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June 15, 2011 | 11:16 AM
Fontana — Roads plagued with potholes wear out the wheels and shocks of the vehicles that bump along them.
Fontana residents are tired of the road problems, and village officials are well aware of it.
During the Village Board's June night meeting, Trustee George Spadoni asked Public Works Director Craig Workman to obtain quotes to repair what are viewed as the village's four worst thoroughfares — Dewey Avenue, Stearns Road, Lake Street and Shabbona Drive.
Spadoni said the village could bond to repair all the roads and pay for it over time. Spadoni said the cost to repair the roads will add little to the village's tax rate and residents would prefer to pay a little more on their tax bill than have roads in terrible conditions.
Biannually, the heads of municipal public work departments file reports with the Department of Transportation, which rates pavement conditions in their communities.
Workman last filed the form in 2009 and said Shabbona Drive was one of the roads that tops the list of those in need of repair. When he fills out the form at the end of this year, he expects Shabbona, Dewey, Stearns and Lake Street to be the lowest rated roads in the village.
In 2009, several other roads also received low ratings on the report including Dade Avenue, Forest Drive, Mayflower Lane, Mohr Road, Juneau Drive, Pottawatomi Drive, Sylvan Drive, Timber Trail Drive and Waubun Drive.
When rating roads that need repair, Workman also considers two other factors — traffic frequency and the condition of underground utilities. When those two conditions are considered, Workman said Shabbona is clearly in the most need of repair.
The condition of roads isn't a new topic at Village Hall.
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When the village's Public Works Committee approved its 2011 budget, it included fixing Shabbona Road, but that expenditure was eventually sliced.
"We made a big mistake by not budgeting for it," Spadoni said during the June meeting.
Road repairs became an agenda item after residents wrote letters to the board citing concerns about the condition of Stearns Road.
During the June meeting, the board reviewed three quotes to repair Stearns Road, which were between $66,000 and $75,000.
If the board wanted to move forward with just repairing Stearns Road, it would need to put the project out to bid. However, board members said the top road repair priority wasn't Stearns Road.
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"It is bad, but Shabbona is a lot worse," Trustee Cynthia Wilson said.
What each road needs
Workman said repairs to Stearns Road are needed from Highway 67 to the Upper Gardens Road.
Problems with Stearns Road began when Highway 67 was under construction in 2006. During construction, traffic was detoured away from Highway 67 onto Stearns Road, Workman said.
In Workman's 2009 report, the two portions of Stearns Road received 8 and 10 rankings, which is on a scale of 1 to 10. However, the condition of the road has quickly taken a turn for the worse.
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Recently, new homes were built on the road and the heavy truck traffic expedited the deterioration of the street.
Workman said potholes are filled, but that is just a temporary solution.
Repairs to Dewey Street are needed from South Main Street to Well No. 1, Workman said.
Workman said Dewey Avenue and Stearns Road simply need repairs to the street, but Lake Street and Shabbona Drive also need sewer and stormwater replacement.
On Shabbona Drive, repairs are needed from about Sauganash Drive to the Castle Gate entrance. The water main on Shabbona needs to be replaced, Workman said.
Workman said he is seeking clarification from the Village Board on what parts of Lake Street they want included in the a cost estimate. According to a road condition report, two portions of the road, which were last paved in 1982, are in the most need of repair. However, the condition of the portion of Lake Street from Bay View Avenue to Kinzie Avenue was ranked high on the report.
However, Workman said in his opinion, the worst section of the road is near the beach house, which has heavy pedestrian traffic.
"It's not safe for pedestrians," Workman said.
Stormwater and sewer replacement is also needed along Lake Street.
Residents react
Residents near Stearns Road have written irate letters to complain about the street's condition.
"The road is despicable," Donald West wrote to the board. "In some cases, you have to drive on the other side of the road to miss the bumps."
West went on to write that the road is highly traveled by cars, trucks and semis.
"Come on Village Board, come and inspect the road for yourselves," West wrote. "It is as I have described it, a total disaster. This is an emergency and must be attended to now."
Petersen empathized with residents and said he travels on Shabbona at least twice daily and he knows the road is in bad condition.
"I hate Shabbona. It is a mess," Petersen said.
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Anaerobic ammonium-oxidizing (anammox) bacteria perform a key step in global nitrogen cycling. These bacteria make use of an organelle to oxidize ammonia anaerobically to nitrogen (N2) and so contribute ~50% of the nitrogen in the atmosphere. It is currently unknown which proteins constitute the organellar proteome and how anammox bacteria are able to specifically target organellar and cell-envelope proteins to their correct final destinations. Experimental approaches are complicated by the absence of pure cultures and genetic accessibility. However, the genome of the anammox bacterium Candidatus "Kuenenia stuttgartiensis" has recently been sequenced. Here, we make use of these genome data to predict the organellar sub-proteome and address the molecular basis of protein sorting in anammox bacteria.
Two training sets representing organellar (30 proteins) and cell envelope (59 proteins) proteins were constructed based on previous experimental evidence and comparative genomics. Random forest (RF) classifiers trained on these two sets could differentiate between organellar and cell envelope proteins with ~89% accuracy using 400 features consisting of frequencies of two adjacent amino acid combinations. A physicochemically distinct organellar sub-proteome containing 562 proteins was predicted with the best RF classifier. This set included almost all catabolic and respiratory factors encoded in the genome. Apparently, the cytoplasmic membrane performs no catabolic functions. We predict that the Tat-translocation system is located exclusively in the organellar membrane, whereas the Sec-translocation system is located on both the organellar and cytoplasmic membranes. Canonical signal peptides were predicted and validated experimentally, but a specific (N- or C-terminal) signal that could be used for protein targeting to the organelle remained elusive.
A physicochemically distinct organellar sub-proteome was predicted from the genome of the anammox bacterium K. stuttgartiensis. This result provides strong in silico support for the existing experimental evidence for the existence of an organelle in this bacterium, and is an important step forward in unravelling a geochemically relevant case of cytoplasmic differentiation in bacteria. The predicted dual location of the Sec-translocation system and the apparent absence of a specific N- or C-terminal signal in the organellar proteins suggests that additional chaperones may be necessary that act on an as-yet unknown property of the targeted proteins.
Anaerobic ammonium-oxidizing (anammox) bacteria convert ammonium and nitrite into nitrogen and are major players in the biogeochemical nitrogen cycle [1-4]. They comprise a monophyletic taxon within the Planctomycetes phylum. Like other Planctomycetes, they possess an unusual cellular architecture with a diderm cell envelope and a compartmentalized cytoplasm [5,6]. More specifically, the cells of anammox bacteria contain a single organelle-like intracytoplasmic compartment bounded by a single bilayer membrane. This compartment is known as the anammoxosome, and was proposed to be the site at which the anammox reaction takes place [7]. This reaction is thought to be performed mainly by cytochrome c enzymes [8]. Within anammox cells, such enzymes have been shown to be present exclusively inside the anammoxosome [9,10].
If indeed the anammoxosome is a separate compartment in which a distinct and substantial part of the proteome is localized, this would present a situation unique to bacteria. In a thorough electron tomographical study it was reported that, unlike for example the magnetosomes of magnetotactic bacteria [11] and the chlorosomes of green photosynthetic bacteria [12], the anammoxosome has no detectable membrane links with the cell envelope during its biogenesis [13]. Furthermore, anammoxosomes divide separately from the cell envelope during cell division [14].
This leads to two questions regarding the cell biology of anammox bacteria: Firstly, which proteins are targeted to the anammoxosome besides the cytochrome c enzymes? Secondly, by what mechanism are these proteins specifically targeted to the anammoxosome?
One possible answer to the second question is that anammoxosomal proteins might contain specific sorting signals such as targeting motifs, domains or signal peptides [15-19]. For example, in Salmonella, several effectors were reported to contain multifunctional motifs or domains that are responsible for translocation and localization of the effector traits [20]. Moreover, some cases have recently been discovered in which modulation of Sec-signal peptide sequences result in different protein localizations [21,22]. Most strikingly, in cyanobacteria, signal peptides from proteins targeted to the thylakoid differ from signal peptides of proteins targeted to the cell envelope [23-25].
