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State Attorney Ed Brodsky has no plans to fire Eric Robinson as his campaign treasurer. But he stressed the Venice accountant only works with the prosecutor’s political campaign, not the State Attorney’s Office itself.
“I employ his agency to comply with accounting,” Brodsky, a Sarasota Republican, said. “In fact I work with someone there other than Eric Robinson. And I am very comfortable with the relationship I have with the agency.”
He spoke to Florida Politics about the matter a day after news broke of an investigation of Robinson for “potential election criminal misconduct.”
Details of that investigation remain scant. The matter became public after Brodsky asked Gov. Ron DeSantis to reassign the investigation to another judicial circuit because of his own relationship to the treasurer.
DeSantis on Sept. 22 issued an executive order redirecting the matter out of Brodsky’s 12th Judicial Circuit and to State Attorney Andrew Warren’s 13th Judicial Circuit in Tampa.
Warren, a Democrat, hasn’t released any information about the ongoing investigation.
“We have accepted the executive assignment and are reviewing the case to determine what charges, if any, are appropriate,” said Grayson Kamm, communications director for Warren’s office. “We are not able to comment on active, ongoing investigations.”
Brodksy also said he could not go into detail. But he made clear the investigation was triggered by a letter sent to the State Attorney’s office by a private citizen, not by any action by law enforcement.
“The complaint this gentleman made has nothing to do with anything out of our office, and it has nothing to do with me,” he said. “It was a citizen’s complaint, and I felt I didn’t have any action I could take without any appearance of a conflict of interest.”
He also said there was an error in the Governor’s order on the case, specifically that it stated Robinson had worked professionally for the State Attorney’s Office. The order should have said only that Robinson worked for the State Attorney, Brodsky.
The letter was received by Brodsky’s office in the past few weeks, he said, and he began the process of requesting a transfer immediately.
Robinson recently lost a reelection bid for the Sarasota County School Board. Brodsky could not say if the complaint was received before the August election.
The State Attorney said he wanted to clarify the situation in part because of attacks by his own Democratic challenger, Betsy Young. On Monday, Young called for Brodsky to fire Robinson to erase suspicion the investigation may involve the State Attorney’s office.
“My retort is that Betsy Young has demonstrated time and time again she is willing to weigh in on subjects and express opinions on things she knows absolutely nothing about,” he said.
Robinson, for his part, said he knows no details of the complaint, but that any past accusations against him have ultimately been dismissed as unfounded. The accountant, who runs a private practice along with Republican Party of Florida chairman Joe Gruters, remains one of the most used Republican campaign treasurers in Florida.


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THIS is an open letter from Dr Steven J Hatfill, a former White House adviser on Covid-19 to President Trump.
This is the story of possibly the greatest corruption scandal in our country’s history.
This is the story of how petty bureaucrats and drug companies unjustly discredited an inexpensive FDA-approved drug that would have prevented Covid-19 hospitalisations and deaths for political spite and financial gain. These perpetrators, who now hold more significant positions in government, need to be held accountable for over 500,000 American deaths and the disruption of our economy.
This is the story of how a few key government officials failed to implement our well-formulated National Pandemic Plan and their weaponisation of a dangerously complicit mainstream media.
I tell this story because we, the People, deserve answers – those responsible need to be held accountable for their actions. The Covid-19 pandemic was a proverbial ‘warning shot across our bow’. Therefore we must forever prioritise pandemic preparedness as critical to our national security.
We deserve better. We deserve the truth. We deserve answers. We deserve JUSTICE.
I hope my words and timeline will help clarify what has happened, who is involved, and what action must be taken to protect our health and freedom.
I am Dr Steven Hatfill. I am a specialist physician, recognised virologist, biological weapons expert and I worked as an outside adviser to the Executive Office of the President of the United States from February 2020 through the inaugural transition period of 2021. My statements are not speculation because I had a front-row seat from the very beginning of the pandemic. My subsequent published papers and articles have been painstakingly referenced and fact-checked.
Some will tell you this is just another conspiracy theory, so I ask you to read on and judge for yourselves.
2005 The United States creates its first National Pandemic Plan, outlining the actions to combat a serious respiratory viral disease pandemic. This is based on the previous Biological Weapons Improved Response Plan.
2008 President Obama closes the White House Global Health Security Office.
2013 After the SARS and MERS outbreaks, Chinese virologists collect hundreds of samples from bats for study at the Wuhan Institute of Virology (WIV). Collaborative research begins on the coronaviruses, including gain of function (GOF) experiments (research which involves increasing the capacity of a pathogen to cause illness) in China and the University of North Carolina at Chapel Hill.
2014 The West Africa Ebola virus outbreak occurs. Dr Anthony Fauci MD [director of the National Institute of Allergy and Infectious Diseases (NIAID)] promotes a single layer of gloves for nursing Ebola patients and other inadequate protective measures. His instructions endanger the lives of health care workers, and national guidelines for protection must be urgently updated.
2014 United States’ Centers for Disease Control and Prevention (CDC) and the World Health Organisation (WHO) ban GOF research. The ban was implemented for 2014-2017.
2015 Chinese virologists and the University of North Carolina at Chapel Hill conduct unauthorised GOF experiments on coronaviruses.
2017 The 2005 Health and Human Services (HHS) National Pandemic Plan is updated. The specific responsibilities of local authorities, states and the federal government have been clearly stated. The national plan is to use early, outpatient antiviral drug treatment, home quarantine, and case contact tracing to cover the ‘vaccine gap’ (the time needed to develop a vaccine to combat any pathogen).
December 2019 Chinese authorities report the first outbreak of Covid-19 in Wuhan. On January 21, 2020, the first recognised case of Covid-19 occurs in the United States.
February 2020 Dr Steven Hatfill MD, a specialist physician and recognised virologist, is brought into the White House as an outside medical adviser.
Some members of the Covid-19 Task Force are considering the use of hydroxychloroquine (HCQ), a safe and effective FDA-approved drug, to control the rapidly spreading pandemic. The drug proves to prevent hospitalisation if taken when early symptoms of Covid-19 arise. The drug is cost-effective at 60 cents per tablet with Covid-19 treatment consisting of 11 tablets taken over five days. The FDA considers HCQ to be a safer drug than Tylenol.
March 23, 2020 The director of the Biological Advanced Research Development Authority (BARDA), Rick Bright PhD, is instructed by his superiors to work with the FDA to establish an Investigational New Drug (IND) authorisation for HCQ.
March 24, 2020 The next day, the director of Drug Evaluation and Research at the Food and Drug Administration (FDA), Dr Janet Woodcock MD, contacts Rick Bright at BARDA. Dr Woodcock wrongly advises Bright that HCQ is a dangerous drug requiring an Emergency Use Authorisation (EUA). Its use should be limited to hospital patients. Bright and Dr Woodcock promote this course of action, despite the early clinical data showing HCQ was the most effective in outpatients if given early during the initial infection, in effect eliminating hospitalisation. The FDA issues a EUA for HCQ for hospital use only.
In a legal document, Rick Bright makes a blatant admission of insubordination to multiple layers of leadership, including the White House, HHS Secretary Azar, and Dr Robert Kadlec, MD, the Assistant HHS Secretary for Preparedness and Readiness. Bright states the following in his whistleblower complaint: ‘. . . instead of a Nationwide Expanded Access IND protocol. Implementing the EUA was a compromise position, to rein in HHS leadership’s initial campaign to make the drugs available to the public outside of a hospital setting’. Question: When is it ever acceptable to ‘compromise’ public health during a rampaging pandemic?
April 4, 2020 Dr Anthony Fauci MD, the member of the Covid-19 Task Force responsible for informing the President of the best course of action for pandemic control, appears to be unaware of the National Pandemic Plan. In a heated White House Situation Room meeting, Dr Fauci refused to consider the use of HCQ for Covid-19 treatment. He dismisses the ever-accumulating HCQ efficacy reports from China, South Korea and France as simply ‘anecdotal’.
April 22, 2020 Rick Bright is fired as BARDA director for his insubordination. Before his dismissal, he falsely informed the press that HCQ is a dangerous drug.
Dr Anthony Fauci, director of NIAID, who is not a virologist, disregards the National Pandemic Plan, which included outreach programmes with physicians using HCQ for outpatient treatment and prevention to bring the Covid-19 pandemic under control.
Dr Fauci brushes off the accumulating evidence and diverts millions of federal funds into a programme to test and manufacture an experimental drug named Remdesivir.
Remdesivir must be administered via IV and only in hospital, instead of early community treatment as prescribed in the original pandemic plan. Dr Fauci changes the plan to promote ‘societal lockdowns’ and push the development of highly experimental mRNA vaccines by multinational pharmaceutical corporations.
Note: Members of Fauci’s Covid-19 treatment panel have ties to Gilead Sciences, Inc. (Foster City, CA), the company that holds the patent for Remdesivir.
May 16, 2020 In Phase 1 clinical trials conducted by Gilead Sciences in co-operation with China and Japan, Remdesivir failed: ‘No statistically clinical effect, with severe adverse reactions’.
Note: On October 16, 2020, the WHO concludes that Remdesivir is an ineffective drug and does not recommend its use to treat Covid-19.
May 20, 2020 Shortly after becoming Senior Medical Adviser to the FDA Commissioner, Dr Janet Woodcock recused herself over future decisions concerning vaccines, citing a conflict of interest.
June 15, 2020 Dr Fauci ignores the data that HCQ works if the drug is administered during the first five days of infection. Its EUA is revoked, despite the overwhelming evidence of its effectiveness. The FDA claims that HCQ is causing fatal heart rhythms in hospitalised patients when the Covid-19 virus itself is the cause.
June 29, 2020 Dr Fauci recommends a $1.6billion purchase of Remdesivir despite the drug’s Phase 1 failure in China. Question: Why did Dr Fauci discredit HCQ, leaving us defenseless, and order $1.6billion of an ineffective and toxic drug?
Note: Dr Fauci’s actions pave the way for the fast-track development of experimental mRNA vaccines (and their subsequent patents), which can receive a EUA only if there are no other approved and effective medicines, like outpatient HCQ.
After widespread lockdowns and millions of global deaths, the experimental mRNA vaccines are granted a EUA and released to the public. As of the date of this letter, the pandemic still prevails, and there is no FDA approved outpatient treatment for Covid-19.
A call to action for prevention, justice and reform
The actions of Dr Anthony Fauci, Dr Janet Woodcock and Rick Bright, PhD, must be independently investigated, and they must be held accountable.
All conflicts of interest and the interactions between government officials and pharmaceutical companies, including the publication of faulty research papers in respected medical journals, must be investigated, and they must be held accountable.
Immediately reinstate HCQ as an FDA-approved drug for Covid-19.
The US Pandemic Plan must be immediately reinstated as initially crafted.
Establish an outside independent United States Pandemic Response Department, with board powers including oversight.
On June 2, 2021, the distribution of my content was cancelled by PR NEWSWIRE, and I have been banned from further distribution. Their action is a brazen attempt to censor and block the true story and silence our voice as citizens.
What can you do?
1. Contact your local, state, and federal representatives and demand answers. They would have you believe that you serve them, but you employ them. Hold them accountable.
2. Cancel biased media subscriptions. Your money fuels their disinformation campaigns.
3. Read the references for this letter and the complete uncensored and referenced articles at www.drstevenhatfill.com.
Please ACT NOW, and together we can create a better future for America and the world.
Dr Steven J Hatfill

