Merck, Darmstadt, Germany) and 5% (v/v) sterile horse blood (Oxoid)], at 37 °C for 48 h in anaerobiosis (10% H2, 10% CO2, and 80% N2).
Experimental assays
Figure 1 depicts an overview of the experimental design. P. gingivalis ATCC 33277 was grown in modified Brain Heart Infusion (BHI) medium (Becton, Dickinson and Company, USA) in anaerobiosis at 37 °C for 24 h [13]. Bacteria were harvested in their late exponential growth phase [0.8; standard deviation (SD) =0.1 of optical density at 550 nm] and added to fresh modified BHI medium in order to obtain a pure culture containing 108 colony-forming units per milliliter (CFU/mL). Two experimental groups were carried out, free-floating P. gingivalis cells either growing in a pure planktonic environment or when they are placed in the presence of a P. gingivalis mono-species biofilm. For that, the planktonic bacterial culture prepared was placed in pre-sterilized polystyrene well tissue culture plates (Greiner Bio-one, Frickenhausen, Germany) under two environmental conditions:
Test group: the bacterial suspension of P. gingivalis was deposited in the culture plates containing sterile ceramic calcium hydroxyapatite (HA) discs [7-mm in diameter and 1.8 mm in thickness (SD = 0.2); (Clarkson Chromatography Products, Williamsport, PA, USA)].
Control group: the bacterial suspension of P. gingivalis was deposited in the plates without HA discs.
Overview of experimental design. Porphyromonas gingivalis ATCC 33277 strain was maintained on blood agar plates and grown in modified Brain Heart Infusion (BHI) medium. After 24 h, optical density was measured, and a pure culture containing 108 colony forming units per milliliter (CFU/mL) was set. Two culture conditions were then prepared: Test cells, depositing P. gingivalis cells in presence of Hydroxyapatite (HA) disc, and Control cells, depositing P. gingivalis cells in the wells without HA discs. After 96 h of incubation of multi-well plates under anaerobic conditions, free floating P. gingivalis cells from both test and control condition were harvested, examined by CLSM, processed and total RNA extracted and purified. Agilent Oligo Microarrays 8x15K (074976) for P. gingivalis ATCC 33277 were used for hybridizations, (this slides also contents probes against the whole genome of P. gingivalis W83), and RT-qPCR analyses were performed to confirm the results. The experiments were repeated three times and each experimental condition was pooled into the three biological replicates and processed for the transcriptomic analysis. (Images for Fig. 1 were taken from https://smart.servier.com/ under a creative commons licence)
Plates were incubated in anaerobic conditions at 37 °C for 96 h. Wells containing only culture medium were also evaluated to assure sterility and lack of contamination.
After 96 h of incubation, free-floating P. gingivalis cells from both groups were harvested and processed for the transcriptomic analysis, with the goal of obtaining approximately 20 μg of total RNA for each replicate. Each pooled sample corresponded to one biological replicate and was processed in the same manner.
Confocal laser scanning microscopy (CLSM)
Test and control group samples were analyzed by confocal laser scanning microscopy (CLSM) after 96 h of growth. 100 μL of free-floating P. gingivalis cells from both test and control groups were deposited in a slide and were stained with the fluorochrome Syto9 (wave lengths 515–530 nm) for 9 min at room temperature to obtain an optimum fluorescence signal (Molecular Probes B. V., Leiden, The Netherlands). Samples were then covered with a coverslip, with the aim of minimizing the movement of bacteria. CLSM clearly showed the absence of sessile phenotype in the bottom of the plate of control group samples, while a sessile phenotype was developed on the surface of hydroxyapatite discs of test group. For that, after completely removing the free-floating P. gingivalis cells from control wells, these ones were sequentially rinsed with 2 mL of sterile PBS three times (time per rinse, 10 s), in order to remove unbound bacteria. In the same way, after completely removing the free-floating P. gingivalis cells from test wells, HA discs from test group were rinsed by immersion in 2 mL of sterile Phosphate-Buffered Saline (PBS) three times (time per rinse, 10 s). Then, the bottom of the plate and the HA discs were stained with the fluorochrome for 9 min at room temperature. Samples were observed with CLSM [Ix83 Olympus® inverted microscope coupled to an Olympus® FV1200 Confocal System (Olympus; Shinjuku, Tokyo, Japan) using × 63 water-immersion lenses (Olympus) The CLSM control software was set to take a z-series of scans (xyz) of 0.5 μm thickness (8 bits, 1024 × 1024 pixels). Image stacks were analyzed with the Olympus® software (Olympus).
After 96 h of incubation under anaerobic conditions, free-floating P. gingivalis cells from both test and control groups were harvested, and total RNA was isolated using the TRIzol® Max Bacterial RNA Isolation Kit (Ambion, Life Technologies, Carlsbad, CA, USA) as detail in Romero-Lastra et al. (2017) [13]. Briefly, P. gingivalis cell pellets from both groups were resuspended in Max Bacterial Reagent® (Ambion) and after temperature shock treatment to help the break of the cell wall, consisting on 4 min incubation at 95 °C and cooling on ice for 10 min, TRIzol® reagent (Ambion) was added. Total RNA was extracted using the chloroform protocol and isopropanol precipitation. Isolated RNA was then washed in 75% ethanol and resuspended in 50 μl RNase-free water (Water PCR grade, Roche Diagnostic GmbH; Mannheim, Germany). To remove any contaminating DNA, samples were then treated with DNase I (Ambion), and purified using columns of RNeasy Mini kit (Qiagen) according to the manufacturer's protocol.
RNA concentration was measured by NanoDrop ND1000 spectrophotometer (NanoDropTechnologies; Thermo Scientific™, LLC, Wilmington, DE, USA), and RNA integrity was assessed using an automated electrophoresis device (Agilent 2100 Bioanalyzer, Agilent Technologies, Santa Clara, CA, USA). An A260/A280 ratio of at least 2.0 was considered appropriate for the experiments.
cDNA synthesis and transcriptomic protocol
All experiments were done in triplicates. The fluorescently labeling was performed using SuperScript Indirect cDNA Labeling System (Invitrogen; Carlsbad, CA, USA) as described in Romero-Lastra et al. (2017) [13]. Preparation of probes and hybridization was performed as described in the manufacturer's instructions [One-Color Microarray Based Gene Expression Analysis Manual Ver. 6.5 (Agilent Technologies)].
Slides specific for the strain P. gingivalis ATCC 33277 [Agilent Oligo Microarrays 8x15K (074976)] were used. The array also contents the whole genome of P. gingivalis W83.
Microarray and data analysis
Images from Cy3 one-color microarrays (Agilent) were taken, corrected and analyzed following the protocol detailed in Romero-Lastra et al. (2017) [13].
LIMMA language with "normexp" and loess methods were used to treat background correction and normalization [31, 32]. Log-ratio values were used for consistency among arrays [31]. Differentially expressed genes were determined using linear models and Bayes moderated t-statistic; Benjamani and Hochberg method was used to correct false discovery rate p-values [31, 32], and was controlled to be lower than 5% and a cutoff of fold change (increase or decrease) up to 1.5 between the two situations. Expression ratios were expressed as means of the fold changes of the three biological replicates and Standard Deviation (SD). Hybridizations and statistical analysis were performed by the Genomics Unit at the National Center of Biotechnology at the Universidad Autónoma of Madrid (Spain).
Confirmation of microarray data by reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
Microarray results were confirmed by RT-qPCR selecting 8 genes differentially expressed, four genes from the up-regulated group and four from the down-regulated one. Universal Probe Library Roche software tool (Roche Diagnostics) was used to design specific primers (Table 1). P. gingivalis 16S rRNA gene was used as a loading control.
Table 1 Primers used for reverse transcription quantitative polymerase chain reaction (RT-qPCR)
RT-qPCR was performed from the cDNA generated from 1 μg of total RNA from each sample, using the High Capacity cDNA Archive Kit (Applied Biosystems, ThermoFisher Scientific; MA, USA) in a final reaction volume of 10 μL. The qPCR reactions were performed in triplicate using 5 μL per well of each cDNA, and 3 μL of a mix composed by 0.4 μM of each primer, 5x HOT FIREPol1 EvaGreen1 qPCR Mix Plus (ROX), and nuclease-free water, to reach a final volume of 8 μL in 384-well optical plates and following the standards protocols provided by ThermoFisher Scientific in an Applied Biosystems ABI PRISM 7900HT apparatus (95 °C 10 min, 40 cycles of 95 °C 15 s and 60 °C 60 s, and a final standard melting curve dissociation protocol). The results of differentially expressed genes were analysed using the Expression Suite Software Version 1.1 and Comparative Ct Method (ΔΔCt) was applied [33].
By CLSM analysis it was verified that in the control group, after 96 h of incubation, free-floating P. gingivalis cells had not evolved to biofilm phenotype (Fig. 2 a). It could be observed that no sessile phenotype was formed on the bottom of the plate, observing only some cellular debris (Fig. 2 c). In contrast, in the test samples, free-floating P. gingivalis cells (Fig. 2 b) were in presence of biofilm evolved on HA disc (Fig. 2d).
Representative confocal micrographs of P. gingivalis depicting 2D maximum projection images after 96 h of incubation of the two experimental groups designed, (a) planktonic P. gingivalis cells growing in a pure planktonic environment and (b) free-floating P. gingivalis cells placed in the presence of a P. gingivalis mono-species biofilm. Image (c) corroborate the absence of sessile phenotype on the bottom of the plate of control group samples, only faint debris could be observed adhered. Image (d) shows a sessile phenotype evolved on the surface of hydroxyapatite discs form the test group. Specimens were stained with Syto9 fluorochrome (Molecular Probes B. V., Leiden, The Netherlands)
The Fig. 3 depicts the genes with differential expression in P. gingivalis ATCC 33277 resulting from microarray-based transcriptome analyses, when comparing the two planktonic states, either in presence or absence of a bacterial biofilm. Differentially expressed genes with 1.5 fold change (up or down) and p-value < 0.05 were plotted, X-axis represents log10 expression of pure planktonic state (in absence of a biofilm) and Y-axis shows the log10 expression genes of cells in the presence a growing biofilm.