Progress in the experimental investigation of the cell biology of anammox bacteria is slow because these bacteria grow exceptionally slowly (with a doubling time of two weeks), and are not available in pure culture. However, the genome of the anammox bacterium Candidatus "Kuenenia stuttgartiensis" was recently assembled from a community genome [8].
Using these genome sequence data, it might be possible to answer the first question. Interestingly, it has been shown experimentally that the anammoxosome may be more acidic than both the cytoplasm and the cell envelope [26]. We reasoned that such a physicochemical difference could be reflected in the amino acid composition of the anammoxosomal sub-proteome [27], and that this difference could be used to predict this sub-proteome in silico. Therefore, a Random forest (RF) classifier was trained on two sets of anammoxosomal (set A) and cell-envelope (set P) protein sequences, constructed based on existing experimental evidence and comparative genomics. The best RF classifier was successful at predicting the targeting of proteins to the anammoxosome. This approach was complemented by the analysis of the encoded protein translocation machinery. Finally, the predicted signal peptides of the two sub-proteomes were analyzed and compared to unravel the molecular basis of protein sorting in anammox bacteria.
Construction of training sets
We constructed two sets of amino acid sequences from soluble proteins with functions that were known to be specific to either the anammoxosome or the cell envelope (Additional file 1: sheet S1). The anammoxosomal set (termed 'set A') contained the sequences of 30 proteins, including the 26 most highly expressed cytochrome c proteins of K. stuttgartiensis [8,10] and 4 orthologues of these proteins from another anammox bacterium, Candidatus "Scalindua marina" (data kindly provided by M. Jetten and M. Kuypers). The cell-envelope set (termed 'set P') contained the sequences of 59 proteins that were homologous to proteins with an experimentally validated function specific for the periplasm, cell envelope, or extracellular environment. All proteins of both sets contained a predicted N-terminal signal peptide that can putatively be recognized by the Sec translocon. TatFind [28] and PilFind [29] predicted no Tat-system or Type IV system-secreted proteins in these two sets.
Additional file 1. Experimental training sets. The experimental training sets and the K. stuttgartiensis proteome subsets based on signal peptide predictions.
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Training of the Random forest classifier
To detect the overall compositional differences between the anammoxosomal and cell-envelope protein sets, we constructed Random forest (RF) classifiers based on amino acid composition, using set A and set P as the training sets. Among commonly used analytical machine-learning techniques [30-35], the RF algorithm has shown its power in classifying proteins based on noisy amino acid composition [36-40]. To balance class sizes of sets A and P, set P was first randomized into two sets (P1 and P2) to arrive at three equally sized protein sets (see Material and Methods).
In total 3000 three-set (A, P1, P2) RF classifiers were trained, based on different combinations of single or two adjacent amino acids frequencies (average out-of-bag (OOB) error 24.4% with standard deviation of 9.7%). The best-performing RF model for discriminating set A from set P was selected based on the highest accuracy (89%) and the best anammoxosomal protein (set A) recall (90%). This RF model had been made using two-adjacent-amino-acids combination frequencies from full-length protein sequences as the input (Figure 1). The top 10 most important adjacent amino acid residue combinations associated with the recognition of set A were GP, TS, ID, YS, TF, LD, YG, IG, GN and IT. The highest accuracy of RFs trained on other types of input data (with single amino acid frequencies or with other regions of the protein sequences, e.g. signal peptides) ranged from ~75% to 84%, and the recall of anammoxosomal protein sequences ranged from 60% to 83%, respectively (Figure 1). No tests on combinations of more than two amino acids were attempted because this required prohibitively long computation time. During the training process, the RF algorithm chose the most representative features of each class by random bagging with overlaps, which is similar to jack-knife cross validations. Within the training process, RF accurately determines classification error-rate (out-of-bag errors; OOB error) [40-42]. These OOB errors were used as a non-biased indicator of the performance of the classifiers.
Figure 1. Performance comparison of the RF model trained on different types of input data. 500 RF models with randomly generated P1 and P2 sets, to correct for class A and P inbalance, were trained on each of the following 6 types of data: the full-length amino acid sequences, the signal peptides (SP) and the mature protein amino acid sequences, each analyzed with either the residue frequency of single amino acids or the frequency of 2 adjacent amino acids. When the 6 top-performing models of each input type are compared, the model trained with full-length protein sequences with the 2 adjacent amino acids combination shows the highest overall accuracy (89%) and A protein recall (90%).
Identification of translocated and membrane proteins
All translocated proteins and membrane proteins encoded in the genome of K. stuttgartiensis were classified based on the predicted presence of transmembrane helices and/or signal peptides. Prediction of signal peptides was not straightforward because anammox bacteria are evolutionarily only distantly related to proteins of those organisms that were used to train the predictors (e.g. Proteobacteria or Gram-positive bacteria). For this reason, 15 different available signal-peptide prediction algorithms were applied to the open-reading frames predicted for the K. stuttgartiensis genome (Figure 2). Positive predictions were combined into a single majority vote decision for each protein. Among 4663 open-reading frames, 594 membrane proteins and 344 translocated soluble proteins with signal peptides were predicted (Additional file 1: sheet S2). Nine of the signal-peptide carrying proteins were predicted to be Tat-translocated by TatFind, and ten proteins were predicted to be secreted by the Type IV secretion system by PilFind.
Figure 2. Signal peptide predictions on the whole proteome of K. stuttgartiensis. Signal peptide predictions on the whole proteome (4663 proteins) of K. stuttgartiensis by fifteen signal peptide prediction algorithms. The y-axis shows the number of proteins predicted to carry a signal peptide. Abbreviations: G+: predict option of Gram-positive; G-: predict option of Gram-negative; Euk: predict option of Eukaryote. The number of predicted Tat and Type IV prepilin substrates using TatFind and PilFind were nine and ten, respectively.
Experimental evidence was obtained to confirm the signal peptide cleavage sites of some exemplary proteins by protein mass-spectrometry. For seven K. stuttgartiensis mature proteins, including two from set A and one from set P, N-terminally non-tryptic peptides were identified that matched exactly to sequences after a putative SP1-cleavage site at the end of a canonical Sec signal peptide (Figure 3), indicating that the signal peptides of both anammoxosomal and cell-envelope proteins were predicted correctly and are functional.
Figure 3. Experimental validation of signal peptides in the Candidatus Kuenenia stuttgartiensis proteins. Identification of cleavage sites from seven Candidatus Kuenenia stuttgartiensis mature proteins. The peptides that were identified in the tryptic digest are coloured red; their N-terminal sides were non-tryptic. Underlined sequences represent the putative signal peptides, and the putative SPase 1 recognition sites adjacent to the non-tryptic side of the peptides are printed in bold. The left column indicates whether the protein is present in either the A or the P training set.
Prediction of the anammoxosomal sub-proteome
We then used our RF classifier to predict the destination of the 938 translocated and membrane proteins. Of these proteins, approximately 60% (562 proteins) was predicted to be anammoxosomal (Table 1, and Additional file 2: sheet S2) after removal of four predicted type IV secretion system substrates.
Table 1. Composition of the predicted organellar proteome of K. stuttgartiensis.
Additional file 2. Predicted anammoxosomal proteome. Details on the anammoxosomal proteome prediction.
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Importantly, the predicted anammoxosomal sub-proteome formed a functionally consistent and cohesive set. First, the cytochrome c maturation machinery [43] was predicted to be anammoxosomal as 6 out of the 8 encoding genes were predicted to be targeted to the anammoxosome (Additional file 2: sheet S4), consistent with the recent proteomics study of Karlsson et al. [9]. Secondly, the destination of proteins encoded in the same genetic neighbourhood (i.e. putative operons) was generally consistent. This makes sense because such proteins are usually subunits of a protein complex or otherwise functionally associated (Additional file 2: sheet S5). Thirdly, we found that only 15 of the 562 predicted anammoxosomal proteins (2.7%) had SMART or PFAM functional domains [44] (e-value < 0.01) which are on functional grounds incompatible with an anammoxosomal location (Additional file 2: sheet S2).