domain: medical


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5 Tips for beginning Photographers | Ashley Cook Photography |
5 Tips fop Beginning Photographers
With Black Friday and Christmas fast approaching, there’s a good chance lots of people will be getting new “fancy cameras”! If the next day you decide you want to dive head first into photography, great! Let me be the first to welcome you into the club. Photography is all about art and how you see the world. Sure, there are “rules” of photography that can be followed (likes the rule of 3rds), but that is also the cool thing about art… your art is yours and my art is mine. No one can tell you yours is wrong because if it’s what you want, than it’s your art not theirs.
With all of that being said, I thought I would put together 5 simple, basic things to get you started on your way to becoming a photographer.
- READ YOUR MANUAL- No, I am not kidding. That book that came with your camera is not a coffee coaster. Grab your camera and book and plan on spending a few hours getting to know your camera. I remember highlighting and flagging things in my book that I thought I would need to know and come back to at a later date.
- GET OUT OF AUTOMATIC MODE- I know the first thing you’ll want to do is take your camera, put it in auto, and starting taking photos. However, I think you will quickly learn that while your new “fancy camera” has all of these bells and whistles, your photos aren’t turning out how you want them to. That is because cameras are fancy, but stupid! When you’re in automatic mode you are letting the camera tell you how it sees. Switching it to manual mode lets you tell the camera how you want it to see.
- APERTURE, SHUTTER SPEED, ISO- These three things are some of the most important things you can learn about photography and your camera (see #4 for the other most important thing). These three things act together in order for your image to be properly exposed. Here is a rough break down of what they are and what they do.
- Aperture (or F stop) is how open or closed your lens is. The lower the F stop the more open your lens will be, meaning the more light that will be brought into your camera. Here is a chart to better help you understand:
- Shutter speed refers to how fast your shutter will open and close. The faster the shutter speed, the more still your subject will appear. The slower the shutter speed, the more blur a moving subject will have. The chart and photos below will help you better understand.
For the image on the right I slowed my shutter speed to 1/30th of a second. In both images my subject was moving, but with the slower shutter speed you can see her movement.
- ISO refers to film speed. The lower your ISO is, the less light that is brought in and the less grain your image will have. The higher your ISO is, more light that will be brought into your camera. This will also result in more grain. The images below were taken on the same day both using only natural light. The image on the left was taken when plenty of light natural light was available, around 5:00pm during the summer, resulting in little to no grain. The image on the right was taken long after sunset, with almost no light available (other than the headlight from a passing car). This forced me to up my ISO to 8000 resulting in a very grainy image. 4. LIGHTING! As a natural light photographer I consider lighting to be one of the most important things. Learn to see the difference between good, bad, even and harsh lighting. I plan to go even more in depth on this subject at a later date. Until then, here are some examples of the different types of natural light. I am sure these aren’t the “technical” terms for lighting, but it’s how I see light 🙂
5. PRACTICE, PRACTICE, PRACTICE! Ask people you know to let you practice on them. And DON’T GIVE UP! Keep pushing through when you get frustrated. Read every book, blog and article you can get your hands on. Watch every “how to” video. Take a class, workshops or mentorships and then practice, practice and keep practicing!
There are so many other things to learn about photography, but I feel like those are some of the most important starting factors. If you have questions, don’t hesitate to ask!

domain: artistic


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Projecttitel: Effect of processing on digestion & immunogenity of proteins in infant nutrition
Missie: Gewaardeerd, gezond en veilig voedsel
MMIP: Gezonde voeding, een makkelijke keuze (D2)
Looptijd: 2019 – 2022
Projectleider: Kasper Hettinga
Milk proteins fulfil a major role in nutrition and development of the immune system in infants. In the developing infant, where the immune system is still immature, it is important to supply foods that offer an optimal potential to establish tolerance to food proteins, including milk proteins. Milk proteins are often the only source of protein after birth and remain one of the main protein sources throughout the first years of life. Industrial preparation of infant formula, however, includes processing which may affect milk protein functionality, for example, heating steps are applied to ensure microbiological safety. This heating may modify milk proteins, which can have consequences for the overall digestibility and thereby have a nutritional and immunological impact. In addition, other components present in the product matrix (including lipids and carbohydrates) may influence these protein modifications.
In this project, variations in processing conditions (dry & wet heating at different intensities), as well as effects of the overall matrix composition, on protein digestion & immunogenicity will be studied. The novelty of this project is the direct link of protein processing with protein modification, digestion and immunogenicity, which is both scientifically and practically relevant. Previous research mostly focused on the effects of a specific modification, i.e. advanced glycation, whereas earlier stages of glycation and non-glycation protein modifications (including amongst others oxidation and protein aggregation) have been insufficiently studied. To study the variation in protein digestion caused by heating, both in vitro and in vivo measurements will be performed. We have recently developed in vitro infant digestion models that reflect the infants digestive physiology at different stages during development. In infants, especially gastric digestion of proteins is more limited compared to that of adults, which may affect the potency of the digesta to initiate intestinal immunological reactions. As gastric digestion can differ depending on the extent of heating of proteins, processing of dairy products can, through this mechanism, impact immune development in infants. Although the effect of protein processing on digestion has been studied, as well as the immunological consequences of processing, an in-depth integrated analysis of the relation between these three steps has not been performed, especially when it comes to milk proteins and infant digestion specifically. Moreover modifications beyond advanced glycation have not been studied. Gastric digestion of diary proteins partly depends on structure formation and breakdown (clotting) under gastric conditions. Better understanding of protein degradation in vivo may be achieved through in vivo monitoring using magnetic resonance imaging (MRI). We will employ dedicated MRI techniques to validate and inform in vitro digestion models, effectively bridging the gap between in vitro models and real life digestion processes. For the companies involved in this project, obtaining detailed insight in the effect of processing on the digestion and immunological consequences of protein allows further optimisation of their processes to ensure products with optimal properties to support infant health.
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Keywords: Paneth cellClose
Jianbo Liu, Kaixuan Liu, Shiquan Chai, Runze Xu, Ying Wang, Yundi Zhang, Shuanglian Wang, Chuanyong Liu, Bing Xue
Journal: Clinical Science
Clin Sci (Lond) (2023) 137 (1): 109–127.
Published: 05 January 2023
... and cyclin B1 in organoids. After G-1 treatment in vivo or in vitro , Paneth cell–derived Wnt3, Wnt3 effector β-catenin and Wnt target genes c-Myc and Cyclin D1 increased in ileum or organoids. Once blocking the secretion of Wnt3 from Paneth cells, the effects of G-1 on organoids growth, ISCs marker genes...
Includes: Supplementary data

domain: literature


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Luis Moro – An Invitation
This is my personal invitation to you. Join the Planet Generation. Together we can transform the world.
I’m extending this invitation to you because I believe we both want to make a difference. The time has come. I’m asking you to unite with millions of other people and together let’s take the actions that will Transform the planet.
I’ve personally taken on leading the launch of the planet generation with a team of leaders committed to keep our Vision and Mission alive until it becomes an active global commitment. We don’t know what that will look like. But we do know we need you to unite to make it a reality.
I believe most people want peace on earth. We want a world that works for everyone. And we want to live fully self-expressed with people we love.
I believe you want that. You really do want a version of that for yourself and your life.
If you do, then I ask that you sign up and support how ever you can. Let’s get in action.
At the very least I ask that you “Speak Peace” for the rest of your life.
Our goal is that we all take on making a difference as a lifestyle choice. We can all make a living, making a difference. Our jobs, our recreation, our lifestyle can be one where we make a difference for the whole of humanity.
Overtime many of us committed to Peace and Transforming the planet will be providing opportunities for you to participate and make a difference for yourself, your community, our planet.
So please be patient with us and participate how you can as we begin the journey of transforming the planet with actions that make a difference. We are the Planet Generation.
Luis A. Moro
The Planet Generation

domain: sociology


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I am calling this stitching done for now. In a certain way I enjoyed stitching this but mostly I lost interest in it. I think I need my stitching to be something not just random. I definitely prefer to fill in a shape or create a mandala. I am looking forward to starting a different project for November.
I am linking with https://kathysquilts.blogspot.ca/search/label/Slow%20Sunday%20Stitching%202017
As a self-taught artist, I have developed a practice of experimenting with my own ideas, which seem boundless. Curiosity and research continually drive me daily. For over 20 years I have taken an explorative approach towards art making. My optimistic approach to life is represented in my art as I find beauty in even the most difficult circumstances. This beautiful, imperfect life I live is meant to be shared through my creativity, resourcefulness and curiosity. I hope to influence my audience with joy.

domain: hobby


Posted by: Laura Hammond
Tue 24th January 2023
A proposal to build a new affordable housing development and community centre in Blackwell is set to be discussed by Bolsover District Council.
The site in question – on Woburn Close – is already home to bungalows, an assisted living building and Blackwell Community Centre.
However, an application put forward by Bolsover District Council and Blackwell Parish Council seeks permission to demolish the existing buildings and replace them with new ones.
The proposal is to build 22 new homes – four houses and 18 bungalows -, an independent living building comprising 20 flats and a new community centre.
According to a design and access statement attached to the application, the existing development, built in the 1970s, is “below current standards in terms of construction, sustainability, amenity and being fit for purpose”.
A statement in the document from Bolsover District Council explains further: “This scheme is part of the council’s Bolsover Homes project which is aimed at building new council houses for affordable rents across the district. The scheme more efficiently uses the council’s land by replacing end of life, unfit for purpose dwellings.
“The proposed new homes exceed today’s standard whilst benefitting the district’s economy through training, skills development and local supply chain.
“As the intention is for the residential element of this site to be delivered as 100 per cent affordable housing, the local housing need to this area has been used when identifying the best unit types and mix of sizes.”
According to the design and access statement, the new community centre will be funded, owned and managed by Blackwell Parish Council but the land will be leased from the district council.
A report due before the planning committee recommends the application for approval.
Bolsover District Council’s planning committee will meet at 10am on Wednesday (January 25). You can find more information here.
Picture credit – Google Maps
Charity van gets a ‘bling’ makeover
Students in full voice for the big sing
Singing group now set to meet in the great outdoors
Before and after pictures of restored station benches
Match report - Scunthorpe United 1-0 Alfreton Town
“We know you’re fed up of this issue and we’re sorry for this” says council about potholes


