text:
So this isn't a bad idea that I came up with after reading a comment from Blueleaf34.
This page will be for anyone who wants to post a link to their latest Stick Nodes animation, artwork, or even your Youtube channel. Everyone is encouraged to comment on others' work and post their own stuff as well.
Keep top-level comments dedicated to only showing off your stuff – be sure every top-level comment contains some sort of link to your work, sub-comments can be the typical lol-nonsense.
Pro-tip: Actually use the "Website" field when you leave a comment! It will make your name clickable so people can check out your Youtube channel, website, etc…


text:
In the US: All episodes available on Hulu
We should probably be giving Shut Eye a medal, since it's doing such a public service - revealing all the tricks of the trade used by psychics to fleece their customers. But good thoughts alone aren't enough to make a good TV programme, so unfortunately for Shut Eye, we have to evaluate it on when it's watchable or not.
The first episode set the scene pretty well, with star Jeffrey Donovan playing a former Las Vegas magician now working as a fake psychic in LA under the purview of a bunch of Gypsies, including Isabella Rossellini. Well versed in the arts of cold reading and setting people up, one day he gets a bump on the noggin from a client's disgruntled boyfriend and winds up having proper psychic visions. Will he use his new powers for good or for evil, we wonder at the end of the episode?
Evil, it turns out. Didn't see that coming, did you?
The casting of Donovan as the lead is a genius move, since he's able to recycle two of his old routines for the role. In episode two, the show becomes full on psychic Burn Notice, with Donovan giving us (and his mark) the rundown on the mystic art of psychically stealing people's money. By episode three, he's mining Touching Evil for sympathetic, dazed, brain-damaged and odd, as he starts using his new found powers to tell people the hard truths they probably don't want to hear.
As you might have deduced from that run-down, Shut Eye is as odd a show as its lead character, since it is by turns comedic and then deeply serious and violent. More problematically, it keeps piling more and more details onto to the plot, almost in an apparent attempt to confuse us while it steals our watches. As well as the Gypsies and their bizarre activities - including poetry recitals and love ceremonies - there's Dexter's David Zayas as a gang boss customer of Donovan, who's as quick to throw someone in a deep fat fryer as he is to fix Donovan's floorboards. There's Donovan's hard-edged wife, KaDee Strickland, who wants him to regain his former manhood while she's simultaneously sleeping with another woman. There's Donovan's son, his supposed ADHD and his school issues. There's The Wire's Sonja Sohn as a police officer who's chasing after Donovan. There's thirtysomething's Mel Harris as Donovan's main mark, who sometimes wakes up with a rooster and a tree branch in her bed. There's even a kooky doctor - Susan Misner (Billions, The Americans) - trying to help unclog Donovan's subconsciousness using Mozart and drugs.
And so on.
It makes for a show that says an awful lot without really taking the time to say anything worthwhile, not even about fake psychics because they might be real, it turns out.
I probably won't be bothering with the rest of Shut Eye, despite its funnier and more psychedelic qualities. Donovan's worth his enormous salary for this gig, but the gig itself could probably have done with a rethink about exactly what story it wanted to tell.
Barrometer rating: 4
TMINE's prediction: Unlikely to get a second season

subset: commoncrawl


text:
Extra practice makes for a perfect cricketing duel
BEFORE the Delhi Test, Sachin Tendulkar spent hours in the nets facing a shaved and taped tennis ball fired at him by the coach. Concerned about his technique against swinging and rising deliveries, and aware that the pitch was likely to be faster than forecast, he wanted to be prepared. To that end, he ironed out the kinks that had crept into his game. Twice he had lost his wicket to loose strokes and once to an outswinger. It was not good enough.
He has always been a diligent player, has always attended to details. He knew, too, that his team had to take its chance to put away the Australians and meant to play his part in that. Not for him the delirium of Diwali.
Before the Delhi Test, Brett Lee spent hours hardening his body and improving his stamina. Every day he went into the gym for a prolonged session. Aware that he had fallen behind in his preparations, he went running every evening, pounding the streets, clearing his mind. Dismayed by his inability to give the attack the leadership it needed, he went flat out in the nets, reaching full pace, striving for wickets. In Mohali, he had tried to bowl within himself, conserving energy and relying on skill, and it had not worked. Enough of that. He knew that his captain was helpless unless his senior players answered his call.
Tendulkar and Lee were the chief protagonists in a rousing opening day featuring an organised and purposeful but still limited Australian side and a home outfit loath to surrender its advantage. Not that the pair were constantly in direct conflict but they set the standard for their comrades. And their exchanges were compelling. Cricket offers few sights better than a fast bowler striving with might and main to remove a great batsman. It is the most naked of cricketing contests.
Tendulkar was majestic. Indeed, he has seldom batted better. Called to the crease after Rahul Dravid had indulged in an indiscretion that imperils his position, Tendulkar swiftly settled into his work. Immediately, it was obvious that his mind was alert and his feet were moving quickly into position. As usual, he broke his duck with a neat tuck to leg. Lots of players can improvise on the front foot but none are as creative as the Indian when stepping back. Several times he retreated, examined the ball and, finding nothing untoward, directed it into a gap. Often he was happy to take a single, a currency he has never undervalued. Now and then he pressed for more, once leaning back to guide the ball over the slips, a daring offering previously reserved for one-day matches. It was an astonishing stroke to play on the first morning of a vital Test, and a bad sign for the visitors.
His work off the front foot was just as impressive. Not once in his double hundred at the SCG in 2003-04 did he drive through the covers. Now he placed the ball between mid-off and extra cover and opened the blade a second time to beat point.
Part of his mastery lies in his use of the face of the bat. Most batsmen are delighted to middle the ball. As always, Tendulkar also ran superbly between the wickets, often taking the pace off the ball or else angling it towards a weak hand. It was professional batsmanship.
Had the Australians wilted or set accommodating fields, India's favourite son would have taken complete control. Instead, Ricky Ponting, a sharper and more commanding figure than in Mohali, set interesting, inviting fields, blocking Tendulkar's most productive strokes with an unusually deep backward point and trying to cut off his supply past square leg as well. Nor was Ponting afraid to change tack. Having probed Tendulkar's back foot game, the Australians switched to full and straight attack in search of a leg before wicket decision and afterwards sought to stretch his patience. Australia persisted and eventually the batsman erred and hope was restored.
Lee began with two erratic deliveries and thereafter was more his old self. Besides fitness work, he had also been thinking about his technique.
Pausing at the top of his mark to gather himself, he ran in hard and smoothly and generated plenty of pace, averaging 10kmh more than in the Punjab. He bowled intelligently as well, changing his line as required, and forcing the batsmen to guard their stumps.
Needing an early wicket as a child needs milk, Lee beat Virender Sehwag with a fast break-backer and celebrated with a broad smile. Continuing to blend full deliveries and flyers, he threatened to make further inroads but was kept out. But he had set the example and his fellow pacemen also worked hard in the common cause. The attack lacked but not effort. Reward came as Tendulkar nibbled at Mitchell Johnson. In Mohali the visitor had fallen back in disarray. Now they stayed in the contest until their luck changed.
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text: FERNDALE, Wash., May 12 /PRNewswire-FirstCall/ -- International Absorbents Inc. ("IAX" or the "Company") (NYSE Amex: IAX), a leading developer and producer of environmentally friendly pet care and industrial products, announced today that at a special meeting of shareholders held on May 12, 2010, its shareholders adopted the arrangement agreement entered into with IAX Acquisition Corporation and IAX Canada Acquisition Company Inc., under which IAX Acquisition Corporation, through its wholly owned subsidiary, IAX Canada Acquisition Company Inc., will acquire all of IAX's outstanding common shares for cash at a price of US$4.75 per share. IAX Acquisition Corporation is a newly formed acquisition company established by Kinderhook Industries, LLC. As of the record date there were 6,410,282 shares outstanding of which approximately 3,309,897 or 52% shares entitled to vote were cast at the special meeting. Of the votes cast 3,153,611 or 95% voted to adopt the arrangement agreement; 149,236 votes or 5% voted against. The transaction remains subject to regulatory approvals and other closing conditions. About International Absorbents Inc International Absorbents Inc. develops, manufactures and markets patented and proprietary cost-effective consumer and commercial products derived from recycled, renewable materials. These environmentally safe products outperform conventional products used in a broad range of consumer and industrial applications, including retail/commercial pet bedding and litter, oil and hazardous liquid spill cleanup and control, oil/water filtration, and packaging. The Company's shares are listed on NYSE Amex under the symbol IAX. Further information is available at www.absorbent.com. About Kinderhook Industries LLC Kinderhook Industries is a New York based private equity firm with $600 million of committed capital. Kinderhook's investment philosophy is based on partnering with exceptional management and adding our operating partner network and capital across a variety of industries to support growth of smaller middle-market companies throughout North America. For additional information, visit www.kinderhook.com. Cautionary Statement Regarding Forward-Looking Statements A number of the matters discussed in this press release that are not historical or current facts that deal with potential future circumstances and developments, including, without limitation, statements referring to the ability of the parties to satisfy the closing conditions and consummate the proposed arrangement, are forward-looking statements. The words "believe," "expect," "intend," "estimate," "assume" and "anticipate," variations of such words and similar expressions identify forward-looking statements, but their absence does not mean that a statement is not forward-looking. The discussion of such matters is qualified by the inherent risks and uncertainties surrounding future expectations generally, and it is important to note that actual results could differ materially from those projected due to various risk factors, including the risk that the transaction does not close or that the closing is delayed. Additional information concerning factors that could cause actual results to differ materially from those in the forward-looking statements is contained from time to time in the Company's reports filed with the SEC, including, but not limited to, the Company's annual report on Form 10-K for the fiscal year ended January 31, 2010 and its quarterly reports on Form 10-Q. This press release speaks only as of its date, and the Company disclaims any duty to update the information herein except as required by law. CONTACT: Chuck Tait of International Absorbents Inc., 1-866-514-6559, email@example.com SOURCE International Absorbents Inc.