Differential gene expression by comparative microarray analyses (represented in log10) when comparing planktonic Porphyromonas gingivalis ATCC 33277 cells either in presence of a growing biofilm or in absence of a biofilm. Control planktonic cell gene expression (X-axis) is plotted against test cells (Y-axis) with a 1.5 fold change (up or down) and p-value < 0.05. Down regulated genes (green) and up-regulated genes (red) are shown in the figure
The differentially regulated genes of P. gingivalis cells under both test and control groups are depicted in Table 2. Expression ratios were expressed as means of the fold changes of the three biological replicates and Standard Deviation (SD). Although the gene expression was not markedly different between both groups, a total of 28 out of 1909 genes (1.5%) were differentially expressed by free-floating P. gingivalis cells growing in the presence of a biofilm.
Table 2 Differentially expressed genes in free-floating Porphyromonas gingivalis ATCC 33277 cells either in presence of a growing biofilm or in pure planktonic growth (cutoff ratio ± 1.5-fold change, p-value < 0.05) for the microarray analysis
From these differentially expressed genes, 21 transcripts were found significantly increased (Table 2). These included genes related to iron acquisition and storage, among them the PGN_0557 (hmuR) gene, which encodes for the outer-membrane hemin utilization receptor involved in the uptake of both free hemin and heme bound to hemoproteins. Also, the gene PGN_0493 appeared overexpressed, which encodes for a receptor Hma, implicated in heme uptake. Similarly, the PGN_1494 gene, which encodes for the oxygen-independent coproporphyrinogen-III oxidase, or the genes PGN_1058 (ftn) and PGN_0604, which are related with intracellular iron-storage proteins, were differentially expressed. P. gingivalis, under these test conditions overexpressed genes encoding for transposases (PG_0009 and PG_0942), in particular the ISPg5 transposase Orf1, and the gene PGN_0780 (prtQ), which encodes a protease with peptidase activity, belonging to the PrtC family of genes encoding collagenase-like proteases. The gene PGN_1312, which encodes a transcriptional regulator of arginine metabolism and the gene PG_0437 involved in the biosynthesis of polysaccharides, was also up-regulated.
Conversely, in the test conditions seven transcripts demonstrated significant down-regulation, being these genes related to adhesion or virulence (PGN_0183 (fimC), PGN_0181, PG_2130, PG_2131, PG_1712) (Table 2).
RT-qPCR was used for the assessment of the microarray results from the selected genes (4 from the up-regulated and 4 from the down-regulated group). Figure 4 shows a high correlation between the log2 ratio of microarray and RT-qPCR results in the two studied conditions (R2 = 0.8477).
Graph depicting the correlation between the log2 ratio Microarray and Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR) gene expression ratios when test and control conditions were compared. The RT-qPCR log2 values were plotted against the microarray data log2 values (R2 = 0.8477)
This in vitro investigation has shown that a 1.5% of genes (28 out of 1909 genes, at 1.5-fold change or more, p-value < 0.05) were differentially expressed by P. gingivalis cells when in the presence of a biofilm. Several studies have investigated the differential gene expression of the periodontal pathogen P. gingivalis when growing under different conditions, basically under planktonic or biofilm conditions, and have reported distinct genetic expression [13,14,15,16,17,18,19]. Among them, our research group reported differences in gene expression when P. gingivalis ATCC 33277 grew either in planktonic or in biofilms states, finding that 4.8% of genes were differentially expressed when growing in biofilm. These up-regulated genes were mainly related to the cell envelope, transport, and binding or outer membranes proteins, while the down-regulated genes were mainly genes related to transposases or oxidative stress [13]. Most of the previous studies, however, have not elucidated the differential gene expression when this bacterium is in transition between both states. That is, whether planktonic P. gingivalis within an environment of biofilm growth, may undertake gene expression changes that would facilitate its adaptation to the developing biofilm environment.
The present study has tried to reproduce experimentally the situation in which free-floating P. gingivalis cells when in the presence of a P. gingivalis mono-species biofilm will develop a differential genetic expression when compared with similar planktonic P. gingivalis cells but without any biofilm influence. To define differential expression, apart from statistical significance (p < 0.05), a threshold of 1.5-fold (up or down) in the average expression ratio was selected, which has been previously used in microarray analysis of gene expression in P. gingivalis and was considered biologically relevant [14, 18, 26]. Under these experimental conditions, a total of 28 out of 1909 genes (1.5%) of P. gingivalis were differentially expressed when cells from the test group were compared with the control.
Among the differentially expressed genes, the gene PGN_0557 (hmuR) showed up-regulation [+ 1.67 (SD = 0.13)], which encodes a major hemin uptake protein, but also a potential adhesin. The role of this adhesin PGN_0557 (hmuR) has been demonstrated by Kuboniwa et al. (2009) [17], using an in vitro biofilm model of three bacterial species (F. nucleatum subsp. nucleatum, P. gingivalis ATCC 33277 and Streptococcus gordonii DL1), demonstrating that the lack of PGN_0557 (hmuR) gene in P. gingivalis resulted in a 70% reduction of community formation. In addition, the four genes involved in iron transport (PGN_1058 (ftn), PGN_0604, PGN_1494 and PGN_0493) appeared up-regulated, what may indicate that these genes may contribute to biofilm formation by protecting P. gingivalis from oxidative stresses generated by intracellular free iron. One of these genes, PGN_0493, encodes for the receptor Hma, which has also been implicated in heme uptake. Hagan and Mobley (2009) [34] demonstrated that iron acquisition, mediated by specific outer membrane receptors, was critical for the colonization of the urinary tract by Escherichia coli. Similarly, heme acquisition facilitated by the receptor Hma was a pre-requisite for the development of kidney infection by E. coli. Therefore, the results obtained may indicate that P. gingivalis would use similar colonization mechanisms to develop biofilm state.
Under these experimental conditions, the gene PG_2094 was up-regulated [+ 1.91 (SD = 0.32)]. It is classified as a hypothetical protein but with certain homology with the LuxR family transcriptional regulator of P. gingivalis SJD2, involved in the quorum sensing system in bacteria [35]. This might suggest a promotion of cell communication in P. gingivalis when these cells are in the presence of a biofilm. However, nowadays, although the homology with this domain is preserved, a plausible function was assigned: translocation/assembly module TamB. Bacteria export proteins across the cell envelope using diverse systems. The secretion mechanisms fulfill general cellular functions but are also essential for pathogenic bacteria during the interaction with the host cells. Selkrig et al. (2012) described a new translocation and assembly module (TAM) that promotes efficient secretion of autotransporters in proteobacteria. Functional analysis of the TAM in several bacteria, Salmonella enterica, Citrobacter rodentium or E. coli demonstrated that TamB is an integral inner membrane protein that forms the translocation and assembly module or TAM complex [36] with the outer membrane protein, TamA, an Omp85-family protein. The discovery of the TAM provides a new target for the development of therapies to inhibit colonization by bacterial pathogens [37].
It must be noted that, involved in inter-species communication and quorum sensing, LuxS-dependent signaling regulatory genes have been also related to stress, protease modulation and haemagglutination or in hemin uptake [24,25,26,27, 38]. Studies using LuxS-deficient mutants of P. gingivalis have reported an altered expression of genes involved in hemin uptake, specifically up-regulation of the genes for a TonB-linked hemin binding protein, HmuR and the iron storage protein ferritin [27, 39, 40]. In the present study, and under these experimental conditions, genes related with the synthesis of both proteins were up-regulated [PGN_0557 (hmuR) [+ 1.67 (SD = 0.13)], PGN_0604 [+ 1.78 (SD = 0.08)] and PGN_1058 (ftn) [+ 1.74 (SD = 0.12)]. In low level conditions of hemin and iron, HmuR production is suppressed by LuxS signaling, and thus the requirement of ferritin for iron storage should be reduced [40, 41]. Instead, when P. gingivalis has availability of heme/iron, as occurred in the conditions used in vitro with 5.0 mg/L hemin concentration or in vivo when the established bacterial community starts to destroy the periodontal tissue, reduced Autoinducer-2 (AI-2) expression, removes the repression of LuxS over hmuR gene, and subsequently of ferritin, which facilitate tight control and ensure adaptability to environmental conditions [40,41,42].
Free-floating P. gingivalis cells in the presence of a biofilm also overexpressed the genes PG_0009 and PG_0942, which encode for transposases, in particular the insertion sequence (IS) elements and ISPg5 transposase Orf1. These results are in agreement with those reported by Califano et al. (2000) [43], that described how ISPg5 and others IS elements could contribute to the diversity of P. gingivalis strains, as a mode of adapting to specific ecological determinants. This differential regulation in transposases genes and transposon functions has also been reported by our research group [13], in the comparative gene expression analysis of P. gingivalis ATCC 33277 in biofilm versus planktonic cells, and has been attributed to an adaptation to the changes to the new phenotypic state. The results from this current investigation could indicate that P. gingivalis begins to adapt to different environmental conditions, and may gradually adopt a sessile phenotype growth.
Among other genes related to a biofilm formation, PG_0437 showed + 1.89 fold changed expression (SD = 0.42). This gene encodes a polysaccharide outer membrane protein exporter, which is involved in polysaccharide biosynthesis. Haft et al. (2006) reported that the overexpression of these proteins occurred preferentially in bacteria from sediments, soils and biofilms [44], so that these results may imply that, under the experimental conditions used, P. gingivalis cells would use this system when adapting to evolve to a biofilm state.
The genes PGN_1312 and PGN_0780 (prtQ) encoding proteases were also up-regulated in the test group. P. gingivalis cells in the presence and, possibly evolving into a biofilm, may develop peptidase activity, with the purpose of inactivating host defense mechanisms, what may be relevant when this pathogen is forming a biofilm [45, 46].