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Brand new and original cookers
3 burners gas 1 electric burner
Has oven for baking and meat roasting
Strong and durable
12 months warranty
call or wasap072********* To make your order.
Double deep fryer thickened cylinder electric deep fryer 2 baskets, stainless steel chicken chips fryer for french fries home kitchen restaurant countertop food cooking, 12.7qt/12l 5000w max silver
shop with us today
Dual tank fryer with large capacity 】: the dual tank design makes this fryer super functional. It is ideal for both commercial uses and home uses, such as lower volume restaurant, cafes, mobile catering, fast food stands, sna...

domain: food


text:
The disappearance of a small forest was the first sign of approaching evil.
One morning, the villagers awoke to find a solid wall of fog where the forest should have been. The brave few that ventured into the pitch-black mist were never heard from again...
The next day, the swirling mists swallowed up the lake. It was not long before an impenetrable wall of the blackest fog enshrouded the village itself in perpetual darkness. And the black fog wasn't finished yet...
By ancient covenant, the Five Kingdoms had forbidden all alliances between kingdoms. Now they were forced to abandon their centuries-long isolation. The Five Kings met, for the first time, in a council of war. But no one, king or peasant, could offer a plan to defeat the fog.
Terrifying rumors spread throughout the land. People whispered tales of an ageless evil reawakened...
Created and Maintained by: X_countryguy
Lost Kingdoms © 2002

domain: library


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Like a person together will be very happy; love a person together will be inexplicable loss. Like a person is always happy; love a person, you will often cry. Like a person when you think of him will smile; love a person when you think of him will be in a daze to the sky. Like a person is to see his advantages; love a person is to contain his shortcomings. ------Like is a mood; love is a feeling.
amghpzp.cnLife in the world is like a colt over the knee. But I can remember every word you say when I live forever.No matter how good a person is, if he doesn't want to accompany you, he is a passer-by.
Remember what should be remembered and forget what should be forgotten. Change what can be changed and accept what cannot be changed!Yuzhu once recorded that the Phoenix visitors did not see the water flowing in the air.You can't change yesterday, but if you worry too much, tomorrow will ruin today.
There is a kind of forbearance, which is actually a kind of power, and there is a kind of silence, which is actually a shocking confession.At both ends of life we stand on the shore of each other.
I always wonder if my memory lives on the other side of the street and my ring dies on the other side.
amghpzp.cnWhat hurts most is that the person who made you feel special yesterday makes you feel dispensable today.Life will round us in order to let us roll further.
The outside decides two people together, and the inside decides how long two people stay together.If you don't understand, you are afraid to pretend to understand.
Don't be afraid of imperfection, just think of yourself as perfect.Love ends one day like a TV play.
auctor, nisi elit consequat ipsum, nec sem nibh id elit.Aenean sollicitudin, lorem quis bibendum auctor, nisi elit eneaollicitudin, lorem quis bibendum auctor,onsepsuec Aenean sollicitudin, lorem quis bibendum auctor, nisi elit consequat ipsum, nec sem nibh id elit.Aenean sollicitudin, lorem quis bibendum auctor, nisi elit eneaollicitudin, lorem quis bibendum auctor,onsepsuec nibh id elit.Aenean sollicitudin, lorem quis bibendum auctor, nisi elit eneaollicitudin, lorem quis bibendum
My heart is full of sunshine, and my pure heart is like a drop of water. No matter how misty and rainy the outside world is, I'll clear myself. That drop of water without pollution is a kind of spirit and a kind of faith, which makes this drop of water always clear and pure.See More

domain: psychology


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Railways has always been known as the Lifeline that Connects Bharat. Indian Railways is the largest rail network in Asia and the 2nd largest in the World. It has 115,000 km of track length. It runs 12,617 trains to carry over 23 million passengers daily connecting more than 7,172 stations. Railways runs more than 7,421 freight trains carrying 3 million tonnes (MT) of freight every day or a billion tonnes annually. With over 1.4 million employees on its rolls, Indian Railways is the world’s seventh largest employer. Such a mammoth enterprise! Despite the many challenges facing it, there is no doubt that our Railways contribute to Nation building every minute of the day.
Ever since the Narendra Modi led Government took over, there have been many facets of the railways which have been improved to make it more people and employee friendly. Some of the stories are so heart-warming that one cannot stop thanking the Railway authorities for making this happen. And one cannot but appreciate the BJP Government which made this possible, making the best use of technology & administrative powers. While almost all earlier Railway Ministers paid special attention to the area they belonged to, the Hon. Railway Ministers of the BJP Government have gone out of their way to see to it that they gave equal weightage to all of Bharat & probably more to those areas which were earlier not reached out to – the Maoist affected areas and the North East. Another change which was very much needed but never addressed was the electrification through Solar panels. But all this another time… Today let us look at the Humane side of the Railways…
BIO-TOILETS & MECHANIZED CLEANING:
Cleanliness was the scourge of the Railways. Seeing human waste on the railway tracks with flies hovering over it is something that most Bharatiyas will remember. Thanks to the bio-toilets this has almost disappeared from our tracks. And something that will make the readers smile – The Railway Ministry has advanced its earlier plan to fit bio-toilets in its entire fleet of coaches from 2021-2022 to 2019. Note: Work on this started in 2011, but the speed with which the work is being completed deserves a loud round of applause.
My mother-in-law, a 85year old lady used to always dread travelling by train (she hates going by road too because of her weak knees) to go to her village. This was because she found it difficult to climb the bridge to go to the other platform. Escalators in Andheri Station made her and many other elderly people happy. Of course it has also helped other commuters with luggage. And for those who cannot use the escalators, Lifts have been installed in major stations. These small citizen-friendly measures which were never thought of earlier, have made life simpler.
It seems unbelievable that the couple with children sitting next to us, could actually be child-traffickers. Would you believe it then if you read that around 35,000 children were rescued in the last four years from railway stations or trains who had either left home on their own or were being trafficked and exploited by others. In Delhi alone, 820 trafficked children have been rescued from railway stations from January to May 2018. Some of these children have run away from home wanting to make it big in cities. But most of them are literally transported from villages, towns and cities to big metros, to push them into begging or into the flesh trade. Project Muskaan of the Railways has helped many such children. Special awareness campaigns have been held to even sensitise Railway employees, Police and stall staff members to this evil.
Another great help that Railway authorities are able to do thanks to the best use of Technology, especially Twitter & Whatsapp, is the uniting of children who have separated from their parents due to large crowds – https://twitter.com/rpfwcr/status/1006057075061895168
IMPROVING LIVES THANKS TO THE FREE WI-FI:
Last month a star was born – Sreenath K, a coolie (railway porter) cleared the written test of Kerala Public Service Commission. What was special about this was that he prepared for these exams thanks to his phone, earphones and the Free Railway WI-FI at Ernakulam Station. The WiFi service, launched in 2016 as part of Prime Minister Narendra Modi’s Digital India initiative, provides free Internet at Railway Stations. The Net is full of stories of people who are able to keep in touch with their families who live far away, fend off boredom or even use tutorials & learning materials, thanks to the Wi-Fi services of the Railways.
Now this has to be the most humane gesture that Railways have to be praised for. One way that this has become possible is because of the elimination of unmanned railway crossings. Derailments have reduced thanks to strict security measures. Despite trains having travelled an additional 74 million train km in 2017-2018 as compared to the last five years, accidents were at the lowest in 57 years.
When Sagar Kanjilal, his wife Banashree and their ailing two-month-old boy were travelling in an air-conditioned coach of Yesvantpur-Howrah Duronto Express (12246) on June 5, 2018 night, they were faced with a life-threatening situation – the last oxygen cylinder was about to get empty before they would reach Vijayawada Station which was far away. The on-board TTE Mihir Kumar sent an SOS to PVS Jagga Rao, chief ticket inspector in Vijayawada. Mr. Rao continuously monitored the control office & saw to it that the train reached Vijaywada Station in just twenty minutes before the tank got empty. The railway authorities had kept fresh oxygen cylinders ready for the little boy so that he could use them till he reached Howrah railway station. The train reached on time & and ambulance was ready at the station to take him to the hospital.
In another instance when Mr. Tulsi collapsed on coming out of a washroom, a fellow passenger on the train (12321 Howrah Mail), Bhuvaneshwar Shaw, tweeted about the emergency to the Railway Minister and his brother, Jharkhande called the 182 helpline to ask for medical help. In less than an hour, when the train pulled into Dadar station, a medical team along with RPF and the deputy station master were there to attend to Tulsi. He was then taken to the emergency medical room available at Dadar and was stabilised.
TRANSPORTING WATER TO LATUR:
Water – the most important requirement of Mankind. In 2016, when the Latur region of Maharashtra was reeling under a water crisis, Railways came to the rescue. A total of 25.95 crore litres of water was transported to the parched Marathwada region.
There is absolutely no doubt that the efficiency of the Indian Railways, the administration, the facilities provided, all have improved since 2014. While many of us feel that this is the moral obligation of the Government, the fact is that this Government has gone out of its way to fulfil the aspirations of a growing Nation. Today we take these positive changes for granted, but just once if one were to peek into the past, one would have to agree that every facility that the Railways granted us in the past was given to us as a Dole, a Favour – but today, it is given to us as a Right. We have to appreciate this change. Yes, there is a lot to be done, especially in the area of Punctuality. The present Government is tackling this very seriously, which is obvious when I learn that the punctuality of trains is now being recorded through data-loggers whereas, earlier it was done manually. Now, even if a train is late by a minute, it is counted as a train arriving late. One cannot have any doubt that the Indian Railways is well on its way to being on par with services in other advanced Nations. It may take a little while, but the intentions are clear & the will and backing of the Government is there. What makes me feel more proud is the evidence of warmth and Humane treatment to the commuters.