text:
Cisco Aironet Access Points IOS Recovery
Cisco Aironet Access Points IOS Recovery.
Configuration Steps
- Cisco AP (1200 Serials and 1300 Serials) only can carry one IOS.
- "boot system flash:/xxx.bin" does not work for AP. Command line take the statement, but "sh run" does not show it. "sh boot" will reflect the IOS which is specified.
- Never use "copy tftp flash:" in Cisco AP. It does not work.
Cisco 1310G IOS Upgrade Procedure
Following is the proper command that you should use for a regular IOS upgrade with a .tar file on these Aironet devices:
ap#archive download-sw /overwrite /force-reload tftp://IP Address of the PC/the name of the IOS image you downloaded, even with the .tar extension.
Note: The IOS image name of the .tar file is case sensitive, and it should be complete.
Recovery Steps
Using the CLI
Follow the steps below to reload the wireless device image using the CLI. When the wireless device begins to boot, you interrupt the boot process and use boot loader commands to load an image from a TFTP server to replace the image in the wireless device.
Note Your wireless device configuration is not changed when using the CLI to reload the image file.
Step 1 Open the CLI using a Telnet session or a connection to the wireless device console port.
Step 2 Reboot the wireless device by removing power and reapplying power.
Step 3 Let the wireless device boot until it begins to inflate the image. When you see these lines on the CLI, press Esc:
Loading "flash:/c350-k9w7-mx.v122_13_ja.20031010/c350-k9w7-mx.v122_13_ja.20031010″ …########################################################################### ################################################################################ ################################################################################ ####################
Step 4 When the AP: command prompt appears, enter the set command to assign an IP address,
subnet mask, and default gateway to the wireless device.
Note You must use upper-case characters when you enter the IP-ADDR, NETMASK, and DEFAULT_ROUTER
options with the set command.
Your entries might look like this example: ap: set IP_ADDR 192.168.133.160 ap: set NETMASK 255.255.255.0 ap: set DEFAULT_ROUTER 192.168.133.1
Step 5 Enter the tftp_init command to prepare the wireless device for TFTP. ap: tftp_init
Step 6 Enter the tar command to load and inflate the new image from your TFTP server. The command must include this information:
•the -xtract option, which inflates the image when it is loaded •the IP address of your TFTP server •the directory on the TFTP server that contains the image •the name of the image •the destination for the image (the wireless device Flash)
Your entry might look like this example:
ap: tar -xtract tftp://192.168.130.222/images/c350-k9w7-tar.122-13.JA1.tar flash:
Step 7 When the display becomes full, the CLI pauses and displays <!–
-->--MORE--. Press the spacebar to continue.
extracting info (229 bytes) c350-k9w7-mx.122-13.JA1/ (directory) 0 (bytes) c350-k9w7-mx.122-13.JA1/html/ (directory) 0 (bytes) c350-k9w7-mx.122-13.JA1/html/level1/ (directory) 0 (bytes) extracting c350-k9w7-mx.122-13.JA1/html/level1/appsui.js (558 bytes) extracting c350-k9w7-mx.122-13.JA1/html/level1/back.htm (205 bytes) extracting c350-k9w7-mx.122-13.JA1/html/level1/cookies.js (5027 bytes). extracting c350-k9w7-mx.122-13.JA1/html/level1/forms.js (15704 bytes)… extracting c350-k9w7-mx.122-13.JA1/html/level1/sitewide.js (14621 bytes)… extracting c350-k9w7-mx.122-13.JA1/html/level1/config.js (2554 bytes) extracting c350-k9w7-mx.122-13.JA1/html/level1/stylesheet.css (3215 bytes) c350-k9w7-mx.122-13.JA1/html/level1/images/ (directory) 0 (bytes) extracting c350-k9w7-mx.122-13.JA1/html/level1/images/ap_title_appname.gif (1422 bytes) extracting c350-k9w7-mx.122-13.JA1/html/level1/images/apps_button_1st.gif (1171 bytes) extracting c350-k9w7-mx.122-13.JA1/html/level1/images/apps_button_cbottom.gif (318 bytes) extracting c350-k9w7-mx.122-13.JA1/html/level1/images/apps_button_current.gif (348 bytes) extracting c350-k9w7-mx.122-13.JA1/html/level1/images/apps_button_last.gif (386 bytes) extracting c350-k9w7-mx.122-13.JA1/html/level1/images/apps_button_last_filler.gif (327 bytes) extracting c350-k9w7-mx.122-13.JA1/html/level1/images/apps_button_last_flat.gif (318 bytes) extracting c350-k9w7-mx.122-13.JA1/html/level1/images/apps_button_nth.gif (1177 bytes) extracting c350-k9w7-mx.122-13.JA1/html/level1/images/apps_leftnav_dkgreen.gif (869 bytes) – MORE –
Note Keep pressing spacebar to complete decompress the file. If you do not press the spacebar to continue, the process eventually times out and the wireless device stops inflating the image.
Step 8 Enter the set BOOT command to designate the new image as the image that the wireless device uses when it reboots. The wireless device creates a directory for the image that has the same name as the image, and you must include the directory in the command. Your entry might look like this example:
ap: set BOOT flash:/c350-k9w7-mx.122-13.JA1/c350-k9w7-mx.122-13.JA1
Step 9 Enter the set command to check your bootloader entries.
ap: set
Step 10 Enter the boot command to reboot the wireless device. When the wireless device reboots, it loads the new image.
ap: boot.
Question: User is using 1310, its in bridge: mode after he lost the password and deleted all files from it as per some pw recovery link he followed.
He assigned an IP, SM, DR and the device isn't accessible to be pinged etc. Step 6 just times out. The lights are red, yellow, red and orange (left to right top to bottom).
Also when i do this i get that:
bridge: dir flash0
unable to stat flash0/: permission denied
Answer: After going from steps 1 to 5 you need to start your tftp server and make sure that your firewall and antivirus is turned off as it blocks the content carried from tftp server to the device... also make sure that the path to the file in the tftp server is correct.. once the process of tftp upload is done than you can check the file by "set" command...
The issue use had was due to the unpingable nature of the bridge: mode. User was under impression that it was not connecting, along with folder vs. file name error it made him think there was no connection. Above mentioned advice was correct, after following it, process was successful.
- Password Recovery Procedure for Cisco Aironet Equipment
- Cisco Aironet 1300 Series - Home Page
- Cisco Aironet 1300 Series
- Cisco IOS Software Configuration Guide for Cisco Aironet Access Points, 12.2(15)JA
- Troubleshooting
- Cisco IOS Software Configuration Guide for Cisco Aironet Access Points for Cisco IOS Releases 15.2(2)JA, 12.4(25d)JA and 12.3(8)JEE - Troubleshooting
- Upgrade Cisco IOS on an Autonomous Access Point
- Upgrade Cisco Aironet AP1200 to IOS


text:
While there's been an explosion in the amount of digital signage being displayed almost everywhere these days, it still suffers from the same problems that have affected more traditional forms of signage. No matter what the message being displayed, it generally has very little to do with anything that might be happening that day.
But as part of an effort to overcome that limitation, Scala, a provider of digital signage systems, is embracing Big Data in the cloud. The provider of a digital signage system has rolled out Scala Advanced Analytics. It's a predictive analytics application for retailers that aggregates market data and live third-party data streams in a way that allows retailers and other organizations to display digital messages that are informed by new events, personal data or general demographic information.
For example, if it's particularly cold, then advertisements about a warm vacation spot that might be close to your heart begin appearing a lot more regularly. If the stock market drops a few hundred points, advertisements about safer investments could suddenly become ubiquitous. The system might also take note of a sale on a particular item, or a general increase in sales volume on some other item, and then conclude that an advertisement on a related product might be the most appropriate thing to display. If it recognizes you, the system might decide to display that message at the register. If it doesn't recognize you, it may choose to display a message in key locations throughout the store or mall that it knows will likely get your attention.
Over time, Dave Palermo, vice president of global marketing for Scala, says the system will collect enough data to accurately guess what different classes of people in different geographies might be inclined to buy on any given day. Delivered as a cloud service, Palermo says Scala Advanced Analytics is designed to aggregate thousands of data streams that can all be used to optimize the marketing messages being delivered via Scala's digital signage systems.
A lot of people realize that, to a limited degree, this kind of "personalization" is already happening online. But what most people don't realize is how this practice can be extended across a lot of different media outlets. As it happens, digital signage presents a fairly controlled environment that will allow marketers to begin experimenting with how far predictive analytics can be used to affect purchasing behavior in the offline world. The degree to which that capability ultimately influences buying patterns remains to be seen. But with more online data than ever being shared with geo-location services via our smartphones, it's becoming clear that, for better or worse, the world around us is increasingly becoming a reflection of what we both consciously and unconsciously project.
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text:
7-Day Dinner Plan to Eat for a Week and Hardly Cook at All
By Hilary Meyer, December 12, 2013 - 9:26am
Hilary asks: What's your secret for minimal cooking during the week but still eating healthy meals?
Cook and season twice the protein or main meal (eg, meat, lasagna, chili) on a night you are not as busy. Cool and freeze half for another meal. Or make two complete meals and refrigerate the second meal for the next day. When my kids were busy in sports, boy scouts, etc., I would make two complete meals and put the second meal in the fridge for the next night when time would be short.