Conversely, several genes encoding proteins involved in bacterial adhesion, invasion or virulence, were identified as down-regulated in free-floating P. gingivalis cells in the presence of a biofilm (PGN_0183 (fimC), PGN_0181, PG_2130, PG_2131, PG_1712). In fact, P. gingivalis fimbriae have been identified as one of its major colonization factors [47,48,49,50,51,52,53]. Although these results demonstrating the down-regulation of the genes encoding for the formation of fimbriae may appear contradictory, these genes encode only minor components of the fimbriae proteins FimA and Mfa1. In the comparative gene expression analysis of P. gingivalis ATCC 33277, in planktonic versus mature biofilms states, carried out by our research group, it was reported that fimD, one of the minor components of the fimbriae A, appeared down-regulated in biofilm state [13]. Similarly, Krogfelt and Klemm (1988) [54] showed that a clone of E. coli lacking the genes encoding these minor component proteins did produce fimbriae consisting of pure Fim A protein, indicating that these minor protein components were not necessary for the structural integrity of the fimbriae. These results are also coincident with those reported by Nagano et al. (2012), which demonstrated that despite a lack of fimC, the FimA protein was still produced and polymerized to form fimbriae [53]; likewise Nishiyama et al. (2007) reported that the complete deletion of PGN_0181 did not affect the formation of FimA fimbriae by P. gingivalis ATCC 33277 [55]. Similarly, previous works demonstrated that P. gingivalis autoaggregation, and its subsequent biofilm initiation, are probably due to FimA [56, 57], this autoaggregation is intensified by a loss of short fimbriae, however, other authors claim that Mfa fimbriae is essential in the process of colony formation on solid surfaces [57, 58]. These reports appear to indicate the assay- and context-dependency in assessing the role of each fimbrial type.
PG_1712 gene encoding the protein alpha-1,2-mannosidase was also down-regulated in test group. This protein belongs to the glycosidic hydrolase family 92, cleaving mannose, which is an important sugar in the synthesis of glycoproteins by Gram-negative bacterium as P. gingivalis. Five genes hydrolyzing mannan have been reported in P. gingivalis (PG_0032, PG_0902, PG_0973, PG_1711, and PG_1712), although the resulting enzymes have not been well characterized, thus making it difficult to interpretate these results. Rangarajan et al. (2013) reported that α- and β-mannosidases from P. gingivalis did not have an effect on the biosynthesis of O-LPS and A-LPS or in the secretion of Arg-gingipains [59].
This in vitro investigation has demonstrated that 28 genes (1.5%) were differentially expressed (up-regulated or down-regulated) when comparing free-floating P. gingivalis placed in the presence of a P. gingivalis mono-species biofilm versus cells growing in a pure planktonic environment. Most of these genes are related to the metabolism of iron, bacterial adhesion, invasion, virulence and quorum-sensing system. Although the differential gene expression between P. gingivalis planktonic cells growing under the test or control conditions may seem limited, the thorough understanding of the genes and its regulatory pathways involved in the transition between planktonic and biofilm states may provide important insights in the prevention of biofilm formation and consequently of periodontal diseases.
Brain Heart Infusion culture medium
CFU/mL:
Colony-forming units per milliliter
CLSM:
Confocal Laser Scanning Microscopy
Deoxyribonucleic acid
HA:
Reverse Transcription Quantitative Polymerase Chain Reaction
Filoche S, Wong L, Sissons CH. Oral biofilms: emerging concepts in microbial ecology. J Dent Res. 2010;89(1):8–18.
Socransky SS, Haffajee AD. The bacterial etiology of destructive periodontal disease: current concepts. J Periodontol. 1992;63(4 Suppl):322–31.
Holt SC, Ebersole JL. Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia: the "red complex", a prototype polybacterial pathogenic consortium in periodontitis. Periodontology 2000. 2005;38:72–122.
Kolenbrander PE, Palmer RJ Jr, Periasamy S, Jakubovics NS. Oral multispecies biofilm development and the key role of cell-cell distance. Nat Rev Microbiol. 2010;8(7):471–80.
Donlan RM, Costerton JW. Biofilms: survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev. 2002;15(2):167–93.
Davies D. Understanding biofilm resistance to antibacterial agents. Nat Rev Drug Discov. 2003;2(2):114–22.
Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms: a common cause of persistent infections. Science (New York, NY). 1999;284(5418):1318–22.
Hoiby N, Ciofu O, Johansen HK, Song ZJ, Moser C, Jensen PO, Molin S, Givskov M, Tolker-Nielsen T, Bjarnsholt T. The clinical impact of bacterial biofilms. International journal of oral science. 2011;3(2):55–65.
Whiteley M, Bangera MG, Bumgarner RE, Parsek MR, Teitzel GM, Lory S, Greenberg EP. Gene expression in Pseudomonas aeruginosa biofilms. Nature. 2001;413(6858):860–4.
Schembri MA, Kjaergaard K, Klemm P. Global gene expression in Escherichia coli biofilms. Mol Microbiol. 2003;48(1):253–67.
Beenken KE, Dunman PM, McAleese F, Macapagal D, Murphy E, Projan SJ, Blevins JS, Smeltzer MS. Global gene expression in Staphylococcus aureus biofilms. J Bacteriol. 2004;186(14):4665–84.
Dotsch A, Eckweiler D, Schniederjans M, Zimmermann A, Jensen V, Scharfe M, Geffers R, Haussler S. The Pseudomonas aeruginosa transcriptome in planktonic cultures and static biofilms using RNA sequencing. PLoS One. 2012;7(2):e31092.
Romero-Lastra P, Sanchez MC, Ribeiro-Vidal H, Llama-Palacios A, Figuero E, Herrera D, Sanz M. Comparative gene expression analysis of Porphyromonas gingivalis ATCC 33277 in planktonic and biofilms states. PLoS One. 2017;12(4):e0174669.
Lo AW, Seers CA, Boyce JD, Dashper SG, Slakeski N, Lissel JP, Reynolds EC. Comparative transcriptomic analysis of Porphyromonas gingivalis biofilm and planktonic cells. BMC Microbiol. 2009;9:18.
Kuboniwa M, Houser JR, Hendrickson EL, Wang Q, Alghamdi SA, Sakanaka A: Metabolic crosstalk regulates Porphyromonas gingivalis colonization and virulence during oral polymicrobial infection 2017, 2(11):1493–1499.
Bao K, Belibasakis GN, Thurnheer T, Aduse-Opoku J, Curtis MA, Bostanci N. Role of Porphyromonas gingivalis gingipains in multi-species biofilm formation. BMC Microbiol. 2014;14:258.
Kuboniwa M, Hendrickson EL, Xia Q, Wang T, Xie H, Hackett M, Lamont RJ. Proteomics of Porphyromonas gingivalis within a model oral microbial community. BMC Microbiol. 2009;9:98.
Yamamoto R, Noiri Y, Yamaguchi M, Asahi Y, Maezono H, Ebisu S. Time course of gene expression during Porphyromonas gingivalis strain ATCC 33277 biofilm formation. Appl Environ Microbiol. 2011;77(18):6733–6.
Redanz S, Standar K, Podbielski A, Kreikemeyer B. A five-species transcriptome array for oral mixed-biofilm studies. PLoS One. 2011;6(12):e27827.
Ang CS, Veith PD, Dashper SG, Reynolds EC. Application of 16O/18O reverse proteolytic labeling to determine the effect of biofilm culture on the cell envelope proteome of Porphyromonas gingivalis W50. Proteomics. 2008;8(8):1645–60.
Xie H, Chung WO, Park Y, Lamont RJ. Regulation of the Porphyromonas gingivalis fimA (Fimbrillin) gene. Infect Immun. 2000;68(12):6574–9.
Dashper SG, Ang CS, Veith PD, Mitchell HL, Lo AW, Seers CA, Walsh KA, Slakeski N, Chen D, Lissel JP, et al. Response of Porphyromonas gingivalis to heme limitation in continuous culture. J Bacteriol. 2009;191(3):1044–55.
Yamaguchi M, Sato K, Yukitake H, Noiri Y, Ebisu S, Nakayama K. A Porphyromonas gingivalis mutant defective in a putative glycosyltransferase exhibits defective biosynthesis of the polysaccharide portions of lipopolysaccharide, decreased gingipain activities, strong autoaggregation, and increased biofilm formation. Infect Immun. 2010;78(9):3801–12.
Shrout JD, Tolker-Nielsen T, Givskov M, Parsek MR. The contribution of cell-cell signaling and motility to bacterial biofilm formation. MRS Bull. 2011;36(5):367–73.
Burgess NA, Kirke DF, Williams P, Winzer K, Hardie KR, Meyers NL, Aduse-Opoku J, Curtis MA, Camara M. LuxS-dependent quorum sensing in Porphyromonas gingivalis modulates protease and haemagglutinin activities but is not essential for virulence. Microbiology (Reading, England). 2002;148(Pt 3):763–72.
Yuan L, Hillman JD, Progulske-Fox A. Microarray analysis of quorum-sensing-regulated genes in Porphyromonas gingivalis. Infect Immun. 2005;73(7):4146–54.
Chung WO, Park Y, Lamont RJ, McNab R, Barbieri B, Demuth DR. Signaling system in Porphyromonas gingivalis based on a LuxS protein. J Bacteriol. 2001;183(13):3903–9.
Marsh PD. Are dental diseases examples of ecological catastrophes? Microbiology (Reading, England). 2003;149(Pt 2):279–94.
Stanley NR, Lazazzera BA. Environmental signals and regulatory pathways that influence biofilm formation. Mol Microbiol. 2004;52(4):917–24.
Stoodley P, Sauer K, Davies DG, Costerton JW. Biofilms as complex differentiated communities. Annu Rev Microbiol. 2002;56:187–209.
Smyth GK, Speed T. Normalization of cDNA microarray data. Methods (San Diego, Calif). 2003;31(4):265–73.
Smyth GK: Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Statistical applications in genetics and molecular biology 2004, 3:Article3.
Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc. 2008;3(6):1101–8.