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Correa Grocery Bag - Grey
PORTER YOSHIDA & CO. Correa Grocery Bag – Grey: Shoulder strap in a multicolored pattern with two carabiners at the ends. Featuring an outdoor mood, it is designed to be attached to the label’s very own grocery bag. A Japanese classic, the accessories by Porter-Yoshida & Co stand out for their aesthetics and quality: in fact the timeless design and functionality of each product blend perfectly with the “Made in Japan” production, characterized, in this case, by the use of nylon, which guarantees its impermeability.
- Adjustable Strap
- Aluminium Metal Clips and Adjuster
Made in Japan
Composition & materials: 100% Polyproplene – 8mmPP plain cord pattern, zinc die casting

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Quick & Easy Potato Recipes: 30 Scrumptious Recipes for Breakfast Potatoes, Potato Side Dishes and More
Potato and Cheese Snowballs
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Get a scrumptious, crunchy-cheesy fast food taste at home with Potato and Cheese Snowballs! This is truly a recipe that the whole family will enjoy!
What You'll Need
- 6 tablespoons (3/4-stick) butter, divided
- 1 (30-ounce) package frozen shredded hash brown potatoes, thawed
- 1 medium onion, chopped
- 1 tablespoon minced garlic
- 1 cup (4 ounces) shredded mozzarella cheese
- 1/4 teaspoon Italian seasoning
- 1 teaspoon salt
- 1/4 teaspoon black pepper
What to Do
- In a large nonstick skillet, melt 3 tablespoons butter over medium heat. Add the potatoes, and cook, turning frequently, for 12 to 15 minutes, until browned. Place in a large bowl and set aside.
- In the same skillet, melt the remaining 3 tablespoons butter then add the onions and garlic; saute the onions and garlic until golden, being careful not to burn. Stir the onions and garlic into the potatoes. Add the remaining ingredients; mix well.
- Preheat the oven to 400 degrees F. Coat a baking sheet with nonstick cooking spray.
- Using an ice cream scoop, scoop the potatoes into 12 equal-sized scoops, packing tightly, and place the scoops on the baking sheet.
- Bake for 18 to 20 minutes, or until browned and heated through.
These are great served right away, or they can be cooled, covered, and refrigerated overnight to serve for brunch or with dinner the next day. Just reheat in a 300 degrees F. oven for 10 to 15 minutes, or until heated through.
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Since I plan on doing a few house-centric resolutions this year that will require a bit of saving, why not get back into the cooking groove? I used to cook every day. Homemade tortillas. Cream puffs. Cinnamon buns. Roast chicken. Stuffed porkchops. Now? Nothin’. Maybe some pasta, or something in a crockpot. That’s just sad.
What has put me in this mood? That’d be a who: Oprah. Got an e-mail from her Web site this morning with a list of New Year’s cooking resolutions. And I thought it would be a very good idea if I adopted a few of ’em. With a twist or two, of course.
So, here are my cooking resolutions for this year. Because really, it’s time I got to rattlin’ those pots & pans.
1) Master 3 new dishes so you can make them (& make ’em delicious) without a recipe
I really need to do this. I can’t seem to come up with anything when someone says “hey, let’s make something….” Maybe perfect the Greek Chicken/Lamb (which is kinda like cheating but I Don’t Care), and then figure out a cool veggie/vegan dish. Then a balls-out bellybuster? Yeah, probably pasta.
2) USE the cookbooks I have
I have great cookbooks. I’ve even tabbed certain recipes in ’em. I never use them. This year? Picking a tab once a month and going for it.
3) Make Your Own Mayonnaise. Again.
Dude. It’s been over 20 years since you made mayo from scratch. Remember how damn easy it was? Then you up and started grad school and got all lazy and stuff. So it’s got egg yolks. It’s dead easy, and it’s absolutely delicious. Why not show someone — my curiously cooking-shy sister, perhaps — how to do it? Then *really* homemade tuna salad for everyone!
4) Cook something with shells
Ulp. As much as I love me some seafood, I fear cooking ’em. How hard could it be? Y’know, not ending the evening in the hospital with a disease named after me? Only time will tell. I’m doing this, dammit. Probably with beer. Because beer is awesome.
5) FINALLY learn how to make risotto!
Because I’ve been yammering on and on about this for years. Time to put up or stuff my piehole with substandard frozen packets.
6) Make biscuits once a week for a month. Or more.
I used to make biscuits just about every day. I even received compliments on them from real Southerners (as opposed to my “Just Below the Mason Dixon Line” self). Make ’em whole wheat to healthy ’em up, but make ’em.
Okay, this list is on the interwebs. And as we all know, if it’s here, it’s true.
My lord, what have I done….


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U-Boat 8465/A/MT Men's Red Darkmoon SS/A Watch - Specification
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A watch brand, based in Italy, producing from almost 20 years handcrafted and limited edition pieces, all of them designed by Italo Fontana. Unique and tailormade watches, all guaranteed by finest...


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Video: Man shoots flamethrower from top of city bus; NYPD investigating
NYPD officials said they "are aware" of the video and an investigation is ongoing
By John Annese, Clayton Guse and Thomas Tracy
New York Daily News
NEW YORK — A wanna-be rapper from Brooklyn wasn’t going to lose his shot at blasting a flamethrower from atop a city bus for a guerrilla music video — even though the illicit nighttime stunt put more than two dozen passengers in harm’s way.
Streams of fire burst into the air and down the back of the MTA bus in a shocking video that emerged on social media in recent days.
In a scene that could have been ripped from the movie Mad Max: Fury Road, the 57-second clip shows rapper Dupree G.O.D jumping shirtless from the top of an ice cream truck onto a B26 bus at Franklin and Putnam Aves. in Bedford- Stuyvesant.
He then fires off his hand held flamethrower, shooting blazing arcs in the air before running to the back of the bus and shooting the flames downward, torching the ground as the bus starts to move.
Witnesses cheer him on as he keeps shooting flames in the air.
“What is you doing, n-----?” one man asked.
The man then jumps down, almost landing in a line of flames he lit.
“We outside, n-----!” the man says before walking off.
Dupree G.O.D also posted overhead video of the dangerous Nov. 8 stunt to his Instagram page.
“'Wutang' music video shoot was insane. It was a movie. It was f---ing epic,” he wrote on Instagram. “Much everyone to all who came out. We shut Franklin Avenue Down. With amazing energy. We are in this together. We The ILLest. Video coming.”
City transit officials were not impressed.
“We don’t even need to say how absurd, dangerous and just plain stupid this was. The reckless individual who torched over the top of an occupied bus put New Yorkers, including the bus operator, in life-threatening peril,” MTA spokesman Tim Minton said Tuesday. "We are grateful the courageous actions of the operator in moving riders to safety minimized risk, and are cooperating fully with the NYPD investigation.”
The bus was ferrying 25 passengers during the 5:30 p.m. incident, MTA officials said. None were reported injured, and all the passengers were transferred to a different bus at the next stop, officials said.
The B26 bus line starts in downtown Brooklyn and travels down Halsey St. through Bedford-Stuyvesent before ending at Wyckoff St. and Palmetto St. in Bushwick.
An NYPD spokeswoman said that the department was “aware of the video.”
“It is being investigated by the 79th Precinct Detective Squad,” the spokeswoman said.
Dupree G.O.D did not return a message seeking comment Tuesday.
We are aware of a video circulating social media that shows a man using a flame thrower on top of a bus in Brooklyn.— NYPD NEWS (@NYPDnews) November 17, 2020
The incident is currently under investigation. We have the utmost confidence in our @NYPDDetectives that this case will be solved quickly. pic.twitter.com/KPnMlYJb1g
(c)2020 New York Daily News


