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A Beginner's Guide to Email Automation
As a business owner, ongoing email communication with your customers is essential. You need to let them know about upcoming sales and promotions, send out order updates, or even touch base with a personalized birthday message.
At the same time, composing each one of these emails one by one would be a daunting, time-consuming task, especially as your customer base grows. That's why it's never too early to begin your email automation efforts using the email marketing tips below.
Overview: What is email automation?
Automating emails allows you to send customers and subscribers emails based on predefined events or "triggers." These can include an account setup welcome message or other information, including an order confirmation, order shipped/delivered, sales events, suggested products, or requests for product reviews.
What are the benefits of automating your emails?
Not only will email automation save you the time of individually composing and sending each message, but the personalization options available and the recipient metrics generated will also help you maintain and grow your customer base.
1. Customer relationship management
Most businesses spend more time trying to acquire new customers than retaining the ones they already have, even though it costs up to seven times more to attract a new customer than to keep an existing one.
Automated emails will keep you front and center with your current customers by providing the timely information they want.
2. List segmentation
The days of the email blast, a single email sent to everyone in your contact management system, are long gone because one size does not fit all.
Instead, you can combine automated email campaigns with customer segmentation to target recipients by gender, location, education level, buying patterns, or other demographic information. This is not limited to online retail sales but also applies to business-to-business (B2B) email marketing.
3. Performance metrics
Marketing used to be a black box: You paid for newspaper ads or sent out direct mail pieces, but who really knew how effective they were, or weren't? Email marketing has changed all that. Now you can track metrics, including how many emails were opened, forwarded, and which links back to your site were clicked.
How to set up email automation for your small business
The key to effective email automation is planning, everything from selecting the best email marketing software, all the way to choosing which metrics to focus on. By being thoughtful upfront in your approach to setting up your email automation, you'll produce actionable results more quickly and save time in the future.
Step 1: Choose an automated email service provider
The first thing you'll discover is that there are dozens of companies that say they provide the "best" email marketing services. While many of their features are similar, you'll want to do some careful research, hence, our suggested options below, to determine the right fit for your needs.
Pro tip: You probably don't need an enterprise software solution with all the bells and whistles that costs hundreds of dollars a month right off the bat. Instead, choose a provider that will allow you to start small and then scale up as your needs grow.
Step 2: Formulate your email strategy
In addition, you want to begin with a simple email strategy. That is, you don't need to have half a dozen or more different emails you send out from the get-go. Instead, choose one or two types of triggers and resulting messages to focus on as you become familiar with the ins and outs of the software you're using.
Pro tip: One standard message you'll want to send out is a welcome email when a customer sets up an account at your business. In addition, include a coupon/discount for them to use so you can begin generating follow-up sales from existing customers.
Step 3: Map out your workflow
Quality and consistency are critical to establish and maintain credibility with your customers. That includes everything from how to start an email, to its layout and design, to careful proofreading. Plus, you'll need to decide exactly who will be responsible for completing each element.
Pro tip: Use Gantt charts, which can be produced with most project management software, to build a visual representation of the workflow for every type of email, identify who is responsible for each task, and create a production timeline with milestones.
Step 4: Build segmented lists
Both customer and market segmentation are key to maximizing the return from your overall email list. You don't pay much attention to direct mail pieces that show up in your physical mailbox because they're hardly ever that relevant.
List segmentation will allow you, however, to personalize emails to groups of customers based on their prior behaviors, preferences, and demographics.
Pro tip: When it comes to segmented lists as opposed to a welcome email that is sent out uniformly, focus on your customers who have made the most purchases. Research shows that each successive purchase by a customer increases the odds they will make another one in the future.
Step 5: Analyze the metrics
Email marketing automation also allows you to collect a variety of different information about the recipients of your emails and how they interact with them. Basic metrics include open rates, bounce rates, clickthroughs, and forwards/sharing.
Pro tip: A/B testing allows you to test two iterations of the same thing to determine which one performs better. For example, you can use two different email subject lines for the same email to determine which one results in the highest open rate. Then, you can model future subject lines on the one that was most successful.
The best email marketing software for setting up email automation
It's easy to become overwhelmed by all the email services on the market, so here's a brief overview of some popular and highly rated providers. Plus, when you take a look at their sites, you'll find a variety of email marketing examples that can provide ideas and inspiration for your own efforts.
They also have resources on effective email formats and how to create an email newsletter.
1. Mailchimp
Mailchimp is an industry leader suitable for both novices and experienced marketers. You can choose from automation templates that include first-time customer welcome, customer loyalty rewards, order notifications, abandoned shopping cart, purchase follow-up, and customer engagement.
There's a free option all the way to a $299/month premium plan.
2. Campaign Monitor
Campaign Monitor is also suitable for beginners and experienced marketers, but its interface is a bit dated compared to its competitors. It does offer robust email automation, which it calls "journeys," that can be set up even by novices, thanks to a series of questions based on industry, goals, and customers to target.
Plans range from $9 to $149/month.
3. Constant Contact
Constant Contact offers an impressive combination of usability and functionality. At the same time, however, its list segmentation capabilities are limited, and unlike Mailchimp, there's no free option for you to try out. Plans start at $20/month.
Begin your email automation now
Email automation is not a business requirement you might consider implementing at some point in the future. Instead, small business email marketing is a necessity you need to begin using as soon as possible in order to reap its tangible rewards.
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Women's Historical past
Typically, it is arduous to know whether you are truly getting a great deal on something. We know that some of the Morgies have already been diagnosed with eczema, sarcoidosis, lyme disease, and various other ailments, and a few of these people have forgone treatment and opted for a self-prognosis of Morgellons. The typical woman generates 300 pounds of tampon trash in her lifetime.
Musicians Buddy Holly and John Lennon grew to become synonymous with the kinds of eye-glasses they wore to the point that thick, black horn-rimmed glasses are often known as "Buddy Holly glasses" and completely round metallic eyeglass frames called "John Lennon (or Harry Potter ) Glasses." British comedic actor Eric Sykes was known in the United Kingdom for carrying thick, square, horn-rimmed glasses, which had been, in fact, a complicated hearing assist that alleviated his deafness by permitting him to "hear" vibrations.
We research the very best costs on-line and in retailer, all so it can save you money and save time. In the event you're simply on the hunt for a deal or wish to know what main sales are occurring, check out our High 20 Offers web page for discounts and concepts. Worth Pushed (23{4cabc7684090e8bc2e1fe690a4ed76e184bc5b172fd48cf957ca354434245ec4}) With fastened budgets for grocery shopping, shoppers in this group are more likely to make a shopping checklist, examine prices and go out of their strategy to discover the shops with the lowest prices.
Your eyes have to bend mild rays so the image could be focused sharply on …

subset: commoncrawl


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WPForms – GetResponse
You'll be able to expand your email subscriber base by using the WPForms GetResponse addon, which enables you to generate registration forms for your GetResponse newsletter directly within WordPress.
GetResponse is a great option for email marketing; do you use it? Do you want to make signup pages for GetResponse in WordPress that are more intelligent?
Our goal at WPForms was to make it as simple as possible for our customers to generate unlimited signup forms for their newsletters using GetResponse.
Through the integration provided by our GetResponse addon, you will have the ability to rapidly generate a newsletter registration form and connect it to your GetResponse account.
The GetResponse addon gives you the ability to choose which email list and segment the subscriber should be added to, giving you complete flexibility in your marketing strategy.
You will be able to construct hyper-segmented email lists for optimum engagement and results by combining this feature with our intelligent conditional logic.
You are able to use our GetResponse addon with any of your other forms, in addition to the subscription forms, including your regular contact form, a form to request a quotation, and pretty much any other form you have.
This makes it possible for you to provide your users with numerous opportunities to sign up for your newsletter, allowing you to achieve the greatest possible number of subscriptions.
GetResponse provides you with the capacity to map form fields to a variety of combine fields, which means that you retain complete control over the mapping process.


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Genre: Fantasy
The Demon Kiss (Blackthorn Hunters Academy Book 1)
A young woman with a secret heritage. A man who made a deal with the devil. And an academy where the students are just as dangerous as the demons... Forge your weapons and cast spells in a school where demons are hunted and the hunters themselves become prey.


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Kyle H on Tue, 28 Jun 2005 08:14:52 -0500 (CDT) |
[Date Prev] [Date Next] [Thread Prev] [Thread Next] [Date Index] [Thread Index]
Re: [eia] Third round of Berlin |
Am I right in thinking that the Austrian stack retreats all the way back to the open space between Brunn and Vienna? Since there are no Austrian depots, the stack must retreat towards Vienna. The closest space would be Wittenberg, but there is an unbesieged corps (Saxony) there. So the stack routes to Theresienstadt, but there is a garrisoned enemy depot there. So the stack routs to Prague, but there is an unbesieged enemy corps there. So the stack routs to the open space between Brunn and Vienna. Is this correct? kdh _______________________________________________ eia mailing list eia@xxxxxxxxx http://lists.ellipsis.cx/mailman/listinfo/eia