Finally, the predicted anammoxosomal sub-proteome was consistent with the proposed biological role of the anammoxosome [7,10,45]. If the main catabolism of anammox bacteria takes place in the anammoxosome, the respiratory complexes should be associated with this compartment. Indeed, all 14 major respiratory complexes encoded in the K. stuttgartiensis genome were predicted to reside in the anammoxosomal membrane (Additional file 2: sheet S6). Moreover, three out of four ammonium transporters, all nitrite transporters, and all nitrite/nitrate antiporters were predicted to be anammoxosomal (Additional file 2: sheet S7). In contrast, importers of essential trace elements and amino acids, as well as multidrug-efflux proteins were predicted to be located on the outside of the cells, as expected. A single putative copper ATP transporter was predicted to be anammoxosomal consistent with the anammoxosomal destination of some enzymes dependent on copper (or other metal cations).
Overall, the RF classifier predicts that in anammox bacteria the cytoplasmic membrane is mainly used for transport and that essentially all catabolic functions (the anammox reaction, respiration and ATP synthesis) are associated with the intracytoplasmic organelle.
Mechanism of protein translocation
The next point we addressed is the molecular basis for protein sorting in anammox bacteria. We reasoned that comparison of predicted protein features to those of reference bacteria could provide the first clues to how such a sorting system could function.
Homology searches showed that the typical bacterial protein translocation system components, including the Sec-translocation system (SecYEG, SecA and YidC proteins) [46-48], Tat-translocation system (TatA/B and TatC proteins) [49] and type I [50], II [51] and IV [52] signal peptidases were encoded by the genome of K. stuttgartiensis. All components were present in single gene copy only (Table 2).
Table 2. Protein sorting components encoded in the K. stuttgartiensis genome
The identified components of the Sec- and Tat-systems appeared canonical, except for the presence of a C-terminal FecR domain (PF04773) in TatC. This signal-transducing domain is absent in any other TatC protein identified so far. Topology predictions of the TatC-FecR protein (by Phobius [53], TMHMM [54], and HMMTOP [55]) unanimously showed that the FecR domain is non-cytoplasmic. Because of the uniqueness of such a domain combination, it is tempting to speculate that the FecR-like domain may somehow have a role in the evolutionary solution that has been found by anammox bacteria for protein sorting to the anammoxosome.
Interestingly, the RF classifier results for Sec components were ambiguous (only 3 out of 6 subunits were predicted to be anammoxosomal), whereas the Tat system was predicted to be completely anammoxosomal (Additional file 2: sheet S8). Consistently, 7 out of the 9 Tat-substrates predicted in the K. stuttgartiensis genome were also predicted to be anammoxosomal. These include a multi-copper oxidase SufI (kuste4301), a putative superoxide dismutase (kustd1303), two Rieske subunits of the bc1 complex (kuste3096 and kuste4569), and a few hypothetical proteins. The NarG nitrate reductase subunit is very probably a false positive, as has been noted earlier [56].
In conclusion, the encoded protein translocation machinery shows that no duplication of the Sec- or Tat-systems has taken place to facilitate separate translocation routes, and that both systems may be involved in protein sorting towards the anammoxosome.
The role of signal peptides in protein sorting
Regardless of the protein translocation machinery used, the targeting of proteins to specific subcellular locations is often accomplished by modulation of N- or C-terminal signal peptides, in eukaryotes [15-19] and prokaryotes [23-25] alike. Therefore, we compared the N- and C-termini of the two training sets A and P to identify a possible distinctive property or amino acid motif that could be used to differentiate both sets of protein sequences.
Direct alignment of the signal peptides resulted in alignments which only had high quality (similarity level higher than 30%) in the h-regions (TMH). Therefore, the n-, h-, and c-regions of the signal peptides from the amino acid sequences from both training sets were first extracted manually, and were used to create a series of ungapped alignments which were aligned at: (1) the N-terminus; (2) the h-region start; (3) the c-region start; and (4) the putative cleavage site. The resulting segmental alignments were then joined in the order corresponding to the original signal peptide architecture. The sequence logo [57] of the ungapped alignments of the N-termini illustrates that in general the signal peptides of both sets were canonical type I signal peptides (Figure 4) and most of the proteins had SPase I cleavage sites with a clear AxA motif. These alignments were further investigated by comparing the search results of Hidden Markov Models [58] of both sets in a sliding window approach (Additional file 3), but no discriminating motif was found in either set A or set P. MEME motif searches [59] and statistical analysis of amino acid frequencies also did not yield any significant differences. Finally, we trained RF classifiers using only the N-terminus (30 amino acids) of the two sets of proteins, with the frequencies of two adjacent amino acid combinations as also used for the full protein RF classifiers. The resulting RF classifiers showed much lower accuracy than the models built based on the complete protein sequences, suggesting that less distinctive features were encoded in the N-terminus of these 2 groups of proteins (Figure 1).
Additional file 3. Experimental and computational analysis of the signal peptides. Methodological details of signal peptide mass spectrometry and analysis on signal peptides using motif searches, statistical analysis and HMMs. Supplemental figure S2 and S3 depict the signal peptide predictions by the 15 predictors on the training sets and weblogos of K. stuttgartiensis TMHs showing phenylalanine overrepresentation compared to E. coli.
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Figure 4. Sequence composition of the signal peptides of the anammoxosome and cell envelope protein sets. Weblogos of the signal peptides of protein sets A (anammoxosomal) and P (cell envelope) are shown. Both the hydrophobic h-regions (residues -6 to -17) and the signal peptidase AxA consensus (residues -1 to -3) preceding the cleavage site are clearly visible. The weblogos were created from sequences aligned to the cleavage site, using Weblogo [47].
An interesting side observation was that the h-regions of the predicted signal peptides from K. stuttgartiensis proteins contained significantly more phenylalanine residues (2.23 on average for both training sets) than found in E. coli TMHs (1.64 on average, Additional file 3: Supplemental Figure S3). We speculate that this difference may be related to the affinity of trans-membrane helices to the unique ladderane membranes of anammox bacteria that have an exceptionally high density to prevent diffusion [60].
Yet in conclusion, it is unlikely that the N- or C- terminus plays a role in protein targeting of anammox bacteria.
The anammoxosome of anammox bacteria is one of the best documented cases of organellar biogenesis in Bacteria; electron tomography has shown that this bacterial organelle divides separately from the cytoplasmic membrane, and is not connected to this membrane during a complete cell cycle [14]. Cytochrome c proteins were detected exclusively inside the anammoxosome [10] and an intracytoplasmic pH gradient was shown to exist [28]. Because experimental investigation of anammox bacteria appeared to be difficult and the genome of K. stuttgartiensis has been sequenced recently, an in silico analysis was therefore obviously the next step forwards in unravelling this interesting biological phenomenon.
In the present study a Random forest classifier was trained with two sets of protein sequences. The best RF classifier predicted an organellar sub-proteome of 562 proteins that was internally consistent and made functional sense. The best classifier used two-adjacent-amino-acids combination frequency as the input. According to their grand average of hydropathy (GRAVY) [61] and aliphatic index [62], the anammoxosomal proteins tend to be more hydropathic and more aliphatic (Figure 5). This could be a consequence of the different physico-chemical characteristics (e.g. different pH [26]) inside the anammoxosome. As computing power is likely to increase, the accuracy of the RF classifier may be improved in follow-up studies, e.g. by using combinations of more than 2 amino acids.
Figure 5. Physicochemical differences between anammoxosomal and cell envelope proteins. Two physicochemical parameters are plotted against each other: GRAVY index (grand average of hydropathy) and aliphatic index (relative volume occupied by aliphatic side chains of I, L, V, and A), which can both be calculated from amino acid compositions. These two parameters separate sets A and P into two largely distinct clusters. Purple dots: set P. Blue dots: set A.
The anammoxosomal training set consisted only of amino acid sequences of cytochrome c proteins. It is possible that the classification was biased by the uniqueness of the protein types in this set. However, the cytochrome c protein family is only defined by the presence of a conserved CXXCH heme-binding motif (which we excluded from the RF input data) in an alpha-helical domain. Yet the remainder of the amino acid sequences were vastly variable, some polypeptides even containing regions with different folds or domains [63,64]. In fact, motif searches showed that no conserved sequence patterns could be found in set A except for the heme-binding motif, and the pairwise sequence identities of proteins in this set were all below 80%, with only 11 of them above 50%.