— Anonymous
12/18/2013 - 7:32pm
Why would you choose a white sandwich bun for a healthy meal?
— Anonymous
12/30/2013 - 4:48pm
Sorry Hillary, but that is your big tip? Make 2 casseroles, freeze or refrigerate 1 and then eat it 3 days later? My family would never go for that.
— Nicole_682
12/30/2013 - 10:13pm


text:
CANADA STOCKS-TSX flat in light Easter volume; TransCanada, Barrick down
TORONTO, April 21 (Reuters) - Canada's main stock index closed little changed on Monday in quiet Easter holiday trading, with TransCanada Corp shares hit by a delay in the Keystone XL pipeline project, and Barrick Gold Corp helping offset gains in other groups. The Toronto Stock Exchange's S&P/TSX composite index finished the session down 6.71 points, or 0.05 percent, at 14,493.68. (Reporting by Solarina Ho; Editing by Meredith Mazzilli)
© Thomson Reuters 2017 All rights reserved.


text:
The surface-exposed NadA adhesin produced by a subset of capsular serogroup B strains of Neisseria meningitidis is currently being considered as a vaccine candidate to prevent invasive disease caused by a hypervirulent lineage of meningococci. Levels of NadA are known to be controlled by both transcriptional regulatory factors and a component of human saliva, 4-hydroxyphenylacetic acid. Herein, we confirmed the capacity of a DNA-binding protein termed FarR to negatively control nadA expression. We also found that a known transcriptional regulator of farR in N. gonorrhoeae termed MtrR can have a negative regulatory impact on farR and nadA expression, especially when over-expressed. MtrR-mediated repression of nadA was found to be direct, and its binding to a target DNA sequence containing the nadA promoter influenced formation and/or stability of FarR::nadA complexes. The complexity of the multi-layered regulation of nadA uncovered during this investigation suggests that N. meningitidis modulates NadA adhesin protein levels for the purpose of interacting with host cells yet avoiding antibody directed against surface exposed epitopes.
Citation: Cloward JM, Shafer WM (2013) MtrR Control of a Transcriptional Regulatory Pathway in Neisseria meningitidis That Influences Expression of a Gene (nadA) Encoding a Vaccine Candidate. PLoS ONE 8(2): e56097. https://doi.org/10.1371/journal.pone.0056097
Editor: Ben Adler, Monash University, Australia
Received: August 24, 2012; Accepted: January 7, 2013; Published: February 8, 2013
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: This work was supported by NIH grant AI021150-28 and a VA Merit award to W.M.S. W.M.S. is the recipient of a Senior Research Career Scientist Award from the Department of Veterans Affairs. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Neisseria meningitidis is a Gram-negative obligate human pathogen that colonizes the nasopharynx in 10–35% of adults [1]. For reasons not currently understood, commensal meningococcal (MC) colonization develops into an invasive disease causing septicemia and meningitis in 0.5 per 100,000 persons in the United States and up to 1,000 per 100,000 persons in sub-Saharan African epidemics [2]. The speed of disease progression results in up to 10–15% mortality even with antibiotic therapy [3], while often leaving survivors with permanent neurological complications [4]. Vaccines against the capsular polysaccharide of the most common disease-associated serotypes (A, C, W135, and Y) are available, leaving the hypervirulent and immune-evasive serotype B as a current focus for vaccine research [5].
Adhesion to the mucosal surface of the nasopharynx is the first step in successful colonization, mediated by a variety of factors, with type IV pili [6], [7], [8] and Opa and Opc proteins [9], [10] produced in the greatest abundance. Recently, a non-fimbrial "Oca" family (Oligomeric coiled-coil adhesin) neisserial adhesin termed NadA was identified in 50% of hypervirulent MC capsular serogroup B lineages [11], but not in other capsular serogroup strains. Comprised of a leader peptide, globular "head" domain, α-helix intermediate region, and a C-terminal membrane anchor, NadA forms highly stable multimeric coiled-coil structures along the helical stalk, positioning the globular "head" for host cell interaction [12]. Importantly for consideration as a vaccine candidate, recombinant NadA lacking the C-terminal anchor elicits a bactericidal antibody response with epitopes accessible in encapsulated MC. Although nadA allele sequences differ between strains, varied antigen expression, not diversity, influences immune sera titer levels and protection [11]. Accordingly, the identification of factors influencing NadA levels at the gene expression level is critical for optimizing the efficacy of a NadA-targeted vaccine. Furthermore, understanding nadA expression may offer clues into the signals involved in converting a passive co-inhabitant of the human mucosal lining into an invasive and fatal septic infection.
MC uses a multi-tiered approach to control nadA expression. Maximum levels of the NadA protein are observed in stationary-phase in a growth-dependent manner [11], with expression of nadA varying widely among MC strains [13], [14]. Upstream from the promoter are multiple tetranucleotide (TAAA) repeats whose number corresponds with varied nadA expression [13], [15]. These repeats are phase variable, likely caused by slipped-strand mispairings during replication [16]. Several regulatory proteins bind to the nadA promoter (Figure 1), including integration host factor (IHF) and ferric uptake regulatory protein (Fur), though nadA expression is unchanged in a Fur null mutant [14]. Recently, a MarR-family transcriptional regulator, termed FarR and NadR in separate publications [14], [17], was identified as a repressor of nadA, further expanding the list of nadA regulatory factors. This DNA-binding protein was first identified in the gonococcus (GC) and was shown to repress expression of the farAB-encoded efflux pump that is responsible for high levels of fatty acid resistance [18]. In contrast, MC FarR does not affect fatty acid resistance through FarAB, perhaps due to naturally high fatty acid resistance expressed by this pathogen [19]. Interestingly, however, MC FarR does bind to its farAB promoter region with relatively high affinity and represses farAB expression as shown by RT-PCR [20]. Because FarR regulates expression of farAB in both MC and GC, while nadA is present only in a subset of MC populations, we will continue to use the nomenclature of FarR for the repressor of nadA based on its more universal activity on farAB in both GC and MC.
Representations of the nadA (A), farR (B), and mtrR (C) promoter regions showing regulatory protein binding sites, intergenic sequences of interest, and primer annealing locations with oligonucleotide sizes. White arrows represent each open reading frame with the translation start codon noted by "ATG" and a vertical line to indicate if a primer overlaps the start codon. Grey arrows represent the respective primers including oligonucleotide sizes with the following nomenclature: DNA probe for EMSAs - "orf"_prom_F & R; promoter region with start codon fused with lacZ - "orf"_F_Bam and "orf"_R_lacZ. IHF and Fur are binding sites for integration host factor and ferric uptake regulatory protein, respectively.
The small molecule 4-hydroxyphenylacetic acid (4HPA) was identified as an inducer or de-repressor of nadA by relieving the DNA-binding activity of FarR [14]. Being a colonizer of the oropharynx, MC is washed in saliva, in which 4HPA is a common metabolite [21], possibly leading to increased expression of nadA and subsequent invasive disease. Curiously, FarR-controlled targets in GC are directly and indirectly regulated by the TetR family regulator MtrR. Repression of farR by MtrR indirectly up-regulates farAB [18], while the gene encoding glutamine synthetase (glnA) is directly regulated by both FarR and MtrR [22]. Therefore, we questioned whether MtrR similarly affects nadA expression in MC, adding to the growing list of regulatory factors targeting nadA. Here we confirm that FarR is the primary repressor of nadA, yet MtrR, when expressed at elevated levels, directly represses nadA as well. Furthermore, DNA-binding and DNase I protection assays suggest that MtrR influences FarR binding at the nadA promoter similar to the phenomenon seen in glnA expression in GC [22], suggesting a higher complexity to Neisserial regulatory schemes that is conserved across species.
Results and Discussion
Control of nadA, farR, and mtrR expression in MC strain M7
Co-regulation and competitive regulation between FarR and MtrR has been shown previously for multiple targets in GC [18], [22] but not MC. Given previous observations that in MC FarR can regulate nadA, we asked if MC MtrR can control farR expression. We also tested if MC MtrR could control nadA directly. To investigate possible influences of MtrR on nadA and/or farR expression in MC, the expression profiles for these genes were determined using a promoterless lacZ-fusion expression system (Figure 1) employed previously to monitor gene expression in GC [23]. Using translational lacZ fusions to each gene's promoter (ranging from 427 to 524 bp; Figure 1), mtrR expression was compared to farR and nadA across multiple growth phases of broth–grown cultures or from overnight, agar-grown cultures (Figure 1) of MC strain M7, which is a capsule-deficient mutant of strain NMB that is used for biosafety purposes [24]. This was done because earlier work [25] reported that farR was maximally expressed between late-log and stationary phase, while nadA expression peaks at stationary phase [11]. Importantly, the lacZ fusions did not significantly impact growth rates in broth for the M7-derived strains (Figure 2A), suggesting that any expression profile differences are not growth rate-dependent. Aliquots from different growth phases (Figure 2A; boxed A, B, and C) were assessed for β-galactosidase activity and compared against the activity of overnight cultures grown on GCB agar plates (Figure 2B). The results showed that agar-grown MC had higher levels of expression for all three genes compared to broth-grown strains, and this was especially true for nadA. With respect to agar-grown cultures, we noted that nadA expression was considerably greater than farR or mtrR with the latter being the most poorly expressed gene (Figure 2B). Based on these results, all subsequent gene expression studies were performed on cultures grown overnight on GCB agar plates.
(A) Growth curve of strain M7 expressing lacZ fused to nadA (solid line; circle timepoints), farR (dotted line; triangle timepoints), and mtrR (dashed line; square timepoints) promoter regions measured by OD600 optical density. Boxed A, B, and C; timepoints for sample harvest. (B) Specific activity of β-galatosidase activity of lacZ fusions as indicated. Samples harvested from liquid culture at various growth phases (A, B, C) were compared with O/N growth on agar plates. Inset; magnified view of mtrR-lacZ expression. NS, not significant; **, P<0.01.