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NLC Jobs National Logistics Cell 2021 Latest Jobs
NLC National Logistics Cell Department announced jobs 2021 NLC Department invites application all over Pakistan. the posting of these posting in all over Pakistan . The source of these jobs is daily Express newspaper of Date 11th July 2021 multiple number of post in this Jobs Ads. There are many type of post mention below . in this We will notify to all here government Jobs 2021 in all over Pakistan .The location of this job All over in Pakistan. Interested candidate can easily check all detail in below in this page. These announced in official Website and daily Express Date 11th July 2021 and the the last Date is 26 july 2021
Here In this page we informed to all of you here a chance to get a honorable jobs in NLC National Logistic cell Department is All over Pakistan . The confident and suitable candidate of can Apply for these posting.
Source: | Express Newspaper |
Posted Date: | 11July 2021 |
Department: | National Logistic Cell |
No of Posts: | Multiple . |
Education: | Middle , Matric , Intermediate , Bachelor |
Job Location: | Pakistan |
Address: | Human Resource Branch Headquarters Tolling NLC sawan Campus Rawalpindi |
Last Date: | 26 July 2021 |
National Logistic cell invites Application from all over Pakistan . These job posted in Official Website and the and daily Express newspaper in Dated 11/07/2021 and the last Date of these job in 26 july 2021. These jobs are Different types likes Assistant Toll plaza manager CCTV Camera Monitor Cook Fire Fighter Lady Searcher LTV Driver HTV Driver Security Guard Security Supervisor Senior Security Supervisor Shift Incharge Sweeper Toll Manager and many Other
The eligibility and term condition for and the these post are different candidate can check in official ads official website of Below in this post. .
The Education is required According to post Bachelor Master Middle matric primary and relevant Diploma and Experience where applicable to post .
check in official #ads. The age limit# According to post below Interested Candidate Can easily Apply within due date announced of jobs Advertisement.
CCTV Camera Monitor
Cook Fire Fighter
Lady Searcher
LTV Driver
HTV Driver
Security Guard
Security Supervisor
Senior Security
Shift Incharge
Sweeper Toll Manager
- Educational Certificates
- Experience Certificate
- Domicile copy
- 2 x Photographs
- Application procedure mention in below ads
- All details given in official Add .
- Original Document will produce to interview time.
- No TA/DA will be given.
- The Age limit is According TO POST
- Army retired can also apply.
- Govt employee can apply through proper chennal.
- Incomplete application not accepted.
- The last Date of Apply is/23/07/2021
HR Branch Headquarter tolling palaza Rawalpindii


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Tim and Rinny Launch Coaching Business Leveraging TriDot
March 21, 2023
Rinny's roster has limited availability, and Tim & Rinny's World is open to all.
DALLAS, TX – March 21, 2023 /ENDURANCE SPORTSWIRE/ – Mirinda ("Rinny") Carfrae and Tim O'Donnell announced the launch of their coaching business that will leverage TriDot's Optimized Triathlon Training®, technology driven by data and artificial intelligence. Their coaching business will focus on triathlon enhancing the lives of athletes through proper life balance, adventure, and family.
"I've had so many wonderful coaches over my career in triathlon, and now I want to give back to the athletes in the sport that gave me so much," says Mirinda Carfrae, 4x IRONMAN World Champion who recently announced retirement from professional triathlon racing. "Leveraging the world's best training technology, TriDot, allows me to spend my time doing what appeals most to me about coaching, which is to guide and support athletes."
Rinny is opening her 1:1 coaching roster to a limited number of athletes. All triathletes are welcome to be part of Tim & Rinny's World, which offers TriDot's top-tier training functionality, interaction with Tim & Rinny through exclusive social media groups, periodic virtual and in-person events, and access to Tim & Rinny's partner discounts. Learn more about either option by visiting their website. Tim will continue racing this year and will not open his roster until after this season.
TriDot delivers Optimized Triathlon Training® powered by data & artificial intelligence that produces better results in less time. It can be used with or without a coach and has subscription levels to fit any budget. Embraced by tens of thousands of athletes of all levels and hundreds of coaches worldwide, including elite coaches like Mark Allen (6x IRONMAN Champion) and Michellie Jones (Olympic Silver Medalist and IRONMAN Champion), and now Tim & Rinny, TriDot is the result of over 18 years of ongoing research and development. In 2022 alone, TriDot was featured in Forbes, Tech Times, Triathlete magazine, Men's Journal, NY Weekly, and LA Weekly.
"We couldn't be more excited to welcome the power couple of Tim & Rinny into the world of triathlon coaching and to support them with TriDot's cutting-edge coaching platform and community of elite coaches," says Jeff Booher, CEO of Predictive Fitness, parent company of TriDot. "They have such high integrity and a passion to help others improve and succeed, a perfect alignment with our company values."
TriDot is a software-as-a-service (SaaS) technology and performance science division of Predictive Fitness, Inc. that provides Optimized Triathlon Training® for triathletes. The platform of choice for top coaches like Mark Allen, Michellie Jones, Timothy O'Donnell and Mirinda Carfrae, TriDot handles the analytics and training program design, giving coaches more time to work with their athletes. Its patents-pending technology uses each athlete's biometrics and training data along with its own proprietary big data and artificial intelligence engine to design and optimize training for athletes, with or without a coach. It produces substantially greater results in up to 30% less training time. www.TriDot.com
Originally from Australia, and now residing in Boulder, Colorado, 4x World Champion Mirinda "Rinny" Carfrae is a one of the world's most recognizable triathletes. Rinny was a threat at every race she entered. In 2023, she retires as one of the greatest triathletes of all time, with over 50 wins at major events throughout the world and seven podium performances at the IRONMAN® World Championship in the span of a decade. Rinny's passion for the sport will shift to coaching with TriDot, allowing her to connect with new athletes on an innovative platform that uses technology to optimize feedback on training and racing for fast results. Interested athletes can inquire directly from her coaching website. This next stage of her career will be supported by her long-term partners GU Energy, Garmin, Hyperice, MasterSpas, Athletic Greens and Inside Tracker. Although not ruling out future running races, retirement from professional triathlon racing will allow Rinny more cherished time with family, especially with her active kids Isabella (5) and Finn (2).


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Heaven's Prisoners by James Lee Burke PDF Download
Download Heaven's Prisoners by James Lee Burke PDF Novel Free. Heaven's Prisoners is the fiction, thriller, literature, redemption, thriller and mystery novel that offers the story of a former Vietnam vet and a detective.
Description of Heaven's Prisoners by James Lee Burke PDF
Heaven's Prisoners is the thriller, mystery, fiction, literature, redemption and mystery novel that describes the story of a detective who is trying his best to save the city from violence. James Lee Burke is the author of this superb novel. He is the bestselling author in the New York Times. Dave Robicheaux, Molly Alafiar and Rocque are the prominent characters of the story. Dave fought for his country in the Vietnam War for several years. He is known for his clean and quick missions. Dave is one of the high rated soldiers of his badge and he never disappointed his people and country.
After retiring from the army he joined the police as a homicide detective. He worked few years in New Orleans and he makes his impact in society. The people are happy with his performance and Dave is known as a specialist in dealing with the mob bosses. Now, he is going to Louisiana with his wife after the posting. The street crimes and gang rivalries is on the top and dozens of people are killed in a few months. Dave was hopeful to control the crimes and then a beautiful little girl enters into Dave and his wife's life after the plane crash. Rocque's wife is suspicious of the last few days and she is planning some great violence in the city. Will Dave able to stop her before she begins?
Details About Heaven's Prisoners by James Lee Burke
- Name: Heaven's Prisoners: A Novel (Dave Robicheaux Mysteries)
- Authors: James Lee Burke
- Publish Date: April 1, 1988
- Language: English
- Genre: Fiction, Thriller, Literature, Mystery
- Format: PDF/ePub
- Size: 1 MB
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Utah Sweet Savings may earn a small commission via affiliate links in this post. Read our full disclosure policy here.
If this FurReal Pet is on your list this Christmas, Amazon and Walmart both have it at its lowest price yet! My kids love this toy!
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Easy Price Comparison: Target is selling this for $49.


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@FPA Forums Team @FxMaster - your attention here pleaseI am pretty sure this is the motel where eaglefx.com has registered as its address.
eaglefx.com is one of the bigger advertisers here at fpa.
obviously, fpa is a straight up bucket shop.. and i want to promise you that bucket shop = you gonna lose yo money... they might process some withdrawls, but it is an exit scheme... AND FPA ACCEPTS THEIR ADVERTISING DOLLARS?
View attachment 71518
View attachment 71519
View attachment 71520
That was obviously a typo.obviously, fpa is a straight up bucket shop
nope.Instead of screenshotting something from BabyPips as proof, you should double-check the information first.
So far, EagleFX has a good record in the reviews.
EagleFX is not tagged in any Scam Alerts threads.