Hagan EC, Mobley HL. Haem acquisition is facilitated by a novel receptor Hma and required by uropathogenic Escherichia coli for kidney infection. Mol Microbiol. 2009;71(1):79–91.
Fuqua WC, Winans SC, Greenberg EP. Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators. J Bacteriol. 1994;176(2):269–75.
Josts I, Grinter R, Kelly SM, Mosbahi K, Roszak A, Cogdell R, Smith BO, Byron O, Walker D. Recombinant expression, purification, crystallization and preliminary X-ray diffraction analysis of the C-terminal DUF490(963-1138) domain of TamB from Escherichia coli. Acta crystallographica Section F, Structural biology communications. 2014;70(Pt 9):1272–5.
Selkrig J, Mosbahi K, Webb CT, Belousoff MJ, Perry AJ, Wells TJ, Morris F, Leyton DL, Totsika M, Phan MD, et al. Discovery of an archetypal protein transport system in bacterial outer membranes. Nat Struct Mol Biol. 2012;19(5):506–10 s501.
McNab R, Ford SK, El-Sabaeny A, Barbieri B, Cook GS, Lamont RJ. LuxS-based signaling in Streptococcus gordonii: autoinducer 2 controls carbohydrate metabolism and biofilm formation with Porphyromonas gingivalis. J Bacteriol. 2003;185(1):274–84.
Hirano T, Beck DA, Demuth DR, Hackett M, Lamont RJ. Deep sequencing of Porphyromonas gingivalis and comparative transcriptome analysis of a LuxS mutant. Front Cell Infect Microbiol. 2012;2:79.
James CE, Hasegawa Y, Park Y, Yeung V, Tribble GD, Kuboniwa M, Demuth DR, Lamont RJ. LuxS involvement in the regulation of genes coding for hemin and iron acquisition systems in Porphyromonas gingivalis. Infect Immun. 2006;74(7):3834–44.
McNab R, Lamont RJ. Microbial dinner-party conversations: the role of LuxS in interspecies communication. J Med Microbiol. 2003;52(Pt 7):541–5.
Butler C, Mitchell H, Dashper S, Reynolds E. The Porphyromonas gingivalis ferric uptake regulator orthologue does not regulate iron homeostasis. Genomics data. 2015;5:167–8.
Califano JV, Kitten T, Lewis JP, Macrina FL, Fleischmann RD, Fraser CM, Duncan MJ, Dewhirst FE. Characterization of Porphyromonas gingivalis insertion sequence-like element ISPg5. Infect Immun. 2000;68(9):5247–53.
Haft DH, Paulsen IT, Ward N, Selengut JD. Exopolysaccharide-associated protein sorting in environmental organisms: the PEP-CTERM/EpsH system. Application of a novel phylogenetic profiling heuristic. BMC biology. 2006;4:29.
Kato T, Takahashi N, Kuramitsu HK. Sequence analysis and characterization of the Porphyromonas gingivalis prtC gene, which expresses a novel collagenase activity. J Bacteriol. 1992;174(12):3889–95.
Brunner J, Wittink FR, Jonker MJ, de Jong M, Breit TM, Laine ML, de Soet JJ, Crielaard W. The core genome of the anaerobic oral pathogenic bacterium Porphyromonas gingivalis. BMC Microbiol. 2010;10:252.
Lamont RJ, Jenkinson HF. Life below the gum line: pathogenic mechanisms of Porphyromonas gingivalis. Microbiology and molecular biology reviews : MMBR. 1998;62(4):1244–63.
Kolenbrander PE, Andersen RN, Blehert DS, Egland PG, Foster JS, Palmer RJ Jr. Communication among oral bacteria. Microbiology and molecular biology reviews : MMBR. 2002;66(3):486–505 table of contents.
Jenkinson HF, Lamont RJ. Oral microbial communities in sickness and in health. Trends Microbiol. 2005;13(12):589–95.
Andrian E, Grenier D, Rouabhia M. Porphyromonas gingivalis-epithelial cell interactions in periodontitis. J Dent Res. 2006;85(5):392–403.
Hasegawa Y, Iwami J, Sato K, Park Y, Nishikawa K, Atsumi T, Moriguchi K, Murakami Y, Lamont RJ, Nakamura H, et al. Anchoring and length regulation of Porphyromonas gingivalis Mfa1 fimbriae by the downstream gene product Mfa2. Microbiology (Reading, England). 2009;155(Pt 10):3333–47.
Yoshimura F, Murakami Y, Nishikawa K, Hasegawa Y, Kawaminami S. Surface components of Porphyromonas gingivalis. J Periodontal Res. 2009;44(1):1–12.
Nagano K, Abiko Y, Yoshida Y, Yoshimura F. Porphyromonas gingivalis FimA fimbriae: roles of the fim gene cluster in the fimbrial assembly and antigenic heterogeneity among fimA genotypes. Journal of Oral Biosciences. 2012;54(3):160–3.
Krogfelt KA, Klemm P. Investigation of minor components of Escherichia coli type 1 fimbriae: protein chemical and immunological aspects. Microb Pathog. 1988;4(3):231–8.
Nishiyama S, Murakami Y, Nagata H, Shizukuishi S, Kawagishi I, Yoshimura F. Involvement of minor components associated with the FimA fimbriae of Porphyromonas gingivalis in adhesive functions. Microbiology (Reading, England). 2007;153(Pt 6):1916–25.
Kuramitsu H, Tokuda M, Yoneda M, Duncan M, Cho MI. Multiple colonization defects in a cysteine protease mutant of Porphyromonas gingivalis. J Periodontal Res. 1997;32(1 Pt 2):140–2.
Umemoto T, Hamada N. Characterization of biologically active cell surface components of a periodontal pathogen. The roles of major and minor fimbriae of Porphyromonas gingivalis. J Periodontol. 2003;74(1):119–22.
Lin X, Wu J, Xie H. Porphyromonas gingivalis minor fimbriae are required for cell-cell interactions. Infect Immun. 2006;74(10):6011–5.
Rangarajan M, Aduse-Opoku J, Hashim A, Paramonov N, Curtis MA. Characterization of the alpha- and beta-mannosidases of Porphyromonas gingivalis. J Bacteriol. 2013;195(23):5297–307.
Project was supported by Extraordinary Chair of DENTAID-UCM. We thank G. García and L. Almonacid from the Centro Nacional de Biotecnología (Universidad Autónoma, Madrid, Spain) for performance of hybridizations and statistical analysis of the microarrays and RT-qPCR. We thank C. Pérez from the Centre of Microscopy and Cytometry from Universidad Complutense (Madrid, Spain).
This work was funded by the Cátedra Dentaid-UCM. None of the funders had any role in designing and/or conducting of the study; collection, management, analysis and interpretation of the data; and preparation, review or approval of the manuscript.
The datasets generated and analyzed during the current study are available in the Gene Expression Omnibus (GEO) repository, using the accession GSE122623.
URL: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE122623
Laboratory of Dental Research, University Complutense, Madrid, Spain
María C. Sánchez, Patricia Romero-Lastra, Honorato Ribeiro-Vidal & Arancha Llama-Palacios
Etiology and Therapy of Periodontal Diseases (ETEP) Research Group, University Complutense, Madrid, Spain
María C. Sánchez, Honorato Ribeiro-Vidal, Arancha Llama-Palacios, Elena Figuero, David Herrera & Mariano Sanz
Department of Dental Clinical Specialities (DDCS), Faculty of Odontology, Plaza Ramón y Cajal s/n Ciudad Universitaria, 28040, Madrid, Spain
Mariano Sanz
María C. Sánchez
Patricia Romero-Lastra
Honorato Ribeiro-Vidal
Arancha Llama-Palacios
Elena Figuero
David Herrera
Conceptualization: MCS PR-L EF DH MS. Funding acquisition: DH MS. Investigation: PR-L MCS HR-V AL-P. Methodology: MCS PR-L HR-V AL-P. Supervision: MCS EF DH MS. Writing - original draft: MCS PR-L. Writing - review & editing: EF DH MS. We confirm that the final version manuscript has been read and approved by all named authors.
Correspondence to Mariano Sanz.
Sánchez, M.C., Romero-Lastra, P., Ribeiro-Vidal, H. et al. Comparative gene expression analysis of planktonic Porphyromonas gingivalis ATCC 33277 in the presence of a growing biofilm versus planktonic cells. BMC Microbiol 19, 58 (2019). https://doi.org/10.1186/s12866-019-1423-9
Porphyromonas gingivalis
Microarray hybridization
Gene transcription
Transcriptomics
RT-qPCR
Planktonic growth


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Album Review: Eidola, "To Speak, To Listen"
July 13, 2017 Thomas Hatton Reviews One comment
Eidola released their third album, To Speak, To Listen, on June 2 of this year. Eidola has definitely made a name for themselves as an up-and-coming band to know in the progressive metal scene. Their previous two releases were met with much acclaim from both modern metal review sites and progressive metal sites as well. Eidola is one of the artists featured on the Blue Swan Records label, which was started by Dance Gavin Dance's Will Swan. Because of this association, Eidola has been grouped together with the other artists on the label, and further given the genre label of Swancore for their sound- which, like any label, is clumsy and potentially limiting. However, according to fans of this subgenre of progressive metal/post-hardcore, Swancore can be identified by highly technical, math-rock like technicality, punk-ish aggression, and high pitched/screaming vocals throughout. All three of these things are present in Eidola's releases, but this album is much more than those labels.
From the second that you put the record on, you're able to hear a band that is overflowing with energy and comfortable with highly technical music and challenging instrumental passages. Eidola has both singing and screaming (by Andrew Wells and Matthew Dommer, respectively) in most of the songs. Wells' vocals are precise and clear, and serve as a melodic constant to the intricate, heavy, and even cacophonous music throughout the album. Dommer's screams serve as an aggressive counterpoint to Well's melodic singing.