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Manchester City came from two goals down to end their International Champions Cup campaign with a 3-2 victory over Bayern Munich at the Hard Rock Stadium in Florida. Bayern Munich's Kingsley Coman (R) controls the ball next to Manchester City's Cameron Humphreys during the International Champions Cup friendly match between FC Bayern Munich and Manchester City at Hard Rock Stadium in Miami, Florida, on July 28, 2018.
Man City vs Bayern: Last 5 Champions League meetings. City play Bayern Munich in the Champions League for the sixth time in just three years at the Etihad Satdium tonight. Bayern Munich 2-0 Man City. By Alistair. Mario Gomez scored twice as Manchester City were given a rude awakening by Bayern Munich in the Champions League. City had two penalty appeals in the.
Champions League, Bayern Munich v Manchester City - Latest score, news update, live match report and result from the Group E clash at Allianz Arena on Wednesday September 17, 2014, kick-off 19:45. Man City 3-2 Bayern Munich: Aguero treble. City's challenge looked over after Aguero's early penalty was cancelled out by Xabi Alonso and Robert Lewandowski, but City star struck twice late on.
City can now go through so long as they better CSKA's result against Bayern - but if City lose their game against Roma, or it ends in a 0-0 draw, they would go out of the competition. Hoffenheim vs Bayern Munich German Bundesliga Match Bayern Munich continue their 2019-20 season into the new years with games coming thick and fast as they juggle three different competitions. They are well in with a shout with German League title chasing RB Leipzig by 4 points only while they face Chelsea in the Champions League in February. Get a report of the Tottenham Hotspur vs. Bayern Munich 2019-20 UEFA Champions League,. Pep Guardiola gives his reaction to the European ban imposed on his Man City side by UEFA for the first time.
Once again, a City team mixed with youth and experience produced a very impressive display, showcasing some beautiful football in parts, creating chance after chance, and a calmness beyond their years to claw us back into the contest. READ: Match report | City 3-2 Bayern Munich. MATCH GALLERY: City v Bayern City signed off the International Champions Cup campaign with a superb comeback victory, as Bernardo Silva came off the bench to bag a brace in the Blues' triumph over Bayern Munich. Man City youngsters tame Bayern Munich to give a bright vision of the future. German champions shocked as Manchester City fight back from two down to win - and show glimpses of a golden future.
International Champions Cup Match Report for Bayern München v Manchester City on 28 July 2018,. Bayern Munich 2 Manchester City 3:. Man City storm back to take win over Bayern. Man City vs Bayern Munich Highlights 2018. Bayern Munich vs Manchester City 2-3 City Highlights 2018.
Watch the highlights of Bayern vs City! Sub now:. meeting between FC Bayern München and Manchester City was also a reunion for the men from Munich with Pep. Man City 2-0 Chelsea | Aguero. Bayern-Man. City UEFA Youth League 2014/15 Group stage. an der Grünwalderstrasse - Munich. 2014/15, Group stage - Group E Manchester City fans started their crunch tie against Bayern Munich booing the Champions League anthem.. Manchester City Man City vs. Bayern Munich: Goals, Highlights from Champions League Match
Wolverhampton vs Man City – Premier League – 25.08.2018. FC Bayern Munich vs Man City 2-3 Highlights & Match Replay Video Links. In addition to Bayern Munich vs Manchester City 2-3 Highlights, you can watch and: Bayern Munich 2-3 Manchester City Extended Highlights. Bayern Munich 2-3 Manchester City Full Match Replay 1st half. Bayern Munich vs Man City: Live stream, TV channel, kick-off time and team news for International Champions Cup clash. Pep Guardiola's Premier League champions continue their US tour with a tough.
Bernardo Silva Double Gives Manchester City ICC Win vs. Bayern Munich Adam Wells @ adamwells1985. Featured Columnist July 29, 2018 0 Comments. When Man City Bid for Messi. Sergio Aguero's last-gasp heroics earned Manchester City a thrilling 3-2 win over 10-man Bayern Munich to boost their hopes of reaching the last 16 of the Champions League.
Bayern Munich 2-3 Man City highlights and reaction as Bernardo Silva runs riot. Manchester City signed off from their pre-season tour of the US with a win after losses to Borussia Dortmund and. Get a report of the Bayern Munich vs. Manchester City 2018 International Champions Cup football match.. Manchester City/Man City via Getty Images. Bayern were already 2-0 ahead by then. Manchester City will face Borussia Dortmund, Liverpool and Bayern Munich in this summer’s International Champions Cup, as we found out on Saturday. The official announcement was made today which.
Manchester City 3-2 Bayern Munich [HD] Highlights + Goals. Monaco 6 x 6 Manchester City UCL 16/17 Extended Goals. Lionel Messi vs Bayern Munich (UCL) (Home) 2014-15 English Commentary. FC Bayern Munich First team squad First team squad Women's team The FC Bayern Women represent the club in the Bundesliga and Women's Champions League. Legends team The FC Bayern Legends team was founded in the summer of 2006 with the aim of bringing former players... Matches Fixtures Standings Stats Pep Guardiola says he was 'incredibly happy' at Bayern Munich, but insists he will see out his contract at Manchester City. Guardiola has been linked to the vacant manager's job at the Allianz.
Manchester City vs Bayern Munich 3-2 extended highlights. Champions League group stage 25.11.2014. English Commentary Juventus vs Bayern Munich 2-2 - https:/... Bayern Munich head for home after being edged 3-2 by Manchester City in their final. Man City won the English Premier League title with a 19-point gap to second-place Manchester United. FC Bayern Munich was founded by members of a Munich gymnastics club (MTV 1879). When a congregation of members of MTV 1879 decided on 27 February 1900 that the footballers of the club would not be allowed to join the German Football Association (DFB), 11 members of the football division left the congregation and on the same evening founded Fußball-Club Bayern München.
Bayern-Man. City UEFA Champions League 2013/14 Group stage. Football Arena Munich - Munich.. Football Arena Munich, Munich. Referee. David Fernández Borbalán (ESP) Assistant referees. Manchester City's defeat by Bayern Munich in the Champions League was overshadowed by Carlos Tevez refusing to go on as a substitute when called on by Roberto Mancini
Manchester City are back in action on Sunday, when they will face Bayern Munich in the International Champions Cup Bayern Munich vs Manchester City: TV channel, live stream, squad news & preview. Man City v Bayern Munich, UEFA Champions League, Score:3-2, Corner:6-0, 1x2 odds: , Prediction/Tips: goal under 3.0,corner under 10.5,Bayern Munich -0.5
Get statistics for the Bayern Munich vs. Manchester City 2018 International Champions Cup football match. Skip to navigation < >. Man United scrape past AC Milan on penalties. UEFA Champions League match Man City vs Bay Munich (2 Oct 2013). Preview and stats followed by live commentary, video highlights and match report.
International Champions Cup 2018 - Bayern Munich vs Manchester City - Full Match HD 90MinTV. Loading... Unsubscribe from 90MinTV?. Man City vs Real Madrid 4-1. Match facts; Bayern start a Champions League campaign with a win for the 14th time, a record they share with Barcelona. Man City have lost three matches in a row in Europe for the first time. Despite allowing 10-man Bayern Munich back into the game, two late goals from Sergio Agüero, to add to his first-half penalty, gave Manchester City a dramatic 3-2 win in the Champions League
Manchester City staged a superb comeback to end Bayern Munich's record run of 10 consecutive Champions League wins but they still finished as Group D runners-up behind the German champions. We invite you to check Bayern Munich and Manchester City match statistict (Tab Stats->Match statistics), where you will find out who is the best scorer.Also you can check the statistics of possession, shots, corners, offsides, and many other applications. In tab Goals stats (tab Stats->Goals stats) check how many loses and scored goals per game, how many games won and lost to zero. Sergio Aguero struck twice in the closing minutes to complete a hat-trick and revive Manchester City's Champions League hopes with a 3-2 win over 10-man Bayern Munich.
Bayern Munich vs Manchester City prediction.. Man City play their home games at the Etihad Stadium and the stadium capacity is 55,097. Manchester United are regarded as Manchester City’s biggest rivals and the likes of Kun Aguero, Carlos Tevez, Shaun Wright-Phillips,. Bayern-Man. City UEFA Youth League 2013/14 Group stage. Städtischesstadion - Munich. 2013/14, Group stage - Group D
Joe Hart's poor form continued as Manchester City's England goalkeeper suffered a nightmare performance in this comprehensive Champions League defeat by champions Bayern Munich at Etihad Stadium. Once again, a City team mixed with youth and experience produced a very impressive display, showcasing some beautiful football in parts, creating chance after chance, and a calmness beyond their years to claw us back into the contest. READ: Match report | City 3-2 Bayern Munich. MATCH GALLERY: City v Bayern
On Saturday, July 28 Bayern Munich and Manchester City will face off at Hard Rock Stadium in Miami for an International Champions Cup match. This is the last of three matches in a week for Bayern. Man City lose to Bayern Munich in Guardiola's first game. Pep Guardiola's first game in charge of Manchester City ended in defeat at his old club Bayern Munich as a goal from Erdal Ozturk gave.

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Camping & Holiday Parks for Couples Newquay, Cornwall
Here at Monkey Tree Holiday Park & Campsite near Newquay Cornwall, we take a lot of pride in creating and providing enjoyable holidays for couples and families.
We have an in house entertainment team who are on hand throughout the season to ensure that everyone has a great holiday. Monkey Tree Holiday Park & Campsite is great for couples holidays with plenty of activities on offer, a selection of restaurants and bars to visit and plenty of quiet places to relax.
Stay up to date with our Special Offers for Couples
To make a Booking Enquiry please click here
Or call Reception on 01872 572032 / Email us here

domain: travel


Minister for Foreign Affairs Pekka Haavisto will visit the capital of Senegal, Dakar, from 2 to 3 January. In Dakar, he will meet Senegalese Foreign Minister Aïssata Tall Sall and other state leaders.
The meetings will focus on bilateral relations between Finland and Senegal as well as topical international questions. Finland opened an embassy in Dakar in 2022. It is Finland’s first embassy in the French-speaking West Africa.
“It is important that Finland has closer relations with West Africa. We are affected by developments in the region, which also offers many opportunities for cooperation. Senegal is an important partner for Finland and for the EU in western Africa,” says Minister for Foreign Affairs Haavisto.
In Dakar, Foreign Minister Haavisto will meet members of Senegal’s National Assembly and representatives of research institutes, international organisations and civil society organisations. They will discuss the situation in Senegal and more widely in West Africa and the Sahel region.
During his visit, Foreign Minister Haavisto will inaugurate Finland’s Embassy in Dakar.
Distributed by APO Group on behalf of Ministry of Foreign Affairs of Finland.


Sones exhaust fan. 10 sones is the sound of a refrigerator. What do sones refer to in exhaust fan sound ratings. 1300-Watt heater for instant heat when you want it. Stainless steel 6 duct collar. BV Ultra-Quiet 20 Sones Exhaust Fan. 20 sones is the noise level in a normal office workplace.
But it is not quite that simple. Bathroom Fan Sone Scale The loudness of fan noise is measured in sones and a sone scale will help get the correct loudness for your specific fan. A bathroom fans loudness is measured in sones. As a general statement an exhaust fan with a lower sone level rating is an overall higher quality fan when compared to an exhaust fan with a higher sone level rating. ECM motor with SmartFlow Technology simplifies the selection process and ensures optimum performance to meet all your exhaust fan needs.
Integrated dimmable LED with color adjustable CCT lighting. 20 sone super quiet performance. For quiet bathroom ventilation the fan should be rated at 10 sones or less. What are Good Sone Ratings. Getting a bathroom exhaust fan with low sones. Pick a Flow 3 speed motor gives you access to 50 80 and 110 CFM for faster clearing speeds. Like other fans bathroom exhaust fans have noise levels that are rated in terms of sonesThese ratings are sometimes listed along with the other technical specifications of the fan making it possible to compare them against each other.