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Background: The main purified compound from soy protein isolates is genistein, but this purified phytoestrogen fails to recapitulate all the features of the soy-based diet that is associated with lower incidence of prostate cancer. Materials and Methods: Rat and human prostate cancer cell lines were treated with either soy protein isolates or purified genistein. In vitro cell growth was correlated with the associated genomic expression profiles using cDNA arrays. The data was subsequently bioinformatically analyzed within and across species to identify common changes in expression profiles associated with the soy protein or genistein treatments. Results: Gene expression profiling and data mining noted genes specific to soy; however, biological pathways showed the same gene regulation profiles between genistein and soy. Conclusion: Genistein is likely the major contributor to the effect of soy proteins on cellular pathways; however, the expression of different genes using soy protein isolates suggests complexity in the many compounds found in whole soy protein.
Prostate cancer is the most common solid tumor and the second leading cause of death among American men (1). Additionally, the risk of prostate cancer is 1 in 6 and the risk of death due to metastatic prostate cancer is 1 in 30 (2). Prostate cancer is a heterogeneous disease ranging from asymptomatic to a rapidly fatal systemic malignancy (3). Aggressive prostate cancer is significantly less prevalent among Asian men and more in African American men (4). Another common description of prostate cancer is that of hereditary versus sporadic cancers (3). This division occurs at the epidemiologic level but fails to distinguish their behavior or molecular activity. While some genes have been identified that confer a reasonable level of risk for prostate cancer in families, most prostate cancers appear to be sporadic. The sporadic tumors most likely arise from the interactions of multiple genes and environmental effects that in combination lead to increased prostate cancer risk and tumor formation (5). Factors such as diet are thought to cause a fair majority of the genetic alterations leading to cancer.
The incidence and mortality of prostate cancer vary widely in different regions of the world. Some research points to high rates of prostate cancer attributed to food products, most commonly animal fats. On the contrary, decreased incidences were found in those populations with increased intake of fruits, vegetables, and vitamins (5). A large number of different chemicals in foods has been implicated in cancer prevention. Particularly in regard to prostate cancer, the administration of anti-oxidants (e.g. selenium, vitamin E, lycopene) and the consumption of foods rich in carcinogen-detoxification enzyme inducers (e.g. vegetables containing sulforaphane) have been the focus of recent studies and clinical trials (5). Epidemiological studies show that the risk for death from prostate cancer in the United States is elevated compared to other countries, especially Asian countries. It has been suggested that this result may be rooted in the environmental affect on diet rather than genetic profile (6). This is based on the increased incidence of prostate cancer in Asian men that have altered their food consumption and become accustomed to an American diet.
Soy protein in particular is consumed in much higher quantities in Asian cultures than in regular American diets. High consumption of foods containing soy results in high plasma, urine, and prostatic fluid concentrations of phytoestrogens, including genistein, and may play a role in the lower incidence in hormone-dependent cancer in Asian countries (4-8). Phytoestrogens are plant compounds found to have weak estrogenic affect on cells by showing a chemical structure similar to 17β-estradiol. Lignands, flavonoids, and isoflavonoids make up the different categories of phytoestrogens. The isoflavonoids that are highly studied include genistein, daidzein, formonentin, and biochanin A. However, genistein and daidzein are thought to be the most important isoflavones in prostate cancer. Many different foods carry varying concentrations of these phytoestrogens, but soy holds the highest concentration. It is this source of protein that is consumed in high quantities by Asian peoples in their native countries and may play a part in the lower incidence of hormone-dependent cancer in those countries (5).
Previously, Dr. Morris Pollard had performed studies showing decreased cancer development in Lobound-Wistar rats that consumed a soy diet compared to controls (10-12). Other animal studies, such as those with the TRAMP mouse model, have shown dose-dependent reduction in the incidence of prostate cancer in relation to increasing levels of genistein (4). Similar affects have been shown in treating tumors of LNCaP androgen-sensitive prostate cancer cell line in SCID mice fed low-fat and high soy diets (13). Thus using various animal models, soy continues to recapitulate the effect seen in the earlier epidemiological studies showing the decreased incidence of hormone-dependent prostate cancer in association with high levels of soy consumption, showing probable cause for human studies. Though multiple different rat models are promising, human studies have shown mixed results.
This current research seeks to elucidate the underlying molecular basis of the effects of soy protein on prostate cancer, while providing potential biomarkers for future evaluation of clinical trials using soy proteins. The studies include solublizing soy protein isolates into cell culture medium, applying this medium to human and rat prostate cancer cell lines, and observing changes in cell proliferation through growth curves. In addition, RNA was isolated and examined using cDNA microarray studies to identify changes in gene transcription. This information will provide a basis for developing a molecular explanation of soy protein anticancer affect.
Materials and Methods
Cell culture and soy or genistein treatment. The study used human and rat prostate cancer cells lines PC3 (human) and PAIII (rat). The PAIII cell line was selected due to its origin and commonality to the Lobund-Wistar rat prostate cancer (metastatic origin). Soy protein isolates have been obtained from Dr. Morris Pollard (Notre Dame, IN, USA), and are from the identical lot used in the associated Lobund-Wistar rat studies (10-12). The material was originally obtained from Harlan TekLad Diets in Madison, Wisconsin. Genistein was obtained from Sigma (St. Louis, MO, USA) (MDL Number: MFCD00016952). Cell lines were received from ATCC, Rockford, MD, USA (LNCap), from Drs. Paul Lindholm and Andre Kadjacsy-Balla (MCW, Milwaukee, WI, USA) (PC3), or Dr. Morris Pollard and Mark Suckow (Notre Dame, South Bend, IN, USA) (PA-III). These cells were cultured in RPMI supplemented with 10% fetal calf serum, 10 mM glutamine, and 10mM sodium pyruvate, and passaged 1:8 or 1:10 when the cells reached 70-80% confluence with trypsin-EDTA passaged before use in the experimental protocols. Soy protein was solubulized in a 0.005% NaOH and water solution. After solublization and sterile filtering, the soy protein was added to the medium of the cancer cells. These cells were then treated with various log concentrations (from 10 to 1000 mg/mL) of the soy protein isolates and their growth rates were measured by growth curves on PAIII, PC3 and LNCaP cell lines (Figure 1a, 2a, and 3a, respectively). From these growth curves using log concentration, the concentration of 100 mg/mL was selected for this study. These preliminary growth curve studies were also performed with genistein, a well-studied soy protein isolate. All growth curves were performed in triplicate and the average number of cells is displayed for each data point. Concentrations of 20 mM and 100 mM genistein were used for growth curve studies in both cell lines (Figure 1b, 2b, 3b, respectively).
RNA isolation and quantization. RNA was isolated from cells using Trizol (cat #15596018; Invitrogen, Carlsbad, CA, USA) and subsequently examined for quality using agarose gel electrophoresis and Gelstar nucleic acid stain against known RNA standards and failed to demonstrate significant degradation based on the presence of high molecular weight RNA species, and intact 28S and 18S ribosomal RNA bands. RNA, DNA and protein were isolated from the cells treated with either 100 mg/mL soy protein isolates or 20 mM genistein grown in 80% confluence over 5 days.
DNA library preparation and amplification. Libraries were prepared as previous described (14, 15). Sequence-verified rat and human libraries (Research Genetics, Huntsville, AL, USA, and University of Iowa cDNA clone set, IA), consisting of 41,472 human clones and 36,000 rat clones were used as a source of probe DNA. Subsets of 200 randomly selected clones had previously been chosen from these libraries, resequenced locally and demonstrated clone accuracy of 92%. We have opted to reformat libraries from 96 to 384-format for culture growth/archiving, PCR, purification, and printing. This has reduced the number of plates of our 41,472 human clone library from 432 to a more manageable 108, and the rat clone library from 375 to 94. The library was reformatted and subsequently manipulated using slot pin replicator tools (VP Scientific, San Diego, CA, USA). Cultures were grown in 150 μl Terrific Broth (Sigma, St. Louis, MO, USA) supplemented with 100 mg/ml ampicillin in 384 deep-well plates (Matrix Technologies, Hudson, NH, USA) sealed with air pore tape sheets (Qiagen, Valencia, CA, USA) and incubated with shaking for 14-16 h. Clone inserts were amplified in duplicate in 384-well format from 0.5 μl bacterial culture diluted 1:8 in sterile distilled water or from 0.5 μl purified plasmid (controls only) using 0.26 μM of each vector primer (SK865 5'-fluorescein-GTC CGT ATG TTG TGT GGA A-3' and SK536: 5'-fluorescein-GCG AAA GGG GGA TGT GCT G-3') (Integrated DNA Technologies, Coralville, IA, USA) in a 20 μl reaction consisting of 10 mM Tris-HCl pH 8.3, 3.0 mM MgCl2, 50 mM KCl, 0.2 mM each dNTP (Amersham, Piscataway, NJ, USA), 1 M betaine, and 0.50 U Taq polymerase (Roche, Indianapolis, IN, USA). Reactions were amplified with a touchdown thermal profile consisting of 94°C for 5 min; 20 cycles of 94°C for 1 min, 60°C for 1 min (minus 0.5° per cycle), 72°C for 1 min; and 15 cycles of 94°C for 5 min; 20 cycles 94°C for 1 min, 55°C for 1 min, 72°C for 1 min; terminated with a 7 min hold at 72°C. PCR reactions analyzed for single products by 1% agarose gel electrophoresis analysis. Products from replicate plates were pooled and then purified by size exclusion filtration using the Multiscreen 384 PCR filter plates (Millipore, Bedford, MA, USA) to remove unincorporated primer and PCR reaction components. Forty wells of each 384-well probe plate were quantified by the PicoGreen assay (Molecular Probes, Eugene, OR, USA) according to the manufacturer's instructions. After quantification, all plates were dried down, and reconstituted at 150 ng/μl in 3% DMSO/1.5M betaine.
Array slide fabrication. Array slide preparation was performed as previously described (14, 15). A single printing array containing 19,200 elements (human) or 2 arrays of 9,600 (rat), were printed on poly-L-lysine coated slides prepared in-house (1-2 arrays/slide) as previously described (14). Printing was conducted with a GeneMachines Omni Grid printer (San Carlos, CA, USA) with 16 or 32 Telechem International SMP3 pins (Sunnyvale, CA, USA) at 40% humidity and 22°C. To control pin contact force and duration, the instrument was set with the following Z motion parameters, velocity: 7 cm/s, acceleration: 100 cm/s2, deceleration: 100 cm/s2. All slides were post-processed using the previously described non-aqueous protocol (13). Slide coating was performed as described previously (http://cmgm.stanford.edu/pbrown/mguide/index.html). Image files on all arrays were collected after blocking (fluorescein), and again after hybridization (Cy3 and Cy5) with a ScanArray 5000 (GSI Lumonics, Billerica, MA, USA) (Figure 4a and 4b).
Experimental design and bioinformatics-based data analysis. Microarray study design and analysis was performed in methods similar to what has been previously described for selenium studies from our lab (16). The microarray study experimental design utilized two biological replicates for each comparison with each replicate incorporating a Cy3/Cy5 dye flip. In addition, self-self hybridizations were performed for each sample to ensure experimental accuracy and evaluate expression bias. Comparisons were organized in a loop design for either human or rat prostate cancer cell lines, or were run as two-sample comparisons of baseline untreated control and soy or genistein treated cells. Array image TIFF files were analyzed with Gleams software (Nutec Sciences, Atlanta, GA, USA). Additional TIFF file analysis, data normalization, clustering, and principle components analysis was performed using the Spotfinder, MIDAS and MultiExperiment Viewer Software from The Institute for Genomic Research (TIGR, Rockville, MD, USA; http://www.tigr.org/software/tm4/) and used default values set in the MCW Practical Guide to TIGR Software Use (M.Datta, unpublished). In brief, image expression data was used as channel intensity minus background and intensity thresholds were set at a value of 300. Images were analyzed as dye flip pairs normalized using MIDAS with LocFit based LOWESS normalization and slice analysis set at two standard deviation cutoffs and a sample data population of 500 (16). Samples were then averaged across two dye flip replicate pairs with removal of zero/dropped values using locally developed averaging software from the BEAR microarray suite (17). These final averaged values were subsequently annotated using the BEAR suite annotator and used for pattern identification and correlation with gene homologs. Homologous genes were identified from the NCBI homologene database ftp files (ftp://ftp.ncbi.nih.gov/pub/HomoloGene/) and parsed using local scripts and databases present in the Bioinformatics Research Center (http://brc.mcw.edu). Additional data mining to identify references in the biomedical literature associated with specific genes and user chosen search terms was performed using the locally developed GeneInfo data tool (16). Raw data files, along with analyzed data subsets are available for use and study after contacting the corresponding author.
RNA expression profiling was performed by assessing the most closely related genes between rat and human cell lines (Figures 5a and 5b) for microarray analysis. After imaging, data acquisition, normalization, and slice analysis, experimental samples were filtered based on self-self hybridization data and the final sample replicates were averaged. Genes were filtered based on desired patterns of expression (up-regulation or down-regulation at various conditions) and the results were examined. Final data mining was performed using BEAR GeneInfo with the gene names and supplemental search terms searched against PubMed to identify associated references (17). The Genebank ID's generated were matched with corresponding SwissProt ID's in order to perform GeneMap analysis of related biochemical pathways to explain the proteins growth inhibition (18, 19).
Generation of common genes and homologs. Sequence validated gene libraries for both the rat and human DNAs were obtained from Research Genetics (Huntsville, AL, USA), and were supplemented with additional DNA samples obtained from the University of Iowa rat clone sequencing program (14, 15). The majority of the rat DNAs, and a subset of the human DNAs were resequenced by Dr. J. Quackenbush at TIGR through a joint Program in Genomic Applications consortium. The GeneBank accession numbers for the 19,200 individual human or rat clones present in the recent slide printings were used to query the NCBI Unigene database to return the associated Unigene IDs. Unigene IDs were returned for virtually all identified clones, and were placed in an Oracle database where they were compared with the downloaded NCBI Homologene data set (build 106) of rat, mouse, and human homologs. Of the 19,200 clones, 5,740 genes were identified with homologues present on both the rat and human slides. This homolog set was used for the subsequent comparisons across species.
Affect on growth. Soy and genistein were used in cell culture studies to show their affect on cell proliferation. The cell lines chosen for these experiments allowed us to compare androgen-sensitive prostate cancer cells (LNCaP) and androgen-insensitive cells (PC3 and PAIII). Previously, the main effect of soy and genistein was to influence cell proliferation mainly through the androgen receptor due to the phytoestrogen component of soy and genistein (8). The effect of decreased cell proliferation has been described in other studies, yet never compared to that with whole soy protein (20, 21, 22, 23). The cell proliferation was studied by manually counting the cells and observing the growth along with the dose-related affect of the compounds.
The initial growth curve of genistein was similar across the 3 cell lines used in this study (LNCaP, PAIII, and PC3) in that the cell proliferation decreased without visual evidence of cell death. Soy, on the other hand, had the most affect at decreasing the growth of the LNCaP cell in particular (Figure 3a), while having less of an affect on the PAIII and PC3 cells (Figure 1a and 2a, respectively). The dose-related effect on cell proliferation from treatment with genistein was stronger. Genistein was used in doses of 20 mM and 100 mM to observe the influence of different concentrations on cell growth. The 100 mM dose caused the cells across all cell lines to stop proliferating without associated cell death (Figures 1b, 2b, and 3b). The 20 mM dose of genistein demonstrated a less dramatic inhibition of cell growth. For this reason in particular, we chose to use the 20 mM Genistein concentration for the RNA extraction and further study through cDNA microarray. As for soy isolate studies, the soy powder was solubilized at the highest concentration possible that did not lead to precipitate formation (saturation) which was 100 μg/mL. Overall, genistein decreased proliferation in all cell lines; whereas solubilized soy protein decreased proliferation exclusively in the LNCaP cell line.
Identification of differentially expressed genes in PC3 and PAIII prostate cancer cells treated with soy or genistein. Differential gene regulation was compared between two cells lines, one rat (PAIII) and one human (PC3). These cell lines were chosen for their similarity (non-androgen dependent) and for cross species analysis. We compared differentially expressed genes after treatment with soy in both rat and human cell lines. To confirm the genomic similarity of the responses in the two species, we compared the differential expression for homologous rat and human genes to see if they were similar. Graphing of the results demonstrated that the differentially expressed genes fell on a straight line, indicative of a linear relationship between the expression changes (Figure 5a) Thus the genes showed similar changes across species. A similar comparison for rat and human genes differentially expressed by genistein yielded similar results (Figure 5b). This technique also yielded similar results when performed in previous studies in our lab comparing Selenium response across genomic species (16). The human and rat genes from the microarray expression studies were compared with respect to soy (Table I). In this table, 10 genes that were similar in regulation among rat and human were selected due to the number of Medline listed references described in the right of Table I. The references were identified using the GeneInfo program. Five of these genes were up-regulated and 5 were down-regulated with regard to the soy to control ratios. The Medline function of the program helps to identify those genes that have been studied in the literature and helps to identify those genes whose importance in prostate cancer has been noted.
Differentially expressed genes in rat and human cell lines with respect to genistein (Table II) showed 13 genes of interest: 7 genes were up-regulated and 6 genes were down-regulated with respect to the genistein versus control ratios. These results identified different genes from those seen with soy. The genes are displayed in summary in Table III showing overexpression of genes with respect to genistein or soy and their correlative Medline search reference lists. These are the genes that are up-regulated with treatment of soy product showing the differences in soy versus genistein. A total of 66 genes are shown in this Table, 48 genes are overexpressed with respect to genistein and 18 genes are overexpressed with respect to soy alone. Table IV separates all the differentially regulated genes only affected by soy.
Examination of pathways affected by soy and genistein. Steroid metabolism. We used the GenMaPP analysis program and annotated cellular pathway maps from KEGG (Figure 6) to observe the influence of the phytoestrogen genistein within the steroid pathway and compare this to soy to determine any gene regulation to add to this established mechanism. Analysis of the overlaid expression data demonstrated that 12 gene were differentially regulated. All 12 genes were up-regulated with respect to both genistein and soy treatments. There were no genes regulated solely to genistein or soy.
Apoptosis. As mentioned previously, apoptosis was not a major feature of the cells in cell culture exposed to the treatment of genistein or soy. The apoptosis pathway is an important regulator of cell proliferation and was explored for its importance in tumor production and proliferation. Gene expression data, with respect to known cellular pathways, again using the GenMaPP analysis program and annotated pathway maps from KEGG (Figure 7) demonstrated that 8 genes were differentially regulated in the apoptosis pathway. This includes 2 genes that were down-regulated with respect to both genistein and soy treatments and 5 genes were up-regulated. One gene was up-regulated with respect to genistein and not soy (BAK).
Cell cycle. The observed changes in cell growth curves could be caused by changes in cell cycle proteins and their regulation. For this reason we examined or gene expression data with respect to known cell cycle pathways using the GenMaPP analysis program (www.genmapp.org) and annotated cell cycle pathway maps from KEGG (Figure 8). Analysis of the overlaid expression data demonstrated that 16 cell cycle genes were differentially expressed. This included 12 genes that were down-regulated and 4 genes that were up-regulated. Of note, all genes that demonstrated differential expression were differentially expressed with both genistein and soy treatments.
Wnt signaling. Wnt signaling pathway controls the regulation of Beta-catenin, which is a well studied protein known to have nuclear effects and facilitates cell proliferation by the genes it promotes (24). As described above, the Wnt pathway has shown to be differentially regulated in prostate cancer in addition to describe beta-catenin mutations (25). In order to visualize the regulation as it pertains to soy and/or genistein, we used a KEGG file with the GenMaPP program to display the regulation patterns (Figure 9). Analysis of the overlaid expression data demonstrated that 11 genes were differentially expressed. This included 7 genes that were down-regulated and 4 genes that were up-regulated. Of note, all genes that demonstrated differential expression were differentially expressed in both genistein and soy treatments.
TGF-beta. We again used the GenMaPP analysis program and annotated pathway maps from KEGG (Figure 10). Analysis of the overlaid expression data demonstrated that 11 genes were differentially expressed genes. This included 7 genes that were down-regulated and 4 genes that were up-regulated. Of note, all genes that demonstrated differential expression were differentially expressed in both genistein and soy treatments.
Nuclear receptors. The KEGG pathway maps (Figure 11) demonstrated that 9 genes were differentially expressed genes. This included 3 genes that were down-regulated and 5 genes that were up-regulated. One gene was up-regulated with respect of treatment of genistein but not soy.
Cellular proliferation. We are interested in the clinical changes caused by soy treatment on prostate cancer development and progression. Our ability to identify changes caused by soy that correlate with clinical results of reduced tumor formation is of utmost importance. These common clinical effects have been seen in humans through epidemiologic and small early clinical studies, as well as in studies using the rat model of prostate cancer. By combining data across species and focusing on the common clinical effects and their associated gene expression changes, we hope to find specific genes that play a significant role in the clinical effects of soy on prostate cancer. Cell culture of PAIII, PC3, and LNCaP allowed visualization of growth patterns of treated soy or genistein compared to control. Genistein displayed a predictable and strong inhibitory affect on proliferation of PAIII, PC3, and LNCaP cells in cell culture (Figure 1b, 2b, 3b). The inhibiton was less dramatic when treated with soy compared to genistein. Thus it appears most likely the majority of the inhibitory affect on proliferation are due to genistein within soy. This is in agreement with the published data on genistein and the main active ingredient in soy (19, 20). While genistein is postulated to work through steroid hormone receptors, the finding of decreased cell proliferation in both androgen-sensitive LNCaP and the androgen-insensitive PC3 cells implies that the effects of genistein may occur through more than just the androgen signaling pathway. Soy protein-treated cells did not show as a dramatic inhibition on prostate cancer cells as isolated genistein, though an influence was noted. The cell proliferation of the soy treated cells showed a decreased slope; though, not significant the finding was consistent over multiple studies (Figures 1a, 2a, and 3a). The LNCaP cell line had an interesting finding in that the soy protein had a stronger affect on cell proliferation (Figure 3a). A possible explanation for this finding is that LNCaP, as an androgen-sensitive cell line, may represent an earlier, less aggressive stage of prostate cancer, and thus the component where soy may have its best chemopreventative benifit. This would suggest that soy protein acts earlier in the progression of prostate cancer and may be effective in blunting the proliferation of the tumor and slowing the course of the disease. While the main impact of both proliferation and gene expression changes appear to be the affect of genistein in the soy, other effects are identified. The differential gene expression shows that soy isolate may propagate through the genes modulated only by soy, which may affect prostate cancer cells by a mechanism other than proliferation.
Examination of pathways affected by soy and genistein. Steroid metabolism. The weak oestrogenic affect of isoflavones are the most reproducible and relevant pathways that have been recognized (8, 11, 22). The interaction of steroid affects, in particular the relationship between estrogens and androgens, has been a mechanism and treatment focus for prostate cancer for over 60 years (26, 27). This association relates back to the use of orchiectomy in the early treatment of advanced prostate cancer. Hormone therapy, including medical orchiectomy through the use of steroid enzyme inhibitors and receptor blockers, is still a major treatment option for prostate cancer (28). Genistein recapitulates the affect of hormonal therapy, but on a much weaker scale and has few side-effects (23). In studying the affect of genistein on prostate cancer cells, gene microarrays have led to multiple findings that allow specific and relevant pathways to be explored. Takahashi, Lavigne, and Wang described in gene array data that the androgen receptor-mediated pathways were modulated by genistein even at the smallest concentration tested (29). They concluded that the androgen receptor mediated pathways were the most relevant mechanism to target for genistein studies. Betick et al. also described that the androgen receptors were down-regulated in LNCaP cells in response to physiologic treatments of genistein (30).
Due to these previous studies describing the effect of genistein on the steroid hormone metabolism pathway, we wanted to compare genistein and soy isolate for this pathway as well. Steroid metabolism is an important aspect of prostate cancer that may impact tumor aggressiveness and prognosis. The GenMapp pathways from this study shows that the vast majority of gene regulation changes express upreguation using soy or genistein (Figure 6). In particular, the majority of the changes in the metabolic enzymes are of up-regulation. The genistein/soy based inhibition of testosterone and DHT could cause feedback expression increases in the enzymes responsible for testosterone production. This hypothesis supports one of the most prominent models by which soy is thought to exert its chemopreventive effects, through androgen pathway inhibition (29, 31).
Apoptosis. Steroid metabolism plays a very intricate and vital role in the overall affect of soy isolates, yet the direct cytotoxic affect has only recently been evaluated. One mechanism of involves the role of proteasomes in apoptosis regulation (32). Preliminary data from Kazi et al. and others found that in the LNCaP cell line, genistein-mediated proteasome inhibition was accompanied by induction of apoptosis (8, 32). Genistein has also been implicated in the induction of caspases, in particular caspase-3, to mediate apoptosis via CPP32 (33). Another manuscript also noted that genistein may induce apoptosis through topoisomerase II (8). In addition, Davis, Singh, and Bhuiyan. described the affect of genistein on cellular proliferation and subsequent apoptosis. In their studies genistein results in the down-regulation of cyclin B and up-regulation in p21 inducing apoptosis after placing the cells in cycle arrest (34). Some studies suggest genistein does not necessarily cause apoptosis, but sensitizes the cancer cells to inhance chemotherapeutic agents (35). The effects of genistein may be dose-dependent because when comparing different concentrations of genistein, higher doses may cause apoptosis while lower doses may not.
We examined soy and genistein differential expression data in PC3 and PAIII and displayed them in a GenMapp KEGG Figure (Figure 7). The majority of the apoptotic pathway components are down-regulated by both soy and genistein and was demonstrated in the cell culture where the treated cells were viable but not proliferating, possibly in G0 arrest. This suggested that the decrease in proliferation was an intrinsic function of the cells and not cell death. Thus while apoptosis is not enhanced with the use of soy or genistein alone, it may predispose the cells to G0 arrest and subsequent apoptosis by other means.
Cell cycle. Hormonal regulation by genistein also plays a role in cell cycle modulation by decreasing cellular proliferation. Wang, Clubbs, and Bomser showed that genistein alters cell proliferation via estrogen dependent kinases as well as extracellular kinases (22). Therefore, they added to the understanding that genistein has both hormonal-dependent and -independent mechanisms affecting the prostate tumor proliferation. Another venue in which soy protein and its isolates may affect cells other than androgens and direct cytotoxicity is modulation of the cell cycle by acting on the cyclins. Soy isolates can alter the cell cycle by modulating TGF-1β or cyclins directly (34, 36). The cell cycle shows diffuse down-regulation in cells when treated with soy or genistein (Figure 8). These results have also been shown in tissue culture and confirms that soy and genistein down-regulate the cell cycle and proliferation of prostate cancer cells.
WNT signaling. Wnts and growth factors have been shown to be differentially expressed in normal prostate tissue and prostate cancer (24). Various Wnt are up-regulated in cancer and have been associated with higher Gleason scores (37). Wnt inhibitory factors are frequently down-regulated possibly causing decreased active GSK3β and increased soluble β-catenin. This leads to cell proliferation in PC3 cells (38). GSK3 is a protein that will phosphorylate β-catenin, therefore, targeting the β-catenin protein for ubiqination and destruction. GSK3 inhibition leads to increased β-catenin concentration and transport into the nucleus activating genes associated with cell proliferation (24). β-Catenin is indirectly associated with the Akt pathway via GSK3 and is also involved in IGF-1-induced androgen receptor activation, thus linking androgens and the Wnt pathway (39). β-Catenin has also been implicated in prostate cancer due to various mutations in the gene noted in prostate cancer (40). These mutations may block β-catenin protein phosphorylation and ubiquitin-related destruction, leading to continued cell proliferation (25). These mutations in β-catenin have been shown in a limited amount of prostate cancer samples and may be a late finding in cancer progression. Verras and Sun concluded that 20-38 % of hormone refractory prostate cancers show β-catenin nuclear localization greater than that would be caused by β-catenin mutation alone, indicating the Wnt pathway as a possible source of this β-catenin activation (40). Soluble β-catenin is only one of the sources of β-catenin, which also has a structural cell function. This other pool of β-catenin allows the stabilization of E-cadherin and thus may be involved in tumor cell metastatic potential (30). Figure 9 shows the GeneMaPP displaying β-catenin to be down-regulated by both soy and genistein. The frizzled receptor is up-regulated by both. The up-regulation of the frizzled 3 protein is likely a feedback mechanism to increase the frizzled receptors due to down-regulation of β-catenin and decreased cell proliferation. Of note, the JNK protein, which is important in the apoptotic pathway, is down-regulated as well. This again suggests that the decrease in proliferation was an intrinsic cellular mechanism not due to apoptosis.
TGF-beta. The TGF-β signaling pathway plays a central role in epithelial cell growth and acts through multiple intracellular pathways. Upon receptor binding, Smad2/3 proteins become phosphorylated and associates with Smad4 (41, 42). This Smad complex translocates to the nucleus, binds to DNA and regulates transcription of specific genes (41). The TGF-β pathway is interconnected with the androgen receptors, VEGF, pathway, JNK/JUN pathway and Wnt pathway through β-catenin and other cofactors including the Smads proteins. Recent data from Sheehan Kallakury. and Ross described that smad4 expression correlates with grade and stage of prostate cancer (42). We have found that the majority of the the TGF-β signaling pathway are inhibited by soy and genistein. Smad 2 in particular is down-regulated with the inhibitory Smad6 being up-regulated (Figure 10). These findings describe that inhibition of the TGF-β pathway correlates with inhibition of cellular proliferation suggesting the value of further researched in this mechanism.
Nuclear receptors. Nuclear receptors play and intricate part in guiding cell proliferation and production as the result of a changing environment. By studying these receptors, insight into the action of soy and/or genistein on nuclear factors and how they regulate cell function is of great importance to explain mechanism of action. Figure 11 contains some important nuclear receptors and their function in cell signaling and proliferation. Of note, the vitamin D3 nuclear receptor is up-regulated with soy and genistein. Tokar and Weber have recently described that vitamin D3 (cholecalciferol) inhibits cellular changes associated with malignant transformation and invasion of prostatic neoplasia by up-regulating nuclear receptors (43). The connection between vitamin D and androgen receptors is controversial but may suggest an important connection between soy and vitamin D. In our study, the orphan nuclear receptor NR1D2 is the only nuclear receptor solely influenced by genistein and not soy (Figure 11). The NR1D2 receptor is up-regulated by genistein and is linked to the RARB gene, which encodes retinoic acid receptor beta (44). The retinoic acid receptors are also differentially regulated by soy and genistein. Of note the NR2B1 orphan nuclear receptor is complexed in a herterodimer with constitutive androstane receptor (CAR) causing it to be continually expressed (45). Figure 11 shows soy and genistein up-regulate this CAR/RXR heterodimer causing the retinoic acid signaling mechanism to be continually expressed, possibly decreasing cancer growth.
Soy protein has been shown to decrease the incidence of clinically significant prostate cancer in both human studies and in animal models of prostate cancer. We have used gene expression microarrays and cross-species gene analysis to identify gene pathways that may be influenced by soy protein.. While the majority of the features we found regarding pathway regulation were common to soy and its purified factor genistein, some genes were identified via microarray analysis possibly due to other soy protein components. The soy protein isolate is made of many proteins, in which only a few have been investigated, and more importantly it is how these proteins interact with one another that is more difficult to investigate. The use of soy and genistein play an important role in chemoprevention and pharmacology in designing treatment modalities to reduce the risk for prostate cancer.
NCI funding through Julia White, MD in the Department of Radiation Oncology at the Medical College of Wisconsin, Milwaukee, WI, USA, who had no involvement in the study design, in the collection, analysis and interpretation of data, in the writing of the manuscript, or in the decision to submit the manuscript for publication.
Mark Suckow and Morris Pollard at the Lobund Laboratory at the University of Notre Dame for supplying the PA-III rat cell line and the soy protein isolate for investigation. Paul Lindhol and Andre Kadjacsy-Balla for supplying the PC-3 human cell line.
- Received March 1, 2010.
- Accepted March 12, 2010.
- Copyright© 2010 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved


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Not again
U of R wrestler Kirk Ackerman is disappointed with second place
Ed Kapp
Sports Writer
Going into the 2011-12 CIS season, fourth-year Cougars wrestler Kirk Ackerman hoped above all else to help his teammates claim a national-championship banner.
Just a few weeks prior to the year-end tournament, Ackerman and the Cougars men's wrestling squad – who were the No. 1 ranked team in Canada for a good part of the season – finished in first place at the Canada West championships.
Once at the tournament in late-February, the Cougars men's squad – who were ranked third in the nation going into the event –¬ continued to impress in Thunder Bay, the host city of the CIS championships.
Four members of the Cougars qualified for gold-medal matches – including victories by Connor Malloy and Steven Schneider.
But Malloy's and Schneider's victories in the final round of the tournament ultimately weren't enough.
"We were tied with Concordia going into the second day and we had guys in big matches for big points and we ended up losing to Concordia by two points," recounted Ackerman, 21, who went into the tournament as the No. 2-ranked 61 kilogram wrestler in Canada and won his first three matches before losing in the final match of the event.
Although the Cougars were ranked third in the nation going into the tournament – and thus surprised some by their showing – Ackerman and his squad claimed a second-place finish, emphasizing that his experience at the nationals was, for all intents and purposes, a disappointing one.
"It was just a state of disbelief, really," Ackerman said. "We had gotten all that way again and did all that work again and fell short, again."
"If somebody was satisfied with that, then they probably shouldn't have been there."
While Ackerman noted that his experience will stay with him for quite some time – "I don't even remember speaking for a few hours after, to be honest," he offered – he will soon have the opportunity to redeem himself on the mat.
In the coming weeks, the Balfour Collegiate graduate is to compete at the 2012 Senior National Wrestling Championships at Brock University in St. Catharine's, Ont.
Although Ackerman is quick to point out he isn't looking past the upcoming competition in Ontario, he is admittedly looking forward to traveling to Orlando in late-March to try to qualify for Team Canada's 2012 Olympic wrestling team.
For Ackerman – who has dreamt of competing at the Olympics since he first took to the mat around 15 years ago – representing Canada at the Olympic Games would be far more than just another tournament.
"It definitely gives you a tingly feeling, like being a little kid on Christmas, again," said Ackerman, who has won more provincial and national competitions than he can remember and has represented Saskatchewan on the mat across Central and North America.
"[Qualifying for the Olympics] would mean the world to me and it would mean a lot to, probably, to more people than I know," Ackerman added. "I know it would mean a lot to my team – I know my whole team is rooting for me – it would mean a lot to all the alumni that I grew up with, my coaches, Leo, and Danny. I'm sure [they] would be happier than I would be. And, of course, my family would be really happy."