For the prediction of translocated proteins, a combination of 15 existing signal peptide prediction algorithms was used. These 15 signal peptide predictors reported immensely different predictions (Figure 2) on the presence or absence of a signal peptide in K. stuttgartiensis proteins. Considering the fact that the Gram-negative predictors were mainly trained with sequences from Proteobacteria, which are only very distantly related to anammox bacteria, it is highly unlikely that the reported accuracies of these predictors of 91-95% [65-68] can be valid for Kuenenia proteins. Moreover, among all employed algorithms, the SignalP-HMM algorithm trained on eukaryotes showed the highest true-positive rate by predicting signal peptides in 68 out of 69 of the proteins in set A. This indicates that anammox signal peptides are more similar to those of eukaryotes than to those of Gram-negative or Gram-positive model bacteria.
Analysis of the encoded protein translocation machinery provided some clues as to how this bacterium targets translocated proteins to their proper destination. Because this machinery is non-redundant, an additional layer of chaperoning would be required to achieve specificity. Moreover, because no sorting signal was apparent at the N- or C-termini of the proteins, such chaperones could act on the physicochemical characteristics observed in the amino acid sequence. Alternatively, the signal may act at the level of the messenger RNA that could determine the fate of the protein even before translation starts.
In case of the Sec translocase, some subunits (SecE, SecG and YidC) were predicted to be anammoxosomal, while some others (SecY, SecA, SecDF,YajC) were not. According to the presence of the seemingly canonical Sec-signal peptides on both cell-envelope-targeted proteins and anammoxosome-targeted proteins, it is likely that the Sec translocase has a dual localization on both the anammoxosomal and the periplasmic membranes. However, recent studies have also shown that under certain conditions Sec-system exported proteins could as well be translocated by the Tat-system [69,70], and therefore we cannot exclude the possibility that the Tat system translocates more than just the predicted twin-arginine-motif-carrying substrates.
An alternative hypothesis for anammoxosomal targeting could be primary translocation to the periplasm through the Sec pathway and secondary retro-translocation to the anammoxosome (or vice versa) through vesicles. However, it must be noted that the application of 3D electron microscopy (tomography) did not reveal any such vesicles in growing or dividing cells [13].
In order to further elucidate the anammox protein targeting problem, more experiments, such as immunolocalization of the Tat- and Sec-translocase subunits and quantitative proteomics approaches [71,72], comparing protein concentration levels in purified anammox cells and solutions enriched in anammoxosomes (a purification method which has been described earlier [60]), are required. The present study provides a clear hypothesis to future experiments: in anammox bacteria, catabolism and respiration are strictly organellar, leaving only transport functions for the cytoplasmic membrane.
The anammoxosome of anammox bacteria is one of the best documented cases of organellar biogenesis in bacteria. Experiments have shown that several key enzymes catalyzing the anammox reaction are present exclusively inside the anammoxosome. The present study makes use of physicochemical characteristics of predicted protein sequences to predict a 90% accurate sub-proteome that constitutes this bacterial organelle. Meanwhile, the mechanism of protein sorting remained largely elusive.
The predicted sub-proteome has been deposited into a freely accessible Microsoft-Excel database (Additional file 2).
Genome sequence of Candidatus K. stuttgartiensis
The complete predicted proteome of K. stuttgartiensis was compiled from all annotated protein sequences (4663 ORFs, accessed 11-02-2008) encoded by K. stuttgartiensis genome fragments kustA - kustE (GenBank accession nrs.: CT030148, CT573074, CT573073, CT573072, CT573071). The proteome has been deposited in the peptidome database (accession number PSE111).
Sets of putatively anammoxosome- and cell-envelope-targeted proteins
The set of amino acid sequences of putative anammoxosomal proteins ("set A") was first constructed from the cytochrome c proteins with a peptide coverage of more than 10% in an experimental analysis of the K. stuttgartiensis proteome (Kartal et al., unpublished data). Next, local BlastP searches were performed with the Scalindu marina metagenome using these protein sequences as queries in order to extend the set A. Reciprocal best Blast hits with the K. stuttgartiensis genome that share an identical gene context with the set A proteins were identified as orthologues and added to set A. By similar methods a set of amino acid sequences of putative cell-envelope and/or excreted proteins ("set P") was also constructed. This set consisted of proteins from the K. stuttgartiensis genome with high similarity to proteins with a validated function in the periplasm, cell envelope or extracellular environment [73-89].
Predicted integral transmembrane proteins (predicted using Phobius [53], combined with manual inspection) were removed from the sets. The translation start sites of the selected proteins were manually checked and corrected when necessary. Pairwise identities of the sequences from both sets were calculated with MatGAT [90] and redundant protein sequences (with pairwise identity higher than 85%) were removed.
Prediction of signal peptide-carrying proteins in Candidatus K. stuttgartiensis
Signal peptide predictions on the whole K. stuttgartiensis proteome were performed by the Gram-negative, Gram-positive and eukaryote versions of the algorithm PrediSi [67], SignalP-HMM and SignalP-NN [66], Gram-negative and Gram-positive versions of Signal-3L [68] and Signal-CF [65], and the general versions of RPSP [91] and Phobius [53]. All positive predictions from all algorithms were combined into a majority vote decision. Trans-membrane helices (TMH) were predicted by Phobius, TMHMM [54], and HMMTOP [55]. Tat-secreted proteins were identified with TatFind [34]. The same algorithms were also run on all protein sequences of sets A and P (see below). The n-, h-, and c-regions of the predicted signal peptides were determined manually based on expert knowledge with help of the predictions by Phobius and SignalP. No attempt was made to differentiate between signal peptides (which are cleaved by a SPase) and signal anchors (which are not cleaved) for these sets.
Random Forest classification based on mature protein amino acid composition
The Random Forest classifier package (version 4.5-28) [42,92] from the R environment (version 2.8.1) [42,93] was used to train RF classifiers for the separation of anammoxosomal (set A) and cell-envelope (set P) proteins.
In order to reduce the bias due to class size imbalance (larger size of set P), this set was randomly separated into two subsets (set P1 and P2) with sizes similar to that of set A. As input for the RF algorithm, features were determined based on the frequencies (occurrences divided by the sequence length) of amino acids, including the frequencies of two-adjacent-amino-acids, from (i) the SPs, (ii) the full-length amino acid sequences and (iii) the mature protein amino acid sequences of sets A, set P1 and set P2 (except cysteines and histidines, which constitute the heme c binding motif). A three-class (P1, P2, and A) RF model was trained with 1000 trees per forest using each set of input data at each round of P set randomization. The randomization training process was repeated 500 times for each set of input data, after which the votes for classes P1 and P2 were pooled into one merged set P and the overall classification or out-of-bag (OOB) errors were calculated.
The best RF model was selected based on overall accuracy and A protein recall. When a tie situation occurred with the overall accuracy, the model with higher A protein recall was preferred.
Identification of Sec signal peptide cleavage sites by mass-spectrometry
Mass-spectrometry experiments were performed to identify the Sec-signal peptide cleavage sites in proteins from sets A and P. A detailed methodology of these experiments is described in the Additional file 3. The resulting mass spectrometric data files were searched against a database containing the K. stuttgartiensis proteins and known contaminants like human keratins and trypsin using Mascot (Matrix Science Inc., USA, version 2.2) [94]. Variant sequences were modified at the N-terminus by deleting amino acid 1 to 50 and were added to the database in order to search for the Sec-signal peptide cleavage site. The resulting peptide hits were validated using an in-house developed script which selects peptides based on peptide score, the number of variable modifications, the expectation value, and the modified delta score. From the list of validated peptides, a non-redundant N-terminal peptide list was manually extracted by the criteria of: (1), whether the protein was identified with ≥ 3 peptides; (2), whether the peptide was the first detectable peptide (based on calculated m/z values of in silico predicted tryptic peptides in relationship with the m/z detection limits of the mass spectrometer) of the protein; (3), whether the peptide was semi-tryptic (with the non-tryptic side at the N-terminus).