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Appearance Schedule Announced
Archived Press Release – Release Date: April 17, 2006
Ferndale, PA – Mary A. Shafer, author of Devastation on the Delaware: Stories and Images of the Deadly Flood of 1955 announces a busy schedule of appearances in the Delaware Valley on both sides of the river during the month of April. Following a recent slide talk and book signing at the Burlington County Library in Westampton, New Jersey, Shafer is gearing up for four more appearances by the end of the month.
This Thursday, April 20, the author will be presenting a digital slide show, followed by an audience question-and-answer session, and will be selling and signing copies of the book at the Phillipsburg Public Library at 200 Frost Avenue in Phillipsburg, NJ. For more information, contact Linda Paff at 908-454-3712, or visit the library’s website at http://www.pburglib.com.
From 1:00-3:00 p.m. this Saturday, April 22, the author will be signing copies of her book at the Hobby Hangout at 3701 William Penn Hwy. in Easton, PA. For more information, call 610-252-6871.
The Haycock Historical Society will be hosting the author’s digital slide show, Q&A and a book signing at 7:30 p.m. next Thursday, April 27. The event will be held at St. Paul’s Lutheran Church, 837 Old Bethlehem Road in Applebachsville, PA. Contact Chris Handschin via email at chandschin AT netcarrier.com for more information.
Finally, Shafer will be appearing to sell and sign her book during this year’s annual ShadFest celebration in Lambertville, NJ. The author will be found in the Word Forge Books booth on Saturday and Sunday, April 29-30, from Noon-5:00 p.m. More ShadFest information is available on the Web at http://www.lambertville.org/main.php?page=shadfest.
# # #
Mary Shafer, author – 610-847-2456 or floodbook AT thewordforge.com
Or directly contact the author


Attachment 32647I have a metal high seat which I am now not likely to ever need again so am asking for £50.00. It can be taken down but involves a lot of nuts and bolts and to re-assemble it would be like tackling a chinese puzzle. I have it in two pieces and am sure it will go on a roof rack in that condition but a trailer may make it easier for collection, posting it is out of the question. I have attached a picture of it leant against a door with the bottom section propped up in front of it with the extending metal bracing stay and fixing strap attached to it. The top half assembled is 6ft to the seat and the bottom section is 4ft so when erected is 10ft to the seat. I must emphasise that it is collection only. I am having difficulty attaching any pictures so an e mail to me might enable me to send a picture. Just checked my profile, it seems you may have to PM me first to get my E Mail addy.