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Philip Swenson
I'm an associate professor at the College of William & Mary. I do research in Ethics, Metaphysics, Agency, and Philosophy of Religion. I earned my PhD at the University of California-Riverside. I also spent time at Notre Dame and Rutgers before coming to William & Mary. Here is an interview in which I discuss moral luck. You can contact me at email@example.com
"Equal Moral Opportunity: A Solution to the Problem of Moral Luck" forthcoming in Australasian Journal of Philosophy
"God and Moral Knowledge" (with Dustin Crummett) A New Theist Response to the New Atheists. Routledge: (2020)
"How to be an Actualist and Blame People" (with Travis Timmerman) Oxford Studies in Agency and Responsibility (2019)
"Luckily, We Are Only Responsible for What We Could Have Avoided" Midwest Studies in Philosophy (2019)
"Moral Responsibility without General Ability" (with Taylor W. Cyr) Philosophical Quarterly (2019)
"Gun Control, the Right to Self-Defense, and Reasonable Beneficence to All" (with Dustin Crummett) Ergo (2019)
"Bundle Theory and the Identity of Indiscernibles" (with Bradley Rettler) Res Philosophica (2019)
"Frankfurt Cases: The Fine-grained Response Revisited" (with Justin Capes) Philosophical Studies (2017)
"Subjective Deontology and the Duty to Gather Information" Ethics (2016)
"Ability, Foreknowledge and Explanatory Dependence" Australasian Journal of Philosophy (2016)
"The Frankfurt Cases and Responsibility for Omissions" Philosophical Quarterly (2016)
"A Challenge for Frankfurt-Style Compatibilists" Philosophical Studies (2015)
"Reasons-Responsiveness and Degrees of Responsibility" (with D. Justin Coates) Philosophical Studies (2013)
"Ability-Based Objections to No-Best-World Arguments" (with Brian Kierland) Philosophical Studies (2013)
"Skepticism about the Argument from Divine Hiddenness" (with Justin P. McBrayer) Religious Studies (2012)
Symposium Contributions, Encyclopedia Articles, and Book Reviews:
"Incompatibilist (Nondeterministic) Theories of Free Will" (with Randolph Clarke and Justin Capes) Stanford Encyclopedia of Philosophy
"Fischer on Foreknowledge and Explanatory Dependence" European Journal for Philosophy of Religion (2017) [Contribution to a book symposium on John Martin Fischer's Our Fate: Essays on God and Free Will.]
"Does it Matter which Explanation is Best?" (with Brian Cutter) Syndicate Philosophy (2017) [Contribution to a book symposium on Kevin McCain's Evidentialism and Epistemic Justification.]
Review of Libertarian Free Will: Contemporary Debates, edited by David Palmer, Notre Dame Philosophical Reviews (2015)