The first few songs are straightforward and somewhat similar sonically. "Telestai" and "Amplissimus Machina" were both released as videos for the album, and make strong statements as standalone songs, but are more typically what you would hear from their label mates. This is not to say that the songs are derivative. They are solid tracks that showcase Eidola's strengths- technicality, melody, spirit, aggression. But the turning point of the album is the ballad, "Loti", which is both tender and atmospheric, and also aggressive. The subsequent songs, while less straightforward as the first few songs, seem to be Eidola flexing their progressive metal side. The band takes the listener on a unique, dense journey through the metaphysical. Whether it is the spoken word poetry heard in the fantastic "Sri Vishnu Yantra", or the double bass heavy finish on "Transcendentium Part II- Fourth Temple", the second half of the album seems to reconnect with the adventurous spirit of the previous albums, while simultaneously channeling a new, more realized sound and energy. The band delivers spirited, strong,and instrumentally impressive passages, chock full of equal parts polyphony and emotion.
From a narrative perspective, the lyrics and references are highly introspective and philosophical on this album. From interviews, it is clear that this album represents, in the 3 album story arc, enlightenment. From a technical perspective- What I see is a progression from the visceral edge of The Great Glass Elephant, to the chunky but technical Degeneraterra, to the clear and crisp soundscapes on To Speak, To Listen. Even if these production decisions were not intentional, they add on another layer of narrative progression from album to album that manifests even in the sonic quality of the music. Eidola's latest album is a strong, dense, and powerful musical statement, and their recognition and praise for this latest release is deserving.
I have been listening to progressive metal for a few decades now. When prog metal became a thing, it meant that a band was going to sound like Fates Warning or Queensryche or Dream Theater. Over time, boundary pushing acts would combine the aggression of metal with other diverse interests. I am really pleased that bands like Eidola are drawing from new, diverse interests to create music that is just as technically challenging as the prog bands of before, but incorporates fresh influences that range from hardcore to punk (which might have even seemed antithetical before).
Eidola's latest album, To Speak, To Listen, was released through Blue Swan Records on June 2, 2017. Order the album here.
andrew wellsblue swan recordseidolamatthew dommerto speak to listen
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Winter Strider Race Series! The Factory & @sevenspokesbike shop have teamed up starting this weekend in Minneapolis to bring you THREE Strider races this winter, gearing up for the finale Snow Cup at Buck Hill this March 2019. We will be featuring races for 1-5 year olds and 6-10 year olds, and it's FREE to … Continue reading Winter Strider Race Series at The Factory!
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Could the proposed Oxford-Cambridge Expressway help the higher education sector?
By Rob Sadler
This blog was kindly contributed by Rob Sadler, the Head of the Cambridge office for Savills.
Plans to reduce journey times between Oxford and Cambridge by 40 minutes via the creation of a new Expressway between the M40 at Oxford and the M1 at Milton Keynes are currently being developed, with passionate individuals on both sides of the debate on whether it will help or hinder the surrounding areas.
Whatever the arguments, better infrastructure links between the two cities, whether that is in the form of increased rail capacity or road improvements, have the potential to impact on the higher education sector across the entire area known as the 'Oxford-Cambridge Innovation Arc'. This is the corridor of land encompassing Oxford, Milton Keynes and Cambridge. This area is one of the most dynamic and innovative places in Europe today and is already home to 3.7 million people and generates over two million jobs.
Taken from https://www.gov.uk/government/news/corridor-announced-to-unlock-full-potential-of-englands-economic-heartland
The specific proposals for the Expressway involve a new dual carriageway being built between the A34 near Oxford and the A14 near Cambridge via, or near, Milton Keynes, with links into the existing local road network. If it goes ahead, the Expressway, alongside other infrastructure improvements such as introducing a new direct train line, is forecast to contribute to the Arc area growing by a further two million people and at least a further million jobs by 2050.
Lifted from Highways England: https://highwaysengland.co.uk/oxford-to-cambridge-expressway-home/
In terms of higher education, the Arc is bookended by Oxford Brookes University and the University of Oxford at one end, and Cambridge University and Anglia Ruskin University at the other. But other institutions in the Arc, and therefore potentially affected by the Expressway, include the University of Bedfordshire, Buckinghamshire New University, Cranfield University, the University of Buckingham, the University of Northampton and The Open University. Another university is set to join them by 2023: MK:U will have 5,000 undergraduate students, focused on science, technology and engineering, supported by Cranfield University in the centre, forming an important part of the plans to redevelop parts of the town, including delivering 4,000 new homes and additional business and commercial space.
The Expressway will also bring thousands more people within a shorter drive of the Arc's multitude of specialist tech and science innovation clusters, some of which are founded and owned by the Oxford or Cambridge colleges.
The 'knowledge intensive' industries which occupy these clusters and many other locations in the region, dominate the local employment landscape and together form one of the world's foremost science, technology and business environments, fed by the talent and innovation nurtured by the nearby higher education institutions. They are an integral part both of the local economy and that of UK Plc with the region contributing an estimated £111 billion annually to the national economy.
But there are significant variations in the sharing of intelligence and resources across the Arc. Many higher education institutions and companies currently have stronger links with their peers in London and Birmingham than with other locations in the corridor, as present transport links are stronger along the north-south 'backbone' of England rather than across the east-west 'waistline'.
With the Expressway forecast to cut journey times significantly, this should change. Intra-regional links between all these higher education institutions will be strengthened and a closer academic community formed, facilitating linkages in terms of research, teaching and learning and knowledge transfer.
Crucially, the Expressway should also allow institutions to attract and retain the best staff on a global scale by expanding the choice of where people can live. At the moment, housing is limited across the region and very expensive. In Cambridge and Oxford homes are 13 and 11 times greater than workplace based earnings respectively, but even the more affordable towns and cities of Milton Keynes, Northampton, and Bedford still have house price to earnings ratios of over eight, well above the UK average. The Expressway will open up new sites for housing development and enable secondary satellite towns to grow, with the result of hopefully lowering housing costs. Increasing the number of affordable locations within a commutable distance of all the higher education institutions within the Arc should aid recruitment and retention.
For many, an Expressway is about cutting journey times between the key centres within the region and easing congestion, but it has an important strategic role to play on both a national and worldwide stage. From a global perspective it has the potential to support a growing ecosystem of learning, research and innovation so it continues to rival other world-leading hubs. Getting it right is therefore crucial.
Read more about the Oxford-Cambridge innovation arc here.
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2021 Challenge: Te uruparenga | Responsiveness
In the Public Service Act, responsiveness is described as what the public service does to understand and meet people's needs and aspirations, including how it works with New Zealanders when designing policies and services.
In the context of future Open Government Partnership commitments, we are interested in building on the work we have been doing on public participation in policy development and service design:
What do you think this public participation should look like?
Here's some more ideas on this theme that came out of our 2020 workshops: OGP NAP4 Ideas Responsiveness
The Public Service delivers government services across New Zealand. The way that policies are developed and how services are designed impacts on every New Zealander in some way.
New Zealand has developed three previous OGP National Action Plans with commitments to support greater responsiveness. For example, in National Action Plan 3:
Commitment 5: Public participation in policy development – aims to assist the New Zealand public sector to develop a deeper and more consistent understanding of what good engagement with the public means
Commitment 6: Service design – aims to develop an assessment model to support implementation of the all-of-government Digital Service Design Standard by public sector agencies
In 2018 we received nearly 100 ideas that expressed in various ways that government and New Zealanders should engage the public more in shaping policy decisions and the design of public services. National Action Plan 3 explains what happened to those ideas. Many ideas:
pointed out the importance of ensuring that policies and services reflected the needs, aspirations, and expectations of people of different cultures, ages, genders and localities
suggested that the best way of doing this was to work with people at all stages of the policy and service design and improve communication of what government is doing
We've heard there were much closer and more productive working relationships between government agencies and civil society partners during the COVID-19 response that worked better for people[1].
[1] "There were significant improvements in relationships with government and other funders as a result of the COVID-19 response. This included greater flexibility in contract requirements, faster and more responsive decision-making, and a sense of real partnership and being treated with respect." – ComVoices 2020 State of the Community and Voluntary Sector Survey Snapshot.
2 ideas matching your filter
citizen-centred services
participation in decision-making
Establish a Policy Horizons joint operational agreement
1 comment April 23, 2021 at 01:55PM
Public surveying
5.0 (1 vote)
0 comments April 17, 2021 at 09:35AM


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Умар Иса аль-Абдулджаббар (; ), или просто Умар Иса — катарский футболист, полузащитник футбольного клуба «Катар СК».

Клубная карьера
Воспитанник академии футбольного клуба «Катар СК». Дебютировал за основной состав клуба 11 декабря 2017 года в матче кубка Старс-лиги с «Аль-Харитиятом», выйдя на поле на 69-й минуте встречи, которая завершилась разгромным поражением 1:6. 4 августа 2018 года сыграл свою первую игру в чемпионате Катара. В поединке с «Аль-Гарафой» Умар Иса появился на поле на 63-й минуте, заменив Али Буджалуфа, отметившегося забитым мячом.

Клубная статистика

Примечания

Футболисты Катара
Игроки ФК «Катар СК»

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Last-second schedule scramble has Flashes visiting rival Zips Friday night
Allen Moff
Record-Courier
The Kent State men's basketball team spent Thursday morning at practice in preparation for a scheduled Mid-American Conference opener at Eastern Michigan on Saturday. At 2:30 Thursday afternoon, the Golden Flashes found out that game was off and they'd instead be playing at bitter backyard rival Akron on Friday at 7 p.m.
Welcome to college basketball 2020-21, pandemic style.
The MAC issued a release Thursday afternoon stating that the Flashes-Eagles contest scheduled for Saturday had been postponed, presumably due to COVID-19 issues involving Eastern Michigan. The release also announced that Kent State (3-1, 0-0 MAC) would play at Akron (3-1, 1-0) on Friday. The game will tip at 7 p.m., and be broadcast live on CBS Sports Network.
The Flashes are the only team in the MAC that has not played a conference game, and has now had three league contests postponed due to COVID-19 issues.