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NKX3.1 is a homeobox gene located at chromosome 8p21.2, and one copy is frequently deleted in prostate carcinoma. Prior studies of NKX3.1 mRNA and protein in human prostate cancer and prostatic intraepithelial neoplasia (PIN) have been conflicting, and expression in focal prostate atrophy lesions has not been investigated. Immunohistochemical staining for NKX3.1 on human tissue microarrays was decreased in most focal atrophy and PIN lesions. In carcinoma, staining was inversely correlated with Gleason grade. Fluorescence in situ hybridization showed that no cases of atrophy had loss or gain of 8p, 8 centromere, or 8q24 (C-MYC) and only 12% of high-grade PIN lesions harbored loss of 8p. By contrast, NKX3.1 staining in carcinoma was correlated with 8p loss and allelic loss was inversely related to Gleason pattern. Quantitative reverse transcription-PCR for NKX3.1 mRNA using microdissected atrophy revealed a concordance with protein in five of seven cases. In carcinoma, mRNA levels were decreased in 6 of 12 cases but mRNA levels correlated with protein levels in only 4 of 12 cases, indicating translational or post-translational control. In summary, NKX3.1 protein is reduced in focal atrophy and PIN but is not related to 8p allelic loss in these lesions. Therefore, whereas genetic disruption of NKX3.1 in mice leads to PIN, nongenetic mechanisms reduce NKX3.1 protein levels early in human prostate carcinogenesis, which may facilitate both proliferation and DNA damage in atrophic and PIN cells. Monoallelic deletions on chromosome 8p are associated with more advanced invasive and aggressive disease. (Cancer Res 2006; 66(22): 10683-90)
- focal prostatic atrophy
- proliferative inflammatory atrophy
- prostatic carcinoma
Prostatic adenocarcinoma is one of the most common malignancies, representing the second leading cause of cancer death in American men ( 1). NKX3.1 is located on chromosome 8p21.2 within a region that shows loss of heterozygosity (LOH) in prostate cancer in approximately 50% to 85% of cases ( 2, 3). Given that mutations in the remaining allele of NKX3.1 have not been detected ( 4, 5), NKX3.1 may function as a haploinsufficient tumor suppressor gene. That loss of one allele of NKX3.1 occurs early in prostate carcinogenesis is evidenced by the finding that LOH on chromosome 8p has been reported to occur in high-grade prostatic intraepithelial neoplasia (PIN), a lesion that is a putative precursor to many invasive prostatic carcinomas ( 6), at a frequency between 20% and 80% ( 7– 9).
Targeted disruption of Nkx3.1 in mice results in abnormal prostate ductal morphogenesis and protein secretion ( 10– 12). Although Nkx3.1 homozygous mutant mice do not develop invasive carcinoma, epithelial hyperplasia and PIN lesions arise with age. Compound mutant mouse studies indicate that cooperativity exists between Nkx3.1 and the tumor suppressors Pten and Cdkn1b (encoding p27; refs. 13– 17). These compound mutants develop PIN lesions that progress to invasive carcinomas and at times to metastatic disease. Because the effects are seen in NKX3.1 heterozygotes, haploinsufficiency of Nkx3.1 also seems to play a role in tumor progression.
Several studies have analyzed NKX3.1 protein expression by immunohistochemistry in human prostate cancer specimens. In a study of PIN and carcinoma, loss of NKX3.1 protein staining was reported in ∼20% of PIN lesions and ∼40% of carcinomas, and loss of expression correlated with high Gleason score, advanced tumor stage, the presence of metastatic disease, and hormone-refractory disease ( 18). However, a more recent study reported that NKX3.1 protein was highly expressed in adenocarcinoma, irrespective of tumor grade ( 19). Asatiani et al. ( 20) revisited the question of NKX3.1 protein expression in prostate cancer and reported that complete loss of staining was very rare (∼5% of cases) but that the intensity of staining in carcinoma was reduced compared with normal epithelium. The reduction of intensity correlated with the combination of loss of one copy of NKX3.1 and hypermethylation of cytosine residues in specific CpG dinucleotides near the putative NKX3.1 promoter region as well as with Gleason grade. Thus, when examining the body of data as a whole, there is still substantial controversy about NKX3.1 protein expression in human prostate cancer.
In terms of NKX3.1 mRNA expression levels in human prostate tumors, there have also been conflicting results. Semiquantitative reverse transcription-PCR (RT-PCR) analyses of benign and malignant tissues revealed overexpression of NKX3.1 mRNA in 31% of cancer specimens, decreased expression in 21%, and no change in 48% ( 21). In the study by Xu et al. ( 21), overexpression of NKX3.1 mRNA correlated with metastatic disease. In contrast, other groups have reported NKX3.1 mRNA to be similarly expressed in normal prostate and tumors of various grades ( 5, 19), and this correlated with NKX3.1 protein ( 19, 20). Thus, controversy remains about NKX3.1 mRNA expression in human prostate cancer.
Focal prostatic atrophy is a common finding in older men and encompasses several morphologic variants ( 22). Some of these lesions are proposed as potential precursors to prostate cancer based on their frequent occurrence in proximity to carcinoma ( 23– 26) and molecular alterations (see below). Focal prostate atrophy commonly merges directly with low- and high-grade PIN ( 24, 25, 27), suggesting that some PIN lesions arise directly from atrophy. More rarely, focal atrophy seems to merge directly with small carcinoma lesions ( 23, 27– 29). Two common types of focal prostatic atrophy, simple atrophy and postatrophic hyperplasia (PAH), are often highly proliferative and associated with inflammation ( 24, 30) and are referred to as proliferative inflammatory atrophy (PIA). 6
We have proposed a multistep progression model that leads from normal epithelium to focal prostatic atrophy to PIN and then to carcinoma ( 24, 31). In this model, ongoing injury to the prostate epithelium, as a result of inflammation and/or dietary exposures, results in cell damage and cell death. Cell regeneration ensues and this is manifest morphologically as PA. These atrophic cells are presumed to be responding to increased oxidative and/or nitrosative stress ( 32– 34) and are apparently undergoing tissue repair/renewal ( 35, 36). Many of the atrophic cells possess a phenotype intermediate between basal cells and mature luminal cells ( 33, 36). These cells may then undergo somatic genome alterations, including methylation of the GSTP1 promoter and telomere shortening, leading to neoplastic transformation. There have been no reports, however, in which NKX3.1 protein expression in focal prostate atrophy has been examined. NKX3.1 mRNA expression in prostatic atrophy has only been briefly described as being decreased relative to normal ( 5).
The primary objective of this study was to determine the potential relevance of molecular alterations of NKX3.1 in relation to our progression model of human prostate carcinogenesis. We now report the results of the following: (a) a quantitative analysis of the expression of NKX3.1 protein using a novel anti-NKX3.1 polyclonal antibody on tissue microarrays (TMA) containing matched samples of normal, focal prostate atrophy, PIN, and clinically localized adenocarcinoma of various Gleason grades; (b) a study to correlate NKX3.1 protein levels with chromosomal alterations on chromosome 8 using fluorescence in situ hybridization (FISH) on the same TMA specimens; and (c) a study to determine if NKX3.1 protein levels correlate with mRNA levels using laser capture microdissection of frozen tissue specimens for quantitative RT-PCR.
Materials and Methods
Generation of anti-NKX3.1 rabbit polyclonal antibodies. To derive polyclonal anti-NKX3.1 antibodies, a His-tagged polypeptide containing the NH2-terminal 153 amino acids of human NKX3.1 was expressed in Escherichia coli and purified over a nickel affinity resin followed by anion exchange chromatography. Fractions with >95% pure His-tagged NH2-terminal NKX3.1 were used to immunize rabbits. Immunoglobulins from production bleeds were purified using a protein A column.
Tissues and TMA construction. This study was approved by our institutional internal review board. Tissue specimens were obtained from radical retropubic prostatectomies done at the Johns Hopkins Hospital (Baltimore, MD). Patient ages ranged from 44 to 74 years. Final Gleason sums varied from 5 to 9, with pathologic stages of the tumors ranging from T2N0Mx to T3BN1Mx (Supplementary Table S1).
For NKX3.1 immunostaining, we used five high-density TMAs. Two of these were designed to compare matched normal and atrophy, and the other three were designed to compare matched normal and carcinoma. One of the atrophy arrays consisted of matched normal (n = 4 cores each from 20 patients) and atrophy (n = 4 cores each from 3 separate atrophy lesions per patient for a total of 58 different atrophy lesions from the 20 patients). For the second atrophy array, there were four cores of normal and four cores of atrophy each from 39 patients. The classification of prostate atrophy lesions was according to De Marzo et al. ( 22). Several TMA cores incidentally contained PIN. For the matched cancer and normal TMAs, the arrays were constructed with matched samples from 40 patients per array as described previously ( 34, 37). Images of each TMA core were captured by scanning of the TMA slides using the Automated Cellular Imaging System II (ACIS II) from Clarient, Inc. (Aliso Viejo, CA) and were imported into the TMAJ Images application 7 as described ( 37). Histologic diagnoses were applied to all images used for the analyses, and these diagnoses are listed in Supplementary Table S2.
Immunohistochemistry. Immunohistochemistry was done with the EnVision+ kit (DakoCytomation, Carpinteria, CA). Slides were steamed for 20 minutes in 10 mmol/L citrate buffer (pH 6.0) for antigen retrieval. Slides were incubated with primary antibodies (1:5,000 dilution) overnight at 4°C. For keratin 8 (CK8) staining, slides were prepared as described above and stained with CK8 antibody (1:800 dilution; InnoGenex, San Ramon, CA) at room temperature for 45 minutes.
Evaluation of immunohistochemical staining. The average intensity and area of staining were analyzed on TMA slides using the ACIS II ( 37). Tissue cores containing more than one type of tissue (i.e., atrophy and carcinoma) were not used for data analysis. The area of nuclear staining for NKX3.1, which is epithelial specific, was normalized to the area of keratin 8 staining of the cytoplasm as described ( 37). Statistical analyses were done using Stata 8.0 software (Stata, College Station, TX).
For each TMA core, a mean intensity of staining and an area of staining were assigned by the ACIS. In most cases, there were multiple cores from the same patient showing the same diagnosis, and image analysis values were averaged for statistical analysis.
Fluorescence in situ hybridization. The FISH technique was modified from that described previously ( 38). FISH hybridization was done with the multicolor probes (ProVysion, Vysis, Inc., Downers Grove, IL) to detect and quantify chromosome 8 centromere probe [chromosome enumeration probe 8 (CEP8)] and with two locus-specific probes, the lipoprotein lipase (LPL; 8p21.3) and the c-MYC 8q24 probe. Three of the same TMAs used for NKX3.1 staining were used for FISH, and three additional TMAs were also used for FISH from which FISH data from additional PIN lesions were obtained. Up to 30 nonoverlapping interphase nuclei from each core/region of benign normal-appearing epithelium, atrophy, PIN, and carcinoma were counted for each probe. For each TMA core that was counted by FISH, there was an internet-based image of a H&E-stained adjacent TMA section that was annotated with the pathologist's diagnosis. Normal value studies using prostate tissue have been previously reported ( 38). An inspection of the copy number of each FISH signal in a nucleus was recorded, and the ratios and distributions of each probe (LPL, c-MYC, and CEP8) of a given core were categorized as normal, gain, abnormal increase, or loss. The threshold values for these categories were chosen to minimize the detection of false-positive changes. The normal category required <30% of epithelial nuclei with three or more signals and <60% of epithelial nuclei with zero or one signal for an applied probe. The gain category required ≥30% of epithelial nuclei with three or more signals for an applied probe. The category of abnormal increase of c-MYC (8p24) required ≥30% of epithelial nuclei with three or more signals for c-MYC (8p24) and an overall 8p24/CEP8 ratio of >1.30. The category of loss of CEP8 required ≥60% of epithelial nuclei with zero or one signal for CEP8. The category of loss of 8p required ≥60% of epithelial nuclei with zero or one signal for LPL and the overall LPL/CEP8 ratio of <0.80.
Laser capture microdissection. For each case, two serial 7-μm frozen sections were fixed in 70% ethanol for 1 minute followed by a rinse in diethyl pyrocarbonate–treated water for 30 seconds. Slides were lightly stained with hematoxylin for 45 seconds and dehydrated. Regions of normal epithelium, atrophy, and tumor were microdissected using the AutoPix Automated Laser Capture Microdissection System (Arcturus Engineering, Inc., Mountain View, CA). Immunohistochemistry was also done to compare NKX3.1 staining in regions microdissected for quantitative RT-PCR.
Quantitative RT-PCR. Total RNA was isolated from laser capture microdissected cells with the Absolutely RNA Microprep RNA Isolation kit (Stratagene, La Jolla, CA) following the manufacturer's protocol. On-column DNase I digestion was done during the RNA extraction. RNA was ethanol precipitated and used to generate cDNA with the iScript cDNA Synthesis kit (Bio-Rad, Richmond, CA). Quantitative PCR was done using SYBR Green I and the iCycler (Bio-Rad) with intron-spanning primers for NKX3.1 (5′-GAATCCGTATGCCCCGCTG-AATCT-3′ and 5′-ACCCTTGCCAGTGCGTGTGC-3′) and for TATA-binding protein (TBP; 5′-CACGAACCACGGCACTGATT-3′ and 5′-TTTTCTTGCTGCCAGTCTGGAC-3′). For quantification of NKX3.1 mRNA levels, we normalized to TBP mRNA, which was used as an endogenous control. The relative amount of NKX3.1 mRNA in each experimental sample was determined using the ΔΔCt method ( 39). Two independent triplicate quantitative PCR experiments were done for each case.
Visual/qualitative assessment of NKX3.1 immunostaining. The specificity of the newly generated polyclonal antibody is shown as supplementary data (Supplementary Fig. S1). In normal epithelium, NKX3.1 staining was intense in the nuclei of most secretory luminal cells ( Fig. 1A and B ), with very faint or negative cytoplasmic staining. The pattern of nuclear staining, as seen at high power, was granular, with both fine granules and more clumped granules seen. Luminal cells throughout a given prostate acinus did show some variability in the intensity of nuclear staining. Although many acini showed negative staining in basal cells, many acini contained weak staining in the nuclei of basal cells ( Figs. 1B and 3A). There was no staining in nonepithelial tissues in the prostate.
Compared with normal epithelium, most atrophy lesions contained reduced NKX3.1 protein ( Fig. 1C and D). Both the intensity of staining and the percentage of cells staining were decreased albeit to a variable extent. Often, the entire lesion was reduced, but in other cases only part of the lesion, or even individual acini, showed reduced staining. At times, some cells in atrophy showed strong staining that was similar to normal epithelium ( Fig. 1D, arrowhead). The granular pattern of nuclear staining in atrophy was similar to that seen in normal-appearing epithelium as was the general lack of cytoplasmic staining.
NKX3.1 immunostaining was seen in all cases of PIN, but the intensity in most cells was usually less than in the normal-appearing luminal cells ( Fig. 2B ), and there were a variable number of cells in the luminal compartment with very faint nuclear staining. Cytoplasmic staining in PIN was generally similar to that in normal epithelium, although at times it was somewhat more prominent in PIN than in normal.