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Maverick Burger
Free tracks, space for history: MAVERICK BURGER is ready to take off…
The challenge at high altitude ,between the endless skies, begins today. .
The challenge at high altitude ,between the endless skies, begins today. .
For just one month at the Cantiere is ready to speed #MaverickBurger, the new temporary inspired by the return to the cinema of "Top Gun Maverick", the sequel to the most followed and loved action film of all time.
Clear the concept of height and discover the ingredients: the only goal will be to reduce hunger!
The ingredients: Burger Macina Cantiere 200 gr. smoked buffalo D.O.P., crunchy capocollo I.G.P., olive pate, sun-dried tomatoes, green salad, green bun "Maverick", to be combined with the "airplane" Squadron 303 vodka theme.


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With coursework, activities, internships and job-searching, college students have a lot on their plates. Although the majority of their time is focused on learning, it's also important to prioritize setting up a solid financial footing for the near and long-term future.
Having a basic understanding of personal financial management can add tremendous value to your future success. College students can take several steps to improve their financial stability odds throughout a lifetime, starting with mastering the arts of saving and building credit.
The How and Why Behind Saving
Smart money habits are easier to maintain the earlier they're started. Saving money is a necessary part of every adult's financial life, but the reason why it's so important may not be inherently clear to college students. On top of not realizing the "why" behind saving, most aren't sure where to start a healthy savings habit. First and foremost, having ample savings offers several advantages, not the least of which is being able to cover an unexpected bill or a costly emergency.
The idea of an unplanned expense showing up is likely not a substantial concern for most college students, but it is relatively common. From emergency car repairs to an unplanned doctor's visit, unexpected costs can come from a handful of different places. Having savings on hand provides the monetary cushion necessary to cover these costs without going into debt. Once this is understood, creating a plan for how to save is the next best financial move.
Creating a Budget
To get started on the right path toward savings, college students can and should focus on what they have available to spend each month. Establishing a realistic but flexible budget is the foundation for this type of financial management. When initially setting up a budget, try to keep it simple. First add up how much money is coming in. Then, add up expenses that you need to cover each week or month, including rent, dining out, gas, school supplies and groceries. Subtract these expenses from what's coming in, and voila, you can see what you have available to spend or save. Don't worry—there are also a handful of budgeting apps and websites available for little or no cost that can do this work for you.
- Many financial experts suggest following a 50/30/20 rule. This approach recommends spending 50% on basic needs, 30% on wants, and 20% on savings. If you are not close to these suggested amounts, or you have a cash-flow deficit each month, it is a good idea to take a close, hard look at income and expenses. Correcting a cash flow deficit requires earning more money or spending less, neither of which may be accomplished easily as a college student. Consider what you can do to create a surplus, like leaning more on financial aid or taking on additional part-time work between classes to create a stronger financial foundation each month.
If you are fortunate enough to have a surplus, it's time to start saving. Calculate what you can easily save each month, then establish a no-cost savings account where you can deposit the extra funds. Automate the process by setting up systematic transfers every pay period or month, and be sure to increase the amount of savings when there is a decrease in expenses or an increase in income. This simple step lays the foundation for a healthy financial future.
Building Credit 101
Above and beyond establishing healthy savings habits early on, college students must also know about credit. Having a strong credit score and credit report provides opportunities for affordable borrowing in the future. But before you can build good credit, it's essential to understand what credit is and how to calculate it.
Your credit is a representation of your financial past and present. Within a credit report, creditors like student loan lenders, credit card and auto loan companies provide information about current or past accounts. These details include how much you owe, the monthly payment, and the payment track record. Missed or late payments and other damaging marks, including bankruptcy or judgments against an individual for non-payment, are included in a credit report. Based on these data points, a person is assigned a credit score. The higher the score, the better an individual has managed credit accounts in the past.
Credit does not happen naturally—it has to be built. A smart place for college students to start the process of building credit is by applying for a student credit card and managing it responsibly. This type of card often has minimal to no fees and low-interest rates, like the Teachers Student Visa Credit Card. It may also come with a low credit limit to help keep spending on the card to a minimum. As long as they pay the card balance each month, college students can begin building a track record of on-time payments that positively impacts their credit score moving forward.
Additionally, taking out student loans can help college students, not only in offsetting the out-of-pocket costs of earning a degree but also on the credit-building front. A person's credit report includes student loans, just like credit cards and other debts. Again, as long as payments are made on-time, student loans can help build a healthy credit history for college students.
Bringing it All Together
Being a college student comes with a slew of responsibilities, and financial management should fall somewhere on the priority list. Start with saving whenever possible, and do so with the help of a realistic budget. Follow that up with credit-building activities like getting a student credit card or utilizing a student loan. Combining these habits surrounding saving, budgeting, and credit proves immensely beneficial throughout one's life. Although you probably won't master these financial principles overnight, taking small steps to build savings, and establish and manage credit consistently can have a substantial impact on your financial health and well-being in the future.
Smart money habits are the building blocks to smart (and happy) living!