"The only thing I'll say about all of this is that I believe this season every team should make the MAC Tournament, not just the top eight," said KSU head coach Rob Senderoff. "When games are postponed and rescheduled on short notice like this and there's so much scrambling going on to get games in, it just makes sense to me to let everyone compete for a chance to make the NCAA Tournament this year. But obviously those decisions aren't for me to make.
"Now we'll get ready for Akron. Our kids haven't played in 18 days. They'll be excited to play."
With that, Senderoff politely asked to leave the call so he could start preparing for the Zips.
The Flashes returned to practice on Wednesday for the first time since mid-December, when the program was forced to pause due to COVID-19 issues. Scheduled home games against Akron (Dec. 19) and Bowling Green (Dec. 30) were postponed.
"We practiced hard (on Wednesday), got up and down the floor. Conditioning certainly needs to improve, as is expected, and we've got to take better care of the basketball. But we had good energy," said Senderoff. "There's still plenty of room for improvement. We've got to continue to work each day."
Kent State was at full strength on Wednesday for the first time since senior forward Tervell Beck suffered an injury during practice in mid-November. Beck is one of only three returnees who averaged over 12 minutes per game last season, along with sophomore point guard Giovanni Santiago and senior forward Danny Pippen.
"As you would expect after being off for six weeks he was a little rusty. It will probably take him a little bit of time to get his wind and get back to where he was. He was having a great preseason," said Senderoff. "It was good to have him back out with the team for sure."
Beck's status for Friday's game remains uncertain.
"I don't want to put him out there unless he's 100 percent, or close to it," said Senderoff. "We'll see how he progresses over the next couple of days and go from there."
The Flashes were making major progress as a team early in the season despite their overall inexperience, winning three of four games. The lone loss was an overtime setback at No. 16 Virginia.
Akron's program has been paused twice due to COVID-19 issues. The Zips were not able to open the season until Dec. 12. After experiencing some typical early season ups and downs during its first three contests, Akron thrashed Ohio 90-70 in its MAC opener on Dec. 22 at J.A.R. Arena.
Who: Kent State (3-1, 0-0) vs. Akron (3-1, 1-0)
When/Where: Friday, 7 p.m./James A. Rhodes Arena, Akron.
Radio/TV: Kent State Radio/CBS Sports Network
Last Year's Records: Kent State 20-12, 9-9. Akron 24-7, 14-4.
Flashes To Watch: Danny Pippen, 6-9, Sr., F (17 points, 6.3 rebounds. 3.5 assists). Mike Nuga, 6-2, Sr., G (17.8 points, 5 rebounds, 2 steals). Justyn Hamilton, 6-11, Sr., F (13 points, 9.5 rebounds). Giovanni Santiago, 6-1, So., G (7.8 points, 2.5 rebounds, 3 assists).
Zips To Watch: Loren Cristian Jackson, 5-8, Sr., G (24 points, 8.5 rebounds). Enrique Freeman, 6-7, So., F (9.75 points, 9.75 rebounds). Maishe Dailey, 6-7, Sr., G (9.5 points, 4.25 rebounds). Bryan Trimble, Jr., 6-2, Jr., G (9.25 points, 2 rebounds).
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Vol. 24, No. 4: December-January 2019
Newsletter Dec'19
inspiration / Motivation
Recording Tips / Methods
Songwriting / Composition
The Power To Release
The Rediscovered (Archive)
In Review (Archive)
iTunes Downloads
Join Us (Free)
Twilight Odyssey
Guitar Nine
Guitarists and Bands , The Undiscovered
Old School Metal Rises Up
The ghost of Iron Maiden (wait, aren't Iron Maiden still alive?) is what first strikes you upon first listen to "Gettysburg", the opening track on the group Twilight Odyssey's three song demo CD-R. Not so much for the intertwined, twin guitar leads Maiden was noted for (though the group does sport two guitarists, founder Ben Asaro and Dan Castro), but for the galloping rhythms and overall '80s-metal vibe. The intro and rhythmic riffage heard on song two, "The Endless Days Of A Stranger", will also resonate familiarly in the ears of Maiden aficionados. Getting Pamela Berlinghof on vocals was a masterstroke, as she can wail with the best of them, in the Leather Leone tradition. Asaro makes sure the guitars get plenty of sonic space, offering extended solo sections and overall heaviness. Here is a group just needing an outside production boost coupled with the songwriting experience to shake off their influences to go to the next level - look for their debut CD on Italy's Steelheart Records.
A love for music was instilled in guitarist Ben Asaro from a very early age, listening to the Beatles and other artists from the 1950s and 1960s through his parent's record collection. He became aware of guitar music about the age of 9 or 10 by listening to classical guitarist John Williams' Bach Lute Suites. Ben started taking guitar lessons as a teen, and was able to attend master classes with guitarists such as Pat Martino, Tal Farlow and Arlen Roth. Musically, he strives to write songs that are guitar-heavy, melodic and memorable, but still have a hard edge to them.
Currently, Twilight Odyssey is seeking management, and are recording their 10-track debut album for Steelheart Records in a New York studio.
Ben Asaro
359A 14th Street
E-mail: info@twilight-odyssey.com
Web site: www.twilight-odyssey.com
Guitar Nine & More
Dan McAvinchey
mcavinchey.org
The Rediscovered
The Undiscovered
It's free to become a member and participate! Join now!
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Compartmentalizing the American Alliance with Israel from the Israeli-Palestinian Conflict
Yaël Mizrahi-Arnaud and Abe Silberstein
Is it time for the United States to reevaluate its alliance with Israel in light of the latter's continued occupation of Palestinian territory?
With the election now behind us, President-elect Joe Biden and his team face several foreign policy challenges left unaddressed or exacerbated by the outgoing Trump administration, including in the Middle East. On first glance, the Israeli-Palestinian conflict does not appear to be one of them. From a high-level perspective, a Biden administration is likely to shift the focus of U.S. policy away from the Middle East and toward the Asia-Pacific region, where a rising and revisionist China threatens Washington's interests and allies, and possibly Europe, where they will inherit a Trump legacy of distrust and recriminations. In crafting its Middle East policy specifically, the Biden team is not inclined to upset America's strongest ally in the region over a conflict that has frustrated generations of diplomats.
Still, we believe this model of tacit approval of Israel's occupation is a terrible mistake. Continuing it would be a missed opportunity to correct the pro-occupation excesses of the outgoing administration while strengthening the U.S.-Israeli alliance in the longer term. However, contrary to those who suggest a new effort to conclude a final status agreement between Israel and the Palestinians, we submit the United States would at best be wasting its time expending much effort on diplomacy in this conflict. Instead, a Biden administration should work to extract the United States from its dominant mediating role, for which it is ill-suited, and clearly compartmentalize its strategic relationship with Israel from that nation's conflict with the Palestinians. This would greatly benefit America's foreign policy and national security interests.
In brief, compartmentalization will clearly delineate U.S. priorities in the Middle East by closing the chapter on failed meditation between Israel and the Palestinians; improve American relations with the Palestinian Authority, an essential governing and counter-terrorism partner that has too often been treated as an afterthought in the traditional U.S.-Israeli relationship; and create a political framework in which the American alliance with Israel can better withstand ideological pressures. For the reasons listed above, we also believe separating the U.S.-Israeli alliance from the Israeli-Palestinian conflict will improve the prospects for a final status agreement.
Implementing this change, first conceptualized in European policy circles as "differentiation," will involve a number of politically difficult measures, as well as some more modest ones that may already be on the president-elect's radar. Domestic limitations, including an overwhelmingly pro-Israel majority in Congress as well as Biden's own deeply positive feelings toward the Jewish state, restrict the extent to which compartmentalization is possible right now. In formulating our suggestions, we kept political feasibility front and center. Those who, for sundry reasons, want to see the U.S.-Israeli relationship changed entirely will invariably find few substantive differences between our vision and the status quo. However, as we will show, compartmentalization would be a major and meaningful break from the past.
Failure of Traditional U.S. Policy
In addition to advancing the U.S.-Israeli relationship, American diplomats have spent the last forty-some years in the pursuit of a just settlement to the Israeli-Palestinian conflict. This goal took on preeminent importance in American foreign policy as it fit squarely within U.S. national security objectives of the last half-century, which included stabilizing the Middle East to ensure the free flow of oil and countering Soviet influence in the region. Its position as mediator was justified under the theory that, due to the "special relationship" between the United States and Israel, America was uniquely positioned to leverage its ties to extract concessions from Israel — ultimately "delivering" peace. However, this has not come to fruition, and negotiations mediated by the United States have too often looked like a joint American-Israeli effort to reach an agreement with the Palestinians. One former American negotiator, Aaron David Miller, described America's role as that of "Israel's lawyer."
American policy has generally followed this pattern, and it's hard to understand why anyone expected otherwise. Israel is a strong, prosperous, and democratic ally whose steady presence in an unstable region benefits American foreign policy and national security interests. After decades of negotiations, it should be clear at this point that no U.S. administration will pressure Israel into making major territorial concessions it is not comfortable making. When failure happens, there is an interest in portraying Israel as a willing partner and the Palestinians as less willing, despite evidence of a far more complex picture.
An additional, unintended consequence of American zeal to reach an agreement has been the advancement of right-wing parties that favor a "Greater Israel," as they have capitalized on the opportunity to put pressure on the government and channel public discontent against perceived American "meddling." This effectively pushes the needle backwards, moving away from a comprehensive and just settlement to the conflict, while hardening beliefs which encourage harmful policies. This should be the primary lesson of the Obama years, which saw the Israeli right gain dominance in the country: Faced with such hostile conditions, it is better not to attempt the big, flashy efforts toward peace at all.
A New Way Forward: Strategic Compartmentalization
Before elucidating what separation of the U.S. strategic relationship with Israel from the occupation would look like, it is important to establish why the United States — and Biden in particular — should begin to pivot away from its traditionally active role in resolving the Israeli-Palestinian conflict.