Having established that the lacZ fusion technology could be employed to monitor gene expression in strain M7, we next asked if MtrR can regulate farR as it does in GC and if, in turn, it can modulate nadA expression. We first asked if loss of MtrR impacts MC farR expression levels and for this purpose constructed an M7 farR-lacZ fusion strain (427 bp promoter region; Figure 1) in an mtrR null mutant that carries the non-polar aphA-3 cassette within the mtrR coding sequence. With this fusion strain, we noted a small but significant (p<0.01; Figure 3) increase in farR-lacZ expression, which was reversed by complementation when the M7 mtrR allele was expressed ectopically under its own promoter or an IPTG-inducible promoter. Having observed a significant, albeit modest, influence of MtrR on farR-lacZ expression, we next asked if nadA expression, known to be negatively controlled by FarR [17], would be impacted due to loss of MtrR using a nadA-lacZ fusion (514 bp promoter region; Figure 1). We first confirmed that loss of FarR significantly increased nadA-lacZ expression (strain JC5AZ; Figure 4A), and this was reversed by complementation with the wild-type farR gene from strain M7 (strain JC6AZ). Interestingly, loss of MtrR alone (strain JC2AZ) resulted in a consistent, albeit small, increase in nadA-lacZ expression, though not to significant levels, with restoration of nadA to wild-type levels when mtrR was expressed ectopically under its own promoter (strain JC3AZ; Figure 4B). Furthermore, complementation with the wild-type mtrR sequence under the control of an IPTG-inducible promoter significantly (p<0.01) reduced nadA-lacZ expression by 26% (strain JC4AZ; Figure 4B). Based on these results, we concluded that FarR-mediated repression of nadA-lacZ was greater than that mediated by MtrR, but that MtrR could impact nadA expression by a FarR-independent mechanism. In order to further test this, we compared nadA-lacZ expression in a ΔfarR/ΔmtrR double mutant (strain JC7AZ) against the single ΔfarR mutant (strain JC5AZ). Here, no significant impact on nadA-lacZ expression was observed, further supporting a dominant repression of nadA by FarR. However, IPTG-induction of mtrR in the absence of FarR (strain JC9AZ) did result in a significant reduction in nadA (p<0.01) compared to both the ΔfarR/ΔmtrR double mutant (strain JC7AZ) and ΔfarR mutant (strain JC5AZ; Figure 4C). Taken together, the data suggested that MtrR, when overexpressed, has an influence on nadA expression that is independent from FarR-modulated expression in MC strain M7. What effects MtrR may have on nadA expression in other MC strains that differ in either mtrR or nadA expression is not yet clear and the subject of future study.
Specific activity of β-galactosidase activity of farR-lacZ in the strains M7 wild-type, ΔmtrR, and ΔmtrR complemented with the native and inducible-promoter alleles (superscript asterisk), respectively. NS, not significant; *, P<0.05; **, P<0.01.
(A–C) Specific activity of β-galactosidase activity of nadA-lacZ in various M7 backgrounds as indicated. Strains over-expressing MtrR marked with a superscript asterisk. NS, not significant; *, P<0.05; **, P<0.01. (D) Western immunoblot analysis of NadA, FarR, and MtrR levels. Protein samples grown overnight on GC agar plates, collected, and analyzed by electropheoresis through 6% (NadA) or 12% (FarR and MtrR) SDS-PAGE gels followed by immunoblot with the respective antisera. Molecular weight standards are listed to the left. Coomassie-stained gel provided for protein level comparison. Arrows represent minor immunoreactive bands used to determine NadA steady-state differences across strains.
Western blot analysis was performed to confirm the results observed with translational nadA promoter-lacZ fusions (Figure 4D). As expected, FarR was absent in the farR null mutant strains (JC5AZ, JC7AZ, and JC9AZ), but was present in the complemented mutants that expressed farR ectopically (strains JC6AZ and JC8AZ); although FarR levels were reduced in the complemented strains, repression of nadA was still evident (Figure 4A, C, D; arrow). NadA migrates in its multimeric form even under denaturing/reducing conditions [11], complicating discrimination of differences in NadA protein levels when analyzing the dominant band alone; therefore, minor NadA-dependent bands, observed elsewhere [20], were used for analysis of steady state levels of NadA (Figure 4D; arrows). Despite repeated attempts, MtrR could not be detected by immunoblot in any strain except when over-expressed (strains JC4AZ and JC9AZ), suggesting that MtrR is maintained at low levels in N. meningitidis. This low level of MtrR may explain why enhanced expression of farR in the mtrR null mutant was modest (Figure 4) and why its ability to repress nadA could only be observed when mtrR was over-expressed ectopically (strain JC4AZ; Figure 4B). In agreement with the data obtained with the nadA promoter translational fusions, steady-state levels of NadA exceeded wild-type in the absence of FarR (Figure 4D; arrows point to minor bands used for comparison). Unfortunately, differences in NadA levels were not discernible when comparing +mtrR to ΔmtrR strains in the +farR (strains M7AZ, JC2AZ, JC3AZ, and JC4AZ) or ΔfarR (strains JC5AZ, JC7AZ and JC9AZ) backgrounds (Figure 4D). Assessing differences in steady-state levels of NadA in other clinical MC strains following the loss of mtrR requires further study.
FarR and MtrR binding to a target DNA sequence upstream of nadA
The data obtained using translational fusion strains bearing an over-expressed wild-type mtrR allele in an mtrR null mutant background indicated that elevated levels of MtrR can exert negative regulatory influences on nadA expression in MC strain M7. Based on this hypothesis, we asked if MtrR can bind target DNA sequences upstream of nadA by employing electrophoretic mobility shift assays (EMSA) using FarR or MtrR fused to maltose-binding protein (MBP) at their C-terminus;maps describing the various DNA probes used are shown in Figure 1. As described previously [14], FarR was found to bind the target nadA sequence (457 bp) in a specific manner with at least three DNA-protein complexes observed (Figure 5A, arrows). Furthermore, the results from binding specificity assays suggested that FarR has greater affinity for the nadA-promoter than to its own (443 bp) or farAB promoter-bearing sequences (497 bp and shown in Figure 5B); importantly, a probe lacking a FarR-binding site (rnpB; 609 bp) was unable to compete with the labeled nadA probe for complexing with FarR.
(A) Successive increases of FarR-MBP incubated with 10 ng nadA promoter region to assess binding by gel-shift analysis. Arrows; various complexes of DNA and FarR-MBP. (B) Competition assays. 32P-labeled nadA promoter (384 bp) was incubated with 0.5 µg FarR-MBP and competed with unlabeled nadA, farR (333 bp), farAB (435 bp), and rnpB (354 bp) at 25, 50, and 100 times molar excess of labeled probe (lanes 3 through 14). The competing probe used is listed below each panel. Arrow; 32P-labeled probe competed away from FarR-MBP by unlabeled probe. Lane 1, labeled probe alone; lane 2, labeled probe and 0.5 µg FarR-MBP without competitor.
Having verified that the nadA probe could be recognized in a specific manner with an MC DNA-binding protein (FarR), we next asked if nadA could bind MtrR. First, we confirmed by EMSA the DNA-binding capacity of the MtrR-MBP fusion protein by evaluating its ability to bind a known target DNA sequence, namely the promoter-bearing region upstream of MC mtrCDE (811 bp). In GC, MtrR is a repressor of the mtrCDE-encoded antimicrobial efflux pump by virtue of its binding between the −10 and −35 promoter elements [26], [27]. We found that as little as 1.0 µg of MC MtrR-MBP incubated with MC mtrCDE promoter-bearing probe resulted in virtually a complete shift (Figure 6A; arrow) of the target sequence. Importantly, a similar shifting of the nadA probe by MtrR was observed (Figure 6B; arrow). Specificity of MtrR-binding to the nadA probe was confirmed by use of competitive EMSA. Although the heterologous unlabeled rnpB promoter-bearing probe did to some extent compete for binding, especially at a high concentration (100×), the mtrCDE and nadA unlabeled probes proved to be more effective competitors at a lower concentration (25×) with the mtrCDE probe appearing to be superior (Figure 6C).
Successive increases of MtrR-MBP incubated with 10 ng mtrCDE (A) or nadA (B) promoter regions to assess binding by gel-shift analysis. Arrow; primary complex of DNA and MtrR-MBP. (C) Competition assays. 32P-labeled nadA promoter (384 bp) was incubated with 1.0 µg MtrR-MBP and competed with unlabeled nadA, mtrCDE (552 bp), and rnpB (354 bp) at 25, 50, and 100 times molar excess of labeled probe (lanes 3 through 14). The competing probe used is listed below each panel. Arrow; 32P-labeled probe competed away from MtrR-MBP by unlabeled probe. Lane 1, labeled probe alone; lane 2, labeled probe and 0.5 µg MtrR-MBP without competitor.
The ability of both FarR and MtrR to bind the nadA promoter-bearing region in a specific manner was reminiscent of their ability to bind the DNA sequence upstream of glnA of GC (22). As the binding of either protein to the glnA target can impact binding of the other we asked if a similar situation might exist for the MC-derived nadA target. To test this possibility, we pre-incubated the nadA probe with a fixed concentration of one protein and then introduced increasing amounts of the second protein. In the absence of a competing protein, both MtrR and FarR exhibited a distinct shift of the probe (Figure 7; arrowhead and arrows, respectively). As FarR-MBP concentrations increased following pre-incubation of nadA target DNA with MtrR-MBP, the MtrR shift remained relatively unchanged, suggesting that FarR-MBP does not compete with MtrR-MBP. In contrast, increasing amounts of MtrR-MBP changed the electrophoretic mobility of FarR-MBP:DNA complexes, suggesting that MtrR-MBP can significantly influence the formation or stability of FarR::nadA complexes.