Most carcinomas showed reduced, albeit variable, staining for NKX3.1 ( Fig. 3B and F ). Many carcinomas showed very mild reductions in intensity of staining, with occasional increases in the number of entirely negatively staining cells. Others showed a more marked reduction in intensity in many of the cells, with more cells staining completely negatively ( Fig. 3F). In rare cases, mitotic figures were seen, and these cells were clearly negative for NKX3.1 staining (data not shown). Cytoplasmic staining in cancer was generally similar to that seen in normal epithelium, although like PIN there were several cases with slightly stronger cytoplasmic staining than that seen in normal-appearing epithelium.
Quantitative image analysis of NKX3.1 staining. Automated image analysis 8 showed that, compared with normal epithelium, the intensity of staining in atrophy lesions was reduced significantly ( Fig. 4A ). The area of nuclear staining in atrophy compared with normal epithelium was even more reduced than the intensity values ( Fig. 4C; Table 1 ). There was no difference in intensity or area of NKX3.1 staining when stratified by type of atrophy (e.g., simple atrophy versus PAH versus simple atrophy with cyst formation; data not shown).
The intensity of NKX3.1 staining in PIN lesions was also reduced significantly in >75% of lesions when compared with normal-appearing epithelium ( Fig. 4B; Table 1). PIN lesions also showed a reduction in area of staining, yet this reduction was not statistically significant ( Table 1B).
In carcinoma, the intensity of NKX3.1 staining was significantly reduced compared with normal, and similar to PIN lesions, the area of staining in carcinoma was reduced but this difference was not significant ( Table 1). In terms of tumor grade, we analyzed the intensity and area of staining for NKX3.1 in the TMA spots in relation to the Gleason score for the prostatectomy case. The intensity for NKX3.1 staining was decreased in all Gleason scores represented; however, the decrease was greater in higher-grade tumors (Gleason scores 8-9; Table 1; Fig. 4B). In terms of area of staining in carcinoma, there was a significant reduction compared with normal only in cases with higher-grade tumors (Gleason sum 8-9; Fig. 4D; Table 1). When the data were analyzed by Gleason pattern of the individual tissue cores, similar results were obtained—those spots with higher grade were generally found to have less intense NKX3.1 staining and less area of staining (data not shown). In multivariate regression analysis, including both Gleason score and NKX3.1 area of staining, only Gleason score remained significant at predicting pathologic stage (data not shown).
Because NKX3.1 is a tumor suppressor with known inhibitory effects on cell proliferation ( 13, 40), we determined whether cells in atrophy that were proliferating, as assessed by Ki67 staining, contained lower levels of NKX3.1. Double-label immunofluorescence was done on one TMA containing atrophy and matched normal and one containing carcinoma and matched normal. By immunofluorescence, there were clear gradations in staining for both makers (Supplementary Fig. S2). In general, when a cell was positive for any amount of Ki67, it tended to be negative or only weakly positive, for NKX3.1, compared with other cells in the same lesions (Supplementary Fig. S2). In cells with the most Ki67 staining, NKX3.1 was virtually always negative. This inverse relation between Ki67 and NKX3.1 suggests that NKX3.1 protein is regulated in a cell cycle–dependent manner.
NKX3.1 mRNA expression. To evaluate the levels of NKX3.1 mRNA, quantitative real-time PCR using RNA prepared from laser microdissected frozen tissue sections was done. Eight atrophy lesions and matched normal-appearing tissues were evaluated. Adjacent sections were also immunostained with the anti-NKX3.1 antibody (in seven of the cases). Three of eight of the atrophy cases microdissected showed a decrease in relative NKX3.1 mRNA abundance (cases A, G, and J; Table 2 ). In the two of these three cases (cases A and G), immunohistochemical analysis revealed that the protein levels were also reduced. Five cases of atrophy displayed no change or increased NKX3.1 mRNA levels compared with matched normal specimens (cases C, D, E, F, and H), yet two had reduced protein levels (40%; cases E and F). There were no cases of atrophy where the mRNA was decreased but the protein staining was intact. Overall, therefore, mRNA and protein levels in focal prostate atrophy lesions were concordant in five of seven cases that were evaluated ( Table 2). The two cases that were not concordant showed that, despite normal mRNA levels, protein staining was decreased.
In carcinoma, the NKX3.1 mRNA was reduced in 6 of 12 cases, was normal in 5 cases, and was increased in 1 case ( Table 2). Of the six cases with reduced mRNA that were stained by immunohistochemistry, two (33%; cases D and F) showed reduced protein staining. Of the six cases with normal or elevated mRNA levels, however, four of these also showed reduced protein staining (66%; cases I, K, L, and N). There were three cases of carcinoma in which the mRNA levels were reduced, yet the protein staining seemed intact. Overall, therefore, only four of nine cases of carcinoma that could be evaluated were concordant between mRNA and protein.
Relation of NKX3.1 protein and chromosome 8p deletion. Three of the same TMAs used above were subjected to FISH. Two of the TMAs consisted of matched atrophy and normal-appearing epithelium and the other was from matched normal and adenocarcinoma. A total of 466 TMA spots from 33 patients were analyzed, including 359 cores of normal and 245 cores of atrophy. Although one atrophy and one normal TMA core were close to the cutoff for deletion of 8p, none of the normal or atrophic regions were found to meet our strict criteria for loss or gain of 8p, 8 centromere, or C-MYC. As in previous studies, ( 26, 41), we also compared the fraction of cells with three or more chromosome 8 centromere signals in atrophy and matched normal in one of the TMAs. Although there was a significant increase in the fraction of cells in atrophy that harbored three or more signals for chromosome 8 centromere compared with normal (2.4% for atrophy versus 1.2%; P = 0.024, Wilcoxon rank sum), no TMA cores containing prostate atrophy were considered to have gained chromosome 8 centromere in a clonal fashion.
In carcinoma, deletion of 8p sequences occurred in 52% of TMA cores and much more frequently in those that were poorly differentiated (74% for Gleason patterns 4-5) compared with those cores that were more well differentiated (33% for Gleason pattern 3; Supplementary Table S4). This increase in the fraction of high-grade lesions with 8p deletion is consistent with other previous studies ( 42– 44). In addition, both reduced intensity and reduced area of NKX3.1 staining correlated with LOH ( Fig. 5 ; P < 0.001 for area and intensity, Kruskal-Wallis).
On these three TMAs, there were six high-grade and two low-grade PIN lesions. Of these, there was one deletion of one allele on 8p in a high-grade lesion (one of six, 17%) and no gains or losses of 8 centromere or 8q24 in any of the PIN lesions. Three additional TMAs containing matched cancer and normal prostate were evaluated and these contained 19 high-grade PIN lesions and 8 low-grade PIN lesions and none of these had gain of 8 centromere or 8q24. Of these, there were two cases with deletion of one allele on 8p in two of the high-grade lesions and none in the low-grade PIN. Thus, overall, 3 of 25 (12%) high-grade and 0 of 10 (0%) low-grade PIN lesions had loss of 8p.
Immunohistochemical analyses of atrophy showed a profound decrease in both staining intensity and percentage area of staining for NKX3.1. These observations fit well with the known functions of NKX3.1 in growth control and with a multistep progression model that leads from normal epithelium to PIA to PIN and then to carcinoma ( 24, 31). The diminished expression of NKX3.1 observed in PIA may reflect a response to oxidative stress. By contrast, because lack of NKX3.1 results in increased oxidative DNA damage by controlling expression of antioxidant enzymes ( 45), reduced NKX3.1 in PIA may contribute to increased oxidant damage in these lesions.
The decreased level of NKX3.1 in PIA may reflect the intermediate cell phenotype that we have proposed for many atrophic luminal cells in PIA ( 36). The levels in most cells that were positive were greater than that seen in basal cells but less than that seen in mature columnar luminal cells. The intermediate level of androgen receptor in PIA ( 24) and the androgen responsiveness of NKX3.1 fit well with this model.
The molecular basis of diminished NKX3.1 in PIA is not clear. Allelic loss does not seem to play a role because many of the atrophic lesions had reduced NKX3.1 protein but none had deletion on chromosome 8p21.3 in the region of the LPL probe. It is still possible, albeit unlikely, that there are small deletions of chromosome 8p21.2 in the NKX3.1 locus itself that would not be detected by the LPL probe. Additional studies using more refined FISH probes will be necessary to rule this out. Quantitative analyses of NKX3.1 steady-state mRNA levels revealed concordance in five of seven cases examined between NKX3.1 mRNA and protein levels in PIA, suggesting that control of NKX3.1 protein levels in prostate atrophy is related to mRNA levels in most cases. These results agree with Ornstein et al. ( 5), who examined NKX3.1 mRNA levels in frozen prostate tissues by in situ hybridization with a radiolabeled probe and reported decreased signal in “atrophic” regions, albeit the number of cases with atrophy was not indicated. The data are consistent with regulation of NKX3.1 mRNA in atrophy at the level of transcription, mRNA processing, or mRNA stability. In two cases, mRNA was normal and protein was decreased. Thus, regulation of NKX3.1 protein levels likely occurs at multiple steps, including at the level of translation and/or protein stability. The serine-threonine kinase CK2 has recently been shown to play a central role in the stability of NKX3.1 by protecting it from proteasomal degradation ( 46).
It is not clear precisely why there are increased signals for chromosome 8 centromere in PIA. One potential explanation is that because the fraction of cells is still very low compared with cases that show gain in a clonal fashion (i.e., >30% of cells have three or more signals), it is possible that this finding simply reflects the known increase in proliferative fraction in prostate atrophy compared with normal epithelium. Cells that have traversed into late S phase or G2-M would have double the number of chromosomes and thus would be expected to show an increase in centromeric FISH signals. In the single previous study that mentioned prostate atrophy using FISH for chromosome 8p, two of seven prostate atrophy lesions had allelic loss ( 41). The most likely explanation for our lack of finding LOH on 8p in atrophy is that we used a more stringent statistical cutoff to indicate loss of the 8p probe ( 41).
NKX3.1 staining intensity was significantly diminished (P = 0.0064) in PIN lesions compared with normal epithelium, in agreement with a recent study ( 20). In this study, we report the novel finding that reduced NKX3.1 protein expression in PIN does not correlate with loss of chromosome 8p because most PIN (>75%) lesions showed decreased protein expression but only 12% showed loss of 8p. Thus, loss of NKX3.1 protein seems to precede genetic loss in prostate neoplasia. These data help to clarify the temporal ordering of molecular alterations in human prostate carcinogenesis and indicate that additional mechanisms of down-regulation of NKX3.1, other than gene deletion, are important in human PIN.
Our study revealed a statistically significant reduction in NKX3.1 immunostaining in prostate cancer compared with normal epithelium, in agreement with several previous reports ( 18, 20). The studies reported here showed a clear correlation between reduced NKX3.1 protein expression and high Gleason grade, which also agrees with recent study by Asatiani ( 20).
Unlike what we observed in most PIA lesions, a general lack of concordance between the mRNA and protein accumulation in carcinoma was found. In some tumors, both mRNA and protein were diminished to a similar extent, in agreement with a recent report ( 19). In others, however, reduced protein accumulation was observed in lesions with a normal mRNA level. A third class of lesions showed reduced NKX3.1 staining despite having a greater-than-normal level of mRNA. Increased NKX3.1 mRNA levels have been previously reported in a subset of cases ( 21). The most likely scenario is that multiple mechanisms operate to down-regulate NKX3.1 protein in normal, atrophic, and neoplastic prostate epithelial cells.
One mechanism of NKX3.1 regulation is at the level of gene dosage. Our data show a strong correlation between 8p loss and NKX3.1 level in carcinoma but not in atrophy or PIN. This suggests that loss of one NKX3.1 allele may be sufficient to result in a measurable decrease in protein accumulation that may result in a state permissive for cell cycle progression. This correlation seen in our study in carcinoma agrees with a recent study that analyzed cases with 8p21 loss ( 20). Although, in that study, the authors also reported that the methylation status of individual CpG dinucleotides in sequences upstream of the transcription start site, when combined with loss of 8p sequences, was the best predictor of decreased protein levels. However, there is currently no functional evidence linking the putative sites of methylation to the transcriptional control of NKX3.1. An enhancer-containing region well downstream of the mouse NKX3.1 gene has recently been reported to be necessary and sufficient for prostate-restricted gene expression in vivo ( 47). It will be important to examine the methylation status of its cognate in the human NKX3.1 locus.
In summary, we report that NKX3.1 protein is decreased in PIA/PA in the human prostate. In most cases, this relates to steady-state mRNA levels but does not relate to gene dosage. In PIN lesions, NKX3.1 was reduced but this too did not correlate with loss of chromosome 8p sequences. Indeed, only 12% of high-grade PIN lesions, and no low-grade PIN lesions, showed 8p21.3 loss by FISH, which is a lower value than even the lowest (20%) that has been reported previously. By contrast, 33% of Gleason pattern 3 and 74% of Gleason pattern 4/5 carcinomas contained 8p deletions, and the deletions correlated with NKX3.1 protein in carcinoma. Thus, deletion of sequences on chromosome 8p21.3 may be involved more with progression of invasive disease than in initiation of disease. Because NKX3.1 may protect cells from oxidative stress, these results support a model whereby low levels of NKX3.1 facilitate both proliferation and DNA damage in atrophic prostate epithelial cells and in PIN cells, increasing the risk of carcinogenesis. By the same mechanisms, reduced NKX3.1 in carcinoma may facilitate disease progression.
Grant support: National Cancer Institute (NCI) National Research Service Award grant CA94818-04 (C.R. Bethel), Public Health Services NIH/NCI grant R01CA84997 and NIH/NCI Specialized Program of Research Excellence in Prostate Cancer grant P50CA58236 (Johns Hopkins), and The Donald and Susan Sturm Foundation (A.M. De Marzo); NIH/NIDDK grant R01DK54067 and DAMD-03-1-0091 from the Congressionally Directed Medical Research Program (C.J. Bieberich).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Dr. Alan K. Meeker for thoughtful discussions and the Johns Hopkins Brady Urological Institute Prostate Specimen Repository for prostate TMAs.
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).
↵6 For similar lesions without inflammation, the term proliferative atrophy (PA) may be used. In this article, most of the lesions examined represent simple atrophy and PAH. Therefore, we use the terms focal prostate atrophy and PIA/PA interchangeably.
↵8 Only nuclear staining was evaluated for image analysis.
- Received March 14, 2006.
- Revision received June 19, 2006.
- Accepted September 20, 2006.
- ©2006 American Association for Cancer Research.


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