For starters, the focus of the Biden administration's foreign policy will not be in the Middle East. If the Obama administration's "pivot to Asia" was a far-sighted aspiration, it will be something closer to an urgent strategic priority for the incoming president. As if to illustrate the point to the United States, 15 Asian countries — including all 10 Association of Southeast Asian Nations members, South Korea, Japan, New Zealand, Australia, and China — signed the far-reaching Regional Comprehensive Economic Partnership agreement that excludes Washington. It will not be easy for Biden, if he even wishes, to revive the American role in the Trans-Pacific Partnership, and so his foreign policy team will be working overtime to explore ways in which to shore up American influence in the Asia Pacific region and counter malign Chinese activities intended to disrupt a rules-based order. Similarly, the issues of coronavirus vaccine distribution, climate change, and protecting the American homeland from kinetic and cyber threats will rank far above yet another ambitious and improbable gambit for peace between Israel and the Palestinians. Where the Middle East does come into play, the Biden administration's time will be occupied by the Iran nuclear issue, which may necessitate a political confrontation with Israel over a matter far more important to U.S. national security interests than the future of the Palestinians.
While one should expect the Middle East to produce unwanted work for American policymakers, the chance of an emergency involving the fundamentals of the Israeli-Palestinian conflict — territory, the status of Palestinian refugees, and major holy sites in Jerusalem — is rather remote. The United States may conceivably be asked to help mediate a ceasefire should a hot war arise from the Hamas-controlled strip, but neither the Israeli nor Palestinian leadership seems ready or able to spend the political capital needed to reach a final status accord. As the Obama administration learned in its peace efforts from 2013 to 2014, Washington cannot fill the demand gap alone.
Biden also has a powerful political reason to initiate a separation of the U.S.-Israeli relationship, which is of immense strategic value to the country, from the Israeli occupation, which is not. We do not accept the predictions of triumphalists or alarmists that progressives such as Rep. Alexandria Ocasio-Cortez and Rep. Ilhan Omar represent the future of foreign policy thinking in the Democratic Party, particularly on Israel. We do, however, realize that this bloc's influence is steadily growing and that its constituents — including Palestinian Americans — will need to be accommodated. Some Israeli analysts and commentators already worry the "radical wing" of the party will push Biden to the left on Israel. From our anecdotal experience as individuals embedded in the progressive foreign policy discourse, we can confirm that harsh and valid criticism of Israel's human rights record in the occupied territories is common, and that American "complicity" is often decried. For someone who rightly values the U.S.-Israeli partnership, it would be wise for Biden to lead the way in crafting a palatable Democratic position in favor of the alliance that could endure inside the party. We are aware that compartmentalization will likely not placate the leftmost wing of the party, but it will provide a compromise position — support for the relationship with Israel combined with a position on the occupation that is congruent with international laws and norms — which will greatly narrow the gulf between the party's progressive values and its support for the alliance with Israel.
Painting the Policy Picture
Given the decades-long history and extent of relations between the United States and Israel, there are innumerable ways for the United States to draw the line between the alliance with Israel and the Israeli-Palestinian conflict. Below are four broad policy suggestions that could go a long way in achieving this objective.
Strengthen the Bilateral Relationship with the Palestinians
In November 2016, then-Israeli Education Minister Naftali Bennett welcomed the incoming President Donald Trump with the jubilant declaration of "the era of the Palestinian state is over." While never explicitly spelled out in these terms, the Trump administration's policy has consistently alienated the Palestinians and exacerbated the tenuous and fragile legitimacy of the Palestinian Authority. Shortly before the move of the Israeli embassy to Jerusalem in 2017, the Palestinian leadership cut off communication with the Trump administration, a choice that left them even more isolated amidst burgeoning relations between Israel and the Gulf states.
The Biden administration is entering a delicate and unsustainable environment: A fractured and unpopular Palestinian leadership, an economic crisis, and increasing international isolation has left the Palestinians vulnerable and without any easy options. Aware of this precariousness, the Biden team has announced some steps they will take to rectify the damage the Trump administration wrought to the U.S.-Palestinian relationship, including reinstating U.S. economic and humanitarian aid, reestablishing a U.S. consulate that deals with Palestinian affairs in East Jerusalem, and working to reopen the Palestine Liberation Organization mission in Washington.
Signaling their desire for a reset in U.S.-Palestinian relations, the Palestinian Authority announced a resumption in relations with Israel, which restores the security cooperation, as well as the Palestinian acceptance of tax revenues, which Israel collects on the Palestinian's behalf. Israel promised in return to abide by previously signed agreements.
However, these moves would only represent a return to the status quo ante. While the conditions for a two-state solution remain distant, another avenue the Biden administration could take to compartmentalize its strategic relationship with Israel from the occupation is by advancing a U.S.-Palestinian bilateral relationship, one which does not abut the Israeli-Palestinian conflict but stands on its own two feet.
A component of such a bilateral relationship already exists in the United States Security Coordinator for Israel and the Palestinian Authority. Though limited to the security sphere, this apparatus may serve as a model for a more comprehensive U.S.-Palestinian relationship. Created in 2005, the security coordinator is responsible for enabling security sector assistance and reform to the Palestinians, which includes train-and-equip programs of different elements of the Palestinian security forces, capacity building for the interior ministry, senior leadership training, as well as facilitating cooperation between Israeli and Palestinian forces. The coordinator position is typically held by a three-star general. Though technically confined to the security realm, the coordinator's responsibility extends beyond these limitations as he is often the highest-level American official on the ground. With skin in the game, the coordinator becomes the staunchest advocate for the stability and security of Palestinian society, an issue critical both to Palestinian and Israeli interests.
Embrace Diplomatic Distinction Between Israel and the Settlements Where It Is Missing
This month, Mike Pompeo became the first U.S. Secretary of State to cross the Green Line separating Israel from the occupied territories and make a diplomatic visit to Israeli settlements. Although the occasion seemed personal (a West Bank winery had named a new label after him), it was in fact the culmination of the Trump administration's settlement-friendly policies. In 2019, the State Department officially withdrew its legal finding that settlements violated international law — a legal consensus accepted by most of the world. Shortly after, with the release of the Trump peace plan, a phased final status proposal that was widely viewed as unacceptable to the Palestinians and overly favorable to Israel, the White House effectively granted its blessing to long-term Israeli control of around one-third of the West Bank. While Biden is expected to take the Trump plan off the table, his record and that of his foreign policy team indicate a reluctance to initiate a public disagreement with Israel. Last spring, on a call with donors to Democratic Majority for Israel, a pro-Israel group, Biden's foreign policy adviser and now secretary of state-designate, Antony Blinken, said that a Biden administration would aim for as little "daylight" as possible between the United States and Israel. By reversing course on Trump's blurring of the Green Line, including American recognition of Israeli sovereignty over the Golan Heights, the Biden administration would be taking the first crucial step to separating American priorities from Israel's settlement project.
However, a reversion to the pre-Trump status quo will not be enough. The Obama administration, like its predecessors, too often allowed the U.S.-Israeli relationship to be portrayed as American backing of the occupation. If the Biden administration is to pursue a policy that differentiates Israel from the occupied territories, it should seek to draw additional lines in its bilateral relationship with Israel and in international institutions. For example, the memorandum of understanding detailing U.S. military aid to Israel contains no clause requiring American aid not be used in ways that advance or protect the settlements. While practically difficult to enforce, insisting on such a requirement to make Israel accede to such restrictions — as it regularly does when dealing with Europe — would send a powerful message that America's interests do not cross, and legitimize actions taken across, the Green Line. The new administration should also follow the precedent set by Obama in his final weeks in office by abstaining on non-punitive U.N. Security Council resolutions that concern Israeli activities in the occupied territories.
Withdraw the Trump Plan, But Do Not Replace It with Another American Proposal
In December 2016, then-Secretary of State John Kerry gave a 70-minute speech at the State Department, where he spelled out six major principles for an Israeli-Palestinian final status agreement. The speech provoked pushback in Jerusalem, with Prime Minister Benjamin Netanyahu expressing "deep disappointment," and Palestinians saying it was too narrow. Fast forward to January 2020, with the release of the Trump plan, and you have the Israelis cheering on an American proposal and Palestinians retrenching further into self-isolation.
This dynamic needs to end. Articulating American parameters for a final status agreement amounts to empty rhetoric, is no longer constructive, and will do more harm than good. It makes little sense for the United States to put down terms for an agreement that has no chance of being realized, and doing so risks triggering the ire of uncompromising forces on each side. While Biden should take the Trump plan off the table, he should not replace it with another detailed framework. The position of the United States should be support for a negotiated two-state solution roughly along the pre-1967 borders, but the details can only be determined in negotiations.
Stop Discouraging Other Actors from Giving Mediation a Try
Since the early 1990s, and perhaps even before then, the United States has jealously guarded its role as the sole "honest broker" who can achieve a final status agreement between Israel and the Palestinians. Other countries certainly play a role in bringing the sides together; since becoming the principal financial backer of Hamas, Qatar has worked with Israel to ensure funds reach Gaza to prop up the government there. In the past, Egypt and Jordan have played mediating roles, and it was Norway — not the United States — which hosted the secret talks that led to the Oslo Accords. However, with Israel's encouragement, the United States tends to resist attempts by other international actors to insert themselves on the final status of the conflict. Most recently, the 2016 French Initiative foundered in the face of Israeli opposition and American reluctance to legitimize it.
Beyond putting much helpful distance between the United States and Israel's conflict with the Palestinians, giving space to other potential parties could even advance the cause of peace. The Abraham Accords, which have seen the United Arab Emirates and Bahrain establish normal relations with Israel, carve out such an opening with regional partners. Whereas previously these states were committed to the Arab Peace Initiative — which envisioned peace with the Palestinians before normal relations with the Arab world — they can now play a public and constructive role in future final status talks.
The Upshot: A More Sensible Policy for an Uncertain Future
In 2022, Israel will mark 55 years of occupation of the West Bank and East Jerusalem (it unilaterally withdrew from Gaza in 2005). With little hope on the horizon for a two-state solution, the new administration should chart a path forward for the U.S.-Israeli relationship that will not involve American approval for a status quo in the occupied territories that appears to be largely sustainable for Israel. Although the U.S.-Israeli alliance should not be downgraded or harmed, it is time for the United States to begin distancing itself from a settlement project that serves no discernible American interest, and in fact threatens regional stability by delegitimizing another American partner, the Palestinian Authority.