Shown is an EMSA evaluating the binding of MtrR–MBP and FarR-MBP to 32P-labeled nadA promoter based on order of protein introduction. Lane assignments from left to right: probe alone; probe plus 1.0 mg MtrR-MBP and increasing amounts of FarR-MBP (0, 0.13, 0.25, 0.50, 1.0, 2.5, and 5.0 mg); probe alone; probe plus 0.5 mg MtrR-MBP and increasing amounts of FarR-MBP (0, 0.13, 0.25, 0.50, 1.0, 2.5, and 5.0 mg). The arrowhead shows the position of MtrR:DNA complex lacking FarR while the arrows show positions of FarR:DNA complexes lacking MtrR.
In order to learn the mechanism by which MtrR could influence the formation of FarR::nadA complexes, we used DNase I protection assays to determine if their respective binding sites might be in close proximity. We confirmed FarR-MBP-binding to the three sites (data not shown) described by Metruccio et al. [14], which include the −10 promoter and TAAA phase-variable regions (Figure 8B). In repeated DNase I protection assays, clear evidence for a sequence(s) capable of recognizing MtrR could not be obtained. However, in these experiments a DNase I hypersensitive site was identified positioned at the end of the phase-variable TAAA repeats (Figure 8B; asterisk at nucleotide position 209). The presence of this suggests an interaction of MtrR with a sequence upstream of nadA that could influence binding of FarR.
(A) Increasing amounts of MtrR-MBP (0, 1, 5, 10 mg) were incubated with the nadA promoter prior to DNase I incubation. Site of DNase I hypersensitivity is denoted with an asterisk. The nucleotide sequence (G, A, T, C) is listed adjacent to the lanes. (B) Nucleotide sequence of the nadA promoter. Colored boxes, FarR-MBP binding sites 1, 2 & 3; Asterisk, DNase I hypersensitive site. The transcription start site (+1) and translational start site (ATG, bold and underlined) are indicated.
While N. meningitidis colonizes up to 35% of humans [1], fewer than 1% of the population develops an invasive infection [2], suggesting that the bacterium focuses on a more commensal lifecycle. The ability to effectively transition between passive residence and active infection relies on tight transcriptional regulation involving an array of external and internal control systems. In GC, the transcriptional regulators FarR and MtrR have been well-characterized for their role in antimicrobial resistance, allowing for host persistence [28]. FarR represses expression of the fatty-acid efflux pump FarAB [29]; MtrR represses expression of the mtrCDE, which encodes an antimicrobial efflux pump [26], [30]. Recently, MC FarR was shown to repress expression of nadA [17], whose gene product is a highly immunogenic adhesin and invasin associated with hypervirulent strains of serotype B MC [11], [12]. Interestingly, GC MtrR has also been shown to repress GC FarR, thereby influencing transcription of farAB [18]. As this regulation of a regulator is not unique to GC [22], we explored whether MtrR likewise modulates farR in MC, thus affecting nadA expression.
Our results suggest that MC employs a dual-repressor approach to control nadA expression. Using lacZ translational fusions, EMSA, and DNase I protection assays, we confirmed earlier work [14], [17], [25] that FarR is a negative regulator of nadA due to its ability to bind target DNA upstream of the coding region (Figure 7B) and affect subsequent expression (Figure 4A). Complicating this regulatory scheme, our results indicate that MtrR can exert an influence on nadA directly by interacting with the upstream DNA sequence and indirectly through its ability to reduce farR expression (Figure 3). With respect to the first mode of MtrR regulation over nadA, our DNA-binding studies indicate that MtrR can bind upstream of this gene in a specific manner (Figure 6C) and can impact the formation and/or stability of FarR::DNA complexes (Figure 7) when its level exceeded that of FarR. The stronger influence of FarR on nadA expression is likely due to its ability to recognize three target sites (Figure 7B) while under the conditions employed in the DNase I protection assay. A possible site for MtrR binding could only be surmised by the presence of a DNase I hypersensitive site (Figure 7A). Interestingly, this site is positioned within a tract of tetranucleotide repeats and a FarR-binding site (Figure 7B).
Under what conditions might MtrR-mediated regulation of nadA have biologic relevance given the strong regulatory action of FarR? We propose several potential mechanisms: The development of mutations impacting FarR regulation of nadA would require alternative mechanisms of transcriptional regulation that could in part be fulfilled by MtrR. Thus, mutations in FarR that reduce its DNA-binding activity or mutations in FarR-binding sites could enhance nadA expression unless other regulatory processes are available. Alternatively, mutations that enhance MtrR levels or interactions with nadA-binding sites might repress nadA expression above that seen by FarR alone. Precedent for clinical isolates of Neisseria bearing regulatory mutations impacting gene expression exists in that gonococcal strains isolated from patients frequently contain mutations in mtrR and these can cause dysregulation of the mtrCDE-encoded efflux pump operon. Furthermore, cis-acting regulatory mutations can influence transcription of mtrR and/or mtrCDE or directly enhance expression of the mtrCDE-encoded efflux pump [28], [31], [32], [33]. In M7, mtrR expression is typically low, yet overexpression of mtrR results in almost 30% repression of nadA (Figure 4B and D). Accordingly, it will be important to evaluate MC clinical isolates to determine if they may develop mutations impacting nadA expression directly or indirectly; the latter being due to mutations in farR or mtrR. With the expression of nadA varying significantly between MC strains [13], [14], mutations affecting farR and/or mtrR expression may have more profound effects on nadA expression than those observed here. Deletion of the Correia element or IHF binding site upstream from mtrR affects expression of mtrCDE, which is an MtrR target [32]; Enriquez et al. observed several MC isolates with Correia element deletions upstream from mtrR, including one serotype B, suggesting that these mutations are not an exception [34]. We propose that this multi-layered regulation of nadA, which now includes direct regulation by MtrR, reflects an effort by MC to balance levels of the NadA adhesion important for interacting with host cells yet avoiding potentially protective antibody responses.
Materials and Methods
Bacterial strains and growth conditions
All N. meningitidis strains listed in Table 1 are derivations of strain M7 constructed for this study; M7 is a stable capsule-negative variant of strain NMB and was used for biosafety purposes. MC were cultured on GCB agar (Difco Laboratories, Detroit, MI) with defined supplements I and II [35] at 37°C under 3.8% (vol/vol) CO2. For growth-phase analysis, MC were grown in a shaking incubator at 37°C in GCB broth with sodium bicarbonate and defined supplements I and II as previously described [35].
Strain construction and verification
For construction of strain JC2, overlapping PCR products were generated to replace mtrR with aphA-3 [36] by allelic exchange at the native N. meningitidis locus, conferring kanamycin resistance. Specifically, primers mtrC_R_out_5′ and mtrR_R_Kan_5′ovhg generated product A; kan_F_mtrR_5′ovhg and kan_R_mtrR_3′ovhg generated product B; and mtrR_F_Kan_3′ovhg and NMB1718_FWD generated product C (Table 2). Products A, B, and C were used as template with flanking primers mtrC_R_out_5′ and NMB1718_FWD (Table 2) to generate the final PCR product used for transformation of wild-type M7 with selection on kanamycin at 50 µg ml−1. The substitution of mtrR with aphA-3 was verified by PCR (data not shown) and Western blot (Figure 4). For construction of JC5, PCR products generated by FarR_prom_F and FarR _Sma_R (Table 2) were subcloned into vector pCR®2.1 (Invitrogen) generating pJC5α with selection on ampicillin at 100 µg ml−1. PCR products from FarR_ Sma_F & FarR_pmalC_Xba_R (Table 2) were purified, digested with SmaI, and ligated into pJC5α at the SmaI site, generating pJC5β, and sequenced for accuracy. Finally, pHP45Ω was digested with SmaI to liberate the spc cassette then ligated into the SmaI site of pJC5β generating pJC5 used to transform M7 with selection on spectinomycin at 60 µg ml−1. The interruption of farR with spc was verified by PCR (data not shown) and Western blot (Figure 4). Complementation of JC2 and JC5 was accomplished by delivering the parent gene ectopically using vector pGCC3 or pGCC4 [37]. Briefly, primers mtrR_F_Pac_GC3 and mtrR_R_Pme or mtrR_F_Pac_GC4 and mtrR_R_Pme (Table 2) were used to amplify the mtrR allele then digested with appropriate restriction enzymes and ligated into pGCC3 and pGCC4, respectively. Similarly, farR_F_Pac_GC3 and farR_R_Pme (Table 2) were used to amplify the farR allele for subsequent digestion and ligation into pGCC3. All constructs were verified by sequencing prior to transformation. Transformants were selected on erythromycin at 1 µg ml−1.