Prediction of the anammoxosomal sub-proteome of K. stuttgartiensis
The Candidatus K. stuttgartiensis proteome was separated into the translocated and non-translocated sets by combining several signal-peptide predictors as follows: firstly, a majority vote of the prediction from 15 signal-peptide prediction algorithms (see above) was used to predict sets of proteins containing SPs (511) and proteins without SPs (4152). Secondly, the proteins without SPs were subdivided into sets of soluble cytoplasmic proteins (3724 proteins, set 4) and SP-less transmembrane proteins (427 proteins, set 3), using predictions of TMHMM 2.0 [54]. Finally the SP-containing proteins were considered subdivided into soluble proteins and proteins containing TMHs, by assessing which proteins had TMHs predicted by a Phobius constrained prediction (constraint: N-terminus = signal peptide). This resulted in a predicted set of 344 SP-containing soluble proteins (set 1) and a predicted set of 167 SP-containing transmembrane proteins (set 2) (Additional file 1: sheet S2). The TMHs (including TMH topology) were predicted by either a constrained Phobius search (for sets 1 and 2) or the TMHMM output (for set 3). Protein sequence composition data of subset 1-3 were then used to predict the anammoxosome proteome.
Identification and sequence analysis of the protein translocation system components in the genome of Candidatus Kuenenia stuttgartiensis
Genes encoding translocation-associated proteins SecY, SecE, SecG, SecA, SecDF, YajC, YidC, TatA, TatC, SPI, SPII, SPIV, FtsY, and Ffh were identified in the K. stuttgartiensis genome by finding reciprocal best Blast hits using BlastP [95] with the well-studied proteins involved in inner membrane translocation in Escherichia coli K12 as queries. Orthologues from S. marina were identified by performing BlastP analysis on a metagenomic database (M. Jetten and M.Kuypers, unpublished results) constructed with a S. marina enrichment culture [96], using the above-identified K. stuttgartiensis protein sequences as a query.
Homologues of the Sec- and Tat- translocation system components from other bacterial species were found by PSI-Blast [97] searches using the Escherichia coli K12 proteins as queries on the GenBank http://www.ncbi.nlm.nih.gov/ webcite genomic data from all bacterial phyla. The K. stuttgartiensis protein translocation-associated proteins were aligned to at least 4 non-planctomycete sequences using Muscle [98].
Analysis of N-terminal signal peptides
Multiple sequence alignments were made by Muscle 3.6 using standard parameters. Motif searches were performed using MEME [59], first with the criterion of zero or one motif per sequence, then with one motif per sequence. Phylogenetic footprinting [99] was performed by aligning the signal peptides of K. stuttgartiensis and S. marina orthologues from set A together with their putative orthologues (based on reciprocal best BlastP hits) from the sequences of planctomycete KSU-1 obtained by Shimamura et al. [100,101]. The protein physicochemical properties, including the prevalence of general amino acid classes (ILV, FWY, AILVMFWYC, AGS, ST, GNP, DE, DN, KR, EQ, DENQ, HKR, DENQHKR, and DENQHKRST), aliphatic index [68], and GRAVY index (grand average of hydropathy, according to the Kyte/Doolittle scale) [61] of the proteins were calculated by custom Python scripts. Statistical analysis on these parameters was performed by calculating the average, standard deviation, and average deviation of the data.
List of Abbreviations
HMM: hidden Markov model; AA: amino acid; RF: random forest; SP: signal peptide; TM: transmembrane; TMH: transmembrane helix; ORF: open reading frame; OOB: out-of-bag error estimate.
Authors' contributions
MM carried out the sequence analysis and training set construction, performed biological interpretation of RF results and carried out in silico signal peptide analyses. MZ trained the Random Forest algorithm and performed the RF predictions. MM and MZ drafted the manuscript. SAFT advised in the Random Forest training. HW and JC carried out the proteomics analysis and supplied methods for the manuscript. MS initially conceived of the study. Both MS and RS participated in its coordination and helped to draft and finalize the manuscript. All authors read and approved the final manuscript.
M Zhou is funded by the BioRange programme of the Netherlands Bioinformatics Centre (NBIC), which is supported by a BSIK grant through the Netherlands Genomics Initiative (NGI). We thank L. van Niftrik, B. Dutilh, H. op den Camp, and A. Pol for stimulating discussions. B. Kartal and K. van de Pas Schoonen are acknowledged for maintaining the anammox enrichment cultures, for performing the protein extraction for proteomics and providing unpublished proteome data. M. Strous is supported by a VIDI grant from NWO. Preliminary genome data of Candidatus "Scalindua marina" were kindly provided by M. Jetten and M. Kuypers.
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subset: commoncrawl


text:
HILLSBORO, Ore. Washington County is publishing what it believes are frivolous 911 calls each week in a new social media campaign called You called 911 for that?!
The No. 1 call this week featured a man frustrated because his debit card did not work at a gas station that was actually closed:
A person called in to report that his debit card wouldn't work at a gas station, which was closed at the time of the call. You called 911 for that?!
The caller said Cantonese (a Chinese dialect) was his main language but he did speak in broken English. He said he recently moved to America. The dispatcher said she would get a translator on the line but then a gas station worker arrived and helped.
The Washington County Consolidated Communications Agency said it has a limited number of 911 phone lines and circulated a press release Wednesday which stated that it's critical that those lines are kept available to those that have actual emergencies.
The agency hopes that by highlighting these calls it can help educate the public and direct people to the non-emergency numbers.
The campaign prompted some lively comments on the WCCCA Facebook page, where one commenter thought the effort wasn't well thought out.
Cindy Davidson commented:
Don't think you are just asking for people to call you with ridiculous calls so they can get chosen for the week? Also, how exactly does shaming people on a Facebook page, that most people don't know exists, help reduce the calls?
The runner up this week was a woman who called 911 because a teenager was smoking on the MAX platform. The platform was a No Smoking Area she says. She was concerned about having an allergy attack. She believed that smoking on the platform was a felony.
The campaign has also gotten lively comments on the KGW Facebook page as well. Some commenters have said the callers shouldn't have dialed 911. Others say it's not appropriate to single out someone who doesn't speak English well.
Join the conversation on our Facebook page
WCCAPerformance Manager Mark Chandler said the dispatch center serves a diverse community and deals with language barriers all the time. Chandler said the caller was able to clearly say that he was having trouble pumping gas. Chandler went on to say that having problems with pumping gas was not an appropriate use of 911.
Chandler said that the campaign's purpose was to remind the public that 9-1-1 should be used for police, fire or medical emergencies when life or property is in jeopardy and immediate assistance is necessary.
Chandler release more top contenders for the pick of the week to KGW Wednesday afternoon.
The police non-emergency numbers are:
- Washington County - 503-629-0111
- Multnomah County - 503-823-3333
- Clackamas County - 503-655-8211
- Clark County- (360) 397-2211
Additional calls released by WCCCAbelow
Man loses cat
A man calls to make a civil rights complaint against an officer


text:
Do you like this article?
|Posted by:
|Garima M.
|User Rating
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|Phrozen, shivandhni, wassup28, MamtaB, tazeen_29, CrystalSaya, Fattoom, minaalM, Nikhi., Yuvika_15, luvkasu, ILoveDrama, wadi..doll, sadffi, iMadz, samicute, pop77, Fatima_Q, Mariaa.RD, Craziest_MG, oblivious., -Eris-, Roopal_ArDi, star_fan
well whateva may be d reason behind them not talking , but i guess FA now knows who is ruling Bollywood:p
SK all d way, luv u heaps..
their dad's were work partners 'Salim Javed' whle salman's mom was a hindu i think and farhan's mom is parsi Honey Irani...I don't think they are cousins...Farhan and Farah are cousins...bt im happy they are getting along as their fathers are not on talking terms...as for d 'rumour this' rumour that', its all rubbish...no concrete source..can't they just have fun without an ulterior motive?? stop cooking up stories guys.
are they cousins? This is a NEWS to me.. :S! newways i am happy that slaman and farhaan are coming toghther..! love salman khan and also admire farhaan akhtar
They are cousins? I dint know tat . I hope he writer somethin specially for salman
I wish they both become friends again!!!!!!! Wnt 2 C dem 2gether again...lov u Salman!!!!!
So what if Farhan attended Salman's b'day party...??
So what if they are planning to work together...??
Why does IF have to spice up such silly things ?
Com'on yaar it was Salman's b'day...atleast keep his b'day party away from such nonsense rumours.
i wish salman and srk become friends.want to see them together.