For Israel, the strategic relationship with the United States will remain a valuable asset. Unlike a few decades ago, Israel today is an economic juggernaut that is not dependent on the United States to survive or defend itself. If it wishes to pursue an ill-advised policy in the occupied territories, it has the capacity to do so without using American aid for that purpose. Compartmentalization efforts are not designed to be coercive and will not disrupt intelligence and defense cooperation, and may be even less likely to trigger the kind of backlash that has previously met active peacemaking efforts.
Similarly, compartmentalization protects the aspects of the U.S.-Israeli relationship that are mutually beneficial. For Palestinians, compartmentalization will mean that America is freed from the diplomatic tug-of-war attempting to bring both sides towards agreement, and can focus on buttressing the many challenges facing the Palestinians, some of which are a consequence of Israeli actions, and some of which are of their own making.
The United States is Israel's ally, not its lawyer, and American policy should reflect that.
Yaël Mizrahi-Arnaud is a research fellow at the Forum for Regional Thinking and a Ph.D. student in history and Jewish studies at New York University. She previously worked in policy research capacities at the Brookings Institution and the Council on Foreign Relations.
Abe Silberstein is a writer and commentator on Israel and U.S.-Israeli relations. His work has previously appeared in The New York Times, Haaretz, The Jerusalem Post, and the Tel Aviv Review of Books. Abe is currently finishing his master's degree in international policy at New York University's Robert F. Wagner Graduate School of Public Service.
Image: Matty Stern/U.S. Embassy Jerusalem


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Connecting-Africa
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Habituation, ecotourism and research for conservation of western gorillas in Central African Republic - Bai Hokou
http://hal.archives-ouvertes.fr/docs/00/48/04/02/PDF/Masi_Gorilla_Gazette_2009.pdf
Masi, Shelly
The south-western region of the Central African Republic reminds one of a paradisiacal habitat, harbouring an exceptional faunal diversity: there are forest elephants, forest buffaloes, sitatungas, bongos, several duiker species, two species of forest pigs, and ten primate species besides gorillas and chimpanzees. As a consequence of the tremendous decline of western gorilla populations in Gabon and Republic of Congo due to Ebola outbreaks, the IUCN recently classified western gorillas as "critically endangered". Under the direction of the Dzanga-Sangha Protected Areas, it consists of 1) the Dzanga-Ndoki National Park, integrally protected, and 2) the Dzanga-Sangha Dense Forest Special Reserve where local people can carry out legal forest activities. Until now Dzanga-Sangha Protected Areas has greatly contributed to the protection of western gorillas and forest elephants, preserving their habitat and supporting ecological and behavioral scientific studies. Gorilla tourism draws national and international attention to problems involved in protecting the ecosystem where gorillas live, and increases support for higher conservation efforts and the livelihood of local people as the case in Rwanda and Uganda. However, developing gorilla ecotourism in Central Africa has been more difficult since western gorillas have been particularly challenging to habituate to human presence, much more so than mountain gorillas. The success of Bai Hokou in habituating western gorillas is also due to rigorous application of rules aimed at minimizing risks of disease transmission from humans to gorillas. The recent success of habituation of western gorillas is starting to gradually fill the gap in our knowledge on western gorilla socioecology. Until recently, information based on direct observation of gorilla behavioral ecology was limited to few, specific habitats, such as forest clearings, or to the well studied population of mountain gorillas. However, mountain and lowland habitats differ dramatically and accordingly the socio-ecology of western and mountain gorillas. Long term research and monitoring of habituated groups of western gorillas are fundamental to increase our understanding of their nutritional and habitat requirements, their life history and their social dynamics. In Bai Hokou four western gorilla groups are currently identified and/or daily followed. Currently we are continuing our research to obtain a complete picture of western gorilla food choice.
[SDV:AEN] Life Sciences/Food and Nutrition; [SDE] Environmental Sciences
non peer-reviewed article
Gorilla Gazette
hal:http://hal.archives-ouvertes.fr/hal-00480402/en/
France - Hyper Article en Ligne (HAL)
Added to C-A: 2010-07-01;10:11:31
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Teens aren't as bad as they seem
Emily Bauman, Staff Reporter
Accusations of bad habits, reckless behavior, and don't forget about everyone's favorite accusation, technology-obsessed, Millennials aged from teen to young adults, often get a bad reputation. Yet, in comparison to other generations, they aren't doing so bad. Teenagers are not doing badly, they are actually doing good.
"Teens are lazy" is a common complaint, but it's not that they're lazy, it's that they are focused on unexpected things.
Rather than individual gain, Millennials tend to invest in organizations that prioritize the greater good more than any previous generation according to the United States Treasury.
Teens want their work to be meaningful, thus the large following of the Black Lives Matters, LGBT community, feminism movements and more.
Today's teens are also making meaningful changes to lifestyles.
According to the CDC, teens in 1991 who were a part of Generation X had a drinking rate of 81.6%. Since then, Millennials have dropped the rate to 63.2%.
The CDC also shows that teens in Generation X had a cigarette smoking rate of 70.1% which Millennials have dropped to 32.3%
Millennials have started promoting healthier lifestyles rather than drinking and partying.
The complaint that Millennials are addicted to technology isn't even something that has been proven. The CDC shows that 42.8% of the previous generation's teens watched three or more hours of television each night, while Millennials decreased this numbers to 24.7%.
It's obvious that in recent years there has been an increase in technology use. But why is that so bad?
Using technology often requires a basic understanding of how it works. The increase in technology has lead to an increase in math, science and computer interests among teens. It's these teens that will one day invent a new product or start a new company.
"A generation ago, we were worried to death about teen pregnancy. Before that, we were panicking about teen drinking and driving," wrote Kyle Smith in The New York Post.
Sparking an interest in technology may mean that kids are staying in more and going out less, and that's not necessarily a bad thing.
Millennials are doing things differently, but that doesn't mean they are doing anything wrong. They are studying new things and entertaining themselves with less dangerous activities while promoting a greater good. Today's teens really aren't that bad.
Required classes: Setting us up for success or failure?
Verbal participation shouldn't determine grades
Iran protests go largely unnoticed by West
Questioning Trump's ability to deliver on campaign promises
Comprehensive sex ed is essential for schools
Real world issues rear-ended by Kardashian's caboose
Hazing isn't harmless
Young voters should become more educated
Spring horror movies fail to scare
Benefits of choosing year round schooling


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Impending Power Plant Closure Means Trying Times Ahead for Coronach
Written by Tanner Wallace-Scribner Monday, Oct 07 2019, 12:51 PM
The Poplar River Coal Mine and Power Plant currently employ approximately 300 people.
The Town of Coronach is entering a stage of uncertainty as the eventual shutdown of the Poplar River Coal Mine and Power Plant looms ahead of them.
Built-in the late 1970s, the plant will reach the end of its lifetime by 2030, leaving approximately 300 people without jobs, and even more affected by the closure.
Catherine MacKay Wilson, Coronach's Town Administrator, explains why the shutdown is taking place.
"It's really more about the lifetime of the plant and its equipment than it is about the coal emissions. But it's up to Sask Power to make a change in terms of an injection of funds in order to change it so that it could keep running in a clean way to meet the emission standards in the future."
A closure would mean a huge shift in the area's economy and population.
"The landowners in the area are very affected by this in terms of income and infrastructure support across the RM. And throughout the town, the local businesses and their jobs will be as well. The dynamics will completely change if we lose 300 jobs between the plant and the mine."
MacKay Wilson says that some of the employees may choose to retire and say in the area but that many will be forced to relocate themselves and their families in order to find work.
"[That many families leaving] would impact the health centre support that we get here as well as our school funding. We're very fortunate to have a local pharmacy and grocery store as well as an amazing sportsplex. So we're trying to prepare to do what we can to not lose those amenities if a lot of people leave."
The potential closure is still pretty far down the road but the MacKay Wilson says the town is working hard on making sure that they are prepared by then. They held town meetings on October 6th and 7th in Coronach, Rockglen, Big Beaver.
The hope for these meetings is to identify priorities for the communities in the area and how they can diversify their economic basis in the upcoming years.
"We're trying to do everything we can...to look forward and be positive about what we can do to develop our tourism and opportunities for small businesses and entrepreneurs. Maybe do some Mainstreet improvements and see if would attract more people. We're a very safe, small, and sweet community and we just have to find a way of telling the bigger Saskatchewan population about that."
The Town has received federal funding to conduct this socio-economic study and MacKay Wilson says this has been a huge help in the transition process.
"I think that it is really important for communities to reach out to the government as much as they can and work as partners to overcome whatever challenges they are facing."
Despite the time of uncertainty ahead of them, MacKay Wilson is still positive that the area can make it through the coming changes and be better for the experience.
"We're really resilient here. This is about revitalizing Coronach and sharing all the amazing things that Cononach has. We're really positive about the opportunities we have been given."
Restrictions on Memorial Drive South Begin Tomorrow


Found 5 item(s) authored in "1979".
Kurt E. Brassel, Robin Fegeas.
Author(s): Arthur Appel, F. James Rohlf, Arthur J. Stein.
Proceedings: Siggraph 79, pp. 151--157, 1979.
The haloed line effect is a technique where when a line in three-dimensional space passes in front of another line, a gap is produced in the projection of the more distant line. The gap is produced as if an opaque halo surrounded the closer line. This method for approximate hidden-line elimination is advantageous because explicit surface equations are not necessary. The relative depth of lines, axes, curves and lettering is easily perceived. This technique is especially suitable for the display of finite element grids, three-dimensional contour maps and ruled surfaces. When the lines or curves on a surface are closer than the gap size, the gaps produced close up to produce a complete hidden-line-elimination. A simple but efficient implementation is described which can be used in the rendering of a variety of three-dimensional situations.