Construction of lacZ reporter fusions, β-galactosidase assay and immunoblot analysis
All lacZ fusions used in this study were prepared in pLES94 and performed as described previously [18], [23] using appropriate primers. For nadA-lacZ, primers nadA_F_Bam and nadA_lacZ_R were used; for farR-lacZ, primers farR_F_Bam and farR_lacZ_R were used (Table 2); and for mtrR-lacZ, primers mtrR_F_Bam and mtrR_lacZ_R were used (Table 2) Constructs encoding a lacZ fusion were grown overnight on GCB agar with supplements, 1 mM IPTG, and 5 mM 4-hydroxyphenylacetic acid, when appropriate. Cells were harvested directly from plates or used to inoculate GCB broth with appropriate supplements and 1 mM IPTG then grown through stationary phase for growth-phase analysis of protein expression. Cells collected from overnight plates were resuspended in phosphate-buffered saline pH 7.2 (PBS), centrifuged for 2 min at 13,000 rpm, and stored overnight at −20°C after the supernatant was removed. From liquid cultures, 5 mL aliquots were removed at the indicated growth phases (Figure 1) and centrifuged for 15 min at 5,000 rpm. After removing the supernatant, cell pellets were resuspended in PBS, centrifuged for 2 min at 13,000 rpm, and stored overnight at −20°C. To determine β-galactosidase specific activities, cell pellets were resuspended in 50/50 PBS and Z-buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgCl2, 50 mM β-Mercaptoethanol; pH 7.0). After addition of 20 µL 0.1% SDS and 40 µL chloroform, samples were vortexed and incubated at room temperature for 5 min. Protein concentrations were quantified by Nanodrop1000 (Nanodrop Technologies,Wilmington, DE). To 200 µL of Z-buffer, 30 µL of each cell lysate and 70 µL of ONPG (2-Nitrophenyl-β-D-galactopyranoside; Sigma, St. Louis, MO) at a concentration of 4 mg ml−1 in Z-buffer was added. Following a color change, the reaction was stopped with 500 µL of 1 M Na2CO3 solution. The reactions were centrifuged at room temperature for 5 min at 13,000 rpm to remove cell debris and 200 µL of supernatant was transferred to a 96-well microtiter plate and analyzed at 420 nM by a PerkinElmer Victor X3 microplate reader. For data analysis, specific activity was calculated using the formula: {[(OD420 * v)/(4500 nL nmoles−1 cm−1×1 cm)]/t/mg protein} with v being the volume used and t being the reaction time. All reactions were performed in triplicate and repeated at least 3 times. Statistical analysis was performed using multivariate ANOVA followed by Tukey HSD post-hoc pairwise comparison using SAS 9.2 software (The SAS Institute, Cary, NC).
Verification of protein absence, overexpression, and comparison between strains were assessed by immunoblot. Total protein was quantified by Nanodrop1000 (Nanodrop Technologies) prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation [38] and Western immunoblotting [39]. Rabbit polyclonal antibodies were used at the following dilutions: anti-NadA, 1∶2,000; anti-FarR, 1∶5,000; and anti-MtrR, 1∶1,000. Anti-rabbit alkaline phosphatase-conjugated secondary antibody (Bio-Rad Laboratories, Hercules, CA) was used at 1∶7,500.
Purification of MtrR and FarR
Fusion and purification of N. meningitidis M7 MtrR and FarR to maltose-binding protein (MBP) were performed per manufacturer's guidelines (New England Biolabs, Beverly, MA) and as described previously [27] with some exceptions. Primers mtrR_pmalC_F & mtrR_pmalC_Xba_R and primers farR_pmalC_F & farR_pmalC_Xba_R were used to PCR amplify the mtrR and farR alleles, respectively, before XbaI digestion and blunt-end ligation into pMal-c2 (New England Biolabs). Both constructs were sequenced for accuracy. Factor Xa digestion was not performed due to protein stability issues; therefore, all DNA-binding investigations utilizing these proteins maintained an intact MBP fusion, which has been successful in prior investigations [27]. Analysis of eluted fractions by SDS-PAGE revealed a 65-kDa band consistent with the expected size an MtrR-MBP fusion (data not shown). For consistency, N. meningitidis M7 FarR was likewise fused to MBP and purified, in which the purified protein band migrated to about 60 kDa (data not shown).
DNA-binding studies
Electrophoretic mobility shift assays (EMSA) and competitive EMSAs using FarR-MBP and MtrR-MBP were performed essentially as previously described [22], [27], [40] with some modifications. Briefly, unlabeled and 32P-labeled probes were generated with the following primers: nadA, nadA_prom_F and nadA_prom_R; mtrCDE, KH9_3 and mtrR_R_out_5′; farR, farR_prom_F and farR_prom_R; farAB, farAB_prom_F and farAB_prom_R; and rnpB, rnpB1F and rnpB1R (Table 2). Appropriate PCR products were end labeled with [γ-32P] dATP using T4 polynucelotide kinase (New England Biolabs, Beverly, MA). The labeled products (10 ng) were each incubated with purified FarR-MBP or MtrR-MBP in a 30 µL reaction volume [10 mM Tris-HCl (pH 7.5), 0.5 mM dithiothreitol, 0.5 mM EDTA, 1 mM MgCl2, 50 mM NaCl, 0.05 µg/mL poly(dI-dC)] at room temperature for 30 min. Loading buffer (Epicentre, Madison, WI) was added to each sample then separated by 6% polyacrylamide gel at 4°C, followed by autoradiography. Competitive EMSAs were performed similarly, but unlabeled probes generated from the same primer sets as labeled probes were included.
DNase I protection assays were performed as previously described [22], [27], [40] with slight modifications. Target DNA probes were generated with PCR pimers nadA_prom_F and nadA_prom_R (Table 2). The 5′ end was labeled with T4 polynucleotide kinase as described above for EMSA probes. Purified MtrR-MBP was incubated with target DNA for 15 min at 37°C. Dnase I in loading buffer [10 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 4 mM CaCl2, 1 mM dithiothreitol, 40% glycerol] was added to each reaction for 1 min at 37°C. The reactions were stopped with DNase I stop buffer (95% ethanol and 7.5 mM ammonium acetate), plunged in an ethanol dry-ice bath for 15 min, and precipitated overnight at −80°C. Pellets were washed in 70% (vol/vol) ethanol, dried, and resuspended in loading buffer (Epicentre). Resuspended reaction mixtures were loaded on 6% denaturing polyacrylamide gel and resolved by autoradiography.
We thank Virginia Stringer for excellent technical assistance, Lane Pucko for help with manuscript preparation, and Oliver Kurzai for the gift of antiserum to FarR and NadA.
Author Contributions
Conceived and designed the experiments: JMC WMS. Performed the experiments: JMC. Analyzed the data: JMC WMS. Contributed reagents/materials/analysis tools: JMC WMS. Wrote the paper: JMC WMS.
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Responsible mining
Responsibly undertaken, gold mining and its associated activities can have a transformative effect on socio-economic development in countries where gold is found. When produced in conformance to high social, environmental and safety standards, gold provides employment opportunities, improved infrastructure and tax revenues. It can also drive foreign direct investment and generate foreign exchange. In 2013 the industry made an economic contribution of more than US$171 billion to the top 15 gold-mining economies.
Maximising the development potential of mining requires continuous attention and discussion. Transparency is critical – both around the operational activities and around the economics of gold mining.
Respect for local societies and human rights are of particular importance for responsible gold-mining companies. Many companies undertake their own social responsibility programmes, as well as sign up to international standards such as the Voluntary Principles on Security and Human Rights.
Responsible mining also means addressing concerns that resources could fund unlawful activity, particularly when operating in areas affected by armed conflict, such as civil war or militia activity. The World Gold Council, working with our Members, developed the Conflict-Free Gold Standard, a common approach by which gold producers can assess and provide assurance that their gold has been extracted in a manner that does not cause, support or benefit unlawful armed conflict or contribute to serious human rights abuses or breaches of international humanitarian law.
Managing the environmental impact of operations is also a vital element of responsible mining. Responsible companies take great care in designing and following policies that preserve the local biodiversity and water quality.
The wellbeing of mine staff is also a leading concern of responsible gold-mining companies, who implement rigorous safety standards to complement national and international laws and guidelines.
The World Gold Council undertakes research to better understand the role that responsible mining plays in contributing to social and economic advancement. We are committed to supporting the gold mining industry in making a positive contribution to sustainable development.


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Failed inspection
I hired a home inspector to get an opinion on my ridge vent problem here is what he found.
My attic he says is around 1800-2000 SQ with 10 foot ceilings, an attic of that size cannot use ridge vents its too big.
Also he found some of the vents along the soffits were not perforated, (I thought they would come from the factory with holes in the vents,)
He suggest installing 2 attic fans
What do you guys think?
the guys who are really up on this are going to want to know
1. what the ridge vent problem was
2. what kind of ridge vent it is
3. how long your ridge is for the quoted sq/ft
We had to go in the attic a month ago and the day was mild, 72* and we noticed how hot the attic was, so I bought a remote access thermometer and started logging the temps daily morning afternoon and night, on hot days the attic was 25* hotter in the a.m., up to 45* hotter in the afternoons, highest temp logged was 139*.
the brand is Cobra
The ridge vent runs the entire peak of the roof.
Your inspector is nuts. A cobra ridge vent will work fine running the length of a gable roofed house. Chances are you have an installation problem with either too small of a slot at the ridge, felt blocking the vent or a problem with the soffit vents - either blocked by insulation or not enough of the vented soffits. My bet is on insulation blocking the soffit vent air from flowing between the rafters.
Tell us the length of the ridge vent in feet.
Home inspector is incorrect. That is not "too large" of an attic for ridge vent.
By far the majority of inspectors I have met are, jack of all trades & master of none persons, so find a local roofer willing to give you a free estimate on correcting your ventilation situation.
Is anyone on the forum near chicago? the length of the main ridge on peak of house is appx 65 feet, Gary
ridge vent
Regardless, I'd invest in a large power vent with a built-in thermometer trip switch. Make sure it is located on the cool/North side of the roof/peak or which ever has the most shade. But like jogr stated, check for insulation stuffed up too tight to the ridge.
65 feet of ridgevent on 2000sq ft? holy hannah, it should have no problem at all. the above post on intake airflow was also my guess. either not enough intake, blocked vents due to blow in insulation going everywhere or a mis-installed ridge vent.