What Farhan has to do with SRK and Salman stupid school fight? Lolz
Of course, he would go if he is invited! :))
Edited on:03 January 2012 at 5:43am
Really wish that they become friends again...!!!!!!!!!!!!
r they guys counis????????????????????????????happy to know
Food lover Nirbhay Wadhwa speaks about his food choices and much more...
Rajeev in conversation with us talks how television has become strong ...
Over 3000 Entries in Less than 24 Hours for the 'Bajrangi Bhaijaan' ... 2
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New Engagement!
Noach Evers (Amersfoort, Holland) and Devorah Leah Lubin (Los Angeles/London)
Noach Evers (Amersfoort, Holland) and Devorah Leah Lubin (Los Angeles/London)
Jewish members of the U.S. Military put on Tefilin and say a prayer with the assistance of several Lubavitch Chassidim, circa early 1970s. Can you identify anyone in the photo?
Gliding down the Seine under a soft drizzle, the lone flame of the traditional Havdalah candle seemed to illuminate not just the boat, but the entire "City of Light." Havdalah is the ceremony marking the end of Shabbat, and for the 500 young Jewish participants who traveled together to Paris, Berlin and the grounds of Auschwitz in Poland as part of Chabad-Lubavitch in Russia's "Eurostars" program, the burning torch's symbolism was apt.
Zalman and Mushka (nee Wolowik) Korf (Crown Heights)
Hundreds gathered for a rally Friday morning at Pride of Lynn Cemetery, where a Holocaust memorial was discovered desecrated with pieces of raw pork earlier this week. "We stand united against all forms of evil," said Rabbi Yossi Lipsker of Chabad Lubavitch of the North Shore, who organized the ceremony reconsecrating the memorial. "We gather here, Jews, Christians, Muslims, and people of all faiths."
8,000 bags – each containing a water bottle and muffin – were distributed to weary Jerusalemites who walked to the Kotel to spend the first night of Shavuos studying Torah.
With sadness we inform you of the passing this past Friday of Reb Sholom Dovber Alperowitz, OBM, in Los Angeles, CA. He was 84 years old.
Children's entertainer Chaim Fogelman and musician Fitche Benshimon put the finishing touches to 'Mitzvah Boulevard – Songs,' which will be released this week, G-d willing.
Levi and Tany (nee Rosenberg) Swimmer (Johannesburg, South Africa)
A week before Shavuos, brothers Yair, Nechemia and Rachamim Ladayev, all three active members of the Chabad community in France, arrived in Zhitomir in western Ukraine to facilitate the distribution of Tefilin and Mezuzos.
Two Jewish teenagers were moderately wounded in a stabbing attack on the first night of Shavuos (Motzei Shabbos) on their way to the Kotel. The attack occurred in the area of Sha'ar Shechem in the old city of Jerusalem.
CrownHeights.info and Lubavitch Archives present a unique photo of the Rebbe getting an Aliyah in 1987.
Yitzy and Miriam (nee Eber) Seabag (Passaic, NJ)
Yossi and Chanie (nee Bruchstat) Cousin (Crown Heights)
Levi and Chaya (nee Tifenbrun) Levitin (Crown Heights)
Aron Meir and Rosy Nechama (nee Michaan) Shloush (S. Paulo, Brazil)
This year we celebrate the 60-year anniversary of the first Tahalucha, which took place on Shvuos 5715/1955.
Alternate side parking (street cleaning) regulations will be suspended on Monday, May 25, for Shavuos. Parking meter regulations are also suspended due to Memorial Day.
On the first night of Shavuos at the Besht, three renowned lecturers will lead discussions in Torah: 12:15am – Rabbi Chaim Schochet – How do we integrate into our lives the Rebbe's bracha of kabolas hatorah bsimcha ub'pnimiyus? 1:15am – Rabbi Sholom Dovid Geisinsky – The significance of Shavous in a Shnas Hashmita. 2:15am – Rabbi Zalman Goldberg – Matan Torah for Egyptians.
On Sunday, the first day of Shavuos, there will be various minyonim for Krias Hatorah – Aseres Hadibros around Crown Heights, which were arranged for women and children who could not make it to Shul during Shachris.
Mendy and Sarah (nee Glick) Korf (Crown Heights)
The Supreme Court of the State of Michigan on Wednesday handed down a ruling in which they dismissed a lawsuit brought by the head Shliach of the state, Rabbi Berel Shemtov, against the Chabad Torah Center in West Bloomfield, led by Rabbi Elimelech Silberberg.


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Welcome to Clark's Trading Post
Home of Clark's Trained Bears and the White Mountain Central RR
Tickets include all rides, all shows, all day!
- Our gift shop opens May 16. The park opens weekends May 23-June 14.
- Daily entertainment starts June 19.
- See 2015 season calendar for details.
We can BEARLY wait to see you at Clark's!
The Clark Family invites you to enjoy a wonderful day of Family Fun. Where else can you watch a famous Black Bear Show, ride a Steam Train, be chased by the Infamous Wolfman, and be amazed by Chinese Acrobats - all in the same day!
You can cool off on the Water Blaster Boats, scale the walls of the Old Man Climbing Tower and learn to glide on a Segway. Merlin will turn your world upside-down and the Tuttle House tour will keep you wondering, "What just happened?!?" You can dress up for an antique-style photo and mine for gemstones and fossils.
There are five museums to enjoy on the Victorian Main Street and four Specialty Shops to browse where you are sure to find a treasure or two. Explore our website and see all the excitement that awaits your next visit. We can BEARLY wait to see you!!


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Email: firstname.lastname@example.com | Twitter: @WWLTV
NEW ORLEANS -- The search is currently on for suspects behind several violent crimes in both Orleans and Jefferson parishes over the weekend.
The New Orleans Police Department and the Jefferson Parish Sheriff's Office had their hands busy after four people were killed, including a pregnant woman, her unborn child, and a 15-year-old boy, Corey Martin.
In Orleans Parish, a 17-year-old male was shot multiple times in the 1800 block of St. Roch Avenue. Police say the shooting happened around 6:45 p.m. Sunday, and that the unidentified teen was taken to the hospital and is currently in critical condition.
New Orleans police are also investigating the murder of a 15-year-old Martin in the 9th Ward. It happened around 7:15 p.m. Sunday in the 1400 block of Bartholomew Street. Police say he was shot multiple times and died on the scene.
These two latest shooting incidents come on the heels of a very successful recruitment fair for the New Orleans Police Department over the weekend, where dozens of people showed up to learn more about the police department, and how to become a member.
View New Orleans Murder Map - 2014 in a larger map
Superintendent Ronal Serpas, Mayor Mitch Landrieu, and other police and city officials also attended Saturday's event. The NOPD's goal is to reach 1,575 officers.
In Jefferson Parish, deputies are investigating the death of an unidentified man, as well as the death of a pregnant woman and her unborn baby, both of which occurred very early Sunday morning on the Westbank.
Jefferson Parish Sheriff's Office is desperate for clues in a homicide that claimed the life of pregnant woman and her baby. Deputies say the murder happened in the 600 block of Whitney Avenue in Gretna. JPSO officials say they received a call shortly before 1 a.m. Sunday morning, from a man who found 23-year-old Katherine Martinez dead in her apartment. She was apparently stabbed to death and deputies say that neither she, nor her baby were able to be saved.
Then just blocks away, a man was found shot to death near the parking lot of an apartment complex in the 300 block of Ruby Street late Saturday night. Deputies say he was shot multiple times. They do not believe this shooting was related to the death of Martinez and her baby.
Both NOPD and JPSO are asking for the public's help Monday in these incidents over the weekend. If you have any information that can help investigators in either parish, you are urged to call Crimestoppers at (504) 822-1111.
View Jefferson Parish Murder Map -- 2014 in a larger map


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From the article: Crochet Myths and Misconceptions
Have you encountered any myths or misconceptions about crochet? For those of you who crochet in public, what's the most aggravating thing anyone's ever said about your crochet? Also, what's your response to this myth, misconception or aggravating comment?