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September 8, 1939 - October 15, 2022
In loving memory ~
Ann passed away peacefully with her family by her side on October 15, 2022 in Nanaimo after a short battle with cancer.
Ann was born and raised in Nanaimo, BC. Ann met Garry in Qualicum Beach in 1958, they wed in 1960 and were happily married for 62 years. Ann loved and was loved by her husband Garry and their three sons. Rory (Ann), Doug (Donna), and Kerry (Susan). There are many happy
memories of their years raising their three sons. She enjoyed watching and supporting them in their love of athletics.
Ann worked for Telus for 25 years, retiring at the age of 50. Her husband Garry was a Port Alberni Businessman and both were fortunate to retire at 50 years and enjoyed a long retirement together travelling. They enjoyed their winter home together in Arizona for 28 years, met many
lifetime friends and played a lot of golf together.
One of their favorite trips was to Australia where they cruised from Sidney Australia to Vancouver.
Ann and Garry enjoyed many cruises together. They also enjoyed a month travelling through Alaska and completed two trips across Canada. No matter where they traveled, Ann could not wait to return to the jewel of Canada, Vancouver Island where both Ann and Garry were born.
Ann's wishes were for no service or celebration of life but preferred a small gathering with family. A special goodbye to her dear friend Donna-Lou, all her golf and mah-jong friends, all of whom she loved.
A special good bye to her dear friend Betty, who Ann volunteered with at Loaves and Fishes in Nanaimo.
Ann was able to be at home thanks to the loving care provided by Garry and Family.
Our thanks to the doctor and care givers during her short stay in the palliative care unit in Nanaimo.


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10 Feb What is it like to be fat?
I'm not sure I am ready to write this, so that probably means it is time. I'm leaping before I look. And, while I'm fairly certain there is an abyss below, staying where I am is no longer an option. So, hang on for the ride, it's a long one.
When I was a young child, I was thin and athletic. I played all the sportsballs. I ran around outside and explored the wooded areas around my house. I rode my Mongoose Californian BMX bike everywhere. When I moved into my high school years, though, I became more sedentary. I began to gain weight quickly. Adolescence is awkward for everyone. But, a more than pudgy know-it-all teenage nerd with a bad complexion is about as awkward as it gets. For the most part, but not entirely, I was able to avoid bullying, but there were always the random comments about being fat. And, they always cut to the core, piling on to an already insecure little kid.
I had hoped that when I went to college the narrative would change – a fresh start, a new me. I had never heard of the Freshman 15 until I got to college, even then I just thought it was the latest and greatest alternative band on MTV's 120 Minutes. I continued to gain weight, and my awkwardness did not subside. I was deathly terrified of girls and stayed sequestered in the safe little bubble of nerdery of my college debate team.
When I went to law school, I again prayed for things to change. And, well, they did. I had fallen in love with a girl who was in love with someone else. So, an arduous depression gripped my soul. As I sat at home and pined away, I just had no real interest in eating. I started to run and began to feel a little bit better. I lost a lot of weight. But, then, I started drinking more than usual and, perhaps, may have engaged in other unhealthy activities. I did not inhale, though. The weight came creeping back.
After law school, I moved to Houston to start my career. I met a group of wonderful friends. We worked hard and partied even harder. Like, really hard. Honestly, I cannot fathom how we all came out of that period alive, but we did. Still, I was grossly obese. I remember stepping on a friend's scale and it said "249". I thought, uh, dude, your scale is broken. (Narrator: It wasn't broken.) When the woman at Star Pizza knows you on a personal basis, you might have a problem.
I am going to fast-forward to a time where, in my first marriage, my then-wife said matter-of-factly: "I think you have an eating problem." We had a new baby who was not sleeping through the night. I would wake up, rock her to sleep, then head into the kitchen at 2 am and inhale an extra 500 calories. I was already consuming my fair share of food during the day. So, I went to an Overeater's Anonymous meeting. I took to the 12 Steps. I worked the program as best I could and saw results. I did a lot of personal work. I lost 65 pounds. I finished a full marathon and a half-Ironman triathlon. I was in the best shape of my life, physically and emotionally. I stuck with it for over three years.
There were a number of things that got me away from the program (a nasty divorce, a knee surgery and extreme anxiety). The weight came back over the years. Now, I am back to square 1, a few pounds over where I was when I entered the OA program. I have a new career, a wonderful partner, and life is great except for, you guessed it, how I feel about my body. There's somewhat of a myth surrounding men and their emotions. The "dad bod" is the latest fad. But, men, much like women, are conscious of their physiques. This morning, as I drove to meet some clients, I saw a sign for a male weight loss program. The billboard played on the old joke of "Dickydoo Disease." What's that, you say? When your belly sticks out further than your dickydoo. I suffer from a chronic strain of the disease. I have almost all of my life.
I realize I am an N of 1. But, here's what is like for me to be fat. There is the endless cycle of shame which I think burdens almost everyone who suffers from an eating disorder. I eat, feel shame, then emotionally eat to ease the shame, then feel more shame. Wash, rinse, repeat. I rarely look at myself in the mirror. I feel uncomfortable naked. Hell, I feel uncomfortable in my clothes. They are tight and constricting. The other day, I was walking down the halls of the courthouse to a hearing. My sport coat was at least one size too small. All I heard in my head was Chris Farley, saying "Fat Guy in a Little Coat." I don't think I can handle going up another waist size. I hate to be in pictures. I fear others judge me as much as I judge myself. I am out of shape. I'm unhealthy. I'm tired. Really tired. I'm burdened… all the time, physically and emotionally.
Mostly, though, I feel helpless. I wake up every day and say, "Today is the day I will eat well." Some days I do, but some days I don't. But, hell, there's always tomorrow, right? This, my friends, is the refrain of an addict. There is always some reason to start tomorrow. Or Monday. Or the first of the year. Or the first of the month. Or the 15th of the month. Rarely, however, it is time to make a change right now. This is an endless cycle, a never-ending treadmill (the type which doesn't burn calories).
"Hi, my name is Blaine and I am a compulsive overeater." I'm speaking this into the Universe because it is time. My brother's heart attack was a giant thump on the head for me. I am not immune. I am not sure what things will look like in the near future for me. My desire is that this post works some magic and propels me on the journey to a better me. I hope you will follow along with me. I know I could use the help.


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February 19, 2024
As Naples' first professional sports team prepares for its inaugural season, the team is asking for your input on its crest, colors, and name.
According to the USL Naples' spokesperson, they are committed to making the city their home and growing the community spirit before the first season even starts.
Fox 4's Bella Line went to its stadium at Paradise Coast Sports Complex to hear from some of their youngest fans.


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Enter the desired query in the Google search bar to see trend keywords. In most cases, these are useful. But if you don't care what people are searching for, you can easily get rid of trend searches on Google on iPhone and Mac. Let me show you how.
What are Trend Search Candidates on Google?
Let's explain this with two examples. Enter the word Apple in a Google search to see some suggestions such as Apple iPhone, your local Apple Store, and Apple's stock price.
Similarly, searching for a celebrity will show its salary, net worth, girlfriend, age, Instagram account, and more.
If you don't like them appearing in your search, here's how to turn them off for a clean experience.
How to get rid of trend search from Google on iPhone
In Google Chrome
Are you one of those who like Google Chrome as your default iPhone browser? If yes, here's how to disable trend search in the world's most popular browsers:
- Open Chrome and tap Your profile picture It is in the upper right.
- Toggle off Autocomplete search and URL..
- Finally, tap Done..
Professional tips: Want to temporarily turn off trend search on Google?Tap 3 dot icons Tap at the bottom right New secret tab.. Browsing in private mode where you won't see trend searches or suggestions.
In Safari
If you prefer the iPhone's built-in browser, see How to turn off Google's suggestions.
- Open Setting Tap Safari..
- Toggle off Search engine suggestions..
- option: You can go one step further and turn it off Safari suggestions..
On the Google website in any browser
- Go to Google.com or, in the case of India, go to a country-specific Google URL such as Google.co.in.
- Tap Hamburger icon It is on the upper left.
- Tap Setting..
- [トレンド検索のオートコンプリート]so,[人気のある検索を表示しない。
- 最後に、をタップします セーブ。
- クリック 3つの垂直ドット 右上で選択します 設定。
- click Sync and Google services..
- Toggle off Autocomplete search and URL..
Report Google's autocomplete forecast
If you don't like a particular forecast, you can report it instead of turning it off altogether. Google may take action and remove it from the predictions if it violates their terms.
Here's how to report Google's autocomplete forecasts:
- When you find a suggestion on Google.com, press and hold it.
- Tap Report this..
- Select and tap the appropriate feedback Send..
If you are using the Google app (I) Button to report forecast.
This is a way to turn off trend search and forecasting on Google on iPhone and Mac. We hope you find this quick guide useful.
Check next:


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Many of us look forward to Memorial Day for a big sale or a party to celebrate the passionate summer. However, we must remember this holiday meaning of a tribute to those who have sacrificed their lives for our freedom. Related: Different ways to Do to Celebrate Earth Day 2021 Enjoy this weekend to enjoy….
April is the month when we pay more attention to environmental issues to treat the planet well. Celebrating Earth Day 2023 is the day when you can convey the message of your responsibility to protect the environment and practice new habits if needed. We can start celebrating this day and helping save Earth, and we….
More recently, American adults have turned to alternative health aids like yoga and even healing crystals. In fact, you may have heard people talk about and show off these beautiful stones. There are several types of crystals that have specific healing powers for the mind, body, and soul. They are said to promote the flow….
Rice is a staple in many countries worldwide, but it's also an important component in a vegetarian's diet. It mainly offers healthy carbohydrates, but other kinds of rice (such as brown rice) have even more nutritious benefits. These include diabetes prevention, heart and digestive support, and cancer risk reduction. Switching to the vegan lifestyle can….
We are recommended to wear a regular nose and mouth mask, hand washing, and social distancing will help reduce the spread of COVID-19 in the community to protect ourselves and our loved ones. You should wear a mask whenever you come into contact with others from work and when going out. Wearing a mask not….
Today, when we suffer from disrupted supply chains and lockdown measures, gardening or agriculture is a great way to grow your own tasty and healthy crops and enjoy the time outside. In fact, gardening is good for the soul, it has a therapeutic effect on many levels. Just being around nature can make you feel….
We all know a crafted gift will be extremely impressive and meaningful to the recipient. The good news is that in this age of online retail, you can shop for a variety of handmade gifts or purchase the tools to make handmade gifts from the comfort of your own home. This means that crafting is….
We live faster and faster with so many things to do in a day. That is why expectations about what can be done during the day and time tracking become even more important for everyone, whether you are a freelancer or a CEO. That's why TimeQube was created, giving you an innovative approach to efficient….
We have just experienced the year 2020 with many difficulties caused by Coronavirus. Don't forget that you are a lover of the environment for Covid 19. The New Year is a great time to set new goals for yourself to have a better life. So what's better to do next year than helping the planet!….
You may be one of the many people who are now transitioning towards an eco-friendly lifestyle. Aside from small daily lifestyle changes, responsible shopping is very essential. Today, Eco Bravo will make it easier for you to make sustainable shopping and truly deserve the money. About Eco Bravo Eco Bravo is a small business mother….


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Parkinson's and Alzheimer's
After many decades in the neurology field, I have come to believe that neurodegenerative conditions like "Parkinson's" and "Alzheimer's" are marketing terms organized around selling products for frailty. There is some heterogeneity in the way we fall apart, but they all have in common underlying tissue and complexity changes that can be understood in basic biological terms as a consequence of impaired growth and repair.
This first article is on rapamycin, an mTOR inhibitor used in anti aging studies (at some point I will review this promising approach to healthy aging). The article makes a good argument that, for treating Alzheimer's, we should be treating aging itself.
"Over the past two decades, research on the biology of aging, referred to as geroscience, has made substantial progress in elucidating the genetic, molecular, and biochemical mechanisms of aging . A small number of "hallmarks of aging," driven by the activity of genes that regulate aging, have been identified that play important and evolutionarily conserved roles in the decline in function and increase in disease associated with old age. By targeting genes that regulate aging and the molecular processes that they represent, researchers have been able to increase life span and delay age-associated decline in every laboratory animal where this has been attempted. In principle, targeting these same processes should also be effective at delaying the onset of specific age-related diseases, including but not limited to AD, and, in some cases, perhaps even reversing specific disease-related pathologies." https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6762017
Glutathione, through its components NAC and glycine, are known to affect growth processes (mTOR and AMPK, corresponding to anabolism and catabolism) which are more directly affected by the experimental approaches of rapamycin and senolytics. Importantly, supplementation with the precursors of glutathione do not result in elevations of glutathione, but rather a return to youthful levels.
The first demonstration that glutathione can be returned to youthful levels by dietary supplementation of precursors came only in September.
I write the noncommercial blog unchronic.ghost.io for people I care about who want to try a supplement to broadly improve their health in old age and are interested in what I do. There is only a smidgen of empirical human evidence, but lots of basic science supporting the Baylor approach of supplementing glutathione.
I suggest to people that they try the "Recipe" for one month, then continue for four months if they improve. As always, this is my personal opinion regarding what those over 50 might consider, not medical advice for you personally.
When you look at this approach to aging for specific effects, for example for early Parkinson's, you also see benefits. (Try also Googling NAC, glycine, theanine, melatonin, or pregnenolone and Covid risk, Alzheimer's, inflammatory arthritis, chronic sinusitis, atherosclerosis, type 2 diabetes, immune senescence, skin wrinkles, etc.)


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[PICTURES] Aagha Ali and Hina Altaf get married in a private ceremony on Jumma tul Wida
Well, that's a surprise!
Showbiz sweethearts Aagha Ali and Hina Altaf surprised their fans by announcing the news of their wedding. They tied the knot in a private marriage ceremony on the 22nd of May, on the occasion of Jumma tul Wida.
Aagha and Hina announced the news via posts on their Instagram accounts. Aagha was previously engaged to 'Sabaat' lead Sarah Khan but they broke it off nearly one year ago.
Following their split, he was rumored to be dating Hina as both were seen together quite often.
Aagha shared their journey in a post announcing their wedding.
"and then the lockdown happened … I missed you like a part of me was missing!!! I realized and was sure that I want to spend the rest of my life you … and here we go!", he wrote.
Hina also shared a sweet love note for her husband.
Here are some of the pictures from the event:
What are your views on this? Share with us in the comments bar below.


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UNDERCOVER Boss Mark Taylor was forced to blow his cover and apologise over shocking working conditions at his company in tonight's episode of the ITV show.
Pickfords' managing director was shown the brutal living conditions some employees faced when working on overnight jobs for Britain's largest and oldest removal and storage firm.
Disguised as 'Dave' Mark was paired with driver Frankie who had worked there for 29 years following in his father's footsteps, and who also recruited his own son when he turned 16.
Hard-working Frankie told the director some home truths about the multi million pound business.
He revealed on night jobs the moving team were forced to "rough it out" in the back of the van as there was only one bed in the truck's cabin.
Horrified Mark said: "They should put you up in a hotel surely?" as Frankie explained they get a small allowance to cover expenses but he didn't want to "scare him on his first day" by revealing how much.
The camera then panned to a pile of raggy old blankets on the hard floor, and shocked Mark later confessed: "I can't have people sleeping in the back of the vehicle. I wasn't aware that was happening. I need to change that."
In another eye-opening scene he met porter called Anthony, nicknamed "the whippet", because of his speed when hauling boxes up and down stairs.
The young lad said he wanted to eventually become a driver but he couldn't afford lessons on his low wage.
The lads can earn more picking litter…
Mark Taylor
Having seen Anthony's enthusiasm for the job, Mark told the cameras: "I'm shocked that the lads can earn more picking litter. Looking at the job they do I think we need to look at our pay scales.
"Pickfords have got to hold onto these people. They've invested and we've invested time in them. To lose them would be the worst thing ever."
The final straw came over at the Barking depot where the director discovered the team had been working out of a shipping container on an industrial estate after the office was shut for logistical reasons.
Pickford staff refused to talk about the discovery on camera saying they felt underappreciated and fed up.
Shaking his head in shame the boss gasped: "It's worse than I thought.
"I need to fix this … I need to sort this out. I'm physically hurt by it."
Mark went in search of Frankie and revealed his true identity, vowing to turn things around for Pickford employees.
He said: "This experience has taught me we need to have a better level of pay and give them a direct career path. It's not just about the money it's about praising people, listening, they know they're being looked out for."
"I've always known the value of my front line workers but they are so much better than I thought they were."
Mark awarded Anthony a pay rise, driving lessons and his first car paid for by the company.
Frankie was given £10,000 as recognition for his hard work, and staff at the Barking depot all received a £1000 bonus to make up for the conditions they'd been working in.


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Second-quarter surge carries top-ranked Raiders to victory in milestone moment for Orne
QUINCY — Eric Orne's milestone moment came at home … sort of.
The Quincy Notre Dame girls basketball coach reached the 500 career victory plateau Friday night with a 58-42 victory over Mascoutah in the opening game of the four-day, 16-game Rumble on the River at John Wood Community College's Student Activity Center.
Although the game took place in the Gem City and came on the heels of the Raiders playing five consecutive road games, it technically wasn't a home game since it wasn't at The Pit.
That doesn't diminish the outcome or the significance of it.
The Raiders went on a 13-2 run over the final four minutes of the first half to erase a two-point deficit and extended the lead to as many as 18 points in the third quarter to take pressure off chasing Orne's milestone victory.
Now in his 21st season overall, Orne owns a 500-117 record with four state championships and seven state trophies. His teams have won 20 or more games 15 times, and at 14-1 already, this year's team is well on its way to reaching that mark before the end of January.
Neither team led by more than three points and there were seven ties in the first 12 minutes with QND's Sage Stratton scoring off a Jenna Durst assist to tie the game at 22 with 4:04 remaining in the first half. Stratton made the subsequent free throw to finish a three-point play and give QND the lead.
That kickstarted the game-turning run. Blair Eftink scored the Raiders' next three field goals as the lead reached 33-24 going to halftime.
After Mascoutah's Kate Schneider scored off a turnover to open the second half, QND went on an 11-0 run with four different players scoring.
Abbey Schreacke led the Raiders with 20 points and six rebounds, while Eftink finished with 16 points and Stratton had 10 points and five assists.
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