Intakes are blocked: http://www.fureyco.com/content/image...ng_The_Air.pdf
The math- 150 min. or 300 min. with v.b.: http://www.airvent.com/homeowner/pro...it-specs.shtml
The intakes are way undersized if not continuous or minimum of 9 NFVA per foot: http://www.lomanco.com/intakemain.html
Interesting reads: http://www.advancedenergy.org/buildi...%20Studies.pdf
Be safe, Gary
65 feet of ridge vent is plenty if the slot is cut wide enough and not blocked with roofing felt or shingles. When you are up in the attic can you see the size of the slot and verify it's not blocked?
Can you see how much of the soffit is perforated and see if the soffit vent airflow is blocked? Do you have soffit vent chutes to keep insulation from blocking the vents?
DO NOT INSTALL POWER VENTS OR OTHER ROOF VENTS. They will ruin the effect of the soffit/ridge vent combination and cost needless money. What you have should work but it might need some blockage removal.
Grumpy and Ed the roofer are in the Chicago area.
The first thing we need to know is: "What is the "problem" you are trying to "solve"?
As Ed notes, the venting potential of ridge vents (and lower venting such as soffit vents as well) depends a good deal on roof configuration, it's not possible to have an informed opinion without seeing at least pictures of the roof/attic.
One concern I have is that if the recommendation for "2 attic fans" was for powered roof ventilation (fans drawing air from the attic and exhausting it to the exterior through the roof) it's unlikely a typical unmodified 2000 ft/2 attic provides sufficient make-up air for even one.
If not, you are going to be depressurizing the attic. In which case when the fan(s) are running the pressure differential (added to the inherent stack effect) will be drawing (perhaps a lot of) conditioned air from the spaces below into the attic, and worst case could be causing back-drafting of gas appliances in conditioned spaces.
It's *really* hard to find roofers, home inspectors or anyone else who really understands this stuff because there are so many variables: roof design, roof coverings, ventilation and insulation and vapor retarders/barriers (at a minimum) are all involved, and IMO except in the case of the simplest open attics under gable or hip roofs specification of solutions to such problems is a bit of a black art - you start by trying the simplest and least expensive solutions (that you believe won't make things worse) first, and then proceed on from there.
Tell me this, on a bright sunny day, if you go into your attic, turn off the lights, and see some day light being reflected in from your ridge vent, should this light be continuous throughout the entire length of the ridge? when I go up there I see some light here and there but certainly not consistent the with the whole length of the ridge which leads me to beleve the opening was not cut wide enough for the entire path,
Michael, the problem I'm trying to solve is if I have a properly installed ridge vent and eve vents to achieve the right air flow.
If the current attic ventilation is not creating problems or failing to solve them, then question then becomes: "Why change things?"
This is not a theoretical question - for example I just inspected a large turn-of-the-century three story Victorian home in Evanston that had *no* attic ventilation... and more than a hundred years later. no signs of problems as a result!
This is an extreme example, and I'm certainly not recommending that people build unconditioned attics with no ventilation.
But in the case of this Victorian while you can report that the ventilation is (radically) sub-optimal by current standards, you also have to ask yourself "What is the functional defect?
Which is why I am asking, "What is the functional problem you are trying to correct?"
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I carry if it's legal. No exception.
This is a discussion on Some of my more anti-friends are becoming more "accepting" within the Concealed Carry Issues & Discussions forums, part of the Defensive Carry Discussions category; *Cliffs at bottom* I am relatively open about my CHP and the fact that I carry "on occasion" (I carry as much as possible but ...
*Cliffs at bottom*
I am relatively open about my CHP and the fact that I carry "on occasion" (I carry as much as possible but they don't need to know that). So most of my good friends know. This has caused the usual "why?" questions and the typical non-understanding of some of my more anti-friends. All are "accepting" and don't give me any real grief about it.
Occasionally they will razz me about being "paranoid" and whatnot--all in jest. Well, recently they have started to become much more "accepting" of my gun. Since April the VT police have been doing a little CYA with email announcments about crime on or around campus. The last couple of weeks have been rather eventful around these parts:At approximately 7:00 pm, on 11/12/07, a female subject was walking on
the East side of Turner Street near Burger King, in the Town of
Blacksburg. She was assaulted by an unknown male, approximately 6'0",
170-180 lbs, wearing all black clothing and a black mask. The suspect
left the scene and has not been identified.A composite of the suspect in this case has been completed based on
information from the victim. To see this composite drawing, go to:
Suspect is a white male, approximately 5 foot 7 inches to 6 foot tall,
last seen wearing a dark hat, dark windbreaker/fleece jacket, and
loose-fitting blue jeans.
Suspect was driving a small, red, four-door car, possibly a hatchback.
Suspect was armed with a small sized, black handgun. The suspect is
right-handed and may be concealing the weapon in a waist level pocket or
in his waistband.t approximately 7:00 pm tonight (November 8, 2007), a person was
observed with a hand gun in the Stadium Parking Lot. A person reported
that he parked in the Stadium Lot and observed a subject next to a car
with a handgun. The subject pointed the weapon at the victim and then
fled the scene in the vehicle headed toward Airport Road. The weapon
was not fired and the victim was not harmed.
The suspect was described as a white male, approximately 6 feet tall,
170 to 200 pounds, 22 to 25 years old, and was wearing a black jacket
and black hat.
The weapon was described as a small dark colored hand gun.
The vehicle was described as red and small.
The community is urged to utilize all personal safety measures.
Anyone with information about the suspect or the incident is urged to
contact the Virginia Tech Police Department at 231-6411.On Wednesday, Nov. 7, 2007, at approximately 6:15 p.m., an individual was robbed and stabbed with a knife in the area of the 600 block on North Main Street.
The incident occurred in the alley behind Taco Bell, Bogens and Campus Exxon.
The suspect is described as a white male 5 foot 8 inches tall, medium build, wearing a grey hooded sweatshirt and light colored pants. The suspect was last seen running towards Progress Street.
We encourage everyone to be cautious and to use personal safety measures.
If you have any information about this incident please notify the Blacksburg Police Department at 961-1150.October 26, 2007
The Virginia Tech Police department is seeking your assistance in obtaining information about an incident that occurred on October 26, 2007 in the Chicken Hill parking area (located near Lane Stadium where Southgate Drive crosses Tech Center Drive) at approximately 11:30 p.m.
A student reported that while walking through the Chicken Hill lot, he was approached by four males and was subsequently punched and robbed. There were no weapons presented.
The males were described as white and older than college age. They may have been tailgating in the area of the first three rows of the Chicken Hill lot.
No other information is available at this time.
If you have any information, or have seen these individuals, please notify the Virginia Tech Police at 231-6411.This is an update concerning the male who, on two prior occasions, has approached young women and attempted to get them in the car with him by offering a ride. The first incident was at the Newman Library on Monday, August 27th. The second incident occurred in B-Lot on August 29th.
A student reported an encounter with a person on Giles Road, in the town of Blacksburg, that may be related to the two prior incidents on campus. The incident occurred on October 17th at approximately 4:00 pm. The student stated that a male stopped and offered her a ride. When she declined the offer, the male attempted to grab her and she fled.
The male is described as having a dark black complexion, medium build, appeared to be in his 30s, and was clean shaven with short hair. He was wearing a colorful hat and a black collared shirt. The vehicle is described as an 80s model tan or grey 4 door, with a boxy shape, similar to a Toyota.
If you have any information, or have seen a person or vehicle that matches the above description, please notify the Blacksburg Police Department at 961-1150 or the Virginia Tech Police at 231-6411.Well since we have been getting bombarded with these emails my friends that use to razz me if I was carrying at "safe places" have started changing their tune. A couple actually said "I hope you are carrying" and "its a shame the guy that got stabbed wasn't carrying" or "what does it take to get a CHP? Can I come shoot with you some time?".This is an update concerning the incident outside of Newman Library on Monday, August 27.
Information has been obtained that the possible suspect has changed his appearance.
A student reported an encounter with a person in the Commuter B parking lot between Prices Fork Road and Perry Street today. The student was walking to her vehicle when she was approached by a male in a vehicle. The male offered the student a ride to her vehicle. The student declined and the person drove away.
The male is described as a black male, dark complexion, late thirties to early forties, shaved head and clean shaven face who spoke with an accent. The vehicle is described as a 1990s small sedan and is green or tan. It is possible that this person is the same person involved in the incident outside of Newman Library and that he has changed his appearance.
If you have any information concerning this person, please contact Virginia Tech Police at 231-6411.
One of my friends is looking to get a new handgun as soon as he can afford it.
--Friends would mess with me about my chp
--Guy gets stabbed at Taco Bell next to campus
--Guy gets a gun pointed at him in the student parking lot
--Guy gets robbed in a parking lot on campus
--Girl gets assaulted at burger king
--Guy has been trying to abduct girls on and around campus
--Friends now "hope" I am carrying and some are now interested in getting a CHP/gun.
I carry if it's legal. No exception.
Les Baer 45
Sig Man
N.R.A. Patron Life Member
Too bad you don't see the one VCU sends/sent out while I was there about "incidents" around/on campus. Different place, same bs. Too bad VCU is specifically mentioned as CHP off-limits in VA State Code...
Vis consili expers mole ruit sua.
Good direction for your friends. Help them keep going there.....take them to the range, help them pick out a gun, etc. Your friends will make good pro-gun voters one day, and what an effective way to overwhelm the anti's.
Helpful hints on pushing back and strengthening the 2A:
Inform them you no longer believe in guns, no longer carry a gun and have seen the light of religion.
That aught to get a reaction.
It sounds like Blacksburg is a pretty rough neighborhood!