You Just Never Know
- I first learned to knot, but just couldn't get the needles to work for me, so I learned to crochet. My grandmother taught me both. I did a lot of crochet in high school and one of my teachers asked me to help host a crochet after school club. It was one of the most participated clubs that we had. We had most of the cheerleaders in the group! After I raised my family and had the time again, I started crocheting again and have a group of people where I live in a twice a week group. Crocheting just brings out the best in people and it's a needle art that will never die.
- —Guest DCMerkle
- best comment and i have had many was at age 12 when crocheting in the park with some older friends was and this was over heard "Poor thing,If they keep teaching her that old lady stuff she'll never get married."
- —Guest Mandy
Not Irritating Responses
- While I sell my handcrafts, I crochet. A lot of people will ask questions or make comments as a way to start a conversation. There are a lot of lonely people out there.
- —Guest Glenda
Nope, not the same.
- It irritated me to no end when this one guy told me I was stuck up because I corrected people who asked what I was knitting. I couldn't understand his attitude. It's not that I think less of knitting, I actually have learned to knit since the last I saw this guy, I just figure that if someone is interested enough to ask, they might be interested in knowing the accurate term for the activity.
- —Guest Cardea
myth conceptions - irritating response
- I am in my 30s and my mum taught me to crochet when I was preg with my first child and i've been doing it ever since. Although I am Canadian I am now settled in Ireland and there is a huge uptake in the art here at the minute. Groups are popping up everywhere it's great to see. My daughter is 12 and has been at it for a good few years now, she takes great delight in making stuff for all her wee friends at school she found knitting too ackward (my husbands aunt tried to teach her) but really loves to crochet. It is most def a craft for everyone!
- —Guest Mikki
Aggravating comments
- I agree with those who mentioned folks expecting us to crochet great stuff for free or just the price of the yarn. I just finished twin blankets, caps and flip flops for babies expected in Florida, and others want sets, too... for nothing. I don't think so. This was a commissioned project for a relative, with yarn cost and something extra; I do not want to make more, especially for nothing or a bare minimum for yarn. Also, I quit crocheting on city buses because people wanted to talk; I wanted to work on my projects, since I crochet to calm my nerves. The worst was one lady who makes beaded bracelets and wanted to trade 2 bracelets for one bottle-holder, when I said I'd make one for $10. I don't want her bracelets. She said she sells them for $5; so sell 2 and commission the bottle-holder!
- —Guest Runeshadow
Crocheting is first alphabetically
- My Library has had a group, called People who knit, when I crochet there new visitors are surprised and say, I would have come sooner if I knew crochet was allowed. Now that we are called the Twisted Stitchers, the library is stalling at giving us space twice a month. No one else want the space we want, and other nearby libraries host Crocheting and Knitting every week. But we are going thru heck getting a room twice a month.
- —Guest patrogers
- yeh i crochet in public but it don't bother me they watch how fast i am and yes ask what are u making most times they say ohh can u make me one lol
- —Guest judy
most annoying
- I find the two most annoying things to be 'what are you knitting?' and 'X needs one, how about making x one?' Sorry, I am NOT knitting and secondly, if x wants one THAT bad, x can buy it. Its not a cheap craft, and custom orders definitely aren't going to be cheaper! You are paying for my time!
- —Guest hookysticks
myths, misconceptions about crochet
- The most aggravating comment is "what are you making with that needle?" I thought it was pretty obvious I was using a hook. The next most aggravating is "Would you make me one like that? I'll pay you for it." Sure, this one took me 50 hours to make so if my time is worth $5hr it will only cost you $250 plus the cost of the yarn. "But I can get one for $50 at Macy's!" Fine, go buy it.
- —Guest BL DeBushe
Conversation Opener
- I have been approached by other women who crochet, asking what I'm making and where can they get the pattern. It's always nice to exchange a few words. And yes, there are those who don't seem to comprehend that there is a difference between crocheting and knitting. I've pretty much given up on explaining the difference, especially when I perceive that "Whatever, it's all the same" attitude. MY attitude is: "Whatever, YOU are obviously not observant!"
- —Guest Anniem
Make me something!
- I get the same thing when I crochet in public. I picked up crochet at my mom and both grandmother's knees, watching them. I started when I was 5 and I am now 51, so I can go pretty fast when working a familiar pattern. People will always comment on that speed and then ask who I am making whatever for. On hearing my reply, they -quite often- suddenly seem to decide they would like a for themselves or their mom or sister's new baby... then they ask the deadly question. How much would x be? When I start describing the options to them for this item, they invariably pick something that is an expert-level pattern with some exceptionally expensive yarn... and then they think I should sell it to them for 10 bucks. "But you just do this for a HOBBY don't you?" -- drives me nuts. If I was going to do this to donate my time and materials-money, it would be for a worthy charity, not someone who just decided to walk up and invade my privacy.
- —Guest Charmain
I could never do that!
- I am almost always told how clever I am. Then I am told "oh, I could never learn to do that!" If I offer to teach them they always say no. I have also told people that crocheting is much easier than knitting (I think so, anyway), and if they had a bad experience with knitting then crocheting might be just the craft for them. I guess nobody ever tells me I look to young because I'm not :-).
- —Guest mardou2
- I'm not sure where these people are you are 'attracting', but everyone that has commented on my knitting or crocheting has been very sweet. Most wanting to know how or things like: How long have I been doing it, or if anyone can learn. I tell them one of my son's crochets and my two if my granddaughters, ages 7, 9. I have a 14 year old granddaughter, and a 16 year old grandson that is learning as well. They also like to sew.
- —Guest Peggy
aggravating comments I hear
- " They usually ask "What are you making/doing?. Then they turn to their friends and say "I don't like to sit and stitch like this for long, who has the patience and time for this. " They also ask if I have help at home to spend time to make/do something like this - in short they think I have lot of time to waste.
- —Guest queenie vaz
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Extraordinary Garage Makeovers
See how the Garage Mahal team transforms ordinary garages into new and improved spaces.
Get Social With Us
We love to DIY. You love to DIY. Let's get together.More DIY Social
Discover Made + Remade
See the latest DIY projects, catch up on trends and meet more cool people who love to create.Make It. Fix It. Learn It. Find It.
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- April
- Spring Home Maintenance Guide
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Welcome to the
Nashua Soup Kitchen & Shelter
We welcome you to come volunteer!
We welcome your donations from our Wish List.
We welcome your financial contributions.
We welcome you to come visit us.
Schedule a tour, see what we're all about.
We welcome you to become part of the
Nashua Soup Kitchen & Shelter in 2015!
Race results......
for our 22nd Annual
Nashua Soup Kitchen & Shelter
Run/Walk for Food & Shelter
sponsored by Toyota of Nashua
Volunteers Needed
We do have Saturday mornings
(7:30AM - 11:30AM) available!
To see our v
olunteer times and descriptions
Click here
The Nashua Soup Kitchen & Shelter
has moved to our new location!
Please visit us at
2 Quincy Street, Nashua
our phone number
is still 603-889-7770
We'd like to share a link to a fantastic Telegraph Editorial regarding a recent event held at our new facility!
Please click here to read.
Did you know you can make a donation
to honor someone special......
or to remember someone
who has passed away?
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The NSKS serves the Greater Nashua area. Community support is critical to the NSKS. Over 70% of the funds for operating programs for those in need come from private contributions. We are grateful for your support.
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We are excited that you are considering a career with Wilmington College! Wilmington College is a career-oriented liberal arts institution affiliated with the Society of Friends (Quakers). Our beautiful main campus is located in southwestern Ohio. We offer a comprehensive compensation and benefits package for full time positions including group medical, dental, vision, life and disability, TIAA-CREF, tuition remission, paid vacation and holidays.
We encourage you to review our website and learn about our history, what we are doing today and how we are preparing for tomorrow. It is an exciting time here at Wilmington College.
Interested applicants should submit a detailed cover letter and resume unless otherwise indicated in the position information below. Please specify position of interest in the letter of application and subject line of email (if submitting electronically). For positions requiring degrees, unofficial transcripts of college work are helpful. Submit materials to the Director of Human Resources, Wilmington College, Pyle Center 1187, 1870 Quaker Way, Wilmington, OH 45177-2499 or email to email@example.com or follow instructions in the position information below.
Wilmington College is an equal opportunity employer and will not discriminate unlawfully in employment matters on the basis of race, religion, gender, color, ancestry, national origin, age, disability or any other category protected by law. EOE
Wilmington College conducts background investigations including criminal, work history and credential confirmation on all employees as a condition of continued employment. Some positions may require additional motor vehicle record review and drug screening.