"Naked and Starving as They are We Cannot Enough Admire the Incomparable Patience and Fidelity of the Soldiery" George Washington, Valley Forge, 1777.
Good for them. Sounds like they are starting to open their eyes and want to take responsibility for their own safety.
"A well-educated electorate, being necessary to the continuance of a free state, the right of the people to keep and read books shall not be infringed."
Is this hard to understand? Then why does it get unintelligible to some people when 5 little words are changed?
I've had to deal with a lot of anti friends and even family. Some know I carry. Some don't. The friends I have that are anti... some are slowly coming around. Others would prefer I leave the gun at home. Too bad for them. I've turned quite a few anti's myself. Too bad my family doesn't get it. My mom will go shooting with me but refuses to accept that I carry. Its a tough situation sometimes but I'm glad your friends are powering through it.
The Gunsite Blog
ITFT / Quick Kill Review"It is enough to note, as we have observed, that the American people have considered the handgun to be the quintessential self-defense weapon." - Justice Scalia, SCOTUS - DC v Heller - 26 JUN 2008
You have "friends" that are anti's??????????????
J/K Good work, continue to be the encourager and maybe you can convert them.
Lex et Libertas Semper Vigilo, Fortis, Paratus, et Fidelis!
"Not only do the people who put their lives on the line to protect the rest of us deserve better, we all deserve better than to have our own security undermined by those who undermine law enforcement." -Thomas Sowell
Certainly sounds like a rough area! Nice to see that once folks become aware of their surroundings they start taking a more realistic approach to protecting themselves.
The ultimate result of shielding men from the effects of folly is to fill the world with fools.
-Herbert Spencer
NRA Life Member
That was my very first thought.
Tell them you sold all your guns and the evil bullets and went to a Brady seminar, drank their free bottled vitamin water, and are now a vegetarian pacifist and are a believer that bad things really as per Brady stats only happen to bad people and gun owners.
I'd pay money to see video of their reaction to that.
- Janq
"Killers who are not deterred by laws against murder are not going to be deterred by laws against guns. " - Robert A. Levy
"A license to carry a concealed weapon does not make you a free-lance policeman." - Florida Div. of Licensing
Good now take them to the range and let them shoot a few rounds, bet they take up the sport too.
"The sword dose not cause the murder, and the maker of the sword dose not bear sin" Rabbi Solomon ben Isaac 11th century
Just talked to my friend that wants to get a gun. We are going up to an indoor range to shoot this thursday..and let him rent a few different models.
Sounds to me as if you are the most level headed and logical thinking of the group of people you hang with.
That is not always a good thing. But if they are starting to come around to the proper way of thinking, then you are doing good work and setting a good example.
,=====o00o _
l_--- \___l---[]lllllll[]
(o)_)-o- (o)_)--o-)_)
Of all my friends, only two know I carry (they carry as well)..I really don't know if any of my other friends are anti or not..as for them knowing that I carry, thats a #51!!


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- Stylist:
- Matthew James
Feb 13, 2010 - I went to Matt for a cut on February 13, 2010. My previous haircut was awful and I needed a rescue haircut desperately. Matt is a fantastic hairdresser. He listens to what you have to say and offers lots of suggestions. The cut is now perfect and I love y hair again. He is lovely and listens to what you ask, he understands hair and explains what he is doing all the time. I have now found the hairdresser I trust.


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Hey guys...I really enjoy your forum and frequent it often to check in on any good racing stories....oh ya, and cause that one guy who you all attempted to bann crack's me up....rsxsucks or whatever his name is. I never post so I thought I'd contribute today.
I own a 2002 S4 with a simple cat back exhaust and GIAC X-Chip. I see a few REX's around Fort Wayne here...a few silver one's and a kid here own's a sweet blue REX. I kinda know the kid who owns the blue REX...I raced him earlier this year on the highway...actually...his friend was driving his car for him...we took them out onto the highway and did a few runs from 60 on. He had just put a large intercooler on the car and I think bigger turbo (not sure). I only had my chip at the time. Anywhos...from 60 on up to about 120? we were pretty much dead even. I didn't downshift the first time we attempted it...I think I was in Fifth gear and we hung bumper to bumper. The second time I downshifted to third and very very slowly walked away from him. Well, that was the only time I had a chance to run against a REX...every other time I see one around they look at me like I'm crazy or don't acknowledge that I want to play. I just don't understand...maybe cause it's during the day when I see the other REX's...I should go strolling at night more often. Anywhos, thanks for the great forum and stories and I love the Scoobie...it's such a flipping nice car and it's potential is insane! If any of you reading this live in Fort Wayne...the next time you see a black S4 with mirrored windows pull up alongside of ya...let's take em for a spin! Good day to everyone


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Discussion in 'Fantasy Sports Central' started by webz, Nov 28, 2011.
The league will be in safe hands [INSERT EVIL LAUGH HERE].
UHm, nice reminder Laimbeer.....
LOL. WebZ is the commish.
Yeah but I thought I clearly elected you "Roscoe's Nanny" Anyway, I like my team. Autodrafting could have gone much worse...
On one hand I'm kicking myself for missing out my #1 pick. I should have at least set that one up but I was sure I'd be around for the draft. On the other hand, I got an nice team.
Didn't set anything up, and then didn't make the draft until the very late rounds. By that time, I just let autopick finish doing it's thing, because I had no idea what it's strategy might me. I dropped DeMarcus Cousins, and then apparently used my 2nd pick to get him back. What?! Oh well. Maybe this will be his breakout year.
I've seen you draft before, Rascoe. You are better off autodrafting!
Separate names with a comma.


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If you've been reading Moderncat for a while, you know that I try to avoid clay litter for a couple of big reasons. First, it is not bio-degradable nor is it a renewable resource, making it an environmental nightmare. Second, silica, found in all clay litter, is a known carcinogen when inhaled (by both cats and humans), and sodium bentonite found in clumping clay litter, may cause serious health problems for your cats when ingested by grooming after using the litter box.
Clay litter is, however, still the most commonly used cat litter and it's tough to find non-clay alternatives that 1) are available everywhere, 2) are affordable, and 3) perform well. Performance features I look for in a good cat litter include:
- Clumpability – Is it easy to scoop?
- Odor control – How well does it control odor?
- Texture – Do the cats like it and will they use it?
- Dust – How much dust is produced when pouring and scooping?
- Scent – What does the litter smell like? Natural smell or chemical perfume?
- Trackability – Does the litter track easily out of the box or is it heavy enough to stay contained in the box?
- Longevity – How long can you use the same litter (scooping and adding a little new litter) without needing to dump the entire box and refill?
- Weight – How heavy is the bag to lift and get home from the store?
So, back to the cost issue, I have a lot of cats and a lot of litter boxes, so I go through A LOT of litter. At the moment, I'm most concerned with finding a non-clay litter that is affordable and I've been experimenting a little. I found out that chicken crumble — used for feeding chickens — is pretty much the same stuff that the commercial corn cat litters are made out of. I went to my local feed store and bought a bag and, guess what? It works! I did a little searching online and this is a common thing to use. The commercial corn litters run about $1/lb and the chicken feed is about half that.
Now, this is not a perfect solution. One thing I learned is that all corn chicken feed will have bugs living in it. They may be dormant, but in a warm and damp climate, they are likely to hatch and crawl around in the chicken feed. Yeah, gross, I know. So the chicken feed litter may not work for everyone. I happen to have all of my litter boxes out on the catio, so if there are any bugs, they won't get in the house, at least they haven't so far the few times I've noticed them. I also live in the desert, which for most of the year is ideal for using chicken feed litter. I have read horror stories about bug infestations inside, so be warned.
Other than the bug issue, the chicken feed works pretty well. Clumping is good, the texture is perfect and the cats love it. It is pretty dusty and has a "farm-y" smell, but so do the commercial corn litters. All corn litters, I've found, break down over time and do have to be completely dumped and replaced fairly frequently, depending on how many cats you have using the box.
As far as I can tell, the commercial corn litters cost twice as much because they treat the corn (probably by freezing it) to kill the bugs for you, plus they add some clumping and odor control agents. I suppose you could try freezing the chicken feed yourself, but I'm not sure how long it would need to remain frozen, and you'd need a really big freezer. If anyone has tips for additives (baking soda, maybe?) to help with clumping or odor, please leave a comment on this post to share your ideas.
I've been purchasing 40 or 50 lb. bags of chicken feed at a local feed store. Since raising chickens is really "in" right now, you can probably find chicken feed at more and more regular pet supply stores. I bought a sturdy plastic bin on a rolling card with a lid that seals for storing the feed. The feed stores also have nice big scoops that work well for transferring the litter to the box. I've been saving quite a bit of money on litter and I'm happy with the results.
Another budget-friendly clay litter alternative I've read about, but I haven't tried myself, is to use wood pellets made for stoves. These are apparently similar to the wood pellet litters available in pet stores. Others have reported excellent results. I would say that you want to look for wood pellets that are not treated with any chemical additives.
I know I'm always posting designer litter boxes that cost hundreds of dollars, but sometimes you just need plain old utility. I've found that cement mixing tubs, available at any home improvement store, work REALLY well for litter boxes. They are designed for easy mixing and scooping, so they have a cuved bottom and no sharp corners inside — perfect for easily scooping litter. They come in different sizes, the small tubs are around 24″ x 20″ x 6″ and cost $6 or $7 and the large tubs measure 24″ x 36″ x 8″ and only cost about $13. I have a large one tucked away on the catio and it is absolutely perfect for a multi-cat household.
So there you have a few budget-friendly cat litter ideas. Maybe not so stylish, but in this economy, we sometimes need to focus on function. Please feel free to leave your own cost-saving litter ideas in the comments!