Wilmington College invites applications for a tenure-track Assistant Professor of Accounting faculty position to teach undergraduate courses effective August 2015.
Teaching responsibilities will include both introductory and upper level courses in accounting and finance. Other responsibilities will include advising majors, contributing to the development of new programs/courses, and serving on college committees. Faculty also interact with students as mentors on research projects and by meeting with prospective students.
Wilmington College's "hands-on learning, hands-on living" motto prepares students to succeed in the accounting profession and/or business community. Therefore, we seek to hire faculty with experience in the accounting and/or finance fields, who will give the students hands-on experience in the classroom, and those who desire to and who are committed to supporting our students as they prepare for a career in accounting. Commitment to high-quality instruction in a student-centered environment is expected.
Ph.D. in Accounting or Finance required for tenure-track, master's with CPA required, (master's only candidates will be considered for non-tenure track) with at least three years of professional experience. The position requires evidence of effective teaching, professional development, and a commitment to service.
Wilmington College is a career-oriented liberal arts institution affiliated with the Society of Friends (Quakers). The college is located in southwestern Ohio and its enrollment consists of nearly 1200 traditional and non-traditional students. Additional information about Wilmington College can be found at www.wilmington.edu. Send resume, letter of application, and names and contact information for three references to: Wilmington College, Director of Human Resources, Pyle Center Box 1187, 1870 Quaker Way, Wilmington, Ohio, 45177-2499. E-mail submissions to firstname.lastname@example.com are encouraged. Review of applications will begin immediately and continue until position is filled. EOE.
Wilmington College (Wilmington, Ohio)
Wilmington College invites applications from, and nominations of, qualified candidates for the position of Director, S. Arthur Watson Library. The Director reports to the Vice President for Academic Affairs and supports the Main Campus as well as academic programs in Cincinnati. The successful candidate will demonstrate a 21st-century vision for leadership in the faculty/student focused environment of the private college academic library. The Director works to continually improve the integration of the Library into the academic life of the college, its faculty and students, aligning services and collections with the curriculum. The Director has overall responsibility for the collections, services, programs, policies and procedures, assessments/reports, staff development/evaluation, and the learning environment of the library and associated media center.
Watson Library currently manages collections through a shared Innovative Interfaces Sierra ILS and participates in campus online personnel and budget management online systems. Watson Library houses the College Archive and Special Collections (Society of Friends historical collections and records deposited with the library by the Wilmington Yearly Meeting and the Ohio Valley Yearly Meeting). These unique collections contain rare documents, photographs, books and maps related to local and Quaker history.
The successful candidate will effectively communicate a vision for leveraging and promoting current member relationships with the Ohio Private Academic Libraries (OPAL) and OhioLINK consortia, integrating emerging technologies, building cooperative campus partnerships that enhance the teaching/learning environment as well as programming opportunities, and promoting and enhancing digital collections.
Qualifications and Characteristics of the Successful Candidate:
Required: MLS from an ALA accredited program; five years of progressively responsible leadership and supervisory experience in a library; proven ability to manage budgets and projects; excellent written, oral, and online communication; proficiency in the use of general MSOffice products and standard library online applications. Important: Commitment to team-development and collaboration at the departmental, college, and consortium levels; demonstrated knowledge of current practices and trends in information research in the disciplines, collection development, information technology, scholarly communication, copyright in academic settings, and the design of learning environments (online and in-house); analytic, strategic and innovative thinking skills; creative, engaged and committed to outreach. Includes evening and some weekend hours; some in-state travel.
Wilmington College is a career-oriented liberal arts institution affiliated with the Society of Friends (Quakers). The main campus is located in southwestern Ohio; two additional locations serve adult learners in the greater Cincinnati area. Information about Wilmington College may be found at http://www.wilmington.edu/.
Send resume, letter of application, and names and contact information for three references to the Director of Human Resources, 1870 Quaker Way, Pyle Center 1187, Wilmington, Ohio, 45177. E-mail submissions to email@example.com are encouraged. Review of applications will begin immediately and continue until position is filled. EOE
Description: Reporting to the Vice President for College Advancement, the Director of Planned Giving will be a member of a team of dynamic professionals responsible for development and aggressive implementation of a strategy to increase the number of planned and major gifts. Responsible for identifying, cultivating, soliciting and stewarding planned and major gifts for Wilmington College. Working closely with the major gifts team, providing vision and leadership in the development and implementation of a planned giving program. Position requires making personal contact with college donors and prospects. The successful candidate will have knowledge of deferred gift vehicles and be able to communicate with confidence appropriate options to prospective donors. Manage and expand a portfolio of planned and major gift prospects. Knowledge of Microsoft Word, Excel, PowerPoint, and planned giving software is a must. Willingness to travel a must.
Requires a Bachelor's degree and at least 3 years experience in planned giving. Experience in higher education fundraising and development preferred.
Contact Info: Please send your letter of application specifying your position interest, resume and list of three references to: Director of Human Resources, Wilmington College, 1870 Quaker Way, Box 1187, Wilmington, OH 45177. Email submission of application materials to firstname.lastname@example.com is encouraged.
Wilmington College is a career-oriented liberal arts institution affiliated with the Religious Society of Friends (Quakers). The main campus of Wilmington College is located in southwestern Ohio, while two additional locations serve adult learners in the greater Cincinnati area. Additional information about Wilmington College may be found at www.wilmington.edu/. EOE.
There are no support staff positions available at this time.


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Ship Upgrades
Do you need to always upgrade to a bigger ship?
If so...would purchasing a Commander rank ship from the Z store be a waste of ZEN?
Since you will need to "discard" it once you get to rank Captain or higher...
...or am I missing something here?
The only reasons to buy one is you want an advantage for the short time you'll be in that rank, have a particularly favored ship, or want a discount on the fleet version later on.
Would it be "certain death" to use a Commander Rank ship till you hit level 50?
Yes, it pretty much would unless you already know the game. You're missing out on a lot of HP and Boff capabilities. The Heavy Cruiser (for example) has less hull than the Reconnaissance Science Vessel (the weakest Rear Admiral ship, hull-wise), and won't allow you to take advantage of your Commander-grade officer powers.
Also, you do NOT get discounts for fleet retrofits if you purchase a lower-tier version (the Commander Thunderchild does not give you a discount on the Fleet Akira, for example).
There is a small advantage in grabbing the lower-tier/rank C-Store ships, in that whatever console that comes with it can be taking off and put on the higher rank ships. The Thunderchild's Point Defense console is very handy, particularly when leveling a character, being able to put a lot hurt in several directions. The ship itself can be used for maybe one rank higher if you keep it well geared, but not beyond that.
A commander rank ship CAN survive in mission play (episodes NOT STFS ) all the way up to Admiral
Do not turn up in one for PVP or STF at Ranks above captain you WILL die
but if you only run missions
no problem
I have used the Thinderchild Cmdr ship in nm STFs and survived.you can use the Excelsior I imagine as well.
MOST Cmdr rank ships won't survive in ESTFs. The Borg just do too much damage. One of the only ships I would dare even try it in is the Cmdr Excel, since it's pretty tanky, but tbh, I hate that ship, so I will stick to my Oddy and Chel'gret thanks much.
In normal STFs, you can win in a Type 8 Shuttle. That's how pitiful the enemies are. Also normals are facerolls requiring very little brainpower and only mk VI weapons. Basically Normal STFs are a joke, and should NEVER be used as a test on what works and what doesn't. And anyone who sticks to just normals shouldn't post on what works and what doesn't.
Is there a reason to grab Cmdr level ships from the C-store? YES. The unique consoles they come with are sometimes VERY awesome (especially on the KDF side). Other than that though, not really. You're better off getting a VA ship.
And for the record, you only get a discount on a fleet ship if you have the VA c-store ship, NOT the Cmdr level one.
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Helen L Mulholland
Chatham, MA
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Joseph John Brozina killed by falling tree during Wednesday's stormComment
A Fauquier County man, Joseph John Brozina, 52, was working to clear damaged trees from his Midland home when he was hit in the head by one. He was pronounced dead on the scene.
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